screening and confirmatory methods for the determination...

5th Thermo Scientific High Resolution GC/MS Meeting on POPsBarcelona, April 29-30, 2010

Screening and Confirmatory Methodsfor the Determination of PCDD/Fs and PCBs

in Feed and Food

European Union Reference Laboratory for Dioxins and PCBs in Feed and FoodState Institute for Chemical and Veterinary Analysis of Food (CVUA)State Institute for Chemical and Veterinary Analysis of Food (CVUA)

Freiburg, Germany

Alexander Kotz, Rainer Malisch, Johannes Hädrich and Kerstin Wahl

State Institute for Chemical and Veterinary Analysis of Food

CVUA Freiburg1 / Screening and Confirmatory methods

Content

European Union Reference Laboratory (EU-RL)p y ( )

Methods for determination of PCDD/Fs and DL-PCBs

Confirmatory methods Confirmatory methods

Screening methods

Comparison of results

Results of proficiency tests



EU-RL for Dioxins and PCBsin Feed and Food

CRL for Dioxins and PCBs in Feed and Foodappointed according to Regulation (EC) 882/2004 of the Europeanappointed according to Regulation (EC) 882/2004 of the European Parliament and of the Council (29 April 2004) on official controls performed to ensure the verification of compliance with feed and food law, animal health and welfare rules Regulation (EC) 882/2004 applies from 1 January 2006, lays

down the general tasks, duties and requirements for Community Reference Laboratories for feed and food and for animal health

Treaty of Lisbon entered into force on 1 December 2009 European Community was replaced

b th E U i hi h d itby the European Union which suceeds itand takes over all its rights and applications



EU-RL for Dioxins and PCBsin Feed and Food

Change of the name Community Reference Laboratory (CRL) to ...

European Union Reference Laboratory (EU-RL)for Dioxins and PCBs in Feed and Food

[email protected]



Confirmatory and Screening MethodsEU Regulationsg

Application of methods for analysisCommission Regulation (EC) No 152/2009 (Feed) No 1883/2006 (Food)Commission Regulation (EC) No 152/2009 (Feed), No 1883/2006 (Food)

Screening methods: Methods used to detect the presence of PCDD/Fs and DL-PCBs at the level

of interest, capacity for high sample throughput with the aim to avoid false-compliant results

e.g. GC/MS methods, bioanalytical methods Confirmation of results in samples with significant level with a confirmatory

method

Confirmatory methods:Methods providing full or complementary information unequivocally Methods providing full or complementary information, unequivocally identification and quantification of PCDD/Fs and DL-PCBs at the level of interest

e.g. GC/HRMS methods



Screening methods

Screening methods applied in routine analysis of food and feed

Physical-chemical screening methods with these detection systems: GC-MS/MS GC-MS, GC-ECD (applied only for indicator PCBs and some

mono-ortho-PCBs)

Bio analytical screening methods with these detection systems: Bio-analytical screening methods with these detection systems: CALUX bioassay EROD bioassay Ah-receptor-PCR assayAh receptor PCR assay



Confirmatory methods

Confirmatory methods applied in routine analysis of feed and food

Only physical-chemical methods with these detection systems: GC/HRMS (Double-focusing mass spectrometer)

M i l CALUX Bi f i d Mainly CALUX-Bioassay for screening and GC/HRMS as confirmatory method appliedin routine analysis of PCDD/Fs and DL-PCBsi f d d f d t lin food and feed control



Extraction, Clean-up and DetectionScreening and Confirmatory methodsg y

Food or feed sample

E t ti f

Sample preparation and pre-treatment

Extraction ofanalytes of interest

Clean-up of extractRemoval of interfering co-extractives

Detection of analytes of interest

In principle identical steps for CALUX screening and GC/HRMS



p p p g

Sample pre-treatment and extraction

Sample pre-treatment: Grinding and homogenization Drying of sample, mixing with drying agent, ...

Extraction Addition of internal standards only for GC/HRMS

Quantitative extraction of analytes of interest Quantitative extraction of analytes of interest Co-extraction of interfering substances:

CALUX: GC/HRMS:Substances with agonistic/antagonistic Effects

Other contaminants, lipids, waxes interfering in GC separation or MS measurement



Clean-up

Removal of interfering matrix

CALUX:Removing of interfering substances (lipids, ...) and co-extractives with

GC/HRMS:Removing of interfering substances ...- with higher molecular weight (lipids, waxes, ...)

ith bl l l i ht ( li h tico extractives with agonistic/antagonistic Effects (e.g. other aromatic hydrocarbons)

- with comparable molecular weight (aliphatic hydrocarbons)

- components with comparable physical-chemical properties

Sulphuric acid/silica, GPC, ...

Separation of PCDD/Fs and PCBs (also from other contaminants) Florisil, alumina, carbon

Concentration of final extract



Detection

CALUX: Transfer of PCDD/Fs and PCBs from non-polar organic solvents to Transfer of PCDD/Fs and PCBs from non-polar organic solvents to

DMSO and culture medium Exposing of CALUX-cells with extract Measuring of luminescence

Comparison with standard curve or reference samples Estimation of the TEQ

GC/HRMS: GC-separation of PCDD/Fs and PCBs (from interfering substances) HRMS-detection

Calculation of results with isotope dilution analysis Concentrations of individual congeners, calculation of WHO-TEQ



Comparison of extraction and clean-upCALUX and GC/HRMS

Comparable steps for extraction and clean-up

Focus on different interfering substances(co extractives solvents adsorbents)(co-extractives, solvents, adsorbents)

Separate optimization of extraction and clean-up p p pprocess for GC/HRMS and CALUX necessary



Content

European Union Reference Laboratory (EU-RL)p y ( )

Methods for determination of PCDD/Fs and DL-PCBs

Confirmatory methods Confirmatory methods

Screening methods

Comparison of results

Results of proficiency tests



Comparison of resultsCalculations based on WHO-TEFs and CALUX-REPs

Calculation of WHO-TEQ and CALUX-TEQ on the b i fbasis of ... WHO-TEF 1998 and 2005 DR-CALUX-REP XDS-CALUX-REP

f l f d i i h diff PCDD/F... for several food matrices with different PCDD/F and DL-PCB congener patterns



TEFs and REPsPCDD/F (1)( )

WHO 1998 WHO 2005 Behnisch et al. (2003) Scippo et al.(2004) Carbonnelle et al (2004)TEF 1998 TEF 2005 DR-CALUX-REP 2003 DR-CALUX-REP 2004 XDS-CALUX-REP 2004

2,3,7,8-TCDD 1 1 1 1 12,3,7,8 TCDD 1 1 1 1 11,2,3,7,8-PeCDD 1 1 0.54 0.5 0.731,2,3,4,7,8-HxCDD 0.1 0.1 0.3 0.1 0.0751,2,3,6,7,8-HxCDD 0.1 0.1 0.14 0.06 0.0981,2,3,7,8,9-HxCDD 0.1 0.1 0.066 0.05 0.0611,2,3,4,6,7,8-HpCDD 0.01 0.01 0.046 0.03 0.031OCDD 0.0001 0.0003 0.00047 0.0005 0.000342,3,7,8-TCDF 0.1 0.1 0.32 0.4 0.0671,2,3,7,8-PeCDF 0.05 0.03 0.21 0.1 0.142,3,4,7,8-PeCDF 0.5 0.3 0.5 0.4 0.581,2,3,4,7,8-HxCDF 0.1 0.1 0.13 0.07 0.131 2 3 6 7 8 H CDF 0 1 0 1 0 039 0 08 0 141,2,3,6,7,8-HxCDF 0.1 0.1 0.039 0.08 0.142,3,4,6,7,8-HxCDF 0.1 0.1 0.11 0.1 0.111,2,3,7,8,9-HxCDF 0.1 0.1 0.18 0.1 0.311,2,3,4,6,7,8-HpCDF 0.01 0.01 0.029 0.01 0.0241,2,3,4,7,8,9-HpCDF 0.01 0.01 0.041 0.04 0.04OCDF 0 0001 0 0003 0 0065 0 004 0OCDF 0.0001 0.0003 0.0065 0.004 0



TEFs and REPsPCDD/F (2)( )

1

0.6

0.8

0.2

0.4DR-CALUX 2004DR-CALUX 2003XDS-CALUX 2004WHO-TEF 1998

2,3,7,8-

TCDD

1,2,3,7,

8-PeC

DD

2 3,4,7,8

-HxC

DD

3,6,7,8

-HxC

DD 7,8

,9-HxC

DD 6,7

,8-HpC

DD OCDD

3 7,8-TCDF

8-PeC

DF 8-P

eCDF

HxCDF

HxCDF

xCDF

CDF CDF

DF

F

0

WHO-TEF 2005

1

1,2,3

1,2,3,

1,2,3,7

1,2,3,4,

6,

2,3,7

1,2,3,7,

82,3

,4,7,8-

1,2,3,4,

7,8-H

1,2,3,6,

7,8-H

x2,3

,4,6,7,8

-HxC

1,2,3,7,

8,9-H

xCD

1,2,3,4,

6,7,8-

HpCD

1,2,3,4,

7,8,9-

HpCDF

OCDF



TEFs and REPsDL-PCB (1)( )

WHO 1998 WHO 2005 Behnisch et al. (2003) Scippo et al.(2004) Carbonnelle et al (2004)TEF 1998 TEF 2005 DR-CALUX-REP 2003 DR-CALUX-REP 2004 XDS-CALUX-REP 2004TEF 1998 TEF 2005 DR-CALUX-REP 2003 DR-CALUX-REP 2004 XDS-CALUX-REP 2004

PCB 77 0.0001 0.0001 0.0013 0.0004 0.0014PCB 81 0.0001 0.0003 0.0042 0.002 0.0045PCB 126 0.1 0.1 0.067 0.04 0.04PCB 169 0.01 0.03 0.0034 0.0008 0.0011

PCB 105 0.0001 0.00003 0.000012 0.000001PCB 114 0.0005 0.00003 0.000048 0.00002 0.00014PCB 118 0.0001 0.00003 0.000005 0.000001PCB 123 0.0001 0.00003 0.00024 0.0000003PCB 156 0.0005 0.00003 0.00021 0.00002 0.00014PCB 157 0.0005 0.00003 0.00008 0.000001PCB 167 0.00001 0.00003 0.000006 0.0000003PCB 189 0.0001 0.00003 0.000007 0.0000002



TEFs and REPsDL-PCB (2)( )

0.1

0 04

0.06

0.08

DR-CALUX 2004DR-CALUX 2003XDS CALUX 20040.001

77 1

0

0.02

0.04 XDS-CALUX 2004WHO-TEF 1998WHO-TEF 2005

0.0006

0.0008

DR-CALUX 2004DR CALUX 2003

PCB 77PCB 81

PCB 126

PCB 169

PCB 105

PCB 114

PCB 118

PCB 123

PCB 156

PCB 157

PCB 167

PCB 189

0

0.0002

0.0004DR-CALUX 2003XDS-CALUX 2004WHO-TEF 1998WHO-TEF 2005



Matrices and available data

Matrices: Hen‘s eggs (n = 690) Fish (n = 253) Cow‘s milk (n = 252)

Results for individual PCDD/F and DL-PCB congeners availablecongeners available

Matrix

Hen‘s eggs (pg/g lipid) WHO-Total-TEQ ≥ 2 WHO-Total-TEQ < 2

Fish (pg/g fw) WHO-Total-TEQ ≥ 1 WHO-Total-TEQ < 1

Cow‘s milk (pg/g lipid) WHO-Total-TEQ ≥ 1 WHO-Total-TEQ < 1



(pg g p )

Hen‘s eggs (1)Comparison of Total-TEQp

100

120

TEQ

[%]

Eggs (DR-CALUX 2004 – TEF 1998)Comparison of „Recovery Total-CALUX-TEQ“ and Contribution of WHO PCDD/F

60

80

O-P

CDD/

F-TE

Q to

WHO

-Tot

al-T TEQ and Contribution of WHO-PCDD/F-

TEQ to WHO-Total-TEQ

20

40

Cont

ribut

ion

of W

HO

80

100

120

O-T

otal

-TEQ

[%]

Eggs (XDS-CALUX 2004 – TEF 1998)

00 20 40 60 80 100 120 140 160 180 200

Recovery Total-CALUX-TEQ [%]

40

60

80n

of W

HO-P

CD

D/F

-TEQ

to W

HO

0

20

0 20 40 60 80 100 120 140 160 180 200

Cont

ribut

ion




Hen‘s eggs (2)Comparison of Total-TEQ

1000

p

Eggs (DR-CALUX 2004 – TEF 1998)Comparison of „Recovery Total-CALUX-

100

g/g

fat]

TEQ“ and WHO-Total-TEQ

10

PCD

D/F

-PC

B-T

EQ

[pg

ML Hen‘s eggs

1

WH

O-P

0.10 20 40 60 80 100 120 140 160 180 200




Cow‘s milkComparison of Total-TEQ

100

120

EQ

[%]

p

Cow‘s milk (DR-CALUX 2004 – TEF 1998) Comparison of „Recovery Total-CALUX-TEQ“ and Contribution of WHO-PCDD/F-

60

80

CDD

/F-T

EQ to

WH

O-T

otal

-TE

120

Cow‘s milk (XDS CALUX 2004 TEF 1998)


20

40

Cont

ribut

ion

of W

HO

-PC

80

100

to W

HO

-Tot

al-T

EQ

[%]

Cow‘s milk (XDS-CALUX 2004 – TEF 1998)

00 20 40 60 80 100 120 140


40

60

utio

n of

WH

O-P

CD

D/F-

TEQ

t

0

20

0 20 40 60 80 100 120 140Recovery Total-CALUX-TEQ [%]

Con

trib



Recovery Total CALUX TEQ [%]

Fish (1)Comparison of Total-TEQ

100

120

EQ

[%]

p

Fish (DR-CALUX 2004 – TEF 1998) Comparison of „Recovery Total-CALUX-TEQ“ and Contribution of WHO-PCDD/F-

120

60

80

CD

D/F

-TE

Q to

WH

O-T

otal

-TE


80

100

120

WHO

-Tot

al-T

EQ [%

]

20

40

Con

trib

utio

n of

WHO

-PC

Fish (XDS-CALUX 2004 – TEF 1998)

Eel

40

60

ion

of W

HO-P

CDD

/F-T

EQ to

W0

0 20 40 60 80 100 120Recovery Total-CALUX-TEQ [%]

0

20

0 20 40 60 80 100 120Recovery Total CALUX TEQ [%]

Con

tribu

ti

Eel




Fish (2)Comparison of Total-TEQ

1000

p

Fish (DR-CALUX 2004 – TEF 1998)Comparison of „Recovery Total-CALUX-TEQ“ and WHO Total TEQ

10

100

g fw

]

EelTEQ and WHO-Total-TEQ

ML Eel

1

10

CD

D/F

-PC

B-T

EQ

[pg/

g

ML Fish

0.01

0.1

WH

O-P

C

0.0010 20 40 60 80 100 120 140




Fish (3)Comparison of Total-TEQ (TEF 1998 and 2005)p ( )

100

120

TEQ

[%]

Fish (XDS-CALUX 2004 – TEF 1998) Comparison of „Recovery Total-CALUX-TEQ“ and Contribution of WHO-PCDD/F-

120

Fish (XDS-CALUX 2004 – TEF 2005)60

80

O-P

CDD

/F-T

EQ to

WHO

-Tot

al-T


80

100

EQ

to W

HO

-Tot

al-T

EQ

[%]

20

40

Con

tribu

tion

of W

HO

Eel

40

60

ribut

ion

of W

HO

-PCD

D/F

-TE

00 20 40 60 80 100 120


0

20

0 20 40 60 80 100 120Recovery Total-CALUX-TEQ [%]

Cont

r



y Q [%]

Comparison of Total-TEQCALUX-TEQ – WHO-TEQ

Recovery of Total-CALUX-TEQ < 100 % d t WHO T t l TEQcompared to WHO-Total-TEQ

Range: about 20 – 100 %(Shift to higher recoveries for comparison with WHO-TEF 2005)

Comparison of theoretical values with results of fi i t tproficiency tests



Proficiency testsorganized by the EU-RL for Dioxins and PCBsg y

Guar gum PT 2008 PCDD/Fs and PCP PCDD/Fs and PCP 54 participating labs, 9 reporting results for bioassays

Fish oil PT 2008Fish oil PT 2008 PCDD/Fs and PCBs 51 participating labs, 7 reporting results for bioassays

Pork sausage PT 2009 PCDD/Fs and PCBs 78 participating labs, 7 reporting results for bioassays

Concentrations of PCDD/Fs and PCBs in the range of action and maximum levels for the matrices



Guar gum PT – Sample A (1)

WHO-PCDD/F-TEQ (WHO-TEF 1998)Sample A

6

8

10 Consensus value for WHO-PCDD/F-TEQ:1.2 ng/kg product

0

2

4

e (σ

= 1

0 %

)

-6

-4

-2

Z-sc

ore

-10

-8

Laboratory code

CALUX-results for PCDD/F

CALUX-results for PCDD/F+PCB



Guar gum PT – Sample A (2)

Overestimation of more than a factor of 2ith bi (i i t GC/MS lt )with bioassay (in comparison to GC/MS results)

Highest contribution of 1 2 3 4 6 7 8 HpCDD to the Highest contribution of 1,2,3,4,6,7,8-HpCDD to the WHO-TEQ (ca. 60 %)

CALUX-REP values more than a factor of 3 higher than WHO-TEF 1998/2005

WHO TEF (1998 2005) 0 01WHO-TEF (1998, 2005): 0.01CALUX-REP: 0.03 – 0.046



Fish oil PT

WHO-PCDD/F-PCB-TEQ (upper bound)Fish oil 2008

Consensus value: 8.50 pg/g lipid weight

3

4

5

0

1

2

re (σ

= 1

0 %

)

-3

-2

-1Z-sc

or

-5

-4

Laboratory code




Pork sausage PT

WHO-PCDD/F-PCB-TEQ (upper bound) Pork Sausage 2009

Consensus value: 3.37 pg/g lipid weight

3

4

5

0

1

2

(σ =

10

%)

3

-2

-1

Z-sc

ore

-5

-4

-3

Laboratory code




Fish oil PT, Pork sausage PT

Tendency of bioassay results for underestimation of th i d WHO T t l TEQthe assigned WHO-Total-TEQ (derived from GC/MS results)

Contribution of PCB 126 to WHO-Total-TEQ ca. 60 % Qfor both matrices

CALUX REP values about a factor of 2 lower than CALUX-REP values about a factor of 2 lower than WHO-TEF 1998

WHO-TEF (1998 2005): 0 1WHO TEF (1998, 2005): 0.1CALUX-REP: 0.04 – 0.67



Differences between estimated CALUX-TEQ and WHO-TEQestimated CALUX TEQ and WHO TEQ

Significant differences of published CALUX-REP and WHO TEF valuesand WHO-TEF values Tendency to underestimation Correction factor necessary

Re-evaluation of the CALUX-REP-values Evaluation of the effect of agonistic/antagonisticEvaluation of the effect of agonistic/antagonistic

co-extractives Contribution of other contaminants with dioxin-like

activity (e g PBDD/F PXDD/F) to the totalactivity (e.g. PBDD/F, PXDD/F) to the total response



Thank you very much fortt ti !your attention !



Acknowledgements

Thanks to our colleagues at the CRL/CVUA Freiburg Kornelia Adamovic, Indra Gerteisen, Renate Tritschler, Jutta Schächtele, Stefan Leswal and Helmut Winterhalter, for their technical and scientific support.



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