Determination of 209 Congeners and Congener Groups by HRGC/HRMS using a Single GC Column: Details of EPA Method 1668A Brian Fowler Axys Analytical Services Ltd. Sidney, BC presented at the EPA 23rd Annual conference Pittsburgh, PA May 16, 2000

Toxic Chlorobiphenyls – WHO 1998 * WHO 1994 Toxic CB List, but not included in 1998 toxicCBs

Features of Draft 1668 Method (1995) Determination of 13 toxic congeners (1994 WHO list), Cl-4 to Cl-7 HRGC/HRMS “Dioxin” type method (high sensitivity/selectivity) HRMS operated at 10,000 mass resolution in selected ion mode Primary Column: 30m SPB-Octyl for 11 CBs, plus 156/157 Confirmation: 30m DB-1 for 156 and Labeled congeners used as Internal standards Allows determination of toxic CBs in a single fraction. Quantitation by Isotope dilution gives recovery corrected concentrations for 13 target congeners

HR Coplanar CB methods HRMS Dioxin C labeled CBs for 10 toxic congeners Draft 1668 method (1995) 1668A method (1999) Draft CB method Standards Formulation, Cal with 209 Congeners 1668 NYS DEC Modified 209 Method (1998) Peer review process Additional labeled CBs Modify GC & HRMS conditions Octyl column Toxic CB separation Test methods for 209 CB with Aroclors Development of quant methods for 209 Evolution of Method 1668

Changes from Draft Method to 1668A GC and HRMS conditions expanded to acquire all 209 congeners Primary column 30m SPB-Octyl column for all 209 congeners, Linearity calibration & QC/QA based on 12 toxics Single point calibration used to determine RTs and RFs for remaining 197 CBs 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 Final extract spiked with 5 labeled CBs as instrument injection internal standards Total of 35 C-13 labeled CBs employed

Distribution of Labeled Standards Retention Time # Chlorines LOC-LQI Injection Clean-up

Resolution Statistics and Separation Criteria

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

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

Resolution Specification for CBs #34 and # x/y < 40%, for similar quantities of 34 and 23 y x

Calibration Standards for Method 1668A  Five native CB congener mixes CBs in each mix completely resolved on Octyl Includes all 209 congeners for initial peak assignment, Periodic confirmation of elution order  Toxic CBs + 13C-LOC/LQIS Linearity, (5 or 6 point ICAL) Calibration verification (CAL Ver) Initial and ongoing precision and recovery (IPR, OPR)  209 Congener Mix GC Resolution test Retention times and response factors (RT/RF Update)

Linearity Standards, Draft Method and 1668A Applied only for the Toxic CBs (13 CBs in Draft, 11 CBs in 1668A)

Considerations for Reporting 209 Congeners Resolved individual congeners selected to ensure consistent list through normal changes in GC performance: 129 individual congeners. Unresolved pairs, or groups of isomers selected to allow for some column degradation. Congeners listed by level of chlorination (LOC) in order of increasing IUPAC, and toxics listed separately. Concentration for unresolved isomer groups reported only on the entry for the lowest congener number with no concentration given on lines for other multiplet components. NB Unique Elution Order differs from historical phases (DB-5, DB-1).

Interference from Higher Homologues Chlorine loss from a parent ion results in lower mass M-Cl and M-2Cl fragments and results in some response at mass monitored for lower homologues When a higher homologue co-elutes with a lower CB interference can occur Important to monitor for contribution form co-eluting higher homologues, critical step for toxics Observed for 126 by 166/128, 169 by 190 and 198 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

The Power of a Full Congener Method Full CB congener data sets provide many options: Toxic equivalents ( with alternate TEFs or congener lists) Congener groups, (e.g. for Risk assessment) or select subset of CBs from custom lists, Reliable Homologue totals, the sum of CBs at each level of chlorination, each CB specifically targeted. Aroclor equivalents based on a sub-set of marker peaks Determination of CBs not present in Aroclors Dechlorinated and specific process congeners Rich data sets for chemometric analysis

Application of Method to Real Samples Aroclors include about 140 CBs. Congeners absent from Aroclors are indicators of other sources, dechlorination etc. Analysis of water, and suspended sediments from NY Harbour, we have routinely detected CBs in 140+ domains, including many non-Aroclor CBs. Review of frequency of detected congeners in real samples it’s difficult to find a congener that is never detected. IUPAC #11 (3,3’-dichlorobiphenyl) although “absent” in Aroclors (<0.05%), detected widely in NY wastewater effluents accounting for 0.01% to 0.05% of total PCB, rising to 0.92%. Anomalous abundance indicates non-Aroclor source of CB#11.

Strengths and Weaknesses of 1668A Strong points 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 should allow method to be readily implemented by HR-MS groups. Weak points Octyl GC phase less stable and the column requires more attention than alternate column phases (DB-1 or DB-5). On Octyl, co-elution of higher homologues can interfere and raise detection limits for toxics with 126 and 169.

Summary The development of 1668A marks a significant advance in PCB analytical methods and provides a sensitive congener specific method that includes all 209 congeners either as individual congeners or as members of a group of unresolved isomers. The WHO toxic congeners are determined at trace levels in the presence of the major congeners in single instrument run. The Method has evolved to use best technology currently available, and is limited mainly by congener separation and column performance. The Octyl column is good but we look forward to the day an even better column is devised.