Analysis of PCB Congeners in SRRTTF Program – Analytical Limits and

ConsiderationsSRRTTF PCB Workshop

Spokane WA, Jan.13-14, 2015Presented by Richard Grace

Director – Sales, Service, Marketing AXYS Analytical Services Ltd.

Agenda

About AXYS Some PCB Basics

– Methods, Aroclors, Congeners

– Total PCBs

Basics on HRMS, and 1668 PCBs Limits of PCBs Measurements

– Identification, blanks

AXYS Focus

Focus – Multi Target Ultra Trace Organic Analysis (pg or ng levels)

– POPs (all matrices)

– Emerging Contaminants

– HRMS, LC MS/MS, GC/MS, GC-ECD instrumentation

– Targeted Metabolomics 3 Analytical Areas

– Environmental (All matrices associated with Bioaccumulation cycle

– LifeScience – Bio-monitoring and Metabolomics

– Method Development (i.e. EPA 1668,1614,1694,1698,1699)

AXYS Analytical Services Ltd

Located Victoria British Columbia 100 Employees Largest Ultra-Trace Lab in NA 65% in U.S. ISO 17025 + NELAP Accreditation

AXYS and PCB Congeners

Since early eighties in Arctic “baseline” investigations (all matrices in bioaccumulation, high volume sampling), HRMS capable in 1984.

Full 209 congener analysis using Isotope Dilution / DB-1 column (HRMS and GC/MS platforms) in early 90s.

EPA Method 1668A Source – Process and Validation (2001 to 2003) – Published 2003 by U.S. EPA Office of Water

Active in 1668B review.

1 of 2 labs to review / validate EPA 1668C for publication 2009

Some PCB Basics

• Biphenyl Rings with CL substitution•209 possibilities or “congeners”•10 homologue possibilities indicating # of Cl (eg. Mono to deca) per PCB or level of chlorination•12 congeners are “WHO Toxic” congeners with Dioxin Like AH receptor affects

Some PCB Basics

Aroclors – variety of technical PCB products sold by Monsanto (now Solutia) from early 30s to early 80s, each composed of 1-7 primary congeners + trace levels of congeners

Named by wt.% Cl – Aroclor 1242 (12 carbons, 42% chlorination by weight)– Lightest by MW – Aroclor 1221, 1232

– Heaviest by MW Aroclor 1268,1270,1272 (primarily 209,206 congeners)

Used as technical standards for calibration and QC control in many PCB methods , primarily GC/ECD

Conversion of EPA 1668C Congener Values to Aroclors (Basis for NELAP Accreditation as Domestic PE Providers Use Aroclor Standards)

Aroclor 1016 = the sum* of PCBs 8, 18/30, 31, 28/20 concentrations multiplied by 2.7

Aroclor 1221 = the sum of PCBs 1, 3, 8 concentrations multiplied by 1.4

Aroclor 1232 = the sum of PCBs 1, 3, 18/30 concentrations multiplied by 3.4

Aroclor 1242 = the sum of PCBs 8, 18/30, 31, 28/20 concentrations multiplied by 3.0

Aroclor 1248 = the sum of PCBs 44/47/65, 49/69, 66 concentrations multiplied by 6.1

Aroclor 1254 = the sum of PCBs 86/87/97/108/119/125, 99 concentrations multiplied by 8.0

Aroclor 1260 = the sum of PCBs 183/185, 180/193, 170 concentrations multiplied by 5.0

PCBs – Multiple Production Sources – More Technical Products Than Aroclors

Widespread Use (1930s to 1980s) plus Global Transport -Ubiquitous

Properties of PCBs

Stable, resistant to breakdown, half-life of 8-20 yrs.

Breakdown largely by de-chlorination to lower MW congeners

High KOW, low water solubility. Lypophyllic and bioaccumulative

Many toxicological end points

In solids, tend to partition to solids. Increasing affinity for solids as MW increases.

Low volatility (varies by MW) but are transported long range

Stockholm POP, banned, targeted for elimination

General trend in environment – decreasing prevalence, shift to lower MW

028 044 049 052 066 074 087 099 101 105 110 118 128 138 146 149 151 153 156 157 167 170 172 177 178 180 183 187 189 194 195 196 199 206 209

12-1920-39

40-5960+

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

PP

B (

ng

/g w

ho

le w

eig

ht)

PCB Congener

12-19 20-39 40-59 60+Age Group:

The Geometric Means of 35 PCBs (whole-weight in ng/g) from NHANES 2003-2004 by Age Group for all Sex, Race/Ethnicity in the U.S. Population

The Mean Percent Congener Contribution to the Sum of the 35 PCBs (Whole-Weight in ng/g) for NHANES 2003-2004 by Age Group – Shift to Lower MW PCBs Over Time

“Total PCBs” and Analytical Measurements

Sum of PCBs measured

Aroclor measurements – total of selected Aroclors only

– Not all Aroclors measured (1268,1270, 1272)

– Aroclors that degrade or weather, not measured

– Aroclor pattern required for identification? Aroclors overlap

– Many other PCB sources

Congener Measurements– Often short list of congeners present in Aroclors

PCB Metabolites (OH, ME) not included in totals

Lake Ontario Mass Balance - % of PCBs by CongenerLake Ontario Mass Balance - % of PCBs by Congener

PCB CongenerIUPAC ID

PCB-11IADN & US/DS

PCB-1IADN

PCB-37US/DS

% of Lake Ontario Surface Water PCBs

209 Congener Method Needed – EPA 1668A

Released Aug. 2003

EPA 1600 Series method – performance based – analytical variation allowed provided performance specifications met

Multi-matrix HRGC / HRMS method, similar to EPA 1613B

Isotope Dilution / Recovery Correction Qunatification

Designed to meet EPA CWA criteria 128 pg/L Total PCBs

169 reported congeners and congener co-elutions

Default SPB Octyl GC Column

Analytical Result is Congener Data – where QC is applied

Updated 2009 – EPA 1668C

AXYS EPA 1668C Analysis Flow Diagram – Aqueous and High Vol. / Passive Matrices

EPA 1668A/C Reported Values Congeners reported as “analytical” result, low pg absolute

Blank limited if DLs good

Mathematical Calculations from Congeners

– Total PCBs, Homologue totals, TEQs

Requires isotope dilution / recovery correction quantification (C labelled surrogates) from spikes in each unknown and QC sample

Requires 2 masses (+/- 15%) from native and surrogate for positive identification (NDR may be EMPC)

General reporting structure – unflagged above LMCL, “J” flagged value below LMCL if 2.5 to 1 S:N ration met for ratioing congener

1668 QC Notes

QC Criteria must meet method guidance for performance (DLs, blank criteria, many specific criteria)

SRRTTF work requires better DLs and blank control than method minimum levels. Not instrument limited.

Validated by lab performing analysis– EPA Tier 1 validation (IPR, MDL, MS/MSD)

– Sets control ranges for recovery of natives and surrogates (what is normal at known accuracy)

External Confirmation of Proficiency / Accuracy– PE programs and CRMs/ SRMs limited

– Requires participation in international round robins / calibrations for real matrices

AXYS MLA 010 (EPA 1668A/C) QA Specifications

QC Parameter Specification

Analysis Duplicate Must agree to within ±20% of the mean (applicable to concentrations 10 times the DL)1

Procedural Blank

Analyte concentrations in blank samples for PCB congeners 77, 81, 114, 123, 126 and 169 must be less than 2 pg/congener/sample, and concentrations of PCB congeners 156, 157, 167 and 189 must be less than 10 pg/congener/sample. Concentrations of all other individual PCB congeners or coelutions must be less than 50 pg/congener/sample in blank samples. The sum of all 209 congeners should be less than 300 pg/sample. Higher levels are acceptable where sample concentrations exceed 10 times the blank levels.

Sample Specific Detection Limit

Typical sample specific detection limits, determined from chromatographic noise, are in the range of 0.5 to 2.0 pg.

Initial Calibration

For 6-point calibration, a relative standard deviation of the RRF’s 20% for all compounds Ion ratios for all congeners must be within ±15% of theoretical for CS 0.2.Minimum S:N ratio 10:1 for all calibration standards. For CS0.2, S:N ratio may be as low as 3:1 for di-PCBs and nona-PCBs.

Continuing CAL VER Refer to the tables above.

Analyte/Surrogate RatiosResponse must be within the calibrated range of the instrument. Coders may use data from more than one chromatogram to get the responses in the calibrated range.

Ion RatiosIon ratios must fall within ±15% of the theoretical values for positive identification of all targets in the calibration standards and samples.

SensitivityMinimum S:N ratio 10:1 for all calibration standards. For CS0.2, S:N ratio may be as low as 3:1. for di-PCBs and nona-PCBs.

Comparisons of Common PCB Methods vs. Positive Identification

Method + Method + EU Point EU Point RatingRating

HRGC/HRMS HRGC/HRMS (8 points)(8 points)

HRGC/GCMSHRGC/GCMS(0-4 points)(0-4 points)

HRGC/ECDHRGC/ECD(0 Points)(0 Points)

DetailsDetails EI (Electron EI (Electron Impact) / Impact) / Voltage SIRVoltage SIR

MID detection MID detection by GCMS in EI by GCMS in EI modemode

ScanScan

QuantificatiQuantificationon

1313C-labeled C-labeled standards for standards for isotope dilutionisotope dilution

Labeled Labeled standards standards possiblepossible

Labeled Labeled standards standards not possiblenot possible

SpecificitySpecificity High: Monitor High: Monitor two to six ions two to six ions from parent ion from parent ion cluster, cluster, compare to compare to theoretical theoretical ratio. Multiple ratio. Multiple cleanups. cleanups. 10,000+ RP.10,000+ RP.

Medium: Medium: Monitor 2-6 Monitor 2-6 masses per masses per congener. congener. Compare to Compare to theoretical theoretical ratio. Less ratio. Less cleanup. cleanup. 100-400 RP100-400 RP

Low: Low: Identification Identification based on GC based on GC retention time retention time and halogen and halogen response. response. Limited Limited cleanup. cleanup. Other Other halogenated halogenated compounds compounds may interfere. may interfere.

SensitivitySensitivity Best (Lowest) Best (Lowest) detection limitdetection limit

Highest Highest detection detection limitslimits

Medium Medium detection detection limitslimits

TechniqueTechnique # of Ions (native plus # of Ions (native plus labeled surrogate labeled surrogate

using isotope dilution)using isotope dilution)

# of Points# of Points

GC/MS (EI or CI)GC/MS (EI or CI) NN NN

LC/MSLC/MS NN NN

GC MS/MSGC MS/MS 1 precursor, 2 daughter1 precursor, 2 daughter 44

LC MS/MSLC MS/MS 1 precursor, 2 daughter1 precursor, 2 daughter 44

LC MS/MSLC MS/MS 2 precursor, each with one 2 precursor, each with one daughterdaughter

55

HRMSHRMS NN 2N2N

EU 657 – Analytical Method - Point Criteria for Positive Identification if Using Internal Standard

(4 Points Required for Positive Identification)

Isotope Dilution / & Surrogate Recovery

Isotope dilution/recovery correction quantitation– Increased accuracy, avoids matrix bias– Allows more extensive clean-up process– Reduction of detection limits

Labelled standard addition at start of analysis means Isotope dilution/surrogate recovery correction throughout extraction and clean-up

Recovery standards added at instrument This correction compensates for loss of target analytes

during the extraction and cleanup process for every sample Extra standards, not reported for diagnostic monitoring –

clean-up standards, field standards

23

Low Level PCBs in 1L Effluent Sample – all specs. met (PCB 67 @ 1.2 pg/L, PCB 63 @ 1.6 pg/LLSDL @ 0.55 pg/L, Reported “J” flaggedPrimary and Confirmation Ion Channels

AXYS EPA 1668C Reporting Limits based on Low Method Calibration Limit (CS0.2)

Matrix WATER/EFFLUENT

Units pg/L

Sample size 2.36L 4L 60L

CL1-PCB-1 1.69 (4@1L) 1.00 0.07

CL1-PCB-2 1.69 1.00 0.07

CL1-PCB-3 1.69 1.00 0.07

CL2-PCB-4 1.69 1.00 0.07

CL2-PCB-5 1.69 1.00 0.07

CL2-PCB-6 1.69 1.00 0.07

CL2-PCB-7 1.69 1.00 0.07

CL2-PCB-8 1.69 1.00 0.07

CL2-PCB-9 1.69 1.00 0.07

CL2-PCB-10 1.69 1.00 0.07

CL2-PCB-11 1.69 1.00 0.07

CL2-PCB-12/13 1.69 1.00 0.07

AXYS 1668C EDLs or SDLs Prorated for Sample Sizes Considered in SRRTTFF Analysis

Matrix WATER/EFFLUENT

Units pg/L

Sample size 2.36L 4L 60L

CL1-PCB-1 0.42 0.25 0.02

CL1-PCB-2 0.42 0.25 0.02

CL1-PCB-3 0.42 0.25 0.02

CL2-PCB-4 0.85 0.50 0.03

CL2-PCB-5 0.85 0.50 0.03

CL2-PCB-6 0.85 0.50 0.03

CL2-PCB-7 0.85 0.50 0.03

CL2-PCB-8 0.85 0.50 0.03

CL2-PCB-9 0.85 0.50 0.03

CL2-PCB-10 0.85 0.50 0.03

CL2-PCB-11 0.85 0.50 0.03

CL2-PCB-12/13 0.85 0.50 0.03

Surrogates PCB 1 and 3 @ 50 pg (CS-3)In 12 hr cal. ver. Primary and Confirmation Ion Channel

Interferences – Minor Dimer IssuePrimary and Confirmation Ion Channel + PFK Channel – Quantitation Not Affected

Interferences - Major DimerPCB 1 poor chromatography, PCB 3 pushed out of window (NQ)

206 Interference @ 55.26 Inflates Surrogate Recovery to 237%, Result NQ’d, Re-Run diluted to Resolve. High Level TissueInterference Unknown – PCB 207,208 Unaffected

Blanks in 1668A/C

Any 1668 analysis will be able to see blank levels for some congeners in EPA 1668A/C

Lower detection limits will increase amount of blank concentrations detected

Multiple Conventions for Reporting of Blank QC Method Acceptance Criteria – exceedences “B” flagged

All applied at congener level

Blank contamination from laboratory for method blanks

Method blank + field + container for field blanks

Method blank + media+ sampler for high vol. and passives

Blank Control Chart Basics

I Chart: PBDPE-209 blank (solids, tissue, and aqueous) Jan to Jul 2009

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AXYS Method Blanks and SRRTTF

Supplied to SRRTTF in two forms

– Mean plus 2 standard deviations of all method blanks (aqueous matrix) at LMCL (EPA 1668A convention)

– Mean plus 2 standard deviationsof all method blanks (aqueous matrix)

Produces very different specs.

Congener specs. not added, below blank limits PCBs behave independently

AXYS default spec. 300 pg per sample, with maxs. Set on select congeners

SRRTTF Limit for 2.36L samples appears to be approx. 50 pg/L

Blank Limits for AXYS MLA 010 – Varying with Sample Size, Type of Reporting Limit Used in Data, ND = 0, Mean + 2 Sigma of Reporting Limit Used

EPA 1668 Convention @ RL Cut-Off CLAM

Samples Size (L) 1 2.36 4 55 60 90

Method Blank Total PCB (pg/L) 20.66 8.754 5.165 0.376 0.344 0.230

AXYS 1668 Convention @ SDL Cut-Off CLAM

Samples Size (L) 1 2.36 4 55 60 90

Method Blank Total PCB (pg/L) 117.616 49.837 29.404 2.138 1.960 1.307

PCB Total Control Chart – All Matrices

PCB 118 Control Chart – Solids

PCB 7 – Specific Contract

Matrix Values – PCB 7 Aqueous

PCB 7 - Tissue

PCB 7 – Solid

PCB 7 – All matrices