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![Page 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,](https://reader034.fdocuments.us/reader034/viewer/2022050819/56649f275503460f94c3f620/html5/thumbnails/1.jpg)
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
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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
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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
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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
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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
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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
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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
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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
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209 PCB Congener merged TIC
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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.
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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.
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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
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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
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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.
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Aroclor 1242, 1254, 1260; congeners used to quantify
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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.
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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
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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
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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
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PCB 81 in Fish Tissue
SPB-Octyl Column
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DB-1 Column
PCB 81 in Fish Tissue
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PCB 81 in Soil
DB-1 Column
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SPB-Octyl Column
PCB 123 in Soil
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DB-1 Column
PCB 123 in Soil
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SPB-Octyl Column
PCB 123 in Serum
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DB-1 Column
PCB 123 in Serum
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SPB-Octyl Column
PCB 126 in Serum
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DB-1 Column
PCB 126 in Serum
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SPB-Octyl Column
PCB 126 in Soil
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DB-1 Column
PCB 126 in Soil
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SPB-Octyl Column
Typical PCB 169
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DB-1 Column
Typical PCB 169
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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
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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.
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• 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
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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.