Document 91N9LV3qK720oEbVVRvev3aE6

3M 3M ENVIRONMENTAL LABORATORY PROJECT E05-0210; INTERIM REPORT #20 Interim Report #20: Analysis of Water Samples from Northern Alabama Potable Water Systems, February 2006 Study Title A n a lysis o f Per flu o r o b u ta n e s u lfo n a te (PFBS), Pe r flu o r o h e x a n e s u lfo n a te (PFHS), AND PERFLUOROOCTANESULFONATE (PFOS) IN WATER, SOIL, SEDIMENT, FISH, C la m s , V e g e t a t io n , S m all Ma m m a l Liver a n d S m all Ma m m a l S erum Usin g LC/MS/MS for the 3M Decatur Monitoring Program Data Requirement EPA TSCA Good Laboratory Practice Standards 40 CFR 792 Author Susan T. Wolf 3M Environmental Laboratory Interim Report Completion Date Date of signing Performing Laboratory 3M Environmental Laboratory Building 2-3E-09 Building 260-5N-17 935 Bush Ave. Maplewood, MN 55144 St. Paul, M N 55106 Project Identification E05-0210 Total Number of Pages 119 IflC C R E D ITE D I Certificate #2052-01 The testing reported herein meet the requirements of ISO/IEC 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). Page 1 of 119 3M ENVIRONMENTAL LABORATORY PROJECT E05-0210; INTERIM REPORT #20 This page has been reserved for specific country requirements. Page 2 of 119 3M ENVIRONMENTAL LABORATORY PROJECT E05-0210; INTERIM REPORT #20 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, February 2006 Interim Study Identification Number: E05-0210 Interim Report #20 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 Certificate #2052-01 The testing reported herein meet the requirements of ISO/IEC 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). Page 3 of 119 3M ENVIRONMENTAL LABORATORY PROJECT E05-0210; INTERIM REPORT #20 Quality A ssurance 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, February 2006 Study Identification Number: E05-0210 Interim Report #20 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 May 18,2006 January 5, 2007 Phase Extraction In-Phase Audit Data and Report Date Reported to Management Study Director August 1,2006 QAU Representative Date Page 4 of 119 3M ENVIRONMENTAL LABORATORY PROJECT E05-0210; INTERIM REPORT #20 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 Method Summaries...................................................................................................................11 Sample Collection.................................................................................................................... 11 Preparatory and Analytical Methods........................................................................................ 11 Extraction.................................................................................................................................. 11 Analysis..................................................................................................................................... 11 Analytical Results..................................................................................................................... 13 Calibration................................................................................................................................. 13 Limit of Quantitation (LOQ)....................................................................................................... 13 Blanks....................................................................................................................................... 13 Solvent Blank............................................................................................................................ 13 Method Blank............................................................................................................................ 13 Trip Blank.................................................................................................................................. 13 System Suitability..................................................................................................................... 13 Continuing Calibration.............................................................................................................. 14 Lab Control Spikes (LCSs).......................................................................................................14 Sample Duplicates................................................................................................................... 14 Field Matrix Spikes................................................................................................................... 14 Data Summary and Discussion................................................................................................ 14 Page 5 of 119 3 M ENVIRONMENTAL LABORATORY PROJECT E05-0210; INTERIM REPORT #20 Statistical Methods and Calculations........................................................................................ 19 Accuracy and Precision Equations............................................................................................19 Determination of Analytical Uncertainty....................................................................................19 Statement of Conclusion.......................................................................................................... 20 References............................................................................................................................... 20 List of Attachments................................................................................................................... 20 Signature Page......................................................................................................................... 21 List of Tables Table 1. Sample Results Summary......................................................................................................9 Table 2. Sample Description Codes....................................................................................................10 Table 3. Study Reference Substances................................................................................................10 Table 4. Sample Collection Information.............................................................................................. 11 Table 5. Instrument Parameters..........................................................................................................12 Table 6. Liquid Chromatography Gradient Program........................................................................... 12 Table 7. Mass Transitions................................................................................................................... 12 Table 8. Lab Control Spike Results..................................................................................................... 14 Table 9. Sample and QC Results........................................................................................................16 Table 10. Analytical Method Uncertainty......................................................................................16 Page 6 of 119 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 PROJECT E05-0210; INTERIM REPORT #20 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 E x p e rim e n ta l Initiation: M arch 1, 2 0 0 6 In te rim E x p e rim e n ta l C o m p le tio n : A u g u s t 27, 2006 Interim S tu dy C om pletion: D a te o f 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. Page 7 of 119 3M ENVIRONMENTAL LABORATORY PROJECT E05-0210; INTERIM REPORT #20 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 March 1,2006. Samples were submitted for analysis as part of 3M Environmental Laboratory project number E05-0210 (3M Decatur Fluorochemical GLP Monitoring Program, Interim Report #20: Analysis of Water Samples from Northern Alabama Potable Water Systems, February 2006). Water samples were analyzed for perfluorobutane sulfonate (PFBS), perfluorohexane sulfonate (PFHS), and perfluorooctane sulfonate (PFOS) 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/IEC 17025-1999 scope of accreditation and the results reported herein were compliant with this accreditation. The analytical start date for this interim report was August 25, 2006 and the analytical termination date was August 27, 2006. 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 (0.05 ng/mL) and high field spike (0.5 ng/mL). Each empty container was marked with a "fill to here" line to produce a final sample volume of 450 mL. Containers designated for field matrix samples were fortified with an appropriate matrix spike solution containing the three target analytes prior to being sent to the field for sample collection. For the samples and field matrix spikes collected and analyzed for this interim report, PFOA [1,2 13C] was not included as a surrogate. This is a deviation from Protocol P0001130 Amendment #3. A record of deviation has been issued as is included in Attachment C. 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 0.0250 ng/mL, 0.0250 ng/mL, and 0.0250 ng/mL, respectively. All results for quality control samples prepared and analyzed with the samples will be reported and discussed elsewhere in this report. Page 8 of 119 Table 1. Sample Results Summary*1' 3M ENVIRONMENTAL LABORATORY PROJECT E05-0210; INTERIM REPORT #20 3M LIMS ID Sample Description E05-0210-94967 DPWS PW FW03 0 060301 E05-0210-94968 DPWS PW FW03 DB 060301 Average Sample/Sample Duplicate %RPD E05-0210-94971 WMEL PW FW03 060301 E05-0210-94972 WMEL PW FW03 DB 060301 Average Sample/Sample Duplicate %RPD E05-0210-94975 MSTP PW FW03 0 060301 E05-0210-94976 MSTP PW FW03 DB 060301 Average Sample/Sample Duplicate %RPD E 0 5 - 0 2 10-94979 TVATP PW FW03 0 060301 E05-0210-94980 TVATP PW FW03 DB 060301 Average Sample/Sample Duplicate %RPD E05-0210-94983 FWTP PW FW03 0 060301 E05-0210-94984 FWTP PW FW03 DB 060301 Average Sample/Sample Duplicate %RPD E05-0210-94987 E05-0210-94988 SWTP PW FW03 0 060301 SWTP PW FW03 DB 060301 Average Sample/Sample Duplicate %RPD PFBS Concentration (ng/mL) <0.0250 <0.0250 <0.0250 NA 0.0596 0.0575 0.0586 3.5 <0.0250 <0.0250 <0.025&3> NA <0.0250 <0.0250 <0.0250^ NA <0.0250 <0.0250 <0.025&s> NA <0.0250 <0.0250 <0.025C/>) NA PFHS Concentration (ng/mL) <0.0250 <0.0250 <0.0250 NA <0.0250 <0.0250 <0.0250TM NA <0.0250 <0.0250 <0.0250 NA <0.0250 <0.0250 <0.0250 NA <0.0250 <0.0250 <0.0250 NA <0.0250 <0.0250 <0.0250 NA PFOS Concentration (ng/mL) <0.0250 <0.0250 <0.0250 NA 0.102 0.105 0.104 2.8 0.0430 0.0446 0.0438 3.4 0.0441 0.0410 0.0425 7.4 0.0448 0.0432 0.0440 3.7 0.0448 0.0428 0.0438 4.4 NA: Not applicable. %RPD values not determined when concentrations for the sample and/or sample duplicate are below the stated LOQ. (1) The analytical method uncertainties associated with the reported results are as follows: PFBS 100% 17%, PFHS 100% 15%, and PFOS 100% 22%, based on control chart data of laboratory control samples. 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 method uncertainty for PFHS on this sample is 100% 41 % due to the low field matrix spike recovery. Additional discussion is provided on the Data Summary and Discussion section of the report. (3) The analytical method uncertainty for PFBS on this sample is 100% 33% due to the field matrix spike recoveries. Additional discussion is provided on the Data Summary and Discussion section of the report. (4) The analytical method uncertainty for PFBS on this sample is 100% 38% due to the low field matrix spike recovery. Additional discussion is provided on the Data Summary and Discussion section of the report. (5) The analytical method uncertainty for PFBS on this sample is 100% 48% due to the low field matrix spike recovery. Additional discussion is provided on the Data Summary and Discussion section of the report. (6) The analytical method uncertainty for PFBS on this sample is 100% 40% due to the low field matrix spike recovery. Additional discussion is provided on the Data Summary and Discussion section of the report. Page 9 of 119 3M ENVIRONMENTAL LABORATORY PROJECT E05-0210; INTERIM REPORT #20 T est Samples Water samples from six locations were received at the 3M Environmental Laboratory on March 3, 2006 from Tim Frinak of Weston Solutions, Inc. A total of twenty-seven containers were submitted for analysis. The samples were logged in by 3M Environmental Laboratory professional services personnel and placed in refrigerated storage on March 3, 2006 until they were removed for extraction on August 25, 2006. Table 2 below provides the codes for the given sample descriptions. Table 2. Sample Description Codes. Sample Description Abbreviation Definition DPWS WMEL Decatur Potable Water System West Morqan/East Lawrence Water Treatment Plant, Lawrence County, AL SWTP MSTP Sheffield Water Treatment Plant, Sheffield AL Muscle Shoals Treatment Plant, Muscle Shoals, AL FWTP Florence Water Treatment Plant, Florence, AL TVATP Tennessee Valley Authority Treatment Plant, Muscle Shoals, AL PW Potable Water FW Finished Water 0 Matrix Sample DB Duplicate LS Low Spike HS High Spike Reference Substances Table 3 lists the pertinent information regarding the reference substance used for this study. Table 3. Study Reference Substances.___________________ ___________________ Reference Substance PFBS PFHS PFOS Chemical Name Perfluorobutanesulfonate Perfluorohexanesulfonate Perfluorooctanesulfonate Chemical Formula C4F9SO3-IO CeF^SOjK* c 8f 17s o 3 k * Identifier <1)S o u r c e Expiration Date Storage Conditions Chemical Lot Number TCR Number CAS # 29420-49-3 3M 1/17/2007 Frozen 2 TCR-281 (99131-039) CAS # 3871-99-6 3M 10/18/2006 Frozen NB# 120067-69 TCR-83 (SE036) 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% 86.4% (2>Solubility 54,400 ppm No information available (1 ) Documentation regarding synthesis of the test substances is located at the source. 680 ppm (2) All test substances are believed to be soluble in water at the levels to be investigated. No visual precipitates observed. Page 10 o f 119 3M ENVIRONMENTAL LABORATORY PROJECTE05-0210; INTERIM REPORT #20 Method Summaries Sample Collection Samples were collected on March 1,2006 in NalgeneTM (low-density polyethylene) bottles prepared at the 3M Environmental Laboratory on February 20, 2006. Containers designated for field matrix spike samples were spiked in the laboratory with a known volume of an appropriate matrix spiking solution containing the 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 matrix spike levels for the trip blanks were the same as the samples. Table 4 below details the spike amounts for the four bottles comprising the sample location set. Table 4. Sample Collection Information mBottle Description Nominal Final Volume Collected (mL) Final Concentration (ng/mL) PFBS PFHS PFOS Sample Sample Dup 450 NA NA NA 450 NA NA NA Low Field Matrix Spike (LS) 450 0.0499 0.0502 0.0496 High Field Matrix Spike (HS) 450 0.499 0.502 0.496 (1) The sample description was assigned in the field by Weston Solutions personnel. 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). Samples were initially extracted on May 18 and 25, 2006 and analyzed on May 22 and 26. Because associated laboratory quality control samples did not meet method criteria, samples were re-extracted along with a new calibration curve and all associated quality control samples on August 25, 2006. The second set of extracts were analyzed on August 27, 2006. All results reported here are from the second extraction set. Analysis All sample and quality control extracts were analyzed for PFBS, PFHS, and PFOS 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. Page 11 of 119 3M ENVIRONMENTAL LABORATORY PROJECT E05-0210; INTERIM REPORT #20 Table 5. Instrument Parameters Instrum ent Name Liquid Chromatograph Guard column Analytical column Injection Volume Mass Spectrometer Electrode Ion Source Polarity Software ETSGinger Agilent 1100 Betasll C18 (2.1 mm X 100 mm), 5 up Betasil C18 (2.1 mm X 100 mm), 5 pm 5 pL Applied Biosystems API 5000 Z-spray Turbo Spray Negative Analyst 1.4.1 Table 6. Liquid Chromatography Gradient Program Step Number 0 1 2 3 4 5 Total Time (min) 0 1.0 11.0 13.5 14.0 17.0 Flow Rate (fdJmin) 300 300 300 300 300 300 Percent A (2 mM ammonium acetate) 60.0 60.0 10.0 10.0 60.0 60.0 Percent B (Methanol) 40.0 40.0 90.0 90.0 40.0 40.0 Table 7. Mass Transitions Analyte Mass Transition Q1/Q3 Dwell Time (msec) (,)P FB S 299/80 150 2 9 9 /9 9 150 ,1|P F H S 399/130 150 399/99 150 399/80 150 <1>P F O S 499/130 150 499/99 150 499/80 150 (1) The individual transitions were summed to produce a "total ion chromatogram" (TIC) for the given analyte. The resultant TIC was used for quantitation. Page 12 of 119 3M ENVIRONMENTAL LABORATORY PROJECT E05-0210; INTERIM REPORT #20 A nalytical Results Calibration Calibration standards were prepared by spiking known amounts of stock solutions containing the target analytes 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 twelve spiked standards ranging from 0.025 ng/ml_ to 10 ng/mL (nominal) were prepared. A quadratic, 1/x weighted, calibration curve was used to fit the data. The data was not forced through zero during the fitting process. Calculating the standard concentration using the peak area counts and the resultant calibration curve confirmed 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). The correlation coefficient was greater than 0.999 for all analytes. Lim it o f 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 at least twice those of the method blank(s). This will be presented and discussed in more detail in the section below regarding method blanks. The LOQ for PFBS, PFHS, and PFOS was 0.0248 ng/mL, 0.0252 ng/mL, and 0.0249 ng/mL, respectively. Blanks Three types of blanks were prepared and analyzed with the samples: method blanks, solvent blanks, and field/trip blanks. Each blank type is described below. Solvent 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 with each extraction set 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). All area counts for the method blanks were less than half the area counts of the calibration standard used to establish the LOQ. Trip Blank Prior to sample collection, one sample container was filled with 450 mL of ASTM Type I water, 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 account for any storage conditions and/or holding time issues that the samples may experience. The resultant field blank concentration for PFBS, PFHS, and PFOS were all <0.0250 ng/mL. System Suitability Five replicate injections of the 1.0 ppb (nominal concentration) extracted calibration standard were analyzed at the beginning and end of the analytical sequence to demonstrate overall system suitability. The relative standard deviation (RSD) of the peak area counts and the RSD of the peak retention times were less than 5% and 2%, respectively, for all analytes which met method criteria. Page 13 of 119 3 M ENVIRONMENTAL LABORATORY PROJECT E05-0210; INTERIM REPORT #20 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 analyte CCV recoveries met method criteria of 25%. Lab Control Spikes (LCSs) Replicate low (0.05 ng/mL nominal concentration) and high (0.5 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. All LCS recoveries met method criteria for both accuracy and precision. Table 8. Lab Control Spike Results*1'. PFBS PFHS PFOS Lab ID Spiked Cone. (ng/mL) Calc. Cone. (ng/mL) % Recovery Spiked Cone. (ng/mL) Calc. Cone. (ng/mL) % Recovery Spiked Cone. (ng/mL) Calc. Cone. (ng/mL) % Recovery LCS-060825-1 LCS-060825-2 LCS-060825-3 LCS-060825-4 LCS-060825-5 LCS-060825-6 0.0999 0.0999 0.0999 0.999 0.999 0.999 0.109 0.108 0.111 1.00 1.09 1.14 109 0.100 0.111 108 0.100 0.111 111 0.100 0.108 100 1.00 1.05 109 1.00 1.10 114 1.00 1.16 111 0.0999 0.103 111 0.0999 0.104 108 0.0999 0.107 105 0.999 1.02 110 0.999 1.07 116 0.999 1.15 103 104 107 102 107 116 Average %Recovery 109% 4.3% 110% 3.3% 106%4.6% %RSD (1) Table displays rounded values for all concentration and percent recovery values (3 significant figures) and %RSD (2 significant figures) using EPA rounding rules. Reported values may vary slightly from the raw data. Sample Duplicates B ecau se a field sam p le duplicate (se p a ra te container) w a s collected a t e ach sam pling location, duplicate/replicate extractions of a given sample were not performed. Overall method precision was determined using LCSs. Field Matrix Spikes Low (nominal concentration of 0.05 ng/mL) and high (nominal concentration 0.5 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. D a ta S u m m a r y a n d D is c u s s io n Table 9 below summarizes the sample results and field matrix spike recoveries for all of the sampling locations plus the trip blank. Field matrix spikes in general, produced recoveries Page 14 of 119 3M ENVIRONMENTAL LABORATORY PROJECT E05-0210; INTERIM REPORT#20 within the acceptance criteria of 100%30%. Sample location WMEL PW FW03 had a low field matrix spike recovery for PFHS of 159% and a high field matrix spike recovery of 133%. The sample and sample duplicate results for PFHS were <0.0250 ng/mL. The analytical method uncertainty for PFHS on this sample is 100%41 %. Sample location MSTP PW FW03 had a low field matrix spike recovery for PFBS of 131% and a high field matrix spike recovery of 133%. The sample and sample duplicate results for PFBS were <0.0250 ng/mL. The analytical method uncertainty for PFBS on this sample is 100%33%. This sample also had a high field matrix spike recovery for PFHS of 134%. As the sample result for PFHS was <0.0250 ng/mL, and the low field matrix spike recovery of 120% was within the acceptance criteria, the sample is accurate to the stated method uncertainty for PFHS. Sample location TVATP PW FW03 had a low field matrix spike recovery for PFBS of 138% and a high field matrix spike recovery of 125%. The sample and sample duplicate results for PFBS were <0.0250 ng/mL. The analytical method uncertainty for PFBS on this sample is 100%38%. Sample location FWTP PW FW03 had a low field matrix spike recovery for PFBS of 148% and a high field matrix spike recovery of 129%. The sample and sample duplicate results for PFBS were <0.0250 ng/mL. The analytical method uncertainty for PFBS on this sample is 100%48%. Sample location SWTP PW FW03 had a low field matrix spike recovery for PFBS of 140% and a high field matrix spike recovery of 128%. The sample and sample duplicate results for PFBS were <0.0250 ng/mL. The analytical method uncertainty for PFBS on this sample is 100%40%. Page 15 of 119 3M ENVIRONMENTAL LABORATORY PROJECT E05-0210; INTERIM REPORT #20 Table 9. Sample and QC Results <1>. Location #1 DPWSPWFW03 PFBS PFHS PFOS 3M LIMS ID Description E05-0210-94967 DPWS PW FW03 0 060301 E05-0210-94968 DPWS PW FW03 DB 060301 E05-0210-94969 DPWS PW FW03 LS 060301 E05-0210-94970 DPWS PW FW03 HS 060301 Average Sample Concentration (ng/mL) %RPDm Cone. (ng/mL) %Recovery <0.0250 <0.0250 0.0612 0.626 <0.0250 NA NA 123 126 Cone. (ng/mL) %Recovery <0.0250 <0.0250 0.0622 0.617 <0.0250 NA NA 124 123 Cone. (ng/mL) %Recovery <0.0250 <0.0250 0.0496 0.493 <0.0250 NA NA 99.4 98.8 Location #2 WMEL PWFW03 PFBS 3M LIMS ID Description E05-0210-94971 WMEL PW FW03 0 060301 E05-0210-94972 WMEL PW FW03 DB 060301 E05-0210-94973 WMEL PW FW03 LS 060301 E05-0210-94974 WMEL PW FW03 HS 060301 Average Sample Concentration (ng/mL) %RPDfv Cone. (ng/mL) %Recovery 0.0596 NA 0.0575 0.123 0.673 NA 130 124 0.0586 3.5% PFHS PFOS Cone. (ng/mL) %Recovery <0.0250 NA <0.0250 0.0801 NA 159(3) 0.666 133(3) <0.0250(3) Cone. (ng/mL) %Recovery 0.102 NA 0.105 NA 0.159 110 0.590 97.5 0.104 2.8% Location #3 MSTP PWFW03 PFBS 3M LIMS ID Description E05-0210-94975 MSTP PW FW03 0 060301 E05-0210-94976 MSTP PW FW03 DB 060301 E05-0210-94977 MSTP PW FW03 LS 060301 E05-0210-94978 MSTP PW FW03 HS 060301 Average Sample Concentration (ng/mL) %RPDf2> Cone. (ng/mL) %Recovery <0.0250 NA <0.0250 0.0650 NA 131(4) 0.661 133(4) <0.025&f) PFHS Cone. (ng/mL) %Recovery <0.0250 NA <0.0250 0.0602 0.672 NA 120 134(4> <0.0250*4) PFOS Cone. (ng/mL) %Recovery 0.0431 NA 0.0446 0.0923 0.569 NA 97.2 105 0.0438 3.4% Page 16 of 119 3M ENVIRONMENTAL LABORATORY PROJECT E05-0210; INTERIM REPORT #20 Table 9 Continued. Location #4 TVATP PWFW03 PFBS PFHS PFOS 3M LIMS ID Description E05-0210-94979 TVATP PW FW03 0 060301 E05-0210-94980 TVATP PW FW03 DB 060301 E05-0210-94981 TVATP PW FW03 LS 060301 E05-0210-94982 TVATP PW FW03 HS 060301 Average Sample Concentration (ng/mL) %RPDf2> Cone. (ng/mL) %Recovery <0.0250 NA <0.0250 0.0687 0.620 NA 138(S) 125 <0.0250*5) Cone. (ng/mL) %Recovery <0.0250 NA <0.0250 NA 0.0626 0.627 125 125 <0.0250 Cone. (ng/mL) %Recovery 0.0441 NA 0.0410 0.0921 NA 99.4 0.512 94.1 0.0425 7.1% Location #5 FWTP PW FW03 PFBS PFHS PFOS 3M LIMS ID Description E05-0210-94983 FWTP PW FW03 0 060301 E05-0210-94984 FWTP PW FW03 DB 060301 E05-0210-94985 FWTP PW FW03 LS 060301 E05-0210-94986 FWTP PW FW03 HS 060301 Average Sample Concentration (ng/mL) %RPCf2> Cone. (ng/mL) %Recovery <0.0250 <0.0250 0.0737 0.642 NA NA 148<6) 129 0.0250 m Cone. (ng/mL) %Recovery <0.0250 NA <0.0250 0.0628 0.649 NA 125 129 <0.0250 Cone. (ng/mL) %Recovery 0.0448 NA 0.0432 0.0977 0.547 NA 108 101 0.0440 + 3.7% Location #6 SWTP PWFW03 PFBS PFHS PFOS 3M LIMS ID Description E05-0210-94987 SWTP PW FW03 0 060301 E05-0210-94988 SWTP PW FW03 DB 060301 E05-0210-94989 SWTP PW FW03 LS 060301 E05-0210-94990 SWTP PW FW03 HS 060301 Average Sample Concentration (ng/mL) %>RPDm Cone. (ng/mL) %Recovery <0.0250 NA <0.0250 NA 0.0696 140(7) 0.632 128 <0.025&7) Cone. (ng/mL) %Recovery <0.0250 NA <0.0250 0.0636 0.629 NA 127 125 <0.0250 Cone. (ng/mL) %Recovery 0.0448 NA 0.0428 0.0962 0.542 NA 105 108 0.0438 4.4% Page 17 of 119 3M ENVIRONMENTAL LABORATORY PROJECT E05-0210; INTERIM REPORT #20 Table 9 Continued. Trip Blank PFBS PFHS PFOS 3M LIMS ID E05-0210-94964 E05-0210-94965 E05-0210-94966 Description DPW SPW TRIP 0 060301 DPWS PW TRIP LS 060301 DPWS PW TRIP HS 060301 Cone. (ng/mL) < 0.0250 0.0507 0.599 %Recovery NA 102 121 Cone. (ng/mL) <0.0250 0.0533 0.603 YoRecovery NA 106 120 Cone. (ng/mL) < 0.0250 0.0422 0.478 YoRecovery NA 84.5 95.8 (1) The analytical method uncertainties associated with the reported results are as follows: PFBS 100% 17%, PFHS 100% 15%, and PFOS 100% 22%, based on control chart data of laboratory control samples. 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) Not applicable. %RPD values not determined when concentrations for the sample and/or sample duplicate are below the stated LOQ. (3) Sample location WMEL PW FW03 had a low field matrix spike recovery for PFHS of 159% and a high field matrix spike recovery of 133%. The sample and sample duplicate results for PFHS were <0.0250 ng/mL. The analytical method uncertainty for PFHS on this sample is 100%41%. (4) Sample location MSTP PW FW03 had a low field matrix spike recovery for PFBS of 131% and a high field matrix spike recovery of 133%. The sample and sample duplicate results for PFBS were <0.0250 ng/mL. The analytical method uncertainty for PFBS on this sample is 100%33%. This sample also had a high field matrix spike recovery for PFHS of 134%. As the sample result for PFHS was <0.0250 ng/mL, and the low field matrix spike recovery of 120% was within the acceptance criteria, the sample is accurate to the stated method uncertainty for PFHS. (5) Sample location TVATP PW FW03 had a low field matrix spike recovery for PFBS of 138% and a high field matrix spike recovery of 125%. The sample and sample duplicate results for PFBS were <0.0250 ng/mL. The analytical method uncertainty for PFBS on this sample is 100%38%. (6) Sample location FWTP PW FW03 had a low field matrix spike recovery for PFBS of 148% and a high field matrix spike recovery of 129%. The sample and sample duplicate results for PFBS were <0.0250 ng/mL. The analytical method uncertainty for PFBS on this sample is 100%48%. (7) Sample location SWTP PW FW03 had a low field matrix spike recovery for PFBS of 140% and a high field matrix spike recovery of 128%. The sample and sample duplicate results for PFBS were <0.0250 ng/mL. The analytical method uncertainty for PFBS on this sample is 100%40%. Page 18 of 119 3M ENVIRONMENTAL LABORATORY PROJECT E05-0210; INTERIM REPORT #20 Statistical M ethods 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 LCS/Surrogate Percent Recovery -C---a--l-c--u--l-a--t-e--d---C---o--n--c--e--n--t-r--a--t-i-o--n- . .100% Spike Concentration (Spiked Sample Concentration - Average Concentration : Field Sample & Field Sample Dup.) . 100Sample Spike Recovery % Spike Concentraton . . . _ . . . , standard deviation of replicates . ,, % RSD (Relative Standard Deviation) = -------------------------------------------- * 100% replicate average > , / i r. t v *. > Absolute difference between sample duplicates . . . . . . % RPD (Relative Percent Difference) = ------------------------------------------------ ------------------ * 100% average sample concentration Determination o f Analytical Uncertainty Analytical uncertainty is based on historical QC data that is control charted and used to evaluate method accuracy and precision. The method uncertainty is calculated following ETS-12-012.2. The standard deviation is calculated for the set of accuracy results (in %) obtained for the QC samples. The expanded uncertainty is calculated by multiplying the standard deviation by factor of 2, which correspond with a confidence level of 95%. A minimum of twenty data points are needed to determine method uncertainty by this method. This method of determining method uncertainty was applied to all analytes. Page 19 of 119 Table 10. Analytical Method Uncertainty Analyte PFBS PFHS PFOS Number of data points used for determining uncertainty 120 121 249 Standard Deviation 8.38 7.45 10.8 3M ENVIRONMENTAL LABORATORY PROJECT E05-0210; INTERIM REPORT #20 Method Uncertainty 100% 17% 100% 15% 100% 22% Statement of Conclusion Sample results for this project were presented in Table 1. Laboratory control spikes were used to determine the method accuracy and precision for the target analytes for this data set. The accuracy and precision were then used to estimate the analytical uncertainty for each of the analytes (PFBS: 1005.2%; PFHS: 1009.3%, and PFOS: 100+11%). Recoveries of field matrix spiked samples demonstrated that the overall analytical method was appropriate for the matrix collected. References Rorabacher, David B. "Statistical Treatment for Rejection of Deviant Values: Critical Values of Dixon's `Q' Parameter and Related Subrange Ratios at the 95% Confidence Level." Anal. Chem. 1991, 63, 139-146. List of Attachments Attachment A: Selected Chromatograms and Calibration Curves Attachment B: Extraction and Analytical Methods Attachment C: Protocol and Protocol Amendments Attachment D: Protocol and/or Method Deviations Page 20 of 119 3M ENVIRONMENTAL LABORATORY PROJECT E05-0210; INTERIM REPORT #20 Signature Page We certify that this report is a true and complete representation of the data for this study: William K. Reagen, Ph.D., 3M Environmental Laboratory Manager Date Page 21 of 119 3M ENVIRONMENTAL LABORATORY PROJECT E05-0210; INTERIM REPORT #20 Attachment A: Sample Chromatograms and Calibration C urves Page 22 of 119 *Ginger AG01330509 Sample Nam*: "g060827a017" Snpte 10: "EC-060825-0081 Peak Name: "plb$" Masses): "299.0/99.0 amu,299.0/80.0 Comment "1.0 ppb extracted curve point" Annotation: Sample Index: 17 Sample Type: Standard Concentration: 0.9990 Calculated Cone: 0.9877 ng/mL ng/mL 82e4 ' Acq. Date: Acq. Time: 8/27/2006 3:48:40 PH 8.0*4 Modified: Window: Expected RT: Relative RT: Yes 30.0 4.86 No sec min Int. Type: Manual Retention Time: 4.882 min i: 1709323 counts Height: 82100. cps art Time: 4.40 min End Time: 5.35 min I Sample Name: "g 06 08 27 a0 ir Sample ID: "EC-060825-008" File: "g060827a.wiT Peak Name: "pfhs" Mass(es): "399.0/99.0 amu,399.0/80.0 amu,399.0/130.0 amu" I Comment: "1.0 ppb extracted curve point" Annotation:"" Sample Index: 17 Sample Type: Standard Concentration: Calculated Cone 1.000 1.009 ng/mL ng/mL 2.6e5 Acq. Date: Acq. Time: 8/27/2006 3:48:40 PM 2.6*5 Modified: RT Window: Expected RT: Use Relative RT Yes 30.0 8.67 No sec min Int. Type: Retention Time: Manual 8.675 min Area: 1675489 counts Height: 262000. cps Start Time: 8.26 min End Time: 9.07 min 2.5e5 2.5e5 2.4e5 2.4*5 2.3e5 2.3e5 2.2e5 2.2e5 2.1*5 2.1 5 2.0e5 2.0e5 1.9e5 1.9e5 1.8eS 1.8e5 1.7e5 1.7eS 1.6e5 1.6e5 1.5e5 1.5e5 1.4e5 1.4e5 1.3e5 1.3e5 1.2e5 1.2e5 1.1*5 1.1e5 1.0e5 9.5e4 9.0e4 8.5e4 8.0*4 7.5e4 7.0*4 6.5e4 6.0e4 5.5*4 5.0*4 4.5e4 4.0e4 3.5*4 3.0*4 2.5e4 2.0e4 1.5e4 1.0*4 5000.0 3.4 3.6 3.8 4.0 4.2 4.4 4.6 4.8 5.0 5.2 5.4 5.6 5.8 6.0 6.2 147 156 164 173 182 190 199 207 216 225 233 242 250 259 268 ________________________ Time, min________________________ 0.0 Printing Time: 1:48:47 PM Results Name: g060827a.rdb I Sample Name: "g060827a017" Peak Name: "pfos" Comment " 1.0 ppb Sample ID: "EC-060825-0081 File: "g060827a.wifr Sample Ind<PROJSI05-0210; INTERIM REPORT #2$# Sample Typ< Concentration: 0.9990 ng/mL 1.8e5 Calculated Cone: 1.006 ng/mL Acq. Date: Acq. Time: 8/27/2006 3:48:40 PM 1.8e5 Modified: No Proc. Algorithm: IntelliQuan Noise Percentage: 50 Base. Sub. Window:: 1.00 i Peak-Split. Factor Report Largest Pea: 1.7*5 Min. Peak Height: Min. Peak width: Smoothing Width: RT Window: points 1.6e5 Expected RT: Use Relative RT: Area : Height: Start Time: End Time: 1411930 counts 181000. cps 9.72 rain 10.7 min 1.4e5 1.4e5 1.3e5 1.3e5 1.2e5 1.2*5 1.1e5 1.1e5 1.0e5 9.5e4 9.0e4 8.5*4 8.0e4 7.5e4 7.0e4 6.5*4 6.0e4 5.5e4 5.0e4 4.5*4 4.0*4 3.5*4 3.0*4 2.5*4 2.0*4 1.5*4 1.0*4 5000.0 0.0 Page 23 qF119i0* 110 431 452 474 n-s 495 Page 1 of 1 Ginger AG01330509 Sample Nairn: *g060827a024` Sample ID: -M B-060825-r F la:'g060827a.arir Peak Name: "pfbs" Masses): *299.0/99.0 amu,299.0/80.0 amu" Comment "Method Btank-1" Annotation: *" :Sample Index: 24 Sample Type: Unknown Concentration: N/A Calculated Cone: <0 Acq. Date: 8/27/2006 Acq. Time: 5:54:01 PM Modified: No Proc. Algorithm: IntelliQuan - IQA Min. Peak Height: 0.00 cps Min. Peak Midth: 0.00 sec Smoothing Width: 0 points RT Window: 30.0 sec Expected RT: 4.86 min Relative RT: No 235 Int. Type: Retention Time: Valley ISample Name: "g060827a024" Sample ID: "MB-060825-1" File: g060fl27a.wffr Peak Name: "pfhs" Mass(es): "399.0/99.0 amu,399.0/80.0 amu.399.0/130.0 amu" Comment: "Method Blank-1" Annotation: "* Sample Index: 24 Sam ple Type Unknown C on centration: C alc u lated Cone: N/A Acq. Date: A cq. Time: ifie d : RT Window: E xp ected RT: Use R e la tiv e RT: No I n t. Type: R eten tion Tim e: A rea: H eight: Tim e: End Time: Manual 8.648 min 2115 counts 332. cps 8.58 min 8.74 min Results Name: g060827a.rdb I M i m m m m m t m m T O R YSample Name: "g060827a024" Sample ID: "MB-060825-r Fite: "g060827a.wrfT Comment: "Method HZit ITpP'ROJfcX E05-0210; INTERIM REPORT #20 Concentration: N/A Calculated Cone: <0 Acq. Date: 8/27/2006 Acq. Time: 5:54:01 PM Modified: No Proc. Algorithm: IntelliQuan - MQII Noise Percentage: 50 Base. Sub. Window: 1.00 min Peak-Split.Factor: 5 Report Largest Peak: No Min. Peak Height: 0.00 cps Min. Peak Width: 0.00 sec Smoothing Width: 0 point RT Window: 30.0sec Expected RT: Use Relative RT No Int. Type: Retention Time: Area: Height : Start Time: End Time: Base To Base 10.349 min 3005 counts 644. cps 10.3 min 10.5 min Printing Time: 1:49:50 PM Page 1 of 1 Ginger AG01330509 I Sample Name: "g060827a038" Sample ID: "LCS-060825-4" File: "g060827a.wir Peak Name: "plbs" Masses): "299.0/99.0 amu,299.0/80.0 amu' Comment "1 ppb Lab Control Spike" Annotation: SSaammppllee TIynpdee:x: 38 QC Concentration: 0.9990 n Calculated Cone: 1.002 n Acq. Date: 8/27/2006 Acq. Time: 10:04:46 PM Modified: RT Window: Expected RT: Relative RT: Yes 30.0 4.86 No Height : End Time: Manual e: 4.843 min 1733925 counts 82900. cps 4.30 min 5.37 min 3.4 3.6 3.6 4.0 4.2 4.4 4.6 4.8 5.0 5.2 5.4 5.6 5.8 6.0 6.2 __1_4_7__1_56__1_6_4__1_7_3__1_8_2__19__0_1_9_9Ti2m0e7, m21in6_2_2_5__2_3_3__2_4_2__25__0_2_5_9__2_6_8_ j Sample Name: "g060827a038" Sample ID: TCS-060825- I Peak Name: "pfhs" Mass(es): "399.0/99.0 amu,399.0/80.0 I Comment "1 ppb Lab Control Spike" Annotation: " Sample Index: 38 Sample Type: QC Concentration: 1.000 ng/mL Calculated Cone: 1.049 ng/mL A cq. Date : 8/27/2006 2.6e5 Acq. Time : 10:04:46 PM 2.6e5 Modified: RT Window: Expected RT: Use Relative RT: Yes 30.0 8.67 No sec min 2.5e5 2.5e5 Int. Type: Manual Retention Time: 8.667 min Area: 1741795 counts Height: 269000. cps Start Time: 8.23 min End Tie: 9.09 min 2.4e5 2.4e52.3e5: 2.3e5- 2.2e5- 2.2e5 2.1e5 2.1e5- 2.0e5 2.0e5- 1.9e5- 1.9e5 1.8e5- 1.8e5- 1.7e5 1.7e5- 1.6e5- 1.6e5- 1.5e5- 1.5e5- 1.4e5: & 1.4e5: I 1.3e5 I 1.3e5: 1.2e5- 1.2e5- 1.1e5- 1.1e5 1.0e5 9.5e4- 9.0e4- B.5e4 8.0e4- 7.5e4- 7.0e4: 6.5e4 6.0e4- 5.5e4- 5.0e4- 4.564: 4.0e4: 3.5e4- 3.064 2.5e4- 2.064- 1.5e4- 1.0e4 5000.0- o.oJ Printing Time: 1:50:42 PM Results Name: g060827a.rdb Page 1 of 1 Ginger AG01330509 Sample Name: g080827a051` Sample ID: "E05-0210-94941" I Peak Name: "ptos" Mass(es): "299.0/99.0 amu,299.0/80.0 amu" > Comment: "WMEL PW FW03 0 060301" Annotation: Sample Index: 51 Sample Type: Unknown Concentration: N/A Calculated Cone: 0.05961 ng/mL Acq. Date: 8/28/2006 Acq. Time: 1:57:46 AN Nodified: RT Window: Expected R T : Relative RT: Yes . Type: Manual Retention Time: 4.838 min Area: 120505 counts Height: 6000. cps Start Time: I sample Name: "g060827a051" Sampte ID: "E05-0210-94941" Peak Name: "pfhs" Mass(es): "399.0/99.0 amu,399.0/80.0 Comment'V M E L P W F W 0 3 0 060301" Annotation Sample Index: 51 Sample Type: Unknown Concentration: N/A Calculated Cone: 0.01718 ng/mL 6600 Acq. Date: Acq. Time: 8/28/2006 1:57:46 AN 6500 6400 Modified: Yes Proc. Algorithm: IntelliQuan 6300 Noise Percentage: 50 6200 Base. Sub. Nindow: 1.00 Peak-Split. Factor:5 6100 Report Largest Peak: Min. Peak Height: Min. Peak Width: No 0.00 0.00 6000 5900 Smoothing Width: RT Window: 0 30.0 se 5800 Expected RT: 5700 Use R e lati RT: No 5600 I n t . Type: R etention Ti A rea : H eight: S t a r t Time: End Time : Base To Base 8.652 min 43387 counts 6590. cps 5500 5400 5300 5200 5100 5000 4900 4800 4700 4600 4500 4400 4300 4200 4100 4000 3900 3800 3700 3600 3500 3400 3300 3200 3100 3000 2900 2800 2700 2600 2500 2400 2300 2200 2100 2000 1900 1800 1700 1600 1500 1400 1300 1200 1100 1000 900 8.41 3.4 3.6 3.6 4.0 4.2 4.4 4.6 4.8 5.0 5.2 5.4 5.6 5.8 6.0 6.2 147 156 164 173 182 190 199 207 216 225 233 242 250 259 268 Ttnm.min Printing Time: 1:51:28 PM ; VA 7.5 8.0 8.5 9.0 323 345 366 388 ____________________ Tana, min Results Name: g060827a.rdb I^^^^m m m am ^m m bwTORYSample Name: "g060827a051" Sampie MD: "E05-0210-94941" File: "g060827a.wiT IZII ITpPROJX E05-0210; INTERIM REPORT # ^ s Acq. Date: Acq. Time: 8/28/2006 1:57:46 AM Modified: No Proc. Algorithm: IntelliQuan - MQII Noise Percentage 50 Base. Sub. Window: 1.00 m Peak-Split. Factor: 5 Report Largest P ak: No Min. Peak Height 0.00 cps Min. Peak Width: 0.00 Smoothing Width: points RT Window: 30.0 sec Expected RT: 10.4 min Use Relative RT: No Int. Type: Base To Base Retention Time: 10.349 min Area: 154062 counts Height : 17400. cps Start Time: 9.72 min End Time: 10.6 min 1.7e4 1.7*4 1.6e4 1.6e4 1.5e4 1.5e4 1.4*4 1.4e4 1.3*4 1.3*4 1.264 1.264 1.164 1.164 1.064 9500.0 fr 9000.0 2? s 6500.0 8000.0 7500.0 7000.0 6500.0 6000.0 5500.0 5000.0 4500.0 4000.0 3500.0 3000.0 2500.0 2000.0 1500.0 1000.0 500.0 0.0 Page 1 of 1 3M ENVIRONMENTAL LABORATORY PROJECT E05-0210; INTERIM REPORT #20 Attachment B: Extraction and A nalytical Methods Page 27 of 119 3M ENVIRONMENTAL LABORATORY PROJECT E05-0210; INTERIM REPORT #20 3M Environm ental Laboratoiy Method Determination of Perfiuorinated Acids, Aicohois, 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 Osyasy>3 Date ETS-8-154.1 Page 1 of 17 Determination of Perfiuorinated Compounds in Water Using SPE and LC/MS. Page 28 of 119 3M ENVIRONMENTAL LABORATORY PROJECT E05-0210; INTERIM REPORT #20 1 Scope and Application This method was validated for the collection, extraction, and analytical procedure for the determination of Perfluorooctane sulfonate (PFOS), Perfluorooctane sulfonylamide (FOSA), and Perfluorooctanoate (PFOA) in groundwater, surface water, and drinking water samples. This method may also be applied to the determination of other perfluorinated acids, alcohols, amides, and sulfonates in similar matrices, as long as the defined Q C elements are satisfied and with the understanding that the method is not validated for compounds outside the scope of the original protocol This method is based in part on the report "Method of Analysis for the Determination of Perfluorooctane sulfonate (PFOS), Perfluorooctane sulfonylamide (PFOSA), and Perfluorooctanoate (POAA) in Water'' (see Section 17), as developed and validated by Exygen Research (formerly Centre Analytical Laboratories, Inc.). 2 Method Summary W ater samples are collected from a site of interest and shipped cold to an analytical facility. Perfluorinated acids, alcohols, amides, and sulfonates are extracted from 40m L water samples using C 18 solid phase extraction (SPE) cartridges. The compounds are eluted from the C18 cartridge, using methanol. Separation, identification, and measurement are accomplished by high performance liquid chromatography/ tandem mass spectrometry (HPLC /M S /M S ) analysis. High performance liquid chromatography/mass spectrometry (HPLC/M S) may be used if the defined Q C elements are satisfied. The concentration of each identified component is measured by comparing the MS response of the quantitation ion produced by that compound to the M S response of the quantitation ion produced by the sam e compound in an extracted calibration standard (external standard). 3 Definitions____________________________________________ __ 3.1 Analytical Sam ple A portion of an extracted Laboratory Sample prepared for analysis. 3.2 Calibration Standard A solution prepared from the Working Standard (W S) and extracted according to this method. The calibration standard solutions are used to calibrate the instrument response with respect to analyte concentration. 3.3 Duplicate Sample (DS) A DS is a separate aliquot of a sample, taken in the analytical laboratory that is extracted and analyzed separately with identical procedures. Analysis of DSs compared to that of the first aliquot give a measure of the precision associated with laboratory procedures, but not with sample collection, preservation, or storage procedures. 3.4 Field Blank Control Sample (FB) A STM Type I water placed in a sample container in the laboratory and treated as a sample in all respects, including exposure to sampling site conditions, storage, preservation and all analytical procedures. The purpose of the FB is to determine if test substances or other interferences are present in the field environment. ETS-8-154.1 Page 2 of 17 Determination of Perfluorinated Compounds in Water Using SPE and LC/MS. Page 29 of 119 3M ENVIRONMENTAL LABORATORY PROJECT E05-0210; INTERIM REPORT #20 3.5 Field Duplicate (FD) A sample collected in duplicate at the same time as the sample and placed under identical circumstances and treated exactly the same throughout field and laboratory procedures. Analysis of FD compared to that of the first sample gives a measure of the precision associated with sample collection, preservation and storage, as well as with laboratory procedures. 3.6 Field Matrix Spike (FMS) A sample collected in duplicate to which known quantities of the target analytes are added in the field at the time of sample collection. Alternatively, the known quantity of target analytes may be added to the sample bottle in the laboratory before the bottles are sent to the field. A known, specific volume of sample must be added to sample container without rinsing. This may be accomplished by making a "fill to this level" line on the outside of the sample container. The FM S should be spiked at approximately 5 0 -1 5 0 % of the expected analyte concentration in the sample. If the expected range of analyte concentrations is unknown, a low and a high spike may be prepared to increase the likelihood that a spike at an appropriate range is made. The FM S is analyzed to ascertain if any matrix effects, interferences, or stability issues may complicate the interpretation of the sample analysis. 3.7 Field Spike Control Sample (FSCS) An aliquot of ASTM Type I water to which known quantities of the target analytes are added in the field at the time of sample collection (at an appropriate concentration to be determined by the project lead) or in the laboratory prior to the shipment of the collection bottles. The FSCS is extracted and analyzed exactly like a sample to determine whether a loss of analyte could be attributed to sample storage and/or shipment. A low and high FSCS may be appropriate when expected sample concentrations are not known. 3.8 Laboratory Control Sample (LCS) An aliquot of A STM Type I water to which known quantities of the target analytes are added in the laboratory. Two levels are included, one at the LLOQ (approx. 25 pg/mL), the other at a concentration of approx. 1 0 0 -2 5 0 pg/mL or another concentration to be determined by the project lead. The LCS is extracted and analyzed exactly like a laboratory sample to determine whether the methodology is in control, and whether the laboratory is capable of making accurate measurements at the required method detection limit and higher. 3.9 Laboratory Sample A portion of a sample received from the field for testing. 3.10 Limit of Detection (LOD) The LOD is the lowest concentration of an analyte that can be measured and reported with 99% confidence that the analyte concentration is greater than zero. If required, the LOD m ay be determined in several ways, including signal-to-noise ratio and statistical calculations. 3.11 Lim it o f Q uantitation (LOQ) The LOQ for a dataset is the lowest concentration (LLOQ) or highest concentration (ULO Q ) that can be reliably achieved within the specified limits of precision and accuracy during routine operating conditions. Note: For many analytes, the LLOQ analyte concentration is selected as the lowest non-zero standard in the calibration curve to 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 in Water Using SPE and LC/MS. Page 30 of 119 3M ENVIRONMENTAL LABORATORY PROJECT E05-0210; INTERIM REPORT *20 A matrix spike is an aliquot of a sample, to which known quantities of target analytes are added in the laboratory. The MS is extracted and analyzed exactly like a laboratory sample to determine whether the sample matrix contributes bias to the analytical results. The background concentrations of the analytes in the sample matrix must be determined in a separate aliquot and the measured values in the MS corrected for background concentrations. 3.13 Method Blank An aliquot of A STM Type I water that is treated exactly like a laboratory sample including exposure to all glassware, equipment, solvents, and reagents that are used with other laboratory samples. The method blank is used to determine if test substances or other interferences are present in the laboratory environment, the reagents, or the apparatus. 3.14 Method Detection Limit (MDL) Determination A MDL is the statistically calculated minimum amount of an analyte that can be measured with 99% confidence that the reported value is greater than zero. One of several processes that may be used to establish a LOD value is found in 4 0 C FR Part 136 Appendix B. 3.15 Sample A sample is a small portion collected from a larger quantity of material intended to represent the original source material. 3.16 Spiking Stock Standard (SSS) A solution prepared from stock standards used to prepare the working standard. 3.17 Stock Standard (SS) A concentrated solution of a single analyte prepared in the laboratory with an assayed reference compound. 3.18 Working Standard (WS) A solution of several analytes prepared in the laboratory from SSs and diluted as needed to prepare calibration standards and other required analyte solutions. 4 Warnings and Cautions 4.1 Health and Safety The acute and chronic toxicity of the standards for this method have not been precisely determined; however, each should be treated as a potential health hazard. Unknown samples may contain high concentrations of volatile toxic compounds. Sample containers should be opened in a hood and handled with gloves to prevent exposure. The laboratory is responsible for maintaining a safe work environment and a current awareness of local regulations regarding the handling of the chemicals used in this method. A reference file of material safety data sheets (M SDS) should be available to all personnel involved in these analyses. 4.2 Cautions None 5 Interferences ETS-8-154.1 Page 4 of 17 Determination of Perfluorinated Compounds in Water Using SPE and LC/MS. Page 31 of 119 3M ENVIRONMENTAL LABORATORY PROJECT E05-0210; INTERIM REPORT #20 During extraction and analysis, major potential contaminant sources are reagents and solid phase extraction devices. All materials used in the analyses shall be demonstrated to be free from interferences under conditions of analysis by running method blanks. Parts and supplies that contain Teflon should be avoided due to the possibility of interference and/or contamination. These may include, but are not limited to: wash bottles, Teflon lined caps, autovial caps, HPLC parts, etc. The use of disposable micropipettes or pipettes to aliquot standard solutions is recommended to make calibration standards and matrix spikes. 6 Instrumentation, Supplies, and Materials Note: Brand names, suppliers, and part numbers are for illustrative purposes only. Equivalent performance may be achieved using apparatus and materials other than those specified here, but demonstration of equivalent performance that meets the requirements of this method is the responsibility of the laboratory performing the analysis. 6.1 Instrum entation Balance, analytical (display at least 0.0001 g), Mettler HPLC/M S/M S or HPLC/M S system, as described in Section 10. 6.2 Supplies and Materials. Sample collection bottles-- LDPE (e.g., NalgeneTM) narrow-mouth bottles with screw cap. Note: Do not use Teflon bottles or Teflon lined caps. Coolers for sample shipment. Ice for sample shipment. Vacuum pump, Buchi. Visiprep vacuum manifold, Supelco. Sep Pak V ac 6cc (1g) tC18 cartridges (part # W A T 036795),Waters. 50m L disposable polypropylene centrifuge tubes, VW R. 15mL disposable polypropylene centrifuge tubes, VW R. Disposable micropipettes (5 0 -1 00pL, 100-200pL), Drummond. Class A pipettes and volumetric flasks, various. Hypercarb drop-in guard column (4mm) (p a rt# 8 4 4017-400), Keystone. Stand-alone drop-in guard cartridge holder, Keystone. 125mL LDPE narrow-mouth bottles, Nalgene. 2mL clear HPLC vial kit (cat # 5 181-3 4 0 0 ), Agilent/Hewlett Packard. Standard lab equipment (graduated cylinders, disposable tubes, etc.), various. 7 Reagents and Standards__________________________________________ __ Note: Suppliers and catalog numbers are for illustrative purposes only. Equivalent performance may be achieved using chemicals obtained from other suppliers. Do not use a lesser grade of chemical than those listed. ETS-8-154.1 Page 5 of 17 Determination of Perfluorinated Compounds in Water Using SPE and LC/MS. Page 32 of 119 3M ENVIRONMENTAL LABORATORY PROJECT E05-0210; INTERIM REPORT #20 7.1 Chem icals Methanol (M eO H), HPLC grade, JT Baker, Catalog No. JT9093-2. Ammonium Acetate, Reagent grade, Sigma-Aldrich, Catalog No. A -7 3 3 0 . ASTM Type I Water, prepared in-house. Sodium Thiosulfate, Reagent grade, JT Baker. 7.2 Standards Potassium perfluorooctane sulfonate Perfluorooctane sulfonylamide Ammonium perfluorooctanoate Others as required. 7.3 Reagent Preparation 250mg/m L sodium thiosulfate solution -- Dissolve 25g of sodium thiosulfate in 100mL reagent water. 40% methanol wash solution - Measure 400m L methanol and adjust volume to 1.0L with reagent water. 100mM ammonium acetate solution (Analysis)-- Weigh 7.71 g of ammonium acetate and dissolve in 1.0L of reagent water. Dilute the 10OmM solution by a factor of 50 to m ake the 2m M ammonium acetate solution used for mobile phase A. Note: Alternative volumes may be prepared as long as the ratios of the solvent to solute ratios are maintained. 7.4 Spiking Stock Standard (SSS) Preparation The following standard preparation procedure serves as an example and may be changed to suit the needs of a particular study. For example, pL volumes may be spiked into volumetric flasks when diluting stock solutions to appropriate levels. 100|jg/m L each PFO S, PFO SA, and PO AA SSSs-- Weigh out 10mg of analytical standard (corrected for percent salt and purity-- i.e., 10 mg C8F 17S03K purity 90% = 8.35m g C8F 17S03~) and dilute to lOOmLwith methanol in a 100mL volumetric flask. Transfer to a 125mL LDPE bottle or other suitable container. Prepare a separate solution for each analyte. Solutions may be stored in a refrigerator at 42C for a maximum period of 6 months from the date of preparation. 1pg/m L m ixed SSS-- Add 1.OmL each of the 100pg/mL SSSs (from 7.4.1) to a 100mL volumetric flask and bring up to volume with methanol. 0.1pg/m L m ixed SS S-- Add 10.0mL of the 1.Opg/mL-mixed solution (from 7.4.2) to a 100mL volumetric flask and bring up to volume with methanol. O.Olpg/mL m ixed SSS-- Add 10.0mL of the O.lpg/mL-mixed solution (from 7.4.3) to a 100mL volumetric flask and bring up to volume with methanol. Storage Conditions-- Store all SSSs in a refrigerator at 42C for a maximum period of 6 months from the date of preparation. 7.5 Calibration Standards The following standard preparation procedure serves as an example and may be changed to suit the needs of a particular study, provided the concentrations are calculated correctly. ETS-8-154.1 Page 6 of 17 Determination of Perfluorinated Compounds in Water Using SPE and LC/MS. Page 33 of 119 3M ENVIRONMENTAL LABORATORY PROJECT E05-0210; INTERIM REPORT #20 100|jg/m L each PFOS, PFO SA, and PO AA stock standard solutions--Weigh out 10mg of analytical standard (corrected for percent salt and purity) and dilute to 100mL with methanol in a 100mL volumetric flask. Transfer to a 125mL LDPE bottle or other suitable container. Prepare a separate solution for each analyte. Store solutions in a refrigerator at 42C for a maximum period of 6 months from the date of preparation. 1pg/mL W orking Standard--Add I.OmL each of the 100pg/ml_ SS solutions (from 7.5.1) to a 100mL volumetric flask and bring up to volume with methanol. 0.1 pg/m L W orking Standard --Add 10.0mL of the 1.0pg/mL mixed solution (from 7.5.2) to a 100mL volumetric flask and bring up to volume with methanol. O.Olpg/mL W orking Standard --Add 10.0mL of the 0.1pg/mL mixed solution (from 7.5.3) to a 100mL volumetric flask and bring up to volume with methanol. Storage Conditions--Store all WSs in a refrigerator at 42C for a maximum period of 6 months from the date of preparation. Calibration Standard-- Prepare calibration solutions in ASTM Type I using the following table as a guideline: Final Calibration Concentration Volume of Standard Volume, mL , of WS, pg/mL WS, ml of ASTM Type 1Water 0.0 0 40 0 .0 1 0 100 40 0 .0 1 0 200 40 0 .0 1 0 400 40 0 .1 0 100 40 0 .1 0 200 40 0 .1 0 300 40 0 .1 0 400 40 1 .0 1 0 0 40 1 .0 400 40 1 .0 1 0 0 0 40 Final Concentration of Calibration Standard, pg/mL, in ASTM Type I Water 0 25 50 100 250 500 750 1000 2500 10000 25000 The standards are processed through the extraction procedure (Section 11), identical to the laboratory samples. The concentration of the calibration standard in the final extract is equal to 8X the initial concentration, due to the concentration of the standard during the extraction process. Storage Conditions--Store all extracted calibration standards in 15mL polypropylene tubes at 42C, for a maximum period of two weeks from the date of preparation 8 Sample Collection and Handling Note: Sampling equipment, including automatic samplers, must be free of Teflon tubing, gaskets, and other parts that may leach interfering analytes into the water sample. Automatic samplers that composite samples overtime should use refrigerated polypropylene sample containers if possible. Sample bottles should not be rinsed before sample collection. Labeling: Each sample bottle must display information regarding the collection of that sample, the individual collecting the sample, and any matrix spike that has been added to the sample. ETS-8-154.1 Page 7 of 17 Determination of Perfluorinated Compounds in Water Using SPE and LC/MS. Page 34 of 119 3M ENVIRONMENTAL LABORATORY PROJECT E05-0210; INTERIM REPORT #20 This includes the volume and concentration of any spiking solution added and the volume and identification of any preservatives added in the field. Spiking: The spiking scheme will be clearly outlined in the sampling plan, including whether the samples will be spiked in the field or in the laboratory prior to the shipment of the bottles to the site. If spiking is to be performed in the field, materials and specific instructions will be included in the sampling kit. Be sure to clearly label each bottle with spiking information if applicable. Tap W ater: Open the tap and allow the system to flush until the water temperature (1510C) has stabilized (usually about two minutes). Adjust the flow to about 500ml_/min and collect samples from the flowing stream. Ground W ater: Purge the well of standing water using a pump or a bailer. Collect the sample directly from the pump or from the bailer. Surface Water: When sampling from an open body of water, fill the sample container with water from a representative area. Sam ple Dechlorination: All samples should be iced or refrigerated at 42C and kept in the dark from the time of collection until extraction. Residual chlorine should be eliminated by adding 200pL of a 250mg/ml_ sodium thiosulfate solution to each tap-water sample and associated FB and FSCS (which may be placed in each bottle before leaving for the sampling site or done in the field.). Holding Tim e (HT): Results of the time/storage study of all target analytes showed that the three compounds are stable for 14 days iri water samples when the samples are dechlorinated and stored as described in the previous section (see also references in section 17). Therefore, laboratory samples must be extracted within 14 days and the extracts analyzed within 30 days of sample collection. If the HT exceeds 14 days, great care is used when evaluating field spikes to avoid misrepresentation of the sample concentration. 8.1 Field Blanks Process a Field Blank Control Sample (FB) along with each sample set (samples collected from the same general sample site at approximately the same time). At the laboratory, prior to sample collection, fill a sample container with ASTM Type I water, seal, and ship the FB to the sampling site along with the empty sample containers. Return the FB to the laboratory with the filled sample bottles. When sodium thiosulfate is added to samples, use the same procedure to preserve the FB. 8.2 Field Duplicates C o lle c t a F ie ld D u p lic a te (F D ) fo r e v e ry te n (1 0 ) s a m p le s c o lle c te d o r p e r e a c h s a m p lin g se t, if less than 10 samples are collected. Separate FDs must be collected for each type of water sample (ground, tap, etc.) collected. Collect the FD immediately after the sample. Preserve, store and ship FD using the same procedures as used for the samples. 8.3 Field Spike Control Sample (FSCS) A Field Spike Control Sample (FSCS) must be prepared for each sample shipment. If multiple coolers are used to ship a set of samples, each cooler must contain a FSCS. At the laboratory, fill a sample container with 100mL of ASTM Type I water. Seal and ship to the sampling site along with the empty sample containers and FBs. Samples may either be spiked in the field or in the laboratory prior to shipment. The method employed should be consistent throughout the study. If the samples are to be spiked in the field, be sure to send appropriate supplies and instructions for the field personnel to follow. ETS-8-154.1 Page 8 of 17 Determination of Perfluorinated Compounds in Water Using SPE and LC/MS. Page 35 of 119 3M ENVIRONMENTAL LABORATORY PROJECT E05-0210; INTERIM REPORT #20 Seal and gently invert the FSCS to mix. Store and ship the FSCS using the same procedures as used for the samples Provide information on sample collection, preservation, shipment and storage. List applicable holding times. Include sample stability and extract storage requirements. Reference the method used for sample preparation, if applicable. 8.4 Field Matrix Spike (FMS) A Field Matrix Spike (FMS) must be prepared for each sampling location. One unspiked sample from the same location must accompany the FMS to determine endogenous levels in the sample. The samples should be clearly identifiable as being from the same location. Samples may either be spiked in the field or in the laboratory prior to shipment. The method employed should be consistent throughout the study. If the samples are to be spiked in the field, be sure to send appropriate supplies and instructions for the field personnel to follow. 9 Quality Control and Data Quality Objectives Analytical results of the FB, FMS, FD, and FSCS should be evaluated at the conclusion of the study to help interpret the quality of sample data. Analytical results for these control/duplicate samples must be reported with the sample data. 9.1 Solvent Blanks Solvent blanks are analyzed with each sample set to determine contamination or carryover. Aliquots of methanol represent the solvent used for the standard curve and the sample extraction. Solvent blanks should have area counts that are less than 50% of the area count of the lowest calibration standard. Solvent blanks should be analyzed prior to and following each calibration curve, each set of system suitability samples, and after no more than 10 unknown sample extracts. If instrument carryover is a problem consecutive solvent blanks may be necessary. In this case the area counts of the solvent blanks should return to <50% of the lowest calibration standard prior to the injection of further standards or samples. 9.2 Method Blanks A method blank consists of an aliquot of ASTM Type I water, equal in volume to the samples, and extracted in the same manner as the samples. At least two method blanks should be prepared and analyzed each day that extractions are performed for a particular study or project. When analyzed the area counts of these samples must be less than 50% of the area count of the lowest calibration standard. 9.3 Sample Replicates All samples, including field spikes, trip blanks, etc., should be extracted at least in duplicate, and in triplicate if difficulties were encountered in the sampling and/or holding conditions of the samples. The relative percent difference (RPD) of duplicate samples or relative standard deviation (RSD) should be less than 15% for the precision of sample preparation and analysis to be considered in control. 9.4 Matrix Spike Matrix spikes are prepared for each sample type and analyzed to determine the matrix effect on the recovery efficiency. Matrix spike recoveries should fall within 25% of expected values. If the matrix spikes fail, evaluate the lab control spikes. If the LCS are within acceptance criteria there may be matrix issues in the samples. Discuss these in the final report. ETS-8-154.1 Page 9 of 17 Determination of Perfluorinated Compounds in Water Using SPE and LC/MS. Page 36 of 119 3M ENVIRONMENTAL LABORATORY PROJECT E05-0210; INTERIM REPORT #20 Matrix spike duplicates are prepared periodically to measure the precision associated with the analysis. Analyze a matrix spike and matrix spike duplicate (if prepared) in the same run as the original sample. Matrix spike and matrix spike duplicate concentrations should fall in the mid-range of the initial calibration curve or should be prepared at 1.5-5 times the endogenous concentration of the analyte. Spike concentrations should fall in the low-range of the initial calibration curve if extremely low-levels are expected. Generally two or more levels are prepared, one in the low range of the curve and one in the m d-range. This avoids the need to pre-screen unknown samples prior to preparation. 9.5 Laboratory Control Spike Lab control spikes are prepared for each study to ensure recovery of the target analytes. These should be prepared at a minimum of 2 levels and in duplicate or triplicate. Recovery of these samples should be within 25% of expected values, and the RPD (or RSD) be ^ 15%. If recoveries fall outside these limits the samples should be addressed in the final report. 10 Calibration and Standardization 10.1 Instrum ent Setup Note: In this example, a MicroMass UltimaTM Liquid Chromatography Tandem Mass Spectrometer (LC/MS/MS) is used. Other brands of LC/MS/MSs as well as single quadrupole mass spectrometers (LC/MS) may be used as long as the method criteria are met. Brand names, suppliers, part numbers, and models are for illustrative purposes only. Equivalent performance may be achieved using apparatus and materials other than those specified here, but demonstration of equivalent performance that meets the requirements of this method is the responsibility of the laboratory. The operator must optimize and document the equipment and settings used. Establish the LC/MS/MS system and operating conditions equivalent to the following: Mass Spec: Micromass Ultima (Micromass) Interface: Electrospray (Micromass) Mode: Electrospray Negative, Multiple Response Monitoring (MRM) Harvard infusion pump (Harvard Instruments), fortuning Computer: COMPAQ Professional Workstation AP200 Software: Windows NT, MassLynx 3.3 HPLC: Hewlett Packard (HP) Series 1100 HP Quaternary Pump HP Vacuum Degasser HP Autosampler HP Column Oven Note: A 4 x 10mm Hypercarb drop-in guard cartridge (Keystone, part# 844017-400) may be attached on-line after the purge valve and before the sample injector port to trap any residue contaminants that may be in the mobile phase and/or HPLC system. HPLC Column: Genesis C8 (Jones Chromatography), 2.1mm x 50mm, 4pm ETS-8-154.1 Page 10 of 17 Determination of Perfluorinated Compounds in Water Using SPE and LC/MS. Page 37 of 119 3M ENVIRONMENTAL LABORATORY PROJECT E05-0210; INTERIM REPORT #20 Column Temperature: 35C Injection Volume: 15pL Mobile Phase (A): 2mM Ammonium Acetate in ASTM Type I water (See 7.3.1 ) Mobile Phase (B): Methanol Tim e, m in Percent M obile Percent M obile Phase A Phase B Flow Rate, m L/m in 0.0 60 40 0.3 met. 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 HPLC gradients may be used as long as the method criteria are It may be necessary to adjust the HPLC gradient in order to optimize instrument performance. Columns with different dimensions (e.g. 2.1mm x 30mm) and columns from different manufacturers (Keystone Betasil C18 etc.) may be used. Ions M onitored: A n a ly te Prim ary Ion Product Ion PFOA PFOS FOSA 413 499 498 169 99 78 Approxim ate Retention Tim e (m inutes) 5.0 5.2 5.8 Other product ions may be chosen at the discretion of the analyst, although m/z 99 is suggested for PFOS. Use of the suggested primary ion is recommended. Retention times may vary slightly, on a day-to-day basis, depending on the batch of mobile phase etc. Drift in retention times is acceptable within an analytical run, as long as the drift continues through the entire analysis and the standards are interspersed throughout the analytical run. 10.2 Tune File Parameters The following values are provided as an example. Actual values may vary from instrument to instrument. Also, these values may be changed from time to time in order to optimize for greatest sensitivity. A n a ly te PFOA PFOS FOSA Dwell, sec 0.2-0.4 0.2-0.4 0.2-0.4 Collision 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 in Water Using SPE and LC/MS. Page 38 of 119 3M ENVIRONMENTAL LABORATORY PROJECT E05-0210; INTERIM REPORT #20 Source Capillary Hexapole 1 Aperture 1 Hexapole 2 Source Block Temp. Desolvation Temp. A n a ly ze r LM Res 1 HM Res 1 IEnergy 1 Entrance Exit LM Res 2 HM Res 2 IEnergy 2 Multiplier Gas Fl^ws Cone G>as Desolvation Pressures 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 in Water Using SPE and LC/MS. Page 39 of 119 3M ENVIRONMENTAL LABORATORY PROJECT E05-0210; INTERIM REPORT #20 10.3 Calibration Curve Analyze the standard curves prior to each set of samples. The validated method specifies that the standard curve should be plotted using a linear fit, weighted 1/x or unweighted. However, the standard curve may also be plotted by quadratic fit (y = aye + bx + c), weighted 1/x or unweighted, using suitable software. The calibration curves may include but should not be forced through zero. The mathematical method used to calculate the calibration curve should be applied consistently throughout a study. Any change should be thoroughly documented in the raw data. If the calibration curve does not meet acceptance criteria perform routine maintenance or prepare a new standard curve (if necessary) and reanalyze. For purposes of accuracy when quantitating low levels of analyte, it may be necessary to use the low end of the calibration curve rather than the full range. For example, when attempting to quantitate approximately 50 pg/mL of analyte, generate a calibration curve consisting of the standards from 25 pg/mL to 1000 pg/mL rather than the full range of the curve (25 pg/mL to 25000 pg/mL). This will reduce inaccuracy attributed to linear regression weighting of high concentration standards. High and/or low points may be excluded from the calibration curves to provide a better fit over the linear range appropriate to the data or because they did not meet the pre-determined acceptance criteria. Low-level curve points should also be excluded if their area counts are not at least twice that of the method and/or solvent blanks. Any curve point may be rejected due to a bad injection or failing to meet accuracy requirements of 25% (and 30% for the LLOQ). Justification for exclusion of calibration curve points will be noted in the raw data. A minimum of 6 points will be used to construct the calibration curve. 10.4 Continuing Calibration Verification (CCV) Continuing calibration verifications (C C V) are analyzed to verify the accuracy of the calibration curve. Analyze a mid-range calibration standard, one of the sam e standards used to construct the calibration curve, at a minimum after every tenth sample, not including solvent blanks, with a minimum of one per sample set. Calibration verification injections must be within 25% to be considered acceptable. The calibration curve and the last passing C C V will then bracket acceptable samples. Multiple C C V levels may be used. 10.5 System Suitability A minimum of three system suitability samples will be injected at the beginning and end of each analytical run. Typically these samples are run prior to the calibration curve. T he system suitability injections must have area counts with an RSD of 5% and a retention time RSD of 2% when evaluated independently. 11 Procedures 11.1 Extraction Scheme Allow samples to equilibrate to room temperature. Thoroughly mix samples by gently inverting the sample bottle. Measure 40m L of sample into 50mL polypropylene centrifuge tubes (Spike the Matrix spikes as required*, replace lid and mix well). Note: * Samples may need to be prescreened to determine an appropriate matrix spike level (typically 5 0 -1 5 0 % of sample concentration). Alternatively the samples could be spiked at more than one level, allowing for the inappropriate spike level to be eliminated. ETS-8-154.1 Page 13 of 17 Determination of Peffluorinated Compounds in Water Using SPE and LC/MS. Page 40 of 119 3M ENVIRONMENTAL LABORATORY PROJECT E05-0210; INTERIM REPORT #20 Condition the C 18 SP E cartridges (1g, 6mL) by passing approximately 10mL methanol followed by approximately 50m L ASTM Type I water (flow rate approximately 2 drop/sec). Do not let column run dry. Note: For the following steps, maintain a ~1 drop/sec flow rate. Do not allow the column to run dry at any time. Load the analytical sample onto the C 18 SP E cartridge. Discard eluate. Ten mL of the 4 0% methanol in water wash mixture is passed through the C 1 8 SP E cartridge to rinse away potential interferences and then discarded. This step must be omitted if perfluorinated compounds with chain lengths less than C 8 are targeted since these will be lost during this wash step. Elute with exactly 5mL of 100% methanol. Collect eluate into graduated 15mL polypropylene centrifuge tubes. This is the target elution fraction (final volume approximately 4.5 mL as not all of the solvent will leave the SPE column. This will not affect the calculations in any way since the curve is also extracted). Analyze a portion of the target elution fraction eluent using negative electrospray HPLC /M S/M S or HPLC/MS. Note: Samples are concentrated by a factor of eight during the extraction; Initial Vol = 40m L Final Vol. = 5mL. Samples are stable at room temperature for at least 24 hours. Analytical samples may be stored in a refrigerator at 42C until analysis. Standardization o f C18 SP E colum ns-- If poor recoveries are observed, it m ay be necessary to standardize the C18 SPE columns in the following manner before analyzing samples. Use a standard with an analyte concentration between 1000 and 4000 pg/mL. Repeat the extraction scheme from the beginning up through the eluting with ~5m L 100% methanol. After the eluting with ~5m L 100% methanol step, collect an additional post-elution fraction by eluting with an additional 5mL of 100% methanol. Analyze both fractions by H PLC/M S/M S or HPLC/MS. If the target fraction contains a minimum of 85% of the respective analytes, it may be considered acceptable. If the wash contains significant standard (>15% ), either the wash volume or percentage of M eO H should be decreased. If the post-elution fraction contains significant standard (>15% ), the target elution volume should be increased. 11.2 Sample Analysis Set up analysis sample queue. Inject the sam e volume (between 5 -2 5 p L ) of each standard, analytical sample and blank into the instrument. All samples with a concentration > ULOQ must be diluted and reanalyzed. If dilution of the final extract fails to produce acceptable results (e.g. poor M S recoveries) dilute the original sample and re-extract. 12 Data Analysis and Calculations____________________ _______ _________ Calculate the analytical sample (extract) concentration from the standard curve using the following equation: ETS-8-154.1 Page 14 of 17 Determination of Perfluorinated Compounds in Water Using SPE and LC/MS. Page 41 of 119 3M ENVIRONMENTAL LABORATORY PROJECT E05-0210; INTERIM REPORT #20 Extract Concentration, pg/mL = (Peak area - intercept) (slope) Calculate the percent recovery of the FSCS using the following equation: FSCS % ree. = (FS C S cone.. pq/mL) x 100 (Cone, added, pg/mL) Calculate the percent recovery of the MSs using the following equation: M S % rec. = (M S cone., pg/mL - Sample cone., pg/mL) x 100 (Cone, added, pg/mL) 13 Method Performance Note: Any method performance parameters that are not achieved must be considered in the evaluation of the data. Nonconformance to any specified parameters must be described and discussed in any reporting of the data. If criteria listed in this method performance section are not met, maintenance may be performed on the system and samples reanalyzed, or other actions taken as determined by the analyst. Document all actions in the raw data. If data are to be reported when performance criteria have not been met, the data must be footnoted on tables and discussed in the text of the report. 13.1 System Suitability A minimum of three system suitability samples will be injected at the beginning and end of each analytical run. Typically these samples are run prior to the calibration curve. The system suitability injections must have area counts with an RSD of S5% and a retention time RSD of 2% when evaluated independently. 13.2 Quantitation Calibration Curve: The coefficient of determination (r2) value for the calibration curve must be greater than or equal to 0.990. Each point in the curve must be within 25% of the theoretical concentration with the exception of the LLOQ, which may be within 30% . D em onstration o f Specificity: Specificity is demonstrated by chromatographic retention time (within 3% of standard) and the mass spectral response of unique ions. 13.3 Sensitivity S o lvent Blanks and M ethod Blanks: Solvent and method blank area counts must be < 50% that of the lowest standard used in the calibration curve. Lim its o f Q uantitation (LOQ): The lower LOQ (LLOQ) is the lowest non-zero active standard in the calibration curve; the peak area of the LLOQ must be at least 2 X that of the extraction blank. By definition, the measured value of the LLOQ must be within 30% of the theoretical value. ETS-8-154.1 Page 15 of 17 Determination of Perfluorinated Compounds in Water Using SPE and LC/MS. Page 42 of 119 3M ENVIRONMENTAL LABORATORY PROJECT E05-0210; INTERIM REPORT #20 13.4 Accuracy C C V Perform ance: Calibration verification injections must be within 25% to be considered acceptable. The calibration curve and the last passing C C V will then bracket acceptable samples. Multiple C C V levels may be used. M atrix Spikes: Matrix spike percent recoveries must be within 25% of the spiked concentration. If matrix effects are suspected, evaluate the LCS results to determine if a matrix effects are present and if the method is in control based on compliant LCS results. Discuss all results in the analytical report. 13.5 Precision R eproducibility: Reproducibility of the method is defined by the results of duplicate or triplicate analysis of samples. A RPD or RSD of 15% will be considered acceptable. System Suitability: The system suitability injections must have area counts with an R SD of 5% and a retention time RSD of 2% when evaluated independently. 14 Pollution Prevention and Waste Management Sample extract waste and flammable solvent is discarded in high BTU containers, and glass pipette waste is discarded in broken glass containers located in the laboratory. 15 Records Each data package generated for a study must have the following information included: study or project number, acquisition method, integration method, sample name, extraction date, dilution factor (if applicable), and analyst. Print the tune page, sample list, and acquisition method to include in the appropriate study folder. Copy these pages and tape into the instrument run log. Plot the calibration curves as described in this method, then print these graphs and store in the study folder. Print data integration summary, integration method, and chromatograms and store in the study folder. Summarize data using suitable software and store in the study folder. 16 Attachments None. 17 References "Method of Analysis for the Determination of Perfluorooctane sulfonate (PFOS), Perfluorooctane sulfonylamide (PFOSA), and Perfluorooctanoate (POAA) in Water'', E. Wickremesinhe and J. Flaherty, Study Number 02 3 -0 0 2 , Centre Analytical Laboratories, Inc., State College, Pennsylvania, January 2000. Validation report for the "Method of Analysis for the Determination of Perfluorooctane sulfonate (PFOS), Perfluorooctane sulfonylamide (PFOSA), and Perfluorooctanoate (POAA) in W ater", E. Wickremesinhe and J. Flaherty, Study Number 0 2 3 -0 0 2 , Centre Analytical Laboratories, Inc., State College, Pennsylvania. ETS-8-154.1 Page 16 of 17 Determination of Perfluorinated Compounds in Water Using SPE and LC/MS. Page 43 of 119 3M ENVIRONMENTAL LABORATORY PROJECT E05-0210; INTERIM REPORT #20 18 Affected Documents None. 19 Revisions Revision Number 1 Revision Num ber U p d a ted to the n e w form at. C h an g ed Title. S e c tio n 1: S ta te s th e validation o f 3 a n alytes, re m o v e s re fe re n c e to E P A d o c u m e n t th a t's no lo n g e r applicable. Section 2: Provided for the extraction o f m ore than the 3 validated analytes, allows the use o f a L C /M S system, not only the L S /M S /M S previously m entioned. Section 3: R evised definitions for field m atrix spike, field control spike, LLO Q , m ethod blank, a n d M DL. Section 5: R e w o rd e d the interferences, ad d e d recom m endation to use disposable pipettes. Section 6: R ecategorized and p ared down. Section 7: C h an g ed storage tim e to 6 m onths. A d d e d m o re calibration points to the table. Section 8:A dded statem ent addressing labeling requirem ents and spiking procedures. E xpanded section 8.8. Section 9: N e w Section S ection 10: C h an g ed som e o f the p ara m e te rs in the tables. A llo w ed fo r u se o f different instrumentation. A d ded inform ation from section 12 o f previous version, extensively revised. Section 11 (section 9 in previous version): Clarification o f w ash step, stated e xact volum e o f eluate is 5 mL, revised standardization process, rem o ved req uirem ent to use L C /M S /M S . Section 1 2 (section 13 in previous version: no changes Section 13 (section 14 in previous version): E xtensively rewritten. Section 14 (section 15 in previous version): no changes Section 15 (section 16 in previous version): M ino r changes to recording requirem ents. ` Section 16 (section 17 in previous version): R em o ved attachm ent. Section 17 (section 18 in previous version): R e m o v e d reference to EP A docum ent that no longer applied to this SO P. Section 18: N e w section. Revision Date 7 ETS-8-154.1 Page 17 of 17 Determination of Perfluorinated Compounds in Water Using SPE and LC/MS. Page 44 of 119 3M ENVIRONMENTAL LABORATORY PROJECT E05-0210; INTERIM REPORT #20 Attachment C: Protocol and Protocol A mendments Page 45 of 119 3M ENVIRONMENTAL LABORATORY PROJECT E05-0210; INTERIM REPORT #20 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 Perform ing Laboratory: E xygen Research 3058 R esearch D rive State C ollege, P A 16801 Phone: (814) 272-1039 Sponsor R epresentative: M ichael A . Santoro D irector o f R egulatory A ffairs 3M B uilding 0236 -0 1 -B -1 0 St. Paul, M N 5 5 1 4 4 Phone: (6 5 1 )7 3 3 -6 3 7 4 Page I o f 65 Page 46 of 119 3M ENVIRONMENTAL LABORATORY PROJECT E05-0210; INTERIM REPORT #20 Exygen Protocol Number: POOOl 131 DISTRIBUTION: 1) Jaisim ha Kesari, Study Director, W eston Solutions 2) John M. Flaherty, Principal Investigator, Exygen Research 3) M ichael A. Santoro, Sponsor Representative, 3M Company 4) Exygen Research Quality Assurance Unit Page 2 o f 65 Page 47 of 119 3M ENVIRONMENTAL LABORATORY PROJECT E05-0210; INTERIM REPORT #20 Exygen Protocol Number: P0001131 PROTOCOL APPROVAL S tu dy T itle: A nalysis o f Perfluorobutanesulfonate (PFBS), Perfluorohexanesulfonate (PFH S), and Perfluorooctanesulfonate (PFO S) in W ater, S oil, Sedim ent, Fish, Clam s, Vegetation, Sm all M ammal Livers and Sm all M am m al Serum U sing LC/M S/M S for the 3M Decatur M onitoring Program Exygen Protocol Number: P 0 0 0 1 131 M ichael A. Santoro, Sponsor Representative 3M Comparfy ibhn M. Flaherty, Principal Investigator Exygen Research Richard A . Gr: :zim, Prsident, F acility M anagem ent Exygen Rese: ch Lydw Shaffer, Techni Exygen Research ead, Quality Assurance Unit u n fa axi /M r D ate 2 $ - Q c l- D ate / /z q /o ) Date Page 3 o f 65 Page 48 of 119 3M ENVIRONMENTAL LABORATORY PROJECT E05-0210; INTERIM REPORT #20 Exygen Protocol Number: P0001131 TABLE OF CONTENTS TITLE PAGE..................................................................................................................................................... 1 DISTRIBUTION.................................................................................................................................................2 PROTOCOL APPROVAL................................................................................................................................. 3 TABLE OF CONTENTS................................................................................................................................... 4 INTRODUCTION...............................................................................................................................................5 TEST MATERIALS...........................................................................................................................................5 OBJECTIVE.......................................................................................................................................................6 TESTING FACILITY.........................................................................................................................................6 STUDY DIRECTOR...........................................................................................................................................7 SPONSOR REPRESENTATIVE....................................................................................................................... 7 PRINCIPAL INVESTIGATOR......................................................................................................................... 7 PROPOSED EXPERIMENTAL START AND TERMINATION DATES...................................................... 7 IDENTIFICATION AND JUSTIFICATION OF THE TEST SYSTEM.......................................................... 8 SAMPLE PROCUREMENT, RECEIPT AND RETENTION.......................................................................... 8 SAMPLE IDENTIFICATION........................................................................................................................... 9 ANALYTICAL PROCEDURE SUMMARY.................................................................................................... 9 VERIFICATION OF ANALYTICAL PROCEDURE....................................................................................... 9 METHOD FOR CONTROL OF BIAS...............................................................................................................11 STATISTICAL METHODS............................................................................................................................... 11 GLP STATEMENT............................................................................................................................................ 11 REPORT .............................................................................................................................................................11 SAFETY AND HEALTH................................................................................................................................... 12 AMENDMENTS TO PROTOCOL....................................................................................................................13 DATA RECORD KEEPING..............................................................................................................................13 QUALITY ASSURANCE..................................................................................................................................14 RETENTION OF DATA AND ARCHIVING...................................................................................................14 APPENDIX I, ANALYTICAL METHODS.......................................................................................................15 Page 4 o f 65 Page 49 of 119 - 3M ENVIRONMENTAL LABORATORY PROJECT E05-0210; INTERIM REPORT #20 Exygen Protocol Number: P0001131 INTRODUCTION The purpose o f this study is to perform analysis for perfluorobutanesulfonate (PFBS), perfluorohexanesulfonate (PFH S) and perfluorooctanesulfonate (PFO S) in water, soil, sedim ent, fish, clam s, vegetation, sm all mammal livers and sm all m ammal serum using LC/M S/M S for the 3M Decatur M onitoring Program. The study w ill be audited for com pliance with EPA T SC A G ood Laboratory Practice Standards 40 CFR 792 by the Q uality A ssurance U nit o f Exygen Research. TEST MATERIALS The test materials are perfluorobutanesulfonate (PFBS), perfluorohexanesulfonate (PFH S) and perfluorooctanesulfonate (PFO S) and are all supplied by 3M. PFBS Chem ical Name: Perfluorobutanesulfonate M olecular W eight: 338 supplied as the potassium salt (C4F9SO 3I O Lot Number: 101 Purity: 96.7% Transitions Monitored: 299 - 99 Structure: FF FF PFHS Chem ical Name: Perfluorohexanesulfonate M olecu lar W eight: 4 3 8 supp lied as the p otassiu m salt (C F n SO a'K*) Lot Number: SE036 Purity: 98.6% Transitions Monitored: 399 - 80 Structure: FFF FFF Page 5 o f 65 Page 50 of 119 3M ENVIRONMENTAL LABORATORY PROJECT E05-0210; INTERIM REPORT #20 Exygen Protocol Number: P000113 1 PFOS Chem ical Name: Perfluorooctanesulfonate M olecular W eight: 538 supplied as the potassium salt (C 8F 17SO 3T O Lot Number: 217 Purity: 86.9% Transitions M onitored: 4 9 9 --> 99 Structure: OBJECTIVE The purpose o f this study is to perform analysis for perfluorobutanesulfonate (PFBS), perfluorohexanesulfonate (PFHS) and perfluorooctanesulfonate (PFO S) in water, soil, sedim ent, fish, clam s, vegetation, sm all m am m al livers and sm all m ammal serum for the 3M Decatur M onitoring Program using the current versions o f the follow ing E xygen analytical methods: V0001780: V0001781: V0001782: V0001783: V0001784: V0001785: V0001786: "M ethod o f Analysis for the Determination o f Perfluorooctanoic A cid (PFO A) in Water by L C /M S/M S" "M ethod o f A nalysis for the D eterm ination o f Perfluorooctanoic A cid (PFO A) in Soil by LC /M S/M S" "M ethod o f A nalysis for the Determination o f Perfluorooctanoic A cid (PFO A) in Sedim ent by LC /M S/M S" "M ethod o f A nalysis for the D eterm ination o f Perfluorooctanoic A cid (PFO A) in Fish and Clam s by L C /M S/M S" "Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in V egetation by LC/MS/MS" "M ethod o f A nalysis for the Determ ination o f Perfluorooctanoic A cid (PFO A) in Sm all Mammal Liver by L C /M S/M S" "M ethod o f A nalysis for the Determ ination o f Perfluorooctanoic A cid (PFO A) in Sm all Mammal Serum by LC /M S/M S" TESTING FACILITY Exygen Research 3058 Research Drive State C ollege, PA 16801 Phone: (814) 272-1039 Page 6 o f 65 Page 51 of 119 3M ENVIRONMENTAL LABORATORY PROJECT E05-0210; INTERIM REPORT #20 Exygen Protocol Number: P0001131 STUDY DIRECTOR Jaisimha Kesari P.E., DEE W eston Solutions, Inc. 1400 W eston W ay W est Chester, PA 19380 Phone: (610) 701-3761 Fax: (610) 701-7401 j .k esari@ w eston solu tion s.com SPONSOR REPRESENTATIVE M ichael A. Santoro 3M Company Director o f Regulatory Affairs 3M Building 0236-01-B -10 St. Paul, M N 55144 Phone: (651) 733-6374 PRINCIPAL INVESTIGATOR John M . Flaherty Exygen Research 3058 Research Drive State C ollege, PA 16801 Phone: (814) 272-1039 john.flaherty@ exygen.com PROPOSED EXPERIMENTAL START AND TERMINATION DATES It is p roposed that the analytical portion o f this stu d y b e con d u cted from O ctober 01, 2 0 0 4 to D ecem ber 31, 2005. The actual experim ental start and termination dates w ill be included in the final report. Page 7 o f 65 Page 52 of 119 3M ENVIRONMENTAL LABORATORY PROJECT E05-0210; INTERIM REPORT #20 Exygen Protocol Number: P0001131 IDENTIFICATION AND JUSTIFICATION OF THE TEST SYSTEM The follow in g are the test system s for this study: W ater (groundwater and surface water) Soil Sedim ent Fish Clams Vegetation Sm all M ammal Liver Sm all Mammal Serum The sam ples w ill be collected by W eston Solutions. The control sam ples will be purchased and prepared by the testing facility. Purchase and processing details for the control sam ples w ill be included in the final report associated with this study. The test system s were chosen to access the environm ental impact o f PFBS, PFHS and PFOS in the Decatur, Alabam a area. SAMPLE PROCUREMENT, RECEIPT AND RETENTION Water, soil, sedim ent, fish, clam, vegetation, sm all m am m al liver and sm all m am m al serum sam ples w ill b e received at E xygen directly from W eston Solutions. The details o f sam ple procurem ent for this study are outlined in the 3M work plan entitled "Phase 2 W ork Plan for Sam pling Environmental M edia." The number and types o f sam ples collected w ill vary depending availability in the field. The total number o f sam ples received and analyzed for each matrix w ill be documented in the final report associated with this study. W ater, soil, and sedim ent sam ples w ill be used as received w ithout further p rocessin g at E xygen. T h ese sam ples w ill be stored refrigerated at 2C -8C . Fish, clam , vegetation and sm all m ammal liver sam ples w ill be processed accord in g to the appropriate analytical m ethod (se e A p p en d ix I). T h ese sam ples w ill b e stored frozen at < -10C. S m all m am m al w h o le blood sam ples w ill be centrifuged in the field at the tim e o f collection and the serum fraction w ill be used for the study. Sm all m am m al serum w ill be stored frozen at < -10C. The receipt and processing o f the sam ples w ill be docum ented in the final report and raw data associated with the study. Page 8 o f 65 Page 53 of 119 3M ENVIRONMENTAL LABORATORY PROJECT E05-0210; INTERIM REPORT #20 Exygen Protocol Number: P0001131 SAMPLE IDENTIFICATION Prior to analysis, each sam ple w ill be assigned a laboratory sam ple reference number. The reference number w ill be unique and w ill distinguish each laboratory sam ple that is processed throughout the analytical procedure. Chromatographic data w ill be identified by the laboratory sam ple reference number. Sam ple storage conditions and locations w ill be docum ented throughout the study. ANALYTICAL PROCEDURE SUMMARY References: V 0001780: "M ethod o f A nalysis for the Determ ination o f Perfluorooctanoic A cid (PFO A) in Water by LC /M S/M S" V 0001781: "M ethod o f A nalysis for the Determ ination o f Perfluorooctanoic A cid (PFO A) in Soil by LC /M S/M S" V 0001782: "M ethod o f A nalysis for the Determ ination o f Perfluorooctanoic A cid (PFO A ) in Sedim ent by L C /M S/M S" V 0 0 0 1 7 8 3 : "M ethod o f A n a ly sis for the D eterm ination o f P erflu orooctan oic A cid (PFO A ) in Fish and Clam s by L C /M S/M S" V 0 0 01784: "M ethod o f A nalysis for the D eterm ination o f P erfluorooctanoic A cid (PFO A) in Vegetation by L C /M S/M S" V 0001785: "M ethod o f A nalysis for the D eterm ination o f Perfluorooctanoic A cid (PFO A) in Sm all M am m al Liver by L C /M S/M S" V 0001786: "M ethod o f A nalysis for the Determ ination o f Perfluorooctanoic A cid (PFO A) in Sm all M am m al Serum by L C /M S/M S" The above m ethods use analytical conditions capable o f separating the isom ers o f PFBS, PFHS and PFOS. The final report w ill include the isom ers sum m ed into total PFBS, total PFHS, and total PFOS found. VERIFICATION OF ANALYTICAL PROCEDURE A laboratory control sam ple w ill be used for the preparation o f fortified control sam ples. The test substance w ill be m ade into solutions as per the m ethod, and added to the matrices via a micropipette. For water sam pling, Exygen w ill supply one bottle per sam ple collected. The bottles w ill be 500 mL precleaned Sci/Spec Premier w ide mouth HDPE bottles. These bottles have been routinely used for fluorochem ical sam ple Page 9 o f 65 Page 54 of 119 3M ENVIRONMENTAL LABORATORY PROJECT E05-0210; INTERIM REPORT #20 Exygen Protocol Number: POOOl 131 collection at the testing facility and have been sh ow n to be free o f PFBS, PFHS and PFO S. Sam ples w ill be added to each container to a volum etric fill line at 2 00 m L. A field duplicate, a lo w field spike and a high field spike o f each sam ple w ill be collected. The low and high field spike bottles will contain PFBS, PFHS and PFOS as w ell as perfluorooctanoic acid (PFO A) and 1.2 -1 3 C p erflu orooctan oic acid ( 13C P F O A ). P F O A and 13C P F O A are included in the solutions used to spike the sam ples. The results for PFOA and 13C P F O A w ill not be reported in this study. E x y g e n w ill su p p ly o n e field blank (control water) and two field blank spikes (control water fortified with PFB S, PFHS and PFOS at a low and high level) for every tw enty sam ples collected. At the testing facility, each water sam ple (excluding field duplicates and field spikes) w ill be extracted in duplicate and w ill also be fortified at a low and high concentration w ith PFB S, PFH S and PFOS and processed through the described procedure to determ ine m ethod accuracy and to check for bias. For soil, sedim ent, clams, and vegetation, Exygen w ill supply one 500 mL precleaned Sci/Spec Premier w ide mouth HDPE bottle per sam ple collected or a zip-seal bag. A ll containers/bags used for sam ple collection w ill be shipped to the sam ple location. Sam ples w ill be added to each container or bag in the field. At the testing facility, each sam ple w ill be extracted in duplicate and w ill also b e fortified at a k n ow n concentration w ith P F B S , P F H S and PFO S at both a low and high level and processed through the described procedure to determ ine m ethod accuracy and to check for bias. For sm all m ammal liver, Exygen w ill supply a 50 m L polypropylene centrifuge tube. For sm all m am m al serum, E xygen w ill supply a collection kit for each sam ple containing serum separator tubes (red top), vacutainers, needle holders and needles, transfer pipettes, and polypropylene tubes. At the testing facility, each liver and serum sam ple w ill be extracted in duplicate and w ill a lso b e fortified at a k now n concentration w ith P F B S , P F H S and PF O S at both a low and high level and processed through the described procedure to determine method accuracy and to check for bias. L ow and high spiking levels for each matrix are defined below : M atrix Low Spiking Level H igh Spiking Level W ater 500 ng/L 5000 ng/L Soil . 4 n g /g 40 ng/g S ed im ent 4 ng/g 40 ng/g Fish 1 0 n g /g 100 ng/g Clam s 10 ng/g 100 ng/g V egetation 10 ng/g 100 ng/g Sm all M am m al Liver 10 ng/g 100 ng/g Small M ammal Serum 10 ng/mL 100 ng/m L Page 10 o f 65 Page 55 of 119 3M ENVIRONMENTAL LABORATORY PROJECT E05-0210; INTERIM REPORT #20 Exygen Protocol Number: P0001131 R ecoveries are anticipated to be betw een 70% and 130% o f the fortified levels; however, the exact precision and accuracy w ill be determined by the analysis o f the quality control sam ples described above. A statement o f accuracy w ill be included in the final report. METHOD FOR CONTROL OF BIAS Control o f bias w ill be addressed by taking representative sub-sam ples from a hom ogeneous m ixture o f each matrix from untreated control sam ples, and by analyzing at least tw o lev els o f fortifications. STATISTICAL METHODS Statistics w ill be lim ited to those specified in the subject m ethods and to the calculation o f average recoveries, as applicable. GLP STATEMENT A ll aspects o f this study shall be performed and reported in com pliance with E PA T SC A G ood Laboratory Practice Standards 4 0 C FR 792. The final report or data package (supplied to the Sponsor) shall contain a statem ent that the study w as conducted in com pliance w ith current and applicable GLP standards and w ill outline any deviations in the study from those standards. This statement w ill be signed by the Study Director and Sponsor R ep resen tative. REPORT A final report w ill be prepared by the principal investigator or their designee at the conclusion o f the study. The report w ill include, but w ill not be limited to, the following: The nam e and address o f the Study Director, Sponsor R epresentative, and o f the testing facility. A statement o f GLP com pliance (any related docum entation, such as chain-of-custody records, m ust be in the study records). Page 11 o f 65 Page 56 of 119 3M ENVIRONMENTAL LABORATORY PROJECT E05-0210; INTERIM REPORT #20 Exygen Protocol Number: P0001131 The signed and dated statement by the E xygen Research Quality Assurance Unit regarding dates o f study inspections and dates findings w ere reported to the Study Director and M anagem ent. A description o f the exact analytical conditions em ployed in the study, i f the subject m ethod was follow ed exactly, it is necessary to include only a copy o f the analytical method. A ny m odifications to this m ethod w ill be incorporated into the report. I f the m ethod is photo-reduced, the project number and page number must be included on each page. Description o f the instrumentation used and operating conditions. A ll results from all sets analyzed. Control and fortified sam ples w ill be identified and the data table w ill include sam ple num ber and fortification level. Representative chromatograms for each analyte in each matrix, including chrom atogram s o f a standard and a control sam p le, and a chrom atogram at a fortification level. The location o f the analyte peaks w ill be clearly identified in all chromatograms. A ll circum stances that m ay have affected the quality or integrity o f the data w ill be docum ented in the report. L ocations w here raw data and the final report are to be archived. A dditions or corrections to the final report shall be in the form o f an am endm ent signed by the Study Director. The am endm ent shall clearly identify that part o f the report that is being altered and the reasons for the alterations. The amendment w ill be signed and dated by the Study Director and the Sponsor Representative. All applicable requirements for reporting o f study results as per 40 CFR 792.185. SAFETY AND HEALTH Laboratory personnel w ill practice good sanitation and health habits. Every reasonable precaution shall be taken to prevent inadvertent exposure o f personnel and the environment to the test or reference substance(s). Page 12 o f 65 Page 57 of 119 3M ENVIRONMENTAL LABORATORY PROJECT E05-0210; INTERIM REPORT #20 Exygen Protocol Number: P0001131 AMENDMENTS TO PROTOCOL A ll significant changes to the analytical protocol outlined here w ill be expressed in writing, signed and dated by the Study D irector and Sponsor Representative. Am endm ents usually w ill be issued prior to initiation o f study plan change. H ow ever, w hen a change is required w ithout sufficient tim e for the issue o f a written amendment, that change m ay be effected verbally with supporting docum entation signed and dated by the Study D irector and fo llo w e d w ith a w ritten am endm ent as so o n as p o ssib le. In this ca se, the effective date o f the written amendment w ill be the date o f the documented change. C opies o f the signed am endm ents w ill be appended to all distributed study plan copies. The original am endm ent w ill be m aintained with the original study plan. A n y deviations from the study plan or from the analytical m ethod as provided w ill be documented and reported prom ptly to the Sponsor R ep resen tative. DATA RECORD KEEPING Records to be maintained include the follow ing (as appropriate): Sam ple tracking sheet(s) Sam ple receipt records, storage history, and chains o f custody H istory and preparation o f standards (stock, fortification, calibration) Description o f any m odifications to the method Instrument run sheets, bench-sheets or logs A nalytical data tables A ll chromatographic and instrumental conditions Sam ple extraction and analysis dates A com plete listing o f study personnel, signatures and initials Chronological presentation o f all study correspondence A ny other documentation necessary for the reconstruction o f the study Chromatograms- A ll chromatograms w ill contain the follow ing: Sam ple identification, injection date, arrow or other indication o f the area o f interest, and injection number corresponding to the run. A dditionally, fortifications w ill include the am ount o f analyte added and the sam ple number o f the sam ple that w as fortified. Analytical standard chromatograms w ill additionally include the concentration (e.g., pg/mL). Page 13 o f 65 Page 58 of 119 3M ENVIRONMENTAL LABORATORY PROJECT E05-0210; INTERIM REPORT #20 Exygen Protocol Number: P0001131 A s part o f the docum entation the follow in g sheets w ill b e included in each analytical set: a run sheet listing the sam ples to b e run in the set, and an instrument conditions sheet describing the instrument type and operating conditions. QUALITY ASSURANCE T he Q A U nit o f Exygen Research w ill inspect the study at intervals adequate to assure com p lia n ce w ith G L P 's, and w ill report the fin d in g s o f audits to the Study Director, Exygen M anagement, and the Sponsor Representative. RETENTION OF DATA AND ARCHIVING A ll hard cop y raw data, including, but not lim ited to, the original chrom atogram s, worksheets, correspondence, and results shall be included w ith the data package submitted to the Study Director. T hese w ill be archived w ith the original study plan, am endm ents, final report, and all pertinent information from the Sponsor. The testing facility shall keep all electronic raw data and any instrument, equipm ent, and storage logs for the period o f tim e specified in 40 CFR 792.195. An exact copy o f the materials submitted to the study director w ill also be kept at E xygen Research. Exygen w ill obtain perm ission from the study director before discarding or returning sam ples. Page 14 o f 65 Page 59 of 119 3M ENVIRONMENTAL LABORATORY PROJECT E05-0210; INTERIM REPORT #20 Exygen Protocol Number: P0001131 A P P E N D IX I ANALYTICAL METHODS V0001780: V0001781: V0001782: V0001783: V0001784: V0001785: V0001786: "M ethod o f Analysis for the Determination o f Perfluorooctanoic A cid (PFO A) in Water by LC /M S/M S" "M ethod o f A nalysis for the Determination o f Perfluorooctanoic A cid (PFO A) in Soil by LC /M S/M S" "M ethod o f A nalysis for the Determination o f Perfluorooctanoic A cid (PFO A) in Sedim ent by LC /M S/M S" "M ethod o f A nalysis for the Determ ination o f Perfluorooctanoic A cid (PFO A) in Fish and Clam s by LC /M S/M S" "M ethod o f Analysis for the Determination o f Perfluorooctanoic A cid (PFO A) in Vegetation b y LC /M S/M S" "M ethod o f A nalysis for the D eterm ination o f P erfluorooctanoic A cid (PFO A) in Sm all M am m al Liver b y L C /M S/M S" "M ethod o f A nalysis for the Determination o f Perfluorooctanoic A cid (PFO A) in Sm all M am m al Serum by L C /M S/M S" Page 15 o f 65 Page 60 of 119 3M ENVIRONMENTAL LABORATORY PROJECTE05-0210; INTERIM REPORT#20 Exygen Protocol Number: P000113 1 ANALYTICAL METHOD Method Number: V0001780 Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Water byLC/MS/MS Analytical Testing Facility: Exygen Research 3058 Research Drive State College, PA 16801 Approved By: Paul Connolly ' Technical Leader, LC-MS, Exygen Research Date 'John Flaherty / Vice President, Operations, Exygen Research Date Total Pages: 7 Page 16 o f 65 Page 61 of 119 3M ENVIRONMENTAL LABORATORY PROJECT E05-0210; INTERIM REPORT #20 Exygen Protocol Number: P0001131 Exygea Research Method Number V0001780 _________________________ANALYTICAL METHOD_________________________ Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Water by LC/MS/MS 1.0 Scope This method is to be employed for the isolation and quantitation of perfluorooctanoic acid by High Performance Liquid Chromatography coupled to a tandem M a s s 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 safety precautions. 3.0 Sample Requirement 3.1 At least 40 mL of 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 for extraction 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 this 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 of 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 (SO-1OOuL, 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 of 7 Page 17 o f 65 Page 62 of 119 3M ENVIRONMENTAL LABORATORY PROJECT E05-0210; INTERIM REPORT #20 Exygen Protocol Number: P0001131 Exygen Research Method Number VOOOI78 ANALYTICAL METHOD_________________________ Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Water by LC/MS/MS 5.12 SPE vacuum manifold. 5.13 Centrifuge capable of 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(.t (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 RunTime: - 2 3 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. 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. P age 3ol ' Page 18 o f 65 Page 63 of 119 3M ENVIRONMENTAL LABORATORY PROJECT E05-0210; INTERIM REPORT #20 Exygen Protocol Number: P0001131 Exygen Research Method Number V0001780 ANALYTICAL METHOD_________________________ Method of Analysis for the Determination of 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 of -100 pg/mL of 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 10 pg/mL fortification solution of PFOA is prepared by bringing 10 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 1.0 pg/mL fortification solution of PFOA is prepared by bringing 10 mL of the 10 pg/mL solution to a final volume oflOO with methanol in a 125 mL LDPE bottle. 9.1.4 A 0.1 pg/mL fortification solution of PFOA is prepared by bringing 10 mL of the 1.0 pg/mL solution to a final volume of 100 with methanol in a 125 mL LDPE bottle. 9.1.5 A 0.01 pg/mL fortification solution of PFOA is prepared by bringing 10 mL of the 0.1 pg/mL solution to a final volume of 100 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 of 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 of Fortification Solution (ppb) 0 Volume (UL) 0 Fortified Control Calibration Sample (mL) Standard (ppt)* 40 0 Standard ID (example) XCmmddyy-0 10 100 10 200 10 400 100 100 100 200 100 400 40 40 40 40 40 40 25 XCmmddyy-1 50 XCmmddyy-2 100 XCmmddyy-3 250 XCmmddyv-4 500 XCmmddyy-5 1000 XCmmddyy-6 * The extracted concentration of the calibration standard is equal to 8x its initial concentration, due to the concentration of the standard during the extraction (SPE). XC = extracted calibration standard. Page 4 o! ^ Page 19 o f 65 Page 64 of 119 3M ENVIRONMENTAL LABORATORY PROJECT E05-0210; INTERIM REPORT #20 Exygen Protocol Number: P0001131 Exygen Research Method Number VOOOI780 _________________________ANALYTICAL METHOD_________________________ Method ofAnalysis for the DeterminationofPerfluorooctanoic Acid (PFOA) in Water by LC/MS/MS 9.2.3 9.2.4 9.2.5 A zero standard solution (reagent blank) must be prepared with each set of standards extracted. Store all extracted calibration standards in 15-mL polypropylene tubes at 2C to 6C, up to two weeks. Alternate volumes and concentrations of standards may be prepared as needed. 10.0 Batch Set Up 10.1 Each batch of samples extracted (typically 20 or less) must include at least one reagent control (method blank using HPLC water) and two reagent controls fortified at known concentrations (lab control spike) to verity 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 Measure 40 mL of sample or a portion of sample diluted to 40 mL with water into 50 mL polypropylene centrifuge tubes (fortify as needed, replace lid and mix well). 11.2 Condition the Cu SPE cartridges (1 g, 6 mL) by passing 10 mL methanol followed by 5 mL of HPLC water (~ 2 drop/sec). Do not let column run dry 11.3 Load sample on conditioned C u SPE cartridge. Discard eluate. 11.4 Elute with ~5 mL 100% methanol. Collect 5 mL of eluate into graduated 15 mL polypropylene centrifuge tubes (final volume * 5 mL). 11.5 Analyze samples using electrospray LC/MS/MS. 12.0 Chromatography 12.1 Inject the same amount of each standard, sample and fortified sample into the LC/MS/MS system. A calibration standard must precede and follow all analyzed samples. 12.2 Standards of PFOA corresponding to at least five or more concentration levels must be included in an analytical set. 12.3 An entire set of extracted calibration standards must be included at the beginning and at the end of a sample set. Extracted standards must be interspersed between every 5-10 samples. As an alternative, an entire set of extracted calibration standards may be injected at the beginning of a set followed by extracted calibration standards interspersed every 5-10 samples (to account for a second set of 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 curves are generated for the analyte by linear regression using l/x weighting of peak area Page 5 of 7 Page 20 o f 65 Page 65 of 119 3M ENVIRONMENTAL LABORATORY PROJECT E05-0210; INTERIM REPORT #20 Exygen Protocol Number: P0001131 Exygen Research Method Number VOOO1780 | ANALYTICAL METHOD j Method of Analysis for the Determination of 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 parent of 413 amu. The 413 amu parent corresponds to the PFOA anion, while 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 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 of 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 of the calibration curve. However, the total number of extracted calibration standards that could be excluded must not exceed 20% of the total number of extracted standards injected. 13.5 The correlation coefficient (R) for calibration curves generated must be 0.992 (RJ 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 of 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/L. based on peak area) using the standard curve (linear regression parameters) 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 Page 21 o f 65 Page 66 of 119 3M ENVIRONMENTAL LABORA TORY PROJECT E05-0210; INTERIM REPORT #20 Exygen Protocol Number: P0001131 Exygen Research Method Number VOOO1780 | ANALYTICAL METHOD ~ Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Water by LC/MS/MS 14.2 For samples fortified with known amounts of PFOA prior to extraction, use the following equation to calculate the percent recovery. Recovery (%) " [total analyte found (ng/L) - analyte found in control (ng/L)] analyte added (ng/L) Page 7 of 7 Page 22 oj 65 Page 67 of 119 3M ENVIRONMENTAL LABORATORY PROJECT E05-0210; INTERIM REPORT #20 Exygen Protocol Number: POOOl 131 ANALYTICAL METHOD MethodNumber: 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: "ELA C_lL Paul Connolly ' Technical Leader, LC-MS, Exygen Research Date Date Total Pages: 7 Page 23 o f 65 Page 68 of 119 3M ENVIRONMENTAL LABORATORY PROJECT E05-0210; INTERIM REPORT #20 Exygen Protocol Number: P0001131 Exygen Research Method Number V 0001781 ANALYTICAL METHOD________________________ Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Soil by LC/MS/MS 1.0 Scope This method is to be employed for the isolation and quantitation of perfluorooctanoic acid by High Performance Liquid Chromatography coupled to a tandem Mass 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 chemical for proper safety precautions. 3.0 Sample Requirement 3.1 At least 15 g of 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 room 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 for this 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 (50-100uL, 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) tC l8 SPE cartridges. 5.12 SPE vacuum manifold. 5.13 Ultrasonic bath. Page 2 o f 7 Page 24 o f 65 Page 69 of 119 3M ENVIRONMENTAL LABORATORY PROJECT E05-0210; INTERIM REPORT # 2 0 Exygen Protocol Number: P0001131 Exygen Research Method Number V0001781 ________________________ANALYTICAL METHOD________________________ Method of Analysis for the Determination of 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. 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 fmin) 0.0 1.0 8.0 20.0 22.5 %A 65 65 25 25 65 Flow Rate % B (mL/miq) 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 RunTime: - 2 3 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 o f water. Alternate volumes may be prepared. Page 3 of 7 Page 25 o f 65 Page 70 of 119 3M ENVIRONMENTAL LABORATORY PROJECT E05-0210; INTERIM REPORT #20 Exygen Protocol Number: P0001131 Exygen Research Method Number VOOO1781 ANALYTICAL METHOD________________________ Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Soil by LC/MS/MS 9.0 Standard Preparation 9.1 Standard Stock/Fortification Solution 9.1.1 Prepare a stock solution of ~100 pg/mL of 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 10 pg/mL fortification solution of PFOA is prepared by bringing 10 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 1.0 pg/mL fortification solution of PFOA is prepared by bringing 10 mL of the 10 pg/mL solution to a final volume of 100 with methanol in a 125 mL LDPE bottle. 9.1.4 A 0.1 pg/mL fortification solution of PFOA is prepared by bringing 10 mL of he 1.0 pg/mL solution to a final volume of 100 with methanol in a 125 mL LDPE bottle. 9.1.5 A 0.01 pg/mL fortification solution of PFOA is prepared by bringing 10 mL of the 0.1 pg/mL solution to a final volume of 100 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 of 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 of Fortification Volume Fortified Control Calibration Standard ID Solution (ppb) 0 10 10 10 100 100 100 (UL) 0 100 200 400 100 200 400 Sample (mL) 40 40 40 40 40 40 40 Standard (ppt)* 0 25 50 100 250 500 1000 (example) XCmmddyy-0 XCmmddyy-1 XCmmddyy-2 XCmmddyy-3 XCmmddyy-4 X C m m ddyy-5 XCmmddyy-6 * The extracted concentration of the calibration standard is equal to 8x its initial concentration, due to the concentration of the standard during the extraction (SPE). XC - extracted calibration standard. Page 4 of 7 Page 26 oj 65 Page 71 of 119 3M ENVIRONMENTAL LABORATORY PROJECT E05-0210; INTERIM REPORT #20 Exygen Protocol Number: P0001131 Exygen Research Method Number VOOO1781 ________________________ ANALYTICAL METHOD________________________ Method of Analysis for the Determination of 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 each set of standards extracted. Store all extracted calibration standards in 15-mL polypropylene lubes at 2C to 6C, up to two weeks. Alternate volumes and concentrations of standards may be prepared as needed. 10.0 Batch Set Up 10.1 Each batch of samples extracted (typically 20 or less) must include at least one reagent control (method blank using 5 mL of 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 of sample into 50 mL polypropylene centrifuge tubes (fortify as needed, replace lid and mix well). 11.2 Add 5 mL of 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 ~10 minutes at ~3000 rpm. 11.6 Condition the Cu SPE cartridges (1 g, 6 mL) by passing 10 mL methanol followed by 5 mL of HPLC water (~ 2 drop/sec). Do not let column run dry 11.7 Load (decant) the sample on the conditioned C|g 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 of each standard, sample and fortified sample into the LC/MS/MS system. A calibration standard must precede and follow all analyzed samples. 12.2 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 of extracted calibration standards must be included at the beginning and at the end of a sample set. Extracted standards must be interspersed between every 5-10 samples. As an alternative, an entire set of Page 5 of 7 Page 27 o f 65 Page 72 of 119 3M ENVIRONMENTAL LABORATORY PROJECT E05-0210; INTERIM REPORT #20 Exygen Protocol Number: P0001131 Exygen Research Method Number V 0001781 | ANALYTICAL METHOD Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Soil by LC/MS/MS ' extracted calibration standards may be injected at the beginning of a set followed by extracted calibration standards interspersed every 5-10 samples (to account for a second set of 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 curves arc 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 of 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 50 ng/L, 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 of 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 of the calibration curve. However, the total number of extracted calibration standards that could be excluded must not exceed 20% of the total number of extracted standards injected. 13.5 The correlation coefficient (R) for calibration curves generated must be 0.992 (RJ 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 of 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. Page 6 of 7 Page 28 o f 65 Page 73 of 119 3M ENVIRONMENTAL LABORATORY PROJECT E05-0210; INTERIM REPORT #20 Exygen Protocol Number: P0001131 Exygen Research Method Number VOOOI781 ________________________ ANALYTICAL METHOD________________________ Method of Analysis for the Determination of Periluorooctanoic Acid (PFOA) in Soil bv LC/MS/MS I J 14.0 Calculations 14.1 Use the following equation to calculate the amount of PFOA found (in ng/L, based on peak area) using the standard curve (linear regression parameters) 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 of PFOA prior to extraction, use the following equation to calculate the percent recovery. Recovery (%) * [ total analyte found (ng/L) - analyte found in control (ng/L)] f ^ analyte added (ng/L) 14.3 Use the following equation to convert the amount of PFOA found in ng/L to ng/g (ppb). PFOA found (ppb) " iPFOA found (ng/L) x volume extracted (0.04D1 sample weight (3 g) 14.4 Use the following equation to calculate the amount of PFOA found in ppb based on dry weight. PFOA found (ppb) dry weight = PFOA found (ppb) x [100% / total solids(%)] Page 7 of 7 Page 29 o f 65 Page 74 o f 119 3M ENVIRONMENTAL LABORATORY PROJECT E05-0210; INTERIM REPORT #20 Exygen Protocol Number: P0001131 ANALYTICAL METHOD Method Number: V0001782 Method of Analyst* 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 \/m a u y ohn Flaherty Vice President, Operations, Exygen Research lO llb M Date Date Total Pages: 7 Page 30 o f 65 Page 75 of 119 3M ENVIRONMENTAL LABORATORY PROJECT E05-0210; INTERIM REPORT #20 Exygen Protocol Number: P0001131 Exygen Research Method Number VOOOI782 | ANALYTICAL METHOD__________________________ j Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Sediment by LC/MS/MS 1.0 Scope This method is to be employed for the isolation and quantitation of perfluorooctanoic acid by High Performance Liquid Chromatography coupled to a tandem Mass 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 of 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 room 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 for this 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 of 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 (50-1OOuL, 100-200uL). 5.7 1 25 -m L L D P E narrow -m outh bottles. 5.8 2 mLclear HPLCvial 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 Page 3 1 o f 65 Page 76 of 119 3M ENVIRONMENTAL LABORATORY PROJECT E05-0210; INTERIM REPORT #20 Exygen Protocol Number: P0001131 Exygen Research Method Number VOOOI782 | ANALYTICAL METHOD j Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Sediment by LC/MS/MS 5.13 Vortexer. 5.14 Wrist-action shaker. 5.15 Centrifuge capable of 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 (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 mJz 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. Page 3 of 7 Page 32 o f 65 - - .. -....Page 7-7-of 149- 3M ENVIRONMENTAL LABORATORY PROJECT E05-0210; INTERIM REPORT#20 Exygen Protocol Number: P000113 1 Exygen Research Method Number V0001782 l ______________ ANALYTICAL METHOD__________________________ j Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Sediment bs LC/MS/MS 8.2 Extraction Solutions 8.2.1 1% acetic acid in water is prepared by adding 10 mL of acetic acid 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 o f -100 pg/mL of 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 10 pg/mL fortification solution of PFOA is prepared by bringing 10 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 1.0 pg/mL fortification solution of PFOA is prepared by bringing 10 mL of the 10 pg/mL solution to a final volume of 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 of the 1.0 pg/mL solution to a final volume of 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 of the 0.1 pg/mL solution to a final volume of 100 with methanol in a 125 mL LDPE bottle. 9.1.6 The stock and 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 of 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 be Concentration of Fortification Solution (ng/mL) too 100 100 10 5 2 Volume (mL) to 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 7 Page 33 o f 65 Page 78 of 119 3M ENVIRONMENTAL LABORATORY PROJECT E05-0210; INTERIM REPORT #20 Exygen Protocol Number: P0001131 Exygen Research Method Number V0001782 | ANALYTICAL METHOD | Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Sediment by LC/MS/MS 9.2.3 9.2.4 Store all calibration standards in 123-mL LDPE narrow-mouth bottles at 2C to 6C, up to six months. Alternate volumes and concentrations of standards may be prepared as needed. 10.0 Batch Set Up 10.1 Each batch of 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 sample into SO mL polypropylene centrifuge tubes (fonify as needed, replace lid and mix well). 11.2 Add 35 mL of 1% acetic acid, cap, vortex and shake on a wrist action shaker for -60 minutes. 11.3 Centrifuge the tubes at -3000 rpm for -20 minutes. 11.4 Condition the Cu SPE cartridges (1 g, 6 mL) by passing 10 mL methanol followed by 20 mL of HPLC water (~ 2 drop/sec). Do not let column run dry 11.5 Load (decant) the sample on the conditioned C u SPE cartridge. Discard eluate. 11.6 Add 20 mL of methanol to the sediment left in the bottom of the 50 mL centrifuge tube. Cap, vortex and shake on a wrist action shaker for -30 minutes. 11.7 Centrifuge the tubes at ~3000 rpm for ~20 minutes. 11.8 Decant the methanol onto the same SPE cartridge. Collect the eluate. 11.9 Wash the column with 4 mL of methanol. Collect the eluate and add it to the eluate collected in step 11.8. 11.10 Condition a second Cu SPE cartridge (1 g, 6 mL) by passing 10 mL methanol followed by 20 mL of HPLC water (~ 2 drop/sec). Do not let column run dry 11.11 Add the methanol to -200 mL of water and load on the second conditioned SPE cartridge. 11.12 Elute with ~5 mL 100% methanol. Collect 5 mL of eluate into graduated 15 mL polypropylene centrifuge tubes (final volume = 5 mL). 11.13 A n a lyze samples using electrospray L C /M S /M S . Page 5 ot' 7 Page 34 o f 65 Page 79 of 119 3M ENVIRONMENTAL LABORATORY PROJECT E05-0210; INTERIM REPORT #20 Exygen Protocol Number: P0001131 Exygen Research Method Number V 0001782 | ANALYTICAL METHOD j Method o f Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Sediment by LC/MS/MS 12.0 Chromatography 12.1 Inject the same amount of each standard, sample and fortified sample into the LC/MS/MS system. A calibration standard must precede and follow all analyzed samples. 12.2 Standards of PFOA corresponding to at least five or more concentration levels must be included in an analytical set. 12.3 An entire set of extracted calibration standards must be included 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 arc 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 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 0.2 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 of 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 of the calibration curve. However, the total number of extracted calibration standards that could be excluded must not exceed 20% of the total number of extracted standards injected. 13.5 The correlation coefficient (R) for calibration curves generated must 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 of samples should be reanalyzed. Page 6 of 7 Page 35 o f 65 Page 80 of 119 3M ENVIRONMENTAL LABORATORY PROJECT E05-0210; INTERIM REPORT #20 Exygen Protocol Number: POOO113 1 Exygen Re*earth Method Number V0001782 _________________________ ANALYTICAL METHOD__________________________ Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Sediment bv LC/MS/MS ' 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/m L, 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) x DF slope DF = factor by which the final volume was diluted, if necessary. 14.2 For samples fortified with known amounts of 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)] 1QQ analyte added (ng/mL) 14.3 Use the following equation to convert the amount of PFOA found in ng/mL to ng/g (ppb). PFOA found (ppb) - fPFOA found (ng/mL) x final volume (5 mL)l sample weight (S g) 14.4 Use the following equation (if necessary) to calculate the amount of PFOA found in ppb based on dry weight. P F O A found (ppb ) d ry w eight -- P F O A found (p p b ) x [1 0 0 % / total solids(% )] Page 7 of 7 Page 36 of 65 Page 81 of 119 3M ENVIRONMENTAL LABORATORY PROJECT E05-0210; INTERIM REPORT #20 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: Paul Connolly Technical Leader, LC-MS, Exygen Research Date John Flaherty ' Vice President, Operations, Exygen Research Date j Total Pages: 8 Page 37 oj 65 Page 82 o f 148 3M ENVIRONMENTAL LABORA TORY PROJECT E05-0210; INTERIM REPORT #20 Exygen Protocol Number: P0001131 Exygen Research Method Number V000I783 ________________________ ANALYTICAL m e t h o d _________________________ Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Fish and Clams by LC/MS/MS 1.0 Scope This method is to be employed for the isolation and quantitation of perfluorooctanoic acid by High Performance Liquid Chromatography coupled to a tandem M a s s 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 proper safety precautions. 3.0 Sample Requirement 3.1 At least 20 g of test sample for extraction. 3.2 Samples should be processed before extraction. Place the frozen sample in a food processor and homogenize with dry ice. Place the samples in containers and leave open in frozen storage overnight to allow for carbon dioxide sublimation. Seal and place the samples in frozen storage until time of analysis. 3.3 Sample collection procedures will be specified in the sampling plan for this project. 4.0 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 of 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. Page 2 of S Page 38 of 65 Page 83 of 119 3M ENVIRONMENTAL LABORATORY PROJECT E05-0210; INTERIM REPORT #20 Exygen Protocol Number: P0001131 Exygen Research Method Number VOOOI783 ________________________ ANALYTICAL METHOD_________________________ Method of Analysis for the Determination of 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-mLLDPE 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, 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: limfi-toin) 0.0 1.0 8.0 20.0 22.5 %A 65 65 25 25 65 Flow Rate % B (ml/roin) 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 Page 39 o f 65 Page 84 of 1-19- 3M ENVIRONMENTAL LABORATORY PROJECT E05-0210; INTERIM REPORT #20 Exygen Protocol Number: P0001131 Exygen Research , M ethod Number V0001783 ANALYTICAL METHOD | Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Fish and Clams by LC/MS/MS 7.0 MS/MS System 7.1 Mode: Electrospray Negative MRM mode, monitoring 4 13 --> 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 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 of 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 of PFOA is prepared by bringing I mL of the 100 pg/mL solution to a final volume of 100 with methanol in a 125 mL LDPE bottle. A 0.1 pg/mL fortification solution of PFOA is prepared by bringing 10 mL of the 1.0 pg/mL solution to a final volume of 100 with methanol in a 125 mL LDPE bottle. A 0.01 pg/mL fortification solution of PFOA is prepared by bringing tO m L o f the 0.1 |ig /m L solution to a fin a l vo lu m e 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 of 6 months from the date of preparation. Page 4 of 8 Page 40 o f 65 Page 85 of 119 3M ENVIRONMENTAL LABORATORY PROJECT E05-0210; INTERIM REPORT #20 Exygen Protocol Number: P0001131 Exygen Research Method Number V0001783 ________________________ ANALYTICAL m e t h o d _________________________ Method of Analysis for the Determination of 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 Solution lue/mL) (mL) Diluted to (mL) Concentration (Ug/mL) l.O 5.0 100 0.05 l.O 2.5 100 1.0 1.0 100 0.025 0.01 0.05 0.025 10 10 100 100 0.005 0.0025 0.1 0.005 10 10 100 100 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 of standards may be prepared as needed. 10.0 Batch Set Up 10.1 Each batch of 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-NHj. 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 of nitrogen. Page 5 of S Page 41 o f 65 Page 86of119 3M ENVIRONMENTAL LABORATORY PROJECT E05-0210; INTERIM REPORT #20 Exygen Protocol Number: P000113 1 Exygen Research Method Number V 0001783 ANALYTICAL METHOD Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Fish and Clams by LC/MS/MS 11.7 Centrifuge the 50 mL polypropylene tubes containing sample at -2000 rpm for ~10 minutes. 11.8 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. 11.9 Add 10 mL of acetonitrile to the sample in the 50 mL centrifuge tube. Homogenize the frozen fat phase using a tissumizer for -30 seconds and rinse the tissumizer with -10 mL of acetonitrile into the tube. 11.10 Shake the sample again for -10 minutes on a wrist-action shaker. 11.11 Place the tubes in a freezer for - 1 hour more. 11.12 Centrifuge the 50 mL polypropylene tubes containing sample at -2000 rpm for -10 minutes. 11.13 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. 11.14 Pass 20 mL of acetonitrile through the SPE column and combine the eluate in the same pear-shaped flask. 11.15 Add 3-4 drops of 1-octanol to the extract in the pear-shaped flask and evaporate at reduced pressure using a rotary evaporator (at < 40C). 11.16 Make the final volume, by adding 2 mL of 2% ascorbic acid in methanol to the pear-shaped flask and swirl to mix/dissolve. 11.17 Transfer the extracts to HPLC vials using disposable pipets. 11.18 Analyze samples using electrospray LC/MS/MS. 12.0 Chromatography 12.1 Inject the same amount of each standard, sample and fortified sample into the LC/MS/MS system. A calibration standard must precede and follow all analyzed samples. 12.2 Standards of PFOA corresponding to at least five or more concentration levels must be included in an analytical set. 12.3 An entire set of 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 o f 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. Page 6 o f 8 Page 42 o f 65 Page 87 of 119... 3M ENVIRONMENTAL LABORATORY PROJECT E05-0210; INTERIM REPORT #20 Exygen Protocol Number: P0001131 Exygen Research Method Number V0001783 ________________________ ANALYTICAL m e t h o d _________________________ Method of Analysis for the Determination of Perfluorooctanoic 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 of 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 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 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% of their known values. If a control spike falls outside the acceptable limits, the entire set of samples should be re-extracted. 13.4 Any calibration standard found to be a statistical outlier by using the Huge Error Test, may be excluded from the calculation of the calibration curve However, the total number of calibration standards that could be excluded must not exceed 20% of the total number of standards injected. 13.5 The 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 of 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/ntL, 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 of 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 Page 43 o f 65 ... Page 88 of 140 3M ENVIRONMENTAL LABORATORY PROJECT E05-0210; INTERIM REPORT #20 Exygen Protocol Number: POOOl 131 Exygen Research Method Number VOOO1783 ________________________ ANALYTICAL m e t h o d _________________________ Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Fish and Clams by LC/MS/MS 14.3 For samples fortified with known amounts of 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)] ^ analyte added (ng/g) Page 8 of 8 Page 44 o f 65 ----------- Page 89 of 149- 3M ENVIRONMENTAL LABORATORY PROJECT E05-0210; INTERIM REPORT #20 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: Paul Connolly Technical Leader, LC-MS, Exygen Research Date 6hn Flaherty Vice President, Operations, Exygen Research Total Pages: 7 Page 45 o f 65 Page 90 o f 119 3M ENVIRONMENTAL LABORATORY PROJECT E05-0210; INTERIM REPORT #20 Exygen Protocol Number: P0001131 Exygen Research Method Number V00017X4 I ANALYTICAL METHOD Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Vegetation by LC/MS/MS 1.0 Scope This method is to be employed for the isolation and quantitation of perfluorooctanoic acid by High Performance Liquid Chromatography coupled to a tandem Mass 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 for proper safety precautions. 3.0 Sample Requirement 3.1 At least 20 g of test sample for extraction. 3.2 Samples should be processed before extraction. Place the frozen sample in a food processor and homogenize with dry ice. Place the samples in containers and leave open in frozen storage overnight to allow for carbon dioxide sublimation. Seal and place the samples in frozen storage until time of analysis. 3.3 Sample collection procedures will be specified in the sampling plan for this project. 4.0 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 of pumping up to 2 solvents equipped with a variable volume injector capable of injecting 5-200 \iL 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. Page 2 o f ' Page 46 o f 65 Page 91 of 119 3M ENVIRONMENTAL LABORATORY PROJECT E05-0210; INTERIM REPORT #20 Exygen Protocol Number: P0001131 Exygen Research Method Number VOOO1784 _________________________ ANALYTICAL METHOD__________________________ Method of Analysis for the Determination of 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 (50-100uL, 100-200uL). 5.9 125-mL LDPE narrow-mouth bottles. 5.10 2 mLclearHPLC 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 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, 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 fmin) 0.0 1.0 8.0 20.0 22.5 %A 65 65 25 25 65 Flow Rate % B imL/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 of7 Page 47 o f 65 .. ~Page~92-of-14&-- 3M ENVIRONMENTAL LABORA TORY PROJECT E05-0210; INTERIM REPORT #20 Exygen Protocol Number: P0001131 Exygen Research Method Number V000I784 _____________________ ANALYTICAL m e t h o d __________________________ Method of Analysis for the Determination of 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 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. 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 of methanol. 30% Dimethyldichlorosilane in toluene is prepared by bringing 3 mL of dimethyldichlorosilane to a final volume of 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 of ~ 100 pg/mL of 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 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. A 0.1 pg/mL fortification solution of PFOA is prepared by bringing 10 mL of the 1.0 pg/mL solution to a final volume of 100 with methanol in a 125 mL LDPE bottle. A 0.01 pg/mL fortification solution of PFOA is prepared by bringing 10 mL of the 0.1 pg/mL solution to a final volume of 100 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 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 o! " Page 48 o f 65 Page 93 of 119 3M ENVIRONMENTAL LABORATORY PROJECT E05-0210; INTERIM REPORT #20 Exygen Protocol Number: P000113 1 Exygen Research Method Number V0001784 _________________________ a n a l y t ic a l m e t h o d __________________________ Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Vegetation byLC/MS/MS 9.2.2 The following is a typical example: additional concentrations may be prepared as needed. Concentration Final of Fortification Volume Diluted to Concentration Solution (nnlmL) (mL) (mL) (u/mL) l.O 5.0 100 0.05 l.O 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 of standards may be prepared as needed. 10.0 Batch Set Up 10.1 Each batch of samples extracted (typically 20 or less) must include at least one untreated control and two untreated controls fortified at k now 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 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 Centrifuge the 50 mL polypropylene tubes containing sample at -2000 rpm for-10 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-NHj. 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% dim ethyldichlorosilane in toluene solution. R inse the flask w ith toluene once, followed by methanol (three times). Dry the flasks completely before use, either by air-drying or with a stream of nitrogen. 11.7 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. Page 5 of ' Page 49 o f 65 Page 94 of 119 3M ENVIRONMENTAL LABORATORY PROJECT E05-0210; INTERIM REPORT #20 Exygen Protocol Number: P0001131 Exygen Research Method Number V0001 ' 84 _________________________ ANALYTICAL m e t h o d __________________________1 Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Vegetation by LC/MS/MS 11.8 Add 20 mL of 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 of 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 of each standard, sample and fortified sample into the LC/MS/MS system. A calibration standard must precede and follow all analyzed samples. 12.2 Standards of PFOA corresponding to at least five or more concentration levels must be included in an analytical set. 12.3 An entire set of extracted calibration standards must be included at the beginning and at the end of a sample set. Extracted standards must be interspersed between every 5-10 samples. As an alternative, an entire set of extracted calibration standards may be injected at the beginning of a set followed by extracted calibration standards interspersed every 5-10 samples (to account for a second set of 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 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 of carbon dioxide. Page 6 of 7 Page 50 o f 65 Page 95 of 119-- 3M ENVIRONMENTAL LABORATORY PROJECT E05-0210; INTERIM REPORT #20 Exygen Protocol Number: P0001131 Exygen Research Method Number V0001784 ANALYTICAL m e t h o d Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Vegetation by LC/MS/MS 13.2 Method blanks must not contain PFOA at levels greater than the LOQ. tf 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% of their known values. If a control spike falls outside the acceptable limits, the entire set of samples should be re-extracted. 13.4 Any calibration standard found to be a statistical outlier by using the Huge Error Test, may be excluded from the calculation of the calibration curve. However, the total number of calibration standards that could be excluded must not exceed 20% of the total number of standards injected. 13.5 The correlation coefficient (R) for calibration curves generated must be 0.992 (RJ 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 of 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/niL, 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 of PFOA found in ng/mL to ng/g (ppb). PFOA found (ppb) = 1PFOA 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 of 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)] ^ analyte added (ng/g) Page 7 oi 7 P age 51 o f 65 ... Page 96 o i 149- 3M ENVIRONMENTAL LABORATORY PROJECT E05-0210; INTERIM REPORT 20 Exygen Protocol Number: POOOl 131 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: TU C ----- ____________________ Paul Connolly I Technical Leader, LC-MS, Exygen Research ___ l frifrf e M Date a / /? ! d - //. John Flaherty / Vice President., Onperatioins, Exygen Research Date Total Pages: 7 P age 52 o f 65 - ...Page 97 of 119 -- 3M ENVIRONMENTAL LABORATORY PROJECT E05-0210; INTERIM REPORT #20 Exygen Protocol Number: P0001131 Exygen Research Method Number V 000178$ | AiSALVTICAL METHOD Method of Analysis for the Determination of 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 of perfluorooctanoic acid by High Performance Liquid Chromatography coupled to a tandem Mass 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 for proper safeiv precautions. 3.0 Sample Requirement 3.1 At least 5 g of test sample for extraction. 3.2 Samples should be processed before extraction. Place the frozen sample in a food processor and homogenize with dry ice. Place the samples in containers and leave open in frozen storage overnight to allow for carbon dioxide sublimation. Seal and place the samples in frozen storage until time of analysis. Alternately, if there is an insufficient amount of sample (-less than 5 g), then no processing is necessary and the sample can be used as supplied. 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 of 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. 5.3 15 mL disposable polypropylene centrifuge tubes. 5.6 Disposable micropipets (50-100uL, 100-200uL). 5.7 125-mL LDPE narrow-mouth bottles. 5.8 2 mL clear HPLC vial kit. Page 2 o f Page 53 o f 65 Page 98of449- 3M ENVIRONMENTAL LABORATORY PROJECT E05-0210; INTERIM REPORT #20 Exygen Protocol Number: P0001131 Exygen Research | Method Number V 0001785 AKAi7vTICALMTHOD Method of Analysis for the Determination of 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: T im e (m io) 0.0 l.o 8.0 20.0 22.5 %A 65 65 25 25 65 Flow Rate fmL/minl 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 o p tim ize the M S M S system. Page .1 of 7 P age 54 o f 65 Page 99 o f 119 3M ENVIRONMENTAL LABORATORY PROJECT E05-0210; INTERIM REPORT #20 Exygen Protocol Number: P0001131 Exygen Research Method Number V 0001785 _______________________ a n a l y t ic a l m e t h o d ________________________ Method of Analysis for the Determination ofPerfluorooctanoic Acid (PFOA) in Small Mammal Liver by LC/MS/MS 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. 9.0 Standard Preparation 9.1 Standard Stock/Fortification Solution 9.1.1 Prepare a stock solution o f -100 pg/mL of 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 I mL of the 100 pg/mL solution to a final volume of 100 w ith 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 of the 1.0 pg/mL solution to a final volume of 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 of 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 he prepared as needed. Concentration of Fortification Volume Diluted to Final Concentration Solution (ng/mL) (mL) (mL) (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 12S-mL LDPE narrow-mouth bottles at 2C to 6C, up to six months. 9.2.4 Alternate volumes and concentrations of standards may be prepared as needed. Page 4 of 7 Page 55 o f 65 Page 100 of 119 3M ENVIRONMENTAL LABORATORY PROJECT E05-0210; INTERIM REPORT #20 Exygen Protocol Number: P0001131 Exygen Research Method Number V0001785 _______________________ ANALYTICAL METHOD________________________ Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Small Mammal Liver by LC/MS/MS 10.0 Batch Set Up 10.1 Each batch of samples extracted (typically 20 or 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 1 g of sample into a 50 mL polypropylene centrifuge tubes (fortify as needed, replace lid and mix well). Note that alternate weights of liver may b e measured depending on the sample size available for use. 11.2 Add water to the sample for a final volume of 10 mL. 11.3 Homogenize sample using a tissuemizer for ~1 minute. 11.4 Transfer 1 mL of the sample using a disposable pipette into a 15 mL disposable centrifuge tube. 11.5 Add 5 mL of acetonitrile and shake for ~20 minutes on a wrist-action shaker. 11.6 Centrifuge the tubes at ~3000 rpm for ~S minutes. 11.7 Decant the supernatant into a 50 mL disposable centrifuge tube and add 35 mL of water. 11.8 Condition the Cu SPE cartridges (1 g, 6 mL) by passing 10 mL methanol followed by 5 mL of HPLC water (~ 2 drop/sec). Do not let column run dry 11.9 Load the sample on conditioned C u SPE cartridge. Discard eluate. 11.10 Elute with ~2 mL of methanol. Collect 2 mL of 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 of PFOA corresponding to at least five or more concentration levels must be included in an analytical set. 12.3 An entire set of 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 -1 0 sam ples (to account fo r a second set o f 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 Page 5 of 7 P age 56 o f 65 Page 101 of 110~ 3M ENVIRONMENTAL LABORATORY PROJECT E05-0210; INTERIM REPORT #20 Exygen Protocol Number: POOOl 131 Exygen Research Method Number V0001785 | ANALYTICAL METHOD Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Small Mammal Liver 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 parent of 413 amu. The 413 amu parent corresponds to the PFOA anion, while 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/g, 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 of 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 of the calibration curve. However, the total number of calibration standards that could be excluded must not exceed 20% of the total number of standards injected. 13.5 The 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 of 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/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) x DF x aliquot factor slope DF factor by which the final volume was diluted, if necessary. Aliquot factor - 1 0 Page 6 of 7 Page 57 o f 65 3M ENVIRONMENTAL LABORATORY PROJECT E05-0210; INTERIM REPORT #20 Exygen Protocol Number: P0001131 Exygen Research Method Number VOOOI7S5 ANALYTICAL m e t h o d Method of Analysts for the Determination of Perfluorooctanoic Acid (PFOA) in Small Mammal Liver by LC/MS/MS 14.2 For samples fortified with known amounts of 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 of PFOA found in ng/mL io ng/g (ppb). PFOA found (ppb) fPFOA found (ng/mL) x final volume (mL)1 sample weight (g) Page 7 of 7 P age 58 of 65 Page 103 of 119 - 3M ENVIRONMENTAL LABORATORY PROJECT E05-0210; INTERIM REPORT #20 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: Paul Connolly Technical Leader, LC-MS, Exygen Research m l UtlQ 'l Date 41Z /7 ? /jj/ Jb hrn Flaherty / Viicc<e President, Operations, Exygen Research Date Total Pages: 7 Page 59 o f 65 Page 104 o f 119.. 3M ENVIRONMENTAL LABORA TORY PROJECT E05-0210; INTERIM REPORT #20 Exygen Protocol Number: P0001131 Exygen Research Method Number VOOO1786 _______________________ ANALYTICAL METHOD________________________ Method of Analysis for the Determination of 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 perfluorooctanoic acid by High Performance Liquid Chromatography coupled to a tandem M a s s Spectrometric Detector (LC/MS/MS) in small mammal serum. 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 1 mL of test sample for extraction. 3.2 No sample processing is needed for serum samples. However, frozen serum samples must to allowed to completely thaw to room temperature before use. 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 4.3 Perfluorooctanoic Acid - Sigma-Aldrich 3.0 Instrument and Equipment 3.1 A high performance liquid chromatograph capable of pumping up to 2 solvents equipped with a variable volume injector capable of injecting S-200 pL connected to a tandem Mass Spectrometer (LC/MS/MS). 3.2 A device to collect raw data for peak integration and quantitation. 3.3 Analytical balance capable of reading to 0.00001 g. 3.4 30 mL disposable polypropylene centrifuge tubes. 5.5 13 mL disposable polypropylene centrifuge tubes. 5.6 Disposable micropipets (50-1 OOuL, 100-200uL). 5.7 125-mL LDPE narrow-mouth bottles. 3.8 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 o f 1 P age 60 o f 65 -- -Page 1Q5 of 119 3M ENVIRONMENTAL LABORA TORY PROJECT E05-0210; INTERIM REPORT #20 Exygen Protocol Number: POOOl 131 Exygen Research Method Number V 0001786 | ANALYTICAL METHOD Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Small Mammal Serum by LC/MS/MS I 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: Tiro? (m in) 0.0 1.0 8.0 20.0 22.5 %A 65 65 25 25 65 Flow Rate % B imL/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 of 7 Page 61 o f 65 Page 106 of 119 3M ENVIRONMENTAL LABORATORY PROJECT E05-0210; INTERIM REPORT #20 Exygen Protocol Number: P0001131 Exygen Research Method Number VOOO1786 | ANALYTICAL METHOD Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Small Mammal Serum by LC/MS/MS j 9.0 Standard Preparation 9.1 Standard Stock/Fortification Solution 9.1.1 Prepare a stock solution of ~100 pg/mL of PFOA by weighing 1mg 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 I mL of the 100 pg/mL solution to a final volume of 100 with methanol in a 125 mL LDPE bottle. 9.1.3 A 0.1 pg/mL fortification solution of PFOA is prepared by bringing 10 mL of the 1.0 pg/mL solution to a final volume o f 100 with methanol in a 125 mL LDPE bottle. 9.1.4 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 of 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 Volume Solution (ng/mL) (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 of 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 of 7 P age 62 o f 65 Page 107-of~tt& 3M ENVIRONMENTAL LABORATORY PROJECT E05-0210; INTERIM REPORT #20 Exygen Protocol Number: P0001131 Exygen Research Method Number V0001786 ANALYTICAL METHOD Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Small Mammal Serum by LC/MS/MS I 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 of 20 mL. Cap tightly 11.3 Vortex for ~1 minute. 11.4 Transfer 1 mL of the sample using a disposable pipette into a 15 mL disposable centrifuge tube. 11.5 Add 5 mL of 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 of water. 11.8 Condition the Cu SPE cartridges (1 g, 6 mL) by passing 10 mL methanol followed by 5 mL of HPLC water (~ 2 drop/sec). Do not let column run dry 11.9 Load the sample on conditioned Cu SPE cartridge. Discard eluate. 11.10 Elute with ~2 mL of methanol. Collect 2 mL of 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 of each standard, sample and fortified sample into the LC/MS/MS system. A calibration standard must precede and follow all analyzed samples. 12.2 Standards of PFOA corresponding to at least five or more concentration levels must be included in an analytical set. 12.3 An entire set of calibration standards must be included at the beginning and at the end of a sample set. Standards must be interspersed between every 5-Id samples. As an alternative, an entire set of calibration standards may be injected at the beginning of a set followed by calibration standards 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 I/x weighting o f 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 ol'7 P age 63 o f 65 3M ENVIRONMENTAL LABORATORY PROJECT E05-0210; INTERIM REPORT #20 Exygen Protocol Number: P0001131 Exygen Research Method Number V 0001786 | ................. ANALYTICAL METHOD ~ Method of Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Small Mammal Serum by LC/MS/MS 13.0 Acceptance Criteria 13.1 Chromatogram must show a peak of 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 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 of 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 of calibration standards that could be excluded must not exceed 20% of the total number of standards injected. 13.5 The correlation coefficient (R) for calibration curves generated must he 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 of 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/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) x DF x aliquot factor slope DF * factor by which the final volume was diluted, if necessary. Aliquot factor * 20 1 4.2 F o r sam ples fo rtifie d w ith k n o w n am ounts o f P F O A p r io r to e xtra c tio n , 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)____________________________ Page 6 of 7 P age 64 o f 65 Page 109 of 119 3M ENVIRONMENTAL LABORATORY PROJECT E05-0210; INTERIM REPORT *20 Exygen Protocol Number: P0001131 Exygen Research Method Number V 0001786 a n a ly tica l m eth o d Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in S m a ll 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) * fPFOA found fng/mLl x final volume (mL)l sample volume (mL) Page 7 of 7 Page 65 o j 65 Page 110of119~ c MAY. 3 0 . 2 0 0 6 12:21PM HEM EHSR 236 IB 3M ENVIRON LAB 260 5 66'l 133 'l o 3M ENVIRONMENTAL LABORATORY Protocol Brygen P O O O -m rf< m < m d Y m m P S ^ ^ ffq Study Title Analysis of Perfluorobutanesulfcnate (PFBS), Perltuorohexanesulfonate (PFHS), and Perfluoraodanesulfbnate (PFOS) inWater, Soil, Sediment Fish, Clams. Vegetation. Small Mammal Liver and Small Mammal Serum Using LC/MS/MS forthe 3MDecatur Monitoring Program PROTOCOL AMENDMENT NO, / " i>r >~ Amendment Date: May 26, 2006 Performing Laboratory 3M Environmental, Health, and Safety Operations 3M Environmental Laboratory 935 BushAvenue St. Paul, MN 55106 Laboratory Project identification E05-0210 0 / it E05.(fl9' ^ GLP Protocol Amendment Page 1of^ 'age 111 of 119 MAY. 3 0 . 2 0 0 6 1 2 :21P M CHEM EHSK 256 lb 3M ENVIRON LAB 260 5 05 > io j ly ju 05/26 '06 13:54 06p. 5 _ 3M ENVIRONMENTAL LABORATORY Protocol Sxygon m Q (M S m pM (^H m fm w ^0210 This amendment modifies the following portion(s) of the protocol; Protocol reads: . P rincipal, In vestig ato r John M, Flaherty Exygen Research 3058 Research Drive State College, PA J0801 Phone: <814)272-1039 Johi]J!ahert>@?xygen.cora Am end to read: Principal Investig ato r fo r Wo rk Co nducted a t B cygen R esea rc h John M. Flaherty R*ygen Research 3058 Research Drive State College, PA 10801 Phone: (814)272-103? JohiLFlaherfy@exygeii.cofn 2006P r i n c i p a l In v e t i c a t o a f o k W o k k C o n o u c t e o a t 3 M p r i o r t o M a y 9 , Michelle D. MaHnsky 3M JEnviromnerrtal Laboratory Building 2-3E-00 935 Bush Avenue St. Paul. MN 55106 Phons (051) 778-6207 mmafinskv@MMM r .n y P r in c ip a l In v ^ t ic a t o r (In t e r im ) FOr W o r k C o n o u c t e o a t 3 M A f t e r M a y 9 , 2 0 0 6 Susan T. W olf 3M Environmental Laboratoiy Building 2-3E-09 935 Bush Avenue S t Paul, MN 55106 Phone (651) 778-4048 stwoJfi@MMM.CQM Reason: D 'U O E 0 5 -^ 0 5 T (c< .. s tw GLP Protocol Amendment ^ S& *^ P $ l1 2 o f1 1 9 MAY. 30.2006 12:21PM CHfcfl L U S K 2 .5 0 ID 3M \EJ NJ V I I RON I --> LAI BS SS2J6 0 5 05/26 '06 13:55 I# ) . 9371 07P. 6 Protocol Exygen AmendmentApproval o 2 .10 EO5-Q20 ST*-' GLP Protocol Amendment 119 Protocol Exygen P0001131; 3M Study Number E05-0210 3M E N V IR O N M ^ m d m m m i PROJECT E05-0210; INTERIM REPORT #20 S tudy Title A n alysis o f P e r flu o r o b u ta n e s u lfo n a te (PFBS), Per flu o r o h e x a n e s u lfo n a te (PFHS), an d Per flu o r o o c ta n e s u lfo n a te (PFOS) in W a t e r , S o il , S e d im e n t, Fis h , C la m s , V e g e ta tio n , S m all Ma m m a l Liv e r a n d S m a ll Ma m m a l S erum Usin g LC/MS/MS fo r t h e 3M Dec atu r M o n ito r in g P ro g ram PROTOCOL AMENDMENT NO. 9 Am endm ent D ate: July 7, 2006 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 GLP Protocol Amendment #9 Page 1 o f 3 Page 114 of 119 Protocol Exygen P0001131; 3 M Study Num ber E05-0210 3M ENVIRONM ENTAlAftftftfffi #Q PROJECT E05-0210; INTERIM REPORT #20 This amendment modifies the following portion(s) of the protocol: The following modification to ETS 8-154.1 will be incorporated into the study: (1) Section 11.1 of the method states that the column will not be allowed to run dry at any time during the extraction process. The current procedure is as follows: Load the analytical sample onto the C18 SPE cartridge. One all the sample has loaded onto the cartridge, isolate the cartridge from the vacuum and wait until all samples on the vacuum manifold have been loaded. Discard eluate. Open manifold valves and pull a vacuum on the SPE cartridges for approximately 3 minutes to remove as much residual water from the SPE cartridge. A new revision o f ETS 8-154 will incorporate the modification 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 if they 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 o f this investigation. E05-0210 GLP Protocol Amendment #9 Page 2 o f 3 Page 115 of 119 Protocol Exygen P 0 0 f f $ i Amendment Approval Amendment #9 3 ^ ^ -- ^ ______________________ l J 'jS a f William K. Reagan, Ph.D. 3M Environmental Laboratory Manager Date Ji/ M c d _____________________________________________________ M Jaisimha Kesari, Weston Solutions, Study Director ob Date E05-0210 GLP Protocol Amendment #9 Page 3 o f 3 Page 116 of 119 Attachment D: Method Deviations 3M ENVIRONMENTAL LABORATORY PROJECT E05-0210; INTERIM REPORT #20 Page 117 of 119 3M Confidential 3M e n v ir o n m e n t a l l a b o r a t o r y R ecord of Nonconformance / OfVfYiff'0' i n t e r i m r e p o r t *2 Study / Project No. E05-0209 Interim Report#20 Deviation type SOP (Check one) 0 Protocol Protocol Requirements: 1. Identification Date(s) of Occurrence: Document Number: February 20, 2006 Exygen Protol #P0000760 Amendment #4 (3M) Equipment Procedure Method GPO Other: II. Description (3) ETS 8-154.1 makes no mention o f sim ple surrogate spikes; however, all samples will be spiked with a radiolabeled surrogate, PFOA [1,2 13CJ. 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 in the final report. Actual pracedure/process: PFOA[1,2 13C] was not included as a surrogate spike in any of the sample/sample duplicates, or field matrix spikes collected for this project. Surrogate recoveries from the other water projects and previous interim reports for E05-0209 indicate that there are solubility/stabitity issues with this surrogate in water. The 3M Environmental Lab has started an official investigation {which is not part of E05-0209) to determine the root cause for the low recoveries. Until this situation is fully understood and a method modification has been incorporated to eliminate or circumvent the issue, PFOA[1,2 13C] will not be added as s u r r o g a t e ...................... .................... ..................................___ _ ........................................ _ ___________ _ til. Actions Taken _ (such as amendment issued, SOP revision, etc.)_____________________ Corrective Action ( Yes 0 No) Reference: Acceptability of the nonconforming work: Surrogates were originally used as an additional quality control element in conjunction with lab control spikes and field matrix spikes. The surrogate recovery was never intended to be the sole confirmation of that the analytical method was appropriate for the given matrix. Non-inclusion of the surrogate spike has no impact on the quality and integrity of the data presented here. Actions: Halting of Work Client Notification Work Recall Withholding of Report 0 Other: Non-inclusion of the surrogate will be addressed in the final report. Recorded by: / --tK. "T > D>a te;;- / Authorization: Date: Sponsor Approval (QLP Protocols): i / 'c y / Date: iW - ,/ Technical Manager Approval (if halting of work or work recall are indicated): Date: ! A 'A K Ar IV. Authorization to Resume Work ___ Where baiting o f work occurred, resumption of work m ust first be approved by Technical M anagem ent technical Manager Approval: Date: p jA a M- Deviation No. L (assigned by Study Director or Project Lead at the end of study or project) Protocol Deviation Page 1 of 1 Page 118 of 119 3M Confidential Record of Nonconformance / Demiamom<ental LABORATORY .. .................................... - PROJECT E05-0210! INTERIM REPORTW2Q I. Identification________________________ Study / Project No. Date(s) of Occurrence: Document Number: E05-0210 Interim Report#20 February 20, 2006 Exygen Protol #P0001131 Amendment #3 (3M) Deviation type SOP (Check one) El Protocol Equipment Procedure Method GPO________________ Other:_________________ II. Description________________________ Protocol Requirements: (3) ETS 8-154.1 makes no mention of sample surrogate spikes; however, all samples will be spiked with a radiolabeled surrogate, PFOA [1 ,2 13CJ. 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 in the final report. Actual procedure/process: PFOA[1,2 13C] was not included as a surrogate spike in any of the sample/sample duplicates, or field matrix spikes collected for this project. Surrogate recoveries from the other water projects and previous j interim reports for E05-0210 indicate that there are solubility/stability issues with this surrogate in water, j The 3M Environmental Lab has started an official investigation (which is not part o f E05-0210) to j determine the root cause for the low recoveries. Until this situation is fully understood and a method j modification has been incorporated to eliminate or circumvent the issue, PFOA[1,2 13C] w ill not be l added as surrogate._____________________________________________________________________ Ilf. Actions Taken [_________________________________ (such a s a m e n d m e n t issued, S O P revision, etc .)_________________________________ i Corrective Action ( Yes 0 No) Reference: Acceptability of the nonconforming work: Surrogates were originally used as an additional quality control element in conjunction with lab control : spikes and field matrix spikes. The surrogate recovery was never intended to be the sole confirmation of that the analytical method was appropriate for the given matrix. Non-inclusion of the surrogate spike jj has no impact on the quality and integrity of the data presented here. Actions: Halting of Work Client Notification Work Recall W ithholding of Report 0 Other: Non-inclusion of the surrogate will be addressed in the final report. Recorded by:- / Date: 0 , Authorization: Date: Sponsor Approval & S J /g/ o 6 Date: o B /lu /p i Technical M anager Approval (if halting of work or work recall are indicated): Date: fir IV. Authorization to Resume Work W here halting o f w ork occurred, resum ption o f work m ust first b e app ro ved b y T echnical M an ag em en t Technical Manager Approval: Date: f ir ' rJ A r Deviation No. Z (assigned by Study Director o r Project Lead at the end o f study o r project) Protocol Deviation Page 119 of 119 Page 1 of 1