State-of-the-art in POPs Analysis: Outcomes of the DIFFERENCE and DIAC projects. Pim Leonards Netherlands Institute for Fisheries Research (RIVO)

Transcription

1 State-of-the-art in POPs Analysis: Outcomes of the DIFFERENCE and DIAC projects Pim Leonards Netherlands Institute for Fisheries Research (RIVO)

2 Dioxins in Food and Feed-Reference Methods and New Certified Reference Materials DIFFERENCE Dioxin Analysis by Comprehensive Multi- Dimensional Gas Chromatography (GCxGC) DIAC COMPETITIVE AND SUSTAINABLE GROWTH (GROWTH) PROGRAMME

3 Background and Societal Needs Belgian chicken incident 1999 Need for dioxin analysis capacity Need for cheap and reliable screening and confirmatory methods New EU MRLs, 1 July 2002

4 Objectives DIAC Optimisation of GCxGC-ECD system for dioxin analysis Selection of best modulator Optimisation of quantification comparison with HRMS Test of alternative detection method: ToF-MS Simplification of extraction and clean-up

5 DIAC Workplan WP 1, task 1 Mini-workshop WP 1, task 2 Optimisation of GCxGC WP 2, task 1 Quantification standard solution/ cleaned sample WP 2, task 2 Quantification real-life samples GCxGC vs. GC-HRMS WP 3, tasks 1, 2 Simplification extraction/ clean-up methods WP 4 Alternative MS detection method WP 5 Dissemination of results Workshop for dioxin users Final report

6 DIFFERENCE Objectives Selection of relevant food and feed matrices Preparation of candidate CRMs Feasibility of certification Optimisation of bio-analytical and chemical methods for dioxin analysis Validation and standardisation Optimisation of extraction and clean-up Standardised protocols for use in Europe

7 DIFFERENCE Workplan WP 1. Preparation materials WP 3. Development & optimisation methods WP 5. Extraction & clean-up CALUX CALUX Ah-PCR MEA SFE GCXGC LR-MS/MS GCXGC LR-MS/MS PLE PLE WP 2. Homogeneity & stability test WP 6. Method validation & standardisation Interlaboratory tests & optimisation methods Partners WP 7. final report WP 4. Interlaboratory studies (feasibility on certification)

8 WHO Dioxins, Furans and dioxin-like PCBs PCDDs PCDFs Dioxin-like PCBs (+IUPAC nos.) 2,3,7,8-TCDD 2,3,7,8-TCDF 3,3',4,4'-TCB (77) 1,2,3,7,8-PcCDD 1,2,3,7,8-PcCDF 3,4,4',5-TCB (81) 1,2,3,4,7,8-HxCDD 2,3,4,7,8-PcCDF 3,3',4,4',5 -PeCB (126) 1,2,3,6,7,8-HxCDD 1,2,3,4,7,8-HxCDF 3,3',4,4',5,5'-HxCB (169) 1,2,3,7,8,9-HxCDD 1,2,3,6,7,8-HxCDF 2,3,3',4,4'-PeCB (105) 1,2,3,4,6,7,8-HpCDD 2,3,4,6,7,8-HxCDF 2,3,4,4',5-PeCB (114) OCDD 1,2,3,7,8,9-HxCDF 2,3',4,4',5-PeCB (118) 1,2,3,4,6,7,8-HpCDF 2',3,4,4',5-PeCB (123) 1,2,3,4,7,8,9-HpCDF 2,3,3'4,4',5-HxCB (156) OCDF 2,3,3',4,4',5'-HxCB (157) 2,3',4,4',5,5'-HxCB (167) 2,3,3',4,4',5,5'-HpCB (189)

9 EU Requirements for Dioxin and dl- PCB Analysis False Negative Rate Screening Methods <1% Confirmatory Methods Trueness -20 to +20% CV <30% <15%

10 Quality Criteria (2002/69/EC) Performance of a method 1 8 pg TEQ LOQ (confirmatory method): range 1/5 level of interest High sensitivity and low limits of detection High selectivity (specificity) High accuracy (trueness and precision) LOQ at 1pgTEQ/g fat Up to 1pgTEQ Interferences PCN, PCB, PCDE r&r

11 EU: sensitivity requirements for food (pg diox./g fat) Pork Ruminants Milk Liver 0 6 Veg. oil Poultry Fish oil Eggs Fish: 4 pg dioxins/g product

12 GCxGC studies GCxGC-ECD GCxGC-ToF-MS Various first and second column combinations Comparison five modulator types RIVO, The Netherlands Jacob de Boer, Peter Korytár, Pim Leonards, Stefan van Leeuwen Umeå University, Sweden Conny Danielsson, Peter Haglund, Mikael Harju, Karin Wiberg Free University, The Netherlands Udo Brinkman, Maria Kristenson, René Vreuls University of Bordeaux, France Hélène Budzinski, Ana Blanc IQS, Barcelona, Spain Jordi Díaz-Ferrero

13 Principles of GCxGC Injector Selection of proper column combinationdetector Selection of modulator 5 different modulators: Modulator SWEEPER LMCS Quad N 2 (l) jet Dual CO 2 jet 1 st column Loop CO 2 2 nd column

14 Cryogenic modulation carrier gas CO 2 1. Trapping stationary phase 1 stationary phase 2 1 st st column nd column 2 nd CO 2 2. Release CO 2 3. Trapping and Separation

15 Cryogenic modulator

16 How does GCxGC work?

17 How does GCxGC work?

18 How does GCxGC work? s 10 s

19 How does GCxGC work? s 10 s

20 2D plots 46 min 10 s s 43 2nd dimension 0 s 10 s 2nd dimension 0 1st dimension 43 46

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22 Cod liver: WHO-PCB separation DB1 x HT-8 28,31 * 3Cl * ,69 * 44 * * 5Cl 61,74 4Cl * 95 * 56,60 6Cl * * * I.S * nd dimension retention time [s] * Cl * * * * * * 153 * 138, * * * * Cl 180 7Cl * * * * * * * * 8Cl * * st dimension retention time [min]

23 Milk: PCB fraction (DB-XLB x LC-50) 2 nd dimension retention time [s] st dimension retention time [min]

24 Milk: Dioxins (DB-XLB x LC-50) 5D1 2 nd dimension retention time [s] 4F1 4D1 5F1 5F2 6F3 6D3 6F2 6D2 6F1 6D1 6F4 7F1 7F2 OCDF 7D1 OCDD 1 st dimension retention time [min]

25 Improved clean-up and solvent grade 2nd dimension retention time [s] A 4F1 4D1 5F1 5F2 5D1 6F3 6D3 6F2 6D2 7F1 6F1 6D1 6F4 7F2 2nd dimension retention time [s] B 4F1 4D1 5F1 5F2 5D1 6F4 6F3 6D3 6F2 6D1 6F1 6D2 7F1 7F2 7D1 7D1 OCDD 1 st dimension retention time [min] 1 st dimension retention time [min]

26 Sewage sludge with improved clean-up 6F4 2 nd dimension retention time [s] 4F1 4D1 5F1 5F2 5D1 6F3 6F2 6D1 6F1 6D3 6D2 7F1 7D1 7F2 1 st dimension retention time [min]

27 Modulator comparison and column selection Cryogenic modulation with CO 2 had best performance Most suitable column combinations: DB-XLB x LC50 DB-1 x 90% cyanopropyl HT5 x BPX 50 Other phases less suitable because of: high background levels (bleeding of column) not all critical congener pairs (with different TEF values) could be separated from each other

28 Integration and identification example: PeCDF Standard Area: Area: Area: Sample Area: Area: Area:

29 Accuracy 30% 20% 10% 0% -10% -20% -30% -40% -50% -60% Deviation from GC-HRMS Pork fat Hake Salmon Tuna Trout Cod Spiked milk Spiked milk 2 Fly ash Milk Feedingstuff Feedingstuff 2 Sewage sludge Sewage sludge 2 Sediment Herring oil Spiked milk Vegetable oil Eel Compound feed Fish oil -70% DIAC 1 DIAC 2 DIAC 3 DIFF CERT

30 Conclusions GCxGC-ECD High selectivity High sensitivity but: Multi clean-up/fractionation steps are needed Integration of peaks is time-consuming various retention time markers in GCxGC plane Improved software requirement

31 GC-LRMS/MS GC-ITMS/MS (GCQ/Polaris) MS/MS mode Electron impact (EI) U B UNIVERSITAT DE BARCELONA Department of Analytical Chemistry University of Barcelona Barcelona (Spain) J. Malavia M.T. Galceran F.J. Santos Mass Spectrometry- Dioxin Laboratory Department of Ecotechnologies IIQAB-CSIC, Barcelona (Spain) M. Ábalos E. Abad J. Rivera

32 Clean Fish Extract: Dioxins RT: SM:9G Relative Abundance m/z m/z ,2,3,7,8-PeCDF m/z m/z m/z m/z ,2,3,7,8-PeCDD C 12-1,2,3,7,8-PeCDD 2,3,4,7,8-PeCDF 13 C 12-1,2,3,7,8-PeCDF 13 C 12-2,3,4,7,8-PeCDF Time (min) NL: 7.85E2 m/z= F: + c SRM ms @1.50 [ ] MS y030121_13 NL: 1.39E4 m/z= m/z m/z F: + c SRM ms @1.50 [ ] MS y030121_13 NL: 1.02E2 m/z= F: + c SRM ms @1.30 [ ] MS y030121_13 NL: 9.80E3 m/z= F: + c SRM ms @1.30 [ ] MS y030121_13

33 Fish oil: HRMS vs. LRMS/MS 5.0 PCDD/Fs Fish oil (Herring) Concentration (pg/g) ,3,7,8-TCDD 1,2,3,7,8-PeCDD 1,2,3,4,7,8-HxCDD 1,2,3,6,7,8-HxCDD 1,2,3,7,8,9-HxCDD 1,2,3,4,6,7,8-HpCDD 1,2,3,4,6,7,8,9-OCDD 2,3,7,8-TCDF 1,2,3,7,8-PeCDF 2,3,4,7,8-PeCDF 1,2,3,4,7,8-HxCDF 1,2,3,6,7,8-HxCDF 2,3,4,6,7,8-HxCDF 1,2,3,7,8,9-HxCDF 1,2,3,4,6,7,8-HpCDF 1,2,3,4,7,8,9-HpCDF 1,2,3,4,6,7,8,9-OCDF GC-HRMS GC-ITMS/MS

34 Problems with clean-up Relative Abundance Insufficient clean-up Suitable clean-up PCB 156 m/z 359.8? PCB 167 PCB PCB C PCB C PCB 138 m/z 371.9? Time (min) Time (min) Relative Abundance 13 C 12 -PCB C 12 -PCB 157 Hexa-mono-ortho-PCBs

35 Conclusions GC-ITMS/MS Low detection limits and high selectivity Appropriate clean-up and fractionation method needed Further studies needed in order to prove the general applicability of the GC-ITMS/MS for the analysis of PCDD/Fs and dioxin-like PCBs

36 DR-CALUX studies Scientific Institute of Public Health, Beernaert H., Carbonnelle S., Hanot V., Hellebosch L., Roos P., Van Loco J., Van Overmeire I., Van Wouwe N. and I. Windal Vrije Universiteit Brussel, Baeyens W., Sanctorum H. and C. Schroijen Université de Liège, De Pauw E., Eppe G. and M. Scippo Federal Agency for Safety of the Food Chain, Behets S., Fontaine A. and H. Vanderperren Flemish Institute for Technological Research Koppen G., Schoeters G. and R. Van Cleuvenbergen RIKILT Institute of Food Safety, Bovee T., Hoogenboom R. and W. Traag Xenobiotic Detection Systems,Brown D., Chu M., Clark G. and Gordon J.

37 DR-CALUX assay

38 CALUX activity Cells respond to all compounds of the sample extract that activate the AhR (dioxin-like activity) both the solvent and the sample contaminants Total dioxin-like activity (Total TEQ of a sample) Separation of dioxins and PCBs from many other compounds

39 Accuracy of CALUX results DIOXIN fraction PCB fraction Calux GC-HRMS Calux GC-HRMS milk fish oil chicken feed pork chicken tissue clay egg herring milk fish oil chicken feed pork chicken tissue clay egg herring

40 REP TEF DIOXIN fraction PCB fraction WHO-TEF 6 CALUXmeas CALUXmeas WHO-TEF 4 CALUX-REP 4 CALUX-REP

41 DR-Calux vs GC-HRMS in fish oil DIOXIN fraction PCB fraction CALUX y = 2,0943x + 1,6936 R 2 = 0,9519 c CALUX y = 0,0658x + 0,9952 R 2 = 0,7287 c GC-HRMS GC-HRMS

42 Conclusions DR-CALUX High sensitivity Accuracy is lower Fast and cheap method Screening method

43 Pressurized liquid extraction (PLE) PLE extraction: Dionex ASE 200 and ASE 300 Non-selective ASE with external clean-up Selective ASE with on-line clean-up Sulphuric acid silica Integrated carbon fractionation Erland Björklund Lund University Lund Sweden Christoph von Holst JRC Geel Belgium Peter Haglund, Umeå University Umeå Sweden

44 Selective PLE fat retainer n-heptane Filter SFE support Matrix / Na 2 SO 4 / Sand Filter Fat Retainer Na 2 SO 4 H 2 SO 4 /Silica gel Florisil Basic alumina Neutral alumina Acidic alumina PCBs (+ Fat) Björklund, Müller, von Holst, Anal. Chem. 2001, 73, 4050 Sporring, Björklund, J. Chrom. A 2004, 1040, 155

45 Fat retainers Ratio fat/fat retainer: Recovery about 100% Coextracted fat: 500 mg fat, 1-3 mg fat left Colour: clear florisil and sulphuric acid silica only Reaction with H2SO4: No reaction with sulphuric acid silica Conclusion sulphuric acid silica preferred

46 Clean-up of fat using selective ASE ASE300, 34mL cells, triglycerides 0,5g (n=3, s.e.m.) 5-20g silica C 50 C 100 C 50 C 100 C 50 C 100 C 50 C Retained fat (%) Pentane Hexane Heptane ,100 0,100 0,075 0,075 0,050 0,050 0,025 0,025 Fat / fat retainer ratio (FFR) Sporring, Björklund, J. Chrom. A 2004, 1040, 155

47 Traditional extraction/clean-up vs. PLE pgteq/g Oil pgteq/g Oil Vegetable oil Lab A Lab B PLE Fish oil (n=6) Lab A Lab B PLE PCDD/F PCB Total-TEQ

48 Individual congeners Vegetable Oil (pg/ (pg/ g oil) g oil) TCDD TCDF 12378F 23478F HxF1 HxF2 HxF3 HxF TCDD TCDF 12378F 23478F HxF1 HxF2 HxF3 HxF4 0.1 Lab A Lab B PLE Fish Oil (pg/ g oil)

49 Fish Oil: Non-ortho PCBs and PCDD/Fs 6 s TeCDD/F PeCDD/F HxCDD/F HpCDD/F OCDD min.

50 Selective PLE with carbon Dionex ASE200; 33 ml cell Na 2 SO 4 3g Fish oil mixed in Na 2 SO 4 Normal PLE parameters Three consecutive extractions (fractions) Matrix/Na 2 SO 4 Na 2 SO % Carbon/Celite Na 2 SO 4

51 Integrated carbon fractionation of PCBs and Dioxins 1. Heptane 2. Heptane:DCM (1:1) 3. Toluene Carbon/Celite Carbon/Celite Carbon/Celite Bulk PCBs Mono-ortho- PCBs Non-ortho-PCBs and PCDD/Fs

52 Elution profile integrated carbon fractionation Fish oil Fat recovery (%) Fraction 1 98,7 Fraction 2 0,7 Fraction 3 0,1 % Bulk-PCBs Mon-ortho PCBs Non-ortho PCBs PCDDs and PCDFs 1. Heptane 2B. Acetone/heptane (2.5:1) 2A. DCM/heptane (1:1) 3.Toluene

53 Conclusions PLE Fast method Integrated carbon PLE cost efficient method Less labour intensive than traditional method Attractive alternative for traditional dioxin method

54 Validation studies VITO (Flemish Institute for Technological Research) R. Van Cleuvenbergen RIVO S. Van Leeuwen, J. de Boer Other partners from DIFFERENCE and participants outside DIFFERENCE

55 Method validation Interlaboratory studies Verification of calibration curves Verification of analytical process Verification of matrix effects during quantification Repeatability, within & between lab reproducibility (ISO 5725) Detection Capability Selectivity Ruggedness Standards, quality control solution, clean fish extract Vegetable oils with spikes of PCBs, PCNs, PCDE s Fish oil, milk, vegetable oil, vegetable oils with spikes, cereal based feed, chicken, vegetable feed, egg, fish, pork

56 DIFFERENCE: (Candidate) CRMs for Dioxin Analysis

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58 Fish Oil: z-scores FISH OIL: dioxin TEQ (upperbound) z-score F G G G G F G G F F F C F C C C B B C J B B C J J A B J J J I B I A I A I I I A A A Lab

59 Spiked Milk MILK: dioxin TEQ (upperbound) z-score CALUX: signal suppression due to high spike of mono-ortho CB-118? -2 F F F F G F G C C G G A C C F C C G G J J J J J A J A I A I A I A I I I MILK: PCB TEQ (upperbound) -3 LAB CALUX CALUX CALUX CALUX CALUX CALUX GC-HRMS GC-HRMS GC-HRMS GC-HRMS GC-HRMS GC-HRMS GC-HRMS GC-HRMS GC*GC GC-HRMS GC*GC GC*GC GC-LRMS GC*GC GC*GC GC-HRMS GC-LRMS GC*GC GC-LRMS GC-HRMS GC-LRMS GC-LRMS GC-HRMS GC-LRMS GC-HRMS GC-HRMS GC-HRMS GC-HRMS GC-HRMS GC-HRMS AAAAAAJ J J J J J F F I F I I G I I F G I G F GG F GCCCCCC GCxGC: overestimation due to combination LOQ and reporting upperbounds LAB z-score

60 Maximum residue level Precision requirement Precision (%) for spiked vegetable oil Within-Lab Reproducibility Within-Lab Reproducibility RSD (%) 50,0% 40,0% 30,0% 20,0% GC-HRMS RSD (%) 50,0% 40,0% 30,0% 20,0% CALUX 10,0% 10,0% 0,0% Conc (pg TEQ/g) 0,0% Conc (pg TEQ/g) Within-Lab Reproducibility Within-Lab Reproducibility RSD (%) 50,0% 40,0% 30,0% 20,0% GC-LRMS RSD (%) 50,0% 40,0% 30,0% 20,0% GCxGC-ECD 10,0% 10,0% 0,0% Conc (pg TEQ/g) 0,0% Conc (pg TEQ/g)

61 Discussion Sens. Accur. Precis. Technique Remarks GC-HRMS Confirmatory method GC-LRMS Potential alternative for HRMS? GCxGC-ECD +/- +/- + Improved software required CALUX + +/- + Screening technique

62 Conclusions GCxGC-ECD (and GCxGC-ToF-MS), and GC-ion-trap MS/MS may serve as alternative (routine) methods for dioxin analysis CALUX is the alternative for times of crisis, but corrections for recovery are essential The use of PLE will significantly reduce the extraction and clean-up time

63 Acknowledgement All DIFFERENCE and DIAC partners Thank you for your attention