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 2003Boston

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

Toxic Chlorinated Biphenyls – WHO 1998

Cl-4 Cl-5 Cl-6 Cl-7Non-ortho 77 126 169

81Mono-ortho 105 156 189

114 157118 167123

Di-ortho 170 *180 *

* WHO 1994 toxic CB list, but not included in WHO 1998 toxic CB list

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

Distribution of Labeled Standards

0

1

2

3

4

5

6

7

8

9

10

10.00 20.00 30.00 40.00 50.00 60.00 70.00

Retention Time

# C

hlo

rin

es LOC

Injection

Clean-up

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

Octyl DB-5 DB-1 Length (m) 30 60 30 Domains 159 160 156 Resolved congeners

125 120 116

Co-elutions with WHO Toxic CBs

Isomeric CBs 2 +1 Cl CBs 6 11 10 +2 Cl CBs 3 2 2

QUANTIFICATION RANGE SIX POINT CALIBRATION SERIES

Solution

Congener Concentration

ng/mL

Concentration in Sample, pg/g *

CS-0.2 0.2 0.4 CS-1 1.0 2.0 CS-2 5.0 10 CS-3 50 100 CS-4 400 800 CS-5 2000 4000 Extended Range 20000 *Assuming 10 gram sample, 20 uL final volume

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

209 PCB Congener merged TIC

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.

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.

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

Typical results

COMPOUND IUPACNO.

CONC.FOUND (pg/g)

3,3',4,4' - TeCB 77 12703,4,4',5 - TeCB 81 57

2,3,3',4,4' - PeCB 105 200002,3,4,4',5 - PeCB 114 15502,3',4,4',5 - PeCB 118 511002',3,4,4',5 - PeCB 123 10903,3',4,4',5 - PeCB 126 103

2,3,3',4,4',5 - HxCB 156 44702,3,3',4,4',5' - HxCB 157 8902,3',4,4',5,5' - HxCB 167 19103,3',4,4',5,5' - HxCB 169 3.2

2,2',3,3',4,4',5 - HpCB 170 106002,2',3,4,4',5,5' - HpCB 180 308002,3,3',4,4',5,5' - HpCB 189 370

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.

Aroclor 1242, 1254, 1260; congeners used to quantify

Considerations for Reporting Toxic EquivalentsGC 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.

a b a

b30% valleya=b

80% valleya=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

Considerations for Reporting Toxic EquivalentsMS 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

PCBPCB # Cl’s# Cl’s SPB Octyl (# Cl’s)SPB Octyl (# Cl’s) DB-1 (# Cl’s)DB-1 (# Cl’s)

8181 44 110/115 (5)110/115 (5) 87/117/125 (5)87/117/125 (5)

123123 55 109 (5)109 (5) 107/109/124 107/109/124 (5)(5)

126126 55 128/166 (6)128/166 (6) 129 (6)129 (6)

169169 66 190, 198 (7, 8)190, 198 (7, 8)

Toxic Congeners Affected by Interfering Non-Toxic Congeners

PCB 81 in Fish Tissue

SPB-Octyl Column

DB-1 Column

PCB 81 in Fish Tissue

PCB 81 in Soil

DB-1 Column

SPB-Octyl Column

PCB 123 in Soil

DB-1 Column

PCB 123 in Soil

SPB-Octyl Column

PCB 123 in Serum

DB-1 Column

PCB 123 in Serum

SPB-Octyl Column

PCB 126 in Serum

DB-1 Column

PCB 126 in Serum

SPB-Octyl Column

PCB 126 in Soil

DB-1 Column

PCB 126 in Soil

SPB-Octyl Column

Typical PCB 169

DB-1 Column

Typical PCB 169

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

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.

• 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

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.