Document LZ989nBOqapv6dzgxDvqama5

3 Environmental Laboratory E05-0210 Interim Report#15 Interim Report #15: Analysis of Water Samples from Northern Alabama Potable Water Systems 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 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 Certificate #2052-01 Performing Laboratory 3MEnvironmental Laboratory Building 2-3E-09 935 BushAve. St. Paul, MN 55106 Project Identification E05-0210 Total Number of Pages 123 The testing reported herein meet the requirements of ISO/IED 17025-1999 "General Requirements for the Competence of Testing and Calibration Laboratories", in accordance with the A2LA Certificate #2052-01. Testing that complies with this International Standard also operate in accordance with ISO 9001/ISO 9002 (1994). E05-0210 Interim Report#15 Northern Alabama Potable Water Systems Page 1 o f 123 This page has been reserved for specific country requirements. 3M Environmental Laboratory E05-0210 Interim Report#15 E05-0210 Interim Report#15 Northern Alabama Potable Water Systems Page 2 o f 123 Environmental Laboratory E05-0210 Interim Report#15 GLP Compliance Statement 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 Interim Study: Analysis of Water Samples from Northern Alabama Potable Water Systems Interim Study Identification Number: E05-0210 Interim Report#15 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 GLP compliance: None Sponsor Representative Jaisimha Kesari P. E., DEE, Weston Solutions, Inc. Study Director Date Certificate #2052-01 T h e testing reported herein m eet the requirem ents o f IS O /IE D 1 7 0 2 5 -19 9 9 "G eneral Requirem ents fo r the C om petence o f Testing and C alibration Laboratories", in accordance w ith the A 2LA C ertificate # 2 0 5 2 -0 1 . Testing that com plies w ith this International Standard also operate in accordance w ith IS O 9 00 1 /IS O 9 00 2 (1 9 9 4 ). 05-0210 Interim ReporHt15 Northern Alabama Potable Water Systems Page 3 of 123 3M Environmental Laboratory E05-0210 Interim Report#! 5 Quality Assurance Statement 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 Interim Study: Analysis of Water Samples from Northern Alabama Potable Water Systems Interim Study Identification Number: E05-0210 Interim Report#! 5 This study was audited by the 3M Environmental Laboratory Quality Assurance Unit (QAU), as indicated in the following table. The findings were reported to the study director and laboratory management. Inspection Dates Decem ber 1 -2 ,2 0 0 5 Phase D a ta /R e p o rt D ate R eported to M anagem ent Study Director Dec. 12, 2005 Dec. 12, 2005 QAU Representative 3-3 /-O T Date E0S-0210 Interim Report#15 Northern Alabama Potable Water Systems Page 4 o f 123 3M Environmental Laboratory E05-0210 Interim Report#15 Table of Contents GLP Compliance Statement.............................................................................................................. 3 Quality Assurance Statement............................................................................................................ 4 Table of Contents.............................................................................................................................. 5 List of Tables......................................................................................................................................6 Study Information.............................................................................................................................. 7 Summary and Introduction................................................................................................................. 8 Test Samples................................................................................................................................... 10 Reference Substances..................................................................................................................... 10 Control Substance............................................................................................................................11 Method Summaries..........................................................................................................................11 Sample Collection................................................................................................................ 11 Preparatory and Analytical Methods..................................................................................... 12 Extraction............................................................................................................................. 12 Analysis................................................................................................................................ 12 Analytical Results............................................................................................................................ 13 Calibration............................................................................................................................ 13 Limit of Quantitation (LOQ)................................................................................................... 13 Blanks.................................................................................................................................. 14 Solvent Blank........................................................................................................................14 Method Blank........................................................................................................................14 Trip Blank............................................................................................................................. 15 System Suitability................................................................................................................. 15 Continuing Calibration...........................................................................................................15 Lab Control Spikes (LCSs)................................................................................................... 15 Sample Duplicates............................................................................................................... 17 Surrogates............................................................................................................................17 E05-0210 Interim Report#15 Northern Alabama Potable Water Systems Page 5 o f 123 3M Environmental Laboratory E05-0210 Interim Report#15 Field Matrix Spikes............................................................................................................... 17 Data Summary and Discussion........................................................................................................17 Statistical Methods and Calculations................................................................................................ 20 Accuracy and Precision Equations........................................................................................ 20 Determination of Analytical Uncertainty................................................................................ 20 Statement of Conclusion.................................................................................................................. 21 List of Attachments.......................................................................................................................... 21 Signature Page................................................................................................................................ 22 List of Tables Table 1. Sample Results Summary................................................................................................... 9 Table 2. Study Reference Substance.............................................................................................. 10 Table 3. Study Control Substance (Surrogate).................................................................................11 Table 4. Sample Collection Information........................................................................................... 12 Table 5. Instrument Parameters...................................................................................................... 12 Table 6. Liquid Chromatography Gradient Program.........................................................................13 Table 7. Mass Transitions............................................................................................................... 13 Table 8. Method Blank Area Counts................................................................................................ 14 Table 9. Surrogate Spiked Method Blank Recoveries...................................................................... 14 Table 10. Lab Control Spike (LCS) Results..................................................................................... 16 Table 11. Detailed Sample and QC Results.................................................................................... 18 E05-0210 Interim Report#15 Northern Alabama Potable Water Systems Page 6 o f 123 Study Information Sponsor 3M Company Sponsor Representative Michael A. Santoro 3M Company 3M Building 0236-01-B-10 St. Paul, MN 55144 Study Director Jaisimha Kesari, P.E., DEE Weston Solutions, Inc. 1400 Weston Way West Chester, PA 19380 3M Environmental Laboratory E05-0210 Interim Report#15 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: October 11,2005 Interim Experimental Completion: October 28, 2005 Interim Study Completion: Date of interim report signing Location of Archives All original raw data, protocol, and analytical report have been archived at the 3M Environmental Laboratory 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. E05-0210 Interim Report#15 Northern Alabama Potable Water Systems Page 7 o f 123 3M Environmental Laboratory E05-0210 Interim Report#15 Summary and Introduction The 3M Environmental Laboratory extracted and analyzed water samples collected from six Northern Alabama potable water systems by Weston Solutions personnel on October 4 and 5, 2005. Samples were submitted for analysis as part of 3M Environmental Laboratory project number E05-0210 (3M Decatur Fluorochemical GLP Monitoring Program, Interim Report #15: Analysis of Water Samples from Northern Alabama Potable Water Systems). Water samples were analyzed for perfluorooctane sulfonate (PFOS), perfluorohexane sulfonate (PFHS), and perfluorobutane sulfonate (PFBS) using 3M Environmental Laboratory Method ETS 8-154.1 "Determination of Perfluorinated Acids, Alcohols, Amides, and Sulfonates in Water by Solid Phase Extractions and High Performance Liquid Chromatography/Mass Spectrometry" in accordance with Exygen Research Protocol P0001131 (Attachment C). ETS 8-154.1 falls under the 3M Environmental Laboratory's current ISO/IED 17025-1999 scope of accreditation and the results reported herein were complient with this accreditation. The analytical start date for this interim report was October 11,2005 and the analytical termination date was October 28, 2005. Sample containers were prepared at the 3M Environmental Laboratory. Sample containers for each sampling location included a field sample, field sample duplicate, low field spike (0.1 ng/mL) and high field spike (1.0 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 three target analytes prior to being sent to the field for sample collection. Table 1 below summarizes the sample results. The average between the sample and the sample duplicate is provided along with the relative percent difference (%RPD) if applicable. The limit of quantitation (LOQ) of PFBS, PFHS, and PFOS for the sample set was 0.0249 ng/mL, 0.0247 ng/mL, and 0.0247 ng/mL, respectively. All results for quality control samples prepared and analyzed with the samples will be reported and discussed elsewhere in this report. E05-0210 Interim Report#15 Northern Alabama Potable Water Systems Page 8 o f 123 Table 1. Sample Results Summary. 3M Environmental Laboratory E05-0210 Interim Report#15 3M LIMS ID E05-0210-89707 E05-0210-89708 E05-0210-89714 E05-0210-89715 E05-0210-89718 E05-0210-89719 E05-0210-89722 E05-0210-89723 E05-0210-89726 E05-0210-89727 E05-0210-89730 E05-0210-89731 Sample Description DPWS PW FW01 0 051004 DPWS PW FW01 DB 051004 Average Sample/Sample Duplicate %RPD WMEL PW FW01 0 051004 WMEL PW FW01 DB 051004 Average Sample/Sample Duplicate %RPD MSTP PW FW01 0 051004 MSTP PW FW01 DB 051004 Average Sample/Sample Duplicate %RPD FWTP PW FW01 0 051004 FWTP PW FW01 DB 051004 Average Sample/Sample Duplicate %RPD TVATP PW FW01 0 051004 TVATP PW FW01 DB 051004 Average Sample/Sample Duplicate %RPD SWTP PW FW01 0 051005 SWTP PW FW01 DB 051005 Average Sample/Sample Duplicate %RPD (1)PFBS Concentration (ng/mL) <0.0249 <0.0249 <0.0249 (4)n a 0.0374 0.0383 (5)0.0378 2.4 <0.0249 <0.0249 <0.0249 (4)n a <0.0249 <0.0249 <0.0249 (4)n a <0.0249 <0.0249 <0.0249 (4)n a <0.0249 <0.0249 <0.0249 (4)n a (2)p f h s Concentration (ng/mL) <0.0247 <0.0247 <0.0247 (4)n a <0.0247 <0.0247 <0.0247 (4)n a <0.0247 <0.0247 <0.0247 (4)n a <0.0247 <0.0247 <0.0247 (4)n a <0.0247 <0.0247 <0.0247 (4)n a <0.0247 <0.0247 <0.0247 (4)n a (3)p f o s Concentration (ng/mL) <0.0247 <0.0247 <0.0247 (4)n a 0.0616 0.0819 0.0718 28 0.0258 <0.0247 (5)0.0258 NA <0.0247 <0.0247 <0.0247 (4)n a <0.0247 <0.0247 <0.0247 (4)n a 0.0398 <0.0247 (5)0.0398 NA (1) The analytical uncertainty of the PFBS resuts is 1006.2% based on method accuracy and precision. All results and averages are presented with three significant figures, %RPD to two significant figures. Sample concentrations, averages, and %RPD values may vary slightly from the raw data. Samples with concentrations less than the LOQ of 0.0249 ng/mL may have detectable amounts of PFBS present; however, quantified concentrations less than the established LOQ are not assigned. (2) The analytical uncertainty of the PFHS resuts is 1004.5% based on method accuracy and precision. All results and averages are presented with three significant figures, %RPD to two significant figures. Sample concentrations, averages, and %RPD values may vary slightly from the raw data. Samples with concentrations less than the LOQ of 0.0247 ng/mL may have detectable amounts of PFHS present; however, quantified concentrations less than the established LOQ are not assigned. (3) The analytical uncertainty of the PFOS resuts is 1009.7% based on method accuracy and precision. All results and averages are presented with three significant figures, %RPD to two significant figures. Sample concentrations, averages, and %RPD values may vary slightly from the raw data. Samples with concentrations less than the LOQ of 0.0247 ng/mL may have detectable amounts of PFOS present; however, quantified concentrations less than the established LOQ are not assigned. (4) NA: Not applicable. %RPD values not determined when concentrations for the sample and/or sample duplicate are below the stated LOQ. (5) The average value listed is the concentration for the sample or sample duplicate that produced a value above the LOQ. A true average and %RPD between the sample and sample duplicate was not determined. E05-0210 Interim Report#15 Northern Alabama Potable Water Systems Page 9 o f 123 3M Environmental Laboratory E05-0210 Interim Report#15 Test Samples Water samples from six locations were received on October 6, 2005 from Tim Frinak of Weston Solutions, Inc. Twenty-seven containers were submitted for analysis. The samples were logged in by 3M Environmental Laboratory professional services personnel and placed in refrigerated storage prior to extraction on October 6, 2005. Reference Substances Table 2 lists the pertinent information regarding the reference substance used for this study. Table 2. Study Reference Substance. Reference Substance PFBS PFHS PFOS Chemical Name Perfluorobutanesulfonate Perfluorohexanesulfonate Perfluorooctanesulfonate Chemical Formula Identifier (1)Source Expiration Date Storage Conditions Chemical Lot Number TCR Number C4 Fg SC>3 - K+ CAS # 29420-49-3 3M 1/17/2007 Frozen 2 TCR-282 (99131-040) Ca F1 3 SO3 K CAS # 3871-99-6 3M 10/18/2006 Frozen NB# 120067-69 TCR-83 (SE036) C8 F1 7 SC3 'K CAS # 2795-39-3 3M 8/31/2006 Frozen 171 TCR-696 Physical Description White Powder White Powder White powder Purity 97.3% 98.6% (2)Solubility 54,400 ppm No Information available (1) Documentation regarding synthesis of the test substances is located at the source. 86.4% 680 ppm (2) All test substances are believed to be soluble in water at the levels to be investigated. No visual precipitates observed. E05-0210 Interim Report#15 Northern Alabama Potable Water Systems Page 10 o f 123 3M Environmental Laboratory E05-0210 Interim Report#15 Control Substance PFOA [1,2 13C] was analyzed as a surrogate against a multi-level extracted calibration curve. A known amount of PFOA [1,2 13C] 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). Control Substance PFOA [1,2 13C] Chemical Name Chemical Formula Identifier Source Expiration Date Perfluorooctanoic Acid C6F 5[13C]F2[13C]OOH N/A Perkin Elmer 03/29/2009 Storage Conditions Frozen Chemical Lot Number TCR Number Physical Description Purity 3507-195 TCR-744 White powder >97% Method Summaries Sample Collection Samples were collected on October 4-5, 2005 in non-rinsed NalgeneTM (low-density polyethylene) bottles prepared at the 3M Environmental Laboratory on October 3, 2005. Prior to sample collection, all bottles were spiked in the laboratory with a known volume of a surrogate solution or appropriate matrix spiking solution containing the surrogate and the three target analytes. Four collection bottles were associated with each individual sampling location: sample, sample duplicate, low level field matrix spike (LS), and high level field matrix spike (HS). Additionally, one "Trip Blank" sample set was prepared. A Trip Blank set consisted of three individual bottles: trip blank sample, trip blank low spike, and trip blank high spike. The surrogate and matrix spike levels for the trip blanks were the same as the samples. Table 4 details the spike amounts for the four bottles comprising the sample location set. E05-0210 Interim Report#15 Northern Alabama Potable Water Systems Page 11 o f 123 3M Environmental Laboratory E05-0210 Interim Report#15 Table 4. Sample Collection Information. <vBottle Description Nominal Final Volume Collected (mL) PFBS Final Spike Concentration <ng/mL) PFHS PFOS PFOA [ 1 ,2 13C] (Surrogate) Sample 450 NA NA NA Sample Dup 450 NA NA NA Low Field Matrix Spike (LS) 450 0.0998 0.0987 0.0992 High Field Matrix Spike (HS) 450 0.998 0.987 0.992 (1) The sample description was assigned in the field by Weston Solutions personnel. 0.500 0.500 0.0998 0.998 Preparatory and Analytical Methods Extraction All samples, calibration standards, and associated quality control samples were extracted using the procedure outlined in ETS-8-154.1 "Determination of Perfluorinated Acids, Alcohols, Amides, and Sulfonates in Water by Solid Phase Extractions and High Performance Liquid Chromatography/Mass Spectrometry". Briefly, 40 mL of sample was loaded onto a pre-conditioned Waters C18 solid phase extraction (SPE) cartridge (Sep-Pak, 6 cc) using a vacuum manifold. The loaded cartridges were then eluted with 5 mL of methanol. This extraction procedure concentrates the samples by a factor of eight. (Initial volume = 40 mL, final volume = 5 mL). Analysis All sample and quality control extracts were analyzed for PFBS, PFHS, PFOS, and PFOA [1,2 13C] using high performance liquid chromatography/ tandem mass spectrometry (HPLC/MS/MS). Pertinent instrument parameters, the liquid chromatography gradient program, and the specific mass transitions analyzed are described in the tables below. Table 5. Instrument Parameters Instrument Name Liquid Chromatograph Guard column Analytical column Injection Volume Mass Spectrometer Electrode Ion Source Polarity Software ETSOllie Agilent 1100 Betasil C18 (2.1 mm X 100 mm),, 5 um Betasil C18 (2.1 mm X 100 mm), 5 um 5 uL Applied Biosystems API 4000 Q Trap Z-spray Turbo Spray Negative Analyst 1.4.1 E05-0210 Interim Report#15 Northern Alabama Potable Water Systems Page 12 o f 123 3M Environmental Laboratory E05-0210 Interim Report#15 Table 6. Liquid Chromatography Gradient Program Step Number 0 1 2 3 4 5 Total Time (min) 0 1.0 14.5 15.5 16.5 20.0 Flow Rate (pL/min) 300 300 300 300 300 300 Percent A (2 m M ammonium acetate) 80.0 80.0 10.0 10.0 80.0 80.0 Percent B (Methanol) 20.0 20.0 90.0 90.0 20.0 20.0 Table 7. Mass Transitions Analyte (1)PFBS (1)PFHS (1)PFOS PFOA [1,2 13C] Mass Transition Q1/Q3 299/80 299/99 399/80 399/99 499/130 499/99 499/80 415/370 Dwell Time (msec) 100 100 100 100 100 100 100 100 (1) All three transitions were summed to produce a "total ion chromatogram" (TIC). The TIC was used for quantitation. Analytical Results Calibration Calibration standards were prepared by spiking known amounts of stock solutions containing PFBS, PFHS, PFOS, and PFOA [1,2 13C] into 40 mL of ASTM type I water. Each spiked water standard was then extracted in the same manner as the collected samples. A total of ten spiked standards ranging from 0.025 ng/mL to 25 ng/mL (nominal) were prepared. A quadratic, 1/x weighted, calibration curve was used to fit the data for each analyte. The data were not forced through zero during the fitting process. Calculating the standard concentration using the peak area counts and the resultant calibration curve confirmed accuracy of each curve point. Each extracted calibration standard used to generate the final calibration curve met the method calibration accuracy requirement of 10025% (10030% for the LOQ standard). Coefficients of determination (r2) 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(s). This will be presented and discussed in more detail in the section below regarding method blanks. E05-0210 Interim Report#15 Northern Alabama Potable Water Systems Page 13 o f 123 3M Environmental Laboratory E05-0210 Interim Report#15 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 Blank Several methanol solvent blanks were analyzed to assess system contamination and/or instrument carryover. Analyte peak area counts in all solvent blank samples were less than half the area counts of the calibration standard used to establish the LOQ. Method Blank Eight method blanks were prepared by loading 40 mL of ASTM Type I water onto a C18 SPE cartridge and eluting with 5 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.). Four of the method blanks prepared were spiked with a known amount of PFOA [1,2 13C] surrogate prior to extraction. Table 8 lists the analyte area counts for the method blanks and the corresponding LOQ standard. Surrogate recoveries for the spiked method blanks are presented in Table 9. Table 8. Method Blank Area Counts. PFBS PFHS PFOS PFOA [ 1 ,2 13C] Surrogate Sample ID Sample Comment Area Counts MB-051011-1 Method Blank-1 8487 MB-051011-2 Method Blank-2 5792 MB-051011-3 Method Blank-3 6269 MB-051011-4 Method Blank-4 6884 MB-051011-5 Method Blank-5 7210 MB-051011-6 Method Blank-6 6501 MB-051011-7 Method Blank-7 7573 MB-051011-8 Method Blank-8 6964 2*Area Counts of Highest Method Blank 16974 Area Counts of the LOQ Standard 90226 LOQ Concentration (0.0249 ng/mL) (1) Method Blanks spiked with 1 ng/mL surrogate. 8788 8039 6871 9454 10691 7848 6191 6207 21382 82409 (0.0247 ng/mL) 40536 2255 41506 1284 37883 1377 33212 985 36261 (1)703106 42680 (1)741777 37869 (1)715325 31373 (1)737959 85360 (2)4510 123012 81116 (0.0247 ng/mL) (0.0998 ng/mL) (2) Method Blanks spiked with surrogate were excluded from LOQ verification. Table 9. Surrogate Spiked Method Blank Recoveries. Sample Name O051020a 031 O051020a 032 O051020a 033 O051020a 034 Sample ID MB-051011-5 MB-051011-6 MB-051011-7 MB-051011-8 Spiked Concentration (ng/mL) 1.015 1.015 1.015 1.015 Calculated Conc. (ng/mL) 0.919 0.972 0.935 0.966 %Recovery 90.5 95.8 92.1 95.2 E05-0210 Interim Report#15 Northern Alabama Potable Water Systems Page 14 o f 123 3M Environmental Laboratory E05-0210 Interim Report#15 Trip Blank Prior to sample collection, one sample container was filled with 450 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 field/trip 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 concentrations for PFBS, PFHS, and PFOS for the field/trip blank were <0.0249 ng/mL, <0.0247 ng/mL, and <0.0247 ng/mL, respectively. System Suitability A 5.0 ppb (nominal concentration) extracted calibration standard was analyzed in triplicate at the beginning and end of the analytical sequence to demonstrate overall system suitability. For all analytes, the relative standard deviation (RSD) of the analyte peak area counts and the RSD of the peak retention times were less than 5% and 2%, respectively, which met method criteria. 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 were still in control. ETS-8-154.1 states that all CCV recoveries must be within 25% for the data to be acceptable. All CCV recoveries met method criteria of 25% for all analytes. Lab Control Spikes (LCSs) Replicate low (0.25 ng/mL nominal concentration) and high (7.5 ng/mL nominal concentration) lab control spikes were prepared and extracted on the same day as the samples. LCSs were prepared by spiking known amounts of the analytes into 40 mL of ASTM Type I water to produce the desired concentration. The spiked water samples were extracted and analyzed in the same manner as the samples. Individual LCS results, along with the average and percent RSD, for the extraction set are presented in the data table below. LCS recoveries for all analytes met method criteria for both accuracy and precision. E05-0210 Interim Report#15 Northern Alabama Potable Water Systems Page 15 o f 123 3M Environmental Laboratory E05-0210 Interim Report#15 Table 10. Lab Control Spike (LCS) Results. (1)PFBS (1)PFHS (1)PFOS (1PFOA [1,2 13C] Sample Description Lab ID Spiked Conc. (ng/mL) Calc. Conc. (ng/mL) % Recovery Spiked Conc. (ng/mL) Calc. Conc. (ng/mL) % Recovery Spiked Conc. (ng/mL) Calc. Conc. (ng/mL) % Recovery Spiked Conc. (ng/mL) Calc. Conc. (ng/mL) % Recovery 0.25 ppb LCS LCS-051011-1 0.249 0.254 102 0.247 0.241 97.6 0.248 0.221 89.1 0.249 0.239 96.0 0.25 ppb LCS LCS-051011-2 0.249 0.249 100.0 0.247 0.244 98.8 0.248 0.227 91.5 0.249 0.238 95.6 0.25 ppb LCS LCS-051011-3 0.249 0.259 104 0.247 0.242 98.0 0.248 0.233 94.0 0.249 0.274 110 7.5 ppb LCS LCS-051011-4 7.48 6.97 93.2 7.40 6.97 94.2 7.44 6.92 93.0 7.48 6.75 90.2 7.5 ppb LCS LCS-051011-5 7.48 7.18 96.0 7.40 7.32 98.9 7.44 7.32 98.4 7.48 6.36 85.0 7.5 ppb LCS LCS-051011-6 7.48 7.04 94.1 7.40 7.14 96.5 7.44 7.07 95.0 7.48 7.10 94.9 Average %Recovery %RSD 98.2%4.5% 97.2%1.8% 93.5%3.4% 95.2%8.8% (1) Table displays rounded values for all concentration and percent recovery values (3 significant figures). %RSD values reported to two significant figures. Recovery and %RSD values may vary slightly from the values in the raw data. E05-0210 Interim Report#15 Northern Alabama Potable Water Systems Page 16 o f 123 3M Environmental Laboratory E05-0210 Interim Report#15 Sample Duplicates Because a field sample duplicate (separate container) was collected at each sampling location, duplicate/replicate extractions of a given sample were not performed. Overall method precision was determined using LCSs. Surrogates Although not specified in the ETS 8-154.1, PFOA [1,2 13C] was added to all samples and sample spikes as a surrogate to evaluate overall method performance. The final PFOA [1,2 13C] concentration for samples and sample duplicates was 0.500 ng/mL. Surrogate recoveries are reported in the next section with sample data and will be discussed in further detail in the Data Summary and Discussion section below. Field Matrix Spikes Low (nominal concentration of 0.1 ng/mL) and high (nominal concentration 1.0 ng/mL) field matrix spikes were collected at each sampling point to verify that the analytical method is applicable to the collected matrix. Field matrix spike recoveries within 10030% 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. Data Summary and Discussion Table 11 below summarizes the sample results, field matrix spike recoveries, and surrogate spike recoveries for all of the sampling locations plus the trip blank. Matrix spike recoveries for PFBS, PFHS, and PFOS were within method acceptance criteria of 10030% for all sampling locations with one exception. E05-0210-89716 (low level PFOS matrix spike associated with WMEL PW FW01) produced a recovery of 54.7%. However, the recovery of the high level matrix spike associated with location was 90.1%. Because the high level spike met method criteria, the sample results have been reported and are considered accurate within the stated method accuracy of 10030%. PFOA[1,2 13C] surrogate recoveries for sample/sample duplicate measurements ranged from 32.6% to 40.4%. The low and high field matrix spike surrogate recovery ranged from 18.2% to 35.2%. At this time, there is no explanation for the low surrogate recoveries. The 3M Environmental Laboratory will be investigating this in detail in a future study. Reported PFBS, PFHS,and PFOS sample concentrations have NOT been corrected for surrogate recovery as the field matrix spikes of the target analyte were within 10030%. E05-0210 Interim Report#15 Northern Alabama Potable Water Systems Page 17 o f 123 3M Environmental Laboratory E05-0210 Interim Report#15 Table 11. Detailed Sample and QC Results. 3M LIMS ID E05-0210-89707 E05-0210-89708 E05-0210-89709 E05-0210-89710 E05-0210-89714 E05-0210-89715 E05-0210-89716 E05-0210-89717 E05-0210-89718 E05-0210-89719 E05-0210-89720 E05-0210-89721 E05-0210-89722 E05-0210-89723 E05-0210-89724 E05-0210-89725 Description DPWS PW FW01 0 051004 DPWS PW FW01 DB 051004 DPWS PW FW01 LS 051004 DPWS PW FW01 HS 051004 Average %RPD WMEL PW FW01 0 051004 WMEL PW FW01 DB 051004 WMEL PW FW01 LS 051004 WMEL PW FW01 HS 051004 Average %RPD MSTP PW FW01 0 051004 MSTP PW FW01 DB 051004 MSTP PW FW01 LS 051004 MSTP PW FW01 HS 051004 Average %RPD FWTP PW FW01 0 051004 FWTP PW FW01 DB 051004 FWTP PW FW01 LS 051004 FWTP PW FW01 HS 051004 Average %RPD (1)PFBS Conc. (ng/mL) <0.0249 <0.0249 0.0991 1.02 (4)<0.0249 NA 0.0374 0.0383 0.137 1.06 0.0378 2.4 <0.0249 <0.0249 0.115 1.05 <0.0249 (4)n a <0.0249 <0.0249 0.121 1.05 <0.0249 (4)<n a PFBS %Recovery NA NA 99.3 102 NA NA 99.3 102 NA NA 115 105 NA NA 121 105 (2)p f h s Conc. (ng/mL) <0.0247 <0.0247 0.0963 0.975 (4)<0.0247 NA <0.0247 <0.0247 0.100 0.942 <0.0247 (4)n a <0.0247 <0.0247 0.0985 0.936 <0.0247 (4)n a <0.0247 <0.0247 0.0955 0.959 <0.0247 (4)<n a PFHS %Recovery NA NA 97.6 98.8 NA NA 101 95.4 NA NA 99.8 94.8 NA NA 96.8 97.2 (3>p f o s Conc. (ng/mL) <0.0247 <0.0247 0.0933 0.908 (4)<0.0247 NA 0.0616 0.0819 0.126 0.966 0.0718 28 0.0258 <0.0247 0.131 0.892 0.0258 (5)n a <0.0247 <0.0247 0.109 0.942 <0.0247 (4)<n a PFOS %Recovery NA NA 94.1 91.5 NA NA 54.7 90.1 NA NA 106 87.3 NA NA 110 95.0 Surrogate Conc. (ng/mL) 0.182 0.186 0.0351 0.202 0.181 0.169 0.0239 0.199 0.174 0.196 0.0288 0.223 0.202 0.184 0.0326 0.196 PFOA [1 ,2 13C] %Recovery 36.4 37.2 35.2 20.2 36.1 33.8 23.9 19.9 34.8 39.2 28.8 22.3 40.4 36.8 32.7 19.6 E05-0210 Interim Report#15 Northern Alabama Potable Water Systems Page 18 o f 123 3M Environmental Laboratory E05-0210 Interim Report#15 Table 11. Continued. 3M LIMS ID E05-0210-89726 E05-0210-89727 E05-0210-89728 E05-0210-89729 E05-0210-89730 E05-0210-89731 E05-0210-89732 E05-0210-89733 E05-0210-89711 E05-0210-89712 E05-0210-89713 Description TVATP PW FW01 0 051004 TVATP PW FW01 DB 051004 TVATP PW FW01 LS 051004 TVATP PW FW01 HS 051004 Average %RPD SWTP PW FW01 0 051005 SWTP PW FW01 DB 051005 SWTP PW FW01 LS 051005 SWTP PW FW01 HS 051005 Average %RPD DPWS PW Trip 0 051004 DPWS PW Trip LS 051004 DPWS PW Trip HS 051004 (1)PFBS Conc. (ng/mL) <0.0249 <0.0249 0.121 1.00 <0.0249 (4)n a <0.0249 <0.0249 0.112 0.985 <0.0249 (4)NA < 0.0249 0.0929 1.02 PFBS %Recovery NA NA 121 100 NA NA 112 98.7 NA 93.1 102 (2p f h s Conc. (ng/mL) <0.0247 <0.0247 0.0908 0.912 <0.0247 (4)NA <0.0247 <0.0247 0.0902 0.916 <0.0247 (4)NA < 0.0247 0.0900 0.964 PFHS %Recovery NA NA 92.0 92.4 NA NA 91.4 92.8 NA 91.2 97.7 (3)p f o s Conc. (ng/mL) <0.0247 <0.0247 0.110 0.882 <0.0247 (4)NA 0.0398 <0.0247 0.0967 0.892 0.0398 (5)n a < 0.0247 0.0743 0.904 PFOS %Recovery NA NA 111 88.9 Surrogate Conc. (ng/mL) 0.175 0.186 0.0301 0.220 PFOA [1 ,2 13C] %Recovery 35.0 37.2 30.2 22.0 NA 0.194 NA 0.172 97.6 0.0296 89.9 0.182 38.8 34.4 29.6 18.2 NA 0.163 75.0 0.0247 91.1 0.192 32.6 24.7 19.2 (1) The analytical uncertainty of the PFBS resuts is 1006.2% based on method accuracy and precision. All results and averages are presented with three significant figures, %RPD to two significant figures. Sample concentrations, averages, and %RPD values may vary slightly from the raw data. (2) The analytical uncertainty of the PFHS resuts is 1004.5% based on method accuracy and precision. All results and averages are presented with three significant figures, %RPD to two significant figures. Sample concentrations, averages, and %RPD values may vary slightly from the raw data. (3) The analytical uncertainty of the PFOS resuts is 1009.7% based on method accuracy and precision. All results and averages are presented with three significant figures, %RPD to two significant figures. Sample concentrations, averages, and %RPD values may vary slightly from the raw data. (4) NA: Not applicable. %RPD values not determined when concentrations for the sample and sample duplicate are below the stated LOQ. (5) The average value listed is the concentration for the sample or sample duplicate that produced a value above the LOQ. A true average and %RPD between the sample and sample duplicate was not determined. E05-0210 Interim Report#15 Northern Alabama Potable Water Systems Page 19 o f 123 3M Environmental Laboratory E05-0210 Interim Report#15 Statistical Methods and 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, 1/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 Calculated Concentration * . LCS/Surrogate Percent Recovery -------------------------------------* 100% Spike Concentration Sample Spike Recovery - (Spiked Sample Concentration - Average Concentration : Field Sample & Field Sample Dup.) * 1 0 0 % Spike Concentraton % RncS,Dn (,DRel1ative 0St.and.ard Devi.a.t.ion.) ---s--t-a-n--d--a-r--d--d--e--v-i-a--t-i-o-n---o--f--r-e-p--l-ic--a--t-e-s- * 10n0n%n. replicate average n. _.n r. ._. . .. n . . Absolute difference between sample duplicates , , nnn. % RPD (Relative Percent Difference) -------------------------------------------------- -------- -----------*100% 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 uncertainty for a given analyte. The measured precision (%RSD) is then used to determine the range of the accuracy. The example below shows how the analytical uncertainty for PFOS was determined for this sample set. Example: 93.50 (accuracy)*0.03374 (precision)=3.155 (accuracy range) 93.50+ 3.155=96.66; 93.50- 3.155 = 90.35 Thus, LCS accuracy results range from 90.35% to 96.66%. The absolute difference of the low and high ends of this range, when compared to 100%, are then calculated. 100%-96.66% = 3.34% 100%-90.35%= 9.65%. The most conservative (largest) absolute difference is then used as the analytical uncertainty for the given analyte. Therefore, the analytical uncertainty for PFOS as defined here, is given as 1009.7% for the data associated with this project. Using this same approach, the analytical uncertainties for PFBS and PFHS were determined to be 1006.2% and 1004.5%, respectively. E05-0210 Interim Report#15 Northern Alabama Potable Water Systems Page 20 o f 123 3M Environmental Laboratory E05-0210 Interim Report#15 Statement of Conclusion PFBS, PFHS, and PFOS results for this project were presented in Table 1. Laboratory control spikes were used to determine the method accuracy and precision for the three target analytes for this data set. The accuracy and precision were then used to estimate the analytical uncertainty for the results (PFBS 1006.2%, PFHS 1004.5%, and PFOS 1009.7%). Recoveries of field matrix spiked samples demonstrated that the overall analytical method was appropriate for the matrix collected. Consequently, sample results are considered accurate to within 10030%. List of Attachments Attachment A: Sample Chromatograms and Calibration Curves Attachment B: Extraction and Analytical Methods Attachment C: Protocol and Protocol Amendments E05-0210 Interim Report#15 Northern Alabama Potable Water Systems Page 21 o f 123 3M Environmental Laboratory E05-0210 Interim Report#15 Signature Page We certify that this report is a true and complete representation of the data for this study: (Mm M & Michael A. Santoro^/ Sponsor Representative ih i/ o i Date E05-0210 Interim Report#!5 Northern Alabama Potable Water Systems Page 22 of !23 3M Environmental Laboratory E05-0210 Interim Report#15 Attachment A: Sample Chromatograms and Calibration Curves E05-0210 Interim Report#15 Northern Alabama Potable Water Systems Page 23 o f 123 *Ollie U00110402 3M Environmental Laboratory E05-0210 Interim Report#15 Results Name: o051020a.rdb *Ollie U00110402 Results Name: o051020a.rdb Printing Date: Tuesday, November 29, 2005 Printing Time: 09:40:15 AM Page 28 of 88 E05-0210 Interim Report#15 Northern Alabama Potable Water Systems Page 24 o f 123 *Ollie U00110402 impleID:"LC-S-0510TI-3" 3M Environmental Laboratory E05-0210 Interim Report#15 Results Name: o051020a.rdb Printing Date: Tuesday, November 29, 2005 Printing Time: 09:40:15 AM *Ollie U00110402 Page 40 of 88 Results Name: o051020a.rdb Printing Date: Tuesday, November 29, 2005 Printing Time: 09:40:15 AM Page 44 of 88 E05-0210 Interim Report#15 Northern Alabama Potable Water Systems Page 25 o f 123 *Ollie U00110402 3M Environmental Laboratory E05-0210 Interim Report#15 Results Name: o051020a.rdb *Ollie U00110402 Results Name: o051020a.rdb Printing Date: Tuesday, November 29, 2005 Printing Time: 09:43:07 AM Page 1 of 1 E05-0210 Interim Report#15 Northern Alabama Potable Water Systems Page 26 o f 123 *Ollie U00110402 3M Environmental Laboratory E05-0210 Interim Report#15 Results Name: o051020a.rdb Printing Date: Tuesday, November 29, 2005 Printing Time: 09:35:05 AM *Ollie U00110402 D:"MB-051011-2" File:'0051020a.Wff' .98.9amu.398.9i&0.0amu398.9H29.9amu' Page 20 of 88 Results Name: o051020a.rdb Printing Date: Tuesday, November 29, 2005 Printing Time: 09:38:26 AM Page 28 of 88 E05-0210 Interim Report#15 Northern Alabama Potable Water Systems Page 27 o f 123 3M Environmental Laboratory E05-0210 Interim Report#15 E05-0210 Interim Report#15 Northern Alabama Potable Water Systems Page 28 o f 123 *Ollie U00110402 3M Environmental Laboratory E05-0210 Interim Report#15 Results Name: o051020a.rdb *Ollie U00110402 Results Name: o051020a.rdb E05-0210 Interim Report#15 Northern Alabama Potable Water Systems Page 29 o f 123 *Ollie U00110402 3M Environmental Laboratory E05-0210 Interim Report#15 Results Name: o051020a.rdb *Ollie U00110402 Results Name: o051020a.rdb E05-0210 Interim Report#15 Northern Alabama Potable Water Systems Page 30 o f 123 *Ollie U00110402 3M Environmental Laboratory E05-0210 Interim Report#15 Results Name: o051020a.rdb *Ollie U00110402 Results Name: o051020a.rdb E05-0210 Interim Report#15 Northern Alabama Potable Water Systems Page 31 o f 123 *Ollie U00110402 3M Environmental Laboratory E05-0210 Interim Report#15 Results Name: o051020a.rdb Printing Time: 09:41:31 AM *Ollie U00110402 Results Name: o051020a.rdb Printing Time: 09:44:09 AM E05-0210 Interim Report#15 Northern Alabama Potable Water Systems Page 32 o f 123 *Ollie U00110402 ' File:"o051^O3.wff' 3M Environmental Laboratory E05-0210 Interim Report#15 Results Name: o051020a.rdb 40C0.C0 Printing Date: Tuesday, November 29, 2005 Printing Time: 09:42:32 AM *Ollie U00110402 Page 20 of 88 Results Name: o051020a.rdb Printing Date: Tuesday, November 29, 2005 Printing Time: 09:40:15 AM Page 28 of 88 E05-0210 Interim Report#15 Northern Alabama Potable Water Systems Page 33 o f 123 *Ollie U00110402 3M Environmental Laboratory E05-0210 Interim Report#15 Results Name: o051020a.rdb *Ollie U00110402 " File:"o351CG0a.wff' Results Name: o051020a.i Printing Date: Tuesday, November 29, 2005 Printing Time: 09:42:32 AM Page 44 of 88 E05-0210 Interim Report#15 Northern Alabama Potable Water Systems Page 34 o f 123 3M Environmental Laboratory E05-0210 Interim Report#15 E05-0210 Interim Report#15 Northern Alabama Potable Water Systems Page 35 o f 123 3M Environmental Laboratory E05-0210 Interim Report#15 ATTACHMENT B: Extraction and Analytical Methods E05-0210 Interim Report#15 Northern Alabama Potable Water Systems Page 36 o f 123 3M Environmental Laboratory E05-0210 Interim Report#15 3M Environmental Laboratory Method Determination of Perftuorinaied Acids, Alcohols, Amides, and Sulfonates In Water By Solid Phase Extraction and High Performance Liquid Chromatography/Mass Spectrometry Method Number: ETS-8-154.1 Adoption Date: 28 Apr 2000 Revision Date: 5 May, 2003 Effective Date: 5 May, 2003 Approved By: William K. Reagen Manager Date ETS-8-154.1 Page 1of 17 Determination of Perfluorinated Compounds inWater Using SPE and LC/MS. E05-0210 Interim Report#15 Northern Alabama Potable Water Systems Page 37 o f 123 3M Environmental Laboratory E05-0210 Interim Report#15 1 Scope and Application This methodwas validatedfor the collection, extraction, and analytical procedurefor the determination of Perfluorooctane sulfonate (PFOS), Perfluorooctanesulfonylamide (FOSA), and Perfluorooctanoate (PFOA) ingroundwater, surfacewater, and drinkingwater samples. This method mayalso beappliedtothe determination of other perfluorinated acids, alcohols, amides, andsulfonates insimilar matrices, as long asthe defined QCelements aresatisfied andwiththe understandingthat the method is not validatedfor compounds outsidethescope of the original protocol This method is based in part onthe report "Method of Analysisfor the Determination of Perfluorooctanesulfonate (PFOS), Perfluorooctanesulfonylamide (PFOSA), and Perfluorooctanoate (POAA) inWater" (see Section 17), as developed andvalidated by Exygen Research (formerlyCentreAnalytical Laboratories, Inc.). 2 Method Summary Water samples are collectedfromasite of interest andshipped coldto an analytical facility. Perfluorinated acids, alcohols, amides, andsulfonates are extractedfrom40mLwater samples using C18solid phase extraction (SPE) cartridges. The compounds are elutedfromthe C18 cartridge, using methanol. Separation, identification, and measurement are accomplished by high performance liquid chromatography/ tandem massspectrometry (HPLC/MS/MS) analysis. High performance liquidchromatography/mass spectrometry (HPLC/MS) may be usedifthe defined QCelements aresatisfied. The concentration of each identified component is measured by comparingthe MS response of the quantitation ion produced bythat compoundtothe MSresponse of the quantitation ion produced bythesame compound inan extracted calibration standard (external standard). 3 Definitions 3.1 Analytical Sample A portion of an extracted Laboratory Sample preparedfor analysis. 3.2 Calibration Standard Asolution preparedfromtheWorking Standard (WS) and extracted accordingtothis method. The calibration standardsolutions are usedto calibratethe instrument responsewith respect to analyte concentration. 3.3 Duplicate Sample (DS) A DSisaseparate aliquot of asample, taken inthe analytical laboratorythat is extracted and analyzedseparatelywith identical procedures. Analysis of DSs comparedtothat ofthefirst aliquot give ameasure ofthe precision associatedwith laboratory procedures, but not with sample collection, preservation, or storage procedures. 3.4 Field Blank Control Sample (FB) ASTMType Iwater placed inasample container inthe laboratoryandtreated as asample inall respects, including exposuretosampling siteconditions, storage, preservation and all analytical procedures. The purpose ofthe FBisto determine iftest substances or other interferences are present inthefield environment. ETS-8-154.1 Page 2 of 17 Determination of Perfluorinated Compounds inWater Using SPE and LC/MS. E05-0210 Interim Report#15 Northern Alabama Potable Water Systems Page 38 o f 123 3M Environmental Laboratory E05-0210 Interim Report#15 3.5 Field Duplicate (FD) Asample collected induplicate at the sametime asthesample and placed under identical circumstances andtreated exactlythesamethroughout field and laboratory procedures. Analysis of FDcomparedtothat of thefirst sample gives a measure of the precision associatedwith sample collection, preservation andstorage, aswell aswith laboratory procedures. 3.6 Field Matrix Spike (FMS) Asample collected induplicatetowhich known quantities ofthetarget analytes are added inthe field atthetime of sample collection. Alternatively, the known quantity oftarget analytes may be addedtothesample bottleinthe laboratory beforethe bottles aresent tothefield. A known, specificvolume of sample must beaddedtosample containerwithout rinsing. This may be accomplished by making a"fill tothis level" line onthe outside ofthesample container. The FMS should bespiked at approximately 50-150% ofthe expected analyte concentration inthe sample. Ifthe expected range of analyte concentrations is unknown, a lowand ahighspike may be preparedto increasethe likelihoodthat aspike at an appropriate range is made. The FMSis analyzedto ascertain if any matrix effects, interferences, or stability issues may complicatethe interpretation of thesample analysis. 3.7 Field Spike Control Sample (FSCS) An aliquot ofASTMType Iwater towhich known quantities of thetarget analytes are added inthe field atthetime of sample collection (at an appropriate concentrationto be determined bythe project lead) or inthe laboratory priortotheshipment ofthe collection bottles. The FSCS is extracted and analyzed exactly like asampleto determinewhether aloss of analyte could be attributedtosamplestorage and/or shipment. A lowand high FSCS may beappropriatewhen expectedsample concentrations are not known. 3.8 Laboratory Control Sample (LCS) An aliquot ofASTMType Iwater towhich known quantities of thetarget analytes are added inthe laboratory. Two levels areincluded, one at the LLOQ(approx. 25 pg/mL), the other at a concentration of approx. 100-250 pg/mLor another concentrationto be determined bythe project lead. The LCSis extracted and analyzed exactly likealaboratorysampleto determinewhether the methodology is incontrol, andwhether the laboratory is capable of making accurate measurements atthe required method detection limit and higher. 3.9 Laboratory Sample A portion of asample receivedfromthefieldfor testing. 3.10 Limit of Detection (LOD) The LODisthe lowest concentration of an analytethat can be measured and reportedwith 99% confidencethat the analyte concentration is greaterthan zero. If required, the LOD may be determined inseveral ways, including signal-to-noise ratio andstatistical calculations. 3.11 Limit of Quantitation (LOQ) The LOQfor adataset isthe lowest concentration (LLOQ) or highest concentration (ULOQ) that can be reliably achievedwithinthe specified limits of precision and accuracy during routine operating conditions. Note: For many analytes, the LLOQanalyte concentration isselected asthe lowest non-zero standard inthe calibration curveto simplify data reporting. Sample LLOQs are matrix-dependent. 3.12 Matrix Spike (MS) ETS-8-154.1 Page 3 of 17 Determination of Perfluorinated Compounds inWater Using SPE and LC/MS. E05-0210 Interim Report#15 Northern Alabama Potable Water Systems Page 39 o f 123 3M Environmental Laboratory E05-0210 Interim Report#15 A matrixspike isan aliquot of asample, towhich known quantities of target analytes are added in the laboratory. The MSisextracted and analyzed exactlylike alaboratorysampleto determine whether thesample matrix contributes biastothe analytical results. The background concentrations ofthe analytes inthesample matrix must bedetermined inaseparate aliquot and the measuredvalues inthe MScorrectedfor background concentrations. 3.13 Method Blank An aliquot ofASTMTypeI water that istreated exactly like alaboratorysample including exposureto all glassware, equipment, solvents, and reagentsthat are usedwith other laboratory samples. The method blankis usedto determine iftest substances or other interferences are present inthe laboratory environment, the reagents, orthe apparatus. 3.14 Method Detection Limit (MDL) Determination A MDL isthestatistically calculated minimum amount of an analytethat can be measuredwith 99%confidencethat the reportedvalue isgreaterthan zero. One of several processesthat may be usedto establish a LODvalue isfound in40 CFR Part 136Appendix B. 3.15 Sample Asample is asmall portion collectedfrom alarger quantity of material intendedto representthe original source material. 3.16 Spiking Stock Standard (SSS) Asolution preparedfromstockstandards usedto preparetheworkingstandard. 3.17 Stock Standard (SS) A concentratedsolution of asingle analyte prepared inthe laboratorywith an assayed reference compound. 3.18 Working Standard (WS) Asolution of several analytes prepared inthe laboratoryfrom SSs and diluted as neededto prepare calibration standards and other required analyte solutions. 4 Warnings and Cautions 4.1 Health and Safety The acute and chronictoxicity ofthe standardsfor this method have not been precisely determined; however, each should betreated as a potential health hazard. Unknown samples maycontain high concentrations ofvolatiletoxic compounds. Sample containersshould beopened ina hood and handledwith glovesto prevent exposure. The laboratory is responsiblefor maintaining asafework environment and acurrent awareness of local regulations regardingthe handling of the chemicals usedinthis method. A referencefileof material safety datasheets (MSDS) should be availableto all personnel involved inthese analyses. 4.2 Cautions None 5 Interferences ETS-8-154.1 Page 4 of 17 Determination of Perfluorinated Compounds inWater Using SPE and LC/MS. E05-0210 Interim Report#15 Northern Alabama Potable Water Systems Page 40 o f 123 3M Environmental Laboratory E05-0210 Interim Report#15 During extraction and analysis, major potential contaminant sources are reagents and solid phase extraction devices. All materials used inthe analysesshall bedemonstratedto befreefrominterferences under conditions of analysis by running method blanks. Parts andsuppliesthat contain Teflonshould beavoided duetothe possibility of interference and/or contamination. These may include, but are not limitedto: wash bottles, Teflonlinedcaps, autovial caps, HPLC parts, etc. The useof disposable micropipettes or pipettesto aliquot standardsolutions is recommendedto make calibration standards and matrixspikes. 6 Instrumentation, Supplies, and Materials Note: Brand names, suppliers, and part numbers arefor illustrative purposes only. Equivalent performance may beachieved usingapparatus and materials otherthanthosespecified here, but demonstration of equivalent performancethat meetsthe requirements of this method isthe responsibilityof the laboratory performingthe analysis. 6.1 Instrumentation Balance, analytical (display at least 0.0001g), Mettler HPLC/MS/MSor HPLC/MSsystem, as described inSection 10. 6.2 Supplies and Materials. Sample collection bottles--LDPE (e.g., NalgeneTM) narrow-mouth bottleswith screwcap. Note: Do not useTeflon bottles or Teflon lined caps. Coolersfor sample shipment. Icefor sampleshipment. Vacuum pump, Buchi. Visiprepvacuum manifold, Supelco. Sep PakVac 6cc (1g) tC18 cartridges (part#WAT 036795),Waters. 50mLdisposable polypropylene centrifugetubes, VWR. 15mLdisposable polypropylene centrifugetubes, VWR. Disposable micropipettes (50-100pL, 100-200pL), Drummond. ClassA pipettes andvolumetricflasks, various. Hypercarbdrop-in guard column (4mm) (part # 844017-400), Keystone. Stand-alone drop-in guard cartridge holder, Keystone. 125mL LDPE narrow-mouth bottles, Nalgene. 2mL clear HPLCvial kit (cat # 5181-3400), Agilent/Hewlett Packard. Standard labequipment (graduated cylinders, disposabletubes, etc.), various. 7 Reagents and Standards Note: Suppliers and catalog numbers arefor illustrative purposes only. Equivalent performance may beachieved usingchemicals obtainedfrom other suppliers. Do not usealesser grade of chemical than those listed. ETS-8-154.1 Page 5 of 17 Determination of Perfluorinated Compounds inWater Using SPE and LC/MS. E05-0210 Interim Report#15 Northern Alabama Potable Water Systems Page 41 o f 123 3M Environmental Laboratory E05-0210 Interim Report#15 7.1 Chemicals Methanol (MeOH), HPLCgrade, JT Baker, Catalog No. JT9093-2. AmmoniumAcetate, Reagent grade, Sigma-Aldrich, Catalog No. A-7330. ASTMType IWater, prepared in-house. SodiumThiosulfate, Reagent grade, JT Baker. 7.2 Standards Potassium perfluorooctanesulfonate Perfluorooctane sulfonylamide Ammonium perfluorooctanoate Others as required. 7.3 Reagent Preparation 250mg/mLsodiumthiosulfatesolution--Dissolve25g of sodiumthiosulfate in 100mL reagent water. 40%methanol wash solution - Measure400mL methanol and adjust volumeto 1.0Lwith reagent water. 100mMammoniumacetate solution (Analysis)--Weigh 7.71g of ammoniumacetate and dissolve in 1.0Lof reagent water. Dilutethe 100mMsolution byafactor of 50to makethe 2mMammonium acetatesolution usedfor mobile phaseA. Note: Alternativevolumes may be prepared as long asthe ratios ofthe solvent tosolute ratios are maintained. 7.4 Spiking Stock Standard (SSS) Preparation Thefollowingstandard preparation procedureserves as an example and may bechangedto suit the needs of aparticular study. For example, pLvolumes may bespiked intovolumetricflasks when dilutingstocksolutionsto appropriate levels. 100pg/m L each PFO S, PFO S A , and P O A A S S S s--Weigh out 10mg of analytical standard (correctedfor percent salt and purity--i.e., 10 mg C8F17S03Kpurity90%=8.35mg C8F17S03-) and diluteto 100mLwith methanol ina 100mLvolumetricflask. Transfer to a 125mL LDPE bottle or other suitable container. Prepare aseparatesolutionfor each analyte. Solutions may bestored in a refrigerator at 42Cfor amaximum period of 6 monthsfromthe dateof preparation. 1pg/m L m ixed SS S--Add 1.0mLeach ofthe 100pg/mLSSSs (from7.4.1) to a 100mLvolumetric flask and bring uptovolumewith methanol. 0.1pg/m L m ixed SS S --Add 10.0mLof the 1.0pg/mL-mixedsolution (from7.4.2) to a 100mL volumetricflask and bring uptovolumewith methanol. 0.01pg/m L m ixed SS S --Add 10.0mLof the 0.1pg/mL-mixed solution (from7.4.3) to a 100mL volumetricflask and bring uptovolumewith methanol. S torage Conditions--Store all SSSs in a refrigerator at 42Cfor a maximum period of 6 monthsfromthe dateof preparation. 7.5 Calibration Standards Thefollowingstandard preparation procedureserves as an example and may bechangedto suit the needs of aparticular study, providedthe concentrations are calculated correctly. ETS-8-154.1 Page 6 of 17 Determination of Perfluorinated Compounds inWater Using SPE and LC/MS. E05-0210 Interim Report#15 Northern Alabama Potable Water Systems Page 42 o f 123 3M Environmental Laboratory E05-0210 Interim Report#15 1 0 0p g /m L each P FO S , P F O S A , and P O A A s to c k sta n d a rd so lu tio n s--Weigh out 10mg of analytical standard (correctedfor percent salt and purity) and diluteto 100mLwith methanol ina 100mLvolumetricflask. Transfer to a 125mL LDPE bottleor other suitable container. Prepare a separatesolutionfor each analyte. Storesolutions inarefrigerator at 42Cfor amaximum period of 6monthsfromthe date of preparation. 1pg/m L W orking Standard--Add 1.0mLeach of the 100pg/mL SSsolutions (from7.5.1) to a 100mLvolumetricflaskand bring uptovolumewith methanol. 0.1pg/m L W orking S tandard --Add 10.0mL of the 1.0pg/mL mixedsolution (from7.5.2) to a 100mLvolumetricflaskand bring uptovolumewith methanol. 0.01p g/m L W orking S tandard --Add 10.0mL of the 0.1pg/mL mixed solution (from7.5.3) to a 100mLvolumetricflaskand bring uptovolumewith methanol. S torage Conditions--Store all WSs ina refrigerator at 42Cfor a maximum period of 6 monthsfromthe dateof preparation. C alibration Standard--Prepare calibration solutions inASTMType I usingthefollowingtable as a guideline: Final Calibration Concentration Volume of Standard Volume, mL , of WS, pg/mL WS, pL of ASTM Type I Water 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 0.10 400 40 40 1.0 100 40 1.0 400 40 1.0 1000 40 Final Concentration of Calibration Standard, pg/mL, in ASTM Type I Water 0 25 50 100 250 500 750 1000 2500 10000 25000 Thestandards are processedthroughthe extraction procedure (Section 11), identical tothe laboratorysamples. The concentration ofthe calibration standard inthefinal extract isequal to 8X the initial concentration, duetothe concentration of thestandard duringthe extraction process. Storage Conditions--Store all extracted calibration standards in 15mL polypropylenetubes at 42C, for a maximum periodoftwoweeksfromthe date of preparation 8 Sample Collection and Handling Note: Sampling equipment, including automatic samplers, must befree of Teflon tubing, gaskets, and other partsthat may leach interfering analytes intothewater sample. Automatic samplersthat compositesamples overtimeshould use refrigerated polypropylenesample containers if possible. Sample bottles should not be rinsed beforesample collection. Labeling: Each sample bottle must display information regardingthe collection ofthat sample, the individual collectingthesample, and any matrixspikethat has been addedtothesample. ETS-8-154.1 Page 7 of 17 Determination of Perfluorinated Compounds inWater Using SPE and LC/MS. E05-0210 Interim Report#15 Northern Alabama Potable Water Systems Page 43 o f 123 3M Environmental Laboratory E05-0210 Interim Report#15 This includesthevolume and concentration of anyspikingsolution added andthevolume and identification of any preservatives added inthefield. Spiking: The spikingschemewill beclearly outlined inthe sampling plan, includingwhether the sampleswill bespiked inthefield or inthe laboratory priortotheshipment ofthe bottlestothe site. If spiking isto be performed inthefield, materials andspecific instructionswill be included in thesampling kit. Besureto clearly label each bottlewith spiking information if applicable. T ap W ater: Open thetap and allowthe systemtoflush until thewater temperature (15o10C) hasstabilized (usually about two minutes). Adjusttheflowto about 500mL/min and collect samples fromthe flowing stream. G round W ater: Purgethewell of standingwater using a pumpor a bailer. Collect the sample directlyfromthe pump orfromthe bailer. Surface W ater: When samplingfrom an open body ofwater, fill thesample container withwater froma representative area. S am ple D echlorination: All samples should be iced or refrigerated at 42Cand kept inthe darkfromthetime of collection until extraction. Residual chlorineshould beeliminated byadding 200pL of a250mg/mLsodiumthiosulfatesolution to eachtap-water sample and associated FB and FSCS(which may be placed ineach bottle before leavingfor thesampling site or done inthe field.). Holding T im e (HT): Results of thetime/storage study of all target analytes showedthat thethree compounds arestablefor 14days inwater samples when thesamples aredechlorinated and stored as described inthe previoussection (see also references insection 17). Therefore, laboratorysamples must beextractedwithin 14days andthe extracts analyzedwithin 30days of sample collection. Ifthe HTexceeds 14days, great care is usedwhen evaluatingfieldspikesto avoid misrepresentation ofthesample concentration. 8.1 Field Blanks Process aField Blank Control Sample (FB) alongwith each sample set (samples collectedfrom thesame general sample siteat approximatelythesametime). At the laboratory, priortosample collection, fill asample containerwith ASTMType Iwater, seal, andshipthe FBtothe sampling site alongwiththe emptysample containers. Returnthe FBtothe laboratorywiththefilledsample bottles. When sodiumthiosulfate is addedto samples, usethe same procedureto preservethe FB. 8.2 Field Duplicates Collect a Field Duplicate (FD) for everyten (10) samples collected or per eachsamplingset, if lessthan 10samples are collected. Separate FDs must becollectedfor eachtype of water sample (ground, tap, etc.) collected. Collectthe FDimmediately afterthesample. Preserve, store andship FDusingthesame procedures as usedfor thesamples. 8.3 Field Spike Control Sample (FSCS) A Field Spike Control Sample (FSCS) must be preparedfor each sampleshipment. If multiple coolers are usedto shipaset of samples, each cooler must contain a FSCS. At the laboratory, fill asample container with 100mLof ASTMType Iwater. Seal and shiptothe samplingsite alongwiththe emptysample containers and FBs. Samples may either bespiked in thefield or inthe laboratory priortoshipment. The method employedshould beconsistent throughoutthestudy. Ifthe samples areto bespiked inthefield, besureto send appropriate supplies and instructionsfor thefield personnel tofollow. ETS-8-154.1 Page 8 of 17 Determination of Perfluorinated Compounds inWater Using SPE and LC/MS. E05-0210 Interim Report#15 Northern Alabama Potable Water Systems Page 44 o f 123 3M Environmental Laboratory E05-0210 Interim Report#15 Seal and gently invert the FSCSto mix. Store andshipthe FSCS usingthe same procedures as usedfor the samples Provide information onsample collection, preservation, shipment andstorage. List applicable holdingtimes. Includesample stability and extract storage requirements. Referencethe method usedfor sample preparation, if applicable. 8.4 Field Matrix Spike (FMS) A Field Matrix Spike (FMS) must be preparedfor each sampling location. One unspikedsample fromthesame location must accompanythe FMSto determine endogenous levels inthe sample. Thesamples should beclearly identifiable as beingfromthesame location. Samples mayeither bespiked inthefield or inthe laboratory priortoshipment. The method employed should be consistent throughout thestudy. Ifthesamples areto bespiked inthefield, besuretosend appropriate supplies and instructionsfor thefield personnel tofollow. 9 Quality Control and Data Quality Objectives Analytical results ofthe FB, FMS, FD, and FSCSshould beevaluated at the conclusion ofthe studyto helpinterpret the quality of sample data. Analytical resultsfor these control/duplicate samples must be reportedwiththe sample data. 9.1 Solvent Blanks Solvent blanks are analyzedwith eachsample set to determine contamination or carryover. Aliquots of methanol represent thesolvent usedfor thestandard curve andthesample extraction. Solvent blanksshould have area countsthat are lessthan 50%ofthe area count ofthe lowest calibrationstandard. Solvent blanksshould beanalyzed priorto andfollowing each calibration curve, eachset of systemsuitabilitysamples, and after no morethan 10unknown sample extracts. If instrument carryover isa problemconsecutivesolvent blanks may be necessary. Inthis casethe area counts of thesolvent blanksshould returnto <50%of the lowest calibration standard priortothe injection offurther standards or samples. 9.2 Method Blanks A method blankconsists of an aliquot ofASTMType Iwater, equal involumetothesamples, and extracted inthe same manner asthesamples. At least two method blanks should be prepared and analyzed each daythat extractions are performedfor aparticular study or project. When analyzedthe area counts ofthesesamples must belessthan 50%of the area count of the lowest calibrationstandard. 9.3 Sample Replicates All samples, includingfieldspikes, trip blanks, etc., should beextracted at least induplicate, and in triplicate if difficultieswere encountered inthesampling and/or holdingconditions ofthe samples. The relative percent difference (RPD) of duplicatesamples or relativestandard deviation (RSD) should be lessthan 15%for the precision of sample preparation and analysisto beconsidered in control. 9.4 Matrix Spike Matrixspikes are preparedfor each sampletype and analyzedto determinethe matrix effect on the recovery efficiency. Matrixspike recoveriesshouldfall within 25%of expectedvalues. Ifthe matrix spikesfail, evaluatethe labcontrol spikes. Ifthe LCSarewithin acceptance criteriathere may be matrix issues inthesamples. Discussthese inthefinal report. ETS-8-154.1 Page 9 of 17 Determination of Perfluorinated Compounds inWater Using SPE and LC/MS. E05-0210 Interim Report#15 Northern Alabama Potable Water Systems Page 45 o f 123 3M Environmental Laboratory E05-0210 Interim Report#15 Matrixspike duplicates are prepared periodicallyto measurethe precision associatedwiththe analysis. Analyze amatrixspike and matrixspike duplicate (if prepared) inthesame run asthe original sample. Matrixspike and matrixspike duplicate concentrations shouldfall inthe mid-range ofthe initial calibration curve or should be prepared at 1.5-5times the endogenous concentration of the analyte. Spike concentrationsshouldfall inthe low-range of the initial calibration curve if extremely low-levels are expected. Generallytwo or more levels are prepared, one inthe low range ofthe curve and one inthe mid-range. This avoidsthe needto pre-screen unknown samples priorto preparation. 9.5 Laboratory Control Spike Labcontrol spikes are preparedfor each studyto ensure recovery ofthetarget analytes. These should be prepared at a minimumof 2 levels and induplicate ortriplicate. Recovery of these samples should bewithin 25%of expectedvalues, andthe RPD(or RSD) bes 15%. If recoveriesfall outsidethese limitsthesamples should be addressed inthefinal report. 10 Calibration and Standardization 10.1 Instrument Setup Note: Inthis example, a MicroMass UltimaTMLiquid ChromatographyTandem Mass Spectrometer (LC/MS/MS) is used. Other brands of LC/MS/MSs aswell assingle quadrupole massspectrometers (LC/MS) may be used as long asthe method criteriaare met. Brand names, suppliers, part numbers, and models arefor illustrative purposes only. Equivalent performance may beachieved usingapparatus and materials otherthan thosespecified here, but demonstration of equivalent performancethat meetsthe requirements ofthis method isthe responsibilityof the laboratory. The operator must optimize and document the equipment and settings used. Establishthe LC/MS/MSsystem andoperating conditions equivalent tothefollowing: Mass Spec: Micromass Ultima(Micromass) Interface: Electrospray(Micromass) Mode: Electrospray Negative, Multiple Response Monitoring (MRM) Harvard infusion pump(Harvard Instruments), for tuning Computer: COMPAQ Professional WorkstationAP200 Software: Windows NT, MassLynx 3.3 HPLC: Hewlett Packard (HP) Series 1100 HPQuaternary Pump HPVacuum Degasser HPAutosampler HPColumn Oven Note: A4x 10mm Hypercarbdrop-in guard cartridge (Keystone, part # 844017-400) may be attached on-line afterthe purgevalve and beforethesample injector port totrapany residue contaminants that may be inthe mobile phase and/or HPLCsystem. HPLCColumn: Genesis C8 (Jones Chromatography), 2.1mmx 50mm, 4pm ETS-8-154.1 Page 10 of 17 Determination of Perfluorinated Compounds inWater Using SPE and LC/MS. E05-0210 Interim Report#15 Northern Alabama Potable Water Systems Page 46 o f 123 3M Environmental Laboratory E05-0210 Interim Report#15 Column Temperature: 35C InjectionVolume: 15pL Mobile Phase (A): 2mMAmmoniumAcetate inASTMType Iwater (See7.3.1) Mobile Phase (B): Methanol Tim e, m in P ercent M obile Percent M obile Phase A Phase B Flow Rate, m L/m in 0.0 60 40 0.3 0.4 60 40 0.3 1.0 10 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 13.5 60 40 0.4 14.0 60 40 0.3 Note: Other HPLCgradients may be used as long asthe method criteria are met. It may be necessaryto adjust the HPLCgradient inorder to optimize instrument performance. Columnswith different dimensions (e.g. 2.1mmx 30mm) and columnsfrom different manufacturers (Keystone Betasil C18 etc.) may be used. Ions M onitored: A nalyte P rim ary Ion P ro d u ct Ion PFOA PFOS FOSA 413 499 498 169 99 78 A pproxim ate R etention T im e (m inutes) 5.0 5.2 5.8 Other product ions may be chosen at the discretion ofthe analyst, although m/z 99 issuggested for PFOS. Useofthesuggested primaryion is recommended. Retentiontimes mayvaryslightly, on aday-to-day basis, depending onthe batch of mobile phase etc. Drift in retentiontimes is acceptablewithin an analytical run, as long asthe drift continuesthroughthe entireanalysis and the standards areinterspersedthroughout the analytical run. 10.2 Tune File Parameters Thefollowingvalues are provided as an example. Actual values mayvaryfrominstrument to instrument. Also, thesevalues may be changedfromtimetotime inorderto optimizefor greatest sensitivity. A nalyte PFOA PFOS FOSA Dw ell, sec 0.2-0.4 0.2-0.4 0.2-0.4 C ollision Energy, eV 10-25 30-60 20-50 Cone, V 20-30 50-80 30-60 ETS-8-154.1 Page 11 of 17 Determination of Perfluorinated Compounds inWater Using SPE and LC/MS. E05-0210 Interim Report#15 Northern Alabama Potable Water Systems Page 47 o f 123 3M Environmental Laboratory E05-0210 Interim Report#15 Source Capillary Hexapole 1 Aperture 1 Hexapole 2 Source Block Temp. Desolvation Temp. A nalyzer LM Res 1 HM Res 1 lEnergy 1 Entrance Exit LM Res 2 HM Res 2 lEnergy 2 Multiplier G as Flow s Cone Gas Desolvation P re s s u re s Gas Cell Set 2.6-3.5kV 0.5V 0.2V 0.8V 100-150C 250-400C Set 12.5-15.0V 12.5-15.0V 0.7V -2V 1V 11.0V 11.0V 1.0V 650V Set 150L/hr 700L/hr Set 3.0e-3mbar ETS-8-154.1 Page 12 of 17 Determination of Perfluorinated Compounds inWater Using SPE and LC/MS. E05-0210 Interim Report#15 Northern Alabama Potable Water Systems Page 48 o f 123 3M Environmental Laboratory E05-0210 Interim Report#15 10.3 Calibration Curve Analyzethestandard curves priorto each set of samples. Thevalidated method specifiesthat thestandard curveshould be plotted using alinear fit, weighted 1/x or unweighted. However, the standard curve may also be plotted by quadraticfit (y=ax'2+bx +c), weighted 1/x or unweighted, usingsuitable software. The calibration curves may include but should not beforcedthrough zero. The mathematical method usedto calculatethe calibration curveshould be applied consistentlythroughout astudy. Any changeshould bethoroughly documented inthe rawdata. Ifthe calibration curve does not meet acceptance criteria performroutine maintenance or prepare a newstandard curve (if necessary) and reanalyze. For purposes of accuracywhen quantitating lowlevels of analyte, it may be necessaryto usethe lowend of the calibration curve ratherthan thefull range. For example, when attemptingto quantitate approximately 50 pg/mLof analyte, generate acalibration curve consisting ofthe standardsfrom25 pg/mLto 1000 pg/mL ratherthanthefull range ofthe curve (25 pg/mLto 25000 pg/mL). Thiswill reduce inaccuracy attributedto linear regression weighting of high concentration standards. High and/or lowpoints may beexcludedfromthe calibration curvesto provide abetterfit overthe linear range appropriatetothe data or becausethey did not meet the pre-determined acceptance criteria. Low-level curve pointsshould also beexcluded if their area counts are not at least twice that ofthe method and/or solvent blanks. Any curve point may be rejected dueto a badinjection orfailingto meet accuracy requirements of 25%(and 30%for the LLOQ). Justificationfor exclusion of calibration curve pointswill be noted inthe rawdata. A minimumof 6 pointswill be usedto construct the calibration curve. 10.4 Continuing Calibration Verification (CCV) Continuing calibration verifications (CCV) are analyzedtoverifythe accuracy of the calibration curve. Analyze amid-range calibrationstandard, one ofthe same standards usedto construct the calibration curve, at aminimumafter everytenth sample, not includingsolvent blanks, with a minimumof one per sampleset. Calibration verification injections must bewithin 25%to be considered acceptable. The calibration curve andthe last passing CCVwill then bracket acceptablesamples. Multiple CCVlevels may be used. 10.5 System Suitability A minimumofthreesystemsuitabilitysampleswill be injected at the beginning and end of each analytical run. Typicallythesesamples are run priortothe calibration curve. Thesystem suitability injections must have areacountswith an RSDof s5%and aretentiontime RSDof s2% when evaluated independently. 11 Procedures 11.1 Extraction Scheme Allowsamples to equilibrateto roomtemperature. Thoroughly mixsamples bygently invertingthe sample bottle. Measure40mL of sample into50mL polypropylene centrifugetubes (Spikethe Matrix spikes as required*, replace lidand mixwell). Note: * Samples may needto be prescreenedto determine an appropriate matrix spike level (typically 50-150%of sample concentration). Alternativelythe samples could bespiked at more than one level, allowingfor the inappropriatespike level to beeliminated. ETS-8-154.1 Page 13 of 17 Determination of Perfluorinated Compounds inWater Using SPE and LC/MS. E05-0210 Interim Report#15 Northern Alabama Potable Water Systems Page 49 o f 123 3M Environmental Laboratory E05-0210 Interim Report#15 Condition the C18 SPEcartridges (1g, 6mL) by passing approximately 10mL methanol followed byapproximately 50mLASTMType Iwater (flowrateapproximately2 drop/sec). Do not let column rundry. Note: Forthefollowing steps, maintain a~1drop/secflowrate. Do not allowthe columnto run dry at anytime. Loadthe analytical sample ontothe C18 SPEcartridge. Discard eluate. Ten mLof the40%methanol inwater wash mixture is passedthroughthe C18 SPEcartridgeto rinseaway potential interferences andthen discarded. This step must beomitted if perfluorinated compoundswith chain lengths lessthan C8 aretargeted sincethesewill be lost duringthis wash step. Elutewith exactly 5mLof 100%methanol. Collect eluate intograduated 15mL polypropylene centrifugetubes. This isthetarget elutionfraction (final volume approximately4.5 mLas not all of thesolvent will leavethe SPEcolumn. Thiswill not affectthe calculations inanyway sincethe curve isalso extracted). Analyze aportion ofthetarget elutionfraction eluent using negative electrospray HPLC/MS/MS or HPLC/MS. Note: Samples are concentrated by afactor of eight duringthe extraction; Initial Vol =40mL ^ Final Vol. =5mL. Samples arestable at roomtemperaturefor at least 24 hours. Analytical samples may bestored inarefrigerator at 42C until analysis. S tandardization o f C 18 S P E co lum ns--If poor recoveries are observed, it may be necessaryto standardizethe C18 SPEcolumns inthefollowing manner before analyzing samples. Use astandardwith an analyte concentration between 1000and4000 pg/mL. Repeat the extraction schemefromthe beginning upthrough the elutingwith ~5mL 100%methanol. After the elutingwith ~5mL 100%methanol step, collect an additional post-elutionfraction by elutingwith an additional 5mLof 100%methanol. Analyze bothfractions by HPLC/MS/MSor HPLC/MS. Ifthetarget fraction contains a minimumof 85%of the respective analytes, it may beconsidered acceptable. Ifthewash containssignificant standard (>15%), eitherthewashvolume or percentage of MeOH should be decreased. Ifthe post-elutionfraction contains significant standard (>15%), thetarget elutionvolume should be increased. 11.2 Sample Analysis Set upanalysis sample queue. Inject thesamevolume (between 5-25pL) of each standard, analytical sample and blankintothe instrument. All samples with aconcentration >ULOQmust bediluted and reanalyzed. If dilution ofthefinal extract failsto produce acceptable results (e.g. poor MS recoveries) dilutethe original sample and re-extract. 12 Data Analysis and Calculations Calculatethe analytical sample (extract) concentrationfromthestandard curve usingthefollowing equation: ETS-8-154.1 Page 14 of 17 Determination of Perfluorinated Compounds inWater Using SPE and LC/MS. E05-0210 Interim Report#15 Northern Alabama Potable Water Systems Page 50 o f 123 3M Environmental Laboratory E05-0210 Interim Report#15 Extract Concentration, pg/mL=(Peak area- intercept) (slope) Calculatethe percent recoveryofthe FSCS usingthefollowing equation: FSCS %rec. =(FSCS conc., pg/mL) x 100 (Conc. added, pg/mL) Calculatethe percent recoveryofthe MSs usingthefollowing equation: MS%rec. =(MSconc., pg/mL- Sample conc., pg/mL) x 100 (Conc. added, pg/mL) 13 Method Performance Note: Any method performance parameters that are not achieved must beconsidered inthe evaluation ofthe data. Nonconformanceto anyspecified parameters must bedescribed and discussed inany reporting ofthe data. If criterialistedinthis method performancesection are not met, maintenance may be performed onthesystemandsamples reanalyzed, or other actionstaken as determined bythe analyst. Document all actions inthe rawdata. If data areto be reportedwhen performance criteria have not been met, the data must be footnoted ontables and discussed inthetext ofthe report. 13.1 System Suitability A minimumofthreesystemsuitabilitysamples will be injected at the beginning and end of each analytical run. Typicallythesesamples are run priortothe calibration curve. The system suitability injections must havearea countswith an RSDof s5%and aretentiontime RSDof s2% when evaluated independently. 13.2 Quantitation C alibration Curve: The coefficient of determination (r2)valuefor the calibration curve must be greaterthan or equal to 0.990. Each point inthe curve must bewithin25%of thetheoretical concentration withthe exception ofthe LLOQ, which may bewithin30%. D em onstration o f Specificity: Specificity isdemonstrated by chromatographic retention time (within 3%of standard) andthe massspectral response of unique ions. 13.3 Sensitivity S o lv en t B lanks and M ethod Blanks: Solvent and method blankarea counts must be <50% that ofthe lowest standard used inthe calibration curve. Lim its o f Q uantitation (LO Q ): The lower LOQ(LLOQ) isthe lowest non-zero activestandard in the calibration curve; the peak area of the LLOQmust beat least 2Xthat of the extraction blank. Bydefinition, the measuredvalue ofthe LLOQmust bewithin 30%ofthetheoretical value. ETS-8-154.1 Page 15 of 17 Determination of Perfluorinated Compounds inWater Using SPE and LC/MS. E05-0210 Interim Report#15 Northern Alabama Potable Water Systems Page 51 o f 123 3M Environmental Laboratory E05-0210 Interim Report#15 13.4 Accuracy C C V Perform ance: Calibrationverification injections must bewithin25%to be considered acceptable. The calibration curve andthe last passing CCVwill then bracket acceptablesamples. Multiple CCVlevels may be used. M atrix Spikes: Matrix spike percent recoveries must bewithin 25%of the spiked concentration. If matrix effects aresuspected, evaluatethe LCS resultsto determine if amatrix effects are present andifthe method is incontrol based on compliant LCS results. Discuss all results inthe analytical report. 13.5 Precision Reproducibility: Reproducibility of the method is defined bythe results of duplicate or triplicate analysis of samples. A RPDor RSDof s 15%will beconsidered acceptable. System Suitability: The systemsuitability injections must have area counts with an RSDof s5% and a retentiontime RSDof s2%when evaluated independently. 14 Pollution Prevention and Waste Management Sample extract waste andflammablesolvent isdiscarded in high BTUcontainers, and glass pipettewaste isdiscarded in broken glass containers located inthe laboratory. 15 Records Each data package generatedfor astudy must havethefollowing information included: study or project number, acquisition method, integration method, sample name, extraction date, dilution factor (if applicable), and analyst. Print thetune page, sample list, and acquisition methodto include inthe appropriatestudyfolder. Copythese pages andtape intothe instrument runlog. Plot the calibration curves as described inthis method, then print these graphs andstore inthe studyfolder. Print data integration summary, integration method, and chromatograms andstore inthestudy folder. Summarize data usingsuitablesoftware andstore inthe studyfolder. 16 Attachments None. 17 References "Method ofAnalysisfor the Determination of Perfluorooctanesulfonate (PFOS), Perfluorooctane sulfonylamide (PFOSA), and Perfluorooctanoate (POAA) inWater", E. Wickremesinhe andJ. Flaherty, Study Number 023-002, CentreAnalytical Laboratories, Inc., State College, Pennsylvania, January2000. Validation report for the"Method ofAnalysis for the Determination of Perfluorooctane sulfonate (PFOS), Perfluorooctanesulfonylamide (PFOSA), and Perfluorooctanoate (POAA) inWater", E. Wickremesinhe andJ. Flaherty, Study Number 023-002, CentreAnalytical Laboratories, Inc., State College, Pennsylvania. ETS-8-154.1 Page 16 of 17 Determination of Perfluorinated Compounds inWater Using SPE and LC/MS. E05-0210 Interim Report#15 Northern Alabama Potable Water Systems Page 52 o f 123 3M Environmental Laboratory E05-0210 Interim Report#15 18 Affected Documents None. 19 Revisions Revision Number 1 Revision Number U p d a te d to th e n e w form at. C h a n g e d Title. S e ctio n 1: S ta te s th e va lid a tio n o f 3 an alyte s, re m o ve s re fe re n ce to E P A d o cum e nt th a t's no lo n g e r applicable. S ection 2: P ro vid e d fo r the extraction o f m ore than the 3 valida ted analytes, allow s the use o f a LC /M S system , n o t on ly the LS /M S /M S pre vio u sly m entioned. S ection 3: R e v is e d d e finitions fo r fie ld m a trix spike, fie ld co n tro l spike, LLO Q , m e th o d blank, a n d MDL. S ection 5: R e w o rd e d the interferences, ad de d reco m m en datio n to use dispo sable pipettes. S ection 6: R e ca te g o rize d a n d p a re d down. S ectio n 7: C h a n g e d sto ra g e tim e to 6 m on th s. A d d e d m o re ca lib ra tio n p o in ts to the table. S ection 8 :A d ded statem e nt a d dre ssing la be lin g re q u ire m e n ts an d spiking p ro ce d u re s. E x p a n d e d se ctio n 8.8. S ection 9: N e w S ection S ection 10: C h a n g e d som e o f the p a ra m e te rs in the tables. A llo w e d fo r use o f diffe re nt in stru m en ta tion . A d d e d in fo rm a tio n from sectio n 12 o f p re vio u s version, e xte n sive ly revised. S e ctio n 11 (se ctio n 9 in p re v io u s ve rsio n ): C la rific a tio n o f w a sh step, s ta te d e xa ct volum e o f e lua te is 5 m L, re v is e d sta n d a rd iza tio n process, re m o ve d req uire m en t to use LC /M S /M S . S ection 12 (sectio n 13 in p re vio u s version: n o changes S ection 13 (section 14 in p re vio u s version ): E xte n sive ly rew ritten. S ection 14 (sectio n 15 in p re vio u s version): no cha ng es S ection 15 (sectio n 16 in p re vio u s version): M in o r cha ng es to re co rd in g requirem ents. S ection 16 (sectio n 17 in p re vio u s version): R e m o ve d attachm ent. S ection 17 (sectio n 18 in p re vio u s version ): R e m o ve d re fe re n ce to EPA do cum e nt tha t no lo n g e r ap p lie d to this SOP. S ectio n 18: N e w section. Revision Date 7 ETS-8-154.1 Page 17 of 17 Determination of Perfluorinated Compounds inWater Using SPE and LC/MS. E05-0210 Interim Report#15 Northern Alabama Potable Water Systems Page 53 o f 123 3M Environmental Laboratory E05-0210 Interim Report#15 ATTACHMENT C: P P Ar o t o c o l a n d r o t o c o l m e n d m e n t s E05-0210 Interim Report#15 Northern Alabama Potable Water Systems Page 54 o f 123 3M Environmental Laboratory E05-0210 Interim Report#15 Exygen Protocol Number: P0001131 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: P0001131 Performing Laboratory: Exygen Research 3058 Research Drive State College, PA 16801 Phone: (814) 272-1039 Sponsor Representative: Michael A. Santoro Director of Regulatory Affairs 3M Building 0236-01-B-10 St. Paul, MN 55144 Phone: (651) 733-6374 E05-0210 Interim Report#15 Northern Alabama Potable Water Systems Page / oj 65 Page 55 o f 123 3M Environmental Laboratory E05-0210 Interim Report#15 Exygen Protocol Number: P0001131 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 E05-0210 Interim Report#15 Northern Alabama Potable Water Systems Page 2 o f 65 Page 56 o f 123 3M Environmental Laboratory E05-0210 Interim Report#15 Exygen Protocol Number: P0001131 PROTOCOL APPROVAL Study Title: Analysis of 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 Exygen Protocol Number: P0001131 __________ ____________ Michael A. Santoro, Sponsor Representative 3M Compaify bhn M. Flaherty, Principal Investigator Exygen Research Richard A. Grafzzini, Pi^sident, Facility Management Exygen Research Lydua Shaffer, Technip^i^ead, Quality Assurance Unit Exygen Research uno )at Date -ocr- 'zf Date //zq /os Date E05-0210 Interim Report#15 Northern Alabama Potable Water Systems Page 3 o f 65 Page 57 o f 123 3M Environmental Laboratory E05-0210 Interim Report#15 Exygen Protocol Number: P0001131 TABLE OF CONTENTS TITLE PAGE............................................................................................... DISTRIBUTION.......................................................................................... PROTOCOL APPROVAL.......................................................................... TABLE OF CONTENTS............................................................................ INTRODUCTION........................................................................................ TEST MATERIALS.................................................................................... OBJECTIVE................................................................................................ TESTING FACILITY.................................................................................. STUDY DIRECTOR.................................................................................... SPONSOR REPRESENTATIVE................................................................ PRINCIPAL INVESTIGATOR.................................................................. PROPOSED EXPERIMENTAL START AND TERMINATION DATES IDENTIFICATION AND JUSTIFICATION OF THE TEST SYSTEM .... SAMPLE PROCUREMENT, RECEIPT AND RETENTION................... SAMPLE IDENTIFICATION.................................................................... ANALYTICAL PROCEDURE SUMMARY............................................. VERIFICATION OF ANALYTICAL PROCEDURE................................ METHOD FOR CONTROL OF BIAS....................................................... STATISTICAL METHODS....................................................................... GLP STATEMENT..................................................................................... REPORT...................................................................................................... SAFETY AND HEALTH........................................................................... AMENDMENTS TO PROTOCOL............................................................ DATA RECORD KEEPING...................................................................... QUALITY ASSURANCE.......................................................................... RETENTION OF DATA AND ARCHIVING........................................... APPENDIX I, ANALYTICAL METHODS............................................... 1 2 -> 4 5 6 7 7 7 7 8 8 9 9 9 11 11 11 11 12 13 13 14 14 15 E05-0210 Interim Report#15 Northern Alabama Potable Water Systems Page 4 o f 65 Page 58 o f 123 3M Environmental Laboratory E05-0210 Interim Report#15 Exygen Protocol Number: P0001131 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 Molecular Weight: 338 supplied as the potassium salt (C4FqS 0 3'K+) Lot Number: 101 Purity: 96.7% Transitions Monitored: 299 -> 99 Structure: PFHS Chemical Name: Perfluorohexanesulfonate Molecular Weight: 438 supplied as the potassium salt (C6Fi3S0 3 'K+) Lot Number: SE036 Purity: 98.6% Transitions Monitored: 399 - 80 Structure: E05-0210 Interim Report#15 Northern Alabama Potable Water Systems Page 5 o f 65 Page 59 o f 123 3M Environmental Laboratory E05-0210 Interim Report#15 Exygen Protocol Number: P0001 PFOS Chemical Name: Perfluorooctanesulfonate Molecular Weight: 538 supplied as the potassium salt (C8Fi7S 0 3'K+) Lot Number: 217 Purity: 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: "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" V0001782: "Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Sediment by LC/MS/MS" V0001783: "Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Fish and Clams by LC/MS/MS" V0001784: "Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Vegetation by LC/MS/MS" V0001785: "Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Small Mammal Liver by LC/MS/MS" V0001786: "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 o f 65 E05-0210 Interim Report#15 Northern Alabama Potable Water Systems Page 60 o f 123 3M Environmental Laboratory E05-0210 Interim Report#15 Exygen Protocol Number: P0001131 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 of Regulatory Affairs 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 j ohn. flaherty@exygen. com PROPOSED EXPERIMENTAL START AND TERMINATION 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. E05-0210 Interim Report#15 Northern Alabama Potable Water Systems Page 7 o f 65 Page 61 o f 123 3M Environmental Laboratory E05-0210 Interim Report#15 Exygen Protocol Number: P0001131 IDENTIFICATION AND JUSTIFICATION OF THE TEST SYSTEM The following are the test systems for this study: Water (groundwater and surface water) Soil Sediment Fish Clams Vegetation Small Mammal Liver 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. SAMPLE PROCUREMENT, RECEIPT AND RETENTION Water, soil, sediment, fish, clam, vegetation, small mammal liver and small mammal serum samples will be received at Exygen directly from Weston Solutions. The details of sample procurement 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 2C-8C. 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 < -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 < -10C. The receipt and processing of the samples will be documented in the final report and raw data associated with the study. E05-0210 Interim Report#15 Northern Alabama Potable Water Systems Page 8 o f 65 Page 62 o f 123 3M Environmental Laboratory E05-0210 Interim Report#15 Exygen Protocol Number: P0001131 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: V0001780: "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" V0001782: "Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Sediment by LC/MS/MS" V0001783: "Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Fish and Clams by LC/MS/MS" V0001784: "Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Vegetation by LC/MS/MS" V0001785: "Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Small Mammal Liver by LC/MS/MS" V0001786: "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 bottles will be 500 mL precleaned Sci/Spec Premier wide mouth HDPE bottles. These bottles have been routinely used for fluorochemical sample Page 9 o f 65 E05-0210 Interim Report#15 Northern Alabama Potable Water Systems Page 63 o f 123 3M Environmental Laboratory E05-0210 Interim Report#15 Exygen Protocol Number: P0001 131 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 (13C PFOA). PFOA and 13C PFOA are included in the solutions used to spike the samples. The results for PFOA and 13C 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 containers/bags 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 centrifuge 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: Matrix Low Spiking Level High Spiking Level Water 500 ng/L 5000 ng/L Soil 4 ng/g 40 ng/g Sediment 4 ng/g 40 ng/g Fish 10 ng/g 100 ng/g Clams 10 ng/g 100 ng/g Vegetation 10 ng/g 100 ng/g Small Mammal Liver 10 ng/g 100 ng/g Small Mammal Serum 10 ng/mL 100 ng/mL E05-0210 'Interim Report#15 Northern Alabama Potable Water Systems Page 10 o f 65 Page 64 o f 123 3M Environmental Laboratory E05-0210 Interim Report#15 Exygen Protocol Number: P0001131 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 final 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). E05-0210 Interim Report#15 Northern Alabama Potable Water Systems Page II o f 65 Page 65 o f 123 3M Environmental Laboratory E05-0210 Interim Report#15 Exygen Protocol Number: P0001131 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. All applicable requirements for reporting of study results as per 40 CFR 792.185. SAFETY AND HEALTH Laboratory personnel 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). E05-0210 Interim Report#15 Northern Alabama Potable Water Systems Page 12 o f 65 Page 66 o f 123 3M Environmental Laboratory E05-0210 Interim Report#15 Exygen Protocol Number: P0001131 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 sufficient 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 from 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): Sample tracking sheet(s) Sample receipt records, storage history, and chains of custody History and preparation of standards (stock, fortification, calibration) Description of any modifications to the method Instrument run sheets, bench-sheets or logs Analytical data tables All chromatographic and instrumental conditions Sample extraction and analysis dates A complete listing of study personnel, signatures and initials Chronological presentation of all study correspondence Any other documentation necessary for the reconstruction of the study Chromatograms- All chromatograms will contain the following: Sample identification, injection date, arrow or other indication of the area of interest, and injection number corresponding to 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). Page 13 o f 65 E05-0210 Interim Report#15 Northern Alabama Potable Water Systems Page 67 o f 123 3M Environmental Laboratory E05-0210 Interim Report#15 Exygen Protocol Number: P0001131 As part of the documentation the 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. E05-0210 Interim Report#15 Northern Alabama Potable Water Systems Page 4 o f 65 Page 68 o f 123 3M Environmental Laboratory E05-0210 Interim Report#15 Exygen Protocol Number: P0001131 APPENDIX I ANALYTICAL METHODS V0001780: "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" V0001782: "Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Sediment by LC/MS/MS" V0001783: "Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Fish and Clams by LC/MS/MS" V0001784: "Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Vegetation by LC/MS/MS" V0001785: "Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Small Mammal Liver by LC/MS/MS" V0001786: "Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Small Mammal Serum by LC/MS/MS" E05-0210 Interim Report#15 Northern Alabama Potable Water Systems Page 15 o f 65 Page 69 o f 123 3M Environmental Laboratory E05-0210 Interim Report#15 Exygen Protocol Number: P0001131 ANALYTICAL METHOD Method Number: V0001780 Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Water by LC/MS/MS Analytical Testing Facility: Exygen Research 3058 Research Drive State College, PA 16801 Approved By: c Paul Connolly Technical Leader, LC-MS, Exygen Research 7 ,1 fl? fl x X / lohn Flaherty / ' VV iircoe PD rr<easoiidflenntt , OH npperr ations, Exygen Research <0)zA/oii Date Date Total Pages: 7 E05-0210 Interim Report#15 Northern Alabama Potable Water Systems Page 16 o f 65 Page 70 o f 123 3M Environmental Laboratory E05-0210 Interim Report#15 Exygen Protocol Number: P0001131 Exygen Research Method Number V0001780 | ANALYTICAL METHOD Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in W a te r b y 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 M ass Spectrometric Detector (LC/MS/MS) in water. 2.0 Safety 2.1 Always observe safe laboratory practices. 2.2 Consult the appropriate MSDS before handling any chemical for proper s a fe ty precautions. 3.0 Sample Requirement 3.1 At least 40 mL o f test sample for extraction. 3.2 No sample processing is needed for water samples. 3.3 Samples stored refrigerated should be allowed to equilibrate to room temperature. 3.4 All samples must be thoroughly mixed before being sampled fo r e x tra c tio n . 3.5 Any samples containing particles should be centrifuged at -3000 rpm for -5 minutes and the supernatant used for the extraction. 3.6 Sample collection procedures will be specified in the sampling plan for th is project. 4.0 Reagents and Standards 4.1 Water - HPLC grade 4.2 Methanol - HPLC grade 4.3 Ammonium Acetate - A.C.S. Reagent Grade 4.4 Perfluorooctanoic Acid - Sigma-Aldrich 5.0 Instrument and Equipment 5.1 A high performance liquid chromatograph capable o f pumping up to 2 solvents equipped with a variable volume injector capable of injecting 5-200 pL connected 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 0.00001 g. 5.4 50 mL disposable polypropylene centrifuge tubes. 5.5 15 mL disposable polypropylene centrifuge tubes. 5.6 Disposable micropipets (50-1 OOuL, 100-200uL). 5.7 125-mLLDPE narrow-mouth bottles. 5.8 2 mL clear HPLC vial kit. 5.9 Disposable pipettes. 5.10 Autopipettes (100-1000 pL and 10-100 pL), with disposable tips. 5.11 Waters Sep Pak Vac 6 cc (lg) tC18 SPE cartridges. Page 2 o f 7 E05-0210 Interim Report#15 Northern Alabama Potable Water Systems Page 17 o f 65 Page 71 o f 123 3M Environmental Laboratory E05-0210 Interim Report#15 Exygen Protocol Number: P0001131 Exygen Research Method Number V0001780 ANALYTICAL METHOD Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Water b v LC/MS/MS ' 5.12 SPE vacuum manifold. 5.13 Centrifuge capable o f spinning 50 mL polypropylene tubes at 3000 rpm. 6.0 Chromatographic System 6.1 Analytical Column: Fluophase RP (Keystone Scientific), 2.1 mm x 50 mm, 5 p (P/N: 82505-052130) 6.2 Temperature: 30C 6.3 Mobile Phase (A) : 2 mM Ammonium Acetate in Water 6.4 Mobile Phase (B) : Methanol 6.5 Gradient Program: Time (min) 0.0 1.0 8.0 20.0 22.5 %A 65 65 25 25 65 Flow Rate % B (mL/min) 35 0.3 35 0.3 75 0.3 75 0.3 35 0.3 6.6 Injection Volume: 15 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 to optimize the HPLC system. 7.0 MS/MS System 7.1 Mode: Electrospray Negative MRM mode, monitoring 4 1 3 - 369 m /z. The above conditions are intended as a guide and may be changed in order to optimize the MSMS system. 8.0 Preparation o f Solutions 8.1 Mobile Phase 8.1.1 2 mM ammonium acetate in water is prepared by adding 0.154 g o f ammonium acetate to 1000 mL o f water. Alternate volumes may be prepared. Page 3 of ' E05-0210 Interim Report#15 Northern Alabama Potable Water Systems Page 18 o f 65 Page 72 o f 123 3M Environmental Laboratory E05-0210 Interim Report#15 Exygen Protocol Number: P0001131 Exygen Research Method Number V0001780 | ANALYTICAL METHOD Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Water by LC/MS/MS 9.0 Standard Preparation 9.1 Standard Stock/Fortification Solution 9.1.1 Prepare a stock solution o f ~100 pg/mL o f PFOA by weighing 10 m g of analytical standard (corrected for purity) and dilute to 100 mL with methanol in a 125-mL LDPE bottle. 9.1.2 A 10 pg/mL fortification solution o f PFOA is prepared by bringing 10 mL of the 100 pg/mL solution to a final volume of 100 with m ethanol in a 125 mL LDPE bottle. 9.1.3 A 1.0 pg/mL fortification solution o f PFOA is prepared by bringing 10 mL of the 10 pg/mL solution to a final volume of 100 with methanol m a 125 mL LDPE bottle. 9.1.4 A 0.1 pg/mL fortification solution o f PFOA is prepared by bringing 10 mL of the 1.0 pg/mL solution to a final volume o f 100 with methanol in a 125 mL LDPE bottle. 9.1.5 A 0.01 pg/mL fortification solution o f PFOA is prepared by bringing 10 mL o f 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 fortification solutions are to be stored in a refrigerator at approximately 4C and are stable for a maximum period of 6 months from the date o f preparation. 9.2 Standard Calibration Solutions 9.2.1 9.2.2 LC/MS/MS calibration standards are prepared in HPLC water. The calibration standards are processed through the extraction procedure, identical to samples. The following is a typical example: additional concentrations may be prepared as needed. Final Concentration Fortification Volume of Concentration of Calibration o f Fortification Volume Fortified Control 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 XCmmddyy-2 10 400 40 100 XCmmddyy-3 100 100 40 250 XCmmddyy-4 100 200 40 500 XCmmddyy-5 100 400 40 1000 XCmmddyy-6 * The extracted concentration o f the calibration standard is equal to 8x its initial concentration, due to the concentration o f the standard during the extraction (SPE). XC = extracted calibration standard. Page 4 of ^ E05-0210 Interim Report#15 Northern Alabama Potable Water Systems Page 19 o f 65 Page 73 o f 123 3M Environmental Laboratory E05-0210 Interim Report#15 Exygen Protocol Number: P0001131 Exygen Research Method Number V0001780 ANALYTICAL METHOD Method o f Analysis for the Determination o f Perfluorooctanoic Acid (P F O A ) in W a te r b y LC/MS/MS 9.2.3 9.2.4 9.2.5 A zero standard solution (reagent blank) must be prepared w ith ea c h set o f standards extracted. Store all extracted calibration standards in 15-mL polypropylene tubes at 2C to 6C, up to two weeks. Alternate volumes and concentrations o f standards may be prepared as needed. 10.0 Batch Set Up 10.1 Each batch o f samples extracted (typically 20 or less) must in c lu d e at least one reagent control (method blank using H P L C w a te r) a n d tw o re a g e n t controls fortified at known concentrations (la b c o n tr o l s p ik e ) to v e rify procedural recovery for the batch. 10.2 Requirements for field and laboratory duplicates and spikes will be s p e c ifie d in the quality assurance plan for this project. 11.0 Sample Extraction 11.1 Measure 40 mL o f sample or a portion o f sample diluted to 40 mL with w a te r into 50 mL polypropylene centrifuge tubes (fortify as needed, replace lid an d mix well). 11.2 Condition the Cig SPE cartridges (1 g, 6 mL) by passing 10 mL m e th a n o l followed by 5 mL o f HPLC water (~ 2 drop/sec). Do not let column ru n d ry 11.3 Load sample on conditioned Cig SPE cartridge. Discard eluate. 11.4 Elute with ~5 mL 100% methanol. Collect 5 mL o f eluate into g ra d u a te d 15 mL polypropylene centrifuge tubes (final volume = 5 m L ). 11.5 Analyze samples using electrospray LC/MS/MS. 12.0 Chromatography 12.1 Inject the same amount o f each standard, sample and fortified sample in to th e LC/MS/MS system. A calibration standard must precede and fo llo w all analyzed samples. 12.2 Standards o f PFOA corresponding to at least five or more concentration le v e ls must be included in an analytical set. 12.3 An entire set o f extracted calibration standards must be in c lu d e d at th e beginning and at the end o f a sample set. E x tr a c te d s ta n d a rd s m u s t be interspersed between every 5-10 samples. As a n a l te r n a tiv e , a n e n tir e set o f extracted calibration standards may be injected at th e b e g in n in g o f a set followed by extracted calibration standards interspersed e v e ry 5 -1 0 s a m p le s (to account for a second set o f extracted standards). In either c a s e , e x tra c te d calibration standards must be the first and last injection in a s a m p le set 12.4 Use linear standard curves for quantitation. Linear standard c u rv e s are generated for the analyte by linear regression using 1/x weighting o f p e a k a re a Page 5 of E05-0210 Interim Report#15 Northern Alabama Potable Water Systems Page 20 o f 65 Page 74 o f 123 3M Environmental Laboratory E05-0210 Interim Report#15 Exygen Protocol Number: P0001131 Exygen Research Method Number V0001780 | ANALYTICAL METHOD Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Water by LC/MS/MS 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 responses should be further diluted and reanalyzed. 13.0 Acceptance Criteria 13.1 Chromatogram must show a peak of a daughter ion at 369 amu from a p a re n t o f 413 amu. The 413 amu parent corresponds to the PFOA anion, w h ile the daughter ion (369 amu) represents the loss of carbon dioxide 13.2 Method blanks must not contain PFOA at levels greater than the LOQ. If a blank contains PFOA at levels greater than 50 ng/L, then a new blank s a m p le must be obtained and the entire set must be re-extracted. 13.3 Recoveries o f control spikes and matrix spikes must be between 7 0 -1 3 0 % o f their known values. If a control spike falls outside the acceptable lim its, th e entire set o f samples should be re-extracted. Any matrix s p ik e o u ts id e 7 0 130% should be evaluated by the analyst to determine if r e - e x tr a c tio n is warranted. 13.4 Any calibration standard found to be a statistical outlier by using the H u g e Error Test, may be excluded from the calculation o f the calibration c u rv e . However, the total number o f extracted calibration standards that c o u ld be excluded must not exceed 20% o f the total number o f extracted standards injected. 13.5 The correlation coefficient (R) for calibration curves generated must be >0.992 (R2 >0.985). If calibration results fall outside these limits, th e n appropriate steps must be taken to adjust instrument operation, an d th e standards or the relevant set of samples should be reanalyzed. 13.6 Retention times between standards and samples m u s t n o t d rift m o re than 4 % within an analytical run. If retention time drift exceeds th is lim it w ith in an analytical run then the set must be reanalyzed. 14.0 Calculations 14.1 Use the following equation to calculate the amount o f PFOA found (in n g /L , based on peak area) using the standard curve (linear regression p a ra m e te rs ) generated by the Mass Lynx software program: PFOA found (ng/L) = (Peak area - intercept) x DF slope DF = factor by which the final volume was diluted, if necessary. Page 6 of 7 E05-0210 Interim Report#15 Northern Alabama Potable Water Systems Page 21 o f 65 Page 75 o f 123 3M Environmental Laboratory E05-0210 Interim Report#15 Exygen Protocol Number: P0001131 Exygen Research Method Number V0001780 | ANALYTICAL METHOD Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in W a te r b y LC/MS/MS 14.2 For samples fortified with known amounts o f PFOA prior to e x tr a c tio n , u se the following equation to calculate the percent recovery. Recovery (%) = [ total analyte found (ng/L) - analyte found in control (ng/L)] ;1QQ analyte added (ng/L) Page 7 o f ' E05-0210 Interim Report#15 Northern Alabama Potable Water Systems Page 22 o f 65 Page 76 o f 123 3M Environmental Laboratory E05-0210 Interim Report#15 Exygen Protocol Number: P0001131 ANALYTICAL METHOD Method Number: V0001781 Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Soil by LC/MS/MS Analytical Testing Facility: Exygen Research 3058 Research Drive State College, PA 16801 Approved By: Paul Connolly Technical Leader, LC-MS, Exygen Research I Date Total Pages: 7 E05-0210 Interim Report#15 Northern Alabama Potable Water Systems Page 23 o f 65 Page 77 o f 123 3M Environmental Laboratory E05-0210 Interim Report#15 Exygen Protocol Number: P0001131 Exygen Research Method Number V0001781 1 ANALYTICAL METHOD Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in S o il b y LC/MS/MS 1.0 Scope This method is to be employed for the isolation and quantitation o f p e r f lu o ro o c ta n o ic acid by High Performance Liquid Chromatography coupled to a ta n d e m M a ss Spectrometric Detector (LC/MS/MS) in soil. 2.0 Safety 2.1 Always observe safe laboratory practices. 2.2 Consult the appropriate MSDS before handling any c h e m ic a l fo r p r o p e r sa fe ty precautions. 3.0 Sample Requirement 3.1 At least 15 g o f test sample for extraction. 3.2 No sample processing is needed for soil samples. 3 .3 Samples stored refrigerated should be allowed to equilibrate to ro o m temperature. 3.4 All samples must be thoroughly mixed before being sampled fo r e x tra c tio n . 3 .5 Sample collection procedures will be specified in the s a m p lin g p la n fo r th is project. 4.0 Reagents and Standards 4.1 Water - HPLC grade 4.2 Methanol - HPLC grade 4.3 Ammonium Acetate - A.C.S. Reagent Grade 4.4 Perfluorooctanoic Acid - Sigma-Aldrich 5.0 Instrument and Equipment 5.1 A high performance liquid chromatograph capable o f p u m p in g up to 2 solvents equipped with a variable volume injector capable o f in je c tin g 5 -2 0 0 pL connected to a tandem Mass Spectrometer (LC/MS/MS). 5.2 A device to collect raw data for peak integration and q u a n tita tio n . 5.3 Analytical balance capable o f reading to 0.00001 g. 5.4 50 mL disposable polypropylene centrifuge tubes. 5.5 15 mL disposable polypropylene centrifuge tubes. 5.6 Disposable micropipets (50-1 OOuL, 100-200uL). 5.7 125-mL LDPE narrow-mouth bottles. 5.8 2 mL clear HPLC vial kit. 5.9 Disposable pipettes. 5.10 Autopipettes (100-1000 pL and 10-100 pL), with disposable tips. 5.11 Waters Sep Pak Vac 6 cc (lg) tC18 SPE cartridges. 5.12 SPE vacuum manifold. 5.13 Ultrasonic bath. Page 2 o f 7 E05-0210 Interim Report#15 Northern Alabama Potable Water Systems Page 24 o f 65 Page 78 o f 123 3M Environmental Laboratory E05-0210 Interim Report#15 Exygen Protocol Number: P0001131 Exygen Research Method Number V0001781 | ANALYTICAL METHOD Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Soil by LC/MS/MS 5.14 Wrist-action shaker. 5.15 Centrifuge capable of spinning 50 mL polypropylene tubes at 5000 rpm. 6.0 Chromatographic System 6.1 Analytical Column: Fluophase RP (Keystone Scientific), 2.1 mm x 50 mm, 5 p (P/N: 82505-052130) 6.2 Temperature: 30C 6.3 Mobile Phase (A) : 2 mM Ammonium Acetate in Water 6.4 Mobile Phase (B) : Methanol 6.5 Gradient Program: Time (min) 0.0 1.0 8.0 20.0 22.5 %A 65 65 25 25 65 Flow Rate % B (mL/min) 35 0.3 35 0.3 75 0.3 75 0.3 35 0.3 6.6 Injection Volume: 15 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 to optimize the HPLC system. 7.0 MS/MS System 7.1 Mode: Electrospray Negative MRM mode, monitoring 413 369 m/'z for PFOA. The above conditions are intended as a guide and may be changed in order to optimize the MSMS system. 8.0 Preparation of Solutions 8.1 Mobile Phase 8.1.1 2 mM ammonium acetate in water is prepared by adding 0.154 g of ammonium acetate to 1000 mL of water. Alternate volumes may be prepared. Page 3 uf 7 E05-0210 Interim Report#15 Northern Alabama Potable Water Systems Page 25 o f 65 Page 79 o f 123 3M Environmental Laboratory E05-0210 Interim Report#15 Exygen Protocol Number: P0001131 Exygen Research Method Number V0001781 ANALYTICAL METHOD Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Soil b y LC/MS/MS 9.0 Standard Preparation 9.1 Standard Stock/Fortification Solution 9.1.1 Prepare a stock solution o f -1 0 0 pg/mL o f PFOA by weighing 10 mg o f analytical standard (corrected for purity) and dilute to 1 0 0 m L w ith methanol in a 125-mL LDPE bottle. 9.1.2 A 10 pg/mL fortification solution o f PFOA is prepared b y b rin g in g 10 mL o f the 100 pg/mL solution to a final volume o f 100 w ith m ethanol in a 125 mL LDPE bottle. 9.1.3 A 1.0 pg/mL fortification solution o f PFOA is prepared by bringing 10 mL of the 10 pg/mL solution to a final volume o f 100 with methanol m a 125 mL LDPE bottle. 9.1.4 A 0.1 pg/mL fortification solution o f PFOA is prepared by bringing 10 mL o f Jhe 1.0 pg/mL solution to a final volume o f 100 with methanol in a 125 mL LDPE bottle. 9.1.5 A 0.01 pg/mL fortification solution o f PFOA is prepared by bringing 10 mL o f the 0.1 pg/mL solution to a final volume of 100 w ith methanol in a 125 mL LDPE bottle. 9.1.6 The stock and fortification solutions are to be stored in a refrigerator at approximately 4C and are stable for a maximum period of 6 months from the date o f preparation. 9.2 Standard Calibration Solutions 9.2.1 9.2.2 LC/MS/MS calibration standards are prepared in HPLC water. The calibration standards are processed through the extraction procedure, identical to samples. The following is a typical example: additional concentrations may be prepared as needed. Final Concentration Fortification Volume of Concentration of Calibration o f Fortification Volume Fortified Control 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 XCmmddyy-2 10 400 40 100 XCmmddyy-3 100 100 40 250 XCmmddyy-4 100 200 40 500 XCmmddyy-5 100 400 40 1000 XCmmddyy-6 * The extracted concentration o f the calibration standard is equal to 8x its initial concentration, due to the concentration o f the standard during the extraction (SPE). XC = extracted calibration standard. Page 4 of 7 E05-0210 Interim Report#15 Northern Alabama Potable Water Systems Page 26 o f 65 Page 80 o f 123 3M Environmental Laboratory E05-0210 Interim Report#15 Exygen Protocol Number: P0001131 Exygen Research Method Number V0001781 | ANALYTICAL METHOD Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Soil by LC/MS/MS 9.2.3 9.2.4 9.2.5 A zero standard solution (reagent blank) must be prepared with ea ch set o f standards extracted. Store all extracted calibration standards in 15-mL polypropylene tu b e s at 2C to 6C, up to two weeks. Alternate volumes and concentrations o f standards may be prepared as needed. 10.0 Batch Set Up 10.1 Each batch o f samples extracted (typically 20 or less) must include at least one reagent control (method blank using 5 mL o f methanol) and two reagent 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 Sample Extraction 11.1 Weigh 5 g o f sample into 50 mL polypropylene centrifuge tubes (fortify as needed, replace lid and mix well). 11.2 Add 5 mL o f methanol and shake on a wrist action shaker for ~15 minutes. 11.3 Transfer the tubes to 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 Centrifuge for -1 0 minutes at -3000 rpm. 11.6 Condition the Cig SPE cartridges (1 g, 6 mL) by passing 10 mL methanol followed by 5 mL o f HPLC water (~ 2 drop/sec). Do not let column run dry 11.7 Load (decant) the sample on the conditioned C)8 SPE cartridge. Discard eluate. 11.8 Elute with -5 mL 100% methanol. Collect 5 mL of eluate into graduated 15 mL polypropylene centrifuge tubes (final volume = 5 mL). 11.9 Analyze samples using electrospray LC/MS/MS. 12.0 Chromatography 12.1 Inject the same amount o f each standard, sample and fortified sample into the LC/MS/MS system. A calibration standard must precede and follow all analyzed samples. 12.2 Standards of PFOA corresponding to at least five or more concentration levels must be included in an analytical set. 12.3 An entire set o f extracted calibration standards must be included at the beginning and at the end o f a sample set. Extracted standards must be interspersed between every 5-10 samples. As an alternative, an entire set of Page 5 of 7 E05-0210 Interim Report#15 Northern Alabama Potable Water Systems Page 27 o f 65 Page 81 o f 123 3M Environmental Laboratory E05-0210 Interim Report#15 Exygen Protocol Number: P0001131 Exygen Research Method Number V0001781 P ANALYTICAL METHOD Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in S o il b y LC/MS/MS ' extracted calibration standards may be injected at the beginning o f a set followed by extracted calibration standards interspersed every 5 -1 0 s a m p le s (to account for a second set o f extracted standards). In e ith e r c a s e , e x tra c te d calibration standards must be the first and last injection in a s a m p le set. 12.4 Use linear standard curves for quantitation. L in e a r s ta n d a rd c u rv e s arc generated for the analyte by linear regression using 1/x w e ig h tin g o f p e a k a re a versus calibration standard concentration using MassLynx 3 .3 (o r e q u iv a le n t) software system. 12 .5 Sample response should not exceed standard responses. A n y s a m p le s th a t exceed standard responses should be further diluted and re a n a ly z e d . 13.0 Acceptance Criteria 13.1 Chromatogram must show a peak o f a daughter ion at 369 amu from a p a re n t of 413 amu. The 413 amu parent corresponds to the PFOA anion, w h ile th e daughter ion (369 amu) represents the loss o f carbon dioxide. 1 3 .2 Method blanks must not contain PFOA at levels g r e a te r th a n th e L O Q . I f a blank contains PFOA at levels greater than 5 0 ng/L, th e n a n e w b la n k s a m p le must be obtained and the entire set must be re-extracted. 13.3 Recoveries o f control spikes and matrix spikes must be between 7 0 -1 3 0 % o f their known values. If a control spike falls outside the acceptable lim its, th e entire set o f samples should be re-extracted. Any matrix spike o u ts id e 7 0 130% should be evaluated by the analyst to determine i f r e - e x tr a c tio n is warranted. 13.4 Any calibration standard found to be a statistical outlier by u s in g th e H u g e Error Test, may be excluded from the calculation o f the c a lib r a tio n c u rv e . However, the total number o f extracted calibration standards th a t c o u ld be excluded must not exceed 2 0 % o f the total n u m b e r o f e x tr a c te d s ta n d a rd s injected. 1 3 .5 The correlation coefficient (R ) for calibration c u r v e s g e n e ra te d m u s t be > 0 .9 9 2 ( R 2 0 .9 8 5 ) . If calibration results fall o u ts id e th e s e lim its , th e n appropriate steps must be taken to adjust in s tr u m e n t o p e r a tio n , a n d th e standards or the relevant set of samples should be reanalyzed. 13.6 Retention times between standards and samples must not drift m o re th a n 4 % within an analytical run. If retention time drift exceeds th is lim it w ith in an analytical run then the set must be reanalyzed. Page 6 of 7 E05-0210 Interim Report#15 Northern Alabama Potable Water Systems Page 28 o f 65 Page 82 o f 123 3M Environmental Laboratory E05-0210 Interim Report#15 Exygen Protocol Number: P0001131 Exygen Research Method NumberV0001781 ANALYTICAL METHOD Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in S o il bv LC/MS/MS ' \ 14.0 Calculations 14.1 Use the following equation to calculate the amount o f PFOA found (in ng/L, based on peak area) using the standard curve (linear regression p a ra m e te rs) generated by the Mass Lynx software program: PFOA found (ng/L) = (Peak area - intercept) x DF slope DF = factor by which the final volume was diluted, if necessary. 14.2 For samples fortified with known amounts o f PFOA prior to extraction, u se the following equation to calculate the percent recovery. Recovery (%) = [ total analyte found (ng/L) - analyte found in control (ng/L)] ^ analyte added (ng/L) 14.3 Use the following equation to convert the amount o f PFOA found in ng/L to ng/g (ppb). PFOA found (ppb) = fPFOA found (ng/L) x volume extracted t'0.04L)l sample weight (5 g) 14.4 Use the following equation to calculate the amount o f PFOA found in p p b based on dry weight. PFOA found (ppb) dry weight = PFOA found (ppb) x [100% / total solids(%)] Page 7 of 7 E05-0210 Interim Report#15 Northern Alabama Potable Water Systems Page 29 o f 65 Page 83 o f 123 3M Environmental Laboratory E05-0210 Interim Report#15 Exygen Protocol Number: P0001131 ANALYTICAL METHOD Method Number: V0001782 Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Sediment by LC/MS/MS Analytical Testing Facility: Exygen Research 3058 Research Drive State College, PA 16801 Approved By: C - j L ______ Paul Connolly I Technical Leader, LC-MS, Exygen Research j/n f b l / ohn Flaherty Vice President, Operations, Exygen Research __ lolWo'/ Date Date Total Pages: 7 E05-0210 Interim Report#15 Northern Alabama Potable Water Systems Page 30 o f 65 Page 84 o f 123 3M Environmental Laboratory E05-0210 Interim Report#15 Exygen Protocol Number: P0001131 Exygen Research Method Number V 0 0 0 1782 | ANALYTICAL METHOD j Method of Analysis for the Determination o f Perfluorooctanoic Acid (P F O A ) in S e d im e n t bv 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 M a ss Spectrometric Detector (LC/MS/MS) in sediment. 2.0 Safety 2.1 Always observe safe laboratory practices. 2.2 Consult the appropriate MSDS before handling any chemical for proper safety precautions. 3.0 Sample Requirement 3.1 At least 30 g o f test sample for extraction. 3.2 No sample processing is needed for sediment samples. 3.3 Samples stored refrigerated should be allowed to equilibrate to ro o m temperature. 3.4 All samples must be thoroughly mixed before being sampled for extraction. 3.5 Sample collection procedures will be specified in the sampling plan fo r th is project. 4.0 Reagents and Standards 4.1 Water - HPLC grade 4.2 Methanol - HPLC grade 4.3 Acetic Acid - Reagent grade 4.4 Ammonium Acetate - A.C.S. Reagent Grade 4.5 Perfluorooctanoic Acid - Sigma-Aldrich 5.0 Instrument and Equipment 5.1 A high performance liquid chromatograph capable o f pumping up to 2 solvents equipped with a variable volume injector capable of injecting 5-200 pL connected 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 o f reading to 0.00001 g. 5.4 50 mL disposable polypropylene centrifuge tubes. 5.5 15 mL disposable polypropylene centrifuge tubes. 5.6 Disposable micropipets (50-1 OOuL, 100-200uL). 5.7 125-mL LDPE narrow-mouth bottles. 5.8 2 mL clear HPLC vial kit. 5.9 Disposable pipettes. 5.10 Autopipettes (100-1000 pL and 10-100 pL), with disposable tips. 5.11 Waters Sep Pak Vac 6 cc (lg) tC18 SPE cartridges. 5.12 SPE vacuum manifold. Page 2 of 7 E05-0210 Interim Report#15 Northern Alabama Potable Water Systems Page 31 o f 65 Page 85 o f 123 3M Environmental Laboratory E05-0210 Interim Report#15 Exygen Protocol Number: P0001131 Exygen Research Method Number V 0001782 ANALYTICAL METHOD \ Method of Analysis for the Determination of Perfluorooctanoic Acid (P F O A ) in S e d im e n t b \ LC/MS/MS ' 5.13 Vortexer. 5.14 Wrist-action shaker. 5.15 Centrifuge capable o f spinning 50 mL polypropylene tubes at 3000 rpm. 6.0 Chromatographic System 6.1 Analytical Column: Fluophase RP (Keystone Scientific), 2.1 mm x 50 mm, 5p (P/N: 82505-052130) 6.2 Temperature: 30C 6.3 Mobile Phase (A) : 2 mM Ammonium Acetate in Water 6.4 Mobile Phase (B) : Methanol 6.5 Gradient Program: Time (min) 0.0 l.O 8.0 20.0 22.5 %A 65 65 25 25 65 Flow Rate % B (mL/min) 35 0.3 35 0.3 75 0.3 75 0.3 35 0.3 6.6 Injection Volume: 15 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 to optimize the HPLC system. 7.0 MS/MS System 7.1 Mode: Electrospray Negative MRM mode, monitoring 413 - 369 m /z for PFOA. The above conditions are intended as a guide and may be changed in order to optimize the MSMS system. 8.0 Preparation o f Solutions 8.1 Mobile Phase 8.1.1 2 mM ammonium acetate in water is prepared by adding 0.154 g of ammonium acetate to 1000 mL o f water. Page 3 of 7 E05-0210 Interim Report#15 Northern Alabama Potable Water Systems Page 32 o f 65 ......Page 86 o f 123 3M Environmental Laboratory E05-0210 Interim Report#15 Exygen Protocol Number: P0001 131 Exygen Research Method Number V0001782 ANALYTICAL METHOD Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Sediment bv LC/MS/MS 8.2 Extraction Solutions 8.2.1 1% acetic acid in water is prepared by adding 10 mL o f acetic acid to 1000 mL o f water. Alternate volumes may be prepared. 9.0 Standard Preparation 9.1 Standard Stock/Fortification Solution 9.1.1 Prepare a stock solution o f -1 0 0 pg/mL o f PFOA by weighing 10 mg of analytical standard (corrected for purity) and dilute to 100 mL w ith methanol in a 125-mL LDPE bottle. 9.1.2 A 10 pg/mL fortification solution o f PFOA is prepared by bringing 10 mL o f the 100 pg/mL solution to a final volume o f 100 with methanol in a 125 mL LDPE bottle. 9.1.3 A 1.0 pg/mL fortification solution o f PFOA is prepared by bringing 10 mL o f the 10 pg/mL solution to a final volume o f 100 with methanol in a 125 mL LDPE bottle. 9.1.4 A 0.1 pg/mL fortification solution o f PFOA is prepared by bringing 10 mL o f the 1.0 pg/mL solution to a final volume o f 100 with methanol in a 125 mL LDPE bottle. 9.1.5 A 0.01 pg/mL fortification solution o f PFOA is prepared by bringing 10 mL o f the 0.1 pg/mL solution to a final volume o f 100 w ith methanol in a 125 mL LDPE bottle. 9.1.6 The stock and fortification solutions are to be stored in a refrigerator at approximately 4C and are stable for a maximum period of 6 months from the date o f preparation. 9.2 Standard Calibration Solutions 9.2.1 LC/MS/MS calibration standards are prepared in methanol via dilution of the 0.1 pg/mL fortification solution. 9.2.2 The following is a typical example: additional concentrations may b e Concentration of Fortification Solution (ng/mL) 100 100 100 10 5 2 Volume (mL) 10 5 2 10 10 10 Diluted to (mL) 100 100 100 100 100 100 Final Concentration (ng/mL) 10.0 5.0 2.0 1.0 0.5 0.2 Page 4 of : E05-0210 Interim Report#15 Northern Alabama Potable Water Systems Page 33 o f 65 Page 87 o f 123 3M Environmental Laboratory E05-0210 Interim Report#15 Exygen Protocol Number: P0001131 Exygen Research Method Number V0001782 | ANALYTICAL METHOD 1 Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Sediment by LC/MS/MS 9.2.3 9.2.4 Store all calibration standards in 125-mL LDPE narrow-mouth bottles at 2C to 6C, up to six months. Alternate volumes and concentrations o f standards may be prepared as needed. 10.0 Batch Set Up 10.1 Each batch o f samples extracted (typically 20 or less) must include at least one untreated control and two untreated controls fortified at k n o w n 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 Sample Extraction 11.1 11.2 11.3 11.4 11.5 1 1 .6 11.7 11.8 11.9 11.10 11.11 11.12 11.13 Weigh 5 g o f sample into 50 mL polypropylene centrifuge tu b e s (fo rtify as needed, replace lid and mix well). Add 35 mL o f 1% acetic acid, cap, vortex and shake on a wrist a c tio n s h a k e r for ~60 minutes. Centrifuge the tubes at -3000 rpm for -2 0 minutes. Condition the Cig SPE cartridges (1 g, 6 mL) by passing 10 mL methanol followed by 20 mL o f HPLC water (~ 2 drop/sec). Do not let column run d ry Load (decant) the sample on the conditioned Cig SPE cartridge. D is c a rd eluate. Add 2 0 mL o f methanol to the sediment left in the bottom o f th e 5 0 m L centrifuge tube. Cap, vortex and shake on a wrist action s h a k e r fo r - 3 0 minutes. Centrifuge the tubes at -3000 rpm for -2 0 minutes. Decant the methanol onto the same SPE cartridge. Collect the eluate. Wash the column with 4 mL o f methanol. Collect the eluate and add it to th e eluate collected in step 11.8. Condition a second Cig SPE cartridge (1 g, 6 mL) by passing 10 mL m e th a n o l followed by 20 mL o f HPLC water (~ 2 drop/sec). Do not let column ru n d ry Add the methanol to -2 0 0 mL o f water and load on the second c o n d itio n e d SPE cartridge. Elute with -5 mL 100% methanol. Collect 5 mL o f eluate into g ra d u a te d 15 mL polypropylene centrifuge tubes (final volume = 5 mL). Analyze samples using electrospray LC/MS/MS. Page 5 of 7 E05-0210 Interim Report#15 Northern Alabama Potable Water Systems Page 34 o f 65 Page 88 o f 123 3M Environmental Laboratory E05-0210 Interim Report#15 Exygen Protocol Number: P0001131 Exygen Research Method Number V0001782 | ANALYTICAL METHOD [ Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Sediment bv LC/MS/MS 12.0 Chromatography 12.1 Inject the same amount o f each standard, sample and fortified sample into th e LC/MS/MS system. A calibration standard must precede and follow all analyzed samples. 12.2 Standards o f PFOA corresponding to at least five or more concentration le v e ls must be included in an analytical set. 12.3 An entire set o f extracted calibration standards must be included at th e beginning and at the end o f a sample set. Standards must be interspersed between every 5-10 samples. As an alternative, an entire set of calibration standards may be injected at the beginning o f a set followed by calibration standards interspersed 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 sample set. 12.4 Use linear standard curves for quantitation. Linear standard curves are generated for the analyte by linear regression using 1/x weighting 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 responses should be further diluted and reanalyzed. 13.0 Acceptance Criteria 13.1 Chromatogram must show a peak o f a daughter ion at 369 amu from a parent of 413 amu. The 413 amu parent corresponds to the PFOA anion, while the daughter ion (369 amu) represents the loss o f carbon dioxide. 13.2 Method blanks must not contain PFOA at levels greater than the LOQ. If a blank contains PFOA at levels greater than 0.2 ng/mL, then a new blank sample must be obtained and the entire set must be re-extracted. 13.3 Recoveries o f 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 o f samples should be re-extracted. Any matrix spike outside 70 130% 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 o f the calibration curve However, the total number o f extracted calibration standards that could be excluded must not exceed 20% o f the total number of extracted standards injected. 13.5 The correlation coefficient (R) for calibration curves generated must be >0.992 (R2 >0.985). If calibration results fall outside these limits, th e n appropriate steps must be taken to adjust instrument operation, and th e standards or the relevant set of samples should be reanalyzed. Page 6 of 7 E05-0210 Interim Report#15 Northern Alabama Potable Water Systems Page 35 o f 65 Page 89 o f 123 3M Environmental Laboratory E05-0210 Interim Report#15 Exygen Protocol Number: P0001131 Exygen Research Method Number V0001782 ANALYTICAL METHOD Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Sediment by LC/MS/MS 13.6 Retention times between standards and samples must not drift m o re th a n 4 % within an analytical run. If retention time drift exceeds this lim it w ith in an analytical run then the set must be reanalyzed. 14.0 Calculations 14.1 Use the following equation to calculate the amount o f P F O A fo u n d (in n g /m L , based on peak area) using the standard curve (linear regression p a ra m e te rs ) generated by the Mass Lynx software program: PFOA found (ng/mL) = (Peak area - intercept) x D F slope DF = factor by which the final volume was diluted, if necessary. 14.2 For samples fortified with known amounts o f PFOA prior to e x tr a c tio n , use the following equation to calculate the percent recovery. Recovery (%) = [ total analyte found (ng/mL) - analyte found in control (ng/mL)] ^ ]QQ analyte added (ng/mL) 14.3 Use the following equation to convert the amount o f PFOA found in n g /m L to ng/g (ppb). PFOA found (ppb) = fPFOA found (ng/mL) x final volume (5 mL)l sample weight (5 g) 14.4 Use the following equation (if necessary) to calculate the amount o f PFOA found in ppb based on dry weight. PFOA found (ppb) dry weight = PFOA found (ppb) x [100% / to ta l s o lid s (% )] Page 7 of 7 E05-0210 Interim Report#15 Northern Alabama Potable Water Systems Page 36 o f 65 Page 90 o f 123 3M Environmental Laboratory E05-0210 Interim Report#15 Exygen Protocol Number: P0001131 ANALYTICAL METHOD Method Number: V0001783 Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Fish and Clams by LC/MS/MS Analytical Testing Facility: Exygen Research 3058 Research Drive State College, PA 16801 Approved By: TA1 Paul Connolly Technical Leader, LC-MS, Exygen Research '/#> / d Y *ohn Flaherty Vice President, Operations, Exygen Research Date A>/*V>Y Date Total Pages: 8 E05-0210 Interim Report#15 Northern Alabama Potable Water Systems Page 37 o f 65 Page 91 o f 123 3M Environmental Laboratory E05-0210 Interim Report#15 Exygen Protocol Number: P0001131 Exygen Research Method Number V0001783 | ANALYTICAL m e t h o d Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in F is h an d Clams by LC/MS/MS 1.0 Scope This method is to be employed for the isolation and quantitation o f p e rf lu o ro o c ta n o ic acid by High Performance Liquid Chromatography c o u p le d to a ta n d e m M a ss Spectrometric Detector (LC/MS/MS) in fish and clams. 2.0 Safety 2.1 Always observe safe laboratory practices. 2.2 Consult the appropriate MSDS before handling any chemical for p ro p e r sa fe ty precautions. 3.0 Sample Requirement 3.1 At least 20 g o f test sample for extraction. 3 .2 Samples should be processed before extraction. Place the frozen s a m p le in a food processor and homogenize with dry ice. Place the s a m p le s in c o n ta in e rs and leave open in frozen storage overnight to allow fo r c a rb o n d io x id e sublimation. Seal and place the samples in frozen s to r a g e u n til tim e o f analysis. 3.3 Sample collection procedures will be specified in the sampling plan for th is project. 4.0 Reagents and Standards 4.1 Water - HPLC grade 4.2 Acetonitrile - HPLC grade 4.3 Carbon (120-400 mesh) - Reagent grade 4.4 Methanol - HPLC grade 4.5 Silica gel (60-200 mesh) - Reagent grade 4.6 Florisil (60-100 mesh) - Reagent grade 4.7 Superclean LC-NH2 - Reagent grade 4.8 1-Octanol - HPLC grade 4.9 L-Ascorbic acid - Reagent grade 4.10 Dimethyldichlorosilane - Reagent grade 4.11 Toluene - Reagent grade 4.12 Ammonium Acetate - A.C.S. Reagent Grade 4.13 Perfluorooctanoic Acid - Sigma-Aldrich 5.0 Instrument and Equipment 5.1 A high performance liquid chromatograph capable o f p u m p in g up to 2 solvents equipped with a variable volume injector capable o f in je c tin g 5 -2 0 0 pL connected 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 o f reading to 0.00001 g. Page 2 of S E05-0210 Interim Report#15 Northern Alabama Potable Water Systems Page 38 o f 65 Page 92 o f 123 3M Environmental Laboratory E05-0210 Interim Report#15 Exygen Protocol Number: P0001131 Exygen Research Method NumberV0001783 1 ANALYTICAL METHOD Method of Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Fish and Clams by LC/MS/MS 5.4 Rotary evaporator. 5.5 Tissumizer. 5.6 125 mL pear-shaped flasks. 5.7 50 mL disposable polypropylene centrifuge tubes. 5.8 15 mL disposable polypropylene centrifuge tubes. 5.9 Disposable micropipets (50-1 OOuL, 100-200uL). 5.10 125-mL LDPE narrow-mouth bottles. 5.11 2 mL clear HPLC vial kit. 5.12 Disposable pipettes. 5.13 Autopipettes (100-1000 pL and 10-100 pL), with disposable tips. 5.14 SPE tubes (20mL) (Supelco cat. no. N057177). 5.15 Wrist action shaker. 5.16 Centrifuge capable of spinning 50 mL polypropylene tubes at 2000 rpm. 6.0 Chromatographic System 6.1 Analytical Column: Fluophase RP (Keystone Scientific), 2.1 mm x 50 mm, 5 p (P/N: 82505-052130) 6.2 Temperature: 30C 6.3 Mobile Phase (A) : 2 mM Ammonium Acetate in Water 6.4 Mobile Phase (B) : Methanol 6.5 Gradient Program: Time (min) 0.0 1.0 8.0 20.0 22.5 %A 65 65 25 25 65 Flow Rate % B (mL/min) 35 0.3 35 0.3 75 0.3 75 0.3 35 0.3 6.6 Injection Volume: 15 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 to optimize the HPLC system. Page 3 of 8 E05-0210 Interim Report#15 Northern Alabama Potable Water Systems Page 39 o f 65 Page 93 o f 123 3M Environmental Laboratory E05-0210 Interim Report#15 Exygen Protocol Number: P0001131 Exygen Research Method Number VOOO1783 ANALYTICAL METHOD Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Fish and Clams by LC/MS/MS 7.0 MS/MS System 7.1 Mode: Electrospray Negative MRM mode, monitoring 413 - 3 6 9 m /z for PFOA. The above conditions are intended as a guide and may be changed in order to optimize the MSMS system. 8.0 Preparation o f Solutions 8.1 Mobile Phase 8.1.1 2 mM ammonium acetate in water is prepared by adding 0.154 g o f ammonium acetate to 1000 mL o f water. 8.2 Extraction Solutions 8.2.1 8.2.2 2% ascorbic acid in methanol is prepared by dissolving 2 g of ascorbic acid in 100 mL o f methanol. 30% Dimethyldichlorosilane in toluene is prepared by bringing 3 mL of dimethyldichlorosilane to a final volume o f 10 mL with toluene. Alternate volumes may be prepared. 9.0 Standard Preparation 9.1 Standard Stock/Fortification Solution 9.1.1 9.1.2 9.1.3 9.1.4 9.1.5 Prepare a stock solution o f -100 pg/mL o f PFOA by weighing 10 mg of analytical standard (corrected for purity) and dilute to 100 mL with methanol in a 125-mL LDPE bottle. A 1.0 pg/mL fortification solution o f PFOA is prepared by bnnging 1 mL o f the 100 pg/mL solution to a final volume of 100 with m ethanol in a 125 mL LDPE bottle. A 0.1 pg/mL fortification solution o f PFOA is prepared by bringing 10 mL o f the 1.0 pg/mL solution to a final volume o f 100 with m ethanol in a 125 mL LDPE bottle. A 0.01 pg/mL fortification solution o f PFOA is prepared by bringing 10 mL o f the 0.1 pg/mL solution to a final volume of 100 with methanol in a 125 mL LDPE bottle. The stock and fortification solutions are to be stored in a refrigerator at approximately 4C and are stable for a maximum period of 6 months from the date o f preparation. Page 4 of 8 E05-0210 Interim Report#15 Northern Alabama Potable Water Systems Page 40 o f 65 Page 94 o f 123 3M Environmental Laboratory E05-0210 Interim Report#15 Exygen Protocol Number: P0001131 Exygen Research Method Number V 0 0 0 1783 ANALYTICAL METHOD Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Fish and Clams by LC/MS/MS 9.2 Standard Calibration Solutions 9.2.1 9.2.2 LC/MS/MS calibration standards are prepared in methanol via dilution of the 1.0 pg/mL fortification solution. The following is a typical example: additional concentrations may be prepared as needed. Concentration Final of Fortification Volume Diluted to Concentration Solution (ug/mL) (mL) (mL) (pg/mL) 1.0 5.0 100 0.05 1.0 2.5 100 0.025 1.0 1.0 100 0.01 0.05 10 100 0.005 0.025 10 100 0.0025 0.1 10 100 0.001 0.005 10 100 0.0005 9.2.3 Store all calibration standards in 125-mL LDPE narrow-mouth bottles at 2C to 6C, up to six months. 9.2.4 Alternate volumes and concentrations o f standards may be prepared as needed. 10.0 Batch Set Up 10.1 Each batch o f samples extracted (typically 20 or less) must include at least one untreated control and two untreated 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 Sample Extraction 11.1 Weigh 5 g of frozen sample into 50 mL polypropylene centrifuge tubes (fortify as needed, replace lid and mix well). 11.2 Add 30 mL of acetonitrile and shake on a wrist action shaker for ~15 minutes. 11.3 Place the tubes in a freezer for ~1 hour. 11.4 Pack and condition the SPE tubes 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 g carbon, and 1 g LC-NH2. Condition the columns with 20 mL of methanol, then 20 mL of acetonitrile. Discard all washes. Do not allow the column to dry. 11.6 Silanize the 125 mL pear-shaped flasks by rinsing with the 30% dimethyldichlorosilane in toluene solution. Rinse the flask with toluene once, followed by methanol (three times). Dry the flasks completely before use, either by air-drying or with a stream o f nitrogen. Page 5 of S E05-0210 Interim Report#15 Northern Alabama Potable Water Systems Page 41 o f 65 Page 95 o f 123. 3M Environmental Laboratory E05-0210 Interim Report#15 Exygen Protocol Number: P0001131 Exygen Research Method Number V0001783 ANALYTICAL METHOD ~ Method o f Analysis for the Determination o f Perfluorooctanoic A c id (P F O A ) in F ish 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 - 2 0 0 0 rpm 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 o f acetonitrile to the sample in the 50 mL centrifuge tube. Homogenize the frozen fat phase using a tissumizer for -3 0 seconds a n d rin se the tissumizer with -1 0 mL o f acetonitrile into the tube. Shake the sample again for -1 0 minutes on a wrist-action shaker. Place the tubes in a freezer for - 1 hour more. Centrifuge the 50 mL polypropylene tubes containing sample at -2000 rpm for-1 0 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 in itia l extraction. Pass 20 mL o f acetonitrile through the SPE column and combine the eluate in the same pear-shaped flask. Add 3-4 drops o f 1-octanol to the extract in the pear-shaped flask and evaporate at reduced pressure using a rotary evaporator (at < 40C). Make the final volume, by adding 2 mL o f 2% ascorbic acid in methanol to the pear-shaped flask and swirl to mix/dissolve. Transfer the extracts to HPLC vials using disposable pipets. Analyze samples using electrospray LC/MS/MS. 12.0 Chromatography 12.1 Inject the same amount o f each standard, sample and fortified sample into the LC/MS/MS system. A calibration standard must precede and follow all analyzed samples. 12.2 Standards o f PFOA corresponding to at least five or more concentration levels must be included in an analytical set. 12.3 An entire set o f calibration standards must be included at the beginning and at the end of a sample set. Standards must be interspersed between every 5-10 samples. As an alternative, an entire set of calibration standards may be injected at the beginning o f a set followed by calibration standards interspersed 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 sample set. 12.4 Use linear standard curves for quantitation. Linear standard curves are generated for the analyte by linear regression using 1/x weighting of peak area versus calibration standard concentration using MassLynx 3.3 (or equivalent) software system. Page 6 of 8 E05-0210 Interim Report#15 Northern Alabama Potable Water Systems Page 42 o f 65 Page 96 o f 123 3M Environmental Laboratory E05-0210 Interim Report#15 Exygen Protocol Number: P 0001131 Exygen Research Method Number V 0 0 0 1783 | ANALYTICAL METHOD Method o f Analysis for the Determination ofPerfluorooctanoic Acid (PFOA) in Fish and Clams by LC/MS/MS 12.5 Sample response should not exceed standard responses. Any samples that exceed standard responses should be further diluted and reanalyzed. 13.0 Acceptance Criteria 13.1 Chromatogram must show a peak o f a daughter ion at 369 amu from a parent o f 413 amu. The 413 amu parent corresponds to the PFOA anion, while the daughter ion (369 amu) represents the loss o f carbon dioxide. 13.2 Method blanks must not contain PFOA at levels greater than the LOQ. If a blank contains PFOA 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 Recoveries of control spikes and matrix spikes must be between 70-130% o f their known values. If a control spike falls outside the acceptable limits, the entire set o f samples should be re-extracted. 13.4 Any calibration standard found to be a statistical outlier by using the Huge Error Test, may be excluded from the calculation o f the calibration curve However, the total number o f calibration standards that could be excluded must not exceed 20% o f the total number o f standards injected. 13.5 The correlation coefficient (R) for calibration curves generated must be >0.992 (R2 >0.985). If calibration results fall outside these limits, then appropriate steps must be taken to adjust instrument operation, and the standards or the relevant set o f samples should be reanalyzed. 13.6 Retention times between standards and samples must not drift more than 4 % within an analytical run. If retention time drift exceeds this lim it w ith in an analytical run then the set must be reanalyzed. 14.0 Calculations 14.1 Use the following equation to calculate the amount of PFOA found (in ng/mL, based on peak area) using the standard curve (linear regression parameters) generated by the Mass Lynx software program: PFOA found (ng/mL) = (Peak area - intercept) slope 14.2 Use the following equation to convert the amount o f PFOA found in ng/mL to ng/g (ppb). PFOA found (ppb) = [PFOA found (ng/mL) x final volume (mL) x DF1 sample weight (g) DF = factor by which the final volume was diluted, if necessary. Page 7 of 8 E05-0210 Interim Report#15 Northern Alabama Potable Water Systems Page 43 o f 65 ... Page 97 o f 123 3M Environmental Laboratory E05-0210 Interim Report#15 Exygen Protocol Number: P0001131 Exygen Research Method Number VOOO1783 1 a x a l y t ic a l .m e t h o d Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Fish and Clams by LC/MS/MS 14.3 For samples fortified with known amounts o f PFOA prior to e x tr a c tio n , use the following equation to calculate the percent recovery. Recovery (%) = [ total analyte found (ng/g) - analyte found in control (ng/g)] ^ analyte added (ng/g) Page 8 of 8 E05-0210 Interim Report#15 Northern Alabama Potable Water Systems Page 44 o f 65 .... Page 98 o f 123. 3M Environmental Laboratory E05-0210 Interim Report#15 Exygen Protocol Number: P0001131 ANALYTICAL METHOD Method Number: V0001784 Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Vegetation by LC/MS/MS Analytical Testing Facility: Exygen Research 3058 Research Drive State College, PA 16801 Approved By: TLA CAL Pauilll CP nornmnnollllyv Technical Leader, LC-MS, Exygen Research n,//r>/fuC/ t>hn Flaherty Vice President, Operations, Exygen Research Date Date Total Pages: 7 E05-0210 Interim Report#15 Northern Alabama Potable Water Systems Page 45 o f 65 Page 99 o f 123 3M Environmental Laboratory E05-0210 Interim Report#15 Exygen Protocol Number: P0001131 Exygen Research Method Number V0O01 784 1 ANALYTICAL METHOD Method of Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Vegetation by LC/MS/MS 1.0 Scope This method is to be employed for the isolation and quantitation o f p e r f lu o ro o c ta n o ic acid by High Performance Liquid Chromatography c o u p le d to a ta n d e m Mass Spectrometric Detector (LC/MS/MS) in vegetation. 2.0 Safety 2.1 Always observe safe laboratory practices. 2.2 Consult the appropriate MSDS before handling any chemical fo r p r o p e r s a fe ty precautions. 3.0 Sample Requirement 3.1 At least 20 g o f test sample for extraction. 3.2 Samples should be processed before extraction. Place the fro z e n sa m p le in a food processor and homogenize with dry ice. Place the samples in c o n ta in e rs and leave open in frozen storage overnight to allow fo r c a rb o n d io x id e sublimation. Seal and place the samples in frozen storage u n til tim e of analysis. 3 .3 Sample collection procedures will be specified in the s a m p lin g p la n for this project. 4.0 Reagents and Standards 4.1 Water - HPLC grade 4.2 Acetonitrile - HPLC grade 4.3 Carbon (120-400 mesh) - Reagent grade 4.4 Methanol - HPLC grade 4.5 Silica gel (60-200 mesh) - Reagent grade 4.6 Florisil (60-100 mesh) - Reagent grade 4.7 Superclean LC-NH2 - Reagent grade 4.8 1-Octanol - HPLC grade 4.9 L-Ascorbic acid - Reagent grade 4.10 Dimethyldichlorosilane - Reagent grade 4.11 Toluene - Reagent grade 4.12 Ammonium Acetate - A.C.S. Reagent Grade 4.13 Perfluorooctanoic Acid - Sigma-Aldrich 5.0 Instrument and Equipment 5.1 A high performance liquid chromatograph capable o f pumping up to 2 solvents equipped with a variable volume injector capable of injecting 5-200 pL connected 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 o f reading to 0.00001 g. Page 2 o f 7 E05-0210 Interim Report#15 Northern Alabama Potable Water Systems Page 46 o f 65 Page 100 o f 123 3M Environmental Laboratory E05-0210 Interim Report#15 Exygen Protocol Number: P0001131 Exygen Research Method Number V 0 0 0 1784 ANALYTICAL METHOD Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Vegetation by LC/MS/MS 5.4 Rotary evaporator. 5.5 125 mL pear-shaped flasks. 5.6 50 mL disposable polypropylene centrifuge tubes. 5.7 15 mL disposable polypropylene centrifuge tubes. 5.8 Disposable micropipets (50-1 OOuL, 100-200uL). 5.9 125-mLLDPE narrow-mouth bottles. 5.10 2 mL clear HPLC vial kit. 5.11 Disposable pipettes. 5.12 Autopipettes (100-1000 pL and 10-100 pL), with disposable tips. 5.13 SPE tubes (20mL) (Supelco cat. no. N057177). 5.14 Wrist action shaker. 5.15 Centrifuge capable o f spinning 50 mL polypropylene tubes at 2000 rpm. 6.0 Chromatographic System 6.1 Analytical Column: Fluophase RP (Keystone Scientific), 2.1 mm x 50 mm, 5p (P/N: 82505-052130) 6.2 Temperature: 30C 6.3 Mobile Phase (A) : 2 mM Ammonium Acetate in Water 6.4 Mobile Phase (B) : Methanol 6.5 Gradient Program: Time (min) 0.0 1.0 8.0 20.0 22.5 %A 65 65 25 25 65 Flow Rate % B (mL/min) 35 0.3 35 0.3 75 0.3 75 0.3 35 0.3 6.6 Injection Volume: 15 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 to optimize the HPLC system. 7.0 MS/MS System 7.1 Mode: Electrospray Negative MRM mode, monitoring 413 --> 369 m/z for PFOA. Page 3 of 7 E05-0210 Interim Report#15 Northern Alabama Potable Water Systems Page 47 o f 65 Page 101 o f 123 3M Environmental Laboratory E05-0210 Interim Report#15 Exygen Protocol Number: P0001131 Exygen Research Method Number V0001784 | ANALYTICAL m e t h o d Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Vegetation by LC/MS/MS The above conditions are intended as a guide and may be changed in order to optimize the MSMS system. 8.0 Preparation o f Solutions 8.1 Mobile Phase 8.1.1 2 mM ammonium acetate in water is prepared by adding 0 .1 5 4 g o f ammonium acetate to 1000 mL o f water. 8.2 Extraction Solutions 8.2.1 8.2.2 2% ascorbic acid in methanol is prepared by dissolving 2 g of ascorbic acid in 100 mL o f methanol. 30% Dimethyldichlorosilane in toluene is prepared by bringing 3 m L o f dimethyldichlorosilane to a final volume o f 10 mL with toluene. Alternate volumes may be prepared. 9.0 Standard Preparation 9.1 Standard Stock/Fortification Solution 9.1.1 9.1.2 9.1.3 9.1.4 9.1.5 Prepare a stock solution o f ~100 pg/mL o f PFOA by weighing 10 m g o f analytical standard (corrected for purity) and dilute to 100 m L w ith methanol in a 125-mL LDPE bottle. A 1.0 pg/mL fortification solution o f PFOA is prepared by bringing 1 mL o f the 100 pg/mL solution to a final volume o f 100 w ith m ethanol in a 125 mL LDPE bottle. A 0.1 pg/mL fortification solution o f PFOA is prepared by bringing 10 mL o f the 1.0 pg/mL solution to a final volume o f 100 with methanol in a 125 mL LDPE bottle. A 0.01 pg/mL fortification solution o f PFOA is prepared b y b rin g in g 10 mL of the 0.1 pg/mL solution to a final volume o f 100 w ith methanol in a 125 mL LDPE bottle. The stock and fortification solutions are to be stored in a refrigerator at approximately 4C and are stable for a maximum period o f 6 m o n th s from the date of preparation. 9.2 Standard Calibration Solutions 9.2.1 LC/MS/MS calibration standards are prepared in methanol via dilution of the 1.0 pg/mL fortification solution. Page 4 ol E05-0210 Interim Report#15 Northern Alabama Potable Water Systems Page 48 o f 65 Page 102 o f 123 3M Environmental Laboratory E05-0210 Interim Report#15 Exygen Protocol Number: P0001131 Exygen Research Method Number V0001784 | AiNa l y h c 'a l 'm e t h o d Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Vegetation by LC/MS/MS 9.2.2 The following is a typical example: additional concentrations may be prepared as needed. Concentration of Fortification Solution (ug/mL) 1.0 1.0 1.0 0.05 0.025 0.1 0.005 Volume (mL) 5.0 2.5 1.0 10 10 10 10 Diluted to (mL) 100 100 100 100 100 100 100 Final Concentration (pg/mL) 0.05 0.025 0.01 0.005 0.0025 0.001 0.0005 9.2.3 Store all calibration standards in 125-mL LDPE narrow-mouth bottles at 2C to 6C, up to six months. 9.2.4 Alternate volumes and concentrations o f standards may be prepared as needed. 10.0 Batch Set Up 10.1 Each batch o f samples extracted (typically 20 or less) must include at least one untreated control and two untreated controls fortified at k n o w n concentrations (lab control spike) to verify procedural recovery for th e b a tc h 10.2 Requirements for field and laboratory duplicates and spikes will be s p e c ifie d in the quality assurance plan for this project. 11.0 Sample Extraction 11.1 Weigh 5 g o f frozen sample into 50 mL polypropylene centrifuge tu b e s (fortify as needed, replace lid and mix well). 11.2 Add 30 mL of acetonitrile and shake on a wrist action shaker for -15 minutes. 11.3 Centrifuge the 50 mL polypropylene tubes containing sample at - 2 0 0 0 rpm for-1 0 minutes. 11.4 Pack and condition the SPE tubes 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 g carbon, and 1 g LC-NH2. Condition the columns with 20 mL of methanol, then 20 mL o f acetonitrile. Discard all washes. Do not allow the column to dry. 11.6 Silanize the 125 mL pear-shaped flasks by rinsing with th e 30/) dimethyldichlorosilane in toluene solution. Rinse the flask with to lu e n e o n c e , followed by methanol (three times). Dry the flasks completely before use, either by air-drying or with a stream o f nitrogen. 11.7 Decant the extract on to a conditioned SPE column fitted inside the mouth 01 ' the pear-shaped flask. Collect the eluate in the 125 mL silanized pear-shape flask. Page 5 o f " E05-0210 Interim Report#15 Northern Alabama Potable Water Systems Page 49 o f 65 Page 103 o f 123 3M Environmental Laboratory E05-0210 Interim Report#15 Exygen Protocol Number: P0001131 Exygen Research Method Number V 0 0 0 1?84 | ANALYTICAL ViF t HOD Method of Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in V e g e ta tio n by LC/MS/MS 11.8 Add 20 mL o f acetonitrile to the sample in the 50 mL centrifuge tube. 11.9 Shake the sample again for ~10 minutes on a wrist-action shaker. 11.10 Centrifuge the 50 mL polypropylene tubes containing sample at -2000 rpm for ~5 minutes. 11.11 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. 11.12 Repeat steps 11.8 through 11.11 again. 11.13 Add 3-4 drops o f 1-octanol to the extract in the pear-shaped flask and evaporate at reduced pressure using a rotary evaporator (at < 40C). 11.14 Make the final volume, by adding 2 mL o f 2% ascorbic acid in methanol to the pear-shaped flask and swirl to mix/dissolve. 11.15 Transfer the extracts to HPLC vials using disposable pipets. 11.16 Analyze samples using electrospray LC/MS/MS. 12.0 Chromatography 12.1 Inject the same amount o f each standard, sample and fortified sample into the LC/MS/MS system. A calibration standard must precede and fo llo w all analyzed samples. 12.2 Standards of PFOA corresponding to at least five or more concentration le v e ls must be included in an analytical set. 12.3 An entire set o f extracted calibration standards must be included at th e beginning and at the end o f a sample set. Extracted standards m u st be interspersed between every 5-10 samples. As an alternative, an entire set o f extracted calibration standards may be injected at the beginning o f a set followed by extracted calibration standards interspersed every 5 -1 0 s a m p le s (to account for a second set o f extracted standards). In either case, extracted calibration standards must be the first and last injection in a sample set. 12.4 Use linear standard curves for quantitation. Linear standard c u rv e s are generated for the analyte by linear regression using 1/x weighting o f p e a k a re a versus calibration standard concentration using MassLynx 3.3 ( o r e q u iv a le n t) software system. 12.5 Sample response should not exceed standard responses. Any samples th at exceed standard responses should be further diluted and reanalyzed. 13.0 Acceptance Criteria 13.1 Chromatogram must show a peak o f a daughter ion at 369 amu from a p a re n t o f 413 amu. The 413 amu parent corresponds to the PFOA a n io n , w h ile the daughter ion (369 amu) represents the loss o f carbon dioxide. Page 6 of 7 E05-0210 Interim Report#15 Northern Alabama Potable Water Systems Page 50 o f 65 Page 104 o f 123 3M Environmental Laboratory E05-0210 Interim Report#15 Exygen Protocol Number: P0001 131 Exygen Research Method Number V 0 0 0 1784 I ANALYTICAL m e t h o d Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Vegetation by LC/MS/MS 13.2 Method blanks must not contain PFOA at levels greater than the LOQ. If a blank contains PFOA at levels greater than 0.5 ppb, then a new blank sa m p le must be obtained and the entire set must be re-extracted. 13.3 Recoveries o f control spikes and matrix spikes must be between 7 0 -1 3 0 % o f their known values. If a control spike falls outside the acceptable lim its, th e entire set o f samples should be re-extracted. 13.4 Any calibration standard found to be a statistical outlier by using th e H u g e Error Test, may be excluded from the calculation of the calibration c u rv e . However, the total number o f calibration standards that could be e x c lu d e d must not exceed 20% of the total number o f standards injected. 13.5 The correlation coefficient (R ) for calibration curves generated m u st be > 0 .9 9 2 (R 2 > 0 .9 8 5 ). If calibration results fall outside these lim its , th e n appropriate steps must be taken to adjust instrument operation, a n d th e standards or the relevant set o f samples should be reanalyzed. 13.6 Retention times between standards and samples must not drift more than 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 calculate the amount o f PFOA found (in ng/mL, based on peak area) using the standard curve (linear regression parameters) generated by the Mass Lynx software program: PFOA found (ng/mL) = (Peak area - intercept) slope 14.2 Use the following equation to convert the amount o f PFOA found in ng/mL to ng/g (ppb). PFOA found (ppb) = rPFOA found (ng/mL) x final volume (mL) x DF1 sample weight (g) DF = factor by which the final volume was diluted, if necessary. 14.3 For samples fortified with known amounts o f PFOA prior to extraction, use the following equation to calculate the percent recovery. Recovery (%) = [ total analyte found (ng/g) - analyte found in control (ng/g)] QQ analyte added (ng/g) Page 7 oi' 7 E05-0210 Interim Report#15 Northern Alabama Potable Water Systems Page 51 o f 65 Page 105 o f 123 3M Environmental Laboratory E05-0210 Interim Report#15 Exygen Protocol Number: P00113 1 ANALYTICAL METHOD Method Number: V0001785 Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Small Mammal Liver by LC/MS/MS Analytical Testing Facility: Exygen Research 3058 Research Drive State College, PA 16801 Approved By: Paul Connolly Technical Leader, LC-MS, Exygen Research a// f i / Z /o 'm . Flaherty ' Vice President, Operations, Exygen Research Date Date Total Pages: 7 E05-0210 Interim Report#15 Northern Alabama Potable Water Systems Page 52 o f 65 .... Page 106 of 123 3M Environmental Laboratory E05-0210 Interim Report#15 Exygen Protocol Number: P0001131 Exygen Research Method Number V 0001785 A ^LY TIC A L^M ET H O D Method of Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Small Mammal Liver by LC/MS/MS 1.0 Scope This method is to be employed for the isolation and quantitation o f p e r f lu o ro o c ta n o ic acid by High Performance Liquid Chromatography coupled to a ta n d e m M ass Spectrometric Detector (LC/MS/MS) in small mammal liver. 2.0 Safety 2.1 Always observe safe laboratory practices. 2.2 Consult the appropriate MSDS before handling any chemical fo r p r o p e r sa fe ty precautions. 3.0 Sample Requirement 3.1 At least 5 g o f test sample for extraction. 3 .2 Samples should be processed before extraction. Place the fro z e n s a m p le in a food processor and homogenize with dry ice. Place the s a m p le s in c o n ta in e rs and leave open in frozen storage overnight to allow for c a rb o n d io x id e sublimation. Seal and place the samples in f r o z e n s to ra g e u n til tim e o f analysis. Alternately, if there is an insufficient amount o f s a m p le ( - l e s s th a n 5 g), then no processing is necessary and the s a m p le c a n b e u s e d a s s u p p lie d . 3.3 Sample collection procedures will be specified in the sampling plan for this project. 4.0 Reagents and Standards 4.1 Water - HPLC grade 4.2 Methanol - HPLC grade 4.3 Acetonitrile - HPLC grade 4.4 Ammonium Acetate - A.C.S. Reagent Grade 4.5 Perfluorooctanoic Acid - Sigma-Aldrich 5.0 Instrument and Equipment 5.1 A high performance liquid chromatograph capable o f pumping up to 2 solvents equipped with a variable volume injector c a p a b le o f in je c tin g 5 -2 0 0 pL connected to a tandem Mass Spectrometer (LC/MS/MS). 5.2 A device to collect raw data for peak integration and q u a n tita tio n . 5.3 Analytical balance capable of reading to 0.00001 g. 5.4 50 mL disposable polypropylene centrifuge tubes. 5.5 15 mL disposable polypropylene centrifuge tubes. 5.6 Disposable micropipets (50-1 OOuL, 100-200uL). 5.7 125-mLLDPE narrow-mouth bottles. 5.8 2 mL clear HPLC vial kit. Page 2 of"7 E05-0210 Interim Report#T5 Northern Alabama Potable Water Systems Page 53 o f 65 Page 107 o f 123 3M Environmental Laboratory E05-0210 Interim Report#15 Exygen Protocol Number: P0001131 Exygen Research Method Number V0001785 | AN Ai7v T IC ^l"m TH Q D Method of Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Small Mammal Liver by LC/MS/MS 5.9 Disposable pipettes. 5.10 Autopipettes (100-1000 pL and 10-100 pL), with disposable tips. 5.11 Waters Sep Pak Vac 6 cc (lg) tC18 SPE cartridges. 5.12 SPE vacuum manifold. 5.13 Tissuemizer. 5.14 Wrist-action shaker. 5.15 Centrifuge capable of spinning 15 mL polypropylene tubes at 3000 rpm. 6.0 Chromatographic System 6.1 Analytical Column: Fluophase RP (Keystone Scientific), 2.1 mm x 50 mm, 5p (P/N: 82505-052130) 6.2 Temperature: 30C 6.3 Mobile Phase ( A ) : 2 mM Ammonium Acetate in Water 6.4 Mobile Phase ( B ) : Methanol 6.5 Gradient Program: Time (min) 0.0 1.0 8.0 20.0 22.5 %A 65 65 25 25 65 Flow Rate % B (mL/min) 35 0.3 35 0.3 75 0.3 75 0.3 35 0.3 6.6 Injection Volume: 15 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 to optimize the HPLC system. 7.0 MS/MS System 7.1 Mode: Electrospray Negative MRM mode, monitoring 413 --^ 369 m/z for PFOA. The above conditions are intended as a guide and may be changed in order to optimize the MSMS system. Page 3 o f 7 E05-0210 Interim Report#15 Northern Alabama Potable Water Systems Page 54 u f 65 Page 108 o f 123 3M Environmental Laboratory E05-0210 Interim Report#15 Exygen Protocol Number: P0001131 Exygen Research Method Number V0001785 1 AN vLYT1CAl "m TH QD Method of Analysis for the Determination ofPerfluorooctanoic Acid (PFOA) in Small Mammal Liver by LC/MS/MS 8.0 Preparation o f Solutions 8.1 Mobile Phase 8.1.1 2 mM ammonium acetate in water is prepared by adding 0.154 g of ammonium acetate to 1000 mL of water. Alternate volumes may be prepared. 9.0 Standard Preparation 9.1 Standard Stock/Fortification Solution 9.1.1 Prepare a stock solution of -100 pg/mL o f PFOA by weighing 10 mg of analytical standard (corrected for purity) and dilute to 100 mL with methanol in a 125-mL LDPE bottle. 9.1.2 A 1.0 pg/mL fortification solution of PFOA is prepared by bringing 1 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 o f PFOA is prepared by bringing 10 mL o f the 1.0 pg/mL solution to a final volume o f 100 with methanol in a 125 mL LDPE bottle. 9.1.4 The stock and fortification solutions are to be stored in a refrigerator at approximately 4C and are stable for a maximum period of 6 months from the date o f preparation. 9.2 Standard Calibration Solutions 9.2.1 9.2.2 LC/MS/MS calibration standards are prepared in methanol via dilution of the 0.1 pg/mL fortification solution. The following is a typical example: additional concentrations may be prepared as needed. Concentration of Fortification Solution (ng/mL) Volume (mL) Diluted to (mL) Final Concentration (ng/mL) 100 5.0 100 100 2.0 100 100 1.0 100 5.0 10 100 2.0 10 100 1.0 10 100 5.0 2.0 1.0 0.5 0.2 0.1 9.2.3 Store all calibration standards in 125-mL LDPE narrow-mouth bottles at 2C to 6C, up to six months. 9.2.4 Alternate volumes and concentrations o f standards may be prepared as needed. Page 4 o f 7 E05-0210 inferimKeport#15. Northern Alabama Potable Water Systems Page 55 o f 65 Page 109 o f 123 3M Environmental Laboratory E05-0210 Interim Report#15 Exygen Protocol Number: P0001131 Exygen Research Method Number V0001785 | ANALYTICAL METHOD Method o f Analysis for the Determination ofPerfluorooctanoic Acid (PFOA) in Small Mammal Liver by LC/MS/MS j 10.0 Batch Set Up 10.1 Each batch o f samples extracted (typically 2 0 or less) must in c lu d e at le ast one untreated control and two untreated controls fortified at k n o w n concentrations (lab control spike) to verify procedural recovery fo r th e b a tc h . 10.2 Requirements for field and laboratory duplicates and spikes will be specified in the quality assurance plan for this project. 11.0 Sample Extraction 11.1 Weigh 1 g of sample into a 50 mL polypropylene centrifuge tubes (fo rtify as needed, replace lid and mix well). Note that alternate weights o f liv e r m a y be measured depending on the sample size available for use. 11.2 Add water to the sample for a final volume o f 10 mL. 11.3 Homogenize sample using a tissuemizer for ~ 1 minute. 11.4 Transfer 1 mL o f the sample using a disposable pipette into a 15 mL disposable centrifuge tube. 11.5 Add 5 mL o f acetonitrile and shake for ~20 minutes on a wrist-action sh a k e r. 11.6 Centrifuge the tubes at -3000 rpm for -5 minutes. 11.7 Decant the supernatant into a 50 mL disposable centrifuge tube and add 35 mL o f water. 11.8 Condition the C18 SPE cartridges (1 g, 6 mL) by passing 10 mL m e th a n o l followed by 5 mL o f HPLC water (~ 2 drop/sec). Do not let column run dry 11.9 Load the sample on conditioned Cig SPE cartridge. Discard eluate. 11.10 Elute with - 2 mL of methanol. Collect 2 mL o f eluate into a graduated 15 mL polypropylene centrifuge tube (final volume = 2 mL). 11.11 Analyze samples using electrospray LC/MS/MS. 12.0 Chromatography 12.1 Inject the same amount o f each standard, sample and fortified sample into the LC/MS/MS system. A calibration standard must precede and follow all analyzed samples. 12.2 Standards o f PFOA corresponding to at least five or more concentration lev els must be included in an analytical set. 12.3 An entire set o f calibration standards must be included at th e b e g in n in g and at the end of a sample set. Standards must be interspersed b e tw e e n e v e ry 5 -1 0 samples. As an alternative, an entire set o f c a lib r a tio n s ta n d a rd s m ay be injected at the beginning o f a set followed b y c a lib r a tio n s ta n d a rd s interspersed every 5-10 samples (to account for a second set o f s ta n d a rd s ). In either case, calibration standards must be the first and la st in je c tio n in a sample set. 12.4 Use linear standard curves for quantitation. Linear standard c u rv e s are generated for the analyte by linear regression using 1/x weighting of p e a k a re a Page 5 of 7 E05-0210 Interim Report#15 Northern Alabama Potable Water Systems Page 56 o f 65 Page 110 o f 123 3M Environmental Laboratory E05-0210 Interim Report#15 Exygen Protocol Number: P0001131 Exygen Research Method Number V0001785 | ANALYTICAL METHOD Method of Analysis for the Determination ofPerfluorooctanoic Acid (PFOA) in Small Mammal Liver by LC/MS/MS versus calibration standard concentration using MassLynx 3.3 (or e q u iv a le n t) software system. 12.5 Sample response should not exceed standard responses. Any samples that exceed standard responses should be further diluted and reanalyzed. 13.0 Acceptance Criteria 13.1 Chromatogram must show a peak o f a daughter ion at 369 amu from a parent o f 413 amu. The 413 amu parent corresponds to the PFOA anion, while the daughter ion (369 amu) represents the loss o f carbon dioxide. 13.2 Method blanks must not contain PFOA at levels greater than the LOQ. If a blank contains PFOA at levels greater than 10 ng/g, then a new blank sample must be obtained and the entire set must be re-extracted. 13.3 Recoveries o f 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 o f samples should be re-extracted. Any matrix spike outside 70 130% 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 H u g e Error Test, may be excluded from the calculation o f the calibration c u rv e. However, the total number o f calibration standards that could be excluded must not exceed 20% o f the total number o f standards injected. 13.5 The correlation coefficient (R) for calibration curves generated must b e >0.992 (R2 >0.985). If calibration results fall outside these limits, th e n appropriate steps must be taken to adjust instrument operation, and th e standards or the relevant set o f samples should be reanalyzed. 13.6 Retention times between standards and samples must not drift more than 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 calculate the amount of PFOA found (in ng/rnL, based on peak area) using the standard curve (linear regression parameters) generated by the Mass Lynx software program: PFOA found (ng/mL) = fPeak area - intercept) x DF x aliquot factor slope DF = factor by which the final volume was diluted, if necessary. Aliquot factor = 10 Page 6 o f 7 E05-0210 Interim Report#15 Northern Alabama Potable Water Systems Page 57 o f 65 Page 111 o f 123 3M Environmental Laboratory E05-0210 Interim Report#15 Exygen Protocol Number: P0001131 Exygen Research Method Number V0001785 | ANALY t l l AL METHOD Method of Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Small Mammal Liver by LC/MS/MS j 14.2 For samples fortified with known amounts o f PFOA prior to extraction, use the following equation to calculate the percent recovery. Recovery (%) = [ total analyte found (ng/mL) - analyte found in control (ng/mL)] analyte added (ng/mL) 14.3 Use the following equation to convert the amount o f PFOA found in ng/mL to ng/g (ppb). PFOA found (ppb) = [PFOA found (ng/mL) x final volume (mL)l sample weight (g) Page 7 of 7 E05-0210 Interim Report#15. Northern Alabama Potable Water Systems Page 58 o f 65 Page 112 o f 123 3M Environmental Laboratory E05-0210 Interim Report#15 Exygen Protocol Number: P0001131 ANALYTICAL METHOD Method Number: V0001786 Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Small Mammal Serum by LC/MS/MS Analytical Testing Facility: Exygen Research 3058 Research Drive State College, PA 16801 Approved By: ^ --V Paul Connolly Technical Leader, LC-MS, Exygen Research J /V / /Z John Flaherty ' Vice President, Operations, Exygen Research \ q|z > (c n { Date Date Total Pages: 7 E05-0210 Interim Report#15 Northern Alabama Potable Water Systems Page 59 o f 65 Page 113 o f 123 3M Environmental Laboratory E05-0210 Interim Report#15 Exygen Protocol Number: P0001131 Exygen Research Method Number V0001786 | ANALYTICAL METHOD Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Small Mammal Serum by LC/MS/MS 1.0 Scope This method is to be employed for the isolation and quantitation of p e r f lu o ro o c ta n o ic acid by High Performance Liquid Chromatography coupled to a ta n d e m M ass Spectrometric Detector (LC/MS/MS) in small mammal s e ru m . 2.0 Safety 2.1 Always observe safe laboratory practices. 2.2 Consult the appropriate MSDS before handling any chemical fo r p ro p e r sa fe ty precautions. 3.0 Sample Requirement 3.1 At least 1 mL o f test sample for extraction. 3.2 No sample processing is needed for serum samples. However, fro z e n s e ru m samples must to allowed to completely thaw to room temperature b e fo re u se. 3.3 Sample collection procedures will be specified in the sampling plan for this project. 4.0 Reagents and Standards 4.1 Water - HPLC grade 4.2 Methanol - HPLC grade 4.3 Acetonitrile - HPLC grade 4.4 Ammonium Acetate - A.C.S. Reagent Grade 4.5 Perfluorooctanoic Acid - Sigma-Aldrich 5.0 Instrument and Equipment 5.1 A high performance liquid chromatograph capable o f p u m p in g up to 2 solvents equipped with a variable volume injector capable of in je c tin g 5 -2 0 0 p L connected 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 o f reading to 0.00001 g. 5.4 50 mL disposable polypropylene centrifuge tubes. 5.5 15 mL disposable polypropylene centrifuge tubes. 5.6 Disposable micropipets (50-1 OOuL, 100-200uL). 5.7 125-mLLDPE narrow-mouth bottles. 5.8 2 mL clear HPLC vial kit. 5.9 Disposable pipettes. 5.10 Autopipettes (100-1000 pL and 10-100 pL), with disposable tips. 5.11 Waters Sep Pak Vac 6 cc (lg ) tC18 SPE cartridges. 5.12 SPE vacuum manifold. 5.13 Vortexer. Page 2 of 7 E05-0210 Interim Report#15 Northern Alabama Potable Water Systems Page 60 o f 65 Page 114 o f 123 3M Environmental Laboratory E05-0210 Interim Report#15 Exygen Protocol Number: P0001131 Exygen Research Method Number V0001786 | AiN/vlYTlCALMETHOD Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in S m a ll Mammal Serum by LC/MS/MS 5.14 Wrist-action shaker. 5.15 Centrifuge capable o f spinning 15 mL polypropylene tubes at 3000 rpm. 6.0 Chromatographic System 6.1 Analytical Column: Fluophase RP (Keystone Scientific), 2.1 mm x 50 mm, 5p (P/N: 82505-052130) 6.2 Temperature: 30C 6.3 Mobile Phase ( A ) : 2 mM Ammonium Acetate in Water 6.4 Mobile Phase ( B ) : Methanol 6.5 Gradient Program: Time (min) 0.0 1.0 8.0 20.0 22.5 %A 65 65 25 25 65 Flow Rate % B (mL/min) 35 0.3 35 0.3 75 0.3 75 0.3 35 0.3 6.6 Injection Volume: 15 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 o rd e r to optimize the HPLC system. 7.0 MS/MS System 7.1 Mode: Electrospray Negative MRM mode, monitoring 4 1 3 -> 3 6 9 m Iz for PFOA. The above conditions are intended as a guide and may be changed in o rd e r to optimize the MSMS system. 8.0 Preparation o f Solutions 8.1 Mobile Phase 8.1.1 2 mM ammonium acetate in water is prepared by adding 0.154 g o f ammonium acetate to 1000 mL of water. Alternate volumes may be prepared. Page 3 of 7 E05-0210 Interim Reprt#f5 Northern Alabama Potable Water Systems Page 61 o f 65 Page 115 o f 123 3M Environmental Laboratory E05-0210 Interim Report#15 Exygen Protocol Number: P0001131 Exygen Research Method Number V0001786 ANALYTICAL METHOD Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Small Mammal Serum by LC/MS/MS 9.0 Standard Preparation 9.1 Standard Stock/Fortification Solution 9.1.1 Prepare a stock solution o f -1 0 0 pg/mL o f PFOA by weighing 10 m g of analytical standard (corrected for purity) and dilute to 100 m L w ith methanol in a 125-mL LDPE bottle. 9.1.2 A 1.0 pg/mL fortification solution o f PFOA is prepared by bringing 1 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 o f PFOA is prepared by bringing 10 mL o f the 1.0 pg/mL solution to a final volume o f 100 with methanol in a 125 mL LDPE bottle. 9.1.4 The stock and fortification solutions are to be stored in a refrigerator at approximately 4C and are stable for a maximum period of 6 months from the date o f preparation. 9.2 Standard Calibration Solutions 9.2.1 9.2.2 LC/MS/MS calibration standards are prepared in methanol via dilution of the 0.1 pg/mL fortification solution. The following is a typical example: additional concentrations may b e prepared as needed. Concentration of Fortification Solution (ng/mL) 100 100 100 5.0 2.0 1.0 Volume (mL) 5.0 2.0 1.0 10 10 10 Diluted to (mL) 100 100 100 100 100 100 Final Concentration (ng/mL) 5.0 2.0 1.0 0.5 0.2 0.1 9.2.3 Store all calibration standards in 125-mL LDPE narrow-mouth b o ttle s at 2C to 6C, up to six months. 9.2.4 Alternate volumes and concentrations o f standards may be prepared as needed. 10.0 Batch Set Up 10.1 Each batch o f samples extracted (typically 20 or less) must include at least one untreated control and two untreated 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. Page 4 o f 7 E05-0210 Interim Report#15 Northern Alabama Potable Water Systems Page 62 o f 65 Page 116~f 123 3M Environmental Laboratory E05-0210 Interim Report#15 Exygen Protocol Number: P0001131 Exygen Research Method Number VOOO1786 I ANALYTICAL m e t h o d Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Small Mammal Serum by LC/MS/MS 11.0 Sample Extraction 11.1 Measure 1 mL o f sample into a 50 mL polypropylene centrifuge tubes (fortify as needed, replace lid and mix well). Note that alternate volumes of serum may be measured depending on the sample size available for use. 11.2 Add water to the sample for a final volume o f 20 mL. Cap tightly 11.3 Vortex for ~1 minute. 11.4 Transfer 1 mL o f the sample using a disposable pipette into a 15 mL disposable centrifuge tube. 11.5 Add 5 mL o f acetonitrile and shake for ~20 minutes on a wrist-action shaker. 11.6 Centrifuge the tubes at -3000 rpm for -5 minutes. 11.7 Decant the supernatant into a 50 mL disposable centrifuge tube and add 35 mL o f water. 11.8 Condition the Cig SPE cartridges (1 g, 6 mL) by passing 10 mL methanol followed by 5 mL o f HPLC water (~ 2 drop/sec). Do not let column run dry 11.9 Load the sample on conditioned Cis SPE cartridge. Discard eluate. 11.10 Elute with - 2 mL o f methanol. Collect 2 mL o f eluate into a graduated 15 mL polypropylene centrifuge tube (final volume = 2 mL). 11.11 Analyze samples using electrospray LC/MS/MS. 12.0 Chromatography 12.1 Inject the same amount o f each standard, sample and fortified sample into the LC/MS/MS system. A calibration standard must precede and follow all analyzed samples. 12.2 Standards o f PFOA corresponding to at least five or more concentration levels must be included in an analytical set. 12.3 An entire set o f calibration standards must be included at the beginning and at the end of a sample set. Standards must be interspersed between every 5-10 samples. As an alternative, an entire set of calibration standards may be injected at the beginning of a set followed by calibration standards interspersed 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 sample set. 12.4 Use linear standard curves for quantitation. Linear standard curves are generated for the analyte by linear regression using 1/x weighting 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 responses should be further diluted and reanalyzed. Page 5 o f 7 E 05-02 f01n trim R p o rt# f5 Northern Alabama Potable Water Systems Page 63 o f 65 PageYY7ofY23 3M Environmental Laboratory E05-0210 Interim Report#15 Exygen Protocol Number: P0001131 Exygen Research Method Number V0001786 | ANAUmCALMKTHOD Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in S m a ll Mammal Serum by LC/MS/MS 13.0 Acceptance Criteria 13.1 Chromatogram must show a peak o f a daughter ion at 369 amu from a p a re n t o f 413 amu. The 413 amu parent corresponds to the PFOA anion, w h ile the daughter ion (369 amu) represents the loss of carbon dioxide. 13.2 Method blanks 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 Recoveries 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 o f samples should be re-extracted. Any matrix spike outside 70 130% 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 H u g e Error Test, may be excluded from the calculation o f the calibration c u rv e However, the total number o f calibration standards that could be e x c lu d e d must not exceed 20% o f the total number o f standards injected. 13.5 The correlation coefficient (R ) for calibration curves generated m u st be > 0 .9 9 2 (R 2 > 0 .9 8 5 ). If calibration results fall outside these lim its , th e n appropriate steps must be taken to adjust instrument operation, a n d the standards or the relevant set of samples should be reanalyzed. 13.6 Retention times between standards and samples must not d rift m o re than 4 % within an analytical run. If retention time drift exceeds this lim it w ith in an analytical run then the set must be reanalyzed. 14.0 Calculations 14.1 Use the following equation to calculate the amount o f PFOA found (in ng/mL, based on peak area) using the standard curve (linear regression p a ra m e te rs ) generated by the Mass Lynx software program: PFOA found (ng/mL) = (Peak area - intercept) x DF x aliquot factor slope DF = factor by which the final volume was diluted, if necessary. Aliquot factor = 20 14.2 For samples fortified with known amounts o f PFOA prior to extraction, use the following equation to calculate the percent recovery. Recovery (%) = [ total analyte found (ng/mL) - analyte found in control (ng/mL)] QQ analyte added (ng/mL)_____________________ _________ -- Page 6 o f 7 E05-0210 Interim Report#15 Northern Alabama Potable Water Systems Page 64 o f 65 Page 118 o f 123 3M Environmental Laboratory E05-0210 Interim Report#15 Exygen Protocol Number: P0001131 Exygen Research Method Number V 0001786 I ANa LVTICAL METHOD ~ Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Small Mammal Serum by LC/MS/MS 14.3 Use the following equation to convert the amount of PFOA found in ng/mL to ppb. PFOA found (ppb) = IPFOA found (ng/mL) x final volume (mL)1 sample volume (mL) Page 7 o f' E05-0210 Interim Report#15 Northern Alabama Potable Water Systems Page 65 o f 65 Page 119 o f 123 3M Environmental Laboratory E05-0210 Interim Report#15 Protocol Exygen P0001131; 3M Study Number E05-0210 Amendment 3 Study Title An a l y s is o f Pe r f l u o r o b u t a n e s u l f o n a t e (PFBS), Pe r f l u o r o h e x a n e s u l f o n a t e (PFHS), a n d PERFLUOROOCTANESULFONATE (PFOS) IN WATER, SO IL, SEDIMENT, FISH, CLAMS, Ve g e t a t io n , Sm a l l Ma m m a l Liv e r a n d Sm a l l Ma m m a l Se r u m Us in g LC/MS/MS f o r t h e 3M D M Pe c a t u r o n it o r in g r o g r a m 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 E05-0210 Interim Report#15 GLP Protocol Amendment #3 Northern Alabama Potable Water Systems Page 1 of 4 Page 120 o f 123 3M Environmental Laboratory E05-0210 Interim Report#15 Protocol Exygen P0001131; 3M Study Number E05-0210 Amendment 3 This amendment modifies the following portion(s) of the protocol: P :r o t o c o l r e a d s T Fe s t i n g a c i l i t y Exygen Research 3058 Research Drive State College, PA15801 Phone: (814) 272-1039 A :m e n d t o r e a d T Fe s t in g a c il it ie s Exygen Research 3058 Research Drive State College, PA15801 Phone: (814) 272-1039 3M Environmental Laboratory Building 2-3E-09 935 Bush Avenue St. Paul, MN 55106 Re a s o n : Addition of 3M Environmental Laboratory as a testing facility allows selected water samples to be sent to the 3Mfacility for analysis of PFBs, PFHS, and PFOS using the most current version of ETS 8-154 "Determination of Perfluorinated Acids, Alcohols, Amides, and Sulfonates inWater by Solid Phase Extractions and High Performance Liquid Chromatography/Mass Spectrometry". The following modifications to ETS 8-154.1 will be incorporated into the study: (1) 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 8 154.1 calls for a 40 mL extraction volume with a 5 mL elution volume which results in an eight fold sample concentration. The actual volumes used and the resulting overall concentration factor will be given inthe final report. Associated method quality control samples will demonstrate that the volume modifications do not impact method accuracy and precision. (2) A solvent (unextracted) calibration curve may be analyzed with the extracted calibration curve to ascertain extraction efficiency. Samples will be analyzed using the extracted curve unless otherwise stated inthe final report. Data fromthe solvent calibration curve will not be included or discussed inthe final report unless it is used to strengthen experimental observations/explanations. (3) ETS 8-154.1 makes no mention of sample surrogate spikes; however, all samples will be spiked with at least one of the following radiolabeled surrogates, PFOA [1,2 13C] (perfluorooctanoic acid), PFOS [18O2] and/or PFNA [1,2 13C] (perfluorononanoic acid - C9).The concentration of the surrogate spike may vary depending on the collection event, but typical samples are spiked with a nominal concentration of 0.05 ng/mL. Surrogate spike recoveries will be documented and discussed inthe final report. Surrogate recoveries between 10025%will be deemed as meeting method criteria for accuracy. (4) 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 blankvalue is used as a "zero point" inthe final calibration curve, then a brief explanation inthe final report or inthe raw data as a Note to File must be E05-0210 GLP Protocol Amendment #3 Page 2 of 4 E05-0210 Interim Report#15 Northern Alabama Potable Water Systems Page 121 o f 123 3M Environmental Laboratory E05-0210 Interim Report#15 Protocol Exygen P0001131; 3M Study Number E05-0210 Amendment 3 included to describe why that specific method blankwas selected as the zero point (closest to the average value, most conservative (largest) value, etc.) (5) The overall analytical uncertainty for each target (non-surrogate) analyte will be estimated using both the method accuracy and precision. The method accuracy and precision will be calculated using data from laboratory contorl 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 howthe analytical uncertainty was determined will be provided inthe final report and/or rawdata. A new revision of ETS 8-154 may incorporate all or several of the modifications listed 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 ifthey are listed above. 3M Environmental Laboratory management will approve all documented deviations to the 3M Environmental Laboratory quality system (SOPs, methods, etc.) that occur during the course of this investigation. E05-0210 E05-0210 Interim Report#15 GLP Protocol Amendment #3 Northern Alabama Potable Water Systems Page 3 of 4 Page 122 o f 123 3M Environmental Laboratory E05-0210 Interim Report#15 Protocol Exygen P0001131; 3M Study Number E05-0210 Amendment 3 Amendment Approval MichaelA. Santoro, 3M , Sponsor Representative J^yP ^ Date William K. Reagen, Ph.D. 3M Environmental Laboratory Manager Date Jaisimha Kesari, Weston Solutions, Study Director Date E05-0210 GLP Protocol Amendment #3 Page 4 of 4 E05-0210 Interim Report#15 Northern Alabama Potable Water Systems Page 123 of 123