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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
State Institute for Chemical and Veterinary Analysis of Food
CVUA Freiburg2 / Screening and Confirmatory methods
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
State Institute for Chemical and Veterinary Analysis of Food
CVUA Freiburg3 / Screening and Confirmatory methods
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
www.eurl-dioxin-freiburg.eueurl-dioxin@cvuafr.bwl.de
State Institute for Chemical and Veterinary Analysis of Food
CVUA Freiburg4 / Screening and Confirmatory methods
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
State Institute for Chemical and Veterinary Analysis of Food
CVUA Freiburg5 / Screening and Confirmatory 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
State Institute for Chemical and Veterinary Analysis of Food
CVUA Freiburg6 / Screening and Confirmatory methods
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
State Institute for Chemical and Veterinary Analysis of Food
CVUA Freiburg7 / Screening and Confirmatory methods
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
State Institute for Chemical and Veterinary Analysis of Food
CVUA Freiburg8 / Screening and Confirmatory methods
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
State Institute for Chemical and Veterinary Analysis of Food
CVUA Freiburg9 / Screening and Confirmatory methods
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
State Institute for Chemical and Veterinary Analysis of Food
CVUA Freiburg10 / Screening and Confirmatory methods
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
State Institute for Chemical and Veterinary Analysis of Food
CVUA Freiburg11 / Screening and Confirmatory methods
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
State Institute for Chemical and Veterinary Analysis of Food
CVUA Freiburg12 / 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
State Institute for Chemical and Veterinary Analysis of Food
CVUA Freiburg13 / Screening and Confirmatory methods
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
State Institute for Chemical and Veterinary Analysis of Food
CVUA Freiburg14 / Screening and Confirmatory methods
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
State Institute for Chemical and Veterinary Analysis of Food
CVUA Freiburg15 / Screening and Confirmatory methods
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
State Institute for Chemical and Veterinary Analysis of Food
CVUA Freiburg16 / Screening and Confirmatory methods
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
State Institute for Chemical and Veterinary Analysis of Food
CVUA Freiburg17 / Screening and Confirmatory methods
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
State Institute for Chemical and Veterinary Analysis of Food
CVUA Freiburg18 / Screening and Confirmatory methods
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
State Institute for Chemical and Veterinary Analysis of Food
CVUA Freiburg19 / Screening and Confirmatory methods
(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
State Institute for Chemical and Veterinary Analysis of Food
CVUA Freiburg20 / Screening and Confirmatory methods
Recovery Total-CALUX-TEQ [%]
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
Recovery Total-CALUX-TEQ [%]
State Institute for Chemical and Veterinary Analysis of Food
CVUA Freiburg21 / Screening and Confirmatory methods
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)
TEQ to WHO-Total-TEQ
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
Recovery Total-CALUX-TEQ [%]
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
State Institute for Chemical and Veterinary Analysis of Food
CVUA Freiburg22 / Screening and Confirmatory methods
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
TEQ to WHO-Total-TEQ
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
State Institute for Chemical and Veterinary Analysis of Food
CVUA Freiburg23 / Screening and Confirmatory methods
Recovery Total-CALUX-TEQ [%]
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
Recovery Total-CALUX-TEQ [%]
State Institute for Chemical and Veterinary Analysis of Food
CVUA Freiburg24 / Screening and Confirmatory methods
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
TEQ to WHO-Total-TEQ
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
Recovery Total-CALUX-TEQ [%]
0
20
0 20 40 60 80 100 120Recovery Total-CALUX-TEQ [%]
Cont
r
State Institute for Chemical and Veterinary Analysis of Food
CVUA Freiburg25 / Screening and Confirmatory methods
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
State Institute for Chemical and Veterinary Analysis of Food
CVUA Freiburg26 / Screening and Confirmatory methods
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
State Institute for Chemical and Veterinary Analysis of Food
CVUA Freiburg27 / Screening and Confirmatory methods
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
State Institute for Chemical and Veterinary Analysis of Food
CVUA Freiburg28 / Screening and Confirmatory methods
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
State Institute for Chemical and Veterinary Analysis of Food
CVUA Freiburg29 / Screening and Confirmatory methods
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
CALUX-results for PCDD/F+PCB
State Institute for Chemical and Veterinary Analysis of Food
CVUA Freiburg30 / Screening and Confirmatory methods
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
CALUX-results for PCDD/F+PCB
State Institute for Chemical and Veterinary Analysis of Food
CVUA Freiburg31 / Screening and Confirmatory methods
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
State Institute for Chemical and Veterinary Analysis of Food
CVUA Freiburg32 / Screening and Confirmatory methods
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
State Institute for Chemical and Veterinary Analysis of Food
CVUA Freiburg33 / Screening and Confirmatory methods
Thank you very much fortt ti !your attention !
State Institute for Chemical and Veterinary Analysis of Food
CVUA Freiburg34 / Screening and Confirmatory methods
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.
State Institute for Chemical and Veterinary Analysis of Food
CVUA Freiburg35 / Screening and Confirmatory methods
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