EUR 25245 EN - 2012

Report on the 8th inter-laboratory

comparison organised by the European

Union Reference LaboratoryFour marker PAHs in a dry extract of St Johns wort

Radoslav Lizak Szilard Szilagyi Philippe Verlinde and Thomas Wenzl

The mission of the JRC-IRMM is to promote a common and reliable European measurement system in support of EU policies European Commission Joint Research Centre Institute for Reference Materials and Measurements Contact information Thomas Wenzl Address Retieseweg 111 B-2440 Geel E-mail jrc-irmm-crl-paheceuropaeu Tel +32 (0)14 571 320 Fax +32 (0)14 571 783 httpirmmjrceceuropaeu httpwwwjrceceuropaeu Legal Notice Neither the European Commission nor any person acting on behalf of the Commission is responsible for the use which might be made of this publication

Europe Direct is a service to help you find answers to your questions about the European Union

Freephone number ()

00 800 6 7 8 9 10 11

() Certain mobile telephone operators do not allow access to 00 800 numbers or these calls may be billed

A great deal of additional information on the European Union is available on the Internet It can be accessed through the Europa server httpeuropaeu JRC 69083 EUR 25245 EN ISBN 978-92-79-23337-1 ISSN 1831-9424 doi 10278759387 Luxembourg Publications Office of the European Union 2012 copy European Union 2012 Reproduction is authorised provided the source is acknowledged

The mission of the JRC-IRMM is to promote a common and reliable European measurement system in support of EU policies European Commission Joint Research Centre Institute for Reference Materials and Measurements Contact information Thomas Wenzl Address Retieseweg 111 B-2440 Geel E-mail jrc-irmm-crl-paheceuropaeu Tel +32 (0)14 571 320 Fax +32 (0)14 571 783 httpirmmjrceceuropaeu httpwwwjrceceuropaeu Legal Notice Neither the European Commission nor any person acting on behalf of the Commission is responsible for the use which might be made of this publication

Europe Direct is a service to help you find answers to your questions about the European Union

Freephone number ()

00 800 6 7 8 9 10 11

() Certain mobile telephone operators do not allow access to 00 800 numbers or these calls may be billed

A great deal of additional information on the European Union is available on the Internet It can be accessed through the Europa server httpeuropaeu JRC 69083 EUR 25245 EN ISBN 978-92-79-23337-1 ISSN 1831-9424 doi 10278759387 Luxembourg Publications Office of the European Union 2012 copy European Union 2012 Reproduction is authorised provided the source is acknowledged

EU-RL-PAH-08

Report on the 8th inter-laboratory

comparison organised by the European Union Reference Laboratory for Polycyclic Aromatic

Hydrocarbons

Four marker PAHs in a dry extract of St Johns wort

Radoslav Lizak Szilard Szilagyi Philippe Verlinde and Thomas Wenzl

EC-JRC-IRMM 06022012

Coordinator

2

Table of Contents

1 Summary 3

2 Introduction 4

3 Scope 5

4 Participating Laboratories 6

5 Time frame 7

6 Confidentiality 8

7 Test materials 8

71 Preparation of test materials 8

72 Assigned values and standard deviation for proficiency assessment 8

73 Homogeneity and stability 11

74 Sample dispatch 12

8 Design of the proficiency test 12

9 Evaluation of the results 13

91 General 13

92 Evaluation criteria 13

93 Performance of participants 14

94 Evaluation of the influence of analysis method 22

95 Evaluation of the influence of calibration 24

96 Evaluation of compliance with legislation 26

10 Follow-up actions for underperforming laboratories 27

11 Conclusions 28

12 Acknowledgements 28

13 References 29

14 ANNEXES 31

3

1 Summary

This report presents the results of the eighth inter-laboratory comparison (ILC) organised by the

European Union Reference Laboratory for Polycyclic Aromatic Hydrocarbons (EU-RL PAHs) on the

determination of the four marker PAHs benz[a]anthracene (BAA) benzo[a]pyrene (BAP)

benzo[b]fluoranthene (BBF) and chrysene (CHR) in a dry extract of St Johns wort It was conducted

in accordance with ISO Standard 17043

In agreement with National Reference Laboratories (NRLs) the test material used in this exercise was

a commercial product naturally incurred with PAHs

Both officially nominated National Reference Laboratories and official food control laboratories

(OCLs) of the EU Member States were admitted as participants Twenty-five NRLs and 23 OCLs

subscribed for participation One of the NRLs did not report results

The participants were free to choose the method for the analysis of the materials The four marker

PAHs were chosen as target analytes as limits for the sum of these four contaminants were recently

introduced in European legislation The determination of the mentioned four PAHs was mandatory for

the participants who had also to report the sum of the four analytes The performance of the

participating laboratories in the determination of the target PAHs in the test material was expressed by

both z-scores and zeta-scores

Participants also received a solution of PAHs in solvent of their choice (either toluene or acetonitrile)

with undisclosed content for the verification of their instrument calibration

A summary of the performance of the participants in the determination of the four marker PAHs in the

food supplement test material is given in the following table

Participant

group

Reporting

laboratories

Calculated

z-scores

z-scores

2

z-scores

2

Calculated

zeta-scores

zeta-scores

2

zeta-scores

2

NRLs 24 116 83 72 111 68 61

OCLs 23 109 70 64 51 29 57

In some cases a bias was discovered in particular chrysene caused problems for laboratories applying

gas chromatography with mass spectrometric detection

4

2 Introduction

The Institute for Reference Materials and Measurements (IRMM) of the European Commissions Joint

Research Centre hosts the European Union Reference Laboratory for Polycyclic Aromatic

Hydrocarbons in Food (EU-RL-PAH) One of its core tasks is to organise inter-laboratory comparisons

(ILCs) for the National Reference Laboratories (NRLs) [1 2]

Polycyclic aromatic hydrocarbons (PAHs) constitute a large class of organic substances The chemical

structure of PAHs consists of two or more fused aromatic rings PAHs may be formed during the

incomplete combustion of organic compounds and can be found in the environment In food PAHs

may be formed during industrial food processing and domestic food preparation such as smoking

drying roasting baking frying or grilling

In 2002 the European Commissionrsquos Scientific Committee on Food identified 15 individual PAHs as

being of major concern for human health These 15 EU priority PAHs should be monitored in food to

enable long-term exposure assessments and to verify the validity of the use of the concentrations of

benzo[a]pyrene (BAP) as a marker for a ldquototal-PAH contentrdquo [3] The toxicological importance of

these compounds was confirmed in 2010 by the International Agency for Research on Cancer (IARC)

which classified BAP as carcinogen to human beings (IARC group 1) and the other three target PAHs

as possibly carcinogenic to human beings (group 2b) [4]

As a consequence the European Commission (EC) issued Commission Regulation (EC) No

18812006 setting maximum levels of benzo[a]pyrene in food and Commission Regulation (EC) No

3332007 laying down sampling methods and performance criteria for methods of analysis for the

official control of benzo[a]pyrene levels in foodstuffs [5 6]

To evaluate the suitability of BaP as a marker for occurrence and toxicity of PAHs in food the

European Commission asked the European Food Safety Authority (EFSA) for a review of the previous

risk assessment on PAHs carried out by the Scientific Committee on Food (SCF)

The scientific opinion on polycyclic aromatic hydrocarbons in food was published by EFSAs Panel on

Contaminants in the Food Chain in June 2008 [7] The Contaminants Panel concluded that

benzo[a]pyrene was not a suitable indicator for the occurrence of PAHs in food and that based on the

currently available data relating to occurrence and toxicity four (PAH4) or eight substances (PAH8)

were the most suitable indicators of PAHs in food with PAH8 not providing much added value

compared to PAH4 Following these conclusions an approach for risk management was agreed in the

Standing Committee on the Food Chain and Animal Health It was agreed that maximum levels should

be set for four PAHs (PAH4) - BAA BAP BBF and CHR In addition maximum levels for BAP

would be maintained to ensure comparability of data Consequently maximum levels for the sum of

the four PAHs were included in Commission Regulation (EU) No 8352011 which amends

Commission Regulation (EC) No 18812006 [8] Coherently also the Commission Regulation (EC)

5

No 3332007 [5] laying down performance criteria of analysis methods for the official control of food

was extended to all four EU marker PAHs [9]The target PAHs are listed in 5Table 1 together with the

acronyms used in this report and with their chemical structures

Table 1 Names and structures of the four EU marker PAHs

1 Benz[a]anthracene (BAA)

3 Benzo[b]fluoranthene

(BBF)

2 Benzo[a]pyrene (BAP)

4 Chrysene

(CHR)

3 Scope

As specified in Regulation (EC) No 8822004 on official controls performed to ensure the verification

of compliance with food and feed law animal health and animal welfare rules [2] one of the core

duties of EU-RLs is to organise inter-laboratory comparison tests (ILCs)

This inter-laboratory comparison study aimed to evaluate the comparability of analysis results reported

by National Reference Laboratories (NRLs) and EU official food control laboratories (OCLs) for the

four EU marker PAHs in a commercial food supplement sample and to assess the influence of

standard preparation and instrument calibration on the performance of individual participants The

appropriateness of the reported measurement uncertainty was also tested as this parameter is important

in the compliance assessment of food with EU maximum levels

The ILC was designed and evaluated according to ISO Standard 17043 and the International

Harmonized Protocol for the Proficiency Testing of Analytical Chemistry Laboratories further

denoted as Harmonized Protocol [10 11]

The IRMM is a PT provider accredited according to ISO Standard 17043 [10]

2

Table of Contents

1 Summary 3

2 Introduction 4

3 Scope 5

4 Participating Laboratories 6

5 Time frame 7

6 Confidentiality 8

7 Test materials 8

71 Preparation of test materials 8

72 Assigned values and standard deviation for proficiency assessment 8

73 Homogeneity and stability 11

74 Sample dispatch 12

8 Design of the proficiency test 12

9 Evaluation of the results 13

91 General 13

92 Evaluation criteria 13

93 Performance of participants 14

94 Evaluation of the influence of analysis method 22

95 Evaluation of the influence of calibration 24

96 Evaluation of compliance with legislation 26

10 Follow-up actions for underperforming laboratories 27

11 Conclusions 28

12 Acknowledgements 28

13 References 29

14 ANNEXES 31

3

1 Summary

This report presents the results of the eighth inter-laboratory comparison (ILC) organised by the

European Union Reference Laboratory for Polycyclic Aromatic Hydrocarbons (EU-RL PAHs) on the

determination of the four marker PAHs benz[a]anthracene (BAA) benzo[a]pyrene (BAP)

benzo[b]fluoranthene (BBF) and chrysene (CHR) in a dry extract of St Johns wort It was conducted

in accordance with ISO Standard 17043

In agreement with National Reference Laboratories (NRLs) the test material used in this exercise was

a commercial product naturally incurred with PAHs

Both officially nominated National Reference Laboratories and official food control laboratories

(OCLs) of the EU Member States were admitted as participants Twenty-five NRLs and 23 OCLs

subscribed for participation One of the NRLs did not report results

The participants were free to choose the method for the analysis of the materials The four marker

PAHs were chosen as target analytes as limits for the sum of these four contaminants were recently

introduced in European legislation The determination of the mentioned four PAHs was mandatory for

the participants who had also to report the sum of the four analytes The performance of the

participating laboratories in the determination of the target PAHs in the test material was expressed by

both z-scores and zeta-scores

Participants also received a solution of PAHs in solvent of their choice (either toluene or acetonitrile)

with undisclosed content for the verification of their instrument calibration

A summary of the performance of the participants in the determination of the four marker PAHs in the

food supplement test material is given in the following table

Participant

group

Reporting

laboratories

Calculated

z-scores

z-scores

2

z-scores

2

Calculated

zeta-scores

zeta-scores

2

zeta-scores

2

NRLs 24 116 83 72 111 68 61

OCLs 23 109 70 64 51 29 57

In some cases a bias was discovered in particular chrysene caused problems for laboratories applying

gas chromatography with mass spectrometric detection

4

2 Introduction

The Institute for Reference Materials and Measurements (IRMM) of the European Commissions Joint

Research Centre hosts the European Union Reference Laboratory for Polycyclic Aromatic

Hydrocarbons in Food (EU-RL-PAH) One of its core tasks is to organise inter-laboratory comparisons

(ILCs) for the National Reference Laboratories (NRLs) [1 2]

Polycyclic aromatic hydrocarbons (PAHs) constitute a large class of organic substances The chemical

structure of PAHs consists of two or more fused aromatic rings PAHs may be formed during the

incomplete combustion of organic compounds and can be found in the environment In food PAHs

may be formed during industrial food processing and domestic food preparation such as smoking

drying roasting baking frying or grilling

In 2002 the European Commissionrsquos Scientific Committee on Food identified 15 individual PAHs as

being of major concern for human health These 15 EU priority PAHs should be monitored in food to

enable long-term exposure assessments and to verify the validity of the use of the concentrations of

benzo[a]pyrene (BAP) as a marker for a ldquototal-PAH contentrdquo [3] The toxicological importance of

these compounds was confirmed in 2010 by the International Agency for Research on Cancer (IARC)

which classified BAP as carcinogen to human beings (IARC group 1) and the other three target PAHs

as possibly carcinogenic to human beings (group 2b) [4]

As a consequence the European Commission (EC) issued Commission Regulation (EC) No

18812006 setting maximum levels of benzo[a]pyrene in food and Commission Regulation (EC) No

3332007 laying down sampling methods and performance criteria for methods of analysis for the

official control of benzo[a]pyrene levels in foodstuffs [5 6]

To evaluate the suitability of BaP as a marker for occurrence and toxicity of PAHs in food the

European Commission asked the European Food Safety Authority (EFSA) for a review of the previous

risk assessment on PAHs carried out by the Scientific Committee on Food (SCF)

The scientific opinion on polycyclic aromatic hydrocarbons in food was published by EFSAs Panel on

Contaminants in the Food Chain in June 2008 [7] The Contaminants Panel concluded that

benzo[a]pyrene was not a suitable indicator for the occurrence of PAHs in food and that based on the

currently available data relating to occurrence and toxicity four (PAH4) or eight substances (PAH8)

were the most suitable indicators of PAHs in food with PAH8 not providing much added value

compared to PAH4 Following these conclusions an approach for risk management was agreed in the

Standing Committee on the Food Chain and Animal Health It was agreed that maximum levels should

be set for four PAHs (PAH4) - BAA BAP BBF and CHR In addition maximum levels for BAP

would be maintained to ensure comparability of data Consequently maximum levels for the sum of

the four PAHs were included in Commission Regulation (EU) No 8352011 which amends

Commission Regulation (EC) No 18812006 [8] Coherently also the Commission Regulation (EC)

5

No 3332007 [5] laying down performance criteria of analysis methods for the official control of food

was extended to all four EU marker PAHs [9]The target PAHs are listed in 5Table 1 together with the

acronyms used in this report and with their chemical structures

Table 1 Names and structures of the four EU marker PAHs

1 Benz[a]anthracene (BAA)

3 Benzo[b]fluoranthene

(BBF)

2 Benzo[a]pyrene (BAP)

4 Chrysene

(CHR)

3 Scope

As specified in Regulation (EC) No 8822004 on official controls performed to ensure the verification

of compliance with food and feed law animal health and animal welfare rules [2] one of the core

duties of EU-RLs is to organise inter-laboratory comparison tests (ILCs)

This inter-laboratory comparison study aimed to evaluate the comparability of analysis results reported

by National Reference Laboratories (NRLs) and EU official food control laboratories (OCLs) for the

four EU marker PAHs in a commercial food supplement sample and to assess the influence of

standard preparation and instrument calibration on the performance of individual participants The

appropriateness of the reported measurement uncertainty was also tested as this parameter is important

in the compliance assessment of food with EU maximum levels

The ILC was designed and evaluated according to ISO Standard 17043 and the International

Harmonized Protocol for the Proficiency Testing of Analytical Chemistry Laboratories further

denoted as Harmonized Protocol [10 11]

The IRMM is a PT provider accredited according to ISO Standard 17043 [10]

3

1 Summary

This report presents the results of the eighth inter-laboratory comparison (ILC) organised by the

European Union Reference Laboratory for Polycyclic Aromatic Hydrocarbons (EU-RL PAHs) on the

determination of the four marker PAHs benz[a]anthracene (BAA) benzo[a]pyrene (BAP)

benzo[b]fluoranthene (BBF) and chrysene (CHR) in a dry extract of St Johns wort It was conducted

in accordance with ISO Standard 17043

In agreement with National Reference Laboratories (NRLs) the test material used in this exercise was

a commercial product naturally incurred with PAHs

Both officially nominated National Reference Laboratories and official food control laboratories

(OCLs) of the EU Member States were admitted as participants Twenty-five NRLs and 23 OCLs

subscribed for participation One of the NRLs did not report results

The participants were free to choose the method for the analysis of the materials The four marker

PAHs were chosen as target analytes as limits for the sum of these four contaminants were recently

introduced in European legislation The determination of the mentioned four PAHs was mandatory for

the participants who had also to report the sum of the four analytes The performance of the

participating laboratories in the determination of the target PAHs in the test material was expressed by

both z-scores and zeta-scores

Participants also received a solution of PAHs in solvent of their choice (either toluene or acetonitrile)

with undisclosed content for the verification of their instrument calibration

A summary of the performance of the participants in the determination of the four marker PAHs in the

food supplement test material is given in the following table

Participant

group

Reporting

laboratories

Calculated

z-scores

z-scores

2

z-scores

2

Calculated

zeta-scores

zeta-scores

2

zeta-scores

2

NRLs 24 116 83 72 111 68 61

OCLs 23 109 70 64 51 29 57

In some cases a bias was discovered in particular chrysene caused problems for laboratories applying

gas chromatography with mass spectrometric detection

4

2 Introduction

The Institute for Reference Materials and Measurements (IRMM) of the European Commissions Joint

Research Centre hosts the European Union Reference Laboratory for Polycyclic Aromatic

Hydrocarbons in Food (EU-RL-PAH) One of its core tasks is to organise inter-laboratory comparisons

(ILCs) for the National Reference Laboratories (NRLs) [1 2]

Polycyclic aromatic hydrocarbons (PAHs) constitute a large class of organic substances The chemical

structure of PAHs consists of two or more fused aromatic rings PAHs may be formed during the

incomplete combustion of organic compounds and can be found in the environment In food PAHs

may be formed during industrial food processing and domestic food preparation such as smoking

drying roasting baking frying or grilling

In 2002 the European Commissionrsquos Scientific Committee on Food identified 15 individual PAHs as

being of major concern for human health These 15 EU priority PAHs should be monitored in food to

enable long-term exposure assessments and to verify the validity of the use of the concentrations of

benzo[a]pyrene (BAP) as a marker for a ldquototal-PAH contentrdquo [3] The toxicological importance of

these compounds was confirmed in 2010 by the International Agency for Research on Cancer (IARC)

which classified BAP as carcinogen to human beings (IARC group 1) and the other three target PAHs

as possibly carcinogenic to human beings (group 2b) [4]

As a consequence the European Commission (EC) issued Commission Regulation (EC) No

18812006 setting maximum levels of benzo[a]pyrene in food and Commission Regulation (EC) No

3332007 laying down sampling methods and performance criteria for methods of analysis for the

official control of benzo[a]pyrene levels in foodstuffs [5 6]

To evaluate the suitability of BaP as a marker for occurrence and toxicity of PAHs in food the

European Commission asked the European Food Safety Authority (EFSA) for a review of the previous

risk assessment on PAHs carried out by the Scientific Committee on Food (SCF)

The scientific opinion on polycyclic aromatic hydrocarbons in food was published by EFSAs Panel on

Contaminants in the Food Chain in June 2008 [7] The Contaminants Panel concluded that

benzo[a]pyrene was not a suitable indicator for the occurrence of PAHs in food and that based on the

currently available data relating to occurrence and toxicity four (PAH4) or eight substances (PAH8)

were the most suitable indicators of PAHs in food with PAH8 not providing much added value

compared to PAH4 Following these conclusions an approach for risk management was agreed in the

Standing Committee on the Food Chain and Animal Health It was agreed that maximum levels should

be set for four PAHs (PAH4) - BAA BAP BBF and CHR In addition maximum levels for BAP

would be maintained to ensure comparability of data Consequently maximum levels for the sum of

the four PAHs were included in Commission Regulation (EU) No 8352011 which amends

Commission Regulation (EC) No 18812006 [8] Coherently also the Commission Regulation (EC)

5

No 3332007 [5] laying down performance criteria of analysis methods for the official control of food

was extended to all four EU marker PAHs [9]The target PAHs are listed in 5Table 1 together with the

acronyms used in this report and with their chemical structures

Table 1 Names and structures of the four EU marker PAHs

1 Benz[a]anthracene (BAA)

3 Benzo[b]fluoranthene

(BBF)

2 Benzo[a]pyrene (BAP)

4 Chrysene

(CHR)

3 Scope

As specified in Regulation (EC) No 8822004 on official controls performed to ensure the verification

of compliance with food and feed law animal health and animal welfare rules [2] one of the core

duties of EU-RLs is to organise inter-laboratory comparison tests (ILCs)

This inter-laboratory comparison study aimed to evaluate the comparability of analysis results reported

by National Reference Laboratories (NRLs) and EU official food control laboratories (OCLs) for the

four EU marker PAHs in a commercial food supplement sample and to assess the influence of

standard preparation and instrument calibration on the performance of individual participants The

appropriateness of the reported measurement uncertainty was also tested as this parameter is important

in the compliance assessment of food with EU maximum levels

The ILC was designed and evaluated according to ISO Standard 17043 and the International

Harmonized Protocol for the Proficiency Testing of Analytical Chemistry Laboratories further

denoted as Harmonized Protocol [10 11]

The IRMM is a PT provider accredited according to ISO Standard 17043 [10]

4

2 Introduction

The Institute for Reference Materials and Measurements (IRMM) of the European Commissions Joint

Research Centre hosts the European Union Reference Laboratory for Polycyclic Aromatic

Hydrocarbons in Food (EU-RL-PAH) One of its core tasks is to organise inter-laboratory comparisons

(ILCs) for the National Reference Laboratories (NRLs) [1 2]

Polycyclic aromatic hydrocarbons (PAHs) constitute a large class of organic substances The chemical

structure of PAHs consists of two or more fused aromatic rings PAHs may be formed during the

incomplete combustion of organic compounds and can be found in the environment In food PAHs

may be formed during industrial food processing and domestic food preparation such as smoking

drying roasting baking frying or grilling

In 2002 the European Commissionrsquos Scientific Committee on Food identified 15 individual PAHs as

being of major concern for human health These 15 EU priority PAHs should be monitored in food to

enable long-term exposure assessments and to verify the validity of the use of the concentrations of

benzo[a]pyrene (BAP) as a marker for a ldquototal-PAH contentrdquo [3] The toxicological importance of

these compounds was confirmed in 2010 by the International Agency for Research on Cancer (IARC)

which classified BAP as carcinogen to human beings (IARC group 1) and the other three target PAHs

as possibly carcinogenic to human beings (group 2b) [4]

As a consequence the European Commission (EC) issued Commission Regulation (EC) No

18812006 setting maximum levels of benzo[a]pyrene in food and Commission Regulation (EC) No

3332007 laying down sampling methods and performance criteria for methods of analysis for the

official control of benzo[a]pyrene levels in foodstuffs [5 6]

To evaluate the suitability of BaP as a marker for occurrence and toxicity of PAHs in food the

European Commission asked the European Food Safety Authority (EFSA) for a review of the previous

risk assessment on PAHs carried out by the Scientific Committee on Food (SCF)

The scientific opinion on polycyclic aromatic hydrocarbons in food was published by EFSAs Panel on

Contaminants in the Food Chain in June 2008 [7] The Contaminants Panel concluded that

benzo[a]pyrene was not a suitable indicator for the occurrence of PAHs in food and that based on the

currently available data relating to occurrence and toxicity four (PAH4) or eight substances (PAH8)

were the most suitable indicators of PAHs in food with PAH8 not providing much added value

compared to PAH4 Following these conclusions an approach for risk management was agreed in the

Standing Committee on the Food Chain and Animal Health It was agreed that maximum levels should

be set for four PAHs (PAH4) - BAA BAP BBF and CHR In addition maximum levels for BAP

would be maintained to ensure comparability of data Consequently maximum levels for the sum of

the four PAHs were included in Commission Regulation (EU) No 8352011 which amends

Commission Regulation (EC) No 18812006 [8] Coherently also the Commission Regulation (EC)

5

No 3332007 [5] laying down performance criteria of analysis methods for the official control of food

was extended to all four EU marker PAHs [9]The target PAHs are listed in 5Table 1 together with the

acronyms used in this report and with their chemical structures

Table 1 Names and structures of the four EU marker PAHs

1 Benz[a]anthracene (BAA)

3 Benzo[b]fluoranthene

(BBF)

2 Benzo[a]pyrene (BAP)

4 Chrysene

(CHR)

3 Scope

As specified in Regulation (EC) No 8822004 on official controls performed to ensure the verification

of compliance with food and feed law animal health and animal welfare rules [2] one of the core

duties of EU-RLs is to organise inter-laboratory comparison tests (ILCs)

This inter-laboratory comparison study aimed to evaluate the comparability of analysis results reported

by National Reference Laboratories (NRLs) and EU official food control laboratories (OCLs) for the

four EU marker PAHs in a commercial food supplement sample and to assess the influence of

standard preparation and instrument calibration on the performance of individual participants The

appropriateness of the reported measurement uncertainty was also tested as this parameter is important

in the compliance assessment of food with EU maximum levels

The ILC was designed and evaluated according to ISO Standard 17043 and the International

Harmonized Protocol for the Proficiency Testing of Analytical Chemistry Laboratories further

denoted as Harmonized Protocol [10 11]

The IRMM is a PT provider accredited according to ISO Standard 17043 [10]

5

No 3332007 [5] laying down performance criteria of analysis methods for the official control of food

was extended to all four EU marker PAHs [9]The target PAHs are listed in 5Table 1 together with the

acronyms used in this report and with their chemical structures

Table 1 Names and structures of the four EU marker PAHs

1 Benz[a]anthracene (BAA)

3 Benzo[b]fluoranthene

(BBF)

2 Benzo[a]pyrene (BAP)

4 Chrysene

(CHR)

3 Scope

As specified in Regulation (EC) No 8822004 on official controls performed to ensure the verification

of compliance with food and feed law animal health and animal welfare rules [2] one of the core

duties of EU-RLs is to organise inter-laboratory comparison tests (ILCs)

This inter-laboratory comparison study aimed to evaluate the comparability of analysis results reported

by National Reference Laboratories (NRLs) and EU official food control laboratories (OCLs) for the

four EU marker PAHs in a commercial food supplement sample and to assess the influence of

standard preparation and instrument calibration on the performance of individual participants The

appropriateness of the reported measurement uncertainty was also tested as this parameter is important

in the compliance assessment of food with EU maximum levels

The ILC was designed and evaluated according to ISO Standard 17043 and the International

Harmonized Protocol for the Proficiency Testing of Analytical Chemistry Laboratories further

denoted as Harmonized Protocol [10 11]

The IRMM is a PT provider accredited according to ISO Standard 17043 [10]

6

4 Participating Laboratories

Officially nominated National Reference Laboratories (NRLs) and official food control laboratories of

the EU Member States were admitted as participants The participants are listed in Table 2 and

Table 3

Table 2 List of participating National Reference Laboratories

Institute Country

AGES GmbH AUSTRIA

WIV-ISP (Scientific Institute of Public Health) BELGIUM

SGL - State General Laboratory Environmental and other Food Contamination Laboratory

CYPRUS

State Veterinary Institute Prague CZECH REPUBLIC

Danish Plant Directorate Laboratory for Feed and Fertilizers DENMARK

Technical University of Denmark (DTU) DENMARK

Tartu Laboratory of Health Protection Inspectorate ESTONIA

Finnish Food Safety Authority Evira FINLAND

LABERCA Laboratoire dEtude des Reacutesidus et des Contaminants dans les Aliments

FRANCE

Bundesamt fuumlr Verbraucherschutz und Lebensmittelsicherheit (BVL) GERMANY

General Chemical State Laboratory GREECE

Central Agricultural Office Food amp Feed Safety Directorate Feed Investigation NRL

HUNGARY

Central Agricultural Office Food amp Feed Safety Directorate Food Residues Toxicological Dept

HUNGARY

Public Analysts Laboratory Dublin IRELAND

Istituto superiore di sanitagrave ITALY

Institut of Food Safety Animal Health and Environment LATVIA

National Food and Veterinary Risk Assessment Institute LITHUANIA

National Institute of Public Health - National Institute of Hygiene POLAND

Instituto Nacional de Recursos Bioloacutegicos PORTUGAL

State Veterinary and Food Institute Dolny Kubin SLOVAKIA

Zavod za zdravstveno varstvo Maribor SLOVENIA

Centro Nacional de Alimentacioacuten SPAIN

Livsmedelsverket (SLV) SWEDEN

RIKILT The NETHERLANDS

Food Environment Research Agency UNITED KINGDOM

One of the 25 NRLs did not report results for this PT

7

Table 3 List of participating Official Food Control Laboratories

Institute Country

Federal Laboratory for the Safety of the Food Chain BELGIUM

LARECO SA BELGIUM

Laboratorium ECCA NV BELGIUM

MTT Agrifood Research Finland FINLAND

LEAV - Laboratoire de lEnvironnement et de lAlimentation FRANCE

LDA56 FRANCE

IDAC FRANCE

Laboraroire Departemental de la Sarthe FRANCE

LDA 22 FRANCE

LDA 26 FRANCE

SCL Ile de France-Massy Laboratory FRANCE

CVUA Freiburg GERMANY

CVUA-Muumlnsterland-Emscher-Lippe GERMANY

Landesbetrieb Hessisches Landeslabor GERMANY

LAVES Lebensmittelinstitut Braunschweig GERMANY

Chemisches Untersuchungsamt Hagen GERMANY

Landesamt fuumlr Verbraucherschutz Sachsen-Anhalt GERMANY

Thuumlringer Landesamt fuumlr Lebensmittelsicherheit und Verbraucherschutz GERMANY

LTZ Augustenberg GERMANY

Landesuntersuchungsanstalt Sachsen GERMANY

Bayerisches Landesamt fuumlr Gesundheit und Lebensmittelsicherheit GERMANY

State Veterinary and Food Institute Košice SLOVAKIA

ANFACO-CECOPESCA SPAIN

5 Time frame

The ILC was agreed with the NRLs at the EU-RL PAH workshop held in Brussels on 6 April 2011 It

was announced on the IRMM web page (see ANNEX 1) and registration was opened on 15 May 2011

(see ANNEX 2) Test samples were dispatched (see ANNEX 3) on the 18 July 2011 and the deadline

for reporting of results was set to finally 22 September 2011

8

6 Confidentiality

The identities of participants are kept confidential unless the participant provides a letter of consent to

the PT organiser giving permission to disclose hisher details and results to a third party

7 Test materials

71 Preparation of test materials

The test material of this PT round was a food supplement in particular a dry extract of St Johns wort

in the following denoted as FS

The test material was prepared from commercial products at the EU-RL PAH laboratories About 60

units containing each 120 pills of a dry extract of St Johns wort were acquired at local stores They

were then ground and homogenised with a Loedige Ploughsharereg mixer Portions of at least 15 g of

the homogenised powder were packed into amber glass screw cap vials

Participants also received a solution of the 15+1 EU priority PAHs in either acetonitrile or toluene

(according to their choice) with disclosed concentrations which served for checking instrument

calibration The technical specifications (see ANNEX 4) of the chosen solution were dispatched

together with the test samples

Besides the standard solution with disclosed content a solution with undisclosed content was prepared

The participants were requested to report the analyte content of this solution to the organiser of the PT

This information was used to identify potential reason for bias

72 Assigned values and standard deviation for proficiency assessment

The assigned values were determined from in-house measurements at the EU-RL PAH applying

bracketing calibration and from measurements conducted by a subcontracted external laboratory The

measurements at the EU-RL were conducted on two different days Four replicate samples were

analysed by bracketing calibration using a commercially available standard solution (Dr Ehrenstorfer

Standard 183) in each of the two analysis sessions The contract laboratory had to perform three

replicate analyses of two different samples each composed of three units of PT test samples The

analyses had to be performed on two different days

9

In cases of almost identical analysis results (difference between own results and results of contract

laboratory lt 5) the results from the in-house measurements were applied as assigned values

(applicable to BAA and BBF) If the difference between the two results exceeded 5 the average of

the two results was applied as assigned value (applicable to BAP and CHR) and the combined

uncertainty of in-house and external measurements was associated with the assigned value

The sum of PAH 4 was calculated from the individually assigned values and the corresponding

uncertainty from the uncertainties of the assigned values according to equation 1 The results of in-

house measurements and the results reported by the contract laboratory as well as the assigned values

of the target PAHs are listed in Table 4

Equation 1 2222CHRBBFBAPBAAsum uuuuu

where usum refers to the standard uncertainty of the sum of the four PAHs and uBAA u BAP uBBF and

uCHR refer to the standard uncertainty of the individual analytes

The standard deviation for proficiency assessment (σP) was set for the four individual analytes equal to

the maximum tolerated standard measurement uncertainty Uf (see Equation 2) as defined by

Commission Regulation (EC) No 3332007 [5] and Commission Regulation (EU) No 8362011 [9]

Equation 2 Uf = 22 )C((LOD2)

where Uf relates to the maximum tolerated standard measurement uncertainty LOD to the required

limit of detection α to a numeric factor depending on the concentration C

The application of Equation 2 with the assigned values listed in Table 4 the maximum tolerated value

of LOD of 030 μgkg and α equal to 02 results in Uf values as reported in Table 4

The Uf values of the individual analytes are propagated in analogy to equation 1 for the determination

of the Uf value for the sum of the four PAHs which was used as standard deviation for proficiency

assessment for the sum parameter

10

Table 4 Analyte contents of the food supplement test material

Average of two

sessions bracketing calibration

External evaluation

Assigned value

and σP

Cont microgkg 298 297 298U microgkg 024 041 024P microgkg 062

Cont microgkg 157 174 165U microgkg 020 016 026P microgkg 036

Cont microgkg 292 289 292U microgkg 028 034 028P microgkg 060

Cont microgkg 424 389 407U microgkg 057 057 081P microgkg 083

Cont microgkg 1162U microgkg 093P microgkg 125

Sum PAH4

BAA

BAP

BBF

CHR

from chemical analysis Sum of the four analytes contents for the SUM

p standard deviation for proficiency assessment For the individual analytes is equal to the uncertainty function - Uf

according to Ref [9]

U expanded uncertainty of the assigned value (k=2)

The gravimetrical preparation concentrations were used as assigned values for the standard solutions

with undisclosed content Table 5 contains the assigned values and associated expanded uncertainties

of the standard solutions with undisclosed content

11

Table 5 Analyte contents of the standard solutions with undisclosed content

Content U (k=2) Content U (k=2) microgkg microgkg microgkg microgkg

benz[a ]anthracene 4317 039 4764 043benzo[c ]fluorene 7342 061 8052 067chrysene 2904 031 3179 034cyclopenta[cd ]pyrene 2558 050 2818 055benzo[a ]pyrene 2897 038 3186 042benzo[b ]fluoranthene 2926 026 3199 029benzo[ghi ]perylene 2214 023 2438 026benzo[j ]fluoranthene 4353 038 4763 041benzo[k ]fluoranthene 2143 017 2380 019dibenzo[ae ]pyrene 5735 084 6326 093dibenzo[ah ]anthracene 2215 032 2445 035dibenzo[ah ]pyrene 2169 029 2394 032dibenzo[ai ]pyrene 3691 100 4035 109dibenzo[al ]pyrene 4627 057 5104 063indeno[123-cd ]pyrene 3793 036 4176 0395-methylchrysene 8589 087 9443 096

Solution in toluene Solution in acetonitrile

Prep conc concentration from gravimetrical preparation U expanded uncertainty of the assigned value k coverage

factor

73 Homogeneity and stability

Homogeneity of the food supplements test sample was evaluated according to ISO Standard 13528

Ten units of the St Johns wort extract test material were selected randomly and analysed by isotope

dilution gas chromatography mass spectrometry (GC-MS) The test material was rated sufficiently

homogeneous (see ANNEX 5)

The contributions of the uncertainties of homogeneity (relative uncertainties below 1 ) to the total

uncertainties of the assigned values were found negligible and were therefore not considered in the

calculations

The stability of the test materials was evaluated applying an isochronous experimental set up This

comprised the storage of test samples both at room temperature and cooled in the fridge The content

values of the cooled sample were used as reference for the stability evaluation The materials were

stored from dispatch of test samples to the participants until expiry of the deadline for reporting of

results Afterwards the samples were analysed under repeatability conditions and results were

compared The content values of the cool-stored sample and the sample stored at room temperature

were not statistically significantly different Hence stability of the samples over the whole study period

can be assumed The analysis data are presented in ANNEX 5

12

The uncertainty of stability of the test material was considered negligible compared to the uncertainty

of the characterisation measurements Therefore this parameter was not taken into account in the

determination of the uncertainties of the assigned values

74 Sample dispatch

Samples were dispatched at room temperature Each participant received at least

- one ampoule of the solution of the 15+1 EU priority PAHs in the chosen solvent with disclosed

content (2 ml)

- one ampoule of the 15+1 EU priority PAHs in the chosen solvent with undisclosed content (2

ml)

- one screw cap vial with the food supplement test material

Each parcel contained a sample receipt form the outline of the study an appropriate material safety

data sheets and a unique code that was required for reporting of results The documents sent to the

participants are presented in ANNEX 6

8 Design of the proficiency test

The design of the PT foresaw triplicate analyses of the test sample and reporting of the individual

results of replicate analyses for the single analytes in the following denoted as FS_REP and

additionally a value for proficiency assessment in the following denoted as final value - FS_FIN

for both the single analytes and the sum of the four marker PAHs Both FS_REP results and FS_FIN

results had to be reported corrected for recovery (and recovery had to be stated in the questionnaire

which participants were asked to fill in together with other parameters of the method applied) the

latter had also to be accompanied by the respective expanded measurement uncertainty The FS_FIN

results were used for performance assessment

Besides analysis results participants were also asked to report details of the applied analysis method

and to provide answers to a questionnaire (see ANNEX 7 for the template and the compiled returned

questionnaires)

13

9 Evaluation of the results

The results reported by participants are listed in ANNEX 8

91 General

The most important evaluation parameter was the performance of the laboratories in the determination

of the target PAHs in the food supplement test material which was expressed by z-scores and zeta-

scores

The compliance of method performance characteristics specified by the participants with provisions

given in legislation was evaluated too

92 Evaluation criteria

The participants were requested to report for all analytes the results of replicate measurements and a

value for proficiency assessment which is the result they wish to be applied for the calculation of

performance indicators z-Scores and zeta-scores were attributed only to these results The individual

results of replicate analyses were not rated

z-Scores

z-scores were calculated based on the FS_FIN values Equation 1 presents the formula for calculation

of z-scores

Equation 1

P

assignedlab Xxz

where z refers to the z-score xlab to the reported ldquovalue for proficiency assessmentrdquo Xassigned to the

assigned value and σP to the standard deviation for proficiency testing

zeta-Scores

In addition to z-scores zeta-scores were calculated In contrast to z-scores zeta-scores describe the

agreement of the reported result with the assigned value within the respective uncertainties

Unsatisfactorily large zeta-scores might be caused by underestimated measurement uncertainties large

bias or a combination of both Zeta-scores were calculated according to Equation 3

14

Equation 3 22assignedlab

assignedlab

uu

Xxzeta

where zeta refers to the zeta-score xlab to the reported ldquovalue for proficiency assessmentrdquo Xassigned to

the assigned value ulab to the measurement uncertainty reported by the laboratory and uassigned to the

uncertainty of the assigned value

The performance of the laboratories was classified according to ISOIEC 170432010 [10] Following

scheme is applied for the interpretation of zeta scores and z-scores

|score| 20 = satisfactory performance

20lt|score| lt 30 = questionable performance

|score| ge 30 = unsatisfactory performance

93 Performance of participants

The 48 participants in the study reported in total 225 results for proficiency evaluation which equals to

about 94 of the 240 possible results About 68 of the reported results were rated as satisfactory

with regard to z-scores

Figure 1 and Figure 2 give an overview of the z-scores assigned to the respective results The larger

the triangles the larger were the differences to the assigned values Red triangles indicate z-scores

above an absolute value of three whereas yellow triangles represent z-scores in the questionable

performance range The corresponding score values are plotted next to the triangles About 40 of the

72 non-satisfactory results were reported by seven laboratories only eg the performance of

participant 3608 was not satisfactory for the majority of target analytes

It is also obvious that most problems were encountered in the determination of chrysene and the sum

of the four analytes Potential reasons for the difficulties related to chrysene are discussed further

down

The ideal situation would be that deviations from the assigned values of the results reported for the

individual analytes are small and randomly distributed around zero This will give a satisfactory

performance statement for the sum parameter Deviations of opposite sign could also cancel out and

lead to a satisfactory performance for the sum parameter (eg participants 5303 5304 and 5305)

However in most cases a single underperformance for one analyte led also to non-satisfactory rating

for the sum parameter

15

The numerical values of the calculated z-scores are compiled in Table 6 and Table 7 z-Scores with an

absolute value of above 2 are given in red bold font

The results of the data evaluation for the individual analytes are given in ANNEX 8

For each analyte the figure shows the individual analysis results of the three replicate determinations

The assigned value is shown as dotted line The blue boxes represent the expanded uncertainties as

reported by participants for the value for proficiency assessment The arithmetic mean of the results

of the individual participants is indicated in the blue boxes by a blue line The satisfactory performance

range is located between the two red lines

The individual results of the replicate measurements and the value for proficiency assessment with

its accompanying expanded measurement uncertainty (k=2) are listed in the tables in ANNEX 8 as

well

16

Figure 1 Graphical presentation of z-scores corresponding to the values for proficiency assessment

reported by the NRLs for the contents of BAA BAP BBF and CHR in the St Johns wort extract test

material

Blue triangles indicate satisfactory performance yellow triangles indicate questionable performance red triangles indicate non-satisfactory performance z-score values are presented above the triangles for the latter two performance categories

ProLab 2011

Z score-3 0 3 -3 0 3 -3 0 3 -3 0 3 -3 0 3

BAA BAP BBF CHR PAH4

La

bo

rato

ry

36003602

3603

36043605

36063607

36083610

36113612

36133614

36153616

36173618

36193620

36213622

36233624

3626

2258

2343

2452

2669

2147

2788

2869

6710

70839

3011

11271

80525

8425

4561

12338

90680

4976

7331

99553

3998

3780

7500

17420

168413

3269

3029

-2348 -2736

-2023 -2981

-2155-3018

-3998

Figure 2 Graphical presentation of z-scores corresponding to the values for proficiency assessment

reported by the OCLs for the contents of BAA BAP BBF and CHR in the St Johns wort extract test

material

Blue triangles indicate satisfactory performance yellow triangles indicate questionable performance red triangles indicate non-satisfactory performance z-score values are presented above the triangles for the latter two performance categories

ProLab 2011

Z score-3 0 3 -3 0 3 -3 0 3 -3 0 3 -3 0 3

BAA BAP BBF CHR PAH4

La

bo

rato

ry

5301

5302

5303

5304

5305

5306

5307

5308

5309

5310

5311

5312

5313

5314

5315

5316

5317

5318

5319

5320

5321

5322

5323

2597

2629

2017

2293

2873

2123

2521

2620

2186

2222

2452

2864

3952

3984

3806

9226

4171 5522

3285

6594

5205

5157

3539

3901

7051

4519

3173

4872

9191

7276

4391

-2435

-2154

-2065

-2340

-2740-3726

-6346

-3505

17

Table 6 Compilation of z-scores calculated from the ldquoresults for proficiency assessment

reported by the NRLs for food supplement test material z-scores outside the

satisfactory range (|z| gt 2) are indicated by red font Values are reproduced as reported

by the laboratories Empty cells denote analytes for which results were not reported

Measurand benz[a]anthracene benzo[a]pyrene benzo[b]fluoranthene chrysene Sum PAH 4

Assigned value

microgkg 298 165 292 407 1162

Standard deviation for proficiency assessment

microgkg 062 036 060 083 125

Laboratory code

Result z-score Result z-score Result z-score Result z-score Result z-score

microgkg microgkg microgkg microgkg microgkg

3600 3 00 17 01 31 03 5 11 128 09

3602 19 -18 124 -11 17 -20 304 -12 79 -30

3603 251 -08 161 -01 256 -06 459 06 1127 -03

3604 213 -14 143 -06 11 -30 429 03 893 -22

3605 291 -01 167 01 247 -07 386 -03 1091 -06

3606 289 -01 167 01 276 -03 451 05 1184 02

3607 438 23 274 30 567 46 819 50 2098 75

3608 714 67 573 113 1036 123 1014 73 3336 174

3610 196 -17 206 11 191 -17 424 02 1138 -02

3611 469 708 308 805 576 907 865 996 2218 1684

3612 31 02 22 15 29 00 61 25 143 21

3613 33 05 18 04 31 03 4 -01 122 05

3614 185 -18 08 -23 127 -27 271 -16 663 -40

3615 376 13 145 -06 4 18 601 23 152 29

3616 2 -16 15 -04 2 -15 47 08 102 -11

3617 362 10 169 01 299 01 422 02 1252 07

3618 32 04 2 10 31 03 45 05 128 09

3619 299 00 207 12 326 06 738 40 157 33

3620 302 01 166 00 276 -03 46 06 1204 03

3621 28 -03 47 84 25 -07 51 12 151 28

3622 322 04 213 13 34 08 488 10 1361 16

3623 33 05 21 12 28 -02 72 38 154 30

3624 18 04

3626 314 03 174 02 292 00 628 27 1409 20

18

Table 7 Compilation of z-scores calculated from the ldquoresults for proficiency assessment

reported by the OCLs for food supplement test material z-scores outside the

satisfactory range (|z| gt 2) are indicated by red font Values are reproduced as reported

by the laboratories Empty cells denote analytes for which results were not reported

Measurand benz[a]anthracene benzo[a]pyrene benzo[b]fluoranthene chrysene Sum PAH 4

Assigned value

microgkg 298 165 292 407 1162

Standard deviation for proficiency assessment

microgkg 062 036 060 083 125

Laboratory code

Result z-score Result z-

score Result z-score Result

z-score

Result z-score

microgkg microgkg microgkg microgkg microgkg

5301 31 02 18 04 29 00 49 10 126 08

5302 316 03 188 06 298 01 679 33 37 -63

5303 2876 -02 1535 -03 2586 -06 6441 29 13438 15

5304 261 -06 146 -05 253 -06 591 22 1251 07

5305 24 -09 16 -01 24 -09 61 25 125 07

5306 22 -13 16 -01 42 21 49 10 129 10

5307 461 27 157 -02 221 -12 524 14 1363 16

5308 16446 -22 107009 -16 229848 -10 213243 -23 72456 -35

5309 17 -21 11 -15 2 -15

5310 305 01 123 -12 313 03 386 -03 1127 -03

5311 459 26 2 10 281 -02 437 04 1367 16

5312 543 40 248 23 299 01 953 66 2042 71

5313 353 09 207 12 328 06 838 52 1726 45

5314 394 16 172 02 404 19 588 22 1558 32

5315 545 40 238 20 444 25 542 16 177 49

5316 235 -10 17 01 273 -03 282 -15 96 -16

5317 222 -12 269 29 192 -17 545 17 1229 05

5318 067 -37 117 -13 279 -02 357 -06 82 -27

5319 147 -24 216 14 396 17 465 07 1225 05

5320 534 38 316 42 625 55 834 52 2309 92

5321 12 -12

5322 87 92 17 01 33 06 7 35 207 73

5323 33 05 2 10 45 26 73 39 171 44

19

Table 8 and Table 9 present the respective zeta-scores As for the z-scores data outside the

satisfactory performance range are given in red bold font Zeta scores were only calculated for

laboratories that provided measurement uncertainty data as well as a numerical value for the coverage

factor Zeta scores were not calculated when the reported uncertainty value exceeded the content value

which is probably linked to mixing of reporting units (microgkg and ) However the participants are

reminded that the measurement uncertainty shall be expressed in the same unit as the content value

The assessment of the performance of the participants based on the reported measurement uncertainty

gave a less favourable picture compared to z-scores Only 60 of the zeta-scores are within the

satisfactory performance range The distribution of satisfactory performance ratings between NRLs

and OCLs is not balanced It was higher for the NRLs

It has to be noted that the magnitude of the zeta-scores were for some participants much higher than

the z-scores attributed to the same results Consequently the laboratories perform according to fitness-

for-purpose criterion specified in legislation which forms the basis for the z-scores but seem to have

difficulties in deducing realistic measurement uncertainty values Eg the relative standard

measurement uncertainties reported by the NRLs ranged from 25 to 20 with a median value of

about 10 Only two OCLs reported measurement uncertainties that exceeded the maximum tolerable

uncertainty (Uf) The respective data is highlighted in yellow in Table 9

However the EU-RL PAHs will continue to pay special attention to measurement uncertainty in future

ILCs as it has major implications on the assessment of compliance of food with European legislation

20

Table 8 Compilation of zeta-scores calculated from the ldquoresults for proficiency assessment

reported by the NRLs for test material St Johns wort the reported measurement

uncertainty and the uncertainty of the analyte content of the test material zeta-scores

outside the satisfactory range (|zeta| gt 2) are indicated by red bold font Empty cells

denote analytes for which either results or measurement uncertainties were not

reported

21

Table 9 Compilation of zeta-scores calculated from the ldquoresults for proficiency assessment

reported by the OCLs for test material St Johns wort the reported measurement

uncertainty and the uncertainty of the analyte content of the test material zeta-scores

outside the satisfactory range (|zeta| gt 2) are indicated by red bold font yellow

highlighted cells ugtUf Empty cells denote analytes for which results were not

reported

22

94 Evaluation of the influence of analysis method

The information on the applied analysis method (ANNEX 11) was used to group the performance

indicators attributed to the reported results according to the applied analysis technique Two groups of

data were formed They represent results obtained by high performance liquid chromatography-

fluorescence detection (HPLC-FD about 40 ) and by gas chromatography-mass spectrometry (GC-

MS about 60 ) respectively Kernel density plots were constructed based on the respective z-scores

Figure 3A to 3E show the different Kernel density plots

Figure 3 Kernel density plots of z-scores attributed to the results for proficiency assessment

which were reported for the food supplements test sample Results were classified

according to the applied analysis technique

A) benz[a]anthracene

B) benzo[a]pyrene

23

Figure 3 continued

C) benzo[b]fluoranthene

D) chrysene

E) sum of four marker PAHs

The kernel density plots for BAP and BBF are almost identical and centred at or close to a z-score

value of zero as can be seen in Figure 3B and 3C Therefore bias introduced by the type of analysis

method is unlikely for these two analytes The situation is different for BAA and CHR In case of

BAA the kernel density distribution of z-scores based on HPLC-FD is shifted to negative values

24

(Figure 3A) which indicates a tendency to underestimate the BAA content in this particular

samplematrix when applying HPLC-FD This might be reasoned by separationintegration problems

between BAA and CHR peaks A few participants (from the HPLC-FD subgroup) that got negative

z-scores for BAA overestimated the CHR content This caused the slight shift of the density trace for

HPLC-FD towards negative values Removing this data from the evaluation would result in a density

trace with the mode at zero

The opposite is the case for CHR (Figure 3D) The density distribution of the z-scores attributed to

HPLC-FD data is centred almost at zero However the distribution for data based on GC-MS is shifted

to positive values indicating strong positive bias This bias might be caused by a lack of selectivity of

the analysis method The test material contains triphenylene which is isobaric to chrysene

Consequently it cannot be distinguished from chrysene by means of mass spectrometry These two

compounds have to be separated chromatographically which is not easy They coelute on unpolar and

on most mid-polar capillary columns Columns that allow their separation were commercialised only

recently and might not be available in all laboratories Therefore laboratories which determine the

four EU marker PAHs by means of GC-MS shall pay attention to the chromatographic separation of

triphenylene and chrysene A lack of chromatographic resolution might lead to strong positive bias of

GC-MS methods and to false conclusions on the marketability of the tested sample

The chromatographic separation of chrysene and triphenylene is not a problem with HPLC which is

reflected in the position of the density plot for chrysene However attention has to be paid in HPLC-

FD analysis to a proper clean up of the sample extract in order to eliminate interferences that show

similar retention as BAA and CHR

95 Evaluation of the influence of calibration

The participants received together with the food supplement test sample two standard solutions in

depending of their choice either acetonitrile or toluene The analyte content of one solution was

disclosed to the participants whereas the content of the other solution was kept undisclosed The

content values of the latter are presented in Table 5 Analysis results for the solution with undisclosed

content had to be reported to the PT organiser The respective data are presented in Annex 9

Percent deviations of the reported results from the assigned values were calculated for both the food

supplement test sample and the standard solution with undisclosed analyte content Youden plots were

prepared in order to visualize the influence of instrument calibration on the results reported for the

food supplement sample Two plots are discussed in the following The whole set of Youden plots are

given in Annex 9

25

Figure 4 Youden plot of the percent deviations of values reported for benzo[a]pyrene from the

assigned values of the food supplement test material and the standard solution in

toluene with undisclosed content

Figure 4 reveals that a number of participants reported results with similar percent deviations from the

assigned values of both the results for the food supplement material and the standard solution in

toluene with undisclosed content This correlation indicates that over- respectively underestimation of

the analyte content of the food supplement material might be caused by biased instrument calibration

This finding underpins the importance of thorough calibration standard preparation However the data

was not in all cases conclusive as some laboratories performed well for the food supplement samples

but reported biased results for the standard solution with undisclosed content (eg Figure 4 participants

3620 and 5305) This fact cannot be explained yet

Figure 5 presents the respective Youden plot for chrysene in the food supplement test material and the

standard solution in toluene with undisclosed content From this graph it can be concluded that the

overestimation of the chrysene content of the food supplement material was not caused by instrument

calibration The results for the solvent solution of many laboratories which underperformed for the

26

food supplement sample agreed well with the assigned value This supports the conclusion that the

overestimation is caused by the determination of an interferent most probably triphenylene

Figure 5 Youden plot of the percent deviations of values reported for chrysene from the

assigned values of the food supplement test material and the standard solution in

toluene with undisclosed content

96 Evaluation of compliance with legislation

The method performance data reported for the four marker PAHs were evaluated for compliance with

the provisions given in Commission Regulation (EC) No 8362011 Table 18 in ANNEX 10 gives an

overview on the results of the evaluation Data were not reported in case of empty cells Non-

compliant data are highlighted in Table 18 It should be noted that Regulation (EU) No 8362011

specifies LOD and LOQ with two decimals This was not respected by all participants which

hampered the compliance assessment

27

In summary it can be stated that the majority of participants reported method performance

characteristics for the determination of the four marker PAHs in the food supplement test sample that

are compliant with legislation However some laboratories need to improve their analysis methods

The laboratories in question are urged to adapt to the provisions given in the new legislation

10 Follow-up actions for underperforming laboratories

All laboratories that got questionable or non-satisfactory performance ratings are urged to perform

root cause analysis and to implement corrective actions

As agreed during the EU-RL PAH workshop 2011 the EU-RL PAH will set up follow-up measures in

due time for all NRLs that received for at least one of the four PAHs (BAA BAP BBF and CHR) z-

scores gt 3 as required by Regulation (EC) 8822004 and by the Protocol for management of

underperformance in comparative testing andor lack of collaboration of National Reference

Laboratories (NRLs) with European Union reference laboratories (EU-RLs) activities

These laboratories shall perform as an immediate action root-cause-analysis and shall report to the

EU-RL PAH in writing the identified cause for their underperformance and corrective actions they are

going to take Additionally they shall participate to an independent (non-EU-RL) proficiency test on

the determination of PAHs in food and shall communicate the outcome of this exercise to the EU-RL

PAH

28

11 Conclusions

Fourty seven participants reported results back to the EU-RL The performance of the majority of

participants was good In total 153 out of 225 attributed z-scores were below an absolute value of two

which equals to almost 68 However this percentage is much below the percentages of satisfactory

performance gained in previous studies This might be the consequence of the higher level of difficulty

with respect to matrix composition In addition bias can be concluded from the pattern of performance

indicators of some laboratories

In general the determination of chrysene caused most difficulties to the participants

zeta-Scores were calculated besides z-scores They indicate the agreement of the reported result with

the assigned value with respect to the stated measurement uncertainty The outcome of this rating was

worse than for the z-scores which indicates that the measurement uncertainty estimates were not

realistic Therefore participants underperforming with regard to zeta scores are urged to adapt their

measurement uncertainty statements

The great majority of participants in this inter-laboratory comparison applied analytical methods which

complied with EU legislation with regard to performance characteristics

12 Acknowledgements

The organisers would like to thank Mrs Beatriz de la Calle and Mr Franz Ulberth (all from IRMM

Geel Belgium) for their accurate revision of this report Mr Hakan Emteborg (from IRMM) for

ampouling of the standard solutions and all NRLs and OCLs for their cooperation

29

13 References

1 EU COMMISSION REGULATION (EC) No 7762006 of 23 May 2006 amending Annex VII to Regulation

(EC) No 8822004 of the European Parliament and of the Council as regards Community reference

laboratories Official Journal of the European Union 2006 L 136 p 3-8

Available from

httpeur-lexeuropaeuLexUriServLexUriServdouri=OJL200613600030008ENPDF

2 EU Regulation (EC) No 8822004 of the European Parliament and of the Council of 29 April 2004 on

official controls performed to ensure the verification of compliance with feed and food law animal health

and animal welfare rules Official Journal of the European Communities 2004 L191 p 1-52 Available

from

httpeur-lexeuropaeuLexUriServLexUriServdouri=OJL200419100010052ENPDF

3 EU Opinion of the Scientific Committee on Food on the risks to human health of Polycyclic Aromatic

Hydrocarbons in food (2002) Available from

httpeuropaeuintcommfoodfsscscfout153_enpdf

4 International Agency for Research on Cancer IARC Monographs on the Evaluation of

Carcinogenic Risks to Humans Some Non-heterocyclic Polycyclic Aromatic Hydrocarbons and Some

Related Exposures Volume 92 (2010) Available from

httpmonographsiarcfrENGMonographsvol92mono92pdf

5 EU Commission Regulation (EC) No 3332007 of 28 March 2007 laying down the methods of sampling

and analysis for the official control of the levels of lead cadmium mercury inorganic tin 3-MCPD and

benzo(a)pyrene in foodstuffs Official Journal of the European Union 2007 L 88 p 29-38 Available

from

httpeur-lexeuropaeuLexUriServLexUriServdouri=OJL200708800290038ENPDF

6 EU Commission Regulation (EC) No 18812006 of 19 December 2006 setting maximum levels for certain

contaminants in foodstuffs Official Journal of the European Union 2006 L 364 p 5-24 Available from

httpeur-lexeuropaeuLexUriServLexUriServdouri=OJL200636400050024ENPDF

7 EFSA Scientific Opinion of the Panel on Contaminants in the Food Chain on a request from the European

Commission on Polycyclic Aromatic Hydrocarbons in Food The EFSA Journal 2008 724 p 1-114

Available from

httpwwwefsaeuropaeuEFSAefsa_locale-1178620753812_1211902034842htm

30

8 EU Commission Regulation (EU) No 8352011 amending Regulation (EC) No 18812006 as regards

maximum levels for polycyclic aromatic hydrocarbons in foodstuffs Official Journal of the European

Union 2011 L 215 p 4-8

httpeur-lexeuropaeuLexUriServLexUriServdouri=OJL201121500040008ENPDF

9 EU Commission Regulation (EU) No 8362011 amending Regulation (EC) No 3332007 laying down the methods of sampling and analysis for the official control of the levels of lead cadmium mercury inorganic tin 3-MCPD and benzo(a)pyrene in foodstuffs Official Journal of the European Union 2011 L 215 p 9-16 httpeur-lexeuropaeuLexUriServLexUriServdouri=OJL201121500090016ENPDF

10 ISOIEC 170432010 (2010) Conformity assessment -- General requirements for proficiency testing

providers Geneva Switzerland

11 Thompson M Ellison SLR and Wood R The International Harmonized Protocol for the Proficiency

Testing of Analytical Chemistry Laboratories Pure Appl Chem (2006) 78(1) p 145ndash196

Available from httpmediaiupacorgpublicationspac2006pdf7801x0145pdf

31

14 ANNEXES

ANNEX 1 ndash Announcement of the PT on the IRMM webpage

ANNEX 2 ndash Announcement via e-mail and invitation

ANNEX 3 ndash Announcement of sample dispatch

ANNEX 4 ndash Technical specifications of the calibration solutions

ANNEX 5 ndash Homogeneity and stability of the test material

ANNEX 6 ndash Acknowledgement of sample receipt

ANNEX 7 ndash Questionnaire on details of analysis method

ANNEX 8 ndash Results reported for the food supplement sample

ANNEX 9 ndash Results reported for the standard solutions with undisclosed analyte content

ANNEX 10 ndash Method performance characteristics reported by participants

ANNEX 11 ndash Details of analysis methods reported by participants

32

ANNEX 1 ndash Announcement of the PT on the IRMM webpage

33

ANNEX 2 - Announcement via e-mail and invitation

34

ANNEX 3 - Announcement sample dispatch

Dear MadameSir

The test samples for the proficiency test on the determination of four PAHs in a food supplement (dry extract of St Johns wort) were

dispatched today

Explanation on the design of the study as well as instructions for sample storage and for reporting of results are in the parcel You will

also find your participant key in the parcel which is required for reporting of results

Please check the parcel after arrival for completeness and report the receipt of the test samples as well as their status to us by applying

the sample receipt form which is in the parcel

You are kindly requested to inform us by email if you do not receive the parcel by end of this week

My colleague Donata Lerda will then take care of it

With best regards

Thomas Wenzl

35

ANNEX 4 - Specifications of standard solutions with disclosed concentration

36

ANNEX 4 - continued

37

ANNEX 5 - Homogeneity and stability of the test material

Homogeneity of the test material

Benz[a]anthracene - BAA

Benzo[a]pyrene - BAP

38

ANNEX 5 - continued

Benzo[b]fluoranthene - BBF

Chrysene - CHR

39

ANNEX 5 - continued

Stability of the test material

Table 10 Content of the four target analytes in test materials stored under different

temperatures

BaA BaP BbF CHR

microgkg microgkg microgkg microgkg

Content 31 16 31 69 Storage at room temperature

U (k=2) 05 02 05 17

Content 31 15 29 68 Storage at reference temperature

U (k=2) 04 03 04 10

The applied capillary column does not separate CHR from triphenylene

40

ANNEX 6 ndash Documents sent to participants

41

ANNEX 6 - continued

42

ANNEX 7 Questionnaire on details of analysis method

43

ANNEX 7 - continued

44

ANNEX 7 - continued

45

ANNEX 8 - Results reported by participants for the food supplements test sample Table 11 Analysis results reported by the participants for the content of benz[a]anthracene (BAA) in the food supplements test material Values are presented as reported

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

3600 31 29 29 3 05 2

3602 199 193 178 19 021

3603 253 253 246 251 022 2

3604 199 209 23 213 071 2

3605 301 3 271 291 076 2

3606 302 288 278 289 064 2

3607 456 416 443 438 096 2

3608 796 695 651 714 149 2

3610 182 192 214 196 039 2

3611 414 504 488 469 96 2

3612 3 34 28 31 06 2

3613 32 33 34 33 07 2

3614 182 182 191 185 037 2

3615 3884 3822 3565 376 0752 2

3616 2 2 2 2 01 2

3617 366 37 35 362 046 2

3618 32 32 32 32 05 2

3619 299 305 297 299 048 2

3620 304 294 302 302 045 2

3621 29 26 29 28 04 2

3622 343 325 318 322 052 2

3623 35 3 33 33 07 2

3624

3626 318 31 314 314 126 2

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

5301 31 3 32 31 06 2

5302 314 319 315 316 063 2

5303 3102 2716 2822 2876 27 62

5304 261 263 26 261 57 2

5305 23 22 26 24 20

5306 22 23 23 22 11 2

5307 488 461 317 461

5308 168633 168544 1562025 1644598

5309 16 19 16 17

5310 3 304 311 305 015

5311 477 452 448 459 15 2

5312 506 63 494 543 134 2

5313 353 301 404 353

5314 431 373 378 394 5 096

5315 596 531 51 545 11 2

5316 224 231 235 235 022 2

5317 216 23 221 222 007 2

5318 064 07 067 01 2

5319 111 184 146 147 029 2

5320 515 525 564 534 48

5321 21 5322 13 87 9 87 35 2

5323 38 33 35 33 16 2

46

Figure 6 Results of replicate determinations (indicated by triangles) of benz[a]anthracene (BAA) in the food supplement test material

Horizontal blue lines represent the arithmetic mean value of replicate measurements and blue bars the reported expanded measurement uncertainty (k=2) The assigned value is

plotted as dotted line Red solid lines mark the range of satisfactory performance (|z|le2) Mean values are given for results outside the displayed data range

ProLab 2011

Laboratory

53

1853

1953

0853

0936

1436

0236

1036

1653

2136

0453

1753

0653

1653

0536

0353

0436

2153

0336

0636

0536

0036

2036

1953

1036

1253

0136

2653

0236

1836

2336

2236

1353

1353

2336

1736

1553

1453

0736

0753

1153

2053

1253

1536

0853

2236

11

an

aly

te c

on

ten

t (micro

gk

g)

10

95

9

85

8

75

7

65

6

55

5

45

4

35

3

25

2

15

1

05

0

102

3 468

7

Sample St Johns wort extract replicatesMeasurand Benz[a]anthraceneNo of laboratories 46

Assigned value 298 microgkg (Reference value)Target sd 062 microgkgTolerance limits 174 - 422 microgkg (|Z score| lt 200)

Assig

ned va

lue

Limit of tolerance

Limit of tolerance

47

Table 12 Analysis results reported by the participants for the content of benzo[a]pyrene (BAP) in the food supplements test material Values are presented as reported

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

3600 16 17 17 17 03 2

3602 118 123 13 124 015

3603 165 162 157 161 015 2

3604 134 154 139 143 04 2

3605 164 166 172 167 057 2

3606 181 159 161 167 031 2

3607 279 266 279 274 055 2

3608 57 687 461 573 226 2

3610 226 216 183 206 042 2

3611 289 325 311 308 36 2

3612 24 22 2 22 05 2

3613 17 18 19 18 05 2

3614 08 08 08 08 016 2

3615 1545 1393 1425 145 029 2

3616 16 16 14 15 03 2

3617 177 171 158 169 023 2

3618 2 2 2 2 02 2

3619 207 207 206 207 075 2

3620 165 169 163 166 017 2

3621 44 47 49 47 05 2

3622 212 208 222 213 038 2

3623 2 21 22 21 03 2

3624 18 178 182 18 031 2

3626 172 173 178 174 07 2

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

5301 18 18 18 18 04 2

5302 188 19 187 188 038 2

5303 1691 1426 149 1535 19 80

5304 145 142 15 146 69 2

5305 15 17 18 16 15

5306 16 17 17 16 08 2

5307 164 157 153 157

5308 102669 109324 109035 107009

5309 1 1 12 11

5310 122 122 124 123 006

5311 199 184 215 2 15 2

5312 258 248 239 248 05 2

5313 199 178 245 207

5314 174 171 172 172 11 0948

5315 253 248 213 238 05 2

5316 177 171 173 17 016 2

5317 255 277 276 269 012 2

5318 114 12 117 02 2

5319 19 228 231 216 043 2

5320 303 3 344 316 78

5321 12 12 12 12

5322 13 17 24 17 35 2

5323 2 2 19 2 1 2

48

Figure 7 Results of replicate determinations (indicated by triangles) of benzo[a]pyrene (BAP) in the food supplement test material

Horizontal blue lines represent the arithmetic mean value of replicate measurements and blue bars the reported expanded measurement uncertainty (k=2) The assigned value is

plotted as dotted line Red solid lines mark the range of satisfactory performance (|z|le2) Mean values are given for results outside the displayed data range

ProLab 2011

Laboratory

36

1453

0953

0853

1853

2153

1036

0236

0436

1553

0436

1653

0353

0736

0336

2036

0053

0553

0636

0636

0536

1753

1453

1636

2636

1336

2453

0153

2253

0253

2353

1136

1836

1953

1336

1036

2336

2253

1936

1253

1553

1253

1736

0753

2036

2136

0836

11

an

aly

te c

on

ten

t (micro

gk

g)

55

5

45

4

35

3

25

2

15

1

05

0

573

308

3

Sample St Johns wort extract replicatesMeasurand Benzo[a]pyreneNo of laboratories 48

Assigned value 165 microgkg (Reference value)Target sd 036 microgkgTolerance limits 093 - 237 microgkg (|Z score| lt 200)

Assig

ned va

lue

Limit of tolerance

Limit of tolerance

49

Table 13 Analysis results reported by the participants for the content of benzo[b]fluoranthene (BBF) in the food supplements test material Values are presented as reported

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

3600 32 3 3 31 09 2

3602 173 175 16 17 017

3603 265 252 25 256 025 2

3604 116 108 105 11 028 2

3605 286 225 232 247 074 2

3606 294 273 261 276 046 2

3607 579 579 544 567 113 2

3608 103 1201 876 1036 326 2

3610 173 196 204 191 038 2

3611 546 618 565 576 75 2

3612 3 28 28 29 06 2

3613 28 31 32 31 06 2

3614 135 111 135 127 022 2

3615 4187 3678 4111 4 08 2

3616 2 2 2 2 01 2

3617 33 27 299 035 2

3618 31 31 31 31 05 2

3619 326 324 312 326 064 2

3620 277 28 272 276 069 2

3621 26 25 23 25 04 2

3622 346 336 333 34 052 2

3623 26 28 29 28 02 2

3624

3626 288 28 307 292 117 2

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

5301 29 3 29 29 06 2

5302 297 3 296 298 06 2

5303 2705 2444 2608 2586 22 78

5304 245 254 26 253 85 2

5305 23 23 26 24 15

5306 42 43 4 42 21 2

5307 221 216 241 221

5308 232169 246654 240722 229848

5309 19 22 19 2 0

5310 3 315 325 313 016

5311 262 277 304 281 15 2

5312 27 289 337 299 075 2

5313 321 284 378 328

5314 423 404 385 404 34 083

5315 42 506 416 444 09 2

5316 265 267 277 273 018 2

5317 185 197 195 192 006 2

5318 276 281 279 04 2

5319 396 389 403 396 079 2

5320 623 618 635 625 14

5321 31

5322 24 33 49 33 35 2

5323 38 45 35 45 27 2

50

Figure 8 Results of replicate determinations (indicated by triangles) of benzo[b]fluoranthene (BBF) in the food supplement test material

Horizontal blue lines represent the arithmetic mean value of replicate measurements and blue bars the reported expanded measurement uncertainty (k=2) The assigned value is

plotted as dotted line Red solid lines mark the range of satisfactory performance (|z|le2) Mean values are given for results outside the displayed data range

ProLab 2011

Laboratory

36

0436

1436

0236

1053

1736

1653

0953

0753

0853

0536

2136

0553

0436

0353

0353

1636

0636

2036

2353

1853

1136

1236

2653

0153

0253

1236

1736

1336

0036

1853

2153

1036

1953

1336

2253

2253

2353

1936

1553

1453

0653

1536

0753

2036

0836

11

an

aly

te c

on

ten

t (micro

gk

g)

75

7

65

6

55

5

45

4

35

3

25

2

15

1

05

0

103

6

576

3

Sample St Johns wort extract replicatesMeasurand Benzo[b]fluorantheneNo of laboratories 46

Assigned value 292 microgkg (Reference value)Target sd 060 microgkgTolerance limits 172 - 412 microgkg (|Z score| lt 200)

Assig

ned va

lue

Limit of tolerance

Limit of tolerance

51

Table 14 Analysis results reported by the participants for the content of chrysene (CHR) in the food supplements test material Values are presented as reported

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

3600 5 49 5 5 07 2

3602 32 304 29 304 036

3603 462 469 447 459 048 2

3604 438 428 42 429 119 2

3605 365 41 381 386 085 2

3606 494 411 45 451 124 2

3607 897 774 787 819 18 2

3608 986 887 1168 1014 284 2

3610 418 392 462 424 085 2

3611 833 893 87 865 61 2

3612 6 64 58 61 12 2

3613 41 39 4 4 08 2

3614 272 254 288 271 054 2

3615 605 6028 5964 601 12 2

3616 47 45 49 47 05 2

3617 445 431 389 422 065 2

3618 45 45 45 45 06 2

3619 738 747 72 738 118 2

3620 463 472 446 46 092 2

3621 53 49 51 51 06 2

3622 478 513 505 488 08 2

3623 73 73 72 72 05 2

3624

3626 63 622 634 628 251 2

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

5301 49 49 49 49 1 2

5302 687 683 666 679 136 2

5303 6777 6181 6382 6441 24 70

5304 592 581 6 591 85 2

5305 6 58 64 61 20

5306 49 5 51 49 25 2

5307 524 5 574 524

5308 211488 216782 221146 213243

5309

5310 372 393 394 386 019

5311 43 459 422 437 15 2

5312 934 943 981 953 191 2

5313 793 73 99 838

5314 636 561 565 588 18 0818

5315 547 583 499 542 11 2

5316 292 286 285 282 06 2

5317 52 598 517 545 046 2

5318 368 345 357 05 2

5319 449 471 474 465 116 2

5320 76 924 818 834 99

5321 33

5322 7 7 76 7 35 2

5323 87 73 77 73 37 2

52

Figure 9 Results of replicate determinations (indicated by triangles) of chrysene (CHR) in the food supplement test material

Horizontal blue lines represent the arithmetic mean value of replicate measurements and blue bars the reported expanded measurement uncertainty (k=2) The assigned value is

plotted as dotted line Red solid lines mark the range of satisfactory performance (|z|le2) Mean values are given for results outside the displayed data range

ProLab 2011

Laboratory

53

0836

1453

1636

0253

2153

1836

0553

1036

1336

1736

1036

0453

1136

1836

0636

0336

2053

1936

1653

0136

0036

2253

0636

2153

0753

1553

1753

1453

0436

1536

1253

0536

2653

0353

0253

2236

2336

1953

2336

0753

2053

1353

1236

0836

11

an

aly

te c

on

ten

t (micro

gk

g)

13

12

11

10

9

8

7

6

5

4

3

2

1

0

865

3

Sample St Johns wort extract replicatesMeasurand ChryseneNo of laboratories 45

Assigned value 407 microgkg (Reference value)Target sd 083 microgkgTolerance limits 241 - 573 microgkg (|Z score| lt 200)

Assig

ned va

lue

Limit of tolerance

Limit of tolerance

53

Table 15 Analysis results reported by the participants for the sum of the four marker PAHs in the food supplements test material Values are presented as reported

Laboratory code

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg

3600 128 24 2

3602 79 126

3603 1127 096 2

3604 893 258 2

3605 1091 305 2

3606 1184 238 2

3607 2098 42 2

3608 3336 325 2

3610 1138 17 2

3611 2218 259 2

3612 143 29 2

3613 122 2 2

3614 663 129 2

3615 152 304 2

3616 102 03 2

3617 1252 169 2

3618 128 18 2

3619 157 305 2

3620 1204 12 2

3621 151 19 2

3622 1361 115 2

3623 154 09 2

3624

3626 1409 56 2

Laboratory code

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg

5301 126

5302 37 074 2

5303 13438 92

5304 1251 85 2

5305 125 20

5306 129 65 2

5307 1363

5308 7245598

5309

5310 1127 056

5311 1367 15 2

5312 2042 408 2

5313 1726

5314 1558

5315 177 35 2

5316 96 073 2

5317 1229 066 2

5318 82 05 2

5319 1225 267 2

5320 2309 41

5321

5322 207 35 2

5323 171 103 2

54

ANNEX 9 Results reported for the standard solutions with undisclosed analyte content Table 16 Analysis results reported by the participants for the four marker PAHs in the standard solution in toluene with undisclosed analyte

content Values are presented as reported U measurement uncertainty k coverage factor

benz[a]anthracene benzo[a]pyrene benzo[b]fluoranthene chrysene Laboratory

code Unit Result U k Result U k Result U k Result U k 3600 microgl 38 67 2 25 45 2 211 68 2 275 38 2

3605 microgl 367 184 2 2482 124 2 2592 13 2 235 118 2

3606 microgkg 3763 176 2 2609 142 2 2721 118 2 2539 134 2

3607 microgkg 3706 815 2 2824 565 2 2591 518 2 2587 569 2

3612 microgl 38 08 2 237 11 2 244 18 2 257 02 2

3614 microgl 324 65 2 209 38 2 215 37 2 21 42 2

3616 microgkg 318 07 2 224 14 2 231 26 2 216 1 2

3618 microgkg 417 17 2 284 08 2 292 06 2 297 03 2

3619 microgkg 47 742 2 274 468 2 228 387 2 274 434 2

3620 microgl 495 74 2 294 29 2 321 8 2 321 64 2

3621 microgl 241 22 2 143 13 2 188 16 2 145 14 2

3623 microgkg 476 25 2 311 53 2 269 3 2 297 03 2

3626 microgl 359 38 2 235 23 2 238 27 2 24 18 2

5301 microgkg 426 29 299 289

5302 microgkg 436 87 2 296 59 2 296 59 2 289 58 2

5303 microgl 3721 27 102 22838 19 99 23756 22 99 25486 24 98

5304 microgkg 38 29 2 225 26 2 257 37 2 258 52 2

5305 microgkg 305 225 245 29

5306 microgkg 316 63 2 247 49 2 65 13 2 22 44 2

5307 microgkg 3658 2559 241 2414

5308 microgkg 16854 109324 246654 216782

5311 microgl 374 20 2 279 15 2 268 15 2 272 15 2

5313 microgl 3408 2165 2275 2373

5314 microgkg 395 5 2875 11 3614 34 2905 18

5320 microgl 3267 368 1986 571 28 683 2427 512

5322 microgl 282 35 2 225 35 2 241 35 2 21 35 2

55

Figure 10 Youden plots of the percent deviation of the reported values from the assigned values for the food supplement test sample and the

standard solution in toluene with undisclosed content

(The identity of the respective measurand is given above the figure Results exceeding a difference of more than 100 were omitted)

56

Figure 10 - continued

57

Table 17 Analysis results reported by the participants for the four marker PAHs in the standard solution in acetonitrile with undisclosed

analyte content

Values are presented as reported U measurement uncertainty k coverage factor

benz[a]anthracene benzo[a]pyrene benzo[b]fluoranthene chrysene

Laboratory

code Unit Result U k Result U k Result U k Result U k

3602 microgkg 3675 247 247 244

3603 microgkg 358 086 2 236 028 2 235 028 2 236 057 2

3604 microgl 475 29 2 19 12 2 194 13 2 258 12 2

3608 microgl 3623 078 2 2385 023 2 2343 18 2 2296 092 2

3610 microgkg 369 185 2 25 125 2 251 126 2 244 122 2

3611 microgkg 48 02 2 323 02 2 328 04 2 317 03 2

3613 microgl 332 31 2 261 3 2 249 25 2 239 24 2

3615 microgl 415 2635 2729 2689

3617 microgkg 37 09 2 244 06 2 284 07 2 239 06 2

3622 microgl 37 12 2 25 23 2 257 21 2 238 1 2

3624 microgkg 402 104 2 255 084 2 265 096 2 253 07 2

5306 microgkg 563 113 2 371 74 2 359 72 2 333 67 2

5308 microgkg 168633 102669 232169 211488

5309 microgl 402 268 27

5310 microgl 315 158 25 125 25 125 288 144

5312 microgl 428 043 2 282 028 2 272 027 2 275 028 2

5315 microgkg 387 08 2 256 05 2 251 05 2 253 05 2

5316 microgl 3839 342 2 2485 191 2 2559 168 2 239 51 2

5317 microgl 2466 043 2 2212 034 2 2472 016 2 2146 068 2

5319 microgl 414 41 2 279 28 2 268 27 2 26 31 2

5321 microgkg 476 316 326 328

5322 microgl 2547 35 2

5323 microgkg 405 203 2 239 119 2 255 153 2 201 11 2

58

Figure 11 Youden plots of the percent deviation of the reported values from the assigned values for the food supplement test sample and the

standard solution in acetonitrile with undisclosed content

(The identity of the respective measurand is given above the figure Results exceeding a difference of more than 100 were omitted)

59

Figure 11 - continued

60

ANNEX 10 Table 18 Method performance parameters reported by the participants

Laboratory code

Analyte LOD LOQ Recovery Analyte LOD LOQ Recovery

microgkg microgkg microgkg microgkg 3600 BaA 03 06 978 BaP 03 07 9483602 BaA 007 021 926 BaP 005 015 993603 BaA 0011 05 987 BaP 0005 051 9553604 BaA 02 04 86 BaP 02 04 883605 BaA 0025 005 118 BaP 0025 005 1083606 BaA 001 003 65 BaP 001 003 703607 BaA 021 071 74 BaP 027 089 943608 BaA 016 048 8213 BaP 015 046 83683610 BaA 02 05 85 BaP 005 02 853611 BaA BaP 3612 BaA 1 117 BaP 1 1203613 BaA 01 03 90 BaP 01 03 903614 BaA 02 05 96 BaP 02 05 933615 BaA 005 015 110 BaP 005 015 1053616 BaA 01 05 108 BaP 01 05 753617 BaA 018 037 405 BaP 009 018 3873618 BaA 01 03 63 BaP 01 03 593619 BaA 005 005 59 BaP 033 033 543620 BaA 0009 0018 64 BaP 0005 001 553621 BaA 007 02 84 BaP 007 02 923622 BaA 01 03 1101 BaP 01 03 9853623 BaA 02 08 105 BaP 02 08 1013624 BaA BaP 01 033 8513625 BaA BaP 3626 BaA BaP 5301 BaA 003 01 102 BaP 003 01 1005302 BaA 01 03 78 BaP 01 03 865303 BaA 003 01 62 BaP 003 01 805304 BaA 1 1 70 BaP 1 1 955305 BaA 02 05 85 BaP 02 05 855306 BaA 03 1 699 BaP 03 1 905307 BaA 6537 BaP 81165308 BaA 0088 0265 99 BaP 00262 00785 1005309 BaA 01 08 92 BaP 01 08 925310 BaA 01 03 BaP 01 03 905311 BaA 002 005 80 BaP 002 004 915312 BaA 03 09 105 BaP 03 09 1055313 BaA 04 08 BaP 04 08 5314 BaA 001 002 96 BaP 001 002 9485315 BaA 01 03 50 - 100 BaP 01 03 50 - 100 5316 BaA 002 011 827 BaP 004 019 10445317 BaA 05 1 76 BaP 05 1 755318 BaA BaP 5319 BaA 009 029 951 BaP 011 041 995320 BaA 12 18 946 BaP 06 1 8345321 BaA 03 05 BaP 05 1 5322 BaA 1 2 100 BaP 1 2 1005323 BaA 26 BaP 26

61

ANNEX 10 Table 18 - continued

Laboratory code Analyte LOD LOQ Recovery Analyte LOD LOQ Recovery

microgkg microgkg microgkg microgkg 3600 BbF 03 05 96 CHR 02 03 10153602 BbF 015 045 947 CHR 003 009 9193603 BbF 0014 05 1012 CHR 0004 05 10033604 BbF 02 04 85 CHR 02 04 873605 BbF 005 01 94 CHR 0025 005 863606 BbF 001 003 70 CHR 001 003 653607 BbF 018 06 100 CHR 014 028 943608 BbF 015 046 7979 CHR 015 045 71253610 BbF 005 02 85 CHR 02 05 853611 BbF CHR 3612 BbF 1 118 CHR 1 1173613 BbF 01 03 90 CHR 01 03 903614 BbF 01 04 96 CHR 02 05 953615 BbF 008 025 109 CHR 003 01 1083616 BbF 01 05 78 CHR 01 05 723617 BbF 035 07 38 CHR 012 024 4163618 BbF 01 03 62 CHR 01 03 623619 BbF 016 016 63 CHR 01 01 593620 BbF 0003 0006 55 CHR 001 002 653621 BbF 007 02 103 CHR 007 02 893622 BbF 02 06 963 CHR 03 09 1043623 BbF 02 08 106 CHR 02 08 1193624 BbF CHR 3625 BbF CHR 3626 BbF CHR 5301 BbF 003 01 96 CHR 003 01 1145302 BbF 01 03 84 CHR 01 03 835303 BbF 003 01 78 CHR 003 01 705304 BbF 1 1 86 CHR 1 1 705305 BbF 02 05 90 CHR 02 05 955306 BbF 03 1 1435 CHR 03 1 1185307 BbF 8116 CHR 65375308 BbF 0079 0238 99 CHR 009 026 1005309 BbF 01 08 92 CHR 5310 BbF 07 2 CHR 01 03 5311 BbF 002 004 95 CHR 002 004 895312 BbF 03 09 105 CHR 03 09 1055313 BbF 04 08 CHR 04 08 5314 BbF 001 002 83 CHR 001 002 8185315 BbF 02 05 50 -100 CHR 02 05 50 - 100 5316 BbF 005 024 814 CHR 001 007 9595317 BbF 05 1 73 CHR 05 1 785318 BbF CHR 5319 BbF 011 038 946 CHR 013 043 9675320 BbF 07 09 1194 CHR 23 32 10455321 BbF 05 1 CHR 05 1 5322 BbF 1 2 100 CHR 1 2 1005323 BbF 26 CHR 26

62

ANNEX 11 Table 19 Details of analysis method reported by the participants Data are presented as reported

Laboratory code

Analysis technique

Instrument calibration

Internal standards applied

Sample clean up

Details of sample

clean up

Extraction technique

Details of sample

extraction

3600 6) GC-MS

2) Internal standardisation with labelled substances

Deuterated PAH mix 9 (Ehrenstorfer)

1) Saponification 2) LiquidLiquid partitioning

D) Other Liquidliquid partitioning

3602 2) HPLC-UV-FLD

1) External calibration

3) Gel permeation chromatography

A) Pressurised liquid extraction (PLE)

3603 1) HPLC-FLD

1) External calibration

3) Gel permeation chromatography

D) Other 1 minute with dichloromethane in vortex

3604 2) HPLC-UV-FLD

2) Internal standardisation with labelled substances

DiP D14

5) Donor acceptor complex chromatography

D) Other LL

3605 1) HPLC-FLD

1) External calibration

3) Gel permeation chromatography

C) Soxhlet extraction

3606 7) GC-MSMS

2) Internal standardisation with labelled substances

BaP 13C4 CHR 13C6 BaA 13C6 BbF 13C6

4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

3607 6) GC-MS

2) Internal standardisation with labelled substances

BaA-d12 CHR-d12 BbF-d12 BkF-d12 BaP-d12 ICP-d12 BgP-d12 DiP-d14

1) Saponification

D) Other solvent partitioning

3608 1) HPLC-FLD

1) External calibration

6) Other Liquid extraction

D) Other

Extraction in a rotary agitator and centrifugation

3610 2) HPLC-UV-FLD

1) External calibration

4) Solid phase extraction

B) Sonication

3611

3612 6) GC-MS

2) Internal standardisation with labelled substances

chrysene-d12 dibenzo(ah)anthracene-d14 dibenzo(ai)pyrene-d14

4) Solid phase extraction

B) Sonication

3613 5) LC-MSMS

3) Internal standardisation with unlabelled substances

I use Chrysene-D12 3) Gel permeation chromatography

D) Other extraction with hexan

63

Table 19 - continued

Laboratory code

Analysis technique

Instrument calibration

Internal standards applied

Sample clean up Details of sample

clean up

Extraction technique

Details of sample

extraction

3614 6) GC-MS 1) External calibration

3) Gel permeation chromatography

B) Sonication

3615 1) HPLC-FLD

1) External calibration

6) Other PLE GPC

A) Pressurised liquid extraction (PLE)

3616 6) GC-MS

2) Internal standardisation with labelled substances

Benzo(a)pyrene-13C4 Benzo(a)anthracene-13C6 Benzo(b)fluoranthene-13C6 Chrysene-13C6

2) LiquidLiquid partitioning 4) Solid phase extraction

B) Sonication

3617 1) HPLC-FLD

1) External calibration

4) Solid phase extraction

C) Soxhlet extraction

3618 6) GC-MS

2) Internal standardisation with labelled substances

deuterated standards

1) Saponification 4) Solid phase extraction

D) Other Handshaken with cyclohexane

3619 6) GC-MS

2) Internal standardisation with labelled substances

C13 Labelled US EPA 16

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

D) Other Homegenisation amp saponiofication

3620 9) GC-HRMS

2) Internal standardisation with labelled substances

mix of deuterated PAHs (BAA-D12 CHR-D12 BAP-D12 BBF-D12)

3) Gel permeation chromatography 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

3621 6) GC-MS

2) Internal standardisation with labelled substances

Chrysene D12 Benzo(a)pyrene D12 Benzo(b)fluoranthene D12 Benzo(a)anthracene

2) LiquidLiquid partitioning 4) Solid phase extraction

B) Sonication

3622 1) HPLC-FLD

4) Standard addition method

4) Solid phase extraction

D) Other

Extraction with cyclohexane by heating under reflux

3623 7) GC-MSMS

2) Internal standardisation with labelled substances

C13 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

3624 2) HPLC-UV-FLD

1) External calibration

2) LiquidLiquid partitioning

D) Other hot solvent extraction

3626

64

Table 19 - continued

Laboratory code

Analysis technique

Instrument calibration

Internal standards

applied

Sample clean up

Details of sample

clean up

Extraction technique

Details of sample

extraction

5301 7) GC-MSMS

2) Internal standardisation with labelled substances

EACH SUBSTANCE C13

4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5302 7) GC-MSMS

2) Internal standardisation with labelled substances

Isotope labelled PAHs 13C for each analyte

4) Solid phase extraction

C) Soxhlet extraction

5303 7) GC-MSMS

3) Internal standardisation with unlabelled substances

HAP C13 4) Solid phase extraction

D) Other automate ASE200 DIONEX

5304 7) GC-MSMS

2) Internal standardisation with labelled substances

with C13 for each HAP

2) LiquidLiquid partitioning 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5305 7) GC-MSMS

2) Internal standardisation with labelled substances

C13 isotopiques internal standards

4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5306 7) GC-MSMS

2) Internal standardisation with labelled substances

C13 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5307 6) GC-MS

2) Internal standardisation with labelled substances

1) Saponification 2) LiquidLiquid partitioning

D) Other solvent extraction

5308 1) HPLC-FLD 1) External calibration

4) Solid phase extraction

B) Sonication

5309 1) HPLC-FLD 1) External calibration

4) Solid phase extraction

D) Other only SPE

5310 1) HPLC-FLD 1) External calibration

1) Saponification 2) LiquidLiquid partitioning 3) Gel permeation chromatography

C) Soxhlet extraction

5311 6) GC-MS

2) Internal standardisation with labelled substances

13 C Chrysene 13 C Benz(a)pyrene

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

B) Sonication

5312 1) HPLC-FLD

3) Internal standardisation with unlabelled substances

Benzo(b)chrysen

2) LiquidLiquid partitioning 4) Solid phase extraction

Quechers D) Other Quechers

65

Table 19 - continued

Laboratory code

Analysis technique

Instrument calibration

Internal standards applied

Sample clean up Details of sample

clean up

Extraction technique

Details of sample extraction

5313 6) GC-MS

2) Internal standardisation with labelled substances

Benz(a)anthracene D12 Benzo(b)fluranthene D12 Benzo(a)pyrene D12 Chrysene D12

1) Saponification 2) LiquidLiquid partitioning 3) Gel permeation chromatography

A) Pressurised liquid extraction (PLE)

5314 6) GC-MS

2) Internal standardisation with labelled substances

Benzo(a)pyren d12 2) LiquidLiquid partitioning

D) Other Extraction with

5315 1) HPLC-FLD

3) Internal standardisation with unlabelled substances

Benz(b)chrysen

1) Saponification 2) LiquidLiquid partitioning

D) Other shaker

5316 1) HPLC-FLD

1) External calibration

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

D) Other

LiquidLiquid extraction with cyclohexane and dimethylformamide

5317 7) GC-MSMS

1) External calibration

3) Gel permeation chromatography 6) Other

silica gel D) Other VDLUFA online method

5318

5319 1) HPLC-FLD

3) Internal standardisation with unlabelled substances

Benzo(b)chrysene 3) Gel permeation chromatography

A) Pressurised liquid extraction (PLE)

5320 6) GC-MS

3) Internal standardisation with unlabelled substances

22-binaphthyl

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

D) Other 3 hours refluxing

5321 1) HPLC-FLD

2) Internal standardisation with labelled substances

6-meacutethylchrysegravene

1) Saponification 2) LiquidLiquid partitioning

B) Sonication

5322 6) GC-MS

2) Internal standardisation with labelled substances

naftalene-d8 acenaphtalene-d10 phenanthrene-d10 chrysene-d12 perylene-d12 benzo(e)anthracene-d12

1) Saponification 2) LiquidLiquid partitioning 6) Other

clean-up with silica packed column

C) Soxhlet extraction

5323 1) HPLC-FLD

1) External calibration

4) Solid phase extraction 6) Other

Accelerated Solvent Extraction

A) Pressurised liquid extraction (PLE)

66

European Commission EUR 25245 EN ndash Joint Research Centre ndash Institute for Reference Materials and Measurements Title Report on the 8th inter-laboratory comparison organised by the European Union Reference Laboratory for Polycyclic Aromatic Hydrocarbons - Four marker PAHs in a dry extract of St Johns wort Author(s) Radoslav Lizak Szilard Szilagyi Philippe Verlinde and Thomas WENZL Luxembourg Publications Office of the European Union 2012 ndash 66 pp ndash 201 x 297 cm EUR ndash Scientific and Technical Research series ndash ISSN 1831-9424 ISBN 978-92-79-23337-1 doi 10278759387 Abstract The proficiency test here reported concerned the determination of the contents of four marker polycyclic aromatic hydrocarbons (PAHs) and of their sum in an food supplements test sample The set of marker PAHs consists of benz[a]anthacene benzo[a]pyrene benzo[b]fluoranthene and chrysene Participants to these PT were National Reference Laboratories for PAHs (NRLs-PAHs) and EU official food control laboratories The number of participants was in total 48

The PT was organised according ISO Standard 170432010

The test material used was a commercial dry extract of St Johns wort Participants also received a solution of the same PAHs either in acetonitrile or in toluene for checking their instrument calibration

The results from participants were rated with z-scores and zeta-scores About 68 of the reported results were attributed with z-scores with an absolute value of below two which is the threshold for satisfactory performance

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science and technology for the Union Close to the policy-making process it serves

the common interest of the Member States while being independent of special

interests whether private or national

L

A-N

A-252

45-EN

-N

• 1 Summary
• 2 Introduction
• 3 Scope
• 4 Participating Laboratories
• 5 Time frame
• 6 Confidentiality
• 7 Test materials
• • 71 1Preparation of test materials
  • 72 Assigned values and standard deviation for proficiency assessment
  • 73 Homogeneity and stability
  • 74 Sample dispatch
  • • 8 Design of the proficiency test
    • 9 Evaluation of the results
    • • 91 2General
      • 92 21BEvaluation criteria
      • 93 Performance of participants
      • 94 Evaluation of the influence of analysis method
      • 95 Evaluation of the influence of calibration
      • 96 26Evaluation of compliance with legislation
      • • 10 Follow-up actions for underperforming laboratories
        • 11 1Conclusions
        • 12 1Acknowledgements
        • 13 References

7

Table 3 List of participating Official Food Control Laboratories

Institute Country

Federal Laboratory for the Safety of the Food Chain BELGIUM

LARECO SA BELGIUM

Laboratorium ECCA NV BELGIUM

MTT Agrifood Research Finland FINLAND

LEAV - Laboratoire de lEnvironnement et de lAlimentation FRANCE

LDA56 FRANCE

IDAC FRANCE

Laboraroire Departemental de la Sarthe FRANCE

LDA 22 FRANCE

LDA 26 FRANCE

SCL Ile de France-Massy Laboratory FRANCE

CVUA Freiburg GERMANY

CVUA-Muumlnsterland-Emscher-Lippe GERMANY

Landesbetrieb Hessisches Landeslabor GERMANY

LAVES Lebensmittelinstitut Braunschweig GERMANY

Chemisches Untersuchungsamt Hagen GERMANY

Landesamt fuumlr Verbraucherschutz Sachsen-Anhalt GERMANY

Thuumlringer Landesamt fuumlr Lebensmittelsicherheit und Verbraucherschutz GERMANY

LTZ Augustenberg GERMANY

Landesuntersuchungsanstalt Sachsen GERMANY

Bayerisches Landesamt fuumlr Gesundheit und Lebensmittelsicherheit GERMANY

State Veterinary and Food Institute Košice SLOVAKIA

ANFACO-CECOPESCA SPAIN

5 Time frame

The ILC was agreed with the NRLs at the EU-RL PAH workshop held in Brussels on 6 April 2011 It

was announced on the IRMM web page (see ANNEX 1) and registration was opened on 15 May 2011

(see ANNEX 2) Test samples were dispatched (see ANNEX 3) on the 18 July 2011 and the deadline

for reporting of results was set to finally 22 September 2011

8

6 Confidentiality

The identities of participants are kept confidential unless the participant provides a letter of consent to

the PT organiser giving permission to disclose hisher details and results to a third party

7 Test materials

71 Preparation of test materials

The test material of this PT round was a food supplement in particular a dry extract of St Johns wort

in the following denoted as FS

The test material was prepared from commercial products at the EU-RL PAH laboratories About 60

units containing each 120 pills of a dry extract of St Johns wort were acquired at local stores They

were then ground and homogenised with a Loedige Ploughsharereg mixer Portions of at least 15 g of

the homogenised powder were packed into amber glass screw cap vials

Participants also received a solution of the 15+1 EU priority PAHs in either acetonitrile or toluene

(according to their choice) with disclosed concentrations which served for checking instrument

calibration The technical specifications (see ANNEX 4) of the chosen solution were dispatched

together with the test samples

Besides the standard solution with disclosed content a solution with undisclosed content was prepared

The participants were requested to report the analyte content of this solution to the organiser of the PT

This information was used to identify potential reason for bias

72 Assigned values and standard deviation for proficiency assessment

The assigned values were determined from in-house measurements at the EU-RL PAH applying

bracketing calibration and from measurements conducted by a subcontracted external laboratory The

measurements at the EU-RL were conducted on two different days Four replicate samples were

analysed by bracketing calibration using a commercially available standard solution (Dr Ehrenstorfer

Standard 183) in each of the two analysis sessions The contract laboratory had to perform three

replicate analyses of two different samples each composed of three units of PT test samples The

analyses had to be performed on two different days

9

In cases of almost identical analysis results (difference between own results and results of contract

laboratory lt 5) the results from the in-house measurements were applied as assigned values

(applicable to BAA and BBF) If the difference between the two results exceeded 5 the average of

the two results was applied as assigned value (applicable to BAP and CHR) and the combined

uncertainty of in-house and external measurements was associated with the assigned value

The sum of PAH 4 was calculated from the individually assigned values and the corresponding

uncertainty from the uncertainties of the assigned values according to equation 1 The results of in-

house measurements and the results reported by the contract laboratory as well as the assigned values

of the target PAHs are listed in Table 4

Equation 1 2222CHRBBFBAPBAAsum uuuuu

where usum refers to the standard uncertainty of the sum of the four PAHs and uBAA u BAP uBBF and

uCHR refer to the standard uncertainty of the individual analytes

The standard deviation for proficiency assessment (σP) was set for the four individual analytes equal to

the maximum tolerated standard measurement uncertainty Uf (see Equation 2) as defined by

Commission Regulation (EC) No 3332007 [5] and Commission Regulation (EU) No 8362011 [9]

Equation 2 Uf = 22 )C((LOD2)

where Uf relates to the maximum tolerated standard measurement uncertainty LOD to the required

limit of detection α to a numeric factor depending on the concentration C

The application of Equation 2 with the assigned values listed in Table 4 the maximum tolerated value

of LOD of 030 μgkg and α equal to 02 results in Uf values as reported in Table 4

The Uf values of the individual analytes are propagated in analogy to equation 1 for the determination

of the Uf value for the sum of the four PAHs which was used as standard deviation for proficiency

assessment for the sum parameter

10

Table 4 Analyte contents of the food supplement test material

Average of two

sessions bracketing calibration

External evaluation

Assigned value

and σP

Cont microgkg 298 297 298U microgkg 024 041 024P microgkg 062

Cont microgkg 157 174 165U microgkg 020 016 026P microgkg 036

Cont microgkg 292 289 292U microgkg 028 034 028P microgkg 060

Cont microgkg 424 389 407U microgkg 057 057 081P microgkg 083

Cont microgkg 1162U microgkg 093P microgkg 125

Sum PAH4

BAA

BAP

BBF

CHR

from chemical analysis Sum of the four analytes contents for the SUM

p standard deviation for proficiency assessment For the individual analytes is equal to the uncertainty function - Uf

according to Ref [9]

U expanded uncertainty of the assigned value (k=2)

The gravimetrical preparation concentrations were used as assigned values for the standard solutions

with undisclosed content Table 5 contains the assigned values and associated expanded uncertainties

of the standard solutions with undisclosed content

11

Table 5 Analyte contents of the standard solutions with undisclosed content

Content U (k=2) Content U (k=2) microgkg microgkg microgkg microgkg

benz[a ]anthracene 4317 039 4764 043benzo[c ]fluorene 7342 061 8052 067chrysene 2904 031 3179 034cyclopenta[cd ]pyrene 2558 050 2818 055benzo[a ]pyrene 2897 038 3186 042benzo[b ]fluoranthene 2926 026 3199 029benzo[ghi ]perylene 2214 023 2438 026benzo[j ]fluoranthene 4353 038 4763 041benzo[k ]fluoranthene 2143 017 2380 019dibenzo[ae ]pyrene 5735 084 6326 093dibenzo[ah ]anthracene 2215 032 2445 035dibenzo[ah ]pyrene 2169 029 2394 032dibenzo[ai ]pyrene 3691 100 4035 109dibenzo[al ]pyrene 4627 057 5104 063indeno[123-cd ]pyrene 3793 036 4176 0395-methylchrysene 8589 087 9443 096

Solution in toluene Solution in acetonitrile

Prep conc concentration from gravimetrical preparation U expanded uncertainty of the assigned value k coverage

factor

73 Homogeneity and stability

Homogeneity of the food supplements test sample was evaluated according to ISO Standard 13528

Ten units of the St Johns wort extract test material were selected randomly and analysed by isotope

dilution gas chromatography mass spectrometry (GC-MS) The test material was rated sufficiently

homogeneous (see ANNEX 5)

The contributions of the uncertainties of homogeneity (relative uncertainties below 1 ) to the total

uncertainties of the assigned values were found negligible and were therefore not considered in the

calculations

The stability of the test materials was evaluated applying an isochronous experimental set up This

comprised the storage of test samples both at room temperature and cooled in the fridge The content

values of the cooled sample were used as reference for the stability evaluation The materials were

stored from dispatch of test samples to the participants until expiry of the deadline for reporting of

results Afterwards the samples were analysed under repeatability conditions and results were

compared The content values of the cool-stored sample and the sample stored at room temperature

were not statistically significantly different Hence stability of the samples over the whole study period

can be assumed The analysis data are presented in ANNEX 5

12

The uncertainty of stability of the test material was considered negligible compared to the uncertainty

of the characterisation measurements Therefore this parameter was not taken into account in the

determination of the uncertainties of the assigned values

74 Sample dispatch

Samples were dispatched at room temperature Each participant received at least

- one ampoule of the solution of the 15+1 EU priority PAHs in the chosen solvent with disclosed

content (2 ml)

- one ampoule of the 15+1 EU priority PAHs in the chosen solvent with undisclosed content (2

ml)

- one screw cap vial with the food supplement test material

Each parcel contained a sample receipt form the outline of the study an appropriate material safety

data sheets and a unique code that was required for reporting of results The documents sent to the

participants are presented in ANNEX 6

8 Design of the proficiency test

The design of the PT foresaw triplicate analyses of the test sample and reporting of the individual

results of replicate analyses for the single analytes in the following denoted as FS_REP and

additionally a value for proficiency assessment in the following denoted as final value - FS_FIN

for both the single analytes and the sum of the four marker PAHs Both FS_REP results and FS_FIN

results had to be reported corrected for recovery (and recovery had to be stated in the questionnaire

which participants were asked to fill in together with other parameters of the method applied) the

latter had also to be accompanied by the respective expanded measurement uncertainty The FS_FIN

results were used for performance assessment

Besides analysis results participants were also asked to report details of the applied analysis method

and to provide answers to a questionnaire (see ANNEX 7 for the template and the compiled returned

questionnaires)

13

9 Evaluation of the results

The results reported by participants are listed in ANNEX 8

91 General

The most important evaluation parameter was the performance of the laboratories in the determination

of the target PAHs in the food supplement test material which was expressed by z-scores and zeta-

scores

The compliance of method performance characteristics specified by the participants with provisions

given in legislation was evaluated too

92 Evaluation criteria

The participants were requested to report for all analytes the results of replicate measurements and a

value for proficiency assessment which is the result they wish to be applied for the calculation of

performance indicators z-Scores and zeta-scores were attributed only to these results The individual

results of replicate analyses were not rated

z-Scores

z-scores were calculated based on the FS_FIN values Equation 1 presents the formula for calculation

of z-scores

Equation 1

P

assignedlab Xxz

where z refers to the z-score xlab to the reported ldquovalue for proficiency assessmentrdquo Xassigned to the

assigned value and σP to the standard deviation for proficiency testing

zeta-Scores

In addition to z-scores zeta-scores were calculated In contrast to z-scores zeta-scores describe the

agreement of the reported result with the assigned value within the respective uncertainties

Unsatisfactorily large zeta-scores might be caused by underestimated measurement uncertainties large

bias or a combination of both Zeta-scores were calculated according to Equation 3

14

Equation 3 22assignedlab

assignedlab

uu

Xxzeta

where zeta refers to the zeta-score xlab to the reported ldquovalue for proficiency assessmentrdquo Xassigned to

the assigned value ulab to the measurement uncertainty reported by the laboratory and uassigned to the

uncertainty of the assigned value

The performance of the laboratories was classified according to ISOIEC 170432010 [10] Following

scheme is applied for the interpretation of zeta scores and z-scores

|score| 20 = satisfactory performance

20lt|score| lt 30 = questionable performance

|score| ge 30 = unsatisfactory performance

93 Performance of participants

The 48 participants in the study reported in total 225 results for proficiency evaluation which equals to

about 94 of the 240 possible results About 68 of the reported results were rated as satisfactory

with regard to z-scores

Figure 1 and Figure 2 give an overview of the z-scores assigned to the respective results The larger

the triangles the larger were the differences to the assigned values Red triangles indicate z-scores

above an absolute value of three whereas yellow triangles represent z-scores in the questionable

performance range The corresponding score values are plotted next to the triangles About 40 of the

72 non-satisfactory results were reported by seven laboratories only eg the performance of

participant 3608 was not satisfactory for the majority of target analytes

It is also obvious that most problems were encountered in the determination of chrysene and the sum

of the four analytes Potential reasons for the difficulties related to chrysene are discussed further

down

The ideal situation would be that deviations from the assigned values of the results reported for the

individual analytes are small and randomly distributed around zero This will give a satisfactory

performance statement for the sum parameter Deviations of opposite sign could also cancel out and

lead to a satisfactory performance for the sum parameter (eg participants 5303 5304 and 5305)

However in most cases a single underperformance for one analyte led also to non-satisfactory rating

for the sum parameter

15

The numerical values of the calculated z-scores are compiled in Table 6 and Table 7 z-Scores with an

absolute value of above 2 are given in red bold font

The results of the data evaluation for the individual analytes are given in ANNEX 8

For each analyte the figure shows the individual analysis results of the three replicate determinations

The assigned value is shown as dotted line The blue boxes represent the expanded uncertainties as

reported by participants for the value for proficiency assessment The arithmetic mean of the results

of the individual participants is indicated in the blue boxes by a blue line The satisfactory performance

range is located between the two red lines

The individual results of the replicate measurements and the value for proficiency assessment with

its accompanying expanded measurement uncertainty (k=2) are listed in the tables in ANNEX 8 as

well

16

Figure 1 Graphical presentation of z-scores corresponding to the values for proficiency assessment

reported by the NRLs for the contents of BAA BAP BBF and CHR in the St Johns wort extract test

material

Blue triangles indicate satisfactory performance yellow triangles indicate questionable performance red triangles indicate non-satisfactory performance z-score values are presented above the triangles for the latter two performance categories

ProLab 2011

Z score-3 0 3 -3 0 3 -3 0 3 -3 0 3 -3 0 3

BAA BAP BBF CHR PAH4

La

bo

rato

ry

36003602

3603

36043605

36063607

36083610

36113612

36133614

36153616

36173618

36193620

36213622

36233624

3626

2258

2343

2452

2669

2147

2788

2869

6710

70839

3011

11271

80525

8425

4561

12338

90680

4976

7331

99553

3998

3780

7500

17420

168413

3269

3029

-2348 -2736

-2023 -2981

-2155-3018

-3998

Figure 2 Graphical presentation of z-scores corresponding to the values for proficiency assessment

reported by the OCLs for the contents of BAA BAP BBF and CHR in the St Johns wort extract test

material

Blue triangles indicate satisfactory performance yellow triangles indicate questionable performance red triangles indicate non-satisfactory performance z-score values are presented above the triangles for the latter two performance categories

ProLab 2011

Z score-3 0 3 -3 0 3 -3 0 3 -3 0 3 -3 0 3

BAA BAP BBF CHR PAH4

La

bo

rato

ry

5301

5302

5303

5304

5305

5306

5307

5308

5309

5310

5311

5312

5313

5314

5315

5316

5317

5318

5319

5320

5321

5322

5323

2597

2629

2017

2293

2873

2123

2521

2620

2186

2222

2452

2864

3952

3984

3806

9226

4171 5522

3285

6594

5205

5157

3539

3901

7051

4519

3173

4872

9191

7276

4391

-2435

-2154

-2065

-2340

-2740-3726

-6346

-3505

17

Table 6 Compilation of z-scores calculated from the ldquoresults for proficiency assessment

reported by the NRLs for food supplement test material z-scores outside the

satisfactory range (|z| gt 2) are indicated by red font Values are reproduced as reported

by the laboratories Empty cells denote analytes for which results were not reported

Measurand benz[a]anthracene benzo[a]pyrene benzo[b]fluoranthene chrysene Sum PAH 4

Assigned value

microgkg 298 165 292 407 1162

Standard deviation for proficiency assessment

microgkg 062 036 060 083 125

Laboratory code

Result z-score Result z-score Result z-score Result z-score Result z-score

microgkg microgkg microgkg microgkg microgkg

3600 3 00 17 01 31 03 5 11 128 09

3602 19 -18 124 -11 17 -20 304 -12 79 -30

3603 251 -08 161 -01 256 -06 459 06 1127 -03

3604 213 -14 143 -06 11 -30 429 03 893 -22

3605 291 -01 167 01 247 -07 386 -03 1091 -06

3606 289 -01 167 01 276 -03 451 05 1184 02

3607 438 23 274 30 567 46 819 50 2098 75

3608 714 67 573 113 1036 123 1014 73 3336 174

3610 196 -17 206 11 191 -17 424 02 1138 -02

3611 469 708 308 805 576 907 865 996 2218 1684

3612 31 02 22 15 29 00 61 25 143 21

3613 33 05 18 04 31 03 4 -01 122 05

3614 185 -18 08 -23 127 -27 271 -16 663 -40

3615 376 13 145 -06 4 18 601 23 152 29

3616 2 -16 15 -04 2 -15 47 08 102 -11

3617 362 10 169 01 299 01 422 02 1252 07

3618 32 04 2 10 31 03 45 05 128 09

3619 299 00 207 12 326 06 738 40 157 33

3620 302 01 166 00 276 -03 46 06 1204 03

3621 28 -03 47 84 25 -07 51 12 151 28

3622 322 04 213 13 34 08 488 10 1361 16

3623 33 05 21 12 28 -02 72 38 154 30

3624 18 04

3626 314 03 174 02 292 00 628 27 1409 20

18

Table 7 Compilation of z-scores calculated from the ldquoresults for proficiency assessment

reported by the OCLs for food supplement test material z-scores outside the

satisfactory range (|z| gt 2) are indicated by red font Values are reproduced as reported

by the laboratories Empty cells denote analytes for which results were not reported

Measurand benz[a]anthracene benzo[a]pyrene benzo[b]fluoranthene chrysene Sum PAH 4

Assigned value

microgkg 298 165 292 407 1162

Standard deviation for proficiency assessment

microgkg 062 036 060 083 125

Laboratory code

Result z-score Result z-

score Result z-score Result

z-score

Result z-score

microgkg microgkg microgkg microgkg microgkg

5301 31 02 18 04 29 00 49 10 126 08

5302 316 03 188 06 298 01 679 33 37 -63

5303 2876 -02 1535 -03 2586 -06 6441 29 13438 15

5304 261 -06 146 -05 253 -06 591 22 1251 07

5305 24 -09 16 -01 24 -09 61 25 125 07

5306 22 -13 16 -01 42 21 49 10 129 10

5307 461 27 157 -02 221 -12 524 14 1363 16

5308 16446 -22 107009 -16 229848 -10 213243 -23 72456 -35

5309 17 -21 11 -15 2 -15

5310 305 01 123 -12 313 03 386 -03 1127 -03

5311 459 26 2 10 281 -02 437 04 1367 16

5312 543 40 248 23 299 01 953 66 2042 71

5313 353 09 207 12 328 06 838 52 1726 45

5314 394 16 172 02 404 19 588 22 1558 32

5315 545 40 238 20 444 25 542 16 177 49

5316 235 -10 17 01 273 -03 282 -15 96 -16

5317 222 -12 269 29 192 -17 545 17 1229 05

5318 067 -37 117 -13 279 -02 357 -06 82 -27

5319 147 -24 216 14 396 17 465 07 1225 05

5320 534 38 316 42 625 55 834 52 2309 92

5321 12 -12

5322 87 92 17 01 33 06 7 35 207 73

5323 33 05 2 10 45 26 73 39 171 44

19

Table 8 and Table 9 present the respective zeta-scores As for the z-scores data outside the

satisfactory performance range are given in red bold font Zeta scores were only calculated for

laboratories that provided measurement uncertainty data as well as a numerical value for the coverage

factor Zeta scores were not calculated when the reported uncertainty value exceeded the content value

which is probably linked to mixing of reporting units (microgkg and ) However the participants are

reminded that the measurement uncertainty shall be expressed in the same unit as the content value

The assessment of the performance of the participants based on the reported measurement uncertainty

gave a less favourable picture compared to z-scores Only 60 of the zeta-scores are within the

satisfactory performance range The distribution of satisfactory performance ratings between NRLs

and OCLs is not balanced It was higher for the NRLs

It has to be noted that the magnitude of the zeta-scores were for some participants much higher than

the z-scores attributed to the same results Consequently the laboratories perform according to fitness-

for-purpose criterion specified in legislation which forms the basis for the z-scores but seem to have

difficulties in deducing realistic measurement uncertainty values Eg the relative standard

measurement uncertainties reported by the NRLs ranged from 25 to 20 with a median value of

about 10 Only two OCLs reported measurement uncertainties that exceeded the maximum tolerable

uncertainty (Uf) The respective data is highlighted in yellow in Table 9

However the EU-RL PAHs will continue to pay special attention to measurement uncertainty in future

ILCs as it has major implications on the assessment of compliance of food with European legislation

20

Table 8 Compilation of zeta-scores calculated from the ldquoresults for proficiency assessment

reported by the NRLs for test material St Johns wort the reported measurement

uncertainty and the uncertainty of the analyte content of the test material zeta-scores

outside the satisfactory range (|zeta| gt 2) are indicated by red bold font Empty cells

denote analytes for which either results or measurement uncertainties were not

reported

21

Table 9 Compilation of zeta-scores calculated from the ldquoresults for proficiency assessment

reported by the OCLs for test material St Johns wort the reported measurement

uncertainty and the uncertainty of the analyte content of the test material zeta-scores

outside the satisfactory range (|zeta| gt 2) are indicated by red bold font yellow

highlighted cells ugtUf Empty cells denote analytes for which results were not

reported

22

94 Evaluation of the influence of analysis method

The information on the applied analysis method (ANNEX 11) was used to group the performance

indicators attributed to the reported results according to the applied analysis technique Two groups of

data were formed They represent results obtained by high performance liquid chromatography-

fluorescence detection (HPLC-FD about 40 ) and by gas chromatography-mass spectrometry (GC-

MS about 60 ) respectively Kernel density plots were constructed based on the respective z-scores

Figure 3A to 3E show the different Kernel density plots

Figure 3 Kernel density plots of z-scores attributed to the results for proficiency assessment

which were reported for the food supplements test sample Results were classified

according to the applied analysis technique

A) benz[a]anthracene

B) benzo[a]pyrene

23

Figure 3 continued

C) benzo[b]fluoranthene

D) chrysene

E) sum of four marker PAHs

The kernel density plots for BAP and BBF are almost identical and centred at or close to a z-score

value of zero as can be seen in Figure 3B and 3C Therefore bias introduced by the type of analysis

method is unlikely for these two analytes The situation is different for BAA and CHR In case of

BAA the kernel density distribution of z-scores based on HPLC-FD is shifted to negative values

24

(Figure 3A) which indicates a tendency to underestimate the BAA content in this particular

samplematrix when applying HPLC-FD This might be reasoned by separationintegration problems

between BAA and CHR peaks A few participants (from the HPLC-FD subgroup) that got negative

z-scores for BAA overestimated the CHR content This caused the slight shift of the density trace for

HPLC-FD towards negative values Removing this data from the evaluation would result in a density

trace with the mode at zero

The opposite is the case for CHR (Figure 3D) The density distribution of the z-scores attributed to

HPLC-FD data is centred almost at zero However the distribution for data based on GC-MS is shifted

to positive values indicating strong positive bias This bias might be caused by a lack of selectivity of

the analysis method The test material contains triphenylene which is isobaric to chrysene

Consequently it cannot be distinguished from chrysene by means of mass spectrometry These two

compounds have to be separated chromatographically which is not easy They coelute on unpolar and

on most mid-polar capillary columns Columns that allow their separation were commercialised only

recently and might not be available in all laboratories Therefore laboratories which determine the

four EU marker PAHs by means of GC-MS shall pay attention to the chromatographic separation of

triphenylene and chrysene A lack of chromatographic resolution might lead to strong positive bias of

GC-MS methods and to false conclusions on the marketability of the tested sample

The chromatographic separation of chrysene and triphenylene is not a problem with HPLC which is

reflected in the position of the density plot for chrysene However attention has to be paid in HPLC-

FD analysis to a proper clean up of the sample extract in order to eliminate interferences that show

similar retention as BAA and CHR

95 Evaluation of the influence of calibration

The participants received together with the food supplement test sample two standard solutions in

depending of their choice either acetonitrile or toluene The analyte content of one solution was

disclosed to the participants whereas the content of the other solution was kept undisclosed The

content values of the latter are presented in Table 5 Analysis results for the solution with undisclosed

content had to be reported to the PT organiser The respective data are presented in Annex 9

Percent deviations of the reported results from the assigned values were calculated for both the food

supplement test sample and the standard solution with undisclosed analyte content Youden plots were

prepared in order to visualize the influence of instrument calibration on the results reported for the

food supplement sample Two plots are discussed in the following The whole set of Youden plots are

given in Annex 9

25

Figure 4 Youden plot of the percent deviations of values reported for benzo[a]pyrene from the

assigned values of the food supplement test material and the standard solution in

toluene with undisclosed content

Figure 4 reveals that a number of participants reported results with similar percent deviations from the

assigned values of both the results for the food supplement material and the standard solution in

toluene with undisclosed content This correlation indicates that over- respectively underestimation of

the analyte content of the food supplement material might be caused by biased instrument calibration

This finding underpins the importance of thorough calibration standard preparation However the data

was not in all cases conclusive as some laboratories performed well for the food supplement samples

but reported biased results for the standard solution with undisclosed content (eg Figure 4 participants

3620 and 5305) This fact cannot be explained yet

Figure 5 presents the respective Youden plot for chrysene in the food supplement test material and the

standard solution in toluene with undisclosed content From this graph it can be concluded that the

overestimation of the chrysene content of the food supplement material was not caused by instrument

calibration The results for the solvent solution of many laboratories which underperformed for the

26

food supplement sample agreed well with the assigned value This supports the conclusion that the

overestimation is caused by the determination of an interferent most probably triphenylene

Figure 5 Youden plot of the percent deviations of values reported for chrysene from the

assigned values of the food supplement test material and the standard solution in

toluene with undisclosed content

96 Evaluation of compliance with legislation

The method performance data reported for the four marker PAHs were evaluated for compliance with

the provisions given in Commission Regulation (EC) No 8362011 Table 18 in ANNEX 10 gives an

overview on the results of the evaluation Data were not reported in case of empty cells Non-

compliant data are highlighted in Table 18 It should be noted that Regulation (EU) No 8362011

specifies LOD and LOQ with two decimals This was not respected by all participants which

hampered the compliance assessment

27

In summary it can be stated that the majority of participants reported method performance

characteristics for the determination of the four marker PAHs in the food supplement test sample that

are compliant with legislation However some laboratories need to improve their analysis methods

The laboratories in question are urged to adapt to the provisions given in the new legislation

10 Follow-up actions for underperforming laboratories

All laboratories that got questionable or non-satisfactory performance ratings are urged to perform

root cause analysis and to implement corrective actions

As agreed during the EU-RL PAH workshop 2011 the EU-RL PAH will set up follow-up measures in

due time for all NRLs that received for at least one of the four PAHs (BAA BAP BBF and CHR) z-

scores gt 3 as required by Regulation (EC) 8822004 and by the Protocol for management of

underperformance in comparative testing andor lack of collaboration of National Reference

Laboratories (NRLs) with European Union reference laboratories (EU-RLs) activities

These laboratories shall perform as an immediate action root-cause-analysis and shall report to the

EU-RL PAH in writing the identified cause for their underperformance and corrective actions they are

going to take Additionally they shall participate to an independent (non-EU-RL) proficiency test on

the determination of PAHs in food and shall communicate the outcome of this exercise to the EU-RL

PAH

28

11 Conclusions

Fourty seven participants reported results back to the EU-RL The performance of the majority of

participants was good In total 153 out of 225 attributed z-scores were below an absolute value of two

which equals to almost 68 However this percentage is much below the percentages of satisfactory

performance gained in previous studies This might be the consequence of the higher level of difficulty

with respect to matrix composition In addition bias can be concluded from the pattern of performance

indicators of some laboratories

In general the determination of chrysene caused most difficulties to the participants

zeta-Scores were calculated besides z-scores They indicate the agreement of the reported result with

the assigned value with respect to the stated measurement uncertainty The outcome of this rating was

worse than for the z-scores which indicates that the measurement uncertainty estimates were not

realistic Therefore participants underperforming with regard to zeta scores are urged to adapt their

measurement uncertainty statements

The great majority of participants in this inter-laboratory comparison applied analytical methods which

complied with EU legislation with regard to performance characteristics

12 Acknowledgements

The organisers would like to thank Mrs Beatriz de la Calle and Mr Franz Ulberth (all from IRMM

Geel Belgium) for their accurate revision of this report Mr Hakan Emteborg (from IRMM) for

ampouling of the standard solutions and all NRLs and OCLs for their cooperation

29

13 References

1 EU COMMISSION REGULATION (EC) No 7762006 of 23 May 2006 amending Annex VII to Regulation

(EC) No 8822004 of the European Parliament and of the Council as regards Community reference

laboratories Official Journal of the European Union 2006 L 136 p 3-8

Available from

httpeur-lexeuropaeuLexUriServLexUriServdouri=OJL200613600030008ENPDF

2 EU Regulation (EC) No 8822004 of the European Parliament and of the Council of 29 April 2004 on

official controls performed to ensure the verification of compliance with feed and food law animal health

and animal welfare rules Official Journal of the European Communities 2004 L191 p 1-52 Available

from

httpeur-lexeuropaeuLexUriServLexUriServdouri=OJL200419100010052ENPDF

3 EU Opinion of the Scientific Committee on Food on the risks to human health of Polycyclic Aromatic

Hydrocarbons in food (2002) Available from

httpeuropaeuintcommfoodfsscscfout153_enpdf

4 International Agency for Research on Cancer IARC Monographs on the Evaluation of

Carcinogenic Risks to Humans Some Non-heterocyclic Polycyclic Aromatic Hydrocarbons and Some

Related Exposures Volume 92 (2010) Available from

httpmonographsiarcfrENGMonographsvol92mono92pdf

5 EU Commission Regulation (EC) No 3332007 of 28 March 2007 laying down the methods of sampling

and analysis for the official control of the levels of lead cadmium mercury inorganic tin 3-MCPD and

benzo(a)pyrene in foodstuffs Official Journal of the European Union 2007 L 88 p 29-38 Available

from

httpeur-lexeuropaeuLexUriServLexUriServdouri=OJL200708800290038ENPDF

6 EU Commission Regulation (EC) No 18812006 of 19 December 2006 setting maximum levels for certain

contaminants in foodstuffs Official Journal of the European Union 2006 L 364 p 5-24 Available from

httpeur-lexeuropaeuLexUriServLexUriServdouri=OJL200636400050024ENPDF

7 EFSA Scientific Opinion of the Panel on Contaminants in the Food Chain on a request from the European

Commission on Polycyclic Aromatic Hydrocarbons in Food The EFSA Journal 2008 724 p 1-114

Available from

httpwwwefsaeuropaeuEFSAefsa_locale-1178620753812_1211902034842htm

30

8 EU Commission Regulation (EU) No 8352011 amending Regulation (EC) No 18812006 as regards

maximum levels for polycyclic aromatic hydrocarbons in foodstuffs Official Journal of the European

Union 2011 L 215 p 4-8

httpeur-lexeuropaeuLexUriServLexUriServdouri=OJL201121500040008ENPDF

9 EU Commission Regulation (EU) No 8362011 amending Regulation (EC) No 3332007 laying down the methods of sampling and analysis for the official control of the levels of lead cadmium mercury inorganic tin 3-MCPD and benzo(a)pyrene in foodstuffs Official Journal of the European Union 2011 L 215 p 9-16 httpeur-lexeuropaeuLexUriServLexUriServdouri=OJL201121500090016ENPDF

10 ISOIEC 170432010 (2010) Conformity assessment -- General requirements for proficiency testing

providers Geneva Switzerland

11 Thompson M Ellison SLR and Wood R The International Harmonized Protocol for the Proficiency

Testing of Analytical Chemistry Laboratories Pure Appl Chem (2006) 78(1) p 145ndash196

Available from httpmediaiupacorgpublicationspac2006pdf7801x0145pdf

31

14 ANNEXES

ANNEX 1 ndash Announcement of the PT on the IRMM webpage

ANNEX 2 ndash Announcement via e-mail and invitation

ANNEX 3 ndash Announcement of sample dispatch

ANNEX 4 ndash Technical specifications of the calibration solutions

ANNEX 5 ndash Homogeneity and stability of the test material

ANNEX 6 ndash Acknowledgement of sample receipt

ANNEX 7 ndash Questionnaire on details of analysis method

ANNEX 8 ndash Results reported for the food supplement sample

ANNEX 9 ndash Results reported for the standard solutions with undisclosed analyte content

ANNEX 10 ndash Method performance characteristics reported by participants

ANNEX 11 ndash Details of analysis methods reported by participants

32

ANNEX 1 ndash Announcement of the PT on the IRMM webpage

33

ANNEX 2 - Announcement via e-mail and invitation

34

ANNEX 3 - Announcement sample dispatch

Dear MadameSir

The test samples for the proficiency test on the determination of four PAHs in a food supplement (dry extract of St Johns wort) were

dispatched today

Explanation on the design of the study as well as instructions for sample storage and for reporting of results are in the parcel You will

also find your participant key in the parcel which is required for reporting of results

Please check the parcel after arrival for completeness and report the receipt of the test samples as well as their status to us by applying

the sample receipt form which is in the parcel

You are kindly requested to inform us by email if you do not receive the parcel by end of this week

My colleague Donata Lerda will then take care of it

With best regards

Thomas Wenzl

35

ANNEX 4 - Specifications of standard solutions with disclosed concentration

36

ANNEX 4 - continued

37

ANNEX 5 - Homogeneity and stability of the test material

Homogeneity of the test material

Benz[a]anthracene - BAA

Benzo[a]pyrene - BAP

38

ANNEX 5 - continued

Benzo[b]fluoranthene - BBF

Chrysene - CHR

39

ANNEX 5 - continued

Stability of the test material

Table 10 Content of the four target analytes in test materials stored under different

temperatures

BaA BaP BbF CHR

microgkg microgkg microgkg microgkg

Content 31 16 31 69 Storage at room temperature

U (k=2) 05 02 05 17

Content 31 15 29 68 Storage at reference temperature

U (k=2) 04 03 04 10

The applied capillary column does not separate CHR from triphenylene

40

ANNEX 6 ndash Documents sent to participants

41

ANNEX 6 - continued

42

ANNEX 7 Questionnaire on details of analysis method

43

ANNEX 7 - continued

44

ANNEX 7 - continued

45

ANNEX 8 - Results reported by participants for the food supplements test sample Table 11 Analysis results reported by the participants for the content of benz[a]anthracene (BAA) in the food supplements test material Values are presented as reported

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

3600 31 29 29 3 05 2

3602 199 193 178 19 021

3603 253 253 246 251 022 2

3604 199 209 23 213 071 2

3605 301 3 271 291 076 2

3606 302 288 278 289 064 2

3607 456 416 443 438 096 2

3608 796 695 651 714 149 2

3610 182 192 214 196 039 2

3611 414 504 488 469 96 2

3612 3 34 28 31 06 2

3613 32 33 34 33 07 2

3614 182 182 191 185 037 2

3615 3884 3822 3565 376 0752 2

3616 2 2 2 2 01 2

3617 366 37 35 362 046 2

3618 32 32 32 32 05 2

3619 299 305 297 299 048 2

3620 304 294 302 302 045 2

3621 29 26 29 28 04 2

3622 343 325 318 322 052 2

3623 35 3 33 33 07 2

3624

3626 318 31 314 314 126 2

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

5301 31 3 32 31 06 2

5302 314 319 315 316 063 2

5303 3102 2716 2822 2876 27 62

5304 261 263 26 261 57 2

5305 23 22 26 24 20

5306 22 23 23 22 11 2

5307 488 461 317 461

5308 168633 168544 1562025 1644598

5309 16 19 16 17

5310 3 304 311 305 015

5311 477 452 448 459 15 2

5312 506 63 494 543 134 2

5313 353 301 404 353

5314 431 373 378 394 5 096

5315 596 531 51 545 11 2

5316 224 231 235 235 022 2

5317 216 23 221 222 007 2

5318 064 07 067 01 2

5319 111 184 146 147 029 2

5320 515 525 564 534 48

5321 21 5322 13 87 9 87 35 2

5323 38 33 35 33 16 2

46

Figure 6 Results of replicate determinations (indicated by triangles) of benz[a]anthracene (BAA) in the food supplement test material

Horizontal blue lines represent the arithmetic mean value of replicate measurements and blue bars the reported expanded measurement uncertainty (k=2) The assigned value is

plotted as dotted line Red solid lines mark the range of satisfactory performance (|z|le2) Mean values are given for results outside the displayed data range

ProLab 2011

Laboratory

53

1853

1953

0853

0936

1436

0236

1036

1653

2136

0453

1753

0653

1653

0536

0353

0436

2153

0336

0636

0536

0036

2036

1953

1036

1253

0136

2653

0236

1836

2336

2236

1353

1353

2336

1736

1553

1453

0736

0753

1153

2053

1253

1536

0853

2236

11

an

aly

te c

on

ten

t (micro

gk

g)

10

95

9

85

8

75

7

65

6

55

5

45

4

35

3

25

2

15

1

05

0

102

3 468

7

Sample St Johns wort extract replicatesMeasurand Benz[a]anthraceneNo of laboratories 46

Assigned value 298 microgkg (Reference value)Target sd 062 microgkgTolerance limits 174 - 422 microgkg (|Z score| lt 200)

Assig

ned va

lue

Limit of tolerance

Limit of tolerance

47

Table 12 Analysis results reported by the participants for the content of benzo[a]pyrene (BAP) in the food supplements test material Values are presented as reported

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

3600 16 17 17 17 03 2

3602 118 123 13 124 015

3603 165 162 157 161 015 2

3604 134 154 139 143 04 2

3605 164 166 172 167 057 2

3606 181 159 161 167 031 2

3607 279 266 279 274 055 2

3608 57 687 461 573 226 2

3610 226 216 183 206 042 2

3611 289 325 311 308 36 2

3612 24 22 2 22 05 2

3613 17 18 19 18 05 2

3614 08 08 08 08 016 2

3615 1545 1393 1425 145 029 2

3616 16 16 14 15 03 2

3617 177 171 158 169 023 2

3618 2 2 2 2 02 2

3619 207 207 206 207 075 2

3620 165 169 163 166 017 2

3621 44 47 49 47 05 2

3622 212 208 222 213 038 2

3623 2 21 22 21 03 2

3624 18 178 182 18 031 2

3626 172 173 178 174 07 2

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

5301 18 18 18 18 04 2

5302 188 19 187 188 038 2

5303 1691 1426 149 1535 19 80

5304 145 142 15 146 69 2

5305 15 17 18 16 15

5306 16 17 17 16 08 2

5307 164 157 153 157

5308 102669 109324 109035 107009

5309 1 1 12 11

5310 122 122 124 123 006

5311 199 184 215 2 15 2

5312 258 248 239 248 05 2

5313 199 178 245 207

5314 174 171 172 172 11 0948

5315 253 248 213 238 05 2

5316 177 171 173 17 016 2

5317 255 277 276 269 012 2

5318 114 12 117 02 2

5319 19 228 231 216 043 2

5320 303 3 344 316 78

5321 12 12 12 12

5322 13 17 24 17 35 2

5323 2 2 19 2 1 2

48

Figure 7 Results of replicate determinations (indicated by triangles) of benzo[a]pyrene (BAP) in the food supplement test material

Horizontal blue lines represent the arithmetic mean value of replicate measurements and blue bars the reported expanded measurement uncertainty (k=2) The assigned value is

plotted as dotted line Red solid lines mark the range of satisfactory performance (|z|le2) Mean values are given for results outside the displayed data range

ProLab 2011

Laboratory

36

1453

0953

0853

1853

2153

1036

0236

0436

1553

0436

1653

0353

0736

0336

2036

0053

0553

0636

0636

0536

1753

1453

1636

2636

1336

2453

0153

2253

0253

2353

1136

1836

1953

1336

1036

2336

2253

1936

1253

1553

1253

1736

0753

2036

2136

0836

11

an

aly

te c

on

ten

t (micro

gk

g)

55

5

45

4

35

3

25

2

15

1

05

0

573

308

3

Sample St Johns wort extract replicatesMeasurand Benzo[a]pyreneNo of laboratories 48

Assigned value 165 microgkg (Reference value)Target sd 036 microgkgTolerance limits 093 - 237 microgkg (|Z score| lt 200)

Assig

ned va

lue

Limit of tolerance

Limit of tolerance

49

Table 13 Analysis results reported by the participants for the content of benzo[b]fluoranthene (BBF) in the food supplements test material Values are presented as reported

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

3600 32 3 3 31 09 2

3602 173 175 16 17 017

3603 265 252 25 256 025 2

3604 116 108 105 11 028 2

3605 286 225 232 247 074 2

3606 294 273 261 276 046 2

3607 579 579 544 567 113 2

3608 103 1201 876 1036 326 2

3610 173 196 204 191 038 2

3611 546 618 565 576 75 2

3612 3 28 28 29 06 2

3613 28 31 32 31 06 2

3614 135 111 135 127 022 2

3615 4187 3678 4111 4 08 2

3616 2 2 2 2 01 2

3617 33 27 299 035 2

3618 31 31 31 31 05 2

3619 326 324 312 326 064 2

3620 277 28 272 276 069 2

3621 26 25 23 25 04 2

3622 346 336 333 34 052 2

3623 26 28 29 28 02 2

3624

3626 288 28 307 292 117 2

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

5301 29 3 29 29 06 2

5302 297 3 296 298 06 2

5303 2705 2444 2608 2586 22 78

5304 245 254 26 253 85 2

5305 23 23 26 24 15

5306 42 43 4 42 21 2

5307 221 216 241 221

5308 232169 246654 240722 229848

5309 19 22 19 2 0

5310 3 315 325 313 016

5311 262 277 304 281 15 2

5312 27 289 337 299 075 2

5313 321 284 378 328

5314 423 404 385 404 34 083

5315 42 506 416 444 09 2

5316 265 267 277 273 018 2

5317 185 197 195 192 006 2

5318 276 281 279 04 2

5319 396 389 403 396 079 2

5320 623 618 635 625 14

5321 31

5322 24 33 49 33 35 2

5323 38 45 35 45 27 2

50

Figure 8 Results of replicate determinations (indicated by triangles) of benzo[b]fluoranthene (BBF) in the food supplement test material

Horizontal blue lines represent the arithmetic mean value of replicate measurements and blue bars the reported expanded measurement uncertainty (k=2) The assigned value is

plotted as dotted line Red solid lines mark the range of satisfactory performance (|z|le2) Mean values are given for results outside the displayed data range

ProLab 2011

Laboratory

36

0436

1436

0236

1053

1736

1653

0953

0753

0853

0536

2136

0553

0436

0353

0353

1636

0636

2036

2353

1853

1136

1236

2653

0153

0253

1236

1736

1336

0036

1853

2153

1036

1953

1336

2253

2253

2353

1936

1553

1453

0653

1536

0753

2036

0836

11

an

aly

te c

on

ten

t (micro

gk

g)

75

7

65

6

55

5

45

4

35

3

25

2

15

1

05

0

103

6

576

3

Sample St Johns wort extract replicatesMeasurand Benzo[b]fluorantheneNo of laboratories 46

Assigned value 292 microgkg (Reference value)Target sd 060 microgkgTolerance limits 172 - 412 microgkg (|Z score| lt 200)

Assig

ned va

lue

Limit of tolerance

Limit of tolerance

51

Table 14 Analysis results reported by the participants for the content of chrysene (CHR) in the food supplements test material Values are presented as reported

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

3600 5 49 5 5 07 2

3602 32 304 29 304 036

3603 462 469 447 459 048 2

3604 438 428 42 429 119 2

3605 365 41 381 386 085 2

3606 494 411 45 451 124 2

3607 897 774 787 819 18 2

3608 986 887 1168 1014 284 2

3610 418 392 462 424 085 2

3611 833 893 87 865 61 2

3612 6 64 58 61 12 2

3613 41 39 4 4 08 2

3614 272 254 288 271 054 2

3615 605 6028 5964 601 12 2

3616 47 45 49 47 05 2

3617 445 431 389 422 065 2

3618 45 45 45 45 06 2

3619 738 747 72 738 118 2

3620 463 472 446 46 092 2

3621 53 49 51 51 06 2

3622 478 513 505 488 08 2

3623 73 73 72 72 05 2

3624

3626 63 622 634 628 251 2

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

5301 49 49 49 49 1 2

5302 687 683 666 679 136 2

5303 6777 6181 6382 6441 24 70

5304 592 581 6 591 85 2

5305 6 58 64 61 20

5306 49 5 51 49 25 2

5307 524 5 574 524

5308 211488 216782 221146 213243

5309

5310 372 393 394 386 019

5311 43 459 422 437 15 2

5312 934 943 981 953 191 2

5313 793 73 99 838

5314 636 561 565 588 18 0818

5315 547 583 499 542 11 2

5316 292 286 285 282 06 2

5317 52 598 517 545 046 2

5318 368 345 357 05 2

5319 449 471 474 465 116 2

5320 76 924 818 834 99

5321 33

5322 7 7 76 7 35 2

5323 87 73 77 73 37 2

52

Figure 9 Results of replicate determinations (indicated by triangles) of chrysene (CHR) in the food supplement test material

Horizontal blue lines represent the arithmetic mean value of replicate measurements and blue bars the reported expanded measurement uncertainty (k=2) The assigned value is

plotted as dotted line Red solid lines mark the range of satisfactory performance (|z|le2) Mean values are given for results outside the displayed data range

ProLab 2011

Laboratory

53

0836

1453

1636

0253

2153

1836

0553

1036

1336

1736

1036

0453

1136

1836

0636

0336

2053

1936

1653

0136

0036

2253

0636

2153

0753

1553

1753

1453

0436

1536

1253

0536

2653

0353

0253

2236

2336

1953

2336

0753

2053

1353

1236

0836

11

an

aly

te c

on

ten

t (micro

gk

g)

13

12

11

10

9

8

7

6

5

4

3

2

1

0

865

3

Sample St Johns wort extract replicatesMeasurand ChryseneNo of laboratories 45

Assigned value 407 microgkg (Reference value)Target sd 083 microgkgTolerance limits 241 - 573 microgkg (|Z score| lt 200)

Assig

ned va

lue

Limit of tolerance

Limit of tolerance

53

Table 15 Analysis results reported by the participants for the sum of the four marker PAHs in the food supplements test material Values are presented as reported

Laboratory code

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg

3600 128 24 2

3602 79 126

3603 1127 096 2

3604 893 258 2

3605 1091 305 2

3606 1184 238 2

3607 2098 42 2

3608 3336 325 2

3610 1138 17 2

3611 2218 259 2

3612 143 29 2

3613 122 2 2

3614 663 129 2

3615 152 304 2

3616 102 03 2

3617 1252 169 2

3618 128 18 2

3619 157 305 2

3620 1204 12 2

3621 151 19 2

3622 1361 115 2

3623 154 09 2

3624

3626 1409 56 2

Laboratory code

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg

5301 126

5302 37 074 2

5303 13438 92

5304 1251 85 2

5305 125 20

5306 129 65 2

5307 1363

5308 7245598

5309

5310 1127 056

5311 1367 15 2

5312 2042 408 2

5313 1726

5314 1558

5315 177 35 2

5316 96 073 2

5317 1229 066 2

5318 82 05 2

5319 1225 267 2

5320 2309 41

5321

5322 207 35 2

5323 171 103 2

54

ANNEX 9 Results reported for the standard solutions with undisclosed analyte content Table 16 Analysis results reported by the participants for the four marker PAHs in the standard solution in toluene with undisclosed analyte

content Values are presented as reported U measurement uncertainty k coverage factor

benz[a]anthracene benzo[a]pyrene benzo[b]fluoranthene chrysene Laboratory

code Unit Result U k Result U k Result U k Result U k 3600 microgl 38 67 2 25 45 2 211 68 2 275 38 2

3605 microgl 367 184 2 2482 124 2 2592 13 2 235 118 2

3606 microgkg 3763 176 2 2609 142 2 2721 118 2 2539 134 2

3607 microgkg 3706 815 2 2824 565 2 2591 518 2 2587 569 2

3612 microgl 38 08 2 237 11 2 244 18 2 257 02 2

3614 microgl 324 65 2 209 38 2 215 37 2 21 42 2

3616 microgkg 318 07 2 224 14 2 231 26 2 216 1 2

3618 microgkg 417 17 2 284 08 2 292 06 2 297 03 2

3619 microgkg 47 742 2 274 468 2 228 387 2 274 434 2

3620 microgl 495 74 2 294 29 2 321 8 2 321 64 2

3621 microgl 241 22 2 143 13 2 188 16 2 145 14 2

3623 microgkg 476 25 2 311 53 2 269 3 2 297 03 2

3626 microgl 359 38 2 235 23 2 238 27 2 24 18 2

5301 microgkg 426 29 299 289

5302 microgkg 436 87 2 296 59 2 296 59 2 289 58 2

5303 microgl 3721 27 102 22838 19 99 23756 22 99 25486 24 98

5304 microgkg 38 29 2 225 26 2 257 37 2 258 52 2

5305 microgkg 305 225 245 29

5306 microgkg 316 63 2 247 49 2 65 13 2 22 44 2

5307 microgkg 3658 2559 241 2414

5308 microgkg 16854 109324 246654 216782

5311 microgl 374 20 2 279 15 2 268 15 2 272 15 2

5313 microgl 3408 2165 2275 2373

5314 microgkg 395 5 2875 11 3614 34 2905 18

5320 microgl 3267 368 1986 571 28 683 2427 512

5322 microgl 282 35 2 225 35 2 241 35 2 21 35 2

55

Figure 10 Youden plots of the percent deviation of the reported values from the assigned values for the food supplement test sample and the

standard solution in toluene with undisclosed content

(The identity of the respective measurand is given above the figure Results exceeding a difference of more than 100 were omitted)

56

Figure 10 - continued

57

Table 17 Analysis results reported by the participants for the four marker PAHs in the standard solution in acetonitrile with undisclosed

analyte content

Values are presented as reported U measurement uncertainty k coverage factor

benz[a]anthracene benzo[a]pyrene benzo[b]fluoranthene chrysene

Laboratory

code Unit Result U k Result U k Result U k Result U k

3602 microgkg 3675 247 247 244

3603 microgkg 358 086 2 236 028 2 235 028 2 236 057 2

3604 microgl 475 29 2 19 12 2 194 13 2 258 12 2

3608 microgl 3623 078 2 2385 023 2 2343 18 2 2296 092 2

3610 microgkg 369 185 2 25 125 2 251 126 2 244 122 2

3611 microgkg 48 02 2 323 02 2 328 04 2 317 03 2

3613 microgl 332 31 2 261 3 2 249 25 2 239 24 2

3615 microgl 415 2635 2729 2689

3617 microgkg 37 09 2 244 06 2 284 07 2 239 06 2

3622 microgl 37 12 2 25 23 2 257 21 2 238 1 2

3624 microgkg 402 104 2 255 084 2 265 096 2 253 07 2

5306 microgkg 563 113 2 371 74 2 359 72 2 333 67 2

5308 microgkg 168633 102669 232169 211488

5309 microgl 402 268 27

5310 microgl 315 158 25 125 25 125 288 144

5312 microgl 428 043 2 282 028 2 272 027 2 275 028 2

5315 microgkg 387 08 2 256 05 2 251 05 2 253 05 2

5316 microgl 3839 342 2 2485 191 2 2559 168 2 239 51 2

5317 microgl 2466 043 2 2212 034 2 2472 016 2 2146 068 2

5319 microgl 414 41 2 279 28 2 268 27 2 26 31 2

5321 microgkg 476 316 326 328

5322 microgl 2547 35 2

5323 microgkg 405 203 2 239 119 2 255 153 2 201 11 2

58

Figure 11 Youden plots of the percent deviation of the reported values from the assigned values for the food supplement test sample and the

standard solution in acetonitrile with undisclosed content

(The identity of the respective measurand is given above the figure Results exceeding a difference of more than 100 were omitted)

59

Figure 11 - continued

60

ANNEX 10 Table 18 Method performance parameters reported by the participants

Laboratory code

Analyte LOD LOQ Recovery Analyte LOD LOQ Recovery

microgkg microgkg microgkg microgkg 3600 BaA 03 06 978 BaP 03 07 9483602 BaA 007 021 926 BaP 005 015 993603 BaA 0011 05 987 BaP 0005 051 9553604 BaA 02 04 86 BaP 02 04 883605 BaA 0025 005 118 BaP 0025 005 1083606 BaA 001 003 65 BaP 001 003 703607 BaA 021 071 74 BaP 027 089 943608 BaA 016 048 8213 BaP 015 046 83683610 BaA 02 05 85 BaP 005 02 853611 BaA BaP 3612 BaA 1 117 BaP 1 1203613 BaA 01 03 90 BaP 01 03 903614 BaA 02 05 96 BaP 02 05 933615 BaA 005 015 110 BaP 005 015 1053616 BaA 01 05 108 BaP 01 05 753617 BaA 018 037 405 BaP 009 018 3873618 BaA 01 03 63 BaP 01 03 593619 BaA 005 005 59 BaP 033 033 543620 BaA 0009 0018 64 BaP 0005 001 553621 BaA 007 02 84 BaP 007 02 923622 BaA 01 03 1101 BaP 01 03 9853623 BaA 02 08 105 BaP 02 08 1013624 BaA BaP 01 033 8513625 BaA BaP 3626 BaA BaP 5301 BaA 003 01 102 BaP 003 01 1005302 BaA 01 03 78 BaP 01 03 865303 BaA 003 01 62 BaP 003 01 805304 BaA 1 1 70 BaP 1 1 955305 BaA 02 05 85 BaP 02 05 855306 BaA 03 1 699 BaP 03 1 905307 BaA 6537 BaP 81165308 BaA 0088 0265 99 BaP 00262 00785 1005309 BaA 01 08 92 BaP 01 08 925310 BaA 01 03 BaP 01 03 905311 BaA 002 005 80 BaP 002 004 915312 BaA 03 09 105 BaP 03 09 1055313 BaA 04 08 BaP 04 08 5314 BaA 001 002 96 BaP 001 002 9485315 BaA 01 03 50 - 100 BaP 01 03 50 - 100 5316 BaA 002 011 827 BaP 004 019 10445317 BaA 05 1 76 BaP 05 1 755318 BaA BaP 5319 BaA 009 029 951 BaP 011 041 995320 BaA 12 18 946 BaP 06 1 8345321 BaA 03 05 BaP 05 1 5322 BaA 1 2 100 BaP 1 2 1005323 BaA 26 BaP 26

61

ANNEX 10 Table 18 - continued

Laboratory code Analyte LOD LOQ Recovery Analyte LOD LOQ Recovery

microgkg microgkg microgkg microgkg 3600 BbF 03 05 96 CHR 02 03 10153602 BbF 015 045 947 CHR 003 009 9193603 BbF 0014 05 1012 CHR 0004 05 10033604 BbF 02 04 85 CHR 02 04 873605 BbF 005 01 94 CHR 0025 005 863606 BbF 001 003 70 CHR 001 003 653607 BbF 018 06 100 CHR 014 028 943608 BbF 015 046 7979 CHR 015 045 71253610 BbF 005 02 85 CHR 02 05 853611 BbF CHR 3612 BbF 1 118 CHR 1 1173613 BbF 01 03 90 CHR 01 03 903614 BbF 01 04 96 CHR 02 05 953615 BbF 008 025 109 CHR 003 01 1083616 BbF 01 05 78 CHR 01 05 723617 BbF 035 07 38 CHR 012 024 4163618 BbF 01 03 62 CHR 01 03 623619 BbF 016 016 63 CHR 01 01 593620 BbF 0003 0006 55 CHR 001 002 653621 BbF 007 02 103 CHR 007 02 893622 BbF 02 06 963 CHR 03 09 1043623 BbF 02 08 106 CHR 02 08 1193624 BbF CHR 3625 BbF CHR 3626 BbF CHR 5301 BbF 003 01 96 CHR 003 01 1145302 BbF 01 03 84 CHR 01 03 835303 BbF 003 01 78 CHR 003 01 705304 BbF 1 1 86 CHR 1 1 705305 BbF 02 05 90 CHR 02 05 955306 BbF 03 1 1435 CHR 03 1 1185307 BbF 8116 CHR 65375308 BbF 0079 0238 99 CHR 009 026 1005309 BbF 01 08 92 CHR 5310 BbF 07 2 CHR 01 03 5311 BbF 002 004 95 CHR 002 004 895312 BbF 03 09 105 CHR 03 09 1055313 BbF 04 08 CHR 04 08 5314 BbF 001 002 83 CHR 001 002 8185315 BbF 02 05 50 -100 CHR 02 05 50 - 100 5316 BbF 005 024 814 CHR 001 007 9595317 BbF 05 1 73 CHR 05 1 785318 BbF CHR 5319 BbF 011 038 946 CHR 013 043 9675320 BbF 07 09 1194 CHR 23 32 10455321 BbF 05 1 CHR 05 1 5322 BbF 1 2 100 CHR 1 2 1005323 BbF 26 CHR 26

62

ANNEX 11 Table 19 Details of analysis method reported by the participants Data are presented as reported

Laboratory code

Analysis technique

Instrument calibration

Internal standards applied

Sample clean up

Details of sample

clean up

Extraction technique

Details of sample

extraction

3600 6) GC-MS

2) Internal standardisation with labelled substances

Deuterated PAH mix 9 (Ehrenstorfer)

1) Saponification 2) LiquidLiquid partitioning

D) Other Liquidliquid partitioning

3602 2) HPLC-UV-FLD

1) External calibration

3) Gel permeation chromatography

A) Pressurised liquid extraction (PLE)

3603 1) HPLC-FLD

1) External calibration

3) Gel permeation chromatography

D) Other 1 minute with dichloromethane in vortex

3604 2) HPLC-UV-FLD

2) Internal standardisation with labelled substances

DiP D14

5) Donor acceptor complex chromatography

D) Other LL

3605 1) HPLC-FLD

1) External calibration

3) Gel permeation chromatography

C) Soxhlet extraction

3606 7) GC-MSMS

2) Internal standardisation with labelled substances

BaP 13C4 CHR 13C6 BaA 13C6 BbF 13C6

4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

3607 6) GC-MS

2) Internal standardisation with labelled substances

BaA-d12 CHR-d12 BbF-d12 BkF-d12 BaP-d12 ICP-d12 BgP-d12 DiP-d14

1) Saponification

D) Other solvent partitioning

3608 1) HPLC-FLD

1) External calibration

6) Other Liquid extraction

D) Other

Extraction in a rotary agitator and centrifugation

3610 2) HPLC-UV-FLD

1) External calibration

4) Solid phase extraction

B) Sonication

3611

3612 6) GC-MS

2) Internal standardisation with labelled substances

chrysene-d12 dibenzo(ah)anthracene-d14 dibenzo(ai)pyrene-d14

4) Solid phase extraction

B) Sonication

3613 5) LC-MSMS

3) Internal standardisation with unlabelled substances

I use Chrysene-D12 3) Gel permeation chromatography

D) Other extraction with hexan

63

Table 19 - continued

Laboratory code

Analysis technique

Instrument calibration

Internal standards applied

Sample clean up Details of sample

clean up

Extraction technique

Details of sample

extraction

3614 6) GC-MS 1) External calibration

3) Gel permeation chromatography

B) Sonication

3615 1) HPLC-FLD

1) External calibration

6) Other PLE GPC

A) Pressurised liquid extraction (PLE)

3616 6) GC-MS

2) Internal standardisation with labelled substances

Benzo(a)pyrene-13C4 Benzo(a)anthracene-13C6 Benzo(b)fluoranthene-13C6 Chrysene-13C6

2) LiquidLiquid partitioning 4) Solid phase extraction

B) Sonication

3617 1) HPLC-FLD

1) External calibration

4) Solid phase extraction

C) Soxhlet extraction

3618 6) GC-MS

2) Internal standardisation with labelled substances

deuterated standards

1) Saponification 4) Solid phase extraction

D) Other Handshaken with cyclohexane

3619 6) GC-MS

2) Internal standardisation with labelled substances

C13 Labelled US EPA 16

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

D) Other Homegenisation amp saponiofication

3620 9) GC-HRMS

2) Internal standardisation with labelled substances

mix of deuterated PAHs (BAA-D12 CHR-D12 BAP-D12 BBF-D12)

3) Gel permeation chromatography 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

3621 6) GC-MS

2) Internal standardisation with labelled substances

Chrysene D12 Benzo(a)pyrene D12 Benzo(b)fluoranthene D12 Benzo(a)anthracene

2) LiquidLiquid partitioning 4) Solid phase extraction

B) Sonication

3622 1) HPLC-FLD

4) Standard addition method

4) Solid phase extraction

D) Other

Extraction with cyclohexane by heating under reflux

3623 7) GC-MSMS

2) Internal standardisation with labelled substances

C13 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

3624 2) HPLC-UV-FLD

1) External calibration

2) LiquidLiquid partitioning

D) Other hot solvent extraction

3626

64

Table 19 - continued

Laboratory code

Analysis technique

Instrument calibration

Internal standards

applied

Sample clean up

Details of sample

clean up

Extraction technique

Details of sample

extraction

5301 7) GC-MSMS

2) Internal standardisation with labelled substances

EACH SUBSTANCE C13

4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5302 7) GC-MSMS

2) Internal standardisation with labelled substances

Isotope labelled PAHs 13C for each analyte

4) Solid phase extraction

C) Soxhlet extraction

5303 7) GC-MSMS

3) Internal standardisation with unlabelled substances

HAP C13 4) Solid phase extraction

D) Other automate ASE200 DIONEX

5304 7) GC-MSMS

2) Internal standardisation with labelled substances

with C13 for each HAP

2) LiquidLiquid partitioning 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5305 7) GC-MSMS

2) Internal standardisation with labelled substances

C13 isotopiques internal standards

4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5306 7) GC-MSMS

2) Internal standardisation with labelled substances

C13 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5307 6) GC-MS

2) Internal standardisation with labelled substances

1) Saponification 2) LiquidLiquid partitioning

D) Other solvent extraction

5308 1) HPLC-FLD 1) External calibration

4) Solid phase extraction

B) Sonication

5309 1) HPLC-FLD 1) External calibration

4) Solid phase extraction

D) Other only SPE

5310 1) HPLC-FLD 1) External calibration

1) Saponification 2) LiquidLiquid partitioning 3) Gel permeation chromatography

C) Soxhlet extraction

5311 6) GC-MS

2) Internal standardisation with labelled substances

13 C Chrysene 13 C Benz(a)pyrene

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

B) Sonication

5312 1) HPLC-FLD

3) Internal standardisation with unlabelled substances

Benzo(b)chrysen

2) LiquidLiquid partitioning 4) Solid phase extraction

Quechers D) Other Quechers

65

Table 19 - continued

Laboratory code

Analysis technique

Instrument calibration

Internal standards applied

Sample clean up Details of sample

clean up

Extraction technique

Details of sample extraction

5313 6) GC-MS

2) Internal standardisation with labelled substances

Benz(a)anthracene D12 Benzo(b)fluranthene D12 Benzo(a)pyrene D12 Chrysene D12

1) Saponification 2) LiquidLiquid partitioning 3) Gel permeation chromatography

A) Pressurised liquid extraction (PLE)

5314 6) GC-MS

2) Internal standardisation with labelled substances

Benzo(a)pyren d12 2) LiquidLiquid partitioning

D) Other Extraction with

5315 1) HPLC-FLD

3) Internal standardisation with unlabelled substances

Benz(b)chrysen

1) Saponification 2) LiquidLiquid partitioning

D) Other shaker

5316 1) HPLC-FLD

1) External calibration

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

D) Other

LiquidLiquid extraction with cyclohexane and dimethylformamide

5317 7) GC-MSMS

1) External calibration

3) Gel permeation chromatography 6) Other

silica gel D) Other VDLUFA online method

5318

5319 1) HPLC-FLD

3) Internal standardisation with unlabelled substances

Benzo(b)chrysene 3) Gel permeation chromatography

A) Pressurised liquid extraction (PLE)

5320 6) GC-MS

3) Internal standardisation with unlabelled substances

22-binaphthyl

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

D) Other 3 hours refluxing

5321 1) HPLC-FLD

2) Internal standardisation with labelled substances

6-meacutethylchrysegravene

1) Saponification 2) LiquidLiquid partitioning

B) Sonication

5322 6) GC-MS

2) Internal standardisation with labelled substances

naftalene-d8 acenaphtalene-d10 phenanthrene-d10 chrysene-d12 perylene-d12 benzo(e)anthracene-d12

1) Saponification 2) LiquidLiquid partitioning 6) Other

clean-up with silica packed column

C) Soxhlet extraction

5323 1) HPLC-FLD

1) External calibration

4) Solid phase extraction 6) Other

Accelerated Solvent Extraction

A) Pressurised liquid extraction (PLE)

66

European Commission EUR 25245 EN ndash Joint Research Centre ndash Institute for Reference Materials and Measurements Title Report on the 8th inter-laboratory comparison organised by the European Union Reference Laboratory for Polycyclic Aromatic Hydrocarbons - Four marker PAHs in a dry extract of St Johns wort Author(s) Radoslav Lizak Szilard Szilagyi Philippe Verlinde and Thomas WENZL Luxembourg Publications Office of the European Union 2012 ndash 66 pp ndash 201 x 297 cm EUR ndash Scientific and Technical Research series ndash ISSN 1831-9424 ISBN 978-92-79-23337-1 doi 10278759387 Abstract The proficiency test here reported concerned the determination of the contents of four marker polycyclic aromatic hydrocarbons (PAHs) and of their sum in an food supplements test sample The set of marker PAHs consists of benz[a]anthacene benzo[a]pyrene benzo[b]fluoranthene and chrysene Participants to these PT were National Reference Laboratories for PAHs (NRLs-PAHs) and EU official food control laboratories The number of participants was in total 48

The PT was organised according ISO Standard 170432010

The test material used was a commercial dry extract of St Johns wort Participants also received a solution of the same PAHs either in acetonitrile or in toluene for checking their instrument calibration

The results from participants were rated with z-scores and zeta-scores About 68 of the reported results were attributed with z-scores with an absolute value of below two which is the threshold for satisfactory performance

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science and technology for the Union Close to the policy-making process it serves

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interests whether private or national

L

A-N

A-252

45-EN

-N

• 1 Summary
• 2 Introduction
• 3 Scope
• 4 Participating Laboratories
• 5 Time frame
• 6 Confidentiality
• 7 Test materials
• • 71 1Preparation of test materials
  • 72 Assigned values and standard deviation for proficiency assessment
  • 73 Homogeneity and stability
  • 74 Sample dispatch
  • • 8 Design of the proficiency test
    • 9 Evaluation of the results
    • • 91 2General
      • 92 21BEvaluation criteria
      • 93 Performance of participants
      • 94 Evaluation of the influence of analysis method
      • 95 Evaluation of the influence of calibration
      • 96 26Evaluation of compliance with legislation
      • • 10 Follow-up actions for underperforming laboratories
        • 11 1Conclusions
        • 12 1Acknowledgements
        • 13 References

8

6 Confidentiality

The identities of participants are kept confidential unless the participant provides a letter of consent to

the PT organiser giving permission to disclose hisher details and results to a third party

7 Test materials

71 Preparation of test materials

The test material of this PT round was a food supplement in particular a dry extract of St Johns wort

in the following denoted as FS

The test material was prepared from commercial products at the EU-RL PAH laboratories About 60

units containing each 120 pills of a dry extract of St Johns wort were acquired at local stores They

were then ground and homogenised with a Loedige Ploughsharereg mixer Portions of at least 15 g of

the homogenised powder were packed into amber glass screw cap vials

Participants also received a solution of the 15+1 EU priority PAHs in either acetonitrile or toluene

(according to their choice) with disclosed concentrations which served for checking instrument

calibration The technical specifications (see ANNEX 4) of the chosen solution were dispatched

together with the test samples

Besides the standard solution with disclosed content a solution with undisclosed content was prepared

The participants were requested to report the analyte content of this solution to the organiser of the PT

This information was used to identify potential reason for bias

72 Assigned values and standard deviation for proficiency assessment

The assigned values were determined from in-house measurements at the EU-RL PAH applying

bracketing calibration and from measurements conducted by a subcontracted external laboratory The

measurements at the EU-RL were conducted on two different days Four replicate samples were

analysed by bracketing calibration using a commercially available standard solution (Dr Ehrenstorfer

Standard 183) in each of the two analysis sessions The contract laboratory had to perform three

replicate analyses of two different samples each composed of three units of PT test samples The

analyses had to be performed on two different days

9

In cases of almost identical analysis results (difference between own results and results of contract

laboratory lt 5) the results from the in-house measurements were applied as assigned values

(applicable to BAA and BBF) If the difference between the two results exceeded 5 the average of

the two results was applied as assigned value (applicable to BAP and CHR) and the combined

uncertainty of in-house and external measurements was associated with the assigned value

The sum of PAH 4 was calculated from the individually assigned values and the corresponding

uncertainty from the uncertainties of the assigned values according to equation 1 The results of in-

house measurements and the results reported by the contract laboratory as well as the assigned values

of the target PAHs are listed in Table 4

Equation 1 2222CHRBBFBAPBAAsum uuuuu

where usum refers to the standard uncertainty of the sum of the four PAHs and uBAA u BAP uBBF and

uCHR refer to the standard uncertainty of the individual analytes

The standard deviation for proficiency assessment (σP) was set for the four individual analytes equal to

the maximum tolerated standard measurement uncertainty Uf (see Equation 2) as defined by

Commission Regulation (EC) No 3332007 [5] and Commission Regulation (EU) No 8362011 [9]

Equation 2 Uf = 22 )C((LOD2)

where Uf relates to the maximum tolerated standard measurement uncertainty LOD to the required

limit of detection α to a numeric factor depending on the concentration C

The application of Equation 2 with the assigned values listed in Table 4 the maximum tolerated value

of LOD of 030 μgkg and α equal to 02 results in Uf values as reported in Table 4

The Uf values of the individual analytes are propagated in analogy to equation 1 for the determination

of the Uf value for the sum of the four PAHs which was used as standard deviation for proficiency

assessment for the sum parameter

10

Table 4 Analyte contents of the food supplement test material

Average of two

sessions bracketing calibration

External evaluation

Assigned value

and σP

Cont microgkg 298 297 298U microgkg 024 041 024P microgkg 062

Cont microgkg 157 174 165U microgkg 020 016 026P microgkg 036

Cont microgkg 292 289 292U microgkg 028 034 028P microgkg 060

Cont microgkg 424 389 407U microgkg 057 057 081P microgkg 083

Cont microgkg 1162U microgkg 093P microgkg 125

Sum PAH4

BAA

BAP

BBF

CHR

from chemical analysis Sum of the four analytes contents for the SUM

p standard deviation for proficiency assessment For the individual analytes is equal to the uncertainty function - Uf

according to Ref [9]

U expanded uncertainty of the assigned value (k=2)

The gravimetrical preparation concentrations were used as assigned values for the standard solutions

with undisclosed content Table 5 contains the assigned values and associated expanded uncertainties

of the standard solutions with undisclosed content

11

Table 5 Analyte contents of the standard solutions with undisclosed content

Content U (k=2) Content U (k=2) microgkg microgkg microgkg microgkg

benz[a ]anthracene 4317 039 4764 043benzo[c ]fluorene 7342 061 8052 067chrysene 2904 031 3179 034cyclopenta[cd ]pyrene 2558 050 2818 055benzo[a ]pyrene 2897 038 3186 042benzo[b ]fluoranthene 2926 026 3199 029benzo[ghi ]perylene 2214 023 2438 026benzo[j ]fluoranthene 4353 038 4763 041benzo[k ]fluoranthene 2143 017 2380 019dibenzo[ae ]pyrene 5735 084 6326 093dibenzo[ah ]anthracene 2215 032 2445 035dibenzo[ah ]pyrene 2169 029 2394 032dibenzo[ai ]pyrene 3691 100 4035 109dibenzo[al ]pyrene 4627 057 5104 063indeno[123-cd ]pyrene 3793 036 4176 0395-methylchrysene 8589 087 9443 096

Solution in toluene Solution in acetonitrile

Prep conc concentration from gravimetrical preparation U expanded uncertainty of the assigned value k coverage

factor

73 Homogeneity and stability

Homogeneity of the food supplements test sample was evaluated according to ISO Standard 13528

Ten units of the St Johns wort extract test material were selected randomly and analysed by isotope

dilution gas chromatography mass spectrometry (GC-MS) The test material was rated sufficiently

homogeneous (see ANNEX 5)

The contributions of the uncertainties of homogeneity (relative uncertainties below 1 ) to the total

uncertainties of the assigned values were found negligible and were therefore not considered in the

calculations

The stability of the test materials was evaluated applying an isochronous experimental set up This

comprised the storage of test samples both at room temperature and cooled in the fridge The content

values of the cooled sample were used as reference for the stability evaluation The materials were

stored from dispatch of test samples to the participants until expiry of the deadline for reporting of

results Afterwards the samples were analysed under repeatability conditions and results were

compared The content values of the cool-stored sample and the sample stored at room temperature

were not statistically significantly different Hence stability of the samples over the whole study period

can be assumed The analysis data are presented in ANNEX 5

12

The uncertainty of stability of the test material was considered negligible compared to the uncertainty

of the characterisation measurements Therefore this parameter was not taken into account in the

determination of the uncertainties of the assigned values

74 Sample dispatch

Samples were dispatched at room temperature Each participant received at least

- one ampoule of the solution of the 15+1 EU priority PAHs in the chosen solvent with disclosed

content (2 ml)

- one ampoule of the 15+1 EU priority PAHs in the chosen solvent with undisclosed content (2

ml)

- one screw cap vial with the food supplement test material

Each parcel contained a sample receipt form the outline of the study an appropriate material safety

data sheets and a unique code that was required for reporting of results The documents sent to the

participants are presented in ANNEX 6

8 Design of the proficiency test

The design of the PT foresaw triplicate analyses of the test sample and reporting of the individual

results of replicate analyses for the single analytes in the following denoted as FS_REP and

additionally a value for proficiency assessment in the following denoted as final value - FS_FIN

for both the single analytes and the sum of the four marker PAHs Both FS_REP results and FS_FIN

results had to be reported corrected for recovery (and recovery had to be stated in the questionnaire

which participants were asked to fill in together with other parameters of the method applied) the

latter had also to be accompanied by the respective expanded measurement uncertainty The FS_FIN

results were used for performance assessment

Besides analysis results participants were also asked to report details of the applied analysis method

and to provide answers to a questionnaire (see ANNEX 7 for the template and the compiled returned

questionnaires)

13

9 Evaluation of the results

The results reported by participants are listed in ANNEX 8

91 General

The most important evaluation parameter was the performance of the laboratories in the determination

of the target PAHs in the food supplement test material which was expressed by z-scores and zeta-

scores

The compliance of method performance characteristics specified by the participants with provisions

given in legislation was evaluated too

92 Evaluation criteria

The participants were requested to report for all analytes the results of replicate measurements and a

value for proficiency assessment which is the result they wish to be applied for the calculation of

performance indicators z-Scores and zeta-scores were attributed only to these results The individual

results of replicate analyses were not rated

z-Scores

z-scores were calculated based on the FS_FIN values Equation 1 presents the formula for calculation

of z-scores

Equation 1

P

assignedlab Xxz

where z refers to the z-score xlab to the reported ldquovalue for proficiency assessmentrdquo Xassigned to the

assigned value and σP to the standard deviation for proficiency testing

zeta-Scores

In addition to z-scores zeta-scores were calculated In contrast to z-scores zeta-scores describe the

agreement of the reported result with the assigned value within the respective uncertainties

Unsatisfactorily large zeta-scores might be caused by underestimated measurement uncertainties large

bias or a combination of both Zeta-scores were calculated according to Equation 3

14

Equation 3 22assignedlab

assignedlab

uu

Xxzeta

where zeta refers to the zeta-score xlab to the reported ldquovalue for proficiency assessmentrdquo Xassigned to

the assigned value ulab to the measurement uncertainty reported by the laboratory and uassigned to the

uncertainty of the assigned value

The performance of the laboratories was classified according to ISOIEC 170432010 [10] Following

scheme is applied for the interpretation of zeta scores and z-scores

|score| 20 = satisfactory performance

20lt|score| lt 30 = questionable performance

|score| ge 30 = unsatisfactory performance

93 Performance of participants

The 48 participants in the study reported in total 225 results for proficiency evaluation which equals to

about 94 of the 240 possible results About 68 of the reported results were rated as satisfactory

with regard to z-scores

Figure 1 and Figure 2 give an overview of the z-scores assigned to the respective results The larger

the triangles the larger were the differences to the assigned values Red triangles indicate z-scores

above an absolute value of three whereas yellow triangles represent z-scores in the questionable

performance range The corresponding score values are plotted next to the triangles About 40 of the

72 non-satisfactory results were reported by seven laboratories only eg the performance of

participant 3608 was not satisfactory for the majority of target analytes

It is also obvious that most problems were encountered in the determination of chrysene and the sum

of the four analytes Potential reasons for the difficulties related to chrysene are discussed further

down

The ideal situation would be that deviations from the assigned values of the results reported for the

individual analytes are small and randomly distributed around zero This will give a satisfactory

performance statement for the sum parameter Deviations of opposite sign could also cancel out and

lead to a satisfactory performance for the sum parameter (eg participants 5303 5304 and 5305)

However in most cases a single underperformance for one analyte led also to non-satisfactory rating

for the sum parameter

15

The numerical values of the calculated z-scores are compiled in Table 6 and Table 7 z-Scores with an

absolute value of above 2 are given in red bold font

The results of the data evaluation for the individual analytes are given in ANNEX 8

For each analyte the figure shows the individual analysis results of the three replicate determinations

The assigned value is shown as dotted line The blue boxes represent the expanded uncertainties as

reported by participants for the value for proficiency assessment The arithmetic mean of the results

of the individual participants is indicated in the blue boxes by a blue line The satisfactory performance

range is located between the two red lines

The individual results of the replicate measurements and the value for proficiency assessment with

its accompanying expanded measurement uncertainty (k=2) are listed in the tables in ANNEX 8 as

well

16

Figure 1 Graphical presentation of z-scores corresponding to the values for proficiency assessment

reported by the NRLs for the contents of BAA BAP BBF and CHR in the St Johns wort extract test

material

Blue triangles indicate satisfactory performance yellow triangles indicate questionable performance red triangles indicate non-satisfactory performance z-score values are presented above the triangles for the latter two performance categories

ProLab 2011

Z score-3 0 3 -3 0 3 -3 0 3 -3 0 3 -3 0 3

BAA BAP BBF CHR PAH4

La

bo

rato

ry

36003602

3603

36043605

36063607

36083610

36113612

36133614

36153616

36173618

36193620

36213622

36233624

3626

2258

2343

2452

2669

2147

2788

2869

6710

70839

3011

11271

80525

8425

4561

12338

90680

4976

7331

99553

3998

3780

7500

17420

168413

3269

3029

-2348 -2736

-2023 -2981

-2155-3018

-3998

Figure 2 Graphical presentation of z-scores corresponding to the values for proficiency assessment

reported by the OCLs for the contents of BAA BAP BBF and CHR in the St Johns wort extract test

material

Blue triangles indicate satisfactory performance yellow triangles indicate questionable performance red triangles indicate non-satisfactory performance z-score values are presented above the triangles for the latter two performance categories

ProLab 2011

Z score-3 0 3 -3 0 3 -3 0 3 -3 0 3 -3 0 3

BAA BAP BBF CHR PAH4

La

bo

rato

ry

5301

5302

5303

5304

5305

5306

5307

5308

5309

5310

5311

5312

5313

5314

5315

5316

5317

5318

5319

5320

5321

5322

5323

2597

2629

2017

2293

2873

2123

2521

2620

2186

2222

2452

2864

3952

3984

3806

9226

4171 5522

3285

6594

5205

5157

3539

3901

7051

4519

3173

4872

9191

7276

4391

-2435

-2154

-2065

-2340

-2740-3726

-6346

-3505

17

Table 6 Compilation of z-scores calculated from the ldquoresults for proficiency assessment

reported by the NRLs for food supplement test material z-scores outside the

satisfactory range (|z| gt 2) are indicated by red font Values are reproduced as reported

by the laboratories Empty cells denote analytes for which results were not reported

Measurand benz[a]anthracene benzo[a]pyrene benzo[b]fluoranthene chrysene Sum PAH 4

Assigned value

microgkg 298 165 292 407 1162

Standard deviation for proficiency assessment

microgkg 062 036 060 083 125

Laboratory code

Result z-score Result z-score Result z-score Result z-score Result z-score

microgkg microgkg microgkg microgkg microgkg

3600 3 00 17 01 31 03 5 11 128 09

3602 19 -18 124 -11 17 -20 304 -12 79 -30

3603 251 -08 161 -01 256 -06 459 06 1127 -03

3604 213 -14 143 -06 11 -30 429 03 893 -22

3605 291 -01 167 01 247 -07 386 -03 1091 -06

3606 289 -01 167 01 276 -03 451 05 1184 02

3607 438 23 274 30 567 46 819 50 2098 75

3608 714 67 573 113 1036 123 1014 73 3336 174

3610 196 -17 206 11 191 -17 424 02 1138 -02

3611 469 708 308 805 576 907 865 996 2218 1684

3612 31 02 22 15 29 00 61 25 143 21

3613 33 05 18 04 31 03 4 -01 122 05

3614 185 -18 08 -23 127 -27 271 -16 663 -40

3615 376 13 145 -06 4 18 601 23 152 29

3616 2 -16 15 -04 2 -15 47 08 102 -11

3617 362 10 169 01 299 01 422 02 1252 07

3618 32 04 2 10 31 03 45 05 128 09

3619 299 00 207 12 326 06 738 40 157 33

3620 302 01 166 00 276 -03 46 06 1204 03

3621 28 -03 47 84 25 -07 51 12 151 28

3622 322 04 213 13 34 08 488 10 1361 16

3623 33 05 21 12 28 -02 72 38 154 30

3624 18 04

3626 314 03 174 02 292 00 628 27 1409 20

18

Table 7 Compilation of z-scores calculated from the ldquoresults for proficiency assessment

reported by the OCLs for food supplement test material z-scores outside the

satisfactory range (|z| gt 2) are indicated by red font Values are reproduced as reported

by the laboratories Empty cells denote analytes for which results were not reported

Measurand benz[a]anthracene benzo[a]pyrene benzo[b]fluoranthene chrysene Sum PAH 4

Assigned value

microgkg 298 165 292 407 1162

Standard deviation for proficiency assessment

microgkg 062 036 060 083 125

Laboratory code

Result z-score Result z-

score Result z-score Result

z-score

Result z-score

microgkg microgkg microgkg microgkg microgkg

5301 31 02 18 04 29 00 49 10 126 08

5302 316 03 188 06 298 01 679 33 37 -63

5303 2876 -02 1535 -03 2586 -06 6441 29 13438 15

5304 261 -06 146 -05 253 -06 591 22 1251 07

5305 24 -09 16 -01 24 -09 61 25 125 07

5306 22 -13 16 -01 42 21 49 10 129 10

5307 461 27 157 -02 221 -12 524 14 1363 16

5308 16446 -22 107009 -16 229848 -10 213243 -23 72456 -35

5309 17 -21 11 -15 2 -15

5310 305 01 123 -12 313 03 386 -03 1127 -03

5311 459 26 2 10 281 -02 437 04 1367 16

5312 543 40 248 23 299 01 953 66 2042 71

5313 353 09 207 12 328 06 838 52 1726 45

5314 394 16 172 02 404 19 588 22 1558 32

5315 545 40 238 20 444 25 542 16 177 49

5316 235 -10 17 01 273 -03 282 -15 96 -16

5317 222 -12 269 29 192 -17 545 17 1229 05

5318 067 -37 117 -13 279 -02 357 -06 82 -27

5319 147 -24 216 14 396 17 465 07 1225 05

5320 534 38 316 42 625 55 834 52 2309 92

5321 12 -12

5322 87 92 17 01 33 06 7 35 207 73

5323 33 05 2 10 45 26 73 39 171 44

19

Table 8 and Table 9 present the respective zeta-scores As for the z-scores data outside the

satisfactory performance range are given in red bold font Zeta scores were only calculated for

laboratories that provided measurement uncertainty data as well as a numerical value for the coverage

factor Zeta scores were not calculated when the reported uncertainty value exceeded the content value

which is probably linked to mixing of reporting units (microgkg and ) However the participants are

reminded that the measurement uncertainty shall be expressed in the same unit as the content value

The assessment of the performance of the participants based on the reported measurement uncertainty

gave a less favourable picture compared to z-scores Only 60 of the zeta-scores are within the

satisfactory performance range The distribution of satisfactory performance ratings between NRLs

and OCLs is not balanced It was higher for the NRLs

It has to be noted that the magnitude of the zeta-scores were for some participants much higher than

the z-scores attributed to the same results Consequently the laboratories perform according to fitness-

for-purpose criterion specified in legislation which forms the basis for the z-scores but seem to have

difficulties in deducing realistic measurement uncertainty values Eg the relative standard

measurement uncertainties reported by the NRLs ranged from 25 to 20 with a median value of

about 10 Only two OCLs reported measurement uncertainties that exceeded the maximum tolerable

uncertainty (Uf) The respective data is highlighted in yellow in Table 9

However the EU-RL PAHs will continue to pay special attention to measurement uncertainty in future

ILCs as it has major implications on the assessment of compliance of food with European legislation

20

Table 8 Compilation of zeta-scores calculated from the ldquoresults for proficiency assessment

reported by the NRLs for test material St Johns wort the reported measurement

uncertainty and the uncertainty of the analyte content of the test material zeta-scores

outside the satisfactory range (|zeta| gt 2) are indicated by red bold font Empty cells

denote analytes for which either results or measurement uncertainties were not

reported

21

Table 9 Compilation of zeta-scores calculated from the ldquoresults for proficiency assessment

reported by the OCLs for test material St Johns wort the reported measurement

uncertainty and the uncertainty of the analyte content of the test material zeta-scores

outside the satisfactory range (|zeta| gt 2) are indicated by red bold font yellow

highlighted cells ugtUf Empty cells denote analytes for which results were not

reported

22

94 Evaluation of the influence of analysis method

The information on the applied analysis method (ANNEX 11) was used to group the performance

indicators attributed to the reported results according to the applied analysis technique Two groups of

data were formed They represent results obtained by high performance liquid chromatography-

fluorescence detection (HPLC-FD about 40 ) and by gas chromatography-mass spectrometry (GC-

MS about 60 ) respectively Kernel density plots were constructed based on the respective z-scores

Figure 3A to 3E show the different Kernel density plots

Figure 3 Kernel density plots of z-scores attributed to the results for proficiency assessment

which were reported for the food supplements test sample Results were classified

according to the applied analysis technique

A) benz[a]anthracene

B) benzo[a]pyrene

23

Figure 3 continued

C) benzo[b]fluoranthene

D) chrysene

E) sum of four marker PAHs

The kernel density plots for BAP and BBF are almost identical and centred at or close to a z-score

value of zero as can be seen in Figure 3B and 3C Therefore bias introduced by the type of analysis

method is unlikely for these two analytes The situation is different for BAA and CHR In case of

BAA the kernel density distribution of z-scores based on HPLC-FD is shifted to negative values

24

(Figure 3A) which indicates a tendency to underestimate the BAA content in this particular

samplematrix when applying HPLC-FD This might be reasoned by separationintegration problems

between BAA and CHR peaks A few participants (from the HPLC-FD subgroup) that got negative

z-scores for BAA overestimated the CHR content This caused the slight shift of the density trace for

HPLC-FD towards negative values Removing this data from the evaluation would result in a density

trace with the mode at zero

The opposite is the case for CHR (Figure 3D) The density distribution of the z-scores attributed to

HPLC-FD data is centred almost at zero However the distribution for data based on GC-MS is shifted

to positive values indicating strong positive bias This bias might be caused by a lack of selectivity of

the analysis method The test material contains triphenylene which is isobaric to chrysene

Consequently it cannot be distinguished from chrysene by means of mass spectrometry These two

compounds have to be separated chromatographically which is not easy They coelute on unpolar and

on most mid-polar capillary columns Columns that allow their separation were commercialised only

recently and might not be available in all laboratories Therefore laboratories which determine the

four EU marker PAHs by means of GC-MS shall pay attention to the chromatographic separation of

triphenylene and chrysene A lack of chromatographic resolution might lead to strong positive bias of

GC-MS methods and to false conclusions on the marketability of the tested sample

The chromatographic separation of chrysene and triphenylene is not a problem with HPLC which is

reflected in the position of the density plot for chrysene However attention has to be paid in HPLC-

FD analysis to a proper clean up of the sample extract in order to eliminate interferences that show

similar retention as BAA and CHR

95 Evaluation of the influence of calibration

The participants received together with the food supplement test sample two standard solutions in

depending of their choice either acetonitrile or toluene The analyte content of one solution was

disclosed to the participants whereas the content of the other solution was kept undisclosed The

content values of the latter are presented in Table 5 Analysis results for the solution with undisclosed

content had to be reported to the PT organiser The respective data are presented in Annex 9

Percent deviations of the reported results from the assigned values were calculated for both the food

supplement test sample and the standard solution with undisclosed analyte content Youden plots were

prepared in order to visualize the influence of instrument calibration on the results reported for the

food supplement sample Two plots are discussed in the following The whole set of Youden plots are

given in Annex 9

25

Figure 4 Youden plot of the percent deviations of values reported for benzo[a]pyrene from the

assigned values of the food supplement test material and the standard solution in

toluene with undisclosed content

Figure 4 reveals that a number of participants reported results with similar percent deviations from the

assigned values of both the results for the food supplement material and the standard solution in

toluene with undisclosed content This correlation indicates that over- respectively underestimation of

the analyte content of the food supplement material might be caused by biased instrument calibration

This finding underpins the importance of thorough calibration standard preparation However the data

was not in all cases conclusive as some laboratories performed well for the food supplement samples

but reported biased results for the standard solution with undisclosed content (eg Figure 4 participants

3620 and 5305) This fact cannot be explained yet

Figure 5 presents the respective Youden plot for chrysene in the food supplement test material and the

standard solution in toluene with undisclosed content From this graph it can be concluded that the

overestimation of the chrysene content of the food supplement material was not caused by instrument

calibration The results for the solvent solution of many laboratories which underperformed for the

26

food supplement sample agreed well with the assigned value This supports the conclusion that the

overestimation is caused by the determination of an interferent most probably triphenylene

Figure 5 Youden plot of the percent deviations of values reported for chrysene from the

assigned values of the food supplement test material and the standard solution in

toluene with undisclosed content

96 Evaluation of compliance with legislation

The method performance data reported for the four marker PAHs were evaluated for compliance with

the provisions given in Commission Regulation (EC) No 8362011 Table 18 in ANNEX 10 gives an

overview on the results of the evaluation Data were not reported in case of empty cells Non-

compliant data are highlighted in Table 18 It should be noted that Regulation (EU) No 8362011

specifies LOD and LOQ with two decimals This was not respected by all participants which

hampered the compliance assessment

27

In summary it can be stated that the majority of participants reported method performance

characteristics for the determination of the four marker PAHs in the food supplement test sample that

are compliant with legislation However some laboratories need to improve their analysis methods

The laboratories in question are urged to adapt to the provisions given in the new legislation

10 Follow-up actions for underperforming laboratories

All laboratories that got questionable or non-satisfactory performance ratings are urged to perform

root cause analysis and to implement corrective actions

As agreed during the EU-RL PAH workshop 2011 the EU-RL PAH will set up follow-up measures in

due time for all NRLs that received for at least one of the four PAHs (BAA BAP BBF and CHR) z-

scores gt 3 as required by Regulation (EC) 8822004 and by the Protocol for management of

underperformance in comparative testing andor lack of collaboration of National Reference

Laboratories (NRLs) with European Union reference laboratories (EU-RLs) activities

These laboratories shall perform as an immediate action root-cause-analysis and shall report to the

EU-RL PAH in writing the identified cause for their underperformance and corrective actions they are

going to take Additionally they shall participate to an independent (non-EU-RL) proficiency test on

the determination of PAHs in food and shall communicate the outcome of this exercise to the EU-RL

PAH

28

11 Conclusions

Fourty seven participants reported results back to the EU-RL The performance of the majority of

participants was good In total 153 out of 225 attributed z-scores were below an absolute value of two

which equals to almost 68 However this percentage is much below the percentages of satisfactory

performance gained in previous studies This might be the consequence of the higher level of difficulty

with respect to matrix composition In addition bias can be concluded from the pattern of performance

indicators of some laboratories

In general the determination of chrysene caused most difficulties to the participants

zeta-Scores were calculated besides z-scores They indicate the agreement of the reported result with

the assigned value with respect to the stated measurement uncertainty The outcome of this rating was

worse than for the z-scores which indicates that the measurement uncertainty estimates were not

realistic Therefore participants underperforming with regard to zeta scores are urged to adapt their

measurement uncertainty statements

The great majority of participants in this inter-laboratory comparison applied analytical methods which

complied with EU legislation with regard to performance characteristics

12 Acknowledgements

The organisers would like to thank Mrs Beatriz de la Calle and Mr Franz Ulberth (all from IRMM

Geel Belgium) for their accurate revision of this report Mr Hakan Emteborg (from IRMM) for

ampouling of the standard solutions and all NRLs and OCLs for their cooperation

29

13 References

1 EU COMMISSION REGULATION (EC) No 7762006 of 23 May 2006 amending Annex VII to Regulation

(EC) No 8822004 of the European Parliament and of the Council as regards Community reference

laboratories Official Journal of the European Union 2006 L 136 p 3-8

Available from

httpeur-lexeuropaeuLexUriServLexUriServdouri=OJL200613600030008ENPDF

2 EU Regulation (EC) No 8822004 of the European Parliament and of the Council of 29 April 2004 on

official controls performed to ensure the verification of compliance with feed and food law animal health

and animal welfare rules Official Journal of the European Communities 2004 L191 p 1-52 Available

from

httpeur-lexeuropaeuLexUriServLexUriServdouri=OJL200419100010052ENPDF

3 EU Opinion of the Scientific Committee on Food on the risks to human health of Polycyclic Aromatic

Hydrocarbons in food (2002) Available from

httpeuropaeuintcommfoodfsscscfout153_enpdf

4 International Agency for Research on Cancer IARC Monographs on the Evaluation of

Carcinogenic Risks to Humans Some Non-heterocyclic Polycyclic Aromatic Hydrocarbons and Some

Related Exposures Volume 92 (2010) Available from

httpmonographsiarcfrENGMonographsvol92mono92pdf

5 EU Commission Regulation (EC) No 3332007 of 28 March 2007 laying down the methods of sampling

and analysis for the official control of the levels of lead cadmium mercury inorganic tin 3-MCPD and

benzo(a)pyrene in foodstuffs Official Journal of the European Union 2007 L 88 p 29-38 Available

from

httpeur-lexeuropaeuLexUriServLexUriServdouri=OJL200708800290038ENPDF

6 EU Commission Regulation (EC) No 18812006 of 19 December 2006 setting maximum levels for certain

contaminants in foodstuffs Official Journal of the European Union 2006 L 364 p 5-24 Available from

httpeur-lexeuropaeuLexUriServLexUriServdouri=OJL200636400050024ENPDF

7 EFSA Scientific Opinion of the Panel on Contaminants in the Food Chain on a request from the European

Commission on Polycyclic Aromatic Hydrocarbons in Food The EFSA Journal 2008 724 p 1-114

Available from

httpwwwefsaeuropaeuEFSAefsa_locale-1178620753812_1211902034842htm

30

8 EU Commission Regulation (EU) No 8352011 amending Regulation (EC) No 18812006 as regards

maximum levels for polycyclic aromatic hydrocarbons in foodstuffs Official Journal of the European

Union 2011 L 215 p 4-8

httpeur-lexeuropaeuLexUriServLexUriServdouri=OJL201121500040008ENPDF

9 EU Commission Regulation (EU) No 8362011 amending Regulation (EC) No 3332007 laying down the methods of sampling and analysis for the official control of the levels of lead cadmium mercury inorganic tin 3-MCPD and benzo(a)pyrene in foodstuffs Official Journal of the European Union 2011 L 215 p 9-16 httpeur-lexeuropaeuLexUriServLexUriServdouri=OJL201121500090016ENPDF

10 ISOIEC 170432010 (2010) Conformity assessment -- General requirements for proficiency testing

providers Geneva Switzerland

11 Thompson M Ellison SLR and Wood R The International Harmonized Protocol for the Proficiency

Testing of Analytical Chemistry Laboratories Pure Appl Chem (2006) 78(1) p 145ndash196

Available from httpmediaiupacorgpublicationspac2006pdf7801x0145pdf

31

14 ANNEXES

ANNEX 1 ndash Announcement of the PT on the IRMM webpage

ANNEX 2 ndash Announcement via e-mail and invitation

ANNEX 3 ndash Announcement of sample dispatch

ANNEX 4 ndash Technical specifications of the calibration solutions

ANNEX 5 ndash Homogeneity and stability of the test material

ANNEX 6 ndash Acknowledgement of sample receipt

ANNEX 7 ndash Questionnaire on details of analysis method

ANNEX 8 ndash Results reported for the food supplement sample

ANNEX 9 ndash Results reported for the standard solutions with undisclosed analyte content

ANNEX 10 ndash Method performance characteristics reported by participants

ANNEX 11 ndash Details of analysis methods reported by participants

32

ANNEX 1 ndash Announcement of the PT on the IRMM webpage

33

ANNEX 2 - Announcement via e-mail and invitation

34

ANNEX 3 - Announcement sample dispatch

Dear MadameSir

The test samples for the proficiency test on the determination of four PAHs in a food supplement (dry extract of St Johns wort) were

dispatched today

Explanation on the design of the study as well as instructions for sample storage and for reporting of results are in the parcel You will

also find your participant key in the parcel which is required for reporting of results

Please check the parcel after arrival for completeness and report the receipt of the test samples as well as their status to us by applying

the sample receipt form which is in the parcel

You are kindly requested to inform us by email if you do not receive the parcel by end of this week

My colleague Donata Lerda will then take care of it

With best regards

Thomas Wenzl

35

ANNEX 4 - Specifications of standard solutions with disclosed concentration

36

ANNEX 4 - continued

37

ANNEX 5 - Homogeneity and stability of the test material

Homogeneity of the test material

Benz[a]anthracene - BAA

Benzo[a]pyrene - BAP

38

ANNEX 5 - continued

Benzo[b]fluoranthene - BBF

Chrysene - CHR

39

ANNEX 5 - continued

Stability of the test material

Table 10 Content of the four target analytes in test materials stored under different

temperatures

BaA BaP BbF CHR

microgkg microgkg microgkg microgkg

Content 31 16 31 69 Storage at room temperature

U (k=2) 05 02 05 17

Content 31 15 29 68 Storage at reference temperature

U (k=2) 04 03 04 10

The applied capillary column does not separate CHR from triphenylene

40

ANNEX 6 ndash Documents sent to participants

41

ANNEX 6 - continued

42

ANNEX 7 Questionnaire on details of analysis method

43

ANNEX 7 - continued

44

ANNEX 7 - continued

45

ANNEX 8 - Results reported by participants for the food supplements test sample Table 11 Analysis results reported by the participants for the content of benz[a]anthracene (BAA) in the food supplements test material Values are presented as reported

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

3600 31 29 29 3 05 2

3602 199 193 178 19 021

3603 253 253 246 251 022 2

3604 199 209 23 213 071 2

3605 301 3 271 291 076 2

3606 302 288 278 289 064 2

3607 456 416 443 438 096 2

3608 796 695 651 714 149 2

3610 182 192 214 196 039 2

3611 414 504 488 469 96 2

3612 3 34 28 31 06 2

3613 32 33 34 33 07 2

3614 182 182 191 185 037 2

3615 3884 3822 3565 376 0752 2

3616 2 2 2 2 01 2

3617 366 37 35 362 046 2

3618 32 32 32 32 05 2

3619 299 305 297 299 048 2

3620 304 294 302 302 045 2

3621 29 26 29 28 04 2

3622 343 325 318 322 052 2

3623 35 3 33 33 07 2

3624

3626 318 31 314 314 126 2

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

5301 31 3 32 31 06 2

5302 314 319 315 316 063 2

5303 3102 2716 2822 2876 27 62

5304 261 263 26 261 57 2

5305 23 22 26 24 20

5306 22 23 23 22 11 2

5307 488 461 317 461

5308 168633 168544 1562025 1644598

5309 16 19 16 17

5310 3 304 311 305 015

5311 477 452 448 459 15 2

5312 506 63 494 543 134 2

5313 353 301 404 353

5314 431 373 378 394 5 096

5315 596 531 51 545 11 2

5316 224 231 235 235 022 2

5317 216 23 221 222 007 2

5318 064 07 067 01 2

5319 111 184 146 147 029 2

5320 515 525 564 534 48

5321 21 5322 13 87 9 87 35 2

5323 38 33 35 33 16 2

46

Figure 6 Results of replicate determinations (indicated by triangles) of benz[a]anthracene (BAA) in the food supplement test material

Horizontal blue lines represent the arithmetic mean value of replicate measurements and blue bars the reported expanded measurement uncertainty (k=2) The assigned value is

plotted as dotted line Red solid lines mark the range of satisfactory performance (|z|le2) Mean values are given for results outside the displayed data range

ProLab 2011

Laboratory

53

1853

1953

0853

0936

1436

0236

1036

1653

2136

0453

1753

0653

1653

0536

0353

0436

2153

0336

0636

0536

0036

2036

1953

1036

1253

0136

2653

0236

1836

2336

2236

1353

1353

2336

1736

1553

1453

0736

0753

1153

2053

1253

1536

0853

2236

11

an

aly

te c

on

ten

t (micro

gk

g)

10

95

9

85

8

75

7

65

6

55

5

45

4

35

3

25

2

15

1

05

0

102

3 468

7

Sample St Johns wort extract replicatesMeasurand Benz[a]anthraceneNo of laboratories 46

Assigned value 298 microgkg (Reference value)Target sd 062 microgkgTolerance limits 174 - 422 microgkg (|Z score| lt 200)

Assig

ned va

lue

Limit of tolerance

Limit of tolerance

47

Table 12 Analysis results reported by the participants for the content of benzo[a]pyrene (BAP) in the food supplements test material Values are presented as reported

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

3600 16 17 17 17 03 2

3602 118 123 13 124 015

3603 165 162 157 161 015 2

3604 134 154 139 143 04 2

3605 164 166 172 167 057 2

3606 181 159 161 167 031 2

3607 279 266 279 274 055 2

3608 57 687 461 573 226 2

3610 226 216 183 206 042 2

3611 289 325 311 308 36 2

3612 24 22 2 22 05 2

3613 17 18 19 18 05 2

3614 08 08 08 08 016 2

3615 1545 1393 1425 145 029 2

3616 16 16 14 15 03 2

3617 177 171 158 169 023 2

3618 2 2 2 2 02 2

3619 207 207 206 207 075 2

3620 165 169 163 166 017 2

3621 44 47 49 47 05 2

3622 212 208 222 213 038 2

3623 2 21 22 21 03 2

3624 18 178 182 18 031 2

3626 172 173 178 174 07 2

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

5301 18 18 18 18 04 2

5302 188 19 187 188 038 2

5303 1691 1426 149 1535 19 80

5304 145 142 15 146 69 2

5305 15 17 18 16 15

5306 16 17 17 16 08 2

5307 164 157 153 157

5308 102669 109324 109035 107009

5309 1 1 12 11

5310 122 122 124 123 006

5311 199 184 215 2 15 2

5312 258 248 239 248 05 2

5313 199 178 245 207

5314 174 171 172 172 11 0948

5315 253 248 213 238 05 2

5316 177 171 173 17 016 2

5317 255 277 276 269 012 2

5318 114 12 117 02 2

5319 19 228 231 216 043 2

5320 303 3 344 316 78

5321 12 12 12 12

5322 13 17 24 17 35 2

5323 2 2 19 2 1 2

48

Figure 7 Results of replicate determinations (indicated by triangles) of benzo[a]pyrene (BAP) in the food supplement test material

Horizontal blue lines represent the arithmetic mean value of replicate measurements and blue bars the reported expanded measurement uncertainty (k=2) The assigned value is

plotted as dotted line Red solid lines mark the range of satisfactory performance (|z|le2) Mean values are given for results outside the displayed data range

ProLab 2011

Laboratory

36

1453

0953

0853

1853

2153

1036

0236

0436

1553

0436

1653

0353

0736

0336

2036

0053

0553

0636

0636

0536

1753

1453

1636

2636

1336

2453

0153

2253

0253

2353

1136

1836

1953

1336

1036

2336

2253

1936

1253

1553

1253

1736

0753

2036

2136

0836

11

an

aly

te c

on

ten

t (micro

gk

g)

55

5

45

4

35

3

25

2

15

1

05

0

573

308

3

Sample St Johns wort extract replicatesMeasurand Benzo[a]pyreneNo of laboratories 48

Assigned value 165 microgkg (Reference value)Target sd 036 microgkgTolerance limits 093 - 237 microgkg (|Z score| lt 200)

Assig

ned va

lue

Limit of tolerance

Limit of tolerance

49

Table 13 Analysis results reported by the participants for the content of benzo[b]fluoranthene (BBF) in the food supplements test material Values are presented as reported

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

3600 32 3 3 31 09 2

3602 173 175 16 17 017

3603 265 252 25 256 025 2

3604 116 108 105 11 028 2

3605 286 225 232 247 074 2

3606 294 273 261 276 046 2

3607 579 579 544 567 113 2

3608 103 1201 876 1036 326 2

3610 173 196 204 191 038 2

3611 546 618 565 576 75 2

3612 3 28 28 29 06 2

3613 28 31 32 31 06 2

3614 135 111 135 127 022 2

3615 4187 3678 4111 4 08 2

3616 2 2 2 2 01 2

3617 33 27 299 035 2

3618 31 31 31 31 05 2

3619 326 324 312 326 064 2

3620 277 28 272 276 069 2

3621 26 25 23 25 04 2

3622 346 336 333 34 052 2

3623 26 28 29 28 02 2

3624

3626 288 28 307 292 117 2

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

5301 29 3 29 29 06 2

5302 297 3 296 298 06 2

5303 2705 2444 2608 2586 22 78

5304 245 254 26 253 85 2

5305 23 23 26 24 15

5306 42 43 4 42 21 2

5307 221 216 241 221

5308 232169 246654 240722 229848

5309 19 22 19 2 0

5310 3 315 325 313 016

5311 262 277 304 281 15 2

5312 27 289 337 299 075 2

5313 321 284 378 328

5314 423 404 385 404 34 083

5315 42 506 416 444 09 2

5316 265 267 277 273 018 2

5317 185 197 195 192 006 2

5318 276 281 279 04 2

5319 396 389 403 396 079 2

5320 623 618 635 625 14

5321 31

5322 24 33 49 33 35 2

5323 38 45 35 45 27 2

50

Figure 8 Results of replicate determinations (indicated by triangles) of benzo[b]fluoranthene (BBF) in the food supplement test material

Horizontal blue lines represent the arithmetic mean value of replicate measurements and blue bars the reported expanded measurement uncertainty (k=2) The assigned value is

plotted as dotted line Red solid lines mark the range of satisfactory performance (|z|le2) Mean values are given for results outside the displayed data range

ProLab 2011

Laboratory

36

0436

1436

0236

1053

1736

1653

0953

0753

0853

0536

2136

0553

0436

0353

0353

1636

0636

2036

2353

1853

1136

1236

2653

0153

0253

1236

1736

1336

0036

1853

2153

1036

1953

1336

2253

2253

2353

1936

1553

1453

0653

1536

0753

2036

0836

11

an

aly

te c

on

ten

t (micro

gk

g)

75

7

65

6

55

5

45

4

35

3

25

2

15

1

05

0

103

6

576

3

Sample St Johns wort extract replicatesMeasurand Benzo[b]fluorantheneNo of laboratories 46

Assigned value 292 microgkg (Reference value)Target sd 060 microgkgTolerance limits 172 - 412 microgkg (|Z score| lt 200)

Assig

ned va

lue

Limit of tolerance

Limit of tolerance

51

Table 14 Analysis results reported by the participants for the content of chrysene (CHR) in the food supplements test material Values are presented as reported

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

3600 5 49 5 5 07 2

3602 32 304 29 304 036

3603 462 469 447 459 048 2

3604 438 428 42 429 119 2

3605 365 41 381 386 085 2

3606 494 411 45 451 124 2

3607 897 774 787 819 18 2

3608 986 887 1168 1014 284 2

3610 418 392 462 424 085 2

3611 833 893 87 865 61 2

3612 6 64 58 61 12 2

3613 41 39 4 4 08 2

3614 272 254 288 271 054 2

3615 605 6028 5964 601 12 2

3616 47 45 49 47 05 2

3617 445 431 389 422 065 2

3618 45 45 45 45 06 2

3619 738 747 72 738 118 2

3620 463 472 446 46 092 2

3621 53 49 51 51 06 2

3622 478 513 505 488 08 2

3623 73 73 72 72 05 2

3624

3626 63 622 634 628 251 2

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

5301 49 49 49 49 1 2

5302 687 683 666 679 136 2

5303 6777 6181 6382 6441 24 70

5304 592 581 6 591 85 2

5305 6 58 64 61 20

5306 49 5 51 49 25 2

5307 524 5 574 524

5308 211488 216782 221146 213243

5309

5310 372 393 394 386 019

5311 43 459 422 437 15 2

5312 934 943 981 953 191 2

5313 793 73 99 838

5314 636 561 565 588 18 0818

5315 547 583 499 542 11 2

5316 292 286 285 282 06 2

5317 52 598 517 545 046 2

5318 368 345 357 05 2

5319 449 471 474 465 116 2

5320 76 924 818 834 99

5321 33

5322 7 7 76 7 35 2

5323 87 73 77 73 37 2

52

Figure 9 Results of replicate determinations (indicated by triangles) of chrysene (CHR) in the food supplement test material

Horizontal blue lines represent the arithmetic mean value of replicate measurements and blue bars the reported expanded measurement uncertainty (k=2) The assigned value is

plotted as dotted line Red solid lines mark the range of satisfactory performance (|z|le2) Mean values are given for results outside the displayed data range

ProLab 2011

Laboratory

53

0836

1453

1636

0253

2153

1836

0553

1036

1336

1736

1036

0453

1136

1836

0636

0336

2053

1936

1653

0136

0036

2253

0636

2153

0753

1553

1753

1453

0436

1536

1253

0536

2653

0353

0253

2236

2336

1953

2336

0753

2053

1353

1236

0836

11

an

aly

te c

on

ten

t (micro

gk

g)

13

12

11

10

9

8

7

6

5

4

3

2

1

0

865

3

Sample St Johns wort extract replicatesMeasurand ChryseneNo of laboratories 45

Assigned value 407 microgkg (Reference value)Target sd 083 microgkgTolerance limits 241 - 573 microgkg (|Z score| lt 200)

Assig

ned va

lue

Limit of tolerance

Limit of tolerance

53

Table 15 Analysis results reported by the participants for the sum of the four marker PAHs in the food supplements test material Values are presented as reported

Laboratory code

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg

3600 128 24 2

3602 79 126

3603 1127 096 2

3604 893 258 2

3605 1091 305 2

3606 1184 238 2

3607 2098 42 2

3608 3336 325 2

3610 1138 17 2

3611 2218 259 2

3612 143 29 2

3613 122 2 2

3614 663 129 2

3615 152 304 2

3616 102 03 2

3617 1252 169 2

3618 128 18 2

3619 157 305 2

3620 1204 12 2

3621 151 19 2

3622 1361 115 2

3623 154 09 2

3624

3626 1409 56 2

Laboratory code

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg

5301 126

5302 37 074 2

5303 13438 92

5304 1251 85 2

5305 125 20

5306 129 65 2

5307 1363

5308 7245598

5309

5310 1127 056

5311 1367 15 2

5312 2042 408 2

5313 1726

5314 1558

5315 177 35 2

5316 96 073 2

5317 1229 066 2

5318 82 05 2

5319 1225 267 2

5320 2309 41

5321

5322 207 35 2

5323 171 103 2

54

ANNEX 9 Results reported for the standard solutions with undisclosed analyte content Table 16 Analysis results reported by the participants for the four marker PAHs in the standard solution in toluene with undisclosed analyte

content Values are presented as reported U measurement uncertainty k coverage factor

benz[a]anthracene benzo[a]pyrene benzo[b]fluoranthene chrysene Laboratory

code Unit Result U k Result U k Result U k Result U k 3600 microgl 38 67 2 25 45 2 211 68 2 275 38 2

3605 microgl 367 184 2 2482 124 2 2592 13 2 235 118 2

3606 microgkg 3763 176 2 2609 142 2 2721 118 2 2539 134 2

3607 microgkg 3706 815 2 2824 565 2 2591 518 2 2587 569 2

3612 microgl 38 08 2 237 11 2 244 18 2 257 02 2

3614 microgl 324 65 2 209 38 2 215 37 2 21 42 2

3616 microgkg 318 07 2 224 14 2 231 26 2 216 1 2

3618 microgkg 417 17 2 284 08 2 292 06 2 297 03 2

3619 microgkg 47 742 2 274 468 2 228 387 2 274 434 2

3620 microgl 495 74 2 294 29 2 321 8 2 321 64 2

3621 microgl 241 22 2 143 13 2 188 16 2 145 14 2

3623 microgkg 476 25 2 311 53 2 269 3 2 297 03 2

3626 microgl 359 38 2 235 23 2 238 27 2 24 18 2

5301 microgkg 426 29 299 289

5302 microgkg 436 87 2 296 59 2 296 59 2 289 58 2

5303 microgl 3721 27 102 22838 19 99 23756 22 99 25486 24 98

5304 microgkg 38 29 2 225 26 2 257 37 2 258 52 2

5305 microgkg 305 225 245 29

5306 microgkg 316 63 2 247 49 2 65 13 2 22 44 2

5307 microgkg 3658 2559 241 2414

5308 microgkg 16854 109324 246654 216782

5311 microgl 374 20 2 279 15 2 268 15 2 272 15 2

5313 microgl 3408 2165 2275 2373

5314 microgkg 395 5 2875 11 3614 34 2905 18

5320 microgl 3267 368 1986 571 28 683 2427 512

5322 microgl 282 35 2 225 35 2 241 35 2 21 35 2

55

Figure 10 Youden plots of the percent deviation of the reported values from the assigned values for the food supplement test sample and the

standard solution in toluene with undisclosed content

(The identity of the respective measurand is given above the figure Results exceeding a difference of more than 100 were omitted)

56

Figure 10 - continued

57

Table 17 Analysis results reported by the participants for the four marker PAHs in the standard solution in acetonitrile with undisclosed

analyte content

Values are presented as reported U measurement uncertainty k coverage factor

benz[a]anthracene benzo[a]pyrene benzo[b]fluoranthene chrysene

Laboratory

code Unit Result U k Result U k Result U k Result U k

3602 microgkg 3675 247 247 244

3603 microgkg 358 086 2 236 028 2 235 028 2 236 057 2

3604 microgl 475 29 2 19 12 2 194 13 2 258 12 2

3608 microgl 3623 078 2 2385 023 2 2343 18 2 2296 092 2

3610 microgkg 369 185 2 25 125 2 251 126 2 244 122 2

3611 microgkg 48 02 2 323 02 2 328 04 2 317 03 2

3613 microgl 332 31 2 261 3 2 249 25 2 239 24 2

3615 microgl 415 2635 2729 2689

3617 microgkg 37 09 2 244 06 2 284 07 2 239 06 2

3622 microgl 37 12 2 25 23 2 257 21 2 238 1 2

3624 microgkg 402 104 2 255 084 2 265 096 2 253 07 2

5306 microgkg 563 113 2 371 74 2 359 72 2 333 67 2

5308 microgkg 168633 102669 232169 211488

5309 microgl 402 268 27

5310 microgl 315 158 25 125 25 125 288 144

5312 microgl 428 043 2 282 028 2 272 027 2 275 028 2

5315 microgkg 387 08 2 256 05 2 251 05 2 253 05 2

5316 microgl 3839 342 2 2485 191 2 2559 168 2 239 51 2

5317 microgl 2466 043 2 2212 034 2 2472 016 2 2146 068 2

5319 microgl 414 41 2 279 28 2 268 27 2 26 31 2

5321 microgkg 476 316 326 328

5322 microgl 2547 35 2

5323 microgkg 405 203 2 239 119 2 255 153 2 201 11 2

58

Figure 11 Youden plots of the percent deviation of the reported values from the assigned values for the food supplement test sample and the

standard solution in acetonitrile with undisclosed content

(The identity of the respective measurand is given above the figure Results exceeding a difference of more than 100 were omitted)

59

Figure 11 - continued

60

ANNEX 10 Table 18 Method performance parameters reported by the participants

Laboratory code

Analyte LOD LOQ Recovery Analyte LOD LOQ Recovery

microgkg microgkg microgkg microgkg 3600 BaA 03 06 978 BaP 03 07 9483602 BaA 007 021 926 BaP 005 015 993603 BaA 0011 05 987 BaP 0005 051 9553604 BaA 02 04 86 BaP 02 04 883605 BaA 0025 005 118 BaP 0025 005 1083606 BaA 001 003 65 BaP 001 003 703607 BaA 021 071 74 BaP 027 089 943608 BaA 016 048 8213 BaP 015 046 83683610 BaA 02 05 85 BaP 005 02 853611 BaA BaP 3612 BaA 1 117 BaP 1 1203613 BaA 01 03 90 BaP 01 03 903614 BaA 02 05 96 BaP 02 05 933615 BaA 005 015 110 BaP 005 015 1053616 BaA 01 05 108 BaP 01 05 753617 BaA 018 037 405 BaP 009 018 3873618 BaA 01 03 63 BaP 01 03 593619 BaA 005 005 59 BaP 033 033 543620 BaA 0009 0018 64 BaP 0005 001 553621 BaA 007 02 84 BaP 007 02 923622 BaA 01 03 1101 BaP 01 03 9853623 BaA 02 08 105 BaP 02 08 1013624 BaA BaP 01 033 8513625 BaA BaP 3626 BaA BaP 5301 BaA 003 01 102 BaP 003 01 1005302 BaA 01 03 78 BaP 01 03 865303 BaA 003 01 62 BaP 003 01 805304 BaA 1 1 70 BaP 1 1 955305 BaA 02 05 85 BaP 02 05 855306 BaA 03 1 699 BaP 03 1 905307 BaA 6537 BaP 81165308 BaA 0088 0265 99 BaP 00262 00785 1005309 BaA 01 08 92 BaP 01 08 925310 BaA 01 03 BaP 01 03 905311 BaA 002 005 80 BaP 002 004 915312 BaA 03 09 105 BaP 03 09 1055313 BaA 04 08 BaP 04 08 5314 BaA 001 002 96 BaP 001 002 9485315 BaA 01 03 50 - 100 BaP 01 03 50 - 100 5316 BaA 002 011 827 BaP 004 019 10445317 BaA 05 1 76 BaP 05 1 755318 BaA BaP 5319 BaA 009 029 951 BaP 011 041 995320 BaA 12 18 946 BaP 06 1 8345321 BaA 03 05 BaP 05 1 5322 BaA 1 2 100 BaP 1 2 1005323 BaA 26 BaP 26

61

ANNEX 10 Table 18 - continued

Laboratory code Analyte LOD LOQ Recovery Analyte LOD LOQ Recovery

microgkg microgkg microgkg microgkg 3600 BbF 03 05 96 CHR 02 03 10153602 BbF 015 045 947 CHR 003 009 9193603 BbF 0014 05 1012 CHR 0004 05 10033604 BbF 02 04 85 CHR 02 04 873605 BbF 005 01 94 CHR 0025 005 863606 BbF 001 003 70 CHR 001 003 653607 BbF 018 06 100 CHR 014 028 943608 BbF 015 046 7979 CHR 015 045 71253610 BbF 005 02 85 CHR 02 05 853611 BbF CHR 3612 BbF 1 118 CHR 1 1173613 BbF 01 03 90 CHR 01 03 903614 BbF 01 04 96 CHR 02 05 953615 BbF 008 025 109 CHR 003 01 1083616 BbF 01 05 78 CHR 01 05 723617 BbF 035 07 38 CHR 012 024 4163618 BbF 01 03 62 CHR 01 03 623619 BbF 016 016 63 CHR 01 01 593620 BbF 0003 0006 55 CHR 001 002 653621 BbF 007 02 103 CHR 007 02 893622 BbF 02 06 963 CHR 03 09 1043623 BbF 02 08 106 CHR 02 08 1193624 BbF CHR 3625 BbF CHR 3626 BbF CHR 5301 BbF 003 01 96 CHR 003 01 1145302 BbF 01 03 84 CHR 01 03 835303 BbF 003 01 78 CHR 003 01 705304 BbF 1 1 86 CHR 1 1 705305 BbF 02 05 90 CHR 02 05 955306 BbF 03 1 1435 CHR 03 1 1185307 BbF 8116 CHR 65375308 BbF 0079 0238 99 CHR 009 026 1005309 BbF 01 08 92 CHR 5310 BbF 07 2 CHR 01 03 5311 BbF 002 004 95 CHR 002 004 895312 BbF 03 09 105 CHR 03 09 1055313 BbF 04 08 CHR 04 08 5314 BbF 001 002 83 CHR 001 002 8185315 BbF 02 05 50 -100 CHR 02 05 50 - 100 5316 BbF 005 024 814 CHR 001 007 9595317 BbF 05 1 73 CHR 05 1 785318 BbF CHR 5319 BbF 011 038 946 CHR 013 043 9675320 BbF 07 09 1194 CHR 23 32 10455321 BbF 05 1 CHR 05 1 5322 BbF 1 2 100 CHR 1 2 1005323 BbF 26 CHR 26

62

ANNEX 11 Table 19 Details of analysis method reported by the participants Data are presented as reported

Laboratory code

Analysis technique

Instrument calibration

Internal standards applied

Sample clean up

Details of sample

clean up

Extraction technique

Details of sample

extraction

3600 6) GC-MS

2) Internal standardisation with labelled substances

Deuterated PAH mix 9 (Ehrenstorfer)

1) Saponification 2) LiquidLiquid partitioning

D) Other Liquidliquid partitioning

3602 2) HPLC-UV-FLD

1) External calibration

3) Gel permeation chromatography

A) Pressurised liquid extraction (PLE)

3603 1) HPLC-FLD

1) External calibration

3) Gel permeation chromatography

D) Other 1 minute with dichloromethane in vortex

3604 2) HPLC-UV-FLD

2) Internal standardisation with labelled substances

DiP D14

5) Donor acceptor complex chromatography

D) Other LL

3605 1) HPLC-FLD

1) External calibration

3) Gel permeation chromatography

C) Soxhlet extraction

3606 7) GC-MSMS

2) Internal standardisation with labelled substances

BaP 13C4 CHR 13C6 BaA 13C6 BbF 13C6

4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

3607 6) GC-MS

2) Internal standardisation with labelled substances

BaA-d12 CHR-d12 BbF-d12 BkF-d12 BaP-d12 ICP-d12 BgP-d12 DiP-d14

1) Saponification

D) Other solvent partitioning

3608 1) HPLC-FLD

1) External calibration

6) Other Liquid extraction

D) Other

Extraction in a rotary agitator and centrifugation

3610 2) HPLC-UV-FLD

1) External calibration

4) Solid phase extraction

B) Sonication

3611

3612 6) GC-MS

2) Internal standardisation with labelled substances

chrysene-d12 dibenzo(ah)anthracene-d14 dibenzo(ai)pyrene-d14

4) Solid phase extraction

B) Sonication

3613 5) LC-MSMS

3) Internal standardisation with unlabelled substances

I use Chrysene-D12 3) Gel permeation chromatography

D) Other extraction with hexan

63

Table 19 - continued

Laboratory code

Analysis technique

Instrument calibration

Internal standards applied

Sample clean up Details of sample

clean up

Extraction technique

Details of sample

extraction

3614 6) GC-MS 1) External calibration

3) Gel permeation chromatography

B) Sonication

3615 1) HPLC-FLD

1) External calibration

6) Other PLE GPC

A) Pressurised liquid extraction (PLE)

3616 6) GC-MS

2) Internal standardisation with labelled substances

Benzo(a)pyrene-13C4 Benzo(a)anthracene-13C6 Benzo(b)fluoranthene-13C6 Chrysene-13C6

2) LiquidLiquid partitioning 4) Solid phase extraction

B) Sonication

3617 1) HPLC-FLD

1) External calibration

4) Solid phase extraction

C) Soxhlet extraction

3618 6) GC-MS

2) Internal standardisation with labelled substances

deuterated standards

1) Saponification 4) Solid phase extraction

D) Other Handshaken with cyclohexane

3619 6) GC-MS

2) Internal standardisation with labelled substances

C13 Labelled US EPA 16

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

D) Other Homegenisation amp saponiofication

3620 9) GC-HRMS

2) Internal standardisation with labelled substances

mix of deuterated PAHs (BAA-D12 CHR-D12 BAP-D12 BBF-D12)

3) Gel permeation chromatography 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

3621 6) GC-MS

2) Internal standardisation with labelled substances

Chrysene D12 Benzo(a)pyrene D12 Benzo(b)fluoranthene D12 Benzo(a)anthracene

2) LiquidLiquid partitioning 4) Solid phase extraction

B) Sonication

3622 1) HPLC-FLD

4) Standard addition method

4) Solid phase extraction

D) Other

Extraction with cyclohexane by heating under reflux

3623 7) GC-MSMS

2) Internal standardisation with labelled substances

C13 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

3624 2) HPLC-UV-FLD

1) External calibration

2) LiquidLiquid partitioning

D) Other hot solvent extraction

3626

64

Table 19 - continued

Laboratory code

Analysis technique

Instrument calibration

Internal standards

applied

Sample clean up

Details of sample

clean up

Extraction technique

Details of sample

extraction

5301 7) GC-MSMS

2) Internal standardisation with labelled substances

EACH SUBSTANCE C13

4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5302 7) GC-MSMS

2) Internal standardisation with labelled substances

Isotope labelled PAHs 13C for each analyte

4) Solid phase extraction

C) Soxhlet extraction

5303 7) GC-MSMS

3) Internal standardisation with unlabelled substances

HAP C13 4) Solid phase extraction

D) Other automate ASE200 DIONEX

5304 7) GC-MSMS

2) Internal standardisation with labelled substances

with C13 for each HAP

2) LiquidLiquid partitioning 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5305 7) GC-MSMS

2) Internal standardisation with labelled substances

C13 isotopiques internal standards

4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5306 7) GC-MSMS

2) Internal standardisation with labelled substances

C13 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5307 6) GC-MS

2) Internal standardisation with labelled substances

1) Saponification 2) LiquidLiquid partitioning

D) Other solvent extraction

5308 1) HPLC-FLD 1) External calibration

4) Solid phase extraction

B) Sonication

5309 1) HPLC-FLD 1) External calibration

4) Solid phase extraction

D) Other only SPE

5310 1) HPLC-FLD 1) External calibration

1) Saponification 2) LiquidLiquid partitioning 3) Gel permeation chromatography

C) Soxhlet extraction

5311 6) GC-MS

2) Internal standardisation with labelled substances

13 C Chrysene 13 C Benz(a)pyrene

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

B) Sonication

5312 1) HPLC-FLD

3) Internal standardisation with unlabelled substances

Benzo(b)chrysen

2) LiquidLiquid partitioning 4) Solid phase extraction

Quechers D) Other Quechers

65

Table 19 - continued

Laboratory code

Analysis technique

Instrument calibration

Internal standards applied

Sample clean up Details of sample

clean up

Extraction technique

Details of sample extraction

5313 6) GC-MS

2) Internal standardisation with labelled substances

Benz(a)anthracene D12 Benzo(b)fluranthene D12 Benzo(a)pyrene D12 Chrysene D12

1) Saponification 2) LiquidLiquid partitioning 3) Gel permeation chromatography

A) Pressurised liquid extraction (PLE)

5314 6) GC-MS

2) Internal standardisation with labelled substances

Benzo(a)pyren d12 2) LiquidLiquid partitioning

D) Other Extraction with

5315 1) HPLC-FLD

3) Internal standardisation with unlabelled substances

Benz(b)chrysen

1) Saponification 2) LiquidLiquid partitioning

D) Other shaker

5316 1) HPLC-FLD

1) External calibration

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

D) Other

LiquidLiquid extraction with cyclohexane and dimethylformamide

5317 7) GC-MSMS

1) External calibration

3) Gel permeation chromatography 6) Other

silica gel D) Other VDLUFA online method

5318

5319 1) HPLC-FLD

3) Internal standardisation with unlabelled substances

Benzo(b)chrysene 3) Gel permeation chromatography

A) Pressurised liquid extraction (PLE)

5320 6) GC-MS

3) Internal standardisation with unlabelled substances

22-binaphthyl

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

D) Other 3 hours refluxing

5321 1) HPLC-FLD

2) Internal standardisation with labelled substances

6-meacutethylchrysegravene

1) Saponification 2) LiquidLiquid partitioning

B) Sonication

5322 6) GC-MS

2) Internal standardisation with labelled substances

naftalene-d8 acenaphtalene-d10 phenanthrene-d10 chrysene-d12 perylene-d12 benzo(e)anthracene-d12

1) Saponification 2) LiquidLiquid partitioning 6) Other

clean-up with silica packed column

C) Soxhlet extraction

5323 1) HPLC-FLD

1) External calibration

4) Solid phase extraction 6) Other

Accelerated Solvent Extraction

A) Pressurised liquid extraction (PLE)

66

European Commission EUR 25245 EN ndash Joint Research Centre ndash Institute for Reference Materials and Measurements Title Report on the 8th inter-laboratory comparison organised by the European Union Reference Laboratory for Polycyclic Aromatic Hydrocarbons - Four marker PAHs in a dry extract of St Johns wort Author(s) Radoslav Lizak Szilard Szilagyi Philippe Verlinde and Thomas WENZL Luxembourg Publications Office of the European Union 2012 ndash 66 pp ndash 201 x 297 cm EUR ndash Scientific and Technical Research series ndash ISSN 1831-9424 ISBN 978-92-79-23337-1 doi 10278759387 Abstract The proficiency test here reported concerned the determination of the contents of four marker polycyclic aromatic hydrocarbons (PAHs) and of their sum in an food supplements test sample The set of marker PAHs consists of benz[a]anthacene benzo[a]pyrene benzo[b]fluoranthene and chrysene Participants to these PT were National Reference Laboratories for PAHs (NRLs-PAHs) and EU official food control laboratories The number of participants was in total 48

The PT was organised according ISO Standard 170432010

The test material used was a commercial dry extract of St Johns wort Participants also received a solution of the same PAHs either in acetonitrile or in toluene for checking their instrument calibration

The results from participants were rated with z-scores and zeta-scores About 68 of the reported results were attributed with z-scores with an absolute value of below two which is the threshold for satisfactory performance

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interests whether private or national

L

A-N

A-252

45-EN

-N

• 1 Summary
• 2 Introduction
• 3 Scope
• 4 Participating Laboratories
• 5 Time frame
• 6 Confidentiality
• 7 Test materials
• • 71 1Preparation of test materials
  • 72 Assigned values and standard deviation for proficiency assessment
  • 73 Homogeneity and stability
  • 74 Sample dispatch
  • • 8 Design of the proficiency test
    • 9 Evaluation of the results
    • • 91 2General
      • 92 21BEvaluation criteria
      • 93 Performance of participants
      • 94 Evaluation of the influence of analysis method
      • 95 Evaluation of the influence of calibration
      • 96 26Evaluation of compliance with legislation
      • • 10 Follow-up actions for underperforming laboratories
        • 11 1Conclusions
        • 12 1Acknowledgements
        • 13 References

9

In cases of almost identical analysis results (difference between own results and results of contract

laboratory lt 5) the results from the in-house measurements were applied as assigned values

(applicable to BAA and BBF) If the difference between the two results exceeded 5 the average of

the two results was applied as assigned value (applicable to BAP and CHR) and the combined

uncertainty of in-house and external measurements was associated with the assigned value

The sum of PAH 4 was calculated from the individually assigned values and the corresponding

uncertainty from the uncertainties of the assigned values according to equation 1 The results of in-

house measurements and the results reported by the contract laboratory as well as the assigned values

of the target PAHs are listed in Table 4

Equation 1 2222CHRBBFBAPBAAsum uuuuu

where usum refers to the standard uncertainty of the sum of the four PAHs and uBAA u BAP uBBF and

uCHR refer to the standard uncertainty of the individual analytes

The standard deviation for proficiency assessment (σP) was set for the four individual analytes equal to

the maximum tolerated standard measurement uncertainty Uf (see Equation 2) as defined by

Commission Regulation (EC) No 3332007 [5] and Commission Regulation (EU) No 8362011 [9]

Equation 2 Uf = 22 )C((LOD2)

where Uf relates to the maximum tolerated standard measurement uncertainty LOD to the required

limit of detection α to a numeric factor depending on the concentration C

The application of Equation 2 with the assigned values listed in Table 4 the maximum tolerated value

of LOD of 030 μgkg and α equal to 02 results in Uf values as reported in Table 4

The Uf values of the individual analytes are propagated in analogy to equation 1 for the determination

of the Uf value for the sum of the four PAHs which was used as standard deviation for proficiency

assessment for the sum parameter

10

Table 4 Analyte contents of the food supplement test material

Average of two

sessions bracketing calibration

External evaluation

Assigned value

and σP

Cont microgkg 298 297 298U microgkg 024 041 024P microgkg 062

Cont microgkg 157 174 165U microgkg 020 016 026P microgkg 036

Cont microgkg 292 289 292U microgkg 028 034 028P microgkg 060

Cont microgkg 424 389 407U microgkg 057 057 081P microgkg 083

Cont microgkg 1162U microgkg 093P microgkg 125

Sum PAH4

BAA

BAP

BBF

CHR

from chemical analysis Sum of the four analytes contents for the SUM

p standard deviation for proficiency assessment For the individual analytes is equal to the uncertainty function - Uf

according to Ref [9]

U expanded uncertainty of the assigned value (k=2)

The gravimetrical preparation concentrations were used as assigned values for the standard solutions

with undisclosed content Table 5 contains the assigned values and associated expanded uncertainties

of the standard solutions with undisclosed content

11

Table 5 Analyte contents of the standard solutions with undisclosed content

Content U (k=2) Content U (k=2) microgkg microgkg microgkg microgkg

benz[a ]anthracene 4317 039 4764 043benzo[c ]fluorene 7342 061 8052 067chrysene 2904 031 3179 034cyclopenta[cd ]pyrene 2558 050 2818 055benzo[a ]pyrene 2897 038 3186 042benzo[b ]fluoranthene 2926 026 3199 029benzo[ghi ]perylene 2214 023 2438 026benzo[j ]fluoranthene 4353 038 4763 041benzo[k ]fluoranthene 2143 017 2380 019dibenzo[ae ]pyrene 5735 084 6326 093dibenzo[ah ]anthracene 2215 032 2445 035dibenzo[ah ]pyrene 2169 029 2394 032dibenzo[ai ]pyrene 3691 100 4035 109dibenzo[al ]pyrene 4627 057 5104 063indeno[123-cd ]pyrene 3793 036 4176 0395-methylchrysene 8589 087 9443 096

Solution in toluene Solution in acetonitrile

Prep conc concentration from gravimetrical preparation U expanded uncertainty of the assigned value k coverage

factor

73 Homogeneity and stability

Homogeneity of the food supplements test sample was evaluated according to ISO Standard 13528

Ten units of the St Johns wort extract test material were selected randomly and analysed by isotope

dilution gas chromatography mass spectrometry (GC-MS) The test material was rated sufficiently

homogeneous (see ANNEX 5)

The contributions of the uncertainties of homogeneity (relative uncertainties below 1 ) to the total

uncertainties of the assigned values were found negligible and were therefore not considered in the

calculations

The stability of the test materials was evaluated applying an isochronous experimental set up This

comprised the storage of test samples both at room temperature and cooled in the fridge The content

values of the cooled sample were used as reference for the stability evaluation The materials were

stored from dispatch of test samples to the participants until expiry of the deadline for reporting of

results Afterwards the samples were analysed under repeatability conditions and results were

compared The content values of the cool-stored sample and the sample stored at room temperature

were not statistically significantly different Hence stability of the samples over the whole study period

can be assumed The analysis data are presented in ANNEX 5

12

The uncertainty of stability of the test material was considered negligible compared to the uncertainty

of the characterisation measurements Therefore this parameter was not taken into account in the

determination of the uncertainties of the assigned values

74 Sample dispatch

Samples were dispatched at room temperature Each participant received at least

- one ampoule of the solution of the 15+1 EU priority PAHs in the chosen solvent with disclosed

content (2 ml)

- one ampoule of the 15+1 EU priority PAHs in the chosen solvent with undisclosed content (2

ml)

- one screw cap vial with the food supplement test material

Each parcel contained a sample receipt form the outline of the study an appropriate material safety

data sheets and a unique code that was required for reporting of results The documents sent to the

participants are presented in ANNEX 6

8 Design of the proficiency test

The design of the PT foresaw triplicate analyses of the test sample and reporting of the individual

results of replicate analyses for the single analytes in the following denoted as FS_REP and

additionally a value for proficiency assessment in the following denoted as final value - FS_FIN

for both the single analytes and the sum of the four marker PAHs Both FS_REP results and FS_FIN

results had to be reported corrected for recovery (and recovery had to be stated in the questionnaire

which participants were asked to fill in together with other parameters of the method applied) the

latter had also to be accompanied by the respective expanded measurement uncertainty The FS_FIN

results were used for performance assessment

Besides analysis results participants were also asked to report details of the applied analysis method

and to provide answers to a questionnaire (see ANNEX 7 for the template and the compiled returned

questionnaires)

13

9 Evaluation of the results

The results reported by participants are listed in ANNEX 8

91 General

The most important evaluation parameter was the performance of the laboratories in the determination

of the target PAHs in the food supplement test material which was expressed by z-scores and zeta-

scores

The compliance of method performance characteristics specified by the participants with provisions

given in legislation was evaluated too

92 Evaluation criteria

The participants were requested to report for all analytes the results of replicate measurements and a

value for proficiency assessment which is the result they wish to be applied for the calculation of

performance indicators z-Scores and zeta-scores were attributed only to these results The individual

results of replicate analyses were not rated

z-Scores

z-scores were calculated based on the FS_FIN values Equation 1 presents the formula for calculation

of z-scores

Equation 1

P

assignedlab Xxz

where z refers to the z-score xlab to the reported ldquovalue for proficiency assessmentrdquo Xassigned to the

assigned value and σP to the standard deviation for proficiency testing

zeta-Scores

In addition to z-scores zeta-scores were calculated In contrast to z-scores zeta-scores describe the

agreement of the reported result with the assigned value within the respective uncertainties

Unsatisfactorily large zeta-scores might be caused by underestimated measurement uncertainties large

bias or a combination of both Zeta-scores were calculated according to Equation 3

14

Equation 3 22assignedlab

assignedlab

uu

Xxzeta

where zeta refers to the zeta-score xlab to the reported ldquovalue for proficiency assessmentrdquo Xassigned to

the assigned value ulab to the measurement uncertainty reported by the laboratory and uassigned to the

uncertainty of the assigned value

The performance of the laboratories was classified according to ISOIEC 170432010 [10] Following

scheme is applied for the interpretation of zeta scores and z-scores

|score| 20 = satisfactory performance

20lt|score| lt 30 = questionable performance

|score| ge 30 = unsatisfactory performance

93 Performance of participants

The 48 participants in the study reported in total 225 results for proficiency evaluation which equals to

about 94 of the 240 possible results About 68 of the reported results were rated as satisfactory

with regard to z-scores

Figure 1 and Figure 2 give an overview of the z-scores assigned to the respective results The larger

the triangles the larger were the differences to the assigned values Red triangles indicate z-scores

above an absolute value of three whereas yellow triangles represent z-scores in the questionable

performance range The corresponding score values are plotted next to the triangles About 40 of the

72 non-satisfactory results were reported by seven laboratories only eg the performance of

participant 3608 was not satisfactory for the majority of target analytes

It is also obvious that most problems were encountered in the determination of chrysene and the sum

of the four analytes Potential reasons for the difficulties related to chrysene are discussed further

down

The ideal situation would be that deviations from the assigned values of the results reported for the

individual analytes are small and randomly distributed around zero This will give a satisfactory

performance statement for the sum parameter Deviations of opposite sign could also cancel out and

lead to a satisfactory performance for the sum parameter (eg participants 5303 5304 and 5305)

However in most cases a single underperformance for one analyte led also to non-satisfactory rating

for the sum parameter

15

The numerical values of the calculated z-scores are compiled in Table 6 and Table 7 z-Scores with an

absolute value of above 2 are given in red bold font

The results of the data evaluation for the individual analytes are given in ANNEX 8

For each analyte the figure shows the individual analysis results of the three replicate determinations

The assigned value is shown as dotted line The blue boxes represent the expanded uncertainties as

reported by participants for the value for proficiency assessment The arithmetic mean of the results

of the individual participants is indicated in the blue boxes by a blue line The satisfactory performance

range is located between the two red lines

The individual results of the replicate measurements and the value for proficiency assessment with

its accompanying expanded measurement uncertainty (k=2) are listed in the tables in ANNEX 8 as

well

16

Figure 1 Graphical presentation of z-scores corresponding to the values for proficiency assessment

reported by the NRLs for the contents of BAA BAP BBF and CHR in the St Johns wort extract test

material

Blue triangles indicate satisfactory performance yellow triangles indicate questionable performance red triangles indicate non-satisfactory performance z-score values are presented above the triangles for the latter two performance categories

ProLab 2011

Z score-3 0 3 -3 0 3 -3 0 3 -3 0 3 -3 0 3

BAA BAP BBF CHR PAH4

La

bo

rato

ry

36003602

3603

36043605

36063607

36083610

36113612

36133614

36153616

36173618

36193620

36213622

36233624

3626

2258

2343

2452

2669

2147

2788

2869

6710

70839

3011

11271

80525

8425

4561

12338

90680

4976

7331

99553

3998

3780

7500

17420

168413

3269

3029

-2348 -2736

-2023 -2981

-2155-3018

-3998

Figure 2 Graphical presentation of z-scores corresponding to the values for proficiency assessment

reported by the OCLs for the contents of BAA BAP BBF and CHR in the St Johns wort extract test

material

Blue triangles indicate satisfactory performance yellow triangles indicate questionable performance red triangles indicate non-satisfactory performance z-score values are presented above the triangles for the latter two performance categories

ProLab 2011

Z score-3 0 3 -3 0 3 -3 0 3 -3 0 3 -3 0 3

BAA BAP BBF CHR PAH4

La

bo

rato

ry

5301

5302

5303

5304

5305

5306

5307

5308

5309

5310

5311

5312

5313

5314

5315

5316

5317

5318

5319

5320

5321

5322

5323

2597

2629

2017

2293

2873

2123

2521

2620

2186

2222

2452

2864

3952

3984

3806

9226

4171 5522

3285

6594

5205

5157

3539

3901

7051

4519

3173

4872

9191

7276

4391

-2435

-2154

-2065

-2340

-2740-3726

-6346

-3505

17

Table 6 Compilation of z-scores calculated from the ldquoresults for proficiency assessment

reported by the NRLs for food supplement test material z-scores outside the

satisfactory range (|z| gt 2) are indicated by red font Values are reproduced as reported

by the laboratories Empty cells denote analytes for which results were not reported

Measurand benz[a]anthracene benzo[a]pyrene benzo[b]fluoranthene chrysene Sum PAH 4

Assigned value

microgkg 298 165 292 407 1162

Standard deviation for proficiency assessment

microgkg 062 036 060 083 125

Laboratory code

Result z-score Result z-score Result z-score Result z-score Result z-score

microgkg microgkg microgkg microgkg microgkg

3600 3 00 17 01 31 03 5 11 128 09

3602 19 -18 124 -11 17 -20 304 -12 79 -30

3603 251 -08 161 -01 256 -06 459 06 1127 -03

3604 213 -14 143 -06 11 -30 429 03 893 -22

3605 291 -01 167 01 247 -07 386 -03 1091 -06

3606 289 -01 167 01 276 -03 451 05 1184 02

3607 438 23 274 30 567 46 819 50 2098 75

3608 714 67 573 113 1036 123 1014 73 3336 174

3610 196 -17 206 11 191 -17 424 02 1138 -02

3611 469 708 308 805 576 907 865 996 2218 1684

3612 31 02 22 15 29 00 61 25 143 21

3613 33 05 18 04 31 03 4 -01 122 05

3614 185 -18 08 -23 127 -27 271 -16 663 -40

3615 376 13 145 -06 4 18 601 23 152 29

3616 2 -16 15 -04 2 -15 47 08 102 -11

3617 362 10 169 01 299 01 422 02 1252 07

3618 32 04 2 10 31 03 45 05 128 09

3619 299 00 207 12 326 06 738 40 157 33

3620 302 01 166 00 276 -03 46 06 1204 03

3621 28 -03 47 84 25 -07 51 12 151 28

3622 322 04 213 13 34 08 488 10 1361 16

3623 33 05 21 12 28 -02 72 38 154 30

3624 18 04

3626 314 03 174 02 292 00 628 27 1409 20

18

Table 7 Compilation of z-scores calculated from the ldquoresults for proficiency assessment

reported by the OCLs for food supplement test material z-scores outside the

satisfactory range (|z| gt 2) are indicated by red font Values are reproduced as reported

by the laboratories Empty cells denote analytes for which results were not reported

Measurand benz[a]anthracene benzo[a]pyrene benzo[b]fluoranthene chrysene Sum PAH 4

Assigned value

microgkg 298 165 292 407 1162

Standard deviation for proficiency assessment

microgkg 062 036 060 083 125

Laboratory code

Result z-score Result z-

score Result z-score Result

z-score

Result z-score

microgkg microgkg microgkg microgkg microgkg

5301 31 02 18 04 29 00 49 10 126 08

5302 316 03 188 06 298 01 679 33 37 -63

5303 2876 -02 1535 -03 2586 -06 6441 29 13438 15

5304 261 -06 146 -05 253 -06 591 22 1251 07

5305 24 -09 16 -01 24 -09 61 25 125 07

5306 22 -13 16 -01 42 21 49 10 129 10

5307 461 27 157 -02 221 -12 524 14 1363 16

5308 16446 -22 107009 -16 229848 -10 213243 -23 72456 -35

5309 17 -21 11 -15 2 -15

5310 305 01 123 -12 313 03 386 -03 1127 -03

5311 459 26 2 10 281 -02 437 04 1367 16

5312 543 40 248 23 299 01 953 66 2042 71

5313 353 09 207 12 328 06 838 52 1726 45

5314 394 16 172 02 404 19 588 22 1558 32

5315 545 40 238 20 444 25 542 16 177 49

5316 235 -10 17 01 273 -03 282 -15 96 -16

5317 222 -12 269 29 192 -17 545 17 1229 05

5318 067 -37 117 -13 279 -02 357 -06 82 -27

5319 147 -24 216 14 396 17 465 07 1225 05

5320 534 38 316 42 625 55 834 52 2309 92

5321 12 -12

5322 87 92 17 01 33 06 7 35 207 73

5323 33 05 2 10 45 26 73 39 171 44

19

Table 8 and Table 9 present the respective zeta-scores As for the z-scores data outside the

satisfactory performance range are given in red bold font Zeta scores were only calculated for

laboratories that provided measurement uncertainty data as well as a numerical value for the coverage

factor Zeta scores were not calculated when the reported uncertainty value exceeded the content value

which is probably linked to mixing of reporting units (microgkg and ) However the participants are

reminded that the measurement uncertainty shall be expressed in the same unit as the content value

The assessment of the performance of the participants based on the reported measurement uncertainty

gave a less favourable picture compared to z-scores Only 60 of the zeta-scores are within the

satisfactory performance range The distribution of satisfactory performance ratings between NRLs

and OCLs is not balanced It was higher for the NRLs

It has to be noted that the magnitude of the zeta-scores were for some participants much higher than

the z-scores attributed to the same results Consequently the laboratories perform according to fitness-

for-purpose criterion specified in legislation which forms the basis for the z-scores but seem to have

difficulties in deducing realistic measurement uncertainty values Eg the relative standard

measurement uncertainties reported by the NRLs ranged from 25 to 20 with a median value of

about 10 Only two OCLs reported measurement uncertainties that exceeded the maximum tolerable

uncertainty (Uf) The respective data is highlighted in yellow in Table 9

However the EU-RL PAHs will continue to pay special attention to measurement uncertainty in future

ILCs as it has major implications on the assessment of compliance of food with European legislation

20

Table 8 Compilation of zeta-scores calculated from the ldquoresults for proficiency assessment

reported by the NRLs for test material St Johns wort the reported measurement

uncertainty and the uncertainty of the analyte content of the test material zeta-scores

outside the satisfactory range (|zeta| gt 2) are indicated by red bold font Empty cells

denote analytes for which either results or measurement uncertainties were not

reported

21

Table 9 Compilation of zeta-scores calculated from the ldquoresults for proficiency assessment

reported by the OCLs for test material St Johns wort the reported measurement

uncertainty and the uncertainty of the analyte content of the test material zeta-scores

outside the satisfactory range (|zeta| gt 2) are indicated by red bold font yellow

highlighted cells ugtUf Empty cells denote analytes for which results were not

reported

22

94 Evaluation of the influence of analysis method

The information on the applied analysis method (ANNEX 11) was used to group the performance

indicators attributed to the reported results according to the applied analysis technique Two groups of

data were formed They represent results obtained by high performance liquid chromatography-

fluorescence detection (HPLC-FD about 40 ) and by gas chromatography-mass spectrometry (GC-

MS about 60 ) respectively Kernel density plots were constructed based on the respective z-scores

Figure 3A to 3E show the different Kernel density plots

Figure 3 Kernel density plots of z-scores attributed to the results for proficiency assessment

which were reported for the food supplements test sample Results were classified

according to the applied analysis technique

A) benz[a]anthracene

B) benzo[a]pyrene

23

Figure 3 continued

C) benzo[b]fluoranthene

D) chrysene

E) sum of four marker PAHs

The kernel density plots for BAP and BBF are almost identical and centred at or close to a z-score

value of zero as can be seen in Figure 3B and 3C Therefore bias introduced by the type of analysis

method is unlikely for these two analytes The situation is different for BAA and CHR In case of

BAA the kernel density distribution of z-scores based on HPLC-FD is shifted to negative values

24

(Figure 3A) which indicates a tendency to underestimate the BAA content in this particular

samplematrix when applying HPLC-FD This might be reasoned by separationintegration problems

between BAA and CHR peaks A few participants (from the HPLC-FD subgroup) that got negative

z-scores for BAA overestimated the CHR content This caused the slight shift of the density trace for

HPLC-FD towards negative values Removing this data from the evaluation would result in a density

trace with the mode at zero

The opposite is the case for CHR (Figure 3D) The density distribution of the z-scores attributed to

HPLC-FD data is centred almost at zero However the distribution for data based on GC-MS is shifted

to positive values indicating strong positive bias This bias might be caused by a lack of selectivity of

the analysis method The test material contains triphenylene which is isobaric to chrysene

Consequently it cannot be distinguished from chrysene by means of mass spectrometry These two

compounds have to be separated chromatographically which is not easy They coelute on unpolar and

on most mid-polar capillary columns Columns that allow their separation were commercialised only

recently and might not be available in all laboratories Therefore laboratories which determine the

four EU marker PAHs by means of GC-MS shall pay attention to the chromatographic separation of

triphenylene and chrysene A lack of chromatographic resolution might lead to strong positive bias of

GC-MS methods and to false conclusions on the marketability of the tested sample

The chromatographic separation of chrysene and triphenylene is not a problem with HPLC which is

reflected in the position of the density plot for chrysene However attention has to be paid in HPLC-

FD analysis to a proper clean up of the sample extract in order to eliminate interferences that show

similar retention as BAA and CHR

95 Evaluation of the influence of calibration

The participants received together with the food supplement test sample two standard solutions in

depending of their choice either acetonitrile or toluene The analyte content of one solution was

disclosed to the participants whereas the content of the other solution was kept undisclosed The

content values of the latter are presented in Table 5 Analysis results for the solution with undisclosed

content had to be reported to the PT organiser The respective data are presented in Annex 9

Percent deviations of the reported results from the assigned values were calculated for both the food

supplement test sample and the standard solution with undisclosed analyte content Youden plots were

prepared in order to visualize the influence of instrument calibration on the results reported for the

food supplement sample Two plots are discussed in the following The whole set of Youden plots are

given in Annex 9

25

Figure 4 Youden plot of the percent deviations of values reported for benzo[a]pyrene from the

assigned values of the food supplement test material and the standard solution in

toluene with undisclosed content

Figure 4 reveals that a number of participants reported results with similar percent deviations from the

assigned values of both the results for the food supplement material and the standard solution in

toluene with undisclosed content This correlation indicates that over- respectively underestimation of

the analyte content of the food supplement material might be caused by biased instrument calibration

This finding underpins the importance of thorough calibration standard preparation However the data

was not in all cases conclusive as some laboratories performed well for the food supplement samples

but reported biased results for the standard solution with undisclosed content (eg Figure 4 participants

3620 and 5305) This fact cannot be explained yet

Figure 5 presents the respective Youden plot for chrysene in the food supplement test material and the

standard solution in toluene with undisclosed content From this graph it can be concluded that the

overestimation of the chrysene content of the food supplement material was not caused by instrument

calibration The results for the solvent solution of many laboratories which underperformed for the

26

food supplement sample agreed well with the assigned value This supports the conclusion that the

overestimation is caused by the determination of an interferent most probably triphenylene

Figure 5 Youden plot of the percent deviations of values reported for chrysene from the

assigned values of the food supplement test material and the standard solution in

toluene with undisclosed content

96 Evaluation of compliance with legislation

The method performance data reported for the four marker PAHs were evaluated for compliance with

the provisions given in Commission Regulation (EC) No 8362011 Table 18 in ANNEX 10 gives an

overview on the results of the evaluation Data were not reported in case of empty cells Non-

compliant data are highlighted in Table 18 It should be noted that Regulation (EU) No 8362011

specifies LOD and LOQ with two decimals This was not respected by all participants which

hampered the compliance assessment

27

In summary it can be stated that the majority of participants reported method performance

characteristics for the determination of the four marker PAHs in the food supplement test sample that

are compliant with legislation However some laboratories need to improve their analysis methods

The laboratories in question are urged to adapt to the provisions given in the new legislation

10 Follow-up actions for underperforming laboratories

All laboratories that got questionable or non-satisfactory performance ratings are urged to perform

root cause analysis and to implement corrective actions

As agreed during the EU-RL PAH workshop 2011 the EU-RL PAH will set up follow-up measures in

due time for all NRLs that received for at least one of the four PAHs (BAA BAP BBF and CHR) z-

scores gt 3 as required by Regulation (EC) 8822004 and by the Protocol for management of

underperformance in comparative testing andor lack of collaboration of National Reference

Laboratories (NRLs) with European Union reference laboratories (EU-RLs) activities

These laboratories shall perform as an immediate action root-cause-analysis and shall report to the

EU-RL PAH in writing the identified cause for their underperformance and corrective actions they are

going to take Additionally they shall participate to an independent (non-EU-RL) proficiency test on

the determination of PAHs in food and shall communicate the outcome of this exercise to the EU-RL

PAH

28

11 Conclusions

Fourty seven participants reported results back to the EU-RL The performance of the majority of

participants was good In total 153 out of 225 attributed z-scores were below an absolute value of two

which equals to almost 68 However this percentage is much below the percentages of satisfactory

performance gained in previous studies This might be the consequence of the higher level of difficulty

with respect to matrix composition In addition bias can be concluded from the pattern of performance

indicators of some laboratories

In general the determination of chrysene caused most difficulties to the participants

zeta-Scores were calculated besides z-scores They indicate the agreement of the reported result with

the assigned value with respect to the stated measurement uncertainty The outcome of this rating was

worse than for the z-scores which indicates that the measurement uncertainty estimates were not

realistic Therefore participants underperforming with regard to zeta scores are urged to adapt their

measurement uncertainty statements

The great majority of participants in this inter-laboratory comparison applied analytical methods which

complied with EU legislation with regard to performance characteristics

12 Acknowledgements

The organisers would like to thank Mrs Beatriz de la Calle and Mr Franz Ulberth (all from IRMM

Geel Belgium) for their accurate revision of this report Mr Hakan Emteborg (from IRMM) for

ampouling of the standard solutions and all NRLs and OCLs for their cooperation

29

13 References

1 EU COMMISSION REGULATION (EC) No 7762006 of 23 May 2006 amending Annex VII to Regulation

(EC) No 8822004 of the European Parliament and of the Council as regards Community reference

laboratories Official Journal of the European Union 2006 L 136 p 3-8

Available from

httpeur-lexeuropaeuLexUriServLexUriServdouri=OJL200613600030008ENPDF

2 EU Regulation (EC) No 8822004 of the European Parliament and of the Council of 29 April 2004 on

official controls performed to ensure the verification of compliance with feed and food law animal health

and animal welfare rules Official Journal of the European Communities 2004 L191 p 1-52 Available

from

httpeur-lexeuropaeuLexUriServLexUriServdouri=OJL200419100010052ENPDF

3 EU Opinion of the Scientific Committee on Food on the risks to human health of Polycyclic Aromatic

Hydrocarbons in food (2002) Available from

httpeuropaeuintcommfoodfsscscfout153_enpdf

4 International Agency for Research on Cancer IARC Monographs on the Evaluation of

Carcinogenic Risks to Humans Some Non-heterocyclic Polycyclic Aromatic Hydrocarbons and Some

Related Exposures Volume 92 (2010) Available from

httpmonographsiarcfrENGMonographsvol92mono92pdf

5 EU Commission Regulation (EC) No 3332007 of 28 March 2007 laying down the methods of sampling

and analysis for the official control of the levels of lead cadmium mercury inorganic tin 3-MCPD and

benzo(a)pyrene in foodstuffs Official Journal of the European Union 2007 L 88 p 29-38 Available

from

httpeur-lexeuropaeuLexUriServLexUriServdouri=OJL200708800290038ENPDF

6 EU Commission Regulation (EC) No 18812006 of 19 December 2006 setting maximum levels for certain

contaminants in foodstuffs Official Journal of the European Union 2006 L 364 p 5-24 Available from

httpeur-lexeuropaeuLexUriServLexUriServdouri=OJL200636400050024ENPDF

7 EFSA Scientific Opinion of the Panel on Contaminants in the Food Chain on a request from the European

Commission on Polycyclic Aromatic Hydrocarbons in Food The EFSA Journal 2008 724 p 1-114

Available from

httpwwwefsaeuropaeuEFSAefsa_locale-1178620753812_1211902034842htm

30

8 EU Commission Regulation (EU) No 8352011 amending Regulation (EC) No 18812006 as regards

maximum levels for polycyclic aromatic hydrocarbons in foodstuffs Official Journal of the European

Union 2011 L 215 p 4-8

httpeur-lexeuropaeuLexUriServLexUriServdouri=OJL201121500040008ENPDF

9 EU Commission Regulation (EU) No 8362011 amending Regulation (EC) No 3332007 laying down the methods of sampling and analysis for the official control of the levels of lead cadmium mercury inorganic tin 3-MCPD and benzo(a)pyrene in foodstuffs Official Journal of the European Union 2011 L 215 p 9-16 httpeur-lexeuropaeuLexUriServLexUriServdouri=OJL201121500090016ENPDF

10 ISOIEC 170432010 (2010) Conformity assessment -- General requirements for proficiency testing

providers Geneva Switzerland

11 Thompson M Ellison SLR and Wood R The International Harmonized Protocol for the Proficiency

Testing of Analytical Chemistry Laboratories Pure Appl Chem (2006) 78(1) p 145ndash196

Available from httpmediaiupacorgpublicationspac2006pdf7801x0145pdf

31

14 ANNEXES

ANNEX 1 ndash Announcement of the PT on the IRMM webpage

ANNEX 2 ndash Announcement via e-mail and invitation

ANNEX 3 ndash Announcement of sample dispatch

ANNEX 4 ndash Technical specifications of the calibration solutions

ANNEX 5 ndash Homogeneity and stability of the test material

ANNEX 6 ndash Acknowledgement of sample receipt

ANNEX 7 ndash Questionnaire on details of analysis method

ANNEX 8 ndash Results reported for the food supplement sample

ANNEX 9 ndash Results reported for the standard solutions with undisclosed analyte content

ANNEX 10 ndash Method performance characteristics reported by participants

ANNEX 11 ndash Details of analysis methods reported by participants

32

ANNEX 1 ndash Announcement of the PT on the IRMM webpage

33

ANNEX 2 - Announcement via e-mail and invitation

34

ANNEX 3 - Announcement sample dispatch

Dear MadameSir

The test samples for the proficiency test on the determination of four PAHs in a food supplement (dry extract of St Johns wort) were

dispatched today

Explanation on the design of the study as well as instructions for sample storage and for reporting of results are in the parcel You will

also find your participant key in the parcel which is required for reporting of results

Please check the parcel after arrival for completeness and report the receipt of the test samples as well as their status to us by applying

the sample receipt form which is in the parcel

You are kindly requested to inform us by email if you do not receive the parcel by end of this week

My colleague Donata Lerda will then take care of it

With best regards

Thomas Wenzl

35

ANNEX 4 - Specifications of standard solutions with disclosed concentration

36

ANNEX 4 - continued

37

ANNEX 5 - Homogeneity and stability of the test material

Homogeneity of the test material

Benz[a]anthracene - BAA

Benzo[a]pyrene - BAP

38

ANNEX 5 - continued

Benzo[b]fluoranthene - BBF

Chrysene - CHR

39

ANNEX 5 - continued

Stability of the test material

Table 10 Content of the four target analytes in test materials stored under different

temperatures

BaA BaP BbF CHR

microgkg microgkg microgkg microgkg

Content 31 16 31 69 Storage at room temperature

U (k=2) 05 02 05 17

Content 31 15 29 68 Storage at reference temperature

U (k=2) 04 03 04 10

The applied capillary column does not separate CHR from triphenylene

40

ANNEX 6 ndash Documents sent to participants

41

ANNEX 6 - continued

42

ANNEX 7 Questionnaire on details of analysis method

43

ANNEX 7 - continued

44

ANNEX 7 - continued

45

ANNEX 8 - Results reported by participants for the food supplements test sample Table 11 Analysis results reported by the participants for the content of benz[a]anthracene (BAA) in the food supplements test material Values are presented as reported

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

3600 31 29 29 3 05 2

3602 199 193 178 19 021

3603 253 253 246 251 022 2

3604 199 209 23 213 071 2

3605 301 3 271 291 076 2

3606 302 288 278 289 064 2

3607 456 416 443 438 096 2

3608 796 695 651 714 149 2

3610 182 192 214 196 039 2

3611 414 504 488 469 96 2

3612 3 34 28 31 06 2

3613 32 33 34 33 07 2

3614 182 182 191 185 037 2

3615 3884 3822 3565 376 0752 2

3616 2 2 2 2 01 2

3617 366 37 35 362 046 2

3618 32 32 32 32 05 2

3619 299 305 297 299 048 2

3620 304 294 302 302 045 2

3621 29 26 29 28 04 2

3622 343 325 318 322 052 2

3623 35 3 33 33 07 2

3624

3626 318 31 314 314 126 2

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

5301 31 3 32 31 06 2

5302 314 319 315 316 063 2

5303 3102 2716 2822 2876 27 62

5304 261 263 26 261 57 2

5305 23 22 26 24 20

5306 22 23 23 22 11 2

5307 488 461 317 461

5308 168633 168544 1562025 1644598

5309 16 19 16 17

5310 3 304 311 305 015

5311 477 452 448 459 15 2

5312 506 63 494 543 134 2

5313 353 301 404 353

5314 431 373 378 394 5 096

5315 596 531 51 545 11 2

5316 224 231 235 235 022 2

5317 216 23 221 222 007 2

5318 064 07 067 01 2

5319 111 184 146 147 029 2

5320 515 525 564 534 48

5321 21 5322 13 87 9 87 35 2

5323 38 33 35 33 16 2

46

Figure 6 Results of replicate determinations (indicated by triangles) of benz[a]anthracene (BAA) in the food supplement test material

Horizontal blue lines represent the arithmetic mean value of replicate measurements and blue bars the reported expanded measurement uncertainty (k=2) The assigned value is

plotted as dotted line Red solid lines mark the range of satisfactory performance (|z|le2) Mean values are given for results outside the displayed data range

ProLab 2011

Laboratory

53

1853

1953

0853

0936

1436

0236

1036

1653

2136

0453

1753

0653

1653

0536

0353

0436

2153

0336

0636

0536

0036

2036

1953

1036

1253

0136

2653

0236

1836

2336

2236

1353

1353

2336

1736

1553

1453

0736

0753

1153

2053

1253

1536

0853

2236

11

an

aly

te c

on

ten

t (micro

gk

g)

10

95

9

85

8

75

7

65

6

55

5

45

4

35

3

25

2

15

1

05

0

102

3 468

7

Sample St Johns wort extract replicatesMeasurand Benz[a]anthraceneNo of laboratories 46

Assigned value 298 microgkg (Reference value)Target sd 062 microgkgTolerance limits 174 - 422 microgkg (|Z score| lt 200)

Assig

ned va

lue

Limit of tolerance

Limit of tolerance

47

Table 12 Analysis results reported by the participants for the content of benzo[a]pyrene (BAP) in the food supplements test material Values are presented as reported

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

3600 16 17 17 17 03 2

3602 118 123 13 124 015

3603 165 162 157 161 015 2

3604 134 154 139 143 04 2

3605 164 166 172 167 057 2

3606 181 159 161 167 031 2

3607 279 266 279 274 055 2

3608 57 687 461 573 226 2

3610 226 216 183 206 042 2

3611 289 325 311 308 36 2

3612 24 22 2 22 05 2

3613 17 18 19 18 05 2

3614 08 08 08 08 016 2

3615 1545 1393 1425 145 029 2

3616 16 16 14 15 03 2

3617 177 171 158 169 023 2

3618 2 2 2 2 02 2

3619 207 207 206 207 075 2

3620 165 169 163 166 017 2

3621 44 47 49 47 05 2

3622 212 208 222 213 038 2

3623 2 21 22 21 03 2

3624 18 178 182 18 031 2

3626 172 173 178 174 07 2

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

5301 18 18 18 18 04 2

5302 188 19 187 188 038 2

5303 1691 1426 149 1535 19 80

5304 145 142 15 146 69 2

5305 15 17 18 16 15

5306 16 17 17 16 08 2

5307 164 157 153 157

5308 102669 109324 109035 107009

5309 1 1 12 11

5310 122 122 124 123 006

5311 199 184 215 2 15 2

5312 258 248 239 248 05 2

5313 199 178 245 207

5314 174 171 172 172 11 0948

5315 253 248 213 238 05 2

5316 177 171 173 17 016 2

5317 255 277 276 269 012 2

5318 114 12 117 02 2

5319 19 228 231 216 043 2

5320 303 3 344 316 78

5321 12 12 12 12

5322 13 17 24 17 35 2

5323 2 2 19 2 1 2

48

Figure 7 Results of replicate determinations (indicated by triangles) of benzo[a]pyrene (BAP) in the food supplement test material

Horizontal blue lines represent the arithmetic mean value of replicate measurements and blue bars the reported expanded measurement uncertainty (k=2) The assigned value is

plotted as dotted line Red solid lines mark the range of satisfactory performance (|z|le2) Mean values are given for results outside the displayed data range

ProLab 2011

Laboratory

36

1453

0953

0853

1853

2153

1036

0236

0436

1553

0436

1653

0353

0736

0336

2036

0053

0553

0636

0636

0536

1753

1453

1636

2636

1336

2453

0153

2253

0253

2353

1136

1836

1953

1336

1036

2336

2253

1936

1253

1553

1253

1736

0753

2036

2136

0836

11

an

aly

te c

on

ten

t (micro

gk

g)

55

5

45

4

35

3

25

2

15

1

05

0

573

308

3

Sample St Johns wort extract replicatesMeasurand Benzo[a]pyreneNo of laboratories 48

Assigned value 165 microgkg (Reference value)Target sd 036 microgkgTolerance limits 093 - 237 microgkg (|Z score| lt 200)

Assig

ned va

lue

Limit of tolerance

Limit of tolerance

49

Table 13 Analysis results reported by the participants for the content of benzo[b]fluoranthene (BBF) in the food supplements test material Values are presented as reported

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

3600 32 3 3 31 09 2

3602 173 175 16 17 017

3603 265 252 25 256 025 2

3604 116 108 105 11 028 2

3605 286 225 232 247 074 2

3606 294 273 261 276 046 2

3607 579 579 544 567 113 2

3608 103 1201 876 1036 326 2

3610 173 196 204 191 038 2

3611 546 618 565 576 75 2

3612 3 28 28 29 06 2

3613 28 31 32 31 06 2

3614 135 111 135 127 022 2

3615 4187 3678 4111 4 08 2

3616 2 2 2 2 01 2

3617 33 27 299 035 2

3618 31 31 31 31 05 2

3619 326 324 312 326 064 2

3620 277 28 272 276 069 2

3621 26 25 23 25 04 2

3622 346 336 333 34 052 2

3623 26 28 29 28 02 2

3624

3626 288 28 307 292 117 2

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

5301 29 3 29 29 06 2

5302 297 3 296 298 06 2

5303 2705 2444 2608 2586 22 78

5304 245 254 26 253 85 2

5305 23 23 26 24 15

5306 42 43 4 42 21 2

5307 221 216 241 221

5308 232169 246654 240722 229848

5309 19 22 19 2 0

5310 3 315 325 313 016

5311 262 277 304 281 15 2

5312 27 289 337 299 075 2

5313 321 284 378 328

5314 423 404 385 404 34 083

5315 42 506 416 444 09 2

5316 265 267 277 273 018 2

5317 185 197 195 192 006 2

5318 276 281 279 04 2

5319 396 389 403 396 079 2

5320 623 618 635 625 14

5321 31

5322 24 33 49 33 35 2

5323 38 45 35 45 27 2

50

Figure 8 Results of replicate determinations (indicated by triangles) of benzo[b]fluoranthene (BBF) in the food supplement test material

Horizontal blue lines represent the arithmetic mean value of replicate measurements and blue bars the reported expanded measurement uncertainty (k=2) The assigned value is

plotted as dotted line Red solid lines mark the range of satisfactory performance (|z|le2) Mean values are given for results outside the displayed data range

ProLab 2011

Laboratory

36

0436

1436

0236

1053

1736

1653

0953

0753

0853

0536

2136

0553

0436

0353

0353

1636

0636

2036

2353

1853

1136

1236

2653

0153

0253

1236

1736

1336

0036

1853

2153

1036

1953

1336

2253

2253

2353

1936

1553

1453

0653

1536

0753

2036

0836

11

an

aly

te c

on

ten

t (micro

gk

g)

75

7

65

6

55

5

45

4

35

3

25

2

15

1

05

0

103

6

576

3

Sample St Johns wort extract replicatesMeasurand Benzo[b]fluorantheneNo of laboratories 46

Assigned value 292 microgkg (Reference value)Target sd 060 microgkgTolerance limits 172 - 412 microgkg (|Z score| lt 200)

Assig

ned va

lue

Limit of tolerance

Limit of tolerance

51

Table 14 Analysis results reported by the participants for the content of chrysene (CHR) in the food supplements test material Values are presented as reported

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

3600 5 49 5 5 07 2

3602 32 304 29 304 036

3603 462 469 447 459 048 2

3604 438 428 42 429 119 2

3605 365 41 381 386 085 2

3606 494 411 45 451 124 2

3607 897 774 787 819 18 2

3608 986 887 1168 1014 284 2

3610 418 392 462 424 085 2

3611 833 893 87 865 61 2

3612 6 64 58 61 12 2

3613 41 39 4 4 08 2

3614 272 254 288 271 054 2

3615 605 6028 5964 601 12 2

3616 47 45 49 47 05 2

3617 445 431 389 422 065 2

3618 45 45 45 45 06 2

3619 738 747 72 738 118 2

3620 463 472 446 46 092 2

3621 53 49 51 51 06 2

3622 478 513 505 488 08 2

3623 73 73 72 72 05 2

3624

3626 63 622 634 628 251 2

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

5301 49 49 49 49 1 2

5302 687 683 666 679 136 2

5303 6777 6181 6382 6441 24 70

5304 592 581 6 591 85 2

5305 6 58 64 61 20

5306 49 5 51 49 25 2

5307 524 5 574 524

5308 211488 216782 221146 213243

5309

5310 372 393 394 386 019

5311 43 459 422 437 15 2

5312 934 943 981 953 191 2

5313 793 73 99 838

5314 636 561 565 588 18 0818

5315 547 583 499 542 11 2

5316 292 286 285 282 06 2

5317 52 598 517 545 046 2

5318 368 345 357 05 2

5319 449 471 474 465 116 2

5320 76 924 818 834 99

5321 33

5322 7 7 76 7 35 2

5323 87 73 77 73 37 2

52

Figure 9 Results of replicate determinations (indicated by triangles) of chrysene (CHR) in the food supplement test material

Horizontal blue lines represent the arithmetic mean value of replicate measurements and blue bars the reported expanded measurement uncertainty (k=2) The assigned value is

plotted as dotted line Red solid lines mark the range of satisfactory performance (|z|le2) Mean values are given for results outside the displayed data range

ProLab 2011

Laboratory

53

0836

1453

1636

0253

2153

1836

0553

1036

1336

1736

1036

0453

1136

1836

0636

0336

2053

1936

1653

0136

0036

2253

0636

2153

0753

1553

1753

1453

0436

1536

1253

0536

2653

0353

0253

2236

2336

1953

2336

0753

2053

1353

1236

0836

11

an

aly

te c

on

ten

t (micro

gk

g)

13

12

11

10

9

8

7

6

5

4

3

2

1

0

865

3

Sample St Johns wort extract replicatesMeasurand ChryseneNo of laboratories 45

Assigned value 407 microgkg (Reference value)Target sd 083 microgkgTolerance limits 241 - 573 microgkg (|Z score| lt 200)

Assig

ned va

lue

Limit of tolerance

Limit of tolerance

53

Table 15 Analysis results reported by the participants for the sum of the four marker PAHs in the food supplements test material Values are presented as reported

Laboratory code

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg

3600 128 24 2

3602 79 126

3603 1127 096 2

3604 893 258 2

3605 1091 305 2

3606 1184 238 2

3607 2098 42 2

3608 3336 325 2

3610 1138 17 2

3611 2218 259 2

3612 143 29 2

3613 122 2 2

3614 663 129 2

3615 152 304 2

3616 102 03 2

3617 1252 169 2

3618 128 18 2

3619 157 305 2

3620 1204 12 2

3621 151 19 2

3622 1361 115 2

3623 154 09 2

3624

3626 1409 56 2

Laboratory code

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg

5301 126

5302 37 074 2

5303 13438 92

5304 1251 85 2

5305 125 20

5306 129 65 2

5307 1363

5308 7245598

5309

5310 1127 056

5311 1367 15 2

5312 2042 408 2

5313 1726

5314 1558

5315 177 35 2

5316 96 073 2

5317 1229 066 2

5318 82 05 2

5319 1225 267 2

5320 2309 41

5321

5322 207 35 2

5323 171 103 2

54

ANNEX 9 Results reported for the standard solutions with undisclosed analyte content Table 16 Analysis results reported by the participants for the four marker PAHs in the standard solution in toluene with undisclosed analyte

content Values are presented as reported U measurement uncertainty k coverage factor

benz[a]anthracene benzo[a]pyrene benzo[b]fluoranthene chrysene Laboratory

code Unit Result U k Result U k Result U k Result U k 3600 microgl 38 67 2 25 45 2 211 68 2 275 38 2

3605 microgl 367 184 2 2482 124 2 2592 13 2 235 118 2

3606 microgkg 3763 176 2 2609 142 2 2721 118 2 2539 134 2

3607 microgkg 3706 815 2 2824 565 2 2591 518 2 2587 569 2

3612 microgl 38 08 2 237 11 2 244 18 2 257 02 2

3614 microgl 324 65 2 209 38 2 215 37 2 21 42 2

3616 microgkg 318 07 2 224 14 2 231 26 2 216 1 2

3618 microgkg 417 17 2 284 08 2 292 06 2 297 03 2

3619 microgkg 47 742 2 274 468 2 228 387 2 274 434 2

3620 microgl 495 74 2 294 29 2 321 8 2 321 64 2

3621 microgl 241 22 2 143 13 2 188 16 2 145 14 2

3623 microgkg 476 25 2 311 53 2 269 3 2 297 03 2

3626 microgl 359 38 2 235 23 2 238 27 2 24 18 2

5301 microgkg 426 29 299 289

5302 microgkg 436 87 2 296 59 2 296 59 2 289 58 2

5303 microgl 3721 27 102 22838 19 99 23756 22 99 25486 24 98

5304 microgkg 38 29 2 225 26 2 257 37 2 258 52 2

5305 microgkg 305 225 245 29

5306 microgkg 316 63 2 247 49 2 65 13 2 22 44 2

5307 microgkg 3658 2559 241 2414

5308 microgkg 16854 109324 246654 216782

5311 microgl 374 20 2 279 15 2 268 15 2 272 15 2

5313 microgl 3408 2165 2275 2373

5314 microgkg 395 5 2875 11 3614 34 2905 18

5320 microgl 3267 368 1986 571 28 683 2427 512

5322 microgl 282 35 2 225 35 2 241 35 2 21 35 2

55

Figure 10 Youden plots of the percent deviation of the reported values from the assigned values for the food supplement test sample and the

standard solution in toluene with undisclosed content

(The identity of the respective measurand is given above the figure Results exceeding a difference of more than 100 were omitted)

56

Figure 10 - continued

57

Table 17 Analysis results reported by the participants for the four marker PAHs in the standard solution in acetonitrile with undisclosed

analyte content

Values are presented as reported U measurement uncertainty k coverage factor

benz[a]anthracene benzo[a]pyrene benzo[b]fluoranthene chrysene

Laboratory

code Unit Result U k Result U k Result U k Result U k

3602 microgkg 3675 247 247 244

3603 microgkg 358 086 2 236 028 2 235 028 2 236 057 2

3604 microgl 475 29 2 19 12 2 194 13 2 258 12 2

3608 microgl 3623 078 2 2385 023 2 2343 18 2 2296 092 2

3610 microgkg 369 185 2 25 125 2 251 126 2 244 122 2

3611 microgkg 48 02 2 323 02 2 328 04 2 317 03 2

3613 microgl 332 31 2 261 3 2 249 25 2 239 24 2

3615 microgl 415 2635 2729 2689

3617 microgkg 37 09 2 244 06 2 284 07 2 239 06 2

3622 microgl 37 12 2 25 23 2 257 21 2 238 1 2

3624 microgkg 402 104 2 255 084 2 265 096 2 253 07 2

5306 microgkg 563 113 2 371 74 2 359 72 2 333 67 2

5308 microgkg 168633 102669 232169 211488

5309 microgl 402 268 27

5310 microgl 315 158 25 125 25 125 288 144

5312 microgl 428 043 2 282 028 2 272 027 2 275 028 2

5315 microgkg 387 08 2 256 05 2 251 05 2 253 05 2

5316 microgl 3839 342 2 2485 191 2 2559 168 2 239 51 2

5317 microgl 2466 043 2 2212 034 2 2472 016 2 2146 068 2

5319 microgl 414 41 2 279 28 2 268 27 2 26 31 2

5321 microgkg 476 316 326 328

5322 microgl 2547 35 2

5323 microgkg 405 203 2 239 119 2 255 153 2 201 11 2

58

Figure 11 Youden plots of the percent deviation of the reported values from the assigned values for the food supplement test sample and the

standard solution in acetonitrile with undisclosed content

(The identity of the respective measurand is given above the figure Results exceeding a difference of more than 100 were omitted)

59

Figure 11 - continued

60

ANNEX 10 Table 18 Method performance parameters reported by the participants

Laboratory code

Analyte LOD LOQ Recovery Analyte LOD LOQ Recovery

microgkg microgkg microgkg microgkg 3600 BaA 03 06 978 BaP 03 07 9483602 BaA 007 021 926 BaP 005 015 993603 BaA 0011 05 987 BaP 0005 051 9553604 BaA 02 04 86 BaP 02 04 883605 BaA 0025 005 118 BaP 0025 005 1083606 BaA 001 003 65 BaP 001 003 703607 BaA 021 071 74 BaP 027 089 943608 BaA 016 048 8213 BaP 015 046 83683610 BaA 02 05 85 BaP 005 02 853611 BaA BaP 3612 BaA 1 117 BaP 1 1203613 BaA 01 03 90 BaP 01 03 903614 BaA 02 05 96 BaP 02 05 933615 BaA 005 015 110 BaP 005 015 1053616 BaA 01 05 108 BaP 01 05 753617 BaA 018 037 405 BaP 009 018 3873618 BaA 01 03 63 BaP 01 03 593619 BaA 005 005 59 BaP 033 033 543620 BaA 0009 0018 64 BaP 0005 001 553621 BaA 007 02 84 BaP 007 02 923622 BaA 01 03 1101 BaP 01 03 9853623 BaA 02 08 105 BaP 02 08 1013624 BaA BaP 01 033 8513625 BaA BaP 3626 BaA BaP 5301 BaA 003 01 102 BaP 003 01 1005302 BaA 01 03 78 BaP 01 03 865303 BaA 003 01 62 BaP 003 01 805304 BaA 1 1 70 BaP 1 1 955305 BaA 02 05 85 BaP 02 05 855306 BaA 03 1 699 BaP 03 1 905307 BaA 6537 BaP 81165308 BaA 0088 0265 99 BaP 00262 00785 1005309 BaA 01 08 92 BaP 01 08 925310 BaA 01 03 BaP 01 03 905311 BaA 002 005 80 BaP 002 004 915312 BaA 03 09 105 BaP 03 09 1055313 BaA 04 08 BaP 04 08 5314 BaA 001 002 96 BaP 001 002 9485315 BaA 01 03 50 - 100 BaP 01 03 50 - 100 5316 BaA 002 011 827 BaP 004 019 10445317 BaA 05 1 76 BaP 05 1 755318 BaA BaP 5319 BaA 009 029 951 BaP 011 041 995320 BaA 12 18 946 BaP 06 1 8345321 BaA 03 05 BaP 05 1 5322 BaA 1 2 100 BaP 1 2 1005323 BaA 26 BaP 26

61

ANNEX 10 Table 18 - continued

Laboratory code Analyte LOD LOQ Recovery Analyte LOD LOQ Recovery

microgkg microgkg microgkg microgkg 3600 BbF 03 05 96 CHR 02 03 10153602 BbF 015 045 947 CHR 003 009 9193603 BbF 0014 05 1012 CHR 0004 05 10033604 BbF 02 04 85 CHR 02 04 873605 BbF 005 01 94 CHR 0025 005 863606 BbF 001 003 70 CHR 001 003 653607 BbF 018 06 100 CHR 014 028 943608 BbF 015 046 7979 CHR 015 045 71253610 BbF 005 02 85 CHR 02 05 853611 BbF CHR 3612 BbF 1 118 CHR 1 1173613 BbF 01 03 90 CHR 01 03 903614 BbF 01 04 96 CHR 02 05 953615 BbF 008 025 109 CHR 003 01 1083616 BbF 01 05 78 CHR 01 05 723617 BbF 035 07 38 CHR 012 024 4163618 BbF 01 03 62 CHR 01 03 623619 BbF 016 016 63 CHR 01 01 593620 BbF 0003 0006 55 CHR 001 002 653621 BbF 007 02 103 CHR 007 02 893622 BbF 02 06 963 CHR 03 09 1043623 BbF 02 08 106 CHR 02 08 1193624 BbF CHR 3625 BbF CHR 3626 BbF CHR 5301 BbF 003 01 96 CHR 003 01 1145302 BbF 01 03 84 CHR 01 03 835303 BbF 003 01 78 CHR 003 01 705304 BbF 1 1 86 CHR 1 1 705305 BbF 02 05 90 CHR 02 05 955306 BbF 03 1 1435 CHR 03 1 1185307 BbF 8116 CHR 65375308 BbF 0079 0238 99 CHR 009 026 1005309 BbF 01 08 92 CHR 5310 BbF 07 2 CHR 01 03 5311 BbF 002 004 95 CHR 002 004 895312 BbF 03 09 105 CHR 03 09 1055313 BbF 04 08 CHR 04 08 5314 BbF 001 002 83 CHR 001 002 8185315 BbF 02 05 50 -100 CHR 02 05 50 - 100 5316 BbF 005 024 814 CHR 001 007 9595317 BbF 05 1 73 CHR 05 1 785318 BbF CHR 5319 BbF 011 038 946 CHR 013 043 9675320 BbF 07 09 1194 CHR 23 32 10455321 BbF 05 1 CHR 05 1 5322 BbF 1 2 100 CHR 1 2 1005323 BbF 26 CHR 26

62

ANNEX 11 Table 19 Details of analysis method reported by the participants Data are presented as reported

Laboratory code

Analysis technique

Instrument calibration

Internal standards applied

Sample clean up

Details of sample

clean up

Extraction technique

Details of sample

extraction

3600 6) GC-MS

2) Internal standardisation with labelled substances

Deuterated PAH mix 9 (Ehrenstorfer)

1) Saponification 2) LiquidLiquid partitioning

D) Other Liquidliquid partitioning

3602 2) HPLC-UV-FLD

1) External calibration

3) Gel permeation chromatography

A) Pressurised liquid extraction (PLE)

3603 1) HPLC-FLD

1) External calibration

3) Gel permeation chromatography

D) Other 1 minute with dichloromethane in vortex

3604 2) HPLC-UV-FLD

2) Internal standardisation with labelled substances

DiP D14

5) Donor acceptor complex chromatography

D) Other LL

3605 1) HPLC-FLD

1) External calibration

3) Gel permeation chromatography

C) Soxhlet extraction

3606 7) GC-MSMS

2) Internal standardisation with labelled substances

BaP 13C4 CHR 13C6 BaA 13C6 BbF 13C6

4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

3607 6) GC-MS

2) Internal standardisation with labelled substances

BaA-d12 CHR-d12 BbF-d12 BkF-d12 BaP-d12 ICP-d12 BgP-d12 DiP-d14

1) Saponification

D) Other solvent partitioning

3608 1) HPLC-FLD

1) External calibration

6) Other Liquid extraction

D) Other

Extraction in a rotary agitator and centrifugation

3610 2) HPLC-UV-FLD

1) External calibration

4) Solid phase extraction

B) Sonication

3611

3612 6) GC-MS

2) Internal standardisation with labelled substances

chrysene-d12 dibenzo(ah)anthracene-d14 dibenzo(ai)pyrene-d14

4) Solid phase extraction

B) Sonication

3613 5) LC-MSMS

3) Internal standardisation with unlabelled substances

I use Chrysene-D12 3) Gel permeation chromatography

D) Other extraction with hexan

63

Table 19 - continued

Laboratory code

Analysis technique

Instrument calibration

Internal standards applied

Sample clean up Details of sample

clean up

Extraction technique

Details of sample

extraction

3614 6) GC-MS 1) External calibration

3) Gel permeation chromatography

B) Sonication

3615 1) HPLC-FLD

1) External calibration

6) Other PLE GPC

A) Pressurised liquid extraction (PLE)

3616 6) GC-MS

2) Internal standardisation with labelled substances

Benzo(a)pyrene-13C4 Benzo(a)anthracene-13C6 Benzo(b)fluoranthene-13C6 Chrysene-13C6

2) LiquidLiquid partitioning 4) Solid phase extraction

B) Sonication

3617 1) HPLC-FLD

1) External calibration

4) Solid phase extraction

C) Soxhlet extraction

3618 6) GC-MS

2) Internal standardisation with labelled substances

deuterated standards

1) Saponification 4) Solid phase extraction

D) Other Handshaken with cyclohexane

3619 6) GC-MS

2) Internal standardisation with labelled substances

C13 Labelled US EPA 16

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

D) Other Homegenisation amp saponiofication

3620 9) GC-HRMS

2) Internal standardisation with labelled substances

mix of deuterated PAHs (BAA-D12 CHR-D12 BAP-D12 BBF-D12)

3) Gel permeation chromatography 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

3621 6) GC-MS

2) Internal standardisation with labelled substances

Chrysene D12 Benzo(a)pyrene D12 Benzo(b)fluoranthene D12 Benzo(a)anthracene

2) LiquidLiquid partitioning 4) Solid phase extraction

B) Sonication

3622 1) HPLC-FLD

4) Standard addition method

4) Solid phase extraction

D) Other

Extraction with cyclohexane by heating under reflux

3623 7) GC-MSMS

2) Internal standardisation with labelled substances

C13 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

3624 2) HPLC-UV-FLD

1) External calibration

2) LiquidLiquid partitioning

D) Other hot solvent extraction

3626

64

Table 19 - continued

Laboratory code

Analysis technique

Instrument calibration

Internal standards

applied

Sample clean up

Details of sample

clean up

Extraction technique

Details of sample

extraction

5301 7) GC-MSMS

2) Internal standardisation with labelled substances

EACH SUBSTANCE C13

4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5302 7) GC-MSMS

2) Internal standardisation with labelled substances

Isotope labelled PAHs 13C for each analyte

4) Solid phase extraction

C) Soxhlet extraction

5303 7) GC-MSMS

3) Internal standardisation with unlabelled substances

HAP C13 4) Solid phase extraction

D) Other automate ASE200 DIONEX

5304 7) GC-MSMS

2) Internal standardisation with labelled substances

with C13 for each HAP

2) LiquidLiquid partitioning 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5305 7) GC-MSMS

2) Internal standardisation with labelled substances

C13 isotopiques internal standards

4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5306 7) GC-MSMS

2) Internal standardisation with labelled substances

C13 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5307 6) GC-MS

2) Internal standardisation with labelled substances

1) Saponification 2) LiquidLiquid partitioning

D) Other solvent extraction

5308 1) HPLC-FLD 1) External calibration

4) Solid phase extraction

B) Sonication

5309 1) HPLC-FLD 1) External calibration

4) Solid phase extraction

D) Other only SPE

5310 1) HPLC-FLD 1) External calibration

1) Saponification 2) LiquidLiquid partitioning 3) Gel permeation chromatography

C) Soxhlet extraction

5311 6) GC-MS

2) Internal standardisation with labelled substances

13 C Chrysene 13 C Benz(a)pyrene

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

B) Sonication

5312 1) HPLC-FLD

3) Internal standardisation with unlabelled substances

Benzo(b)chrysen

2) LiquidLiquid partitioning 4) Solid phase extraction

Quechers D) Other Quechers

65

Table 19 - continued

Laboratory code

Analysis technique

Instrument calibration

Internal standards applied

Sample clean up Details of sample

clean up

Extraction technique

Details of sample extraction

5313 6) GC-MS

2) Internal standardisation with labelled substances

Benz(a)anthracene D12 Benzo(b)fluranthene D12 Benzo(a)pyrene D12 Chrysene D12

1) Saponification 2) LiquidLiquid partitioning 3) Gel permeation chromatography

A) Pressurised liquid extraction (PLE)

5314 6) GC-MS

2) Internal standardisation with labelled substances

Benzo(a)pyren d12 2) LiquidLiquid partitioning

D) Other Extraction with

5315 1) HPLC-FLD

3) Internal standardisation with unlabelled substances

Benz(b)chrysen

1) Saponification 2) LiquidLiquid partitioning

D) Other shaker

5316 1) HPLC-FLD

1) External calibration

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

D) Other

LiquidLiquid extraction with cyclohexane and dimethylformamide

5317 7) GC-MSMS

1) External calibration

3) Gel permeation chromatography 6) Other

silica gel D) Other VDLUFA online method

5318

5319 1) HPLC-FLD

3) Internal standardisation with unlabelled substances

Benzo(b)chrysene 3) Gel permeation chromatography

A) Pressurised liquid extraction (PLE)

5320 6) GC-MS

3) Internal standardisation with unlabelled substances

22-binaphthyl

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

D) Other 3 hours refluxing

5321 1) HPLC-FLD

2) Internal standardisation with labelled substances

6-meacutethylchrysegravene

1) Saponification 2) LiquidLiquid partitioning

B) Sonication

5322 6) GC-MS

2) Internal standardisation with labelled substances

naftalene-d8 acenaphtalene-d10 phenanthrene-d10 chrysene-d12 perylene-d12 benzo(e)anthracene-d12

1) Saponification 2) LiquidLiquid partitioning 6) Other

clean-up with silica packed column

C) Soxhlet extraction

5323 1) HPLC-FLD

1) External calibration

4) Solid phase extraction 6) Other

Accelerated Solvent Extraction

A) Pressurised liquid extraction (PLE)

66

European Commission EUR 25245 EN ndash Joint Research Centre ndash Institute for Reference Materials and Measurements Title Report on the 8th inter-laboratory comparison organised by the European Union Reference Laboratory for Polycyclic Aromatic Hydrocarbons - Four marker PAHs in a dry extract of St Johns wort Author(s) Radoslav Lizak Szilard Szilagyi Philippe Verlinde and Thomas WENZL Luxembourg Publications Office of the European Union 2012 ndash 66 pp ndash 201 x 297 cm EUR ndash Scientific and Technical Research series ndash ISSN 1831-9424 ISBN 978-92-79-23337-1 doi 10278759387 Abstract The proficiency test here reported concerned the determination of the contents of four marker polycyclic aromatic hydrocarbons (PAHs) and of their sum in an food supplements test sample The set of marker PAHs consists of benz[a]anthacene benzo[a]pyrene benzo[b]fluoranthene and chrysene Participants to these PT were National Reference Laboratories for PAHs (NRLs-PAHs) and EU official food control laboratories The number of participants was in total 48

The PT was organised according ISO Standard 170432010

The test material used was a commercial dry extract of St Johns wort Participants also received a solution of the same PAHs either in acetonitrile or in toluene for checking their instrument calibration

The results from participants were rated with z-scores and zeta-scores About 68 of the reported results were attributed with z-scores with an absolute value of below two which is the threshold for satisfactory performance

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service of the European Commission the JRC functions as a reference centre of

science and technology for the Union Close to the policy-making process it serves

the common interest of the Member States while being independent of special

interests whether private or national

L

A-N

A-252

45-EN

-N

• 1 Summary
• 2 Introduction
• 3 Scope
• 4 Participating Laboratories
• 5 Time frame
• 6 Confidentiality
• 7 Test materials
• • 71 1Preparation of test materials
  • 72 Assigned values and standard deviation for proficiency assessment
  • 73 Homogeneity and stability
  • 74 Sample dispatch
  • • 8 Design of the proficiency test
    • 9 Evaluation of the results
    • • 91 2General
      • 92 21BEvaluation criteria
      • 93 Performance of participants
      • 94 Evaluation of the influence of analysis method
      • 95 Evaluation of the influence of calibration
      • 96 26Evaluation of compliance with legislation
      • • 10 Follow-up actions for underperforming laboratories
        • 11 1Conclusions
        • 12 1Acknowledgements
        • 13 References

10

Table 4 Analyte contents of the food supplement test material

Average of two

sessions bracketing calibration

External evaluation

Assigned value

and σP

Cont microgkg 298 297 298U microgkg 024 041 024P microgkg 062

Cont microgkg 157 174 165U microgkg 020 016 026P microgkg 036

Cont microgkg 292 289 292U microgkg 028 034 028P microgkg 060

Cont microgkg 424 389 407U microgkg 057 057 081P microgkg 083

Cont microgkg 1162U microgkg 093P microgkg 125

Sum PAH4

BAA

BAP

BBF

CHR

from chemical analysis Sum of the four analytes contents for the SUM

p standard deviation for proficiency assessment For the individual analytes is equal to the uncertainty function - Uf

according to Ref [9]

U expanded uncertainty of the assigned value (k=2)

The gravimetrical preparation concentrations were used as assigned values for the standard solutions

with undisclosed content Table 5 contains the assigned values and associated expanded uncertainties

of the standard solutions with undisclosed content

11

Table 5 Analyte contents of the standard solutions with undisclosed content

Content U (k=2) Content U (k=2) microgkg microgkg microgkg microgkg

benz[a ]anthracene 4317 039 4764 043benzo[c ]fluorene 7342 061 8052 067chrysene 2904 031 3179 034cyclopenta[cd ]pyrene 2558 050 2818 055benzo[a ]pyrene 2897 038 3186 042benzo[b ]fluoranthene 2926 026 3199 029benzo[ghi ]perylene 2214 023 2438 026benzo[j ]fluoranthene 4353 038 4763 041benzo[k ]fluoranthene 2143 017 2380 019dibenzo[ae ]pyrene 5735 084 6326 093dibenzo[ah ]anthracene 2215 032 2445 035dibenzo[ah ]pyrene 2169 029 2394 032dibenzo[ai ]pyrene 3691 100 4035 109dibenzo[al ]pyrene 4627 057 5104 063indeno[123-cd ]pyrene 3793 036 4176 0395-methylchrysene 8589 087 9443 096

Solution in toluene Solution in acetonitrile

Prep conc concentration from gravimetrical preparation U expanded uncertainty of the assigned value k coverage

factor

73 Homogeneity and stability

Homogeneity of the food supplements test sample was evaluated according to ISO Standard 13528

Ten units of the St Johns wort extract test material were selected randomly and analysed by isotope

dilution gas chromatography mass spectrometry (GC-MS) The test material was rated sufficiently

homogeneous (see ANNEX 5)

The contributions of the uncertainties of homogeneity (relative uncertainties below 1 ) to the total

uncertainties of the assigned values were found negligible and were therefore not considered in the

calculations

The stability of the test materials was evaluated applying an isochronous experimental set up This

comprised the storage of test samples both at room temperature and cooled in the fridge The content

values of the cooled sample were used as reference for the stability evaluation The materials were

stored from dispatch of test samples to the participants until expiry of the deadline for reporting of

results Afterwards the samples were analysed under repeatability conditions and results were

compared The content values of the cool-stored sample and the sample stored at room temperature

were not statistically significantly different Hence stability of the samples over the whole study period

can be assumed The analysis data are presented in ANNEX 5

12

The uncertainty of stability of the test material was considered negligible compared to the uncertainty

of the characterisation measurements Therefore this parameter was not taken into account in the

determination of the uncertainties of the assigned values

74 Sample dispatch

Samples were dispatched at room temperature Each participant received at least

- one ampoule of the solution of the 15+1 EU priority PAHs in the chosen solvent with disclosed

content (2 ml)

- one ampoule of the 15+1 EU priority PAHs in the chosen solvent with undisclosed content (2

ml)

- one screw cap vial with the food supplement test material

Each parcel contained a sample receipt form the outline of the study an appropriate material safety

data sheets and a unique code that was required for reporting of results The documents sent to the

participants are presented in ANNEX 6

8 Design of the proficiency test

The design of the PT foresaw triplicate analyses of the test sample and reporting of the individual

results of replicate analyses for the single analytes in the following denoted as FS_REP and

additionally a value for proficiency assessment in the following denoted as final value - FS_FIN

for both the single analytes and the sum of the four marker PAHs Both FS_REP results and FS_FIN

results had to be reported corrected for recovery (and recovery had to be stated in the questionnaire

which participants were asked to fill in together with other parameters of the method applied) the

latter had also to be accompanied by the respective expanded measurement uncertainty The FS_FIN

results were used for performance assessment

Besides analysis results participants were also asked to report details of the applied analysis method

and to provide answers to a questionnaire (see ANNEX 7 for the template and the compiled returned

questionnaires)

13

9 Evaluation of the results

The results reported by participants are listed in ANNEX 8

91 General

The most important evaluation parameter was the performance of the laboratories in the determination

of the target PAHs in the food supplement test material which was expressed by z-scores and zeta-

scores

The compliance of method performance characteristics specified by the participants with provisions

given in legislation was evaluated too

92 Evaluation criteria

The participants were requested to report for all analytes the results of replicate measurements and a

value for proficiency assessment which is the result they wish to be applied for the calculation of

performance indicators z-Scores and zeta-scores were attributed only to these results The individual

results of replicate analyses were not rated

z-Scores

z-scores were calculated based on the FS_FIN values Equation 1 presents the formula for calculation

of z-scores

Equation 1

P

assignedlab Xxz

where z refers to the z-score xlab to the reported ldquovalue for proficiency assessmentrdquo Xassigned to the

assigned value and σP to the standard deviation for proficiency testing

zeta-Scores

In addition to z-scores zeta-scores were calculated In contrast to z-scores zeta-scores describe the

agreement of the reported result with the assigned value within the respective uncertainties

Unsatisfactorily large zeta-scores might be caused by underestimated measurement uncertainties large

bias or a combination of both Zeta-scores were calculated according to Equation 3

14

Equation 3 22assignedlab

assignedlab

uu

Xxzeta

where zeta refers to the zeta-score xlab to the reported ldquovalue for proficiency assessmentrdquo Xassigned to

the assigned value ulab to the measurement uncertainty reported by the laboratory and uassigned to the

uncertainty of the assigned value

The performance of the laboratories was classified according to ISOIEC 170432010 [10] Following

scheme is applied for the interpretation of zeta scores and z-scores

|score| 20 = satisfactory performance

20lt|score| lt 30 = questionable performance

|score| ge 30 = unsatisfactory performance

93 Performance of participants

The 48 participants in the study reported in total 225 results for proficiency evaluation which equals to

about 94 of the 240 possible results About 68 of the reported results were rated as satisfactory

with regard to z-scores

Figure 1 and Figure 2 give an overview of the z-scores assigned to the respective results The larger

the triangles the larger were the differences to the assigned values Red triangles indicate z-scores

above an absolute value of three whereas yellow triangles represent z-scores in the questionable

performance range The corresponding score values are plotted next to the triangles About 40 of the

72 non-satisfactory results were reported by seven laboratories only eg the performance of

participant 3608 was not satisfactory for the majority of target analytes

It is also obvious that most problems were encountered in the determination of chrysene and the sum

of the four analytes Potential reasons for the difficulties related to chrysene are discussed further

down

The ideal situation would be that deviations from the assigned values of the results reported for the

individual analytes are small and randomly distributed around zero This will give a satisfactory

performance statement for the sum parameter Deviations of opposite sign could also cancel out and

lead to a satisfactory performance for the sum parameter (eg participants 5303 5304 and 5305)

However in most cases a single underperformance for one analyte led also to non-satisfactory rating

for the sum parameter

15

The numerical values of the calculated z-scores are compiled in Table 6 and Table 7 z-Scores with an

absolute value of above 2 are given in red bold font

The results of the data evaluation for the individual analytes are given in ANNEX 8

For each analyte the figure shows the individual analysis results of the three replicate determinations

The assigned value is shown as dotted line The blue boxes represent the expanded uncertainties as

reported by participants for the value for proficiency assessment The arithmetic mean of the results

of the individual participants is indicated in the blue boxes by a blue line The satisfactory performance

range is located between the two red lines

The individual results of the replicate measurements and the value for proficiency assessment with

its accompanying expanded measurement uncertainty (k=2) are listed in the tables in ANNEX 8 as

well

16

Figure 1 Graphical presentation of z-scores corresponding to the values for proficiency assessment

reported by the NRLs for the contents of BAA BAP BBF and CHR in the St Johns wort extract test

material

Blue triangles indicate satisfactory performance yellow triangles indicate questionable performance red triangles indicate non-satisfactory performance z-score values are presented above the triangles for the latter two performance categories

ProLab 2011

Z score-3 0 3 -3 0 3 -3 0 3 -3 0 3 -3 0 3

BAA BAP BBF CHR PAH4

La

bo

rato

ry

36003602

3603

36043605

36063607

36083610

36113612

36133614

36153616

36173618

36193620

36213622

36233624

3626

2258

2343

2452

2669

2147

2788

2869

6710

70839

3011

11271

80525

8425

4561

12338

90680

4976

7331

99553

3998

3780

7500

17420

168413

3269

3029

-2348 -2736

-2023 -2981

-2155-3018

-3998

Figure 2 Graphical presentation of z-scores corresponding to the values for proficiency assessment

reported by the OCLs for the contents of BAA BAP BBF and CHR in the St Johns wort extract test

material

Blue triangles indicate satisfactory performance yellow triangles indicate questionable performance red triangles indicate non-satisfactory performance z-score values are presented above the triangles for the latter two performance categories

ProLab 2011

Z score-3 0 3 -3 0 3 -3 0 3 -3 0 3 -3 0 3

BAA BAP BBF CHR PAH4

La

bo

rato

ry

5301

5302

5303

5304

5305

5306

5307

5308

5309

5310

5311

5312

5313

5314

5315

5316

5317

5318

5319

5320

5321

5322

5323

2597

2629

2017

2293

2873

2123

2521

2620

2186

2222

2452

2864

3952

3984

3806

9226

4171 5522

3285

6594

5205

5157

3539

3901

7051

4519

3173

4872

9191

7276

4391

-2435

-2154

-2065

-2340

-2740-3726

-6346

-3505

17

Table 6 Compilation of z-scores calculated from the ldquoresults for proficiency assessment

reported by the NRLs for food supplement test material z-scores outside the

satisfactory range (|z| gt 2) are indicated by red font Values are reproduced as reported

by the laboratories Empty cells denote analytes for which results were not reported

Measurand benz[a]anthracene benzo[a]pyrene benzo[b]fluoranthene chrysene Sum PAH 4

Assigned value

microgkg 298 165 292 407 1162

Standard deviation for proficiency assessment

microgkg 062 036 060 083 125

Laboratory code

Result z-score Result z-score Result z-score Result z-score Result z-score

microgkg microgkg microgkg microgkg microgkg

3600 3 00 17 01 31 03 5 11 128 09

3602 19 -18 124 -11 17 -20 304 -12 79 -30

3603 251 -08 161 -01 256 -06 459 06 1127 -03

3604 213 -14 143 -06 11 -30 429 03 893 -22

3605 291 -01 167 01 247 -07 386 -03 1091 -06

3606 289 -01 167 01 276 -03 451 05 1184 02

3607 438 23 274 30 567 46 819 50 2098 75

3608 714 67 573 113 1036 123 1014 73 3336 174

3610 196 -17 206 11 191 -17 424 02 1138 -02

3611 469 708 308 805 576 907 865 996 2218 1684

3612 31 02 22 15 29 00 61 25 143 21

3613 33 05 18 04 31 03 4 -01 122 05

3614 185 -18 08 -23 127 -27 271 -16 663 -40

3615 376 13 145 -06 4 18 601 23 152 29

3616 2 -16 15 -04 2 -15 47 08 102 -11

3617 362 10 169 01 299 01 422 02 1252 07

3618 32 04 2 10 31 03 45 05 128 09

3619 299 00 207 12 326 06 738 40 157 33

3620 302 01 166 00 276 -03 46 06 1204 03

3621 28 -03 47 84 25 -07 51 12 151 28

3622 322 04 213 13 34 08 488 10 1361 16

3623 33 05 21 12 28 -02 72 38 154 30

3624 18 04

3626 314 03 174 02 292 00 628 27 1409 20

18

Table 7 Compilation of z-scores calculated from the ldquoresults for proficiency assessment

reported by the OCLs for food supplement test material z-scores outside the

satisfactory range (|z| gt 2) are indicated by red font Values are reproduced as reported

by the laboratories Empty cells denote analytes for which results were not reported

Measurand benz[a]anthracene benzo[a]pyrene benzo[b]fluoranthene chrysene Sum PAH 4

Assigned value

microgkg 298 165 292 407 1162

Standard deviation for proficiency assessment

microgkg 062 036 060 083 125

Laboratory code

Result z-score Result z-

score Result z-score Result

z-score

Result z-score

microgkg microgkg microgkg microgkg microgkg

5301 31 02 18 04 29 00 49 10 126 08

5302 316 03 188 06 298 01 679 33 37 -63

5303 2876 -02 1535 -03 2586 -06 6441 29 13438 15

5304 261 -06 146 -05 253 -06 591 22 1251 07

5305 24 -09 16 -01 24 -09 61 25 125 07

5306 22 -13 16 -01 42 21 49 10 129 10

5307 461 27 157 -02 221 -12 524 14 1363 16

5308 16446 -22 107009 -16 229848 -10 213243 -23 72456 -35

5309 17 -21 11 -15 2 -15

5310 305 01 123 -12 313 03 386 -03 1127 -03

5311 459 26 2 10 281 -02 437 04 1367 16

5312 543 40 248 23 299 01 953 66 2042 71

5313 353 09 207 12 328 06 838 52 1726 45

5314 394 16 172 02 404 19 588 22 1558 32

5315 545 40 238 20 444 25 542 16 177 49

5316 235 -10 17 01 273 -03 282 -15 96 -16

5317 222 -12 269 29 192 -17 545 17 1229 05

5318 067 -37 117 -13 279 -02 357 -06 82 -27

5319 147 -24 216 14 396 17 465 07 1225 05

5320 534 38 316 42 625 55 834 52 2309 92

5321 12 -12

5322 87 92 17 01 33 06 7 35 207 73

5323 33 05 2 10 45 26 73 39 171 44

19

Table 8 and Table 9 present the respective zeta-scores As for the z-scores data outside the

satisfactory performance range are given in red bold font Zeta scores were only calculated for

laboratories that provided measurement uncertainty data as well as a numerical value for the coverage

factor Zeta scores were not calculated when the reported uncertainty value exceeded the content value

which is probably linked to mixing of reporting units (microgkg and ) However the participants are

reminded that the measurement uncertainty shall be expressed in the same unit as the content value

The assessment of the performance of the participants based on the reported measurement uncertainty

gave a less favourable picture compared to z-scores Only 60 of the zeta-scores are within the

satisfactory performance range The distribution of satisfactory performance ratings between NRLs

and OCLs is not balanced It was higher for the NRLs

It has to be noted that the magnitude of the zeta-scores were for some participants much higher than

the z-scores attributed to the same results Consequently the laboratories perform according to fitness-

for-purpose criterion specified in legislation which forms the basis for the z-scores but seem to have

difficulties in deducing realistic measurement uncertainty values Eg the relative standard

measurement uncertainties reported by the NRLs ranged from 25 to 20 with a median value of

about 10 Only two OCLs reported measurement uncertainties that exceeded the maximum tolerable

uncertainty (Uf) The respective data is highlighted in yellow in Table 9

However the EU-RL PAHs will continue to pay special attention to measurement uncertainty in future

ILCs as it has major implications on the assessment of compliance of food with European legislation

20

Table 8 Compilation of zeta-scores calculated from the ldquoresults for proficiency assessment

reported by the NRLs for test material St Johns wort the reported measurement

uncertainty and the uncertainty of the analyte content of the test material zeta-scores

outside the satisfactory range (|zeta| gt 2) are indicated by red bold font Empty cells

denote analytes for which either results or measurement uncertainties were not

reported

21

Table 9 Compilation of zeta-scores calculated from the ldquoresults for proficiency assessment

reported by the OCLs for test material St Johns wort the reported measurement

uncertainty and the uncertainty of the analyte content of the test material zeta-scores

outside the satisfactory range (|zeta| gt 2) are indicated by red bold font yellow

highlighted cells ugtUf Empty cells denote analytes for which results were not

reported

22

94 Evaluation of the influence of analysis method

The information on the applied analysis method (ANNEX 11) was used to group the performance

indicators attributed to the reported results according to the applied analysis technique Two groups of

data were formed They represent results obtained by high performance liquid chromatography-

fluorescence detection (HPLC-FD about 40 ) and by gas chromatography-mass spectrometry (GC-

MS about 60 ) respectively Kernel density plots were constructed based on the respective z-scores

Figure 3A to 3E show the different Kernel density plots

Figure 3 Kernel density plots of z-scores attributed to the results for proficiency assessment

which were reported for the food supplements test sample Results were classified

according to the applied analysis technique

A) benz[a]anthracene

B) benzo[a]pyrene

23

Figure 3 continued

C) benzo[b]fluoranthene

D) chrysene

E) sum of four marker PAHs

The kernel density plots for BAP and BBF are almost identical and centred at or close to a z-score

value of zero as can be seen in Figure 3B and 3C Therefore bias introduced by the type of analysis

method is unlikely for these two analytes The situation is different for BAA and CHR In case of

BAA the kernel density distribution of z-scores based on HPLC-FD is shifted to negative values

24

(Figure 3A) which indicates a tendency to underestimate the BAA content in this particular

samplematrix when applying HPLC-FD This might be reasoned by separationintegration problems

between BAA and CHR peaks A few participants (from the HPLC-FD subgroup) that got negative

z-scores for BAA overestimated the CHR content This caused the slight shift of the density trace for

HPLC-FD towards negative values Removing this data from the evaluation would result in a density

trace with the mode at zero

The opposite is the case for CHR (Figure 3D) The density distribution of the z-scores attributed to

HPLC-FD data is centred almost at zero However the distribution for data based on GC-MS is shifted

to positive values indicating strong positive bias This bias might be caused by a lack of selectivity of

the analysis method The test material contains triphenylene which is isobaric to chrysene

Consequently it cannot be distinguished from chrysene by means of mass spectrometry These two

compounds have to be separated chromatographically which is not easy They coelute on unpolar and

on most mid-polar capillary columns Columns that allow their separation were commercialised only

recently and might not be available in all laboratories Therefore laboratories which determine the

four EU marker PAHs by means of GC-MS shall pay attention to the chromatographic separation of

triphenylene and chrysene A lack of chromatographic resolution might lead to strong positive bias of

GC-MS methods and to false conclusions on the marketability of the tested sample

The chromatographic separation of chrysene and triphenylene is not a problem with HPLC which is

reflected in the position of the density plot for chrysene However attention has to be paid in HPLC-

FD analysis to a proper clean up of the sample extract in order to eliminate interferences that show

similar retention as BAA and CHR

95 Evaluation of the influence of calibration

The participants received together with the food supplement test sample two standard solutions in

depending of their choice either acetonitrile or toluene The analyte content of one solution was

disclosed to the participants whereas the content of the other solution was kept undisclosed The

content values of the latter are presented in Table 5 Analysis results for the solution with undisclosed

content had to be reported to the PT organiser The respective data are presented in Annex 9

Percent deviations of the reported results from the assigned values were calculated for both the food

supplement test sample and the standard solution with undisclosed analyte content Youden plots were

prepared in order to visualize the influence of instrument calibration on the results reported for the

food supplement sample Two plots are discussed in the following The whole set of Youden plots are

given in Annex 9

25

Figure 4 Youden plot of the percent deviations of values reported for benzo[a]pyrene from the

assigned values of the food supplement test material and the standard solution in

toluene with undisclosed content

Figure 4 reveals that a number of participants reported results with similar percent deviations from the

assigned values of both the results for the food supplement material and the standard solution in

toluene with undisclosed content This correlation indicates that over- respectively underestimation of

the analyte content of the food supplement material might be caused by biased instrument calibration

This finding underpins the importance of thorough calibration standard preparation However the data

was not in all cases conclusive as some laboratories performed well for the food supplement samples

but reported biased results for the standard solution with undisclosed content (eg Figure 4 participants

3620 and 5305) This fact cannot be explained yet

Figure 5 presents the respective Youden plot for chrysene in the food supplement test material and the

standard solution in toluene with undisclosed content From this graph it can be concluded that the

overestimation of the chrysene content of the food supplement material was not caused by instrument

calibration The results for the solvent solution of many laboratories which underperformed for the

26

food supplement sample agreed well with the assigned value This supports the conclusion that the

overestimation is caused by the determination of an interferent most probably triphenylene

Figure 5 Youden plot of the percent deviations of values reported for chrysene from the

assigned values of the food supplement test material and the standard solution in

toluene with undisclosed content

96 Evaluation of compliance with legislation

The method performance data reported for the four marker PAHs were evaluated for compliance with

the provisions given in Commission Regulation (EC) No 8362011 Table 18 in ANNEX 10 gives an

overview on the results of the evaluation Data were not reported in case of empty cells Non-

compliant data are highlighted in Table 18 It should be noted that Regulation (EU) No 8362011

specifies LOD and LOQ with two decimals This was not respected by all participants which

hampered the compliance assessment

27

In summary it can be stated that the majority of participants reported method performance

characteristics for the determination of the four marker PAHs in the food supplement test sample that

are compliant with legislation However some laboratories need to improve their analysis methods

The laboratories in question are urged to adapt to the provisions given in the new legislation

10 Follow-up actions for underperforming laboratories

All laboratories that got questionable or non-satisfactory performance ratings are urged to perform

root cause analysis and to implement corrective actions

As agreed during the EU-RL PAH workshop 2011 the EU-RL PAH will set up follow-up measures in

due time for all NRLs that received for at least one of the four PAHs (BAA BAP BBF and CHR) z-

scores gt 3 as required by Regulation (EC) 8822004 and by the Protocol for management of

underperformance in comparative testing andor lack of collaboration of National Reference

Laboratories (NRLs) with European Union reference laboratories (EU-RLs) activities

These laboratories shall perform as an immediate action root-cause-analysis and shall report to the

EU-RL PAH in writing the identified cause for their underperformance and corrective actions they are

going to take Additionally they shall participate to an independent (non-EU-RL) proficiency test on

the determination of PAHs in food and shall communicate the outcome of this exercise to the EU-RL

PAH

28

11 Conclusions

Fourty seven participants reported results back to the EU-RL The performance of the majority of

participants was good In total 153 out of 225 attributed z-scores were below an absolute value of two

which equals to almost 68 However this percentage is much below the percentages of satisfactory

performance gained in previous studies This might be the consequence of the higher level of difficulty

with respect to matrix composition In addition bias can be concluded from the pattern of performance

indicators of some laboratories

In general the determination of chrysene caused most difficulties to the participants

zeta-Scores were calculated besides z-scores They indicate the agreement of the reported result with

the assigned value with respect to the stated measurement uncertainty The outcome of this rating was

worse than for the z-scores which indicates that the measurement uncertainty estimates were not

realistic Therefore participants underperforming with regard to zeta scores are urged to adapt their

measurement uncertainty statements

The great majority of participants in this inter-laboratory comparison applied analytical methods which

complied with EU legislation with regard to performance characteristics

12 Acknowledgements

The organisers would like to thank Mrs Beatriz de la Calle and Mr Franz Ulberth (all from IRMM

Geel Belgium) for their accurate revision of this report Mr Hakan Emteborg (from IRMM) for

ampouling of the standard solutions and all NRLs and OCLs for their cooperation

29

13 References

1 EU COMMISSION REGULATION (EC) No 7762006 of 23 May 2006 amending Annex VII to Regulation

(EC) No 8822004 of the European Parliament and of the Council as regards Community reference

laboratories Official Journal of the European Union 2006 L 136 p 3-8

Available from

httpeur-lexeuropaeuLexUriServLexUriServdouri=OJL200613600030008ENPDF

2 EU Regulation (EC) No 8822004 of the European Parliament and of the Council of 29 April 2004 on

official controls performed to ensure the verification of compliance with feed and food law animal health

and animal welfare rules Official Journal of the European Communities 2004 L191 p 1-52 Available

from

httpeur-lexeuropaeuLexUriServLexUriServdouri=OJL200419100010052ENPDF

3 EU Opinion of the Scientific Committee on Food on the risks to human health of Polycyclic Aromatic

Hydrocarbons in food (2002) Available from

httpeuropaeuintcommfoodfsscscfout153_enpdf

4 International Agency for Research on Cancer IARC Monographs on the Evaluation of

Carcinogenic Risks to Humans Some Non-heterocyclic Polycyclic Aromatic Hydrocarbons and Some

Related Exposures Volume 92 (2010) Available from

httpmonographsiarcfrENGMonographsvol92mono92pdf

5 EU Commission Regulation (EC) No 3332007 of 28 March 2007 laying down the methods of sampling

and analysis for the official control of the levels of lead cadmium mercury inorganic tin 3-MCPD and

benzo(a)pyrene in foodstuffs Official Journal of the European Union 2007 L 88 p 29-38 Available

from

httpeur-lexeuropaeuLexUriServLexUriServdouri=OJL200708800290038ENPDF

6 EU Commission Regulation (EC) No 18812006 of 19 December 2006 setting maximum levels for certain

contaminants in foodstuffs Official Journal of the European Union 2006 L 364 p 5-24 Available from

httpeur-lexeuropaeuLexUriServLexUriServdouri=OJL200636400050024ENPDF

7 EFSA Scientific Opinion of the Panel on Contaminants in the Food Chain on a request from the European

Commission on Polycyclic Aromatic Hydrocarbons in Food The EFSA Journal 2008 724 p 1-114

Available from

httpwwwefsaeuropaeuEFSAefsa_locale-1178620753812_1211902034842htm

30

8 EU Commission Regulation (EU) No 8352011 amending Regulation (EC) No 18812006 as regards

maximum levels for polycyclic aromatic hydrocarbons in foodstuffs Official Journal of the European

Union 2011 L 215 p 4-8

httpeur-lexeuropaeuLexUriServLexUriServdouri=OJL201121500040008ENPDF

9 EU Commission Regulation (EU) No 8362011 amending Regulation (EC) No 3332007 laying down the methods of sampling and analysis for the official control of the levels of lead cadmium mercury inorganic tin 3-MCPD and benzo(a)pyrene in foodstuffs Official Journal of the European Union 2011 L 215 p 9-16 httpeur-lexeuropaeuLexUriServLexUriServdouri=OJL201121500090016ENPDF

10 ISOIEC 170432010 (2010) Conformity assessment -- General requirements for proficiency testing

providers Geneva Switzerland

11 Thompson M Ellison SLR and Wood R The International Harmonized Protocol for the Proficiency

Testing of Analytical Chemistry Laboratories Pure Appl Chem (2006) 78(1) p 145ndash196

Available from httpmediaiupacorgpublicationspac2006pdf7801x0145pdf

31

14 ANNEXES

ANNEX 1 ndash Announcement of the PT on the IRMM webpage

ANNEX 2 ndash Announcement via e-mail and invitation

ANNEX 3 ndash Announcement of sample dispatch

ANNEX 4 ndash Technical specifications of the calibration solutions

ANNEX 5 ndash Homogeneity and stability of the test material

ANNEX 6 ndash Acknowledgement of sample receipt

ANNEX 7 ndash Questionnaire on details of analysis method

ANNEX 8 ndash Results reported for the food supplement sample

ANNEX 9 ndash Results reported for the standard solutions with undisclosed analyte content

ANNEX 10 ndash Method performance characteristics reported by participants

ANNEX 11 ndash Details of analysis methods reported by participants

32

ANNEX 1 ndash Announcement of the PT on the IRMM webpage

33

ANNEX 2 - Announcement via e-mail and invitation

34

ANNEX 3 - Announcement sample dispatch

Dear MadameSir

The test samples for the proficiency test on the determination of four PAHs in a food supplement (dry extract of St Johns wort) were

dispatched today

Explanation on the design of the study as well as instructions for sample storage and for reporting of results are in the parcel You will

also find your participant key in the parcel which is required for reporting of results

Please check the parcel after arrival for completeness and report the receipt of the test samples as well as their status to us by applying

the sample receipt form which is in the parcel

You are kindly requested to inform us by email if you do not receive the parcel by end of this week

My colleague Donata Lerda will then take care of it

With best regards

Thomas Wenzl

35

ANNEX 4 - Specifications of standard solutions with disclosed concentration

36

ANNEX 4 - continued

37

ANNEX 5 - Homogeneity and stability of the test material

Homogeneity of the test material

Benz[a]anthracene - BAA

Benzo[a]pyrene - BAP

38

ANNEX 5 - continued

Benzo[b]fluoranthene - BBF

Chrysene - CHR

39

ANNEX 5 - continued

Stability of the test material

Table 10 Content of the four target analytes in test materials stored under different

temperatures

BaA BaP BbF CHR

microgkg microgkg microgkg microgkg

Content 31 16 31 69 Storage at room temperature

U (k=2) 05 02 05 17

Content 31 15 29 68 Storage at reference temperature

U (k=2) 04 03 04 10

The applied capillary column does not separate CHR from triphenylene

40

ANNEX 6 ndash Documents sent to participants

41

ANNEX 6 - continued

42

ANNEX 7 Questionnaire on details of analysis method

43

ANNEX 7 - continued

44

ANNEX 7 - continued

45

ANNEX 8 - Results reported by participants for the food supplements test sample Table 11 Analysis results reported by the participants for the content of benz[a]anthracene (BAA) in the food supplements test material Values are presented as reported

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

3600 31 29 29 3 05 2

3602 199 193 178 19 021

3603 253 253 246 251 022 2

3604 199 209 23 213 071 2

3605 301 3 271 291 076 2

3606 302 288 278 289 064 2

3607 456 416 443 438 096 2

3608 796 695 651 714 149 2

3610 182 192 214 196 039 2

3611 414 504 488 469 96 2

3612 3 34 28 31 06 2

3613 32 33 34 33 07 2

3614 182 182 191 185 037 2

3615 3884 3822 3565 376 0752 2

3616 2 2 2 2 01 2

3617 366 37 35 362 046 2

3618 32 32 32 32 05 2

3619 299 305 297 299 048 2

3620 304 294 302 302 045 2

3621 29 26 29 28 04 2

3622 343 325 318 322 052 2

3623 35 3 33 33 07 2

3624

3626 318 31 314 314 126 2

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

5301 31 3 32 31 06 2

5302 314 319 315 316 063 2

5303 3102 2716 2822 2876 27 62

5304 261 263 26 261 57 2

5305 23 22 26 24 20

5306 22 23 23 22 11 2

5307 488 461 317 461

5308 168633 168544 1562025 1644598

5309 16 19 16 17

5310 3 304 311 305 015

5311 477 452 448 459 15 2

5312 506 63 494 543 134 2

5313 353 301 404 353

5314 431 373 378 394 5 096

5315 596 531 51 545 11 2

5316 224 231 235 235 022 2

5317 216 23 221 222 007 2

5318 064 07 067 01 2

5319 111 184 146 147 029 2

5320 515 525 564 534 48

5321 21 5322 13 87 9 87 35 2

5323 38 33 35 33 16 2

46

Figure 6 Results of replicate determinations (indicated by triangles) of benz[a]anthracene (BAA) in the food supplement test material

Horizontal blue lines represent the arithmetic mean value of replicate measurements and blue bars the reported expanded measurement uncertainty (k=2) The assigned value is

plotted as dotted line Red solid lines mark the range of satisfactory performance (|z|le2) Mean values are given for results outside the displayed data range

ProLab 2011

Laboratory

53

1853

1953

0853

0936

1436

0236

1036

1653

2136

0453

1753

0653

1653

0536

0353

0436

2153

0336

0636

0536

0036

2036

1953

1036

1253

0136

2653

0236

1836

2336

2236

1353

1353

2336

1736

1553

1453

0736

0753

1153

2053

1253

1536

0853

2236

11

an

aly

te c

on

ten

t (micro

gk

g)

10

95

9

85

8

75

7

65

6

55

5

45

4

35

3

25

2

15

1

05

0

102

3 468

7

Sample St Johns wort extract replicatesMeasurand Benz[a]anthraceneNo of laboratories 46

Assigned value 298 microgkg (Reference value)Target sd 062 microgkgTolerance limits 174 - 422 microgkg (|Z score| lt 200)

Assig

ned va

lue

Limit of tolerance

Limit of tolerance

47

Table 12 Analysis results reported by the participants for the content of benzo[a]pyrene (BAP) in the food supplements test material Values are presented as reported

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

3600 16 17 17 17 03 2

3602 118 123 13 124 015

3603 165 162 157 161 015 2

3604 134 154 139 143 04 2

3605 164 166 172 167 057 2

3606 181 159 161 167 031 2

3607 279 266 279 274 055 2

3608 57 687 461 573 226 2

3610 226 216 183 206 042 2

3611 289 325 311 308 36 2

3612 24 22 2 22 05 2

3613 17 18 19 18 05 2

3614 08 08 08 08 016 2

3615 1545 1393 1425 145 029 2

3616 16 16 14 15 03 2

3617 177 171 158 169 023 2

3618 2 2 2 2 02 2

3619 207 207 206 207 075 2

3620 165 169 163 166 017 2

3621 44 47 49 47 05 2

3622 212 208 222 213 038 2

3623 2 21 22 21 03 2

3624 18 178 182 18 031 2

3626 172 173 178 174 07 2

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

5301 18 18 18 18 04 2

5302 188 19 187 188 038 2

5303 1691 1426 149 1535 19 80

5304 145 142 15 146 69 2

5305 15 17 18 16 15

5306 16 17 17 16 08 2

5307 164 157 153 157

5308 102669 109324 109035 107009

5309 1 1 12 11

5310 122 122 124 123 006

5311 199 184 215 2 15 2

5312 258 248 239 248 05 2

5313 199 178 245 207

5314 174 171 172 172 11 0948

5315 253 248 213 238 05 2

5316 177 171 173 17 016 2

5317 255 277 276 269 012 2

5318 114 12 117 02 2

5319 19 228 231 216 043 2

5320 303 3 344 316 78

5321 12 12 12 12

5322 13 17 24 17 35 2

5323 2 2 19 2 1 2

48

Figure 7 Results of replicate determinations (indicated by triangles) of benzo[a]pyrene (BAP) in the food supplement test material

Horizontal blue lines represent the arithmetic mean value of replicate measurements and blue bars the reported expanded measurement uncertainty (k=2) The assigned value is

plotted as dotted line Red solid lines mark the range of satisfactory performance (|z|le2) Mean values are given for results outside the displayed data range

ProLab 2011

Laboratory

36

1453

0953

0853

1853

2153

1036

0236

0436

1553

0436

1653

0353

0736

0336

2036

0053

0553

0636

0636

0536

1753

1453

1636

2636

1336

2453

0153

2253

0253

2353

1136

1836

1953

1336

1036

2336

2253

1936

1253

1553

1253

1736

0753

2036

2136

0836

11

an

aly

te c

on

ten

t (micro

gk

g)

55

5

45

4

35

3

25

2

15

1

05

0

573

308

3

Sample St Johns wort extract replicatesMeasurand Benzo[a]pyreneNo of laboratories 48

Assigned value 165 microgkg (Reference value)Target sd 036 microgkgTolerance limits 093 - 237 microgkg (|Z score| lt 200)

Assig

ned va

lue

Limit of tolerance

Limit of tolerance

49

Table 13 Analysis results reported by the participants for the content of benzo[b]fluoranthene (BBF) in the food supplements test material Values are presented as reported

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

3600 32 3 3 31 09 2

3602 173 175 16 17 017

3603 265 252 25 256 025 2

3604 116 108 105 11 028 2

3605 286 225 232 247 074 2

3606 294 273 261 276 046 2

3607 579 579 544 567 113 2

3608 103 1201 876 1036 326 2

3610 173 196 204 191 038 2

3611 546 618 565 576 75 2

3612 3 28 28 29 06 2

3613 28 31 32 31 06 2

3614 135 111 135 127 022 2

3615 4187 3678 4111 4 08 2

3616 2 2 2 2 01 2

3617 33 27 299 035 2

3618 31 31 31 31 05 2

3619 326 324 312 326 064 2

3620 277 28 272 276 069 2

3621 26 25 23 25 04 2

3622 346 336 333 34 052 2

3623 26 28 29 28 02 2

3624

3626 288 28 307 292 117 2

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

5301 29 3 29 29 06 2

5302 297 3 296 298 06 2

5303 2705 2444 2608 2586 22 78

5304 245 254 26 253 85 2

5305 23 23 26 24 15

5306 42 43 4 42 21 2

5307 221 216 241 221

5308 232169 246654 240722 229848

5309 19 22 19 2 0

5310 3 315 325 313 016

5311 262 277 304 281 15 2

5312 27 289 337 299 075 2

5313 321 284 378 328

5314 423 404 385 404 34 083

5315 42 506 416 444 09 2

5316 265 267 277 273 018 2

5317 185 197 195 192 006 2

5318 276 281 279 04 2

5319 396 389 403 396 079 2

5320 623 618 635 625 14

5321 31

5322 24 33 49 33 35 2

5323 38 45 35 45 27 2

50

Figure 8 Results of replicate determinations (indicated by triangles) of benzo[b]fluoranthene (BBF) in the food supplement test material

Horizontal blue lines represent the arithmetic mean value of replicate measurements and blue bars the reported expanded measurement uncertainty (k=2) The assigned value is

plotted as dotted line Red solid lines mark the range of satisfactory performance (|z|le2) Mean values are given for results outside the displayed data range

ProLab 2011

Laboratory

36

0436

1436

0236

1053

1736

1653

0953

0753

0853

0536

2136

0553

0436

0353

0353

1636

0636

2036

2353

1853

1136

1236

2653

0153

0253

1236

1736

1336

0036

1853

2153

1036

1953

1336

2253

2253

2353

1936

1553

1453

0653

1536

0753

2036

0836

11

an

aly

te c

on

ten

t (micro

gk

g)

75

7

65

6

55

5

45

4

35

3

25

2

15

1

05

0

103

6

576

3

Sample St Johns wort extract replicatesMeasurand Benzo[b]fluorantheneNo of laboratories 46

Assigned value 292 microgkg (Reference value)Target sd 060 microgkgTolerance limits 172 - 412 microgkg (|Z score| lt 200)

Assig

ned va

lue

Limit of tolerance

Limit of tolerance

51

Table 14 Analysis results reported by the participants for the content of chrysene (CHR) in the food supplements test material Values are presented as reported

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

3600 5 49 5 5 07 2

3602 32 304 29 304 036

3603 462 469 447 459 048 2

3604 438 428 42 429 119 2

3605 365 41 381 386 085 2

3606 494 411 45 451 124 2

3607 897 774 787 819 18 2

3608 986 887 1168 1014 284 2

3610 418 392 462 424 085 2

3611 833 893 87 865 61 2

3612 6 64 58 61 12 2

3613 41 39 4 4 08 2

3614 272 254 288 271 054 2

3615 605 6028 5964 601 12 2

3616 47 45 49 47 05 2

3617 445 431 389 422 065 2

3618 45 45 45 45 06 2

3619 738 747 72 738 118 2

3620 463 472 446 46 092 2

3621 53 49 51 51 06 2

3622 478 513 505 488 08 2

3623 73 73 72 72 05 2

3624

3626 63 622 634 628 251 2

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

5301 49 49 49 49 1 2

5302 687 683 666 679 136 2

5303 6777 6181 6382 6441 24 70

5304 592 581 6 591 85 2

5305 6 58 64 61 20

5306 49 5 51 49 25 2

5307 524 5 574 524

5308 211488 216782 221146 213243

5309

5310 372 393 394 386 019

5311 43 459 422 437 15 2

5312 934 943 981 953 191 2

5313 793 73 99 838

5314 636 561 565 588 18 0818

5315 547 583 499 542 11 2

5316 292 286 285 282 06 2

5317 52 598 517 545 046 2

5318 368 345 357 05 2

5319 449 471 474 465 116 2

5320 76 924 818 834 99

5321 33

5322 7 7 76 7 35 2

5323 87 73 77 73 37 2

52

Figure 9 Results of replicate determinations (indicated by triangles) of chrysene (CHR) in the food supplement test material

Horizontal blue lines represent the arithmetic mean value of replicate measurements and blue bars the reported expanded measurement uncertainty (k=2) The assigned value is

plotted as dotted line Red solid lines mark the range of satisfactory performance (|z|le2) Mean values are given for results outside the displayed data range

ProLab 2011

Laboratory

53

0836

1453

1636

0253

2153

1836

0553

1036

1336

1736

1036

0453

1136

1836

0636

0336

2053

1936

1653

0136

0036

2253

0636

2153

0753

1553

1753

1453

0436

1536

1253

0536

2653

0353

0253

2236

2336

1953

2336

0753

2053

1353

1236

0836

11

an

aly

te c

on

ten

t (micro

gk

g)

13

12

11

10

9

8

7

6

5

4

3

2

1

0

865

3

Sample St Johns wort extract replicatesMeasurand ChryseneNo of laboratories 45

Assigned value 407 microgkg (Reference value)Target sd 083 microgkgTolerance limits 241 - 573 microgkg (|Z score| lt 200)

Assig

ned va

lue

Limit of tolerance

Limit of tolerance

53

Table 15 Analysis results reported by the participants for the sum of the four marker PAHs in the food supplements test material Values are presented as reported

Laboratory code

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg

3600 128 24 2

3602 79 126

3603 1127 096 2

3604 893 258 2

3605 1091 305 2

3606 1184 238 2

3607 2098 42 2

3608 3336 325 2

3610 1138 17 2

3611 2218 259 2

3612 143 29 2

3613 122 2 2

3614 663 129 2

3615 152 304 2

3616 102 03 2

3617 1252 169 2

3618 128 18 2

3619 157 305 2

3620 1204 12 2

3621 151 19 2

3622 1361 115 2

3623 154 09 2

3624

3626 1409 56 2

Laboratory code

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg

5301 126

5302 37 074 2

5303 13438 92

5304 1251 85 2

5305 125 20

5306 129 65 2

5307 1363

5308 7245598

5309

5310 1127 056

5311 1367 15 2

5312 2042 408 2

5313 1726

5314 1558

5315 177 35 2

5316 96 073 2

5317 1229 066 2

5318 82 05 2

5319 1225 267 2

5320 2309 41

5321

5322 207 35 2

5323 171 103 2

54

ANNEX 9 Results reported for the standard solutions with undisclosed analyte content Table 16 Analysis results reported by the participants for the four marker PAHs in the standard solution in toluene with undisclosed analyte

content Values are presented as reported U measurement uncertainty k coverage factor

benz[a]anthracene benzo[a]pyrene benzo[b]fluoranthene chrysene Laboratory

code Unit Result U k Result U k Result U k Result U k 3600 microgl 38 67 2 25 45 2 211 68 2 275 38 2

3605 microgl 367 184 2 2482 124 2 2592 13 2 235 118 2

3606 microgkg 3763 176 2 2609 142 2 2721 118 2 2539 134 2

3607 microgkg 3706 815 2 2824 565 2 2591 518 2 2587 569 2

3612 microgl 38 08 2 237 11 2 244 18 2 257 02 2

3614 microgl 324 65 2 209 38 2 215 37 2 21 42 2

3616 microgkg 318 07 2 224 14 2 231 26 2 216 1 2

3618 microgkg 417 17 2 284 08 2 292 06 2 297 03 2

3619 microgkg 47 742 2 274 468 2 228 387 2 274 434 2

3620 microgl 495 74 2 294 29 2 321 8 2 321 64 2

3621 microgl 241 22 2 143 13 2 188 16 2 145 14 2

3623 microgkg 476 25 2 311 53 2 269 3 2 297 03 2

3626 microgl 359 38 2 235 23 2 238 27 2 24 18 2

5301 microgkg 426 29 299 289

5302 microgkg 436 87 2 296 59 2 296 59 2 289 58 2

5303 microgl 3721 27 102 22838 19 99 23756 22 99 25486 24 98

5304 microgkg 38 29 2 225 26 2 257 37 2 258 52 2

5305 microgkg 305 225 245 29

5306 microgkg 316 63 2 247 49 2 65 13 2 22 44 2

5307 microgkg 3658 2559 241 2414

5308 microgkg 16854 109324 246654 216782

5311 microgl 374 20 2 279 15 2 268 15 2 272 15 2

5313 microgl 3408 2165 2275 2373

5314 microgkg 395 5 2875 11 3614 34 2905 18

5320 microgl 3267 368 1986 571 28 683 2427 512

5322 microgl 282 35 2 225 35 2 241 35 2 21 35 2

55

Figure 10 Youden plots of the percent deviation of the reported values from the assigned values for the food supplement test sample and the

standard solution in toluene with undisclosed content

(The identity of the respective measurand is given above the figure Results exceeding a difference of more than 100 were omitted)

56

Figure 10 - continued

57

Table 17 Analysis results reported by the participants for the four marker PAHs in the standard solution in acetonitrile with undisclosed

analyte content

Values are presented as reported U measurement uncertainty k coverage factor

benz[a]anthracene benzo[a]pyrene benzo[b]fluoranthene chrysene

Laboratory

code Unit Result U k Result U k Result U k Result U k

3602 microgkg 3675 247 247 244

3603 microgkg 358 086 2 236 028 2 235 028 2 236 057 2

3604 microgl 475 29 2 19 12 2 194 13 2 258 12 2

3608 microgl 3623 078 2 2385 023 2 2343 18 2 2296 092 2

3610 microgkg 369 185 2 25 125 2 251 126 2 244 122 2

3611 microgkg 48 02 2 323 02 2 328 04 2 317 03 2

3613 microgl 332 31 2 261 3 2 249 25 2 239 24 2

3615 microgl 415 2635 2729 2689

3617 microgkg 37 09 2 244 06 2 284 07 2 239 06 2

3622 microgl 37 12 2 25 23 2 257 21 2 238 1 2

3624 microgkg 402 104 2 255 084 2 265 096 2 253 07 2

5306 microgkg 563 113 2 371 74 2 359 72 2 333 67 2

5308 microgkg 168633 102669 232169 211488

5309 microgl 402 268 27

5310 microgl 315 158 25 125 25 125 288 144

5312 microgl 428 043 2 282 028 2 272 027 2 275 028 2

5315 microgkg 387 08 2 256 05 2 251 05 2 253 05 2

5316 microgl 3839 342 2 2485 191 2 2559 168 2 239 51 2

5317 microgl 2466 043 2 2212 034 2 2472 016 2 2146 068 2

5319 microgl 414 41 2 279 28 2 268 27 2 26 31 2

5321 microgkg 476 316 326 328

5322 microgl 2547 35 2

5323 microgkg 405 203 2 239 119 2 255 153 2 201 11 2

58

Figure 11 Youden plots of the percent deviation of the reported values from the assigned values for the food supplement test sample and the

standard solution in acetonitrile with undisclosed content

(The identity of the respective measurand is given above the figure Results exceeding a difference of more than 100 were omitted)

59

Figure 11 - continued

60

ANNEX 10 Table 18 Method performance parameters reported by the participants

Laboratory code

Analyte LOD LOQ Recovery Analyte LOD LOQ Recovery

microgkg microgkg microgkg microgkg 3600 BaA 03 06 978 BaP 03 07 9483602 BaA 007 021 926 BaP 005 015 993603 BaA 0011 05 987 BaP 0005 051 9553604 BaA 02 04 86 BaP 02 04 883605 BaA 0025 005 118 BaP 0025 005 1083606 BaA 001 003 65 BaP 001 003 703607 BaA 021 071 74 BaP 027 089 943608 BaA 016 048 8213 BaP 015 046 83683610 BaA 02 05 85 BaP 005 02 853611 BaA BaP 3612 BaA 1 117 BaP 1 1203613 BaA 01 03 90 BaP 01 03 903614 BaA 02 05 96 BaP 02 05 933615 BaA 005 015 110 BaP 005 015 1053616 BaA 01 05 108 BaP 01 05 753617 BaA 018 037 405 BaP 009 018 3873618 BaA 01 03 63 BaP 01 03 593619 BaA 005 005 59 BaP 033 033 543620 BaA 0009 0018 64 BaP 0005 001 553621 BaA 007 02 84 BaP 007 02 923622 BaA 01 03 1101 BaP 01 03 9853623 BaA 02 08 105 BaP 02 08 1013624 BaA BaP 01 033 8513625 BaA BaP 3626 BaA BaP 5301 BaA 003 01 102 BaP 003 01 1005302 BaA 01 03 78 BaP 01 03 865303 BaA 003 01 62 BaP 003 01 805304 BaA 1 1 70 BaP 1 1 955305 BaA 02 05 85 BaP 02 05 855306 BaA 03 1 699 BaP 03 1 905307 BaA 6537 BaP 81165308 BaA 0088 0265 99 BaP 00262 00785 1005309 BaA 01 08 92 BaP 01 08 925310 BaA 01 03 BaP 01 03 905311 BaA 002 005 80 BaP 002 004 915312 BaA 03 09 105 BaP 03 09 1055313 BaA 04 08 BaP 04 08 5314 BaA 001 002 96 BaP 001 002 9485315 BaA 01 03 50 - 100 BaP 01 03 50 - 100 5316 BaA 002 011 827 BaP 004 019 10445317 BaA 05 1 76 BaP 05 1 755318 BaA BaP 5319 BaA 009 029 951 BaP 011 041 995320 BaA 12 18 946 BaP 06 1 8345321 BaA 03 05 BaP 05 1 5322 BaA 1 2 100 BaP 1 2 1005323 BaA 26 BaP 26

61

ANNEX 10 Table 18 - continued

Laboratory code Analyte LOD LOQ Recovery Analyte LOD LOQ Recovery

microgkg microgkg microgkg microgkg 3600 BbF 03 05 96 CHR 02 03 10153602 BbF 015 045 947 CHR 003 009 9193603 BbF 0014 05 1012 CHR 0004 05 10033604 BbF 02 04 85 CHR 02 04 873605 BbF 005 01 94 CHR 0025 005 863606 BbF 001 003 70 CHR 001 003 653607 BbF 018 06 100 CHR 014 028 943608 BbF 015 046 7979 CHR 015 045 71253610 BbF 005 02 85 CHR 02 05 853611 BbF CHR 3612 BbF 1 118 CHR 1 1173613 BbF 01 03 90 CHR 01 03 903614 BbF 01 04 96 CHR 02 05 953615 BbF 008 025 109 CHR 003 01 1083616 BbF 01 05 78 CHR 01 05 723617 BbF 035 07 38 CHR 012 024 4163618 BbF 01 03 62 CHR 01 03 623619 BbF 016 016 63 CHR 01 01 593620 BbF 0003 0006 55 CHR 001 002 653621 BbF 007 02 103 CHR 007 02 893622 BbF 02 06 963 CHR 03 09 1043623 BbF 02 08 106 CHR 02 08 1193624 BbF CHR 3625 BbF CHR 3626 BbF CHR 5301 BbF 003 01 96 CHR 003 01 1145302 BbF 01 03 84 CHR 01 03 835303 BbF 003 01 78 CHR 003 01 705304 BbF 1 1 86 CHR 1 1 705305 BbF 02 05 90 CHR 02 05 955306 BbF 03 1 1435 CHR 03 1 1185307 BbF 8116 CHR 65375308 BbF 0079 0238 99 CHR 009 026 1005309 BbF 01 08 92 CHR 5310 BbF 07 2 CHR 01 03 5311 BbF 002 004 95 CHR 002 004 895312 BbF 03 09 105 CHR 03 09 1055313 BbF 04 08 CHR 04 08 5314 BbF 001 002 83 CHR 001 002 8185315 BbF 02 05 50 -100 CHR 02 05 50 - 100 5316 BbF 005 024 814 CHR 001 007 9595317 BbF 05 1 73 CHR 05 1 785318 BbF CHR 5319 BbF 011 038 946 CHR 013 043 9675320 BbF 07 09 1194 CHR 23 32 10455321 BbF 05 1 CHR 05 1 5322 BbF 1 2 100 CHR 1 2 1005323 BbF 26 CHR 26

62

ANNEX 11 Table 19 Details of analysis method reported by the participants Data are presented as reported

Laboratory code

Analysis technique

Instrument calibration

Internal standards applied

Sample clean up

Details of sample

clean up

Extraction technique

Details of sample

extraction

3600 6) GC-MS

2) Internal standardisation with labelled substances

Deuterated PAH mix 9 (Ehrenstorfer)

1) Saponification 2) LiquidLiquid partitioning

D) Other Liquidliquid partitioning

3602 2) HPLC-UV-FLD

1) External calibration

3) Gel permeation chromatography

A) Pressurised liquid extraction (PLE)

3603 1) HPLC-FLD

1) External calibration

3) Gel permeation chromatography

D) Other 1 minute with dichloromethane in vortex

3604 2) HPLC-UV-FLD

2) Internal standardisation with labelled substances

DiP D14

5) Donor acceptor complex chromatography

D) Other LL

3605 1) HPLC-FLD

1) External calibration

3) Gel permeation chromatography

C) Soxhlet extraction

3606 7) GC-MSMS

2) Internal standardisation with labelled substances

BaP 13C4 CHR 13C6 BaA 13C6 BbF 13C6

4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

3607 6) GC-MS

2) Internal standardisation with labelled substances

BaA-d12 CHR-d12 BbF-d12 BkF-d12 BaP-d12 ICP-d12 BgP-d12 DiP-d14

1) Saponification

D) Other solvent partitioning

3608 1) HPLC-FLD

1) External calibration

6) Other Liquid extraction

D) Other

Extraction in a rotary agitator and centrifugation

3610 2) HPLC-UV-FLD

1) External calibration

4) Solid phase extraction

B) Sonication

3611

3612 6) GC-MS

2) Internal standardisation with labelled substances

chrysene-d12 dibenzo(ah)anthracene-d14 dibenzo(ai)pyrene-d14

4) Solid phase extraction

B) Sonication

3613 5) LC-MSMS

3) Internal standardisation with unlabelled substances

I use Chrysene-D12 3) Gel permeation chromatography

D) Other extraction with hexan

63

Table 19 - continued

Laboratory code

Analysis technique

Instrument calibration

Internal standards applied

Sample clean up Details of sample

clean up

Extraction technique

Details of sample

extraction

3614 6) GC-MS 1) External calibration

3) Gel permeation chromatography

B) Sonication

3615 1) HPLC-FLD

1) External calibration

6) Other PLE GPC

A) Pressurised liquid extraction (PLE)

3616 6) GC-MS

2) Internal standardisation with labelled substances

Benzo(a)pyrene-13C4 Benzo(a)anthracene-13C6 Benzo(b)fluoranthene-13C6 Chrysene-13C6

2) LiquidLiquid partitioning 4) Solid phase extraction

B) Sonication

3617 1) HPLC-FLD

1) External calibration

4) Solid phase extraction

C) Soxhlet extraction

3618 6) GC-MS

2) Internal standardisation with labelled substances

deuterated standards

1) Saponification 4) Solid phase extraction

D) Other Handshaken with cyclohexane

3619 6) GC-MS

2) Internal standardisation with labelled substances

C13 Labelled US EPA 16

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

D) Other Homegenisation amp saponiofication

3620 9) GC-HRMS

2) Internal standardisation with labelled substances

mix of deuterated PAHs (BAA-D12 CHR-D12 BAP-D12 BBF-D12)

3) Gel permeation chromatography 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

3621 6) GC-MS

2) Internal standardisation with labelled substances

Chrysene D12 Benzo(a)pyrene D12 Benzo(b)fluoranthene D12 Benzo(a)anthracene

2) LiquidLiquid partitioning 4) Solid phase extraction

B) Sonication

3622 1) HPLC-FLD

4) Standard addition method

4) Solid phase extraction

D) Other

Extraction with cyclohexane by heating under reflux

3623 7) GC-MSMS

2) Internal standardisation with labelled substances

C13 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

3624 2) HPLC-UV-FLD

1) External calibration

2) LiquidLiquid partitioning

D) Other hot solvent extraction

3626

64

Table 19 - continued

Laboratory code

Analysis technique

Instrument calibration

Internal standards

applied

Sample clean up

Details of sample

clean up

Extraction technique

Details of sample

extraction

5301 7) GC-MSMS

2) Internal standardisation with labelled substances

EACH SUBSTANCE C13

4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5302 7) GC-MSMS

2) Internal standardisation with labelled substances

Isotope labelled PAHs 13C for each analyte

4) Solid phase extraction

C) Soxhlet extraction

5303 7) GC-MSMS

3) Internal standardisation with unlabelled substances

HAP C13 4) Solid phase extraction

D) Other automate ASE200 DIONEX

5304 7) GC-MSMS

2) Internal standardisation with labelled substances

with C13 for each HAP

2) LiquidLiquid partitioning 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5305 7) GC-MSMS

2) Internal standardisation with labelled substances

C13 isotopiques internal standards

4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5306 7) GC-MSMS

2) Internal standardisation with labelled substances

C13 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5307 6) GC-MS

2) Internal standardisation with labelled substances

1) Saponification 2) LiquidLiquid partitioning

D) Other solvent extraction

5308 1) HPLC-FLD 1) External calibration

4) Solid phase extraction

B) Sonication

5309 1) HPLC-FLD 1) External calibration

4) Solid phase extraction

D) Other only SPE

5310 1) HPLC-FLD 1) External calibration

1) Saponification 2) LiquidLiquid partitioning 3) Gel permeation chromatography

C) Soxhlet extraction

5311 6) GC-MS

2) Internal standardisation with labelled substances

13 C Chrysene 13 C Benz(a)pyrene

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

B) Sonication

5312 1) HPLC-FLD

3) Internal standardisation with unlabelled substances

Benzo(b)chrysen

2) LiquidLiquid partitioning 4) Solid phase extraction

Quechers D) Other Quechers

65

Table 19 - continued

Laboratory code

Analysis technique

Instrument calibration

Internal standards applied

Sample clean up Details of sample

clean up

Extraction technique

Details of sample extraction

5313 6) GC-MS

2) Internal standardisation with labelled substances

Benz(a)anthracene D12 Benzo(b)fluranthene D12 Benzo(a)pyrene D12 Chrysene D12

1) Saponification 2) LiquidLiquid partitioning 3) Gel permeation chromatography

A) Pressurised liquid extraction (PLE)

5314 6) GC-MS

2) Internal standardisation with labelled substances

Benzo(a)pyren d12 2) LiquidLiquid partitioning

D) Other Extraction with

5315 1) HPLC-FLD

3) Internal standardisation with unlabelled substances

Benz(b)chrysen

1) Saponification 2) LiquidLiquid partitioning

D) Other shaker

5316 1) HPLC-FLD

1) External calibration

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

D) Other

LiquidLiquid extraction with cyclohexane and dimethylformamide

5317 7) GC-MSMS

1) External calibration

3) Gel permeation chromatography 6) Other

silica gel D) Other VDLUFA online method

5318

5319 1) HPLC-FLD

3) Internal standardisation with unlabelled substances

Benzo(b)chrysene 3) Gel permeation chromatography

A) Pressurised liquid extraction (PLE)

5320 6) GC-MS

3) Internal standardisation with unlabelled substances

22-binaphthyl

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

D) Other 3 hours refluxing

5321 1) HPLC-FLD

2) Internal standardisation with labelled substances

6-meacutethylchrysegravene

1) Saponification 2) LiquidLiquid partitioning

B) Sonication

5322 6) GC-MS

2) Internal standardisation with labelled substances

naftalene-d8 acenaphtalene-d10 phenanthrene-d10 chrysene-d12 perylene-d12 benzo(e)anthracene-d12

1) Saponification 2) LiquidLiquid partitioning 6) Other

clean-up with silica packed column

C) Soxhlet extraction

5323 1) HPLC-FLD

1) External calibration

4) Solid phase extraction 6) Other

Accelerated Solvent Extraction

A) Pressurised liquid extraction (PLE)

66

European Commission EUR 25245 EN ndash Joint Research Centre ndash Institute for Reference Materials and Measurements Title Report on the 8th inter-laboratory comparison organised by the European Union Reference Laboratory for Polycyclic Aromatic Hydrocarbons - Four marker PAHs in a dry extract of St Johns wort Author(s) Radoslav Lizak Szilard Szilagyi Philippe Verlinde and Thomas WENZL Luxembourg Publications Office of the European Union 2012 ndash 66 pp ndash 201 x 297 cm EUR ndash Scientific and Technical Research series ndash ISSN 1831-9424 ISBN 978-92-79-23337-1 doi 10278759387 Abstract The proficiency test here reported concerned the determination of the contents of four marker polycyclic aromatic hydrocarbons (PAHs) and of their sum in an food supplements test sample The set of marker PAHs consists of benz[a]anthacene benzo[a]pyrene benzo[b]fluoranthene and chrysene Participants to these PT were National Reference Laboratories for PAHs (NRLs-PAHs) and EU official food control laboratories The number of participants was in total 48

The PT was organised according ISO Standard 170432010

The test material used was a commercial dry extract of St Johns wort Participants also received a solution of the same PAHs either in acetonitrile or in toluene for checking their instrument calibration

The results from participants were rated with z-scores and zeta-scores About 68 of the reported results were attributed with z-scores with an absolute value of below two which is the threshold for satisfactory performance

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L

A-N

A-252

45-EN

-N

• 1 Summary
• 2 Introduction
• 3 Scope
• 4 Participating Laboratories
• 5 Time frame
• 6 Confidentiality
• 7 Test materials
• • 71 1Preparation of test materials
  • 72 Assigned values and standard deviation for proficiency assessment
  • 73 Homogeneity and stability
  • 74 Sample dispatch
  • • 8 Design of the proficiency test
    • 9 Evaluation of the results
    • • 91 2General
      • 92 21BEvaluation criteria
      • 93 Performance of participants
      • 94 Evaluation of the influence of analysis method
      • 95 Evaluation of the influence of calibration
      • 96 26Evaluation of compliance with legislation
      • • 10 Follow-up actions for underperforming laboratories
        • 11 1Conclusions
        • 12 1Acknowledgements
        • 13 References

11

Table 5 Analyte contents of the standard solutions with undisclosed content

Content U (k=2) Content U (k=2) microgkg microgkg microgkg microgkg

benz[a ]anthracene 4317 039 4764 043benzo[c ]fluorene 7342 061 8052 067chrysene 2904 031 3179 034cyclopenta[cd ]pyrene 2558 050 2818 055benzo[a ]pyrene 2897 038 3186 042benzo[b ]fluoranthene 2926 026 3199 029benzo[ghi ]perylene 2214 023 2438 026benzo[j ]fluoranthene 4353 038 4763 041benzo[k ]fluoranthene 2143 017 2380 019dibenzo[ae ]pyrene 5735 084 6326 093dibenzo[ah ]anthracene 2215 032 2445 035dibenzo[ah ]pyrene 2169 029 2394 032dibenzo[ai ]pyrene 3691 100 4035 109dibenzo[al ]pyrene 4627 057 5104 063indeno[123-cd ]pyrene 3793 036 4176 0395-methylchrysene 8589 087 9443 096

Solution in toluene Solution in acetonitrile

Prep conc concentration from gravimetrical preparation U expanded uncertainty of the assigned value k coverage

factor

73 Homogeneity and stability

Homogeneity of the food supplements test sample was evaluated according to ISO Standard 13528

Ten units of the St Johns wort extract test material were selected randomly and analysed by isotope

dilution gas chromatography mass spectrometry (GC-MS) The test material was rated sufficiently

homogeneous (see ANNEX 5)

The contributions of the uncertainties of homogeneity (relative uncertainties below 1 ) to the total

uncertainties of the assigned values were found negligible and were therefore not considered in the

calculations

The stability of the test materials was evaluated applying an isochronous experimental set up This

comprised the storage of test samples both at room temperature and cooled in the fridge The content

values of the cooled sample were used as reference for the stability evaluation The materials were

stored from dispatch of test samples to the participants until expiry of the deadline for reporting of

results Afterwards the samples were analysed under repeatability conditions and results were

compared The content values of the cool-stored sample and the sample stored at room temperature

were not statistically significantly different Hence stability of the samples over the whole study period

can be assumed The analysis data are presented in ANNEX 5

12

The uncertainty of stability of the test material was considered negligible compared to the uncertainty

of the characterisation measurements Therefore this parameter was not taken into account in the

determination of the uncertainties of the assigned values

74 Sample dispatch

Samples were dispatched at room temperature Each participant received at least

- one ampoule of the solution of the 15+1 EU priority PAHs in the chosen solvent with disclosed

content (2 ml)

- one ampoule of the 15+1 EU priority PAHs in the chosen solvent with undisclosed content (2

ml)

- one screw cap vial with the food supplement test material

Each parcel contained a sample receipt form the outline of the study an appropriate material safety

data sheets and a unique code that was required for reporting of results The documents sent to the

participants are presented in ANNEX 6

8 Design of the proficiency test

The design of the PT foresaw triplicate analyses of the test sample and reporting of the individual

results of replicate analyses for the single analytes in the following denoted as FS_REP and

additionally a value for proficiency assessment in the following denoted as final value - FS_FIN

for both the single analytes and the sum of the four marker PAHs Both FS_REP results and FS_FIN

results had to be reported corrected for recovery (and recovery had to be stated in the questionnaire

which participants were asked to fill in together with other parameters of the method applied) the

latter had also to be accompanied by the respective expanded measurement uncertainty The FS_FIN

results were used for performance assessment

Besides analysis results participants were also asked to report details of the applied analysis method

and to provide answers to a questionnaire (see ANNEX 7 for the template and the compiled returned

questionnaires)

13

9 Evaluation of the results

The results reported by participants are listed in ANNEX 8

91 General

The most important evaluation parameter was the performance of the laboratories in the determination

of the target PAHs in the food supplement test material which was expressed by z-scores and zeta-

scores

The compliance of method performance characteristics specified by the participants with provisions

given in legislation was evaluated too

92 Evaluation criteria

The participants were requested to report for all analytes the results of replicate measurements and a

value for proficiency assessment which is the result they wish to be applied for the calculation of

performance indicators z-Scores and zeta-scores were attributed only to these results The individual

results of replicate analyses were not rated

z-Scores

z-scores were calculated based on the FS_FIN values Equation 1 presents the formula for calculation

of z-scores

Equation 1

P

assignedlab Xxz

where z refers to the z-score xlab to the reported ldquovalue for proficiency assessmentrdquo Xassigned to the

assigned value and σP to the standard deviation for proficiency testing

zeta-Scores

In addition to z-scores zeta-scores were calculated In contrast to z-scores zeta-scores describe the

agreement of the reported result with the assigned value within the respective uncertainties

Unsatisfactorily large zeta-scores might be caused by underestimated measurement uncertainties large

bias or a combination of both Zeta-scores were calculated according to Equation 3

14

Equation 3 22assignedlab

assignedlab

uu

Xxzeta

where zeta refers to the zeta-score xlab to the reported ldquovalue for proficiency assessmentrdquo Xassigned to

the assigned value ulab to the measurement uncertainty reported by the laboratory and uassigned to the

uncertainty of the assigned value

The performance of the laboratories was classified according to ISOIEC 170432010 [10] Following

scheme is applied for the interpretation of zeta scores and z-scores

|score| 20 = satisfactory performance

20lt|score| lt 30 = questionable performance

|score| ge 30 = unsatisfactory performance

93 Performance of participants

The 48 participants in the study reported in total 225 results for proficiency evaluation which equals to

about 94 of the 240 possible results About 68 of the reported results were rated as satisfactory

with regard to z-scores

Figure 1 and Figure 2 give an overview of the z-scores assigned to the respective results The larger

the triangles the larger were the differences to the assigned values Red triangles indicate z-scores

above an absolute value of three whereas yellow triangles represent z-scores in the questionable

performance range The corresponding score values are plotted next to the triangles About 40 of the

72 non-satisfactory results were reported by seven laboratories only eg the performance of

participant 3608 was not satisfactory for the majority of target analytes

It is also obvious that most problems were encountered in the determination of chrysene and the sum

of the four analytes Potential reasons for the difficulties related to chrysene are discussed further

down

The ideal situation would be that deviations from the assigned values of the results reported for the

individual analytes are small and randomly distributed around zero This will give a satisfactory

performance statement for the sum parameter Deviations of opposite sign could also cancel out and

lead to a satisfactory performance for the sum parameter (eg participants 5303 5304 and 5305)

However in most cases a single underperformance for one analyte led also to non-satisfactory rating

for the sum parameter

15

The numerical values of the calculated z-scores are compiled in Table 6 and Table 7 z-Scores with an

absolute value of above 2 are given in red bold font

The results of the data evaluation for the individual analytes are given in ANNEX 8

For each analyte the figure shows the individual analysis results of the three replicate determinations

The assigned value is shown as dotted line The blue boxes represent the expanded uncertainties as

reported by participants for the value for proficiency assessment The arithmetic mean of the results

of the individual participants is indicated in the blue boxes by a blue line The satisfactory performance

range is located between the two red lines

The individual results of the replicate measurements and the value for proficiency assessment with

its accompanying expanded measurement uncertainty (k=2) are listed in the tables in ANNEX 8 as

well

16

Figure 1 Graphical presentation of z-scores corresponding to the values for proficiency assessment

reported by the NRLs for the contents of BAA BAP BBF and CHR in the St Johns wort extract test

material

Blue triangles indicate satisfactory performance yellow triangles indicate questionable performance red triangles indicate non-satisfactory performance z-score values are presented above the triangles for the latter two performance categories

ProLab 2011

Z score-3 0 3 -3 0 3 -3 0 3 -3 0 3 -3 0 3

BAA BAP BBF CHR PAH4

La

bo

rato

ry

36003602

3603

36043605

36063607

36083610

36113612

36133614

36153616

36173618

36193620

36213622

36233624

3626

2258

2343

2452

2669

2147

2788

2869

6710

70839

3011

11271

80525

8425

4561

12338

90680

4976

7331

99553

3998

3780

7500

17420

168413

3269

3029

-2348 -2736

-2023 -2981

-2155-3018

-3998

Figure 2 Graphical presentation of z-scores corresponding to the values for proficiency assessment

reported by the OCLs for the contents of BAA BAP BBF and CHR in the St Johns wort extract test

material

Blue triangles indicate satisfactory performance yellow triangles indicate questionable performance red triangles indicate non-satisfactory performance z-score values are presented above the triangles for the latter two performance categories

ProLab 2011

Z score-3 0 3 -3 0 3 -3 0 3 -3 0 3 -3 0 3

BAA BAP BBF CHR PAH4

La

bo

rato

ry

5301

5302

5303

5304

5305

5306

5307

5308

5309

5310

5311

5312

5313

5314

5315

5316

5317

5318

5319

5320

5321

5322

5323

2597

2629

2017

2293

2873

2123

2521

2620

2186

2222

2452

2864

3952

3984

3806

9226

4171 5522

3285

6594

5205

5157

3539

3901

7051

4519

3173

4872

9191

7276

4391

-2435

-2154

-2065

-2340

-2740-3726

-6346

-3505

17

Table 6 Compilation of z-scores calculated from the ldquoresults for proficiency assessment

reported by the NRLs for food supplement test material z-scores outside the

satisfactory range (|z| gt 2) are indicated by red font Values are reproduced as reported

by the laboratories Empty cells denote analytes for which results were not reported

Measurand benz[a]anthracene benzo[a]pyrene benzo[b]fluoranthene chrysene Sum PAH 4

Assigned value

microgkg 298 165 292 407 1162

Standard deviation for proficiency assessment

microgkg 062 036 060 083 125

Laboratory code

Result z-score Result z-score Result z-score Result z-score Result z-score

microgkg microgkg microgkg microgkg microgkg

3600 3 00 17 01 31 03 5 11 128 09

3602 19 -18 124 -11 17 -20 304 -12 79 -30

3603 251 -08 161 -01 256 -06 459 06 1127 -03

3604 213 -14 143 -06 11 -30 429 03 893 -22

3605 291 -01 167 01 247 -07 386 -03 1091 -06

3606 289 -01 167 01 276 -03 451 05 1184 02

3607 438 23 274 30 567 46 819 50 2098 75

3608 714 67 573 113 1036 123 1014 73 3336 174

3610 196 -17 206 11 191 -17 424 02 1138 -02

3611 469 708 308 805 576 907 865 996 2218 1684

3612 31 02 22 15 29 00 61 25 143 21

3613 33 05 18 04 31 03 4 -01 122 05

3614 185 -18 08 -23 127 -27 271 -16 663 -40

3615 376 13 145 -06 4 18 601 23 152 29

3616 2 -16 15 -04 2 -15 47 08 102 -11

3617 362 10 169 01 299 01 422 02 1252 07

3618 32 04 2 10 31 03 45 05 128 09

3619 299 00 207 12 326 06 738 40 157 33

3620 302 01 166 00 276 -03 46 06 1204 03

3621 28 -03 47 84 25 -07 51 12 151 28

3622 322 04 213 13 34 08 488 10 1361 16

3623 33 05 21 12 28 -02 72 38 154 30

3624 18 04

3626 314 03 174 02 292 00 628 27 1409 20

18

Table 7 Compilation of z-scores calculated from the ldquoresults for proficiency assessment

reported by the OCLs for food supplement test material z-scores outside the

satisfactory range (|z| gt 2) are indicated by red font Values are reproduced as reported

by the laboratories Empty cells denote analytes for which results were not reported

Measurand benz[a]anthracene benzo[a]pyrene benzo[b]fluoranthene chrysene Sum PAH 4

Assigned value

microgkg 298 165 292 407 1162

Standard deviation for proficiency assessment

microgkg 062 036 060 083 125

Laboratory code

Result z-score Result z-

score Result z-score Result

z-score

Result z-score

microgkg microgkg microgkg microgkg microgkg

5301 31 02 18 04 29 00 49 10 126 08

5302 316 03 188 06 298 01 679 33 37 -63

5303 2876 -02 1535 -03 2586 -06 6441 29 13438 15

5304 261 -06 146 -05 253 -06 591 22 1251 07

5305 24 -09 16 -01 24 -09 61 25 125 07

5306 22 -13 16 -01 42 21 49 10 129 10

5307 461 27 157 -02 221 -12 524 14 1363 16

5308 16446 -22 107009 -16 229848 -10 213243 -23 72456 -35

5309 17 -21 11 -15 2 -15

5310 305 01 123 -12 313 03 386 -03 1127 -03

5311 459 26 2 10 281 -02 437 04 1367 16

5312 543 40 248 23 299 01 953 66 2042 71

5313 353 09 207 12 328 06 838 52 1726 45

5314 394 16 172 02 404 19 588 22 1558 32

5315 545 40 238 20 444 25 542 16 177 49

5316 235 -10 17 01 273 -03 282 -15 96 -16

5317 222 -12 269 29 192 -17 545 17 1229 05

5318 067 -37 117 -13 279 -02 357 -06 82 -27

5319 147 -24 216 14 396 17 465 07 1225 05

5320 534 38 316 42 625 55 834 52 2309 92

5321 12 -12

5322 87 92 17 01 33 06 7 35 207 73

5323 33 05 2 10 45 26 73 39 171 44

19

Table 8 and Table 9 present the respective zeta-scores As for the z-scores data outside the

satisfactory performance range are given in red bold font Zeta scores were only calculated for

laboratories that provided measurement uncertainty data as well as a numerical value for the coverage

factor Zeta scores were not calculated when the reported uncertainty value exceeded the content value

which is probably linked to mixing of reporting units (microgkg and ) However the participants are

reminded that the measurement uncertainty shall be expressed in the same unit as the content value

The assessment of the performance of the participants based on the reported measurement uncertainty

gave a less favourable picture compared to z-scores Only 60 of the zeta-scores are within the

satisfactory performance range The distribution of satisfactory performance ratings between NRLs

and OCLs is not balanced It was higher for the NRLs

It has to be noted that the magnitude of the zeta-scores were for some participants much higher than

the z-scores attributed to the same results Consequently the laboratories perform according to fitness-

for-purpose criterion specified in legislation which forms the basis for the z-scores but seem to have

difficulties in deducing realistic measurement uncertainty values Eg the relative standard

measurement uncertainties reported by the NRLs ranged from 25 to 20 with a median value of

about 10 Only two OCLs reported measurement uncertainties that exceeded the maximum tolerable

uncertainty (Uf) The respective data is highlighted in yellow in Table 9

However the EU-RL PAHs will continue to pay special attention to measurement uncertainty in future

ILCs as it has major implications on the assessment of compliance of food with European legislation

20

Table 8 Compilation of zeta-scores calculated from the ldquoresults for proficiency assessment

reported by the NRLs for test material St Johns wort the reported measurement

uncertainty and the uncertainty of the analyte content of the test material zeta-scores

outside the satisfactory range (|zeta| gt 2) are indicated by red bold font Empty cells

denote analytes for which either results or measurement uncertainties were not

reported

21

Table 9 Compilation of zeta-scores calculated from the ldquoresults for proficiency assessment

reported by the OCLs for test material St Johns wort the reported measurement

uncertainty and the uncertainty of the analyte content of the test material zeta-scores

outside the satisfactory range (|zeta| gt 2) are indicated by red bold font yellow

highlighted cells ugtUf Empty cells denote analytes for which results were not

reported

22

94 Evaluation of the influence of analysis method

The information on the applied analysis method (ANNEX 11) was used to group the performance

indicators attributed to the reported results according to the applied analysis technique Two groups of

data were formed They represent results obtained by high performance liquid chromatography-

fluorescence detection (HPLC-FD about 40 ) and by gas chromatography-mass spectrometry (GC-

MS about 60 ) respectively Kernel density plots were constructed based on the respective z-scores

Figure 3A to 3E show the different Kernel density plots

Figure 3 Kernel density plots of z-scores attributed to the results for proficiency assessment

which were reported for the food supplements test sample Results were classified

according to the applied analysis technique

A) benz[a]anthracene

B) benzo[a]pyrene

23

Figure 3 continued

C) benzo[b]fluoranthene

D) chrysene

E) sum of four marker PAHs

The kernel density plots for BAP and BBF are almost identical and centred at or close to a z-score

value of zero as can be seen in Figure 3B and 3C Therefore bias introduced by the type of analysis

method is unlikely for these two analytes The situation is different for BAA and CHR In case of

BAA the kernel density distribution of z-scores based on HPLC-FD is shifted to negative values

24

(Figure 3A) which indicates a tendency to underestimate the BAA content in this particular

samplematrix when applying HPLC-FD This might be reasoned by separationintegration problems

between BAA and CHR peaks A few participants (from the HPLC-FD subgroup) that got negative

z-scores for BAA overestimated the CHR content This caused the slight shift of the density trace for

HPLC-FD towards negative values Removing this data from the evaluation would result in a density

trace with the mode at zero

The opposite is the case for CHR (Figure 3D) The density distribution of the z-scores attributed to

HPLC-FD data is centred almost at zero However the distribution for data based on GC-MS is shifted

to positive values indicating strong positive bias This bias might be caused by a lack of selectivity of

the analysis method The test material contains triphenylene which is isobaric to chrysene

Consequently it cannot be distinguished from chrysene by means of mass spectrometry These two

compounds have to be separated chromatographically which is not easy They coelute on unpolar and

on most mid-polar capillary columns Columns that allow their separation were commercialised only

recently and might not be available in all laboratories Therefore laboratories which determine the

four EU marker PAHs by means of GC-MS shall pay attention to the chromatographic separation of

triphenylene and chrysene A lack of chromatographic resolution might lead to strong positive bias of

GC-MS methods and to false conclusions on the marketability of the tested sample

The chromatographic separation of chrysene and triphenylene is not a problem with HPLC which is

reflected in the position of the density plot for chrysene However attention has to be paid in HPLC-

FD analysis to a proper clean up of the sample extract in order to eliminate interferences that show

similar retention as BAA and CHR

95 Evaluation of the influence of calibration

The participants received together with the food supplement test sample two standard solutions in

depending of their choice either acetonitrile or toluene The analyte content of one solution was

disclosed to the participants whereas the content of the other solution was kept undisclosed The

content values of the latter are presented in Table 5 Analysis results for the solution with undisclosed

content had to be reported to the PT organiser The respective data are presented in Annex 9

Percent deviations of the reported results from the assigned values were calculated for both the food

supplement test sample and the standard solution with undisclosed analyte content Youden plots were

prepared in order to visualize the influence of instrument calibration on the results reported for the

food supplement sample Two plots are discussed in the following The whole set of Youden plots are

given in Annex 9

25

Figure 4 Youden plot of the percent deviations of values reported for benzo[a]pyrene from the

assigned values of the food supplement test material and the standard solution in

toluene with undisclosed content

Figure 4 reveals that a number of participants reported results with similar percent deviations from the

assigned values of both the results for the food supplement material and the standard solution in

toluene with undisclosed content This correlation indicates that over- respectively underestimation of

the analyte content of the food supplement material might be caused by biased instrument calibration

This finding underpins the importance of thorough calibration standard preparation However the data

was not in all cases conclusive as some laboratories performed well for the food supplement samples

but reported biased results for the standard solution with undisclosed content (eg Figure 4 participants

3620 and 5305) This fact cannot be explained yet

Figure 5 presents the respective Youden plot for chrysene in the food supplement test material and the

standard solution in toluene with undisclosed content From this graph it can be concluded that the

overestimation of the chrysene content of the food supplement material was not caused by instrument

calibration The results for the solvent solution of many laboratories which underperformed for the

26

food supplement sample agreed well with the assigned value This supports the conclusion that the

overestimation is caused by the determination of an interferent most probably triphenylene

Figure 5 Youden plot of the percent deviations of values reported for chrysene from the

assigned values of the food supplement test material and the standard solution in

toluene with undisclosed content

96 Evaluation of compliance with legislation

The method performance data reported for the four marker PAHs were evaluated for compliance with

the provisions given in Commission Regulation (EC) No 8362011 Table 18 in ANNEX 10 gives an

overview on the results of the evaluation Data were not reported in case of empty cells Non-

compliant data are highlighted in Table 18 It should be noted that Regulation (EU) No 8362011

specifies LOD and LOQ with two decimals This was not respected by all participants which

hampered the compliance assessment

27

In summary it can be stated that the majority of participants reported method performance

characteristics for the determination of the four marker PAHs in the food supplement test sample that

are compliant with legislation However some laboratories need to improve their analysis methods

The laboratories in question are urged to adapt to the provisions given in the new legislation

10 Follow-up actions for underperforming laboratories

All laboratories that got questionable or non-satisfactory performance ratings are urged to perform

root cause analysis and to implement corrective actions

As agreed during the EU-RL PAH workshop 2011 the EU-RL PAH will set up follow-up measures in

due time for all NRLs that received for at least one of the four PAHs (BAA BAP BBF and CHR) z-

scores gt 3 as required by Regulation (EC) 8822004 and by the Protocol for management of

underperformance in comparative testing andor lack of collaboration of National Reference

Laboratories (NRLs) with European Union reference laboratories (EU-RLs) activities

These laboratories shall perform as an immediate action root-cause-analysis and shall report to the

EU-RL PAH in writing the identified cause for their underperformance and corrective actions they are

going to take Additionally they shall participate to an independent (non-EU-RL) proficiency test on

the determination of PAHs in food and shall communicate the outcome of this exercise to the EU-RL

PAH

28

11 Conclusions

Fourty seven participants reported results back to the EU-RL The performance of the majority of

participants was good In total 153 out of 225 attributed z-scores were below an absolute value of two

which equals to almost 68 However this percentage is much below the percentages of satisfactory

performance gained in previous studies This might be the consequence of the higher level of difficulty

with respect to matrix composition In addition bias can be concluded from the pattern of performance

indicators of some laboratories

In general the determination of chrysene caused most difficulties to the participants

zeta-Scores were calculated besides z-scores They indicate the agreement of the reported result with

the assigned value with respect to the stated measurement uncertainty The outcome of this rating was

worse than for the z-scores which indicates that the measurement uncertainty estimates were not

realistic Therefore participants underperforming with regard to zeta scores are urged to adapt their

measurement uncertainty statements

The great majority of participants in this inter-laboratory comparison applied analytical methods which

complied with EU legislation with regard to performance characteristics

12 Acknowledgements

The organisers would like to thank Mrs Beatriz de la Calle and Mr Franz Ulberth (all from IRMM

Geel Belgium) for their accurate revision of this report Mr Hakan Emteborg (from IRMM) for

ampouling of the standard solutions and all NRLs and OCLs for their cooperation

29

13 References

1 EU COMMISSION REGULATION (EC) No 7762006 of 23 May 2006 amending Annex VII to Regulation

(EC) No 8822004 of the European Parliament and of the Council as regards Community reference

laboratories Official Journal of the European Union 2006 L 136 p 3-8

Available from

httpeur-lexeuropaeuLexUriServLexUriServdouri=OJL200613600030008ENPDF

2 EU Regulation (EC) No 8822004 of the European Parliament and of the Council of 29 April 2004 on

official controls performed to ensure the verification of compliance with feed and food law animal health

and animal welfare rules Official Journal of the European Communities 2004 L191 p 1-52 Available

from

httpeur-lexeuropaeuLexUriServLexUriServdouri=OJL200419100010052ENPDF

3 EU Opinion of the Scientific Committee on Food on the risks to human health of Polycyclic Aromatic

Hydrocarbons in food (2002) Available from

httpeuropaeuintcommfoodfsscscfout153_enpdf

4 International Agency for Research on Cancer IARC Monographs on the Evaluation of

Carcinogenic Risks to Humans Some Non-heterocyclic Polycyclic Aromatic Hydrocarbons and Some

Related Exposures Volume 92 (2010) Available from

httpmonographsiarcfrENGMonographsvol92mono92pdf

5 EU Commission Regulation (EC) No 3332007 of 28 March 2007 laying down the methods of sampling

and analysis for the official control of the levels of lead cadmium mercury inorganic tin 3-MCPD and

benzo(a)pyrene in foodstuffs Official Journal of the European Union 2007 L 88 p 29-38 Available

from

httpeur-lexeuropaeuLexUriServLexUriServdouri=OJL200708800290038ENPDF

6 EU Commission Regulation (EC) No 18812006 of 19 December 2006 setting maximum levels for certain

contaminants in foodstuffs Official Journal of the European Union 2006 L 364 p 5-24 Available from

httpeur-lexeuropaeuLexUriServLexUriServdouri=OJL200636400050024ENPDF

7 EFSA Scientific Opinion of the Panel on Contaminants in the Food Chain on a request from the European

Commission on Polycyclic Aromatic Hydrocarbons in Food The EFSA Journal 2008 724 p 1-114

Available from

httpwwwefsaeuropaeuEFSAefsa_locale-1178620753812_1211902034842htm

30

8 EU Commission Regulation (EU) No 8352011 amending Regulation (EC) No 18812006 as regards

maximum levels for polycyclic aromatic hydrocarbons in foodstuffs Official Journal of the European

Union 2011 L 215 p 4-8

httpeur-lexeuropaeuLexUriServLexUriServdouri=OJL201121500040008ENPDF

9 EU Commission Regulation (EU) No 8362011 amending Regulation (EC) No 3332007 laying down the methods of sampling and analysis for the official control of the levels of lead cadmium mercury inorganic tin 3-MCPD and benzo(a)pyrene in foodstuffs Official Journal of the European Union 2011 L 215 p 9-16 httpeur-lexeuropaeuLexUriServLexUriServdouri=OJL201121500090016ENPDF

10 ISOIEC 170432010 (2010) Conformity assessment -- General requirements for proficiency testing

providers Geneva Switzerland

11 Thompson M Ellison SLR and Wood R The International Harmonized Protocol for the Proficiency

Testing of Analytical Chemistry Laboratories Pure Appl Chem (2006) 78(1) p 145ndash196

Available from httpmediaiupacorgpublicationspac2006pdf7801x0145pdf

31

14 ANNEXES

ANNEX 1 ndash Announcement of the PT on the IRMM webpage

ANNEX 2 ndash Announcement via e-mail and invitation

ANNEX 3 ndash Announcement of sample dispatch

ANNEX 4 ndash Technical specifications of the calibration solutions

ANNEX 5 ndash Homogeneity and stability of the test material

ANNEX 6 ndash Acknowledgement of sample receipt

ANNEX 7 ndash Questionnaire on details of analysis method

ANNEX 8 ndash Results reported for the food supplement sample

ANNEX 9 ndash Results reported for the standard solutions with undisclosed analyte content

ANNEX 10 ndash Method performance characteristics reported by participants

ANNEX 11 ndash Details of analysis methods reported by participants

32

ANNEX 1 ndash Announcement of the PT on the IRMM webpage

33

ANNEX 2 - Announcement via e-mail and invitation

34

ANNEX 3 - Announcement sample dispatch

Dear MadameSir

The test samples for the proficiency test on the determination of four PAHs in a food supplement (dry extract of St Johns wort) were

dispatched today

Explanation on the design of the study as well as instructions for sample storage and for reporting of results are in the parcel You will

also find your participant key in the parcel which is required for reporting of results

Please check the parcel after arrival for completeness and report the receipt of the test samples as well as their status to us by applying

the sample receipt form which is in the parcel

You are kindly requested to inform us by email if you do not receive the parcel by end of this week

My colleague Donata Lerda will then take care of it

With best regards

Thomas Wenzl

35

ANNEX 4 - Specifications of standard solutions with disclosed concentration

36

ANNEX 4 - continued

37

ANNEX 5 - Homogeneity and stability of the test material

Homogeneity of the test material

Benz[a]anthracene - BAA

Benzo[a]pyrene - BAP

38

ANNEX 5 - continued

Benzo[b]fluoranthene - BBF

Chrysene - CHR

39

ANNEX 5 - continued

Stability of the test material

Table 10 Content of the four target analytes in test materials stored under different

temperatures

BaA BaP BbF CHR

microgkg microgkg microgkg microgkg

Content 31 16 31 69 Storage at room temperature

U (k=2) 05 02 05 17

Content 31 15 29 68 Storage at reference temperature

U (k=2) 04 03 04 10

The applied capillary column does not separate CHR from triphenylene

40

ANNEX 6 ndash Documents sent to participants

41

ANNEX 6 - continued

42

ANNEX 7 Questionnaire on details of analysis method

43

ANNEX 7 - continued

44

ANNEX 7 - continued

45

ANNEX 8 - Results reported by participants for the food supplements test sample Table 11 Analysis results reported by the participants for the content of benz[a]anthracene (BAA) in the food supplements test material Values are presented as reported

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

3600 31 29 29 3 05 2

3602 199 193 178 19 021

3603 253 253 246 251 022 2

3604 199 209 23 213 071 2

3605 301 3 271 291 076 2

3606 302 288 278 289 064 2

3607 456 416 443 438 096 2

3608 796 695 651 714 149 2

3610 182 192 214 196 039 2

3611 414 504 488 469 96 2

3612 3 34 28 31 06 2

3613 32 33 34 33 07 2

3614 182 182 191 185 037 2

3615 3884 3822 3565 376 0752 2

3616 2 2 2 2 01 2

3617 366 37 35 362 046 2

3618 32 32 32 32 05 2

3619 299 305 297 299 048 2

3620 304 294 302 302 045 2

3621 29 26 29 28 04 2

3622 343 325 318 322 052 2

3623 35 3 33 33 07 2

3624

3626 318 31 314 314 126 2

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

5301 31 3 32 31 06 2

5302 314 319 315 316 063 2

5303 3102 2716 2822 2876 27 62

5304 261 263 26 261 57 2

5305 23 22 26 24 20

5306 22 23 23 22 11 2

5307 488 461 317 461

5308 168633 168544 1562025 1644598

5309 16 19 16 17

5310 3 304 311 305 015

5311 477 452 448 459 15 2

5312 506 63 494 543 134 2

5313 353 301 404 353

5314 431 373 378 394 5 096

5315 596 531 51 545 11 2

5316 224 231 235 235 022 2

5317 216 23 221 222 007 2

5318 064 07 067 01 2

5319 111 184 146 147 029 2

5320 515 525 564 534 48

5321 21 5322 13 87 9 87 35 2

5323 38 33 35 33 16 2

46

Figure 6 Results of replicate determinations (indicated by triangles) of benz[a]anthracene (BAA) in the food supplement test material

Horizontal blue lines represent the arithmetic mean value of replicate measurements and blue bars the reported expanded measurement uncertainty (k=2) The assigned value is

plotted as dotted line Red solid lines mark the range of satisfactory performance (|z|le2) Mean values are given for results outside the displayed data range

ProLab 2011

Laboratory

53

1853

1953

0853

0936

1436

0236

1036

1653

2136

0453

1753

0653

1653

0536

0353

0436

2153

0336

0636

0536

0036

2036

1953

1036

1253

0136

2653

0236

1836

2336

2236

1353

1353

2336

1736

1553

1453

0736

0753

1153

2053

1253

1536

0853

2236

11

an

aly

te c

on

ten

t (micro

gk

g)

10

95

9

85

8

75

7

65

6

55

5

45

4

35

3

25

2

15

1

05

0

102

3 468

7

Sample St Johns wort extract replicatesMeasurand Benz[a]anthraceneNo of laboratories 46

Assigned value 298 microgkg (Reference value)Target sd 062 microgkgTolerance limits 174 - 422 microgkg (|Z score| lt 200)

Assig

ned va

lue

Limit of tolerance

Limit of tolerance

47

Table 12 Analysis results reported by the participants for the content of benzo[a]pyrene (BAP) in the food supplements test material Values are presented as reported

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

3600 16 17 17 17 03 2

3602 118 123 13 124 015

3603 165 162 157 161 015 2

3604 134 154 139 143 04 2

3605 164 166 172 167 057 2

3606 181 159 161 167 031 2

3607 279 266 279 274 055 2

3608 57 687 461 573 226 2

3610 226 216 183 206 042 2

3611 289 325 311 308 36 2

3612 24 22 2 22 05 2

3613 17 18 19 18 05 2

3614 08 08 08 08 016 2

3615 1545 1393 1425 145 029 2

3616 16 16 14 15 03 2

3617 177 171 158 169 023 2

3618 2 2 2 2 02 2

3619 207 207 206 207 075 2

3620 165 169 163 166 017 2

3621 44 47 49 47 05 2

3622 212 208 222 213 038 2

3623 2 21 22 21 03 2

3624 18 178 182 18 031 2

3626 172 173 178 174 07 2

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

5301 18 18 18 18 04 2

5302 188 19 187 188 038 2

5303 1691 1426 149 1535 19 80

5304 145 142 15 146 69 2

5305 15 17 18 16 15

5306 16 17 17 16 08 2

5307 164 157 153 157

5308 102669 109324 109035 107009

5309 1 1 12 11

5310 122 122 124 123 006

5311 199 184 215 2 15 2

5312 258 248 239 248 05 2

5313 199 178 245 207

5314 174 171 172 172 11 0948

5315 253 248 213 238 05 2

5316 177 171 173 17 016 2

5317 255 277 276 269 012 2

5318 114 12 117 02 2

5319 19 228 231 216 043 2

5320 303 3 344 316 78

5321 12 12 12 12

5322 13 17 24 17 35 2

5323 2 2 19 2 1 2

48

Figure 7 Results of replicate determinations (indicated by triangles) of benzo[a]pyrene (BAP) in the food supplement test material

Horizontal blue lines represent the arithmetic mean value of replicate measurements and blue bars the reported expanded measurement uncertainty (k=2) The assigned value is

plotted as dotted line Red solid lines mark the range of satisfactory performance (|z|le2) Mean values are given for results outside the displayed data range

ProLab 2011

Laboratory

36

1453

0953

0853

1853

2153

1036

0236

0436

1553

0436

1653

0353

0736

0336

2036

0053

0553

0636

0636

0536

1753

1453

1636

2636

1336

2453

0153

2253

0253

2353

1136

1836

1953

1336

1036

2336

2253

1936

1253

1553

1253

1736

0753

2036

2136

0836

11

an

aly

te c

on

ten

t (micro

gk

g)

55

5

45

4

35

3

25

2

15

1

05

0

573

308

3

Sample St Johns wort extract replicatesMeasurand Benzo[a]pyreneNo of laboratories 48

Assigned value 165 microgkg (Reference value)Target sd 036 microgkgTolerance limits 093 - 237 microgkg (|Z score| lt 200)

Assig

ned va

lue

Limit of tolerance

Limit of tolerance

49

Table 13 Analysis results reported by the participants for the content of benzo[b]fluoranthene (BBF) in the food supplements test material Values are presented as reported

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

3600 32 3 3 31 09 2

3602 173 175 16 17 017

3603 265 252 25 256 025 2

3604 116 108 105 11 028 2

3605 286 225 232 247 074 2

3606 294 273 261 276 046 2

3607 579 579 544 567 113 2

3608 103 1201 876 1036 326 2

3610 173 196 204 191 038 2

3611 546 618 565 576 75 2

3612 3 28 28 29 06 2

3613 28 31 32 31 06 2

3614 135 111 135 127 022 2

3615 4187 3678 4111 4 08 2

3616 2 2 2 2 01 2

3617 33 27 299 035 2

3618 31 31 31 31 05 2

3619 326 324 312 326 064 2

3620 277 28 272 276 069 2

3621 26 25 23 25 04 2

3622 346 336 333 34 052 2

3623 26 28 29 28 02 2

3624

3626 288 28 307 292 117 2

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

5301 29 3 29 29 06 2

5302 297 3 296 298 06 2

5303 2705 2444 2608 2586 22 78

5304 245 254 26 253 85 2

5305 23 23 26 24 15

5306 42 43 4 42 21 2

5307 221 216 241 221

5308 232169 246654 240722 229848

5309 19 22 19 2 0

5310 3 315 325 313 016

5311 262 277 304 281 15 2

5312 27 289 337 299 075 2

5313 321 284 378 328

5314 423 404 385 404 34 083

5315 42 506 416 444 09 2

5316 265 267 277 273 018 2

5317 185 197 195 192 006 2

5318 276 281 279 04 2

5319 396 389 403 396 079 2

5320 623 618 635 625 14

5321 31

5322 24 33 49 33 35 2

5323 38 45 35 45 27 2

50

Figure 8 Results of replicate determinations (indicated by triangles) of benzo[b]fluoranthene (BBF) in the food supplement test material

Horizontal blue lines represent the arithmetic mean value of replicate measurements and blue bars the reported expanded measurement uncertainty (k=2) The assigned value is

plotted as dotted line Red solid lines mark the range of satisfactory performance (|z|le2) Mean values are given for results outside the displayed data range

ProLab 2011

Laboratory

36

0436

1436

0236

1053

1736

1653

0953

0753

0853

0536

2136

0553

0436

0353

0353

1636

0636

2036

2353

1853

1136

1236

2653

0153

0253

1236

1736

1336

0036

1853

2153

1036

1953

1336

2253

2253

2353

1936

1553

1453

0653

1536

0753

2036

0836

11

an

aly

te c

on

ten

t (micro

gk

g)

75

7

65

6

55

5

45

4

35

3

25

2

15

1

05

0

103

6

576

3

Sample St Johns wort extract replicatesMeasurand Benzo[b]fluorantheneNo of laboratories 46

Assigned value 292 microgkg (Reference value)Target sd 060 microgkgTolerance limits 172 - 412 microgkg (|Z score| lt 200)

Assig

ned va

lue

Limit of tolerance

Limit of tolerance

51

Table 14 Analysis results reported by the participants for the content of chrysene (CHR) in the food supplements test material Values are presented as reported

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

3600 5 49 5 5 07 2

3602 32 304 29 304 036

3603 462 469 447 459 048 2

3604 438 428 42 429 119 2

3605 365 41 381 386 085 2

3606 494 411 45 451 124 2

3607 897 774 787 819 18 2

3608 986 887 1168 1014 284 2

3610 418 392 462 424 085 2

3611 833 893 87 865 61 2

3612 6 64 58 61 12 2

3613 41 39 4 4 08 2

3614 272 254 288 271 054 2

3615 605 6028 5964 601 12 2

3616 47 45 49 47 05 2

3617 445 431 389 422 065 2

3618 45 45 45 45 06 2

3619 738 747 72 738 118 2

3620 463 472 446 46 092 2

3621 53 49 51 51 06 2

3622 478 513 505 488 08 2

3623 73 73 72 72 05 2

3624

3626 63 622 634 628 251 2

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

5301 49 49 49 49 1 2

5302 687 683 666 679 136 2

5303 6777 6181 6382 6441 24 70

5304 592 581 6 591 85 2

5305 6 58 64 61 20

5306 49 5 51 49 25 2

5307 524 5 574 524

5308 211488 216782 221146 213243

5309

5310 372 393 394 386 019

5311 43 459 422 437 15 2

5312 934 943 981 953 191 2

5313 793 73 99 838

5314 636 561 565 588 18 0818

5315 547 583 499 542 11 2

5316 292 286 285 282 06 2

5317 52 598 517 545 046 2

5318 368 345 357 05 2

5319 449 471 474 465 116 2

5320 76 924 818 834 99

5321 33

5322 7 7 76 7 35 2

5323 87 73 77 73 37 2

52

Figure 9 Results of replicate determinations (indicated by triangles) of chrysene (CHR) in the food supplement test material

Horizontal blue lines represent the arithmetic mean value of replicate measurements and blue bars the reported expanded measurement uncertainty (k=2) The assigned value is

plotted as dotted line Red solid lines mark the range of satisfactory performance (|z|le2) Mean values are given for results outside the displayed data range

ProLab 2011

Laboratory

53

0836

1453

1636

0253

2153

1836

0553

1036

1336

1736

1036

0453

1136

1836

0636

0336

2053

1936

1653

0136

0036

2253

0636

2153

0753

1553

1753

1453

0436

1536

1253

0536

2653

0353

0253

2236

2336

1953

2336

0753

2053

1353

1236

0836

11

an

aly

te c

on

ten

t (micro

gk

g)

13

12

11

10

9

8

7

6

5

4

3

2

1

0

865

3

Sample St Johns wort extract replicatesMeasurand ChryseneNo of laboratories 45

Assigned value 407 microgkg (Reference value)Target sd 083 microgkgTolerance limits 241 - 573 microgkg (|Z score| lt 200)

Assig

ned va

lue

Limit of tolerance

Limit of tolerance

53

Table 15 Analysis results reported by the participants for the sum of the four marker PAHs in the food supplements test material Values are presented as reported

Laboratory code

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg

3600 128 24 2

3602 79 126

3603 1127 096 2

3604 893 258 2

3605 1091 305 2

3606 1184 238 2

3607 2098 42 2

3608 3336 325 2

3610 1138 17 2

3611 2218 259 2

3612 143 29 2

3613 122 2 2

3614 663 129 2

3615 152 304 2

3616 102 03 2

3617 1252 169 2

3618 128 18 2

3619 157 305 2

3620 1204 12 2

3621 151 19 2

3622 1361 115 2

3623 154 09 2

3624

3626 1409 56 2

Laboratory code

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg

5301 126

5302 37 074 2

5303 13438 92

5304 1251 85 2

5305 125 20

5306 129 65 2

5307 1363

5308 7245598

5309

5310 1127 056

5311 1367 15 2

5312 2042 408 2

5313 1726

5314 1558

5315 177 35 2

5316 96 073 2

5317 1229 066 2

5318 82 05 2

5319 1225 267 2

5320 2309 41

5321

5322 207 35 2

5323 171 103 2

54

ANNEX 9 Results reported for the standard solutions with undisclosed analyte content Table 16 Analysis results reported by the participants for the four marker PAHs in the standard solution in toluene with undisclosed analyte

content Values are presented as reported U measurement uncertainty k coverage factor

benz[a]anthracene benzo[a]pyrene benzo[b]fluoranthene chrysene Laboratory

code Unit Result U k Result U k Result U k Result U k 3600 microgl 38 67 2 25 45 2 211 68 2 275 38 2

3605 microgl 367 184 2 2482 124 2 2592 13 2 235 118 2

3606 microgkg 3763 176 2 2609 142 2 2721 118 2 2539 134 2

3607 microgkg 3706 815 2 2824 565 2 2591 518 2 2587 569 2

3612 microgl 38 08 2 237 11 2 244 18 2 257 02 2

3614 microgl 324 65 2 209 38 2 215 37 2 21 42 2

3616 microgkg 318 07 2 224 14 2 231 26 2 216 1 2

3618 microgkg 417 17 2 284 08 2 292 06 2 297 03 2

3619 microgkg 47 742 2 274 468 2 228 387 2 274 434 2

3620 microgl 495 74 2 294 29 2 321 8 2 321 64 2

3621 microgl 241 22 2 143 13 2 188 16 2 145 14 2

3623 microgkg 476 25 2 311 53 2 269 3 2 297 03 2

3626 microgl 359 38 2 235 23 2 238 27 2 24 18 2

5301 microgkg 426 29 299 289

5302 microgkg 436 87 2 296 59 2 296 59 2 289 58 2

5303 microgl 3721 27 102 22838 19 99 23756 22 99 25486 24 98

5304 microgkg 38 29 2 225 26 2 257 37 2 258 52 2

5305 microgkg 305 225 245 29

5306 microgkg 316 63 2 247 49 2 65 13 2 22 44 2

5307 microgkg 3658 2559 241 2414

5308 microgkg 16854 109324 246654 216782

5311 microgl 374 20 2 279 15 2 268 15 2 272 15 2

5313 microgl 3408 2165 2275 2373

5314 microgkg 395 5 2875 11 3614 34 2905 18

5320 microgl 3267 368 1986 571 28 683 2427 512

5322 microgl 282 35 2 225 35 2 241 35 2 21 35 2

55

Figure 10 Youden plots of the percent deviation of the reported values from the assigned values for the food supplement test sample and the

standard solution in toluene with undisclosed content

(The identity of the respective measurand is given above the figure Results exceeding a difference of more than 100 were omitted)

56

Figure 10 - continued

57

Table 17 Analysis results reported by the participants for the four marker PAHs in the standard solution in acetonitrile with undisclosed

analyte content

Values are presented as reported U measurement uncertainty k coverage factor

benz[a]anthracene benzo[a]pyrene benzo[b]fluoranthene chrysene

Laboratory

code Unit Result U k Result U k Result U k Result U k

3602 microgkg 3675 247 247 244

3603 microgkg 358 086 2 236 028 2 235 028 2 236 057 2

3604 microgl 475 29 2 19 12 2 194 13 2 258 12 2

3608 microgl 3623 078 2 2385 023 2 2343 18 2 2296 092 2

3610 microgkg 369 185 2 25 125 2 251 126 2 244 122 2

3611 microgkg 48 02 2 323 02 2 328 04 2 317 03 2

3613 microgl 332 31 2 261 3 2 249 25 2 239 24 2

3615 microgl 415 2635 2729 2689

3617 microgkg 37 09 2 244 06 2 284 07 2 239 06 2

3622 microgl 37 12 2 25 23 2 257 21 2 238 1 2

3624 microgkg 402 104 2 255 084 2 265 096 2 253 07 2

5306 microgkg 563 113 2 371 74 2 359 72 2 333 67 2

5308 microgkg 168633 102669 232169 211488

5309 microgl 402 268 27

5310 microgl 315 158 25 125 25 125 288 144

5312 microgl 428 043 2 282 028 2 272 027 2 275 028 2

5315 microgkg 387 08 2 256 05 2 251 05 2 253 05 2

5316 microgl 3839 342 2 2485 191 2 2559 168 2 239 51 2

5317 microgl 2466 043 2 2212 034 2 2472 016 2 2146 068 2

5319 microgl 414 41 2 279 28 2 268 27 2 26 31 2

5321 microgkg 476 316 326 328

5322 microgl 2547 35 2

5323 microgkg 405 203 2 239 119 2 255 153 2 201 11 2

58

Figure 11 Youden plots of the percent deviation of the reported values from the assigned values for the food supplement test sample and the

standard solution in acetonitrile with undisclosed content

(The identity of the respective measurand is given above the figure Results exceeding a difference of more than 100 were omitted)

59

Figure 11 - continued

60

ANNEX 10 Table 18 Method performance parameters reported by the participants

Laboratory code

Analyte LOD LOQ Recovery Analyte LOD LOQ Recovery

microgkg microgkg microgkg microgkg 3600 BaA 03 06 978 BaP 03 07 9483602 BaA 007 021 926 BaP 005 015 993603 BaA 0011 05 987 BaP 0005 051 9553604 BaA 02 04 86 BaP 02 04 883605 BaA 0025 005 118 BaP 0025 005 1083606 BaA 001 003 65 BaP 001 003 703607 BaA 021 071 74 BaP 027 089 943608 BaA 016 048 8213 BaP 015 046 83683610 BaA 02 05 85 BaP 005 02 853611 BaA BaP 3612 BaA 1 117 BaP 1 1203613 BaA 01 03 90 BaP 01 03 903614 BaA 02 05 96 BaP 02 05 933615 BaA 005 015 110 BaP 005 015 1053616 BaA 01 05 108 BaP 01 05 753617 BaA 018 037 405 BaP 009 018 3873618 BaA 01 03 63 BaP 01 03 593619 BaA 005 005 59 BaP 033 033 543620 BaA 0009 0018 64 BaP 0005 001 553621 BaA 007 02 84 BaP 007 02 923622 BaA 01 03 1101 BaP 01 03 9853623 BaA 02 08 105 BaP 02 08 1013624 BaA BaP 01 033 8513625 BaA BaP 3626 BaA BaP 5301 BaA 003 01 102 BaP 003 01 1005302 BaA 01 03 78 BaP 01 03 865303 BaA 003 01 62 BaP 003 01 805304 BaA 1 1 70 BaP 1 1 955305 BaA 02 05 85 BaP 02 05 855306 BaA 03 1 699 BaP 03 1 905307 BaA 6537 BaP 81165308 BaA 0088 0265 99 BaP 00262 00785 1005309 BaA 01 08 92 BaP 01 08 925310 BaA 01 03 BaP 01 03 905311 BaA 002 005 80 BaP 002 004 915312 BaA 03 09 105 BaP 03 09 1055313 BaA 04 08 BaP 04 08 5314 BaA 001 002 96 BaP 001 002 9485315 BaA 01 03 50 - 100 BaP 01 03 50 - 100 5316 BaA 002 011 827 BaP 004 019 10445317 BaA 05 1 76 BaP 05 1 755318 BaA BaP 5319 BaA 009 029 951 BaP 011 041 995320 BaA 12 18 946 BaP 06 1 8345321 BaA 03 05 BaP 05 1 5322 BaA 1 2 100 BaP 1 2 1005323 BaA 26 BaP 26

61

ANNEX 10 Table 18 - continued

Laboratory code Analyte LOD LOQ Recovery Analyte LOD LOQ Recovery

microgkg microgkg microgkg microgkg 3600 BbF 03 05 96 CHR 02 03 10153602 BbF 015 045 947 CHR 003 009 9193603 BbF 0014 05 1012 CHR 0004 05 10033604 BbF 02 04 85 CHR 02 04 873605 BbF 005 01 94 CHR 0025 005 863606 BbF 001 003 70 CHR 001 003 653607 BbF 018 06 100 CHR 014 028 943608 BbF 015 046 7979 CHR 015 045 71253610 BbF 005 02 85 CHR 02 05 853611 BbF CHR 3612 BbF 1 118 CHR 1 1173613 BbF 01 03 90 CHR 01 03 903614 BbF 01 04 96 CHR 02 05 953615 BbF 008 025 109 CHR 003 01 1083616 BbF 01 05 78 CHR 01 05 723617 BbF 035 07 38 CHR 012 024 4163618 BbF 01 03 62 CHR 01 03 623619 BbF 016 016 63 CHR 01 01 593620 BbF 0003 0006 55 CHR 001 002 653621 BbF 007 02 103 CHR 007 02 893622 BbF 02 06 963 CHR 03 09 1043623 BbF 02 08 106 CHR 02 08 1193624 BbF CHR 3625 BbF CHR 3626 BbF CHR 5301 BbF 003 01 96 CHR 003 01 1145302 BbF 01 03 84 CHR 01 03 835303 BbF 003 01 78 CHR 003 01 705304 BbF 1 1 86 CHR 1 1 705305 BbF 02 05 90 CHR 02 05 955306 BbF 03 1 1435 CHR 03 1 1185307 BbF 8116 CHR 65375308 BbF 0079 0238 99 CHR 009 026 1005309 BbF 01 08 92 CHR 5310 BbF 07 2 CHR 01 03 5311 BbF 002 004 95 CHR 002 004 895312 BbF 03 09 105 CHR 03 09 1055313 BbF 04 08 CHR 04 08 5314 BbF 001 002 83 CHR 001 002 8185315 BbF 02 05 50 -100 CHR 02 05 50 - 100 5316 BbF 005 024 814 CHR 001 007 9595317 BbF 05 1 73 CHR 05 1 785318 BbF CHR 5319 BbF 011 038 946 CHR 013 043 9675320 BbF 07 09 1194 CHR 23 32 10455321 BbF 05 1 CHR 05 1 5322 BbF 1 2 100 CHR 1 2 1005323 BbF 26 CHR 26

62

ANNEX 11 Table 19 Details of analysis method reported by the participants Data are presented as reported

Laboratory code

Analysis technique

Instrument calibration

Internal standards applied

Sample clean up

Details of sample

clean up

Extraction technique

Details of sample

extraction

3600 6) GC-MS

2) Internal standardisation with labelled substances

Deuterated PAH mix 9 (Ehrenstorfer)

1) Saponification 2) LiquidLiquid partitioning

D) Other Liquidliquid partitioning

3602 2) HPLC-UV-FLD

1) External calibration

3) Gel permeation chromatography

A) Pressurised liquid extraction (PLE)

3603 1) HPLC-FLD

1) External calibration

3) Gel permeation chromatography

D) Other 1 minute with dichloromethane in vortex

3604 2) HPLC-UV-FLD

2) Internal standardisation with labelled substances

DiP D14

5) Donor acceptor complex chromatography

D) Other LL

3605 1) HPLC-FLD

1) External calibration

3) Gel permeation chromatography

C) Soxhlet extraction

3606 7) GC-MSMS

2) Internal standardisation with labelled substances

BaP 13C4 CHR 13C6 BaA 13C6 BbF 13C6

4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

3607 6) GC-MS

2) Internal standardisation with labelled substances

BaA-d12 CHR-d12 BbF-d12 BkF-d12 BaP-d12 ICP-d12 BgP-d12 DiP-d14

1) Saponification

D) Other solvent partitioning

3608 1) HPLC-FLD

1) External calibration

6) Other Liquid extraction

D) Other

Extraction in a rotary agitator and centrifugation

3610 2) HPLC-UV-FLD

1) External calibration

4) Solid phase extraction

B) Sonication

3611

3612 6) GC-MS

2) Internal standardisation with labelled substances

chrysene-d12 dibenzo(ah)anthracene-d14 dibenzo(ai)pyrene-d14

4) Solid phase extraction

B) Sonication

3613 5) LC-MSMS

3) Internal standardisation with unlabelled substances

I use Chrysene-D12 3) Gel permeation chromatography

D) Other extraction with hexan

63

Table 19 - continued

Laboratory code

Analysis technique

Instrument calibration

Internal standards applied

Sample clean up Details of sample

clean up

Extraction technique

Details of sample

extraction

3614 6) GC-MS 1) External calibration

3) Gel permeation chromatography

B) Sonication

3615 1) HPLC-FLD

1) External calibration

6) Other PLE GPC

A) Pressurised liquid extraction (PLE)

3616 6) GC-MS

2) Internal standardisation with labelled substances

Benzo(a)pyrene-13C4 Benzo(a)anthracene-13C6 Benzo(b)fluoranthene-13C6 Chrysene-13C6

2) LiquidLiquid partitioning 4) Solid phase extraction

B) Sonication

3617 1) HPLC-FLD

1) External calibration

4) Solid phase extraction

C) Soxhlet extraction

3618 6) GC-MS

2) Internal standardisation with labelled substances

deuterated standards

1) Saponification 4) Solid phase extraction

D) Other Handshaken with cyclohexane

3619 6) GC-MS

2) Internal standardisation with labelled substances

C13 Labelled US EPA 16

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

D) Other Homegenisation amp saponiofication

3620 9) GC-HRMS

2) Internal standardisation with labelled substances

mix of deuterated PAHs (BAA-D12 CHR-D12 BAP-D12 BBF-D12)

3) Gel permeation chromatography 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

3621 6) GC-MS

2) Internal standardisation with labelled substances

Chrysene D12 Benzo(a)pyrene D12 Benzo(b)fluoranthene D12 Benzo(a)anthracene

2) LiquidLiquid partitioning 4) Solid phase extraction

B) Sonication

3622 1) HPLC-FLD

4) Standard addition method

4) Solid phase extraction

D) Other

Extraction with cyclohexane by heating under reflux

3623 7) GC-MSMS

2) Internal standardisation with labelled substances

C13 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

3624 2) HPLC-UV-FLD

1) External calibration

2) LiquidLiquid partitioning

D) Other hot solvent extraction

3626

64

Table 19 - continued

Laboratory code

Analysis technique

Instrument calibration

Internal standards

applied

Sample clean up

Details of sample

clean up

Extraction technique

Details of sample

extraction

5301 7) GC-MSMS

2) Internal standardisation with labelled substances

EACH SUBSTANCE C13

4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5302 7) GC-MSMS

2) Internal standardisation with labelled substances

Isotope labelled PAHs 13C for each analyte

4) Solid phase extraction

C) Soxhlet extraction

5303 7) GC-MSMS

3) Internal standardisation with unlabelled substances

HAP C13 4) Solid phase extraction

D) Other automate ASE200 DIONEX

5304 7) GC-MSMS

2) Internal standardisation with labelled substances

with C13 for each HAP

2) LiquidLiquid partitioning 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5305 7) GC-MSMS

2) Internal standardisation with labelled substances

C13 isotopiques internal standards

4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5306 7) GC-MSMS

2) Internal standardisation with labelled substances

C13 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5307 6) GC-MS

2) Internal standardisation with labelled substances

1) Saponification 2) LiquidLiquid partitioning

D) Other solvent extraction

5308 1) HPLC-FLD 1) External calibration

4) Solid phase extraction

B) Sonication

5309 1) HPLC-FLD 1) External calibration

4) Solid phase extraction

D) Other only SPE

5310 1) HPLC-FLD 1) External calibration

1) Saponification 2) LiquidLiquid partitioning 3) Gel permeation chromatography

C) Soxhlet extraction

5311 6) GC-MS

2) Internal standardisation with labelled substances

13 C Chrysene 13 C Benz(a)pyrene

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

B) Sonication

5312 1) HPLC-FLD

3) Internal standardisation with unlabelled substances

Benzo(b)chrysen

2) LiquidLiquid partitioning 4) Solid phase extraction

Quechers D) Other Quechers

65

Table 19 - continued

Laboratory code

Analysis technique

Instrument calibration

Internal standards applied

Sample clean up Details of sample

clean up

Extraction technique

Details of sample extraction

5313 6) GC-MS

2) Internal standardisation with labelled substances

Benz(a)anthracene D12 Benzo(b)fluranthene D12 Benzo(a)pyrene D12 Chrysene D12

1) Saponification 2) LiquidLiquid partitioning 3) Gel permeation chromatography

A) Pressurised liquid extraction (PLE)

5314 6) GC-MS

2) Internal standardisation with labelled substances

Benzo(a)pyren d12 2) LiquidLiquid partitioning

D) Other Extraction with

5315 1) HPLC-FLD

3) Internal standardisation with unlabelled substances

Benz(b)chrysen

1) Saponification 2) LiquidLiquid partitioning

D) Other shaker

5316 1) HPLC-FLD

1) External calibration

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

D) Other

LiquidLiquid extraction with cyclohexane and dimethylformamide

5317 7) GC-MSMS

1) External calibration

3) Gel permeation chromatography 6) Other

silica gel D) Other VDLUFA online method

5318

5319 1) HPLC-FLD

3) Internal standardisation with unlabelled substances

Benzo(b)chrysene 3) Gel permeation chromatography

A) Pressurised liquid extraction (PLE)

5320 6) GC-MS

3) Internal standardisation with unlabelled substances

22-binaphthyl

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

D) Other 3 hours refluxing

5321 1) HPLC-FLD

2) Internal standardisation with labelled substances

6-meacutethylchrysegravene

1) Saponification 2) LiquidLiquid partitioning

B) Sonication

5322 6) GC-MS

2) Internal standardisation with labelled substances

naftalene-d8 acenaphtalene-d10 phenanthrene-d10 chrysene-d12 perylene-d12 benzo(e)anthracene-d12

1) Saponification 2) LiquidLiquid partitioning 6) Other

clean-up with silica packed column

C) Soxhlet extraction

5323 1) HPLC-FLD

1) External calibration

4) Solid phase extraction 6) Other

Accelerated Solvent Extraction

A) Pressurised liquid extraction (PLE)

66

European Commission EUR 25245 EN ndash Joint Research Centre ndash Institute for Reference Materials and Measurements Title Report on the 8th inter-laboratory comparison organised by the European Union Reference Laboratory for Polycyclic Aromatic Hydrocarbons - Four marker PAHs in a dry extract of St Johns wort Author(s) Radoslav Lizak Szilard Szilagyi Philippe Verlinde and Thomas WENZL Luxembourg Publications Office of the European Union 2012 ndash 66 pp ndash 201 x 297 cm EUR ndash Scientific and Technical Research series ndash ISSN 1831-9424 ISBN 978-92-79-23337-1 doi 10278759387 Abstract The proficiency test here reported concerned the determination of the contents of four marker polycyclic aromatic hydrocarbons (PAHs) and of their sum in an food supplements test sample The set of marker PAHs consists of benz[a]anthacene benzo[a]pyrene benzo[b]fluoranthene and chrysene Participants to these PT were National Reference Laboratories for PAHs (NRLs-PAHs) and EU official food control laboratories The number of participants was in total 48

The PT was organised according ISO Standard 170432010

The test material used was a commercial dry extract of St Johns wort Participants also received a solution of the same PAHs either in acetonitrile or in toluene for checking their instrument calibration

The results from participants were rated with z-scores and zeta-scores About 68 of the reported results were attributed with z-scores with an absolute value of below two which is the threshold for satisfactory performance

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the common interest of the Member States while being independent of special

interests whether private or national

L

A-N

A-252

45-EN

-N

• 1 Summary
• 2 Introduction
• 3 Scope
• 4 Participating Laboratories
• 5 Time frame
• 6 Confidentiality
• 7 Test materials
• • 71 1Preparation of test materials
  • 72 Assigned values and standard deviation for proficiency assessment
  • 73 Homogeneity and stability
  • 74 Sample dispatch
  • • 8 Design of the proficiency test
    • 9 Evaluation of the results
    • • 91 2General
      • 92 21BEvaluation criteria
      • 93 Performance of participants
      • 94 Evaluation of the influence of analysis method
      • 95 Evaluation of the influence of calibration
      • 96 26Evaluation of compliance with legislation
      • • 10 Follow-up actions for underperforming laboratories
        • 11 1Conclusions
        • 12 1Acknowledgements
        • 13 References

12

The uncertainty of stability of the test material was considered negligible compared to the uncertainty

of the characterisation measurements Therefore this parameter was not taken into account in the

determination of the uncertainties of the assigned values

74 Sample dispatch

Samples were dispatched at room temperature Each participant received at least

- one ampoule of the solution of the 15+1 EU priority PAHs in the chosen solvent with disclosed

content (2 ml)

- one ampoule of the 15+1 EU priority PAHs in the chosen solvent with undisclosed content (2

ml)

- one screw cap vial with the food supplement test material

Each parcel contained a sample receipt form the outline of the study an appropriate material safety

data sheets and a unique code that was required for reporting of results The documents sent to the

participants are presented in ANNEX 6

8 Design of the proficiency test

The design of the PT foresaw triplicate analyses of the test sample and reporting of the individual

results of replicate analyses for the single analytes in the following denoted as FS_REP and

additionally a value for proficiency assessment in the following denoted as final value - FS_FIN

for both the single analytes and the sum of the four marker PAHs Both FS_REP results and FS_FIN

results had to be reported corrected for recovery (and recovery had to be stated in the questionnaire

which participants were asked to fill in together with other parameters of the method applied) the

latter had also to be accompanied by the respective expanded measurement uncertainty The FS_FIN

results were used for performance assessment

Besides analysis results participants were also asked to report details of the applied analysis method

and to provide answers to a questionnaire (see ANNEX 7 for the template and the compiled returned

questionnaires)

13

9 Evaluation of the results

The results reported by participants are listed in ANNEX 8

91 General

The most important evaluation parameter was the performance of the laboratories in the determination

of the target PAHs in the food supplement test material which was expressed by z-scores and zeta-

scores

The compliance of method performance characteristics specified by the participants with provisions

given in legislation was evaluated too

92 Evaluation criteria

The participants were requested to report for all analytes the results of replicate measurements and a

value for proficiency assessment which is the result they wish to be applied for the calculation of

performance indicators z-Scores and zeta-scores were attributed only to these results The individual

results of replicate analyses were not rated

z-Scores

z-scores were calculated based on the FS_FIN values Equation 1 presents the formula for calculation

of z-scores

Equation 1

P

assignedlab Xxz

where z refers to the z-score xlab to the reported ldquovalue for proficiency assessmentrdquo Xassigned to the

assigned value and σP to the standard deviation for proficiency testing

zeta-Scores

In addition to z-scores zeta-scores were calculated In contrast to z-scores zeta-scores describe the

agreement of the reported result with the assigned value within the respective uncertainties

Unsatisfactorily large zeta-scores might be caused by underestimated measurement uncertainties large

bias or a combination of both Zeta-scores were calculated according to Equation 3

14

Equation 3 22assignedlab

assignedlab

uu

Xxzeta

where zeta refers to the zeta-score xlab to the reported ldquovalue for proficiency assessmentrdquo Xassigned to

the assigned value ulab to the measurement uncertainty reported by the laboratory and uassigned to the

uncertainty of the assigned value

The performance of the laboratories was classified according to ISOIEC 170432010 [10] Following

scheme is applied for the interpretation of zeta scores and z-scores

|score| 20 = satisfactory performance

20lt|score| lt 30 = questionable performance

|score| ge 30 = unsatisfactory performance

93 Performance of participants

The 48 participants in the study reported in total 225 results for proficiency evaluation which equals to

about 94 of the 240 possible results About 68 of the reported results were rated as satisfactory

with regard to z-scores

Figure 1 and Figure 2 give an overview of the z-scores assigned to the respective results The larger

the triangles the larger were the differences to the assigned values Red triangles indicate z-scores

above an absolute value of three whereas yellow triangles represent z-scores in the questionable

performance range The corresponding score values are plotted next to the triangles About 40 of the

72 non-satisfactory results were reported by seven laboratories only eg the performance of

participant 3608 was not satisfactory for the majority of target analytes

It is also obvious that most problems were encountered in the determination of chrysene and the sum

of the four analytes Potential reasons for the difficulties related to chrysene are discussed further

down

The ideal situation would be that deviations from the assigned values of the results reported for the

individual analytes are small and randomly distributed around zero This will give a satisfactory

performance statement for the sum parameter Deviations of opposite sign could also cancel out and

lead to a satisfactory performance for the sum parameter (eg participants 5303 5304 and 5305)

However in most cases a single underperformance for one analyte led also to non-satisfactory rating

for the sum parameter

15

The numerical values of the calculated z-scores are compiled in Table 6 and Table 7 z-Scores with an

absolute value of above 2 are given in red bold font

The results of the data evaluation for the individual analytes are given in ANNEX 8

For each analyte the figure shows the individual analysis results of the three replicate determinations

The assigned value is shown as dotted line The blue boxes represent the expanded uncertainties as

reported by participants for the value for proficiency assessment The arithmetic mean of the results

of the individual participants is indicated in the blue boxes by a blue line The satisfactory performance

range is located between the two red lines

The individual results of the replicate measurements and the value for proficiency assessment with

its accompanying expanded measurement uncertainty (k=2) are listed in the tables in ANNEX 8 as

well

16

Figure 1 Graphical presentation of z-scores corresponding to the values for proficiency assessment

reported by the NRLs for the contents of BAA BAP BBF and CHR in the St Johns wort extract test

material

Blue triangles indicate satisfactory performance yellow triangles indicate questionable performance red triangles indicate non-satisfactory performance z-score values are presented above the triangles for the latter two performance categories

ProLab 2011

Z score-3 0 3 -3 0 3 -3 0 3 -3 0 3 -3 0 3

BAA BAP BBF CHR PAH4

La

bo

rato

ry

36003602

3603

36043605

36063607

36083610

36113612

36133614

36153616

36173618

36193620

36213622

36233624

3626

2258

2343

2452

2669

2147

2788

2869

6710

70839

3011

11271

80525

8425

4561

12338

90680

4976

7331

99553

3998

3780

7500

17420

168413

3269

3029

-2348 -2736

-2023 -2981

-2155-3018

-3998

Figure 2 Graphical presentation of z-scores corresponding to the values for proficiency assessment

reported by the OCLs for the contents of BAA BAP BBF and CHR in the St Johns wort extract test

material

Blue triangles indicate satisfactory performance yellow triangles indicate questionable performance red triangles indicate non-satisfactory performance z-score values are presented above the triangles for the latter two performance categories

ProLab 2011

Z score-3 0 3 -3 0 3 -3 0 3 -3 0 3 -3 0 3

BAA BAP BBF CHR PAH4

La

bo

rato

ry

5301

5302

5303

5304

5305

5306

5307

5308

5309

5310

5311

5312

5313

5314

5315

5316

5317

5318

5319

5320

5321

5322

5323

2597

2629

2017

2293

2873

2123

2521

2620

2186

2222

2452

2864

3952

3984

3806

9226

4171 5522

3285

6594

5205

5157

3539

3901

7051

4519

3173

4872

9191

7276

4391

-2435

-2154

-2065

-2340

-2740-3726

-6346

-3505

17

Table 6 Compilation of z-scores calculated from the ldquoresults for proficiency assessment

reported by the NRLs for food supplement test material z-scores outside the

satisfactory range (|z| gt 2) are indicated by red font Values are reproduced as reported

by the laboratories Empty cells denote analytes for which results were not reported

Measurand benz[a]anthracene benzo[a]pyrene benzo[b]fluoranthene chrysene Sum PAH 4

Assigned value

microgkg 298 165 292 407 1162

Standard deviation for proficiency assessment

microgkg 062 036 060 083 125

Laboratory code

Result z-score Result z-score Result z-score Result z-score Result z-score

microgkg microgkg microgkg microgkg microgkg

3600 3 00 17 01 31 03 5 11 128 09

3602 19 -18 124 -11 17 -20 304 -12 79 -30

3603 251 -08 161 -01 256 -06 459 06 1127 -03

3604 213 -14 143 -06 11 -30 429 03 893 -22

3605 291 -01 167 01 247 -07 386 -03 1091 -06

3606 289 -01 167 01 276 -03 451 05 1184 02

3607 438 23 274 30 567 46 819 50 2098 75

3608 714 67 573 113 1036 123 1014 73 3336 174

3610 196 -17 206 11 191 -17 424 02 1138 -02

3611 469 708 308 805 576 907 865 996 2218 1684

3612 31 02 22 15 29 00 61 25 143 21

3613 33 05 18 04 31 03 4 -01 122 05

3614 185 -18 08 -23 127 -27 271 -16 663 -40

3615 376 13 145 -06 4 18 601 23 152 29

3616 2 -16 15 -04 2 -15 47 08 102 -11

3617 362 10 169 01 299 01 422 02 1252 07

3618 32 04 2 10 31 03 45 05 128 09

3619 299 00 207 12 326 06 738 40 157 33

3620 302 01 166 00 276 -03 46 06 1204 03

3621 28 -03 47 84 25 -07 51 12 151 28

3622 322 04 213 13 34 08 488 10 1361 16

3623 33 05 21 12 28 -02 72 38 154 30

3624 18 04

3626 314 03 174 02 292 00 628 27 1409 20

18

Table 7 Compilation of z-scores calculated from the ldquoresults for proficiency assessment

reported by the OCLs for food supplement test material z-scores outside the

satisfactory range (|z| gt 2) are indicated by red font Values are reproduced as reported

by the laboratories Empty cells denote analytes for which results were not reported

Measurand benz[a]anthracene benzo[a]pyrene benzo[b]fluoranthene chrysene Sum PAH 4

Assigned value

microgkg 298 165 292 407 1162

Standard deviation for proficiency assessment

microgkg 062 036 060 083 125

Laboratory code

Result z-score Result z-

score Result z-score Result

z-score

Result z-score

microgkg microgkg microgkg microgkg microgkg

5301 31 02 18 04 29 00 49 10 126 08

5302 316 03 188 06 298 01 679 33 37 -63

5303 2876 -02 1535 -03 2586 -06 6441 29 13438 15

5304 261 -06 146 -05 253 -06 591 22 1251 07

5305 24 -09 16 -01 24 -09 61 25 125 07

5306 22 -13 16 -01 42 21 49 10 129 10

5307 461 27 157 -02 221 -12 524 14 1363 16

5308 16446 -22 107009 -16 229848 -10 213243 -23 72456 -35

5309 17 -21 11 -15 2 -15

5310 305 01 123 -12 313 03 386 -03 1127 -03

5311 459 26 2 10 281 -02 437 04 1367 16

5312 543 40 248 23 299 01 953 66 2042 71

5313 353 09 207 12 328 06 838 52 1726 45

5314 394 16 172 02 404 19 588 22 1558 32

5315 545 40 238 20 444 25 542 16 177 49

5316 235 -10 17 01 273 -03 282 -15 96 -16

5317 222 -12 269 29 192 -17 545 17 1229 05

5318 067 -37 117 -13 279 -02 357 -06 82 -27

5319 147 -24 216 14 396 17 465 07 1225 05

5320 534 38 316 42 625 55 834 52 2309 92

5321 12 -12

5322 87 92 17 01 33 06 7 35 207 73

5323 33 05 2 10 45 26 73 39 171 44

19

Table 8 and Table 9 present the respective zeta-scores As for the z-scores data outside the

satisfactory performance range are given in red bold font Zeta scores were only calculated for

laboratories that provided measurement uncertainty data as well as a numerical value for the coverage

factor Zeta scores were not calculated when the reported uncertainty value exceeded the content value

which is probably linked to mixing of reporting units (microgkg and ) However the participants are

reminded that the measurement uncertainty shall be expressed in the same unit as the content value

The assessment of the performance of the participants based on the reported measurement uncertainty

gave a less favourable picture compared to z-scores Only 60 of the zeta-scores are within the

satisfactory performance range The distribution of satisfactory performance ratings between NRLs

and OCLs is not balanced It was higher for the NRLs

It has to be noted that the magnitude of the zeta-scores were for some participants much higher than

the z-scores attributed to the same results Consequently the laboratories perform according to fitness-

for-purpose criterion specified in legislation which forms the basis for the z-scores but seem to have

difficulties in deducing realistic measurement uncertainty values Eg the relative standard

measurement uncertainties reported by the NRLs ranged from 25 to 20 with a median value of

about 10 Only two OCLs reported measurement uncertainties that exceeded the maximum tolerable

uncertainty (Uf) The respective data is highlighted in yellow in Table 9

However the EU-RL PAHs will continue to pay special attention to measurement uncertainty in future

ILCs as it has major implications on the assessment of compliance of food with European legislation

20

Table 8 Compilation of zeta-scores calculated from the ldquoresults for proficiency assessment

reported by the NRLs for test material St Johns wort the reported measurement

uncertainty and the uncertainty of the analyte content of the test material zeta-scores

outside the satisfactory range (|zeta| gt 2) are indicated by red bold font Empty cells

denote analytes for which either results or measurement uncertainties were not

reported

21

Table 9 Compilation of zeta-scores calculated from the ldquoresults for proficiency assessment

reported by the OCLs for test material St Johns wort the reported measurement

uncertainty and the uncertainty of the analyte content of the test material zeta-scores

outside the satisfactory range (|zeta| gt 2) are indicated by red bold font yellow

highlighted cells ugtUf Empty cells denote analytes for which results were not

reported

22

94 Evaluation of the influence of analysis method

The information on the applied analysis method (ANNEX 11) was used to group the performance

indicators attributed to the reported results according to the applied analysis technique Two groups of

data were formed They represent results obtained by high performance liquid chromatography-

fluorescence detection (HPLC-FD about 40 ) and by gas chromatography-mass spectrometry (GC-

MS about 60 ) respectively Kernel density plots were constructed based on the respective z-scores

Figure 3A to 3E show the different Kernel density plots

Figure 3 Kernel density plots of z-scores attributed to the results for proficiency assessment

which were reported for the food supplements test sample Results were classified

according to the applied analysis technique

A) benz[a]anthracene

B) benzo[a]pyrene

23

Figure 3 continued

C) benzo[b]fluoranthene

D) chrysene

E) sum of four marker PAHs

The kernel density plots for BAP and BBF are almost identical and centred at or close to a z-score

value of zero as can be seen in Figure 3B and 3C Therefore bias introduced by the type of analysis

method is unlikely for these two analytes The situation is different for BAA and CHR In case of

BAA the kernel density distribution of z-scores based on HPLC-FD is shifted to negative values

24

(Figure 3A) which indicates a tendency to underestimate the BAA content in this particular

samplematrix when applying HPLC-FD This might be reasoned by separationintegration problems

between BAA and CHR peaks A few participants (from the HPLC-FD subgroup) that got negative

z-scores for BAA overestimated the CHR content This caused the slight shift of the density trace for

HPLC-FD towards negative values Removing this data from the evaluation would result in a density

trace with the mode at zero

The opposite is the case for CHR (Figure 3D) The density distribution of the z-scores attributed to

HPLC-FD data is centred almost at zero However the distribution for data based on GC-MS is shifted

to positive values indicating strong positive bias This bias might be caused by a lack of selectivity of

the analysis method The test material contains triphenylene which is isobaric to chrysene

Consequently it cannot be distinguished from chrysene by means of mass spectrometry These two

compounds have to be separated chromatographically which is not easy They coelute on unpolar and

on most mid-polar capillary columns Columns that allow their separation were commercialised only

recently and might not be available in all laboratories Therefore laboratories which determine the

four EU marker PAHs by means of GC-MS shall pay attention to the chromatographic separation of

triphenylene and chrysene A lack of chromatographic resolution might lead to strong positive bias of

GC-MS methods and to false conclusions on the marketability of the tested sample

The chromatographic separation of chrysene and triphenylene is not a problem with HPLC which is

reflected in the position of the density plot for chrysene However attention has to be paid in HPLC-

FD analysis to a proper clean up of the sample extract in order to eliminate interferences that show

similar retention as BAA and CHR

95 Evaluation of the influence of calibration

The participants received together with the food supplement test sample two standard solutions in

depending of their choice either acetonitrile or toluene The analyte content of one solution was

disclosed to the participants whereas the content of the other solution was kept undisclosed The

content values of the latter are presented in Table 5 Analysis results for the solution with undisclosed

content had to be reported to the PT organiser The respective data are presented in Annex 9

Percent deviations of the reported results from the assigned values were calculated for both the food

supplement test sample and the standard solution with undisclosed analyte content Youden plots were

prepared in order to visualize the influence of instrument calibration on the results reported for the

food supplement sample Two plots are discussed in the following The whole set of Youden plots are

given in Annex 9

25

Figure 4 Youden plot of the percent deviations of values reported for benzo[a]pyrene from the

assigned values of the food supplement test material and the standard solution in

toluene with undisclosed content

Figure 4 reveals that a number of participants reported results with similar percent deviations from the

assigned values of both the results for the food supplement material and the standard solution in

toluene with undisclosed content This correlation indicates that over- respectively underestimation of

the analyte content of the food supplement material might be caused by biased instrument calibration

This finding underpins the importance of thorough calibration standard preparation However the data

was not in all cases conclusive as some laboratories performed well for the food supplement samples

but reported biased results for the standard solution with undisclosed content (eg Figure 4 participants

3620 and 5305) This fact cannot be explained yet

Figure 5 presents the respective Youden plot for chrysene in the food supplement test material and the

standard solution in toluene with undisclosed content From this graph it can be concluded that the

overestimation of the chrysene content of the food supplement material was not caused by instrument

calibration The results for the solvent solution of many laboratories which underperformed for the

26

food supplement sample agreed well with the assigned value This supports the conclusion that the

overestimation is caused by the determination of an interferent most probably triphenylene

Figure 5 Youden plot of the percent deviations of values reported for chrysene from the

assigned values of the food supplement test material and the standard solution in

toluene with undisclosed content

96 Evaluation of compliance with legislation

The method performance data reported for the four marker PAHs were evaluated for compliance with

the provisions given in Commission Regulation (EC) No 8362011 Table 18 in ANNEX 10 gives an

overview on the results of the evaluation Data were not reported in case of empty cells Non-

compliant data are highlighted in Table 18 It should be noted that Regulation (EU) No 8362011

specifies LOD and LOQ with two decimals This was not respected by all participants which

hampered the compliance assessment

27

In summary it can be stated that the majority of participants reported method performance

characteristics for the determination of the four marker PAHs in the food supplement test sample that

are compliant with legislation However some laboratories need to improve their analysis methods

The laboratories in question are urged to adapt to the provisions given in the new legislation

10 Follow-up actions for underperforming laboratories

All laboratories that got questionable or non-satisfactory performance ratings are urged to perform

root cause analysis and to implement corrective actions

As agreed during the EU-RL PAH workshop 2011 the EU-RL PAH will set up follow-up measures in

due time for all NRLs that received for at least one of the four PAHs (BAA BAP BBF and CHR) z-

scores gt 3 as required by Regulation (EC) 8822004 and by the Protocol for management of

underperformance in comparative testing andor lack of collaboration of National Reference

Laboratories (NRLs) with European Union reference laboratories (EU-RLs) activities

These laboratories shall perform as an immediate action root-cause-analysis and shall report to the

EU-RL PAH in writing the identified cause for their underperformance and corrective actions they are

going to take Additionally they shall participate to an independent (non-EU-RL) proficiency test on

the determination of PAHs in food and shall communicate the outcome of this exercise to the EU-RL

PAH

28

11 Conclusions

Fourty seven participants reported results back to the EU-RL The performance of the majority of

participants was good In total 153 out of 225 attributed z-scores were below an absolute value of two

which equals to almost 68 However this percentage is much below the percentages of satisfactory

performance gained in previous studies This might be the consequence of the higher level of difficulty

with respect to matrix composition In addition bias can be concluded from the pattern of performance

indicators of some laboratories

In general the determination of chrysene caused most difficulties to the participants

zeta-Scores were calculated besides z-scores They indicate the agreement of the reported result with

the assigned value with respect to the stated measurement uncertainty The outcome of this rating was

worse than for the z-scores which indicates that the measurement uncertainty estimates were not

realistic Therefore participants underperforming with regard to zeta scores are urged to adapt their

measurement uncertainty statements

The great majority of participants in this inter-laboratory comparison applied analytical methods which

complied with EU legislation with regard to performance characteristics

12 Acknowledgements

The organisers would like to thank Mrs Beatriz de la Calle and Mr Franz Ulberth (all from IRMM

Geel Belgium) for their accurate revision of this report Mr Hakan Emteborg (from IRMM) for

ampouling of the standard solutions and all NRLs and OCLs for their cooperation

29

13 References

1 EU COMMISSION REGULATION (EC) No 7762006 of 23 May 2006 amending Annex VII to Regulation

(EC) No 8822004 of the European Parliament and of the Council as regards Community reference

laboratories Official Journal of the European Union 2006 L 136 p 3-8

Available from

httpeur-lexeuropaeuLexUriServLexUriServdouri=OJL200613600030008ENPDF

2 EU Regulation (EC) No 8822004 of the European Parliament and of the Council of 29 April 2004 on

official controls performed to ensure the verification of compliance with feed and food law animal health

and animal welfare rules Official Journal of the European Communities 2004 L191 p 1-52 Available

from

httpeur-lexeuropaeuLexUriServLexUriServdouri=OJL200419100010052ENPDF

3 EU Opinion of the Scientific Committee on Food on the risks to human health of Polycyclic Aromatic

Hydrocarbons in food (2002) Available from

httpeuropaeuintcommfoodfsscscfout153_enpdf

4 International Agency for Research on Cancer IARC Monographs on the Evaluation of

Carcinogenic Risks to Humans Some Non-heterocyclic Polycyclic Aromatic Hydrocarbons and Some

Related Exposures Volume 92 (2010) Available from

httpmonographsiarcfrENGMonographsvol92mono92pdf

5 EU Commission Regulation (EC) No 3332007 of 28 March 2007 laying down the methods of sampling

and analysis for the official control of the levels of lead cadmium mercury inorganic tin 3-MCPD and

benzo(a)pyrene in foodstuffs Official Journal of the European Union 2007 L 88 p 29-38 Available

from

httpeur-lexeuropaeuLexUriServLexUriServdouri=OJL200708800290038ENPDF

6 EU Commission Regulation (EC) No 18812006 of 19 December 2006 setting maximum levels for certain

contaminants in foodstuffs Official Journal of the European Union 2006 L 364 p 5-24 Available from

httpeur-lexeuropaeuLexUriServLexUriServdouri=OJL200636400050024ENPDF

7 EFSA Scientific Opinion of the Panel on Contaminants in the Food Chain on a request from the European

Commission on Polycyclic Aromatic Hydrocarbons in Food The EFSA Journal 2008 724 p 1-114

Available from

httpwwwefsaeuropaeuEFSAefsa_locale-1178620753812_1211902034842htm

30

8 EU Commission Regulation (EU) No 8352011 amending Regulation (EC) No 18812006 as regards

maximum levels for polycyclic aromatic hydrocarbons in foodstuffs Official Journal of the European

Union 2011 L 215 p 4-8

httpeur-lexeuropaeuLexUriServLexUriServdouri=OJL201121500040008ENPDF

9 EU Commission Regulation (EU) No 8362011 amending Regulation (EC) No 3332007 laying down the methods of sampling and analysis for the official control of the levels of lead cadmium mercury inorganic tin 3-MCPD and benzo(a)pyrene in foodstuffs Official Journal of the European Union 2011 L 215 p 9-16 httpeur-lexeuropaeuLexUriServLexUriServdouri=OJL201121500090016ENPDF

10 ISOIEC 170432010 (2010) Conformity assessment -- General requirements for proficiency testing

providers Geneva Switzerland

11 Thompson M Ellison SLR and Wood R The International Harmonized Protocol for the Proficiency

Testing of Analytical Chemistry Laboratories Pure Appl Chem (2006) 78(1) p 145ndash196

Available from httpmediaiupacorgpublicationspac2006pdf7801x0145pdf

31

14 ANNEXES

ANNEX 1 ndash Announcement of the PT on the IRMM webpage

ANNEX 2 ndash Announcement via e-mail and invitation

ANNEX 3 ndash Announcement of sample dispatch

ANNEX 4 ndash Technical specifications of the calibration solutions

ANNEX 5 ndash Homogeneity and stability of the test material

ANNEX 6 ndash Acknowledgement of sample receipt

ANNEX 7 ndash Questionnaire on details of analysis method

ANNEX 8 ndash Results reported for the food supplement sample

ANNEX 9 ndash Results reported for the standard solutions with undisclosed analyte content

ANNEX 10 ndash Method performance characteristics reported by participants

ANNEX 11 ndash Details of analysis methods reported by participants

32

ANNEX 1 ndash Announcement of the PT on the IRMM webpage

33

ANNEX 2 - Announcement via e-mail and invitation

34

ANNEX 3 - Announcement sample dispatch

Dear MadameSir

The test samples for the proficiency test on the determination of four PAHs in a food supplement (dry extract of St Johns wort) were

dispatched today

Explanation on the design of the study as well as instructions for sample storage and for reporting of results are in the parcel You will

also find your participant key in the parcel which is required for reporting of results

Please check the parcel after arrival for completeness and report the receipt of the test samples as well as their status to us by applying

the sample receipt form which is in the parcel

You are kindly requested to inform us by email if you do not receive the parcel by end of this week

My colleague Donata Lerda will then take care of it

With best regards

Thomas Wenzl

35

ANNEX 4 - Specifications of standard solutions with disclosed concentration

36

ANNEX 4 - continued

37

ANNEX 5 - Homogeneity and stability of the test material

Homogeneity of the test material

Benz[a]anthracene - BAA

Benzo[a]pyrene - BAP

38

ANNEX 5 - continued

Benzo[b]fluoranthene - BBF

Chrysene - CHR

39

ANNEX 5 - continued

Stability of the test material

Table 10 Content of the four target analytes in test materials stored under different

temperatures

BaA BaP BbF CHR

microgkg microgkg microgkg microgkg

Content 31 16 31 69 Storage at room temperature

U (k=2) 05 02 05 17

Content 31 15 29 68 Storage at reference temperature

U (k=2) 04 03 04 10

The applied capillary column does not separate CHR from triphenylene

40

ANNEX 6 ndash Documents sent to participants

41

ANNEX 6 - continued

42

ANNEX 7 Questionnaire on details of analysis method

43

ANNEX 7 - continued

44

ANNEX 7 - continued

45

ANNEX 8 - Results reported by participants for the food supplements test sample Table 11 Analysis results reported by the participants for the content of benz[a]anthracene (BAA) in the food supplements test material Values are presented as reported

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

3600 31 29 29 3 05 2

3602 199 193 178 19 021

3603 253 253 246 251 022 2

3604 199 209 23 213 071 2

3605 301 3 271 291 076 2

3606 302 288 278 289 064 2

3607 456 416 443 438 096 2

3608 796 695 651 714 149 2

3610 182 192 214 196 039 2

3611 414 504 488 469 96 2

3612 3 34 28 31 06 2

3613 32 33 34 33 07 2

3614 182 182 191 185 037 2

3615 3884 3822 3565 376 0752 2

3616 2 2 2 2 01 2

3617 366 37 35 362 046 2

3618 32 32 32 32 05 2

3619 299 305 297 299 048 2

3620 304 294 302 302 045 2

3621 29 26 29 28 04 2

3622 343 325 318 322 052 2

3623 35 3 33 33 07 2

3624

3626 318 31 314 314 126 2

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

5301 31 3 32 31 06 2

5302 314 319 315 316 063 2

5303 3102 2716 2822 2876 27 62

5304 261 263 26 261 57 2

5305 23 22 26 24 20

5306 22 23 23 22 11 2

5307 488 461 317 461

5308 168633 168544 1562025 1644598

5309 16 19 16 17

5310 3 304 311 305 015

5311 477 452 448 459 15 2

5312 506 63 494 543 134 2

5313 353 301 404 353

5314 431 373 378 394 5 096

5315 596 531 51 545 11 2

5316 224 231 235 235 022 2

5317 216 23 221 222 007 2

5318 064 07 067 01 2

5319 111 184 146 147 029 2

5320 515 525 564 534 48

5321 21 5322 13 87 9 87 35 2

5323 38 33 35 33 16 2

46

Figure 6 Results of replicate determinations (indicated by triangles) of benz[a]anthracene (BAA) in the food supplement test material

Horizontal blue lines represent the arithmetic mean value of replicate measurements and blue bars the reported expanded measurement uncertainty (k=2) The assigned value is

plotted as dotted line Red solid lines mark the range of satisfactory performance (|z|le2) Mean values are given for results outside the displayed data range

ProLab 2011

Laboratory

53

1853

1953

0853

0936

1436

0236

1036

1653

2136

0453

1753

0653

1653

0536

0353

0436

2153

0336

0636

0536

0036

2036

1953

1036

1253

0136

2653

0236

1836

2336

2236

1353

1353

2336

1736

1553

1453

0736

0753

1153

2053

1253

1536

0853

2236

11

an

aly

te c

on

ten

t (micro

gk

g)

10

95

9

85

8

75

7

65

6

55

5

45

4

35

3

25

2

15

1

05

0

102

3 468

7

Sample St Johns wort extract replicatesMeasurand Benz[a]anthraceneNo of laboratories 46

Assigned value 298 microgkg (Reference value)Target sd 062 microgkgTolerance limits 174 - 422 microgkg (|Z score| lt 200)

Assig

ned va

lue

Limit of tolerance

Limit of tolerance

47

Table 12 Analysis results reported by the participants for the content of benzo[a]pyrene (BAP) in the food supplements test material Values are presented as reported

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

3600 16 17 17 17 03 2

3602 118 123 13 124 015

3603 165 162 157 161 015 2

3604 134 154 139 143 04 2

3605 164 166 172 167 057 2

3606 181 159 161 167 031 2

3607 279 266 279 274 055 2

3608 57 687 461 573 226 2

3610 226 216 183 206 042 2

3611 289 325 311 308 36 2

3612 24 22 2 22 05 2

3613 17 18 19 18 05 2

3614 08 08 08 08 016 2

3615 1545 1393 1425 145 029 2

3616 16 16 14 15 03 2

3617 177 171 158 169 023 2

3618 2 2 2 2 02 2

3619 207 207 206 207 075 2

3620 165 169 163 166 017 2

3621 44 47 49 47 05 2

3622 212 208 222 213 038 2

3623 2 21 22 21 03 2

3624 18 178 182 18 031 2

3626 172 173 178 174 07 2

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

5301 18 18 18 18 04 2

5302 188 19 187 188 038 2

5303 1691 1426 149 1535 19 80

5304 145 142 15 146 69 2

5305 15 17 18 16 15

5306 16 17 17 16 08 2

5307 164 157 153 157

5308 102669 109324 109035 107009

5309 1 1 12 11

5310 122 122 124 123 006

5311 199 184 215 2 15 2

5312 258 248 239 248 05 2

5313 199 178 245 207

5314 174 171 172 172 11 0948

5315 253 248 213 238 05 2

5316 177 171 173 17 016 2

5317 255 277 276 269 012 2

5318 114 12 117 02 2

5319 19 228 231 216 043 2

5320 303 3 344 316 78

5321 12 12 12 12

5322 13 17 24 17 35 2

5323 2 2 19 2 1 2

48

Figure 7 Results of replicate determinations (indicated by triangles) of benzo[a]pyrene (BAP) in the food supplement test material

Horizontal blue lines represent the arithmetic mean value of replicate measurements and blue bars the reported expanded measurement uncertainty (k=2) The assigned value is

plotted as dotted line Red solid lines mark the range of satisfactory performance (|z|le2) Mean values are given for results outside the displayed data range

ProLab 2011

Laboratory

36

1453

0953

0853

1853

2153

1036

0236

0436

1553

0436

1653

0353

0736

0336

2036

0053

0553

0636

0636

0536

1753

1453

1636

2636

1336

2453

0153

2253

0253

2353

1136

1836

1953

1336

1036

2336

2253

1936

1253

1553

1253

1736

0753

2036

2136

0836

11

an

aly

te c

on

ten

t (micro

gk

g)

55

5

45

4

35

3

25

2

15

1

05

0

573

308

3

Sample St Johns wort extract replicatesMeasurand Benzo[a]pyreneNo of laboratories 48

Assigned value 165 microgkg (Reference value)Target sd 036 microgkgTolerance limits 093 - 237 microgkg (|Z score| lt 200)

Assig

ned va

lue

Limit of tolerance

Limit of tolerance

49

Table 13 Analysis results reported by the participants for the content of benzo[b]fluoranthene (BBF) in the food supplements test material Values are presented as reported

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

3600 32 3 3 31 09 2

3602 173 175 16 17 017

3603 265 252 25 256 025 2

3604 116 108 105 11 028 2

3605 286 225 232 247 074 2

3606 294 273 261 276 046 2

3607 579 579 544 567 113 2

3608 103 1201 876 1036 326 2

3610 173 196 204 191 038 2

3611 546 618 565 576 75 2

3612 3 28 28 29 06 2

3613 28 31 32 31 06 2

3614 135 111 135 127 022 2

3615 4187 3678 4111 4 08 2

3616 2 2 2 2 01 2

3617 33 27 299 035 2

3618 31 31 31 31 05 2

3619 326 324 312 326 064 2

3620 277 28 272 276 069 2

3621 26 25 23 25 04 2

3622 346 336 333 34 052 2

3623 26 28 29 28 02 2

3624

3626 288 28 307 292 117 2

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

5301 29 3 29 29 06 2

5302 297 3 296 298 06 2

5303 2705 2444 2608 2586 22 78

5304 245 254 26 253 85 2

5305 23 23 26 24 15

5306 42 43 4 42 21 2

5307 221 216 241 221

5308 232169 246654 240722 229848

5309 19 22 19 2 0

5310 3 315 325 313 016

5311 262 277 304 281 15 2

5312 27 289 337 299 075 2

5313 321 284 378 328

5314 423 404 385 404 34 083

5315 42 506 416 444 09 2

5316 265 267 277 273 018 2

5317 185 197 195 192 006 2

5318 276 281 279 04 2

5319 396 389 403 396 079 2

5320 623 618 635 625 14

5321 31

5322 24 33 49 33 35 2

5323 38 45 35 45 27 2

50

Figure 8 Results of replicate determinations (indicated by triangles) of benzo[b]fluoranthene (BBF) in the food supplement test material

Horizontal blue lines represent the arithmetic mean value of replicate measurements and blue bars the reported expanded measurement uncertainty (k=2) The assigned value is

plotted as dotted line Red solid lines mark the range of satisfactory performance (|z|le2) Mean values are given for results outside the displayed data range

ProLab 2011

Laboratory

36

0436

1436

0236

1053

1736

1653

0953

0753

0853

0536

2136

0553

0436

0353

0353

1636

0636

2036

2353

1853

1136

1236

2653

0153

0253

1236

1736

1336

0036

1853

2153

1036

1953

1336

2253

2253

2353

1936

1553

1453

0653

1536

0753

2036

0836

11

an

aly

te c

on

ten

t (micro

gk

g)

75

7

65

6

55

5

45

4

35

3

25

2

15

1

05

0

103

6

576

3

Sample St Johns wort extract replicatesMeasurand Benzo[b]fluorantheneNo of laboratories 46

Assigned value 292 microgkg (Reference value)Target sd 060 microgkgTolerance limits 172 - 412 microgkg (|Z score| lt 200)

Assig

ned va

lue

Limit of tolerance

Limit of tolerance

51

Table 14 Analysis results reported by the participants for the content of chrysene (CHR) in the food supplements test material Values are presented as reported

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

3600 5 49 5 5 07 2

3602 32 304 29 304 036

3603 462 469 447 459 048 2

3604 438 428 42 429 119 2

3605 365 41 381 386 085 2

3606 494 411 45 451 124 2

3607 897 774 787 819 18 2

3608 986 887 1168 1014 284 2

3610 418 392 462 424 085 2

3611 833 893 87 865 61 2

3612 6 64 58 61 12 2

3613 41 39 4 4 08 2

3614 272 254 288 271 054 2

3615 605 6028 5964 601 12 2

3616 47 45 49 47 05 2

3617 445 431 389 422 065 2

3618 45 45 45 45 06 2

3619 738 747 72 738 118 2

3620 463 472 446 46 092 2

3621 53 49 51 51 06 2

3622 478 513 505 488 08 2

3623 73 73 72 72 05 2

3624

3626 63 622 634 628 251 2

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

5301 49 49 49 49 1 2

5302 687 683 666 679 136 2

5303 6777 6181 6382 6441 24 70

5304 592 581 6 591 85 2

5305 6 58 64 61 20

5306 49 5 51 49 25 2

5307 524 5 574 524

5308 211488 216782 221146 213243

5309

5310 372 393 394 386 019

5311 43 459 422 437 15 2

5312 934 943 981 953 191 2

5313 793 73 99 838

5314 636 561 565 588 18 0818

5315 547 583 499 542 11 2

5316 292 286 285 282 06 2

5317 52 598 517 545 046 2

5318 368 345 357 05 2

5319 449 471 474 465 116 2

5320 76 924 818 834 99

5321 33

5322 7 7 76 7 35 2

5323 87 73 77 73 37 2

52

Figure 9 Results of replicate determinations (indicated by triangles) of chrysene (CHR) in the food supplement test material

Horizontal blue lines represent the arithmetic mean value of replicate measurements and blue bars the reported expanded measurement uncertainty (k=2) The assigned value is

plotted as dotted line Red solid lines mark the range of satisfactory performance (|z|le2) Mean values are given for results outside the displayed data range

ProLab 2011

Laboratory

53

0836

1453

1636

0253

2153

1836

0553

1036

1336

1736

1036

0453

1136

1836

0636

0336

2053

1936

1653

0136

0036

2253

0636

2153

0753

1553

1753

1453

0436

1536

1253

0536

2653

0353

0253

2236

2336

1953

2336

0753

2053

1353

1236

0836

11

an

aly

te c

on

ten

t (micro

gk

g)

13

12

11

10

9

8

7

6

5

4

3

2

1

0

865

3

Sample St Johns wort extract replicatesMeasurand ChryseneNo of laboratories 45

Assigned value 407 microgkg (Reference value)Target sd 083 microgkgTolerance limits 241 - 573 microgkg (|Z score| lt 200)

Assig

ned va

lue

Limit of tolerance

Limit of tolerance

53

Table 15 Analysis results reported by the participants for the sum of the four marker PAHs in the food supplements test material Values are presented as reported

Laboratory code

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg

3600 128 24 2

3602 79 126

3603 1127 096 2

3604 893 258 2

3605 1091 305 2

3606 1184 238 2

3607 2098 42 2

3608 3336 325 2

3610 1138 17 2

3611 2218 259 2

3612 143 29 2

3613 122 2 2

3614 663 129 2

3615 152 304 2

3616 102 03 2

3617 1252 169 2

3618 128 18 2

3619 157 305 2

3620 1204 12 2

3621 151 19 2

3622 1361 115 2

3623 154 09 2

3624

3626 1409 56 2

Laboratory code

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg

5301 126

5302 37 074 2

5303 13438 92

5304 1251 85 2

5305 125 20

5306 129 65 2

5307 1363

5308 7245598

5309

5310 1127 056

5311 1367 15 2

5312 2042 408 2

5313 1726

5314 1558

5315 177 35 2

5316 96 073 2

5317 1229 066 2

5318 82 05 2

5319 1225 267 2

5320 2309 41

5321

5322 207 35 2

5323 171 103 2

54

ANNEX 9 Results reported for the standard solutions with undisclosed analyte content Table 16 Analysis results reported by the participants for the four marker PAHs in the standard solution in toluene with undisclosed analyte

content Values are presented as reported U measurement uncertainty k coverage factor

benz[a]anthracene benzo[a]pyrene benzo[b]fluoranthene chrysene Laboratory

code Unit Result U k Result U k Result U k Result U k 3600 microgl 38 67 2 25 45 2 211 68 2 275 38 2

3605 microgl 367 184 2 2482 124 2 2592 13 2 235 118 2

3606 microgkg 3763 176 2 2609 142 2 2721 118 2 2539 134 2

3607 microgkg 3706 815 2 2824 565 2 2591 518 2 2587 569 2

3612 microgl 38 08 2 237 11 2 244 18 2 257 02 2

3614 microgl 324 65 2 209 38 2 215 37 2 21 42 2

3616 microgkg 318 07 2 224 14 2 231 26 2 216 1 2

3618 microgkg 417 17 2 284 08 2 292 06 2 297 03 2

3619 microgkg 47 742 2 274 468 2 228 387 2 274 434 2

3620 microgl 495 74 2 294 29 2 321 8 2 321 64 2

3621 microgl 241 22 2 143 13 2 188 16 2 145 14 2

3623 microgkg 476 25 2 311 53 2 269 3 2 297 03 2

3626 microgl 359 38 2 235 23 2 238 27 2 24 18 2

5301 microgkg 426 29 299 289

5302 microgkg 436 87 2 296 59 2 296 59 2 289 58 2

5303 microgl 3721 27 102 22838 19 99 23756 22 99 25486 24 98

5304 microgkg 38 29 2 225 26 2 257 37 2 258 52 2

5305 microgkg 305 225 245 29

5306 microgkg 316 63 2 247 49 2 65 13 2 22 44 2

5307 microgkg 3658 2559 241 2414

5308 microgkg 16854 109324 246654 216782

5311 microgl 374 20 2 279 15 2 268 15 2 272 15 2

5313 microgl 3408 2165 2275 2373

5314 microgkg 395 5 2875 11 3614 34 2905 18

5320 microgl 3267 368 1986 571 28 683 2427 512

5322 microgl 282 35 2 225 35 2 241 35 2 21 35 2

55

Figure 10 Youden plots of the percent deviation of the reported values from the assigned values for the food supplement test sample and the

standard solution in toluene with undisclosed content

(The identity of the respective measurand is given above the figure Results exceeding a difference of more than 100 were omitted)

56

Figure 10 - continued

57

Table 17 Analysis results reported by the participants for the four marker PAHs in the standard solution in acetonitrile with undisclosed

analyte content

Values are presented as reported U measurement uncertainty k coverage factor

benz[a]anthracene benzo[a]pyrene benzo[b]fluoranthene chrysene

Laboratory

code Unit Result U k Result U k Result U k Result U k

3602 microgkg 3675 247 247 244

3603 microgkg 358 086 2 236 028 2 235 028 2 236 057 2

3604 microgl 475 29 2 19 12 2 194 13 2 258 12 2

3608 microgl 3623 078 2 2385 023 2 2343 18 2 2296 092 2

3610 microgkg 369 185 2 25 125 2 251 126 2 244 122 2

3611 microgkg 48 02 2 323 02 2 328 04 2 317 03 2

3613 microgl 332 31 2 261 3 2 249 25 2 239 24 2

3615 microgl 415 2635 2729 2689

3617 microgkg 37 09 2 244 06 2 284 07 2 239 06 2

3622 microgl 37 12 2 25 23 2 257 21 2 238 1 2

3624 microgkg 402 104 2 255 084 2 265 096 2 253 07 2

5306 microgkg 563 113 2 371 74 2 359 72 2 333 67 2

5308 microgkg 168633 102669 232169 211488

5309 microgl 402 268 27

5310 microgl 315 158 25 125 25 125 288 144

5312 microgl 428 043 2 282 028 2 272 027 2 275 028 2

5315 microgkg 387 08 2 256 05 2 251 05 2 253 05 2

5316 microgl 3839 342 2 2485 191 2 2559 168 2 239 51 2

5317 microgl 2466 043 2 2212 034 2 2472 016 2 2146 068 2

5319 microgl 414 41 2 279 28 2 268 27 2 26 31 2

5321 microgkg 476 316 326 328

5322 microgl 2547 35 2

5323 microgkg 405 203 2 239 119 2 255 153 2 201 11 2

58

Figure 11 Youden plots of the percent deviation of the reported values from the assigned values for the food supplement test sample and the

standard solution in acetonitrile with undisclosed content

(The identity of the respective measurand is given above the figure Results exceeding a difference of more than 100 were omitted)

59

Figure 11 - continued

60

ANNEX 10 Table 18 Method performance parameters reported by the participants

Laboratory code

Analyte LOD LOQ Recovery Analyte LOD LOQ Recovery

microgkg microgkg microgkg microgkg 3600 BaA 03 06 978 BaP 03 07 9483602 BaA 007 021 926 BaP 005 015 993603 BaA 0011 05 987 BaP 0005 051 9553604 BaA 02 04 86 BaP 02 04 883605 BaA 0025 005 118 BaP 0025 005 1083606 BaA 001 003 65 BaP 001 003 703607 BaA 021 071 74 BaP 027 089 943608 BaA 016 048 8213 BaP 015 046 83683610 BaA 02 05 85 BaP 005 02 853611 BaA BaP 3612 BaA 1 117 BaP 1 1203613 BaA 01 03 90 BaP 01 03 903614 BaA 02 05 96 BaP 02 05 933615 BaA 005 015 110 BaP 005 015 1053616 BaA 01 05 108 BaP 01 05 753617 BaA 018 037 405 BaP 009 018 3873618 BaA 01 03 63 BaP 01 03 593619 BaA 005 005 59 BaP 033 033 543620 BaA 0009 0018 64 BaP 0005 001 553621 BaA 007 02 84 BaP 007 02 923622 BaA 01 03 1101 BaP 01 03 9853623 BaA 02 08 105 BaP 02 08 1013624 BaA BaP 01 033 8513625 BaA BaP 3626 BaA BaP 5301 BaA 003 01 102 BaP 003 01 1005302 BaA 01 03 78 BaP 01 03 865303 BaA 003 01 62 BaP 003 01 805304 BaA 1 1 70 BaP 1 1 955305 BaA 02 05 85 BaP 02 05 855306 BaA 03 1 699 BaP 03 1 905307 BaA 6537 BaP 81165308 BaA 0088 0265 99 BaP 00262 00785 1005309 BaA 01 08 92 BaP 01 08 925310 BaA 01 03 BaP 01 03 905311 BaA 002 005 80 BaP 002 004 915312 BaA 03 09 105 BaP 03 09 1055313 BaA 04 08 BaP 04 08 5314 BaA 001 002 96 BaP 001 002 9485315 BaA 01 03 50 - 100 BaP 01 03 50 - 100 5316 BaA 002 011 827 BaP 004 019 10445317 BaA 05 1 76 BaP 05 1 755318 BaA BaP 5319 BaA 009 029 951 BaP 011 041 995320 BaA 12 18 946 BaP 06 1 8345321 BaA 03 05 BaP 05 1 5322 BaA 1 2 100 BaP 1 2 1005323 BaA 26 BaP 26

61

ANNEX 10 Table 18 - continued

Laboratory code Analyte LOD LOQ Recovery Analyte LOD LOQ Recovery

microgkg microgkg microgkg microgkg 3600 BbF 03 05 96 CHR 02 03 10153602 BbF 015 045 947 CHR 003 009 9193603 BbF 0014 05 1012 CHR 0004 05 10033604 BbF 02 04 85 CHR 02 04 873605 BbF 005 01 94 CHR 0025 005 863606 BbF 001 003 70 CHR 001 003 653607 BbF 018 06 100 CHR 014 028 943608 BbF 015 046 7979 CHR 015 045 71253610 BbF 005 02 85 CHR 02 05 853611 BbF CHR 3612 BbF 1 118 CHR 1 1173613 BbF 01 03 90 CHR 01 03 903614 BbF 01 04 96 CHR 02 05 953615 BbF 008 025 109 CHR 003 01 1083616 BbF 01 05 78 CHR 01 05 723617 BbF 035 07 38 CHR 012 024 4163618 BbF 01 03 62 CHR 01 03 623619 BbF 016 016 63 CHR 01 01 593620 BbF 0003 0006 55 CHR 001 002 653621 BbF 007 02 103 CHR 007 02 893622 BbF 02 06 963 CHR 03 09 1043623 BbF 02 08 106 CHR 02 08 1193624 BbF CHR 3625 BbF CHR 3626 BbF CHR 5301 BbF 003 01 96 CHR 003 01 1145302 BbF 01 03 84 CHR 01 03 835303 BbF 003 01 78 CHR 003 01 705304 BbF 1 1 86 CHR 1 1 705305 BbF 02 05 90 CHR 02 05 955306 BbF 03 1 1435 CHR 03 1 1185307 BbF 8116 CHR 65375308 BbF 0079 0238 99 CHR 009 026 1005309 BbF 01 08 92 CHR 5310 BbF 07 2 CHR 01 03 5311 BbF 002 004 95 CHR 002 004 895312 BbF 03 09 105 CHR 03 09 1055313 BbF 04 08 CHR 04 08 5314 BbF 001 002 83 CHR 001 002 8185315 BbF 02 05 50 -100 CHR 02 05 50 - 100 5316 BbF 005 024 814 CHR 001 007 9595317 BbF 05 1 73 CHR 05 1 785318 BbF CHR 5319 BbF 011 038 946 CHR 013 043 9675320 BbF 07 09 1194 CHR 23 32 10455321 BbF 05 1 CHR 05 1 5322 BbF 1 2 100 CHR 1 2 1005323 BbF 26 CHR 26

62

ANNEX 11 Table 19 Details of analysis method reported by the participants Data are presented as reported

Laboratory code

Analysis technique

Instrument calibration

Internal standards applied

Sample clean up

Details of sample

clean up

Extraction technique

Details of sample

extraction

3600 6) GC-MS

2) Internal standardisation with labelled substances

Deuterated PAH mix 9 (Ehrenstorfer)

1) Saponification 2) LiquidLiquid partitioning

D) Other Liquidliquid partitioning

3602 2) HPLC-UV-FLD

1) External calibration

3) Gel permeation chromatography

A) Pressurised liquid extraction (PLE)

3603 1) HPLC-FLD

1) External calibration

3) Gel permeation chromatography

D) Other 1 minute with dichloromethane in vortex

3604 2) HPLC-UV-FLD

2) Internal standardisation with labelled substances

DiP D14

5) Donor acceptor complex chromatography

D) Other LL

3605 1) HPLC-FLD

1) External calibration

3) Gel permeation chromatography

C) Soxhlet extraction

3606 7) GC-MSMS

2) Internal standardisation with labelled substances

BaP 13C4 CHR 13C6 BaA 13C6 BbF 13C6

4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

3607 6) GC-MS

2) Internal standardisation with labelled substances

BaA-d12 CHR-d12 BbF-d12 BkF-d12 BaP-d12 ICP-d12 BgP-d12 DiP-d14

1) Saponification

D) Other solvent partitioning

3608 1) HPLC-FLD

1) External calibration

6) Other Liquid extraction

D) Other

Extraction in a rotary agitator and centrifugation

3610 2) HPLC-UV-FLD

1) External calibration

4) Solid phase extraction

B) Sonication

3611

3612 6) GC-MS

2) Internal standardisation with labelled substances

chrysene-d12 dibenzo(ah)anthracene-d14 dibenzo(ai)pyrene-d14

4) Solid phase extraction

B) Sonication

3613 5) LC-MSMS

3) Internal standardisation with unlabelled substances

I use Chrysene-D12 3) Gel permeation chromatography

D) Other extraction with hexan

63

Table 19 - continued

Laboratory code

Analysis technique

Instrument calibration

Internal standards applied

Sample clean up Details of sample

clean up

Extraction technique

Details of sample

extraction

3614 6) GC-MS 1) External calibration

3) Gel permeation chromatography

B) Sonication

3615 1) HPLC-FLD

1) External calibration

6) Other PLE GPC

A) Pressurised liquid extraction (PLE)

3616 6) GC-MS

2) Internal standardisation with labelled substances

Benzo(a)pyrene-13C4 Benzo(a)anthracene-13C6 Benzo(b)fluoranthene-13C6 Chrysene-13C6

2) LiquidLiquid partitioning 4) Solid phase extraction

B) Sonication

3617 1) HPLC-FLD

1) External calibration

4) Solid phase extraction

C) Soxhlet extraction

3618 6) GC-MS

2) Internal standardisation with labelled substances

deuterated standards

1) Saponification 4) Solid phase extraction

D) Other Handshaken with cyclohexane

3619 6) GC-MS

2) Internal standardisation with labelled substances

C13 Labelled US EPA 16

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

D) Other Homegenisation amp saponiofication

3620 9) GC-HRMS

2) Internal standardisation with labelled substances

mix of deuterated PAHs (BAA-D12 CHR-D12 BAP-D12 BBF-D12)

3) Gel permeation chromatography 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

3621 6) GC-MS

2) Internal standardisation with labelled substances

Chrysene D12 Benzo(a)pyrene D12 Benzo(b)fluoranthene D12 Benzo(a)anthracene

2) LiquidLiquid partitioning 4) Solid phase extraction

B) Sonication

3622 1) HPLC-FLD

4) Standard addition method

4) Solid phase extraction

D) Other

Extraction with cyclohexane by heating under reflux

3623 7) GC-MSMS

2) Internal standardisation with labelled substances

C13 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

3624 2) HPLC-UV-FLD

1) External calibration

2) LiquidLiquid partitioning

D) Other hot solvent extraction

3626

64

Table 19 - continued

Laboratory code

Analysis technique

Instrument calibration

Internal standards

applied

Sample clean up

Details of sample

clean up

Extraction technique

Details of sample

extraction

5301 7) GC-MSMS

2) Internal standardisation with labelled substances

EACH SUBSTANCE C13

4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5302 7) GC-MSMS

2) Internal standardisation with labelled substances

Isotope labelled PAHs 13C for each analyte

4) Solid phase extraction

C) Soxhlet extraction

5303 7) GC-MSMS

3) Internal standardisation with unlabelled substances

HAP C13 4) Solid phase extraction

D) Other automate ASE200 DIONEX

5304 7) GC-MSMS

2) Internal standardisation with labelled substances

with C13 for each HAP

2) LiquidLiquid partitioning 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5305 7) GC-MSMS

2) Internal standardisation with labelled substances

C13 isotopiques internal standards

4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5306 7) GC-MSMS

2) Internal standardisation with labelled substances

C13 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5307 6) GC-MS

2) Internal standardisation with labelled substances

1) Saponification 2) LiquidLiquid partitioning

D) Other solvent extraction

5308 1) HPLC-FLD 1) External calibration

4) Solid phase extraction

B) Sonication

5309 1) HPLC-FLD 1) External calibration

4) Solid phase extraction

D) Other only SPE

5310 1) HPLC-FLD 1) External calibration

1) Saponification 2) LiquidLiquid partitioning 3) Gel permeation chromatography

C) Soxhlet extraction

5311 6) GC-MS

2) Internal standardisation with labelled substances

13 C Chrysene 13 C Benz(a)pyrene

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

B) Sonication

5312 1) HPLC-FLD

3) Internal standardisation with unlabelled substances

Benzo(b)chrysen

2) LiquidLiquid partitioning 4) Solid phase extraction

Quechers D) Other Quechers

65

Table 19 - continued

Laboratory code

Analysis technique

Instrument calibration

Internal standards applied

Sample clean up Details of sample

clean up

Extraction technique

Details of sample extraction

5313 6) GC-MS

2) Internal standardisation with labelled substances

Benz(a)anthracene D12 Benzo(b)fluranthene D12 Benzo(a)pyrene D12 Chrysene D12

1) Saponification 2) LiquidLiquid partitioning 3) Gel permeation chromatography

A) Pressurised liquid extraction (PLE)

5314 6) GC-MS

2) Internal standardisation with labelled substances

Benzo(a)pyren d12 2) LiquidLiquid partitioning

D) Other Extraction with

5315 1) HPLC-FLD

3) Internal standardisation with unlabelled substances

Benz(b)chrysen

1) Saponification 2) LiquidLiquid partitioning

D) Other shaker

5316 1) HPLC-FLD

1) External calibration

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

D) Other

LiquidLiquid extraction with cyclohexane and dimethylformamide

5317 7) GC-MSMS

1) External calibration

3) Gel permeation chromatography 6) Other

silica gel D) Other VDLUFA online method

5318

5319 1) HPLC-FLD

3) Internal standardisation with unlabelled substances

Benzo(b)chrysene 3) Gel permeation chromatography

A) Pressurised liquid extraction (PLE)

5320 6) GC-MS

3) Internal standardisation with unlabelled substances

22-binaphthyl

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

D) Other 3 hours refluxing

5321 1) HPLC-FLD

2) Internal standardisation with labelled substances

6-meacutethylchrysegravene

1) Saponification 2) LiquidLiquid partitioning

B) Sonication

5322 6) GC-MS

2) Internal standardisation with labelled substances

naftalene-d8 acenaphtalene-d10 phenanthrene-d10 chrysene-d12 perylene-d12 benzo(e)anthracene-d12

1) Saponification 2) LiquidLiquid partitioning 6) Other

clean-up with silica packed column

C) Soxhlet extraction

5323 1) HPLC-FLD

1) External calibration

4) Solid phase extraction 6) Other

Accelerated Solvent Extraction

A) Pressurised liquid extraction (PLE)

66

European Commission EUR 25245 EN ndash Joint Research Centre ndash Institute for Reference Materials and Measurements Title Report on the 8th inter-laboratory comparison organised by the European Union Reference Laboratory for Polycyclic Aromatic Hydrocarbons - Four marker PAHs in a dry extract of St Johns wort Author(s) Radoslav Lizak Szilard Szilagyi Philippe Verlinde and Thomas WENZL Luxembourg Publications Office of the European Union 2012 ndash 66 pp ndash 201 x 297 cm EUR ndash Scientific and Technical Research series ndash ISSN 1831-9424 ISBN 978-92-79-23337-1 doi 10278759387 Abstract The proficiency test here reported concerned the determination of the contents of four marker polycyclic aromatic hydrocarbons (PAHs) and of their sum in an food supplements test sample The set of marker PAHs consists of benz[a]anthacene benzo[a]pyrene benzo[b]fluoranthene and chrysene Participants to these PT were National Reference Laboratories for PAHs (NRLs-PAHs) and EU official food control laboratories The number of participants was in total 48

The PT was organised according ISO Standard 170432010

The test material used was a commercial dry extract of St Johns wort Participants also received a solution of the same PAHs either in acetonitrile or in toluene for checking their instrument calibration

The results from participants were rated with z-scores and zeta-scores About 68 of the reported results were attributed with z-scores with an absolute value of below two which is the threshold for satisfactory performance

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interests whether private or national

L

A-N

A-252

45-EN

-N

• 1 Summary
• 2 Introduction
• 3 Scope
• 4 Participating Laboratories
• 5 Time frame
• 6 Confidentiality
• 7 Test materials
• • 71 1Preparation of test materials
  • 72 Assigned values and standard deviation for proficiency assessment
  • 73 Homogeneity and stability
  • 74 Sample dispatch
  • • 8 Design of the proficiency test
    • 9 Evaluation of the results
    • • 91 2General
      • 92 21BEvaluation criteria
      • 93 Performance of participants
      • 94 Evaluation of the influence of analysis method
      • 95 Evaluation of the influence of calibration
      • 96 26Evaluation of compliance with legislation
      • • 10 Follow-up actions for underperforming laboratories
        • 11 1Conclusions
        • 12 1Acknowledgements
        • 13 References

13

9 Evaluation of the results

The results reported by participants are listed in ANNEX 8

91 General

The most important evaluation parameter was the performance of the laboratories in the determination

of the target PAHs in the food supplement test material which was expressed by z-scores and zeta-

scores

The compliance of method performance characteristics specified by the participants with provisions

given in legislation was evaluated too

92 Evaluation criteria

The participants were requested to report for all analytes the results of replicate measurements and a

value for proficiency assessment which is the result they wish to be applied for the calculation of

performance indicators z-Scores and zeta-scores were attributed only to these results The individual

results of replicate analyses were not rated

z-Scores

z-scores were calculated based on the FS_FIN values Equation 1 presents the formula for calculation

of z-scores

Equation 1

P

assignedlab Xxz

where z refers to the z-score xlab to the reported ldquovalue for proficiency assessmentrdquo Xassigned to the

assigned value and σP to the standard deviation for proficiency testing

zeta-Scores

In addition to z-scores zeta-scores were calculated In contrast to z-scores zeta-scores describe the

agreement of the reported result with the assigned value within the respective uncertainties

Unsatisfactorily large zeta-scores might be caused by underestimated measurement uncertainties large

bias or a combination of both Zeta-scores were calculated according to Equation 3

14

Equation 3 22assignedlab

assignedlab

uu

Xxzeta

where zeta refers to the zeta-score xlab to the reported ldquovalue for proficiency assessmentrdquo Xassigned to

the assigned value ulab to the measurement uncertainty reported by the laboratory and uassigned to the

uncertainty of the assigned value

The performance of the laboratories was classified according to ISOIEC 170432010 [10] Following

scheme is applied for the interpretation of zeta scores and z-scores

|score| 20 = satisfactory performance

20lt|score| lt 30 = questionable performance

|score| ge 30 = unsatisfactory performance

93 Performance of participants

The 48 participants in the study reported in total 225 results for proficiency evaluation which equals to

about 94 of the 240 possible results About 68 of the reported results were rated as satisfactory

with regard to z-scores

Figure 1 and Figure 2 give an overview of the z-scores assigned to the respective results The larger

the triangles the larger were the differences to the assigned values Red triangles indicate z-scores

above an absolute value of three whereas yellow triangles represent z-scores in the questionable

performance range The corresponding score values are plotted next to the triangles About 40 of the

72 non-satisfactory results were reported by seven laboratories only eg the performance of

participant 3608 was not satisfactory for the majority of target analytes

It is also obvious that most problems were encountered in the determination of chrysene and the sum

of the four analytes Potential reasons for the difficulties related to chrysene are discussed further

down

The ideal situation would be that deviations from the assigned values of the results reported for the

individual analytes are small and randomly distributed around zero This will give a satisfactory

performance statement for the sum parameter Deviations of opposite sign could also cancel out and

lead to a satisfactory performance for the sum parameter (eg participants 5303 5304 and 5305)

However in most cases a single underperformance for one analyte led also to non-satisfactory rating

for the sum parameter

15

The numerical values of the calculated z-scores are compiled in Table 6 and Table 7 z-Scores with an

absolute value of above 2 are given in red bold font

The results of the data evaluation for the individual analytes are given in ANNEX 8

For each analyte the figure shows the individual analysis results of the three replicate determinations

The assigned value is shown as dotted line The blue boxes represent the expanded uncertainties as

reported by participants for the value for proficiency assessment The arithmetic mean of the results

of the individual participants is indicated in the blue boxes by a blue line The satisfactory performance

range is located between the two red lines

The individual results of the replicate measurements and the value for proficiency assessment with

its accompanying expanded measurement uncertainty (k=2) are listed in the tables in ANNEX 8 as

well

16

Figure 1 Graphical presentation of z-scores corresponding to the values for proficiency assessment

reported by the NRLs for the contents of BAA BAP BBF and CHR in the St Johns wort extract test

material

Blue triangles indicate satisfactory performance yellow triangles indicate questionable performance red triangles indicate non-satisfactory performance z-score values are presented above the triangles for the latter two performance categories

ProLab 2011

Z score-3 0 3 -3 0 3 -3 0 3 -3 0 3 -3 0 3

BAA BAP BBF CHR PAH4

La

bo

rato

ry

36003602

3603

36043605

36063607

36083610

36113612

36133614

36153616

36173618

36193620

36213622

36233624

3626

2258

2343

2452

2669

2147

2788

2869

6710

70839

3011

11271

80525

8425

4561

12338

90680

4976

7331

99553

3998

3780

7500

17420

168413

3269

3029

-2348 -2736

-2023 -2981

-2155-3018

-3998

Figure 2 Graphical presentation of z-scores corresponding to the values for proficiency assessment

reported by the OCLs for the contents of BAA BAP BBF and CHR in the St Johns wort extract test

material

Blue triangles indicate satisfactory performance yellow triangles indicate questionable performance red triangles indicate non-satisfactory performance z-score values are presented above the triangles for the latter two performance categories

ProLab 2011

Z score-3 0 3 -3 0 3 -3 0 3 -3 0 3 -3 0 3

BAA BAP BBF CHR PAH4

La

bo

rato

ry

5301

5302

5303

5304

5305

5306

5307

5308

5309

5310

5311

5312

5313

5314

5315

5316

5317

5318

5319

5320

5321

5322

5323

2597

2629

2017

2293

2873

2123

2521

2620

2186

2222

2452

2864

3952

3984

3806

9226

4171 5522

3285

6594

5205

5157

3539

3901

7051

4519

3173

4872

9191

7276

4391

-2435

-2154

-2065

-2340

-2740-3726

-6346

-3505

17

Table 6 Compilation of z-scores calculated from the ldquoresults for proficiency assessment

reported by the NRLs for food supplement test material z-scores outside the

satisfactory range (|z| gt 2) are indicated by red font Values are reproduced as reported

by the laboratories Empty cells denote analytes for which results were not reported

Measurand benz[a]anthracene benzo[a]pyrene benzo[b]fluoranthene chrysene Sum PAH 4

Assigned value

microgkg 298 165 292 407 1162

Standard deviation for proficiency assessment

microgkg 062 036 060 083 125

Laboratory code

Result z-score Result z-score Result z-score Result z-score Result z-score

microgkg microgkg microgkg microgkg microgkg

3600 3 00 17 01 31 03 5 11 128 09

3602 19 -18 124 -11 17 -20 304 -12 79 -30

3603 251 -08 161 -01 256 -06 459 06 1127 -03

3604 213 -14 143 -06 11 -30 429 03 893 -22

3605 291 -01 167 01 247 -07 386 -03 1091 -06

3606 289 -01 167 01 276 -03 451 05 1184 02

3607 438 23 274 30 567 46 819 50 2098 75

3608 714 67 573 113 1036 123 1014 73 3336 174

3610 196 -17 206 11 191 -17 424 02 1138 -02

3611 469 708 308 805 576 907 865 996 2218 1684

3612 31 02 22 15 29 00 61 25 143 21

3613 33 05 18 04 31 03 4 -01 122 05

3614 185 -18 08 -23 127 -27 271 -16 663 -40

3615 376 13 145 -06 4 18 601 23 152 29

3616 2 -16 15 -04 2 -15 47 08 102 -11

3617 362 10 169 01 299 01 422 02 1252 07

3618 32 04 2 10 31 03 45 05 128 09

3619 299 00 207 12 326 06 738 40 157 33

3620 302 01 166 00 276 -03 46 06 1204 03

3621 28 -03 47 84 25 -07 51 12 151 28

3622 322 04 213 13 34 08 488 10 1361 16

3623 33 05 21 12 28 -02 72 38 154 30

3624 18 04

3626 314 03 174 02 292 00 628 27 1409 20

18

Table 7 Compilation of z-scores calculated from the ldquoresults for proficiency assessment

reported by the OCLs for food supplement test material z-scores outside the

satisfactory range (|z| gt 2) are indicated by red font Values are reproduced as reported

by the laboratories Empty cells denote analytes for which results were not reported

Measurand benz[a]anthracene benzo[a]pyrene benzo[b]fluoranthene chrysene Sum PAH 4

Assigned value

microgkg 298 165 292 407 1162

Standard deviation for proficiency assessment

microgkg 062 036 060 083 125

Laboratory code

Result z-score Result z-

score Result z-score Result

z-score

Result z-score

microgkg microgkg microgkg microgkg microgkg

5301 31 02 18 04 29 00 49 10 126 08

5302 316 03 188 06 298 01 679 33 37 -63

5303 2876 -02 1535 -03 2586 -06 6441 29 13438 15

5304 261 -06 146 -05 253 -06 591 22 1251 07

5305 24 -09 16 -01 24 -09 61 25 125 07

5306 22 -13 16 -01 42 21 49 10 129 10

5307 461 27 157 -02 221 -12 524 14 1363 16

5308 16446 -22 107009 -16 229848 -10 213243 -23 72456 -35

5309 17 -21 11 -15 2 -15

5310 305 01 123 -12 313 03 386 -03 1127 -03

5311 459 26 2 10 281 -02 437 04 1367 16

5312 543 40 248 23 299 01 953 66 2042 71

5313 353 09 207 12 328 06 838 52 1726 45

5314 394 16 172 02 404 19 588 22 1558 32

5315 545 40 238 20 444 25 542 16 177 49

5316 235 -10 17 01 273 -03 282 -15 96 -16

5317 222 -12 269 29 192 -17 545 17 1229 05

5318 067 -37 117 -13 279 -02 357 -06 82 -27

5319 147 -24 216 14 396 17 465 07 1225 05

5320 534 38 316 42 625 55 834 52 2309 92

5321 12 -12

5322 87 92 17 01 33 06 7 35 207 73

5323 33 05 2 10 45 26 73 39 171 44

19

Table 8 and Table 9 present the respective zeta-scores As for the z-scores data outside the

satisfactory performance range are given in red bold font Zeta scores were only calculated for

laboratories that provided measurement uncertainty data as well as a numerical value for the coverage

factor Zeta scores were not calculated when the reported uncertainty value exceeded the content value

which is probably linked to mixing of reporting units (microgkg and ) However the participants are

reminded that the measurement uncertainty shall be expressed in the same unit as the content value

The assessment of the performance of the participants based on the reported measurement uncertainty

gave a less favourable picture compared to z-scores Only 60 of the zeta-scores are within the

satisfactory performance range The distribution of satisfactory performance ratings between NRLs

and OCLs is not balanced It was higher for the NRLs

It has to be noted that the magnitude of the zeta-scores were for some participants much higher than

the z-scores attributed to the same results Consequently the laboratories perform according to fitness-

for-purpose criterion specified in legislation which forms the basis for the z-scores but seem to have

difficulties in deducing realistic measurement uncertainty values Eg the relative standard

measurement uncertainties reported by the NRLs ranged from 25 to 20 with a median value of

about 10 Only two OCLs reported measurement uncertainties that exceeded the maximum tolerable

uncertainty (Uf) The respective data is highlighted in yellow in Table 9

However the EU-RL PAHs will continue to pay special attention to measurement uncertainty in future

ILCs as it has major implications on the assessment of compliance of food with European legislation

20

Table 8 Compilation of zeta-scores calculated from the ldquoresults for proficiency assessment

reported by the NRLs for test material St Johns wort the reported measurement

uncertainty and the uncertainty of the analyte content of the test material zeta-scores

outside the satisfactory range (|zeta| gt 2) are indicated by red bold font Empty cells

denote analytes for which either results or measurement uncertainties were not

reported

21

Table 9 Compilation of zeta-scores calculated from the ldquoresults for proficiency assessment

reported by the OCLs for test material St Johns wort the reported measurement

uncertainty and the uncertainty of the analyte content of the test material zeta-scores

outside the satisfactory range (|zeta| gt 2) are indicated by red bold font yellow

highlighted cells ugtUf Empty cells denote analytes for which results were not

reported

22

94 Evaluation of the influence of analysis method

The information on the applied analysis method (ANNEX 11) was used to group the performance

indicators attributed to the reported results according to the applied analysis technique Two groups of

data were formed They represent results obtained by high performance liquid chromatography-

fluorescence detection (HPLC-FD about 40 ) and by gas chromatography-mass spectrometry (GC-

MS about 60 ) respectively Kernel density plots were constructed based on the respective z-scores

Figure 3A to 3E show the different Kernel density plots

Figure 3 Kernel density plots of z-scores attributed to the results for proficiency assessment

which were reported for the food supplements test sample Results were classified

according to the applied analysis technique

A) benz[a]anthracene

B) benzo[a]pyrene

23

Figure 3 continued

C) benzo[b]fluoranthene

D) chrysene

E) sum of four marker PAHs

The kernel density plots for BAP and BBF are almost identical and centred at or close to a z-score

value of zero as can be seen in Figure 3B and 3C Therefore bias introduced by the type of analysis

method is unlikely for these two analytes The situation is different for BAA and CHR In case of

BAA the kernel density distribution of z-scores based on HPLC-FD is shifted to negative values

24

(Figure 3A) which indicates a tendency to underestimate the BAA content in this particular

samplematrix when applying HPLC-FD This might be reasoned by separationintegration problems

between BAA and CHR peaks A few participants (from the HPLC-FD subgroup) that got negative

z-scores for BAA overestimated the CHR content This caused the slight shift of the density trace for

HPLC-FD towards negative values Removing this data from the evaluation would result in a density

trace with the mode at zero

The opposite is the case for CHR (Figure 3D) The density distribution of the z-scores attributed to

HPLC-FD data is centred almost at zero However the distribution for data based on GC-MS is shifted

to positive values indicating strong positive bias This bias might be caused by a lack of selectivity of

the analysis method The test material contains triphenylene which is isobaric to chrysene

Consequently it cannot be distinguished from chrysene by means of mass spectrometry These two

compounds have to be separated chromatographically which is not easy They coelute on unpolar and

on most mid-polar capillary columns Columns that allow their separation were commercialised only

recently and might not be available in all laboratories Therefore laboratories which determine the

four EU marker PAHs by means of GC-MS shall pay attention to the chromatographic separation of

triphenylene and chrysene A lack of chromatographic resolution might lead to strong positive bias of

GC-MS methods and to false conclusions on the marketability of the tested sample

The chromatographic separation of chrysene and triphenylene is not a problem with HPLC which is

reflected in the position of the density plot for chrysene However attention has to be paid in HPLC-

FD analysis to a proper clean up of the sample extract in order to eliminate interferences that show

similar retention as BAA and CHR

95 Evaluation of the influence of calibration

The participants received together with the food supplement test sample two standard solutions in

depending of their choice either acetonitrile or toluene The analyte content of one solution was

disclosed to the participants whereas the content of the other solution was kept undisclosed The

content values of the latter are presented in Table 5 Analysis results for the solution with undisclosed

content had to be reported to the PT organiser The respective data are presented in Annex 9

Percent deviations of the reported results from the assigned values were calculated for both the food

supplement test sample and the standard solution with undisclosed analyte content Youden plots were

prepared in order to visualize the influence of instrument calibration on the results reported for the

food supplement sample Two plots are discussed in the following The whole set of Youden plots are

given in Annex 9

25

Figure 4 Youden plot of the percent deviations of values reported for benzo[a]pyrene from the

assigned values of the food supplement test material and the standard solution in

toluene with undisclosed content

Figure 4 reveals that a number of participants reported results with similar percent deviations from the

assigned values of both the results for the food supplement material and the standard solution in

toluene with undisclosed content This correlation indicates that over- respectively underestimation of

the analyte content of the food supplement material might be caused by biased instrument calibration

This finding underpins the importance of thorough calibration standard preparation However the data

was not in all cases conclusive as some laboratories performed well for the food supplement samples

but reported biased results for the standard solution with undisclosed content (eg Figure 4 participants

3620 and 5305) This fact cannot be explained yet

Figure 5 presents the respective Youden plot for chrysene in the food supplement test material and the

standard solution in toluene with undisclosed content From this graph it can be concluded that the

overestimation of the chrysene content of the food supplement material was not caused by instrument

calibration The results for the solvent solution of many laboratories which underperformed for the

26

food supplement sample agreed well with the assigned value This supports the conclusion that the

overestimation is caused by the determination of an interferent most probably triphenylene

Figure 5 Youden plot of the percent deviations of values reported for chrysene from the

assigned values of the food supplement test material and the standard solution in

toluene with undisclosed content

96 Evaluation of compliance with legislation

The method performance data reported for the four marker PAHs were evaluated for compliance with

the provisions given in Commission Regulation (EC) No 8362011 Table 18 in ANNEX 10 gives an

overview on the results of the evaluation Data were not reported in case of empty cells Non-

compliant data are highlighted in Table 18 It should be noted that Regulation (EU) No 8362011

specifies LOD and LOQ with two decimals This was not respected by all participants which

hampered the compliance assessment

27

In summary it can be stated that the majority of participants reported method performance

characteristics for the determination of the four marker PAHs in the food supplement test sample that

are compliant with legislation However some laboratories need to improve their analysis methods

The laboratories in question are urged to adapt to the provisions given in the new legislation

10 Follow-up actions for underperforming laboratories

All laboratories that got questionable or non-satisfactory performance ratings are urged to perform

root cause analysis and to implement corrective actions

As agreed during the EU-RL PAH workshop 2011 the EU-RL PAH will set up follow-up measures in

due time for all NRLs that received for at least one of the four PAHs (BAA BAP BBF and CHR) z-

scores gt 3 as required by Regulation (EC) 8822004 and by the Protocol for management of

underperformance in comparative testing andor lack of collaboration of National Reference

Laboratories (NRLs) with European Union reference laboratories (EU-RLs) activities

These laboratories shall perform as an immediate action root-cause-analysis and shall report to the

EU-RL PAH in writing the identified cause for their underperformance and corrective actions they are

going to take Additionally they shall participate to an independent (non-EU-RL) proficiency test on

the determination of PAHs in food and shall communicate the outcome of this exercise to the EU-RL

PAH

28

11 Conclusions

Fourty seven participants reported results back to the EU-RL The performance of the majority of

participants was good In total 153 out of 225 attributed z-scores were below an absolute value of two

which equals to almost 68 However this percentage is much below the percentages of satisfactory

performance gained in previous studies This might be the consequence of the higher level of difficulty

with respect to matrix composition In addition bias can be concluded from the pattern of performance

indicators of some laboratories

In general the determination of chrysene caused most difficulties to the participants

zeta-Scores were calculated besides z-scores They indicate the agreement of the reported result with

the assigned value with respect to the stated measurement uncertainty The outcome of this rating was

worse than for the z-scores which indicates that the measurement uncertainty estimates were not

realistic Therefore participants underperforming with regard to zeta scores are urged to adapt their

measurement uncertainty statements

The great majority of participants in this inter-laboratory comparison applied analytical methods which

complied with EU legislation with regard to performance characteristics

12 Acknowledgements

The organisers would like to thank Mrs Beatriz de la Calle and Mr Franz Ulberth (all from IRMM

Geel Belgium) for their accurate revision of this report Mr Hakan Emteborg (from IRMM) for

ampouling of the standard solutions and all NRLs and OCLs for their cooperation

29

13 References

1 EU COMMISSION REGULATION (EC) No 7762006 of 23 May 2006 amending Annex VII to Regulation

(EC) No 8822004 of the European Parliament and of the Council as regards Community reference

laboratories Official Journal of the European Union 2006 L 136 p 3-8

Available from

httpeur-lexeuropaeuLexUriServLexUriServdouri=OJL200613600030008ENPDF

2 EU Regulation (EC) No 8822004 of the European Parliament and of the Council of 29 April 2004 on

official controls performed to ensure the verification of compliance with feed and food law animal health

and animal welfare rules Official Journal of the European Communities 2004 L191 p 1-52 Available

from

httpeur-lexeuropaeuLexUriServLexUriServdouri=OJL200419100010052ENPDF

3 EU Opinion of the Scientific Committee on Food on the risks to human health of Polycyclic Aromatic

Hydrocarbons in food (2002) Available from

httpeuropaeuintcommfoodfsscscfout153_enpdf

4 International Agency for Research on Cancer IARC Monographs on the Evaluation of

Carcinogenic Risks to Humans Some Non-heterocyclic Polycyclic Aromatic Hydrocarbons and Some

Related Exposures Volume 92 (2010) Available from

httpmonographsiarcfrENGMonographsvol92mono92pdf

5 EU Commission Regulation (EC) No 3332007 of 28 March 2007 laying down the methods of sampling

and analysis for the official control of the levels of lead cadmium mercury inorganic tin 3-MCPD and

benzo(a)pyrene in foodstuffs Official Journal of the European Union 2007 L 88 p 29-38 Available

from

httpeur-lexeuropaeuLexUriServLexUriServdouri=OJL200708800290038ENPDF

6 EU Commission Regulation (EC) No 18812006 of 19 December 2006 setting maximum levels for certain

contaminants in foodstuffs Official Journal of the European Union 2006 L 364 p 5-24 Available from

httpeur-lexeuropaeuLexUriServLexUriServdouri=OJL200636400050024ENPDF

7 EFSA Scientific Opinion of the Panel on Contaminants in the Food Chain on a request from the European

Commission on Polycyclic Aromatic Hydrocarbons in Food The EFSA Journal 2008 724 p 1-114

Available from

httpwwwefsaeuropaeuEFSAefsa_locale-1178620753812_1211902034842htm

30

8 EU Commission Regulation (EU) No 8352011 amending Regulation (EC) No 18812006 as regards

maximum levels for polycyclic aromatic hydrocarbons in foodstuffs Official Journal of the European

Union 2011 L 215 p 4-8

httpeur-lexeuropaeuLexUriServLexUriServdouri=OJL201121500040008ENPDF

9 EU Commission Regulation (EU) No 8362011 amending Regulation (EC) No 3332007 laying down the methods of sampling and analysis for the official control of the levels of lead cadmium mercury inorganic tin 3-MCPD and benzo(a)pyrene in foodstuffs Official Journal of the European Union 2011 L 215 p 9-16 httpeur-lexeuropaeuLexUriServLexUriServdouri=OJL201121500090016ENPDF

10 ISOIEC 170432010 (2010) Conformity assessment -- General requirements for proficiency testing

providers Geneva Switzerland

11 Thompson M Ellison SLR and Wood R The International Harmonized Protocol for the Proficiency

Testing of Analytical Chemistry Laboratories Pure Appl Chem (2006) 78(1) p 145ndash196

Available from httpmediaiupacorgpublicationspac2006pdf7801x0145pdf

31

14 ANNEXES

ANNEX 1 ndash Announcement of the PT on the IRMM webpage

ANNEX 2 ndash Announcement via e-mail and invitation

ANNEX 3 ndash Announcement of sample dispatch

ANNEX 4 ndash Technical specifications of the calibration solutions

ANNEX 5 ndash Homogeneity and stability of the test material

ANNEX 6 ndash Acknowledgement of sample receipt

ANNEX 7 ndash Questionnaire on details of analysis method

ANNEX 8 ndash Results reported for the food supplement sample

ANNEX 9 ndash Results reported for the standard solutions with undisclosed analyte content

ANNEX 10 ndash Method performance characteristics reported by participants

ANNEX 11 ndash Details of analysis methods reported by participants

32

ANNEX 1 ndash Announcement of the PT on the IRMM webpage

33

ANNEX 2 - Announcement via e-mail and invitation

34

ANNEX 3 - Announcement sample dispatch

Dear MadameSir

The test samples for the proficiency test on the determination of four PAHs in a food supplement (dry extract of St Johns wort) were

dispatched today

Explanation on the design of the study as well as instructions for sample storage and for reporting of results are in the parcel You will

also find your participant key in the parcel which is required for reporting of results

Please check the parcel after arrival for completeness and report the receipt of the test samples as well as their status to us by applying

the sample receipt form which is in the parcel

You are kindly requested to inform us by email if you do not receive the parcel by end of this week

My colleague Donata Lerda will then take care of it

With best regards

Thomas Wenzl

35

ANNEX 4 - Specifications of standard solutions with disclosed concentration

36

ANNEX 4 - continued

37

ANNEX 5 - Homogeneity and stability of the test material

Homogeneity of the test material

Benz[a]anthracene - BAA

Benzo[a]pyrene - BAP

38

ANNEX 5 - continued

Benzo[b]fluoranthene - BBF

Chrysene - CHR

39

ANNEX 5 - continued

Stability of the test material

Table 10 Content of the four target analytes in test materials stored under different

temperatures

BaA BaP BbF CHR

microgkg microgkg microgkg microgkg

Content 31 16 31 69 Storage at room temperature

U (k=2) 05 02 05 17

Content 31 15 29 68 Storage at reference temperature

U (k=2) 04 03 04 10

The applied capillary column does not separate CHR from triphenylene

40

ANNEX 6 ndash Documents sent to participants

41

ANNEX 6 - continued

42

ANNEX 7 Questionnaire on details of analysis method

43

ANNEX 7 - continued

44

ANNEX 7 - continued

45

ANNEX 8 - Results reported by participants for the food supplements test sample Table 11 Analysis results reported by the participants for the content of benz[a]anthracene (BAA) in the food supplements test material Values are presented as reported

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

3600 31 29 29 3 05 2

3602 199 193 178 19 021

3603 253 253 246 251 022 2

3604 199 209 23 213 071 2

3605 301 3 271 291 076 2

3606 302 288 278 289 064 2

3607 456 416 443 438 096 2

3608 796 695 651 714 149 2

3610 182 192 214 196 039 2

3611 414 504 488 469 96 2

3612 3 34 28 31 06 2

3613 32 33 34 33 07 2

3614 182 182 191 185 037 2

3615 3884 3822 3565 376 0752 2

3616 2 2 2 2 01 2

3617 366 37 35 362 046 2

3618 32 32 32 32 05 2

3619 299 305 297 299 048 2

3620 304 294 302 302 045 2

3621 29 26 29 28 04 2

3622 343 325 318 322 052 2

3623 35 3 33 33 07 2

3624

3626 318 31 314 314 126 2

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

5301 31 3 32 31 06 2

5302 314 319 315 316 063 2

5303 3102 2716 2822 2876 27 62

5304 261 263 26 261 57 2

5305 23 22 26 24 20

5306 22 23 23 22 11 2

5307 488 461 317 461

5308 168633 168544 1562025 1644598

5309 16 19 16 17

5310 3 304 311 305 015

5311 477 452 448 459 15 2

5312 506 63 494 543 134 2

5313 353 301 404 353

5314 431 373 378 394 5 096

5315 596 531 51 545 11 2

5316 224 231 235 235 022 2

5317 216 23 221 222 007 2

5318 064 07 067 01 2

5319 111 184 146 147 029 2

5320 515 525 564 534 48

5321 21 5322 13 87 9 87 35 2

5323 38 33 35 33 16 2

46

Figure 6 Results of replicate determinations (indicated by triangles) of benz[a]anthracene (BAA) in the food supplement test material

Horizontal blue lines represent the arithmetic mean value of replicate measurements and blue bars the reported expanded measurement uncertainty (k=2) The assigned value is

plotted as dotted line Red solid lines mark the range of satisfactory performance (|z|le2) Mean values are given for results outside the displayed data range

ProLab 2011

Laboratory

53

1853

1953

0853

0936

1436

0236

1036

1653

2136

0453

1753

0653

1653

0536

0353

0436

2153

0336

0636

0536

0036

2036

1953

1036

1253

0136

2653

0236

1836

2336

2236

1353

1353

2336

1736

1553

1453

0736

0753

1153

2053

1253

1536

0853

2236

11

an

aly

te c

on

ten

t (micro

gk

g)

10

95

9

85

8

75

7

65

6

55

5

45

4

35

3

25

2

15

1

05

0

102

3 468

7

Sample St Johns wort extract replicatesMeasurand Benz[a]anthraceneNo of laboratories 46

Assigned value 298 microgkg (Reference value)Target sd 062 microgkgTolerance limits 174 - 422 microgkg (|Z score| lt 200)

Assig

ned va

lue

Limit of tolerance

Limit of tolerance

47

Table 12 Analysis results reported by the participants for the content of benzo[a]pyrene (BAP) in the food supplements test material Values are presented as reported

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

3600 16 17 17 17 03 2

3602 118 123 13 124 015

3603 165 162 157 161 015 2

3604 134 154 139 143 04 2

3605 164 166 172 167 057 2

3606 181 159 161 167 031 2

3607 279 266 279 274 055 2

3608 57 687 461 573 226 2

3610 226 216 183 206 042 2

3611 289 325 311 308 36 2

3612 24 22 2 22 05 2

3613 17 18 19 18 05 2

3614 08 08 08 08 016 2

3615 1545 1393 1425 145 029 2

3616 16 16 14 15 03 2

3617 177 171 158 169 023 2

3618 2 2 2 2 02 2

3619 207 207 206 207 075 2

3620 165 169 163 166 017 2

3621 44 47 49 47 05 2

3622 212 208 222 213 038 2

3623 2 21 22 21 03 2

3624 18 178 182 18 031 2

3626 172 173 178 174 07 2

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

5301 18 18 18 18 04 2

5302 188 19 187 188 038 2

5303 1691 1426 149 1535 19 80

5304 145 142 15 146 69 2

5305 15 17 18 16 15

5306 16 17 17 16 08 2

5307 164 157 153 157

5308 102669 109324 109035 107009

5309 1 1 12 11

5310 122 122 124 123 006

5311 199 184 215 2 15 2

5312 258 248 239 248 05 2

5313 199 178 245 207

5314 174 171 172 172 11 0948

5315 253 248 213 238 05 2

5316 177 171 173 17 016 2

5317 255 277 276 269 012 2

5318 114 12 117 02 2

5319 19 228 231 216 043 2

5320 303 3 344 316 78

5321 12 12 12 12

5322 13 17 24 17 35 2

5323 2 2 19 2 1 2

48

Figure 7 Results of replicate determinations (indicated by triangles) of benzo[a]pyrene (BAP) in the food supplement test material

Horizontal blue lines represent the arithmetic mean value of replicate measurements and blue bars the reported expanded measurement uncertainty (k=2) The assigned value is

plotted as dotted line Red solid lines mark the range of satisfactory performance (|z|le2) Mean values are given for results outside the displayed data range

ProLab 2011

Laboratory

36

1453

0953

0853

1853

2153

1036

0236

0436

1553

0436

1653

0353

0736

0336

2036

0053

0553

0636

0636

0536

1753

1453

1636

2636

1336

2453

0153

2253

0253

2353

1136

1836

1953

1336

1036

2336

2253

1936

1253

1553

1253

1736

0753

2036

2136

0836

11

an

aly

te c

on

ten

t (micro

gk

g)

55

5

45

4

35

3

25

2

15

1

05

0

573

308

3

Sample St Johns wort extract replicatesMeasurand Benzo[a]pyreneNo of laboratories 48

Assigned value 165 microgkg (Reference value)Target sd 036 microgkgTolerance limits 093 - 237 microgkg (|Z score| lt 200)

Assig

ned va

lue

Limit of tolerance

Limit of tolerance

49

Table 13 Analysis results reported by the participants for the content of benzo[b]fluoranthene (BBF) in the food supplements test material Values are presented as reported

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

3600 32 3 3 31 09 2

3602 173 175 16 17 017

3603 265 252 25 256 025 2

3604 116 108 105 11 028 2

3605 286 225 232 247 074 2

3606 294 273 261 276 046 2

3607 579 579 544 567 113 2

3608 103 1201 876 1036 326 2

3610 173 196 204 191 038 2

3611 546 618 565 576 75 2

3612 3 28 28 29 06 2

3613 28 31 32 31 06 2

3614 135 111 135 127 022 2

3615 4187 3678 4111 4 08 2

3616 2 2 2 2 01 2

3617 33 27 299 035 2

3618 31 31 31 31 05 2

3619 326 324 312 326 064 2

3620 277 28 272 276 069 2

3621 26 25 23 25 04 2

3622 346 336 333 34 052 2

3623 26 28 29 28 02 2

3624

3626 288 28 307 292 117 2

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

5301 29 3 29 29 06 2

5302 297 3 296 298 06 2

5303 2705 2444 2608 2586 22 78

5304 245 254 26 253 85 2

5305 23 23 26 24 15

5306 42 43 4 42 21 2

5307 221 216 241 221

5308 232169 246654 240722 229848

5309 19 22 19 2 0

5310 3 315 325 313 016

5311 262 277 304 281 15 2

5312 27 289 337 299 075 2

5313 321 284 378 328

5314 423 404 385 404 34 083

5315 42 506 416 444 09 2

5316 265 267 277 273 018 2

5317 185 197 195 192 006 2

5318 276 281 279 04 2

5319 396 389 403 396 079 2

5320 623 618 635 625 14

5321 31

5322 24 33 49 33 35 2

5323 38 45 35 45 27 2

50

Figure 8 Results of replicate determinations (indicated by triangles) of benzo[b]fluoranthene (BBF) in the food supplement test material

Horizontal blue lines represent the arithmetic mean value of replicate measurements and blue bars the reported expanded measurement uncertainty (k=2) The assigned value is

plotted as dotted line Red solid lines mark the range of satisfactory performance (|z|le2) Mean values are given for results outside the displayed data range

ProLab 2011

Laboratory

36

0436

1436

0236

1053

1736

1653

0953

0753

0853

0536

2136

0553

0436

0353

0353

1636

0636

2036

2353

1853

1136

1236

2653

0153

0253

1236

1736

1336

0036

1853

2153

1036

1953

1336

2253

2253

2353

1936

1553

1453

0653

1536

0753

2036

0836

11

an

aly

te c

on

ten

t (micro

gk

g)

75

7

65

6

55

5

45

4

35

3

25

2

15

1

05

0

103

6

576

3

Sample St Johns wort extract replicatesMeasurand Benzo[b]fluorantheneNo of laboratories 46

Assigned value 292 microgkg (Reference value)Target sd 060 microgkgTolerance limits 172 - 412 microgkg (|Z score| lt 200)

Assig

ned va

lue

Limit of tolerance

Limit of tolerance

51

Table 14 Analysis results reported by the participants for the content of chrysene (CHR) in the food supplements test material Values are presented as reported

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

3600 5 49 5 5 07 2

3602 32 304 29 304 036

3603 462 469 447 459 048 2

3604 438 428 42 429 119 2

3605 365 41 381 386 085 2

3606 494 411 45 451 124 2

3607 897 774 787 819 18 2

3608 986 887 1168 1014 284 2

3610 418 392 462 424 085 2

3611 833 893 87 865 61 2

3612 6 64 58 61 12 2

3613 41 39 4 4 08 2

3614 272 254 288 271 054 2

3615 605 6028 5964 601 12 2

3616 47 45 49 47 05 2

3617 445 431 389 422 065 2

3618 45 45 45 45 06 2

3619 738 747 72 738 118 2

3620 463 472 446 46 092 2

3621 53 49 51 51 06 2

3622 478 513 505 488 08 2

3623 73 73 72 72 05 2

3624

3626 63 622 634 628 251 2

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

5301 49 49 49 49 1 2

5302 687 683 666 679 136 2

5303 6777 6181 6382 6441 24 70

5304 592 581 6 591 85 2

5305 6 58 64 61 20

5306 49 5 51 49 25 2

5307 524 5 574 524

5308 211488 216782 221146 213243

5309

5310 372 393 394 386 019

5311 43 459 422 437 15 2

5312 934 943 981 953 191 2

5313 793 73 99 838

5314 636 561 565 588 18 0818

5315 547 583 499 542 11 2

5316 292 286 285 282 06 2

5317 52 598 517 545 046 2

5318 368 345 357 05 2

5319 449 471 474 465 116 2

5320 76 924 818 834 99

5321 33

5322 7 7 76 7 35 2

5323 87 73 77 73 37 2

52

Figure 9 Results of replicate determinations (indicated by triangles) of chrysene (CHR) in the food supplement test material

Horizontal blue lines represent the arithmetic mean value of replicate measurements and blue bars the reported expanded measurement uncertainty (k=2) The assigned value is

plotted as dotted line Red solid lines mark the range of satisfactory performance (|z|le2) Mean values are given for results outside the displayed data range

ProLab 2011

Laboratory

53

0836

1453

1636

0253

2153

1836

0553

1036

1336

1736

1036

0453

1136

1836

0636

0336

2053

1936

1653

0136

0036

2253

0636

2153

0753

1553

1753

1453

0436

1536

1253

0536

2653

0353

0253

2236

2336

1953

2336

0753

2053

1353

1236

0836

11

an

aly

te c

on

ten

t (micro

gk

g)

13

12

11

10

9

8

7

6

5

4

3

2

1

0

865

3

Sample St Johns wort extract replicatesMeasurand ChryseneNo of laboratories 45

Assigned value 407 microgkg (Reference value)Target sd 083 microgkgTolerance limits 241 - 573 microgkg (|Z score| lt 200)

Assig

ned va

lue

Limit of tolerance

Limit of tolerance

53

Table 15 Analysis results reported by the participants for the sum of the four marker PAHs in the food supplements test material Values are presented as reported

Laboratory code

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg

3600 128 24 2

3602 79 126

3603 1127 096 2

3604 893 258 2

3605 1091 305 2

3606 1184 238 2

3607 2098 42 2

3608 3336 325 2

3610 1138 17 2

3611 2218 259 2

3612 143 29 2

3613 122 2 2

3614 663 129 2

3615 152 304 2

3616 102 03 2

3617 1252 169 2

3618 128 18 2

3619 157 305 2

3620 1204 12 2

3621 151 19 2

3622 1361 115 2

3623 154 09 2

3624

3626 1409 56 2

Laboratory code

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg

5301 126

5302 37 074 2

5303 13438 92

5304 1251 85 2

5305 125 20

5306 129 65 2

5307 1363

5308 7245598

5309

5310 1127 056

5311 1367 15 2

5312 2042 408 2

5313 1726

5314 1558

5315 177 35 2

5316 96 073 2

5317 1229 066 2

5318 82 05 2

5319 1225 267 2

5320 2309 41

5321

5322 207 35 2

5323 171 103 2

54

ANNEX 9 Results reported for the standard solutions with undisclosed analyte content Table 16 Analysis results reported by the participants for the four marker PAHs in the standard solution in toluene with undisclosed analyte

content Values are presented as reported U measurement uncertainty k coverage factor

benz[a]anthracene benzo[a]pyrene benzo[b]fluoranthene chrysene Laboratory

code Unit Result U k Result U k Result U k Result U k 3600 microgl 38 67 2 25 45 2 211 68 2 275 38 2

3605 microgl 367 184 2 2482 124 2 2592 13 2 235 118 2

3606 microgkg 3763 176 2 2609 142 2 2721 118 2 2539 134 2

3607 microgkg 3706 815 2 2824 565 2 2591 518 2 2587 569 2

3612 microgl 38 08 2 237 11 2 244 18 2 257 02 2

3614 microgl 324 65 2 209 38 2 215 37 2 21 42 2

3616 microgkg 318 07 2 224 14 2 231 26 2 216 1 2

3618 microgkg 417 17 2 284 08 2 292 06 2 297 03 2

3619 microgkg 47 742 2 274 468 2 228 387 2 274 434 2

3620 microgl 495 74 2 294 29 2 321 8 2 321 64 2

3621 microgl 241 22 2 143 13 2 188 16 2 145 14 2

3623 microgkg 476 25 2 311 53 2 269 3 2 297 03 2

3626 microgl 359 38 2 235 23 2 238 27 2 24 18 2

5301 microgkg 426 29 299 289

5302 microgkg 436 87 2 296 59 2 296 59 2 289 58 2

5303 microgl 3721 27 102 22838 19 99 23756 22 99 25486 24 98

5304 microgkg 38 29 2 225 26 2 257 37 2 258 52 2

5305 microgkg 305 225 245 29

5306 microgkg 316 63 2 247 49 2 65 13 2 22 44 2

5307 microgkg 3658 2559 241 2414

5308 microgkg 16854 109324 246654 216782

5311 microgl 374 20 2 279 15 2 268 15 2 272 15 2

5313 microgl 3408 2165 2275 2373

5314 microgkg 395 5 2875 11 3614 34 2905 18

5320 microgl 3267 368 1986 571 28 683 2427 512

5322 microgl 282 35 2 225 35 2 241 35 2 21 35 2

55

Figure 10 Youden plots of the percent deviation of the reported values from the assigned values for the food supplement test sample and the

standard solution in toluene with undisclosed content

(The identity of the respective measurand is given above the figure Results exceeding a difference of more than 100 were omitted)

56

Figure 10 - continued

57

Table 17 Analysis results reported by the participants for the four marker PAHs in the standard solution in acetonitrile with undisclosed

analyte content

Values are presented as reported U measurement uncertainty k coverage factor

benz[a]anthracene benzo[a]pyrene benzo[b]fluoranthene chrysene

Laboratory

code Unit Result U k Result U k Result U k Result U k

3602 microgkg 3675 247 247 244

3603 microgkg 358 086 2 236 028 2 235 028 2 236 057 2

3604 microgl 475 29 2 19 12 2 194 13 2 258 12 2

3608 microgl 3623 078 2 2385 023 2 2343 18 2 2296 092 2

3610 microgkg 369 185 2 25 125 2 251 126 2 244 122 2

3611 microgkg 48 02 2 323 02 2 328 04 2 317 03 2

3613 microgl 332 31 2 261 3 2 249 25 2 239 24 2

3615 microgl 415 2635 2729 2689

3617 microgkg 37 09 2 244 06 2 284 07 2 239 06 2

3622 microgl 37 12 2 25 23 2 257 21 2 238 1 2

3624 microgkg 402 104 2 255 084 2 265 096 2 253 07 2

5306 microgkg 563 113 2 371 74 2 359 72 2 333 67 2

5308 microgkg 168633 102669 232169 211488

5309 microgl 402 268 27

5310 microgl 315 158 25 125 25 125 288 144

5312 microgl 428 043 2 282 028 2 272 027 2 275 028 2

5315 microgkg 387 08 2 256 05 2 251 05 2 253 05 2

5316 microgl 3839 342 2 2485 191 2 2559 168 2 239 51 2

5317 microgl 2466 043 2 2212 034 2 2472 016 2 2146 068 2

5319 microgl 414 41 2 279 28 2 268 27 2 26 31 2

5321 microgkg 476 316 326 328

5322 microgl 2547 35 2

5323 microgkg 405 203 2 239 119 2 255 153 2 201 11 2

58

Figure 11 Youden plots of the percent deviation of the reported values from the assigned values for the food supplement test sample and the

standard solution in acetonitrile with undisclosed content

(The identity of the respective measurand is given above the figure Results exceeding a difference of more than 100 were omitted)

59

Figure 11 - continued

60

ANNEX 10 Table 18 Method performance parameters reported by the participants

Laboratory code

Analyte LOD LOQ Recovery Analyte LOD LOQ Recovery

microgkg microgkg microgkg microgkg 3600 BaA 03 06 978 BaP 03 07 9483602 BaA 007 021 926 BaP 005 015 993603 BaA 0011 05 987 BaP 0005 051 9553604 BaA 02 04 86 BaP 02 04 883605 BaA 0025 005 118 BaP 0025 005 1083606 BaA 001 003 65 BaP 001 003 703607 BaA 021 071 74 BaP 027 089 943608 BaA 016 048 8213 BaP 015 046 83683610 BaA 02 05 85 BaP 005 02 853611 BaA BaP 3612 BaA 1 117 BaP 1 1203613 BaA 01 03 90 BaP 01 03 903614 BaA 02 05 96 BaP 02 05 933615 BaA 005 015 110 BaP 005 015 1053616 BaA 01 05 108 BaP 01 05 753617 BaA 018 037 405 BaP 009 018 3873618 BaA 01 03 63 BaP 01 03 593619 BaA 005 005 59 BaP 033 033 543620 BaA 0009 0018 64 BaP 0005 001 553621 BaA 007 02 84 BaP 007 02 923622 BaA 01 03 1101 BaP 01 03 9853623 BaA 02 08 105 BaP 02 08 1013624 BaA BaP 01 033 8513625 BaA BaP 3626 BaA BaP 5301 BaA 003 01 102 BaP 003 01 1005302 BaA 01 03 78 BaP 01 03 865303 BaA 003 01 62 BaP 003 01 805304 BaA 1 1 70 BaP 1 1 955305 BaA 02 05 85 BaP 02 05 855306 BaA 03 1 699 BaP 03 1 905307 BaA 6537 BaP 81165308 BaA 0088 0265 99 BaP 00262 00785 1005309 BaA 01 08 92 BaP 01 08 925310 BaA 01 03 BaP 01 03 905311 BaA 002 005 80 BaP 002 004 915312 BaA 03 09 105 BaP 03 09 1055313 BaA 04 08 BaP 04 08 5314 BaA 001 002 96 BaP 001 002 9485315 BaA 01 03 50 - 100 BaP 01 03 50 - 100 5316 BaA 002 011 827 BaP 004 019 10445317 BaA 05 1 76 BaP 05 1 755318 BaA BaP 5319 BaA 009 029 951 BaP 011 041 995320 BaA 12 18 946 BaP 06 1 8345321 BaA 03 05 BaP 05 1 5322 BaA 1 2 100 BaP 1 2 1005323 BaA 26 BaP 26

61

ANNEX 10 Table 18 - continued

Laboratory code Analyte LOD LOQ Recovery Analyte LOD LOQ Recovery

microgkg microgkg microgkg microgkg 3600 BbF 03 05 96 CHR 02 03 10153602 BbF 015 045 947 CHR 003 009 9193603 BbF 0014 05 1012 CHR 0004 05 10033604 BbF 02 04 85 CHR 02 04 873605 BbF 005 01 94 CHR 0025 005 863606 BbF 001 003 70 CHR 001 003 653607 BbF 018 06 100 CHR 014 028 943608 BbF 015 046 7979 CHR 015 045 71253610 BbF 005 02 85 CHR 02 05 853611 BbF CHR 3612 BbF 1 118 CHR 1 1173613 BbF 01 03 90 CHR 01 03 903614 BbF 01 04 96 CHR 02 05 953615 BbF 008 025 109 CHR 003 01 1083616 BbF 01 05 78 CHR 01 05 723617 BbF 035 07 38 CHR 012 024 4163618 BbF 01 03 62 CHR 01 03 623619 BbF 016 016 63 CHR 01 01 593620 BbF 0003 0006 55 CHR 001 002 653621 BbF 007 02 103 CHR 007 02 893622 BbF 02 06 963 CHR 03 09 1043623 BbF 02 08 106 CHR 02 08 1193624 BbF CHR 3625 BbF CHR 3626 BbF CHR 5301 BbF 003 01 96 CHR 003 01 1145302 BbF 01 03 84 CHR 01 03 835303 BbF 003 01 78 CHR 003 01 705304 BbF 1 1 86 CHR 1 1 705305 BbF 02 05 90 CHR 02 05 955306 BbF 03 1 1435 CHR 03 1 1185307 BbF 8116 CHR 65375308 BbF 0079 0238 99 CHR 009 026 1005309 BbF 01 08 92 CHR 5310 BbF 07 2 CHR 01 03 5311 BbF 002 004 95 CHR 002 004 895312 BbF 03 09 105 CHR 03 09 1055313 BbF 04 08 CHR 04 08 5314 BbF 001 002 83 CHR 001 002 8185315 BbF 02 05 50 -100 CHR 02 05 50 - 100 5316 BbF 005 024 814 CHR 001 007 9595317 BbF 05 1 73 CHR 05 1 785318 BbF CHR 5319 BbF 011 038 946 CHR 013 043 9675320 BbF 07 09 1194 CHR 23 32 10455321 BbF 05 1 CHR 05 1 5322 BbF 1 2 100 CHR 1 2 1005323 BbF 26 CHR 26

62

ANNEX 11 Table 19 Details of analysis method reported by the participants Data are presented as reported

Laboratory code

Analysis technique

Instrument calibration

Internal standards applied

Sample clean up

Details of sample

clean up

Extraction technique

Details of sample

extraction

3600 6) GC-MS

2) Internal standardisation with labelled substances

Deuterated PAH mix 9 (Ehrenstorfer)

1) Saponification 2) LiquidLiquid partitioning

D) Other Liquidliquid partitioning

3602 2) HPLC-UV-FLD

1) External calibration

3) Gel permeation chromatography

A) Pressurised liquid extraction (PLE)

3603 1) HPLC-FLD

1) External calibration

3) Gel permeation chromatography

D) Other 1 minute with dichloromethane in vortex

3604 2) HPLC-UV-FLD

2) Internal standardisation with labelled substances

DiP D14

5) Donor acceptor complex chromatography

D) Other LL

3605 1) HPLC-FLD

1) External calibration

3) Gel permeation chromatography

C) Soxhlet extraction

3606 7) GC-MSMS

2) Internal standardisation with labelled substances

BaP 13C4 CHR 13C6 BaA 13C6 BbF 13C6

4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

3607 6) GC-MS

2) Internal standardisation with labelled substances

BaA-d12 CHR-d12 BbF-d12 BkF-d12 BaP-d12 ICP-d12 BgP-d12 DiP-d14

1) Saponification

D) Other solvent partitioning

3608 1) HPLC-FLD

1) External calibration

6) Other Liquid extraction

D) Other

Extraction in a rotary agitator and centrifugation

3610 2) HPLC-UV-FLD

1) External calibration

4) Solid phase extraction

B) Sonication

3611

3612 6) GC-MS

2) Internal standardisation with labelled substances

chrysene-d12 dibenzo(ah)anthracene-d14 dibenzo(ai)pyrene-d14

4) Solid phase extraction

B) Sonication

3613 5) LC-MSMS

3) Internal standardisation with unlabelled substances

I use Chrysene-D12 3) Gel permeation chromatography

D) Other extraction with hexan

63

Table 19 - continued

Laboratory code

Analysis technique

Instrument calibration

Internal standards applied

Sample clean up Details of sample

clean up

Extraction technique

Details of sample

extraction

3614 6) GC-MS 1) External calibration

3) Gel permeation chromatography

B) Sonication

3615 1) HPLC-FLD

1) External calibration

6) Other PLE GPC

A) Pressurised liquid extraction (PLE)

3616 6) GC-MS

2) Internal standardisation with labelled substances

Benzo(a)pyrene-13C4 Benzo(a)anthracene-13C6 Benzo(b)fluoranthene-13C6 Chrysene-13C6

2) LiquidLiquid partitioning 4) Solid phase extraction

B) Sonication

3617 1) HPLC-FLD

1) External calibration

4) Solid phase extraction

C) Soxhlet extraction

3618 6) GC-MS

2) Internal standardisation with labelled substances

deuterated standards

1) Saponification 4) Solid phase extraction

D) Other Handshaken with cyclohexane

3619 6) GC-MS

2) Internal standardisation with labelled substances

C13 Labelled US EPA 16

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

D) Other Homegenisation amp saponiofication

3620 9) GC-HRMS

2) Internal standardisation with labelled substances

mix of deuterated PAHs (BAA-D12 CHR-D12 BAP-D12 BBF-D12)

3) Gel permeation chromatography 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

3621 6) GC-MS

2) Internal standardisation with labelled substances

Chrysene D12 Benzo(a)pyrene D12 Benzo(b)fluoranthene D12 Benzo(a)anthracene

2) LiquidLiquid partitioning 4) Solid phase extraction

B) Sonication

3622 1) HPLC-FLD

4) Standard addition method

4) Solid phase extraction

D) Other

Extraction with cyclohexane by heating under reflux

3623 7) GC-MSMS

2) Internal standardisation with labelled substances

C13 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

3624 2) HPLC-UV-FLD

1) External calibration

2) LiquidLiquid partitioning

D) Other hot solvent extraction

3626

64

Table 19 - continued

Laboratory code

Analysis technique

Instrument calibration

Internal standards

applied

Sample clean up

Details of sample

clean up

Extraction technique

Details of sample

extraction

5301 7) GC-MSMS

2) Internal standardisation with labelled substances

EACH SUBSTANCE C13

4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5302 7) GC-MSMS

2) Internal standardisation with labelled substances

Isotope labelled PAHs 13C for each analyte

4) Solid phase extraction

C) Soxhlet extraction

5303 7) GC-MSMS

3) Internal standardisation with unlabelled substances

HAP C13 4) Solid phase extraction

D) Other automate ASE200 DIONEX

5304 7) GC-MSMS

2) Internal standardisation with labelled substances

with C13 for each HAP

2) LiquidLiquid partitioning 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5305 7) GC-MSMS

2) Internal standardisation with labelled substances

C13 isotopiques internal standards

4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5306 7) GC-MSMS

2) Internal standardisation with labelled substances

C13 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5307 6) GC-MS

2) Internal standardisation with labelled substances

1) Saponification 2) LiquidLiquid partitioning

D) Other solvent extraction

5308 1) HPLC-FLD 1) External calibration

4) Solid phase extraction

B) Sonication

5309 1) HPLC-FLD 1) External calibration

4) Solid phase extraction

D) Other only SPE

5310 1) HPLC-FLD 1) External calibration

1) Saponification 2) LiquidLiquid partitioning 3) Gel permeation chromatography

C) Soxhlet extraction

5311 6) GC-MS

2) Internal standardisation with labelled substances

13 C Chrysene 13 C Benz(a)pyrene

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

B) Sonication

5312 1) HPLC-FLD

3) Internal standardisation with unlabelled substances

Benzo(b)chrysen

2) LiquidLiquid partitioning 4) Solid phase extraction

Quechers D) Other Quechers

65

Table 19 - continued

Laboratory code

Analysis technique

Instrument calibration

Internal standards applied

Sample clean up Details of sample

clean up

Extraction technique

Details of sample extraction

5313 6) GC-MS

2) Internal standardisation with labelled substances

Benz(a)anthracene D12 Benzo(b)fluranthene D12 Benzo(a)pyrene D12 Chrysene D12

1) Saponification 2) LiquidLiquid partitioning 3) Gel permeation chromatography

A) Pressurised liquid extraction (PLE)

5314 6) GC-MS

2) Internal standardisation with labelled substances

Benzo(a)pyren d12 2) LiquidLiquid partitioning

D) Other Extraction with

5315 1) HPLC-FLD

3) Internal standardisation with unlabelled substances

Benz(b)chrysen

1) Saponification 2) LiquidLiquid partitioning

D) Other shaker

5316 1) HPLC-FLD

1) External calibration

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

D) Other

LiquidLiquid extraction with cyclohexane and dimethylformamide

5317 7) GC-MSMS

1) External calibration

3) Gel permeation chromatography 6) Other

silica gel D) Other VDLUFA online method

5318

5319 1) HPLC-FLD

3) Internal standardisation with unlabelled substances

Benzo(b)chrysene 3) Gel permeation chromatography

A) Pressurised liquid extraction (PLE)

5320 6) GC-MS

3) Internal standardisation with unlabelled substances

22-binaphthyl

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

D) Other 3 hours refluxing

5321 1) HPLC-FLD

2) Internal standardisation with labelled substances

6-meacutethylchrysegravene

1) Saponification 2) LiquidLiquid partitioning

B) Sonication

5322 6) GC-MS

2) Internal standardisation with labelled substances

naftalene-d8 acenaphtalene-d10 phenanthrene-d10 chrysene-d12 perylene-d12 benzo(e)anthracene-d12

1) Saponification 2) LiquidLiquid partitioning 6) Other

clean-up with silica packed column

C) Soxhlet extraction

5323 1) HPLC-FLD

1) External calibration

4) Solid phase extraction 6) Other

Accelerated Solvent Extraction

A) Pressurised liquid extraction (PLE)

66

European Commission EUR 25245 EN ndash Joint Research Centre ndash Institute for Reference Materials and Measurements Title Report on the 8th inter-laboratory comparison organised by the European Union Reference Laboratory for Polycyclic Aromatic Hydrocarbons - Four marker PAHs in a dry extract of St Johns wort Author(s) Radoslav Lizak Szilard Szilagyi Philippe Verlinde and Thomas WENZL Luxembourg Publications Office of the European Union 2012 ndash 66 pp ndash 201 x 297 cm EUR ndash Scientific and Technical Research series ndash ISSN 1831-9424 ISBN 978-92-79-23337-1 doi 10278759387 Abstract The proficiency test here reported concerned the determination of the contents of four marker polycyclic aromatic hydrocarbons (PAHs) and of their sum in an food supplements test sample The set of marker PAHs consists of benz[a]anthacene benzo[a]pyrene benzo[b]fluoranthene and chrysene Participants to these PT were National Reference Laboratories for PAHs (NRLs-PAHs) and EU official food control laboratories The number of participants was in total 48

The PT was organised according ISO Standard 170432010

The test material used was a commercial dry extract of St Johns wort Participants also received a solution of the same PAHs either in acetonitrile or in toluene for checking their instrument calibration

The results from participants were rated with z-scores and zeta-scores About 68 of the reported results were attributed with z-scores with an absolute value of below two which is the threshold for satisfactory performance

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science and technology for the Union Close to the policy-making process it serves

the common interest of the Member States while being independent of special

interests whether private or national

L

A-N

A-252

45-EN

-N

• 1 Summary
• 2 Introduction
• 3 Scope
• 4 Participating Laboratories
• 5 Time frame
• 6 Confidentiality
• 7 Test materials
• • 71 1Preparation of test materials
  • 72 Assigned values and standard deviation for proficiency assessment
  • 73 Homogeneity and stability
  • 74 Sample dispatch
  • • 8 Design of the proficiency test
    • 9 Evaluation of the results
    • • 91 2General
      • 92 21BEvaluation criteria
      • 93 Performance of participants
      • 94 Evaluation of the influence of analysis method
      • 95 Evaluation of the influence of calibration
      • 96 26Evaluation of compliance with legislation
      • • 10 Follow-up actions for underperforming laboratories
        • 11 1Conclusions
        • 12 1Acknowledgements
        • 13 References

14

Equation 3 22assignedlab

assignedlab

uu

Xxzeta

where zeta refers to the zeta-score xlab to the reported ldquovalue for proficiency assessmentrdquo Xassigned to

the assigned value ulab to the measurement uncertainty reported by the laboratory and uassigned to the

uncertainty of the assigned value

The performance of the laboratories was classified according to ISOIEC 170432010 [10] Following

scheme is applied for the interpretation of zeta scores and z-scores

|score| 20 = satisfactory performance

20lt|score| lt 30 = questionable performance

|score| ge 30 = unsatisfactory performance

93 Performance of participants

The 48 participants in the study reported in total 225 results for proficiency evaluation which equals to

about 94 of the 240 possible results About 68 of the reported results were rated as satisfactory

with regard to z-scores

Figure 1 and Figure 2 give an overview of the z-scores assigned to the respective results The larger

the triangles the larger were the differences to the assigned values Red triangles indicate z-scores

above an absolute value of three whereas yellow triangles represent z-scores in the questionable

performance range The corresponding score values are plotted next to the triangles About 40 of the

72 non-satisfactory results were reported by seven laboratories only eg the performance of

participant 3608 was not satisfactory for the majority of target analytes

It is also obvious that most problems were encountered in the determination of chrysene and the sum

of the four analytes Potential reasons for the difficulties related to chrysene are discussed further

down

The ideal situation would be that deviations from the assigned values of the results reported for the

individual analytes are small and randomly distributed around zero This will give a satisfactory

performance statement for the sum parameter Deviations of opposite sign could also cancel out and

lead to a satisfactory performance for the sum parameter (eg participants 5303 5304 and 5305)

However in most cases a single underperformance for one analyte led also to non-satisfactory rating

for the sum parameter

15

The numerical values of the calculated z-scores are compiled in Table 6 and Table 7 z-Scores with an

absolute value of above 2 are given in red bold font

The results of the data evaluation for the individual analytes are given in ANNEX 8

For each analyte the figure shows the individual analysis results of the three replicate determinations

The assigned value is shown as dotted line The blue boxes represent the expanded uncertainties as

reported by participants for the value for proficiency assessment The arithmetic mean of the results

of the individual participants is indicated in the blue boxes by a blue line The satisfactory performance

range is located between the two red lines

The individual results of the replicate measurements and the value for proficiency assessment with

its accompanying expanded measurement uncertainty (k=2) are listed in the tables in ANNEX 8 as

well

16

Figure 1 Graphical presentation of z-scores corresponding to the values for proficiency assessment

reported by the NRLs for the contents of BAA BAP BBF and CHR in the St Johns wort extract test

material

Blue triangles indicate satisfactory performance yellow triangles indicate questionable performance red triangles indicate non-satisfactory performance z-score values are presented above the triangles for the latter two performance categories

ProLab 2011

Z score-3 0 3 -3 0 3 -3 0 3 -3 0 3 -3 0 3

BAA BAP BBF CHR PAH4

La

bo

rato

ry

36003602

3603

36043605

36063607

36083610

36113612

36133614

36153616

36173618

36193620

36213622

36233624

3626

2258

2343

2452

2669

2147

2788

2869

6710

70839

3011

11271

80525

8425

4561

12338

90680

4976

7331

99553

3998

3780

7500

17420

168413

3269

3029

-2348 -2736

-2023 -2981

-2155-3018

-3998

Figure 2 Graphical presentation of z-scores corresponding to the values for proficiency assessment

reported by the OCLs for the contents of BAA BAP BBF and CHR in the St Johns wort extract test

material

Blue triangles indicate satisfactory performance yellow triangles indicate questionable performance red triangles indicate non-satisfactory performance z-score values are presented above the triangles for the latter two performance categories

ProLab 2011

Z score-3 0 3 -3 0 3 -3 0 3 -3 0 3 -3 0 3

BAA BAP BBF CHR PAH4

La

bo

rato

ry

5301

5302

5303

5304

5305

5306

5307

5308

5309

5310

5311

5312

5313

5314

5315

5316

5317

5318

5319

5320

5321

5322

5323

2597

2629

2017

2293

2873

2123

2521

2620

2186

2222

2452

2864

3952

3984

3806

9226

4171 5522

3285

6594

5205

5157

3539

3901

7051

4519

3173

4872

9191

7276

4391

-2435

-2154

-2065

-2340

-2740-3726

-6346

-3505

17

Table 6 Compilation of z-scores calculated from the ldquoresults for proficiency assessment

reported by the NRLs for food supplement test material z-scores outside the

satisfactory range (|z| gt 2) are indicated by red font Values are reproduced as reported

by the laboratories Empty cells denote analytes for which results were not reported

Measurand benz[a]anthracene benzo[a]pyrene benzo[b]fluoranthene chrysene Sum PAH 4

Assigned value

microgkg 298 165 292 407 1162

Standard deviation for proficiency assessment

microgkg 062 036 060 083 125

Laboratory code

Result z-score Result z-score Result z-score Result z-score Result z-score

microgkg microgkg microgkg microgkg microgkg

3600 3 00 17 01 31 03 5 11 128 09

3602 19 -18 124 -11 17 -20 304 -12 79 -30

3603 251 -08 161 -01 256 -06 459 06 1127 -03

3604 213 -14 143 -06 11 -30 429 03 893 -22

3605 291 -01 167 01 247 -07 386 -03 1091 -06

3606 289 -01 167 01 276 -03 451 05 1184 02

3607 438 23 274 30 567 46 819 50 2098 75

3608 714 67 573 113 1036 123 1014 73 3336 174

3610 196 -17 206 11 191 -17 424 02 1138 -02

3611 469 708 308 805 576 907 865 996 2218 1684

3612 31 02 22 15 29 00 61 25 143 21

3613 33 05 18 04 31 03 4 -01 122 05

3614 185 -18 08 -23 127 -27 271 -16 663 -40

3615 376 13 145 -06 4 18 601 23 152 29

3616 2 -16 15 -04 2 -15 47 08 102 -11

3617 362 10 169 01 299 01 422 02 1252 07

3618 32 04 2 10 31 03 45 05 128 09

3619 299 00 207 12 326 06 738 40 157 33

3620 302 01 166 00 276 -03 46 06 1204 03

3621 28 -03 47 84 25 -07 51 12 151 28

3622 322 04 213 13 34 08 488 10 1361 16

3623 33 05 21 12 28 -02 72 38 154 30

3624 18 04

3626 314 03 174 02 292 00 628 27 1409 20

18

Table 7 Compilation of z-scores calculated from the ldquoresults for proficiency assessment

reported by the OCLs for food supplement test material z-scores outside the

satisfactory range (|z| gt 2) are indicated by red font Values are reproduced as reported

by the laboratories Empty cells denote analytes for which results were not reported

Measurand benz[a]anthracene benzo[a]pyrene benzo[b]fluoranthene chrysene Sum PAH 4

Assigned value

microgkg 298 165 292 407 1162

Standard deviation for proficiency assessment

microgkg 062 036 060 083 125

Laboratory code

Result z-score Result z-

score Result z-score Result

z-score

Result z-score

microgkg microgkg microgkg microgkg microgkg

5301 31 02 18 04 29 00 49 10 126 08

5302 316 03 188 06 298 01 679 33 37 -63

5303 2876 -02 1535 -03 2586 -06 6441 29 13438 15

5304 261 -06 146 -05 253 -06 591 22 1251 07

5305 24 -09 16 -01 24 -09 61 25 125 07

5306 22 -13 16 -01 42 21 49 10 129 10

5307 461 27 157 -02 221 -12 524 14 1363 16

5308 16446 -22 107009 -16 229848 -10 213243 -23 72456 -35

5309 17 -21 11 -15 2 -15

5310 305 01 123 -12 313 03 386 -03 1127 -03

5311 459 26 2 10 281 -02 437 04 1367 16

5312 543 40 248 23 299 01 953 66 2042 71

5313 353 09 207 12 328 06 838 52 1726 45

5314 394 16 172 02 404 19 588 22 1558 32

5315 545 40 238 20 444 25 542 16 177 49

5316 235 -10 17 01 273 -03 282 -15 96 -16

5317 222 -12 269 29 192 -17 545 17 1229 05

5318 067 -37 117 -13 279 -02 357 -06 82 -27

5319 147 -24 216 14 396 17 465 07 1225 05

5320 534 38 316 42 625 55 834 52 2309 92

5321 12 -12

5322 87 92 17 01 33 06 7 35 207 73

5323 33 05 2 10 45 26 73 39 171 44

19

Table 8 and Table 9 present the respective zeta-scores As for the z-scores data outside the

satisfactory performance range are given in red bold font Zeta scores were only calculated for

laboratories that provided measurement uncertainty data as well as a numerical value for the coverage

factor Zeta scores were not calculated when the reported uncertainty value exceeded the content value

which is probably linked to mixing of reporting units (microgkg and ) However the participants are

reminded that the measurement uncertainty shall be expressed in the same unit as the content value

The assessment of the performance of the participants based on the reported measurement uncertainty

gave a less favourable picture compared to z-scores Only 60 of the zeta-scores are within the

satisfactory performance range The distribution of satisfactory performance ratings between NRLs

and OCLs is not balanced It was higher for the NRLs

It has to be noted that the magnitude of the zeta-scores were for some participants much higher than

the z-scores attributed to the same results Consequently the laboratories perform according to fitness-

for-purpose criterion specified in legislation which forms the basis for the z-scores but seem to have

difficulties in deducing realistic measurement uncertainty values Eg the relative standard

measurement uncertainties reported by the NRLs ranged from 25 to 20 with a median value of

about 10 Only two OCLs reported measurement uncertainties that exceeded the maximum tolerable

uncertainty (Uf) The respective data is highlighted in yellow in Table 9

However the EU-RL PAHs will continue to pay special attention to measurement uncertainty in future

ILCs as it has major implications on the assessment of compliance of food with European legislation

20

Table 8 Compilation of zeta-scores calculated from the ldquoresults for proficiency assessment

reported by the NRLs for test material St Johns wort the reported measurement

uncertainty and the uncertainty of the analyte content of the test material zeta-scores

outside the satisfactory range (|zeta| gt 2) are indicated by red bold font Empty cells

denote analytes for which either results or measurement uncertainties were not

reported

21

Table 9 Compilation of zeta-scores calculated from the ldquoresults for proficiency assessment

reported by the OCLs for test material St Johns wort the reported measurement

uncertainty and the uncertainty of the analyte content of the test material zeta-scores

outside the satisfactory range (|zeta| gt 2) are indicated by red bold font yellow

highlighted cells ugtUf Empty cells denote analytes for which results were not

reported

22

94 Evaluation of the influence of analysis method

The information on the applied analysis method (ANNEX 11) was used to group the performance

indicators attributed to the reported results according to the applied analysis technique Two groups of

data were formed They represent results obtained by high performance liquid chromatography-

fluorescence detection (HPLC-FD about 40 ) and by gas chromatography-mass spectrometry (GC-

MS about 60 ) respectively Kernel density plots were constructed based on the respective z-scores

Figure 3A to 3E show the different Kernel density plots

Figure 3 Kernel density plots of z-scores attributed to the results for proficiency assessment

which were reported for the food supplements test sample Results were classified

according to the applied analysis technique

A) benz[a]anthracene

B) benzo[a]pyrene

23

Figure 3 continued

C) benzo[b]fluoranthene

D) chrysene

E) sum of four marker PAHs

The kernel density plots for BAP and BBF are almost identical and centred at or close to a z-score

value of zero as can be seen in Figure 3B and 3C Therefore bias introduced by the type of analysis

method is unlikely for these two analytes The situation is different for BAA and CHR In case of

BAA the kernel density distribution of z-scores based on HPLC-FD is shifted to negative values

24

(Figure 3A) which indicates a tendency to underestimate the BAA content in this particular

samplematrix when applying HPLC-FD This might be reasoned by separationintegration problems

between BAA and CHR peaks A few participants (from the HPLC-FD subgroup) that got negative

z-scores for BAA overestimated the CHR content This caused the slight shift of the density trace for

HPLC-FD towards negative values Removing this data from the evaluation would result in a density

trace with the mode at zero

The opposite is the case for CHR (Figure 3D) The density distribution of the z-scores attributed to

HPLC-FD data is centred almost at zero However the distribution for data based on GC-MS is shifted

to positive values indicating strong positive bias This bias might be caused by a lack of selectivity of

the analysis method The test material contains triphenylene which is isobaric to chrysene

Consequently it cannot be distinguished from chrysene by means of mass spectrometry These two

compounds have to be separated chromatographically which is not easy They coelute on unpolar and

on most mid-polar capillary columns Columns that allow their separation were commercialised only

recently and might not be available in all laboratories Therefore laboratories which determine the

four EU marker PAHs by means of GC-MS shall pay attention to the chromatographic separation of

triphenylene and chrysene A lack of chromatographic resolution might lead to strong positive bias of

GC-MS methods and to false conclusions on the marketability of the tested sample

The chromatographic separation of chrysene and triphenylene is not a problem with HPLC which is

reflected in the position of the density plot for chrysene However attention has to be paid in HPLC-

FD analysis to a proper clean up of the sample extract in order to eliminate interferences that show

similar retention as BAA and CHR

95 Evaluation of the influence of calibration

The participants received together with the food supplement test sample two standard solutions in

depending of their choice either acetonitrile or toluene The analyte content of one solution was

disclosed to the participants whereas the content of the other solution was kept undisclosed The

content values of the latter are presented in Table 5 Analysis results for the solution with undisclosed

content had to be reported to the PT organiser The respective data are presented in Annex 9

Percent deviations of the reported results from the assigned values were calculated for both the food

supplement test sample and the standard solution with undisclosed analyte content Youden plots were

prepared in order to visualize the influence of instrument calibration on the results reported for the

food supplement sample Two plots are discussed in the following The whole set of Youden plots are

given in Annex 9

25

Figure 4 Youden plot of the percent deviations of values reported for benzo[a]pyrene from the

assigned values of the food supplement test material and the standard solution in

toluene with undisclosed content

Figure 4 reveals that a number of participants reported results with similar percent deviations from the

assigned values of both the results for the food supplement material and the standard solution in

toluene with undisclosed content This correlation indicates that over- respectively underestimation of

the analyte content of the food supplement material might be caused by biased instrument calibration

This finding underpins the importance of thorough calibration standard preparation However the data

was not in all cases conclusive as some laboratories performed well for the food supplement samples

but reported biased results for the standard solution with undisclosed content (eg Figure 4 participants

3620 and 5305) This fact cannot be explained yet

Figure 5 presents the respective Youden plot for chrysene in the food supplement test material and the

standard solution in toluene with undisclosed content From this graph it can be concluded that the

overestimation of the chrysene content of the food supplement material was not caused by instrument

calibration The results for the solvent solution of many laboratories which underperformed for the

26

food supplement sample agreed well with the assigned value This supports the conclusion that the

overestimation is caused by the determination of an interferent most probably triphenylene

Figure 5 Youden plot of the percent deviations of values reported for chrysene from the

assigned values of the food supplement test material and the standard solution in

toluene with undisclosed content

96 Evaluation of compliance with legislation

The method performance data reported for the four marker PAHs were evaluated for compliance with

the provisions given in Commission Regulation (EC) No 8362011 Table 18 in ANNEX 10 gives an

overview on the results of the evaluation Data were not reported in case of empty cells Non-

compliant data are highlighted in Table 18 It should be noted that Regulation (EU) No 8362011

specifies LOD and LOQ with two decimals This was not respected by all participants which

hampered the compliance assessment

27

In summary it can be stated that the majority of participants reported method performance

characteristics for the determination of the four marker PAHs in the food supplement test sample that

are compliant with legislation However some laboratories need to improve their analysis methods

The laboratories in question are urged to adapt to the provisions given in the new legislation

10 Follow-up actions for underperforming laboratories

All laboratories that got questionable or non-satisfactory performance ratings are urged to perform

root cause analysis and to implement corrective actions

As agreed during the EU-RL PAH workshop 2011 the EU-RL PAH will set up follow-up measures in

due time for all NRLs that received for at least one of the four PAHs (BAA BAP BBF and CHR) z-

scores gt 3 as required by Regulation (EC) 8822004 and by the Protocol for management of

underperformance in comparative testing andor lack of collaboration of National Reference

Laboratories (NRLs) with European Union reference laboratories (EU-RLs) activities

These laboratories shall perform as an immediate action root-cause-analysis and shall report to the

EU-RL PAH in writing the identified cause for their underperformance and corrective actions they are

going to take Additionally they shall participate to an independent (non-EU-RL) proficiency test on

the determination of PAHs in food and shall communicate the outcome of this exercise to the EU-RL

PAH

28

11 Conclusions

Fourty seven participants reported results back to the EU-RL The performance of the majority of

participants was good In total 153 out of 225 attributed z-scores were below an absolute value of two

which equals to almost 68 However this percentage is much below the percentages of satisfactory

performance gained in previous studies This might be the consequence of the higher level of difficulty

with respect to matrix composition In addition bias can be concluded from the pattern of performance

indicators of some laboratories

In general the determination of chrysene caused most difficulties to the participants

zeta-Scores were calculated besides z-scores They indicate the agreement of the reported result with

the assigned value with respect to the stated measurement uncertainty The outcome of this rating was

worse than for the z-scores which indicates that the measurement uncertainty estimates were not

realistic Therefore participants underperforming with regard to zeta scores are urged to adapt their

measurement uncertainty statements

The great majority of participants in this inter-laboratory comparison applied analytical methods which

complied with EU legislation with regard to performance characteristics

12 Acknowledgements

The organisers would like to thank Mrs Beatriz de la Calle and Mr Franz Ulberth (all from IRMM

Geel Belgium) for their accurate revision of this report Mr Hakan Emteborg (from IRMM) for

ampouling of the standard solutions and all NRLs and OCLs for their cooperation

29

13 References

1 EU COMMISSION REGULATION (EC) No 7762006 of 23 May 2006 amending Annex VII to Regulation

(EC) No 8822004 of the European Parliament and of the Council as regards Community reference

laboratories Official Journal of the European Union 2006 L 136 p 3-8

Available from

httpeur-lexeuropaeuLexUriServLexUriServdouri=OJL200613600030008ENPDF

2 EU Regulation (EC) No 8822004 of the European Parliament and of the Council of 29 April 2004 on

official controls performed to ensure the verification of compliance with feed and food law animal health

and animal welfare rules Official Journal of the European Communities 2004 L191 p 1-52 Available

from

httpeur-lexeuropaeuLexUriServLexUriServdouri=OJL200419100010052ENPDF

3 EU Opinion of the Scientific Committee on Food on the risks to human health of Polycyclic Aromatic

Hydrocarbons in food (2002) Available from

httpeuropaeuintcommfoodfsscscfout153_enpdf

4 International Agency for Research on Cancer IARC Monographs on the Evaluation of

Carcinogenic Risks to Humans Some Non-heterocyclic Polycyclic Aromatic Hydrocarbons and Some

Related Exposures Volume 92 (2010) Available from

httpmonographsiarcfrENGMonographsvol92mono92pdf

5 EU Commission Regulation (EC) No 3332007 of 28 March 2007 laying down the methods of sampling

and analysis for the official control of the levels of lead cadmium mercury inorganic tin 3-MCPD and

benzo(a)pyrene in foodstuffs Official Journal of the European Union 2007 L 88 p 29-38 Available

from

httpeur-lexeuropaeuLexUriServLexUriServdouri=OJL200708800290038ENPDF

6 EU Commission Regulation (EC) No 18812006 of 19 December 2006 setting maximum levels for certain

contaminants in foodstuffs Official Journal of the European Union 2006 L 364 p 5-24 Available from

httpeur-lexeuropaeuLexUriServLexUriServdouri=OJL200636400050024ENPDF

7 EFSA Scientific Opinion of the Panel on Contaminants in the Food Chain on a request from the European

Commission on Polycyclic Aromatic Hydrocarbons in Food The EFSA Journal 2008 724 p 1-114

Available from

httpwwwefsaeuropaeuEFSAefsa_locale-1178620753812_1211902034842htm

30

8 EU Commission Regulation (EU) No 8352011 amending Regulation (EC) No 18812006 as regards

maximum levels for polycyclic aromatic hydrocarbons in foodstuffs Official Journal of the European

Union 2011 L 215 p 4-8

httpeur-lexeuropaeuLexUriServLexUriServdouri=OJL201121500040008ENPDF

9 EU Commission Regulation (EU) No 8362011 amending Regulation (EC) No 3332007 laying down the methods of sampling and analysis for the official control of the levels of lead cadmium mercury inorganic tin 3-MCPD and benzo(a)pyrene in foodstuffs Official Journal of the European Union 2011 L 215 p 9-16 httpeur-lexeuropaeuLexUriServLexUriServdouri=OJL201121500090016ENPDF

10 ISOIEC 170432010 (2010) Conformity assessment -- General requirements for proficiency testing

providers Geneva Switzerland

11 Thompson M Ellison SLR and Wood R The International Harmonized Protocol for the Proficiency

Testing of Analytical Chemistry Laboratories Pure Appl Chem (2006) 78(1) p 145ndash196

Available from httpmediaiupacorgpublicationspac2006pdf7801x0145pdf

31

14 ANNEXES

ANNEX 1 ndash Announcement of the PT on the IRMM webpage

ANNEX 2 ndash Announcement via e-mail and invitation

ANNEX 3 ndash Announcement of sample dispatch

ANNEX 4 ndash Technical specifications of the calibration solutions

ANNEX 5 ndash Homogeneity and stability of the test material

ANNEX 6 ndash Acknowledgement of sample receipt

ANNEX 7 ndash Questionnaire on details of analysis method

ANNEX 8 ndash Results reported for the food supplement sample

ANNEX 9 ndash Results reported for the standard solutions with undisclosed analyte content

ANNEX 10 ndash Method performance characteristics reported by participants

ANNEX 11 ndash Details of analysis methods reported by participants

32

ANNEX 1 ndash Announcement of the PT on the IRMM webpage

33

ANNEX 2 - Announcement via e-mail and invitation

34

ANNEX 3 - Announcement sample dispatch

Dear MadameSir

The test samples for the proficiency test on the determination of four PAHs in a food supplement (dry extract of St Johns wort) were

dispatched today

Explanation on the design of the study as well as instructions for sample storage and for reporting of results are in the parcel You will

also find your participant key in the parcel which is required for reporting of results

Please check the parcel after arrival for completeness and report the receipt of the test samples as well as their status to us by applying

the sample receipt form which is in the parcel

You are kindly requested to inform us by email if you do not receive the parcel by end of this week

My colleague Donata Lerda will then take care of it

With best regards

Thomas Wenzl

35

ANNEX 4 - Specifications of standard solutions with disclosed concentration

36

ANNEX 4 - continued

37

ANNEX 5 - Homogeneity and stability of the test material

Homogeneity of the test material

Benz[a]anthracene - BAA

Benzo[a]pyrene - BAP

38

ANNEX 5 - continued

Benzo[b]fluoranthene - BBF

Chrysene - CHR

39

ANNEX 5 - continued

Stability of the test material

Table 10 Content of the four target analytes in test materials stored under different

temperatures

BaA BaP BbF CHR

microgkg microgkg microgkg microgkg

Content 31 16 31 69 Storage at room temperature

U (k=2) 05 02 05 17

Content 31 15 29 68 Storage at reference temperature

U (k=2) 04 03 04 10

The applied capillary column does not separate CHR from triphenylene

40

ANNEX 6 ndash Documents sent to participants

41

ANNEX 6 - continued

42

ANNEX 7 Questionnaire on details of analysis method

43

ANNEX 7 - continued

44

ANNEX 7 - continued

45

ANNEX 8 - Results reported by participants for the food supplements test sample Table 11 Analysis results reported by the participants for the content of benz[a]anthracene (BAA) in the food supplements test material Values are presented as reported

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

3600 31 29 29 3 05 2

3602 199 193 178 19 021

3603 253 253 246 251 022 2

3604 199 209 23 213 071 2

3605 301 3 271 291 076 2

3606 302 288 278 289 064 2

3607 456 416 443 438 096 2

3608 796 695 651 714 149 2

3610 182 192 214 196 039 2

3611 414 504 488 469 96 2

3612 3 34 28 31 06 2

3613 32 33 34 33 07 2

3614 182 182 191 185 037 2

3615 3884 3822 3565 376 0752 2

3616 2 2 2 2 01 2

3617 366 37 35 362 046 2

3618 32 32 32 32 05 2

3619 299 305 297 299 048 2

3620 304 294 302 302 045 2

3621 29 26 29 28 04 2

3622 343 325 318 322 052 2

3623 35 3 33 33 07 2

3624

3626 318 31 314 314 126 2

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

5301 31 3 32 31 06 2

5302 314 319 315 316 063 2

5303 3102 2716 2822 2876 27 62

5304 261 263 26 261 57 2

5305 23 22 26 24 20

5306 22 23 23 22 11 2

5307 488 461 317 461

5308 168633 168544 1562025 1644598

5309 16 19 16 17

5310 3 304 311 305 015

5311 477 452 448 459 15 2

5312 506 63 494 543 134 2

5313 353 301 404 353

5314 431 373 378 394 5 096

5315 596 531 51 545 11 2

5316 224 231 235 235 022 2

5317 216 23 221 222 007 2

5318 064 07 067 01 2

5319 111 184 146 147 029 2

5320 515 525 564 534 48

5321 21 5322 13 87 9 87 35 2

5323 38 33 35 33 16 2

46

Figure 6 Results of replicate determinations (indicated by triangles) of benz[a]anthracene (BAA) in the food supplement test material

Horizontal blue lines represent the arithmetic mean value of replicate measurements and blue bars the reported expanded measurement uncertainty (k=2) The assigned value is

plotted as dotted line Red solid lines mark the range of satisfactory performance (|z|le2) Mean values are given for results outside the displayed data range

ProLab 2011

Laboratory

53

1853

1953

0853

0936

1436

0236

1036

1653

2136

0453

1753

0653

1653

0536

0353

0436

2153

0336

0636

0536

0036

2036

1953

1036

1253

0136

2653

0236

1836

2336

2236

1353

1353

2336

1736

1553

1453

0736

0753

1153

2053

1253

1536

0853

2236

11

an

aly

te c

on

ten

t (micro

gk

g)

10

95

9

85

8

75

7

65

6

55

5

45

4

35

3

25

2

15

1

05

0

102

3 468

7

Sample St Johns wort extract replicatesMeasurand Benz[a]anthraceneNo of laboratories 46

Assigned value 298 microgkg (Reference value)Target sd 062 microgkgTolerance limits 174 - 422 microgkg (|Z score| lt 200)

Assig

ned va

lue

Limit of tolerance

Limit of tolerance

47

Table 12 Analysis results reported by the participants for the content of benzo[a]pyrene (BAP) in the food supplements test material Values are presented as reported

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

3600 16 17 17 17 03 2

3602 118 123 13 124 015

3603 165 162 157 161 015 2

3604 134 154 139 143 04 2

3605 164 166 172 167 057 2

3606 181 159 161 167 031 2

3607 279 266 279 274 055 2

3608 57 687 461 573 226 2

3610 226 216 183 206 042 2

3611 289 325 311 308 36 2

3612 24 22 2 22 05 2

3613 17 18 19 18 05 2

3614 08 08 08 08 016 2

3615 1545 1393 1425 145 029 2

3616 16 16 14 15 03 2

3617 177 171 158 169 023 2

3618 2 2 2 2 02 2

3619 207 207 206 207 075 2

3620 165 169 163 166 017 2

3621 44 47 49 47 05 2

3622 212 208 222 213 038 2

3623 2 21 22 21 03 2

3624 18 178 182 18 031 2

3626 172 173 178 174 07 2

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

5301 18 18 18 18 04 2

5302 188 19 187 188 038 2

5303 1691 1426 149 1535 19 80

5304 145 142 15 146 69 2

5305 15 17 18 16 15

5306 16 17 17 16 08 2

5307 164 157 153 157

5308 102669 109324 109035 107009

5309 1 1 12 11

5310 122 122 124 123 006

5311 199 184 215 2 15 2

5312 258 248 239 248 05 2

5313 199 178 245 207

5314 174 171 172 172 11 0948

5315 253 248 213 238 05 2

5316 177 171 173 17 016 2

5317 255 277 276 269 012 2

5318 114 12 117 02 2

5319 19 228 231 216 043 2

5320 303 3 344 316 78

5321 12 12 12 12

5322 13 17 24 17 35 2

5323 2 2 19 2 1 2

48

Figure 7 Results of replicate determinations (indicated by triangles) of benzo[a]pyrene (BAP) in the food supplement test material

Horizontal blue lines represent the arithmetic mean value of replicate measurements and blue bars the reported expanded measurement uncertainty (k=2) The assigned value is

plotted as dotted line Red solid lines mark the range of satisfactory performance (|z|le2) Mean values are given for results outside the displayed data range

ProLab 2011

Laboratory

36

1453

0953

0853

1853

2153

1036

0236

0436

1553

0436

1653

0353

0736

0336

2036

0053

0553

0636

0636

0536

1753

1453

1636

2636

1336

2453

0153

2253

0253

2353

1136

1836

1953

1336

1036

2336

2253

1936

1253

1553

1253

1736

0753

2036

2136

0836

11

an

aly

te c

on

ten

t (micro

gk

g)

55

5

45

4

35

3

25

2

15

1

05

0

573

308

3

Sample St Johns wort extract replicatesMeasurand Benzo[a]pyreneNo of laboratories 48

Assigned value 165 microgkg (Reference value)Target sd 036 microgkgTolerance limits 093 - 237 microgkg (|Z score| lt 200)

Assig

ned va

lue

Limit of tolerance

Limit of tolerance

49

Table 13 Analysis results reported by the participants for the content of benzo[b]fluoranthene (BBF) in the food supplements test material Values are presented as reported

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

3600 32 3 3 31 09 2

3602 173 175 16 17 017

3603 265 252 25 256 025 2

3604 116 108 105 11 028 2

3605 286 225 232 247 074 2

3606 294 273 261 276 046 2

3607 579 579 544 567 113 2

3608 103 1201 876 1036 326 2

3610 173 196 204 191 038 2

3611 546 618 565 576 75 2

3612 3 28 28 29 06 2

3613 28 31 32 31 06 2

3614 135 111 135 127 022 2

3615 4187 3678 4111 4 08 2

3616 2 2 2 2 01 2

3617 33 27 299 035 2

3618 31 31 31 31 05 2

3619 326 324 312 326 064 2

3620 277 28 272 276 069 2

3621 26 25 23 25 04 2

3622 346 336 333 34 052 2

3623 26 28 29 28 02 2

3624

3626 288 28 307 292 117 2

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

5301 29 3 29 29 06 2

5302 297 3 296 298 06 2

5303 2705 2444 2608 2586 22 78

5304 245 254 26 253 85 2

5305 23 23 26 24 15

5306 42 43 4 42 21 2

5307 221 216 241 221

5308 232169 246654 240722 229848

5309 19 22 19 2 0

5310 3 315 325 313 016

5311 262 277 304 281 15 2

5312 27 289 337 299 075 2

5313 321 284 378 328

5314 423 404 385 404 34 083

5315 42 506 416 444 09 2

5316 265 267 277 273 018 2

5317 185 197 195 192 006 2

5318 276 281 279 04 2

5319 396 389 403 396 079 2

5320 623 618 635 625 14

5321 31

5322 24 33 49 33 35 2

5323 38 45 35 45 27 2

50

Figure 8 Results of replicate determinations (indicated by triangles) of benzo[b]fluoranthene (BBF) in the food supplement test material

Horizontal blue lines represent the arithmetic mean value of replicate measurements and blue bars the reported expanded measurement uncertainty (k=2) The assigned value is

plotted as dotted line Red solid lines mark the range of satisfactory performance (|z|le2) Mean values are given for results outside the displayed data range

ProLab 2011

Laboratory

36

0436

1436

0236

1053

1736

1653

0953

0753

0853

0536

2136

0553

0436

0353

0353

1636

0636

2036

2353

1853

1136

1236

2653

0153

0253

1236

1736

1336

0036

1853

2153

1036

1953

1336

2253

2253

2353

1936

1553

1453

0653

1536

0753

2036

0836

11

an

aly

te c

on

ten

t (micro

gk

g)

75

7

65

6

55

5

45

4

35

3

25

2

15

1

05

0

103

6

576

3

Sample St Johns wort extract replicatesMeasurand Benzo[b]fluorantheneNo of laboratories 46

Assigned value 292 microgkg (Reference value)Target sd 060 microgkgTolerance limits 172 - 412 microgkg (|Z score| lt 200)

Assig

ned va

lue

Limit of tolerance

Limit of tolerance

51

Table 14 Analysis results reported by the participants for the content of chrysene (CHR) in the food supplements test material Values are presented as reported

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

3600 5 49 5 5 07 2

3602 32 304 29 304 036

3603 462 469 447 459 048 2

3604 438 428 42 429 119 2

3605 365 41 381 386 085 2

3606 494 411 45 451 124 2

3607 897 774 787 819 18 2

3608 986 887 1168 1014 284 2

3610 418 392 462 424 085 2

3611 833 893 87 865 61 2

3612 6 64 58 61 12 2

3613 41 39 4 4 08 2

3614 272 254 288 271 054 2

3615 605 6028 5964 601 12 2

3616 47 45 49 47 05 2

3617 445 431 389 422 065 2

3618 45 45 45 45 06 2

3619 738 747 72 738 118 2

3620 463 472 446 46 092 2

3621 53 49 51 51 06 2

3622 478 513 505 488 08 2

3623 73 73 72 72 05 2

3624

3626 63 622 634 628 251 2

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

5301 49 49 49 49 1 2

5302 687 683 666 679 136 2

5303 6777 6181 6382 6441 24 70

5304 592 581 6 591 85 2

5305 6 58 64 61 20

5306 49 5 51 49 25 2

5307 524 5 574 524

5308 211488 216782 221146 213243

5309

5310 372 393 394 386 019

5311 43 459 422 437 15 2

5312 934 943 981 953 191 2

5313 793 73 99 838

5314 636 561 565 588 18 0818

5315 547 583 499 542 11 2

5316 292 286 285 282 06 2

5317 52 598 517 545 046 2

5318 368 345 357 05 2

5319 449 471 474 465 116 2

5320 76 924 818 834 99

5321 33

5322 7 7 76 7 35 2

5323 87 73 77 73 37 2

52

Figure 9 Results of replicate determinations (indicated by triangles) of chrysene (CHR) in the food supplement test material

Horizontal blue lines represent the arithmetic mean value of replicate measurements and blue bars the reported expanded measurement uncertainty (k=2) The assigned value is

plotted as dotted line Red solid lines mark the range of satisfactory performance (|z|le2) Mean values are given for results outside the displayed data range

ProLab 2011

Laboratory

53

0836

1453

1636

0253

2153

1836

0553

1036

1336

1736

1036

0453

1136

1836

0636

0336

2053

1936

1653

0136

0036

2253

0636

2153

0753

1553

1753

1453

0436

1536

1253

0536

2653

0353

0253

2236

2336

1953

2336

0753

2053

1353

1236

0836

11

an

aly

te c

on

ten

t (micro

gk

g)

13

12

11

10

9

8

7

6

5

4

3

2

1

0

865

3

Sample St Johns wort extract replicatesMeasurand ChryseneNo of laboratories 45

Assigned value 407 microgkg (Reference value)Target sd 083 microgkgTolerance limits 241 - 573 microgkg (|Z score| lt 200)

Assig

ned va

lue

Limit of tolerance

Limit of tolerance

53

Table 15 Analysis results reported by the participants for the sum of the four marker PAHs in the food supplements test material Values are presented as reported

Laboratory code

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg

3600 128 24 2

3602 79 126

3603 1127 096 2

3604 893 258 2

3605 1091 305 2

3606 1184 238 2

3607 2098 42 2

3608 3336 325 2

3610 1138 17 2

3611 2218 259 2

3612 143 29 2

3613 122 2 2

3614 663 129 2

3615 152 304 2

3616 102 03 2

3617 1252 169 2

3618 128 18 2

3619 157 305 2

3620 1204 12 2

3621 151 19 2

3622 1361 115 2

3623 154 09 2

3624

3626 1409 56 2

Laboratory code

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg

5301 126

5302 37 074 2

5303 13438 92

5304 1251 85 2

5305 125 20

5306 129 65 2

5307 1363

5308 7245598

5309

5310 1127 056

5311 1367 15 2

5312 2042 408 2

5313 1726

5314 1558

5315 177 35 2

5316 96 073 2

5317 1229 066 2

5318 82 05 2

5319 1225 267 2

5320 2309 41

5321

5322 207 35 2

5323 171 103 2

54

ANNEX 9 Results reported for the standard solutions with undisclosed analyte content Table 16 Analysis results reported by the participants for the four marker PAHs in the standard solution in toluene with undisclosed analyte

content Values are presented as reported U measurement uncertainty k coverage factor

benz[a]anthracene benzo[a]pyrene benzo[b]fluoranthene chrysene Laboratory

code Unit Result U k Result U k Result U k Result U k 3600 microgl 38 67 2 25 45 2 211 68 2 275 38 2

3605 microgl 367 184 2 2482 124 2 2592 13 2 235 118 2

3606 microgkg 3763 176 2 2609 142 2 2721 118 2 2539 134 2

3607 microgkg 3706 815 2 2824 565 2 2591 518 2 2587 569 2

3612 microgl 38 08 2 237 11 2 244 18 2 257 02 2

3614 microgl 324 65 2 209 38 2 215 37 2 21 42 2

3616 microgkg 318 07 2 224 14 2 231 26 2 216 1 2

3618 microgkg 417 17 2 284 08 2 292 06 2 297 03 2

3619 microgkg 47 742 2 274 468 2 228 387 2 274 434 2

3620 microgl 495 74 2 294 29 2 321 8 2 321 64 2

3621 microgl 241 22 2 143 13 2 188 16 2 145 14 2

3623 microgkg 476 25 2 311 53 2 269 3 2 297 03 2

3626 microgl 359 38 2 235 23 2 238 27 2 24 18 2

5301 microgkg 426 29 299 289

5302 microgkg 436 87 2 296 59 2 296 59 2 289 58 2

5303 microgl 3721 27 102 22838 19 99 23756 22 99 25486 24 98

5304 microgkg 38 29 2 225 26 2 257 37 2 258 52 2

5305 microgkg 305 225 245 29

5306 microgkg 316 63 2 247 49 2 65 13 2 22 44 2

5307 microgkg 3658 2559 241 2414

5308 microgkg 16854 109324 246654 216782

5311 microgl 374 20 2 279 15 2 268 15 2 272 15 2

5313 microgl 3408 2165 2275 2373

5314 microgkg 395 5 2875 11 3614 34 2905 18

5320 microgl 3267 368 1986 571 28 683 2427 512

5322 microgl 282 35 2 225 35 2 241 35 2 21 35 2

55

Figure 10 Youden plots of the percent deviation of the reported values from the assigned values for the food supplement test sample and the

standard solution in toluene with undisclosed content

(The identity of the respective measurand is given above the figure Results exceeding a difference of more than 100 were omitted)

56

Figure 10 - continued

57

Table 17 Analysis results reported by the participants for the four marker PAHs in the standard solution in acetonitrile with undisclosed

analyte content

Values are presented as reported U measurement uncertainty k coverage factor

benz[a]anthracene benzo[a]pyrene benzo[b]fluoranthene chrysene

Laboratory

code Unit Result U k Result U k Result U k Result U k

3602 microgkg 3675 247 247 244

3603 microgkg 358 086 2 236 028 2 235 028 2 236 057 2

3604 microgl 475 29 2 19 12 2 194 13 2 258 12 2

3608 microgl 3623 078 2 2385 023 2 2343 18 2 2296 092 2

3610 microgkg 369 185 2 25 125 2 251 126 2 244 122 2

3611 microgkg 48 02 2 323 02 2 328 04 2 317 03 2

3613 microgl 332 31 2 261 3 2 249 25 2 239 24 2

3615 microgl 415 2635 2729 2689

3617 microgkg 37 09 2 244 06 2 284 07 2 239 06 2

3622 microgl 37 12 2 25 23 2 257 21 2 238 1 2

3624 microgkg 402 104 2 255 084 2 265 096 2 253 07 2

5306 microgkg 563 113 2 371 74 2 359 72 2 333 67 2

5308 microgkg 168633 102669 232169 211488

5309 microgl 402 268 27

5310 microgl 315 158 25 125 25 125 288 144

5312 microgl 428 043 2 282 028 2 272 027 2 275 028 2

5315 microgkg 387 08 2 256 05 2 251 05 2 253 05 2

5316 microgl 3839 342 2 2485 191 2 2559 168 2 239 51 2

5317 microgl 2466 043 2 2212 034 2 2472 016 2 2146 068 2

5319 microgl 414 41 2 279 28 2 268 27 2 26 31 2

5321 microgkg 476 316 326 328

5322 microgl 2547 35 2

5323 microgkg 405 203 2 239 119 2 255 153 2 201 11 2

58

Figure 11 Youden plots of the percent deviation of the reported values from the assigned values for the food supplement test sample and the

standard solution in acetonitrile with undisclosed content

(The identity of the respective measurand is given above the figure Results exceeding a difference of more than 100 were omitted)

59

Figure 11 - continued

60

ANNEX 10 Table 18 Method performance parameters reported by the participants

Laboratory code

Analyte LOD LOQ Recovery Analyte LOD LOQ Recovery

microgkg microgkg microgkg microgkg 3600 BaA 03 06 978 BaP 03 07 9483602 BaA 007 021 926 BaP 005 015 993603 BaA 0011 05 987 BaP 0005 051 9553604 BaA 02 04 86 BaP 02 04 883605 BaA 0025 005 118 BaP 0025 005 1083606 BaA 001 003 65 BaP 001 003 703607 BaA 021 071 74 BaP 027 089 943608 BaA 016 048 8213 BaP 015 046 83683610 BaA 02 05 85 BaP 005 02 853611 BaA BaP 3612 BaA 1 117 BaP 1 1203613 BaA 01 03 90 BaP 01 03 903614 BaA 02 05 96 BaP 02 05 933615 BaA 005 015 110 BaP 005 015 1053616 BaA 01 05 108 BaP 01 05 753617 BaA 018 037 405 BaP 009 018 3873618 BaA 01 03 63 BaP 01 03 593619 BaA 005 005 59 BaP 033 033 543620 BaA 0009 0018 64 BaP 0005 001 553621 BaA 007 02 84 BaP 007 02 923622 BaA 01 03 1101 BaP 01 03 9853623 BaA 02 08 105 BaP 02 08 1013624 BaA BaP 01 033 8513625 BaA BaP 3626 BaA BaP 5301 BaA 003 01 102 BaP 003 01 1005302 BaA 01 03 78 BaP 01 03 865303 BaA 003 01 62 BaP 003 01 805304 BaA 1 1 70 BaP 1 1 955305 BaA 02 05 85 BaP 02 05 855306 BaA 03 1 699 BaP 03 1 905307 BaA 6537 BaP 81165308 BaA 0088 0265 99 BaP 00262 00785 1005309 BaA 01 08 92 BaP 01 08 925310 BaA 01 03 BaP 01 03 905311 BaA 002 005 80 BaP 002 004 915312 BaA 03 09 105 BaP 03 09 1055313 BaA 04 08 BaP 04 08 5314 BaA 001 002 96 BaP 001 002 9485315 BaA 01 03 50 - 100 BaP 01 03 50 - 100 5316 BaA 002 011 827 BaP 004 019 10445317 BaA 05 1 76 BaP 05 1 755318 BaA BaP 5319 BaA 009 029 951 BaP 011 041 995320 BaA 12 18 946 BaP 06 1 8345321 BaA 03 05 BaP 05 1 5322 BaA 1 2 100 BaP 1 2 1005323 BaA 26 BaP 26

61

ANNEX 10 Table 18 - continued

Laboratory code Analyte LOD LOQ Recovery Analyte LOD LOQ Recovery

microgkg microgkg microgkg microgkg 3600 BbF 03 05 96 CHR 02 03 10153602 BbF 015 045 947 CHR 003 009 9193603 BbF 0014 05 1012 CHR 0004 05 10033604 BbF 02 04 85 CHR 02 04 873605 BbF 005 01 94 CHR 0025 005 863606 BbF 001 003 70 CHR 001 003 653607 BbF 018 06 100 CHR 014 028 943608 BbF 015 046 7979 CHR 015 045 71253610 BbF 005 02 85 CHR 02 05 853611 BbF CHR 3612 BbF 1 118 CHR 1 1173613 BbF 01 03 90 CHR 01 03 903614 BbF 01 04 96 CHR 02 05 953615 BbF 008 025 109 CHR 003 01 1083616 BbF 01 05 78 CHR 01 05 723617 BbF 035 07 38 CHR 012 024 4163618 BbF 01 03 62 CHR 01 03 623619 BbF 016 016 63 CHR 01 01 593620 BbF 0003 0006 55 CHR 001 002 653621 BbF 007 02 103 CHR 007 02 893622 BbF 02 06 963 CHR 03 09 1043623 BbF 02 08 106 CHR 02 08 1193624 BbF CHR 3625 BbF CHR 3626 BbF CHR 5301 BbF 003 01 96 CHR 003 01 1145302 BbF 01 03 84 CHR 01 03 835303 BbF 003 01 78 CHR 003 01 705304 BbF 1 1 86 CHR 1 1 705305 BbF 02 05 90 CHR 02 05 955306 BbF 03 1 1435 CHR 03 1 1185307 BbF 8116 CHR 65375308 BbF 0079 0238 99 CHR 009 026 1005309 BbF 01 08 92 CHR 5310 BbF 07 2 CHR 01 03 5311 BbF 002 004 95 CHR 002 004 895312 BbF 03 09 105 CHR 03 09 1055313 BbF 04 08 CHR 04 08 5314 BbF 001 002 83 CHR 001 002 8185315 BbF 02 05 50 -100 CHR 02 05 50 - 100 5316 BbF 005 024 814 CHR 001 007 9595317 BbF 05 1 73 CHR 05 1 785318 BbF CHR 5319 BbF 011 038 946 CHR 013 043 9675320 BbF 07 09 1194 CHR 23 32 10455321 BbF 05 1 CHR 05 1 5322 BbF 1 2 100 CHR 1 2 1005323 BbF 26 CHR 26

62

ANNEX 11 Table 19 Details of analysis method reported by the participants Data are presented as reported

Laboratory code

Analysis technique

Instrument calibration

Internal standards applied

Sample clean up

Details of sample

clean up

Extraction technique

Details of sample

extraction

3600 6) GC-MS

2) Internal standardisation with labelled substances

Deuterated PAH mix 9 (Ehrenstorfer)

1) Saponification 2) LiquidLiquid partitioning

D) Other Liquidliquid partitioning

3602 2) HPLC-UV-FLD

1) External calibration

3) Gel permeation chromatography

A) Pressurised liquid extraction (PLE)

3603 1) HPLC-FLD

1) External calibration

3) Gel permeation chromatography

D) Other 1 minute with dichloromethane in vortex

3604 2) HPLC-UV-FLD

2) Internal standardisation with labelled substances

DiP D14

5) Donor acceptor complex chromatography

D) Other LL

3605 1) HPLC-FLD

1) External calibration

3) Gel permeation chromatography

C) Soxhlet extraction

3606 7) GC-MSMS

2) Internal standardisation with labelled substances

BaP 13C4 CHR 13C6 BaA 13C6 BbF 13C6

4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

3607 6) GC-MS

2) Internal standardisation with labelled substances

BaA-d12 CHR-d12 BbF-d12 BkF-d12 BaP-d12 ICP-d12 BgP-d12 DiP-d14

1) Saponification

D) Other solvent partitioning

3608 1) HPLC-FLD

1) External calibration

6) Other Liquid extraction

D) Other

Extraction in a rotary agitator and centrifugation

3610 2) HPLC-UV-FLD

1) External calibration

4) Solid phase extraction

B) Sonication

3611

3612 6) GC-MS

2) Internal standardisation with labelled substances

chrysene-d12 dibenzo(ah)anthracene-d14 dibenzo(ai)pyrene-d14

4) Solid phase extraction

B) Sonication

3613 5) LC-MSMS

3) Internal standardisation with unlabelled substances

I use Chrysene-D12 3) Gel permeation chromatography

D) Other extraction with hexan

63

Table 19 - continued

Laboratory code

Analysis technique

Instrument calibration

Internal standards applied

Sample clean up Details of sample

clean up

Extraction technique

Details of sample

extraction

3614 6) GC-MS 1) External calibration

3) Gel permeation chromatography

B) Sonication

3615 1) HPLC-FLD

1) External calibration

6) Other PLE GPC

A) Pressurised liquid extraction (PLE)

3616 6) GC-MS

2) Internal standardisation with labelled substances

Benzo(a)pyrene-13C4 Benzo(a)anthracene-13C6 Benzo(b)fluoranthene-13C6 Chrysene-13C6

2) LiquidLiquid partitioning 4) Solid phase extraction

B) Sonication

3617 1) HPLC-FLD

1) External calibration

4) Solid phase extraction

C) Soxhlet extraction

3618 6) GC-MS

2) Internal standardisation with labelled substances

deuterated standards

1) Saponification 4) Solid phase extraction

D) Other Handshaken with cyclohexane

3619 6) GC-MS

2) Internal standardisation with labelled substances

C13 Labelled US EPA 16

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

D) Other Homegenisation amp saponiofication

3620 9) GC-HRMS

2) Internal standardisation with labelled substances

mix of deuterated PAHs (BAA-D12 CHR-D12 BAP-D12 BBF-D12)

3) Gel permeation chromatography 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

3621 6) GC-MS

2) Internal standardisation with labelled substances

Chrysene D12 Benzo(a)pyrene D12 Benzo(b)fluoranthene D12 Benzo(a)anthracene

2) LiquidLiquid partitioning 4) Solid phase extraction

B) Sonication

3622 1) HPLC-FLD

4) Standard addition method

4) Solid phase extraction

D) Other

Extraction with cyclohexane by heating under reflux

3623 7) GC-MSMS

2) Internal standardisation with labelled substances

C13 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

3624 2) HPLC-UV-FLD

1) External calibration

2) LiquidLiquid partitioning

D) Other hot solvent extraction

3626

64

Table 19 - continued

Laboratory code

Analysis technique

Instrument calibration

Internal standards

applied

Sample clean up

Details of sample

clean up

Extraction technique

Details of sample

extraction

5301 7) GC-MSMS

2) Internal standardisation with labelled substances

EACH SUBSTANCE C13

4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5302 7) GC-MSMS

2) Internal standardisation with labelled substances

Isotope labelled PAHs 13C for each analyte

4) Solid phase extraction

C) Soxhlet extraction

5303 7) GC-MSMS

3) Internal standardisation with unlabelled substances

HAP C13 4) Solid phase extraction

D) Other automate ASE200 DIONEX

5304 7) GC-MSMS

2) Internal standardisation with labelled substances

with C13 for each HAP

2) LiquidLiquid partitioning 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5305 7) GC-MSMS

2) Internal standardisation with labelled substances

C13 isotopiques internal standards

4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5306 7) GC-MSMS

2) Internal standardisation with labelled substances

C13 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5307 6) GC-MS

2) Internal standardisation with labelled substances

1) Saponification 2) LiquidLiquid partitioning

D) Other solvent extraction

5308 1) HPLC-FLD 1) External calibration

4) Solid phase extraction

B) Sonication

5309 1) HPLC-FLD 1) External calibration

4) Solid phase extraction

D) Other only SPE

5310 1) HPLC-FLD 1) External calibration

1) Saponification 2) LiquidLiquid partitioning 3) Gel permeation chromatography

C) Soxhlet extraction

5311 6) GC-MS

2) Internal standardisation with labelled substances

13 C Chrysene 13 C Benz(a)pyrene

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

B) Sonication

5312 1) HPLC-FLD

3) Internal standardisation with unlabelled substances

Benzo(b)chrysen

2) LiquidLiquid partitioning 4) Solid phase extraction

Quechers D) Other Quechers

65

Table 19 - continued

Laboratory code

Analysis technique

Instrument calibration

Internal standards applied

Sample clean up Details of sample

clean up

Extraction technique

Details of sample extraction

5313 6) GC-MS

2) Internal standardisation with labelled substances

Benz(a)anthracene D12 Benzo(b)fluranthene D12 Benzo(a)pyrene D12 Chrysene D12

1) Saponification 2) LiquidLiquid partitioning 3) Gel permeation chromatography

A) Pressurised liquid extraction (PLE)

5314 6) GC-MS

2) Internal standardisation with labelled substances

Benzo(a)pyren d12 2) LiquidLiquid partitioning

D) Other Extraction with

5315 1) HPLC-FLD

3) Internal standardisation with unlabelled substances

Benz(b)chrysen

1) Saponification 2) LiquidLiquid partitioning

D) Other shaker

5316 1) HPLC-FLD

1) External calibration

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

D) Other

LiquidLiquid extraction with cyclohexane and dimethylformamide

5317 7) GC-MSMS

1) External calibration

3) Gel permeation chromatography 6) Other

silica gel D) Other VDLUFA online method

5318

5319 1) HPLC-FLD

3) Internal standardisation with unlabelled substances

Benzo(b)chrysene 3) Gel permeation chromatography

A) Pressurised liquid extraction (PLE)

5320 6) GC-MS

3) Internal standardisation with unlabelled substances

22-binaphthyl

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

D) Other 3 hours refluxing

5321 1) HPLC-FLD

2) Internal standardisation with labelled substances

6-meacutethylchrysegravene

1) Saponification 2) LiquidLiquid partitioning

B) Sonication

5322 6) GC-MS

2) Internal standardisation with labelled substances

naftalene-d8 acenaphtalene-d10 phenanthrene-d10 chrysene-d12 perylene-d12 benzo(e)anthracene-d12

1) Saponification 2) LiquidLiquid partitioning 6) Other

clean-up with silica packed column

C) Soxhlet extraction

5323 1) HPLC-FLD

1) External calibration

4) Solid phase extraction 6) Other

Accelerated Solvent Extraction

A) Pressurised liquid extraction (PLE)

66

European Commission EUR 25245 EN ndash Joint Research Centre ndash Institute for Reference Materials and Measurements Title Report on the 8th inter-laboratory comparison organised by the European Union Reference Laboratory for Polycyclic Aromatic Hydrocarbons - Four marker PAHs in a dry extract of St Johns wort Author(s) Radoslav Lizak Szilard Szilagyi Philippe Verlinde and Thomas WENZL Luxembourg Publications Office of the European Union 2012 ndash 66 pp ndash 201 x 297 cm EUR ndash Scientific and Technical Research series ndash ISSN 1831-9424 ISBN 978-92-79-23337-1 doi 10278759387 Abstract The proficiency test here reported concerned the determination of the contents of four marker polycyclic aromatic hydrocarbons (PAHs) and of their sum in an food supplements test sample The set of marker PAHs consists of benz[a]anthacene benzo[a]pyrene benzo[b]fluoranthene and chrysene Participants to these PT were National Reference Laboratories for PAHs (NRLs-PAHs) and EU official food control laboratories The number of participants was in total 48

The PT was organised according ISO Standard 170432010

The test material used was a commercial dry extract of St Johns wort Participants also received a solution of the same PAHs either in acetonitrile or in toluene for checking their instrument calibration

The results from participants were rated with z-scores and zeta-scores About 68 of the reported results were attributed with z-scores with an absolute value of below two which is the threshold for satisfactory performance

How to obtain EU publications

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The mission of the JRC is to provide customer-driven scientific and technical support

for the conception development implementation and monitoring of EU policies As a

service of the European Commission the JRC functions as a reference centre of

science and technology for the Union Close to the policy-making process it serves

the common interest of the Member States while being independent of special

interests whether private or national

L

A-N

A-252

45-EN

-N

• 1 Summary
• 2 Introduction
• 3 Scope
• 4 Participating Laboratories
• 5 Time frame
• 6 Confidentiality
• 7 Test materials
• • 71 1Preparation of test materials
  • 72 Assigned values and standard deviation for proficiency assessment
  • 73 Homogeneity and stability
  • 74 Sample dispatch
  • • 8 Design of the proficiency test
    • 9 Evaluation of the results
    • • 91 2General
      • 92 21BEvaluation criteria
      • 93 Performance of participants
      • 94 Evaluation of the influence of analysis method
      • 95 Evaluation of the influence of calibration
      • 96 26Evaluation of compliance with legislation
      • • 10 Follow-up actions for underperforming laboratories
        • 11 1Conclusions
        • 12 1Acknowledgements
        • 13 References

15

The numerical values of the calculated z-scores are compiled in Table 6 and Table 7 z-Scores with an

absolute value of above 2 are given in red bold font

The results of the data evaluation for the individual analytes are given in ANNEX 8

For each analyte the figure shows the individual analysis results of the three replicate determinations

The assigned value is shown as dotted line The blue boxes represent the expanded uncertainties as

reported by participants for the value for proficiency assessment The arithmetic mean of the results

of the individual participants is indicated in the blue boxes by a blue line The satisfactory performance

range is located between the two red lines

The individual results of the replicate measurements and the value for proficiency assessment with

its accompanying expanded measurement uncertainty (k=2) are listed in the tables in ANNEX 8 as

well

16

Figure 1 Graphical presentation of z-scores corresponding to the values for proficiency assessment

reported by the NRLs for the contents of BAA BAP BBF and CHR in the St Johns wort extract test

material

Blue triangles indicate satisfactory performance yellow triangles indicate questionable performance red triangles indicate non-satisfactory performance z-score values are presented above the triangles for the latter two performance categories

ProLab 2011

Z score-3 0 3 -3 0 3 -3 0 3 -3 0 3 -3 0 3

BAA BAP BBF CHR PAH4

La

bo

rato

ry

36003602

3603

36043605

36063607

36083610

36113612

36133614

36153616

36173618

36193620

36213622

36233624

3626

2258

2343

2452

2669

2147

2788

2869

6710

70839

3011

11271

80525

8425

4561

12338

90680

4976

7331

99553

3998

3780

7500

17420

168413

3269

3029

-2348 -2736

-2023 -2981

-2155-3018

-3998

Figure 2 Graphical presentation of z-scores corresponding to the values for proficiency assessment

reported by the OCLs for the contents of BAA BAP BBF and CHR in the St Johns wort extract test

material

Blue triangles indicate satisfactory performance yellow triangles indicate questionable performance red triangles indicate non-satisfactory performance z-score values are presented above the triangles for the latter two performance categories

ProLab 2011

Z score-3 0 3 -3 0 3 -3 0 3 -3 0 3 -3 0 3

BAA BAP BBF CHR PAH4

La

bo

rato

ry

5301

5302

5303

5304

5305

5306

5307

5308

5309

5310

5311

5312

5313

5314

5315

5316

5317

5318

5319

5320

5321

5322

5323

2597

2629

2017

2293

2873

2123

2521

2620

2186

2222

2452

2864

3952

3984

3806

9226

4171 5522

3285

6594

5205

5157

3539

3901

7051

4519

3173

4872

9191

7276

4391

-2435

-2154

-2065

-2340

-2740-3726

-6346

-3505

17

Table 6 Compilation of z-scores calculated from the ldquoresults for proficiency assessment

reported by the NRLs for food supplement test material z-scores outside the

satisfactory range (|z| gt 2) are indicated by red font Values are reproduced as reported

by the laboratories Empty cells denote analytes for which results were not reported

Measurand benz[a]anthracene benzo[a]pyrene benzo[b]fluoranthene chrysene Sum PAH 4

Assigned value

microgkg 298 165 292 407 1162

Standard deviation for proficiency assessment

microgkg 062 036 060 083 125

Laboratory code

Result z-score Result z-score Result z-score Result z-score Result z-score

microgkg microgkg microgkg microgkg microgkg

3600 3 00 17 01 31 03 5 11 128 09

3602 19 -18 124 -11 17 -20 304 -12 79 -30

3603 251 -08 161 -01 256 -06 459 06 1127 -03

3604 213 -14 143 -06 11 -30 429 03 893 -22

3605 291 -01 167 01 247 -07 386 -03 1091 -06

3606 289 -01 167 01 276 -03 451 05 1184 02

3607 438 23 274 30 567 46 819 50 2098 75

3608 714 67 573 113 1036 123 1014 73 3336 174

3610 196 -17 206 11 191 -17 424 02 1138 -02

3611 469 708 308 805 576 907 865 996 2218 1684

3612 31 02 22 15 29 00 61 25 143 21

3613 33 05 18 04 31 03 4 -01 122 05

3614 185 -18 08 -23 127 -27 271 -16 663 -40

3615 376 13 145 -06 4 18 601 23 152 29

3616 2 -16 15 -04 2 -15 47 08 102 -11

3617 362 10 169 01 299 01 422 02 1252 07

3618 32 04 2 10 31 03 45 05 128 09

3619 299 00 207 12 326 06 738 40 157 33

3620 302 01 166 00 276 -03 46 06 1204 03

3621 28 -03 47 84 25 -07 51 12 151 28

3622 322 04 213 13 34 08 488 10 1361 16

3623 33 05 21 12 28 -02 72 38 154 30

3624 18 04

3626 314 03 174 02 292 00 628 27 1409 20

18

Table 7 Compilation of z-scores calculated from the ldquoresults for proficiency assessment

reported by the OCLs for food supplement test material z-scores outside the

satisfactory range (|z| gt 2) are indicated by red font Values are reproduced as reported

by the laboratories Empty cells denote analytes for which results were not reported

Measurand benz[a]anthracene benzo[a]pyrene benzo[b]fluoranthene chrysene Sum PAH 4

Assigned value

microgkg 298 165 292 407 1162

Standard deviation for proficiency assessment

microgkg 062 036 060 083 125

Laboratory code

Result z-score Result z-

score Result z-score Result

z-score

Result z-score

microgkg microgkg microgkg microgkg microgkg

5301 31 02 18 04 29 00 49 10 126 08

5302 316 03 188 06 298 01 679 33 37 -63

5303 2876 -02 1535 -03 2586 -06 6441 29 13438 15

5304 261 -06 146 -05 253 -06 591 22 1251 07

5305 24 -09 16 -01 24 -09 61 25 125 07

5306 22 -13 16 -01 42 21 49 10 129 10

5307 461 27 157 -02 221 -12 524 14 1363 16

5308 16446 -22 107009 -16 229848 -10 213243 -23 72456 -35

5309 17 -21 11 -15 2 -15

5310 305 01 123 -12 313 03 386 -03 1127 -03

5311 459 26 2 10 281 -02 437 04 1367 16

5312 543 40 248 23 299 01 953 66 2042 71

5313 353 09 207 12 328 06 838 52 1726 45

5314 394 16 172 02 404 19 588 22 1558 32

5315 545 40 238 20 444 25 542 16 177 49

5316 235 -10 17 01 273 -03 282 -15 96 -16

5317 222 -12 269 29 192 -17 545 17 1229 05

5318 067 -37 117 -13 279 -02 357 -06 82 -27

5319 147 -24 216 14 396 17 465 07 1225 05

5320 534 38 316 42 625 55 834 52 2309 92

5321 12 -12

5322 87 92 17 01 33 06 7 35 207 73

5323 33 05 2 10 45 26 73 39 171 44

19

Table 8 and Table 9 present the respective zeta-scores As for the z-scores data outside the

satisfactory performance range are given in red bold font Zeta scores were only calculated for

laboratories that provided measurement uncertainty data as well as a numerical value for the coverage

factor Zeta scores were not calculated when the reported uncertainty value exceeded the content value

which is probably linked to mixing of reporting units (microgkg and ) However the participants are

reminded that the measurement uncertainty shall be expressed in the same unit as the content value

The assessment of the performance of the participants based on the reported measurement uncertainty

gave a less favourable picture compared to z-scores Only 60 of the zeta-scores are within the

satisfactory performance range The distribution of satisfactory performance ratings between NRLs

and OCLs is not balanced It was higher for the NRLs

It has to be noted that the magnitude of the zeta-scores were for some participants much higher than

the z-scores attributed to the same results Consequently the laboratories perform according to fitness-

for-purpose criterion specified in legislation which forms the basis for the z-scores but seem to have

difficulties in deducing realistic measurement uncertainty values Eg the relative standard

measurement uncertainties reported by the NRLs ranged from 25 to 20 with a median value of

about 10 Only two OCLs reported measurement uncertainties that exceeded the maximum tolerable

uncertainty (Uf) The respective data is highlighted in yellow in Table 9

However the EU-RL PAHs will continue to pay special attention to measurement uncertainty in future

ILCs as it has major implications on the assessment of compliance of food with European legislation

20

Table 8 Compilation of zeta-scores calculated from the ldquoresults for proficiency assessment

reported by the NRLs for test material St Johns wort the reported measurement

uncertainty and the uncertainty of the analyte content of the test material zeta-scores

outside the satisfactory range (|zeta| gt 2) are indicated by red bold font Empty cells

denote analytes for which either results or measurement uncertainties were not

reported

21

Table 9 Compilation of zeta-scores calculated from the ldquoresults for proficiency assessment

reported by the OCLs for test material St Johns wort the reported measurement

uncertainty and the uncertainty of the analyte content of the test material zeta-scores

outside the satisfactory range (|zeta| gt 2) are indicated by red bold font yellow

highlighted cells ugtUf Empty cells denote analytes for which results were not

reported

22

94 Evaluation of the influence of analysis method

The information on the applied analysis method (ANNEX 11) was used to group the performance

indicators attributed to the reported results according to the applied analysis technique Two groups of

data were formed They represent results obtained by high performance liquid chromatography-

fluorescence detection (HPLC-FD about 40 ) and by gas chromatography-mass spectrometry (GC-

MS about 60 ) respectively Kernel density plots were constructed based on the respective z-scores

Figure 3A to 3E show the different Kernel density plots

Figure 3 Kernel density plots of z-scores attributed to the results for proficiency assessment

which were reported for the food supplements test sample Results were classified

according to the applied analysis technique

A) benz[a]anthracene

B) benzo[a]pyrene

23

Figure 3 continued

C) benzo[b]fluoranthene

D) chrysene

E) sum of four marker PAHs

The kernel density plots for BAP and BBF are almost identical and centred at or close to a z-score

value of zero as can be seen in Figure 3B and 3C Therefore bias introduced by the type of analysis

method is unlikely for these two analytes The situation is different for BAA and CHR In case of

BAA the kernel density distribution of z-scores based on HPLC-FD is shifted to negative values

24

(Figure 3A) which indicates a tendency to underestimate the BAA content in this particular

samplematrix when applying HPLC-FD This might be reasoned by separationintegration problems

between BAA and CHR peaks A few participants (from the HPLC-FD subgroup) that got negative

z-scores for BAA overestimated the CHR content This caused the slight shift of the density trace for

HPLC-FD towards negative values Removing this data from the evaluation would result in a density

trace with the mode at zero

The opposite is the case for CHR (Figure 3D) The density distribution of the z-scores attributed to

HPLC-FD data is centred almost at zero However the distribution for data based on GC-MS is shifted

to positive values indicating strong positive bias This bias might be caused by a lack of selectivity of

the analysis method The test material contains triphenylene which is isobaric to chrysene

Consequently it cannot be distinguished from chrysene by means of mass spectrometry These two

compounds have to be separated chromatographically which is not easy They coelute on unpolar and

on most mid-polar capillary columns Columns that allow their separation were commercialised only

recently and might not be available in all laboratories Therefore laboratories which determine the

four EU marker PAHs by means of GC-MS shall pay attention to the chromatographic separation of

triphenylene and chrysene A lack of chromatographic resolution might lead to strong positive bias of

GC-MS methods and to false conclusions on the marketability of the tested sample

The chromatographic separation of chrysene and triphenylene is not a problem with HPLC which is

reflected in the position of the density plot for chrysene However attention has to be paid in HPLC-

FD analysis to a proper clean up of the sample extract in order to eliminate interferences that show

similar retention as BAA and CHR

95 Evaluation of the influence of calibration

The participants received together with the food supplement test sample two standard solutions in

depending of their choice either acetonitrile or toluene The analyte content of one solution was

disclosed to the participants whereas the content of the other solution was kept undisclosed The

content values of the latter are presented in Table 5 Analysis results for the solution with undisclosed

content had to be reported to the PT organiser The respective data are presented in Annex 9

Percent deviations of the reported results from the assigned values were calculated for both the food

supplement test sample and the standard solution with undisclosed analyte content Youden plots were

prepared in order to visualize the influence of instrument calibration on the results reported for the

food supplement sample Two plots are discussed in the following The whole set of Youden plots are

given in Annex 9

25

Figure 4 Youden plot of the percent deviations of values reported for benzo[a]pyrene from the

assigned values of the food supplement test material and the standard solution in

toluene with undisclosed content

Figure 4 reveals that a number of participants reported results with similar percent deviations from the

assigned values of both the results for the food supplement material and the standard solution in

toluene with undisclosed content This correlation indicates that over- respectively underestimation of

the analyte content of the food supplement material might be caused by biased instrument calibration

This finding underpins the importance of thorough calibration standard preparation However the data

was not in all cases conclusive as some laboratories performed well for the food supplement samples

but reported biased results for the standard solution with undisclosed content (eg Figure 4 participants

3620 and 5305) This fact cannot be explained yet

Figure 5 presents the respective Youden plot for chrysene in the food supplement test material and the

standard solution in toluene with undisclosed content From this graph it can be concluded that the

overestimation of the chrysene content of the food supplement material was not caused by instrument

calibration The results for the solvent solution of many laboratories which underperformed for the

26

food supplement sample agreed well with the assigned value This supports the conclusion that the

overestimation is caused by the determination of an interferent most probably triphenylene

Figure 5 Youden plot of the percent deviations of values reported for chrysene from the

assigned values of the food supplement test material and the standard solution in

toluene with undisclosed content

96 Evaluation of compliance with legislation

The method performance data reported for the four marker PAHs were evaluated for compliance with

the provisions given in Commission Regulation (EC) No 8362011 Table 18 in ANNEX 10 gives an

overview on the results of the evaluation Data were not reported in case of empty cells Non-

compliant data are highlighted in Table 18 It should be noted that Regulation (EU) No 8362011

specifies LOD and LOQ with two decimals This was not respected by all participants which

hampered the compliance assessment

27

In summary it can be stated that the majority of participants reported method performance

characteristics for the determination of the four marker PAHs in the food supplement test sample that

are compliant with legislation However some laboratories need to improve their analysis methods

The laboratories in question are urged to adapt to the provisions given in the new legislation

10 Follow-up actions for underperforming laboratories

All laboratories that got questionable or non-satisfactory performance ratings are urged to perform

root cause analysis and to implement corrective actions

As agreed during the EU-RL PAH workshop 2011 the EU-RL PAH will set up follow-up measures in

due time for all NRLs that received for at least one of the four PAHs (BAA BAP BBF and CHR) z-

scores gt 3 as required by Regulation (EC) 8822004 and by the Protocol for management of

underperformance in comparative testing andor lack of collaboration of National Reference

Laboratories (NRLs) with European Union reference laboratories (EU-RLs) activities

These laboratories shall perform as an immediate action root-cause-analysis and shall report to the

EU-RL PAH in writing the identified cause for their underperformance and corrective actions they are

going to take Additionally they shall participate to an independent (non-EU-RL) proficiency test on

the determination of PAHs in food and shall communicate the outcome of this exercise to the EU-RL

PAH

28

11 Conclusions

Fourty seven participants reported results back to the EU-RL The performance of the majority of

participants was good In total 153 out of 225 attributed z-scores were below an absolute value of two

which equals to almost 68 However this percentage is much below the percentages of satisfactory

performance gained in previous studies This might be the consequence of the higher level of difficulty

with respect to matrix composition In addition bias can be concluded from the pattern of performance

indicators of some laboratories

In general the determination of chrysene caused most difficulties to the participants

zeta-Scores were calculated besides z-scores They indicate the agreement of the reported result with

the assigned value with respect to the stated measurement uncertainty The outcome of this rating was

worse than for the z-scores which indicates that the measurement uncertainty estimates were not

realistic Therefore participants underperforming with regard to zeta scores are urged to adapt their

measurement uncertainty statements

The great majority of participants in this inter-laboratory comparison applied analytical methods which

complied with EU legislation with regard to performance characteristics

12 Acknowledgements

The organisers would like to thank Mrs Beatriz de la Calle and Mr Franz Ulberth (all from IRMM

Geel Belgium) for their accurate revision of this report Mr Hakan Emteborg (from IRMM) for

ampouling of the standard solutions and all NRLs and OCLs for their cooperation

29

13 References

1 EU COMMISSION REGULATION (EC) No 7762006 of 23 May 2006 amending Annex VII to Regulation

(EC) No 8822004 of the European Parliament and of the Council as regards Community reference

laboratories Official Journal of the European Union 2006 L 136 p 3-8

Available from

httpeur-lexeuropaeuLexUriServLexUriServdouri=OJL200613600030008ENPDF

2 EU Regulation (EC) No 8822004 of the European Parliament and of the Council of 29 April 2004 on

official controls performed to ensure the verification of compliance with feed and food law animal health

and animal welfare rules Official Journal of the European Communities 2004 L191 p 1-52 Available

from

httpeur-lexeuropaeuLexUriServLexUriServdouri=OJL200419100010052ENPDF

3 EU Opinion of the Scientific Committee on Food on the risks to human health of Polycyclic Aromatic

Hydrocarbons in food (2002) Available from

httpeuropaeuintcommfoodfsscscfout153_enpdf

4 International Agency for Research on Cancer IARC Monographs on the Evaluation of

Carcinogenic Risks to Humans Some Non-heterocyclic Polycyclic Aromatic Hydrocarbons and Some

Related Exposures Volume 92 (2010) Available from

httpmonographsiarcfrENGMonographsvol92mono92pdf

5 EU Commission Regulation (EC) No 3332007 of 28 March 2007 laying down the methods of sampling

and analysis for the official control of the levels of lead cadmium mercury inorganic tin 3-MCPD and

benzo(a)pyrene in foodstuffs Official Journal of the European Union 2007 L 88 p 29-38 Available

from

httpeur-lexeuropaeuLexUriServLexUriServdouri=OJL200708800290038ENPDF

6 EU Commission Regulation (EC) No 18812006 of 19 December 2006 setting maximum levels for certain

contaminants in foodstuffs Official Journal of the European Union 2006 L 364 p 5-24 Available from

httpeur-lexeuropaeuLexUriServLexUriServdouri=OJL200636400050024ENPDF

7 EFSA Scientific Opinion of the Panel on Contaminants in the Food Chain on a request from the European

Commission on Polycyclic Aromatic Hydrocarbons in Food The EFSA Journal 2008 724 p 1-114

Available from

httpwwwefsaeuropaeuEFSAefsa_locale-1178620753812_1211902034842htm

30

8 EU Commission Regulation (EU) No 8352011 amending Regulation (EC) No 18812006 as regards

maximum levels for polycyclic aromatic hydrocarbons in foodstuffs Official Journal of the European

Union 2011 L 215 p 4-8

httpeur-lexeuropaeuLexUriServLexUriServdouri=OJL201121500040008ENPDF

9 EU Commission Regulation (EU) No 8362011 amending Regulation (EC) No 3332007 laying down the methods of sampling and analysis for the official control of the levels of lead cadmium mercury inorganic tin 3-MCPD and benzo(a)pyrene in foodstuffs Official Journal of the European Union 2011 L 215 p 9-16 httpeur-lexeuropaeuLexUriServLexUriServdouri=OJL201121500090016ENPDF

10 ISOIEC 170432010 (2010) Conformity assessment -- General requirements for proficiency testing

providers Geneva Switzerland

11 Thompson M Ellison SLR and Wood R The International Harmonized Protocol for the Proficiency

Testing of Analytical Chemistry Laboratories Pure Appl Chem (2006) 78(1) p 145ndash196

Available from httpmediaiupacorgpublicationspac2006pdf7801x0145pdf

31

14 ANNEXES

ANNEX 1 ndash Announcement of the PT on the IRMM webpage

ANNEX 2 ndash Announcement via e-mail and invitation

ANNEX 3 ndash Announcement of sample dispatch

ANNEX 4 ndash Technical specifications of the calibration solutions

ANNEX 5 ndash Homogeneity and stability of the test material

ANNEX 6 ndash Acknowledgement of sample receipt

ANNEX 7 ndash Questionnaire on details of analysis method

ANNEX 8 ndash Results reported for the food supplement sample

ANNEX 9 ndash Results reported for the standard solutions with undisclosed analyte content

ANNEX 10 ndash Method performance characteristics reported by participants

ANNEX 11 ndash Details of analysis methods reported by participants

32

ANNEX 1 ndash Announcement of the PT on the IRMM webpage

33

ANNEX 2 - Announcement via e-mail and invitation

34

ANNEX 3 - Announcement sample dispatch

Dear MadameSir

The test samples for the proficiency test on the determination of four PAHs in a food supplement (dry extract of St Johns wort) were

dispatched today

Explanation on the design of the study as well as instructions for sample storage and for reporting of results are in the parcel You will

also find your participant key in the parcel which is required for reporting of results

Please check the parcel after arrival for completeness and report the receipt of the test samples as well as their status to us by applying

the sample receipt form which is in the parcel

You are kindly requested to inform us by email if you do not receive the parcel by end of this week

My colleague Donata Lerda will then take care of it

With best regards

Thomas Wenzl

35

ANNEX 4 - Specifications of standard solutions with disclosed concentration

36

ANNEX 4 - continued

37

ANNEX 5 - Homogeneity and stability of the test material

Homogeneity of the test material

Benz[a]anthracene - BAA

Benzo[a]pyrene - BAP

38

ANNEX 5 - continued

Benzo[b]fluoranthene - BBF

Chrysene - CHR

39

ANNEX 5 - continued

Stability of the test material

Table 10 Content of the four target analytes in test materials stored under different

temperatures

BaA BaP BbF CHR

microgkg microgkg microgkg microgkg

Content 31 16 31 69 Storage at room temperature

U (k=2) 05 02 05 17

Content 31 15 29 68 Storage at reference temperature

U (k=2) 04 03 04 10

The applied capillary column does not separate CHR from triphenylene

40

ANNEX 6 ndash Documents sent to participants

41

ANNEX 6 - continued

42

ANNEX 7 Questionnaire on details of analysis method

43

ANNEX 7 - continued

44

ANNEX 7 - continued

45

ANNEX 8 - Results reported by participants for the food supplements test sample Table 11 Analysis results reported by the participants for the content of benz[a]anthracene (BAA) in the food supplements test material Values are presented as reported

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

3600 31 29 29 3 05 2

3602 199 193 178 19 021

3603 253 253 246 251 022 2

3604 199 209 23 213 071 2

3605 301 3 271 291 076 2

3606 302 288 278 289 064 2

3607 456 416 443 438 096 2

3608 796 695 651 714 149 2

3610 182 192 214 196 039 2

3611 414 504 488 469 96 2

3612 3 34 28 31 06 2

3613 32 33 34 33 07 2

3614 182 182 191 185 037 2

3615 3884 3822 3565 376 0752 2

3616 2 2 2 2 01 2

3617 366 37 35 362 046 2

3618 32 32 32 32 05 2

3619 299 305 297 299 048 2

3620 304 294 302 302 045 2

3621 29 26 29 28 04 2

3622 343 325 318 322 052 2

3623 35 3 33 33 07 2

3624

3626 318 31 314 314 126 2

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

5301 31 3 32 31 06 2

5302 314 319 315 316 063 2

5303 3102 2716 2822 2876 27 62

5304 261 263 26 261 57 2

5305 23 22 26 24 20

5306 22 23 23 22 11 2

5307 488 461 317 461

5308 168633 168544 1562025 1644598

5309 16 19 16 17

5310 3 304 311 305 015

5311 477 452 448 459 15 2

5312 506 63 494 543 134 2

5313 353 301 404 353

5314 431 373 378 394 5 096

5315 596 531 51 545 11 2

5316 224 231 235 235 022 2

5317 216 23 221 222 007 2

5318 064 07 067 01 2

5319 111 184 146 147 029 2

5320 515 525 564 534 48

5321 21 5322 13 87 9 87 35 2

5323 38 33 35 33 16 2

46

Figure 6 Results of replicate determinations (indicated by triangles) of benz[a]anthracene (BAA) in the food supplement test material

Horizontal blue lines represent the arithmetic mean value of replicate measurements and blue bars the reported expanded measurement uncertainty (k=2) The assigned value is

plotted as dotted line Red solid lines mark the range of satisfactory performance (|z|le2) Mean values are given for results outside the displayed data range

ProLab 2011

Laboratory

53

1853

1953

0853

0936

1436

0236

1036

1653

2136

0453

1753

0653

1653

0536

0353

0436

2153

0336

0636

0536

0036

2036

1953

1036

1253

0136

2653

0236

1836

2336

2236

1353

1353

2336

1736

1553

1453

0736

0753

1153

2053

1253

1536

0853

2236

11

an

aly

te c

on

ten

t (micro

gk

g)

10

95

9

85

8

75

7

65

6

55

5

45

4

35

3

25

2

15

1

05

0

102

3 468

7

Sample St Johns wort extract replicatesMeasurand Benz[a]anthraceneNo of laboratories 46

Assigned value 298 microgkg (Reference value)Target sd 062 microgkgTolerance limits 174 - 422 microgkg (|Z score| lt 200)

Assig

ned va

lue

Limit of tolerance

Limit of tolerance

47

Table 12 Analysis results reported by the participants for the content of benzo[a]pyrene (BAP) in the food supplements test material Values are presented as reported

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

3600 16 17 17 17 03 2

3602 118 123 13 124 015

3603 165 162 157 161 015 2

3604 134 154 139 143 04 2

3605 164 166 172 167 057 2

3606 181 159 161 167 031 2

3607 279 266 279 274 055 2

3608 57 687 461 573 226 2

3610 226 216 183 206 042 2

3611 289 325 311 308 36 2

3612 24 22 2 22 05 2

3613 17 18 19 18 05 2

3614 08 08 08 08 016 2

3615 1545 1393 1425 145 029 2

3616 16 16 14 15 03 2

3617 177 171 158 169 023 2

3618 2 2 2 2 02 2

3619 207 207 206 207 075 2

3620 165 169 163 166 017 2

3621 44 47 49 47 05 2

3622 212 208 222 213 038 2

3623 2 21 22 21 03 2

3624 18 178 182 18 031 2

3626 172 173 178 174 07 2

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

5301 18 18 18 18 04 2

5302 188 19 187 188 038 2

5303 1691 1426 149 1535 19 80

5304 145 142 15 146 69 2

5305 15 17 18 16 15

5306 16 17 17 16 08 2

5307 164 157 153 157

5308 102669 109324 109035 107009

5309 1 1 12 11

5310 122 122 124 123 006

5311 199 184 215 2 15 2

5312 258 248 239 248 05 2

5313 199 178 245 207

5314 174 171 172 172 11 0948

5315 253 248 213 238 05 2

5316 177 171 173 17 016 2

5317 255 277 276 269 012 2

5318 114 12 117 02 2

5319 19 228 231 216 043 2

5320 303 3 344 316 78

5321 12 12 12 12

5322 13 17 24 17 35 2

5323 2 2 19 2 1 2

48

Figure 7 Results of replicate determinations (indicated by triangles) of benzo[a]pyrene (BAP) in the food supplement test material

Horizontal blue lines represent the arithmetic mean value of replicate measurements and blue bars the reported expanded measurement uncertainty (k=2) The assigned value is

plotted as dotted line Red solid lines mark the range of satisfactory performance (|z|le2) Mean values are given for results outside the displayed data range

ProLab 2011

Laboratory

36

1453

0953

0853

1853

2153

1036

0236

0436

1553

0436

1653

0353

0736

0336

2036

0053

0553

0636

0636

0536

1753

1453

1636

2636

1336

2453

0153

2253

0253

2353

1136

1836

1953

1336

1036

2336

2253

1936

1253

1553

1253

1736

0753

2036

2136

0836

11

an

aly

te c

on

ten

t (micro

gk

g)

55

5

45

4

35

3

25

2

15

1

05

0

573

308

3

Sample St Johns wort extract replicatesMeasurand Benzo[a]pyreneNo of laboratories 48

Assigned value 165 microgkg (Reference value)Target sd 036 microgkgTolerance limits 093 - 237 microgkg (|Z score| lt 200)

Assig

ned va

lue

Limit of tolerance

Limit of tolerance

49

Table 13 Analysis results reported by the participants for the content of benzo[b]fluoranthene (BBF) in the food supplements test material Values are presented as reported

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

3600 32 3 3 31 09 2

3602 173 175 16 17 017

3603 265 252 25 256 025 2

3604 116 108 105 11 028 2

3605 286 225 232 247 074 2

3606 294 273 261 276 046 2

3607 579 579 544 567 113 2

3608 103 1201 876 1036 326 2

3610 173 196 204 191 038 2

3611 546 618 565 576 75 2

3612 3 28 28 29 06 2

3613 28 31 32 31 06 2

3614 135 111 135 127 022 2

3615 4187 3678 4111 4 08 2

3616 2 2 2 2 01 2

3617 33 27 299 035 2

3618 31 31 31 31 05 2

3619 326 324 312 326 064 2

3620 277 28 272 276 069 2

3621 26 25 23 25 04 2

3622 346 336 333 34 052 2

3623 26 28 29 28 02 2

3624

3626 288 28 307 292 117 2

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

5301 29 3 29 29 06 2

5302 297 3 296 298 06 2

5303 2705 2444 2608 2586 22 78

5304 245 254 26 253 85 2

5305 23 23 26 24 15

5306 42 43 4 42 21 2

5307 221 216 241 221

5308 232169 246654 240722 229848

5309 19 22 19 2 0

5310 3 315 325 313 016

5311 262 277 304 281 15 2

5312 27 289 337 299 075 2

5313 321 284 378 328

5314 423 404 385 404 34 083

5315 42 506 416 444 09 2

5316 265 267 277 273 018 2

5317 185 197 195 192 006 2

5318 276 281 279 04 2

5319 396 389 403 396 079 2

5320 623 618 635 625 14

5321 31

5322 24 33 49 33 35 2

5323 38 45 35 45 27 2

50

Figure 8 Results of replicate determinations (indicated by triangles) of benzo[b]fluoranthene (BBF) in the food supplement test material

Horizontal blue lines represent the arithmetic mean value of replicate measurements and blue bars the reported expanded measurement uncertainty (k=2) The assigned value is

plotted as dotted line Red solid lines mark the range of satisfactory performance (|z|le2) Mean values are given for results outside the displayed data range

ProLab 2011

Laboratory

36

0436

1436

0236

1053

1736

1653

0953

0753

0853

0536

2136

0553

0436

0353

0353

1636

0636

2036

2353

1853

1136

1236

2653

0153

0253

1236

1736

1336

0036

1853

2153

1036

1953

1336

2253

2253

2353

1936

1553

1453

0653

1536

0753

2036

0836

11

an

aly

te c

on

ten

t (micro

gk

g)

75

7

65

6

55

5

45

4

35

3

25

2

15

1

05

0

103

6

576

3

Sample St Johns wort extract replicatesMeasurand Benzo[b]fluorantheneNo of laboratories 46

Assigned value 292 microgkg (Reference value)Target sd 060 microgkgTolerance limits 172 - 412 microgkg (|Z score| lt 200)

Assig

ned va

lue

Limit of tolerance

Limit of tolerance

51

Table 14 Analysis results reported by the participants for the content of chrysene (CHR) in the food supplements test material Values are presented as reported

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

3600 5 49 5 5 07 2

3602 32 304 29 304 036

3603 462 469 447 459 048 2

3604 438 428 42 429 119 2

3605 365 41 381 386 085 2

3606 494 411 45 451 124 2

3607 897 774 787 819 18 2

3608 986 887 1168 1014 284 2

3610 418 392 462 424 085 2

3611 833 893 87 865 61 2

3612 6 64 58 61 12 2

3613 41 39 4 4 08 2

3614 272 254 288 271 054 2

3615 605 6028 5964 601 12 2

3616 47 45 49 47 05 2

3617 445 431 389 422 065 2

3618 45 45 45 45 06 2

3619 738 747 72 738 118 2

3620 463 472 446 46 092 2

3621 53 49 51 51 06 2

3622 478 513 505 488 08 2

3623 73 73 72 72 05 2

3624

3626 63 622 634 628 251 2

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

5301 49 49 49 49 1 2

5302 687 683 666 679 136 2

5303 6777 6181 6382 6441 24 70

5304 592 581 6 591 85 2

5305 6 58 64 61 20

5306 49 5 51 49 25 2

5307 524 5 574 524

5308 211488 216782 221146 213243

5309

5310 372 393 394 386 019

5311 43 459 422 437 15 2

5312 934 943 981 953 191 2

5313 793 73 99 838

5314 636 561 565 588 18 0818

5315 547 583 499 542 11 2

5316 292 286 285 282 06 2

5317 52 598 517 545 046 2

5318 368 345 357 05 2

5319 449 471 474 465 116 2

5320 76 924 818 834 99

5321 33

5322 7 7 76 7 35 2

5323 87 73 77 73 37 2

52

Figure 9 Results of replicate determinations (indicated by triangles) of chrysene (CHR) in the food supplement test material

Horizontal blue lines represent the arithmetic mean value of replicate measurements and blue bars the reported expanded measurement uncertainty (k=2) The assigned value is

plotted as dotted line Red solid lines mark the range of satisfactory performance (|z|le2) Mean values are given for results outside the displayed data range

ProLab 2011

Laboratory

53

0836

1453

1636

0253

2153

1836

0553

1036

1336

1736

1036

0453

1136

1836

0636

0336

2053

1936

1653

0136

0036

2253

0636

2153

0753

1553

1753

1453

0436

1536

1253

0536

2653

0353

0253

2236

2336

1953

2336

0753

2053

1353

1236

0836

11

an

aly

te c

on

ten

t (micro

gk

g)

13

12

11

10

9

8

7

6

5

4

3

2

1

0

865

3

Sample St Johns wort extract replicatesMeasurand ChryseneNo of laboratories 45

Assigned value 407 microgkg (Reference value)Target sd 083 microgkgTolerance limits 241 - 573 microgkg (|Z score| lt 200)

Assig

ned va

lue

Limit of tolerance

Limit of tolerance

53

Table 15 Analysis results reported by the participants for the sum of the four marker PAHs in the food supplements test material Values are presented as reported

Laboratory code

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg

3600 128 24 2

3602 79 126

3603 1127 096 2

3604 893 258 2

3605 1091 305 2

3606 1184 238 2

3607 2098 42 2

3608 3336 325 2

3610 1138 17 2

3611 2218 259 2

3612 143 29 2

3613 122 2 2

3614 663 129 2

3615 152 304 2

3616 102 03 2

3617 1252 169 2

3618 128 18 2

3619 157 305 2

3620 1204 12 2

3621 151 19 2

3622 1361 115 2

3623 154 09 2

3624

3626 1409 56 2

Laboratory code

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg

5301 126

5302 37 074 2

5303 13438 92

5304 1251 85 2

5305 125 20

5306 129 65 2

5307 1363

5308 7245598

5309

5310 1127 056

5311 1367 15 2

5312 2042 408 2

5313 1726

5314 1558

5315 177 35 2

5316 96 073 2

5317 1229 066 2

5318 82 05 2

5319 1225 267 2

5320 2309 41

5321

5322 207 35 2

5323 171 103 2

54

ANNEX 9 Results reported for the standard solutions with undisclosed analyte content Table 16 Analysis results reported by the participants for the four marker PAHs in the standard solution in toluene with undisclosed analyte

content Values are presented as reported U measurement uncertainty k coverage factor

benz[a]anthracene benzo[a]pyrene benzo[b]fluoranthene chrysene Laboratory

code Unit Result U k Result U k Result U k Result U k 3600 microgl 38 67 2 25 45 2 211 68 2 275 38 2

3605 microgl 367 184 2 2482 124 2 2592 13 2 235 118 2

3606 microgkg 3763 176 2 2609 142 2 2721 118 2 2539 134 2

3607 microgkg 3706 815 2 2824 565 2 2591 518 2 2587 569 2

3612 microgl 38 08 2 237 11 2 244 18 2 257 02 2

3614 microgl 324 65 2 209 38 2 215 37 2 21 42 2

3616 microgkg 318 07 2 224 14 2 231 26 2 216 1 2

3618 microgkg 417 17 2 284 08 2 292 06 2 297 03 2

3619 microgkg 47 742 2 274 468 2 228 387 2 274 434 2

3620 microgl 495 74 2 294 29 2 321 8 2 321 64 2

3621 microgl 241 22 2 143 13 2 188 16 2 145 14 2

3623 microgkg 476 25 2 311 53 2 269 3 2 297 03 2

3626 microgl 359 38 2 235 23 2 238 27 2 24 18 2

5301 microgkg 426 29 299 289

5302 microgkg 436 87 2 296 59 2 296 59 2 289 58 2

5303 microgl 3721 27 102 22838 19 99 23756 22 99 25486 24 98

5304 microgkg 38 29 2 225 26 2 257 37 2 258 52 2

5305 microgkg 305 225 245 29

5306 microgkg 316 63 2 247 49 2 65 13 2 22 44 2

5307 microgkg 3658 2559 241 2414

5308 microgkg 16854 109324 246654 216782

5311 microgl 374 20 2 279 15 2 268 15 2 272 15 2

5313 microgl 3408 2165 2275 2373

5314 microgkg 395 5 2875 11 3614 34 2905 18

5320 microgl 3267 368 1986 571 28 683 2427 512

5322 microgl 282 35 2 225 35 2 241 35 2 21 35 2

55

Figure 10 Youden plots of the percent deviation of the reported values from the assigned values for the food supplement test sample and the

standard solution in toluene with undisclosed content

(The identity of the respective measurand is given above the figure Results exceeding a difference of more than 100 were omitted)

56

Figure 10 - continued

57

Table 17 Analysis results reported by the participants for the four marker PAHs in the standard solution in acetonitrile with undisclosed

analyte content

Values are presented as reported U measurement uncertainty k coverage factor

benz[a]anthracene benzo[a]pyrene benzo[b]fluoranthene chrysene

Laboratory

code Unit Result U k Result U k Result U k Result U k

3602 microgkg 3675 247 247 244

3603 microgkg 358 086 2 236 028 2 235 028 2 236 057 2

3604 microgl 475 29 2 19 12 2 194 13 2 258 12 2

3608 microgl 3623 078 2 2385 023 2 2343 18 2 2296 092 2

3610 microgkg 369 185 2 25 125 2 251 126 2 244 122 2

3611 microgkg 48 02 2 323 02 2 328 04 2 317 03 2

3613 microgl 332 31 2 261 3 2 249 25 2 239 24 2

3615 microgl 415 2635 2729 2689

3617 microgkg 37 09 2 244 06 2 284 07 2 239 06 2

3622 microgl 37 12 2 25 23 2 257 21 2 238 1 2

3624 microgkg 402 104 2 255 084 2 265 096 2 253 07 2

5306 microgkg 563 113 2 371 74 2 359 72 2 333 67 2

5308 microgkg 168633 102669 232169 211488

5309 microgl 402 268 27

5310 microgl 315 158 25 125 25 125 288 144

5312 microgl 428 043 2 282 028 2 272 027 2 275 028 2

5315 microgkg 387 08 2 256 05 2 251 05 2 253 05 2

5316 microgl 3839 342 2 2485 191 2 2559 168 2 239 51 2

5317 microgl 2466 043 2 2212 034 2 2472 016 2 2146 068 2

5319 microgl 414 41 2 279 28 2 268 27 2 26 31 2

5321 microgkg 476 316 326 328

5322 microgl 2547 35 2

5323 microgkg 405 203 2 239 119 2 255 153 2 201 11 2

58

Figure 11 Youden plots of the percent deviation of the reported values from the assigned values for the food supplement test sample and the

standard solution in acetonitrile with undisclosed content

(The identity of the respective measurand is given above the figure Results exceeding a difference of more than 100 were omitted)

59

Figure 11 - continued

60

ANNEX 10 Table 18 Method performance parameters reported by the participants

Laboratory code

Analyte LOD LOQ Recovery Analyte LOD LOQ Recovery

microgkg microgkg microgkg microgkg 3600 BaA 03 06 978 BaP 03 07 9483602 BaA 007 021 926 BaP 005 015 993603 BaA 0011 05 987 BaP 0005 051 9553604 BaA 02 04 86 BaP 02 04 883605 BaA 0025 005 118 BaP 0025 005 1083606 BaA 001 003 65 BaP 001 003 703607 BaA 021 071 74 BaP 027 089 943608 BaA 016 048 8213 BaP 015 046 83683610 BaA 02 05 85 BaP 005 02 853611 BaA BaP 3612 BaA 1 117 BaP 1 1203613 BaA 01 03 90 BaP 01 03 903614 BaA 02 05 96 BaP 02 05 933615 BaA 005 015 110 BaP 005 015 1053616 BaA 01 05 108 BaP 01 05 753617 BaA 018 037 405 BaP 009 018 3873618 BaA 01 03 63 BaP 01 03 593619 BaA 005 005 59 BaP 033 033 543620 BaA 0009 0018 64 BaP 0005 001 553621 BaA 007 02 84 BaP 007 02 923622 BaA 01 03 1101 BaP 01 03 9853623 BaA 02 08 105 BaP 02 08 1013624 BaA BaP 01 033 8513625 BaA BaP 3626 BaA BaP 5301 BaA 003 01 102 BaP 003 01 1005302 BaA 01 03 78 BaP 01 03 865303 BaA 003 01 62 BaP 003 01 805304 BaA 1 1 70 BaP 1 1 955305 BaA 02 05 85 BaP 02 05 855306 BaA 03 1 699 BaP 03 1 905307 BaA 6537 BaP 81165308 BaA 0088 0265 99 BaP 00262 00785 1005309 BaA 01 08 92 BaP 01 08 925310 BaA 01 03 BaP 01 03 905311 BaA 002 005 80 BaP 002 004 915312 BaA 03 09 105 BaP 03 09 1055313 BaA 04 08 BaP 04 08 5314 BaA 001 002 96 BaP 001 002 9485315 BaA 01 03 50 - 100 BaP 01 03 50 - 100 5316 BaA 002 011 827 BaP 004 019 10445317 BaA 05 1 76 BaP 05 1 755318 BaA BaP 5319 BaA 009 029 951 BaP 011 041 995320 BaA 12 18 946 BaP 06 1 8345321 BaA 03 05 BaP 05 1 5322 BaA 1 2 100 BaP 1 2 1005323 BaA 26 BaP 26

61

ANNEX 10 Table 18 - continued

Laboratory code Analyte LOD LOQ Recovery Analyte LOD LOQ Recovery

microgkg microgkg microgkg microgkg 3600 BbF 03 05 96 CHR 02 03 10153602 BbF 015 045 947 CHR 003 009 9193603 BbF 0014 05 1012 CHR 0004 05 10033604 BbF 02 04 85 CHR 02 04 873605 BbF 005 01 94 CHR 0025 005 863606 BbF 001 003 70 CHR 001 003 653607 BbF 018 06 100 CHR 014 028 943608 BbF 015 046 7979 CHR 015 045 71253610 BbF 005 02 85 CHR 02 05 853611 BbF CHR 3612 BbF 1 118 CHR 1 1173613 BbF 01 03 90 CHR 01 03 903614 BbF 01 04 96 CHR 02 05 953615 BbF 008 025 109 CHR 003 01 1083616 BbF 01 05 78 CHR 01 05 723617 BbF 035 07 38 CHR 012 024 4163618 BbF 01 03 62 CHR 01 03 623619 BbF 016 016 63 CHR 01 01 593620 BbF 0003 0006 55 CHR 001 002 653621 BbF 007 02 103 CHR 007 02 893622 BbF 02 06 963 CHR 03 09 1043623 BbF 02 08 106 CHR 02 08 1193624 BbF CHR 3625 BbF CHR 3626 BbF CHR 5301 BbF 003 01 96 CHR 003 01 1145302 BbF 01 03 84 CHR 01 03 835303 BbF 003 01 78 CHR 003 01 705304 BbF 1 1 86 CHR 1 1 705305 BbF 02 05 90 CHR 02 05 955306 BbF 03 1 1435 CHR 03 1 1185307 BbF 8116 CHR 65375308 BbF 0079 0238 99 CHR 009 026 1005309 BbF 01 08 92 CHR 5310 BbF 07 2 CHR 01 03 5311 BbF 002 004 95 CHR 002 004 895312 BbF 03 09 105 CHR 03 09 1055313 BbF 04 08 CHR 04 08 5314 BbF 001 002 83 CHR 001 002 8185315 BbF 02 05 50 -100 CHR 02 05 50 - 100 5316 BbF 005 024 814 CHR 001 007 9595317 BbF 05 1 73 CHR 05 1 785318 BbF CHR 5319 BbF 011 038 946 CHR 013 043 9675320 BbF 07 09 1194 CHR 23 32 10455321 BbF 05 1 CHR 05 1 5322 BbF 1 2 100 CHR 1 2 1005323 BbF 26 CHR 26

62

ANNEX 11 Table 19 Details of analysis method reported by the participants Data are presented as reported

Laboratory code

Analysis technique

Instrument calibration

Internal standards applied

Sample clean up

Details of sample

clean up

Extraction technique

Details of sample

extraction

3600 6) GC-MS

2) Internal standardisation with labelled substances

Deuterated PAH mix 9 (Ehrenstorfer)

1) Saponification 2) LiquidLiquid partitioning

D) Other Liquidliquid partitioning

3602 2) HPLC-UV-FLD

1) External calibration

3) Gel permeation chromatography

A) Pressurised liquid extraction (PLE)

3603 1) HPLC-FLD

1) External calibration

3) Gel permeation chromatography

D) Other 1 minute with dichloromethane in vortex

3604 2) HPLC-UV-FLD

2) Internal standardisation with labelled substances

DiP D14

5) Donor acceptor complex chromatography

D) Other LL

3605 1) HPLC-FLD

1) External calibration

3) Gel permeation chromatography

C) Soxhlet extraction

3606 7) GC-MSMS

2) Internal standardisation with labelled substances

BaP 13C4 CHR 13C6 BaA 13C6 BbF 13C6

4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

3607 6) GC-MS

2) Internal standardisation with labelled substances

BaA-d12 CHR-d12 BbF-d12 BkF-d12 BaP-d12 ICP-d12 BgP-d12 DiP-d14

1) Saponification

D) Other solvent partitioning

3608 1) HPLC-FLD

1) External calibration

6) Other Liquid extraction

D) Other

Extraction in a rotary agitator and centrifugation

3610 2) HPLC-UV-FLD

1) External calibration

4) Solid phase extraction

B) Sonication

3611

3612 6) GC-MS

2) Internal standardisation with labelled substances

chrysene-d12 dibenzo(ah)anthracene-d14 dibenzo(ai)pyrene-d14

4) Solid phase extraction

B) Sonication

3613 5) LC-MSMS

3) Internal standardisation with unlabelled substances

I use Chrysene-D12 3) Gel permeation chromatography

D) Other extraction with hexan

63

Table 19 - continued

Laboratory code

Analysis technique

Instrument calibration

Internal standards applied

Sample clean up Details of sample

clean up

Extraction technique

Details of sample

extraction

3614 6) GC-MS 1) External calibration

3) Gel permeation chromatography

B) Sonication

3615 1) HPLC-FLD

1) External calibration

6) Other PLE GPC

A) Pressurised liquid extraction (PLE)

3616 6) GC-MS

2) Internal standardisation with labelled substances

Benzo(a)pyrene-13C4 Benzo(a)anthracene-13C6 Benzo(b)fluoranthene-13C6 Chrysene-13C6

2) LiquidLiquid partitioning 4) Solid phase extraction

B) Sonication

3617 1) HPLC-FLD

1) External calibration

4) Solid phase extraction

C) Soxhlet extraction

3618 6) GC-MS

2) Internal standardisation with labelled substances

deuterated standards

1) Saponification 4) Solid phase extraction

D) Other Handshaken with cyclohexane

3619 6) GC-MS

2) Internal standardisation with labelled substances

C13 Labelled US EPA 16

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

D) Other Homegenisation amp saponiofication

3620 9) GC-HRMS

2) Internal standardisation with labelled substances

mix of deuterated PAHs (BAA-D12 CHR-D12 BAP-D12 BBF-D12)

3) Gel permeation chromatography 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

3621 6) GC-MS

2) Internal standardisation with labelled substances

Chrysene D12 Benzo(a)pyrene D12 Benzo(b)fluoranthene D12 Benzo(a)anthracene

2) LiquidLiquid partitioning 4) Solid phase extraction

B) Sonication

3622 1) HPLC-FLD

4) Standard addition method

4) Solid phase extraction

D) Other

Extraction with cyclohexane by heating under reflux

3623 7) GC-MSMS

2) Internal standardisation with labelled substances

C13 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

3624 2) HPLC-UV-FLD

1) External calibration

2) LiquidLiquid partitioning

D) Other hot solvent extraction

3626

64

Table 19 - continued

Laboratory code

Analysis technique

Instrument calibration

Internal standards

applied

Sample clean up

Details of sample

clean up

Extraction technique

Details of sample

extraction

5301 7) GC-MSMS

2) Internal standardisation with labelled substances

EACH SUBSTANCE C13

4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5302 7) GC-MSMS

2) Internal standardisation with labelled substances

Isotope labelled PAHs 13C for each analyte

4) Solid phase extraction

C) Soxhlet extraction

5303 7) GC-MSMS

3) Internal standardisation with unlabelled substances

HAP C13 4) Solid phase extraction

D) Other automate ASE200 DIONEX

5304 7) GC-MSMS

2) Internal standardisation with labelled substances

with C13 for each HAP

2) LiquidLiquid partitioning 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5305 7) GC-MSMS

2) Internal standardisation with labelled substances

C13 isotopiques internal standards

4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5306 7) GC-MSMS

2) Internal standardisation with labelled substances

C13 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5307 6) GC-MS

2) Internal standardisation with labelled substances

1) Saponification 2) LiquidLiquid partitioning

D) Other solvent extraction

5308 1) HPLC-FLD 1) External calibration

4) Solid phase extraction

B) Sonication

5309 1) HPLC-FLD 1) External calibration

4) Solid phase extraction

D) Other only SPE

5310 1) HPLC-FLD 1) External calibration

1) Saponification 2) LiquidLiquid partitioning 3) Gel permeation chromatography

C) Soxhlet extraction

5311 6) GC-MS

2) Internal standardisation with labelled substances

13 C Chrysene 13 C Benz(a)pyrene

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

B) Sonication

5312 1) HPLC-FLD

3) Internal standardisation with unlabelled substances

Benzo(b)chrysen

2) LiquidLiquid partitioning 4) Solid phase extraction

Quechers D) Other Quechers

65

Table 19 - continued

Laboratory code

Analysis technique

Instrument calibration

Internal standards applied

Sample clean up Details of sample

clean up

Extraction technique

Details of sample extraction

5313 6) GC-MS

2) Internal standardisation with labelled substances

Benz(a)anthracene D12 Benzo(b)fluranthene D12 Benzo(a)pyrene D12 Chrysene D12

1) Saponification 2) LiquidLiquid partitioning 3) Gel permeation chromatography

A) Pressurised liquid extraction (PLE)

5314 6) GC-MS

2) Internal standardisation with labelled substances

Benzo(a)pyren d12 2) LiquidLiquid partitioning

D) Other Extraction with

5315 1) HPLC-FLD

3) Internal standardisation with unlabelled substances

Benz(b)chrysen

1) Saponification 2) LiquidLiquid partitioning

D) Other shaker

5316 1) HPLC-FLD

1) External calibration

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

D) Other

LiquidLiquid extraction with cyclohexane and dimethylformamide

5317 7) GC-MSMS

1) External calibration

3) Gel permeation chromatography 6) Other

silica gel D) Other VDLUFA online method

5318

5319 1) HPLC-FLD

3) Internal standardisation with unlabelled substances

Benzo(b)chrysene 3) Gel permeation chromatography

A) Pressurised liquid extraction (PLE)

5320 6) GC-MS

3) Internal standardisation with unlabelled substances

22-binaphthyl

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

D) Other 3 hours refluxing

5321 1) HPLC-FLD

2) Internal standardisation with labelled substances

6-meacutethylchrysegravene

1) Saponification 2) LiquidLiquid partitioning

B) Sonication

5322 6) GC-MS

2) Internal standardisation with labelled substances

naftalene-d8 acenaphtalene-d10 phenanthrene-d10 chrysene-d12 perylene-d12 benzo(e)anthracene-d12

1) Saponification 2) LiquidLiquid partitioning 6) Other

clean-up with silica packed column

C) Soxhlet extraction

5323 1) HPLC-FLD

1) External calibration

4) Solid phase extraction 6) Other

Accelerated Solvent Extraction

A) Pressurised liquid extraction (PLE)

66

European Commission EUR 25245 EN ndash Joint Research Centre ndash Institute for Reference Materials and Measurements Title Report on the 8th inter-laboratory comparison organised by the European Union Reference Laboratory for Polycyclic Aromatic Hydrocarbons - Four marker PAHs in a dry extract of St Johns wort Author(s) Radoslav Lizak Szilard Szilagyi Philippe Verlinde and Thomas WENZL Luxembourg Publications Office of the European Union 2012 ndash 66 pp ndash 201 x 297 cm EUR ndash Scientific and Technical Research series ndash ISSN 1831-9424 ISBN 978-92-79-23337-1 doi 10278759387 Abstract The proficiency test here reported concerned the determination of the contents of four marker polycyclic aromatic hydrocarbons (PAHs) and of their sum in an food supplements test sample The set of marker PAHs consists of benz[a]anthacene benzo[a]pyrene benzo[b]fluoranthene and chrysene Participants to these PT were National Reference Laboratories for PAHs (NRLs-PAHs) and EU official food control laboratories The number of participants was in total 48

The PT was organised according ISO Standard 170432010

The test material used was a commercial dry extract of St Johns wort Participants also received a solution of the same PAHs either in acetonitrile or in toluene for checking their instrument calibration

The results from participants were rated with z-scores and zeta-scores About 68 of the reported results were attributed with z-scores with an absolute value of below two which is the threshold for satisfactory performance

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interests whether private or national

L

A-N

A-252

45-EN

-N

• 1 Summary
• 2 Introduction
• 3 Scope
• 4 Participating Laboratories
• 5 Time frame
• 6 Confidentiality
• 7 Test materials
• • 71 1Preparation of test materials
  • 72 Assigned values and standard deviation for proficiency assessment
  • 73 Homogeneity and stability
  • 74 Sample dispatch
  • • 8 Design of the proficiency test
    • 9 Evaluation of the results
    • • 91 2General
      • 92 21BEvaluation criteria
      • 93 Performance of participants
      • 94 Evaluation of the influence of analysis method
      • 95 Evaluation of the influence of calibration
      • 96 26Evaluation of compliance with legislation
      • • 10 Follow-up actions for underperforming laboratories
        • 11 1Conclusions
        • 12 1Acknowledgements
        • 13 References

16

Figure 1 Graphical presentation of z-scores corresponding to the values for proficiency assessment

reported by the NRLs for the contents of BAA BAP BBF and CHR in the St Johns wort extract test

material

Blue triangles indicate satisfactory performance yellow triangles indicate questionable performance red triangles indicate non-satisfactory performance z-score values are presented above the triangles for the latter two performance categories

ProLab 2011

Z score-3 0 3 -3 0 3 -3 0 3 -3 0 3 -3 0 3

BAA BAP BBF CHR PAH4

La

bo

rato

ry

36003602

3603

36043605

36063607

36083610

36113612

36133614

36153616

36173618

36193620

36213622

36233624

3626

2258

2343

2452

2669

2147

2788

2869

6710

70839

3011

11271

80525

8425

4561

12338

90680

4976

7331

99553

3998

3780

7500

17420

168413

3269

3029

-2348 -2736

-2023 -2981

-2155-3018

-3998

Figure 2 Graphical presentation of z-scores corresponding to the values for proficiency assessment

reported by the OCLs for the contents of BAA BAP BBF and CHR in the St Johns wort extract test

material

Blue triangles indicate satisfactory performance yellow triangles indicate questionable performance red triangles indicate non-satisfactory performance z-score values are presented above the triangles for the latter two performance categories

ProLab 2011

Z score-3 0 3 -3 0 3 -3 0 3 -3 0 3 -3 0 3

BAA BAP BBF CHR PAH4

La

bo

rato

ry

5301

5302

5303

5304

5305

5306

5307

5308

5309

5310

5311

5312

5313

5314

5315

5316

5317

5318

5319

5320

5321

5322

5323

2597

2629

2017

2293

2873

2123

2521

2620

2186

2222

2452

2864

3952

3984

3806

9226

4171 5522

3285

6594

5205

5157

3539

3901

7051

4519

3173

4872

9191

7276

4391

-2435

-2154

-2065

-2340

-2740-3726

-6346

-3505

17

Table 6 Compilation of z-scores calculated from the ldquoresults for proficiency assessment

reported by the NRLs for food supplement test material z-scores outside the

satisfactory range (|z| gt 2) are indicated by red font Values are reproduced as reported

by the laboratories Empty cells denote analytes for which results were not reported

Measurand benz[a]anthracene benzo[a]pyrene benzo[b]fluoranthene chrysene Sum PAH 4

Assigned value

microgkg 298 165 292 407 1162

Standard deviation for proficiency assessment

microgkg 062 036 060 083 125

Laboratory code

Result z-score Result z-score Result z-score Result z-score Result z-score

microgkg microgkg microgkg microgkg microgkg

3600 3 00 17 01 31 03 5 11 128 09

3602 19 -18 124 -11 17 -20 304 -12 79 -30

3603 251 -08 161 -01 256 -06 459 06 1127 -03

3604 213 -14 143 -06 11 -30 429 03 893 -22

3605 291 -01 167 01 247 -07 386 -03 1091 -06

3606 289 -01 167 01 276 -03 451 05 1184 02

3607 438 23 274 30 567 46 819 50 2098 75

3608 714 67 573 113 1036 123 1014 73 3336 174

3610 196 -17 206 11 191 -17 424 02 1138 -02

3611 469 708 308 805 576 907 865 996 2218 1684

3612 31 02 22 15 29 00 61 25 143 21

3613 33 05 18 04 31 03 4 -01 122 05

3614 185 -18 08 -23 127 -27 271 -16 663 -40

3615 376 13 145 -06 4 18 601 23 152 29

3616 2 -16 15 -04 2 -15 47 08 102 -11

3617 362 10 169 01 299 01 422 02 1252 07

3618 32 04 2 10 31 03 45 05 128 09

3619 299 00 207 12 326 06 738 40 157 33

3620 302 01 166 00 276 -03 46 06 1204 03

3621 28 -03 47 84 25 -07 51 12 151 28

3622 322 04 213 13 34 08 488 10 1361 16

3623 33 05 21 12 28 -02 72 38 154 30

3624 18 04

3626 314 03 174 02 292 00 628 27 1409 20

18

Table 7 Compilation of z-scores calculated from the ldquoresults for proficiency assessment

reported by the OCLs for food supplement test material z-scores outside the

satisfactory range (|z| gt 2) are indicated by red font Values are reproduced as reported

by the laboratories Empty cells denote analytes for which results were not reported

Measurand benz[a]anthracene benzo[a]pyrene benzo[b]fluoranthene chrysene Sum PAH 4

Assigned value

microgkg 298 165 292 407 1162

Standard deviation for proficiency assessment

microgkg 062 036 060 083 125

Laboratory code

Result z-score Result z-

score Result z-score Result

z-score

Result z-score

microgkg microgkg microgkg microgkg microgkg

5301 31 02 18 04 29 00 49 10 126 08

5302 316 03 188 06 298 01 679 33 37 -63

5303 2876 -02 1535 -03 2586 -06 6441 29 13438 15

5304 261 -06 146 -05 253 -06 591 22 1251 07

5305 24 -09 16 -01 24 -09 61 25 125 07

5306 22 -13 16 -01 42 21 49 10 129 10

5307 461 27 157 -02 221 -12 524 14 1363 16

5308 16446 -22 107009 -16 229848 -10 213243 -23 72456 -35

5309 17 -21 11 -15 2 -15

5310 305 01 123 -12 313 03 386 -03 1127 -03

5311 459 26 2 10 281 -02 437 04 1367 16

5312 543 40 248 23 299 01 953 66 2042 71

5313 353 09 207 12 328 06 838 52 1726 45

5314 394 16 172 02 404 19 588 22 1558 32

5315 545 40 238 20 444 25 542 16 177 49

5316 235 -10 17 01 273 -03 282 -15 96 -16

5317 222 -12 269 29 192 -17 545 17 1229 05

5318 067 -37 117 -13 279 -02 357 -06 82 -27

5319 147 -24 216 14 396 17 465 07 1225 05

5320 534 38 316 42 625 55 834 52 2309 92

5321 12 -12

5322 87 92 17 01 33 06 7 35 207 73

5323 33 05 2 10 45 26 73 39 171 44

19

Table 8 and Table 9 present the respective zeta-scores As for the z-scores data outside the

satisfactory performance range are given in red bold font Zeta scores were only calculated for

laboratories that provided measurement uncertainty data as well as a numerical value for the coverage

factor Zeta scores were not calculated when the reported uncertainty value exceeded the content value

which is probably linked to mixing of reporting units (microgkg and ) However the participants are

reminded that the measurement uncertainty shall be expressed in the same unit as the content value

The assessment of the performance of the participants based on the reported measurement uncertainty

gave a less favourable picture compared to z-scores Only 60 of the zeta-scores are within the

satisfactory performance range The distribution of satisfactory performance ratings between NRLs

and OCLs is not balanced It was higher for the NRLs

It has to be noted that the magnitude of the zeta-scores were for some participants much higher than

the z-scores attributed to the same results Consequently the laboratories perform according to fitness-

for-purpose criterion specified in legislation which forms the basis for the z-scores but seem to have

difficulties in deducing realistic measurement uncertainty values Eg the relative standard

measurement uncertainties reported by the NRLs ranged from 25 to 20 with a median value of

about 10 Only two OCLs reported measurement uncertainties that exceeded the maximum tolerable

uncertainty (Uf) The respective data is highlighted in yellow in Table 9

However the EU-RL PAHs will continue to pay special attention to measurement uncertainty in future

ILCs as it has major implications on the assessment of compliance of food with European legislation

20

Table 8 Compilation of zeta-scores calculated from the ldquoresults for proficiency assessment

reported by the NRLs for test material St Johns wort the reported measurement

uncertainty and the uncertainty of the analyte content of the test material zeta-scores

outside the satisfactory range (|zeta| gt 2) are indicated by red bold font Empty cells

denote analytes for which either results or measurement uncertainties were not

reported

21

Table 9 Compilation of zeta-scores calculated from the ldquoresults for proficiency assessment

reported by the OCLs for test material St Johns wort the reported measurement

uncertainty and the uncertainty of the analyte content of the test material zeta-scores

outside the satisfactory range (|zeta| gt 2) are indicated by red bold font yellow

highlighted cells ugtUf Empty cells denote analytes for which results were not

reported

22

94 Evaluation of the influence of analysis method

The information on the applied analysis method (ANNEX 11) was used to group the performance

indicators attributed to the reported results according to the applied analysis technique Two groups of

data were formed They represent results obtained by high performance liquid chromatography-

fluorescence detection (HPLC-FD about 40 ) and by gas chromatography-mass spectrometry (GC-

MS about 60 ) respectively Kernel density plots were constructed based on the respective z-scores

Figure 3A to 3E show the different Kernel density plots

Figure 3 Kernel density plots of z-scores attributed to the results for proficiency assessment

which were reported for the food supplements test sample Results were classified

according to the applied analysis technique

A) benz[a]anthracene

B) benzo[a]pyrene

23

Figure 3 continued

C) benzo[b]fluoranthene

D) chrysene

E) sum of four marker PAHs

The kernel density plots for BAP and BBF are almost identical and centred at or close to a z-score

value of zero as can be seen in Figure 3B and 3C Therefore bias introduced by the type of analysis

method is unlikely for these two analytes The situation is different for BAA and CHR In case of

BAA the kernel density distribution of z-scores based on HPLC-FD is shifted to negative values

24

(Figure 3A) which indicates a tendency to underestimate the BAA content in this particular

samplematrix when applying HPLC-FD This might be reasoned by separationintegration problems

between BAA and CHR peaks A few participants (from the HPLC-FD subgroup) that got negative

z-scores for BAA overestimated the CHR content This caused the slight shift of the density trace for

HPLC-FD towards negative values Removing this data from the evaluation would result in a density

trace with the mode at zero

The opposite is the case for CHR (Figure 3D) The density distribution of the z-scores attributed to

HPLC-FD data is centred almost at zero However the distribution for data based on GC-MS is shifted

to positive values indicating strong positive bias This bias might be caused by a lack of selectivity of

the analysis method The test material contains triphenylene which is isobaric to chrysene

Consequently it cannot be distinguished from chrysene by means of mass spectrometry These two

compounds have to be separated chromatographically which is not easy They coelute on unpolar and

on most mid-polar capillary columns Columns that allow their separation were commercialised only

recently and might not be available in all laboratories Therefore laboratories which determine the

four EU marker PAHs by means of GC-MS shall pay attention to the chromatographic separation of

triphenylene and chrysene A lack of chromatographic resolution might lead to strong positive bias of

GC-MS methods and to false conclusions on the marketability of the tested sample

The chromatographic separation of chrysene and triphenylene is not a problem with HPLC which is

reflected in the position of the density plot for chrysene However attention has to be paid in HPLC-

FD analysis to a proper clean up of the sample extract in order to eliminate interferences that show

similar retention as BAA and CHR

95 Evaluation of the influence of calibration

The participants received together with the food supplement test sample two standard solutions in

depending of their choice either acetonitrile or toluene The analyte content of one solution was

disclosed to the participants whereas the content of the other solution was kept undisclosed The

content values of the latter are presented in Table 5 Analysis results for the solution with undisclosed

content had to be reported to the PT organiser The respective data are presented in Annex 9

Percent deviations of the reported results from the assigned values were calculated for both the food

supplement test sample and the standard solution with undisclosed analyte content Youden plots were

prepared in order to visualize the influence of instrument calibration on the results reported for the

food supplement sample Two plots are discussed in the following The whole set of Youden plots are

given in Annex 9

25

Figure 4 Youden plot of the percent deviations of values reported for benzo[a]pyrene from the

assigned values of the food supplement test material and the standard solution in

toluene with undisclosed content

Figure 4 reveals that a number of participants reported results with similar percent deviations from the

assigned values of both the results for the food supplement material and the standard solution in

toluene with undisclosed content This correlation indicates that over- respectively underestimation of

the analyte content of the food supplement material might be caused by biased instrument calibration

This finding underpins the importance of thorough calibration standard preparation However the data

was not in all cases conclusive as some laboratories performed well for the food supplement samples

but reported biased results for the standard solution with undisclosed content (eg Figure 4 participants

3620 and 5305) This fact cannot be explained yet

Figure 5 presents the respective Youden plot for chrysene in the food supplement test material and the

standard solution in toluene with undisclosed content From this graph it can be concluded that the

overestimation of the chrysene content of the food supplement material was not caused by instrument

calibration The results for the solvent solution of many laboratories which underperformed for the

26

food supplement sample agreed well with the assigned value This supports the conclusion that the

overestimation is caused by the determination of an interferent most probably triphenylene

Figure 5 Youden plot of the percent deviations of values reported for chrysene from the

assigned values of the food supplement test material and the standard solution in

toluene with undisclosed content

96 Evaluation of compliance with legislation

The method performance data reported for the four marker PAHs were evaluated for compliance with

the provisions given in Commission Regulation (EC) No 8362011 Table 18 in ANNEX 10 gives an

overview on the results of the evaluation Data were not reported in case of empty cells Non-

compliant data are highlighted in Table 18 It should be noted that Regulation (EU) No 8362011

specifies LOD and LOQ with two decimals This was not respected by all participants which

hampered the compliance assessment

27

In summary it can be stated that the majority of participants reported method performance

characteristics for the determination of the four marker PAHs in the food supplement test sample that

are compliant with legislation However some laboratories need to improve their analysis methods

The laboratories in question are urged to adapt to the provisions given in the new legislation

10 Follow-up actions for underperforming laboratories

All laboratories that got questionable or non-satisfactory performance ratings are urged to perform

root cause analysis and to implement corrective actions

As agreed during the EU-RL PAH workshop 2011 the EU-RL PAH will set up follow-up measures in

due time for all NRLs that received for at least one of the four PAHs (BAA BAP BBF and CHR) z-

scores gt 3 as required by Regulation (EC) 8822004 and by the Protocol for management of

underperformance in comparative testing andor lack of collaboration of National Reference

Laboratories (NRLs) with European Union reference laboratories (EU-RLs) activities

These laboratories shall perform as an immediate action root-cause-analysis and shall report to the

EU-RL PAH in writing the identified cause for their underperformance and corrective actions they are

going to take Additionally they shall participate to an independent (non-EU-RL) proficiency test on

the determination of PAHs in food and shall communicate the outcome of this exercise to the EU-RL

PAH

28

11 Conclusions

Fourty seven participants reported results back to the EU-RL The performance of the majority of

participants was good In total 153 out of 225 attributed z-scores were below an absolute value of two

which equals to almost 68 However this percentage is much below the percentages of satisfactory

performance gained in previous studies This might be the consequence of the higher level of difficulty

with respect to matrix composition In addition bias can be concluded from the pattern of performance

indicators of some laboratories

In general the determination of chrysene caused most difficulties to the participants

zeta-Scores were calculated besides z-scores They indicate the agreement of the reported result with

the assigned value with respect to the stated measurement uncertainty The outcome of this rating was

worse than for the z-scores which indicates that the measurement uncertainty estimates were not

realistic Therefore participants underperforming with regard to zeta scores are urged to adapt their

measurement uncertainty statements

The great majority of participants in this inter-laboratory comparison applied analytical methods which

complied with EU legislation with regard to performance characteristics

12 Acknowledgements

The organisers would like to thank Mrs Beatriz de la Calle and Mr Franz Ulberth (all from IRMM

Geel Belgium) for their accurate revision of this report Mr Hakan Emteborg (from IRMM) for

ampouling of the standard solutions and all NRLs and OCLs for their cooperation

29

13 References

1 EU COMMISSION REGULATION (EC) No 7762006 of 23 May 2006 amending Annex VII to Regulation

(EC) No 8822004 of the European Parliament and of the Council as regards Community reference

laboratories Official Journal of the European Union 2006 L 136 p 3-8

Available from

httpeur-lexeuropaeuLexUriServLexUriServdouri=OJL200613600030008ENPDF

2 EU Regulation (EC) No 8822004 of the European Parliament and of the Council of 29 April 2004 on

official controls performed to ensure the verification of compliance with feed and food law animal health

and animal welfare rules Official Journal of the European Communities 2004 L191 p 1-52 Available

from

httpeur-lexeuropaeuLexUriServLexUriServdouri=OJL200419100010052ENPDF

3 EU Opinion of the Scientific Committee on Food on the risks to human health of Polycyclic Aromatic

Hydrocarbons in food (2002) Available from

httpeuropaeuintcommfoodfsscscfout153_enpdf

4 International Agency for Research on Cancer IARC Monographs on the Evaluation of

Carcinogenic Risks to Humans Some Non-heterocyclic Polycyclic Aromatic Hydrocarbons and Some

Related Exposures Volume 92 (2010) Available from

httpmonographsiarcfrENGMonographsvol92mono92pdf

5 EU Commission Regulation (EC) No 3332007 of 28 March 2007 laying down the methods of sampling

and analysis for the official control of the levels of lead cadmium mercury inorganic tin 3-MCPD and

benzo(a)pyrene in foodstuffs Official Journal of the European Union 2007 L 88 p 29-38 Available

from

httpeur-lexeuropaeuLexUriServLexUriServdouri=OJL200708800290038ENPDF

6 EU Commission Regulation (EC) No 18812006 of 19 December 2006 setting maximum levels for certain

contaminants in foodstuffs Official Journal of the European Union 2006 L 364 p 5-24 Available from

httpeur-lexeuropaeuLexUriServLexUriServdouri=OJL200636400050024ENPDF

7 EFSA Scientific Opinion of the Panel on Contaminants in the Food Chain on a request from the European

Commission on Polycyclic Aromatic Hydrocarbons in Food The EFSA Journal 2008 724 p 1-114

Available from

httpwwwefsaeuropaeuEFSAefsa_locale-1178620753812_1211902034842htm

30

8 EU Commission Regulation (EU) No 8352011 amending Regulation (EC) No 18812006 as regards

maximum levels for polycyclic aromatic hydrocarbons in foodstuffs Official Journal of the European

Union 2011 L 215 p 4-8

httpeur-lexeuropaeuLexUriServLexUriServdouri=OJL201121500040008ENPDF

9 EU Commission Regulation (EU) No 8362011 amending Regulation (EC) No 3332007 laying down the methods of sampling and analysis for the official control of the levels of lead cadmium mercury inorganic tin 3-MCPD and benzo(a)pyrene in foodstuffs Official Journal of the European Union 2011 L 215 p 9-16 httpeur-lexeuropaeuLexUriServLexUriServdouri=OJL201121500090016ENPDF

10 ISOIEC 170432010 (2010) Conformity assessment -- General requirements for proficiency testing

providers Geneva Switzerland

11 Thompson M Ellison SLR and Wood R The International Harmonized Protocol for the Proficiency

Testing of Analytical Chemistry Laboratories Pure Appl Chem (2006) 78(1) p 145ndash196

Available from httpmediaiupacorgpublicationspac2006pdf7801x0145pdf

31

14 ANNEXES

ANNEX 1 ndash Announcement of the PT on the IRMM webpage

ANNEX 2 ndash Announcement via e-mail and invitation

ANNEX 3 ndash Announcement of sample dispatch

ANNEX 4 ndash Technical specifications of the calibration solutions

ANNEX 5 ndash Homogeneity and stability of the test material

ANNEX 6 ndash Acknowledgement of sample receipt

ANNEX 7 ndash Questionnaire on details of analysis method

ANNEX 8 ndash Results reported for the food supplement sample

ANNEX 9 ndash Results reported for the standard solutions with undisclosed analyte content

ANNEX 10 ndash Method performance characteristics reported by participants

ANNEX 11 ndash Details of analysis methods reported by participants

32

ANNEX 1 ndash Announcement of the PT on the IRMM webpage

33

ANNEX 2 - Announcement via e-mail and invitation

34

ANNEX 3 - Announcement sample dispatch

Dear MadameSir

The test samples for the proficiency test on the determination of four PAHs in a food supplement (dry extract of St Johns wort) were

dispatched today

Explanation on the design of the study as well as instructions for sample storage and for reporting of results are in the parcel You will

also find your participant key in the parcel which is required for reporting of results

Please check the parcel after arrival for completeness and report the receipt of the test samples as well as their status to us by applying

the sample receipt form which is in the parcel

You are kindly requested to inform us by email if you do not receive the parcel by end of this week

My colleague Donata Lerda will then take care of it

With best regards

Thomas Wenzl

35

ANNEX 4 - Specifications of standard solutions with disclosed concentration

36

ANNEX 4 - continued

37

ANNEX 5 - Homogeneity and stability of the test material

Homogeneity of the test material

Benz[a]anthracene - BAA

Benzo[a]pyrene - BAP

38

ANNEX 5 - continued

Benzo[b]fluoranthene - BBF

Chrysene - CHR

39

ANNEX 5 - continued

Stability of the test material

Table 10 Content of the four target analytes in test materials stored under different

temperatures

BaA BaP BbF CHR

microgkg microgkg microgkg microgkg

Content 31 16 31 69 Storage at room temperature

U (k=2) 05 02 05 17

Content 31 15 29 68 Storage at reference temperature

U (k=2) 04 03 04 10

The applied capillary column does not separate CHR from triphenylene

40

ANNEX 6 ndash Documents sent to participants

41

ANNEX 6 - continued

42

ANNEX 7 Questionnaire on details of analysis method

43

ANNEX 7 - continued

44

ANNEX 7 - continued

45

ANNEX 8 - Results reported by participants for the food supplements test sample Table 11 Analysis results reported by the participants for the content of benz[a]anthracene (BAA) in the food supplements test material Values are presented as reported

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

3600 31 29 29 3 05 2

3602 199 193 178 19 021

3603 253 253 246 251 022 2

3604 199 209 23 213 071 2

3605 301 3 271 291 076 2

3606 302 288 278 289 064 2

3607 456 416 443 438 096 2

3608 796 695 651 714 149 2

3610 182 192 214 196 039 2

3611 414 504 488 469 96 2

3612 3 34 28 31 06 2

3613 32 33 34 33 07 2

3614 182 182 191 185 037 2

3615 3884 3822 3565 376 0752 2

3616 2 2 2 2 01 2

3617 366 37 35 362 046 2

3618 32 32 32 32 05 2

3619 299 305 297 299 048 2

3620 304 294 302 302 045 2

3621 29 26 29 28 04 2

3622 343 325 318 322 052 2

3623 35 3 33 33 07 2

3624

3626 318 31 314 314 126 2

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

5301 31 3 32 31 06 2

5302 314 319 315 316 063 2

5303 3102 2716 2822 2876 27 62

5304 261 263 26 261 57 2

5305 23 22 26 24 20

5306 22 23 23 22 11 2

5307 488 461 317 461

5308 168633 168544 1562025 1644598

5309 16 19 16 17

5310 3 304 311 305 015

5311 477 452 448 459 15 2

5312 506 63 494 543 134 2

5313 353 301 404 353

5314 431 373 378 394 5 096

5315 596 531 51 545 11 2

5316 224 231 235 235 022 2

5317 216 23 221 222 007 2

5318 064 07 067 01 2

5319 111 184 146 147 029 2

5320 515 525 564 534 48

5321 21 5322 13 87 9 87 35 2

5323 38 33 35 33 16 2

46

Figure 6 Results of replicate determinations (indicated by triangles) of benz[a]anthracene (BAA) in the food supplement test material

Horizontal blue lines represent the arithmetic mean value of replicate measurements and blue bars the reported expanded measurement uncertainty (k=2) The assigned value is

plotted as dotted line Red solid lines mark the range of satisfactory performance (|z|le2) Mean values are given for results outside the displayed data range

ProLab 2011

Laboratory

53

1853

1953

0853

0936

1436

0236

1036

1653

2136

0453

1753

0653

1653

0536

0353

0436

2153

0336

0636

0536

0036

2036

1953

1036

1253

0136

2653

0236

1836

2336

2236

1353

1353

2336

1736

1553

1453

0736

0753

1153

2053

1253

1536

0853

2236

11

an

aly

te c

on

ten

t (micro

gk

g)

10

95

9

85

8

75

7

65

6

55

5

45

4

35

3

25

2

15

1

05

0

102

3 468

7

Sample St Johns wort extract replicatesMeasurand Benz[a]anthraceneNo of laboratories 46

Assigned value 298 microgkg (Reference value)Target sd 062 microgkgTolerance limits 174 - 422 microgkg (|Z score| lt 200)

Assig

ned va

lue

Limit of tolerance

Limit of tolerance

47

Table 12 Analysis results reported by the participants for the content of benzo[a]pyrene (BAP) in the food supplements test material Values are presented as reported

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

3600 16 17 17 17 03 2

3602 118 123 13 124 015

3603 165 162 157 161 015 2

3604 134 154 139 143 04 2

3605 164 166 172 167 057 2

3606 181 159 161 167 031 2

3607 279 266 279 274 055 2

3608 57 687 461 573 226 2

3610 226 216 183 206 042 2

3611 289 325 311 308 36 2

3612 24 22 2 22 05 2

3613 17 18 19 18 05 2

3614 08 08 08 08 016 2

3615 1545 1393 1425 145 029 2

3616 16 16 14 15 03 2

3617 177 171 158 169 023 2

3618 2 2 2 2 02 2

3619 207 207 206 207 075 2

3620 165 169 163 166 017 2

3621 44 47 49 47 05 2

3622 212 208 222 213 038 2

3623 2 21 22 21 03 2

3624 18 178 182 18 031 2

3626 172 173 178 174 07 2

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

5301 18 18 18 18 04 2

5302 188 19 187 188 038 2

5303 1691 1426 149 1535 19 80

5304 145 142 15 146 69 2

5305 15 17 18 16 15

5306 16 17 17 16 08 2

5307 164 157 153 157

5308 102669 109324 109035 107009

5309 1 1 12 11

5310 122 122 124 123 006

5311 199 184 215 2 15 2

5312 258 248 239 248 05 2

5313 199 178 245 207

5314 174 171 172 172 11 0948

5315 253 248 213 238 05 2

5316 177 171 173 17 016 2

5317 255 277 276 269 012 2

5318 114 12 117 02 2

5319 19 228 231 216 043 2

5320 303 3 344 316 78

5321 12 12 12 12

5322 13 17 24 17 35 2

5323 2 2 19 2 1 2

48

Figure 7 Results of replicate determinations (indicated by triangles) of benzo[a]pyrene (BAP) in the food supplement test material

Horizontal blue lines represent the arithmetic mean value of replicate measurements and blue bars the reported expanded measurement uncertainty (k=2) The assigned value is

plotted as dotted line Red solid lines mark the range of satisfactory performance (|z|le2) Mean values are given for results outside the displayed data range

ProLab 2011

Laboratory

36

1453

0953

0853

1853

2153

1036

0236

0436

1553

0436

1653

0353

0736

0336

2036

0053

0553

0636

0636

0536

1753

1453

1636

2636

1336

2453

0153

2253

0253

2353

1136

1836

1953

1336

1036

2336

2253

1936

1253

1553

1253

1736

0753

2036

2136

0836

11

an

aly

te c

on

ten

t (micro

gk

g)

55

5

45

4

35

3

25

2

15

1

05

0

573

308

3

Sample St Johns wort extract replicatesMeasurand Benzo[a]pyreneNo of laboratories 48

Assigned value 165 microgkg (Reference value)Target sd 036 microgkgTolerance limits 093 - 237 microgkg (|Z score| lt 200)

Assig

ned va

lue

Limit of tolerance

Limit of tolerance

49

Table 13 Analysis results reported by the participants for the content of benzo[b]fluoranthene (BBF) in the food supplements test material Values are presented as reported

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

3600 32 3 3 31 09 2

3602 173 175 16 17 017

3603 265 252 25 256 025 2

3604 116 108 105 11 028 2

3605 286 225 232 247 074 2

3606 294 273 261 276 046 2

3607 579 579 544 567 113 2

3608 103 1201 876 1036 326 2

3610 173 196 204 191 038 2

3611 546 618 565 576 75 2

3612 3 28 28 29 06 2

3613 28 31 32 31 06 2

3614 135 111 135 127 022 2

3615 4187 3678 4111 4 08 2

3616 2 2 2 2 01 2

3617 33 27 299 035 2

3618 31 31 31 31 05 2

3619 326 324 312 326 064 2

3620 277 28 272 276 069 2

3621 26 25 23 25 04 2

3622 346 336 333 34 052 2

3623 26 28 29 28 02 2

3624

3626 288 28 307 292 117 2

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

5301 29 3 29 29 06 2

5302 297 3 296 298 06 2

5303 2705 2444 2608 2586 22 78

5304 245 254 26 253 85 2

5305 23 23 26 24 15

5306 42 43 4 42 21 2

5307 221 216 241 221

5308 232169 246654 240722 229848

5309 19 22 19 2 0

5310 3 315 325 313 016

5311 262 277 304 281 15 2

5312 27 289 337 299 075 2

5313 321 284 378 328

5314 423 404 385 404 34 083

5315 42 506 416 444 09 2

5316 265 267 277 273 018 2

5317 185 197 195 192 006 2

5318 276 281 279 04 2

5319 396 389 403 396 079 2

5320 623 618 635 625 14

5321 31

5322 24 33 49 33 35 2

5323 38 45 35 45 27 2

50

Figure 8 Results of replicate determinations (indicated by triangles) of benzo[b]fluoranthene (BBF) in the food supplement test material

Horizontal blue lines represent the arithmetic mean value of replicate measurements and blue bars the reported expanded measurement uncertainty (k=2) The assigned value is

plotted as dotted line Red solid lines mark the range of satisfactory performance (|z|le2) Mean values are given for results outside the displayed data range

ProLab 2011

Laboratory

36

0436

1436

0236

1053

1736

1653

0953

0753

0853

0536

2136

0553

0436

0353

0353

1636

0636

2036

2353

1853

1136

1236

2653

0153

0253

1236

1736

1336

0036

1853

2153

1036

1953

1336

2253

2253

2353

1936

1553

1453

0653

1536

0753

2036

0836

11

an

aly

te c

on

ten

t (micro

gk

g)

75

7

65

6

55

5

45

4

35

3

25

2

15

1

05

0

103

6

576

3

Sample St Johns wort extract replicatesMeasurand Benzo[b]fluorantheneNo of laboratories 46

Assigned value 292 microgkg (Reference value)Target sd 060 microgkgTolerance limits 172 - 412 microgkg (|Z score| lt 200)

Assig

ned va

lue

Limit of tolerance

Limit of tolerance

51

Table 14 Analysis results reported by the participants for the content of chrysene (CHR) in the food supplements test material Values are presented as reported

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

3600 5 49 5 5 07 2

3602 32 304 29 304 036

3603 462 469 447 459 048 2

3604 438 428 42 429 119 2

3605 365 41 381 386 085 2

3606 494 411 45 451 124 2

3607 897 774 787 819 18 2

3608 986 887 1168 1014 284 2

3610 418 392 462 424 085 2

3611 833 893 87 865 61 2

3612 6 64 58 61 12 2

3613 41 39 4 4 08 2

3614 272 254 288 271 054 2

3615 605 6028 5964 601 12 2

3616 47 45 49 47 05 2

3617 445 431 389 422 065 2

3618 45 45 45 45 06 2

3619 738 747 72 738 118 2

3620 463 472 446 46 092 2

3621 53 49 51 51 06 2

3622 478 513 505 488 08 2

3623 73 73 72 72 05 2

3624

3626 63 622 634 628 251 2

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

5301 49 49 49 49 1 2

5302 687 683 666 679 136 2

5303 6777 6181 6382 6441 24 70

5304 592 581 6 591 85 2

5305 6 58 64 61 20

5306 49 5 51 49 25 2

5307 524 5 574 524

5308 211488 216782 221146 213243

5309

5310 372 393 394 386 019

5311 43 459 422 437 15 2

5312 934 943 981 953 191 2

5313 793 73 99 838

5314 636 561 565 588 18 0818

5315 547 583 499 542 11 2

5316 292 286 285 282 06 2

5317 52 598 517 545 046 2

5318 368 345 357 05 2

5319 449 471 474 465 116 2

5320 76 924 818 834 99

5321 33

5322 7 7 76 7 35 2

5323 87 73 77 73 37 2

52

Figure 9 Results of replicate determinations (indicated by triangles) of chrysene (CHR) in the food supplement test material

Horizontal blue lines represent the arithmetic mean value of replicate measurements and blue bars the reported expanded measurement uncertainty (k=2) The assigned value is

plotted as dotted line Red solid lines mark the range of satisfactory performance (|z|le2) Mean values are given for results outside the displayed data range

ProLab 2011

Laboratory

53

0836

1453

1636

0253

2153

1836

0553

1036

1336

1736

1036

0453

1136

1836

0636

0336

2053

1936

1653

0136

0036

2253

0636

2153

0753

1553

1753

1453

0436

1536

1253

0536

2653

0353

0253

2236

2336

1953

2336

0753

2053

1353

1236

0836

11

an

aly

te c

on

ten

t (micro

gk

g)

13

12

11

10

9

8

7

6

5

4

3

2

1

0

865

3

Sample St Johns wort extract replicatesMeasurand ChryseneNo of laboratories 45

Assigned value 407 microgkg (Reference value)Target sd 083 microgkgTolerance limits 241 - 573 microgkg (|Z score| lt 200)

Assig

ned va

lue

Limit of tolerance

Limit of tolerance

53

Table 15 Analysis results reported by the participants for the sum of the four marker PAHs in the food supplements test material Values are presented as reported

Laboratory code

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg

3600 128 24 2

3602 79 126

3603 1127 096 2

3604 893 258 2

3605 1091 305 2

3606 1184 238 2

3607 2098 42 2

3608 3336 325 2

3610 1138 17 2

3611 2218 259 2

3612 143 29 2

3613 122 2 2

3614 663 129 2

3615 152 304 2

3616 102 03 2

3617 1252 169 2

3618 128 18 2

3619 157 305 2

3620 1204 12 2

3621 151 19 2

3622 1361 115 2

3623 154 09 2

3624

3626 1409 56 2

Laboratory code

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg

5301 126

5302 37 074 2

5303 13438 92

5304 1251 85 2

5305 125 20

5306 129 65 2

5307 1363

5308 7245598

5309

5310 1127 056

5311 1367 15 2

5312 2042 408 2

5313 1726

5314 1558

5315 177 35 2

5316 96 073 2

5317 1229 066 2

5318 82 05 2

5319 1225 267 2

5320 2309 41

5321

5322 207 35 2

5323 171 103 2

54

ANNEX 9 Results reported for the standard solutions with undisclosed analyte content Table 16 Analysis results reported by the participants for the four marker PAHs in the standard solution in toluene with undisclosed analyte

content Values are presented as reported U measurement uncertainty k coverage factor

benz[a]anthracene benzo[a]pyrene benzo[b]fluoranthene chrysene Laboratory

code Unit Result U k Result U k Result U k Result U k 3600 microgl 38 67 2 25 45 2 211 68 2 275 38 2

3605 microgl 367 184 2 2482 124 2 2592 13 2 235 118 2

3606 microgkg 3763 176 2 2609 142 2 2721 118 2 2539 134 2

3607 microgkg 3706 815 2 2824 565 2 2591 518 2 2587 569 2

3612 microgl 38 08 2 237 11 2 244 18 2 257 02 2

3614 microgl 324 65 2 209 38 2 215 37 2 21 42 2

3616 microgkg 318 07 2 224 14 2 231 26 2 216 1 2

3618 microgkg 417 17 2 284 08 2 292 06 2 297 03 2

3619 microgkg 47 742 2 274 468 2 228 387 2 274 434 2

3620 microgl 495 74 2 294 29 2 321 8 2 321 64 2

3621 microgl 241 22 2 143 13 2 188 16 2 145 14 2

3623 microgkg 476 25 2 311 53 2 269 3 2 297 03 2

3626 microgl 359 38 2 235 23 2 238 27 2 24 18 2

5301 microgkg 426 29 299 289

5302 microgkg 436 87 2 296 59 2 296 59 2 289 58 2

5303 microgl 3721 27 102 22838 19 99 23756 22 99 25486 24 98

5304 microgkg 38 29 2 225 26 2 257 37 2 258 52 2

5305 microgkg 305 225 245 29

5306 microgkg 316 63 2 247 49 2 65 13 2 22 44 2

5307 microgkg 3658 2559 241 2414

5308 microgkg 16854 109324 246654 216782

5311 microgl 374 20 2 279 15 2 268 15 2 272 15 2

5313 microgl 3408 2165 2275 2373

5314 microgkg 395 5 2875 11 3614 34 2905 18

5320 microgl 3267 368 1986 571 28 683 2427 512

5322 microgl 282 35 2 225 35 2 241 35 2 21 35 2

55

Figure 10 Youden plots of the percent deviation of the reported values from the assigned values for the food supplement test sample and the

standard solution in toluene with undisclosed content

(The identity of the respective measurand is given above the figure Results exceeding a difference of more than 100 were omitted)

56

Figure 10 - continued

57

Table 17 Analysis results reported by the participants for the four marker PAHs in the standard solution in acetonitrile with undisclosed

analyte content

Values are presented as reported U measurement uncertainty k coverage factor

benz[a]anthracene benzo[a]pyrene benzo[b]fluoranthene chrysene

Laboratory

code Unit Result U k Result U k Result U k Result U k

3602 microgkg 3675 247 247 244

3603 microgkg 358 086 2 236 028 2 235 028 2 236 057 2

3604 microgl 475 29 2 19 12 2 194 13 2 258 12 2

3608 microgl 3623 078 2 2385 023 2 2343 18 2 2296 092 2

3610 microgkg 369 185 2 25 125 2 251 126 2 244 122 2

3611 microgkg 48 02 2 323 02 2 328 04 2 317 03 2

3613 microgl 332 31 2 261 3 2 249 25 2 239 24 2

3615 microgl 415 2635 2729 2689

3617 microgkg 37 09 2 244 06 2 284 07 2 239 06 2

3622 microgl 37 12 2 25 23 2 257 21 2 238 1 2

3624 microgkg 402 104 2 255 084 2 265 096 2 253 07 2

5306 microgkg 563 113 2 371 74 2 359 72 2 333 67 2

5308 microgkg 168633 102669 232169 211488

5309 microgl 402 268 27

5310 microgl 315 158 25 125 25 125 288 144

5312 microgl 428 043 2 282 028 2 272 027 2 275 028 2

5315 microgkg 387 08 2 256 05 2 251 05 2 253 05 2

5316 microgl 3839 342 2 2485 191 2 2559 168 2 239 51 2

5317 microgl 2466 043 2 2212 034 2 2472 016 2 2146 068 2

5319 microgl 414 41 2 279 28 2 268 27 2 26 31 2

5321 microgkg 476 316 326 328

5322 microgl 2547 35 2

5323 microgkg 405 203 2 239 119 2 255 153 2 201 11 2

58

Figure 11 Youden plots of the percent deviation of the reported values from the assigned values for the food supplement test sample and the

standard solution in acetonitrile with undisclosed content

(The identity of the respective measurand is given above the figure Results exceeding a difference of more than 100 were omitted)

59

Figure 11 - continued

60

ANNEX 10 Table 18 Method performance parameters reported by the participants

Laboratory code

Analyte LOD LOQ Recovery Analyte LOD LOQ Recovery

microgkg microgkg microgkg microgkg 3600 BaA 03 06 978 BaP 03 07 9483602 BaA 007 021 926 BaP 005 015 993603 BaA 0011 05 987 BaP 0005 051 9553604 BaA 02 04 86 BaP 02 04 883605 BaA 0025 005 118 BaP 0025 005 1083606 BaA 001 003 65 BaP 001 003 703607 BaA 021 071 74 BaP 027 089 943608 BaA 016 048 8213 BaP 015 046 83683610 BaA 02 05 85 BaP 005 02 853611 BaA BaP 3612 BaA 1 117 BaP 1 1203613 BaA 01 03 90 BaP 01 03 903614 BaA 02 05 96 BaP 02 05 933615 BaA 005 015 110 BaP 005 015 1053616 BaA 01 05 108 BaP 01 05 753617 BaA 018 037 405 BaP 009 018 3873618 BaA 01 03 63 BaP 01 03 593619 BaA 005 005 59 BaP 033 033 543620 BaA 0009 0018 64 BaP 0005 001 553621 BaA 007 02 84 BaP 007 02 923622 BaA 01 03 1101 BaP 01 03 9853623 BaA 02 08 105 BaP 02 08 1013624 BaA BaP 01 033 8513625 BaA BaP 3626 BaA BaP 5301 BaA 003 01 102 BaP 003 01 1005302 BaA 01 03 78 BaP 01 03 865303 BaA 003 01 62 BaP 003 01 805304 BaA 1 1 70 BaP 1 1 955305 BaA 02 05 85 BaP 02 05 855306 BaA 03 1 699 BaP 03 1 905307 BaA 6537 BaP 81165308 BaA 0088 0265 99 BaP 00262 00785 1005309 BaA 01 08 92 BaP 01 08 925310 BaA 01 03 BaP 01 03 905311 BaA 002 005 80 BaP 002 004 915312 BaA 03 09 105 BaP 03 09 1055313 BaA 04 08 BaP 04 08 5314 BaA 001 002 96 BaP 001 002 9485315 BaA 01 03 50 - 100 BaP 01 03 50 - 100 5316 BaA 002 011 827 BaP 004 019 10445317 BaA 05 1 76 BaP 05 1 755318 BaA BaP 5319 BaA 009 029 951 BaP 011 041 995320 BaA 12 18 946 BaP 06 1 8345321 BaA 03 05 BaP 05 1 5322 BaA 1 2 100 BaP 1 2 1005323 BaA 26 BaP 26

61

ANNEX 10 Table 18 - continued

Laboratory code Analyte LOD LOQ Recovery Analyte LOD LOQ Recovery

microgkg microgkg microgkg microgkg 3600 BbF 03 05 96 CHR 02 03 10153602 BbF 015 045 947 CHR 003 009 9193603 BbF 0014 05 1012 CHR 0004 05 10033604 BbF 02 04 85 CHR 02 04 873605 BbF 005 01 94 CHR 0025 005 863606 BbF 001 003 70 CHR 001 003 653607 BbF 018 06 100 CHR 014 028 943608 BbF 015 046 7979 CHR 015 045 71253610 BbF 005 02 85 CHR 02 05 853611 BbF CHR 3612 BbF 1 118 CHR 1 1173613 BbF 01 03 90 CHR 01 03 903614 BbF 01 04 96 CHR 02 05 953615 BbF 008 025 109 CHR 003 01 1083616 BbF 01 05 78 CHR 01 05 723617 BbF 035 07 38 CHR 012 024 4163618 BbF 01 03 62 CHR 01 03 623619 BbF 016 016 63 CHR 01 01 593620 BbF 0003 0006 55 CHR 001 002 653621 BbF 007 02 103 CHR 007 02 893622 BbF 02 06 963 CHR 03 09 1043623 BbF 02 08 106 CHR 02 08 1193624 BbF CHR 3625 BbF CHR 3626 BbF CHR 5301 BbF 003 01 96 CHR 003 01 1145302 BbF 01 03 84 CHR 01 03 835303 BbF 003 01 78 CHR 003 01 705304 BbF 1 1 86 CHR 1 1 705305 BbF 02 05 90 CHR 02 05 955306 BbF 03 1 1435 CHR 03 1 1185307 BbF 8116 CHR 65375308 BbF 0079 0238 99 CHR 009 026 1005309 BbF 01 08 92 CHR 5310 BbF 07 2 CHR 01 03 5311 BbF 002 004 95 CHR 002 004 895312 BbF 03 09 105 CHR 03 09 1055313 BbF 04 08 CHR 04 08 5314 BbF 001 002 83 CHR 001 002 8185315 BbF 02 05 50 -100 CHR 02 05 50 - 100 5316 BbF 005 024 814 CHR 001 007 9595317 BbF 05 1 73 CHR 05 1 785318 BbF CHR 5319 BbF 011 038 946 CHR 013 043 9675320 BbF 07 09 1194 CHR 23 32 10455321 BbF 05 1 CHR 05 1 5322 BbF 1 2 100 CHR 1 2 1005323 BbF 26 CHR 26

62

ANNEX 11 Table 19 Details of analysis method reported by the participants Data are presented as reported

Laboratory code

Analysis technique

Instrument calibration

Internal standards applied

Sample clean up

Details of sample

clean up

Extraction technique

Details of sample

extraction

3600 6) GC-MS

2) Internal standardisation with labelled substances

Deuterated PAH mix 9 (Ehrenstorfer)

1) Saponification 2) LiquidLiquid partitioning

D) Other Liquidliquid partitioning

3602 2) HPLC-UV-FLD

1) External calibration

3) Gel permeation chromatography

A) Pressurised liquid extraction (PLE)

3603 1) HPLC-FLD

1) External calibration

3) Gel permeation chromatography

D) Other 1 minute with dichloromethane in vortex

3604 2) HPLC-UV-FLD

2) Internal standardisation with labelled substances

DiP D14

5) Donor acceptor complex chromatography

D) Other LL

3605 1) HPLC-FLD

1) External calibration

3) Gel permeation chromatography

C) Soxhlet extraction

3606 7) GC-MSMS

2) Internal standardisation with labelled substances

BaP 13C4 CHR 13C6 BaA 13C6 BbF 13C6

4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

3607 6) GC-MS

2) Internal standardisation with labelled substances

BaA-d12 CHR-d12 BbF-d12 BkF-d12 BaP-d12 ICP-d12 BgP-d12 DiP-d14

1) Saponification

D) Other solvent partitioning

3608 1) HPLC-FLD

1) External calibration

6) Other Liquid extraction

D) Other

Extraction in a rotary agitator and centrifugation

3610 2) HPLC-UV-FLD

1) External calibration

4) Solid phase extraction

B) Sonication

3611

3612 6) GC-MS

2) Internal standardisation with labelled substances

chrysene-d12 dibenzo(ah)anthracene-d14 dibenzo(ai)pyrene-d14

4) Solid phase extraction

B) Sonication

3613 5) LC-MSMS

3) Internal standardisation with unlabelled substances

I use Chrysene-D12 3) Gel permeation chromatography

D) Other extraction with hexan

63

Table 19 - continued

Laboratory code

Analysis technique

Instrument calibration

Internal standards applied

Sample clean up Details of sample

clean up

Extraction technique

Details of sample

extraction

3614 6) GC-MS 1) External calibration

3) Gel permeation chromatography

B) Sonication

3615 1) HPLC-FLD

1) External calibration

6) Other PLE GPC

A) Pressurised liquid extraction (PLE)

3616 6) GC-MS

2) Internal standardisation with labelled substances

Benzo(a)pyrene-13C4 Benzo(a)anthracene-13C6 Benzo(b)fluoranthene-13C6 Chrysene-13C6

2) LiquidLiquid partitioning 4) Solid phase extraction

B) Sonication

3617 1) HPLC-FLD

1) External calibration

4) Solid phase extraction

C) Soxhlet extraction

3618 6) GC-MS

2) Internal standardisation with labelled substances

deuterated standards

1) Saponification 4) Solid phase extraction

D) Other Handshaken with cyclohexane

3619 6) GC-MS

2) Internal standardisation with labelled substances

C13 Labelled US EPA 16

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

D) Other Homegenisation amp saponiofication

3620 9) GC-HRMS

2) Internal standardisation with labelled substances

mix of deuterated PAHs (BAA-D12 CHR-D12 BAP-D12 BBF-D12)

3) Gel permeation chromatography 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

3621 6) GC-MS

2) Internal standardisation with labelled substances

Chrysene D12 Benzo(a)pyrene D12 Benzo(b)fluoranthene D12 Benzo(a)anthracene

2) LiquidLiquid partitioning 4) Solid phase extraction

B) Sonication

3622 1) HPLC-FLD

4) Standard addition method

4) Solid phase extraction

D) Other

Extraction with cyclohexane by heating under reflux

3623 7) GC-MSMS

2) Internal standardisation with labelled substances

C13 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

3624 2) HPLC-UV-FLD

1) External calibration

2) LiquidLiquid partitioning

D) Other hot solvent extraction

3626

64

Table 19 - continued

Laboratory code

Analysis technique

Instrument calibration

Internal standards

applied

Sample clean up

Details of sample

clean up

Extraction technique

Details of sample

extraction

5301 7) GC-MSMS

2) Internal standardisation with labelled substances

EACH SUBSTANCE C13

4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5302 7) GC-MSMS

2) Internal standardisation with labelled substances

Isotope labelled PAHs 13C for each analyte

4) Solid phase extraction

C) Soxhlet extraction

5303 7) GC-MSMS

3) Internal standardisation with unlabelled substances

HAP C13 4) Solid phase extraction

D) Other automate ASE200 DIONEX

5304 7) GC-MSMS

2) Internal standardisation with labelled substances

with C13 for each HAP

2) LiquidLiquid partitioning 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5305 7) GC-MSMS

2) Internal standardisation with labelled substances

C13 isotopiques internal standards

4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5306 7) GC-MSMS

2) Internal standardisation with labelled substances

C13 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5307 6) GC-MS

2) Internal standardisation with labelled substances

1) Saponification 2) LiquidLiquid partitioning

D) Other solvent extraction

5308 1) HPLC-FLD 1) External calibration

4) Solid phase extraction

B) Sonication

5309 1) HPLC-FLD 1) External calibration

4) Solid phase extraction

D) Other only SPE

5310 1) HPLC-FLD 1) External calibration

1) Saponification 2) LiquidLiquid partitioning 3) Gel permeation chromatography

C) Soxhlet extraction

5311 6) GC-MS

2) Internal standardisation with labelled substances

13 C Chrysene 13 C Benz(a)pyrene

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

B) Sonication

5312 1) HPLC-FLD

3) Internal standardisation with unlabelled substances

Benzo(b)chrysen

2) LiquidLiquid partitioning 4) Solid phase extraction

Quechers D) Other Quechers

65

Table 19 - continued

Laboratory code

Analysis technique

Instrument calibration

Internal standards applied

Sample clean up Details of sample

clean up

Extraction technique

Details of sample extraction

5313 6) GC-MS

2) Internal standardisation with labelled substances

Benz(a)anthracene D12 Benzo(b)fluranthene D12 Benzo(a)pyrene D12 Chrysene D12

1) Saponification 2) LiquidLiquid partitioning 3) Gel permeation chromatography

A) Pressurised liquid extraction (PLE)

5314 6) GC-MS

2) Internal standardisation with labelled substances

Benzo(a)pyren d12 2) LiquidLiquid partitioning

D) Other Extraction with

5315 1) HPLC-FLD

3) Internal standardisation with unlabelled substances

Benz(b)chrysen

1) Saponification 2) LiquidLiquid partitioning

D) Other shaker

5316 1) HPLC-FLD

1) External calibration

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

D) Other

LiquidLiquid extraction with cyclohexane and dimethylformamide

5317 7) GC-MSMS

1) External calibration

3) Gel permeation chromatography 6) Other

silica gel D) Other VDLUFA online method

5318

5319 1) HPLC-FLD

3) Internal standardisation with unlabelled substances

Benzo(b)chrysene 3) Gel permeation chromatography

A) Pressurised liquid extraction (PLE)

5320 6) GC-MS

3) Internal standardisation with unlabelled substances

22-binaphthyl

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

D) Other 3 hours refluxing

5321 1) HPLC-FLD

2) Internal standardisation with labelled substances

6-meacutethylchrysegravene

1) Saponification 2) LiquidLiquid partitioning

B) Sonication

5322 6) GC-MS

2) Internal standardisation with labelled substances

naftalene-d8 acenaphtalene-d10 phenanthrene-d10 chrysene-d12 perylene-d12 benzo(e)anthracene-d12

1) Saponification 2) LiquidLiquid partitioning 6) Other

clean-up with silica packed column

C) Soxhlet extraction

5323 1) HPLC-FLD

1) External calibration

4) Solid phase extraction 6) Other

Accelerated Solvent Extraction

A) Pressurised liquid extraction (PLE)

66

European Commission EUR 25245 EN ndash Joint Research Centre ndash Institute for Reference Materials and Measurements Title Report on the 8th inter-laboratory comparison organised by the European Union Reference Laboratory for Polycyclic Aromatic Hydrocarbons - Four marker PAHs in a dry extract of St Johns wort Author(s) Radoslav Lizak Szilard Szilagyi Philippe Verlinde and Thomas WENZL Luxembourg Publications Office of the European Union 2012 ndash 66 pp ndash 201 x 297 cm EUR ndash Scientific and Technical Research series ndash ISSN 1831-9424 ISBN 978-92-79-23337-1 doi 10278759387 Abstract The proficiency test here reported concerned the determination of the contents of four marker polycyclic aromatic hydrocarbons (PAHs) and of their sum in an food supplements test sample The set of marker PAHs consists of benz[a]anthacene benzo[a]pyrene benzo[b]fluoranthene and chrysene Participants to these PT were National Reference Laboratories for PAHs (NRLs-PAHs) and EU official food control laboratories The number of participants was in total 48

The PT was organised according ISO Standard 170432010

The test material used was a commercial dry extract of St Johns wort Participants also received a solution of the same PAHs either in acetonitrile or in toluene for checking their instrument calibration

The results from participants were rated with z-scores and zeta-scores About 68 of the reported results were attributed with z-scores with an absolute value of below two which is the threshold for satisfactory performance

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interests whether private or national

L

A-N

A-252

45-EN

-N

• 1 Summary
• 2 Introduction
• 3 Scope
• 4 Participating Laboratories
• 5 Time frame
• 6 Confidentiality
• 7 Test materials
• • 71 1Preparation of test materials
  • 72 Assigned values and standard deviation for proficiency assessment
  • 73 Homogeneity and stability
  • 74 Sample dispatch
  • • 8 Design of the proficiency test
    • 9 Evaluation of the results
    • • 91 2General
      • 92 21BEvaluation criteria
      • 93 Performance of participants
      • 94 Evaluation of the influence of analysis method
      • 95 Evaluation of the influence of calibration
      • 96 26Evaluation of compliance with legislation
      • • 10 Follow-up actions for underperforming laboratories
        • 11 1Conclusions
        • 12 1Acknowledgements
        • 13 References

17

Table 6 Compilation of z-scores calculated from the ldquoresults for proficiency assessment

reported by the NRLs for food supplement test material z-scores outside the

satisfactory range (|z| gt 2) are indicated by red font Values are reproduced as reported

by the laboratories Empty cells denote analytes for which results were not reported

Measurand benz[a]anthracene benzo[a]pyrene benzo[b]fluoranthene chrysene Sum PAH 4

Assigned value

microgkg 298 165 292 407 1162

Standard deviation for proficiency assessment

microgkg 062 036 060 083 125

Laboratory code

Result z-score Result z-score Result z-score Result z-score Result z-score

microgkg microgkg microgkg microgkg microgkg

3600 3 00 17 01 31 03 5 11 128 09

3602 19 -18 124 -11 17 -20 304 -12 79 -30

3603 251 -08 161 -01 256 -06 459 06 1127 -03

3604 213 -14 143 -06 11 -30 429 03 893 -22

3605 291 -01 167 01 247 -07 386 -03 1091 -06

3606 289 -01 167 01 276 -03 451 05 1184 02

3607 438 23 274 30 567 46 819 50 2098 75

3608 714 67 573 113 1036 123 1014 73 3336 174

3610 196 -17 206 11 191 -17 424 02 1138 -02

3611 469 708 308 805 576 907 865 996 2218 1684

3612 31 02 22 15 29 00 61 25 143 21

3613 33 05 18 04 31 03 4 -01 122 05

3614 185 -18 08 -23 127 -27 271 -16 663 -40

3615 376 13 145 -06 4 18 601 23 152 29

3616 2 -16 15 -04 2 -15 47 08 102 -11

3617 362 10 169 01 299 01 422 02 1252 07

3618 32 04 2 10 31 03 45 05 128 09

3619 299 00 207 12 326 06 738 40 157 33

3620 302 01 166 00 276 -03 46 06 1204 03

3621 28 -03 47 84 25 -07 51 12 151 28

3622 322 04 213 13 34 08 488 10 1361 16

3623 33 05 21 12 28 -02 72 38 154 30

3624 18 04

3626 314 03 174 02 292 00 628 27 1409 20

18

Table 7 Compilation of z-scores calculated from the ldquoresults for proficiency assessment

reported by the OCLs for food supplement test material z-scores outside the

satisfactory range (|z| gt 2) are indicated by red font Values are reproduced as reported

by the laboratories Empty cells denote analytes for which results were not reported

Measurand benz[a]anthracene benzo[a]pyrene benzo[b]fluoranthene chrysene Sum PAH 4

Assigned value

microgkg 298 165 292 407 1162

Standard deviation for proficiency assessment

microgkg 062 036 060 083 125

Laboratory code

Result z-score Result z-

score Result z-score Result

z-score

Result z-score

microgkg microgkg microgkg microgkg microgkg

5301 31 02 18 04 29 00 49 10 126 08

5302 316 03 188 06 298 01 679 33 37 -63

5303 2876 -02 1535 -03 2586 -06 6441 29 13438 15

5304 261 -06 146 -05 253 -06 591 22 1251 07

5305 24 -09 16 -01 24 -09 61 25 125 07

5306 22 -13 16 -01 42 21 49 10 129 10

5307 461 27 157 -02 221 -12 524 14 1363 16

5308 16446 -22 107009 -16 229848 -10 213243 -23 72456 -35

5309 17 -21 11 -15 2 -15

5310 305 01 123 -12 313 03 386 -03 1127 -03

5311 459 26 2 10 281 -02 437 04 1367 16

5312 543 40 248 23 299 01 953 66 2042 71

5313 353 09 207 12 328 06 838 52 1726 45

5314 394 16 172 02 404 19 588 22 1558 32

5315 545 40 238 20 444 25 542 16 177 49

5316 235 -10 17 01 273 -03 282 -15 96 -16

5317 222 -12 269 29 192 -17 545 17 1229 05

5318 067 -37 117 -13 279 -02 357 -06 82 -27

5319 147 -24 216 14 396 17 465 07 1225 05

5320 534 38 316 42 625 55 834 52 2309 92

5321 12 -12

5322 87 92 17 01 33 06 7 35 207 73

5323 33 05 2 10 45 26 73 39 171 44

19

Table 8 and Table 9 present the respective zeta-scores As for the z-scores data outside the

satisfactory performance range are given in red bold font Zeta scores were only calculated for

laboratories that provided measurement uncertainty data as well as a numerical value for the coverage

factor Zeta scores were not calculated when the reported uncertainty value exceeded the content value

which is probably linked to mixing of reporting units (microgkg and ) However the participants are

reminded that the measurement uncertainty shall be expressed in the same unit as the content value

The assessment of the performance of the participants based on the reported measurement uncertainty

gave a less favourable picture compared to z-scores Only 60 of the zeta-scores are within the

satisfactory performance range The distribution of satisfactory performance ratings between NRLs

and OCLs is not balanced It was higher for the NRLs

It has to be noted that the magnitude of the zeta-scores were for some participants much higher than

the z-scores attributed to the same results Consequently the laboratories perform according to fitness-

for-purpose criterion specified in legislation which forms the basis for the z-scores but seem to have

difficulties in deducing realistic measurement uncertainty values Eg the relative standard

measurement uncertainties reported by the NRLs ranged from 25 to 20 with a median value of

about 10 Only two OCLs reported measurement uncertainties that exceeded the maximum tolerable

uncertainty (Uf) The respective data is highlighted in yellow in Table 9

However the EU-RL PAHs will continue to pay special attention to measurement uncertainty in future

ILCs as it has major implications on the assessment of compliance of food with European legislation

20

Table 8 Compilation of zeta-scores calculated from the ldquoresults for proficiency assessment

reported by the NRLs for test material St Johns wort the reported measurement

uncertainty and the uncertainty of the analyte content of the test material zeta-scores

outside the satisfactory range (|zeta| gt 2) are indicated by red bold font Empty cells

denote analytes for which either results or measurement uncertainties were not

reported

21

Table 9 Compilation of zeta-scores calculated from the ldquoresults for proficiency assessment

reported by the OCLs for test material St Johns wort the reported measurement

uncertainty and the uncertainty of the analyte content of the test material zeta-scores

outside the satisfactory range (|zeta| gt 2) are indicated by red bold font yellow

highlighted cells ugtUf Empty cells denote analytes for which results were not

reported

22

94 Evaluation of the influence of analysis method

The information on the applied analysis method (ANNEX 11) was used to group the performance

indicators attributed to the reported results according to the applied analysis technique Two groups of

data were formed They represent results obtained by high performance liquid chromatography-

fluorescence detection (HPLC-FD about 40 ) and by gas chromatography-mass spectrometry (GC-

MS about 60 ) respectively Kernel density plots were constructed based on the respective z-scores

Figure 3A to 3E show the different Kernel density plots

Figure 3 Kernel density plots of z-scores attributed to the results for proficiency assessment

which were reported for the food supplements test sample Results were classified

according to the applied analysis technique

A) benz[a]anthracene

B) benzo[a]pyrene

23

Figure 3 continued

C) benzo[b]fluoranthene

D) chrysene

E) sum of four marker PAHs

The kernel density plots for BAP and BBF are almost identical and centred at or close to a z-score

value of zero as can be seen in Figure 3B and 3C Therefore bias introduced by the type of analysis

method is unlikely for these two analytes The situation is different for BAA and CHR In case of

BAA the kernel density distribution of z-scores based on HPLC-FD is shifted to negative values

24

(Figure 3A) which indicates a tendency to underestimate the BAA content in this particular

samplematrix when applying HPLC-FD This might be reasoned by separationintegration problems

between BAA and CHR peaks A few participants (from the HPLC-FD subgroup) that got negative

z-scores for BAA overestimated the CHR content This caused the slight shift of the density trace for

HPLC-FD towards negative values Removing this data from the evaluation would result in a density

trace with the mode at zero

The opposite is the case for CHR (Figure 3D) The density distribution of the z-scores attributed to

HPLC-FD data is centred almost at zero However the distribution for data based on GC-MS is shifted

to positive values indicating strong positive bias This bias might be caused by a lack of selectivity of

the analysis method The test material contains triphenylene which is isobaric to chrysene

Consequently it cannot be distinguished from chrysene by means of mass spectrometry These two

compounds have to be separated chromatographically which is not easy They coelute on unpolar and

on most mid-polar capillary columns Columns that allow their separation were commercialised only

recently and might not be available in all laboratories Therefore laboratories which determine the

four EU marker PAHs by means of GC-MS shall pay attention to the chromatographic separation of

triphenylene and chrysene A lack of chromatographic resolution might lead to strong positive bias of

GC-MS methods and to false conclusions on the marketability of the tested sample

The chromatographic separation of chrysene and triphenylene is not a problem with HPLC which is

reflected in the position of the density plot for chrysene However attention has to be paid in HPLC-

FD analysis to a proper clean up of the sample extract in order to eliminate interferences that show

similar retention as BAA and CHR

95 Evaluation of the influence of calibration

The participants received together with the food supplement test sample two standard solutions in

depending of their choice either acetonitrile or toluene The analyte content of one solution was

disclosed to the participants whereas the content of the other solution was kept undisclosed The

content values of the latter are presented in Table 5 Analysis results for the solution with undisclosed

content had to be reported to the PT organiser The respective data are presented in Annex 9

Percent deviations of the reported results from the assigned values were calculated for both the food

supplement test sample and the standard solution with undisclosed analyte content Youden plots were

prepared in order to visualize the influence of instrument calibration on the results reported for the

food supplement sample Two plots are discussed in the following The whole set of Youden plots are

given in Annex 9

25

Figure 4 Youden plot of the percent deviations of values reported for benzo[a]pyrene from the

assigned values of the food supplement test material and the standard solution in

toluene with undisclosed content

Figure 4 reveals that a number of participants reported results with similar percent deviations from the

assigned values of both the results for the food supplement material and the standard solution in

toluene with undisclosed content This correlation indicates that over- respectively underestimation of

the analyte content of the food supplement material might be caused by biased instrument calibration

This finding underpins the importance of thorough calibration standard preparation However the data

was not in all cases conclusive as some laboratories performed well for the food supplement samples

but reported biased results for the standard solution with undisclosed content (eg Figure 4 participants

3620 and 5305) This fact cannot be explained yet

Figure 5 presents the respective Youden plot for chrysene in the food supplement test material and the

standard solution in toluene with undisclosed content From this graph it can be concluded that the

overestimation of the chrysene content of the food supplement material was not caused by instrument

calibration The results for the solvent solution of many laboratories which underperformed for the

26

food supplement sample agreed well with the assigned value This supports the conclusion that the

overestimation is caused by the determination of an interferent most probably triphenylene

Figure 5 Youden plot of the percent deviations of values reported for chrysene from the

assigned values of the food supplement test material and the standard solution in

toluene with undisclosed content

96 Evaluation of compliance with legislation

The method performance data reported for the four marker PAHs were evaluated for compliance with

the provisions given in Commission Regulation (EC) No 8362011 Table 18 in ANNEX 10 gives an

overview on the results of the evaluation Data were not reported in case of empty cells Non-

compliant data are highlighted in Table 18 It should be noted that Regulation (EU) No 8362011

specifies LOD and LOQ with two decimals This was not respected by all participants which

hampered the compliance assessment

27

In summary it can be stated that the majority of participants reported method performance

characteristics for the determination of the four marker PAHs in the food supplement test sample that

are compliant with legislation However some laboratories need to improve their analysis methods

The laboratories in question are urged to adapt to the provisions given in the new legislation

10 Follow-up actions for underperforming laboratories

All laboratories that got questionable or non-satisfactory performance ratings are urged to perform

root cause analysis and to implement corrective actions

As agreed during the EU-RL PAH workshop 2011 the EU-RL PAH will set up follow-up measures in

due time for all NRLs that received for at least one of the four PAHs (BAA BAP BBF and CHR) z-

scores gt 3 as required by Regulation (EC) 8822004 and by the Protocol for management of

underperformance in comparative testing andor lack of collaboration of National Reference

Laboratories (NRLs) with European Union reference laboratories (EU-RLs) activities

These laboratories shall perform as an immediate action root-cause-analysis and shall report to the

EU-RL PAH in writing the identified cause for their underperformance and corrective actions they are

going to take Additionally they shall participate to an independent (non-EU-RL) proficiency test on

the determination of PAHs in food and shall communicate the outcome of this exercise to the EU-RL

PAH

28

11 Conclusions

Fourty seven participants reported results back to the EU-RL The performance of the majority of

participants was good In total 153 out of 225 attributed z-scores were below an absolute value of two

which equals to almost 68 However this percentage is much below the percentages of satisfactory

performance gained in previous studies This might be the consequence of the higher level of difficulty

with respect to matrix composition In addition bias can be concluded from the pattern of performance

indicators of some laboratories

In general the determination of chrysene caused most difficulties to the participants

zeta-Scores were calculated besides z-scores They indicate the agreement of the reported result with

the assigned value with respect to the stated measurement uncertainty The outcome of this rating was

worse than for the z-scores which indicates that the measurement uncertainty estimates were not

realistic Therefore participants underperforming with regard to zeta scores are urged to adapt their

measurement uncertainty statements

The great majority of participants in this inter-laboratory comparison applied analytical methods which

complied with EU legislation with regard to performance characteristics

12 Acknowledgements

The organisers would like to thank Mrs Beatriz de la Calle and Mr Franz Ulberth (all from IRMM

Geel Belgium) for their accurate revision of this report Mr Hakan Emteborg (from IRMM) for

ampouling of the standard solutions and all NRLs and OCLs for their cooperation

29

13 References

1 EU COMMISSION REGULATION (EC) No 7762006 of 23 May 2006 amending Annex VII to Regulation

(EC) No 8822004 of the European Parliament and of the Council as regards Community reference

laboratories Official Journal of the European Union 2006 L 136 p 3-8

Available from

httpeur-lexeuropaeuLexUriServLexUriServdouri=OJL200613600030008ENPDF

2 EU Regulation (EC) No 8822004 of the European Parliament and of the Council of 29 April 2004 on

official controls performed to ensure the verification of compliance with feed and food law animal health

and animal welfare rules Official Journal of the European Communities 2004 L191 p 1-52 Available

from

httpeur-lexeuropaeuLexUriServLexUriServdouri=OJL200419100010052ENPDF

3 EU Opinion of the Scientific Committee on Food on the risks to human health of Polycyclic Aromatic

Hydrocarbons in food (2002) Available from

httpeuropaeuintcommfoodfsscscfout153_enpdf

4 International Agency for Research on Cancer IARC Monographs on the Evaluation of

Carcinogenic Risks to Humans Some Non-heterocyclic Polycyclic Aromatic Hydrocarbons and Some

Related Exposures Volume 92 (2010) Available from

httpmonographsiarcfrENGMonographsvol92mono92pdf

5 EU Commission Regulation (EC) No 3332007 of 28 March 2007 laying down the methods of sampling

and analysis for the official control of the levels of lead cadmium mercury inorganic tin 3-MCPD and

benzo(a)pyrene in foodstuffs Official Journal of the European Union 2007 L 88 p 29-38 Available

from

httpeur-lexeuropaeuLexUriServLexUriServdouri=OJL200708800290038ENPDF

6 EU Commission Regulation (EC) No 18812006 of 19 December 2006 setting maximum levels for certain

contaminants in foodstuffs Official Journal of the European Union 2006 L 364 p 5-24 Available from

httpeur-lexeuropaeuLexUriServLexUriServdouri=OJL200636400050024ENPDF

7 EFSA Scientific Opinion of the Panel on Contaminants in the Food Chain on a request from the European

Commission on Polycyclic Aromatic Hydrocarbons in Food The EFSA Journal 2008 724 p 1-114

Available from

httpwwwefsaeuropaeuEFSAefsa_locale-1178620753812_1211902034842htm

30

8 EU Commission Regulation (EU) No 8352011 amending Regulation (EC) No 18812006 as regards

maximum levels for polycyclic aromatic hydrocarbons in foodstuffs Official Journal of the European

Union 2011 L 215 p 4-8

httpeur-lexeuropaeuLexUriServLexUriServdouri=OJL201121500040008ENPDF

9 EU Commission Regulation (EU) No 8362011 amending Regulation (EC) No 3332007 laying down the methods of sampling and analysis for the official control of the levels of lead cadmium mercury inorganic tin 3-MCPD and benzo(a)pyrene in foodstuffs Official Journal of the European Union 2011 L 215 p 9-16 httpeur-lexeuropaeuLexUriServLexUriServdouri=OJL201121500090016ENPDF

10 ISOIEC 170432010 (2010) Conformity assessment -- General requirements for proficiency testing

providers Geneva Switzerland

11 Thompson M Ellison SLR and Wood R The International Harmonized Protocol for the Proficiency

Testing of Analytical Chemistry Laboratories Pure Appl Chem (2006) 78(1) p 145ndash196

Available from httpmediaiupacorgpublicationspac2006pdf7801x0145pdf

31

14 ANNEXES

ANNEX 1 ndash Announcement of the PT on the IRMM webpage

ANNEX 2 ndash Announcement via e-mail and invitation

ANNEX 3 ndash Announcement of sample dispatch

ANNEX 4 ndash Technical specifications of the calibration solutions

ANNEX 5 ndash Homogeneity and stability of the test material

ANNEX 6 ndash Acknowledgement of sample receipt

ANNEX 7 ndash Questionnaire on details of analysis method

ANNEX 8 ndash Results reported for the food supplement sample

ANNEX 9 ndash Results reported for the standard solutions with undisclosed analyte content

ANNEX 10 ndash Method performance characteristics reported by participants

ANNEX 11 ndash Details of analysis methods reported by participants

32

ANNEX 1 ndash Announcement of the PT on the IRMM webpage

33

ANNEX 2 - Announcement via e-mail and invitation

34

ANNEX 3 - Announcement sample dispatch

Dear MadameSir

The test samples for the proficiency test on the determination of four PAHs in a food supplement (dry extract of St Johns wort) were

dispatched today

Explanation on the design of the study as well as instructions for sample storage and for reporting of results are in the parcel You will

also find your participant key in the parcel which is required for reporting of results

Please check the parcel after arrival for completeness and report the receipt of the test samples as well as their status to us by applying

the sample receipt form which is in the parcel

You are kindly requested to inform us by email if you do not receive the parcel by end of this week

My colleague Donata Lerda will then take care of it

With best regards

Thomas Wenzl

35

ANNEX 4 - Specifications of standard solutions with disclosed concentration

36

ANNEX 4 - continued

37

ANNEX 5 - Homogeneity and stability of the test material

Homogeneity of the test material

Benz[a]anthracene - BAA

Benzo[a]pyrene - BAP

38

ANNEX 5 - continued

Benzo[b]fluoranthene - BBF

Chrysene - CHR

39

ANNEX 5 - continued

Stability of the test material

Table 10 Content of the four target analytes in test materials stored under different

temperatures

BaA BaP BbF CHR

microgkg microgkg microgkg microgkg

Content 31 16 31 69 Storage at room temperature

U (k=2) 05 02 05 17

Content 31 15 29 68 Storage at reference temperature

U (k=2) 04 03 04 10

The applied capillary column does not separate CHR from triphenylene

40

ANNEX 6 ndash Documents sent to participants

41

ANNEX 6 - continued

42

ANNEX 7 Questionnaire on details of analysis method

43

ANNEX 7 - continued

44

ANNEX 7 - continued

45

ANNEX 8 - Results reported by participants for the food supplements test sample Table 11 Analysis results reported by the participants for the content of benz[a]anthracene (BAA) in the food supplements test material Values are presented as reported

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

3600 31 29 29 3 05 2

3602 199 193 178 19 021

3603 253 253 246 251 022 2

3604 199 209 23 213 071 2

3605 301 3 271 291 076 2

3606 302 288 278 289 064 2

3607 456 416 443 438 096 2

3608 796 695 651 714 149 2

3610 182 192 214 196 039 2

3611 414 504 488 469 96 2

3612 3 34 28 31 06 2

3613 32 33 34 33 07 2

3614 182 182 191 185 037 2

3615 3884 3822 3565 376 0752 2

3616 2 2 2 2 01 2

3617 366 37 35 362 046 2

3618 32 32 32 32 05 2

3619 299 305 297 299 048 2

3620 304 294 302 302 045 2

3621 29 26 29 28 04 2

3622 343 325 318 322 052 2

3623 35 3 33 33 07 2

3624

3626 318 31 314 314 126 2

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

5301 31 3 32 31 06 2

5302 314 319 315 316 063 2

5303 3102 2716 2822 2876 27 62

5304 261 263 26 261 57 2

5305 23 22 26 24 20

5306 22 23 23 22 11 2

5307 488 461 317 461

5308 168633 168544 1562025 1644598

5309 16 19 16 17

5310 3 304 311 305 015

5311 477 452 448 459 15 2

5312 506 63 494 543 134 2

5313 353 301 404 353

5314 431 373 378 394 5 096

5315 596 531 51 545 11 2

5316 224 231 235 235 022 2

5317 216 23 221 222 007 2

5318 064 07 067 01 2

5319 111 184 146 147 029 2

5320 515 525 564 534 48

5321 21 5322 13 87 9 87 35 2

5323 38 33 35 33 16 2

46

Figure 6 Results of replicate determinations (indicated by triangles) of benz[a]anthracene (BAA) in the food supplement test material

Horizontal blue lines represent the arithmetic mean value of replicate measurements and blue bars the reported expanded measurement uncertainty (k=2) The assigned value is

plotted as dotted line Red solid lines mark the range of satisfactory performance (|z|le2) Mean values are given for results outside the displayed data range

ProLab 2011

Laboratory

53

1853

1953

0853

0936

1436

0236

1036

1653

2136

0453

1753

0653

1653

0536

0353

0436

2153

0336

0636

0536

0036

2036

1953

1036

1253

0136

2653

0236

1836

2336

2236

1353

1353

2336

1736

1553

1453

0736

0753

1153

2053

1253

1536

0853

2236

11

an

aly

te c

on

ten

t (micro

gk

g)

10

95

9

85

8

75

7

65

6

55

5

45

4

35

3

25

2

15

1

05

0

102

3 468

7

Sample St Johns wort extract replicatesMeasurand Benz[a]anthraceneNo of laboratories 46

Assigned value 298 microgkg (Reference value)Target sd 062 microgkgTolerance limits 174 - 422 microgkg (|Z score| lt 200)

Assig

ned va

lue

Limit of tolerance

Limit of tolerance

47

Table 12 Analysis results reported by the participants for the content of benzo[a]pyrene (BAP) in the food supplements test material Values are presented as reported

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

3600 16 17 17 17 03 2

3602 118 123 13 124 015

3603 165 162 157 161 015 2

3604 134 154 139 143 04 2

3605 164 166 172 167 057 2

3606 181 159 161 167 031 2

3607 279 266 279 274 055 2

3608 57 687 461 573 226 2

3610 226 216 183 206 042 2

3611 289 325 311 308 36 2

3612 24 22 2 22 05 2

3613 17 18 19 18 05 2

3614 08 08 08 08 016 2

3615 1545 1393 1425 145 029 2

3616 16 16 14 15 03 2

3617 177 171 158 169 023 2

3618 2 2 2 2 02 2

3619 207 207 206 207 075 2

3620 165 169 163 166 017 2

3621 44 47 49 47 05 2

3622 212 208 222 213 038 2

3623 2 21 22 21 03 2

3624 18 178 182 18 031 2

3626 172 173 178 174 07 2

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

5301 18 18 18 18 04 2

5302 188 19 187 188 038 2

5303 1691 1426 149 1535 19 80

5304 145 142 15 146 69 2

5305 15 17 18 16 15

5306 16 17 17 16 08 2

5307 164 157 153 157

5308 102669 109324 109035 107009

5309 1 1 12 11

5310 122 122 124 123 006

5311 199 184 215 2 15 2

5312 258 248 239 248 05 2

5313 199 178 245 207

5314 174 171 172 172 11 0948

5315 253 248 213 238 05 2

5316 177 171 173 17 016 2

5317 255 277 276 269 012 2

5318 114 12 117 02 2

5319 19 228 231 216 043 2

5320 303 3 344 316 78

5321 12 12 12 12

5322 13 17 24 17 35 2

5323 2 2 19 2 1 2

48

Figure 7 Results of replicate determinations (indicated by triangles) of benzo[a]pyrene (BAP) in the food supplement test material

Horizontal blue lines represent the arithmetic mean value of replicate measurements and blue bars the reported expanded measurement uncertainty (k=2) The assigned value is

plotted as dotted line Red solid lines mark the range of satisfactory performance (|z|le2) Mean values are given for results outside the displayed data range

ProLab 2011

Laboratory

36

1453

0953

0853

1853

2153

1036

0236

0436

1553

0436

1653

0353

0736

0336

2036

0053

0553

0636

0636

0536

1753

1453

1636

2636

1336

2453

0153

2253

0253

2353

1136

1836

1953

1336

1036

2336

2253

1936

1253

1553

1253

1736

0753

2036

2136

0836

11

an

aly

te c

on

ten

t (micro

gk

g)

55

5

45

4

35

3

25

2

15

1

05

0

573

308

3

Sample St Johns wort extract replicatesMeasurand Benzo[a]pyreneNo of laboratories 48

Assigned value 165 microgkg (Reference value)Target sd 036 microgkgTolerance limits 093 - 237 microgkg (|Z score| lt 200)

Assig

ned va

lue

Limit of tolerance

Limit of tolerance

49

Table 13 Analysis results reported by the participants for the content of benzo[b]fluoranthene (BBF) in the food supplements test material Values are presented as reported

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

3600 32 3 3 31 09 2

3602 173 175 16 17 017

3603 265 252 25 256 025 2

3604 116 108 105 11 028 2

3605 286 225 232 247 074 2

3606 294 273 261 276 046 2

3607 579 579 544 567 113 2

3608 103 1201 876 1036 326 2

3610 173 196 204 191 038 2

3611 546 618 565 576 75 2

3612 3 28 28 29 06 2

3613 28 31 32 31 06 2

3614 135 111 135 127 022 2

3615 4187 3678 4111 4 08 2

3616 2 2 2 2 01 2

3617 33 27 299 035 2

3618 31 31 31 31 05 2

3619 326 324 312 326 064 2

3620 277 28 272 276 069 2

3621 26 25 23 25 04 2

3622 346 336 333 34 052 2

3623 26 28 29 28 02 2

3624

3626 288 28 307 292 117 2

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

5301 29 3 29 29 06 2

5302 297 3 296 298 06 2

5303 2705 2444 2608 2586 22 78

5304 245 254 26 253 85 2

5305 23 23 26 24 15

5306 42 43 4 42 21 2

5307 221 216 241 221

5308 232169 246654 240722 229848

5309 19 22 19 2 0

5310 3 315 325 313 016

5311 262 277 304 281 15 2

5312 27 289 337 299 075 2

5313 321 284 378 328

5314 423 404 385 404 34 083

5315 42 506 416 444 09 2

5316 265 267 277 273 018 2

5317 185 197 195 192 006 2

5318 276 281 279 04 2

5319 396 389 403 396 079 2

5320 623 618 635 625 14

5321 31

5322 24 33 49 33 35 2

5323 38 45 35 45 27 2

50

Figure 8 Results of replicate determinations (indicated by triangles) of benzo[b]fluoranthene (BBF) in the food supplement test material

Horizontal blue lines represent the arithmetic mean value of replicate measurements and blue bars the reported expanded measurement uncertainty (k=2) The assigned value is

plotted as dotted line Red solid lines mark the range of satisfactory performance (|z|le2) Mean values are given for results outside the displayed data range

ProLab 2011

Laboratory

36

0436

1436

0236

1053

1736

1653

0953

0753

0853

0536

2136

0553

0436

0353

0353

1636

0636

2036

2353

1853

1136

1236

2653

0153

0253

1236

1736

1336

0036

1853

2153

1036

1953

1336

2253

2253

2353

1936

1553

1453

0653

1536

0753

2036

0836

11

an

aly

te c

on

ten

t (micro

gk

g)

75

7

65

6

55

5

45

4

35

3

25

2

15

1

05

0

103

6

576

3

Sample St Johns wort extract replicatesMeasurand Benzo[b]fluorantheneNo of laboratories 46

Assigned value 292 microgkg (Reference value)Target sd 060 microgkgTolerance limits 172 - 412 microgkg (|Z score| lt 200)

Assig

ned va

lue

Limit of tolerance

Limit of tolerance

51

Table 14 Analysis results reported by the participants for the content of chrysene (CHR) in the food supplements test material Values are presented as reported

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

3600 5 49 5 5 07 2

3602 32 304 29 304 036

3603 462 469 447 459 048 2

3604 438 428 42 429 119 2

3605 365 41 381 386 085 2

3606 494 411 45 451 124 2

3607 897 774 787 819 18 2

3608 986 887 1168 1014 284 2

3610 418 392 462 424 085 2

3611 833 893 87 865 61 2

3612 6 64 58 61 12 2

3613 41 39 4 4 08 2

3614 272 254 288 271 054 2

3615 605 6028 5964 601 12 2

3616 47 45 49 47 05 2

3617 445 431 389 422 065 2

3618 45 45 45 45 06 2

3619 738 747 72 738 118 2

3620 463 472 446 46 092 2

3621 53 49 51 51 06 2

3622 478 513 505 488 08 2

3623 73 73 72 72 05 2

3624

3626 63 622 634 628 251 2

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

5301 49 49 49 49 1 2

5302 687 683 666 679 136 2

5303 6777 6181 6382 6441 24 70

5304 592 581 6 591 85 2

5305 6 58 64 61 20

5306 49 5 51 49 25 2

5307 524 5 574 524

5308 211488 216782 221146 213243

5309

5310 372 393 394 386 019

5311 43 459 422 437 15 2

5312 934 943 981 953 191 2

5313 793 73 99 838

5314 636 561 565 588 18 0818

5315 547 583 499 542 11 2

5316 292 286 285 282 06 2

5317 52 598 517 545 046 2

5318 368 345 357 05 2

5319 449 471 474 465 116 2

5320 76 924 818 834 99

5321 33

5322 7 7 76 7 35 2

5323 87 73 77 73 37 2

52

Figure 9 Results of replicate determinations (indicated by triangles) of chrysene (CHR) in the food supplement test material

Horizontal blue lines represent the arithmetic mean value of replicate measurements and blue bars the reported expanded measurement uncertainty (k=2) The assigned value is

plotted as dotted line Red solid lines mark the range of satisfactory performance (|z|le2) Mean values are given for results outside the displayed data range

ProLab 2011

Laboratory

53

0836

1453

1636

0253

2153

1836

0553

1036

1336

1736

1036

0453

1136

1836

0636

0336

2053

1936

1653

0136

0036

2253

0636

2153

0753

1553

1753

1453

0436

1536

1253

0536

2653

0353

0253

2236

2336

1953

2336

0753

2053

1353

1236

0836

11

an

aly

te c

on

ten

t (micro

gk

g)

13

12

11

10

9

8

7

6

5

4

3

2

1

0

865

3

Sample St Johns wort extract replicatesMeasurand ChryseneNo of laboratories 45

Assigned value 407 microgkg (Reference value)Target sd 083 microgkgTolerance limits 241 - 573 microgkg (|Z score| lt 200)

Assig

ned va

lue

Limit of tolerance

Limit of tolerance

53

Table 15 Analysis results reported by the participants for the sum of the four marker PAHs in the food supplements test material Values are presented as reported

Laboratory code

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg

3600 128 24 2

3602 79 126

3603 1127 096 2

3604 893 258 2

3605 1091 305 2

3606 1184 238 2

3607 2098 42 2

3608 3336 325 2

3610 1138 17 2

3611 2218 259 2

3612 143 29 2

3613 122 2 2

3614 663 129 2

3615 152 304 2

3616 102 03 2

3617 1252 169 2

3618 128 18 2

3619 157 305 2

3620 1204 12 2

3621 151 19 2

3622 1361 115 2

3623 154 09 2

3624

3626 1409 56 2

Laboratory code

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg

5301 126

5302 37 074 2

5303 13438 92

5304 1251 85 2

5305 125 20

5306 129 65 2

5307 1363

5308 7245598

5309

5310 1127 056

5311 1367 15 2

5312 2042 408 2

5313 1726

5314 1558

5315 177 35 2

5316 96 073 2

5317 1229 066 2

5318 82 05 2

5319 1225 267 2

5320 2309 41

5321

5322 207 35 2

5323 171 103 2

54

ANNEX 9 Results reported for the standard solutions with undisclosed analyte content Table 16 Analysis results reported by the participants for the four marker PAHs in the standard solution in toluene with undisclosed analyte

content Values are presented as reported U measurement uncertainty k coverage factor

benz[a]anthracene benzo[a]pyrene benzo[b]fluoranthene chrysene Laboratory

code Unit Result U k Result U k Result U k Result U k 3600 microgl 38 67 2 25 45 2 211 68 2 275 38 2

3605 microgl 367 184 2 2482 124 2 2592 13 2 235 118 2

3606 microgkg 3763 176 2 2609 142 2 2721 118 2 2539 134 2

3607 microgkg 3706 815 2 2824 565 2 2591 518 2 2587 569 2

3612 microgl 38 08 2 237 11 2 244 18 2 257 02 2

3614 microgl 324 65 2 209 38 2 215 37 2 21 42 2

3616 microgkg 318 07 2 224 14 2 231 26 2 216 1 2

3618 microgkg 417 17 2 284 08 2 292 06 2 297 03 2

3619 microgkg 47 742 2 274 468 2 228 387 2 274 434 2

3620 microgl 495 74 2 294 29 2 321 8 2 321 64 2

3621 microgl 241 22 2 143 13 2 188 16 2 145 14 2

3623 microgkg 476 25 2 311 53 2 269 3 2 297 03 2

3626 microgl 359 38 2 235 23 2 238 27 2 24 18 2

5301 microgkg 426 29 299 289

5302 microgkg 436 87 2 296 59 2 296 59 2 289 58 2

5303 microgl 3721 27 102 22838 19 99 23756 22 99 25486 24 98

5304 microgkg 38 29 2 225 26 2 257 37 2 258 52 2

5305 microgkg 305 225 245 29

5306 microgkg 316 63 2 247 49 2 65 13 2 22 44 2

5307 microgkg 3658 2559 241 2414

5308 microgkg 16854 109324 246654 216782

5311 microgl 374 20 2 279 15 2 268 15 2 272 15 2

5313 microgl 3408 2165 2275 2373

5314 microgkg 395 5 2875 11 3614 34 2905 18

5320 microgl 3267 368 1986 571 28 683 2427 512

5322 microgl 282 35 2 225 35 2 241 35 2 21 35 2

55

Figure 10 Youden plots of the percent deviation of the reported values from the assigned values for the food supplement test sample and the

standard solution in toluene with undisclosed content

(The identity of the respective measurand is given above the figure Results exceeding a difference of more than 100 were omitted)

56

Figure 10 - continued

57

Table 17 Analysis results reported by the participants for the four marker PAHs in the standard solution in acetonitrile with undisclosed

analyte content

Values are presented as reported U measurement uncertainty k coverage factor

benz[a]anthracene benzo[a]pyrene benzo[b]fluoranthene chrysene

Laboratory

code Unit Result U k Result U k Result U k Result U k

3602 microgkg 3675 247 247 244

3603 microgkg 358 086 2 236 028 2 235 028 2 236 057 2

3604 microgl 475 29 2 19 12 2 194 13 2 258 12 2

3608 microgl 3623 078 2 2385 023 2 2343 18 2 2296 092 2

3610 microgkg 369 185 2 25 125 2 251 126 2 244 122 2

3611 microgkg 48 02 2 323 02 2 328 04 2 317 03 2

3613 microgl 332 31 2 261 3 2 249 25 2 239 24 2

3615 microgl 415 2635 2729 2689

3617 microgkg 37 09 2 244 06 2 284 07 2 239 06 2

3622 microgl 37 12 2 25 23 2 257 21 2 238 1 2

3624 microgkg 402 104 2 255 084 2 265 096 2 253 07 2

5306 microgkg 563 113 2 371 74 2 359 72 2 333 67 2

5308 microgkg 168633 102669 232169 211488

5309 microgl 402 268 27

5310 microgl 315 158 25 125 25 125 288 144

5312 microgl 428 043 2 282 028 2 272 027 2 275 028 2

5315 microgkg 387 08 2 256 05 2 251 05 2 253 05 2

5316 microgl 3839 342 2 2485 191 2 2559 168 2 239 51 2

5317 microgl 2466 043 2 2212 034 2 2472 016 2 2146 068 2

5319 microgl 414 41 2 279 28 2 268 27 2 26 31 2

5321 microgkg 476 316 326 328

5322 microgl 2547 35 2

5323 microgkg 405 203 2 239 119 2 255 153 2 201 11 2

58

Figure 11 Youden plots of the percent deviation of the reported values from the assigned values for the food supplement test sample and the

standard solution in acetonitrile with undisclosed content

(The identity of the respective measurand is given above the figure Results exceeding a difference of more than 100 were omitted)

59

Figure 11 - continued

60

ANNEX 10 Table 18 Method performance parameters reported by the participants

Laboratory code

Analyte LOD LOQ Recovery Analyte LOD LOQ Recovery

microgkg microgkg microgkg microgkg 3600 BaA 03 06 978 BaP 03 07 9483602 BaA 007 021 926 BaP 005 015 993603 BaA 0011 05 987 BaP 0005 051 9553604 BaA 02 04 86 BaP 02 04 883605 BaA 0025 005 118 BaP 0025 005 1083606 BaA 001 003 65 BaP 001 003 703607 BaA 021 071 74 BaP 027 089 943608 BaA 016 048 8213 BaP 015 046 83683610 BaA 02 05 85 BaP 005 02 853611 BaA BaP 3612 BaA 1 117 BaP 1 1203613 BaA 01 03 90 BaP 01 03 903614 BaA 02 05 96 BaP 02 05 933615 BaA 005 015 110 BaP 005 015 1053616 BaA 01 05 108 BaP 01 05 753617 BaA 018 037 405 BaP 009 018 3873618 BaA 01 03 63 BaP 01 03 593619 BaA 005 005 59 BaP 033 033 543620 BaA 0009 0018 64 BaP 0005 001 553621 BaA 007 02 84 BaP 007 02 923622 BaA 01 03 1101 BaP 01 03 9853623 BaA 02 08 105 BaP 02 08 1013624 BaA BaP 01 033 8513625 BaA BaP 3626 BaA BaP 5301 BaA 003 01 102 BaP 003 01 1005302 BaA 01 03 78 BaP 01 03 865303 BaA 003 01 62 BaP 003 01 805304 BaA 1 1 70 BaP 1 1 955305 BaA 02 05 85 BaP 02 05 855306 BaA 03 1 699 BaP 03 1 905307 BaA 6537 BaP 81165308 BaA 0088 0265 99 BaP 00262 00785 1005309 BaA 01 08 92 BaP 01 08 925310 BaA 01 03 BaP 01 03 905311 BaA 002 005 80 BaP 002 004 915312 BaA 03 09 105 BaP 03 09 1055313 BaA 04 08 BaP 04 08 5314 BaA 001 002 96 BaP 001 002 9485315 BaA 01 03 50 - 100 BaP 01 03 50 - 100 5316 BaA 002 011 827 BaP 004 019 10445317 BaA 05 1 76 BaP 05 1 755318 BaA BaP 5319 BaA 009 029 951 BaP 011 041 995320 BaA 12 18 946 BaP 06 1 8345321 BaA 03 05 BaP 05 1 5322 BaA 1 2 100 BaP 1 2 1005323 BaA 26 BaP 26

61

ANNEX 10 Table 18 - continued

Laboratory code Analyte LOD LOQ Recovery Analyte LOD LOQ Recovery

microgkg microgkg microgkg microgkg 3600 BbF 03 05 96 CHR 02 03 10153602 BbF 015 045 947 CHR 003 009 9193603 BbF 0014 05 1012 CHR 0004 05 10033604 BbF 02 04 85 CHR 02 04 873605 BbF 005 01 94 CHR 0025 005 863606 BbF 001 003 70 CHR 001 003 653607 BbF 018 06 100 CHR 014 028 943608 BbF 015 046 7979 CHR 015 045 71253610 BbF 005 02 85 CHR 02 05 853611 BbF CHR 3612 BbF 1 118 CHR 1 1173613 BbF 01 03 90 CHR 01 03 903614 BbF 01 04 96 CHR 02 05 953615 BbF 008 025 109 CHR 003 01 1083616 BbF 01 05 78 CHR 01 05 723617 BbF 035 07 38 CHR 012 024 4163618 BbF 01 03 62 CHR 01 03 623619 BbF 016 016 63 CHR 01 01 593620 BbF 0003 0006 55 CHR 001 002 653621 BbF 007 02 103 CHR 007 02 893622 BbF 02 06 963 CHR 03 09 1043623 BbF 02 08 106 CHR 02 08 1193624 BbF CHR 3625 BbF CHR 3626 BbF CHR 5301 BbF 003 01 96 CHR 003 01 1145302 BbF 01 03 84 CHR 01 03 835303 BbF 003 01 78 CHR 003 01 705304 BbF 1 1 86 CHR 1 1 705305 BbF 02 05 90 CHR 02 05 955306 BbF 03 1 1435 CHR 03 1 1185307 BbF 8116 CHR 65375308 BbF 0079 0238 99 CHR 009 026 1005309 BbF 01 08 92 CHR 5310 BbF 07 2 CHR 01 03 5311 BbF 002 004 95 CHR 002 004 895312 BbF 03 09 105 CHR 03 09 1055313 BbF 04 08 CHR 04 08 5314 BbF 001 002 83 CHR 001 002 8185315 BbF 02 05 50 -100 CHR 02 05 50 - 100 5316 BbF 005 024 814 CHR 001 007 9595317 BbF 05 1 73 CHR 05 1 785318 BbF CHR 5319 BbF 011 038 946 CHR 013 043 9675320 BbF 07 09 1194 CHR 23 32 10455321 BbF 05 1 CHR 05 1 5322 BbF 1 2 100 CHR 1 2 1005323 BbF 26 CHR 26

62

ANNEX 11 Table 19 Details of analysis method reported by the participants Data are presented as reported

Laboratory code

Analysis technique

Instrument calibration

Internal standards applied

Sample clean up

Details of sample

clean up

Extraction technique

Details of sample

extraction

3600 6) GC-MS

2) Internal standardisation with labelled substances

Deuterated PAH mix 9 (Ehrenstorfer)

1) Saponification 2) LiquidLiquid partitioning

D) Other Liquidliquid partitioning

3602 2) HPLC-UV-FLD

1) External calibration

3) Gel permeation chromatography

A) Pressurised liquid extraction (PLE)

3603 1) HPLC-FLD

1) External calibration

3) Gel permeation chromatography

D) Other 1 minute with dichloromethane in vortex

3604 2) HPLC-UV-FLD

2) Internal standardisation with labelled substances

DiP D14

5) Donor acceptor complex chromatography

D) Other LL

3605 1) HPLC-FLD

1) External calibration

3) Gel permeation chromatography

C) Soxhlet extraction

3606 7) GC-MSMS

2) Internal standardisation with labelled substances

BaP 13C4 CHR 13C6 BaA 13C6 BbF 13C6

4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

3607 6) GC-MS

2) Internal standardisation with labelled substances

BaA-d12 CHR-d12 BbF-d12 BkF-d12 BaP-d12 ICP-d12 BgP-d12 DiP-d14

1) Saponification

D) Other solvent partitioning

3608 1) HPLC-FLD

1) External calibration

6) Other Liquid extraction

D) Other

Extraction in a rotary agitator and centrifugation

3610 2) HPLC-UV-FLD

1) External calibration

4) Solid phase extraction

B) Sonication

3611

3612 6) GC-MS

2) Internal standardisation with labelled substances

chrysene-d12 dibenzo(ah)anthracene-d14 dibenzo(ai)pyrene-d14

4) Solid phase extraction

B) Sonication

3613 5) LC-MSMS

3) Internal standardisation with unlabelled substances

I use Chrysene-D12 3) Gel permeation chromatography

D) Other extraction with hexan

63

Table 19 - continued

Laboratory code

Analysis technique

Instrument calibration

Internal standards applied

Sample clean up Details of sample

clean up

Extraction technique

Details of sample

extraction

3614 6) GC-MS 1) External calibration

3) Gel permeation chromatography

B) Sonication

3615 1) HPLC-FLD

1) External calibration

6) Other PLE GPC

A) Pressurised liquid extraction (PLE)

3616 6) GC-MS

2) Internal standardisation with labelled substances

Benzo(a)pyrene-13C4 Benzo(a)anthracene-13C6 Benzo(b)fluoranthene-13C6 Chrysene-13C6

2) LiquidLiquid partitioning 4) Solid phase extraction

B) Sonication

3617 1) HPLC-FLD

1) External calibration

4) Solid phase extraction

C) Soxhlet extraction

3618 6) GC-MS

2) Internal standardisation with labelled substances

deuterated standards

1) Saponification 4) Solid phase extraction

D) Other Handshaken with cyclohexane

3619 6) GC-MS

2) Internal standardisation with labelled substances

C13 Labelled US EPA 16

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

D) Other Homegenisation amp saponiofication

3620 9) GC-HRMS

2) Internal standardisation with labelled substances

mix of deuterated PAHs (BAA-D12 CHR-D12 BAP-D12 BBF-D12)

3) Gel permeation chromatography 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

3621 6) GC-MS

2) Internal standardisation with labelled substances

Chrysene D12 Benzo(a)pyrene D12 Benzo(b)fluoranthene D12 Benzo(a)anthracene

2) LiquidLiquid partitioning 4) Solid phase extraction

B) Sonication

3622 1) HPLC-FLD

4) Standard addition method

4) Solid phase extraction

D) Other

Extraction with cyclohexane by heating under reflux

3623 7) GC-MSMS

2) Internal standardisation with labelled substances

C13 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

3624 2) HPLC-UV-FLD

1) External calibration

2) LiquidLiquid partitioning

D) Other hot solvent extraction

3626

64

Table 19 - continued

Laboratory code

Analysis technique

Instrument calibration

Internal standards

applied

Sample clean up

Details of sample

clean up

Extraction technique

Details of sample

extraction

5301 7) GC-MSMS

2) Internal standardisation with labelled substances

EACH SUBSTANCE C13

4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5302 7) GC-MSMS

2) Internal standardisation with labelled substances

Isotope labelled PAHs 13C for each analyte

4) Solid phase extraction

C) Soxhlet extraction

5303 7) GC-MSMS

3) Internal standardisation with unlabelled substances

HAP C13 4) Solid phase extraction

D) Other automate ASE200 DIONEX

5304 7) GC-MSMS

2) Internal standardisation with labelled substances

with C13 for each HAP

2) LiquidLiquid partitioning 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5305 7) GC-MSMS

2) Internal standardisation with labelled substances

C13 isotopiques internal standards

4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5306 7) GC-MSMS

2) Internal standardisation with labelled substances

C13 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5307 6) GC-MS

2) Internal standardisation with labelled substances

1) Saponification 2) LiquidLiquid partitioning

D) Other solvent extraction

5308 1) HPLC-FLD 1) External calibration

4) Solid phase extraction

B) Sonication

5309 1) HPLC-FLD 1) External calibration

4) Solid phase extraction

D) Other only SPE

5310 1) HPLC-FLD 1) External calibration

1) Saponification 2) LiquidLiquid partitioning 3) Gel permeation chromatography

C) Soxhlet extraction

5311 6) GC-MS

2) Internal standardisation with labelled substances

13 C Chrysene 13 C Benz(a)pyrene

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

B) Sonication

5312 1) HPLC-FLD

3) Internal standardisation with unlabelled substances

Benzo(b)chrysen

2) LiquidLiquid partitioning 4) Solid phase extraction

Quechers D) Other Quechers

65

Table 19 - continued

Laboratory code

Analysis technique

Instrument calibration

Internal standards applied

Sample clean up Details of sample

clean up

Extraction technique

Details of sample extraction

5313 6) GC-MS

2) Internal standardisation with labelled substances

Benz(a)anthracene D12 Benzo(b)fluranthene D12 Benzo(a)pyrene D12 Chrysene D12

1) Saponification 2) LiquidLiquid partitioning 3) Gel permeation chromatography

A) Pressurised liquid extraction (PLE)

5314 6) GC-MS

2) Internal standardisation with labelled substances

Benzo(a)pyren d12 2) LiquidLiquid partitioning

D) Other Extraction with

5315 1) HPLC-FLD

3) Internal standardisation with unlabelled substances

Benz(b)chrysen

1) Saponification 2) LiquidLiquid partitioning

D) Other shaker

5316 1) HPLC-FLD

1) External calibration

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

D) Other

LiquidLiquid extraction with cyclohexane and dimethylformamide

5317 7) GC-MSMS

1) External calibration

3) Gel permeation chromatography 6) Other

silica gel D) Other VDLUFA online method

5318

5319 1) HPLC-FLD

3) Internal standardisation with unlabelled substances

Benzo(b)chrysene 3) Gel permeation chromatography

A) Pressurised liquid extraction (PLE)

5320 6) GC-MS

3) Internal standardisation with unlabelled substances

22-binaphthyl

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

D) Other 3 hours refluxing

5321 1) HPLC-FLD

2) Internal standardisation with labelled substances

6-meacutethylchrysegravene

1) Saponification 2) LiquidLiquid partitioning

B) Sonication

5322 6) GC-MS

2) Internal standardisation with labelled substances

naftalene-d8 acenaphtalene-d10 phenanthrene-d10 chrysene-d12 perylene-d12 benzo(e)anthracene-d12

1) Saponification 2) LiquidLiquid partitioning 6) Other

clean-up with silica packed column

C) Soxhlet extraction

5323 1) HPLC-FLD

1) External calibration

4) Solid phase extraction 6) Other

Accelerated Solvent Extraction

A) Pressurised liquid extraction (PLE)

66

European Commission EUR 25245 EN ndash Joint Research Centre ndash Institute for Reference Materials and Measurements Title Report on the 8th inter-laboratory comparison organised by the European Union Reference Laboratory for Polycyclic Aromatic Hydrocarbons - Four marker PAHs in a dry extract of St Johns wort Author(s) Radoslav Lizak Szilard Szilagyi Philippe Verlinde and Thomas WENZL Luxembourg Publications Office of the European Union 2012 ndash 66 pp ndash 201 x 297 cm EUR ndash Scientific and Technical Research series ndash ISSN 1831-9424 ISBN 978-92-79-23337-1 doi 10278759387 Abstract The proficiency test here reported concerned the determination of the contents of four marker polycyclic aromatic hydrocarbons (PAHs) and of their sum in an food supplements test sample The set of marker PAHs consists of benz[a]anthacene benzo[a]pyrene benzo[b]fluoranthene and chrysene Participants to these PT were National Reference Laboratories for PAHs (NRLs-PAHs) and EU official food control laboratories The number of participants was in total 48

The PT was organised according ISO Standard 170432010

The test material used was a commercial dry extract of St Johns wort Participants also received a solution of the same PAHs either in acetonitrile or in toluene for checking their instrument calibration

The results from participants were rated with z-scores and zeta-scores About 68 of the reported results were attributed with z-scores with an absolute value of below two which is the threshold for satisfactory performance

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The mission of the JRC is to provide customer-driven scientific and technical support

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science and technology for the Union Close to the policy-making process it serves

the common interest of the Member States while being independent of special

interests whether private or national

L

A-N

A-252

45-EN

-N

• 1 Summary
• 2 Introduction
• 3 Scope
• 4 Participating Laboratories
• 5 Time frame
• 6 Confidentiality
• 7 Test materials
• • 71 1Preparation of test materials
  • 72 Assigned values and standard deviation for proficiency assessment
  • 73 Homogeneity and stability
  • 74 Sample dispatch
  • • 8 Design of the proficiency test
    • 9 Evaluation of the results
    • • 91 2General
      • 92 21BEvaluation criteria
      • 93 Performance of participants
      • 94 Evaluation of the influence of analysis method
      • 95 Evaluation of the influence of calibration
      • 96 26Evaluation of compliance with legislation
      • • 10 Follow-up actions for underperforming laboratories
        • 11 1Conclusions
        • 12 1Acknowledgements
        • 13 References

18

Table 7 Compilation of z-scores calculated from the ldquoresults for proficiency assessment

reported by the OCLs for food supplement test material z-scores outside the

satisfactory range (|z| gt 2) are indicated by red font Values are reproduced as reported

by the laboratories Empty cells denote analytes for which results were not reported

Measurand benz[a]anthracene benzo[a]pyrene benzo[b]fluoranthene chrysene Sum PAH 4

Assigned value

microgkg 298 165 292 407 1162

Standard deviation for proficiency assessment

microgkg 062 036 060 083 125

Laboratory code

Result z-score Result z-

score Result z-score Result

z-score

Result z-score

microgkg microgkg microgkg microgkg microgkg

5301 31 02 18 04 29 00 49 10 126 08

5302 316 03 188 06 298 01 679 33 37 -63

5303 2876 -02 1535 -03 2586 -06 6441 29 13438 15

5304 261 -06 146 -05 253 -06 591 22 1251 07

5305 24 -09 16 -01 24 -09 61 25 125 07

5306 22 -13 16 -01 42 21 49 10 129 10

5307 461 27 157 -02 221 -12 524 14 1363 16

5308 16446 -22 107009 -16 229848 -10 213243 -23 72456 -35

5309 17 -21 11 -15 2 -15

5310 305 01 123 -12 313 03 386 -03 1127 -03

5311 459 26 2 10 281 -02 437 04 1367 16

5312 543 40 248 23 299 01 953 66 2042 71

5313 353 09 207 12 328 06 838 52 1726 45

5314 394 16 172 02 404 19 588 22 1558 32

5315 545 40 238 20 444 25 542 16 177 49

5316 235 -10 17 01 273 -03 282 -15 96 -16

5317 222 -12 269 29 192 -17 545 17 1229 05

5318 067 -37 117 -13 279 -02 357 -06 82 -27

5319 147 -24 216 14 396 17 465 07 1225 05

5320 534 38 316 42 625 55 834 52 2309 92

5321 12 -12

5322 87 92 17 01 33 06 7 35 207 73

5323 33 05 2 10 45 26 73 39 171 44

19

Table 8 and Table 9 present the respective zeta-scores As for the z-scores data outside the

satisfactory performance range are given in red bold font Zeta scores were only calculated for

laboratories that provided measurement uncertainty data as well as a numerical value for the coverage

factor Zeta scores were not calculated when the reported uncertainty value exceeded the content value

which is probably linked to mixing of reporting units (microgkg and ) However the participants are

reminded that the measurement uncertainty shall be expressed in the same unit as the content value

The assessment of the performance of the participants based on the reported measurement uncertainty

gave a less favourable picture compared to z-scores Only 60 of the zeta-scores are within the

satisfactory performance range The distribution of satisfactory performance ratings between NRLs

and OCLs is not balanced It was higher for the NRLs

It has to be noted that the magnitude of the zeta-scores were for some participants much higher than

the z-scores attributed to the same results Consequently the laboratories perform according to fitness-

for-purpose criterion specified in legislation which forms the basis for the z-scores but seem to have

difficulties in deducing realistic measurement uncertainty values Eg the relative standard

measurement uncertainties reported by the NRLs ranged from 25 to 20 with a median value of

about 10 Only two OCLs reported measurement uncertainties that exceeded the maximum tolerable

uncertainty (Uf) The respective data is highlighted in yellow in Table 9

However the EU-RL PAHs will continue to pay special attention to measurement uncertainty in future

ILCs as it has major implications on the assessment of compliance of food with European legislation

20

Table 8 Compilation of zeta-scores calculated from the ldquoresults for proficiency assessment

reported by the NRLs for test material St Johns wort the reported measurement

uncertainty and the uncertainty of the analyte content of the test material zeta-scores

outside the satisfactory range (|zeta| gt 2) are indicated by red bold font Empty cells

denote analytes for which either results or measurement uncertainties were not

reported

21

Table 9 Compilation of zeta-scores calculated from the ldquoresults for proficiency assessment

reported by the OCLs for test material St Johns wort the reported measurement

uncertainty and the uncertainty of the analyte content of the test material zeta-scores

outside the satisfactory range (|zeta| gt 2) are indicated by red bold font yellow

highlighted cells ugtUf Empty cells denote analytes for which results were not

reported

22

94 Evaluation of the influence of analysis method

The information on the applied analysis method (ANNEX 11) was used to group the performance

indicators attributed to the reported results according to the applied analysis technique Two groups of

data were formed They represent results obtained by high performance liquid chromatography-

fluorescence detection (HPLC-FD about 40 ) and by gas chromatography-mass spectrometry (GC-

MS about 60 ) respectively Kernel density plots were constructed based on the respective z-scores

Figure 3A to 3E show the different Kernel density plots

Figure 3 Kernel density plots of z-scores attributed to the results for proficiency assessment

which were reported for the food supplements test sample Results were classified

according to the applied analysis technique

A) benz[a]anthracene

B) benzo[a]pyrene

23

Figure 3 continued

C) benzo[b]fluoranthene

D) chrysene

E) sum of four marker PAHs

The kernel density plots for BAP and BBF are almost identical and centred at or close to a z-score

value of zero as can be seen in Figure 3B and 3C Therefore bias introduced by the type of analysis

method is unlikely for these two analytes The situation is different for BAA and CHR In case of

BAA the kernel density distribution of z-scores based on HPLC-FD is shifted to negative values

24

(Figure 3A) which indicates a tendency to underestimate the BAA content in this particular

samplematrix when applying HPLC-FD This might be reasoned by separationintegration problems

between BAA and CHR peaks A few participants (from the HPLC-FD subgroup) that got negative

z-scores for BAA overestimated the CHR content This caused the slight shift of the density trace for

HPLC-FD towards negative values Removing this data from the evaluation would result in a density

trace with the mode at zero

The opposite is the case for CHR (Figure 3D) The density distribution of the z-scores attributed to

HPLC-FD data is centred almost at zero However the distribution for data based on GC-MS is shifted

to positive values indicating strong positive bias This bias might be caused by a lack of selectivity of

the analysis method The test material contains triphenylene which is isobaric to chrysene

Consequently it cannot be distinguished from chrysene by means of mass spectrometry These two

compounds have to be separated chromatographically which is not easy They coelute on unpolar and

on most mid-polar capillary columns Columns that allow their separation were commercialised only

recently and might not be available in all laboratories Therefore laboratories which determine the

four EU marker PAHs by means of GC-MS shall pay attention to the chromatographic separation of

triphenylene and chrysene A lack of chromatographic resolution might lead to strong positive bias of

GC-MS methods and to false conclusions on the marketability of the tested sample

The chromatographic separation of chrysene and triphenylene is not a problem with HPLC which is

reflected in the position of the density plot for chrysene However attention has to be paid in HPLC-

FD analysis to a proper clean up of the sample extract in order to eliminate interferences that show

similar retention as BAA and CHR

95 Evaluation of the influence of calibration

The participants received together with the food supplement test sample two standard solutions in

depending of their choice either acetonitrile or toluene The analyte content of one solution was

disclosed to the participants whereas the content of the other solution was kept undisclosed The

content values of the latter are presented in Table 5 Analysis results for the solution with undisclosed

content had to be reported to the PT organiser The respective data are presented in Annex 9

Percent deviations of the reported results from the assigned values were calculated for both the food

supplement test sample and the standard solution with undisclosed analyte content Youden plots were

prepared in order to visualize the influence of instrument calibration on the results reported for the

food supplement sample Two plots are discussed in the following The whole set of Youden plots are

given in Annex 9

25

Figure 4 Youden plot of the percent deviations of values reported for benzo[a]pyrene from the

assigned values of the food supplement test material and the standard solution in

toluene with undisclosed content

Figure 4 reveals that a number of participants reported results with similar percent deviations from the

assigned values of both the results for the food supplement material and the standard solution in

toluene with undisclosed content This correlation indicates that over- respectively underestimation of

the analyte content of the food supplement material might be caused by biased instrument calibration

This finding underpins the importance of thorough calibration standard preparation However the data

was not in all cases conclusive as some laboratories performed well for the food supplement samples

but reported biased results for the standard solution with undisclosed content (eg Figure 4 participants

3620 and 5305) This fact cannot be explained yet

Figure 5 presents the respective Youden plot for chrysene in the food supplement test material and the

standard solution in toluene with undisclosed content From this graph it can be concluded that the

overestimation of the chrysene content of the food supplement material was not caused by instrument

calibration The results for the solvent solution of many laboratories which underperformed for the

26

food supplement sample agreed well with the assigned value This supports the conclusion that the

overestimation is caused by the determination of an interferent most probably triphenylene

Figure 5 Youden plot of the percent deviations of values reported for chrysene from the

assigned values of the food supplement test material and the standard solution in

toluene with undisclosed content

96 Evaluation of compliance with legislation

The method performance data reported for the four marker PAHs were evaluated for compliance with

the provisions given in Commission Regulation (EC) No 8362011 Table 18 in ANNEX 10 gives an

overview on the results of the evaluation Data were not reported in case of empty cells Non-

compliant data are highlighted in Table 18 It should be noted that Regulation (EU) No 8362011

specifies LOD and LOQ with two decimals This was not respected by all participants which

hampered the compliance assessment

27

In summary it can be stated that the majority of participants reported method performance

characteristics for the determination of the four marker PAHs in the food supplement test sample that

are compliant with legislation However some laboratories need to improve their analysis methods

The laboratories in question are urged to adapt to the provisions given in the new legislation

10 Follow-up actions for underperforming laboratories

All laboratories that got questionable or non-satisfactory performance ratings are urged to perform

root cause analysis and to implement corrective actions

As agreed during the EU-RL PAH workshop 2011 the EU-RL PAH will set up follow-up measures in

due time for all NRLs that received for at least one of the four PAHs (BAA BAP BBF and CHR) z-

scores gt 3 as required by Regulation (EC) 8822004 and by the Protocol for management of

underperformance in comparative testing andor lack of collaboration of National Reference

Laboratories (NRLs) with European Union reference laboratories (EU-RLs) activities

These laboratories shall perform as an immediate action root-cause-analysis and shall report to the

EU-RL PAH in writing the identified cause for their underperformance and corrective actions they are

going to take Additionally they shall participate to an independent (non-EU-RL) proficiency test on

the determination of PAHs in food and shall communicate the outcome of this exercise to the EU-RL

PAH

28

11 Conclusions

Fourty seven participants reported results back to the EU-RL The performance of the majority of

participants was good In total 153 out of 225 attributed z-scores were below an absolute value of two

which equals to almost 68 However this percentage is much below the percentages of satisfactory

performance gained in previous studies This might be the consequence of the higher level of difficulty

with respect to matrix composition In addition bias can be concluded from the pattern of performance

indicators of some laboratories

In general the determination of chrysene caused most difficulties to the participants

zeta-Scores were calculated besides z-scores They indicate the agreement of the reported result with

the assigned value with respect to the stated measurement uncertainty The outcome of this rating was

worse than for the z-scores which indicates that the measurement uncertainty estimates were not

realistic Therefore participants underperforming with regard to zeta scores are urged to adapt their

measurement uncertainty statements

The great majority of participants in this inter-laboratory comparison applied analytical methods which

complied with EU legislation with regard to performance characteristics

12 Acknowledgements

The organisers would like to thank Mrs Beatriz de la Calle and Mr Franz Ulberth (all from IRMM

Geel Belgium) for their accurate revision of this report Mr Hakan Emteborg (from IRMM) for

ampouling of the standard solutions and all NRLs and OCLs for their cooperation

29

13 References

1 EU COMMISSION REGULATION (EC) No 7762006 of 23 May 2006 amending Annex VII to Regulation

(EC) No 8822004 of the European Parliament and of the Council as regards Community reference

laboratories Official Journal of the European Union 2006 L 136 p 3-8

Available from

httpeur-lexeuropaeuLexUriServLexUriServdouri=OJL200613600030008ENPDF

2 EU Regulation (EC) No 8822004 of the European Parliament and of the Council of 29 April 2004 on

official controls performed to ensure the verification of compliance with feed and food law animal health

and animal welfare rules Official Journal of the European Communities 2004 L191 p 1-52 Available

from

httpeur-lexeuropaeuLexUriServLexUriServdouri=OJL200419100010052ENPDF

3 EU Opinion of the Scientific Committee on Food on the risks to human health of Polycyclic Aromatic

Hydrocarbons in food (2002) Available from

httpeuropaeuintcommfoodfsscscfout153_enpdf

4 International Agency for Research on Cancer IARC Monographs on the Evaluation of

Carcinogenic Risks to Humans Some Non-heterocyclic Polycyclic Aromatic Hydrocarbons and Some

Related Exposures Volume 92 (2010) Available from

httpmonographsiarcfrENGMonographsvol92mono92pdf

5 EU Commission Regulation (EC) No 3332007 of 28 March 2007 laying down the methods of sampling

and analysis for the official control of the levels of lead cadmium mercury inorganic tin 3-MCPD and

benzo(a)pyrene in foodstuffs Official Journal of the European Union 2007 L 88 p 29-38 Available

from

httpeur-lexeuropaeuLexUriServLexUriServdouri=OJL200708800290038ENPDF

6 EU Commission Regulation (EC) No 18812006 of 19 December 2006 setting maximum levels for certain

contaminants in foodstuffs Official Journal of the European Union 2006 L 364 p 5-24 Available from

httpeur-lexeuropaeuLexUriServLexUriServdouri=OJL200636400050024ENPDF

7 EFSA Scientific Opinion of the Panel on Contaminants in the Food Chain on a request from the European

Commission on Polycyclic Aromatic Hydrocarbons in Food The EFSA Journal 2008 724 p 1-114

Available from

httpwwwefsaeuropaeuEFSAefsa_locale-1178620753812_1211902034842htm

30

8 EU Commission Regulation (EU) No 8352011 amending Regulation (EC) No 18812006 as regards

maximum levels for polycyclic aromatic hydrocarbons in foodstuffs Official Journal of the European

Union 2011 L 215 p 4-8

httpeur-lexeuropaeuLexUriServLexUriServdouri=OJL201121500040008ENPDF

9 EU Commission Regulation (EU) No 8362011 amending Regulation (EC) No 3332007 laying down the methods of sampling and analysis for the official control of the levels of lead cadmium mercury inorganic tin 3-MCPD and benzo(a)pyrene in foodstuffs Official Journal of the European Union 2011 L 215 p 9-16 httpeur-lexeuropaeuLexUriServLexUriServdouri=OJL201121500090016ENPDF

10 ISOIEC 170432010 (2010) Conformity assessment -- General requirements for proficiency testing

providers Geneva Switzerland

11 Thompson M Ellison SLR and Wood R The International Harmonized Protocol for the Proficiency

Testing of Analytical Chemistry Laboratories Pure Appl Chem (2006) 78(1) p 145ndash196

Available from httpmediaiupacorgpublicationspac2006pdf7801x0145pdf

31

14 ANNEXES

ANNEX 1 ndash Announcement of the PT on the IRMM webpage

ANNEX 2 ndash Announcement via e-mail and invitation

ANNEX 3 ndash Announcement of sample dispatch

ANNEX 4 ndash Technical specifications of the calibration solutions

ANNEX 5 ndash Homogeneity and stability of the test material

ANNEX 6 ndash Acknowledgement of sample receipt

ANNEX 7 ndash Questionnaire on details of analysis method

ANNEX 8 ndash Results reported for the food supplement sample

ANNEX 9 ndash Results reported for the standard solutions with undisclosed analyte content

ANNEX 10 ndash Method performance characteristics reported by participants

ANNEX 11 ndash Details of analysis methods reported by participants

32

ANNEX 1 ndash Announcement of the PT on the IRMM webpage

33

ANNEX 2 - Announcement via e-mail and invitation

34

ANNEX 3 - Announcement sample dispatch

Dear MadameSir

The test samples for the proficiency test on the determination of four PAHs in a food supplement (dry extract of St Johns wort) were

dispatched today

Explanation on the design of the study as well as instructions for sample storage and for reporting of results are in the parcel You will

also find your participant key in the parcel which is required for reporting of results

Please check the parcel after arrival for completeness and report the receipt of the test samples as well as their status to us by applying

the sample receipt form which is in the parcel

You are kindly requested to inform us by email if you do not receive the parcel by end of this week

My colleague Donata Lerda will then take care of it

With best regards

Thomas Wenzl

35

ANNEX 4 - Specifications of standard solutions with disclosed concentration

36

ANNEX 4 - continued

37

ANNEX 5 - Homogeneity and stability of the test material

Homogeneity of the test material

Benz[a]anthracene - BAA

Benzo[a]pyrene - BAP

38

ANNEX 5 - continued

Benzo[b]fluoranthene - BBF

Chrysene - CHR

39

ANNEX 5 - continued

Stability of the test material

Table 10 Content of the four target analytes in test materials stored under different

temperatures

BaA BaP BbF CHR

microgkg microgkg microgkg microgkg

Content 31 16 31 69 Storage at room temperature

U (k=2) 05 02 05 17

Content 31 15 29 68 Storage at reference temperature

U (k=2) 04 03 04 10

The applied capillary column does not separate CHR from triphenylene

40

ANNEX 6 ndash Documents sent to participants

41

ANNEX 6 - continued

42

ANNEX 7 Questionnaire on details of analysis method

43

ANNEX 7 - continued

44

ANNEX 7 - continued

45

ANNEX 8 - Results reported by participants for the food supplements test sample Table 11 Analysis results reported by the participants for the content of benz[a]anthracene (BAA) in the food supplements test material Values are presented as reported

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

3600 31 29 29 3 05 2

3602 199 193 178 19 021

3603 253 253 246 251 022 2

3604 199 209 23 213 071 2

3605 301 3 271 291 076 2

3606 302 288 278 289 064 2

3607 456 416 443 438 096 2

3608 796 695 651 714 149 2

3610 182 192 214 196 039 2

3611 414 504 488 469 96 2

3612 3 34 28 31 06 2

3613 32 33 34 33 07 2

3614 182 182 191 185 037 2

3615 3884 3822 3565 376 0752 2

3616 2 2 2 2 01 2

3617 366 37 35 362 046 2

3618 32 32 32 32 05 2

3619 299 305 297 299 048 2

3620 304 294 302 302 045 2

3621 29 26 29 28 04 2

3622 343 325 318 322 052 2

3623 35 3 33 33 07 2

3624

3626 318 31 314 314 126 2

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

5301 31 3 32 31 06 2

5302 314 319 315 316 063 2

5303 3102 2716 2822 2876 27 62

5304 261 263 26 261 57 2

5305 23 22 26 24 20

5306 22 23 23 22 11 2

5307 488 461 317 461

5308 168633 168544 1562025 1644598

5309 16 19 16 17

5310 3 304 311 305 015

5311 477 452 448 459 15 2

5312 506 63 494 543 134 2

5313 353 301 404 353

5314 431 373 378 394 5 096

5315 596 531 51 545 11 2

5316 224 231 235 235 022 2

5317 216 23 221 222 007 2

5318 064 07 067 01 2

5319 111 184 146 147 029 2

5320 515 525 564 534 48

5321 21 5322 13 87 9 87 35 2

5323 38 33 35 33 16 2

46

Figure 6 Results of replicate determinations (indicated by triangles) of benz[a]anthracene (BAA) in the food supplement test material

Horizontal blue lines represent the arithmetic mean value of replicate measurements and blue bars the reported expanded measurement uncertainty (k=2) The assigned value is

plotted as dotted line Red solid lines mark the range of satisfactory performance (|z|le2) Mean values are given for results outside the displayed data range

ProLab 2011

Laboratory

53

1853

1953

0853

0936

1436

0236

1036

1653

2136

0453

1753

0653

1653

0536

0353

0436

2153

0336

0636

0536

0036

2036

1953

1036

1253

0136

2653

0236

1836

2336

2236

1353

1353

2336

1736

1553

1453

0736

0753

1153

2053

1253

1536

0853

2236

11

an

aly

te c

on

ten

t (micro

gk

g)

10

95

9

85

8

75

7

65

6

55

5

45

4

35

3

25

2

15

1

05

0

102

3 468

7

Sample St Johns wort extract replicatesMeasurand Benz[a]anthraceneNo of laboratories 46

Assigned value 298 microgkg (Reference value)Target sd 062 microgkgTolerance limits 174 - 422 microgkg (|Z score| lt 200)

Assig

ned va

lue

Limit of tolerance

Limit of tolerance

47

Table 12 Analysis results reported by the participants for the content of benzo[a]pyrene (BAP) in the food supplements test material Values are presented as reported

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

3600 16 17 17 17 03 2

3602 118 123 13 124 015

3603 165 162 157 161 015 2

3604 134 154 139 143 04 2

3605 164 166 172 167 057 2

3606 181 159 161 167 031 2

3607 279 266 279 274 055 2

3608 57 687 461 573 226 2

3610 226 216 183 206 042 2

3611 289 325 311 308 36 2

3612 24 22 2 22 05 2

3613 17 18 19 18 05 2

3614 08 08 08 08 016 2

3615 1545 1393 1425 145 029 2

3616 16 16 14 15 03 2

3617 177 171 158 169 023 2

3618 2 2 2 2 02 2

3619 207 207 206 207 075 2

3620 165 169 163 166 017 2

3621 44 47 49 47 05 2

3622 212 208 222 213 038 2

3623 2 21 22 21 03 2

3624 18 178 182 18 031 2

3626 172 173 178 174 07 2

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

5301 18 18 18 18 04 2

5302 188 19 187 188 038 2

5303 1691 1426 149 1535 19 80

5304 145 142 15 146 69 2

5305 15 17 18 16 15

5306 16 17 17 16 08 2

5307 164 157 153 157

5308 102669 109324 109035 107009

5309 1 1 12 11

5310 122 122 124 123 006

5311 199 184 215 2 15 2

5312 258 248 239 248 05 2

5313 199 178 245 207

5314 174 171 172 172 11 0948

5315 253 248 213 238 05 2

5316 177 171 173 17 016 2

5317 255 277 276 269 012 2

5318 114 12 117 02 2

5319 19 228 231 216 043 2

5320 303 3 344 316 78

5321 12 12 12 12

5322 13 17 24 17 35 2

5323 2 2 19 2 1 2

48

Figure 7 Results of replicate determinations (indicated by triangles) of benzo[a]pyrene (BAP) in the food supplement test material

Horizontal blue lines represent the arithmetic mean value of replicate measurements and blue bars the reported expanded measurement uncertainty (k=2) The assigned value is

plotted as dotted line Red solid lines mark the range of satisfactory performance (|z|le2) Mean values are given for results outside the displayed data range

ProLab 2011

Laboratory

36

1453

0953

0853

1853

2153

1036

0236

0436

1553

0436

1653

0353

0736

0336

2036

0053

0553

0636

0636

0536

1753

1453

1636

2636

1336

2453

0153

2253

0253

2353

1136

1836

1953

1336

1036

2336

2253

1936

1253

1553

1253

1736

0753

2036

2136

0836

11

an

aly

te c

on

ten

t (micro

gk

g)

55

5

45

4

35

3

25

2

15

1

05

0

573

308

3

Sample St Johns wort extract replicatesMeasurand Benzo[a]pyreneNo of laboratories 48

Assigned value 165 microgkg (Reference value)Target sd 036 microgkgTolerance limits 093 - 237 microgkg (|Z score| lt 200)

Assig

ned va

lue

Limit of tolerance

Limit of tolerance

49

Table 13 Analysis results reported by the participants for the content of benzo[b]fluoranthene (BBF) in the food supplements test material Values are presented as reported

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

3600 32 3 3 31 09 2

3602 173 175 16 17 017

3603 265 252 25 256 025 2

3604 116 108 105 11 028 2

3605 286 225 232 247 074 2

3606 294 273 261 276 046 2

3607 579 579 544 567 113 2

3608 103 1201 876 1036 326 2

3610 173 196 204 191 038 2

3611 546 618 565 576 75 2

3612 3 28 28 29 06 2

3613 28 31 32 31 06 2

3614 135 111 135 127 022 2

3615 4187 3678 4111 4 08 2

3616 2 2 2 2 01 2

3617 33 27 299 035 2

3618 31 31 31 31 05 2

3619 326 324 312 326 064 2

3620 277 28 272 276 069 2

3621 26 25 23 25 04 2

3622 346 336 333 34 052 2

3623 26 28 29 28 02 2

3624

3626 288 28 307 292 117 2

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

5301 29 3 29 29 06 2

5302 297 3 296 298 06 2

5303 2705 2444 2608 2586 22 78

5304 245 254 26 253 85 2

5305 23 23 26 24 15

5306 42 43 4 42 21 2

5307 221 216 241 221

5308 232169 246654 240722 229848

5309 19 22 19 2 0

5310 3 315 325 313 016

5311 262 277 304 281 15 2

5312 27 289 337 299 075 2

5313 321 284 378 328

5314 423 404 385 404 34 083

5315 42 506 416 444 09 2

5316 265 267 277 273 018 2

5317 185 197 195 192 006 2

5318 276 281 279 04 2

5319 396 389 403 396 079 2

5320 623 618 635 625 14

5321 31

5322 24 33 49 33 35 2

5323 38 45 35 45 27 2

50

Figure 8 Results of replicate determinations (indicated by triangles) of benzo[b]fluoranthene (BBF) in the food supplement test material

Horizontal blue lines represent the arithmetic mean value of replicate measurements and blue bars the reported expanded measurement uncertainty (k=2) The assigned value is

plotted as dotted line Red solid lines mark the range of satisfactory performance (|z|le2) Mean values are given for results outside the displayed data range

ProLab 2011

Laboratory

36

0436

1436

0236

1053

1736

1653

0953

0753

0853

0536

2136

0553

0436

0353

0353

1636

0636

2036

2353

1853

1136

1236

2653

0153

0253

1236

1736

1336

0036

1853

2153

1036

1953

1336

2253

2253

2353

1936

1553

1453

0653

1536

0753

2036

0836

11

an

aly

te c

on

ten

t (micro

gk

g)

75

7

65

6

55

5

45

4

35

3

25

2

15

1

05

0

103

6

576

3

Sample St Johns wort extract replicatesMeasurand Benzo[b]fluorantheneNo of laboratories 46

Assigned value 292 microgkg (Reference value)Target sd 060 microgkgTolerance limits 172 - 412 microgkg (|Z score| lt 200)

Assig

ned va

lue

Limit of tolerance

Limit of tolerance

51

Table 14 Analysis results reported by the participants for the content of chrysene (CHR) in the food supplements test material Values are presented as reported

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

3600 5 49 5 5 07 2

3602 32 304 29 304 036

3603 462 469 447 459 048 2

3604 438 428 42 429 119 2

3605 365 41 381 386 085 2

3606 494 411 45 451 124 2

3607 897 774 787 819 18 2

3608 986 887 1168 1014 284 2

3610 418 392 462 424 085 2

3611 833 893 87 865 61 2

3612 6 64 58 61 12 2

3613 41 39 4 4 08 2

3614 272 254 288 271 054 2

3615 605 6028 5964 601 12 2

3616 47 45 49 47 05 2

3617 445 431 389 422 065 2

3618 45 45 45 45 06 2

3619 738 747 72 738 118 2

3620 463 472 446 46 092 2

3621 53 49 51 51 06 2

3622 478 513 505 488 08 2

3623 73 73 72 72 05 2

3624

3626 63 622 634 628 251 2

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

5301 49 49 49 49 1 2

5302 687 683 666 679 136 2

5303 6777 6181 6382 6441 24 70

5304 592 581 6 591 85 2

5305 6 58 64 61 20

5306 49 5 51 49 25 2

5307 524 5 574 524

5308 211488 216782 221146 213243

5309

5310 372 393 394 386 019

5311 43 459 422 437 15 2

5312 934 943 981 953 191 2

5313 793 73 99 838

5314 636 561 565 588 18 0818

5315 547 583 499 542 11 2

5316 292 286 285 282 06 2

5317 52 598 517 545 046 2

5318 368 345 357 05 2

5319 449 471 474 465 116 2

5320 76 924 818 834 99

5321 33

5322 7 7 76 7 35 2

5323 87 73 77 73 37 2

52

Figure 9 Results of replicate determinations (indicated by triangles) of chrysene (CHR) in the food supplement test material

Horizontal blue lines represent the arithmetic mean value of replicate measurements and blue bars the reported expanded measurement uncertainty (k=2) The assigned value is

plotted as dotted line Red solid lines mark the range of satisfactory performance (|z|le2) Mean values are given for results outside the displayed data range

ProLab 2011

Laboratory

53

0836

1453

1636

0253

2153

1836

0553

1036

1336

1736

1036

0453

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0336

2053

1936

1653

0136

0036

2253

0636

2153

0753

1553

1753

1453

0436

1536

1253

0536

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0353

0253

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2336

1953

2336

0753

2053

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0836

11

an

aly

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ten

t (micro

gk

g)

13

12

11

10

9

8

7

6

5

4

3

2

1

0

865

3

Sample St Johns wort extract replicatesMeasurand ChryseneNo of laboratories 45

Assigned value 407 microgkg (Reference value)Target sd 083 microgkgTolerance limits 241 - 573 microgkg (|Z score| lt 200)

Assig

ned va

lue

Limit of tolerance

Limit of tolerance

53

Table 15 Analysis results reported by the participants for the sum of the four marker PAHs in the food supplements test material Values are presented as reported

Laboratory code

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg

3600 128 24 2

3602 79 126

3603 1127 096 2

3604 893 258 2

3605 1091 305 2

3606 1184 238 2

3607 2098 42 2

3608 3336 325 2

3610 1138 17 2

3611 2218 259 2

3612 143 29 2

3613 122 2 2

3614 663 129 2

3615 152 304 2

3616 102 03 2

3617 1252 169 2

3618 128 18 2

3619 157 305 2

3620 1204 12 2

3621 151 19 2

3622 1361 115 2

3623 154 09 2

3624

3626 1409 56 2

Laboratory code

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg

5301 126

5302 37 074 2

5303 13438 92

5304 1251 85 2

5305 125 20

5306 129 65 2

5307 1363

5308 7245598

5309

5310 1127 056

5311 1367 15 2

5312 2042 408 2

5313 1726

5314 1558

5315 177 35 2

5316 96 073 2

5317 1229 066 2

5318 82 05 2

5319 1225 267 2

5320 2309 41

5321

5322 207 35 2

5323 171 103 2

54

ANNEX 9 Results reported for the standard solutions with undisclosed analyte content Table 16 Analysis results reported by the participants for the four marker PAHs in the standard solution in toluene with undisclosed analyte

content Values are presented as reported U measurement uncertainty k coverage factor

benz[a]anthracene benzo[a]pyrene benzo[b]fluoranthene chrysene Laboratory

code Unit Result U k Result U k Result U k Result U k 3600 microgl 38 67 2 25 45 2 211 68 2 275 38 2

3605 microgl 367 184 2 2482 124 2 2592 13 2 235 118 2

3606 microgkg 3763 176 2 2609 142 2 2721 118 2 2539 134 2

3607 microgkg 3706 815 2 2824 565 2 2591 518 2 2587 569 2

3612 microgl 38 08 2 237 11 2 244 18 2 257 02 2

3614 microgl 324 65 2 209 38 2 215 37 2 21 42 2

3616 microgkg 318 07 2 224 14 2 231 26 2 216 1 2

3618 microgkg 417 17 2 284 08 2 292 06 2 297 03 2

3619 microgkg 47 742 2 274 468 2 228 387 2 274 434 2

3620 microgl 495 74 2 294 29 2 321 8 2 321 64 2

3621 microgl 241 22 2 143 13 2 188 16 2 145 14 2

3623 microgkg 476 25 2 311 53 2 269 3 2 297 03 2

3626 microgl 359 38 2 235 23 2 238 27 2 24 18 2

5301 microgkg 426 29 299 289

5302 microgkg 436 87 2 296 59 2 296 59 2 289 58 2

5303 microgl 3721 27 102 22838 19 99 23756 22 99 25486 24 98

5304 microgkg 38 29 2 225 26 2 257 37 2 258 52 2

5305 microgkg 305 225 245 29

5306 microgkg 316 63 2 247 49 2 65 13 2 22 44 2

5307 microgkg 3658 2559 241 2414

5308 microgkg 16854 109324 246654 216782

5311 microgl 374 20 2 279 15 2 268 15 2 272 15 2

5313 microgl 3408 2165 2275 2373

5314 microgkg 395 5 2875 11 3614 34 2905 18

5320 microgl 3267 368 1986 571 28 683 2427 512

5322 microgl 282 35 2 225 35 2 241 35 2 21 35 2

55

Figure 10 Youden plots of the percent deviation of the reported values from the assigned values for the food supplement test sample and the

standard solution in toluene with undisclosed content

(The identity of the respective measurand is given above the figure Results exceeding a difference of more than 100 were omitted)

56

Figure 10 - continued

57

Table 17 Analysis results reported by the participants for the four marker PAHs in the standard solution in acetonitrile with undisclosed

analyte content

Values are presented as reported U measurement uncertainty k coverage factor

benz[a]anthracene benzo[a]pyrene benzo[b]fluoranthene chrysene

Laboratory

code Unit Result U k Result U k Result U k Result U k

3602 microgkg 3675 247 247 244

3603 microgkg 358 086 2 236 028 2 235 028 2 236 057 2

3604 microgl 475 29 2 19 12 2 194 13 2 258 12 2

3608 microgl 3623 078 2 2385 023 2 2343 18 2 2296 092 2

3610 microgkg 369 185 2 25 125 2 251 126 2 244 122 2

3611 microgkg 48 02 2 323 02 2 328 04 2 317 03 2

3613 microgl 332 31 2 261 3 2 249 25 2 239 24 2

3615 microgl 415 2635 2729 2689

3617 microgkg 37 09 2 244 06 2 284 07 2 239 06 2

3622 microgl 37 12 2 25 23 2 257 21 2 238 1 2

3624 microgkg 402 104 2 255 084 2 265 096 2 253 07 2

5306 microgkg 563 113 2 371 74 2 359 72 2 333 67 2

5308 microgkg 168633 102669 232169 211488

5309 microgl 402 268 27

5310 microgl 315 158 25 125 25 125 288 144

5312 microgl 428 043 2 282 028 2 272 027 2 275 028 2

5315 microgkg 387 08 2 256 05 2 251 05 2 253 05 2

5316 microgl 3839 342 2 2485 191 2 2559 168 2 239 51 2

5317 microgl 2466 043 2 2212 034 2 2472 016 2 2146 068 2

5319 microgl 414 41 2 279 28 2 268 27 2 26 31 2

5321 microgkg 476 316 326 328

5322 microgl 2547 35 2

5323 microgkg 405 203 2 239 119 2 255 153 2 201 11 2

58

Figure 11 Youden plots of the percent deviation of the reported values from the assigned values for the food supplement test sample and the

standard solution in acetonitrile with undisclosed content

(The identity of the respective measurand is given above the figure Results exceeding a difference of more than 100 were omitted)

59

Figure 11 - continued

60

ANNEX 10 Table 18 Method performance parameters reported by the participants

Laboratory code

Analyte LOD LOQ Recovery Analyte LOD LOQ Recovery

microgkg microgkg microgkg microgkg 3600 BaA 03 06 978 BaP 03 07 9483602 BaA 007 021 926 BaP 005 015 993603 BaA 0011 05 987 BaP 0005 051 9553604 BaA 02 04 86 BaP 02 04 883605 BaA 0025 005 118 BaP 0025 005 1083606 BaA 001 003 65 BaP 001 003 703607 BaA 021 071 74 BaP 027 089 943608 BaA 016 048 8213 BaP 015 046 83683610 BaA 02 05 85 BaP 005 02 853611 BaA BaP 3612 BaA 1 117 BaP 1 1203613 BaA 01 03 90 BaP 01 03 903614 BaA 02 05 96 BaP 02 05 933615 BaA 005 015 110 BaP 005 015 1053616 BaA 01 05 108 BaP 01 05 753617 BaA 018 037 405 BaP 009 018 3873618 BaA 01 03 63 BaP 01 03 593619 BaA 005 005 59 BaP 033 033 543620 BaA 0009 0018 64 BaP 0005 001 553621 BaA 007 02 84 BaP 007 02 923622 BaA 01 03 1101 BaP 01 03 9853623 BaA 02 08 105 BaP 02 08 1013624 BaA BaP 01 033 8513625 BaA BaP 3626 BaA BaP 5301 BaA 003 01 102 BaP 003 01 1005302 BaA 01 03 78 BaP 01 03 865303 BaA 003 01 62 BaP 003 01 805304 BaA 1 1 70 BaP 1 1 955305 BaA 02 05 85 BaP 02 05 855306 BaA 03 1 699 BaP 03 1 905307 BaA 6537 BaP 81165308 BaA 0088 0265 99 BaP 00262 00785 1005309 BaA 01 08 92 BaP 01 08 925310 BaA 01 03 BaP 01 03 905311 BaA 002 005 80 BaP 002 004 915312 BaA 03 09 105 BaP 03 09 1055313 BaA 04 08 BaP 04 08 5314 BaA 001 002 96 BaP 001 002 9485315 BaA 01 03 50 - 100 BaP 01 03 50 - 100 5316 BaA 002 011 827 BaP 004 019 10445317 BaA 05 1 76 BaP 05 1 755318 BaA BaP 5319 BaA 009 029 951 BaP 011 041 995320 BaA 12 18 946 BaP 06 1 8345321 BaA 03 05 BaP 05 1 5322 BaA 1 2 100 BaP 1 2 1005323 BaA 26 BaP 26

61

ANNEX 10 Table 18 - continued

Laboratory code Analyte LOD LOQ Recovery Analyte LOD LOQ Recovery

microgkg microgkg microgkg microgkg 3600 BbF 03 05 96 CHR 02 03 10153602 BbF 015 045 947 CHR 003 009 9193603 BbF 0014 05 1012 CHR 0004 05 10033604 BbF 02 04 85 CHR 02 04 873605 BbF 005 01 94 CHR 0025 005 863606 BbF 001 003 70 CHR 001 003 653607 BbF 018 06 100 CHR 014 028 943608 BbF 015 046 7979 CHR 015 045 71253610 BbF 005 02 85 CHR 02 05 853611 BbF CHR 3612 BbF 1 118 CHR 1 1173613 BbF 01 03 90 CHR 01 03 903614 BbF 01 04 96 CHR 02 05 953615 BbF 008 025 109 CHR 003 01 1083616 BbF 01 05 78 CHR 01 05 723617 BbF 035 07 38 CHR 012 024 4163618 BbF 01 03 62 CHR 01 03 623619 BbF 016 016 63 CHR 01 01 593620 BbF 0003 0006 55 CHR 001 002 653621 BbF 007 02 103 CHR 007 02 893622 BbF 02 06 963 CHR 03 09 1043623 BbF 02 08 106 CHR 02 08 1193624 BbF CHR 3625 BbF CHR 3626 BbF CHR 5301 BbF 003 01 96 CHR 003 01 1145302 BbF 01 03 84 CHR 01 03 835303 BbF 003 01 78 CHR 003 01 705304 BbF 1 1 86 CHR 1 1 705305 BbF 02 05 90 CHR 02 05 955306 BbF 03 1 1435 CHR 03 1 1185307 BbF 8116 CHR 65375308 BbF 0079 0238 99 CHR 009 026 1005309 BbF 01 08 92 CHR 5310 BbF 07 2 CHR 01 03 5311 BbF 002 004 95 CHR 002 004 895312 BbF 03 09 105 CHR 03 09 1055313 BbF 04 08 CHR 04 08 5314 BbF 001 002 83 CHR 001 002 8185315 BbF 02 05 50 -100 CHR 02 05 50 - 100 5316 BbF 005 024 814 CHR 001 007 9595317 BbF 05 1 73 CHR 05 1 785318 BbF CHR 5319 BbF 011 038 946 CHR 013 043 9675320 BbF 07 09 1194 CHR 23 32 10455321 BbF 05 1 CHR 05 1 5322 BbF 1 2 100 CHR 1 2 1005323 BbF 26 CHR 26

62

ANNEX 11 Table 19 Details of analysis method reported by the participants Data are presented as reported

Laboratory code

Analysis technique

Instrument calibration

Internal standards applied

Sample clean up

Details of sample

clean up

Extraction technique

Details of sample

extraction

3600 6) GC-MS

2) Internal standardisation with labelled substances

Deuterated PAH mix 9 (Ehrenstorfer)

1) Saponification 2) LiquidLiquid partitioning

D) Other Liquidliquid partitioning

3602 2) HPLC-UV-FLD

1) External calibration

3) Gel permeation chromatography

A) Pressurised liquid extraction (PLE)

3603 1) HPLC-FLD

1) External calibration

3) Gel permeation chromatography

D) Other 1 minute with dichloromethane in vortex

3604 2) HPLC-UV-FLD

2) Internal standardisation with labelled substances

DiP D14

5) Donor acceptor complex chromatography

D) Other LL

3605 1) HPLC-FLD

1) External calibration

3) Gel permeation chromatography

C) Soxhlet extraction

3606 7) GC-MSMS

2) Internal standardisation with labelled substances

BaP 13C4 CHR 13C6 BaA 13C6 BbF 13C6

4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

3607 6) GC-MS

2) Internal standardisation with labelled substances

BaA-d12 CHR-d12 BbF-d12 BkF-d12 BaP-d12 ICP-d12 BgP-d12 DiP-d14

1) Saponification

D) Other solvent partitioning

3608 1) HPLC-FLD

1) External calibration

6) Other Liquid extraction

D) Other

Extraction in a rotary agitator and centrifugation

3610 2) HPLC-UV-FLD

1) External calibration

4) Solid phase extraction

B) Sonication

3611

3612 6) GC-MS

2) Internal standardisation with labelled substances

chrysene-d12 dibenzo(ah)anthracene-d14 dibenzo(ai)pyrene-d14

4) Solid phase extraction

B) Sonication

3613 5) LC-MSMS

3) Internal standardisation with unlabelled substances

I use Chrysene-D12 3) Gel permeation chromatography

D) Other extraction with hexan

63

Table 19 - continued

Laboratory code

Analysis technique

Instrument calibration

Internal standards applied

Sample clean up Details of sample

clean up

Extraction technique

Details of sample

extraction

3614 6) GC-MS 1) External calibration

3) Gel permeation chromatography

B) Sonication

3615 1) HPLC-FLD

1) External calibration

6) Other PLE GPC

A) Pressurised liquid extraction (PLE)

3616 6) GC-MS

2) Internal standardisation with labelled substances

Benzo(a)pyrene-13C4 Benzo(a)anthracene-13C6 Benzo(b)fluoranthene-13C6 Chrysene-13C6

2) LiquidLiquid partitioning 4) Solid phase extraction

B) Sonication

3617 1) HPLC-FLD

1) External calibration

4) Solid phase extraction

C) Soxhlet extraction

3618 6) GC-MS

2) Internal standardisation with labelled substances

deuterated standards

1) Saponification 4) Solid phase extraction

D) Other Handshaken with cyclohexane

3619 6) GC-MS

2) Internal standardisation with labelled substances

C13 Labelled US EPA 16

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

D) Other Homegenisation amp saponiofication

3620 9) GC-HRMS

2) Internal standardisation with labelled substances

mix of deuterated PAHs (BAA-D12 CHR-D12 BAP-D12 BBF-D12)

3) Gel permeation chromatography 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

3621 6) GC-MS

2) Internal standardisation with labelled substances

Chrysene D12 Benzo(a)pyrene D12 Benzo(b)fluoranthene D12 Benzo(a)anthracene

2) LiquidLiquid partitioning 4) Solid phase extraction

B) Sonication

3622 1) HPLC-FLD

4) Standard addition method

4) Solid phase extraction

D) Other

Extraction with cyclohexane by heating under reflux

3623 7) GC-MSMS

2) Internal standardisation with labelled substances

C13 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

3624 2) HPLC-UV-FLD

1) External calibration

2) LiquidLiquid partitioning

D) Other hot solvent extraction

3626

64

Table 19 - continued

Laboratory code

Analysis technique

Instrument calibration

Internal standards

applied

Sample clean up

Details of sample

clean up

Extraction technique

Details of sample

extraction

5301 7) GC-MSMS

2) Internal standardisation with labelled substances

EACH SUBSTANCE C13

4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5302 7) GC-MSMS

2) Internal standardisation with labelled substances

Isotope labelled PAHs 13C for each analyte

4) Solid phase extraction

C) Soxhlet extraction

5303 7) GC-MSMS

3) Internal standardisation with unlabelled substances

HAP C13 4) Solid phase extraction

D) Other automate ASE200 DIONEX

5304 7) GC-MSMS

2) Internal standardisation with labelled substances

with C13 for each HAP

2) LiquidLiquid partitioning 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5305 7) GC-MSMS

2) Internal standardisation with labelled substances

C13 isotopiques internal standards

4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5306 7) GC-MSMS

2) Internal standardisation with labelled substances

C13 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5307 6) GC-MS

2) Internal standardisation with labelled substances

1) Saponification 2) LiquidLiquid partitioning

D) Other solvent extraction

5308 1) HPLC-FLD 1) External calibration

4) Solid phase extraction

B) Sonication

5309 1) HPLC-FLD 1) External calibration

4) Solid phase extraction

D) Other only SPE

5310 1) HPLC-FLD 1) External calibration

1) Saponification 2) LiquidLiquid partitioning 3) Gel permeation chromatography

C) Soxhlet extraction

5311 6) GC-MS

2) Internal standardisation with labelled substances

13 C Chrysene 13 C Benz(a)pyrene

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

B) Sonication

5312 1) HPLC-FLD

3) Internal standardisation with unlabelled substances

Benzo(b)chrysen

2) LiquidLiquid partitioning 4) Solid phase extraction

Quechers D) Other Quechers

65

Table 19 - continued

Laboratory code

Analysis technique

Instrument calibration

Internal standards applied

Sample clean up Details of sample

clean up

Extraction technique

Details of sample extraction

5313 6) GC-MS

2) Internal standardisation with labelled substances

Benz(a)anthracene D12 Benzo(b)fluranthene D12 Benzo(a)pyrene D12 Chrysene D12

1) Saponification 2) LiquidLiquid partitioning 3) Gel permeation chromatography

A) Pressurised liquid extraction (PLE)

5314 6) GC-MS

2) Internal standardisation with labelled substances

Benzo(a)pyren d12 2) LiquidLiquid partitioning

D) Other Extraction with

5315 1) HPLC-FLD

3) Internal standardisation with unlabelled substances

Benz(b)chrysen

1) Saponification 2) LiquidLiquid partitioning

D) Other shaker

5316 1) HPLC-FLD

1) External calibration

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

D) Other

LiquidLiquid extraction with cyclohexane and dimethylformamide

5317 7) GC-MSMS

1) External calibration

3) Gel permeation chromatography 6) Other

silica gel D) Other VDLUFA online method

5318

5319 1) HPLC-FLD

3) Internal standardisation with unlabelled substances

Benzo(b)chrysene 3) Gel permeation chromatography

A) Pressurised liquid extraction (PLE)

5320 6) GC-MS

3) Internal standardisation with unlabelled substances

22-binaphthyl

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

D) Other 3 hours refluxing

5321 1) HPLC-FLD

2) Internal standardisation with labelled substances

6-meacutethylchrysegravene

1) Saponification 2) LiquidLiquid partitioning

B) Sonication

5322 6) GC-MS

2) Internal standardisation with labelled substances

naftalene-d8 acenaphtalene-d10 phenanthrene-d10 chrysene-d12 perylene-d12 benzo(e)anthracene-d12

1) Saponification 2) LiquidLiquid partitioning 6) Other

clean-up with silica packed column

C) Soxhlet extraction

5323 1) HPLC-FLD

1) External calibration

4) Solid phase extraction 6) Other

Accelerated Solvent Extraction

A) Pressurised liquid extraction (PLE)

66

European Commission EUR 25245 EN ndash Joint Research Centre ndash Institute for Reference Materials and Measurements Title Report on the 8th inter-laboratory comparison organised by the European Union Reference Laboratory for Polycyclic Aromatic Hydrocarbons - Four marker PAHs in a dry extract of St Johns wort Author(s) Radoslav Lizak Szilard Szilagyi Philippe Verlinde and Thomas WENZL Luxembourg Publications Office of the European Union 2012 ndash 66 pp ndash 201 x 297 cm EUR ndash Scientific and Technical Research series ndash ISSN 1831-9424 ISBN 978-92-79-23337-1 doi 10278759387 Abstract The proficiency test here reported concerned the determination of the contents of four marker polycyclic aromatic hydrocarbons (PAHs) and of their sum in an food supplements test sample The set of marker PAHs consists of benz[a]anthacene benzo[a]pyrene benzo[b]fluoranthene and chrysene Participants to these PT were National Reference Laboratories for PAHs (NRLs-PAHs) and EU official food control laboratories The number of participants was in total 48

The PT was organised according ISO Standard 170432010

The test material used was a commercial dry extract of St Johns wort Participants also received a solution of the same PAHs either in acetonitrile or in toluene for checking their instrument calibration

The results from participants were rated with z-scores and zeta-scores About 68 of the reported results were attributed with z-scores with an absolute value of below two which is the threshold for satisfactory performance

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interests whether private or national

L

A-N

A-252

45-EN

-N

• 1 Summary
• 2 Introduction
• 3 Scope
• 4 Participating Laboratories
• 5 Time frame
• 6 Confidentiality
• 7 Test materials
• • 71 1Preparation of test materials
  • 72 Assigned values and standard deviation for proficiency assessment
  • 73 Homogeneity and stability
  • 74 Sample dispatch
  • • 8 Design of the proficiency test
    • 9 Evaluation of the results
    • • 91 2General
      • 92 21BEvaluation criteria
      • 93 Performance of participants
      • 94 Evaluation of the influence of analysis method
      • 95 Evaluation of the influence of calibration
      • 96 26Evaluation of compliance with legislation
      • • 10 Follow-up actions for underperforming laboratories
        • 11 1Conclusions
        • 12 1Acknowledgements
        • 13 References

19

Table 8 and Table 9 present the respective zeta-scores As for the z-scores data outside the

satisfactory performance range are given in red bold font Zeta scores were only calculated for

laboratories that provided measurement uncertainty data as well as a numerical value for the coverage

factor Zeta scores were not calculated when the reported uncertainty value exceeded the content value

which is probably linked to mixing of reporting units (microgkg and ) However the participants are

reminded that the measurement uncertainty shall be expressed in the same unit as the content value

The assessment of the performance of the participants based on the reported measurement uncertainty

gave a less favourable picture compared to z-scores Only 60 of the zeta-scores are within the

satisfactory performance range The distribution of satisfactory performance ratings between NRLs

and OCLs is not balanced It was higher for the NRLs

It has to be noted that the magnitude of the zeta-scores were for some participants much higher than

the z-scores attributed to the same results Consequently the laboratories perform according to fitness-

for-purpose criterion specified in legislation which forms the basis for the z-scores but seem to have

difficulties in deducing realistic measurement uncertainty values Eg the relative standard

measurement uncertainties reported by the NRLs ranged from 25 to 20 with a median value of

about 10 Only two OCLs reported measurement uncertainties that exceeded the maximum tolerable

uncertainty (Uf) The respective data is highlighted in yellow in Table 9

However the EU-RL PAHs will continue to pay special attention to measurement uncertainty in future

ILCs as it has major implications on the assessment of compliance of food with European legislation

20

Table 8 Compilation of zeta-scores calculated from the ldquoresults for proficiency assessment

reported by the NRLs for test material St Johns wort the reported measurement

uncertainty and the uncertainty of the analyte content of the test material zeta-scores

outside the satisfactory range (|zeta| gt 2) are indicated by red bold font Empty cells

denote analytes for which either results or measurement uncertainties were not

reported

21

Table 9 Compilation of zeta-scores calculated from the ldquoresults for proficiency assessment

reported by the OCLs for test material St Johns wort the reported measurement

uncertainty and the uncertainty of the analyte content of the test material zeta-scores

outside the satisfactory range (|zeta| gt 2) are indicated by red bold font yellow

highlighted cells ugtUf Empty cells denote analytes for which results were not

reported

22

94 Evaluation of the influence of analysis method

The information on the applied analysis method (ANNEX 11) was used to group the performance

indicators attributed to the reported results according to the applied analysis technique Two groups of

data were formed They represent results obtained by high performance liquid chromatography-

fluorescence detection (HPLC-FD about 40 ) and by gas chromatography-mass spectrometry (GC-

MS about 60 ) respectively Kernel density plots were constructed based on the respective z-scores

Figure 3A to 3E show the different Kernel density plots

Figure 3 Kernel density plots of z-scores attributed to the results for proficiency assessment

which were reported for the food supplements test sample Results were classified

according to the applied analysis technique

A) benz[a]anthracene

B) benzo[a]pyrene

23

Figure 3 continued

C) benzo[b]fluoranthene

D) chrysene

E) sum of four marker PAHs

The kernel density plots for BAP and BBF are almost identical and centred at or close to a z-score

value of zero as can be seen in Figure 3B and 3C Therefore bias introduced by the type of analysis

method is unlikely for these two analytes The situation is different for BAA and CHR In case of

BAA the kernel density distribution of z-scores based on HPLC-FD is shifted to negative values

24

(Figure 3A) which indicates a tendency to underestimate the BAA content in this particular

samplematrix when applying HPLC-FD This might be reasoned by separationintegration problems

between BAA and CHR peaks A few participants (from the HPLC-FD subgroup) that got negative

z-scores for BAA overestimated the CHR content This caused the slight shift of the density trace for

HPLC-FD towards negative values Removing this data from the evaluation would result in a density

trace with the mode at zero

The opposite is the case for CHR (Figure 3D) The density distribution of the z-scores attributed to

HPLC-FD data is centred almost at zero However the distribution for data based on GC-MS is shifted

to positive values indicating strong positive bias This bias might be caused by a lack of selectivity of

the analysis method The test material contains triphenylene which is isobaric to chrysene

Consequently it cannot be distinguished from chrysene by means of mass spectrometry These two

compounds have to be separated chromatographically which is not easy They coelute on unpolar and

on most mid-polar capillary columns Columns that allow their separation were commercialised only

recently and might not be available in all laboratories Therefore laboratories which determine the

four EU marker PAHs by means of GC-MS shall pay attention to the chromatographic separation of

triphenylene and chrysene A lack of chromatographic resolution might lead to strong positive bias of

GC-MS methods and to false conclusions on the marketability of the tested sample

The chromatographic separation of chrysene and triphenylene is not a problem with HPLC which is

reflected in the position of the density plot for chrysene However attention has to be paid in HPLC-

FD analysis to a proper clean up of the sample extract in order to eliminate interferences that show

similar retention as BAA and CHR

95 Evaluation of the influence of calibration

The participants received together with the food supplement test sample two standard solutions in

depending of their choice either acetonitrile or toluene The analyte content of one solution was

disclosed to the participants whereas the content of the other solution was kept undisclosed The

content values of the latter are presented in Table 5 Analysis results for the solution with undisclosed

content had to be reported to the PT organiser The respective data are presented in Annex 9

Percent deviations of the reported results from the assigned values were calculated for both the food

supplement test sample and the standard solution with undisclosed analyte content Youden plots were

prepared in order to visualize the influence of instrument calibration on the results reported for the

food supplement sample Two plots are discussed in the following The whole set of Youden plots are

given in Annex 9

25

Figure 4 Youden plot of the percent deviations of values reported for benzo[a]pyrene from the

assigned values of the food supplement test material and the standard solution in

toluene with undisclosed content

Figure 4 reveals that a number of participants reported results with similar percent deviations from the

assigned values of both the results for the food supplement material and the standard solution in

toluene with undisclosed content This correlation indicates that over- respectively underestimation of

the analyte content of the food supplement material might be caused by biased instrument calibration

This finding underpins the importance of thorough calibration standard preparation However the data

was not in all cases conclusive as some laboratories performed well for the food supplement samples

but reported biased results for the standard solution with undisclosed content (eg Figure 4 participants

3620 and 5305) This fact cannot be explained yet

Figure 5 presents the respective Youden plot for chrysene in the food supplement test material and the

standard solution in toluene with undisclosed content From this graph it can be concluded that the

overestimation of the chrysene content of the food supplement material was not caused by instrument

calibration The results for the solvent solution of many laboratories which underperformed for the

26

food supplement sample agreed well with the assigned value This supports the conclusion that the

overestimation is caused by the determination of an interferent most probably triphenylene

Figure 5 Youden plot of the percent deviations of values reported for chrysene from the

assigned values of the food supplement test material and the standard solution in

toluene with undisclosed content

96 Evaluation of compliance with legislation

The method performance data reported for the four marker PAHs were evaluated for compliance with

the provisions given in Commission Regulation (EC) No 8362011 Table 18 in ANNEX 10 gives an

overview on the results of the evaluation Data were not reported in case of empty cells Non-

compliant data are highlighted in Table 18 It should be noted that Regulation (EU) No 8362011

specifies LOD and LOQ with two decimals This was not respected by all participants which

hampered the compliance assessment

27

In summary it can be stated that the majority of participants reported method performance

characteristics for the determination of the four marker PAHs in the food supplement test sample that

are compliant with legislation However some laboratories need to improve their analysis methods

The laboratories in question are urged to adapt to the provisions given in the new legislation

10 Follow-up actions for underperforming laboratories

All laboratories that got questionable or non-satisfactory performance ratings are urged to perform

root cause analysis and to implement corrective actions

As agreed during the EU-RL PAH workshop 2011 the EU-RL PAH will set up follow-up measures in

due time for all NRLs that received for at least one of the four PAHs (BAA BAP BBF and CHR) z-

scores gt 3 as required by Regulation (EC) 8822004 and by the Protocol for management of

underperformance in comparative testing andor lack of collaboration of National Reference

Laboratories (NRLs) with European Union reference laboratories (EU-RLs) activities

These laboratories shall perform as an immediate action root-cause-analysis and shall report to the

EU-RL PAH in writing the identified cause for their underperformance and corrective actions they are

going to take Additionally they shall participate to an independent (non-EU-RL) proficiency test on

the determination of PAHs in food and shall communicate the outcome of this exercise to the EU-RL

PAH

28

11 Conclusions

Fourty seven participants reported results back to the EU-RL The performance of the majority of

participants was good In total 153 out of 225 attributed z-scores were below an absolute value of two

which equals to almost 68 However this percentage is much below the percentages of satisfactory

performance gained in previous studies This might be the consequence of the higher level of difficulty

with respect to matrix composition In addition bias can be concluded from the pattern of performance

indicators of some laboratories

In general the determination of chrysene caused most difficulties to the participants

zeta-Scores were calculated besides z-scores They indicate the agreement of the reported result with

the assigned value with respect to the stated measurement uncertainty The outcome of this rating was

worse than for the z-scores which indicates that the measurement uncertainty estimates were not

realistic Therefore participants underperforming with regard to zeta scores are urged to adapt their

measurement uncertainty statements

The great majority of participants in this inter-laboratory comparison applied analytical methods which

complied with EU legislation with regard to performance characteristics

12 Acknowledgements

The organisers would like to thank Mrs Beatriz de la Calle and Mr Franz Ulberth (all from IRMM

Geel Belgium) for their accurate revision of this report Mr Hakan Emteborg (from IRMM) for

ampouling of the standard solutions and all NRLs and OCLs for their cooperation

29

13 References

1 EU COMMISSION REGULATION (EC) No 7762006 of 23 May 2006 amending Annex VII to Regulation

(EC) No 8822004 of the European Parliament and of the Council as regards Community reference

laboratories Official Journal of the European Union 2006 L 136 p 3-8

Available from

httpeur-lexeuropaeuLexUriServLexUriServdouri=OJL200613600030008ENPDF

2 EU Regulation (EC) No 8822004 of the European Parliament and of the Council of 29 April 2004 on

official controls performed to ensure the verification of compliance with feed and food law animal health

and animal welfare rules Official Journal of the European Communities 2004 L191 p 1-52 Available

from

httpeur-lexeuropaeuLexUriServLexUriServdouri=OJL200419100010052ENPDF

3 EU Opinion of the Scientific Committee on Food on the risks to human health of Polycyclic Aromatic

Hydrocarbons in food (2002) Available from

httpeuropaeuintcommfoodfsscscfout153_enpdf

4 International Agency for Research on Cancer IARC Monographs on the Evaluation of

Carcinogenic Risks to Humans Some Non-heterocyclic Polycyclic Aromatic Hydrocarbons and Some

Related Exposures Volume 92 (2010) Available from

httpmonographsiarcfrENGMonographsvol92mono92pdf

5 EU Commission Regulation (EC) No 3332007 of 28 March 2007 laying down the methods of sampling

and analysis for the official control of the levels of lead cadmium mercury inorganic tin 3-MCPD and

benzo(a)pyrene in foodstuffs Official Journal of the European Union 2007 L 88 p 29-38 Available

from

httpeur-lexeuropaeuLexUriServLexUriServdouri=OJL200708800290038ENPDF

6 EU Commission Regulation (EC) No 18812006 of 19 December 2006 setting maximum levels for certain

contaminants in foodstuffs Official Journal of the European Union 2006 L 364 p 5-24 Available from

httpeur-lexeuropaeuLexUriServLexUriServdouri=OJL200636400050024ENPDF

7 EFSA Scientific Opinion of the Panel on Contaminants in the Food Chain on a request from the European

Commission on Polycyclic Aromatic Hydrocarbons in Food The EFSA Journal 2008 724 p 1-114

Available from

httpwwwefsaeuropaeuEFSAefsa_locale-1178620753812_1211902034842htm

30

8 EU Commission Regulation (EU) No 8352011 amending Regulation (EC) No 18812006 as regards

maximum levels for polycyclic aromatic hydrocarbons in foodstuffs Official Journal of the European

Union 2011 L 215 p 4-8

httpeur-lexeuropaeuLexUriServLexUriServdouri=OJL201121500040008ENPDF

9 EU Commission Regulation (EU) No 8362011 amending Regulation (EC) No 3332007 laying down the methods of sampling and analysis for the official control of the levels of lead cadmium mercury inorganic tin 3-MCPD and benzo(a)pyrene in foodstuffs Official Journal of the European Union 2011 L 215 p 9-16 httpeur-lexeuropaeuLexUriServLexUriServdouri=OJL201121500090016ENPDF

10 ISOIEC 170432010 (2010) Conformity assessment -- General requirements for proficiency testing

providers Geneva Switzerland

11 Thompson M Ellison SLR and Wood R The International Harmonized Protocol for the Proficiency

Testing of Analytical Chemistry Laboratories Pure Appl Chem (2006) 78(1) p 145ndash196

Available from httpmediaiupacorgpublicationspac2006pdf7801x0145pdf

31

14 ANNEXES

ANNEX 1 ndash Announcement of the PT on the IRMM webpage

ANNEX 2 ndash Announcement via e-mail and invitation

ANNEX 3 ndash Announcement of sample dispatch

ANNEX 4 ndash Technical specifications of the calibration solutions

ANNEX 5 ndash Homogeneity and stability of the test material

ANNEX 6 ndash Acknowledgement of sample receipt

ANNEX 7 ndash Questionnaire on details of analysis method

ANNEX 8 ndash Results reported for the food supplement sample

ANNEX 9 ndash Results reported for the standard solutions with undisclosed analyte content

ANNEX 10 ndash Method performance characteristics reported by participants

ANNEX 11 ndash Details of analysis methods reported by participants

32

ANNEX 1 ndash Announcement of the PT on the IRMM webpage

33

ANNEX 2 - Announcement via e-mail and invitation

34

ANNEX 3 - Announcement sample dispatch

Dear MadameSir

The test samples for the proficiency test on the determination of four PAHs in a food supplement (dry extract of St Johns wort) were

dispatched today

Explanation on the design of the study as well as instructions for sample storage and for reporting of results are in the parcel You will

also find your participant key in the parcel which is required for reporting of results

Please check the parcel after arrival for completeness and report the receipt of the test samples as well as their status to us by applying

the sample receipt form which is in the parcel

You are kindly requested to inform us by email if you do not receive the parcel by end of this week

My colleague Donata Lerda will then take care of it

With best regards

Thomas Wenzl

35

ANNEX 4 - Specifications of standard solutions with disclosed concentration

36

ANNEX 4 - continued

37

ANNEX 5 - Homogeneity and stability of the test material

Homogeneity of the test material

Benz[a]anthracene - BAA

Benzo[a]pyrene - BAP

38

ANNEX 5 - continued

Benzo[b]fluoranthene - BBF

Chrysene - CHR

39

ANNEX 5 - continued

Stability of the test material

Table 10 Content of the four target analytes in test materials stored under different

temperatures

BaA BaP BbF CHR

microgkg microgkg microgkg microgkg

Content 31 16 31 69 Storage at room temperature

U (k=2) 05 02 05 17

Content 31 15 29 68 Storage at reference temperature

U (k=2) 04 03 04 10

The applied capillary column does not separate CHR from triphenylene

40

ANNEX 6 ndash Documents sent to participants

41

ANNEX 6 - continued

42

ANNEX 7 Questionnaire on details of analysis method

43

ANNEX 7 - continued

44

ANNEX 7 - continued

45

ANNEX 8 - Results reported by participants for the food supplements test sample Table 11 Analysis results reported by the participants for the content of benz[a]anthracene (BAA) in the food supplements test material Values are presented as reported

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

3600 31 29 29 3 05 2

3602 199 193 178 19 021

3603 253 253 246 251 022 2

3604 199 209 23 213 071 2

3605 301 3 271 291 076 2

3606 302 288 278 289 064 2

3607 456 416 443 438 096 2

3608 796 695 651 714 149 2

3610 182 192 214 196 039 2

3611 414 504 488 469 96 2

3612 3 34 28 31 06 2

3613 32 33 34 33 07 2

3614 182 182 191 185 037 2

3615 3884 3822 3565 376 0752 2

3616 2 2 2 2 01 2

3617 366 37 35 362 046 2

3618 32 32 32 32 05 2

3619 299 305 297 299 048 2

3620 304 294 302 302 045 2

3621 29 26 29 28 04 2

3622 343 325 318 322 052 2

3623 35 3 33 33 07 2

3624

3626 318 31 314 314 126 2

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

5301 31 3 32 31 06 2

5302 314 319 315 316 063 2

5303 3102 2716 2822 2876 27 62

5304 261 263 26 261 57 2

5305 23 22 26 24 20

5306 22 23 23 22 11 2

5307 488 461 317 461

5308 168633 168544 1562025 1644598

5309 16 19 16 17

5310 3 304 311 305 015

5311 477 452 448 459 15 2

5312 506 63 494 543 134 2

5313 353 301 404 353

5314 431 373 378 394 5 096

5315 596 531 51 545 11 2

5316 224 231 235 235 022 2

5317 216 23 221 222 007 2

5318 064 07 067 01 2

5319 111 184 146 147 029 2

5320 515 525 564 534 48

5321 21 5322 13 87 9 87 35 2

5323 38 33 35 33 16 2

46

Figure 6 Results of replicate determinations (indicated by triangles) of benz[a]anthracene (BAA) in the food supplement test material

Horizontal blue lines represent the arithmetic mean value of replicate measurements and blue bars the reported expanded measurement uncertainty (k=2) The assigned value is

plotted as dotted line Red solid lines mark the range of satisfactory performance (|z|le2) Mean values are given for results outside the displayed data range

ProLab 2011

Laboratory

53

1853

1953

0853

0936

1436

0236

1036

1653

2136

0453

1753

0653

1653

0536

0353

0436

2153

0336

0636

0536

0036

2036

1953

1036

1253

0136

2653

0236

1836

2336

2236

1353

1353

2336

1736

1553

1453

0736

0753

1153

2053

1253

1536

0853

2236

11

an

aly

te c

on

ten

t (micro

gk

g)

10

95

9

85

8

75

7

65

6

55

5

45

4

35

3

25

2

15

1

05

0

102

3 468

7

Sample St Johns wort extract replicatesMeasurand Benz[a]anthraceneNo of laboratories 46

Assigned value 298 microgkg (Reference value)Target sd 062 microgkgTolerance limits 174 - 422 microgkg (|Z score| lt 200)

Assig

ned va

lue

Limit of tolerance

Limit of tolerance

47

Table 12 Analysis results reported by the participants for the content of benzo[a]pyrene (BAP) in the food supplements test material Values are presented as reported

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

3600 16 17 17 17 03 2

3602 118 123 13 124 015

3603 165 162 157 161 015 2

3604 134 154 139 143 04 2

3605 164 166 172 167 057 2

3606 181 159 161 167 031 2

3607 279 266 279 274 055 2

3608 57 687 461 573 226 2

3610 226 216 183 206 042 2

3611 289 325 311 308 36 2

3612 24 22 2 22 05 2

3613 17 18 19 18 05 2

3614 08 08 08 08 016 2

3615 1545 1393 1425 145 029 2

3616 16 16 14 15 03 2

3617 177 171 158 169 023 2

3618 2 2 2 2 02 2

3619 207 207 206 207 075 2

3620 165 169 163 166 017 2

3621 44 47 49 47 05 2

3622 212 208 222 213 038 2

3623 2 21 22 21 03 2

3624 18 178 182 18 031 2

3626 172 173 178 174 07 2

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

5301 18 18 18 18 04 2

5302 188 19 187 188 038 2

5303 1691 1426 149 1535 19 80

5304 145 142 15 146 69 2

5305 15 17 18 16 15

5306 16 17 17 16 08 2

5307 164 157 153 157

5308 102669 109324 109035 107009

5309 1 1 12 11

5310 122 122 124 123 006

5311 199 184 215 2 15 2

5312 258 248 239 248 05 2

5313 199 178 245 207

5314 174 171 172 172 11 0948

5315 253 248 213 238 05 2

5316 177 171 173 17 016 2

5317 255 277 276 269 012 2

5318 114 12 117 02 2

5319 19 228 231 216 043 2

5320 303 3 344 316 78

5321 12 12 12 12

5322 13 17 24 17 35 2

5323 2 2 19 2 1 2

48

Figure 7 Results of replicate determinations (indicated by triangles) of benzo[a]pyrene (BAP) in the food supplement test material

Horizontal blue lines represent the arithmetic mean value of replicate measurements and blue bars the reported expanded measurement uncertainty (k=2) The assigned value is

plotted as dotted line Red solid lines mark the range of satisfactory performance (|z|le2) Mean values are given for results outside the displayed data range

ProLab 2011

Laboratory

36

1453

0953

0853

1853

2153

1036

0236

0436

1553

0436

1653

0353

0736

0336

2036

0053

0553

0636

0636

0536

1753

1453

1636

2636

1336

2453

0153

2253

0253

2353

1136

1836

1953

1336

1036

2336

2253

1936

1253

1553

1253

1736

0753

2036

2136

0836

11

an

aly

te c

on

ten

t (micro

gk

g)

55

5

45

4

35

3

25

2

15

1

05

0

573

308

3

Sample St Johns wort extract replicatesMeasurand Benzo[a]pyreneNo of laboratories 48

Assigned value 165 microgkg (Reference value)Target sd 036 microgkgTolerance limits 093 - 237 microgkg (|Z score| lt 200)

Assig

ned va

lue

Limit of tolerance

Limit of tolerance

49

Table 13 Analysis results reported by the participants for the content of benzo[b]fluoranthene (BBF) in the food supplements test material Values are presented as reported

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

3600 32 3 3 31 09 2

3602 173 175 16 17 017

3603 265 252 25 256 025 2

3604 116 108 105 11 028 2

3605 286 225 232 247 074 2

3606 294 273 261 276 046 2

3607 579 579 544 567 113 2

3608 103 1201 876 1036 326 2

3610 173 196 204 191 038 2

3611 546 618 565 576 75 2

3612 3 28 28 29 06 2

3613 28 31 32 31 06 2

3614 135 111 135 127 022 2

3615 4187 3678 4111 4 08 2

3616 2 2 2 2 01 2

3617 33 27 299 035 2

3618 31 31 31 31 05 2

3619 326 324 312 326 064 2

3620 277 28 272 276 069 2

3621 26 25 23 25 04 2

3622 346 336 333 34 052 2

3623 26 28 29 28 02 2

3624

3626 288 28 307 292 117 2

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

5301 29 3 29 29 06 2

5302 297 3 296 298 06 2

5303 2705 2444 2608 2586 22 78

5304 245 254 26 253 85 2

5305 23 23 26 24 15

5306 42 43 4 42 21 2

5307 221 216 241 221

5308 232169 246654 240722 229848

5309 19 22 19 2 0

5310 3 315 325 313 016

5311 262 277 304 281 15 2

5312 27 289 337 299 075 2

5313 321 284 378 328

5314 423 404 385 404 34 083

5315 42 506 416 444 09 2

5316 265 267 277 273 018 2

5317 185 197 195 192 006 2

5318 276 281 279 04 2

5319 396 389 403 396 079 2

5320 623 618 635 625 14

5321 31

5322 24 33 49 33 35 2

5323 38 45 35 45 27 2

50

Figure 8 Results of replicate determinations (indicated by triangles) of benzo[b]fluoranthene (BBF) in the food supplement test material

Horizontal blue lines represent the arithmetic mean value of replicate measurements and blue bars the reported expanded measurement uncertainty (k=2) The assigned value is

plotted as dotted line Red solid lines mark the range of satisfactory performance (|z|le2) Mean values are given for results outside the displayed data range

ProLab 2011

Laboratory

36

0436

1436

0236

1053

1736

1653

0953

0753

0853

0536

2136

0553

0436

0353

0353

1636

0636

2036

2353

1853

1136

1236

2653

0153

0253

1236

1736

1336

0036

1853

2153

1036

1953

1336

2253

2253

2353

1936

1553

1453

0653

1536

0753

2036

0836

11

an

aly

te c

on

ten

t (micro

gk

g)

75

7

65

6

55

5

45

4

35

3

25

2

15

1

05

0

103

6

576

3

Sample St Johns wort extract replicatesMeasurand Benzo[b]fluorantheneNo of laboratories 46

Assigned value 292 microgkg (Reference value)Target sd 060 microgkgTolerance limits 172 - 412 microgkg (|Z score| lt 200)

Assig

ned va

lue

Limit of tolerance

Limit of tolerance

51

Table 14 Analysis results reported by the participants for the content of chrysene (CHR) in the food supplements test material Values are presented as reported

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

3600 5 49 5 5 07 2

3602 32 304 29 304 036

3603 462 469 447 459 048 2

3604 438 428 42 429 119 2

3605 365 41 381 386 085 2

3606 494 411 45 451 124 2

3607 897 774 787 819 18 2

3608 986 887 1168 1014 284 2

3610 418 392 462 424 085 2

3611 833 893 87 865 61 2

3612 6 64 58 61 12 2

3613 41 39 4 4 08 2

3614 272 254 288 271 054 2

3615 605 6028 5964 601 12 2

3616 47 45 49 47 05 2

3617 445 431 389 422 065 2

3618 45 45 45 45 06 2

3619 738 747 72 738 118 2

3620 463 472 446 46 092 2

3621 53 49 51 51 06 2

3622 478 513 505 488 08 2

3623 73 73 72 72 05 2

3624

3626 63 622 634 628 251 2

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

5301 49 49 49 49 1 2

5302 687 683 666 679 136 2

5303 6777 6181 6382 6441 24 70

5304 592 581 6 591 85 2

5305 6 58 64 61 20

5306 49 5 51 49 25 2

5307 524 5 574 524

5308 211488 216782 221146 213243

5309

5310 372 393 394 386 019

5311 43 459 422 437 15 2

5312 934 943 981 953 191 2

5313 793 73 99 838

5314 636 561 565 588 18 0818

5315 547 583 499 542 11 2

5316 292 286 285 282 06 2

5317 52 598 517 545 046 2

5318 368 345 357 05 2

5319 449 471 474 465 116 2

5320 76 924 818 834 99

5321 33

5322 7 7 76 7 35 2

5323 87 73 77 73 37 2

52

Figure 9 Results of replicate determinations (indicated by triangles) of chrysene (CHR) in the food supplement test material

Horizontal blue lines represent the arithmetic mean value of replicate measurements and blue bars the reported expanded measurement uncertainty (k=2) The assigned value is

plotted as dotted line Red solid lines mark the range of satisfactory performance (|z|le2) Mean values are given for results outside the displayed data range

ProLab 2011

Laboratory

53

0836

1453

1636

0253

2153

1836

0553

1036

1336

1736

1036

0453

1136

1836

0636

0336

2053

1936

1653

0136

0036

2253

0636

2153

0753

1553

1753

1453

0436

1536

1253

0536

2653

0353

0253

2236

2336

1953

2336

0753

2053

1353

1236

0836

11

an

aly

te c

on

ten

t (micro

gk

g)

13

12

11

10

9

8

7

6

5

4

3

2

1

0

865

3

Sample St Johns wort extract replicatesMeasurand ChryseneNo of laboratories 45

Assigned value 407 microgkg (Reference value)Target sd 083 microgkgTolerance limits 241 - 573 microgkg (|Z score| lt 200)

Assig

ned va

lue

Limit of tolerance

Limit of tolerance

53

Table 15 Analysis results reported by the participants for the sum of the four marker PAHs in the food supplements test material Values are presented as reported

Laboratory code

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg

3600 128 24 2

3602 79 126

3603 1127 096 2

3604 893 258 2

3605 1091 305 2

3606 1184 238 2

3607 2098 42 2

3608 3336 325 2

3610 1138 17 2

3611 2218 259 2

3612 143 29 2

3613 122 2 2

3614 663 129 2

3615 152 304 2

3616 102 03 2

3617 1252 169 2

3618 128 18 2

3619 157 305 2

3620 1204 12 2

3621 151 19 2

3622 1361 115 2

3623 154 09 2

3624

3626 1409 56 2

Laboratory code

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg

5301 126

5302 37 074 2

5303 13438 92

5304 1251 85 2

5305 125 20

5306 129 65 2

5307 1363

5308 7245598

5309

5310 1127 056

5311 1367 15 2

5312 2042 408 2

5313 1726

5314 1558

5315 177 35 2

5316 96 073 2

5317 1229 066 2

5318 82 05 2

5319 1225 267 2

5320 2309 41

5321

5322 207 35 2

5323 171 103 2

54

ANNEX 9 Results reported for the standard solutions with undisclosed analyte content Table 16 Analysis results reported by the participants for the four marker PAHs in the standard solution in toluene with undisclosed analyte

content Values are presented as reported U measurement uncertainty k coverage factor

benz[a]anthracene benzo[a]pyrene benzo[b]fluoranthene chrysene Laboratory

code Unit Result U k Result U k Result U k Result U k 3600 microgl 38 67 2 25 45 2 211 68 2 275 38 2

3605 microgl 367 184 2 2482 124 2 2592 13 2 235 118 2

3606 microgkg 3763 176 2 2609 142 2 2721 118 2 2539 134 2

3607 microgkg 3706 815 2 2824 565 2 2591 518 2 2587 569 2

3612 microgl 38 08 2 237 11 2 244 18 2 257 02 2

3614 microgl 324 65 2 209 38 2 215 37 2 21 42 2

3616 microgkg 318 07 2 224 14 2 231 26 2 216 1 2

3618 microgkg 417 17 2 284 08 2 292 06 2 297 03 2

3619 microgkg 47 742 2 274 468 2 228 387 2 274 434 2

3620 microgl 495 74 2 294 29 2 321 8 2 321 64 2

3621 microgl 241 22 2 143 13 2 188 16 2 145 14 2

3623 microgkg 476 25 2 311 53 2 269 3 2 297 03 2

3626 microgl 359 38 2 235 23 2 238 27 2 24 18 2

5301 microgkg 426 29 299 289

5302 microgkg 436 87 2 296 59 2 296 59 2 289 58 2

5303 microgl 3721 27 102 22838 19 99 23756 22 99 25486 24 98

5304 microgkg 38 29 2 225 26 2 257 37 2 258 52 2

5305 microgkg 305 225 245 29

5306 microgkg 316 63 2 247 49 2 65 13 2 22 44 2

5307 microgkg 3658 2559 241 2414

5308 microgkg 16854 109324 246654 216782

5311 microgl 374 20 2 279 15 2 268 15 2 272 15 2

5313 microgl 3408 2165 2275 2373

5314 microgkg 395 5 2875 11 3614 34 2905 18

5320 microgl 3267 368 1986 571 28 683 2427 512

5322 microgl 282 35 2 225 35 2 241 35 2 21 35 2

55

Figure 10 Youden plots of the percent deviation of the reported values from the assigned values for the food supplement test sample and the

standard solution in toluene with undisclosed content

(The identity of the respective measurand is given above the figure Results exceeding a difference of more than 100 were omitted)

56

Figure 10 - continued

57

Table 17 Analysis results reported by the participants for the four marker PAHs in the standard solution in acetonitrile with undisclosed

analyte content

Values are presented as reported U measurement uncertainty k coverage factor

benz[a]anthracene benzo[a]pyrene benzo[b]fluoranthene chrysene

Laboratory

code Unit Result U k Result U k Result U k Result U k

3602 microgkg 3675 247 247 244

3603 microgkg 358 086 2 236 028 2 235 028 2 236 057 2

3604 microgl 475 29 2 19 12 2 194 13 2 258 12 2

3608 microgl 3623 078 2 2385 023 2 2343 18 2 2296 092 2

3610 microgkg 369 185 2 25 125 2 251 126 2 244 122 2

3611 microgkg 48 02 2 323 02 2 328 04 2 317 03 2

3613 microgl 332 31 2 261 3 2 249 25 2 239 24 2

3615 microgl 415 2635 2729 2689

3617 microgkg 37 09 2 244 06 2 284 07 2 239 06 2

3622 microgl 37 12 2 25 23 2 257 21 2 238 1 2

3624 microgkg 402 104 2 255 084 2 265 096 2 253 07 2

5306 microgkg 563 113 2 371 74 2 359 72 2 333 67 2

5308 microgkg 168633 102669 232169 211488

5309 microgl 402 268 27

5310 microgl 315 158 25 125 25 125 288 144

5312 microgl 428 043 2 282 028 2 272 027 2 275 028 2

5315 microgkg 387 08 2 256 05 2 251 05 2 253 05 2

5316 microgl 3839 342 2 2485 191 2 2559 168 2 239 51 2

5317 microgl 2466 043 2 2212 034 2 2472 016 2 2146 068 2

5319 microgl 414 41 2 279 28 2 268 27 2 26 31 2

5321 microgkg 476 316 326 328

5322 microgl 2547 35 2

5323 microgkg 405 203 2 239 119 2 255 153 2 201 11 2

58

Figure 11 Youden plots of the percent deviation of the reported values from the assigned values for the food supplement test sample and the

standard solution in acetonitrile with undisclosed content

(The identity of the respective measurand is given above the figure Results exceeding a difference of more than 100 were omitted)

59

Figure 11 - continued

60

ANNEX 10 Table 18 Method performance parameters reported by the participants

Laboratory code

Analyte LOD LOQ Recovery Analyte LOD LOQ Recovery

microgkg microgkg microgkg microgkg 3600 BaA 03 06 978 BaP 03 07 9483602 BaA 007 021 926 BaP 005 015 993603 BaA 0011 05 987 BaP 0005 051 9553604 BaA 02 04 86 BaP 02 04 883605 BaA 0025 005 118 BaP 0025 005 1083606 BaA 001 003 65 BaP 001 003 703607 BaA 021 071 74 BaP 027 089 943608 BaA 016 048 8213 BaP 015 046 83683610 BaA 02 05 85 BaP 005 02 853611 BaA BaP 3612 BaA 1 117 BaP 1 1203613 BaA 01 03 90 BaP 01 03 903614 BaA 02 05 96 BaP 02 05 933615 BaA 005 015 110 BaP 005 015 1053616 BaA 01 05 108 BaP 01 05 753617 BaA 018 037 405 BaP 009 018 3873618 BaA 01 03 63 BaP 01 03 593619 BaA 005 005 59 BaP 033 033 543620 BaA 0009 0018 64 BaP 0005 001 553621 BaA 007 02 84 BaP 007 02 923622 BaA 01 03 1101 BaP 01 03 9853623 BaA 02 08 105 BaP 02 08 1013624 BaA BaP 01 033 8513625 BaA BaP 3626 BaA BaP 5301 BaA 003 01 102 BaP 003 01 1005302 BaA 01 03 78 BaP 01 03 865303 BaA 003 01 62 BaP 003 01 805304 BaA 1 1 70 BaP 1 1 955305 BaA 02 05 85 BaP 02 05 855306 BaA 03 1 699 BaP 03 1 905307 BaA 6537 BaP 81165308 BaA 0088 0265 99 BaP 00262 00785 1005309 BaA 01 08 92 BaP 01 08 925310 BaA 01 03 BaP 01 03 905311 BaA 002 005 80 BaP 002 004 915312 BaA 03 09 105 BaP 03 09 1055313 BaA 04 08 BaP 04 08 5314 BaA 001 002 96 BaP 001 002 9485315 BaA 01 03 50 - 100 BaP 01 03 50 - 100 5316 BaA 002 011 827 BaP 004 019 10445317 BaA 05 1 76 BaP 05 1 755318 BaA BaP 5319 BaA 009 029 951 BaP 011 041 995320 BaA 12 18 946 BaP 06 1 8345321 BaA 03 05 BaP 05 1 5322 BaA 1 2 100 BaP 1 2 1005323 BaA 26 BaP 26

61

ANNEX 10 Table 18 - continued

Laboratory code Analyte LOD LOQ Recovery Analyte LOD LOQ Recovery

microgkg microgkg microgkg microgkg 3600 BbF 03 05 96 CHR 02 03 10153602 BbF 015 045 947 CHR 003 009 9193603 BbF 0014 05 1012 CHR 0004 05 10033604 BbF 02 04 85 CHR 02 04 873605 BbF 005 01 94 CHR 0025 005 863606 BbF 001 003 70 CHR 001 003 653607 BbF 018 06 100 CHR 014 028 943608 BbF 015 046 7979 CHR 015 045 71253610 BbF 005 02 85 CHR 02 05 853611 BbF CHR 3612 BbF 1 118 CHR 1 1173613 BbF 01 03 90 CHR 01 03 903614 BbF 01 04 96 CHR 02 05 953615 BbF 008 025 109 CHR 003 01 1083616 BbF 01 05 78 CHR 01 05 723617 BbF 035 07 38 CHR 012 024 4163618 BbF 01 03 62 CHR 01 03 623619 BbF 016 016 63 CHR 01 01 593620 BbF 0003 0006 55 CHR 001 002 653621 BbF 007 02 103 CHR 007 02 893622 BbF 02 06 963 CHR 03 09 1043623 BbF 02 08 106 CHR 02 08 1193624 BbF CHR 3625 BbF CHR 3626 BbF CHR 5301 BbF 003 01 96 CHR 003 01 1145302 BbF 01 03 84 CHR 01 03 835303 BbF 003 01 78 CHR 003 01 705304 BbF 1 1 86 CHR 1 1 705305 BbF 02 05 90 CHR 02 05 955306 BbF 03 1 1435 CHR 03 1 1185307 BbF 8116 CHR 65375308 BbF 0079 0238 99 CHR 009 026 1005309 BbF 01 08 92 CHR 5310 BbF 07 2 CHR 01 03 5311 BbF 002 004 95 CHR 002 004 895312 BbF 03 09 105 CHR 03 09 1055313 BbF 04 08 CHR 04 08 5314 BbF 001 002 83 CHR 001 002 8185315 BbF 02 05 50 -100 CHR 02 05 50 - 100 5316 BbF 005 024 814 CHR 001 007 9595317 BbF 05 1 73 CHR 05 1 785318 BbF CHR 5319 BbF 011 038 946 CHR 013 043 9675320 BbF 07 09 1194 CHR 23 32 10455321 BbF 05 1 CHR 05 1 5322 BbF 1 2 100 CHR 1 2 1005323 BbF 26 CHR 26

62

ANNEX 11 Table 19 Details of analysis method reported by the participants Data are presented as reported

Laboratory code

Analysis technique

Instrument calibration

Internal standards applied

Sample clean up

Details of sample

clean up

Extraction technique

Details of sample

extraction

3600 6) GC-MS

2) Internal standardisation with labelled substances

Deuterated PAH mix 9 (Ehrenstorfer)

1) Saponification 2) LiquidLiquid partitioning

D) Other Liquidliquid partitioning

3602 2) HPLC-UV-FLD

1) External calibration

3) Gel permeation chromatography

A) Pressurised liquid extraction (PLE)

3603 1) HPLC-FLD

1) External calibration

3) Gel permeation chromatography

D) Other 1 minute with dichloromethane in vortex

3604 2) HPLC-UV-FLD

2) Internal standardisation with labelled substances

DiP D14

5) Donor acceptor complex chromatography

D) Other LL

3605 1) HPLC-FLD

1) External calibration

3) Gel permeation chromatography

C) Soxhlet extraction

3606 7) GC-MSMS

2) Internal standardisation with labelled substances

BaP 13C4 CHR 13C6 BaA 13C6 BbF 13C6

4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

3607 6) GC-MS

2) Internal standardisation with labelled substances

BaA-d12 CHR-d12 BbF-d12 BkF-d12 BaP-d12 ICP-d12 BgP-d12 DiP-d14

1) Saponification

D) Other solvent partitioning

3608 1) HPLC-FLD

1) External calibration

6) Other Liquid extraction

D) Other

Extraction in a rotary agitator and centrifugation

3610 2) HPLC-UV-FLD

1) External calibration

4) Solid phase extraction

B) Sonication

3611

3612 6) GC-MS

2) Internal standardisation with labelled substances

chrysene-d12 dibenzo(ah)anthracene-d14 dibenzo(ai)pyrene-d14

4) Solid phase extraction

B) Sonication

3613 5) LC-MSMS

3) Internal standardisation with unlabelled substances

I use Chrysene-D12 3) Gel permeation chromatography

D) Other extraction with hexan

63

Table 19 - continued

Laboratory code

Analysis technique

Instrument calibration

Internal standards applied

Sample clean up Details of sample

clean up

Extraction technique

Details of sample

extraction

3614 6) GC-MS 1) External calibration

3) Gel permeation chromatography

B) Sonication

3615 1) HPLC-FLD

1) External calibration

6) Other PLE GPC

A) Pressurised liquid extraction (PLE)

3616 6) GC-MS

2) Internal standardisation with labelled substances

Benzo(a)pyrene-13C4 Benzo(a)anthracene-13C6 Benzo(b)fluoranthene-13C6 Chrysene-13C6

2) LiquidLiquid partitioning 4) Solid phase extraction

B) Sonication

3617 1) HPLC-FLD

1) External calibration

4) Solid phase extraction

C) Soxhlet extraction

3618 6) GC-MS

2) Internal standardisation with labelled substances

deuterated standards

1) Saponification 4) Solid phase extraction

D) Other Handshaken with cyclohexane

3619 6) GC-MS

2) Internal standardisation with labelled substances

C13 Labelled US EPA 16

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

D) Other Homegenisation amp saponiofication

3620 9) GC-HRMS

2) Internal standardisation with labelled substances

mix of deuterated PAHs (BAA-D12 CHR-D12 BAP-D12 BBF-D12)

3) Gel permeation chromatography 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

3621 6) GC-MS

2) Internal standardisation with labelled substances

Chrysene D12 Benzo(a)pyrene D12 Benzo(b)fluoranthene D12 Benzo(a)anthracene

2) LiquidLiquid partitioning 4) Solid phase extraction

B) Sonication

3622 1) HPLC-FLD

4) Standard addition method

4) Solid phase extraction

D) Other

Extraction with cyclohexane by heating under reflux

3623 7) GC-MSMS

2) Internal standardisation with labelled substances

C13 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

3624 2) HPLC-UV-FLD

1) External calibration

2) LiquidLiquid partitioning

D) Other hot solvent extraction

3626

64

Table 19 - continued

Laboratory code

Analysis technique

Instrument calibration

Internal standards

applied

Sample clean up

Details of sample

clean up

Extraction technique

Details of sample

extraction

5301 7) GC-MSMS

2) Internal standardisation with labelled substances

EACH SUBSTANCE C13

4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5302 7) GC-MSMS

2) Internal standardisation with labelled substances

Isotope labelled PAHs 13C for each analyte

4) Solid phase extraction

C) Soxhlet extraction

5303 7) GC-MSMS

3) Internal standardisation with unlabelled substances

HAP C13 4) Solid phase extraction

D) Other automate ASE200 DIONEX

5304 7) GC-MSMS

2) Internal standardisation with labelled substances

with C13 for each HAP

2) LiquidLiquid partitioning 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5305 7) GC-MSMS

2) Internal standardisation with labelled substances

C13 isotopiques internal standards

4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5306 7) GC-MSMS

2) Internal standardisation with labelled substances

C13 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5307 6) GC-MS

2) Internal standardisation with labelled substances

1) Saponification 2) LiquidLiquid partitioning

D) Other solvent extraction

5308 1) HPLC-FLD 1) External calibration

4) Solid phase extraction

B) Sonication

5309 1) HPLC-FLD 1) External calibration

4) Solid phase extraction

D) Other only SPE

5310 1) HPLC-FLD 1) External calibration

1) Saponification 2) LiquidLiquid partitioning 3) Gel permeation chromatography

C) Soxhlet extraction

5311 6) GC-MS

2) Internal standardisation with labelled substances

13 C Chrysene 13 C Benz(a)pyrene

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

B) Sonication

5312 1) HPLC-FLD

3) Internal standardisation with unlabelled substances

Benzo(b)chrysen

2) LiquidLiquid partitioning 4) Solid phase extraction

Quechers D) Other Quechers

65

Table 19 - continued

Laboratory code

Analysis technique

Instrument calibration

Internal standards applied

Sample clean up Details of sample

clean up

Extraction technique

Details of sample extraction

5313 6) GC-MS

2) Internal standardisation with labelled substances

Benz(a)anthracene D12 Benzo(b)fluranthene D12 Benzo(a)pyrene D12 Chrysene D12

1) Saponification 2) LiquidLiquid partitioning 3) Gel permeation chromatography

A) Pressurised liquid extraction (PLE)

5314 6) GC-MS

2) Internal standardisation with labelled substances

Benzo(a)pyren d12 2) LiquidLiquid partitioning

D) Other Extraction with

5315 1) HPLC-FLD

3) Internal standardisation with unlabelled substances

Benz(b)chrysen

1) Saponification 2) LiquidLiquid partitioning

D) Other shaker

5316 1) HPLC-FLD

1) External calibration

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

D) Other

LiquidLiquid extraction with cyclohexane and dimethylformamide

5317 7) GC-MSMS

1) External calibration

3) Gel permeation chromatography 6) Other

silica gel D) Other VDLUFA online method

5318

5319 1) HPLC-FLD

3) Internal standardisation with unlabelled substances

Benzo(b)chrysene 3) Gel permeation chromatography

A) Pressurised liquid extraction (PLE)

5320 6) GC-MS

3) Internal standardisation with unlabelled substances

22-binaphthyl

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

D) Other 3 hours refluxing

5321 1) HPLC-FLD

2) Internal standardisation with labelled substances

6-meacutethylchrysegravene

1) Saponification 2) LiquidLiquid partitioning

B) Sonication

5322 6) GC-MS

2) Internal standardisation with labelled substances

naftalene-d8 acenaphtalene-d10 phenanthrene-d10 chrysene-d12 perylene-d12 benzo(e)anthracene-d12

1) Saponification 2) LiquidLiquid partitioning 6) Other

clean-up with silica packed column

C) Soxhlet extraction

5323 1) HPLC-FLD

1) External calibration

4) Solid phase extraction 6) Other

Accelerated Solvent Extraction

A) Pressurised liquid extraction (PLE)

66

European Commission EUR 25245 EN ndash Joint Research Centre ndash Institute for Reference Materials and Measurements Title Report on the 8th inter-laboratory comparison organised by the European Union Reference Laboratory for Polycyclic Aromatic Hydrocarbons - Four marker PAHs in a dry extract of St Johns wort Author(s) Radoslav Lizak Szilard Szilagyi Philippe Verlinde and Thomas WENZL Luxembourg Publications Office of the European Union 2012 ndash 66 pp ndash 201 x 297 cm EUR ndash Scientific and Technical Research series ndash ISSN 1831-9424 ISBN 978-92-79-23337-1 doi 10278759387 Abstract The proficiency test here reported concerned the determination of the contents of four marker polycyclic aromatic hydrocarbons (PAHs) and of their sum in an food supplements test sample The set of marker PAHs consists of benz[a]anthacene benzo[a]pyrene benzo[b]fluoranthene and chrysene Participants to these PT were National Reference Laboratories for PAHs (NRLs-PAHs) and EU official food control laboratories The number of participants was in total 48

The PT was organised according ISO Standard 170432010

The test material used was a commercial dry extract of St Johns wort Participants also received a solution of the same PAHs either in acetonitrile or in toluene for checking their instrument calibration

The results from participants were rated with z-scores and zeta-scores About 68 of the reported results were attributed with z-scores with an absolute value of below two which is the threshold for satisfactory performance

How to obtain EU publications

Our priced publications are available from EU Bookshop (httpbookshopeuropaeu) where you can place

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sending a fax to (352) 29 29-42758

The mission of the JRC is to provide customer-driven scientific and technical support

for the conception development implementation and monitoring of EU policies As a

service of the European Commission the JRC functions as a reference centre of

science and technology for the Union Close to the policy-making process it serves

the common interest of the Member States while being independent of special

interests whether private or national

L

A-N

A-252

45-EN

-N

• 1 Summary
• 2 Introduction
• 3 Scope
• 4 Participating Laboratories
• 5 Time frame
• 6 Confidentiality
• 7 Test materials
• • 71 1Preparation of test materials
  • 72 Assigned values and standard deviation for proficiency assessment
  • 73 Homogeneity and stability
  • 74 Sample dispatch
  • • 8 Design of the proficiency test
    • 9 Evaluation of the results
    • • 91 2General
      • 92 21BEvaluation criteria
      • 93 Performance of participants
      • 94 Evaluation of the influence of analysis method
      • 95 Evaluation of the influence of calibration
      • 96 26Evaluation of compliance with legislation
      • • 10 Follow-up actions for underperforming laboratories
        • 11 1Conclusions
        • 12 1Acknowledgements
        • 13 References

20

Table 8 Compilation of zeta-scores calculated from the ldquoresults for proficiency assessment

reported by the NRLs for test material St Johns wort the reported measurement

uncertainty and the uncertainty of the analyte content of the test material zeta-scores

outside the satisfactory range (|zeta| gt 2) are indicated by red bold font Empty cells

denote analytes for which either results or measurement uncertainties were not

reported

21

Table 9 Compilation of zeta-scores calculated from the ldquoresults for proficiency assessment

reported by the OCLs for test material St Johns wort the reported measurement

uncertainty and the uncertainty of the analyte content of the test material zeta-scores

outside the satisfactory range (|zeta| gt 2) are indicated by red bold font yellow

highlighted cells ugtUf Empty cells denote analytes for which results were not

reported

22

94 Evaluation of the influence of analysis method

The information on the applied analysis method (ANNEX 11) was used to group the performance

indicators attributed to the reported results according to the applied analysis technique Two groups of

data were formed They represent results obtained by high performance liquid chromatography-

fluorescence detection (HPLC-FD about 40 ) and by gas chromatography-mass spectrometry (GC-

MS about 60 ) respectively Kernel density plots were constructed based on the respective z-scores

Figure 3A to 3E show the different Kernel density plots

Figure 3 Kernel density plots of z-scores attributed to the results for proficiency assessment

which were reported for the food supplements test sample Results were classified

according to the applied analysis technique

A) benz[a]anthracene

B) benzo[a]pyrene

23

Figure 3 continued

C) benzo[b]fluoranthene

D) chrysene

E) sum of four marker PAHs

The kernel density plots for BAP and BBF are almost identical and centred at or close to a z-score

value of zero as can be seen in Figure 3B and 3C Therefore bias introduced by the type of analysis

method is unlikely for these two analytes The situation is different for BAA and CHR In case of

BAA the kernel density distribution of z-scores based on HPLC-FD is shifted to negative values

24

(Figure 3A) which indicates a tendency to underestimate the BAA content in this particular

samplematrix when applying HPLC-FD This might be reasoned by separationintegration problems

between BAA and CHR peaks A few participants (from the HPLC-FD subgroup) that got negative

z-scores for BAA overestimated the CHR content This caused the slight shift of the density trace for

HPLC-FD towards negative values Removing this data from the evaluation would result in a density

trace with the mode at zero

The opposite is the case for CHR (Figure 3D) The density distribution of the z-scores attributed to

HPLC-FD data is centred almost at zero However the distribution for data based on GC-MS is shifted

to positive values indicating strong positive bias This bias might be caused by a lack of selectivity of

the analysis method The test material contains triphenylene which is isobaric to chrysene

Consequently it cannot be distinguished from chrysene by means of mass spectrometry These two

compounds have to be separated chromatographically which is not easy They coelute on unpolar and

on most mid-polar capillary columns Columns that allow their separation were commercialised only

recently and might not be available in all laboratories Therefore laboratories which determine the

four EU marker PAHs by means of GC-MS shall pay attention to the chromatographic separation of

triphenylene and chrysene A lack of chromatographic resolution might lead to strong positive bias of

GC-MS methods and to false conclusions on the marketability of the tested sample

The chromatographic separation of chrysene and triphenylene is not a problem with HPLC which is

reflected in the position of the density plot for chrysene However attention has to be paid in HPLC-

FD analysis to a proper clean up of the sample extract in order to eliminate interferences that show

similar retention as BAA and CHR

95 Evaluation of the influence of calibration

The participants received together with the food supplement test sample two standard solutions in

depending of their choice either acetonitrile or toluene The analyte content of one solution was

disclosed to the participants whereas the content of the other solution was kept undisclosed The

content values of the latter are presented in Table 5 Analysis results for the solution with undisclosed

content had to be reported to the PT organiser The respective data are presented in Annex 9

Percent deviations of the reported results from the assigned values were calculated for both the food

supplement test sample and the standard solution with undisclosed analyte content Youden plots were

prepared in order to visualize the influence of instrument calibration on the results reported for the

food supplement sample Two plots are discussed in the following The whole set of Youden plots are

given in Annex 9

25

Figure 4 Youden plot of the percent deviations of values reported for benzo[a]pyrene from the

assigned values of the food supplement test material and the standard solution in

toluene with undisclosed content

Figure 4 reveals that a number of participants reported results with similar percent deviations from the

assigned values of both the results for the food supplement material and the standard solution in

toluene with undisclosed content This correlation indicates that over- respectively underestimation of

the analyte content of the food supplement material might be caused by biased instrument calibration

This finding underpins the importance of thorough calibration standard preparation However the data

was not in all cases conclusive as some laboratories performed well for the food supplement samples

but reported biased results for the standard solution with undisclosed content (eg Figure 4 participants

3620 and 5305) This fact cannot be explained yet

Figure 5 presents the respective Youden plot for chrysene in the food supplement test material and the

standard solution in toluene with undisclosed content From this graph it can be concluded that the

overestimation of the chrysene content of the food supplement material was not caused by instrument

calibration The results for the solvent solution of many laboratories which underperformed for the

26

food supplement sample agreed well with the assigned value This supports the conclusion that the

overestimation is caused by the determination of an interferent most probably triphenylene

Figure 5 Youden plot of the percent deviations of values reported for chrysene from the

assigned values of the food supplement test material and the standard solution in

toluene with undisclosed content

96 Evaluation of compliance with legislation

The method performance data reported for the four marker PAHs were evaluated for compliance with

the provisions given in Commission Regulation (EC) No 8362011 Table 18 in ANNEX 10 gives an

overview on the results of the evaluation Data were not reported in case of empty cells Non-

compliant data are highlighted in Table 18 It should be noted that Regulation (EU) No 8362011

specifies LOD and LOQ with two decimals This was not respected by all participants which

hampered the compliance assessment

27

In summary it can be stated that the majority of participants reported method performance

characteristics for the determination of the four marker PAHs in the food supplement test sample that

are compliant with legislation However some laboratories need to improve their analysis methods

The laboratories in question are urged to adapt to the provisions given in the new legislation

10 Follow-up actions for underperforming laboratories

All laboratories that got questionable or non-satisfactory performance ratings are urged to perform

root cause analysis and to implement corrective actions

As agreed during the EU-RL PAH workshop 2011 the EU-RL PAH will set up follow-up measures in

due time for all NRLs that received for at least one of the four PAHs (BAA BAP BBF and CHR) z-

scores gt 3 as required by Regulation (EC) 8822004 and by the Protocol for management of

underperformance in comparative testing andor lack of collaboration of National Reference

Laboratories (NRLs) with European Union reference laboratories (EU-RLs) activities

These laboratories shall perform as an immediate action root-cause-analysis and shall report to the

EU-RL PAH in writing the identified cause for their underperformance and corrective actions they are

going to take Additionally they shall participate to an independent (non-EU-RL) proficiency test on

the determination of PAHs in food and shall communicate the outcome of this exercise to the EU-RL

PAH

28

11 Conclusions

Fourty seven participants reported results back to the EU-RL The performance of the majority of

participants was good In total 153 out of 225 attributed z-scores were below an absolute value of two

which equals to almost 68 However this percentage is much below the percentages of satisfactory

performance gained in previous studies This might be the consequence of the higher level of difficulty

with respect to matrix composition In addition bias can be concluded from the pattern of performance

indicators of some laboratories

In general the determination of chrysene caused most difficulties to the participants

zeta-Scores were calculated besides z-scores They indicate the agreement of the reported result with

the assigned value with respect to the stated measurement uncertainty The outcome of this rating was

worse than for the z-scores which indicates that the measurement uncertainty estimates were not

realistic Therefore participants underperforming with regard to zeta scores are urged to adapt their

measurement uncertainty statements

The great majority of participants in this inter-laboratory comparison applied analytical methods which

complied with EU legislation with regard to performance characteristics

12 Acknowledgements

The organisers would like to thank Mrs Beatriz de la Calle and Mr Franz Ulberth (all from IRMM

Geel Belgium) for their accurate revision of this report Mr Hakan Emteborg (from IRMM) for

ampouling of the standard solutions and all NRLs and OCLs for their cooperation

29

13 References

1 EU COMMISSION REGULATION (EC) No 7762006 of 23 May 2006 amending Annex VII to Regulation

(EC) No 8822004 of the European Parliament and of the Council as regards Community reference

laboratories Official Journal of the European Union 2006 L 136 p 3-8

Available from

httpeur-lexeuropaeuLexUriServLexUriServdouri=OJL200613600030008ENPDF

2 EU Regulation (EC) No 8822004 of the European Parliament and of the Council of 29 April 2004 on

official controls performed to ensure the verification of compliance with feed and food law animal health

and animal welfare rules Official Journal of the European Communities 2004 L191 p 1-52 Available

from

httpeur-lexeuropaeuLexUriServLexUriServdouri=OJL200419100010052ENPDF

3 EU Opinion of the Scientific Committee on Food on the risks to human health of Polycyclic Aromatic

Hydrocarbons in food (2002) Available from

httpeuropaeuintcommfoodfsscscfout153_enpdf

4 International Agency for Research on Cancer IARC Monographs on the Evaluation of

Carcinogenic Risks to Humans Some Non-heterocyclic Polycyclic Aromatic Hydrocarbons and Some

Related Exposures Volume 92 (2010) Available from

httpmonographsiarcfrENGMonographsvol92mono92pdf

5 EU Commission Regulation (EC) No 3332007 of 28 March 2007 laying down the methods of sampling

and analysis for the official control of the levels of lead cadmium mercury inorganic tin 3-MCPD and

benzo(a)pyrene in foodstuffs Official Journal of the European Union 2007 L 88 p 29-38 Available

from

httpeur-lexeuropaeuLexUriServLexUriServdouri=OJL200708800290038ENPDF

6 EU Commission Regulation (EC) No 18812006 of 19 December 2006 setting maximum levels for certain

contaminants in foodstuffs Official Journal of the European Union 2006 L 364 p 5-24 Available from

httpeur-lexeuropaeuLexUriServLexUriServdouri=OJL200636400050024ENPDF

7 EFSA Scientific Opinion of the Panel on Contaminants in the Food Chain on a request from the European

Commission on Polycyclic Aromatic Hydrocarbons in Food The EFSA Journal 2008 724 p 1-114

Available from

httpwwwefsaeuropaeuEFSAefsa_locale-1178620753812_1211902034842htm

30

8 EU Commission Regulation (EU) No 8352011 amending Regulation (EC) No 18812006 as regards

maximum levels for polycyclic aromatic hydrocarbons in foodstuffs Official Journal of the European

Union 2011 L 215 p 4-8

httpeur-lexeuropaeuLexUriServLexUriServdouri=OJL201121500040008ENPDF

9 EU Commission Regulation (EU) No 8362011 amending Regulation (EC) No 3332007 laying down the methods of sampling and analysis for the official control of the levels of lead cadmium mercury inorganic tin 3-MCPD and benzo(a)pyrene in foodstuffs Official Journal of the European Union 2011 L 215 p 9-16 httpeur-lexeuropaeuLexUriServLexUriServdouri=OJL201121500090016ENPDF

10 ISOIEC 170432010 (2010) Conformity assessment -- General requirements for proficiency testing

providers Geneva Switzerland

11 Thompson M Ellison SLR and Wood R The International Harmonized Protocol for the Proficiency

Testing of Analytical Chemistry Laboratories Pure Appl Chem (2006) 78(1) p 145ndash196

Available from httpmediaiupacorgpublicationspac2006pdf7801x0145pdf

31

14 ANNEXES

ANNEX 1 ndash Announcement of the PT on the IRMM webpage

ANNEX 2 ndash Announcement via e-mail and invitation

ANNEX 3 ndash Announcement of sample dispatch

ANNEX 4 ndash Technical specifications of the calibration solutions

ANNEX 5 ndash Homogeneity and stability of the test material

ANNEX 6 ndash Acknowledgement of sample receipt

ANNEX 7 ndash Questionnaire on details of analysis method

ANNEX 8 ndash Results reported for the food supplement sample

ANNEX 9 ndash Results reported for the standard solutions with undisclosed analyte content

ANNEX 10 ndash Method performance characteristics reported by participants

ANNEX 11 ndash Details of analysis methods reported by participants

32

ANNEX 1 ndash Announcement of the PT on the IRMM webpage

33

ANNEX 2 - Announcement via e-mail and invitation

34

ANNEX 3 - Announcement sample dispatch

Dear MadameSir

The test samples for the proficiency test on the determination of four PAHs in a food supplement (dry extract of St Johns wort) were

dispatched today

Explanation on the design of the study as well as instructions for sample storage and for reporting of results are in the parcel You will

also find your participant key in the parcel which is required for reporting of results

Please check the parcel after arrival for completeness and report the receipt of the test samples as well as their status to us by applying

the sample receipt form which is in the parcel

You are kindly requested to inform us by email if you do not receive the parcel by end of this week

My colleague Donata Lerda will then take care of it

With best regards

Thomas Wenzl

35

ANNEX 4 - Specifications of standard solutions with disclosed concentration

36

ANNEX 4 - continued

37

ANNEX 5 - Homogeneity and stability of the test material

Homogeneity of the test material

Benz[a]anthracene - BAA

Benzo[a]pyrene - BAP

38

ANNEX 5 - continued

Benzo[b]fluoranthene - BBF

Chrysene - CHR

39

ANNEX 5 - continued

Stability of the test material

Table 10 Content of the four target analytes in test materials stored under different

temperatures

BaA BaP BbF CHR

microgkg microgkg microgkg microgkg

Content 31 16 31 69 Storage at room temperature

U (k=2) 05 02 05 17

Content 31 15 29 68 Storage at reference temperature

U (k=2) 04 03 04 10

The applied capillary column does not separate CHR from triphenylene

40

ANNEX 6 ndash Documents sent to participants

41

ANNEX 6 - continued

42

ANNEX 7 Questionnaire on details of analysis method

43

ANNEX 7 - continued

44

ANNEX 7 - continued

45

ANNEX 8 - Results reported by participants for the food supplements test sample Table 11 Analysis results reported by the participants for the content of benz[a]anthracene (BAA) in the food supplements test material Values are presented as reported

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

3600 31 29 29 3 05 2

3602 199 193 178 19 021

3603 253 253 246 251 022 2

3604 199 209 23 213 071 2

3605 301 3 271 291 076 2

3606 302 288 278 289 064 2

3607 456 416 443 438 096 2

3608 796 695 651 714 149 2

3610 182 192 214 196 039 2

3611 414 504 488 469 96 2

3612 3 34 28 31 06 2

3613 32 33 34 33 07 2

3614 182 182 191 185 037 2

3615 3884 3822 3565 376 0752 2

3616 2 2 2 2 01 2

3617 366 37 35 362 046 2

3618 32 32 32 32 05 2

3619 299 305 297 299 048 2

3620 304 294 302 302 045 2

3621 29 26 29 28 04 2

3622 343 325 318 322 052 2

3623 35 3 33 33 07 2

3624

3626 318 31 314 314 126 2

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

5301 31 3 32 31 06 2

5302 314 319 315 316 063 2

5303 3102 2716 2822 2876 27 62

5304 261 263 26 261 57 2

5305 23 22 26 24 20

5306 22 23 23 22 11 2

5307 488 461 317 461

5308 168633 168544 1562025 1644598

5309 16 19 16 17

5310 3 304 311 305 015

5311 477 452 448 459 15 2

5312 506 63 494 543 134 2

5313 353 301 404 353

5314 431 373 378 394 5 096

5315 596 531 51 545 11 2

5316 224 231 235 235 022 2

5317 216 23 221 222 007 2

5318 064 07 067 01 2

5319 111 184 146 147 029 2

5320 515 525 564 534 48

5321 21 5322 13 87 9 87 35 2

5323 38 33 35 33 16 2

46

Figure 6 Results of replicate determinations (indicated by triangles) of benz[a]anthracene (BAA) in the food supplement test material

Horizontal blue lines represent the arithmetic mean value of replicate measurements and blue bars the reported expanded measurement uncertainty (k=2) The assigned value is

plotted as dotted line Red solid lines mark the range of satisfactory performance (|z|le2) Mean values are given for results outside the displayed data range

ProLab 2011

Laboratory

53

1853

1953

0853

0936

1436

0236

1036

1653

2136

0453

1753

0653

1653

0536

0353

0436

2153

0336

0636

0536

0036

2036

1953

1036

1253

0136

2653

0236

1836

2336

2236

1353

1353

2336

1736

1553

1453

0736

0753

1153

2053

1253

1536

0853

2236

11

an

aly

te c

on

ten

t (micro

gk

g)

10

95

9

85

8

75

7

65

6

55

5

45

4

35

3

25

2

15

1

05

0

102

3 468

7

Sample St Johns wort extract replicatesMeasurand Benz[a]anthraceneNo of laboratories 46

Assigned value 298 microgkg (Reference value)Target sd 062 microgkgTolerance limits 174 - 422 microgkg (|Z score| lt 200)

Assig

ned va

lue

Limit of tolerance

Limit of tolerance

47

Table 12 Analysis results reported by the participants for the content of benzo[a]pyrene (BAP) in the food supplements test material Values are presented as reported

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

3600 16 17 17 17 03 2

3602 118 123 13 124 015

3603 165 162 157 161 015 2

3604 134 154 139 143 04 2

3605 164 166 172 167 057 2

3606 181 159 161 167 031 2

3607 279 266 279 274 055 2

3608 57 687 461 573 226 2

3610 226 216 183 206 042 2

3611 289 325 311 308 36 2

3612 24 22 2 22 05 2

3613 17 18 19 18 05 2

3614 08 08 08 08 016 2

3615 1545 1393 1425 145 029 2

3616 16 16 14 15 03 2

3617 177 171 158 169 023 2

3618 2 2 2 2 02 2

3619 207 207 206 207 075 2

3620 165 169 163 166 017 2

3621 44 47 49 47 05 2

3622 212 208 222 213 038 2

3623 2 21 22 21 03 2

3624 18 178 182 18 031 2

3626 172 173 178 174 07 2

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

5301 18 18 18 18 04 2

5302 188 19 187 188 038 2

5303 1691 1426 149 1535 19 80

5304 145 142 15 146 69 2

5305 15 17 18 16 15

5306 16 17 17 16 08 2

5307 164 157 153 157

5308 102669 109324 109035 107009

5309 1 1 12 11

5310 122 122 124 123 006

5311 199 184 215 2 15 2

5312 258 248 239 248 05 2

5313 199 178 245 207

5314 174 171 172 172 11 0948

5315 253 248 213 238 05 2

5316 177 171 173 17 016 2

5317 255 277 276 269 012 2

5318 114 12 117 02 2

5319 19 228 231 216 043 2

5320 303 3 344 316 78

5321 12 12 12 12

5322 13 17 24 17 35 2

5323 2 2 19 2 1 2

48

Figure 7 Results of replicate determinations (indicated by triangles) of benzo[a]pyrene (BAP) in the food supplement test material

Horizontal blue lines represent the arithmetic mean value of replicate measurements and blue bars the reported expanded measurement uncertainty (k=2) The assigned value is

plotted as dotted line Red solid lines mark the range of satisfactory performance (|z|le2) Mean values are given for results outside the displayed data range

ProLab 2011

Laboratory

36

1453

0953

0853

1853

2153

1036

0236

0436

1553

0436

1653

0353

0736

0336

2036

0053

0553

0636

0636

0536

1753

1453

1636

2636

1336

2453

0153

2253

0253

2353

1136

1836

1953

1336

1036

2336

2253

1936

1253

1553

1253

1736

0753

2036

2136

0836

11

an

aly

te c

on

ten

t (micro

gk

g)

55

5

45

4

35

3

25

2

15

1

05

0

573

308

3

Sample St Johns wort extract replicatesMeasurand Benzo[a]pyreneNo of laboratories 48

Assigned value 165 microgkg (Reference value)Target sd 036 microgkgTolerance limits 093 - 237 microgkg (|Z score| lt 200)

Assig

ned va

lue

Limit of tolerance

Limit of tolerance

49

Table 13 Analysis results reported by the participants for the content of benzo[b]fluoranthene (BBF) in the food supplements test material Values are presented as reported

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

3600 32 3 3 31 09 2

3602 173 175 16 17 017

3603 265 252 25 256 025 2

3604 116 108 105 11 028 2

3605 286 225 232 247 074 2

3606 294 273 261 276 046 2

3607 579 579 544 567 113 2

3608 103 1201 876 1036 326 2

3610 173 196 204 191 038 2

3611 546 618 565 576 75 2

3612 3 28 28 29 06 2

3613 28 31 32 31 06 2

3614 135 111 135 127 022 2

3615 4187 3678 4111 4 08 2

3616 2 2 2 2 01 2

3617 33 27 299 035 2

3618 31 31 31 31 05 2

3619 326 324 312 326 064 2

3620 277 28 272 276 069 2

3621 26 25 23 25 04 2

3622 346 336 333 34 052 2

3623 26 28 29 28 02 2

3624

3626 288 28 307 292 117 2

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

5301 29 3 29 29 06 2

5302 297 3 296 298 06 2

5303 2705 2444 2608 2586 22 78

5304 245 254 26 253 85 2

5305 23 23 26 24 15

5306 42 43 4 42 21 2

5307 221 216 241 221

5308 232169 246654 240722 229848

5309 19 22 19 2 0

5310 3 315 325 313 016

5311 262 277 304 281 15 2

5312 27 289 337 299 075 2

5313 321 284 378 328

5314 423 404 385 404 34 083

5315 42 506 416 444 09 2

5316 265 267 277 273 018 2

5317 185 197 195 192 006 2

5318 276 281 279 04 2

5319 396 389 403 396 079 2

5320 623 618 635 625 14

5321 31

5322 24 33 49 33 35 2

5323 38 45 35 45 27 2

50

Figure 8 Results of replicate determinations (indicated by triangles) of benzo[b]fluoranthene (BBF) in the food supplement test material

Horizontal blue lines represent the arithmetic mean value of replicate measurements and blue bars the reported expanded measurement uncertainty (k=2) The assigned value is

plotted as dotted line Red solid lines mark the range of satisfactory performance (|z|le2) Mean values are given for results outside the displayed data range

ProLab 2011

Laboratory

36

0436

1436

0236

1053

1736

1653

0953

0753

0853

0536

2136

0553

0436

0353

0353

1636

0636

2036

2353

1853

1136

1236

2653

0153

0253

1236

1736

1336

0036

1853

2153

1036

1953

1336

2253

2253

2353

1936

1553

1453

0653

1536

0753

2036

0836

11

an

aly

te c

on

ten

t (micro

gk

g)

75

7

65

6

55

5

45

4

35

3

25

2

15

1

05

0

103

6

576

3

Sample St Johns wort extract replicatesMeasurand Benzo[b]fluorantheneNo of laboratories 46

Assigned value 292 microgkg (Reference value)Target sd 060 microgkgTolerance limits 172 - 412 microgkg (|Z score| lt 200)

Assig

ned va

lue

Limit of tolerance

Limit of tolerance

51

Table 14 Analysis results reported by the participants for the content of chrysene (CHR) in the food supplements test material Values are presented as reported

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

3600 5 49 5 5 07 2

3602 32 304 29 304 036

3603 462 469 447 459 048 2

3604 438 428 42 429 119 2

3605 365 41 381 386 085 2

3606 494 411 45 451 124 2

3607 897 774 787 819 18 2

3608 986 887 1168 1014 284 2

3610 418 392 462 424 085 2

3611 833 893 87 865 61 2

3612 6 64 58 61 12 2

3613 41 39 4 4 08 2

3614 272 254 288 271 054 2

3615 605 6028 5964 601 12 2

3616 47 45 49 47 05 2

3617 445 431 389 422 065 2

3618 45 45 45 45 06 2

3619 738 747 72 738 118 2

3620 463 472 446 46 092 2

3621 53 49 51 51 06 2

3622 478 513 505 488 08 2

3623 73 73 72 72 05 2

3624

3626 63 622 634 628 251 2

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

5301 49 49 49 49 1 2

5302 687 683 666 679 136 2

5303 6777 6181 6382 6441 24 70

5304 592 581 6 591 85 2

5305 6 58 64 61 20

5306 49 5 51 49 25 2

5307 524 5 574 524

5308 211488 216782 221146 213243

5309

5310 372 393 394 386 019

5311 43 459 422 437 15 2

5312 934 943 981 953 191 2

5313 793 73 99 838

5314 636 561 565 588 18 0818

5315 547 583 499 542 11 2

5316 292 286 285 282 06 2

5317 52 598 517 545 046 2

5318 368 345 357 05 2

5319 449 471 474 465 116 2

5320 76 924 818 834 99

5321 33

5322 7 7 76 7 35 2

5323 87 73 77 73 37 2

52

Figure 9 Results of replicate determinations (indicated by triangles) of chrysene (CHR) in the food supplement test material

Horizontal blue lines represent the arithmetic mean value of replicate measurements and blue bars the reported expanded measurement uncertainty (k=2) The assigned value is

plotted as dotted line Red solid lines mark the range of satisfactory performance (|z|le2) Mean values are given for results outside the displayed data range

ProLab 2011

Laboratory

53

0836

1453

1636

0253

2153

1836

0553

1036

1336

1736

1036

0453

1136

1836

0636

0336

2053

1936

1653

0136

0036

2253

0636

2153

0753

1553

1753

1453

0436

1536

1253

0536

2653

0353

0253

2236

2336

1953

2336

0753

2053

1353

1236

0836

11

an

aly

te c

on

ten

t (micro

gk

g)

13

12

11

10

9

8

7

6

5

4

3

2

1

0

865

3

Sample St Johns wort extract replicatesMeasurand ChryseneNo of laboratories 45

Assigned value 407 microgkg (Reference value)Target sd 083 microgkgTolerance limits 241 - 573 microgkg (|Z score| lt 200)

Assig

ned va

lue

Limit of tolerance

Limit of tolerance

53

Table 15 Analysis results reported by the participants for the sum of the four marker PAHs in the food supplements test material Values are presented as reported

Laboratory code

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg

3600 128 24 2

3602 79 126

3603 1127 096 2

3604 893 258 2

3605 1091 305 2

3606 1184 238 2

3607 2098 42 2

3608 3336 325 2

3610 1138 17 2

3611 2218 259 2

3612 143 29 2

3613 122 2 2

3614 663 129 2

3615 152 304 2

3616 102 03 2

3617 1252 169 2

3618 128 18 2

3619 157 305 2

3620 1204 12 2

3621 151 19 2

3622 1361 115 2

3623 154 09 2

3624

3626 1409 56 2

Laboratory code

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg

5301 126

5302 37 074 2

5303 13438 92

5304 1251 85 2

5305 125 20

5306 129 65 2

5307 1363

5308 7245598

5309

5310 1127 056

5311 1367 15 2

5312 2042 408 2

5313 1726

5314 1558

5315 177 35 2

5316 96 073 2

5317 1229 066 2

5318 82 05 2

5319 1225 267 2

5320 2309 41

5321

5322 207 35 2

5323 171 103 2

54

ANNEX 9 Results reported for the standard solutions with undisclosed analyte content Table 16 Analysis results reported by the participants for the four marker PAHs in the standard solution in toluene with undisclosed analyte

content Values are presented as reported U measurement uncertainty k coverage factor

benz[a]anthracene benzo[a]pyrene benzo[b]fluoranthene chrysene Laboratory

code Unit Result U k Result U k Result U k Result U k 3600 microgl 38 67 2 25 45 2 211 68 2 275 38 2

3605 microgl 367 184 2 2482 124 2 2592 13 2 235 118 2

3606 microgkg 3763 176 2 2609 142 2 2721 118 2 2539 134 2

3607 microgkg 3706 815 2 2824 565 2 2591 518 2 2587 569 2

3612 microgl 38 08 2 237 11 2 244 18 2 257 02 2

3614 microgl 324 65 2 209 38 2 215 37 2 21 42 2

3616 microgkg 318 07 2 224 14 2 231 26 2 216 1 2

3618 microgkg 417 17 2 284 08 2 292 06 2 297 03 2

3619 microgkg 47 742 2 274 468 2 228 387 2 274 434 2

3620 microgl 495 74 2 294 29 2 321 8 2 321 64 2

3621 microgl 241 22 2 143 13 2 188 16 2 145 14 2

3623 microgkg 476 25 2 311 53 2 269 3 2 297 03 2

3626 microgl 359 38 2 235 23 2 238 27 2 24 18 2

5301 microgkg 426 29 299 289

5302 microgkg 436 87 2 296 59 2 296 59 2 289 58 2

5303 microgl 3721 27 102 22838 19 99 23756 22 99 25486 24 98

5304 microgkg 38 29 2 225 26 2 257 37 2 258 52 2

5305 microgkg 305 225 245 29

5306 microgkg 316 63 2 247 49 2 65 13 2 22 44 2

5307 microgkg 3658 2559 241 2414

5308 microgkg 16854 109324 246654 216782

5311 microgl 374 20 2 279 15 2 268 15 2 272 15 2

5313 microgl 3408 2165 2275 2373

5314 microgkg 395 5 2875 11 3614 34 2905 18

5320 microgl 3267 368 1986 571 28 683 2427 512

5322 microgl 282 35 2 225 35 2 241 35 2 21 35 2

55

Figure 10 Youden plots of the percent deviation of the reported values from the assigned values for the food supplement test sample and the

standard solution in toluene with undisclosed content

(The identity of the respective measurand is given above the figure Results exceeding a difference of more than 100 were omitted)

56

Figure 10 - continued

57

Table 17 Analysis results reported by the participants for the four marker PAHs in the standard solution in acetonitrile with undisclosed

analyte content

Values are presented as reported U measurement uncertainty k coverage factor

benz[a]anthracene benzo[a]pyrene benzo[b]fluoranthene chrysene

Laboratory

code Unit Result U k Result U k Result U k Result U k

3602 microgkg 3675 247 247 244

3603 microgkg 358 086 2 236 028 2 235 028 2 236 057 2

3604 microgl 475 29 2 19 12 2 194 13 2 258 12 2

3608 microgl 3623 078 2 2385 023 2 2343 18 2 2296 092 2

3610 microgkg 369 185 2 25 125 2 251 126 2 244 122 2

3611 microgkg 48 02 2 323 02 2 328 04 2 317 03 2

3613 microgl 332 31 2 261 3 2 249 25 2 239 24 2

3615 microgl 415 2635 2729 2689

3617 microgkg 37 09 2 244 06 2 284 07 2 239 06 2

3622 microgl 37 12 2 25 23 2 257 21 2 238 1 2

3624 microgkg 402 104 2 255 084 2 265 096 2 253 07 2

5306 microgkg 563 113 2 371 74 2 359 72 2 333 67 2

5308 microgkg 168633 102669 232169 211488

5309 microgl 402 268 27

5310 microgl 315 158 25 125 25 125 288 144

5312 microgl 428 043 2 282 028 2 272 027 2 275 028 2

5315 microgkg 387 08 2 256 05 2 251 05 2 253 05 2

5316 microgl 3839 342 2 2485 191 2 2559 168 2 239 51 2

5317 microgl 2466 043 2 2212 034 2 2472 016 2 2146 068 2

5319 microgl 414 41 2 279 28 2 268 27 2 26 31 2

5321 microgkg 476 316 326 328

5322 microgl 2547 35 2

5323 microgkg 405 203 2 239 119 2 255 153 2 201 11 2

58

Figure 11 Youden plots of the percent deviation of the reported values from the assigned values for the food supplement test sample and the

standard solution in acetonitrile with undisclosed content

(The identity of the respective measurand is given above the figure Results exceeding a difference of more than 100 were omitted)

59

Figure 11 - continued

60

ANNEX 10 Table 18 Method performance parameters reported by the participants

Laboratory code

Analyte LOD LOQ Recovery Analyte LOD LOQ Recovery

microgkg microgkg microgkg microgkg 3600 BaA 03 06 978 BaP 03 07 9483602 BaA 007 021 926 BaP 005 015 993603 BaA 0011 05 987 BaP 0005 051 9553604 BaA 02 04 86 BaP 02 04 883605 BaA 0025 005 118 BaP 0025 005 1083606 BaA 001 003 65 BaP 001 003 703607 BaA 021 071 74 BaP 027 089 943608 BaA 016 048 8213 BaP 015 046 83683610 BaA 02 05 85 BaP 005 02 853611 BaA BaP 3612 BaA 1 117 BaP 1 1203613 BaA 01 03 90 BaP 01 03 903614 BaA 02 05 96 BaP 02 05 933615 BaA 005 015 110 BaP 005 015 1053616 BaA 01 05 108 BaP 01 05 753617 BaA 018 037 405 BaP 009 018 3873618 BaA 01 03 63 BaP 01 03 593619 BaA 005 005 59 BaP 033 033 543620 BaA 0009 0018 64 BaP 0005 001 553621 BaA 007 02 84 BaP 007 02 923622 BaA 01 03 1101 BaP 01 03 9853623 BaA 02 08 105 BaP 02 08 1013624 BaA BaP 01 033 8513625 BaA BaP 3626 BaA BaP 5301 BaA 003 01 102 BaP 003 01 1005302 BaA 01 03 78 BaP 01 03 865303 BaA 003 01 62 BaP 003 01 805304 BaA 1 1 70 BaP 1 1 955305 BaA 02 05 85 BaP 02 05 855306 BaA 03 1 699 BaP 03 1 905307 BaA 6537 BaP 81165308 BaA 0088 0265 99 BaP 00262 00785 1005309 BaA 01 08 92 BaP 01 08 925310 BaA 01 03 BaP 01 03 905311 BaA 002 005 80 BaP 002 004 915312 BaA 03 09 105 BaP 03 09 1055313 BaA 04 08 BaP 04 08 5314 BaA 001 002 96 BaP 001 002 9485315 BaA 01 03 50 - 100 BaP 01 03 50 - 100 5316 BaA 002 011 827 BaP 004 019 10445317 BaA 05 1 76 BaP 05 1 755318 BaA BaP 5319 BaA 009 029 951 BaP 011 041 995320 BaA 12 18 946 BaP 06 1 8345321 BaA 03 05 BaP 05 1 5322 BaA 1 2 100 BaP 1 2 1005323 BaA 26 BaP 26

61

ANNEX 10 Table 18 - continued

Laboratory code Analyte LOD LOQ Recovery Analyte LOD LOQ Recovery

microgkg microgkg microgkg microgkg 3600 BbF 03 05 96 CHR 02 03 10153602 BbF 015 045 947 CHR 003 009 9193603 BbF 0014 05 1012 CHR 0004 05 10033604 BbF 02 04 85 CHR 02 04 873605 BbF 005 01 94 CHR 0025 005 863606 BbF 001 003 70 CHR 001 003 653607 BbF 018 06 100 CHR 014 028 943608 BbF 015 046 7979 CHR 015 045 71253610 BbF 005 02 85 CHR 02 05 853611 BbF CHR 3612 BbF 1 118 CHR 1 1173613 BbF 01 03 90 CHR 01 03 903614 BbF 01 04 96 CHR 02 05 953615 BbF 008 025 109 CHR 003 01 1083616 BbF 01 05 78 CHR 01 05 723617 BbF 035 07 38 CHR 012 024 4163618 BbF 01 03 62 CHR 01 03 623619 BbF 016 016 63 CHR 01 01 593620 BbF 0003 0006 55 CHR 001 002 653621 BbF 007 02 103 CHR 007 02 893622 BbF 02 06 963 CHR 03 09 1043623 BbF 02 08 106 CHR 02 08 1193624 BbF CHR 3625 BbF CHR 3626 BbF CHR 5301 BbF 003 01 96 CHR 003 01 1145302 BbF 01 03 84 CHR 01 03 835303 BbF 003 01 78 CHR 003 01 705304 BbF 1 1 86 CHR 1 1 705305 BbF 02 05 90 CHR 02 05 955306 BbF 03 1 1435 CHR 03 1 1185307 BbF 8116 CHR 65375308 BbF 0079 0238 99 CHR 009 026 1005309 BbF 01 08 92 CHR 5310 BbF 07 2 CHR 01 03 5311 BbF 002 004 95 CHR 002 004 895312 BbF 03 09 105 CHR 03 09 1055313 BbF 04 08 CHR 04 08 5314 BbF 001 002 83 CHR 001 002 8185315 BbF 02 05 50 -100 CHR 02 05 50 - 100 5316 BbF 005 024 814 CHR 001 007 9595317 BbF 05 1 73 CHR 05 1 785318 BbF CHR 5319 BbF 011 038 946 CHR 013 043 9675320 BbF 07 09 1194 CHR 23 32 10455321 BbF 05 1 CHR 05 1 5322 BbF 1 2 100 CHR 1 2 1005323 BbF 26 CHR 26

62

ANNEX 11 Table 19 Details of analysis method reported by the participants Data are presented as reported

Laboratory code

Analysis technique

Instrument calibration

Internal standards applied

Sample clean up

Details of sample

clean up

Extraction technique

Details of sample

extraction

3600 6) GC-MS

2) Internal standardisation with labelled substances

Deuterated PAH mix 9 (Ehrenstorfer)

1) Saponification 2) LiquidLiquid partitioning

D) Other Liquidliquid partitioning

3602 2) HPLC-UV-FLD

1) External calibration

3) Gel permeation chromatography

A) Pressurised liquid extraction (PLE)

3603 1) HPLC-FLD

1) External calibration

3) Gel permeation chromatography

D) Other 1 minute with dichloromethane in vortex

3604 2) HPLC-UV-FLD

2) Internal standardisation with labelled substances

DiP D14

5) Donor acceptor complex chromatography

D) Other LL

3605 1) HPLC-FLD

1) External calibration

3) Gel permeation chromatography

C) Soxhlet extraction

3606 7) GC-MSMS

2) Internal standardisation with labelled substances

BaP 13C4 CHR 13C6 BaA 13C6 BbF 13C6

4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

3607 6) GC-MS

2) Internal standardisation with labelled substances

BaA-d12 CHR-d12 BbF-d12 BkF-d12 BaP-d12 ICP-d12 BgP-d12 DiP-d14

1) Saponification

D) Other solvent partitioning

3608 1) HPLC-FLD

1) External calibration

6) Other Liquid extraction

D) Other

Extraction in a rotary agitator and centrifugation

3610 2) HPLC-UV-FLD

1) External calibration

4) Solid phase extraction

B) Sonication

3611

3612 6) GC-MS

2) Internal standardisation with labelled substances

chrysene-d12 dibenzo(ah)anthracene-d14 dibenzo(ai)pyrene-d14

4) Solid phase extraction

B) Sonication

3613 5) LC-MSMS

3) Internal standardisation with unlabelled substances

I use Chrysene-D12 3) Gel permeation chromatography

D) Other extraction with hexan

63

Table 19 - continued

Laboratory code

Analysis technique

Instrument calibration

Internal standards applied

Sample clean up Details of sample

clean up

Extraction technique

Details of sample

extraction

3614 6) GC-MS 1) External calibration

3) Gel permeation chromatography

B) Sonication

3615 1) HPLC-FLD

1) External calibration

6) Other PLE GPC

A) Pressurised liquid extraction (PLE)

3616 6) GC-MS

2) Internal standardisation with labelled substances

Benzo(a)pyrene-13C4 Benzo(a)anthracene-13C6 Benzo(b)fluoranthene-13C6 Chrysene-13C6

2) LiquidLiquid partitioning 4) Solid phase extraction

B) Sonication

3617 1) HPLC-FLD

1) External calibration

4) Solid phase extraction

C) Soxhlet extraction

3618 6) GC-MS

2) Internal standardisation with labelled substances

deuterated standards

1) Saponification 4) Solid phase extraction

D) Other Handshaken with cyclohexane

3619 6) GC-MS

2) Internal standardisation with labelled substances

C13 Labelled US EPA 16

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

D) Other Homegenisation amp saponiofication

3620 9) GC-HRMS

2) Internal standardisation with labelled substances

mix of deuterated PAHs (BAA-D12 CHR-D12 BAP-D12 BBF-D12)

3) Gel permeation chromatography 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

3621 6) GC-MS

2) Internal standardisation with labelled substances

Chrysene D12 Benzo(a)pyrene D12 Benzo(b)fluoranthene D12 Benzo(a)anthracene

2) LiquidLiquid partitioning 4) Solid phase extraction

B) Sonication

3622 1) HPLC-FLD

4) Standard addition method

4) Solid phase extraction

D) Other

Extraction with cyclohexane by heating under reflux

3623 7) GC-MSMS

2) Internal standardisation with labelled substances

C13 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

3624 2) HPLC-UV-FLD

1) External calibration

2) LiquidLiquid partitioning

D) Other hot solvent extraction

3626

64

Table 19 - continued

Laboratory code

Analysis technique

Instrument calibration

Internal standards

applied

Sample clean up

Details of sample

clean up

Extraction technique

Details of sample

extraction

5301 7) GC-MSMS

2) Internal standardisation with labelled substances

EACH SUBSTANCE C13

4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5302 7) GC-MSMS

2) Internal standardisation with labelled substances

Isotope labelled PAHs 13C for each analyte

4) Solid phase extraction

C) Soxhlet extraction

5303 7) GC-MSMS

3) Internal standardisation with unlabelled substances

HAP C13 4) Solid phase extraction

D) Other automate ASE200 DIONEX

5304 7) GC-MSMS

2) Internal standardisation with labelled substances

with C13 for each HAP

2) LiquidLiquid partitioning 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5305 7) GC-MSMS

2) Internal standardisation with labelled substances

C13 isotopiques internal standards

4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5306 7) GC-MSMS

2) Internal standardisation with labelled substances

C13 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5307 6) GC-MS

2) Internal standardisation with labelled substances

1) Saponification 2) LiquidLiquid partitioning

D) Other solvent extraction

5308 1) HPLC-FLD 1) External calibration

4) Solid phase extraction

B) Sonication

5309 1) HPLC-FLD 1) External calibration

4) Solid phase extraction

D) Other only SPE

5310 1) HPLC-FLD 1) External calibration

1) Saponification 2) LiquidLiquid partitioning 3) Gel permeation chromatography

C) Soxhlet extraction

5311 6) GC-MS

2) Internal standardisation with labelled substances

13 C Chrysene 13 C Benz(a)pyrene

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

B) Sonication

5312 1) HPLC-FLD

3) Internal standardisation with unlabelled substances

Benzo(b)chrysen

2) LiquidLiquid partitioning 4) Solid phase extraction

Quechers D) Other Quechers

65

Table 19 - continued

Laboratory code

Analysis technique

Instrument calibration

Internal standards applied

Sample clean up Details of sample

clean up

Extraction technique

Details of sample extraction

5313 6) GC-MS

2) Internal standardisation with labelled substances

Benz(a)anthracene D12 Benzo(b)fluranthene D12 Benzo(a)pyrene D12 Chrysene D12

1) Saponification 2) LiquidLiquid partitioning 3) Gel permeation chromatography

A) Pressurised liquid extraction (PLE)

5314 6) GC-MS

2) Internal standardisation with labelled substances

Benzo(a)pyren d12 2) LiquidLiquid partitioning

D) Other Extraction with

5315 1) HPLC-FLD

3) Internal standardisation with unlabelled substances

Benz(b)chrysen

1) Saponification 2) LiquidLiquid partitioning

D) Other shaker

5316 1) HPLC-FLD

1) External calibration

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

D) Other

LiquidLiquid extraction with cyclohexane and dimethylformamide

5317 7) GC-MSMS

1) External calibration

3) Gel permeation chromatography 6) Other

silica gel D) Other VDLUFA online method

5318

5319 1) HPLC-FLD

3) Internal standardisation with unlabelled substances

Benzo(b)chrysene 3) Gel permeation chromatography

A) Pressurised liquid extraction (PLE)

5320 6) GC-MS

3) Internal standardisation with unlabelled substances

22-binaphthyl

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

D) Other 3 hours refluxing

5321 1) HPLC-FLD

2) Internal standardisation with labelled substances

6-meacutethylchrysegravene

1) Saponification 2) LiquidLiquid partitioning

B) Sonication

5322 6) GC-MS

2) Internal standardisation with labelled substances

naftalene-d8 acenaphtalene-d10 phenanthrene-d10 chrysene-d12 perylene-d12 benzo(e)anthracene-d12

1) Saponification 2) LiquidLiquid partitioning 6) Other

clean-up with silica packed column

C) Soxhlet extraction

5323 1) HPLC-FLD

1) External calibration

4) Solid phase extraction 6) Other

Accelerated Solvent Extraction

A) Pressurised liquid extraction (PLE)

66

European Commission EUR 25245 EN ndash Joint Research Centre ndash Institute for Reference Materials and Measurements Title Report on the 8th inter-laboratory comparison organised by the European Union Reference Laboratory for Polycyclic Aromatic Hydrocarbons - Four marker PAHs in a dry extract of St Johns wort Author(s) Radoslav Lizak Szilard Szilagyi Philippe Verlinde and Thomas WENZL Luxembourg Publications Office of the European Union 2012 ndash 66 pp ndash 201 x 297 cm EUR ndash Scientific and Technical Research series ndash ISSN 1831-9424 ISBN 978-92-79-23337-1 doi 10278759387 Abstract The proficiency test here reported concerned the determination of the contents of four marker polycyclic aromatic hydrocarbons (PAHs) and of their sum in an food supplements test sample The set of marker PAHs consists of benz[a]anthacene benzo[a]pyrene benzo[b]fluoranthene and chrysene Participants to these PT were National Reference Laboratories for PAHs (NRLs-PAHs) and EU official food control laboratories The number of participants was in total 48

The PT was organised according ISO Standard 170432010

The test material used was a commercial dry extract of St Johns wort Participants also received a solution of the same PAHs either in acetonitrile or in toluene for checking their instrument calibration

The results from participants were rated with z-scores and zeta-scores About 68 of the reported results were attributed with z-scores with an absolute value of below two which is the threshold for satisfactory performance

How to obtain EU publications

Our priced publications are available from EU Bookshop (httpbookshopeuropaeu) where you can place

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The mission of the JRC is to provide customer-driven scientific and technical support

for the conception development implementation and monitoring of EU policies As a

service of the European Commission the JRC functions as a reference centre of

science and technology for the Union Close to the policy-making process it serves

the common interest of the Member States while being independent of special

interests whether private or national

L

A-N

A-252

45-EN

-N

• 1 Summary
• 2 Introduction
• 3 Scope
• 4 Participating Laboratories
• 5 Time frame
• 6 Confidentiality
• 7 Test materials
• • 71 1Preparation of test materials
  • 72 Assigned values and standard deviation for proficiency assessment
  • 73 Homogeneity and stability
  • 74 Sample dispatch
  • • 8 Design of the proficiency test
    • 9 Evaluation of the results
    • • 91 2General
      • 92 21BEvaluation criteria
      • 93 Performance of participants
      • 94 Evaluation of the influence of analysis method
      • 95 Evaluation of the influence of calibration
      • 96 26Evaluation of compliance with legislation
      • • 10 Follow-up actions for underperforming laboratories
        • 11 1Conclusions
        • 12 1Acknowledgements
        • 13 References

21

Table 9 Compilation of zeta-scores calculated from the ldquoresults for proficiency assessment

reported by the OCLs for test material St Johns wort the reported measurement

uncertainty and the uncertainty of the analyte content of the test material zeta-scores

outside the satisfactory range (|zeta| gt 2) are indicated by red bold font yellow

highlighted cells ugtUf Empty cells denote analytes for which results were not

reported

22

94 Evaluation of the influence of analysis method

The information on the applied analysis method (ANNEX 11) was used to group the performance

indicators attributed to the reported results according to the applied analysis technique Two groups of

data were formed They represent results obtained by high performance liquid chromatography-

fluorescence detection (HPLC-FD about 40 ) and by gas chromatography-mass spectrometry (GC-

MS about 60 ) respectively Kernel density plots were constructed based on the respective z-scores

Figure 3A to 3E show the different Kernel density plots

Figure 3 Kernel density plots of z-scores attributed to the results for proficiency assessment

which were reported for the food supplements test sample Results were classified

according to the applied analysis technique

A) benz[a]anthracene

B) benzo[a]pyrene

23

Figure 3 continued

C) benzo[b]fluoranthene

D) chrysene

E) sum of four marker PAHs

The kernel density plots for BAP and BBF are almost identical and centred at or close to a z-score

value of zero as can be seen in Figure 3B and 3C Therefore bias introduced by the type of analysis

method is unlikely for these two analytes The situation is different for BAA and CHR In case of

BAA the kernel density distribution of z-scores based on HPLC-FD is shifted to negative values

24

(Figure 3A) which indicates a tendency to underestimate the BAA content in this particular

samplematrix when applying HPLC-FD This might be reasoned by separationintegration problems

between BAA and CHR peaks A few participants (from the HPLC-FD subgroup) that got negative

z-scores for BAA overestimated the CHR content This caused the slight shift of the density trace for

HPLC-FD towards negative values Removing this data from the evaluation would result in a density

trace with the mode at zero

The opposite is the case for CHR (Figure 3D) The density distribution of the z-scores attributed to

HPLC-FD data is centred almost at zero However the distribution for data based on GC-MS is shifted

to positive values indicating strong positive bias This bias might be caused by a lack of selectivity of

the analysis method The test material contains triphenylene which is isobaric to chrysene

Consequently it cannot be distinguished from chrysene by means of mass spectrometry These two

compounds have to be separated chromatographically which is not easy They coelute on unpolar and

on most mid-polar capillary columns Columns that allow their separation were commercialised only

recently and might not be available in all laboratories Therefore laboratories which determine the

four EU marker PAHs by means of GC-MS shall pay attention to the chromatographic separation of

triphenylene and chrysene A lack of chromatographic resolution might lead to strong positive bias of

GC-MS methods and to false conclusions on the marketability of the tested sample

The chromatographic separation of chrysene and triphenylene is not a problem with HPLC which is

reflected in the position of the density plot for chrysene However attention has to be paid in HPLC-

FD analysis to a proper clean up of the sample extract in order to eliminate interferences that show

similar retention as BAA and CHR

95 Evaluation of the influence of calibration

The participants received together with the food supplement test sample two standard solutions in

depending of their choice either acetonitrile or toluene The analyte content of one solution was

disclosed to the participants whereas the content of the other solution was kept undisclosed The

content values of the latter are presented in Table 5 Analysis results for the solution with undisclosed

content had to be reported to the PT organiser The respective data are presented in Annex 9

Percent deviations of the reported results from the assigned values were calculated for both the food

supplement test sample and the standard solution with undisclosed analyte content Youden plots were

prepared in order to visualize the influence of instrument calibration on the results reported for the

food supplement sample Two plots are discussed in the following The whole set of Youden plots are

given in Annex 9

25

Figure 4 Youden plot of the percent deviations of values reported for benzo[a]pyrene from the

assigned values of the food supplement test material and the standard solution in

toluene with undisclosed content

Figure 4 reveals that a number of participants reported results with similar percent deviations from the

assigned values of both the results for the food supplement material and the standard solution in

toluene with undisclosed content This correlation indicates that over- respectively underestimation of

the analyte content of the food supplement material might be caused by biased instrument calibration

This finding underpins the importance of thorough calibration standard preparation However the data

was not in all cases conclusive as some laboratories performed well for the food supplement samples

but reported biased results for the standard solution with undisclosed content (eg Figure 4 participants

3620 and 5305) This fact cannot be explained yet

Figure 5 presents the respective Youden plot for chrysene in the food supplement test material and the

standard solution in toluene with undisclosed content From this graph it can be concluded that the

overestimation of the chrysene content of the food supplement material was not caused by instrument

calibration The results for the solvent solution of many laboratories which underperformed for the

26

food supplement sample agreed well with the assigned value This supports the conclusion that the

overestimation is caused by the determination of an interferent most probably triphenylene

Figure 5 Youden plot of the percent deviations of values reported for chrysene from the

assigned values of the food supplement test material and the standard solution in

toluene with undisclosed content

96 Evaluation of compliance with legislation

The method performance data reported for the four marker PAHs were evaluated for compliance with

the provisions given in Commission Regulation (EC) No 8362011 Table 18 in ANNEX 10 gives an

overview on the results of the evaluation Data were not reported in case of empty cells Non-

compliant data are highlighted in Table 18 It should be noted that Regulation (EU) No 8362011

specifies LOD and LOQ with two decimals This was not respected by all participants which

hampered the compliance assessment

27

In summary it can be stated that the majority of participants reported method performance

characteristics for the determination of the four marker PAHs in the food supplement test sample that

are compliant with legislation However some laboratories need to improve their analysis methods

The laboratories in question are urged to adapt to the provisions given in the new legislation

10 Follow-up actions for underperforming laboratories

All laboratories that got questionable or non-satisfactory performance ratings are urged to perform

root cause analysis and to implement corrective actions

As agreed during the EU-RL PAH workshop 2011 the EU-RL PAH will set up follow-up measures in

due time for all NRLs that received for at least one of the four PAHs (BAA BAP BBF and CHR) z-

scores gt 3 as required by Regulation (EC) 8822004 and by the Protocol for management of

underperformance in comparative testing andor lack of collaboration of National Reference

Laboratories (NRLs) with European Union reference laboratories (EU-RLs) activities

These laboratories shall perform as an immediate action root-cause-analysis and shall report to the

EU-RL PAH in writing the identified cause for their underperformance and corrective actions they are

going to take Additionally they shall participate to an independent (non-EU-RL) proficiency test on

the determination of PAHs in food and shall communicate the outcome of this exercise to the EU-RL

PAH

28

11 Conclusions

Fourty seven participants reported results back to the EU-RL The performance of the majority of

participants was good In total 153 out of 225 attributed z-scores were below an absolute value of two

which equals to almost 68 However this percentage is much below the percentages of satisfactory

performance gained in previous studies This might be the consequence of the higher level of difficulty

with respect to matrix composition In addition bias can be concluded from the pattern of performance

indicators of some laboratories

In general the determination of chrysene caused most difficulties to the participants

zeta-Scores were calculated besides z-scores They indicate the agreement of the reported result with

the assigned value with respect to the stated measurement uncertainty The outcome of this rating was

worse than for the z-scores which indicates that the measurement uncertainty estimates were not

realistic Therefore participants underperforming with regard to zeta scores are urged to adapt their

measurement uncertainty statements

The great majority of participants in this inter-laboratory comparison applied analytical methods which

complied with EU legislation with regard to performance characteristics

12 Acknowledgements

The organisers would like to thank Mrs Beatriz de la Calle and Mr Franz Ulberth (all from IRMM

Geel Belgium) for their accurate revision of this report Mr Hakan Emteborg (from IRMM) for

ampouling of the standard solutions and all NRLs and OCLs for their cooperation

29

13 References

1 EU COMMISSION REGULATION (EC) No 7762006 of 23 May 2006 amending Annex VII to Regulation

(EC) No 8822004 of the European Parliament and of the Council as regards Community reference

laboratories Official Journal of the European Union 2006 L 136 p 3-8

Available from

httpeur-lexeuropaeuLexUriServLexUriServdouri=OJL200613600030008ENPDF

2 EU Regulation (EC) No 8822004 of the European Parliament and of the Council of 29 April 2004 on

official controls performed to ensure the verification of compliance with feed and food law animal health

and animal welfare rules Official Journal of the European Communities 2004 L191 p 1-52 Available

from

httpeur-lexeuropaeuLexUriServLexUriServdouri=OJL200419100010052ENPDF

3 EU Opinion of the Scientific Committee on Food on the risks to human health of Polycyclic Aromatic

Hydrocarbons in food (2002) Available from

httpeuropaeuintcommfoodfsscscfout153_enpdf

4 International Agency for Research on Cancer IARC Monographs on the Evaluation of

Carcinogenic Risks to Humans Some Non-heterocyclic Polycyclic Aromatic Hydrocarbons and Some

Related Exposures Volume 92 (2010) Available from

httpmonographsiarcfrENGMonographsvol92mono92pdf

5 EU Commission Regulation (EC) No 3332007 of 28 March 2007 laying down the methods of sampling

and analysis for the official control of the levels of lead cadmium mercury inorganic tin 3-MCPD and

benzo(a)pyrene in foodstuffs Official Journal of the European Union 2007 L 88 p 29-38 Available

from

httpeur-lexeuropaeuLexUriServLexUriServdouri=OJL200708800290038ENPDF

6 EU Commission Regulation (EC) No 18812006 of 19 December 2006 setting maximum levels for certain

contaminants in foodstuffs Official Journal of the European Union 2006 L 364 p 5-24 Available from

httpeur-lexeuropaeuLexUriServLexUriServdouri=OJL200636400050024ENPDF

7 EFSA Scientific Opinion of the Panel on Contaminants in the Food Chain on a request from the European

Commission on Polycyclic Aromatic Hydrocarbons in Food The EFSA Journal 2008 724 p 1-114

Available from

httpwwwefsaeuropaeuEFSAefsa_locale-1178620753812_1211902034842htm

30

8 EU Commission Regulation (EU) No 8352011 amending Regulation (EC) No 18812006 as regards

maximum levels for polycyclic aromatic hydrocarbons in foodstuffs Official Journal of the European

Union 2011 L 215 p 4-8

httpeur-lexeuropaeuLexUriServLexUriServdouri=OJL201121500040008ENPDF

9 EU Commission Regulation (EU) No 8362011 amending Regulation (EC) No 3332007 laying down the methods of sampling and analysis for the official control of the levels of lead cadmium mercury inorganic tin 3-MCPD and benzo(a)pyrene in foodstuffs Official Journal of the European Union 2011 L 215 p 9-16 httpeur-lexeuropaeuLexUriServLexUriServdouri=OJL201121500090016ENPDF

10 ISOIEC 170432010 (2010) Conformity assessment -- General requirements for proficiency testing

providers Geneva Switzerland

11 Thompson M Ellison SLR and Wood R The International Harmonized Protocol for the Proficiency

Testing of Analytical Chemistry Laboratories Pure Appl Chem (2006) 78(1) p 145ndash196

Available from httpmediaiupacorgpublicationspac2006pdf7801x0145pdf

31

14 ANNEXES

ANNEX 1 ndash Announcement of the PT on the IRMM webpage

ANNEX 2 ndash Announcement via e-mail and invitation

ANNEX 3 ndash Announcement of sample dispatch

ANNEX 4 ndash Technical specifications of the calibration solutions

ANNEX 5 ndash Homogeneity and stability of the test material

ANNEX 6 ndash Acknowledgement of sample receipt

ANNEX 7 ndash Questionnaire on details of analysis method

ANNEX 8 ndash Results reported for the food supplement sample

ANNEX 9 ndash Results reported for the standard solutions with undisclosed analyte content

ANNEX 10 ndash Method performance characteristics reported by participants

ANNEX 11 ndash Details of analysis methods reported by participants

32

ANNEX 1 ndash Announcement of the PT on the IRMM webpage

33

ANNEX 2 - Announcement via e-mail and invitation

34

ANNEX 3 - Announcement sample dispatch

Dear MadameSir

The test samples for the proficiency test on the determination of four PAHs in a food supplement (dry extract of St Johns wort) were

dispatched today

Explanation on the design of the study as well as instructions for sample storage and for reporting of results are in the parcel You will

also find your participant key in the parcel which is required for reporting of results

Please check the parcel after arrival for completeness and report the receipt of the test samples as well as their status to us by applying

the sample receipt form which is in the parcel

You are kindly requested to inform us by email if you do not receive the parcel by end of this week

My colleague Donata Lerda will then take care of it

With best regards

Thomas Wenzl

35

ANNEX 4 - Specifications of standard solutions with disclosed concentration

36

ANNEX 4 - continued

37

ANNEX 5 - Homogeneity and stability of the test material

Homogeneity of the test material

Benz[a]anthracene - BAA

Benzo[a]pyrene - BAP

38

ANNEX 5 - continued

Benzo[b]fluoranthene - BBF

Chrysene - CHR

39

ANNEX 5 - continued

Stability of the test material

Table 10 Content of the four target analytes in test materials stored under different

temperatures

BaA BaP BbF CHR

microgkg microgkg microgkg microgkg

Content 31 16 31 69 Storage at room temperature

U (k=2) 05 02 05 17

Content 31 15 29 68 Storage at reference temperature

U (k=2) 04 03 04 10

The applied capillary column does not separate CHR from triphenylene

40

ANNEX 6 ndash Documents sent to participants

41

ANNEX 6 - continued

42

ANNEX 7 Questionnaire on details of analysis method

43

ANNEX 7 - continued

44

ANNEX 7 - continued

45

ANNEX 8 - Results reported by participants for the food supplements test sample Table 11 Analysis results reported by the participants for the content of benz[a]anthracene (BAA) in the food supplements test material Values are presented as reported

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

3600 31 29 29 3 05 2

3602 199 193 178 19 021

3603 253 253 246 251 022 2

3604 199 209 23 213 071 2

3605 301 3 271 291 076 2

3606 302 288 278 289 064 2

3607 456 416 443 438 096 2

3608 796 695 651 714 149 2

3610 182 192 214 196 039 2

3611 414 504 488 469 96 2

3612 3 34 28 31 06 2

3613 32 33 34 33 07 2

3614 182 182 191 185 037 2

3615 3884 3822 3565 376 0752 2

3616 2 2 2 2 01 2

3617 366 37 35 362 046 2

3618 32 32 32 32 05 2

3619 299 305 297 299 048 2

3620 304 294 302 302 045 2

3621 29 26 29 28 04 2

3622 343 325 318 322 052 2

3623 35 3 33 33 07 2

3624

3626 318 31 314 314 126 2

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

5301 31 3 32 31 06 2

5302 314 319 315 316 063 2

5303 3102 2716 2822 2876 27 62

5304 261 263 26 261 57 2

5305 23 22 26 24 20

5306 22 23 23 22 11 2

5307 488 461 317 461

5308 168633 168544 1562025 1644598

5309 16 19 16 17

5310 3 304 311 305 015

5311 477 452 448 459 15 2

5312 506 63 494 543 134 2

5313 353 301 404 353

5314 431 373 378 394 5 096

5315 596 531 51 545 11 2

5316 224 231 235 235 022 2

5317 216 23 221 222 007 2

5318 064 07 067 01 2

5319 111 184 146 147 029 2

5320 515 525 564 534 48

5321 21 5322 13 87 9 87 35 2

5323 38 33 35 33 16 2

46

Figure 6 Results of replicate determinations (indicated by triangles) of benz[a]anthracene (BAA) in the food supplement test material

Horizontal blue lines represent the arithmetic mean value of replicate measurements and blue bars the reported expanded measurement uncertainty (k=2) The assigned value is

plotted as dotted line Red solid lines mark the range of satisfactory performance (|z|le2) Mean values are given for results outside the displayed data range

ProLab 2011

Laboratory

53

1853

1953

0853

0936

1436

0236

1036

1653

2136

0453

1753

0653

1653

0536

0353

0436

2153

0336

0636

0536

0036

2036

1953

1036

1253

0136

2653

0236

1836

2336

2236

1353

1353

2336

1736

1553

1453

0736

0753

1153

2053

1253

1536

0853

2236

11

an

aly

te c

on

ten

t (micro

gk

g)

10

95

9

85

8

75

7

65

6

55

5

45

4

35

3

25

2

15

1

05

0

102

3 468

7

Sample St Johns wort extract replicatesMeasurand Benz[a]anthraceneNo of laboratories 46

Assigned value 298 microgkg (Reference value)Target sd 062 microgkgTolerance limits 174 - 422 microgkg (|Z score| lt 200)

Assig

ned va

lue

Limit of tolerance

Limit of tolerance

47

Table 12 Analysis results reported by the participants for the content of benzo[a]pyrene (BAP) in the food supplements test material Values are presented as reported

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

3600 16 17 17 17 03 2

3602 118 123 13 124 015

3603 165 162 157 161 015 2

3604 134 154 139 143 04 2

3605 164 166 172 167 057 2

3606 181 159 161 167 031 2

3607 279 266 279 274 055 2

3608 57 687 461 573 226 2

3610 226 216 183 206 042 2

3611 289 325 311 308 36 2

3612 24 22 2 22 05 2

3613 17 18 19 18 05 2

3614 08 08 08 08 016 2

3615 1545 1393 1425 145 029 2

3616 16 16 14 15 03 2

3617 177 171 158 169 023 2

3618 2 2 2 2 02 2

3619 207 207 206 207 075 2

3620 165 169 163 166 017 2

3621 44 47 49 47 05 2

3622 212 208 222 213 038 2

3623 2 21 22 21 03 2

3624 18 178 182 18 031 2

3626 172 173 178 174 07 2

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

5301 18 18 18 18 04 2

5302 188 19 187 188 038 2

5303 1691 1426 149 1535 19 80

5304 145 142 15 146 69 2

5305 15 17 18 16 15

5306 16 17 17 16 08 2

5307 164 157 153 157

5308 102669 109324 109035 107009

5309 1 1 12 11

5310 122 122 124 123 006

5311 199 184 215 2 15 2

5312 258 248 239 248 05 2

5313 199 178 245 207

5314 174 171 172 172 11 0948

5315 253 248 213 238 05 2

5316 177 171 173 17 016 2

5317 255 277 276 269 012 2

5318 114 12 117 02 2

5319 19 228 231 216 043 2

5320 303 3 344 316 78

5321 12 12 12 12

5322 13 17 24 17 35 2

5323 2 2 19 2 1 2

48

Figure 7 Results of replicate determinations (indicated by triangles) of benzo[a]pyrene (BAP) in the food supplement test material

Horizontal blue lines represent the arithmetic mean value of replicate measurements and blue bars the reported expanded measurement uncertainty (k=2) The assigned value is

plotted as dotted line Red solid lines mark the range of satisfactory performance (|z|le2) Mean values are given for results outside the displayed data range

ProLab 2011

Laboratory

36

1453

0953

0853

1853

2153

1036

0236

0436

1553

0436

1653

0353

0736

0336

2036

0053

0553

0636

0636

0536

1753

1453

1636

2636

1336

2453

0153

2253

0253

2353

1136

1836

1953

1336

1036

2336

2253

1936

1253

1553

1253

1736

0753

2036

2136

0836

11

an

aly

te c

on

ten

t (micro

gk

g)

55

5

45

4

35

3

25

2

15

1

05

0

573

308

3

Sample St Johns wort extract replicatesMeasurand Benzo[a]pyreneNo of laboratories 48

Assigned value 165 microgkg (Reference value)Target sd 036 microgkgTolerance limits 093 - 237 microgkg (|Z score| lt 200)

Assig

ned va

lue

Limit of tolerance

Limit of tolerance

49

Table 13 Analysis results reported by the participants for the content of benzo[b]fluoranthene (BBF) in the food supplements test material Values are presented as reported

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

3600 32 3 3 31 09 2

3602 173 175 16 17 017

3603 265 252 25 256 025 2

3604 116 108 105 11 028 2

3605 286 225 232 247 074 2

3606 294 273 261 276 046 2

3607 579 579 544 567 113 2

3608 103 1201 876 1036 326 2

3610 173 196 204 191 038 2

3611 546 618 565 576 75 2

3612 3 28 28 29 06 2

3613 28 31 32 31 06 2

3614 135 111 135 127 022 2

3615 4187 3678 4111 4 08 2

3616 2 2 2 2 01 2

3617 33 27 299 035 2

3618 31 31 31 31 05 2

3619 326 324 312 326 064 2

3620 277 28 272 276 069 2

3621 26 25 23 25 04 2

3622 346 336 333 34 052 2

3623 26 28 29 28 02 2

3624

3626 288 28 307 292 117 2

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

5301 29 3 29 29 06 2

5302 297 3 296 298 06 2

5303 2705 2444 2608 2586 22 78

5304 245 254 26 253 85 2

5305 23 23 26 24 15

5306 42 43 4 42 21 2

5307 221 216 241 221

5308 232169 246654 240722 229848

5309 19 22 19 2 0

5310 3 315 325 313 016

5311 262 277 304 281 15 2

5312 27 289 337 299 075 2

5313 321 284 378 328

5314 423 404 385 404 34 083

5315 42 506 416 444 09 2

5316 265 267 277 273 018 2

5317 185 197 195 192 006 2

5318 276 281 279 04 2

5319 396 389 403 396 079 2

5320 623 618 635 625 14

5321 31

5322 24 33 49 33 35 2

5323 38 45 35 45 27 2

50

Figure 8 Results of replicate determinations (indicated by triangles) of benzo[b]fluoranthene (BBF) in the food supplement test material

Horizontal blue lines represent the arithmetic mean value of replicate measurements and blue bars the reported expanded measurement uncertainty (k=2) The assigned value is

plotted as dotted line Red solid lines mark the range of satisfactory performance (|z|le2) Mean values are given for results outside the displayed data range

ProLab 2011

Laboratory

36

0436

1436

0236

1053

1736

1653

0953

0753

0853

0536

2136

0553

0436

0353

0353

1636

0636

2036

2353

1853

1136

1236

2653

0153

0253

1236

1736

1336

0036

1853

2153

1036

1953

1336

2253

2253

2353

1936

1553

1453

0653

1536

0753

2036

0836

11

an

aly

te c

on

ten

t (micro

gk

g)

75

7

65

6

55

5

45

4

35

3

25

2

15

1

05

0

103

6

576

3

Sample St Johns wort extract replicatesMeasurand Benzo[b]fluorantheneNo of laboratories 46

Assigned value 292 microgkg (Reference value)Target sd 060 microgkgTolerance limits 172 - 412 microgkg (|Z score| lt 200)

Assig

ned va

lue

Limit of tolerance

Limit of tolerance

51

Table 14 Analysis results reported by the participants for the content of chrysene (CHR) in the food supplements test material Values are presented as reported

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

3600 5 49 5 5 07 2

3602 32 304 29 304 036

3603 462 469 447 459 048 2

3604 438 428 42 429 119 2

3605 365 41 381 386 085 2

3606 494 411 45 451 124 2

3607 897 774 787 819 18 2

3608 986 887 1168 1014 284 2

3610 418 392 462 424 085 2

3611 833 893 87 865 61 2

3612 6 64 58 61 12 2

3613 41 39 4 4 08 2

3614 272 254 288 271 054 2

3615 605 6028 5964 601 12 2

3616 47 45 49 47 05 2

3617 445 431 389 422 065 2

3618 45 45 45 45 06 2

3619 738 747 72 738 118 2

3620 463 472 446 46 092 2

3621 53 49 51 51 06 2

3622 478 513 505 488 08 2

3623 73 73 72 72 05 2

3624

3626 63 622 634 628 251 2

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

5301 49 49 49 49 1 2

5302 687 683 666 679 136 2

5303 6777 6181 6382 6441 24 70

5304 592 581 6 591 85 2

5305 6 58 64 61 20

5306 49 5 51 49 25 2

5307 524 5 574 524

5308 211488 216782 221146 213243

5309

5310 372 393 394 386 019

5311 43 459 422 437 15 2

5312 934 943 981 953 191 2

5313 793 73 99 838

5314 636 561 565 588 18 0818

5315 547 583 499 542 11 2

5316 292 286 285 282 06 2

5317 52 598 517 545 046 2

5318 368 345 357 05 2

5319 449 471 474 465 116 2

5320 76 924 818 834 99

5321 33

5322 7 7 76 7 35 2

5323 87 73 77 73 37 2

52

Figure 9 Results of replicate determinations (indicated by triangles) of chrysene (CHR) in the food supplement test material

Horizontal blue lines represent the arithmetic mean value of replicate measurements and blue bars the reported expanded measurement uncertainty (k=2) The assigned value is

plotted as dotted line Red solid lines mark the range of satisfactory performance (|z|le2) Mean values are given for results outside the displayed data range

ProLab 2011

Laboratory

53

0836

1453

1636

0253

2153

1836

0553

1036

1336

1736

1036

0453

1136

1836

0636

0336

2053

1936

1653

0136

0036

2253

0636

2153

0753

1553

1753

1453

0436

1536

1253

0536

2653

0353

0253

2236

2336

1953

2336

0753

2053

1353

1236

0836

11

an

aly

te c

on

ten

t (micro

gk

g)

13

12

11

10

9

8

7

6

5

4

3

2

1

0

865

3

Sample St Johns wort extract replicatesMeasurand ChryseneNo of laboratories 45

Assigned value 407 microgkg (Reference value)Target sd 083 microgkgTolerance limits 241 - 573 microgkg (|Z score| lt 200)

Assig

ned va

lue

Limit of tolerance

Limit of tolerance

53

Table 15 Analysis results reported by the participants for the sum of the four marker PAHs in the food supplements test material Values are presented as reported

Laboratory code

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg

3600 128 24 2

3602 79 126

3603 1127 096 2

3604 893 258 2

3605 1091 305 2

3606 1184 238 2

3607 2098 42 2

3608 3336 325 2

3610 1138 17 2

3611 2218 259 2

3612 143 29 2

3613 122 2 2

3614 663 129 2

3615 152 304 2

3616 102 03 2

3617 1252 169 2

3618 128 18 2

3619 157 305 2

3620 1204 12 2

3621 151 19 2

3622 1361 115 2

3623 154 09 2

3624

3626 1409 56 2

Laboratory code

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg

5301 126

5302 37 074 2

5303 13438 92

5304 1251 85 2

5305 125 20

5306 129 65 2

5307 1363

5308 7245598

5309

5310 1127 056

5311 1367 15 2

5312 2042 408 2

5313 1726

5314 1558

5315 177 35 2

5316 96 073 2

5317 1229 066 2

5318 82 05 2

5319 1225 267 2

5320 2309 41

5321

5322 207 35 2

5323 171 103 2

54

ANNEX 9 Results reported for the standard solutions with undisclosed analyte content Table 16 Analysis results reported by the participants for the four marker PAHs in the standard solution in toluene with undisclosed analyte

content Values are presented as reported U measurement uncertainty k coverage factor

benz[a]anthracene benzo[a]pyrene benzo[b]fluoranthene chrysene Laboratory

code Unit Result U k Result U k Result U k Result U k 3600 microgl 38 67 2 25 45 2 211 68 2 275 38 2

3605 microgl 367 184 2 2482 124 2 2592 13 2 235 118 2

3606 microgkg 3763 176 2 2609 142 2 2721 118 2 2539 134 2

3607 microgkg 3706 815 2 2824 565 2 2591 518 2 2587 569 2

3612 microgl 38 08 2 237 11 2 244 18 2 257 02 2

3614 microgl 324 65 2 209 38 2 215 37 2 21 42 2

3616 microgkg 318 07 2 224 14 2 231 26 2 216 1 2

3618 microgkg 417 17 2 284 08 2 292 06 2 297 03 2

3619 microgkg 47 742 2 274 468 2 228 387 2 274 434 2

3620 microgl 495 74 2 294 29 2 321 8 2 321 64 2

3621 microgl 241 22 2 143 13 2 188 16 2 145 14 2

3623 microgkg 476 25 2 311 53 2 269 3 2 297 03 2

3626 microgl 359 38 2 235 23 2 238 27 2 24 18 2

5301 microgkg 426 29 299 289

5302 microgkg 436 87 2 296 59 2 296 59 2 289 58 2

5303 microgl 3721 27 102 22838 19 99 23756 22 99 25486 24 98

5304 microgkg 38 29 2 225 26 2 257 37 2 258 52 2

5305 microgkg 305 225 245 29

5306 microgkg 316 63 2 247 49 2 65 13 2 22 44 2

5307 microgkg 3658 2559 241 2414

5308 microgkg 16854 109324 246654 216782

5311 microgl 374 20 2 279 15 2 268 15 2 272 15 2

5313 microgl 3408 2165 2275 2373

5314 microgkg 395 5 2875 11 3614 34 2905 18

5320 microgl 3267 368 1986 571 28 683 2427 512

5322 microgl 282 35 2 225 35 2 241 35 2 21 35 2

55

Figure 10 Youden plots of the percent deviation of the reported values from the assigned values for the food supplement test sample and the

standard solution in toluene with undisclosed content

(The identity of the respective measurand is given above the figure Results exceeding a difference of more than 100 were omitted)

56

Figure 10 - continued

57

Table 17 Analysis results reported by the participants for the four marker PAHs in the standard solution in acetonitrile with undisclosed

analyte content

Values are presented as reported U measurement uncertainty k coverage factor

benz[a]anthracene benzo[a]pyrene benzo[b]fluoranthene chrysene

Laboratory

code Unit Result U k Result U k Result U k Result U k

3602 microgkg 3675 247 247 244

3603 microgkg 358 086 2 236 028 2 235 028 2 236 057 2

3604 microgl 475 29 2 19 12 2 194 13 2 258 12 2

3608 microgl 3623 078 2 2385 023 2 2343 18 2 2296 092 2

3610 microgkg 369 185 2 25 125 2 251 126 2 244 122 2

3611 microgkg 48 02 2 323 02 2 328 04 2 317 03 2

3613 microgl 332 31 2 261 3 2 249 25 2 239 24 2

3615 microgl 415 2635 2729 2689

3617 microgkg 37 09 2 244 06 2 284 07 2 239 06 2

3622 microgl 37 12 2 25 23 2 257 21 2 238 1 2

3624 microgkg 402 104 2 255 084 2 265 096 2 253 07 2

5306 microgkg 563 113 2 371 74 2 359 72 2 333 67 2

5308 microgkg 168633 102669 232169 211488

5309 microgl 402 268 27

5310 microgl 315 158 25 125 25 125 288 144

5312 microgl 428 043 2 282 028 2 272 027 2 275 028 2

5315 microgkg 387 08 2 256 05 2 251 05 2 253 05 2

5316 microgl 3839 342 2 2485 191 2 2559 168 2 239 51 2

5317 microgl 2466 043 2 2212 034 2 2472 016 2 2146 068 2

5319 microgl 414 41 2 279 28 2 268 27 2 26 31 2

5321 microgkg 476 316 326 328

5322 microgl 2547 35 2

5323 microgkg 405 203 2 239 119 2 255 153 2 201 11 2

58

Figure 11 Youden plots of the percent deviation of the reported values from the assigned values for the food supplement test sample and the

standard solution in acetonitrile with undisclosed content

(The identity of the respective measurand is given above the figure Results exceeding a difference of more than 100 were omitted)

59

Figure 11 - continued

60

ANNEX 10 Table 18 Method performance parameters reported by the participants

Laboratory code

Analyte LOD LOQ Recovery Analyte LOD LOQ Recovery

microgkg microgkg microgkg microgkg 3600 BaA 03 06 978 BaP 03 07 9483602 BaA 007 021 926 BaP 005 015 993603 BaA 0011 05 987 BaP 0005 051 9553604 BaA 02 04 86 BaP 02 04 883605 BaA 0025 005 118 BaP 0025 005 1083606 BaA 001 003 65 BaP 001 003 703607 BaA 021 071 74 BaP 027 089 943608 BaA 016 048 8213 BaP 015 046 83683610 BaA 02 05 85 BaP 005 02 853611 BaA BaP 3612 BaA 1 117 BaP 1 1203613 BaA 01 03 90 BaP 01 03 903614 BaA 02 05 96 BaP 02 05 933615 BaA 005 015 110 BaP 005 015 1053616 BaA 01 05 108 BaP 01 05 753617 BaA 018 037 405 BaP 009 018 3873618 BaA 01 03 63 BaP 01 03 593619 BaA 005 005 59 BaP 033 033 543620 BaA 0009 0018 64 BaP 0005 001 553621 BaA 007 02 84 BaP 007 02 923622 BaA 01 03 1101 BaP 01 03 9853623 BaA 02 08 105 BaP 02 08 1013624 BaA BaP 01 033 8513625 BaA BaP 3626 BaA BaP 5301 BaA 003 01 102 BaP 003 01 1005302 BaA 01 03 78 BaP 01 03 865303 BaA 003 01 62 BaP 003 01 805304 BaA 1 1 70 BaP 1 1 955305 BaA 02 05 85 BaP 02 05 855306 BaA 03 1 699 BaP 03 1 905307 BaA 6537 BaP 81165308 BaA 0088 0265 99 BaP 00262 00785 1005309 BaA 01 08 92 BaP 01 08 925310 BaA 01 03 BaP 01 03 905311 BaA 002 005 80 BaP 002 004 915312 BaA 03 09 105 BaP 03 09 1055313 BaA 04 08 BaP 04 08 5314 BaA 001 002 96 BaP 001 002 9485315 BaA 01 03 50 - 100 BaP 01 03 50 - 100 5316 BaA 002 011 827 BaP 004 019 10445317 BaA 05 1 76 BaP 05 1 755318 BaA BaP 5319 BaA 009 029 951 BaP 011 041 995320 BaA 12 18 946 BaP 06 1 8345321 BaA 03 05 BaP 05 1 5322 BaA 1 2 100 BaP 1 2 1005323 BaA 26 BaP 26

61

ANNEX 10 Table 18 - continued

Laboratory code Analyte LOD LOQ Recovery Analyte LOD LOQ Recovery

microgkg microgkg microgkg microgkg 3600 BbF 03 05 96 CHR 02 03 10153602 BbF 015 045 947 CHR 003 009 9193603 BbF 0014 05 1012 CHR 0004 05 10033604 BbF 02 04 85 CHR 02 04 873605 BbF 005 01 94 CHR 0025 005 863606 BbF 001 003 70 CHR 001 003 653607 BbF 018 06 100 CHR 014 028 943608 BbF 015 046 7979 CHR 015 045 71253610 BbF 005 02 85 CHR 02 05 853611 BbF CHR 3612 BbF 1 118 CHR 1 1173613 BbF 01 03 90 CHR 01 03 903614 BbF 01 04 96 CHR 02 05 953615 BbF 008 025 109 CHR 003 01 1083616 BbF 01 05 78 CHR 01 05 723617 BbF 035 07 38 CHR 012 024 4163618 BbF 01 03 62 CHR 01 03 623619 BbF 016 016 63 CHR 01 01 593620 BbF 0003 0006 55 CHR 001 002 653621 BbF 007 02 103 CHR 007 02 893622 BbF 02 06 963 CHR 03 09 1043623 BbF 02 08 106 CHR 02 08 1193624 BbF CHR 3625 BbF CHR 3626 BbF CHR 5301 BbF 003 01 96 CHR 003 01 1145302 BbF 01 03 84 CHR 01 03 835303 BbF 003 01 78 CHR 003 01 705304 BbF 1 1 86 CHR 1 1 705305 BbF 02 05 90 CHR 02 05 955306 BbF 03 1 1435 CHR 03 1 1185307 BbF 8116 CHR 65375308 BbF 0079 0238 99 CHR 009 026 1005309 BbF 01 08 92 CHR 5310 BbF 07 2 CHR 01 03 5311 BbF 002 004 95 CHR 002 004 895312 BbF 03 09 105 CHR 03 09 1055313 BbF 04 08 CHR 04 08 5314 BbF 001 002 83 CHR 001 002 8185315 BbF 02 05 50 -100 CHR 02 05 50 - 100 5316 BbF 005 024 814 CHR 001 007 9595317 BbF 05 1 73 CHR 05 1 785318 BbF CHR 5319 BbF 011 038 946 CHR 013 043 9675320 BbF 07 09 1194 CHR 23 32 10455321 BbF 05 1 CHR 05 1 5322 BbF 1 2 100 CHR 1 2 1005323 BbF 26 CHR 26

62

ANNEX 11 Table 19 Details of analysis method reported by the participants Data are presented as reported

Laboratory code

Analysis technique

Instrument calibration

Internal standards applied

Sample clean up

Details of sample

clean up

Extraction technique

Details of sample

extraction

3600 6) GC-MS

2) Internal standardisation with labelled substances

Deuterated PAH mix 9 (Ehrenstorfer)

1) Saponification 2) LiquidLiquid partitioning

D) Other Liquidliquid partitioning

3602 2) HPLC-UV-FLD

1) External calibration

3) Gel permeation chromatography

A) Pressurised liquid extraction (PLE)

3603 1) HPLC-FLD

1) External calibration

3) Gel permeation chromatography

D) Other 1 minute with dichloromethane in vortex

3604 2) HPLC-UV-FLD

2) Internal standardisation with labelled substances

DiP D14

5) Donor acceptor complex chromatography

D) Other LL

3605 1) HPLC-FLD

1) External calibration

3) Gel permeation chromatography

C) Soxhlet extraction

3606 7) GC-MSMS

2) Internal standardisation with labelled substances

BaP 13C4 CHR 13C6 BaA 13C6 BbF 13C6

4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

3607 6) GC-MS

2) Internal standardisation with labelled substances

BaA-d12 CHR-d12 BbF-d12 BkF-d12 BaP-d12 ICP-d12 BgP-d12 DiP-d14

1) Saponification

D) Other solvent partitioning

3608 1) HPLC-FLD

1) External calibration

6) Other Liquid extraction

D) Other

Extraction in a rotary agitator and centrifugation

3610 2) HPLC-UV-FLD

1) External calibration

4) Solid phase extraction

B) Sonication

3611

3612 6) GC-MS

2) Internal standardisation with labelled substances

chrysene-d12 dibenzo(ah)anthracene-d14 dibenzo(ai)pyrene-d14

4) Solid phase extraction

B) Sonication

3613 5) LC-MSMS

3) Internal standardisation with unlabelled substances

I use Chrysene-D12 3) Gel permeation chromatography

D) Other extraction with hexan

63

Table 19 - continued

Laboratory code

Analysis technique

Instrument calibration

Internal standards applied

Sample clean up Details of sample

clean up

Extraction technique

Details of sample

extraction

3614 6) GC-MS 1) External calibration

3) Gel permeation chromatography

B) Sonication

3615 1) HPLC-FLD

1) External calibration

6) Other PLE GPC

A) Pressurised liquid extraction (PLE)

3616 6) GC-MS

2) Internal standardisation with labelled substances

Benzo(a)pyrene-13C4 Benzo(a)anthracene-13C6 Benzo(b)fluoranthene-13C6 Chrysene-13C6

2) LiquidLiquid partitioning 4) Solid phase extraction

B) Sonication

3617 1) HPLC-FLD

1) External calibration

4) Solid phase extraction

C) Soxhlet extraction

3618 6) GC-MS

2) Internal standardisation with labelled substances

deuterated standards

1) Saponification 4) Solid phase extraction

D) Other Handshaken with cyclohexane

3619 6) GC-MS

2) Internal standardisation with labelled substances

C13 Labelled US EPA 16

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

D) Other Homegenisation amp saponiofication

3620 9) GC-HRMS

2) Internal standardisation with labelled substances

mix of deuterated PAHs (BAA-D12 CHR-D12 BAP-D12 BBF-D12)

3) Gel permeation chromatography 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

3621 6) GC-MS

2) Internal standardisation with labelled substances

Chrysene D12 Benzo(a)pyrene D12 Benzo(b)fluoranthene D12 Benzo(a)anthracene

2) LiquidLiquid partitioning 4) Solid phase extraction

B) Sonication

3622 1) HPLC-FLD

4) Standard addition method

4) Solid phase extraction

D) Other

Extraction with cyclohexane by heating under reflux

3623 7) GC-MSMS

2) Internal standardisation with labelled substances

C13 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

3624 2) HPLC-UV-FLD

1) External calibration

2) LiquidLiquid partitioning

D) Other hot solvent extraction

3626

64

Table 19 - continued

Laboratory code

Analysis technique

Instrument calibration

Internal standards

applied

Sample clean up

Details of sample

clean up

Extraction technique

Details of sample

extraction

5301 7) GC-MSMS

2) Internal standardisation with labelled substances

EACH SUBSTANCE C13

4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5302 7) GC-MSMS

2) Internal standardisation with labelled substances

Isotope labelled PAHs 13C for each analyte

4) Solid phase extraction

C) Soxhlet extraction

5303 7) GC-MSMS

3) Internal standardisation with unlabelled substances

HAP C13 4) Solid phase extraction

D) Other automate ASE200 DIONEX

5304 7) GC-MSMS

2) Internal standardisation with labelled substances

with C13 for each HAP

2) LiquidLiquid partitioning 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5305 7) GC-MSMS

2) Internal standardisation with labelled substances

C13 isotopiques internal standards

4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5306 7) GC-MSMS

2) Internal standardisation with labelled substances

C13 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5307 6) GC-MS

2) Internal standardisation with labelled substances

1) Saponification 2) LiquidLiquid partitioning

D) Other solvent extraction

5308 1) HPLC-FLD 1) External calibration

4) Solid phase extraction

B) Sonication

5309 1) HPLC-FLD 1) External calibration

4) Solid phase extraction

D) Other only SPE

5310 1) HPLC-FLD 1) External calibration

1) Saponification 2) LiquidLiquid partitioning 3) Gel permeation chromatography

C) Soxhlet extraction

5311 6) GC-MS

2) Internal standardisation with labelled substances

13 C Chrysene 13 C Benz(a)pyrene

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

B) Sonication

5312 1) HPLC-FLD

3) Internal standardisation with unlabelled substances

Benzo(b)chrysen

2) LiquidLiquid partitioning 4) Solid phase extraction

Quechers D) Other Quechers

65

Table 19 - continued

Laboratory code

Analysis technique

Instrument calibration

Internal standards applied

Sample clean up Details of sample

clean up

Extraction technique

Details of sample extraction

5313 6) GC-MS

2) Internal standardisation with labelled substances

Benz(a)anthracene D12 Benzo(b)fluranthene D12 Benzo(a)pyrene D12 Chrysene D12

1) Saponification 2) LiquidLiquid partitioning 3) Gel permeation chromatography

A) Pressurised liquid extraction (PLE)

5314 6) GC-MS

2) Internal standardisation with labelled substances

Benzo(a)pyren d12 2) LiquidLiquid partitioning

D) Other Extraction with

5315 1) HPLC-FLD

3) Internal standardisation with unlabelled substances

Benz(b)chrysen

1) Saponification 2) LiquidLiquid partitioning

D) Other shaker

5316 1) HPLC-FLD

1) External calibration

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

D) Other

LiquidLiquid extraction with cyclohexane and dimethylformamide

5317 7) GC-MSMS

1) External calibration

3) Gel permeation chromatography 6) Other

silica gel D) Other VDLUFA online method

5318

5319 1) HPLC-FLD

3) Internal standardisation with unlabelled substances

Benzo(b)chrysene 3) Gel permeation chromatography

A) Pressurised liquid extraction (PLE)

5320 6) GC-MS

3) Internal standardisation with unlabelled substances

22-binaphthyl

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

D) Other 3 hours refluxing

5321 1) HPLC-FLD

2) Internal standardisation with labelled substances

6-meacutethylchrysegravene

1) Saponification 2) LiquidLiquid partitioning

B) Sonication

5322 6) GC-MS

2) Internal standardisation with labelled substances

naftalene-d8 acenaphtalene-d10 phenanthrene-d10 chrysene-d12 perylene-d12 benzo(e)anthracene-d12

1) Saponification 2) LiquidLiquid partitioning 6) Other

clean-up with silica packed column

C) Soxhlet extraction

5323 1) HPLC-FLD

1) External calibration

4) Solid phase extraction 6) Other

Accelerated Solvent Extraction

A) Pressurised liquid extraction (PLE)

66

European Commission EUR 25245 EN ndash Joint Research Centre ndash Institute for Reference Materials and Measurements Title Report on the 8th inter-laboratory comparison organised by the European Union Reference Laboratory for Polycyclic Aromatic Hydrocarbons - Four marker PAHs in a dry extract of St Johns wort Author(s) Radoslav Lizak Szilard Szilagyi Philippe Verlinde and Thomas WENZL Luxembourg Publications Office of the European Union 2012 ndash 66 pp ndash 201 x 297 cm EUR ndash Scientific and Technical Research series ndash ISSN 1831-9424 ISBN 978-92-79-23337-1 doi 10278759387 Abstract The proficiency test here reported concerned the determination of the contents of four marker polycyclic aromatic hydrocarbons (PAHs) and of their sum in an food supplements test sample The set of marker PAHs consists of benz[a]anthacene benzo[a]pyrene benzo[b]fluoranthene and chrysene Participants to these PT were National Reference Laboratories for PAHs (NRLs-PAHs) and EU official food control laboratories The number of participants was in total 48

The PT was organised according ISO Standard 170432010

The test material used was a commercial dry extract of St Johns wort Participants also received a solution of the same PAHs either in acetonitrile or in toluene for checking their instrument calibration

The results from participants were rated with z-scores and zeta-scores About 68 of the reported results were attributed with z-scores with an absolute value of below two which is the threshold for satisfactory performance

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interests whether private or national

L

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• 1 Summary
• 2 Introduction
• 3 Scope
• 4 Participating Laboratories
• 5 Time frame
• 6 Confidentiality
• 7 Test materials
• • 71 1Preparation of test materials
  • 72 Assigned values and standard deviation for proficiency assessment
  • 73 Homogeneity and stability
  • 74 Sample dispatch
  • • 8 Design of the proficiency test
    • 9 Evaluation of the results
    • • 91 2General
      • 92 21BEvaluation criteria
      • 93 Performance of participants
      • 94 Evaluation of the influence of analysis method
      • 95 Evaluation of the influence of calibration
      • 96 26Evaluation of compliance with legislation
      • • 10 Follow-up actions for underperforming laboratories
        • 11 1Conclusions
        • 12 1Acknowledgements
        • 13 References

22

94 Evaluation of the influence of analysis method

The information on the applied analysis method (ANNEX 11) was used to group the performance

indicators attributed to the reported results according to the applied analysis technique Two groups of

data were formed They represent results obtained by high performance liquid chromatography-

fluorescence detection (HPLC-FD about 40 ) and by gas chromatography-mass spectrometry (GC-

MS about 60 ) respectively Kernel density plots were constructed based on the respective z-scores

Figure 3A to 3E show the different Kernel density plots

Figure 3 Kernel density plots of z-scores attributed to the results for proficiency assessment

which were reported for the food supplements test sample Results were classified

according to the applied analysis technique

A) benz[a]anthracene

B) benzo[a]pyrene

23

Figure 3 continued

C) benzo[b]fluoranthene

D) chrysene

E) sum of four marker PAHs

The kernel density plots for BAP and BBF are almost identical and centred at or close to a z-score

value of zero as can be seen in Figure 3B and 3C Therefore bias introduced by the type of analysis

method is unlikely for these two analytes The situation is different for BAA and CHR In case of

BAA the kernel density distribution of z-scores based on HPLC-FD is shifted to negative values

24

(Figure 3A) which indicates a tendency to underestimate the BAA content in this particular

samplematrix when applying HPLC-FD This might be reasoned by separationintegration problems

between BAA and CHR peaks A few participants (from the HPLC-FD subgroup) that got negative

z-scores for BAA overestimated the CHR content This caused the slight shift of the density trace for

HPLC-FD towards negative values Removing this data from the evaluation would result in a density

trace with the mode at zero

The opposite is the case for CHR (Figure 3D) The density distribution of the z-scores attributed to

HPLC-FD data is centred almost at zero However the distribution for data based on GC-MS is shifted

to positive values indicating strong positive bias This bias might be caused by a lack of selectivity of

the analysis method The test material contains triphenylene which is isobaric to chrysene

Consequently it cannot be distinguished from chrysene by means of mass spectrometry These two

compounds have to be separated chromatographically which is not easy They coelute on unpolar and

on most mid-polar capillary columns Columns that allow their separation were commercialised only

recently and might not be available in all laboratories Therefore laboratories which determine the

four EU marker PAHs by means of GC-MS shall pay attention to the chromatographic separation of

triphenylene and chrysene A lack of chromatographic resolution might lead to strong positive bias of

GC-MS methods and to false conclusions on the marketability of the tested sample

The chromatographic separation of chrysene and triphenylene is not a problem with HPLC which is

reflected in the position of the density plot for chrysene However attention has to be paid in HPLC-

FD analysis to a proper clean up of the sample extract in order to eliminate interferences that show

similar retention as BAA and CHR

95 Evaluation of the influence of calibration

The participants received together with the food supplement test sample two standard solutions in

depending of their choice either acetonitrile or toluene The analyte content of one solution was

disclosed to the participants whereas the content of the other solution was kept undisclosed The

content values of the latter are presented in Table 5 Analysis results for the solution with undisclosed

content had to be reported to the PT organiser The respective data are presented in Annex 9

Percent deviations of the reported results from the assigned values were calculated for both the food

supplement test sample and the standard solution with undisclosed analyte content Youden plots were

prepared in order to visualize the influence of instrument calibration on the results reported for the

food supplement sample Two plots are discussed in the following The whole set of Youden plots are

given in Annex 9

25

Figure 4 Youden plot of the percent deviations of values reported for benzo[a]pyrene from the

assigned values of the food supplement test material and the standard solution in

toluene with undisclosed content

Figure 4 reveals that a number of participants reported results with similar percent deviations from the

assigned values of both the results for the food supplement material and the standard solution in

toluene with undisclosed content This correlation indicates that over- respectively underestimation of

the analyte content of the food supplement material might be caused by biased instrument calibration

This finding underpins the importance of thorough calibration standard preparation However the data

was not in all cases conclusive as some laboratories performed well for the food supplement samples

but reported biased results for the standard solution with undisclosed content (eg Figure 4 participants

3620 and 5305) This fact cannot be explained yet

Figure 5 presents the respective Youden plot for chrysene in the food supplement test material and the

standard solution in toluene with undisclosed content From this graph it can be concluded that the

overestimation of the chrysene content of the food supplement material was not caused by instrument

calibration The results for the solvent solution of many laboratories which underperformed for the

26

food supplement sample agreed well with the assigned value This supports the conclusion that the

overestimation is caused by the determination of an interferent most probably triphenylene

Figure 5 Youden plot of the percent deviations of values reported for chrysene from the

assigned values of the food supplement test material and the standard solution in

toluene with undisclosed content

96 Evaluation of compliance with legislation

The method performance data reported for the four marker PAHs were evaluated for compliance with

the provisions given in Commission Regulation (EC) No 8362011 Table 18 in ANNEX 10 gives an

overview on the results of the evaluation Data were not reported in case of empty cells Non-

compliant data are highlighted in Table 18 It should be noted that Regulation (EU) No 8362011

specifies LOD and LOQ with two decimals This was not respected by all participants which

hampered the compliance assessment

27

In summary it can be stated that the majority of participants reported method performance

characteristics for the determination of the four marker PAHs in the food supplement test sample that

are compliant with legislation However some laboratories need to improve their analysis methods

The laboratories in question are urged to adapt to the provisions given in the new legislation

10 Follow-up actions for underperforming laboratories

All laboratories that got questionable or non-satisfactory performance ratings are urged to perform

root cause analysis and to implement corrective actions

As agreed during the EU-RL PAH workshop 2011 the EU-RL PAH will set up follow-up measures in

due time for all NRLs that received for at least one of the four PAHs (BAA BAP BBF and CHR) z-

scores gt 3 as required by Regulation (EC) 8822004 and by the Protocol for management of

underperformance in comparative testing andor lack of collaboration of National Reference

Laboratories (NRLs) with European Union reference laboratories (EU-RLs) activities

These laboratories shall perform as an immediate action root-cause-analysis and shall report to the

EU-RL PAH in writing the identified cause for their underperformance and corrective actions they are

going to take Additionally they shall participate to an independent (non-EU-RL) proficiency test on

the determination of PAHs in food and shall communicate the outcome of this exercise to the EU-RL

PAH

28

11 Conclusions

Fourty seven participants reported results back to the EU-RL The performance of the majority of

participants was good In total 153 out of 225 attributed z-scores were below an absolute value of two

which equals to almost 68 However this percentage is much below the percentages of satisfactory

performance gained in previous studies This might be the consequence of the higher level of difficulty

with respect to matrix composition In addition bias can be concluded from the pattern of performance

indicators of some laboratories

In general the determination of chrysene caused most difficulties to the participants

zeta-Scores were calculated besides z-scores They indicate the agreement of the reported result with

the assigned value with respect to the stated measurement uncertainty The outcome of this rating was

worse than for the z-scores which indicates that the measurement uncertainty estimates were not

realistic Therefore participants underperforming with regard to zeta scores are urged to adapt their

measurement uncertainty statements

The great majority of participants in this inter-laboratory comparison applied analytical methods which

complied with EU legislation with regard to performance characteristics

12 Acknowledgements

The organisers would like to thank Mrs Beatriz de la Calle and Mr Franz Ulberth (all from IRMM

Geel Belgium) for their accurate revision of this report Mr Hakan Emteborg (from IRMM) for

ampouling of the standard solutions and all NRLs and OCLs for their cooperation

29

13 References

1 EU COMMISSION REGULATION (EC) No 7762006 of 23 May 2006 amending Annex VII to Regulation

(EC) No 8822004 of the European Parliament and of the Council as regards Community reference

laboratories Official Journal of the European Union 2006 L 136 p 3-8

Available from

httpeur-lexeuropaeuLexUriServLexUriServdouri=OJL200613600030008ENPDF

2 EU Regulation (EC) No 8822004 of the European Parliament and of the Council of 29 April 2004 on

official controls performed to ensure the verification of compliance with feed and food law animal health

and animal welfare rules Official Journal of the European Communities 2004 L191 p 1-52 Available

from

httpeur-lexeuropaeuLexUriServLexUriServdouri=OJL200419100010052ENPDF

3 EU Opinion of the Scientific Committee on Food on the risks to human health of Polycyclic Aromatic

Hydrocarbons in food (2002) Available from

httpeuropaeuintcommfoodfsscscfout153_enpdf

4 International Agency for Research on Cancer IARC Monographs on the Evaluation of

Carcinogenic Risks to Humans Some Non-heterocyclic Polycyclic Aromatic Hydrocarbons and Some

Related Exposures Volume 92 (2010) Available from

httpmonographsiarcfrENGMonographsvol92mono92pdf

5 EU Commission Regulation (EC) No 3332007 of 28 March 2007 laying down the methods of sampling

and analysis for the official control of the levels of lead cadmium mercury inorganic tin 3-MCPD and

benzo(a)pyrene in foodstuffs Official Journal of the European Union 2007 L 88 p 29-38 Available

from

httpeur-lexeuropaeuLexUriServLexUriServdouri=OJL200708800290038ENPDF

6 EU Commission Regulation (EC) No 18812006 of 19 December 2006 setting maximum levels for certain

contaminants in foodstuffs Official Journal of the European Union 2006 L 364 p 5-24 Available from

httpeur-lexeuropaeuLexUriServLexUriServdouri=OJL200636400050024ENPDF

7 EFSA Scientific Opinion of the Panel on Contaminants in the Food Chain on a request from the European

Commission on Polycyclic Aromatic Hydrocarbons in Food The EFSA Journal 2008 724 p 1-114

Available from

httpwwwefsaeuropaeuEFSAefsa_locale-1178620753812_1211902034842htm

30

8 EU Commission Regulation (EU) No 8352011 amending Regulation (EC) No 18812006 as regards

maximum levels for polycyclic aromatic hydrocarbons in foodstuffs Official Journal of the European

Union 2011 L 215 p 4-8

httpeur-lexeuropaeuLexUriServLexUriServdouri=OJL201121500040008ENPDF

9 EU Commission Regulation (EU) No 8362011 amending Regulation (EC) No 3332007 laying down the methods of sampling and analysis for the official control of the levels of lead cadmium mercury inorganic tin 3-MCPD and benzo(a)pyrene in foodstuffs Official Journal of the European Union 2011 L 215 p 9-16 httpeur-lexeuropaeuLexUriServLexUriServdouri=OJL201121500090016ENPDF

10 ISOIEC 170432010 (2010) Conformity assessment -- General requirements for proficiency testing

providers Geneva Switzerland

11 Thompson M Ellison SLR and Wood R The International Harmonized Protocol for the Proficiency

Testing of Analytical Chemistry Laboratories Pure Appl Chem (2006) 78(1) p 145ndash196

Available from httpmediaiupacorgpublicationspac2006pdf7801x0145pdf

31

14 ANNEXES

ANNEX 1 ndash Announcement of the PT on the IRMM webpage

ANNEX 2 ndash Announcement via e-mail and invitation

ANNEX 3 ndash Announcement of sample dispatch

ANNEX 4 ndash Technical specifications of the calibration solutions

ANNEX 5 ndash Homogeneity and stability of the test material

ANNEX 6 ndash Acknowledgement of sample receipt

ANNEX 7 ndash Questionnaire on details of analysis method

ANNEX 8 ndash Results reported for the food supplement sample

ANNEX 9 ndash Results reported for the standard solutions with undisclosed analyte content

ANNEX 10 ndash Method performance characteristics reported by participants

ANNEX 11 ndash Details of analysis methods reported by participants

32

ANNEX 1 ndash Announcement of the PT on the IRMM webpage

33

ANNEX 2 - Announcement via e-mail and invitation

34

ANNEX 3 - Announcement sample dispatch

Dear MadameSir

The test samples for the proficiency test on the determination of four PAHs in a food supplement (dry extract of St Johns wort) were

dispatched today

Explanation on the design of the study as well as instructions for sample storage and for reporting of results are in the parcel You will

also find your participant key in the parcel which is required for reporting of results

Please check the parcel after arrival for completeness and report the receipt of the test samples as well as their status to us by applying

the sample receipt form which is in the parcel

You are kindly requested to inform us by email if you do not receive the parcel by end of this week

My colleague Donata Lerda will then take care of it

With best regards

Thomas Wenzl

35

ANNEX 4 - Specifications of standard solutions with disclosed concentration

36

ANNEX 4 - continued

37

ANNEX 5 - Homogeneity and stability of the test material

Homogeneity of the test material

Benz[a]anthracene - BAA

Benzo[a]pyrene - BAP

38

ANNEX 5 - continued

Benzo[b]fluoranthene - BBF

Chrysene - CHR

39

ANNEX 5 - continued

Stability of the test material

Table 10 Content of the four target analytes in test materials stored under different

temperatures

BaA BaP BbF CHR

microgkg microgkg microgkg microgkg

Content 31 16 31 69 Storage at room temperature

U (k=2) 05 02 05 17

Content 31 15 29 68 Storage at reference temperature

U (k=2) 04 03 04 10

The applied capillary column does not separate CHR from triphenylene

40

ANNEX 6 ndash Documents sent to participants

41

ANNEX 6 - continued

42

ANNEX 7 Questionnaire on details of analysis method

43

ANNEX 7 - continued

44

ANNEX 7 - continued

45

ANNEX 8 - Results reported by participants for the food supplements test sample Table 11 Analysis results reported by the participants for the content of benz[a]anthracene (BAA) in the food supplements test material Values are presented as reported

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

3600 31 29 29 3 05 2

3602 199 193 178 19 021

3603 253 253 246 251 022 2

3604 199 209 23 213 071 2

3605 301 3 271 291 076 2

3606 302 288 278 289 064 2

3607 456 416 443 438 096 2

3608 796 695 651 714 149 2

3610 182 192 214 196 039 2

3611 414 504 488 469 96 2

3612 3 34 28 31 06 2

3613 32 33 34 33 07 2

3614 182 182 191 185 037 2

3615 3884 3822 3565 376 0752 2

3616 2 2 2 2 01 2

3617 366 37 35 362 046 2

3618 32 32 32 32 05 2

3619 299 305 297 299 048 2

3620 304 294 302 302 045 2

3621 29 26 29 28 04 2

3622 343 325 318 322 052 2

3623 35 3 33 33 07 2

3624

3626 318 31 314 314 126 2

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

5301 31 3 32 31 06 2

5302 314 319 315 316 063 2

5303 3102 2716 2822 2876 27 62

5304 261 263 26 261 57 2

5305 23 22 26 24 20

5306 22 23 23 22 11 2

5307 488 461 317 461

5308 168633 168544 1562025 1644598

5309 16 19 16 17

5310 3 304 311 305 015

5311 477 452 448 459 15 2

5312 506 63 494 543 134 2

5313 353 301 404 353

5314 431 373 378 394 5 096

5315 596 531 51 545 11 2

5316 224 231 235 235 022 2

5317 216 23 221 222 007 2

5318 064 07 067 01 2

5319 111 184 146 147 029 2

5320 515 525 564 534 48

5321 21 5322 13 87 9 87 35 2

5323 38 33 35 33 16 2

46

Figure 6 Results of replicate determinations (indicated by triangles) of benz[a]anthracene (BAA) in the food supplement test material

Horizontal blue lines represent the arithmetic mean value of replicate measurements and blue bars the reported expanded measurement uncertainty (k=2) The assigned value is

plotted as dotted line Red solid lines mark the range of satisfactory performance (|z|le2) Mean values are given for results outside the displayed data range

ProLab 2011

Laboratory

53

1853

1953

0853

0936

1436

0236

1036

1653

2136

0453

1753

0653

1653

0536

0353

0436

2153

0336

0636

0536

0036

2036

1953

1036

1253

0136

2653

0236

1836

2336

2236

1353

1353

2336

1736

1553

1453

0736

0753

1153

2053

1253

1536

0853

2236

11

an

aly

te c

on

ten

t (micro

gk

g)

10

95

9

85

8

75

7

65

6

55

5

45

4

35

3

25

2

15

1

05

0

102

3 468

7

Sample St Johns wort extract replicatesMeasurand Benz[a]anthraceneNo of laboratories 46

Assigned value 298 microgkg (Reference value)Target sd 062 microgkgTolerance limits 174 - 422 microgkg (|Z score| lt 200)

Assig

ned va

lue

Limit of tolerance

Limit of tolerance

47

Table 12 Analysis results reported by the participants for the content of benzo[a]pyrene (BAP) in the food supplements test material Values are presented as reported

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

3600 16 17 17 17 03 2

3602 118 123 13 124 015

3603 165 162 157 161 015 2

3604 134 154 139 143 04 2

3605 164 166 172 167 057 2

3606 181 159 161 167 031 2

3607 279 266 279 274 055 2

3608 57 687 461 573 226 2

3610 226 216 183 206 042 2

3611 289 325 311 308 36 2

3612 24 22 2 22 05 2

3613 17 18 19 18 05 2

3614 08 08 08 08 016 2

3615 1545 1393 1425 145 029 2

3616 16 16 14 15 03 2

3617 177 171 158 169 023 2

3618 2 2 2 2 02 2

3619 207 207 206 207 075 2

3620 165 169 163 166 017 2

3621 44 47 49 47 05 2

3622 212 208 222 213 038 2

3623 2 21 22 21 03 2

3624 18 178 182 18 031 2

3626 172 173 178 174 07 2

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

5301 18 18 18 18 04 2

5302 188 19 187 188 038 2

5303 1691 1426 149 1535 19 80

5304 145 142 15 146 69 2

5305 15 17 18 16 15

5306 16 17 17 16 08 2

5307 164 157 153 157

5308 102669 109324 109035 107009

5309 1 1 12 11

5310 122 122 124 123 006

5311 199 184 215 2 15 2

5312 258 248 239 248 05 2

5313 199 178 245 207

5314 174 171 172 172 11 0948

5315 253 248 213 238 05 2

5316 177 171 173 17 016 2

5317 255 277 276 269 012 2

5318 114 12 117 02 2

5319 19 228 231 216 043 2

5320 303 3 344 316 78

5321 12 12 12 12

5322 13 17 24 17 35 2

5323 2 2 19 2 1 2

48

Figure 7 Results of replicate determinations (indicated by triangles) of benzo[a]pyrene (BAP) in the food supplement test material

Horizontal blue lines represent the arithmetic mean value of replicate measurements and blue bars the reported expanded measurement uncertainty (k=2) The assigned value is

plotted as dotted line Red solid lines mark the range of satisfactory performance (|z|le2) Mean values are given for results outside the displayed data range

ProLab 2011

Laboratory

36

1453

0953

0853

1853

2153

1036

0236

0436

1553

0436

1653

0353

0736

0336

2036

0053

0553

0636

0636

0536

1753

1453

1636

2636

1336

2453

0153

2253

0253

2353

1136

1836

1953

1336

1036

2336

2253

1936

1253

1553

1253

1736

0753

2036

2136

0836

11

an

aly

te c

on

ten

t (micro

gk

g)

55

5

45

4

35

3

25

2

15

1

05

0

573

308

3

Sample St Johns wort extract replicatesMeasurand Benzo[a]pyreneNo of laboratories 48

Assigned value 165 microgkg (Reference value)Target sd 036 microgkgTolerance limits 093 - 237 microgkg (|Z score| lt 200)

Assig

ned va

lue

Limit of tolerance

Limit of tolerance

49

Table 13 Analysis results reported by the participants for the content of benzo[b]fluoranthene (BBF) in the food supplements test material Values are presented as reported

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

3600 32 3 3 31 09 2

3602 173 175 16 17 017

3603 265 252 25 256 025 2

3604 116 108 105 11 028 2

3605 286 225 232 247 074 2

3606 294 273 261 276 046 2

3607 579 579 544 567 113 2

3608 103 1201 876 1036 326 2

3610 173 196 204 191 038 2

3611 546 618 565 576 75 2

3612 3 28 28 29 06 2

3613 28 31 32 31 06 2

3614 135 111 135 127 022 2

3615 4187 3678 4111 4 08 2

3616 2 2 2 2 01 2

3617 33 27 299 035 2

3618 31 31 31 31 05 2

3619 326 324 312 326 064 2

3620 277 28 272 276 069 2

3621 26 25 23 25 04 2

3622 346 336 333 34 052 2

3623 26 28 29 28 02 2

3624

3626 288 28 307 292 117 2

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

5301 29 3 29 29 06 2

5302 297 3 296 298 06 2

5303 2705 2444 2608 2586 22 78

5304 245 254 26 253 85 2

5305 23 23 26 24 15

5306 42 43 4 42 21 2

5307 221 216 241 221

5308 232169 246654 240722 229848

5309 19 22 19 2 0

5310 3 315 325 313 016

5311 262 277 304 281 15 2

5312 27 289 337 299 075 2

5313 321 284 378 328

5314 423 404 385 404 34 083

5315 42 506 416 444 09 2

5316 265 267 277 273 018 2

5317 185 197 195 192 006 2

5318 276 281 279 04 2

5319 396 389 403 396 079 2

5320 623 618 635 625 14

5321 31

5322 24 33 49 33 35 2

5323 38 45 35 45 27 2

50

Figure 8 Results of replicate determinations (indicated by triangles) of benzo[b]fluoranthene (BBF) in the food supplement test material

Horizontal blue lines represent the arithmetic mean value of replicate measurements and blue bars the reported expanded measurement uncertainty (k=2) The assigned value is

plotted as dotted line Red solid lines mark the range of satisfactory performance (|z|le2) Mean values are given for results outside the displayed data range

ProLab 2011

Laboratory

36

0436

1436

0236

1053

1736

1653

0953

0753

0853

0536

2136

0553

0436

0353

0353

1636

0636

2036

2353

1853

1136

1236

2653

0153

0253

1236

1736

1336

0036

1853

2153

1036

1953

1336

2253

2253

2353

1936

1553

1453

0653

1536

0753

2036

0836

11

an

aly

te c

on

ten

t (micro

gk

g)

75

7

65

6

55

5

45

4

35

3

25

2

15

1

05

0

103

6

576

3

Sample St Johns wort extract replicatesMeasurand Benzo[b]fluorantheneNo of laboratories 46

Assigned value 292 microgkg (Reference value)Target sd 060 microgkgTolerance limits 172 - 412 microgkg (|Z score| lt 200)

Assig

ned va

lue

Limit of tolerance

Limit of tolerance

51

Table 14 Analysis results reported by the participants for the content of chrysene (CHR) in the food supplements test material Values are presented as reported

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

3600 5 49 5 5 07 2

3602 32 304 29 304 036

3603 462 469 447 459 048 2

3604 438 428 42 429 119 2

3605 365 41 381 386 085 2

3606 494 411 45 451 124 2

3607 897 774 787 819 18 2

3608 986 887 1168 1014 284 2

3610 418 392 462 424 085 2

3611 833 893 87 865 61 2

3612 6 64 58 61 12 2

3613 41 39 4 4 08 2

3614 272 254 288 271 054 2

3615 605 6028 5964 601 12 2

3616 47 45 49 47 05 2

3617 445 431 389 422 065 2

3618 45 45 45 45 06 2

3619 738 747 72 738 118 2

3620 463 472 446 46 092 2

3621 53 49 51 51 06 2

3622 478 513 505 488 08 2

3623 73 73 72 72 05 2

3624

3626 63 622 634 628 251 2

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

5301 49 49 49 49 1 2

5302 687 683 666 679 136 2

5303 6777 6181 6382 6441 24 70

5304 592 581 6 591 85 2

5305 6 58 64 61 20

5306 49 5 51 49 25 2

5307 524 5 574 524

5308 211488 216782 221146 213243

5309

5310 372 393 394 386 019

5311 43 459 422 437 15 2

5312 934 943 981 953 191 2

5313 793 73 99 838

5314 636 561 565 588 18 0818

5315 547 583 499 542 11 2

5316 292 286 285 282 06 2

5317 52 598 517 545 046 2

5318 368 345 357 05 2

5319 449 471 474 465 116 2

5320 76 924 818 834 99

5321 33

5322 7 7 76 7 35 2

5323 87 73 77 73 37 2

52

Figure 9 Results of replicate determinations (indicated by triangles) of chrysene (CHR) in the food supplement test material

Horizontal blue lines represent the arithmetic mean value of replicate measurements and blue bars the reported expanded measurement uncertainty (k=2) The assigned value is

plotted as dotted line Red solid lines mark the range of satisfactory performance (|z|le2) Mean values are given for results outside the displayed data range

ProLab 2011

Laboratory

53

0836

1453

1636

0253

2153

1836

0553

1036

1336

1736

1036

0453

1136

1836

0636

0336

2053

1936

1653

0136

0036

2253

0636

2153

0753

1553

1753

1453

0436

1536

1253

0536

2653

0353

0253

2236

2336

1953

2336

0753

2053

1353

1236

0836

11

an

aly

te c

on

ten

t (micro

gk

g)

13

12

11

10

9

8

7

6

5

4

3

2

1

0

865

3

Sample St Johns wort extract replicatesMeasurand ChryseneNo of laboratories 45

Assigned value 407 microgkg (Reference value)Target sd 083 microgkgTolerance limits 241 - 573 microgkg (|Z score| lt 200)

Assig

ned va

lue

Limit of tolerance

Limit of tolerance

53

Table 15 Analysis results reported by the participants for the sum of the four marker PAHs in the food supplements test material Values are presented as reported

Laboratory code

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg

3600 128 24 2

3602 79 126

3603 1127 096 2

3604 893 258 2

3605 1091 305 2

3606 1184 238 2

3607 2098 42 2

3608 3336 325 2

3610 1138 17 2

3611 2218 259 2

3612 143 29 2

3613 122 2 2

3614 663 129 2

3615 152 304 2

3616 102 03 2

3617 1252 169 2

3618 128 18 2

3619 157 305 2

3620 1204 12 2

3621 151 19 2

3622 1361 115 2

3623 154 09 2

3624

3626 1409 56 2

Laboratory code

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg

5301 126

5302 37 074 2

5303 13438 92

5304 1251 85 2

5305 125 20

5306 129 65 2

5307 1363

5308 7245598

5309

5310 1127 056

5311 1367 15 2

5312 2042 408 2

5313 1726

5314 1558

5315 177 35 2

5316 96 073 2

5317 1229 066 2

5318 82 05 2

5319 1225 267 2

5320 2309 41

5321

5322 207 35 2

5323 171 103 2

54

ANNEX 9 Results reported for the standard solutions with undisclosed analyte content Table 16 Analysis results reported by the participants for the four marker PAHs in the standard solution in toluene with undisclosed analyte

content Values are presented as reported U measurement uncertainty k coverage factor

benz[a]anthracene benzo[a]pyrene benzo[b]fluoranthene chrysene Laboratory

code Unit Result U k Result U k Result U k Result U k 3600 microgl 38 67 2 25 45 2 211 68 2 275 38 2

3605 microgl 367 184 2 2482 124 2 2592 13 2 235 118 2

3606 microgkg 3763 176 2 2609 142 2 2721 118 2 2539 134 2

3607 microgkg 3706 815 2 2824 565 2 2591 518 2 2587 569 2

3612 microgl 38 08 2 237 11 2 244 18 2 257 02 2

3614 microgl 324 65 2 209 38 2 215 37 2 21 42 2

3616 microgkg 318 07 2 224 14 2 231 26 2 216 1 2

3618 microgkg 417 17 2 284 08 2 292 06 2 297 03 2

3619 microgkg 47 742 2 274 468 2 228 387 2 274 434 2

3620 microgl 495 74 2 294 29 2 321 8 2 321 64 2

3621 microgl 241 22 2 143 13 2 188 16 2 145 14 2

3623 microgkg 476 25 2 311 53 2 269 3 2 297 03 2

3626 microgl 359 38 2 235 23 2 238 27 2 24 18 2

5301 microgkg 426 29 299 289

5302 microgkg 436 87 2 296 59 2 296 59 2 289 58 2

5303 microgl 3721 27 102 22838 19 99 23756 22 99 25486 24 98

5304 microgkg 38 29 2 225 26 2 257 37 2 258 52 2

5305 microgkg 305 225 245 29

5306 microgkg 316 63 2 247 49 2 65 13 2 22 44 2

5307 microgkg 3658 2559 241 2414

5308 microgkg 16854 109324 246654 216782

5311 microgl 374 20 2 279 15 2 268 15 2 272 15 2

5313 microgl 3408 2165 2275 2373

5314 microgkg 395 5 2875 11 3614 34 2905 18

5320 microgl 3267 368 1986 571 28 683 2427 512

5322 microgl 282 35 2 225 35 2 241 35 2 21 35 2

55

Figure 10 Youden plots of the percent deviation of the reported values from the assigned values for the food supplement test sample and the

standard solution in toluene with undisclosed content

(The identity of the respective measurand is given above the figure Results exceeding a difference of more than 100 were omitted)

56

Figure 10 - continued

57

Table 17 Analysis results reported by the participants for the four marker PAHs in the standard solution in acetonitrile with undisclosed

analyte content

Values are presented as reported U measurement uncertainty k coverage factor

benz[a]anthracene benzo[a]pyrene benzo[b]fluoranthene chrysene

Laboratory

code Unit Result U k Result U k Result U k Result U k

3602 microgkg 3675 247 247 244

3603 microgkg 358 086 2 236 028 2 235 028 2 236 057 2

3604 microgl 475 29 2 19 12 2 194 13 2 258 12 2

3608 microgl 3623 078 2 2385 023 2 2343 18 2 2296 092 2

3610 microgkg 369 185 2 25 125 2 251 126 2 244 122 2

3611 microgkg 48 02 2 323 02 2 328 04 2 317 03 2

3613 microgl 332 31 2 261 3 2 249 25 2 239 24 2

3615 microgl 415 2635 2729 2689

3617 microgkg 37 09 2 244 06 2 284 07 2 239 06 2

3622 microgl 37 12 2 25 23 2 257 21 2 238 1 2

3624 microgkg 402 104 2 255 084 2 265 096 2 253 07 2

5306 microgkg 563 113 2 371 74 2 359 72 2 333 67 2

5308 microgkg 168633 102669 232169 211488

5309 microgl 402 268 27

5310 microgl 315 158 25 125 25 125 288 144

5312 microgl 428 043 2 282 028 2 272 027 2 275 028 2

5315 microgkg 387 08 2 256 05 2 251 05 2 253 05 2

5316 microgl 3839 342 2 2485 191 2 2559 168 2 239 51 2

5317 microgl 2466 043 2 2212 034 2 2472 016 2 2146 068 2

5319 microgl 414 41 2 279 28 2 268 27 2 26 31 2

5321 microgkg 476 316 326 328

5322 microgl 2547 35 2

5323 microgkg 405 203 2 239 119 2 255 153 2 201 11 2

58

Figure 11 Youden plots of the percent deviation of the reported values from the assigned values for the food supplement test sample and the

standard solution in acetonitrile with undisclosed content

(The identity of the respective measurand is given above the figure Results exceeding a difference of more than 100 were omitted)

59

Figure 11 - continued

60

ANNEX 10 Table 18 Method performance parameters reported by the participants

Laboratory code

Analyte LOD LOQ Recovery Analyte LOD LOQ Recovery

microgkg microgkg microgkg microgkg 3600 BaA 03 06 978 BaP 03 07 9483602 BaA 007 021 926 BaP 005 015 993603 BaA 0011 05 987 BaP 0005 051 9553604 BaA 02 04 86 BaP 02 04 883605 BaA 0025 005 118 BaP 0025 005 1083606 BaA 001 003 65 BaP 001 003 703607 BaA 021 071 74 BaP 027 089 943608 BaA 016 048 8213 BaP 015 046 83683610 BaA 02 05 85 BaP 005 02 853611 BaA BaP 3612 BaA 1 117 BaP 1 1203613 BaA 01 03 90 BaP 01 03 903614 BaA 02 05 96 BaP 02 05 933615 BaA 005 015 110 BaP 005 015 1053616 BaA 01 05 108 BaP 01 05 753617 BaA 018 037 405 BaP 009 018 3873618 BaA 01 03 63 BaP 01 03 593619 BaA 005 005 59 BaP 033 033 543620 BaA 0009 0018 64 BaP 0005 001 553621 BaA 007 02 84 BaP 007 02 923622 BaA 01 03 1101 BaP 01 03 9853623 BaA 02 08 105 BaP 02 08 1013624 BaA BaP 01 033 8513625 BaA BaP 3626 BaA BaP 5301 BaA 003 01 102 BaP 003 01 1005302 BaA 01 03 78 BaP 01 03 865303 BaA 003 01 62 BaP 003 01 805304 BaA 1 1 70 BaP 1 1 955305 BaA 02 05 85 BaP 02 05 855306 BaA 03 1 699 BaP 03 1 905307 BaA 6537 BaP 81165308 BaA 0088 0265 99 BaP 00262 00785 1005309 BaA 01 08 92 BaP 01 08 925310 BaA 01 03 BaP 01 03 905311 BaA 002 005 80 BaP 002 004 915312 BaA 03 09 105 BaP 03 09 1055313 BaA 04 08 BaP 04 08 5314 BaA 001 002 96 BaP 001 002 9485315 BaA 01 03 50 - 100 BaP 01 03 50 - 100 5316 BaA 002 011 827 BaP 004 019 10445317 BaA 05 1 76 BaP 05 1 755318 BaA BaP 5319 BaA 009 029 951 BaP 011 041 995320 BaA 12 18 946 BaP 06 1 8345321 BaA 03 05 BaP 05 1 5322 BaA 1 2 100 BaP 1 2 1005323 BaA 26 BaP 26

61

ANNEX 10 Table 18 - continued

Laboratory code Analyte LOD LOQ Recovery Analyte LOD LOQ Recovery

microgkg microgkg microgkg microgkg 3600 BbF 03 05 96 CHR 02 03 10153602 BbF 015 045 947 CHR 003 009 9193603 BbF 0014 05 1012 CHR 0004 05 10033604 BbF 02 04 85 CHR 02 04 873605 BbF 005 01 94 CHR 0025 005 863606 BbF 001 003 70 CHR 001 003 653607 BbF 018 06 100 CHR 014 028 943608 BbF 015 046 7979 CHR 015 045 71253610 BbF 005 02 85 CHR 02 05 853611 BbF CHR 3612 BbF 1 118 CHR 1 1173613 BbF 01 03 90 CHR 01 03 903614 BbF 01 04 96 CHR 02 05 953615 BbF 008 025 109 CHR 003 01 1083616 BbF 01 05 78 CHR 01 05 723617 BbF 035 07 38 CHR 012 024 4163618 BbF 01 03 62 CHR 01 03 623619 BbF 016 016 63 CHR 01 01 593620 BbF 0003 0006 55 CHR 001 002 653621 BbF 007 02 103 CHR 007 02 893622 BbF 02 06 963 CHR 03 09 1043623 BbF 02 08 106 CHR 02 08 1193624 BbF CHR 3625 BbF CHR 3626 BbF CHR 5301 BbF 003 01 96 CHR 003 01 1145302 BbF 01 03 84 CHR 01 03 835303 BbF 003 01 78 CHR 003 01 705304 BbF 1 1 86 CHR 1 1 705305 BbF 02 05 90 CHR 02 05 955306 BbF 03 1 1435 CHR 03 1 1185307 BbF 8116 CHR 65375308 BbF 0079 0238 99 CHR 009 026 1005309 BbF 01 08 92 CHR 5310 BbF 07 2 CHR 01 03 5311 BbF 002 004 95 CHR 002 004 895312 BbF 03 09 105 CHR 03 09 1055313 BbF 04 08 CHR 04 08 5314 BbF 001 002 83 CHR 001 002 8185315 BbF 02 05 50 -100 CHR 02 05 50 - 100 5316 BbF 005 024 814 CHR 001 007 9595317 BbF 05 1 73 CHR 05 1 785318 BbF CHR 5319 BbF 011 038 946 CHR 013 043 9675320 BbF 07 09 1194 CHR 23 32 10455321 BbF 05 1 CHR 05 1 5322 BbF 1 2 100 CHR 1 2 1005323 BbF 26 CHR 26

62

ANNEX 11 Table 19 Details of analysis method reported by the participants Data are presented as reported

Laboratory code

Analysis technique

Instrument calibration

Internal standards applied

Sample clean up

Details of sample

clean up

Extraction technique

Details of sample

extraction

3600 6) GC-MS

2) Internal standardisation with labelled substances

Deuterated PAH mix 9 (Ehrenstorfer)

1) Saponification 2) LiquidLiquid partitioning

D) Other Liquidliquid partitioning

3602 2) HPLC-UV-FLD

1) External calibration

3) Gel permeation chromatography

A) Pressurised liquid extraction (PLE)

3603 1) HPLC-FLD

1) External calibration

3) Gel permeation chromatography

D) Other 1 minute with dichloromethane in vortex

3604 2) HPLC-UV-FLD

2) Internal standardisation with labelled substances

DiP D14

5) Donor acceptor complex chromatography

D) Other LL

3605 1) HPLC-FLD

1) External calibration

3) Gel permeation chromatography

C) Soxhlet extraction

3606 7) GC-MSMS

2) Internal standardisation with labelled substances

BaP 13C4 CHR 13C6 BaA 13C6 BbF 13C6

4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

3607 6) GC-MS

2) Internal standardisation with labelled substances

BaA-d12 CHR-d12 BbF-d12 BkF-d12 BaP-d12 ICP-d12 BgP-d12 DiP-d14

1) Saponification

D) Other solvent partitioning

3608 1) HPLC-FLD

1) External calibration

6) Other Liquid extraction

D) Other

Extraction in a rotary agitator and centrifugation

3610 2) HPLC-UV-FLD

1) External calibration

4) Solid phase extraction

B) Sonication

3611

3612 6) GC-MS

2) Internal standardisation with labelled substances

chrysene-d12 dibenzo(ah)anthracene-d14 dibenzo(ai)pyrene-d14

4) Solid phase extraction

B) Sonication

3613 5) LC-MSMS

3) Internal standardisation with unlabelled substances

I use Chrysene-D12 3) Gel permeation chromatography

D) Other extraction with hexan

63

Table 19 - continued

Laboratory code

Analysis technique

Instrument calibration

Internal standards applied

Sample clean up Details of sample

clean up

Extraction technique

Details of sample

extraction

3614 6) GC-MS 1) External calibration

3) Gel permeation chromatography

B) Sonication

3615 1) HPLC-FLD

1) External calibration

6) Other PLE GPC

A) Pressurised liquid extraction (PLE)

3616 6) GC-MS

2) Internal standardisation with labelled substances

Benzo(a)pyrene-13C4 Benzo(a)anthracene-13C6 Benzo(b)fluoranthene-13C6 Chrysene-13C6

2) LiquidLiquid partitioning 4) Solid phase extraction

B) Sonication

3617 1) HPLC-FLD

1) External calibration

4) Solid phase extraction

C) Soxhlet extraction

3618 6) GC-MS

2) Internal standardisation with labelled substances

deuterated standards

1) Saponification 4) Solid phase extraction

D) Other Handshaken with cyclohexane

3619 6) GC-MS

2) Internal standardisation with labelled substances

C13 Labelled US EPA 16

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

D) Other Homegenisation amp saponiofication

3620 9) GC-HRMS

2) Internal standardisation with labelled substances

mix of deuterated PAHs (BAA-D12 CHR-D12 BAP-D12 BBF-D12)

3) Gel permeation chromatography 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

3621 6) GC-MS

2) Internal standardisation with labelled substances

Chrysene D12 Benzo(a)pyrene D12 Benzo(b)fluoranthene D12 Benzo(a)anthracene

2) LiquidLiquid partitioning 4) Solid phase extraction

B) Sonication

3622 1) HPLC-FLD

4) Standard addition method

4) Solid phase extraction

D) Other

Extraction with cyclohexane by heating under reflux

3623 7) GC-MSMS

2) Internal standardisation with labelled substances

C13 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

3624 2) HPLC-UV-FLD

1) External calibration

2) LiquidLiquid partitioning

D) Other hot solvent extraction

3626

64

Table 19 - continued

Laboratory code

Analysis technique

Instrument calibration

Internal standards

applied

Sample clean up

Details of sample

clean up

Extraction technique

Details of sample

extraction

5301 7) GC-MSMS

2) Internal standardisation with labelled substances

EACH SUBSTANCE C13

4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5302 7) GC-MSMS

2) Internal standardisation with labelled substances

Isotope labelled PAHs 13C for each analyte

4) Solid phase extraction

C) Soxhlet extraction

5303 7) GC-MSMS

3) Internal standardisation with unlabelled substances

HAP C13 4) Solid phase extraction

D) Other automate ASE200 DIONEX

5304 7) GC-MSMS

2) Internal standardisation with labelled substances

with C13 for each HAP

2) LiquidLiquid partitioning 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5305 7) GC-MSMS

2) Internal standardisation with labelled substances

C13 isotopiques internal standards

4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5306 7) GC-MSMS

2) Internal standardisation with labelled substances

C13 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5307 6) GC-MS

2) Internal standardisation with labelled substances

1) Saponification 2) LiquidLiquid partitioning

D) Other solvent extraction

5308 1) HPLC-FLD 1) External calibration

4) Solid phase extraction

B) Sonication

5309 1) HPLC-FLD 1) External calibration

4) Solid phase extraction

D) Other only SPE

5310 1) HPLC-FLD 1) External calibration

1) Saponification 2) LiquidLiquid partitioning 3) Gel permeation chromatography

C) Soxhlet extraction

5311 6) GC-MS

2) Internal standardisation with labelled substances

13 C Chrysene 13 C Benz(a)pyrene

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

B) Sonication

5312 1) HPLC-FLD

3) Internal standardisation with unlabelled substances

Benzo(b)chrysen

2) LiquidLiquid partitioning 4) Solid phase extraction

Quechers D) Other Quechers

65

Table 19 - continued

Laboratory code

Analysis technique

Instrument calibration

Internal standards applied

Sample clean up Details of sample

clean up

Extraction technique

Details of sample extraction

5313 6) GC-MS

2) Internal standardisation with labelled substances

Benz(a)anthracene D12 Benzo(b)fluranthene D12 Benzo(a)pyrene D12 Chrysene D12

1) Saponification 2) LiquidLiquid partitioning 3) Gel permeation chromatography

A) Pressurised liquid extraction (PLE)

5314 6) GC-MS

2) Internal standardisation with labelled substances

Benzo(a)pyren d12 2) LiquidLiquid partitioning

D) Other Extraction with

5315 1) HPLC-FLD

3) Internal standardisation with unlabelled substances

Benz(b)chrysen

1) Saponification 2) LiquidLiquid partitioning

D) Other shaker

5316 1) HPLC-FLD

1) External calibration

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

D) Other

LiquidLiquid extraction with cyclohexane and dimethylformamide

5317 7) GC-MSMS

1) External calibration

3) Gel permeation chromatography 6) Other

silica gel D) Other VDLUFA online method

5318

5319 1) HPLC-FLD

3) Internal standardisation with unlabelled substances

Benzo(b)chrysene 3) Gel permeation chromatography

A) Pressurised liquid extraction (PLE)

5320 6) GC-MS

3) Internal standardisation with unlabelled substances

22-binaphthyl

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

D) Other 3 hours refluxing

5321 1) HPLC-FLD

2) Internal standardisation with labelled substances

6-meacutethylchrysegravene

1) Saponification 2) LiquidLiquid partitioning

B) Sonication

5322 6) GC-MS

2) Internal standardisation with labelled substances

naftalene-d8 acenaphtalene-d10 phenanthrene-d10 chrysene-d12 perylene-d12 benzo(e)anthracene-d12

1) Saponification 2) LiquidLiquid partitioning 6) Other

clean-up with silica packed column

C) Soxhlet extraction

5323 1) HPLC-FLD

1) External calibration

4) Solid phase extraction 6) Other

Accelerated Solvent Extraction

A) Pressurised liquid extraction (PLE)

66

European Commission EUR 25245 EN ndash Joint Research Centre ndash Institute for Reference Materials and Measurements Title Report on the 8th inter-laboratory comparison organised by the European Union Reference Laboratory for Polycyclic Aromatic Hydrocarbons - Four marker PAHs in a dry extract of St Johns wort Author(s) Radoslav Lizak Szilard Szilagyi Philippe Verlinde and Thomas WENZL Luxembourg Publications Office of the European Union 2012 ndash 66 pp ndash 201 x 297 cm EUR ndash Scientific and Technical Research series ndash ISSN 1831-9424 ISBN 978-92-79-23337-1 doi 10278759387 Abstract The proficiency test here reported concerned the determination of the contents of four marker polycyclic aromatic hydrocarbons (PAHs) and of their sum in an food supplements test sample The set of marker PAHs consists of benz[a]anthacene benzo[a]pyrene benzo[b]fluoranthene and chrysene Participants to these PT were National Reference Laboratories for PAHs (NRLs-PAHs) and EU official food control laboratories The number of participants was in total 48

The PT was organised according ISO Standard 170432010

The test material used was a commercial dry extract of St Johns wort Participants also received a solution of the same PAHs either in acetonitrile or in toluene for checking their instrument calibration

The results from participants were rated with z-scores and zeta-scores About 68 of the reported results were attributed with z-scores with an absolute value of below two which is the threshold for satisfactory performance

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The mission of the JRC is to provide customer-driven scientific and technical support

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service of the European Commission the JRC functions as a reference centre of

science and technology for the Union Close to the policy-making process it serves

the common interest of the Member States while being independent of special

interests whether private or national

L

A-N

A-252

45-EN

-N

• 1 Summary
• 2 Introduction
• 3 Scope
• 4 Participating Laboratories
• 5 Time frame
• 6 Confidentiality
• 7 Test materials
• • 71 1Preparation of test materials
  • 72 Assigned values and standard deviation for proficiency assessment
  • 73 Homogeneity and stability
  • 74 Sample dispatch
  • • 8 Design of the proficiency test
    • 9 Evaluation of the results
    • • 91 2General
      • 92 21BEvaluation criteria
      • 93 Performance of participants
      • 94 Evaluation of the influence of analysis method
      • 95 Evaluation of the influence of calibration
      • 96 26Evaluation of compliance with legislation
      • • 10 Follow-up actions for underperforming laboratories
        • 11 1Conclusions
        • 12 1Acknowledgements
        • 13 References

23

Figure 3 continued

C) benzo[b]fluoranthene

D) chrysene

E) sum of four marker PAHs

The kernel density plots for BAP and BBF are almost identical and centred at or close to a z-score

value of zero as can be seen in Figure 3B and 3C Therefore bias introduced by the type of analysis

method is unlikely for these two analytes The situation is different for BAA and CHR In case of

BAA the kernel density distribution of z-scores based on HPLC-FD is shifted to negative values

24

(Figure 3A) which indicates a tendency to underestimate the BAA content in this particular

samplematrix when applying HPLC-FD This might be reasoned by separationintegration problems

between BAA and CHR peaks A few participants (from the HPLC-FD subgroup) that got negative

z-scores for BAA overestimated the CHR content This caused the slight shift of the density trace for

HPLC-FD towards negative values Removing this data from the evaluation would result in a density

trace with the mode at zero

The opposite is the case for CHR (Figure 3D) The density distribution of the z-scores attributed to

HPLC-FD data is centred almost at zero However the distribution for data based on GC-MS is shifted

to positive values indicating strong positive bias This bias might be caused by a lack of selectivity of

the analysis method The test material contains triphenylene which is isobaric to chrysene

Consequently it cannot be distinguished from chrysene by means of mass spectrometry These two

compounds have to be separated chromatographically which is not easy They coelute on unpolar and

on most mid-polar capillary columns Columns that allow their separation were commercialised only

recently and might not be available in all laboratories Therefore laboratories which determine the

four EU marker PAHs by means of GC-MS shall pay attention to the chromatographic separation of

triphenylene and chrysene A lack of chromatographic resolution might lead to strong positive bias of

GC-MS methods and to false conclusions on the marketability of the tested sample

The chromatographic separation of chrysene and triphenylene is not a problem with HPLC which is

reflected in the position of the density plot for chrysene However attention has to be paid in HPLC-

FD analysis to a proper clean up of the sample extract in order to eliminate interferences that show

similar retention as BAA and CHR

95 Evaluation of the influence of calibration

The participants received together with the food supplement test sample two standard solutions in

depending of their choice either acetonitrile or toluene The analyte content of one solution was

disclosed to the participants whereas the content of the other solution was kept undisclosed The

content values of the latter are presented in Table 5 Analysis results for the solution with undisclosed

content had to be reported to the PT organiser The respective data are presented in Annex 9

Percent deviations of the reported results from the assigned values were calculated for both the food

supplement test sample and the standard solution with undisclosed analyte content Youden plots were

prepared in order to visualize the influence of instrument calibration on the results reported for the

food supplement sample Two plots are discussed in the following The whole set of Youden plots are

given in Annex 9

25

Figure 4 Youden plot of the percent deviations of values reported for benzo[a]pyrene from the

assigned values of the food supplement test material and the standard solution in

toluene with undisclosed content

Figure 4 reveals that a number of participants reported results with similar percent deviations from the

assigned values of both the results for the food supplement material and the standard solution in

toluene with undisclosed content This correlation indicates that over- respectively underestimation of

the analyte content of the food supplement material might be caused by biased instrument calibration

This finding underpins the importance of thorough calibration standard preparation However the data

was not in all cases conclusive as some laboratories performed well for the food supplement samples

but reported biased results for the standard solution with undisclosed content (eg Figure 4 participants

3620 and 5305) This fact cannot be explained yet

Figure 5 presents the respective Youden plot for chrysene in the food supplement test material and the

standard solution in toluene with undisclosed content From this graph it can be concluded that the

overestimation of the chrysene content of the food supplement material was not caused by instrument

calibration The results for the solvent solution of many laboratories which underperformed for the

26

food supplement sample agreed well with the assigned value This supports the conclusion that the

overestimation is caused by the determination of an interferent most probably triphenylene

Figure 5 Youden plot of the percent deviations of values reported for chrysene from the

assigned values of the food supplement test material and the standard solution in

toluene with undisclosed content

96 Evaluation of compliance with legislation

The method performance data reported for the four marker PAHs were evaluated for compliance with

the provisions given in Commission Regulation (EC) No 8362011 Table 18 in ANNEX 10 gives an

overview on the results of the evaluation Data were not reported in case of empty cells Non-

compliant data are highlighted in Table 18 It should be noted that Regulation (EU) No 8362011

specifies LOD and LOQ with two decimals This was not respected by all participants which

hampered the compliance assessment

27

In summary it can be stated that the majority of participants reported method performance

characteristics for the determination of the four marker PAHs in the food supplement test sample that

are compliant with legislation However some laboratories need to improve their analysis methods

The laboratories in question are urged to adapt to the provisions given in the new legislation

10 Follow-up actions for underperforming laboratories

All laboratories that got questionable or non-satisfactory performance ratings are urged to perform

root cause analysis and to implement corrective actions

As agreed during the EU-RL PAH workshop 2011 the EU-RL PAH will set up follow-up measures in

due time for all NRLs that received for at least one of the four PAHs (BAA BAP BBF and CHR) z-

scores gt 3 as required by Regulation (EC) 8822004 and by the Protocol for management of

underperformance in comparative testing andor lack of collaboration of National Reference

Laboratories (NRLs) with European Union reference laboratories (EU-RLs) activities

These laboratories shall perform as an immediate action root-cause-analysis and shall report to the

EU-RL PAH in writing the identified cause for their underperformance and corrective actions they are

going to take Additionally they shall participate to an independent (non-EU-RL) proficiency test on

the determination of PAHs in food and shall communicate the outcome of this exercise to the EU-RL

PAH

28

11 Conclusions

Fourty seven participants reported results back to the EU-RL The performance of the majority of

participants was good In total 153 out of 225 attributed z-scores were below an absolute value of two

which equals to almost 68 However this percentage is much below the percentages of satisfactory

performance gained in previous studies This might be the consequence of the higher level of difficulty

with respect to matrix composition In addition bias can be concluded from the pattern of performance

indicators of some laboratories

In general the determination of chrysene caused most difficulties to the participants

zeta-Scores were calculated besides z-scores They indicate the agreement of the reported result with

the assigned value with respect to the stated measurement uncertainty The outcome of this rating was

worse than for the z-scores which indicates that the measurement uncertainty estimates were not

realistic Therefore participants underperforming with regard to zeta scores are urged to adapt their

measurement uncertainty statements

The great majority of participants in this inter-laboratory comparison applied analytical methods which

complied with EU legislation with regard to performance characteristics

12 Acknowledgements

The organisers would like to thank Mrs Beatriz de la Calle and Mr Franz Ulberth (all from IRMM

Geel Belgium) for their accurate revision of this report Mr Hakan Emteborg (from IRMM) for

ampouling of the standard solutions and all NRLs and OCLs for their cooperation

29

13 References

1 EU COMMISSION REGULATION (EC) No 7762006 of 23 May 2006 amending Annex VII to Regulation

(EC) No 8822004 of the European Parliament and of the Council as regards Community reference

laboratories Official Journal of the European Union 2006 L 136 p 3-8

Available from

httpeur-lexeuropaeuLexUriServLexUriServdouri=OJL200613600030008ENPDF

2 EU Regulation (EC) No 8822004 of the European Parliament and of the Council of 29 April 2004 on

official controls performed to ensure the verification of compliance with feed and food law animal health

and animal welfare rules Official Journal of the European Communities 2004 L191 p 1-52 Available

from

httpeur-lexeuropaeuLexUriServLexUriServdouri=OJL200419100010052ENPDF

3 EU Opinion of the Scientific Committee on Food on the risks to human health of Polycyclic Aromatic

Hydrocarbons in food (2002) Available from

httpeuropaeuintcommfoodfsscscfout153_enpdf

4 International Agency for Research on Cancer IARC Monographs on the Evaluation of

Carcinogenic Risks to Humans Some Non-heterocyclic Polycyclic Aromatic Hydrocarbons and Some

Related Exposures Volume 92 (2010) Available from

httpmonographsiarcfrENGMonographsvol92mono92pdf

5 EU Commission Regulation (EC) No 3332007 of 28 March 2007 laying down the methods of sampling

and analysis for the official control of the levels of lead cadmium mercury inorganic tin 3-MCPD and

benzo(a)pyrene in foodstuffs Official Journal of the European Union 2007 L 88 p 29-38 Available

from

httpeur-lexeuropaeuLexUriServLexUriServdouri=OJL200708800290038ENPDF

6 EU Commission Regulation (EC) No 18812006 of 19 December 2006 setting maximum levels for certain

contaminants in foodstuffs Official Journal of the European Union 2006 L 364 p 5-24 Available from

httpeur-lexeuropaeuLexUriServLexUriServdouri=OJL200636400050024ENPDF

7 EFSA Scientific Opinion of the Panel on Contaminants in the Food Chain on a request from the European

Commission on Polycyclic Aromatic Hydrocarbons in Food The EFSA Journal 2008 724 p 1-114

Available from

httpwwwefsaeuropaeuEFSAefsa_locale-1178620753812_1211902034842htm

30

8 EU Commission Regulation (EU) No 8352011 amending Regulation (EC) No 18812006 as regards

maximum levels for polycyclic aromatic hydrocarbons in foodstuffs Official Journal of the European

Union 2011 L 215 p 4-8

httpeur-lexeuropaeuLexUriServLexUriServdouri=OJL201121500040008ENPDF

9 EU Commission Regulation (EU) No 8362011 amending Regulation (EC) No 3332007 laying down the methods of sampling and analysis for the official control of the levels of lead cadmium mercury inorganic tin 3-MCPD and benzo(a)pyrene in foodstuffs Official Journal of the European Union 2011 L 215 p 9-16 httpeur-lexeuropaeuLexUriServLexUriServdouri=OJL201121500090016ENPDF

10 ISOIEC 170432010 (2010) Conformity assessment -- General requirements for proficiency testing

providers Geneva Switzerland

11 Thompson M Ellison SLR and Wood R The International Harmonized Protocol for the Proficiency

Testing of Analytical Chemistry Laboratories Pure Appl Chem (2006) 78(1) p 145ndash196

Available from httpmediaiupacorgpublicationspac2006pdf7801x0145pdf

31

14 ANNEXES

ANNEX 1 ndash Announcement of the PT on the IRMM webpage

ANNEX 2 ndash Announcement via e-mail and invitation

ANNEX 3 ndash Announcement of sample dispatch

ANNEX 4 ndash Technical specifications of the calibration solutions

ANNEX 5 ndash Homogeneity and stability of the test material

ANNEX 6 ndash Acknowledgement of sample receipt

ANNEX 7 ndash Questionnaire on details of analysis method

ANNEX 8 ndash Results reported for the food supplement sample

ANNEX 9 ndash Results reported for the standard solutions with undisclosed analyte content

ANNEX 10 ndash Method performance characteristics reported by participants

ANNEX 11 ndash Details of analysis methods reported by participants

32

ANNEX 1 ndash Announcement of the PT on the IRMM webpage

33

ANNEX 2 - Announcement via e-mail and invitation

34

ANNEX 3 - Announcement sample dispatch

Dear MadameSir

The test samples for the proficiency test on the determination of four PAHs in a food supplement (dry extract of St Johns wort) were

dispatched today

Explanation on the design of the study as well as instructions for sample storage and for reporting of results are in the parcel You will

also find your participant key in the parcel which is required for reporting of results

Please check the parcel after arrival for completeness and report the receipt of the test samples as well as their status to us by applying

the sample receipt form which is in the parcel

You are kindly requested to inform us by email if you do not receive the parcel by end of this week

My colleague Donata Lerda will then take care of it

With best regards

Thomas Wenzl

35

ANNEX 4 - Specifications of standard solutions with disclosed concentration

36

ANNEX 4 - continued

37

ANNEX 5 - Homogeneity and stability of the test material

Homogeneity of the test material

Benz[a]anthracene - BAA

Benzo[a]pyrene - BAP

38

ANNEX 5 - continued

Benzo[b]fluoranthene - BBF

Chrysene - CHR

39

ANNEX 5 - continued

Stability of the test material

Table 10 Content of the four target analytes in test materials stored under different

temperatures

BaA BaP BbF CHR

microgkg microgkg microgkg microgkg

Content 31 16 31 69 Storage at room temperature

U (k=2) 05 02 05 17

Content 31 15 29 68 Storage at reference temperature

U (k=2) 04 03 04 10

The applied capillary column does not separate CHR from triphenylene

40

ANNEX 6 ndash Documents sent to participants

41

ANNEX 6 - continued

42

ANNEX 7 Questionnaire on details of analysis method

43

ANNEX 7 - continued

44

ANNEX 7 - continued

45

ANNEX 8 - Results reported by participants for the food supplements test sample Table 11 Analysis results reported by the participants for the content of benz[a]anthracene (BAA) in the food supplements test material Values are presented as reported

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

3600 31 29 29 3 05 2

3602 199 193 178 19 021

3603 253 253 246 251 022 2

3604 199 209 23 213 071 2

3605 301 3 271 291 076 2

3606 302 288 278 289 064 2

3607 456 416 443 438 096 2

3608 796 695 651 714 149 2

3610 182 192 214 196 039 2

3611 414 504 488 469 96 2

3612 3 34 28 31 06 2

3613 32 33 34 33 07 2

3614 182 182 191 185 037 2

3615 3884 3822 3565 376 0752 2

3616 2 2 2 2 01 2

3617 366 37 35 362 046 2

3618 32 32 32 32 05 2

3619 299 305 297 299 048 2

3620 304 294 302 302 045 2

3621 29 26 29 28 04 2

3622 343 325 318 322 052 2

3623 35 3 33 33 07 2

3624

3626 318 31 314 314 126 2

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

5301 31 3 32 31 06 2

5302 314 319 315 316 063 2

5303 3102 2716 2822 2876 27 62

5304 261 263 26 261 57 2

5305 23 22 26 24 20

5306 22 23 23 22 11 2

5307 488 461 317 461

5308 168633 168544 1562025 1644598

5309 16 19 16 17

5310 3 304 311 305 015

5311 477 452 448 459 15 2

5312 506 63 494 543 134 2

5313 353 301 404 353

5314 431 373 378 394 5 096

5315 596 531 51 545 11 2

5316 224 231 235 235 022 2

5317 216 23 221 222 007 2

5318 064 07 067 01 2

5319 111 184 146 147 029 2

5320 515 525 564 534 48

5321 21 5322 13 87 9 87 35 2

5323 38 33 35 33 16 2

46

Figure 6 Results of replicate determinations (indicated by triangles) of benz[a]anthracene (BAA) in the food supplement test material

Horizontal blue lines represent the arithmetic mean value of replicate measurements and blue bars the reported expanded measurement uncertainty (k=2) The assigned value is

plotted as dotted line Red solid lines mark the range of satisfactory performance (|z|le2) Mean values are given for results outside the displayed data range

ProLab 2011

Laboratory

53

1853

1953

0853

0936

1436

0236

1036

1653

2136

0453

1753

0653

1653

0536

0353

0436

2153

0336

0636

0536

0036

2036

1953

1036

1253

0136

2653

0236

1836

2336

2236

1353

1353

2336

1736

1553

1453

0736

0753

1153

2053

1253

1536

0853

2236

11

an

aly

te c

on

ten

t (micro

gk

g)

10

95

9

85

8

75

7

65

6

55

5

45

4

35

3

25

2

15

1

05

0

102

3 468

7

Sample St Johns wort extract replicatesMeasurand Benz[a]anthraceneNo of laboratories 46

Assigned value 298 microgkg (Reference value)Target sd 062 microgkgTolerance limits 174 - 422 microgkg (|Z score| lt 200)

Assig

ned va

lue

Limit of tolerance

Limit of tolerance

47

Table 12 Analysis results reported by the participants for the content of benzo[a]pyrene (BAP) in the food supplements test material Values are presented as reported

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

3600 16 17 17 17 03 2

3602 118 123 13 124 015

3603 165 162 157 161 015 2

3604 134 154 139 143 04 2

3605 164 166 172 167 057 2

3606 181 159 161 167 031 2

3607 279 266 279 274 055 2

3608 57 687 461 573 226 2

3610 226 216 183 206 042 2

3611 289 325 311 308 36 2

3612 24 22 2 22 05 2

3613 17 18 19 18 05 2

3614 08 08 08 08 016 2

3615 1545 1393 1425 145 029 2

3616 16 16 14 15 03 2

3617 177 171 158 169 023 2

3618 2 2 2 2 02 2

3619 207 207 206 207 075 2

3620 165 169 163 166 017 2

3621 44 47 49 47 05 2

3622 212 208 222 213 038 2

3623 2 21 22 21 03 2

3624 18 178 182 18 031 2

3626 172 173 178 174 07 2

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

5301 18 18 18 18 04 2

5302 188 19 187 188 038 2

5303 1691 1426 149 1535 19 80

5304 145 142 15 146 69 2

5305 15 17 18 16 15

5306 16 17 17 16 08 2

5307 164 157 153 157

5308 102669 109324 109035 107009

5309 1 1 12 11

5310 122 122 124 123 006

5311 199 184 215 2 15 2

5312 258 248 239 248 05 2

5313 199 178 245 207

5314 174 171 172 172 11 0948

5315 253 248 213 238 05 2

5316 177 171 173 17 016 2

5317 255 277 276 269 012 2

5318 114 12 117 02 2

5319 19 228 231 216 043 2

5320 303 3 344 316 78

5321 12 12 12 12

5322 13 17 24 17 35 2

5323 2 2 19 2 1 2

48

Figure 7 Results of replicate determinations (indicated by triangles) of benzo[a]pyrene (BAP) in the food supplement test material

Horizontal blue lines represent the arithmetic mean value of replicate measurements and blue bars the reported expanded measurement uncertainty (k=2) The assigned value is

plotted as dotted line Red solid lines mark the range of satisfactory performance (|z|le2) Mean values are given for results outside the displayed data range

ProLab 2011

Laboratory

36

1453

0953

0853

1853

2153

1036

0236

0436

1553

0436

1653

0353

0736

0336

2036

0053

0553

0636

0636

0536

1753

1453

1636

2636

1336

2453

0153

2253

0253

2353

1136

1836

1953

1336

1036

2336

2253

1936

1253

1553

1253

1736

0753

2036

2136

0836

11

an

aly

te c

on

ten

t (micro

gk

g)

55

5

45

4

35

3

25

2

15

1

05

0

573

308

3

Sample St Johns wort extract replicatesMeasurand Benzo[a]pyreneNo of laboratories 48

Assigned value 165 microgkg (Reference value)Target sd 036 microgkgTolerance limits 093 - 237 microgkg (|Z score| lt 200)

Assig

ned va

lue

Limit of tolerance

Limit of tolerance

49

Table 13 Analysis results reported by the participants for the content of benzo[b]fluoranthene (BBF) in the food supplements test material Values are presented as reported

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

3600 32 3 3 31 09 2

3602 173 175 16 17 017

3603 265 252 25 256 025 2

3604 116 108 105 11 028 2

3605 286 225 232 247 074 2

3606 294 273 261 276 046 2

3607 579 579 544 567 113 2

3608 103 1201 876 1036 326 2

3610 173 196 204 191 038 2

3611 546 618 565 576 75 2

3612 3 28 28 29 06 2

3613 28 31 32 31 06 2

3614 135 111 135 127 022 2

3615 4187 3678 4111 4 08 2

3616 2 2 2 2 01 2

3617 33 27 299 035 2

3618 31 31 31 31 05 2

3619 326 324 312 326 064 2

3620 277 28 272 276 069 2

3621 26 25 23 25 04 2

3622 346 336 333 34 052 2

3623 26 28 29 28 02 2

3624

3626 288 28 307 292 117 2

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

5301 29 3 29 29 06 2

5302 297 3 296 298 06 2

5303 2705 2444 2608 2586 22 78

5304 245 254 26 253 85 2

5305 23 23 26 24 15

5306 42 43 4 42 21 2

5307 221 216 241 221

5308 232169 246654 240722 229848

5309 19 22 19 2 0

5310 3 315 325 313 016

5311 262 277 304 281 15 2

5312 27 289 337 299 075 2

5313 321 284 378 328

5314 423 404 385 404 34 083

5315 42 506 416 444 09 2

5316 265 267 277 273 018 2

5317 185 197 195 192 006 2

5318 276 281 279 04 2

5319 396 389 403 396 079 2

5320 623 618 635 625 14

5321 31

5322 24 33 49 33 35 2

5323 38 45 35 45 27 2

50

Figure 8 Results of replicate determinations (indicated by triangles) of benzo[b]fluoranthene (BBF) in the food supplement test material

Horizontal blue lines represent the arithmetic mean value of replicate measurements and blue bars the reported expanded measurement uncertainty (k=2) The assigned value is

plotted as dotted line Red solid lines mark the range of satisfactory performance (|z|le2) Mean values are given for results outside the displayed data range

ProLab 2011

Laboratory

36

0436

1436

0236

1053

1736

1653

0953

0753

0853

0536

2136

0553

0436

0353

0353

1636

0636

2036

2353

1853

1136

1236

2653

0153

0253

1236

1736

1336

0036

1853

2153

1036

1953

1336

2253

2253

2353

1936

1553

1453

0653

1536

0753

2036

0836

11

an

aly

te c

on

ten

t (micro

gk

g)

75

7

65

6

55

5

45

4

35

3

25

2

15

1

05

0

103

6

576

3

Sample St Johns wort extract replicatesMeasurand Benzo[b]fluorantheneNo of laboratories 46

Assigned value 292 microgkg (Reference value)Target sd 060 microgkgTolerance limits 172 - 412 microgkg (|Z score| lt 200)

Assig

ned va

lue

Limit of tolerance

Limit of tolerance

51

Table 14 Analysis results reported by the participants for the content of chrysene (CHR) in the food supplements test material Values are presented as reported

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

3600 5 49 5 5 07 2

3602 32 304 29 304 036

3603 462 469 447 459 048 2

3604 438 428 42 429 119 2

3605 365 41 381 386 085 2

3606 494 411 45 451 124 2

3607 897 774 787 819 18 2

3608 986 887 1168 1014 284 2

3610 418 392 462 424 085 2

3611 833 893 87 865 61 2

3612 6 64 58 61 12 2

3613 41 39 4 4 08 2

3614 272 254 288 271 054 2

3615 605 6028 5964 601 12 2

3616 47 45 49 47 05 2

3617 445 431 389 422 065 2

3618 45 45 45 45 06 2

3619 738 747 72 738 118 2

3620 463 472 446 46 092 2

3621 53 49 51 51 06 2

3622 478 513 505 488 08 2

3623 73 73 72 72 05 2

3624

3626 63 622 634 628 251 2

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

5301 49 49 49 49 1 2

5302 687 683 666 679 136 2

5303 6777 6181 6382 6441 24 70

5304 592 581 6 591 85 2

5305 6 58 64 61 20

5306 49 5 51 49 25 2

5307 524 5 574 524

5308 211488 216782 221146 213243

5309

5310 372 393 394 386 019

5311 43 459 422 437 15 2

5312 934 943 981 953 191 2

5313 793 73 99 838

5314 636 561 565 588 18 0818

5315 547 583 499 542 11 2

5316 292 286 285 282 06 2

5317 52 598 517 545 046 2

5318 368 345 357 05 2

5319 449 471 474 465 116 2

5320 76 924 818 834 99

5321 33

5322 7 7 76 7 35 2

5323 87 73 77 73 37 2

52

Figure 9 Results of replicate determinations (indicated by triangles) of chrysene (CHR) in the food supplement test material

Horizontal blue lines represent the arithmetic mean value of replicate measurements and blue bars the reported expanded measurement uncertainty (k=2) The assigned value is

plotted as dotted line Red solid lines mark the range of satisfactory performance (|z|le2) Mean values are given for results outside the displayed data range

ProLab 2011

Laboratory

53

0836

1453

1636

0253

2153

1836

0553

1036

1336

1736

1036

0453

1136

1836

0636

0336

2053

1936

1653

0136

0036

2253

0636

2153

0753

1553

1753

1453

0436

1536

1253

0536

2653

0353

0253

2236

2336

1953

2336

0753

2053

1353

1236

0836

11

an

aly

te c

on

ten

t (micro

gk

g)

13

12

11

10

9

8

7

6

5

4

3

2

1

0

865

3

Sample St Johns wort extract replicatesMeasurand ChryseneNo of laboratories 45

Assigned value 407 microgkg (Reference value)Target sd 083 microgkgTolerance limits 241 - 573 microgkg (|Z score| lt 200)

Assig

ned va

lue

Limit of tolerance

Limit of tolerance

53

Table 15 Analysis results reported by the participants for the sum of the four marker PAHs in the food supplements test material Values are presented as reported

Laboratory code

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg

3600 128 24 2

3602 79 126

3603 1127 096 2

3604 893 258 2

3605 1091 305 2

3606 1184 238 2

3607 2098 42 2

3608 3336 325 2

3610 1138 17 2

3611 2218 259 2

3612 143 29 2

3613 122 2 2

3614 663 129 2

3615 152 304 2

3616 102 03 2

3617 1252 169 2

3618 128 18 2

3619 157 305 2

3620 1204 12 2

3621 151 19 2

3622 1361 115 2

3623 154 09 2

3624

3626 1409 56 2

Laboratory code

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg

5301 126

5302 37 074 2

5303 13438 92

5304 1251 85 2

5305 125 20

5306 129 65 2

5307 1363

5308 7245598

5309

5310 1127 056

5311 1367 15 2

5312 2042 408 2

5313 1726

5314 1558

5315 177 35 2

5316 96 073 2

5317 1229 066 2

5318 82 05 2

5319 1225 267 2

5320 2309 41

5321

5322 207 35 2

5323 171 103 2

54

ANNEX 9 Results reported for the standard solutions with undisclosed analyte content Table 16 Analysis results reported by the participants for the four marker PAHs in the standard solution in toluene with undisclosed analyte

content Values are presented as reported U measurement uncertainty k coverage factor

benz[a]anthracene benzo[a]pyrene benzo[b]fluoranthene chrysene Laboratory

code Unit Result U k Result U k Result U k Result U k 3600 microgl 38 67 2 25 45 2 211 68 2 275 38 2

3605 microgl 367 184 2 2482 124 2 2592 13 2 235 118 2

3606 microgkg 3763 176 2 2609 142 2 2721 118 2 2539 134 2

3607 microgkg 3706 815 2 2824 565 2 2591 518 2 2587 569 2

3612 microgl 38 08 2 237 11 2 244 18 2 257 02 2

3614 microgl 324 65 2 209 38 2 215 37 2 21 42 2

3616 microgkg 318 07 2 224 14 2 231 26 2 216 1 2

3618 microgkg 417 17 2 284 08 2 292 06 2 297 03 2

3619 microgkg 47 742 2 274 468 2 228 387 2 274 434 2

3620 microgl 495 74 2 294 29 2 321 8 2 321 64 2

3621 microgl 241 22 2 143 13 2 188 16 2 145 14 2

3623 microgkg 476 25 2 311 53 2 269 3 2 297 03 2

3626 microgl 359 38 2 235 23 2 238 27 2 24 18 2

5301 microgkg 426 29 299 289

5302 microgkg 436 87 2 296 59 2 296 59 2 289 58 2

5303 microgl 3721 27 102 22838 19 99 23756 22 99 25486 24 98

5304 microgkg 38 29 2 225 26 2 257 37 2 258 52 2

5305 microgkg 305 225 245 29

5306 microgkg 316 63 2 247 49 2 65 13 2 22 44 2

5307 microgkg 3658 2559 241 2414

5308 microgkg 16854 109324 246654 216782

5311 microgl 374 20 2 279 15 2 268 15 2 272 15 2

5313 microgl 3408 2165 2275 2373

5314 microgkg 395 5 2875 11 3614 34 2905 18

5320 microgl 3267 368 1986 571 28 683 2427 512

5322 microgl 282 35 2 225 35 2 241 35 2 21 35 2

55

Figure 10 Youden plots of the percent deviation of the reported values from the assigned values for the food supplement test sample and the

standard solution in toluene with undisclosed content

(The identity of the respective measurand is given above the figure Results exceeding a difference of more than 100 were omitted)

56

Figure 10 - continued

57

Table 17 Analysis results reported by the participants for the four marker PAHs in the standard solution in acetonitrile with undisclosed

analyte content

Values are presented as reported U measurement uncertainty k coverage factor

benz[a]anthracene benzo[a]pyrene benzo[b]fluoranthene chrysene

Laboratory

code Unit Result U k Result U k Result U k Result U k

3602 microgkg 3675 247 247 244

3603 microgkg 358 086 2 236 028 2 235 028 2 236 057 2

3604 microgl 475 29 2 19 12 2 194 13 2 258 12 2

3608 microgl 3623 078 2 2385 023 2 2343 18 2 2296 092 2

3610 microgkg 369 185 2 25 125 2 251 126 2 244 122 2

3611 microgkg 48 02 2 323 02 2 328 04 2 317 03 2

3613 microgl 332 31 2 261 3 2 249 25 2 239 24 2

3615 microgl 415 2635 2729 2689

3617 microgkg 37 09 2 244 06 2 284 07 2 239 06 2

3622 microgl 37 12 2 25 23 2 257 21 2 238 1 2

3624 microgkg 402 104 2 255 084 2 265 096 2 253 07 2

5306 microgkg 563 113 2 371 74 2 359 72 2 333 67 2

5308 microgkg 168633 102669 232169 211488

5309 microgl 402 268 27

5310 microgl 315 158 25 125 25 125 288 144

5312 microgl 428 043 2 282 028 2 272 027 2 275 028 2

5315 microgkg 387 08 2 256 05 2 251 05 2 253 05 2

5316 microgl 3839 342 2 2485 191 2 2559 168 2 239 51 2

5317 microgl 2466 043 2 2212 034 2 2472 016 2 2146 068 2

5319 microgl 414 41 2 279 28 2 268 27 2 26 31 2

5321 microgkg 476 316 326 328

5322 microgl 2547 35 2

5323 microgkg 405 203 2 239 119 2 255 153 2 201 11 2

58

Figure 11 Youden plots of the percent deviation of the reported values from the assigned values for the food supplement test sample and the

standard solution in acetonitrile with undisclosed content

(The identity of the respective measurand is given above the figure Results exceeding a difference of more than 100 were omitted)

59

Figure 11 - continued

60

ANNEX 10 Table 18 Method performance parameters reported by the participants

Laboratory code

Analyte LOD LOQ Recovery Analyte LOD LOQ Recovery

microgkg microgkg microgkg microgkg 3600 BaA 03 06 978 BaP 03 07 9483602 BaA 007 021 926 BaP 005 015 993603 BaA 0011 05 987 BaP 0005 051 9553604 BaA 02 04 86 BaP 02 04 883605 BaA 0025 005 118 BaP 0025 005 1083606 BaA 001 003 65 BaP 001 003 703607 BaA 021 071 74 BaP 027 089 943608 BaA 016 048 8213 BaP 015 046 83683610 BaA 02 05 85 BaP 005 02 853611 BaA BaP 3612 BaA 1 117 BaP 1 1203613 BaA 01 03 90 BaP 01 03 903614 BaA 02 05 96 BaP 02 05 933615 BaA 005 015 110 BaP 005 015 1053616 BaA 01 05 108 BaP 01 05 753617 BaA 018 037 405 BaP 009 018 3873618 BaA 01 03 63 BaP 01 03 593619 BaA 005 005 59 BaP 033 033 543620 BaA 0009 0018 64 BaP 0005 001 553621 BaA 007 02 84 BaP 007 02 923622 BaA 01 03 1101 BaP 01 03 9853623 BaA 02 08 105 BaP 02 08 1013624 BaA BaP 01 033 8513625 BaA BaP 3626 BaA BaP 5301 BaA 003 01 102 BaP 003 01 1005302 BaA 01 03 78 BaP 01 03 865303 BaA 003 01 62 BaP 003 01 805304 BaA 1 1 70 BaP 1 1 955305 BaA 02 05 85 BaP 02 05 855306 BaA 03 1 699 BaP 03 1 905307 BaA 6537 BaP 81165308 BaA 0088 0265 99 BaP 00262 00785 1005309 BaA 01 08 92 BaP 01 08 925310 BaA 01 03 BaP 01 03 905311 BaA 002 005 80 BaP 002 004 915312 BaA 03 09 105 BaP 03 09 1055313 BaA 04 08 BaP 04 08 5314 BaA 001 002 96 BaP 001 002 9485315 BaA 01 03 50 - 100 BaP 01 03 50 - 100 5316 BaA 002 011 827 BaP 004 019 10445317 BaA 05 1 76 BaP 05 1 755318 BaA BaP 5319 BaA 009 029 951 BaP 011 041 995320 BaA 12 18 946 BaP 06 1 8345321 BaA 03 05 BaP 05 1 5322 BaA 1 2 100 BaP 1 2 1005323 BaA 26 BaP 26

61

ANNEX 10 Table 18 - continued

Laboratory code Analyte LOD LOQ Recovery Analyte LOD LOQ Recovery

microgkg microgkg microgkg microgkg 3600 BbF 03 05 96 CHR 02 03 10153602 BbF 015 045 947 CHR 003 009 9193603 BbF 0014 05 1012 CHR 0004 05 10033604 BbF 02 04 85 CHR 02 04 873605 BbF 005 01 94 CHR 0025 005 863606 BbF 001 003 70 CHR 001 003 653607 BbF 018 06 100 CHR 014 028 943608 BbF 015 046 7979 CHR 015 045 71253610 BbF 005 02 85 CHR 02 05 853611 BbF CHR 3612 BbF 1 118 CHR 1 1173613 BbF 01 03 90 CHR 01 03 903614 BbF 01 04 96 CHR 02 05 953615 BbF 008 025 109 CHR 003 01 1083616 BbF 01 05 78 CHR 01 05 723617 BbF 035 07 38 CHR 012 024 4163618 BbF 01 03 62 CHR 01 03 623619 BbF 016 016 63 CHR 01 01 593620 BbF 0003 0006 55 CHR 001 002 653621 BbF 007 02 103 CHR 007 02 893622 BbF 02 06 963 CHR 03 09 1043623 BbF 02 08 106 CHR 02 08 1193624 BbF CHR 3625 BbF CHR 3626 BbF CHR 5301 BbF 003 01 96 CHR 003 01 1145302 BbF 01 03 84 CHR 01 03 835303 BbF 003 01 78 CHR 003 01 705304 BbF 1 1 86 CHR 1 1 705305 BbF 02 05 90 CHR 02 05 955306 BbF 03 1 1435 CHR 03 1 1185307 BbF 8116 CHR 65375308 BbF 0079 0238 99 CHR 009 026 1005309 BbF 01 08 92 CHR 5310 BbF 07 2 CHR 01 03 5311 BbF 002 004 95 CHR 002 004 895312 BbF 03 09 105 CHR 03 09 1055313 BbF 04 08 CHR 04 08 5314 BbF 001 002 83 CHR 001 002 8185315 BbF 02 05 50 -100 CHR 02 05 50 - 100 5316 BbF 005 024 814 CHR 001 007 9595317 BbF 05 1 73 CHR 05 1 785318 BbF CHR 5319 BbF 011 038 946 CHR 013 043 9675320 BbF 07 09 1194 CHR 23 32 10455321 BbF 05 1 CHR 05 1 5322 BbF 1 2 100 CHR 1 2 1005323 BbF 26 CHR 26

62

ANNEX 11 Table 19 Details of analysis method reported by the participants Data are presented as reported

Laboratory code

Analysis technique

Instrument calibration

Internal standards applied

Sample clean up

Details of sample

clean up

Extraction technique

Details of sample

extraction

3600 6) GC-MS

2) Internal standardisation with labelled substances

Deuterated PAH mix 9 (Ehrenstorfer)

1) Saponification 2) LiquidLiquid partitioning

D) Other Liquidliquid partitioning

3602 2) HPLC-UV-FLD

1) External calibration

3) Gel permeation chromatography

A) Pressurised liquid extraction (PLE)

3603 1) HPLC-FLD

1) External calibration

3) Gel permeation chromatography

D) Other 1 minute with dichloromethane in vortex

3604 2) HPLC-UV-FLD

2) Internal standardisation with labelled substances

DiP D14

5) Donor acceptor complex chromatography

D) Other LL

3605 1) HPLC-FLD

1) External calibration

3) Gel permeation chromatography

C) Soxhlet extraction

3606 7) GC-MSMS

2) Internal standardisation with labelled substances

BaP 13C4 CHR 13C6 BaA 13C6 BbF 13C6

4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

3607 6) GC-MS

2) Internal standardisation with labelled substances

BaA-d12 CHR-d12 BbF-d12 BkF-d12 BaP-d12 ICP-d12 BgP-d12 DiP-d14

1) Saponification

D) Other solvent partitioning

3608 1) HPLC-FLD

1) External calibration

6) Other Liquid extraction

D) Other

Extraction in a rotary agitator and centrifugation

3610 2) HPLC-UV-FLD

1) External calibration

4) Solid phase extraction

B) Sonication

3611

3612 6) GC-MS

2) Internal standardisation with labelled substances

chrysene-d12 dibenzo(ah)anthracene-d14 dibenzo(ai)pyrene-d14

4) Solid phase extraction

B) Sonication

3613 5) LC-MSMS

3) Internal standardisation with unlabelled substances

I use Chrysene-D12 3) Gel permeation chromatography

D) Other extraction with hexan

63

Table 19 - continued

Laboratory code

Analysis technique

Instrument calibration

Internal standards applied

Sample clean up Details of sample

clean up

Extraction technique

Details of sample

extraction

3614 6) GC-MS 1) External calibration

3) Gel permeation chromatography

B) Sonication

3615 1) HPLC-FLD

1) External calibration

6) Other PLE GPC

A) Pressurised liquid extraction (PLE)

3616 6) GC-MS

2) Internal standardisation with labelled substances

Benzo(a)pyrene-13C4 Benzo(a)anthracene-13C6 Benzo(b)fluoranthene-13C6 Chrysene-13C6

2) LiquidLiquid partitioning 4) Solid phase extraction

B) Sonication

3617 1) HPLC-FLD

1) External calibration

4) Solid phase extraction

C) Soxhlet extraction

3618 6) GC-MS

2) Internal standardisation with labelled substances

deuterated standards

1) Saponification 4) Solid phase extraction

D) Other Handshaken with cyclohexane

3619 6) GC-MS

2) Internal standardisation with labelled substances

C13 Labelled US EPA 16

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

D) Other Homegenisation amp saponiofication

3620 9) GC-HRMS

2) Internal standardisation with labelled substances

mix of deuterated PAHs (BAA-D12 CHR-D12 BAP-D12 BBF-D12)

3) Gel permeation chromatography 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

3621 6) GC-MS

2) Internal standardisation with labelled substances

Chrysene D12 Benzo(a)pyrene D12 Benzo(b)fluoranthene D12 Benzo(a)anthracene

2) LiquidLiquid partitioning 4) Solid phase extraction

B) Sonication

3622 1) HPLC-FLD

4) Standard addition method

4) Solid phase extraction

D) Other

Extraction with cyclohexane by heating under reflux

3623 7) GC-MSMS

2) Internal standardisation with labelled substances

C13 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

3624 2) HPLC-UV-FLD

1) External calibration

2) LiquidLiquid partitioning

D) Other hot solvent extraction

3626

64

Table 19 - continued

Laboratory code

Analysis technique

Instrument calibration

Internal standards

applied

Sample clean up

Details of sample

clean up

Extraction technique

Details of sample

extraction

5301 7) GC-MSMS

2) Internal standardisation with labelled substances

EACH SUBSTANCE C13

4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5302 7) GC-MSMS

2) Internal standardisation with labelled substances

Isotope labelled PAHs 13C for each analyte

4) Solid phase extraction

C) Soxhlet extraction

5303 7) GC-MSMS

3) Internal standardisation with unlabelled substances

HAP C13 4) Solid phase extraction

D) Other automate ASE200 DIONEX

5304 7) GC-MSMS

2) Internal standardisation with labelled substances

with C13 for each HAP

2) LiquidLiquid partitioning 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5305 7) GC-MSMS

2) Internal standardisation with labelled substances

C13 isotopiques internal standards

4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5306 7) GC-MSMS

2) Internal standardisation with labelled substances

C13 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5307 6) GC-MS

2) Internal standardisation with labelled substances

1) Saponification 2) LiquidLiquid partitioning

D) Other solvent extraction

5308 1) HPLC-FLD 1) External calibration

4) Solid phase extraction

B) Sonication

5309 1) HPLC-FLD 1) External calibration

4) Solid phase extraction

D) Other only SPE

5310 1) HPLC-FLD 1) External calibration

1) Saponification 2) LiquidLiquid partitioning 3) Gel permeation chromatography

C) Soxhlet extraction

5311 6) GC-MS

2) Internal standardisation with labelled substances

13 C Chrysene 13 C Benz(a)pyrene

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

B) Sonication

5312 1) HPLC-FLD

3) Internal standardisation with unlabelled substances

Benzo(b)chrysen

2) LiquidLiquid partitioning 4) Solid phase extraction

Quechers D) Other Quechers

65

Table 19 - continued

Laboratory code

Analysis technique

Instrument calibration

Internal standards applied

Sample clean up Details of sample

clean up

Extraction technique

Details of sample extraction

5313 6) GC-MS

2) Internal standardisation with labelled substances

Benz(a)anthracene D12 Benzo(b)fluranthene D12 Benzo(a)pyrene D12 Chrysene D12

1) Saponification 2) LiquidLiquid partitioning 3) Gel permeation chromatography

A) Pressurised liquid extraction (PLE)

5314 6) GC-MS

2) Internal standardisation with labelled substances

Benzo(a)pyren d12 2) LiquidLiquid partitioning

D) Other Extraction with

5315 1) HPLC-FLD

3) Internal standardisation with unlabelled substances

Benz(b)chrysen

1) Saponification 2) LiquidLiquid partitioning

D) Other shaker

5316 1) HPLC-FLD

1) External calibration

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

D) Other

LiquidLiquid extraction with cyclohexane and dimethylformamide

5317 7) GC-MSMS

1) External calibration

3) Gel permeation chromatography 6) Other

silica gel D) Other VDLUFA online method

5318

5319 1) HPLC-FLD

3) Internal standardisation with unlabelled substances

Benzo(b)chrysene 3) Gel permeation chromatography

A) Pressurised liquid extraction (PLE)

5320 6) GC-MS

3) Internal standardisation with unlabelled substances

22-binaphthyl

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

D) Other 3 hours refluxing

5321 1) HPLC-FLD

2) Internal standardisation with labelled substances

6-meacutethylchrysegravene

1) Saponification 2) LiquidLiquid partitioning

B) Sonication

5322 6) GC-MS

2) Internal standardisation with labelled substances

naftalene-d8 acenaphtalene-d10 phenanthrene-d10 chrysene-d12 perylene-d12 benzo(e)anthracene-d12

1) Saponification 2) LiquidLiquid partitioning 6) Other

clean-up with silica packed column

C) Soxhlet extraction

5323 1) HPLC-FLD

1) External calibration

4) Solid phase extraction 6) Other

Accelerated Solvent Extraction

A) Pressurised liquid extraction (PLE)

66

European Commission EUR 25245 EN ndash Joint Research Centre ndash Institute for Reference Materials and Measurements Title Report on the 8th inter-laboratory comparison organised by the European Union Reference Laboratory for Polycyclic Aromatic Hydrocarbons - Four marker PAHs in a dry extract of St Johns wort Author(s) Radoslav Lizak Szilard Szilagyi Philippe Verlinde and Thomas WENZL Luxembourg Publications Office of the European Union 2012 ndash 66 pp ndash 201 x 297 cm EUR ndash Scientific and Technical Research series ndash ISSN 1831-9424 ISBN 978-92-79-23337-1 doi 10278759387 Abstract The proficiency test here reported concerned the determination of the contents of four marker polycyclic aromatic hydrocarbons (PAHs) and of their sum in an food supplements test sample The set of marker PAHs consists of benz[a]anthacene benzo[a]pyrene benzo[b]fluoranthene and chrysene Participants to these PT were National Reference Laboratories for PAHs (NRLs-PAHs) and EU official food control laboratories The number of participants was in total 48

The PT was organised according ISO Standard 170432010

The test material used was a commercial dry extract of St Johns wort Participants also received a solution of the same PAHs either in acetonitrile or in toluene for checking their instrument calibration

The results from participants were rated with z-scores and zeta-scores About 68 of the reported results were attributed with z-scores with an absolute value of below two which is the threshold for satisfactory performance

How to obtain EU publications

Our priced publications are available from EU Bookshop (httpbookshopeuropaeu) where you can place

an order with the sales agent of your choice

The Publications Office has a worldwide network of sales agents You can obtain their contact details by

sending a fax to (352) 29 29-42758

The mission of the JRC is to provide customer-driven scientific and technical support

for the conception development implementation and monitoring of EU policies As a

service of the European Commission the JRC functions as a reference centre of

science and technology for the Union Close to the policy-making process it serves

the common interest of the Member States while being independent of special

interests whether private or national

L

A-N

A-252

45-EN

-N

• 1 Summary
• 2 Introduction
• 3 Scope
• 4 Participating Laboratories
• 5 Time frame
• 6 Confidentiality
• 7 Test materials
• • 71 1Preparation of test materials
  • 72 Assigned values and standard deviation for proficiency assessment
  • 73 Homogeneity and stability
  • 74 Sample dispatch
  • • 8 Design of the proficiency test
    • 9 Evaluation of the results
    • • 91 2General
      • 92 21BEvaluation criteria
      • 93 Performance of participants
      • 94 Evaluation of the influence of analysis method
      • 95 Evaluation of the influence of calibration
      • 96 26Evaluation of compliance with legislation
      • • 10 Follow-up actions for underperforming laboratories
        • 11 1Conclusions
        • 12 1Acknowledgements
        • 13 References

24

(Figure 3A) which indicates a tendency to underestimate the BAA content in this particular

samplematrix when applying HPLC-FD This might be reasoned by separationintegration problems

between BAA and CHR peaks A few participants (from the HPLC-FD subgroup) that got negative

z-scores for BAA overestimated the CHR content This caused the slight shift of the density trace for

HPLC-FD towards negative values Removing this data from the evaluation would result in a density

trace with the mode at zero

The opposite is the case for CHR (Figure 3D) The density distribution of the z-scores attributed to

HPLC-FD data is centred almost at zero However the distribution for data based on GC-MS is shifted

to positive values indicating strong positive bias This bias might be caused by a lack of selectivity of

the analysis method The test material contains triphenylene which is isobaric to chrysene

Consequently it cannot be distinguished from chrysene by means of mass spectrometry These two

compounds have to be separated chromatographically which is not easy They coelute on unpolar and

on most mid-polar capillary columns Columns that allow their separation were commercialised only

recently and might not be available in all laboratories Therefore laboratories which determine the

four EU marker PAHs by means of GC-MS shall pay attention to the chromatographic separation of

triphenylene and chrysene A lack of chromatographic resolution might lead to strong positive bias of

GC-MS methods and to false conclusions on the marketability of the tested sample

The chromatographic separation of chrysene and triphenylene is not a problem with HPLC which is

reflected in the position of the density plot for chrysene However attention has to be paid in HPLC-

FD analysis to a proper clean up of the sample extract in order to eliminate interferences that show

similar retention as BAA and CHR

95 Evaluation of the influence of calibration

The participants received together with the food supplement test sample two standard solutions in

depending of their choice either acetonitrile or toluene The analyte content of one solution was

disclosed to the participants whereas the content of the other solution was kept undisclosed The

content values of the latter are presented in Table 5 Analysis results for the solution with undisclosed

content had to be reported to the PT organiser The respective data are presented in Annex 9

Percent deviations of the reported results from the assigned values were calculated for both the food

supplement test sample and the standard solution with undisclosed analyte content Youden plots were

prepared in order to visualize the influence of instrument calibration on the results reported for the

food supplement sample Two plots are discussed in the following The whole set of Youden plots are

given in Annex 9

25

Figure 4 Youden plot of the percent deviations of values reported for benzo[a]pyrene from the

assigned values of the food supplement test material and the standard solution in

toluene with undisclosed content

Figure 4 reveals that a number of participants reported results with similar percent deviations from the

assigned values of both the results for the food supplement material and the standard solution in

toluene with undisclosed content This correlation indicates that over- respectively underestimation of

the analyte content of the food supplement material might be caused by biased instrument calibration

This finding underpins the importance of thorough calibration standard preparation However the data

was not in all cases conclusive as some laboratories performed well for the food supplement samples

but reported biased results for the standard solution with undisclosed content (eg Figure 4 participants

3620 and 5305) This fact cannot be explained yet

Figure 5 presents the respective Youden plot for chrysene in the food supplement test material and the

standard solution in toluene with undisclosed content From this graph it can be concluded that the

overestimation of the chrysene content of the food supplement material was not caused by instrument

calibration The results for the solvent solution of many laboratories which underperformed for the

26

food supplement sample agreed well with the assigned value This supports the conclusion that the

overestimation is caused by the determination of an interferent most probably triphenylene

Figure 5 Youden plot of the percent deviations of values reported for chrysene from the

assigned values of the food supplement test material and the standard solution in

toluene with undisclosed content

96 Evaluation of compliance with legislation

The method performance data reported for the four marker PAHs were evaluated for compliance with

the provisions given in Commission Regulation (EC) No 8362011 Table 18 in ANNEX 10 gives an

overview on the results of the evaluation Data were not reported in case of empty cells Non-

compliant data are highlighted in Table 18 It should be noted that Regulation (EU) No 8362011

specifies LOD and LOQ with two decimals This was not respected by all participants which

hampered the compliance assessment

27

In summary it can be stated that the majority of participants reported method performance

characteristics for the determination of the four marker PAHs in the food supplement test sample that

are compliant with legislation However some laboratories need to improve their analysis methods

The laboratories in question are urged to adapt to the provisions given in the new legislation

10 Follow-up actions for underperforming laboratories

All laboratories that got questionable or non-satisfactory performance ratings are urged to perform

root cause analysis and to implement corrective actions

As agreed during the EU-RL PAH workshop 2011 the EU-RL PAH will set up follow-up measures in

due time for all NRLs that received for at least one of the four PAHs (BAA BAP BBF and CHR) z-

scores gt 3 as required by Regulation (EC) 8822004 and by the Protocol for management of

underperformance in comparative testing andor lack of collaboration of National Reference

Laboratories (NRLs) with European Union reference laboratories (EU-RLs) activities

These laboratories shall perform as an immediate action root-cause-analysis and shall report to the

EU-RL PAH in writing the identified cause for their underperformance and corrective actions they are

going to take Additionally they shall participate to an independent (non-EU-RL) proficiency test on

the determination of PAHs in food and shall communicate the outcome of this exercise to the EU-RL

PAH

28

11 Conclusions

Fourty seven participants reported results back to the EU-RL The performance of the majority of

participants was good In total 153 out of 225 attributed z-scores were below an absolute value of two

which equals to almost 68 However this percentage is much below the percentages of satisfactory

performance gained in previous studies This might be the consequence of the higher level of difficulty

with respect to matrix composition In addition bias can be concluded from the pattern of performance

indicators of some laboratories

In general the determination of chrysene caused most difficulties to the participants

zeta-Scores were calculated besides z-scores They indicate the agreement of the reported result with

the assigned value with respect to the stated measurement uncertainty The outcome of this rating was

worse than for the z-scores which indicates that the measurement uncertainty estimates were not

realistic Therefore participants underperforming with regard to zeta scores are urged to adapt their

measurement uncertainty statements

The great majority of participants in this inter-laboratory comparison applied analytical methods which

complied with EU legislation with regard to performance characteristics

12 Acknowledgements

The organisers would like to thank Mrs Beatriz de la Calle and Mr Franz Ulberth (all from IRMM

Geel Belgium) for their accurate revision of this report Mr Hakan Emteborg (from IRMM) for

ampouling of the standard solutions and all NRLs and OCLs for their cooperation

29

13 References

1 EU COMMISSION REGULATION (EC) No 7762006 of 23 May 2006 amending Annex VII to Regulation

(EC) No 8822004 of the European Parliament and of the Council as regards Community reference

laboratories Official Journal of the European Union 2006 L 136 p 3-8

Available from

httpeur-lexeuropaeuLexUriServLexUriServdouri=OJL200613600030008ENPDF

2 EU Regulation (EC) No 8822004 of the European Parliament and of the Council of 29 April 2004 on

official controls performed to ensure the verification of compliance with feed and food law animal health

and animal welfare rules Official Journal of the European Communities 2004 L191 p 1-52 Available

from

httpeur-lexeuropaeuLexUriServLexUriServdouri=OJL200419100010052ENPDF

3 EU Opinion of the Scientific Committee on Food on the risks to human health of Polycyclic Aromatic

Hydrocarbons in food (2002) Available from

httpeuropaeuintcommfoodfsscscfout153_enpdf

4 International Agency for Research on Cancer IARC Monographs on the Evaluation of

Carcinogenic Risks to Humans Some Non-heterocyclic Polycyclic Aromatic Hydrocarbons and Some

Related Exposures Volume 92 (2010) Available from

httpmonographsiarcfrENGMonographsvol92mono92pdf

5 EU Commission Regulation (EC) No 3332007 of 28 March 2007 laying down the methods of sampling

and analysis for the official control of the levels of lead cadmium mercury inorganic tin 3-MCPD and

benzo(a)pyrene in foodstuffs Official Journal of the European Union 2007 L 88 p 29-38 Available

from

httpeur-lexeuropaeuLexUriServLexUriServdouri=OJL200708800290038ENPDF

6 EU Commission Regulation (EC) No 18812006 of 19 December 2006 setting maximum levels for certain

contaminants in foodstuffs Official Journal of the European Union 2006 L 364 p 5-24 Available from

httpeur-lexeuropaeuLexUriServLexUriServdouri=OJL200636400050024ENPDF

7 EFSA Scientific Opinion of the Panel on Contaminants in the Food Chain on a request from the European

Commission on Polycyclic Aromatic Hydrocarbons in Food The EFSA Journal 2008 724 p 1-114

Available from

httpwwwefsaeuropaeuEFSAefsa_locale-1178620753812_1211902034842htm

30

8 EU Commission Regulation (EU) No 8352011 amending Regulation (EC) No 18812006 as regards

maximum levels for polycyclic aromatic hydrocarbons in foodstuffs Official Journal of the European

Union 2011 L 215 p 4-8

httpeur-lexeuropaeuLexUriServLexUriServdouri=OJL201121500040008ENPDF

9 EU Commission Regulation (EU) No 8362011 amending Regulation (EC) No 3332007 laying down the methods of sampling and analysis for the official control of the levels of lead cadmium mercury inorganic tin 3-MCPD and benzo(a)pyrene in foodstuffs Official Journal of the European Union 2011 L 215 p 9-16 httpeur-lexeuropaeuLexUriServLexUriServdouri=OJL201121500090016ENPDF

10 ISOIEC 170432010 (2010) Conformity assessment -- General requirements for proficiency testing

providers Geneva Switzerland

11 Thompson M Ellison SLR and Wood R The International Harmonized Protocol for the Proficiency

Testing of Analytical Chemistry Laboratories Pure Appl Chem (2006) 78(1) p 145ndash196

Available from httpmediaiupacorgpublicationspac2006pdf7801x0145pdf

31

14 ANNEXES

ANNEX 1 ndash Announcement of the PT on the IRMM webpage

ANNEX 2 ndash Announcement via e-mail and invitation

ANNEX 3 ndash Announcement of sample dispatch

ANNEX 4 ndash Technical specifications of the calibration solutions

ANNEX 5 ndash Homogeneity and stability of the test material

ANNEX 6 ndash Acknowledgement of sample receipt

ANNEX 7 ndash Questionnaire on details of analysis method

ANNEX 8 ndash Results reported for the food supplement sample

ANNEX 9 ndash Results reported for the standard solutions with undisclosed analyte content

ANNEX 10 ndash Method performance characteristics reported by participants

ANNEX 11 ndash Details of analysis methods reported by participants

32

ANNEX 1 ndash Announcement of the PT on the IRMM webpage

33

ANNEX 2 - Announcement via e-mail and invitation

34

ANNEX 3 - Announcement sample dispatch

Dear MadameSir

The test samples for the proficiency test on the determination of four PAHs in a food supplement (dry extract of St Johns wort) were

dispatched today

Explanation on the design of the study as well as instructions for sample storage and for reporting of results are in the parcel You will

also find your participant key in the parcel which is required for reporting of results

Please check the parcel after arrival for completeness and report the receipt of the test samples as well as their status to us by applying

the sample receipt form which is in the parcel

You are kindly requested to inform us by email if you do not receive the parcel by end of this week

My colleague Donata Lerda will then take care of it

With best regards

Thomas Wenzl

35

ANNEX 4 - Specifications of standard solutions with disclosed concentration

36

ANNEX 4 - continued

37

ANNEX 5 - Homogeneity and stability of the test material

Homogeneity of the test material

Benz[a]anthracene - BAA

Benzo[a]pyrene - BAP

38

ANNEX 5 - continued

Benzo[b]fluoranthene - BBF

Chrysene - CHR

39

ANNEX 5 - continued

Stability of the test material

Table 10 Content of the four target analytes in test materials stored under different

temperatures

BaA BaP BbF CHR

microgkg microgkg microgkg microgkg

Content 31 16 31 69 Storage at room temperature

U (k=2) 05 02 05 17

Content 31 15 29 68 Storage at reference temperature

U (k=2) 04 03 04 10

The applied capillary column does not separate CHR from triphenylene

40

ANNEX 6 ndash Documents sent to participants

41

ANNEX 6 - continued

42

ANNEX 7 Questionnaire on details of analysis method

43

ANNEX 7 - continued

44

ANNEX 7 - continued

45

ANNEX 8 - Results reported by participants for the food supplements test sample Table 11 Analysis results reported by the participants for the content of benz[a]anthracene (BAA) in the food supplements test material Values are presented as reported

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

3600 31 29 29 3 05 2

3602 199 193 178 19 021

3603 253 253 246 251 022 2

3604 199 209 23 213 071 2

3605 301 3 271 291 076 2

3606 302 288 278 289 064 2

3607 456 416 443 438 096 2

3608 796 695 651 714 149 2

3610 182 192 214 196 039 2

3611 414 504 488 469 96 2

3612 3 34 28 31 06 2

3613 32 33 34 33 07 2

3614 182 182 191 185 037 2

3615 3884 3822 3565 376 0752 2

3616 2 2 2 2 01 2

3617 366 37 35 362 046 2

3618 32 32 32 32 05 2

3619 299 305 297 299 048 2

3620 304 294 302 302 045 2

3621 29 26 29 28 04 2

3622 343 325 318 322 052 2

3623 35 3 33 33 07 2

3624

3626 318 31 314 314 126 2

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

5301 31 3 32 31 06 2

5302 314 319 315 316 063 2

5303 3102 2716 2822 2876 27 62

5304 261 263 26 261 57 2

5305 23 22 26 24 20

5306 22 23 23 22 11 2

5307 488 461 317 461

5308 168633 168544 1562025 1644598

5309 16 19 16 17

5310 3 304 311 305 015

5311 477 452 448 459 15 2

5312 506 63 494 543 134 2

5313 353 301 404 353

5314 431 373 378 394 5 096

5315 596 531 51 545 11 2

5316 224 231 235 235 022 2

5317 216 23 221 222 007 2

5318 064 07 067 01 2

5319 111 184 146 147 029 2

5320 515 525 564 534 48

5321 21 5322 13 87 9 87 35 2

5323 38 33 35 33 16 2

46

Figure 6 Results of replicate determinations (indicated by triangles) of benz[a]anthracene (BAA) in the food supplement test material

Horizontal blue lines represent the arithmetic mean value of replicate measurements and blue bars the reported expanded measurement uncertainty (k=2) The assigned value is

plotted as dotted line Red solid lines mark the range of satisfactory performance (|z|le2) Mean values are given for results outside the displayed data range

ProLab 2011

Laboratory

53

1853

1953

0853

0936

1436

0236

1036

1653

2136

0453

1753

0653

1653

0536

0353

0436

2153

0336

0636

0536

0036

2036

1953

1036

1253

0136

2653

0236

1836

2336

2236

1353

1353

2336

1736

1553

1453

0736

0753

1153

2053

1253

1536

0853

2236

11

an

aly

te c

on

ten

t (micro

gk

g)

10

95

9

85

8

75

7

65

6

55

5

45

4

35

3

25

2

15

1

05

0

102

3 468

7

Sample St Johns wort extract replicatesMeasurand Benz[a]anthraceneNo of laboratories 46

Assigned value 298 microgkg (Reference value)Target sd 062 microgkgTolerance limits 174 - 422 microgkg (|Z score| lt 200)

Assig

ned va

lue

Limit of tolerance

Limit of tolerance

47

Table 12 Analysis results reported by the participants for the content of benzo[a]pyrene (BAP) in the food supplements test material Values are presented as reported

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

3600 16 17 17 17 03 2

3602 118 123 13 124 015

3603 165 162 157 161 015 2

3604 134 154 139 143 04 2

3605 164 166 172 167 057 2

3606 181 159 161 167 031 2

3607 279 266 279 274 055 2

3608 57 687 461 573 226 2

3610 226 216 183 206 042 2

3611 289 325 311 308 36 2

3612 24 22 2 22 05 2

3613 17 18 19 18 05 2

3614 08 08 08 08 016 2

3615 1545 1393 1425 145 029 2

3616 16 16 14 15 03 2

3617 177 171 158 169 023 2

3618 2 2 2 2 02 2

3619 207 207 206 207 075 2

3620 165 169 163 166 017 2

3621 44 47 49 47 05 2

3622 212 208 222 213 038 2

3623 2 21 22 21 03 2

3624 18 178 182 18 031 2

3626 172 173 178 174 07 2

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

5301 18 18 18 18 04 2

5302 188 19 187 188 038 2

5303 1691 1426 149 1535 19 80

5304 145 142 15 146 69 2

5305 15 17 18 16 15

5306 16 17 17 16 08 2

5307 164 157 153 157

5308 102669 109324 109035 107009

5309 1 1 12 11

5310 122 122 124 123 006

5311 199 184 215 2 15 2

5312 258 248 239 248 05 2

5313 199 178 245 207

5314 174 171 172 172 11 0948

5315 253 248 213 238 05 2

5316 177 171 173 17 016 2

5317 255 277 276 269 012 2

5318 114 12 117 02 2

5319 19 228 231 216 043 2

5320 303 3 344 316 78

5321 12 12 12 12

5322 13 17 24 17 35 2

5323 2 2 19 2 1 2

48

Figure 7 Results of replicate determinations (indicated by triangles) of benzo[a]pyrene (BAP) in the food supplement test material

Horizontal blue lines represent the arithmetic mean value of replicate measurements and blue bars the reported expanded measurement uncertainty (k=2) The assigned value is

plotted as dotted line Red solid lines mark the range of satisfactory performance (|z|le2) Mean values are given for results outside the displayed data range

ProLab 2011

Laboratory

36

1453

0953

0853

1853

2153

1036

0236

0436

1553

0436

1653

0353

0736

0336

2036

0053

0553

0636

0636

0536

1753

1453

1636

2636

1336

2453

0153

2253

0253

2353

1136

1836

1953

1336

1036

2336

2253

1936

1253

1553

1253

1736

0753

2036

2136

0836

11

an

aly

te c

on

ten

t (micro

gk

g)

55

5

45

4

35

3

25

2

15

1

05

0

573

308

3

Sample St Johns wort extract replicatesMeasurand Benzo[a]pyreneNo of laboratories 48

Assigned value 165 microgkg (Reference value)Target sd 036 microgkgTolerance limits 093 - 237 microgkg (|Z score| lt 200)

Assig

ned va

lue

Limit of tolerance

Limit of tolerance

49

Table 13 Analysis results reported by the participants for the content of benzo[b]fluoranthene (BBF) in the food supplements test material Values are presented as reported

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

3600 32 3 3 31 09 2

3602 173 175 16 17 017

3603 265 252 25 256 025 2

3604 116 108 105 11 028 2

3605 286 225 232 247 074 2

3606 294 273 261 276 046 2

3607 579 579 544 567 113 2

3608 103 1201 876 1036 326 2

3610 173 196 204 191 038 2

3611 546 618 565 576 75 2

3612 3 28 28 29 06 2

3613 28 31 32 31 06 2

3614 135 111 135 127 022 2

3615 4187 3678 4111 4 08 2

3616 2 2 2 2 01 2

3617 33 27 299 035 2

3618 31 31 31 31 05 2

3619 326 324 312 326 064 2

3620 277 28 272 276 069 2

3621 26 25 23 25 04 2

3622 346 336 333 34 052 2

3623 26 28 29 28 02 2

3624

3626 288 28 307 292 117 2

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

5301 29 3 29 29 06 2

5302 297 3 296 298 06 2

5303 2705 2444 2608 2586 22 78

5304 245 254 26 253 85 2

5305 23 23 26 24 15

5306 42 43 4 42 21 2

5307 221 216 241 221

5308 232169 246654 240722 229848

5309 19 22 19 2 0

5310 3 315 325 313 016

5311 262 277 304 281 15 2

5312 27 289 337 299 075 2

5313 321 284 378 328

5314 423 404 385 404 34 083

5315 42 506 416 444 09 2

5316 265 267 277 273 018 2

5317 185 197 195 192 006 2

5318 276 281 279 04 2

5319 396 389 403 396 079 2

5320 623 618 635 625 14

5321 31

5322 24 33 49 33 35 2

5323 38 45 35 45 27 2

50

Figure 8 Results of replicate determinations (indicated by triangles) of benzo[b]fluoranthene (BBF) in the food supplement test material

Horizontal blue lines represent the arithmetic mean value of replicate measurements and blue bars the reported expanded measurement uncertainty (k=2) The assigned value is

plotted as dotted line Red solid lines mark the range of satisfactory performance (|z|le2) Mean values are given for results outside the displayed data range

ProLab 2011

Laboratory

36

0436

1436

0236

1053

1736

1653

0953

0753

0853

0536

2136

0553

0436

0353

0353

1636

0636

2036

2353

1853

1136

1236

2653

0153

0253

1236

1736

1336

0036

1853

2153

1036

1953

1336

2253

2253

2353

1936

1553

1453

0653

1536

0753

2036

0836

11

an

aly

te c

on

ten

t (micro

gk

g)

75

7

65

6

55

5

45

4

35

3

25

2

15

1

05

0

103

6

576

3

Sample St Johns wort extract replicatesMeasurand Benzo[b]fluorantheneNo of laboratories 46

Assigned value 292 microgkg (Reference value)Target sd 060 microgkgTolerance limits 172 - 412 microgkg (|Z score| lt 200)

Assig

ned va

lue

Limit of tolerance

Limit of tolerance

51

Table 14 Analysis results reported by the participants for the content of chrysene (CHR) in the food supplements test material Values are presented as reported

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

3600 5 49 5 5 07 2

3602 32 304 29 304 036

3603 462 469 447 459 048 2

3604 438 428 42 429 119 2

3605 365 41 381 386 085 2

3606 494 411 45 451 124 2

3607 897 774 787 819 18 2

3608 986 887 1168 1014 284 2

3610 418 392 462 424 085 2

3611 833 893 87 865 61 2

3612 6 64 58 61 12 2

3613 41 39 4 4 08 2

3614 272 254 288 271 054 2

3615 605 6028 5964 601 12 2

3616 47 45 49 47 05 2

3617 445 431 389 422 065 2

3618 45 45 45 45 06 2

3619 738 747 72 738 118 2

3620 463 472 446 46 092 2

3621 53 49 51 51 06 2

3622 478 513 505 488 08 2

3623 73 73 72 72 05 2

3624

3626 63 622 634 628 251 2

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

5301 49 49 49 49 1 2

5302 687 683 666 679 136 2

5303 6777 6181 6382 6441 24 70

5304 592 581 6 591 85 2

5305 6 58 64 61 20

5306 49 5 51 49 25 2

5307 524 5 574 524

5308 211488 216782 221146 213243

5309

5310 372 393 394 386 019

5311 43 459 422 437 15 2

5312 934 943 981 953 191 2

5313 793 73 99 838

5314 636 561 565 588 18 0818

5315 547 583 499 542 11 2

5316 292 286 285 282 06 2

5317 52 598 517 545 046 2

5318 368 345 357 05 2

5319 449 471 474 465 116 2

5320 76 924 818 834 99

5321 33

5322 7 7 76 7 35 2

5323 87 73 77 73 37 2

52

Figure 9 Results of replicate determinations (indicated by triangles) of chrysene (CHR) in the food supplement test material

Horizontal blue lines represent the arithmetic mean value of replicate measurements and blue bars the reported expanded measurement uncertainty (k=2) The assigned value is

plotted as dotted line Red solid lines mark the range of satisfactory performance (|z|le2) Mean values are given for results outside the displayed data range

ProLab 2011

Laboratory

53

0836

1453

1636

0253

2153

1836

0553

1036

1336

1736

1036

0453

1136

1836

0636

0336

2053

1936

1653

0136

0036

2253

0636

2153

0753

1553

1753

1453

0436

1536

1253

0536

2653

0353

0253

2236

2336

1953

2336

0753

2053

1353

1236

0836

11

an

aly

te c

on

ten

t (micro

gk

g)

13

12

11

10

9

8

7

6

5

4

3

2

1

0

865

3

Sample St Johns wort extract replicatesMeasurand ChryseneNo of laboratories 45

Assigned value 407 microgkg (Reference value)Target sd 083 microgkgTolerance limits 241 - 573 microgkg (|Z score| lt 200)

Assig

ned va

lue

Limit of tolerance

Limit of tolerance

53

Table 15 Analysis results reported by the participants for the sum of the four marker PAHs in the food supplements test material Values are presented as reported

Laboratory code

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg

3600 128 24 2

3602 79 126

3603 1127 096 2

3604 893 258 2

3605 1091 305 2

3606 1184 238 2

3607 2098 42 2

3608 3336 325 2

3610 1138 17 2

3611 2218 259 2

3612 143 29 2

3613 122 2 2

3614 663 129 2

3615 152 304 2

3616 102 03 2

3617 1252 169 2

3618 128 18 2

3619 157 305 2

3620 1204 12 2

3621 151 19 2

3622 1361 115 2

3623 154 09 2

3624

3626 1409 56 2

Laboratory code

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg

5301 126

5302 37 074 2

5303 13438 92

5304 1251 85 2

5305 125 20

5306 129 65 2

5307 1363

5308 7245598

5309

5310 1127 056

5311 1367 15 2

5312 2042 408 2

5313 1726

5314 1558

5315 177 35 2

5316 96 073 2

5317 1229 066 2

5318 82 05 2

5319 1225 267 2

5320 2309 41

5321

5322 207 35 2

5323 171 103 2

54

ANNEX 9 Results reported for the standard solutions with undisclosed analyte content Table 16 Analysis results reported by the participants for the four marker PAHs in the standard solution in toluene with undisclosed analyte

content Values are presented as reported U measurement uncertainty k coverage factor

benz[a]anthracene benzo[a]pyrene benzo[b]fluoranthene chrysene Laboratory

code Unit Result U k Result U k Result U k Result U k 3600 microgl 38 67 2 25 45 2 211 68 2 275 38 2

3605 microgl 367 184 2 2482 124 2 2592 13 2 235 118 2

3606 microgkg 3763 176 2 2609 142 2 2721 118 2 2539 134 2

3607 microgkg 3706 815 2 2824 565 2 2591 518 2 2587 569 2

3612 microgl 38 08 2 237 11 2 244 18 2 257 02 2

3614 microgl 324 65 2 209 38 2 215 37 2 21 42 2

3616 microgkg 318 07 2 224 14 2 231 26 2 216 1 2

3618 microgkg 417 17 2 284 08 2 292 06 2 297 03 2

3619 microgkg 47 742 2 274 468 2 228 387 2 274 434 2

3620 microgl 495 74 2 294 29 2 321 8 2 321 64 2

3621 microgl 241 22 2 143 13 2 188 16 2 145 14 2

3623 microgkg 476 25 2 311 53 2 269 3 2 297 03 2

3626 microgl 359 38 2 235 23 2 238 27 2 24 18 2

5301 microgkg 426 29 299 289

5302 microgkg 436 87 2 296 59 2 296 59 2 289 58 2

5303 microgl 3721 27 102 22838 19 99 23756 22 99 25486 24 98

5304 microgkg 38 29 2 225 26 2 257 37 2 258 52 2

5305 microgkg 305 225 245 29

5306 microgkg 316 63 2 247 49 2 65 13 2 22 44 2

5307 microgkg 3658 2559 241 2414

5308 microgkg 16854 109324 246654 216782

5311 microgl 374 20 2 279 15 2 268 15 2 272 15 2

5313 microgl 3408 2165 2275 2373

5314 microgkg 395 5 2875 11 3614 34 2905 18

5320 microgl 3267 368 1986 571 28 683 2427 512

5322 microgl 282 35 2 225 35 2 241 35 2 21 35 2

55

Figure 10 Youden plots of the percent deviation of the reported values from the assigned values for the food supplement test sample and the

standard solution in toluene with undisclosed content

(The identity of the respective measurand is given above the figure Results exceeding a difference of more than 100 were omitted)

56

Figure 10 - continued

57

Table 17 Analysis results reported by the participants for the four marker PAHs in the standard solution in acetonitrile with undisclosed

analyte content

Values are presented as reported U measurement uncertainty k coverage factor

benz[a]anthracene benzo[a]pyrene benzo[b]fluoranthene chrysene

Laboratory

code Unit Result U k Result U k Result U k Result U k

3602 microgkg 3675 247 247 244

3603 microgkg 358 086 2 236 028 2 235 028 2 236 057 2

3604 microgl 475 29 2 19 12 2 194 13 2 258 12 2

3608 microgl 3623 078 2 2385 023 2 2343 18 2 2296 092 2

3610 microgkg 369 185 2 25 125 2 251 126 2 244 122 2

3611 microgkg 48 02 2 323 02 2 328 04 2 317 03 2

3613 microgl 332 31 2 261 3 2 249 25 2 239 24 2

3615 microgl 415 2635 2729 2689

3617 microgkg 37 09 2 244 06 2 284 07 2 239 06 2

3622 microgl 37 12 2 25 23 2 257 21 2 238 1 2

3624 microgkg 402 104 2 255 084 2 265 096 2 253 07 2

5306 microgkg 563 113 2 371 74 2 359 72 2 333 67 2

5308 microgkg 168633 102669 232169 211488

5309 microgl 402 268 27

5310 microgl 315 158 25 125 25 125 288 144

5312 microgl 428 043 2 282 028 2 272 027 2 275 028 2

5315 microgkg 387 08 2 256 05 2 251 05 2 253 05 2

5316 microgl 3839 342 2 2485 191 2 2559 168 2 239 51 2

5317 microgl 2466 043 2 2212 034 2 2472 016 2 2146 068 2

5319 microgl 414 41 2 279 28 2 268 27 2 26 31 2

5321 microgkg 476 316 326 328

5322 microgl 2547 35 2

5323 microgkg 405 203 2 239 119 2 255 153 2 201 11 2

58

Figure 11 Youden plots of the percent deviation of the reported values from the assigned values for the food supplement test sample and the

standard solution in acetonitrile with undisclosed content

(The identity of the respective measurand is given above the figure Results exceeding a difference of more than 100 were omitted)

59

Figure 11 - continued

60

ANNEX 10 Table 18 Method performance parameters reported by the participants

Laboratory code

Analyte LOD LOQ Recovery Analyte LOD LOQ Recovery

microgkg microgkg microgkg microgkg 3600 BaA 03 06 978 BaP 03 07 9483602 BaA 007 021 926 BaP 005 015 993603 BaA 0011 05 987 BaP 0005 051 9553604 BaA 02 04 86 BaP 02 04 883605 BaA 0025 005 118 BaP 0025 005 1083606 BaA 001 003 65 BaP 001 003 703607 BaA 021 071 74 BaP 027 089 943608 BaA 016 048 8213 BaP 015 046 83683610 BaA 02 05 85 BaP 005 02 853611 BaA BaP 3612 BaA 1 117 BaP 1 1203613 BaA 01 03 90 BaP 01 03 903614 BaA 02 05 96 BaP 02 05 933615 BaA 005 015 110 BaP 005 015 1053616 BaA 01 05 108 BaP 01 05 753617 BaA 018 037 405 BaP 009 018 3873618 BaA 01 03 63 BaP 01 03 593619 BaA 005 005 59 BaP 033 033 543620 BaA 0009 0018 64 BaP 0005 001 553621 BaA 007 02 84 BaP 007 02 923622 BaA 01 03 1101 BaP 01 03 9853623 BaA 02 08 105 BaP 02 08 1013624 BaA BaP 01 033 8513625 BaA BaP 3626 BaA BaP 5301 BaA 003 01 102 BaP 003 01 1005302 BaA 01 03 78 BaP 01 03 865303 BaA 003 01 62 BaP 003 01 805304 BaA 1 1 70 BaP 1 1 955305 BaA 02 05 85 BaP 02 05 855306 BaA 03 1 699 BaP 03 1 905307 BaA 6537 BaP 81165308 BaA 0088 0265 99 BaP 00262 00785 1005309 BaA 01 08 92 BaP 01 08 925310 BaA 01 03 BaP 01 03 905311 BaA 002 005 80 BaP 002 004 915312 BaA 03 09 105 BaP 03 09 1055313 BaA 04 08 BaP 04 08 5314 BaA 001 002 96 BaP 001 002 9485315 BaA 01 03 50 - 100 BaP 01 03 50 - 100 5316 BaA 002 011 827 BaP 004 019 10445317 BaA 05 1 76 BaP 05 1 755318 BaA BaP 5319 BaA 009 029 951 BaP 011 041 995320 BaA 12 18 946 BaP 06 1 8345321 BaA 03 05 BaP 05 1 5322 BaA 1 2 100 BaP 1 2 1005323 BaA 26 BaP 26

61

ANNEX 10 Table 18 - continued

Laboratory code Analyte LOD LOQ Recovery Analyte LOD LOQ Recovery

microgkg microgkg microgkg microgkg 3600 BbF 03 05 96 CHR 02 03 10153602 BbF 015 045 947 CHR 003 009 9193603 BbF 0014 05 1012 CHR 0004 05 10033604 BbF 02 04 85 CHR 02 04 873605 BbF 005 01 94 CHR 0025 005 863606 BbF 001 003 70 CHR 001 003 653607 BbF 018 06 100 CHR 014 028 943608 BbF 015 046 7979 CHR 015 045 71253610 BbF 005 02 85 CHR 02 05 853611 BbF CHR 3612 BbF 1 118 CHR 1 1173613 BbF 01 03 90 CHR 01 03 903614 BbF 01 04 96 CHR 02 05 953615 BbF 008 025 109 CHR 003 01 1083616 BbF 01 05 78 CHR 01 05 723617 BbF 035 07 38 CHR 012 024 4163618 BbF 01 03 62 CHR 01 03 623619 BbF 016 016 63 CHR 01 01 593620 BbF 0003 0006 55 CHR 001 002 653621 BbF 007 02 103 CHR 007 02 893622 BbF 02 06 963 CHR 03 09 1043623 BbF 02 08 106 CHR 02 08 1193624 BbF CHR 3625 BbF CHR 3626 BbF CHR 5301 BbF 003 01 96 CHR 003 01 1145302 BbF 01 03 84 CHR 01 03 835303 BbF 003 01 78 CHR 003 01 705304 BbF 1 1 86 CHR 1 1 705305 BbF 02 05 90 CHR 02 05 955306 BbF 03 1 1435 CHR 03 1 1185307 BbF 8116 CHR 65375308 BbF 0079 0238 99 CHR 009 026 1005309 BbF 01 08 92 CHR 5310 BbF 07 2 CHR 01 03 5311 BbF 002 004 95 CHR 002 004 895312 BbF 03 09 105 CHR 03 09 1055313 BbF 04 08 CHR 04 08 5314 BbF 001 002 83 CHR 001 002 8185315 BbF 02 05 50 -100 CHR 02 05 50 - 100 5316 BbF 005 024 814 CHR 001 007 9595317 BbF 05 1 73 CHR 05 1 785318 BbF CHR 5319 BbF 011 038 946 CHR 013 043 9675320 BbF 07 09 1194 CHR 23 32 10455321 BbF 05 1 CHR 05 1 5322 BbF 1 2 100 CHR 1 2 1005323 BbF 26 CHR 26

62

ANNEX 11 Table 19 Details of analysis method reported by the participants Data are presented as reported

Laboratory code

Analysis technique

Instrument calibration

Internal standards applied

Sample clean up

Details of sample

clean up

Extraction technique

Details of sample

extraction

3600 6) GC-MS

2) Internal standardisation with labelled substances

Deuterated PAH mix 9 (Ehrenstorfer)

1) Saponification 2) LiquidLiquid partitioning

D) Other Liquidliquid partitioning

3602 2) HPLC-UV-FLD

1) External calibration

3) Gel permeation chromatography

A) Pressurised liquid extraction (PLE)

3603 1) HPLC-FLD

1) External calibration

3) Gel permeation chromatography

D) Other 1 minute with dichloromethane in vortex

3604 2) HPLC-UV-FLD

2) Internal standardisation with labelled substances

DiP D14

5) Donor acceptor complex chromatography

D) Other LL

3605 1) HPLC-FLD

1) External calibration

3) Gel permeation chromatography

C) Soxhlet extraction

3606 7) GC-MSMS

2) Internal standardisation with labelled substances

BaP 13C4 CHR 13C6 BaA 13C6 BbF 13C6

4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

3607 6) GC-MS

2) Internal standardisation with labelled substances

BaA-d12 CHR-d12 BbF-d12 BkF-d12 BaP-d12 ICP-d12 BgP-d12 DiP-d14

1) Saponification

D) Other solvent partitioning

3608 1) HPLC-FLD

1) External calibration

6) Other Liquid extraction

D) Other

Extraction in a rotary agitator and centrifugation

3610 2) HPLC-UV-FLD

1) External calibration

4) Solid phase extraction

B) Sonication

3611

3612 6) GC-MS

2) Internal standardisation with labelled substances

chrysene-d12 dibenzo(ah)anthracene-d14 dibenzo(ai)pyrene-d14

4) Solid phase extraction

B) Sonication

3613 5) LC-MSMS

3) Internal standardisation with unlabelled substances

I use Chrysene-D12 3) Gel permeation chromatography

D) Other extraction with hexan

63

Table 19 - continued

Laboratory code

Analysis technique

Instrument calibration

Internal standards applied

Sample clean up Details of sample

clean up

Extraction technique

Details of sample

extraction

3614 6) GC-MS 1) External calibration

3) Gel permeation chromatography

B) Sonication

3615 1) HPLC-FLD

1) External calibration

6) Other PLE GPC

A) Pressurised liquid extraction (PLE)

3616 6) GC-MS

2) Internal standardisation with labelled substances

Benzo(a)pyrene-13C4 Benzo(a)anthracene-13C6 Benzo(b)fluoranthene-13C6 Chrysene-13C6

2) LiquidLiquid partitioning 4) Solid phase extraction

B) Sonication

3617 1) HPLC-FLD

1) External calibration

4) Solid phase extraction

C) Soxhlet extraction

3618 6) GC-MS

2) Internal standardisation with labelled substances

deuterated standards

1) Saponification 4) Solid phase extraction

D) Other Handshaken with cyclohexane

3619 6) GC-MS

2) Internal standardisation with labelled substances

C13 Labelled US EPA 16

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

D) Other Homegenisation amp saponiofication

3620 9) GC-HRMS

2) Internal standardisation with labelled substances

mix of deuterated PAHs (BAA-D12 CHR-D12 BAP-D12 BBF-D12)

3) Gel permeation chromatography 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

3621 6) GC-MS

2) Internal standardisation with labelled substances

Chrysene D12 Benzo(a)pyrene D12 Benzo(b)fluoranthene D12 Benzo(a)anthracene

2) LiquidLiquid partitioning 4) Solid phase extraction

B) Sonication

3622 1) HPLC-FLD

4) Standard addition method

4) Solid phase extraction

D) Other

Extraction with cyclohexane by heating under reflux

3623 7) GC-MSMS

2) Internal standardisation with labelled substances

C13 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

3624 2) HPLC-UV-FLD

1) External calibration

2) LiquidLiquid partitioning

D) Other hot solvent extraction

3626

64

Table 19 - continued

Laboratory code

Analysis technique

Instrument calibration

Internal standards

applied

Sample clean up

Details of sample

clean up

Extraction technique

Details of sample

extraction

5301 7) GC-MSMS

2) Internal standardisation with labelled substances

EACH SUBSTANCE C13

4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5302 7) GC-MSMS

2) Internal standardisation with labelled substances

Isotope labelled PAHs 13C for each analyte

4) Solid phase extraction

C) Soxhlet extraction

5303 7) GC-MSMS

3) Internal standardisation with unlabelled substances

HAP C13 4) Solid phase extraction

D) Other automate ASE200 DIONEX

5304 7) GC-MSMS

2) Internal standardisation with labelled substances

with C13 for each HAP

2) LiquidLiquid partitioning 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5305 7) GC-MSMS

2) Internal standardisation with labelled substances

C13 isotopiques internal standards

4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5306 7) GC-MSMS

2) Internal standardisation with labelled substances

C13 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5307 6) GC-MS

2) Internal standardisation with labelled substances

1) Saponification 2) LiquidLiquid partitioning

D) Other solvent extraction

5308 1) HPLC-FLD 1) External calibration

4) Solid phase extraction

B) Sonication

5309 1) HPLC-FLD 1) External calibration

4) Solid phase extraction

D) Other only SPE

5310 1) HPLC-FLD 1) External calibration

1) Saponification 2) LiquidLiquid partitioning 3) Gel permeation chromatography

C) Soxhlet extraction

5311 6) GC-MS

2) Internal standardisation with labelled substances

13 C Chrysene 13 C Benz(a)pyrene

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

B) Sonication

5312 1) HPLC-FLD

3) Internal standardisation with unlabelled substances

Benzo(b)chrysen

2) LiquidLiquid partitioning 4) Solid phase extraction

Quechers D) Other Quechers

65

Table 19 - continued

Laboratory code

Analysis technique

Instrument calibration

Internal standards applied

Sample clean up Details of sample

clean up

Extraction technique

Details of sample extraction

5313 6) GC-MS

2) Internal standardisation with labelled substances

Benz(a)anthracene D12 Benzo(b)fluranthene D12 Benzo(a)pyrene D12 Chrysene D12

1) Saponification 2) LiquidLiquid partitioning 3) Gel permeation chromatography

A) Pressurised liquid extraction (PLE)

5314 6) GC-MS

2) Internal standardisation with labelled substances

Benzo(a)pyren d12 2) LiquidLiquid partitioning

D) Other Extraction with

5315 1) HPLC-FLD

3) Internal standardisation with unlabelled substances

Benz(b)chrysen

1) Saponification 2) LiquidLiquid partitioning

D) Other shaker

5316 1) HPLC-FLD

1) External calibration

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

D) Other

LiquidLiquid extraction with cyclohexane and dimethylformamide

5317 7) GC-MSMS

1) External calibration

3) Gel permeation chromatography 6) Other

silica gel D) Other VDLUFA online method

5318

5319 1) HPLC-FLD

3) Internal standardisation with unlabelled substances

Benzo(b)chrysene 3) Gel permeation chromatography

A) Pressurised liquid extraction (PLE)

5320 6) GC-MS

3) Internal standardisation with unlabelled substances

22-binaphthyl

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

D) Other 3 hours refluxing

5321 1) HPLC-FLD

2) Internal standardisation with labelled substances

6-meacutethylchrysegravene

1) Saponification 2) LiquidLiquid partitioning

B) Sonication

5322 6) GC-MS

2) Internal standardisation with labelled substances

naftalene-d8 acenaphtalene-d10 phenanthrene-d10 chrysene-d12 perylene-d12 benzo(e)anthracene-d12

1) Saponification 2) LiquidLiquid partitioning 6) Other

clean-up with silica packed column

C) Soxhlet extraction

5323 1) HPLC-FLD

1) External calibration

4) Solid phase extraction 6) Other

Accelerated Solvent Extraction

A) Pressurised liquid extraction (PLE)

66

European Commission EUR 25245 EN ndash Joint Research Centre ndash Institute for Reference Materials and Measurements Title Report on the 8th inter-laboratory comparison organised by the European Union Reference Laboratory for Polycyclic Aromatic Hydrocarbons - Four marker PAHs in a dry extract of St Johns wort Author(s) Radoslav Lizak Szilard Szilagyi Philippe Verlinde and Thomas WENZL Luxembourg Publications Office of the European Union 2012 ndash 66 pp ndash 201 x 297 cm EUR ndash Scientific and Technical Research series ndash ISSN 1831-9424 ISBN 978-92-79-23337-1 doi 10278759387 Abstract The proficiency test here reported concerned the determination of the contents of four marker polycyclic aromatic hydrocarbons (PAHs) and of their sum in an food supplements test sample The set of marker PAHs consists of benz[a]anthacene benzo[a]pyrene benzo[b]fluoranthene and chrysene Participants to these PT were National Reference Laboratories for PAHs (NRLs-PAHs) and EU official food control laboratories The number of participants was in total 48

The PT was organised according ISO Standard 170432010

The test material used was a commercial dry extract of St Johns wort Participants also received a solution of the same PAHs either in acetonitrile or in toluene for checking their instrument calibration

The results from participants were rated with z-scores and zeta-scores About 68 of the reported results were attributed with z-scores with an absolute value of below two which is the threshold for satisfactory performance

How to obtain EU publications

Our priced publications are available from EU Bookshop (httpbookshopeuropaeu) where you can place

an order with the sales agent of your choice

The Publications Office has a worldwide network of sales agents You can obtain their contact details by

sending a fax to (352) 29 29-42758

The mission of the JRC is to provide customer-driven scientific and technical support

for the conception development implementation and monitoring of EU policies As a

service of the European Commission the JRC functions as a reference centre of

science and technology for the Union Close to the policy-making process it serves

the common interest of the Member States while being independent of special

interests whether private or national

L

A-N

A-252

45-EN

-N

• 1 Summary
• 2 Introduction
• 3 Scope
• 4 Participating Laboratories
• 5 Time frame
• 6 Confidentiality
• 7 Test materials
• • 71 1Preparation of test materials
  • 72 Assigned values and standard deviation for proficiency assessment
  • 73 Homogeneity and stability
  • 74 Sample dispatch
  • • 8 Design of the proficiency test
    • 9 Evaluation of the results
    • • 91 2General
      • 92 21BEvaluation criteria
      • 93 Performance of participants
      • 94 Evaluation of the influence of analysis method
      • 95 Evaluation of the influence of calibration
      • 96 26Evaluation of compliance with legislation
      • • 10 Follow-up actions for underperforming laboratories
        • 11 1Conclusions
        • 12 1Acknowledgements
        • 13 References

25

Figure 4 Youden plot of the percent deviations of values reported for benzo[a]pyrene from the

assigned values of the food supplement test material and the standard solution in

toluene with undisclosed content

Figure 4 reveals that a number of participants reported results with similar percent deviations from the

assigned values of both the results for the food supplement material and the standard solution in

toluene with undisclosed content This correlation indicates that over- respectively underestimation of

the analyte content of the food supplement material might be caused by biased instrument calibration

This finding underpins the importance of thorough calibration standard preparation However the data

was not in all cases conclusive as some laboratories performed well for the food supplement samples

but reported biased results for the standard solution with undisclosed content (eg Figure 4 participants

3620 and 5305) This fact cannot be explained yet

Figure 5 presents the respective Youden plot for chrysene in the food supplement test material and the

standard solution in toluene with undisclosed content From this graph it can be concluded that the

overestimation of the chrysene content of the food supplement material was not caused by instrument

calibration The results for the solvent solution of many laboratories which underperformed for the

26

food supplement sample agreed well with the assigned value This supports the conclusion that the

overestimation is caused by the determination of an interferent most probably triphenylene

Figure 5 Youden plot of the percent deviations of values reported for chrysene from the

assigned values of the food supplement test material and the standard solution in

toluene with undisclosed content

96 Evaluation of compliance with legislation

The method performance data reported for the four marker PAHs were evaluated for compliance with

the provisions given in Commission Regulation (EC) No 8362011 Table 18 in ANNEX 10 gives an

overview on the results of the evaluation Data were not reported in case of empty cells Non-

compliant data are highlighted in Table 18 It should be noted that Regulation (EU) No 8362011

specifies LOD and LOQ with two decimals This was not respected by all participants which

hampered the compliance assessment

27

In summary it can be stated that the majority of participants reported method performance

characteristics for the determination of the four marker PAHs in the food supplement test sample that

are compliant with legislation However some laboratories need to improve their analysis methods

The laboratories in question are urged to adapt to the provisions given in the new legislation

10 Follow-up actions for underperforming laboratories

All laboratories that got questionable or non-satisfactory performance ratings are urged to perform

root cause analysis and to implement corrective actions

As agreed during the EU-RL PAH workshop 2011 the EU-RL PAH will set up follow-up measures in

due time for all NRLs that received for at least one of the four PAHs (BAA BAP BBF and CHR) z-

scores gt 3 as required by Regulation (EC) 8822004 and by the Protocol for management of

underperformance in comparative testing andor lack of collaboration of National Reference

Laboratories (NRLs) with European Union reference laboratories (EU-RLs) activities

These laboratories shall perform as an immediate action root-cause-analysis and shall report to the

EU-RL PAH in writing the identified cause for their underperformance and corrective actions they are

going to take Additionally they shall participate to an independent (non-EU-RL) proficiency test on

the determination of PAHs in food and shall communicate the outcome of this exercise to the EU-RL

PAH

28

11 Conclusions

Fourty seven participants reported results back to the EU-RL The performance of the majority of

participants was good In total 153 out of 225 attributed z-scores were below an absolute value of two

which equals to almost 68 However this percentage is much below the percentages of satisfactory

performance gained in previous studies This might be the consequence of the higher level of difficulty

with respect to matrix composition In addition bias can be concluded from the pattern of performance

indicators of some laboratories

In general the determination of chrysene caused most difficulties to the participants

zeta-Scores were calculated besides z-scores They indicate the agreement of the reported result with

the assigned value with respect to the stated measurement uncertainty The outcome of this rating was

worse than for the z-scores which indicates that the measurement uncertainty estimates were not

realistic Therefore participants underperforming with regard to zeta scores are urged to adapt their

measurement uncertainty statements

The great majority of participants in this inter-laboratory comparison applied analytical methods which

complied with EU legislation with regard to performance characteristics

12 Acknowledgements

The organisers would like to thank Mrs Beatriz de la Calle and Mr Franz Ulberth (all from IRMM

Geel Belgium) for their accurate revision of this report Mr Hakan Emteborg (from IRMM) for

ampouling of the standard solutions and all NRLs and OCLs for their cooperation

29

13 References

1 EU COMMISSION REGULATION (EC) No 7762006 of 23 May 2006 amending Annex VII to Regulation

(EC) No 8822004 of the European Parliament and of the Council as regards Community reference

laboratories Official Journal of the European Union 2006 L 136 p 3-8

Available from

httpeur-lexeuropaeuLexUriServLexUriServdouri=OJL200613600030008ENPDF

2 EU Regulation (EC) No 8822004 of the European Parliament and of the Council of 29 April 2004 on

official controls performed to ensure the verification of compliance with feed and food law animal health

and animal welfare rules Official Journal of the European Communities 2004 L191 p 1-52 Available

from

httpeur-lexeuropaeuLexUriServLexUriServdouri=OJL200419100010052ENPDF

3 EU Opinion of the Scientific Committee on Food on the risks to human health of Polycyclic Aromatic

Hydrocarbons in food (2002) Available from

httpeuropaeuintcommfoodfsscscfout153_enpdf

4 International Agency for Research on Cancer IARC Monographs on the Evaluation of

Carcinogenic Risks to Humans Some Non-heterocyclic Polycyclic Aromatic Hydrocarbons and Some

Related Exposures Volume 92 (2010) Available from

httpmonographsiarcfrENGMonographsvol92mono92pdf

5 EU Commission Regulation (EC) No 3332007 of 28 March 2007 laying down the methods of sampling

and analysis for the official control of the levels of lead cadmium mercury inorganic tin 3-MCPD and

benzo(a)pyrene in foodstuffs Official Journal of the European Union 2007 L 88 p 29-38 Available

from

httpeur-lexeuropaeuLexUriServLexUriServdouri=OJL200708800290038ENPDF

6 EU Commission Regulation (EC) No 18812006 of 19 December 2006 setting maximum levels for certain

contaminants in foodstuffs Official Journal of the European Union 2006 L 364 p 5-24 Available from

httpeur-lexeuropaeuLexUriServLexUriServdouri=OJL200636400050024ENPDF

7 EFSA Scientific Opinion of the Panel on Contaminants in the Food Chain on a request from the European

Commission on Polycyclic Aromatic Hydrocarbons in Food The EFSA Journal 2008 724 p 1-114

Available from

httpwwwefsaeuropaeuEFSAefsa_locale-1178620753812_1211902034842htm

30

8 EU Commission Regulation (EU) No 8352011 amending Regulation (EC) No 18812006 as regards

maximum levels for polycyclic aromatic hydrocarbons in foodstuffs Official Journal of the European

Union 2011 L 215 p 4-8

httpeur-lexeuropaeuLexUriServLexUriServdouri=OJL201121500040008ENPDF

9 EU Commission Regulation (EU) No 8362011 amending Regulation (EC) No 3332007 laying down the methods of sampling and analysis for the official control of the levels of lead cadmium mercury inorganic tin 3-MCPD and benzo(a)pyrene in foodstuffs Official Journal of the European Union 2011 L 215 p 9-16 httpeur-lexeuropaeuLexUriServLexUriServdouri=OJL201121500090016ENPDF

10 ISOIEC 170432010 (2010) Conformity assessment -- General requirements for proficiency testing

providers Geneva Switzerland

11 Thompson M Ellison SLR and Wood R The International Harmonized Protocol for the Proficiency

Testing of Analytical Chemistry Laboratories Pure Appl Chem (2006) 78(1) p 145ndash196

Available from httpmediaiupacorgpublicationspac2006pdf7801x0145pdf

31

14 ANNEXES

ANNEX 1 ndash Announcement of the PT on the IRMM webpage

ANNEX 2 ndash Announcement via e-mail and invitation

ANNEX 3 ndash Announcement of sample dispatch

ANNEX 4 ndash Technical specifications of the calibration solutions

ANNEX 5 ndash Homogeneity and stability of the test material

ANNEX 6 ndash Acknowledgement of sample receipt

ANNEX 7 ndash Questionnaire on details of analysis method

ANNEX 8 ndash Results reported for the food supplement sample

ANNEX 9 ndash Results reported for the standard solutions with undisclosed analyte content

ANNEX 10 ndash Method performance characteristics reported by participants

ANNEX 11 ndash Details of analysis methods reported by participants

32

ANNEX 1 ndash Announcement of the PT on the IRMM webpage

33

ANNEX 2 - Announcement via e-mail and invitation

34

ANNEX 3 - Announcement sample dispatch

Dear MadameSir

The test samples for the proficiency test on the determination of four PAHs in a food supplement (dry extract of St Johns wort) were

dispatched today

Explanation on the design of the study as well as instructions for sample storage and for reporting of results are in the parcel You will

also find your participant key in the parcel which is required for reporting of results

Please check the parcel after arrival for completeness and report the receipt of the test samples as well as their status to us by applying

the sample receipt form which is in the parcel

You are kindly requested to inform us by email if you do not receive the parcel by end of this week

My colleague Donata Lerda will then take care of it

With best regards

Thomas Wenzl

35

ANNEX 4 - Specifications of standard solutions with disclosed concentration

36

ANNEX 4 - continued

37

ANNEX 5 - Homogeneity and stability of the test material

Homogeneity of the test material

Benz[a]anthracene - BAA

Benzo[a]pyrene - BAP

38

ANNEX 5 - continued

Benzo[b]fluoranthene - BBF

Chrysene - CHR

39

ANNEX 5 - continued

Stability of the test material

Table 10 Content of the four target analytes in test materials stored under different

temperatures

BaA BaP BbF CHR

microgkg microgkg microgkg microgkg

Content 31 16 31 69 Storage at room temperature

U (k=2) 05 02 05 17

Content 31 15 29 68 Storage at reference temperature

U (k=2) 04 03 04 10

The applied capillary column does not separate CHR from triphenylene

40

ANNEX 6 ndash Documents sent to participants

41

ANNEX 6 - continued

42

ANNEX 7 Questionnaire on details of analysis method

43

ANNEX 7 - continued

44

ANNEX 7 - continued

45

ANNEX 8 - Results reported by participants for the food supplements test sample Table 11 Analysis results reported by the participants for the content of benz[a]anthracene (BAA) in the food supplements test material Values are presented as reported

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

3600 31 29 29 3 05 2

3602 199 193 178 19 021

3603 253 253 246 251 022 2

3604 199 209 23 213 071 2

3605 301 3 271 291 076 2

3606 302 288 278 289 064 2

3607 456 416 443 438 096 2

3608 796 695 651 714 149 2

3610 182 192 214 196 039 2

3611 414 504 488 469 96 2

3612 3 34 28 31 06 2

3613 32 33 34 33 07 2

3614 182 182 191 185 037 2

3615 3884 3822 3565 376 0752 2

3616 2 2 2 2 01 2

3617 366 37 35 362 046 2

3618 32 32 32 32 05 2

3619 299 305 297 299 048 2

3620 304 294 302 302 045 2

3621 29 26 29 28 04 2

3622 343 325 318 322 052 2

3623 35 3 33 33 07 2

3624

3626 318 31 314 314 126 2

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

5301 31 3 32 31 06 2

5302 314 319 315 316 063 2

5303 3102 2716 2822 2876 27 62

5304 261 263 26 261 57 2

5305 23 22 26 24 20

5306 22 23 23 22 11 2

5307 488 461 317 461

5308 168633 168544 1562025 1644598

5309 16 19 16 17

5310 3 304 311 305 015

5311 477 452 448 459 15 2

5312 506 63 494 543 134 2

5313 353 301 404 353

5314 431 373 378 394 5 096

5315 596 531 51 545 11 2

5316 224 231 235 235 022 2

5317 216 23 221 222 007 2

5318 064 07 067 01 2

5319 111 184 146 147 029 2

5320 515 525 564 534 48

5321 21 5322 13 87 9 87 35 2

5323 38 33 35 33 16 2

46

Figure 6 Results of replicate determinations (indicated by triangles) of benz[a]anthracene (BAA) in the food supplement test material

Horizontal blue lines represent the arithmetic mean value of replicate measurements and blue bars the reported expanded measurement uncertainty (k=2) The assigned value is

plotted as dotted line Red solid lines mark the range of satisfactory performance (|z|le2) Mean values are given for results outside the displayed data range

ProLab 2011

Laboratory

53

1853

1953

0853

0936

1436

0236

1036

1653

2136

0453

1753

0653

1653

0536

0353

0436

2153

0336

0636

0536

0036

2036

1953

1036

1253

0136

2653

0236

1836

2336

2236

1353

1353

2336

1736

1553

1453

0736

0753

1153

2053

1253

1536

0853

2236

11

an

aly

te c

on

ten

t (micro

gk

g)

10

95

9

85

8

75

7

65

6

55

5

45

4

35

3

25

2

15

1

05

0

102

3 468

7

Sample St Johns wort extract replicatesMeasurand Benz[a]anthraceneNo of laboratories 46

Assigned value 298 microgkg (Reference value)Target sd 062 microgkgTolerance limits 174 - 422 microgkg (|Z score| lt 200)

Assig

ned va

lue

Limit of tolerance

Limit of tolerance

47

Table 12 Analysis results reported by the participants for the content of benzo[a]pyrene (BAP) in the food supplements test material Values are presented as reported

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

3600 16 17 17 17 03 2

3602 118 123 13 124 015

3603 165 162 157 161 015 2

3604 134 154 139 143 04 2

3605 164 166 172 167 057 2

3606 181 159 161 167 031 2

3607 279 266 279 274 055 2

3608 57 687 461 573 226 2

3610 226 216 183 206 042 2

3611 289 325 311 308 36 2

3612 24 22 2 22 05 2

3613 17 18 19 18 05 2

3614 08 08 08 08 016 2

3615 1545 1393 1425 145 029 2

3616 16 16 14 15 03 2

3617 177 171 158 169 023 2

3618 2 2 2 2 02 2

3619 207 207 206 207 075 2

3620 165 169 163 166 017 2

3621 44 47 49 47 05 2

3622 212 208 222 213 038 2

3623 2 21 22 21 03 2

3624 18 178 182 18 031 2

3626 172 173 178 174 07 2

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

5301 18 18 18 18 04 2

5302 188 19 187 188 038 2

5303 1691 1426 149 1535 19 80

5304 145 142 15 146 69 2

5305 15 17 18 16 15

5306 16 17 17 16 08 2

5307 164 157 153 157

5308 102669 109324 109035 107009

5309 1 1 12 11

5310 122 122 124 123 006

5311 199 184 215 2 15 2

5312 258 248 239 248 05 2

5313 199 178 245 207

5314 174 171 172 172 11 0948

5315 253 248 213 238 05 2

5316 177 171 173 17 016 2

5317 255 277 276 269 012 2

5318 114 12 117 02 2

5319 19 228 231 216 043 2

5320 303 3 344 316 78

5321 12 12 12 12

5322 13 17 24 17 35 2

5323 2 2 19 2 1 2

48

Figure 7 Results of replicate determinations (indicated by triangles) of benzo[a]pyrene (BAP) in the food supplement test material

Horizontal blue lines represent the arithmetic mean value of replicate measurements and blue bars the reported expanded measurement uncertainty (k=2) The assigned value is

plotted as dotted line Red solid lines mark the range of satisfactory performance (|z|le2) Mean values are given for results outside the displayed data range

ProLab 2011

Laboratory

36

1453

0953

0853

1853

2153

1036

0236

0436

1553

0436

1653

0353

0736

0336

2036

0053

0553

0636

0636

0536

1753

1453

1636

2636

1336

2453

0153

2253

0253

2353

1136

1836

1953

1336

1036

2336

2253

1936

1253

1553

1253

1736

0753

2036

2136

0836

11

an

aly

te c

on

ten

t (micro

gk

g)

55

5

45

4

35

3

25

2

15

1

05

0

573

308

3

Sample St Johns wort extract replicatesMeasurand Benzo[a]pyreneNo of laboratories 48

Assigned value 165 microgkg (Reference value)Target sd 036 microgkgTolerance limits 093 - 237 microgkg (|Z score| lt 200)

Assig

ned va

lue

Limit of tolerance

Limit of tolerance

49

Table 13 Analysis results reported by the participants for the content of benzo[b]fluoranthene (BBF) in the food supplements test material Values are presented as reported

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

3600 32 3 3 31 09 2

3602 173 175 16 17 017

3603 265 252 25 256 025 2

3604 116 108 105 11 028 2

3605 286 225 232 247 074 2

3606 294 273 261 276 046 2

3607 579 579 544 567 113 2

3608 103 1201 876 1036 326 2

3610 173 196 204 191 038 2

3611 546 618 565 576 75 2

3612 3 28 28 29 06 2

3613 28 31 32 31 06 2

3614 135 111 135 127 022 2

3615 4187 3678 4111 4 08 2

3616 2 2 2 2 01 2

3617 33 27 299 035 2

3618 31 31 31 31 05 2

3619 326 324 312 326 064 2

3620 277 28 272 276 069 2

3621 26 25 23 25 04 2

3622 346 336 333 34 052 2

3623 26 28 29 28 02 2

3624

3626 288 28 307 292 117 2

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

5301 29 3 29 29 06 2

5302 297 3 296 298 06 2

5303 2705 2444 2608 2586 22 78

5304 245 254 26 253 85 2

5305 23 23 26 24 15

5306 42 43 4 42 21 2

5307 221 216 241 221

5308 232169 246654 240722 229848

5309 19 22 19 2 0

5310 3 315 325 313 016

5311 262 277 304 281 15 2

5312 27 289 337 299 075 2

5313 321 284 378 328

5314 423 404 385 404 34 083

5315 42 506 416 444 09 2

5316 265 267 277 273 018 2

5317 185 197 195 192 006 2

5318 276 281 279 04 2

5319 396 389 403 396 079 2

5320 623 618 635 625 14

5321 31

5322 24 33 49 33 35 2

5323 38 45 35 45 27 2

50

Figure 8 Results of replicate determinations (indicated by triangles) of benzo[b]fluoranthene (BBF) in the food supplement test material

Horizontal blue lines represent the arithmetic mean value of replicate measurements and blue bars the reported expanded measurement uncertainty (k=2) The assigned value is

plotted as dotted line Red solid lines mark the range of satisfactory performance (|z|le2) Mean values are given for results outside the displayed data range

ProLab 2011

Laboratory

36

0436

1436

0236

1053

1736

1653

0953

0753

0853

0536

2136

0553

0436

0353

0353

1636

0636

2036

2353

1853

1136

1236

2653

0153

0253

1236

1736

1336

0036

1853

2153

1036

1953

1336

2253

2253

2353

1936

1553

1453

0653

1536

0753

2036

0836

11

an

aly

te c

on

ten

t (micro

gk

g)

75

7

65

6

55

5

45

4

35

3

25

2

15

1

05

0

103

6

576

3

Sample St Johns wort extract replicatesMeasurand Benzo[b]fluorantheneNo of laboratories 46

Assigned value 292 microgkg (Reference value)Target sd 060 microgkgTolerance limits 172 - 412 microgkg (|Z score| lt 200)

Assig

ned va

lue

Limit of tolerance

Limit of tolerance

51

Table 14 Analysis results reported by the participants for the content of chrysene (CHR) in the food supplements test material Values are presented as reported

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

3600 5 49 5 5 07 2

3602 32 304 29 304 036

3603 462 469 447 459 048 2

3604 438 428 42 429 119 2

3605 365 41 381 386 085 2

3606 494 411 45 451 124 2

3607 897 774 787 819 18 2

3608 986 887 1168 1014 284 2

3610 418 392 462 424 085 2

3611 833 893 87 865 61 2

3612 6 64 58 61 12 2

3613 41 39 4 4 08 2

3614 272 254 288 271 054 2

3615 605 6028 5964 601 12 2

3616 47 45 49 47 05 2

3617 445 431 389 422 065 2

3618 45 45 45 45 06 2

3619 738 747 72 738 118 2

3620 463 472 446 46 092 2

3621 53 49 51 51 06 2

3622 478 513 505 488 08 2

3623 73 73 72 72 05 2

3624

3626 63 622 634 628 251 2

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

5301 49 49 49 49 1 2

5302 687 683 666 679 136 2

5303 6777 6181 6382 6441 24 70

5304 592 581 6 591 85 2

5305 6 58 64 61 20

5306 49 5 51 49 25 2

5307 524 5 574 524

5308 211488 216782 221146 213243

5309

5310 372 393 394 386 019

5311 43 459 422 437 15 2

5312 934 943 981 953 191 2

5313 793 73 99 838

5314 636 561 565 588 18 0818

5315 547 583 499 542 11 2

5316 292 286 285 282 06 2

5317 52 598 517 545 046 2

5318 368 345 357 05 2

5319 449 471 474 465 116 2

5320 76 924 818 834 99

5321 33

5322 7 7 76 7 35 2

5323 87 73 77 73 37 2

52

Figure 9 Results of replicate determinations (indicated by triangles) of chrysene (CHR) in the food supplement test material

Horizontal blue lines represent the arithmetic mean value of replicate measurements and blue bars the reported expanded measurement uncertainty (k=2) The assigned value is

plotted as dotted line Red solid lines mark the range of satisfactory performance (|z|le2) Mean values are given for results outside the displayed data range

ProLab 2011

Laboratory

53

0836

1453

1636

0253

2153

1836

0553

1036

1336

1736

1036

0453

1136

1836

0636

0336

2053

1936

1653

0136

0036

2253

0636

2153

0753

1553

1753

1453

0436

1536

1253

0536

2653

0353

0253

2236

2336

1953

2336

0753

2053

1353

1236

0836

11

an

aly

te c

on

ten

t (micro

gk

g)

13

12

11

10

9

8

7

6

5

4

3

2

1

0

865

3

Sample St Johns wort extract replicatesMeasurand ChryseneNo of laboratories 45

Assigned value 407 microgkg (Reference value)Target sd 083 microgkgTolerance limits 241 - 573 microgkg (|Z score| lt 200)

Assig

ned va

lue

Limit of tolerance

Limit of tolerance

53

Table 15 Analysis results reported by the participants for the sum of the four marker PAHs in the food supplements test material Values are presented as reported

Laboratory code

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg

3600 128 24 2

3602 79 126

3603 1127 096 2

3604 893 258 2

3605 1091 305 2

3606 1184 238 2

3607 2098 42 2

3608 3336 325 2

3610 1138 17 2

3611 2218 259 2

3612 143 29 2

3613 122 2 2

3614 663 129 2

3615 152 304 2

3616 102 03 2

3617 1252 169 2

3618 128 18 2

3619 157 305 2

3620 1204 12 2

3621 151 19 2

3622 1361 115 2

3623 154 09 2

3624

3626 1409 56 2

Laboratory code

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg

5301 126

5302 37 074 2

5303 13438 92

5304 1251 85 2

5305 125 20

5306 129 65 2

5307 1363

5308 7245598

5309

5310 1127 056

5311 1367 15 2

5312 2042 408 2

5313 1726

5314 1558

5315 177 35 2

5316 96 073 2

5317 1229 066 2

5318 82 05 2

5319 1225 267 2

5320 2309 41

5321

5322 207 35 2

5323 171 103 2

54

ANNEX 9 Results reported for the standard solutions with undisclosed analyte content Table 16 Analysis results reported by the participants for the four marker PAHs in the standard solution in toluene with undisclosed analyte

content Values are presented as reported U measurement uncertainty k coverage factor

benz[a]anthracene benzo[a]pyrene benzo[b]fluoranthene chrysene Laboratory

code Unit Result U k Result U k Result U k Result U k 3600 microgl 38 67 2 25 45 2 211 68 2 275 38 2

3605 microgl 367 184 2 2482 124 2 2592 13 2 235 118 2

3606 microgkg 3763 176 2 2609 142 2 2721 118 2 2539 134 2

3607 microgkg 3706 815 2 2824 565 2 2591 518 2 2587 569 2

3612 microgl 38 08 2 237 11 2 244 18 2 257 02 2

3614 microgl 324 65 2 209 38 2 215 37 2 21 42 2

3616 microgkg 318 07 2 224 14 2 231 26 2 216 1 2

3618 microgkg 417 17 2 284 08 2 292 06 2 297 03 2

3619 microgkg 47 742 2 274 468 2 228 387 2 274 434 2

3620 microgl 495 74 2 294 29 2 321 8 2 321 64 2

3621 microgl 241 22 2 143 13 2 188 16 2 145 14 2

3623 microgkg 476 25 2 311 53 2 269 3 2 297 03 2

3626 microgl 359 38 2 235 23 2 238 27 2 24 18 2

5301 microgkg 426 29 299 289

5302 microgkg 436 87 2 296 59 2 296 59 2 289 58 2

5303 microgl 3721 27 102 22838 19 99 23756 22 99 25486 24 98

5304 microgkg 38 29 2 225 26 2 257 37 2 258 52 2

5305 microgkg 305 225 245 29

5306 microgkg 316 63 2 247 49 2 65 13 2 22 44 2

5307 microgkg 3658 2559 241 2414

5308 microgkg 16854 109324 246654 216782

5311 microgl 374 20 2 279 15 2 268 15 2 272 15 2

5313 microgl 3408 2165 2275 2373

5314 microgkg 395 5 2875 11 3614 34 2905 18

5320 microgl 3267 368 1986 571 28 683 2427 512

5322 microgl 282 35 2 225 35 2 241 35 2 21 35 2

55

Figure 10 Youden plots of the percent deviation of the reported values from the assigned values for the food supplement test sample and the

standard solution in toluene with undisclosed content

(The identity of the respective measurand is given above the figure Results exceeding a difference of more than 100 were omitted)

56

Figure 10 - continued

57

Table 17 Analysis results reported by the participants for the four marker PAHs in the standard solution in acetonitrile with undisclosed

analyte content

Values are presented as reported U measurement uncertainty k coverage factor

benz[a]anthracene benzo[a]pyrene benzo[b]fluoranthene chrysene

Laboratory

code Unit Result U k Result U k Result U k Result U k

3602 microgkg 3675 247 247 244

3603 microgkg 358 086 2 236 028 2 235 028 2 236 057 2

3604 microgl 475 29 2 19 12 2 194 13 2 258 12 2

3608 microgl 3623 078 2 2385 023 2 2343 18 2 2296 092 2

3610 microgkg 369 185 2 25 125 2 251 126 2 244 122 2

3611 microgkg 48 02 2 323 02 2 328 04 2 317 03 2

3613 microgl 332 31 2 261 3 2 249 25 2 239 24 2

3615 microgl 415 2635 2729 2689

3617 microgkg 37 09 2 244 06 2 284 07 2 239 06 2

3622 microgl 37 12 2 25 23 2 257 21 2 238 1 2

3624 microgkg 402 104 2 255 084 2 265 096 2 253 07 2

5306 microgkg 563 113 2 371 74 2 359 72 2 333 67 2

5308 microgkg 168633 102669 232169 211488

5309 microgl 402 268 27

5310 microgl 315 158 25 125 25 125 288 144

5312 microgl 428 043 2 282 028 2 272 027 2 275 028 2

5315 microgkg 387 08 2 256 05 2 251 05 2 253 05 2

5316 microgl 3839 342 2 2485 191 2 2559 168 2 239 51 2

5317 microgl 2466 043 2 2212 034 2 2472 016 2 2146 068 2

5319 microgl 414 41 2 279 28 2 268 27 2 26 31 2

5321 microgkg 476 316 326 328

5322 microgl 2547 35 2

5323 microgkg 405 203 2 239 119 2 255 153 2 201 11 2

58

Figure 11 Youden plots of the percent deviation of the reported values from the assigned values for the food supplement test sample and the

standard solution in acetonitrile with undisclosed content

(The identity of the respective measurand is given above the figure Results exceeding a difference of more than 100 were omitted)

59

Figure 11 - continued

60

ANNEX 10 Table 18 Method performance parameters reported by the participants

Laboratory code

Analyte LOD LOQ Recovery Analyte LOD LOQ Recovery

microgkg microgkg microgkg microgkg 3600 BaA 03 06 978 BaP 03 07 9483602 BaA 007 021 926 BaP 005 015 993603 BaA 0011 05 987 BaP 0005 051 9553604 BaA 02 04 86 BaP 02 04 883605 BaA 0025 005 118 BaP 0025 005 1083606 BaA 001 003 65 BaP 001 003 703607 BaA 021 071 74 BaP 027 089 943608 BaA 016 048 8213 BaP 015 046 83683610 BaA 02 05 85 BaP 005 02 853611 BaA BaP 3612 BaA 1 117 BaP 1 1203613 BaA 01 03 90 BaP 01 03 903614 BaA 02 05 96 BaP 02 05 933615 BaA 005 015 110 BaP 005 015 1053616 BaA 01 05 108 BaP 01 05 753617 BaA 018 037 405 BaP 009 018 3873618 BaA 01 03 63 BaP 01 03 593619 BaA 005 005 59 BaP 033 033 543620 BaA 0009 0018 64 BaP 0005 001 553621 BaA 007 02 84 BaP 007 02 923622 BaA 01 03 1101 BaP 01 03 9853623 BaA 02 08 105 BaP 02 08 1013624 BaA BaP 01 033 8513625 BaA BaP 3626 BaA BaP 5301 BaA 003 01 102 BaP 003 01 1005302 BaA 01 03 78 BaP 01 03 865303 BaA 003 01 62 BaP 003 01 805304 BaA 1 1 70 BaP 1 1 955305 BaA 02 05 85 BaP 02 05 855306 BaA 03 1 699 BaP 03 1 905307 BaA 6537 BaP 81165308 BaA 0088 0265 99 BaP 00262 00785 1005309 BaA 01 08 92 BaP 01 08 925310 BaA 01 03 BaP 01 03 905311 BaA 002 005 80 BaP 002 004 915312 BaA 03 09 105 BaP 03 09 1055313 BaA 04 08 BaP 04 08 5314 BaA 001 002 96 BaP 001 002 9485315 BaA 01 03 50 - 100 BaP 01 03 50 - 100 5316 BaA 002 011 827 BaP 004 019 10445317 BaA 05 1 76 BaP 05 1 755318 BaA BaP 5319 BaA 009 029 951 BaP 011 041 995320 BaA 12 18 946 BaP 06 1 8345321 BaA 03 05 BaP 05 1 5322 BaA 1 2 100 BaP 1 2 1005323 BaA 26 BaP 26

61

ANNEX 10 Table 18 - continued

Laboratory code Analyte LOD LOQ Recovery Analyte LOD LOQ Recovery

microgkg microgkg microgkg microgkg 3600 BbF 03 05 96 CHR 02 03 10153602 BbF 015 045 947 CHR 003 009 9193603 BbF 0014 05 1012 CHR 0004 05 10033604 BbF 02 04 85 CHR 02 04 873605 BbF 005 01 94 CHR 0025 005 863606 BbF 001 003 70 CHR 001 003 653607 BbF 018 06 100 CHR 014 028 943608 BbF 015 046 7979 CHR 015 045 71253610 BbF 005 02 85 CHR 02 05 853611 BbF CHR 3612 BbF 1 118 CHR 1 1173613 BbF 01 03 90 CHR 01 03 903614 BbF 01 04 96 CHR 02 05 953615 BbF 008 025 109 CHR 003 01 1083616 BbF 01 05 78 CHR 01 05 723617 BbF 035 07 38 CHR 012 024 4163618 BbF 01 03 62 CHR 01 03 623619 BbF 016 016 63 CHR 01 01 593620 BbF 0003 0006 55 CHR 001 002 653621 BbF 007 02 103 CHR 007 02 893622 BbF 02 06 963 CHR 03 09 1043623 BbF 02 08 106 CHR 02 08 1193624 BbF CHR 3625 BbF CHR 3626 BbF CHR 5301 BbF 003 01 96 CHR 003 01 1145302 BbF 01 03 84 CHR 01 03 835303 BbF 003 01 78 CHR 003 01 705304 BbF 1 1 86 CHR 1 1 705305 BbF 02 05 90 CHR 02 05 955306 BbF 03 1 1435 CHR 03 1 1185307 BbF 8116 CHR 65375308 BbF 0079 0238 99 CHR 009 026 1005309 BbF 01 08 92 CHR 5310 BbF 07 2 CHR 01 03 5311 BbF 002 004 95 CHR 002 004 895312 BbF 03 09 105 CHR 03 09 1055313 BbF 04 08 CHR 04 08 5314 BbF 001 002 83 CHR 001 002 8185315 BbF 02 05 50 -100 CHR 02 05 50 - 100 5316 BbF 005 024 814 CHR 001 007 9595317 BbF 05 1 73 CHR 05 1 785318 BbF CHR 5319 BbF 011 038 946 CHR 013 043 9675320 BbF 07 09 1194 CHR 23 32 10455321 BbF 05 1 CHR 05 1 5322 BbF 1 2 100 CHR 1 2 1005323 BbF 26 CHR 26

62

ANNEX 11 Table 19 Details of analysis method reported by the participants Data are presented as reported

Laboratory code

Analysis technique

Instrument calibration

Internal standards applied

Sample clean up

Details of sample

clean up

Extraction technique

Details of sample

extraction

3600 6) GC-MS

2) Internal standardisation with labelled substances

Deuterated PAH mix 9 (Ehrenstorfer)

1) Saponification 2) LiquidLiquid partitioning

D) Other Liquidliquid partitioning

3602 2) HPLC-UV-FLD

1) External calibration

3) Gel permeation chromatography

A) Pressurised liquid extraction (PLE)

3603 1) HPLC-FLD

1) External calibration

3) Gel permeation chromatography

D) Other 1 minute with dichloromethane in vortex

3604 2) HPLC-UV-FLD

2) Internal standardisation with labelled substances

DiP D14

5) Donor acceptor complex chromatography

D) Other LL

3605 1) HPLC-FLD

1) External calibration

3) Gel permeation chromatography

C) Soxhlet extraction

3606 7) GC-MSMS

2) Internal standardisation with labelled substances

BaP 13C4 CHR 13C6 BaA 13C6 BbF 13C6

4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

3607 6) GC-MS

2) Internal standardisation with labelled substances

BaA-d12 CHR-d12 BbF-d12 BkF-d12 BaP-d12 ICP-d12 BgP-d12 DiP-d14

1) Saponification

D) Other solvent partitioning

3608 1) HPLC-FLD

1) External calibration

6) Other Liquid extraction

D) Other

Extraction in a rotary agitator and centrifugation

3610 2) HPLC-UV-FLD

1) External calibration

4) Solid phase extraction

B) Sonication

3611

3612 6) GC-MS

2) Internal standardisation with labelled substances

chrysene-d12 dibenzo(ah)anthracene-d14 dibenzo(ai)pyrene-d14

4) Solid phase extraction

B) Sonication

3613 5) LC-MSMS

3) Internal standardisation with unlabelled substances

I use Chrysene-D12 3) Gel permeation chromatography

D) Other extraction with hexan

63

Table 19 - continued

Laboratory code

Analysis technique

Instrument calibration

Internal standards applied

Sample clean up Details of sample

clean up

Extraction technique

Details of sample

extraction

3614 6) GC-MS 1) External calibration

3) Gel permeation chromatography

B) Sonication

3615 1) HPLC-FLD

1) External calibration

6) Other PLE GPC

A) Pressurised liquid extraction (PLE)

3616 6) GC-MS

2) Internal standardisation with labelled substances

Benzo(a)pyrene-13C4 Benzo(a)anthracene-13C6 Benzo(b)fluoranthene-13C6 Chrysene-13C6

2) LiquidLiquid partitioning 4) Solid phase extraction

B) Sonication

3617 1) HPLC-FLD

1) External calibration

4) Solid phase extraction

C) Soxhlet extraction

3618 6) GC-MS

2) Internal standardisation with labelled substances

deuterated standards

1) Saponification 4) Solid phase extraction

D) Other Handshaken with cyclohexane

3619 6) GC-MS

2) Internal standardisation with labelled substances

C13 Labelled US EPA 16

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

D) Other Homegenisation amp saponiofication

3620 9) GC-HRMS

2) Internal standardisation with labelled substances

mix of deuterated PAHs (BAA-D12 CHR-D12 BAP-D12 BBF-D12)

3) Gel permeation chromatography 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

3621 6) GC-MS

2) Internal standardisation with labelled substances

Chrysene D12 Benzo(a)pyrene D12 Benzo(b)fluoranthene D12 Benzo(a)anthracene

2) LiquidLiquid partitioning 4) Solid phase extraction

B) Sonication

3622 1) HPLC-FLD

4) Standard addition method

4) Solid phase extraction

D) Other

Extraction with cyclohexane by heating under reflux

3623 7) GC-MSMS

2) Internal standardisation with labelled substances

C13 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

3624 2) HPLC-UV-FLD

1) External calibration

2) LiquidLiquid partitioning

D) Other hot solvent extraction

3626

64

Table 19 - continued

Laboratory code

Analysis technique

Instrument calibration

Internal standards

applied

Sample clean up

Details of sample

clean up

Extraction technique

Details of sample

extraction

5301 7) GC-MSMS

2) Internal standardisation with labelled substances

EACH SUBSTANCE C13

4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5302 7) GC-MSMS

2) Internal standardisation with labelled substances

Isotope labelled PAHs 13C for each analyte

4) Solid phase extraction

C) Soxhlet extraction

5303 7) GC-MSMS

3) Internal standardisation with unlabelled substances

HAP C13 4) Solid phase extraction

D) Other automate ASE200 DIONEX

5304 7) GC-MSMS

2) Internal standardisation with labelled substances

with C13 for each HAP

2) LiquidLiquid partitioning 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5305 7) GC-MSMS

2) Internal standardisation with labelled substances

C13 isotopiques internal standards

4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5306 7) GC-MSMS

2) Internal standardisation with labelled substances

C13 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5307 6) GC-MS

2) Internal standardisation with labelled substances

1) Saponification 2) LiquidLiquid partitioning

D) Other solvent extraction

5308 1) HPLC-FLD 1) External calibration

4) Solid phase extraction

B) Sonication

5309 1) HPLC-FLD 1) External calibration

4) Solid phase extraction

D) Other only SPE

5310 1) HPLC-FLD 1) External calibration

1) Saponification 2) LiquidLiquid partitioning 3) Gel permeation chromatography

C) Soxhlet extraction

5311 6) GC-MS

2) Internal standardisation with labelled substances

13 C Chrysene 13 C Benz(a)pyrene

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

B) Sonication

5312 1) HPLC-FLD

3) Internal standardisation with unlabelled substances

Benzo(b)chrysen

2) LiquidLiquid partitioning 4) Solid phase extraction

Quechers D) Other Quechers

65

Table 19 - continued

Laboratory code

Analysis technique

Instrument calibration

Internal standards applied

Sample clean up Details of sample

clean up

Extraction technique

Details of sample extraction

5313 6) GC-MS

2) Internal standardisation with labelled substances

Benz(a)anthracene D12 Benzo(b)fluranthene D12 Benzo(a)pyrene D12 Chrysene D12

1) Saponification 2) LiquidLiquid partitioning 3) Gel permeation chromatography

A) Pressurised liquid extraction (PLE)

5314 6) GC-MS

2) Internal standardisation with labelled substances

Benzo(a)pyren d12 2) LiquidLiquid partitioning

D) Other Extraction with

5315 1) HPLC-FLD

3) Internal standardisation with unlabelled substances

Benz(b)chrysen

1) Saponification 2) LiquidLiquid partitioning

D) Other shaker

5316 1) HPLC-FLD

1) External calibration

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

D) Other

LiquidLiquid extraction with cyclohexane and dimethylformamide

5317 7) GC-MSMS

1) External calibration

3) Gel permeation chromatography 6) Other

silica gel D) Other VDLUFA online method

5318

5319 1) HPLC-FLD

3) Internal standardisation with unlabelled substances

Benzo(b)chrysene 3) Gel permeation chromatography

A) Pressurised liquid extraction (PLE)

5320 6) GC-MS

3) Internal standardisation with unlabelled substances

22-binaphthyl

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

D) Other 3 hours refluxing

5321 1) HPLC-FLD

2) Internal standardisation with labelled substances

6-meacutethylchrysegravene

1) Saponification 2) LiquidLiquid partitioning

B) Sonication

5322 6) GC-MS

2) Internal standardisation with labelled substances

naftalene-d8 acenaphtalene-d10 phenanthrene-d10 chrysene-d12 perylene-d12 benzo(e)anthracene-d12

1) Saponification 2) LiquidLiquid partitioning 6) Other

clean-up with silica packed column

C) Soxhlet extraction

5323 1) HPLC-FLD

1) External calibration

4) Solid phase extraction 6) Other

Accelerated Solvent Extraction

A) Pressurised liquid extraction (PLE)

66

European Commission EUR 25245 EN ndash Joint Research Centre ndash Institute for Reference Materials and Measurements Title Report on the 8th inter-laboratory comparison organised by the European Union Reference Laboratory for Polycyclic Aromatic Hydrocarbons - Four marker PAHs in a dry extract of St Johns wort Author(s) Radoslav Lizak Szilard Szilagyi Philippe Verlinde and Thomas WENZL Luxembourg Publications Office of the European Union 2012 ndash 66 pp ndash 201 x 297 cm EUR ndash Scientific and Technical Research series ndash ISSN 1831-9424 ISBN 978-92-79-23337-1 doi 10278759387 Abstract The proficiency test here reported concerned the determination of the contents of four marker polycyclic aromatic hydrocarbons (PAHs) and of their sum in an food supplements test sample The set of marker PAHs consists of benz[a]anthacene benzo[a]pyrene benzo[b]fluoranthene and chrysene Participants to these PT were National Reference Laboratories for PAHs (NRLs-PAHs) and EU official food control laboratories The number of participants was in total 48

The PT was organised according ISO Standard 170432010

The test material used was a commercial dry extract of St Johns wort Participants also received a solution of the same PAHs either in acetonitrile or in toluene for checking their instrument calibration

The results from participants were rated with z-scores and zeta-scores About 68 of the reported results were attributed with z-scores with an absolute value of below two which is the threshold for satisfactory performance

How to obtain EU publications

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The mission of the JRC is to provide customer-driven scientific and technical support

for the conception development implementation and monitoring of EU policies As a

service of the European Commission the JRC functions as a reference centre of

science and technology for the Union Close to the policy-making process it serves

the common interest of the Member States while being independent of special

interests whether private or national

L

A-N

A-252

45-EN

-N

• 1 Summary
• 2 Introduction
• 3 Scope
• 4 Participating Laboratories
• 5 Time frame
• 6 Confidentiality
• 7 Test materials
• • 71 1Preparation of test materials
  • 72 Assigned values and standard deviation for proficiency assessment
  • 73 Homogeneity and stability
  • 74 Sample dispatch
  • • 8 Design of the proficiency test
    • 9 Evaluation of the results
    • • 91 2General
      • 92 21BEvaluation criteria
      • 93 Performance of participants
      • 94 Evaluation of the influence of analysis method
      • 95 Evaluation of the influence of calibration
      • 96 26Evaluation of compliance with legislation
      • • 10 Follow-up actions for underperforming laboratories
        • 11 1Conclusions
        • 12 1Acknowledgements
        • 13 References

26

food supplement sample agreed well with the assigned value This supports the conclusion that the

overestimation is caused by the determination of an interferent most probably triphenylene

Figure 5 Youden plot of the percent deviations of values reported for chrysene from the

assigned values of the food supplement test material and the standard solution in

toluene with undisclosed content

96 Evaluation of compliance with legislation

The method performance data reported for the four marker PAHs were evaluated for compliance with

the provisions given in Commission Regulation (EC) No 8362011 Table 18 in ANNEX 10 gives an

overview on the results of the evaluation Data were not reported in case of empty cells Non-

compliant data are highlighted in Table 18 It should be noted that Regulation (EU) No 8362011

specifies LOD and LOQ with two decimals This was not respected by all participants which

hampered the compliance assessment

27

In summary it can be stated that the majority of participants reported method performance

characteristics for the determination of the four marker PAHs in the food supplement test sample that

are compliant with legislation However some laboratories need to improve their analysis methods

The laboratories in question are urged to adapt to the provisions given in the new legislation

10 Follow-up actions for underperforming laboratories

All laboratories that got questionable or non-satisfactory performance ratings are urged to perform

root cause analysis and to implement corrective actions

As agreed during the EU-RL PAH workshop 2011 the EU-RL PAH will set up follow-up measures in

due time for all NRLs that received for at least one of the four PAHs (BAA BAP BBF and CHR) z-

scores gt 3 as required by Regulation (EC) 8822004 and by the Protocol for management of

underperformance in comparative testing andor lack of collaboration of National Reference

Laboratories (NRLs) with European Union reference laboratories (EU-RLs) activities

These laboratories shall perform as an immediate action root-cause-analysis and shall report to the

EU-RL PAH in writing the identified cause for their underperformance and corrective actions they are

going to take Additionally they shall participate to an independent (non-EU-RL) proficiency test on

the determination of PAHs in food and shall communicate the outcome of this exercise to the EU-RL

PAH

28

11 Conclusions

Fourty seven participants reported results back to the EU-RL The performance of the majority of

participants was good In total 153 out of 225 attributed z-scores were below an absolute value of two

which equals to almost 68 However this percentage is much below the percentages of satisfactory

performance gained in previous studies This might be the consequence of the higher level of difficulty

with respect to matrix composition In addition bias can be concluded from the pattern of performance

indicators of some laboratories

In general the determination of chrysene caused most difficulties to the participants

zeta-Scores were calculated besides z-scores They indicate the agreement of the reported result with

the assigned value with respect to the stated measurement uncertainty The outcome of this rating was

worse than for the z-scores which indicates that the measurement uncertainty estimates were not

realistic Therefore participants underperforming with regard to zeta scores are urged to adapt their

measurement uncertainty statements

The great majority of participants in this inter-laboratory comparison applied analytical methods which

complied with EU legislation with regard to performance characteristics

12 Acknowledgements

The organisers would like to thank Mrs Beatriz de la Calle and Mr Franz Ulberth (all from IRMM

Geel Belgium) for their accurate revision of this report Mr Hakan Emteborg (from IRMM) for

ampouling of the standard solutions and all NRLs and OCLs for their cooperation

29

13 References

1 EU COMMISSION REGULATION (EC) No 7762006 of 23 May 2006 amending Annex VII to Regulation

(EC) No 8822004 of the European Parliament and of the Council as regards Community reference

laboratories Official Journal of the European Union 2006 L 136 p 3-8

Available from

httpeur-lexeuropaeuLexUriServLexUriServdouri=OJL200613600030008ENPDF

2 EU Regulation (EC) No 8822004 of the European Parliament and of the Council of 29 April 2004 on

official controls performed to ensure the verification of compliance with feed and food law animal health

and animal welfare rules Official Journal of the European Communities 2004 L191 p 1-52 Available

from

httpeur-lexeuropaeuLexUriServLexUriServdouri=OJL200419100010052ENPDF

3 EU Opinion of the Scientific Committee on Food on the risks to human health of Polycyclic Aromatic

Hydrocarbons in food (2002) Available from

httpeuropaeuintcommfoodfsscscfout153_enpdf

4 International Agency for Research on Cancer IARC Monographs on the Evaluation of

Carcinogenic Risks to Humans Some Non-heterocyclic Polycyclic Aromatic Hydrocarbons and Some

Related Exposures Volume 92 (2010) Available from

httpmonographsiarcfrENGMonographsvol92mono92pdf

5 EU Commission Regulation (EC) No 3332007 of 28 March 2007 laying down the methods of sampling

and analysis for the official control of the levels of lead cadmium mercury inorganic tin 3-MCPD and

benzo(a)pyrene in foodstuffs Official Journal of the European Union 2007 L 88 p 29-38 Available

from

httpeur-lexeuropaeuLexUriServLexUriServdouri=OJL200708800290038ENPDF

6 EU Commission Regulation (EC) No 18812006 of 19 December 2006 setting maximum levels for certain

contaminants in foodstuffs Official Journal of the European Union 2006 L 364 p 5-24 Available from

httpeur-lexeuropaeuLexUriServLexUriServdouri=OJL200636400050024ENPDF

7 EFSA Scientific Opinion of the Panel on Contaminants in the Food Chain on a request from the European

Commission on Polycyclic Aromatic Hydrocarbons in Food The EFSA Journal 2008 724 p 1-114

Available from

httpwwwefsaeuropaeuEFSAefsa_locale-1178620753812_1211902034842htm

30

8 EU Commission Regulation (EU) No 8352011 amending Regulation (EC) No 18812006 as regards

maximum levels for polycyclic aromatic hydrocarbons in foodstuffs Official Journal of the European

Union 2011 L 215 p 4-8

httpeur-lexeuropaeuLexUriServLexUriServdouri=OJL201121500040008ENPDF

9 EU Commission Regulation (EU) No 8362011 amending Regulation (EC) No 3332007 laying down the methods of sampling and analysis for the official control of the levels of lead cadmium mercury inorganic tin 3-MCPD and benzo(a)pyrene in foodstuffs Official Journal of the European Union 2011 L 215 p 9-16 httpeur-lexeuropaeuLexUriServLexUriServdouri=OJL201121500090016ENPDF

10 ISOIEC 170432010 (2010) Conformity assessment -- General requirements for proficiency testing

providers Geneva Switzerland

11 Thompson M Ellison SLR and Wood R The International Harmonized Protocol for the Proficiency

Testing of Analytical Chemistry Laboratories Pure Appl Chem (2006) 78(1) p 145ndash196

Available from httpmediaiupacorgpublicationspac2006pdf7801x0145pdf

31

14 ANNEXES

ANNEX 1 ndash Announcement of the PT on the IRMM webpage

ANNEX 2 ndash Announcement via e-mail and invitation

ANNEX 3 ndash Announcement of sample dispatch

ANNEX 4 ndash Technical specifications of the calibration solutions

ANNEX 5 ndash Homogeneity and stability of the test material

ANNEX 6 ndash Acknowledgement of sample receipt

ANNEX 7 ndash Questionnaire on details of analysis method

ANNEX 8 ndash Results reported for the food supplement sample

ANNEX 9 ndash Results reported for the standard solutions with undisclosed analyte content

ANNEX 10 ndash Method performance characteristics reported by participants

ANNEX 11 ndash Details of analysis methods reported by participants

32

ANNEX 1 ndash Announcement of the PT on the IRMM webpage

33

ANNEX 2 - Announcement via e-mail and invitation

34

ANNEX 3 - Announcement sample dispatch

Dear MadameSir

The test samples for the proficiency test on the determination of four PAHs in a food supplement (dry extract of St Johns wort) were

dispatched today

Explanation on the design of the study as well as instructions for sample storage and for reporting of results are in the parcel You will

also find your participant key in the parcel which is required for reporting of results

Please check the parcel after arrival for completeness and report the receipt of the test samples as well as their status to us by applying

the sample receipt form which is in the parcel

You are kindly requested to inform us by email if you do not receive the parcel by end of this week

My colleague Donata Lerda will then take care of it

With best regards

Thomas Wenzl

35

ANNEX 4 - Specifications of standard solutions with disclosed concentration

36

ANNEX 4 - continued

37

ANNEX 5 - Homogeneity and stability of the test material

Homogeneity of the test material

Benz[a]anthracene - BAA

Benzo[a]pyrene - BAP

38

ANNEX 5 - continued

Benzo[b]fluoranthene - BBF

Chrysene - CHR

39

ANNEX 5 - continued

Stability of the test material

Table 10 Content of the four target analytes in test materials stored under different

temperatures

BaA BaP BbF CHR

microgkg microgkg microgkg microgkg

Content 31 16 31 69 Storage at room temperature

U (k=2) 05 02 05 17

Content 31 15 29 68 Storage at reference temperature

U (k=2) 04 03 04 10

The applied capillary column does not separate CHR from triphenylene

40

ANNEX 6 ndash Documents sent to participants

41

ANNEX 6 - continued

42

ANNEX 7 Questionnaire on details of analysis method

43

ANNEX 7 - continued

44

ANNEX 7 - continued

45

ANNEX 8 - Results reported by participants for the food supplements test sample Table 11 Analysis results reported by the participants for the content of benz[a]anthracene (BAA) in the food supplements test material Values are presented as reported

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

3600 31 29 29 3 05 2

3602 199 193 178 19 021

3603 253 253 246 251 022 2

3604 199 209 23 213 071 2

3605 301 3 271 291 076 2

3606 302 288 278 289 064 2

3607 456 416 443 438 096 2

3608 796 695 651 714 149 2

3610 182 192 214 196 039 2

3611 414 504 488 469 96 2

3612 3 34 28 31 06 2

3613 32 33 34 33 07 2

3614 182 182 191 185 037 2

3615 3884 3822 3565 376 0752 2

3616 2 2 2 2 01 2

3617 366 37 35 362 046 2

3618 32 32 32 32 05 2

3619 299 305 297 299 048 2

3620 304 294 302 302 045 2

3621 29 26 29 28 04 2

3622 343 325 318 322 052 2

3623 35 3 33 33 07 2

3624

3626 318 31 314 314 126 2

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

5301 31 3 32 31 06 2

5302 314 319 315 316 063 2

5303 3102 2716 2822 2876 27 62

5304 261 263 26 261 57 2

5305 23 22 26 24 20

5306 22 23 23 22 11 2

5307 488 461 317 461

5308 168633 168544 1562025 1644598

5309 16 19 16 17

5310 3 304 311 305 015

5311 477 452 448 459 15 2

5312 506 63 494 543 134 2

5313 353 301 404 353

5314 431 373 378 394 5 096

5315 596 531 51 545 11 2

5316 224 231 235 235 022 2

5317 216 23 221 222 007 2

5318 064 07 067 01 2

5319 111 184 146 147 029 2

5320 515 525 564 534 48

5321 21 5322 13 87 9 87 35 2

5323 38 33 35 33 16 2

46

Figure 6 Results of replicate determinations (indicated by triangles) of benz[a]anthracene (BAA) in the food supplement test material

Horizontal blue lines represent the arithmetic mean value of replicate measurements and blue bars the reported expanded measurement uncertainty (k=2) The assigned value is

plotted as dotted line Red solid lines mark the range of satisfactory performance (|z|le2) Mean values are given for results outside the displayed data range

ProLab 2011

Laboratory

53

1853

1953

0853

0936

1436

0236

1036

1653

2136

0453

1753

0653

1653

0536

0353

0436

2153

0336

0636

0536

0036

2036

1953

1036

1253

0136

2653

0236

1836

2336

2236

1353

1353

2336

1736

1553

1453

0736

0753

1153

2053

1253

1536

0853

2236

11

an

aly

te c

on

ten

t (micro

gk

g)

10

95

9

85

8

75

7

65

6

55

5

45

4

35

3

25

2

15

1

05

0

102

3 468

7

Sample St Johns wort extract replicatesMeasurand Benz[a]anthraceneNo of laboratories 46

Assigned value 298 microgkg (Reference value)Target sd 062 microgkgTolerance limits 174 - 422 microgkg (|Z score| lt 200)

Assig

ned va

lue

Limit of tolerance

Limit of tolerance

47

Table 12 Analysis results reported by the participants for the content of benzo[a]pyrene (BAP) in the food supplements test material Values are presented as reported

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

3600 16 17 17 17 03 2

3602 118 123 13 124 015

3603 165 162 157 161 015 2

3604 134 154 139 143 04 2

3605 164 166 172 167 057 2

3606 181 159 161 167 031 2

3607 279 266 279 274 055 2

3608 57 687 461 573 226 2

3610 226 216 183 206 042 2

3611 289 325 311 308 36 2

3612 24 22 2 22 05 2

3613 17 18 19 18 05 2

3614 08 08 08 08 016 2

3615 1545 1393 1425 145 029 2

3616 16 16 14 15 03 2

3617 177 171 158 169 023 2

3618 2 2 2 2 02 2

3619 207 207 206 207 075 2

3620 165 169 163 166 017 2

3621 44 47 49 47 05 2

3622 212 208 222 213 038 2

3623 2 21 22 21 03 2

3624 18 178 182 18 031 2

3626 172 173 178 174 07 2

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

5301 18 18 18 18 04 2

5302 188 19 187 188 038 2

5303 1691 1426 149 1535 19 80

5304 145 142 15 146 69 2

5305 15 17 18 16 15

5306 16 17 17 16 08 2

5307 164 157 153 157

5308 102669 109324 109035 107009

5309 1 1 12 11

5310 122 122 124 123 006

5311 199 184 215 2 15 2

5312 258 248 239 248 05 2

5313 199 178 245 207

5314 174 171 172 172 11 0948

5315 253 248 213 238 05 2

5316 177 171 173 17 016 2

5317 255 277 276 269 012 2

5318 114 12 117 02 2

5319 19 228 231 216 043 2

5320 303 3 344 316 78

5321 12 12 12 12

5322 13 17 24 17 35 2

5323 2 2 19 2 1 2

48

Figure 7 Results of replicate determinations (indicated by triangles) of benzo[a]pyrene (BAP) in the food supplement test material

Horizontal blue lines represent the arithmetic mean value of replicate measurements and blue bars the reported expanded measurement uncertainty (k=2) The assigned value is

plotted as dotted line Red solid lines mark the range of satisfactory performance (|z|le2) Mean values are given for results outside the displayed data range

ProLab 2011

Laboratory

36

1453

0953

0853

1853

2153

1036

0236

0436

1553

0436

1653

0353

0736

0336

2036

0053

0553

0636

0636

0536

1753

1453

1636

2636

1336

2453

0153

2253

0253

2353

1136

1836

1953

1336

1036

2336

2253

1936

1253

1553

1253

1736

0753

2036

2136

0836

11

an

aly

te c

on

ten

t (micro

gk

g)

55

5

45

4

35

3

25

2

15

1

05

0

573

308

3

Sample St Johns wort extract replicatesMeasurand Benzo[a]pyreneNo of laboratories 48

Assigned value 165 microgkg (Reference value)Target sd 036 microgkgTolerance limits 093 - 237 microgkg (|Z score| lt 200)

Assig

ned va

lue

Limit of tolerance

Limit of tolerance

49

Table 13 Analysis results reported by the participants for the content of benzo[b]fluoranthene (BBF) in the food supplements test material Values are presented as reported

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

3600 32 3 3 31 09 2

3602 173 175 16 17 017

3603 265 252 25 256 025 2

3604 116 108 105 11 028 2

3605 286 225 232 247 074 2

3606 294 273 261 276 046 2

3607 579 579 544 567 113 2

3608 103 1201 876 1036 326 2

3610 173 196 204 191 038 2

3611 546 618 565 576 75 2

3612 3 28 28 29 06 2

3613 28 31 32 31 06 2

3614 135 111 135 127 022 2

3615 4187 3678 4111 4 08 2

3616 2 2 2 2 01 2

3617 33 27 299 035 2

3618 31 31 31 31 05 2

3619 326 324 312 326 064 2

3620 277 28 272 276 069 2

3621 26 25 23 25 04 2

3622 346 336 333 34 052 2

3623 26 28 29 28 02 2

3624

3626 288 28 307 292 117 2

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

5301 29 3 29 29 06 2

5302 297 3 296 298 06 2

5303 2705 2444 2608 2586 22 78

5304 245 254 26 253 85 2

5305 23 23 26 24 15

5306 42 43 4 42 21 2

5307 221 216 241 221

5308 232169 246654 240722 229848

5309 19 22 19 2 0

5310 3 315 325 313 016

5311 262 277 304 281 15 2

5312 27 289 337 299 075 2

5313 321 284 378 328

5314 423 404 385 404 34 083

5315 42 506 416 444 09 2

5316 265 267 277 273 018 2

5317 185 197 195 192 006 2

5318 276 281 279 04 2

5319 396 389 403 396 079 2

5320 623 618 635 625 14

5321 31

5322 24 33 49 33 35 2

5323 38 45 35 45 27 2

50

Figure 8 Results of replicate determinations (indicated by triangles) of benzo[b]fluoranthene (BBF) in the food supplement test material

Horizontal blue lines represent the arithmetic mean value of replicate measurements and blue bars the reported expanded measurement uncertainty (k=2) The assigned value is

plotted as dotted line Red solid lines mark the range of satisfactory performance (|z|le2) Mean values are given for results outside the displayed data range

ProLab 2011

Laboratory

36

0436

1436

0236

1053

1736

1653

0953

0753

0853

0536

2136

0553

0436

0353

0353

1636

0636

2036

2353

1853

1136

1236

2653

0153

0253

1236

1736

1336

0036

1853

2153

1036

1953

1336

2253

2253

2353

1936

1553

1453

0653

1536

0753

2036

0836

11

an

aly

te c

on

ten

t (micro

gk

g)

75

7

65

6

55

5

45

4

35

3

25

2

15

1

05

0

103

6

576

3

Sample St Johns wort extract replicatesMeasurand Benzo[b]fluorantheneNo of laboratories 46

Assigned value 292 microgkg (Reference value)Target sd 060 microgkgTolerance limits 172 - 412 microgkg (|Z score| lt 200)

Assig

ned va

lue

Limit of tolerance

Limit of tolerance

51

Table 14 Analysis results reported by the participants for the content of chrysene (CHR) in the food supplements test material Values are presented as reported

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

3600 5 49 5 5 07 2

3602 32 304 29 304 036

3603 462 469 447 459 048 2

3604 438 428 42 429 119 2

3605 365 41 381 386 085 2

3606 494 411 45 451 124 2

3607 897 774 787 819 18 2

3608 986 887 1168 1014 284 2

3610 418 392 462 424 085 2

3611 833 893 87 865 61 2

3612 6 64 58 61 12 2

3613 41 39 4 4 08 2

3614 272 254 288 271 054 2

3615 605 6028 5964 601 12 2

3616 47 45 49 47 05 2

3617 445 431 389 422 065 2

3618 45 45 45 45 06 2

3619 738 747 72 738 118 2

3620 463 472 446 46 092 2

3621 53 49 51 51 06 2

3622 478 513 505 488 08 2

3623 73 73 72 72 05 2

3624

3626 63 622 634 628 251 2

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

5301 49 49 49 49 1 2

5302 687 683 666 679 136 2

5303 6777 6181 6382 6441 24 70

5304 592 581 6 591 85 2

5305 6 58 64 61 20

5306 49 5 51 49 25 2

5307 524 5 574 524

5308 211488 216782 221146 213243

5309

5310 372 393 394 386 019

5311 43 459 422 437 15 2

5312 934 943 981 953 191 2

5313 793 73 99 838

5314 636 561 565 588 18 0818

5315 547 583 499 542 11 2

5316 292 286 285 282 06 2

5317 52 598 517 545 046 2

5318 368 345 357 05 2

5319 449 471 474 465 116 2

5320 76 924 818 834 99

5321 33

5322 7 7 76 7 35 2

5323 87 73 77 73 37 2

52

Figure 9 Results of replicate determinations (indicated by triangles) of chrysene (CHR) in the food supplement test material

Horizontal blue lines represent the arithmetic mean value of replicate measurements and blue bars the reported expanded measurement uncertainty (k=2) The assigned value is

plotted as dotted line Red solid lines mark the range of satisfactory performance (|z|le2) Mean values are given for results outside the displayed data range

ProLab 2011

Laboratory

53

0836

1453

1636

0253

2153

1836

0553

1036

1336

1736

1036

0453

1136

1836

0636

0336

2053

1936

1653

0136

0036

2253

0636

2153

0753

1553

1753

1453

0436

1536

1253

0536

2653

0353

0253

2236

2336

1953

2336

0753

2053

1353

1236

0836

11

an

aly

te c

on

ten

t (micro

gk

g)

13

12

11

10

9

8

7

6

5

4

3

2

1

0

865

3

Sample St Johns wort extract replicatesMeasurand ChryseneNo of laboratories 45

Assigned value 407 microgkg (Reference value)Target sd 083 microgkgTolerance limits 241 - 573 microgkg (|Z score| lt 200)

Assig

ned va

lue

Limit of tolerance

Limit of tolerance

53

Table 15 Analysis results reported by the participants for the sum of the four marker PAHs in the food supplements test material Values are presented as reported

Laboratory code

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg

3600 128 24 2

3602 79 126

3603 1127 096 2

3604 893 258 2

3605 1091 305 2

3606 1184 238 2

3607 2098 42 2

3608 3336 325 2

3610 1138 17 2

3611 2218 259 2

3612 143 29 2

3613 122 2 2

3614 663 129 2

3615 152 304 2

3616 102 03 2

3617 1252 169 2

3618 128 18 2

3619 157 305 2

3620 1204 12 2

3621 151 19 2

3622 1361 115 2

3623 154 09 2

3624

3626 1409 56 2

Laboratory code

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg

5301 126

5302 37 074 2

5303 13438 92

5304 1251 85 2

5305 125 20

5306 129 65 2

5307 1363

5308 7245598

5309

5310 1127 056

5311 1367 15 2

5312 2042 408 2

5313 1726

5314 1558

5315 177 35 2

5316 96 073 2

5317 1229 066 2

5318 82 05 2

5319 1225 267 2

5320 2309 41

5321

5322 207 35 2

5323 171 103 2

54

ANNEX 9 Results reported for the standard solutions with undisclosed analyte content Table 16 Analysis results reported by the participants for the four marker PAHs in the standard solution in toluene with undisclosed analyte

content Values are presented as reported U measurement uncertainty k coverage factor

benz[a]anthracene benzo[a]pyrene benzo[b]fluoranthene chrysene Laboratory

code Unit Result U k Result U k Result U k Result U k 3600 microgl 38 67 2 25 45 2 211 68 2 275 38 2

3605 microgl 367 184 2 2482 124 2 2592 13 2 235 118 2

3606 microgkg 3763 176 2 2609 142 2 2721 118 2 2539 134 2

3607 microgkg 3706 815 2 2824 565 2 2591 518 2 2587 569 2

3612 microgl 38 08 2 237 11 2 244 18 2 257 02 2

3614 microgl 324 65 2 209 38 2 215 37 2 21 42 2

3616 microgkg 318 07 2 224 14 2 231 26 2 216 1 2

3618 microgkg 417 17 2 284 08 2 292 06 2 297 03 2

3619 microgkg 47 742 2 274 468 2 228 387 2 274 434 2

3620 microgl 495 74 2 294 29 2 321 8 2 321 64 2

3621 microgl 241 22 2 143 13 2 188 16 2 145 14 2

3623 microgkg 476 25 2 311 53 2 269 3 2 297 03 2

3626 microgl 359 38 2 235 23 2 238 27 2 24 18 2

5301 microgkg 426 29 299 289

5302 microgkg 436 87 2 296 59 2 296 59 2 289 58 2

5303 microgl 3721 27 102 22838 19 99 23756 22 99 25486 24 98

5304 microgkg 38 29 2 225 26 2 257 37 2 258 52 2

5305 microgkg 305 225 245 29

5306 microgkg 316 63 2 247 49 2 65 13 2 22 44 2

5307 microgkg 3658 2559 241 2414

5308 microgkg 16854 109324 246654 216782

5311 microgl 374 20 2 279 15 2 268 15 2 272 15 2

5313 microgl 3408 2165 2275 2373

5314 microgkg 395 5 2875 11 3614 34 2905 18

5320 microgl 3267 368 1986 571 28 683 2427 512

5322 microgl 282 35 2 225 35 2 241 35 2 21 35 2

55

Figure 10 Youden plots of the percent deviation of the reported values from the assigned values for the food supplement test sample and the

standard solution in toluene with undisclosed content

(The identity of the respective measurand is given above the figure Results exceeding a difference of more than 100 were omitted)

56

Figure 10 - continued

57

Table 17 Analysis results reported by the participants for the four marker PAHs in the standard solution in acetonitrile with undisclosed

analyte content

Values are presented as reported U measurement uncertainty k coverage factor

benz[a]anthracene benzo[a]pyrene benzo[b]fluoranthene chrysene

Laboratory

code Unit Result U k Result U k Result U k Result U k

3602 microgkg 3675 247 247 244

3603 microgkg 358 086 2 236 028 2 235 028 2 236 057 2

3604 microgl 475 29 2 19 12 2 194 13 2 258 12 2

3608 microgl 3623 078 2 2385 023 2 2343 18 2 2296 092 2

3610 microgkg 369 185 2 25 125 2 251 126 2 244 122 2

3611 microgkg 48 02 2 323 02 2 328 04 2 317 03 2

3613 microgl 332 31 2 261 3 2 249 25 2 239 24 2

3615 microgl 415 2635 2729 2689

3617 microgkg 37 09 2 244 06 2 284 07 2 239 06 2

3622 microgl 37 12 2 25 23 2 257 21 2 238 1 2

3624 microgkg 402 104 2 255 084 2 265 096 2 253 07 2

5306 microgkg 563 113 2 371 74 2 359 72 2 333 67 2

5308 microgkg 168633 102669 232169 211488

5309 microgl 402 268 27

5310 microgl 315 158 25 125 25 125 288 144

5312 microgl 428 043 2 282 028 2 272 027 2 275 028 2

5315 microgkg 387 08 2 256 05 2 251 05 2 253 05 2

5316 microgl 3839 342 2 2485 191 2 2559 168 2 239 51 2

5317 microgl 2466 043 2 2212 034 2 2472 016 2 2146 068 2

5319 microgl 414 41 2 279 28 2 268 27 2 26 31 2

5321 microgkg 476 316 326 328

5322 microgl 2547 35 2

5323 microgkg 405 203 2 239 119 2 255 153 2 201 11 2

58

Figure 11 Youden plots of the percent deviation of the reported values from the assigned values for the food supplement test sample and the

standard solution in acetonitrile with undisclosed content

(The identity of the respective measurand is given above the figure Results exceeding a difference of more than 100 were omitted)

59

Figure 11 - continued

60

ANNEX 10 Table 18 Method performance parameters reported by the participants

Laboratory code

Analyte LOD LOQ Recovery Analyte LOD LOQ Recovery

microgkg microgkg microgkg microgkg 3600 BaA 03 06 978 BaP 03 07 9483602 BaA 007 021 926 BaP 005 015 993603 BaA 0011 05 987 BaP 0005 051 9553604 BaA 02 04 86 BaP 02 04 883605 BaA 0025 005 118 BaP 0025 005 1083606 BaA 001 003 65 BaP 001 003 703607 BaA 021 071 74 BaP 027 089 943608 BaA 016 048 8213 BaP 015 046 83683610 BaA 02 05 85 BaP 005 02 853611 BaA BaP 3612 BaA 1 117 BaP 1 1203613 BaA 01 03 90 BaP 01 03 903614 BaA 02 05 96 BaP 02 05 933615 BaA 005 015 110 BaP 005 015 1053616 BaA 01 05 108 BaP 01 05 753617 BaA 018 037 405 BaP 009 018 3873618 BaA 01 03 63 BaP 01 03 593619 BaA 005 005 59 BaP 033 033 543620 BaA 0009 0018 64 BaP 0005 001 553621 BaA 007 02 84 BaP 007 02 923622 BaA 01 03 1101 BaP 01 03 9853623 BaA 02 08 105 BaP 02 08 1013624 BaA BaP 01 033 8513625 BaA BaP 3626 BaA BaP 5301 BaA 003 01 102 BaP 003 01 1005302 BaA 01 03 78 BaP 01 03 865303 BaA 003 01 62 BaP 003 01 805304 BaA 1 1 70 BaP 1 1 955305 BaA 02 05 85 BaP 02 05 855306 BaA 03 1 699 BaP 03 1 905307 BaA 6537 BaP 81165308 BaA 0088 0265 99 BaP 00262 00785 1005309 BaA 01 08 92 BaP 01 08 925310 BaA 01 03 BaP 01 03 905311 BaA 002 005 80 BaP 002 004 915312 BaA 03 09 105 BaP 03 09 1055313 BaA 04 08 BaP 04 08 5314 BaA 001 002 96 BaP 001 002 9485315 BaA 01 03 50 - 100 BaP 01 03 50 - 100 5316 BaA 002 011 827 BaP 004 019 10445317 BaA 05 1 76 BaP 05 1 755318 BaA BaP 5319 BaA 009 029 951 BaP 011 041 995320 BaA 12 18 946 BaP 06 1 8345321 BaA 03 05 BaP 05 1 5322 BaA 1 2 100 BaP 1 2 1005323 BaA 26 BaP 26

61

ANNEX 10 Table 18 - continued

Laboratory code Analyte LOD LOQ Recovery Analyte LOD LOQ Recovery

microgkg microgkg microgkg microgkg 3600 BbF 03 05 96 CHR 02 03 10153602 BbF 015 045 947 CHR 003 009 9193603 BbF 0014 05 1012 CHR 0004 05 10033604 BbF 02 04 85 CHR 02 04 873605 BbF 005 01 94 CHR 0025 005 863606 BbF 001 003 70 CHR 001 003 653607 BbF 018 06 100 CHR 014 028 943608 BbF 015 046 7979 CHR 015 045 71253610 BbF 005 02 85 CHR 02 05 853611 BbF CHR 3612 BbF 1 118 CHR 1 1173613 BbF 01 03 90 CHR 01 03 903614 BbF 01 04 96 CHR 02 05 953615 BbF 008 025 109 CHR 003 01 1083616 BbF 01 05 78 CHR 01 05 723617 BbF 035 07 38 CHR 012 024 4163618 BbF 01 03 62 CHR 01 03 623619 BbF 016 016 63 CHR 01 01 593620 BbF 0003 0006 55 CHR 001 002 653621 BbF 007 02 103 CHR 007 02 893622 BbF 02 06 963 CHR 03 09 1043623 BbF 02 08 106 CHR 02 08 1193624 BbF CHR 3625 BbF CHR 3626 BbF CHR 5301 BbF 003 01 96 CHR 003 01 1145302 BbF 01 03 84 CHR 01 03 835303 BbF 003 01 78 CHR 003 01 705304 BbF 1 1 86 CHR 1 1 705305 BbF 02 05 90 CHR 02 05 955306 BbF 03 1 1435 CHR 03 1 1185307 BbF 8116 CHR 65375308 BbF 0079 0238 99 CHR 009 026 1005309 BbF 01 08 92 CHR 5310 BbF 07 2 CHR 01 03 5311 BbF 002 004 95 CHR 002 004 895312 BbF 03 09 105 CHR 03 09 1055313 BbF 04 08 CHR 04 08 5314 BbF 001 002 83 CHR 001 002 8185315 BbF 02 05 50 -100 CHR 02 05 50 - 100 5316 BbF 005 024 814 CHR 001 007 9595317 BbF 05 1 73 CHR 05 1 785318 BbF CHR 5319 BbF 011 038 946 CHR 013 043 9675320 BbF 07 09 1194 CHR 23 32 10455321 BbF 05 1 CHR 05 1 5322 BbF 1 2 100 CHR 1 2 1005323 BbF 26 CHR 26

62

ANNEX 11 Table 19 Details of analysis method reported by the participants Data are presented as reported

Laboratory code

Analysis technique

Instrument calibration

Internal standards applied

Sample clean up

Details of sample

clean up

Extraction technique

Details of sample

extraction

3600 6) GC-MS

2) Internal standardisation with labelled substances

Deuterated PAH mix 9 (Ehrenstorfer)

1) Saponification 2) LiquidLiquid partitioning

D) Other Liquidliquid partitioning

3602 2) HPLC-UV-FLD

1) External calibration

3) Gel permeation chromatography

A) Pressurised liquid extraction (PLE)

3603 1) HPLC-FLD

1) External calibration

3) Gel permeation chromatography

D) Other 1 minute with dichloromethane in vortex

3604 2) HPLC-UV-FLD

2) Internal standardisation with labelled substances

DiP D14

5) Donor acceptor complex chromatography

D) Other LL

3605 1) HPLC-FLD

1) External calibration

3) Gel permeation chromatography

C) Soxhlet extraction

3606 7) GC-MSMS

2) Internal standardisation with labelled substances

BaP 13C4 CHR 13C6 BaA 13C6 BbF 13C6

4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

3607 6) GC-MS

2) Internal standardisation with labelled substances

BaA-d12 CHR-d12 BbF-d12 BkF-d12 BaP-d12 ICP-d12 BgP-d12 DiP-d14

1) Saponification

D) Other solvent partitioning

3608 1) HPLC-FLD

1) External calibration

6) Other Liquid extraction

D) Other

Extraction in a rotary agitator and centrifugation

3610 2) HPLC-UV-FLD

1) External calibration

4) Solid phase extraction

B) Sonication

3611

3612 6) GC-MS

2) Internal standardisation with labelled substances

chrysene-d12 dibenzo(ah)anthracene-d14 dibenzo(ai)pyrene-d14

4) Solid phase extraction

B) Sonication

3613 5) LC-MSMS

3) Internal standardisation with unlabelled substances

I use Chrysene-D12 3) Gel permeation chromatography

D) Other extraction with hexan

63

Table 19 - continued

Laboratory code

Analysis technique

Instrument calibration

Internal standards applied

Sample clean up Details of sample

clean up

Extraction technique

Details of sample

extraction

3614 6) GC-MS 1) External calibration

3) Gel permeation chromatography

B) Sonication

3615 1) HPLC-FLD

1) External calibration

6) Other PLE GPC

A) Pressurised liquid extraction (PLE)

3616 6) GC-MS

2) Internal standardisation with labelled substances

Benzo(a)pyrene-13C4 Benzo(a)anthracene-13C6 Benzo(b)fluoranthene-13C6 Chrysene-13C6

2) LiquidLiquid partitioning 4) Solid phase extraction

B) Sonication

3617 1) HPLC-FLD

1) External calibration

4) Solid phase extraction

C) Soxhlet extraction

3618 6) GC-MS

2) Internal standardisation with labelled substances

deuterated standards

1) Saponification 4) Solid phase extraction

D) Other Handshaken with cyclohexane

3619 6) GC-MS

2) Internal standardisation with labelled substances

C13 Labelled US EPA 16

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

D) Other Homegenisation amp saponiofication

3620 9) GC-HRMS

2) Internal standardisation with labelled substances

mix of deuterated PAHs (BAA-D12 CHR-D12 BAP-D12 BBF-D12)

3) Gel permeation chromatography 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

3621 6) GC-MS

2) Internal standardisation with labelled substances

Chrysene D12 Benzo(a)pyrene D12 Benzo(b)fluoranthene D12 Benzo(a)anthracene

2) LiquidLiquid partitioning 4) Solid phase extraction

B) Sonication

3622 1) HPLC-FLD

4) Standard addition method

4) Solid phase extraction

D) Other

Extraction with cyclohexane by heating under reflux

3623 7) GC-MSMS

2) Internal standardisation with labelled substances

C13 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

3624 2) HPLC-UV-FLD

1) External calibration

2) LiquidLiquid partitioning

D) Other hot solvent extraction

3626

64

Table 19 - continued

Laboratory code

Analysis technique

Instrument calibration

Internal standards

applied

Sample clean up

Details of sample

clean up

Extraction technique

Details of sample

extraction

5301 7) GC-MSMS

2) Internal standardisation with labelled substances

EACH SUBSTANCE C13

4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5302 7) GC-MSMS

2) Internal standardisation with labelled substances

Isotope labelled PAHs 13C for each analyte

4) Solid phase extraction

C) Soxhlet extraction

5303 7) GC-MSMS

3) Internal standardisation with unlabelled substances

HAP C13 4) Solid phase extraction

D) Other automate ASE200 DIONEX

5304 7) GC-MSMS

2) Internal standardisation with labelled substances

with C13 for each HAP

2) LiquidLiquid partitioning 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5305 7) GC-MSMS

2) Internal standardisation with labelled substances

C13 isotopiques internal standards

4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5306 7) GC-MSMS

2) Internal standardisation with labelled substances

C13 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5307 6) GC-MS

2) Internal standardisation with labelled substances

1) Saponification 2) LiquidLiquid partitioning

D) Other solvent extraction

5308 1) HPLC-FLD 1) External calibration

4) Solid phase extraction

B) Sonication

5309 1) HPLC-FLD 1) External calibration

4) Solid phase extraction

D) Other only SPE

5310 1) HPLC-FLD 1) External calibration

1) Saponification 2) LiquidLiquid partitioning 3) Gel permeation chromatography

C) Soxhlet extraction

5311 6) GC-MS

2) Internal standardisation with labelled substances

13 C Chrysene 13 C Benz(a)pyrene

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

B) Sonication

5312 1) HPLC-FLD

3) Internal standardisation with unlabelled substances

Benzo(b)chrysen

2) LiquidLiquid partitioning 4) Solid phase extraction

Quechers D) Other Quechers

65

Table 19 - continued

Laboratory code

Analysis technique

Instrument calibration

Internal standards applied

Sample clean up Details of sample

clean up

Extraction technique

Details of sample extraction

5313 6) GC-MS

2) Internal standardisation with labelled substances

Benz(a)anthracene D12 Benzo(b)fluranthene D12 Benzo(a)pyrene D12 Chrysene D12

1) Saponification 2) LiquidLiquid partitioning 3) Gel permeation chromatography

A) Pressurised liquid extraction (PLE)

5314 6) GC-MS

2) Internal standardisation with labelled substances

Benzo(a)pyren d12 2) LiquidLiquid partitioning

D) Other Extraction with

5315 1) HPLC-FLD

3) Internal standardisation with unlabelled substances

Benz(b)chrysen

1) Saponification 2) LiquidLiquid partitioning

D) Other shaker

5316 1) HPLC-FLD

1) External calibration

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

D) Other

LiquidLiquid extraction with cyclohexane and dimethylformamide

5317 7) GC-MSMS

1) External calibration

3) Gel permeation chromatography 6) Other

silica gel D) Other VDLUFA online method

5318

5319 1) HPLC-FLD

3) Internal standardisation with unlabelled substances

Benzo(b)chrysene 3) Gel permeation chromatography

A) Pressurised liquid extraction (PLE)

5320 6) GC-MS

3) Internal standardisation with unlabelled substances

22-binaphthyl

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

D) Other 3 hours refluxing

5321 1) HPLC-FLD

2) Internal standardisation with labelled substances

6-meacutethylchrysegravene

1) Saponification 2) LiquidLiquid partitioning

B) Sonication

5322 6) GC-MS

2) Internal standardisation with labelled substances

naftalene-d8 acenaphtalene-d10 phenanthrene-d10 chrysene-d12 perylene-d12 benzo(e)anthracene-d12

1) Saponification 2) LiquidLiquid partitioning 6) Other

clean-up with silica packed column

C) Soxhlet extraction

5323 1) HPLC-FLD

1) External calibration

4) Solid phase extraction 6) Other

Accelerated Solvent Extraction

A) Pressurised liquid extraction (PLE)

66

European Commission EUR 25245 EN ndash Joint Research Centre ndash Institute for Reference Materials and Measurements Title Report on the 8th inter-laboratory comparison organised by the European Union Reference Laboratory for Polycyclic Aromatic Hydrocarbons - Four marker PAHs in a dry extract of St Johns wort Author(s) Radoslav Lizak Szilard Szilagyi Philippe Verlinde and Thomas WENZL Luxembourg Publications Office of the European Union 2012 ndash 66 pp ndash 201 x 297 cm EUR ndash Scientific and Technical Research series ndash ISSN 1831-9424 ISBN 978-92-79-23337-1 doi 10278759387 Abstract The proficiency test here reported concerned the determination of the contents of four marker polycyclic aromatic hydrocarbons (PAHs) and of their sum in an food supplements test sample The set of marker PAHs consists of benz[a]anthacene benzo[a]pyrene benzo[b]fluoranthene and chrysene Participants to these PT were National Reference Laboratories for PAHs (NRLs-PAHs) and EU official food control laboratories The number of participants was in total 48

The PT was organised according ISO Standard 170432010

The test material used was a commercial dry extract of St Johns wort Participants also received a solution of the same PAHs either in acetonitrile or in toluene for checking their instrument calibration

The results from participants were rated with z-scores and zeta-scores About 68 of the reported results were attributed with z-scores with an absolute value of below two which is the threshold for satisfactory performance

How to obtain EU publications

Our priced publications are available from EU Bookshop (httpbookshopeuropaeu) where you can place

an order with the sales agent of your choice

The Publications Office has a worldwide network of sales agents You can obtain their contact details by

sending a fax to (352) 29 29-42758

The mission of the JRC is to provide customer-driven scientific and technical support

for the conception development implementation and monitoring of EU policies As a

service of the European Commission the JRC functions as a reference centre of

science and technology for the Union Close to the policy-making process it serves

the common interest of the Member States while being independent of special

interests whether private or national

L

A-N

A-252

45-EN

-N

• 1 Summary
• 2 Introduction
• 3 Scope
• 4 Participating Laboratories
• 5 Time frame
• 6 Confidentiality
• 7 Test materials
• • 71 1Preparation of test materials
  • 72 Assigned values and standard deviation for proficiency assessment
  • 73 Homogeneity and stability
  • 74 Sample dispatch
  • • 8 Design of the proficiency test
    • 9 Evaluation of the results
    • • 91 2General
      • 92 21BEvaluation criteria
      • 93 Performance of participants
      • 94 Evaluation of the influence of analysis method
      • 95 Evaluation of the influence of calibration
      • 96 26Evaluation of compliance with legislation
      • • 10 Follow-up actions for underperforming laboratories
        • 11 1Conclusions
        • 12 1Acknowledgements
        • 13 References

27

In summary it can be stated that the majority of participants reported method performance

characteristics for the determination of the four marker PAHs in the food supplement test sample that

are compliant with legislation However some laboratories need to improve their analysis methods

The laboratories in question are urged to adapt to the provisions given in the new legislation

10 Follow-up actions for underperforming laboratories

All laboratories that got questionable or non-satisfactory performance ratings are urged to perform

root cause analysis and to implement corrective actions

As agreed during the EU-RL PAH workshop 2011 the EU-RL PAH will set up follow-up measures in

due time for all NRLs that received for at least one of the four PAHs (BAA BAP BBF and CHR) z-

scores gt 3 as required by Regulation (EC) 8822004 and by the Protocol for management of

underperformance in comparative testing andor lack of collaboration of National Reference

Laboratories (NRLs) with European Union reference laboratories (EU-RLs) activities

These laboratories shall perform as an immediate action root-cause-analysis and shall report to the

EU-RL PAH in writing the identified cause for their underperformance and corrective actions they are

going to take Additionally they shall participate to an independent (non-EU-RL) proficiency test on

the determination of PAHs in food and shall communicate the outcome of this exercise to the EU-RL

PAH

28

11 Conclusions

Fourty seven participants reported results back to the EU-RL The performance of the majority of

participants was good In total 153 out of 225 attributed z-scores were below an absolute value of two

which equals to almost 68 However this percentage is much below the percentages of satisfactory

performance gained in previous studies This might be the consequence of the higher level of difficulty

with respect to matrix composition In addition bias can be concluded from the pattern of performance

indicators of some laboratories

In general the determination of chrysene caused most difficulties to the participants

zeta-Scores were calculated besides z-scores They indicate the agreement of the reported result with

the assigned value with respect to the stated measurement uncertainty The outcome of this rating was

worse than for the z-scores which indicates that the measurement uncertainty estimates were not

realistic Therefore participants underperforming with regard to zeta scores are urged to adapt their

measurement uncertainty statements

The great majority of participants in this inter-laboratory comparison applied analytical methods which

complied with EU legislation with regard to performance characteristics

12 Acknowledgements

The organisers would like to thank Mrs Beatriz de la Calle and Mr Franz Ulberth (all from IRMM

Geel Belgium) for their accurate revision of this report Mr Hakan Emteborg (from IRMM) for

ampouling of the standard solutions and all NRLs and OCLs for their cooperation

29

13 References

1 EU COMMISSION REGULATION (EC) No 7762006 of 23 May 2006 amending Annex VII to Regulation

(EC) No 8822004 of the European Parliament and of the Council as regards Community reference

laboratories Official Journal of the European Union 2006 L 136 p 3-8

Available from

httpeur-lexeuropaeuLexUriServLexUriServdouri=OJL200613600030008ENPDF

2 EU Regulation (EC) No 8822004 of the European Parliament and of the Council of 29 April 2004 on

official controls performed to ensure the verification of compliance with feed and food law animal health

and animal welfare rules Official Journal of the European Communities 2004 L191 p 1-52 Available

from

httpeur-lexeuropaeuLexUriServLexUriServdouri=OJL200419100010052ENPDF

3 EU Opinion of the Scientific Committee on Food on the risks to human health of Polycyclic Aromatic

Hydrocarbons in food (2002) Available from

httpeuropaeuintcommfoodfsscscfout153_enpdf

4 International Agency for Research on Cancer IARC Monographs on the Evaluation of

Carcinogenic Risks to Humans Some Non-heterocyclic Polycyclic Aromatic Hydrocarbons and Some

Related Exposures Volume 92 (2010) Available from

httpmonographsiarcfrENGMonographsvol92mono92pdf

5 EU Commission Regulation (EC) No 3332007 of 28 March 2007 laying down the methods of sampling

and analysis for the official control of the levels of lead cadmium mercury inorganic tin 3-MCPD and

benzo(a)pyrene in foodstuffs Official Journal of the European Union 2007 L 88 p 29-38 Available

from

httpeur-lexeuropaeuLexUriServLexUriServdouri=OJL200708800290038ENPDF

6 EU Commission Regulation (EC) No 18812006 of 19 December 2006 setting maximum levels for certain

contaminants in foodstuffs Official Journal of the European Union 2006 L 364 p 5-24 Available from

httpeur-lexeuropaeuLexUriServLexUriServdouri=OJL200636400050024ENPDF

7 EFSA Scientific Opinion of the Panel on Contaminants in the Food Chain on a request from the European

Commission on Polycyclic Aromatic Hydrocarbons in Food The EFSA Journal 2008 724 p 1-114

Available from

httpwwwefsaeuropaeuEFSAefsa_locale-1178620753812_1211902034842htm

30

8 EU Commission Regulation (EU) No 8352011 amending Regulation (EC) No 18812006 as regards

maximum levels for polycyclic aromatic hydrocarbons in foodstuffs Official Journal of the European

Union 2011 L 215 p 4-8

httpeur-lexeuropaeuLexUriServLexUriServdouri=OJL201121500040008ENPDF

9 EU Commission Regulation (EU) No 8362011 amending Regulation (EC) No 3332007 laying down the methods of sampling and analysis for the official control of the levels of lead cadmium mercury inorganic tin 3-MCPD and benzo(a)pyrene in foodstuffs Official Journal of the European Union 2011 L 215 p 9-16 httpeur-lexeuropaeuLexUriServLexUriServdouri=OJL201121500090016ENPDF

10 ISOIEC 170432010 (2010) Conformity assessment -- General requirements for proficiency testing

providers Geneva Switzerland

11 Thompson M Ellison SLR and Wood R The International Harmonized Protocol for the Proficiency

Testing of Analytical Chemistry Laboratories Pure Appl Chem (2006) 78(1) p 145ndash196

Available from httpmediaiupacorgpublicationspac2006pdf7801x0145pdf

31

14 ANNEXES

ANNEX 1 ndash Announcement of the PT on the IRMM webpage

ANNEX 2 ndash Announcement via e-mail and invitation

ANNEX 3 ndash Announcement of sample dispatch

ANNEX 4 ndash Technical specifications of the calibration solutions

ANNEX 5 ndash Homogeneity and stability of the test material

ANNEX 6 ndash Acknowledgement of sample receipt

ANNEX 7 ndash Questionnaire on details of analysis method

ANNEX 8 ndash Results reported for the food supplement sample

ANNEX 9 ndash Results reported for the standard solutions with undisclosed analyte content

ANNEX 10 ndash Method performance characteristics reported by participants

ANNEX 11 ndash Details of analysis methods reported by participants

32

ANNEX 1 ndash Announcement of the PT on the IRMM webpage

33

ANNEX 2 - Announcement via e-mail and invitation

34

ANNEX 3 - Announcement sample dispatch

Dear MadameSir

The test samples for the proficiency test on the determination of four PAHs in a food supplement (dry extract of St Johns wort) were

dispatched today

Explanation on the design of the study as well as instructions for sample storage and for reporting of results are in the parcel You will

also find your participant key in the parcel which is required for reporting of results

Please check the parcel after arrival for completeness and report the receipt of the test samples as well as their status to us by applying

the sample receipt form which is in the parcel

You are kindly requested to inform us by email if you do not receive the parcel by end of this week

My colleague Donata Lerda will then take care of it

With best regards

Thomas Wenzl

35

ANNEX 4 - Specifications of standard solutions with disclosed concentration

36

ANNEX 4 - continued

37

ANNEX 5 - Homogeneity and stability of the test material

Homogeneity of the test material

Benz[a]anthracene - BAA

Benzo[a]pyrene - BAP

38

ANNEX 5 - continued

Benzo[b]fluoranthene - BBF

Chrysene - CHR

39

ANNEX 5 - continued

Stability of the test material

Table 10 Content of the four target analytes in test materials stored under different

temperatures

BaA BaP BbF CHR

microgkg microgkg microgkg microgkg

Content 31 16 31 69 Storage at room temperature

U (k=2) 05 02 05 17

Content 31 15 29 68 Storage at reference temperature

U (k=2) 04 03 04 10

The applied capillary column does not separate CHR from triphenylene

40

ANNEX 6 ndash Documents sent to participants

41

ANNEX 6 - continued

42

ANNEX 7 Questionnaire on details of analysis method

43

ANNEX 7 - continued

44

ANNEX 7 - continued

45

ANNEX 8 - Results reported by participants for the food supplements test sample Table 11 Analysis results reported by the participants for the content of benz[a]anthracene (BAA) in the food supplements test material Values are presented as reported

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

3600 31 29 29 3 05 2

3602 199 193 178 19 021

3603 253 253 246 251 022 2

3604 199 209 23 213 071 2

3605 301 3 271 291 076 2

3606 302 288 278 289 064 2

3607 456 416 443 438 096 2

3608 796 695 651 714 149 2

3610 182 192 214 196 039 2

3611 414 504 488 469 96 2

3612 3 34 28 31 06 2

3613 32 33 34 33 07 2

3614 182 182 191 185 037 2

3615 3884 3822 3565 376 0752 2

3616 2 2 2 2 01 2

3617 366 37 35 362 046 2

3618 32 32 32 32 05 2

3619 299 305 297 299 048 2

3620 304 294 302 302 045 2

3621 29 26 29 28 04 2

3622 343 325 318 322 052 2

3623 35 3 33 33 07 2

3624

3626 318 31 314 314 126 2

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

5301 31 3 32 31 06 2

5302 314 319 315 316 063 2

5303 3102 2716 2822 2876 27 62

5304 261 263 26 261 57 2

5305 23 22 26 24 20

5306 22 23 23 22 11 2

5307 488 461 317 461

5308 168633 168544 1562025 1644598

5309 16 19 16 17

5310 3 304 311 305 015

5311 477 452 448 459 15 2

5312 506 63 494 543 134 2

5313 353 301 404 353

5314 431 373 378 394 5 096

5315 596 531 51 545 11 2

5316 224 231 235 235 022 2

5317 216 23 221 222 007 2

5318 064 07 067 01 2

5319 111 184 146 147 029 2

5320 515 525 564 534 48

5321 21 5322 13 87 9 87 35 2

5323 38 33 35 33 16 2

46

Figure 6 Results of replicate determinations (indicated by triangles) of benz[a]anthracene (BAA) in the food supplement test material

Horizontal blue lines represent the arithmetic mean value of replicate measurements and blue bars the reported expanded measurement uncertainty (k=2) The assigned value is

plotted as dotted line Red solid lines mark the range of satisfactory performance (|z|le2) Mean values are given for results outside the displayed data range

ProLab 2011

Laboratory

53

1853

1953

0853

0936

1436

0236

1036

1653

2136

0453

1753

0653

1653

0536

0353

0436

2153

0336

0636

0536

0036

2036

1953

1036

1253

0136

2653

0236

1836

2336

2236

1353

1353

2336

1736

1553

1453

0736

0753

1153

2053

1253

1536

0853

2236

11

an

aly

te c

on

ten

t (micro

gk

g)

10

95

9

85

8

75

7

65

6

55

5

45

4

35

3

25

2

15

1

05

0

102

3 468

7

Sample St Johns wort extract replicatesMeasurand Benz[a]anthraceneNo of laboratories 46

Assigned value 298 microgkg (Reference value)Target sd 062 microgkgTolerance limits 174 - 422 microgkg (|Z score| lt 200)

Assig

ned va

lue

Limit of tolerance

Limit of tolerance

47

Table 12 Analysis results reported by the participants for the content of benzo[a]pyrene (BAP) in the food supplements test material Values are presented as reported

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

3600 16 17 17 17 03 2

3602 118 123 13 124 015

3603 165 162 157 161 015 2

3604 134 154 139 143 04 2

3605 164 166 172 167 057 2

3606 181 159 161 167 031 2

3607 279 266 279 274 055 2

3608 57 687 461 573 226 2

3610 226 216 183 206 042 2

3611 289 325 311 308 36 2

3612 24 22 2 22 05 2

3613 17 18 19 18 05 2

3614 08 08 08 08 016 2

3615 1545 1393 1425 145 029 2

3616 16 16 14 15 03 2

3617 177 171 158 169 023 2

3618 2 2 2 2 02 2

3619 207 207 206 207 075 2

3620 165 169 163 166 017 2

3621 44 47 49 47 05 2

3622 212 208 222 213 038 2

3623 2 21 22 21 03 2

3624 18 178 182 18 031 2

3626 172 173 178 174 07 2

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

5301 18 18 18 18 04 2

5302 188 19 187 188 038 2

5303 1691 1426 149 1535 19 80

5304 145 142 15 146 69 2

5305 15 17 18 16 15

5306 16 17 17 16 08 2

5307 164 157 153 157

5308 102669 109324 109035 107009

5309 1 1 12 11

5310 122 122 124 123 006

5311 199 184 215 2 15 2

5312 258 248 239 248 05 2

5313 199 178 245 207

5314 174 171 172 172 11 0948

5315 253 248 213 238 05 2

5316 177 171 173 17 016 2

5317 255 277 276 269 012 2

5318 114 12 117 02 2

5319 19 228 231 216 043 2

5320 303 3 344 316 78

5321 12 12 12 12

5322 13 17 24 17 35 2

5323 2 2 19 2 1 2

48

Figure 7 Results of replicate determinations (indicated by triangles) of benzo[a]pyrene (BAP) in the food supplement test material

Horizontal blue lines represent the arithmetic mean value of replicate measurements and blue bars the reported expanded measurement uncertainty (k=2) The assigned value is

plotted as dotted line Red solid lines mark the range of satisfactory performance (|z|le2) Mean values are given for results outside the displayed data range

ProLab 2011

Laboratory

36

1453

0953

0853

1853

2153

1036

0236

0436

1553

0436

1653

0353

0736

0336

2036

0053

0553

0636

0636

0536

1753

1453

1636

2636

1336

2453

0153

2253

0253

2353

1136

1836

1953

1336

1036

2336

2253

1936

1253

1553

1253

1736

0753

2036

2136

0836

11

an

aly

te c

on

ten

t (micro

gk

g)

55

5

45

4

35

3

25

2

15

1

05

0

573

308

3

Sample St Johns wort extract replicatesMeasurand Benzo[a]pyreneNo of laboratories 48

Assigned value 165 microgkg (Reference value)Target sd 036 microgkgTolerance limits 093 - 237 microgkg (|Z score| lt 200)

Assig

ned va

lue

Limit of tolerance

Limit of tolerance

49

Table 13 Analysis results reported by the participants for the content of benzo[b]fluoranthene (BBF) in the food supplements test material Values are presented as reported

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

3600 32 3 3 31 09 2

3602 173 175 16 17 017

3603 265 252 25 256 025 2

3604 116 108 105 11 028 2

3605 286 225 232 247 074 2

3606 294 273 261 276 046 2

3607 579 579 544 567 113 2

3608 103 1201 876 1036 326 2

3610 173 196 204 191 038 2

3611 546 618 565 576 75 2

3612 3 28 28 29 06 2

3613 28 31 32 31 06 2

3614 135 111 135 127 022 2

3615 4187 3678 4111 4 08 2

3616 2 2 2 2 01 2

3617 33 27 299 035 2

3618 31 31 31 31 05 2

3619 326 324 312 326 064 2

3620 277 28 272 276 069 2

3621 26 25 23 25 04 2

3622 346 336 333 34 052 2

3623 26 28 29 28 02 2

3624

3626 288 28 307 292 117 2

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

5301 29 3 29 29 06 2

5302 297 3 296 298 06 2

5303 2705 2444 2608 2586 22 78

5304 245 254 26 253 85 2

5305 23 23 26 24 15

5306 42 43 4 42 21 2

5307 221 216 241 221

5308 232169 246654 240722 229848

5309 19 22 19 2 0

5310 3 315 325 313 016

5311 262 277 304 281 15 2

5312 27 289 337 299 075 2

5313 321 284 378 328

5314 423 404 385 404 34 083

5315 42 506 416 444 09 2

5316 265 267 277 273 018 2

5317 185 197 195 192 006 2

5318 276 281 279 04 2

5319 396 389 403 396 079 2

5320 623 618 635 625 14

5321 31

5322 24 33 49 33 35 2

5323 38 45 35 45 27 2

50

Figure 8 Results of replicate determinations (indicated by triangles) of benzo[b]fluoranthene (BBF) in the food supplement test material

Horizontal blue lines represent the arithmetic mean value of replicate measurements and blue bars the reported expanded measurement uncertainty (k=2) The assigned value is

plotted as dotted line Red solid lines mark the range of satisfactory performance (|z|le2) Mean values are given for results outside the displayed data range

ProLab 2011

Laboratory

36

0436

1436

0236

1053

1736

1653

0953

0753

0853

0536

2136

0553

0436

0353

0353

1636

0636

2036

2353

1853

1136

1236

2653

0153

0253

1236

1736

1336

0036

1853

2153

1036

1953

1336

2253

2253

2353

1936

1553

1453

0653

1536

0753

2036

0836

11

an

aly

te c

on

ten

t (micro

gk

g)

75

7

65

6

55

5

45

4

35

3

25

2

15

1

05

0

103

6

576

3

Sample St Johns wort extract replicatesMeasurand Benzo[b]fluorantheneNo of laboratories 46

Assigned value 292 microgkg (Reference value)Target sd 060 microgkgTolerance limits 172 - 412 microgkg (|Z score| lt 200)

Assig

ned va

lue

Limit of tolerance

Limit of tolerance

51

Table 14 Analysis results reported by the participants for the content of chrysene (CHR) in the food supplements test material Values are presented as reported

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

3600 5 49 5 5 07 2

3602 32 304 29 304 036

3603 462 469 447 459 048 2

3604 438 428 42 429 119 2

3605 365 41 381 386 085 2

3606 494 411 45 451 124 2

3607 897 774 787 819 18 2

3608 986 887 1168 1014 284 2

3610 418 392 462 424 085 2

3611 833 893 87 865 61 2

3612 6 64 58 61 12 2

3613 41 39 4 4 08 2

3614 272 254 288 271 054 2

3615 605 6028 5964 601 12 2

3616 47 45 49 47 05 2

3617 445 431 389 422 065 2

3618 45 45 45 45 06 2

3619 738 747 72 738 118 2

3620 463 472 446 46 092 2

3621 53 49 51 51 06 2

3622 478 513 505 488 08 2

3623 73 73 72 72 05 2

3624

3626 63 622 634 628 251 2

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

5301 49 49 49 49 1 2

5302 687 683 666 679 136 2

5303 6777 6181 6382 6441 24 70

5304 592 581 6 591 85 2

5305 6 58 64 61 20

5306 49 5 51 49 25 2

5307 524 5 574 524

5308 211488 216782 221146 213243

5309

5310 372 393 394 386 019

5311 43 459 422 437 15 2

5312 934 943 981 953 191 2

5313 793 73 99 838

5314 636 561 565 588 18 0818

5315 547 583 499 542 11 2

5316 292 286 285 282 06 2

5317 52 598 517 545 046 2

5318 368 345 357 05 2

5319 449 471 474 465 116 2

5320 76 924 818 834 99

5321 33

5322 7 7 76 7 35 2

5323 87 73 77 73 37 2

52

Figure 9 Results of replicate determinations (indicated by triangles) of chrysene (CHR) in the food supplement test material

Horizontal blue lines represent the arithmetic mean value of replicate measurements and blue bars the reported expanded measurement uncertainty (k=2) The assigned value is

plotted as dotted line Red solid lines mark the range of satisfactory performance (|z|le2) Mean values are given for results outside the displayed data range

ProLab 2011

Laboratory

53

0836

1453

1636

0253

2153

1836

0553

1036

1336

1736

1036

0453

1136

1836

0636

0336

2053

1936

1653

0136

0036

2253

0636

2153

0753

1553

1753

1453

0436

1536

1253

0536

2653

0353

0253

2236

2336

1953

2336

0753

2053

1353

1236

0836

11

an

aly

te c

on

ten

t (micro

gk

g)

13

12

11

10

9

8

7

6

5

4

3

2

1

0

865

3

Sample St Johns wort extract replicatesMeasurand ChryseneNo of laboratories 45

Assigned value 407 microgkg (Reference value)Target sd 083 microgkgTolerance limits 241 - 573 microgkg (|Z score| lt 200)

Assig

ned va

lue

Limit of tolerance

Limit of tolerance

53

Table 15 Analysis results reported by the participants for the sum of the four marker PAHs in the food supplements test material Values are presented as reported

Laboratory code

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg

3600 128 24 2

3602 79 126

3603 1127 096 2

3604 893 258 2

3605 1091 305 2

3606 1184 238 2

3607 2098 42 2

3608 3336 325 2

3610 1138 17 2

3611 2218 259 2

3612 143 29 2

3613 122 2 2

3614 663 129 2

3615 152 304 2

3616 102 03 2

3617 1252 169 2

3618 128 18 2

3619 157 305 2

3620 1204 12 2

3621 151 19 2

3622 1361 115 2

3623 154 09 2

3624

3626 1409 56 2

Laboratory code

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg

5301 126

5302 37 074 2

5303 13438 92

5304 1251 85 2

5305 125 20

5306 129 65 2

5307 1363

5308 7245598

5309

5310 1127 056

5311 1367 15 2

5312 2042 408 2

5313 1726

5314 1558

5315 177 35 2

5316 96 073 2

5317 1229 066 2

5318 82 05 2

5319 1225 267 2

5320 2309 41

5321

5322 207 35 2

5323 171 103 2

54

ANNEX 9 Results reported for the standard solutions with undisclosed analyte content Table 16 Analysis results reported by the participants for the four marker PAHs in the standard solution in toluene with undisclosed analyte

content Values are presented as reported U measurement uncertainty k coverage factor

benz[a]anthracene benzo[a]pyrene benzo[b]fluoranthene chrysene Laboratory

code Unit Result U k Result U k Result U k Result U k 3600 microgl 38 67 2 25 45 2 211 68 2 275 38 2

3605 microgl 367 184 2 2482 124 2 2592 13 2 235 118 2

3606 microgkg 3763 176 2 2609 142 2 2721 118 2 2539 134 2

3607 microgkg 3706 815 2 2824 565 2 2591 518 2 2587 569 2

3612 microgl 38 08 2 237 11 2 244 18 2 257 02 2

3614 microgl 324 65 2 209 38 2 215 37 2 21 42 2

3616 microgkg 318 07 2 224 14 2 231 26 2 216 1 2

3618 microgkg 417 17 2 284 08 2 292 06 2 297 03 2

3619 microgkg 47 742 2 274 468 2 228 387 2 274 434 2

3620 microgl 495 74 2 294 29 2 321 8 2 321 64 2

3621 microgl 241 22 2 143 13 2 188 16 2 145 14 2

3623 microgkg 476 25 2 311 53 2 269 3 2 297 03 2

3626 microgl 359 38 2 235 23 2 238 27 2 24 18 2

5301 microgkg 426 29 299 289

5302 microgkg 436 87 2 296 59 2 296 59 2 289 58 2

5303 microgl 3721 27 102 22838 19 99 23756 22 99 25486 24 98

5304 microgkg 38 29 2 225 26 2 257 37 2 258 52 2

5305 microgkg 305 225 245 29

5306 microgkg 316 63 2 247 49 2 65 13 2 22 44 2

5307 microgkg 3658 2559 241 2414

5308 microgkg 16854 109324 246654 216782

5311 microgl 374 20 2 279 15 2 268 15 2 272 15 2

5313 microgl 3408 2165 2275 2373

5314 microgkg 395 5 2875 11 3614 34 2905 18

5320 microgl 3267 368 1986 571 28 683 2427 512

5322 microgl 282 35 2 225 35 2 241 35 2 21 35 2

55

Figure 10 Youden plots of the percent deviation of the reported values from the assigned values for the food supplement test sample and the

standard solution in toluene with undisclosed content

(The identity of the respective measurand is given above the figure Results exceeding a difference of more than 100 were omitted)

56

Figure 10 - continued

57

Table 17 Analysis results reported by the participants for the four marker PAHs in the standard solution in acetonitrile with undisclosed

analyte content

Values are presented as reported U measurement uncertainty k coverage factor

benz[a]anthracene benzo[a]pyrene benzo[b]fluoranthene chrysene

Laboratory

code Unit Result U k Result U k Result U k Result U k

3602 microgkg 3675 247 247 244

3603 microgkg 358 086 2 236 028 2 235 028 2 236 057 2

3604 microgl 475 29 2 19 12 2 194 13 2 258 12 2

3608 microgl 3623 078 2 2385 023 2 2343 18 2 2296 092 2

3610 microgkg 369 185 2 25 125 2 251 126 2 244 122 2

3611 microgkg 48 02 2 323 02 2 328 04 2 317 03 2

3613 microgl 332 31 2 261 3 2 249 25 2 239 24 2

3615 microgl 415 2635 2729 2689

3617 microgkg 37 09 2 244 06 2 284 07 2 239 06 2

3622 microgl 37 12 2 25 23 2 257 21 2 238 1 2

3624 microgkg 402 104 2 255 084 2 265 096 2 253 07 2

5306 microgkg 563 113 2 371 74 2 359 72 2 333 67 2

5308 microgkg 168633 102669 232169 211488

5309 microgl 402 268 27

5310 microgl 315 158 25 125 25 125 288 144

5312 microgl 428 043 2 282 028 2 272 027 2 275 028 2

5315 microgkg 387 08 2 256 05 2 251 05 2 253 05 2

5316 microgl 3839 342 2 2485 191 2 2559 168 2 239 51 2

5317 microgl 2466 043 2 2212 034 2 2472 016 2 2146 068 2

5319 microgl 414 41 2 279 28 2 268 27 2 26 31 2

5321 microgkg 476 316 326 328

5322 microgl 2547 35 2

5323 microgkg 405 203 2 239 119 2 255 153 2 201 11 2

58

Figure 11 Youden plots of the percent deviation of the reported values from the assigned values for the food supplement test sample and the

standard solution in acetonitrile with undisclosed content

(The identity of the respective measurand is given above the figure Results exceeding a difference of more than 100 were omitted)

59

Figure 11 - continued

60

ANNEX 10 Table 18 Method performance parameters reported by the participants

Laboratory code

Analyte LOD LOQ Recovery Analyte LOD LOQ Recovery

microgkg microgkg microgkg microgkg 3600 BaA 03 06 978 BaP 03 07 9483602 BaA 007 021 926 BaP 005 015 993603 BaA 0011 05 987 BaP 0005 051 9553604 BaA 02 04 86 BaP 02 04 883605 BaA 0025 005 118 BaP 0025 005 1083606 BaA 001 003 65 BaP 001 003 703607 BaA 021 071 74 BaP 027 089 943608 BaA 016 048 8213 BaP 015 046 83683610 BaA 02 05 85 BaP 005 02 853611 BaA BaP 3612 BaA 1 117 BaP 1 1203613 BaA 01 03 90 BaP 01 03 903614 BaA 02 05 96 BaP 02 05 933615 BaA 005 015 110 BaP 005 015 1053616 BaA 01 05 108 BaP 01 05 753617 BaA 018 037 405 BaP 009 018 3873618 BaA 01 03 63 BaP 01 03 593619 BaA 005 005 59 BaP 033 033 543620 BaA 0009 0018 64 BaP 0005 001 553621 BaA 007 02 84 BaP 007 02 923622 BaA 01 03 1101 BaP 01 03 9853623 BaA 02 08 105 BaP 02 08 1013624 BaA BaP 01 033 8513625 BaA BaP 3626 BaA BaP 5301 BaA 003 01 102 BaP 003 01 1005302 BaA 01 03 78 BaP 01 03 865303 BaA 003 01 62 BaP 003 01 805304 BaA 1 1 70 BaP 1 1 955305 BaA 02 05 85 BaP 02 05 855306 BaA 03 1 699 BaP 03 1 905307 BaA 6537 BaP 81165308 BaA 0088 0265 99 BaP 00262 00785 1005309 BaA 01 08 92 BaP 01 08 925310 BaA 01 03 BaP 01 03 905311 BaA 002 005 80 BaP 002 004 915312 BaA 03 09 105 BaP 03 09 1055313 BaA 04 08 BaP 04 08 5314 BaA 001 002 96 BaP 001 002 9485315 BaA 01 03 50 - 100 BaP 01 03 50 - 100 5316 BaA 002 011 827 BaP 004 019 10445317 BaA 05 1 76 BaP 05 1 755318 BaA BaP 5319 BaA 009 029 951 BaP 011 041 995320 BaA 12 18 946 BaP 06 1 8345321 BaA 03 05 BaP 05 1 5322 BaA 1 2 100 BaP 1 2 1005323 BaA 26 BaP 26

61

ANNEX 10 Table 18 - continued

Laboratory code Analyte LOD LOQ Recovery Analyte LOD LOQ Recovery

microgkg microgkg microgkg microgkg 3600 BbF 03 05 96 CHR 02 03 10153602 BbF 015 045 947 CHR 003 009 9193603 BbF 0014 05 1012 CHR 0004 05 10033604 BbF 02 04 85 CHR 02 04 873605 BbF 005 01 94 CHR 0025 005 863606 BbF 001 003 70 CHR 001 003 653607 BbF 018 06 100 CHR 014 028 943608 BbF 015 046 7979 CHR 015 045 71253610 BbF 005 02 85 CHR 02 05 853611 BbF CHR 3612 BbF 1 118 CHR 1 1173613 BbF 01 03 90 CHR 01 03 903614 BbF 01 04 96 CHR 02 05 953615 BbF 008 025 109 CHR 003 01 1083616 BbF 01 05 78 CHR 01 05 723617 BbF 035 07 38 CHR 012 024 4163618 BbF 01 03 62 CHR 01 03 623619 BbF 016 016 63 CHR 01 01 593620 BbF 0003 0006 55 CHR 001 002 653621 BbF 007 02 103 CHR 007 02 893622 BbF 02 06 963 CHR 03 09 1043623 BbF 02 08 106 CHR 02 08 1193624 BbF CHR 3625 BbF CHR 3626 BbF CHR 5301 BbF 003 01 96 CHR 003 01 1145302 BbF 01 03 84 CHR 01 03 835303 BbF 003 01 78 CHR 003 01 705304 BbF 1 1 86 CHR 1 1 705305 BbF 02 05 90 CHR 02 05 955306 BbF 03 1 1435 CHR 03 1 1185307 BbF 8116 CHR 65375308 BbF 0079 0238 99 CHR 009 026 1005309 BbF 01 08 92 CHR 5310 BbF 07 2 CHR 01 03 5311 BbF 002 004 95 CHR 002 004 895312 BbF 03 09 105 CHR 03 09 1055313 BbF 04 08 CHR 04 08 5314 BbF 001 002 83 CHR 001 002 8185315 BbF 02 05 50 -100 CHR 02 05 50 - 100 5316 BbF 005 024 814 CHR 001 007 9595317 BbF 05 1 73 CHR 05 1 785318 BbF CHR 5319 BbF 011 038 946 CHR 013 043 9675320 BbF 07 09 1194 CHR 23 32 10455321 BbF 05 1 CHR 05 1 5322 BbF 1 2 100 CHR 1 2 1005323 BbF 26 CHR 26

62

ANNEX 11 Table 19 Details of analysis method reported by the participants Data are presented as reported

Laboratory code

Analysis technique

Instrument calibration

Internal standards applied

Sample clean up

Details of sample

clean up

Extraction technique

Details of sample

extraction

3600 6) GC-MS

2) Internal standardisation with labelled substances

Deuterated PAH mix 9 (Ehrenstorfer)

1) Saponification 2) LiquidLiquid partitioning

D) Other Liquidliquid partitioning

3602 2) HPLC-UV-FLD

1) External calibration

3) Gel permeation chromatography

A) Pressurised liquid extraction (PLE)

3603 1) HPLC-FLD

1) External calibration

3) Gel permeation chromatography

D) Other 1 minute with dichloromethane in vortex

3604 2) HPLC-UV-FLD

2) Internal standardisation with labelled substances

DiP D14

5) Donor acceptor complex chromatography

D) Other LL

3605 1) HPLC-FLD

1) External calibration

3) Gel permeation chromatography

C) Soxhlet extraction

3606 7) GC-MSMS

2) Internal standardisation with labelled substances

BaP 13C4 CHR 13C6 BaA 13C6 BbF 13C6

4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

3607 6) GC-MS

2) Internal standardisation with labelled substances

BaA-d12 CHR-d12 BbF-d12 BkF-d12 BaP-d12 ICP-d12 BgP-d12 DiP-d14

1) Saponification

D) Other solvent partitioning

3608 1) HPLC-FLD

1) External calibration

6) Other Liquid extraction

D) Other

Extraction in a rotary agitator and centrifugation

3610 2) HPLC-UV-FLD

1) External calibration

4) Solid phase extraction

B) Sonication

3611

3612 6) GC-MS

2) Internal standardisation with labelled substances

chrysene-d12 dibenzo(ah)anthracene-d14 dibenzo(ai)pyrene-d14

4) Solid phase extraction

B) Sonication

3613 5) LC-MSMS

3) Internal standardisation with unlabelled substances

I use Chrysene-D12 3) Gel permeation chromatography

D) Other extraction with hexan

63

Table 19 - continued

Laboratory code

Analysis technique

Instrument calibration

Internal standards applied

Sample clean up Details of sample

clean up

Extraction technique

Details of sample

extraction

3614 6) GC-MS 1) External calibration

3) Gel permeation chromatography

B) Sonication

3615 1) HPLC-FLD

1) External calibration

6) Other PLE GPC

A) Pressurised liquid extraction (PLE)

3616 6) GC-MS

2) Internal standardisation with labelled substances

Benzo(a)pyrene-13C4 Benzo(a)anthracene-13C6 Benzo(b)fluoranthene-13C6 Chrysene-13C6

2) LiquidLiquid partitioning 4) Solid phase extraction

B) Sonication

3617 1) HPLC-FLD

1) External calibration

4) Solid phase extraction

C) Soxhlet extraction

3618 6) GC-MS

2) Internal standardisation with labelled substances

deuterated standards

1) Saponification 4) Solid phase extraction

D) Other Handshaken with cyclohexane

3619 6) GC-MS

2) Internal standardisation with labelled substances

C13 Labelled US EPA 16

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

D) Other Homegenisation amp saponiofication

3620 9) GC-HRMS

2) Internal standardisation with labelled substances

mix of deuterated PAHs (BAA-D12 CHR-D12 BAP-D12 BBF-D12)

3) Gel permeation chromatography 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

3621 6) GC-MS

2) Internal standardisation with labelled substances

Chrysene D12 Benzo(a)pyrene D12 Benzo(b)fluoranthene D12 Benzo(a)anthracene

2) LiquidLiquid partitioning 4) Solid phase extraction

B) Sonication

3622 1) HPLC-FLD

4) Standard addition method

4) Solid phase extraction

D) Other

Extraction with cyclohexane by heating under reflux

3623 7) GC-MSMS

2) Internal standardisation with labelled substances

C13 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

3624 2) HPLC-UV-FLD

1) External calibration

2) LiquidLiquid partitioning

D) Other hot solvent extraction

3626

64

Table 19 - continued

Laboratory code

Analysis technique

Instrument calibration

Internal standards

applied

Sample clean up

Details of sample

clean up

Extraction technique

Details of sample

extraction

5301 7) GC-MSMS

2) Internal standardisation with labelled substances

EACH SUBSTANCE C13

4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5302 7) GC-MSMS

2) Internal standardisation with labelled substances

Isotope labelled PAHs 13C for each analyte

4) Solid phase extraction

C) Soxhlet extraction

5303 7) GC-MSMS

3) Internal standardisation with unlabelled substances

HAP C13 4) Solid phase extraction

D) Other automate ASE200 DIONEX

5304 7) GC-MSMS

2) Internal standardisation with labelled substances

with C13 for each HAP

2) LiquidLiquid partitioning 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5305 7) GC-MSMS

2) Internal standardisation with labelled substances

C13 isotopiques internal standards

4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5306 7) GC-MSMS

2) Internal standardisation with labelled substances

C13 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5307 6) GC-MS

2) Internal standardisation with labelled substances

1) Saponification 2) LiquidLiquid partitioning

D) Other solvent extraction

5308 1) HPLC-FLD 1) External calibration

4) Solid phase extraction

B) Sonication

5309 1) HPLC-FLD 1) External calibration

4) Solid phase extraction

D) Other only SPE

5310 1) HPLC-FLD 1) External calibration

1) Saponification 2) LiquidLiquid partitioning 3) Gel permeation chromatography

C) Soxhlet extraction

5311 6) GC-MS

2) Internal standardisation with labelled substances

13 C Chrysene 13 C Benz(a)pyrene

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

B) Sonication

5312 1) HPLC-FLD

3) Internal standardisation with unlabelled substances

Benzo(b)chrysen

2) LiquidLiquid partitioning 4) Solid phase extraction

Quechers D) Other Quechers

65

Table 19 - continued

Laboratory code

Analysis technique

Instrument calibration

Internal standards applied

Sample clean up Details of sample

clean up

Extraction technique

Details of sample extraction

5313 6) GC-MS

2) Internal standardisation with labelled substances

Benz(a)anthracene D12 Benzo(b)fluranthene D12 Benzo(a)pyrene D12 Chrysene D12

1) Saponification 2) LiquidLiquid partitioning 3) Gel permeation chromatography

A) Pressurised liquid extraction (PLE)

5314 6) GC-MS

2) Internal standardisation with labelled substances

Benzo(a)pyren d12 2) LiquidLiquid partitioning

D) Other Extraction with

5315 1) HPLC-FLD

3) Internal standardisation with unlabelled substances

Benz(b)chrysen

1) Saponification 2) LiquidLiquid partitioning

D) Other shaker

5316 1) HPLC-FLD

1) External calibration

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

D) Other

LiquidLiquid extraction with cyclohexane and dimethylformamide

5317 7) GC-MSMS

1) External calibration

3) Gel permeation chromatography 6) Other

silica gel D) Other VDLUFA online method

5318

5319 1) HPLC-FLD

3) Internal standardisation with unlabelled substances

Benzo(b)chrysene 3) Gel permeation chromatography

A) Pressurised liquid extraction (PLE)

5320 6) GC-MS

3) Internal standardisation with unlabelled substances

22-binaphthyl

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

D) Other 3 hours refluxing

5321 1) HPLC-FLD

2) Internal standardisation with labelled substances

6-meacutethylchrysegravene

1) Saponification 2) LiquidLiquid partitioning

B) Sonication

5322 6) GC-MS

2) Internal standardisation with labelled substances

naftalene-d8 acenaphtalene-d10 phenanthrene-d10 chrysene-d12 perylene-d12 benzo(e)anthracene-d12

1) Saponification 2) LiquidLiquid partitioning 6) Other

clean-up with silica packed column

C) Soxhlet extraction

5323 1) HPLC-FLD

1) External calibration

4) Solid phase extraction 6) Other

Accelerated Solvent Extraction

A) Pressurised liquid extraction (PLE)

66

European Commission EUR 25245 EN ndash Joint Research Centre ndash Institute for Reference Materials and Measurements Title Report on the 8th inter-laboratory comparison organised by the European Union Reference Laboratory for Polycyclic Aromatic Hydrocarbons - Four marker PAHs in a dry extract of St Johns wort Author(s) Radoslav Lizak Szilard Szilagyi Philippe Verlinde and Thomas WENZL Luxembourg Publications Office of the European Union 2012 ndash 66 pp ndash 201 x 297 cm EUR ndash Scientific and Technical Research series ndash ISSN 1831-9424 ISBN 978-92-79-23337-1 doi 10278759387 Abstract The proficiency test here reported concerned the determination of the contents of four marker polycyclic aromatic hydrocarbons (PAHs) and of their sum in an food supplements test sample The set of marker PAHs consists of benz[a]anthacene benzo[a]pyrene benzo[b]fluoranthene and chrysene Participants to these PT were National Reference Laboratories for PAHs (NRLs-PAHs) and EU official food control laboratories The number of participants was in total 48

The PT was organised according ISO Standard 170432010

The test material used was a commercial dry extract of St Johns wort Participants also received a solution of the same PAHs either in acetonitrile or in toluene for checking their instrument calibration

The results from participants were rated with z-scores and zeta-scores About 68 of the reported results were attributed with z-scores with an absolute value of below two which is the threshold for satisfactory performance

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L

A-N

A-252

45-EN

-N

• 1 Summary
• 2 Introduction
• 3 Scope
• 4 Participating Laboratories
• 5 Time frame
• 6 Confidentiality
• 7 Test materials
• • 71 1Preparation of test materials
  • 72 Assigned values and standard deviation for proficiency assessment
  • 73 Homogeneity and stability
  • 74 Sample dispatch
  • • 8 Design of the proficiency test
    • 9 Evaluation of the results
    • • 91 2General
      • 92 21BEvaluation criteria
      • 93 Performance of participants
      • 94 Evaluation of the influence of analysis method
      • 95 Evaluation of the influence of calibration
      • 96 26Evaluation of compliance with legislation
      • • 10 Follow-up actions for underperforming laboratories
        • 11 1Conclusions
        • 12 1Acknowledgements
        • 13 References

28

11 Conclusions

Fourty seven participants reported results back to the EU-RL The performance of the majority of

participants was good In total 153 out of 225 attributed z-scores were below an absolute value of two

which equals to almost 68 However this percentage is much below the percentages of satisfactory

performance gained in previous studies This might be the consequence of the higher level of difficulty

with respect to matrix composition In addition bias can be concluded from the pattern of performance

indicators of some laboratories

In general the determination of chrysene caused most difficulties to the participants

zeta-Scores were calculated besides z-scores They indicate the agreement of the reported result with

the assigned value with respect to the stated measurement uncertainty The outcome of this rating was

worse than for the z-scores which indicates that the measurement uncertainty estimates were not

realistic Therefore participants underperforming with regard to zeta scores are urged to adapt their

measurement uncertainty statements

The great majority of participants in this inter-laboratory comparison applied analytical methods which

complied with EU legislation with regard to performance characteristics

12 Acknowledgements

The organisers would like to thank Mrs Beatriz de la Calle and Mr Franz Ulberth (all from IRMM

Geel Belgium) for their accurate revision of this report Mr Hakan Emteborg (from IRMM) for

ampouling of the standard solutions and all NRLs and OCLs for their cooperation

29

13 References

1 EU COMMISSION REGULATION (EC) No 7762006 of 23 May 2006 amending Annex VII to Regulation

(EC) No 8822004 of the European Parliament and of the Council as regards Community reference

laboratories Official Journal of the European Union 2006 L 136 p 3-8

Available from

httpeur-lexeuropaeuLexUriServLexUriServdouri=OJL200613600030008ENPDF

2 EU Regulation (EC) No 8822004 of the European Parliament and of the Council of 29 April 2004 on

official controls performed to ensure the verification of compliance with feed and food law animal health

and animal welfare rules Official Journal of the European Communities 2004 L191 p 1-52 Available

from

httpeur-lexeuropaeuLexUriServLexUriServdouri=OJL200419100010052ENPDF

3 EU Opinion of the Scientific Committee on Food on the risks to human health of Polycyclic Aromatic

Hydrocarbons in food (2002) Available from

httpeuropaeuintcommfoodfsscscfout153_enpdf

4 International Agency for Research on Cancer IARC Monographs on the Evaluation of

Carcinogenic Risks to Humans Some Non-heterocyclic Polycyclic Aromatic Hydrocarbons and Some

Related Exposures Volume 92 (2010) Available from

httpmonographsiarcfrENGMonographsvol92mono92pdf

5 EU Commission Regulation (EC) No 3332007 of 28 March 2007 laying down the methods of sampling

and analysis for the official control of the levels of lead cadmium mercury inorganic tin 3-MCPD and

benzo(a)pyrene in foodstuffs Official Journal of the European Union 2007 L 88 p 29-38 Available

from

httpeur-lexeuropaeuLexUriServLexUriServdouri=OJL200708800290038ENPDF

6 EU Commission Regulation (EC) No 18812006 of 19 December 2006 setting maximum levels for certain

contaminants in foodstuffs Official Journal of the European Union 2006 L 364 p 5-24 Available from

httpeur-lexeuropaeuLexUriServLexUriServdouri=OJL200636400050024ENPDF

7 EFSA Scientific Opinion of the Panel on Contaminants in the Food Chain on a request from the European

Commission on Polycyclic Aromatic Hydrocarbons in Food The EFSA Journal 2008 724 p 1-114

Available from

httpwwwefsaeuropaeuEFSAefsa_locale-1178620753812_1211902034842htm

30

8 EU Commission Regulation (EU) No 8352011 amending Regulation (EC) No 18812006 as regards

maximum levels for polycyclic aromatic hydrocarbons in foodstuffs Official Journal of the European

Union 2011 L 215 p 4-8

httpeur-lexeuropaeuLexUriServLexUriServdouri=OJL201121500040008ENPDF

9 EU Commission Regulation (EU) No 8362011 amending Regulation (EC) No 3332007 laying down the methods of sampling and analysis for the official control of the levels of lead cadmium mercury inorganic tin 3-MCPD and benzo(a)pyrene in foodstuffs Official Journal of the European Union 2011 L 215 p 9-16 httpeur-lexeuropaeuLexUriServLexUriServdouri=OJL201121500090016ENPDF

10 ISOIEC 170432010 (2010) Conformity assessment -- General requirements for proficiency testing

providers Geneva Switzerland

11 Thompson M Ellison SLR and Wood R The International Harmonized Protocol for the Proficiency

Testing of Analytical Chemistry Laboratories Pure Appl Chem (2006) 78(1) p 145ndash196

Available from httpmediaiupacorgpublicationspac2006pdf7801x0145pdf

31

14 ANNEXES

ANNEX 1 ndash Announcement of the PT on the IRMM webpage

ANNEX 2 ndash Announcement via e-mail and invitation

ANNEX 3 ndash Announcement of sample dispatch

ANNEX 4 ndash Technical specifications of the calibration solutions

ANNEX 5 ndash Homogeneity and stability of the test material

ANNEX 6 ndash Acknowledgement of sample receipt

ANNEX 7 ndash Questionnaire on details of analysis method

ANNEX 8 ndash Results reported for the food supplement sample

ANNEX 9 ndash Results reported for the standard solutions with undisclosed analyte content

ANNEX 10 ndash Method performance characteristics reported by participants

ANNEX 11 ndash Details of analysis methods reported by participants

32

ANNEX 1 ndash Announcement of the PT on the IRMM webpage

33

ANNEX 2 - Announcement via e-mail and invitation

34

ANNEX 3 - Announcement sample dispatch

Dear MadameSir

The test samples for the proficiency test on the determination of four PAHs in a food supplement (dry extract of St Johns wort) were

dispatched today

Explanation on the design of the study as well as instructions for sample storage and for reporting of results are in the parcel You will

also find your participant key in the parcel which is required for reporting of results

Please check the parcel after arrival for completeness and report the receipt of the test samples as well as their status to us by applying

the sample receipt form which is in the parcel

You are kindly requested to inform us by email if you do not receive the parcel by end of this week

My colleague Donata Lerda will then take care of it

With best regards

Thomas Wenzl

35

ANNEX 4 - Specifications of standard solutions with disclosed concentration

36

ANNEX 4 - continued

37

ANNEX 5 - Homogeneity and stability of the test material

Homogeneity of the test material

Benz[a]anthracene - BAA

Benzo[a]pyrene - BAP

38

ANNEX 5 - continued

Benzo[b]fluoranthene - BBF

Chrysene - CHR

39

ANNEX 5 - continued

Stability of the test material

Table 10 Content of the four target analytes in test materials stored under different

temperatures

BaA BaP BbF CHR

microgkg microgkg microgkg microgkg

Content 31 16 31 69 Storage at room temperature

U (k=2) 05 02 05 17

Content 31 15 29 68 Storage at reference temperature

U (k=2) 04 03 04 10

The applied capillary column does not separate CHR from triphenylene

40

ANNEX 6 ndash Documents sent to participants

41

ANNEX 6 - continued

42

ANNEX 7 Questionnaire on details of analysis method

43

ANNEX 7 - continued

44

ANNEX 7 - continued

45

ANNEX 8 - Results reported by participants for the food supplements test sample Table 11 Analysis results reported by the participants for the content of benz[a]anthracene (BAA) in the food supplements test material Values are presented as reported

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

3600 31 29 29 3 05 2

3602 199 193 178 19 021

3603 253 253 246 251 022 2

3604 199 209 23 213 071 2

3605 301 3 271 291 076 2

3606 302 288 278 289 064 2

3607 456 416 443 438 096 2

3608 796 695 651 714 149 2

3610 182 192 214 196 039 2

3611 414 504 488 469 96 2

3612 3 34 28 31 06 2

3613 32 33 34 33 07 2

3614 182 182 191 185 037 2

3615 3884 3822 3565 376 0752 2

3616 2 2 2 2 01 2

3617 366 37 35 362 046 2

3618 32 32 32 32 05 2

3619 299 305 297 299 048 2

3620 304 294 302 302 045 2

3621 29 26 29 28 04 2

3622 343 325 318 322 052 2

3623 35 3 33 33 07 2

3624

3626 318 31 314 314 126 2

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

5301 31 3 32 31 06 2

5302 314 319 315 316 063 2

5303 3102 2716 2822 2876 27 62

5304 261 263 26 261 57 2

5305 23 22 26 24 20

5306 22 23 23 22 11 2

5307 488 461 317 461

5308 168633 168544 1562025 1644598

5309 16 19 16 17

5310 3 304 311 305 015

5311 477 452 448 459 15 2

5312 506 63 494 543 134 2

5313 353 301 404 353

5314 431 373 378 394 5 096

5315 596 531 51 545 11 2

5316 224 231 235 235 022 2

5317 216 23 221 222 007 2

5318 064 07 067 01 2

5319 111 184 146 147 029 2

5320 515 525 564 534 48

5321 21 5322 13 87 9 87 35 2

5323 38 33 35 33 16 2

46

Figure 6 Results of replicate determinations (indicated by triangles) of benz[a]anthracene (BAA) in the food supplement test material

Horizontal blue lines represent the arithmetic mean value of replicate measurements and blue bars the reported expanded measurement uncertainty (k=2) The assigned value is

plotted as dotted line Red solid lines mark the range of satisfactory performance (|z|le2) Mean values are given for results outside the displayed data range

ProLab 2011

Laboratory

53

1853

1953

0853

0936

1436

0236

1036

1653

2136

0453

1753

0653

1653

0536

0353

0436

2153

0336

0636

0536

0036

2036

1953

1036

1253

0136

2653

0236

1836

2336

2236

1353

1353

2336

1736

1553

1453

0736

0753

1153

2053

1253

1536

0853

2236

11

an

aly

te c

on

ten

t (micro

gk

g)

10

95

9

85

8

75

7

65

6

55

5

45

4

35

3

25

2

15

1

05

0

102

3 468

7

Sample St Johns wort extract replicatesMeasurand Benz[a]anthraceneNo of laboratories 46

Assigned value 298 microgkg (Reference value)Target sd 062 microgkgTolerance limits 174 - 422 microgkg (|Z score| lt 200)

Assig

ned va

lue

Limit of tolerance

Limit of tolerance

47

Table 12 Analysis results reported by the participants for the content of benzo[a]pyrene (BAP) in the food supplements test material Values are presented as reported

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

3600 16 17 17 17 03 2

3602 118 123 13 124 015

3603 165 162 157 161 015 2

3604 134 154 139 143 04 2

3605 164 166 172 167 057 2

3606 181 159 161 167 031 2

3607 279 266 279 274 055 2

3608 57 687 461 573 226 2

3610 226 216 183 206 042 2

3611 289 325 311 308 36 2

3612 24 22 2 22 05 2

3613 17 18 19 18 05 2

3614 08 08 08 08 016 2

3615 1545 1393 1425 145 029 2

3616 16 16 14 15 03 2

3617 177 171 158 169 023 2

3618 2 2 2 2 02 2

3619 207 207 206 207 075 2

3620 165 169 163 166 017 2

3621 44 47 49 47 05 2

3622 212 208 222 213 038 2

3623 2 21 22 21 03 2

3624 18 178 182 18 031 2

3626 172 173 178 174 07 2

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

5301 18 18 18 18 04 2

5302 188 19 187 188 038 2

5303 1691 1426 149 1535 19 80

5304 145 142 15 146 69 2

5305 15 17 18 16 15

5306 16 17 17 16 08 2

5307 164 157 153 157

5308 102669 109324 109035 107009

5309 1 1 12 11

5310 122 122 124 123 006

5311 199 184 215 2 15 2

5312 258 248 239 248 05 2

5313 199 178 245 207

5314 174 171 172 172 11 0948

5315 253 248 213 238 05 2

5316 177 171 173 17 016 2

5317 255 277 276 269 012 2

5318 114 12 117 02 2

5319 19 228 231 216 043 2

5320 303 3 344 316 78

5321 12 12 12 12

5322 13 17 24 17 35 2

5323 2 2 19 2 1 2

48

Figure 7 Results of replicate determinations (indicated by triangles) of benzo[a]pyrene (BAP) in the food supplement test material

Horizontal blue lines represent the arithmetic mean value of replicate measurements and blue bars the reported expanded measurement uncertainty (k=2) The assigned value is

plotted as dotted line Red solid lines mark the range of satisfactory performance (|z|le2) Mean values are given for results outside the displayed data range

ProLab 2011

Laboratory

36

1453

0953

0853

1853

2153

1036

0236

0436

1553

0436

1653

0353

0736

0336

2036

0053

0553

0636

0636

0536

1753

1453

1636

2636

1336

2453

0153

2253

0253

2353

1136

1836

1953

1336

1036

2336

2253

1936

1253

1553

1253

1736

0753

2036

2136

0836

11

an

aly

te c

on

ten

t (micro

gk

g)

55

5

45

4

35

3

25

2

15

1

05

0

573

308

3

Sample St Johns wort extract replicatesMeasurand Benzo[a]pyreneNo of laboratories 48

Assigned value 165 microgkg (Reference value)Target sd 036 microgkgTolerance limits 093 - 237 microgkg (|Z score| lt 200)

Assig

ned va

lue

Limit of tolerance

Limit of tolerance

49

Table 13 Analysis results reported by the participants for the content of benzo[b]fluoranthene (BBF) in the food supplements test material Values are presented as reported

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

3600 32 3 3 31 09 2

3602 173 175 16 17 017

3603 265 252 25 256 025 2

3604 116 108 105 11 028 2

3605 286 225 232 247 074 2

3606 294 273 261 276 046 2

3607 579 579 544 567 113 2

3608 103 1201 876 1036 326 2

3610 173 196 204 191 038 2

3611 546 618 565 576 75 2

3612 3 28 28 29 06 2

3613 28 31 32 31 06 2

3614 135 111 135 127 022 2

3615 4187 3678 4111 4 08 2

3616 2 2 2 2 01 2

3617 33 27 299 035 2

3618 31 31 31 31 05 2

3619 326 324 312 326 064 2

3620 277 28 272 276 069 2

3621 26 25 23 25 04 2

3622 346 336 333 34 052 2

3623 26 28 29 28 02 2

3624

3626 288 28 307 292 117 2

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

5301 29 3 29 29 06 2

5302 297 3 296 298 06 2

5303 2705 2444 2608 2586 22 78

5304 245 254 26 253 85 2

5305 23 23 26 24 15

5306 42 43 4 42 21 2

5307 221 216 241 221

5308 232169 246654 240722 229848

5309 19 22 19 2 0

5310 3 315 325 313 016

5311 262 277 304 281 15 2

5312 27 289 337 299 075 2

5313 321 284 378 328

5314 423 404 385 404 34 083

5315 42 506 416 444 09 2

5316 265 267 277 273 018 2

5317 185 197 195 192 006 2

5318 276 281 279 04 2

5319 396 389 403 396 079 2

5320 623 618 635 625 14

5321 31

5322 24 33 49 33 35 2

5323 38 45 35 45 27 2

50

Figure 8 Results of replicate determinations (indicated by triangles) of benzo[b]fluoranthene (BBF) in the food supplement test material

Horizontal blue lines represent the arithmetic mean value of replicate measurements and blue bars the reported expanded measurement uncertainty (k=2) The assigned value is

plotted as dotted line Red solid lines mark the range of satisfactory performance (|z|le2) Mean values are given for results outside the displayed data range

ProLab 2011

Laboratory

36

0436

1436

0236

1053

1736

1653

0953

0753

0853

0536

2136

0553

0436

0353

0353

1636

0636

2036

2353

1853

1136

1236

2653

0153

0253

1236

1736

1336

0036

1853

2153

1036

1953

1336

2253

2253

2353

1936

1553

1453

0653

1536

0753

2036

0836

11

an

aly

te c

on

ten

t (micro

gk

g)

75

7

65

6

55

5

45

4

35

3

25

2

15

1

05

0

103

6

576

3

Sample St Johns wort extract replicatesMeasurand Benzo[b]fluorantheneNo of laboratories 46

Assigned value 292 microgkg (Reference value)Target sd 060 microgkgTolerance limits 172 - 412 microgkg (|Z score| lt 200)

Assig

ned va

lue

Limit of tolerance

Limit of tolerance

51

Table 14 Analysis results reported by the participants for the content of chrysene (CHR) in the food supplements test material Values are presented as reported

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

3600 5 49 5 5 07 2

3602 32 304 29 304 036

3603 462 469 447 459 048 2

3604 438 428 42 429 119 2

3605 365 41 381 386 085 2

3606 494 411 45 451 124 2

3607 897 774 787 819 18 2

3608 986 887 1168 1014 284 2

3610 418 392 462 424 085 2

3611 833 893 87 865 61 2

3612 6 64 58 61 12 2

3613 41 39 4 4 08 2

3614 272 254 288 271 054 2

3615 605 6028 5964 601 12 2

3616 47 45 49 47 05 2

3617 445 431 389 422 065 2

3618 45 45 45 45 06 2

3619 738 747 72 738 118 2

3620 463 472 446 46 092 2

3621 53 49 51 51 06 2

3622 478 513 505 488 08 2

3623 73 73 72 72 05 2

3624

3626 63 622 634 628 251 2

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

5301 49 49 49 49 1 2

5302 687 683 666 679 136 2

5303 6777 6181 6382 6441 24 70

5304 592 581 6 591 85 2

5305 6 58 64 61 20

5306 49 5 51 49 25 2

5307 524 5 574 524

5308 211488 216782 221146 213243

5309

5310 372 393 394 386 019

5311 43 459 422 437 15 2

5312 934 943 981 953 191 2

5313 793 73 99 838

5314 636 561 565 588 18 0818

5315 547 583 499 542 11 2

5316 292 286 285 282 06 2

5317 52 598 517 545 046 2

5318 368 345 357 05 2

5319 449 471 474 465 116 2

5320 76 924 818 834 99

5321 33

5322 7 7 76 7 35 2

5323 87 73 77 73 37 2

52

Figure 9 Results of replicate determinations (indicated by triangles) of chrysene (CHR) in the food supplement test material

Horizontal blue lines represent the arithmetic mean value of replicate measurements and blue bars the reported expanded measurement uncertainty (k=2) The assigned value is

plotted as dotted line Red solid lines mark the range of satisfactory performance (|z|le2) Mean values are given for results outside the displayed data range

ProLab 2011

Laboratory

53

0836

1453

1636

0253

2153

1836

0553

1036

1336

1736

1036

0453

1136

1836

0636

0336

2053

1936

1653

0136

0036

2253

0636

2153

0753

1553

1753

1453

0436

1536

1253

0536

2653

0353

0253

2236

2336

1953

2336

0753

2053

1353

1236

0836

11

an

aly

te c

on

ten

t (micro

gk

g)

13

12

11

10

9

8

7

6

5

4

3

2

1

0

865

3

Sample St Johns wort extract replicatesMeasurand ChryseneNo of laboratories 45

Assigned value 407 microgkg (Reference value)Target sd 083 microgkgTolerance limits 241 - 573 microgkg (|Z score| lt 200)

Assig

ned va

lue

Limit of tolerance

Limit of tolerance

53

Table 15 Analysis results reported by the participants for the sum of the four marker PAHs in the food supplements test material Values are presented as reported

Laboratory code

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg

3600 128 24 2

3602 79 126

3603 1127 096 2

3604 893 258 2

3605 1091 305 2

3606 1184 238 2

3607 2098 42 2

3608 3336 325 2

3610 1138 17 2

3611 2218 259 2

3612 143 29 2

3613 122 2 2

3614 663 129 2

3615 152 304 2

3616 102 03 2

3617 1252 169 2

3618 128 18 2

3619 157 305 2

3620 1204 12 2

3621 151 19 2

3622 1361 115 2

3623 154 09 2

3624

3626 1409 56 2

Laboratory code

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg

5301 126

5302 37 074 2

5303 13438 92

5304 1251 85 2

5305 125 20

5306 129 65 2

5307 1363

5308 7245598

5309

5310 1127 056

5311 1367 15 2

5312 2042 408 2

5313 1726

5314 1558

5315 177 35 2

5316 96 073 2

5317 1229 066 2

5318 82 05 2

5319 1225 267 2

5320 2309 41

5321

5322 207 35 2

5323 171 103 2

54

ANNEX 9 Results reported for the standard solutions with undisclosed analyte content Table 16 Analysis results reported by the participants for the four marker PAHs in the standard solution in toluene with undisclosed analyte

content Values are presented as reported U measurement uncertainty k coverage factor

benz[a]anthracene benzo[a]pyrene benzo[b]fluoranthene chrysene Laboratory

code Unit Result U k Result U k Result U k Result U k 3600 microgl 38 67 2 25 45 2 211 68 2 275 38 2

3605 microgl 367 184 2 2482 124 2 2592 13 2 235 118 2

3606 microgkg 3763 176 2 2609 142 2 2721 118 2 2539 134 2

3607 microgkg 3706 815 2 2824 565 2 2591 518 2 2587 569 2

3612 microgl 38 08 2 237 11 2 244 18 2 257 02 2

3614 microgl 324 65 2 209 38 2 215 37 2 21 42 2

3616 microgkg 318 07 2 224 14 2 231 26 2 216 1 2

3618 microgkg 417 17 2 284 08 2 292 06 2 297 03 2

3619 microgkg 47 742 2 274 468 2 228 387 2 274 434 2

3620 microgl 495 74 2 294 29 2 321 8 2 321 64 2

3621 microgl 241 22 2 143 13 2 188 16 2 145 14 2

3623 microgkg 476 25 2 311 53 2 269 3 2 297 03 2

3626 microgl 359 38 2 235 23 2 238 27 2 24 18 2

5301 microgkg 426 29 299 289

5302 microgkg 436 87 2 296 59 2 296 59 2 289 58 2

5303 microgl 3721 27 102 22838 19 99 23756 22 99 25486 24 98

5304 microgkg 38 29 2 225 26 2 257 37 2 258 52 2

5305 microgkg 305 225 245 29

5306 microgkg 316 63 2 247 49 2 65 13 2 22 44 2

5307 microgkg 3658 2559 241 2414

5308 microgkg 16854 109324 246654 216782

5311 microgl 374 20 2 279 15 2 268 15 2 272 15 2

5313 microgl 3408 2165 2275 2373

5314 microgkg 395 5 2875 11 3614 34 2905 18

5320 microgl 3267 368 1986 571 28 683 2427 512

5322 microgl 282 35 2 225 35 2 241 35 2 21 35 2

55

Figure 10 Youden plots of the percent deviation of the reported values from the assigned values for the food supplement test sample and the

standard solution in toluene with undisclosed content

(The identity of the respective measurand is given above the figure Results exceeding a difference of more than 100 were omitted)

56

Figure 10 - continued

57

Table 17 Analysis results reported by the participants for the four marker PAHs in the standard solution in acetonitrile with undisclosed

analyte content

Values are presented as reported U measurement uncertainty k coverage factor

benz[a]anthracene benzo[a]pyrene benzo[b]fluoranthene chrysene

Laboratory

code Unit Result U k Result U k Result U k Result U k

3602 microgkg 3675 247 247 244

3603 microgkg 358 086 2 236 028 2 235 028 2 236 057 2

3604 microgl 475 29 2 19 12 2 194 13 2 258 12 2

3608 microgl 3623 078 2 2385 023 2 2343 18 2 2296 092 2

3610 microgkg 369 185 2 25 125 2 251 126 2 244 122 2

3611 microgkg 48 02 2 323 02 2 328 04 2 317 03 2

3613 microgl 332 31 2 261 3 2 249 25 2 239 24 2

3615 microgl 415 2635 2729 2689

3617 microgkg 37 09 2 244 06 2 284 07 2 239 06 2

3622 microgl 37 12 2 25 23 2 257 21 2 238 1 2

3624 microgkg 402 104 2 255 084 2 265 096 2 253 07 2

5306 microgkg 563 113 2 371 74 2 359 72 2 333 67 2

5308 microgkg 168633 102669 232169 211488

5309 microgl 402 268 27

5310 microgl 315 158 25 125 25 125 288 144

5312 microgl 428 043 2 282 028 2 272 027 2 275 028 2

5315 microgkg 387 08 2 256 05 2 251 05 2 253 05 2

5316 microgl 3839 342 2 2485 191 2 2559 168 2 239 51 2

5317 microgl 2466 043 2 2212 034 2 2472 016 2 2146 068 2

5319 microgl 414 41 2 279 28 2 268 27 2 26 31 2

5321 microgkg 476 316 326 328

5322 microgl 2547 35 2

5323 microgkg 405 203 2 239 119 2 255 153 2 201 11 2

58

Figure 11 Youden plots of the percent deviation of the reported values from the assigned values for the food supplement test sample and the

standard solution in acetonitrile with undisclosed content

(The identity of the respective measurand is given above the figure Results exceeding a difference of more than 100 were omitted)

59

Figure 11 - continued

60

ANNEX 10 Table 18 Method performance parameters reported by the participants

Laboratory code

Analyte LOD LOQ Recovery Analyte LOD LOQ Recovery

microgkg microgkg microgkg microgkg 3600 BaA 03 06 978 BaP 03 07 9483602 BaA 007 021 926 BaP 005 015 993603 BaA 0011 05 987 BaP 0005 051 9553604 BaA 02 04 86 BaP 02 04 883605 BaA 0025 005 118 BaP 0025 005 1083606 BaA 001 003 65 BaP 001 003 703607 BaA 021 071 74 BaP 027 089 943608 BaA 016 048 8213 BaP 015 046 83683610 BaA 02 05 85 BaP 005 02 853611 BaA BaP 3612 BaA 1 117 BaP 1 1203613 BaA 01 03 90 BaP 01 03 903614 BaA 02 05 96 BaP 02 05 933615 BaA 005 015 110 BaP 005 015 1053616 BaA 01 05 108 BaP 01 05 753617 BaA 018 037 405 BaP 009 018 3873618 BaA 01 03 63 BaP 01 03 593619 BaA 005 005 59 BaP 033 033 543620 BaA 0009 0018 64 BaP 0005 001 553621 BaA 007 02 84 BaP 007 02 923622 BaA 01 03 1101 BaP 01 03 9853623 BaA 02 08 105 BaP 02 08 1013624 BaA BaP 01 033 8513625 BaA BaP 3626 BaA BaP 5301 BaA 003 01 102 BaP 003 01 1005302 BaA 01 03 78 BaP 01 03 865303 BaA 003 01 62 BaP 003 01 805304 BaA 1 1 70 BaP 1 1 955305 BaA 02 05 85 BaP 02 05 855306 BaA 03 1 699 BaP 03 1 905307 BaA 6537 BaP 81165308 BaA 0088 0265 99 BaP 00262 00785 1005309 BaA 01 08 92 BaP 01 08 925310 BaA 01 03 BaP 01 03 905311 BaA 002 005 80 BaP 002 004 915312 BaA 03 09 105 BaP 03 09 1055313 BaA 04 08 BaP 04 08 5314 BaA 001 002 96 BaP 001 002 9485315 BaA 01 03 50 - 100 BaP 01 03 50 - 100 5316 BaA 002 011 827 BaP 004 019 10445317 BaA 05 1 76 BaP 05 1 755318 BaA BaP 5319 BaA 009 029 951 BaP 011 041 995320 BaA 12 18 946 BaP 06 1 8345321 BaA 03 05 BaP 05 1 5322 BaA 1 2 100 BaP 1 2 1005323 BaA 26 BaP 26

61

ANNEX 10 Table 18 - continued

Laboratory code Analyte LOD LOQ Recovery Analyte LOD LOQ Recovery

microgkg microgkg microgkg microgkg 3600 BbF 03 05 96 CHR 02 03 10153602 BbF 015 045 947 CHR 003 009 9193603 BbF 0014 05 1012 CHR 0004 05 10033604 BbF 02 04 85 CHR 02 04 873605 BbF 005 01 94 CHR 0025 005 863606 BbF 001 003 70 CHR 001 003 653607 BbF 018 06 100 CHR 014 028 943608 BbF 015 046 7979 CHR 015 045 71253610 BbF 005 02 85 CHR 02 05 853611 BbF CHR 3612 BbF 1 118 CHR 1 1173613 BbF 01 03 90 CHR 01 03 903614 BbF 01 04 96 CHR 02 05 953615 BbF 008 025 109 CHR 003 01 1083616 BbF 01 05 78 CHR 01 05 723617 BbF 035 07 38 CHR 012 024 4163618 BbF 01 03 62 CHR 01 03 623619 BbF 016 016 63 CHR 01 01 593620 BbF 0003 0006 55 CHR 001 002 653621 BbF 007 02 103 CHR 007 02 893622 BbF 02 06 963 CHR 03 09 1043623 BbF 02 08 106 CHR 02 08 1193624 BbF CHR 3625 BbF CHR 3626 BbF CHR 5301 BbF 003 01 96 CHR 003 01 1145302 BbF 01 03 84 CHR 01 03 835303 BbF 003 01 78 CHR 003 01 705304 BbF 1 1 86 CHR 1 1 705305 BbF 02 05 90 CHR 02 05 955306 BbF 03 1 1435 CHR 03 1 1185307 BbF 8116 CHR 65375308 BbF 0079 0238 99 CHR 009 026 1005309 BbF 01 08 92 CHR 5310 BbF 07 2 CHR 01 03 5311 BbF 002 004 95 CHR 002 004 895312 BbF 03 09 105 CHR 03 09 1055313 BbF 04 08 CHR 04 08 5314 BbF 001 002 83 CHR 001 002 8185315 BbF 02 05 50 -100 CHR 02 05 50 - 100 5316 BbF 005 024 814 CHR 001 007 9595317 BbF 05 1 73 CHR 05 1 785318 BbF CHR 5319 BbF 011 038 946 CHR 013 043 9675320 BbF 07 09 1194 CHR 23 32 10455321 BbF 05 1 CHR 05 1 5322 BbF 1 2 100 CHR 1 2 1005323 BbF 26 CHR 26

62

ANNEX 11 Table 19 Details of analysis method reported by the participants Data are presented as reported

Laboratory code

Analysis technique

Instrument calibration

Internal standards applied

Sample clean up

Details of sample

clean up

Extraction technique

Details of sample

extraction

3600 6) GC-MS

2) Internal standardisation with labelled substances

Deuterated PAH mix 9 (Ehrenstorfer)

1) Saponification 2) LiquidLiquid partitioning

D) Other Liquidliquid partitioning

3602 2) HPLC-UV-FLD

1) External calibration

3) Gel permeation chromatography

A) Pressurised liquid extraction (PLE)

3603 1) HPLC-FLD

1) External calibration

3) Gel permeation chromatography

D) Other 1 minute with dichloromethane in vortex

3604 2) HPLC-UV-FLD

2) Internal standardisation with labelled substances

DiP D14

5) Donor acceptor complex chromatography

D) Other LL

3605 1) HPLC-FLD

1) External calibration

3) Gel permeation chromatography

C) Soxhlet extraction

3606 7) GC-MSMS

2) Internal standardisation with labelled substances

BaP 13C4 CHR 13C6 BaA 13C6 BbF 13C6

4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

3607 6) GC-MS

2) Internal standardisation with labelled substances

BaA-d12 CHR-d12 BbF-d12 BkF-d12 BaP-d12 ICP-d12 BgP-d12 DiP-d14

1) Saponification

D) Other solvent partitioning

3608 1) HPLC-FLD

1) External calibration

6) Other Liquid extraction

D) Other

Extraction in a rotary agitator and centrifugation

3610 2) HPLC-UV-FLD

1) External calibration

4) Solid phase extraction

B) Sonication

3611

3612 6) GC-MS

2) Internal standardisation with labelled substances

chrysene-d12 dibenzo(ah)anthracene-d14 dibenzo(ai)pyrene-d14

4) Solid phase extraction

B) Sonication

3613 5) LC-MSMS

3) Internal standardisation with unlabelled substances

I use Chrysene-D12 3) Gel permeation chromatography

D) Other extraction with hexan

63

Table 19 - continued

Laboratory code

Analysis technique

Instrument calibration

Internal standards applied

Sample clean up Details of sample

clean up

Extraction technique

Details of sample

extraction

3614 6) GC-MS 1) External calibration

3) Gel permeation chromatography

B) Sonication

3615 1) HPLC-FLD

1) External calibration

6) Other PLE GPC

A) Pressurised liquid extraction (PLE)

3616 6) GC-MS

2) Internal standardisation with labelled substances

Benzo(a)pyrene-13C4 Benzo(a)anthracene-13C6 Benzo(b)fluoranthene-13C6 Chrysene-13C6

2) LiquidLiquid partitioning 4) Solid phase extraction

B) Sonication

3617 1) HPLC-FLD

1) External calibration

4) Solid phase extraction

C) Soxhlet extraction

3618 6) GC-MS

2) Internal standardisation with labelled substances

deuterated standards

1) Saponification 4) Solid phase extraction

D) Other Handshaken with cyclohexane

3619 6) GC-MS

2) Internal standardisation with labelled substances

C13 Labelled US EPA 16

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

D) Other Homegenisation amp saponiofication

3620 9) GC-HRMS

2) Internal standardisation with labelled substances

mix of deuterated PAHs (BAA-D12 CHR-D12 BAP-D12 BBF-D12)

3) Gel permeation chromatography 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

3621 6) GC-MS

2) Internal standardisation with labelled substances

Chrysene D12 Benzo(a)pyrene D12 Benzo(b)fluoranthene D12 Benzo(a)anthracene

2) LiquidLiquid partitioning 4) Solid phase extraction

B) Sonication

3622 1) HPLC-FLD

4) Standard addition method

4) Solid phase extraction

D) Other

Extraction with cyclohexane by heating under reflux

3623 7) GC-MSMS

2) Internal standardisation with labelled substances

C13 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

3624 2) HPLC-UV-FLD

1) External calibration

2) LiquidLiquid partitioning

D) Other hot solvent extraction

3626

64

Table 19 - continued

Laboratory code

Analysis technique

Instrument calibration

Internal standards

applied

Sample clean up

Details of sample

clean up

Extraction technique

Details of sample

extraction

5301 7) GC-MSMS

2) Internal standardisation with labelled substances

EACH SUBSTANCE C13

4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5302 7) GC-MSMS

2) Internal standardisation with labelled substances

Isotope labelled PAHs 13C for each analyte

4) Solid phase extraction

C) Soxhlet extraction

5303 7) GC-MSMS

3) Internal standardisation with unlabelled substances

HAP C13 4) Solid phase extraction

D) Other automate ASE200 DIONEX

5304 7) GC-MSMS

2) Internal standardisation with labelled substances

with C13 for each HAP

2) LiquidLiquid partitioning 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5305 7) GC-MSMS

2) Internal standardisation with labelled substances

C13 isotopiques internal standards

4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5306 7) GC-MSMS

2) Internal standardisation with labelled substances

C13 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5307 6) GC-MS

2) Internal standardisation with labelled substances

1) Saponification 2) LiquidLiquid partitioning

D) Other solvent extraction

5308 1) HPLC-FLD 1) External calibration

4) Solid phase extraction

B) Sonication

5309 1) HPLC-FLD 1) External calibration

4) Solid phase extraction

D) Other only SPE

5310 1) HPLC-FLD 1) External calibration

1) Saponification 2) LiquidLiquid partitioning 3) Gel permeation chromatography

C) Soxhlet extraction

5311 6) GC-MS

2) Internal standardisation with labelled substances

13 C Chrysene 13 C Benz(a)pyrene

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

B) Sonication

5312 1) HPLC-FLD

3) Internal standardisation with unlabelled substances

Benzo(b)chrysen

2) LiquidLiquid partitioning 4) Solid phase extraction

Quechers D) Other Quechers

65

Table 19 - continued

Laboratory code

Analysis technique

Instrument calibration

Internal standards applied

Sample clean up Details of sample

clean up

Extraction technique

Details of sample extraction

5313 6) GC-MS

2) Internal standardisation with labelled substances

Benz(a)anthracene D12 Benzo(b)fluranthene D12 Benzo(a)pyrene D12 Chrysene D12

1) Saponification 2) LiquidLiquid partitioning 3) Gel permeation chromatography

A) Pressurised liquid extraction (PLE)

5314 6) GC-MS

2) Internal standardisation with labelled substances

Benzo(a)pyren d12 2) LiquidLiquid partitioning

D) Other Extraction with

5315 1) HPLC-FLD

3) Internal standardisation with unlabelled substances

Benz(b)chrysen

1) Saponification 2) LiquidLiquid partitioning

D) Other shaker

5316 1) HPLC-FLD

1) External calibration

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

D) Other

LiquidLiquid extraction with cyclohexane and dimethylformamide

5317 7) GC-MSMS

1) External calibration

3) Gel permeation chromatography 6) Other

silica gel D) Other VDLUFA online method

5318

5319 1) HPLC-FLD

3) Internal standardisation with unlabelled substances

Benzo(b)chrysene 3) Gel permeation chromatography

A) Pressurised liquid extraction (PLE)

5320 6) GC-MS

3) Internal standardisation with unlabelled substances

22-binaphthyl

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

D) Other 3 hours refluxing

5321 1) HPLC-FLD

2) Internal standardisation with labelled substances

6-meacutethylchrysegravene

1) Saponification 2) LiquidLiquid partitioning

B) Sonication

5322 6) GC-MS

2) Internal standardisation with labelled substances

naftalene-d8 acenaphtalene-d10 phenanthrene-d10 chrysene-d12 perylene-d12 benzo(e)anthracene-d12

1) Saponification 2) LiquidLiquid partitioning 6) Other

clean-up with silica packed column

C) Soxhlet extraction

5323 1) HPLC-FLD

1) External calibration

4) Solid phase extraction 6) Other

Accelerated Solvent Extraction

A) Pressurised liquid extraction (PLE)

66

European Commission EUR 25245 EN ndash Joint Research Centre ndash Institute for Reference Materials and Measurements Title Report on the 8th inter-laboratory comparison organised by the European Union Reference Laboratory for Polycyclic Aromatic Hydrocarbons - Four marker PAHs in a dry extract of St Johns wort Author(s) Radoslav Lizak Szilard Szilagyi Philippe Verlinde and Thomas WENZL Luxembourg Publications Office of the European Union 2012 ndash 66 pp ndash 201 x 297 cm EUR ndash Scientific and Technical Research series ndash ISSN 1831-9424 ISBN 978-92-79-23337-1 doi 10278759387 Abstract The proficiency test here reported concerned the determination of the contents of four marker polycyclic aromatic hydrocarbons (PAHs) and of their sum in an food supplements test sample The set of marker PAHs consists of benz[a]anthacene benzo[a]pyrene benzo[b]fluoranthene and chrysene Participants to these PT were National Reference Laboratories for PAHs (NRLs-PAHs) and EU official food control laboratories The number of participants was in total 48

The PT was organised according ISO Standard 170432010

The test material used was a commercial dry extract of St Johns wort Participants also received a solution of the same PAHs either in acetonitrile or in toluene for checking their instrument calibration

The results from participants were rated with z-scores and zeta-scores About 68 of the reported results were attributed with z-scores with an absolute value of below two which is the threshold for satisfactory performance

How to obtain EU publications

Our priced publications are available from EU Bookshop (httpbookshopeuropaeu) where you can place

an order with the sales agent of your choice

The Publications Office has a worldwide network of sales agents You can obtain their contact details by

sending a fax to (352) 29 29-42758

The mission of the JRC is to provide customer-driven scientific and technical support

for the conception development implementation and monitoring of EU policies As a

service of the European Commission the JRC functions as a reference centre of

science and technology for the Union Close to the policy-making process it serves

the common interest of the Member States while being independent of special

interests whether private or national

L

A-N

A-252

45-EN

-N

• 1 Summary
• 2 Introduction
• 3 Scope
• 4 Participating Laboratories
• 5 Time frame
• 6 Confidentiality
• 7 Test materials
• • 71 1Preparation of test materials
  • 72 Assigned values and standard deviation for proficiency assessment
  • 73 Homogeneity and stability
  • 74 Sample dispatch
  • • 8 Design of the proficiency test
    • 9 Evaluation of the results
    • • 91 2General
      • 92 21BEvaluation criteria
      • 93 Performance of participants
      • 94 Evaluation of the influence of analysis method
      • 95 Evaluation of the influence of calibration
      • 96 26Evaluation of compliance with legislation
      • • 10 Follow-up actions for underperforming laboratories
        • 11 1Conclusions
        • 12 1Acknowledgements
        • 13 References

29

13 References

1 EU COMMISSION REGULATION (EC) No 7762006 of 23 May 2006 amending Annex VII to Regulation

(EC) No 8822004 of the European Parliament and of the Council as regards Community reference

laboratories Official Journal of the European Union 2006 L 136 p 3-8

Available from

httpeur-lexeuropaeuLexUriServLexUriServdouri=OJL200613600030008ENPDF

2 EU Regulation (EC) No 8822004 of the European Parliament and of the Council of 29 April 2004 on

official controls performed to ensure the verification of compliance with feed and food law animal health

and animal welfare rules Official Journal of the European Communities 2004 L191 p 1-52 Available

from

httpeur-lexeuropaeuLexUriServLexUriServdouri=OJL200419100010052ENPDF

3 EU Opinion of the Scientific Committee on Food on the risks to human health of Polycyclic Aromatic

Hydrocarbons in food (2002) Available from

httpeuropaeuintcommfoodfsscscfout153_enpdf

4 International Agency for Research on Cancer IARC Monographs on the Evaluation of

Carcinogenic Risks to Humans Some Non-heterocyclic Polycyclic Aromatic Hydrocarbons and Some

Related Exposures Volume 92 (2010) Available from

httpmonographsiarcfrENGMonographsvol92mono92pdf

5 EU Commission Regulation (EC) No 3332007 of 28 March 2007 laying down the methods of sampling

and analysis for the official control of the levels of lead cadmium mercury inorganic tin 3-MCPD and

benzo(a)pyrene in foodstuffs Official Journal of the European Union 2007 L 88 p 29-38 Available

from

httpeur-lexeuropaeuLexUriServLexUriServdouri=OJL200708800290038ENPDF

6 EU Commission Regulation (EC) No 18812006 of 19 December 2006 setting maximum levels for certain

contaminants in foodstuffs Official Journal of the European Union 2006 L 364 p 5-24 Available from

httpeur-lexeuropaeuLexUriServLexUriServdouri=OJL200636400050024ENPDF

7 EFSA Scientific Opinion of the Panel on Contaminants in the Food Chain on a request from the European

Commission on Polycyclic Aromatic Hydrocarbons in Food The EFSA Journal 2008 724 p 1-114

Available from

httpwwwefsaeuropaeuEFSAefsa_locale-1178620753812_1211902034842htm

30

8 EU Commission Regulation (EU) No 8352011 amending Regulation (EC) No 18812006 as regards

maximum levels for polycyclic aromatic hydrocarbons in foodstuffs Official Journal of the European

Union 2011 L 215 p 4-8

httpeur-lexeuropaeuLexUriServLexUriServdouri=OJL201121500040008ENPDF

9 EU Commission Regulation (EU) No 8362011 amending Regulation (EC) No 3332007 laying down the methods of sampling and analysis for the official control of the levels of lead cadmium mercury inorganic tin 3-MCPD and benzo(a)pyrene in foodstuffs Official Journal of the European Union 2011 L 215 p 9-16 httpeur-lexeuropaeuLexUriServLexUriServdouri=OJL201121500090016ENPDF

10 ISOIEC 170432010 (2010) Conformity assessment -- General requirements for proficiency testing

providers Geneva Switzerland

11 Thompson M Ellison SLR and Wood R The International Harmonized Protocol for the Proficiency

Testing of Analytical Chemistry Laboratories Pure Appl Chem (2006) 78(1) p 145ndash196

Available from httpmediaiupacorgpublicationspac2006pdf7801x0145pdf

31

14 ANNEXES

ANNEX 1 ndash Announcement of the PT on the IRMM webpage

ANNEX 2 ndash Announcement via e-mail and invitation

ANNEX 3 ndash Announcement of sample dispatch

ANNEX 4 ndash Technical specifications of the calibration solutions

ANNEX 5 ndash Homogeneity and stability of the test material

ANNEX 6 ndash Acknowledgement of sample receipt

ANNEX 7 ndash Questionnaire on details of analysis method

ANNEX 8 ndash Results reported for the food supplement sample

ANNEX 9 ndash Results reported for the standard solutions with undisclosed analyte content

ANNEX 10 ndash Method performance characteristics reported by participants

ANNEX 11 ndash Details of analysis methods reported by participants

32

ANNEX 1 ndash Announcement of the PT on the IRMM webpage

33

ANNEX 2 - Announcement via e-mail and invitation

34

ANNEX 3 - Announcement sample dispatch

Dear MadameSir

The test samples for the proficiency test on the determination of four PAHs in a food supplement (dry extract of St Johns wort) were

dispatched today

Explanation on the design of the study as well as instructions for sample storage and for reporting of results are in the parcel You will

also find your participant key in the parcel which is required for reporting of results

Please check the parcel after arrival for completeness and report the receipt of the test samples as well as their status to us by applying

the sample receipt form which is in the parcel

You are kindly requested to inform us by email if you do not receive the parcel by end of this week

My colleague Donata Lerda will then take care of it

With best regards

Thomas Wenzl

35

ANNEX 4 - Specifications of standard solutions with disclosed concentration

36

ANNEX 4 - continued

37

ANNEX 5 - Homogeneity and stability of the test material

Homogeneity of the test material

Benz[a]anthracene - BAA

Benzo[a]pyrene - BAP

38

ANNEX 5 - continued

Benzo[b]fluoranthene - BBF

Chrysene - CHR

39

ANNEX 5 - continued

Stability of the test material

Table 10 Content of the four target analytes in test materials stored under different

temperatures

BaA BaP BbF CHR

microgkg microgkg microgkg microgkg

Content 31 16 31 69 Storage at room temperature

U (k=2) 05 02 05 17

Content 31 15 29 68 Storage at reference temperature

U (k=2) 04 03 04 10

The applied capillary column does not separate CHR from triphenylene

40

ANNEX 6 ndash Documents sent to participants

41

ANNEX 6 - continued

42

ANNEX 7 Questionnaire on details of analysis method

43

ANNEX 7 - continued

44

ANNEX 7 - continued

45

ANNEX 8 - Results reported by participants for the food supplements test sample Table 11 Analysis results reported by the participants for the content of benz[a]anthracene (BAA) in the food supplements test material Values are presented as reported

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

3600 31 29 29 3 05 2

3602 199 193 178 19 021

3603 253 253 246 251 022 2

3604 199 209 23 213 071 2

3605 301 3 271 291 076 2

3606 302 288 278 289 064 2

3607 456 416 443 438 096 2

3608 796 695 651 714 149 2

3610 182 192 214 196 039 2

3611 414 504 488 469 96 2

3612 3 34 28 31 06 2

3613 32 33 34 33 07 2

3614 182 182 191 185 037 2

3615 3884 3822 3565 376 0752 2

3616 2 2 2 2 01 2

3617 366 37 35 362 046 2

3618 32 32 32 32 05 2

3619 299 305 297 299 048 2

3620 304 294 302 302 045 2

3621 29 26 29 28 04 2

3622 343 325 318 322 052 2

3623 35 3 33 33 07 2

3624

3626 318 31 314 314 126 2

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

5301 31 3 32 31 06 2

5302 314 319 315 316 063 2

5303 3102 2716 2822 2876 27 62

5304 261 263 26 261 57 2

5305 23 22 26 24 20

5306 22 23 23 22 11 2

5307 488 461 317 461

5308 168633 168544 1562025 1644598

5309 16 19 16 17

5310 3 304 311 305 015

5311 477 452 448 459 15 2

5312 506 63 494 543 134 2

5313 353 301 404 353

5314 431 373 378 394 5 096

5315 596 531 51 545 11 2

5316 224 231 235 235 022 2

5317 216 23 221 222 007 2

5318 064 07 067 01 2

5319 111 184 146 147 029 2

5320 515 525 564 534 48

5321 21 5322 13 87 9 87 35 2

5323 38 33 35 33 16 2

46

Figure 6 Results of replicate determinations (indicated by triangles) of benz[a]anthracene (BAA) in the food supplement test material

Horizontal blue lines represent the arithmetic mean value of replicate measurements and blue bars the reported expanded measurement uncertainty (k=2) The assigned value is

plotted as dotted line Red solid lines mark the range of satisfactory performance (|z|le2) Mean values are given for results outside the displayed data range

ProLab 2011

Laboratory

53

1853

1953

0853

0936

1436

0236

1036

1653

2136

0453

1753

0653

1653

0536

0353

0436

2153

0336

0636

0536

0036

2036

1953

1036

1253

0136

2653

0236

1836

2336

2236

1353

1353

2336

1736

1553

1453

0736

0753

1153

2053

1253

1536

0853

2236

11

an

aly

te c

on

ten

t (micro

gk

g)

10

95

9

85

8

75

7

65

6

55

5

45

4

35

3

25

2

15

1

05

0

102

3 468

7

Sample St Johns wort extract replicatesMeasurand Benz[a]anthraceneNo of laboratories 46

Assigned value 298 microgkg (Reference value)Target sd 062 microgkgTolerance limits 174 - 422 microgkg (|Z score| lt 200)

Assig

ned va

lue

Limit of tolerance

Limit of tolerance

47

Table 12 Analysis results reported by the participants for the content of benzo[a]pyrene (BAP) in the food supplements test material Values are presented as reported

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

3600 16 17 17 17 03 2

3602 118 123 13 124 015

3603 165 162 157 161 015 2

3604 134 154 139 143 04 2

3605 164 166 172 167 057 2

3606 181 159 161 167 031 2

3607 279 266 279 274 055 2

3608 57 687 461 573 226 2

3610 226 216 183 206 042 2

3611 289 325 311 308 36 2

3612 24 22 2 22 05 2

3613 17 18 19 18 05 2

3614 08 08 08 08 016 2

3615 1545 1393 1425 145 029 2

3616 16 16 14 15 03 2

3617 177 171 158 169 023 2

3618 2 2 2 2 02 2

3619 207 207 206 207 075 2

3620 165 169 163 166 017 2

3621 44 47 49 47 05 2

3622 212 208 222 213 038 2

3623 2 21 22 21 03 2

3624 18 178 182 18 031 2

3626 172 173 178 174 07 2

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

5301 18 18 18 18 04 2

5302 188 19 187 188 038 2

5303 1691 1426 149 1535 19 80

5304 145 142 15 146 69 2

5305 15 17 18 16 15

5306 16 17 17 16 08 2

5307 164 157 153 157

5308 102669 109324 109035 107009

5309 1 1 12 11

5310 122 122 124 123 006

5311 199 184 215 2 15 2

5312 258 248 239 248 05 2

5313 199 178 245 207

5314 174 171 172 172 11 0948

5315 253 248 213 238 05 2

5316 177 171 173 17 016 2

5317 255 277 276 269 012 2

5318 114 12 117 02 2

5319 19 228 231 216 043 2

5320 303 3 344 316 78

5321 12 12 12 12

5322 13 17 24 17 35 2

5323 2 2 19 2 1 2

48

Figure 7 Results of replicate determinations (indicated by triangles) of benzo[a]pyrene (BAP) in the food supplement test material

Horizontal blue lines represent the arithmetic mean value of replicate measurements and blue bars the reported expanded measurement uncertainty (k=2) The assigned value is

plotted as dotted line Red solid lines mark the range of satisfactory performance (|z|le2) Mean values are given for results outside the displayed data range

ProLab 2011

Laboratory

36

1453

0953

0853

1853

2153

1036

0236

0436

1553

0436

1653

0353

0736

0336

2036

0053

0553

0636

0636

0536

1753

1453

1636

2636

1336

2453

0153

2253

0253

2353

1136

1836

1953

1336

1036

2336

2253

1936

1253

1553

1253

1736

0753

2036

2136

0836

11

an

aly

te c

on

ten

t (micro

gk

g)

55

5

45

4

35

3

25

2

15

1

05

0

573

308

3

Sample St Johns wort extract replicatesMeasurand Benzo[a]pyreneNo of laboratories 48

Assigned value 165 microgkg (Reference value)Target sd 036 microgkgTolerance limits 093 - 237 microgkg (|Z score| lt 200)

Assig

ned va

lue

Limit of tolerance

Limit of tolerance

49

Table 13 Analysis results reported by the participants for the content of benzo[b]fluoranthene (BBF) in the food supplements test material Values are presented as reported

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

3600 32 3 3 31 09 2

3602 173 175 16 17 017

3603 265 252 25 256 025 2

3604 116 108 105 11 028 2

3605 286 225 232 247 074 2

3606 294 273 261 276 046 2

3607 579 579 544 567 113 2

3608 103 1201 876 1036 326 2

3610 173 196 204 191 038 2

3611 546 618 565 576 75 2

3612 3 28 28 29 06 2

3613 28 31 32 31 06 2

3614 135 111 135 127 022 2

3615 4187 3678 4111 4 08 2

3616 2 2 2 2 01 2

3617 33 27 299 035 2

3618 31 31 31 31 05 2

3619 326 324 312 326 064 2

3620 277 28 272 276 069 2

3621 26 25 23 25 04 2

3622 346 336 333 34 052 2

3623 26 28 29 28 02 2

3624

3626 288 28 307 292 117 2

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

5301 29 3 29 29 06 2

5302 297 3 296 298 06 2

5303 2705 2444 2608 2586 22 78

5304 245 254 26 253 85 2

5305 23 23 26 24 15

5306 42 43 4 42 21 2

5307 221 216 241 221

5308 232169 246654 240722 229848

5309 19 22 19 2 0

5310 3 315 325 313 016

5311 262 277 304 281 15 2

5312 27 289 337 299 075 2

5313 321 284 378 328

5314 423 404 385 404 34 083

5315 42 506 416 444 09 2

5316 265 267 277 273 018 2

5317 185 197 195 192 006 2

5318 276 281 279 04 2

5319 396 389 403 396 079 2

5320 623 618 635 625 14

5321 31

5322 24 33 49 33 35 2

5323 38 45 35 45 27 2

50

Figure 8 Results of replicate determinations (indicated by triangles) of benzo[b]fluoranthene (BBF) in the food supplement test material

Horizontal blue lines represent the arithmetic mean value of replicate measurements and blue bars the reported expanded measurement uncertainty (k=2) The assigned value is

plotted as dotted line Red solid lines mark the range of satisfactory performance (|z|le2) Mean values are given for results outside the displayed data range

ProLab 2011

Laboratory

36

0436

1436

0236

1053

1736

1653

0953

0753

0853

0536

2136

0553

0436

0353

0353

1636

0636

2036

2353

1853

1136

1236

2653

0153

0253

1236

1736

1336

0036

1853

2153

1036

1953

1336

2253

2253

2353

1936

1553

1453

0653

1536

0753

2036

0836

11

an

aly

te c

on

ten

t (micro

gk

g)

75

7

65

6

55

5

45

4

35

3

25

2

15

1

05

0

103

6

576

3

Sample St Johns wort extract replicatesMeasurand Benzo[b]fluorantheneNo of laboratories 46

Assigned value 292 microgkg (Reference value)Target sd 060 microgkgTolerance limits 172 - 412 microgkg (|Z score| lt 200)

Assig

ned va

lue

Limit of tolerance

Limit of tolerance

51

Table 14 Analysis results reported by the participants for the content of chrysene (CHR) in the food supplements test material Values are presented as reported

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

3600 5 49 5 5 07 2

3602 32 304 29 304 036

3603 462 469 447 459 048 2

3604 438 428 42 429 119 2

3605 365 41 381 386 085 2

3606 494 411 45 451 124 2

3607 897 774 787 819 18 2

3608 986 887 1168 1014 284 2

3610 418 392 462 424 085 2

3611 833 893 87 865 61 2

3612 6 64 58 61 12 2

3613 41 39 4 4 08 2

3614 272 254 288 271 054 2

3615 605 6028 5964 601 12 2

3616 47 45 49 47 05 2

3617 445 431 389 422 065 2

3618 45 45 45 45 06 2

3619 738 747 72 738 118 2

3620 463 472 446 46 092 2

3621 53 49 51 51 06 2

3622 478 513 505 488 08 2

3623 73 73 72 72 05 2

3624

3626 63 622 634 628 251 2

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

5301 49 49 49 49 1 2

5302 687 683 666 679 136 2

5303 6777 6181 6382 6441 24 70

5304 592 581 6 591 85 2

5305 6 58 64 61 20

5306 49 5 51 49 25 2

5307 524 5 574 524

5308 211488 216782 221146 213243

5309

5310 372 393 394 386 019

5311 43 459 422 437 15 2

5312 934 943 981 953 191 2

5313 793 73 99 838

5314 636 561 565 588 18 0818

5315 547 583 499 542 11 2

5316 292 286 285 282 06 2

5317 52 598 517 545 046 2

5318 368 345 357 05 2

5319 449 471 474 465 116 2

5320 76 924 818 834 99

5321 33

5322 7 7 76 7 35 2

5323 87 73 77 73 37 2

52

Figure 9 Results of replicate determinations (indicated by triangles) of chrysene (CHR) in the food supplement test material

Horizontal blue lines represent the arithmetic mean value of replicate measurements and blue bars the reported expanded measurement uncertainty (k=2) The assigned value is

plotted as dotted line Red solid lines mark the range of satisfactory performance (|z|le2) Mean values are given for results outside the displayed data range

ProLab 2011

Laboratory

53

0836

1453

1636

0253

2153

1836

0553

1036

1336

1736

1036

0453

1136

1836

0636

0336

2053

1936

1653

0136

0036

2253

0636

2153

0753

1553

1753

1453

0436

1536

1253

0536

2653

0353

0253

2236

2336

1953

2336

0753

2053

1353

1236

0836

11

an

aly

te c

on

ten

t (micro

gk

g)

13

12

11

10

9

8

7

6

5

4

3

2

1

0

865

3

Sample St Johns wort extract replicatesMeasurand ChryseneNo of laboratories 45

Assigned value 407 microgkg (Reference value)Target sd 083 microgkgTolerance limits 241 - 573 microgkg (|Z score| lt 200)

Assig

ned va

lue

Limit of tolerance

Limit of tolerance

53

Table 15 Analysis results reported by the participants for the sum of the four marker PAHs in the food supplements test material Values are presented as reported

Laboratory code

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg

3600 128 24 2

3602 79 126

3603 1127 096 2

3604 893 258 2

3605 1091 305 2

3606 1184 238 2

3607 2098 42 2

3608 3336 325 2

3610 1138 17 2

3611 2218 259 2

3612 143 29 2

3613 122 2 2

3614 663 129 2

3615 152 304 2

3616 102 03 2

3617 1252 169 2

3618 128 18 2

3619 157 305 2

3620 1204 12 2

3621 151 19 2

3622 1361 115 2

3623 154 09 2

3624

3626 1409 56 2

Laboratory code

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg

5301 126

5302 37 074 2

5303 13438 92

5304 1251 85 2

5305 125 20

5306 129 65 2

5307 1363

5308 7245598

5309

5310 1127 056

5311 1367 15 2

5312 2042 408 2

5313 1726

5314 1558

5315 177 35 2

5316 96 073 2

5317 1229 066 2

5318 82 05 2

5319 1225 267 2

5320 2309 41

5321

5322 207 35 2

5323 171 103 2

54

ANNEX 9 Results reported for the standard solutions with undisclosed analyte content Table 16 Analysis results reported by the participants for the four marker PAHs in the standard solution in toluene with undisclosed analyte

content Values are presented as reported U measurement uncertainty k coverage factor

benz[a]anthracene benzo[a]pyrene benzo[b]fluoranthene chrysene Laboratory

code Unit Result U k Result U k Result U k Result U k 3600 microgl 38 67 2 25 45 2 211 68 2 275 38 2

3605 microgl 367 184 2 2482 124 2 2592 13 2 235 118 2

3606 microgkg 3763 176 2 2609 142 2 2721 118 2 2539 134 2

3607 microgkg 3706 815 2 2824 565 2 2591 518 2 2587 569 2

3612 microgl 38 08 2 237 11 2 244 18 2 257 02 2

3614 microgl 324 65 2 209 38 2 215 37 2 21 42 2

3616 microgkg 318 07 2 224 14 2 231 26 2 216 1 2

3618 microgkg 417 17 2 284 08 2 292 06 2 297 03 2

3619 microgkg 47 742 2 274 468 2 228 387 2 274 434 2

3620 microgl 495 74 2 294 29 2 321 8 2 321 64 2

3621 microgl 241 22 2 143 13 2 188 16 2 145 14 2

3623 microgkg 476 25 2 311 53 2 269 3 2 297 03 2

3626 microgl 359 38 2 235 23 2 238 27 2 24 18 2

5301 microgkg 426 29 299 289

5302 microgkg 436 87 2 296 59 2 296 59 2 289 58 2

5303 microgl 3721 27 102 22838 19 99 23756 22 99 25486 24 98

5304 microgkg 38 29 2 225 26 2 257 37 2 258 52 2

5305 microgkg 305 225 245 29

5306 microgkg 316 63 2 247 49 2 65 13 2 22 44 2

5307 microgkg 3658 2559 241 2414

5308 microgkg 16854 109324 246654 216782

5311 microgl 374 20 2 279 15 2 268 15 2 272 15 2

5313 microgl 3408 2165 2275 2373

5314 microgkg 395 5 2875 11 3614 34 2905 18

5320 microgl 3267 368 1986 571 28 683 2427 512

5322 microgl 282 35 2 225 35 2 241 35 2 21 35 2

55

Figure 10 Youden plots of the percent deviation of the reported values from the assigned values for the food supplement test sample and the

standard solution in toluene with undisclosed content

(The identity of the respective measurand is given above the figure Results exceeding a difference of more than 100 were omitted)

56

Figure 10 - continued

57

Table 17 Analysis results reported by the participants for the four marker PAHs in the standard solution in acetonitrile with undisclosed

analyte content

Values are presented as reported U measurement uncertainty k coverage factor

benz[a]anthracene benzo[a]pyrene benzo[b]fluoranthene chrysene

Laboratory

code Unit Result U k Result U k Result U k Result U k

3602 microgkg 3675 247 247 244

3603 microgkg 358 086 2 236 028 2 235 028 2 236 057 2

3604 microgl 475 29 2 19 12 2 194 13 2 258 12 2

3608 microgl 3623 078 2 2385 023 2 2343 18 2 2296 092 2

3610 microgkg 369 185 2 25 125 2 251 126 2 244 122 2

3611 microgkg 48 02 2 323 02 2 328 04 2 317 03 2

3613 microgl 332 31 2 261 3 2 249 25 2 239 24 2

3615 microgl 415 2635 2729 2689

3617 microgkg 37 09 2 244 06 2 284 07 2 239 06 2

3622 microgl 37 12 2 25 23 2 257 21 2 238 1 2

3624 microgkg 402 104 2 255 084 2 265 096 2 253 07 2

5306 microgkg 563 113 2 371 74 2 359 72 2 333 67 2

5308 microgkg 168633 102669 232169 211488

5309 microgl 402 268 27

5310 microgl 315 158 25 125 25 125 288 144

5312 microgl 428 043 2 282 028 2 272 027 2 275 028 2

5315 microgkg 387 08 2 256 05 2 251 05 2 253 05 2

5316 microgl 3839 342 2 2485 191 2 2559 168 2 239 51 2

5317 microgl 2466 043 2 2212 034 2 2472 016 2 2146 068 2

5319 microgl 414 41 2 279 28 2 268 27 2 26 31 2

5321 microgkg 476 316 326 328

5322 microgl 2547 35 2

5323 microgkg 405 203 2 239 119 2 255 153 2 201 11 2

58

Figure 11 Youden plots of the percent deviation of the reported values from the assigned values for the food supplement test sample and the

standard solution in acetonitrile with undisclosed content

(The identity of the respective measurand is given above the figure Results exceeding a difference of more than 100 were omitted)

59

Figure 11 - continued

60

ANNEX 10 Table 18 Method performance parameters reported by the participants

Laboratory code

Analyte LOD LOQ Recovery Analyte LOD LOQ Recovery

microgkg microgkg microgkg microgkg 3600 BaA 03 06 978 BaP 03 07 9483602 BaA 007 021 926 BaP 005 015 993603 BaA 0011 05 987 BaP 0005 051 9553604 BaA 02 04 86 BaP 02 04 883605 BaA 0025 005 118 BaP 0025 005 1083606 BaA 001 003 65 BaP 001 003 703607 BaA 021 071 74 BaP 027 089 943608 BaA 016 048 8213 BaP 015 046 83683610 BaA 02 05 85 BaP 005 02 853611 BaA BaP 3612 BaA 1 117 BaP 1 1203613 BaA 01 03 90 BaP 01 03 903614 BaA 02 05 96 BaP 02 05 933615 BaA 005 015 110 BaP 005 015 1053616 BaA 01 05 108 BaP 01 05 753617 BaA 018 037 405 BaP 009 018 3873618 BaA 01 03 63 BaP 01 03 593619 BaA 005 005 59 BaP 033 033 543620 BaA 0009 0018 64 BaP 0005 001 553621 BaA 007 02 84 BaP 007 02 923622 BaA 01 03 1101 BaP 01 03 9853623 BaA 02 08 105 BaP 02 08 1013624 BaA BaP 01 033 8513625 BaA BaP 3626 BaA BaP 5301 BaA 003 01 102 BaP 003 01 1005302 BaA 01 03 78 BaP 01 03 865303 BaA 003 01 62 BaP 003 01 805304 BaA 1 1 70 BaP 1 1 955305 BaA 02 05 85 BaP 02 05 855306 BaA 03 1 699 BaP 03 1 905307 BaA 6537 BaP 81165308 BaA 0088 0265 99 BaP 00262 00785 1005309 BaA 01 08 92 BaP 01 08 925310 BaA 01 03 BaP 01 03 905311 BaA 002 005 80 BaP 002 004 915312 BaA 03 09 105 BaP 03 09 1055313 BaA 04 08 BaP 04 08 5314 BaA 001 002 96 BaP 001 002 9485315 BaA 01 03 50 - 100 BaP 01 03 50 - 100 5316 BaA 002 011 827 BaP 004 019 10445317 BaA 05 1 76 BaP 05 1 755318 BaA BaP 5319 BaA 009 029 951 BaP 011 041 995320 BaA 12 18 946 BaP 06 1 8345321 BaA 03 05 BaP 05 1 5322 BaA 1 2 100 BaP 1 2 1005323 BaA 26 BaP 26

61

ANNEX 10 Table 18 - continued

Laboratory code Analyte LOD LOQ Recovery Analyte LOD LOQ Recovery

microgkg microgkg microgkg microgkg 3600 BbF 03 05 96 CHR 02 03 10153602 BbF 015 045 947 CHR 003 009 9193603 BbF 0014 05 1012 CHR 0004 05 10033604 BbF 02 04 85 CHR 02 04 873605 BbF 005 01 94 CHR 0025 005 863606 BbF 001 003 70 CHR 001 003 653607 BbF 018 06 100 CHR 014 028 943608 BbF 015 046 7979 CHR 015 045 71253610 BbF 005 02 85 CHR 02 05 853611 BbF CHR 3612 BbF 1 118 CHR 1 1173613 BbF 01 03 90 CHR 01 03 903614 BbF 01 04 96 CHR 02 05 953615 BbF 008 025 109 CHR 003 01 1083616 BbF 01 05 78 CHR 01 05 723617 BbF 035 07 38 CHR 012 024 4163618 BbF 01 03 62 CHR 01 03 623619 BbF 016 016 63 CHR 01 01 593620 BbF 0003 0006 55 CHR 001 002 653621 BbF 007 02 103 CHR 007 02 893622 BbF 02 06 963 CHR 03 09 1043623 BbF 02 08 106 CHR 02 08 1193624 BbF CHR 3625 BbF CHR 3626 BbF CHR 5301 BbF 003 01 96 CHR 003 01 1145302 BbF 01 03 84 CHR 01 03 835303 BbF 003 01 78 CHR 003 01 705304 BbF 1 1 86 CHR 1 1 705305 BbF 02 05 90 CHR 02 05 955306 BbF 03 1 1435 CHR 03 1 1185307 BbF 8116 CHR 65375308 BbF 0079 0238 99 CHR 009 026 1005309 BbF 01 08 92 CHR 5310 BbF 07 2 CHR 01 03 5311 BbF 002 004 95 CHR 002 004 895312 BbF 03 09 105 CHR 03 09 1055313 BbF 04 08 CHR 04 08 5314 BbF 001 002 83 CHR 001 002 8185315 BbF 02 05 50 -100 CHR 02 05 50 - 100 5316 BbF 005 024 814 CHR 001 007 9595317 BbF 05 1 73 CHR 05 1 785318 BbF CHR 5319 BbF 011 038 946 CHR 013 043 9675320 BbF 07 09 1194 CHR 23 32 10455321 BbF 05 1 CHR 05 1 5322 BbF 1 2 100 CHR 1 2 1005323 BbF 26 CHR 26

62

ANNEX 11 Table 19 Details of analysis method reported by the participants Data are presented as reported

Laboratory code

Analysis technique

Instrument calibration

Internal standards applied

Sample clean up

Details of sample

clean up

Extraction technique

Details of sample

extraction

3600 6) GC-MS

2) Internal standardisation with labelled substances

Deuterated PAH mix 9 (Ehrenstorfer)

1) Saponification 2) LiquidLiquid partitioning

D) Other Liquidliquid partitioning

3602 2) HPLC-UV-FLD

1) External calibration

3) Gel permeation chromatography

A) Pressurised liquid extraction (PLE)

3603 1) HPLC-FLD

1) External calibration

3) Gel permeation chromatography

D) Other 1 minute with dichloromethane in vortex

3604 2) HPLC-UV-FLD

2) Internal standardisation with labelled substances

DiP D14

5) Donor acceptor complex chromatography

D) Other LL

3605 1) HPLC-FLD

1) External calibration

3) Gel permeation chromatography

C) Soxhlet extraction

3606 7) GC-MSMS

2) Internal standardisation with labelled substances

BaP 13C4 CHR 13C6 BaA 13C6 BbF 13C6

4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

3607 6) GC-MS

2) Internal standardisation with labelled substances

BaA-d12 CHR-d12 BbF-d12 BkF-d12 BaP-d12 ICP-d12 BgP-d12 DiP-d14

1) Saponification

D) Other solvent partitioning

3608 1) HPLC-FLD

1) External calibration

6) Other Liquid extraction

D) Other

Extraction in a rotary agitator and centrifugation

3610 2) HPLC-UV-FLD

1) External calibration

4) Solid phase extraction

B) Sonication

3611

3612 6) GC-MS

2) Internal standardisation with labelled substances

chrysene-d12 dibenzo(ah)anthracene-d14 dibenzo(ai)pyrene-d14

4) Solid phase extraction

B) Sonication

3613 5) LC-MSMS

3) Internal standardisation with unlabelled substances

I use Chrysene-D12 3) Gel permeation chromatography

D) Other extraction with hexan

63

Table 19 - continued

Laboratory code

Analysis technique

Instrument calibration

Internal standards applied

Sample clean up Details of sample

clean up

Extraction technique

Details of sample

extraction

3614 6) GC-MS 1) External calibration

3) Gel permeation chromatography

B) Sonication

3615 1) HPLC-FLD

1) External calibration

6) Other PLE GPC

A) Pressurised liquid extraction (PLE)

3616 6) GC-MS

2) Internal standardisation with labelled substances

Benzo(a)pyrene-13C4 Benzo(a)anthracene-13C6 Benzo(b)fluoranthene-13C6 Chrysene-13C6

2) LiquidLiquid partitioning 4) Solid phase extraction

B) Sonication

3617 1) HPLC-FLD

1) External calibration

4) Solid phase extraction

C) Soxhlet extraction

3618 6) GC-MS

2) Internal standardisation with labelled substances

deuterated standards

1) Saponification 4) Solid phase extraction

D) Other Handshaken with cyclohexane

3619 6) GC-MS

2) Internal standardisation with labelled substances

C13 Labelled US EPA 16

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

D) Other Homegenisation amp saponiofication

3620 9) GC-HRMS

2) Internal standardisation with labelled substances

mix of deuterated PAHs (BAA-D12 CHR-D12 BAP-D12 BBF-D12)

3) Gel permeation chromatography 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

3621 6) GC-MS

2) Internal standardisation with labelled substances

Chrysene D12 Benzo(a)pyrene D12 Benzo(b)fluoranthene D12 Benzo(a)anthracene

2) LiquidLiquid partitioning 4) Solid phase extraction

B) Sonication

3622 1) HPLC-FLD

4) Standard addition method

4) Solid phase extraction

D) Other

Extraction with cyclohexane by heating under reflux

3623 7) GC-MSMS

2) Internal standardisation with labelled substances

C13 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

3624 2) HPLC-UV-FLD

1) External calibration

2) LiquidLiquid partitioning

D) Other hot solvent extraction

3626

64

Table 19 - continued

Laboratory code

Analysis technique

Instrument calibration

Internal standards

applied

Sample clean up

Details of sample

clean up

Extraction technique

Details of sample

extraction

5301 7) GC-MSMS

2) Internal standardisation with labelled substances

EACH SUBSTANCE C13

4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5302 7) GC-MSMS

2) Internal standardisation with labelled substances

Isotope labelled PAHs 13C for each analyte

4) Solid phase extraction

C) Soxhlet extraction

5303 7) GC-MSMS

3) Internal standardisation with unlabelled substances

HAP C13 4) Solid phase extraction

D) Other automate ASE200 DIONEX

5304 7) GC-MSMS

2) Internal standardisation with labelled substances

with C13 for each HAP

2) LiquidLiquid partitioning 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5305 7) GC-MSMS

2) Internal standardisation with labelled substances

C13 isotopiques internal standards

4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5306 7) GC-MSMS

2) Internal standardisation with labelled substances

C13 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5307 6) GC-MS

2) Internal standardisation with labelled substances

1) Saponification 2) LiquidLiquid partitioning

D) Other solvent extraction

5308 1) HPLC-FLD 1) External calibration

4) Solid phase extraction

B) Sonication

5309 1) HPLC-FLD 1) External calibration

4) Solid phase extraction

D) Other only SPE

5310 1) HPLC-FLD 1) External calibration

1) Saponification 2) LiquidLiquid partitioning 3) Gel permeation chromatography

C) Soxhlet extraction

5311 6) GC-MS

2) Internal standardisation with labelled substances

13 C Chrysene 13 C Benz(a)pyrene

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

B) Sonication

5312 1) HPLC-FLD

3) Internal standardisation with unlabelled substances

Benzo(b)chrysen

2) LiquidLiquid partitioning 4) Solid phase extraction

Quechers D) Other Quechers

65

Table 19 - continued

Laboratory code

Analysis technique

Instrument calibration

Internal standards applied

Sample clean up Details of sample

clean up

Extraction technique

Details of sample extraction

5313 6) GC-MS

2) Internal standardisation with labelled substances

Benz(a)anthracene D12 Benzo(b)fluranthene D12 Benzo(a)pyrene D12 Chrysene D12

1) Saponification 2) LiquidLiquid partitioning 3) Gel permeation chromatography

A) Pressurised liquid extraction (PLE)

5314 6) GC-MS

2) Internal standardisation with labelled substances

Benzo(a)pyren d12 2) LiquidLiquid partitioning

D) Other Extraction with

5315 1) HPLC-FLD

3) Internal standardisation with unlabelled substances

Benz(b)chrysen

1) Saponification 2) LiquidLiquid partitioning

D) Other shaker

5316 1) HPLC-FLD

1) External calibration

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

D) Other

LiquidLiquid extraction with cyclohexane and dimethylformamide

5317 7) GC-MSMS

1) External calibration

3) Gel permeation chromatography 6) Other

silica gel D) Other VDLUFA online method

5318

5319 1) HPLC-FLD

3) Internal standardisation with unlabelled substances

Benzo(b)chrysene 3) Gel permeation chromatography

A) Pressurised liquid extraction (PLE)

5320 6) GC-MS

3) Internal standardisation with unlabelled substances

22-binaphthyl

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

D) Other 3 hours refluxing

5321 1) HPLC-FLD

2) Internal standardisation with labelled substances

6-meacutethylchrysegravene

1) Saponification 2) LiquidLiquid partitioning

B) Sonication

5322 6) GC-MS

2) Internal standardisation with labelled substances

naftalene-d8 acenaphtalene-d10 phenanthrene-d10 chrysene-d12 perylene-d12 benzo(e)anthracene-d12

1) Saponification 2) LiquidLiquid partitioning 6) Other

clean-up with silica packed column

C) Soxhlet extraction

5323 1) HPLC-FLD

1) External calibration

4) Solid phase extraction 6) Other

Accelerated Solvent Extraction

A) Pressurised liquid extraction (PLE)

66

European Commission EUR 25245 EN ndash Joint Research Centre ndash Institute for Reference Materials and Measurements Title Report on the 8th inter-laboratory comparison organised by the European Union Reference Laboratory for Polycyclic Aromatic Hydrocarbons - Four marker PAHs in a dry extract of St Johns wort Author(s) Radoslav Lizak Szilard Szilagyi Philippe Verlinde and Thomas WENZL Luxembourg Publications Office of the European Union 2012 ndash 66 pp ndash 201 x 297 cm EUR ndash Scientific and Technical Research series ndash ISSN 1831-9424 ISBN 978-92-79-23337-1 doi 10278759387 Abstract The proficiency test here reported concerned the determination of the contents of four marker polycyclic aromatic hydrocarbons (PAHs) and of their sum in an food supplements test sample The set of marker PAHs consists of benz[a]anthacene benzo[a]pyrene benzo[b]fluoranthene and chrysene Participants to these PT were National Reference Laboratories for PAHs (NRLs-PAHs) and EU official food control laboratories The number of participants was in total 48

The PT was organised according ISO Standard 170432010

The test material used was a commercial dry extract of St Johns wort Participants also received a solution of the same PAHs either in acetonitrile or in toluene for checking their instrument calibration

The results from participants were rated with z-scores and zeta-scores About 68 of the reported results were attributed with z-scores with an absolute value of below two which is the threshold for satisfactory performance

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science and technology for the Union Close to the policy-making process it serves

the common interest of the Member States while being independent of special

interests whether private or national

L

A-N

A-252

45-EN

-N

• 1 Summary
• 2 Introduction
• 3 Scope
• 4 Participating Laboratories
• 5 Time frame
• 6 Confidentiality
• 7 Test materials
• • 71 1Preparation of test materials
  • 72 Assigned values and standard deviation for proficiency assessment
  • 73 Homogeneity and stability
  • 74 Sample dispatch
  • • 8 Design of the proficiency test
    • 9 Evaluation of the results
    • • 91 2General
      • 92 21BEvaluation criteria
      • 93 Performance of participants
      • 94 Evaluation of the influence of analysis method
      • 95 Evaluation of the influence of calibration
      • 96 26Evaluation of compliance with legislation
      • • 10 Follow-up actions for underperforming laboratories
        • 11 1Conclusions
        • 12 1Acknowledgements
        • 13 References

30

8 EU Commission Regulation (EU) No 8352011 amending Regulation (EC) No 18812006 as regards

maximum levels for polycyclic aromatic hydrocarbons in foodstuffs Official Journal of the European

Union 2011 L 215 p 4-8

httpeur-lexeuropaeuLexUriServLexUriServdouri=OJL201121500040008ENPDF

9 EU Commission Regulation (EU) No 8362011 amending Regulation (EC) No 3332007 laying down the methods of sampling and analysis for the official control of the levels of lead cadmium mercury inorganic tin 3-MCPD and benzo(a)pyrene in foodstuffs Official Journal of the European Union 2011 L 215 p 9-16 httpeur-lexeuropaeuLexUriServLexUriServdouri=OJL201121500090016ENPDF

10 ISOIEC 170432010 (2010) Conformity assessment -- General requirements for proficiency testing

providers Geneva Switzerland

11 Thompson M Ellison SLR and Wood R The International Harmonized Protocol for the Proficiency

Testing of Analytical Chemistry Laboratories Pure Appl Chem (2006) 78(1) p 145ndash196

Available from httpmediaiupacorgpublicationspac2006pdf7801x0145pdf

31

14 ANNEXES

ANNEX 1 ndash Announcement of the PT on the IRMM webpage

ANNEX 2 ndash Announcement via e-mail and invitation

ANNEX 3 ndash Announcement of sample dispatch

ANNEX 4 ndash Technical specifications of the calibration solutions

ANNEX 5 ndash Homogeneity and stability of the test material

ANNEX 6 ndash Acknowledgement of sample receipt

ANNEX 7 ndash Questionnaire on details of analysis method

ANNEX 8 ndash Results reported for the food supplement sample

ANNEX 9 ndash Results reported for the standard solutions with undisclosed analyte content

ANNEX 10 ndash Method performance characteristics reported by participants

ANNEX 11 ndash Details of analysis methods reported by participants

32

ANNEX 1 ndash Announcement of the PT on the IRMM webpage

33

ANNEX 2 - Announcement via e-mail and invitation

34

ANNEX 3 - Announcement sample dispatch

Dear MadameSir

The test samples for the proficiency test on the determination of four PAHs in a food supplement (dry extract of St Johns wort) were

dispatched today

Explanation on the design of the study as well as instructions for sample storage and for reporting of results are in the parcel You will

also find your participant key in the parcel which is required for reporting of results

Please check the parcel after arrival for completeness and report the receipt of the test samples as well as their status to us by applying

the sample receipt form which is in the parcel

You are kindly requested to inform us by email if you do not receive the parcel by end of this week

My colleague Donata Lerda will then take care of it

With best regards

Thomas Wenzl

35

ANNEX 4 - Specifications of standard solutions with disclosed concentration

36

ANNEX 4 - continued

37

ANNEX 5 - Homogeneity and stability of the test material

Homogeneity of the test material

Benz[a]anthracene - BAA

Benzo[a]pyrene - BAP

38

ANNEX 5 - continued

Benzo[b]fluoranthene - BBF

Chrysene - CHR

39

ANNEX 5 - continued

Stability of the test material

Table 10 Content of the four target analytes in test materials stored under different

temperatures

BaA BaP BbF CHR

microgkg microgkg microgkg microgkg

Content 31 16 31 69 Storage at room temperature

U (k=2) 05 02 05 17

Content 31 15 29 68 Storage at reference temperature

U (k=2) 04 03 04 10

The applied capillary column does not separate CHR from triphenylene

40

ANNEX 6 ndash Documents sent to participants

41

ANNEX 6 - continued

42

ANNEX 7 Questionnaire on details of analysis method

43

ANNEX 7 - continued

44

ANNEX 7 - continued

45

ANNEX 8 - Results reported by participants for the food supplements test sample Table 11 Analysis results reported by the participants for the content of benz[a]anthracene (BAA) in the food supplements test material Values are presented as reported

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

3600 31 29 29 3 05 2

3602 199 193 178 19 021

3603 253 253 246 251 022 2

3604 199 209 23 213 071 2

3605 301 3 271 291 076 2

3606 302 288 278 289 064 2

3607 456 416 443 438 096 2

3608 796 695 651 714 149 2

3610 182 192 214 196 039 2

3611 414 504 488 469 96 2

3612 3 34 28 31 06 2

3613 32 33 34 33 07 2

3614 182 182 191 185 037 2

3615 3884 3822 3565 376 0752 2

3616 2 2 2 2 01 2

3617 366 37 35 362 046 2

3618 32 32 32 32 05 2

3619 299 305 297 299 048 2

3620 304 294 302 302 045 2

3621 29 26 29 28 04 2

3622 343 325 318 322 052 2

3623 35 3 33 33 07 2

3624

3626 318 31 314 314 126 2

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

5301 31 3 32 31 06 2

5302 314 319 315 316 063 2

5303 3102 2716 2822 2876 27 62

5304 261 263 26 261 57 2

5305 23 22 26 24 20

5306 22 23 23 22 11 2

5307 488 461 317 461

5308 168633 168544 1562025 1644598

5309 16 19 16 17

5310 3 304 311 305 015

5311 477 452 448 459 15 2

5312 506 63 494 543 134 2

5313 353 301 404 353

5314 431 373 378 394 5 096

5315 596 531 51 545 11 2

5316 224 231 235 235 022 2

5317 216 23 221 222 007 2

5318 064 07 067 01 2

5319 111 184 146 147 029 2

5320 515 525 564 534 48

5321 21 5322 13 87 9 87 35 2

5323 38 33 35 33 16 2

46

Figure 6 Results of replicate determinations (indicated by triangles) of benz[a]anthracene (BAA) in the food supplement test material

Horizontal blue lines represent the arithmetic mean value of replicate measurements and blue bars the reported expanded measurement uncertainty (k=2) The assigned value is

plotted as dotted line Red solid lines mark the range of satisfactory performance (|z|le2) Mean values are given for results outside the displayed data range

ProLab 2011

Laboratory

53

1853

1953

0853

0936

1436

0236

1036

1653

2136

0453

1753

0653

1653

0536

0353

0436

2153

0336

0636

0536

0036

2036

1953

1036

1253

0136

2653

0236

1836

2336

2236

1353

1353

2336

1736

1553

1453

0736

0753

1153

2053

1253

1536

0853

2236

11

an

aly

te c

on

ten

t (micro

gk

g)

10

95

9

85

8

75

7

65

6

55

5

45

4

35

3

25

2

15

1

05

0

102

3 468

7

Sample St Johns wort extract replicatesMeasurand Benz[a]anthraceneNo of laboratories 46

Assigned value 298 microgkg (Reference value)Target sd 062 microgkgTolerance limits 174 - 422 microgkg (|Z score| lt 200)

Assig

ned va

lue

Limit of tolerance

Limit of tolerance

47

Table 12 Analysis results reported by the participants for the content of benzo[a]pyrene (BAP) in the food supplements test material Values are presented as reported

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

3600 16 17 17 17 03 2

3602 118 123 13 124 015

3603 165 162 157 161 015 2

3604 134 154 139 143 04 2

3605 164 166 172 167 057 2

3606 181 159 161 167 031 2

3607 279 266 279 274 055 2

3608 57 687 461 573 226 2

3610 226 216 183 206 042 2

3611 289 325 311 308 36 2

3612 24 22 2 22 05 2

3613 17 18 19 18 05 2

3614 08 08 08 08 016 2

3615 1545 1393 1425 145 029 2

3616 16 16 14 15 03 2

3617 177 171 158 169 023 2

3618 2 2 2 2 02 2

3619 207 207 206 207 075 2

3620 165 169 163 166 017 2

3621 44 47 49 47 05 2

3622 212 208 222 213 038 2

3623 2 21 22 21 03 2

3624 18 178 182 18 031 2

3626 172 173 178 174 07 2

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

5301 18 18 18 18 04 2

5302 188 19 187 188 038 2

5303 1691 1426 149 1535 19 80

5304 145 142 15 146 69 2

5305 15 17 18 16 15

5306 16 17 17 16 08 2

5307 164 157 153 157

5308 102669 109324 109035 107009

5309 1 1 12 11

5310 122 122 124 123 006

5311 199 184 215 2 15 2

5312 258 248 239 248 05 2

5313 199 178 245 207

5314 174 171 172 172 11 0948

5315 253 248 213 238 05 2

5316 177 171 173 17 016 2

5317 255 277 276 269 012 2

5318 114 12 117 02 2

5319 19 228 231 216 043 2

5320 303 3 344 316 78

5321 12 12 12 12

5322 13 17 24 17 35 2

5323 2 2 19 2 1 2

48

Figure 7 Results of replicate determinations (indicated by triangles) of benzo[a]pyrene (BAP) in the food supplement test material

Horizontal blue lines represent the arithmetic mean value of replicate measurements and blue bars the reported expanded measurement uncertainty (k=2) The assigned value is

plotted as dotted line Red solid lines mark the range of satisfactory performance (|z|le2) Mean values are given for results outside the displayed data range

ProLab 2011

Laboratory

36

1453

0953

0853

1853

2153

1036

0236

0436

1553

0436

1653

0353

0736

0336

2036

0053

0553

0636

0636

0536

1753

1453

1636

2636

1336

2453

0153

2253

0253

2353

1136

1836

1953

1336

1036

2336

2253

1936

1253

1553

1253

1736

0753

2036

2136

0836

11

an

aly

te c

on

ten

t (micro

gk

g)

55

5

45

4

35

3

25

2

15

1

05

0

573

308

3

Sample St Johns wort extract replicatesMeasurand Benzo[a]pyreneNo of laboratories 48

Assigned value 165 microgkg (Reference value)Target sd 036 microgkgTolerance limits 093 - 237 microgkg (|Z score| lt 200)

Assig

ned va

lue

Limit of tolerance

Limit of tolerance

49

Table 13 Analysis results reported by the participants for the content of benzo[b]fluoranthene (BBF) in the food supplements test material Values are presented as reported

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

3600 32 3 3 31 09 2

3602 173 175 16 17 017

3603 265 252 25 256 025 2

3604 116 108 105 11 028 2

3605 286 225 232 247 074 2

3606 294 273 261 276 046 2

3607 579 579 544 567 113 2

3608 103 1201 876 1036 326 2

3610 173 196 204 191 038 2

3611 546 618 565 576 75 2

3612 3 28 28 29 06 2

3613 28 31 32 31 06 2

3614 135 111 135 127 022 2

3615 4187 3678 4111 4 08 2

3616 2 2 2 2 01 2

3617 33 27 299 035 2

3618 31 31 31 31 05 2

3619 326 324 312 326 064 2

3620 277 28 272 276 069 2

3621 26 25 23 25 04 2

3622 346 336 333 34 052 2

3623 26 28 29 28 02 2

3624

3626 288 28 307 292 117 2

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

5301 29 3 29 29 06 2

5302 297 3 296 298 06 2

5303 2705 2444 2608 2586 22 78

5304 245 254 26 253 85 2

5305 23 23 26 24 15

5306 42 43 4 42 21 2

5307 221 216 241 221

5308 232169 246654 240722 229848

5309 19 22 19 2 0

5310 3 315 325 313 016

5311 262 277 304 281 15 2

5312 27 289 337 299 075 2

5313 321 284 378 328

5314 423 404 385 404 34 083

5315 42 506 416 444 09 2

5316 265 267 277 273 018 2

5317 185 197 195 192 006 2

5318 276 281 279 04 2

5319 396 389 403 396 079 2

5320 623 618 635 625 14

5321 31

5322 24 33 49 33 35 2

5323 38 45 35 45 27 2

50

Figure 8 Results of replicate determinations (indicated by triangles) of benzo[b]fluoranthene (BBF) in the food supplement test material

Horizontal blue lines represent the arithmetic mean value of replicate measurements and blue bars the reported expanded measurement uncertainty (k=2) The assigned value is

plotted as dotted line Red solid lines mark the range of satisfactory performance (|z|le2) Mean values are given for results outside the displayed data range

ProLab 2011

Laboratory

36

0436

1436

0236

1053

1736

1653

0953

0753

0853

0536

2136

0553

0436

0353

0353

1636

0636

2036

2353

1853

1136

1236

2653

0153

0253

1236

1736

1336

0036

1853

2153

1036

1953

1336

2253

2253

2353

1936

1553

1453

0653

1536

0753

2036

0836

11

an

aly

te c

on

ten

t (micro

gk

g)

75

7

65

6

55

5

45

4

35

3

25

2

15

1

05

0

103

6

576

3

Sample St Johns wort extract replicatesMeasurand Benzo[b]fluorantheneNo of laboratories 46

Assigned value 292 microgkg (Reference value)Target sd 060 microgkgTolerance limits 172 - 412 microgkg (|Z score| lt 200)

Assig

ned va

lue

Limit of tolerance

Limit of tolerance

51

Table 14 Analysis results reported by the participants for the content of chrysene (CHR) in the food supplements test material Values are presented as reported

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

3600 5 49 5 5 07 2

3602 32 304 29 304 036

3603 462 469 447 459 048 2

3604 438 428 42 429 119 2

3605 365 41 381 386 085 2

3606 494 411 45 451 124 2

3607 897 774 787 819 18 2

3608 986 887 1168 1014 284 2

3610 418 392 462 424 085 2

3611 833 893 87 865 61 2

3612 6 64 58 61 12 2

3613 41 39 4 4 08 2

3614 272 254 288 271 054 2

3615 605 6028 5964 601 12 2

3616 47 45 49 47 05 2

3617 445 431 389 422 065 2

3618 45 45 45 45 06 2

3619 738 747 72 738 118 2

3620 463 472 446 46 092 2

3621 53 49 51 51 06 2

3622 478 513 505 488 08 2

3623 73 73 72 72 05 2

3624

3626 63 622 634 628 251 2

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

5301 49 49 49 49 1 2

5302 687 683 666 679 136 2

5303 6777 6181 6382 6441 24 70

5304 592 581 6 591 85 2

5305 6 58 64 61 20

5306 49 5 51 49 25 2

5307 524 5 574 524

5308 211488 216782 221146 213243

5309

5310 372 393 394 386 019

5311 43 459 422 437 15 2

5312 934 943 981 953 191 2

5313 793 73 99 838

5314 636 561 565 588 18 0818

5315 547 583 499 542 11 2

5316 292 286 285 282 06 2

5317 52 598 517 545 046 2

5318 368 345 357 05 2

5319 449 471 474 465 116 2

5320 76 924 818 834 99

5321 33

5322 7 7 76 7 35 2

5323 87 73 77 73 37 2

52

Figure 9 Results of replicate determinations (indicated by triangles) of chrysene (CHR) in the food supplement test material

Horizontal blue lines represent the arithmetic mean value of replicate measurements and blue bars the reported expanded measurement uncertainty (k=2) The assigned value is

plotted as dotted line Red solid lines mark the range of satisfactory performance (|z|le2) Mean values are given for results outside the displayed data range

ProLab 2011

Laboratory

53

0836

1453

1636

0253

2153

1836

0553

1036

1336

1736

1036

0453

1136

1836

0636

0336

2053

1936

1653

0136

0036

2253

0636

2153

0753

1553

1753

1453

0436

1536

1253

0536

2653

0353

0253

2236

2336

1953

2336

0753

2053

1353

1236

0836

11

an

aly

te c

on

ten

t (micro

gk

g)

13

12

11

10

9

8

7

6

5

4

3

2

1

0

865

3

Sample St Johns wort extract replicatesMeasurand ChryseneNo of laboratories 45

Assigned value 407 microgkg (Reference value)Target sd 083 microgkgTolerance limits 241 - 573 microgkg (|Z score| lt 200)

Assig

ned va

lue

Limit of tolerance

Limit of tolerance

53

Table 15 Analysis results reported by the participants for the sum of the four marker PAHs in the food supplements test material Values are presented as reported

Laboratory code

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg

3600 128 24 2

3602 79 126

3603 1127 096 2

3604 893 258 2

3605 1091 305 2

3606 1184 238 2

3607 2098 42 2

3608 3336 325 2

3610 1138 17 2

3611 2218 259 2

3612 143 29 2

3613 122 2 2

3614 663 129 2

3615 152 304 2

3616 102 03 2

3617 1252 169 2

3618 128 18 2

3619 157 305 2

3620 1204 12 2

3621 151 19 2

3622 1361 115 2

3623 154 09 2

3624

3626 1409 56 2

Laboratory code

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg

5301 126

5302 37 074 2

5303 13438 92

5304 1251 85 2

5305 125 20

5306 129 65 2

5307 1363

5308 7245598

5309

5310 1127 056

5311 1367 15 2

5312 2042 408 2

5313 1726

5314 1558

5315 177 35 2

5316 96 073 2

5317 1229 066 2

5318 82 05 2

5319 1225 267 2

5320 2309 41

5321

5322 207 35 2

5323 171 103 2

54

ANNEX 9 Results reported for the standard solutions with undisclosed analyte content Table 16 Analysis results reported by the participants for the four marker PAHs in the standard solution in toluene with undisclosed analyte

content Values are presented as reported U measurement uncertainty k coverage factor

benz[a]anthracene benzo[a]pyrene benzo[b]fluoranthene chrysene Laboratory

code Unit Result U k Result U k Result U k Result U k 3600 microgl 38 67 2 25 45 2 211 68 2 275 38 2

3605 microgl 367 184 2 2482 124 2 2592 13 2 235 118 2

3606 microgkg 3763 176 2 2609 142 2 2721 118 2 2539 134 2

3607 microgkg 3706 815 2 2824 565 2 2591 518 2 2587 569 2

3612 microgl 38 08 2 237 11 2 244 18 2 257 02 2

3614 microgl 324 65 2 209 38 2 215 37 2 21 42 2

3616 microgkg 318 07 2 224 14 2 231 26 2 216 1 2

3618 microgkg 417 17 2 284 08 2 292 06 2 297 03 2

3619 microgkg 47 742 2 274 468 2 228 387 2 274 434 2

3620 microgl 495 74 2 294 29 2 321 8 2 321 64 2

3621 microgl 241 22 2 143 13 2 188 16 2 145 14 2

3623 microgkg 476 25 2 311 53 2 269 3 2 297 03 2

3626 microgl 359 38 2 235 23 2 238 27 2 24 18 2

5301 microgkg 426 29 299 289

5302 microgkg 436 87 2 296 59 2 296 59 2 289 58 2

5303 microgl 3721 27 102 22838 19 99 23756 22 99 25486 24 98

5304 microgkg 38 29 2 225 26 2 257 37 2 258 52 2

5305 microgkg 305 225 245 29

5306 microgkg 316 63 2 247 49 2 65 13 2 22 44 2

5307 microgkg 3658 2559 241 2414

5308 microgkg 16854 109324 246654 216782

5311 microgl 374 20 2 279 15 2 268 15 2 272 15 2

5313 microgl 3408 2165 2275 2373

5314 microgkg 395 5 2875 11 3614 34 2905 18

5320 microgl 3267 368 1986 571 28 683 2427 512

5322 microgl 282 35 2 225 35 2 241 35 2 21 35 2

55

Figure 10 Youden plots of the percent deviation of the reported values from the assigned values for the food supplement test sample and the

standard solution in toluene with undisclosed content

(The identity of the respective measurand is given above the figure Results exceeding a difference of more than 100 were omitted)

56

Figure 10 - continued

57

Table 17 Analysis results reported by the participants for the four marker PAHs in the standard solution in acetonitrile with undisclosed

analyte content

Values are presented as reported U measurement uncertainty k coverage factor

benz[a]anthracene benzo[a]pyrene benzo[b]fluoranthene chrysene

Laboratory

code Unit Result U k Result U k Result U k Result U k

3602 microgkg 3675 247 247 244

3603 microgkg 358 086 2 236 028 2 235 028 2 236 057 2

3604 microgl 475 29 2 19 12 2 194 13 2 258 12 2

3608 microgl 3623 078 2 2385 023 2 2343 18 2 2296 092 2

3610 microgkg 369 185 2 25 125 2 251 126 2 244 122 2

3611 microgkg 48 02 2 323 02 2 328 04 2 317 03 2

3613 microgl 332 31 2 261 3 2 249 25 2 239 24 2

3615 microgl 415 2635 2729 2689

3617 microgkg 37 09 2 244 06 2 284 07 2 239 06 2

3622 microgl 37 12 2 25 23 2 257 21 2 238 1 2

3624 microgkg 402 104 2 255 084 2 265 096 2 253 07 2

5306 microgkg 563 113 2 371 74 2 359 72 2 333 67 2

5308 microgkg 168633 102669 232169 211488

5309 microgl 402 268 27

5310 microgl 315 158 25 125 25 125 288 144

5312 microgl 428 043 2 282 028 2 272 027 2 275 028 2

5315 microgkg 387 08 2 256 05 2 251 05 2 253 05 2

5316 microgl 3839 342 2 2485 191 2 2559 168 2 239 51 2

5317 microgl 2466 043 2 2212 034 2 2472 016 2 2146 068 2

5319 microgl 414 41 2 279 28 2 268 27 2 26 31 2

5321 microgkg 476 316 326 328

5322 microgl 2547 35 2

5323 microgkg 405 203 2 239 119 2 255 153 2 201 11 2

58

Figure 11 Youden plots of the percent deviation of the reported values from the assigned values for the food supplement test sample and the

standard solution in acetonitrile with undisclosed content

(The identity of the respective measurand is given above the figure Results exceeding a difference of more than 100 were omitted)

59

Figure 11 - continued

60

ANNEX 10 Table 18 Method performance parameters reported by the participants

Laboratory code

Analyte LOD LOQ Recovery Analyte LOD LOQ Recovery

microgkg microgkg microgkg microgkg 3600 BaA 03 06 978 BaP 03 07 9483602 BaA 007 021 926 BaP 005 015 993603 BaA 0011 05 987 BaP 0005 051 9553604 BaA 02 04 86 BaP 02 04 883605 BaA 0025 005 118 BaP 0025 005 1083606 BaA 001 003 65 BaP 001 003 703607 BaA 021 071 74 BaP 027 089 943608 BaA 016 048 8213 BaP 015 046 83683610 BaA 02 05 85 BaP 005 02 853611 BaA BaP 3612 BaA 1 117 BaP 1 1203613 BaA 01 03 90 BaP 01 03 903614 BaA 02 05 96 BaP 02 05 933615 BaA 005 015 110 BaP 005 015 1053616 BaA 01 05 108 BaP 01 05 753617 BaA 018 037 405 BaP 009 018 3873618 BaA 01 03 63 BaP 01 03 593619 BaA 005 005 59 BaP 033 033 543620 BaA 0009 0018 64 BaP 0005 001 553621 BaA 007 02 84 BaP 007 02 923622 BaA 01 03 1101 BaP 01 03 9853623 BaA 02 08 105 BaP 02 08 1013624 BaA BaP 01 033 8513625 BaA BaP 3626 BaA BaP 5301 BaA 003 01 102 BaP 003 01 1005302 BaA 01 03 78 BaP 01 03 865303 BaA 003 01 62 BaP 003 01 805304 BaA 1 1 70 BaP 1 1 955305 BaA 02 05 85 BaP 02 05 855306 BaA 03 1 699 BaP 03 1 905307 BaA 6537 BaP 81165308 BaA 0088 0265 99 BaP 00262 00785 1005309 BaA 01 08 92 BaP 01 08 925310 BaA 01 03 BaP 01 03 905311 BaA 002 005 80 BaP 002 004 915312 BaA 03 09 105 BaP 03 09 1055313 BaA 04 08 BaP 04 08 5314 BaA 001 002 96 BaP 001 002 9485315 BaA 01 03 50 - 100 BaP 01 03 50 - 100 5316 BaA 002 011 827 BaP 004 019 10445317 BaA 05 1 76 BaP 05 1 755318 BaA BaP 5319 BaA 009 029 951 BaP 011 041 995320 BaA 12 18 946 BaP 06 1 8345321 BaA 03 05 BaP 05 1 5322 BaA 1 2 100 BaP 1 2 1005323 BaA 26 BaP 26

61

ANNEX 10 Table 18 - continued

Laboratory code Analyte LOD LOQ Recovery Analyte LOD LOQ Recovery

microgkg microgkg microgkg microgkg 3600 BbF 03 05 96 CHR 02 03 10153602 BbF 015 045 947 CHR 003 009 9193603 BbF 0014 05 1012 CHR 0004 05 10033604 BbF 02 04 85 CHR 02 04 873605 BbF 005 01 94 CHR 0025 005 863606 BbF 001 003 70 CHR 001 003 653607 BbF 018 06 100 CHR 014 028 943608 BbF 015 046 7979 CHR 015 045 71253610 BbF 005 02 85 CHR 02 05 853611 BbF CHR 3612 BbF 1 118 CHR 1 1173613 BbF 01 03 90 CHR 01 03 903614 BbF 01 04 96 CHR 02 05 953615 BbF 008 025 109 CHR 003 01 1083616 BbF 01 05 78 CHR 01 05 723617 BbF 035 07 38 CHR 012 024 4163618 BbF 01 03 62 CHR 01 03 623619 BbF 016 016 63 CHR 01 01 593620 BbF 0003 0006 55 CHR 001 002 653621 BbF 007 02 103 CHR 007 02 893622 BbF 02 06 963 CHR 03 09 1043623 BbF 02 08 106 CHR 02 08 1193624 BbF CHR 3625 BbF CHR 3626 BbF CHR 5301 BbF 003 01 96 CHR 003 01 1145302 BbF 01 03 84 CHR 01 03 835303 BbF 003 01 78 CHR 003 01 705304 BbF 1 1 86 CHR 1 1 705305 BbF 02 05 90 CHR 02 05 955306 BbF 03 1 1435 CHR 03 1 1185307 BbF 8116 CHR 65375308 BbF 0079 0238 99 CHR 009 026 1005309 BbF 01 08 92 CHR 5310 BbF 07 2 CHR 01 03 5311 BbF 002 004 95 CHR 002 004 895312 BbF 03 09 105 CHR 03 09 1055313 BbF 04 08 CHR 04 08 5314 BbF 001 002 83 CHR 001 002 8185315 BbF 02 05 50 -100 CHR 02 05 50 - 100 5316 BbF 005 024 814 CHR 001 007 9595317 BbF 05 1 73 CHR 05 1 785318 BbF CHR 5319 BbF 011 038 946 CHR 013 043 9675320 BbF 07 09 1194 CHR 23 32 10455321 BbF 05 1 CHR 05 1 5322 BbF 1 2 100 CHR 1 2 1005323 BbF 26 CHR 26

62

ANNEX 11 Table 19 Details of analysis method reported by the participants Data are presented as reported

Laboratory code

Analysis technique

Instrument calibration

Internal standards applied

Sample clean up

Details of sample

clean up

Extraction technique

Details of sample

extraction

3600 6) GC-MS

2) Internal standardisation with labelled substances

Deuterated PAH mix 9 (Ehrenstorfer)

1) Saponification 2) LiquidLiquid partitioning

D) Other Liquidliquid partitioning

3602 2) HPLC-UV-FLD

1) External calibration

3) Gel permeation chromatography

A) Pressurised liquid extraction (PLE)

3603 1) HPLC-FLD

1) External calibration

3) Gel permeation chromatography

D) Other 1 minute with dichloromethane in vortex

3604 2) HPLC-UV-FLD

2) Internal standardisation with labelled substances

DiP D14

5) Donor acceptor complex chromatography

D) Other LL

3605 1) HPLC-FLD

1) External calibration

3) Gel permeation chromatography

C) Soxhlet extraction

3606 7) GC-MSMS

2) Internal standardisation with labelled substances

BaP 13C4 CHR 13C6 BaA 13C6 BbF 13C6

4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

3607 6) GC-MS

2) Internal standardisation with labelled substances

BaA-d12 CHR-d12 BbF-d12 BkF-d12 BaP-d12 ICP-d12 BgP-d12 DiP-d14

1) Saponification

D) Other solvent partitioning

3608 1) HPLC-FLD

1) External calibration

6) Other Liquid extraction

D) Other

Extraction in a rotary agitator and centrifugation

3610 2) HPLC-UV-FLD

1) External calibration

4) Solid phase extraction

B) Sonication

3611

3612 6) GC-MS

2) Internal standardisation with labelled substances

chrysene-d12 dibenzo(ah)anthracene-d14 dibenzo(ai)pyrene-d14

4) Solid phase extraction

B) Sonication

3613 5) LC-MSMS

3) Internal standardisation with unlabelled substances

I use Chrysene-D12 3) Gel permeation chromatography

D) Other extraction with hexan

63

Table 19 - continued

Laboratory code

Analysis technique

Instrument calibration

Internal standards applied

Sample clean up Details of sample

clean up

Extraction technique

Details of sample

extraction

3614 6) GC-MS 1) External calibration

3) Gel permeation chromatography

B) Sonication

3615 1) HPLC-FLD

1) External calibration

6) Other PLE GPC

A) Pressurised liquid extraction (PLE)

3616 6) GC-MS

2) Internal standardisation with labelled substances

Benzo(a)pyrene-13C4 Benzo(a)anthracene-13C6 Benzo(b)fluoranthene-13C6 Chrysene-13C6

2) LiquidLiquid partitioning 4) Solid phase extraction

B) Sonication

3617 1) HPLC-FLD

1) External calibration

4) Solid phase extraction

C) Soxhlet extraction

3618 6) GC-MS

2) Internal standardisation with labelled substances

deuterated standards

1) Saponification 4) Solid phase extraction

D) Other Handshaken with cyclohexane

3619 6) GC-MS

2) Internal standardisation with labelled substances

C13 Labelled US EPA 16

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

D) Other Homegenisation amp saponiofication

3620 9) GC-HRMS

2) Internal standardisation with labelled substances

mix of deuterated PAHs (BAA-D12 CHR-D12 BAP-D12 BBF-D12)

3) Gel permeation chromatography 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

3621 6) GC-MS

2) Internal standardisation with labelled substances

Chrysene D12 Benzo(a)pyrene D12 Benzo(b)fluoranthene D12 Benzo(a)anthracene

2) LiquidLiquid partitioning 4) Solid phase extraction

B) Sonication

3622 1) HPLC-FLD

4) Standard addition method

4) Solid phase extraction

D) Other

Extraction with cyclohexane by heating under reflux

3623 7) GC-MSMS

2) Internal standardisation with labelled substances

C13 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

3624 2) HPLC-UV-FLD

1) External calibration

2) LiquidLiquid partitioning

D) Other hot solvent extraction

3626

64

Table 19 - continued

Laboratory code

Analysis technique

Instrument calibration

Internal standards

applied

Sample clean up

Details of sample

clean up

Extraction technique

Details of sample

extraction

5301 7) GC-MSMS

2) Internal standardisation with labelled substances

EACH SUBSTANCE C13

4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5302 7) GC-MSMS

2) Internal standardisation with labelled substances

Isotope labelled PAHs 13C for each analyte

4) Solid phase extraction

C) Soxhlet extraction

5303 7) GC-MSMS

3) Internal standardisation with unlabelled substances

HAP C13 4) Solid phase extraction

D) Other automate ASE200 DIONEX

5304 7) GC-MSMS

2) Internal standardisation with labelled substances

with C13 for each HAP

2) LiquidLiquid partitioning 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5305 7) GC-MSMS

2) Internal standardisation with labelled substances

C13 isotopiques internal standards

4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5306 7) GC-MSMS

2) Internal standardisation with labelled substances

C13 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5307 6) GC-MS

2) Internal standardisation with labelled substances

1) Saponification 2) LiquidLiquid partitioning

D) Other solvent extraction

5308 1) HPLC-FLD 1) External calibration

4) Solid phase extraction

B) Sonication

5309 1) HPLC-FLD 1) External calibration

4) Solid phase extraction

D) Other only SPE

5310 1) HPLC-FLD 1) External calibration

1) Saponification 2) LiquidLiquid partitioning 3) Gel permeation chromatography

C) Soxhlet extraction

5311 6) GC-MS

2) Internal standardisation with labelled substances

13 C Chrysene 13 C Benz(a)pyrene

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

B) Sonication

5312 1) HPLC-FLD

3) Internal standardisation with unlabelled substances

Benzo(b)chrysen

2) LiquidLiquid partitioning 4) Solid phase extraction

Quechers D) Other Quechers

65

Table 19 - continued

Laboratory code

Analysis technique

Instrument calibration

Internal standards applied

Sample clean up Details of sample

clean up

Extraction technique

Details of sample extraction

5313 6) GC-MS

2) Internal standardisation with labelled substances

Benz(a)anthracene D12 Benzo(b)fluranthene D12 Benzo(a)pyrene D12 Chrysene D12

1) Saponification 2) LiquidLiquid partitioning 3) Gel permeation chromatography

A) Pressurised liquid extraction (PLE)

5314 6) GC-MS

2) Internal standardisation with labelled substances

Benzo(a)pyren d12 2) LiquidLiquid partitioning

D) Other Extraction with

5315 1) HPLC-FLD

3) Internal standardisation with unlabelled substances

Benz(b)chrysen

1) Saponification 2) LiquidLiquid partitioning

D) Other shaker

5316 1) HPLC-FLD

1) External calibration

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

D) Other

LiquidLiquid extraction with cyclohexane and dimethylformamide

5317 7) GC-MSMS

1) External calibration

3) Gel permeation chromatography 6) Other

silica gel D) Other VDLUFA online method

5318

5319 1) HPLC-FLD

3) Internal standardisation with unlabelled substances

Benzo(b)chrysene 3) Gel permeation chromatography

A) Pressurised liquid extraction (PLE)

5320 6) GC-MS

3) Internal standardisation with unlabelled substances

22-binaphthyl

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

D) Other 3 hours refluxing

5321 1) HPLC-FLD

2) Internal standardisation with labelled substances

6-meacutethylchrysegravene

1) Saponification 2) LiquidLiquid partitioning

B) Sonication

5322 6) GC-MS

2) Internal standardisation with labelled substances

naftalene-d8 acenaphtalene-d10 phenanthrene-d10 chrysene-d12 perylene-d12 benzo(e)anthracene-d12

1) Saponification 2) LiquidLiquid partitioning 6) Other

clean-up with silica packed column

C) Soxhlet extraction

5323 1) HPLC-FLD

1) External calibration

4) Solid phase extraction 6) Other

Accelerated Solvent Extraction

A) Pressurised liquid extraction (PLE)

66

European Commission EUR 25245 EN ndash Joint Research Centre ndash Institute for Reference Materials and Measurements Title Report on the 8th inter-laboratory comparison organised by the European Union Reference Laboratory for Polycyclic Aromatic Hydrocarbons - Four marker PAHs in a dry extract of St Johns wort Author(s) Radoslav Lizak Szilard Szilagyi Philippe Verlinde and Thomas WENZL Luxembourg Publications Office of the European Union 2012 ndash 66 pp ndash 201 x 297 cm EUR ndash Scientific and Technical Research series ndash ISSN 1831-9424 ISBN 978-92-79-23337-1 doi 10278759387 Abstract The proficiency test here reported concerned the determination of the contents of four marker polycyclic aromatic hydrocarbons (PAHs) and of their sum in an food supplements test sample The set of marker PAHs consists of benz[a]anthacene benzo[a]pyrene benzo[b]fluoranthene and chrysene Participants to these PT were National Reference Laboratories for PAHs (NRLs-PAHs) and EU official food control laboratories The number of participants was in total 48

The PT was organised according ISO Standard 170432010

The test material used was a commercial dry extract of St Johns wort Participants also received a solution of the same PAHs either in acetonitrile or in toluene for checking their instrument calibration

The results from participants were rated with z-scores and zeta-scores About 68 of the reported results were attributed with z-scores with an absolute value of below two which is the threshold for satisfactory performance

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science and technology for the Union Close to the policy-making process it serves

the common interest of the Member States while being independent of special

interests whether private or national

L

A-N

A-252

45-EN

-N

• 1 Summary
• 2 Introduction
• 3 Scope
• 4 Participating Laboratories
• 5 Time frame
• 6 Confidentiality
• 7 Test materials
• • 71 1Preparation of test materials
  • 72 Assigned values and standard deviation for proficiency assessment
  • 73 Homogeneity and stability
  • 74 Sample dispatch
  • • 8 Design of the proficiency test
    • 9 Evaluation of the results
    • • 91 2General
      • 92 21BEvaluation criteria
      • 93 Performance of participants
      • 94 Evaluation of the influence of analysis method
      • 95 Evaluation of the influence of calibration
      • 96 26Evaluation of compliance with legislation
      • • 10 Follow-up actions for underperforming laboratories
        • 11 1Conclusions
        • 12 1Acknowledgements
        • 13 References

31

14 ANNEXES

ANNEX 1 ndash Announcement of the PT on the IRMM webpage

ANNEX 2 ndash Announcement via e-mail and invitation

ANNEX 3 ndash Announcement of sample dispatch

ANNEX 4 ndash Technical specifications of the calibration solutions

ANNEX 5 ndash Homogeneity and stability of the test material

ANNEX 6 ndash Acknowledgement of sample receipt

ANNEX 7 ndash Questionnaire on details of analysis method

ANNEX 8 ndash Results reported for the food supplement sample

ANNEX 9 ndash Results reported for the standard solutions with undisclosed analyte content

ANNEX 10 ndash Method performance characteristics reported by participants

ANNEX 11 ndash Details of analysis methods reported by participants

32

ANNEX 1 ndash Announcement of the PT on the IRMM webpage

33

ANNEX 2 - Announcement via e-mail and invitation

34

ANNEX 3 - Announcement sample dispatch

Dear MadameSir

The test samples for the proficiency test on the determination of four PAHs in a food supplement (dry extract of St Johns wort) were

dispatched today

Explanation on the design of the study as well as instructions for sample storage and for reporting of results are in the parcel You will

also find your participant key in the parcel which is required for reporting of results

Please check the parcel after arrival for completeness and report the receipt of the test samples as well as their status to us by applying

the sample receipt form which is in the parcel

You are kindly requested to inform us by email if you do not receive the parcel by end of this week

My colleague Donata Lerda will then take care of it

With best regards

Thomas Wenzl

35

ANNEX 4 - Specifications of standard solutions with disclosed concentration

36

ANNEX 4 - continued

37

ANNEX 5 - Homogeneity and stability of the test material

Homogeneity of the test material

Benz[a]anthracene - BAA

Benzo[a]pyrene - BAP

38

ANNEX 5 - continued

Benzo[b]fluoranthene - BBF

Chrysene - CHR

39

ANNEX 5 - continued

Stability of the test material

Table 10 Content of the four target analytes in test materials stored under different

temperatures

BaA BaP BbF CHR

microgkg microgkg microgkg microgkg

Content 31 16 31 69 Storage at room temperature

U (k=2) 05 02 05 17

Content 31 15 29 68 Storage at reference temperature

U (k=2) 04 03 04 10

The applied capillary column does not separate CHR from triphenylene

40

ANNEX 6 ndash Documents sent to participants

41

ANNEX 6 - continued

42

ANNEX 7 Questionnaire on details of analysis method

43

ANNEX 7 - continued

44

ANNEX 7 - continued

45

ANNEX 8 - Results reported by participants for the food supplements test sample Table 11 Analysis results reported by the participants for the content of benz[a]anthracene (BAA) in the food supplements test material Values are presented as reported

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

3600 31 29 29 3 05 2

3602 199 193 178 19 021

3603 253 253 246 251 022 2

3604 199 209 23 213 071 2

3605 301 3 271 291 076 2

3606 302 288 278 289 064 2

3607 456 416 443 438 096 2

3608 796 695 651 714 149 2

3610 182 192 214 196 039 2

3611 414 504 488 469 96 2

3612 3 34 28 31 06 2

3613 32 33 34 33 07 2

3614 182 182 191 185 037 2

3615 3884 3822 3565 376 0752 2

3616 2 2 2 2 01 2

3617 366 37 35 362 046 2

3618 32 32 32 32 05 2

3619 299 305 297 299 048 2

3620 304 294 302 302 045 2

3621 29 26 29 28 04 2

3622 343 325 318 322 052 2

3623 35 3 33 33 07 2

3624

3626 318 31 314 314 126 2

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

5301 31 3 32 31 06 2

5302 314 319 315 316 063 2

5303 3102 2716 2822 2876 27 62

5304 261 263 26 261 57 2

5305 23 22 26 24 20

5306 22 23 23 22 11 2

5307 488 461 317 461

5308 168633 168544 1562025 1644598

5309 16 19 16 17

5310 3 304 311 305 015

5311 477 452 448 459 15 2

5312 506 63 494 543 134 2

5313 353 301 404 353

5314 431 373 378 394 5 096

5315 596 531 51 545 11 2

5316 224 231 235 235 022 2

5317 216 23 221 222 007 2

5318 064 07 067 01 2

5319 111 184 146 147 029 2

5320 515 525 564 534 48

5321 21 5322 13 87 9 87 35 2

5323 38 33 35 33 16 2

46

Figure 6 Results of replicate determinations (indicated by triangles) of benz[a]anthracene (BAA) in the food supplement test material

Horizontal blue lines represent the arithmetic mean value of replicate measurements and blue bars the reported expanded measurement uncertainty (k=2) The assigned value is

plotted as dotted line Red solid lines mark the range of satisfactory performance (|z|le2) Mean values are given for results outside the displayed data range

ProLab 2011

Laboratory

53

1853

1953

0853

0936

1436

0236

1036

1653

2136

0453

1753

0653

1653

0536

0353

0436

2153

0336

0636

0536

0036

2036

1953

1036

1253

0136

2653

0236

1836

2336

2236

1353

1353

2336

1736

1553

1453

0736

0753

1153

2053

1253

1536

0853

2236

11

an

aly

te c

on

ten

t (micro

gk

g)

10

95

9

85

8

75

7

65

6

55

5

45

4

35

3

25

2

15

1

05

0

102

3 468

7

Sample St Johns wort extract replicatesMeasurand Benz[a]anthraceneNo of laboratories 46

Assigned value 298 microgkg (Reference value)Target sd 062 microgkgTolerance limits 174 - 422 microgkg (|Z score| lt 200)

Assig

ned va

lue

Limit of tolerance

Limit of tolerance

47

Table 12 Analysis results reported by the participants for the content of benzo[a]pyrene (BAP) in the food supplements test material Values are presented as reported

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

3600 16 17 17 17 03 2

3602 118 123 13 124 015

3603 165 162 157 161 015 2

3604 134 154 139 143 04 2

3605 164 166 172 167 057 2

3606 181 159 161 167 031 2

3607 279 266 279 274 055 2

3608 57 687 461 573 226 2

3610 226 216 183 206 042 2

3611 289 325 311 308 36 2

3612 24 22 2 22 05 2

3613 17 18 19 18 05 2

3614 08 08 08 08 016 2

3615 1545 1393 1425 145 029 2

3616 16 16 14 15 03 2

3617 177 171 158 169 023 2

3618 2 2 2 2 02 2

3619 207 207 206 207 075 2

3620 165 169 163 166 017 2

3621 44 47 49 47 05 2

3622 212 208 222 213 038 2

3623 2 21 22 21 03 2

3624 18 178 182 18 031 2

3626 172 173 178 174 07 2

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

5301 18 18 18 18 04 2

5302 188 19 187 188 038 2

5303 1691 1426 149 1535 19 80

5304 145 142 15 146 69 2

5305 15 17 18 16 15

5306 16 17 17 16 08 2

5307 164 157 153 157

5308 102669 109324 109035 107009

5309 1 1 12 11

5310 122 122 124 123 006

5311 199 184 215 2 15 2

5312 258 248 239 248 05 2

5313 199 178 245 207

5314 174 171 172 172 11 0948

5315 253 248 213 238 05 2

5316 177 171 173 17 016 2

5317 255 277 276 269 012 2

5318 114 12 117 02 2

5319 19 228 231 216 043 2

5320 303 3 344 316 78

5321 12 12 12 12

5322 13 17 24 17 35 2

5323 2 2 19 2 1 2

48

Figure 7 Results of replicate determinations (indicated by triangles) of benzo[a]pyrene (BAP) in the food supplement test material

Horizontal blue lines represent the arithmetic mean value of replicate measurements and blue bars the reported expanded measurement uncertainty (k=2) The assigned value is

plotted as dotted line Red solid lines mark the range of satisfactory performance (|z|le2) Mean values are given for results outside the displayed data range

ProLab 2011

Laboratory

36

1453

0953

0853

1853

2153

1036

0236

0436

1553

0436

1653

0353

0736

0336

2036

0053

0553

0636

0636

0536

1753

1453

1636

2636

1336

2453

0153

2253

0253

2353

1136

1836

1953

1336

1036

2336

2253

1936

1253

1553

1253

1736

0753

2036

2136

0836

11

an

aly

te c

on

ten

t (micro

gk

g)

55

5

45

4

35

3

25

2

15

1

05

0

573

308

3

Sample St Johns wort extract replicatesMeasurand Benzo[a]pyreneNo of laboratories 48

Assigned value 165 microgkg (Reference value)Target sd 036 microgkgTolerance limits 093 - 237 microgkg (|Z score| lt 200)

Assig

ned va

lue

Limit of tolerance

Limit of tolerance

49

Table 13 Analysis results reported by the participants for the content of benzo[b]fluoranthene (BBF) in the food supplements test material Values are presented as reported

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

3600 32 3 3 31 09 2

3602 173 175 16 17 017

3603 265 252 25 256 025 2

3604 116 108 105 11 028 2

3605 286 225 232 247 074 2

3606 294 273 261 276 046 2

3607 579 579 544 567 113 2

3608 103 1201 876 1036 326 2

3610 173 196 204 191 038 2

3611 546 618 565 576 75 2

3612 3 28 28 29 06 2

3613 28 31 32 31 06 2

3614 135 111 135 127 022 2

3615 4187 3678 4111 4 08 2

3616 2 2 2 2 01 2

3617 33 27 299 035 2

3618 31 31 31 31 05 2

3619 326 324 312 326 064 2

3620 277 28 272 276 069 2

3621 26 25 23 25 04 2

3622 346 336 333 34 052 2

3623 26 28 29 28 02 2

3624

3626 288 28 307 292 117 2

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

5301 29 3 29 29 06 2

5302 297 3 296 298 06 2

5303 2705 2444 2608 2586 22 78

5304 245 254 26 253 85 2

5305 23 23 26 24 15

5306 42 43 4 42 21 2

5307 221 216 241 221

5308 232169 246654 240722 229848

5309 19 22 19 2 0

5310 3 315 325 313 016

5311 262 277 304 281 15 2

5312 27 289 337 299 075 2

5313 321 284 378 328

5314 423 404 385 404 34 083

5315 42 506 416 444 09 2

5316 265 267 277 273 018 2

5317 185 197 195 192 006 2

5318 276 281 279 04 2

5319 396 389 403 396 079 2

5320 623 618 635 625 14

5321 31

5322 24 33 49 33 35 2

5323 38 45 35 45 27 2

50

Figure 8 Results of replicate determinations (indicated by triangles) of benzo[b]fluoranthene (BBF) in the food supplement test material

Horizontal blue lines represent the arithmetic mean value of replicate measurements and blue bars the reported expanded measurement uncertainty (k=2) The assigned value is

plotted as dotted line Red solid lines mark the range of satisfactory performance (|z|le2) Mean values are given for results outside the displayed data range

ProLab 2011

Laboratory

36

0436

1436

0236

1053

1736

1653

0953

0753

0853

0536

2136

0553

0436

0353

0353

1636

0636

2036

2353

1853

1136

1236

2653

0153

0253

1236

1736

1336

0036

1853

2153

1036

1953

1336

2253

2253

2353

1936

1553

1453

0653

1536

0753

2036

0836

11

an

aly

te c

on

ten

t (micro

gk

g)

75

7

65

6

55

5

45

4

35

3

25

2

15

1

05

0

103

6

576

3

Sample St Johns wort extract replicatesMeasurand Benzo[b]fluorantheneNo of laboratories 46

Assigned value 292 microgkg (Reference value)Target sd 060 microgkgTolerance limits 172 - 412 microgkg (|Z score| lt 200)

Assig

ned va

lue

Limit of tolerance

Limit of tolerance

51

Table 14 Analysis results reported by the participants for the content of chrysene (CHR) in the food supplements test material Values are presented as reported

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

3600 5 49 5 5 07 2

3602 32 304 29 304 036

3603 462 469 447 459 048 2

3604 438 428 42 429 119 2

3605 365 41 381 386 085 2

3606 494 411 45 451 124 2

3607 897 774 787 819 18 2

3608 986 887 1168 1014 284 2

3610 418 392 462 424 085 2

3611 833 893 87 865 61 2

3612 6 64 58 61 12 2

3613 41 39 4 4 08 2

3614 272 254 288 271 054 2

3615 605 6028 5964 601 12 2

3616 47 45 49 47 05 2

3617 445 431 389 422 065 2

3618 45 45 45 45 06 2

3619 738 747 72 738 118 2

3620 463 472 446 46 092 2

3621 53 49 51 51 06 2

3622 478 513 505 488 08 2

3623 73 73 72 72 05 2

3624

3626 63 622 634 628 251 2

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

5301 49 49 49 49 1 2

5302 687 683 666 679 136 2

5303 6777 6181 6382 6441 24 70

5304 592 581 6 591 85 2

5305 6 58 64 61 20

5306 49 5 51 49 25 2

5307 524 5 574 524

5308 211488 216782 221146 213243

5309

5310 372 393 394 386 019

5311 43 459 422 437 15 2

5312 934 943 981 953 191 2

5313 793 73 99 838

5314 636 561 565 588 18 0818

5315 547 583 499 542 11 2

5316 292 286 285 282 06 2

5317 52 598 517 545 046 2

5318 368 345 357 05 2

5319 449 471 474 465 116 2

5320 76 924 818 834 99

5321 33

5322 7 7 76 7 35 2

5323 87 73 77 73 37 2

52

Figure 9 Results of replicate determinations (indicated by triangles) of chrysene (CHR) in the food supplement test material

Horizontal blue lines represent the arithmetic mean value of replicate measurements and blue bars the reported expanded measurement uncertainty (k=2) The assigned value is

plotted as dotted line Red solid lines mark the range of satisfactory performance (|z|le2) Mean values are given for results outside the displayed data range

ProLab 2011

Laboratory

53

0836

1453

1636

0253

2153

1836

0553

1036

1336

1736

1036

0453

1136

1836

0636

0336

2053

1936

1653

0136

0036

2253

0636

2153

0753

1553

1753

1453

0436

1536

1253

0536

2653

0353

0253

2236

2336

1953

2336

0753

2053

1353

1236

0836

11

an

aly

te c

on

ten

t (micro

gk

g)

13

12

11

10

9

8

7

6

5

4

3

2

1

0

865

3

Sample St Johns wort extract replicatesMeasurand ChryseneNo of laboratories 45

Assigned value 407 microgkg (Reference value)Target sd 083 microgkgTolerance limits 241 - 573 microgkg (|Z score| lt 200)

Assig

ned va

lue

Limit of tolerance

Limit of tolerance

53

Table 15 Analysis results reported by the participants for the sum of the four marker PAHs in the food supplements test material Values are presented as reported

Laboratory code

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg

3600 128 24 2

3602 79 126

3603 1127 096 2

3604 893 258 2

3605 1091 305 2

3606 1184 238 2

3607 2098 42 2

3608 3336 325 2

3610 1138 17 2

3611 2218 259 2

3612 143 29 2

3613 122 2 2

3614 663 129 2

3615 152 304 2

3616 102 03 2

3617 1252 169 2

3618 128 18 2

3619 157 305 2

3620 1204 12 2

3621 151 19 2

3622 1361 115 2

3623 154 09 2

3624

3626 1409 56 2

Laboratory code

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg

5301 126

5302 37 074 2

5303 13438 92

5304 1251 85 2

5305 125 20

5306 129 65 2

5307 1363

5308 7245598

5309

5310 1127 056

5311 1367 15 2

5312 2042 408 2

5313 1726

5314 1558

5315 177 35 2

5316 96 073 2

5317 1229 066 2

5318 82 05 2

5319 1225 267 2

5320 2309 41

5321

5322 207 35 2

5323 171 103 2

54

ANNEX 9 Results reported for the standard solutions with undisclosed analyte content Table 16 Analysis results reported by the participants for the four marker PAHs in the standard solution in toluene with undisclosed analyte

content Values are presented as reported U measurement uncertainty k coverage factor

benz[a]anthracene benzo[a]pyrene benzo[b]fluoranthene chrysene Laboratory

code Unit Result U k Result U k Result U k Result U k 3600 microgl 38 67 2 25 45 2 211 68 2 275 38 2

3605 microgl 367 184 2 2482 124 2 2592 13 2 235 118 2

3606 microgkg 3763 176 2 2609 142 2 2721 118 2 2539 134 2

3607 microgkg 3706 815 2 2824 565 2 2591 518 2 2587 569 2

3612 microgl 38 08 2 237 11 2 244 18 2 257 02 2

3614 microgl 324 65 2 209 38 2 215 37 2 21 42 2

3616 microgkg 318 07 2 224 14 2 231 26 2 216 1 2

3618 microgkg 417 17 2 284 08 2 292 06 2 297 03 2

3619 microgkg 47 742 2 274 468 2 228 387 2 274 434 2

3620 microgl 495 74 2 294 29 2 321 8 2 321 64 2

3621 microgl 241 22 2 143 13 2 188 16 2 145 14 2

3623 microgkg 476 25 2 311 53 2 269 3 2 297 03 2

3626 microgl 359 38 2 235 23 2 238 27 2 24 18 2

5301 microgkg 426 29 299 289

5302 microgkg 436 87 2 296 59 2 296 59 2 289 58 2

5303 microgl 3721 27 102 22838 19 99 23756 22 99 25486 24 98

5304 microgkg 38 29 2 225 26 2 257 37 2 258 52 2

5305 microgkg 305 225 245 29

5306 microgkg 316 63 2 247 49 2 65 13 2 22 44 2

5307 microgkg 3658 2559 241 2414

5308 microgkg 16854 109324 246654 216782

5311 microgl 374 20 2 279 15 2 268 15 2 272 15 2

5313 microgl 3408 2165 2275 2373

5314 microgkg 395 5 2875 11 3614 34 2905 18

5320 microgl 3267 368 1986 571 28 683 2427 512

5322 microgl 282 35 2 225 35 2 241 35 2 21 35 2

55

Figure 10 Youden plots of the percent deviation of the reported values from the assigned values for the food supplement test sample and the

standard solution in toluene with undisclosed content

(The identity of the respective measurand is given above the figure Results exceeding a difference of more than 100 were omitted)

56

Figure 10 - continued

57

Table 17 Analysis results reported by the participants for the four marker PAHs in the standard solution in acetonitrile with undisclosed

analyte content

Values are presented as reported U measurement uncertainty k coverage factor

benz[a]anthracene benzo[a]pyrene benzo[b]fluoranthene chrysene

Laboratory

code Unit Result U k Result U k Result U k Result U k

3602 microgkg 3675 247 247 244

3603 microgkg 358 086 2 236 028 2 235 028 2 236 057 2

3604 microgl 475 29 2 19 12 2 194 13 2 258 12 2

3608 microgl 3623 078 2 2385 023 2 2343 18 2 2296 092 2

3610 microgkg 369 185 2 25 125 2 251 126 2 244 122 2

3611 microgkg 48 02 2 323 02 2 328 04 2 317 03 2

3613 microgl 332 31 2 261 3 2 249 25 2 239 24 2

3615 microgl 415 2635 2729 2689

3617 microgkg 37 09 2 244 06 2 284 07 2 239 06 2

3622 microgl 37 12 2 25 23 2 257 21 2 238 1 2

3624 microgkg 402 104 2 255 084 2 265 096 2 253 07 2

5306 microgkg 563 113 2 371 74 2 359 72 2 333 67 2

5308 microgkg 168633 102669 232169 211488

5309 microgl 402 268 27

5310 microgl 315 158 25 125 25 125 288 144

5312 microgl 428 043 2 282 028 2 272 027 2 275 028 2

5315 microgkg 387 08 2 256 05 2 251 05 2 253 05 2

5316 microgl 3839 342 2 2485 191 2 2559 168 2 239 51 2

5317 microgl 2466 043 2 2212 034 2 2472 016 2 2146 068 2

5319 microgl 414 41 2 279 28 2 268 27 2 26 31 2

5321 microgkg 476 316 326 328

5322 microgl 2547 35 2

5323 microgkg 405 203 2 239 119 2 255 153 2 201 11 2

58

Figure 11 Youden plots of the percent deviation of the reported values from the assigned values for the food supplement test sample and the

standard solution in acetonitrile with undisclosed content

(The identity of the respective measurand is given above the figure Results exceeding a difference of more than 100 were omitted)

59

Figure 11 - continued

60

ANNEX 10 Table 18 Method performance parameters reported by the participants

Laboratory code

Analyte LOD LOQ Recovery Analyte LOD LOQ Recovery

microgkg microgkg microgkg microgkg 3600 BaA 03 06 978 BaP 03 07 9483602 BaA 007 021 926 BaP 005 015 993603 BaA 0011 05 987 BaP 0005 051 9553604 BaA 02 04 86 BaP 02 04 883605 BaA 0025 005 118 BaP 0025 005 1083606 BaA 001 003 65 BaP 001 003 703607 BaA 021 071 74 BaP 027 089 943608 BaA 016 048 8213 BaP 015 046 83683610 BaA 02 05 85 BaP 005 02 853611 BaA BaP 3612 BaA 1 117 BaP 1 1203613 BaA 01 03 90 BaP 01 03 903614 BaA 02 05 96 BaP 02 05 933615 BaA 005 015 110 BaP 005 015 1053616 BaA 01 05 108 BaP 01 05 753617 BaA 018 037 405 BaP 009 018 3873618 BaA 01 03 63 BaP 01 03 593619 BaA 005 005 59 BaP 033 033 543620 BaA 0009 0018 64 BaP 0005 001 553621 BaA 007 02 84 BaP 007 02 923622 BaA 01 03 1101 BaP 01 03 9853623 BaA 02 08 105 BaP 02 08 1013624 BaA BaP 01 033 8513625 BaA BaP 3626 BaA BaP 5301 BaA 003 01 102 BaP 003 01 1005302 BaA 01 03 78 BaP 01 03 865303 BaA 003 01 62 BaP 003 01 805304 BaA 1 1 70 BaP 1 1 955305 BaA 02 05 85 BaP 02 05 855306 BaA 03 1 699 BaP 03 1 905307 BaA 6537 BaP 81165308 BaA 0088 0265 99 BaP 00262 00785 1005309 BaA 01 08 92 BaP 01 08 925310 BaA 01 03 BaP 01 03 905311 BaA 002 005 80 BaP 002 004 915312 BaA 03 09 105 BaP 03 09 1055313 BaA 04 08 BaP 04 08 5314 BaA 001 002 96 BaP 001 002 9485315 BaA 01 03 50 - 100 BaP 01 03 50 - 100 5316 BaA 002 011 827 BaP 004 019 10445317 BaA 05 1 76 BaP 05 1 755318 BaA BaP 5319 BaA 009 029 951 BaP 011 041 995320 BaA 12 18 946 BaP 06 1 8345321 BaA 03 05 BaP 05 1 5322 BaA 1 2 100 BaP 1 2 1005323 BaA 26 BaP 26

61

ANNEX 10 Table 18 - continued

Laboratory code Analyte LOD LOQ Recovery Analyte LOD LOQ Recovery

microgkg microgkg microgkg microgkg 3600 BbF 03 05 96 CHR 02 03 10153602 BbF 015 045 947 CHR 003 009 9193603 BbF 0014 05 1012 CHR 0004 05 10033604 BbF 02 04 85 CHR 02 04 873605 BbF 005 01 94 CHR 0025 005 863606 BbF 001 003 70 CHR 001 003 653607 BbF 018 06 100 CHR 014 028 943608 BbF 015 046 7979 CHR 015 045 71253610 BbF 005 02 85 CHR 02 05 853611 BbF CHR 3612 BbF 1 118 CHR 1 1173613 BbF 01 03 90 CHR 01 03 903614 BbF 01 04 96 CHR 02 05 953615 BbF 008 025 109 CHR 003 01 1083616 BbF 01 05 78 CHR 01 05 723617 BbF 035 07 38 CHR 012 024 4163618 BbF 01 03 62 CHR 01 03 623619 BbF 016 016 63 CHR 01 01 593620 BbF 0003 0006 55 CHR 001 002 653621 BbF 007 02 103 CHR 007 02 893622 BbF 02 06 963 CHR 03 09 1043623 BbF 02 08 106 CHR 02 08 1193624 BbF CHR 3625 BbF CHR 3626 BbF CHR 5301 BbF 003 01 96 CHR 003 01 1145302 BbF 01 03 84 CHR 01 03 835303 BbF 003 01 78 CHR 003 01 705304 BbF 1 1 86 CHR 1 1 705305 BbF 02 05 90 CHR 02 05 955306 BbF 03 1 1435 CHR 03 1 1185307 BbF 8116 CHR 65375308 BbF 0079 0238 99 CHR 009 026 1005309 BbF 01 08 92 CHR 5310 BbF 07 2 CHR 01 03 5311 BbF 002 004 95 CHR 002 004 895312 BbF 03 09 105 CHR 03 09 1055313 BbF 04 08 CHR 04 08 5314 BbF 001 002 83 CHR 001 002 8185315 BbF 02 05 50 -100 CHR 02 05 50 - 100 5316 BbF 005 024 814 CHR 001 007 9595317 BbF 05 1 73 CHR 05 1 785318 BbF CHR 5319 BbF 011 038 946 CHR 013 043 9675320 BbF 07 09 1194 CHR 23 32 10455321 BbF 05 1 CHR 05 1 5322 BbF 1 2 100 CHR 1 2 1005323 BbF 26 CHR 26

62

ANNEX 11 Table 19 Details of analysis method reported by the participants Data are presented as reported

Laboratory code

Analysis technique

Instrument calibration

Internal standards applied

Sample clean up

Details of sample

clean up

Extraction technique

Details of sample

extraction

3600 6) GC-MS

2) Internal standardisation with labelled substances

Deuterated PAH mix 9 (Ehrenstorfer)

1) Saponification 2) LiquidLiquid partitioning

D) Other Liquidliquid partitioning

3602 2) HPLC-UV-FLD

1) External calibration

3) Gel permeation chromatography

A) Pressurised liquid extraction (PLE)

3603 1) HPLC-FLD

1) External calibration

3) Gel permeation chromatography

D) Other 1 minute with dichloromethane in vortex

3604 2) HPLC-UV-FLD

2) Internal standardisation with labelled substances

DiP D14

5) Donor acceptor complex chromatography

D) Other LL

3605 1) HPLC-FLD

1) External calibration

3) Gel permeation chromatography

C) Soxhlet extraction

3606 7) GC-MSMS

2) Internal standardisation with labelled substances

BaP 13C4 CHR 13C6 BaA 13C6 BbF 13C6

4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

3607 6) GC-MS

2) Internal standardisation with labelled substances

BaA-d12 CHR-d12 BbF-d12 BkF-d12 BaP-d12 ICP-d12 BgP-d12 DiP-d14

1) Saponification

D) Other solvent partitioning

3608 1) HPLC-FLD

1) External calibration

6) Other Liquid extraction

D) Other

Extraction in a rotary agitator and centrifugation

3610 2) HPLC-UV-FLD

1) External calibration

4) Solid phase extraction

B) Sonication

3611

3612 6) GC-MS

2) Internal standardisation with labelled substances

chrysene-d12 dibenzo(ah)anthracene-d14 dibenzo(ai)pyrene-d14

4) Solid phase extraction

B) Sonication

3613 5) LC-MSMS

3) Internal standardisation with unlabelled substances

I use Chrysene-D12 3) Gel permeation chromatography

D) Other extraction with hexan

63

Table 19 - continued

Laboratory code

Analysis technique

Instrument calibration

Internal standards applied

Sample clean up Details of sample

clean up

Extraction technique

Details of sample

extraction

3614 6) GC-MS 1) External calibration

3) Gel permeation chromatography

B) Sonication

3615 1) HPLC-FLD

1) External calibration

6) Other PLE GPC

A) Pressurised liquid extraction (PLE)

3616 6) GC-MS

2) Internal standardisation with labelled substances

Benzo(a)pyrene-13C4 Benzo(a)anthracene-13C6 Benzo(b)fluoranthene-13C6 Chrysene-13C6

2) LiquidLiquid partitioning 4) Solid phase extraction

B) Sonication

3617 1) HPLC-FLD

1) External calibration

4) Solid phase extraction

C) Soxhlet extraction

3618 6) GC-MS

2) Internal standardisation with labelled substances

deuterated standards

1) Saponification 4) Solid phase extraction

D) Other Handshaken with cyclohexane

3619 6) GC-MS

2) Internal standardisation with labelled substances

C13 Labelled US EPA 16

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

D) Other Homegenisation amp saponiofication

3620 9) GC-HRMS

2) Internal standardisation with labelled substances

mix of deuterated PAHs (BAA-D12 CHR-D12 BAP-D12 BBF-D12)

3) Gel permeation chromatography 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

3621 6) GC-MS

2) Internal standardisation with labelled substances

Chrysene D12 Benzo(a)pyrene D12 Benzo(b)fluoranthene D12 Benzo(a)anthracene

2) LiquidLiquid partitioning 4) Solid phase extraction

B) Sonication

3622 1) HPLC-FLD

4) Standard addition method

4) Solid phase extraction

D) Other

Extraction with cyclohexane by heating under reflux

3623 7) GC-MSMS

2) Internal standardisation with labelled substances

C13 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

3624 2) HPLC-UV-FLD

1) External calibration

2) LiquidLiquid partitioning

D) Other hot solvent extraction

3626

64

Table 19 - continued

Laboratory code

Analysis technique

Instrument calibration

Internal standards

applied

Sample clean up

Details of sample

clean up

Extraction technique

Details of sample

extraction

5301 7) GC-MSMS

2) Internal standardisation with labelled substances

EACH SUBSTANCE C13

4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5302 7) GC-MSMS

2) Internal standardisation with labelled substances

Isotope labelled PAHs 13C for each analyte

4) Solid phase extraction

C) Soxhlet extraction

5303 7) GC-MSMS

3) Internal standardisation with unlabelled substances

HAP C13 4) Solid phase extraction

D) Other automate ASE200 DIONEX

5304 7) GC-MSMS

2) Internal standardisation with labelled substances

with C13 for each HAP

2) LiquidLiquid partitioning 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5305 7) GC-MSMS

2) Internal standardisation with labelled substances

C13 isotopiques internal standards

4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5306 7) GC-MSMS

2) Internal standardisation with labelled substances

C13 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5307 6) GC-MS

2) Internal standardisation with labelled substances

1) Saponification 2) LiquidLiquid partitioning

D) Other solvent extraction

5308 1) HPLC-FLD 1) External calibration

4) Solid phase extraction

B) Sonication

5309 1) HPLC-FLD 1) External calibration

4) Solid phase extraction

D) Other only SPE

5310 1) HPLC-FLD 1) External calibration

1) Saponification 2) LiquidLiquid partitioning 3) Gel permeation chromatography

C) Soxhlet extraction

5311 6) GC-MS

2) Internal standardisation with labelled substances

13 C Chrysene 13 C Benz(a)pyrene

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

B) Sonication

5312 1) HPLC-FLD

3) Internal standardisation with unlabelled substances

Benzo(b)chrysen

2) LiquidLiquid partitioning 4) Solid phase extraction

Quechers D) Other Quechers

65

Table 19 - continued

Laboratory code

Analysis technique

Instrument calibration

Internal standards applied

Sample clean up Details of sample

clean up

Extraction technique

Details of sample extraction

5313 6) GC-MS

2) Internal standardisation with labelled substances

Benz(a)anthracene D12 Benzo(b)fluranthene D12 Benzo(a)pyrene D12 Chrysene D12

1) Saponification 2) LiquidLiquid partitioning 3) Gel permeation chromatography

A) Pressurised liquid extraction (PLE)

5314 6) GC-MS

2) Internal standardisation with labelled substances

Benzo(a)pyren d12 2) LiquidLiquid partitioning

D) Other Extraction with

5315 1) HPLC-FLD

3) Internal standardisation with unlabelled substances

Benz(b)chrysen

1) Saponification 2) LiquidLiquid partitioning

D) Other shaker

5316 1) HPLC-FLD

1) External calibration

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

D) Other

LiquidLiquid extraction with cyclohexane and dimethylformamide

5317 7) GC-MSMS

1) External calibration

3) Gel permeation chromatography 6) Other

silica gel D) Other VDLUFA online method

5318

5319 1) HPLC-FLD

3) Internal standardisation with unlabelled substances

Benzo(b)chrysene 3) Gel permeation chromatography

A) Pressurised liquid extraction (PLE)

5320 6) GC-MS

3) Internal standardisation with unlabelled substances

22-binaphthyl

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

D) Other 3 hours refluxing

5321 1) HPLC-FLD

2) Internal standardisation with labelled substances

6-meacutethylchrysegravene

1) Saponification 2) LiquidLiquid partitioning

B) Sonication

5322 6) GC-MS

2) Internal standardisation with labelled substances

naftalene-d8 acenaphtalene-d10 phenanthrene-d10 chrysene-d12 perylene-d12 benzo(e)anthracene-d12

1) Saponification 2) LiquidLiquid partitioning 6) Other

clean-up with silica packed column

C) Soxhlet extraction

5323 1) HPLC-FLD

1) External calibration

4) Solid phase extraction 6) Other

Accelerated Solvent Extraction

A) Pressurised liquid extraction (PLE)

66

European Commission EUR 25245 EN ndash Joint Research Centre ndash Institute for Reference Materials and Measurements Title Report on the 8th inter-laboratory comparison organised by the European Union Reference Laboratory for Polycyclic Aromatic Hydrocarbons - Four marker PAHs in a dry extract of St Johns wort Author(s) Radoslav Lizak Szilard Szilagyi Philippe Verlinde and Thomas WENZL Luxembourg Publications Office of the European Union 2012 ndash 66 pp ndash 201 x 297 cm EUR ndash Scientific and Technical Research series ndash ISSN 1831-9424 ISBN 978-92-79-23337-1 doi 10278759387 Abstract The proficiency test here reported concerned the determination of the contents of four marker polycyclic aromatic hydrocarbons (PAHs) and of their sum in an food supplements test sample The set of marker PAHs consists of benz[a]anthacene benzo[a]pyrene benzo[b]fluoranthene and chrysene Participants to these PT were National Reference Laboratories for PAHs (NRLs-PAHs) and EU official food control laboratories The number of participants was in total 48

The PT was organised according ISO Standard 170432010

The test material used was a commercial dry extract of St Johns wort Participants also received a solution of the same PAHs either in acetonitrile or in toluene for checking their instrument calibration

The results from participants were rated with z-scores and zeta-scores About 68 of the reported results were attributed with z-scores with an absolute value of below two which is the threshold for satisfactory performance

How to obtain EU publications

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sending a fax to (352) 29 29-42758

The mission of the JRC is to provide customer-driven scientific and technical support

for the conception development implementation and monitoring of EU policies As a

service of the European Commission the JRC functions as a reference centre of

science and technology for the Union Close to the policy-making process it serves

the common interest of the Member States while being independent of special

interests whether private or national

L

A-N

A-252

45-EN

-N

• 1 Summary
• 2 Introduction
• 3 Scope
• 4 Participating Laboratories
• 5 Time frame
• 6 Confidentiality
• 7 Test materials
• • 71 1Preparation of test materials
  • 72 Assigned values and standard deviation for proficiency assessment
  • 73 Homogeneity and stability
  • 74 Sample dispatch
  • • 8 Design of the proficiency test
    • 9 Evaluation of the results
    • • 91 2General
      • 92 21BEvaluation criteria
      • 93 Performance of participants
      • 94 Evaluation of the influence of analysis method
      • 95 Evaluation of the influence of calibration
      • 96 26Evaluation of compliance with legislation
      • • 10 Follow-up actions for underperforming laboratories
        • 11 1Conclusions
        • 12 1Acknowledgements
        • 13 References

32

ANNEX 1 ndash Announcement of the PT on the IRMM webpage

33

ANNEX 2 - Announcement via e-mail and invitation

34

ANNEX 3 - Announcement sample dispatch

Dear MadameSir

The test samples for the proficiency test on the determination of four PAHs in a food supplement (dry extract of St Johns wort) were

dispatched today

Explanation on the design of the study as well as instructions for sample storage and for reporting of results are in the parcel You will

also find your participant key in the parcel which is required for reporting of results

Please check the parcel after arrival for completeness and report the receipt of the test samples as well as their status to us by applying

the sample receipt form which is in the parcel

You are kindly requested to inform us by email if you do not receive the parcel by end of this week

My colleague Donata Lerda will then take care of it

With best regards

Thomas Wenzl

35

ANNEX 4 - Specifications of standard solutions with disclosed concentration

36

ANNEX 4 - continued

37

ANNEX 5 - Homogeneity and stability of the test material

Homogeneity of the test material

Benz[a]anthracene - BAA

Benzo[a]pyrene - BAP

38

ANNEX 5 - continued

Benzo[b]fluoranthene - BBF

Chrysene - CHR

39

ANNEX 5 - continued

Stability of the test material

Table 10 Content of the four target analytes in test materials stored under different

temperatures

BaA BaP BbF CHR

microgkg microgkg microgkg microgkg

Content 31 16 31 69 Storage at room temperature

U (k=2) 05 02 05 17

Content 31 15 29 68 Storage at reference temperature

U (k=2) 04 03 04 10

The applied capillary column does not separate CHR from triphenylene

40

ANNEX 6 ndash Documents sent to participants

41

ANNEX 6 - continued

42

ANNEX 7 Questionnaire on details of analysis method

43

ANNEX 7 - continued

44

ANNEX 7 - continued

45

ANNEX 8 - Results reported by participants for the food supplements test sample Table 11 Analysis results reported by the participants for the content of benz[a]anthracene (BAA) in the food supplements test material Values are presented as reported

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

3600 31 29 29 3 05 2

3602 199 193 178 19 021

3603 253 253 246 251 022 2

3604 199 209 23 213 071 2

3605 301 3 271 291 076 2

3606 302 288 278 289 064 2

3607 456 416 443 438 096 2

3608 796 695 651 714 149 2

3610 182 192 214 196 039 2

3611 414 504 488 469 96 2

3612 3 34 28 31 06 2

3613 32 33 34 33 07 2

3614 182 182 191 185 037 2

3615 3884 3822 3565 376 0752 2

3616 2 2 2 2 01 2

3617 366 37 35 362 046 2

3618 32 32 32 32 05 2

3619 299 305 297 299 048 2

3620 304 294 302 302 045 2

3621 29 26 29 28 04 2

3622 343 325 318 322 052 2

3623 35 3 33 33 07 2

3624

3626 318 31 314 314 126 2

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

5301 31 3 32 31 06 2

5302 314 319 315 316 063 2

5303 3102 2716 2822 2876 27 62

5304 261 263 26 261 57 2

5305 23 22 26 24 20

5306 22 23 23 22 11 2

5307 488 461 317 461

5308 168633 168544 1562025 1644598

5309 16 19 16 17

5310 3 304 311 305 015

5311 477 452 448 459 15 2

5312 506 63 494 543 134 2

5313 353 301 404 353

5314 431 373 378 394 5 096

5315 596 531 51 545 11 2

5316 224 231 235 235 022 2

5317 216 23 221 222 007 2

5318 064 07 067 01 2

5319 111 184 146 147 029 2

5320 515 525 564 534 48

5321 21 5322 13 87 9 87 35 2

5323 38 33 35 33 16 2

46

Figure 6 Results of replicate determinations (indicated by triangles) of benz[a]anthracene (BAA) in the food supplement test material

Horizontal blue lines represent the arithmetic mean value of replicate measurements and blue bars the reported expanded measurement uncertainty (k=2) The assigned value is

plotted as dotted line Red solid lines mark the range of satisfactory performance (|z|le2) Mean values are given for results outside the displayed data range

ProLab 2011

Laboratory

53

1853

1953

0853

0936

1436

0236

1036

1653

2136

0453

1753

0653

1653

0536

0353

0436

2153

0336

0636

0536

0036

2036

1953

1036

1253

0136

2653

0236

1836

2336

2236

1353

1353

2336

1736

1553

1453

0736

0753

1153

2053

1253

1536

0853

2236

11

an

aly

te c

on

ten

t (micro

gk

g)

10

95

9

85

8

75

7

65

6

55

5

45

4

35

3

25

2

15

1

05

0

102

3 468

7

Sample St Johns wort extract replicatesMeasurand Benz[a]anthraceneNo of laboratories 46

Assigned value 298 microgkg (Reference value)Target sd 062 microgkgTolerance limits 174 - 422 microgkg (|Z score| lt 200)

Assig

ned va

lue

Limit of tolerance

Limit of tolerance

47

Table 12 Analysis results reported by the participants for the content of benzo[a]pyrene (BAP) in the food supplements test material Values are presented as reported

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

3600 16 17 17 17 03 2

3602 118 123 13 124 015

3603 165 162 157 161 015 2

3604 134 154 139 143 04 2

3605 164 166 172 167 057 2

3606 181 159 161 167 031 2

3607 279 266 279 274 055 2

3608 57 687 461 573 226 2

3610 226 216 183 206 042 2

3611 289 325 311 308 36 2

3612 24 22 2 22 05 2

3613 17 18 19 18 05 2

3614 08 08 08 08 016 2

3615 1545 1393 1425 145 029 2

3616 16 16 14 15 03 2

3617 177 171 158 169 023 2

3618 2 2 2 2 02 2

3619 207 207 206 207 075 2

3620 165 169 163 166 017 2

3621 44 47 49 47 05 2

3622 212 208 222 213 038 2

3623 2 21 22 21 03 2

3624 18 178 182 18 031 2

3626 172 173 178 174 07 2

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

5301 18 18 18 18 04 2

5302 188 19 187 188 038 2

5303 1691 1426 149 1535 19 80

5304 145 142 15 146 69 2

5305 15 17 18 16 15

5306 16 17 17 16 08 2

5307 164 157 153 157

5308 102669 109324 109035 107009

5309 1 1 12 11

5310 122 122 124 123 006

5311 199 184 215 2 15 2

5312 258 248 239 248 05 2

5313 199 178 245 207

5314 174 171 172 172 11 0948

5315 253 248 213 238 05 2

5316 177 171 173 17 016 2

5317 255 277 276 269 012 2

5318 114 12 117 02 2

5319 19 228 231 216 043 2

5320 303 3 344 316 78

5321 12 12 12 12

5322 13 17 24 17 35 2

5323 2 2 19 2 1 2

48

Figure 7 Results of replicate determinations (indicated by triangles) of benzo[a]pyrene (BAP) in the food supplement test material

Horizontal blue lines represent the arithmetic mean value of replicate measurements and blue bars the reported expanded measurement uncertainty (k=2) The assigned value is

plotted as dotted line Red solid lines mark the range of satisfactory performance (|z|le2) Mean values are given for results outside the displayed data range

ProLab 2011

Laboratory

36

1453

0953

0853

1853

2153

1036

0236

0436

1553

0436

1653

0353

0736

0336

2036

0053

0553

0636

0636

0536

1753

1453

1636

2636

1336

2453

0153

2253

0253

2353

1136

1836

1953

1336

1036

2336

2253

1936

1253

1553

1253

1736

0753

2036

2136

0836

11

an

aly

te c

on

ten

t (micro

gk

g)

55

5

45

4

35

3

25

2

15

1

05

0

573

308

3

Sample St Johns wort extract replicatesMeasurand Benzo[a]pyreneNo of laboratories 48

Assigned value 165 microgkg (Reference value)Target sd 036 microgkgTolerance limits 093 - 237 microgkg (|Z score| lt 200)

Assig

ned va

lue

Limit of tolerance

Limit of tolerance

49

Table 13 Analysis results reported by the participants for the content of benzo[b]fluoranthene (BBF) in the food supplements test material Values are presented as reported

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

3600 32 3 3 31 09 2

3602 173 175 16 17 017

3603 265 252 25 256 025 2

3604 116 108 105 11 028 2

3605 286 225 232 247 074 2

3606 294 273 261 276 046 2

3607 579 579 544 567 113 2

3608 103 1201 876 1036 326 2

3610 173 196 204 191 038 2

3611 546 618 565 576 75 2

3612 3 28 28 29 06 2

3613 28 31 32 31 06 2

3614 135 111 135 127 022 2

3615 4187 3678 4111 4 08 2

3616 2 2 2 2 01 2

3617 33 27 299 035 2

3618 31 31 31 31 05 2

3619 326 324 312 326 064 2

3620 277 28 272 276 069 2

3621 26 25 23 25 04 2

3622 346 336 333 34 052 2

3623 26 28 29 28 02 2

3624

3626 288 28 307 292 117 2

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

5301 29 3 29 29 06 2

5302 297 3 296 298 06 2

5303 2705 2444 2608 2586 22 78

5304 245 254 26 253 85 2

5305 23 23 26 24 15

5306 42 43 4 42 21 2

5307 221 216 241 221

5308 232169 246654 240722 229848

5309 19 22 19 2 0

5310 3 315 325 313 016

5311 262 277 304 281 15 2

5312 27 289 337 299 075 2

5313 321 284 378 328

5314 423 404 385 404 34 083

5315 42 506 416 444 09 2

5316 265 267 277 273 018 2

5317 185 197 195 192 006 2

5318 276 281 279 04 2

5319 396 389 403 396 079 2

5320 623 618 635 625 14

5321 31

5322 24 33 49 33 35 2

5323 38 45 35 45 27 2

50

Figure 8 Results of replicate determinations (indicated by triangles) of benzo[b]fluoranthene (BBF) in the food supplement test material

Horizontal blue lines represent the arithmetic mean value of replicate measurements and blue bars the reported expanded measurement uncertainty (k=2) The assigned value is

plotted as dotted line Red solid lines mark the range of satisfactory performance (|z|le2) Mean values are given for results outside the displayed data range

ProLab 2011

Laboratory

36

0436

1436

0236

1053

1736

1653

0953

0753

0853

0536

2136

0553

0436

0353

0353

1636

0636

2036

2353

1853

1136

1236

2653

0153

0253

1236

1736

1336

0036

1853

2153

1036

1953

1336

2253

2253

2353

1936

1553

1453

0653

1536

0753

2036

0836

11

an

aly

te c

on

ten

t (micro

gk

g)

75

7

65

6

55

5

45

4

35

3

25

2

15

1

05

0

103

6

576

3

Sample St Johns wort extract replicatesMeasurand Benzo[b]fluorantheneNo of laboratories 46

Assigned value 292 microgkg (Reference value)Target sd 060 microgkgTolerance limits 172 - 412 microgkg (|Z score| lt 200)

Assig

ned va

lue

Limit of tolerance

Limit of tolerance

51

Table 14 Analysis results reported by the participants for the content of chrysene (CHR) in the food supplements test material Values are presented as reported

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

3600 5 49 5 5 07 2

3602 32 304 29 304 036

3603 462 469 447 459 048 2

3604 438 428 42 429 119 2

3605 365 41 381 386 085 2

3606 494 411 45 451 124 2

3607 897 774 787 819 18 2

3608 986 887 1168 1014 284 2

3610 418 392 462 424 085 2

3611 833 893 87 865 61 2

3612 6 64 58 61 12 2

3613 41 39 4 4 08 2

3614 272 254 288 271 054 2

3615 605 6028 5964 601 12 2

3616 47 45 49 47 05 2

3617 445 431 389 422 065 2

3618 45 45 45 45 06 2

3619 738 747 72 738 118 2

3620 463 472 446 46 092 2

3621 53 49 51 51 06 2

3622 478 513 505 488 08 2

3623 73 73 72 72 05 2

3624

3626 63 622 634 628 251 2

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

5301 49 49 49 49 1 2

5302 687 683 666 679 136 2

5303 6777 6181 6382 6441 24 70

5304 592 581 6 591 85 2

5305 6 58 64 61 20

5306 49 5 51 49 25 2

5307 524 5 574 524

5308 211488 216782 221146 213243

5309

5310 372 393 394 386 019

5311 43 459 422 437 15 2

5312 934 943 981 953 191 2

5313 793 73 99 838

5314 636 561 565 588 18 0818

5315 547 583 499 542 11 2

5316 292 286 285 282 06 2

5317 52 598 517 545 046 2

5318 368 345 357 05 2

5319 449 471 474 465 116 2

5320 76 924 818 834 99

5321 33

5322 7 7 76 7 35 2

5323 87 73 77 73 37 2

52

Figure 9 Results of replicate determinations (indicated by triangles) of chrysene (CHR) in the food supplement test material

Horizontal blue lines represent the arithmetic mean value of replicate measurements and blue bars the reported expanded measurement uncertainty (k=2) The assigned value is

plotted as dotted line Red solid lines mark the range of satisfactory performance (|z|le2) Mean values are given for results outside the displayed data range

ProLab 2011

Laboratory

53

0836

1453

1636

0253

2153

1836

0553

1036

1336

1736

1036

0453

1136

1836

0636

0336

2053

1936

1653

0136

0036

2253

0636

2153

0753

1553

1753

1453

0436

1536

1253

0536

2653

0353

0253

2236

2336

1953

2336

0753

2053

1353

1236

0836

11

an

aly

te c

on

ten

t (micro

gk

g)

13

12

11

10

9

8

7

6

5

4

3

2

1

0

865

3

Sample St Johns wort extract replicatesMeasurand ChryseneNo of laboratories 45

Assigned value 407 microgkg (Reference value)Target sd 083 microgkgTolerance limits 241 - 573 microgkg (|Z score| lt 200)

Assig

ned va

lue

Limit of tolerance

Limit of tolerance

53

Table 15 Analysis results reported by the participants for the sum of the four marker PAHs in the food supplements test material Values are presented as reported

Laboratory code

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg

3600 128 24 2

3602 79 126

3603 1127 096 2

3604 893 258 2

3605 1091 305 2

3606 1184 238 2

3607 2098 42 2

3608 3336 325 2

3610 1138 17 2

3611 2218 259 2

3612 143 29 2

3613 122 2 2

3614 663 129 2

3615 152 304 2

3616 102 03 2

3617 1252 169 2

3618 128 18 2

3619 157 305 2

3620 1204 12 2

3621 151 19 2

3622 1361 115 2

3623 154 09 2

3624

3626 1409 56 2

Laboratory code

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg

5301 126

5302 37 074 2

5303 13438 92

5304 1251 85 2

5305 125 20

5306 129 65 2

5307 1363

5308 7245598

5309

5310 1127 056

5311 1367 15 2

5312 2042 408 2

5313 1726

5314 1558

5315 177 35 2

5316 96 073 2

5317 1229 066 2

5318 82 05 2

5319 1225 267 2

5320 2309 41

5321

5322 207 35 2

5323 171 103 2

54

ANNEX 9 Results reported for the standard solutions with undisclosed analyte content Table 16 Analysis results reported by the participants for the four marker PAHs in the standard solution in toluene with undisclosed analyte

content Values are presented as reported U measurement uncertainty k coverage factor

benz[a]anthracene benzo[a]pyrene benzo[b]fluoranthene chrysene Laboratory

code Unit Result U k Result U k Result U k Result U k 3600 microgl 38 67 2 25 45 2 211 68 2 275 38 2

3605 microgl 367 184 2 2482 124 2 2592 13 2 235 118 2

3606 microgkg 3763 176 2 2609 142 2 2721 118 2 2539 134 2

3607 microgkg 3706 815 2 2824 565 2 2591 518 2 2587 569 2

3612 microgl 38 08 2 237 11 2 244 18 2 257 02 2

3614 microgl 324 65 2 209 38 2 215 37 2 21 42 2

3616 microgkg 318 07 2 224 14 2 231 26 2 216 1 2

3618 microgkg 417 17 2 284 08 2 292 06 2 297 03 2

3619 microgkg 47 742 2 274 468 2 228 387 2 274 434 2

3620 microgl 495 74 2 294 29 2 321 8 2 321 64 2

3621 microgl 241 22 2 143 13 2 188 16 2 145 14 2

3623 microgkg 476 25 2 311 53 2 269 3 2 297 03 2

3626 microgl 359 38 2 235 23 2 238 27 2 24 18 2

5301 microgkg 426 29 299 289

5302 microgkg 436 87 2 296 59 2 296 59 2 289 58 2

5303 microgl 3721 27 102 22838 19 99 23756 22 99 25486 24 98

5304 microgkg 38 29 2 225 26 2 257 37 2 258 52 2

5305 microgkg 305 225 245 29

5306 microgkg 316 63 2 247 49 2 65 13 2 22 44 2

5307 microgkg 3658 2559 241 2414

5308 microgkg 16854 109324 246654 216782

5311 microgl 374 20 2 279 15 2 268 15 2 272 15 2

5313 microgl 3408 2165 2275 2373

5314 microgkg 395 5 2875 11 3614 34 2905 18

5320 microgl 3267 368 1986 571 28 683 2427 512

5322 microgl 282 35 2 225 35 2 241 35 2 21 35 2

55

Figure 10 Youden plots of the percent deviation of the reported values from the assigned values for the food supplement test sample and the

standard solution in toluene with undisclosed content

(The identity of the respective measurand is given above the figure Results exceeding a difference of more than 100 were omitted)

56

Figure 10 - continued

57

Table 17 Analysis results reported by the participants for the four marker PAHs in the standard solution in acetonitrile with undisclosed

analyte content

Values are presented as reported U measurement uncertainty k coverage factor

benz[a]anthracene benzo[a]pyrene benzo[b]fluoranthene chrysene

Laboratory

code Unit Result U k Result U k Result U k Result U k

3602 microgkg 3675 247 247 244

3603 microgkg 358 086 2 236 028 2 235 028 2 236 057 2

3604 microgl 475 29 2 19 12 2 194 13 2 258 12 2

3608 microgl 3623 078 2 2385 023 2 2343 18 2 2296 092 2

3610 microgkg 369 185 2 25 125 2 251 126 2 244 122 2

3611 microgkg 48 02 2 323 02 2 328 04 2 317 03 2

3613 microgl 332 31 2 261 3 2 249 25 2 239 24 2

3615 microgl 415 2635 2729 2689

3617 microgkg 37 09 2 244 06 2 284 07 2 239 06 2

3622 microgl 37 12 2 25 23 2 257 21 2 238 1 2

3624 microgkg 402 104 2 255 084 2 265 096 2 253 07 2

5306 microgkg 563 113 2 371 74 2 359 72 2 333 67 2

5308 microgkg 168633 102669 232169 211488

5309 microgl 402 268 27

5310 microgl 315 158 25 125 25 125 288 144

5312 microgl 428 043 2 282 028 2 272 027 2 275 028 2

5315 microgkg 387 08 2 256 05 2 251 05 2 253 05 2

5316 microgl 3839 342 2 2485 191 2 2559 168 2 239 51 2

5317 microgl 2466 043 2 2212 034 2 2472 016 2 2146 068 2

5319 microgl 414 41 2 279 28 2 268 27 2 26 31 2

5321 microgkg 476 316 326 328

5322 microgl 2547 35 2

5323 microgkg 405 203 2 239 119 2 255 153 2 201 11 2

58

Figure 11 Youden plots of the percent deviation of the reported values from the assigned values for the food supplement test sample and the

standard solution in acetonitrile with undisclosed content

(The identity of the respective measurand is given above the figure Results exceeding a difference of more than 100 were omitted)

59

Figure 11 - continued

60

ANNEX 10 Table 18 Method performance parameters reported by the participants

Laboratory code

Analyte LOD LOQ Recovery Analyte LOD LOQ Recovery

microgkg microgkg microgkg microgkg 3600 BaA 03 06 978 BaP 03 07 9483602 BaA 007 021 926 BaP 005 015 993603 BaA 0011 05 987 BaP 0005 051 9553604 BaA 02 04 86 BaP 02 04 883605 BaA 0025 005 118 BaP 0025 005 1083606 BaA 001 003 65 BaP 001 003 703607 BaA 021 071 74 BaP 027 089 943608 BaA 016 048 8213 BaP 015 046 83683610 BaA 02 05 85 BaP 005 02 853611 BaA BaP 3612 BaA 1 117 BaP 1 1203613 BaA 01 03 90 BaP 01 03 903614 BaA 02 05 96 BaP 02 05 933615 BaA 005 015 110 BaP 005 015 1053616 BaA 01 05 108 BaP 01 05 753617 BaA 018 037 405 BaP 009 018 3873618 BaA 01 03 63 BaP 01 03 593619 BaA 005 005 59 BaP 033 033 543620 BaA 0009 0018 64 BaP 0005 001 553621 BaA 007 02 84 BaP 007 02 923622 BaA 01 03 1101 BaP 01 03 9853623 BaA 02 08 105 BaP 02 08 1013624 BaA BaP 01 033 8513625 BaA BaP 3626 BaA BaP 5301 BaA 003 01 102 BaP 003 01 1005302 BaA 01 03 78 BaP 01 03 865303 BaA 003 01 62 BaP 003 01 805304 BaA 1 1 70 BaP 1 1 955305 BaA 02 05 85 BaP 02 05 855306 BaA 03 1 699 BaP 03 1 905307 BaA 6537 BaP 81165308 BaA 0088 0265 99 BaP 00262 00785 1005309 BaA 01 08 92 BaP 01 08 925310 BaA 01 03 BaP 01 03 905311 BaA 002 005 80 BaP 002 004 915312 BaA 03 09 105 BaP 03 09 1055313 BaA 04 08 BaP 04 08 5314 BaA 001 002 96 BaP 001 002 9485315 BaA 01 03 50 - 100 BaP 01 03 50 - 100 5316 BaA 002 011 827 BaP 004 019 10445317 BaA 05 1 76 BaP 05 1 755318 BaA BaP 5319 BaA 009 029 951 BaP 011 041 995320 BaA 12 18 946 BaP 06 1 8345321 BaA 03 05 BaP 05 1 5322 BaA 1 2 100 BaP 1 2 1005323 BaA 26 BaP 26

61

ANNEX 10 Table 18 - continued

Laboratory code Analyte LOD LOQ Recovery Analyte LOD LOQ Recovery

microgkg microgkg microgkg microgkg 3600 BbF 03 05 96 CHR 02 03 10153602 BbF 015 045 947 CHR 003 009 9193603 BbF 0014 05 1012 CHR 0004 05 10033604 BbF 02 04 85 CHR 02 04 873605 BbF 005 01 94 CHR 0025 005 863606 BbF 001 003 70 CHR 001 003 653607 BbF 018 06 100 CHR 014 028 943608 BbF 015 046 7979 CHR 015 045 71253610 BbF 005 02 85 CHR 02 05 853611 BbF CHR 3612 BbF 1 118 CHR 1 1173613 BbF 01 03 90 CHR 01 03 903614 BbF 01 04 96 CHR 02 05 953615 BbF 008 025 109 CHR 003 01 1083616 BbF 01 05 78 CHR 01 05 723617 BbF 035 07 38 CHR 012 024 4163618 BbF 01 03 62 CHR 01 03 623619 BbF 016 016 63 CHR 01 01 593620 BbF 0003 0006 55 CHR 001 002 653621 BbF 007 02 103 CHR 007 02 893622 BbF 02 06 963 CHR 03 09 1043623 BbF 02 08 106 CHR 02 08 1193624 BbF CHR 3625 BbF CHR 3626 BbF CHR 5301 BbF 003 01 96 CHR 003 01 1145302 BbF 01 03 84 CHR 01 03 835303 BbF 003 01 78 CHR 003 01 705304 BbF 1 1 86 CHR 1 1 705305 BbF 02 05 90 CHR 02 05 955306 BbF 03 1 1435 CHR 03 1 1185307 BbF 8116 CHR 65375308 BbF 0079 0238 99 CHR 009 026 1005309 BbF 01 08 92 CHR 5310 BbF 07 2 CHR 01 03 5311 BbF 002 004 95 CHR 002 004 895312 BbF 03 09 105 CHR 03 09 1055313 BbF 04 08 CHR 04 08 5314 BbF 001 002 83 CHR 001 002 8185315 BbF 02 05 50 -100 CHR 02 05 50 - 100 5316 BbF 005 024 814 CHR 001 007 9595317 BbF 05 1 73 CHR 05 1 785318 BbF CHR 5319 BbF 011 038 946 CHR 013 043 9675320 BbF 07 09 1194 CHR 23 32 10455321 BbF 05 1 CHR 05 1 5322 BbF 1 2 100 CHR 1 2 1005323 BbF 26 CHR 26

62

ANNEX 11 Table 19 Details of analysis method reported by the participants Data are presented as reported

Laboratory code

Analysis technique

Instrument calibration

Internal standards applied

Sample clean up

Details of sample

clean up

Extraction technique

Details of sample

extraction

3600 6) GC-MS

2) Internal standardisation with labelled substances

Deuterated PAH mix 9 (Ehrenstorfer)

1) Saponification 2) LiquidLiquid partitioning

D) Other Liquidliquid partitioning

3602 2) HPLC-UV-FLD

1) External calibration

3) Gel permeation chromatography

A) Pressurised liquid extraction (PLE)

3603 1) HPLC-FLD

1) External calibration

3) Gel permeation chromatography

D) Other 1 minute with dichloromethane in vortex

3604 2) HPLC-UV-FLD

2) Internal standardisation with labelled substances

DiP D14

5) Donor acceptor complex chromatography

D) Other LL

3605 1) HPLC-FLD

1) External calibration

3) Gel permeation chromatography

C) Soxhlet extraction

3606 7) GC-MSMS

2) Internal standardisation with labelled substances

BaP 13C4 CHR 13C6 BaA 13C6 BbF 13C6

4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

3607 6) GC-MS

2) Internal standardisation with labelled substances

BaA-d12 CHR-d12 BbF-d12 BkF-d12 BaP-d12 ICP-d12 BgP-d12 DiP-d14

1) Saponification

D) Other solvent partitioning

3608 1) HPLC-FLD

1) External calibration

6) Other Liquid extraction

D) Other

Extraction in a rotary agitator and centrifugation

3610 2) HPLC-UV-FLD

1) External calibration

4) Solid phase extraction

B) Sonication

3611

3612 6) GC-MS

2) Internal standardisation with labelled substances

chrysene-d12 dibenzo(ah)anthracene-d14 dibenzo(ai)pyrene-d14

4) Solid phase extraction

B) Sonication

3613 5) LC-MSMS

3) Internal standardisation with unlabelled substances

I use Chrysene-D12 3) Gel permeation chromatography

D) Other extraction with hexan

63

Table 19 - continued

Laboratory code

Analysis technique

Instrument calibration

Internal standards applied

Sample clean up Details of sample

clean up

Extraction technique

Details of sample

extraction

3614 6) GC-MS 1) External calibration

3) Gel permeation chromatography

B) Sonication

3615 1) HPLC-FLD

1) External calibration

6) Other PLE GPC

A) Pressurised liquid extraction (PLE)

3616 6) GC-MS

2) Internal standardisation with labelled substances

Benzo(a)pyrene-13C4 Benzo(a)anthracene-13C6 Benzo(b)fluoranthene-13C6 Chrysene-13C6

2) LiquidLiquid partitioning 4) Solid phase extraction

B) Sonication

3617 1) HPLC-FLD

1) External calibration

4) Solid phase extraction

C) Soxhlet extraction

3618 6) GC-MS

2) Internal standardisation with labelled substances

deuterated standards

1) Saponification 4) Solid phase extraction

D) Other Handshaken with cyclohexane

3619 6) GC-MS

2) Internal standardisation with labelled substances

C13 Labelled US EPA 16

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

D) Other Homegenisation amp saponiofication

3620 9) GC-HRMS

2) Internal standardisation with labelled substances

mix of deuterated PAHs (BAA-D12 CHR-D12 BAP-D12 BBF-D12)

3) Gel permeation chromatography 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

3621 6) GC-MS

2) Internal standardisation with labelled substances

Chrysene D12 Benzo(a)pyrene D12 Benzo(b)fluoranthene D12 Benzo(a)anthracene

2) LiquidLiquid partitioning 4) Solid phase extraction

B) Sonication

3622 1) HPLC-FLD

4) Standard addition method

4) Solid phase extraction

D) Other

Extraction with cyclohexane by heating under reflux

3623 7) GC-MSMS

2) Internal standardisation with labelled substances

C13 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

3624 2) HPLC-UV-FLD

1) External calibration

2) LiquidLiquid partitioning

D) Other hot solvent extraction

3626

64

Table 19 - continued

Laboratory code

Analysis technique

Instrument calibration

Internal standards

applied

Sample clean up

Details of sample

clean up

Extraction technique

Details of sample

extraction

5301 7) GC-MSMS

2) Internal standardisation with labelled substances

EACH SUBSTANCE C13

4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5302 7) GC-MSMS

2) Internal standardisation with labelled substances

Isotope labelled PAHs 13C for each analyte

4) Solid phase extraction

C) Soxhlet extraction

5303 7) GC-MSMS

3) Internal standardisation with unlabelled substances

HAP C13 4) Solid phase extraction

D) Other automate ASE200 DIONEX

5304 7) GC-MSMS

2) Internal standardisation with labelled substances

with C13 for each HAP

2) LiquidLiquid partitioning 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5305 7) GC-MSMS

2) Internal standardisation with labelled substances

C13 isotopiques internal standards

4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5306 7) GC-MSMS

2) Internal standardisation with labelled substances

C13 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5307 6) GC-MS

2) Internal standardisation with labelled substances

1) Saponification 2) LiquidLiquid partitioning

D) Other solvent extraction

5308 1) HPLC-FLD 1) External calibration

4) Solid phase extraction

B) Sonication

5309 1) HPLC-FLD 1) External calibration

4) Solid phase extraction

D) Other only SPE

5310 1) HPLC-FLD 1) External calibration

1) Saponification 2) LiquidLiquid partitioning 3) Gel permeation chromatography

C) Soxhlet extraction

5311 6) GC-MS

2) Internal standardisation with labelled substances

13 C Chrysene 13 C Benz(a)pyrene

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

B) Sonication

5312 1) HPLC-FLD

3) Internal standardisation with unlabelled substances

Benzo(b)chrysen

2) LiquidLiquid partitioning 4) Solid phase extraction

Quechers D) Other Quechers

65

Table 19 - continued

Laboratory code

Analysis technique

Instrument calibration

Internal standards applied

Sample clean up Details of sample

clean up

Extraction technique

Details of sample extraction

5313 6) GC-MS

2) Internal standardisation with labelled substances

Benz(a)anthracene D12 Benzo(b)fluranthene D12 Benzo(a)pyrene D12 Chrysene D12

1) Saponification 2) LiquidLiquid partitioning 3) Gel permeation chromatography

A) Pressurised liquid extraction (PLE)

5314 6) GC-MS

2) Internal standardisation with labelled substances

Benzo(a)pyren d12 2) LiquidLiquid partitioning

D) Other Extraction with

5315 1) HPLC-FLD

3) Internal standardisation with unlabelled substances

Benz(b)chrysen

1) Saponification 2) LiquidLiquid partitioning

D) Other shaker

5316 1) HPLC-FLD

1) External calibration

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

D) Other

LiquidLiquid extraction with cyclohexane and dimethylformamide

5317 7) GC-MSMS

1) External calibration

3) Gel permeation chromatography 6) Other

silica gel D) Other VDLUFA online method

5318

5319 1) HPLC-FLD

3) Internal standardisation with unlabelled substances

Benzo(b)chrysene 3) Gel permeation chromatography

A) Pressurised liquid extraction (PLE)

5320 6) GC-MS

3) Internal standardisation with unlabelled substances

22-binaphthyl

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

D) Other 3 hours refluxing

5321 1) HPLC-FLD

2) Internal standardisation with labelled substances

6-meacutethylchrysegravene

1) Saponification 2) LiquidLiquid partitioning

B) Sonication

5322 6) GC-MS

2) Internal standardisation with labelled substances

naftalene-d8 acenaphtalene-d10 phenanthrene-d10 chrysene-d12 perylene-d12 benzo(e)anthracene-d12

1) Saponification 2) LiquidLiquid partitioning 6) Other

clean-up with silica packed column

C) Soxhlet extraction

5323 1) HPLC-FLD

1) External calibration

4) Solid phase extraction 6) Other

Accelerated Solvent Extraction

A) Pressurised liquid extraction (PLE)

66

European Commission EUR 25245 EN ndash Joint Research Centre ndash Institute for Reference Materials and Measurements Title Report on the 8th inter-laboratory comparison organised by the European Union Reference Laboratory for Polycyclic Aromatic Hydrocarbons - Four marker PAHs in a dry extract of St Johns wort Author(s) Radoslav Lizak Szilard Szilagyi Philippe Verlinde and Thomas WENZL Luxembourg Publications Office of the European Union 2012 ndash 66 pp ndash 201 x 297 cm EUR ndash Scientific and Technical Research series ndash ISSN 1831-9424 ISBN 978-92-79-23337-1 doi 10278759387 Abstract The proficiency test here reported concerned the determination of the contents of four marker polycyclic aromatic hydrocarbons (PAHs) and of their sum in an food supplements test sample The set of marker PAHs consists of benz[a]anthacene benzo[a]pyrene benzo[b]fluoranthene and chrysene Participants to these PT were National Reference Laboratories for PAHs (NRLs-PAHs) and EU official food control laboratories The number of participants was in total 48

The PT was organised according ISO Standard 170432010

The test material used was a commercial dry extract of St Johns wort Participants also received a solution of the same PAHs either in acetonitrile or in toluene for checking their instrument calibration

The results from participants were rated with z-scores and zeta-scores About 68 of the reported results were attributed with z-scores with an absolute value of below two which is the threshold for satisfactory performance

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interests whether private or national

L

A-N

A-252

45-EN

-N

• 1 Summary
• 2 Introduction
• 3 Scope
• 4 Participating Laboratories
• 5 Time frame
• 6 Confidentiality
• 7 Test materials
• • 71 1Preparation of test materials
  • 72 Assigned values and standard deviation for proficiency assessment
  • 73 Homogeneity and stability
  • 74 Sample dispatch
  • • 8 Design of the proficiency test
    • 9 Evaluation of the results
    • • 91 2General
      • 92 21BEvaluation criteria
      • 93 Performance of participants
      • 94 Evaluation of the influence of analysis method
      • 95 Evaluation of the influence of calibration
      • 96 26Evaluation of compliance with legislation
      • • 10 Follow-up actions for underperforming laboratories
        • 11 1Conclusions
        • 12 1Acknowledgements
        • 13 References

33

ANNEX 2 - Announcement via e-mail and invitation

34

ANNEX 3 - Announcement sample dispatch

Dear MadameSir

The test samples for the proficiency test on the determination of four PAHs in a food supplement (dry extract of St Johns wort) were

dispatched today

Explanation on the design of the study as well as instructions for sample storage and for reporting of results are in the parcel You will

also find your participant key in the parcel which is required for reporting of results

Please check the parcel after arrival for completeness and report the receipt of the test samples as well as their status to us by applying

the sample receipt form which is in the parcel

You are kindly requested to inform us by email if you do not receive the parcel by end of this week

My colleague Donata Lerda will then take care of it

With best regards

Thomas Wenzl

35

ANNEX 4 - Specifications of standard solutions with disclosed concentration

36

ANNEX 4 - continued

37

ANNEX 5 - Homogeneity and stability of the test material

Homogeneity of the test material

Benz[a]anthracene - BAA

Benzo[a]pyrene - BAP

38

ANNEX 5 - continued

Benzo[b]fluoranthene - BBF

Chrysene - CHR

39

ANNEX 5 - continued

Stability of the test material

Table 10 Content of the four target analytes in test materials stored under different

temperatures

BaA BaP BbF CHR

microgkg microgkg microgkg microgkg

Content 31 16 31 69 Storage at room temperature

U (k=2) 05 02 05 17

Content 31 15 29 68 Storage at reference temperature

U (k=2) 04 03 04 10

The applied capillary column does not separate CHR from triphenylene

40

ANNEX 6 ndash Documents sent to participants

41

ANNEX 6 - continued

42

ANNEX 7 Questionnaire on details of analysis method

43

ANNEX 7 - continued

44

ANNEX 7 - continued

45

ANNEX 8 - Results reported by participants for the food supplements test sample Table 11 Analysis results reported by the participants for the content of benz[a]anthracene (BAA) in the food supplements test material Values are presented as reported

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

3600 31 29 29 3 05 2

3602 199 193 178 19 021

3603 253 253 246 251 022 2

3604 199 209 23 213 071 2

3605 301 3 271 291 076 2

3606 302 288 278 289 064 2

3607 456 416 443 438 096 2

3608 796 695 651 714 149 2

3610 182 192 214 196 039 2

3611 414 504 488 469 96 2

3612 3 34 28 31 06 2

3613 32 33 34 33 07 2

3614 182 182 191 185 037 2

3615 3884 3822 3565 376 0752 2

3616 2 2 2 2 01 2

3617 366 37 35 362 046 2

3618 32 32 32 32 05 2

3619 299 305 297 299 048 2

3620 304 294 302 302 045 2

3621 29 26 29 28 04 2

3622 343 325 318 322 052 2

3623 35 3 33 33 07 2

3624

3626 318 31 314 314 126 2

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

5301 31 3 32 31 06 2

5302 314 319 315 316 063 2

5303 3102 2716 2822 2876 27 62

5304 261 263 26 261 57 2

5305 23 22 26 24 20

5306 22 23 23 22 11 2

5307 488 461 317 461

5308 168633 168544 1562025 1644598

5309 16 19 16 17

5310 3 304 311 305 015

5311 477 452 448 459 15 2

5312 506 63 494 543 134 2

5313 353 301 404 353

5314 431 373 378 394 5 096

5315 596 531 51 545 11 2

5316 224 231 235 235 022 2

5317 216 23 221 222 007 2

5318 064 07 067 01 2

5319 111 184 146 147 029 2

5320 515 525 564 534 48

5321 21 5322 13 87 9 87 35 2

5323 38 33 35 33 16 2

46

Figure 6 Results of replicate determinations (indicated by triangles) of benz[a]anthracene (BAA) in the food supplement test material

Horizontal blue lines represent the arithmetic mean value of replicate measurements and blue bars the reported expanded measurement uncertainty (k=2) The assigned value is

plotted as dotted line Red solid lines mark the range of satisfactory performance (|z|le2) Mean values are given for results outside the displayed data range

ProLab 2011

Laboratory

53

1853

1953

0853

0936

1436

0236

1036

1653

2136

0453

1753

0653

1653

0536

0353

0436

2153

0336

0636

0536

0036

2036

1953

1036

1253

0136

2653

0236

1836

2336

2236

1353

1353

2336

1736

1553

1453

0736

0753

1153

2053

1253

1536

0853

2236

11

an

aly

te c

on

ten

t (micro

gk

g)

10

95

9

85

8

75

7

65

6

55

5

45

4

35

3

25

2

15

1

05

0

102

3 468

7

Sample St Johns wort extract replicatesMeasurand Benz[a]anthraceneNo of laboratories 46

Assigned value 298 microgkg (Reference value)Target sd 062 microgkgTolerance limits 174 - 422 microgkg (|Z score| lt 200)

Assig

ned va

lue

Limit of tolerance

Limit of tolerance

47

Table 12 Analysis results reported by the participants for the content of benzo[a]pyrene (BAP) in the food supplements test material Values are presented as reported

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

3600 16 17 17 17 03 2

3602 118 123 13 124 015

3603 165 162 157 161 015 2

3604 134 154 139 143 04 2

3605 164 166 172 167 057 2

3606 181 159 161 167 031 2

3607 279 266 279 274 055 2

3608 57 687 461 573 226 2

3610 226 216 183 206 042 2

3611 289 325 311 308 36 2

3612 24 22 2 22 05 2

3613 17 18 19 18 05 2

3614 08 08 08 08 016 2

3615 1545 1393 1425 145 029 2

3616 16 16 14 15 03 2

3617 177 171 158 169 023 2

3618 2 2 2 2 02 2

3619 207 207 206 207 075 2

3620 165 169 163 166 017 2

3621 44 47 49 47 05 2

3622 212 208 222 213 038 2

3623 2 21 22 21 03 2

3624 18 178 182 18 031 2

3626 172 173 178 174 07 2

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

5301 18 18 18 18 04 2

5302 188 19 187 188 038 2

5303 1691 1426 149 1535 19 80

5304 145 142 15 146 69 2

5305 15 17 18 16 15

5306 16 17 17 16 08 2

5307 164 157 153 157

5308 102669 109324 109035 107009

5309 1 1 12 11

5310 122 122 124 123 006

5311 199 184 215 2 15 2

5312 258 248 239 248 05 2

5313 199 178 245 207

5314 174 171 172 172 11 0948

5315 253 248 213 238 05 2

5316 177 171 173 17 016 2

5317 255 277 276 269 012 2

5318 114 12 117 02 2

5319 19 228 231 216 043 2

5320 303 3 344 316 78

5321 12 12 12 12

5322 13 17 24 17 35 2

5323 2 2 19 2 1 2

48

Figure 7 Results of replicate determinations (indicated by triangles) of benzo[a]pyrene (BAP) in the food supplement test material

Horizontal blue lines represent the arithmetic mean value of replicate measurements and blue bars the reported expanded measurement uncertainty (k=2) The assigned value is

plotted as dotted line Red solid lines mark the range of satisfactory performance (|z|le2) Mean values are given for results outside the displayed data range

ProLab 2011

Laboratory

36

1453

0953

0853

1853

2153

1036

0236

0436

1553

0436

1653

0353

0736

0336

2036

0053

0553

0636

0636

0536

1753

1453

1636

2636

1336

2453

0153

2253

0253

2353

1136

1836

1953

1336

1036

2336

2253

1936

1253

1553

1253

1736

0753

2036

2136

0836

11

an

aly

te c

on

ten

t (micro

gk

g)

55

5

45

4

35

3

25

2

15

1

05

0

573

308

3

Sample St Johns wort extract replicatesMeasurand Benzo[a]pyreneNo of laboratories 48

Assigned value 165 microgkg (Reference value)Target sd 036 microgkgTolerance limits 093 - 237 microgkg (|Z score| lt 200)

Assig

ned va

lue

Limit of tolerance

Limit of tolerance

49

Table 13 Analysis results reported by the participants for the content of benzo[b]fluoranthene (BBF) in the food supplements test material Values are presented as reported

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

3600 32 3 3 31 09 2

3602 173 175 16 17 017

3603 265 252 25 256 025 2

3604 116 108 105 11 028 2

3605 286 225 232 247 074 2

3606 294 273 261 276 046 2

3607 579 579 544 567 113 2

3608 103 1201 876 1036 326 2

3610 173 196 204 191 038 2

3611 546 618 565 576 75 2

3612 3 28 28 29 06 2

3613 28 31 32 31 06 2

3614 135 111 135 127 022 2

3615 4187 3678 4111 4 08 2

3616 2 2 2 2 01 2

3617 33 27 299 035 2

3618 31 31 31 31 05 2

3619 326 324 312 326 064 2

3620 277 28 272 276 069 2

3621 26 25 23 25 04 2

3622 346 336 333 34 052 2

3623 26 28 29 28 02 2

3624

3626 288 28 307 292 117 2

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

5301 29 3 29 29 06 2

5302 297 3 296 298 06 2

5303 2705 2444 2608 2586 22 78

5304 245 254 26 253 85 2

5305 23 23 26 24 15

5306 42 43 4 42 21 2

5307 221 216 241 221

5308 232169 246654 240722 229848

5309 19 22 19 2 0

5310 3 315 325 313 016

5311 262 277 304 281 15 2

5312 27 289 337 299 075 2

5313 321 284 378 328

5314 423 404 385 404 34 083

5315 42 506 416 444 09 2

5316 265 267 277 273 018 2

5317 185 197 195 192 006 2

5318 276 281 279 04 2

5319 396 389 403 396 079 2

5320 623 618 635 625 14

5321 31

5322 24 33 49 33 35 2

5323 38 45 35 45 27 2

50

Figure 8 Results of replicate determinations (indicated by triangles) of benzo[b]fluoranthene (BBF) in the food supplement test material

Horizontal blue lines represent the arithmetic mean value of replicate measurements and blue bars the reported expanded measurement uncertainty (k=2) The assigned value is

plotted as dotted line Red solid lines mark the range of satisfactory performance (|z|le2) Mean values are given for results outside the displayed data range

ProLab 2011

Laboratory

36

0436

1436

0236

1053

1736

1653

0953

0753

0853

0536

2136

0553

0436

0353

0353

1636

0636

2036

2353

1853

1136

1236

2653

0153

0253

1236

1736

1336

0036

1853

2153

1036

1953

1336

2253

2253

2353

1936

1553

1453

0653

1536

0753

2036

0836

11

an

aly

te c

on

ten

t (micro

gk

g)

75

7

65

6

55

5

45

4

35

3

25

2

15

1

05

0

103

6

576

3

Sample St Johns wort extract replicatesMeasurand Benzo[b]fluorantheneNo of laboratories 46

Assigned value 292 microgkg (Reference value)Target sd 060 microgkgTolerance limits 172 - 412 microgkg (|Z score| lt 200)

Assig

ned va

lue

Limit of tolerance

Limit of tolerance

51

Table 14 Analysis results reported by the participants for the content of chrysene (CHR) in the food supplements test material Values are presented as reported

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

3600 5 49 5 5 07 2

3602 32 304 29 304 036

3603 462 469 447 459 048 2

3604 438 428 42 429 119 2

3605 365 41 381 386 085 2

3606 494 411 45 451 124 2

3607 897 774 787 819 18 2

3608 986 887 1168 1014 284 2

3610 418 392 462 424 085 2

3611 833 893 87 865 61 2

3612 6 64 58 61 12 2

3613 41 39 4 4 08 2

3614 272 254 288 271 054 2

3615 605 6028 5964 601 12 2

3616 47 45 49 47 05 2

3617 445 431 389 422 065 2

3618 45 45 45 45 06 2

3619 738 747 72 738 118 2

3620 463 472 446 46 092 2

3621 53 49 51 51 06 2

3622 478 513 505 488 08 2

3623 73 73 72 72 05 2

3624

3626 63 622 634 628 251 2

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

5301 49 49 49 49 1 2

5302 687 683 666 679 136 2

5303 6777 6181 6382 6441 24 70

5304 592 581 6 591 85 2

5305 6 58 64 61 20

5306 49 5 51 49 25 2

5307 524 5 574 524

5308 211488 216782 221146 213243

5309

5310 372 393 394 386 019

5311 43 459 422 437 15 2

5312 934 943 981 953 191 2

5313 793 73 99 838

5314 636 561 565 588 18 0818

5315 547 583 499 542 11 2

5316 292 286 285 282 06 2

5317 52 598 517 545 046 2

5318 368 345 357 05 2

5319 449 471 474 465 116 2

5320 76 924 818 834 99

5321 33

5322 7 7 76 7 35 2

5323 87 73 77 73 37 2

52

Figure 9 Results of replicate determinations (indicated by triangles) of chrysene (CHR) in the food supplement test material

Horizontal blue lines represent the arithmetic mean value of replicate measurements and blue bars the reported expanded measurement uncertainty (k=2) The assigned value is

plotted as dotted line Red solid lines mark the range of satisfactory performance (|z|le2) Mean values are given for results outside the displayed data range

ProLab 2011

Laboratory

53

0836

1453

1636

0253

2153

1836

0553

1036

1336

1736

1036

0453

1136

1836

0636

0336

2053

1936

1653

0136

0036

2253

0636

2153

0753

1553

1753

1453

0436

1536

1253

0536

2653

0353

0253

2236

2336

1953

2336

0753

2053

1353

1236

0836

11

an

aly

te c

on

ten

t (micro

gk

g)

13

12

11

10

9

8

7

6

5

4

3

2

1

0

865

3

Sample St Johns wort extract replicatesMeasurand ChryseneNo of laboratories 45

Assigned value 407 microgkg (Reference value)Target sd 083 microgkgTolerance limits 241 - 573 microgkg (|Z score| lt 200)

Assig

ned va

lue

Limit of tolerance

Limit of tolerance

53

Table 15 Analysis results reported by the participants for the sum of the four marker PAHs in the food supplements test material Values are presented as reported

Laboratory code

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg

3600 128 24 2

3602 79 126

3603 1127 096 2

3604 893 258 2

3605 1091 305 2

3606 1184 238 2

3607 2098 42 2

3608 3336 325 2

3610 1138 17 2

3611 2218 259 2

3612 143 29 2

3613 122 2 2

3614 663 129 2

3615 152 304 2

3616 102 03 2

3617 1252 169 2

3618 128 18 2

3619 157 305 2

3620 1204 12 2

3621 151 19 2

3622 1361 115 2

3623 154 09 2

3624

3626 1409 56 2

Laboratory code

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg

5301 126

5302 37 074 2

5303 13438 92

5304 1251 85 2

5305 125 20

5306 129 65 2

5307 1363

5308 7245598

5309

5310 1127 056

5311 1367 15 2

5312 2042 408 2

5313 1726

5314 1558

5315 177 35 2

5316 96 073 2

5317 1229 066 2

5318 82 05 2

5319 1225 267 2

5320 2309 41

5321

5322 207 35 2

5323 171 103 2

54

ANNEX 9 Results reported for the standard solutions with undisclosed analyte content Table 16 Analysis results reported by the participants for the four marker PAHs in the standard solution in toluene with undisclosed analyte

content Values are presented as reported U measurement uncertainty k coverage factor

benz[a]anthracene benzo[a]pyrene benzo[b]fluoranthene chrysene Laboratory

code Unit Result U k Result U k Result U k Result U k 3600 microgl 38 67 2 25 45 2 211 68 2 275 38 2

3605 microgl 367 184 2 2482 124 2 2592 13 2 235 118 2

3606 microgkg 3763 176 2 2609 142 2 2721 118 2 2539 134 2

3607 microgkg 3706 815 2 2824 565 2 2591 518 2 2587 569 2

3612 microgl 38 08 2 237 11 2 244 18 2 257 02 2

3614 microgl 324 65 2 209 38 2 215 37 2 21 42 2

3616 microgkg 318 07 2 224 14 2 231 26 2 216 1 2

3618 microgkg 417 17 2 284 08 2 292 06 2 297 03 2

3619 microgkg 47 742 2 274 468 2 228 387 2 274 434 2

3620 microgl 495 74 2 294 29 2 321 8 2 321 64 2

3621 microgl 241 22 2 143 13 2 188 16 2 145 14 2

3623 microgkg 476 25 2 311 53 2 269 3 2 297 03 2

3626 microgl 359 38 2 235 23 2 238 27 2 24 18 2

5301 microgkg 426 29 299 289

5302 microgkg 436 87 2 296 59 2 296 59 2 289 58 2

5303 microgl 3721 27 102 22838 19 99 23756 22 99 25486 24 98

5304 microgkg 38 29 2 225 26 2 257 37 2 258 52 2

5305 microgkg 305 225 245 29

5306 microgkg 316 63 2 247 49 2 65 13 2 22 44 2

5307 microgkg 3658 2559 241 2414

5308 microgkg 16854 109324 246654 216782

5311 microgl 374 20 2 279 15 2 268 15 2 272 15 2

5313 microgl 3408 2165 2275 2373

5314 microgkg 395 5 2875 11 3614 34 2905 18

5320 microgl 3267 368 1986 571 28 683 2427 512

5322 microgl 282 35 2 225 35 2 241 35 2 21 35 2

55

Figure 10 Youden plots of the percent deviation of the reported values from the assigned values for the food supplement test sample and the

standard solution in toluene with undisclosed content

(The identity of the respective measurand is given above the figure Results exceeding a difference of more than 100 were omitted)

56

Figure 10 - continued

57

Table 17 Analysis results reported by the participants for the four marker PAHs in the standard solution in acetonitrile with undisclosed

analyte content

Values are presented as reported U measurement uncertainty k coverage factor

benz[a]anthracene benzo[a]pyrene benzo[b]fluoranthene chrysene

Laboratory

code Unit Result U k Result U k Result U k Result U k

3602 microgkg 3675 247 247 244

3603 microgkg 358 086 2 236 028 2 235 028 2 236 057 2

3604 microgl 475 29 2 19 12 2 194 13 2 258 12 2

3608 microgl 3623 078 2 2385 023 2 2343 18 2 2296 092 2

3610 microgkg 369 185 2 25 125 2 251 126 2 244 122 2

3611 microgkg 48 02 2 323 02 2 328 04 2 317 03 2

3613 microgl 332 31 2 261 3 2 249 25 2 239 24 2

3615 microgl 415 2635 2729 2689

3617 microgkg 37 09 2 244 06 2 284 07 2 239 06 2

3622 microgl 37 12 2 25 23 2 257 21 2 238 1 2

3624 microgkg 402 104 2 255 084 2 265 096 2 253 07 2

5306 microgkg 563 113 2 371 74 2 359 72 2 333 67 2

5308 microgkg 168633 102669 232169 211488

5309 microgl 402 268 27

5310 microgl 315 158 25 125 25 125 288 144

5312 microgl 428 043 2 282 028 2 272 027 2 275 028 2

5315 microgkg 387 08 2 256 05 2 251 05 2 253 05 2

5316 microgl 3839 342 2 2485 191 2 2559 168 2 239 51 2

5317 microgl 2466 043 2 2212 034 2 2472 016 2 2146 068 2

5319 microgl 414 41 2 279 28 2 268 27 2 26 31 2

5321 microgkg 476 316 326 328

5322 microgl 2547 35 2

5323 microgkg 405 203 2 239 119 2 255 153 2 201 11 2

58

Figure 11 Youden plots of the percent deviation of the reported values from the assigned values for the food supplement test sample and the

standard solution in acetonitrile with undisclosed content

(The identity of the respective measurand is given above the figure Results exceeding a difference of more than 100 were omitted)

59

Figure 11 - continued

60

ANNEX 10 Table 18 Method performance parameters reported by the participants

Laboratory code

Analyte LOD LOQ Recovery Analyte LOD LOQ Recovery

microgkg microgkg microgkg microgkg 3600 BaA 03 06 978 BaP 03 07 9483602 BaA 007 021 926 BaP 005 015 993603 BaA 0011 05 987 BaP 0005 051 9553604 BaA 02 04 86 BaP 02 04 883605 BaA 0025 005 118 BaP 0025 005 1083606 BaA 001 003 65 BaP 001 003 703607 BaA 021 071 74 BaP 027 089 943608 BaA 016 048 8213 BaP 015 046 83683610 BaA 02 05 85 BaP 005 02 853611 BaA BaP 3612 BaA 1 117 BaP 1 1203613 BaA 01 03 90 BaP 01 03 903614 BaA 02 05 96 BaP 02 05 933615 BaA 005 015 110 BaP 005 015 1053616 BaA 01 05 108 BaP 01 05 753617 BaA 018 037 405 BaP 009 018 3873618 BaA 01 03 63 BaP 01 03 593619 BaA 005 005 59 BaP 033 033 543620 BaA 0009 0018 64 BaP 0005 001 553621 BaA 007 02 84 BaP 007 02 923622 BaA 01 03 1101 BaP 01 03 9853623 BaA 02 08 105 BaP 02 08 1013624 BaA BaP 01 033 8513625 BaA BaP 3626 BaA BaP 5301 BaA 003 01 102 BaP 003 01 1005302 BaA 01 03 78 BaP 01 03 865303 BaA 003 01 62 BaP 003 01 805304 BaA 1 1 70 BaP 1 1 955305 BaA 02 05 85 BaP 02 05 855306 BaA 03 1 699 BaP 03 1 905307 BaA 6537 BaP 81165308 BaA 0088 0265 99 BaP 00262 00785 1005309 BaA 01 08 92 BaP 01 08 925310 BaA 01 03 BaP 01 03 905311 BaA 002 005 80 BaP 002 004 915312 BaA 03 09 105 BaP 03 09 1055313 BaA 04 08 BaP 04 08 5314 BaA 001 002 96 BaP 001 002 9485315 BaA 01 03 50 - 100 BaP 01 03 50 - 100 5316 BaA 002 011 827 BaP 004 019 10445317 BaA 05 1 76 BaP 05 1 755318 BaA BaP 5319 BaA 009 029 951 BaP 011 041 995320 BaA 12 18 946 BaP 06 1 8345321 BaA 03 05 BaP 05 1 5322 BaA 1 2 100 BaP 1 2 1005323 BaA 26 BaP 26

61

ANNEX 10 Table 18 - continued

Laboratory code Analyte LOD LOQ Recovery Analyte LOD LOQ Recovery

microgkg microgkg microgkg microgkg 3600 BbF 03 05 96 CHR 02 03 10153602 BbF 015 045 947 CHR 003 009 9193603 BbF 0014 05 1012 CHR 0004 05 10033604 BbF 02 04 85 CHR 02 04 873605 BbF 005 01 94 CHR 0025 005 863606 BbF 001 003 70 CHR 001 003 653607 BbF 018 06 100 CHR 014 028 943608 BbF 015 046 7979 CHR 015 045 71253610 BbF 005 02 85 CHR 02 05 853611 BbF CHR 3612 BbF 1 118 CHR 1 1173613 BbF 01 03 90 CHR 01 03 903614 BbF 01 04 96 CHR 02 05 953615 BbF 008 025 109 CHR 003 01 1083616 BbF 01 05 78 CHR 01 05 723617 BbF 035 07 38 CHR 012 024 4163618 BbF 01 03 62 CHR 01 03 623619 BbF 016 016 63 CHR 01 01 593620 BbF 0003 0006 55 CHR 001 002 653621 BbF 007 02 103 CHR 007 02 893622 BbF 02 06 963 CHR 03 09 1043623 BbF 02 08 106 CHR 02 08 1193624 BbF CHR 3625 BbF CHR 3626 BbF CHR 5301 BbF 003 01 96 CHR 003 01 1145302 BbF 01 03 84 CHR 01 03 835303 BbF 003 01 78 CHR 003 01 705304 BbF 1 1 86 CHR 1 1 705305 BbF 02 05 90 CHR 02 05 955306 BbF 03 1 1435 CHR 03 1 1185307 BbF 8116 CHR 65375308 BbF 0079 0238 99 CHR 009 026 1005309 BbF 01 08 92 CHR 5310 BbF 07 2 CHR 01 03 5311 BbF 002 004 95 CHR 002 004 895312 BbF 03 09 105 CHR 03 09 1055313 BbF 04 08 CHR 04 08 5314 BbF 001 002 83 CHR 001 002 8185315 BbF 02 05 50 -100 CHR 02 05 50 - 100 5316 BbF 005 024 814 CHR 001 007 9595317 BbF 05 1 73 CHR 05 1 785318 BbF CHR 5319 BbF 011 038 946 CHR 013 043 9675320 BbF 07 09 1194 CHR 23 32 10455321 BbF 05 1 CHR 05 1 5322 BbF 1 2 100 CHR 1 2 1005323 BbF 26 CHR 26

62

ANNEX 11 Table 19 Details of analysis method reported by the participants Data are presented as reported

Laboratory code

Analysis technique

Instrument calibration

Internal standards applied

Sample clean up

Details of sample

clean up

Extraction technique

Details of sample

extraction

3600 6) GC-MS

2) Internal standardisation with labelled substances

Deuterated PAH mix 9 (Ehrenstorfer)

1) Saponification 2) LiquidLiquid partitioning

D) Other Liquidliquid partitioning

3602 2) HPLC-UV-FLD

1) External calibration

3) Gel permeation chromatography

A) Pressurised liquid extraction (PLE)

3603 1) HPLC-FLD

1) External calibration

3) Gel permeation chromatography

D) Other 1 minute with dichloromethane in vortex

3604 2) HPLC-UV-FLD

2) Internal standardisation with labelled substances

DiP D14

5) Donor acceptor complex chromatography

D) Other LL

3605 1) HPLC-FLD

1) External calibration

3) Gel permeation chromatography

C) Soxhlet extraction

3606 7) GC-MSMS

2) Internal standardisation with labelled substances

BaP 13C4 CHR 13C6 BaA 13C6 BbF 13C6

4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

3607 6) GC-MS

2) Internal standardisation with labelled substances

BaA-d12 CHR-d12 BbF-d12 BkF-d12 BaP-d12 ICP-d12 BgP-d12 DiP-d14

1) Saponification

D) Other solvent partitioning

3608 1) HPLC-FLD

1) External calibration

6) Other Liquid extraction

D) Other

Extraction in a rotary agitator and centrifugation

3610 2) HPLC-UV-FLD

1) External calibration

4) Solid phase extraction

B) Sonication

3611

3612 6) GC-MS

2) Internal standardisation with labelled substances

chrysene-d12 dibenzo(ah)anthracene-d14 dibenzo(ai)pyrene-d14

4) Solid phase extraction

B) Sonication

3613 5) LC-MSMS

3) Internal standardisation with unlabelled substances

I use Chrysene-D12 3) Gel permeation chromatography

D) Other extraction with hexan

63

Table 19 - continued

Laboratory code

Analysis technique

Instrument calibration

Internal standards applied

Sample clean up Details of sample

clean up

Extraction technique

Details of sample

extraction

3614 6) GC-MS 1) External calibration

3) Gel permeation chromatography

B) Sonication

3615 1) HPLC-FLD

1) External calibration

6) Other PLE GPC

A) Pressurised liquid extraction (PLE)

3616 6) GC-MS

2) Internal standardisation with labelled substances

Benzo(a)pyrene-13C4 Benzo(a)anthracene-13C6 Benzo(b)fluoranthene-13C6 Chrysene-13C6

2) LiquidLiquid partitioning 4) Solid phase extraction

B) Sonication

3617 1) HPLC-FLD

1) External calibration

4) Solid phase extraction

C) Soxhlet extraction

3618 6) GC-MS

2) Internal standardisation with labelled substances

deuterated standards

1) Saponification 4) Solid phase extraction

D) Other Handshaken with cyclohexane

3619 6) GC-MS

2) Internal standardisation with labelled substances

C13 Labelled US EPA 16

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

D) Other Homegenisation amp saponiofication

3620 9) GC-HRMS

2) Internal standardisation with labelled substances

mix of deuterated PAHs (BAA-D12 CHR-D12 BAP-D12 BBF-D12)

3) Gel permeation chromatography 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

3621 6) GC-MS

2) Internal standardisation with labelled substances

Chrysene D12 Benzo(a)pyrene D12 Benzo(b)fluoranthene D12 Benzo(a)anthracene

2) LiquidLiquid partitioning 4) Solid phase extraction

B) Sonication

3622 1) HPLC-FLD

4) Standard addition method

4) Solid phase extraction

D) Other

Extraction with cyclohexane by heating under reflux

3623 7) GC-MSMS

2) Internal standardisation with labelled substances

C13 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

3624 2) HPLC-UV-FLD

1) External calibration

2) LiquidLiquid partitioning

D) Other hot solvent extraction

3626

64

Table 19 - continued

Laboratory code

Analysis technique

Instrument calibration

Internal standards

applied

Sample clean up

Details of sample

clean up

Extraction technique

Details of sample

extraction

5301 7) GC-MSMS

2) Internal standardisation with labelled substances

EACH SUBSTANCE C13

4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5302 7) GC-MSMS

2) Internal standardisation with labelled substances

Isotope labelled PAHs 13C for each analyte

4) Solid phase extraction

C) Soxhlet extraction

5303 7) GC-MSMS

3) Internal standardisation with unlabelled substances

HAP C13 4) Solid phase extraction

D) Other automate ASE200 DIONEX

5304 7) GC-MSMS

2) Internal standardisation with labelled substances

with C13 for each HAP

2) LiquidLiquid partitioning 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5305 7) GC-MSMS

2) Internal standardisation with labelled substances

C13 isotopiques internal standards

4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5306 7) GC-MSMS

2) Internal standardisation with labelled substances

C13 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5307 6) GC-MS

2) Internal standardisation with labelled substances

1) Saponification 2) LiquidLiquid partitioning

D) Other solvent extraction

5308 1) HPLC-FLD 1) External calibration

4) Solid phase extraction

B) Sonication

5309 1) HPLC-FLD 1) External calibration

4) Solid phase extraction

D) Other only SPE

5310 1) HPLC-FLD 1) External calibration

1) Saponification 2) LiquidLiquid partitioning 3) Gel permeation chromatography

C) Soxhlet extraction

5311 6) GC-MS

2) Internal standardisation with labelled substances

13 C Chrysene 13 C Benz(a)pyrene

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

B) Sonication

5312 1) HPLC-FLD

3) Internal standardisation with unlabelled substances

Benzo(b)chrysen

2) LiquidLiquid partitioning 4) Solid phase extraction

Quechers D) Other Quechers

65

Table 19 - continued

Laboratory code

Analysis technique

Instrument calibration

Internal standards applied

Sample clean up Details of sample

clean up

Extraction technique

Details of sample extraction

5313 6) GC-MS

2) Internal standardisation with labelled substances

Benz(a)anthracene D12 Benzo(b)fluranthene D12 Benzo(a)pyrene D12 Chrysene D12

1) Saponification 2) LiquidLiquid partitioning 3) Gel permeation chromatography

A) Pressurised liquid extraction (PLE)

5314 6) GC-MS

2) Internal standardisation with labelled substances

Benzo(a)pyren d12 2) LiquidLiquid partitioning

D) Other Extraction with

5315 1) HPLC-FLD

3) Internal standardisation with unlabelled substances

Benz(b)chrysen

1) Saponification 2) LiquidLiquid partitioning

D) Other shaker

5316 1) HPLC-FLD

1) External calibration

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

D) Other

LiquidLiquid extraction with cyclohexane and dimethylformamide

5317 7) GC-MSMS

1) External calibration

3) Gel permeation chromatography 6) Other

silica gel D) Other VDLUFA online method

5318

5319 1) HPLC-FLD

3) Internal standardisation with unlabelled substances

Benzo(b)chrysene 3) Gel permeation chromatography

A) Pressurised liquid extraction (PLE)

5320 6) GC-MS

3) Internal standardisation with unlabelled substances

22-binaphthyl

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

D) Other 3 hours refluxing

5321 1) HPLC-FLD

2) Internal standardisation with labelled substances

6-meacutethylchrysegravene

1) Saponification 2) LiquidLiquid partitioning

B) Sonication

5322 6) GC-MS

2) Internal standardisation with labelled substances

naftalene-d8 acenaphtalene-d10 phenanthrene-d10 chrysene-d12 perylene-d12 benzo(e)anthracene-d12

1) Saponification 2) LiquidLiquid partitioning 6) Other

clean-up with silica packed column

C) Soxhlet extraction

5323 1) HPLC-FLD

1) External calibration

4) Solid phase extraction 6) Other

Accelerated Solvent Extraction

A) Pressurised liquid extraction (PLE)

66

European Commission EUR 25245 EN ndash Joint Research Centre ndash Institute for Reference Materials and Measurements Title Report on the 8th inter-laboratory comparison organised by the European Union Reference Laboratory for Polycyclic Aromatic Hydrocarbons - Four marker PAHs in a dry extract of St Johns wort Author(s) Radoslav Lizak Szilard Szilagyi Philippe Verlinde and Thomas WENZL Luxembourg Publications Office of the European Union 2012 ndash 66 pp ndash 201 x 297 cm EUR ndash Scientific and Technical Research series ndash ISSN 1831-9424 ISBN 978-92-79-23337-1 doi 10278759387 Abstract The proficiency test here reported concerned the determination of the contents of four marker polycyclic aromatic hydrocarbons (PAHs) and of their sum in an food supplements test sample The set of marker PAHs consists of benz[a]anthacene benzo[a]pyrene benzo[b]fluoranthene and chrysene Participants to these PT were National Reference Laboratories for PAHs (NRLs-PAHs) and EU official food control laboratories The number of participants was in total 48

The PT was organised according ISO Standard 170432010

The test material used was a commercial dry extract of St Johns wort Participants also received a solution of the same PAHs either in acetonitrile or in toluene for checking their instrument calibration

The results from participants were rated with z-scores and zeta-scores About 68 of the reported results were attributed with z-scores with an absolute value of below two which is the threshold for satisfactory performance

How to obtain EU publications

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service of the European Commission the JRC functions as a reference centre of

science and technology for the Union Close to the policy-making process it serves

the common interest of the Member States while being independent of special

interests whether private or national

L

A-N

A-252

45-EN

-N

• 1 Summary
• 2 Introduction
• 3 Scope
• 4 Participating Laboratories
• 5 Time frame
• 6 Confidentiality
• 7 Test materials
• • 71 1Preparation of test materials
  • 72 Assigned values and standard deviation for proficiency assessment
  • 73 Homogeneity and stability
  • 74 Sample dispatch
  • • 8 Design of the proficiency test
    • 9 Evaluation of the results
    • • 91 2General
      • 92 21BEvaluation criteria
      • 93 Performance of participants
      • 94 Evaluation of the influence of analysis method
      • 95 Evaluation of the influence of calibration
      • 96 26Evaluation of compliance with legislation
      • • 10 Follow-up actions for underperforming laboratories
        • 11 1Conclusions
        • 12 1Acknowledgements
        • 13 References

34

ANNEX 3 - Announcement sample dispatch

Dear MadameSir

The test samples for the proficiency test on the determination of four PAHs in a food supplement (dry extract of St Johns wort) were

dispatched today

Explanation on the design of the study as well as instructions for sample storage and for reporting of results are in the parcel You will

also find your participant key in the parcel which is required for reporting of results

Please check the parcel after arrival for completeness and report the receipt of the test samples as well as their status to us by applying

the sample receipt form which is in the parcel

You are kindly requested to inform us by email if you do not receive the parcel by end of this week

My colleague Donata Lerda will then take care of it

With best regards

Thomas Wenzl

35

ANNEX 4 - Specifications of standard solutions with disclosed concentration

36

ANNEX 4 - continued

37

ANNEX 5 - Homogeneity and stability of the test material

Homogeneity of the test material

Benz[a]anthracene - BAA

Benzo[a]pyrene - BAP

38

ANNEX 5 - continued

Benzo[b]fluoranthene - BBF

Chrysene - CHR

39

ANNEX 5 - continued

Stability of the test material

Table 10 Content of the four target analytes in test materials stored under different

temperatures

BaA BaP BbF CHR

microgkg microgkg microgkg microgkg

Content 31 16 31 69 Storage at room temperature

U (k=2) 05 02 05 17

Content 31 15 29 68 Storage at reference temperature

U (k=2) 04 03 04 10

The applied capillary column does not separate CHR from triphenylene

40

ANNEX 6 ndash Documents sent to participants

41

ANNEX 6 - continued

42

ANNEX 7 Questionnaire on details of analysis method

43

ANNEX 7 - continued

44

ANNEX 7 - continued

45

ANNEX 8 - Results reported by participants for the food supplements test sample Table 11 Analysis results reported by the participants for the content of benz[a]anthracene (BAA) in the food supplements test material Values are presented as reported

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

3600 31 29 29 3 05 2

3602 199 193 178 19 021

3603 253 253 246 251 022 2

3604 199 209 23 213 071 2

3605 301 3 271 291 076 2

3606 302 288 278 289 064 2

3607 456 416 443 438 096 2

3608 796 695 651 714 149 2

3610 182 192 214 196 039 2

3611 414 504 488 469 96 2

3612 3 34 28 31 06 2

3613 32 33 34 33 07 2

3614 182 182 191 185 037 2

3615 3884 3822 3565 376 0752 2

3616 2 2 2 2 01 2

3617 366 37 35 362 046 2

3618 32 32 32 32 05 2

3619 299 305 297 299 048 2

3620 304 294 302 302 045 2

3621 29 26 29 28 04 2

3622 343 325 318 322 052 2

3623 35 3 33 33 07 2

3624

3626 318 31 314 314 126 2

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

5301 31 3 32 31 06 2

5302 314 319 315 316 063 2

5303 3102 2716 2822 2876 27 62

5304 261 263 26 261 57 2

5305 23 22 26 24 20

5306 22 23 23 22 11 2

5307 488 461 317 461

5308 168633 168544 1562025 1644598

5309 16 19 16 17

5310 3 304 311 305 015

5311 477 452 448 459 15 2

5312 506 63 494 543 134 2

5313 353 301 404 353

5314 431 373 378 394 5 096

5315 596 531 51 545 11 2

5316 224 231 235 235 022 2

5317 216 23 221 222 007 2

5318 064 07 067 01 2

5319 111 184 146 147 029 2

5320 515 525 564 534 48

5321 21 5322 13 87 9 87 35 2

5323 38 33 35 33 16 2

46

Figure 6 Results of replicate determinations (indicated by triangles) of benz[a]anthracene (BAA) in the food supplement test material

Horizontal blue lines represent the arithmetic mean value of replicate measurements and blue bars the reported expanded measurement uncertainty (k=2) The assigned value is

plotted as dotted line Red solid lines mark the range of satisfactory performance (|z|le2) Mean values are given for results outside the displayed data range

ProLab 2011

Laboratory

53

1853

1953

0853

0936

1436

0236

1036

1653

2136

0453

1753

0653

1653

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0353

0436

2153

0336

0636

0536

0036

2036

1953

1036

1253

0136

2653

0236

1836

2336

2236

1353

1353

2336

1736

1553

1453

0736

0753

1153

2053

1253

1536

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2236

11

an

aly

te c

on

ten

t (micro

gk

g)

10

95

9

85

8

75

7

65

6

55

5

45

4

35

3

25

2

15

1

05

0

102

3 468

7

Sample St Johns wort extract replicatesMeasurand Benz[a]anthraceneNo of laboratories 46

Assigned value 298 microgkg (Reference value)Target sd 062 microgkgTolerance limits 174 - 422 microgkg (|Z score| lt 200)

Assig

ned va

lue

Limit of tolerance

Limit of tolerance

47

Table 12 Analysis results reported by the participants for the content of benzo[a]pyrene (BAP) in the food supplements test material Values are presented as reported

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

3600 16 17 17 17 03 2

3602 118 123 13 124 015

3603 165 162 157 161 015 2

3604 134 154 139 143 04 2

3605 164 166 172 167 057 2

3606 181 159 161 167 031 2

3607 279 266 279 274 055 2

3608 57 687 461 573 226 2

3610 226 216 183 206 042 2

3611 289 325 311 308 36 2

3612 24 22 2 22 05 2

3613 17 18 19 18 05 2

3614 08 08 08 08 016 2

3615 1545 1393 1425 145 029 2

3616 16 16 14 15 03 2

3617 177 171 158 169 023 2

3618 2 2 2 2 02 2

3619 207 207 206 207 075 2

3620 165 169 163 166 017 2

3621 44 47 49 47 05 2

3622 212 208 222 213 038 2

3623 2 21 22 21 03 2

3624 18 178 182 18 031 2

3626 172 173 178 174 07 2

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

5301 18 18 18 18 04 2

5302 188 19 187 188 038 2

5303 1691 1426 149 1535 19 80

5304 145 142 15 146 69 2

5305 15 17 18 16 15

5306 16 17 17 16 08 2

5307 164 157 153 157

5308 102669 109324 109035 107009

5309 1 1 12 11

5310 122 122 124 123 006

5311 199 184 215 2 15 2

5312 258 248 239 248 05 2

5313 199 178 245 207

5314 174 171 172 172 11 0948

5315 253 248 213 238 05 2

5316 177 171 173 17 016 2

5317 255 277 276 269 012 2

5318 114 12 117 02 2

5319 19 228 231 216 043 2

5320 303 3 344 316 78

5321 12 12 12 12

5322 13 17 24 17 35 2

5323 2 2 19 2 1 2

48

Figure 7 Results of replicate determinations (indicated by triangles) of benzo[a]pyrene (BAP) in the food supplement test material

Horizontal blue lines represent the arithmetic mean value of replicate measurements and blue bars the reported expanded measurement uncertainty (k=2) The assigned value is

plotted as dotted line Red solid lines mark the range of satisfactory performance (|z|le2) Mean values are given for results outside the displayed data range

ProLab 2011

Laboratory

36

1453

0953

0853

1853

2153

1036

0236

0436

1553

0436

1653

0353

0736

0336

2036

0053

0553

0636

0636

0536

1753

1453

1636

2636

1336

2453

0153

2253

0253

2353

1136

1836

1953

1336

1036

2336

2253

1936

1253

1553

1253

1736

0753

2036

2136

0836

11

an

aly

te c

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ten

t (micro

gk

g)

55

5

45

4

35

3

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2

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1

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0

573

308

3

Sample St Johns wort extract replicatesMeasurand Benzo[a]pyreneNo of laboratories 48

Assigned value 165 microgkg (Reference value)Target sd 036 microgkgTolerance limits 093 - 237 microgkg (|Z score| lt 200)

Assig

ned va

lue

Limit of tolerance

Limit of tolerance

49

Table 13 Analysis results reported by the participants for the content of benzo[b]fluoranthene (BBF) in the food supplements test material Values are presented as reported

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

3600 32 3 3 31 09 2

3602 173 175 16 17 017

3603 265 252 25 256 025 2

3604 116 108 105 11 028 2

3605 286 225 232 247 074 2

3606 294 273 261 276 046 2

3607 579 579 544 567 113 2

3608 103 1201 876 1036 326 2

3610 173 196 204 191 038 2

3611 546 618 565 576 75 2

3612 3 28 28 29 06 2

3613 28 31 32 31 06 2

3614 135 111 135 127 022 2

3615 4187 3678 4111 4 08 2

3616 2 2 2 2 01 2

3617 33 27 299 035 2

3618 31 31 31 31 05 2

3619 326 324 312 326 064 2

3620 277 28 272 276 069 2

3621 26 25 23 25 04 2

3622 346 336 333 34 052 2

3623 26 28 29 28 02 2

3624

3626 288 28 307 292 117 2

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

5301 29 3 29 29 06 2

5302 297 3 296 298 06 2

5303 2705 2444 2608 2586 22 78

5304 245 254 26 253 85 2

5305 23 23 26 24 15

5306 42 43 4 42 21 2

5307 221 216 241 221

5308 232169 246654 240722 229848

5309 19 22 19 2 0

5310 3 315 325 313 016

5311 262 277 304 281 15 2

5312 27 289 337 299 075 2

5313 321 284 378 328

5314 423 404 385 404 34 083

5315 42 506 416 444 09 2

5316 265 267 277 273 018 2

5317 185 197 195 192 006 2

5318 276 281 279 04 2

5319 396 389 403 396 079 2

5320 623 618 635 625 14

5321 31

5322 24 33 49 33 35 2

5323 38 45 35 45 27 2

50

Figure 8 Results of replicate determinations (indicated by triangles) of benzo[b]fluoranthene (BBF) in the food supplement test material

Horizontal blue lines represent the arithmetic mean value of replicate measurements and blue bars the reported expanded measurement uncertainty (k=2) The assigned value is

plotted as dotted line Red solid lines mark the range of satisfactory performance (|z|le2) Mean values are given for results outside the displayed data range

ProLab 2011

Laboratory

36

0436

1436

0236

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1736

1653

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05

0

103

6

576

3

Sample St Johns wort extract replicatesMeasurand Benzo[b]fluorantheneNo of laboratories 46

Assigned value 292 microgkg (Reference value)Target sd 060 microgkgTolerance limits 172 - 412 microgkg (|Z score| lt 200)

Assig

ned va

lue

Limit of tolerance

Limit of tolerance

51

Table 14 Analysis results reported by the participants for the content of chrysene (CHR) in the food supplements test material Values are presented as reported

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

3600 5 49 5 5 07 2

3602 32 304 29 304 036

3603 462 469 447 459 048 2

3604 438 428 42 429 119 2

3605 365 41 381 386 085 2

3606 494 411 45 451 124 2

3607 897 774 787 819 18 2

3608 986 887 1168 1014 284 2

3610 418 392 462 424 085 2

3611 833 893 87 865 61 2

3612 6 64 58 61 12 2

3613 41 39 4 4 08 2

3614 272 254 288 271 054 2

3615 605 6028 5964 601 12 2

3616 47 45 49 47 05 2

3617 445 431 389 422 065 2

3618 45 45 45 45 06 2

3619 738 747 72 738 118 2

3620 463 472 446 46 092 2

3621 53 49 51 51 06 2

3622 478 513 505 488 08 2

3623 73 73 72 72 05 2

3624

3626 63 622 634 628 251 2

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

5301 49 49 49 49 1 2

5302 687 683 666 679 136 2

5303 6777 6181 6382 6441 24 70

5304 592 581 6 591 85 2

5305 6 58 64 61 20

5306 49 5 51 49 25 2

5307 524 5 574 524

5308 211488 216782 221146 213243

5309

5310 372 393 394 386 019

5311 43 459 422 437 15 2

5312 934 943 981 953 191 2

5313 793 73 99 838

5314 636 561 565 588 18 0818

5315 547 583 499 542 11 2

5316 292 286 285 282 06 2

5317 52 598 517 545 046 2

5318 368 345 357 05 2

5319 449 471 474 465 116 2

5320 76 924 818 834 99

5321 33

5322 7 7 76 7 35 2

5323 87 73 77 73 37 2

52

Figure 9 Results of replicate determinations (indicated by triangles) of chrysene (CHR) in the food supplement test material

Horizontal blue lines represent the arithmetic mean value of replicate measurements and blue bars the reported expanded measurement uncertainty (k=2) The assigned value is

plotted as dotted line Red solid lines mark the range of satisfactory performance (|z|le2) Mean values are given for results outside the displayed data range

ProLab 2011

Laboratory

53

0836

1453

1636

0253

2153

1836

0553

1036

1336

1736

1036

0453

1136

1836

0636

0336

2053

1936

1653

0136

0036

2253

0636

2153

0753

1553

1753

1453

0436

1536

1253

0536

2653

0353

0253

2236

2336

1953

2336

0753

2053

1353

1236

0836

11

an

aly

te c

on

ten

t (micro

gk

g)

13

12

11

10

9

8

7

6

5

4

3

2

1

0

865

3

Sample St Johns wort extract replicatesMeasurand ChryseneNo of laboratories 45

Assigned value 407 microgkg (Reference value)Target sd 083 microgkgTolerance limits 241 - 573 microgkg (|Z score| lt 200)

Assig

ned va

lue

Limit of tolerance

Limit of tolerance

53

Table 15 Analysis results reported by the participants for the sum of the four marker PAHs in the food supplements test material Values are presented as reported

Laboratory code

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg

3600 128 24 2

3602 79 126

3603 1127 096 2

3604 893 258 2

3605 1091 305 2

3606 1184 238 2

3607 2098 42 2

3608 3336 325 2

3610 1138 17 2

3611 2218 259 2

3612 143 29 2

3613 122 2 2

3614 663 129 2

3615 152 304 2

3616 102 03 2

3617 1252 169 2

3618 128 18 2

3619 157 305 2

3620 1204 12 2

3621 151 19 2

3622 1361 115 2

3623 154 09 2

3624

3626 1409 56 2

Laboratory code

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg

5301 126

5302 37 074 2

5303 13438 92

5304 1251 85 2

5305 125 20

5306 129 65 2

5307 1363

5308 7245598

5309

5310 1127 056

5311 1367 15 2

5312 2042 408 2

5313 1726

5314 1558

5315 177 35 2

5316 96 073 2

5317 1229 066 2

5318 82 05 2

5319 1225 267 2

5320 2309 41

5321

5322 207 35 2

5323 171 103 2

54

ANNEX 9 Results reported for the standard solutions with undisclosed analyte content Table 16 Analysis results reported by the participants for the four marker PAHs in the standard solution in toluene with undisclosed analyte

content Values are presented as reported U measurement uncertainty k coverage factor

benz[a]anthracene benzo[a]pyrene benzo[b]fluoranthene chrysene Laboratory

code Unit Result U k Result U k Result U k Result U k 3600 microgl 38 67 2 25 45 2 211 68 2 275 38 2

3605 microgl 367 184 2 2482 124 2 2592 13 2 235 118 2

3606 microgkg 3763 176 2 2609 142 2 2721 118 2 2539 134 2

3607 microgkg 3706 815 2 2824 565 2 2591 518 2 2587 569 2

3612 microgl 38 08 2 237 11 2 244 18 2 257 02 2

3614 microgl 324 65 2 209 38 2 215 37 2 21 42 2

3616 microgkg 318 07 2 224 14 2 231 26 2 216 1 2

3618 microgkg 417 17 2 284 08 2 292 06 2 297 03 2

3619 microgkg 47 742 2 274 468 2 228 387 2 274 434 2

3620 microgl 495 74 2 294 29 2 321 8 2 321 64 2

3621 microgl 241 22 2 143 13 2 188 16 2 145 14 2

3623 microgkg 476 25 2 311 53 2 269 3 2 297 03 2

3626 microgl 359 38 2 235 23 2 238 27 2 24 18 2

5301 microgkg 426 29 299 289

5302 microgkg 436 87 2 296 59 2 296 59 2 289 58 2

5303 microgl 3721 27 102 22838 19 99 23756 22 99 25486 24 98

5304 microgkg 38 29 2 225 26 2 257 37 2 258 52 2

5305 microgkg 305 225 245 29

5306 microgkg 316 63 2 247 49 2 65 13 2 22 44 2

5307 microgkg 3658 2559 241 2414

5308 microgkg 16854 109324 246654 216782

5311 microgl 374 20 2 279 15 2 268 15 2 272 15 2

5313 microgl 3408 2165 2275 2373

5314 microgkg 395 5 2875 11 3614 34 2905 18

5320 microgl 3267 368 1986 571 28 683 2427 512

5322 microgl 282 35 2 225 35 2 241 35 2 21 35 2

55

Figure 10 Youden plots of the percent deviation of the reported values from the assigned values for the food supplement test sample and the

standard solution in toluene with undisclosed content

(The identity of the respective measurand is given above the figure Results exceeding a difference of more than 100 were omitted)

56

Figure 10 - continued

57

Table 17 Analysis results reported by the participants for the four marker PAHs in the standard solution in acetonitrile with undisclosed

analyte content

Values are presented as reported U measurement uncertainty k coverage factor

benz[a]anthracene benzo[a]pyrene benzo[b]fluoranthene chrysene

Laboratory

code Unit Result U k Result U k Result U k Result U k

3602 microgkg 3675 247 247 244

3603 microgkg 358 086 2 236 028 2 235 028 2 236 057 2

3604 microgl 475 29 2 19 12 2 194 13 2 258 12 2

3608 microgl 3623 078 2 2385 023 2 2343 18 2 2296 092 2

3610 microgkg 369 185 2 25 125 2 251 126 2 244 122 2

3611 microgkg 48 02 2 323 02 2 328 04 2 317 03 2

3613 microgl 332 31 2 261 3 2 249 25 2 239 24 2

3615 microgl 415 2635 2729 2689

3617 microgkg 37 09 2 244 06 2 284 07 2 239 06 2

3622 microgl 37 12 2 25 23 2 257 21 2 238 1 2

3624 microgkg 402 104 2 255 084 2 265 096 2 253 07 2

5306 microgkg 563 113 2 371 74 2 359 72 2 333 67 2

5308 microgkg 168633 102669 232169 211488

5309 microgl 402 268 27

5310 microgl 315 158 25 125 25 125 288 144

5312 microgl 428 043 2 282 028 2 272 027 2 275 028 2

5315 microgkg 387 08 2 256 05 2 251 05 2 253 05 2

5316 microgl 3839 342 2 2485 191 2 2559 168 2 239 51 2

5317 microgl 2466 043 2 2212 034 2 2472 016 2 2146 068 2

5319 microgl 414 41 2 279 28 2 268 27 2 26 31 2

5321 microgkg 476 316 326 328

5322 microgl 2547 35 2

5323 microgkg 405 203 2 239 119 2 255 153 2 201 11 2

58

Figure 11 Youden plots of the percent deviation of the reported values from the assigned values for the food supplement test sample and the

standard solution in acetonitrile with undisclosed content

(The identity of the respective measurand is given above the figure Results exceeding a difference of more than 100 were omitted)

59

Figure 11 - continued

60

ANNEX 10 Table 18 Method performance parameters reported by the participants

Laboratory code

Analyte LOD LOQ Recovery Analyte LOD LOQ Recovery

microgkg microgkg microgkg microgkg 3600 BaA 03 06 978 BaP 03 07 9483602 BaA 007 021 926 BaP 005 015 993603 BaA 0011 05 987 BaP 0005 051 9553604 BaA 02 04 86 BaP 02 04 883605 BaA 0025 005 118 BaP 0025 005 1083606 BaA 001 003 65 BaP 001 003 703607 BaA 021 071 74 BaP 027 089 943608 BaA 016 048 8213 BaP 015 046 83683610 BaA 02 05 85 BaP 005 02 853611 BaA BaP 3612 BaA 1 117 BaP 1 1203613 BaA 01 03 90 BaP 01 03 903614 BaA 02 05 96 BaP 02 05 933615 BaA 005 015 110 BaP 005 015 1053616 BaA 01 05 108 BaP 01 05 753617 BaA 018 037 405 BaP 009 018 3873618 BaA 01 03 63 BaP 01 03 593619 BaA 005 005 59 BaP 033 033 543620 BaA 0009 0018 64 BaP 0005 001 553621 BaA 007 02 84 BaP 007 02 923622 BaA 01 03 1101 BaP 01 03 9853623 BaA 02 08 105 BaP 02 08 1013624 BaA BaP 01 033 8513625 BaA BaP 3626 BaA BaP 5301 BaA 003 01 102 BaP 003 01 1005302 BaA 01 03 78 BaP 01 03 865303 BaA 003 01 62 BaP 003 01 805304 BaA 1 1 70 BaP 1 1 955305 BaA 02 05 85 BaP 02 05 855306 BaA 03 1 699 BaP 03 1 905307 BaA 6537 BaP 81165308 BaA 0088 0265 99 BaP 00262 00785 1005309 BaA 01 08 92 BaP 01 08 925310 BaA 01 03 BaP 01 03 905311 BaA 002 005 80 BaP 002 004 915312 BaA 03 09 105 BaP 03 09 1055313 BaA 04 08 BaP 04 08 5314 BaA 001 002 96 BaP 001 002 9485315 BaA 01 03 50 - 100 BaP 01 03 50 - 100 5316 BaA 002 011 827 BaP 004 019 10445317 BaA 05 1 76 BaP 05 1 755318 BaA BaP 5319 BaA 009 029 951 BaP 011 041 995320 BaA 12 18 946 BaP 06 1 8345321 BaA 03 05 BaP 05 1 5322 BaA 1 2 100 BaP 1 2 1005323 BaA 26 BaP 26

61

ANNEX 10 Table 18 - continued

Laboratory code Analyte LOD LOQ Recovery Analyte LOD LOQ Recovery

microgkg microgkg microgkg microgkg 3600 BbF 03 05 96 CHR 02 03 10153602 BbF 015 045 947 CHR 003 009 9193603 BbF 0014 05 1012 CHR 0004 05 10033604 BbF 02 04 85 CHR 02 04 873605 BbF 005 01 94 CHR 0025 005 863606 BbF 001 003 70 CHR 001 003 653607 BbF 018 06 100 CHR 014 028 943608 BbF 015 046 7979 CHR 015 045 71253610 BbF 005 02 85 CHR 02 05 853611 BbF CHR 3612 BbF 1 118 CHR 1 1173613 BbF 01 03 90 CHR 01 03 903614 BbF 01 04 96 CHR 02 05 953615 BbF 008 025 109 CHR 003 01 1083616 BbF 01 05 78 CHR 01 05 723617 BbF 035 07 38 CHR 012 024 4163618 BbF 01 03 62 CHR 01 03 623619 BbF 016 016 63 CHR 01 01 593620 BbF 0003 0006 55 CHR 001 002 653621 BbF 007 02 103 CHR 007 02 893622 BbF 02 06 963 CHR 03 09 1043623 BbF 02 08 106 CHR 02 08 1193624 BbF CHR 3625 BbF CHR 3626 BbF CHR 5301 BbF 003 01 96 CHR 003 01 1145302 BbF 01 03 84 CHR 01 03 835303 BbF 003 01 78 CHR 003 01 705304 BbF 1 1 86 CHR 1 1 705305 BbF 02 05 90 CHR 02 05 955306 BbF 03 1 1435 CHR 03 1 1185307 BbF 8116 CHR 65375308 BbF 0079 0238 99 CHR 009 026 1005309 BbF 01 08 92 CHR 5310 BbF 07 2 CHR 01 03 5311 BbF 002 004 95 CHR 002 004 895312 BbF 03 09 105 CHR 03 09 1055313 BbF 04 08 CHR 04 08 5314 BbF 001 002 83 CHR 001 002 8185315 BbF 02 05 50 -100 CHR 02 05 50 - 100 5316 BbF 005 024 814 CHR 001 007 9595317 BbF 05 1 73 CHR 05 1 785318 BbF CHR 5319 BbF 011 038 946 CHR 013 043 9675320 BbF 07 09 1194 CHR 23 32 10455321 BbF 05 1 CHR 05 1 5322 BbF 1 2 100 CHR 1 2 1005323 BbF 26 CHR 26

62

ANNEX 11 Table 19 Details of analysis method reported by the participants Data are presented as reported

Laboratory code

Analysis technique

Instrument calibration

Internal standards applied

Sample clean up

Details of sample

clean up

Extraction technique

Details of sample

extraction

3600 6) GC-MS

2) Internal standardisation with labelled substances

Deuterated PAH mix 9 (Ehrenstorfer)

1) Saponification 2) LiquidLiquid partitioning

D) Other Liquidliquid partitioning

3602 2) HPLC-UV-FLD

1) External calibration

3) Gel permeation chromatography

A) Pressurised liquid extraction (PLE)

3603 1) HPLC-FLD

1) External calibration

3) Gel permeation chromatography

D) Other 1 minute with dichloromethane in vortex

3604 2) HPLC-UV-FLD

2) Internal standardisation with labelled substances

DiP D14

5) Donor acceptor complex chromatography

D) Other LL

3605 1) HPLC-FLD

1) External calibration

3) Gel permeation chromatography

C) Soxhlet extraction

3606 7) GC-MSMS

2) Internal standardisation with labelled substances

BaP 13C4 CHR 13C6 BaA 13C6 BbF 13C6

4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

3607 6) GC-MS

2) Internal standardisation with labelled substances

BaA-d12 CHR-d12 BbF-d12 BkF-d12 BaP-d12 ICP-d12 BgP-d12 DiP-d14

1) Saponification

D) Other solvent partitioning

3608 1) HPLC-FLD

1) External calibration

6) Other Liquid extraction

D) Other

Extraction in a rotary agitator and centrifugation

3610 2) HPLC-UV-FLD

1) External calibration

4) Solid phase extraction

B) Sonication

3611

3612 6) GC-MS

2) Internal standardisation with labelled substances

chrysene-d12 dibenzo(ah)anthracene-d14 dibenzo(ai)pyrene-d14

4) Solid phase extraction

B) Sonication

3613 5) LC-MSMS

3) Internal standardisation with unlabelled substances

I use Chrysene-D12 3) Gel permeation chromatography

D) Other extraction with hexan

63

Table 19 - continued

Laboratory code

Analysis technique

Instrument calibration

Internal standards applied

Sample clean up Details of sample

clean up

Extraction technique

Details of sample

extraction

3614 6) GC-MS 1) External calibration

3) Gel permeation chromatography

B) Sonication

3615 1) HPLC-FLD

1) External calibration

6) Other PLE GPC

A) Pressurised liquid extraction (PLE)

3616 6) GC-MS

2) Internal standardisation with labelled substances

Benzo(a)pyrene-13C4 Benzo(a)anthracene-13C6 Benzo(b)fluoranthene-13C6 Chrysene-13C6

2) LiquidLiquid partitioning 4) Solid phase extraction

B) Sonication

3617 1) HPLC-FLD

1) External calibration

4) Solid phase extraction

C) Soxhlet extraction

3618 6) GC-MS

2) Internal standardisation with labelled substances

deuterated standards

1) Saponification 4) Solid phase extraction

D) Other Handshaken with cyclohexane

3619 6) GC-MS

2) Internal standardisation with labelled substances

C13 Labelled US EPA 16

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

D) Other Homegenisation amp saponiofication

3620 9) GC-HRMS

2) Internal standardisation with labelled substances

mix of deuterated PAHs (BAA-D12 CHR-D12 BAP-D12 BBF-D12)

3) Gel permeation chromatography 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

3621 6) GC-MS

2) Internal standardisation with labelled substances

Chrysene D12 Benzo(a)pyrene D12 Benzo(b)fluoranthene D12 Benzo(a)anthracene

2) LiquidLiquid partitioning 4) Solid phase extraction

B) Sonication

3622 1) HPLC-FLD

4) Standard addition method

4) Solid phase extraction

D) Other

Extraction with cyclohexane by heating under reflux

3623 7) GC-MSMS

2) Internal standardisation with labelled substances

C13 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

3624 2) HPLC-UV-FLD

1) External calibration

2) LiquidLiquid partitioning

D) Other hot solvent extraction

3626

64

Table 19 - continued

Laboratory code

Analysis technique

Instrument calibration

Internal standards

applied

Sample clean up

Details of sample

clean up

Extraction technique

Details of sample

extraction

5301 7) GC-MSMS

2) Internal standardisation with labelled substances

EACH SUBSTANCE C13

4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5302 7) GC-MSMS

2) Internal standardisation with labelled substances

Isotope labelled PAHs 13C for each analyte

4) Solid phase extraction

C) Soxhlet extraction

5303 7) GC-MSMS

3) Internal standardisation with unlabelled substances

HAP C13 4) Solid phase extraction

D) Other automate ASE200 DIONEX

5304 7) GC-MSMS

2) Internal standardisation with labelled substances

with C13 for each HAP

2) LiquidLiquid partitioning 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5305 7) GC-MSMS

2) Internal standardisation with labelled substances

C13 isotopiques internal standards

4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5306 7) GC-MSMS

2) Internal standardisation with labelled substances

C13 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5307 6) GC-MS

2) Internal standardisation with labelled substances

1) Saponification 2) LiquidLiquid partitioning

D) Other solvent extraction

5308 1) HPLC-FLD 1) External calibration

4) Solid phase extraction

B) Sonication

5309 1) HPLC-FLD 1) External calibration

4) Solid phase extraction

D) Other only SPE

5310 1) HPLC-FLD 1) External calibration

1) Saponification 2) LiquidLiquid partitioning 3) Gel permeation chromatography

C) Soxhlet extraction

5311 6) GC-MS

2) Internal standardisation with labelled substances

13 C Chrysene 13 C Benz(a)pyrene

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

B) Sonication

5312 1) HPLC-FLD

3) Internal standardisation with unlabelled substances

Benzo(b)chrysen

2) LiquidLiquid partitioning 4) Solid phase extraction

Quechers D) Other Quechers

65

Table 19 - continued

Laboratory code

Analysis technique

Instrument calibration

Internal standards applied

Sample clean up Details of sample

clean up

Extraction technique

Details of sample extraction

5313 6) GC-MS

2) Internal standardisation with labelled substances

Benz(a)anthracene D12 Benzo(b)fluranthene D12 Benzo(a)pyrene D12 Chrysene D12

1) Saponification 2) LiquidLiquid partitioning 3) Gel permeation chromatography

A) Pressurised liquid extraction (PLE)

5314 6) GC-MS

2) Internal standardisation with labelled substances

Benzo(a)pyren d12 2) LiquidLiquid partitioning

D) Other Extraction with

5315 1) HPLC-FLD

3) Internal standardisation with unlabelled substances

Benz(b)chrysen

1) Saponification 2) LiquidLiquid partitioning

D) Other shaker

5316 1) HPLC-FLD

1) External calibration

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

D) Other

LiquidLiquid extraction with cyclohexane and dimethylformamide

5317 7) GC-MSMS

1) External calibration

3) Gel permeation chromatography 6) Other

silica gel D) Other VDLUFA online method

5318

5319 1) HPLC-FLD

3) Internal standardisation with unlabelled substances

Benzo(b)chrysene 3) Gel permeation chromatography

A) Pressurised liquid extraction (PLE)

5320 6) GC-MS

3) Internal standardisation with unlabelled substances

22-binaphthyl

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

D) Other 3 hours refluxing

5321 1) HPLC-FLD

2) Internal standardisation with labelled substances

6-meacutethylchrysegravene

1) Saponification 2) LiquidLiquid partitioning

B) Sonication

5322 6) GC-MS

2) Internal standardisation with labelled substances

naftalene-d8 acenaphtalene-d10 phenanthrene-d10 chrysene-d12 perylene-d12 benzo(e)anthracene-d12

1) Saponification 2) LiquidLiquid partitioning 6) Other

clean-up with silica packed column

C) Soxhlet extraction

5323 1) HPLC-FLD

1) External calibration

4) Solid phase extraction 6) Other

Accelerated Solvent Extraction

A) Pressurised liquid extraction (PLE)

66

European Commission EUR 25245 EN ndash Joint Research Centre ndash Institute for Reference Materials and Measurements Title Report on the 8th inter-laboratory comparison organised by the European Union Reference Laboratory for Polycyclic Aromatic Hydrocarbons - Four marker PAHs in a dry extract of St Johns wort Author(s) Radoslav Lizak Szilard Szilagyi Philippe Verlinde and Thomas WENZL Luxembourg Publications Office of the European Union 2012 ndash 66 pp ndash 201 x 297 cm EUR ndash Scientific and Technical Research series ndash ISSN 1831-9424 ISBN 978-92-79-23337-1 doi 10278759387 Abstract The proficiency test here reported concerned the determination of the contents of four marker polycyclic aromatic hydrocarbons (PAHs) and of their sum in an food supplements test sample The set of marker PAHs consists of benz[a]anthacene benzo[a]pyrene benzo[b]fluoranthene and chrysene Participants to these PT were National Reference Laboratories for PAHs (NRLs-PAHs) and EU official food control laboratories The number of participants was in total 48

The PT was organised according ISO Standard 170432010

The test material used was a commercial dry extract of St Johns wort Participants also received a solution of the same PAHs either in acetonitrile or in toluene for checking their instrument calibration

The results from participants were rated with z-scores and zeta-scores About 68 of the reported results were attributed with z-scores with an absolute value of below two which is the threshold for satisfactory performance

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interests whether private or national

L

A-N

A-252

45-EN

-N

• 1 Summary
• 2 Introduction
• 3 Scope
• 4 Participating Laboratories
• 5 Time frame
• 6 Confidentiality
• 7 Test materials
• • 71 1Preparation of test materials
  • 72 Assigned values and standard deviation for proficiency assessment
  • 73 Homogeneity and stability
  • 74 Sample dispatch
  • • 8 Design of the proficiency test
    • 9 Evaluation of the results
    • • 91 2General
      • 92 21BEvaluation criteria
      • 93 Performance of participants
      • 94 Evaluation of the influence of analysis method
      • 95 Evaluation of the influence of calibration
      • 96 26Evaluation of compliance with legislation
      • • 10 Follow-up actions for underperforming laboratories
        • 11 1Conclusions
        • 12 1Acknowledgements
        • 13 References

35

ANNEX 4 - Specifications of standard solutions with disclosed concentration

36

ANNEX 4 - continued

37

ANNEX 5 - Homogeneity and stability of the test material

Homogeneity of the test material

Benz[a]anthracene - BAA

Benzo[a]pyrene - BAP

38

ANNEX 5 - continued

Benzo[b]fluoranthene - BBF

Chrysene - CHR

39

ANNEX 5 - continued

Stability of the test material

Table 10 Content of the four target analytes in test materials stored under different

temperatures

BaA BaP BbF CHR

microgkg microgkg microgkg microgkg

Content 31 16 31 69 Storage at room temperature

U (k=2) 05 02 05 17

Content 31 15 29 68 Storage at reference temperature

U (k=2) 04 03 04 10

The applied capillary column does not separate CHR from triphenylene

40

ANNEX 6 ndash Documents sent to participants

41

ANNEX 6 - continued

42

ANNEX 7 Questionnaire on details of analysis method

43

ANNEX 7 - continued

44

ANNEX 7 - continued

45

ANNEX 8 - Results reported by participants for the food supplements test sample Table 11 Analysis results reported by the participants for the content of benz[a]anthracene (BAA) in the food supplements test material Values are presented as reported

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

3600 31 29 29 3 05 2

3602 199 193 178 19 021

3603 253 253 246 251 022 2

3604 199 209 23 213 071 2

3605 301 3 271 291 076 2

3606 302 288 278 289 064 2

3607 456 416 443 438 096 2

3608 796 695 651 714 149 2

3610 182 192 214 196 039 2

3611 414 504 488 469 96 2

3612 3 34 28 31 06 2

3613 32 33 34 33 07 2

3614 182 182 191 185 037 2

3615 3884 3822 3565 376 0752 2

3616 2 2 2 2 01 2

3617 366 37 35 362 046 2

3618 32 32 32 32 05 2

3619 299 305 297 299 048 2

3620 304 294 302 302 045 2

3621 29 26 29 28 04 2

3622 343 325 318 322 052 2

3623 35 3 33 33 07 2

3624

3626 318 31 314 314 126 2

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

5301 31 3 32 31 06 2

5302 314 319 315 316 063 2

5303 3102 2716 2822 2876 27 62

5304 261 263 26 261 57 2

5305 23 22 26 24 20

5306 22 23 23 22 11 2

5307 488 461 317 461

5308 168633 168544 1562025 1644598

5309 16 19 16 17

5310 3 304 311 305 015

5311 477 452 448 459 15 2

5312 506 63 494 543 134 2

5313 353 301 404 353

5314 431 373 378 394 5 096

5315 596 531 51 545 11 2

5316 224 231 235 235 022 2

5317 216 23 221 222 007 2

5318 064 07 067 01 2

5319 111 184 146 147 029 2

5320 515 525 564 534 48

5321 21 5322 13 87 9 87 35 2

5323 38 33 35 33 16 2

46

Figure 6 Results of replicate determinations (indicated by triangles) of benz[a]anthracene (BAA) in the food supplement test material

Horizontal blue lines represent the arithmetic mean value of replicate measurements and blue bars the reported expanded measurement uncertainty (k=2) The assigned value is

plotted as dotted line Red solid lines mark the range of satisfactory performance (|z|le2) Mean values are given for results outside the displayed data range

ProLab 2011

Laboratory

53

1853

1953

0853

0936

1436

0236

1036

1653

2136

0453

1753

0653

1653

0536

0353

0436

2153

0336

0636

0536

0036

2036

1953

1036

1253

0136

2653

0236

1836

2336

2236

1353

1353

2336

1736

1553

1453

0736

0753

1153

2053

1253

1536

0853

2236

11

an

aly

te c

on

ten

t (micro

gk

g)

10

95

9

85

8

75

7

65

6

55

5

45

4

35

3

25

2

15

1

05

0

102

3 468

7

Sample St Johns wort extract replicatesMeasurand Benz[a]anthraceneNo of laboratories 46

Assigned value 298 microgkg (Reference value)Target sd 062 microgkgTolerance limits 174 - 422 microgkg (|Z score| lt 200)

Assig

ned va

lue

Limit of tolerance

Limit of tolerance

47

Table 12 Analysis results reported by the participants for the content of benzo[a]pyrene (BAP) in the food supplements test material Values are presented as reported

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

3600 16 17 17 17 03 2

3602 118 123 13 124 015

3603 165 162 157 161 015 2

3604 134 154 139 143 04 2

3605 164 166 172 167 057 2

3606 181 159 161 167 031 2

3607 279 266 279 274 055 2

3608 57 687 461 573 226 2

3610 226 216 183 206 042 2

3611 289 325 311 308 36 2

3612 24 22 2 22 05 2

3613 17 18 19 18 05 2

3614 08 08 08 08 016 2

3615 1545 1393 1425 145 029 2

3616 16 16 14 15 03 2

3617 177 171 158 169 023 2

3618 2 2 2 2 02 2

3619 207 207 206 207 075 2

3620 165 169 163 166 017 2

3621 44 47 49 47 05 2

3622 212 208 222 213 038 2

3623 2 21 22 21 03 2

3624 18 178 182 18 031 2

3626 172 173 178 174 07 2

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

5301 18 18 18 18 04 2

5302 188 19 187 188 038 2

5303 1691 1426 149 1535 19 80

5304 145 142 15 146 69 2

5305 15 17 18 16 15

5306 16 17 17 16 08 2

5307 164 157 153 157

5308 102669 109324 109035 107009

5309 1 1 12 11

5310 122 122 124 123 006

5311 199 184 215 2 15 2

5312 258 248 239 248 05 2

5313 199 178 245 207

5314 174 171 172 172 11 0948

5315 253 248 213 238 05 2

5316 177 171 173 17 016 2

5317 255 277 276 269 012 2

5318 114 12 117 02 2

5319 19 228 231 216 043 2

5320 303 3 344 316 78

5321 12 12 12 12

5322 13 17 24 17 35 2

5323 2 2 19 2 1 2

48

Figure 7 Results of replicate determinations (indicated by triangles) of benzo[a]pyrene (BAP) in the food supplement test material

Horizontal blue lines represent the arithmetic mean value of replicate measurements and blue bars the reported expanded measurement uncertainty (k=2) The assigned value is

plotted as dotted line Red solid lines mark the range of satisfactory performance (|z|le2) Mean values are given for results outside the displayed data range

ProLab 2011

Laboratory

36

1453

0953

0853

1853

2153

1036

0236

0436

1553

0436

1653

0353

0736

0336

2036

0053

0553

0636

0636

0536

1753

1453

1636

2636

1336

2453

0153

2253

0253

2353

1136

1836

1953

1336

1036

2336

2253

1936

1253

1553

1253

1736

0753

2036

2136

0836

11

an

aly

te c

on

ten

t (micro

gk

g)

55

5

45

4

35

3

25

2

15

1

05

0

573

308

3

Sample St Johns wort extract replicatesMeasurand Benzo[a]pyreneNo of laboratories 48

Assigned value 165 microgkg (Reference value)Target sd 036 microgkgTolerance limits 093 - 237 microgkg (|Z score| lt 200)

Assig

ned va

lue

Limit of tolerance

Limit of tolerance

49

Table 13 Analysis results reported by the participants for the content of benzo[b]fluoranthene (BBF) in the food supplements test material Values are presented as reported

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

3600 32 3 3 31 09 2

3602 173 175 16 17 017

3603 265 252 25 256 025 2

3604 116 108 105 11 028 2

3605 286 225 232 247 074 2

3606 294 273 261 276 046 2

3607 579 579 544 567 113 2

3608 103 1201 876 1036 326 2

3610 173 196 204 191 038 2

3611 546 618 565 576 75 2

3612 3 28 28 29 06 2

3613 28 31 32 31 06 2

3614 135 111 135 127 022 2

3615 4187 3678 4111 4 08 2

3616 2 2 2 2 01 2

3617 33 27 299 035 2

3618 31 31 31 31 05 2

3619 326 324 312 326 064 2

3620 277 28 272 276 069 2

3621 26 25 23 25 04 2

3622 346 336 333 34 052 2

3623 26 28 29 28 02 2

3624

3626 288 28 307 292 117 2

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

5301 29 3 29 29 06 2

5302 297 3 296 298 06 2

5303 2705 2444 2608 2586 22 78

5304 245 254 26 253 85 2

5305 23 23 26 24 15

5306 42 43 4 42 21 2

5307 221 216 241 221

5308 232169 246654 240722 229848

5309 19 22 19 2 0

5310 3 315 325 313 016

5311 262 277 304 281 15 2

5312 27 289 337 299 075 2

5313 321 284 378 328

5314 423 404 385 404 34 083

5315 42 506 416 444 09 2

5316 265 267 277 273 018 2

5317 185 197 195 192 006 2

5318 276 281 279 04 2

5319 396 389 403 396 079 2

5320 623 618 635 625 14

5321 31

5322 24 33 49 33 35 2

5323 38 45 35 45 27 2

50

Figure 8 Results of replicate determinations (indicated by triangles) of benzo[b]fluoranthene (BBF) in the food supplement test material

Horizontal blue lines represent the arithmetic mean value of replicate measurements and blue bars the reported expanded measurement uncertainty (k=2) The assigned value is

plotted as dotted line Red solid lines mark the range of satisfactory performance (|z|le2) Mean values are given for results outside the displayed data range

ProLab 2011

Laboratory

36

0436

1436

0236

1053

1736

1653

0953

0753

0853

0536

2136

0553

0436

0353

0353

1636

0636

2036

2353

1853

1136

1236

2653

0153

0253

1236

1736

1336

0036

1853

2153

1036

1953

1336

2253

2253

2353

1936

1553

1453

0653

1536

0753

2036

0836

11

an

aly

te c

on

ten

t (micro

gk

g)

75

7

65

6

55

5

45

4

35

3

25

2

15

1

05

0

103

6

576

3

Sample St Johns wort extract replicatesMeasurand Benzo[b]fluorantheneNo of laboratories 46

Assigned value 292 microgkg (Reference value)Target sd 060 microgkgTolerance limits 172 - 412 microgkg (|Z score| lt 200)

Assig

ned va

lue

Limit of tolerance

Limit of tolerance

51

Table 14 Analysis results reported by the participants for the content of chrysene (CHR) in the food supplements test material Values are presented as reported

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

3600 5 49 5 5 07 2

3602 32 304 29 304 036

3603 462 469 447 459 048 2

3604 438 428 42 429 119 2

3605 365 41 381 386 085 2

3606 494 411 45 451 124 2

3607 897 774 787 819 18 2

3608 986 887 1168 1014 284 2

3610 418 392 462 424 085 2

3611 833 893 87 865 61 2

3612 6 64 58 61 12 2

3613 41 39 4 4 08 2

3614 272 254 288 271 054 2

3615 605 6028 5964 601 12 2

3616 47 45 49 47 05 2

3617 445 431 389 422 065 2

3618 45 45 45 45 06 2

3619 738 747 72 738 118 2

3620 463 472 446 46 092 2

3621 53 49 51 51 06 2

3622 478 513 505 488 08 2

3623 73 73 72 72 05 2

3624

3626 63 622 634 628 251 2

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

5301 49 49 49 49 1 2

5302 687 683 666 679 136 2

5303 6777 6181 6382 6441 24 70

5304 592 581 6 591 85 2

5305 6 58 64 61 20

5306 49 5 51 49 25 2

5307 524 5 574 524

5308 211488 216782 221146 213243

5309

5310 372 393 394 386 019

5311 43 459 422 437 15 2

5312 934 943 981 953 191 2

5313 793 73 99 838

5314 636 561 565 588 18 0818

5315 547 583 499 542 11 2

5316 292 286 285 282 06 2

5317 52 598 517 545 046 2

5318 368 345 357 05 2

5319 449 471 474 465 116 2

5320 76 924 818 834 99

5321 33

5322 7 7 76 7 35 2

5323 87 73 77 73 37 2

52

Figure 9 Results of replicate determinations (indicated by triangles) of chrysene (CHR) in the food supplement test material

Horizontal blue lines represent the arithmetic mean value of replicate measurements and blue bars the reported expanded measurement uncertainty (k=2) The assigned value is

plotted as dotted line Red solid lines mark the range of satisfactory performance (|z|le2) Mean values are given for results outside the displayed data range

ProLab 2011

Laboratory

53

0836

1453

1636

0253

2153

1836

0553

1036

1336

1736

1036

0453

1136

1836

0636

0336

2053

1936

1653

0136

0036

2253

0636

2153

0753

1553

1753

1453

0436

1536

1253

0536

2653

0353

0253

2236

2336

1953

2336

0753

2053

1353

1236

0836

11

an

aly

te c

on

ten

t (micro

gk

g)

13

12

11

10

9

8

7

6

5

4

3

2

1

0

865

3

Sample St Johns wort extract replicatesMeasurand ChryseneNo of laboratories 45

Assigned value 407 microgkg (Reference value)Target sd 083 microgkgTolerance limits 241 - 573 microgkg (|Z score| lt 200)

Assig

ned va

lue

Limit of tolerance

Limit of tolerance

53

Table 15 Analysis results reported by the participants for the sum of the four marker PAHs in the food supplements test material Values are presented as reported

Laboratory code

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg

3600 128 24 2

3602 79 126

3603 1127 096 2

3604 893 258 2

3605 1091 305 2

3606 1184 238 2

3607 2098 42 2

3608 3336 325 2

3610 1138 17 2

3611 2218 259 2

3612 143 29 2

3613 122 2 2

3614 663 129 2

3615 152 304 2

3616 102 03 2

3617 1252 169 2

3618 128 18 2

3619 157 305 2

3620 1204 12 2

3621 151 19 2

3622 1361 115 2

3623 154 09 2

3624

3626 1409 56 2

Laboratory code

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg

5301 126

5302 37 074 2

5303 13438 92

5304 1251 85 2

5305 125 20

5306 129 65 2

5307 1363

5308 7245598

5309

5310 1127 056

5311 1367 15 2

5312 2042 408 2

5313 1726

5314 1558

5315 177 35 2

5316 96 073 2

5317 1229 066 2

5318 82 05 2

5319 1225 267 2

5320 2309 41

5321

5322 207 35 2

5323 171 103 2

54

ANNEX 9 Results reported for the standard solutions with undisclosed analyte content Table 16 Analysis results reported by the participants for the four marker PAHs in the standard solution in toluene with undisclosed analyte

content Values are presented as reported U measurement uncertainty k coverage factor

benz[a]anthracene benzo[a]pyrene benzo[b]fluoranthene chrysene Laboratory

code Unit Result U k Result U k Result U k Result U k 3600 microgl 38 67 2 25 45 2 211 68 2 275 38 2

3605 microgl 367 184 2 2482 124 2 2592 13 2 235 118 2

3606 microgkg 3763 176 2 2609 142 2 2721 118 2 2539 134 2

3607 microgkg 3706 815 2 2824 565 2 2591 518 2 2587 569 2

3612 microgl 38 08 2 237 11 2 244 18 2 257 02 2

3614 microgl 324 65 2 209 38 2 215 37 2 21 42 2

3616 microgkg 318 07 2 224 14 2 231 26 2 216 1 2

3618 microgkg 417 17 2 284 08 2 292 06 2 297 03 2

3619 microgkg 47 742 2 274 468 2 228 387 2 274 434 2

3620 microgl 495 74 2 294 29 2 321 8 2 321 64 2

3621 microgl 241 22 2 143 13 2 188 16 2 145 14 2

3623 microgkg 476 25 2 311 53 2 269 3 2 297 03 2

3626 microgl 359 38 2 235 23 2 238 27 2 24 18 2

5301 microgkg 426 29 299 289

5302 microgkg 436 87 2 296 59 2 296 59 2 289 58 2

5303 microgl 3721 27 102 22838 19 99 23756 22 99 25486 24 98

5304 microgkg 38 29 2 225 26 2 257 37 2 258 52 2

5305 microgkg 305 225 245 29

5306 microgkg 316 63 2 247 49 2 65 13 2 22 44 2

5307 microgkg 3658 2559 241 2414

5308 microgkg 16854 109324 246654 216782

5311 microgl 374 20 2 279 15 2 268 15 2 272 15 2

5313 microgl 3408 2165 2275 2373

5314 microgkg 395 5 2875 11 3614 34 2905 18

5320 microgl 3267 368 1986 571 28 683 2427 512

5322 microgl 282 35 2 225 35 2 241 35 2 21 35 2

55

Figure 10 Youden plots of the percent deviation of the reported values from the assigned values for the food supplement test sample and the

standard solution in toluene with undisclosed content

(The identity of the respective measurand is given above the figure Results exceeding a difference of more than 100 were omitted)

56

Figure 10 - continued

57

Table 17 Analysis results reported by the participants for the four marker PAHs in the standard solution in acetonitrile with undisclosed

analyte content

Values are presented as reported U measurement uncertainty k coverage factor

benz[a]anthracene benzo[a]pyrene benzo[b]fluoranthene chrysene

Laboratory

code Unit Result U k Result U k Result U k Result U k

3602 microgkg 3675 247 247 244

3603 microgkg 358 086 2 236 028 2 235 028 2 236 057 2

3604 microgl 475 29 2 19 12 2 194 13 2 258 12 2

3608 microgl 3623 078 2 2385 023 2 2343 18 2 2296 092 2

3610 microgkg 369 185 2 25 125 2 251 126 2 244 122 2

3611 microgkg 48 02 2 323 02 2 328 04 2 317 03 2

3613 microgl 332 31 2 261 3 2 249 25 2 239 24 2

3615 microgl 415 2635 2729 2689

3617 microgkg 37 09 2 244 06 2 284 07 2 239 06 2

3622 microgl 37 12 2 25 23 2 257 21 2 238 1 2

3624 microgkg 402 104 2 255 084 2 265 096 2 253 07 2

5306 microgkg 563 113 2 371 74 2 359 72 2 333 67 2

5308 microgkg 168633 102669 232169 211488

5309 microgl 402 268 27

5310 microgl 315 158 25 125 25 125 288 144

5312 microgl 428 043 2 282 028 2 272 027 2 275 028 2

5315 microgkg 387 08 2 256 05 2 251 05 2 253 05 2

5316 microgl 3839 342 2 2485 191 2 2559 168 2 239 51 2

5317 microgl 2466 043 2 2212 034 2 2472 016 2 2146 068 2

5319 microgl 414 41 2 279 28 2 268 27 2 26 31 2

5321 microgkg 476 316 326 328

5322 microgl 2547 35 2

5323 microgkg 405 203 2 239 119 2 255 153 2 201 11 2

58

Figure 11 Youden plots of the percent deviation of the reported values from the assigned values for the food supplement test sample and the

standard solution in acetonitrile with undisclosed content

(The identity of the respective measurand is given above the figure Results exceeding a difference of more than 100 were omitted)

59

Figure 11 - continued

60

ANNEX 10 Table 18 Method performance parameters reported by the participants

Laboratory code

Analyte LOD LOQ Recovery Analyte LOD LOQ Recovery

microgkg microgkg microgkg microgkg 3600 BaA 03 06 978 BaP 03 07 9483602 BaA 007 021 926 BaP 005 015 993603 BaA 0011 05 987 BaP 0005 051 9553604 BaA 02 04 86 BaP 02 04 883605 BaA 0025 005 118 BaP 0025 005 1083606 BaA 001 003 65 BaP 001 003 703607 BaA 021 071 74 BaP 027 089 943608 BaA 016 048 8213 BaP 015 046 83683610 BaA 02 05 85 BaP 005 02 853611 BaA BaP 3612 BaA 1 117 BaP 1 1203613 BaA 01 03 90 BaP 01 03 903614 BaA 02 05 96 BaP 02 05 933615 BaA 005 015 110 BaP 005 015 1053616 BaA 01 05 108 BaP 01 05 753617 BaA 018 037 405 BaP 009 018 3873618 BaA 01 03 63 BaP 01 03 593619 BaA 005 005 59 BaP 033 033 543620 BaA 0009 0018 64 BaP 0005 001 553621 BaA 007 02 84 BaP 007 02 923622 BaA 01 03 1101 BaP 01 03 9853623 BaA 02 08 105 BaP 02 08 1013624 BaA BaP 01 033 8513625 BaA BaP 3626 BaA BaP 5301 BaA 003 01 102 BaP 003 01 1005302 BaA 01 03 78 BaP 01 03 865303 BaA 003 01 62 BaP 003 01 805304 BaA 1 1 70 BaP 1 1 955305 BaA 02 05 85 BaP 02 05 855306 BaA 03 1 699 BaP 03 1 905307 BaA 6537 BaP 81165308 BaA 0088 0265 99 BaP 00262 00785 1005309 BaA 01 08 92 BaP 01 08 925310 BaA 01 03 BaP 01 03 905311 BaA 002 005 80 BaP 002 004 915312 BaA 03 09 105 BaP 03 09 1055313 BaA 04 08 BaP 04 08 5314 BaA 001 002 96 BaP 001 002 9485315 BaA 01 03 50 - 100 BaP 01 03 50 - 100 5316 BaA 002 011 827 BaP 004 019 10445317 BaA 05 1 76 BaP 05 1 755318 BaA BaP 5319 BaA 009 029 951 BaP 011 041 995320 BaA 12 18 946 BaP 06 1 8345321 BaA 03 05 BaP 05 1 5322 BaA 1 2 100 BaP 1 2 1005323 BaA 26 BaP 26

61

ANNEX 10 Table 18 - continued

Laboratory code Analyte LOD LOQ Recovery Analyte LOD LOQ Recovery

microgkg microgkg microgkg microgkg 3600 BbF 03 05 96 CHR 02 03 10153602 BbF 015 045 947 CHR 003 009 9193603 BbF 0014 05 1012 CHR 0004 05 10033604 BbF 02 04 85 CHR 02 04 873605 BbF 005 01 94 CHR 0025 005 863606 BbF 001 003 70 CHR 001 003 653607 BbF 018 06 100 CHR 014 028 943608 BbF 015 046 7979 CHR 015 045 71253610 BbF 005 02 85 CHR 02 05 853611 BbF CHR 3612 BbF 1 118 CHR 1 1173613 BbF 01 03 90 CHR 01 03 903614 BbF 01 04 96 CHR 02 05 953615 BbF 008 025 109 CHR 003 01 1083616 BbF 01 05 78 CHR 01 05 723617 BbF 035 07 38 CHR 012 024 4163618 BbF 01 03 62 CHR 01 03 623619 BbF 016 016 63 CHR 01 01 593620 BbF 0003 0006 55 CHR 001 002 653621 BbF 007 02 103 CHR 007 02 893622 BbF 02 06 963 CHR 03 09 1043623 BbF 02 08 106 CHR 02 08 1193624 BbF CHR 3625 BbF CHR 3626 BbF CHR 5301 BbF 003 01 96 CHR 003 01 1145302 BbF 01 03 84 CHR 01 03 835303 BbF 003 01 78 CHR 003 01 705304 BbF 1 1 86 CHR 1 1 705305 BbF 02 05 90 CHR 02 05 955306 BbF 03 1 1435 CHR 03 1 1185307 BbF 8116 CHR 65375308 BbF 0079 0238 99 CHR 009 026 1005309 BbF 01 08 92 CHR 5310 BbF 07 2 CHR 01 03 5311 BbF 002 004 95 CHR 002 004 895312 BbF 03 09 105 CHR 03 09 1055313 BbF 04 08 CHR 04 08 5314 BbF 001 002 83 CHR 001 002 8185315 BbF 02 05 50 -100 CHR 02 05 50 - 100 5316 BbF 005 024 814 CHR 001 007 9595317 BbF 05 1 73 CHR 05 1 785318 BbF CHR 5319 BbF 011 038 946 CHR 013 043 9675320 BbF 07 09 1194 CHR 23 32 10455321 BbF 05 1 CHR 05 1 5322 BbF 1 2 100 CHR 1 2 1005323 BbF 26 CHR 26

62

ANNEX 11 Table 19 Details of analysis method reported by the participants Data are presented as reported

Laboratory code

Analysis technique

Instrument calibration

Internal standards applied

Sample clean up

Details of sample

clean up

Extraction technique

Details of sample

extraction

3600 6) GC-MS

2) Internal standardisation with labelled substances

Deuterated PAH mix 9 (Ehrenstorfer)

1) Saponification 2) LiquidLiquid partitioning

D) Other Liquidliquid partitioning

3602 2) HPLC-UV-FLD

1) External calibration

3) Gel permeation chromatography

A) Pressurised liquid extraction (PLE)

3603 1) HPLC-FLD

1) External calibration

3) Gel permeation chromatography

D) Other 1 minute with dichloromethane in vortex

3604 2) HPLC-UV-FLD

2) Internal standardisation with labelled substances

DiP D14

5) Donor acceptor complex chromatography

D) Other LL

3605 1) HPLC-FLD

1) External calibration

3) Gel permeation chromatography

C) Soxhlet extraction

3606 7) GC-MSMS

2) Internal standardisation with labelled substances

BaP 13C4 CHR 13C6 BaA 13C6 BbF 13C6

4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

3607 6) GC-MS

2) Internal standardisation with labelled substances

BaA-d12 CHR-d12 BbF-d12 BkF-d12 BaP-d12 ICP-d12 BgP-d12 DiP-d14

1) Saponification

D) Other solvent partitioning

3608 1) HPLC-FLD

1) External calibration

6) Other Liquid extraction

D) Other

Extraction in a rotary agitator and centrifugation

3610 2) HPLC-UV-FLD

1) External calibration

4) Solid phase extraction

B) Sonication

3611

3612 6) GC-MS

2) Internal standardisation with labelled substances

chrysene-d12 dibenzo(ah)anthracene-d14 dibenzo(ai)pyrene-d14

4) Solid phase extraction

B) Sonication

3613 5) LC-MSMS

3) Internal standardisation with unlabelled substances

I use Chrysene-D12 3) Gel permeation chromatography

D) Other extraction with hexan

63

Table 19 - continued

Laboratory code

Analysis technique

Instrument calibration

Internal standards applied

Sample clean up Details of sample

clean up

Extraction technique

Details of sample

extraction

3614 6) GC-MS 1) External calibration

3) Gel permeation chromatography

B) Sonication

3615 1) HPLC-FLD

1) External calibration

6) Other PLE GPC

A) Pressurised liquid extraction (PLE)

3616 6) GC-MS

2) Internal standardisation with labelled substances

Benzo(a)pyrene-13C4 Benzo(a)anthracene-13C6 Benzo(b)fluoranthene-13C6 Chrysene-13C6

2) LiquidLiquid partitioning 4) Solid phase extraction

B) Sonication

3617 1) HPLC-FLD

1) External calibration

4) Solid phase extraction

C) Soxhlet extraction

3618 6) GC-MS

2) Internal standardisation with labelled substances

deuterated standards

1) Saponification 4) Solid phase extraction

D) Other Handshaken with cyclohexane

3619 6) GC-MS

2) Internal standardisation with labelled substances

C13 Labelled US EPA 16

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

D) Other Homegenisation amp saponiofication

3620 9) GC-HRMS

2) Internal standardisation with labelled substances

mix of deuterated PAHs (BAA-D12 CHR-D12 BAP-D12 BBF-D12)

3) Gel permeation chromatography 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

3621 6) GC-MS

2) Internal standardisation with labelled substances

Chrysene D12 Benzo(a)pyrene D12 Benzo(b)fluoranthene D12 Benzo(a)anthracene

2) LiquidLiquid partitioning 4) Solid phase extraction

B) Sonication

3622 1) HPLC-FLD

4) Standard addition method

4) Solid phase extraction

D) Other

Extraction with cyclohexane by heating under reflux

3623 7) GC-MSMS

2) Internal standardisation with labelled substances

C13 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

3624 2) HPLC-UV-FLD

1) External calibration

2) LiquidLiquid partitioning

D) Other hot solvent extraction

3626

64

Table 19 - continued

Laboratory code

Analysis technique

Instrument calibration

Internal standards

applied

Sample clean up

Details of sample

clean up

Extraction technique

Details of sample

extraction

5301 7) GC-MSMS

2) Internal standardisation with labelled substances

EACH SUBSTANCE C13

4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5302 7) GC-MSMS

2) Internal standardisation with labelled substances

Isotope labelled PAHs 13C for each analyte

4) Solid phase extraction

C) Soxhlet extraction

5303 7) GC-MSMS

3) Internal standardisation with unlabelled substances

HAP C13 4) Solid phase extraction

D) Other automate ASE200 DIONEX

5304 7) GC-MSMS

2) Internal standardisation with labelled substances

with C13 for each HAP

2) LiquidLiquid partitioning 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5305 7) GC-MSMS

2) Internal standardisation with labelled substances

C13 isotopiques internal standards

4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5306 7) GC-MSMS

2) Internal standardisation with labelled substances

C13 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5307 6) GC-MS

2) Internal standardisation with labelled substances

1) Saponification 2) LiquidLiquid partitioning

D) Other solvent extraction

5308 1) HPLC-FLD 1) External calibration

4) Solid phase extraction

B) Sonication

5309 1) HPLC-FLD 1) External calibration

4) Solid phase extraction

D) Other only SPE

5310 1) HPLC-FLD 1) External calibration

1) Saponification 2) LiquidLiquid partitioning 3) Gel permeation chromatography

C) Soxhlet extraction

5311 6) GC-MS

2) Internal standardisation with labelled substances

13 C Chrysene 13 C Benz(a)pyrene

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

B) Sonication

5312 1) HPLC-FLD

3) Internal standardisation with unlabelled substances

Benzo(b)chrysen

2) LiquidLiquid partitioning 4) Solid phase extraction

Quechers D) Other Quechers

65

Table 19 - continued

Laboratory code

Analysis technique

Instrument calibration

Internal standards applied

Sample clean up Details of sample

clean up

Extraction technique

Details of sample extraction

5313 6) GC-MS

2) Internal standardisation with labelled substances

Benz(a)anthracene D12 Benzo(b)fluranthene D12 Benzo(a)pyrene D12 Chrysene D12

1) Saponification 2) LiquidLiquid partitioning 3) Gel permeation chromatography

A) Pressurised liquid extraction (PLE)

5314 6) GC-MS

2) Internal standardisation with labelled substances

Benzo(a)pyren d12 2) LiquidLiquid partitioning

D) Other Extraction with

5315 1) HPLC-FLD

3) Internal standardisation with unlabelled substances

Benz(b)chrysen

1) Saponification 2) LiquidLiquid partitioning

D) Other shaker

5316 1) HPLC-FLD

1) External calibration

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

D) Other

LiquidLiquid extraction with cyclohexane and dimethylformamide

5317 7) GC-MSMS

1) External calibration

3) Gel permeation chromatography 6) Other

silica gel D) Other VDLUFA online method

5318

5319 1) HPLC-FLD

3) Internal standardisation with unlabelled substances

Benzo(b)chrysene 3) Gel permeation chromatography

A) Pressurised liquid extraction (PLE)

5320 6) GC-MS

3) Internal standardisation with unlabelled substances

22-binaphthyl

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

D) Other 3 hours refluxing

5321 1) HPLC-FLD

2) Internal standardisation with labelled substances

6-meacutethylchrysegravene

1) Saponification 2) LiquidLiquid partitioning

B) Sonication

5322 6) GC-MS

2) Internal standardisation with labelled substances

naftalene-d8 acenaphtalene-d10 phenanthrene-d10 chrysene-d12 perylene-d12 benzo(e)anthracene-d12

1) Saponification 2) LiquidLiquid partitioning 6) Other

clean-up with silica packed column

C) Soxhlet extraction

5323 1) HPLC-FLD

1) External calibration

4) Solid phase extraction 6) Other

Accelerated Solvent Extraction

A) Pressurised liquid extraction (PLE)

66

European Commission EUR 25245 EN ndash Joint Research Centre ndash Institute for Reference Materials and Measurements Title Report on the 8th inter-laboratory comparison organised by the European Union Reference Laboratory for Polycyclic Aromatic Hydrocarbons - Four marker PAHs in a dry extract of St Johns wort Author(s) Radoslav Lizak Szilard Szilagyi Philippe Verlinde and Thomas WENZL Luxembourg Publications Office of the European Union 2012 ndash 66 pp ndash 201 x 297 cm EUR ndash Scientific and Technical Research series ndash ISSN 1831-9424 ISBN 978-92-79-23337-1 doi 10278759387 Abstract The proficiency test here reported concerned the determination of the contents of four marker polycyclic aromatic hydrocarbons (PAHs) and of their sum in an food supplements test sample The set of marker PAHs consists of benz[a]anthacene benzo[a]pyrene benzo[b]fluoranthene and chrysene Participants to these PT were National Reference Laboratories for PAHs (NRLs-PAHs) and EU official food control laboratories The number of participants was in total 48

The PT was organised according ISO Standard 170432010

The test material used was a commercial dry extract of St Johns wort Participants also received a solution of the same PAHs either in acetonitrile or in toluene for checking their instrument calibration

The results from participants were rated with z-scores and zeta-scores About 68 of the reported results were attributed with z-scores with an absolute value of below two which is the threshold for satisfactory performance

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interests whether private or national

L

A-N

A-252

45-EN

-N

• 1 Summary
• 2 Introduction
• 3 Scope
• 4 Participating Laboratories
• 5 Time frame
• 6 Confidentiality
• 7 Test materials
• • 71 1Preparation of test materials
  • 72 Assigned values and standard deviation for proficiency assessment
  • 73 Homogeneity and stability
  • 74 Sample dispatch
  • • 8 Design of the proficiency test
    • 9 Evaluation of the results
    • • 91 2General
      • 92 21BEvaluation criteria
      • 93 Performance of participants
      • 94 Evaluation of the influence of analysis method
      • 95 Evaluation of the influence of calibration
      • 96 26Evaluation of compliance with legislation
      • • 10 Follow-up actions for underperforming laboratories
        • 11 1Conclusions
        • 12 1Acknowledgements
        • 13 References

36

ANNEX 4 - continued

37

ANNEX 5 - Homogeneity and stability of the test material

Homogeneity of the test material

Benz[a]anthracene - BAA

Benzo[a]pyrene - BAP

38

ANNEX 5 - continued

Benzo[b]fluoranthene - BBF

Chrysene - CHR

39

ANNEX 5 - continued

Stability of the test material

Table 10 Content of the four target analytes in test materials stored under different

temperatures

BaA BaP BbF CHR

microgkg microgkg microgkg microgkg

Content 31 16 31 69 Storage at room temperature

U (k=2) 05 02 05 17

Content 31 15 29 68 Storage at reference temperature

U (k=2) 04 03 04 10

The applied capillary column does not separate CHR from triphenylene

40

ANNEX 6 ndash Documents sent to participants

41

ANNEX 6 - continued

42

ANNEX 7 Questionnaire on details of analysis method

43

ANNEX 7 - continued

44

ANNEX 7 - continued

45

ANNEX 8 - Results reported by participants for the food supplements test sample Table 11 Analysis results reported by the participants for the content of benz[a]anthracene (BAA) in the food supplements test material Values are presented as reported

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

3600 31 29 29 3 05 2

3602 199 193 178 19 021

3603 253 253 246 251 022 2

3604 199 209 23 213 071 2

3605 301 3 271 291 076 2

3606 302 288 278 289 064 2

3607 456 416 443 438 096 2

3608 796 695 651 714 149 2

3610 182 192 214 196 039 2

3611 414 504 488 469 96 2

3612 3 34 28 31 06 2

3613 32 33 34 33 07 2

3614 182 182 191 185 037 2

3615 3884 3822 3565 376 0752 2

3616 2 2 2 2 01 2

3617 366 37 35 362 046 2

3618 32 32 32 32 05 2

3619 299 305 297 299 048 2

3620 304 294 302 302 045 2

3621 29 26 29 28 04 2

3622 343 325 318 322 052 2

3623 35 3 33 33 07 2

3624

3626 318 31 314 314 126 2

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

5301 31 3 32 31 06 2

5302 314 319 315 316 063 2

5303 3102 2716 2822 2876 27 62

5304 261 263 26 261 57 2

5305 23 22 26 24 20

5306 22 23 23 22 11 2

5307 488 461 317 461

5308 168633 168544 1562025 1644598

5309 16 19 16 17

5310 3 304 311 305 015

5311 477 452 448 459 15 2

5312 506 63 494 543 134 2

5313 353 301 404 353

5314 431 373 378 394 5 096

5315 596 531 51 545 11 2

5316 224 231 235 235 022 2

5317 216 23 221 222 007 2

5318 064 07 067 01 2

5319 111 184 146 147 029 2

5320 515 525 564 534 48

5321 21 5322 13 87 9 87 35 2

5323 38 33 35 33 16 2

46

Figure 6 Results of replicate determinations (indicated by triangles) of benz[a]anthracene (BAA) in the food supplement test material

Horizontal blue lines represent the arithmetic mean value of replicate measurements and blue bars the reported expanded measurement uncertainty (k=2) The assigned value is

plotted as dotted line Red solid lines mark the range of satisfactory performance (|z|le2) Mean values are given for results outside the displayed data range

ProLab 2011

Laboratory

53

1853

1953

0853

0936

1436

0236

1036

1653

2136

0453

1753

0653

1653

0536

0353

0436

2153

0336

0636

0536

0036

2036

1953

1036

1253

0136

2653

0236

1836

2336

2236

1353

1353

2336

1736

1553

1453

0736

0753

1153

2053

1253

1536

0853

2236

11

an

aly

te c

on

ten

t (micro

gk

g)

10

95

9

85

8

75

7

65

6

55

5

45

4

35

3

25

2

15

1

05

0

102

3 468

7

Sample St Johns wort extract replicatesMeasurand Benz[a]anthraceneNo of laboratories 46

Assigned value 298 microgkg (Reference value)Target sd 062 microgkgTolerance limits 174 - 422 microgkg (|Z score| lt 200)

Assig

ned va

lue

Limit of tolerance

Limit of tolerance

47

Table 12 Analysis results reported by the participants for the content of benzo[a]pyrene (BAP) in the food supplements test material Values are presented as reported

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

3600 16 17 17 17 03 2

3602 118 123 13 124 015

3603 165 162 157 161 015 2

3604 134 154 139 143 04 2

3605 164 166 172 167 057 2

3606 181 159 161 167 031 2

3607 279 266 279 274 055 2

3608 57 687 461 573 226 2

3610 226 216 183 206 042 2

3611 289 325 311 308 36 2

3612 24 22 2 22 05 2

3613 17 18 19 18 05 2

3614 08 08 08 08 016 2

3615 1545 1393 1425 145 029 2

3616 16 16 14 15 03 2

3617 177 171 158 169 023 2

3618 2 2 2 2 02 2

3619 207 207 206 207 075 2

3620 165 169 163 166 017 2

3621 44 47 49 47 05 2

3622 212 208 222 213 038 2

3623 2 21 22 21 03 2

3624 18 178 182 18 031 2

3626 172 173 178 174 07 2

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

5301 18 18 18 18 04 2

5302 188 19 187 188 038 2

5303 1691 1426 149 1535 19 80

5304 145 142 15 146 69 2

5305 15 17 18 16 15

5306 16 17 17 16 08 2

5307 164 157 153 157

5308 102669 109324 109035 107009

5309 1 1 12 11

5310 122 122 124 123 006

5311 199 184 215 2 15 2

5312 258 248 239 248 05 2

5313 199 178 245 207

5314 174 171 172 172 11 0948

5315 253 248 213 238 05 2

5316 177 171 173 17 016 2

5317 255 277 276 269 012 2

5318 114 12 117 02 2

5319 19 228 231 216 043 2

5320 303 3 344 316 78

5321 12 12 12 12

5322 13 17 24 17 35 2

5323 2 2 19 2 1 2

48

Figure 7 Results of replicate determinations (indicated by triangles) of benzo[a]pyrene (BAP) in the food supplement test material

Horizontal blue lines represent the arithmetic mean value of replicate measurements and blue bars the reported expanded measurement uncertainty (k=2) The assigned value is

plotted as dotted line Red solid lines mark the range of satisfactory performance (|z|le2) Mean values are given for results outside the displayed data range

ProLab 2011

Laboratory

36

1453

0953

0853

1853

2153

1036

0236

0436

1553

0436

1653

0353

0736

0336

2036

0053

0553

0636

0636

0536

1753

1453

1636

2636

1336

2453

0153

2253

0253

2353

1136

1836

1953

1336

1036

2336

2253

1936

1253

1553

1253

1736

0753

2036

2136

0836

11

an

aly

te c

on

ten

t (micro

gk

g)

55

5

45

4

35

3

25

2

15

1

05

0

573

308

3

Sample St Johns wort extract replicatesMeasurand Benzo[a]pyreneNo of laboratories 48

Assigned value 165 microgkg (Reference value)Target sd 036 microgkgTolerance limits 093 - 237 microgkg (|Z score| lt 200)

Assig

ned va

lue

Limit of tolerance

Limit of tolerance

49

Table 13 Analysis results reported by the participants for the content of benzo[b]fluoranthene (BBF) in the food supplements test material Values are presented as reported

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

3600 32 3 3 31 09 2

3602 173 175 16 17 017

3603 265 252 25 256 025 2

3604 116 108 105 11 028 2

3605 286 225 232 247 074 2

3606 294 273 261 276 046 2

3607 579 579 544 567 113 2

3608 103 1201 876 1036 326 2

3610 173 196 204 191 038 2

3611 546 618 565 576 75 2

3612 3 28 28 29 06 2

3613 28 31 32 31 06 2

3614 135 111 135 127 022 2

3615 4187 3678 4111 4 08 2

3616 2 2 2 2 01 2

3617 33 27 299 035 2

3618 31 31 31 31 05 2

3619 326 324 312 326 064 2

3620 277 28 272 276 069 2

3621 26 25 23 25 04 2

3622 346 336 333 34 052 2

3623 26 28 29 28 02 2

3624

3626 288 28 307 292 117 2

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

5301 29 3 29 29 06 2

5302 297 3 296 298 06 2

5303 2705 2444 2608 2586 22 78

5304 245 254 26 253 85 2

5305 23 23 26 24 15

5306 42 43 4 42 21 2

5307 221 216 241 221

5308 232169 246654 240722 229848

5309 19 22 19 2 0

5310 3 315 325 313 016

5311 262 277 304 281 15 2

5312 27 289 337 299 075 2

5313 321 284 378 328

5314 423 404 385 404 34 083

5315 42 506 416 444 09 2

5316 265 267 277 273 018 2

5317 185 197 195 192 006 2

5318 276 281 279 04 2

5319 396 389 403 396 079 2

5320 623 618 635 625 14

5321 31

5322 24 33 49 33 35 2

5323 38 45 35 45 27 2

50

Figure 8 Results of replicate determinations (indicated by triangles) of benzo[b]fluoranthene (BBF) in the food supplement test material

Horizontal blue lines represent the arithmetic mean value of replicate measurements and blue bars the reported expanded measurement uncertainty (k=2) The assigned value is

plotted as dotted line Red solid lines mark the range of satisfactory performance (|z|le2) Mean values are given for results outside the displayed data range

ProLab 2011

Laboratory

36

0436

1436

0236

1053

1736

1653

0953

0753

0853

0536

2136

0553

0436

0353

0353

1636

0636

2036

2353

1853

1136

1236

2653

0153

0253

1236

1736

1336

0036

1853

2153

1036

1953

1336

2253

2253

2353

1936

1553

1453

0653

1536

0753

2036

0836

11

an

aly

te c

on

ten

t (micro

gk

g)

75

7

65

6

55

5

45

4

35

3

25

2

15

1

05

0

103

6

576

3

Sample St Johns wort extract replicatesMeasurand Benzo[b]fluorantheneNo of laboratories 46

Assigned value 292 microgkg (Reference value)Target sd 060 microgkgTolerance limits 172 - 412 microgkg (|Z score| lt 200)

Assig

ned va

lue

Limit of tolerance

Limit of tolerance

51

Table 14 Analysis results reported by the participants for the content of chrysene (CHR) in the food supplements test material Values are presented as reported

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

3600 5 49 5 5 07 2

3602 32 304 29 304 036

3603 462 469 447 459 048 2

3604 438 428 42 429 119 2

3605 365 41 381 386 085 2

3606 494 411 45 451 124 2

3607 897 774 787 819 18 2

3608 986 887 1168 1014 284 2

3610 418 392 462 424 085 2

3611 833 893 87 865 61 2

3612 6 64 58 61 12 2

3613 41 39 4 4 08 2

3614 272 254 288 271 054 2

3615 605 6028 5964 601 12 2

3616 47 45 49 47 05 2

3617 445 431 389 422 065 2

3618 45 45 45 45 06 2

3619 738 747 72 738 118 2

3620 463 472 446 46 092 2

3621 53 49 51 51 06 2

3622 478 513 505 488 08 2

3623 73 73 72 72 05 2

3624

3626 63 622 634 628 251 2

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

5301 49 49 49 49 1 2

5302 687 683 666 679 136 2

5303 6777 6181 6382 6441 24 70

5304 592 581 6 591 85 2

5305 6 58 64 61 20

5306 49 5 51 49 25 2

5307 524 5 574 524

5308 211488 216782 221146 213243

5309

5310 372 393 394 386 019

5311 43 459 422 437 15 2

5312 934 943 981 953 191 2

5313 793 73 99 838

5314 636 561 565 588 18 0818

5315 547 583 499 542 11 2

5316 292 286 285 282 06 2

5317 52 598 517 545 046 2

5318 368 345 357 05 2

5319 449 471 474 465 116 2

5320 76 924 818 834 99

5321 33

5322 7 7 76 7 35 2

5323 87 73 77 73 37 2

52

Figure 9 Results of replicate determinations (indicated by triangles) of chrysene (CHR) in the food supplement test material

Horizontal blue lines represent the arithmetic mean value of replicate measurements and blue bars the reported expanded measurement uncertainty (k=2) The assigned value is

plotted as dotted line Red solid lines mark the range of satisfactory performance (|z|le2) Mean values are given for results outside the displayed data range

ProLab 2011

Laboratory

53

0836

1453

1636

0253

2153

1836

0553

1036

1336

1736

1036

0453

1136

1836

0636

0336

2053

1936

1653

0136

0036

2253

0636

2153

0753

1553

1753

1453

0436

1536

1253

0536

2653

0353

0253

2236

2336

1953

2336

0753

2053

1353

1236

0836

11

an

aly

te c

on

ten

t (micro

gk

g)

13

12

11

10

9

8

7

6

5

4

3

2

1

0

865

3

Sample St Johns wort extract replicatesMeasurand ChryseneNo of laboratories 45

Assigned value 407 microgkg (Reference value)Target sd 083 microgkgTolerance limits 241 - 573 microgkg (|Z score| lt 200)

Assig

ned va

lue

Limit of tolerance

Limit of tolerance

53

Table 15 Analysis results reported by the participants for the sum of the four marker PAHs in the food supplements test material Values are presented as reported

Laboratory code

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg

3600 128 24 2

3602 79 126

3603 1127 096 2

3604 893 258 2

3605 1091 305 2

3606 1184 238 2

3607 2098 42 2

3608 3336 325 2

3610 1138 17 2

3611 2218 259 2

3612 143 29 2

3613 122 2 2

3614 663 129 2

3615 152 304 2

3616 102 03 2

3617 1252 169 2

3618 128 18 2

3619 157 305 2

3620 1204 12 2

3621 151 19 2

3622 1361 115 2

3623 154 09 2

3624

3626 1409 56 2

Laboratory code

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg

5301 126

5302 37 074 2

5303 13438 92

5304 1251 85 2

5305 125 20

5306 129 65 2

5307 1363

5308 7245598

5309

5310 1127 056

5311 1367 15 2

5312 2042 408 2

5313 1726

5314 1558

5315 177 35 2

5316 96 073 2

5317 1229 066 2

5318 82 05 2

5319 1225 267 2

5320 2309 41

5321

5322 207 35 2

5323 171 103 2

54

ANNEX 9 Results reported for the standard solutions with undisclosed analyte content Table 16 Analysis results reported by the participants for the four marker PAHs in the standard solution in toluene with undisclosed analyte

content Values are presented as reported U measurement uncertainty k coverage factor

benz[a]anthracene benzo[a]pyrene benzo[b]fluoranthene chrysene Laboratory

code Unit Result U k Result U k Result U k Result U k 3600 microgl 38 67 2 25 45 2 211 68 2 275 38 2

3605 microgl 367 184 2 2482 124 2 2592 13 2 235 118 2

3606 microgkg 3763 176 2 2609 142 2 2721 118 2 2539 134 2

3607 microgkg 3706 815 2 2824 565 2 2591 518 2 2587 569 2

3612 microgl 38 08 2 237 11 2 244 18 2 257 02 2

3614 microgl 324 65 2 209 38 2 215 37 2 21 42 2

3616 microgkg 318 07 2 224 14 2 231 26 2 216 1 2

3618 microgkg 417 17 2 284 08 2 292 06 2 297 03 2

3619 microgkg 47 742 2 274 468 2 228 387 2 274 434 2

3620 microgl 495 74 2 294 29 2 321 8 2 321 64 2

3621 microgl 241 22 2 143 13 2 188 16 2 145 14 2

3623 microgkg 476 25 2 311 53 2 269 3 2 297 03 2

3626 microgl 359 38 2 235 23 2 238 27 2 24 18 2

5301 microgkg 426 29 299 289

5302 microgkg 436 87 2 296 59 2 296 59 2 289 58 2

5303 microgl 3721 27 102 22838 19 99 23756 22 99 25486 24 98

5304 microgkg 38 29 2 225 26 2 257 37 2 258 52 2

5305 microgkg 305 225 245 29

5306 microgkg 316 63 2 247 49 2 65 13 2 22 44 2

5307 microgkg 3658 2559 241 2414

5308 microgkg 16854 109324 246654 216782

5311 microgl 374 20 2 279 15 2 268 15 2 272 15 2

5313 microgl 3408 2165 2275 2373

5314 microgkg 395 5 2875 11 3614 34 2905 18

5320 microgl 3267 368 1986 571 28 683 2427 512

5322 microgl 282 35 2 225 35 2 241 35 2 21 35 2

55

Figure 10 Youden plots of the percent deviation of the reported values from the assigned values for the food supplement test sample and the

standard solution in toluene with undisclosed content

(The identity of the respective measurand is given above the figure Results exceeding a difference of more than 100 were omitted)

56

Figure 10 - continued

57

Table 17 Analysis results reported by the participants for the four marker PAHs in the standard solution in acetonitrile with undisclosed

analyte content

Values are presented as reported U measurement uncertainty k coverage factor

benz[a]anthracene benzo[a]pyrene benzo[b]fluoranthene chrysene

Laboratory

code Unit Result U k Result U k Result U k Result U k

3602 microgkg 3675 247 247 244

3603 microgkg 358 086 2 236 028 2 235 028 2 236 057 2

3604 microgl 475 29 2 19 12 2 194 13 2 258 12 2

3608 microgl 3623 078 2 2385 023 2 2343 18 2 2296 092 2

3610 microgkg 369 185 2 25 125 2 251 126 2 244 122 2

3611 microgkg 48 02 2 323 02 2 328 04 2 317 03 2

3613 microgl 332 31 2 261 3 2 249 25 2 239 24 2

3615 microgl 415 2635 2729 2689

3617 microgkg 37 09 2 244 06 2 284 07 2 239 06 2

3622 microgl 37 12 2 25 23 2 257 21 2 238 1 2

3624 microgkg 402 104 2 255 084 2 265 096 2 253 07 2

5306 microgkg 563 113 2 371 74 2 359 72 2 333 67 2

5308 microgkg 168633 102669 232169 211488

5309 microgl 402 268 27

5310 microgl 315 158 25 125 25 125 288 144

5312 microgl 428 043 2 282 028 2 272 027 2 275 028 2

5315 microgkg 387 08 2 256 05 2 251 05 2 253 05 2

5316 microgl 3839 342 2 2485 191 2 2559 168 2 239 51 2

5317 microgl 2466 043 2 2212 034 2 2472 016 2 2146 068 2

5319 microgl 414 41 2 279 28 2 268 27 2 26 31 2

5321 microgkg 476 316 326 328

5322 microgl 2547 35 2

5323 microgkg 405 203 2 239 119 2 255 153 2 201 11 2

58

Figure 11 Youden plots of the percent deviation of the reported values from the assigned values for the food supplement test sample and the

standard solution in acetonitrile with undisclosed content

(The identity of the respective measurand is given above the figure Results exceeding a difference of more than 100 were omitted)

59

Figure 11 - continued

60

ANNEX 10 Table 18 Method performance parameters reported by the participants

Laboratory code

Analyte LOD LOQ Recovery Analyte LOD LOQ Recovery

microgkg microgkg microgkg microgkg 3600 BaA 03 06 978 BaP 03 07 9483602 BaA 007 021 926 BaP 005 015 993603 BaA 0011 05 987 BaP 0005 051 9553604 BaA 02 04 86 BaP 02 04 883605 BaA 0025 005 118 BaP 0025 005 1083606 BaA 001 003 65 BaP 001 003 703607 BaA 021 071 74 BaP 027 089 943608 BaA 016 048 8213 BaP 015 046 83683610 BaA 02 05 85 BaP 005 02 853611 BaA BaP 3612 BaA 1 117 BaP 1 1203613 BaA 01 03 90 BaP 01 03 903614 BaA 02 05 96 BaP 02 05 933615 BaA 005 015 110 BaP 005 015 1053616 BaA 01 05 108 BaP 01 05 753617 BaA 018 037 405 BaP 009 018 3873618 BaA 01 03 63 BaP 01 03 593619 BaA 005 005 59 BaP 033 033 543620 BaA 0009 0018 64 BaP 0005 001 553621 BaA 007 02 84 BaP 007 02 923622 BaA 01 03 1101 BaP 01 03 9853623 BaA 02 08 105 BaP 02 08 1013624 BaA BaP 01 033 8513625 BaA BaP 3626 BaA BaP 5301 BaA 003 01 102 BaP 003 01 1005302 BaA 01 03 78 BaP 01 03 865303 BaA 003 01 62 BaP 003 01 805304 BaA 1 1 70 BaP 1 1 955305 BaA 02 05 85 BaP 02 05 855306 BaA 03 1 699 BaP 03 1 905307 BaA 6537 BaP 81165308 BaA 0088 0265 99 BaP 00262 00785 1005309 BaA 01 08 92 BaP 01 08 925310 BaA 01 03 BaP 01 03 905311 BaA 002 005 80 BaP 002 004 915312 BaA 03 09 105 BaP 03 09 1055313 BaA 04 08 BaP 04 08 5314 BaA 001 002 96 BaP 001 002 9485315 BaA 01 03 50 - 100 BaP 01 03 50 - 100 5316 BaA 002 011 827 BaP 004 019 10445317 BaA 05 1 76 BaP 05 1 755318 BaA BaP 5319 BaA 009 029 951 BaP 011 041 995320 BaA 12 18 946 BaP 06 1 8345321 BaA 03 05 BaP 05 1 5322 BaA 1 2 100 BaP 1 2 1005323 BaA 26 BaP 26

61

ANNEX 10 Table 18 - continued

Laboratory code Analyte LOD LOQ Recovery Analyte LOD LOQ Recovery

microgkg microgkg microgkg microgkg 3600 BbF 03 05 96 CHR 02 03 10153602 BbF 015 045 947 CHR 003 009 9193603 BbF 0014 05 1012 CHR 0004 05 10033604 BbF 02 04 85 CHR 02 04 873605 BbF 005 01 94 CHR 0025 005 863606 BbF 001 003 70 CHR 001 003 653607 BbF 018 06 100 CHR 014 028 943608 BbF 015 046 7979 CHR 015 045 71253610 BbF 005 02 85 CHR 02 05 853611 BbF CHR 3612 BbF 1 118 CHR 1 1173613 BbF 01 03 90 CHR 01 03 903614 BbF 01 04 96 CHR 02 05 953615 BbF 008 025 109 CHR 003 01 1083616 BbF 01 05 78 CHR 01 05 723617 BbF 035 07 38 CHR 012 024 4163618 BbF 01 03 62 CHR 01 03 623619 BbF 016 016 63 CHR 01 01 593620 BbF 0003 0006 55 CHR 001 002 653621 BbF 007 02 103 CHR 007 02 893622 BbF 02 06 963 CHR 03 09 1043623 BbF 02 08 106 CHR 02 08 1193624 BbF CHR 3625 BbF CHR 3626 BbF CHR 5301 BbF 003 01 96 CHR 003 01 1145302 BbF 01 03 84 CHR 01 03 835303 BbF 003 01 78 CHR 003 01 705304 BbF 1 1 86 CHR 1 1 705305 BbF 02 05 90 CHR 02 05 955306 BbF 03 1 1435 CHR 03 1 1185307 BbF 8116 CHR 65375308 BbF 0079 0238 99 CHR 009 026 1005309 BbF 01 08 92 CHR 5310 BbF 07 2 CHR 01 03 5311 BbF 002 004 95 CHR 002 004 895312 BbF 03 09 105 CHR 03 09 1055313 BbF 04 08 CHR 04 08 5314 BbF 001 002 83 CHR 001 002 8185315 BbF 02 05 50 -100 CHR 02 05 50 - 100 5316 BbF 005 024 814 CHR 001 007 9595317 BbF 05 1 73 CHR 05 1 785318 BbF CHR 5319 BbF 011 038 946 CHR 013 043 9675320 BbF 07 09 1194 CHR 23 32 10455321 BbF 05 1 CHR 05 1 5322 BbF 1 2 100 CHR 1 2 1005323 BbF 26 CHR 26

62

ANNEX 11 Table 19 Details of analysis method reported by the participants Data are presented as reported

Laboratory code

Analysis technique

Instrument calibration

Internal standards applied

Sample clean up

Details of sample

clean up

Extraction technique

Details of sample

extraction

3600 6) GC-MS

2) Internal standardisation with labelled substances

Deuterated PAH mix 9 (Ehrenstorfer)

1) Saponification 2) LiquidLiquid partitioning

D) Other Liquidliquid partitioning

3602 2) HPLC-UV-FLD

1) External calibration

3) Gel permeation chromatography

A) Pressurised liquid extraction (PLE)

3603 1) HPLC-FLD

1) External calibration

3) Gel permeation chromatography

D) Other 1 minute with dichloromethane in vortex

3604 2) HPLC-UV-FLD

2) Internal standardisation with labelled substances

DiP D14

5) Donor acceptor complex chromatography

D) Other LL

3605 1) HPLC-FLD

1) External calibration

3) Gel permeation chromatography

C) Soxhlet extraction

3606 7) GC-MSMS

2) Internal standardisation with labelled substances

BaP 13C4 CHR 13C6 BaA 13C6 BbF 13C6

4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

3607 6) GC-MS

2) Internal standardisation with labelled substances

BaA-d12 CHR-d12 BbF-d12 BkF-d12 BaP-d12 ICP-d12 BgP-d12 DiP-d14

1) Saponification

D) Other solvent partitioning

3608 1) HPLC-FLD

1) External calibration

6) Other Liquid extraction

D) Other

Extraction in a rotary agitator and centrifugation

3610 2) HPLC-UV-FLD

1) External calibration

4) Solid phase extraction

B) Sonication

3611

3612 6) GC-MS

2) Internal standardisation with labelled substances

chrysene-d12 dibenzo(ah)anthracene-d14 dibenzo(ai)pyrene-d14

4) Solid phase extraction

B) Sonication

3613 5) LC-MSMS

3) Internal standardisation with unlabelled substances

I use Chrysene-D12 3) Gel permeation chromatography

D) Other extraction with hexan

63

Table 19 - continued

Laboratory code

Analysis technique

Instrument calibration

Internal standards applied

Sample clean up Details of sample

clean up

Extraction technique

Details of sample

extraction

3614 6) GC-MS 1) External calibration

3) Gel permeation chromatography

B) Sonication

3615 1) HPLC-FLD

1) External calibration

6) Other PLE GPC

A) Pressurised liquid extraction (PLE)

3616 6) GC-MS

2) Internal standardisation with labelled substances

Benzo(a)pyrene-13C4 Benzo(a)anthracene-13C6 Benzo(b)fluoranthene-13C6 Chrysene-13C6

2) LiquidLiquid partitioning 4) Solid phase extraction

B) Sonication

3617 1) HPLC-FLD

1) External calibration

4) Solid phase extraction

C) Soxhlet extraction

3618 6) GC-MS

2) Internal standardisation with labelled substances

deuterated standards

1) Saponification 4) Solid phase extraction

D) Other Handshaken with cyclohexane

3619 6) GC-MS

2) Internal standardisation with labelled substances

C13 Labelled US EPA 16

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

D) Other Homegenisation amp saponiofication

3620 9) GC-HRMS

2) Internal standardisation with labelled substances

mix of deuterated PAHs (BAA-D12 CHR-D12 BAP-D12 BBF-D12)

3) Gel permeation chromatography 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

3621 6) GC-MS

2) Internal standardisation with labelled substances

Chrysene D12 Benzo(a)pyrene D12 Benzo(b)fluoranthene D12 Benzo(a)anthracene

2) LiquidLiquid partitioning 4) Solid phase extraction

B) Sonication

3622 1) HPLC-FLD

4) Standard addition method

4) Solid phase extraction

D) Other

Extraction with cyclohexane by heating under reflux

3623 7) GC-MSMS

2) Internal standardisation with labelled substances

C13 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

3624 2) HPLC-UV-FLD

1) External calibration

2) LiquidLiquid partitioning

D) Other hot solvent extraction

3626

64

Table 19 - continued

Laboratory code

Analysis technique

Instrument calibration

Internal standards

applied

Sample clean up

Details of sample

clean up

Extraction technique

Details of sample

extraction

5301 7) GC-MSMS

2) Internal standardisation with labelled substances

EACH SUBSTANCE C13

4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5302 7) GC-MSMS

2) Internal standardisation with labelled substances

Isotope labelled PAHs 13C for each analyte

4) Solid phase extraction

C) Soxhlet extraction

5303 7) GC-MSMS

3) Internal standardisation with unlabelled substances

HAP C13 4) Solid phase extraction

D) Other automate ASE200 DIONEX

5304 7) GC-MSMS

2) Internal standardisation with labelled substances

with C13 for each HAP

2) LiquidLiquid partitioning 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5305 7) GC-MSMS

2) Internal standardisation with labelled substances

C13 isotopiques internal standards

4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5306 7) GC-MSMS

2) Internal standardisation with labelled substances

C13 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5307 6) GC-MS

2) Internal standardisation with labelled substances

1) Saponification 2) LiquidLiquid partitioning

D) Other solvent extraction

5308 1) HPLC-FLD 1) External calibration

4) Solid phase extraction

B) Sonication

5309 1) HPLC-FLD 1) External calibration

4) Solid phase extraction

D) Other only SPE

5310 1) HPLC-FLD 1) External calibration

1) Saponification 2) LiquidLiquid partitioning 3) Gel permeation chromatography

C) Soxhlet extraction

5311 6) GC-MS

2) Internal standardisation with labelled substances

13 C Chrysene 13 C Benz(a)pyrene

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

B) Sonication

5312 1) HPLC-FLD

3) Internal standardisation with unlabelled substances

Benzo(b)chrysen

2) LiquidLiquid partitioning 4) Solid phase extraction

Quechers D) Other Quechers

65

Table 19 - continued

Laboratory code

Analysis technique

Instrument calibration

Internal standards applied

Sample clean up Details of sample

clean up

Extraction technique

Details of sample extraction

5313 6) GC-MS

2) Internal standardisation with labelled substances

Benz(a)anthracene D12 Benzo(b)fluranthene D12 Benzo(a)pyrene D12 Chrysene D12

1) Saponification 2) LiquidLiquid partitioning 3) Gel permeation chromatography

A) Pressurised liquid extraction (PLE)

5314 6) GC-MS

2) Internal standardisation with labelled substances

Benzo(a)pyren d12 2) LiquidLiquid partitioning

D) Other Extraction with

5315 1) HPLC-FLD

3) Internal standardisation with unlabelled substances

Benz(b)chrysen

1) Saponification 2) LiquidLiquid partitioning

D) Other shaker

5316 1) HPLC-FLD

1) External calibration

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

D) Other

LiquidLiquid extraction with cyclohexane and dimethylformamide

5317 7) GC-MSMS

1) External calibration

3) Gel permeation chromatography 6) Other

silica gel D) Other VDLUFA online method

5318

5319 1) HPLC-FLD

3) Internal standardisation with unlabelled substances

Benzo(b)chrysene 3) Gel permeation chromatography

A) Pressurised liquid extraction (PLE)

5320 6) GC-MS

3) Internal standardisation with unlabelled substances

22-binaphthyl

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

D) Other 3 hours refluxing

5321 1) HPLC-FLD

2) Internal standardisation with labelled substances

6-meacutethylchrysegravene

1) Saponification 2) LiquidLiquid partitioning

B) Sonication

5322 6) GC-MS

2) Internal standardisation with labelled substances

naftalene-d8 acenaphtalene-d10 phenanthrene-d10 chrysene-d12 perylene-d12 benzo(e)anthracene-d12

1) Saponification 2) LiquidLiquid partitioning 6) Other

clean-up with silica packed column

C) Soxhlet extraction

5323 1) HPLC-FLD

1) External calibration

4) Solid phase extraction 6) Other

Accelerated Solvent Extraction

A) Pressurised liquid extraction (PLE)

66

European Commission EUR 25245 EN ndash Joint Research Centre ndash Institute for Reference Materials and Measurements Title Report on the 8th inter-laboratory comparison organised by the European Union Reference Laboratory for Polycyclic Aromatic Hydrocarbons - Four marker PAHs in a dry extract of St Johns wort Author(s) Radoslav Lizak Szilard Szilagyi Philippe Verlinde and Thomas WENZL Luxembourg Publications Office of the European Union 2012 ndash 66 pp ndash 201 x 297 cm EUR ndash Scientific and Technical Research series ndash ISSN 1831-9424 ISBN 978-92-79-23337-1 doi 10278759387 Abstract The proficiency test here reported concerned the determination of the contents of four marker polycyclic aromatic hydrocarbons (PAHs) and of their sum in an food supplements test sample The set of marker PAHs consists of benz[a]anthacene benzo[a]pyrene benzo[b]fluoranthene and chrysene Participants to these PT were National Reference Laboratories for PAHs (NRLs-PAHs) and EU official food control laboratories The number of participants was in total 48

The PT was organised according ISO Standard 170432010

The test material used was a commercial dry extract of St Johns wort Participants also received a solution of the same PAHs either in acetonitrile or in toluene for checking their instrument calibration

The results from participants were rated with z-scores and zeta-scores About 68 of the reported results were attributed with z-scores with an absolute value of below two which is the threshold for satisfactory performance

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interests whether private or national

L

A-N

A-252

45-EN

-N

• 1 Summary
• 2 Introduction
• 3 Scope
• 4 Participating Laboratories
• 5 Time frame
• 6 Confidentiality
• 7 Test materials
• • 71 1Preparation of test materials
  • 72 Assigned values and standard deviation for proficiency assessment
  • 73 Homogeneity and stability
  • 74 Sample dispatch
  • • 8 Design of the proficiency test
    • 9 Evaluation of the results
    • • 91 2General
      • 92 21BEvaluation criteria
      • 93 Performance of participants
      • 94 Evaluation of the influence of analysis method
      • 95 Evaluation of the influence of calibration
      • 96 26Evaluation of compliance with legislation
      • • 10 Follow-up actions for underperforming laboratories
        • 11 1Conclusions
        • 12 1Acknowledgements
        • 13 References

37

ANNEX 5 - Homogeneity and stability of the test material

Homogeneity of the test material

Benz[a]anthracene - BAA

Benzo[a]pyrene - BAP

38

ANNEX 5 - continued

Benzo[b]fluoranthene - BBF

Chrysene - CHR

39

ANNEX 5 - continued

Stability of the test material

Table 10 Content of the four target analytes in test materials stored under different

temperatures

BaA BaP BbF CHR

microgkg microgkg microgkg microgkg

Content 31 16 31 69 Storage at room temperature

U (k=2) 05 02 05 17

Content 31 15 29 68 Storage at reference temperature

U (k=2) 04 03 04 10

The applied capillary column does not separate CHR from triphenylene

40

ANNEX 6 ndash Documents sent to participants

41

ANNEX 6 - continued

42

ANNEX 7 Questionnaire on details of analysis method

43

ANNEX 7 - continued

44

ANNEX 7 - continued

45

ANNEX 8 - Results reported by participants for the food supplements test sample Table 11 Analysis results reported by the participants for the content of benz[a]anthracene (BAA) in the food supplements test material Values are presented as reported

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

3600 31 29 29 3 05 2

3602 199 193 178 19 021

3603 253 253 246 251 022 2

3604 199 209 23 213 071 2

3605 301 3 271 291 076 2

3606 302 288 278 289 064 2

3607 456 416 443 438 096 2

3608 796 695 651 714 149 2

3610 182 192 214 196 039 2

3611 414 504 488 469 96 2

3612 3 34 28 31 06 2

3613 32 33 34 33 07 2

3614 182 182 191 185 037 2

3615 3884 3822 3565 376 0752 2

3616 2 2 2 2 01 2

3617 366 37 35 362 046 2

3618 32 32 32 32 05 2

3619 299 305 297 299 048 2

3620 304 294 302 302 045 2

3621 29 26 29 28 04 2

3622 343 325 318 322 052 2

3623 35 3 33 33 07 2

3624

3626 318 31 314 314 126 2

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

5301 31 3 32 31 06 2

5302 314 319 315 316 063 2

5303 3102 2716 2822 2876 27 62

5304 261 263 26 261 57 2

5305 23 22 26 24 20

5306 22 23 23 22 11 2

5307 488 461 317 461

5308 168633 168544 1562025 1644598

5309 16 19 16 17

5310 3 304 311 305 015

5311 477 452 448 459 15 2

5312 506 63 494 543 134 2

5313 353 301 404 353

5314 431 373 378 394 5 096

5315 596 531 51 545 11 2

5316 224 231 235 235 022 2

5317 216 23 221 222 007 2

5318 064 07 067 01 2

5319 111 184 146 147 029 2

5320 515 525 564 534 48

5321 21 5322 13 87 9 87 35 2

5323 38 33 35 33 16 2

46

Figure 6 Results of replicate determinations (indicated by triangles) of benz[a]anthracene (BAA) in the food supplement test material

Horizontal blue lines represent the arithmetic mean value of replicate measurements and blue bars the reported expanded measurement uncertainty (k=2) The assigned value is

plotted as dotted line Red solid lines mark the range of satisfactory performance (|z|le2) Mean values are given for results outside the displayed data range

ProLab 2011

Laboratory

53

1853

1953

0853

0936

1436

0236

1036

1653

2136

0453

1753

0653

1653

0536

0353

0436

2153

0336

0636

0536

0036

2036

1953

1036

1253

0136

2653

0236

1836

2336

2236

1353

1353

2336

1736

1553

1453

0736

0753

1153

2053

1253

1536

0853

2236

11

an

aly

te c

on

ten

t (micro

gk

g)

10

95

9

85

8

75

7

65

6

55

5

45

4

35

3

25

2

15

1

05

0

102

3 468

7

Sample St Johns wort extract replicatesMeasurand Benz[a]anthraceneNo of laboratories 46

Assigned value 298 microgkg (Reference value)Target sd 062 microgkgTolerance limits 174 - 422 microgkg (|Z score| lt 200)

Assig

ned va

lue

Limit of tolerance

Limit of tolerance

47

Table 12 Analysis results reported by the participants for the content of benzo[a]pyrene (BAP) in the food supplements test material Values are presented as reported

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

3600 16 17 17 17 03 2

3602 118 123 13 124 015

3603 165 162 157 161 015 2

3604 134 154 139 143 04 2

3605 164 166 172 167 057 2

3606 181 159 161 167 031 2

3607 279 266 279 274 055 2

3608 57 687 461 573 226 2

3610 226 216 183 206 042 2

3611 289 325 311 308 36 2

3612 24 22 2 22 05 2

3613 17 18 19 18 05 2

3614 08 08 08 08 016 2

3615 1545 1393 1425 145 029 2

3616 16 16 14 15 03 2

3617 177 171 158 169 023 2

3618 2 2 2 2 02 2

3619 207 207 206 207 075 2

3620 165 169 163 166 017 2

3621 44 47 49 47 05 2

3622 212 208 222 213 038 2

3623 2 21 22 21 03 2

3624 18 178 182 18 031 2

3626 172 173 178 174 07 2

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

5301 18 18 18 18 04 2

5302 188 19 187 188 038 2

5303 1691 1426 149 1535 19 80

5304 145 142 15 146 69 2

5305 15 17 18 16 15

5306 16 17 17 16 08 2

5307 164 157 153 157

5308 102669 109324 109035 107009

5309 1 1 12 11

5310 122 122 124 123 006

5311 199 184 215 2 15 2

5312 258 248 239 248 05 2

5313 199 178 245 207

5314 174 171 172 172 11 0948

5315 253 248 213 238 05 2

5316 177 171 173 17 016 2

5317 255 277 276 269 012 2

5318 114 12 117 02 2

5319 19 228 231 216 043 2

5320 303 3 344 316 78

5321 12 12 12 12

5322 13 17 24 17 35 2

5323 2 2 19 2 1 2

48

Figure 7 Results of replicate determinations (indicated by triangles) of benzo[a]pyrene (BAP) in the food supplement test material

Horizontal blue lines represent the arithmetic mean value of replicate measurements and blue bars the reported expanded measurement uncertainty (k=2) The assigned value is

plotted as dotted line Red solid lines mark the range of satisfactory performance (|z|le2) Mean values are given for results outside the displayed data range

ProLab 2011

Laboratory

36

1453

0953

0853

1853

2153

1036

0236

0436

1553

0436

1653

0353

0736

0336

2036

0053

0553

0636

0636

0536

1753

1453

1636

2636

1336

2453

0153

2253

0253

2353

1136

1836

1953

1336

1036

2336

2253

1936

1253

1553

1253

1736

0753

2036

2136

0836

11

an

aly

te c

on

ten

t (micro

gk

g)

55

5

45

4

35

3

25

2

15

1

05

0

573

308

3

Sample St Johns wort extract replicatesMeasurand Benzo[a]pyreneNo of laboratories 48

Assigned value 165 microgkg (Reference value)Target sd 036 microgkgTolerance limits 093 - 237 microgkg (|Z score| lt 200)

Assig

ned va

lue

Limit of tolerance

Limit of tolerance

49

Table 13 Analysis results reported by the participants for the content of benzo[b]fluoranthene (BBF) in the food supplements test material Values are presented as reported

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

3600 32 3 3 31 09 2

3602 173 175 16 17 017

3603 265 252 25 256 025 2

3604 116 108 105 11 028 2

3605 286 225 232 247 074 2

3606 294 273 261 276 046 2

3607 579 579 544 567 113 2

3608 103 1201 876 1036 326 2

3610 173 196 204 191 038 2

3611 546 618 565 576 75 2

3612 3 28 28 29 06 2

3613 28 31 32 31 06 2

3614 135 111 135 127 022 2

3615 4187 3678 4111 4 08 2

3616 2 2 2 2 01 2

3617 33 27 299 035 2

3618 31 31 31 31 05 2

3619 326 324 312 326 064 2

3620 277 28 272 276 069 2

3621 26 25 23 25 04 2

3622 346 336 333 34 052 2

3623 26 28 29 28 02 2

3624

3626 288 28 307 292 117 2

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

5301 29 3 29 29 06 2

5302 297 3 296 298 06 2

5303 2705 2444 2608 2586 22 78

5304 245 254 26 253 85 2

5305 23 23 26 24 15

5306 42 43 4 42 21 2

5307 221 216 241 221

5308 232169 246654 240722 229848

5309 19 22 19 2 0

5310 3 315 325 313 016

5311 262 277 304 281 15 2

5312 27 289 337 299 075 2

5313 321 284 378 328

5314 423 404 385 404 34 083

5315 42 506 416 444 09 2

5316 265 267 277 273 018 2

5317 185 197 195 192 006 2

5318 276 281 279 04 2

5319 396 389 403 396 079 2

5320 623 618 635 625 14

5321 31

5322 24 33 49 33 35 2

5323 38 45 35 45 27 2

50

Figure 8 Results of replicate determinations (indicated by triangles) of benzo[b]fluoranthene (BBF) in the food supplement test material

Horizontal blue lines represent the arithmetic mean value of replicate measurements and blue bars the reported expanded measurement uncertainty (k=2) The assigned value is

plotted as dotted line Red solid lines mark the range of satisfactory performance (|z|le2) Mean values are given for results outside the displayed data range

ProLab 2011

Laboratory

36

0436

1436

0236

1053

1736

1653

0953

0753

0853

0536

2136

0553

0436

0353

0353

1636

0636

2036

2353

1853

1136

1236

2653

0153

0253

1236

1736

1336

0036

1853

2153

1036

1953

1336

2253

2253

2353

1936

1553

1453

0653

1536

0753

2036

0836

11

an

aly

te c

on

ten

t (micro

gk

g)

75

7

65

6

55

5

45

4

35

3

25

2

15

1

05

0

103

6

576

3

Sample St Johns wort extract replicatesMeasurand Benzo[b]fluorantheneNo of laboratories 46

Assigned value 292 microgkg (Reference value)Target sd 060 microgkgTolerance limits 172 - 412 microgkg (|Z score| lt 200)

Assig

ned va

lue

Limit of tolerance

Limit of tolerance

51

Table 14 Analysis results reported by the participants for the content of chrysene (CHR) in the food supplements test material Values are presented as reported

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

3600 5 49 5 5 07 2

3602 32 304 29 304 036

3603 462 469 447 459 048 2

3604 438 428 42 429 119 2

3605 365 41 381 386 085 2

3606 494 411 45 451 124 2

3607 897 774 787 819 18 2

3608 986 887 1168 1014 284 2

3610 418 392 462 424 085 2

3611 833 893 87 865 61 2

3612 6 64 58 61 12 2

3613 41 39 4 4 08 2

3614 272 254 288 271 054 2

3615 605 6028 5964 601 12 2

3616 47 45 49 47 05 2

3617 445 431 389 422 065 2

3618 45 45 45 45 06 2

3619 738 747 72 738 118 2

3620 463 472 446 46 092 2

3621 53 49 51 51 06 2

3622 478 513 505 488 08 2

3623 73 73 72 72 05 2

3624

3626 63 622 634 628 251 2

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

5301 49 49 49 49 1 2

5302 687 683 666 679 136 2

5303 6777 6181 6382 6441 24 70

5304 592 581 6 591 85 2

5305 6 58 64 61 20

5306 49 5 51 49 25 2

5307 524 5 574 524

5308 211488 216782 221146 213243

5309

5310 372 393 394 386 019

5311 43 459 422 437 15 2

5312 934 943 981 953 191 2

5313 793 73 99 838

5314 636 561 565 588 18 0818

5315 547 583 499 542 11 2

5316 292 286 285 282 06 2

5317 52 598 517 545 046 2

5318 368 345 357 05 2

5319 449 471 474 465 116 2

5320 76 924 818 834 99

5321 33

5322 7 7 76 7 35 2

5323 87 73 77 73 37 2

52

Figure 9 Results of replicate determinations (indicated by triangles) of chrysene (CHR) in the food supplement test material

Horizontal blue lines represent the arithmetic mean value of replicate measurements and blue bars the reported expanded measurement uncertainty (k=2) The assigned value is

plotted as dotted line Red solid lines mark the range of satisfactory performance (|z|le2) Mean values are given for results outside the displayed data range

ProLab 2011

Laboratory

53

0836

1453

1636

0253

2153

1836

0553

1036

1336

1736

1036

0453

1136

1836

0636

0336

2053

1936

1653

0136

0036

2253

0636

2153

0753

1553

1753

1453

0436

1536

1253

0536

2653

0353

0253

2236

2336

1953

2336

0753

2053

1353

1236

0836

11

an

aly

te c

on

ten

t (micro

gk

g)

13

12

11

10

9

8

7

6

5

4

3

2

1

0

865

3

Sample St Johns wort extract replicatesMeasurand ChryseneNo of laboratories 45

Assigned value 407 microgkg (Reference value)Target sd 083 microgkgTolerance limits 241 - 573 microgkg (|Z score| lt 200)

Assig

ned va

lue

Limit of tolerance

Limit of tolerance

53

Table 15 Analysis results reported by the participants for the sum of the four marker PAHs in the food supplements test material Values are presented as reported

Laboratory code

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg

3600 128 24 2

3602 79 126

3603 1127 096 2

3604 893 258 2

3605 1091 305 2

3606 1184 238 2

3607 2098 42 2

3608 3336 325 2

3610 1138 17 2

3611 2218 259 2

3612 143 29 2

3613 122 2 2

3614 663 129 2

3615 152 304 2

3616 102 03 2

3617 1252 169 2

3618 128 18 2

3619 157 305 2

3620 1204 12 2

3621 151 19 2

3622 1361 115 2

3623 154 09 2

3624

3626 1409 56 2

Laboratory code

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg

5301 126

5302 37 074 2

5303 13438 92

5304 1251 85 2

5305 125 20

5306 129 65 2

5307 1363

5308 7245598

5309

5310 1127 056

5311 1367 15 2

5312 2042 408 2

5313 1726

5314 1558

5315 177 35 2

5316 96 073 2

5317 1229 066 2

5318 82 05 2

5319 1225 267 2

5320 2309 41

5321

5322 207 35 2

5323 171 103 2

54

ANNEX 9 Results reported for the standard solutions with undisclosed analyte content Table 16 Analysis results reported by the participants for the four marker PAHs in the standard solution in toluene with undisclosed analyte

content Values are presented as reported U measurement uncertainty k coverage factor

benz[a]anthracene benzo[a]pyrene benzo[b]fluoranthene chrysene Laboratory

code Unit Result U k Result U k Result U k Result U k 3600 microgl 38 67 2 25 45 2 211 68 2 275 38 2

3605 microgl 367 184 2 2482 124 2 2592 13 2 235 118 2

3606 microgkg 3763 176 2 2609 142 2 2721 118 2 2539 134 2

3607 microgkg 3706 815 2 2824 565 2 2591 518 2 2587 569 2

3612 microgl 38 08 2 237 11 2 244 18 2 257 02 2

3614 microgl 324 65 2 209 38 2 215 37 2 21 42 2

3616 microgkg 318 07 2 224 14 2 231 26 2 216 1 2

3618 microgkg 417 17 2 284 08 2 292 06 2 297 03 2

3619 microgkg 47 742 2 274 468 2 228 387 2 274 434 2

3620 microgl 495 74 2 294 29 2 321 8 2 321 64 2

3621 microgl 241 22 2 143 13 2 188 16 2 145 14 2

3623 microgkg 476 25 2 311 53 2 269 3 2 297 03 2

3626 microgl 359 38 2 235 23 2 238 27 2 24 18 2

5301 microgkg 426 29 299 289

5302 microgkg 436 87 2 296 59 2 296 59 2 289 58 2

5303 microgl 3721 27 102 22838 19 99 23756 22 99 25486 24 98

5304 microgkg 38 29 2 225 26 2 257 37 2 258 52 2

5305 microgkg 305 225 245 29

5306 microgkg 316 63 2 247 49 2 65 13 2 22 44 2

5307 microgkg 3658 2559 241 2414

5308 microgkg 16854 109324 246654 216782

5311 microgl 374 20 2 279 15 2 268 15 2 272 15 2

5313 microgl 3408 2165 2275 2373

5314 microgkg 395 5 2875 11 3614 34 2905 18

5320 microgl 3267 368 1986 571 28 683 2427 512

5322 microgl 282 35 2 225 35 2 241 35 2 21 35 2

55

Figure 10 Youden plots of the percent deviation of the reported values from the assigned values for the food supplement test sample and the

standard solution in toluene with undisclosed content

(The identity of the respective measurand is given above the figure Results exceeding a difference of more than 100 were omitted)

56

Figure 10 - continued

57

Table 17 Analysis results reported by the participants for the four marker PAHs in the standard solution in acetonitrile with undisclosed

analyte content

Values are presented as reported U measurement uncertainty k coverage factor

benz[a]anthracene benzo[a]pyrene benzo[b]fluoranthene chrysene

Laboratory

code Unit Result U k Result U k Result U k Result U k

3602 microgkg 3675 247 247 244

3603 microgkg 358 086 2 236 028 2 235 028 2 236 057 2

3604 microgl 475 29 2 19 12 2 194 13 2 258 12 2

3608 microgl 3623 078 2 2385 023 2 2343 18 2 2296 092 2

3610 microgkg 369 185 2 25 125 2 251 126 2 244 122 2

3611 microgkg 48 02 2 323 02 2 328 04 2 317 03 2

3613 microgl 332 31 2 261 3 2 249 25 2 239 24 2

3615 microgl 415 2635 2729 2689

3617 microgkg 37 09 2 244 06 2 284 07 2 239 06 2

3622 microgl 37 12 2 25 23 2 257 21 2 238 1 2

3624 microgkg 402 104 2 255 084 2 265 096 2 253 07 2

5306 microgkg 563 113 2 371 74 2 359 72 2 333 67 2

5308 microgkg 168633 102669 232169 211488

5309 microgl 402 268 27

5310 microgl 315 158 25 125 25 125 288 144

5312 microgl 428 043 2 282 028 2 272 027 2 275 028 2

5315 microgkg 387 08 2 256 05 2 251 05 2 253 05 2

5316 microgl 3839 342 2 2485 191 2 2559 168 2 239 51 2

5317 microgl 2466 043 2 2212 034 2 2472 016 2 2146 068 2

5319 microgl 414 41 2 279 28 2 268 27 2 26 31 2

5321 microgkg 476 316 326 328

5322 microgl 2547 35 2

5323 microgkg 405 203 2 239 119 2 255 153 2 201 11 2

58

Figure 11 Youden plots of the percent deviation of the reported values from the assigned values for the food supplement test sample and the

standard solution in acetonitrile with undisclosed content

(The identity of the respective measurand is given above the figure Results exceeding a difference of more than 100 were omitted)

59

Figure 11 - continued

60

ANNEX 10 Table 18 Method performance parameters reported by the participants

Laboratory code

Analyte LOD LOQ Recovery Analyte LOD LOQ Recovery

microgkg microgkg microgkg microgkg 3600 BaA 03 06 978 BaP 03 07 9483602 BaA 007 021 926 BaP 005 015 993603 BaA 0011 05 987 BaP 0005 051 9553604 BaA 02 04 86 BaP 02 04 883605 BaA 0025 005 118 BaP 0025 005 1083606 BaA 001 003 65 BaP 001 003 703607 BaA 021 071 74 BaP 027 089 943608 BaA 016 048 8213 BaP 015 046 83683610 BaA 02 05 85 BaP 005 02 853611 BaA BaP 3612 BaA 1 117 BaP 1 1203613 BaA 01 03 90 BaP 01 03 903614 BaA 02 05 96 BaP 02 05 933615 BaA 005 015 110 BaP 005 015 1053616 BaA 01 05 108 BaP 01 05 753617 BaA 018 037 405 BaP 009 018 3873618 BaA 01 03 63 BaP 01 03 593619 BaA 005 005 59 BaP 033 033 543620 BaA 0009 0018 64 BaP 0005 001 553621 BaA 007 02 84 BaP 007 02 923622 BaA 01 03 1101 BaP 01 03 9853623 BaA 02 08 105 BaP 02 08 1013624 BaA BaP 01 033 8513625 BaA BaP 3626 BaA BaP 5301 BaA 003 01 102 BaP 003 01 1005302 BaA 01 03 78 BaP 01 03 865303 BaA 003 01 62 BaP 003 01 805304 BaA 1 1 70 BaP 1 1 955305 BaA 02 05 85 BaP 02 05 855306 BaA 03 1 699 BaP 03 1 905307 BaA 6537 BaP 81165308 BaA 0088 0265 99 BaP 00262 00785 1005309 BaA 01 08 92 BaP 01 08 925310 BaA 01 03 BaP 01 03 905311 BaA 002 005 80 BaP 002 004 915312 BaA 03 09 105 BaP 03 09 1055313 BaA 04 08 BaP 04 08 5314 BaA 001 002 96 BaP 001 002 9485315 BaA 01 03 50 - 100 BaP 01 03 50 - 100 5316 BaA 002 011 827 BaP 004 019 10445317 BaA 05 1 76 BaP 05 1 755318 BaA BaP 5319 BaA 009 029 951 BaP 011 041 995320 BaA 12 18 946 BaP 06 1 8345321 BaA 03 05 BaP 05 1 5322 BaA 1 2 100 BaP 1 2 1005323 BaA 26 BaP 26

61

ANNEX 10 Table 18 - continued

Laboratory code Analyte LOD LOQ Recovery Analyte LOD LOQ Recovery

microgkg microgkg microgkg microgkg 3600 BbF 03 05 96 CHR 02 03 10153602 BbF 015 045 947 CHR 003 009 9193603 BbF 0014 05 1012 CHR 0004 05 10033604 BbF 02 04 85 CHR 02 04 873605 BbF 005 01 94 CHR 0025 005 863606 BbF 001 003 70 CHR 001 003 653607 BbF 018 06 100 CHR 014 028 943608 BbF 015 046 7979 CHR 015 045 71253610 BbF 005 02 85 CHR 02 05 853611 BbF CHR 3612 BbF 1 118 CHR 1 1173613 BbF 01 03 90 CHR 01 03 903614 BbF 01 04 96 CHR 02 05 953615 BbF 008 025 109 CHR 003 01 1083616 BbF 01 05 78 CHR 01 05 723617 BbF 035 07 38 CHR 012 024 4163618 BbF 01 03 62 CHR 01 03 623619 BbF 016 016 63 CHR 01 01 593620 BbF 0003 0006 55 CHR 001 002 653621 BbF 007 02 103 CHR 007 02 893622 BbF 02 06 963 CHR 03 09 1043623 BbF 02 08 106 CHR 02 08 1193624 BbF CHR 3625 BbF CHR 3626 BbF CHR 5301 BbF 003 01 96 CHR 003 01 1145302 BbF 01 03 84 CHR 01 03 835303 BbF 003 01 78 CHR 003 01 705304 BbF 1 1 86 CHR 1 1 705305 BbF 02 05 90 CHR 02 05 955306 BbF 03 1 1435 CHR 03 1 1185307 BbF 8116 CHR 65375308 BbF 0079 0238 99 CHR 009 026 1005309 BbF 01 08 92 CHR 5310 BbF 07 2 CHR 01 03 5311 BbF 002 004 95 CHR 002 004 895312 BbF 03 09 105 CHR 03 09 1055313 BbF 04 08 CHR 04 08 5314 BbF 001 002 83 CHR 001 002 8185315 BbF 02 05 50 -100 CHR 02 05 50 - 100 5316 BbF 005 024 814 CHR 001 007 9595317 BbF 05 1 73 CHR 05 1 785318 BbF CHR 5319 BbF 011 038 946 CHR 013 043 9675320 BbF 07 09 1194 CHR 23 32 10455321 BbF 05 1 CHR 05 1 5322 BbF 1 2 100 CHR 1 2 1005323 BbF 26 CHR 26

62

ANNEX 11 Table 19 Details of analysis method reported by the participants Data are presented as reported

Laboratory code

Analysis technique

Instrument calibration

Internal standards applied

Sample clean up

Details of sample

clean up

Extraction technique

Details of sample

extraction

3600 6) GC-MS

2) Internal standardisation with labelled substances

Deuterated PAH mix 9 (Ehrenstorfer)

1) Saponification 2) LiquidLiquid partitioning

D) Other Liquidliquid partitioning

3602 2) HPLC-UV-FLD

1) External calibration

3) Gel permeation chromatography

A) Pressurised liquid extraction (PLE)

3603 1) HPLC-FLD

1) External calibration

3) Gel permeation chromatography

D) Other 1 minute with dichloromethane in vortex

3604 2) HPLC-UV-FLD

2) Internal standardisation with labelled substances

DiP D14

5) Donor acceptor complex chromatography

D) Other LL

3605 1) HPLC-FLD

1) External calibration

3) Gel permeation chromatography

C) Soxhlet extraction

3606 7) GC-MSMS

2) Internal standardisation with labelled substances

BaP 13C4 CHR 13C6 BaA 13C6 BbF 13C6

4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

3607 6) GC-MS

2) Internal standardisation with labelled substances

BaA-d12 CHR-d12 BbF-d12 BkF-d12 BaP-d12 ICP-d12 BgP-d12 DiP-d14

1) Saponification

D) Other solvent partitioning

3608 1) HPLC-FLD

1) External calibration

6) Other Liquid extraction

D) Other

Extraction in a rotary agitator and centrifugation

3610 2) HPLC-UV-FLD

1) External calibration

4) Solid phase extraction

B) Sonication

3611

3612 6) GC-MS

2) Internal standardisation with labelled substances

chrysene-d12 dibenzo(ah)anthracene-d14 dibenzo(ai)pyrene-d14

4) Solid phase extraction

B) Sonication

3613 5) LC-MSMS

3) Internal standardisation with unlabelled substances

I use Chrysene-D12 3) Gel permeation chromatography

D) Other extraction with hexan

63

Table 19 - continued

Laboratory code

Analysis technique

Instrument calibration

Internal standards applied

Sample clean up Details of sample

clean up

Extraction technique

Details of sample

extraction

3614 6) GC-MS 1) External calibration

3) Gel permeation chromatography

B) Sonication

3615 1) HPLC-FLD

1) External calibration

6) Other PLE GPC

A) Pressurised liquid extraction (PLE)

3616 6) GC-MS

2) Internal standardisation with labelled substances

Benzo(a)pyrene-13C4 Benzo(a)anthracene-13C6 Benzo(b)fluoranthene-13C6 Chrysene-13C6

2) LiquidLiquid partitioning 4) Solid phase extraction

B) Sonication

3617 1) HPLC-FLD

1) External calibration

4) Solid phase extraction

C) Soxhlet extraction

3618 6) GC-MS

2) Internal standardisation with labelled substances

deuterated standards

1) Saponification 4) Solid phase extraction

D) Other Handshaken with cyclohexane

3619 6) GC-MS

2) Internal standardisation with labelled substances

C13 Labelled US EPA 16

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

D) Other Homegenisation amp saponiofication

3620 9) GC-HRMS

2) Internal standardisation with labelled substances

mix of deuterated PAHs (BAA-D12 CHR-D12 BAP-D12 BBF-D12)

3) Gel permeation chromatography 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

3621 6) GC-MS

2) Internal standardisation with labelled substances

Chrysene D12 Benzo(a)pyrene D12 Benzo(b)fluoranthene D12 Benzo(a)anthracene

2) LiquidLiquid partitioning 4) Solid phase extraction

B) Sonication

3622 1) HPLC-FLD

4) Standard addition method

4) Solid phase extraction

D) Other

Extraction with cyclohexane by heating under reflux

3623 7) GC-MSMS

2) Internal standardisation with labelled substances

C13 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

3624 2) HPLC-UV-FLD

1) External calibration

2) LiquidLiquid partitioning

D) Other hot solvent extraction

3626

64

Table 19 - continued

Laboratory code

Analysis technique

Instrument calibration

Internal standards

applied

Sample clean up

Details of sample

clean up

Extraction technique

Details of sample

extraction

5301 7) GC-MSMS

2) Internal standardisation with labelled substances

EACH SUBSTANCE C13

4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5302 7) GC-MSMS

2) Internal standardisation with labelled substances

Isotope labelled PAHs 13C for each analyte

4) Solid phase extraction

C) Soxhlet extraction

5303 7) GC-MSMS

3) Internal standardisation with unlabelled substances

HAP C13 4) Solid phase extraction

D) Other automate ASE200 DIONEX

5304 7) GC-MSMS

2) Internal standardisation with labelled substances

with C13 for each HAP

2) LiquidLiquid partitioning 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5305 7) GC-MSMS

2) Internal standardisation with labelled substances

C13 isotopiques internal standards

4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5306 7) GC-MSMS

2) Internal standardisation with labelled substances

C13 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5307 6) GC-MS

2) Internal standardisation with labelled substances

1) Saponification 2) LiquidLiquid partitioning

D) Other solvent extraction

5308 1) HPLC-FLD 1) External calibration

4) Solid phase extraction

B) Sonication

5309 1) HPLC-FLD 1) External calibration

4) Solid phase extraction

D) Other only SPE

5310 1) HPLC-FLD 1) External calibration

1) Saponification 2) LiquidLiquid partitioning 3) Gel permeation chromatography

C) Soxhlet extraction

5311 6) GC-MS

2) Internal standardisation with labelled substances

13 C Chrysene 13 C Benz(a)pyrene

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

B) Sonication

5312 1) HPLC-FLD

3) Internal standardisation with unlabelled substances

Benzo(b)chrysen

2) LiquidLiquid partitioning 4) Solid phase extraction

Quechers D) Other Quechers

65

Table 19 - continued

Laboratory code

Analysis technique

Instrument calibration

Internal standards applied

Sample clean up Details of sample

clean up

Extraction technique

Details of sample extraction

5313 6) GC-MS

2) Internal standardisation with labelled substances

Benz(a)anthracene D12 Benzo(b)fluranthene D12 Benzo(a)pyrene D12 Chrysene D12

1) Saponification 2) LiquidLiquid partitioning 3) Gel permeation chromatography

A) Pressurised liquid extraction (PLE)

5314 6) GC-MS

2) Internal standardisation with labelled substances

Benzo(a)pyren d12 2) LiquidLiquid partitioning

D) Other Extraction with

5315 1) HPLC-FLD

3) Internal standardisation with unlabelled substances

Benz(b)chrysen

1) Saponification 2) LiquidLiquid partitioning

D) Other shaker

5316 1) HPLC-FLD

1) External calibration

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

D) Other

LiquidLiquid extraction with cyclohexane and dimethylformamide

5317 7) GC-MSMS

1) External calibration

3) Gel permeation chromatography 6) Other

silica gel D) Other VDLUFA online method

5318

5319 1) HPLC-FLD

3) Internal standardisation with unlabelled substances

Benzo(b)chrysene 3) Gel permeation chromatography

A) Pressurised liquid extraction (PLE)

5320 6) GC-MS

3) Internal standardisation with unlabelled substances

22-binaphthyl

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

D) Other 3 hours refluxing

5321 1) HPLC-FLD

2) Internal standardisation with labelled substances

6-meacutethylchrysegravene

1) Saponification 2) LiquidLiquid partitioning

B) Sonication

5322 6) GC-MS

2) Internal standardisation with labelled substances

naftalene-d8 acenaphtalene-d10 phenanthrene-d10 chrysene-d12 perylene-d12 benzo(e)anthracene-d12

1) Saponification 2) LiquidLiquid partitioning 6) Other

clean-up with silica packed column

C) Soxhlet extraction

5323 1) HPLC-FLD

1) External calibration

4) Solid phase extraction 6) Other

Accelerated Solvent Extraction

A) Pressurised liquid extraction (PLE)

66

European Commission EUR 25245 EN ndash Joint Research Centre ndash Institute for Reference Materials and Measurements Title Report on the 8th inter-laboratory comparison organised by the European Union Reference Laboratory for Polycyclic Aromatic Hydrocarbons - Four marker PAHs in a dry extract of St Johns wort Author(s) Radoslav Lizak Szilard Szilagyi Philippe Verlinde and Thomas WENZL Luxembourg Publications Office of the European Union 2012 ndash 66 pp ndash 201 x 297 cm EUR ndash Scientific and Technical Research series ndash ISSN 1831-9424 ISBN 978-92-79-23337-1 doi 10278759387 Abstract The proficiency test here reported concerned the determination of the contents of four marker polycyclic aromatic hydrocarbons (PAHs) and of their sum in an food supplements test sample The set of marker PAHs consists of benz[a]anthacene benzo[a]pyrene benzo[b]fluoranthene and chrysene Participants to these PT were National Reference Laboratories for PAHs (NRLs-PAHs) and EU official food control laboratories The number of participants was in total 48

The PT was organised according ISO Standard 170432010

The test material used was a commercial dry extract of St Johns wort Participants also received a solution of the same PAHs either in acetonitrile or in toluene for checking their instrument calibration

The results from participants were rated with z-scores and zeta-scores About 68 of the reported results were attributed with z-scores with an absolute value of below two which is the threshold for satisfactory performance

How to obtain EU publications

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sending a fax to (352) 29 29-42758

The mission of the JRC is to provide customer-driven scientific and technical support

for the conception development implementation and monitoring of EU policies As a

service of the European Commission the JRC functions as a reference centre of

science and technology for the Union Close to the policy-making process it serves

the common interest of the Member States while being independent of special

interests whether private or national

L

A-N

A-252

45-EN

-N

• 1 Summary
• 2 Introduction
• 3 Scope
• 4 Participating Laboratories
• 5 Time frame
• 6 Confidentiality
• 7 Test materials
• • 71 1Preparation of test materials
  • 72 Assigned values and standard deviation for proficiency assessment
  • 73 Homogeneity and stability
  • 74 Sample dispatch
  • • 8 Design of the proficiency test
    • 9 Evaluation of the results
    • • 91 2General
      • 92 21BEvaluation criteria
      • 93 Performance of participants
      • 94 Evaluation of the influence of analysis method
      • 95 Evaluation of the influence of calibration
      • 96 26Evaluation of compliance with legislation
      • • 10 Follow-up actions for underperforming laboratories
        • 11 1Conclusions
        • 12 1Acknowledgements
        • 13 References

38

ANNEX 5 - continued

Benzo[b]fluoranthene - BBF

Chrysene - CHR

39

ANNEX 5 - continued

Stability of the test material

Table 10 Content of the four target analytes in test materials stored under different

temperatures

BaA BaP BbF CHR

microgkg microgkg microgkg microgkg

Content 31 16 31 69 Storage at room temperature

U (k=2) 05 02 05 17

Content 31 15 29 68 Storage at reference temperature

U (k=2) 04 03 04 10

The applied capillary column does not separate CHR from triphenylene

40

ANNEX 6 ndash Documents sent to participants

41

ANNEX 6 - continued

42

ANNEX 7 Questionnaire on details of analysis method

43

ANNEX 7 - continued

44

ANNEX 7 - continued

45

ANNEX 8 - Results reported by participants for the food supplements test sample Table 11 Analysis results reported by the participants for the content of benz[a]anthracene (BAA) in the food supplements test material Values are presented as reported

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

3600 31 29 29 3 05 2

3602 199 193 178 19 021

3603 253 253 246 251 022 2

3604 199 209 23 213 071 2

3605 301 3 271 291 076 2

3606 302 288 278 289 064 2

3607 456 416 443 438 096 2

3608 796 695 651 714 149 2

3610 182 192 214 196 039 2

3611 414 504 488 469 96 2

3612 3 34 28 31 06 2

3613 32 33 34 33 07 2

3614 182 182 191 185 037 2

3615 3884 3822 3565 376 0752 2

3616 2 2 2 2 01 2

3617 366 37 35 362 046 2

3618 32 32 32 32 05 2

3619 299 305 297 299 048 2

3620 304 294 302 302 045 2

3621 29 26 29 28 04 2

3622 343 325 318 322 052 2

3623 35 3 33 33 07 2

3624

3626 318 31 314 314 126 2

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

5301 31 3 32 31 06 2

5302 314 319 315 316 063 2

5303 3102 2716 2822 2876 27 62

5304 261 263 26 261 57 2

5305 23 22 26 24 20

5306 22 23 23 22 11 2

5307 488 461 317 461

5308 168633 168544 1562025 1644598

5309 16 19 16 17

5310 3 304 311 305 015

5311 477 452 448 459 15 2

5312 506 63 494 543 134 2

5313 353 301 404 353

5314 431 373 378 394 5 096

5315 596 531 51 545 11 2

5316 224 231 235 235 022 2

5317 216 23 221 222 007 2

5318 064 07 067 01 2

5319 111 184 146 147 029 2

5320 515 525 564 534 48

5321 21 5322 13 87 9 87 35 2

5323 38 33 35 33 16 2

46

Figure 6 Results of replicate determinations (indicated by triangles) of benz[a]anthracene (BAA) in the food supplement test material

Horizontal blue lines represent the arithmetic mean value of replicate measurements and blue bars the reported expanded measurement uncertainty (k=2) The assigned value is

plotted as dotted line Red solid lines mark the range of satisfactory performance (|z|le2) Mean values are given for results outside the displayed data range

ProLab 2011

Laboratory

53

1853

1953

0853

0936

1436

0236

1036

1653

2136

0453

1753

0653

1653

0536

0353

0436

2153

0336

0636

0536

0036

2036

1953

1036

1253

0136

2653

0236

1836

2336

2236

1353

1353

2336

1736

1553

1453

0736

0753

1153

2053

1253

1536

0853

2236

11

an

aly

te c

on

ten

t (micro

gk

g)

10

95

9

85

8

75

7

65

6

55

5

45

4

35

3

25

2

15

1

05

0

102

3 468

7

Sample St Johns wort extract replicatesMeasurand Benz[a]anthraceneNo of laboratories 46

Assigned value 298 microgkg (Reference value)Target sd 062 microgkgTolerance limits 174 - 422 microgkg (|Z score| lt 200)

Assig

ned va

lue

Limit of tolerance

Limit of tolerance

47

Table 12 Analysis results reported by the participants for the content of benzo[a]pyrene (BAP) in the food supplements test material Values are presented as reported

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

3600 16 17 17 17 03 2

3602 118 123 13 124 015

3603 165 162 157 161 015 2

3604 134 154 139 143 04 2

3605 164 166 172 167 057 2

3606 181 159 161 167 031 2

3607 279 266 279 274 055 2

3608 57 687 461 573 226 2

3610 226 216 183 206 042 2

3611 289 325 311 308 36 2

3612 24 22 2 22 05 2

3613 17 18 19 18 05 2

3614 08 08 08 08 016 2

3615 1545 1393 1425 145 029 2

3616 16 16 14 15 03 2

3617 177 171 158 169 023 2

3618 2 2 2 2 02 2

3619 207 207 206 207 075 2

3620 165 169 163 166 017 2

3621 44 47 49 47 05 2

3622 212 208 222 213 038 2

3623 2 21 22 21 03 2

3624 18 178 182 18 031 2

3626 172 173 178 174 07 2

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

5301 18 18 18 18 04 2

5302 188 19 187 188 038 2

5303 1691 1426 149 1535 19 80

5304 145 142 15 146 69 2

5305 15 17 18 16 15

5306 16 17 17 16 08 2

5307 164 157 153 157

5308 102669 109324 109035 107009

5309 1 1 12 11

5310 122 122 124 123 006

5311 199 184 215 2 15 2

5312 258 248 239 248 05 2

5313 199 178 245 207

5314 174 171 172 172 11 0948

5315 253 248 213 238 05 2

5316 177 171 173 17 016 2

5317 255 277 276 269 012 2

5318 114 12 117 02 2

5319 19 228 231 216 043 2

5320 303 3 344 316 78

5321 12 12 12 12

5322 13 17 24 17 35 2

5323 2 2 19 2 1 2

48

Figure 7 Results of replicate determinations (indicated by triangles) of benzo[a]pyrene (BAP) in the food supplement test material

Horizontal blue lines represent the arithmetic mean value of replicate measurements and blue bars the reported expanded measurement uncertainty (k=2) The assigned value is

plotted as dotted line Red solid lines mark the range of satisfactory performance (|z|le2) Mean values are given for results outside the displayed data range

ProLab 2011

Laboratory

36

1453

0953

0853

1853

2153

1036

0236

0436

1553

0436

1653

0353

0736

0336

2036

0053

0553

0636

0636

0536

1753

1453

1636

2636

1336

2453

0153

2253

0253

2353

1136

1836

1953

1336

1036

2336

2253

1936

1253

1553

1253

1736

0753

2036

2136

0836

11

an

aly

te c

on

ten

t (micro

gk

g)

55

5

45

4

35

3

25

2

15

1

05

0

573

308

3

Sample St Johns wort extract replicatesMeasurand Benzo[a]pyreneNo of laboratories 48

Assigned value 165 microgkg (Reference value)Target sd 036 microgkgTolerance limits 093 - 237 microgkg (|Z score| lt 200)

Assig

ned va

lue

Limit of tolerance

Limit of tolerance

49

Table 13 Analysis results reported by the participants for the content of benzo[b]fluoranthene (BBF) in the food supplements test material Values are presented as reported

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

3600 32 3 3 31 09 2

3602 173 175 16 17 017

3603 265 252 25 256 025 2

3604 116 108 105 11 028 2

3605 286 225 232 247 074 2

3606 294 273 261 276 046 2

3607 579 579 544 567 113 2

3608 103 1201 876 1036 326 2

3610 173 196 204 191 038 2

3611 546 618 565 576 75 2

3612 3 28 28 29 06 2

3613 28 31 32 31 06 2

3614 135 111 135 127 022 2

3615 4187 3678 4111 4 08 2

3616 2 2 2 2 01 2

3617 33 27 299 035 2

3618 31 31 31 31 05 2

3619 326 324 312 326 064 2

3620 277 28 272 276 069 2

3621 26 25 23 25 04 2

3622 346 336 333 34 052 2

3623 26 28 29 28 02 2

3624

3626 288 28 307 292 117 2

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

5301 29 3 29 29 06 2

5302 297 3 296 298 06 2

5303 2705 2444 2608 2586 22 78

5304 245 254 26 253 85 2

5305 23 23 26 24 15

5306 42 43 4 42 21 2

5307 221 216 241 221

5308 232169 246654 240722 229848

5309 19 22 19 2 0

5310 3 315 325 313 016

5311 262 277 304 281 15 2

5312 27 289 337 299 075 2

5313 321 284 378 328

5314 423 404 385 404 34 083

5315 42 506 416 444 09 2

5316 265 267 277 273 018 2

5317 185 197 195 192 006 2

5318 276 281 279 04 2

5319 396 389 403 396 079 2

5320 623 618 635 625 14

5321 31

5322 24 33 49 33 35 2

5323 38 45 35 45 27 2

50

Figure 8 Results of replicate determinations (indicated by triangles) of benzo[b]fluoranthene (BBF) in the food supplement test material

Horizontal blue lines represent the arithmetic mean value of replicate measurements and blue bars the reported expanded measurement uncertainty (k=2) The assigned value is

plotted as dotted line Red solid lines mark the range of satisfactory performance (|z|le2) Mean values are given for results outside the displayed data range

ProLab 2011

Laboratory

36

0436

1436

0236

1053

1736

1653

0953

0753

0853

0536

2136

0553

0436

0353

0353

1636

0636

2036

2353

1853

1136

1236

2653

0153

0253

1236

1736

1336

0036

1853

2153

1036

1953

1336

2253

2253

2353

1936

1553

1453

0653

1536

0753

2036

0836

11

an

aly

te c

on

ten

t (micro

gk

g)

75

7

65

6

55

5

45

4

35

3

25

2

15

1

05

0

103

6

576

3

Sample St Johns wort extract replicatesMeasurand Benzo[b]fluorantheneNo of laboratories 46

Assigned value 292 microgkg (Reference value)Target sd 060 microgkgTolerance limits 172 - 412 microgkg (|Z score| lt 200)

Assig

ned va

lue

Limit of tolerance

Limit of tolerance

51

Table 14 Analysis results reported by the participants for the content of chrysene (CHR) in the food supplements test material Values are presented as reported

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

3600 5 49 5 5 07 2

3602 32 304 29 304 036

3603 462 469 447 459 048 2

3604 438 428 42 429 119 2

3605 365 41 381 386 085 2

3606 494 411 45 451 124 2

3607 897 774 787 819 18 2

3608 986 887 1168 1014 284 2

3610 418 392 462 424 085 2

3611 833 893 87 865 61 2

3612 6 64 58 61 12 2

3613 41 39 4 4 08 2

3614 272 254 288 271 054 2

3615 605 6028 5964 601 12 2

3616 47 45 49 47 05 2

3617 445 431 389 422 065 2

3618 45 45 45 45 06 2

3619 738 747 72 738 118 2

3620 463 472 446 46 092 2

3621 53 49 51 51 06 2

3622 478 513 505 488 08 2

3623 73 73 72 72 05 2

3624

3626 63 622 634 628 251 2

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

5301 49 49 49 49 1 2

5302 687 683 666 679 136 2

5303 6777 6181 6382 6441 24 70

5304 592 581 6 591 85 2

5305 6 58 64 61 20

5306 49 5 51 49 25 2

5307 524 5 574 524

5308 211488 216782 221146 213243

5309

5310 372 393 394 386 019

5311 43 459 422 437 15 2

5312 934 943 981 953 191 2

5313 793 73 99 838

5314 636 561 565 588 18 0818

5315 547 583 499 542 11 2

5316 292 286 285 282 06 2

5317 52 598 517 545 046 2

5318 368 345 357 05 2

5319 449 471 474 465 116 2

5320 76 924 818 834 99

5321 33

5322 7 7 76 7 35 2

5323 87 73 77 73 37 2

52

Figure 9 Results of replicate determinations (indicated by triangles) of chrysene (CHR) in the food supplement test material

Horizontal blue lines represent the arithmetic mean value of replicate measurements and blue bars the reported expanded measurement uncertainty (k=2) The assigned value is

plotted as dotted line Red solid lines mark the range of satisfactory performance (|z|le2) Mean values are given for results outside the displayed data range

ProLab 2011

Laboratory

53

0836

1453

1636

0253

2153

1836

0553

1036

1336

1736

1036

0453

1136

1836

0636

0336

2053

1936

1653

0136

0036

2253

0636

2153

0753

1553

1753

1453

0436

1536

1253

0536

2653

0353

0253

2236

2336

1953

2336

0753

2053

1353

1236

0836

11

an

aly

te c

on

ten

t (micro

gk

g)

13

12

11

10

9

8

7

6

5

4

3

2

1

0

865

3

Sample St Johns wort extract replicatesMeasurand ChryseneNo of laboratories 45

Assigned value 407 microgkg (Reference value)Target sd 083 microgkgTolerance limits 241 - 573 microgkg (|Z score| lt 200)

Assig

ned va

lue

Limit of tolerance

Limit of tolerance

53

Table 15 Analysis results reported by the participants for the sum of the four marker PAHs in the food supplements test material Values are presented as reported

Laboratory code

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg

3600 128 24 2

3602 79 126

3603 1127 096 2

3604 893 258 2

3605 1091 305 2

3606 1184 238 2

3607 2098 42 2

3608 3336 325 2

3610 1138 17 2

3611 2218 259 2

3612 143 29 2

3613 122 2 2

3614 663 129 2

3615 152 304 2

3616 102 03 2

3617 1252 169 2

3618 128 18 2

3619 157 305 2

3620 1204 12 2

3621 151 19 2

3622 1361 115 2

3623 154 09 2

3624

3626 1409 56 2

Laboratory code

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg

5301 126

5302 37 074 2

5303 13438 92

5304 1251 85 2

5305 125 20

5306 129 65 2

5307 1363

5308 7245598

5309

5310 1127 056

5311 1367 15 2

5312 2042 408 2

5313 1726

5314 1558

5315 177 35 2

5316 96 073 2

5317 1229 066 2

5318 82 05 2

5319 1225 267 2

5320 2309 41

5321

5322 207 35 2

5323 171 103 2

54

ANNEX 9 Results reported for the standard solutions with undisclosed analyte content Table 16 Analysis results reported by the participants for the four marker PAHs in the standard solution in toluene with undisclosed analyte

content Values are presented as reported U measurement uncertainty k coverage factor

benz[a]anthracene benzo[a]pyrene benzo[b]fluoranthene chrysene Laboratory

code Unit Result U k Result U k Result U k Result U k 3600 microgl 38 67 2 25 45 2 211 68 2 275 38 2

3605 microgl 367 184 2 2482 124 2 2592 13 2 235 118 2

3606 microgkg 3763 176 2 2609 142 2 2721 118 2 2539 134 2

3607 microgkg 3706 815 2 2824 565 2 2591 518 2 2587 569 2

3612 microgl 38 08 2 237 11 2 244 18 2 257 02 2

3614 microgl 324 65 2 209 38 2 215 37 2 21 42 2

3616 microgkg 318 07 2 224 14 2 231 26 2 216 1 2

3618 microgkg 417 17 2 284 08 2 292 06 2 297 03 2

3619 microgkg 47 742 2 274 468 2 228 387 2 274 434 2

3620 microgl 495 74 2 294 29 2 321 8 2 321 64 2

3621 microgl 241 22 2 143 13 2 188 16 2 145 14 2

3623 microgkg 476 25 2 311 53 2 269 3 2 297 03 2

3626 microgl 359 38 2 235 23 2 238 27 2 24 18 2

5301 microgkg 426 29 299 289

5302 microgkg 436 87 2 296 59 2 296 59 2 289 58 2

5303 microgl 3721 27 102 22838 19 99 23756 22 99 25486 24 98

5304 microgkg 38 29 2 225 26 2 257 37 2 258 52 2

5305 microgkg 305 225 245 29

5306 microgkg 316 63 2 247 49 2 65 13 2 22 44 2

5307 microgkg 3658 2559 241 2414

5308 microgkg 16854 109324 246654 216782

5311 microgl 374 20 2 279 15 2 268 15 2 272 15 2

5313 microgl 3408 2165 2275 2373

5314 microgkg 395 5 2875 11 3614 34 2905 18

5320 microgl 3267 368 1986 571 28 683 2427 512

5322 microgl 282 35 2 225 35 2 241 35 2 21 35 2

55

Figure 10 Youden plots of the percent deviation of the reported values from the assigned values for the food supplement test sample and the

standard solution in toluene with undisclosed content

(The identity of the respective measurand is given above the figure Results exceeding a difference of more than 100 were omitted)

56

Figure 10 - continued

57

Table 17 Analysis results reported by the participants for the four marker PAHs in the standard solution in acetonitrile with undisclosed

analyte content

Values are presented as reported U measurement uncertainty k coverage factor

benz[a]anthracene benzo[a]pyrene benzo[b]fluoranthene chrysene

Laboratory

code Unit Result U k Result U k Result U k Result U k

3602 microgkg 3675 247 247 244

3603 microgkg 358 086 2 236 028 2 235 028 2 236 057 2

3604 microgl 475 29 2 19 12 2 194 13 2 258 12 2

3608 microgl 3623 078 2 2385 023 2 2343 18 2 2296 092 2

3610 microgkg 369 185 2 25 125 2 251 126 2 244 122 2

3611 microgkg 48 02 2 323 02 2 328 04 2 317 03 2

3613 microgl 332 31 2 261 3 2 249 25 2 239 24 2

3615 microgl 415 2635 2729 2689

3617 microgkg 37 09 2 244 06 2 284 07 2 239 06 2

3622 microgl 37 12 2 25 23 2 257 21 2 238 1 2

3624 microgkg 402 104 2 255 084 2 265 096 2 253 07 2

5306 microgkg 563 113 2 371 74 2 359 72 2 333 67 2

5308 microgkg 168633 102669 232169 211488

5309 microgl 402 268 27

5310 microgl 315 158 25 125 25 125 288 144

5312 microgl 428 043 2 282 028 2 272 027 2 275 028 2

5315 microgkg 387 08 2 256 05 2 251 05 2 253 05 2

5316 microgl 3839 342 2 2485 191 2 2559 168 2 239 51 2

5317 microgl 2466 043 2 2212 034 2 2472 016 2 2146 068 2

5319 microgl 414 41 2 279 28 2 268 27 2 26 31 2

5321 microgkg 476 316 326 328

5322 microgl 2547 35 2

5323 microgkg 405 203 2 239 119 2 255 153 2 201 11 2

58

Figure 11 Youden plots of the percent deviation of the reported values from the assigned values for the food supplement test sample and the

standard solution in acetonitrile with undisclosed content

(The identity of the respective measurand is given above the figure Results exceeding a difference of more than 100 were omitted)

59

Figure 11 - continued

60

ANNEX 10 Table 18 Method performance parameters reported by the participants

Laboratory code

Analyte LOD LOQ Recovery Analyte LOD LOQ Recovery

microgkg microgkg microgkg microgkg 3600 BaA 03 06 978 BaP 03 07 9483602 BaA 007 021 926 BaP 005 015 993603 BaA 0011 05 987 BaP 0005 051 9553604 BaA 02 04 86 BaP 02 04 883605 BaA 0025 005 118 BaP 0025 005 1083606 BaA 001 003 65 BaP 001 003 703607 BaA 021 071 74 BaP 027 089 943608 BaA 016 048 8213 BaP 015 046 83683610 BaA 02 05 85 BaP 005 02 853611 BaA BaP 3612 BaA 1 117 BaP 1 1203613 BaA 01 03 90 BaP 01 03 903614 BaA 02 05 96 BaP 02 05 933615 BaA 005 015 110 BaP 005 015 1053616 BaA 01 05 108 BaP 01 05 753617 BaA 018 037 405 BaP 009 018 3873618 BaA 01 03 63 BaP 01 03 593619 BaA 005 005 59 BaP 033 033 543620 BaA 0009 0018 64 BaP 0005 001 553621 BaA 007 02 84 BaP 007 02 923622 BaA 01 03 1101 BaP 01 03 9853623 BaA 02 08 105 BaP 02 08 1013624 BaA BaP 01 033 8513625 BaA BaP 3626 BaA BaP 5301 BaA 003 01 102 BaP 003 01 1005302 BaA 01 03 78 BaP 01 03 865303 BaA 003 01 62 BaP 003 01 805304 BaA 1 1 70 BaP 1 1 955305 BaA 02 05 85 BaP 02 05 855306 BaA 03 1 699 BaP 03 1 905307 BaA 6537 BaP 81165308 BaA 0088 0265 99 BaP 00262 00785 1005309 BaA 01 08 92 BaP 01 08 925310 BaA 01 03 BaP 01 03 905311 BaA 002 005 80 BaP 002 004 915312 BaA 03 09 105 BaP 03 09 1055313 BaA 04 08 BaP 04 08 5314 BaA 001 002 96 BaP 001 002 9485315 BaA 01 03 50 - 100 BaP 01 03 50 - 100 5316 BaA 002 011 827 BaP 004 019 10445317 BaA 05 1 76 BaP 05 1 755318 BaA BaP 5319 BaA 009 029 951 BaP 011 041 995320 BaA 12 18 946 BaP 06 1 8345321 BaA 03 05 BaP 05 1 5322 BaA 1 2 100 BaP 1 2 1005323 BaA 26 BaP 26

61

ANNEX 10 Table 18 - continued

Laboratory code Analyte LOD LOQ Recovery Analyte LOD LOQ Recovery

microgkg microgkg microgkg microgkg 3600 BbF 03 05 96 CHR 02 03 10153602 BbF 015 045 947 CHR 003 009 9193603 BbF 0014 05 1012 CHR 0004 05 10033604 BbF 02 04 85 CHR 02 04 873605 BbF 005 01 94 CHR 0025 005 863606 BbF 001 003 70 CHR 001 003 653607 BbF 018 06 100 CHR 014 028 943608 BbF 015 046 7979 CHR 015 045 71253610 BbF 005 02 85 CHR 02 05 853611 BbF CHR 3612 BbF 1 118 CHR 1 1173613 BbF 01 03 90 CHR 01 03 903614 BbF 01 04 96 CHR 02 05 953615 BbF 008 025 109 CHR 003 01 1083616 BbF 01 05 78 CHR 01 05 723617 BbF 035 07 38 CHR 012 024 4163618 BbF 01 03 62 CHR 01 03 623619 BbF 016 016 63 CHR 01 01 593620 BbF 0003 0006 55 CHR 001 002 653621 BbF 007 02 103 CHR 007 02 893622 BbF 02 06 963 CHR 03 09 1043623 BbF 02 08 106 CHR 02 08 1193624 BbF CHR 3625 BbF CHR 3626 BbF CHR 5301 BbF 003 01 96 CHR 003 01 1145302 BbF 01 03 84 CHR 01 03 835303 BbF 003 01 78 CHR 003 01 705304 BbF 1 1 86 CHR 1 1 705305 BbF 02 05 90 CHR 02 05 955306 BbF 03 1 1435 CHR 03 1 1185307 BbF 8116 CHR 65375308 BbF 0079 0238 99 CHR 009 026 1005309 BbF 01 08 92 CHR 5310 BbF 07 2 CHR 01 03 5311 BbF 002 004 95 CHR 002 004 895312 BbF 03 09 105 CHR 03 09 1055313 BbF 04 08 CHR 04 08 5314 BbF 001 002 83 CHR 001 002 8185315 BbF 02 05 50 -100 CHR 02 05 50 - 100 5316 BbF 005 024 814 CHR 001 007 9595317 BbF 05 1 73 CHR 05 1 785318 BbF CHR 5319 BbF 011 038 946 CHR 013 043 9675320 BbF 07 09 1194 CHR 23 32 10455321 BbF 05 1 CHR 05 1 5322 BbF 1 2 100 CHR 1 2 1005323 BbF 26 CHR 26

62

ANNEX 11 Table 19 Details of analysis method reported by the participants Data are presented as reported

Laboratory code

Analysis technique

Instrument calibration

Internal standards applied

Sample clean up

Details of sample

clean up

Extraction technique

Details of sample

extraction

3600 6) GC-MS

2) Internal standardisation with labelled substances

Deuterated PAH mix 9 (Ehrenstorfer)

1) Saponification 2) LiquidLiquid partitioning

D) Other Liquidliquid partitioning

3602 2) HPLC-UV-FLD

1) External calibration

3) Gel permeation chromatography

A) Pressurised liquid extraction (PLE)

3603 1) HPLC-FLD

1) External calibration

3) Gel permeation chromatography

D) Other 1 minute with dichloromethane in vortex

3604 2) HPLC-UV-FLD

2) Internal standardisation with labelled substances

DiP D14

5) Donor acceptor complex chromatography

D) Other LL

3605 1) HPLC-FLD

1) External calibration

3) Gel permeation chromatography

C) Soxhlet extraction

3606 7) GC-MSMS

2) Internal standardisation with labelled substances

BaP 13C4 CHR 13C6 BaA 13C6 BbF 13C6

4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

3607 6) GC-MS

2) Internal standardisation with labelled substances

BaA-d12 CHR-d12 BbF-d12 BkF-d12 BaP-d12 ICP-d12 BgP-d12 DiP-d14

1) Saponification

D) Other solvent partitioning

3608 1) HPLC-FLD

1) External calibration

6) Other Liquid extraction

D) Other

Extraction in a rotary agitator and centrifugation

3610 2) HPLC-UV-FLD

1) External calibration

4) Solid phase extraction

B) Sonication

3611

3612 6) GC-MS

2) Internal standardisation with labelled substances

chrysene-d12 dibenzo(ah)anthracene-d14 dibenzo(ai)pyrene-d14

4) Solid phase extraction

B) Sonication

3613 5) LC-MSMS

3) Internal standardisation with unlabelled substances

I use Chrysene-D12 3) Gel permeation chromatography

D) Other extraction with hexan

63

Table 19 - continued

Laboratory code

Analysis technique

Instrument calibration

Internal standards applied

Sample clean up Details of sample

clean up

Extraction technique

Details of sample

extraction

3614 6) GC-MS 1) External calibration

3) Gel permeation chromatography

B) Sonication

3615 1) HPLC-FLD

1) External calibration

6) Other PLE GPC

A) Pressurised liquid extraction (PLE)

3616 6) GC-MS

2) Internal standardisation with labelled substances

Benzo(a)pyrene-13C4 Benzo(a)anthracene-13C6 Benzo(b)fluoranthene-13C6 Chrysene-13C6

2) LiquidLiquid partitioning 4) Solid phase extraction

B) Sonication

3617 1) HPLC-FLD

1) External calibration

4) Solid phase extraction

C) Soxhlet extraction

3618 6) GC-MS

2) Internal standardisation with labelled substances

deuterated standards

1) Saponification 4) Solid phase extraction

D) Other Handshaken with cyclohexane

3619 6) GC-MS

2) Internal standardisation with labelled substances

C13 Labelled US EPA 16

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

D) Other Homegenisation amp saponiofication

3620 9) GC-HRMS

2) Internal standardisation with labelled substances

mix of deuterated PAHs (BAA-D12 CHR-D12 BAP-D12 BBF-D12)

3) Gel permeation chromatography 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

3621 6) GC-MS

2) Internal standardisation with labelled substances

Chrysene D12 Benzo(a)pyrene D12 Benzo(b)fluoranthene D12 Benzo(a)anthracene

2) LiquidLiquid partitioning 4) Solid phase extraction

B) Sonication

3622 1) HPLC-FLD

4) Standard addition method

4) Solid phase extraction

D) Other

Extraction with cyclohexane by heating under reflux

3623 7) GC-MSMS

2) Internal standardisation with labelled substances

C13 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

3624 2) HPLC-UV-FLD

1) External calibration

2) LiquidLiquid partitioning

D) Other hot solvent extraction

3626

64

Table 19 - continued

Laboratory code

Analysis technique

Instrument calibration

Internal standards

applied

Sample clean up

Details of sample

clean up

Extraction technique

Details of sample

extraction

5301 7) GC-MSMS

2) Internal standardisation with labelled substances

EACH SUBSTANCE C13

4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5302 7) GC-MSMS

2) Internal standardisation with labelled substances

Isotope labelled PAHs 13C for each analyte

4) Solid phase extraction

C) Soxhlet extraction

5303 7) GC-MSMS

3) Internal standardisation with unlabelled substances

HAP C13 4) Solid phase extraction

D) Other automate ASE200 DIONEX

5304 7) GC-MSMS

2) Internal standardisation with labelled substances

with C13 for each HAP

2) LiquidLiquid partitioning 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5305 7) GC-MSMS

2) Internal standardisation with labelled substances

C13 isotopiques internal standards

4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5306 7) GC-MSMS

2) Internal standardisation with labelled substances

C13 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5307 6) GC-MS

2) Internal standardisation with labelled substances

1) Saponification 2) LiquidLiquid partitioning

D) Other solvent extraction

5308 1) HPLC-FLD 1) External calibration

4) Solid phase extraction

B) Sonication

5309 1) HPLC-FLD 1) External calibration

4) Solid phase extraction

D) Other only SPE

5310 1) HPLC-FLD 1) External calibration

1) Saponification 2) LiquidLiquid partitioning 3) Gel permeation chromatography

C) Soxhlet extraction

5311 6) GC-MS

2) Internal standardisation with labelled substances

13 C Chrysene 13 C Benz(a)pyrene

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

B) Sonication

5312 1) HPLC-FLD

3) Internal standardisation with unlabelled substances

Benzo(b)chrysen

2) LiquidLiquid partitioning 4) Solid phase extraction

Quechers D) Other Quechers

65

Table 19 - continued

Laboratory code

Analysis technique

Instrument calibration

Internal standards applied

Sample clean up Details of sample

clean up

Extraction technique

Details of sample extraction

5313 6) GC-MS

2) Internal standardisation with labelled substances

Benz(a)anthracene D12 Benzo(b)fluranthene D12 Benzo(a)pyrene D12 Chrysene D12

1) Saponification 2) LiquidLiquid partitioning 3) Gel permeation chromatography

A) Pressurised liquid extraction (PLE)

5314 6) GC-MS

2) Internal standardisation with labelled substances

Benzo(a)pyren d12 2) LiquidLiquid partitioning

D) Other Extraction with

5315 1) HPLC-FLD

3) Internal standardisation with unlabelled substances

Benz(b)chrysen

1) Saponification 2) LiquidLiquid partitioning

D) Other shaker

5316 1) HPLC-FLD

1) External calibration

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

D) Other

LiquidLiquid extraction with cyclohexane and dimethylformamide

5317 7) GC-MSMS

1) External calibration

3) Gel permeation chromatography 6) Other

silica gel D) Other VDLUFA online method

5318

5319 1) HPLC-FLD

3) Internal standardisation with unlabelled substances

Benzo(b)chrysene 3) Gel permeation chromatography

A) Pressurised liquid extraction (PLE)

5320 6) GC-MS

3) Internal standardisation with unlabelled substances

22-binaphthyl

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

D) Other 3 hours refluxing

5321 1) HPLC-FLD

2) Internal standardisation with labelled substances

6-meacutethylchrysegravene

1) Saponification 2) LiquidLiquid partitioning

B) Sonication

5322 6) GC-MS

2) Internal standardisation with labelled substances

naftalene-d8 acenaphtalene-d10 phenanthrene-d10 chrysene-d12 perylene-d12 benzo(e)anthracene-d12

1) Saponification 2) LiquidLiquid partitioning 6) Other

clean-up with silica packed column

C) Soxhlet extraction

5323 1) HPLC-FLD

1) External calibration

4) Solid phase extraction 6) Other

Accelerated Solvent Extraction

A) Pressurised liquid extraction (PLE)

66

European Commission EUR 25245 EN ndash Joint Research Centre ndash Institute for Reference Materials and Measurements Title Report on the 8th inter-laboratory comparison organised by the European Union Reference Laboratory for Polycyclic Aromatic Hydrocarbons - Four marker PAHs in a dry extract of St Johns wort Author(s) Radoslav Lizak Szilard Szilagyi Philippe Verlinde and Thomas WENZL Luxembourg Publications Office of the European Union 2012 ndash 66 pp ndash 201 x 297 cm EUR ndash Scientific and Technical Research series ndash ISSN 1831-9424 ISBN 978-92-79-23337-1 doi 10278759387 Abstract The proficiency test here reported concerned the determination of the contents of four marker polycyclic aromatic hydrocarbons (PAHs) and of their sum in an food supplements test sample The set of marker PAHs consists of benz[a]anthacene benzo[a]pyrene benzo[b]fluoranthene and chrysene Participants to these PT were National Reference Laboratories for PAHs (NRLs-PAHs) and EU official food control laboratories The number of participants was in total 48

The PT was organised according ISO Standard 170432010

The test material used was a commercial dry extract of St Johns wort Participants also received a solution of the same PAHs either in acetonitrile or in toluene for checking their instrument calibration

The results from participants were rated with z-scores and zeta-scores About 68 of the reported results were attributed with z-scores with an absolute value of below two which is the threshold for satisfactory performance

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interests whether private or national

L

A-N

A-252

45-EN

-N

• 1 Summary
• 2 Introduction
• 3 Scope
• 4 Participating Laboratories
• 5 Time frame
• 6 Confidentiality
• 7 Test materials
• • 71 1Preparation of test materials
  • 72 Assigned values and standard deviation for proficiency assessment
  • 73 Homogeneity and stability
  • 74 Sample dispatch
  • • 8 Design of the proficiency test
    • 9 Evaluation of the results
    • • 91 2General
      • 92 21BEvaluation criteria
      • 93 Performance of participants
      • 94 Evaluation of the influence of analysis method
      • 95 Evaluation of the influence of calibration
      • 96 26Evaluation of compliance with legislation
      • • 10 Follow-up actions for underperforming laboratories
        • 11 1Conclusions
        • 12 1Acknowledgements
        • 13 References

39

ANNEX 5 - continued

Stability of the test material

Table 10 Content of the four target analytes in test materials stored under different

temperatures

BaA BaP BbF CHR

microgkg microgkg microgkg microgkg

Content 31 16 31 69 Storage at room temperature

U (k=2) 05 02 05 17

Content 31 15 29 68 Storage at reference temperature

U (k=2) 04 03 04 10

The applied capillary column does not separate CHR from triphenylene

40

ANNEX 6 ndash Documents sent to participants

41

ANNEX 6 - continued

42

ANNEX 7 Questionnaire on details of analysis method

43

ANNEX 7 - continued

44

ANNEX 7 - continued

45

ANNEX 8 - Results reported by participants for the food supplements test sample Table 11 Analysis results reported by the participants for the content of benz[a]anthracene (BAA) in the food supplements test material Values are presented as reported

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

3600 31 29 29 3 05 2

3602 199 193 178 19 021

3603 253 253 246 251 022 2

3604 199 209 23 213 071 2

3605 301 3 271 291 076 2

3606 302 288 278 289 064 2

3607 456 416 443 438 096 2

3608 796 695 651 714 149 2

3610 182 192 214 196 039 2

3611 414 504 488 469 96 2

3612 3 34 28 31 06 2

3613 32 33 34 33 07 2

3614 182 182 191 185 037 2

3615 3884 3822 3565 376 0752 2

3616 2 2 2 2 01 2

3617 366 37 35 362 046 2

3618 32 32 32 32 05 2

3619 299 305 297 299 048 2

3620 304 294 302 302 045 2

3621 29 26 29 28 04 2

3622 343 325 318 322 052 2

3623 35 3 33 33 07 2

3624

3626 318 31 314 314 126 2

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

5301 31 3 32 31 06 2

5302 314 319 315 316 063 2

5303 3102 2716 2822 2876 27 62

5304 261 263 26 261 57 2

5305 23 22 26 24 20

5306 22 23 23 22 11 2

5307 488 461 317 461

5308 168633 168544 1562025 1644598

5309 16 19 16 17

5310 3 304 311 305 015

5311 477 452 448 459 15 2

5312 506 63 494 543 134 2

5313 353 301 404 353

5314 431 373 378 394 5 096

5315 596 531 51 545 11 2

5316 224 231 235 235 022 2

5317 216 23 221 222 007 2

5318 064 07 067 01 2

5319 111 184 146 147 029 2

5320 515 525 564 534 48

5321 21 5322 13 87 9 87 35 2

5323 38 33 35 33 16 2

46

Figure 6 Results of replicate determinations (indicated by triangles) of benz[a]anthracene (BAA) in the food supplement test material

Horizontal blue lines represent the arithmetic mean value of replicate measurements and blue bars the reported expanded measurement uncertainty (k=2) The assigned value is

plotted as dotted line Red solid lines mark the range of satisfactory performance (|z|le2) Mean values are given for results outside the displayed data range

ProLab 2011

Laboratory

53

1853

1953

0853

0936

1436

0236

1036

1653

2136

0453

1753

0653

1653

0536

0353

0436

2153

0336

0636

0536

0036

2036

1953

1036

1253

0136

2653

0236

1836

2336

2236

1353

1353

2336

1736

1553

1453

0736

0753

1153

2053

1253

1536

0853

2236

11

an

aly

te c

on

ten

t (micro

gk

g)

10

95

9

85

8

75

7

65

6

55

5

45

4

35

3

25

2

15

1

05

0

102

3 468

7

Sample St Johns wort extract replicatesMeasurand Benz[a]anthraceneNo of laboratories 46

Assigned value 298 microgkg (Reference value)Target sd 062 microgkgTolerance limits 174 - 422 microgkg (|Z score| lt 200)

Assig

ned va

lue

Limit of tolerance

Limit of tolerance

47

Table 12 Analysis results reported by the participants for the content of benzo[a]pyrene (BAP) in the food supplements test material Values are presented as reported

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

3600 16 17 17 17 03 2

3602 118 123 13 124 015

3603 165 162 157 161 015 2

3604 134 154 139 143 04 2

3605 164 166 172 167 057 2

3606 181 159 161 167 031 2

3607 279 266 279 274 055 2

3608 57 687 461 573 226 2

3610 226 216 183 206 042 2

3611 289 325 311 308 36 2

3612 24 22 2 22 05 2

3613 17 18 19 18 05 2

3614 08 08 08 08 016 2

3615 1545 1393 1425 145 029 2

3616 16 16 14 15 03 2

3617 177 171 158 169 023 2

3618 2 2 2 2 02 2

3619 207 207 206 207 075 2

3620 165 169 163 166 017 2

3621 44 47 49 47 05 2

3622 212 208 222 213 038 2

3623 2 21 22 21 03 2

3624 18 178 182 18 031 2

3626 172 173 178 174 07 2

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

5301 18 18 18 18 04 2

5302 188 19 187 188 038 2

5303 1691 1426 149 1535 19 80

5304 145 142 15 146 69 2

5305 15 17 18 16 15

5306 16 17 17 16 08 2

5307 164 157 153 157

5308 102669 109324 109035 107009

5309 1 1 12 11

5310 122 122 124 123 006

5311 199 184 215 2 15 2

5312 258 248 239 248 05 2

5313 199 178 245 207

5314 174 171 172 172 11 0948

5315 253 248 213 238 05 2

5316 177 171 173 17 016 2

5317 255 277 276 269 012 2

5318 114 12 117 02 2

5319 19 228 231 216 043 2

5320 303 3 344 316 78

5321 12 12 12 12

5322 13 17 24 17 35 2

5323 2 2 19 2 1 2

48

Figure 7 Results of replicate determinations (indicated by triangles) of benzo[a]pyrene (BAP) in the food supplement test material

Horizontal blue lines represent the arithmetic mean value of replicate measurements and blue bars the reported expanded measurement uncertainty (k=2) The assigned value is

plotted as dotted line Red solid lines mark the range of satisfactory performance (|z|le2) Mean values are given for results outside the displayed data range

ProLab 2011

Laboratory

36

1453

0953

0853

1853

2153

1036

0236

0436

1553

0436

1653

0353

0736

0336

2036

0053

0553

0636

0636

0536

1753

1453

1636

2636

1336

2453

0153

2253

0253

2353

1136

1836

1953

1336

1036

2336

2253

1936

1253

1553

1253

1736

0753

2036

2136

0836

11

an

aly

te c

on

ten

t (micro

gk

g)

55

5

45

4

35

3

25

2

15

1

05

0

573

308

3

Sample St Johns wort extract replicatesMeasurand Benzo[a]pyreneNo of laboratories 48

Assigned value 165 microgkg (Reference value)Target sd 036 microgkgTolerance limits 093 - 237 microgkg (|Z score| lt 200)

Assig

ned va

lue

Limit of tolerance

Limit of tolerance

49

Table 13 Analysis results reported by the participants for the content of benzo[b]fluoranthene (BBF) in the food supplements test material Values are presented as reported

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

3600 32 3 3 31 09 2

3602 173 175 16 17 017

3603 265 252 25 256 025 2

3604 116 108 105 11 028 2

3605 286 225 232 247 074 2

3606 294 273 261 276 046 2

3607 579 579 544 567 113 2

3608 103 1201 876 1036 326 2

3610 173 196 204 191 038 2

3611 546 618 565 576 75 2

3612 3 28 28 29 06 2

3613 28 31 32 31 06 2

3614 135 111 135 127 022 2

3615 4187 3678 4111 4 08 2

3616 2 2 2 2 01 2

3617 33 27 299 035 2

3618 31 31 31 31 05 2

3619 326 324 312 326 064 2

3620 277 28 272 276 069 2

3621 26 25 23 25 04 2

3622 346 336 333 34 052 2

3623 26 28 29 28 02 2

3624

3626 288 28 307 292 117 2

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

5301 29 3 29 29 06 2

5302 297 3 296 298 06 2

5303 2705 2444 2608 2586 22 78

5304 245 254 26 253 85 2

5305 23 23 26 24 15

5306 42 43 4 42 21 2

5307 221 216 241 221

5308 232169 246654 240722 229848

5309 19 22 19 2 0

5310 3 315 325 313 016

5311 262 277 304 281 15 2

5312 27 289 337 299 075 2

5313 321 284 378 328

5314 423 404 385 404 34 083

5315 42 506 416 444 09 2

5316 265 267 277 273 018 2

5317 185 197 195 192 006 2

5318 276 281 279 04 2

5319 396 389 403 396 079 2

5320 623 618 635 625 14

5321 31

5322 24 33 49 33 35 2

5323 38 45 35 45 27 2

50

Figure 8 Results of replicate determinations (indicated by triangles) of benzo[b]fluoranthene (BBF) in the food supplement test material

Horizontal blue lines represent the arithmetic mean value of replicate measurements and blue bars the reported expanded measurement uncertainty (k=2) The assigned value is

plotted as dotted line Red solid lines mark the range of satisfactory performance (|z|le2) Mean values are given for results outside the displayed data range

ProLab 2011

Laboratory

36

0436

1436

0236

1053

1736

1653

0953

0753

0853

0536

2136

0553

0436

0353

0353

1636

0636

2036

2353

1853

1136

1236

2653

0153

0253

1236

1736

1336

0036

1853

2153

1036

1953

1336

2253

2253

2353

1936

1553

1453

0653

1536

0753

2036

0836

11

an

aly

te c

on

ten

t (micro

gk

g)

75

7

65

6

55

5

45

4

35

3

25

2

15

1

05

0

103

6

576

3

Sample St Johns wort extract replicatesMeasurand Benzo[b]fluorantheneNo of laboratories 46

Assigned value 292 microgkg (Reference value)Target sd 060 microgkgTolerance limits 172 - 412 microgkg (|Z score| lt 200)

Assig

ned va

lue

Limit of tolerance

Limit of tolerance

51

Table 14 Analysis results reported by the participants for the content of chrysene (CHR) in the food supplements test material Values are presented as reported

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

3600 5 49 5 5 07 2

3602 32 304 29 304 036

3603 462 469 447 459 048 2

3604 438 428 42 429 119 2

3605 365 41 381 386 085 2

3606 494 411 45 451 124 2

3607 897 774 787 819 18 2

3608 986 887 1168 1014 284 2

3610 418 392 462 424 085 2

3611 833 893 87 865 61 2

3612 6 64 58 61 12 2

3613 41 39 4 4 08 2

3614 272 254 288 271 054 2

3615 605 6028 5964 601 12 2

3616 47 45 49 47 05 2

3617 445 431 389 422 065 2

3618 45 45 45 45 06 2

3619 738 747 72 738 118 2

3620 463 472 446 46 092 2

3621 53 49 51 51 06 2

3622 478 513 505 488 08 2

3623 73 73 72 72 05 2

3624

3626 63 622 634 628 251 2

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

5301 49 49 49 49 1 2

5302 687 683 666 679 136 2

5303 6777 6181 6382 6441 24 70

5304 592 581 6 591 85 2

5305 6 58 64 61 20

5306 49 5 51 49 25 2

5307 524 5 574 524

5308 211488 216782 221146 213243

5309

5310 372 393 394 386 019

5311 43 459 422 437 15 2

5312 934 943 981 953 191 2

5313 793 73 99 838

5314 636 561 565 588 18 0818

5315 547 583 499 542 11 2

5316 292 286 285 282 06 2

5317 52 598 517 545 046 2

5318 368 345 357 05 2

5319 449 471 474 465 116 2

5320 76 924 818 834 99

5321 33

5322 7 7 76 7 35 2

5323 87 73 77 73 37 2

52

Figure 9 Results of replicate determinations (indicated by triangles) of chrysene (CHR) in the food supplement test material

Horizontal blue lines represent the arithmetic mean value of replicate measurements and blue bars the reported expanded measurement uncertainty (k=2) The assigned value is

plotted as dotted line Red solid lines mark the range of satisfactory performance (|z|le2) Mean values are given for results outside the displayed data range

ProLab 2011

Laboratory

53

0836

1453

1636

0253

2153

1836

0553

1036

1336

1736

1036

0453

1136

1836

0636

0336

2053

1936

1653

0136

0036

2253

0636

2153

0753

1553

1753

1453

0436

1536

1253

0536

2653

0353

0253

2236

2336

1953

2336

0753

2053

1353

1236

0836

11

an

aly

te c

on

ten

t (micro

gk

g)

13

12

11

10

9

8

7

6

5

4

3

2

1

0

865

3

Sample St Johns wort extract replicatesMeasurand ChryseneNo of laboratories 45

Assigned value 407 microgkg (Reference value)Target sd 083 microgkgTolerance limits 241 - 573 microgkg (|Z score| lt 200)

Assig

ned va

lue

Limit of tolerance

Limit of tolerance

53

Table 15 Analysis results reported by the participants for the sum of the four marker PAHs in the food supplements test material Values are presented as reported

Laboratory code

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg

3600 128 24 2

3602 79 126

3603 1127 096 2

3604 893 258 2

3605 1091 305 2

3606 1184 238 2

3607 2098 42 2

3608 3336 325 2

3610 1138 17 2

3611 2218 259 2

3612 143 29 2

3613 122 2 2

3614 663 129 2

3615 152 304 2

3616 102 03 2

3617 1252 169 2

3618 128 18 2

3619 157 305 2

3620 1204 12 2

3621 151 19 2

3622 1361 115 2

3623 154 09 2

3624

3626 1409 56 2

Laboratory code

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg

5301 126

5302 37 074 2

5303 13438 92

5304 1251 85 2

5305 125 20

5306 129 65 2

5307 1363

5308 7245598

5309

5310 1127 056

5311 1367 15 2

5312 2042 408 2

5313 1726

5314 1558

5315 177 35 2

5316 96 073 2

5317 1229 066 2

5318 82 05 2

5319 1225 267 2

5320 2309 41

5321

5322 207 35 2

5323 171 103 2

54

ANNEX 9 Results reported for the standard solutions with undisclosed analyte content Table 16 Analysis results reported by the participants for the four marker PAHs in the standard solution in toluene with undisclosed analyte

content Values are presented as reported U measurement uncertainty k coverage factor

benz[a]anthracene benzo[a]pyrene benzo[b]fluoranthene chrysene Laboratory

code Unit Result U k Result U k Result U k Result U k 3600 microgl 38 67 2 25 45 2 211 68 2 275 38 2

3605 microgl 367 184 2 2482 124 2 2592 13 2 235 118 2

3606 microgkg 3763 176 2 2609 142 2 2721 118 2 2539 134 2

3607 microgkg 3706 815 2 2824 565 2 2591 518 2 2587 569 2

3612 microgl 38 08 2 237 11 2 244 18 2 257 02 2

3614 microgl 324 65 2 209 38 2 215 37 2 21 42 2

3616 microgkg 318 07 2 224 14 2 231 26 2 216 1 2

3618 microgkg 417 17 2 284 08 2 292 06 2 297 03 2

3619 microgkg 47 742 2 274 468 2 228 387 2 274 434 2

3620 microgl 495 74 2 294 29 2 321 8 2 321 64 2

3621 microgl 241 22 2 143 13 2 188 16 2 145 14 2

3623 microgkg 476 25 2 311 53 2 269 3 2 297 03 2

3626 microgl 359 38 2 235 23 2 238 27 2 24 18 2

5301 microgkg 426 29 299 289

5302 microgkg 436 87 2 296 59 2 296 59 2 289 58 2

5303 microgl 3721 27 102 22838 19 99 23756 22 99 25486 24 98

5304 microgkg 38 29 2 225 26 2 257 37 2 258 52 2

5305 microgkg 305 225 245 29

5306 microgkg 316 63 2 247 49 2 65 13 2 22 44 2

5307 microgkg 3658 2559 241 2414

5308 microgkg 16854 109324 246654 216782

5311 microgl 374 20 2 279 15 2 268 15 2 272 15 2

5313 microgl 3408 2165 2275 2373

5314 microgkg 395 5 2875 11 3614 34 2905 18

5320 microgl 3267 368 1986 571 28 683 2427 512

5322 microgl 282 35 2 225 35 2 241 35 2 21 35 2

55

Figure 10 Youden plots of the percent deviation of the reported values from the assigned values for the food supplement test sample and the

standard solution in toluene with undisclosed content

(The identity of the respective measurand is given above the figure Results exceeding a difference of more than 100 were omitted)

56

Figure 10 - continued

57

Table 17 Analysis results reported by the participants for the four marker PAHs in the standard solution in acetonitrile with undisclosed

analyte content

Values are presented as reported U measurement uncertainty k coverage factor

benz[a]anthracene benzo[a]pyrene benzo[b]fluoranthene chrysene

Laboratory

code Unit Result U k Result U k Result U k Result U k

3602 microgkg 3675 247 247 244

3603 microgkg 358 086 2 236 028 2 235 028 2 236 057 2

3604 microgl 475 29 2 19 12 2 194 13 2 258 12 2

3608 microgl 3623 078 2 2385 023 2 2343 18 2 2296 092 2

3610 microgkg 369 185 2 25 125 2 251 126 2 244 122 2

3611 microgkg 48 02 2 323 02 2 328 04 2 317 03 2

3613 microgl 332 31 2 261 3 2 249 25 2 239 24 2

3615 microgl 415 2635 2729 2689

3617 microgkg 37 09 2 244 06 2 284 07 2 239 06 2

3622 microgl 37 12 2 25 23 2 257 21 2 238 1 2

3624 microgkg 402 104 2 255 084 2 265 096 2 253 07 2

5306 microgkg 563 113 2 371 74 2 359 72 2 333 67 2

5308 microgkg 168633 102669 232169 211488

5309 microgl 402 268 27

5310 microgl 315 158 25 125 25 125 288 144

5312 microgl 428 043 2 282 028 2 272 027 2 275 028 2

5315 microgkg 387 08 2 256 05 2 251 05 2 253 05 2

5316 microgl 3839 342 2 2485 191 2 2559 168 2 239 51 2

5317 microgl 2466 043 2 2212 034 2 2472 016 2 2146 068 2

5319 microgl 414 41 2 279 28 2 268 27 2 26 31 2

5321 microgkg 476 316 326 328

5322 microgl 2547 35 2

5323 microgkg 405 203 2 239 119 2 255 153 2 201 11 2

58

Figure 11 Youden plots of the percent deviation of the reported values from the assigned values for the food supplement test sample and the

standard solution in acetonitrile with undisclosed content

(The identity of the respective measurand is given above the figure Results exceeding a difference of more than 100 were omitted)

59

Figure 11 - continued

60

ANNEX 10 Table 18 Method performance parameters reported by the participants

Laboratory code

Analyte LOD LOQ Recovery Analyte LOD LOQ Recovery

microgkg microgkg microgkg microgkg 3600 BaA 03 06 978 BaP 03 07 9483602 BaA 007 021 926 BaP 005 015 993603 BaA 0011 05 987 BaP 0005 051 9553604 BaA 02 04 86 BaP 02 04 883605 BaA 0025 005 118 BaP 0025 005 1083606 BaA 001 003 65 BaP 001 003 703607 BaA 021 071 74 BaP 027 089 943608 BaA 016 048 8213 BaP 015 046 83683610 BaA 02 05 85 BaP 005 02 853611 BaA BaP 3612 BaA 1 117 BaP 1 1203613 BaA 01 03 90 BaP 01 03 903614 BaA 02 05 96 BaP 02 05 933615 BaA 005 015 110 BaP 005 015 1053616 BaA 01 05 108 BaP 01 05 753617 BaA 018 037 405 BaP 009 018 3873618 BaA 01 03 63 BaP 01 03 593619 BaA 005 005 59 BaP 033 033 543620 BaA 0009 0018 64 BaP 0005 001 553621 BaA 007 02 84 BaP 007 02 923622 BaA 01 03 1101 BaP 01 03 9853623 BaA 02 08 105 BaP 02 08 1013624 BaA BaP 01 033 8513625 BaA BaP 3626 BaA BaP 5301 BaA 003 01 102 BaP 003 01 1005302 BaA 01 03 78 BaP 01 03 865303 BaA 003 01 62 BaP 003 01 805304 BaA 1 1 70 BaP 1 1 955305 BaA 02 05 85 BaP 02 05 855306 BaA 03 1 699 BaP 03 1 905307 BaA 6537 BaP 81165308 BaA 0088 0265 99 BaP 00262 00785 1005309 BaA 01 08 92 BaP 01 08 925310 BaA 01 03 BaP 01 03 905311 BaA 002 005 80 BaP 002 004 915312 BaA 03 09 105 BaP 03 09 1055313 BaA 04 08 BaP 04 08 5314 BaA 001 002 96 BaP 001 002 9485315 BaA 01 03 50 - 100 BaP 01 03 50 - 100 5316 BaA 002 011 827 BaP 004 019 10445317 BaA 05 1 76 BaP 05 1 755318 BaA BaP 5319 BaA 009 029 951 BaP 011 041 995320 BaA 12 18 946 BaP 06 1 8345321 BaA 03 05 BaP 05 1 5322 BaA 1 2 100 BaP 1 2 1005323 BaA 26 BaP 26

61

ANNEX 10 Table 18 - continued

Laboratory code Analyte LOD LOQ Recovery Analyte LOD LOQ Recovery

microgkg microgkg microgkg microgkg 3600 BbF 03 05 96 CHR 02 03 10153602 BbF 015 045 947 CHR 003 009 9193603 BbF 0014 05 1012 CHR 0004 05 10033604 BbF 02 04 85 CHR 02 04 873605 BbF 005 01 94 CHR 0025 005 863606 BbF 001 003 70 CHR 001 003 653607 BbF 018 06 100 CHR 014 028 943608 BbF 015 046 7979 CHR 015 045 71253610 BbF 005 02 85 CHR 02 05 853611 BbF CHR 3612 BbF 1 118 CHR 1 1173613 BbF 01 03 90 CHR 01 03 903614 BbF 01 04 96 CHR 02 05 953615 BbF 008 025 109 CHR 003 01 1083616 BbF 01 05 78 CHR 01 05 723617 BbF 035 07 38 CHR 012 024 4163618 BbF 01 03 62 CHR 01 03 623619 BbF 016 016 63 CHR 01 01 593620 BbF 0003 0006 55 CHR 001 002 653621 BbF 007 02 103 CHR 007 02 893622 BbF 02 06 963 CHR 03 09 1043623 BbF 02 08 106 CHR 02 08 1193624 BbF CHR 3625 BbF CHR 3626 BbF CHR 5301 BbF 003 01 96 CHR 003 01 1145302 BbF 01 03 84 CHR 01 03 835303 BbF 003 01 78 CHR 003 01 705304 BbF 1 1 86 CHR 1 1 705305 BbF 02 05 90 CHR 02 05 955306 BbF 03 1 1435 CHR 03 1 1185307 BbF 8116 CHR 65375308 BbF 0079 0238 99 CHR 009 026 1005309 BbF 01 08 92 CHR 5310 BbF 07 2 CHR 01 03 5311 BbF 002 004 95 CHR 002 004 895312 BbF 03 09 105 CHR 03 09 1055313 BbF 04 08 CHR 04 08 5314 BbF 001 002 83 CHR 001 002 8185315 BbF 02 05 50 -100 CHR 02 05 50 - 100 5316 BbF 005 024 814 CHR 001 007 9595317 BbF 05 1 73 CHR 05 1 785318 BbF CHR 5319 BbF 011 038 946 CHR 013 043 9675320 BbF 07 09 1194 CHR 23 32 10455321 BbF 05 1 CHR 05 1 5322 BbF 1 2 100 CHR 1 2 1005323 BbF 26 CHR 26

62

ANNEX 11 Table 19 Details of analysis method reported by the participants Data are presented as reported

Laboratory code

Analysis technique

Instrument calibration

Internal standards applied

Sample clean up

Details of sample

clean up

Extraction technique

Details of sample

extraction

3600 6) GC-MS

2) Internal standardisation with labelled substances

Deuterated PAH mix 9 (Ehrenstorfer)

1) Saponification 2) LiquidLiquid partitioning

D) Other Liquidliquid partitioning

3602 2) HPLC-UV-FLD

1) External calibration

3) Gel permeation chromatography

A) Pressurised liquid extraction (PLE)

3603 1) HPLC-FLD

1) External calibration

3) Gel permeation chromatography

D) Other 1 minute with dichloromethane in vortex

3604 2) HPLC-UV-FLD

2) Internal standardisation with labelled substances

DiP D14

5) Donor acceptor complex chromatography

D) Other LL

3605 1) HPLC-FLD

1) External calibration

3) Gel permeation chromatography

C) Soxhlet extraction

3606 7) GC-MSMS

2) Internal standardisation with labelled substances

BaP 13C4 CHR 13C6 BaA 13C6 BbF 13C6

4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

3607 6) GC-MS

2) Internal standardisation with labelled substances

BaA-d12 CHR-d12 BbF-d12 BkF-d12 BaP-d12 ICP-d12 BgP-d12 DiP-d14

1) Saponification

D) Other solvent partitioning

3608 1) HPLC-FLD

1) External calibration

6) Other Liquid extraction

D) Other

Extraction in a rotary agitator and centrifugation

3610 2) HPLC-UV-FLD

1) External calibration

4) Solid phase extraction

B) Sonication

3611

3612 6) GC-MS

2) Internal standardisation with labelled substances

chrysene-d12 dibenzo(ah)anthracene-d14 dibenzo(ai)pyrene-d14

4) Solid phase extraction

B) Sonication

3613 5) LC-MSMS

3) Internal standardisation with unlabelled substances

I use Chrysene-D12 3) Gel permeation chromatography

D) Other extraction with hexan

63

Table 19 - continued

Laboratory code

Analysis technique

Instrument calibration

Internal standards applied

Sample clean up Details of sample

clean up

Extraction technique

Details of sample

extraction

3614 6) GC-MS 1) External calibration

3) Gel permeation chromatography

B) Sonication

3615 1) HPLC-FLD

1) External calibration

6) Other PLE GPC

A) Pressurised liquid extraction (PLE)

3616 6) GC-MS

2) Internal standardisation with labelled substances

Benzo(a)pyrene-13C4 Benzo(a)anthracene-13C6 Benzo(b)fluoranthene-13C6 Chrysene-13C6

2) LiquidLiquid partitioning 4) Solid phase extraction

B) Sonication

3617 1) HPLC-FLD

1) External calibration

4) Solid phase extraction

C) Soxhlet extraction

3618 6) GC-MS

2) Internal standardisation with labelled substances

deuterated standards

1) Saponification 4) Solid phase extraction

D) Other Handshaken with cyclohexane

3619 6) GC-MS

2) Internal standardisation with labelled substances

C13 Labelled US EPA 16

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

D) Other Homegenisation amp saponiofication

3620 9) GC-HRMS

2) Internal standardisation with labelled substances

mix of deuterated PAHs (BAA-D12 CHR-D12 BAP-D12 BBF-D12)

3) Gel permeation chromatography 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

3621 6) GC-MS

2) Internal standardisation with labelled substances

Chrysene D12 Benzo(a)pyrene D12 Benzo(b)fluoranthene D12 Benzo(a)anthracene

2) LiquidLiquid partitioning 4) Solid phase extraction

B) Sonication

3622 1) HPLC-FLD

4) Standard addition method

4) Solid phase extraction

D) Other

Extraction with cyclohexane by heating under reflux

3623 7) GC-MSMS

2) Internal standardisation with labelled substances

C13 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

3624 2) HPLC-UV-FLD

1) External calibration

2) LiquidLiquid partitioning

D) Other hot solvent extraction

3626

64

Table 19 - continued

Laboratory code

Analysis technique

Instrument calibration

Internal standards

applied

Sample clean up

Details of sample

clean up

Extraction technique

Details of sample

extraction

5301 7) GC-MSMS

2) Internal standardisation with labelled substances

EACH SUBSTANCE C13

4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5302 7) GC-MSMS

2) Internal standardisation with labelled substances

Isotope labelled PAHs 13C for each analyte

4) Solid phase extraction

C) Soxhlet extraction

5303 7) GC-MSMS

3) Internal standardisation with unlabelled substances

HAP C13 4) Solid phase extraction

D) Other automate ASE200 DIONEX

5304 7) GC-MSMS

2) Internal standardisation with labelled substances

with C13 for each HAP

2) LiquidLiquid partitioning 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5305 7) GC-MSMS

2) Internal standardisation with labelled substances

C13 isotopiques internal standards

4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5306 7) GC-MSMS

2) Internal standardisation with labelled substances

C13 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5307 6) GC-MS

2) Internal standardisation with labelled substances

1) Saponification 2) LiquidLiquid partitioning

D) Other solvent extraction

5308 1) HPLC-FLD 1) External calibration

4) Solid phase extraction

B) Sonication

5309 1) HPLC-FLD 1) External calibration

4) Solid phase extraction

D) Other only SPE

5310 1) HPLC-FLD 1) External calibration

1) Saponification 2) LiquidLiquid partitioning 3) Gel permeation chromatography

C) Soxhlet extraction

5311 6) GC-MS

2) Internal standardisation with labelled substances

13 C Chrysene 13 C Benz(a)pyrene

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

B) Sonication

5312 1) HPLC-FLD

3) Internal standardisation with unlabelled substances

Benzo(b)chrysen

2) LiquidLiquid partitioning 4) Solid phase extraction

Quechers D) Other Quechers

65

Table 19 - continued

Laboratory code

Analysis technique

Instrument calibration

Internal standards applied

Sample clean up Details of sample

clean up

Extraction technique

Details of sample extraction

5313 6) GC-MS

2) Internal standardisation with labelled substances

Benz(a)anthracene D12 Benzo(b)fluranthene D12 Benzo(a)pyrene D12 Chrysene D12

1) Saponification 2) LiquidLiquid partitioning 3) Gel permeation chromatography

A) Pressurised liquid extraction (PLE)

5314 6) GC-MS

2) Internal standardisation with labelled substances

Benzo(a)pyren d12 2) LiquidLiquid partitioning

D) Other Extraction with

5315 1) HPLC-FLD

3) Internal standardisation with unlabelled substances

Benz(b)chrysen

1) Saponification 2) LiquidLiquid partitioning

D) Other shaker

5316 1) HPLC-FLD

1) External calibration

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

D) Other

LiquidLiquid extraction with cyclohexane and dimethylformamide

5317 7) GC-MSMS

1) External calibration

3) Gel permeation chromatography 6) Other

silica gel D) Other VDLUFA online method

5318

5319 1) HPLC-FLD

3) Internal standardisation with unlabelled substances

Benzo(b)chrysene 3) Gel permeation chromatography

A) Pressurised liquid extraction (PLE)

5320 6) GC-MS

3) Internal standardisation with unlabelled substances

22-binaphthyl

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

D) Other 3 hours refluxing

5321 1) HPLC-FLD

2) Internal standardisation with labelled substances

6-meacutethylchrysegravene

1) Saponification 2) LiquidLiquid partitioning

B) Sonication

5322 6) GC-MS

2) Internal standardisation with labelled substances

naftalene-d8 acenaphtalene-d10 phenanthrene-d10 chrysene-d12 perylene-d12 benzo(e)anthracene-d12

1) Saponification 2) LiquidLiquid partitioning 6) Other

clean-up with silica packed column

C) Soxhlet extraction

5323 1) HPLC-FLD

1) External calibration

4) Solid phase extraction 6) Other

Accelerated Solvent Extraction

A) Pressurised liquid extraction (PLE)

66

European Commission EUR 25245 EN ndash Joint Research Centre ndash Institute for Reference Materials and Measurements Title Report on the 8th inter-laboratory comparison organised by the European Union Reference Laboratory for Polycyclic Aromatic Hydrocarbons - Four marker PAHs in a dry extract of St Johns wort Author(s) Radoslav Lizak Szilard Szilagyi Philippe Verlinde and Thomas WENZL Luxembourg Publications Office of the European Union 2012 ndash 66 pp ndash 201 x 297 cm EUR ndash Scientific and Technical Research series ndash ISSN 1831-9424 ISBN 978-92-79-23337-1 doi 10278759387 Abstract The proficiency test here reported concerned the determination of the contents of four marker polycyclic aromatic hydrocarbons (PAHs) and of their sum in an food supplements test sample The set of marker PAHs consists of benz[a]anthacene benzo[a]pyrene benzo[b]fluoranthene and chrysene Participants to these PT were National Reference Laboratories for PAHs (NRLs-PAHs) and EU official food control laboratories The number of participants was in total 48

The PT was organised according ISO Standard 170432010

The test material used was a commercial dry extract of St Johns wort Participants also received a solution of the same PAHs either in acetonitrile or in toluene for checking their instrument calibration

The results from participants were rated with z-scores and zeta-scores About 68 of the reported results were attributed with z-scores with an absolute value of below two which is the threshold for satisfactory performance

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interests whether private or national

L

A-N

A-252

45-EN

-N

• 1 Summary
• 2 Introduction
• 3 Scope
• 4 Participating Laboratories
• 5 Time frame
• 6 Confidentiality
• 7 Test materials
• • 71 1Preparation of test materials
  • 72 Assigned values and standard deviation for proficiency assessment
  • 73 Homogeneity and stability
  • 74 Sample dispatch
  • • 8 Design of the proficiency test
    • 9 Evaluation of the results
    • • 91 2General
      • 92 21BEvaluation criteria
      • 93 Performance of participants
      • 94 Evaluation of the influence of analysis method
      • 95 Evaluation of the influence of calibration
      • 96 26Evaluation of compliance with legislation
      • • 10 Follow-up actions for underperforming laboratories
        • 11 1Conclusions
        • 12 1Acknowledgements
        • 13 References

40

ANNEX 6 ndash Documents sent to participants

41

ANNEX 6 - continued

42

ANNEX 7 Questionnaire on details of analysis method

43

ANNEX 7 - continued

44

ANNEX 7 - continued

45

ANNEX 8 - Results reported by participants for the food supplements test sample Table 11 Analysis results reported by the participants for the content of benz[a]anthracene (BAA) in the food supplements test material Values are presented as reported

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

3600 31 29 29 3 05 2

3602 199 193 178 19 021

3603 253 253 246 251 022 2

3604 199 209 23 213 071 2

3605 301 3 271 291 076 2

3606 302 288 278 289 064 2

3607 456 416 443 438 096 2

3608 796 695 651 714 149 2

3610 182 192 214 196 039 2

3611 414 504 488 469 96 2

3612 3 34 28 31 06 2

3613 32 33 34 33 07 2

3614 182 182 191 185 037 2

3615 3884 3822 3565 376 0752 2

3616 2 2 2 2 01 2

3617 366 37 35 362 046 2

3618 32 32 32 32 05 2

3619 299 305 297 299 048 2

3620 304 294 302 302 045 2

3621 29 26 29 28 04 2

3622 343 325 318 322 052 2

3623 35 3 33 33 07 2

3624

3626 318 31 314 314 126 2

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

5301 31 3 32 31 06 2

5302 314 319 315 316 063 2

5303 3102 2716 2822 2876 27 62

5304 261 263 26 261 57 2

5305 23 22 26 24 20

5306 22 23 23 22 11 2

5307 488 461 317 461

5308 168633 168544 1562025 1644598

5309 16 19 16 17

5310 3 304 311 305 015

5311 477 452 448 459 15 2

5312 506 63 494 543 134 2

5313 353 301 404 353

5314 431 373 378 394 5 096

5315 596 531 51 545 11 2

5316 224 231 235 235 022 2

5317 216 23 221 222 007 2

5318 064 07 067 01 2

5319 111 184 146 147 029 2

5320 515 525 564 534 48

5321 21 5322 13 87 9 87 35 2

5323 38 33 35 33 16 2

46

Figure 6 Results of replicate determinations (indicated by triangles) of benz[a]anthracene (BAA) in the food supplement test material

Horizontal blue lines represent the arithmetic mean value of replicate measurements and blue bars the reported expanded measurement uncertainty (k=2) The assigned value is

plotted as dotted line Red solid lines mark the range of satisfactory performance (|z|le2) Mean values are given for results outside the displayed data range

ProLab 2011

Laboratory

53

1853

1953

0853

0936

1436

0236

1036

1653

2136

0453

1753

0653

1653

0536

0353

0436

2153

0336

0636

0536

0036

2036

1953

1036

1253

0136

2653

0236

1836

2336

2236

1353

1353

2336

1736

1553

1453

0736

0753

1153

2053

1253

1536

0853

2236

11

an

aly

te c

on

ten

t (micro

gk

g)

10

95

9

85

8

75

7

65

6

55

5

45

4

35

3

25

2

15

1

05

0

102

3 468

7

Sample St Johns wort extract replicatesMeasurand Benz[a]anthraceneNo of laboratories 46

Assigned value 298 microgkg (Reference value)Target sd 062 microgkgTolerance limits 174 - 422 microgkg (|Z score| lt 200)

Assig

ned va

lue

Limit of tolerance

Limit of tolerance

47

Table 12 Analysis results reported by the participants for the content of benzo[a]pyrene (BAP) in the food supplements test material Values are presented as reported

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

3600 16 17 17 17 03 2

3602 118 123 13 124 015

3603 165 162 157 161 015 2

3604 134 154 139 143 04 2

3605 164 166 172 167 057 2

3606 181 159 161 167 031 2

3607 279 266 279 274 055 2

3608 57 687 461 573 226 2

3610 226 216 183 206 042 2

3611 289 325 311 308 36 2

3612 24 22 2 22 05 2

3613 17 18 19 18 05 2

3614 08 08 08 08 016 2

3615 1545 1393 1425 145 029 2

3616 16 16 14 15 03 2

3617 177 171 158 169 023 2

3618 2 2 2 2 02 2

3619 207 207 206 207 075 2

3620 165 169 163 166 017 2

3621 44 47 49 47 05 2

3622 212 208 222 213 038 2

3623 2 21 22 21 03 2

3624 18 178 182 18 031 2

3626 172 173 178 174 07 2

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

5301 18 18 18 18 04 2

5302 188 19 187 188 038 2

5303 1691 1426 149 1535 19 80

5304 145 142 15 146 69 2

5305 15 17 18 16 15

5306 16 17 17 16 08 2

5307 164 157 153 157

5308 102669 109324 109035 107009

5309 1 1 12 11

5310 122 122 124 123 006

5311 199 184 215 2 15 2

5312 258 248 239 248 05 2

5313 199 178 245 207

5314 174 171 172 172 11 0948

5315 253 248 213 238 05 2

5316 177 171 173 17 016 2

5317 255 277 276 269 012 2

5318 114 12 117 02 2

5319 19 228 231 216 043 2

5320 303 3 344 316 78

5321 12 12 12 12

5322 13 17 24 17 35 2

5323 2 2 19 2 1 2

48

Figure 7 Results of replicate determinations (indicated by triangles) of benzo[a]pyrene (BAP) in the food supplement test material

Horizontal blue lines represent the arithmetic mean value of replicate measurements and blue bars the reported expanded measurement uncertainty (k=2) The assigned value is

plotted as dotted line Red solid lines mark the range of satisfactory performance (|z|le2) Mean values are given for results outside the displayed data range

ProLab 2011

Laboratory

36

1453

0953

0853

1853

2153

1036

0236

0436

1553

0436

1653

0353

0736

0336

2036

0053

0553

0636

0636

0536

1753

1453

1636

2636

1336

2453

0153

2253

0253

2353

1136

1836

1953

1336

1036

2336

2253

1936

1253

1553

1253

1736

0753

2036

2136

0836

11

an

aly

te c

on

ten

t (micro

gk

g)

55

5

45

4

35

3

25

2

15

1

05

0

573

308

3

Sample St Johns wort extract replicatesMeasurand Benzo[a]pyreneNo of laboratories 48

Assigned value 165 microgkg (Reference value)Target sd 036 microgkgTolerance limits 093 - 237 microgkg (|Z score| lt 200)

Assig

ned va

lue

Limit of tolerance

Limit of tolerance

49

Table 13 Analysis results reported by the participants for the content of benzo[b]fluoranthene (BBF) in the food supplements test material Values are presented as reported

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

3600 32 3 3 31 09 2

3602 173 175 16 17 017

3603 265 252 25 256 025 2

3604 116 108 105 11 028 2

3605 286 225 232 247 074 2

3606 294 273 261 276 046 2

3607 579 579 544 567 113 2

3608 103 1201 876 1036 326 2

3610 173 196 204 191 038 2

3611 546 618 565 576 75 2

3612 3 28 28 29 06 2

3613 28 31 32 31 06 2

3614 135 111 135 127 022 2

3615 4187 3678 4111 4 08 2

3616 2 2 2 2 01 2

3617 33 27 299 035 2

3618 31 31 31 31 05 2

3619 326 324 312 326 064 2

3620 277 28 272 276 069 2

3621 26 25 23 25 04 2

3622 346 336 333 34 052 2

3623 26 28 29 28 02 2

3624

3626 288 28 307 292 117 2

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

5301 29 3 29 29 06 2

5302 297 3 296 298 06 2

5303 2705 2444 2608 2586 22 78

5304 245 254 26 253 85 2

5305 23 23 26 24 15

5306 42 43 4 42 21 2

5307 221 216 241 221

5308 232169 246654 240722 229848

5309 19 22 19 2 0

5310 3 315 325 313 016

5311 262 277 304 281 15 2

5312 27 289 337 299 075 2

5313 321 284 378 328

5314 423 404 385 404 34 083

5315 42 506 416 444 09 2

5316 265 267 277 273 018 2

5317 185 197 195 192 006 2

5318 276 281 279 04 2

5319 396 389 403 396 079 2

5320 623 618 635 625 14

5321 31

5322 24 33 49 33 35 2

5323 38 45 35 45 27 2

50

Figure 8 Results of replicate determinations (indicated by triangles) of benzo[b]fluoranthene (BBF) in the food supplement test material

Horizontal blue lines represent the arithmetic mean value of replicate measurements and blue bars the reported expanded measurement uncertainty (k=2) The assigned value is

plotted as dotted line Red solid lines mark the range of satisfactory performance (|z|le2) Mean values are given for results outside the displayed data range

ProLab 2011

Laboratory

36

0436

1436

0236

1053

1736

1653

0953

0753

0853

0536

2136

0553

0436

0353

0353

1636

0636

2036

2353

1853

1136

1236

2653

0153

0253

1236

1736

1336

0036

1853

2153

1036

1953

1336

2253

2253

2353

1936

1553

1453

0653

1536

0753

2036

0836

11

an

aly

te c

on

ten

t (micro

gk

g)

75

7

65

6

55

5

45

4

35

3

25

2

15

1

05

0

103

6

576

3

Sample St Johns wort extract replicatesMeasurand Benzo[b]fluorantheneNo of laboratories 46

Assigned value 292 microgkg (Reference value)Target sd 060 microgkgTolerance limits 172 - 412 microgkg (|Z score| lt 200)

Assig

ned va

lue

Limit of tolerance

Limit of tolerance

51

Table 14 Analysis results reported by the participants for the content of chrysene (CHR) in the food supplements test material Values are presented as reported

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

3600 5 49 5 5 07 2

3602 32 304 29 304 036

3603 462 469 447 459 048 2

3604 438 428 42 429 119 2

3605 365 41 381 386 085 2

3606 494 411 45 451 124 2

3607 897 774 787 819 18 2

3608 986 887 1168 1014 284 2

3610 418 392 462 424 085 2

3611 833 893 87 865 61 2

3612 6 64 58 61 12 2

3613 41 39 4 4 08 2

3614 272 254 288 271 054 2

3615 605 6028 5964 601 12 2

3616 47 45 49 47 05 2

3617 445 431 389 422 065 2

3618 45 45 45 45 06 2

3619 738 747 72 738 118 2

3620 463 472 446 46 092 2

3621 53 49 51 51 06 2

3622 478 513 505 488 08 2

3623 73 73 72 72 05 2

3624

3626 63 622 634 628 251 2

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

5301 49 49 49 49 1 2

5302 687 683 666 679 136 2

5303 6777 6181 6382 6441 24 70

5304 592 581 6 591 85 2

5305 6 58 64 61 20

5306 49 5 51 49 25 2

5307 524 5 574 524

5308 211488 216782 221146 213243

5309

5310 372 393 394 386 019

5311 43 459 422 437 15 2

5312 934 943 981 953 191 2

5313 793 73 99 838

5314 636 561 565 588 18 0818

5315 547 583 499 542 11 2

5316 292 286 285 282 06 2

5317 52 598 517 545 046 2

5318 368 345 357 05 2

5319 449 471 474 465 116 2

5320 76 924 818 834 99

5321 33

5322 7 7 76 7 35 2

5323 87 73 77 73 37 2

52

Figure 9 Results of replicate determinations (indicated by triangles) of chrysene (CHR) in the food supplement test material

Horizontal blue lines represent the arithmetic mean value of replicate measurements and blue bars the reported expanded measurement uncertainty (k=2) The assigned value is

plotted as dotted line Red solid lines mark the range of satisfactory performance (|z|le2) Mean values are given for results outside the displayed data range

ProLab 2011

Laboratory

53

0836

1453

1636

0253

2153

1836

0553

1036

1336

1736

1036

0453

1136

1836

0636

0336

2053

1936

1653

0136

0036

2253

0636

2153

0753

1553

1753

1453

0436

1536

1253

0536

2653

0353

0253

2236

2336

1953

2336

0753

2053

1353

1236

0836

11

an

aly

te c

on

ten

t (micro

gk

g)

13

12

11

10

9

8

7

6

5

4

3

2

1

0

865

3

Sample St Johns wort extract replicatesMeasurand ChryseneNo of laboratories 45

Assigned value 407 microgkg (Reference value)Target sd 083 microgkgTolerance limits 241 - 573 microgkg (|Z score| lt 200)

Assig

ned va

lue

Limit of tolerance

Limit of tolerance

53

Table 15 Analysis results reported by the participants for the sum of the four marker PAHs in the food supplements test material Values are presented as reported

Laboratory code

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg

3600 128 24 2

3602 79 126

3603 1127 096 2

3604 893 258 2

3605 1091 305 2

3606 1184 238 2

3607 2098 42 2

3608 3336 325 2

3610 1138 17 2

3611 2218 259 2

3612 143 29 2

3613 122 2 2

3614 663 129 2

3615 152 304 2

3616 102 03 2

3617 1252 169 2

3618 128 18 2

3619 157 305 2

3620 1204 12 2

3621 151 19 2

3622 1361 115 2

3623 154 09 2

3624

3626 1409 56 2

Laboratory code

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg

5301 126

5302 37 074 2

5303 13438 92

5304 1251 85 2

5305 125 20

5306 129 65 2

5307 1363

5308 7245598

5309

5310 1127 056

5311 1367 15 2

5312 2042 408 2

5313 1726

5314 1558

5315 177 35 2

5316 96 073 2

5317 1229 066 2

5318 82 05 2

5319 1225 267 2

5320 2309 41

5321

5322 207 35 2

5323 171 103 2

54

ANNEX 9 Results reported for the standard solutions with undisclosed analyte content Table 16 Analysis results reported by the participants for the four marker PAHs in the standard solution in toluene with undisclosed analyte

content Values are presented as reported U measurement uncertainty k coverage factor

benz[a]anthracene benzo[a]pyrene benzo[b]fluoranthene chrysene Laboratory

code Unit Result U k Result U k Result U k Result U k 3600 microgl 38 67 2 25 45 2 211 68 2 275 38 2

3605 microgl 367 184 2 2482 124 2 2592 13 2 235 118 2

3606 microgkg 3763 176 2 2609 142 2 2721 118 2 2539 134 2

3607 microgkg 3706 815 2 2824 565 2 2591 518 2 2587 569 2

3612 microgl 38 08 2 237 11 2 244 18 2 257 02 2

3614 microgl 324 65 2 209 38 2 215 37 2 21 42 2

3616 microgkg 318 07 2 224 14 2 231 26 2 216 1 2

3618 microgkg 417 17 2 284 08 2 292 06 2 297 03 2

3619 microgkg 47 742 2 274 468 2 228 387 2 274 434 2

3620 microgl 495 74 2 294 29 2 321 8 2 321 64 2

3621 microgl 241 22 2 143 13 2 188 16 2 145 14 2

3623 microgkg 476 25 2 311 53 2 269 3 2 297 03 2

3626 microgl 359 38 2 235 23 2 238 27 2 24 18 2

5301 microgkg 426 29 299 289

5302 microgkg 436 87 2 296 59 2 296 59 2 289 58 2

5303 microgl 3721 27 102 22838 19 99 23756 22 99 25486 24 98

5304 microgkg 38 29 2 225 26 2 257 37 2 258 52 2

5305 microgkg 305 225 245 29

5306 microgkg 316 63 2 247 49 2 65 13 2 22 44 2

5307 microgkg 3658 2559 241 2414

5308 microgkg 16854 109324 246654 216782

5311 microgl 374 20 2 279 15 2 268 15 2 272 15 2

5313 microgl 3408 2165 2275 2373

5314 microgkg 395 5 2875 11 3614 34 2905 18

5320 microgl 3267 368 1986 571 28 683 2427 512

5322 microgl 282 35 2 225 35 2 241 35 2 21 35 2

55

Figure 10 Youden plots of the percent deviation of the reported values from the assigned values for the food supplement test sample and the

standard solution in toluene with undisclosed content

(The identity of the respective measurand is given above the figure Results exceeding a difference of more than 100 were omitted)

56

Figure 10 - continued

57

Table 17 Analysis results reported by the participants for the four marker PAHs in the standard solution in acetonitrile with undisclosed

analyte content

Values are presented as reported U measurement uncertainty k coverage factor

benz[a]anthracene benzo[a]pyrene benzo[b]fluoranthene chrysene

Laboratory

code Unit Result U k Result U k Result U k Result U k

3602 microgkg 3675 247 247 244

3603 microgkg 358 086 2 236 028 2 235 028 2 236 057 2

3604 microgl 475 29 2 19 12 2 194 13 2 258 12 2

3608 microgl 3623 078 2 2385 023 2 2343 18 2 2296 092 2

3610 microgkg 369 185 2 25 125 2 251 126 2 244 122 2

3611 microgkg 48 02 2 323 02 2 328 04 2 317 03 2

3613 microgl 332 31 2 261 3 2 249 25 2 239 24 2

3615 microgl 415 2635 2729 2689

3617 microgkg 37 09 2 244 06 2 284 07 2 239 06 2

3622 microgl 37 12 2 25 23 2 257 21 2 238 1 2

3624 microgkg 402 104 2 255 084 2 265 096 2 253 07 2

5306 microgkg 563 113 2 371 74 2 359 72 2 333 67 2

5308 microgkg 168633 102669 232169 211488

5309 microgl 402 268 27

5310 microgl 315 158 25 125 25 125 288 144

5312 microgl 428 043 2 282 028 2 272 027 2 275 028 2

5315 microgkg 387 08 2 256 05 2 251 05 2 253 05 2

5316 microgl 3839 342 2 2485 191 2 2559 168 2 239 51 2

5317 microgl 2466 043 2 2212 034 2 2472 016 2 2146 068 2

5319 microgl 414 41 2 279 28 2 268 27 2 26 31 2

5321 microgkg 476 316 326 328

5322 microgl 2547 35 2

5323 microgkg 405 203 2 239 119 2 255 153 2 201 11 2

58

Figure 11 Youden plots of the percent deviation of the reported values from the assigned values for the food supplement test sample and the

standard solution in acetonitrile with undisclosed content

(The identity of the respective measurand is given above the figure Results exceeding a difference of more than 100 were omitted)

59

Figure 11 - continued

60

ANNEX 10 Table 18 Method performance parameters reported by the participants

Laboratory code

Analyte LOD LOQ Recovery Analyte LOD LOQ Recovery

microgkg microgkg microgkg microgkg 3600 BaA 03 06 978 BaP 03 07 9483602 BaA 007 021 926 BaP 005 015 993603 BaA 0011 05 987 BaP 0005 051 9553604 BaA 02 04 86 BaP 02 04 883605 BaA 0025 005 118 BaP 0025 005 1083606 BaA 001 003 65 BaP 001 003 703607 BaA 021 071 74 BaP 027 089 943608 BaA 016 048 8213 BaP 015 046 83683610 BaA 02 05 85 BaP 005 02 853611 BaA BaP 3612 BaA 1 117 BaP 1 1203613 BaA 01 03 90 BaP 01 03 903614 BaA 02 05 96 BaP 02 05 933615 BaA 005 015 110 BaP 005 015 1053616 BaA 01 05 108 BaP 01 05 753617 BaA 018 037 405 BaP 009 018 3873618 BaA 01 03 63 BaP 01 03 593619 BaA 005 005 59 BaP 033 033 543620 BaA 0009 0018 64 BaP 0005 001 553621 BaA 007 02 84 BaP 007 02 923622 BaA 01 03 1101 BaP 01 03 9853623 BaA 02 08 105 BaP 02 08 1013624 BaA BaP 01 033 8513625 BaA BaP 3626 BaA BaP 5301 BaA 003 01 102 BaP 003 01 1005302 BaA 01 03 78 BaP 01 03 865303 BaA 003 01 62 BaP 003 01 805304 BaA 1 1 70 BaP 1 1 955305 BaA 02 05 85 BaP 02 05 855306 BaA 03 1 699 BaP 03 1 905307 BaA 6537 BaP 81165308 BaA 0088 0265 99 BaP 00262 00785 1005309 BaA 01 08 92 BaP 01 08 925310 BaA 01 03 BaP 01 03 905311 BaA 002 005 80 BaP 002 004 915312 BaA 03 09 105 BaP 03 09 1055313 BaA 04 08 BaP 04 08 5314 BaA 001 002 96 BaP 001 002 9485315 BaA 01 03 50 - 100 BaP 01 03 50 - 100 5316 BaA 002 011 827 BaP 004 019 10445317 BaA 05 1 76 BaP 05 1 755318 BaA BaP 5319 BaA 009 029 951 BaP 011 041 995320 BaA 12 18 946 BaP 06 1 8345321 BaA 03 05 BaP 05 1 5322 BaA 1 2 100 BaP 1 2 1005323 BaA 26 BaP 26

61

ANNEX 10 Table 18 - continued

Laboratory code Analyte LOD LOQ Recovery Analyte LOD LOQ Recovery

microgkg microgkg microgkg microgkg 3600 BbF 03 05 96 CHR 02 03 10153602 BbF 015 045 947 CHR 003 009 9193603 BbF 0014 05 1012 CHR 0004 05 10033604 BbF 02 04 85 CHR 02 04 873605 BbF 005 01 94 CHR 0025 005 863606 BbF 001 003 70 CHR 001 003 653607 BbF 018 06 100 CHR 014 028 943608 BbF 015 046 7979 CHR 015 045 71253610 BbF 005 02 85 CHR 02 05 853611 BbF CHR 3612 BbF 1 118 CHR 1 1173613 BbF 01 03 90 CHR 01 03 903614 BbF 01 04 96 CHR 02 05 953615 BbF 008 025 109 CHR 003 01 1083616 BbF 01 05 78 CHR 01 05 723617 BbF 035 07 38 CHR 012 024 4163618 BbF 01 03 62 CHR 01 03 623619 BbF 016 016 63 CHR 01 01 593620 BbF 0003 0006 55 CHR 001 002 653621 BbF 007 02 103 CHR 007 02 893622 BbF 02 06 963 CHR 03 09 1043623 BbF 02 08 106 CHR 02 08 1193624 BbF CHR 3625 BbF CHR 3626 BbF CHR 5301 BbF 003 01 96 CHR 003 01 1145302 BbF 01 03 84 CHR 01 03 835303 BbF 003 01 78 CHR 003 01 705304 BbF 1 1 86 CHR 1 1 705305 BbF 02 05 90 CHR 02 05 955306 BbF 03 1 1435 CHR 03 1 1185307 BbF 8116 CHR 65375308 BbF 0079 0238 99 CHR 009 026 1005309 BbF 01 08 92 CHR 5310 BbF 07 2 CHR 01 03 5311 BbF 002 004 95 CHR 002 004 895312 BbF 03 09 105 CHR 03 09 1055313 BbF 04 08 CHR 04 08 5314 BbF 001 002 83 CHR 001 002 8185315 BbF 02 05 50 -100 CHR 02 05 50 - 100 5316 BbF 005 024 814 CHR 001 007 9595317 BbF 05 1 73 CHR 05 1 785318 BbF CHR 5319 BbF 011 038 946 CHR 013 043 9675320 BbF 07 09 1194 CHR 23 32 10455321 BbF 05 1 CHR 05 1 5322 BbF 1 2 100 CHR 1 2 1005323 BbF 26 CHR 26

62

ANNEX 11 Table 19 Details of analysis method reported by the participants Data are presented as reported

Laboratory code

Analysis technique

Instrument calibration

Internal standards applied

Sample clean up

Details of sample

clean up

Extraction technique

Details of sample

extraction

3600 6) GC-MS

2) Internal standardisation with labelled substances

Deuterated PAH mix 9 (Ehrenstorfer)

1) Saponification 2) LiquidLiquid partitioning

D) Other Liquidliquid partitioning

3602 2) HPLC-UV-FLD

1) External calibration

3) Gel permeation chromatography

A) Pressurised liquid extraction (PLE)

3603 1) HPLC-FLD

1) External calibration

3) Gel permeation chromatography

D) Other 1 minute with dichloromethane in vortex

3604 2) HPLC-UV-FLD

2) Internal standardisation with labelled substances

DiP D14

5) Donor acceptor complex chromatography

D) Other LL

3605 1) HPLC-FLD

1) External calibration

3) Gel permeation chromatography

C) Soxhlet extraction

3606 7) GC-MSMS

2) Internal standardisation with labelled substances

BaP 13C4 CHR 13C6 BaA 13C6 BbF 13C6

4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

3607 6) GC-MS

2) Internal standardisation with labelled substances

BaA-d12 CHR-d12 BbF-d12 BkF-d12 BaP-d12 ICP-d12 BgP-d12 DiP-d14

1) Saponification

D) Other solvent partitioning

3608 1) HPLC-FLD

1) External calibration

6) Other Liquid extraction

D) Other

Extraction in a rotary agitator and centrifugation

3610 2) HPLC-UV-FLD

1) External calibration

4) Solid phase extraction

B) Sonication

3611

3612 6) GC-MS

2) Internal standardisation with labelled substances

chrysene-d12 dibenzo(ah)anthracene-d14 dibenzo(ai)pyrene-d14

4) Solid phase extraction

B) Sonication

3613 5) LC-MSMS

3) Internal standardisation with unlabelled substances

I use Chrysene-D12 3) Gel permeation chromatography

D) Other extraction with hexan

63

Table 19 - continued

Laboratory code

Analysis technique

Instrument calibration

Internal standards applied

Sample clean up Details of sample

clean up

Extraction technique

Details of sample

extraction

3614 6) GC-MS 1) External calibration

3) Gel permeation chromatography

B) Sonication

3615 1) HPLC-FLD

1) External calibration

6) Other PLE GPC

A) Pressurised liquid extraction (PLE)

3616 6) GC-MS

2) Internal standardisation with labelled substances

Benzo(a)pyrene-13C4 Benzo(a)anthracene-13C6 Benzo(b)fluoranthene-13C6 Chrysene-13C6

2) LiquidLiquid partitioning 4) Solid phase extraction

B) Sonication

3617 1) HPLC-FLD

1) External calibration

4) Solid phase extraction

C) Soxhlet extraction

3618 6) GC-MS

2) Internal standardisation with labelled substances

deuterated standards

1) Saponification 4) Solid phase extraction

D) Other Handshaken with cyclohexane

3619 6) GC-MS

2) Internal standardisation with labelled substances

C13 Labelled US EPA 16

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

D) Other Homegenisation amp saponiofication

3620 9) GC-HRMS

2) Internal standardisation with labelled substances

mix of deuterated PAHs (BAA-D12 CHR-D12 BAP-D12 BBF-D12)

3) Gel permeation chromatography 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

3621 6) GC-MS

2) Internal standardisation with labelled substances

Chrysene D12 Benzo(a)pyrene D12 Benzo(b)fluoranthene D12 Benzo(a)anthracene

2) LiquidLiquid partitioning 4) Solid phase extraction

B) Sonication

3622 1) HPLC-FLD

4) Standard addition method

4) Solid phase extraction

D) Other

Extraction with cyclohexane by heating under reflux

3623 7) GC-MSMS

2) Internal standardisation with labelled substances

C13 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

3624 2) HPLC-UV-FLD

1) External calibration

2) LiquidLiquid partitioning

D) Other hot solvent extraction

3626

64

Table 19 - continued

Laboratory code

Analysis technique

Instrument calibration

Internal standards

applied

Sample clean up

Details of sample

clean up

Extraction technique

Details of sample

extraction

5301 7) GC-MSMS

2) Internal standardisation with labelled substances

EACH SUBSTANCE C13

4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5302 7) GC-MSMS

2) Internal standardisation with labelled substances

Isotope labelled PAHs 13C for each analyte

4) Solid phase extraction

C) Soxhlet extraction

5303 7) GC-MSMS

3) Internal standardisation with unlabelled substances

HAP C13 4) Solid phase extraction

D) Other automate ASE200 DIONEX

5304 7) GC-MSMS

2) Internal standardisation with labelled substances

with C13 for each HAP

2) LiquidLiquid partitioning 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5305 7) GC-MSMS

2) Internal standardisation with labelled substances

C13 isotopiques internal standards

4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5306 7) GC-MSMS

2) Internal standardisation with labelled substances

C13 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5307 6) GC-MS

2) Internal standardisation with labelled substances

1) Saponification 2) LiquidLiquid partitioning

D) Other solvent extraction

5308 1) HPLC-FLD 1) External calibration

4) Solid phase extraction

B) Sonication

5309 1) HPLC-FLD 1) External calibration

4) Solid phase extraction

D) Other only SPE

5310 1) HPLC-FLD 1) External calibration

1) Saponification 2) LiquidLiquid partitioning 3) Gel permeation chromatography

C) Soxhlet extraction

5311 6) GC-MS

2) Internal standardisation with labelled substances

13 C Chrysene 13 C Benz(a)pyrene

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

B) Sonication

5312 1) HPLC-FLD

3) Internal standardisation with unlabelled substances

Benzo(b)chrysen

2) LiquidLiquid partitioning 4) Solid phase extraction

Quechers D) Other Quechers

65

Table 19 - continued

Laboratory code

Analysis technique

Instrument calibration

Internal standards applied

Sample clean up Details of sample

clean up

Extraction technique

Details of sample extraction

5313 6) GC-MS

2) Internal standardisation with labelled substances

Benz(a)anthracene D12 Benzo(b)fluranthene D12 Benzo(a)pyrene D12 Chrysene D12

1) Saponification 2) LiquidLiquid partitioning 3) Gel permeation chromatography

A) Pressurised liquid extraction (PLE)

5314 6) GC-MS

2) Internal standardisation with labelled substances

Benzo(a)pyren d12 2) LiquidLiquid partitioning

D) Other Extraction with

5315 1) HPLC-FLD

3) Internal standardisation with unlabelled substances

Benz(b)chrysen

1) Saponification 2) LiquidLiquid partitioning

D) Other shaker

5316 1) HPLC-FLD

1) External calibration

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

D) Other

LiquidLiquid extraction with cyclohexane and dimethylformamide

5317 7) GC-MSMS

1) External calibration

3) Gel permeation chromatography 6) Other

silica gel D) Other VDLUFA online method

5318

5319 1) HPLC-FLD

3) Internal standardisation with unlabelled substances

Benzo(b)chrysene 3) Gel permeation chromatography

A) Pressurised liquid extraction (PLE)

5320 6) GC-MS

3) Internal standardisation with unlabelled substances

22-binaphthyl

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

D) Other 3 hours refluxing

5321 1) HPLC-FLD

2) Internal standardisation with labelled substances

6-meacutethylchrysegravene

1) Saponification 2) LiquidLiquid partitioning

B) Sonication

5322 6) GC-MS

2) Internal standardisation with labelled substances

naftalene-d8 acenaphtalene-d10 phenanthrene-d10 chrysene-d12 perylene-d12 benzo(e)anthracene-d12

1) Saponification 2) LiquidLiquid partitioning 6) Other

clean-up with silica packed column

C) Soxhlet extraction

5323 1) HPLC-FLD

1) External calibration

4) Solid phase extraction 6) Other

Accelerated Solvent Extraction

A) Pressurised liquid extraction (PLE)

66

European Commission EUR 25245 EN ndash Joint Research Centre ndash Institute for Reference Materials and Measurements Title Report on the 8th inter-laboratory comparison organised by the European Union Reference Laboratory for Polycyclic Aromatic Hydrocarbons - Four marker PAHs in a dry extract of St Johns wort Author(s) Radoslav Lizak Szilard Szilagyi Philippe Verlinde and Thomas WENZL Luxembourg Publications Office of the European Union 2012 ndash 66 pp ndash 201 x 297 cm EUR ndash Scientific and Technical Research series ndash ISSN 1831-9424 ISBN 978-92-79-23337-1 doi 10278759387 Abstract The proficiency test here reported concerned the determination of the contents of four marker polycyclic aromatic hydrocarbons (PAHs) and of their sum in an food supplements test sample The set of marker PAHs consists of benz[a]anthacene benzo[a]pyrene benzo[b]fluoranthene and chrysene Participants to these PT were National Reference Laboratories for PAHs (NRLs-PAHs) and EU official food control laboratories The number of participants was in total 48

The PT was organised according ISO Standard 170432010

The test material used was a commercial dry extract of St Johns wort Participants also received a solution of the same PAHs either in acetonitrile or in toluene for checking their instrument calibration

The results from participants were rated with z-scores and zeta-scores About 68 of the reported results were attributed with z-scores with an absolute value of below two which is the threshold for satisfactory performance

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The mission of the JRC is to provide customer-driven scientific and technical support

for the conception development implementation and monitoring of EU policies As a

service of the European Commission the JRC functions as a reference centre of

science and technology for the Union Close to the policy-making process it serves

the common interest of the Member States while being independent of special

interests whether private or national

L

A-N

A-252

45-EN

-N

• 1 Summary
• 2 Introduction
• 3 Scope
• 4 Participating Laboratories
• 5 Time frame
• 6 Confidentiality
• 7 Test materials
• • 71 1Preparation of test materials
  • 72 Assigned values and standard deviation for proficiency assessment
  • 73 Homogeneity and stability
  • 74 Sample dispatch
  • • 8 Design of the proficiency test
    • 9 Evaluation of the results
    • • 91 2General
      • 92 21BEvaluation criteria
      • 93 Performance of participants
      • 94 Evaluation of the influence of analysis method
      • 95 Evaluation of the influence of calibration
      • 96 26Evaluation of compliance with legislation
      • • 10 Follow-up actions for underperforming laboratories
        • 11 1Conclusions
        • 12 1Acknowledgements
        • 13 References

41

ANNEX 6 - continued

42

ANNEX 7 Questionnaire on details of analysis method

43

ANNEX 7 - continued

44

ANNEX 7 - continued

45

ANNEX 8 - Results reported by participants for the food supplements test sample Table 11 Analysis results reported by the participants for the content of benz[a]anthracene (BAA) in the food supplements test material Values are presented as reported

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

3600 31 29 29 3 05 2

3602 199 193 178 19 021

3603 253 253 246 251 022 2

3604 199 209 23 213 071 2

3605 301 3 271 291 076 2

3606 302 288 278 289 064 2

3607 456 416 443 438 096 2

3608 796 695 651 714 149 2

3610 182 192 214 196 039 2

3611 414 504 488 469 96 2

3612 3 34 28 31 06 2

3613 32 33 34 33 07 2

3614 182 182 191 185 037 2

3615 3884 3822 3565 376 0752 2

3616 2 2 2 2 01 2

3617 366 37 35 362 046 2

3618 32 32 32 32 05 2

3619 299 305 297 299 048 2

3620 304 294 302 302 045 2

3621 29 26 29 28 04 2

3622 343 325 318 322 052 2

3623 35 3 33 33 07 2

3624

3626 318 31 314 314 126 2

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

5301 31 3 32 31 06 2

5302 314 319 315 316 063 2

5303 3102 2716 2822 2876 27 62

5304 261 263 26 261 57 2

5305 23 22 26 24 20

5306 22 23 23 22 11 2

5307 488 461 317 461

5308 168633 168544 1562025 1644598

5309 16 19 16 17

5310 3 304 311 305 015

5311 477 452 448 459 15 2

5312 506 63 494 543 134 2

5313 353 301 404 353

5314 431 373 378 394 5 096

5315 596 531 51 545 11 2

5316 224 231 235 235 022 2

5317 216 23 221 222 007 2

5318 064 07 067 01 2

5319 111 184 146 147 029 2

5320 515 525 564 534 48

5321 21 5322 13 87 9 87 35 2

5323 38 33 35 33 16 2

46

Figure 6 Results of replicate determinations (indicated by triangles) of benz[a]anthracene (BAA) in the food supplement test material

Horizontal blue lines represent the arithmetic mean value of replicate measurements and blue bars the reported expanded measurement uncertainty (k=2) The assigned value is

plotted as dotted line Red solid lines mark the range of satisfactory performance (|z|le2) Mean values are given for results outside the displayed data range

ProLab 2011

Laboratory

53

1853

1953

0853

0936

1436

0236

1036

1653

2136

0453

1753

0653

1653

0536

0353

0436

2153

0336

0636

0536

0036

2036

1953

1036

1253

0136

2653

0236

1836

2336

2236

1353

1353

2336

1736

1553

1453

0736

0753

1153

2053

1253

1536

0853

2236

11

an

aly

te c

on

ten

t (micro

gk

g)

10

95

9

85

8

75

7

65

6

55

5

45

4

35

3

25

2

15

1

05

0

102

3 468

7

Sample St Johns wort extract replicatesMeasurand Benz[a]anthraceneNo of laboratories 46

Assigned value 298 microgkg (Reference value)Target sd 062 microgkgTolerance limits 174 - 422 microgkg (|Z score| lt 200)

Assig

ned va

lue

Limit of tolerance

Limit of tolerance

47

Table 12 Analysis results reported by the participants for the content of benzo[a]pyrene (BAP) in the food supplements test material Values are presented as reported

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

3600 16 17 17 17 03 2

3602 118 123 13 124 015

3603 165 162 157 161 015 2

3604 134 154 139 143 04 2

3605 164 166 172 167 057 2

3606 181 159 161 167 031 2

3607 279 266 279 274 055 2

3608 57 687 461 573 226 2

3610 226 216 183 206 042 2

3611 289 325 311 308 36 2

3612 24 22 2 22 05 2

3613 17 18 19 18 05 2

3614 08 08 08 08 016 2

3615 1545 1393 1425 145 029 2

3616 16 16 14 15 03 2

3617 177 171 158 169 023 2

3618 2 2 2 2 02 2

3619 207 207 206 207 075 2

3620 165 169 163 166 017 2

3621 44 47 49 47 05 2

3622 212 208 222 213 038 2

3623 2 21 22 21 03 2

3624 18 178 182 18 031 2

3626 172 173 178 174 07 2

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

5301 18 18 18 18 04 2

5302 188 19 187 188 038 2

5303 1691 1426 149 1535 19 80

5304 145 142 15 146 69 2

5305 15 17 18 16 15

5306 16 17 17 16 08 2

5307 164 157 153 157

5308 102669 109324 109035 107009

5309 1 1 12 11

5310 122 122 124 123 006

5311 199 184 215 2 15 2

5312 258 248 239 248 05 2

5313 199 178 245 207

5314 174 171 172 172 11 0948

5315 253 248 213 238 05 2

5316 177 171 173 17 016 2

5317 255 277 276 269 012 2

5318 114 12 117 02 2

5319 19 228 231 216 043 2

5320 303 3 344 316 78

5321 12 12 12 12

5322 13 17 24 17 35 2

5323 2 2 19 2 1 2

48

Figure 7 Results of replicate determinations (indicated by triangles) of benzo[a]pyrene (BAP) in the food supplement test material

Horizontal blue lines represent the arithmetic mean value of replicate measurements and blue bars the reported expanded measurement uncertainty (k=2) The assigned value is

plotted as dotted line Red solid lines mark the range of satisfactory performance (|z|le2) Mean values are given for results outside the displayed data range

ProLab 2011

Laboratory

36

1453

0953

0853

1853

2153

1036

0236

0436

1553

0436

1653

0353

0736

0336

2036

0053

0553

0636

0636

0536

1753

1453

1636

2636

1336

2453

0153

2253

0253

2353

1136

1836

1953

1336

1036

2336

2253

1936

1253

1553

1253

1736

0753

2036

2136

0836

11

an

aly

te c

on

ten

t (micro

gk

g)

55

5

45

4

35

3

25

2

15

1

05

0

573

308

3

Sample St Johns wort extract replicatesMeasurand Benzo[a]pyreneNo of laboratories 48

Assigned value 165 microgkg (Reference value)Target sd 036 microgkgTolerance limits 093 - 237 microgkg (|Z score| lt 200)

Assig

ned va

lue

Limit of tolerance

Limit of tolerance

49

Table 13 Analysis results reported by the participants for the content of benzo[b]fluoranthene (BBF) in the food supplements test material Values are presented as reported

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

3600 32 3 3 31 09 2

3602 173 175 16 17 017

3603 265 252 25 256 025 2

3604 116 108 105 11 028 2

3605 286 225 232 247 074 2

3606 294 273 261 276 046 2

3607 579 579 544 567 113 2

3608 103 1201 876 1036 326 2

3610 173 196 204 191 038 2

3611 546 618 565 576 75 2

3612 3 28 28 29 06 2

3613 28 31 32 31 06 2

3614 135 111 135 127 022 2

3615 4187 3678 4111 4 08 2

3616 2 2 2 2 01 2

3617 33 27 299 035 2

3618 31 31 31 31 05 2

3619 326 324 312 326 064 2

3620 277 28 272 276 069 2

3621 26 25 23 25 04 2

3622 346 336 333 34 052 2

3623 26 28 29 28 02 2

3624

3626 288 28 307 292 117 2

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

5301 29 3 29 29 06 2

5302 297 3 296 298 06 2

5303 2705 2444 2608 2586 22 78

5304 245 254 26 253 85 2

5305 23 23 26 24 15

5306 42 43 4 42 21 2

5307 221 216 241 221

5308 232169 246654 240722 229848

5309 19 22 19 2 0

5310 3 315 325 313 016

5311 262 277 304 281 15 2

5312 27 289 337 299 075 2

5313 321 284 378 328

5314 423 404 385 404 34 083

5315 42 506 416 444 09 2

5316 265 267 277 273 018 2

5317 185 197 195 192 006 2

5318 276 281 279 04 2

5319 396 389 403 396 079 2

5320 623 618 635 625 14

5321 31

5322 24 33 49 33 35 2

5323 38 45 35 45 27 2

50

Figure 8 Results of replicate determinations (indicated by triangles) of benzo[b]fluoranthene (BBF) in the food supplement test material

Horizontal blue lines represent the arithmetic mean value of replicate measurements and blue bars the reported expanded measurement uncertainty (k=2) The assigned value is

plotted as dotted line Red solid lines mark the range of satisfactory performance (|z|le2) Mean values are given for results outside the displayed data range

ProLab 2011

Laboratory

36

0436

1436

0236

1053

1736

1653

0953

0753

0853

0536

2136

0553

0436

0353

0353

1636

0636

2036

2353

1853

1136

1236

2653

0153

0253

1236

1736

1336

0036

1853

2153

1036

1953

1336

2253

2253

2353

1936

1553

1453

0653

1536

0753

2036

0836

11

an

aly

te c

on

ten

t (micro

gk

g)

75

7

65

6

55

5

45

4

35

3

25

2

15

1

05

0

103

6

576

3

Sample St Johns wort extract replicatesMeasurand Benzo[b]fluorantheneNo of laboratories 46

Assigned value 292 microgkg (Reference value)Target sd 060 microgkgTolerance limits 172 - 412 microgkg (|Z score| lt 200)

Assig

ned va

lue

Limit of tolerance

Limit of tolerance

51

Table 14 Analysis results reported by the participants for the content of chrysene (CHR) in the food supplements test material Values are presented as reported

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

3600 5 49 5 5 07 2

3602 32 304 29 304 036

3603 462 469 447 459 048 2

3604 438 428 42 429 119 2

3605 365 41 381 386 085 2

3606 494 411 45 451 124 2

3607 897 774 787 819 18 2

3608 986 887 1168 1014 284 2

3610 418 392 462 424 085 2

3611 833 893 87 865 61 2

3612 6 64 58 61 12 2

3613 41 39 4 4 08 2

3614 272 254 288 271 054 2

3615 605 6028 5964 601 12 2

3616 47 45 49 47 05 2

3617 445 431 389 422 065 2

3618 45 45 45 45 06 2

3619 738 747 72 738 118 2

3620 463 472 446 46 092 2

3621 53 49 51 51 06 2

3622 478 513 505 488 08 2

3623 73 73 72 72 05 2

3624

3626 63 622 634 628 251 2

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

5301 49 49 49 49 1 2

5302 687 683 666 679 136 2

5303 6777 6181 6382 6441 24 70

5304 592 581 6 591 85 2

5305 6 58 64 61 20

5306 49 5 51 49 25 2

5307 524 5 574 524

5308 211488 216782 221146 213243

5309

5310 372 393 394 386 019

5311 43 459 422 437 15 2

5312 934 943 981 953 191 2

5313 793 73 99 838

5314 636 561 565 588 18 0818

5315 547 583 499 542 11 2

5316 292 286 285 282 06 2

5317 52 598 517 545 046 2

5318 368 345 357 05 2

5319 449 471 474 465 116 2

5320 76 924 818 834 99

5321 33

5322 7 7 76 7 35 2

5323 87 73 77 73 37 2

52

Figure 9 Results of replicate determinations (indicated by triangles) of chrysene (CHR) in the food supplement test material

Horizontal blue lines represent the arithmetic mean value of replicate measurements and blue bars the reported expanded measurement uncertainty (k=2) The assigned value is

plotted as dotted line Red solid lines mark the range of satisfactory performance (|z|le2) Mean values are given for results outside the displayed data range

ProLab 2011

Laboratory

53

0836

1453

1636

0253

2153

1836

0553

1036

1336

1736

1036

0453

1136

1836

0636

0336

2053

1936

1653

0136

0036

2253

0636

2153

0753

1553

1753

1453

0436

1536

1253

0536

2653

0353

0253

2236

2336

1953

2336

0753

2053

1353

1236

0836

11

an

aly

te c

on

ten

t (micro

gk

g)

13

12

11

10

9

8

7

6

5

4

3

2

1

0

865

3

Sample St Johns wort extract replicatesMeasurand ChryseneNo of laboratories 45

Assigned value 407 microgkg (Reference value)Target sd 083 microgkgTolerance limits 241 - 573 microgkg (|Z score| lt 200)

Assig

ned va

lue

Limit of tolerance

Limit of tolerance

53

Table 15 Analysis results reported by the participants for the sum of the four marker PAHs in the food supplements test material Values are presented as reported

Laboratory code

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg

3600 128 24 2

3602 79 126

3603 1127 096 2

3604 893 258 2

3605 1091 305 2

3606 1184 238 2

3607 2098 42 2

3608 3336 325 2

3610 1138 17 2

3611 2218 259 2

3612 143 29 2

3613 122 2 2

3614 663 129 2

3615 152 304 2

3616 102 03 2

3617 1252 169 2

3618 128 18 2

3619 157 305 2

3620 1204 12 2

3621 151 19 2

3622 1361 115 2

3623 154 09 2

3624

3626 1409 56 2

Laboratory code

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg

5301 126

5302 37 074 2

5303 13438 92

5304 1251 85 2

5305 125 20

5306 129 65 2

5307 1363

5308 7245598

5309

5310 1127 056

5311 1367 15 2

5312 2042 408 2

5313 1726

5314 1558

5315 177 35 2

5316 96 073 2

5317 1229 066 2

5318 82 05 2

5319 1225 267 2

5320 2309 41

5321

5322 207 35 2

5323 171 103 2

54

ANNEX 9 Results reported for the standard solutions with undisclosed analyte content Table 16 Analysis results reported by the participants for the four marker PAHs in the standard solution in toluene with undisclosed analyte

content Values are presented as reported U measurement uncertainty k coverage factor

benz[a]anthracene benzo[a]pyrene benzo[b]fluoranthene chrysene Laboratory

code Unit Result U k Result U k Result U k Result U k 3600 microgl 38 67 2 25 45 2 211 68 2 275 38 2

3605 microgl 367 184 2 2482 124 2 2592 13 2 235 118 2

3606 microgkg 3763 176 2 2609 142 2 2721 118 2 2539 134 2

3607 microgkg 3706 815 2 2824 565 2 2591 518 2 2587 569 2

3612 microgl 38 08 2 237 11 2 244 18 2 257 02 2

3614 microgl 324 65 2 209 38 2 215 37 2 21 42 2

3616 microgkg 318 07 2 224 14 2 231 26 2 216 1 2

3618 microgkg 417 17 2 284 08 2 292 06 2 297 03 2

3619 microgkg 47 742 2 274 468 2 228 387 2 274 434 2

3620 microgl 495 74 2 294 29 2 321 8 2 321 64 2

3621 microgl 241 22 2 143 13 2 188 16 2 145 14 2

3623 microgkg 476 25 2 311 53 2 269 3 2 297 03 2

3626 microgl 359 38 2 235 23 2 238 27 2 24 18 2

5301 microgkg 426 29 299 289

5302 microgkg 436 87 2 296 59 2 296 59 2 289 58 2

5303 microgl 3721 27 102 22838 19 99 23756 22 99 25486 24 98

5304 microgkg 38 29 2 225 26 2 257 37 2 258 52 2

5305 microgkg 305 225 245 29

5306 microgkg 316 63 2 247 49 2 65 13 2 22 44 2

5307 microgkg 3658 2559 241 2414

5308 microgkg 16854 109324 246654 216782

5311 microgl 374 20 2 279 15 2 268 15 2 272 15 2

5313 microgl 3408 2165 2275 2373

5314 microgkg 395 5 2875 11 3614 34 2905 18

5320 microgl 3267 368 1986 571 28 683 2427 512

5322 microgl 282 35 2 225 35 2 241 35 2 21 35 2

55

Figure 10 Youden plots of the percent deviation of the reported values from the assigned values for the food supplement test sample and the

standard solution in toluene with undisclosed content

(The identity of the respective measurand is given above the figure Results exceeding a difference of more than 100 were omitted)

56

Figure 10 - continued

57

Table 17 Analysis results reported by the participants for the four marker PAHs in the standard solution in acetonitrile with undisclosed

analyte content

Values are presented as reported U measurement uncertainty k coverage factor

benz[a]anthracene benzo[a]pyrene benzo[b]fluoranthene chrysene

Laboratory

code Unit Result U k Result U k Result U k Result U k

3602 microgkg 3675 247 247 244

3603 microgkg 358 086 2 236 028 2 235 028 2 236 057 2

3604 microgl 475 29 2 19 12 2 194 13 2 258 12 2

3608 microgl 3623 078 2 2385 023 2 2343 18 2 2296 092 2

3610 microgkg 369 185 2 25 125 2 251 126 2 244 122 2

3611 microgkg 48 02 2 323 02 2 328 04 2 317 03 2

3613 microgl 332 31 2 261 3 2 249 25 2 239 24 2

3615 microgl 415 2635 2729 2689

3617 microgkg 37 09 2 244 06 2 284 07 2 239 06 2

3622 microgl 37 12 2 25 23 2 257 21 2 238 1 2

3624 microgkg 402 104 2 255 084 2 265 096 2 253 07 2

5306 microgkg 563 113 2 371 74 2 359 72 2 333 67 2

5308 microgkg 168633 102669 232169 211488

5309 microgl 402 268 27

5310 microgl 315 158 25 125 25 125 288 144

5312 microgl 428 043 2 282 028 2 272 027 2 275 028 2

5315 microgkg 387 08 2 256 05 2 251 05 2 253 05 2

5316 microgl 3839 342 2 2485 191 2 2559 168 2 239 51 2

5317 microgl 2466 043 2 2212 034 2 2472 016 2 2146 068 2

5319 microgl 414 41 2 279 28 2 268 27 2 26 31 2

5321 microgkg 476 316 326 328

5322 microgl 2547 35 2

5323 microgkg 405 203 2 239 119 2 255 153 2 201 11 2

58

Figure 11 Youden plots of the percent deviation of the reported values from the assigned values for the food supplement test sample and the

standard solution in acetonitrile with undisclosed content

(The identity of the respective measurand is given above the figure Results exceeding a difference of more than 100 were omitted)

59

Figure 11 - continued

60

ANNEX 10 Table 18 Method performance parameters reported by the participants

Laboratory code

Analyte LOD LOQ Recovery Analyte LOD LOQ Recovery

microgkg microgkg microgkg microgkg 3600 BaA 03 06 978 BaP 03 07 9483602 BaA 007 021 926 BaP 005 015 993603 BaA 0011 05 987 BaP 0005 051 9553604 BaA 02 04 86 BaP 02 04 883605 BaA 0025 005 118 BaP 0025 005 1083606 BaA 001 003 65 BaP 001 003 703607 BaA 021 071 74 BaP 027 089 943608 BaA 016 048 8213 BaP 015 046 83683610 BaA 02 05 85 BaP 005 02 853611 BaA BaP 3612 BaA 1 117 BaP 1 1203613 BaA 01 03 90 BaP 01 03 903614 BaA 02 05 96 BaP 02 05 933615 BaA 005 015 110 BaP 005 015 1053616 BaA 01 05 108 BaP 01 05 753617 BaA 018 037 405 BaP 009 018 3873618 BaA 01 03 63 BaP 01 03 593619 BaA 005 005 59 BaP 033 033 543620 BaA 0009 0018 64 BaP 0005 001 553621 BaA 007 02 84 BaP 007 02 923622 BaA 01 03 1101 BaP 01 03 9853623 BaA 02 08 105 BaP 02 08 1013624 BaA BaP 01 033 8513625 BaA BaP 3626 BaA BaP 5301 BaA 003 01 102 BaP 003 01 1005302 BaA 01 03 78 BaP 01 03 865303 BaA 003 01 62 BaP 003 01 805304 BaA 1 1 70 BaP 1 1 955305 BaA 02 05 85 BaP 02 05 855306 BaA 03 1 699 BaP 03 1 905307 BaA 6537 BaP 81165308 BaA 0088 0265 99 BaP 00262 00785 1005309 BaA 01 08 92 BaP 01 08 925310 BaA 01 03 BaP 01 03 905311 BaA 002 005 80 BaP 002 004 915312 BaA 03 09 105 BaP 03 09 1055313 BaA 04 08 BaP 04 08 5314 BaA 001 002 96 BaP 001 002 9485315 BaA 01 03 50 - 100 BaP 01 03 50 - 100 5316 BaA 002 011 827 BaP 004 019 10445317 BaA 05 1 76 BaP 05 1 755318 BaA BaP 5319 BaA 009 029 951 BaP 011 041 995320 BaA 12 18 946 BaP 06 1 8345321 BaA 03 05 BaP 05 1 5322 BaA 1 2 100 BaP 1 2 1005323 BaA 26 BaP 26

61

ANNEX 10 Table 18 - continued

Laboratory code Analyte LOD LOQ Recovery Analyte LOD LOQ Recovery

microgkg microgkg microgkg microgkg 3600 BbF 03 05 96 CHR 02 03 10153602 BbF 015 045 947 CHR 003 009 9193603 BbF 0014 05 1012 CHR 0004 05 10033604 BbF 02 04 85 CHR 02 04 873605 BbF 005 01 94 CHR 0025 005 863606 BbF 001 003 70 CHR 001 003 653607 BbF 018 06 100 CHR 014 028 943608 BbF 015 046 7979 CHR 015 045 71253610 BbF 005 02 85 CHR 02 05 853611 BbF CHR 3612 BbF 1 118 CHR 1 1173613 BbF 01 03 90 CHR 01 03 903614 BbF 01 04 96 CHR 02 05 953615 BbF 008 025 109 CHR 003 01 1083616 BbF 01 05 78 CHR 01 05 723617 BbF 035 07 38 CHR 012 024 4163618 BbF 01 03 62 CHR 01 03 623619 BbF 016 016 63 CHR 01 01 593620 BbF 0003 0006 55 CHR 001 002 653621 BbF 007 02 103 CHR 007 02 893622 BbF 02 06 963 CHR 03 09 1043623 BbF 02 08 106 CHR 02 08 1193624 BbF CHR 3625 BbF CHR 3626 BbF CHR 5301 BbF 003 01 96 CHR 003 01 1145302 BbF 01 03 84 CHR 01 03 835303 BbF 003 01 78 CHR 003 01 705304 BbF 1 1 86 CHR 1 1 705305 BbF 02 05 90 CHR 02 05 955306 BbF 03 1 1435 CHR 03 1 1185307 BbF 8116 CHR 65375308 BbF 0079 0238 99 CHR 009 026 1005309 BbF 01 08 92 CHR 5310 BbF 07 2 CHR 01 03 5311 BbF 002 004 95 CHR 002 004 895312 BbF 03 09 105 CHR 03 09 1055313 BbF 04 08 CHR 04 08 5314 BbF 001 002 83 CHR 001 002 8185315 BbF 02 05 50 -100 CHR 02 05 50 - 100 5316 BbF 005 024 814 CHR 001 007 9595317 BbF 05 1 73 CHR 05 1 785318 BbF CHR 5319 BbF 011 038 946 CHR 013 043 9675320 BbF 07 09 1194 CHR 23 32 10455321 BbF 05 1 CHR 05 1 5322 BbF 1 2 100 CHR 1 2 1005323 BbF 26 CHR 26

62

ANNEX 11 Table 19 Details of analysis method reported by the participants Data are presented as reported

Laboratory code

Analysis technique

Instrument calibration

Internal standards applied

Sample clean up

Details of sample

clean up

Extraction technique

Details of sample

extraction

3600 6) GC-MS

2) Internal standardisation with labelled substances

Deuterated PAH mix 9 (Ehrenstorfer)

1) Saponification 2) LiquidLiquid partitioning

D) Other Liquidliquid partitioning

3602 2) HPLC-UV-FLD

1) External calibration

3) Gel permeation chromatography

A) Pressurised liquid extraction (PLE)

3603 1) HPLC-FLD

1) External calibration

3) Gel permeation chromatography

D) Other 1 minute with dichloromethane in vortex

3604 2) HPLC-UV-FLD

2) Internal standardisation with labelled substances

DiP D14

5) Donor acceptor complex chromatography

D) Other LL

3605 1) HPLC-FLD

1) External calibration

3) Gel permeation chromatography

C) Soxhlet extraction

3606 7) GC-MSMS

2) Internal standardisation with labelled substances

BaP 13C4 CHR 13C6 BaA 13C6 BbF 13C6

4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

3607 6) GC-MS

2) Internal standardisation with labelled substances

BaA-d12 CHR-d12 BbF-d12 BkF-d12 BaP-d12 ICP-d12 BgP-d12 DiP-d14

1) Saponification

D) Other solvent partitioning

3608 1) HPLC-FLD

1) External calibration

6) Other Liquid extraction

D) Other

Extraction in a rotary agitator and centrifugation

3610 2) HPLC-UV-FLD

1) External calibration

4) Solid phase extraction

B) Sonication

3611

3612 6) GC-MS

2) Internal standardisation with labelled substances

chrysene-d12 dibenzo(ah)anthracene-d14 dibenzo(ai)pyrene-d14

4) Solid phase extraction

B) Sonication

3613 5) LC-MSMS

3) Internal standardisation with unlabelled substances

I use Chrysene-D12 3) Gel permeation chromatography

D) Other extraction with hexan

63

Table 19 - continued

Laboratory code

Analysis technique

Instrument calibration

Internal standards applied

Sample clean up Details of sample

clean up

Extraction technique

Details of sample

extraction

3614 6) GC-MS 1) External calibration

3) Gel permeation chromatography

B) Sonication

3615 1) HPLC-FLD

1) External calibration

6) Other PLE GPC

A) Pressurised liquid extraction (PLE)

3616 6) GC-MS

2) Internal standardisation with labelled substances

Benzo(a)pyrene-13C4 Benzo(a)anthracene-13C6 Benzo(b)fluoranthene-13C6 Chrysene-13C6

2) LiquidLiquid partitioning 4) Solid phase extraction

B) Sonication

3617 1) HPLC-FLD

1) External calibration

4) Solid phase extraction

C) Soxhlet extraction

3618 6) GC-MS

2) Internal standardisation with labelled substances

deuterated standards

1) Saponification 4) Solid phase extraction

D) Other Handshaken with cyclohexane

3619 6) GC-MS

2) Internal standardisation with labelled substances

C13 Labelled US EPA 16

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

D) Other Homegenisation amp saponiofication

3620 9) GC-HRMS

2) Internal standardisation with labelled substances

mix of deuterated PAHs (BAA-D12 CHR-D12 BAP-D12 BBF-D12)

3) Gel permeation chromatography 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

3621 6) GC-MS

2) Internal standardisation with labelled substances

Chrysene D12 Benzo(a)pyrene D12 Benzo(b)fluoranthene D12 Benzo(a)anthracene

2) LiquidLiquid partitioning 4) Solid phase extraction

B) Sonication

3622 1) HPLC-FLD

4) Standard addition method

4) Solid phase extraction

D) Other

Extraction with cyclohexane by heating under reflux

3623 7) GC-MSMS

2) Internal standardisation with labelled substances

C13 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

3624 2) HPLC-UV-FLD

1) External calibration

2) LiquidLiquid partitioning

D) Other hot solvent extraction

3626

64

Table 19 - continued

Laboratory code

Analysis technique

Instrument calibration

Internal standards

applied

Sample clean up

Details of sample

clean up

Extraction technique

Details of sample

extraction

5301 7) GC-MSMS

2) Internal standardisation with labelled substances

EACH SUBSTANCE C13

4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5302 7) GC-MSMS

2) Internal standardisation with labelled substances

Isotope labelled PAHs 13C for each analyte

4) Solid phase extraction

C) Soxhlet extraction

5303 7) GC-MSMS

3) Internal standardisation with unlabelled substances

HAP C13 4) Solid phase extraction

D) Other automate ASE200 DIONEX

5304 7) GC-MSMS

2) Internal standardisation with labelled substances

with C13 for each HAP

2) LiquidLiquid partitioning 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5305 7) GC-MSMS

2) Internal standardisation with labelled substances

C13 isotopiques internal standards

4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5306 7) GC-MSMS

2) Internal standardisation with labelled substances

C13 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5307 6) GC-MS

2) Internal standardisation with labelled substances

1) Saponification 2) LiquidLiquid partitioning

D) Other solvent extraction

5308 1) HPLC-FLD 1) External calibration

4) Solid phase extraction

B) Sonication

5309 1) HPLC-FLD 1) External calibration

4) Solid phase extraction

D) Other only SPE

5310 1) HPLC-FLD 1) External calibration

1) Saponification 2) LiquidLiquid partitioning 3) Gel permeation chromatography

C) Soxhlet extraction

5311 6) GC-MS

2) Internal standardisation with labelled substances

13 C Chrysene 13 C Benz(a)pyrene

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

B) Sonication

5312 1) HPLC-FLD

3) Internal standardisation with unlabelled substances

Benzo(b)chrysen

2) LiquidLiquid partitioning 4) Solid phase extraction

Quechers D) Other Quechers

65

Table 19 - continued

Laboratory code

Analysis technique

Instrument calibration

Internal standards applied

Sample clean up Details of sample

clean up

Extraction technique

Details of sample extraction

5313 6) GC-MS

2) Internal standardisation with labelled substances

Benz(a)anthracene D12 Benzo(b)fluranthene D12 Benzo(a)pyrene D12 Chrysene D12

1) Saponification 2) LiquidLiquid partitioning 3) Gel permeation chromatography

A) Pressurised liquid extraction (PLE)

5314 6) GC-MS

2) Internal standardisation with labelled substances

Benzo(a)pyren d12 2) LiquidLiquid partitioning

D) Other Extraction with

5315 1) HPLC-FLD

3) Internal standardisation with unlabelled substances

Benz(b)chrysen

1) Saponification 2) LiquidLiquid partitioning

D) Other shaker

5316 1) HPLC-FLD

1) External calibration

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

D) Other

LiquidLiquid extraction with cyclohexane and dimethylformamide

5317 7) GC-MSMS

1) External calibration

3) Gel permeation chromatography 6) Other

silica gel D) Other VDLUFA online method

5318

5319 1) HPLC-FLD

3) Internal standardisation with unlabelled substances

Benzo(b)chrysene 3) Gel permeation chromatography

A) Pressurised liquid extraction (PLE)

5320 6) GC-MS

3) Internal standardisation with unlabelled substances

22-binaphthyl

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

D) Other 3 hours refluxing

5321 1) HPLC-FLD

2) Internal standardisation with labelled substances

6-meacutethylchrysegravene

1) Saponification 2) LiquidLiquid partitioning

B) Sonication

5322 6) GC-MS

2) Internal standardisation with labelled substances

naftalene-d8 acenaphtalene-d10 phenanthrene-d10 chrysene-d12 perylene-d12 benzo(e)anthracene-d12

1) Saponification 2) LiquidLiquid partitioning 6) Other

clean-up with silica packed column

C) Soxhlet extraction

5323 1) HPLC-FLD

1) External calibration

4) Solid phase extraction 6) Other

Accelerated Solvent Extraction

A) Pressurised liquid extraction (PLE)

66

European Commission EUR 25245 EN ndash Joint Research Centre ndash Institute for Reference Materials and Measurements Title Report on the 8th inter-laboratory comparison organised by the European Union Reference Laboratory for Polycyclic Aromatic Hydrocarbons - Four marker PAHs in a dry extract of St Johns wort Author(s) Radoslav Lizak Szilard Szilagyi Philippe Verlinde and Thomas WENZL Luxembourg Publications Office of the European Union 2012 ndash 66 pp ndash 201 x 297 cm EUR ndash Scientific and Technical Research series ndash ISSN 1831-9424 ISBN 978-92-79-23337-1 doi 10278759387 Abstract The proficiency test here reported concerned the determination of the contents of four marker polycyclic aromatic hydrocarbons (PAHs) and of their sum in an food supplements test sample The set of marker PAHs consists of benz[a]anthacene benzo[a]pyrene benzo[b]fluoranthene and chrysene Participants to these PT were National Reference Laboratories for PAHs (NRLs-PAHs) and EU official food control laboratories The number of participants was in total 48

The PT was organised according ISO Standard 170432010

The test material used was a commercial dry extract of St Johns wort Participants also received a solution of the same PAHs either in acetonitrile or in toluene for checking their instrument calibration

The results from participants were rated with z-scores and zeta-scores About 68 of the reported results were attributed with z-scores with an absolute value of below two which is the threshold for satisfactory performance

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interests whether private or national

L

A-N

A-252

45-EN

-N

• 1 Summary
• 2 Introduction
• 3 Scope
• 4 Participating Laboratories
• 5 Time frame
• 6 Confidentiality
• 7 Test materials
• • 71 1Preparation of test materials
  • 72 Assigned values and standard deviation for proficiency assessment
  • 73 Homogeneity and stability
  • 74 Sample dispatch
  • • 8 Design of the proficiency test
    • 9 Evaluation of the results
    • • 91 2General
      • 92 21BEvaluation criteria
      • 93 Performance of participants
      • 94 Evaluation of the influence of analysis method
      • 95 Evaluation of the influence of calibration
      • 96 26Evaluation of compliance with legislation
      • • 10 Follow-up actions for underperforming laboratories
        • 11 1Conclusions
        • 12 1Acknowledgements
        • 13 References

42

ANNEX 7 Questionnaire on details of analysis method

43

ANNEX 7 - continued

44

ANNEX 7 - continued

45

ANNEX 8 - Results reported by participants for the food supplements test sample Table 11 Analysis results reported by the participants for the content of benz[a]anthracene (BAA) in the food supplements test material Values are presented as reported

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

3600 31 29 29 3 05 2

3602 199 193 178 19 021

3603 253 253 246 251 022 2

3604 199 209 23 213 071 2

3605 301 3 271 291 076 2

3606 302 288 278 289 064 2

3607 456 416 443 438 096 2

3608 796 695 651 714 149 2

3610 182 192 214 196 039 2

3611 414 504 488 469 96 2

3612 3 34 28 31 06 2

3613 32 33 34 33 07 2

3614 182 182 191 185 037 2

3615 3884 3822 3565 376 0752 2

3616 2 2 2 2 01 2

3617 366 37 35 362 046 2

3618 32 32 32 32 05 2

3619 299 305 297 299 048 2

3620 304 294 302 302 045 2

3621 29 26 29 28 04 2

3622 343 325 318 322 052 2

3623 35 3 33 33 07 2

3624

3626 318 31 314 314 126 2

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

5301 31 3 32 31 06 2

5302 314 319 315 316 063 2

5303 3102 2716 2822 2876 27 62

5304 261 263 26 261 57 2

5305 23 22 26 24 20

5306 22 23 23 22 11 2

5307 488 461 317 461

5308 168633 168544 1562025 1644598

5309 16 19 16 17

5310 3 304 311 305 015

5311 477 452 448 459 15 2

5312 506 63 494 543 134 2

5313 353 301 404 353

5314 431 373 378 394 5 096

5315 596 531 51 545 11 2

5316 224 231 235 235 022 2

5317 216 23 221 222 007 2

5318 064 07 067 01 2

5319 111 184 146 147 029 2

5320 515 525 564 534 48

5321 21 5322 13 87 9 87 35 2

5323 38 33 35 33 16 2

46

Figure 6 Results of replicate determinations (indicated by triangles) of benz[a]anthracene (BAA) in the food supplement test material

Horizontal blue lines represent the arithmetic mean value of replicate measurements and blue bars the reported expanded measurement uncertainty (k=2) The assigned value is

plotted as dotted line Red solid lines mark the range of satisfactory performance (|z|le2) Mean values are given for results outside the displayed data range

ProLab 2011

Laboratory

53

1853

1953

0853

0936

1436

0236

1036

1653

2136

0453

1753

0653

1653

0536

0353

0436

2153

0336

0636

0536

0036

2036

1953

1036

1253

0136

2653

0236

1836

2336

2236

1353

1353

2336

1736

1553

1453

0736

0753

1153

2053

1253

1536

0853

2236

11

an

aly

te c

on

ten

t (micro

gk

g)

10

95

9

85

8

75

7

65

6

55

5

45

4

35

3

25

2

15

1

05

0

102

3 468

7

Sample St Johns wort extract replicatesMeasurand Benz[a]anthraceneNo of laboratories 46

Assigned value 298 microgkg (Reference value)Target sd 062 microgkgTolerance limits 174 - 422 microgkg (|Z score| lt 200)

Assig

ned va

lue

Limit of tolerance

Limit of tolerance

47

Table 12 Analysis results reported by the participants for the content of benzo[a]pyrene (BAP) in the food supplements test material Values are presented as reported

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

3600 16 17 17 17 03 2

3602 118 123 13 124 015

3603 165 162 157 161 015 2

3604 134 154 139 143 04 2

3605 164 166 172 167 057 2

3606 181 159 161 167 031 2

3607 279 266 279 274 055 2

3608 57 687 461 573 226 2

3610 226 216 183 206 042 2

3611 289 325 311 308 36 2

3612 24 22 2 22 05 2

3613 17 18 19 18 05 2

3614 08 08 08 08 016 2

3615 1545 1393 1425 145 029 2

3616 16 16 14 15 03 2

3617 177 171 158 169 023 2

3618 2 2 2 2 02 2

3619 207 207 206 207 075 2

3620 165 169 163 166 017 2

3621 44 47 49 47 05 2

3622 212 208 222 213 038 2

3623 2 21 22 21 03 2

3624 18 178 182 18 031 2

3626 172 173 178 174 07 2

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

5301 18 18 18 18 04 2

5302 188 19 187 188 038 2

5303 1691 1426 149 1535 19 80

5304 145 142 15 146 69 2

5305 15 17 18 16 15

5306 16 17 17 16 08 2

5307 164 157 153 157

5308 102669 109324 109035 107009

5309 1 1 12 11

5310 122 122 124 123 006

5311 199 184 215 2 15 2

5312 258 248 239 248 05 2

5313 199 178 245 207

5314 174 171 172 172 11 0948

5315 253 248 213 238 05 2

5316 177 171 173 17 016 2

5317 255 277 276 269 012 2

5318 114 12 117 02 2

5319 19 228 231 216 043 2

5320 303 3 344 316 78

5321 12 12 12 12

5322 13 17 24 17 35 2

5323 2 2 19 2 1 2

48

Figure 7 Results of replicate determinations (indicated by triangles) of benzo[a]pyrene (BAP) in the food supplement test material

Horizontal blue lines represent the arithmetic mean value of replicate measurements and blue bars the reported expanded measurement uncertainty (k=2) The assigned value is

plotted as dotted line Red solid lines mark the range of satisfactory performance (|z|le2) Mean values are given for results outside the displayed data range

ProLab 2011

Laboratory

36

1453

0953

0853

1853

2153

1036

0236

0436

1553

0436

1653

0353

0736

0336

2036

0053

0553

0636

0636

0536

1753

1453

1636

2636

1336

2453

0153

2253

0253

2353

1136

1836

1953

1336

1036

2336

2253

1936

1253

1553

1253

1736

0753

2036

2136

0836

11

an

aly

te c

on

ten

t (micro

gk

g)

55

5

45

4

35

3

25

2

15

1

05

0

573

308

3

Sample St Johns wort extract replicatesMeasurand Benzo[a]pyreneNo of laboratories 48

Assigned value 165 microgkg (Reference value)Target sd 036 microgkgTolerance limits 093 - 237 microgkg (|Z score| lt 200)

Assig

ned va

lue

Limit of tolerance

Limit of tolerance

49

Table 13 Analysis results reported by the participants for the content of benzo[b]fluoranthene (BBF) in the food supplements test material Values are presented as reported

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

3600 32 3 3 31 09 2

3602 173 175 16 17 017

3603 265 252 25 256 025 2

3604 116 108 105 11 028 2

3605 286 225 232 247 074 2

3606 294 273 261 276 046 2

3607 579 579 544 567 113 2

3608 103 1201 876 1036 326 2

3610 173 196 204 191 038 2

3611 546 618 565 576 75 2

3612 3 28 28 29 06 2

3613 28 31 32 31 06 2

3614 135 111 135 127 022 2

3615 4187 3678 4111 4 08 2

3616 2 2 2 2 01 2

3617 33 27 299 035 2

3618 31 31 31 31 05 2

3619 326 324 312 326 064 2

3620 277 28 272 276 069 2

3621 26 25 23 25 04 2

3622 346 336 333 34 052 2

3623 26 28 29 28 02 2

3624

3626 288 28 307 292 117 2

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

5301 29 3 29 29 06 2

5302 297 3 296 298 06 2

5303 2705 2444 2608 2586 22 78

5304 245 254 26 253 85 2

5305 23 23 26 24 15

5306 42 43 4 42 21 2

5307 221 216 241 221

5308 232169 246654 240722 229848

5309 19 22 19 2 0

5310 3 315 325 313 016

5311 262 277 304 281 15 2

5312 27 289 337 299 075 2

5313 321 284 378 328

5314 423 404 385 404 34 083

5315 42 506 416 444 09 2

5316 265 267 277 273 018 2

5317 185 197 195 192 006 2

5318 276 281 279 04 2

5319 396 389 403 396 079 2

5320 623 618 635 625 14

5321 31

5322 24 33 49 33 35 2

5323 38 45 35 45 27 2

50

Figure 8 Results of replicate determinations (indicated by triangles) of benzo[b]fluoranthene (BBF) in the food supplement test material

Horizontal blue lines represent the arithmetic mean value of replicate measurements and blue bars the reported expanded measurement uncertainty (k=2) The assigned value is

plotted as dotted line Red solid lines mark the range of satisfactory performance (|z|le2) Mean values are given for results outside the displayed data range

ProLab 2011

Laboratory

36

0436

1436

0236

1053

1736

1653

0953

0753

0853

0536

2136

0553

0436

0353

0353

1636

0636

2036

2353

1853

1136

1236

2653

0153

0253

1236

1736

1336

0036

1853

2153

1036

1953

1336

2253

2253

2353

1936

1553

1453

0653

1536

0753

2036

0836

11

an

aly

te c

on

ten

t (micro

gk

g)

75

7

65

6

55

5

45

4

35

3

25

2

15

1

05

0

103

6

576

3

Sample St Johns wort extract replicatesMeasurand Benzo[b]fluorantheneNo of laboratories 46

Assigned value 292 microgkg (Reference value)Target sd 060 microgkgTolerance limits 172 - 412 microgkg (|Z score| lt 200)

Assig

ned va

lue

Limit of tolerance

Limit of tolerance

51

Table 14 Analysis results reported by the participants for the content of chrysene (CHR) in the food supplements test material Values are presented as reported

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

3600 5 49 5 5 07 2

3602 32 304 29 304 036

3603 462 469 447 459 048 2

3604 438 428 42 429 119 2

3605 365 41 381 386 085 2

3606 494 411 45 451 124 2

3607 897 774 787 819 18 2

3608 986 887 1168 1014 284 2

3610 418 392 462 424 085 2

3611 833 893 87 865 61 2

3612 6 64 58 61 12 2

3613 41 39 4 4 08 2

3614 272 254 288 271 054 2

3615 605 6028 5964 601 12 2

3616 47 45 49 47 05 2

3617 445 431 389 422 065 2

3618 45 45 45 45 06 2

3619 738 747 72 738 118 2

3620 463 472 446 46 092 2

3621 53 49 51 51 06 2

3622 478 513 505 488 08 2

3623 73 73 72 72 05 2

3624

3626 63 622 634 628 251 2

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

5301 49 49 49 49 1 2

5302 687 683 666 679 136 2

5303 6777 6181 6382 6441 24 70

5304 592 581 6 591 85 2

5305 6 58 64 61 20

5306 49 5 51 49 25 2

5307 524 5 574 524

5308 211488 216782 221146 213243

5309

5310 372 393 394 386 019

5311 43 459 422 437 15 2

5312 934 943 981 953 191 2

5313 793 73 99 838

5314 636 561 565 588 18 0818

5315 547 583 499 542 11 2

5316 292 286 285 282 06 2

5317 52 598 517 545 046 2

5318 368 345 357 05 2

5319 449 471 474 465 116 2

5320 76 924 818 834 99

5321 33

5322 7 7 76 7 35 2

5323 87 73 77 73 37 2

52

Figure 9 Results of replicate determinations (indicated by triangles) of chrysene (CHR) in the food supplement test material

Horizontal blue lines represent the arithmetic mean value of replicate measurements and blue bars the reported expanded measurement uncertainty (k=2) The assigned value is

plotted as dotted line Red solid lines mark the range of satisfactory performance (|z|le2) Mean values are given for results outside the displayed data range

ProLab 2011

Laboratory

53

0836

1453

1636

0253

2153

1836

0553

1036

1336

1736

1036

0453

1136

1836

0636

0336

2053

1936

1653

0136

0036

2253

0636

2153

0753

1553

1753

1453

0436

1536

1253

0536

2653

0353

0253

2236

2336

1953

2336

0753

2053

1353

1236

0836

11

an

aly

te c

on

ten

t (micro

gk

g)

13

12

11

10

9

8

7

6

5

4

3

2

1

0

865

3

Sample St Johns wort extract replicatesMeasurand ChryseneNo of laboratories 45

Assigned value 407 microgkg (Reference value)Target sd 083 microgkgTolerance limits 241 - 573 microgkg (|Z score| lt 200)

Assig

ned va

lue

Limit of tolerance

Limit of tolerance

53

Table 15 Analysis results reported by the participants for the sum of the four marker PAHs in the food supplements test material Values are presented as reported

Laboratory code

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg

3600 128 24 2

3602 79 126

3603 1127 096 2

3604 893 258 2

3605 1091 305 2

3606 1184 238 2

3607 2098 42 2

3608 3336 325 2

3610 1138 17 2

3611 2218 259 2

3612 143 29 2

3613 122 2 2

3614 663 129 2

3615 152 304 2

3616 102 03 2

3617 1252 169 2

3618 128 18 2

3619 157 305 2

3620 1204 12 2

3621 151 19 2

3622 1361 115 2

3623 154 09 2

3624

3626 1409 56 2

Laboratory code

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg

5301 126

5302 37 074 2

5303 13438 92

5304 1251 85 2

5305 125 20

5306 129 65 2

5307 1363

5308 7245598

5309

5310 1127 056

5311 1367 15 2

5312 2042 408 2

5313 1726

5314 1558

5315 177 35 2

5316 96 073 2

5317 1229 066 2

5318 82 05 2

5319 1225 267 2

5320 2309 41

5321

5322 207 35 2

5323 171 103 2

54

ANNEX 9 Results reported for the standard solutions with undisclosed analyte content Table 16 Analysis results reported by the participants for the four marker PAHs in the standard solution in toluene with undisclosed analyte

content Values are presented as reported U measurement uncertainty k coverage factor

benz[a]anthracene benzo[a]pyrene benzo[b]fluoranthene chrysene Laboratory

code Unit Result U k Result U k Result U k Result U k 3600 microgl 38 67 2 25 45 2 211 68 2 275 38 2

3605 microgl 367 184 2 2482 124 2 2592 13 2 235 118 2

3606 microgkg 3763 176 2 2609 142 2 2721 118 2 2539 134 2

3607 microgkg 3706 815 2 2824 565 2 2591 518 2 2587 569 2

3612 microgl 38 08 2 237 11 2 244 18 2 257 02 2

3614 microgl 324 65 2 209 38 2 215 37 2 21 42 2

3616 microgkg 318 07 2 224 14 2 231 26 2 216 1 2

3618 microgkg 417 17 2 284 08 2 292 06 2 297 03 2

3619 microgkg 47 742 2 274 468 2 228 387 2 274 434 2

3620 microgl 495 74 2 294 29 2 321 8 2 321 64 2

3621 microgl 241 22 2 143 13 2 188 16 2 145 14 2

3623 microgkg 476 25 2 311 53 2 269 3 2 297 03 2

3626 microgl 359 38 2 235 23 2 238 27 2 24 18 2

5301 microgkg 426 29 299 289

5302 microgkg 436 87 2 296 59 2 296 59 2 289 58 2

5303 microgl 3721 27 102 22838 19 99 23756 22 99 25486 24 98

5304 microgkg 38 29 2 225 26 2 257 37 2 258 52 2

5305 microgkg 305 225 245 29

5306 microgkg 316 63 2 247 49 2 65 13 2 22 44 2

5307 microgkg 3658 2559 241 2414

5308 microgkg 16854 109324 246654 216782

5311 microgl 374 20 2 279 15 2 268 15 2 272 15 2

5313 microgl 3408 2165 2275 2373

5314 microgkg 395 5 2875 11 3614 34 2905 18

5320 microgl 3267 368 1986 571 28 683 2427 512

5322 microgl 282 35 2 225 35 2 241 35 2 21 35 2

55

Figure 10 Youden plots of the percent deviation of the reported values from the assigned values for the food supplement test sample and the

standard solution in toluene with undisclosed content

(The identity of the respective measurand is given above the figure Results exceeding a difference of more than 100 were omitted)

56

Figure 10 - continued

57

Table 17 Analysis results reported by the participants for the four marker PAHs in the standard solution in acetonitrile with undisclosed

analyte content

Values are presented as reported U measurement uncertainty k coverage factor

benz[a]anthracene benzo[a]pyrene benzo[b]fluoranthene chrysene

Laboratory

code Unit Result U k Result U k Result U k Result U k

3602 microgkg 3675 247 247 244

3603 microgkg 358 086 2 236 028 2 235 028 2 236 057 2

3604 microgl 475 29 2 19 12 2 194 13 2 258 12 2

3608 microgl 3623 078 2 2385 023 2 2343 18 2 2296 092 2

3610 microgkg 369 185 2 25 125 2 251 126 2 244 122 2

3611 microgkg 48 02 2 323 02 2 328 04 2 317 03 2

3613 microgl 332 31 2 261 3 2 249 25 2 239 24 2

3615 microgl 415 2635 2729 2689

3617 microgkg 37 09 2 244 06 2 284 07 2 239 06 2

3622 microgl 37 12 2 25 23 2 257 21 2 238 1 2

3624 microgkg 402 104 2 255 084 2 265 096 2 253 07 2

5306 microgkg 563 113 2 371 74 2 359 72 2 333 67 2

5308 microgkg 168633 102669 232169 211488

5309 microgl 402 268 27

5310 microgl 315 158 25 125 25 125 288 144

5312 microgl 428 043 2 282 028 2 272 027 2 275 028 2

5315 microgkg 387 08 2 256 05 2 251 05 2 253 05 2

5316 microgl 3839 342 2 2485 191 2 2559 168 2 239 51 2

5317 microgl 2466 043 2 2212 034 2 2472 016 2 2146 068 2

5319 microgl 414 41 2 279 28 2 268 27 2 26 31 2

5321 microgkg 476 316 326 328

5322 microgl 2547 35 2

5323 microgkg 405 203 2 239 119 2 255 153 2 201 11 2

58

Figure 11 Youden plots of the percent deviation of the reported values from the assigned values for the food supplement test sample and the

standard solution in acetonitrile with undisclosed content

(The identity of the respective measurand is given above the figure Results exceeding a difference of more than 100 were omitted)

59

Figure 11 - continued

60

ANNEX 10 Table 18 Method performance parameters reported by the participants

Laboratory code

Analyte LOD LOQ Recovery Analyte LOD LOQ Recovery

microgkg microgkg microgkg microgkg 3600 BaA 03 06 978 BaP 03 07 9483602 BaA 007 021 926 BaP 005 015 993603 BaA 0011 05 987 BaP 0005 051 9553604 BaA 02 04 86 BaP 02 04 883605 BaA 0025 005 118 BaP 0025 005 1083606 BaA 001 003 65 BaP 001 003 703607 BaA 021 071 74 BaP 027 089 943608 BaA 016 048 8213 BaP 015 046 83683610 BaA 02 05 85 BaP 005 02 853611 BaA BaP 3612 BaA 1 117 BaP 1 1203613 BaA 01 03 90 BaP 01 03 903614 BaA 02 05 96 BaP 02 05 933615 BaA 005 015 110 BaP 005 015 1053616 BaA 01 05 108 BaP 01 05 753617 BaA 018 037 405 BaP 009 018 3873618 BaA 01 03 63 BaP 01 03 593619 BaA 005 005 59 BaP 033 033 543620 BaA 0009 0018 64 BaP 0005 001 553621 BaA 007 02 84 BaP 007 02 923622 BaA 01 03 1101 BaP 01 03 9853623 BaA 02 08 105 BaP 02 08 1013624 BaA BaP 01 033 8513625 BaA BaP 3626 BaA BaP 5301 BaA 003 01 102 BaP 003 01 1005302 BaA 01 03 78 BaP 01 03 865303 BaA 003 01 62 BaP 003 01 805304 BaA 1 1 70 BaP 1 1 955305 BaA 02 05 85 BaP 02 05 855306 BaA 03 1 699 BaP 03 1 905307 BaA 6537 BaP 81165308 BaA 0088 0265 99 BaP 00262 00785 1005309 BaA 01 08 92 BaP 01 08 925310 BaA 01 03 BaP 01 03 905311 BaA 002 005 80 BaP 002 004 915312 BaA 03 09 105 BaP 03 09 1055313 BaA 04 08 BaP 04 08 5314 BaA 001 002 96 BaP 001 002 9485315 BaA 01 03 50 - 100 BaP 01 03 50 - 100 5316 BaA 002 011 827 BaP 004 019 10445317 BaA 05 1 76 BaP 05 1 755318 BaA BaP 5319 BaA 009 029 951 BaP 011 041 995320 BaA 12 18 946 BaP 06 1 8345321 BaA 03 05 BaP 05 1 5322 BaA 1 2 100 BaP 1 2 1005323 BaA 26 BaP 26

61

ANNEX 10 Table 18 - continued

Laboratory code Analyte LOD LOQ Recovery Analyte LOD LOQ Recovery

microgkg microgkg microgkg microgkg 3600 BbF 03 05 96 CHR 02 03 10153602 BbF 015 045 947 CHR 003 009 9193603 BbF 0014 05 1012 CHR 0004 05 10033604 BbF 02 04 85 CHR 02 04 873605 BbF 005 01 94 CHR 0025 005 863606 BbF 001 003 70 CHR 001 003 653607 BbF 018 06 100 CHR 014 028 943608 BbF 015 046 7979 CHR 015 045 71253610 BbF 005 02 85 CHR 02 05 853611 BbF CHR 3612 BbF 1 118 CHR 1 1173613 BbF 01 03 90 CHR 01 03 903614 BbF 01 04 96 CHR 02 05 953615 BbF 008 025 109 CHR 003 01 1083616 BbF 01 05 78 CHR 01 05 723617 BbF 035 07 38 CHR 012 024 4163618 BbF 01 03 62 CHR 01 03 623619 BbF 016 016 63 CHR 01 01 593620 BbF 0003 0006 55 CHR 001 002 653621 BbF 007 02 103 CHR 007 02 893622 BbF 02 06 963 CHR 03 09 1043623 BbF 02 08 106 CHR 02 08 1193624 BbF CHR 3625 BbF CHR 3626 BbF CHR 5301 BbF 003 01 96 CHR 003 01 1145302 BbF 01 03 84 CHR 01 03 835303 BbF 003 01 78 CHR 003 01 705304 BbF 1 1 86 CHR 1 1 705305 BbF 02 05 90 CHR 02 05 955306 BbF 03 1 1435 CHR 03 1 1185307 BbF 8116 CHR 65375308 BbF 0079 0238 99 CHR 009 026 1005309 BbF 01 08 92 CHR 5310 BbF 07 2 CHR 01 03 5311 BbF 002 004 95 CHR 002 004 895312 BbF 03 09 105 CHR 03 09 1055313 BbF 04 08 CHR 04 08 5314 BbF 001 002 83 CHR 001 002 8185315 BbF 02 05 50 -100 CHR 02 05 50 - 100 5316 BbF 005 024 814 CHR 001 007 9595317 BbF 05 1 73 CHR 05 1 785318 BbF CHR 5319 BbF 011 038 946 CHR 013 043 9675320 BbF 07 09 1194 CHR 23 32 10455321 BbF 05 1 CHR 05 1 5322 BbF 1 2 100 CHR 1 2 1005323 BbF 26 CHR 26

62

ANNEX 11 Table 19 Details of analysis method reported by the participants Data are presented as reported

Laboratory code

Analysis technique

Instrument calibration

Internal standards applied

Sample clean up

Details of sample

clean up

Extraction technique

Details of sample

extraction

3600 6) GC-MS

2) Internal standardisation with labelled substances

Deuterated PAH mix 9 (Ehrenstorfer)

1) Saponification 2) LiquidLiquid partitioning

D) Other Liquidliquid partitioning

3602 2) HPLC-UV-FLD

1) External calibration

3) Gel permeation chromatography

A) Pressurised liquid extraction (PLE)

3603 1) HPLC-FLD

1) External calibration

3) Gel permeation chromatography

D) Other 1 minute with dichloromethane in vortex

3604 2) HPLC-UV-FLD

2) Internal standardisation with labelled substances

DiP D14

5) Donor acceptor complex chromatography

D) Other LL

3605 1) HPLC-FLD

1) External calibration

3) Gel permeation chromatography

C) Soxhlet extraction

3606 7) GC-MSMS

2) Internal standardisation with labelled substances

BaP 13C4 CHR 13C6 BaA 13C6 BbF 13C6

4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

3607 6) GC-MS

2) Internal standardisation with labelled substances

BaA-d12 CHR-d12 BbF-d12 BkF-d12 BaP-d12 ICP-d12 BgP-d12 DiP-d14

1) Saponification

D) Other solvent partitioning

3608 1) HPLC-FLD

1) External calibration

6) Other Liquid extraction

D) Other

Extraction in a rotary agitator and centrifugation

3610 2) HPLC-UV-FLD

1) External calibration

4) Solid phase extraction

B) Sonication

3611

3612 6) GC-MS

2) Internal standardisation with labelled substances

chrysene-d12 dibenzo(ah)anthracene-d14 dibenzo(ai)pyrene-d14

4) Solid phase extraction

B) Sonication

3613 5) LC-MSMS

3) Internal standardisation with unlabelled substances

I use Chrysene-D12 3) Gel permeation chromatography

D) Other extraction with hexan

63

Table 19 - continued

Laboratory code

Analysis technique

Instrument calibration

Internal standards applied

Sample clean up Details of sample

clean up

Extraction technique

Details of sample

extraction

3614 6) GC-MS 1) External calibration

3) Gel permeation chromatography

B) Sonication

3615 1) HPLC-FLD

1) External calibration

6) Other PLE GPC

A) Pressurised liquid extraction (PLE)

3616 6) GC-MS

2) Internal standardisation with labelled substances

Benzo(a)pyrene-13C4 Benzo(a)anthracene-13C6 Benzo(b)fluoranthene-13C6 Chrysene-13C6

2) LiquidLiquid partitioning 4) Solid phase extraction

B) Sonication

3617 1) HPLC-FLD

1) External calibration

4) Solid phase extraction

C) Soxhlet extraction

3618 6) GC-MS

2) Internal standardisation with labelled substances

deuterated standards

1) Saponification 4) Solid phase extraction

D) Other Handshaken with cyclohexane

3619 6) GC-MS

2) Internal standardisation with labelled substances

C13 Labelled US EPA 16

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

D) Other Homegenisation amp saponiofication

3620 9) GC-HRMS

2) Internal standardisation with labelled substances

mix of deuterated PAHs (BAA-D12 CHR-D12 BAP-D12 BBF-D12)

3) Gel permeation chromatography 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

3621 6) GC-MS

2) Internal standardisation with labelled substances

Chrysene D12 Benzo(a)pyrene D12 Benzo(b)fluoranthene D12 Benzo(a)anthracene

2) LiquidLiquid partitioning 4) Solid phase extraction

B) Sonication

3622 1) HPLC-FLD

4) Standard addition method

4) Solid phase extraction

D) Other

Extraction with cyclohexane by heating under reflux

3623 7) GC-MSMS

2) Internal standardisation with labelled substances

C13 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

3624 2) HPLC-UV-FLD

1) External calibration

2) LiquidLiquid partitioning

D) Other hot solvent extraction

3626

64

Table 19 - continued

Laboratory code

Analysis technique

Instrument calibration

Internal standards

applied

Sample clean up

Details of sample

clean up

Extraction technique

Details of sample

extraction

5301 7) GC-MSMS

2) Internal standardisation with labelled substances

EACH SUBSTANCE C13

4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5302 7) GC-MSMS

2) Internal standardisation with labelled substances

Isotope labelled PAHs 13C for each analyte

4) Solid phase extraction

C) Soxhlet extraction

5303 7) GC-MSMS

3) Internal standardisation with unlabelled substances

HAP C13 4) Solid phase extraction

D) Other automate ASE200 DIONEX

5304 7) GC-MSMS

2) Internal standardisation with labelled substances

with C13 for each HAP

2) LiquidLiquid partitioning 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5305 7) GC-MSMS

2) Internal standardisation with labelled substances

C13 isotopiques internal standards

4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5306 7) GC-MSMS

2) Internal standardisation with labelled substances

C13 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5307 6) GC-MS

2) Internal standardisation with labelled substances

1) Saponification 2) LiquidLiquid partitioning

D) Other solvent extraction

5308 1) HPLC-FLD 1) External calibration

4) Solid phase extraction

B) Sonication

5309 1) HPLC-FLD 1) External calibration

4) Solid phase extraction

D) Other only SPE

5310 1) HPLC-FLD 1) External calibration

1) Saponification 2) LiquidLiquid partitioning 3) Gel permeation chromatography

C) Soxhlet extraction

5311 6) GC-MS

2) Internal standardisation with labelled substances

13 C Chrysene 13 C Benz(a)pyrene

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

B) Sonication

5312 1) HPLC-FLD

3) Internal standardisation with unlabelled substances

Benzo(b)chrysen

2) LiquidLiquid partitioning 4) Solid phase extraction

Quechers D) Other Quechers

65

Table 19 - continued

Laboratory code

Analysis technique

Instrument calibration

Internal standards applied

Sample clean up Details of sample

clean up

Extraction technique

Details of sample extraction

5313 6) GC-MS

2) Internal standardisation with labelled substances

Benz(a)anthracene D12 Benzo(b)fluranthene D12 Benzo(a)pyrene D12 Chrysene D12

1) Saponification 2) LiquidLiquid partitioning 3) Gel permeation chromatography

A) Pressurised liquid extraction (PLE)

5314 6) GC-MS

2) Internal standardisation with labelled substances

Benzo(a)pyren d12 2) LiquidLiquid partitioning

D) Other Extraction with

5315 1) HPLC-FLD

3) Internal standardisation with unlabelled substances

Benz(b)chrysen

1) Saponification 2) LiquidLiquid partitioning

D) Other shaker

5316 1) HPLC-FLD

1) External calibration

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

D) Other

LiquidLiquid extraction with cyclohexane and dimethylformamide

5317 7) GC-MSMS

1) External calibration

3) Gel permeation chromatography 6) Other

silica gel D) Other VDLUFA online method

5318

5319 1) HPLC-FLD

3) Internal standardisation with unlabelled substances

Benzo(b)chrysene 3) Gel permeation chromatography

A) Pressurised liquid extraction (PLE)

5320 6) GC-MS

3) Internal standardisation with unlabelled substances

22-binaphthyl

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

D) Other 3 hours refluxing

5321 1) HPLC-FLD

2) Internal standardisation with labelled substances

6-meacutethylchrysegravene

1) Saponification 2) LiquidLiquid partitioning

B) Sonication

5322 6) GC-MS

2) Internal standardisation with labelled substances

naftalene-d8 acenaphtalene-d10 phenanthrene-d10 chrysene-d12 perylene-d12 benzo(e)anthracene-d12

1) Saponification 2) LiquidLiquid partitioning 6) Other

clean-up with silica packed column

C) Soxhlet extraction

5323 1) HPLC-FLD

1) External calibration

4) Solid phase extraction 6) Other

Accelerated Solvent Extraction

A) Pressurised liquid extraction (PLE)

66

European Commission EUR 25245 EN ndash Joint Research Centre ndash Institute for Reference Materials and Measurements Title Report on the 8th inter-laboratory comparison organised by the European Union Reference Laboratory for Polycyclic Aromatic Hydrocarbons - Four marker PAHs in a dry extract of St Johns wort Author(s) Radoslav Lizak Szilard Szilagyi Philippe Verlinde and Thomas WENZL Luxembourg Publications Office of the European Union 2012 ndash 66 pp ndash 201 x 297 cm EUR ndash Scientific and Technical Research series ndash ISSN 1831-9424 ISBN 978-92-79-23337-1 doi 10278759387 Abstract The proficiency test here reported concerned the determination of the contents of four marker polycyclic aromatic hydrocarbons (PAHs) and of their sum in an food supplements test sample The set of marker PAHs consists of benz[a]anthacene benzo[a]pyrene benzo[b]fluoranthene and chrysene Participants to these PT were National Reference Laboratories for PAHs (NRLs-PAHs) and EU official food control laboratories The number of participants was in total 48

The PT was organised according ISO Standard 170432010

The test material used was a commercial dry extract of St Johns wort Participants also received a solution of the same PAHs either in acetonitrile or in toluene for checking their instrument calibration

The results from participants were rated with z-scores and zeta-scores About 68 of the reported results were attributed with z-scores with an absolute value of below two which is the threshold for satisfactory performance

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interests whether private or national

L

A-N

A-252

45-EN

-N

• 1 Summary
• 2 Introduction
• 3 Scope
• 4 Participating Laboratories
• 5 Time frame
• 6 Confidentiality
• 7 Test materials
• • 71 1Preparation of test materials
  • 72 Assigned values and standard deviation for proficiency assessment
  • 73 Homogeneity and stability
  • 74 Sample dispatch
  • • 8 Design of the proficiency test
    • 9 Evaluation of the results
    • • 91 2General
      • 92 21BEvaluation criteria
      • 93 Performance of participants
      • 94 Evaluation of the influence of analysis method
      • 95 Evaluation of the influence of calibration
      • 96 26Evaluation of compliance with legislation
      • • 10 Follow-up actions for underperforming laboratories
        • 11 1Conclusions
        • 12 1Acknowledgements
        • 13 References

43

ANNEX 7 - continued

44

ANNEX 7 - continued

45

ANNEX 8 - Results reported by participants for the food supplements test sample Table 11 Analysis results reported by the participants for the content of benz[a]anthracene (BAA) in the food supplements test material Values are presented as reported

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

3600 31 29 29 3 05 2

3602 199 193 178 19 021

3603 253 253 246 251 022 2

3604 199 209 23 213 071 2

3605 301 3 271 291 076 2

3606 302 288 278 289 064 2

3607 456 416 443 438 096 2

3608 796 695 651 714 149 2

3610 182 192 214 196 039 2

3611 414 504 488 469 96 2

3612 3 34 28 31 06 2

3613 32 33 34 33 07 2

3614 182 182 191 185 037 2

3615 3884 3822 3565 376 0752 2

3616 2 2 2 2 01 2

3617 366 37 35 362 046 2

3618 32 32 32 32 05 2

3619 299 305 297 299 048 2

3620 304 294 302 302 045 2

3621 29 26 29 28 04 2

3622 343 325 318 322 052 2

3623 35 3 33 33 07 2

3624

3626 318 31 314 314 126 2

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

5301 31 3 32 31 06 2

5302 314 319 315 316 063 2

5303 3102 2716 2822 2876 27 62

5304 261 263 26 261 57 2

5305 23 22 26 24 20

5306 22 23 23 22 11 2

5307 488 461 317 461

5308 168633 168544 1562025 1644598

5309 16 19 16 17

5310 3 304 311 305 015

5311 477 452 448 459 15 2

5312 506 63 494 543 134 2

5313 353 301 404 353

5314 431 373 378 394 5 096

5315 596 531 51 545 11 2

5316 224 231 235 235 022 2

5317 216 23 221 222 007 2

5318 064 07 067 01 2

5319 111 184 146 147 029 2

5320 515 525 564 534 48

5321 21 5322 13 87 9 87 35 2

5323 38 33 35 33 16 2

46

Figure 6 Results of replicate determinations (indicated by triangles) of benz[a]anthracene (BAA) in the food supplement test material

Horizontal blue lines represent the arithmetic mean value of replicate measurements and blue bars the reported expanded measurement uncertainty (k=2) The assigned value is

plotted as dotted line Red solid lines mark the range of satisfactory performance (|z|le2) Mean values are given for results outside the displayed data range

ProLab 2011

Laboratory

53

1853

1953

0853

0936

1436

0236

1036

1653

2136

0453

1753

0653

1653

0536

0353

0436

2153

0336

0636

0536

0036

2036

1953

1036

1253

0136

2653

0236

1836

2336

2236

1353

1353

2336

1736

1553

1453

0736

0753

1153

2053

1253

1536

0853

2236

11

an

aly

te c

on

ten

t (micro

gk

g)

10

95

9

85

8

75

7

65

6

55

5

45

4

35

3

25

2

15

1

05

0

102

3 468

7

Sample St Johns wort extract replicatesMeasurand Benz[a]anthraceneNo of laboratories 46

Assigned value 298 microgkg (Reference value)Target sd 062 microgkgTolerance limits 174 - 422 microgkg (|Z score| lt 200)

Assig

ned va

lue

Limit of tolerance

Limit of tolerance

47

Table 12 Analysis results reported by the participants for the content of benzo[a]pyrene (BAP) in the food supplements test material Values are presented as reported

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

3600 16 17 17 17 03 2

3602 118 123 13 124 015

3603 165 162 157 161 015 2

3604 134 154 139 143 04 2

3605 164 166 172 167 057 2

3606 181 159 161 167 031 2

3607 279 266 279 274 055 2

3608 57 687 461 573 226 2

3610 226 216 183 206 042 2

3611 289 325 311 308 36 2

3612 24 22 2 22 05 2

3613 17 18 19 18 05 2

3614 08 08 08 08 016 2

3615 1545 1393 1425 145 029 2

3616 16 16 14 15 03 2

3617 177 171 158 169 023 2

3618 2 2 2 2 02 2

3619 207 207 206 207 075 2

3620 165 169 163 166 017 2

3621 44 47 49 47 05 2

3622 212 208 222 213 038 2

3623 2 21 22 21 03 2

3624 18 178 182 18 031 2

3626 172 173 178 174 07 2

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

5301 18 18 18 18 04 2

5302 188 19 187 188 038 2

5303 1691 1426 149 1535 19 80

5304 145 142 15 146 69 2

5305 15 17 18 16 15

5306 16 17 17 16 08 2

5307 164 157 153 157

5308 102669 109324 109035 107009

5309 1 1 12 11

5310 122 122 124 123 006

5311 199 184 215 2 15 2

5312 258 248 239 248 05 2

5313 199 178 245 207

5314 174 171 172 172 11 0948

5315 253 248 213 238 05 2

5316 177 171 173 17 016 2

5317 255 277 276 269 012 2

5318 114 12 117 02 2

5319 19 228 231 216 043 2

5320 303 3 344 316 78

5321 12 12 12 12

5322 13 17 24 17 35 2

5323 2 2 19 2 1 2

48

Figure 7 Results of replicate determinations (indicated by triangles) of benzo[a]pyrene (BAP) in the food supplement test material

Horizontal blue lines represent the arithmetic mean value of replicate measurements and blue bars the reported expanded measurement uncertainty (k=2) The assigned value is

plotted as dotted line Red solid lines mark the range of satisfactory performance (|z|le2) Mean values are given for results outside the displayed data range

ProLab 2011

Laboratory

36

1453

0953

0853

1853

2153

1036

0236

0436

1553

0436

1653

0353

0736

0336

2036

0053

0553

0636

0636

0536

1753

1453

1636

2636

1336

2453

0153

2253

0253

2353

1136

1836

1953

1336

1036

2336

2253

1936

1253

1553

1253

1736

0753

2036

2136

0836

11

an

aly

te c

on

ten

t (micro

gk

g)

55

5

45

4

35

3

25

2

15

1

05

0

573

308

3

Sample St Johns wort extract replicatesMeasurand Benzo[a]pyreneNo of laboratories 48

Assigned value 165 microgkg (Reference value)Target sd 036 microgkgTolerance limits 093 - 237 microgkg (|Z score| lt 200)

Assig

ned va

lue

Limit of tolerance

Limit of tolerance

49

Table 13 Analysis results reported by the participants for the content of benzo[b]fluoranthene (BBF) in the food supplements test material Values are presented as reported

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

3600 32 3 3 31 09 2

3602 173 175 16 17 017

3603 265 252 25 256 025 2

3604 116 108 105 11 028 2

3605 286 225 232 247 074 2

3606 294 273 261 276 046 2

3607 579 579 544 567 113 2

3608 103 1201 876 1036 326 2

3610 173 196 204 191 038 2

3611 546 618 565 576 75 2

3612 3 28 28 29 06 2

3613 28 31 32 31 06 2

3614 135 111 135 127 022 2

3615 4187 3678 4111 4 08 2

3616 2 2 2 2 01 2

3617 33 27 299 035 2

3618 31 31 31 31 05 2

3619 326 324 312 326 064 2

3620 277 28 272 276 069 2

3621 26 25 23 25 04 2

3622 346 336 333 34 052 2

3623 26 28 29 28 02 2

3624

3626 288 28 307 292 117 2

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

5301 29 3 29 29 06 2

5302 297 3 296 298 06 2

5303 2705 2444 2608 2586 22 78

5304 245 254 26 253 85 2

5305 23 23 26 24 15

5306 42 43 4 42 21 2

5307 221 216 241 221

5308 232169 246654 240722 229848

5309 19 22 19 2 0

5310 3 315 325 313 016

5311 262 277 304 281 15 2

5312 27 289 337 299 075 2

5313 321 284 378 328

5314 423 404 385 404 34 083

5315 42 506 416 444 09 2

5316 265 267 277 273 018 2

5317 185 197 195 192 006 2

5318 276 281 279 04 2

5319 396 389 403 396 079 2

5320 623 618 635 625 14

5321 31

5322 24 33 49 33 35 2

5323 38 45 35 45 27 2

50

Figure 8 Results of replicate determinations (indicated by triangles) of benzo[b]fluoranthene (BBF) in the food supplement test material

Horizontal blue lines represent the arithmetic mean value of replicate measurements and blue bars the reported expanded measurement uncertainty (k=2) The assigned value is

plotted as dotted line Red solid lines mark the range of satisfactory performance (|z|le2) Mean values are given for results outside the displayed data range

ProLab 2011

Laboratory

36

0436

1436

0236

1053

1736

1653

0953

0753

0853

0536

2136

0553

0436

0353

0353

1636

0636

2036

2353

1853

1136

1236

2653

0153

0253

1236

1736

1336

0036

1853

2153

1036

1953

1336

2253

2253

2353

1936

1553

1453

0653

1536

0753

2036

0836

11

an

aly

te c

on

ten

t (micro

gk

g)

75

7

65

6

55

5

45

4

35

3

25

2

15

1

05

0

103

6

576

3

Sample St Johns wort extract replicatesMeasurand Benzo[b]fluorantheneNo of laboratories 46

Assigned value 292 microgkg (Reference value)Target sd 060 microgkgTolerance limits 172 - 412 microgkg (|Z score| lt 200)

Assig

ned va

lue

Limit of tolerance

Limit of tolerance

51

Table 14 Analysis results reported by the participants for the content of chrysene (CHR) in the food supplements test material Values are presented as reported

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

3600 5 49 5 5 07 2

3602 32 304 29 304 036

3603 462 469 447 459 048 2

3604 438 428 42 429 119 2

3605 365 41 381 386 085 2

3606 494 411 45 451 124 2

3607 897 774 787 819 18 2

3608 986 887 1168 1014 284 2

3610 418 392 462 424 085 2

3611 833 893 87 865 61 2

3612 6 64 58 61 12 2

3613 41 39 4 4 08 2

3614 272 254 288 271 054 2

3615 605 6028 5964 601 12 2

3616 47 45 49 47 05 2

3617 445 431 389 422 065 2

3618 45 45 45 45 06 2

3619 738 747 72 738 118 2

3620 463 472 446 46 092 2

3621 53 49 51 51 06 2

3622 478 513 505 488 08 2

3623 73 73 72 72 05 2

3624

3626 63 622 634 628 251 2

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

5301 49 49 49 49 1 2

5302 687 683 666 679 136 2

5303 6777 6181 6382 6441 24 70

5304 592 581 6 591 85 2

5305 6 58 64 61 20

5306 49 5 51 49 25 2

5307 524 5 574 524

5308 211488 216782 221146 213243

5309

5310 372 393 394 386 019

5311 43 459 422 437 15 2

5312 934 943 981 953 191 2

5313 793 73 99 838

5314 636 561 565 588 18 0818

5315 547 583 499 542 11 2

5316 292 286 285 282 06 2

5317 52 598 517 545 046 2

5318 368 345 357 05 2

5319 449 471 474 465 116 2

5320 76 924 818 834 99

5321 33

5322 7 7 76 7 35 2

5323 87 73 77 73 37 2

52

Figure 9 Results of replicate determinations (indicated by triangles) of chrysene (CHR) in the food supplement test material

Horizontal blue lines represent the arithmetic mean value of replicate measurements and blue bars the reported expanded measurement uncertainty (k=2) The assigned value is

plotted as dotted line Red solid lines mark the range of satisfactory performance (|z|le2) Mean values are given for results outside the displayed data range

ProLab 2011

Laboratory

53

0836

1453

1636

0253

2153

1836

0553

1036

1336

1736

1036

0453

1136

1836

0636

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2053

1936

1653

0136

0036

2253

0636

2153

0753

1553

1753

1453

0436

1536

1253

0536

2653

0353

0253

2236

2336

1953

2336

0753

2053

1353

1236

0836

11

an

aly

te c

on

ten

t (micro

gk

g)

13

12

11

10

9

8

7

6

5

4

3

2

1

0

865

3

Sample St Johns wort extract replicatesMeasurand ChryseneNo of laboratories 45

Assigned value 407 microgkg (Reference value)Target sd 083 microgkgTolerance limits 241 - 573 microgkg (|Z score| lt 200)

Assig

ned va

lue

Limit of tolerance

Limit of tolerance

53

Table 15 Analysis results reported by the participants for the sum of the four marker PAHs in the food supplements test material Values are presented as reported

Laboratory code

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg

3600 128 24 2

3602 79 126

3603 1127 096 2

3604 893 258 2

3605 1091 305 2

3606 1184 238 2

3607 2098 42 2

3608 3336 325 2

3610 1138 17 2

3611 2218 259 2

3612 143 29 2

3613 122 2 2

3614 663 129 2

3615 152 304 2

3616 102 03 2

3617 1252 169 2

3618 128 18 2

3619 157 305 2

3620 1204 12 2

3621 151 19 2

3622 1361 115 2

3623 154 09 2

3624

3626 1409 56 2

Laboratory code

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg

5301 126

5302 37 074 2

5303 13438 92

5304 1251 85 2

5305 125 20

5306 129 65 2

5307 1363

5308 7245598

5309

5310 1127 056

5311 1367 15 2

5312 2042 408 2

5313 1726

5314 1558

5315 177 35 2

5316 96 073 2

5317 1229 066 2

5318 82 05 2

5319 1225 267 2

5320 2309 41

5321

5322 207 35 2

5323 171 103 2

54

ANNEX 9 Results reported for the standard solutions with undisclosed analyte content Table 16 Analysis results reported by the participants for the four marker PAHs in the standard solution in toluene with undisclosed analyte

content Values are presented as reported U measurement uncertainty k coverage factor

benz[a]anthracene benzo[a]pyrene benzo[b]fluoranthene chrysene Laboratory

code Unit Result U k Result U k Result U k Result U k 3600 microgl 38 67 2 25 45 2 211 68 2 275 38 2

3605 microgl 367 184 2 2482 124 2 2592 13 2 235 118 2

3606 microgkg 3763 176 2 2609 142 2 2721 118 2 2539 134 2

3607 microgkg 3706 815 2 2824 565 2 2591 518 2 2587 569 2

3612 microgl 38 08 2 237 11 2 244 18 2 257 02 2

3614 microgl 324 65 2 209 38 2 215 37 2 21 42 2

3616 microgkg 318 07 2 224 14 2 231 26 2 216 1 2

3618 microgkg 417 17 2 284 08 2 292 06 2 297 03 2

3619 microgkg 47 742 2 274 468 2 228 387 2 274 434 2

3620 microgl 495 74 2 294 29 2 321 8 2 321 64 2

3621 microgl 241 22 2 143 13 2 188 16 2 145 14 2

3623 microgkg 476 25 2 311 53 2 269 3 2 297 03 2

3626 microgl 359 38 2 235 23 2 238 27 2 24 18 2

5301 microgkg 426 29 299 289

5302 microgkg 436 87 2 296 59 2 296 59 2 289 58 2

5303 microgl 3721 27 102 22838 19 99 23756 22 99 25486 24 98

5304 microgkg 38 29 2 225 26 2 257 37 2 258 52 2

5305 microgkg 305 225 245 29

5306 microgkg 316 63 2 247 49 2 65 13 2 22 44 2

5307 microgkg 3658 2559 241 2414

5308 microgkg 16854 109324 246654 216782

5311 microgl 374 20 2 279 15 2 268 15 2 272 15 2

5313 microgl 3408 2165 2275 2373

5314 microgkg 395 5 2875 11 3614 34 2905 18

5320 microgl 3267 368 1986 571 28 683 2427 512

5322 microgl 282 35 2 225 35 2 241 35 2 21 35 2

55

Figure 10 Youden plots of the percent deviation of the reported values from the assigned values for the food supplement test sample and the

standard solution in toluene with undisclosed content

(The identity of the respective measurand is given above the figure Results exceeding a difference of more than 100 were omitted)

56

Figure 10 - continued

57

Table 17 Analysis results reported by the participants for the four marker PAHs in the standard solution in acetonitrile with undisclosed

analyte content

Values are presented as reported U measurement uncertainty k coverage factor

benz[a]anthracene benzo[a]pyrene benzo[b]fluoranthene chrysene

Laboratory

code Unit Result U k Result U k Result U k Result U k

3602 microgkg 3675 247 247 244

3603 microgkg 358 086 2 236 028 2 235 028 2 236 057 2

3604 microgl 475 29 2 19 12 2 194 13 2 258 12 2

3608 microgl 3623 078 2 2385 023 2 2343 18 2 2296 092 2

3610 microgkg 369 185 2 25 125 2 251 126 2 244 122 2

3611 microgkg 48 02 2 323 02 2 328 04 2 317 03 2

3613 microgl 332 31 2 261 3 2 249 25 2 239 24 2

3615 microgl 415 2635 2729 2689

3617 microgkg 37 09 2 244 06 2 284 07 2 239 06 2

3622 microgl 37 12 2 25 23 2 257 21 2 238 1 2

3624 microgkg 402 104 2 255 084 2 265 096 2 253 07 2

5306 microgkg 563 113 2 371 74 2 359 72 2 333 67 2

5308 microgkg 168633 102669 232169 211488

5309 microgl 402 268 27

5310 microgl 315 158 25 125 25 125 288 144

5312 microgl 428 043 2 282 028 2 272 027 2 275 028 2

5315 microgkg 387 08 2 256 05 2 251 05 2 253 05 2

5316 microgl 3839 342 2 2485 191 2 2559 168 2 239 51 2

5317 microgl 2466 043 2 2212 034 2 2472 016 2 2146 068 2

5319 microgl 414 41 2 279 28 2 268 27 2 26 31 2

5321 microgkg 476 316 326 328

5322 microgl 2547 35 2

5323 microgkg 405 203 2 239 119 2 255 153 2 201 11 2

58

Figure 11 Youden plots of the percent deviation of the reported values from the assigned values for the food supplement test sample and the

standard solution in acetonitrile with undisclosed content

(The identity of the respective measurand is given above the figure Results exceeding a difference of more than 100 were omitted)

59

Figure 11 - continued

60

ANNEX 10 Table 18 Method performance parameters reported by the participants

Laboratory code

Analyte LOD LOQ Recovery Analyte LOD LOQ Recovery

microgkg microgkg microgkg microgkg 3600 BaA 03 06 978 BaP 03 07 9483602 BaA 007 021 926 BaP 005 015 993603 BaA 0011 05 987 BaP 0005 051 9553604 BaA 02 04 86 BaP 02 04 883605 BaA 0025 005 118 BaP 0025 005 1083606 BaA 001 003 65 BaP 001 003 703607 BaA 021 071 74 BaP 027 089 943608 BaA 016 048 8213 BaP 015 046 83683610 BaA 02 05 85 BaP 005 02 853611 BaA BaP 3612 BaA 1 117 BaP 1 1203613 BaA 01 03 90 BaP 01 03 903614 BaA 02 05 96 BaP 02 05 933615 BaA 005 015 110 BaP 005 015 1053616 BaA 01 05 108 BaP 01 05 753617 BaA 018 037 405 BaP 009 018 3873618 BaA 01 03 63 BaP 01 03 593619 BaA 005 005 59 BaP 033 033 543620 BaA 0009 0018 64 BaP 0005 001 553621 BaA 007 02 84 BaP 007 02 923622 BaA 01 03 1101 BaP 01 03 9853623 BaA 02 08 105 BaP 02 08 1013624 BaA BaP 01 033 8513625 BaA BaP 3626 BaA BaP 5301 BaA 003 01 102 BaP 003 01 1005302 BaA 01 03 78 BaP 01 03 865303 BaA 003 01 62 BaP 003 01 805304 BaA 1 1 70 BaP 1 1 955305 BaA 02 05 85 BaP 02 05 855306 BaA 03 1 699 BaP 03 1 905307 BaA 6537 BaP 81165308 BaA 0088 0265 99 BaP 00262 00785 1005309 BaA 01 08 92 BaP 01 08 925310 BaA 01 03 BaP 01 03 905311 BaA 002 005 80 BaP 002 004 915312 BaA 03 09 105 BaP 03 09 1055313 BaA 04 08 BaP 04 08 5314 BaA 001 002 96 BaP 001 002 9485315 BaA 01 03 50 - 100 BaP 01 03 50 - 100 5316 BaA 002 011 827 BaP 004 019 10445317 BaA 05 1 76 BaP 05 1 755318 BaA BaP 5319 BaA 009 029 951 BaP 011 041 995320 BaA 12 18 946 BaP 06 1 8345321 BaA 03 05 BaP 05 1 5322 BaA 1 2 100 BaP 1 2 1005323 BaA 26 BaP 26

61

ANNEX 10 Table 18 - continued

Laboratory code Analyte LOD LOQ Recovery Analyte LOD LOQ Recovery

microgkg microgkg microgkg microgkg 3600 BbF 03 05 96 CHR 02 03 10153602 BbF 015 045 947 CHR 003 009 9193603 BbF 0014 05 1012 CHR 0004 05 10033604 BbF 02 04 85 CHR 02 04 873605 BbF 005 01 94 CHR 0025 005 863606 BbF 001 003 70 CHR 001 003 653607 BbF 018 06 100 CHR 014 028 943608 BbF 015 046 7979 CHR 015 045 71253610 BbF 005 02 85 CHR 02 05 853611 BbF CHR 3612 BbF 1 118 CHR 1 1173613 BbF 01 03 90 CHR 01 03 903614 BbF 01 04 96 CHR 02 05 953615 BbF 008 025 109 CHR 003 01 1083616 BbF 01 05 78 CHR 01 05 723617 BbF 035 07 38 CHR 012 024 4163618 BbF 01 03 62 CHR 01 03 623619 BbF 016 016 63 CHR 01 01 593620 BbF 0003 0006 55 CHR 001 002 653621 BbF 007 02 103 CHR 007 02 893622 BbF 02 06 963 CHR 03 09 1043623 BbF 02 08 106 CHR 02 08 1193624 BbF CHR 3625 BbF CHR 3626 BbF CHR 5301 BbF 003 01 96 CHR 003 01 1145302 BbF 01 03 84 CHR 01 03 835303 BbF 003 01 78 CHR 003 01 705304 BbF 1 1 86 CHR 1 1 705305 BbF 02 05 90 CHR 02 05 955306 BbF 03 1 1435 CHR 03 1 1185307 BbF 8116 CHR 65375308 BbF 0079 0238 99 CHR 009 026 1005309 BbF 01 08 92 CHR 5310 BbF 07 2 CHR 01 03 5311 BbF 002 004 95 CHR 002 004 895312 BbF 03 09 105 CHR 03 09 1055313 BbF 04 08 CHR 04 08 5314 BbF 001 002 83 CHR 001 002 8185315 BbF 02 05 50 -100 CHR 02 05 50 - 100 5316 BbF 005 024 814 CHR 001 007 9595317 BbF 05 1 73 CHR 05 1 785318 BbF CHR 5319 BbF 011 038 946 CHR 013 043 9675320 BbF 07 09 1194 CHR 23 32 10455321 BbF 05 1 CHR 05 1 5322 BbF 1 2 100 CHR 1 2 1005323 BbF 26 CHR 26

62

ANNEX 11 Table 19 Details of analysis method reported by the participants Data are presented as reported

Laboratory code

Analysis technique

Instrument calibration

Internal standards applied

Sample clean up

Details of sample

clean up

Extraction technique

Details of sample

extraction

3600 6) GC-MS

2) Internal standardisation with labelled substances

Deuterated PAH mix 9 (Ehrenstorfer)

1) Saponification 2) LiquidLiquid partitioning

D) Other Liquidliquid partitioning

3602 2) HPLC-UV-FLD

1) External calibration

3) Gel permeation chromatography

A) Pressurised liquid extraction (PLE)

3603 1) HPLC-FLD

1) External calibration

3) Gel permeation chromatography

D) Other 1 minute with dichloromethane in vortex

3604 2) HPLC-UV-FLD

2) Internal standardisation with labelled substances

DiP D14

5) Donor acceptor complex chromatography

D) Other LL

3605 1) HPLC-FLD

1) External calibration

3) Gel permeation chromatography

C) Soxhlet extraction

3606 7) GC-MSMS

2) Internal standardisation with labelled substances

BaP 13C4 CHR 13C6 BaA 13C6 BbF 13C6

4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

3607 6) GC-MS

2) Internal standardisation with labelled substances

BaA-d12 CHR-d12 BbF-d12 BkF-d12 BaP-d12 ICP-d12 BgP-d12 DiP-d14

1) Saponification

D) Other solvent partitioning

3608 1) HPLC-FLD

1) External calibration

6) Other Liquid extraction

D) Other

Extraction in a rotary agitator and centrifugation

3610 2) HPLC-UV-FLD

1) External calibration

4) Solid phase extraction

B) Sonication

3611

3612 6) GC-MS

2) Internal standardisation with labelled substances

chrysene-d12 dibenzo(ah)anthracene-d14 dibenzo(ai)pyrene-d14

4) Solid phase extraction

B) Sonication

3613 5) LC-MSMS

3) Internal standardisation with unlabelled substances

I use Chrysene-D12 3) Gel permeation chromatography

D) Other extraction with hexan

63

Table 19 - continued

Laboratory code

Analysis technique

Instrument calibration

Internal standards applied

Sample clean up Details of sample

clean up

Extraction technique

Details of sample

extraction

3614 6) GC-MS 1) External calibration

3) Gel permeation chromatography

B) Sonication

3615 1) HPLC-FLD

1) External calibration

6) Other PLE GPC

A) Pressurised liquid extraction (PLE)

3616 6) GC-MS

2) Internal standardisation with labelled substances

Benzo(a)pyrene-13C4 Benzo(a)anthracene-13C6 Benzo(b)fluoranthene-13C6 Chrysene-13C6

2) LiquidLiquid partitioning 4) Solid phase extraction

B) Sonication

3617 1) HPLC-FLD

1) External calibration

4) Solid phase extraction

C) Soxhlet extraction

3618 6) GC-MS

2) Internal standardisation with labelled substances

deuterated standards

1) Saponification 4) Solid phase extraction

D) Other Handshaken with cyclohexane

3619 6) GC-MS

2) Internal standardisation with labelled substances

C13 Labelled US EPA 16

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

D) Other Homegenisation amp saponiofication

3620 9) GC-HRMS

2) Internal standardisation with labelled substances

mix of deuterated PAHs (BAA-D12 CHR-D12 BAP-D12 BBF-D12)

3) Gel permeation chromatography 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

3621 6) GC-MS

2) Internal standardisation with labelled substances

Chrysene D12 Benzo(a)pyrene D12 Benzo(b)fluoranthene D12 Benzo(a)anthracene

2) LiquidLiquid partitioning 4) Solid phase extraction

B) Sonication

3622 1) HPLC-FLD

4) Standard addition method

4) Solid phase extraction

D) Other

Extraction with cyclohexane by heating under reflux

3623 7) GC-MSMS

2) Internal standardisation with labelled substances

C13 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

3624 2) HPLC-UV-FLD

1) External calibration

2) LiquidLiquid partitioning

D) Other hot solvent extraction

3626

64

Table 19 - continued

Laboratory code

Analysis technique

Instrument calibration

Internal standards

applied

Sample clean up

Details of sample

clean up

Extraction technique

Details of sample

extraction

5301 7) GC-MSMS

2) Internal standardisation with labelled substances

EACH SUBSTANCE C13

4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5302 7) GC-MSMS

2) Internal standardisation with labelled substances

Isotope labelled PAHs 13C for each analyte

4) Solid phase extraction

C) Soxhlet extraction

5303 7) GC-MSMS

3) Internal standardisation with unlabelled substances

HAP C13 4) Solid phase extraction

D) Other automate ASE200 DIONEX

5304 7) GC-MSMS

2) Internal standardisation with labelled substances

with C13 for each HAP

2) LiquidLiquid partitioning 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5305 7) GC-MSMS

2) Internal standardisation with labelled substances

C13 isotopiques internal standards

4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5306 7) GC-MSMS

2) Internal standardisation with labelled substances

C13 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5307 6) GC-MS

2) Internal standardisation with labelled substances

1) Saponification 2) LiquidLiquid partitioning

D) Other solvent extraction

5308 1) HPLC-FLD 1) External calibration

4) Solid phase extraction

B) Sonication

5309 1) HPLC-FLD 1) External calibration

4) Solid phase extraction

D) Other only SPE

5310 1) HPLC-FLD 1) External calibration

1) Saponification 2) LiquidLiquid partitioning 3) Gel permeation chromatography

C) Soxhlet extraction

5311 6) GC-MS

2) Internal standardisation with labelled substances

13 C Chrysene 13 C Benz(a)pyrene

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

B) Sonication

5312 1) HPLC-FLD

3) Internal standardisation with unlabelled substances

Benzo(b)chrysen

2) LiquidLiquid partitioning 4) Solid phase extraction

Quechers D) Other Quechers

65

Table 19 - continued

Laboratory code

Analysis technique

Instrument calibration

Internal standards applied

Sample clean up Details of sample

clean up

Extraction technique

Details of sample extraction

5313 6) GC-MS

2) Internal standardisation with labelled substances

Benz(a)anthracene D12 Benzo(b)fluranthene D12 Benzo(a)pyrene D12 Chrysene D12

1) Saponification 2) LiquidLiquid partitioning 3) Gel permeation chromatography

A) Pressurised liquid extraction (PLE)

5314 6) GC-MS

2) Internal standardisation with labelled substances

Benzo(a)pyren d12 2) LiquidLiquid partitioning

D) Other Extraction with

5315 1) HPLC-FLD

3) Internal standardisation with unlabelled substances

Benz(b)chrysen

1) Saponification 2) LiquidLiquid partitioning

D) Other shaker

5316 1) HPLC-FLD

1) External calibration

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

D) Other

LiquidLiquid extraction with cyclohexane and dimethylformamide

5317 7) GC-MSMS

1) External calibration

3) Gel permeation chromatography 6) Other

silica gel D) Other VDLUFA online method

5318

5319 1) HPLC-FLD

3) Internal standardisation with unlabelled substances

Benzo(b)chrysene 3) Gel permeation chromatography

A) Pressurised liquid extraction (PLE)

5320 6) GC-MS

3) Internal standardisation with unlabelled substances

22-binaphthyl

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

D) Other 3 hours refluxing

5321 1) HPLC-FLD

2) Internal standardisation with labelled substances

6-meacutethylchrysegravene

1) Saponification 2) LiquidLiquid partitioning

B) Sonication

5322 6) GC-MS

2) Internal standardisation with labelled substances

naftalene-d8 acenaphtalene-d10 phenanthrene-d10 chrysene-d12 perylene-d12 benzo(e)anthracene-d12

1) Saponification 2) LiquidLiquid partitioning 6) Other

clean-up with silica packed column

C) Soxhlet extraction

5323 1) HPLC-FLD

1) External calibration

4) Solid phase extraction 6) Other

Accelerated Solvent Extraction

A) Pressurised liquid extraction (PLE)

66

European Commission EUR 25245 EN ndash Joint Research Centre ndash Institute for Reference Materials and Measurements Title Report on the 8th inter-laboratory comparison organised by the European Union Reference Laboratory for Polycyclic Aromatic Hydrocarbons - Four marker PAHs in a dry extract of St Johns wort Author(s) Radoslav Lizak Szilard Szilagyi Philippe Verlinde and Thomas WENZL Luxembourg Publications Office of the European Union 2012 ndash 66 pp ndash 201 x 297 cm EUR ndash Scientific and Technical Research series ndash ISSN 1831-9424 ISBN 978-92-79-23337-1 doi 10278759387 Abstract The proficiency test here reported concerned the determination of the contents of four marker polycyclic aromatic hydrocarbons (PAHs) and of their sum in an food supplements test sample The set of marker PAHs consists of benz[a]anthacene benzo[a]pyrene benzo[b]fluoranthene and chrysene Participants to these PT were National Reference Laboratories for PAHs (NRLs-PAHs) and EU official food control laboratories The number of participants was in total 48

The PT was organised according ISO Standard 170432010

The test material used was a commercial dry extract of St Johns wort Participants also received a solution of the same PAHs either in acetonitrile or in toluene for checking their instrument calibration

The results from participants were rated with z-scores and zeta-scores About 68 of the reported results were attributed with z-scores with an absolute value of below two which is the threshold for satisfactory performance

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science and technology for the Union Close to the policy-making process it serves

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interests whether private or national

L

A-N

A-252

45-EN

-N

• 1 Summary
• 2 Introduction
• 3 Scope
• 4 Participating Laboratories
• 5 Time frame
• 6 Confidentiality
• 7 Test materials
• • 71 1Preparation of test materials
  • 72 Assigned values and standard deviation for proficiency assessment
  • 73 Homogeneity and stability
  • 74 Sample dispatch
  • • 8 Design of the proficiency test
    • 9 Evaluation of the results
    • • 91 2General
      • 92 21BEvaluation criteria
      • 93 Performance of participants
      • 94 Evaluation of the influence of analysis method
      • 95 Evaluation of the influence of calibration
      • 96 26Evaluation of compliance with legislation
      • • 10 Follow-up actions for underperforming laboratories
        • 11 1Conclusions
        • 12 1Acknowledgements
        • 13 References

44

ANNEX 7 - continued

45

ANNEX 8 - Results reported by participants for the food supplements test sample Table 11 Analysis results reported by the participants for the content of benz[a]anthracene (BAA) in the food supplements test material Values are presented as reported

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

3600 31 29 29 3 05 2

3602 199 193 178 19 021

3603 253 253 246 251 022 2

3604 199 209 23 213 071 2

3605 301 3 271 291 076 2

3606 302 288 278 289 064 2

3607 456 416 443 438 096 2

3608 796 695 651 714 149 2

3610 182 192 214 196 039 2

3611 414 504 488 469 96 2

3612 3 34 28 31 06 2

3613 32 33 34 33 07 2

3614 182 182 191 185 037 2

3615 3884 3822 3565 376 0752 2

3616 2 2 2 2 01 2

3617 366 37 35 362 046 2

3618 32 32 32 32 05 2

3619 299 305 297 299 048 2

3620 304 294 302 302 045 2

3621 29 26 29 28 04 2

3622 343 325 318 322 052 2

3623 35 3 33 33 07 2

3624

3626 318 31 314 314 126 2

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

5301 31 3 32 31 06 2

5302 314 319 315 316 063 2

5303 3102 2716 2822 2876 27 62

5304 261 263 26 261 57 2

5305 23 22 26 24 20

5306 22 23 23 22 11 2

5307 488 461 317 461

5308 168633 168544 1562025 1644598

5309 16 19 16 17

5310 3 304 311 305 015

5311 477 452 448 459 15 2

5312 506 63 494 543 134 2

5313 353 301 404 353

5314 431 373 378 394 5 096

5315 596 531 51 545 11 2

5316 224 231 235 235 022 2

5317 216 23 221 222 007 2

5318 064 07 067 01 2

5319 111 184 146 147 029 2

5320 515 525 564 534 48

5321 21 5322 13 87 9 87 35 2

5323 38 33 35 33 16 2

46

Figure 6 Results of replicate determinations (indicated by triangles) of benz[a]anthracene (BAA) in the food supplement test material

Horizontal blue lines represent the arithmetic mean value of replicate measurements and blue bars the reported expanded measurement uncertainty (k=2) The assigned value is

plotted as dotted line Red solid lines mark the range of satisfactory performance (|z|le2) Mean values are given for results outside the displayed data range

ProLab 2011

Laboratory

53

1853

1953

0853

0936

1436

0236

1036

1653

2136

0453

1753

0653

1653

0536

0353

0436

2153

0336

0636

0536

0036

2036

1953

1036

1253

0136

2653

0236

1836

2336

2236

1353

1353

2336

1736

1553

1453

0736

0753

1153

2053

1253

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11

an

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ten

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75

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65

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55

5

45

4

35

3

25

2

15

1

05

0

102

3 468

7

Sample St Johns wort extract replicatesMeasurand Benz[a]anthraceneNo of laboratories 46

Assigned value 298 microgkg (Reference value)Target sd 062 microgkgTolerance limits 174 - 422 microgkg (|Z score| lt 200)

Assig

ned va

lue

Limit of tolerance

Limit of tolerance

47

Table 12 Analysis results reported by the participants for the content of benzo[a]pyrene (BAP) in the food supplements test material Values are presented as reported

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

3600 16 17 17 17 03 2

3602 118 123 13 124 015

3603 165 162 157 161 015 2

3604 134 154 139 143 04 2

3605 164 166 172 167 057 2

3606 181 159 161 167 031 2

3607 279 266 279 274 055 2

3608 57 687 461 573 226 2

3610 226 216 183 206 042 2

3611 289 325 311 308 36 2

3612 24 22 2 22 05 2

3613 17 18 19 18 05 2

3614 08 08 08 08 016 2

3615 1545 1393 1425 145 029 2

3616 16 16 14 15 03 2

3617 177 171 158 169 023 2

3618 2 2 2 2 02 2

3619 207 207 206 207 075 2

3620 165 169 163 166 017 2

3621 44 47 49 47 05 2

3622 212 208 222 213 038 2

3623 2 21 22 21 03 2

3624 18 178 182 18 031 2

3626 172 173 178 174 07 2

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

5301 18 18 18 18 04 2

5302 188 19 187 188 038 2

5303 1691 1426 149 1535 19 80

5304 145 142 15 146 69 2

5305 15 17 18 16 15

5306 16 17 17 16 08 2

5307 164 157 153 157

5308 102669 109324 109035 107009

5309 1 1 12 11

5310 122 122 124 123 006

5311 199 184 215 2 15 2

5312 258 248 239 248 05 2

5313 199 178 245 207

5314 174 171 172 172 11 0948

5315 253 248 213 238 05 2

5316 177 171 173 17 016 2

5317 255 277 276 269 012 2

5318 114 12 117 02 2

5319 19 228 231 216 043 2

5320 303 3 344 316 78

5321 12 12 12 12

5322 13 17 24 17 35 2

5323 2 2 19 2 1 2

48

Figure 7 Results of replicate determinations (indicated by triangles) of benzo[a]pyrene (BAP) in the food supplement test material

Horizontal blue lines represent the arithmetic mean value of replicate measurements and blue bars the reported expanded measurement uncertainty (k=2) The assigned value is

plotted as dotted line Red solid lines mark the range of satisfactory performance (|z|le2) Mean values are given for results outside the displayed data range

ProLab 2011

Laboratory

36

1453

0953

0853

1853

2153

1036

0236

0436

1553

0436

1653

0353

0736

0336

2036

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0636

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0536

1753

1453

1636

2636

1336

2453

0153

2253

0253

2353

1136

1836

1953

1336

1036

2336

2253

1936

1253

1553

1253

1736

0753

2036

2136

0836

11

an

aly

te c

on

ten

t (micro

gk

g)

55

5

45

4

35

3

25

2

15

1

05

0

573

308

3

Sample St Johns wort extract replicatesMeasurand Benzo[a]pyreneNo of laboratories 48

Assigned value 165 microgkg (Reference value)Target sd 036 microgkgTolerance limits 093 - 237 microgkg (|Z score| lt 200)

Assig

ned va

lue

Limit of tolerance

Limit of tolerance

49

Table 13 Analysis results reported by the participants for the content of benzo[b]fluoranthene (BBF) in the food supplements test material Values are presented as reported

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

3600 32 3 3 31 09 2

3602 173 175 16 17 017

3603 265 252 25 256 025 2

3604 116 108 105 11 028 2

3605 286 225 232 247 074 2

3606 294 273 261 276 046 2

3607 579 579 544 567 113 2

3608 103 1201 876 1036 326 2

3610 173 196 204 191 038 2

3611 546 618 565 576 75 2

3612 3 28 28 29 06 2

3613 28 31 32 31 06 2

3614 135 111 135 127 022 2

3615 4187 3678 4111 4 08 2

3616 2 2 2 2 01 2

3617 33 27 299 035 2

3618 31 31 31 31 05 2

3619 326 324 312 326 064 2

3620 277 28 272 276 069 2

3621 26 25 23 25 04 2

3622 346 336 333 34 052 2

3623 26 28 29 28 02 2

3624

3626 288 28 307 292 117 2

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

5301 29 3 29 29 06 2

5302 297 3 296 298 06 2

5303 2705 2444 2608 2586 22 78

5304 245 254 26 253 85 2

5305 23 23 26 24 15

5306 42 43 4 42 21 2

5307 221 216 241 221

5308 232169 246654 240722 229848

5309 19 22 19 2 0

5310 3 315 325 313 016

5311 262 277 304 281 15 2

5312 27 289 337 299 075 2

5313 321 284 378 328

5314 423 404 385 404 34 083

5315 42 506 416 444 09 2

5316 265 267 277 273 018 2

5317 185 197 195 192 006 2

5318 276 281 279 04 2

5319 396 389 403 396 079 2

5320 623 618 635 625 14

5321 31

5322 24 33 49 33 35 2

5323 38 45 35 45 27 2

50

Figure 8 Results of replicate determinations (indicated by triangles) of benzo[b]fluoranthene (BBF) in the food supplement test material

Horizontal blue lines represent the arithmetic mean value of replicate measurements and blue bars the reported expanded measurement uncertainty (k=2) The assigned value is

plotted as dotted line Red solid lines mark the range of satisfactory performance (|z|le2) Mean values are given for results outside the displayed data range

ProLab 2011

Laboratory

36

0436

1436

0236

1053

1736

1653

0953

0753

0853

0536

2136

0553

0436

0353

0353

1636

0636

2036

2353

1853

1136

1236

2653

0153

0253

1236

1736

1336

0036

1853

2153

1036

1953

1336

2253

2253

2353

1936

1553

1453

0653

1536

0753

2036

0836

11

an

aly

te c

on

ten

t (micro

gk

g)

75

7

65

6

55

5

45

4

35

3

25

2

15

1

05

0

103

6

576

3

Sample St Johns wort extract replicatesMeasurand Benzo[b]fluorantheneNo of laboratories 46

Assigned value 292 microgkg (Reference value)Target sd 060 microgkgTolerance limits 172 - 412 microgkg (|Z score| lt 200)

Assig

ned va

lue

Limit of tolerance

Limit of tolerance

51

Table 14 Analysis results reported by the participants for the content of chrysene (CHR) in the food supplements test material Values are presented as reported

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

3600 5 49 5 5 07 2

3602 32 304 29 304 036

3603 462 469 447 459 048 2

3604 438 428 42 429 119 2

3605 365 41 381 386 085 2

3606 494 411 45 451 124 2

3607 897 774 787 819 18 2

3608 986 887 1168 1014 284 2

3610 418 392 462 424 085 2

3611 833 893 87 865 61 2

3612 6 64 58 61 12 2

3613 41 39 4 4 08 2

3614 272 254 288 271 054 2

3615 605 6028 5964 601 12 2

3616 47 45 49 47 05 2

3617 445 431 389 422 065 2

3618 45 45 45 45 06 2

3619 738 747 72 738 118 2

3620 463 472 446 46 092 2

3621 53 49 51 51 06 2

3622 478 513 505 488 08 2

3623 73 73 72 72 05 2

3624

3626 63 622 634 628 251 2

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

5301 49 49 49 49 1 2

5302 687 683 666 679 136 2

5303 6777 6181 6382 6441 24 70

5304 592 581 6 591 85 2

5305 6 58 64 61 20

5306 49 5 51 49 25 2

5307 524 5 574 524

5308 211488 216782 221146 213243

5309

5310 372 393 394 386 019

5311 43 459 422 437 15 2

5312 934 943 981 953 191 2

5313 793 73 99 838

5314 636 561 565 588 18 0818

5315 547 583 499 542 11 2

5316 292 286 285 282 06 2

5317 52 598 517 545 046 2

5318 368 345 357 05 2

5319 449 471 474 465 116 2

5320 76 924 818 834 99

5321 33

5322 7 7 76 7 35 2

5323 87 73 77 73 37 2

52

Figure 9 Results of replicate determinations (indicated by triangles) of chrysene (CHR) in the food supplement test material

Horizontal blue lines represent the arithmetic mean value of replicate measurements and blue bars the reported expanded measurement uncertainty (k=2) The assigned value is

plotted as dotted line Red solid lines mark the range of satisfactory performance (|z|le2) Mean values are given for results outside the displayed data range

ProLab 2011

Laboratory

53

0836

1453

1636

0253

2153

1836

0553

1036

1336

1736

1036

0453

1136

1836

0636

0336

2053

1936

1653

0136

0036

2253

0636

2153

0753

1553

1753

1453

0436

1536

1253

0536

2653

0353

0253

2236

2336

1953

2336

0753

2053

1353

1236

0836

11

an

aly

te c

on

ten

t (micro

gk

g)

13

12

11

10

9

8

7

6

5

4

3

2

1

0

865

3

Sample St Johns wort extract replicatesMeasurand ChryseneNo of laboratories 45

Assigned value 407 microgkg (Reference value)Target sd 083 microgkgTolerance limits 241 - 573 microgkg (|Z score| lt 200)

Assig

ned va

lue

Limit of tolerance

Limit of tolerance

53

Table 15 Analysis results reported by the participants for the sum of the four marker PAHs in the food supplements test material Values are presented as reported

Laboratory code

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg

3600 128 24 2

3602 79 126

3603 1127 096 2

3604 893 258 2

3605 1091 305 2

3606 1184 238 2

3607 2098 42 2

3608 3336 325 2

3610 1138 17 2

3611 2218 259 2

3612 143 29 2

3613 122 2 2

3614 663 129 2

3615 152 304 2

3616 102 03 2

3617 1252 169 2

3618 128 18 2

3619 157 305 2

3620 1204 12 2

3621 151 19 2

3622 1361 115 2

3623 154 09 2

3624

3626 1409 56 2

Laboratory code

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg

5301 126

5302 37 074 2

5303 13438 92

5304 1251 85 2

5305 125 20

5306 129 65 2

5307 1363

5308 7245598

5309

5310 1127 056

5311 1367 15 2

5312 2042 408 2

5313 1726

5314 1558

5315 177 35 2

5316 96 073 2

5317 1229 066 2

5318 82 05 2

5319 1225 267 2

5320 2309 41

5321

5322 207 35 2

5323 171 103 2

54

ANNEX 9 Results reported for the standard solutions with undisclosed analyte content Table 16 Analysis results reported by the participants for the four marker PAHs in the standard solution in toluene with undisclosed analyte

content Values are presented as reported U measurement uncertainty k coverage factor

benz[a]anthracene benzo[a]pyrene benzo[b]fluoranthene chrysene Laboratory

code Unit Result U k Result U k Result U k Result U k 3600 microgl 38 67 2 25 45 2 211 68 2 275 38 2

3605 microgl 367 184 2 2482 124 2 2592 13 2 235 118 2

3606 microgkg 3763 176 2 2609 142 2 2721 118 2 2539 134 2

3607 microgkg 3706 815 2 2824 565 2 2591 518 2 2587 569 2

3612 microgl 38 08 2 237 11 2 244 18 2 257 02 2

3614 microgl 324 65 2 209 38 2 215 37 2 21 42 2

3616 microgkg 318 07 2 224 14 2 231 26 2 216 1 2

3618 microgkg 417 17 2 284 08 2 292 06 2 297 03 2

3619 microgkg 47 742 2 274 468 2 228 387 2 274 434 2

3620 microgl 495 74 2 294 29 2 321 8 2 321 64 2

3621 microgl 241 22 2 143 13 2 188 16 2 145 14 2

3623 microgkg 476 25 2 311 53 2 269 3 2 297 03 2

3626 microgl 359 38 2 235 23 2 238 27 2 24 18 2

5301 microgkg 426 29 299 289

5302 microgkg 436 87 2 296 59 2 296 59 2 289 58 2

5303 microgl 3721 27 102 22838 19 99 23756 22 99 25486 24 98

5304 microgkg 38 29 2 225 26 2 257 37 2 258 52 2

5305 microgkg 305 225 245 29

5306 microgkg 316 63 2 247 49 2 65 13 2 22 44 2

5307 microgkg 3658 2559 241 2414

5308 microgkg 16854 109324 246654 216782

5311 microgl 374 20 2 279 15 2 268 15 2 272 15 2

5313 microgl 3408 2165 2275 2373

5314 microgkg 395 5 2875 11 3614 34 2905 18

5320 microgl 3267 368 1986 571 28 683 2427 512

5322 microgl 282 35 2 225 35 2 241 35 2 21 35 2

55

Figure 10 Youden plots of the percent deviation of the reported values from the assigned values for the food supplement test sample and the

standard solution in toluene with undisclosed content

(The identity of the respective measurand is given above the figure Results exceeding a difference of more than 100 were omitted)

56

Figure 10 - continued

57

Table 17 Analysis results reported by the participants for the four marker PAHs in the standard solution in acetonitrile with undisclosed

analyte content

Values are presented as reported U measurement uncertainty k coverage factor

benz[a]anthracene benzo[a]pyrene benzo[b]fluoranthene chrysene

Laboratory

code Unit Result U k Result U k Result U k Result U k

3602 microgkg 3675 247 247 244

3603 microgkg 358 086 2 236 028 2 235 028 2 236 057 2

3604 microgl 475 29 2 19 12 2 194 13 2 258 12 2

3608 microgl 3623 078 2 2385 023 2 2343 18 2 2296 092 2

3610 microgkg 369 185 2 25 125 2 251 126 2 244 122 2

3611 microgkg 48 02 2 323 02 2 328 04 2 317 03 2

3613 microgl 332 31 2 261 3 2 249 25 2 239 24 2

3615 microgl 415 2635 2729 2689

3617 microgkg 37 09 2 244 06 2 284 07 2 239 06 2

3622 microgl 37 12 2 25 23 2 257 21 2 238 1 2

3624 microgkg 402 104 2 255 084 2 265 096 2 253 07 2

5306 microgkg 563 113 2 371 74 2 359 72 2 333 67 2

5308 microgkg 168633 102669 232169 211488

5309 microgl 402 268 27

5310 microgl 315 158 25 125 25 125 288 144

5312 microgl 428 043 2 282 028 2 272 027 2 275 028 2

5315 microgkg 387 08 2 256 05 2 251 05 2 253 05 2

5316 microgl 3839 342 2 2485 191 2 2559 168 2 239 51 2

5317 microgl 2466 043 2 2212 034 2 2472 016 2 2146 068 2

5319 microgl 414 41 2 279 28 2 268 27 2 26 31 2

5321 microgkg 476 316 326 328

5322 microgl 2547 35 2

5323 microgkg 405 203 2 239 119 2 255 153 2 201 11 2

58

Figure 11 Youden plots of the percent deviation of the reported values from the assigned values for the food supplement test sample and the

standard solution in acetonitrile with undisclosed content

(The identity of the respective measurand is given above the figure Results exceeding a difference of more than 100 were omitted)

59

Figure 11 - continued

60

ANNEX 10 Table 18 Method performance parameters reported by the participants

Laboratory code

Analyte LOD LOQ Recovery Analyte LOD LOQ Recovery

microgkg microgkg microgkg microgkg 3600 BaA 03 06 978 BaP 03 07 9483602 BaA 007 021 926 BaP 005 015 993603 BaA 0011 05 987 BaP 0005 051 9553604 BaA 02 04 86 BaP 02 04 883605 BaA 0025 005 118 BaP 0025 005 1083606 BaA 001 003 65 BaP 001 003 703607 BaA 021 071 74 BaP 027 089 943608 BaA 016 048 8213 BaP 015 046 83683610 BaA 02 05 85 BaP 005 02 853611 BaA BaP 3612 BaA 1 117 BaP 1 1203613 BaA 01 03 90 BaP 01 03 903614 BaA 02 05 96 BaP 02 05 933615 BaA 005 015 110 BaP 005 015 1053616 BaA 01 05 108 BaP 01 05 753617 BaA 018 037 405 BaP 009 018 3873618 BaA 01 03 63 BaP 01 03 593619 BaA 005 005 59 BaP 033 033 543620 BaA 0009 0018 64 BaP 0005 001 553621 BaA 007 02 84 BaP 007 02 923622 BaA 01 03 1101 BaP 01 03 9853623 BaA 02 08 105 BaP 02 08 1013624 BaA BaP 01 033 8513625 BaA BaP 3626 BaA BaP 5301 BaA 003 01 102 BaP 003 01 1005302 BaA 01 03 78 BaP 01 03 865303 BaA 003 01 62 BaP 003 01 805304 BaA 1 1 70 BaP 1 1 955305 BaA 02 05 85 BaP 02 05 855306 BaA 03 1 699 BaP 03 1 905307 BaA 6537 BaP 81165308 BaA 0088 0265 99 BaP 00262 00785 1005309 BaA 01 08 92 BaP 01 08 925310 BaA 01 03 BaP 01 03 905311 BaA 002 005 80 BaP 002 004 915312 BaA 03 09 105 BaP 03 09 1055313 BaA 04 08 BaP 04 08 5314 BaA 001 002 96 BaP 001 002 9485315 BaA 01 03 50 - 100 BaP 01 03 50 - 100 5316 BaA 002 011 827 BaP 004 019 10445317 BaA 05 1 76 BaP 05 1 755318 BaA BaP 5319 BaA 009 029 951 BaP 011 041 995320 BaA 12 18 946 BaP 06 1 8345321 BaA 03 05 BaP 05 1 5322 BaA 1 2 100 BaP 1 2 1005323 BaA 26 BaP 26

61

ANNEX 10 Table 18 - continued

Laboratory code Analyte LOD LOQ Recovery Analyte LOD LOQ Recovery

microgkg microgkg microgkg microgkg 3600 BbF 03 05 96 CHR 02 03 10153602 BbF 015 045 947 CHR 003 009 9193603 BbF 0014 05 1012 CHR 0004 05 10033604 BbF 02 04 85 CHR 02 04 873605 BbF 005 01 94 CHR 0025 005 863606 BbF 001 003 70 CHR 001 003 653607 BbF 018 06 100 CHR 014 028 943608 BbF 015 046 7979 CHR 015 045 71253610 BbF 005 02 85 CHR 02 05 853611 BbF CHR 3612 BbF 1 118 CHR 1 1173613 BbF 01 03 90 CHR 01 03 903614 BbF 01 04 96 CHR 02 05 953615 BbF 008 025 109 CHR 003 01 1083616 BbF 01 05 78 CHR 01 05 723617 BbF 035 07 38 CHR 012 024 4163618 BbF 01 03 62 CHR 01 03 623619 BbF 016 016 63 CHR 01 01 593620 BbF 0003 0006 55 CHR 001 002 653621 BbF 007 02 103 CHR 007 02 893622 BbF 02 06 963 CHR 03 09 1043623 BbF 02 08 106 CHR 02 08 1193624 BbF CHR 3625 BbF CHR 3626 BbF CHR 5301 BbF 003 01 96 CHR 003 01 1145302 BbF 01 03 84 CHR 01 03 835303 BbF 003 01 78 CHR 003 01 705304 BbF 1 1 86 CHR 1 1 705305 BbF 02 05 90 CHR 02 05 955306 BbF 03 1 1435 CHR 03 1 1185307 BbF 8116 CHR 65375308 BbF 0079 0238 99 CHR 009 026 1005309 BbF 01 08 92 CHR 5310 BbF 07 2 CHR 01 03 5311 BbF 002 004 95 CHR 002 004 895312 BbF 03 09 105 CHR 03 09 1055313 BbF 04 08 CHR 04 08 5314 BbF 001 002 83 CHR 001 002 8185315 BbF 02 05 50 -100 CHR 02 05 50 - 100 5316 BbF 005 024 814 CHR 001 007 9595317 BbF 05 1 73 CHR 05 1 785318 BbF CHR 5319 BbF 011 038 946 CHR 013 043 9675320 BbF 07 09 1194 CHR 23 32 10455321 BbF 05 1 CHR 05 1 5322 BbF 1 2 100 CHR 1 2 1005323 BbF 26 CHR 26

62

ANNEX 11 Table 19 Details of analysis method reported by the participants Data are presented as reported

Laboratory code

Analysis technique

Instrument calibration

Internal standards applied

Sample clean up

Details of sample

clean up

Extraction technique

Details of sample

extraction

3600 6) GC-MS

2) Internal standardisation with labelled substances

Deuterated PAH mix 9 (Ehrenstorfer)

1) Saponification 2) LiquidLiquid partitioning

D) Other Liquidliquid partitioning

3602 2) HPLC-UV-FLD

1) External calibration

3) Gel permeation chromatography

A) Pressurised liquid extraction (PLE)

3603 1) HPLC-FLD

1) External calibration

3) Gel permeation chromatography

D) Other 1 minute with dichloromethane in vortex

3604 2) HPLC-UV-FLD

2) Internal standardisation with labelled substances

DiP D14

5) Donor acceptor complex chromatography

D) Other LL

3605 1) HPLC-FLD

1) External calibration

3) Gel permeation chromatography

C) Soxhlet extraction

3606 7) GC-MSMS

2) Internal standardisation with labelled substances

BaP 13C4 CHR 13C6 BaA 13C6 BbF 13C6

4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

3607 6) GC-MS

2) Internal standardisation with labelled substances

BaA-d12 CHR-d12 BbF-d12 BkF-d12 BaP-d12 ICP-d12 BgP-d12 DiP-d14

1) Saponification

D) Other solvent partitioning

3608 1) HPLC-FLD

1) External calibration

6) Other Liquid extraction

D) Other

Extraction in a rotary agitator and centrifugation

3610 2) HPLC-UV-FLD

1) External calibration

4) Solid phase extraction

B) Sonication

3611

3612 6) GC-MS

2) Internal standardisation with labelled substances

chrysene-d12 dibenzo(ah)anthracene-d14 dibenzo(ai)pyrene-d14

4) Solid phase extraction

B) Sonication

3613 5) LC-MSMS

3) Internal standardisation with unlabelled substances

I use Chrysene-D12 3) Gel permeation chromatography

D) Other extraction with hexan

63

Table 19 - continued

Laboratory code

Analysis technique

Instrument calibration

Internal standards applied

Sample clean up Details of sample

clean up

Extraction technique

Details of sample

extraction

3614 6) GC-MS 1) External calibration

3) Gel permeation chromatography

B) Sonication

3615 1) HPLC-FLD

1) External calibration

6) Other PLE GPC

A) Pressurised liquid extraction (PLE)

3616 6) GC-MS

2) Internal standardisation with labelled substances

Benzo(a)pyrene-13C4 Benzo(a)anthracene-13C6 Benzo(b)fluoranthene-13C6 Chrysene-13C6

2) LiquidLiquid partitioning 4) Solid phase extraction

B) Sonication

3617 1) HPLC-FLD

1) External calibration

4) Solid phase extraction

C) Soxhlet extraction

3618 6) GC-MS

2) Internal standardisation with labelled substances

deuterated standards

1) Saponification 4) Solid phase extraction

D) Other Handshaken with cyclohexane

3619 6) GC-MS

2) Internal standardisation with labelled substances

C13 Labelled US EPA 16

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

D) Other Homegenisation amp saponiofication

3620 9) GC-HRMS

2) Internal standardisation with labelled substances

mix of deuterated PAHs (BAA-D12 CHR-D12 BAP-D12 BBF-D12)

3) Gel permeation chromatography 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

3621 6) GC-MS

2) Internal standardisation with labelled substances

Chrysene D12 Benzo(a)pyrene D12 Benzo(b)fluoranthene D12 Benzo(a)anthracene

2) LiquidLiquid partitioning 4) Solid phase extraction

B) Sonication

3622 1) HPLC-FLD

4) Standard addition method

4) Solid phase extraction

D) Other

Extraction with cyclohexane by heating under reflux

3623 7) GC-MSMS

2) Internal standardisation with labelled substances

C13 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

3624 2) HPLC-UV-FLD

1) External calibration

2) LiquidLiquid partitioning

D) Other hot solvent extraction

3626

64

Table 19 - continued

Laboratory code

Analysis technique

Instrument calibration

Internal standards

applied

Sample clean up

Details of sample

clean up

Extraction technique

Details of sample

extraction

5301 7) GC-MSMS

2) Internal standardisation with labelled substances

EACH SUBSTANCE C13

4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5302 7) GC-MSMS

2) Internal standardisation with labelled substances

Isotope labelled PAHs 13C for each analyte

4) Solid phase extraction

C) Soxhlet extraction

5303 7) GC-MSMS

3) Internal standardisation with unlabelled substances

HAP C13 4) Solid phase extraction

D) Other automate ASE200 DIONEX

5304 7) GC-MSMS

2) Internal standardisation with labelled substances

with C13 for each HAP

2) LiquidLiquid partitioning 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5305 7) GC-MSMS

2) Internal standardisation with labelled substances

C13 isotopiques internal standards

4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5306 7) GC-MSMS

2) Internal standardisation with labelled substances

C13 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5307 6) GC-MS

2) Internal standardisation with labelled substances

1) Saponification 2) LiquidLiquid partitioning

D) Other solvent extraction

5308 1) HPLC-FLD 1) External calibration

4) Solid phase extraction

B) Sonication

5309 1) HPLC-FLD 1) External calibration

4) Solid phase extraction

D) Other only SPE

5310 1) HPLC-FLD 1) External calibration

1) Saponification 2) LiquidLiquid partitioning 3) Gel permeation chromatography

C) Soxhlet extraction

5311 6) GC-MS

2) Internal standardisation with labelled substances

13 C Chrysene 13 C Benz(a)pyrene

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

B) Sonication

5312 1) HPLC-FLD

3) Internal standardisation with unlabelled substances

Benzo(b)chrysen

2) LiquidLiquid partitioning 4) Solid phase extraction

Quechers D) Other Quechers

65

Table 19 - continued

Laboratory code

Analysis technique

Instrument calibration

Internal standards applied

Sample clean up Details of sample

clean up

Extraction technique

Details of sample extraction

5313 6) GC-MS

2) Internal standardisation with labelled substances

Benz(a)anthracene D12 Benzo(b)fluranthene D12 Benzo(a)pyrene D12 Chrysene D12

1) Saponification 2) LiquidLiquid partitioning 3) Gel permeation chromatography

A) Pressurised liquid extraction (PLE)

5314 6) GC-MS

2) Internal standardisation with labelled substances

Benzo(a)pyren d12 2) LiquidLiquid partitioning

D) Other Extraction with

5315 1) HPLC-FLD

3) Internal standardisation with unlabelled substances

Benz(b)chrysen

1) Saponification 2) LiquidLiquid partitioning

D) Other shaker

5316 1) HPLC-FLD

1) External calibration

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

D) Other

LiquidLiquid extraction with cyclohexane and dimethylformamide

5317 7) GC-MSMS

1) External calibration

3) Gel permeation chromatography 6) Other

silica gel D) Other VDLUFA online method

5318

5319 1) HPLC-FLD

3) Internal standardisation with unlabelled substances

Benzo(b)chrysene 3) Gel permeation chromatography

A) Pressurised liquid extraction (PLE)

5320 6) GC-MS

3) Internal standardisation with unlabelled substances

22-binaphthyl

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

D) Other 3 hours refluxing

5321 1) HPLC-FLD

2) Internal standardisation with labelled substances

6-meacutethylchrysegravene

1) Saponification 2) LiquidLiquid partitioning

B) Sonication

5322 6) GC-MS

2) Internal standardisation with labelled substances

naftalene-d8 acenaphtalene-d10 phenanthrene-d10 chrysene-d12 perylene-d12 benzo(e)anthracene-d12

1) Saponification 2) LiquidLiquid partitioning 6) Other

clean-up with silica packed column

C) Soxhlet extraction

5323 1) HPLC-FLD

1) External calibration

4) Solid phase extraction 6) Other

Accelerated Solvent Extraction

A) Pressurised liquid extraction (PLE)

66

European Commission EUR 25245 EN ndash Joint Research Centre ndash Institute for Reference Materials and Measurements Title Report on the 8th inter-laboratory comparison organised by the European Union Reference Laboratory for Polycyclic Aromatic Hydrocarbons - Four marker PAHs in a dry extract of St Johns wort Author(s) Radoslav Lizak Szilard Szilagyi Philippe Verlinde and Thomas WENZL Luxembourg Publications Office of the European Union 2012 ndash 66 pp ndash 201 x 297 cm EUR ndash Scientific and Technical Research series ndash ISSN 1831-9424 ISBN 978-92-79-23337-1 doi 10278759387 Abstract The proficiency test here reported concerned the determination of the contents of four marker polycyclic aromatic hydrocarbons (PAHs) and of their sum in an food supplements test sample The set of marker PAHs consists of benz[a]anthacene benzo[a]pyrene benzo[b]fluoranthene and chrysene Participants to these PT were National Reference Laboratories for PAHs (NRLs-PAHs) and EU official food control laboratories The number of participants was in total 48

The PT was organised according ISO Standard 170432010

The test material used was a commercial dry extract of St Johns wort Participants also received a solution of the same PAHs either in acetonitrile or in toluene for checking their instrument calibration

The results from participants were rated with z-scores and zeta-scores About 68 of the reported results were attributed with z-scores with an absolute value of below two which is the threshold for satisfactory performance

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L

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45-EN

-N

• 1 Summary
• 2 Introduction
• 3 Scope
• 4 Participating Laboratories
• 5 Time frame
• 6 Confidentiality
• 7 Test materials
• • 71 1Preparation of test materials
  • 72 Assigned values and standard deviation for proficiency assessment
  • 73 Homogeneity and stability
  • 74 Sample dispatch
  • • 8 Design of the proficiency test
    • 9 Evaluation of the results
    • • 91 2General
      • 92 21BEvaluation criteria
      • 93 Performance of participants
      • 94 Evaluation of the influence of analysis method
      • 95 Evaluation of the influence of calibration
      • 96 26Evaluation of compliance with legislation
      • • 10 Follow-up actions for underperforming laboratories
        • 11 1Conclusions
        • 12 1Acknowledgements
        • 13 References

45

ANNEX 8 - Results reported by participants for the food supplements test sample Table 11 Analysis results reported by the participants for the content of benz[a]anthracene (BAA) in the food supplements test material Values are presented as reported

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

3600 31 29 29 3 05 2

3602 199 193 178 19 021

3603 253 253 246 251 022 2

3604 199 209 23 213 071 2

3605 301 3 271 291 076 2

3606 302 288 278 289 064 2

3607 456 416 443 438 096 2

3608 796 695 651 714 149 2

3610 182 192 214 196 039 2

3611 414 504 488 469 96 2

3612 3 34 28 31 06 2

3613 32 33 34 33 07 2

3614 182 182 191 185 037 2

3615 3884 3822 3565 376 0752 2

3616 2 2 2 2 01 2

3617 366 37 35 362 046 2

3618 32 32 32 32 05 2

3619 299 305 297 299 048 2

3620 304 294 302 302 045 2

3621 29 26 29 28 04 2

3622 343 325 318 322 052 2

3623 35 3 33 33 07 2

3624

3626 318 31 314 314 126 2

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

5301 31 3 32 31 06 2

5302 314 319 315 316 063 2

5303 3102 2716 2822 2876 27 62

5304 261 263 26 261 57 2

5305 23 22 26 24 20

5306 22 23 23 22 11 2

5307 488 461 317 461

5308 168633 168544 1562025 1644598

5309 16 19 16 17

5310 3 304 311 305 015

5311 477 452 448 459 15 2

5312 506 63 494 543 134 2

5313 353 301 404 353

5314 431 373 378 394 5 096

5315 596 531 51 545 11 2

5316 224 231 235 235 022 2

5317 216 23 221 222 007 2

5318 064 07 067 01 2

5319 111 184 146 147 029 2

5320 515 525 564 534 48

5321 21 5322 13 87 9 87 35 2

5323 38 33 35 33 16 2

46

Figure 6 Results of replicate determinations (indicated by triangles) of benz[a]anthracene (BAA) in the food supplement test material

Horizontal blue lines represent the arithmetic mean value of replicate measurements and blue bars the reported expanded measurement uncertainty (k=2) The assigned value is

plotted as dotted line Red solid lines mark the range of satisfactory performance (|z|le2) Mean values are given for results outside the displayed data range

ProLab 2011

Laboratory

53

1853

1953

0853

0936

1436

0236

1036

1653

2136

0453

1753

0653

1653

0536

0353

0436

2153

0336

0636

0536

0036

2036

1953

1036

1253

0136

2653

0236

1836

2336

2236

1353

1353

2336

1736

1553

1453

0736

0753

1153

2053

1253

1536

0853

2236

11

an

aly

te c

on

ten

t (micro

gk

g)

10

95

9

85

8

75

7

65

6

55

5

45

4

35

3

25

2

15

1

05

0

102

3 468

7

Sample St Johns wort extract replicatesMeasurand Benz[a]anthraceneNo of laboratories 46

Assigned value 298 microgkg (Reference value)Target sd 062 microgkgTolerance limits 174 - 422 microgkg (|Z score| lt 200)

Assig

ned va

lue

Limit of tolerance

Limit of tolerance

47

Table 12 Analysis results reported by the participants for the content of benzo[a]pyrene (BAP) in the food supplements test material Values are presented as reported

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

3600 16 17 17 17 03 2

3602 118 123 13 124 015

3603 165 162 157 161 015 2

3604 134 154 139 143 04 2

3605 164 166 172 167 057 2

3606 181 159 161 167 031 2

3607 279 266 279 274 055 2

3608 57 687 461 573 226 2

3610 226 216 183 206 042 2

3611 289 325 311 308 36 2

3612 24 22 2 22 05 2

3613 17 18 19 18 05 2

3614 08 08 08 08 016 2

3615 1545 1393 1425 145 029 2

3616 16 16 14 15 03 2

3617 177 171 158 169 023 2

3618 2 2 2 2 02 2

3619 207 207 206 207 075 2

3620 165 169 163 166 017 2

3621 44 47 49 47 05 2

3622 212 208 222 213 038 2

3623 2 21 22 21 03 2

3624 18 178 182 18 031 2

3626 172 173 178 174 07 2

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

5301 18 18 18 18 04 2

5302 188 19 187 188 038 2

5303 1691 1426 149 1535 19 80

5304 145 142 15 146 69 2

5305 15 17 18 16 15

5306 16 17 17 16 08 2

5307 164 157 153 157

5308 102669 109324 109035 107009

5309 1 1 12 11

5310 122 122 124 123 006

5311 199 184 215 2 15 2

5312 258 248 239 248 05 2

5313 199 178 245 207

5314 174 171 172 172 11 0948

5315 253 248 213 238 05 2

5316 177 171 173 17 016 2

5317 255 277 276 269 012 2

5318 114 12 117 02 2

5319 19 228 231 216 043 2

5320 303 3 344 316 78

5321 12 12 12 12

5322 13 17 24 17 35 2

5323 2 2 19 2 1 2

48

Figure 7 Results of replicate determinations (indicated by triangles) of benzo[a]pyrene (BAP) in the food supplement test material

Horizontal blue lines represent the arithmetic mean value of replicate measurements and blue bars the reported expanded measurement uncertainty (k=2) The assigned value is

plotted as dotted line Red solid lines mark the range of satisfactory performance (|z|le2) Mean values are given for results outside the displayed data range

ProLab 2011

Laboratory

36

1453

0953

0853

1853

2153

1036

0236

0436

1553

0436

1653

0353

0736

0336

2036

0053

0553

0636

0636

0536

1753

1453

1636

2636

1336

2453

0153

2253

0253

2353

1136

1836

1953

1336

1036

2336

2253

1936

1253

1553

1253

1736

0753

2036

2136

0836

11

an

aly

te c

on

ten

t (micro

gk

g)

55

5

45

4

35

3

25

2

15

1

05

0

573

308

3

Sample St Johns wort extract replicatesMeasurand Benzo[a]pyreneNo of laboratories 48

Assigned value 165 microgkg (Reference value)Target sd 036 microgkgTolerance limits 093 - 237 microgkg (|Z score| lt 200)

Assig

ned va

lue

Limit of tolerance

Limit of tolerance

49

Table 13 Analysis results reported by the participants for the content of benzo[b]fluoranthene (BBF) in the food supplements test material Values are presented as reported

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

3600 32 3 3 31 09 2

3602 173 175 16 17 017

3603 265 252 25 256 025 2

3604 116 108 105 11 028 2

3605 286 225 232 247 074 2

3606 294 273 261 276 046 2

3607 579 579 544 567 113 2

3608 103 1201 876 1036 326 2

3610 173 196 204 191 038 2

3611 546 618 565 576 75 2

3612 3 28 28 29 06 2

3613 28 31 32 31 06 2

3614 135 111 135 127 022 2

3615 4187 3678 4111 4 08 2

3616 2 2 2 2 01 2

3617 33 27 299 035 2

3618 31 31 31 31 05 2

3619 326 324 312 326 064 2

3620 277 28 272 276 069 2

3621 26 25 23 25 04 2

3622 346 336 333 34 052 2

3623 26 28 29 28 02 2

3624

3626 288 28 307 292 117 2

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

5301 29 3 29 29 06 2

5302 297 3 296 298 06 2

5303 2705 2444 2608 2586 22 78

5304 245 254 26 253 85 2

5305 23 23 26 24 15

5306 42 43 4 42 21 2

5307 221 216 241 221

5308 232169 246654 240722 229848

5309 19 22 19 2 0

5310 3 315 325 313 016

5311 262 277 304 281 15 2

5312 27 289 337 299 075 2

5313 321 284 378 328

5314 423 404 385 404 34 083

5315 42 506 416 444 09 2

5316 265 267 277 273 018 2

5317 185 197 195 192 006 2

5318 276 281 279 04 2

5319 396 389 403 396 079 2

5320 623 618 635 625 14

5321 31

5322 24 33 49 33 35 2

5323 38 45 35 45 27 2

50

Figure 8 Results of replicate determinations (indicated by triangles) of benzo[b]fluoranthene (BBF) in the food supplement test material

Horizontal blue lines represent the arithmetic mean value of replicate measurements and blue bars the reported expanded measurement uncertainty (k=2) The assigned value is

plotted as dotted line Red solid lines mark the range of satisfactory performance (|z|le2) Mean values are given for results outside the displayed data range

ProLab 2011

Laboratory

36

0436

1436

0236

1053

1736

1653

0953

0753

0853

0536

2136

0553

0436

0353

0353

1636

0636

2036

2353

1853

1136

1236

2653

0153

0253

1236

1736

1336

0036

1853

2153

1036

1953

1336

2253

2253

2353

1936

1553

1453

0653

1536

0753

2036

0836

11

an

aly

te c

on

ten

t (micro

gk

g)

75

7

65

6

55

5

45

4

35

3

25

2

15

1

05

0

103

6

576

3

Sample St Johns wort extract replicatesMeasurand Benzo[b]fluorantheneNo of laboratories 46

Assigned value 292 microgkg (Reference value)Target sd 060 microgkgTolerance limits 172 - 412 microgkg (|Z score| lt 200)

Assig

ned va

lue

Limit of tolerance

Limit of tolerance

51

Table 14 Analysis results reported by the participants for the content of chrysene (CHR) in the food supplements test material Values are presented as reported

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

3600 5 49 5 5 07 2

3602 32 304 29 304 036

3603 462 469 447 459 048 2

3604 438 428 42 429 119 2

3605 365 41 381 386 085 2

3606 494 411 45 451 124 2

3607 897 774 787 819 18 2

3608 986 887 1168 1014 284 2

3610 418 392 462 424 085 2

3611 833 893 87 865 61 2

3612 6 64 58 61 12 2

3613 41 39 4 4 08 2

3614 272 254 288 271 054 2

3615 605 6028 5964 601 12 2

3616 47 45 49 47 05 2

3617 445 431 389 422 065 2

3618 45 45 45 45 06 2

3619 738 747 72 738 118 2

3620 463 472 446 46 092 2

3621 53 49 51 51 06 2

3622 478 513 505 488 08 2

3623 73 73 72 72 05 2

3624

3626 63 622 634 628 251 2

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

5301 49 49 49 49 1 2

5302 687 683 666 679 136 2

5303 6777 6181 6382 6441 24 70

5304 592 581 6 591 85 2

5305 6 58 64 61 20

5306 49 5 51 49 25 2

5307 524 5 574 524

5308 211488 216782 221146 213243

5309

5310 372 393 394 386 019

5311 43 459 422 437 15 2

5312 934 943 981 953 191 2

5313 793 73 99 838

5314 636 561 565 588 18 0818

5315 547 583 499 542 11 2

5316 292 286 285 282 06 2

5317 52 598 517 545 046 2

5318 368 345 357 05 2

5319 449 471 474 465 116 2

5320 76 924 818 834 99

5321 33

5322 7 7 76 7 35 2

5323 87 73 77 73 37 2

52

Figure 9 Results of replicate determinations (indicated by triangles) of chrysene (CHR) in the food supplement test material

Horizontal blue lines represent the arithmetic mean value of replicate measurements and blue bars the reported expanded measurement uncertainty (k=2) The assigned value is

plotted as dotted line Red solid lines mark the range of satisfactory performance (|z|le2) Mean values are given for results outside the displayed data range

ProLab 2011

Laboratory

53

0836

1453

1636

0253

2153

1836

0553

1036

1336

1736

1036

0453

1136

1836

0636

0336

2053

1936

1653

0136

0036

2253

0636

2153

0753

1553

1753

1453

0436

1536

1253

0536

2653

0353

0253

2236

2336

1953

2336

0753

2053

1353

1236

0836

11

an

aly

te c

on

ten

t (micro

gk

g)

13

12

11

10

9

8

7

6

5

4

3

2

1

0

865

3

Sample St Johns wort extract replicatesMeasurand ChryseneNo of laboratories 45

Assigned value 407 microgkg (Reference value)Target sd 083 microgkgTolerance limits 241 - 573 microgkg (|Z score| lt 200)

Assig

ned va

lue

Limit of tolerance

Limit of tolerance

53

Table 15 Analysis results reported by the participants for the sum of the four marker PAHs in the food supplements test material Values are presented as reported

Laboratory code

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg

3600 128 24 2

3602 79 126

3603 1127 096 2

3604 893 258 2

3605 1091 305 2

3606 1184 238 2

3607 2098 42 2

3608 3336 325 2

3610 1138 17 2

3611 2218 259 2

3612 143 29 2

3613 122 2 2

3614 663 129 2

3615 152 304 2

3616 102 03 2

3617 1252 169 2

3618 128 18 2

3619 157 305 2

3620 1204 12 2

3621 151 19 2

3622 1361 115 2

3623 154 09 2

3624

3626 1409 56 2

Laboratory code

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg

5301 126

5302 37 074 2

5303 13438 92

5304 1251 85 2

5305 125 20

5306 129 65 2

5307 1363

5308 7245598

5309

5310 1127 056

5311 1367 15 2

5312 2042 408 2

5313 1726

5314 1558

5315 177 35 2

5316 96 073 2

5317 1229 066 2

5318 82 05 2

5319 1225 267 2

5320 2309 41

5321

5322 207 35 2

5323 171 103 2

54

ANNEX 9 Results reported for the standard solutions with undisclosed analyte content Table 16 Analysis results reported by the participants for the four marker PAHs in the standard solution in toluene with undisclosed analyte

content Values are presented as reported U measurement uncertainty k coverage factor

benz[a]anthracene benzo[a]pyrene benzo[b]fluoranthene chrysene Laboratory

code Unit Result U k Result U k Result U k Result U k 3600 microgl 38 67 2 25 45 2 211 68 2 275 38 2

3605 microgl 367 184 2 2482 124 2 2592 13 2 235 118 2

3606 microgkg 3763 176 2 2609 142 2 2721 118 2 2539 134 2

3607 microgkg 3706 815 2 2824 565 2 2591 518 2 2587 569 2

3612 microgl 38 08 2 237 11 2 244 18 2 257 02 2

3614 microgl 324 65 2 209 38 2 215 37 2 21 42 2

3616 microgkg 318 07 2 224 14 2 231 26 2 216 1 2

3618 microgkg 417 17 2 284 08 2 292 06 2 297 03 2

3619 microgkg 47 742 2 274 468 2 228 387 2 274 434 2

3620 microgl 495 74 2 294 29 2 321 8 2 321 64 2

3621 microgl 241 22 2 143 13 2 188 16 2 145 14 2

3623 microgkg 476 25 2 311 53 2 269 3 2 297 03 2

3626 microgl 359 38 2 235 23 2 238 27 2 24 18 2

5301 microgkg 426 29 299 289

5302 microgkg 436 87 2 296 59 2 296 59 2 289 58 2

5303 microgl 3721 27 102 22838 19 99 23756 22 99 25486 24 98

5304 microgkg 38 29 2 225 26 2 257 37 2 258 52 2

5305 microgkg 305 225 245 29

5306 microgkg 316 63 2 247 49 2 65 13 2 22 44 2

5307 microgkg 3658 2559 241 2414

5308 microgkg 16854 109324 246654 216782

5311 microgl 374 20 2 279 15 2 268 15 2 272 15 2

5313 microgl 3408 2165 2275 2373

5314 microgkg 395 5 2875 11 3614 34 2905 18

5320 microgl 3267 368 1986 571 28 683 2427 512

5322 microgl 282 35 2 225 35 2 241 35 2 21 35 2

55

Figure 10 Youden plots of the percent deviation of the reported values from the assigned values for the food supplement test sample and the

standard solution in toluene with undisclosed content

(The identity of the respective measurand is given above the figure Results exceeding a difference of more than 100 were omitted)

56

Figure 10 - continued

57

Table 17 Analysis results reported by the participants for the four marker PAHs in the standard solution in acetonitrile with undisclosed

analyte content

Values are presented as reported U measurement uncertainty k coverage factor

benz[a]anthracene benzo[a]pyrene benzo[b]fluoranthene chrysene

Laboratory

code Unit Result U k Result U k Result U k Result U k

3602 microgkg 3675 247 247 244

3603 microgkg 358 086 2 236 028 2 235 028 2 236 057 2

3604 microgl 475 29 2 19 12 2 194 13 2 258 12 2

3608 microgl 3623 078 2 2385 023 2 2343 18 2 2296 092 2

3610 microgkg 369 185 2 25 125 2 251 126 2 244 122 2

3611 microgkg 48 02 2 323 02 2 328 04 2 317 03 2

3613 microgl 332 31 2 261 3 2 249 25 2 239 24 2

3615 microgl 415 2635 2729 2689

3617 microgkg 37 09 2 244 06 2 284 07 2 239 06 2

3622 microgl 37 12 2 25 23 2 257 21 2 238 1 2

3624 microgkg 402 104 2 255 084 2 265 096 2 253 07 2

5306 microgkg 563 113 2 371 74 2 359 72 2 333 67 2

5308 microgkg 168633 102669 232169 211488

5309 microgl 402 268 27

5310 microgl 315 158 25 125 25 125 288 144

5312 microgl 428 043 2 282 028 2 272 027 2 275 028 2

5315 microgkg 387 08 2 256 05 2 251 05 2 253 05 2

5316 microgl 3839 342 2 2485 191 2 2559 168 2 239 51 2

5317 microgl 2466 043 2 2212 034 2 2472 016 2 2146 068 2

5319 microgl 414 41 2 279 28 2 268 27 2 26 31 2

5321 microgkg 476 316 326 328

5322 microgl 2547 35 2

5323 microgkg 405 203 2 239 119 2 255 153 2 201 11 2

58

Figure 11 Youden plots of the percent deviation of the reported values from the assigned values for the food supplement test sample and the

standard solution in acetonitrile with undisclosed content

(The identity of the respective measurand is given above the figure Results exceeding a difference of more than 100 were omitted)

59

Figure 11 - continued

60

ANNEX 10 Table 18 Method performance parameters reported by the participants

Laboratory code

Analyte LOD LOQ Recovery Analyte LOD LOQ Recovery

microgkg microgkg microgkg microgkg 3600 BaA 03 06 978 BaP 03 07 9483602 BaA 007 021 926 BaP 005 015 993603 BaA 0011 05 987 BaP 0005 051 9553604 BaA 02 04 86 BaP 02 04 883605 BaA 0025 005 118 BaP 0025 005 1083606 BaA 001 003 65 BaP 001 003 703607 BaA 021 071 74 BaP 027 089 943608 BaA 016 048 8213 BaP 015 046 83683610 BaA 02 05 85 BaP 005 02 853611 BaA BaP 3612 BaA 1 117 BaP 1 1203613 BaA 01 03 90 BaP 01 03 903614 BaA 02 05 96 BaP 02 05 933615 BaA 005 015 110 BaP 005 015 1053616 BaA 01 05 108 BaP 01 05 753617 BaA 018 037 405 BaP 009 018 3873618 BaA 01 03 63 BaP 01 03 593619 BaA 005 005 59 BaP 033 033 543620 BaA 0009 0018 64 BaP 0005 001 553621 BaA 007 02 84 BaP 007 02 923622 BaA 01 03 1101 BaP 01 03 9853623 BaA 02 08 105 BaP 02 08 1013624 BaA BaP 01 033 8513625 BaA BaP 3626 BaA BaP 5301 BaA 003 01 102 BaP 003 01 1005302 BaA 01 03 78 BaP 01 03 865303 BaA 003 01 62 BaP 003 01 805304 BaA 1 1 70 BaP 1 1 955305 BaA 02 05 85 BaP 02 05 855306 BaA 03 1 699 BaP 03 1 905307 BaA 6537 BaP 81165308 BaA 0088 0265 99 BaP 00262 00785 1005309 BaA 01 08 92 BaP 01 08 925310 BaA 01 03 BaP 01 03 905311 BaA 002 005 80 BaP 002 004 915312 BaA 03 09 105 BaP 03 09 1055313 BaA 04 08 BaP 04 08 5314 BaA 001 002 96 BaP 001 002 9485315 BaA 01 03 50 - 100 BaP 01 03 50 - 100 5316 BaA 002 011 827 BaP 004 019 10445317 BaA 05 1 76 BaP 05 1 755318 BaA BaP 5319 BaA 009 029 951 BaP 011 041 995320 BaA 12 18 946 BaP 06 1 8345321 BaA 03 05 BaP 05 1 5322 BaA 1 2 100 BaP 1 2 1005323 BaA 26 BaP 26

61

ANNEX 10 Table 18 - continued

Laboratory code Analyte LOD LOQ Recovery Analyte LOD LOQ Recovery

microgkg microgkg microgkg microgkg 3600 BbF 03 05 96 CHR 02 03 10153602 BbF 015 045 947 CHR 003 009 9193603 BbF 0014 05 1012 CHR 0004 05 10033604 BbF 02 04 85 CHR 02 04 873605 BbF 005 01 94 CHR 0025 005 863606 BbF 001 003 70 CHR 001 003 653607 BbF 018 06 100 CHR 014 028 943608 BbF 015 046 7979 CHR 015 045 71253610 BbF 005 02 85 CHR 02 05 853611 BbF CHR 3612 BbF 1 118 CHR 1 1173613 BbF 01 03 90 CHR 01 03 903614 BbF 01 04 96 CHR 02 05 953615 BbF 008 025 109 CHR 003 01 1083616 BbF 01 05 78 CHR 01 05 723617 BbF 035 07 38 CHR 012 024 4163618 BbF 01 03 62 CHR 01 03 623619 BbF 016 016 63 CHR 01 01 593620 BbF 0003 0006 55 CHR 001 002 653621 BbF 007 02 103 CHR 007 02 893622 BbF 02 06 963 CHR 03 09 1043623 BbF 02 08 106 CHR 02 08 1193624 BbF CHR 3625 BbF CHR 3626 BbF CHR 5301 BbF 003 01 96 CHR 003 01 1145302 BbF 01 03 84 CHR 01 03 835303 BbF 003 01 78 CHR 003 01 705304 BbF 1 1 86 CHR 1 1 705305 BbF 02 05 90 CHR 02 05 955306 BbF 03 1 1435 CHR 03 1 1185307 BbF 8116 CHR 65375308 BbF 0079 0238 99 CHR 009 026 1005309 BbF 01 08 92 CHR 5310 BbF 07 2 CHR 01 03 5311 BbF 002 004 95 CHR 002 004 895312 BbF 03 09 105 CHR 03 09 1055313 BbF 04 08 CHR 04 08 5314 BbF 001 002 83 CHR 001 002 8185315 BbF 02 05 50 -100 CHR 02 05 50 - 100 5316 BbF 005 024 814 CHR 001 007 9595317 BbF 05 1 73 CHR 05 1 785318 BbF CHR 5319 BbF 011 038 946 CHR 013 043 9675320 BbF 07 09 1194 CHR 23 32 10455321 BbF 05 1 CHR 05 1 5322 BbF 1 2 100 CHR 1 2 1005323 BbF 26 CHR 26

62

ANNEX 11 Table 19 Details of analysis method reported by the participants Data are presented as reported

Laboratory code

Analysis technique

Instrument calibration

Internal standards applied

Sample clean up

Details of sample

clean up

Extraction technique

Details of sample

extraction

3600 6) GC-MS

2) Internal standardisation with labelled substances

Deuterated PAH mix 9 (Ehrenstorfer)

1) Saponification 2) LiquidLiquid partitioning

D) Other Liquidliquid partitioning

3602 2) HPLC-UV-FLD

1) External calibration

3) Gel permeation chromatography

A) Pressurised liquid extraction (PLE)

3603 1) HPLC-FLD

1) External calibration

3) Gel permeation chromatography

D) Other 1 minute with dichloromethane in vortex

3604 2) HPLC-UV-FLD

2) Internal standardisation with labelled substances

DiP D14

5) Donor acceptor complex chromatography

D) Other LL

3605 1) HPLC-FLD

1) External calibration

3) Gel permeation chromatography

C) Soxhlet extraction

3606 7) GC-MSMS

2) Internal standardisation with labelled substances

BaP 13C4 CHR 13C6 BaA 13C6 BbF 13C6

4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

3607 6) GC-MS

2) Internal standardisation with labelled substances

BaA-d12 CHR-d12 BbF-d12 BkF-d12 BaP-d12 ICP-d12 BgP-d12 DiP-d14

1) Saponification

D) Other solvent partitioning

3608 1) HPLC-FLD

1) External calibration

6) Other Liquid extraction

D) Other

Extraction in a rotary agitator and centrifugation

3610 2) HPLC-UV-FLD

1) External calibration

4) Solid phase extraction

B) Sonication

3611

3612 6) GC-MS

2) Internal standardisation with labelled substances

chrysene-d12 dibenzo(ah)anthracene-d14 dibenzo(ai)pyrene-d14

4) Solid phase extraction

B) Sonication

3613 5) LC-MSMS

3) Internal standardisation with unlabelled substances

I use Chrysene-D12 3) Gel permeation chromatography

D) Other extraction with hexan

63

Table 19 - continued

Laboratory code

Analysis technique

Instrument calibration

Internal standards applied

Sample clean up Details of sample

clean up

Extraction technique

Details of sample

extraction

3614 6) GC-MS 1) External calibration

3) Gel permeation chromatography

B) Sonication

3615 1) HPLC-FLD

1) External calibration

6) Other PLE GPC

A) Pressurised liquid extraction (PLE)

3616 6) GC-MS

2) Internal standardisation with labelled substances

Benzo(a)pyrene-13C4 Benzo(a)anthracene-13C6 Benzo(b)fluoranthene-13C6 Chrysene-13C6

2) LiquidLiquid partitioning 4) Solid phase extraction

B) Sonication

3617 1) HPLC-FLD

1) External calibration

4) Solid phase extraction

C) Soxhlet extraction

3618 6) GC-MS

2) Internal standardisation with labelled substances

deuterated standards

1) Saponification 4) Solid phase extraction

D) Other Handshaken with cyclohexane

3619 6) GC-MS

2) Internal standardisation with labelled substances

C13 Labelled US EPA 16

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

D) Other Homegenisation amp saponiofication

3620 9) GC-HRMS

2) Internal standardisation with labelled substances

mix of deuterated PAHs (BAA-D12 CHR-D12 BAP-D12 BBF-D12)

3) Gel permeation chromatography 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

3621 6) GC-MS

2) Internal standardisation with labelled substances

Chrysene D12 Benzo(a)pyrene D12 Benzo(b)fluoranthene D12 Benzo(a)anthracene

2) LiquidLiquid partitioning 4) Solid phase extraction

B) Sonication

3622 1) HPLC-FLD

4) Standard addition method

4) Solid phase extraction

D) Other

Extraction with cyclohexane by heating under reflux

3623 7) GC-MSMS

2) Internal standardisation with labelled substances

C13 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

3624 2) HPLC-UV-FLD

1) External calibration

2) LiquidLiquid partitioning

D) Other hot solvent extraction

3626

64

Table 19 - continued

Laboratory code

Analysis technique

Instrument calibration

Internal standards

applied

Sample clean up

Details of sample

clean up

Extraction technique

Details of sample

extraction

5301 7) GC-MSMS

2) Internal standardisation with labelled substances

EACH SUBSTANCE C13

4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5302 7) GC-MSMS

2) Internal standardisation with labelled substances

Isotope labelled PAHs 13C for each analyte

4) Solid phase extraction

C) Soxhlet extraction

5303 7) GC-MSMS

3) Internal standardisation with unlabelled substances

HAP C13 4) Solid phase extraction

D) Other automate ASE200 DIONEX

5304 7) GC-MSMS

2) Internal standardisation with labelled substances

with C13 for each HAP

2) LiquidLiquid partitioning 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5305 7) GC-MSMS

2) Internal standardisation with labelled substances

C13 isotopiques internal standards

4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5306 7) GC-MSMS

2) Internal standardisation with labelled substances

C13 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5307 6) GC-MS

2) Internal standardisation with labelled substances

1) Saponification 2) LiquidLiquid partitioning

D) Other solvent extraction

5308 1) HPLC-FLD 1) External calibration

4) Solid phase extraction

B) Sonication

5309 1) HPLC-FLD 1) External calibration

4) Solid phase extraction

D) Other only SPE

5310 1) HPLC-FLD 1) External calibration

1) Saponification 2) LiquidLiquid partitioning 3) Gel permeation chromatography

C) Soxhlet extraction

5311 6) GC-MS

2) Internal standardisation with labelled substances

13 C Chrysene 13 C Benz(a)pyrene

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

B) Sonication

5312 1) HPLC-FLD

3) Internal standardisation with unlabelled substances

Benzo(b)chrysen

2) LiquidLiquid partitioning 4) Solid phase extraction

Quechers D) Other Quechers

65

Table 19 - continued

Laboratory code

Analysis technique

Instrument calibration

Internal standards applied

Sample clean up Details of sample

clean up

Extraction technique

Details of sample extraction

5313 6) GC-MS

2) Internal standardisation with labelled substances

Benz(a)anthracene D12 Benzo(b)fluranthene D12 Benzo(a)pyrene D12 Chrysene D12

1) Saponification 2) LiquidLiquid partitioning 3) Gel permeation chromatography

A) Pressurised liquid extraction (PLE)

5314 6) GC-MS

2) Internal standardisation with labelled substances

Benzo(a)pyren d12 2) LiquidLiquid partitioning

D) Other Extraction with

5315 1) HPLC-FLD

3) Internal standardisation with unlabelled substances

Benz(b)chrysen

1) Saponification 2) LiquidLiquid partitioning

D) Other shaker

5316 1) HPLC-FLD

1) External calibration

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

D) Other

LiquidLiquid extraction with cyclohexane and dimethylformamide

5317 7) GC-MSMS

1) External calibration

3) Gel permeation chromatography 6) Other

silica gel D) Other VDLUFA online method

5318

5319 1) HPLC-FLD

3) Internal standardisation with unlabelled substances

Benzo(b)chrysene 3) Gel permeation chromatography

A) Pressurised liquid extraction (PLE)

5320 6) GC-MS

3) Internal standardisation with unlabelled substances

22-binaphthyl

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

D) Other 3 hours refluxing

5321 1) HPLC-FLD

2) Internal standardisation with labelled substances

6-meacutethylchrysegravene

1) Saponification 2) LiquidLiquid partitioning

B) Sonication

5322 6) GC-MS

2) Internal standardisation with labelled substances

naftalene-d8 acenaphtalene-d10 phenanthrene-d10 chrysene-d12 perylene-d12 benzo(e)anthracene-d12

1) Saponification 2) LiquidLiquid partitioning 6) Other

clean-up with silica packed column

C) Soxhlet extraction

5323 1) HPLC-FLD

1) External calibration

4) Solid phase extraction 6) Other

Accelerated Solvent Extraction

A) Pressurised liquid extraction (PLE)

66

European Commission EUR 25245 EN ndash Joint Research Centre ndash Institute for Reference Materials and Measurements Title Report on the 8th inter-laboratory comparison organised by the European Union Reference Laboratory for Polycyclic Aromatic Hydrocarbons - Four marker PAHs in a dry extract of St Johns wort Author(s) Radoslav Lizak Szilard Szilagyi Philippe Verlinde and Thomas WENZL Luxembourg Publications Office of the European Union 2012 ndash 66 pp ndash 201 x 297 cm EUR ndash Scientific and Technical Research series ndash ISSN 1831-9424 ISBN 978-92-79-23337-1 doi 10278759387 Abstract The proficiency test here reported concerned the determination of the contents of four marker polycyclic aromatic hydrocarbons (PAHs) and of their sum in an food supplements test sample The set of marker PAHs consists of benz[a]anthacene benzo[a]pyrene benzo[b]fluoranthene and chrysene Participants to these PT were National Reference Laboratories for PAHs (NRLs-PAHs) and EU official food control laboratories The number of participants was in total 48

The PT was organised according ISO Standard 170432010

The test material used was a commercial dry extract of St Johns wort Participants also received a solution of the same PAHs either in acetonitrile or in toluene for checking their instrument calibration

The results from participants were rated with z-scores and zeta-scores About 68 of the reported results were attributed with z-scores with an absolute value of below two which is the threshold for satisfactory performance

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interests whether private or national

L

A-N

A-252

45-EN

-N

• 1 Summary
• 2 Introduction
• 3 Scope
• 4 Participating Laboratories
• 5 Time frame
• 6 Confidentiality
• 7 Test materials
• • 71 1Preparation of test materials
  • 72 Assigned values and standard deviation for proficiency assessment
  • 73 Homogeneity and stability
  • 74 Sample dispatch
  • • 8 Design of the proficiency test
    • 9 Evaluation of the results
    • • 91 2General
      • 92 21BEvaluation criteria
      • 93 Performance of participants
      • 94 Evaluation of the influence of analysis method
      • 95 Evaluation of the influence of calibration
      • 96 26Evaluation of compliance with legislation
      • • 10 Follow-up actions for underperforming laboratories
        • 11 1Conclusions
        • 12 1Acknowledgements
        • 13 References

46

Figure 6 Results of replicate determinations (indicated by triangles) of benz[a]anthracene (BAA) in the food supplement test material

Horizontal blue lines represent the arithmetic mean value of replicate measurements and blue bars the reported expanded measurement uncertainty (k=2) The assigned value is

plotted as dotted line Red solid lines mark the range of satisfactory performance (|z|le2) Mean values are given for results outside the displayed data range

ProLab 2011

Laboratory

53

1853

1953

0853

0936

1436

0236

1036

1653

2136

0453

1753

0653

1653

0536

0353

0436

2153

0336

0636

0536

0036

2036

1953

1036

1253

0136

2653

0236

1836

2336

2236

1353

1353

2336

1736

1553

1453

0736

0753

1153

2053

1253

1536

0853

2236

11

an

aly

te c

on

ten

t (micro

gk

g)

10

95

9

85

8

75

7

65

6

55

5

45

4

35

3

25

2

15

1

05

0

102

3 468

7

Sample St Johns wort extract replicatesMeasurand Benz[a]anthraceneNo of laboratories 46

Assigned value 298 microgkg (Reference value)Target sd 062 microgkgTolerance limits 174 - 422 microgkg (|Z score| lt 200)

Assig

ned va

lue

Limit of tolerance

Limit of tolerance

47

Table 12 Analysis results reported by the participants for the content of benzo[a]pyrene (BAP) in the food supplements test material Values are presented as reported

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

3600 16 17 17 17 03 2

3602 118 123 13 124 015

3603 165 162 157 161 015 2

3604 134 154 139 143 04 2

3605 164 166 172 167 057 2

3606 181 159 161 167 031 2

3607 279 266 279 274 055 2

3608 57 687 461 573 226 2

3610 226 216 183 206 042 2

3611 289 325 311 308 36 2

3612 24 22 2 22 05 2

3613 17 18 19 18 05 2

3614 08 08 08 08 016 2

3615 1545 1393 1425 145 029 2

3616 16 16 14 15 03 2

3617 177 171 158 169 023 2

3618 2 2 2 2 02 2

3619 207 207 206 207 075 2

3620 165 169 163 166 017 2

3621 44 47 49 47 05 2

3622 212 208 222 213 038 2

3623 2 21 22 21 03 2

3624 18 178 182 18 031 2

3626 172 173 178 174 07 2

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

5301 18 18 18 18 04 2

5302 188 19 187 188 038 2

5303 1691 1426 149 1535 19 80

5304 145 142 15 146 69 2

5305 15 17 18 16 15

5306 16 17 17 16 08 2

5307 164 157 153 157

5308 102669 109324 109035 107009

5309 1 1 12 11

5310 122 122 124 123 006

5311 199 184 215 2 15 2

5312 258 248 239 248 05 2

5313 199 178 245 207

5314 174 171 172 172 11 0948

5315 253 248 213 238 05 2

5316 177 171 173 17 016 2

5317 255 277 276 269 012 2

5318 114 12 117 02 2

5319 19 228 231 216 043 2

5320 303 3 344 316 78

5321 12 12 12 12

5322 13 17 24 17 35 2

5323 2 2 19 2 1 2

48

Figure 7 Results of replicate determinations (indicated by triangles) of benzo[a]pyrene (BAP) in the food supplement test material

Horizontal blue lines represent the arithmetic mean value of replicate measurements and blue bars the reported expanded measurement uncertainty (k=2) The assigned value is

plotted as dotted line Red solid lines mark the range of satisfactory performance (|z|le2) Mean values are given for results outside the displayed data range

ProLab 2011

Laboratory

36

1453

0953

0853

1853

2153

1036

0236

0436

1553

0436

1653

0353

0736

0336

2036

0053

0553

0636

0636

0536

1753

1453

1636

2636

1336

2453

0153

2253

0253

2353

1136

1836

1953

1336

1036

2336

2253

1936

1253

1553

1253

1736

0753

2036

2136

0836

11

an

aly

te c

on

ten

t (micro

gk

g)

55

5

45

4

35

3

25

2

15

1

05

0

573

308

3

Sample St Johns wort extract replicatesMeasurand Benzo[a]pyreneNo of laboratories 48

Assigned value 165 microgkg (Reference value)Target sd 036 microgkgTolerance limits 093 - 237 microgkg (|Z score| lt 200)

Assig

ned va

lue

Limit of tolerance

Limit of tolerance

49

Table 13 Analysis results reported by the participants for the content of benzo[b]fluoranthene (BBF) in the food supplements test material Values are presented as reported

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

3600 32 3 3 31 09 2

3602 173 175 16 17 017

3603 265 252 25 256 025 2

3604 116 108 105 11 028 2

3605 286 225 232 247 074 2

3606 294 273 261 276 046 2

3607 579 579 544 567 113 2

3608 103 1201 876 1036 326 2

3610 173 196 204 191 038 2

3611 546 618 565 576 75 2

3612 3 28 28 29 06 2

3613 28 31 32 31 06 2

3614 135 111 135 127 022 2

3615 4187 3678 4111 4 08 2

3616 2 2 2 2 01 2

3617 33 27 299 035 2

3618 31 31 31 31 05 2

3619 326 324 312 326 064 2

3620 277 28 272 276 069 2

3621 26 25 23 25 04 2

3622 346 336 333 34 052 2

3623 26 28 29 28 02 2

3624

3626 288 28 307 292 117 2

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

5301 29 3 29 29 06 2

5302 297 3 296 298 06 2

5303 2705 2444 2608 2586 22 78

5304 245 254 26 253 85 2

5305 23 23 26 24 15

5306 42 43 4 42 21 2

5307 221 216 241 221

5308 232169 246654 240722 229848

5309 19 22 19 2 0

5310 3 315 325 313 016

5311 262 277 304 281 15 2

5312 27 289 337 299 075 2

5313 321 284 378 328

5314 423 404 385 404 34 083

5315 42 506 416 444 09 2

5316 265 267 277 273 018 2

5317 185 197 195 192 006 2

5318 276 281 279 04 2

5319 396 389 403 396 079 2

5320 623 618 635 625 14

5321 31

5322 24 33 49 33 35 2

5323 38 45 35 45 27 2

50

Figure 8 Results of replicate determinations (indicated by triangles) of benzo[b]fluoranthene (BBF) in the food supplement test material

Horizontal blue lines represent the arithmetic mean value of replicate measurements and blue bars the reported expanded measurement uncertainty (k=2) The assigned value is

plotted as dotted line Red solid lines mark the range of satisfactory performance (|z|le2) Mean values are given for results outside the displayed data range

ProLab 2011

Laboratory

36

0436

1436

0236

1053

1736

1653

0953

0753

0853

0536

2136

0553

0436

0353

0353

1636

0636

2036

2353

1853

1136

1236

2653

0153

0253

1236

1736

1336

0036

1853

2153

1036

1953

1336

2253

2253

2353

1936

1553

1453

0653

1536

0753

2036

0836

11

an

aly

te c

on

ten

t (micro

gk

g)

75

7

65

6

55

5

45

4

35

3

25

2

15

1

05

0

103

6

576

3

Sample St Johns wort extract replicatesMeasurand Benzo[b]fluorantheneNo of laboratories 46

Assigned value 292 microgkg (Reference value)Target sd 060 microgkgTolerance limits 172 - 412 microgkg (|Z score| lt 200)

Assig

ned va

lue

Limit of tolerance

Limit of tolerance

51

Table 14 Analysis results reported by the participants for the content of chrysene (CHR) in the food supplements test material Values are presented as reported

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

3600 5 49 5 5 07 2

3602 32 304 29 304 036

3603 462 469 447 459 048 2

3604 438 428 42 429 119 2

3605 365 41 381 386 085 2

3606 494 411 45 451 124 2

3607 897 774 787 819 18 2

3608 986 887 1168 1014 284 2

3610 418 392 462 424 085 2

3611 833 893 87 865 61 2

3612 6 64 58 61 12 2

3613 41 39 4 4 08 2

3614 272 254 288 271 054 2

3615 605 6028 5964 601 12 2

3616 47 45 49 47 05 2

3617 445 431 389 422 065 2

3618 45 45 45 45 06 2

3619 738 747 72 738 118 2

3620 463 472 446 46 092 2

3621 53 49 51 51 06 2

3622 478 513 505 488 08 2

3623 73 73 72 72 05 2

3624

3626 63 622 634 628 251 2

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

5301 49 49 49 49 1 2

5302 687 683 666 679 136 2

5303 6777 6181 6382 6441 24 70

5304 592 581 6 591 85 2

5305 6 58 64 61 20

5306 49 5 51 49 25 2

5307 524 5 574 524

5308 211488 216782 221146 213243

5309

5310 372 393 394 386 019

5311 43 459 422 437 15 2

5312 934 943 981 953 191 2

5313 793 73 99 838

5314 636 561 565 588 18 0818

5315 547 583 499 542 11 2

5316 292 286 285 282 06 2

5317 52 598 517 545 046 2

5318 368 345 357 05 2

5319 449 471 474 465 116 2

5320 76 924 818 834 99

5321 33

5322 7 7 76 7 35 2

5323 87 73 77 73 37 2

52

Figure 9 Results of replicate determinations (indicated by triangles) of chrysene (CHR) in the food supplement test material

Horizontal blue lines represent the arithmetic mean value of replicate measurements and blue bars the reported expanded measurement uncertainty (k=2) The assigned value is

plotted as dotted line Red solid lines mark the range of satisfactory performance (|z|le2) Mean values are given for results outside the displayed data range

ProLab 2011

Laboratory

53

0836

1453

1636

0253

2153

1836

0553

1036

1336

1736

1036

0453

1136

1836

0636

0336

2053

1936

1653

0136

0036

2253

0636

2153

0753

1553

1753

1453

0436

1536

1253

0536

2653

0353

0253

2236

2336

1953

2336

0753

2053

1353

1236

0836

11

an

aly

te c

on

ten

t (micro

gk

g)

13

12

11

10

9

8

7

6

5

4

3

2

1

0

865

3

Sample St Johns wort extract replicatesMeasurand ChryseneNo of laboratories 45

Assigned value 407 microgkg (Reference value)Target sd 083 microgkgTolerance limits 241 - 573 microgkg (|Z score| lt 200)

Assig

ned va

lue

Limit of tolerance

Limit of tolerance

53

Table 15 Analysis results reported by the participants for the sum of the four marker PAHs in the food supplements test material Values are presented as reported

Laboratory code

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg

3600 128 24 2

3602 79 126

3603 1127 096 2

3604 893 258 2

3605 1091 305 2

3606 1184 238 2

3607 2098 42 2

3608 3336 325 2

3610 1138 17 2

3611 2218 259 2

3612 143 29 2

3613 122 2 2

3614 663 129 2

3615 152 304 2

3616 102 03 2

3617 1252 169 2

3618 128 18 2

3619 157 305 2

3620 1204 12 2

3621 151 19 2

3622 1361 115 2

3623 154 09 2

3624

3626 1409 56 2

Laboratory code

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg

5301 126

5302 37 074 2

5303 13438 92

5304 1251 85 2

5305 125 20

5306 129 65 2

5307 1363

5308 7245598

5309

5310 1127 056

5311 1367 15 2

5312 2042 408 2

5313 1726

5314 1558

5315 177 35 2

5316 96 073 2

5317 1229 066 2

5318 82 05 2

5319 1225 267 2

5320 2309 41

5321

5322 207 35 2

5323 171 103 2

54

ANNEX 9 Results reported for the standard solutions with undisclosed analyte content Table 16 Analysis results reported by the participants for the four marker PAHs in the standard solution in toluene with undisclosed analyte

content Values are presented as reported U measurement uncertainty k coverage factor

benz[a]anthracene benzo[a]pyrene benzo[b]fluoranthene chrysene Laboratory

code Unit Result U k Result U k Result U k Result U k 3600 microgl 38 67 2 25 45 2 211 68 2 275 38 2

3605 microgl 367 184 2 2482 124 2 2592 13 2 235 118 2

3606 microgkg 3763 176 2 2609 142 2 2721 118 2 2539 134 2

3607 microgkg 3706 815 2 2824 565 2 2591 518 2 2587 569 2

3612 microgl 38 08 2 237 11 2 244 18 2 257 02 2

3614 microgl 324 65 2 209 38 2 215 37 2 21 42 2

3616 microgkg 318 07 2 224 14 2 231 26 2 216 1 2

3618 microgkg 417 17 2 284 08 2 292 06 2 297 03 2

3619 microgkg 47 742 2 274 468 2 228 387 2 274 434 2

3620 microgl 495 74 2 294 29 2 321 8 2 321 64 2

3621 microgl 241 22 2 143 13 2 188 16 2 145 14 2

3623 microgkg 476 25 2 311 53 2 269 3 2 297 03 2

3626 microgl 359 38 2 235 23 2 238 27 2 24 18 2

5301 microgkg 426 29 299 289

5302 microgkg 436 87 2 296 59 2 296 59 2 289 58 2

5303 microgl 3721 27 102 22838 19 99 23756 22 99 25486 24 98

5304 microgkg 38 29 2 225 26 2 257 37 2 258 52 2

5305 microgkg 305 225 245 29

5306 microgkg 316 63 2 247 49 2 65 13 2 22 44 2

5307 microgkg 3658 2559 241 2414

5308 microgkg 16854 109324 246654 216782

5311 microgl 374 20 2 279 15 2 268 15 2 272 15 2

5313 microgl 3408 2165 2275 2373

5314 microgkg 395 5 2875 11 3614 34 2905 18

5320 microgl 3267 368 1986 571 28 683 2427 512

5322 microgl 282 35 2 225 35 2 241 35 2 21 35 2

55

Figure 10 Youden plots of the percent deviation of the reported values from the assigned values for the food supplement test sample and the

standard solution in toluene with undisclosed content

(The identity of the respective measurand is given above the figure Results exceeding a difference of more than 100 were omitted)

56

Figure 10 - continued

57

Table 17 Analysis results reported by the participants for the four marker PAHs in the standard solution in acetonitrile with undisclosed

analyte content

Values are presented as reported U measurement uncertainty k coverage factor

benz[a]anthracene benzo[a]pyrene benzo[b]fluoranthene chrysene

Laboratory

code Unit Result U k Result U k Result U k Result U k

3602 microgkg 3675 247 247 244

3603 microgkg 358 086 2 236 028 2 235 028 2 236 057 2

3604 microgl 475 29 2 19 12 2 194 13 2 258 12 2

3608 microgl 3623 078 2 2385 023 2 2343 18 2 2296 092 2

3610 microgkg 369 185 2 25 125 2 251 126 2 244 122 2

3611 microgkg 48 02 2 323 02 2 328 04 2 317 03 2

3613 microgl 332 31 2 261 3 2 249 25 2 239 24 2

3615 microgl 415 2635 2729 2689

3617 microgkg 37 09 2 244 06 2 284 07 2 239 06 2

3622 microgl 37 12 2 25 23 2 257 21 2 238 1 2

3624 microgkg 402 104 2 255 084 2 265 096 2 253 07 2

5306 microgkg 563 113 2 371 74 2 359 72 2 333 67 2

5308 microgkg 168633 102669 232169 211488

5309 microgl 402 268 27

5310 microgl 315 158 25 125 25 125 288 144

5312 microgl 428 043 2 282 028 2 272 027 2 275 028 2

5315 microgkg 387 08 2 256 05 2 251 05 2 253 05 2

5316 microgl 3839 342 2 2485 191 2 2559 168 2 239 51 2

5317 microgl 2466 043 2 2212 034 2 2472 016 2 2146 068 2

5319 microgl 414 41 2 279 28 2 268 27 2 26 31 2

5321 microgkg 476 316 326 328

5322 microgl 2547 35 2

5323 microgkg 405 203 2 239 119 2 255 153 2 201 11 2

58

Figure 11 Youden plots of the percent deviation of the reported values from the assigned values for the food supplement test sample and the

standard solution in acetonitrile with undisclosed content

(The identity of the respective measurand is given above the figure Results exceeding a difference of more than 100 were omitted)

59

Figure 11 - continued

60

ANNEX 10 Table 18 Method performance parameters reported by the participants

Laboratory code

Analyte LOD LOQ Recovery Analyte LOD LOQ Recovery

microgkg microgkg microgkg microgkg 3600 BaA 03 06 978 BaP 03 07 9483602 BaA 007 021 926 BaP 005 015 993603 BaA 0011 05 987 BaP 0005 051 9553604 BaA 02 04 86 BaP 02 04 883605 BaA 0025 005 118 BaP 0025 005 1083606 BaA 001 003 65 BaP 001 003 703607 BaA 021 071 74 BaP 027 089 943608 BaA 016 048 8213 BaP 015 046 83683610 BaA 02 05 85 BaP 005 02 853611 BaA BaP 3612 BaA 1 117 BaP 1 1203613 BaA 01 03 90 BaP 01 03 903614 BaA 02 05 96 BaP 02 05 933615 BaA 005 015 110 BaP 005 015 1053616 BaA 01 05 108 BaP 01 05 753617 BaA 018 037 405 BaP 009 018 3873618 BaA 01 03 63 BaP 01 03 593619 BaA 005 005 59 BaP 033 033 543620 BaA 0009 0018 64 BaP 0005 001 553621 BaA 007 02 84 BaP 007 02 923622 BaA 01 03 1101 BaP 01 03 9853623 BaA 02 08 105 BaP 02 08 1013624 BaA BaP 01 033 8513625 BaA BaP 3626 BaA BaP 5301 BaA 003 01 102 BaP 003 01 1005302 BaA 01 03 78 BaP 01 03 865303 BaA 003 01 62 BaP 003 01 805304 BaA 1 1 70 BaP 1 1 955305 BaA 02 05 85 BaP 02 05 855306 BaA 03 1 699 BaP 03 1 905307 BaA 6537 BaP 81165308 BaA 0088 0265 99 BaP 00262 00785 1005309 BaA 01 08 92 BaP 01 08 925310 BaA 01 03 BaP 01 03 905311 BaA 002 005 80 BaP 002 004 915312 BaA 03 09 105 BaP 03 09 1055313 BaA 04 08 BaP 04 08 5314 BaA 001 002 96 BaP 001 002 9485315 BaA 01 03 50 - 100 BaP 01 03 50 - 100 5316 BaA 002 011 827 BaP 004 019 10445317 BaA 05 1 76 BaP 05 1 755318 BaA BaP 5319 BaA 009 029 951 BaP 011 041 995320 BaA 12 18 946 BaP 06 1 8345321 BaA 03 05 BaP 05 1 5322 BaA 1 2 100 BaP 1 2 1005323 BaA 26 BaP 26

61

ANNEX 10 Table 18 - continued

Laboratory code Analyte LOD LOQ Recovery Analyte LOD LOQ Recovery

microgkg microgkg microgkg microgkg 3600 BbF 03 05 96 CHR 02 03 10153602 BbF 015 045 947 CHR 003 009 9193603 BbF 0014 05 1012 CHR 0004 05 10033604 BbF 02 04 85 CHR 02 04 873605 BbF 005 01 94 CHR 0025 005 863606 BbF 001 003 70 CHR 001 003 653607 BbF 018 06 100 CHR 014 028 943608 BbF 015 046 7979 CHR 015 045 71253610 BbF 005 02 85 CHR 02 05 853611 BbF CHR 3612 BbF 1 118 CHR 1 1173613 BbF 01 03 90 CHR 01 03 903614 BbF 01 04 96 CHR 02 05 953615 BbF 008 025 109 CHR 003 01 1083616 BbF 01 05 78 CHR 01 05 723617 BbF 035 07 38 CHR 012 024 4163618 BbF 01 03 62 CHR 01 03 623619 BbF 016 016 63 CHR 01 01 593620 BbF 0003 0006 55 CHR 001 002 653621 BbF 007 02 103 CHR 007 02 893622 BbF 02 06 963 CHR 03 09 1043623 BbF 02 08 106 CHR 02 08 1193624 BbF CHR 3625 BbF CHR 3626 BbF CHR 5301 BbF 003 01 96 CHR 003 01 1145302 BbF 01 03 84 CHR 01 03 835303 BbF 003 01 78 CHR 003 01 705304 BbF 1 1 86 CHR 1 1 705305 BbF 02 05 90 CHR 02 05 955306 BbF 03 1 1435 CHR 03 1 1185307 BbF 8116 CHR 65375308 BbF 0079 0238 99 CHR 009 026 1005309 BbF 01 08 92 CHR 5310 BbF 07 2 CHR 01 03 5311 BbF 002 004 95 CHR 002 004 895312 BbF 03 09 105 CHR 03 09 1055313 BbF 04 08 CHR 04 08 5314 BbF 001 002 83 CHR 001 002 8185315 BbF 02 05 50 -100 CHR 02 05 50 - 100 5316 BbF 005 024 814 CHR 001 007 9595317 BbF 05 1 73 CHR 05 1 785318 BbF CHR 5319 BbF 011 038 946 CHR 013 043 9675320 BbF 07 09 1194 CHR 23 32 10455321 BbF 05 1 CHR 05 1 5322 BbF 1 2 100 CHR 1 2 1005323 BbF 26 CHR 26

62

ANNEX 11 Table 19 Details of analysis method reported by the participants Data are presented as reported

Laboratory code

Analysis technique

Instrument calibration

Internal standards applied

Sample clean up

Details of sample

clean up

Extraction technique

Details of sample

extraction

3600 6) GC-MS

2) Internal standardisation with labelled substances

Deuterated PAH mix 9 (Ehrenstorfer)

1) Saponification 2) LiquidLiquid partitioning

D) Other Liquidliquid partitioning

3602 2) HPLC-UV-FLD

1) External calibration

3) Gel permeation chromatography

A) Pressurised liquid extraction (PLE)

3603 1) HPLC-FLD

1) External calibration

3) Gel permeation chromatography

D) Other 1 minute with dichloromethane in vortex

3604 2) HPLC-UV-FLD

2) Internal standardisation with labelled substances

DiP D14

5) Donor acceptor complex chromatography

D) Other LL

3605 1) HPLC-FLD

1) External calibration

3) Gel permeation chromatography

C) Soxhlet extraction

3606 7) GC-MSMS

2) Internal standardisation with labelled substances

BaP 13C4 CHR 13C6 BaA 13C6 BbF 13C6

4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

3607 6) GC-MS

2) Internal standardisation with labelled substances

BaA-d12 CHR-d12 BbF-d12 BkF-d12 BaP-d12 ICP-d12 BgP-d12 DiP-d14

1) Saponification

D) Other solvent partitioning

3608 1) HPLC-FLD

1) External calibration

6) Other Liquid extraction

D) Other

Extraction in a rotary agitator and centrifugation

3610 2) HPLC-UV-FLD

1) External calibration

4) Solid phase extraction

B) Sonication

3611

3612 6) GC-MS

2) Internal standardisation with labelled substances

chrysene-d12 dibenzo(ah)anthracene-d14 dibenzo(ai)pyrene-d14

4) Solid phase extraction

B) Sonication

3613 5) LC-MSMS

3) Internal standardisation with unlabelled substances

I use Chrysene-D12 3) Gel permeation chromatography

D) Other extraction with hexan

63

Table 19 - continued

Laboratory code

Analysis technique

Instrument calibration

Internal standards applied

Sample clean up Details of sample

clean up

Extraction technique

Details of sample

extraction

3614 6) GC-MS 1) External calibration

3) Gel permeation chromatography

B) Sonication

3615 1) HPLC-FLD

1) External calibration

6) Other PLE GPC

A) Pressurised liquid extraction (PLE)

3616 6) GC-MS

2) Internal standardisation with labelled substances

Benzo(a)pyrene-13C4 Benzo(a)anthracene-13C6 Benzo(b)fluoranthene-13C6 Chrysene-13C6

2) LiquidLiquid partitioning 4) Solid phase extraction

B) Sonication

3617 1) HPLC-FLD

1) External calibration

4) Solid phase extraction

C) Soxhlet extraction

3618 6) GC-MS

2) Internal standardisation with labelled substances

deuterated standards

1) Saponification 4) Solid phase extraction

D) Other Handshaken with cyclohexane

3619 6) GC-MS

2) Internal standardisation with labelled substances

C13 Labelled US EPA 16

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

D) Other Homegenisation amp saponiofication

3620 9) GC-HRMS

2) Internal standardisation with labelled substances

mix of deuterated PAHs (BAA-D12 CHR-D12 BAP-D12 BBF-D12)

3) Gel permeation chromatography 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

3621 6) GC-MS

2) Internal standardisation with labelled substances

Chrysene D12 Benzo(a)pyrene D12 Benzo(b)fluoranthene D12 Benzo(a)anthracene

2) LiquidLiquid partitioning 4) Solid phase extraction

B) Sonication

3622 1) HPLC-FLD

4) Standard addition method

4) Solid phase extraction

D) Other

Extraction with cyclohexane by heating under reflux

3623 7) GC-MSMS

2) Internal standardisation with labelled substances

C13 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

3624 2) HPLC-UV-FLD

1) External calibration

2) LiquidLiquid partitioning

D) Other hot solvent extraction

3626

64

Table 19 - continued

Laboratory code

Analysis technique

Instrument calibration

Internal standards

applied

Sample clean up

Details of sample

clean up

Extraction technique

Details of sample

extraction

5301 7) GC-MSMS

2) Internal standardisation with labelled substances

EACH SUBSTANCE C13

4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5302 7) GC-MSMS

2) Internal standardisation with labelled substances

Isotope labelled PAHs 13C for each analyte

4) Solid phase extraction

C) Soxhlet extraction

5303 7) GC-MSMS

3) Internal standardisation with unlabelled substances

HAP C13 4) Solid phase extraction

D) Other automate ASE200 DIONEX

5304 7) GC-MSMS

2) Internal standardisation with labelled substances

with C13 for each HAP

2) LiquidLiquid partitioning 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5305 7) GC-MSMS

2) Internal standardisation with labelled substances

C13 isotopiques internal standards

4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5306 7) GC-MSMS

2) Internal standardisation with labelled substances

C13 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5307 6) GC-MS

2) Internal standardisation with labelled substances

1) Saponification 2) LiquidLiquid partitioning

D) Other solvent extraction

5308 1) HPLC-FLD 1) External calibration

4) Solid phase extraction

B) Sonication

5309 1) HPLC-FLD 1) External calibration

4) Solid phase extraction

D) Other only SPE

5310 1) HPLC-FLD 1) External calibration

1) Saponification 2) LiquidLiquid partitioning 3) Gel permeation chromatography

C) Soxhlet extraction

5311 6) GC-MS

2) Internal standardisation with labelled substances

13 C Chrysene 13 C Benz(a)pyrene

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

B) Sonication

5312 1) HPLC-FLD

3) Internal standardisation with unlabelled substances

Benzo(b)chrysen

2) LiquidLiquid partitioning 4) Solid phase extraction

Quechers D) Other Quechers

65

Table 19 - continued

Laboratory code

Analysis technique

Instrument calibration

Internal standards applied

Sample clean up Details of sample

clean up

Extraction technique

Details of sample extraction

5313 6) GC-MS

2) Internal standardisation with labelled substances

Benz(a)anthracene D12 Benzo(b)fluranthene D12 Benzo(a)pyrene D12 Chrysene D12

1) Saponification 2) LiquidLiquid partitioning 3) Gel permeation chromatography

A) Pressurised liquid extraction (PLE)

5314 6) GC-MS

2) Internal standardisation with labelled substances

Benzo(a)pyren d12 2) LiquidLiquid partitioning

D) Other Extraction with

5315 1) HPLC-FLD

3) Internal standardisation with unlabelled substances

Benz(b)chrysen

1) Saponification 2) LiquidLiquid partitioning

D) Other shaker

5316 1) HPLC-FLD

1) External calibration

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

D) Other

LiquidLiquid extraction with cyclohexane and dimethylformamide

5317 7) GC-MSMS

1) External calibration

3) Gel permeation chromatography 6) Other

silica gel D) Other VDLUFA online method

5318

5319 1) HPLC-FLD

3) Internal standardisation with unlabelled substances

Benzo(b)chrysene 3) Gel permeation chromatography

A) Pressurised liquid extraction (PLE)

5320 6) GC-MS

3) Internal standardisation with unlabelled substances

22-binaphthyl

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

D) Other 3 hours refluxing

5321 1) HPLC-FLD

2) Internal standardisation with labelled substances

6-meacutethylchrysegravene

1) Saponification 2) LiquidLiquid partitioning

B) Sonication

5322 6) GC-MS

2) Internal standardisation with labelled substances

naftalene-d8 acenaphtalene-d10 phenanthrene-d10 chrysene-d12 perylene-d12 benzo(e)anthracene-d12

1) Saponification 2) LiquidLiquid partitioning 6) Other

clean-up with silica packed column

C) Soxhlet extraction

5323 1) HPLC-FLD

1) External calibration

4) Solid phase extraction 6) Other

Accelerated Solvent Extraction

A) Pressurised liquid extraction (PLE)

66

European Commission EUR 25245 EN ndash Joint Research Centre ndash Institute for Reference Materials and Measurements Title Report on the 8th inter-laboratory comparison organised by the European Union Reference Laboratory for Polycyclic Aromatic Hydrocarbons - Four marker PAHs in a dry extract of St Johns wort Author(s) Radoslav Lizak Szilard Szilagyi Philippe Verlinde and Thomas WENZL Luxembourg Publications Office of the European Union 2012 ndash 66 pp ndash 201 x 297 cm EUR ndash Scientific and Technical Research series ndash ISSN 1831-9424 ISBN 978-92-79-23337-1 doi 10278759387 Abstract The proficiency test here reported concerned the determination of the contents of four marker polycyclic aromatic hydrocarbons (PAHs) and of their sum in an food supplements test sample The set of marker PAHs consists of benz[a]anthacene benzo[a]pyrene benzo[b]fluoranthene and chrysene Participants to these PT were National Reference Laboratories for PAHs (NRLs-PAHs) and EU official food control laboratories The number of participants was in total 48

The PT was organised according ISO Standard 170432010

The test material used was a commercial dry extract of St Johns wort Participants also received a solution of the same PAHs either in acetonitrile or in toluene for checking their instrument calibration

The results from participants were rated with z-scores and zeta-scores About 68 of the reported results were attributed with z-scores with an absolute value of below two which is the threshold for satisfactory performance

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interests whether private or national

L

A-N

A-252

45-EN

-N

• 1 Summary
• 2 Introduction
• 3 Scope
• 4 Participating Laboratories
• 5 Time frame
• 6 Confidentiality
• 7 Test materials
• • 71 1Preparation of test materials
  • 72 Assigned values and standard deviation for proficiency assessment
  • 73 Homogeneity and stability
  • 74 Sample dispatch
  • • 8 Design of the proficiency test
    • 9 Evaluation of the results
    • • 91 2General
      • 92 21BEvaluation criteria
      • 93 Performance of participants
      • 94 Evaluation of the influence of analysis method
      • 95 Evaluation of the influence of calibration
      • 96 26Evaluation of compliance with legislation
      • • 10 Follow-up actions for underperforming laboratories
        • 11 1Conclusions
        • 12 1Acknowledgements
        • 13 References

47

Table 12 Analysis results reported by the participants for the content of benzo[a]pyrene (BAP) in the food supplements test material Values are presented as reported

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

3600 16 17 17 17 03 2

3602 118 123 13 124 015

3603 165 162 157 161 015 2

3604 134 154 139 143 04 2

3605 164 166 172 167 057 2

3606 181 159 161 167 031 2

3607 279 266 279 274 055 2

3608 57 687 461 573 226 2

3610 226 216 183 206 042 2

3611 289 325 311 308 36 2

3612 24 22 2 22 05 2

3613 17 18 19 18 05 2

3614 08 08 08 08 016 2

3615 1545 1393 1425 145 029 2

3616 16 16 14 15 03 2

3617 177 171 158 169 023 2

3618 2 2 2 2 02 2

3619 207 207 206 207 075 2

3620 165 169 163 166 017 2

3621 44 47 49 47 05 2

3622 212 208 222 213 038 2

3623 2 21 22 21 03 2

3624 18 178 182 18 031 2

3626 172 173 178 174 07 2

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

5301 18 18 18 18 04 2

5302 188 19 187 188 038 2

5303 1691 1426 149 1535 19 80

5304 145 142 15 146 69 2

5305 15 17 18 16 15

5306 16 17 17 16 08 2

5307 164 157 153 157

5308 102669 109324 109035 107009

5309 1 1 12 11

5310 122 122 124 123 006

5311 199 184 215 2 15 2

5312 258 248 239 248 05 2

5313 199 178 245 207

5314 174 171 172 172 11 0948

5315 253 248 213 238 05 2

5316 177 171 173 17 016 2

5317 255 277 276 269 012 2

5318 114 12 117 02 2

5319 19 228 231 216 043 2

5320 303 3 344 316 78

5321 12 12 12 12

5322 13 17 24 17 35 2

5323 2 2 19 2 1 2

48

Figure 7 Results of replicate determinations (indicated by triangles) of benzo[a]pyrene (BAP) in the food supplement test material

Horizontal blue lines represent the arithmetic mean value of replicate measurements and blue bars the reported expanded measurement uncertainty (k=2) The assigned value is

plotted as dotted line Red solid lines mark the range of satisfactory performance (|z|le2) Mean values are given for results outside the displayed data range

ProLab 2011

Laboratory

36

1453

0953

0853

1853

2153

1036

0236

0436

1553

0436

1653

0353

0736

0336

2036

0053

0553

0636

0636

0536

1753

1453

1636

2636

1336

2453

0153

2253

0253

2353

1136

1836

1953

1336

1036

2336

2253

1936

1253

1553

1253

1736

0753

2036

2136

0836

11

an

aly

te c

on

ten

t (micro

gk

g)

55

5

45

4

35

3

25

2

15

1

05

0

573

308

3

Sample St Johns wort extract replicatesMeasurand Benzo[a]pyreneNo of laboratories 48

Assigned value 165 microgkg (Reference value)Target sd 036 microgkgTolerance limits 093 - 237 microgkg (|Z score| lt 200)

Assig

ned va

lue

Limit of tolerance

Limit of tolerance

49

Table 13 Analysis results reported by the participants for the content of benzo[b]fluoranthene (BBF) in the food supplements test material Values are presented as reported

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

3600 32 3 3 31 09 2

3602 173 175 16 17 017

3603 265 252 25 256 025 2

3604 116 108 105 11 028 2

3605 286 225 232 247 074 2

3606 294 273 261 276 046 2

3607 579 579 544 567 113 2

3608 103 1201 876 1036 326 2

3610 173 196 204 191 038 2

3611 546 618 565 576 75 2

3612 3 28 28 29 06 2

3613 28 31 32 31 06 2

3614 135 111 135 127 022 2

3615 4187 3678 4111 4 08 2

3616 2 2 2 2 01 2

3617 33 27 299 035 2

3618 31 31 31 31 05 2

3619 326 324 312 326 064 2

3620 277 28 272 276 069 2

3621 26 25 23 25 04 2

3622 346 336 333 34 052 2

3623 26 28 29 28 02 2

3624

3626 288 28 307 292 117 2

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

5301 29 3 29 29 06 2

5302 297 3 296 298 06 2

5303 2705 2444 2608 2586 22 78

5304 245 254 26 253 85 2

5305 23 23 26 24 15

5306 42 43 4 42 21 2

5307 221 216 241 221

5308 232169 246654 240722 229848

5309 19 22 19 2 0

5310 3 315 325 313 016

5311 262 277 304 281 15 2

5312 27 289 337 299 075 2

5313 321 284 378 328

5314 423 404 385 404 34 083

5315 42 506 416 444 09 2

5316 265 267 277 273 018 2

5317 185 197 195 192 006 2

5318 276 281 279 04 2

5319 396 389 403 396 079 2

5320 623 618 635 625 14

5321 31

5322 24 33 49 33 35 2

5323 38 45 35 45 27 2

50

Figure 8 Results of replicate determinations (indicated by triangles) of benzo[b]fluoranthene (BBF) in the food supplement test material

Horizontal blue lines represent the arithmetic mean value of replicate measurements and blue bars the reported expanded measurement uncertainty (k=2) The assigned value is

plotted as dotted line Red solid lines mark the range of satisfactory performance (|z|le2) Mean values are given for results outside the displayed data range

ProLab 2011

Laboratory

36

0436

1436

0236

1053

1736

1653

0953

0753

0853

0536

2136

0553

0436

0353

0353

1636

0636

2036

2353

1853

1136

1236

2653

0153

0253

1236

1736

1336

0036

1853

2153

1036

1953

1336

2253

2253

2353

1936

1553

1453

0653

1536

0753

2036

0836

11

an

aly

te c

on

ten

t (micro

gk

g)

75

7

65

6

55

5

45

4

35

3

25

2

15

1

05

0

103

6

576

3

Sample St Johns wort extract replicatesMeasurand Benzo[b]fluorantheneNo of laboratories 46

Assigned value 292 microgkg (Reference value)Target sd 060 microgkgTolerance limits 172 - 412 microgkg (|Z score| lt 200)

Assig

ned va

lue

Limit of tolerance

Limit of tolerance

51

Table 14 Analysis results reported by the participants for the content of chrysene (CHR) in the food supplements test material Values are presented as reported

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

3600 5 49 5 5 07 2

3602 32 304 29 304 036

3603 462 469 447 459 048 2

3604 438 428 42 429 119 2

3605 365 41 381 386 085 2

3606 494 411 45 451 124 2

3607 897 774 787 819 18 2

3608 986 887 1168 1014 284 2

3610 418 392 462 424 085 2

3611 833 893 87 865 61 2

3612 6 64 58 61 12 2

3613 41 39 4 4 08 2

3614 272 254 288 271 054 2

3615 605 6028 5964 601 12 2

3616 47 45 49 47 05 2

3617 445 431 389 422 065 2

3618 45 45 45 45 06 2

3619 738 747 72 738 118 2

3620 463 472 446 46 092 2

3621 53 49 51 51 06 2

3622 478 513 505 488 08 2

3623 73 73 72 72 05 2

3624

3626 63 622 634 628 251 2

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

5301 49 49 49 49 1 2

5302 687 683 666 679 136 2

5303 6777 6181 6382 6441 24 70

5304 592 581 6 591 85 2

5305 6 58 64 61 20

5306 49 5 51 49 25 2

5307 524 5 574 524

5308 211488 216782 221146 213243

5309

5310 372 393 394 386 019

5311 43 459 422 437 15 2

5312 934 943 981 953 191 2

5313 793 73 99 838

5314 636 561 565 588 18 0818

5315 547 583 499 542 11 2

5316 292 286 285 282 06 2

5317 52 598 517 545 046 2

5318 368 345 357 05 2

5319 449 471 474 465 116 2

5320 76 924 818 834 99

5321 33

5322 7 7 76 7 35 2

5323 87 73 77 73 37 2

52

Figure 9 Results of replicate determinations (indicated by triangles) of chrysene (CHR) in the food supplement test material

Horizontal blue lines represent the arithmetic mean value of replicate measurements and blue bars the reported expanded measurement uncertainty (k=2) The assigned value is

plotted as dotted line Red solid lines mark the range of satisfactory performance (|z|le2) Mean values are given for results outside the displayed data range

ProLab 2011

Laboratory

53

0836

1453

1636

0253

2153

1836

0553

1036

1336

1736

1036

0453

1136

1836

0636

0336

2053

1936

1653

0136

0036

2253

0636

2153

0753

1553

1753

1453

0436

1536

1253

0536

2653

0353

0253

2236

2336

1953

2336

0753

2053

1353

1236

0836

11

an

aly

te c

on

ten

t (micro

gk

g)

13

12

11

10

9

8

7

6

5

4

3

2

1

0

865

3

Sample St Johns wort extract replicatesMeasurand ChryseneNo of laboratories 45

Assigned value 407 microgkg (Reference value)Target sd 083 microgkgTolerance limits 241 - 573 microgkg (|Z score| lt 200)

Assig

ned va

lue

Limit of tolerance

Limit of tolerance

53

Table 15 Analysis results reported by the participants for the sum of the four marker PAHs in the food supplements test material Values are presented as reported

Laboratory code

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg

3600 128 24 2

3602 79 126

3603 1127 096 2

3604 893 258 2

3605 1091 305 2

3606 1184 238 2

3607 2098 42 2

3608 3336 325 2

3610 1138 17 2

3611 2218 259 2

3612 143 29 2

3613 122 2 2

3614 663 129 2

3615 152 304 2

3616 102 03 2

3617 1252 169 2

3618 128 18 2

3619 157 305 2

3620 1204 12 2

3621 151 19 2

3622 1361 115 2

3623 154 09 2

3624

3626 1409 56 2

Laboratory code

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg

5301 126

5302 37 074 2

5303 13438 92

5304 1251 85 2

5305 125 20

5306 129 65 2

5307 1363

5308 7245598

5309

5310 1127 056

5311 1367 15 2

5312 2042 408 2

5313 1726

5314 1558

5315 177 35 2

5316 96 073 2

5317 1229 066 2

5318 82 05 2

5319 1225 267 2

5320 2309 41

5321

5322 207 35 2

5323 171 103 2

54

ANNEX 9 Results reported for the standard solutions with undisclosed analyte content Table 16 Analysis results reported by the participants for the four marker PAHs in the standard solution in toluene with undisclosed analyte

content Values are presented as reported U measurement uncertainty k coverage factor

benz[a]anthracene benzo[a]pyrene benzo[b]fluoranthene chrysene Laboratory

code Unit Result U k Result U k Result U k Result U k 3600 microgl 38 67 2 25 45 2 211 68 2 275 38 2

3605 microgl 367 184 2 2482 124 2 2592 13 2 235 118 2

3606 microgkg 3763 176 2 2609 142 2 2721 118 2 2539 134 2

3607 microgkg 3706 815 2 2824 565 2 2591 518 2 2587 569 2

3612 microgl 38 08 2 237 11 2 244 18 2 257 02 2

3614 microgl 324 65 2 209 38 2 215 37 2 21 42 2

3616 microgkg 318 07 2 224 14 2 231 26 2 216 1 2

3618 microgkg 417 17 2 284 08 2 292 06 2 297 03 2

3619 microgkg 47 742 2 274 468 2 228 387 2 274 434 2

3620 microgl 495 74 2 294 29 2 321 8 2 321 64 2

3621 microgl 241 22 2 143 13 2 188 16 2 145 14 2

3623 microgkg 476 25 2 311 53 2 269 3 2 297 03 2

3626 microgl 359 38 2 235 23 2 238 27 2 24 18 2

5301 microgkg 426 29 299 289

5302 microgkg 436 87 2 296 59 2 296 59 2 289 58 2

5303 microgl 3721 27 102 22838 19 99 23756 22 99 25486 24 98

5304 microgkg 38 29 2 225 26 2 257 37 2 258 52 2

5305 microgkg 305 225 245 29

5306 microgkg 316 63 2 247 49 2 65 13 2 22 44 2

5307 microgkg 3658 2559 241 2414

5308 microgkg 16854 109324 246654 216782

5311 microgl 374 20 2 279 15 2 268 15 2 272 15 2

5313 microgl 3408 2165 2275 2373

5314 microgkg 395 5 2875 11 3614 34 2905 18

5320 microgl 3267 368 1986 571 28 683 2427 512

5322 microgl 282 35 2 225 35 2 241 35 2 21 35 2

55

Figure 10 Youden plots of the percent deviation of the reported values from the assigned values for the food supplement test sample and the

standard solution in toluene with undisclosed content

(The identity of the respective measurand is given above the figure Results exceeding a difference of more than 100 were omitted)

56

Figure 10 - continued

57

Table 17 Analysis results reported by the participants for the four marker PAHs in the standard solution in acetonitrile with undisclosed

analyte content

Values are presented as reported U measurement uncertainty k coverage factor

benz[a]anthracene benzo[a]pyrene benzo[b]fluoranthene chrysene

Laboratory

code Unit Result U k Result U k Result U k Result U k

3602 microgkg 3675 247 247 244

3603 microgkg 358 086 2 236 028 2 235 028 2 236 057 2

3604 microgl 475 29 2 19 12 2 194 13 2 258 12 2

3608 microgl 3623 078 2 2385 023 2 2343 18 2 2296 092 2

3610 microgkg 369 185 2 25 125 2 251 126 2 244 122 2

3611 microgkg 48 02 2 323 02 2 328 04 2 317 03 2

3613 microgl 332 31 2 261 3 2 249 25 2 239 24 2

3615 microgl 415 2635 2729 2689

3617 microgkg 37 09 2 244 06 2 284 07 2 239 06 2

3622 microgl 37 12 2 25 23 2 257 21 2 238 1 2

3624 microgkg 402 104 2 255 084 2 265 096 2 253 07 2

5306 microgkg 563 113 2 371 74 2 359 72 2 333 67 2

5308 microgkg 168633 102669 232169 211488

5309 microgl 402 268 27

5310 microgl 315 158 25 125 25 125 288 144

5312 microgl 428 043 2 282 028 2 272 027 2 275 028 2

5315 microgkg 387 08 2 256 05 2 251 05 2 253 05 2

5316 microgl 3839 342 2 2485 191 2 2559 168 2 239 51 2

5317 microgl 2466 043 2 2212 034 2 2472 016 2 2146 068 2

5319 microgl 414 41 2 279 28 2 268 27 2 26 31 2

5321 microgkg 476 316 326 328

5322 microgl 2547 35 2

5323 microgkg 405 203 2 239 119 2 255 153 2 201 11 2

58

Figure 11 Youden plots of the percent deviation of the reported values from the assigned values for the food supplement test sample and the

standard solution in acetonitrile with undisclosed content

(The identity of the respective measurand is given above the figure Results exceeding a difference of more than 100 were omitted)

59

Figure 11 - continued

60

ANNEX 10 Table 18 Method performance parameters reported by the participants

Laboratory code

Analyte LOD LOQ Recovery Analyte LOD LOQ Recovery

microgkg microgkg microgkg microgkg 3600 BaA 03 06 978 BaP 03 07 9483602 BaA 007 021 926 BaP 005 015 993603 BaA 0011 05 987 BaP 0005 051 9553604 BaA 02 04 86 BaP 02 04 883605 BaA 0025 005 118 BaP 0025 005 1083606 BaA 001 003 65 BaP 001 003 703607 BaA 021 071 74 BaP 027 089 943608 BaA 016 048 8213 BaP 015 046 83683610 BaA 02 05 85 BaP 005 02 853611 BaA BaP 3612 BaA 1 117 BaP 1 1203613 BaA 01 03 90 BaP 01 03 903614 BaA 02 05 96 BaP 02 05 933615 BaA 005 015 110 BaP 005 015 1053616 BaA 01 05 108 BaP 01 05 753617 BaA 018 037 405 BaP 009 018 3873618 BaA 01 03 63 BaP 01 03 593619 BaA 005 005 59 BaP 033 033 543620 BaA 0009 0018 64 BaP 0005 001 553621 BaA 007 02 84 BaP 007 02 923622 BaA 01 03 1101 BaP 01 03 9853623 BaA 02 08 105 BaP 02 08 1013624 BaA BaP 01 033 8513625 BaA BaP 3626 BaA BaP 5301 BaA 003 01 102 BaP 003 01 1005302 BaA 01 03 78 BaP 01 03 865303 BaA 003 01 62 BaP 003 01 805304 BaA 1 1 70 BaP 1 1 955305 BaA 02 05 85 BaP 02 05 855306 BaA 03 1 699 BaP 03 1 905307 BaA 6537 BaP 81165308 BaA 0088 0265 99 BaP 00262 00785 1005309 BaA 01 08 92 BaP 01 08 925310 BaA 01 03 BaP 01 03 905311 BaA 002 005 80 BaP 002 004 915312 BaA 03 09 105 BaP 03 09 1055313 BaA 04 08 BaP 04 08 5314 BaA 001 002 96 BaP 001 002 9485315 BaA 01 03 50 - 100 BaP 01 03 50 - 100 5316 BaA 002 011 827 BaP 004 019 10445317 BaA 05 1 76 BaP 05 1 755318 BaA BaP 5319 BaA 009 029 951 BaP 011 041 995320 BaA 12 18 946 BaP 06 1 8345321 BaA 03 05 BaP 05 1 5322 BaA 1 2 100 BaP 1 2 1005323 BaA 26 BaP 26

61

ANNEX 10 Table 18 - continued

Laboratory code Analyte LOD LOQ Recovery Analyte LOD LOQ Recovery

microgkg microgkg microgkg microgkg 3600 BbF 03 05 96 CHR 02 03 10153602 BbF 015 045 947 CHR 003 009 9193603 BbF 0014 05 1012 CHR 0004 05 10033604 BbF 02 04 85 CHR 02 04 873605 BbF 005 01 94 CHR 0025 005 863606 BbF 001 003 70 CHR 001 003 653607 BbF 018 06 100 CHR 014 028 943608 BbF 015 046 7979 CHR 015 045 71253610 BbF 005 02 85 CHR 02 05 853611 BbF CHR 3612 BbF 1 118 CHR 1 1173613 BbF 01 03 90 CHR 01 03 903614 BbF 01 04 96 CHR 02 05 953615 BbF 008 025 109 CHR 003 01 1083616 BbF 01 05 78 CHR 01 05 723617 BbF 035 07 38 CHR 012 024 4163618 BbF 01 03 62 CHR 01 03 623619 BbF 016 016 63 CHR 01 01 593620 BbF 0003 0006 55 CHR 001 002 653621 BbF 007 02 103 CHR 007 02 893622 BbF 02 06 963 CHR 03 09 1043623 BbF 02 08 106 CHR 02 08 1193624 BbF CHR 3625 BbF CHR 3626 BbF CHR 5301 BbF 003 01 96 CHR 003 01 1145302 BbF 01 03 84 CHR 01 03 835303 BbF 003 01 78 CHR 003 01 705304 BbF 1 1 86 CHR 1 1 705305 BbF 02 05 90 CHR 02 05 955306 BbF 03 1 1435 CHR 03 1 1185307 BbF 8116 CHR 65375308 BbF 0079 0238 99 CHR 009 026 1005309 BbF 01 08 92 CHR 5310 BbF 07 2 CHR 01 03 5311 BbF 002 004 95 CHR 002 004 895312 BbF 03 09 105 CHR 03 09 1055313 BbF 04 08 CHR 04 08 5314 BbF 001 002 83 CHR 001 002 8185315 BbF 02 05 50 -100 CHR 02 05 50 - 100 5316 BbF 005 024 814 CHR 001 007 9595317 BbF 05 1 73 CHR 05 1 785318 BbF CHR 5319 BbF 011 038 946 CHR 013 043 9675320 BbF 07 09 1194 CHR 23 32 10455321 BbF 05 1 CHR 05 1 5322 BbF 1 2 100 CHR 1 2 1005323 BbF 26 CHR 26

62

ANNEX 11 Table 19 Details of analysis method reported by the participants Data are presented as reported

Laboratory code

Analysis technique

Instrument calibration

Internal standards applied

Sample clean up

Details of sample

clean up

Extraction technique

Details of sample

extraction

3600 6) GC-MS

2) Internal standardisation with labelled substances

Deuterated PAH mix 9 (Ehrenstorfer)

1) Saponification 2) LiquidLiquid partitioning

D) Other Liquidliquid partitioning

3602 2) HPLC-UV-FLD

1) External calibration

3) Gel permeation chromatography

A) Pressurised liquid extraction (PLE)

3603 1) HPLC-FLD

1) External calibration

3) Gel permeation chromatography

D) Other 1 minute with dichloromethane in vortex

3604 2) HPLC-UV-FLD

2) Internal standardisation with labelled substances

DiP D14

5) Donor acceptor complex chromatography

D) Other LL

3605 1) HPLC-FLD

1) External calibration

3) Gel permeation chromatography

C) Soxhlet extraction

3606 7) GC-MSMS

2) Internal standardisation with labelled substances

BaP 13C4 CHR 13C6 BaA 13C6 BbF 13C6

4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

3607 6) GC-MS

2) Internal standardisation with labelled substances

BaA-d12 CHR-d12 BbF-d12 BkF-d12 BaP-d12 ICP-d12 BgP-d12 DiP-d14

1) Saponification

D) Other solvent partitioning

3608 1) HPLC-FLD

1) External calibration

6) Other Liquid extraction

D) Other

Extraction in a rotary agitator and centrifugation

3610 2) HPLC-UV-FLD

1) External calibration

4) Solid phase extraction

B) Sonication

3611

3612 6) GC-MS

2) Internal standardisation with labelled substances

chrysene-d12 dibenzo(ah)anthracene-d14 dibenzo(ai)pyrene-d14

4) Solid phase extraction

B) Sonication

3613 5) LC-MSMS

3) Internal standardisation with unlabelled substances

I use Chrysene-D12 3) Gel permeation chromatography

D) Other extraction with hexan

63

Table 19 - continued

Laboratory code

Analysis technique

Instrument calibration

Internal standards applied

Sample clean up Details of sample

clean up

Extraction technique

Details of sample

extraction

3614 6) GC-MS 1) External calibration

3) Gel permeation chromatography

B) Sonication

3615 1) HPLC-FLD

1) External calibration

6) Other PLE GPC

A) Pressurised liquid extraction (PLE)

3616 6) GC-MS

2) Internal standardisation with labelled substances

Benzo(a)pyrene-13C4 Benzo(a)anthracene-13C6 Benzo(b)fluoranthene-13C6 Chrysene-13C6

2) LiquidLiquid partitioning 4) Solid phase extraction

B) Sonication

3617 1) HPLC-FLD

1) External calibration

4) Solid phase extraction

C) Soxhlet extraction

3618 6) GC-MS

2) Internal standardisation with labelled substances

deuterated standards

1) Saponification 4) Solid phase extraction

D) Other Handshaken with cyclohexane

3619 6) GC-MS

2) Internal standardisation with labelled substances

C13 Labelled US EPA 16

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

D) Other Homegenisation amp saponiofication

3620 9) GC-HRMS

2) Internal standardisation with labelled substances

mix of deuterated PAHs (BAA-D12 CHR-D12 BAP-D12 BBF-D12)

3) Gel permeation chromatography 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

3621 6) GC-MS

2) Internal standardisation with labelled substances

Chrysene D12 Benzo(a)pyrene D12 Benzo(b)fluoranthene D12 Benzo(a)anthracene

2) LiquidLiquid partitioning 4) Solid phase extraction

B) Sonication

3622 1) HPLC-FLD

4) Standard addition method

4) Solid phase extraction

D) Other

Extraction with cyclohexane by heating under reflux

3623 7) GC-MSMS

2) Internal standardisation with labelled substances

C13 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

3624 2) HPLC-UV-FLD

1) External calibration

2) LiquidLiquid partitioning

D) Other hot solvent extraction

3626

64

Table 19 - continued

Laboratory code

Analysis technique

Instrument calibration

Internal standards

applied

Sample clean up

Details of sample

clean up

Extraction technique

Details of sample

extraction

5301 7) GC-MSMS

2) Internal standardisation with labelled substances

EACH SUBSTANCE C13

4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5302 7) GC-MSMS

2) Internal standardisation with labelled substances

Isotope labelled PAHs 13C for each analyte

4) Solid phase extraction

C) Soxhlet extraction

5303 7) GC-MSMS

3) Internal standardisation with unlabelled substances

HAP C13 4) Solid phase extraction

D) Other automate ASE200 DIONEX

5304 7) GC-MSMS

2) Internal standardisation with labelled substances

with C13 for each HAP

2) LiquidLiquid partitioning 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5305 7) GC-MSMS

2) Internal standardisation with labelled substances

C13 isotopiques internal standards

4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5306 7) GC-MSMS

2) Internal standardisation with labelled substances

C13 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5307 6) GC-MS

2) Internal standardisation with labelled substances

1) Saponification 2) LiquidLiquid partitioning

D) Other solvent extraction

5308 1) HPLC-FLD 1) External calibration

4) Solid phase extraction

B) Sonication

5309 1) HPLC-FLD 1) External calibration

4) Solid phase extraction

D) Other only SPE

5310 1) HPLC-FLD 1) External calibration

1) Saponification 2) LiquidLiquid partitioning 3) Gel permeation chromatography

C) Soxhlet extraction

5311 6) GC-MS

2) Internal standardisation with labelled substances

13 C Chrysene 13 C Benz(a)pyrene

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

B) Sonication

5312 1) HPLC-FLD

3) Internal standardisation with unlabelled substances

Benzo(b)chrysen

2) LiquidLiquid partitioning 4) Solid phase extraction

Quechers D) Other Quechers

65

Table 19 - continued

Laboratory code

Analysis technique

Instrument calibration

Internal standards applied

Sample clean up Details of sample

clean up

Extraction technique

Details of sample extraction

5313 6) GC-MS

2) Internal standardisation with labelled substances

Benz(a)anthracene D12 Benzo(b)fluranthene D12 Benzo(a)pyrene D12 Chrysene D12

1) Saponification 2) LiquidLiquid partitioning 3) Gel permeation chromatography

A) Pressurised liquid extraction (PLE)

5314 6) GC-MS

2) Internal standardisation with labelled substances

Benzo(a)pyren d12 2) LiquidLiquid partitioning

D) Other Extraction with

5315 1) HPLC-FLD

3) Internal standardisation with unlabelled substances

Benz(b)chrysen

1) Saponification 2) LiquidLiquid partitioning

D) Other shaker

5316 1) HPLC-FLD

1) External calibration

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

D) Other

LiquidLiquid extraction with cyclohexane and dimethylformamide

5317 7) GC-MSMS

1) External calibration

3) Gel permeation chromatography 6) Other

silica gel D) Other VDLUFA online method

5318

5319 1) HPLC-FLD

3) Internal standardisation with unlabelled substances

Benzo(b)chrysene 3) Gel permeation chromatography

A) Pressurised liquid extraction (PLE)

5320 6) GC-MS

3) Internal standardisation with unlabelled substances

22-binaphthyl

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

D) Other 3 hours refluxing

5321 1) HPLC-FLD

2) Internal standardisation with labelled substances

6-meacutethylchrysegravene

1) Saponification 2) LiquidLiquid partitioning

B) Sonication

5322 6) GC-MS

2) Internal standardisation with labelled substances

naftalene-d8 acenaphtalene-d10 phenanthrene-d10 chrysene-d12 perylene-d12 benzo(e)anthracene-d12

1) Saponification 2) LiquidLiquid partitioning 6) Other

clean-up with silica packed column

C) Soxhlet extraction

5323 1) HPLC-FLD

1) External calibration

4) Solid phase extraction 6) Other

Accelerated Solvent Extraction

A) Pressurised liquid extraction (PLE)

66

European Commission EUR 25245 EN ndash Joint Research Centre ndash Institute for Reference Materials and Measurements Title Report on the 8th inter-laboratory comparison organised by the European Union Reference Laboratory for Polycyclic Aromatic Hydrocarbons - Four marker PAHs in a dry extract of St Johns wort Author(s) Radoslav Lizak Szilard Szilagyi Philippe Verlinde and Thomas WENZL Luxembourg Publications Office of the European Union 2012 ndash 66 pp ndash 201 x 297 cm EUR ndash Scientific and Technical Research series ndash ISSN 1831-9424 ISBN 978-92-79-23337-1 doi 10278759387 Abstract The proficiency test here reported concerned the determination of the contents of four marker polycyclic aromatic hydrocarbons (PAHs) and of their sum in an food supplements test sample The set of marker PAHs consists of benz[a]anthacene benzo[a]pyrene benzo[b]fluoranthene and chrysene Participants to these PT were National Reference Laboratories for PAHs (NRLs-PAHs) and EU official food control laboratories The number of participants was in total 48

The PT was organised according ISO Standard 170432010

The test material used was a commercial dry extract of St Johns wort Participants also received a solution of the same PAHs either in acetonitrile or in toluene for checking their instrument calibration

The results from participants were rated with z-scores and zeta-scores About 68 of the reported results were attributed with z-scores with an absolute value of below two which is the threshold for satisfactory performance

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The mission of the JRC is to provide customer-driven scientific and technical support

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service of the European Commission the JRC functions as a reference centre of

science and technology for the Union Close to the policy-making process it serves

the common interest of the Member States while being independent of special

interests whether private or national

L

A-N

A-252

45-EN

-N

• 1 Summary
• 2 Introduction
• 3 Scope
• 4 Participating Laboratories
• 5 Time frame
• 6 Confidentiality
• 7 Test materials
• • 71 1Preparation of test materials
  • 72 Assigned values and standard deviation for proficiency assessment
  • 73 Homogeneity and stability
  • 74 Sample dispatch
  • • 8 Design of the proficiency test
    • 9 Evaluation of the results
    • • 91 2General
      • 92 21BEvaluation criteria
      • 93 Performance of participants
      • 94 Evaluation of the influence of analysis method
      • 95 Evaluation of the influence of calibration
      • 96 26Evaluation of compliance with legislation
      • • 10 Follow-up actions for underperforming laboratories
        • 11 1Conclusions
        • 12 1Acknowledgements
        • 13 References

48

Figure 7 Results of replicate determinations (indicated by triangles) of benzo[a]pyrene (BAP) in the food supplement test material

Horizontal blue lines represent the arithmetic mean value of replicate measurements and blue bars the reported expanded measurement uncertainty (k=2) The assigned value is

plotted as dotted line Red solid lines mark the range of satisfactory performance (|z|le2) Mean values are given for results outside the displayed data range

ProLab 2011

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36

1453

0953

0853

1853

2153

1036

0236

0436

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0436

1653

0353

0736

0336

2036

0053

0553

0636

0636

0536

1753

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2636

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2453

0153

2253

0253

2353

1136

1836

1953

1336

1036

2336

2253

1936

1253

1553

1253

1736

0753

2036

2136

0836

11

an

aly

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ten

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gk

g)

55

5

45

4

35

3

25

2

15

1

05

0

573

308

3

Sample St Johns wort extract replicatesMeasurand Benzo[a]pyreneNo of laboratories 48

Assigned value 165 microgkg (Reference value)Target sd 036 microgkgTolerance limits 093 - 237 microgkg (|Z score| lt 200)

Assig

ned va

lue

Limit of tolerance

Limit of tolerance

49

Table 13 Analysis results reported by the participants for the content of benzo[b]fluoranthene (BBF) in the food supplements test material Values are presented as reported

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

3600 32 3 3 31 09 2

3602 173 175 16 17 017

3603 265 252 25 256 025 2

3604 116 108 105 11 028 2

3605 286 225 232 247 074 2

3606 294 273 261 276 046 2

3607 579 579 544 567 113 2

3608 103 1201 876 1036 326 2

3610 173 196 204 191 038 2

3611 546 618 565 576 75 2

3612 3 28 28 29 06 2

3613 28 31 32 31 06 2

3614 135 111 135 127 022 2

3615 4187 3678 4111 4 08 2

3616 2 2 2 2 01 2

3617 33 27 299 035 2

3618 31 31 31 31 05 2

3619 326 324 312 326 064 2

3620 277 28 272 276 069 2

3621 26 25 23 25 04 2

3622 346 336 333 34 052 2

3623 26 28 29 28 02 2

3624

3626 288 28 307 292 117 2

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

5301 29 3 29 29 06 2

5302 297 3 296 298 06 2

5303 2705 2444 2608 2586 22 78

5304 245 254 26 253 85 2

5305 23 23 26 24 15

5306 42 43 4 42 21 2

5307 221 216 241 221

5308 232169 246654 240722 229848

5309 19 22 19 2 0

5310 3 315 325 313 016

5311 262 277 304 281 15 2

5312 27 289 337 299 075 2

5313 321 284 378 328

5314 423 404 385 404 34 083

5315 42 506 416 444 09 2

5316 265 267 277 273 018 2

5317 185 197 195 192 006 2

5318 276 281 279 04 2

5319 396 389 403 396 079 2

5320 623 618 635 625 14

5321 31

5322 24 33 49 33 35 2

5323 38 45 35 45 27 2

50

Figure 8 Results of replicate determinations (indicated by triangles) of benzo[b]fluoranthene (BBF) in the food supplement test material

Horizontal blue lines represent the arithmetic mean value of replicate measurements and blue bars the reported expanded measurement uncertainty (k=2) The assigned value is

plotted as dotted line Red solid lines mark the range of satisfactory performance (|z|le2) Mean values are given for results outside the displayed data range

ProLab 2011

Laboratory

36

0436

1436

0236

1053

1736

1653

0953

0753

0853

0536

2136

0553

0436

0353

0353

1636

0636

2036

2353

1853

1136

1236

2653

0153

0253

1236

1736

1336

0036

1853

2153

1036

1953

1336

2253

2253

2353

1936

1553

1453

0653

1536

0753

2036

0836

11

an

aly

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on

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gk

g)

75

7

65

6

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45

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35

3

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2

15

1

05

0

103

6

576

3

Sample St Johns wort extract replicatesMeasurand Benzo[b]fluorantheneNo of laboratories 46

Assigned value 292 microgkg (Reference value)Target sd 060 microgkgTolerance limits 172 - 412 microgkg (|Z score| lt 200)

Assig

ned va

lue

Limit of tolerance

Limit of tolerance

51

Table 14 Analysis results reported by the participants for the content of chrysene (CHR) in the food supplements test material Values are presented as reported

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

3600 5 49 5 5 07 2

3602 32 304 29 304 036

3603 462 469 447 459 048 2

3604 438 428 42 429 119 2

3605 365 41 381 386 085 2

3606 494 411 45 451 124 2

3607 897 774 787 819 18 2

3608 986 887 1168 1014 284 2

3610 418 392 462 424 085 2

3611 833 893 87 865 61 2

3612 6 64 58 61 12 2

3613 41 39 4 4 08 2

3614 272 254 288 271 054 2

3615 605 6028 5964 601 12 2

3616 47 45 49 47 05 2

3617 445 431 389 422 065 2

3618 45 45 45 45 06 2

3619 738 747 72 738 118 2

3620 463 472 446 46 092 2

3621 53 49 51 51 06 2

3622 478 513 505 488 08 2

3623 73 73 72 72 05 2

3624

3626 63 622 634 628 251 2

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

5301 49 49 49 49 1 2

5302 687 683 666 679 136 2

5303 6777 6181 6382 6441 24 70

5304 592 581 6 591 85 2

5305 6 58 64 61 20

5306 49 5 51 49 25 2

5307 524 5 574 524

5308 211488 216782 221146 213243

5309

5310 372 393 394 386 019

5311 43 459 422 437 15 2

5312 934 943 981 953 191 2

5313 793 73 99 838

5314 636 561 565 588 18 0818

5315 547 583 499 542 11 2

5316 292 286 285 282 06 2

5317 52 598 517 545 046 2

5318 368 345 357 05 2

5319 449 471 474 465 116 2

5320 76 924 818 834 99

5321 33

5322 7 7 76 7 35 2

5323 87 73 77 73 37 2

52

Figure 9 Results of replicate determinations (indicated by triangles) of chrysene (CHR) in the food supplement test material

Horizontal blue lines represent the arithmetic mean value of replicate measurements and blue bars the reported expanded measurement uncertainty (k=2) The assigned value is

plotted as dotted line Red solid lines mark the range of satisfactory performance (|z|le2) Mean values are given for results outside the displayed data range

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0753

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0253

2236

2336

1953

2336

0753

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1353

1236

0836

11

an

aly

te c

on

ten

t (micro

gk

g)

13

12

11

10

9

8

7

6

5

4

3

2

1

0

865

3

Sample St Johns wort extract replicatesMeasurand ChryseneNo of laboratories 45

Assigned value 407 microgkg (Reference value)Target sd 083 microgkgTolerance limits 241 - 573 microgkg (|Z score| lt 200)

Assig

ned va

lue

Limit of tolerance

Limit of tolerance

53

Table 15 Analysis results reported by the participants for the sum of the four marker PAHs in the food supplements test material Values are presented as reported

Laboratory code

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg

3600 128 24 2

3602 79 126

3603 1127 096 2

3604 893 258 2

3605 1091 305 2

3606 1184 238 2

3607 2098 42 2

3608 3336 325 2

3610 1138 17 2

3611 2218 259 2

3612 143 29 2

3613 122 2 2

3614 663 129 2

3615 152 304 2

3616 102 03 2

3617 1252 169 2

3618 128 18 2

3619 157 305 2

3620 1204 12 2

3621 151 19 2

3622 1361 115 2

3623 154 09 2

3624

3626 1409 56 2

Laboratory code

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg

5301 126

5302 37 074 2

5303 13438 92

5304 1251 85 2

5305 125 20

5306 129 65 2

5307 1363

5308 7245598

5309

5310 1127 056

5311 1367 15 2

5312 2042 408 2

5313 1726

5314 1558

5315 177 35 2

5316 96 073 2

5317 1229 066 2

5318 82 05 2

5319 1225 267 2

5320 2309 41

5321

5322 207 35 2

5323 171 103 2

54

ANNEX 9 Results reported for the standard solutions with undisclosed analyte content Table 16 Analysis results reported by the participants for the four marker PAHs in the standard solution in toluene with undisclosed analyte

content Values are presented as reported U measurement uncertainty k coverage factor

benz[a]anthracene benzo[a]pyrene benzo[b]fluoranthene chrysene Laboratory

code Unit Result U k Result U k Result U k Result U k 3600 microgl 38 67 2 25 45 2 211 68 2 275 38 2

3605 microgl 367 184 2 2482 124 2 2592 13 2 235 118 2

3606 microgkg 3763 176 2 2609 142 2 2721 118 2 2539 134 2

3607 microgkg 3706 815 2 2824 565 2 2591 518 2 2587 569 2

3612 microgl 38 08 2 237 11 2 244 18 2 257 02 2

3614 microgl 324 65 2 209 38 2 215 37 2 21 42 2

3616 microgkg 318 07 2 224 14 2 231 26 2 216 1 2

3618 microgkg 417 17 2 284 08 2 292 06 2 297 03 2

3619 microgkg 47 742 2 274 468 2 228 387 2 274 434 2

3620 microgl 495 74 2 294 29 2 321 8 2 321 64 2

3621 microgl 241 22 2 143 13 2 188 16 2 145 14 2

3623 microgkg 476 25 2 311 53 2 269 3 2 297 03 2

3626 microgl 359 38 2 235 23 2 238 27 2 24 18 2

5301 microgkg 426 29 299 289

5302 microgkg 436 87 2 296 59 2 296 59 2 289 58 2

5303 microgl 3721 27 102 22838 19 99 23756 22 99 25486 24 98

5304 microgkg 38 29 2 225 26 2 257 37 2 258 52 2

5305 microgkg 305 225 245 29

5306 microgkg 316 63 2 247 49 2 65 13 2 22 44 2

5307 microgkg 3658 2559 241 2414

5308 microgkg 16854 109324 246654 216782

5311 microgl 374 20 2 279 15 2 268 15 2 272 15 2

5313 microgl 3408 2165 2275 2373

5314 microgkg 395 5 2875 11 3614 34 2905 18

5320 microgl 3267 368 1986 571 28 683 2427 512

5322 microgl 282 35 2 225 35 2 241 35 2 21 35 2

55

Figure 10 Youden plots of the percent deviation of the reported values from the assigned values for the food supplement test sample and the

standard solution in toluene with undisclosed content

(The identity of the respective measurand is given above the figure Results exceeding a difference of more than 100 were omitted)

56

Figure 10 - continued

57

Table 17 Analysis results reported by the participants for the four marker PAHs in the standard solution in acetonitrile with undisclosed

analyte content

Values are presented as reported U measurement uncertainty k coverage factor

benz[a]anthracene benzo[a]pyrene benzo[b]fluoranthene chrysene

Laboratory

code Unit Result U k Result U k Result U k Result U k

3602 microgkg 3675 247 247 244

3603 microgkg 358 086 2 236 028 2 235 028 2 236 057 2

3604 microgl 475 29 2 19 12 2 194 13 2 258 12 2

3608 microgl 3623 078 2 2385 023 2 2343 18 2 2296 092 2

3610 microgkg 369 185 2 25 125 2 251 126 2 244 122 2

3611 microgkg 48 02 2 323 02 2 328 04 2 317 03 2

3613 microgl 332 31 2 261 3 2 249 25 2 239 24 2

3615 microgl 415 2635 2729 2689

3617 microgkg 37 09 2 244 06 2 284 07 2 239 06 2

3622 microgl 37 12 2 25 23 2 257 21 2 238 1 2

3624 microgkg 402 104 2 255 084 2 265 096 2 253 07 2

5306 microgkg 563 113 2 371 74 2 359 72 2 333 67 2

5308 microgkg 168633 102669 232169 211488

5309 microgl 402 268 27

5310 microgl 315 158 25 125 25 125 288 144

5312 microgl 428 043 2 282 028 2 272 027 2 275 028 2

5315 microgkg 387 08 2 256 05 2 251 05 2 253 05 2

5316 microgl 3839 342 2 2485 191 2 2559 168 2 239 51 2

5317 microgl 2466 043 2 2212 034 2 2472 016 2 2146 068 2

5319 microgl 414 41 2 279 28 2 268 27 2 26 31 2

5321 microgkg 476 316 326 328

5322 microgl 2547 35 2

5323 microgkg 405 203 2 239 119 2 255 153 2 201 11 2

58

Figure 11 Youden plots of the percent deviation of the reported values from the assigned values for the food supplement test sample and the

standard solution in acetonitrile with undisclosed content

(The identity of the respective measurand is given above the figure Results exceeding a difference of more than 100 were omitted)

59

Figure 11 - continued

60

ANNEX 10 Table 18 Method performance parameters reported by the participants

Laboratory code

Analyte LOD LOQ Recovery Analyte LOD LOQ Recovery

microgkg microgkg microgkg microgkg 3600 BaA 03 06 978 BaP 03 07 9483602 BaA 007 021 926 BaP 005 015 993603 BaA 0011 05 987 BaP 0005 051 9553604 BaA 02 04 86 BaP 02 04 883605 BaA 0025 005 118 BaP 0025 005 1083606 BaA 001 003 65 BaP 001 003 703607 BaA 021 071 74 BaP 027 089 943608 BaA 016 048 8213 BaP 015 046 83683610 BaA 02 05 85 BaP 005 02 853611 BaA BaP 3612 BaA 1 117 BaP 1 1203613 BaA 01 03 90 BaP 01 03 903614 BaA 02 05 96 BaP 02 05 933615 BaA 005 015 110 BaP 005 015 1053616 BaA 01 05 108 BaP 01 05 753617 BaA 018 037 405 BaP 009 018 3873618 BaA 01 03 63 BaP 01 03 593619 BaA 005 005 59 BaP 033 033 543620 BaA 0009 0018 64 BaP 0005 001 553621 BaA 007 02 84 BaP 007 02 923622 BaA 01 03 1101 BaP 01 03 9853623 BaA 02 08 105 BaP 02 08 1013624 BaA BaP 01 033 8513625 BaA BaP 3626 BaA BaP 5301 BaA 003 01 102 BaP 003 01 1005302 BaA 01 03 78 BaP 01 03 865303 BaA 003 01 62 BaP 003 01 805304 BaA 1 1 70 BaP 1 1 955305 BaA 02 05 85 BaP 02 05 855306 BaA 03 1 699 BaP 03 1 905307 BaA 6537 BaP 81165308 BaA 0088 0265 99 BaP 00262 00785 1005309 BaA 01 08 92 BaP 01 08 925310 BaA 01 03 BaP 01 03 905311 BaA 002 005 80 BaP 002 004 915312 BaA 03 09 105 BaP 03 09 1055313 BaA 04 08 BaP 04 08 5314 BaA 001 002 96 BaP 001 002 9485315 BaA 01 03 50 - 100 BaP 01 03 50 - 100 5316 BaA 002 011 827 BaP 004 019 10445317 BaA 05 1 76 BaP 05 1 755318 BaA BaP 5319 BaA 009 029 951 BaP 011 041 995320 BaA 12 18 946 BaP 06 1 8345321 BaA 03 05 BaP 05 1 5322 BaA 1 2 100 BaP 1 2 1005323 BaA 26 BaP 26

61

ANNEX 10 Table 18 - continued

Laboratory code Analyte LOD LOQ Recovery Analyte LOD LOQ Recovery

microgkg microgkg microgkg microgkg 3600 BbF 03 05 96 CHR 02 03 10153602 BbF 015 045 947 CHR 003 009 9193603 BbF 0014 05 1012 CHR 0004 05 10033604 BbF 02 04 85 CHR 02 04 873605 BbF 005 01 94 CHR 0025 005 863606 BbF 001 003 70 CHR 001 003 653607 BbF 018 06 100 CHR 014 028 943608 BbF 015 046 7979 CHR 015 045 71253610 BbF 005 02 85 CHR 02 05 853611 BbF CHR 3612 BbF 1 118 CHR 1 1173613 BbF 01 03 90 CHR 01 03 903614 BbF 01 04 96 CHR 02 05 953615 BbF 008 025 109 CHR 003 01 1083616 BbF 01 05 78 CHR 01 05 723617 BbF 035 07 38 CHR 012 024 4163618 BbF 01 03 62 CHR 01 03 623619 BbF 016 016 63 CHR 01 01 593620 BbF 0003 0006 55 CHR 001 002 653621 BbF 007 02 103 CHR 007 02 893622 BbF 02 06 963 CHR 03 09 1043623 BbF 02 08 106 CHR 02 08 1193624 BbF CHR 3625 BbF CHR 3626 BbF CHR 5301 BbF 003 01 96 CHR 003 01 1145302 BbF 01 03 84 CHR 01 03 835303 BbF 003 01 78 CHR 003 01 705304 BbF 1 1 86 CHR 1 1 705305 BbF 02 05 90 CHR 02 05 955306 BbF 03 1 1435 CHR 03 1 1185307 BbF 8116 CHR 65375308 BbF 0079 0238 99 CHR 009 026 1005309 BbF 01 08 92 CHR 5310 BbF 07 2 CHR 01 03 5311 BbF 002 004 95 CHR 002 004 895312 BbF 03 09 105 CHR 03 09 1055313 BbF 04 08 CHR 04 08 5314 BbF 001 002 83 CHR 001 002 8185315 BbF 02 05 50 -100 CHR 02 05 50 - 100 5316 BbF 005 024 814 CHR 001 007 9595317 BbF 05 1 73 CHR 05 1 785318 BbF CHR 5319 BbF 011 038 946 CHR 013 043 9675320 BbF 07 09 1194 CHR 23 32 10455321 BbF 05 1 CHR 05 1 5322 BbF 1 2 100 CHR 1 2 1005323 BbF 26 CHR 26

62

ANNEX 11 Table 19 Details of analysis method reported by the participants Data are presented as reported

Laboratory code

Analysis technique

Instrument calibration

Internal standards applied

Sample clean up

Details of sample

clean up

Extraction technique

Details of sample

extraction

3600 6) GC-MS

2) Internal standardisation with labelled substances

Deuterated PAH mix 9 (Ehrenstorfer)

1) Saponification 2) LiquidLiquid partitioning

D) Other Liquidliquid partitioning

3602 2) HPLC-UV-FLD

1) External calibration

3) Gel permeation chromatography

A) Pressurised liquid extraction (PLE)

3603 1) HPLC-FLD

1) External calibration

3) Gel permeation chromatography

D) Other 1 minute with dichloromethane in vortex

3604 2) HPLC-UV-FLD

2) Internal standardisation with labelled substances

DiP D14

5) Donor acceptor complex chromatography

D) Other LL

3605 1) HPLC-FLD

1) External calibration

3) Gel permeation chromatography

C) Soxhlet extraction

3606 7) GC-MSMS

2) Internal standardisation with labelled substances

BaP 13C4 CHR 13C6 BaA 13C6 BbF 13C6

4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

3607 6) GC-MS

2) Internal standardisation with labelled substances

BaA-d12 CHR-d12 BbF-d12 BkF-d12 BaP-d12 ICP-d12 BgP-d12 DiP-d14

1) Saponification

D) Other solvent partitioning

3608 1) HPLC-FLD

1) External calibration

6) Other Liquid extraction

D) Other

Extraction in a rotary agitator and centrifugation

3610 2) HPLC-UV-FLD

1) External calibration

4) Solid phase extraction

B) Sonication

3611

3612 6) GC-MS

2) Internal standardisation with labelled substances

chrysene-d12 dibenzo(ah)anthracene-d14 dibenzo(ai)pyrene-d14

4) Solid phase extraction

B) Sonication

3613 5) LC-MSMS

3) Internal standardisation with unlabelled substances

I use Chrysene-D12 3) Gel permeation chromatography

D) Other extraction with hexan

63

Table 19 - continued

Laboratory code

Analysis technique

Instrument calibration

Internal standards applied

Sample clean up Details of sample

clean up

Extraction technique

Details of sample

extraction

3614 6) GC-MS 1) External calibration

3) Gel permeation chromatography

B) Sonication

3615 1) HPLC-FLD

1) External calibration

6) Other PLE GPC

A) Pressurised liquid extraction (PLE)

3616 6) GC-MS

2) Internal standardisation with labelled substances

Benzo(a)pyrene-13C4 Benzo(a)anthracene-13C6 Benzo(b)fluoranthene-13C6 Chrysene-13C6

2) LiquidLiquid partitioning 4) Solid phase extraction

B) Sonication

3617 1) HPLC-FLD

1) External calibration

4) Solid phase extraction

C) Soxhlet extraction

3618 6) GC-MS

2) Internal standardisation with labelled substances

deuterated standards

1) Saponification 4) Solid phase extraction

D) Other Handshaken with cyclohexane

3619 6) GC-MS

2) Internal standardisation with labelled substances

C13 Labelled US EPA 16

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

D) Other Homegenisation amp saponiofication

3620 9) GC-HRMS

2) Internal standardisation with labelled substances

mix of deuterated PAHs (BAA-D12 CHR-D12 BAP-D12 BBF-D12)

3) Gel permeation chromatography 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

3621 6) GC-MS

2) Internal standardisation with labelled substances

Chrysene D12 Benzo(a)pyrene D12 Benzo(b)fluoranthene D12 Benzo(a)anthracene

2) LiquidLiquid partitioning 4) Solid phase extraction

B) Sonication

3622 1) HPLC-FLD

4) Standard addition method

4) Solid phase extraction

D) Other

Extraction with cyclohexane by heating under reflux

3623 7) GC-MSMS

2) Internal standardisation with labelled substances

C13 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

3624 2) HPLC-UV-FLD

1) External calibration

2) LiquidLiquid partitioning

D) Other hot solvent extraction

3626

64

Table 19 - continued

Laboratory code

Analysis technique

Instrument calibration

Internal standards

applied

Sample clean up

Details of sample

clean up

Extraction technique

Details of sample

extraction

5301 7) GC-MSMS

2) Internal standardisation with labelled substances

EACH SUBSTANCE C13

4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5302 7) GC-MSMS

2) Internal standardisation with labelled substances

Isotope labelled PAHs 13C for each analyte

4) Solid phase extraction

C) Soxhlet extraction

5303 7) GC-MSMS

3) Internal standardisation with unlabelled substances

HAP C13 4) Solid phase extraction

D) Other automate ASE200 DIONEX

5304 7) GC-MSMS

2) Internal standardisation with labelled substances

with C13 for each HAP

2) LiquidLiquid partitioning 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5305 7) GC-MSMS

2) Internal standardisation with labelled substances

C13 isotopiques internal standards

4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5306 7) GC-MSMS

2) Internal standardisation with labelled substances

C13 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5307 6) GC-MS

2) Internal standardisation with labelled substances

1) Saponification 2) LiquidLiquid partitioning

D) Other solvent extraction

5308 1) HPLC-FLD 1) External calibration

4) Solid phase extraction

B) Sonication

5309 1) HPLC-FLD 1) External calibration

4) Solid phase extraction

D) Other only SPE

5310 1) HPLC-FLD 1) External calibration

1) Saponification 2) LiquidLiquid partitioning 3) Gel permeation chromatography

C) Soxhlet extraction

5311 6) GC-MS

2) Internal standardisation with labelled substances

13 C Chrysene 13 C Benz(a)pyrene

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

B) Sonication

5312 1) HPLC-FLD

3) Internal standardisation with unlabelled substances

Benzo(b)chrysen

2) LiquidLiquid partitioning 4) Solid phase extraction

Quechers D) Other Quechers

65

Table 19 - continued

Laboratory code

Analysis technique

Instrument calibration

Internal standards applied

Sample clean up Details of sample

clean up

Extraction technique

Details of sample extraction

5313 6) GC-MS

2) Internal standardisation with labelled substances

Benz(a)anthracene D12 Benzo(b)fluranthene D12 Benzo(a)pyrene D12 Chrysene D12

1) Saponification 2) LiquidLiquid partitioning 3) Gel permeation chromatography

A) Pressurised liquid extraction (PLE)

5314 6) GC-MS

2) Internal standardisation with labelled substances

Benzo(a)pyren d12 2) LiquidLiquid partitioning

D) Other Extraction with

5315 1) HPLC-FLD

3) Internal standardisation with unlabelled substances

Benz(b)chrysen

1) Saponification 2) LiquidLiquid partitioning

D) Other shaker

5316 1) HPLC-FLD

1) External calibration

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

D) Other

LiquidLiquid extraction with cyclohexane and dimethylformamide

5317 7) GC-MSMS

1) External calibration

3) Gel permeation chromatography 6) Other

silica gel D) Other VDLUFA online method

5318

5319 1) HPLC-FLD

3) Internal standardisation with unlabelled substances

Benzo(b)chrysene 3) Gel permeation chromatography

A) Pressurised liquid extraction (PLE)

5320 6) GC-MS

3) Internal standardisation with unlabelled substances

22-binaphthyl

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

D) Other 3 hours refluxing

5321 1) HPLC-FLD

2) Internal standardisation with labelled substances

6-meacutethylchrysegravene

1) Saponification 2) LiquidLiquid partitioning

B) Sonication

5322 6) GC-MS

2) Internal standardisation with labelled substances

naftalene-d8 acenaphtalene-d10 phenanthrene-d10 chrysene-d12 perylene-d12 benzo(e)anthracene-d12

1) Saponification 2) LiquidLiquid partitioning 6) Other

clean-up with silica packed column

C) Soxhlet extraction

5323 1) HPLC-FLD

1) External calibration

4) Solid phase extraction 6) Other

Accelerated Solvent Extraction

A) Pressurised liquid extraction (PLE)

66

European Commission EUR 25245 EN ndash Joint Research Centre ndash Institute for Reference Materials and Measurements Title Report on the 8th inter-laboratory comparison organised by the European Union Reference Laboratory for Polycyclic Aromatic Hydrocarbons - Four marker PAHs in a dry extract of St Johns wort Author(s) Radoslav Lizak Szilard Szilagyi Philippe Verlinde and Thomas WENZL Luxembourg Publications Office of the European Union 2012 ndash 66 pp ndash 201 x 297 cm EUR ndash Scientific and Technical Research series ndash ISSN 1831-9424 ISBN 978-92-79-23337-1 doi 10278759387 Abstract The proficiency test here reported concerned the determination of the contents of four marker polycyclic aromatic hydrocarbons (PAHs) and of their sum in an food supplements test sample The set of marker PAHs consists of benz[a]anthacene benzo[a]pyrene benzo[b]fluoranthene and chrysene Participants to these PT were National Reference Laboratories for PAHs (NRLs-PAHs) and EU official food control laboratories The number of participants was in total 48

The PT was organised according ISO Standard 170432010

The test material used was a commercial dry extract of St Johns wort Participants also received a solution of the same PAHs either in acetonitrile or in toluene for checking their instrument calibration

The results from participants were rated with z-scores and zeta-scores About 68 of the reported results were attributed with z-scores with an absolute value of below two which is the threshold for satisfactory performance

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science and technology for the Union Close to the policy-making process it serves

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interests whether private or national

L

A-N

A-252

45-EN

-N

• 1 Summary
• 2 Introduction
• 3 Scope
• 4 Participating Laboratories
• 5 Time frame
• 6 Confidentiality
• 7 Test materials
• • 71 1Preparation of test materials
  • 72 Assigned values and standard deviation for proficiency assessment
  • 73 Homogeneity and stability
  • 74 Sample dispatch
  • • 8 Design of the proficiency test
    • 9 Evaluation of the results
    • • 91 2General
      • 92 21BEvaluation criteria
      • 93 Performance of participants
      • 94 Evaluation of the influence of analysis method
      • 95 Evaluation of the influence of calibration
      • 96 26Evaluation of compliance with legislation
      • • 10 Follow-up actions for underperforming laboratories
        • 11 1Conclusions
        • 12 1Acknowledgements
        • 13 References

49

Table 13 Analysis results reported by the participants for the content of benzo[b]fluoranthene (BBF) in the food supplements test material Values are presented as reported

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

3600 32 3 3 31 09 2

3602 173 175 16 17 017

3603 265 252 25 256 025 2

3604 116 108 105 11 028 2

3605 286 225 232 247 074 2

3606 294 273 261 276 046 2

3607 579 579 544 567 113 2

3608 103 1201 876 1036 326 2

3610 173 196 204 191 038 2

3611 546 618 565 576 75 2

3612 3 28 28 29 06 2

3613 28 31 32 31 06 2

3614 135 111 135 127 022 2

3615 4187 3678 4111 4 08 2

3616 2 2 2 2 01 2

3617 33 27 299 035 2

3618 31 31 31 31 05 2

3619 326 324 312 326 064 2

3620 277 28 272 276 069 2

3621 26 25 23 25 04 2

3622 346 336 333 34 052 2

3623 26 28 29 28 02 2

3624

3626 288 28 307 292 117 2

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

5301 29 3 29 29 06 2

5302 297 3 296 298 06 2

5303 2705 2444 2608 2586 22 78

5304 245 254 26 253 85 2

5305 23 23 26 24 15

5306 42 43 4 42 21 2

5307 221 216 241 221

5308 232169 246654 240722 229848

5309 19 22 19 2 0

5310 3 315 325 313 016

5311 262 277 304 281 15 2

5312 27 289 337 299 075 2

5313 321 284 378 328

5314 423 404 385 404 34 083

5315 42 506 416 444 09 2

5316 265 267 277 273 018 2

5317 185 197 195 192 006 2

5318 276 281 279 04 2

5319 396 389 403 396 079 2

5320 623 618 635 625 14

5321 31

5322 24 33 49 33 35 2

5323 38 45 35 45 27 2

50

Figure 8 Results of replicate determinations (indicated by triangles) of benzo[b]fluoranthene (BBF) in the food supplement test material

Horizontal blue lines represent the arithmetic mean value of replicate measurements and blue bars the reported expanded measurement uncertainty (k=2) The assigned value is

plotted as dotted line Red solid lines mark the range of satisfactory performance (|z|le2) Mean values are given for results outside the displayed data range

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1936

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Sample St Johns wort extract replicatesMeasurand Benzo[b]fluorantheneNo of laboratories 46

Assigned value 292 microgkg (Reference value)Target sd 060 microgkgTolerance limits 172 - 412 microgkg (|Z score| lt 200)

Assig

ned va

lue

Limit of tolerance

Limit of tolerance

51

Table 14 Analysis results reported by the participants for the content of chrysene (CHR) in the food supplements test material Values are presented as reported

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

3600 5 49 5 5 07 2

3602 32 304 29 304 036

3603 462 469 447 459 048 2

3604 438 428 42 429 119 2

3605 365 41 381 386 085 2

3606 494 411 45 451 124 2

3607 897 774 787 819 18 2

3608 986 887 1168 1014 284 2

3610 418 392 462 424 085 2

3611 833 893 87 865 61 2

3612 6 64 58 61 12 2

3613 41 39 4 4 08 2

3614 272 254 288 271 054 2

3615 605 6028 5964 601 12 2

3616 47 45 49 47 05 2

3617 445 431 389 422 065 2

3618 45 45 45 45 06 2

3619 738 747 72 738 118 2

3620 463 472 446 46 092 2

3621 53 49 51 51 06 2

3622 478 513 505 488 08 2

3623 73 73 72 72 05 2

3624

3626 63 622 634 628 251 2

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

5301 49 49 49 49 1 2

5302 687 683 666 679 136 2

5303 6777 6181 6382 6441 24 70

5304 592 581 6 591 85 2

5305 6 58 64 61 20

5306 49 5 51 49 25 2

5307 524 5 574 524

5308 211488 216782 221146 213243

5309

5310 372 393 394 386 019

5311 43 459 422 437 15 2

5312 934 943 981 953 191 2

5313 793 73 99 838

5314 636 561 565 588 18 0818

5315 547 583 499 542 11 2

5316 292 286 285 282 06 2

5317 52 598 517 545 046 2

5318 368 345 357 05 2

5319 449 471 474 465 116 2

5320 76 924 818 834 99

5321 33

5322 7 7 76 7 35 2

5323 87 73 77 73 37 2

52

Figure 9 Results of replicate determinations (indicated by triangles) of chrysene (CHR) in the food supplement test material

Horizontal blue lines represent the arithmetic mean value of replicate measurements and blue bars the reported expanded measurement uncertainty (k=2) The assigned value is

plotted as dotted line Red solid lines mark the range of satisfactory performance (|z|le2) Mean values are given for results outside the displayed data range

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0836

1453

1636

0253

2153

1836

0553

1036

1336

1736

1036

0453

1136

1836

0636

0336

2053

1936

1653

0136

0036

2253

0636

2153

0753

1553

1753

1453

0436

1536

1253

0536

2653

0353

0253

2236

2336

1953

2336

0753

2053

1353

1236

0836

11

an

aly

te c

on

ten

t (micro

gk

g)

13

12

11

10

9

8

7

6

5

4

3

2

1

0

865

3

Sample St Johns wort extract replicatesMeasurand ChryseneNo of laboratories 45

Assigned value 407 microgkg (Reference value)Target sd 083 microgkgTolerance limits 241 - 573 microgkg (|Z score| lt 200)

Assig

ned va

lue

Limit of tolerance

Limit of tolerance

53

Table 15 Analysis results reported by the participants for the sum of the four marker PAHs in the food supplements test material Values are presented as reported

Laboratory code

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg

3600 128 24 2

3602 79 126

3603 1127 096 2

3604 893 258 2

3605 1091 305 2

3606 1184 238 2

3607 2098 42 2

3608 3336 325 2

3610 1138 17 2

3611 2218 259 2

3612 143 29 2

3613 122 2 2

3614 663 129 2

3615 152 304 2

3616 102 03 2

3617 1252 169 2

3618 128 18 2

3619 157 305 2

3620 1204 12 2

3621 151 19 2

3622 1361 115 2

3623 154 09 2

3624

3626 1409 56 2

Laboratory code

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg

5301 126

5302 37 074 2

5303 13438 92

5304 1251 85 2

5305 125 20

5306 129 65 2

5307 1363

5308 7245598

5309

5310 1127 056

5311 1367 15 2

5312 2042 408 2

5313 1726

5314 1558

5315 177 35 2

5316 96 073 2

5317 1229 066 2

5318 82 05 2

5319 1225 267 2

5320 2309 41

5321

5322 207 35 2

5323 171 103 2

54

ANNEX 9 Results reported for the standard solutions with undisclosed analyte content Table 16 Analysis results reported by the participants for the four marker PAHs in the standard solution in toluene with undisclosed analyte

content Values are presented as reported U measurement uncertainty k coverage factor

benz[a]anthracene benzo[a]pyrene benzo[b]fluoranthene chrysene Laboratory

code Unit Result U k Result U k Result U k Result U k 3600 microgl 38 67 2 25 45 2 211 68 2 275 38 2

3605 microgl 367 184 2 2482 124 2 2592 13 2 235 118 2

3606 microgkg 3763 176 2 2609 142 2 2721 118 2 2539 134 2

3607 microgkg 3706 815 2 2824 565 2 2591 518 2 2587 569 2

3612 microgl 38 08 2 237 11 2 244 18 2 257 02 2

3614 microgl 324 65 2 209 38 2 215 37 2 21 42 2

3616 microgkg 318 07 2 224 14 2 231 26 2 216 1 2

3618 microgkg 417 17 2 284 08 2 292 06 2 297 03 2

3619 microgkg 47 742 2 274 468 2 228 387 2 274 434 2

3620 microgl 495 74 2 294 29 2 321 8 2 321 64 2

3621 microgl 241 22 2 143 13 2 188 16 2 145 14 2

3623 microgkg 476 25 2 311 53 2 269 3 2 297 03 2

3626 microgl 359 38 2 235 23 2 238 27 2 24 18 2

5301 microgkg 426 29 299 289

5302 microgkg 436 87 2 296 59 2 296 59 2 289 58 2

5303 microgl 3721 27 102 22838 19 99 23756 22 99 25486 24 98

5304 microgkg 38 29 2 225 26 2 257 37 2 258 52 2

5305 microgkg 305 225 245 29

5306 microgkg 316 63 2 247 49 2 65 13 2 22 44 2

5307 microgkg 3658 2559 241 2414

5308 microgkg 16854 109324 246654 216782

5311 microgl 374 20 2 279 15 2 268 15 2 272 15 2

5313 microgl 3408 2165 2275 2373

5314 microgkg 395 5 2875 11 3614 34 2905 18

5320 microgl 3267 368 1986 571 28 683 2427 512

5322 microgl 282 35 2 225 35 2 241 35 2 21 35 2

55

Figure 10 Youden plots of the percent deviation of the reported values from the assigned values for the food supplement test sample and the

standard solution in toluene with undisclosed content

(The identity of the respective measurand is given above the figure Results exceeding a difference of more than 100 were omitted)

56

Figure 10 - continued

57

Table 17 Analysis results reported by the participants for the four marker PAHs in the standard solution in acetonitrile with undisclosed

analyte content

Values are presented as reported U measurement uncertainty k coverage factor

benz[a]anthracene benzo[a]pyrene benzo[b]fluoranthene chrysene

Laboratory

code Unit Result U k Result U k Result U k Result U k

3602 microgkg 3675 247 247 244

3603 microgkg 358 086 2 236 028 2 235 028 2 236 057 2

3604 microgl 475 29 2 19 12 2 194 13 2 258 12 2

3608 microgl 3623 078 2 2385 023 2 2343 18 2 2296 092 2

3610 microgkg 369 185 2 25 125 2 251 126 2 244 122 2

3611 microgkg 48 02 2 323 02 2 328 04 2 317 03 2

3613 microgl 332 31 2 261 3 2 249 25 2 239 24 2

3615 microgl 415 2635 2729 2689

3617 microgkg 37 09 2 244 06 2 284 07 2 239 06 2

3622 microgl 37 12 2 25 23 2 257 21 2 238 1 2

3624 microgkg 402 104 2 255 084 2 265 096 2 253 07 2

5306 microgkg 563 113 2 371 74 2 359 72 2 333 67 2

5308 microgkg 168633 102669 232169 211488

5309 microgl 402 268 27

5310 microgl 315 158 25 125 25 125 288 144

5312 microgl 428 043 2 282 028 2 272 027 2 275 028 2

5315 microgkg 387 08 2 256 05 2 251 05 2 253 05 2

5316 microgl 3839 342 2 2485 191 2 2559 168 2 239 51 2

5317 microgl 2466 043 2 2212 034 2 2472 016 2 2146 068 2

5319 microgl 414 41 2 279 28 2 268 27 2 26 31 2

5321 microgkg 476 316 326 328

5322 microgl 2547 35 2

5323 microgkg 405 203 2 239 119 2 255 153 2 201 11 2

58

Figure 11 Youden plots of the percent deviation of the reported values from the assigned values for the food supplement test sample and the

standard solution in acetonitrile with undisclosed content

(The identity of the respective measurand is given above the figure Results exceeding a difference of more than 100 were omitted)

59

Figure 11 - continued

60

ANNEX 10 Table 18 Method performance parameters reported by the participants

Laboratory code

Analyte LOD LOQ Recovery Analyte LOD LOQ Recovery

microgkg microgkg microgkg microgkg 3600 BaA 03 06 978 BaP 03 07 9483602 BaA 007 021 926 BaP 005 015 993603 BaA 0011 05 987 BaP 0005 051 9553604 BaA 02 04 86 BaP 02 04 883605 BaA 0025 005 118 BaP 0025 005 1083606 BaA 001 003 65 BaP 001 003 703607 BaA 021 071 74 BaP 027 089 943608 BaA 016 048 8213 BaP 015 046 83683610 BaA 02 05 85 BaP 005 02 853611 BaA BaP 3612 BaA 1 117 BaP 1 1203613 BaA 01 03 90 BaP 01 03 903614 BaA 02 05 96 BaP 02 05 933615 BaA 005 015 110 BaP 005 015 1053616 BaA 01 05 108 BaP 01 05 753617 BaA 018 037 405 BaP 009 018 3873618 BaA 01 03 63 BaP 01 03 593619 BaA 005 005 59 BaP 033 033 543620 BaA 0009 0018 64 BaP 0005 001 553621 BaA 007 02 84 BaP 007 02 923622 BaA 01 03 1101 BaP 01 03 9853623 BaA 02 08 105 BaP 02 08 1013624 BaA BaP 01 033 8513625 BaA BaP 3626 BaA BaP 5301 BaA 003 01 102 BaP 003 01 1005302 BaA 01 03 78 BaP 01 03 865303 BaA 003 01 62 BaP 003 01 805304 BaA 1 1 70 BaP 1 1 955305 BaA 02 05 85 BaP 02 05 855306 BaA 03 1 699 BaP 03 1 905307 BaA 6537 BaP 81165308 BaA 0088 0265 99 BaP 00262 00785 1005309 BaA 01 08 92 BaP 01 08 925310 BaA 01 03 BaP 01 03 905311 BaA 002 005 80 BaP 002 004 915312 BaA 03 09 105 BaP 03 09 1055313 BaA 04 08 BaP 04 08 5314 BaA 001 002 96 BaP 001 002 9485315 BaA 01 03 50 - 100 BaP 01 03 50 - 100 5316 BaA 002 011 827 BaP 004 019 10445317 BaA 05 1 76 BaP 05 1 755318 BaA BaP 5319 BaA 009 029 951 BaP 011 041 995320 BaA 12 18 946 BaP 06 1 8345321 BaA 03 05 BaP 05 1 5322 BaA 1 2 100 BaP 1 2 1005323 BaA 26 BaP 26

61

ANNEX 10 Table 18 - continued

Laboratory code Analyte LOD LOQ Recovery Analyte LOD LOQ Recovery

microgkg microgkg microgkg microgkg 3600 BbF 03 05 96 CHR 02 03 10153602 BbF 015 045 947 CHR 003 009 9193603 BbF 0014 05 1012 CHR 0004 05 10033604 BbF 02 04 85 CHR 02 04 873605 BbF 005 01 94 CHR 0025 005 863606 BbF 001 003 70 CHR 001 003 653607 BbF 018 06 100 CHR 014 028 943608 BbF 015 046 7979 CHR 015 045 71253610 BbF 005 02 85 CHR 02 05 853611 BbF CHR 3612 BbF 1 118 CHR 1 1173613 BbF 01 03 90 CHR 01 03 903614 BbF 01 04 96 CHR 02 05 953615 BbF 008 025 109 CHR 003 01 1083616 BbF 01 05 78 CHR 01 05 723617 BbF 035 07 38 CHR 012 024 4163618 BbF 01 03 62 CHR 01 03 623619 BbF 016 016 63 CHR 01 01 593620 BbF 0003 0006 55 CHR 001 002 653621 BbF 007 02 103 CHR 007 02 893622 BbF 02 06 963 CHR 03 09 1043623 BbF 02 08 106 CHR 02 08 1193624 BbF CHR 3625 BbF CHR 3626 BbF CHR 5301 BbF 003 01 96 CHR 003 01 1145302 BbF 01 03 84 CHR 01 03 835303 BbF 003 01 78 CHR 003 01 705304 BbF 1 1 86 CHR 1 1 705305 BbF 02 05 90 CHR 02 05 955306 BbF 03 1 1435 CHR 03 1 1185307 BbF 8116 CHR 65375308 BbF 0079 0238 99 CHR 009 026 1005309 BbF 01 08 92 CHR 5310 BbF 07 2 CHR 01 03 5311 BbF 002 004 95 CHR 002 004 895312 BbF 03 09 105 CHR 03 09 1055313 BbF 04 08 CHR 04 08 5314 BbF 001 002 83 CHR 001 002 8185315 BbF 02 05 50 -100 CHR 02 05 50 - 100 5316 BbF 005 024 814 CHR 001 007 9595317 BbF 05 1 73 CHR 05 1 785318 BbF CHR 5319 BbF 011 038 946 CHR 013 043 9675320 BbF 07 09 1194 CHR 23 32 10455321 BbF 05 1 CHR 05 1 5322 BbF 1 2 100 CHR 1 2 1005323 BbF 26 CHR 26

62

ANNEX 11 Table 19 Details of analysis method reported by the participants Data are presented as reported

Laboratory code

Analysis technique

Instrument calibration

Internal standards applied

Sample clean up

Details of sample

clean up

Extraction technique

Details of sample

extraction

3600 6) GC-MS

2) Internal standardisation with labelled substances

Deuterated PAH mix 9 (Ehrenstorfer)

1) Saponification 2) LiquidLiquid partitioning

D) Other Liquidliquid partitioning

3602 2) HPLC-UV-FLD

1) External calibration

3) Gel permeation chromatography

A) Pressurised liquid extraction (PLE)

3603 1) HPLC-FLD

1) External calibration

3) Gel permeation chromatography

D) Other 1 minute with dichloromethane in vortex

3604 2) HPLC-UV-FLD

2) Internal standardisation with labelled substances

DiP D14

5) Donor acceptor complex chromatography

D) Other LL

3605 1) HPLC-FLD

1) External calibration

3) Gel permeation chromatography

C) Soxhlet extraction

3606 7) GC-MSMS

2) Internal standardisation with labelled substances

BaP 13C4 CHR 13C6 BaA 13C6 BbF 13C6

4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

3607 6) GC-MS

2) Internal standardisation with labelled substances

BaA-d12 CHR-d12 BbF-d12 BkF-d12 BaP-d12 ICP-d12 BgP-d12 DiP-d14

1) Saponification

D) Other solvent partitioning

3608 1) HPLC-FLD

1) External calibration

6) Other Liquid extraction

D) Other

Extraction in a rotary agitator and centrifugation

3610 2) HPLC-UV-FLD

1) External calibration

4) Solid phase extraction

B) Sonication

3611

3612 6) GC-MS

2) Internal standardisation with labelled substances

chrysene-d12 dibenzo(ah)anthracene-d14 dibenzo(ai)pyrene-d14

4) Solid phase extraction

B) Sonication

3613 5) LC-MSMS

3) Internal standardisation with unlabelled substances

I use Chrysene-D12 3) Gel permeation chromatography

D) Other extraction with hexan

63

Table 19 - continued

Laboratory code

Analysis technique

Instrument calibration

Internal standards applied

Sample clean up Details of sample

clean up

Extraction technique

Details of sample

extraction

3614 6) GC-MS 1) External calibration

3) Gel permeation chromatography

B) Sonication

3615 1) HPLC-FLD

1) External calibration

6) Other PLE GPC

A) Pressurised liquid extraction (PLE)

3616 6) GC-MS

2) Internal standardisation with labelled substances

Benzo(a)pyrene-13C4 Benzo(a)anthracene-13C6 Benzo(b)fluoranthene-13C6 Chrysene-13C6

2) LiquidLiquid partitioning 4) Solid phase extraction

B) Sonication

3617 1) HPLC-FLD

1) External calibration

4) Solid phase extraction

C) Soxhlet extraction

3618 6) GC-MS

2) Internal standardisation with labelled substances

deuterated standards

1) Saponification 4) Solid phase extraction

D) Other Handshaken with cyclohexane

3619 6) GC-MS

2) Internal standardisation with labelled substances

C13 Labelled US EPA 16

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

D) Other Homegenisation amp saponiofication

3620 9) GC-HRMS

2) Internal standardisation with labelled substances

mix of deuterated PAHs (BAA-D12 CHR-D12 BAP-D12 BBF-D12)

3) Gel permeation chromatography 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

3621 6) GC-MS

2) Internal standardisation with labelled substances

Chrysene D12 Benzo(a)pyrene D12 Benzo(b)fluoranthene D12 Benzo(a)anthracene

2) LiquidLiquid partitioning 4) Solid phase extraction

B) Sonication

3622 1) HPLC-FLD

4) Standard addition method

4) Solid phase extraction

D) Other

Extraction with cyclohexane by heating under reflux

3623 7) GC-MSMS

2) Internal standardisation with labelled substances

C13 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

3624 2) HPLC-UV-FLD

1) External calibration

2) LiquidLiquid partitioning

D) Other hot solvent extraction

3626

64

Table 19 - continued

Laboratory code

Analysis technique

Instrument calibration

Internal standards

applied

Sample clean up

Details of sample

clean up

Extraction technique

Details of sample

extraction

5301 7) GC-MSMS

2) Internal standardisation with labelled substances

EACH SUBSTANCE C13

4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5302 7) GC-MSMS

2) Internal standardisation with labelled substances

Isotope labelled PAHs 13C for each analyte

4) Solid phase extraction

C) Soxhlet extraction

5303 7) GC-MSMS

3) Internal standardisation with unlabelled substances

HAP C13 4) Solid phase extraction

D) Other automate ASE200 DIONEX

5304 7) GC-MSMS

2) Internal standardisation with labelled substances

with C13 for each HAP

2) LiquidLiquid partitioning 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5305 7) GC-MSMS

2) Internal standardisation with labelled substances

C13 isotopiques internal standards

4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5306 7) GC-MSMS

2) Internal standardisation with labelled substances

C13 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5307 6) GC-MS

2) Internal standardisation with labelled substances

1) Saponification 2) LiquidLiquid partitioning

D) Other solvent extraction

5308 1) HPLC-FLD 1) External calibration

4) Solid phase extraction

B) Sonication

5309 1) HPLC-FLD 1) External calibration

4) Solid phase extraction

D) Other only SPE

5310 1) HPLC-FLD 1) External calibration

1) Saponification 2) LiquidLiquid partitioning 3) Gel permeation chromatography

C) Soxhlet extraction

5311 6) GC-MS

2) Internal standardisation with labelled substances

13 C Chrysene 13 C Benz(a)pyrene

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

B) Sonication

5312 1) HPLC-FLD

3) Internal standardisation with unlabelled substances

Benzo(b)chrysen

2) LiquidLiquid partitioning 4) Solid phase extraction

Quechers D) Other Quechers

65

Table 19 - continued

Laboratory code

Analysis technique

Instrument calibration

Internal standards applied

Sample clean up Details of sample

clean up

Extraction technique

Details of sample extraction

5313 6) GC-MS

2) Internal standardisation with labelled substances

Benz(a)anthracene D12 Benzo(b)fluranthene D12 Benzo(a)pyrene D12 Chrysene D12

1) Saponification 2) LiquidLiquid partitioning 3) Gel permeation chromatography

A) Pressurised liquid extraction (PLE)

5314 6) GC-MS

2) Internal standardisation with labelled substances

Benzo(a)pyren d12 2) LiquidLiquid partitioning

D) Other Extraction with

5315 1) HPLC-FLD

3) Internal standardisation with unlabelled substances

Benz(b)chrysen

1) Saponification 2) LiquidLiquid partitioning

D) Other shaker

5316 1) HPLC-FLD

1) External calibration

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

D) Other

LiquidLiquid extraction with cyclohexane and dimethylformamide

5317 7) GC-MSMS

1) External calibration

3) Gel permeation chromatography 6) Other

silica gel D) Other VDLUFA online method

5318

5319 1) HPLC-FLD

3) Internal standardisation with unlabelled substances

Benzo(b)chrysene 3) Gel permeation chromatography

A) Pressurised liquid extraction (PLE)

5320 6) GC-MS

3) Internal standardisation with unlabelled substances

22-binaphthyl

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

D) Other 3 hours refluxing

5321 1) HPLC-FLD

2) Internal standardisation with labelled substances

6-meacutethylchrysegravene

1) Saponification 2) LiquidLiquid partitioning

B) Sonication

5322 6) GC-MS

2) Internal standardisation with labelled substances

naftalene-d8 acenaphtalene-d10 phenanthrene-d10 chrysene-d12 perylene-d12 benzo(e)anthracene-d12

1) Saponification 2) LiquidLiquid partitioning 6) Other

clean-up with silica packed column

C) Soxhlet extraction

5323 1) HPLC-FLD

1) External calibration

4) Solid phase extraction 6) Other

Accelerated Solvent Extraction

A) Pressurised liquid extraction (PLE)

66

European Commission EUR 25245 EN ndash Joint Research Centre ndash Institute for Reference Materials and Measurements Title Report on the 8th inter-laboratory comparison organised by the European Union Reference Laboratory for Polycyclic Aromatic Hydrocarbons - Four marker PAHs in a dry extract of St Johns wort Author(s) Radoslav Lizak Szilard Szilagyi Philippe Verlinde and Thomas WENZL Luxembourg Publications Office of the European Union 2012 ndash 66 pp ndash 201 x 297 cm EUR ndash Scientific and Technical Research series ndash ISSN 1831-9424 ISBN 978-92-79-23337-1 doi 10278759387 Abstract The proficiency test here reported concerned the determination of the contents of four marker polycyclic aromatic hydrocarbons (PAHs) and of their sum in an food supplements test sample The set of marker PAHs consists of benz[a]anthacene benzo[a]pyrene benzo[b]fluoranthene and chrysene Participants to these PT were National Reference Laboratories for PAHs (NRLs-PAHs) and EU official food control laboratories The number of participants was in total 48

The PT was organised according ISO Standard 170432010

The test material used was a commercial dry extract of St Johns wort Participants also received a solution of the same PAHs either in acetonitrile or in toluene for checking their instrument calibration

The results from participants were rated with z-scores and zeta-scores About 68 of the reported results were attributed with z-scores with an absolute value of below two which is the threshold for satisfactory performance

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science and technology for the Union Close to the policy-making process it serves

the common interest of the Member States while being independent of special

interests whether private or national

L

A-N

A-252

45-EN

-N

• 1 Summary
• 2 Introduction
• 3 Scope
• 4 Participating Laboratories
• 5 Time frame
• 6 Confidentiality
• 7 Test materials
• • 71 1Preparation of test materials
  • 72 Assigned values and standard deviation for proficiency assessment
  • 73 Homogeneity and stability
  • 74 Sample dispatch
  • • 8 Design of the proficiency test
    • 9 Evaluation of the results
    • • 91 2General
      • 92 21BEvaluation criteria
      • 93 Performance of participants
      • 94 Evaluation of the influence of analysis method
      • 95 Evaluation of the influence of calibration
      • 96 26Evaluation of compliance with legislation
      • • 10 Follow-up actions for underperforming laboratories
        • 11 1Conclusions
        • 12 1Acknowledgements
        • 13 References

50

Figure 8 Results of replicate determinations (indicated by triangles) of benzo[b]fluoranthene (BBF) in the food supplement test material

Horizontal blue lines represent the arithmetic mean value of replicate measurements and blue bars the reported expanded measurement uncertainty (k=2) The assigned value is

plotted as dotted line Red solid lines mark the range of satisfactory performance (|z|le2) Mean values are given for results outside the displayed data range

ProLab 2011

Laboratory

36

0436

1436

0236

1053

1736

1653

0953

0753

0853

0536

2136

0553

0436

0353

0353

1636

0636

2036

2353

1853

1136

1236

2653

0153

0253

1236

1736

1336

0036

1853

2153

1036

1953

1336

2253

2253

2353

1936

1553

1453

0653

1536

0753

2036

0836

11

an

aly

te c

on

ten

t (micro

gk

g)

75

7

65

6

55

5

45

4

35

3

25

2

15

1

05

0

103

6

576

3

Sample St Johns wort extract replicatesMeasurand Benzo[b]fluorantheneNo of laboratories 46

Assigned value 292 microgkg (Reference value)Target sd 060 microgkgTolerance limits 172 - 412 microgkg (|Z score| lt 200)

Assig

ned va

lue

Limit of tolerance

Limit of tolerance

51

Table 14 Analysis results reported by the participants for the content of chrysene (CHR) in the food supplements test material Values are presented as reported

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

3600 5 49 5 5 07 2

3602 32 304 29 304 036

3603 462 469 447 459 048 2

3604 438 428 42 429 119 2

3605 365 41 381 386 085 2

3606 494 411 45 451 124 2

3607 897 774 787 819 18 2

3608 986 887 1168 1014 284 2

3610 418 392 462 424 085 2

3611 833 893 87 865 61 2

3612 6 64 58 61 12 2

3613 41 39 4 4 08 2

3614 272 254 288 271 054 2

3615 605 6028 5964 601 12 2

3616 47 45 49 47 05 2

3617 445 431 389 422 065 2

3618 45 45 45 45 06 2

3619 738 747 72 738 118 2

3620 463 472 446 46 092 2

3621 53 49 51 51 06 2

3622 478 513 505 488 08 2

3623 73 73 72 72 05 2

3624

3626 63 622 634 628 251 2

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

5301 49 49 49 49 1 2

5302 687 683 666 679 136 2

5303 6777 6181 6382 6441 24 70

5304 592 581 6 591 85 2

5305 6 58 64 61 20

5306 49 5 51 49 25 2

5307 524 5 574 524

5308 211488 216782 221146 213243

5309

5310 372 393 394 386 019

5311 43 459 422 437 15 2

5312 934 943 981 953 191 2

5313 793 73 99 838

5314 636 561 565 588 18 0818

5315 547 583 499 542 11 2

5316 292 286 285 282 06 2

5317 52 598 517 545 046 2

5318 368 345 357 05 2

5319 449 471 474 465 116 2

5320 76 924 818 834 99

5321 33

5322 7 7 76 7 35 2

5323 87 73 77 73 37 2

52

Figure 9 Results of replicate determinations (indicated by triangles) of chrysene (CHR) in the food supplement test material

Horizontal blue lines represent the arithmetic mean value of replicate measurements and blue bars the reported expanded measurement uncertainty (k=2) The assigned value is

plotted as dotted line Red solid lines mark the range of satisfactory performance (|z|le2) Mean values are given for results outside the displayed data range

ProLab 2011

Laboratory

53

0836

1453

1636

0253

2153

1836

0553

1036

1336

1736

1036

0453

1136

1836

0636

0336

2053

1936

1653

0136

0036

2253

0636

2153

0753

1553

1753

1453

0436

1536

1253

0536

2653

0353

0253

2236

2336

1953

2336

0753

2053

1353

1236

0836

11

an

aly

te c

on

ten

t (micro

gk

g)

13

12

11

10

9

8

7

6

5

4

3

2

1

0

865

3

Sample St Johns wort extract replicatesMeasurand ChryseneNo of laboratories 45

Assigned value 407 microgkg (Reference value)Target sd 083 microgkgTolerance limits 241 - 573 microgkg (|Z score| lt 200)

Assig

ned va

lue

Limit of tolerance

Limit of tolerance

53

Table 15 Analysis results reported by the participants for the sum of the four marker PAHs in the food supplements test material Values are presented as reported

Laboratory code

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg

3600 128 24 2

3602 79 126

3603 1127 096 2

3604 893 258 2

3605 1091 305 2

3606 1184 238 2

3607 2098 42 2

3608 3336 325 2

3610 1138 17 2

3611 2218 259 2

3612 143 29 2

3613 122 2 2

3614 663 129 2

3615 152 304 2

3616 102 03 2

3617 1252 169 2

3618 128 18 2

3619 157 305 2

3620 1204 12 2

3621 151 19 2

3622 1361 115 2

3623 154 09 2

3624

3626 1409 56 2

Laboratory code

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg

5301 126

5302 37 074 2

5303 13438 92

5304 1251 85 2

5305 125 20

5306 129 65 2

5307 1363

5308 7245598

5309

5310 1127 056

5311 1367 15 2

5312 2042 408 2

5313 1726

5314 1558

5315 177 35 2

5316 96 073 2

5317 1229 066 2

5318 82 05 2

5319 1225 267 2

5320 2309 41

5321

5322 207 35 2

5323 171 103 2

54

ANNEX 9 Results reported for the standard solutions with undisclosed analyte content Table 16 Analysis results reported by the participants for the four marker PAHs in the standard solution in toluene with undisclosed analyte

content Values are presented as reported U measurement uncertainty k coverage factor

benz[a]anthracene benzo[a]pyrene benzo[b]fluoranthene chrysene Laboratory

code Unit Result U k Result U k Result U k Result U k 3600 microgl 38 67 2 25 45 2 211 68 2 275 38 2

3605 microgl 367 184 2 2482 124 2 2592 13 2 235 118 2

3606 microgkg 3763 176 2 2609 142 2 2721 118 2 2539 134 2

3607 microgkg 3706 815 2 2824 565 2 2591 518 2 2587 569 2

3612 microgl 38 08 2 237 11 2 244 18 2 257 02 2

3614 microgl 324 65 2 209 38 2 215 37 2 21 42 2

3616 microgkg 318 07 2 224 14 2 231 26 2 216 1 2

3618 microgkg 417 17 2 284 08 2 292 06 2 297 03 2

3619 microgkg 47 742 2 274 468 2 228 387 2 274 434 2

3620 microgl 495 74 2 294 29 2 321 8 2 321 64 2

3621 microgl 241 22 2 143 13 2 188 16 2 145 14 2

3623 microgkg 476 25 2 311 53 2 269 3 2 297 03 2

3626 microgl 359 38 2 235 23 2 238 27 2 24 18 2

5301 microgkg 426 29 299 289

5302 microgkg 436 87 2 296 59 2 296 59 2 289 58 2

5303 microgl 3721 27 102 22838 19 99 23756 22 99 25486 24 98

5304 microgkg 38 29 2 225 26 2 257 37 2 258 52 2

5305 microgkg 305 225 245 29

5306 microgkg 316 63 2 247 49 2 65 13 2 22 44 2

5307 microgkg 3658 2559 241 2414

5308 microgkg 16854 109324 246654 216782

5311 microgl 374 20 2 279 15 2 268 15 2 272 15 2

5313 microgl 3408 2165 2275 2373

5314 microgkg 395 5 2875 11 3614 34 2905 18

5320 microgl 3267 368 1986 571 28 683 2427 512

5322 microgl 282 35 2 225 35 2 241 35 2 21 35 2

55

Figure 10 Youden plots of the percent deviation of the reported values from the assigned values for the food supplement test sample and the

standard solution in toluene with undisclosed content

(The identity of the respective measurand is given above the figure Results exceeding a difference of more than 100 were omitted)

56

Figure 10 - continued

57

Table 17 Analysis results reported by the participants for the four marker PAHs in the standard solution in acetonitrile with undisclosed

analyte content

Values are presented as reported U measurement uncertainty k coverage factor

benz[a]anthracene benzo[a]pyrene benzo[b]fluoranthene chrysene

Laboratory

code Unit Result U k Result U k Result U k Result U k

3602 microgkg 3675 247 247 244

3603 microgkg 358 086 2 236 028 2 235 028 2 236 057 2

3604 microgl 475 29 2 19 12 2 194 13 2 258 12 2

3608 microgl 3623 078 2 2385 023 2 2343 18 2 2296 092 2

3610 microgkg 369 185 2 25 125 2 251 126 2 244 122 2

3611 microgkg 48 02 2 323 02 2 328 04 2 317 03 2

3613 microgl 332 31 2 261 3 2 249 25 2 239 24 2

3615 microgl 415 2635 2729 2689

3617 microgkg 37 09 2 244 06 2 284 07 2 239 06 2

3622 microgl 37 12 2 25 23 2 257 21 2 238 1 2

3624 microgkg 402 104 2 255 084 2 265 096 2 253 07 2

5306 microgkg 563 113 2 371 74 2 359 72 2 333 67 2

5308 microgkg 168633 102669 232169 211488

5309 microgl 402 268 27

5310 microgl 315 158 25 125 25 125 288 144

5312 microgl 428 043 2 282 028 2 272 027 2 275 028 2

5315 microgkg 387 08 2 256 05 2 251 05 2 253 05 2

5316 microgl 3839 342 2 2485 191 2 2559 168 2 239 51 2

5317 microgl 2466 043 2 2212 034 2 2472 016 2 2146 068 2

5319 microgl 414 41 2 279 28 2 268 27 2 26 31 2

5321 microgkg 476 316 326 328

5322 microgl 2547 35 2

5323 microgkg 405 203 2 239 119 2 255 153 2 201 11 2

58

Figure 11 Youden plots of the percent deviation of the reported values from the assigned values for the food supplement test sample and the

standard solution in acetonitrile with undisclosed content

(The identity of the respective measurand is given above the figure Results exceeding a difference of more than 100 were omitted)

59

Figure 11 - continued

60

ANNEX 10 Table 18 Method performance parameters reported by the participants

Laboratory code

Analyte LOD LOQ Recovery Analyte LOD LOQ Recovery

microgkg microgkg microgkg microgkg 3600 BaA 03 06 978 BaP 03 07 9483602 BaA 007 021 926 BaP 005 015 993603 BaA 0011 05 987 BaP 0005 051 9553604 BaA 02 04 86 BaP 02 04 883605 BaA 0025 005 118 BaP 0025 005 1083606 BaA 001 003 65 BaP 001 003 703607 BaA 021 071 74 BaP 027 089 943608 BaA 016 048 8213 BaP 015 046 83683610 BaA 02 05 85 BaP 005 02 853611 BaA BaP 3612 BaA 1 117 BaP 1 1203613 BaA 01 03 90 BaP 01 03 903614 BaA 02 05 96 BaP 02 05 933615 BaA 005 015 110 BaP 005 015 1053616 BaA 01 05 108 BaP 01 05 753617 BaA 018 037 405 BaP 009 018 3873618 BaA 01 03 63 BaP 01 03 593619 BaA 005 005 59 BaP 033 033 543620 BaA 0009 0018 64 BaP 0005 001 553621 BaA 007 02 84 BaP 007 02 923622 BaA 01 03 1101 BaP 01 03 9853623 BaA 02 08 105 BaP 02 08 1013624 BaA BaP 01 033 8513625 BaA BaP 3626 BaA BaP 5301 BaA 003 01 102 BaP 003 01 1005302 BaA 01 03 78 BaP 01 03 865303 BaA 003 01 62 BaP 003 01 805304 BaA 1 1 70 BaP 1 1 955305 BaA 02 05 85 BaP 02 05 855306 BaA 03 1 699 BaP 03 1 905307 BaA 6537 BaP 81165308 BaA 0088 0265 99 BaP 00262 00785 1005309 BaA 01 08 92 BaP 01 08 925310 BaA 01 03 BaP 01 03 905311 BaA 002 005 80 BaP 002 004 915312 BaA 03 09 105 BaP 03 09 1055313 BaA 04 08 BaP 04 08 5314 BaA 001 002 96 BaP 001 002 9485315 BaA 01 03 50 - 100 BaP 01 03 50 - 100 5316 BaA 002 011 827 BaP 004 019 10445317 BaA 05 1 76 BaP 05 1 755318 BaA BaP 5319 BaA 009 029 951 BaP 011 041 995320 BaA 12 18 946 BaP 06 1 8345321 BaA 03 05 BaP 05 1 5322 BaA 1 2 100 BaP 1 2 1005323 BaA 26 BaP 26

61

ANNEX 10 Table 18 - continued

Laboratory code Analyte LOD LOQ Recovery Analyte LOD LOQ Recovery

microgkg microgkg microgkg microgkg 3600 BbF 03 05 96 CHR 02 03 10153602 BbF 015 045 947 CHR 003 009 9193603 BbF 0014 05 1012 CHR 0004 05 10033604 BbF 02 04 85 CHR 02 04 873605 BbF 005 01 94 CHR 0025 005 863606 BbF 001 003 70 CHR 001 003 653607 BbF 018 06 100 CHR 014 028 943608 BbF 015 046 7979 CHR 015 045 71253610 BbF 005 02 85 CHR 02 05 853611 BbF CHR 3612 BbF 1 118 CHR 1 1173613 BbF 01 03 90 CHR 01 03 903614 BbF 01 04 96 CHR 02 05 953615 BbF 008 025 109 CHR 003 01 1083616 BbF 01 05 78 CHR 01 05 723617 BbF 035 07 38 CHR 012 024 4163618 BbF 01 03 62 CHR 01 03 623619 BbF 016 016 63 CHR 01 01 593620 BbF 0003 0006 55 CHR 001 002 653621 BbF 007 02 103 CHR 007 02 893622 BbF 02 06 963 CHR 03 09 1043623 BbF 02 08 106 CHR 02 08 1193624 BbF CHR 3625 BbF CHR 3626 BbF CHR 5301 BbF 003 01 96 CHR 003 01 1145302 BbF 01 03 84 CHR 01 03 835303 BbF 003 01 78 CHR 003 01 705304 BbF 1 1 86 CHR 1 1 705305 BbF 02 05 90 CHR 02 05 955306 BbF 03 1 1435 CHR 03 1 1185307 BbF 8116 CHR 65375308 BbF 0079 0238 99 CHR 009 026 1005309 BbF 01 08 92 CHR 5310 BbF 07 2 CHR 01 03 5311 BbF 002 004 95 CHR 002 004 895312 BbF 03 09 105 CHR 03 09 1055313 BbF 04 08 CHR 04 08 5314 BbF 001 002 83 CHR 001 002 8185315 BbF 02 05 50 -100 CHR 02 05 50 - 100 5316 BbF 005 024 814 CHR 001 007 9595317 BbF 05 1 73 CHR 05 1 785318 BbF CHR 5319 BbF 011 038 946 CHR 013 043 9675320 BbF 07 09 1194 CHR 23 32 10455321 BbF 05 1 CHR 05 1 5322 BbF 1 2 100 CHR 1 2 1005323 BbF 26 CHR 26

62

ANNEX 11 Table 19 Details of analysis method reported by the participants Data are presented as reported

Laboratory code

Analysis technique

Instrument calibration

Internal standards applied

Sample clean up

Details of sample

clean up

Extraction technique

Details of sample

extraction

3600 6) GC-MS

2) Internal standardisation with labelled substances

Deuterated PAH mix 9 (Ehrenstorfer)

1) Saponification 2) LiquidLiquid partitioning

D) Other Liquidliquid partitioning

3602 2) HPLC-UV-FLD

1) External calibration

3) Gel permeation chromatography

A) Pressurised liquid extraction (PLE)

3603 1) HPLC-FLD

1) External calibration

3) Gel permeation chromatography

D) Other 1 minute with dichloromethane in vortex

3604 2) HPLC-UV-FLD

2) Internal standardisation with labelled substances

DiP D14

5) Donor acceptor complex chromatography

D) Other LL

3605 1) HPLC-FLD

1) External calibration

3) Gel permeation chromatography

C) Soxhlet extraction

3606 7) GC-MSMS

2) Internal standardisation with labelled substances

BaP 13C4 CHR 13C6 BaA 13C6 BbF 13C6

4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

3607 6) GC-MS

2) Internal standardisation with labelled substances

BaA-d12 CHR-d12 BbF-d12 BkF-d12 BaP-d12 ICP-d12 BgP-d12 DiP-d14

1) Saponification

D) Other solvent partitioning

3608 1) HPLC-FLD

1) External calibration

6) Other Liquid extraction

D) Other

Extraction in a rotary agitator and centrifugation

3610 2) HPLC-UV-FLD

1) External calibration

4) Solid phase extraction

B) Sonication

3611

3612 6) GC-MS

2) Internal standardisation with labelled substances

chrysene-d12 dibenzo(ah)anthracene-d14 dibenzo(ai)pyrene-d14

4) Solid phase extraction

B) Sonication

3613 5) LC-MSMS

3) Internal standardisation with unlabelled substances

I use Chrysene-D12 3) Gel permeation chromatography

D) Other extraction with hexan

63

Table 19 - continued

Laboratory code

Analysis technique

Instrument calibration

Internal standards applied

Sample clean up Details of sample

clean up

Extraction technique

Details of sample

extraction

3614 6) GC-MS 1) External calibration

3) Gel permeation chromatography

B) Sonication

3615 1) HPLC-FLD

1) External calibration

6) Other PLE GPC

A) Pressurised liquid extraction (PLE)

3616 6) GC-MS

2) Internal standardisation with labelled substances

Benzo(a)pyrene-13C4 Benzo(a)anthracene-13C6 Benzo(b)fluoranthene-13C6 Chrysene-13C6

2) LiquidLiquid partitioning 4) Solid phase extraction

B) Sonication

3617 1) HPLC-FLD

1) External calibration

4) Solid phase extraction

C) Soxhlet extraction

3618 6) GC-MS

2) Internal standardisation with labelled substances

deuterated standards

1) Saponification 4) Solid phase extraction

D) Other Handshaken with cyclohexane

3619 6) GC-MS

2) Internal standardisation with labelled substances

C13 Labelled US EPA 16

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

D) Other Homegenisation amp saponiofication

3620 9) GC-HRMS

2) Internal standardisation with labelled substances

mix of deuterated PAHs (BAA-D12 CHR-D12 BAP-D12 BBF-D12)

3) Gel permeation chromatography 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

3621 6) GC-MS

2) Internal standardisation with labelled substances

Chrysene D12 Benzo(a)pyrene D12 Benzo(b)fluoranthene D12 Benzo(a)anthracene

2) LiquidLiquid partitioning 4) Solid phase extraction

B) Sonication

3622 1) HPLC-FLD

4) Standard addition method

4) Solid phase extraction

D) Other

Extraction with cyclohexane by heating under reflux

3623 7) GC-MSMS

2) Internal standardisation with labelled substances

C13 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

3624 2) HPLC-UV-FLD

1) External calibration

2) LiquidLiquid partitioning

D) Other hot solvent extraction

3626

64

Table 19 - continued

Laboratory code

Analysis technique

Instrument calibration

Internal standards

applied

Sample clean up

Details of sample

clean up

Extraction technique

Details of sample

extraction

5301 7) GC-MSMS

2) Internal standardisation with labelled substances

EACH SUBSTANCE C13

4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5302 7) GC-MSMS

2) Internal standardisation with labelled substances

Isotope labelled PAHs 13C for each analyte

4) Solid phase extraction

C) Soxhlet extraction

5303 7) GC-MSMS

3) Internal standardisation with unlabelled substances

HAP C13 4) Solid phase extraction

D) Other automate ASE200 DIONEX

5304 7) GC-MSMS

2) Internal standardisation with labelled substances

with C13 for each HAP

2) LiquidLiquid partitioning 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5305 7) GC-MSMS

2) Internal standardisation with labelled substances

C13 isotopiques internal standards

4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5306 7) GC-MSMS

2) Internal standardisation with labelled substances

C13 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5307 6) GC-MS

2) Internal standardisation with labelled substances

1) Saponification 2) LiquidLiquid partitioning

D) Other solvent extraction

5308 1) HPLC-FLD 1) External calibration

4) Solid phase extraction

B) Sonication

5309 1) HPLC-FLD 1) External calibration

4) Solid phase extraction

D) Other only SPE

5310 1) HPLC-FLD 1) External calibration

1) Saponification 2) LiquidLiquid partitioning 3) Gel permeation chromatography

C) Soxhlet extraction

5311 6) GC-MS

2) Internal standardisation with labelled substances

13 C Chrysene 13 C Benz(a)pyrene

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

B) Sonication

5312 1) HPLC-FLD

3) Internal standardisation with unlabelled substances

Benzo(b)chrysen

2) LiquidLiquid partitioning 4) Solid phase extraction

Quechers D) Other Quechers

65

Table 19 - continued

Laboratory code

Analysis technique

Instrument calibration

Internal standards applied

Sample clean up Details of sample

clean up

Extraction technique

Details of sample extraction

5313 6) GC-MS

2) Internal standardisation with labelled substances

Benz(a)anthracene D12 Benzo(b)fluranthene D12 Benzo(a)pyrene D12 Chrysene D12

1) Saponification 2) LiquidLiquid partitioning 3) Gel permeation chromatography

A) Pressurised liquid extraction (PLE)

5314 6) GC-MS

2) Internal standardisation with labelled substances

Benzo(a)pyren d12 2) LiquidLiquid partitioning

D) Other Extraction with

5315 1) HPLC-FLD

3) Internal standardisation with unlabelled substances

Benz(b)chrysen

1) Saponification 2) LiquidLiquid partitioning

D) Other shaker

5316 1) HPLC-FLD

1) External calibration

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

D) Other

LiquidLiquid extraction with cyclohexane and dimethylformamide

5317 7) GC-MSMS

1) External calibration

3) Gel permeation chromatography 6) Other

silica gel D) Other VDLUFA online method

5318

5319 1) HPLC-FLD

3) Internal standardisation with unlabelled substances

Benzo(b)chrysene 3) Gel permeation chromatography

A) Pressurised liquid extraction (PLE)

5320 6) GC-MS

3) Internal standardisation with unlabelled substances

22-binaphthyl

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

D) Other 3 hours refluxing

5321 1) HPLC-FLD

2) Internal standardisation with labelled substances

6-meacutethylchrysegravene

1) Saponification 2) LiquidLiquid partitioning

B) Sonication

5322 6) GC-MS

2) Internal standardisation with labelled substances

naftalene-d8 acenaphtalene-d10 phenanthrene-d10 chrysene-d12 perylene-d12 benzo(e)anthracene-d12

1) Saponification 2) LiquidLiquid partitioning 6) Other

clean-up with silica packed column

C) Soxhlet extraction

5323 1) HPLC-FLD

1) External calibration

4) Solid phase extraction 6) Other

Accelerated Solvent Extraction

A) Pressurised liquid extraction (PLE)

66

European Commission EUR 25245 EN ndash Joint Research Centre ndash Institute for Reference Materials and Measurements Title Report on the 8th inter-laboratory comparison organised by the European Union Reference Laboratory for Polycyclic Aromatic Hydrocarbons - Four marker PAHs in a dry extract of St Johns wort Author(s) Radoslav Lizak Szilard Szilagyi Philippe Verlinde and Thomas WENZL Luxembourg Publications Office of the European Union 2012 ndash 66 pp ndash 201 x 297 cm EUR ndash Scientific and Technical Research series ndash ISSN 1831-9424 ISBN 978-92-79-23337-1 doi 10278759387 Abstract The proficiency test here reported concerned the determination of the contents of four marker polycyclic aromatic hydrocarbons (PAHs) and of their sum in an food supplements test sample The set of marker PAHs consists of benz[a]anthacene benzo[a]pyrene benzo[b]fluoranthene and chrysene Participants to these PT were National Reference Laboratories for PAHs (NRLs-PAHs) and EU official food control laboratories The number of participants was in total 48

The PT was organised according ISO Standard 170432010

The test material used was a commercial dry extract of St Johns wort Participants also received a solution of the same PAHs either in acetonitrile or in toluene for checking their instrument calibration

The results from participants were rated with z-scores and zeta-scores About 68 of the reported results were attributed with z-scores with an absolute value of below two which is the threshold for satisfactory performance

How to obtain EU publications

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The mission of the JRC is to provide customer-driven scientific and technical support

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service of the European Commission the JRC functions as a reference centre of

science and technology for the Union Close to the policy-making process it serves

the common interest of the Member States while being independent of special

interests whether private or national

L

A-N

A-252

45-EN

-N

• 1 Summary
• 2 Introduction
• 3 Scope
• 4 Participating Laboratories
• 5 Time frame
• 6 Confidentiality
• 7 Test materials
• • 71 1Preparation of test materials
  • 72 Assigned values and standard deviation for proficiency assessment
  • 73 Homogeneity and stability
  • 74 Sample dispatch
  • • 8 Design of the proficiency test
    • 9 Evaluation of the results
    • • 91 2General
      • 92 21BEvaluation criteria
      • 93 Performance of participants
      • 94 Evaluation of the influence of analysis method
      • 95 Evaluation of the influence of calibration
      • 96 26Evaluation of compliance with legislation
      • • 10 Follow-up actions for underperforming laboratories
        • 11 1Conclusions
        • 12 1Acknowledgements
        • 13 References

51

Table 14 Analysis results reported by the participants for the content of chrysene (CHR) in the food supplements test material Values are presented as reported

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

3600 5 49 5 5 07 2

3602 32 304 29 304 036

3603 462 469 447 459 048 2

3604 438 428 42 429 119 2

3605 365 41 381 386 085 2

3606 494 411 45 451 124 2

3607 897 774 787 819 18 2

3608 986 887 1168 1014 284 2

3610 418 392 462 424 085 2

3611 833 893 87 865 61 2

3612 6 64 58 61 12 2

3613 41 39 4 4 08 2

3614 272 254 288 271 054 2

3615 605 6028 5964 601 12 2

3616 47 45 49 47 05 2

3617 445 431 389 422 065 2

3618 45 45 45 45 06 2

3619 738 747 72 738 118 2

3620 463 472 446 46 092 2

3621 53 49 51 51 06 2

3622 478 513 505 488 08 2

3623 73 73 72 72 05 2

3624

3626 63 622 634 628 251 2

Laboratory code

Replicate 1

Replicate 2

Replicate 3

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg microgkg microgkg microgkg

5301 49 49 49 49 1 2

5302 687 683 666 679 136 2

5303 6777 6181 6382 6441 24 70

5304 592 581 6 591 85 2

5305 6 58 64 61 20

5306 49 5 51 49 25 2

5307 524 5 574 524

5308 211488 216782 221146 213243

5309

5310 372 393 394 386 019

5311 43 459 422 437 15 2

5312 934 943 981 953 191 2

5313 793 73 99 838

5314 636 561 565 588 18 0818

5315 547 583 499 542 11 2

5316 292 286 285 282 06 2

5317 52 598 517 545 046 2

5318 368 345 357 05 2

5319 449 471 474 465 116 2

5320 76 924 818 834 99

5321 33

5322 7 7 76 7 35 2

5323 87 73 77 73 37 2

52

Figure 9 Results of replicate determinations (indicated by triangles) of chrysene (CHR) in the food supplement test material

Horizontal blue lines represent the arithmetic mean value of replicate measurements and blue bars the reported expanded measurement uncertainty (k=2) The assigned value is

plotted as dotted line Red solid lines mark the range of satisfactory performance (|z|le2) Mean values are given for results outside the displayed data range

ProLab 2011

Laboratory

53

0836

1453

1636

0253

2153

1836

0553

1036

1336

1736

1036

0453

1136

1836

0636

0336

2053

1936

1653

0136

0036

2253

0636

2153

0753

1553

1753

1453

0436

1536

1253

0536

2653

0353

0253

2236

2336

1953

2336

0753

2053

1353

1236

0836

11

an

aly

te c

on

ten

t (micro

gk

g)

13

12

11

10

9

8

7

6

5

4

3

2

1

0

865

3

Sample St Johns wort extract replicatesMeasurand ChryseneNo of laboratories 45

Assigned value 407 microgkg (Reference value)Target sd 083 microgkgTolerance limits 241 - 573 microgkg (|Z score| lt 200)

Assig

ned va

lue

Limit of tolerance

Limit of tolerance

53

Table 15 Analysis results reported by the participants for the sum of the four marker PAHs in the food supplements test material Values are presented as reported

Laboratory code

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg

3600 128 24 2

3602 79 126

3603 1127 096 2

3604 893 258 2

3605 1091 305 2

3606 1184 238 2

3607 2098 42 2

3608 3336 325 2

3610 1138 17 2

3611 2218 259 2

3612 143 29 2

3613 122 2 2

3614 663 129 2

3615 152 304 2

3616 102 03 2

3617 1252 169 2

3618 128 18 2

3619 157 305 2

3620 1204 12 2

3621 151 19 2

3622 1361 115 2

3623 154 09 2

3624

3626 1409 56 2

Laboratory code

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg

5301 126

5302 37 074 2

5303 13438 92

5304 1251 85 2

5305 125 20

5306 129 65 2

5307 1363

5308 7245598

5309

5310 1127 056

5311 1367 15 2

5312 2042 408 2

5313 1726

5314 1558

5315 177 35 2

5316 96 073 2

5317 1229 066 2

5318 82 05 2

5319 1225 267 2

5320 2309 41

5321

5322 207 35 2

5323 171 103 2

54

ANNEX 9 Results reported for the standard solutions with undisclosed analyte content Table 16 Analysis results reported by the participants for the four marker PAHs in the standard solution in toluene with undisclosed analyte

content Values are presented as reported U measurement uncertainty k coverage factor

benz[a]anthracene benzo[a]pyrene benzo[b]fluoranthene chrysene Laboratory

code Unit Result U k Result U k Result U k Result U k 3600 microgl 38 67 2 25 45 2 211 68 2 275 38 2

3605 microgl 367 184 2 2482 124 2 2592 13 2 235 118 2

3606 microgkg 3763 176 2 2609 142 2 2721 118 2 2539 134 2

3607 microgkg 3706 815 2 2824 565 2 2591 518 2 2587 569 2

3612 microgl 38 08 2 237 11 2 244 18 2 257 02 2

3614 microgl 324 65 2 209 38 2 215 37 2 21 42 2

3616 microgkg 318 07 2 224 14 2 231 26 2 216 1 2

3618 microgkg 417 17 2 284 08 2 292 06 2 297 03 2

3619 microgkg 47 742 2 274 468 2 228 387 2 274 434 2

3620 microgl 495 74 2 294 29 2 321 8 2 321 64 2

3621 microgl 241 22 2 143 13 2 188 16 2 145 14 2

3623 microgkg 476 25 2 311 53 2 269 3 2 297 03 2

3626 microgl 359 38 2 235 23 2 238 27 2 24 18 2

5301 microgkg 426 29 299 289

5302 microgkg 436 87 2 296 59 2 296 59 2 289 58 2

5303 microgl 3721 27 102 22838 19 99 23756 22 99 25486 24 98

5304 microgkg 38 29 2 225 26 2 257 37 2 258 52 2

5305 microgkg 305 225 245 29

5306 microgkg 316 63 2 247 49 2 65 13 2 22 44 2

5307 microgkg 3658 2559 241 2414

5308 microgkg 16854 109324 246654 216782

5311 microgl 374 20 2 279 15 2 268 15 2 272 15 2

5313 microgl 3408 2165 2275 2373

5314 microgkg 395 5 2875 11 3614 34 2905 18

5320 microgl 3267 368 1986 571 28 683 2427 512

5322 microgl 282 35 2 225 35 2 241 35 2 21 35 2

55

Figure 10 Youden plots of the percent deviation of the reported values from the assigned values for the food supplement test sample and the

standard solution in toluene with undisclosed content

(The identity of the respective measurand is given above the figure Results exceeding a difference of more than 100 were omitted)

56

Figure 10 - continued

57

Table 17 Analysis results reported by the participants for the four marker PAHs in the standard solution in acetonitrile with undisclosed

analyte content

Values are presented as reported U measurement uncertainty k coverage factor

benz[a]anthracene benzo[a]pyrene benzo[b]fluoranthene chrysene

Laboratory

code Unit Result U k Result U k Result U k Result U k

3602 microgkg 3675 247 247 244

3603 microgkg 358 086 2 236 028 2 235 028 2 236 057 2

3604 microgl 475 29 2 19 12 2 194 13 2 258 12 2

3608 microgl 3623 078 2 2385 023 2 2343 18 2 2296 092 2

3610 microgkg 369 185 2 25 125 2 251 126 2 244 122 2

3611 microgkg 48 02 2 323 02 2 328 04 2 317 03 2

3613 microgl 332 31 2 261 3 2 249 25 2 239 24 2

3615 microgl 415 2635 2729 2689

3617 microgkg 37 09 2 244 06 2 284 07 2 239 06 2

3622 microgl 37 12 2 25 23 2 257 21 2 238 1 2

3624 microgkg 402 104 2 255 084 2 265 096 2 253 07 2

5306 microgkg 563 113 2 371 74 2 359 72 2 333 67 2

5308 microgkg 168633 102669 232169 211488

5309 microgl 402 268 27

5310 microgl 315 158 25 125 25 125 288 144

5312 microgl 428 043 2 282 028 2 272 027 2 275 028 2

5315 microgkg 387 08 2 256 05 2 251 05 2 253 05 2

5316 microgl 3839 342 2 2485 191 2 2559 168 2 239 51 2

5317 microgl 2466 043 2 2212 034 2 2472 016 2 2146 068 2

5319 microgl 414 41 2 279 28 2 268 27 2 26 31 2

5321 microgkg 476 316 326 328

5322 microgl 2547 35 2

5323 microgkg 405 203 2 239 119 2 255 153 2 201 11 2

58

Figure 11 Youden plots of the percent deviation of the reported values from the assigned values for the food supplement test sample and the

standard solution in acetonitrile with undisclosed content

(The identity of the respective measurand is given above the figure Results exceeding a difference of more than 100 were omitted)

59

Figure 11 - continued

60

ANNEX 10 Table 18 Method performance parameters reported by the participants

Laboratory code

Analyte LOD LOQ Recovery Analyte LOD LOQ Recovery

microgkg microgkg microgkg microgkg 3600 BaA 03 06 978 BaP 03 07 9483602 BaA 007 021 926 BaP 005 015 993603 BaA 0011 05 987 BaP 0005 051 9553604 BaA 02 04 86 BaP 02 04 883605 BaA 0025 005 118 BaP 0025 005 1083606 BaA 001 003 65 BaP 001 003 703607 BaA 021 071 74 BaP 027 089 943608 BaA 016 048 8213 BaP 015 046 83683610 BaA 02 05 85 BaP 005 02 853611 BaA BaP 3612 BaA 1 117 BaP 1 1203613 BaA 01 03 90 BaP 01 03 903614 BaA 02 05 96 BaP 02 05 933615 BaA 005 015 110 BaP 005 015 1053616 BaA 01 05 108 BaP 01 05 753617 BaA 018 037 405 BaP 009 018 3873618 BaA 01 03 63 BaP 01 03 593619 BaA 005 005 59 BaP 033 033 543620 BaA 0009 0018 64 BaP 0005 001 553621 BaA 007 02 84 BaP 007 02 923622 BaA 01 03 1101 BaP 01 03 9853623 BaA 02 08 105 BaP 02 08 1013624 BaA BaP 01 033 8513625 BaA BaP 3626 BaA BaP 5301 BaA 003 01 102 BaP 003 01 1005302 BaA 01 03 78 BaP 01 03 865303 BaA 003 01 62 BaP 003 01 805304 BaA 1 1 70 BaP 1 1 955305 BaA 02 05 85 BaP 02 05 855306 BaA 03 1 699 BaP 03 1 905307 BaA 6537 BaP 81165308 BaA 0088 0265 99 BaP 00262 00785 1005309 BaA 01 08 92 BaP 01 08 925310 BaA 01 03 BaP 01 03 905311 BaA 002 005 80 BaP 002 004 915312 BaA 03 09 105 BaP 03 09 1055313 BaA 04 08 BaP 04 08 5314 BaA 001 002 96 BaP 001 002 9485315 BaA 01 03 50 - 100 BaP 01 03 50 - 100 5316 BaA 002 011 827 BaP 004 019 10445317 BaA 05 1 76 BaP 05 1 755318 BaA BaP 5319 BaA 009 029 951 BaP 011 041 995320 BaA 12 18 946 BaP 06 1 8345321 BaA 03 05 BaP 05 1 5322 BaA 1 2 100 BaP 1 2 1005323 BaA 26 BaP 26

61

ANNEX 10 Table 18 - continued

Laboratory code Analyte LOD LOQ Recovery Analyte LOD LOQ Recovery

microgkg microgkg microgkg microgkg 3600 BbF 03 05 96 CHR 02 03 10153602 BbF 015 045 947 CHR 003 009 9193603 BbF 0014 05 1012 CHR 0004 05 10033604 BbF 02 04 85 CHR 02 04 873605 BbF 005 01 94 CHR 0025 005 863606 BbF 001 003 70 CHR 001 003 653607 BbF 018 06 100 CHR 014 028 943608 BbF 015 046 7979 CHR 015 045 71253610 BbF 005 02 85 CHR 02 05 853611 BbF CHR 3612 BbF 1 118 CHR 1 1173613 BbF 01 03 90 CHR 01 03 903614 BbF 01 04 96 CHR 02 05 953615 BbF 008 025 109 CHR 003 01 1083616 BbF 01 05 78 CHR 01 05 723617 BbF 035 07 38 CHR 012 024 4163618 BbF 01 03 62 CHR 01 03 623619 BbF 016 016 63 CHR 01 01 593620 BbF 0003 0006 55 CHR 001 002 653621 BbF 007 02 103 CHR 007 02 893622 BbF 02 06 963 CHR 03 09 1043623 BbF 02 08 106 CHR 02 08 1193624 BbF CHR 3625 BbF CHR 3626 BbF CHR 5301 BbF 003 01 96 CHR 003 01 1145302 BbF 01 03 84 CHR 01 03 835303 BbF 003 01 78 CHR 003 01 705304 BbF 1 1 86 CHR 1 1 705305 BbF 02 05 90 CHR 02 05 955306 BbF 03 1 1435 CHR 03 1 1185307 BbF 8116 CHR 65375308 BbF 0079 0238 99 CHR 009 026 1005309 BbF 01 08 92 CHR 5310 BbF 07 2 CHR 01 03 5311 BbF 002 004 95 CHR 002 004 895312 BbF 03 09 105 CHR 03 09 1055313 BbF 04 08 CHR 04 08 5314 BbF 001 002 83 CHR 001 002 8185315 BbF 02 05 50 -100 CHR 02 05 50 - 100 5316 BbF 005 024 814 CHR 001 007 9595317 BbF 05 1 73 CHR 05 1 785318 BbF CHR 5319 BbF 011 038 946 CHR 013 043 9675320 BbF 07 09 1194 CHR 23 32 10455321 BbF 05 1 CHR 05 1 5322 BbF 1 2 100 CHR 1 2 1005323 BbF 26 CHR 26

62

ANNEX 11 Table 19 Details of analysis method reported by the participants Data are presented as reported

Laboratory code

Analysis technique

Instrument calibration

Internal standards applied

Sample clean up

Details of sample

clean up

Extraction technique

Details of sample

extraction

3600 6) GC-MS

2) Internal standardisation with labelled substances

Deuterated PAH mix 9 (Ehrenstorfer)

1) Saponification 2) LiquidLiquid partitioning

D) Other Liquidliquid partitioning

3602 2) HPLC-UV-FLD

1) External calibration

3) Gel permeation chromatography

A) Pressurised liquid extraction (PLE)

3603 1) HPLC-FLD

1) External calibration

3) Gel permeation chromatography

D) Other 1 minute with dichloromethane in vortex

3604 2) HPLC-UV-FLD

2) Internal standardisation with labelled substances

DiP D14

5) Donor acceptor complex chromatography

D) Other LL

3605 1) HPLC-FLD

1) External calibration

3) Gel permeation chromatography

C) Soxhlet extraction

3606 7) GC-MSMS

2) Internal standardisation with labelled substances

BaP 13C4 CHR 13C6 BaA 13C6 BbF 13C6

4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

3607 6) GC-MS

2) Internal standardisation with labelled substances

BaA-d12 CHR-d12 BbF-d12 BkF-d12 BaP-d12 ICP-d12 BgP-d12 DiP-d14

1) Saponification

D) Other solvent partitioning

3608 1) HPLC-FLD

1) External calibration

6) Other Liquid extraction

D) Other

Extraction in a rotary agitator and centrifugation

3610 2) HPLC-UV-FLD

1) External calibration

4) Solid phase extraction

B) Sonication

3611

3612 6) GC-MS

2) Internal standardisation with labelled substances

chrysene-d12 dibenzo(ah)anthracene-d14 dibenzo(ai)pyrene-d14

4) Solid phase extraction

B) Sonication

3613 5) LC-MSMS

3) Internal standardisation with unlabelled substances

I use Chrysene-D12 3) Gel permeation chromatography

D) Other extraction with hexan

63

Table 19 - continued

Laboratory code

Analysis technique

Instrument calibration

Internal standards applied

Sample clean up Details of sample

clean up

Extraction technique

Details of sample

extraction

3614 6) GC-MS 1) External calibration

3) Gel permeation chromatography

B) Sonication

3615 1) HPLC-FLD

1) External calibration

6) Other PLE GPC

A) Pressurised liquid extraction (PLE)

3616 6) GC-MS

2) Internal standardisation with labelled substances

Benzo(a)pyrene-13C4 Benzo(a)anthracene-13C6 Benzo(b)fluoranthene-13C6 Chrysene-13C6

2) LiquidLiquid partitioning 4) Solid phase extraction

B) Sonication

3617 1) HPLC-FLD

1) External calibration

4) Solid phase extraction

C) Soxhlet extraction

3618 6) GC-MS

2) Internal standardisation with labelled substances

deuterated standards

1) Saponification 4) Solid phase extraction

D) Other Handshaken with cyclohexane

3619 6) GC-MS

2) Internal standardisation with labelled substances

C13 Labelled US EPA 16

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

D) Other Homegenisation amp saponiofication

3620 9) GC-HRMS

2) Internal standardisation with labelled substances

mix of deuterated PAHs (BAA-D12 CHR-D12 BAP-D12 BBF-D12)

3) Gel permeation chromatography 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

3621 6) GC-MS

2) Internal standardisation with labelled substances

Chrysene D12 Benzo(a)pyrene D12 Benzo(b)fluoranthene D12 Benzo(a)anthracene

2) LiquidLiquid partitioning 4) Solid phase extraction

B) Sonication

3622 1) HPLC-FLD

4) Standard addition method

4) Solid phase extraction

D) Other

Extraction with cyclohexane by heating under reflux

3623 7) GC-MSMS

2) Internal standardisation with labelled substances

C13 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

3624 2) HPLC-UV-FLD

1) External calibration

2) LiquidLiquid partitioning

D) Other hot solvent extraction

3626

64

Table 19 - continued

Laboratory code

Analysis technique

Instrument calibration

Internal standards

applied

Sample clean up

Details of sample

clean up

Extraction technique

Details of sample

extraction

5301 7) GC-MSMS

2) Internal standardisation with labelled substances

EACH SUBSTANCE C13

4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5302 7) GC-MSMS

2) Internal standardisation with labelled substances

Isotope labelled PAHs 13C for each analyte

4) Solid phase extraction

C) Soxhlet extraction

5303 7) GC-MSMS

3) Internal standardisation with unlabelled substances

HAP C13 4) Solid phase extraction

D) Other automate ASE200 DIONEX

5304 7) GC-MSMS

2) Internal standardisation with labelled substances

with C13 for each HAP

2) LiquidLiquid partitioning 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5305 7) GC-MSMS

2) Internal standardisation with labelled substances

C13 isotopiques internal standards

4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5306 7) GC-MSMS

2) Internal standardisation with labelled substances

C13 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5307 6) GC-MS

2) Internal standardisation with labelled substances

1) Saponification 2) LiquidLiquid partitioning

D) Other solvent extraction

5308 1) HPLC-FLD 1) External calibration

4) Solid phase extraction

B) Sonication

5309 1) HPLC-FLD 1) External calibration

4) Solid phase extraction

D) Other only SPE

5310 1) HPLC-FLD 1) External calibration

1) Saponification 2) LiquidLiquid partitioning 3) Gel permeation chromatography

C) Soxhlet extraction

5311 6) GC-MS

2) Internal standardisation with labelled substances

13 C Chrysene 13 C Benz(a)pyrene

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

B) Sonication

5312 1) HPLC-FLD

3) Internal standardisation with unlabelled substances

Benzo(b)chrysen

2) LiquidLiquid partitioning 4) Solid phase extraction

Quechers D) Other Quechers

65

Table 19 - continued

Laboratory code

Analysis technique

Instrument calibration

Internal standards applied

Sample clean up Details of sample

clean up

Extraction technique

Details of sample extraction

5313 6) GC-MS

2) Internal standardisation with labelled substances

Benz(a)anthracene D12 Benzo(b)fluranthene D12 Benzo(a)pyrene D12 Chrysene D12

1) Saponification 2) LiquidLiquid partitioning 3) Gel permeation chromatography

A) Pressurised liquid extraction (PLE)

5314 6) GC-MS

2) Internal standardisation with labelled substances

Benzo(a)pyren d12 2) LiquidLiquid partitioning

D) Other Extraction with

5315 1) HPLC-FLD

3) Internal standardisation with unlabelled substances

Benz(b)chrysen

1) Saponification 2) LiquidLiquid partitioning

D) Other shaker

5316 1) HPLC-FLD

1) External calibration

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

D) Other

LiquidLiquid extraction with cyclohexane and dimethylformamide

5317 7) GC-MSMS

1) External calibration

3) Gel permeation chromatography 6) Other

silica gel D) Other VDLUFA online method

5318

5319 1) HPLC-FLD

3) Internal standardisation with unlabelled substances

Benzo(b)chrysene 3) Gel permeation chromatography

A) Pressurised liquid extraction (PLE)

5320 6) GC-MS

3) Internal standardisation with unlabelled substances

22-binaphthyl

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

D) Other 3 hours refluxing

5321 1) HPLC-FLD

2) Internal standardisation with labelled substances

6-meacutethylchrysegravene

1) Saponification 2) LiquidLiquid partitioning

B) Sonication

5322 6) GC-MS

2) Internal standardisation with labelled substances

naftalene-d8 acenaphtalene-d10 phenanthrene-d10 chrysene-d12 perylene-d12 benzo(e)anthracene-d12

1) Saponification 2) LiquidLiquid partitioning 6) Other

clean-up with silica packed column

C) Soxhlet extraction

5323 1) HPLC-FLD

1) External calibration

4) Solid phase extraction 6) Other

Accelerated Solvent Extraction

A) Pressurised liquid extraction (PLE)

66

European Commission EUR 25245 EN ndash Joint Research Centre ndash Institute for Reference Materials and Measurements Title Report on the 8th inter-laboratory comparison organised by the European Union Reference Laboratory for Polycyclic Aromatic Hydrocarbons - Four marker PAHs in a dry extract of St Johns wort Author(s) Radoslav Lizak Szilard Szilagyi Philippe Verlinde and Thomas WENZL Luxembourg Publications Office of the European Union 2012 ndash 66 pp ndash 201 x 297 cm EUR ndash Scientific and Technical Research series ndash ISSN 1831-9424 ISBN 978-92-79-23337-1 doi 10278759387 Abstract The proficiency test here reported concerned the determination of the contents of four marker polycyclic aromatic hydrocarbons (PAHs) and of their sum in an food supplements test sample The set of marker PAHs consists of benz[a]anthacene benzo[a]pyrene benzo[b]fluoranthene and chrysene Participants to these PT were National Reference Laboratories for PAHs (NRLs-PAHs) and EU official food control laboratories The number of participants was in total 48

The PT was organised according ISO Standard 170432010

The test material used was a commercial dry extract of St Johns wort Participants also received a solution of the same PAHs either in acetonitrile or in toluene for checking their instrument calibration

The results from participants were rated with z-scores and zeta-scores About 68 of the reported results were attributed with z-scores with an absolute value of below two which is the threshold for satisfactory performance

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science and technology for the Union Close to the policy-making process it serves

the common interest of the Member States while being independent of special

interests whether private or national

L

A-N

A-252

45-EN

-N

• 1 Summary
• 2 Introduction
• 3 Scope
• 4 Participating Laboratories
• 5 Time frame
• 6 Confidentiality
• 7 Test materials
• • 71 1Preparation of test materials
  • 72 Assigned values and standard deviation for proficiency assessment
  • 73 Homogeneity and stability
  • 74 Sample dispatch
  • • 8 Design of the proficiency test
    • 9 Evaluation of the results
    • • 91 2General
      • 92 21BEvaluation criteria
      • 93 Performance of participants
      • 94 Evaluation of the influence of analysis method
      • 95 Evaluation of the influence of calibration
      • 96 26Evaluation of compliance with legislation
      • • 10 Follow-up actions for underperforming laboratories
        • 11 1Conclusions
        • 12 1Acknowledgements
        • 13 References

52

Figure 9 Results of replicate determinations (indicated by triangles) of chrysene (CHR) in the food supplement test material

Horizontal blue lines represent the arithmetic mean value of replicate measurements and blue bars the reported expanded measurement uncertainty (k=2) The assigned value is

plotted as dotted line Red solid lines mark the range of satisfactory performance (|z|le2) Mean values are given for results outside the displayed data range

ProLab 2011

Laboratory

53

0836

1453

1636

0253

2153

1836

0553

1036

1336

1736

1036

0453

1136

1836

0636

0336

2053

1936

1653

0136

0036

2253

0636

2153

0753

1553

1753

1453

0436

1536

1253

0536

2653

0353

0253

2236

2336

1953

2336

0753

2053

1353

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11

an

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on

ten

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gk

g)

13

12

11

10

9

8

7

6

5

4

3

2

1

0

865

3

Sample St Johns wort extract replicatesMeasurand ChryseneNo of laboratories 45

Assigned value 407 microgkg (Reference value)Target sd 083 microgkgTolerance limits 241 - 573 microgkg (|Z score| lt 200)

Assig

ned va

lue

Limit of tolerance

Limit of tolerance

53

Table 15 Analysis results reported by the participants for the sum of the four marker PAHs in the food supplements test material Values are presented as reported

Laboratory code

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg

3600 128 24 2

3602 79 126

3603 1127 096 2

3604 893 258 2

3605 1091 305 2

3606 1184 238 2

3607 2098 42 2

3608 3336 325 2

3610 1138 17 2

3611 2218 259 2

3612 143 29 2

3613 122 2 2

3614 663 129 2

3615 152 304 2

3616 102 03 2

3617 1252 169 2

3618 128 18 2

3619 157 305 2

3620 1204 12 2

3621 151 19 2

3622 1361 115 2

3623 154 09 2

3624

3626 1409 56 2

Laboratory code

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg

5301 126

5302 37 074 2

5303 13438 92

5304 1251 85 2

5305 125 20

5306 129 65 2

5307 1363

5308 7245598

5309

5310 1127 056

5311 1367 15 2

5312 2042 408 2

5313 1726

5314 1558

5315 177 35 2

5316 96 073 2

5317 1229 066 2

5318 82 05 2

5319 1225 267 2

5320 2309 41

5321

5322 207 35 2

5323 171 103 2

54

ANNEX 9 Results reported for the standard solutions with undisclosed analyte content Table 16 Analysis results reported by the participants for the four marker PAHs in the standard solution in toluene with undisclosed analyte

content Values are presented as reported U measurement uncertainty k coverage factor

benz[a]anthracene benzo[a]pyrene benzo[b]fluoranthene chrysene Laboratory

code Unit Result U k Result U k Result U k Result U k 3600 microgl 38 67 2 25 45 2 211 68 2 275 38 2

3605 microgl 367 184 2 2482 124 2 2592 13 2 235 118 2

3606 microgkg 3763 176 2 2609 142 2 2721 118 2 2539 134 2

3607 microgkg 3706 815 2 2824 565 2 2591 518 2 2587 569 2

3612 microgl 38 08 2 237 11 2 244 18 2 257 02 2

3614 microgl 324 65 2 209 38 2 215 37 2 21 42 2

3616 microgkg 318 07 2 224 14 2 231 26 2 216 1 2

3618 microgkg 417 17 2 284 08 2 292 06 2 297 03 2

3619 microgkg 47 742 2 274 468 2 228 387 2 274 434 2

3620 microgl 495 74 2 294 29 2 321 8 2 321 64 2

3621 microgl 241 22 2 143 13 2 188 16 2 145 14 2

3623 microgkg 476 25 2 311 53 2 269 3 2 297 03 2

3626 microgl 359 38 2 235 23 2 238 27 2 24 18 2

5301 microgkg 426 29 299 289

5302 microgkg 436 87 2 296 59 2 296 59 2 289 58 2

5303 microgl 3721 27 102 22838 19 99 23756 22 99 25486 24 98

5304 microgkg 38 29 2 225 26 2 257 37 2 258 52 2

5305 microgkg 305 225 245 29

5306 microgkg 316 63 2 247 49 2 65 13 2 22 44 2

5307 microgkg 3658 2559 241 2414

5308 microgkg 16854 109324 246654 216782

5311 microgl 374 20 2 279 15 2 268 15 2 272 15 2

5313 microgl 3408 2165 2275 2373

5314 microgkg 395 5 2875 11 3614 34 2905 18

5320 microgl 3267 368 1986 571 28 683 2427 512

5322 microgl 282 35 2 225 35 2 241 35 2 21 35 2

55

Figure 10 Youden plots of the percent deviation of the reported values from the assigned values for the food supplement test sample and the

standard solution in toluene with undisclosed content

(The identity of the respective measurand is given above the figure Results exceeding a difference of more than 100 were omitted)

56

Figure 10 - continued

57

Table 17 Analysis results reported by the participants for the four marker PAHs in the standard solution in acetonitrile with undisclosed

analyte content

Values are presented as reported U measurement uncertainty k coverage factor

benz[a]anthracene benzo[a]pyrene benzo[b]fluoranthene chrysene

Laboratory

code Unit Result U k Result U k Result U k Result U k

3602 microgkg 3675 247 247 244

3603 microgkg 358 086 2 236 028 2 235 028 2 236 057 2

3604 microgl 475 29 2 19 12 2 194 13 2 258 12 2

3608 microgl 3623 078 2 2385 023 2 2343 18 2 2296 092 2

3610 microgkg 369 185 2 25 125 2 251 126 2 244 122 2

3611 microgkg 48 02 2 323 02 2 328 04 2 317 03 2

3613 microgl 332 31 2 261 3 2 249 25 2 239 24 2

3615 microgl 415 2635 2729 2689

3617 microgkg 37 09 2 244 06 2 284 07 2 239 06 2

3622 microgl 37 12 2 25 23 2 257 21 2 238 1 2

3624 microgkg 402 104 2 255 084 2 265 096 2 253 07 2

5306 microgkg 563 113 2 371 74 2 359 72 2 333 67 2

5308 microgkg 168633 102669 232169 211488

5309 microgl 402 268 27

5310 microgl 315 158 25 125 25 125 288 144

5312 microgl 428 043 2 282 028 2 272 027 2 275 028 2

5315 microgkg 387 08 2 256 05 2 251 05 2 253 05 2

5316 microgl 3839 342 2 2485 191 2 2559 168 2 239 51 2

5317 microgl 2466 043 2 2212 034 2 2472 016 2 2146 068 2

5319 microgl 414 41 2 279 28 2 268 27 2 26 31 2

5321 microgkg 476 316 326 328

5322 microgl 2547 35 2

5323 microgkg 405 203 2 239 119 2 255 153 2 201 11 2

58

Figure 11 Youden plots of the percent deviation of the reported values from the assigned values for the food supplement test sample and the

standard solution in acetonitrile with undisclosed content

(The identity of the respective measurand is given above the figure Results exceeding a difference of more than 100 were omitted)

59

Figure 11 - continued

60

ANNEX 10 Table 18 Method performance parameters reported by the participants

Laboratory code

Analyte LOD LOQ Recovery Analyte LOD LOQ Recovery

microgkg microgkg microgkg microgkg 3600 BaA 03 06 978 BaP 03 07 9483602 BaA 007 021 926 BaP 005 015 993603 BaA 0011 05 987 BaP 0005 051 9553604 BaA 02 04 86 BaP 02 04 883605 BaA 0025 005 118 BaP 0025 005 1083606 BaA 001 003 65 BaP 001 003 703607 BaA 021 071 74 BaP 027 089 943608 BaA 016 048 8213 BaP 015 046 83683610 BaA 02 05 85 BaP 005 02 853611 BaA BaP 3612 BaA 1 117 BaP 1 1203613 BaA 01 03 90 BaP 01 03 903614 BaA 02 05 96 BaP 02 05 933615 BaA 005 015 110 BaP 005 015 1053616 BaA 01 05 108 BaP 01 05 753617 BaA 018 037 405 BaP 009 018 3873618 BaA 01 03 63 BaP 01 03 593619 BaA 005 005 59 BaP 033 033 543620 BaA 0009 0018 64 BaP 0005 001 553621 BaA 007 02 84 BaP 007 02 923622 BaA 01 03 1101 BaP 01 03 9853623 BaA 02 08 105 BaP 02 08 1013624 BaA BaP 01 033 8513625 BaA BaP 3626 BaA BaP 5301 BaA 003 01 102 BaP 003 01 1005302 BaA 01 03 78 BaP 01 03 865303 BaA 003 01 62 BaP 003 01 805304 BaA 1 1 70 BaP 1 1 955305 BaA 02 05 85 BaP 02 05 855306 BaA 03 1 699 BaP 03 1 905307 BaA 6537 BaP 81165308 BaA 0088 0265 99 BaP 00262 00785 1005309 BaA 01 08 92 BaP 01 08 925310 BaA 01 03 BaP 01 03 905311 BaA 002 005 80 BaP 002 004 915312 BaA 03 09 105 BaP 03 09 1055313 BaA 04 08 BaP 04 08 5314 BaA 001 002 96 BaP 001 002 9485315 BaA 01 03 50 - 100 BaP 01 03 50 - 100 5316 BaA 002 011 827 BaP 004 019 10445317 BaA 05 1 76 BaP 05 1 755318 BaA BaP 5319 BaA 009 029 951 BaP 011 041 995320 BaA 12 18 946 BaP 06 1 8345321 BaA 03 05 BaP 05 1 5322 BaA 1 2 100 BaP 1 2 1005323 BaA 26 BaP 26

61

ANNEX 10 Table 18 - continued

Laboratory code Analyte LOD LOQ Recovery Analyte LOD LOQ Recovery

microgkg microgkg microgkg microgkg 3600 BbF 03 05 96 CHR 02 03 10153602 BbF 015 045 947 CHR 003 009 9193603 BbF 0014 05 1012 CHR 0004 05 10033604 BbF 02 04 85 CHR 02 04 873605 BbF 005 01 94 CHR 0025 005 863606 BbF 001 003 70 CHR 001 003 653607 BbF 018 06 100 CHR 014 028 943608 BbF 015 046 7979 CHR 015 045 71253610 BbF 005 02 85 CHR 02 05 853611 BbF CHR 3612 BbF 1 118 CHR 1 1173613 BbF 01 03 90 CHR 01 03 903614 BbF 01 04 96 CHR 02 05 953615 BbF 008 025 109 CHR 003 01 1083616 BbF 01 05 78 CHR 01 05 723617 BbF 035 07 38 CHR 012 024 4163618 BbF 01 03 62 CHR 01 03 623619 BbF 016 016 63 CHR 01 01 593620 BbF 0003 0006 55 CHR 001 002 653621 BbF 007 02 103 CHR 007 02 893622 BbF 02 06 963 CHR 03 09 1043623 BbF 02 08 106 CHR 02 08 1193624 BbF CHR 3625 BbF CHR 3626 BbF CHR 5301 BbF 003 01 96 CHR 003 01 1145302 BbF 01 03 84 CHR 01 03 835303 BbF 003 01 78 CHR 003 01 705304 BbF 1 1 86 CHR 1 1 705305 BbF 02 05 90 CHR 02 05 955306 BbF 03 1 1435 CHR 03 1 1185307 BbF 8116 CHR 65375308 BbF 0079 0238 99 CHR 009 026 1005309 BbF 01 08 92 CHR 5310 BbF 07 2 CHR 01 03 5311 BbF 002 004 95 CHR 002 004 895312 BbF 03 09 105 CHR 03 09 1055313 BbF 04 08 CHR 04 08 5314 BbF 001 002 83 CHR 001 002 8185315 BbF 02 05 50 -100 CHR 02 05 50 - 100 5316 BbF 005 024 814 CHR 001 007 9595317 BbF 05 1 73 CHR 05 1 785318 BbF CHR 5319 BbF 011 038 946 CHR 013 043 9675320 BbF 07 09 1194 CHR 23 32 10455321 BbF 05 1 CHR 05 1 5322 BbF 1 2 100 CHR 1 2 1005323 BbF 26 CHR 26

62

ANNEX 11 Table 19 Details of analysis method reported by the participants Data are presented as reported

Laboratory code

Analysis technique

Instrument calibration

Internal standards applied

Sample clean up

Details of sample

clean up

Extraction technique

Details of sample

extraction

3600 6) GC-MS

2) Internal standardisation with labelled substances

Deuterated PAH mix 9 (Ehrenstorfer)

1) Saponification 2) LiquidLiquid partitioning

D) Other Liquidliquid partitioning

3602 2) HPLC-UV-FLD

1) External calibration

3) Gel permeation chromatography

A) Pressurised liquid extraction (PLE)

3603 1) HPLC-FLD

1) External calibration

3) Gel permeation chromatography

D) Other 1 minute with dichloromethane in vortex

3604 2) HPLC-UV-FLD

2) Internal standardisation with labelled substances

DiP D14

5) Donor acceptor complex chromatography

D) Other LL

3605 1) HPLC-FLD

1) External calibration

3) Gel permeation chromatography

C) Soxhlet extraction

3606 7) GC-MSMS

2) Internal standardisation with labelled substances

BaP 13C4 CHR 13C6 BaA 13C6 BbF 13C6

4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

3607 6) GC-MS

2) Internal standardisation with labelled substances

BaA-d12 CHR-d12 BbF-d12 BkF-d12 BaP-d12 ICP-d12 BgP-d12 DiP-d14

1) Saponification

D) Other solvent partitioning

3608 1) HPLC-FLD

1) External calibration

6) Other Liquid extraction

D) Other

Extraction in a rotary agitator and centrifugation

3610 2) HPLC-UV-FLD

1) External calibration

4) Solid phase extraction

B) Sonication

3611

3612 6) GC-MS

2) Internal standardisation with labelled substances

chrysene-d12 dibenzo(ah)anthracene-d14 dibenzo(ai)pyrene-d14

4) Solid phase extraction

B) Sonication

3613 5) LC-MSMS

3) Internal standardisation with unlabelled substances

I use Chrysene-D12 3) Gel permeation chromatography

D) Other extraction with hexan

63

Table 19 - continued

Laboratory code

Analysis technique

Instrument calibration

Internal standards applied

Sample clean up Details of sample

clean up

Extraction technique

Details of sample

extraction

3614 6) GC-MS 1) External calibration

3) Gel permeation chromatography

B) Sonication

3615 1) HPLC-FLD

1) External calibration

6) Other PLE GPC

A) Pressurised liquid extraction (PLE)

3616 6) GC-MS

2) Internal standardisation with labelled substances

Benzo(a)pyrene-13C4 Benzo(a)anthracene-13C6 Benzo(b)fluoranthene-13C6 Chrysene-13C6

2) LiquidLiquid partitioning 4) Solid phase extraction

B) Sonication

3617 1) HPLC-FLD

1) External calibration

4) Solid phase extraction

C) Soxhlet extraction

3618 6) GC-MS

2) Internal standardisation with labelled substances

deuterated standards

1) Saponification 4) Solid phase extraction

D) Other Handshaken with cyclohexane

3619 6) GC-MS

2) Internal standardisation with labelled substances

C13 Labelled US EPA 16

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

D) Other Homegenisation amp saponiofication

3620 9) GC-HRMS

2) Internal standardisation with labelled substances

mix of deuterated PAHs (BAA-D12 CHR-D12 BAP-D12 BBF-D12)

3) Gel permeation chromatography 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

3621 6) GC-MS

2) Internal standardisation with labelled substances

Chrysene D12 Benzo(a)pyrene D12 Benzo(b)fluoranthene D12 Benzo(a)anthracene

2) LiquidLiquid partitioning 4) Solid phase extraction

B) Sonication

3622 1) HPLC-FLD

4) Standard addition method

4) Solid phase extraction

D) Other

Extraction with cyclohexane by heating under reflux

3623 7) GC-MSMS

2) Internal standardisation with labelled substances

C13 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

3624 2) HPLC-UV-FLD

1) External calibration

2) LiquidLiquid partitioning

D) Other hot solvent extraction

3626

64

Table 19 - continued

Laboratory code

Analysis technique

Instrument calibration

Internal standards

applied

Sample clean up

Details of sample

clean up

Extraction technique

Details of sample

extraction

5301 7) GC-MSMS

2) Internal standardisation with labelled substances

EACH SUBSTANCE C13

4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5302 7) GC-MSMS

2) Internal standardisation with labelled substances

Isotope labelled PAHs 13C for each analyte

4) Solid phase extraction

C) Soxhlet extraction

5303 7) GC-MSMS

3) Internal standardisation with unlabelled substances

HAP C13 4) Solid phase extraction

D) Other automate ASE200 DIONEX

5304 7) GC-MSMS

2) Internal standardisation with labelled substances

with C13 for each HAP

2) LiquidLiquid partitioning 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5305 7) GC-MSMS

2) Internal standardisation with labelled substances

C13 isotopiques internal standards

4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5306 7) GC-MSMS

2) Internal standardisation with labelled substances

C13 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5307 6) GC-MS

2) Internal standardisation with labelled substances

1) Saponification 2) LiquidLiquid partitioning

D) Other solvent extraction

5308 1) HPLC-FLD 1) External calibration

4) Solid phase extraction

B) Sonication

5309 1) HPLC-FLD 1) External calibration

4) Solid phase extraction

D) Other only SPE

5310 1) HPLC-FLD 1) External calibration

1) Saponification 2) LiquidLiquid partitioning 3) Gel permeation chromatography

C) Soxhlet extraction

5311 6) GC-MS

2) Internal standardisation with labelled substances

13 C Chrysene 13 C Benz(a)pyrene

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

B) Sonication

5312 1) HPLC-FLD

3) Internal standardisation with unlabelled substances

Benzo(b)chrysen

2) LiquidLiquid partitioning 4) Solid phase extraction

Quechers D) Other Quechers

65

Table 19 - continued

Laboratory code

Analysis technique

Instrument calibration

Internal standards applied

Sample clean up Details of sample

clean up

Extraction technique

Details of sample extraction

5313 6) GC-MS

2) Internal standardisation with labelled substances

Benz(a)anthracene D12 Benzo(b)fluranthene D12 Benzo(a)pyrene D12 Chrysene D12

1) Saponification 2) LiquidLiquid partitioning 3) Gel permeation chromatography

A) Pressurised liquid extraction (PLE)

5314 6) GC-MS

2) Internal standardisation with labelled substances

Benzo(a)pyren d12 2) LiquidLiquid partitioning

D) Other Extraction with

5315 1) HPLC-FLD

3) Internal standardisation with unlabelled substances

Benz(b)chrysen

1) Saponification 2) LiquidLiquid partitioning

D) Other shaker

5316 1) HPLC-FLD

1) External calibration

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

D) Other

LiquidLiquid extraction with cyclohexane and dimethylformamide

5317 7) GC-MSMS

1) External calibration

3) Gel permeation chromatography 6) Other

silica gel D) Other VDLUFA online method

5318

5319 1) HPLC-FLD

3) Internal standardisation with unlabelled substances

Benzo(b)chrysene 3) Gel permeation chromatography

A) Pressurised liquid extraction (PLE)

5320 6) GC-MS

3) Internal standardisation with unlabelled substances

22-binaphthyl

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

D) Other 3 hours refluxing

5321 1) HPLC-FLD

2) Internal standardisation with labelled substances

6-meacutethylchrysegravene

1) Saponification 2) LiquidLiquid partitioning

B) Sonication

5322 6) GC-MS

2) Internal standardisation with labelled substances

naftalene-d8 acenaphtalene-d10 phenanthrene-d10 chrysene-d12 perylene-d12 benzo(e)anthracene-d12

1) Saponification 2) LiquidLiquid partitioning 6) Other

clean-up with silica packed column

C) Soxhlet extraction

5323 1) HPLC-FLD

1) External calibration

4) Solid phase extraction 6) Other

Accelerated Solvent Extraction

A) Pressurised liquid extraction (PLE)

66

European Commission EUR 25245 EN ndash Joint Research Centre ndash Institute for Reference Materials and Measurements Title Report on the 8th inter-laboratory comparison organised by the European Union Reference Laboratory for Polycyclic Aromatic Hydrocarbons - Four marker PAHs in a dry extract of St Johns wort Author(s) Radoslav Lizak Szilard Szilagyi Philippe Verlinde and Thomas WENZL Luxembourg Publications Office of the European Union 2012 ndash 66 pp ndash 201 x 297 cm EUR ndash Scientific and Technical Research series ndash ISSN 1831-9424 ISBN 978-92-79-23337-1 doi 10278759387 Abstract The proficiency test here reported concerned the determination of the contents of four marker polycyclic aromatic hydrocarbons (PAHs) and of their sum in an food supplements test sample The set of marker PAHs consists of benz[a]anthacene benzo[a]pyrene benzo[b]fluoranthene and chrysene Participants to these PT were National Reference Laboratories for PAHs (NRLs-PAHs) and EU official food control laboratories The number of participants was in total 48

The PT was organised according ISO Standard 170432010

The test material used was a commercial dry extract of St Johns wort Participants also received a solution of the same PAHs either in acetonitrile or in toluene for checking their instrument calibration

The results from participants were rated with z-scores and zeta-scores About 68 of the reported results were attributed with z-scores with an absolute value of below two which is the threshold for satisfactory performance

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science and technology for the Union Close to the policy-making process it serves

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interests whether private or national

L

A-N

A-252

45-EN

-N

• 1 Summary
• 2 Introduction
• 3 Scope
• 4 Participating Laboratories
• 5 Time frame
• 6 Confidentiality
• 7 Test materials
• • 71 1Preparation of test materials
  • 72 Assigned values and standard deviation for proficiency assessment
  • 73 Homogeneity and stability
  • 74 Sample dispatch
  • • 8 Design of the proficiency test
    • 9 Evaluation of the results
    • • 91 2General
      • 92 21BEvaluation criteria
      • 93 Performance of participants
      • 94 Evaluation of the influence of analysis method
      • 95 Evaluation of the influence of calibration
      • 96 26Evaluation of compliance with legislation
      • • 10 Follow-up actions for underperforming laboratories
        • 11 1Conclusions
        • 12 1Acknowledgements
        • 13 References

53

Table 15 Analysis results reported by the participants for the sum of the four marker PAHs in the food supplements test material Values are presented as reported

Laboratory code

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg

3600 128 24 2

3602 79 126

3603 1127 096 2

3604 893 258 2

3605 1091 305 2

3606 1184 238 2

3607 2098 42 2

3608 3336 325 2

3610 1138 17 2

3611 2218 259 2

3612 143 29 2

3613 122 2 2

3614 663 129 2

3615 152 304 2

3616 102 03 2

3617 1252 169 2

3618 128 18 2

3619 157 305 2

3620 1204 12 2

3621 151 19 2

3622 1361 115 2

3623 154 09 2

3624

3626 1409 56 2

Laboratory code

Value for proficiency

assessment Uncertainty

Coverage factor (k)

microgkg microgkg

5301 126

5302 37 074 2

5303 13438 92

5304 1251 85 2

5305 125 20

5306 129 65 2

5307 1363

5308 7245598

5309

5310 1127 056

5311 1367 15 2

5312 2042 408 2

5313 1726

5314 1558

5315 177 35 2

5316 96 073 2

5317 1229 066 2

5318 82 05 2

5319 1225 267 2

5320 2309 41

5321

5322 207 35 2

5323 171 103 2

54

ANNEX 9 Results reported for the standard solutions with undisclosed analyte content Table 16 Analysis results reported by the participants for the four marker PAHs in the standard solution in toluene with undisclosed analyte

content Values are presented as reported U measurement uncertainty k coverage factor

benz[a]anthracene benzo[a]pyrene benzo[b]fluoranthene chrysene Laboratory

code Unit Result U k Result U k Result U k Result U k 3600 microgl 38 67 2 25 45 2 211 68 2 275 38 2

3605 microgl 367 184 2 2482 124 2 2592 13 2 235 118 2

3606 microgkg 3763 176 2 2609 142 2 2721 118 2 2539 134 2

3607 microgkg 3706 815 2 2824 565 2 2591 518 2 2587 569 2

3612 microgl 38 08 2 237 11 2 244 18 2 257 02 2

3614 microgl 324 65 2 209 38 2 215 37 2 21 42 2

3616 microgkg 318 07 2 224 14 2 231 26 2 216 1 2

3618 microgkg 417 17 2 284 08 2 292 06 2 297 03 2

3619 microgkg 47 742 2 274 468 2 228 387 2 274 434 2

3620 microgl 495 74 2 294 29 2 321 8 2 321 64 2

3621 microgl 241 22 2 143 13 2 188 16 2 145 14 2

3623 microgkg 476 25 2 311 53 2 269 3 2 297 03 2

3626 microgl 359 38 2 235 23 2 238 27 2 24 18 2

5301 microgkg 426 29 299 289

5302 microgkg 436 87 2 296 59 2 296 59 2 289 58 2

5303 microgl 3721 27 102 22838 19 99 23756 22 99 25486 24 98

5304 microgkg 38 29 2 225 26 2 257 37 2 258 52 2

5305 microgkg 305 225 245 29

5306 microgkg 316 63 2 247 49 2 65 13 2 22 44 2

5307 microgkg 3658 2559 241 2414

5308 microgkg 16854 109324 246654 216782

5311 microgl 374 20 2 279 15 2 268 15 2 272 15 2

5313 microgl 3408 2165 2275 2373

5314 microgkg 395 5 2875 11 3614 34 2905 18

5320 microgl 3267 368 1986 571 28 683 2427 512

5322 microgl 282 35 2 225 35 2 241 35 2 21 35 2

55

Figure 10 Youden plots of the percent deviation of the reported values from the assigned values for the food supplement test sample and the

standard solution in toluene with undisclosed content

(The identity of the respective measurand is given above the figure Results exceeding a difference of more than 100 were omitted)

56

Figure 10 - continued

57

Table 17 Analysis results reported by the participants for the four marker PAHs in the standard solution in acetonitrile with undisclosed

analyte content

Values are presented as reported U measurement uncertainty k coverage factor

benz[a]anthracene benzo[a]pyrene benzo[b]fluoranthene chrysene

Laboratory

code Unit Result U k Result U k Result U k Result U k

3602 microgkg 3675 247 247 244

3603 microgkg 358 086 2 236 028 2 235 028 2 236 057 2

3604 microgl 475 29 2 19 12 2 194 13 2 258 12 2

3608 microgl 3623 078 2 2385 023 2 2343 18 2 2296 092 2

3610 microgkg 369 185 2 25 125 2 251 126 2 244 122 2

3611 microgkg 48 02 2 323 02 2 328 04 2 317 03 2

3613 microgl 332 31 2 261 3 2 249 25 2 239 24 2

3615 microgl 415 2635 2729 2689

3617 microgkg 37 09 2 244 06 2 284 07 2 239 06 2

3622 microgl 37 12 2 25 23 2 257 21 2 238 1 2

3624 microgkg 402 104 2 255 084 2 265 096 2 253 07 2

5306 microgkg 563 113 2 371 74 2 359 72 2 333 67 2

5308 microgkg 168633 102669 232169 211488

5309 microgl 402 268 27

5310 microgl 315 158 25 125 25 125 288 144

5312 microgl 428 043 2 282 028 2 272 027 2 275 028 2

5315 microgkg 387 08 2 256 05 2 251 05 2 253 05 2

5316 microgl 3839 342 2 2485 191 2 2559 168 2 239 51 2

5317 microgl 2466 043 2 2212 034 2 2472 016 2 2146 068 2

5319 microgl 414 41 2 279 28 2 268 27 2 26 31 2

5321 microgkg 476 316 326 328

5322 microgl 2547 35 2

5323 microgkg 405 203 2 239 119 2 255 153 2 201 11 2

58

Figure 11 Youden plots of the percent deviation of the reported values from the assigned values for the food supplement test sample and the

standard solution in acetonitrile with undisclosed content

(The identity of the respective measurand is given above the figure Results exceeding a difference of more than 100 were omitted)

59

Figure 11 - continued

60

ANNEX 10 Table 18 Method performance parameters reported by the participants

Laboratory code

Analyte LOD LOQ Recovery Analyte LOD LOQ Recovery

microgkg microgkg microgkg microgkg 3600 BaA 03 06 978 BaP 03 07 9483602 BaA 007 021 926 BaP 005 015 993603 BaA 0011 05 987 BaP 0005 051 9553604 BaA 02 04 86 BaP 02 04 883605 BaA 0025 005 118 BaP 0025 005 1083606 BaA 001 003 65 BaP 001 003 703607 BaA 021 071 74 BaP 027 089 943608 BaA 016 048 8213 BaP 015 046 83683610 BaA 02 05 85 BaP 005 02 853611 BaA BaP 3612 BaA 1 117 BaP 1 1203613 BaA 01 03 90 BaP 01 03 903614 BaA 02 05 96 BaP 02 05 933615 BaA 005 015 110 BaP 005 015 1053616 BaA 01 05 108 BaP 01 05 753617 BaA 018 037 405 BaP 009 018 3873618 BaA 01 03 63 BaP 01 03 593619 BaA 005 005 59 BaP 033 033 543620 BaA 0009 0018 64 BaP 0005 001 553621 BaA 007 02 84 BaP 007 02 923622 BaA 01 03 1101 BaP 01 03 9853623 BaA 02 08 105 BaP 02 08 1013624 BaA BaP 01 033 8513625 BaA BaP 3626 BaA BaP 5301 BaA 003 01 102 BaP 003 01 1005302 BaA 01 03 78 BaP 01 03 865303 BaA 003 01 62 BaP 003 01 805304 BaA 1 1 70 BaP 1 1 955305 BaA 02 05 85 BaP 02 05 855306 BaA 03 1 699 BaP 03 1 905307 BaA 6537 BaP 81165308 BaA 0088 0265 99 BaP 00262 00785 1005309 BaA 01 08 92 BaP 01 08 925310 BaA 01 03 BaP 01 03 905311 BaA 002 005 80 BaP 002 004 915312 BaA 03 09 105 BaP 03 09 1055313 BaA 04 08 BaP 04 08 5314 BaA 001 002 96 BaP 001 002 9485315 BaA 01 03 50 - 100 BaP 01 03 50 - 100 5316 BaA 002 011 827 BaP 004 019 10445317 BaA 05 1 76 BaP 05 1 755318 BaA BaP 5319 BaA 009 029 951 BaP 011 041 995320 BaA 12 18 946 BaP 06 1 8345321 BaA 03 05 BaP 05 1 5322 BaA 1 2 100 BaP 1 2 1005323 BaA 26 BaP 26

61

ANNEX 10 Table 18 - continued

Laboratory code Analyte LOD LOQ Recovery Analyte LOD LOQ Recovery

microgkg microgkg microgkg microgkg 3600 BbF 03 05 96 CHR 02 03 10153602 BbF 015 045 947 CHR 003 009 9193603 BbF 0014 05 1012 CHR 0004 05 10033604 BbF 02 04 85 CHR 02 04 873605 BbF 005 01 94 CHR 0025 005 863606 BbF 001 003 70 CHR 001 003 653607 BbF 018 06 100 CHR 014 028 943608 BbF 015 046 7979 CHR 015 045 71253610 BbF 005 02 85 CHR 02 05 853611 BbF CHR 3612 BbF 1 118 CHR 1 1173613 BbF 01 03 90 CHR 01 03 903614 BbF 01 04 96 CHR 02 05 953615 BbF 008 025 109 CHR 003 01 1083616 BbF 01 05 78 CHR 01 05 723617 BbF 035 07 38 CHR 012 024 4163618 BbF 01 03 62 CHR 01 03 623619 BbF 016 016 63 CHR 01 01 593620 BbF 0003 0006 55 CHR 001 002 653621 BbF 007 02 103 CHR 007 02 893622 BbF 02 06 963 CHR 03 09 1043623 BbF 02 08 106 CHR 02 08 1193624 BbF CHR 3625 BbF CHR 3626 BbF CHR 5301 BbF 003 01 96 CHR 003 01 1145302 BbF 01 03 84 CHR 01 03 835303 BbF 003 01 78 CHR 003 01 705304 BbF 1 1 86 CHR 1 1 705305 BbF 02 05 90 CHR 02 05 955306 BbF 03 1 1435 CHR 03 1 1185307 BbF 8116 CHR 65375308 BbF 0079 0238 99 CHR 009 026 1005309 BbF 01 08 92 CHR 5310 BbF 07 2 CHR 01 03 5311 BbF 002 004 95 CHR 002 004 895312 BbF 03 09 105 CHR 03 09 1055313 BbF 04 08 CHR 04 08 5314 BbF 001 002 83 CHR 001 002 8185315 BbF 02 05 50 -100 CHR 02 05 50 - 100 5316 BbF 005 024 814 CHR 001 007 9595317 BbF 05 1 73 CHR 05 1 785318 BbF CHR 5319 BbF 011 038 946 CHR 013 043 9675320 BbF 07 09 1194 CHR 23 32 10455321 BbF 05 1 CHR 05 1 5322 BbF 1 2 100 CHR 1 2 1005323 BbF 26 CHR 26

62

ANNEX 11 Table 19 Details of analysis method reported by the participants Data are presented as reported

Laboratory code

Analysis technique

Instrument calibration

Internal standards applied

Sample clean up

Details of sample

clean up

Extraction technique

Details of sample

extraction

3600 6) GC-MS

2) Internal standardisation with labelled substances

Deuterated PAH mix 9 (Ehrenstorfer)

1) Saponification 2) LiquidLiquid partitioning

D) Other Liquidliquid partitioning

3602 2) HPLC-UV-FLD

1) External calibration

3) Gel permeation chromatography

A) Pressurised liquid extraction (PLE)

3603 1) HPLC-FLD

1) External calibration

3) Gel permeation chromatography

D) Other 1 minute with dichloromethane in vortex

3604 2) HPLC-UV-FLD

2) Internal standardisation with labelled substances

DiP D14

5) Donor acceptor complex chromatography

D) Other LL

3605 1) HPLC-FLD

1) External calibration

3) Gel permeation chromatography

C) Soxhlet extraction

3606 7) GC-MSMS

2) Internal standardisation with labelled substances

BaP 13C4 CHR 13C6 BaA 13C6 BbF 13C6

4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

3607 6) GC-MS

2) Internal standardisation with labelled substances

BaA-d12 CHR-d12 BbF-d12 BkF-d12 BaP-d12 ICP-d12 BgP-d12 DiP-d14

1) Saponification

D) Other solvent partitioning

3608 1) HPLC-FLD

1) External calibration

6) Other Liquid extraction

D) Other

Extraction in a rotary agitator and centrifugation

3610 2) HPLC-UV-FLD

1) External calibration

4) Solid phase extraction

B) Sonication

3611

3612 6) GC-MS

2) Internal standardisation with labelled substances

chrysene-d12 dibenzo(ah)anthracene-d14 dibenzo(ai)pyrene-d14

4) Solid phase extraction

B) Sonication

3613 5) LC-MSMS

3) Internal standardisation with unlabelled substances

I use Chrysene-D12 3) Gel permeation chromatography

D) Other extraction with hexan

63

Table 19 - continued

Laboratory code

Analysis technique

Instrument calibration

Internal standards applied

Sample clean up Details of sample

clean up

Extraction technique

Details of sample

extraction

3614 6) GC-MS 1) External calibration

3) Gel permeation chromatography

B) Sonication

3615 1) HPLC-FLD

1) External calibration

6) Other PLE GPC

A) Pressurised liquid extraction (PLE)

3616 6) GC-MS

2) Internal standardisation with labelled substances

Benzo(a)pyrene-13C4 Benzo(a)anthracene-13C6 Benzo(b)fluoranthene-13C6 Chrysene-13C6

2) LiquidLiquid partitioning 4) Solid phase extraction

B) Sonication

3617 1) HPLC-FLD

1) External calibration

4) Solid phase extraction

C) Soxhlet extraction

3618 6) GC-MS

2) Internal standardisation with labelled substances

deuterated standards

1) Saponification 4) Solid phase extraction

D) Other Handshaken with cyclohexane

3619 6) GC-MS

2) Internal standardisation with labelled substances

C13 Labelled US EPA 16

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

D) Other Homegenisation amp saponiofication

3620 9) GC-HRMS

2) Internal standardisation with labelled substances

mix of deuterated PAHs (BAA-D12 CHR-D12 BAP-D12 BBF-D12)

3) Gel permeation chromatography 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

3621 6) GC-MS

2) Internal standardisation with labelled substances

Chrysene D12 Benzo(a)pyrene D12 Benzo(b)fluoranthene D12 Benzo(a)anthracene

2) LiquidLiquid partitioning 4) Solid phase extraction

B) Sonication

3622 1) HPLC-FLD

4) Standard addition method

4) Solid phase extraction

D) Other

Extraction with cyclohexane by heating under reflux

3623 7) GC-MSMS

2) Internal standardisation with labelled substances

C13 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

3624 2) HPLC-UV-FLD

1) External calibration

2) LiquidLiquid partitioning

D) Other hot solvent extraction

3626

64

Table 19 - continued

Laboratory code

Analysis technique

Instrument calibration

Internal standards

applied

Sample clean up

Details of sample

clean up

Extraction technique

Details of sample

extraction

5301 7) GC-MSMS

2) Internal standardisation with labelled substances

EACH SUBSTANCE C13

4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5302 7) GC-MSMS

2) Internal standardisation with labelled substances

Isotope labelled PAHs 13C for each analyte

4) Solid phase extraction

C) Soxhlet extraction

5303 7) GC-MSMS

3) Internal standardisation with unlabelled substances

HAP C13 4) Solid phase extraction

D) Other automate ASE200 DIONEX

5304 7) GC-MSMS

2) Internal standardisation with labelled substances

with C13 for each HAP

2) LiquidLiquid partitioning 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5305 7) GC-MSMS

2) Internal standardisation with labelled substances

C13 isotopiques internal standards

4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5306 7) GC-MSMS

2) Internal standardisation with labelled substances

C13 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5307 6) GC-MS

2) Internal standardisation with labelled substances

1) Saponification 2) LiquidLiquid partitioning

D) Other solvent extraction

5308 1) HPLC-FLD 1) External calibration

4) Solid phase extraction

B) Sonication

5309 1) HPLC-FLD 1) External calibration

4) Solid phase extraction

D) Other only SPE

5310 1) HPLC-FLD 1) External calibration

1) Saponification 2) LiquidLiquid partitioning 3) Gel permeation chromatography

C) Soxhlet extraction

5311 6) GC-MS

2) Internal standardisation with labelled substances

13 C Chrysene 13 C Benz(a)pyrene

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

B) Sonication

5312 1) HPLC-FLD

3) Internal standardisation with unlabelled substances

Benzo(b)chrysen

2) LiquidLiquid partitioning 4) Solid phase extraction

Quechers D) Other Quechers

65

Table 19 - continued

Laboratory code

Analysis technique

Instrument calibration

Internal standards applied

Sample clean up Details of sample

clean up

Extraction technique

Details of sample extraction

5313 6) GC-MS

2) Internal standardisation with labelled substances

Benz(a)anthracene D12 Benzo(b)fluranthene D12 Benzo(a)pyrene D12 Chrysene D12

1) Saponification 2) LiquidLiquid partitioning 3) Gel permeation chromatography

A) Pressurised liquid extraction (PLE)

5314 6) GC-MS

2) Internal standardisation with labelled substances

Benzo(a)pyren d12 2) LiquidLiquid partitioning

D) Other Extraction with

5315 1) HPLC-FLD

3) Internal standardisation with unlabelled substances

Benz(b)chrysen

1) Saponification 2) LiquidLiquid partitioning

D) Other shaker

5316 1) HPLC-FLD

1) External calibration

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

D) Other

LiquidLiquid extraction with cyclohexane and dimethylformamide

5317 7) GC-MSMS

1) External calibration

3) Gel permeation chromatography 6) Other

silica gel D) Other VDLUFA online method

5318

5319 1) HPLC-FLD

3) Internal standardisation with unlabelled substances

Benzo(b)chrysene 3) Gel permeation chromatography

A) Pressurised liquid extraction (PLE)

5320 6) GC-MS

3) Internal standardisation with unlabelled substances

22-binaphthyl

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

D) Other 3 hours refluxing

5321 1) HPLC-FLD

2) Internal standardisation with labelled substances

6-meacutethylchrysegravene

1) Saponification 2) LiquidLiquid partitioning

B) Sonication

5322 6) GC-MS

2) Internal standardisation with labelled substances

naftalene-d8 acenaphtalene-d10 phenanthrene-d10 chrysene-d12 perylene-d12 benzo(e)anthracene-d12

1) Saponification 2) LiquidLiquid partitioning 6) Other

clean-up with silica packed column

C) Soxhlet extraction

5323 1) HPLC-FLD

1) External calibration

4) Solid phase extraction 6) Other

Accelerated Solvent Extraction

A) Pressurised liquid extraction (PLE)

66

European Commission EUR 25245 EN ndash Joint Research Centre ndash Institute for Reference Materials and Measurements Title Report on the 8th inter-laboratory comparison organised by the European Union Reference Laboratory for Polycyclic Aromatic Hydrocarbons - Four marker PAHs in a dry extract of St Johns wort Author(s) Radoslav Lizak Szilard Szilagyi Philippe Verlinde and Thomas WENZL Luxembourg Publications Office of the European Union 2012 ndash 66 pp ndash 201 x 297 cm EUR ndash Scientific and Technical Research series ndash ISSN 1831-9424 ISBN 978-92-79-23337-1 doi 10278759387 Abstract The proficiency test here reported concerned the determination of the contents of four marker polycyclic aromatic hydrocarbons (PAHs) and of their sum in an food supplements test sample The set of marker PAHs consists of benz[a]anthacene benzo[a]pyrene benzo[b]fluoranthene and chrysene Participants to these PT were National Reference Laboratories for PAHs (NRLs-PAHs) and EU official food control laboratories The number of participants was in total 48

The PT was organised according ISO Standard 170432010

The test material used was a commercial dry extract of St Johns wort Participants also received a solution of the same PAHs either in acetonitrile or in toluene for checking their instrument calibration

The results from participants were rated with z-scores and zeta-scores About 68 of the reported results were attributed with z-scores with an absolute value of below two which is the threshold for satisfactory performance

How to obtain EU publications

Our priced publications are available from EU Bookshop (httpbookshopeuropaeu) where you can place

an order with the sales agent of your choice

The Publications Office has a worldwide network of sales agents You can obtain their contact details by

sending a fax to (352) 29 29-42758

The mission of the JRC is to provide customer-driven scientific and technical support

for the conception development implementation and monitoring of EU policies As a

service of the European Commission the JRC functions as a reference centre of

science and technology for the Union Close to the policy-making process it serves

the common interest of the Member States while being independent of special

interests whether private or national

L

A-N

A-252

45-EN

-N

• 1 Summary
• 2 Introduction
• 3 Scope
• 4 Participating Laboratories
• 5 Time frame
• 6 Confidentiality
• 7 Test materials
• • 71 1Preparation of test materials
  • 72 Assigned values and standard deviation for proficiency assessment
  • 73 Homogeneity and stability
  • 74 Sample dispatch
  • • 8 Design of the proficiency test
    • 9 Evaluation of the results
    • • 91 2General
      • 92 21BEvaluation criteria
      • 93 Performance of participants
      • 94 Evaluation of the influence of analysis method
      • 95 Evaluation of the influence of calibration
      • 96 26Evaluation of compliance with legislation
      • • 10 Follow-up actions for underperforming laboratories
        • 11 1Conclusions
        • 12 1Acknowledgements
        • 13 References

54

ANNEX 9 Results reported for the standard solutions with undisclosed analyte content Table 16 Analysis results reported by the participants for the four marker PAHs in the standard solution in toluene with undisclosed analyte

content Values are presented as reported U measurement uncertainty k coverage factor

benz[a]anthracene benzo[a]pyrene benzo[b]fluoranthene chrysene Laboratory

code Unit Result U k Result U k Result U k Result U k 3600 microgl 38 67 2 25 45 2 211 68 2 275 38 2

3605 microgl 367 184 2 2482 124 2 2592 13 2 235 118 2

3606 microgkg 3763 176 2 2609 142 2 2721 118 2 2539 134 2

3607 microgkg 3706 815 2 2824 565 2 2591 518 2 2587 569 2

3612 microgl 38 08 2 237 11 2 244 18 2 257 02 2

3614 microgl 324 65 2 209 38 2 215 37 2 21 42 2

3616 microgkg 318 07 2 224 14 2 231 26 2 216 1 2

3618 microgkg 417 17 2 284 08 2 292 06 2 297 03 2

3619 microgkg 47 742 2 274 468 2 228 387 2 274 434 2

3620 microgl 495 74 2 294 29 2 321 8 2 321 64 2

3621 microgl 241 22 2 143 13 2 188 16 2 145 14 2

3623 microgkg 476 25 2 311 53 2 269 3 2 297 03 2

3626 microgl 359 38 2 235 23 2 238 27 2 24 18 2

5301 microgkg 426 29 299 289

5302 microgkg 436 87 2 296 59 2 296 59 2 289 58 2

5303 microgl 3721 27 102 22838 19 99 23756 22 99 25486 24 98

5304 microgkg 38 29 2 225 26 2 257 37 2 258 52 2

5305 microgkg 305 225 245 29

5306 microgkg 316 63 2 247 49 2 65 13 2 22 44 2

5307 microgkg 3658 2559 241 2414

5308 microgkg 16854 109324 246654 216782

5311 microgl 374 20 2 279 15 2 268 15 2 272 15 2

5313 microgl 3408 2165 2275 2373

5314 microgkg 395 5 2875 11 3614 34 2905 18

5320 microgl 3267 368 1986 571 28 683 2427 512

5322 microgl 282 35 2 225 35 2 241 35 2 21 35 2

55

Figure 10 Youden plots of the percent deviation of the reported values from the assigned values for the food supplement test sample and the

standard solution in toluene with undisclosed content

(The identity of the respective measurand is given above the figure Results exceeding a difference of more than 100 were omitted)

56

Figure 10 - continued

57

Table 17 Analysis results reported by the participants for the four marker PAHs in the standard solution in acetonitrile with undisclosed

analyte content

Values are presented as reported U measurement uncertainty k coverage factor

benz[a]anthracene benzo[a]pyrene benzo[b]fluoranthene chrysene

Laboratory

code Unit Result U k Result U k Result U k Result U k

3602 microgkg 3675 247 247 244

3603 microgkg 358 086 2 236 028 2 235 028 2 236 057 2

3604 microgl 475 29 2 19 12 2 194 13 2 258 12 2

3608 microgl 3623 078 2 2385 023 2 2343 18 2 2296 092 2

3610 microgkg 369 185 2 25 125 2 251 126 2 244 122 2

3611 microgkg 48 02 2 323 02 2 328 04 2 317 03 2

3613 microgl 332 31 2 261 3 2 249 25 2 239 24 2

3615 microgl 415 2635 2729 2689

3617 microgkg 37 09 2 244 06 2 284 07 2 239 06 2

3622 microgl 37 12 2 25 23 2 257 21 2 238 1 2

3624 microgkg 402 104 2 255 084 2 265 096 2 253 07 2

5306 microgkg 563 113 2 371 74 2 359 72 2 333 67 2

5308 microgkg 168633 102669 232169 211488

5309 microgl 402 268 27

5310 microgl 315 158 25 125 25 125 288 144

5312 microgl 428 043 2 282 028 2 272 027 2 275 028 2

5315 microgkg 387 08 2 256 05 2 251 05 2 253 05 2

5316 microgl 3839 342 2 2485 191 2 2559 168 2 239 51 2

5317 microgl 2466 043 2 2212 034 2 2472 016 2 2146 068 2

5319 microgl 414 41 2 279 28 2 268 27 2 26 31 2

5321 microgkg 476 316 326 328

5322 microgl 2547 35 2

5323 microgkg 405 203 2 239 119 2 255 153 2 201 11 2

58

Figure 11 Youden plots of the percent deviation of the reported values from the assigned values for the food supplement test sample and the

standard solution in acetonitrile with undisclosed content

(The identity of the respective measurand is given above the figure Results exceeding a difference of more than 100 were omitted)

59

Figure 11 - continued

60

ANNEX 10 Table 18 Method performance parameters reported by the participants

Laboratory code

Analyte LOD LOQ Recovery Analyte LOD LOQ Recovery

microgkg microgkg microgkg microgkg 3600 BaA 03 06 978 BaP 03 07 9483602 BaA 007 021 926 BaP 005 015 993603 BaA 0011 05 987 BaP 0005 051 9553604 BaA 02 04 86 BaP 02 04 883605 BaA 0025 005 118 BaP 0025 005 1083606 BaA 001 003 65 BaP 001 003 703607 BaA 021 071 74 BaP 027 089 943608 BaA 016 048 8213 BaP 015 046 83683610 BaA 02 05 85 BaP 005 02 853611 BaA BaP 3612 BaA 1 117 BaP 1 1203613 BaA 01 03 90 BaP 01 03 903614 BaA 02 05 96 BaP 02 05 933615 BaA 005 015 110 BaP 005 015 1053616 BaA 01 05 108 BaP 01 05 753617 BaA 018 037 405 BaP 009 018 3873618 BaA 01 03 63 BaP 01 03 593619 BaA 005 005 59 BaP 033 033 543620 BaA 0009 0018 64 BaP 0005 001 553621 BaA 007 02 84 BaP 007 02 923622 BaA 01 03 1101 BaP 01 03 9853623 BaA 02 08 105 BaP 02 08 1013624 BaA BaP 01 033 8513625 BaA BaP 3626 BaA BaP 5301 BaA 003 01 102 BaP 003 01 1005302 BaA 01 03 78 BaP 01 03 865303 BaA 003 01 62 BaP 003 01 805304 BaA 1 1 70 BaP 1 1 955305 BaA 02 05 85 BaP 02 05 855306 BaA 03 1 699 BaP 03 1 905307 BaA 6537 BaP 81165308 BaA 0088 0265 99 BaP 00262 00785 1005309 BaA 01 08 92 BaP 01 08 925310 BaA 01 03 BaP 01 03 905311 BaA 002 005 80 BaP 002 004 915312 BaA 03 09 105 BaP 03 09 1055313 BaA 04 08 BaP 04 08 5314 BaA 001 002 96 BaP 001 002 9485315 BaA 01 03 50 - 100 BaP 01 03 50 - 100 5316 BaA 002 011 827 BaP 004 019 10445317 BaA 05 1 76 BaP 05 1 755318 BaA BaP 5319 BaA 009 029 951 BaP 011 041 995320 BaA 12 18 946 BaP 06 1 8345321 BaA 03 05 BaP 05 1 5322 BaA 1 2 100 BaP 1 2 1005323 BaA 26 BaP 26

61

ANNEX 10 Table 18 - continued

Laboratory code Analyte LOD LOQ Recovery Analyte LOD LOQ Recovery

microgkg microgkg microgkg microgkg 3600 BbF 03 05 96 CHR 02 03 10153602 BbF 015 045 947 CHR 003 009 9193603 BbF 0014 05 1012 CHR 0004 05 10033604 BbF 02 04 85 CHR 02 04 873605 BbF 005 01 94 CHR 0025 005 863606 BbF 001 003 70 CHR 001 003 653607 BbF 018 06 100 CHR 014 028 943608 BbF 015 046 7979 CHR 015 045 71253610 BbF 005 02 85 CHR 02 05 853611 BbF CHR 3612 BbF 1 118 CHR 1 1173613 BbF 01 03 90 CHR 01 03 903614 BbF 01 04 96 CHR 02 05 953615 BbF 008 025 109 CHR 003 01 1083616 BbF 01 05 78 CHR 01 05 723617 BbF 035 07 38 CHR 012 024 4163618 BbF 01 03 62 CHR 01 03 623619 BbF 016 016 63 CHR 01 01 593620 BbF 0003 0006 55 CHR 001 002 653621 BbF 007 02 103 CHR 007 02 893622 BbF 02 06 963 CHR 03 09 1043623 BbF 02 08 106 CHR 02 08 1193624 BbF CHR 3625 BbF CHR 3626 BbF CHR 5301 BbF 003 01 96 CHR 003 01 1145302 BbF 01 03 84 CHR 01 03 835303 BbF 003 01 78 CHR 003 01 705304 BbF 1 1 86 CHR 1 1 705305 BbF 02 05 90 CHR 02 05 955306 BbF 03 1 1435 CHR 03 1 1185307 BbF 8116 CHR 65375308 BbF 0079 0238 99 CHR 009 026 1005309 BbF 01 08 92 CHR 5310 BbF 07 2 CHR 01 03 5311 BbF 002 004 95 CHR 002 004 895312 BbF 03 09 105 CHR 03 09 1055313 BbF 04 08 CHR 04 08 5314 BbF 001 002 83 CHR 001 002 8185315 BbF 02 05 50 -100 CHR 02 05 50 - 100 5316 BbF 005 024 814 CHR 001 007 9595317 BbF 05 1 73 CHR 05 1 785318 BbF CHR 5319 BbF 011 038 946 CHR 013 043 9675320 BbF 07 09 1194 CHR 23 32 10455321 BbF 05 1 CHR 05 1 5322 BbF 1 2 100 CHR 1 2 1005323 BbF 26 CHR 26

62

ANNEX 11 Table 19 Details of analysis method reported by the participants Data are presented as reported

Laboratory code

Analysis technique

Instrument calibration

Internal standards applied

Sample clean up

Details of sample

clean up

Extraction technique

Details of sample

extraction

3600 6) GC-MS

2) Internal standardisation with labelled substances

Deuterated PAH mix 9 (Ehrenstorfer)

1) Saponification 2) LiquidLiquid partitioning

D) Other Liquidliquid partitioning

3602 2) HPLC-UV-FLD

1) External calibration

3) Gel permeation chromatography

A) Pressurised liquid extraction (PLE)

3603 1) HPLC-FLD

1) External calibration

3) Gel permeation chromatography

D) Other 1 minute with dichloromethane in vortex

3604 2) HPLC-UV-FLD

2) Internal standardisation with labelled substances

DiP D14

5) Donor acceptor complex chromatography

D) Other LL

3605 1) HPLC-FLD

1) External calibration

3) Gel permeation chromatography

C) Soxhlet extraction

3606 7) GC-MSMS

2) Internal standardisation with labelled substances

BaP 13C4 CHR 13C6 BaA 13C6 BbF 13C6

4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

3607 6) GC-MS

2) Internal standardisation with labelled substances

BaA-d12 CHR-d12 BbF-d12 BkF-d12 BaP-d12 ICP-d12 BgP-d12 DiP-d14

1) Saponification

D) Other solvent partitioning

3608 1) HPLC-FLD

1) External calibration

6) Other Liquid extraction

D) Other

Extraction in a rotary agitator and centrifugation

3610 2) HPLC-UV-FLD

1) External calibration

4) Solid phase extraction

B) Sonication

3611

3612 6) GC-MS

2) Internal standardisation with labelled substances

chrysene-d12 dibenzo(ah)anthracene-d14 dibenzo(ai)pyrene-d14

4) Solid phase extraction

B) Sonication

3613 5) LC-MSMS

3) Internal standardisation with unlabelled substances

I use Chrysene-D12 3) Gel permeation chromatography

D) Other extraction with hexan

63

Table 19 - continued

Laboratory code

Analysis technique

Instrument calibration

Internal standards applied

Sample clean up Details of sample

clean up

Extraction technique

Details of sample

extraction

3614 6) GC-MS 1) External calibration

3) Gel permeation chromatography

B) Sonication

3615 1) HPLC-FLD

1) External calibration

6) Other PLE GPC

A) Pressurised liquid extraction (PLE)

3616 6) GC-MS

2) Internal standardisation with labelled substances

Benzo(a)pyrene-13C4 Benzo(a)anthracene-13C6 Benzo(b)fluoranthene-13C6 Chrysene-13C6

2) LiquidLiquid partitioning 4) Solid phase extraction

B) Sonication

3617 1) HPLC-FLD

1) External calibration

4) Solid phase extraction

C) Soxhlet extraction

3618 6) GC-MS

2) Internal standardisation with labelled substances

deuterated standards

1) Saponification 4) Solid phase extraction

D) Other Handshaken with cyclohexane

3619 6) GC-MS

2) Internal standardisation with labelled substances

C13 Labelled US EPA 16

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

D) Other Homegenisation amp saponiofication

3620 9) GC-HRMS

2) Internal standardisation with labelled substances

mix of deuterated PAHs (BAA-D12 CHR-D12 BAP-D12 BBF-D12)

3) Gel permeation chromatography 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

3621 6) GC-MS

2) Internal standardisation with labelled substances

Chrysene D12 Benzo(a)pyrene D12 Benzo(b)fluoranthene D12 Benzo(a)anthracene

2) LiquidLiquid partitioning 4) Solid phase extraction

B) Sonication

3622 1) HPLC-FLD

4) Standard addition method

4) Solid phase extraction

D) Other

Extraction with cyclohexane by heating under reflux

3623 7) GC-MSMS

2) Internal standardisation with labelled substances

C13 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

3624 2) HPLC-UV-FLD

1) External calibration

2) LiquidLiquid partitioning

D) Other hot solvent extraction

3626

64

Table 19 - continued

Laboratory code

Analysis technique

Instrument calibration

Internal standards

applied

Sample clean up

Details of sample

clean up

Extraction technique

Details of sample

extraction

5301 7) GC-MSMS

2) Internal standardisation with labelled substances

EACH SUBSTANCE C13

4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5302 7) GC-MSMS

2) Internal standardisation with labelled substances

Isotope labelled PAHs 13C for each analyte

4) Solid phase extraction

C) Soxhlet extraction

5303 7) GC-MSMS

3) Internal standardisation with unlabelled substances

HAP C13 4) Solid phase extraction

D) Other automate ASE200 DIONEX

5304 7) GC-MSMS

2) Internal standardisation with labelled substances

with C13 for each HAP

2) LiquidLiquid partitioning 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5305 7) GC-MSMS

2) Internal standardisation with labelled substances

C13 isotopiques internal standards

4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5306 7) GC-MSMS

2) Internal standardisation with labelled substances

C13 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5307 6) GC-MS

2) Internal standardisation with labelled substances

1) Saponification 2) LiquidLiquid partitioning

D) Other solvent extraction

5308 1) HPLC-FLD 1) External calibration

4) Solid phase extraction

B) Sonication

5309 1) HPLC-FLD 1) External calibration

4) Solid phase extraction

D) Other only SPE

5310 1) HPLC-FLD 1) External calibration

1) Saponification 2) LiquidLiquid partitioning 3) Gel permeation chromatography

C) Soxhlet extraction

5311 6) GC-MS

2) Internal standardisation with labelled substances

13 C Chrysene 13 C Benz(a)pyrene

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

B) Sonication

5312 1) HPLC-FLD

3) Internal standardisation with unlabelled substances

Benzo(b)chrysen

2) LiquidLiquid partitioning 4) Solid phase extraction

Quechers D) Other Quechers

65

Table 19 - continued

Laboratory code

Analysis technique

Instrument calibration

Internal standards applied

Sample clean up Details of sample

clean up

Extraction technique

Details of sample extraction

5313 6) GC-MS

2) Internal standardisation with labelled substances

Benz(a)anthracene D12 Benzo(b)fluranthene D12 Benzo(a)pyrene D12 Chrysene D12

1) Saponification 2) LiquidLiquid partitioning 3) Gel permeation chromatography

A) Pressurised liquid extraction (PLE)

5314 6) GC-MS

2) Internal standardisation with labelled substances

Benzo(a)pyren d12 2) LiquidLiquid partitioning

D) Other Extraction with

5315 1) HPLC-FLD

3) Internal standardisation with unlabelled substances

Benz(b)chrysen

1) Saponification 2) LiquidLiquid partitioning

D) Other shaker

5316 1) HPLC-FLD

1) External calibration

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

D) Other

LiquidLiquid extraction with cyclohexane and dimethylformamide

5317 7) GC-MSMS

1) External calibration

3) Gel permeation chromatography 6) Other

silica gel D) Other VDLUFA online method

5318

5319 1) HPLC-FLD

3) Internal standardisation with unlabelled substances

Benzo(b)chrysene 3) Gel permeation chromatography

A) Pressurised liquid extraction (PLE)

5320 6) GC-MS

3) Internal standardisation with unlabelled substances

22-binaphthyl

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

D) Other 3 hours refluxing

5321 1) HPLC-FLD

2) Internal standardisation with labelled substances

6-meacutethylchrysegravene

1) Saponification 2) LiquidLiquid partitioning

B) Sonication

5322 6) GC-MS

2) Internal standardisation with labelled substances

naftalene-d8 acenaphtalene-d10 phenanthrene-d10 chrysene-d12 perylene-d12 benzo(e)anthracene-d12

1) Saponification 2) LiquidLiquid partitioning 6) Other

clean-up with silica packed column

C) Soxhlet extraction

5323 1) HPLC-FLD

1) External calibration

4) Solid phase extraction 6) Other

Accelerated Solvent Extraction

A) Pressurised liquid extraction (PLE)

66

European Commission EUR 25245 EN ndash Joint Research Centre ndash Institute for Reference Materials and Measurements Title Report on the 8th inter-laboratory comparison organised by the European Union Reference Laboratory for Polycyclic Aromatic Hydrocarbons - Four marker PAHs in a dry extract of St Johns wort Author(s) Radoslav Lizak Szilard Szilagyi Philippe Verlinde and Thomas WENZL Luxembourg Publications Office of the European Union 2012 ndash 66 pp ndash 201 x 297 cm EUR ndash Scientific and Technical Research series ndash ISSN 1831-9424 ISBN 978-92-79-23337-1 doi 10278759387 Abstract The proficiency test here reported concerned the determination of the contents of four marker polycyclic aromatic hydrocarbons (PAHs) and of their sum in an food supplements test sample The set of marker PAHs consists of benz[a]anthacene benzo[a]pyrene benzo[b]fluoranthene and chrysene Participants to these PT were National Reference Laboratories for PAHs (NRLs-PAHs) and EU official food control laboratories The number of participants was in total 48

The PT was organised according ISO Standard 170432010

The test material used was a commercial dry extract of St Johns wort Participants also received a solution of the same PAHs either in acetonitrile or in toluene for checking their instrument calibration

The results from participants were rated with z-scores and zeta-scores About 68 of the reported results were attributed with z-scores with an absolute value of below two which is the threshold for satisfactory performance

How to obtain EU publications

Our priced publications are available from EU Bookshop (httpbookshopeuropaeu) where you can place

an order with the sales agent of your choice

The Publications Office has a worldwide network of sales agents You can obtain their contact details by

sending a fax to (352) 29 29-42758

The mission of the JRC is to provide customer-driven scientific and technical support

for the conception development implementation and monitoring of EU policies As a

service of the European Commission the JRC functions as a reference centre of

science and technology for the Union Close to the policy-making process it serves

the common interest of the Member States while being independent of special

interests whether private or national

L

A-N

A-252

45-EN

-N

• 1 Summary
• 2 Introduction
• 3 Scope
• 4 Participating Laboratories
• 5 Time frame
• 6 Confidentiality
• 7 Test materials
• • 71 1Preparation of test materials
  • 72 Assigned values and standard deviation for proficiency assessment
  • 73 Homogeneity and stability
  • 74 Sample dispatch
  • • 8 Design of the proficiency test
    • 9 Evaluation of the results
    • • 91 2General
      • 92 21BEvaluation criteria
      • 93 Performance of participants
      • 94 Evaluation of the influence of analysis method
      • 95 Evaluation of the influence of calibration
      • 96 26Evaluation of compliance with legislation
      • • 10 Follow-up actions for underperforming laboratories
        • 11 1Conclusions
        • 12 1Acknowledgements
        • 13 References

55

Figure 10 Youden plots of the percent deviation of the reported values from the assigned values for the food supplement test sample and the

standard solution in toluene with undisclosed content

(The identity of the respective measurand is given above the figure Results exceeding a difference of more than 100 were omitted)

56

Figure 10 - continued

57

Table 17 Analysis results reported by the participants for the four marker PAHs in the standard solution in acetonitrile with undisclosed

analyte content

Values are presented as reported U measurement uncertainty k coverage factor

benz[a]anthracene benzo[a]pyrene benzo[b]fluoranthene chrysene

Laboratory

code Unit Result U k Result U k Result U k Result U k

3602 microgkg 3675 247 247 244

3603 microgkg 358 086 2 236 028 2 235 028 2 236 057 2

3604 microgl 475 29 2 19 12 2 194 13 2 258 12 2

3608 microgl 3623 078 2 2385 023 2 2343 18 2 2296 092 2

3610 microgkg 369 185 2 25 125 2 251 126 2 244 122 2

3611 microgkg 48 02 2 323 02 2 328 04 2 317 03 2

3613 microgl 332 31 2 261 3 2 249 25 2 239 24 2

3615 microgl 415 2635 2729 2689

3617 microgkg 37 09 2 244 06 2 284 07 2 239 06 2

3622 microgl 37 12 2 25 23 2 257 21 2 238 1 2

3624 microgkg 402 104 2 255 084 2 265 096 2 253 07 2

5306 microgkg 563 113 2 371 74 2 359 72 2 333 67 2

5308 microgkg 168633 102669 232169 211488

5309 microgl 402 268 27

5310 microgl 315 158 25 125 25 125 288 144

5312 microgl 428 043 2 282 028 2 272 027 2 275 028 2

5315 microgkg 387 08 2 256 05 2 251 05 2 253 05 2

5316 microgl 3839 342 2 2485 191 2 2559 168 2 239 51 2

5317 microgl 2466 043 2 2212 034 2 2472 016 2 2146 068 2

5319 microgl 414 41 2 279 28 2 268 27 2 26 31 2

5321 microgkg 476 316 326 328

5322 microgl 2547 35 2

5323 microgkg 405 203 2 239 119 2 255 153 2 201 11 2

58

Figure 11 Youden plots of the percent deviation of the reported values from the assigned values for the food supplement test sample and the

standard solution in acetonitrile with undisclosed content

(The identity of the respective measurand is given above the figure Results exceeding a difference of more than 100 were omitted)

59

Figure 11 - continued

60

ANNEX 10 Table 18 Method performance parameters reported by the participants

Laboratory code

Analyte LOD LOQ Recovery Analyte LOD LOQ Recovery

microgkg microgkg microgkg microgkg 3600 BaA 03 06 978 BaP 03 07 9483602 BaA 007 021 926 BaP 005 015 993603 BaA 0011 05 987 BaP 0005 051 9553604 BaA 02 04 86 BaP 02 04 883605 BaA 0025 005 118 BaP 0025 005 1083606 BaA 001 003 65 BaP 001 003 703607 BaA 021 071 74 BaP 027 089 943608 BaA 016 048 8213 BaP 015 046 83683610 BaA 02 05 85 BaP 005 02 853611 BaA BaP 3612 BaA 1 117 BaP 1 1203613 BaA 01 03 90 BaP 01 03 903614 BaA 02 05 96 BaP 02 05 933615 BaA 005 015 110 BaP 005 015 1053616 BaA 01 05 108 BaP 01 05 753617 BaA 018 037 405 BaP 009 018 3873618 BaA 01 03 63 BaP 01 03 593619 BaA 005 005 59 BaP 033 033 543620 BaA 0009 0018 64 BaP 0005 001 553621 BaA 007 02 84 BaP 007 02 923622 BaA 01 03 1101 BaP 01 03 9853623 BaA 02 08 105 BaP 02 08 1013624 BaA BaP 01 033 8513625 BaA BaP 3626 BaA BaP 5301 BaA 003 01 102 BaP 003 01 1005302 BaA 01 03 78 BaP 01 03 865303 BaA 003 01 62 BaP 003 01 805304 BaA 1 1 70 BaP 1 1 955305 BaA 02 05 85 BaP 02 05 855306 BaA 03 1 699 BaP 03 1 905307 BaA 6537 BaP 81165308 BaA 0088 0265 99 BaP 00262 00785 1005309 BaA 01 08 92 BaP 01 08 925310 BaA 01 03 BaP 01 03 905311 BaA 002 005 80 BaP 002 004 915312 BaA 03 09 105 BaP 03 09 1055313 BaA 04 08 BaP 04 08 5314 BaA 001 002 96 BaP 001 002 9485315 BaA 01 03 50 - 100 BaP 01 03 50 - 100 5316 BaA 002 011 827 BaP 004 019 10445317 BaA 05 1 76 BaP 05 1 755318 BaA BaP 5319 BaA 009 029 951 BaP 011 041 995320 BaA 12 18 946 BaP 06 1 8345321 BaA 03 05 BaP 05 1 5322 BaA 1 2 100 BaP 1 2 1005323 BaA 26 BaP 26

61

ANNEX 10 Table 18 - continued

Laboratory code Analyte LOD LOQ Recovery Analyte LOD LOQ Recovery

microgkg microgkg microgkg microgkg 3600 BbF 03 05 96 CHR 02 03 10153602 BbF 015 045 947 CHR 003 009 9193603 BbF 0014 05 1012 CHR 0004 05 10033604 BbF 02 04 85 CHR 02 04 873605 BbF 005 01 94 CHR 0025 005 863606 BbF 001 003 70 CHR 001 003 653607 BbF 018 06 100 CHR 014 028 943608 BbF 015 046 7979 CHR 015 045 71253610 BbF 005 02 85 CHR 02 05 853611 BbF CHR 3612 BbF 1 118 CHR 1 1173613 BbF 01 03 90 CHR 01 03 903614 BbF 01 04 96 CHR 02 05 953615 BbF 008 025 109 CHR 003 01 1083616 BbF 01 05 78 CHR 01 05 723617 BbF 035 07 38 CHR 012 024 4163618 BbF 01 03 62 CHR 01 03 623619 BbF 016 016 63 CHR 01 01 593620 BbF 0003 0006 55 CHR 001 002 653621 BbF 007 02 103 CHR 007 02 893622 BbF 02 06 963 CHR 03 09 1043623 BbF 02 08 106 CHR 02 08 1193624 BbF CHR 3625 BbF CHR 3626 BbF CHR 5301 BbF 003 01 96 CHR 003 01 1145302 BbF 01 03 84 CHR 01 03 835303 BbF 003 01 78 CHR 003 01 705304 BbF 1 1 86 CHR 1 1 705305 BbF 02 05 90 CHR 02 05 955306 BbF 03 1 1435 CHR 03 1 1185307 BbF 8116 CHR 65375308 BbF 0079 0238 99 CHR 009 026 1005309 BbF 01 08 92 CHR 5310 BbF 07 2 CHR 01 03 5311 BbF 002 004 95 CHR 002 004 895312 BbF 03 09 105 CHR 03 09 1055313 BbF 04 08 CHR 04 08 5314 BbF 001 002 83 CHR 001 002 8185315 BbF 02 05 50 -100 CHR 02 05 50 - 100 5316 BbF 005 024 814 CHR 001 007 9595317 BbF 05 1 73 CHR 05 1 785318 BbF CHR 5319 BbF 011 038 946 CHR 013 043 9675320 BbF 07 09 1194 CHR 23 32 10455321 BbF 05 1 CHR 05 1 5322 BbF 1 2 100 CHR 1 2 1005323 BbF 26 CHR 26

62

ANNEX 11 Table 19 Details of analysis method reported by the participants Data are presented as reported

Laboratory code

Analysis technique

Instrument calibration

Internal standards applied

Sample clean up

Details of sample

clean up

Extraction technique

Details of sample

extraction

3600 6) GC-MS

2) Internal standardisation with labelled substances

Deuterated PAH mix 9 (Ehrenstorfer)

1) Saponification 2) LiquidLiquid partitioning

D) Other Liquidliquid partitioning

3602 2) HPLC-UV-FLD

1) External calibration

3) Gel permeation chromatography

A) Pressurised liquid extraction (PLE)

3603 1) HPLC-FLD

1) External calibration

3) Gel permeation chromatography

D) Other 1 minute with dichloromethane in vortex

3604 2) HPLC-UV-FLD

2) Internal standardisation with labelled substances

DiP D14

5) Donor acceptor complex chromatography

D) Other LL

3605 1) HPLC-FLD

1) External calibration

3) Gel permeation chromatography

C) Soxhlet extraction

3606 7) GC-MSMS

2) Internal standardisation with labelled substances

BaP 13C4 CHR 13C6 BaA 13C6 BbF 13C6

4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

3607 6) GC-MS

2) Internal standardisation with labelled substances

BaA-d12 CHR-d12 BbF-d12 BkF-d12 BaP-d12 ICP-d12 BgP-d12 DiP-d14

1) Saponification

D) Other solvent partitioning

3608 1) HPLC-FLD

1) External calibration

6) Other Liquid extraction

D) Other

Extraction in a rotary agitator and centrifugation

3610 2) HPLC-UV-FLD

1) External calibration

4) Solid phase extraction

B) Sonication

3611

3612 6) GC-MS

2) Internal standardisation with labelled substances

chrysene-d12 dibenzo(ah)anthracene-d14 dibenzo(ai)pyrene-d14

4) Solid phase extraction

B) Sonication

3613 5) LC-MSMS

3) Internal standardisation with unlabelled substances

I use Chrysene-D12 3) Gel permeation chromatography

D) Other extraction with hexan

63

Table 19 - continued

Laboratory code

Analysis technique

Instrument calibration

Internal standards applied

Sample clean up Details of sample

clean up

Extraction technique

Details of sample

extraction

3614 6) GC-MS 1) External calibration

3) Gel permeation chromatography

B) Sonication

3615 1) HPLC-FLD

1) External calibration

6) Other PLE GPC

A) Pressurised liquid extraction (PLE)

3616 6) GC-MS

2) Internal standardisation with labelled substances

Benzo(a)pyrene-13C4 Benzo(a)anthracene-13C6 Benzo(b)fluoranthene-13C6 Chrysene-13C6

2) LiquidLiquid partitioning 4) Solid phase extraction

B) Sonication

3617 1) HPLC-FLD

1) External calibration

4) Solid phase extraction

C) Soxhlet extraction

3618 6) GC-MS

2) Internal standardisation with labelled substances

deuterated standards

1) Saponification 4) Solid phase extraction

D) Other Handshaken with cyclohexane

3619 6) GC-MS

2) Internal standardisation with labelled substances

C13 Labelled US EPA 16

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

D) Other Homegenisation amp saponiofication

3620 9) GC-HRMS

2) Internal standardisation with labelled substances

mix of deuterated PAHs (BAA-D12 CHR-D12 BAP-D12 BBF-D12)

3) Gel permeation chromatography 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

3621 6) GC-MS

2) Internal standardisation with labelled substances

Chrysene D12 Benzo(a)pyrene D12 Benzo(b)fluoranthene D12 Benzo(a)anthracene

2) LiquidLiquid partitioning 4) Solid phase extraction

B) Sonication

3622 1) HPLC-FLD

4) Standard addition method

4) Solid phase extraction

D) Other

Extraction with cyclohexane by heating under reflux

3623 7) GC-MSMS

2) Internal standardisation with labelled substances

C13 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

3624 2) HPLC-UV-FLD

1) External calibration

2) LiquidLiquid partitioning

D) Other hot solvent extraction

3626

64

Table 19 - continued

Laboratory code

Analysis technique

Instrument calibration

Internal standards

applied

Sample clean up

Details of sample

clean up

Extraction technique

Details of sample

extraction

5301 7) GC-MSMS

2) Internal standardisation with labelled substances

EACH SUBSTANCE C13

4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5302 7) GC-MSMS

2) Internal standardisation with labelled substances

Isotope labelled PAHs 13C for each analyte

4) Solid phase extraction

C) Soxhlet extraction

5303 7) GC-MSMS

3) Internal standardisation with unlabelled substances

HAP C13 4) Solid phase extraction

D) Other automate ASE200 DIONEX

5304 7) GC-MSMS

2) Internal standardisation with labelled substances

with C13 for each HAP

2) LiquidLiquid partitioning 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5305 7) GC-MSMS

2) Internal standardisation with labelled substances

C13 isotopiques internal standards

4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5306 7) GC-MSMS

2) Internal standardisation with labelled substances

C13 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5307 6) GC-MS

2) Internal standardisation with labelled substances

1) Saponification 2) LiquidLiquid partitioning

D) Other solvent extraction

5308 1) HPLC-FLD 1) External calibration

4) Solid phase extraction

B) Sonication

5309 1) HPLC-FLD 1) External calibration

4) Solid phase extraction

D) Other only SPE

5310 1) HPLC-FLD 1) External calibration

1) Saponification 2) LiquidLiquid partitioning 3) Gel permeation chromatography

C) Soxhlet extraction

5311 6) GC-MS

2) Internal standardisation with labelled substances

13 C Chrysene 13 C Benz(a)pyrene

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

B) Sonication

5312 1) HPLC-FLD

3) Internal standardisation with unlabelled substances

Benzo(b)chrysen

2) LiquidLiquid partitioning 4) Solid phase extraction

Quechers D) Other Quechers

65

Table 19 - continued

Laboratory code

Analysis technique

Instrument calibration

Internal standards applied

Sample clean up Details of sample

clean up

Extraction technique

Details of sample extraction

5313 6) GC-MS

2) Internal standardisation with labelled substances

Benz(a)anthracene D12 Benzo(b)fluranthene D12 Benzo(a)pyrene D12 Chrysene D12

1) Saponification 2) LiquidLiquid partitioning 3) Gel permeation chromatography

A) Pressurised liquid extraction (PLE)

5314 6) GC-MS

2) Internal standardisation with labelled substances

Benzo(a)pyren d12 2) LiquidLiquid partitioning

D) Other Extraction with

5315 1) HPLC-FLD

3) Internal standardisation with unlabelled substances

Benz(b)chrysen

1) Saponification 2) LiquidLiquid partitioning

D) Other shaker

5316 1) HPLC-FLD

1) External calibration

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

D) Other

LiquidLiquid extraction with cyclohexane and dimethylformamide

5317 7) GC-MSMS

1) External calibration

3) Gel permeation chromatography 6) Other

silica gel D) Other VDLUFA online method

5318

5319 1) HPLC-FLD

3) Internal standardisation with unlabelled substances

Benzo(b)chrysene 3) Gel permeation chromatography

A) Pressurised liquid extraction (PLE)

5320 6) GC-MS

3) Internal standardisation with unlabelled substances

22-binaphthyl

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

D) Other 3 hours refluxing

5321 1) HPLC-FLD

2) Internal standardisation with labelled substances

6-meacutethylchrysegravene

1) Saponification 2) LiquidLiquid partitioning

B) Sonication

5322 6) GC-MS

2) Internal standardisation with labelled substances

naftalene-d8 acenaphtalene-d10 phenanthrene-d10 chrysene-d12 perylene-d12 benzo(e)anthracene-d12

1) Saponification 2) LiquidLiquid partitioning 6) Other

clean-up with silica packed column

C) Soxhlet extraction

5323 1) HPLC-FLD

1) External calibration

4) Solid phase extraction 6) Other

Accelerated Solvent Extraction

A) Pressurised liquid extraction (PLE)

66

European Commission EUR 25245 EN ndash Joint Research Centre ndash Institute for Reference Materials and Measurements Title Report on the 8th inter-laboratory comparison organised by the European Union Reference Laboratory for Polycyclic Aromatic Hydrocarbons - Four marker PAHs in a dry extract of St Johns wort Author(s) Radoslav Lizak Szilard Szilagyi Philippe Verlinde and Thomas WENZL Luxembourg Publications Office of the European Union 2012 ndash 66 pp ndash 201 x 297 cm EUR ndash Scientific and Technical Research series ndash ISSN 1831-9424 ISBN 978-92-79-23337-1 doi 10278759387 Abstract The proficiency test here reported concerned the determination of the contents of four marker polycyclic aromatic hydrocarbons (PAHs) and of their sum in an food supplements test sample The set of marker PAHs consists of benz[a]anthacene benzo[a]pyrene benzo[b]fluoranthene and chrysene Participants to these PT were National Reference Laboratories for PAHs (NRLs-PAHs) and EU official food control laboratories The number of participants was in total 48

The PT was organised according ISO Standard 170432010

The test material used was a commercial dry extract of St Johns wort Participants also received a solution of the same PAHs either in acetonitrile or in toluene for checking their instrument calibration

The results from participants were rated with z-scores and zeta-scores About 68 of the reported results were attributed with z-scores with an absolute value of below two which is the threshold for satisfactory performance

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science and technology for the Union Close to the policy-making process it serves

the common interest of the Member States while being independent of special

interests whether private or national

L

A-N

A-252

45-EN

-N

• 1 Summary
• 2 Introduction
• 3 Scope
• 4 Participating Laboratories
• 5 Time frame
• 6 Confidentiality
• 7 Test materials
• • 71 1Preparation of test materials
  • 72 Assigned values and standard deviation for proficiency assessment
  • 73 Homogeneity and stability
  • 74 Sample dispatch
  • • 8 Design of the proficiency test
    • 9 Evaluation of the results
    • • 91 2General
      • 92 21BEvaluation criteria
      • 93 Performance of participants
      • 94 Evaluation of the influence of analysis method
      • 95 Evaluation of the influence of calibration
      • 96 26Evaluation of compliance with legislation
      • • 10 Follow-up actions for underperforming laboratories
        • 11 1Conclusions
        • 12 1Acknowledgements
        • 13 References

56

Figure 10 - continued

57

Table 17 Analysis results reported by the participants for the four marker PAHs in the standard solution in acetonitrile with undisclosed

analyte content

Values are presented as reported U measurement uncertainty k coverage factor

benz[a]anthracene benzo[a]pyrene benzo[b]fluoranthene chrysene

Laboratory

code Unit Result U k Result U k Result U k Result U k

3602 microgkg 3675 247 247 244

3603 microgkg 358 086 2 236 028 2 235 028 2 236 057 2

3604 microgl 475 29 2 19 12 2 194 13 2 258 12 2

3608 microgl 3623 078 2 2385 023 2 2343 18 2 2296 092 2

3610 microgkg 369 185 2 25 125 2 251 126 2 244 122 2

3611 microgkg 48 02 2 323 02 2 328 04 2 317 03 2

3613 microgl 332 31 2 261 3 2 249 25 2 239 24 2

3615 microgl 415 2635 2729 2689

3617 microgkg 37 09 2 244 06 2 284 07 2 239 06 2

3622 microgl 37 12 2 25 23 2 257 21 2 238 1 2

3624 microgkg 402 104 2 255 084 2 265 096 2 253 07 2

5306 microgkg 563 113 2 371 74 2 359 72 2 333 67 2

5308 microgkg 168633 102669 232169 211488

5309 microgl 402 268 27

5310 microgl 315 158 25 125 25 125 288 144

5312 microgl 428 043 2 282 028 2 272 027 2 275 028 2

5315 microgkg 387 08 2 256 05 2 251 05 2 253 05 2

5316 microgl 3839 342 2 2485 191 2 2559 168 2 239 51 2

5317 microgl 2466 043 2 2212 034 2 2472 016 2 2146 068 2

5319 microgl 414 41 2 279 28 2 268 27 2 26 31 2

5321 microgkg 476 316 326 328

5322 microgl 2547 35 2

5323 microgkg 405 203 2 239 119 2 255 153 2 201 11 2

58

Figure 11 Youden plots of the percent deviation of the reported values from the assigned values for the food supplement test sample and the

standard solution in acetonitrile with undisclosed content

(The identity of the respective measurand is given above the figure Results exceeding a difference of more than 100 were omitted)

59

Figure 11 - continued

60

ANNEX 10 Table 18 Method performance parameters reported by the participants

Laboratory code

Analyte LOD LOQ Recovery Analyte LOD LOQ Recovery

microgkg microgkg microgkg microgkg 3600 BaA 03 06 978 BaP 03 07 9483602 BaA 007 021 926 BaP 005 015 993603 BaA 0011 05 987 BaP 0005 051 9553604 BaA 02 04 86 BaP 02 04 883605 BaA 0025 005 118 BaP 0025 005 1083606 BaA 001 003 65 BaP 001 003 703607 BaA 021 071 74 BaP 027 089 943608 BaA 016 048 8213 BaP 015 046 83683610 BaA 02 05 85 BaP 005 02 853611 BaA BaP 3612 BaA 1 117 BaP 1 1203613 BaA 01 03 90 BaP 01 03 903614 BaA 02 05 96 BaP 02 05 933615 BaA 005 015 110 BaP 005 015 1053616 BaA 01 05 108 BaP 01 05 753617 BaA 018 037 405 BaP 009 018 3873618 BaA 01 03 63 BaP 01 03 593619 BaA 005 005 59 BaP 033 033 543620 BaA 0009 0018 64 BaP 0005 001 553621 BaA 007 02 84 BaP 007 02 923622 BaA 01 03 1101 BaP 01 03 9853623 BaA 02 08 105 BaP 02 08 1013624 BaA BaP 01 033 8513625 BaA BaP 3626 BaA BaP 5301 BaA 003 01 102 BaP 003 01 1005302 BaA 01 03 78 BaP 01 03 865303 BaA 003 01 62 BaP 003 01 805304 BaA 1 1 70 BaP 1 1 955305 BaA 02 05 85 BaP 02 05 855306 BaA 03 1 699 BaP 03 1 905307 BaA 6537 BaP 81165308 BaA 0088 0265 99 BaP 00262 00785 1005309 BaA 01 08 92 BaP 01 08 925310 BaA 01 03 BaP 01 03 905311 BaA 002 005 80 BaP 002 004 915312 BaA 03 09 105 BaP 03 09 1055313 BaA 04 08 BaP 04 08 5314 BaA 001 002 96 BaP 001 002 9485315 BaA 01 03 50 - 100 BaP 01 03 50 - 100 5316 BaA 002 011 827 BaP 004 019 10445317 BaA 05 1 76 BaP 05 1 755318 BaA BaP 5319 BaA 009 029 951 BaP 011 041 995320 BaA 12 18 946 BaP 06 1 8345321 BaA 03 05 BaP 05 1 5322 BaA 1 2 100 BaP 1 2 1005323 BaA 26 BaP 26

61

ANNEX 10 Table 18 - continued

Laboratory code Analyte LOD LOQ Recovery Analyte LOD LOQ Recovery

microgkg microgkg microgkg microgkg 3600 BbF 03 05 96 CHR 02 03 10153602 BbF 015 045 947 CHR 003 009 9193603 BbF 0014 05 1012 CHR 0004 05 10033604 BbF 02 04 85 CHR 02 04 873605 BbF 005 01 94 CHR 0025 005 863606 BbF 001 003 70 CHR 001 003 653607 BbF 018 06 100 CHR 014 028 943608 BbF 015 046 7979 CHR 015 045 71253610 BbF 005 02 85 CHR 02 05 853611 BbF CHR 3612 BbF 1 118 CHR 1 1173613 BbF 01 03 90 CHR 01 03 903614 BbF 01 04 96 CHR 02 05 953615 BbF 008 025 109 CHR 003 01 1083616 BbF 01 05 78 CHR 01 05 723617 BbF 035 07 38 CHR 012 024 4163618 BbF 01 03 62 CHR 01 03 623619 BbF 016 016 63 CHR 01 01 593620 BbF 0003 0006 55 CHR 001 002 653621 BbF 007 02 103 CHR 007 02 893622 BbF 02 06 963 CHR 03 09 1043623 BbF 02 08 106 CHR 02 08 1193624 BbF CHR 3625 BbF CHR 3626 BbF CHR 5301 BbF 003 01 96 CHR 003 01 1145302 BbF 01 03 84 CHR 01 03 835303 BbF 003 01 78 CHR 003 01 705304 BbF 1 1 86 CHR 1 1 705305 BbF 02 05 90 CHR 02 05 955306 BbF 03 1 1435 CHR 03 1 1185307 BbF 8116 CHR 65375308 BbF 0079 0238 99 CHR 009 026 1005309 BbF 01 08 92 CHR 5310 BbF 07 2 CHR 01 03 5311 BbF 002 004 95 CHR 002 004 895312 BbF 03 09 105 CHR 03 09 1055313 BbF 04 08 CHR 04 08 5314 BbF 001 002 83 CHR 001 002 8185315 BbF 02 05 50 -100 CHR 02 05 50 - 100 5316 BbF 005 024 814 CHR 001 007 9595317 BbF 05 1 73 CHR 05 1 785318 BbF CHR 5319 BbF 011 038 946 CHR 013 043 9675320 BbF 07 09 1194 CHR 23 32 10455321 BbF 05 1 CHR 05 1 5322 BbF 1 2 100 CHR 1 2 1005323 BbF 26 CHR 26

62

ANNEX 11 Table 19 Details of analysis method reported by the participants Data are presented as reported

Laboratory code

Analysis technique

Instrument calibration

Internal standards applied

Sample clean up

Details of sample

clean up

Extraction technique

Details of sample

extraction

3600 6) GC-MS

2) Internal standardisation with labelled substances

Deuterated PAH mix 9 (Ehrenstorfer)

1) Saponification 2) LiquidLiquid partitioning

D) Other Liquidliquid partitioning

3602 2) HPLC-UV-FLD

1) External calibration

3) Gel permeation chromatography

A) Pressurised liquid extraction (PLE)

3603 1) HPLC-FLD

1) External calibration

3) Gel permeation chromatography

D) Other 1 minute with dichloromethane in vortex

3604 2) HPLC-UV-FLD

2) Internal standardisation with labelled substances

DiP D14

5) Donor acceptor complex chromatography

D) Other LL

3605 1) HPLC-FLD

1) External calibration

3) Gel permeation chromatography

C) Soxhlet extraction

3606 7) GC-MSMS

2) Internal standardisation with labelled substances

BaP 13C4 CHR 13C6 BaA 13C6 BbF 13C6

4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

3607 6) GC-MS

2) Internal standardisation with labelled substances

BaA-d12 CHR-d12 BbF-d12 BkF-d12 BaP-d12 ICP-d12 BgP-d12 DiP-d14

1) Saponification

D) Other solvent partitioning

3608 1) HPLC-FLD

1) External calibration

6) Other Liquid extraction

D) Other

Extraction in a rotary agitator and centrifugation

3610 2) HPLC-UV-FLD

1) External calibration

4) Solid phase extraction

B) Sonication

3611

3612 6) GC-MS

2) Internal standardisation with labelled substances

chrysene-d12 dibenzo(ah)anthracene-d14 dibenzo(ai)pyrene-d14

4) Solid phase extraction

B) Sonication

3613 5) LC-MSMS

3) Internal standardisation with unlabelled substances

I use Chrysene-D12 3) Gel permeation chromatography

D) Other extraction with hexan

63

Table 19 - continued

Laboratory code

Analysis technique

Instrument calibration

Internal standards applied

Sample clean up Details of sample

clean up

Extraction technique

Details of sample

extraction

3614 6) GC-MS 1) External calibration

3) Gel permeation chromatography

B) Sonication

3615 1) HPLC-FLD

1) External calibration

6) Other PLE GPC

A) Pressurised liquid extraction (PLE)

3616 6) GC-MS

2) Internal standardisation with labelled substances

Benzo(a)pyrene-13C4 Benzo(a)anthracene-13C6 Benzo(b)fluoranthene-13C6 Chrysene-13C6

2) LiquidLiquid partitioning 4) Solid phase extraction

B) Sonication

3617 1) HPLC-FLD

1) External calibration

4) Solid phase extraction

C) Soxhlet extraction

3618 6) GC-MS

2) Internal standardisation with labelled substances

deuterated standards

1) Saponification 4) Solid phase extraction

D) Other Handshaken with cyclohexane

3619 6) GC-MS

2) Internal standardisation with labelled substances

C13 Labelled US EPA 16

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

D) Other Homegenisation amp saponiofication

3620 9) GC-HRMS

2) Internal standardisation with labelled substances

mix of deuterated PAHs (BAA-D12 CHR-D12 BAP-D12 BBF-D12)

3) Gel permeation chromatography 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

3621 6) GC-MS

2) Internal standardisation with labelled substances

Chrysene D12 Benzo(a)pyrene D12 Benzo(b)fluoranthene D12 Benzo(a)anthracene

2) LiquidLiquid partitioning 4) Solid phase extraction

B) Sonication

3622 1) HPLC-FLD

4) Standard addition method

4) Solid phase extraction

D) Other

Extraction with cyclohexane by heating under reflux

3623 7) GC-MSMS

2) Internal standardisation with labelled substances

C13 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

3624 2) HPLC-UV-FLD

1) External calibration

2) LiquidLiquid partitioning

D) Other hot solvent extraction

3626

64

Table 19 - continued

Laboratory code

Analysis technique

Instrument calibration

Internal standards

applied

Sample clean up

Details of sample

clean up

Extraction technique

Details of sample

extraction

5301 7) GC-MSMS

2) Internal standardisation with labelled substances

EACH SUBSTANCE C13

4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5302 7) GC-MSMS

2) Internal standardisation with labelled substances

Isotope labelled PAHs 13C for each analyte

4) Solid phase extraction

C) Soxhlet extraction

5303 7) GC-MSMS

3) Internal standardisation with unlabelled substances

HAP C13 4) Solid phase extraction

D) Other automate ASE200 DIONEX

5304 7) GC-MSMS

2) Internal standardisation with labelled substances

with C13 for each HAP

2) LiquidLiquid partitioning 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5305 7) GC-MSMS

2) Internal standardisation with labelled substances

C13 isotopiques internal standards

4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5306 7) GC-MSMS

2) Internal standardisation with labelled substances

C13 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5307 6) GC-MS

2) Internal standardisation with labelled substances

1) Saponification 2) LiquidLiquid partitioning

D) Other solvent extraction

5308 1) HPLC-FLD 1) External calibration

4) Solid phase extraction

B) Sonication

5309 1) HPLC-FLD 1) External calibration

4) Solid phase extraction

D) Other only SPE

5310 1) HPLC-FLD 1) External calibration

1) Saponification 2) LiquidLiquid partitioning 3) Gel permeation chromatography

C) Soxhlet extraction

5311 6) GC-MS

2) Internal standardisation with labelled substances

13 C Chrysene 13 C Benz(a)pyrene

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

B) Sonication

5312 1) HPLC-FLD

3) Internal standardisation with unlabelled substances

Benzo(b)chrysen

2) LiquidLiquid partitioning 4) Solid phase extraction

Quechers D) Other Quechers

65

Table 19 - continued

Laboratory code

Analysis technique

Instrument calibration

Internal standards applied

Sample clean up Details of sample

clean up

Extraction technique

Details of sample extraction

5313 6) GC-MS

2) Internal standardisation with labelled substances

Benz(a)anthracene D12 Benzo(b)fluranthene D12 Benzo(a)pyrene D12 Chrysene D12

1) Saponification 2) LiquidLiquid partitioning 3) Gel permeation chromatography

A) Pressurised liquid extraction (PLE)

5314 6) GC-MS

2) Internal standardisation with labelled substances

Benzo(a)pyren d12 2) LiquidLiquid partitioning

D) Other Extraction with

5315 1) HPLC-FLD

3) Internal standardisation with unlabelled substances

Benz(b)chrysen

1) Saponification 2) LiquidLiquid partitioning

D) Other shaker

5316 1) HPLC-FLD

1) External calibration

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

D) Other

LiquidLiquid extraction with cyclohexane and dimethylformamide

5317 7) GC-MSMS

1) External calibration

3) Gel permeation chromatography 6) Other

silica gel D) Other VDLUFA online method

5318

5319 1) HPLC-FLD

3) Internal standardisation with unlabelled substances

Benzo(b)chrysene 3) Gel permeation chromatography

A) Pressurised liquid extraction (PLE)

5320 6) GC-MS

3) Internal standardisation with unlabelled substances

22-binaphthyl

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

D) Other 3 hours refluxing

5321 1) HPLC-FLD

2) Internal standardisation with labelled substances

6-meacutethylchrysegravene

1) Saponification 2) LiquidLiquid partitioning

B) Sonication

5322 6) GC-MS

2) Internal standardisation with labelled substances

naftalene-d8 acenaphtalene-d10 phenanthrene-d10 chrysene-d12 perylene-d12 benzo(e)anthracene-d12

1) Saponification 2) LiquidLiquid partitioning 6) Other

clean-up with silica packed column

C) Soxhlet extraction

5323 1) HPLC-FLD

1) External calibration

4) Solid phase extraction 6) Other

Accelerated Solvent Extraction

A) Pressurised liquid extraction (PLE)

66

European Commission EUR 25245 EN ndash Joint Research Centre ndash Institute for Reference Materials and Measurements Title Report on the 8th inter-laboratory comparison organised by the European Union Reference Laboratory for Polycyclic Aromatic Hydrocarbons - Four marker PAHs in a dry extract of St Johns wort Author(s) Radoslav Lizak Szilard Szilagyi Philippe Verlinde and Thomas WENZL Luxembourg Publications Office of the European Union 2012 ndash 66 pp ndash 201 x 297 cm EUR ndash Scientific and Technical Research series ndash ISSN 1831-9424 ISBN 978-92-79-23337-1 doi 10278759387 Abstract The proficiency test here reported concerned the determination of the contents of four marker polycyclic aromatic hydrocarbons (PAHs) and of their sum in an food supplements test sample The set of marker PAHs consists of benz[a]anthacene benzo[a]pyrene benzo[b]fluoranthene and chrysene Participants to these PT were National Reference Laboratories for PAHs (NRLs-PAHs) and EU official food control laboratories The number of participants was in total 48

The PT was organised according ISO Standard 170432010

The test material used was a commercial dry extract of St Johns wort Participants also received a solution of the same PAHs either in acetonitrile or in toluene for checking their instrument calibration

The results from participants were rated with z-scores and zeta-scores About 68 of the reported results were attributed with z-scores with an absolute value of below two which is the threshold for satisfactory performance

How to obtain EU publications

Our priced publications are available from EU Bookshop (httpbookshopeuropaeu) where you can place

an order with the sales agent of your choice

The Publications Office has a worldwide network of sales agents You can obtain their contact details by

sending a fax to (352) 29 29-42758

The mission of the JRC is to provide customer-driven scientific and technical support

for the conception development implementation and monitoring of EU policies As a

service of the European Commission the JRC functions as a reference centre of

science and technology for the Union Close to the policy-making process it serves

the common interest of the Member States while being independent of special

interests whether private or national

L

A-N

A-252

45-EN

-N

• 1 Summary
• 2 Introduction
• 3 Scope
• 4 Participating Laboratories
• 5 Time frame
• 6 Confidentiality
• 7 Test materials
• • 71 1Preparation of test materials
  • 72 Assigned values and standard deviation for proficiency assessment
  • 73 Homogeneity and stability
  • 74 Sample dispatch
  • • 8 Design of the proficiency test
    • 9 Evaluation of the results
    • • 91 2General
      • 92 21BEvaluation criteria
      • 93 Performance of participants
      • 94 Evaluation of the influence of analysis method
      • 95 Evaluation of the influence of calibration
      • 96 26Evaluation of compliance with legislation
      • • 10 Follow-up actions for underperforming laboratories
        • 11 1Conclusions
        • 12 1Acknowledgements
        • 13 References

57

Table 17 Analysis results reported by the participants for the four marker PAHs in the standard solution in acetonitrile with undisclosed

analyte content

Values are presented as reported U measurement uncertainty k coverage factor

benz[a]anthracene benzo[a]pyrene benzo[b]fluoranthene chrysene

Laboratory

code Unit Result U k Result U k Result U k Result U k

3602 microgkg 3675 247 247 244

3603 microgkg 358 086 2 236 028 2 235 028 2 236 057 2

3604 microgl 475 29 2 19 12 2 194 13 2 258 12 2

3608 microgl 3623 078 2 2385 023 2 2343 18 2 2296 092 2

3610 microgkg 369 185 2 25 125 2 251 126 2 244 122 2

3611 microgkg 48 02 2 323 02 2 328 04 2 317 03 2

3613 microgl 332 31 2 261 3 2 249 25 2 239 24 2

3615 microgl 415 2635 2729 2689

3617 microgkg 37 09 2 244 06 2 284 07 2 239 06 2

3622 microgl 37 12 2 25 23 2 257 21 2 238 1 2

3624 microgkg 402 104 2 255 084 2 265 096 2 253 07 2

5306 microgkg 563 113 2 371 74 2 359 72 2 333 67 2

5308 microgkg 168633 102669 232169 211488

5309 microgl 402 268 27

5310 microgl 315 158 25 125 25 125 288 144

5312 microgl 428 043 2 282 028 2 272 027 2 275 028 2

5315 microgkg 387 08 2 256 05 2 251 05 2 253 05 2

5316 microgl 3839 342 2 2485 191 2 2559 168 2 239 51 2

5317 microgl 2466 043 2 2212 034 2 2472 016 2 2146 068 2

5319 microgl 414 41 2 279 28 2 268 27 2 26 31 2

5321 microgkg 476 316 326 328

5322 microgl 2547 35 2

5323 microgkg 405 203 2 239 119 2 255 153 2 201 11 2

58

Figure 11 Youden plots of the percent deviation of the reported values from the assigned values for the food supplement test sample and the

standard solution in acetonitrile with undisclosed content

(The identity of the respective measurand is given above the figure Results exceeding a difference of more than 100 were omitted)

59

Figure 11 - continued

60

ANNEX 10 Table 18 Method performance parameters reported by the participants

Laboratory code

Analyte LOD LOQ Recovery Analyte LOD LOQ Recovery

microgkg microgkg microgkg microgkg 3600 BaA 03 06 978 BaP 03 07 9483602 BaA 007 021 926 BaP 005 015 993603 BaA 0011 05 987 BaP 0005 051 9553604 BaA 02 04 86 BaP 02 04 883605 BaA 0025 005 118 BaP 0025 005 1083606 BaA 001 003 65 BaP 001 003 703607 BaA 021 071 74 BaP 027 089 943608 BaA 016 048 8213 BaP 015 046 83683610 BaA 02 05 85 BaP 005 02 853611 BaA BaP 3612 BaA 1 117 BaP 1 1203613 BaA 01 03 90 BaP 01 03 903614 BaA 02 05 96 BaP 02 05 933615 BaA 005 015 110 BaP 005 015 1053616 BaA 01 05 108 BaP 01 05 753617 BaA 018 037 405 BaP 009 018 3873618 BaA 01 03 63 BaP 01 03 593619 BaA 005 005 59 BaP 033 033 543620 BaA 0009 0018 64 BaP 0005 001 553621 BaA 007 02 84 BaP 007 02 923622 BaA 01 03 1101 BaP 01 03 9853623 BaA 02 08 105 BaP 02 08 1013624 BaA BaP 01 033 8513625 BaA BaP 3626 BaA BaP 5301 BaA 003 01 102 BaP 003 01 1005302 BaA 01 03 78 BaP 01 03 865303 BaA 003 01 62 BaP 003 01 805304 BaA 1 1 70 BaP 1 1 955305 BaA 02 05 85 BaP 02 05 855306 BaA 03 1 699 BaP 03 1 905307 BaA 6537 BaP 81165308 BaA 0088 0265 99 BaP 00262 00785 1005309 BaA 01 08 92 BaP 01 08 925310 BaA 01 03 BaP 01 03 905311 BaA 002 005 80 BaP 002 004 915312 BaA 03 09 105 BaP 03 09 1055313 BaA 04 08 BaP 04 08 5314 BaA 001 002 96 BaP 001 002 9485315 BaA 01 03 50 - 100 BaP 01 03 50 - 100 5316 BaA 002 011 827 BaP 004 019 10445317 BaA 05 1 76 BaP 05 1 755318 BaA BaP 5319 BaA 009 029 951 BaP 011 041 995320 BaA 12 18 946 BaP 06 1 8345321 BaA 03 05 BaP 05 1 5322 BaA 1 2 100 BaP 1 2 1005323 BaA 26 BaP 26

61

ANNEX 10 Table 18 - continued

Laboratory code Analyte LOD LOQ Recovery Analyte LOD LOQ Recovery

microgkg microgkg microgkg microgkg 3600 BbF 03 05 96 CHR 02 03 10153602 BbF 015 045 947 CHR 003 009 9193603 BbF 0014 05 1012 CHR 0004 05 10033604 BbF 02 04 85 CHR 02 04 873605 BbF 005 01 94 CHR 0025 005 863606 BbF 001 003 70 CHR 001 003 653607 BbF 018 06 100 CHR 014 028 943608 BbF 015 046 7979 CHR 015 045 71253610 BbF 005 02 85 CHR 02 05 853611 BbF CHR 3612 BbF 1 118 CHR 1 1173613 BbF 01 03 90 CHR 01 03 903614 BbF 01 04 96 CHR 02 05 953615 BbF 008 025 109 CHR 003 01 1083616 BbF 01 05 78 CHR 01 05 723617 BbF 035 07 38 CHR 012 024 4163618 BbF 01 03 62 CHR 01 03 623619 BbF 016 016 63 CHR 01 01 593620 BbF 0003 0006 55 CHR 001 002 653621 BbF 007 02 103 CHR 007 02 893622 BbF 02 06 963 CHR 03 09 1043623 BbF 02 08 106 CHR 02 08 1193624 BbF CHR 3625 BbF CHR 3626 BbF CHR 5301 BbF 003 01 96 CHR 003 01 1145302 BbF 01 03 84 CHR 01 03 835303 BbF 003 01 78 CHR 003 01 705304 BbF 1 1 86 CHR 1 1 705305 BbF 02 05 90 CHR 02 05 955306 BbF 03 1 1435 CHR 03 1 1185307 BbF 8116 CHR 65375308 BbF 0079 0238 99 CHR 009 026 1005309 BbF 01 08 92 CHR 5310 BbF 07 2 CHR 01 03 5311 BbF 002 004 95 CHR 002 004 895312 BbF 03 09 105 CHR 03 09 1055313 BbF 04 08 CHR 04 08 5314 BbF 001 002 83 CHR 001 002 8185315 BbF 02 05 50 -100 CHR 02 05 50 - 100 5316 BbF 005 024 814 CHR 001 007 9595317 BbF 05 1 73 CHR 05 1 785318 BbF CHR 5319 BbF 011 038 946 CHR 013 043 9675320 BbF 07 09 1194 CHR 23 32 10455321 BbF 05 1 CHR 05 1 5322 BbF 1 2 100 CHR 1 2 1005323 BbF 26 CHR 26

62

ANNEX 11 Table 19 Details of analysis method reported by the participants Data are presented as reported

Laboratory code

Analysis technique

Instrument calibration

Internal standards applied

Sample clean up

Details of sample

clean up

Extraction technique

Details of sample

extraction

3600 6) GC-MS

2) Internal standardisation with labelled substances

Deuterated PAH mix 9 (Ehrenstorfer)

1) Saponification 2) LiquidLiquid partitioning

D) Other Liquidliquid partitioning

3602 2) HPLC-UV-FLD

1) External calibration

3) Gel permeation chromatography

A) Pressurised liquid extraction (PLE)

3603 1) HPLC-FLD

1) External calibration

3) Gel permeation chromatography

D) Other 1 minute with dichloromethane in vortex

3604 2) HPLC-UV-FLD

2) Internal standardisation with labelled substances

DiP D14

5) Donor acceptor complex chromatography

D) Other LL

3605 1) HPLC-FLD

1) External calibration

3) Gel permeation chromatography

C) Soxhlet extraction

3606 7) GC-MSMS

2) Internal standardisation with labelled substances

BaP 13C4 CHR 13C6 BaA 13C6 BbF 13C6

4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

3607 6) GC-MS

2) Internal standardisation with labelled substances

BaA-d12 CHR-d12 BbF-d12 BkF-d12 BaP-d12 ICP-d12 BgP-d12 DiP-d14

1) Saponification

D) Other solvent partitioning

3608 1) HPLC-FLD

1) External calibration

6) Other Liquid extraction

D) Other

Extraction in a rotary agitator and centrifugation

3610 2) HPLC-UV-FLD

1) External calibration

4) Solid phase extraction

B) Sonication

3611

3612 6) GC-MS

2) Internal standardisation with labelled substances

chrysene-d12 dibenzo(ah)anthracene-d14 dibenzo(ai)pyrene-d14

4) Solid phase extraction

B) Sonication

3613 5) LC-MSMS

3) Internal standardisation with unlabelled substances

I use Chrysene-D12 3) Gel permeation chromatography

D) Other extraction with hexan

63

Table 19 - continued

Laboratory code

Analysis technique

Instrument calibration

Internal standards applied

Sample clean up Details of sample

clean up

Extraction technique

Details of sample

extraction

3614 6) GC-MS 1) External calibration

3) Gel permeation chromatography

B) Sonication

3615 1) HPLC-FLD

1) External calibration

6) Other PLE GPC

A) Pressurised liquid extraction (PLE)

3616 6) GC-MS

2) Internal standardisation with labelled substances

Benzo(a)pyrene-13C4 Benzo(a)anthracene-13C6 Benzo(b)fluoranthene-13C6 Chrysene-13C6

2) LiquidLiquid partitioning 4) Solid phase extraction

B) Sonication

3617 1) HPLC-FLD

1) External calibration

4) Solid phase extraction

C) Soxhlet extraction

3618 6) GC-MS

2) Internal standardisation with labelled substances

deuterated standards

1) Saponification 4) Solid phase extraction

D) Other Handshaken with cyclohexane

3619 6) GC-MS

2) Internal standardisation with labelled substances

C13 Labelled US EPA 16

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

D) Other Homegenisation amp saponiofication

3620 9) GC-HRMS

2) Internal standardisation with labelled substances

mix of deuterated PAHs (BAA-D12 CHR-D12 BAP-D12 BBF-D12)

3) Gel permeation chromatography 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

3621 6) GC-MS

2) Internal standardisation with labelled substances

Chrysene D12 Benzo(a)pyrene D12 Benzo(b)fluoranthene D12 Benzo(a)anthracene

2) LiquidLiquid partitioning 4) Solid phase extraction

B) Sonication

3622 1) HPLC-FLD

4) Standard addition method

4) Solid phase extraction

D) Other

Extraction with cyclohexane by heating under reflux

3623 7) GC-MSMS

2) Internal standardisation with labelled substances

C13 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

3624 2) HPLC-UV-FLD

1) External calibration

2) LiquidLiquid partitioning

D) Other hot solvent extraction

3626

64

Table 19 - continued

Laboratory code

Analysis technique

Instrument calibration

Internal standards

applied

Sample clean up

Details of sample

clean up

Extraction technique

Details of sample

extraction

5301 7) GC-MSMS

2) Internal standardisation with labelled substances

EACH SUBSTANCE C13

4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5302 7) GC-MSMS

2) Internal standardisation with labelled substances

Isotope labelled PAHs 13C for each analyte

4) Solid phase extraction

C) Soxhlet extraction

5303 7) GC-MSMS

3) Internal standardisation with unlabelled substances

HAP C13 4) Solid phase extraction

D) Other automate ASE200 DIONEX

5304 7) GC-MSMS

2) Internal standardisation with labelled substances

with C13 for each HAP

2) LiquidLiquid partitioning 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5305 7) GC-MSMS

2) Internal standardisation with labelled substances

C13 isotopiques internal standards

4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5306 7) GC-MSMS

2) Internal standardisation with labelled substances

C13 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5307 6) GC-MS

2) Internal standardisation with labelled substances

1) Saponification 2) LiquidLiquid partitioning

D) Other solvent extraction

5308 1) HPLC-FLD 1) External calibration

4) Solid phase extraction

B) Sonication

5309 1) HPLC-FLD 1) External calibration

4) Solid phase extraction

D) Other only SPE

5310 1) HPLC-FLD 1) External calibration

1) Saponification 2) LiquidLiquid partitioning 3) Gel permeation chromatography

C) Soxhlet extraction

5311 6) GC-MS

2) Internal standardisation with labelled substances

13 C Chrysene 13 C Benz(a)pyrene

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

B) Sonication

5312 1) HPLC-FLD

3) Internal standardisation with unlabelled substances

Benzo(b)chrysen

2) LiquidLiquid partitioning 4) Solid phase extraction

Quechers D) Other Quechers

65

Table 19 - continued

Laboratory code

Analysis technique

Instrument calibration

Internal standards applied

Sample clean up Details of sample

clean up

Extraction technique

Details of sample extraction

5313 6) GC-MS

2) Internal standardisation with labelled substances

Benz(a)anthracene D12 Benzo(b)fluranthene D12 Benzo(a)pyrene D12 Chrysene D12

1) Saponification 2) LiquidLiquid partitioning 3) Gel permeation chromatography

A) Pressurised liquid extraction (PLE)

5314 6) GC-MS

2) Internal standardisation with labelled substances

Benzo(a)pyren d12 2) LiquidLiquid partitioning

D) Other Extraction with

5315 1) HPLC-FLD

3) Internal standardisation with unlabelled substances

Benz(b)chrysen

1) Saponification 2) LiquidLiquid partitioning

D) Other shaker

5316 1) HPLC-FLD

1) External calibration

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

D) Other

LiquidLiquid extraction with cyclohexane and dimethylformamide

5317 7) GC-MSMS

1) External calibration

3) Gel permeation chromatography 6) Other

silica gel D) Other VDLUFA online method

5318

5319 1) HPLC-FLD

3) Internal standardisation with unlabelled substances

Benzo(b)chrysene 3) Gel permeation chromatography

A) Pressurised liquid extraction (PLE)

5320 6) GC-MS

3) Internal standardisation with unlabelled substances

22-binaphthyl

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

D) Other 3 hours refluxing

5321 1) HPLC-FLD

2) Internal standardisation with labelled substances

6-meacutethylchrysegravene

1) Saponification 2) LiquidLiquid partitioning

B) Sonication

5322 6) GC-MS

2) Internal standardisation with labelled substances

naftalene-d8 acenaphtalene-d10 phenanthrene-d10 chrysene-d12 perylene-d12 benzo(e)anthracene-d12

1) Saponification 2) LiquidLiquid partitioning 6) Other

clean-up with silica packed column

C) Soxhlet extraction

5323 1) HPLC-FLD

1) External calibration

4) Solid phase extraction 6) Other

Accelerated Solvent Extraction

A) Pressurised liquid extraction (PLE)

66

European Commission EUR 25245 EN ndash Joint Research Centre ndash Institute for Reference Materials and Measurements Title Report on the 8th inter-laboratory comparison organised by the European Union Reference Laboratory for Polycyclic Aromatic Hydrocarbons - Four marker PAHs in a dry extract of St Johns wort Author(s) Radoslav Lizak Szilard Szilagyi Philippe Verlinde and Thomas WENZL Luxembourg Publications Office of the European Union 2012 ndash 66 pp ndash 201 x 297 cm EUR ndash Scientific and Technical Research series ndash ISSN 1831-9424 ISBN 978-92-79-23337-1 doi 10278759387 Abstract The proficiency test here reported concerned the determination of the contents of four marker polycyclic aromatic hydrocarbons (PAHs) and of their sum in an food supplements test sample The set of marker PAHs consists of benz[a]anthacene benzo[a]pyrene benzo[b]fluoranthene and chrysene Participants to these PT were National Reference Laboratories for PAHs (NRLs-PAHs) and EU official food control laboratories The number of participants was in total 48

The PT was organised according ISO Standard 170432010

The test material used was a commercial dry extract of St Johns wort Participants also received a solution of the same PAHs either in acetonitrile or in toluene for checking their instrument calibration

The results from participants were rated with z-scores and zeta-scores About 68 of the reported results were attributed with z-scores with an absolute value of below two which is the threshold for satisfactory performance

How to obtain EU publications

Our priced publications are available from EU Bookshop (httpbookshopeuropaeu) where you can place

an order with the sales agent of your choice

The Publications Office has a worldwide network of sales agents You can obtain their contact details by

sending a fax to (352) 29 29-42758

The mission of the JRC is to provide customer-driven scientific and technical support

for the conception development implementation and monitoring of EU policies As a

service of the European Commission the JRC functions as a reference centre of

science and technology for the Union Close to the policy-making process it serves

the common interest of the Member States while being independent of special

interests whether private or national

L

A-N

A-252

45-EN

-N

• 1 Summary
• 2 Introduction
• 3 Scope
• 4 Participating Laboratories
• 5 Time frame
• 6 Confidentiality
• 7 Test materials
• • 71 1Preparation of test materials
  • 72 Assigned values and standard deviation for proficiency assessment
  • 73 Homogeneity and stability
  • 74 Sample dispatch
  • • 8 Design of the proficiency test
    • 9 Evaluation of the results
    • • 91 2General
      • 92 21BEvaluation criteria
      • 93 Performance of participants
      • 94 Evaluation of the influence of analysis method
      • 95 Evaluation of the influence of calibration
      • 96 26Evaluation of compliance with legislation
      • • 10 Follow-up actions for underperforming laboratories
        • 11 1Conclusions
        • 12 1Acknowledgements
        • 13 References

58

Figure 11 Youden plots of the percent deviation of the reported values from the assigned values for the food supplement test sample and the

standard solution in acetonitrile with undisclosed content

(The identity of the respective measurand is given above the figure Results exceeding a difference of more than 100 were omitted)

59

Figure 11 - continued

60

ANNEX 10 Table 18 Method performance parameters reported by the participants

Laboratory code

Analyte LOD LOQ Recovery Analyte LOD LOQ Recovery

microgkg microgkg microgkg microgkg 3600 BaA 03 06 978 BaP 03 07 9483602 BaA 007 021 926 BaP 005 015 993603 BaA 0011 05 987 BaP 0005 051 9553604 BaA 02 04 86 BaP 02 04 883605 BaA 0025 005 118 BaP 0025 005 1083606 BaA 001 003 65 BaP 001 003 703607 BaA 021 071 74 BaP 027 089 943608 BaA 016 048 8213 BaP 015 046 83683610 BaA 02 05 85 BaP 005 02 853611 BaA BaP 3612 BaA 1 117 BaP 1 1203613 BaA 01 03 90 BaP 01 03 903614 BaA 02 05 96 BaP 02 05 933615 BaA 005 015 110 BaP 005 015 1053616 BaA 01 05 108 BaP 01 05 753617 BaA 018 037 405 BaP 009 018 3873618 BaA 01 03 63 BaP 01 03 593619 BaA 005 005 59 BaP 033 033 543620 BaA 0009 0018 64 BaP 0005 001 553621 BaA 007 02 84 BaP 007 02 923622 BaA 01 03 1101 BaP 01 03 9853623 BaA 02 08 105 BaP 02 08 1013624 BaA BaP 01 033 8513625 BaA BaP 3626 BaA BaP 5301 BaA 003 01 102 BaP 003 01 1005302 BaA 01 03 78 BaP 01 03 865303 BaA 003 01 62 BaP 003 01 805304 BaA 1 1 70 BaP 1 1 955305 BaA 02 05 85 BaP 02 05 855306 BaA 03 1 699 BaP 03 1 905307 BaA 6537 BaP 81165308 BaA 0088 0265 99 BaP 00262 00785 1005309 BaA 01 08 92 BaP 01 08 925310 BaA 01 03 BaP 01 03 905311 BaA 002 005 80 BaP 002 004 915312 BaA 03 09 105 BaP 03 09 1055313 BaA 04 08 BaP 04 08 5314 BaA 001 002 96 BaP 001 002 9485315 BaA 01 03 50 - 100 BaP 01 03 50 - 100 5316 BaA 002 011 827 BaP 004 019 10445317 BaA 05 1 76 BaP 05 1 755318 BaA BaP 5319 BaA 009 029 951 BaP 011 041 995320 BaA 12 18 946 BaP 06 1 8345321 BaA 03 05 BaP 05 1 5322 BaA 1 2 100 BaP 1 2 1005323 BaA 26 BaP 26

61

ANNEX 10 Table 18 - continued

Laboratory code Analyte LOD LOQ Recovery Analyte LOD LOQ Recovery

microgkg microgkg microgkg microgkg 3600 BbF 03 05 96 CHR 02 03 10153602 BbF 015 045 947 CHR 003 009 9193603 BbF 0014 05 1012 CHR 0004 05 10033604 BbF 02 04 85 CHR 02 04 873605 BbF 005 01 94 CHR 0025 005 863606 BbF 001 003 70 CHR 001 003 653607 BbF 018 06 100 CHR 014 028 943608 BbF 015 046 7979 CHR 015 045 71253610 BbF 005 02 85 CHR 02 05 853611 BbF CHR 3612 BbF 1 118 CHR 1 1173613 BbF 01 03 90 CHR 01 03 903614 BbF 01 04 96 CHR 02 05 953615 BbF 008 025 109 CHR 003 01 1083616 BbF 01 05 78 CHR 01 05 723617 BbF 035 07 38 CHR 012 024 4163618 BbF 01 03 62 CHR 01 03 623619 BbF 016 016 63 CHR 01 01 593620 BbF 0003 0006 55 CHR 001 002 653621 BbF 007 02 103 CHR 007 02 893622 BbF 02 06 963 CHR 03 09 1043623 BbF 02 08 106 CHR 02 08 1193624 BbF CHR 3625 BbF CHR 3626 BbF CHR 5301 BbF 003 01 96 CHR 003 01 1145302 BbF 01 03 84 CHR 01 03 835303 BbF 003 01 78 CHR 003 01 705304 BbF 1 1 86 CHR 1 1 705305 BbF 02 05 90 CHR 02 05 955306 BbF 03 1 1435 CHR 03 1 1185307 BbF 8116 CHR 65375308 BbF 0079 0238 99 CHR 009 026 1005309 BbF 01 08 92 CHR 5310 BbF 07 2 CHR 01 03 5311 BbF 002 004 95 CHR 002 004 895312 BbF 03 09 105 CHR 03 09 1055313 BbF 04 08 CHR 04 08 5314 BbF 001 002 83 CHR 001 002 8185315 BbF 02 05 50 -100 CHR 02 05 50 - 100 5316 BbF 005 024 814 CHR 001 007 9595317 BbF 05 1 73 CHR 05 1 785318 BbF CHR 5319 BbF 011 038 946 CHR 013 043 9675320 BbF 07 09 1194 CHR 23 32 10455321 BbF 05 1 CHR 05 1 5322 BbF 1 2 100 CHR 1 2 1005323 BbF 26 CHR 26

62

ANNEX 11 Table 19 Details of analysis method reported by the participants Data are presented as reported

Laboratory code

Analysis technique

Instrument calibration

Internal standards applied

Sample clean up

Details of sample

clean up

Extraction technique

Details of sample

extraction

3600 6) GC-MS

2) Internal standardisation with labelled substances

Deuterated PAH mix 9 (Ehrenstorfer)

1) Saponification 2) LiquidLiquid partitioning

D) Other Liquidliquid partitioning

3602 2) HPLC-UV-FLD

1) External calibration

3) Gel permeation chromatography

A) Pressurised liquid extraction (PLE)

3603 1) HPLC-FLD

1) External calibration

3) Gel permeation chromatography

D) Other 1 minute with dichloromethane in vortex

3604 2) HPLC-UV-FLD

2) Internal standardisation with labelled substances

DiP D14

5) Donor acceptor complex chromatography

D) Other LL

3605 1) HPLC-FLD

1) External calibration

3) Gel permeation chromatography

C) Soxhlet extraction

3606 7) GC-MSMS

2) Internal standardisation with labelled substances

BaP 13C4 CHR 13C6 BaA 13C6 BbF 13C6

4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

3607 6) GC-MS

2) Internal standardisation with labelled substances

BaA-d12 CHR-d12 BbF-d12 BkF-d12 BaP-d12 ICP-d12 BgP-d12 DiP-d14

1) Saponification

D) Other solvent partitioning

3608 1) HPLC-FLD

1) External calibration

6) Other Liquid extraction

D) Other

Extraction in a rotary agitator and centrifugation

3610 2) HPLC-UV-FLD

1) External calibration

4) Solid phase extraction

B) Sonication

3611

3612 6) GC-MS

2) Internal standardisation with labelled substances

chrysene-d12 dibenzo(ah)anthracene-d14 dibenzo(ai)pyrene-d14

4) Solid phase extraction

B) Sonication

3613 5) LC-MSMS

3) Internal standardisation with unlabelled substances

I use Chrysene-D12 3) Gel permeation chromatography

D) Other extraction with hexan

63

Table 19 - continued

Laboratory code

Analysis technique

Instrument calibration

Internal standards applied

Sample clean up Details of sample

clean up

Extraction technique

Details of sample

extraction

3614 6) GC-MS 1) External calibration

3) Gel permeation chromatography

B) Sonication

3615 1) HPLC-FLD

1) External calibration

6) Other PLE GPC

A) Pressurised liquid extraction (PLE)

3616 6) GC-MS

2) Internal standardisation with labelled substances

Benzo(a)pyrene-13C4 Benzo(a)anthracene-13C6 Benzo(b)fluoranthene-13C6 Chrysene-13C6

2) LiquidLiquid partitioning 4) Solid phase extraction

B) Sonication

3617 1) HPLC-FLD

1) External calibration

4) Solid phase extraction

C) Soxhlet extraction

3618 6) GC-MS

2) Internal standardisation with labelled substances

deuterated standards

1) Saponification 4) Solid phase extraction

D) Other Handshaken with cyclohexane

3619 6) GC-MS

2) Internal standardisation with labelled substances

C13 Labelled US EPA 16

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

D) Other Homegenisation amp saponiofication

3620 9) GC-HRMS

2) Internal standardisation with labelled substances

mix of deuterated PAHs (BAA-D12 CHR-D12 BAP-D12 BBF-D12)

3) Gel permeation chromatography 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

3621 6) GC-MS

2) Internal standardisation with labelled substances

Chrysene D12 Benzo(a)pyrene D12 Benzo(b)fluoranthene D12 Benzo(a)anthracene

2) LiquidLiquid partitioning 4) Solid phase extraction

B) Sonication

3622 1) HPLC-FLD

4) Standard addition method

4) Solid phase extraction

D) Other

Extraction with cyclohexane by heating under reflux

3623 7) GC-MSMS

2) Internal standardisation with labelled substances

C13 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

3624 2) HPLC-UV-FLD

1) External calibration

2) LiquidLiquid partitioning

D) Other hot solvent extraction

3626

64

Table 19 - continued

Laboratory code

Analysis technique

Instrument calibration

Internal standards

applied

Sample clean up

Details of sample

clean up

Extraction technique

Details of sample

extraction

5301 7) GC-MSMS

2) Internal standardisation with labelled substances

EACH SUBSTANCE C13

4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5302 7) GC-MSMS

2) Internal standardisation with labelled substances

Isotope labelled PAHs 13C for each analyte

4) Solid phase extraction

C) Soxhlet extraction

5303 7) GC-MSMS

3) Internal standardisation with unlabelled substances

HAP C13 4) Solid phase extraction

D) Other automate ASE200 DIONEX

5304 7) GC-MSMS

2) Internal standardisation with labelled substances

with C13 for each HAP

2) LiquidLiquid partitioning 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5305 7) GC-MSMS

2) Internal standardisation with labelled substances

C13 isotopiques internal standards

4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5306 7) GC-MSMS

2) Internal standardisation with labelled substances

C13 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5307 6) GC-MS

2) Internal standardisation with labelled substances

1) Saponification 2) LiquidLiquid partitioning

D) Other solvent extraction

5308 1) HPLC-FLD 1) External calibration

4) Solid phase extraction

B) Sonication

5309 1) HPLC-FLD 1) External calibration

4) Solid phase extraction

D) Other only SPE

5310 1) HPLC-FLD 1) External calibration

1) Saponification 2) LiquidLiquid partitioning 3) Gel permeation chromatography

C) Soxhlet extraction

5311 6) GC-MS

2) Internal standardisation with labelled substances

13 C Chrysene 13 C Benz(a)pyrene

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

B) Sonication

5312 1) HPLC-FLD

3) Internal standardisation with unlabelled substances

Benzo(b)chrysen

2) LiquidLiquid partitioning 4) Solid phase extraction

Quechers D) Other Quechers

65

Table 19 - continued

Laboratory code

Analysis technique

Instrument calibration

Internal standards applied

Sample clean up Details of sample

clean up

Extraction technique

Details of sample extraction

5313 6) GC-MS

2) Internal standardisation with labelled substances

Benz(a)anthracene D12 Benzo(b)fluranthene D12 Benzo(a)pyrene D12 Chrysene D12

1) Saponification 2) LiquidLiquid partitioning 3) Gel permeation chromatography

A) Pressurised liquid extraction (PLE)

5314 6) GC-MS

2) Internal standardisation with labelled substances

Benzo(a)pyren d12 2) LiquidLiquid partitioning

D) Other Extraction with

5315 1) HPLC-FLD

3) Internal standardisation with unlabelled substances

Benz(b)chrysen

1) Saponification 2) LiquidLiquid partitioning

D) Other shaker

5316 1) HPLC-FLD

1) External calibration

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

D) Other

LiquidLiquid extraction with cyclohexane and dimethylformamide

5317 7) GC-MSMS

1) External calibration

3) Gel permeation chromatography 6) Other

silica gel D) Other VDLUFA online method

5318

5319 1) HPLC-FLD

3) Internal standardisation with unlabelled substances

Benzo(b)chrysene 3) Gel permeation chromatography

A) Pressurised liquid extraction (PLE)

5320 6) GC-MS

3) Internal standardisation with unlabelled substances

22-binaphthyl

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

D) Other 3 hours refluxing

5321 1) HPLC-FLD

2) Internal standardisation with labelled substances

6-meacutethylchrysegravene

1) Saponification 2) LiquidLiquid partitioning

B) Sonication

5322 6) GC-MS

2) Internal standardisation with labelled substances

naftalene-d8 acenaphtalene-d10 phenanthrene-d10 chrysene-d12 perylene-d12 benzo(e)anthracene-d12

1) Saponification 2) LiquidLiquid partitioning 6) Other

clean-up with silica packed column

C) Soxhlet extraction

5323 1) HPLC-FLD

1) External calibration

4) Solid phase extraction 6) Other

Accelerated Solvent Extraction

A) Pressurised liquid extraction (PLE)

66

European Commission EUR 25245 EN ndash Joint Research Centre ndash Institute for Reference Materials and Measurements Title Report on the 8th inter-laboratory comparison organised by the European Union Reference Laboratory for Polycyclic Aromatic Hydrocarbons - Four marker PAHs in a dry extract of St Johns wort Author(s) Radoslav Lizak Szilard Szilagyi Philippe Verlinde and Thomas WENZL Luxembourg Publications Office of the European Union 2012 ndash 66 pp ndash 201 x 297 cm EUR ndash Scientific and Technical Research series ndash ISSN 1831-9424 ISBN 978-92-79-23337-1 doi 10278759387 Abstract The proficiency test here reported concerned the determination of the contents of four marker polycyclic aromatic hydrocarbons (PAHs) and of their sum in an food supplements test sample The set of marker PAHs consists of benz[a]anthacene benzo[a]pyrene benzo[b]fluoranthene and chrysene Participants to these PT were National Reference Laboratories for PAHs (NRLs-PAHs) and EU official food control laboratories The number of participants was in total 48

The PT was organised according ISO Standard 170432010

The test material used was a commercial dry extract of St Johns wort Participants also received a solution of the same PAHs either in acetonitrile or in toluene for checking their instrument calibration

The results from participants were rated with z-scores and zeta-scores About 68 of the reported results were attributed with z-scores with an absolute value of below two which is the threshold for satisfactory performance

How to obtain EU publications

Our priced publications are available from EU Bookshop (httpbookshopeuropaeu) where you can place

an order with the sales agent of your choice

The Publications Office has a worldwide network of sales agents You can obtain their contact details by

sending a fax to (352) 29 29-42758

The mission of the JRC is to provide customer-driven scientific and technical support

for the conception development implementation and monitoring of EU policies As a

service of the European Commission the JRC functions as a reference centre of

science and technology for the Union Close to the policy-making process it serves

the common interest of the Member States while being independent of special

interests whether private or national

L

A-N

A-252

45-EN

-N

• 1 Summary
• 2 Introduction
• 3 Scope
• 4 Participating Laboratories
• 5 Time frame
• 6 Confidentiality
• 7 Test materials
• • 71 1Preparation of test materials
  • 72 Assigned values and standard deviation for proficiency assessment
  • 73 Homogeneity and stability
  • 74 Sample dispatch
  • • 8 Design of the proficiency test
    • 9 Evaluation of the results
    • • 91 2General
      • 92 21BEvaluation criteria
      • 93 Performance of participants
      • 94 Evaluation of the influence of analysis method
      • 95 Evaluation of the influence of calibration
      • 96 26Evaluation of compliance with legislation
      • • 10 Follow-up actions for underperforming laboratories
        • 11 1Conclusions
        • 12 1Acknowledgements
        • 13 References

59

Figure 11 - continued

60

ANNEX 10 Table 18 Method performance parameters reported by the participants

Laboratory code

Analyte LOD LOQ Recovery Analyte LOD LOQ Recovery

microgkg microgkg microgkg microgkg 3600 BaA 03 06 978 BaP 03 07 9483602 BaA 007 021 926 BaP 005 015 993603 BaA 0011 05 987 BaP 0005 051 9553604 BaA 02 04 86 BaP 02 04 883605 BaA 0025 005 118 BaP 0025 005 1083606 BaA 001 003 65 BaP 001 003 703607 BaA 021 071 74 BaP 027 089 943608 BaA 016 048 8213 BaP 015 046 83683610 BaA 02 05 85 BaP 005 02 853611 BaA BaP 3612 BaA 1 117 BaP 1 1203613 BaA 01 03 90 BaP 01 03 903614 BaA 02 05 96 BaP 02 05 933615 BaA 005 015 110 BaP 005 015 1053616 BaA 01 05 108 BaP 01 05 753617 BaA 018 037 405 BaP 009 018 3873618 BaA 01 03 63 BaP 01 03 593619 BaA 005 005 59 BaP 033 033 543620 BaA 0009 0018 64 BaP 0005 001 553621 BaA 007 02 84 BaP 007 02 923622 BaA 01 03 1101 BaP 01 03 9853623 BaA 02 08 105 BaP 02 08 1013624 BaA BaP 01 033 8513625 BaA BaP 3626 BaA BaP 5301 BaA 003 01 102 BaP 003 01 1005302 BaA 01 03 78 BaP 01 03 865303 BaA 003 01 62 BaP 003 01 805304 BaA 1 1 70 BaP 1 1 955305 BaA 02 05 85 BaP 02 05 855306 BaA 03 1 699 BaP 03 1 905307 BaA 6537 BaP 81165308 BaA 0088 0265 99 BaP 00262 00785 1005309 BaA 01 08 92 BaP 01 08 925310 BaA 01 03 BaP 01 03 905311 BaA 002 005 80 BaP 002 004 915312 BaA 03 09 105 BaP 03 09 1055313 BaA 04 08 BaP 04 08 5314 BaA 001 002 96 BaP 001 002 9485315 BaA 01 03 50 - 100 BaP 01 03 50 - 100 5316 BaA 002 011 827 BaP 004 019 10445317 BaA 05 1 76 BaP 05 1 755318 BaA BaP 5319 BaA 009 029 951 BaP 011 041 995320 BaA 12 18 946 BaP 06 1 8345321 BaA 03 05 BaP 05 1 5322 BaA 1 2 100 BaP 1 2 1005323 BaA 26 BaP 26

61

ANNEX 10 Table 18 - continued

Laboratory code Analyte LOD LOQ Recovery Analyte LOD LOQ Recovery

microgkg microgkg microgkg microgkg 3600 BbF 03 05 96 CHR 02 03 10153602 BbF 015 045 947 CHR 003 009 9193603 BbF 0014 05 1012 CHR 0004 05 10033604 BbF 02 04 85 CHR 02 04 873605 BbF 005 01 94 CHR 0025 005 863606 BbF 001 003 70 CHR 001 003 653607 BbF 018 06 100 CHR 014 028 943608 BbF 015 046 7979 CHR 015 045 71253610 BbF 005 02 85 CHR 02 05 853611 BbF CHR 3612 BbF 1 118 CHR 1 1173613 BbF 01 03 90 CHR 01 03 903614 BbF 01 04 96 CHR 02 05 953615 BbF 008 025 109 CHR 003 01 1083616 BbF 01 05 78 CHR 01 05 723617 BbF 035 07 38 CHR 012 024 4163618 BbF 01 03 62 CHR 01 03 623619 BbF 016 016 63 CHR 01 01 593620 BbF 0003 0006 55 CHR 001 002 653621 BbF 007 02 103 CHR 007 02 893622 BbF 02 06 963 CHR 03 09 1043623 BbF 02 08 106 CHR 02 08 1193624 BbF CHR 3625 BbF CHR 3626 BbF CHR 5301 BbF 003 01 96 CHR 003 01 1145302 BbF 01 03 84 CHR 01 03 835303 BbF 003 01 78 CHR 003 01 705304 BbF 1 1 86 CHR 1 1 705305 BbF 02 05 90 CHR 02 05 955306 BbF 03 1 1435 CHR 03 1 1185307 BbF 8116 CHR 65375308 BbF 0079 0238 99 CHR 009 026 1005309 BbF 01 08 92 CHR 5310 BbF 07 2 CHR 01 03 5311 BbF 002 004 95 CHR 002 004 895312 BbF 03 09 105 CHR 03 09 1055313 BbF 04 08 CHR 04 08 5314 BbF 001 002 83 CHR 001 002 8185315 BbF 02 05 50 -100 CHR 02 05 50 - 100 5316 BbF 005 024 814 CHR 001 007 9595317 BbF 05 1 73 CHR 05 1 785318 BbF CHR 5319 BbF 011 038 946 CHR 013 043 9675320 BbF 07 09 1194 CHR 23 32 10455321 BbF 05 1 CHR 05 1 5322 BbF 1 2 100 CHR 1 2 1005323 BbF 26 CHR 26

62

ANNEX 11 Table 19 Details of analysis method reported by the participants Data are presented as reported

Laboratory code

Analysis technique

Instrument calibration

Internal standards applied

Sample clean up

Details of sample

clean up

Extraction technique

Details of sample

extraction

3600 6) GC-MS

2) Internal standardisation with labelled substances

Deuterated PAH mix 9 (Ehrenstorfer)

1) Saponification 2) LiquidLiquid partitioning

D) Other Liquidliquid partitioning

3602 2) HPLC-UV-FLD

1) External calibration

3) Gel permeation chromatography

A) Pressurised liquid extraction (PLE)

3603 1) HPLC-FLD

1) External calibration

3) Gel permeation chromatography

D) Other 1 minute with dichloromethane in vortex

3604 2) HPLC-UV-FLD

2) Internal standardisation with labelled substances

DiP D14

5) Donor acceptor complex chromatography

D) Other LL

3605 1) HPLC-FLD

1) External calibration

3) Gel permeation chromatography

C) Soxhlet extraction

3606 7) GC-MSMS

2) Internal standardisation with labelled substances

BaP 13C4 CHR 13C6 BaA 13C6 BbF 13C6

4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

3607 6) GC-MS

2) Internal standardisation with labelled substances

BaA-d12 CHR-d12 BbF-d12 BkF-d12 BaP-d12 ICP-d12 BgP-d12 DiP-d14

1) Saponification

D) Other solvent partitioning

3608 1) HPLC-FLD

1) External calibration

6) Other Liquid extraction

D) Other

Extraction in a rotary agitator and centrifugation

3610 2) HPLC-UV-FLD

1) External calibration

4) Solid phase extraction

B) Sonication

3611

3612 6) GC-MS

2) Internal standardisation with labelled substances

chrysene-d12 dibenzo(ah)anthracene-d14 dibenzo(ai)pyrene-d14

4) Solid phase extraction

B) Sonication

3613 5) LC-MSMS

3) Internal standardisation with unlabelled substances

I use Chrysene-D12 3) Gel permeation chromatography

D) Other extraction with hexan

63

Table 19 - continued

Laboratory code

Analysis technique

Instrument calibration

Internal standards applied

Sample clean up Details of sample

clean up

Extraction technique

Details of sample

extraction

3614 6) GC-MS 1) External calibration

3) Gel permeation chromatography

B) Sonication

3615 1) HPLC-FLD

1) External calibration

6) Other PLE GPC

A) Pressurised liquid extraction (PLE)

3616 6) GC-MS

2) Internal standardisation with labelled substances

Benzo(a)pyrene-13C4 Benzo(a)anthracene-13C6 Benzo(b)fluoranthene-13C6 Chrysene-13C6

2) LiquidLiquid partitioning 4) Solid phase extraction

B) Sonication

3617 1) HPLC-FLD

1) External calibration

4) Solid phase extraction

C) Soxhlet extraction

3618 6) GC-MS

2) Internal standardisation with labelled substances

deuterated standards

1) Saponification 4) Solid phase extraction

D) Other Handshaken with cyclohexane

3619 6) GC-MS

2) Internal standardisation with labelled substances

C13 Labelled US EPA 16

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

D) Other Homegenisation amp saponiofication

3620 9) GC-HRMS

2) Internal standardisation with labelled substances

mix of deuterated PAHs (BAA-D12 CHR-D12 BAP-D12 BBF-D12)

3) Gel permeation chromatography 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

3621 6) GC-MS

2) Internal standardisation with labelled substances

Chrysene D12 Benzo(a)pyrene D12 Benzo(b)fluoranthene D12 Benzo(a)anthracene

2) LiquidLiquid partitioning 4) Solid phase extraction

B) Sonication

3622 1) HPLC-FLD

4) Standard addition method

4) Solid phase extraction

D) Other

Extraction with cyclohexane by heating under reflux

3623 7) GC-MSMS

2) Internal standardisation with labelled substances

C13 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

3624 2) HPLC-UV-FLD

1) External calibration

2) LiquidLiquid partitioning

D) Other hot solvent extraction

3626

64

Table 19 - continued

Laboratory code

Analysis technique

Instrument calibration

Internal standards

applied

Sample clean up

Details of sample

clean up

Extraction technique

Details of sample

extraction

5301 7) GC-MSMS

2) Internal standardisation with labelled substances

EACH SUBSTANCE C13

4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5302 7) GC-MSMS

2) Internal standardisation with labelled substances

Isotope labelled PAHs 13C for each analyte

4) Solid phase extraction

C) Soxhlet extraction

5303 7) GC-MSMS

3) Internal standardisation with unlabelled substances

HAP C13 4) Solid phase extraction

D) Other automate ASE200 DIONEX

5304 7) GC-MSMS

2) Internal standardisation with labelled substances

with C13 for each HAP

2) LiquidLiquid partitioning 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5305 7) GC-MSMS

2) Internal standardisation with labelled substances

C13 isotopiques internal standards

4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5306 7) GC-MSMS

2) Internal standardisation with labelled substances

C13 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5307 6) GC-MS

2) Internal standardisation with labelled substances

1) Saponification 2) LiquidLiquid partitioning

D) Other solvent extraction

5308 1) HPLC-FLD 1) External calibration

4) Solid phase extraction

B) Sonication

5309 1) HPLC-FLD 1) External calibration

4) Solid phase extraction

D) Other only SPE

5310 1) HPLC-FLD 1) External calibration

1) Saponification 2) LiquidLiquid partitioning 3) Gel permeation chromatography

C) Soxhlet extraction

5311 6) GC-MS

2) Internal standardisation with labelled substances

13 C Chrysene 13 C Benz(a)pyrene

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

B) Sonication

5312 1) HPLC-FLD

3) Internal standardisation with unlabelled substances

Benzo(b)chrysen

2) LiquidLiquid partitioning 4) Solid phase extraction

Quechers D) Other Quechers

65

Table 19 - continued

Laboratory code

Analysis technique

Instrument calibration

Internal standards applied

Sample clean up Details of sample

clean up

Extraction technique

Details of sample extraction

5313 6) GC-MS

2) Internal standardisation with labelled substances

Benz(a)anthracene D12 Benzo(b)fluranthene D12 Benzo(a)pyrene D12 Chrysene D12

1) Saponification 2) LiquidLiquid partitioning 3) Gel permeation chromatography

A) Pressurised liquid extraction (PLE)

5314 6) GC-MS

2) Internal standardisation with labelled substances

Benzo(a)pyren d12 2) LiquidLiquid partitioning

D) Other Extraction with

5315 1) HPLC-FLD

3) Internal standardisation with unlabelled substances

Benz(b)chrysen

1) Saponification 2) LiquidLiquid partitioning

D) Other shaker

5316 1) HPLC-FLD

1) External calibration

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

D) Other

LiquidLiquid extraction with cyclohexane and dimethylformamide

5317 7) GC-MSMS

1) External calibration

3) Gel permeation chromatography 6) Other

silica gel D) Other VDLUFA online method

5318

5319 1) HPLC-FLD

3) Internal standardisation with unlabelled substances

Benzo(b)chrysene 3) Gel permeation chromatography

A) Pressurised liquid extraction (PLE)

5320 6) GC-MS

3) Internal standardisation with unlabelled substances

22-binaphthyl

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

D) Other 3 hours refluxing

5321 1) HPLC-FLD

2) Internal standardisation with labelled substances

6-meacutethylchrysegravene

1) Saponification 2) LiquidLiquid partitioning

B) Sonication

5322 6) GC-MS

2) Internal standardisation with labelled substances

naftalene-d8 acenaphtalene-d10 phenanthrene-d10 chrysene-d12 perylene-d12 benzo(e)anthracene-d12

1) Saponification 2) LiquidLiquid partitioning 6) Other

clean-up with silica packed column

C) Soxhlet extraction

5323 1) HPLC-FLD

1) External calibration

4) Solid phase extraction 6) Other

Accelerated Solvent Extraction

A) Pressurised liquid extraction (PLE)

66

European Commission EUR 25245 EN ndash Joint Research Centre ndash Institute for Reference Materials and Measurements Title Report on the 8th inter-laboratory comparison organised by the European Union Reference Laboratory for Polycyclic Aromatic Hydrocarbons - Four marker PAHs in a dry extract of St Johns wort Author(s) Radoslav Lizak Szilard Szilagyi Philippe Verlinde and Thomas WENZL Luxembourg Publications Office of the European Union 2012 ndash 66 pp ndash 201 x 297 cm EUR ndash Scientific and Technical Research series ndash ISSN 1831-9424 ISBN 978-92-79-23337-1 doi 10278759387 Abstract The proficiency test here reported concerned the determination of the contents of four marker polycyclic aromatic hydrocarbons (PAHs) and of their sum in an food supplements test sample The set of marker PAHs consists of benz[a]anthacene benzo[a]pyrene benzo[b]fluoranthene and chrysene Participants to these PT were National Reference Laboratories for PAHs (NRLs-PAHs) and EU official food control laboratories The number of participants was in total 48

The PT was organised according ISO Standard 170432010

The test material used was a commercial dry extract of St Johns wort Participants also received a solution of the same PAHs either in acetonitrile or in toluene for checking their instrument calibration

The results from participants were rated with z-scores and zeta-scores About 68 of the reported results were attributed with z-scores with an absolute value of below two which is the threshold for satisfactory performance

How to obtain EU publications

Our priced publications are available from EU Bookshop (httpbookshopeuropaeu) where you can place

an order with the sales agent of your choice

The Publications Office has a worldwide network of sales agents You can obtain their contact details by

sending a fax to (352) 29 29-42758

The mission of the JRC is to provide customer-driven scientific and technical support

for the conception development implementation and monitoring of EU policies As a

service of the European Commission the JRC functions as a reference centre of

science and technology for the Union Close to the policy-making process it serves

the common interest of the Member States while being independent of special

interests whether private or national

L

A-N

A-252

45-EN

-N

• 1 Summary
• 2 Introduction
• 3 Scope
• 4 Participating Laboratories
• 5 Time frame
• 6 Confidentiality
• 7 Test materials
• • 71 1Preparation of test materials
  • 72 Assigned values and standard deviation for proficiency assessment
  • 73 Homogeneity and stability
  • 74 Sample dispatch
  • • 8 Design of the proficiency test
    • 9 Evaluation of the results
    • • 91 2General
      • 92 21BEvaluation criteria
      • 93 Performance of participants
      • 94 Evaluation of the influence of analysis method
      • 95 Evaluation of the influence of calibration
      • 96 26Evaluation of compliance with legislation
      • • 10 Follow-up actions for underperforming laboratories
        • 11 1Conclusions
        • 12 1Acknowledgements
        • 13 References

60

ANNEX 10 Table 18 Method performance parameters reported by the participants

Laboratory code

Analyte LOD LOQ Recovery Analyte LOD LOQ Recovery

microgkg microgkg microgkg microgkg 3600 BaA 03 06 978 BaP 03 07 9483602 BaA 007 021 926 BaP 005 015 993603 BaA 0011 05 987 BaP 0005 051 9553604 BaA 02 04 86 BaP 02 04 883605 BaA 0025 005 118 BaP 0025 005 1083606 BaA 001 003 65 BaP 001 003 703607 BaA 021 071 74 BaP 027 089 943608 BaA 016 048 8213 BaP 015 046 83683610 BaA 02 05 85 BaP 005 02 853611 BaA BaP 3612 BaA 1 117 BaP 1 1203613 BaA 01 03 90 BaP 01 03 903614 BaA 02 05 96 BaP 02 05 933615 BaA 005 015 110 BaP 005 015 1053616 BaA 01 05 108 BaP 01 05 753617 BaA 018 037 405 BaP 009 018 3873618 BaA 01 03 63 BaP 01 03 593619 BaA 005 005 59 BaP 033 033 543620 BaA 0009 0018 64 BaP 0005 001 553621 BaA 007 02 84 BaP 007 02 923622 BaA 01 03 1101 BaP 01 03 9853623 BaA 02 08 105 BaP 02 08 1013624 BaA BaP 01 033 8513625 BaA BaP 3626 BaA BaP 5301 BaA 003 01 102 BaP 003 01 1005302 BaA 01 03 78 BaP 01 03 865303 BaA 003 01 62 BaP 003 01 805304 BaA 1 1 70 BaP 1 1 955305 BaA 02 05 85 BaP 02 05 855306 BaA 03 1 699 BaP 03 1 905307 BaA 6537 BaP 81165308 BaA 0088 0265 99 BaP 00262 00785 1005309 BaA 01 08 92 BaP 01 08 925310 BaA 01 03 BaP 01 03 905311 BaA 002 005 80 BaP 002 004 915312 BaA 03 09 105 BaP 03 09 1055313 BaA 04 08 BaP 04 08 5314 BaA 001 002 96 BaP 001 002 9485315 BaA 01 03 50 - 100 BaP 01 03 50 - 100 5316 BaA 002 011 827 BaP 004 019 10445317 BaA 05 1 76 BaP 05 1 755318 BaA BaP 5319 BaA 009 029 951 BaP 011 041 995320 BaA 12 18 946 BaP 06 1 8345321 BaA 03 05 BaP 05 1 5322 BaA 1 2 100 BaP 1 2 1005323 BaA 26 BaP 26

61

ANNEX 10 Table 18 - continued

Laboratory code Analyte LOD LOQ Recovery Analyte LOD LOQ Recovery

microgkg microgkg microgkg microgkg 3600 BbF 03 05 96 CHR 02 03 10153602 BbF 015 045 947 CHR 003 009 9193603 BbF 0014 05 1012 CHR 0004 05 10033604 BbF 02 04 85 CHR 02 04 873605 BbF 005 01 94 CHR 0025 005 863606 BbF 001 003 70 CHR 001 003 653607 BbF 018 06 100 CHR 014 028 943608 BbF 015 046 7979 CHR 015 045 71253610 BbF 005 02 85 CHR 02 05 853611 BbF CHR 3612 BbF 1 118 CHR 1 1173613 BbF 01 03 90 CHR 01 03 903614 BbF 01 04 96 CHR 02 05 953615 BbF 008 025 109 CHR 003 01 1083616 BbF 01 05 78 CHR 01 05 723617 BbF 035 07 38 CHR 012 024 4163618 BbF 01 03 62 CHR 01 03 623619 BbF 016 016 63 CHR 01 01 593620 BbF 0003 0006 55 CHR 001 002 653621 BbF 007 02 103 CHR 007 02 893622 BbF 02 06 963 CHR 03 09 1043623 BbF 02 08 106 CHR 02 08 1193624 BbF CHR 3625 BbF CHR 3626 BbF CHR 5301 BbF 003 01 96 CHR 003 01 1145302 BbF 01 03 84 CHR 01 03 835303 BbF 003 01 78 CHR 003 01 705304 BbF 1 1 86 CHR 1 1 705305 BbF 02 05 90 CHR 02 05 955306 BbF 03 1 1435 CHR 03 1 1185307 BbF 8116 CHR 65375308 BbF 0079 0238 99 CHR 009 026 1005309 BbF 01 08 92 CHR 5310 BbF 07 2 CHR 01 03 5311 BbF 002 004 95 CHR 002 004 895312 BbF 03 09 105 CHR 03 09 1055313 BbF 04 08 CHR 04 08 5314 BbF 001 002 83 CHR 001 002 8185315 BbF 02 05 50 -100 CHR 02 05 50 - 100 5316 BbF 005 024 814 CHR 001 007 9595317 BbF 05 1 73 CHR 05 1 785318 BbF CHR 5319 BbF 011 038 946 CHR 013 043 9675320 BbF 07 09 1194 CHR 23 32 10455321 BbF 05 1 CHR 05 1 5322 BbF 1 2 100 CHR 1 2 1005323 BbF 26 CHR 26

62

ANNEX 11 Table 19 Details of analysis method reported by the participants Data are presented as reported

Laboratory code

Analysis technique

Instrument calibration

Internal standards applied

Sample clean up

Details of sample

clean up

Extraction technique

Details of sample

extraction

3600 6) GC-MS

2) Internal standardisation with labelled substances

Deuterated PAH mix 9 (Ehrenstorfer)

1) Saponification 2) LiquidLiquid partitioning

D) Other Liquidliquid partitioning

3602 2) HPLC-UV-FLD

1) External calibration

3) Gel permeation chromatography

A) Pressurised liquid extraction (PLE)

3603 1) HPLC-FLD

1) External calibration

3) Gel permeation chromatography

D) Other 1 minute with dichloromethane in vortex

3604 2) HPLC-UV-FLD

2) Internal standardisation with labelled substances

DiP D14

5) Donor acceptor complex chromatography

D) Other LL

3605 1) HPLC-FLD

1) External calibration

3) Gel permeation chromatography

C) Soxhlet extraction

3606 7) GC-MSMS

2) Internal standardisation with labelled substances

BaP 13C4 CHR 13C6 BaA 13C6 BbF 13C6

4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

3607 6) GC-MS

2) Internal standardisation with labelled substances

BaA-d12 CHR-d12 BbF-d12 BkF-d12 BaP-d12 ICP-d12 BgP-d12 DiP-d14

1) Saponification

D) Other solvent partitioning

3608 1) HPLC-FLD

1) External calibration

6) Other Liquid extraction

D) Other

Extraction in a rotary agitator and centrifugation

3610 2) HPLC-UV-FLD

1) External calibration

4) Solid phase extraction

B) Sonication

3611

3612 6) GC-MS

2) Internal standardisation with labelled substances

chrysene-d12 dibenzo(ah)anthracene-d14 dibenzo(ai)pyrene-d14

4) Solid phase extraction

B) Sonication

3613 5) LC-MSMS

3) Internal standardisation with unlabelled substances

I use Chrysene-D12 3) Gel permeation chromatography

D) Other extraction with hexan

63

Table 19 - continued

Laboratory code

Analysis technique

Instrument calibration

Internal standards applied

Sample clean up Details of sample

clean up

Extraction technique

Details of sample

extraction

3614 6) GC-MS 1) External calibration

3) Gel permeation chromatography

B) Sonication

3615 1) HPLC-FLD

1) External calibration

6) Other PLE GPC

A) Pressurised liquid extraction (PLE)

3616 6) GC-MS

2) Internal standardisation with labelled substances

Benzo(a)pyrene-13C4 Benzo(a)anthracene-13C6 Benzo(b)fluoranthene-13C6 Chrysene-13C6

2) LiquidLiquid partitioning 4) Solid phase extraction

B) Sonication

3617 1) HPLC-FLD

1) External calibration

4) Solid phase extraction

C) Soxhlet extraction

3618 6) GC-MS

2) Internal standardisation with labelled substances

deuterated standards

1) Saponification 4) Solid phase extraction

D) Other Handshaken with cyclohexane

3619 6) GC-MS

2) Internal standardisation with labelled substances

C13 Labelled US EPA 16

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

D) Other Homegenisation amp saponiofication

3620 9) GC-HRMS

2) Internal standardisation with labelled substances

mix of deuterated PAHs (BAA-D12 CHR-D12 BAP-D12 BBF-D12)

3) Gel permeation chromatography 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

3621 6) GC-MS

2) Internal standardisation with labelled substances

Chrysene D12 Benzo(a)pyrene D12 Benzo(b)fluoranthene D12 Benzo(a)anthracene

2) LiquidLiquid partitioning 4) Solid phase extraction

B) Sonication

3622 1) HPLC-FLD

4) Standard addition method

4) Solid phase extraction

D) Other

Extraction with cyclohexane by heating under reflux

3623 7) GC-MSMS

2) Internal standardisation with labelled substances

C13 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

3624 2) HPLC-UV-FLD

1) External calibration

2) LiquidLiquid partitioning

D) Other hot solvent extraction

3626

64

Table 19 - continued

Laboratory code

Analysis technique

Instrument calibration

Internal standards

applied

Sample clean up

Details of sample

clean up

Extraction technique

Details of sample

extraction

5301 7) GC-MSMS

2) Internal standardisation with labelled substances

EACH SUBSTANCE C13

4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5302 7) GC-MSMS

2) Internal standardisation with labelled substances

Isotope labelled PAHs 13C for each analyte

4) Solid phase extraction

C) Soxhlet extraction

5303 7) GC-MSMS

3) Internal standardisation with unlabelled substances

HAP C13 4) Solid phase extraction

D) Other automate ASE200 DIONEX

5304 7) GC-MSMS

2) Internal standardisation with labelled substances

with C13 for each HAP

2) LiquidLiquid partitioning 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5305 7) GC-MSMS

2) Internal standardisation with labelled substances

C13 isotopiques internal standards

4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5306 7) GC-MSMS

2) Internal standardisation with labelled substances

C13 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5307 6) GC-MS

2) Internal standardisation with labelled substances

1) Saponification 2) LiquidLiquid partitioning

D) Other solvent extraction

5308 1) HPLC-FLD 1) External calibration

4) Solid phase extraction

B) Sonication

5309 1) HPLC-FLD 1) External calibration

4) Solid phase extraction

D) Other only SPE

5310 1) HPLC-FLD 1) External calibration

1) Saponification 2) LiquidLiquid partitioning 3) Gel permeation chromatography

C) Soxhlet extraction

5311 6) GC-MS

2) Internal standardisation with labelled substances

13 C Chrysene 13 C Benz(a)pyrene

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

B) Sonication

5312 1) HPLC-FLD

3) Internal standardisation with unlabelled substances

Benzo(b)chrysen

2) LiquidLiquid partitioning 4) Solid phase extraction

Quechers D) Other Quechers

65

Table 19 - continued

Laboratory code

Analysis technique

Instrument calibration

Internal standards applied

Sample clean up Details of sample

clean up

Extraction technique

Details of sample extraction

5313 6) GC-MS

2) Internal standardisation with labelled substances

Benz(a)anthracene D12 Benzo(b)fluranthene D12 Benzo(a)pyrene D12 Chrysene D12

1) Saponification 2) LiquidLiquid partitioning 3) Gel permeation chromatography

A) Pressurised liquid extraction (PLE)

5314 6) GC-MS

2) Internal standardisation with labelled substances

Benzo(a)pyren d12 2) LiquidLiquid partitioning

D) Other Extraction with

5315 1) HPLC-FLD

3) Internal standardisation with unlabelled substances

Benz(b)chrysen

1) Saponification 2) LiquidLiquid partitioning

D) Other shaker

5316 1) HPLC-FLD

1) External calibration

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

D) Other

LiquidLiquid extraction with cyclohexane and dimethylformamide

5317 7) GC-MSMS

1) External calibration

3) Gel permeation chromatography 6) Other

silica gel D) Other VDLUFA online method

5318

5319 1) HPLC-FLD

3) Internal standardisation with unlabelled substances

Benzo(b)chrysene 3) Gel permeation chromatography

A) Pressurised liquid extraction (PLE)

5320 6) GC-MS

3) Internal standardisation with unlabelled substances

22-binaphthyl

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

D) Other 3 hours refluxing

5321 1) HPLC-FLD

2) Internal standardisation with labelled substances

6-meacutethylchrysegravene

1) Saponification 2) LiquidLiquid partitioning

B) Sonication

5322 6) GC-MS

2) Internal standardisation with labelled substances

naftalene-d8 acenaphtalene-d10 phenanthrene-d10 chrysene-d12 perylene-d12 benzo(e)anthracene-d12

1) Saponification 2) LiquidLiquid partitioning 6) Other

clean-up with silica packed column

C) Soxhlet extraction

5323 1) HPLC-FLD

1) External calibration

4) Solid phase extraction 6) Other

Accelerated Solvent Extraction

A) Pressurised liquid extraction (PLE)

66

European Commission EUR 25245 EN ndash Joint Research Centre ndash Institute for Reference Materials and Measurements Title Report on the 8th inter-laboratory comparison organised by the European Union Reference Laboratory for Polycyclic Aromatic Hydrocarbons - Four marker PAHs in a dry extract of St Johns wort Author(s) Radoslav Lizak Szilard Szilagyi Philippe Verlinde and Thomas WENZL Luxembourg Publications Office of the European Union 2012 ndash 66 pp ndash 201 x 297 cm EUR ndash Scientific and Technical Research series ndash ISSN 1831-9424 ISBN 978-92-79-23337-1 doi 10278759387 Abstract The proficiency test here reported concerned the determination of the contents of four marker polycyclic aromatic hydrocarbons (PAHs) and of their sum in an food supplements test sample The set of marker PAHs consists of benz[a]anthacene benzo[a]pyrene benzo[b]fluoranthene and chrysene Participants to these PT were National Reference Laboratories for PAHs (NRLs-PAHs) and EU official food control laboratories The number of participants was in total 48

The PT was organised according ISO Standard 170432010

The test material used was a commercial dry extract of St Johns wort Participants also received a solution of the same PAHs either in acetonitrile or in toluene for checking their instrument calibration

The results from participants were rated with z-scores and zeta-scores About 68 of the reported results were attributed with z-scores with an absolute value of below two which is the threshold for satisfactory performance

How to obtain EU publications

Our priced publications are available from EU Bookshop (httpbookshopeuropaeu) where you can place

an order with the sales agent of your choice

The Publications Office has a worldwide network of sales agents You can obtain their contact details by

sending a fax to (352) 29 29-42758

The mission of the JRC is to provide customer-driven scientific and technical support

for the conception development implementation and monitoring of EU policies As a

service of the European Commission the JRC functions as a reference centre of

science and technology for the Union Close to the policy-making process it serves

the common interest of the Member States while being independent of special

interests whether private or national

L

A-N

A-252

45-EN

-N

• 1 Summary
• 2 Introduction
• 3 Scope
• 4 Participating Laboratories
• 5 Time frame
• 6 Confidentiality
• 7 Test materials
• • 71 1Preparation of test materials
  • 72 Assigned values and standard deviation for proficiency assessment
  • 73 Homogeneity and stability
  • 74 Sample dispatch
  • • 8 Design of the proficiency test
    • 9 Evaluation of the results
    • • 91 2General
      • 92 21BEvaluation criteria
      • 93 Performance of participants
      • 94 Evaluation of the influence of analysis method
      • 95 Evaluation of the influence of calibration
      • 96 26Evaluation of compliance with legislation
      • • 10 Follow-up actions for underperforming laboratories
        • 11 1Conclusions
        • 12 1Acknowledgements
        • 13 References

61

ANNEX 10 Table 18 - continued

Laboratory code Analyte LOD LOQ Recovery Analyte LOD LOQ Recovery

microgkg microgkg microgkg microgkg 3600 BbF 03 05 96 CHR 02 03 10153602 BbF 015 045 947 CHR 003 009 9193603 BbF 0014 05 1012 CHR 0004 05 10033604 BbF 02 04 85 CHR 02 04 873605 BbF 005 01 94 CHR 0025 005 863606 BbF 001 003 70 CHR 001 003 653607 BbF 018 06 100 CHR 014 028 943608 BbF 015 046 7979 CHR 015 045 71253610 BbF 005 02 85 CHR 02 05 853611 BbF CHR 3612 BbF 1 118 CHR 1 1173613 BbF 01 03 90 CHR 01 03 903614 BbF 01 04 96 CHR 02 05 953615 BbF 008 025 109 CHR 003 01 1083616 BbF 01 05 78 CHR 01 05 723617 BbF 035 07 38 CHR 012 024 4163618 BbF 01 03 62 CHR 01 03 623619 BbF 016 016 63 CHR 01 01 593620 BbF 0003 0006 55 CHR 001 002 653621 BbF 007 02 103 CHR 007 02 893622 BbF 02 06 963 CHR 03 09 1043623 BbF 02 08 106 CHR 02 08 1193624 BbF CHR 3625 BbF CHR 3626 BbF CHR 5301 BbF 003 01 96 CHR 003 01 1145302 BbF 01 03 84 CHR 01 03 835303 BbF 003 01 78 CHR 003 01 705304 BbF 1 1 86 CHR 1 1 705305 BbF 02 05 90 CHR 02 05 955306 BbF 03 1 1435 CHR 03 1 1185307 BbF 8116 CHR 65375308 BbF 0079 0238 99 CHR 009 026 1005309 BbF 01 08 92 CHR 5310 BbF 07 2 CHR 01 03 5311 BbF 002 004 95 CHR 002 004 895312 BbF 03 09 105 CHR 03 09 1055313 BbF 04 08 CHR 04 08 5314 BbF 001 002 83 CHR 001 002 8185315 BbF 02 05 50 -100 CHR 02 05 50 - 100 5316 BbF 005 024 814 CHR 001 007 9595317 BbF 05 1 73 CHR 05 1 785318 BbF CHR 5319 BbF 011 038 946 CHR 013 043 9675320 BbF 07 09 1194 CHR 23 32 10455321 BbF 05 1 CHR 05 1 5322 BbF 1 2 100 CHR 1 2 1005323 BbF 26 CHR 26

62

ANNEX 11 Table 19 Details of analysis method reported by the participants Data are presented as reported

Laboratory code

Analysis technique

Instrument calibration

Internal standards applied

Sample clean up

Details of sample

clean up

Extraction technique

Details of sample

extraction

3600 6) GC-MS

2) Internal standardisation with labelled substances

Deuterated PAH mix 9 (Ehrenstorfer)

1) Saponification 2) LiquidLiquid partitioning

D) Other Liquidliquid partitioning

3602 2) HPLC-UV-FLD

1) External calibration

3) Gel permeation chromatography

A) Pressurised liquid extraction (PLE)

3603 1) HPLC-FLD

1) External calibration

3) Gel permeation chromatography

D) Other 1 minute with dichloromethane in vortex

3604 2) HPLC-UV-FLD

2) Internal standardisation with labelled substances

DiP D14

5) Donor acceptor complex chromatography

D) Other LL

3605 1) HPLC-FLD

1) External calibration

3) Gel permeation chromatography

C) Soxhlet extraction

3606 7) GC-MSMS

2) Internal standardisation with labelled substances

BaP 13C4 CHR 13C6 BaA 13C6 BbF 13C6

4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

3607 6) GC-MS

2) Internal standardisation with labelled substances

BaA-d12 CHR-d12 BbF-d12 BkF-d12 BaP-d12 ICP-d12 BgP-d12 DiP-d14

1) Saponification

D) Other solvent partitioning

3608 1) HPLC-FLD

1) External calibration

6) Other Liquid extraction

D) Other

Extraction in a rotary agitator and centrifugation

3610 2) HPLC-UV-FLD

1) External calibration

4) Solid phase extraction

B) Sonication

3611

3612 6) GC-MS

2) Internal standardisation with labelled substances

chrysene-d12 dibenzo(ah)anthracene-d14 dibenzo(ai)pyrene-d14

4) Solid phase extraction

B) Sonication

3613 5) LC-MSMS

3) Internal standardisation with unlabelled substances

I use Chrysene-D12 3) Gel permeation chromatography

D) Other extraction with hexan

63

Table 19 - continued

Laboratory code

Analysis technique

Instrument calibration

Internal standards applied

Sample clean up Details of sample

clean up

Extraction technique

Details of sample

extraction

3614 6) GC-MS 1) External calibration

3) Gel permeation chromatography

B) Sonication

3615 1) HPLC-FLD

1) External calibration

6) Other PLE GPC

A) Pressurised liquid extraction (PLE)

3616 6) GC-MS

2) Internal standardisation with labelled substances

Benzo(a)pyrene-13C4 Benzo(a)anthracene-13C6 Benzo(b)fluoranthene-13C6 Chrysene-13C6

2) LiquidLiquid partitioning 4) Solid phase extraction

B) Sonication

3617 1) HPLC-FLD

1) External calibration

4) Solid phase extraction

C) Soxhlet extraction

3618 6) GC-MS

2) Internal standardisation with labelled substances

deuterated standards

1) Saponification 4) Solid phase extraction

D) Other Handshaken with cyclohexane

3619 6) GC-MS

2) Internal standardisation with labelled substances

C13 Labelled US EPA 16

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

D) Other Homegenisation amp saponiofication

3620 9) GC-HRMS

2) Internal standardisation with labelled substances

mix of deuterated PAHs (BAA-D12 CHR-D12 BAP-D12 BBF-D12)

3) Gel permeation chromatography 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

3621 6) GC-MS

2) Internal standardisation with labelled substances

Chrysene D12 Benzo(a)pyrene D12 Benzo(b)fluoranthene D12 Benzo(a)anthracene

2) LiquidLiquid partitioning 4) Solid phase extraction

B) Sonication

3622 1) HPLC-FLD

4) Standard addition method

4) Solid phase extraction

D) Other

Extraction with cyclohexane by heating under reflux

3623 7) GC-MSMS

2) Internal standardisation with labelled substances

C13 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

3624 2) HPLC-UV-FLD

1) External calibration

2) LiquidLiquid partitioning

D) Other hot solvent extraction

3626

64

Table 19 - continued

Laboratory code

Analysis technique

Instrument calibration

Internal standards

applied

Sample clean up

Details of sample

clean up

Extraction technique

Details of sample

extraction

5301 7) GC-MSMS

2) Internal standardisation with labelled substances

EACH SUBSTANCE C13

4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5302 7) GC-MSMS

2) Internal standardisation with labelled substances

Isotope labelled PAHs 13C for each analyte

4) Solid phase extraction

C) Soxhlet extraction

5303 7) GC-MSMS

3) Internal standardisation with unlabelled substances

HAP C13 4) Solid phase extraction

D) Other automate ASE200 DIONEX

5304 7) GC-MSMS

2) Internal standardisation with labelled substances

with C13 for each HAP

2) LiquidLiquid partitioning 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5305 7) GC-MSMS

2) Internal standardisation with labelled substances

C13 isotopiques internal standards

4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5306 7) GC-MSMS

2) Internal standardisation with labelled substances

C13 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5307 6) GC-MS

2) Internal standardisation with labelled substances

1) Saponification 2) LiquidLiquid partitioning

D) Other solvent extraction

5308 1) HPLC-FLD 1) External calibration

4) Solid phase extraction

B) Sonication

5309 1) HPLC-FLD 1) External calibration

4) Solid phase extraction

D) Other only SPE

5310 1) HPLC-FLD 1) External calibration

1) Saponification 2) LiquidLiquid partitioning 3) Gel permeation chromatography

C) Soxhlet extraction

5311 6) GC-MS

2) Internal standardisation with labelled substances

13 C Chrysene 13 C Benz(a)pyrene

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

B) Sonication

5312 1) HPLC-FLD

3) Internal standardisation with unlabelled substances

Benzo(b)chrysen

2) LiquidLiquid partitioning 4) Solid phase extraction

Quechers D) Other Quechers

65

Table 19 - continued

Laboratory code

Analysis technique

Instrument calibration

Internal standards applied

Sample clean up Details of sample

clean up

Extraction technique

Details of sample extraction

5313 6) GC-MS

2) Internal standardisation with labelled substances

Benz(a)anthracene D12 Benzo(b)fluranthene D12 Benzo(a)pyrene D12 Chrysene D12

1) Saponification 2) LiquidLiquid partitioning 3) Gel permeation chromatography

A) Pressurised liquid extraction (PLE)

5314 6) GC-MS

2) Internal standardisation with labelled substances

Benzo(a)pyren d12 2) LiquidLiquid partitioning

D) Other Extraction with

5315 1) HPLC-FLD

3) Internal standardisation with unlabelled substances

Benz(b)chrysen

1) Saponification 2) LiquidLiquid partitioning

D) Other shaker

5316 1) HPLC-FLD

1) External calibration

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

D) Other

LiquidLiquid extraction with cyclohexane and dimethylformamide

5317 7) GC-MSMS

1) External calibration

3) Gel permeation chromatography 6) Other

silica gel D) Other VDLUFA online method

5318

5319 1) HPLC-FLD

3) Internal standardisation with unlabelled substances

Benzo(b)chrysene 3) Gel permeation chromatography

A) Pressurised liquid extraction (PLE)

5320 6) GC-MS

3) Internal standardisation with unlabelled substances

22-binaphthyl

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

D) Other 3 hours refluxing

5321 1) HPLC-FLD

2) Internal standardisation with labelled substances

6-meacutethylchrysegravene

1) Saponification 2) LiquidLiquid partitioning

B) Sonication

5322 6) GC-MS

2) Internal standardisation with labelled substances

naftalene-d8 acenaphtalene-d10 phenanthrene-d10 chrysene-d12 perylene-d12 benzo(e)anthracene-d12

1) Saponification 2) LiquidLiquid partitioning 6) Other

clean-up with silica packed column

C) Soxhlet extraction

5323 1) HPLC-FLD

1) External calibration

4) Solid phase extraction 6) Other

Accelerated Solvent Extraction

A) Pressurised liquid extraction (PLE)

66

European Commission EUR 25245 EN ndash Joint Research Centre ndash Institute for Reference Materials and Measurements Title Report on the 8th inter-laboratory comparison organised by the European Union Reference Laboratory for Polycyclic Aromatic Hydrocarbons - Four marker PAHs in a dry extract of St Johns wort Author(s) Radoslav Lizak Szilard Szilagyi Philippe Verlinde and Thomas WENZL Luxembourg Publications Office of the European Union 2012 ndash 66 pp ndash 201 x 297 cm EUR ndash Scientific and Technical Research series ndash ISSN 1831-9424 ISBN 978-92-79-23337-1 doi 10278759387 Abstract The proficiency test here reported concerned the determination of the contents of four marker polycyclic aromatic hydrocarbons (PAHs) and of their sum in an food supplements test sample The set of marker PAHs consists of benz[a]anthacene benzo[a]pyrene benzo[b]fluoranthene and chrysene Participants to these PT were National Reference Laboratories for PAHs (NRLs-PAHs) and EU official food control laboratories The number of participants was in total 48

The PT was organised according ISO Standard 170432010

The test material used was a commercial dry extract of St Johns wort Participants also received a solution of the same PAHs either in acetonitrile or in toluene for checking their instrument calibration

The results from participants were rated with z-scores and zeta-scores About 68 of the reported results were attributed with z-scores with an absolute value of below two which is the threshold for satisfactory performance

How to obtain EU publications

Our priced publications are available from EU Bookshop (httpbookshopeuropaeu) where you can place

an order with the sales agent of your choice

The Publications Office has a worldwide network of sales agents You can obtain their contact details by

sending a fax to (352) 29 29-42758

The mission of the JRC is to provide customer-driven scientific and technical support

for the conception development implementation and monitoring of EU policies As a

service of the European Commission the JRC functions as a reference centre of

science and technology for the Union Close to the policy-making process it serves

the common interest of the Member States while being independent of special

interests whether private or national

L

A-N

A-252

45-EN

-N

• 1 Summary
• 2 Introduction
• 3 Scope
• 4 Participating Laboratories
• 5 Time frame
• 6 Confidentiality
• 7 Test materials
• • 71 1Preparation of test materials
  • 72 Assigned values and standard deviation for proficiency assessment
  • 73 Homogeneity and stability
  • 74 Sample dispatch
  • • 8 Design of the proficiency test
    • 9 Evaluation of the results
    • • 91 2General
      • 92 21BEvaluation criteria
      • 93 Performance of participants
      • 94 Evaluation of the influence of analysis method
      • 95 Evaluation of the influence of calibration
      • 96 26Evaluation of compliance with legislation
      • • 10 Follow-up actions for underperforming laboratories
        • 11 1Conclusions
        • 12 1Acknowledgements
        • 13 References

62

ANNEX 11 Table 19 Details of analysis method reported by the participants Data are presented as reported

Laboratory code

Analysis technique

Instrument calibration

Internal standards applied

Sample clean up

Details of sample

clean up

Extraction technique

Details of sample

extraction

3600 6) GC-MS

2) Internal standardisation with labelled substances

Deuterated PAH mix 9 (Ehrenstorfer)

1) Saponification 2) LiquidLiquid partitioning

D) Other Liquidliquid partitioning

3602 2) HPLC-UV-FLD

1) External calibration

3) Gel permeation chromatography

A) Pressurised liquid extraction (PLE)

3603 1) HPLC-FLD

1) External calibration

3) Gel permeation chromatography

D) Other 1 minute with dichloromethane in vortex

3604 2) HPLC-UV-FLD

2) Internal standardisation with labelled substances

DiP D14

5) Donor acceptor complex chromatography

D) Other LL

3605 1) HPLC-FLD

1) External calibration

3) Gel permeation chromatography

C) Soxhlet extraction

3606 7) GC-MSMS

2) Internal standardisation with labelled substances

BaP 13C4 CHR 13C6 BaA 13C6 BbF 13C6

4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

3607 6) GC-MS

2) Internal standardisation with labelled substances

BaA-d12 CHR-d12 BbF-d12 BkF-d12 BaP-d12 ICP-d12 BgP-d12 DiP-d14

1) Saponification

D) Other solvent partitioning

3608 1) HPLC-FLD

1) External calibration

6) Other Liquid extraction

D) Other

Extraction in a rotary agitator and centrifugation

3610 2) HPLC-UV-FLD

1) External calibration

4) Solid phase extraction

B) Sonication

3611

3612 6) GC-MS

2) Internal standardisation with labelled substances

chrysene-d12 dibenzo(ah)anthracene-d14 dibenzo(ai)pyrene-d14

4) Solid phase extraction

B) Sonication

3613 5) LC-MSMS

3) Internal standardisation with unlabelled substances

I use Chrysene-D12 3) Gel permeation chromatography

D) Other extraction with hexan

63

Table 19 - continued

Laboratory code

Analysis technique

Instrument calibration

Internal standards applied

Sample clean up Details of sample

clean up

Extraction technique

Details of sample

extraction

3614 6) GC-MS 1) External calibration

3) Gel permeation chromatography

B) Sonication

3615 1) HPLC-FLD

1) External calibration

6) Other PLE GPC

A) Pressurised liquid extraction (PLE)

3616 6) GC-MS

2) Internal standardisation with labelled substances

Benzo(a)pyrene-13C4 Benzo(a)anthracene-13C6 Benzo(b)fluoranthene-13C6 Chrysene-13C6

2) LiquidLiquid partitioning 4) Solid phase extraction

B) Sonication

3617 1) HPLC-FLD

1) External calibration

4) Solid phase extraction

C) Soxhlet extraction

3618 6) GC-MS

2) Internal standardisation with labelled substances

deuterated standards

1) Saponification 4) Solid phase extraction

D) Other Handshaken with cyclohexane

3619 6) GC-MS

2) Internal standardisation with labelled substances

C13 Labelled US EPA 16

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

D) Other Homegenisation amp saponiofication

3620 9) GC-HRMS

2) Internal standardisation with labelled substances

mix of deuterated PAHs (BAA-D12 CHR-D12 BAP-D12 BBF-D12)

3) Gel permeation chromatography 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

3621 6) GC-MS

2) Internal standardisation with labelled substances

Chrysene D12 Benzo(a)pyrene D12 Benzo(b)fluoranthene D12 Benzo(a)anthracene

2) LiquidLiquid partitioning 4) Solid phase extraction

B) Sonication

3622 1) HPLC-FLD

4) Standard addition method

4) Solid phase extraction

D) Other

Extraction with cyclohexane by heating under reflux

3623 7) GC-MSMS

2) Internal standardisation with labelled substances

C13 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

3624 2) HPLC-UV-FLD

1) External calibration

2) LiquidLiquid partitioning

D) Other hot solvent extraction

3626

64

Table 19 - continued

Laboratory code

Analysis technique

Instrument calibration

Internal standards

applied

Sample clean up

Details of sample

clean up

Extraction technique

Details of sample

extraction

5301 7) GC-MSMS

2) Internal standardisation with labelled substances

EACH SUBSTANCE C13

4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5302 7) GC-MSMS

2) Internal standardisation with labelled substances

Isotope labelled PAHs 13C for each analyte

4) Solid phase extraction

C) Soxhlet extraction

5303 7) GC-MSMS

3) Internal standardisation with unlabelled substances

HAP C13 4) Solid phase extraction

D) Other automate ASE200 DIONEX

5304 7) GC-MSMS

2) Internal standardisation with labelled substances

with C13 for each HAP

2) LiquidLiquid partitioning 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5305 7) GC-MSMS

2) Internal standardisation with labelled substances

C13 isotopiques internal standards

4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5306 7) GC-MSMS

2) Internal standardisation with labelled substances

C13 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5307 6) GC-MS

2) Internal standardisation with labelled substances

1) Saponification 2) LiquidLiquid partitioning

D) Other solvent extraction

5308 1) HPLC-FLD 1) External calibration

4) Solid phase extraction

B) Sonication

5309 1) HPLC-FLD 1) External calibration

4) Solid phase extraction

D) Other only SPE

5310 1) HPLC-FLD 1) External calibration

1) Saponification 2) LiquidLiquid partitioning 3) Gel permeation chromatography

C) Soxhlet extraction

5311 6) GC-MS

2) Internal standardisation with labelled substances

13 C Chrysene 13 C Benz(a)pyrene

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

B) Sonication

5312 1) HPLC-FLD

3) Internal standardisation with unlabelled substances

Benzo(b)chrysen

2) LiquidLiquid partitioning 4) Solid phase extraction

Quechers D) Other Quechers

65

Table 19 - continued

Laboratory code

Analysis technique

Instrument calibration

Internal standards applied

Sample clean up Details of sample

clean up

Extraction technique

Details of sample extraction

5313 6) GC-MS

2) Internal standardisation with labelled substances

Benz(a)anthracene D12 Benzo(b)fluranthene D12 Benzo(a)pyrene D12 Chrysene D12

1) Saponification 2) LiquidLiquid partitioning 3) Gel permeation chromatography

A) Pressurised liquid extraction (PLE)

5314 6) GC-MS

2) Internal standardisation with labelled substances

Benzo(a)pyren d12 2) LiquidLiquid partitioning

D) Other Extraction with

5315 1) HPLC-FLD

3) Internal standardisation with unlabelled substances

Benz(b)chrysen

1) Saponification 2) LiquidLiquid partitioning

D) Other shaker

5316 1) HPLC-FLD

1) External calibration

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

D) Other

LiquidLiquid extraction with cyclohexane and dimethylformamide

5317 7) GC-MSMS

1) External calibration

3) Gel permeation chromatography 6) Other

silica gel D) Other VDLUFA online method

5318

5319 1) HPLC-FLD

3) Internal standardisation with unlabelled substances

Benzo(b)chrysene 3) Gel permeation chromatography

A) Pressurised liquid extraction (PLE)

5320 6) GC-MS

3) Internal standardisation with unlabelled substances

22-binaphthyl

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

D) Other 3 hours refluxing

5321 1) HPLC-FLD

2) Internal standardisation with labelled substances

6-meacutethylchrysegravene

1) Saponification 2) LiquidLiquid partitioning

B) Sonication

5322 6) GC-MS

2) Internal standardisation with labelled substances

naftalene-d8 acenaphtalene-d10 phenanthrene-d10 chrysene-d12 perylene-d12 benzo(e)anthracene-d12

1) Saponification 2) LiquidLiquid partitioning 6) Other

clean-up with silica packed column

C) Soxhlet extraction

5323 1) HPLC-FLD

1) External calibration

4) Solid phase extraction 6) Other

Accelerated Solvent Extraction

A) Pressurised liquid extraction (PLE)

66

European Commission EUR 25245 EN ndash Joint Research Centre ndash Institute for Reference Materials and Measurements Title Report on the 8th inter-laboratory comparison organised by the European Union Reference Laboratory for Polycyclic Aromatic Hydrocarbons - Four marker PAHs in a dry extract of St Johns wort Author(s) Radoslav Lizak Szilard Szilagyi Philippe Verlinde and Thomas WENZL Luxembourg Publications Office of the European Union 2012 ndash 66 pp ndash 201 x 297 cm EUR ndash Scientific and Technical Research series ndash ISSN 1831-9424 ISBN 978-92-79-23337-1 doi 10278759387 Abstract The proficiency test here reported concerned the determination of the contents of four marker polycyclic aromatic hydrocarbons (PAHs) and of their sum in an food supplements test sample The set of marker PAHs consists of benz[a]anthacene benzo[a]pyrene benzo[b]fluoranthene and chrysene Participants to these PT were National Reference Laboratories for PAHs (NRLs-PAHs) and EU official food control laboratories The number of participants was in total 48

The PT was organised according ISO Standard 170432010

The test material used was a commercial dry extract of St Johns wort Participants also received a solution of the same PAHs either in acetonitrile or in toluene for checking their instrument calibration

The results from participants were rated with z-scores and zeta-scores About 68 of the reported results were attributed with z-scores with an absolute value of below two which is the threshold for satisfactory performance

How to obtain EU publications

Our priced publications are available from EU Bookshop (httpbookshopeuropaeu) where you can place

an order with the sales agent of your choice

The Publications Office has a worldwide network of sales agents You can obtain their contact details by

sending a fax to (352) 29 29-42758

The mission of the JRC is to provide customer-driven scientific and technical support

for the conception development implementation and monitoring of EU policies As a

service of the European Commission the JRC functions as a reference centre of

science and technology for the Union Close to the policy-making process it serves

the common interest of the Member States while being independent of special

interests whether private or national

L

A-N

A-252

45-EN

-N

• 1 Summary
• 2 Introduction
• 3 Scope
• 4 Participating Laboratories
• 5 Time frame
• 6 Confidentiality
• 7 Test materials
• • 71 1Preparation of test materials
  • 72 Assigned values and standard deviation for proficiency assessment
  • 73 Homogeneity and stability
  • 74 Sample dispatch
  • • 8 Design of the proficiency test
    • 9 Evaluation of the results
    • • 91 2General
      • 92 21BEvaluation criteria
      • 93 Performance of participants
      • 94 Evaluation of the influence of analysis method
      • 95 Evaluation of the influence of calibration
      • 96 26Evaluation of compliance with legislation
      • • 10 Follow-up actions for underperforming laboratories
        • 11 1Conclusions
        • 12 1Acknowledgements
        • 13 References

63

Table 19 - continued

Laboratory code

Analysis technique

Instrument calibration

Internal standards applied

Sample clean up Details of sample

clean up

Extraction technique

Details of sample

extraction

3614 6) GC-MS 1) External calibration

3) Gel permeation chromatography

B) Sonication

3615 1) HPLC-FLD

1) External calibration

6) Other PLE GPC

A) Pressurised liquid extraction (PLE)

3616 6) GC-MS

2) Internal standardisation with labelled substances

Benzo(a)pyrene-13C4 Benzo(a)anthracene-13C6 Benzo(b)fluoranthene-13C6 Chrysene-13C6

2) LiquidLiquid partitioning 4) Solid phase extraction

B) Sonication

3617 1) HPLC-FLD

1) External calibration

4) Solid phase extraction

C) Soxhlet extraction

3618 6) GC-MS

2) Internal standardisation with labelled substances

deuterated standards

1) Saponification 4) Solid phase extraction

D) Other Handshaken with cyclohexane

3619 6) GC-MS

2) Internal standardisation with labelled substances

C13 Labelled US EPA 16

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

D) Other Homegenisation amp saponiofication

3620 9) GC-HRMS

2) Internal standardisation with labelled substances

mix of deuterated PAHs (BAA-D12 CHR-D12 BAP-D12 BBF-D12)

3) Gel permeation chromatography 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

3621 6) GC-MS

2) Internal standardisation with labelled substances

Chrysene D12 Benzo(a)pyrene D12 Benzo(b)fluoranthene D12 Benzo(a)anthracene

2) LiquidLiquid partitioning 4) Solid phase extraction

B) Sonication

3622 1) HPLC-FLD

4) Standard addition method

4) Solid phase extraction

D) Other

Extraction with cyclohexane by heating under reflux

3623 7) GC-MSMS

2) Internal standardisation with labelled substances

C13 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

3624 2) HPLC-UV-FLD

1) External calibration

2) LiquidLiquid partitioning

D) Other hot solvent extraction

3626

64

Table 19 - continued

Laboratory code

Analysis technique

Instrument calibration

Internal standards

applied

Sample clean up

Details of sample

clean up

Extraction technique

Details of sample

extraction

5301 7) GC-MSMS

2) Internal standardisation with labelled substances

EACH SUBSTANCE C13

4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5302 7) GC-MSMS

2) Internal standardisation with labelled substances

Isotope labelled PAHs 13C for each analyte

4) Solid phase extraction

C) Soxhlet extraction

5303 7) GC-MSMS

3) Internal standardisation with unlabelled substances

HAP C13 4) Solid phase extraction

D) Other automate ASE200 DIONEX

5304 7) GC-MSMS

2) Internal standardisation with labelled substances

with C13 for each HAP

2) LiquidLiquid partitioning 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5305 7) GC-MSMS

2) Internal standardisation with labelled substances

C13 isotopiques internal standards

4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5306 7) GC-MSMS

2) Internal standardisation with labelled substances

C13 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5307 6) GC-MS

2) Internal standardisation with labelled substances

1) Saponification 2) LiquidLiquid partitioning

D) Other solvent extraction

5308 1) HPLC-FLD 1) External calibration

4) Solid phase extraction

B) Sonication

5309 1) HPLC-FLD 1) External calibration

4) Solid phase extraction

D) Other only SPE

5310 1) HPLC-FLD 1) External calibration

1) Saponification 2) LiquidLiquid partitioning 3) Gel permeation chromatography

C) Soxhlet extraction

5311 6) GC-MS

2) Internal standardisation with labelled substances

13 C Chrysene 13 C Benz(a)pyrene

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

B) Sonication

5312 1) HPLC-FLD

3) Internal standardisation with unlabelled substances

Benzo(b)chrysen

2) LiquidLiquid partitioning 4) Solid phase extraction

Quechers D) Other Quechers

65

Table 19 - continued

Laboratory code

Analysis technique

Instrument calibration

Internal standards applied

Sample clean up Details of sample

clean up

Extraction technique

Details of sample extraction

5313 6) GC-MS

2) Internal standardisation with labelled substances

Benz(a)anthracene D12 Benzo(b)fluranthene D12 Benzo(a)pyrene D12 Chrysene D12

1) Saponification 2) LiquidLiquid partitioning 3) Gel permeation chromatography

A) Pressurised liquid extraction (PLE)

5314 6) GC-MS

2) Internal standardisation with labelled substances

Benzo(a)pyren d12 2) LiquidLiquid partitioning

D) Other Extraction with

5315 1) HPLC-FLD

3) Internal standardisation with unlabelled substances

Benz(b)chrysen

1) Saponification 2) LiquidLiquid partitioning

D) Other shaker

5316 1) HPLC-FLD

1) External calibration

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

D) Other

LiquidLiquid extraction with cyclohexane and dimethylformamide

5317 7) GC-MSMS

1) External calibration

3) Gel permeation chromatography 6) Other

silica gel D) Other VDLUFA online method

5318

5319 1) HPLC-FLD

3) Internal standardisation with unlabelled substances

Benzo(b)chrysene 3) Gel permeation chromatography

A) Pressurised liquid extraction (PLE)

5320 6) GC-MS

3) Internal standardisation with unlabelled substances

22-binaphthyl

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

D) Other 3 hours refluxing

5321 1) HPLC-FLD

2) Internal standardisation with labelled substances

6-meacutethylchrysegravene

1) Saponification 2) LiquidLiquid partitioning

B) Sonication

5322 6) GC-MS

2) Internal standardisation with labelled substances

naftalene-d8 acenaphtalene-d10 phenanthrene-d10 chrysene-d12 perylene-d12 benzo(e)anthracene-d12

1) Saponification 2) LiquidLiquid partitioning 6) Other

clean-up with silica packed column

C) Soxhlet extraction

5323 1) HPLC-FLD

1) External calibration

4) Solid phase extraction 6) Other

Accelerated Solvent Extraction

A) Pressurised liquid extraction (PLE)

66

European Commission EUR 25245 EN ndash Joint Research Centre ndash Institute for Reference Materials and Measurements Title Report on the 8th inter-laboratory comparison organised by the European Union Reference Laboratory for Polycyclic Aromatic Hydrocarbons - Four marker PAHs in a dry extract of St Johns wort Author(s) Radoslav Lizak Szilard Szilagyi Philippe Verlinde and Thomas WENZL Luxembourg Publications Office of the European Union 2012 ndash 66 pp ndash 201 x 297 cm EUR ndash Scientific and Technical Research series ndash ISSN 1831-9424 ISBN 978-92-79-23337-1 doi 10278759387 Abstract The proficiency test here reported concerned the determination of the contents of four marker polycyclic aromatic hydrocarbons (PAHs) and of their sum in an food supplements test sample The set of marker PAHs consists of benz[a]anthacene benzo[a]pyrene benzo[b]fluoranthene and chrysene Participants to these PT were National Reference Laboratories for PAHs (NRLs-PAHs) and EU official food control laboratories The number of participants was in total 48

The PT was organised according ISO Standard 170432010

The test material used was a commercial dry extract of St Johns wort Participants also received a solution of the same PAHs either in acetonitrile or in toluene for checking their instrument calibration

The results from participants were rated with z-scores and zeta-scores About 68 of the reported results were attributed with z-scores with an absolute value of below two which is the threshold for satisfactory performance

How to obtain EU publications

Our priced publications are available from EU Bookshop (httpbookshopeuropaeu) where you can place

an order with the sales agent of your choice

The Publications Office has a worldwide network of sales agents You can obtain their contact details by

sending a fax to (352) 29 29-42758

The mission of the JRC is to provide customer-driven scientific and technical support

for the conception development implementation and monitoring of EU policies As a

service of the European Commission the JRC functions as a reference centre of

science and technology for the Union Close to the policy-making process it serves

the common interest of the Member States while being independent of special

interests whether private or national

L

A-N

A-252

45-EN

-N

• 1 Summary
• 2 Introduction
• 3 Scope
• 4 Participating Laboratories
• 5 Time frame
• 6 Confidentiality
• 7 Test materials
• • 71 1Preparation of test materials
  • 72 Assigned values and standard deviation for proficiency assessment
  • 73 Homogeneity and stability
  • 74 Sample dispatch
  • • 8 Design of the proficiency test
    • 9 Evaluation of the results
    • • 91 2General
      • 92 21BEvaluation criteria
      • 93 Performance of participants
      • 94 Evaluation of the influence of analysis method
      • 95 Evaluation of the influence of calibration
      • 96 26Evaluation of compliance with legislation
      • • 10 Follow-up actions for underperforming laboratories
        • 11 1Conclusions
        • 12 1Acknowledgements
        • 13 References

64

Table 19 - continued

Laboratory code

Analysis technique

Instrument calibration

Internal standards

applied

Sample clean up

Details of sample

clean up

Extraction technique

Details of sample

extraction

5301 7) GC-MSMS

2) Internal standardisation with labelled substances

EACH SUBSTANCE C13

4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5302 7) GC-MSMS

2) Internal standardisation with labelled substances

Isotope labelled PAHs 13C for each analyte

4) Solid phase extraction

C) Soxhlet extraction

5303 7) GC-MSMS

3) Internal standardisation with unlabelled substances

HAP C13 4) Solid phase extraction

D) Other automate ASE200 DIONEX

5304 7) GC-MSMS

2) Internal standardisation with labelled substances

with C13 for each HAP

2) LiquidLiquid partitioning 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5305 7) GC-MSMS

2) Internal standardisation with labelled substances

C13 isotopiques internal standards

4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5306 7) GC-MSMS

2) Internal standardisation with labelled substances

C13 4) Solid phase extraction

A) Pressurised liquid extraction (PLE)

5307 6) GC-MS

2) Internal standardisation with labelled substances

1) Saponification 2) LiquidLiquid partitioning

D) Other solvent extraction

5308 1) HPLC-FLD 1) External calibration

4) Solid phase extraction

B) Sonication

5309 1) HPLC-FLD 1) External calibration

4) Solid phase extraction

D) Other only SPE

5310 1) HPLC-FLD 1) External calibration

1) Saponification 2) LiquidLiquid partitioning 3) Gel permeation chromatography

C) Soxhlet extraction

5311 6) GC-MS

2) Internal standardisation with labelled substances

13 C Chrysene 13 C Benz(a)pyrene

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

B) Sonication

5312 1) HPLC-FLD

3) Internal standardisation with unlabelled substances

Benzo(b)chrysen

2) LiquidLiquid partitioning 4) Solid phase extraction

Quechers D) Other Quechers

65

Table 19 - continued

Laboratory code

Analysis technique

Instrument calibration

Internal standards applied

Sample clean up Details of sample

clean up

Extraction technique

Details of sample extraction

5313 6) GC-MS

2) Internal standardisation with labelled substances

Benz(a)anthracene D12 Benzo(b)fluranthene D12 Benzo(a)pyrene D12 Chrysene D12

1) Saponification 2) LiquidLiquid partitioning 3) Gel permeation chromatography

A) Pressurised liquid extraction (PLE)

5314 6) GC-MS

2) Internal standardisation with labelled substances

Benzo(a)pyren d12 2) LiquidLiquid partitioning

D) Other Extraction with

5315 1) HPLC-FLD

3) Internal standardisation with unlabelled substances

Benz(b)chrysen

1) Saponification 2) LiquidLiquid partitioning

D) Other shaker

5316 1) HPLC-FLD

1) External calibration

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

D) Other

LiquidLiquid extraction with cyclohexane and dimethylformamide

5317 7) GC-MSMS

1) External calibration

3) Gel permeation chromatography 6) Other

silica gel D) Other VDLUFA online method

5318

5319 1) HPLC-FLD

3) Internal standardisation with unlabelled substances

Benzo(b)chrysene 3) Gel permeation chromatography

A) Pressurised liquid extraction (PLE)

5320 6) GC-MS

3) Internal standardisation with unlabelled substances

22-binaphthyl

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

D) Other 3 hours refluxing

5321 1) HPLC-FLD

2) Internal standardisation with labelled substances

6-meacutethylchrysegravene

1) Saponification 2) LiquidLiquid partitioning

B) Sonication

5322 6) GC-MS

2) Internal standardisation with labelled substances

naftalene-d8 acenaphtalene-d10 phenanthrene-d10 chrysene-d12 perylene-d12 benzo(e)anthracene-d12

1) Saponification 2) LiquidLiquid partitioning 6) Other

clean-up with silica packed column

C) Soxhlet extraction

5323 1) HPLC-FLD

1) External calibration

4) Solid phase extraction 6) Other

Accelerated Solvent Extraction

A) Pressurised liquid extraction (PLE)

66

European Commission EUR 25245 EN ndash Joint Research Centre ndash Institute for Reference Materials and Measurements Title Report on the 8th inter-laboratory comparison organised by the European Union Reference Laboratory for Polycyclic Aromatic Hydrocarbons - Four marker PAHs in a dry extract of St Johns wort Author(s) Radoslav Lizak Szilard Szilagyi Philippe Verlinde and Thomas WENZL Luxembourg Publications Office of the European Union 2012 ndash 66 pp ndash 201 x 297 cm EUR ndash Scientific and Technical Research series ndash ISSN 1831-9424 ISBN 978-92-79-23337-1 doi 10278759387 Abstract The proficiency test here reported concerned the determination of the contents of four marker polycyclic aromatic hydrocarbons (PAHs) and of their sum in an food supplements test sample The set of marker PAHs consists of benz[a]anthacene benzo[a]pyrene benzo[b]fluoranthene and chrysene Participants to these PT were National Reference Laboratories for PAHs (NRLs-PAHs) and EU official food control laboratories The number of participants was in total 48

The PT was organised according ISO Standard 170432010

The test material used was a commercial dry extract of St Johns wort Participants also received a solution of the same PAHs either in acetonitrile or in toluene for checking their instrument calibration

The results from participants were rated with z-scores and zeta-scores About 68 of the reported results were attributed with z-scores with an absolute value of below two which is the threshold for satisfactory performance

How to obtain EU publications

Our priced publications are available from EU Bookshop (httpbookshopeuropaeu) where you can place

an order with the sales agent of your choice

The Publications Office has a worldwide network of sales agents You can obtain their contact details by

sending a fax to (352) 29 29-42758

The mission of the JRC is to provide customer-driven scientific and technical support

for the conception development implementation and monitoring of EU policies As a

service of the European Commission the JRC functions as a reference centre of

science and technology for the Union Close to the policy-making process it serves

the common interest of the Member States while being independent of special

interests whether private or national

L

A-N

A-252

45-EN

-N

• 1 Summary
• 2 Introduction
• 3 Scope
• 4 Participating Laboratories
• 5 Time frame
• 6 Confidentiality
• 7 Test materials
• • 71 1Preparation of test materials
  • 72 Assigned values and standard deviation for proficiency assessment
  • 73 Homogeneity and stability
  • 74 Sample dispatch
  • • 8 Design of the proficiency test
    • 9 Evaluation of the results
    • • 91 2General
      • 92 21BEvaluation criteria
      • 93 Performance of participants
      • 94 Evaluation of the influence of analysis method
      • 95 Evaluation of the influence of calibration
      • 96 26Evaluation of compliance with legislation
      • • 10 Follow-up actions for underperforming laboratories
        • 11 1Conclusions
        • 12 1Acknowledgements
        • 13 References

65

Table 19 - continued

Laboratory code

Analysis technique

Instrument calibration

Internal standards applied

Sample clean up Details of sample

clean up

Extraction technique

Details of sample extraction

5313 6) GC-MS

2) Internal standardisation with labelled substances

Benz(a)anthracene D12 Benzo(b)fluranthene D12 Benzo(a)pyrene D12 Chrysene D12

1) Saponification 2) LiquidLiquid partitioning 3) Gel permeation chromatography

A) Pressurised liquid extraction (PLE)

5314 6) GC-MS

2) Internal standardisation with labelled substances

Benzo(a)pyren d12 2) LiquidLiquid partitioning

D) Other Extraction with

5315 1) HPLC-FLD

3) Internal standardisation with unlabelled substances

Benz(b)chrysen

1) Saponification 2) LiquidLiquid partitioning

D) Other shaker

5316 1) HPLC-FLD

1) External calibration

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

D) Other

LiquidLiquid extraction with cyclohexane and dimethylformamide

5317 7) GC-MSMS

1) External calibration

3) Gel permeation chromatography 6) Other

silica gel D) Other VDLUFA online method

5318

5319 1) HPLC-FLD

3) Internal standardisation with unlabelled substances

Benzo(b)chrysene 3) Gel permeation chromatography

A) Pressurised liquid extraction (PLE)

5320 6) GC-MS

3) Internal standardisation with unlabelled substances

22-binaphthyl

1) Saponification 2) LiquidLiquid partitioning 4) Solid phase extraction

D) Other 3 hours refluxing

5321 1) HPLC-FLD

2) Internal standardisation with labelled substances

6-meacutethylchrysegravene

1) Saponification 2) LiquidLiquid partitioning

B) Sonication

5322 6) GC-MS

2) Internal standardisation with labelled substances

naftalene-d8 acenaphtalene-d10 phenanthrene-d10 chrysene-d12 perylene-d12 benzo(e)anthracene-d12

1) Saponification 2) LiquidLiquid partitioning 6) Other

clean-up with silica packed column

C) Soxhlet extraction

5323 1) HPLC-FLD

1) External calibration

4) Solid phase extraction 6) Other

Accelerated Solvent Extraction

A) Pressurised liquid extraction (PLE)

66

European Commission EUR 25245 EN ndash Joint Research Centre ndash Institute for Reference Materials and Measurements Title Report on the 8th inter-laboratory comparison organised by the European Union Reference Laboratory for Polycyclic Aromatic Hydrocarbons - Four marker PAHs in a dry extract of St Johns wort Author(s) Radoslav Lizak Szilard Szilagyi Philippe Verlinde and Thomas WENZL Luxembourg Publications Office of the European Union 2012 ndash 66 pp ndash 201 x 297 cm EUR ndash Scientific and Technical Research series ndash ISSN 1831-9424 ISBN 978-92-79-23337-1 doi 10278759387 Abstract The proficiency test here reported concerned the determination of the contents of four marker polycyclic aromatic hydrocarbons (PAHs) and of their sum in an food supplements test sample The set of marker PAHs consists of benz[a]anthacene benzo[a]pyrene benzo[b]fluoranthene and chrysene Participants to these PT were National Reference Laboratories for PAHs (NRLs-PAHs) and EU official food control laboratories The number of participants was in total 48

The PT was organised according ISO Standard 170432010

The test material used was a commercial dry extract of St Johns wort Participants also received a solution of the same PAHs either in acetonitrile or in toluene for checking their instrument calibration

The results from participants were rated with z-scores and zeta-scores About 68 of the reported results were attributed with z-scores with an absolute value of below two which is the threshold for satisfactory performance

How to obtain EU publications

Our priced publications are available from EU Bookshop (httpbookshopeuropaeu) where you can place

an order with the sales agent of your choice

The Publications Office has a worldwide network of sales agents You can obtain their contact details by

sending a fax to (352) 29 29-42758

The mission of the JRC is to provide customer-driven scientific and technical support

for the conception development implementation and monitoring of EU policies As a

service of the European Commission the JRC functions as a reference centre of

science and technology for the Union Close to the policy-making process it serves

the common interest of the Member States while being independent of special

interests whether private or national

L

A-N

A-252

45-EN

-N

• 1 Summary
• 2 Introduction
• 3 Scope
• 4 Participating Laboratories
• 5 Time frame
• 6 Confidentiality
• 7 Test materials
• • 71 1Preparation of test materials
  • 72 Assigned values and standard deviation for proficiency assessment
  • 73 Homogeneity and stability
  • 74 Sample dispatch
  • • 8 Design of the proficiency test
    • 9 Evaluation of the results
    • • 91 2General
      • 92 21BEvaluation criteria
      • 93 Performance of participants
      • 94 Evaluation of the influence of analysis method
      • 95 Evaluation of the influence of calibration
      • 96 26Evaluation of compliance with legislation
      • • 10 Follow-up actions for underperforming laboratories
        • 11 1Conclusions
        • 12 1Acknowledgements
        • 13 References

66

European Commission EUR 25245 EN ndash Joint Research Centre ndash Institute for Reference Materials and Measurements Title Report on the 8th inter-laboratory comparison organised by the European Union Reference Laboratory for Polycyclic Aromatic Hydrocarbons - Four marker PAHs in a dry extract of St Johns wort Author(s) Radoslav Lizak Szilard Szilagyi Philippe Verlinde and Thomas WENZL Luxembourg Publications Office of the European Union 2012 ndash 66 pp ndash 201 x 297 cm EUR ndash Scientific and Technical Research series ndash ISSN 1831-9424 ISBN 978-92-79-23337-1 doi 10278759387 Abstract The proficiency test here reported concerned the determination of the contents of four marker polycyclic aromatic hydrocarbons (PAHs) and of their sum in an food supplements test sample The set of marker PAHs consists of benz[a]anthacene benzo[a]pyrene benzo[b]fluoranthene and chrysene Participants to these PT were National Reference Laboratories for PAHs (NRLs-PAHs) and EU official food control laboratories The number of participants was in total 48

The PT was organised according ISO Standard 170432010

The test material used was a commercial dry extract of St Johns wort Participants also received a solution of the same PAHs either in acetonitrile or in toluene for checking their instrument calibration

The results from participants were rated with z-scores and zeta-scores About 68 of the reported results were attributed with z-scores with an absolute value of below two which is the threshold for satisfactory performance

How to obtain EU publications

Our priced publications are available from EU Bookshop (httpbookshopeuropaeu) where you can place

an order with the sales agent of your choice

The Publications Office has a worldwide network of sales agents You can obtain their contact details by

sending a fax to (352) 29 29-42758

The mission of the JRC is to provide customer-driven scientific and technical support

for the conception development implementation and monitoring of EU policies As a

service of the European Commission the JRC functions as a reference centre of

science and technology for the Union Close to the policy-making process it serves

the common interest of the Member States while being independent of special

interests whether private or national

L

A-N

A-252

45-EN

-N

• 1 Summary
• 2 Introduction
• 3 Scope
• 4 Participating Laboratories
• 5 Time frame
• 6 Confidentiality
• 7 Test materials
• • 71 1Preparation of test materials
  • 72 Assigned values and standard deviation for proficiency assessment
  • 73 Homogeneity and stability
  • 74 Sample dispatch
  • • 8 Design of the proficiency test
    • 9 Evaluation of the results
    • • 91 2General
      • 92 21BEvaluation criteria
      • 93 Performance of participants
      • 94 Evaluation of the influence of analysis method
      • 95 Evaluation of the influence of calibration
      • 96 26Evaluation of compliance with legislation
      • • 10 Follow-up actions for underperforming laboratories
        • 11 1Conclusions
        • 12 1Acknowledgements
        • 13 References

How to obtain EU publications

Our priced publications are available from EU Bookshop (httpbookshopeuropaeu) where you can place

an order with the sales agent of your choice

The Publications Office has a worldwide network of sales agents You can obtain their contact details by

sending a fax to (352) 29 29-42758

The mission of the JRC is to provide customer-driven scientific and technical support

for the conception development implementation and monitoring of EU policies As a

service of the European Commission the JRC functions as a reference centre of

science and technology for the Union Close to the policy-making process it serves

the common interest of the Member States while being independent of special

interests whether private or national

L

A-N

A-252

45-EN

-N

• 1 Summary
• 2 Introduction
• 3 Scope
• 4 Participating Laboratories
• 5 Time frame
• 6 Confidentiality
• 7 Test materials
• • 71 1Preparation of test materials
  • 72 Assigned values and standard deviation for proficiency assessment
  • 73 Homogeneity and stability
  • 74 Sample dispatch
  • • 8 Design of the proficiency test
    • 9 Evaluation of the results
    • • 91 2General
      • 92 21BEvaluation criteria
      • 93 Performance of participants
      • 94 Evaluation of the influence of analysis method
      • 95 Evaluation of the influence of calibration
      • 96 26Evaluation of compliance with legislation
      • • 10 Follow-up actions for underperforming laboratories
        • 11 1Conclusions
        • 12 1Acknowledgements
        • 13 References

The mission of the JRC is to provide customer-driven scientific and technical support

for the conception development implementation and monitoring of EU policies As a

service of the European Commission the JRC functions as a reference centre of

science and technology for the Union Close to the policy-making process it serves

the common interest of the Member States while being independent of special

interests whether private or national

L

A-N

A-252

45-EN

-N

• 1 Summary
• 2 Introduction
• 3 Scope
• 4 Participating Laboratories
• 5 Time frame
• 6 Confidentiality
• 7 Test materials
• • 71 1Preparation of test materials
  • 72 Assigned values and standard deviation for proficiency assessment
  • 73 Homogeneity and stability
  • 74 Sample dispatch
  • • 8 Design of the proficiency test
    • 9 Evaluation of the results
    • • 91 2General
      • 92 21BEvaluation criteria
      • 93 Performance of participants
      • 94 Evaluation of the influence of analysis method
      • 95 Evaluation of the influence of calibration
      • 96 26Evaluation of compliance with legislation
      • • 10 Follow-up actions for underperforming laboratories
        • 11 1Conclusions
        • 12 1Acknowledgements
        • 13 References