axys analytical services ltd “ultra trace organic analysis, pops, and emerging contaminants ”...

AXYS ANALYTICAL SERVICES LtdAXYS ANALYTICAL SERVICES Ltd“Ultra Trace Organic Analysis, “Ultra Trace Organic Analysis,

POPs, and Emerging POPs, and Emerging ContaminantsContaminants” ”

For ASU April 8, 2011For ASU April 8, 2011By Richard Grace – Director – Sales, Marketing and Service – AXYS By Richard Grace – Director – Sales, Marketing and Service – AXYS

Analytical Services Ltd. Analytical Services Ltd. [email protected]@axys.com 1-888-373-08811-888-373-0881

OrOr

905-484-2314 905-484-2314

mailto:[email protected]

AGENDAAGENDA

• About AXYSAbout AXYS• Ultra- Trace POPs and Emerging Ultra- Trace POPs and Emerging

Contaminant Analysis BasicsContaminant Analysis Basics• Selecting and Developing MethodsSelecting and Developing Methods

– AttributesAttributes– Accreditation + Quality SystemsAccreditation + Quality Systems– Detection + Reporting LimitsDetection + Reporting Limits– Applicable Levels + Blank LevelsApplicable Levels + Blank Levels– Analyte Lists + Time Analyte Lists + Time

• Some topical items on CECsSome topical items on CECs

About AXYSAbout AXYS• 100 employees located in Victoria B.C. (Vancouver Island)100 employees located in Victoria B.C. (Vancouver Island)

• ISO 17025 Certified, Multiple NELAP accreditations ISO 17025 Certified, Multiple NELAP accreditations

• Focus – Multi Target Ultra Trace Organic Analysis (pg or ng Focus – Multi Target Ultra Trace Organic Analysis (pg or ng levels)levels)– POPsPOPs– Emerging ContaminantsEmerging Contaminants– HRMS, LC MS/MS HRMS, LC MS/MS – some GC/MS, GC-ECD optionssome GC/MS, GC-ECD options– High barrier to entryHigh barrier to entry

• 3 Analytical Areas3 Analytical Areas– Environmental (All matrices associated with Bioaccumulation Environmental (All matrices associated with Bioaccumulation

cycle) cycle) – Bio-monitoringBio-monitoring– Method Development and Validation (i.e. EPA 1668, 1614, 1694, Method Development and Validation (i.e. EPA 1668, 1614, 1694,

1698, 1699) 1698, 1699) – Big “D” , little “R”Big “D” , little “R”

Where is Victoria?Where is Victoria?

AXYS HRMS AnalysisAXYS HRMS Analysis(AXYS Developed EPA Methods in brackets)(AXYS Developed EPA Methods in brackets)

– Chlorinated and Brominated Dioxins and Chlorinated and Brominated Dioxins and FuransFurans

– PCBs (EPA 1668A and 1668C)PCBs (EPA 1668A and 1668C)– OC, OP, ON Pesticides (EPA 1699)OC, OP, ON Pesticides (EPA 1699)– PBDEs (EPA 1614) PBDEs (EPA 1614) – Hormones and Sterols (EPA 1698)Hormones and Sterols (EPA 1698)– Acid Extractable Herbicides Acid Extractable Herbicides – Chlorinated Naphthalenes Chlorinated Naphthalenes – PBBsPBBs– PyrethroidsPyrethroids

AXYS LC MS/MS AnalysisAXYS LC MS/MS Analysis(EPA Reference Methods in Brackets)(EPA Reference Methods in Brackets)

– Perfluoronated Compounds (pending)Perfluoronated Compounds (pending)– Personal Care and Pharmaceutical Personal Care and Pharmaceutical

Products (EPA 1694)Products (EPA 1694)– Hormones (EPA 1645)Hormones (EPA 1645)– Phthalate Esters MetabolitesPhthalate Esters Metabolites– Parabens and metabolitesParabens and metabolites– Bisphenol A and metabolitesBisphenol A and metabolites– Nonyl Phenols and Nonyl Phenol Nonyl Phenols and Nonyl Phenol

EthoxylatesEthoxylates– Variety of Pesticide Panels Variety of Pesticide Panels

Who do we work for?Who do we work for?• 70% U.S., 30% Cdn. + International 70% U.S., 30% Cdn. + International

• Government and Regulatory Authorities (50% of Government and Regulatory Authorities (50% of work)work)– National and State GovernmentsNational and State Governments– POP and CEC AssessmentPOP and CEC Assessment– Legal issuesLegal issues– Method DevelopmentMethod Development

• Private Sector Industry + Consultants (40% of work)Private Sector Industry + Consultants (40% of work)– Assessment and RemediationAssessment and Remediation– Regional and Ambient / Baseline Work / TMDLsRegional and Ambient / Baseline Work / TMDLs– POP and CEC Risk Assessment / LitigationPOP and CEC Risk Assessment / Litigation

• Academic, Legal, NGO – 10% Academic, Legal, NGO – 10%

AXYS AnalysisAXYS Analysis

• Early Days (1980 to 2000)Early Days (1980 to 2000)– Occurrence of POPs in all environmental and Occurrence of POPs in all environmental and

bioaccumulation matricesbioaccumulation matrices– ArcticArctic

• Regulatory Framework, Litigation and Regulatory Framework, Litigation and SettlementsSettlements– POPs in humans, NRD assessmentsPOPs in humans, NRD assessments

• Early Reconnaissance Studies and NGOsEarly Reconnaissance Studies and NGOs– Awareness of CECsAwareness of CECs

• Epidemiology Studies and CECsEpidemiology Studies and CECs– Explosion of programs 2007-8Explosion of programs 2007-8– CEC focusCEC focus

• AXYS does not publish studies, our clients AXYS does not publish studies, our clients dodo– 2-4 year lag vs. time of work2-4 year lag vs. time of work

AXYS Analytical Process (75 Staff)AXYS Analytical Process (75 Staff)

Log-In / Sample Storage Sample Prep. – Homogenization, Compositing, Dissection, Media Prep.

ExtractionCleanup

Reduction / Microvialing

InstrumentationPrimary Validation (before and after transfer to LIMS)

Secondary Validation+ Case Narratives + EDD Validation to Support Reporting

Expanded AXYS Analytical Process Expanded AXYS Analytical Process (15 client services staff)(15 client services staff)

- Definition of Client QAAP / DQO (Review/Agreement) - Definition of Client QAAP / DQO (Review/Agreement) - Creation of Project Instruction Notes- Creation of Project Instruction Notes

- Entry of LIMS / Reporting Instructions (P-Key)- Entry of LIMS / Reporting Instructions (P-Key)- PM schedules and communicates- PM schedules and communicates

AnalysisAnalysis

- Subcontract Analysis Calculated / Reported- Subcontract Analysis Calculated / Reported - Reporting- Reporting

-Electronic Data Deliverable Report (EDD)Electronic Data Deliverable Report (EDD)- Follow-up with ClientFollow-up with Client

AXYS AnalysisAXYS Analysis• All analysis performed with multiple mass All analysis performed with multiple mass

or mass fragment detection per targetor mass fragment detection per target– HRMS (EI or CI)HRMS (EI or CI)– GC/MS (EI or CI in SIM)GC/MS (EI or CI in SIM)– LC MS/MS in MRM mode (multiple LC MS/MS in MRM mode (multiple

transitions where possible) transitions where possible)

• All analysis use labelled standards added at All analysis use labelled standards added at start of analytical process, recovery start of analytical process, recovery corrected vs. recovery standardcorrected vs. recovery standard

• All “active” analysis EPA Tier 1 validation All “active” analysis EPA Tier 1 validation

(IPR, MDL, MS/MSD by EPA Federal Register (IPR, MDL, MS/MSD by EPA Federal Register 40 CFR Part 136, Appendix B, no iteration40 CFR Part 136, Appendix B, no iteration

GCMS Scan Chromatogram in Urine MatrixGCMS Scan Chromatogram in Urine MatrixUltra Trace Detection Not Required for Ultra Trace Detection Not Required for

TargetsTargets

GC/MS with Ultra Trace DLs Not Met GC/MS with Ultra Trace DLs Not Met pp-DDT by GC EI MS SIMpp-DDT by GC EI MS SIM

Meeting Ultra Trace RequirementsMeeting Ultra Trace Requirements13C12 pp DDT and pp DDT by HRMS13C12 pp DDT and pp DDT by HRMS

LC / MS vs. LC MS/MSLC / MS vs. LC MS/MSTIC (Total Ion Chromatogram) vs. MRM Monitoring TIC (Total Ion Chromatogram) vs. MRM Monitoring

Time0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00

%

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16.91

16.39

21.17 m in13.00 14.00 15.00 16.00 17.00

%

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PP7J_222S16

Dehydronefidipine

Ultra Trace Analysis PrinciplesUltra Trace Analysis PrinciplesPositive Identification and Quantification Positive Identification and Quantification

• Performance of multiple ions monitored and reported to Performance of multiple ions monitored and reported to meet response, ratio, and retention criteriameet response, ratio, and retention criteria

• Recovery correction requiredRecovery correction required

• Max. chromatographic separation and selectivity achieved Max. chromatographic separation and selectivity achieved through cleanup, columns, instrument typethrough cleanup, columns, instrument type

• Selectivity achieves low detection limits, ruggednessSelectivity achieves low detection limits, ruggedness

• Common to all CDC and EPA 1600 series methods for POPs Common to all CDC and EPA 1600 series methods for POPs and CECsand CECs

• Basis for EU 657 standard for positive identification and Basis for EU 657 standard for positive identification and quantification of targets with no specified reference quantification of targets with no specified reference methodmethod

• Many bio-monitoring methods would be considered a “non-Many bio-monitoring methods would be considered a “non-positive screen by this standard positive screen by this standard

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

# 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 Point Criteria for Positive EU 657 Point Criteria for Positive Identification if Using Isotope Identification if Using Isotope

DilutionDilution(4 Points Required for a Positive)(4 Points Required for a Positive)

Applying Point System to PBDE Applying Point System to PBDE AnalysisAnalysis

MethodMethod HRGC/HRMS HRGC/HRMS (8+ points)(8+ points)

HRGC/LRMSHRGC/LRMS(1-2 points)(1-2 points)

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

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

NCI (Negative NCI (Negative Chemical Chemical Ionization) /SIMIonization) /SIM

ScanScan

QuantificatioQuantificationn

1313C-labeled C-labeled standards for standards for isotope isotope dilutiondilution

Labeled standards Labeled standards not possiblenot possible

Labeled Labeled standards standards not possiblenot possible

SpecificitySpecificity High: High: Monitor two Monitor two to six ions to six ions from parent from parent ion cluster, ion cluster, compare to compare to theoretical theoretical ratioratio

Medium: Monitor BrMedium: Monitor Br-- ions lost from PBDEs. ions lost from PBDEs. Other brominated Other brominated compounds e.g. PBBs compounds e.g. PBBs may interfere if have may interfere if have same GC retention same GC retention timetime

Low: Low: Identification Identification based on GC based on GC retention time. retention time. Other Other halogenated halogenated compounds may compounds may interfere e.g. interfere e.g. PBBs, PCBs, Cl PBBs, PCBs, Cl pesticides if pesticides if same GC same GC retention time.retention time.

SensitivitySensitivity Best Best (Lowest) (Lowest) detection detection limitlimit

Highest Highest detection limitsdetection limits

Medium Medium detection limitsdetection limits

PFC Methods That Meet CriteriaPFC Methods That Meet CriteriaMRM Transitions and Surrogates UsedMRM Transitions and Surrogates Used

Target AnalyteTarget Analyte MRM TransitionMRM Transition ISIS MRMMRM SurrogateSurrogate

PFBA (C4 Acid)PFBA (C4 Acid) 213>169213>169 13C13C4 4 PFBAPFBA 217>17217>1722

13C4 PFOA13C4 PFOA

417>372417>372

PFPeA (C5 Acid)PFPeA (C5 Acid) 263>219263>219 217>17217>1722

PFHxA (C6 Acid)PFHxA (C6 Acid) 313>269,313>269, 313>119 313>119 13C13C2 2 PFHxAPFHxA 315>27315>2700

PFHpA (C7 Acid)PFHpA (C7 Acid) 363>319363>319, 363>169, 363>169 417>37417>3722

PFOA (C8 Acid)PFOA (C8 Acid) 413>369, 413>219,413>369, 413>219, 413>169413>169 13C2 PFOA13C2 PFOA 415>37415>37

00

PFNA (C9 Acid)PFNA (C9 Acid) 463>419463>419, 463>219, , 463>219, 463>169463>169

13C13C5 5 PFNAPFNA 468>42468>4233

PFDA (C10 Acid)PFDA (C10 Acid) 513>469513>469, 513>269, , 513>269, 513>219513>219

13C13C2 2 PFDAPFDA 515>47515>4700

PFUnA (C11 Acid)PFUnA (C11 Acid) 563>519563>519, 563>269, , 563>269, 563>219563>219

13C13C2 2 PFUnAPFUnA 565>52565>5200

PFDoA (C12 Acid)PFDoA (C12 Acid) 613>569613>569, 613>319, , 613>319, 613>169613>169

13C13C2 2 PFDoAPFDoA 615>57615>5700

PFBS (C4 Sulfonate)PFBS (C4 Sulfonate) 299>80, 299>99299>80, 299>99 303>84303>84 13 C2 FOUEA 13 C2 FOUEA 18 O2 PFOS18 O2 PFOS

PFHS (C6 Sulfonate)PFHS (C6 Sulfonate) 399>80, 399>99399>80, 399>99 18O18O2 2 PFHSPFHS 403>84403>84

PFOS (C8 Sulfonate)PFOS (C8 Sulfonate) 499>80, 499>99,499>80, 499>99, 499>130499>130 13C2 PFOS13C2 PFOS 503>80503>80

FOSA (C8 FOSA (C8 Sulfonamide)Sulfonamide) 498>70498>70 503>80503>80

CEC Surprises – Triggers for ChangeCEC Surprises – Triggers for ChangePFOS Pharmacokinetics and DistributionPFOS Pharmacokinetics and Distribution

• Well absorbed orally (95% within 24 h)Well absorbed orally (95% within 24 h)

• Distributed mainly in serum and liverDistributed mainly in serum and liver

• Not metabolizedNot metabolized

• Fecal and urinary excretionFecal and urinary excretion

• Perception change in early 2000’sPerception change in early 2000’s

• 30 ng/mL serum average in North Americans in 200430 ng/mL serum average in North Americans in 2004

Rat: Plasma t½ = 7.5 days

Monkey: Serum t½ = 200 days

Human: Estimated Serum t½ = ~8.7 years

AXYS PFC Method BasicsAXYS PFC Method Basics

• For water, soils, sediments, tissues, For water, soils, sediments, tissues, serum, airserum, air

• Applies principles of EPA 1600 Applies principles of EPA 1600 series performance based methodsseries performance based methods

• Key AXYS PFC Method AttributesKey AXYS PFC Method Attributes– Recovery CorrectedRecovery Corrected– Matrix Matched Calibration Matrix Matched Calibration – Pre-Concentration by SPE CartridgePre-Concentration by SPE Cartridge– Low detection limits and blank levelsLow detection limits and blank levels– Big “D”, small “R”Big “D”, small “R”– Benchmarked, multiple method Benchmarked, multiple method

validations to insure accuracyvalidations to insure accuracy– LOQ is LMCL point above MDL or LOQ is LMCL point above MDL or

client specific levelclient specific level

Adjust pH to 5.5 ±0.5 With HCOOHSpike labeled surrogates

SPE extraction with WAXCondition: 5 mL 0.3% NH4OH in MeOH

and 5mL0.1 M HCOOH

Wash: with 5 mL of H2O 5 mL & With 1:1 (0.1 M HCOOH: MeOH)

Analyze by (-ESI) LC-MS/MS

Elute: 4 mL of 0.3% NH4OH in MeOHMicrovial 300uL portion

Standard AXYS PFC In Water MethodMLA 060

POPs and CECs AnalysesPOPs and CECs AnalysesAccreditation and BenchmarkingAccreditation and Benchmarking

• POPs and CECs = Environmental ContaminantsPOPs and CECs = Environmental Contaminants

• ISO 17025:2005 is correct quality standardISO 17025:2005 is correct quality standard– ISO 17025: 2005 = ISO 9001:2000 + technical ISO 17025: 2005 = ISO 9001:2000 + technical

requirements (method and statistical QC)requirements (method and statistical QC)– National BodiesNational Bodies– NELAP not ISO 17025NELAP not ISO 17025

• Accreditation by test and environmental matrixAccreditation by test and environmental matrix

– NELAP offers regulatory method programsNELAP offers regulatory method programs– Human matrices not offered in pastHuman matrices not offered in past– CALA / SCC has now made availableCALA / SCC has now made available– Proficiency not available, must go to other forumsProficiency not available, must go to other forums

POPs and CECs – POPs and CECs – Benchmarking AccuracyBenchmarking Accuracy

• No “regulatory” programs or methodsNo “regulatory” programs or methods

• Tools for establishing accuracyTools for establishing accuracy– Inter-calibrations / Round Robins / PE ProgramsInter-calibrations / Round Robins / PE Programs– SRMs / CRMsSRMs / CRMs

• Inter-calibrations availableInter-calibrations available– AMAPAMAP– Dioxin Inter-calibrationsDioxin Inter-calibrations– PFC Inter-calibrationsPFC Inter-calibrations– Some academic programs for endocrine disruption Some academic programs for endocrine disruption

(BPA, PEMs)(BPA, PEMs)

• SRMs / CRMsSRMs / CRMs– NIST developedNIST developed– Available for most legacy (regulated) POPsAvailable for most legacy (regulated) POPs– Some persistent CECs in development (PFCs, PBDEs)Some persistent CECs in development (PFCs, PBDEs)– Not available for most other CECs (BPA, PEM, Parabens Not available for most other CECs (BPA, PEM, Parabens

etc.) etc.)

1st Worldwide Interlaboratory Study on PFCs in Environmental and Human Samples

*Fish Fillet Results PFOS PFOA

Spiked Concentration (ng/g) 37 10

Analytical Results (ng/g)

Minimum 2.8 0.54

Median 40 13

Maximum 295 204

%RSD 125 201

Evaluation of Results

%Satisfactory 17 25

%Questionable - 30

%Unsatisfactory 83 45

*Van Leeuwen et al. Environ. Sci. Technol. (2006) Vol. 40, p. 7854-7860.

Limits of Accuracy in early POPs Limits of Accuracy in early POPs and CEC Analysisand CEC Analysis

• Phthalate Ester metabolitesPhthalate Ester metabolites– Standards accuracy issueStandards accuracy issue– i.e. MCPP standard used for most studies i.e. MCPP standard used for most studies

appears to be 17% of reported valueappears to be 17% of reported value

• PFCs in EggsPFCs in Eggs

• BPABPA– 0.25 to 10 ng/mL gives 90% population 0.25 to 10 ng/mL gives 90% population

coverage in urine (NHANES)coverage in urine (NHANES)– Urine deconjugated BPA 10X blood valuesUrine deconjugated BPA 10X blood values– Some studies show much higher blood values Some studies show much higher blood values

(BPA instrument values)(BPA instrument values)

LC MS/MS False Positive ExampleLC MS/MS False Positive Example• Analysis – PFCs by LC MS/MSAnalysis – PFCs by LC MS/MS

• Matrix – Eggs for human consumptionMatrix – Eggs for human consumption

• Analytical Result – 120 mean ng/g PFOS per Egg Analytical Result – 120 mean ng/g PFOS per Egg detected using LC MS/MS in MRM mode (Carbon detected using LC MS/MS in MRM mode (Carbon labelled PFOS surrogate employed, one transition labelled PFOS surrogate employed, one transition monitored @ specified retention time, UPLC technology monitored @ specified retention time, UPLC technology submitted for publication)submitted for publication)

• Net Result – Paper withdrawn, results were identified Net Result – Paper withdrawn, results were identified

as false positive (TDCA, a bile acid in many animals)as false positive (TDCA, a bile acid in many animals) • Issue Identified – not enough positive identification Issue Identified – not enough positive identification

criteria criteria – Identified need for monitoring multiple transitionsIdentified need for monitoring multiple transitions– Did not focus on chromatographic separationDid not focus on chromatographic separation

Compound MW Parent Ion 1 Ion 2 Ion 3 CE CV

Taurochendeoxycholate 499.29 498.2 79.8 106.8 123.8 55 80

Taurodeoxycholate 499.29 498.2 79.8 106.8 123.8 55 80

Tauroursodeoxycholate

499.29 498.2 79.8 106.8 123.8 55 80

PFOS 499.93 498.9 79.9 98.9 N/A 45/40 80

LC MS/MS InterferencesLC MS/MS InterferencesPFOS and Taurodeoxycholate Acid PFOS and Taurodeoxycholate Acid

(TDCA)(TDCA)Same MW, Common TransitionSame MW, Common Transition

PFOS / TDCA InterferencePFOS / TDCA InterferenceTIC Chromatogram Separation TIC Chromatogram Separation PFOS – Top, 3 TDCA IsomersPFOS – Top, 3 TDCA Isomers

1,,15uL18:19:3202-Sep-2009

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8.64e56.80 6.84

Detection and Reporting LimitsDetection and Reporting Limits

• Select analytes, then required reporting Select analytes, then required reporting limits, then determine sample sizelimits, then determine sample size

• Define Define reportingreporting limits by study limits by study objectivesobjectives– NHANES is an excellent resourceNHANES is an excellent resource– Literature MAY be usefulLiterature MAY be useful

• Be aware of very different languagesBe aware of very different languages– Many Laboratories and PublicationsMany Laboratories and Publications

•Reporting Limit < MDL < Blank LevelReporting Limit < MDL < Blank Level

– ISO 17025 + Reference LaboratoriesISO 17025 + Reference Laboratories•Reporting Limit > MDL > Blank Level (ideal)Reporting Limit > MDL > Blank Level (ideal)

AXYS Reporting LimitsAXYS Reporting Limits• Default Sample Reporting Limits (RL)Default Sample Reporting Limits (RL)

– HRMS and GC/MS – LMCL or 3:1 signal to noise ratio of HRMS and GC/MS – LMCL or 3:1 signal to noise ratio of peakspeaks

– LC MS/MS – LMCL or 3:1 signal to noise ratio if above LMCLLC MS/MS – LMCL or 3:1 signal to noise ratio if above LMCL

• IDL not calculated or important, LMCL response 10-30X IDL not calculated or important, LMCL response 10-30X S/NS/N

• MDL 3-5X < RL (ideally), not used for reportingMDL 3-5X < RL (ideally), not used for reporting

• Dilution increases RL proportionallyDilution increases RL proportionally

• No blank correction, blank levels may influence No blank correction, blank levels may influence reporting limits by prior agreementreporting limits by prior agreement

• Applied “flags” to data quality – e.g. estimated Applied “flags” to data quality – e.g. estimated maximum and estimated minimum probable maximum and estimated minimum probable concentrations (EMPCs)concentrations (EMPCs)

• Should always be discussed with clientShould always be discussed with client

Blank Levels – A dirty secretBlank Levels – A dirty secret

• Many CECs are ubiquitous as in many Many CECs are ubiquitous as in many consumer goods and lab materialsconsumer goods and lab materials

• Blanks limit detection capability in analysis Blanks limit detection capability in analysis such as; such as; – PBDEsPBDEs– PCBsPCBs– PFCsPFCs– BPABPA– CotinineCotinine

• Can control with sample size selectionCan control with sample size selection

• Lab control not in method, but related SOPsLab control not in method, but related SOPs

• Ask for control charts (10 method blanks Ask for control charts (10 method blanks min.)min.)

Blank Control ChartsBlank Control ChartsUse them to set large study stat. limitsUse them to set large study stat. limits

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

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Analyte Lists – Which Ones to Analyte Lists – Which Ones to Measure?Measure?

• AXYS experience that what gets measured continues AXYS experience that what gets measured continues to get measuredto get measured

• Relevant POPs and CECs change over timeRelevant POPs and CECs change over time– Legacy POPsLegacy POPs– Replacement products for Replacement products for

• PFCsPFCs• PBDEs PBDEs • Phthalates Phthalates

– Aging (POPs congener distribution)Aging (POPs congener distribution)• Very little measurement of metabolites and Very little measurement of metabolites and

degradation productsdegradation products• All lack toxicity / epidemiological end point info for All lack toxicity / epidemiological end point info for

initial years of studyinitial years of study• All may be endocrine disruptorsAll may be endocrine disruptors• Studies should change over timeStudies should change over time

The Mean Percent Congener Distribution to The Mean Percent Congener Distribution to the Sum of the 35 PCBs (Whole Weight in the Sum of the 35 PCBs (Whole Weight in

ng/g) for NHANES 2003-2004 by Age Groupng/g) for NHANES 2003-2004 by Age Group

The Mean Percent Congener Distribution to The Mean Percent Congener Distribution to the Sum of the 35 PCBs (Whole Weight in the Sum of the 35 PCBs (Whole Weight in

ng/g) for NHANES 2003-2004 by Age Groupng/g) for NHANES 2003-2004 by Age Group

Lake Ontario % of PCB Concentrations Not Lake Ontario % of PCB Concentrations Not Part of IADN and US/DS Lists – Drives EPA Part of IADN and US/DS Lists – Drives EPA

1668 A,B,C1668 A,B,C

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153 + 168

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118

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40 + 41 + 71

66

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90 + 101 + 113

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21+ 33

16

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Are PFOS and PFOA the Only PFCs?Are PFOS and PFOA the Only PFCs?6 Months of Occurrence Data POTW Aqueous 6 Months of Occurrence Data POTW Aqueous

StreamsStreams

Analytes UnitsNo. of

Samples

No. of Detects

Percent of

Detects

Observed Concentration

Avg Conc. Found

Min Conc. Found

Max Conc. Found

PFBA ng/L 339 234 69 65.5 1.15 8540

PFBS 339 84 24.8 568 2.19 21000

PFDA 339 109 32.2 3.79 1.1 29.1

PFDoA 339 2 0.6 1.45 1.23 1.66

PFHpA 339 233 68.7 52.9 1.03 9610

PFHxA 339 253 74.6 328 1.03 76200

PFHxS 339 117 34.5 1650 2.03 190000

PFNA 339 210 61.9 5.62 1.03 28.1

PFOA 339 276 81.4 74.2 1.38 15600

PFOS 339 175 51.6 445 2.02 25200

PFOSA 339 25 7.4 15.3 1.04 61.7

PFPeA 340 216 63.5 174 1.04 29200

PFUnA 339 5 1.5 2.57 1.18 4.73

6 Months of PFCs in POTW 6 Months of PFCs in POTW BiosolidsBiosolids

Analytes UnitsNo. of

SamplesNo. of

Detects

Percent of

Detects


Avg Conc. Found

Min Conc. Found

Max Conc. Found

PFBA ng/g 123 11 8.9 7.26 1.14 16.2

PFBS 123 14 11.4 9.12 2.45 32.4

PFDA 123 91 74 14.6 0.712 57.5

PFDoA 123 56 45.5 8.08 0.292 58.9

PFHpA 123 12 9.8 6.54 2.01 28.1

PFHxA 123 22 17.9 14 0.727 105

PFHxS 123 25 20.3 32.3 1.82 147

PFNA 123 60 48.8 9.37 0.139 39.2

PFOA 123 55 44.7 17 0.585 256

PFOS 123 85 69.1 476 0.901 7480

PFOSA 123 39 31.7 40.8 0.194 343

PFPeA 123 21 17.1 12.4 0.567 69.6

PFUnA 123 52 42.3 6.44 1.12 35.2

PBDEs from TNSSS – EPA 2008PBDEs from TNSSS – EPA 2008

AnalyteAnalyte Units Units Minimum. Minimum. ConcentratiConcentrati

onon

Mean Mean ConcentratiConcentrati

onon

Maximum Maximum ConcentratiConcentrati

onon

% Solids% Solids %% 0.430.43 93.5393.53

BDE – 28BDE – 28 DryWt. DryWt. ng/kgng/kg

2,2002,200 160,000160,000


73,00073,000 709,174709,174 5,000,0005,000,000


1,8001,800 110,000110,000


3,2003,200 150,000150,000


64,00064,000 716,362716,362 4,000,0004,000,000


13,00013,000 1,100,0001,100,000


1,9001,900 40,00040,000


9,1009,100 52,68552,685 410,000410,000


7,7007,700 440,000440,000


2,1002,100 120,000120,000


150,000150,000 3,462,9423,462,942 17,000,00017,000,000

PBDE’s by EPA 1614 – Moving to 46 congeners PBDE’s by EPA 1614 – Moving to 46 congeners from Top 8 to 11 Congenersfrom Top 8 to 11 Congeners

• Theory – Penta and Octa changes will eliminate Theory – Penta and Octa changes will eliminate most toxic PBDEs, incoming Deca controls will most toxic PBDEs, incoming Deca controls will eliminate 209 congenereliminate 209 congener

• Current Situation – about 850M tons in Current Situation – about 850M tons in environmental inventory in NAenvironmental inventory in NA– Degradation of PBDE containing products giving ongoing Degradation of PBDE containing products giving ongoing

supplysupply– Deca plus new products production risingDeca plus new products production rising– Deca degradation rapid – to other PBDEsDeca degradation rapid – to other PBDEs

• May be in for a long PBDE storyMay be in for a long PBDE story

Legacy POPs and New POPsLegacy POPs and New POPsPFC Beluga Study – Published ES&T June 2009PFC Beluga Study – Published ES&T June 2009

POPs = Persistency, Toxicity, Bioaccumulation + TransportPOPs = Persistency, Toxicity, Bioaccumulation + Transport

Field work: Village of Umiujaq / Nastapoka River Estuary

Study OverviewStudy Overview

• Sample Collections: E. Hudson Bay– Sediment samples (n = 8) petit ponar grabs – Macro-algae (n = 6) – Harvested beluga whales (n=25); 12 females, 12

males, 1 calf

• liver, blubber, blood, milk– Fish: capelin, arctic cod, sculpin, salmon (n = 6

per species)

• Chemical analysis: – Previous Work: PCBs, PBDEs, OC Pesticides– Current Study: PFCs– AXYS Extended PFC List

(Carboxylates, Sulfonates, Sulfonamides)

Mean Values of PFCs in Beluga Mean Values of PFCs in Beluga Blood, Liver, MilkBlood, Liver, Milk

0

25

50

75

100

PFBA(C4)

PFPeA(C5)

PFHxA(C6)

PFHpA(C7)

PFOA(C8)

PFNA(C9)

PFDA(C10)

PFUnA(C11)

PFDoA(C12)

PFTA(C13)

Blood

Liver

Milk

0

100

200

300

400

PFBS PFHxS PFOS PFDS PFOSA N-MeFOSA

N-EtFOSA

N-MeFOSE

N-EtFOSE

PFCs (ng/g) in Arctic TissuePFCs (ng/g) in Arctic Tissue

0

10

20

30

40

50

PFBA(C4)

PFPeA(C5)

PFHxA(C6)

PFHpA(C7)

PFOA(C8)

PFNA(C9)

PFDA(C10)

PFUnA(C11)

PFDoA(C12)

PFTA(C13)

FishMale Beluga

0

50

100

150

200

250

300

PFBS PFHxS PFOS PFDS PFOSA N-MeFOSA

N-EtFOSA

N-MeFOSE

N-EtFOSE

PFC Levels in Trophic MagnificationPFC Levels in Trophic Magnificationvs. Legacy Organohalogensvs. Legacy Organohalogens

ng

.g-1

dry

wt.

0

0.5

1

1.5

2

Sedim

ents

0

5

10

15

20

25

Mac

ro-a

lgae

0

10

20

30

40

50

60

70

80

90

100

Capel

inCod

Salm

on

0

200

400

600

800

1000

1200

1400

1600

1800

2000

Beluga

= PCBs

= OC Pest.

= PBDEs

= PFCs

Beluga

Liver

Contaminants of Emerging Concern – “CECs”Contaminants of Emerging Concern – “CECs”Some New POPs + Many Endocrine Some New POPs + Many Endocrine

DisruptorsDisruptors• Many not persistent but exposure constantMany not persistent but exposure constant

• Many present through consumer goods – ubiquitousMany present through consumer goods – ubiquitous

• Many thought to be endocrine disruptorsMany thought to be endocrine disruptors

• Endocrine System Regulates Changes in Body Endocrine System Regulates Changes in Body – Response to environmentResponse to environment– Orchestrates body development and reproductive changesOrchestrates body development and reproductive changes

• Hormones are “Messengers” to CellsHormones are “Messengers” to Cells– Change cell internal and external chemistryChange cell internal and external chemistry– Many hormones and receptors, many interactions and Many hormones and receptors, many interactions and

outcomesoutcomes

• Endocrine Disruption Occurs when;Endocrine Disruption Occurs when;– Hormone receptors inhibited to not functionHormone receptors inhibited to not function– Hormone receptors triggered by “outside” compoundHormone receptors triggered by “outside” compound– Hormone System “Out of Balance” Hormone System “Out of Balance” – Effect is not a “toxic” effect Effect is not a “toxic” effect

Defining Endocrine Disruption End PointsDefining Endocrine Disruption End Points

• ToxicityToxicity– Typical screening processTypical screening process– Long term, subtle developmental effects not screenedLong term, subtle developmental effects not screened– ““Nonmonotonic Dose Response Curves” (more chemical does Nonmonotonic Dose Response Curves” (more chemical does

not equal more response – example BPA)not equal more response – example BPA)

• Effect May be at Specific Stage of Life CycleEffect May be at Specific Stage of Life Cycle– Requires study over long periods of life cycleRequires study over long periods of life cycle– More difficult with more complex speciesMore difficult with more complex species– Low level studies required, statistically relevant study size Low level studies required, statistically relevant study size

• Limited ToolsLimited Tools– Limited analytical and epidemiological methods at relevant Limited analytical and epidemiological methods at relevant

levelslevels– # of studies / study size – inconclusive data in many cases# of studies / study size – inconclusive data in many cases– Interpretation / vested interests Interpretation / vested interests

Topical End Points for POPs and CECsTopical End Points for POPs and CECs• ReproductiveReproductive

– Reduced fertilityReduced fertility– Reproductive tract abnormalitiesReproductive tract abnormalities– Male / Female Sex ratiosMale / Female Sex ratios

• Brain Development and Behavior IssuesBrain Development and Behavior Issues– Thyroid impairmentThyroid impairment– I.Q. reductionI.Q. reduction– ADHDADHD

• Immune System IssuesImmune System Issues– Increased IllnessIncreased Illness– ““Weaker” organismWeaker” organism

• Cancer, Diabetes, Heart Disease, Cholesterol variations, obesity Cancer, Diabetes, Heart Disease, Cholesterol variations, obesity etc.etc.

• Synergistic and Antagonistic EffectsSynergistic and Antagonistic Effects

An Interesting StudyAn Interesting Study

• Kidd et al 2007Kidd et al 2007– Environmental End Point – hormonal effects / Environmental End Point – hormonal effects /

vitellogenin gene expression on Fathead Minnowsvitellogenin gene expression on Fathead Minnows– Vitellogenin – egg yolk precursor protein expressed by Vitellogenin – egg yolk precursor protein expressed by

female fishfemale fish– Hormonal triggers (synthetic hormones, NP etc.) Hormonal triggers (synthetic hormones, NP etc.)

produce fish vitellogenin production / feminizationproduce fish vitellogenin production / feminization

• Study – Test (5-6 ng 17A ethinyl estradiol addition) Study – Test (5-6 ng 17A ethinyl estradiol addition) vs. Control Lake vs. Control Lake – Year 3 produces complete Fathead Minnow feminizationYear 3 produces complete Fathead Minnow feminization– Fathead population eliminated Fathead population eliminated

• Notes on InterpretationNotes on Interpretation– End Point measured on Fathead MinnowsEnd Point measured on Fathead Minnows– In other species - effects not notedIn other species - effects not noted– Tie to Humans? Tie to Humans?

Kidd K. A. et.al. PNAS 2007;104:8897-8901

Length frequency distributions of fathead minnow captured in trap nets in reference Lake 442 (A) and Lake 260 (B) (amended

with 5–6 ng·L−1 of EE2 in 2001–2003) during the fall of 1999–2005

• ““Reconnaissance” or Screen in Water and Reconnaissance” or Screen in Water and Sediments based on “USGS”, 1694 or Other Sediments based on “USGS”, 1694 or Other ListList

• Occurrence, Levels, and Fate of PPCPs in Occurrence, Levels, and Fate of PPCPs in POTW and Drinking Water ProcessesPOTW and Drinking Water Processes

• Occurrence, Levels, and Fate in POTW and Occurrence, Levels, and Fate in POTW and Water Treatment Systems for New Treatment Water Treatment Systems for New Treatment Systems / Capital Projects Systems / Capital Projects

• Trace back / fingerprinting of sources of Trace back / fingerprinting of sources of PPCPsPPCPs

• Support of Toxicity, Fate, and End Point Support of Toxicity, Fate, and End Point StudiesStudies

Purpose of AXYS “Client” Requests for Purpose of AXYS “Client” Requests for CEC CEC

Analysis – Example PPCPsAnalysis – Example PPCPs

The List – Which PPCP Compounds The List – Which PPCP Compounds to Analyze?to Analyze?

• 3000 potential PPCPs3000 potential PPCPs• Starting Point – USGS Reconnaissance ListStarting Point – USGS Reconnaissance List

– 40 PPCPS + Hormones and Sterols40 PPCPS + Hormones and Sterols• Constant requests from clients for more compounds Constant requests from clients for more compounds

based on literature, other studies, media articlesbased on literature, other studies, media articles• Input from National Regulatory BodiesInput from National Regulatory Bodies

– Lists supplied based on volumes, persistence, toxicity / Lists supplied based on volumes, persistence, toxicity / end point concerns (i.e. endocrine disruption, antibiotic end point concerns (i.e. endocrine disruption, antibiotic resistance)resistance)

– Fit for analysis – standards, surrogates, stability, Fit for analysis – standards, surrogates, stability, appropriate instrumentation appropriate instrumentation

• Framework to manage addition of compounds, Framework to manage addition of compounds, groups to maintain, validate, and improvegroups to maintain, validate, and improve

AXYS PHASE 3 PPCP METHOD (2008/9)AXYS PHASE 3 PPCP METHOD (2008/9)Grouping of Compounds by Analytical Grouping of Compounds by Analytical

Performance Performance EPA Method 1694 Compounds + Extended EPA Method 1694 Compounds + Extended

ListListPPCPc by PPCPc by LC-MS/MSLC-MS/MS

121 Compounds121 Compounds49 labelled 49 labelled SurrogatesSurrogates

List 1 PPCPsList 1 PPCPsAcidic ExtractAcidic Extract

+ve ESI+ve ESI 49 Compounds49 Compounds

11 Surrogates11 Surrogates

List 2 TCY’sList 2 TCY’sAcidic ExtractAcidic Extract

+ve ESI+ve ESI14 Compounds14 Compounds1 Surrogates1 Surrogates

List 4 PPCPsList 4 PPCPsBasic ExtractBasic Extract





-ve ESI-ve ESI12 Compounds12 Compounds9 Surrogates9 Surrogates

Building the MethodsBuilding the Methods

• Built From Back to FrontBuilt From Back to Front– Instrument InfusionInstrument Infusion– Response, Mode, Cone Voltage, TransitionsResponse, Mode, Cone Voltage, Transitions– Retain and Repeat !!!! Retain and Repeat !!!!

• Column SelectionColumn Selection– Phase 1 onlyPhase 1 only

• Selection of SPE CartridgesSelection of SPE Cartridges– Multiple tests in Phase 1 onlyMultiple tests in Phase 1 only– Varying pre-wash, loading, elutionVarying pre-wash, loading, elution

• ExtractionExtraction– Acidic, BasicAcidic, Basic

• ValidationValidation– MDL, IPR, MS/MSDMDL, IPR, MS/MSD

• Troubleshooting + Re-ValidationTroubleshooting + Re-Validation

EPA 1694 PPCPs – Frequency and Range of EPA 1694 PPCPs – Frequency and Range of DetectsDetects

EPA “Targeted National Sewage Sludge Survey”EPA “Targeted National Sewage Sludge Survey”All Results in dry weight ug/KgAll Results in dry weight ug/Kg

ANALYTEANALYTE # # DetectDetect

Minimum Minimum ug/Kgug/Kg

Maximum Maximum ug/Kgug/Kg

4-Epioxytetracycline4-Epioxytetracycline 88 35.735.7 54.954.94-Epitetracycline4-Epitetracycline 8080 47.247.2 4,3804,380Erythromycin-TotalErythromycin-Total 7777 3.13.1 180180FlumequineFlumequine 00 NANA NANAFluoxetineFluoxetine 7979 12.412.4 3,1303,130GemfibrozilGemfibrozil 7676 12.112.1 2,6502,650IbuprofenIbuprofen 5454 99.599.5 11,90011,900IsochlortetracyclineIsochlortetracycline 11 3,1403,140 3,1403,140LinomycinLinomycin 33 13.913.9 33.433.4LomefloxacinLomefloxacin 22 33.333.3 39.839.8MetforminMetformin 66 550550 1,1601,160MiconazoleMiconazole 8080 14.214.2 9,2109,210MinocyclineMinocycline 3232 351351 8,6508,650NaproxenNaproxen 4444 20.920.9 1,0201,020NorfloxacinNorfloxacin 2929 99.399.3 1,2901,290

Are We Measuring the Right Thing?Are We Measuring the Right Thing?Conjugates, Degradation Products, Conjugates, Degradation Products,

MetabolitesMetabolites

• Current StateCurrent State– Analytes primarily native compounds Analytes primarily native compounds – Most compounds “persistent” by constant Most compounds “persistent” by constant

presence presence – Limited degradation products and metabolites Limited degradation products and metabolites – No Chlorination byproductsNo Chlorination byproducts– No conjugatesNo conjugates

• About ConjugatesAbout Conjugates– Many compounds exist as conjugates from urine Many compounds exist as conjugates from urine – Deconjugation may occur at some rate in POTW Deconjugation may occur at some rate in POTW

environments or on consumption environments or on consumption – Limited info but detected in studiesLimited info but detected in studies– Methods from Biomonitoring AvailableMethods from Biomonitoring Available

• I.e. Triclosan, BPA, Parabens, mono-phthalate estersI.e. Triclosan, BPA, Parabens, mono-phthalate esters

Looking for the Correct Analyte FormLooking for the Correct Analyte FormTriclosan Conjugates in Urine vs. Triclosan Conjugates in Urine vs.

Native Triclosan in SerumNative Triclosan in Serum

Analyte UnitsNo. of

SamplesNo. of

Detects

Percent of

Detects


Avg Conc. Found

Min Conc. Found

Max Conc. Found

Total TriclosanIn Urine

ng/mL 62 52 83.9 109 1.33 1980

NativeTriclosanIn Serum

ng/mL 22 0 0 ND ND ND

Degradation Products / Degradation Products / MetabolitesMetabolites

• Degradation ProductsDegradation Products– Many Compounds not Stable Many Compounds not Stable – Fit for TOF LC-MS/MS to identify degradation Fit for TOF LC-MS/MS to identify degradation

productsproducts– Not certain degradation products of less concern Not certain degradation products of less concern

• MetabolitesMetabolites– In Humans and in EnvironmentIn Humans and in Environment– Fit for TOF LC-MS/MS to identify degradation Fit for TOF LC-MS/MS to identify degradation – Not certain degradation products of less concernNot certain degradation products of less concern

Major Cl Transformation ProductsMajor Cl Transformation ProductsTriclosan + Free ChlorineTriclosan + Free Chlorine

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