““ANALYSIS OF CURRENT USE ANALYSIS OF CURRENT USE PESTICIDES IN ENVIRONMENTAL PESTICIDES IN ENVIRONMENTAL AND WASTEWATER SAMPLES BY AND WASTEWATER SAMPLES BY

HIGH RESOLUTION GC WITH HIGH HIGH RESOLUTION GC WITH HIGH RESOLUTION MASS RESOLUTION MASS

SPECTROMETRIC DETECTION”SPECTROMETRIC DETECTION”

Richard GraceRichard Grace, Coreen Hamilton, Million , Coreen Hamilton, Million WoudnehWoudneh

AXYS Analytical Services Ltd. , Sidney, BC, AXYS Analytical Services Ltd. , Sidney, BC, CanadaCanada

AXYS ANALYTICAL SERVICES LTD.AXYS ANALYTICAL SERVICES LTD.

NELAP ACCREDITED / ISO 17025 CERTIFIEDNELAP ACCREDITED / ISO 17025 CERTIFIED

AGENDAAGENDA

Purpose (s) of AnalysisPurpose (s) of Analysis Development HistoryDevelopment History Details of the MethodDetails of the Method Detection Limits and Method QCDetection Limits and Method QC Applications and ResultsApplications and Results

PURPOSE (s) of ANALYSISPURPOSE (s) of ANALYSIS

Analysis of Wide Variety of PesticidesAnalysis of Wide Variety of Pesticides Legacy (OC) PesticidesLegacy (OC) Pesticides Current Use Pesticides (Canada / U.S. Focus)Current Use Pesticides (Canada / U.S. Focus) ““Multi-Residue Pesticide Test” or “MRES”Multi-Residue Pesticide Test” or “MRES”

Improvements in Sensitivity and Improvements in Sensitivity and SelectivitySelectivity Improvement in Detection Limits vs. Improvement in Detection Limits vs.

Reference Methods (10 – 1000X lower Reference Methods (10 – 1000X lower detection limits vs. non 1600 series EPA detection limits vs. non 1600 series EPA methods) – meet ambient criteria where methods) – meet ambient criteria where establishedestablished

Reduction of False Positives and Matrix Reduction of False Positives and Matrix Effects by Use of HRMS and Serial Isotope Effects by Use of HRMS and Serial Isotope Dilution / RRF QuantificationDilution / RRF Quantification

PURPOSE (s) of ANALYSISPURPOSE (s) of ANALYSIS

Applicable to Wide Variety of MatricesApplicable to Wide Variety of Matrices Platform for Analysis in Most Matrices (1600 Platform for Analysis in Most Matrices (1600

series)series) Water – Surface, Ground, Effluent, Potable, Water – Surface, Ground, Effluent, Potable,

High Volume Sampling on XAD ResinHigh Volume Sampling on XAD Resin Solids – Soil, Sediment, POTW BiosolidsSolids – Soil, Sediment, POTW Biosolids Tissue / Serum (current development work)Tissue / Serum (current development work)

Reduction of Pre-Laboratory WorkReduction of Pre-Laboratory Work Reduction in number / size of samplesReduction in number / size of samples Reduction in compositing / homogenization Reduction in compositing / homogenization

for broad reconnaissance of Pesticides for broad reconnaissance of Pesticides

HISTORY of DEVELOPMENTHISTORY of DEVELOPMENT

2003 - Initial Method Development for Environment 2003 - Initial Method Development for Environment Canada (Pacific Region) for Aqueous SamplesCanada (Pacific Region) for Aqueous Samples

2003 – 2005 – Groundwater / Surface Water Survey 2003 – 2005 – Groundwater / Surface Water Survey for Environment Canada in Lower Fraser Valley for Environment Canada in Lower Fraser Valley

2005 – Application to Soils / Sediments2005 – Application to Soils / Sediments 2006 – Application / Validation to POTW Influent / 2006 – Application / Validation to POTW Influent /

Effluent / Biosolids for U.S. EPA Office of WaterEffluent / Biosolids for U.S. EPA Office of Water 2006 – High Volume Water Application / Collection 2006 – High Volume Water Application / Collection

on XAD Resin Developedon XAD Resin Developed 2007 – Further Method Refinement and Validation 2007 – Further Method Refinement and Validation

Under Contract to U.S. EPA Office of WaterUnder Contract to U.S. EPA Office of Water 2007 – Application of Method to Tissue and Serum 2007 – Application of Method to Tissue and Serum

ANALYTESANALYTES

CURRENT LISTCURRENT LIST 35 OC Pesticides and Metabolites35 OC Pesticides and Metabolites 20 Organophosphorous 20 Organophosphorous

CompoundsCompounds 7 Triazine Compounds7 Triazine Compounds 2 Pyrethroid Groups2 Pyrethroid Groups 14 Organonitrogen Compounds 14 Organonitrogen Compounds

RATIONALE for ANALYTE RATIONALE for ANALYTE SELECTIONSELECTION

Current or Historic UseCurrent or Historic Use Availability of Standards and Availability of Standards and

Appropriate SurrogatesAppropriate Surrogates Capture by SPE cartridgeCapture by SPE cartridge Analyte Stability (Matrix and Analyte Stability (Matrix and

Instrument)Instrument) Appropriate monitoring ionsAppropriate monitoring ions Validation of Positive IdentificationValidation of Positive Identification

OVERVIEW of ANALYSISOVERVIEW of ANALYSISSample

1L Water or 1-5 g (dry wt.) soil, Sediment, or Biosolid

Surrogate Addition followed by DCM Extraction (3X)Dry, Filter, Sample Reduction to 100uL

Make-up to 1mL in 1:2:1 ethyl acetate, acetonitrile, toluene

SPE Cartridge Cleanup (1 gram aminopropyl bonded silica –Varian)SPE Steps – condition, load, rinse, elute, reduce, transfer to hexane

Microsilica Column Cleanup – wet, load, rinse, elute, reduce

ANALYSIS by HRGC / HRMSRun 1 – OC, OP, Triazine, and Pyrethroid Analytes

Run 2 – ON Analytes

EXTRACTON DETAILS (Aqueous)EXTRACTON DETAILS (Aqueous)

Weigh 1L bottle, and all steps in transfersWeigh 1L bottle, and all steps in transfers Transfer water to 2L sep. funnel, rinse Transfer water to 2L sep. funnel, rinse

sample container with DCM, transfer DCM sample container with DCM, transfer DCM to 2L sep. funnelto 2L sep. funnel

Add surrogates in acetone (1mL), include Add surrogates in acetone (1mL), include rinsesrinses

Extract through shaking ( 3 x 100 mL DCM)Extract through shaking ( 3 x 100 mL DCM) Combine extracts, then dry with anhydrous Combine extracts, then dry with anhydrous

NaSO4NaSO4 Transfer to round bottom with 1 ml keeper Transfer to round bottom with 1 ml keeper

toluene and roto vap to 1 mL tube, toluene and roto vap to 1 mL tube, concentrate to 100ul by N2concentrate to 100ul by N2

Makeup to 1mL in 1:2:1 ethyl acetate, Makeup to 1mL in 1:2:1 ethyl acetate, acetonitrile, acetonitrile, toluenetoluene

EXTRACTION DETAILS (Solid)EXTRACTION DETAILS (Solid) 1 -5 g dry weight in beaker, add 1 -5 g dry weight in beaker, add

anhydrous powdered sodium sulfate, anhydrous powdered sodium sulfate, equilibrate 30 min.equilibrate 30 min.

Load to soxhlet thimble, add surrogatesLoad to soxhlet thimble, add surrogates Add 300 mL DCM to soxhlet, add Add 300 mL DCM to soxhlet, add

thimblethimble Soxhlet 16 hoursSoxhlet 16 hours RotoVap to 1 ml, dry with sodium RotoVap to 1 ml, dry with sodium

sulfate if necessary, filter through glass sulfate if necessary, filter through glass wool columnwool column

Continue as per aqueous procedure Continue as per aqueous procedure

SPE CLEANUP DETAILSSPE CLEANUP DETAILS

SPE Cartridge Type – 1 g aminopropyl SPE Cartridge Type – 1 g aminopropyl bonded silica columnbonded silica column

Solvent – 1:2:1 ethyl acetone, acetonitrile, Solvent – 1:2:1 ethyl acetone, acetonitrile, toluenetoluene Condition using 2 X 3 mL (column volume)Condition using 2 X 3 mL (column volume) Load sample followed by 1 mL rinseLoad sample followed by 1 mL rinse Elute with 11 mLsElute with 11 mLs

Concentrate to 1 mL by RotoVapConcentrate to 1 mL by RotoVap Transfer to centrifuge tubes with hexane Transfer to centrifuge tubes with hexane

rinserinse Concentrate to 100 uL using gentle Concentrate to 100 uL using gentle

nitrogennitrogen

MICROSILICA COLUMN CLEANUP MICROSILICA COLUMN CLEANUP DETAILSDETAILS

Makeup extract to 1mL in HexaneMakeup extract to 1mL in Hexane Wet microsilica column (Silica AR 100-Wet microsilica column (Silica AR 100-

200 mesh from Mallinckrodt) with 5-10 200 mesh from Mallinckrodt) with 5-10 mLs HexanemLs Hexane

Load sample followed by 500uL hexane Load sample followed by 500uL hexane rinse, 2 x 300uL rinse 10% MeOH/ 90% rinse, 2 x 300uL rinse 10% MeOH/ 90% DCMDCM

Elute with 5 mL 10% MeOH/90% DCM, Elute with 5 mL 10% MeOH/90% DCM, combine with rinsescombine with rinses

Add 5 mL acetone and 1 mL iso-octane, Add 5 mL acetone and 1 mL iso-octane, rotovap to 1 mLrotovap to 1 mL

Transfer to centrifuge tube with hexane Transfer to centrifuge tube with hexane rinses, concentrate to 20 - 300 uL, rinses, concentrate to 20 - 300 uL, transfer to microvial, add recovery transfer to microvial, add recovery standards standards

ANALYSIS DETAILS ANALYSIS DETAILS

All Analysis performed with Micromass All Analysis performed with Micromass Ultima HRMS with Agilent 6890 GC, CTC Ultima HRMS with Agilent 6890 GC, CTC autosampler, alpha data system running autosampler, alpha data system running Opus 6.3 softwareOpus 6.3 software

DB-17 capillary GC column (30m, 0.25 mm DB-17 capillary GC column (30m, 0.25 mm i.d., 0.25 um film thickness, direct couple i.d., 0.25 um film thickness, direct couple to MS sourceto MS source

8,000 static mass resolution in EI mode8,000 static mass resolution in EI mode Multiple Ion detection – two ions for each Multiple Ion detection – two ions for each

target* and surrogate target* and surrogate 20 – 400 uL typical extract volume, 1-2 uL 20 – 400 uL typical extract volume, 1-2 uL

injection volumeinjection volume

INSTRUMENT DETAILS (cont.)INSTRUMENT DETAILS (cont.)

2 runs2 runs Run 1 – OC compounds, OP compounds, Run 1 – OC compounds, OP compounds,

Triazine compounds, PyrethroidsTriazine compounds, Pyrethroids Run 2 – Organonitrogen compoundsRun 2 – Organonitrogen compounds

Injector temperature and ramping is Injector temperature and ramping is key differencekey difference 220C for Run 1 injector temp.220C for Run 1 injector temp. 260C for Run 2 injector temp.260C for Run 2 injector temp.

Bracketed calibration during runBracketed calibration during run Calibration vs. 6 point linearityCalibration vs. 6 point linearity

AQUEOUS DETECTION LIMITSAQUEOUS DETECTION LIMITS

Determined by Federal Register (USA) 40 Determined by Federal Register (USA) 40 CFR Part 136, Appendix B, October CFR Part 136, Appendix B, October 26,198426,1984

OC Pesticides – 0.4 pg/L to 60.7 pg/L, OC Pesticides – 0.4 pg/L to 60.7 pg/L, OP Pesticides - 2.6 to 98.9 pg/L, OP Pesticides - 2.6 to 98.9 pg/L, Triazines – 1.5 to 12.7 pg/L, Triazines – 1.5 to 12.7 pg/L, Pyrethroids – 22 to 45 pg/L, Pyrethroids – 22 to 45 pg/L, Organonitrogen – 0.53 to 9.41 ng/L, Organonitrogen – 0.53 to 9.41 ng/L, Sample Detection Limits (similar to EPA Sample Detection Limits (similar to EPA

1613b) often used to reduce DLs 2-5X 1613b) often used to reduce DLs 2-5X from calculated MDLs in clean matricesfrom calculated MDLs in clean matrices

DETECTION LIMIT NOTESDETECTION LIMIT NOTES

Clean Matrix MDLs follow Aqueous Clean Matrix MDLs follow Aqueous patterns, units in pg/gpatterns, units in pg/g

POTW Effluent generally raises DLs by POTW Effluent generally raises DLs by 10X10X

POTW Influent generally raises DLs by POTW Influent generally raises DLs by 40X40X

POTW Biosolids generally raises DLs by POTW Biosolids generally raises DLs by to 0.2 to 70 ng/gto 0.2 to 70 ng/g

STANDARD METHOD QC – STANDARD METHOD QC – Standards and SurrogatesStandards and Surrogates

Non-labeled standards supplied by Non-labeled standards supplied by Accustandard (78 compounds)Accustandard (78 compounds)

Carbon labeled standards supplied by Carbon labeled standards supplied by Cambridge Isotope LaboratoriesCambridge Isotope Laboratories 21 C13 labeled OC Pesticides21 C13 labeled OC Pesticides 1 permethrin (cis/trans) mix1 permethrin (cis/trans) mix 2 ON Compounds (Alachlor, Metalochlor)2 ON Compounds (Alachlor, Metalochlor) 1 OP Compound (Fonofos)1 OP Compound (Fonofos) 1 Triazine Compound (Atrazine)1 Triazine Compound (Atrazine)

Deuterated surrogates (linuran, Deuterated surrogates (linuran, azinophos methyl, diazinon) supplied azinophos methyl, diazinon) supplied by Dr. Ehrenstorfer Gmbhby Dr. Ehrenstorfer Gmbh

Recovery Standards (C13 PCB 52,138) Recovery Standards (C13 PCB 52,138) supplied by Wellington Laboratories supplied by Wellington Laboratories Inc. Inc.

METHOD QC CRITERIAMETHOD QC CRITERIA

Max. Batch Size (including QC) is 20 Max. Batch Size (including QC) is 20 samples (8 to 14 is more common)samples (8 to 14 is more common)

Each Batch contains: Each Batch contains: method blank method blank duplicate duplicate Laboratory Control Sample (LCS) Laboratory Control Sample (LCS) MS/MSD, SRM,CRM included by request and MS/MSD, SRM,CRM included by request and

applicabilityapplicability LCS Recovery 50-150 most common, LCS Recovery 50-150 most common,

some wider specs. if reproducibility high some wider specs. if reproducibility high

QA ACCEPTANCE CRITERAQA ACCEPTANCE CRITERA

EPA Tier 1 Validation Protocols for Single EPA Tier 1 Validation Protocols for Single Laboratory Method for IPR, MS/MSD, and Laboratory Method for IPR, MS/MSD, and MDL criteriaMDL criteria

Duplicate Criteria – 40% RPD for Analytes Duplicate Criteria – 40% RPD for Analytes 10X above MDL10X above MDL

Bracketing Cal. – 40% RSD for targets Bracketing Cal. – 40% RSD for targets detecteddetected

Cal. Ver – 25% RSD for native analytes with Cal. Ver – 25% RSD for native analytes with exact labeled standards, 35% for others exact labeled standards, 35% for others

Linearity (ICAL) – 25% RSD for native Linearity (ICAL) – 25% RSD for native analytes that have exact labeled standards, analytes that have exact labeled standards, 35% for others35% for others

APPLICATIONS and RESULTSAPPLICATIONS and RESULTS

To date N > 500 samplesTo date N > 500 samples 50 % Groundwater / Surface Water (diverse 50 % Groundwater / Surface Water (diverse

locations)locations) 35% POTW influent/effluent/biosolids35% POTW influent/effluent/biosolids 15% sediments / high volume water / other15% sediments / high volume water / other

Continuous Improvement has led to 5 Continuous Improvement has led to 5 method revisions. Key experiences in; method revisions. Key experiences in; Spiking levels and standards maintenance / Spiking levels and standards maintenance /

groupinggrouping Bench “technique” in extraction / cleanupBench “technique” in extraction / cleanup Sample size and final extract options to limit Sample size and final extract options to limit

matrix effects and maintain instrument matrix effects and maintain instrument cleanlinesscleanliness

Use of Method QC to update acceptance criteria Use of Method QC to update acceptance criteria

APPLICATIONS and RESULTS APPLICATIONS and RESULTS (cont.)(cont.)

““Detects” vary widely dependant Detects” vary widely dependant on matriceson matrices Many compounds are readily Many compounds are readily

degraded by biological activity, degraded by biological activity, photolysis, or low persistency. photolysis, or low persistency. Generally, metabolites are not the Generally, metabolites are not the target of the analysis.target of the analysis.

Compounds detected vary widely Compounds detected vary widely dependant on regiondependant on region Urban vs. ruralUrban vs. rural Type of Agricultural ActivityType of Agricultural Activity Regulatory ActionRegulatory Action

NEXT STEPSNEXT STEPS

Peer Review of draft method by Peer Review of draft method by EPA – Q3/4 2007EPA – Q3/4 2007

Further processing of samples to Further processing of samples to develop further statistical develop further statistical information information

Tissue / Serum ApplicationTissue / Serum Application Multi-Lab Validation?Multi-Lab Validation? Questions? Questions?