1. Application of Method 1668A to the Analysis of Dioxin-Like PCBs and Total PCBs in Human Tissue and Environmental Samples Coreen Hamilton, Todd Fisher, Dale Hoover and Steve Kennedy Axys Analytical Services Ltd. Sidney, BC Presented at Dioxin 2003 Boston

2. Features of Method 1668 A HRGC/HRMS “Dioxin” type method (high sensitivity/selectivity), monitoring all 209 congeners Determination of toxic congeners, Cl-4 to Cl-7 in a single fraction. Primary GC Column: 30m SPB-Octyl for all 209 congeners Confirmation Column : 30m DB-1 Isotope Dilution/Internal Standard quantification procedures results in recovery corrected concentrations for all congeners

3. Toxic Chlorinated Biphenyls – WHO 1998 * WHO 1994 toxic CB list, but not included in WHO 1998 toxic CB list

4. Quantification by Method 1668A Linearity calibration & QC/QA based on 27 toxic congeners Single point calibration used to determine RTs and RFs for remaining 182 CB congeners Prior to extraction, samples spiked with labeled internal standard that includes 27 labeled congeners (C-13) Labeled standard includes WHO toxics and the first and last eluters for each level of chlorination (LOC) Extract spiked with 3 labeled CBs as clean-up standards and final extract spiked with 5 labeled CBs as instrument injection internal standards Total of 35 C-13 labeled CBs employed

5. Distribution of Labeled Standards 0 1 2 3 4 5 6 7 8 9 10 10.0020.0030.0040.0050.0060.0070.00 Retention Time # Chlorines LOC Injection Clean-up

6. PCB Congener Resolution Statistics for SPB-Octyl compared to DB-5 and DB-1

8. The Power of a Full Congener Method Full CB congener data sets provide many options: Dioxin-Like Toxic Equivalents Reliable Homologue totals, the sum of CBs at each level of chlorination, each CB specifically targeted, plus a “Total PCB”. Aroclor equivalents based on a set of marker peaks Determination of CBs not present in Aroclors such as dechlorinated and specific process congeners Rich data sets for chemometric analysis

9. 209 PCB Congener merged TIC

10. Application of Method to Real Samples Aroclors include about 140 CB congeners. Congeners absent from Aroclors are indicators of other sources, dechlorination etc. In analysis of water, tissue, soil and and suspended sediments, we routinely detect CBs in 140+ domains, including many non-Aroclor CBs Review of frequency of detected congeners in real samples indicates that it’s difficult to find a congener that is never detected.

11. Considerations for Reporting 209 Congeners Resolved individual congeners selected to ensure consistent list through normal changes in GC performance: 125 individual congeners. Unresolved pairs, or groups of isomers selected to allow for some column degradation. Quantification of both (low concentration) toxic congeners as well as higher concentration congeners in the same sample extract. Calibration range may be limiting. Background levels and blanks.

12. Typical blank concentrations PCBs 77, 81, 114, 123, 126 and 169 less than 2 picograms absolute PCBs 156/157, 167 and 189 less than 10 picograms absolute All other PCBs less than 50 picograms absolute per congener and total less than 200 pg

13. Typical results

14. Total PCBs and Aroclor Equivalents from Method 1668A Homologue totals determined by summing concentrations of individual detected congeners that meet criteria Total PCBs as sum of homologue totals Aroclor equivalents calculated using marker peaks show good correlation with Aroclor methods.

15. Aroclor 1242, 1254, 1260; congeners used to quantify

16. Considerations for Reporting Toxic Equivalents GC Resolution Resolution of each “toxic congener” required for quantification of toxicity Both GC interference and MS interference must be eliminated. Some congeners with high Toxicity Factors are at much lower concentrations than other congeners and can interfere. GC resolution can be an issue.

17. aba b 30% valley a=b 80% valley a=b 30% valley, a>>b 80% valley, a>>b Peak domain, total area = a +b a and b resolved, gives individual peak areas for a and b Peak Resolution vs Relative Abundance

18. Considerations for Reporting Toxic Equivalents MS Interference from Higher Homologues Fragments (M-Cl and M-2Cl) from co-eluting higher LOC congeners Response of interfering fragments from high concentration congeners can be significant for low concentration toxics Typically M-Cl at 4.5% - 6 % of M in lower mass channel Typically M-2Cl at 1.1% - 1.6% of M in lower channel Need >60000 mass res to completely eliminate M-Cl Need >15000 mass res to completely eliminate M-2Cl

19. PCB # Cl’s SPB Octyl (# Cl’s) DB-1 (# Cl’s) 814 110/115 (5) 87/117/125 (5) 1235 109 (5) 109 (5) 107/109/124 (5) 107/109/124 (5) 1265 128/166 (6) 129 (6) 129 (6) 1696 190, 198 (7, 8) Toxic Congeners Affected by Interfering Non-Toxic Congeners

20. PCB 81 in Fish Tissue SPB-Octyl Column

21. DB-1 Column PCB 81 in Fish Tissue

22. PCB 81 in Soil DB-1 Column

23. SPB-Octyl Column PCB 123 in Soil

24. DB-1 Column PCB 123 in Soil

25. SPB-Octyl Column PCB 123 in Serum

26. DB-1 Column PCB 123 in Serum

27. SPB-Octyl Column PCB 126 in Serum

28. DB-1 Column PCB 126 in Serum

29. SPB-Octyl Column PCB 126 in Soil

30. DB-1 Column PCB 126 in Soil

31. SPB-Octyl Column Typical PCB 169

32. DB-1 Column Typical PCB 169

33. SPB-Octyl DB-1 Comparison Summary Accurate quantification of PCB 169 requires DB-1 column analysis PCBs 81, 123 and 126 can have interfering congeners present on both column systems Recommendations Perform carbon column treatment to isolate toxic congeners and analyze on DB-1 to minimize interferences with PCB 169

34. Strengths of 1668A High sensitivity and selectivity allows the toxic CBs and wide range of other CBs to be accurately measured in complex samples with high confidence. Method is calibrated using all 209 CB congeners Numerous labeled standards provides recovery corrected concentrations and extensive built-in method performance checks and QA/QC. Similarity to 1613 dioxin methods allows method to be readily implemented by HR-MS groups.

35. On Octyl, co-elution of higher homologues can interfere and raise detection limits for toxics with 126 and 169 Confirmation on second GC column and/or carbon cleanup may be required for certain sample matrices. Weaknesses of 1668A

36. Summary Method 1668A provides a sensitive congener specific method that includes all 209 congeners either as individual congeners or as members of a group of unresolved isomers (domains). The WHO toxic congeners are determined at trace levels in the presence of the major congeners in single instrument run for most sample matrices. The Method uses the best technology currently available and can be successfully applied to a variety of sample matrices containing a range of PCB concentrations.