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3M EnvirOnmental Laboratory Pmjeci No. 05-0210 Interim Report #5: Analysis of West MorganlEast Lawrence Municipal Water Samples Study Title ANALYSIS OF PERFLUOROBUTANESULFONATE(PFBS), PERFLUOROHEXANESULFONATE (PFHS), AND PERFLUOROOCTANESULFONATE(PFOS) IN WATER, SOIL, SEDIMENT, FISH, CLAMS, VEGETATION, SMALL MAMMALIVER AND SMALL MAMMALSERUM USING LC/MS/MS FOR THE 3M DECATURMONITORINGPROGRAM Data Requirement EPA TSCA Good Laboratory Practice Standards 40 CFR 792 Author Michelle D. Malinsky, Ph.D 3M Environmental Laboratory Interim Report Completion Date Date of signing Performing Laboratory 3M Environmental Laboratory Building 2-3E-09 935 Bush Ave. St. Paul, MN 55106 Project Identification E05-0210 EO5-0210Interim Report #5 Total Number of Pages 123 Page 1 of 123 This page has been reserved for specific country requirements. 3M Environmental Laboratory Prvject No. EO5-02 10 EO5-0210 Interim Report #5 Page 2 of 123 3M Environmental Laboratory Project No. E050210 GLP COMPLIANCSETATEMENT Study Title: Analysis of Perfluorobutanesulfonate(PFBS), Perfluorohexanesulfonate (PFHS), and Perfluorooctanesulfonate (PFOS) in Water, Soil, Sediment, Fish, Clams,Vegetation, Small Mammal Liver and Small Mammal Serum Using LCIMSIMS for the 3M Decatur Monitoring Program InterimStudy: Analysis of West MorganIEast Lawrence MunicipalWater Samples Study IdentificationNumber: E05-0210 This study was conducted in compliance with Toxic Substances Control Act (TSCA) Good Laboratory Practice (GLP) Standards, 40 CFR 792, with the exceptions listed below: Exceptions to GLPpmpliance: Jaisimha KMariP. E., DSE, Study Director d%/U Date / Michael A. Santdo, 3M Company, Sponsor Representative Date 05-021 0 Interim Report #A5 Page 3 of 123 3M Environmental Laboratory Project No. 05-0210 QUALITYASSURANCSETATEMENT Study Title: Analysis of Pefluorobutanesulfonate(PFBS), Pemuorohexanesulfonate (PFHS), and Perfluorooctanesulfonate(PFOS) in Water, Soil, Sediment, Fish, Clams,Vegetation, Small Mammal Liver and Small Mammal Serum Using LCIMSIMS for the 3M Decatur Monitoring Program Interim Study: Analysis of West Morgan I East Lawrence Municipal Water Samples InterimStudy IdentificationNumber: E05-0210 This study was audited by the 3M Environmental Laboratory Quality Assurance Unit (QAU), as indicatedin the following table. The findings were reportedto the study director and laboratory management. May 6,2005 May 13,2005& May 16,2005 I In-Phase DatalFinal Report I 5 17-2005 51 7-2005 51 7-2005 5 17-2005 I I I */- Date E050210 Interim Report#5 Page 4 of 123 3M nVironmental Laboratory Project No.05-0210 TABLEOF CONTENTS GLP Compliance Statement ...................................................................................................................... 3 Quality Assurance Statement .................................................................................................................... 4 Table of Contents ....................................................................................................................................... 5 Listof Tables............................................................................................................................................... 6 List of Figures ............................................................................................................................................. 7 Study Information ....................................................................................................................................... 8 Summaryand Introduction......................................................................................................................... 9 Test Samples.............................................................................................................................................. 9 ReferenceSubstances............................................................................................................................. 10 Control Substance.................................................................................................................................... 10 Method Summaries .................................................................................................................................. 11 SampleCollection................................................................................................................................ 11 Preparatoryand Analytrcal Methods.................................................................................................. 11 Extraction.............................................................................................................................................. 11 Analysis................................................................................................................................................. 11 Analytical Results ..................................................................................................................................... 12 Calibration............................................................................................................................................. 12 Limitof Quantitation (LOQ).................................................................................................................. 13 Blanks ................................................................................................................................................... 13 Solvent Blanks...................................................................................................................................... 13 Method Blanks..................................................................................................................................... 13 Trip Blanks............................................................................................................................................ 14 System Suitab.tl.lty................................................................................................................................. 14 Continuing Calibration.......................................................................................................................... 14 Lab Control Spikes (LCSs).................................................................................................................. 14 Sample Duplicates ............................................................................................................................... 15 05-0210 Interim Report #5 Page 5 of 123 3M Environmental Laboratory Pmject No.05-0210 Surrogates ............................................................................................................................................ 16 Field Matrix Spikes............................................................................................................................... 16 Data Summary.......................................................................................................................................... 16 Statistical Methods and Calculations....................................................................................................... 18 Accuracy and Precision Equations ..................................................................................................... 18 Determinationof Analytical Uncertainty.............................................................................................. 18 Statement of Conclusion.......................................................................................................................... 19 List of Attachments ................................................................................................................................... 19 SignaturePage......................................................................................................................................... 20 LISTOF TABLES Table 1. Sample ResultsSummary............................................................................................................. 9 Table 2. Study ReferenceSubstance....................................................................................................... 10 Table 3. Study Control Substance (Surrogate)........................................................................................ 10 Table 4. Sample Collection Information.................................................................................................... 11 Table 5. InstrumentParameters................................................................................................................ 12 Table 6. LiquidChromatographyGradient Program................................................................................ 12 Table 7. MassTransitions.......................................................................................................................... 12 Table 8. MethodBlankArea Counts......................................................................................................... 13 Table 9. LabControlSpike Results............................................................................................................ 15 Table 10. Sample Results Location#I: WMEL PW RWOI.................................................................... 17 Table 11. Sample Results Location##2:WMEL PW R N O l .................................................................... 17 Table 12. Sample Results: Trip Blank...................................................................................................... 18 05-0210 lnterim Report #5 Pa98 6 Of 723 3M EnvironmentalLaboratory Project No. 05-0210 LISTOF FIGURES Figure 1. CalibrationCurve: ExtractedPFBSCalibrationStandards inASTM Type IWater................22 Figure2. CalibrationCurve: ExtractedPFHSCalibrationStandardsinASTM Type IWater. ..............23 Figure3. CalibrationCurve: ExtractedPFOS CalibrationStandards inASTM Type IWater. ..............24 Figure4. CalibrationCurve: ExtractedPFOA [1,2 I3C]CalibrationStandardsinASTM Type IWater. 25 Figure5. Total IonChromatogramfor PFBS: 0.0253 ng/mL ExtractedCalibrationStandard.............26 Figure6. Total Ion Chromatogramfor PFHS: 0.0263 ng/mL ExtractedCalibrationStandard..............27 Figure7. Total IonChromatogramof PFOS: 0.0257 ng/mL ExtractedCalibrationStandard...............28 Figure8. Total Ion Chromatogramof PFOA [I,2 "C]: 0.0249 ng/mL ExtractedCalibrationStandard.29 Figure9. PFOS Total Ion Chromatogram: RepresentativeMethodBlank (050506a072; Day 2 Method Blank 3) ....................................................................................................................................... 30 Figure 10. PFOSTotal IonChromatogram: RepresentativeMethanolSolvent Blank (0050506a052).31 Figure 11. PFBS Total Ion Chromatogram: (Location #2; WMEL PW FW 01, Sample lD82438. 005O506a061). ........................................................................................................................... 32 Figure 12. PFHS Total Ion Chromatogram: (Location ##2W; MEL PW FW 01, Sample lD82438, 0050506a061). ........................................................................................................................... 33 Figure 13. PFOSTotal Ion Chromatogram(Locatione;WMEL PW FW 01, Sample lD82438; 0050506a061). ........................................................................................................................... 34 Figure 14. PFOA [ I,2 I3C]- Surrogate Chromatogram: (Location#2; WMEL PW FW 01, Sample lD82438,o050506a061)............................................................................................................ 35 EO5-0210 Interim Report #5 Page 7 of 123 3M Environmental Laboratoy Pmject No. 05-0210 STUDYINFORMATION Sponsor 3M Company Sponsor Representative MichaelA. Santoro 3M Company 3M Building 0236-01-8-10 St. Paul, MN 55144 Study Director Jaisimha Kesari, P.E., DEE Weston Solutions, Inc. 1400 Weston Way West Chester, PA 19380 Study Location Testing Facilities Exygen Research 3058 Research Drive State College, PA 16801 3M Environmental Laboratory 3M Building 2-3E-09 935 Bush Avenue St. Paul, MN 55144 Study Dates Study Initiation: 11/5/2004 Interim Experimental Initiation: May 4,2005 Interim Experimental Completion: May 8,2005 Interim Study Completion: Location of Archives All original raw data, protocol, and analytical report have been archived at the 3M EnvironmentalLaboratory according to 40 CFR Part 792. The test substance and analytical reference standard reserve samples are archived at the 3M Environmental Laboratory according to 40 CFR Part 792. E054210 Interim Repott #5 Page 8 of 123 3M Environmental Laboratory Project No. 05-0210 SUMMARY AND INTRODUCTION The 3M EnvironmentalLaboratoryextracted and analyzed municipal (raw and finished) water samples collected by Weston Solutions personnel on May 3,2005 in Decatur, Alabama for 3M Environmental Laboratory project number E05-0210 (3M Decatur FluorochemicalGLP Monitoring Program). Water samples were analyzed for perfluorobutanesulfonate(PFBS), perfluorohexanesulfonate(PFHS), and perfluorooctanesulfonate (PFOS) using 3M EnvironmentalLaboratory Method ETS 8-154.1 "Determinationof PerfluorinatedAcids, Alcohols, Amides, and Sulfonates in Water by Solid Phase Extractionsand High PerformanceLiquid Chromatography/MassSpectrometry" in accordancewith Exygen Research Protocol PO001131 (AttachmentC). The analyticalstart date for this interim report was May 4, 2005 and the analytical completion date was May 8,2005. Sample containerswere prepared at the 3M EnvironmentalLaboratory. Sample containers for each sampling location included a field sample, field sample duplicate, low field spike (0.10 ng/mL) and high field spike (1.O ng/mL). Each empty container was marked with a "fill to here" line and was fortified with a surrogate spike or an appropriate matrix spike solution containing the surrogate and the target analytes prior to being sent to the field for sample collection. Table 1 below summarizesthe sample results. All results for quality control samples prepared and analyzed with the samples will be reported and discussed elsewhere in this report. Quality control samples were used to establish the analytical uncertainty of the results presented below. Table 1. Sample Results Summary Concentration 0.116 0.0727 0.0754 The analytical uncertainty of the PFBS results is IO&%% based on method accuracy and precision. The lower limit of quantitation (LOQ) for PFBS is 0.00506 nglmL. The analytical uncertaintyof the PFHS results is 100*9% based on methodaccuracy and precision. The lower limit of quantitation (LOQ) for the samples is 0.00525 n g h L for PFHS. The analytical uncertainty of the PFOS results is 100*12% based on method accuracy and precision. The lower limit of quantitation (LOQ) for the samples is 0.0103 ng/mL for PFOS. TESTSAMPLES Water samples from two locations (3M LlMS ID # 82756-82766) were received on ice on May 4, 2005 from Tim Frinak of Weston Solutions, Inc. A total of eleven containers were submittedfor analysis. The samples were logged in by 3M EnvironmentalLaboratory professional services personnel and placed in refrigerated storage after extraction on May 5, 2005. E0542 10 Interim Report #5 Page 9 of 723 3M Environmental Laboratoty Project No. 05-0210 REFERENCSEUBSTANCES Table 2 lists the pertinent information regardingthe reference substance used for this study. Table 2. Study Reference Substance. Chemical Name Chemical Formula Identifier id source Expiration Date Storage Conditions Chemical Lot Number TCR Number Physical Dewiption Purity "Solubility Pertluorobutanesulfonate C,F~SO~K+ CAS # 2942049-3 3M 1011712006 Frozen 101 TCR-116 (99030-023) Whiie Powder 96.7% 54,400 ppm Pertluorohexanesulfonate C813So3'K* CAS # 3871-99-6 3M 1011812006 Frozen NB# 12006749 TCR-83 (SE036) White Powder 98.6% No Information available Pertluorooctanesulfonate C~F~,SGK+ CAS # 2795-39-3 3M 8/31/2006 Frozen 171 TCR-696 White powder I 86.4% 680 ppm CONTROSLUBSTANCE Radiolabelledperfluorooctanoicacid (PFOA [1,2 13C])was analyzed as a surrogate against a multi-level extractedcalibrationcurve. A known amount of PFOA [1,2 I3C]was spiked into each sample container in the laboratory prior to sample collection. Table 3 lists the pertinent information regarding the study control substance. Table 3. Study Control Substance (Surrogate). Chemical Name Chemical Formula Identifier source Expiration Date Storage Conditions Chemical Lot Number I TCRNumber I Physical Description Purity PerfluoroodanoicAcid CsFj5[13C]F2[13C]OOH N/A Perkin Elmer 03/29/2009 Frozen 3507-195 TCR-744 I White powder >97% 05-0210 Interim Report #5 Page 10 of 123 3M Environmental Laboratory Project No. E050210 METHODSUMMARIES Sample Collection Sampleswere collected on May 3,2005 in pre-rinsed NalgeneTM(low-densitypolyethylene) bottles prepared at the 3M EnvironmentalLaboratoryon May 2, 2005. Prior to sample collection, all bottleswere spiked in the laboratorywith a known volume of a surrogate solution and appropriate matrix spiking solution if applicable. Table 4 below details the samples collected and final spike concentrationsadded to each bottle. Table 4. Sample Collection Information 3M UMS Number 82756 82757 82758 82759 82760 82761 82762 82763 82764 82765 82766 SamDle DescriDtion SamDle Comment Vominal Final Volume :ollected (mL WMEL PW RWOl 0 050503 Location 1 Sample 200 WMEL PW RWOl DP 050503 Location 1 Sample Duplicate 200 NMEL PW RWOl LS 050503 Location 1 Low Field Spike 200 WMEL PW RWOIHS 050503 Location 1 High Field Spike 200 WMEL PW F W O l 0 050503 Location 2 Sample 200 WMEL PW F W O l DP 050503 Location 2 Sample Duplicate 200 WMEL PW F W O l LS 050503 Location 2 Low Spike 200 WMEL PW FWOIHS 050503 Location 2 High Spike 200 WMEL W TRIP 0 050503 Trip Blank 200 WMEL W TRIP LS 050503 Trip Blank Low Spike 200 WMEL W TRIP HS 050503 Trip Blank High Spike 200 Final Nominal Spike Concentation (ng/mL) 'FOA [1,2 "C (Surrogate) 0.050 0.050 0.050 0.050 0.050 0.050 0.050 0.050 0.050 0.050 0.050 PFBS, PFHS, PFOS NA NA 0.10 1 .o NA NA 0.10 1 .o NA 0.10 1 .o Preparatory and Analytical Methods Extraction All samples, calibrationstandards, and associatedquality control samples were extracted using a modified procedure of ETS-8-154.1 "Determinationof PerfluorinatedAcids, Alcohols, Amides, and Sulfonates in Water by Solid Phase Extractions and High PerformanceLiquid Chromatography/MassSpectrometry". Briefly, approximatelyeighty mL of sample were loaded onto a pre-conditionedWaters C18 solid phase extraction (SPE) cartridge (SepPak, 6 cc)using a Caliper Life SciencesAutotrace automatedSPE workstation. The same workstation then used positive pressure to elute the loaded cartridges with 2 mL of methanol This extraction procedureconcentratesthe samples by a factor of forty. (Initialvolume = 80 mL, final volume = 2 mL). As written, ETS-8-154.1 calls for a 40 mL and 5 mL extraction and elution volume, respectively. Analysis All sample and quality control extracts were analyzed for PFBS, PFHS, PFOS, and PFOA [I,2 13C]using high performanceliquid chromatography/tandem mass spectrometry (HPLC/MS/MS). Pertinent instrumentparameters,the liquid chromatographygradient program, and the specific mass transitions analyzed are described in the tables below. 05-0210 Interim Report #5 Page 11 of 123 Table 5. Instrument Parameters 3M Environmental Laboratory Project No. 05-0210 Table 6. Liquid Chromatography Gradient Program mM ammonium Table 7. Mass Transitions Anaryte `"PFBS (I'PFHS "'PFOS PFOA [1,2 13C] Mass Transition 41/43 299180 299199 3991130 399199 399180 49911 30 499199 499180 4151370 Dwell Time (msec) 100 100 100 100 100 100 100 100 100 ~~ ~ ANALYTICARLESULTS Calibration Calibrationstandardswere prepared by spiking known amounts of stock solutions containing PFBS, PFHS, PFOS, and PFOA [1,2I3C]into 80 mL of ASTM type Iwater. Each spiked water standard was then extracted in the same manner as the collected samples. A total of ten spiked standards ranging from 0.005 ng/mL to 2.5 nglmL (nominal) were prepared. A quadratic, l l x weighted, calibrationcurve was used to fit the data for each 05-0210 lnterim Report #5 Page 12of 123 3M Environmental Laboratory Project NO.05-0210 analyte. The data was not forced through zero during the fitting process. Calculating the standard concentration using the peak area counts and the resultant calibration curve confirmed the accuracy of each curve point. Each extracted calibration standard used to generate the final calibration curve met the method calibration accuracy requirement of 100i25%. Coefficients of determination(?)were greater than 0.996 for all analytes. Limit of Quantitation (LOQ) The LOQ for this analysis, as defined in ETS-8-154.1, is the lowest non-zero calibration standard in the curve in which the area counts are twice those of the method blank@). For PFOS, the LOQ was 0.0103 ng/mL as it was the lowest calibration standard to fulfill the area count requirement for all method blanks analyzed. For PFBS, PFHS, and PFOA [1,2 I3C], the LOQs were 0.00506, 0.00525, and 0.00499 ng/mL, respectively. Blanks Three types of blanks were prepared and analyzed with the samples: method blanks, solvent blanks, and field/trip blanks. Each blank type is described below. Solvent Blanks Several methanol solvent blanks were analyzed to assess system contamination and/or instrument carryover. Analyte peak area counts in all blank samples were less than half the area counts of the calibration standard used to establish the LOQ. Method Blanks Over the two days in which samples, calibration standards, and quality control samples were extracted, several method blanks were prepared by loading 80 mL of ASTM Type Iwater onto a C18 SPE cartridge and eluting with 2 mL of methanol using the same extraction procedure as the samples. Method blanks were prepared to evaluate the levels of background contamination in the overall extraction process (glassware, SPE cartridges, etc.) Table 8 lists the area counts for the method blanks. Table 8. Method Blank Area Counts Day 1 Day 1 Day 1 Day 1 Day 1 Day 1 Day 1 Day 2 Day 2 Method Blank -2 Method Blank -3 Method Blank -4 Method Blank -5 Method Blank -6 Method Blank -7 Method Blank -8 Method Blank -1 Method Blank -2 PFHS Area Counts 5036 1352 1463 1603 984 5152 2132 5640 2931 2460 2761 2865 75010 (0.00525 ng/mL) PFOS Area Counts 19444 17140 17746 15230 12305 20970 28896 37615 34842 19020 21958 22288 80616 (0.0103 ng/rnL) . DFOA 11.2 ''C] Area Counts 1907 793 670 485 572 1237 666 3283 1250 979 641 1135 25953 (0.00499 ng/rnL) 05-0210 Interim Report #5 Page 73 of 123 3M Environmental Laboratory Project No. 05-0210 Trip Blanks Prior to sample collection, one sample container was filled with 200 mL of ASTM Type I water, spiked with surrogate, sealed, and shipped to the sample collection site along with the empty containers. This sample was analyzed as the fieldltrip blank. The trip blank serves as an additional method blank that accounts for any storage conditions and/or holding time issues that the samples may experience. The resultant field blank concentration for all analytes was than the reported LOQ. System Suitability A 25 ng/mL solvent (unextracted) standard was analyzed in triplicate at the beginning and end of the analytical sequence to demonstrate overall system suitability. For PFBS, PFHS, and PFOS, the relative standard deviation (RSD) of the analyte peak area counts and the RSD of the peak retention times was less than 5% and 2%, respectively, for both opening and closing sets of system suitability samples which met method criteria. For PFOA [1,2 %I, the opening set of system suitabilities met method criteria; however, the peak area RSD was 7.9% for closing set. A method deviation was written for this failure. Continuing Calibration During the course of the analytical sequence, several continuing calibration verification samples (CCVs)were analyzed to confirm that the instrument response and the initial calibration curve was still in control. All CCVs,for all analytes, produced recoverieswithin *25%, which met method criteria. Lab Control Spikes (LCSs) Replicate low (0.025-0.031 ng/mL nominal concentration) and high (1.25 ng/mL nominal concentration) lab control spikes were prepared and extracted each day samples or calibration standards were extracted. LCSs were prepared by spiking known amounts of the analytes into 80 mL of ASTM Type I water to produce the desired concentration. The spiked water samples were then extracted and analyzed in the same manner as the samples. Table 9 provides the LCS recovery results. LCSs were used to evaluate method accuracy and precision which was then used to determine analytical uncertainty for each of the analytes reportedwith the results in Table 1. Informationon how the analytical uncertainty was determined is provided at the end of this report in the "Statistical Methods and Calculations" section. Two low level PFBS LCSs were outside the method acceptance criteria of 100*25% (698% and 134% recovery). The PFBS LCS with 698% is considered an anomaly as only one spiking solution was used and the other analytes for that same sample produced acceptable results. Therefore, this value was excluded when the PFBS LCS statistics were calculated. The sample with 134% recovery was included in statistical calculations as its exceedance of method criteria was not as severe. When the high and low spikes are considered collectively for PFBS, the overall average recovery was 101% with a relative standard deviation (RSD) of 14%, which meets method acceptance criteria for accuracy and precision (100*25%, RSD<15%). For PFHS, PFOS, and PFOA [1,2 %I, the LCSs show excellent accuracy and good precision, at both individual levels, and when considered collectively. 05-0210 Interim Report #5 Page 14 of 123 3M Environmental Laboratory Pmject No. 05-0210 Table 9. Lab Control Spike Results. X S Low-1 (Day 1) -CS Low-2 (Day 1) X S Low-3 (Day 1) X S Low-1 (Day 2) -CS Low-2 (Day 2) (') PFBS Spiked Concentration (na/mL) 0.0316 0.0316 0.0316 0.0253 0.0253 LCS High-1 (Day 1) LCS High-2 (Day 1) LCS High-1 (Day 2) 106% f18% 1.26 1.26 1.26 1.15 90.9 1.26 1.16 /Day I 6 Day 2) Average All Samples 95.8% f 6.2% IOlf14% (') PFHS 105 98.5 116 99.4 94.3 103% f 8.2% 1.31 1.32 103% f 1.6% 103i5.8% (') PFOS (') PFOA [1,2 13C] I I Sample Description Spiked Calculated Concentration Concentration (ng/mL) (ndmL) %Recovery %Recove .CS Low-1 (Day 1) 0.0321 0.0294 91.7 .CS Low-2 (Day 1) 0.0321 0.0264 82.3 .CS Low-3 (Day 1) 0.0321 0.0338 105 .CS Low-1 (Day 2) 0.0257 0.0263 102 .CS Low-2 (Day 2) 0.0257 0.0235 91.4 94.8 4verage Low {Day 16 Day21 LCS High-1 (Day 1) LCS High-2 (Day 1) 94.5% -.e% 1.28 1.4 1.28 1.28 ~1109 100 99.0% f71% LCS High-1 (Day 2) 1.28 1.26 98.7 LCS High-2 (Day 2) 1.28 1.13 88.5 Average High (Day I 6 Day 2) 99.1% f 8.5% 98.136f6.294 I I bverage AMsamples 96.5% f8.9% 98.6 *.FA (1) Tabla displays roundedvalues for all concentrationand percentrecoveryvalues (3 significantiigures). Reaxmyand E05-0210 Interim Report #5 Page 1 5 0 f 123 3M Environmental Laboratory Project No. 05-0210 Surrogates Although not specified in the ETS 8-154.1, PFOA [I,2 I3C]was added to all samples and sample spikes as a surrogate to evaluate overall method performance. The final PFOA [1,2 13C]concentration was 0.0499 nglmL. Surrogate recoveries are reported in the next section with sample data. Field Matrix Spikes Low (nominal concentration of 0.1 nglmL) and high (nominal concentration 1.0 nglmL) field matrix spikes were collected at each sampling point to verify that the analytical method is applicable to the collected matrix. Field matrix spike recoverieswithin method acceptance criteria of 100&25%confirm that "unknown" components in the sample matrix do not interfere with the extraction and analysis of the analytes of interest. Field matrix spikes will be presented in the next section with the sample data. DATASUMMARY The tables below summarize the sample results for the two locations (Table 10: WMEL PW RWOl; Table 11: WMEL PW FWOl) as well as the Trip Blank (Table 12). An average concentration was calculated for the field sample and field sample duplicate. The relative percent difference (%RPD) of the two measurementsis also provided. For Location#1 (WMEL PW RWOl), both the low and high field spike recoveriesfor PFBS and PFHS met method criteria of 1OOi25%. Reproducibilityof the field sample and field sample duplicate (%RPD) for both of these analytes was less than 10%. Furthermore, the field sample, field sample duplicate, and field spikes produced acceptable surrogate recoveries. However, a low recovery was observed for the low field spike for PFOS (36.4%). This low recovery can be partially explained by the high RPD (50%) between the field sample and the field sample duplicate. Because LCS results for PFOS (Table 9) demonstrate excellent precision for PFOS, the discrepancy between the field sample and the field sample duplicate for Location#iIs attributedto variability in the collected matrix and not the analytical method. If the low field spike for PFOS is calculated using only the field duplicate concentration, then the field spike recovery becomes 59.2% which agrees well with the low PFOS field spike recovery (62.9%) observed for Location #2 (WMEL PW FWOl). The high field spike at Location #1 for PFOS did meet method criteria with 87.3% recovery. Similar to Location # l , both the low and high field spikes for PFBS and PFHS and the high spike for PFOS produced acceptable recoveriesfor Location#2 (WMEL PW FWOl). As mentioned previously, the low field spike recovery for PFOS was 62.9% which was outside the method acceptance criteria of 100&25%. The low level PFOS field spike for the Trip Blank (Table 12) was 79.4%. Although this recovery technically meets method acceptance criteria, it is lower than that PFBS and PFHS recoveriesfor the same sample. This result may indicate a holding time issue with PFOS at the low end. If the low level Trip Blank recovery is used to correct the low level field spikes, the recoveries for PFOS increase to 74.7% and 79.2% for Locations#(IWMEL PW RWO1) and #2(WMEL PW FWOl), respectively. Overall accuracy and precision of the surrogate (excluding Location #2 field duplicate) was 99.8% with a RSD of 16%. 05-0210 Interim Report #5 Page 160f 123 Table I O . Sample Results Location #I: WMEL PW RWOI 3M Environmental Laboratory Project No. 05-0210 Average Sample Concentration (nrJmL) f%RPD 0.0926 *so% NA I (1) Table displays roundedvalues for all Concanlrah and parcentrecoveryvalues (3 significantfigures). Recovery and RPD values may vary slightly from the values in the taw data. (2) If only the fielddupliite is usedto calculatefield spike recovery, the low PFOSspike recovery is 59.2%. Ifthis value is then corrected forthe trip blank (holdingtime) low spike recovery,the recoveryis 74.7%. Table 11. Sample ResultsLocation #2: WMEL PW F W O l I I I (~IPFBS I I (~)PFHS I 0050506a064 NA NA 98.7 99.1 1.7% I Surrogate %Recovery Sample Field Dup Field Spike-Low Field Spike-High 82760 82761 82762 82763 0050506a061 0050506a063 0050506a064 0050506a065 0.0727 0.0782 0.140 0.896 NA NA '3'62.9 79.9 0.0504 0.0134 0.0672 0.0596 101 "'26.9 135 120 Average Sample Concentration (nrJmL) f%RPD 0.0754 f7.3% NA (1) Table displays roundedvalues for all concentrationand percentrecoveryvalues (3 significantfigures). Recoveryand RPD values may vary slightly from the values in the raw data. (2) Surrogate result not indudedin overallstatisticsas it is not representativeofthe LCSdata. Laboratoryanomalyor artifadsuspected. (3) PFOSlow spike recovery = 79.2% when COlTected for the recovery of theTrip Blank ( W i n g time) low levelmatrbc spike. 05-02 10 Interim Report #5 Page 17 of 123 Table 12. Sample Results: Trip Blank Concentration 3M Environmental Laboratory Pmject No. 05-02 IO Concentration values may vary sliihtry from the values in the raw data. STATISTICAL METHODASND CALCULATIONS Statistical methods used to interpret sample results include averages and standard deviations. The Analyst software program calculated sample concentrations using resultant analyte peak areas and the established quadratic, l / x weighted, calibration curve. None of the samples analyzed for this interim report required dilution. Sample calculations and equations used to report method accuracy and precision are described below. Accuracy and Precision Equations . LCSlSurrogatePercentRecovery = CalculatedConcentration Spike Concentration . (SpikedSample Concentration-Average Concentration: FieldSample andFieldSampleDup.) Sample Spike Recovery = Spike Concentraton % RSD (RelativeStandardDeviation) = standard deviationof replicates replicate average % RPD (Relative Percent Difference) = Absolute difference between sample duplicates , average sample concentration Determination of Analytical Uncertainty Both the accuracy (percent recovery) and precision (%RSD) of the lab control spikes are used to estimate the analytical uncertaintyfor a given analyte. For example, the overall accuracy and precision for PFOS based on LCS results was 96.5%&8.9%. The measured precision (%RSD) is then used to determine the spread of the accuracy. Example: 96.5*(0.089) = 8.59. 96.5 +8.59 = 105; 96.5 - 8.59 = 87.91 E05-0210 interim Report #5 Page 18 of 723 3M Environmental Laboratory Project No. 05-02 10 Thus, LCS accuracy results range from 87.9% to 105%. The absolute difference of the low and high ends of this range, when compared loo%, are then calculated. 100%-87.9% = 12.1% 105%-100 = 5%. The most conservative(largest)absolute difference is then used as the analyticaluncertainty for the given analyte. Therefore, the analytical uncertaintyfor PFOS is given as 100*12% for this set of data. STATEMENT OF CONCLUSION Sample results listed in Table 1 are considered accurate within the analytical uncertainties listed. Spike recoveries for low level (0.100 nglmL) PFOS field matrix spikes may indicate a holding time issue; however method accuracy and precision demonstrated by lab control spike data indicate the method is control and resultsfor the two sampling locations are valid for all three analytes. LISTOF ATTACHMENTS Attachment A Sample Chromatogramsand Calibration Curves Attachment B: Extractionand Analytical Methods Attachment C: Protocol, Protocol Amendments and Deviations 05-0210 lnterim Report #5 Page 19 of 123 3M Environmental Laboratory Project No. E050210 SIGNATURPEAGE We certify thafthis report is a true and complete representationof the data for this study: JaisimhyKesari, P.E., DEE , Study Director Date I William K. Reagen, Ph.D., Testing Facility Management Michael A. Santafo, Sponsor Representative Date - 7 Date 05-0210 Interim Report #I5 Page 20 of 123 3M Environmental Laboratory Project No. 05-02 10 ATTACHMENAT. SAMPLE CHROMATOGRAMS AND CALIBRATION CURVES E050210 Interim Report #5 Page 21 of 123 Figure 1. Calibration Curve: Extracted PFBS Calibration Standards in ASTM Type I Water. Figure 2. Calibration Curve: Extracted PFHS Calibration Standards in ASTM Type I Water. aO5OSOBaSUltondtCLrdb (PFHS TIC$ "Ouadfati@ Resrelrion c1 t f urciohting): y- -2.19e+OO1 e 2 + 1.53~e+Mx7+ 4.5Je+OW(r= 0.ooSs) Figure 3. CalibrationCuwe: Extracted PFOS CalibrationStandards in ASTM Type I Water. ~ J s u l f o n a t ~ s . r d b ( P F OTSI C ~ " 0 w d l a tRi ~.gr.aonC1 / r f u i ~ h t i a ~ Y i - 8 b 5 . ~ ~ + O1 ~. 3~1~~ x+ 0+ 0 4 ( ~ * 0 ~ 1.06*7 tm*7 0.6080 O.Md 8.5080 8.ma 7.6010 7.0080 3.0080 2.w.B 2.ma 1 .sod 1 .md 5.md , 0.00 0.1 0.2 0.3 0.4 0.6 0.0 0.7 0.8 0.0 I O 1.t 12 1.3 1.4 i s i.a 1.7 ia 1.0 2.0 2.1 2 2 2 3 2.4 2.6 Cono*ntntion. ndmL cs i Figure 4. Calibration Curve: Extracted PFOA [I,2 13C]Calibration Standards in ASTM Type I Water. oOSOM)%rsulfonrtu.rdb(13C PFOA414.Q r36OB). "OuadrJtIb R . p m i r n C 1 I f weightin3 y- -1.12e+MO a'2 + 13r+C@3x * -7b+m(!-Oaoso) 8 8 . . 7 5 6 6 4 ~.6d~ 5.56 6.06 $ 43*0 dC 4.06 3.56 3R6 2.6d 206 1.06 LCJ 5.0.5 " OR 02 0.4 00 0.8 1 .O 12 4 A .le 1.8 2 .o 22 2.4 C o n n n t d o n . n#mL Figure 5. Total Ion Chromatogram for PFBS: 0.0253 nglmL Extracted Calibration Standard. 1dW 1.4~ 1.4r law 1.3~ 12u 1.2u l.1M 1.14 1 .ad em.0 8wo.O s 8ooo.o ii 7m.0 7000.0 c - 06m.o 6000.0 6600.0 ww.0 45oQ.o 4Mo.o Mm.0 3ooo.o ZJm a m.n G3 1m.0 5m 1000.0 ma OR- Figure 6. Total Ion Chromatogram for PFHS: 0.0263 nglmL Extracted Calibration Standard. 1 I 1'12 i3 i4 1834 ii 10 Figure 7. Total ton Chromatogram of PFOS: 0.0257 nglmL Extracted Calibration Standard. 1.54 14.4 1.4.4 1. A d 13.4 1.3.4 '12.4 12e4 1.1.4 1.1.4 1.owl 06M.0 oooo.0 a 8600.0 g 8Mo.0 7500.0 7M)O.O woo a ma 5500.0 6Mo.0 m.0 ma 3500 a 3Ka a 2500.0 nm.0 1500.0 1m.o 500.0 0.0 1 2 3 4 5 0 7 8 0 10 11 12 Time. min 113.86. '3.6% Figure 8. Total Ion Chromatogramof PFOA [1,2 ' k ] : 0.0249 nglmL Extracted CalibrationStandard. ooses0c.w. 7 ~ : ~ ~ 0 ~ 4 ~ 4 ~ ~ 3 ~ ~ ( ~ k ~-nnmoplr~ilnotrwjtr4on1 O4(Y~YCe..Ww~Y1 Area: 57682eou.b Iuplt: MIocpr RJ: 13.(ria 1 B 17.62 2 3 4 6 8 r 8 D IO 11 12 13 14 18 16 17 18 19 Tim.. min 8 a Figure 9. PFOS Total Ion Chromatogram: RepresentativeMethod Blank (050506a072; Day 2 Method Blank 3) a 0 0 5 0 s o ~ o n.PN)S TIC ~ t r o w r ) ~ v O a , . r r , 4 9 1 ~ o w n , 4 8 a ~ ~ ~ wnro. f w f m ~oo~s)(..nlll Area: 2 f % ~ ~ . w~bg & t 2:f41cp~RE 14lmft1 1.47 t Time. mln 13.88. Figure 10. PFOS Total Ion Chromatogram: Representative Methanol Solvent Blank (0050506a052). I OOSOSOC.OSZ-PFOSTI(CI * ~ R ~ ~ ~ ~ ~ L V H S N * , ~ V # O . ~ , C # M Z A S H ~-=nph szd8sfmn-.VH# Aru: 9022cou& 866Cpr RT: 141mi 1 14.01 13.87, G, L I 2 3 4 5 e 7 8 0 10 11 12 13 Time. mln 15 10 17 18 19 Figure 11. PFBS Total Ion Chromatogram: (Location #2; WMEL PW FW 01, Sample 1082438,0050506a061). 6.44i I 3 62.4. 60.4. 46.4. 4.6r4. 4.4e4. 42.4. 4.0.4. 3.8.4- 3.6.4. 3.4.4. 32.4. 0 3.0.4- 0 0 9: 2.8.4. f 2.04*.4: 22.4. 2.0.4. 1.8.4. 1.0H. 1.44. 12.4- 1.0.4- eOm.0. mm0. m . 0- 2ooor). 080 0.0 8.07 '3 1 2 3 4 5 0 7 8 0 Figure 12. PFHS Total Ion Chromatogram: (Location #2; WMEL PW FW 01, Sample lD82438,0050506a061). Tima. min Figure 13. PFOS Total Ion Chromatogram (Location #2; WMEL PW FW 01, Sample lD82438; 0050506a061). I 005066067 .PFOS m: ~ ~ u o w ~ ) ~ I ~ ~ ~ -~u a ~p h C~I o f WM froIa 0L0seosw~m~~ ~ , ~ w ~ ~ ~ ~ u ARC aaico..tr WQM: 311~(sprRE 1 4 7 d ~ 3.844 Do 3.0*4- 3.4.4. - 322.4 3.02.4- 2.84- 2d2.4- 2.w. 22.4. m n E 2.0e4. & ! ! Idd- 1.or). 1.44. 12d- 1.04- 8Mo.O. 6mo.o. m.0. I mOO.0 . 1362 r 0.0' . , 'I 2 3 4 5 8 8 0 '10 11 12 13 15 10 17 18 19 Figure 14. PFOA [1,2 "C] - Surrogate Chromatogram: (Location #2; WMEL PW FW 01, Sample lD82438,0050506aO61), 4.2.4. 4.04. 3.8.4. 1 B 1. a 4- 12.4. 1.04- eoOo.0. bmo.0. 4QJo.O. 20004. 0.0 J 1747 1 2 3 4 6 0 7 8 Q 10 11 12 13 14 16 16 17 18 18 Time. mln ATAICHMENT 3M EnvironmentalLaboratory Project No. 05-02 10 B. EXTRACTIOANND ANALYTICAMLETHOD E050210 Interim Report #5 Page 36 of 123 3M EnvironmentalLaboratory Project No. 05-0210 Method Determinationof PerfluorinatedAcids, Alcohols, Amides, and SulfonatesIn Water By Solid Phase Extraction and High Performance Liquid Chmmatography/Mass Spectrometry Method Number: ETS-8454.1 Adoption Date: 28 Apr 2000 Revision Date: 5 May, 2003 Effective Date: 5 May, 2003 Approved By: William K. Reagen Manager Date ETSS-154.1 Page 1 of 17 Determination of Perfluorinated Compounds in Water Using SPE and LCIMS. E050210 Interim Repotf #5 Page 37 of 123 3M Environmental Laboratoty Pm@f NO.E05-02IO 1 Scope and Application This methodwas validated for the collection, extraction, and analytical procedurefor the determinationof Pertluorooctanesulfonate (PFOS), Perfluorooctanesulfonylamide (FOSA), and Perfluorooctanoate(PFOA) in groundwater, surface water, and drinkingwater samples. This method may also be applied to the determination of other perRuorinated adds, alcohols, amides, and sulfonates in similar matrices, as long as the defined QC elements are satisfied and with the understanding that the method is not validated for compounds outside the scope of the original protocol This method is based in parton the report"Methodof Analysis for the Determinationof Perfluorooctane sulfonate (PFOS), Perfluorooctane sulfonylamide (PFOSA), and Perfluorooctanoate(POAA)inWater" (seeSection 17), as developedand validated by Exygen Research (formerly Centre Analytical Laboratories, Inc.). 2 Method Summary Water samples are dlected from a site of interest and shippedcold to an analyticalfacilii. Perfluorinated acids, alcohols, amides, and sulfonates are extracted from 40mL water samples using C18 solid phase extraction (SPE) cartridges. The compounds are eluted from the C18 cartridge, using methanol. Separation, identification, and measurement are accomplished by highperformance liquidchromatography/tandem mass spectrometry (HPLCIMSIMS)analysis. Highperformanceliquidchrornatographylmassspectrometry(HPLCIMS)may be used if the defined QC elements are satisfied. The concentrationof each identifiedcomponent is measuredby comparing the MS responseof the quantitation ion produced by that compound to the MS response of the quantitation ion produced by the same compound in an extracted calibration standard (external standard). 3 Definitions 3.1 Analytical Sample A portionof an extracted LaboratorySample preparedfor analysis. 3.2 CalibrationStandard A solutionpreparedfrom the Working Standard (WS)and extractedaccordingto this method. The calibrationstandard solutionsare usedto calibrate the instrument responsewith respect to analyte concentration. 3.3 Duplicate Sample (DS) A DS is a separate aliquot of a sample, taken in the analytical laboratory that is extracted and analyzed separately with identical procedures. Analysis of DSs compared to that of the first aliquot give a measureofthe precisionassociatedwith laboratory procedures, but notwith sample collection, preservation, or storage procedures. 3.4 Field Blank Control Sample (FB) ASTM Type I water placed in a sample container in the laboratory and treated as a sample in all respects, including exposure to sampling site conditions, storage, preservationand all analytical procedures. The purposeofthe FB is to determine if test substances or other interferences are present in the field environment. ETS-8-154.1 Page 2 of 17 Determinationof PertluorinatedCompounds in Water Using SPE and LC/MS. 05-02IO Interim Report #5 Page 38 of 123 3M Environmental Laboratory Project No. 05-02 10 3.5 Field Duplicate (FD) A sample collected in duplicate at the same time as the sample and placed under identical circumstances and treated exactly the same throughout field and laboratory procedures. Analysis of FD compared to that of the first sample gives a measure of the precision associated with sample collection. preservation and storage, as well as with laboratory procedures. 3.6 Field Matrix Spike (FMS) A sample collected induplicate to which known quantitiesof the target analytes are added inthe field at the time of sample collection. Alternatively, the known quantity of target analytes may be added to the sample bottle in the laboratory before the bottles are sent to the field. A known. specific volume of sample must be added to sample container without rinsing. This may be accomplishedby makinga "tillto this level" line on the outside of thesample container. The FMS should be spiked at approximately50-1 50% of the expectedanalyte concentratiinin the sample. If the expected range of analyte concentrations is unknown, a low and a high spike may be prepared to increase the likelihood that a spike at an appropriate range is made. The FMS is analyzedto ascertain if any matrix effectsi,nterferences,or stability issuesmay complicatethe interpretation ofthe sample analysis. 3.7 Field Spike Control Sample (FSCS) An aliquotof ASTM Type Iwater to which knownquantities of the target analytes are added in the field at the time of sample collection (at an appropriate concentrationto be determinedbythe project lead)or inthe laboratory priorto the shipment of the collection bottles.The FSCS is extracted and analyzed exactly like a sample to determine whether a loss of analyte could be attributedto sample storage andlor shipment. A low and high FSCSmay be appropriatewhen expeded sample concentratbnsare not known. 3.8 Laboratory Control Sample (LCS) An aliquot of ASTM Type Iwater to which known quantities of the target analytesare added in the laboratory. Two levels are included, one at the LLOQ (approx. 25 PglmL), the other at a concentration of approx. 1W250 WmL or another concentrationto be determinedby the project lead. The LCS is extracted and analyzed exactly like a laboratory sample to determine whether the methodologyis incontrd, and whether the laboratory is capable of makingaccurate measurements at the required method detection limit and higher. 3.9 Laboratory Sample A portionof a sample receivedfrom the field for testing. 3.10 Limit of Detection (LOD) The LOO isthe lowest concentrationof an analytethat can be measuredand reported with 99% confidence that the analyte concentration is greater than zero. If required, the LOD may be determined in several ways, includingsignal-tonoiseratio and statistical calculations. 3.11 Limit of Quantitation (LOQ) The LOQfor a dataset is the lowest concentration(LLOQ)or highest concentration(ULOQ)that can be reliablyachievedwithin the specified limits of precisionand accuracy during routine operating conditions. Note: For many analytes, the LLOQ analyte concentratiin is selected as the lowest non-zero standard in the calibration curve to simplify data reporting. Sample LLOQs are matrixdependent. 3.12 Matrix Spike (MS) ETS-8-154.1 Page 3 of 17 Determinationof PeffluorinatedCompounds in Water Using SPE and LCIMS. EO5-02 10 Interim Report #5 Page 39 of 123 3M Environmental Laboratory Pmject No. 05-0210 A matrix spike is an aliquot of a sample, to which known quantities of target analytes are added in the laboratory.The MS is extracted and analyzedexactly likea laboratorysample to determine whether the sample matrix contributes bias to the analytical results. The background concentrationsof the analytes inthe sample matrix must be determinedin a separate aliquot and the measuredvalues in the MS correctedfor backgrwnd concentrations. 3.13 Method Blank An aliquot of ASTM Type Iwater that is treated exactly like a laboratorysample including exposure to all glassware, equipment, solvents, and reagents that are used with other laboratory samples. The method Mank is used to determine if test substances or other interferences are present in the laboratory environment, the reagents, or the apparatus. 3.14 Method Detection Limit (MDL) Determination A MDL is the statistically calculated minimum amount of an analyte that can be measured with 99% confidencethatthe reported value is greater than zero. One of several processesthat may be used to establish a LODvalue is found in 40 CFR Part 136Appendix B. 3.15 Sample A sample is a small portion collectedfrom a larger quantityof material intendedto representthe original source material. 3.16 Spiking Stock Standard (SSS) A solution preparedfrom stock standards usedto prepare the working standard. 3.17 Stock Standard (SS) A concentratedsolutionof a single analyte preparedinthe laboratorywith an assayed reference compound. 3.18 Working Standard (WS) A solution of several analytes prepared inthe laboratoryfrom SSs and dilutedas neededto prepare calibration standards and other required analyte solutions. 4 Warnings and Cautions 4.1 Health and Safety The acute and chronic toxicity of the standards for this method have not been precisely determined; however, each should be treated as a potential health hazard. Unknown samples may contain high concentrations of volatile toxic compounds. Sample containers should be opened in a hood and handled with gloves to prevent exposure. The laboratory is responsiblefor maintaining a safe work environmentand a current awareness of localregulationsregardingthe handlingof the chemicalsused in this method. A referencefile of material safety data sheets (MSDS) should be available to all personnel involved in these analyses. 4.2 Cautions None 5 Interferences ETS-8-154.1 P a g 4 of 17 Determinationof PertluorinatedCompoundsin Water Using SPE and LCIMS. 05-0210 lnterim Report #5 Page 40 of 123 3M Environmental Laboratory Project No. 05-0210 During extraction and analysis, major potential contaminant sources are reagents and solid phase extraction devices. All materials used in the analyses shall be demonstratedto befree from interferencesunder conditions of analysis by running method blanks. Parts and suppliesthat containTeflom should be avoided due tothe possibility of interference and/or contamination. These may include, but are not limited to: wash bottles, TeRon@' lined caps, autovial caps, HPLC parts, etc. The use of disposable micropipettes or pipettes to aliquot standard solutions is recommended to make calibration standards and matrix spikes. 6 Instrumentation, Supplies, and Materials ~ ~~ ~~ Note: Brand names, suppliers, and part numbers are for illustrative purposbs only. Equivalent performance may be achieved using apparatus and materials other than those specified here, but demonstrationof equivalent performancethat meets the requirementsof this method is the responsibility of the laboratory performing the analysis. 6.1 Instrumentation Balance, analytical (displayat least O.OOOlg), Meffler HPLCIMSIMSor HPLCIMS system, as described in Section IO. 6.2 Supplies and Materials. Samplecollection bottles-LDPE(e.g., NalgeneTMn)am-mouth bottleswith screw cap. Note: Do not use TeRon bottles or Tetlon lined caps. Coolers for sample shipment. Ice for sample shipment. Vacuum pump, Bischi. Viiprep vacuum manifold, Supelco. Sep Pak Vac 6cc (lg) tC18 cartridges (part # WAT 036795),Waters. 50mL disposable polypropylene centrifuge tubes, WVR. 15mL disposable polypropylene centrifuge tubes, VWR. Disposablemicropipettes(5O-lOOpL, 10CL200yL), Drummond. Class A pipettes and volumetric flasks, various. Hypercarbdrop-in guard column (4mm) (part # 844017400), Keystone. Stand-alonedropin guard carbidgeholder, Keystone. 125mL LDPE narrow-mouth bottles, Nalgene. 2mL dear HPLCvial kit (cat# 5181-3400),AgilenWewlett Packard. Standardlab equipment (graduatedcylinders, disposabletubes, etc.), various. 7 Reagents and Standards Note: Suppliers and catalog numbers are for illustrative purposes only. Equivalent performance may be achieved using chemicals obtained from other suppliers. Do not use a lesser grade of chemical than those listed. ETS-8-154.1 Pam 5 of 17 Determinationof Peffluorinated Compoundsin Water Using SPE and LC/MS. EO5-0210 Interim Report #5 Page 41 of 123 3M Envitvnmental Laboratory Project No. 05-02IO 7.1 Chemicals Methanol (MeOH), HPLC grade, JT Baker, Catalog No. JT9093-2. Ammonium Acetate, Reagent grade, Sigma-Aldrich, Catalog No. A-7330. ASTM Type I Water, prepared in-house. Sodium Thiosulfate, Reagent grade, JT Baker. 7.2 Standards Potassium perfluomoctane sulfonate Perfluoroodane sulfonylamide Ammonium perfluoruoctanoate Others as required. 7.3 Reagent Preparation 250mglmL sodium thiosulfate solution-Dissolve 259 of sodium thiosulfate in IOOmL reagent water. 40% methanol wash solution - Measure 400mL methanol and adjust volume to 1.OL with reagent water. 100mM ammonium acetate sdution (Analysis)-Weigh 7.719 of ammonium acetate and dissolve in I.OL of reagentwater. Dilutethe 1OOmMsolution by a factor of 50 to make the 2mM ammonium acetate solution used for mobile phase A. Note: Alternative volumes may be prepared as long as the ratios of the solvent to solute ratios are maintained. 7.4 Spiking Stock Standard (SSS) Preparation The following standard preparation procedure sewes as an example and may be changed to suit the needs of a particular study. For example, p t volumes may be spiked intovolumetric flasks when diluting stock solutions to appropriate levels. IOOpg/mL each PFOS, PFOSA, and POAASSSs-Weigh out 10mgof analytical standard (corectedfor percent salt and p u W . e . , 10 mg C8Fl7SO3K purity90% = 8.35119 c8F17%-) and diluteto 1OOmLwith methanolin a 1OOmL volumetric flask. Transferto a 125mLLDPE bottleor other suitable container. Prepare a separate solutionfor each analyte. Solutions may be stored in a refrigerator at 4"S"C for a maximum periodof 6monthsfrom the date of preparation. 1WmL mixed SSS-Add 1.OmL each of the IOOpg/mL SSSs (ffm7.4.1) to a 1OOmLvolumetric flask and bring up to volume with methanol. O.lpg/mL mixed SSS-Add 10.0mL ofthe l.Opg/mL-mixed solution (from7.4.2) to a IOOmL volumetric flask and bring up to volume with methanol. 0.OlclglmL mixed S S M d d 10.0mL ofthe 0.li.rglmLmixed solution(from 7.4.3) to a 100mL volumetric flask and bring up to volume with methanol. Storage Conditions4tore all SSSs in a refrigerator at 4"SC for a maximum periodof 6 monthsfrom the date of preparation. 7.5 Calibration Standards The followingstandard preparationprocedureserves as an example and may be changedto suit the needs of a particular study, provided the concentrations are calculated correctly. ETS-8-154.1 Page 6 of 17 Determinationof PertluorinatedCompounds in Water Using SPE and LCIMS. E05-0210 Interim Report #5 Page 42 of 123 3M Environmental Laboratory Project No. 05-0210 1001rglmLeach PFOS, PFOSA,and POAA stock standardsolutions-Weigh out 10mgof analytical standard (correctedfor percent salt and purity) and diluteto 100mLwith methanol in a 100mL volumetric flask. Transfer to a 125mL LDPE bottle or other suitable container. Prepare a separate solution for each analyte. Store solutions in a refrigerator at 4"35!'C for a maximum periodof 6months from the date of preparation. 1clglmLWorking Standard-Add 1.OmL each ofthe 100pglmLSS solutions (from 7.5.1) to a 100mL volumetric flask and bring up to volume with methanol. 0.ll.lglmL Working Standard- A d d 10.0mLof the 1.Opg/mL mixedsolution (from 7.5.2) to a IOOmL volumetric flask and bring up to volume with methanol. 0.OlclglmL Working Standard- A d d 10.0mL of the 0.1pg/mL mixedsolution (h 7.5.3) to a 100mL volumetric flask and bring up to volume with methanol. Storage Conditiondtore all WSs in a remeratorat 4WC for a maximum periodof 6 monthsfrom the date of prepamtion. Calibration Standard-Preparecalibrationsolutions inASTM Type Iusingthe following table as a guideline: 0.0 0 40 0.010 100 40 0.010 200 40 0.010 400 40 0.10 100 40 0.10 200 40 0.10 300 40 0.10 400 40 1 .o 100 40 1 .o 400 40 1 .o 1000 40 0 25 50 100 250 500 750 1000 2500 10000 25000 The standardsare pmcessedthrough the extraction procedure(Section 1I), identicalto the laboratorysamples. The concentrationof the calibration standard in the final extract is equalto 8X the initialconcentration, due to the concentrationof the standardduring the extraction process. StorageConditions-Storeall extracted calibration standardsin 15mL polypropylenetubes at 4"35!C, for a maximum period of two weeks from the date of preparation 8 Sample Collection and Handling Note: Samplingequipment, indudingautomatic samplers, must befree of Tdon tubing, gaskets, and other partsthat may leachinterferinganalytes intothe water sample. Automatic samplers that composite samples over time should userefrigerated polypropylenesample containers if possible. Sampb bottles should not be rinsed before sample collection. Labeling: Eachsample bottlemustdisplay informationregardingthe collectionofthat sample, theindividualcollecting the sample, and any matrix spike that has been added to the sample. ETS-8-154.1 Page 7 of 17 Determinationof PerfluorinatedCompounds in Water Using SPE and LC/MS. 05-0270 Interim Repoft #5 Page 43 of 123 3M Environmental Laboratory Pmject No. 05-0210 This indudes the volume and concentration of any spiking solution added and the volume and identification of any preservatives added in the field. Spiking: The spiking scheme will be clearlyoutlined inthe sampling plan, including whether the samples will be spiked inthe field or in the laboratory prior to the Shipmentof the bottlesto the site. If spiking is to be performed inthe field, materials and specific instructions will be indudedin thesampling kit. Be sure to clearly labeleach bottlewith spiking information if applicable. Tap Water: Openthe tap and allow the system to flush untilthe water temperature (15k100C) has stabilized (usually about two minutes). Adjust the flow to about 500mUmin and collect samples from the flowing stream. Ground Water: Purgethe well of standing water usinga pump or a bailer. Collectthe sample directly from the pump or from the bailer. Surface Water: When sampling from an open body of water, fill the sample container with water from a representativearea. Sample Dechlorination:All samples should be icedor refrigeratedat 4+.20Cand kept inthe dark from the time of collection until extraction. Residual chlorine should be eliminated by adding 200vL of a 250mghnL sodiumthiosulfatesolution to each tapwater sample and associated FB and FSCS(which may be placedin each bottlebefmleavingfor thesampling site or done inthe field.). Holding Time (HT): Resultsof the timdstoragestudy of all target analytes showed that thethree compounds are stablefor 14days in water sampleswhen thesamples are dechlorinatedand stored as describedin the previous section (seealso references in section 17).Therefore, laboratory samples must be extracted within 14 days and the extracts analyzed within 30 days of sample collection. If the HT exceeds 14 days, great care is used when evaluating field spikes to avoid misrepresentationof the sample concentration. 8.1 Field Blanks Process a Field Blank Control Sample (FB) along with each sample set (samples collectedfrom the same general sample site at approximately the same time). At the laboratory, prior to sample collection, fill a sample container with ASTM Type I water, seal, and ship the FB to the sampling site along with the empty sample containers. Return the FB to the laboratory with the filled sample bottles. When sodium thiosulfate is added to samples, use the same procedureto preservethe FB. 8.2 Field Duplicates Collect a Field Duplicate (FD) for every ten (IO) samples collected or per each sampling set, if less than 10 samples are collected. SeparateFDs must be collectedfor each type of water sample (ground, tap, etc.) collected. Collect the FD immediately after the sample. Preserve, store and ship FD using the same procedures as used for the samples. 8.3 Field Spike Control Sample (FSCS) A Field Spike Contrd Sample (FSCS) must be preparedfor each sample shipment. If multiple coolers are used to ship a set of samples, each cooler must contain a FSCS. At the laboratory, fill a sample container with IOOmLof ASTM Type Iwater. Seal and ship to the sampling site along with the empty sample containers and FBs. Samples may either be spiked in the field or in the laboratory prior to shipment. The method employed should be consistent throughout the study. If the samples are to be spiked in the field, be sure to send appropriate supplies and instructionsfor the field personnelto follow. ETS-8-154.1 Page 8 of 17 Determination of Perfluorinated Compounds in Water Using SPE and LCIMS. 05-0210 lntenm Report #5 Page 44 of 123 3M Environmental Laboratory Project No. 05-0270 Seal and gently invertthe FSCSto mix. Store and ship the FSCSusingthe same procedures as used for the samples Pmide informationon sample collection, preservation,shipment and storage. List applicable holding times. Include sample stability and extract storage requirements. Reference the method used for sample preparation, if applicable. 8.4 Field Matrix Spike (FMS) A FieldMatrix Spike (FMS) must be preparedfor each sampling location. One unspiked sample from the same location must accompanythe FMSto determine endogenous levels in the sample. The samples should be dearly identifiable as being from the same location. Samples may either be spiked in the field or in the laboratory prior to shipment. The method employed should be consistent throughout the study. If the samples are to be spiked in the field, be sure to send appropriate supplies and instructions for the field personnel to follow. 9 Quality Control and Data Quality Objectives Analytical resub of the FB, FMS, FD, and FSCS should beevaluated at the conclusionof the study to help interpretthe qualityof sample data. Analytical resultsfor these contdduplicate samples must be reported with the sample data. 9.1 Solvent Blanks Solvent blanks are analyzed with each sample set to determine contamination or canyover. Aliquots of methanol represent the solvent used for the standard curve and the sample extraction. Solvent blanks should have area counts that are lessthan 50% ofthe area count of the lowest calibration standard. Solvent blanks should be analyzed prior to and following each calibration curve, each set of system suitability samples, and after no more than 10 unknown sample extracts. If instrument canyover is a problem consecutive solvent blanks may be necessary. In this case the area counts of the solvent blanks should returnto ~ 5 0 %of the lowest calibration standard priorto the injectionof further standards or samples. 9.2 Method Blanks A method blank consists of an aliquot of ASTM Type Iwater, equal in volume to the samples, and extracted in the same manner as the samples. At least two method blanks should be prepared and analyzed each day that extractions are performedfor a particularstudy or project. When analyzed the area counts of these samples must be less than 50% of the area count of the lowest calibration standard. 9.3 Sample Replicates All samples, indudingfield spikes, trip blanks, etc., shouldbe extractedat leastinduplicate, and in triplicate if dfiwltieswere encounteredin the sampling and/or holding condiionsof the samples. The relative percent difference (RPD)of duplicate samples or relativestandard deviation (RSD) should be less than 15%for the precision of sample preparationand analysis to be considered in control. 9.4 Matrix Spike Matrix spikes are prepared for each sample type and analyzed to determine the matrix effect on the recovery efficiency. Matrix spike recoveries should fall within f 25% of expected values. If the matrix spikes fail, evaluate the lab control spikes. If the LCS are within acceptance criteria there may be matrix issues in the samples. Discussthese in the final report. ETs-8-154.1 Page 9 of 17 Determinationof Perfluorinated Compounds in Water Using SPE and LCIMS. 05-02 10 Interim Report #5 Page 45 of 723 3M Environmental Laboratory Project No. 05-0210 Matrix spike duplicates are prepared periodically to measure the precision associated with the analysis. Analyze a matrix spike and matrix spike duplicate (r prepared) in the same run as the original sample. Matrix spike and matrix spike duplicate concentratiinsshouldfall inthe mid-range ofthe initial calibration curve or should be prepared at 1.55 times the endogenous concentration of the analyte. Spike concentrationsshouldfall in the low-rangeof the initial calibration curve if extremelyW e v e l s are expected. Generallytwoor more levelsare prepared, one in the low range of the curve and one in the mid-range. This avoids the need to pre-screen unknown samples prior to preparation. 9.5 Laboratory Control Spike Lab controlspikes are preparedfor each study to ensure recovery of the target analytes. These should be prepared at a minimum of 2 levels and in duplicate or triplicate. Recoveryof these samples should be within f 25% of expectedvalues, and the RPD (orRSD) beS 15%. If recoveriesfall outsidethese limits the samples should be addressed in the final report. 10 Calibration and Standardization 10.1 Instrument Setup Note: Inthis example, a MicroMassUltimaTULiquid ChromatographyTandem Mass Spectrometer (LC/MS/MS) is used. Other brands of LC/MS/MSs as well as single quadrupole mass specErometers (LC/MS) may be used as long as the method criteria are met. Brand names, suppliers, part numbers, and models are for illustrative purposes only. Equivalent performance may be achieved using apparatus and materials other than those specified here, but demonstrationof equivalent performancethat meets the requirements of thismethod is the responsibility of the laboratory. The operator must optimize and document the equipment and settings used. Establish the LC/MS/MS system and operating conditions equivalent to the following: Mass Spec: Micromass Ultima (Micromass) Interface: Electrospray (Micromass) Mode: EledrosprayNegative, Multiple Response Monitoring (MRM) Harvardinfusionpump (HarvardInstruments),for tuning Computer. COMPAQ ProfessionalWorkstationAP200 Soflware: Windows NT, MassLynx 3.3 HPLC: Hewlett Packard (HP) Series 1100 HP Quaternary Pump HP Vacuum Degasser HP Autosampler HP Column Oven Note: A 4 x 10mm Hypercarbdropin guard cartridge (Keystone, part# 844017-400)may be attached on-line after the purge valve and before the sample injector port to trap any residue contaminantsthat may be in the mobile phase and/or HPLC system. HPLC Column: Genesis C8 (Jones Chromatography), 2.lmm x 50mm, 4pm ETs-8-154.1 Page 10 of 17 Determinationof PerfluorinatedCompoundsin Water Using SPE and LCIMS. 05-0210 Interim Report 4'5 Page 46 of 123 3M Environmental Laboratory Pmject No. 05-0210 Column Temperature: 35C InjectionVolume: 15pL Mobile Phase(A): 2mM Ammonium Acetate inASTM Type Iwater (See 7.3.1) Mobile Phase (6): Methanol I 0.0 60 40 0.3 I 0.4 60 I.o 10 40 0.3 90 0.3 7.0 10 90 0.3 7.5 0 100 0.3 9.0 0 100 0.4 9.5 60 40 0.4 I 13.5 14.0 60 60 40 40 0.4 0.3 I Note: Other HPLC gradients may be used as long as the method criteria are met. It may be necessary to adjust the HPLC gradient in order to optimize instrument performance. Columns with different dimensions (e.g. 2.lmm x 30mm) and columns from different manufacturers (Keystone Betasil C18 etc.) may be used. PFOA 41 3 169 5.0 PFOS 499 99 5.2 FOSA 498 78 5.8 Other product ions may be chosen at the discretion ofthe analyst, although m h99 is suggested for PFOS. Useof the suggested primary ion is recommended.Retentiontimes may vary slightly, on a day-tday basis, depending on the batchof mobile phase etc. Drift in retentiontimes is acceptable within an analytical run, as long as the drift continues through the entire analysis and the standards are interspersed throughout the analytical run. 10.2 Tune File Parameters The followingvalues are providedas an example. Actual values may vary from instrument to instrument. Also,these values maybe changedfrom timeto time in order to optimize for greatest sensitivity. PFOA PFOS FOSA 0.29.4 0.2-0.4 0.2-0.4 10-25 30-60 20-50 20-30 50-80 30-60 ETS-8- 154.1 Page 11 of 17 Determinationof PeffluorinatedCompounds in Water Using SPE and LCIMS. 05-0210 Interim Report #5 Page 47 of 123 3M Environmental Laboratory Ptvject No. 05-0210 Capillary Hexapole 1 Aperture 1 Hexapoie 2 Source Block Temp. Desolvation Temp. 2.6-3.5kV 0.5V 0.2v o.av 100-1 50C 250400C LM Res 1 HM Res 1 IEnergy 1 Entrance Exit LM Res 2 HM Res 2 IEnergy 2 Multiplier Cone Gas Desolvation I Gas Cell 12.5-15.OV 12.515.0V 0.N -2v 1v 11.ov 11.ov 1 .ov 650V 150Llhr 7OOUhr ms-8-154.1 Page 12 of 17 Determination of Pertluorinated Compounds in Water Using SPE and LCIMS. E054210 Interim Report #5 Page 48 of 123 3M Environmental Laboratory Project No. 05-0210 10.3 Calibration Curve Analyze the standard curves prior to each set of samples. The validated method specifies that J the standardcurve should be plottedusinga linear ft,wei hted l/x or unweighted. However, the standard curve may also be plottedby quadratic fit (y = a + bx + c), weighted l/x or unweighted, using suitable software. The calibration curves may include but should not be forced through zero. The mathematical method used to calculate the calibration curve should be applied consistently throughout a study. Any change should be thoroughly dowmented in the raw data. If the calibration curve does not meet acceptance criteria perform routine maintenance or prepare a newstandard curve (iinecessary)and reanalyze. For purposesof accuracywhen quantitating low levelsof analyte, it may be necessaryto usethe low end of the calibration curve ratherthan the full range. For example, when attemptingto quantiite approximately50 p g h L of analyte, generate a calibration curve consisting of the standardsfrom 25 PglmLto 1OOO pg/mL rather than the full range of the curve (25 pq/mL to 25OOO pg/mL). This will reduce inaccuracy attributed to linear regression weighting of high concentration standards. High andlor low points may be exduded from the calibration curves to provide a better ft over the linearrange appropriateto the data or becausethey did notmeet the predeterminedacceptance criteria. Low-level curve points should also be exduded if their area counts are not at leasttwice that of the method and/or solvent blanks. Any curve point may be rejected due to a bad injection or failing to meet accuracy requirementsoff 25% (andf 30%for the LLOQ). Justification for exclusion of calibration curve pointswill be noted in the raw data. A minimumof 6 pointswill be used to construct the calibration curve. 10.4 Continuing Calibration Verification (CCV) Continuingcalibrationverifications (CCV) are analyzedto venfy the accuracy of the calibration curve. Analyze a mid-rangecalibrationstandard, one of the same standards used to construct the calibrationcurve, at a minimum after every tenth sample, not includingsolvent blanks, with a minimum of one per sample set. Calibration verification injections must be within f25% to be consideredacceptable. The calibrationcurve and the last passing CCV will then bracket acceptablesamples. MultipleCCV levels may be used. 10.5 System Suitability A minimumof three system su'ibilii samples will be injectedat the beginning and end of each analytical run. Typicallythese samples are run prior to the calibration curve. The system suitabilii injections must have area counts with an RSD of S%and a retentiontime RSD of Q ? when evaluated independently. 11 Procedures 11.1 Extraction Scheme Allow samples to equilibrate to room temperature. Thoroughly mix samples by gently invertingthe sample bottle. Measure 40mL of sample into 50mL polypropylene centrifuge tubes (Spike the Matrix spikes as required*, replace lid and mix well). Note: * Samples may need to be prescreened to determine an appropriate matrix spike level (typically SI50% of sample concentration). Alternatively the samples could be spiked at more than one level, allowing for the inappropriate spike level to be eliminated. ETS-8-154.1 Page 13 of 17 Determinationof PerfluorinatedCompounds in Water Using SPE and LC/MS. 05-0210 Interim Report #5 Page 49 of 123 3M Environmental Laboratory Project No. 05-02 70 Conditionthe C18 SPE cartridges (lg, 6mL) by passing approximatelylOmLmethanolfollowed by approximately50mL ASTM Type Iwater (flow rate approximately2 drop/sec). Do not let column run dry. Note: For the following steps, maintain a -Idrop/sec flow rate. Do not allow the column to run dry at any time. Load the analytical sample onto the C18 SPE cartridge. Discard eluate. Ten mL of the 40% methanolinwater wash mixtureis passedthrough the C18 SPE cartridge to rinse away potential interferences and then discarded. This step must be omitted if perfluorinated compounds with chain lengthslessthan C8 are targeted since these will be lost during this wash step. Elutewith exactly 5mL of 1 0 % methanol. Collect eluate into graduated 15mL polypropylene centrifugetubes. This is the target elution fraction(final volume approximately4.5 mL as not all of the solvent will leave the SPE cdumn. This will not affect the calculations in any way since the curve is also extracted). Analyze a portion of the target elution fractioneluent using negativeelectrospmy HPLCmnWSor + HPLCmnS. Note: Samples are concentrated by a factor of eight during the extraction; Initial Vol = 40mL Final Vol. = 5mL. Samples are stable at room temperature for at least 24 hours. Analytical samples may be stored in a refrigerator at 4"i2OC until analysis. Standardizationof CIS SPE colurnns-lf poor recoveriesare observed, it may be necessaryto standardiiethe C18 SPE cdumns inthe following manner before analyzing samples. Use a standard with an analyte concentration between 1000 and 4000 pg/mL. Repeat the extraction scheme from the beginning up through the eluting with -5mL 100% methanol. M e r the eluting with -5mL 100%methanol step, collectan additional post-elutionfractionby eluting with an additional 5mL of 100% methanol. Analyze both fractions by HPLCIMSIMS or HPLUMS. If the target fraction contains a minimum of 85% of the respective analytes, it may be considered acceptable. If the wash contains significant standard (>15%), either the wash volume or percentage of MeOH should be decreased. If the post-elution fraction contains significant standard (>15%), the target elution volume should be increased. 11.2 Sample Analysis Set up analysis sample queue. Inject the same volume (between 5-25pL) of each standard, analytical sample and blank into the instrument. All samples with a concentration> ULOQ must be diluted and reanalyzed. If dilutionof the final extract fails to produceacceptable results(e.g. poor MS recoveries)dilutethe originalsample and re-?xtract. 12 Data Analysis and Calculations Calculatethe analyticalsample (extract)concentrationfrom the standard curve usingthe following equation: ETS-&154.1 Page 14 of 17 Determinationof PerfluorinatedCompounds in Water Using SPE and LC/MS. 05-02 10 Interim Report #5 Page 50 of 723 3M Environmental Laboratory Project No. 05-02 10 Extract Concentration, pg/mL = - (slope) Calculate the percent recovery of the FSCS using the following equation: FSCS% rec. =/FSCS conc., WmL) x 100 (Conc. added, pg/mL) Calculatethe percent recoveryof the MSs usingthe following equation: MS % rec. = (MS conc., - pg/mL Sample conc., WmL) x 100 (Conc. added, pg/mL) 13 Method Performance Note: Any method performanceparametersthat are not achieved must be considered inthe evaluation of the data. Nonconformance to any speCmed parameters must be described and discussed in any reporting of the data. If criteria listed in this method performancesection are not met, maintenancemay be performed on the system and samples reanalyzed, or other actions taken as determined by the analyst. Document all actions in the raw data. If data are to be reported when performance criteria have not been met, the data must be footnoted on tables and discussed in the text of the report. 13.1 System Suitability A minimum of three system suitabiiity samples will be injected at the beginning and end of each analytical run. Typicallythese samples are run prior to the calibration curve. The system suitability injectionsmust have area counts with an RSD of S%and a retentiontime RSDof S2% when evaluated independently. 13.2 Quantitation Calibration Curve: The coefficient of determination(?)value for the calibration curve must be greater than or equal to 0.990. Each point in the curve must be within 5?5% of the theoretical concentrationwith the exceptionofthe LLOQ, which may bewithin *N%. Demonstration of Specificity: Specificity is demonstrated by chromatographic retention time (within 3%of standard) and the m a s spectral responseof unique ions. 13.3 Sensitivity Solvent Blanks and Method Blanks: Solvent and method blank area counts must be 50% that of the lowest standard used in the calibration curve. Limits of Quantitation (LOQ): The lower LOQ (LLOQ) is the lowest non-zero active standard in the calibration curve; the peak area of the LLOQ must be at least 2X that ofthe extraction blank. By definition, the measured value of the LLOQ must be within 30% of the theoretical value. ETS-8-154.1 Pags 15 of 17 Determinationof PerfluorinatedCompoundsin Water Using SPE and LC/MS. 05-0210 Interim Report #5 Page 51 of 123 3M Environmental Laboratory Project No. 05-0210 13.4 Accuracy CCV Performance:Calibration verification injectionsmust bewithin i25% to be considered acceptable. The calibration curve and the last passing CCV will then bracket acceptable samples. Multiple CCV levels may be used. Matrix Spikes: Matrix spike percent recoveries must be within f 25% of the spiked concentration. If matrix effects are suspected, evaluate the LCS results to determine if a matrix effectsare present and if the method is in control basedon compliant LCS results. Discuss all resultsin theanalytical report. 13.5 Precision Reproducibility: Reproducibility of the method is defined by the results of duplicate or triplicate analysis of samples. A RPD or RSD of S 15% will be considered acceptable. system Suitability: The system suitability injections must have area counts with an RSDof S5% and a retentiontime RSDof S2% when evaluatedindependently. 14 Pollution Prevention and Waste Management Sample extract waste andflammable d e n t is discarded in high BTU containers, and glass pipettewaste is discarded in brokenglass containers locatedinthe laboratory. 15 Records Each data package generated for a study must have the following information included: study or project number, acquisition method, integration method, sample name, extraction date, dilution factor (iiapplicable), and analyst. Print the tune page, sample list, and acquisition method to include in the appropriate study folder. Copy these pages and tape into the instrument run log. Plotthe calibrationcurves as desm'bed in this method, then printthese graphs and store inthe study folder. Print data integrationsummary, integration method, and chromatogramsand store in the study folder. Summarize data using suitable software and store in the study folder. 16 Attachments None. 17 References "Methodof Analysis for the Determinationof Petfluorooctane sulfonate (PFOS), Petfluorooctane sulfonylamide(PFOSA), and Perfluorooctanoate(POAA)inWater", E. Widvemesinheand J. Flaherty, Study Number 023402,CentreAnalytical Laboratories, Inc., State College, Pennsylvania, January 2000. Validation reportfor the "Method of Analysis for the Determinationof Petfluorooctanesulfonate (PFOS), Perfluorooctanesulfonylamide(PFOSA), and Perfluorooctanoate(POAA) in Water", E. Wickremesinhe and J. Flaherty, Study Number 023-002, Centre Analytical Laboratories, Inc., State College, Pennsylvania. ETS-8-154.1 P a g 16 of 17 Determinationof PerfluorinatedCompounds in Water Using SPE and LC/MS. 05-0210 Interim Report #5 Page 52 of 123 3M Environmental Laboratory Project No. 05-0210 18 Affected Documents None. 19 Revisions Revision Number 1 Revision Number Updated to the new format. Changed Title. Section I : States the validationof 3 analyfes,removes referenceto PA document that's no longer applicable. Section 2: Provided for the extraction of more than the 3 validatedanalytes, allows the use of a LCIMS system, not only the LSIMSIMS previously mentioned. Section 3: Revised definitions for field matrix spike, field control spike, LLOQ, method blank, and MOL. Section 5: Reworded the interferences,added recommendation to use disposable pipettes. Section 6: Recategonzed and pared down. Section 7: Changedstorage time to 6months. Added more calibrationpoints to the table. Section 8:Added statement addressing labeling requirements and spiking procedures. Expanded section 8.8. Section 9: New Section Section IO: Changed some of the parameters in the tables. Allowed for use of dflerent instrumentation. Added informationfrom section 12 of previous version, extensively revised. Section 1I (section9 in previous version): Clarificationof wash step, stated exact volume of eluate is 5 mL, revised standardizationprocess, removed requirementto use LCIMSIMS. Section 12 (section 13 in previous version: no changes Section 13 (section 14 in previous version): Extensively rewritten. Section 14 (section 15in previous version):no changes Secfion 15 (secfion 16in previous version):Minor changes to recording requirements. Section 16 (section 17 in previous version): Removed attachment. Section 17 (section 18 in previous version): Removed reference to EPA document that no longer applied to this SOP. Section 18: New section. - Revision Date 7 ETS-8-154.1 P a g 17 of 17 Determinationof PefluorinatedCompounds in Water Using SPE and LCIMS. 05-0270 Interim Report #5 Page 53 of 123 3M Environmental Laboratory Project No. 05-0210 ATTACHMENCT. PROTOTOALND PROTOCOALMENDMENTS 05-0210 Interim Report #5 Page 54 of 123 3M Environmental Laboratory Project No. EO5-0210 Exygen Protocol Number: POOOl 13 1 STUDY PROTOCOL Study Title: Analysis of Perfluorobutanesulfonate (PFBS), Perfluorohexanesulfonate (PFHS), and Perfluorooctanesulfonate (PFOS) in Water, Soil, Sediment, Fish, Clams, Vegetation, Small Mammal Liver and Small Mammal Serum Using LC/MS/MS for the 3M Decatur Monitoring Program Exygen Protocol Number: POOOl 131 Performing Laboratory: Exygen Research 3058 Research Drive State College, PA 16801 Phone: (814)272-1039 h EO5-02 10 Interim Report #5 Sponsor Representative: Michael A. Santoro Director ofRegulatory Affairs 3M Building 0236-01-B-IO St. Paul, MN 55144 Phone: (651) 733-6374 --__ . P q c I cjj -. Page 55 of 123 3M Environmental Laboratory Project No. 05-0210 Exygen Protocol Number: PO001 13 1 c DISTRIBUTION: 1) Jaisimha Kesari, Study Director, Weston Solutions 2) John M. Flaherty, Principal Investigator, Exygen Research 3) Michael A. Santoro, Sponsor Representative, 3M Company 4) Exygen Research Quality Assurance Unit EO5-0210Interim Report #5 Pnge 2 of 65 Page 56 of 123 3M Environmental Laboratory Project No. 05-0210 Exygen Protocol Number: POOOl 13 1 c PROTOCOL APPROVAL Study Title: Analysis ofPerfluorobutanesulfonate(PFBS),Perfluorohexanesulfonate (PF'HS), and Perfluorooctanesulfonate (PFOS) in Water, Soil, Sediment, Fish, Clams, Vegetation, Small Mammal Livers and Small Mammal Serum Using LC/MS/MS for the 3M Decatur Monitoring Program Exygen Protocol Number: POOOl131 APPROVALS Weston Solutions Michael A. Sjdtoro, Sponsor Representative 3M Compady +- /;/I& / Exygen Research Date Exygen R e s e w 05-0210 lnterim Report #5 Page 57 of 123 3M Environmental Laboratory Project No.05-0210 Exygen Protocol Number: PO00 1131 TABLE OF CONTENTS TITLE PAGE .................................................................................................................................................... i DKMUBUTION............................................................................................................................................... 2 PROTOCOL APPROVAL................................................................................................................................ 3 TABLE OF CONTENTS .................................................................................................................................. 4 INTRODUCTION............................................................................................................................................. 5 TEST MATERIALS ......................................................................................................................................... 5 OBJECTIVE ..................................................................................................................................................... 6 TESTING FACILITY....................................................................................................................................... 6 STUDY DIRECTOR......................................................................................................................................... 7 SPONSOR REPRESENTATIVE...................................................................................................................... 7 PRINCIPAL INVESTIGATOR........................................................................................................................ 7 PROPOSED EXPERMENTAL START AND TERMINATION DATES .............................................. IDENTIFICATIONAND JUSTIFICATION OF THE TEST SYSTEM ......................................................... Y - SAMPLE PROCUREMENT, RECEIPT AND RETENTION ......................................................................... 8 SAMPLE IDENTIFICATION .......................................................................................................................... 9 ANALYnCAL PROCEDURE SUMMARY................................................................................................... 9 VERIFICATION OF ANALYTICAL PROCEDURE...................................................................................... 9 METHOD FOR CONTROL OF BIAS............................................................................................................. 1 1 STATISTICALMETHODS ............................................................................................................................. 1 1 GLP STATEMENT .......................................................................................................................................... 1 1 REPORT ........................................................................................................................................................... 11 SAFETY AND HEALTH ................................................................................................................................. 12 AMENDMENTST O PROTOCOL .................................................................................................................. 1-3 DATA RECORD KEEPING ............................................................................................................................ 1-3 QUALITY ASSURANCE ................................................................................................................................ 14 RETENTION OF DATA AND ARCHIVING ................................................................................................. 14 APPENDIX 1. ANALYTICAL METHODS ..................................................................................................... 15 . E050210 Interim Report #5 Page 58 of 123 3M Environmental Laboratory Project No. 05-0210 Exygen Protocol Number: PO00I 131 F INTRODUCTION The purpose of this study is to perform analysis for perfluorobutanesulfonate (PFBS), perfluorohexanesulfonate (PFHS) and perfluorooctanesulfonate (PFOS) in water, soil, sediment, fish, clams, vegetation, small mammal livers and small mammal serum using LC/MS/MS for the 3M Decatur Monitoring Program. The study will be audited for compliance with EPA TSCA Good Laboratory Practice Standards 40 CFR 792 by the Quality Assurance Unit of Exygen Research. TEST MATERIALS The test materials are perfluorobutanesulfonate (PFBS), perfluorohexanesulfonate (PFHS) and perfluorooctanesulfonate (PFOS) and are all supplied by 3M. PFBS Chemical Name: Perfluorobutanesulfonate c Molecular Weight: 338 supplied as the potassium salt (C4F9SOiK+) LotNumber: 101 Purity: 96.7% Transitions Monitored: 299 +99 Structure: PFHS Chemical Name: Perfluorohexanesulfonate Molecular Weight: 438 supplied as the potassium salt ( C ~ F I ~ S O ~ - K ' ) Lot Number: SE036 Purity: 98.6% Transitions Monitored: 399 -# 80 Structure: E054210 Interim Repott #5 Page 59 of 123 3M EnvironmentalLaboratory Project No. EO5-0210 Exygen Protocol Number: PO00I I 3 I LI PFOS Chemical Name: Perfluorooctanesulfonate Molecular Weight: 538 supplied as the potassium salt (C~FUSO~K') Lot Number: 2 17 Punty: 86.9% Transitions Monitored: 499 +99 Structure: OBJECTIVE The purpose of this study is to perform analysis for perfluorobutanesulfonate (PFBS), perfluorohexanesulfonate (PFHS) and perfluorooctanesulfonate (PFOS) in water, soil, sediment, fish, clams, vegetation, small mammal livers and small mammal serum for the 3M Decatur Monitoring Program using the current versions of the following Exygen analytical methods: V0001780: VOOOl78 1 : VOOOl782: V0001783: V0001784: V0001785: V0001786: "Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Water by LC/MS/MS" "Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Soil by LCMS/MS" "Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Sediment by LCMSNS" "Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Fish and Clams by LC/MS/MS" "Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Vegetation by LC/MS/MS" "Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Small Mammal Liver by LC/MS/MS" "Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Small Mammal Serum by LC/MS/MS" TESTING FACILITY Exygen Research 3058 Research Drive State College, PA 16801 Phone: (814) 272-1039 Page 6 of 65 E050210 Interim Report #5 Page 60 of 123 3M EnvironmentalLaboratory Project No. E05-0210 Exygen Protocol Number: PO001 13 I STUDY DIRECTOR Jaisimha Kesari P.E., DEE Weston Solutions, Inc. 1400 Weston Way West Chester, PA 19380 Phone: (610)701-3761 Fax: (610)701-7401 j .kesari@westonsolutions.com SPONSOR REPRESENTATIVE Michael A. Santoro 3M Company Director ofRegulatoryAffairs 3M Building 0236-01-B-10 St. Paul, MN 55144 Phone: (651)733-6374 PRINCIPAL INVESTIGATOR John M.Flaherty Exygen Research 3058 Research Drive State College, PA 16801 Phone: (814)272-1039 john.flaherty@exygen.com PROPOSED EXPERIMENTAL STiR T AND TERMINATIOP DATES It is proposed that the analytical portion of this study be conducted from October 01, 2004 to December 31,2005. The actual experimental start and termination dates will be included in the final report. EO5-0210 Interim Report #5 Page 7 of65 Page 61 of 123 3M Environmental Laboratory Project No. 05-0210 Exygen Protocol Number: PO001 13 1 n IDENTIFICATION AND JUSTIFICATION OF THE TEST SYSTEM The following are the test systems for this study: 0 Water (groundwater and surface water) 0 Soil 0 Sediment 0 Fish Clams 0 Vegetation , 0 Small Mammal Liver 0 Small Mammal Serum The samples will be collected by Weston Solutions. The control samples will be purchased and prepared by the testing facility. Purchase and processing details for the control samples will be included in the final report associated with this study. The test systems were chosen to access the environmental impact of PFBS. PFHS and PFOS in the Decatur, Alabama area. c SAMPLE PROCUREMENT, RECEIPT AND RETENTION Water, soil, sediment, fish, clam, vegetation, small mammal liver and small mammal s e m samples will be received at Exygen directly from Weston Solutions. The details of sampleprocurement for this study are outlined in the 3M work plan entitled "Phase 2 Work Plan for Sampling Environmental Media." The number and types of samples collected will vary depending availability in the field. The total number of samples received and analyzed for each matrix will be documented in the final report associated with this study. Water, soil, and sediment samples will be used as received without further processing at Exygen. These samples will be stored refrigerated at Z"C-8"C. Fish, clam, vegetation and small mammal liver samples will be processed according to the appropriate analytical method (see Appendix I). These samples will be stored frozen at I -10C. Small mammal whole blood samples will be centrifuged in the field at the time of collection and the serum fraction will be used for the study. Small mammal serum will be stored frozen at I-1O"C. The receipt and processing of the samples will be documented in the final L. report and raw data associated with the study. E050210 Interim Report #5 Page 62of 123 3M Environmental Laboratory Project No. 05-0210 Exygen Protocol Number: PO001 131 L SAMPLE IDENTIFICATION Prior to analysis, each sample will be assigned a laboratory sample reference number. The reference number will be unique and will distinguish each laboratory sample that is processed throughout the analytical procedure. Chromatographic data will be identified by the laboratory sample reference number. Sample storage conditions and locations will be documented throughout the study. ANALYTICAL PROCEDURE SUMMARY References: VOOOl780: "Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Water by LCIEVISIMS" V0001781: "Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Soil by LC/MS/MS" VOOOl782: "Method of Analysis for the Determination of Perfluorooctanoic c Acid (PFOA) in Sediment by LCIEVISIEVIS" VOOOl783: "Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Fish and Clams by LC/MS/MS" VOOOl784: "Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Vegetation by LCMSiMS" VOOOl785: "Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Small Mammal Liver by LC/MS/MS" VOOOl786: "Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Small Mammal Serum by LC/MS/MS" The above methods use analytical conditions capable of separating the isomers of PFBS, PFHS and PFOS. The final report will include the isomers summed into total PFBS, total PFHS, and total PFOS found. VERIFICATION OF ANALYTICAL PROCEDURE A laboratory control sample will be used for the preparation of fortified control samples. The test substance will be made into solutions as per the method, and added to the matrices via a micropipette. For water sampling, Exygen will supply one bottle per sample collected. The .h bottles will be 500 mL precleaned Sci/Spec Premier wide mouth HDPE bottles. These bottles have been routinely used for fluorochemical sample -__ EO5-0210 Interim Report #5 Page 63of 123 3M Environmental Laboratory Project No. 05-02 10 Exygen Protocol Number: PO00 1 I3 I c collection at the testing facility and have been shown to be free of PFBS, PFHS and PFOS. Samples will be added to each container to a volumetric fill line at 200 mL. A field duplicate, a low field spike and a high field spike of each sample will be collected. The low and high field spike bottles will contain PFBS, PFHS and PFOS as well as perfluorooctanoic acid (PFOA) and 1.2-13C perfluorooctanoic acid ( ' k PFOA). PFOA and I3C PFOA are included in the solutions used to spike the samples. The results for PFOA and I3C PFOA will not be reported in this study. Exygen will supply one field blank (control water) and two field blank spikes (control water fortified with PFBS, PFHS and PFOS at a low and high level) for every twenty samples collected. At the testing facility, each water sample (excluding field duplicates and field spikes) will be extracted in duplicate and will also be fortified at a low and high concentration with PFBS, PFHS and PFOS and processed through the described procedure to determine method accuracy and to check for bias. For soil, sediment, clams, and vegetation, Exygen will supply one 500 mL precleaned Sci/Spec Premier wide mouth HDPE bottle per sample collected or a zip-seal bag. All containershags used for sample collection will be shipped to the sample location. Samples will be added to each container or bag in the field. At the testing facility, each sample will be extracted in duplicate and will also be fortified at a known concentration with PFBS, PFHS and PFOS at both a low and high level and processed through the described procedure to determine method accuracy and to check for bias. For small mammal liver, Exygen will supply a 50 mL polypropylene centrihge tube. For small mammal serum, Exygen will supply a collection kit for each sample containing serum separator tubes (red top), vacutainers. needle holders and needles, transfer pipettes, and polypropylene tubes. At the testing facility, each liver and serum sample will be extracted in duplicate and will also be fortified at a known concentration with PFBS, PFHS and PFOS at both a low and high level and processed through the described procedure to determine method accuracy and to check for bias. Low and high spiking levels for each matrix are defined below: Soil Sediment Fish Clams Vegetation Small Mammal Liver Small Mammal Serum 500 n d L 4 ng/g 4 ng/g 10nde 10 ng/g 10ng/g 10 ng/g 10 ng/mL 5000 ndL 40 ng/g 40 ng/g 100n d g 100 ng/g 100 ng/g 100 ng/g 100 ng/mL 05-0210 Interim Report #5 Page IO 01'65 -- Page 64 of 123 3M Environmental Laboratow Project No. 05-0210 Exygen Protocol Number:PO001131 L Recoveries are anticipated to be between 70% and 130% of the fortified levels; however, the exact precision and accuracy will be determined by the analysis of the quality control samples described above. A statement of accuracy will be included in the final report. METHOD FOR CONTROL OF BIAS Control of bias will be addressed by taking representative sub-samples from a homogeneous mixture of each matrix from untreated control samples, and by analyzing at least two levels of fortifications. STATISTICAL METHODS Statistics will be limited to those specified in the subject methods and to the calculation of average recoveries, as applicable. GLP STATEMENT All aspects of this study shall be performed and reported in compliance with EPA TSCA Good Laboratory Practice Standards 40 CFR 792. The final report or data package (supplied to the Sponsor) shall contain a statement that the study was conducted in compliance with current and applicable GLP standards and will outline any deviations in the study from those standards. This statement will be signed by the Study Director and Sponsor Representative. REPORT A finaI report will be prepared by the principal investigator or their designee at the conclusion of the study. The report will include, but will not be limited to, the following: The name and address of the Study Director, Sponsor Representative, and of the testing facility. A statement of GLP compliance (any related documentation, such as chain-of-custody records, must be in the study records). E050210 Interim Report #5 Page 65 of 123 3M Environmental Laboratory Project No. 05-0210 Exygen Protocol Number: PO001 131 The signed and dated statement by the Exygen Research Quality Assurance Unit regarding dates of study inspections and dates findings were reported to the Study Director and Management. A description of the exact analytical conditions employed in the study. If the subject method was followed exactly, it is necessary to include only a copy of the analytical method. Any modifications to this method will be incorporated into the report. If the method is photo-reduced, the project number and page number must be included on each page. Description of the instrumentation used and operating conditions. All results from all sets analyzed. Control and fortified samples will be identified and the data table will include sample number and fortification level. Representative chromatograms for each analyte in each matrix, including chromatograms of a standard and a control sample, and a chromatogram at a fortification level. The location of the analyte peaks will be clearly identified in all chromatograms. All circumstances that may have affected the quality or integrity of the data will be documented in the report. Locations where raw data and the final report are to be archived. Additions or corrections to the final report shall be in the form of an amendment signed by the Study Director. The amendment shall clearly identify that part of the report that is being altered and the reasons for the alterations. The amendment will be signed and dated by the Study Director and the Sponsor Representative. .I All applicable requirements for reporting of study results as per 40 CFR 792.185. SAFETY AND HEALTH Laboratorypersonnel will practice good sanitation and health habits. Every reasonable precaution shall be taken to prevent inadvertent exposure of personnel and the environment to the test or reference substance(s). EO5-0210 Interim Report #5 Page I2 of65 Page 66 of 123 3M Environmental Laboratory Project No. 05-0210 Exygen Protocol Number: PO001 13 1 c AMENDMENTS TO PROTOCOL All significant changes to the analytical protocol outlined here will be expressed in writing, signed and dated by the Study Director and Sponsor Representative. Amendments usually will be issued prior to initiation of study plan change. However, when a change is required without suficient time for the issue of a written amendment, that change may be effected verbally with supporting documentation signed and dated by the Study Director and followed with a written amendment as soon as possible. In this case, the effective date of the written amendment will be the date of the documented change. Copies of the signed amendments will be appended to all distributed study plan copies. The original amendment will be maintained with the original study plan. Any deviations fiom the study plan or from the analytical method as provided will be documented and reported promptly to the Sponsor Representative. DATA RECORD KEEPING Records to be maintained include the following (as appropriate): a Sample tracking sheet@) m Sample receipt records, storage history, and chains of custody a History and preparation of standards (stock, fortification, calibration) a Description of any modifications to the method a Instrument run sheets, bench-sheets or logs a Analytical data tables a All chromatographic and instrumental conditions e Sample extraction and analysis dates a A complete listing of study personnel, signatures and initials a Chronological presentation of all study correspondence a Any other documentation necessary for the reconstruction of the study Chromatograms-All chromatogramswill contain the following: Sample identification, injection date, arrow or other indication of the area of interest, and injection number correspondingto the run. Additionally, fortifications will include the amount of analyte added and the sample number of the sample that was fortified. Analytical standard chromatograms will additionally include the concentration (e.g., pg/mL). 05-0210 Interim Report #5 Page 67 of 123 h _- 3M Environmental Laboratory Project No. 05-0210 Exygen Protocol Number: PO001 13 I As part of the documentationthe following sheets will be included in each analytical set: a run sheet listing the samples to be run in the set, and an instrument conditions sheet describing the instrument type and operating conditions. QUALITY ASSURANCE The QA Unit of Exygen Research will inspect the study at intervals adequate to assure compliance with GLP's, and will report the findings of audits to the Study Director, Exygen Management, and the Sponsor Representative. RETENTION OF DATA AND ARCHIVING All hard copy raw data, including, but not limited to, the original chromatograms, worksheets, correspondence, and results shall be included with the data package submitted to the Study Director. These will be archived with the original study plan, amendments, final report, and all pertinent information from the Sponsor. The testing facility shall keep all electronic raw data and any instrument, equipment, and storage logs for the period of time specified in 40 CFR 792.195. An exact copy of the materials submitted to the study director will also be kept at Exygen Research. Exygen will obtain permission from the study director before discarding or returning samples. c 05-0210 Interim Report #5 Page 68 of 123 3M Environmental Laboratory Project No. EO5-0210 Exygen Protocol Number: PO001 131 h APPENDIX I ANALYTICAL METHODS VOOOl780: "Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Water by LC/MS/MS" V0001781: "Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Soil by LC/MS/MS' VOOOl782: "Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Sediment by LC/MS/MS" VOOOl783: "Method of Analysis for the Determination of Perfluorooctanoic -- Acid (PFOA) in Fish and Clams by LC/MS/MS" VOOOl784: "Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Vegetation by LC/MS/MS" VOOOl785: "Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Small Mammal Liver by LC/MS/MS' VOOOl786: "Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Small Mammal Serum by LC/MS/MS" c .. 05-0210 Interim Report #5 Page Is of65 .-.-- Page 69 of 123 3M Environmental Laboratory Project No. 05-0210 Exygen Protocol Number: PO00 I 13 I ANALYTICAL METHOD Method Number: VOOOl780 Method of Anrwlr for the Determinrtlon of Pertluorooctanoic Acid (PFOA)io Water by Lc/Ms/Ms Analytical Testing Facility: Exygm Research 3058 Research Drive State College, PA 16801 Approved B y Paul Comolly I Technical Leader. LC-MS, E x y p Research ' Vice Presid&t. Operations,ExygmRcscarch A Dati?e !&- 05-0210 lnterim Report #5 .-. TotalPages: 7 Page 16 0165 Page 70 of 123 3M Environmental Laboratory Project No. 05-0210 Exygen Protocol Number: PO00 1 13 1 Exygcn Rewarch Method Number VOOO1780 I I ANALYTICAL METHOD Method ofhalysis for the Determination of PertluorooctanoicAcid (PFOA) in Water by LC/Ms/MS 1.0 Scope This method is to be employed for the isolation and quantitation of perfluorooctanoic acid by High Performance Liquid Chromatography coupled to a tandem Mass SptctmmehicDctcctor (LC/MS/MS)in water. 2.0 SafaY 2.1 Alwaysobservesafe laboratory practices. 2.2 Consult the appropriate MSDS before handling any chemical for proper safety precautions. 3.0 SampleRequirement 3.1 At least 40mL of test sample for extraction. 3.2 No sampleproctssing is needed for water samples. 3.3 Samples stored refrigerated should be allowed to equilibrate to room temperame. 3.4 All samples must be thoroughly mixed before being sampled for extraction. 3.5 Any samples containing particles should be centrifuged at -3000 rpm for -5 minutes and the supematant used for the extraction. 3.6 Sample collection procedum will be specified in the sampling plan for this project. 4.0 Reagents and Standards 4.1 Wata - WLC grade - 4.2 Methanol HPLC grade - 4.3 Ammonium Acetate A.C.S. Reagent Grade 4.4 Pcrfluorooctanoic Acid - Sigma-Aldrich 5.0 instrument and Equipment 5.I A high pmfonnauce liquid chromatograph capable of pumping up to 1 solvents equipped with a variable volume injector capable of injecting 5-200 pL COMeCtCd to 8 tandem MW SpCCtrometCI(Lc&fs/Ms). 5.2 A device to collect raw data for peak integration and quantitation. 5.3 Analytical balance capableofreading to O.oooO1 g. 5.4 50 mL disposablepolypropylenecentrifuge tubes. 5.5 15 mL disposable polypropylene centrifuge tubcs. 5.6 Disposable micropipets (50-1OOuL. 100-2OOuL). 5.7 125-mL LDPE narrowmouthbottles. 5.8 2 mL clear HPLC vial kit. 5.9 Disposable pipettes. 5.10 AutopipeneCl(100-1OOOpL and 10-100 pL). with disposable tips. 5.1 I Waters Scp Pak Vac 6 cc (le) tC18 SPE cartridges. 05-0210 Interim Report #5 Page I7 of 65 Page 71 of 123 3M Environmental Laboratory Project No. 05-0210 Exygen Protocol Number: PO001 13I ExpRcseush MeWNumbcr V000178u I I AhALYTlCAL ,METHOD Methodof Analysis for the Determination of PerfluorooctanoicAcid (PFOA)in Water by LCIMSIMS 5.12 SPE vacuum manifold. 5.13 Centrifuge capableof spinningSO mL polypropylenetubes at 3000 tpm. 6.0 chromatographic System 6.1 Analytical Column:F l u o p h e RP (KeystoneScientific), 2.1 m m x SO mm. 511 ( P M 82505-052130) 6.2 Tmperaturc: 30C 6.3 Mobile Phase (A) : 2 mM Ammonium Acetate in Water 6.4 Mobile Phase (B) : Methanol 6.5 Gradient Rogram: Time (mini 0.0 65 1.o 65 8.0 25 20.0 25 22.5 65 a 35 35 75 75 35 Flow Rate fmUmin) 0.3 0.3 0.3 0.3 0.3 - 6.6 Injection Volume: 15 pL (canbe increasedto as much as SO pL). 6.7 Quantitation: Peak Area - c x t d standardcalibration curve. 6.8 Run Time: 23 minutes. The above conditions an intended as a guide and may be changed in order IO optimize the HPLC system. 7.0 MS/MSSystem 7.1 Mode: ElectrosprayNegativeMRM mode.monitoring413 +369 m/z. The above conditions an intended as a guide and may be changed in order IO optimize the MSMS system. 8.0 Preparation of Solutions 8.1 Mobile Phase 8.I . I 2 mM anmoNum acetate in water is p " p d by adding 0.I54 g of ammonium acetateto 1OOO mL of water. Alternate volumcs may be prepared. 05-0210 Interim Report #5 Page 72 of 123 3M Environmental Laboratory Project No. 05-02 10 Exygen Protocol Number: PO001131 c Exygcn Research M c W Number VooOl780 1 ANALYTICQL METHOD i Mahod of Analysis for the Dctcnninationof PerfluorooctanoicAcid (PFOA) in Water by LCIMsiMS 9.0 StandardpnpprOtion 9.1 Standard Stock/Fortification Solution 9.1.1 Prepare a stock solution of-100 pUmL of PFOA by weighing 10 nig of analytical standard (corrected for purity) and dilute lo 100 mL with methanol in a 125-mL LDPE bottle. 9.1.2 A IO p&L fortification solution of PFOA is prepared by bringing I O mL of the 100 pB/mL solution to a final volume of 100 with methanol in a 125 mL LDPE bottle. 9.1.3 A 1.0 pghnL fortification solutionof PFOA is prepared by bringing I O mL ofthe 10 p&lmLsolution to a final volume of 100 with meihanol i n a 125 mL LDPE bottle. 9.1.4 A 0.1 pg/mL fortification solution of PFOA is prepared by bringing I O mLof the 1.0 p@mLsolution to a final volume of 100with methanol in a 125mL LDPE bottle. 9.1.5 A 0.01 p@mL fortification solution of PFOA is prepared by bringing 10 mL of the 0.1 pg/mL solution to a final volume of 100 with methanol in a 125 mL LDPE bottle. 9.1.6 The stock and fortificationsolutions are to be stored in a refrigerator at approximately 4'C and are stable for a maximum period of 6 months from the date of preparation. 9.2 Standard Calibration Solutions 9.2.1 LCIMSMS calibration stludardr are prepared in HPLC water. The calibration standards arc processed through the extraction procedure. identical to samplcs. 9.2.2 The following is a typical example: additional concentrations may be prepared as needed. Final Concentration Fortification Volume of Concentration of Calibration of Fortihation Volume FortifiedControl Calibration Standard ID Solution (ppb) (pL) Sample (mL) Standard (ppt). (example) 0 0 40 0 XCmmddyy-0 10 100 40 25 XCmmddyy-1 10 200 40 50 XCmrnddyy-2 IO 400 40 100 XCmmddyy-3 100 100 40 250 XCmmddyy-4 100 200 40 500 XCmmddyy-5 100 400 40 lo00 XCmmddyy-6 The exhcted concentrationofthe calibration standard is equal to 8x its initial concentration, due to the concentration of the standard during the extraction ISPE) XC =extracted calibration standard. 05-0210 Interim Report #5 Page 19 of 65 Page 73 of 123 3M Environmental Laboratory Project No. 05-0210 Exygen Protocol Number:PO001131 I I ExygenResurcb Metbod Numbcr VOOO1780 ANALYTICAL METHOD Method of Analysis for the Detmninationof Pemuorooctanoic Acid (PFOA) in Water by LC/MS/MS 9.2.3 A m standard solution (reagent blank) m t be o m r e d with each set of standards extracted. 9.2.4 Store all extracted calibration stpndardsin 15-mL polypropylenetubes at 2C to 6OC. up to two weeks. 9.2.5 Alterrmte volumes and ConccntratiOnS of standards may be prepared as needed. 10.0 Batch Sa Up 10.1 Each batch of samples extracted (typically 20 or less) must include at leut om reagent control (method blank using HPLC water) and two rcaycni controls fortified at known concentrations (lab control spike) tu verify procedural remvcryfor the batch. 10.2 Roquimnents for field and laboratory duplicates and spikes will be specifid in the quality assuranceplan for this project. 11.O Sample Extraction 11.1 Measure 40mL of asmple or a portion of sample diluted to 40mL with water into 50 mL. polypropyleneCentriibgC lubes (fortify as nceded, replace lid and mix well). 11.2 Condition the CISSPE cartridges (1 g. 6 mL) by passing 10 mL methanol followed by 5 mLof HPLC water (- 2 dropkc). Do not let column run dry 1I .3 Load sampleon conditioned CI, SPE cartridge. Discard eluate. 11.4 Elute with -5 mL 100% methanol. Collect 5 mL of eluate into graduated IS mL polypropylenecentrifugetubes (final volume = 5 mL). 11.5 Analyze samplesusing dectrosprayLC/MS/MS. 12.0 Chromatography 12.1 Inject the same amount of each s t d a d , sample and fortified sample into the LC/Ms/MS system. A calibration standard must precede and follow all analyzed samples. 12.2 StandardsofPFOA correspondingto at least five or more concentration levels must be included in an analytical set 12.3 An entire set of extracted calibration standards must be included a1 the beginning and at the end of a sample set. Extracted standards must hc i n t v c d between every 5-10 samples. As an alternative. an entire sei 01- extracted calibration standards may be injected at the beginning of a SCI followed by extracted calibration standards interspersed every 5-10 samples (to account for a s a n d set of extracted standards). In either case, extracted calibration standardsmust be the first and last injection in a sample sei. 12.4 Use linear standard curves for quantitation. Linear standard curves are generated for the analyteby linearregnssion using l/x weightingofpeak are& 05-0210 Interim Report #5 Pnge 20 oJ6j Page 74 of 123 3M Environmental Laboratory Project No. EO5-02 10 Exygen Protocol Number: PO001 131 I 1 IEx-n-zmReoeueb ANALYTICAL METHOD MethodNumber VWOI780 1 Methodof Analysis for the Determinationof P e d u o m t a n o i cAcid (PFOA) in Water by LCIMSIMS versus calibration standard concentration using MassLynx 3.3 (or equivalent) sofhvarc system. 12.5 Sample response should not excad standard responses. Any samples that exceed standard reaponres should be liuthcr diluted and reanalyzed. 13.0 Acceptance Criteria 13.1 Chromatogrammust show a peak of a daughter ion at 369 a m u from a parent of 413 amu. The 413 amu parent corresponds to the PFOA anion, while the daughter ion (369m u ) representsthe loss of carbon dioxide. 13.2 Method blanks must not conlain PFOA a1 levels greater than the LOQ. If a blank contaihs PFOA at levels greater than 50 ng/L, then a new blank sample must be obtained and the entire set must be re-extracted. 13.3 Recoveria of control spikes and matrix spikes must be between 70- 130% of their known values. If a control spike falls outside the acceptable limits, the entire set of samples should be re-cxtracted. Any matrix spike outside 70130% should be evaluated by the analyst to determine if re-extraction is warranted. 13.4 Any calibration standard found to be a statistical outlier by using the Huge E m Test, may be excluded from the calculation of the calibration curve However, the total number of extracted calibration standards that could be excludcd must not exceed 20% of the total number of extracted standards injected. 13.5 The correlation coeficient (R) for calibration curves generated must be 20.992 (R' 20.985). If calibration results fall outside these limits, then appropriate steps must be taken to adjust instrument operation, and the standards or the relevant set of samples should be reanalyzed. 13.6 Retention times between standards and samples must not drift more thdn f 4 % within an analylical tun. If retention time drift exceeds this limit within an analytical run then the set must be reanalyzed. 14.0 Calculations 14.1 Use the followingequation to calculate the mount of PFOA found (in ng/L. based on peak area) using the standard curve (linear regression parameters) generatedby the Mas8 Lynx softwareprogram: PFOA found (n&) = Peak arca - intercmtk x DF slope DF = factor by which the final volume was diluted, if necessary. EO5-0210 interim Report #5 Page 21 of 65 Page 75of 123 3M Environmental Laboratory Project No. E05-0210 Exygen Protocol Number: PO001 131 IEx-v-lenR e d ANALYTICAL METHOD Method Numbn VOOOl780 1 Method ofAnalysis for the Determinationof Perfluorooctanoic Acid (PFOA) in Water by LC/Ms/MS 14.2 For samples fortified with known amounts of PFOA prior to extraction, usc the followingequation to calculate the percent recovery. Recovay (%) = I tow Pnalytefound (ng/L) - analytefound in control (ng/L)l x , oo analyte added (ng/L) 05-0210 Interim Report #5 Page 7 of fnge 22 of65 Page 76 of 123 3M Environmental Laboratory Project No. 05-0210 Exygen Protocol Number: PO001 131 ANALYTICAL METHOD Method Number VO00I 78I Method of Anrlyrk for the Determlnatlonof Perfluorooctanoic Acid (PFOA) in Soil by LCIMSIMS Analytical Testing Facility: Exygen Research 3058 Research Drive State College, PA 16801 Approved By: toJu.lo Date Vice President,Opedom,ExygenR-h PA74 Date c 05-0210 Interim Report #5 TotalPages: 7 Page 23 of65 Page 77of 123 3M Environmental Laboratory Project No. 05-0210 Exygen Protocol Number: PO00I 131 Exypen Rctclrcb Mcthod Number VooOl781 1 1 ANALYTICALMETHOD Mahod of Analysis for the Determination of Pcr!luorooctanoic Acid (PFOA) in Soil by LC/MS/MS I .o scope This method is to be mployed for the isolationand quantitation of perfluorooctanoic acid by High PcrCormance Liquid Chromatography coupled to a tandem Mass SpectrometricDetector (LcIMs/MS) in soil. 2.0 safw 2. I Always observe safe laboratory practices. 2.2 Consult the appropriateMSDS before handling any chemical for proper safety precautions. 3.0 SampleRequirement 3.1 At least I5 g of test sample for extraction. 3.2 No sampleprocessingis needed for soil samples. 3.3 Samples stored refrigerated should be allowed to equilibrate IO room temperature. 3.4 All samples must be thoroughlymixed before being sampled for extraction. 3.5 Sample collection procedures will be specified in the sampling plan for this project. 4.0 Reagents and Standards 4.1 Wata-HPLC@e 4.2 Methanol- HPLC vsde 4.3 Ammonium Acetate -ACS. Reagent Grade 4.4 PcrfluorooctanoicAcid - Sigma-Aldrich 5.0 Inshument and Equipment 5.1 A high performance liquid duornatogrqh capable of pumping up to 1 solvents equipped with a variable volume injector capable of injecting S - Z N pL connected to a tandem Mass Spectrometer(LCIMSIMS). 5.2 A device to collect raw data for peak integration and quantiration. 5.3 Analytical balance capable of reading to O.OOOO1 g. 5.4 50 mL disposablepolypropylenecentrifugetubes. 5.5 15 mL disposablepolypropylenecentrifugetubes. 5.6 Disposablemicropipets(SO-1OOuL. 100-2OOuL). 5.7 125-mLLDPE narrow-mouthbottles. 5.8 2 mL clcar HPLC vial kit. 5.9 Disposablepipettes. 5.10 Autopipettes(100.1OOO pL and 10-100 pL), with disposable tips. 5.11 Waters Scp Pak Vac 6 cc ( 1 3 tC18 SPE cartridges. 5.12 SPE vacuum manifold 5.13 Ulh?lsonic bath. 05-0210 Interim Report #5 Page 24 oj 65 Page 78 of 123 c EO5-O210 Interim Report #5 3M Environmental Laboratory Project No. EO5-0210 Exygen Protocol Number: PO001131 E x y p R~esearch M e W ~vabcrvooo I 7nI I 1 ANALYTICAL METHOD Method ofAnalysis for the Determinationof PerflwrooctanoicAcid (F'FOA) in Soil by Lc/Ms/MS 5.14 Wrist-action shaker. 5.1 5 Cenbifugecapableof spinning50 mL polypropylenetubes at 5000 rpm. 6.0 chromatographicsystem 6.1 Analytical Column:FluophaseRP (KeyatoneScientific), 2.1 mm x 50 mm. 5j1 - a (PM: 82505-052130) 6.2 Temperature: 30'C 6.3 Mobile Phase (A) : 2 mM Ammonium Acetate in Water 6.4 Mobile Phase (B): Methanol 6.5 GradientProgram: Flow Rate (mUmi& 0.0 65 35 0.3 1.o 65 35 0.3 8.0 25 75 0.3 20.0 25 75 0.3 22.5 65 35 0.3 - 6.6 InjectionVolume: I5 pL (canbe increased to as much a6 50 pL). 6.7 Quantitation: Peak Area - external standardcalibration c w e . 6.8 Run Time: 23 minutes. The above wnditions are intended u a guide and may be changed in order to optimize the HPLC system. 7.0 MS/MS System 7.1 Mode: ElcctmsprpyNegative MRMmode. monitoring 413 + 369 miz for PFOA. The above conditionsarc intended as a guide and may be changed in order IO optimize the MSMS system. 8.0 Preparation of Solutions 8.1 Mobile Phase 8.1.1 2 mM ammoNm acetate in watcr is prepared by adding 0.154 g o l ammonium acetate to lo00 mL ofwater. Alternatevolumesmay be prepared. Page 3 of Page 25 qf6S Page 79 of 123 3M Environmental Laboratory Project No. 05-0210 Exygen Protocol Number: PO001131 Exyga Rcsurcb Method Number VOOO1781 1 1 ANALYTICAL METHOD Method of Analysis for the Dctaminationof PerfluorooctanoicAcid (F'FOA) in Soil by Lmms 9.0 Standard Preparation 9.1 Standard StoddFortification Solution 9.1.1 Prrpare a stock solution of -100 pg/mL of PFOA by weighing IO tng of analytical StMdard (corrected for purity) and dilute to 100 mL with me(hano1 in a 125-mL LDPE bottle. 9.1.2 A 10 pg/mL fortificationsolution of PFOA is prepared by bringing IO mL of the 100 pghnL solution to a final volume of 100 with methanol in a 125 mL LDPE bottle. 9.1.3 A 1.0 pg/mL fortificationsolution of PFOA is prcparcd by brinlyng 1(1 mL of the IO pg/mL solution to a final volume of 100 with mrihanul 111 a 125 mL LDPE bottle. 9.1.4 A 0.1 pg/mL fortificationsolution of PFOA is prepad by bringing Io d.of the 1.0 p S / d solutionto a final volume of 100 with methanol in a 125 mL LDPE bottle. 9.1.5 A 0.01 pghnL fortification solution of PFOA is prepared by bringing 10 mL of the 0.1 pg/mL solution to a final volume of 100 with methanol in a 125 mL JDPE bottle. 9.1.6 The stock and fortificationsolutions arc to be stored in a refrigerator at epproximstely 4OC and arc stable for a maximum period of 6 months from the date of preporption. 9.2 Standad CalibrationSolutions 9.2.1 LcIMs/Ms calibration standanls are prepared in HPLC wafer. Thc calibration standards arc processed through the extraction procedure. identicalto samples. 9.2.2 The following is a typical example: additional concentrations may be prepared as needed. Final Concenfntion Fortification Volume of Concentration of Calibration of Fortification Volume Fortified Conbol Calibration Standard ID Solution (ppb) (ML) Sample (mL) Standard (ppt). (example)- 0 0 40 IO loo 40 0 XCrnmddyy-0 2s XCNnddyy-l 10 200 40 50 XCmmddyy-2 10 400 40 100 XCmmddyy-3 100 100 40 250 XCmmddyy-4 100 200 40 500 XCmmddyy-5 loo 400 40 loo0 XCmmddyy-6 * The extracted concmlration of the calibrationstandard is equal to 8x its initial conccntmtion, due to the concentrationof the standard during the extraction(SPE). XC = extracted calibrationstandard. Psgc4 of 7 E05-0210 Interim Report #5 Page 26 01'65 ._ Page 80 of 123 3M Environmental Laboratory Project No. 05-0210 Exygen Protocol Number: PO001131 Exy.e_en Resarcb Metbod Number VOOOI78 I L I ANALYTICAL METHOD 1 Method of Analysisfor the Determinationof PerfluorooctanoicAcid (PFOA) in Soil by LC/MS/MS 9.2.3 A a m standard solution (reagent blank) must be Drmared with each set of standardsextracted. 9.2.4 Store a11 extracted calibration standards in 15-mLpolypropylenetubes at 2C to 6C. up to two weeks. 92.5 Alternate volumes and concentrationsof standards may be prepared as needed. 10.0 Batch Set Up 10.1 Each batch of samples extracted (typically 20 or Icss) must include at least one reagent control (method blank using 5 mL of methanol) and two reaptit controls fortified at known concentrations (lab control spike) to verify procedural recovery for the batch. 10.2 Requirements for field and laboratory duplicates and spikes will be specified in the quality assurance plan for this project. 11.0 SampleExtraction 11.1 Weigh 5 g of sample into 50 mL polypmpylene centrifuge tubes (fortify as needed. replace lid and mix well). 11.2 Add 5 mLof methanoland shakeon a wrist action shaker for -1s minutes. 11.3 Transferthe tubcsto an ultrasonic bath and sonicate for -15 minutes. 11.4 Bring the volume up to 40 mL with water in the 50 mL polypropylene centrifuge tube. 11.5 Centrifugefor -10 minutesat -3000 rpm. 11.6 Condition the CISSPE cartridges (1 g, 6 mL) by passing 10 mL methanol followed by 5 mL of HPLC water (- 2 droplsec). Do not let column run dry 11.7 b a d (decant) the sample on the conditioned C,, SPE canridge. Discard eluate. 11.8 Elute with -5 mL 100% methanol. Collect 5 mL of eluate into graduated IS mL polypropylene centrifuge tubcs (final volume = 5 mL). 11.9 Analyze samples using electrospray LC/MS/MS. 12.0 Chromatography 12.1 Inject the same amount of each standard, sample and fortified sample into rhc LC/Ms/MS system. A calibration standard must precede and follow all analyzed samples. 12.2 Standardsof PFOA correspondingto at least five or more concentration levels must be included in an analytical set. 12.3 An entire 6et of extracted calibration standards must be included at the beginning and at thc end of a sample set. Extracted standards must be interspersedbetween every 5-10 samples. As an alternative. an mtin set of 05-0210 Interim Report #5 Page 81 of 123 3M Environmental Laboratory Project No. 05-0210 Exygen Protocol Number: PO001131 Exygca Research Method Numb% V0001781 I 1 ANALYTICAL METHOD Method of Analvsir for the Damninetion of Perfluorooctanoic Acid (PFOAI in Soil bv extracted calibration s t d d a may be injected at the beginning of a set followed by extracted calibration standads interspersed every 5-10 samples (to account for a sccond sct of extracted standards). In either case. extracled calibrationstandarb must be the tint and last injection in a sample set. 12.4 Use linear standard curves for quantitation. Linear standard curves arc generated for the analyte by linear regression using Ilx weighting of peak area versus calibration standard concentration using MassLynx 3.3 (or equivtlcnt i software system. 12.5 Sample response should not excad standard responses. Any samp\es that excced standard responses should be mer diluted and reanalyzed. 13.0 Acceptance Criteria 13.1 Chromatogrammust show a p d of a daughte-r ion a! 369 m u from a parent of 413 m u . The 413 amu parent corresponds to the PFOA anion, while (he daughter ion (369 emu) np-ts the loss of carbon dioxide. 13.2 Method blanks must not contain PFOA at levels greater than the LOQ. If a blank contains PFOA a1 levels greater than 50 ngL. then a new blank sample must be obtained and the entire sct must be re-extracted 13.3 Recoveries of control spikes and matrix spikes must be between 70-130%of their !mown values. If a control spike falls outside the acceptable limits. the entire set of samples should be raextrrctcd. Any matrix spike outside 70130% should be evaluated by the analyst to determine if re-extraction is warranted. 13.4 Any calibration standard found to be a statistical outlier by using the Huge Error Test,may be excluded from the calculation of the calibration cune However, the total number of extracted calibration standards that could be excluded must not exceed 20% of the total number of extracted standards injected. 13.5 The correlation coefficient (R) for calibration curves generated must k 20.992 (R' 20.985). If calibration results fall outside thac limiis. Iheii appropriate steps must be taken to adjust instrument operation. and ihr' standardsor the relevant set of samplesshould be reanalyzed. 13.6 Retention times between standards and samples must not drift more than f 4 %within an analytical run. Ifmtentiontime drift exceeds this limit within an analytical ma then the set must be reanalyzed. Page 6 0 1 7 ,- 05-0210 Interim Report #5 Page 28 oJ6S Page 82 of 123 3M Environmental Laboratory Project No. 05-0210 Exygen Protocol Number: PO001 13 1 Enwen Rcrcuch McthodNwnbcr VwO1781 14.0 Calculations 14.1 Use the following equationto calculate the amount of PFOA found (in ng/L. based on pcak arca) using the standard curve (linear regression parameters) gcncrated by the M a s Lynx sofhvare program: - PFOA found (n@) =lpcak arm intercat) x DF slope -DF factor by which the finalvolume waa diluted, if necessary. 14.2 For samples fortified with known amounts of PFOA prior to extraction, u x the followingequation to calculateh e percent recovery. Recovay (%) 9 14.3 Use the following equation to convert the amount of PFOA found in ng/L to n%l3 @Pb). PFOA found (ppb) * FFOA fot&(WL) x volume extracted I0.04LU sample weight (5 g) 14.4 Use the followin8 equation to calculate the amount of PFOA found in pph based on dry weight. PFOA found (ppb) dry weight - PFOA found (ppb) x [ 100%I total solids(%)] +- 05-0210 Interim Report #5 Page 83 of 123 3M Environmental Laboratory Project No. 05-0210 Exygen Protocol Number: PO001 I3 1 ANALYTICAL METHOD Method Number: VOOO1782 Method of Anrlystr for the Dcterminrtlonof PernucrrooetmoicAcid (PFOA)in Sediment by LCMSMS Analytical Testing Facility: Exygcn Research 3058 Research Drive State College, PA 16801 Approvcd B y ? ? JJcal., Paul Connolly I Technical Leader,LC-MS,EXygm Refearch / Vice President. Opmtions, Exygen Reseprch 4Date 4L- 05-0210 Interim Report #5 TotalPagcs: 7 Page 30 of 65 Page 84 of 123 3M Environmental Laboratory Project No. 05-0210 EXygen Protocol Number: PO001 13 1 Exvmn R-h Method Number VOW1782 1.0 sfope This mcthod is to be employed for the isolation and quantitation of perfluoroocianoic acid by High Performance Liquid chromatography coupled to a tandem Mass SpcctromCtric Detector (Lc/MS/MS) in sediment. 2.0 safety 2.1 Always observe safe laboratory practices. 2.2 Consult the appropriateMSDS before handling any chemical for proper safety precautions. 3.0 SampleRcquiranent 3.1 At least 30 g of test sample for extraction. 3.2 No sampleprocessingis ncedcd for sediment samples. 3.3 Samples stored refrigerated should be allowed IO equilibrate IO room temperature. 3.4 All oampln must be thoroughly mixed before being sampled for extraction. 3.5 Sample collection procedures will be specified in the sampling plan for this project 4.0 Rcagenta and Standards - 4.1 Water -HPLC grade 4.2 Methanol HPU: @de 4.3 Acetic Acid -Reagent grade 4.4 - Ammonium ACaate A.C.S. ReWat Grade 4.5 PafluorooctenoicAcid - Sigma-Aldrich 5.0 InstMnentand Equipment 5.1 A high pcrformauce liquid chromptogrsph capable of pumping up to 2 solvents equipped with a variable volume injector capable of injecting 5-200 pL C O M C C ~to~ a~ tandem Mass Spectrometer(LC/MS/MS). 5.2 A device to collect raw data for peak integration and quantitation. 5.3 Analytical balance capable of reading to O.oooO1 ig. 5.4 50 mL disposablepolypropylene cmtrifusctubes. 5.5 15 mL disposPblepolypropylenecentrifugetubes. 5.6 Disposable micropipets (SO-IOOuL, 100-2OOuL). 5.7 125-mL LDPE narrow-mouthbottles. 5.8 2 mL clearHPLC vial kit. 5.9 Disposablepipettes. 5.10 Autopipettes (1W1OOO pL and 10-100 pL). with disposable tips. 5.11 Waters Sep PakVac 6 cc (le) tC18 SPE cartridges. 5.12 SPE vacuum manifold. Page 2 01 7 EO5-0210Interim Repott #5 Page 85 of 123 3M Environmental Laboratory Project No. E050210 Exygen Protocol Number: PO001 13 1 E x y pRnareh MethodNumber VOOO1782 L ANALYTICAL METHOD I Method ofAnalysk for the Determination of PerfluorooctanoicAcid (PFOA) in Sedimenr h! LC/MS/MS 5.13 vortuter. 5.14 Wtkt-o~ti~n~hrlra. 5.15 Centrifugecapableof spinning 50mL polypropylenetubes at 3000 rpm. 6.0 C h r o r m t O ~ b i Csystan 6.1 Analytical Column:FluophPPeRP (KeystoneScientific), 2.I mm x 50 rnm. 5p (PN 82505-052130) 6.2 Trmpffahue: 30C 6.3 Mobile Phasc (A) : 2 mM Ammonium Acetate in Water 6.4 Mobile Phase (B) : Methanol 6.5 OradientProgram: limG.bw `LB 0.0 65 1 .o 65 8.0 25 20.0 25 22.5 65 oa 35 35 75 75 35 Flow Rate (,mUmin) 0.3 0.3 0.3 0.3 0.3 6.6 hjection Volume: I5 pL (can be increased to as much as 50 pL). - 6.7 Quantitation: Peak Area -uttmalstandardcalibration curve. h 6.8 Run Time: 23 minutes. The aboveconditionsarc intended as a guide and may be changed in order to optimize the HPLC system. 7.0 M W S System 7.1 Mode: ElcctrosprayNegative MRM mode, monitoring 413 +369 m/z for PFOA. The above conditionsarc intended &( a guide and may be changed in order to optimize&e MSMS rystcm. 8.0 Preparationof Solutions 8.1 MobilePhuc 8.1.1 2 mM ammonium pcctptc in water is prepared by adding 0.154 g o f ammonium seetale to 1M)o mL of water. Page 3 01'7 E050210 Interim Repott #5 Page 32 of 65 - .-__ - -- Page 86 of 123 3M Environmental Laboratory Project No. EO5-0210 Egygen Protocol Number: PO001 131 Exma Ruclrch McthodNumber V0001782 1 1 ANALYTICALMETHOD Method ofhalysis for the Determination of PerfluorooctanoicAcid (PFOA) in Scdimeni by LC/MS/MS 8.2 Extraction S O ~ U ~ ~ O M 8.2.1 1% acetic acid in water is prepared by adding 10 mL of acetic acid to lo00 mL of water. Alternate volumes may be prepared. 9.0 Standardpnparation 9.1 Standard StoddFortification Solution 9.1.1 Prepare a stock solution of-100 p&mL of PFOA by weighing 10 mt: of analytical standard (comcted for purity) and dilute to 100 mL with methanol in a 125-mL LDPE bottle. 9.1.2 A 10 pghL fortificationsolution of PFOA is prepared by bringing IO mL of the 100 pglmL solution to a final volume of 100 with mlbncrl in a 125 mL LDPE bottle. 9.1.3 A 1.0 p g h L fortificationsolution ofPFOA is prepared by bringing IO mL ofthe 10 pglmL solution to a final volume of 100 with methanol in a 12s mL LDPE bottle. 9.1.4 A 0.1 pUmL fortificationsolution of PFOA is prepared by bringing II) mL ofthe 1.0 p@mLsolution to a find volumeof 100 with methanol in a 125 mL LDPE bottle. 9.1.5 A 0.01 p&nL fortification solution of PFOA is prepared by bringing 10 mL of the 0.1 &mL solution to a final volume of 100 with methanol in a 125 mL LDPE bottle. 9.1.6 The stock and fortificationsolutionsare to be stored in a refrigerator at appmximately 4'C and am stable for a maximum period of 6 months from the date of preparation. 9.2 Standard Calibration Solutions 9.2.1 Lc/Ms/MS calibrations t a n d d s arc prepared in methanolvia dilution ofthe 0.1 p@L fortificationsolution. 9.2.2 The following is a typical example: additional concentrations may he prepared as needed. Cmcenhation Final of Fortification Volume Diluted to Concentrahon Solution (ndmL) (mL) (mL) (np/mL) 100 10 100 10.0 100 S 100 5.0 100 2 100 2.0 10 10 100 I .o 5 10 100 0.5 2 10 100 0.2 EO5-0210 Interim Report #5 Page 33 of65 Page 87 of 123 3M Environmental Laboratory Project No. 05-0210 Exygen Protocol Number: PO00 1 13 1 9.2.3 Store dl calibration standards in 125-mL LDPE narrow-mouth bottles at 2'C to 6C. up to six months. 9.2.4 Alternate volumes and concentrationsof standards may be prepared as nccded. 10.0 Batch Set Up 10.1 Each batch of ramplea exbactcd (typically 20 or less) must include at least one untreated control and two untreated controls fonifitd at known concentrations(lab control @e) to vaify proceduralrecovery for [he batch. 10.2 Requirements for field and laboratory duplicates and spikes will be specified in the qualityasaurameplan for this project. 11.0 Sample Extraction 11.1 11.2 11.3 11.4 11.5 11.6 11.7 11.8 11.9 11.10 11.11 11.12 11.13 Weigh 5 g of sample into 50 mL polypropylene centrifuge tubes (fortif> as nccded, replace lid and mix well). Add 35 mL of 1% acetic acid, cap, voItcx and shake on a wrist action shaher for -60 minuter. Centrifugethe tubesat -3000 rpm for -20 minutes. Condition the Cia SPE CPltridges (1 8. 6 mL) by passing 10 mL methanol followed by 20 mL of HPLC water (- 2 drop/sec). Do not let column run dry Load (decant) the sample on the conditioned Clr SPE cattridge. Discard eluate. Add 20 mL of methanol to the scdiment left in the bottom of the 50 mL centrifuge tube. Cap, vortex and shake on a wrist action shaker for -30 minutes. Centrifugethe tubesat -3000 rpm for -20 minutes. Decant the methanol onto the same SPE cutridge. Collect the eluate. Wash the column with 4 mL of methanol. Collect the eluate and add it to the eluate collected in step 11.8. Conditiona ucond CISSPE earhidge(1 g,6 mL) by passing 10 mL methanol followed by 20 mL of HPLC water (- 2 droplsec). Do not let column run dry Add the methanol to -200 mL of water and load on the second conditioncd SPE camidge. Elute with -5 mL 100% methanol. Collect 5 mL of eluate into graduated 15 mL polypropylene cmtrifUBetubes (final volume = 5 mL). Analyze samples using clectrosprayLCMSIMS. 05-0210 Interim Report #5 Page 34 of 65 Page 88 of 123 3M Environmental Laboratory Project No. EO5-0210 Exygen Protocol Number: PO001131 1 ANALYTICAL MeTHOD 1 Method ofAnalvsis for the Determinationof PerfluorooctanoicAcid (PFOA) in Sediment bv 12.0 Chromatography 12.1 Inject the same amount of each standard, sample and fortified sample into the LcIMS/MS systan. A calibration standard must precede and follow all d y z e d ramplcs. 12.2 Standardsof PFOA corrcspondiigto a! least five or more concentrationlevels must be included in an analytical set. 12.3 An entire set of extracted calibration standards must be included at the beginniig and at the end of a sample set. Standards must be interspersed W e e n every 5-10 samples. As an alternative, an entire set of calibrdtiun standards may be injected at the beginning of a set followed by calibration standards ~ntmpascdevery 5-10 samples (to account for a second set of standards). In either case. calibration standards must be the first and last injection in a sample set. 12.4 Use linear stvldard curves for quantitation. Linear standard curves arc generated for the d y t e by lineorregression using l/x weighting of peak arc3 versus calibration standard concentration using MassLynx 3.3 (or equivalent) software systan. 12.5 Sample response should not exceed standard responses. Any samples that e x c d standard responses should be hrther diluted and reanalyzed. 13.0 Acceptance Criteria 13.1 chromatogrunmust show a peak of a daughter ion at 369 a m u from a parent of 413 amu. The 413 a m u parcnt corresponds to the PFOA anion. while thc daughterion (369 mu) represent8 the loss of carbon dioxide. 13.2 Method blanks must not contain PFOA at levels greater than the LOQ. If a blank contah PFOA at levels g r a t a than 0.2 nglmL, then a new blank sample must be obtained and the entire set must be re-extracted. 13.3 Recoveries of control spikesand matrix spike8 must be between 70-130% of their known vllues. If a control spike falls outside the acceptable limits, the entire set of samples should be re-attracted. Any matrix spike outside 7013Wh should be evaluated by the analyst to determine if re-extraction is wmted. 13.4 Any calibration standard found to be a statistical outlier by using the Huge Error Test, may be excluded h m the calculation of the calibration curve However, the total number of extracted calibration standards that could be cxcludcd must not exceed 2Wo of the total number of extracted standards injected. 13.5 The cornlation coefficient (R) for calibration curves generated must be 20.992 (R' 20.985). If calibration results fall outside these limits. thcti appropriate steps must be taken to adjust instrument operation. and the standardsor the relevant set of samplesshould be reanalyzed. 05-0210 lnterim Report #5 Page 35 of65 Page 89 of 123 3M Environmental Laboratory Project No. 05-02 10 Exygen Protocol Number: PO00I I3 I c 13.6 Retention times between standards and samples must not drift more ihaii f 4 YOwithin an d y t i c a l run. Ifretention time drift exceeds this limrr within an pnslyticpl runthen the set muat be reanalyzed. 14.0 Calculations 14.1 Usc the followingequation to calculatethe amount of PFOA found (in ng/mL. b a d on peak uea) using the standard curve (linear regression parameters) generatedby the Mors Lynx softwareprogram: PFOA found (n-) - =IpcaLarea intercent) x DF slope DF = factor by which the tinal volume was diluted. if necessary. 14.2 For s p m p l ~fortified with known mmts of PFOA prior to extraction. use R e - the following covay (Vi) equation to calculate the pcrccnt recovery. I total analytefound( n m ) - d y t e foundin control(ng/mL)l ,oo analyte added (ng/mL) 14.3 Use the followingequation to convert the amount of PFOA found in ng/mL Io ns/e @Pb). PFOA found @Pb)=IpFOA found (nn/mU x final volume I5 mL)l m p l e weight (5 g) 14.4 Use tbe following equation (if ncceaspry) to ~&dath~e amount of PFOA found in ppb based on dry weight. PFOA found @pb) dry weight = PFOA found @pb) x [loOslo I total solids(%)l 05-0210 Interim Report #5 Page 36 q/ 65 Page 90 of 123 3M Environmental Laboratory Project No. 05-0210 Exygen ProtocolNumber: PO001131 ANALYTICAL METHOD McthodNumber: V0001783 Method of Anrlyab for the Determlnatlon of PemuoroortDnoic Acid (PFOA) in Fish and C h m by LCMSMS Analytical Testing Facility: Exygen Research 3058 Research Drive State College, PA 16801 Approved By: T J CAR Paul Conaolly Technical Lcsdcr, E - M S , Exygen Reseprch /MY Date 'Vice President, Operations,Exygen Research . .- E05-0210 Interim Report #5 TotalPagcs: 8 Page 91 of 123 3M Environmental Laboratory Project No. EO5-0210 ExygenProtocol Number:PO001131 .- Exygen Research MetbodNumber V0001783 I 1 AKwLYTICAL METHOD Method of Analysis for the Deanof PerfluorooctanoicAcid (PFOA) in Fish and Clams bv LCIMSIMS 1.0 scope This method is to be mployed for the isolation and quantitation of pertluorooctanoic acid by High Performance Liquid chromatography coupled to a tandem Mass SpectrometticDetector (LC/MS/MS)in fish and clams. 2.0 safety 2.1 Always observesafe laboratorypractices. 2.2 Consult the appropriateMSDS before handling any chemical for proper safetr. proalutions. 3.0 Sample Requinment 3.1 At IM 20gof tcst sample for extraction. 3.2 Samples should be ~ I U C C S bPe~fore extraction. Place the frozen sample in a food processor and homogenize with dry ice. Place the samples in containers and leave open in from storage overnight to allow for carbon dioxide sublimation. Seal and place the m p l c s in frozen storage until time of analysis. 3.3 Sample collection procadures will be specified in the sampling plan for this project. 4.0 RcagcntsdStaudarda - 4.1 Wata-HPLCgrode 4.2 Ac~tonihi-l~HPLC @e 4.3 Carbon (12O-400 mesh) Rcagent grade 4.4 M ~ t h m l -HPLCgrade 4.5 Silica gel (60-200mesh)-Reagent grade 4.6 Florisil(60-100 mesh) - Reagent grade 4.7 Superclean LC-NHZ- Reagentgrade 4.8 I-Oetul~l-HPLC grade - 4.9 L-Ascorbic acid Reagent me - 4.10 Dimethyldichlorosilanne Reagent grade 4.11 TO~UU-ICR a g a t grade 4.12 Ammoni~mAcaSte - A.C.S. Reagent Grade 4.13 Pdwrooctanoic Acid - Sigma-Aldrich 5.0 lnrtrument and Equipment 5.1 A high performance liquid chromatograph capable of pumping up IO 2 solvents equipped with a variable volume injector capable of injecting S-XM pL connectod to a tandem Mass Spectrometer (LCIMSMS). 5.2 A device to collCa raw data for peak integrationand quantitation. 5.3 Analytical balance capable of reading to O.oooO1 g. Page 2 018 05-0210 Interim Report #5 . Page 92 of 123 3M Environmental Laboratory Project No. 05-0210 ExygenProtocol Number: PO001131 Exyscn RcKuch Method Numkr V0001783 1 1 ANALITICAL METHOD Method of Analysis for the Determinationof PerfluorooctanoicAcid (PFOA)in Fish and Clams by LcIMS/MS -. 5.4 Rotary evaporator. 5.5 TissumiZCK 5.6 125 mL pear-shapedflasks. 5.7 50 mL disposablepolypropylenecentrifugetubes. 5.8 15 mL disposablepolypropylenecentrifugetubes. 5.9 Disposable micropipets (50-1OOuL. 100-2OOuL). 5.10 125-mL LDPE narrow-mouth bottles. 5.11 2 mL clear HPLC vial kit. 5.12 Disposable pipcttcs. 5.13 Autopipatcr (lOelO00 pL and 10-100pL), with disposable tips. 5.14 SPE tubes (2OmL)(Supelcocat. no. NOS7177). 5.15 Wrist action shaker. 5.16 Centrifugecapableof spinning 50 mL polypropylenetubesat 2000 rpm. 6.0 Cbromatogqbic System 6.1 AnslylicalColumn:FluopheJeRF' (Keystone Scientific), 2.1 mm x SO mm. 511 (PIN: 82505-052130) 6.2 Tan-: 30'C 6.3 Mobile Phase (A) : 2 mM Ammonium Acetate in Water 6.4 Mobile Phase (B) : Methanol 6.5 Gradientprogram: Iid3J.u %A 0.0 65 I .O 65 8.0 25 20.0 25 22.5 65 Flow Rate %Bo 35 0.3 35 0.3 75 0.3 75 0.3 35 0.3 - 6.6 Injection Volume: I5 pL (can be increased to as much as 50 pL). 6.7 Quantitation: Peak Area - external standard calibration curve. 6.8 Run Time: 23 minutes. The above conditions are intended as a guide and may be changed in order io optimize the HPLC system. 05-0210 Interim Repott #5 Page 39 of 65 Page 93 of 123 3M Environmental Laboratory Project No. 05-0210 Exygen Protocol Number: PO001 13 I .- Exygm Rerearch L Method of Anal* Method NumberVOOO1783 I ANALYTICAL METHOD for the Ddmnination of Pduorooctanoic Acid (PFOA) in Fish and Clams by LCIMSIMS -. . 7.0 MS/MSSystan 7.1 Mode: Electrospray Negative MRM mode, monitoring 413 + 369 m u for PFOA. The above conditionsare intended as a @de and may be changed in order to optimize the MSMS system. 8.0 Reparationof Solutions 8.1 Mobile Phase 8.1.1 2 mM ammonium acetate in water is prepared by adding 0.154 g of ammoniumacetateto IO00mL of water. 8.2 Extraaion Solutions 8.2.1 2%ascorbic acid in methanol is prepared by dissolving 2 g of ascorbic acid in 100 mL of methanol. 8.2.2 3099 Dimcthyldichlorosilane in toluene is prepared by bringing 3 inL ofdimethyldichlorosilaneto a final volume of 10 mL with toluene. Altanate volumes may be prepared. 9.0 StandardPrcparation 9.1 Standard StockEortificationSolution 9.1.1 9.1.2 9.1.3 9.1.4 9.1.5 Repam a stock solution of -100 pg/mL of PFOA by weighing I O rng of analfical stsndard (comcted for purity) and dilute to 100 mL with methanol in a 125-mL LDPE bottle. A 1.O pg/mL fortification solution of PFOA is prepared by bringing I mL of the 100 p g h L sohtion to a final volumc of 100 with methanol in a 125 mL LDPE bottle. A 0.1 pdmL fortificationsolution ofPFOA is prepared by bringing 1 0 mL ofthe 1.0pg/mL solution to a find volume of 100 with methanol iii a 125 mL LDPE bottle. A 0.01 pg/mL fortification solution of PFOA io prepared by bringing 10 mL of the 0.1 pg/mL solution to a final volumc of 1 0 0 with methanol in a 125 mL LDPE bottle. The stock and fortificationsolutions arc to be stored in a refrigerator ai approximately 4'C and M stable for a maximum period of 6 months fmm the datc of preparation. 05-02 10 Interim Report #5 Page 94 of 123 3M Environrnental Laboratory Project No. E05-0210 Exygen Protocol Number: PO00113t ExygcnM a r c h Memod Numbcr V0001783 L 1 ANALkTICM. METIOD Method of Analysis for the Determination of PerfluorooctanoicAcid (PFOA) in Fish and Clams by LC!/MS/MS 9.2 Standard CalibrationS O ~ U ~ ~ O M 9.2.1 LLYMSRdS calibrationstandardsare prepared in methanol via dilution of the 1.0 pgmL fortificationsolution. 9.2.2 The following is a typical example: additional concentrations may be prepared as needed. Concentration Final of Fortification Volume Diluted to Concentration SOlUtion(&nL) (mL) (mL) 1 .o 5.0 100 1 .o 2.5 100 1 .o I.o 100 (IrdmL) 0.0s 0.025 0.01 0.05 10 100 0.00s 0.025 10 100 0.002s 0.1 10 100 0.001 0.005 10 100 o.Ooo5 9.2.3 Store dl calibration standards in 12s-mL LDPE narrow-mouth bottles at ZC to 6OC, up to six months. 9.2.4 Alternate volumes and c0ncenIration.s of standards may be prepared as needed. 10.0 Batch Set Up 10.1 Each batch of rampla extracted (typically 20 or Ius) must include at least one untreated control and two untreated controls fortified at known concentration8 (lab control spile) to verify proceduralrecoveryfor the batch. 10.2 Rquircments for field and laboratory duplicates and spikes will be specified in the quality assuranceplan for this pmjcct. 11.0 Sample Extraction 11.1 Weigh 5 g of frozen sample into 50 mL polypropylene centrifuge tubes (fortify aa needed, replace lid and mix well). 11.2 Add 30 mL of acetonitrileand shakeon a wrist action shaker for -IS minutes. 11.3 Place the Nbes ina freaer for -1 hour. 1 1.4 Pack and condition the SPE tu& and silanize the pear-shaped flasks. 11.5 Pack the 20 mL SPE tubes in sequence with 2 g florisil, 2 g silica gel, 2 1: carbon. and 1 g LC-NH,. Condition the columns with 20 mL of methanol. then 20 mL of acetonitrile. Discard all warha. Do not allow the column IO dry. 11.6 Silanize the 125 d pear-shaped flasks by rinsing with the 30% dimctbyldichl~ilanein toluene solution. Rinse the flask with toluene once. followed by methanol (thrcc times). Dry the flasks completely before use. either by air-drying or with a stream of nitrogen. 05-0210 interim Report #5 Page 41 o j 6 5 -~ Page 95 of 123 3M EnvironmentalLaboratory Project No. 05-0210 ExygenProtocol Number: PO001I3 1 Exyga Rncucb M e W N u m b n V0001783 L I ANALITICAL MKTHOD Methodof Analysis for the Determination of Pduomoctanoic Acid (PFOA) in Fish and Clams by LC/Ms/MS 11.7 11.8 11.9 11.10 11.11 11.12 11.13 11.14 11.15 11.16 11.17 11.18 Centrifuge the 50 mL polypropylene tubes containing sample at -2000 rpni for -10 minutes. Decant the extract on to a conditioned SPE column fitted inside the mouth of the pear-shaped flask. Collect the eluate in the 125 mL silanized pear-shape flask. Add 10 mL of acctonitde to the sample in the 50 mL centrifuge tube. Homogenize the frozen fat phase using a tissumizer for -30 reconds and rinsc the tissumizerwith-10 mL of acetonitrileinto the tube. - Shakethe sample again for -10 minuteson a wrist-action shaker. Place the tubes in a frraa for 1 hour more. Ccntrifbge the 50 mL polypropylene tubes containing sample at -2000 rpni for -10 minutes. Decant the extract onto the m e SPE column. Collect the eluate into the same pear-shaped flask and combine with the eluent from the initial CXtractiOn. Pass 20 mL. of acetonitrile through the SPE column and combine the eluate in the lame pe~r-~hnpefldask. Add 3-4 drops of l-octanol to the extract in the pew-shaped flask and evaporate at reduced pressure using a rotary evaporator (at < 4OOC). Make the final volume, by adding 2 of 2% ascorbic acid in methanol io the p~r-shapcdflask and swirl to m i X / d i ~ ~ o l ~ ~ . Transfcr the extracts to HPLC vials using disposable pipas. Analyze samples using electrospray LCIMSIMS. 12.0 Chromatography 12.1 Inject the same amount of each standard. sample and fortified sample into the LClMSlMS system. A calibration stpndard must precede and follow all analyzed samples. 12.2 Standardsof PFOA correspondingto at least five or more concentrationlevels must be included in an analytical set. 12.3 An entire set of calibrationstandards must be included at the beginning and ai the end of a sample set. Standards must be inteqersed between every 5 - ID samples. As an alternative, an entire set of calibration standards may be injected at the beginning of a set followed by calibration standards intrrspascd every 5-10 samples (to account for a sccond set of standards). In either case. calibration standards mu8t be the first and last injection i r il sample w. 12.4 Use linear standard curves for quantitation. Linear standard curves are genaated for the analyte by linear regressionusing I/xweightingof peak ana versus calibration standard concentration using MassLynx 3.3 (or equivalent) software system. Page 6 ui8 05-0210 Interim Repott #5 Page 42 o j 6 5 - Page 96 of 123 3M Environmental Laboratory Project No. 05-0210 Exygen Protocol Number: PO001 13 I ,.- Exygen Rewch Method Number VooO1783 I I ANALYTICAL METHOD Method of AnalyEis for the Determination of Pduorooctanoic Acid (PFOA) in Fish and Clams by L.CiMS/MS 12.5 Sample response should not exceed standard responses. Any samples ihal exceed standnrd responses should be further diluted and reanalyzed. 13.0 Acceptance Criteria 13.1 chroma to^ must show a peak of a daughter ion at 369 amu from a parent of 413 am& Tbe 413 amu parent corresponds to the PFOA anion, while the daughter ion (369 mu) represents the lossof carbon dioxide. 13.2 Method blanks must not contain PFOA at levels greater than the LOQ. If B blank contaim PFOA at levels greater than 0.5 ppb, then a new blank sample must be obtained and tk entire set must be n-extracted. 13.3 Rccovaies of control spikes and matrix spikes must be between 70-130% of their known values. If8 control spike falls outside the acceptable limits. the entin ret Of spm~leshould be n-atracted. 13.4 Any calibration standard found to be a statistical outlier by using the Huge E m r Ttst, may be excluded from the calculation of the calibration curve However, the total number of calibration standards that could be excluded must not exceed 20% of the total number of standards injected. 13.5 The corrclatim coefficient (R) for calibration curves generated must be 20.992 (R* 20985). If calibration resub fall outside these limits. then appropriate steps must be taken to adjust instrument operation. and the stsndardpor the relevant set of samplesshould be reanalyzed. .-..- 13.6 Retention times between standards and samples must not drift more than f 4 % within an analyticalrun. If retention time drift exceeds this limit within an analytical run then the set must be reanalyzed. 14.0 Calculations 14.1 Use the followingequation to calculatethe amount of PFOA found (in nglniL. based on peak prep) using the standard curve (linear regression parameters) generated by the M w Lynx soAwareprogram: PFOA found (ng/mL) = Lpcak area - intercat) slope 14.2 Use the f o l l o e~quation to convert the amount of PFOA found in n g l d IO W'g Wb). PFOA found @pb)=a a l v lume x F sample weight (9) DF = factor by which tk find volume was diluted, if necessary 05-0210 Interim Report #5 Page 43 of155 -- Page 97 of 123 _- c 3M Environmental Laboratory Project No. EO5-0210 Exygen Protocol Number: PO001 13 I Exygea Racueh Merhod Number V000178f I 1 AMl.\T'IC.U METHOD Method of Analysis for the Damnhation of PcrfluorooctanoicAcid (PFOA) in Fish and Clams by LCIMSIMS 14.3 For samples fortified with known amounts of PFOA prior lo extraction. use the following equation to calculate the percent recovery. Rccovery (%) = Itotalanalytcfound(nglg) - d y l efoundin control (ng/g)] ,oo d y t e d3.d W e ) c 05-0210 Interim Report #5 Page 8 v i x Page 44 of 65 Page 98 of 123 3M Environmental Laboratory Project No. 05-0210 Exygen Protocol Number: PO001131 ANALYTICAL METHOD Method Number: V0001784 Metbod of Analysis for the Determination of PerfluorooctanoicAcid (PFOA)in Vegetation by LCIMSIMS Analytical Testing Facility: Exygen Research 3058 Research Drive State College, PA 16801 Approved By: Paul Connolly \ T~hnicaLl -c~cx,LC-MS,E x y e R a m h nbhn Flnhcrtv ' 'Vice Presidck, Operations, Exygen Rtxarch 05-0210 Interim Report #5 -_ TotalPagu: 7 Page 45 of 65 -- Page 99 of 123 3M Environmental Laboratory Project No. 05-0210 Exygen ProtocolNumber:PO001 131 Exygcn R-h Method Numbcr \.MW)I'84 t 1 AXALL"I'1CALMETHOD I Method ofAnalysis for the Determination of Pcrfluomtanoic Acid (PFOA) in Vegetation by LCIMStMS 1.0 scope This mcthod is to be employed for the isolation and quantitation of pcrfluomtanoic acid by High Performance Liquid Chromatography coupled to a tandem Mass SpectrometricDetector (LC/MS/MS) in vegetation. 2.0 safety 2.1 Always obsave safe laboratorypractices. 2.2 Consult the appmpriateMSDS before handling any chemical for proper safely precautions. 3.0 Sample Requirement 3.1 At least 20 g oftest samplefor extraction. 3.2 Samples should be processed before extraction. Place the frozen sample in B food processor aod h0mogeniz.c with dry ice. Place the samples in containers and leave o p in from storage overnight to allow for carbon dioxide sublimation. Seal and place the samples in frozen storage until time of analysis. 3.3 Sample collection procedures will be specified in the sampling plan for this project. 4.0 ReagcntsandStrmdsrdr 4.1 Water-HPLCgradc 4.2 AC~tOnitri-l~HPLC grade 4.3 Carbon (12O-400 mesh) - Reagent grade 4.4 Methanol - HPU: @e 4.5 Silica gel (60-200mesh)- Reagentgrade 4.6 Florisil (&lo0 mesh) - Reagent grade - 4.7 SUPCICICMLC-NHz R w m t grade 4.8 I-OCtan~l- HPLC grade - 4.9 L-Ascorbic acid Reagent grade 4.10 Dimahyldichlomiilane- Reagent grade 4.11 TO~UUI-CReagent grade 4.12 4.13 - Ammonium Acetate- A.C.S. Reagent Grade PafluomoctanoicAcid Sigma-Aldrich 5.0 Instrument and Equipment 5.1 A high performance liquid chmatograph capable of pumping up to 2 solvents equipped with a variable volume injector capable of injecting 5-200 1Lconnected to a taudmMass Spectmmettr(LCIMSIMS). 5.2 A device to collect raw data for peak integrationand quantitation. 5.3 Analytical balance capableofreading to 0.00001 g. 05-02 10 Interim Report #5 Page 46 of65 Page 100 of 123 3M Environmental Laboratory Project No. 05-02 10 Exygen Protocol Number: PO001 131 Exmen Rncueh Melhod Number VOOO I784 Method of Analysis for the Determination of PeffluorooctanoicAcid (PFOA) in Vegetation by LCIMSIMS 5.4 Rotary evaporator. 5.5 125 mLpea-sh?pcd flasks. 5.6 50 mL disposablepolypropylene centrifugeNbes. 5.7 15 mL disposablepolypropylene centrifugetubes. 5.8 Disposablemicmpipets(50-100uL.100-2OOuL). 5.9 125-mL LDPE narrow-mouth bottles. 5. IO 2 mL clear HPLC vial kit. 5.1'1 Disposablepipettea. 5.12 Autopipata (1OO-lOOOpL and 10-100 pL), with disposable tips. 5.13 SPE tubes(2OmL) (Supelcocat. no. N057177). 5.14 Wrist action shaka. 5.15 Centrifugecapableof spinning50 mL polypropylenetubes at 2000 rpm 6.0 ChromatopphicSystan 6.1 Analytical Column: Fluopharc RP (Keystone Scientific), 2.1 mrn x 50 rnm. 511 ( P N 82505-052130) 6.2 Tcmpcraturc: 30C 6.3 Mobile Phase (A) : 2 mM Ammonium Acetate in Water 6.4 Mobile Phase (E): Methanol 6.5 Gradient Program: Time fmin) 0.0 1 .o 8.0 20.0 22.5 %A 65 65 25 25 65 a 35 35 75 75 35 Flow Rate fmllmin) 0.3 0.3 0.3 0.3 0.3 6.6 Injection Volume: I5 pL (canbe increased to as much as 50 pL). 6.7 6.8 - Quantitation: Peak Area -external Run Time: 23 minutes. standard calibration curve. The above conditionsut intendedas a guide and may be changed in order to optimize the HPLC system. 7.0 MS/MSSystem 7.1 Mode: Electrospray Negative MRM mode, monitoring 413 + 369 m/z for PFOA. E050210 Interim Report #5 Page 47 01'65 . ___ Page 101 of 123 3M Environmental Laboratory Project No. 05-0210 Exygen Protocol Number: PO001131 Exygcn Rerurch Method Number VOW I784 I I AKALYTICtkL WTHOD Mahod of Analysis for the Determination of Pduorooctanoic Acid (PFOA) in Vegetation by LCJMSIMS The aboveconditionsarc intended as a guide and may be changed in order to optimize the MSMS system. 8.0 Preparation of Solutions 8.1 MobilePhasc 8.1.1 2 mM ammonium acctnte in water is prepared by adding 0.154 g of ammoNumacetateto loo0 mL of water. 8.2 Extraction Solutions 8.2.1 2%ascorbicacid in methanol ispreparedby dissolving 2 g of ascorbic acid in 100 mL of methanol. 8.2.2 300h Dimcthyldichlorosilanein toluene is prepared by bringing 3 mL of dimethyldichlorosilaneto a tind volume of 10 mL with toluene. Altanate volumes may be prepared. 9.0 StandardPrcparation 9.1 Standard StocWFortificationSolution .- 9.1.) Prepare a stock solution of-100 pdmL of PFOA by weighing IO nig of analytical standard (comctcd for purity) and diluteto 100 mL with methanol in a 125-mL LDPE bottle. 9.1.2 A 1.0 KghL fonification solution of PFOA is prepared by bringing I mL of the 100 pg/mL solution to a final volume of 100 with methanol in a 125 mL LDPE bottle. 9.1.3 A 0.1 pg/mL fortification solution of PFOA is prepared by bringing IO mL ofthe 1.0 p&lmLsolution to a final volume of 100 with methanol in a 125 mL LDPE bottle. 9.1.4 A 0.01 pg/mL fortification solution of PFOA is prepared by bringing 10 mL of the 0.1 pg/mL solution to a final volume of 100 wttli methrnol in a 125 mL LDPE bottle. 9.1.5 Thc stock and fortificationsolutionram to be stored in a refrigeratora[ approximately 4'C and arc stable for a maximum period of 6 months from the &e ofpreparation. 9.2 Standard Calibntion Solutions 9.2.1 LC/Ms/MS calibrationrt.ndPrds arc prepared in methanol via dilution of the 1.O pg/mL fortificationsolution. A EO5-0210 Interim Report #5 Page 48 of65 Page 102 of 123 3M Environmental Laboratory Project No. 05-0210 Exygen Protocol Number: PO001 13 1 c Ex.m-en F&scucb MahodNumber V0001784 I I AhALYlI'ICAL ME'MOD Method of Analysis for the Determination of PerfluorooctanoicAcid (PFOA) in Vegetation by LC/MS/MS 9.2.2 The following is a typical example: additional concentrations may be prepared as needed. Conccntntion Final of Fortification Volume Diluted to Conmuation Solution be'mL) 1 .o I .o 1 .o (d) 5.0 2.5 1 .o (Id) 100 100 100 (pdd) 0.05 0.025 0.01 0.05 10 100 0.005 0.025 10 100 0.0025 0.1 10 100 0.001 0.005 10 100 o.oO05 9.2.3 Store all calibration standards in 125-mL LDPE narrow-mouth bottles at 2'C to 6C. up to six months. 9.2.4 A l m t e volumes and concentrationsof standards may be prepared as needed. 10.0 Batch Set Up 10.1 Each batch of sampla extracted (typically 20 or less) must include at least one untreated control and two untmtcd controls fortified at known concentrations(lab control spike)to verify proceduralrecovery for the bauh 10.2 Requirements for field and laboratory duplicates and spikes will be specilied c in the quality assuranceplan for this project. 11.O SampleExtraction 11.1 Weigh 5 g of frozen sample into 50 mL polypropylene centrifuge tubes (fortifyMneeded, replace lid and mix well). 11.2 Add 30 mLof acetonitrileand shakeon a Wrist action shaker for -15 minutes. 11.3 Centrifuge the 50 mL polypropylene tuba containing sample at -2000 rpin for -10 minutes. 11.4 Pack and condition the SPE tubed and silanize the pear-shaped flasks. 11.5 Pack thc 20 mL SPE tubes in sequence with 2 g florisil. 2 g silica gel. 2 I: carbon. and 1 g LC-NHz. Condition the columns with 20 mL of methanol. then 20 mL of acetonitnle. Discard all washes. Do not allow the column to dry. 11.6 SilnnizC the 125 mL --shaped flasks by nnsing with the 30% dimcthyldichlomiImein toluene solution. Rim the flask with tolucnc oncc. followed by methanol (three times). Dry the flasks completely before use. either by airdrying or with a stream of nitrogen. 11.7 Dccant the extract on to a conditioned SPE column fitted inside the mouth 01' the par-shapedflask. Collect the eluste in the 125 mL silanized pear-shape flask. E050210 Interim Report #5 Page 49 of 65 --_ Page 103 of 123 3M Environmental Laboratory Project No. 05-0210 Exygen Protocol Number: PO00I I3I Exyscn Rcscucb Mcrbod Number VOWI:U4 AhALYrlCAL MUHOD 1 Method of Analvsis for the Detaminationof Perfluomtanoic Acid (PFOA) in Vegeraiion by LCIMSIMS 11.8 11.9 11.10 11.11 11.12 11.13 11.14 11.15 11.16 Add 20 mL of acetonitrile to the samplein the 50 mL centrifuge tube. Shakethe sampleagain for -10 minutes on a wrist-actionshaker. Centrifuge the 50 mL polypmpylcne tubcs containing sample at -2000 rpm for -5 minutes. Decant the extract onto the same SPE column. Collect the eluate into the same pear-shaped flask and combine with the eluent from the initial extraction. Rcpcat steps 11.8 h u g h 11.11 again. Add 3-4 drops of l0cton01 to tht uttract in the pu-shaped flask and evaporate at reduced pres- using a rotary evaporator (at e 40'C). Make the final volume. by adding 2 mL of 2% ascorbic acid in methanol IO the par-shapedflask and swirl to mix/dissolve. Transfer the extracts to HPLC vials using disposable pipets. Analyze samples using electmspray LC/MS/MS. 12.0 Chromatography 12.1 Inject the same amount of each standard, sample and fortified sample into the LC/MS/MS system. A calibration standard must precede and follow all analyzed samples. 12.2 Standardsof PFOA correspondingto at least five or more concentra~ionlevels must be included in an analyticalset 12.3 An entire set of extracted calibration standards must be included at the beginning and at the end of a sample set. Extracted standards must hc interspersed between every 5-10 samples. As an alternative, an entire set of extracted calibration standards may be injected at the beginning of a set followed by extracted dibration standards interspcrscd every 5-10 samples (to account for a second set of extracted standards). In either case. extracted calibration stadads must be the first and last injection in a sample set. 12.4 Use linear standard curves for quantitation. Linear standard curves are generated for the analytc by linear regressionusing I/xweighting of peak area versus calibration standard concentration using MassLynx 3.3 (or equivalent, software system. 12.5 Sample response should not exceed standard responses. Any samples that exceed standard responsts should be M e r diluted and reanalyzed. 13.0 Acccptancc Criteria 13.1 Chromatogram must show a peak of a daughter ion at 369 amu b m a pareni of 413 mu The 413 m u parent comsponds to the PFOA anion. while the daughter ion (369 amu) representsthe loss of carbon dioxide. E050210 Interim Report #5 Page 50 of 65 - Page 104 of 123 3M Environmental Laboratory Project No. 05-02 70 Exygen Protocol Number: PO001 13 1 ExypRcroush hielbod Number VOW I784 I I a - ASALYTICAL METHOD Method ofhalysis for the Danmination of PerfluomtanoicAcid (PFOA) in Vegetation by LC/MSIMS 13.2 Method blanks must not contain PFOA at levels greater than the LOQ. If a blank containsPFOA at levels greater than 0.5 ppb. then a new blank sample must be obtained and the entire set must be re-extracted. 13.3 Rceovcries of control spikes and matrix spikes must be between 70-130% 01' their known valuu. If a control spike falls outside the acceptable limits, the entire set of samples should be re-extracted. 13.4 Any calibration standard found to be a statistical outlier by using the Huge Enor Tut, may be excluded from the calculation of the calibration curve. However, the total number of calibration standards that could be excluded must note x d 20% of the total numtm of standards injected. 13.5 The cornlation cocfficicnt (R) for calibration curves gencrited must he 20.992 (R' 20.985). If calibration rcsultr fall outside these limits. then appropriate steps must be takcn to adjust instrument operation. and the standardsor the relevant set of samples should be reanalyzed. 13.6 Retention times between standard0 and samples must not drift more than f 4 %within m malytical run. Ifretention time drift exceeds this limit within an analytical run then the set must be reanalyzed. 14.0 Calculations 14.1 Use the following equation to calculate theamount of PFOA found (in nJniL, based on peak area) using the standard curve (linear regression parameters) generated by the Mass Lynx software program: - PFOA found (ng/mL)=peakarca intercat) slop 14.2 Use the following equation to convert the mountof PFOA found in ng/mL to n%e @PW. PFOA found(ppb)-=p x fml volume (mL)x DF] sample weight (g) DF = factor by which the final volume was diluted, if necessary. 14.3 For samples fortified with known amounts of PFOA prior to extraction. use the following equation to calculate the pcrcmt recovery. RUXVCIY(Yo) total d y t e found(ng@ - analyte found in control(ng/g)l I oo analyteadded (ng/g) ,- 05-0210 Interim Report #5 Page 705of 723 3M Environmental Laboratory Project No. 05-0210 Exygen Protocol Number: PO001 13 I ANALYTICAL METHOD Method Number: VOOOl785 Method of Analysis for the Determlnatlonof PerlluorooctanoicAcid (PFOA) in Small Mammal Liver by LCMSMS Analytical Testing Facility: ExygenResearch 3058 Research Drive State College.PA 16801 Approved By: 16hn FlaheItV ' /Vice Presideht, Opantions, Exygen Rescarch IO\Wl04 Date /??Ai Date c EO5-0210 Interim Report #5 TotalPage3: 7 Page 52 of65 -- - Page 106 of 123 3M Environmental Laboratory Project No. E050210 Exygen Protocol Number: PO00113 1 Exygen R-h M o w Number VUWl78S 1 1 MALYTICAL METHOD Method of Analysis for the Determination of PerfluorooctanoicAcid (PFOA) in Small Mammal Liver by LCIMS/MS _. 1.0 scope This method is to be anployat for the isolation and quantitation of perfluorooctanoic acid by High Performance Liquid Chromatography coupled to a tandem Mass SpectrometricDetector (LUMSMS) in small mammal liver. 2.0 safety 2.1 Always observe safe laboratory practices. 2.2 Consult the appropriate MSDS before handling any chemical for proper safely precautions. 3.0 Sample Requirement 3.1 At least 5 g of test sample for extraction. 3.2 Samplesshould be processed before extraction Place the frozen sample in a food processor and homogenize with dry ice. Place the samples in containers and leave opcn in frozen storage overnight to allow for carbon dioxide sublimation. Seal and place the samples in frozen storage until time ol` analysis. Alternately, if thae is an insuffkient mount of sample (-less than 5 g), then no processing is necessary and the sample can be used as supplied. 3.3 Sample collcetion procedures will be specified in the sampling plan for this project. 4.0 Reagents and Standards 4.1 Woter-HPLCme 4.2 Methanol - HPLC @C 4.3 Acetonitrile - HPLC grade 4.4 Ammoni~mAC-te - A.C.S. h g m t Grade 4.5 PerfluorooctanoicAcid - Sigma-Aldrich 5.0 Instrument and Equipment 5.1 A high pdonnance liquid cbmatograph capable of pumping up IO 2 solvents equipped with a variable volume injector capable of injecting 5-?0(1 pL connectedto a tandem Mass Spcctrometcr(LC/MS/MS). 5.2 A device to collect raw data for peak integration and quantitation. 5.3 Analytical balance capable of reading to O.oooO1 g. 5.4 50 mL disposablepolypropylene centrifugetubes. 5.5 IS mL disposablepolypropylenecenuifbge tubes. 5.6 Disposable micropipets (5&1ooul, 100-2OOuL). 5.7 125-mLLDPE m w - m o u t h bottles. 5.8 2 mL clear HPLC vial kit. - _-- 05-0210 Interim Report #5 Page 53 01'65 - Page 107of 123 3M Environmental Laboratory Project No. 05-0210 Exygen Protocol Number: PO001 13I Exypn Resarsh Method Number VooOl785 I 1 Mn'AiYTICALMETHOD Method ofAnalysis for the Determination of PcrfluorooctanoicAcid (PFOA) in Small Mammal Liver by Lc/MsIMS 5.9 Disposable pipettes. 5.10 Autopipettes (100-1OOOgL and 10-100 pL), with disposable tips. 5.11 Waters Scp Pak Vac 6 cc (Ig) tC18 SPE cartridges. 5.12 SPE vacuum manifold. 5.13 Tissuemiza. 5.14 Wrist-action &aka. 5.15 Centrihge capableof spinning 15 mL polypropylenetubes at 3000rpm. 6.0 ChromatographicSystem 6.1 Analytical Column: FllLophPseRP (KeystoneScientific), 2.1 mm x 50 mm. 511 ( P N 82505-052130) 6.2 Temperature: 30'C 6.3 Mobile Phasc (A) : 2 mM Ammonium Acetate in Water 6.4 MobilePhare(B) : Methanol 6.5 Gradient Program: Time (mi@ !a 0.0 65 1.o 65 8.0 25 20.0 25 22.5 65 a 35 35 75 15 35 Flow Rate fmUmin) 0.3 0.3 0.3 0.3 0.3 6.6 Injection Volume: I5 pL (can be increased to as much as 50 pL). 6.7 6.8 - Quantitation: Peak Area - Run Time: 23 minutes. mtanal standard calibration curve. The above conditim an intended as a guide and m y be changed in order to optimizethe HPLC system. 7.0 MSMSSystem 7.1 Mode: ElectsosprayNegativeMRMmode. monitoring413 -+369 mlz for PFOA. The above conditions are intmded as a guide and may be changed in order IO optimize the MSMS ayrtan. J 05-0210 Interim Report #5 PageS4of65 Page 108 of 123 3M Environmental Laboratory Project No. E050210 Exygen Protocol Number: PO00113 1 Exygen Rcsurch Method Number V0001785 I AN:LYTICAL METHOD ~~ 7 Method of Analysis for the Determinationof Perfluorooctanoic Acid (PFOA) in Small Mammal Liver by LC/MSIMS 8.0 Preparationof Solutions 8.1 Mobile Phase 8.1.1 2 mM ammonium acetate in water is p r c p a d by adding 0.154 y oE ammonium acetateto 1000 mL ofwater. Alternate volumes may be prepared. 9.0 StandardPrepamtion 9.1 Standard StoddFortification Solution 9.1.1 Prepare a stock solution of -100 pumL of PFOA by weighing IO m g of analytical standard (corrected for purity) and dilute Io 100 mL with methanol in a 125-mLLDPE bottle. 9.1.2 A 1.0pdmL fortificationsolution of PFOA is prepared by brinyiny I mL of the 100 pdmL solution to a final volume of 100 with methanol in a I25 mL LDPE bottle. 9.1.3 A 0.1 pdmL fortificationsolutionof PFOA is p r c p a d by bringing Ill mL ofthe 1.0 pdmL solutionto a final volume of 100 with methanol in a 125 mL LDPE bottle. 9.1.4 The stock and fortificationsolutionsarc to be stored in a refrigerator at approximately4'C and am stable for a maximum period of 6 months from the date of preparation. 9.2 Standard Calibration Solutions 9.2.1 LCIMSIMScalibration standards arc prepared in methanol via dilution of the 0.1 pglmL fortification solution. 9.2.2 The following is a typical example: additional concentrations may he prcparcd as needed. Concentration Final of Fortitication Volume Diluted to Concmbation Solution (ndmL) (mL) (mL) (ng/mL) 100 5.0 100 5.0 100 2.0 100 2.0 100 1.o 100 I .o 5.0 10 I00 0.5 2.0 to I00 0.2 1 .o 10 100 0.1 9.2.3 Store all calibration standards in 125-mL LDPE narrow-mouth bottles at 2C to 6OC, up to six months. 9.2.4 Altanate volumes and concmlrations of standards may be prcpaml as needed. Page 4 of 7 .-. __ 05-0210 Interim Report #5 Page 5.5 of bS Page 109of 123 3M Environmental Laboratory Project No. 05-0210 Exygen Protocol Number:PO00113 1 Exypcn Research Method Number VOOO I765 L I Ah.WYTICAL METHOD Method ofAnalysis for the DeterminationofPerfluorooctanoic Acid (PFOA) in Small Mammal Liver by LC/MS/MS -- 10.0 Batch Set Up 10.1 Each batch of samples extracted (typically 20 or Icss) must include at least one untreated control and two untreated controls fortified at known concentrations(lab control spike)to vcrifyprocedural recovery for the batch 10.2 Requirements for field and laboratory duplicates and spikes w~lbl e specified in the quality assurance plan for this project. 11.0 Sample Exmtion 11.1 11.2 11.3 11.4 11.5 11.6 11.7 11.8 11.9 11.10 11.11 Weigh 1 g of sample into a 50 mL polypropylene centrifuge tubes (fortify as needed. replace lid and mix well). Note that alternate weights of liver may he measureddependingon the samplesize available for use. Add water to the sample for a f d volume of 10 mL. Homogenizesample using a tissuemizer for -1 minute. Transfer 1 mL of the sample using a disposable pipette into a I5 mL disposable Centrifilge tube. Add 5 mL of acctOnitrile and shake for -20 minuter on a wrist-action shaker. Centrifugethe tubes at -3000 rpm for -5 minutes. Decant the supernatant into a SO mL disposable centrifuge tube and add 35 mL of water. Condition the CI, SPE cartridges (1 g, 6 mL) by passing 10 mL methanol followedby 5 mL of HPLC water f- 2 drop/clcc). Do not let column run dry Load the sample on conditioned Cas SPE cartridge. Discard eluate. Elute with -2 mL of methanol. Collect 2 mL of eluate into a graduated I5 mL polypropylene centrifuge tube (final volume = 2 mL). Analyze samplesusing electrosprayLCiMSiMS. 12.0 Chromatogrsphy 12.1 Inject the rsme amount of each standard. sample and fortified sample inlo the LC/MS/MS system. A calibration standard must precede and follow all analyzed samples. 12.2 StandardsofPFOA correspondingto at least five or more concentration levels must be included in manalyticalset. 12.3 An entire set of calibration standards must be included at the beginning and .it the end of a spmple set. Standards must be interspersed between every 5-1 0 samples. As an alternative, an entire set of calibration standards may hc injected a8 the begmning of a set followed by calibration standards intmperscd every 5-10 samples (to account for a second set of standards). In either case. calibration standards must be the first and last injection in a m p l e set. 12.4 Use linear st&ndard curved for quantitation. Linear standard curves are generated for the analyte by linear regression using Ihc weightingof peak area 05-0210 Interim Report #5 - __I_ Page 110 of 123 3M Environmental Laboratory Project No. 05-0210 Exygen Protocol Number: PO001 131 c 1 I Exygcn Rucrrsb M e W Numbn VOW1785 m , u m c LMETHOD Method of Analysis for the Determination of PcrfluorooctanoicAcid (PFOA) in Small Mammal Liver by L U M S M S versus calibrationstandard concabation using MassLynx 3.3 (or cquivalcnt) software system. 12.5 Sample rrsponsc should not excecd standard responses. Any samples that excecd standard responsesshould be further diluted and reanalyzed. 13.0 Acceptance Criteria 13.1 Chromatogram must show a peak of a daughter ion at 369 a m u from a parent of 413 amu. The 413 amu parent corresponds to the PFOA anion. while thc daughterion (369 amu) representsthe loss of carbon dioxide. 13.2 Mahod blanks mwt not contain PFOA at levels greater than the LOQ. If a blank contains PFOA at levels greater than 10 ndg, then a new blank sample must be obtained and the entire set must be re-extracted. 13.3 RecovCries of contml spikes and matrix spikes must be between 70-130% of their known values. If a control spike falls outside the acceptable limits. the entire set of samplca should be n-cxtracted. Any matrix spike outside 70130.h should be evaluated by the analyst to determine if re-extraction is W-tCd. 13.4 Any calibration standard found to be a statistical outlier by using the Huge Error Test, may be excluded h m the calculation of the calibration curve. However, the total number of calibration standards that could be excluded must not excccd 20% ofthe total number of standards injected. 13.5 The comlation coefficient (R) for calibration curves generated must be M.992 (R2 20.985). If calibration results fall outside these limits. then appropriate steps mwt be taken to adjust instrument operation, and the standardsor the relevant set of samples should be reanalyzed. 13.6 Retention tima between standards and samples must not drift more Ihan f 4 % within an analytical run. If retention time drift exceeds this limit within an analytical run then the set must be reanalyzed. 14.0 Calculations 14.1 Use the following equation to calculatethe amount of PFOA found (in ng/mL. bared on peak area) using the standard curve (linear regression parameters) generated by the Mass Lynx soRwareprogram: - - PFOA found(nghnL) intercent1x DF x aliquot factor slope -DF factor by which the final volume was diluted, if necessary. . Aliquot factor 10 Page 6 of 7 - . . ... 05-0210 Interim Report #5 - - ~.. -. . Page 111 of 123 3M Environmental Laboratory Project No. 05-0210 ExygenProtocol Number: PO001 131 I I ExygcoRcseuch McthodNumber V0001765 ANALYI ICAL MErHOD Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA)in Small Mammal Liver by LCMSMS 14.2 For samples fortified with known amounts of PFOA prior IO extraction. use the followiq equationto calculate the percent recovery. - Recovery (%) 1total analyte found (ng/mL) - analytefound in control (nghL)1 analyteadded (nghL) 14.3 Use the following equation to convert the amount of PFOA found in ngmL Io ngle bb). PFOA found @pb) =LpFOA foudh&&xl final volume fmLU sample weight (g) ._ _ _ - 05-0210 Interim Report #5 Page 7 017 __ -.- Page 112of 123 3M Environmental Laboratory Project No. 05-0210 Exygen Protocol Number: PO00I I3 1 ANALYTICAL. METHOD Method Number: VOOOl786 Method of Analpis for the Determln8tlon of PerfluoroocUnoicAcid (PFOA)in Smull M8mmd Serum by LC/MS/MS Analytical Testing Facility: Exygen Research 3058 Research Drive State College, PA 16801 Approved By: Paul Connolly I Tcchnical Leader. LC-MS, Exygen Research 05-0210 Interim Report #5 TotalPages: 7 Puge SY qf '65 - _-.- Page 113 of 123 3M Environmental Laboratory Project No. 05-0210 Exygen Protocol Number: PO001 13 1 Exygen ReKuEh MclhodNumber VOOO1786 I I ANUt'TICAL !METHOD Method of Analysisfor the Determination of Pduorooctanoic Acid (PFOA) in Small Mammal Serum by LC/MS/MS 1.o scope This method is to be employed for the isolation and quantitation of perfluoroocianoic acid by High Performance Liquid Chromatography coupled to a tandem Mass SpectrometricDetector (LC/MS/MS)in small mammal serum. 2.0 Safety 2.I Always obscrvesafe labomtoy practices. 2.2 Consult the appropriateMSDS before handling any chemical for proper safety precautions. 3.0 Sample Requirement 3.1 At least 1 mLof twt sample for cxtrnction. 3.2 No sample processing is needed for serum samples. Howeva, Frozen serum ramplea must to allowed to completelythaw to room temperaturebefore use. 3.3 Sample collection pmcedurcs will be specified in the sampling plan for this project. 4.0 Reagents and Standards 4.1 WIta-HPLC@C 4.2 Mahmol-HPU: grade 4.3 Acetonihile-HPLC grade 4.4 AmmoniumAc&tt -A.C.S. RepgentGiade 4.5 PduorooctanoicAcid - Sigma-Aldrich 5.0 Instrumentand Equipment 5.1 A high performance liquid chromatograph capable of pumping up to 1 solvents quipped with a variable vo~umeinjector capable of injecting 5-200 pL connectedto a tandem Mass Spectrometer(LCMSMS). 5.2 A device to collect raw data for peak integration and quantitation. 5.3 Analytical balance capableof reading to O.OOOO1 g. 5.4 50 rnL disposablepolypropylene centrifugeNbes. 5.5 15 mL disposablepolypropylene centrifugetubes. 5.6 Disposablemicropipets (50-100uL,100-2OOuL). 5.? 125-mLLDPE narrow-mouthbottles. 5.8 2 mL clcsr HPLC vial kit. 5.9 Disposablepipcnts. 5.10 Autopipettes(100-1000 pLand 10-100 pL), with disposable tips. 5.1 1 Waters Sep Pak V u 6 cc Ilg) tC18 SPE cartridges. 5.12 SPE vacuum manifold. 5.13 vortcxa. Page 60 of65 3M Environmental Laboratory Project No. 05-02 10 ExygenProtocol Number: PO001 13 1 Exyeen Raurch Method Number VOOU I 786 1 1 AI\ALYI'KAL METHOD Method of Analysis for the Dctcnnimtion of PerfluorooctanoicAcid (PFOA)in Small Mammal Smun by LCIMSlMS 5.14 Wrist-action shaker. 5.15 Centrifugecapableof spinning I5 mL polypropylenetubes at 3000 rpm. 6.0 Chromatographic System 6.1 Analytical Column: FluophaJeRe (KeystoneScientific). 2.1 mm x 50 mm. 5p (PM: 82505-052130) 6.2 Tempaature: 30'C 6.3 Mobile Phase (A) : 2 mM Ammonium Acetate in Water 6.4 Mobile Phase (B) : Methanol 6.5 GradicntRogram: TlmbiJd &!A 0.0 65 I .o 65 8.0 25 20.0 25 22.5 65 Flow Rate fmUmin) 35 0.3 35 0.3 75 0.3 75 0.3 35 0.3 - 6.6 Injection Volume: 15 (can be increased to as much as 50 pL). 6.7 Quantitation: Peak A m - e x t d standardcalibration curve. 6.8 Run Time: 23 minutes. The above conditions am intended as a guide and may be changed in order to optimize the HPLC system. 7.0 MSMSSystem 7.1 Mode: ElectrosprayNegative MRM mode, monitoring413 +369 m/z for PFOA. The above conditions arc intended as a guide and may be changed in order lo optimize the MSMS system. 8.0 F'rcpadon of Solutions 8.1 Mobile P k 8.1.1 2 mM anunor&m acetate in water is prepared by adding 0.154 g of ammonium acetateto 1OOO mL ofwater. Alternatevolumes may be pnpared. Page 3 of 7 - - 05-0210 Interim Report #5 ..- Page 115 of 123 3M Environmental Laboratory Project No. 05-0210 Exygen Protocol Number: PO001 131 Exygecn Rruarcb MelhodNvmbnV0001786 1 I ANALYTICAL .WTHOD Method of Analysis for the Dttenninationof PerfluorooctanoicAcid (PFOA) in Small Mammal Serum by LCIMSIMS 9.0 Standardpnpantion 9.1 Standard StocWFortification Solution 9.1.1 Preparc a stock solution of -100 p g h L of PFOA by weighiny 10 my of analytical standard (comcted for purity) and dilute to 100 m L *it11 methanolin a 125-mLLDPE bottle. 9.1.2 A 1.0 pg/mL fortificationsolution of PFOA is prepared by bringing I mL of the 100 p&nL solution to a final volume of 100 with methanol in a 125 mL LDPE bottle. 9.1.3 A 0.1 pB/mL fortificationsolution of PFOA is prepared by bringing IO mL of the 1.0 pumL solution to a final volume of 100 with methanol in a 125 mL LDPE bottle. 9.1.4 The stock and fortificationsolutionsan to be stored in a refrigeratorat approximately 4 T and arc stable for a maximum period of 6 months from the date of preparation. 9.2 Standard Calibration Solutions 9.2.1 LC/MS/MS calibration standards arc prepared in methanol via dilution of the 0.1 pg/mL fortificationsolution. 9.2.2 The following is a typical example: additional concentrations may be prepared M needed. Concenlntion Final of Fortification Volumc Diluted lo Concentration Solution (nglmL) (mL) (mL) (nglmL) loo 5 .o 100 5.0 100 2.0 100 2.0 loo 1 .o 100 I .o 5.0 IO I00 0.5 2.0 IO 100 0.2 I .o IO 100 0.1 9.2.3 Store all calibration standards in 125-mL LDPE narrow-mouth bottles at 2'C to 6"C, up to six months. 9.2.4 Alternate volumes and concentrationsof standards may be prepared as needed. 10.0 BatchSaUp 10.1 Each batch of sunplea extra*ed (typically 20 or lw) must include at least one untreated coni701 and two untnatcd coni70I.s fortified at known concentrations(lab control spike)to verify procedural recovery for the batch 10.2 Requirements for field and laboratory duplicates and spikes will be spccilied in the qualityassuranceplan for this project. Page 4 o f ? -.- --__-__- __ 05-0210 lnterim Report #5 Page 62 of45 -- Page 116 of 123 3M Environmental Laboratory Project No. 05-0210 Exygen Protocol Number: PO001131 Exygcn Rcteuch Method Number VOOO I786 1 1 ANALYTICALMETHOD Methodof Analyais for the Detcnninationof Pduorooctanoic Acid (PFOA) in Small Mammal Serum by LCIMSIMS 1 1.O Sample Extraction 11.1 11.2 11.3 11.4 11.5 11.6 11.7 11.8 11.9 11.10 11.11 Measure 1 mL of sample into a 50 mL polypropylenecentritkge tubes (fortify as needed, replace lid and mix well). Note that alternate volumes of serum may be mcaaurcd dependingon the sample size available for use. Add water to the sample for a final volume of 20 mL. Cap tightly vortex far -1 minute. Transfer 1 mL of the sample using a disposable pipette into a 15 mL disposablecentrifugetube. Add 5 mLof acetonitrileand shake for -20 minutes on a wrist-action shaker. Centrifuge the tuba at -3000 rpm for -5 minutes. Decant the supematant into a 50 mL disposable centrifuge tube and add 35 mL of water. Condition the Cia SPE cartridges (1 g, 6 mL) by passing 10 mL methanol followed by 5 mL of HPLC water (- 2 drop/sec). Do not let column run dry Load the sampleon conditioned CIUSPE cartridge. Discard eluate. Elute with -2 mL of methanol. Collect 2 mL of eluate into a graduated I5 mL polypropylene centrifugetube (final volume-2 mL). Analyze samplesusing elec-y LCMISIMS. 12.0 chromatogrsphy 12.1 Inject the same amount of each standard, sample and fortified sample into the LC/MsIMs system. A calibration standard must precede and follow all a n a l p d samples. 12.2 Standards of PFOA correspondingto at least five or more concentrationlevels must be included in an analytical set. 12.3 An entire set of calibrationstandards must be included at the beginning and the end of a sample set. Standards must be interspersed between every 5-11) samples. As an alternative, an entire set of calibration standards may be injected at the beginning of 8 set followed by calibration standards intersper& way 5-10 samples (to account for a second set of standards). In either case, calibration standards must be the fmt and last injection in a sample set. 12.4 Use linear standard curves for quantitation. Linear standard curves are generated for the analyte by linear regression using Ihc weighting of peak arca vcrsus calibration standard concentration using MassLynx 3.3 (or equivalenr) 00mv~0ydosn. 12.5 Sample response should not exceed standard responses. Any samples thai exceed standardrcsponscs should be M e r diluted and reanalyzed. ---I --- . 05-0210 Interim Report #5 Pnge 63 oj'6j - I _ Page 117of 123 3M Environmental Laboratory Project No. EO5-0210 Exygen Protocol Number:PO00113I Exygm Flueuch Method Number VOWl786 1 I ANALYTICAL METHOD Method of Analysis for the Damination of PeriluomoctmoicAcid (PFOA) in Small Mammal Scrum by LCIMSIMS 13.0 Acceptance Criteria 13.1 chromntogrpmmust show a peak of a daughter ion at 369 a m u from a parent of 413 mu. The 413 m u parent corresponds to the PFOA anion, while tlis daughter ion (369 amu) npnsents the loss of carbon dioxide. 13.2 Method blanlts must not contain PFOA at levels greater than the LOQ. If a blank contains PFOA at levels greater than 10 ng/mL, then a new blank sample must be obtained and the entire set must be re-extracted. 13.3 Recovaics of control spikes and matrix spikes must be between 70.130% of their known valucr. If a control spike falls outside the acceptable limits, the entire. set of samples should be rc-cxtractcd. Any matrix spike outside 70130% should be evaluated by the analyst to determine if re-extraction is warranted. 13.4 Any calibration st.ndard found to be a statistical outlier by using the Huge Error Test, may be excluded from L e calculation of the calibration curve However, the total number of calibration standards that could be excluded must not exceed 20% of the total number of standards injected. 13.5 The cornlation coefficient (R) for calibration curves generated must he 20.992 (R' 20.985). If calibration results fall outside these limits. then appropriate steps must be taken to adjust insbumen1 operation. and tllc standardsor the relevant set of samples should be reanalyzed. 13.6 Retention times between standards and samples must not dritl more thaii f 4 %within an analyiical run. If retention time drift exceeds this limit within an analyticalrun then the set must be reanalyzed. 14.0 Calculations 14.1 Use the following equation tu calculatethe amount ofPFOA found (in ng/mL. based on peak area) using the standard curve (linear regression paramelersl generated by the M a s Lynx softwareprogram: PFOA found (ng/mL)=/Peak area - intercmt) x DF x aliquot factor slope DF = faaor by whichthe finalvolume was diluted. if necessary. Aliquot factor = 20 14.2 For samples fortified with knovm 8mounU of PFOA prior to extraction, UEU the following equation to calculatethe pmcnt recovery. Recovery (%) = 05-0210 Interim Report #5 Page 64 of65 -- Page 118 of 123 3M Environmental Laboratory Project No. 05-0210 Exygen Protocol Number: PO001 131 ExygcnW u c h Metbod Numbcr VooO1786 --- AiiALrrIcfi METHOD 1 I Method of Analysis for the Determinationof Perfluorooctanoic Acid (PFOA) in Small Mammal Scrumby LC/MSi?vlS - 14.3 Use the followingequation to convert the amount of PFOA found in n g h L in PPb. PFOA found @pb) =[PFOA found lndmL)x final volume (mLU sample volume (mL) 05-0210 Interim Report #5 Page 65 of 65 . - Page 119 of 123 3M Environmental Laboratoty Project No. 05-0210 Protocol Exygen PO007 737; 3M Study Number 050210 Amendment 3 Study Title ANALYSIS OF PERFLUOROBUTANESULFONATE(PFBS), PERFLUOROHWNESULFONATE (PFHS), AND PERFLUOROOCTANESULFONATE (PFOS) IN WATER, SOIL, SEDIMENT, FISH,CLAMS, VEGETATION, SMALL MAMMALIVERAND SMALL MAMMASLERUM USING LC/MS/MS FOR THE 3M DECATURMONITORINPGROGRAM PROTOCOL AMENDMENT NO. 3 Amendment Date: May 16,2005 Performing Laboratory 3M Environmental, Health, and Safety Operations 3M Environmental Laboratory 935 Bush Avenue St. Paul, MN 55106 Laboratory Project Identification E05-0210 E05-0210 05-0210 Interim Report #5 GLP Protocol Amendment#3 Page 1 of4 Page 120 of 123 3M Environmental Laboratory Prvject No. EO5-02 10 Protocol Exygen PO001 131; 3M Study Number 05-0210 Amendment 3 This amendment modifies the following portion(s) of the protocol: PROTOCOL READS: TESTINGFACILITY Exygen Research 3058 Research Drive State College, PA 15801 Phone: (814) 272-1039 AMENDTO READ: TES77NGF A C K ~ E S Exygen Research 3058 Research Drive State College, PA 15801 Phone: (814) 272-1039 3M Environmental Laboratory Building 2-3E-09 935 Bush Avenue St. Paul, MN 55106 REASON: Addition of 3M EnvironmentalLaboratoryas a testing facility allows selected water samples to be sent to the 3M facility for analysis of PFBS, PFHS, and PFOS using the most current version of ETS 8-154 "Determinationof PerfluorinatedAcids, Alcohols, Amides, and SulfonatesinWater by Solid Phase Extractionsand High PerformanceLiquid Chromatography/MassSpectrometry". The following modificationsto ETS 6-154.1 will be incorporatedinto the study: The sample volume extracted and the final SPE elution volume may be adjusted, at the analyst's discretion, to better achieve the detection limits specified in study objectives. As written, ETS 8154.1 calls for a 40 mL extraction volume with a 5 mL elution volume which results in an eightfold sample concentration. The actual volumes used and the resulting overall concentration factor will be given in the final report. Associated method quality control samples will demonstratethat the volume modificationsdo not impact method accuracy and precision. A solvent (unextracted)calibration curve may be analyzedwith the extracted calibration curve to ascertain extraction efficiency. Samples will be analyzed using the extracted curve unless otherwise stated in the final report. Data from the solvent calibration curve will not be included or discussed in the final report unless it is used to strengthen experimental observationdexplanations. ETS 8-154.1 makes no mention of sample surrogate spikes; however all samples will be spiked - with at least one of the followinq radiolabeledsurrogates, PFOA [1,2 13C] (perfluorooctanoicacid), PFOS [1'02] andlor PFNA [1,2 k](perfluorononanoic acid C9).The concentrationof the surrogate spike may vary depending on the collection event, but typical samples are spiked with a nominalconcentrationof 0.05 nglmL. Surrogate spike recoveries will be documented and discussed in the final report. Surrogate recoveries between 100KZ5% will be deemed as meeting method criteria for accuracy. Several method blanks may be prepared and analyzed to better determine a "representative" area count value for the method blank. The method blank area count is instrumental in determining the method limit of quantitation and understanding the average and range of area counts possible is critical. If a method blank value is used as a "zero point" in the final calibration curve, then a brief explanation in the final report or in the raw data as a Note to File must be E05-0210 GLP Protocol Amendment #3 Page 2 of4 05-0210 lntenm Report #5 Page 121 of 123 3M Environmental Laboratory P m j d No. 05-0210 Protocol Exygen PO001131;3M Study Number 05-0210 Amendment 3 included to describe why that specific method blank was selected as the zero point (closest to the average value, most conservative (largest) value, etc.) (5) The overall analytical uncertaintyfor each target (non-surrogate)analyte will be estimatedusing both the method accuracy and precision. The method accuracy and precision will be calculated using data from laboratory contori spike (LCS) samples prepared and analyzed with the sample set(s). Replicate LCSs must be prepared each day samples and/or other quality control samples are prepared. A sample calculation of how the analytical uncertaintywas determined will be provided in the final report andlor raw data. A new revisionof ETS 8-154 may incorporateall or several of the modificationslisted above. If a new version of ETS 8-154 is issued during the course of this study, sample results must meet the new method requirements regardless if they are listed above. 3M EnvironmentalLaboratorymanagementwill approve all documented deviationsto the 3M EnvironmentalLaboratory quality system (SOPS,methods, etc.) that occur during the course of this investigation. E05-0210 05-02 10 Interim Report #5 GLP Protocol Amendment #3 Page 3 of4 Page 122of 123 3M Environmental Laboratory Pmject No. E050210 Protocol Exygen PO001131; 3M Study Number 05-0210 Amendment 3 Amendment Approval Michael A. Sahtom, 3 M , c Sponsor Representative Date 3M EnvironmentalLaboratory Manager Date E05-0210 05-0210 Interim Report ##5 GLP Protocol Amendment #3 Page 4 of4 Page 123 of 123