Document daa2BV4VwXxjkbXkgOJDXpGJ0

3M ENVIRONMENTALLABORATORY PROJECT05-0210: INTERIMREPORT#20 Interim Report #20: Analysis of Water Samples from Northern Alabama Potable Water Systems, February 2006 Study Title ANALYSISOF PERFLUOROBUTANESULFO(PNFABTSE), PERFLUOROHEXANESULFONATE (PFHS), AND PERFLUOROOCTANESULFONATE(PFOS) IN WATER, SOIL, SEDIMENT, FISH, CLAMS, VEGETATION, SMALL MAMMALIVERAND SMALL MAMMALSERUM USING LC/MS/MS FOR THE 3M DECATURMONITORINGPROGRAM 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, MN 55106 Project Identification E05-0210 Total Number of Pages 119 si!!!!! ACCREDITED Certificate#205201 The testing reported hereinmeet the requirementsof ISOAEC 170251999"General Requirementsfor the Competence of Testing and CalibrationLaboratories",in accordancewith the A2lA Certificate #2052-01.Testing that complies with this International Standard also operate in accordancewith IS0 9001/ISO9002 (1994). Page 1 of 119 3M ENVIRONMENTALLABORATORY PROJECT 05-0210;INTERIM REPORT #20 This page has been reserved for specific country requirements. Page 2 of 119 3M ENVIRONMENTAL LABORATORY PROJECT 05-0210; INTERIM REPORT #20 GLP COMPLIANCSETATEMENT Study Title: Analysis of Perfluorobutanesulfonate(PFBS), Perfluorohexanesulfonate (PFHS), and Perfluorooctanesulfonate(PFOS) in Water, Soil, Sediment, Fish, Clams, Vegetation, Small Mammal Liver and Small Mammal Serum Using 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 MichaelA. f Santor 3M Company, Sponsor Representative Io7 215 1 I Date Jaisimha Kesari P. E., DEE, Study Director Certificate #205241 The testing reportedhereinmeet the requirements of ISO/IEC 170251999"General Requirementsfor the Competence of Testing and CalibrationLaboratories", in accordancewith the MIACertificate#2052-01. Testingthat complieswith this International Standard also operate in accordance with IS0 9001/ISO9002 (1994). Page 3 of 119 3M ENVIRONMENTAL LABORATORY PROJECT 05-0210; INTERIM REPORT #20 QUALITY ASSURANCSETATEMENT 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 IdentificationNumber: 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 I January 5,2007 Phase Extraction In-Phase Audit I DataandReport 1 I Date Reported to Management Study Director August 1,2006 I I QAU Representative Date Page 4 of 119 3M ENVIRONMENTAL LABORATORY PROJECT 05-0210; INTERIM REPORT #20 TABLEOF 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 ReferenceSubstances ................................................................................................................ I O MethodSummaries...................................................................................................................... 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 Suitab.il.ity......................................................................................................................... 13 ContinuingCalibration.................................................................................................................. 14 Lab Control Spikes (LCSs) .......................................................................................................... 14 Sample Duplicates ................................ .................................................................................. 14 Field Matrix Spikes....................................................................................................................... 14 Data Summaryand Discussion................................................................................................... 14 Page 5of 119 3M ENVIRONMENTAL LABORATORY PROJECT 05-0210; INTERIM REPORT #20 Statistical Methodsand Calculations........................................................................................... 19 Accuracy and PrecisionEquations.............................................................................................. 19 Determinationof Analytical Uncertainty...................................................................................... 19 Statementof Conclusion.............................................................................................................. 20 References................................................................................................................................... 20 List of Attachments....................................................................................................................... 20 Signature Page............................................................................................................................. 21 LISTOF TABLES Table 1. Sample Results Summary............................................................................................................. 9 Table 2. Sample DescriptionCodes......................................................................................................... 10 Table 3. Study ReferenceSubstances..................................................................................................... 10 Table 4. Sample Collection Information.................................................................................................... 11 Table 5. Instrument Parameters................................................................................................................ 12 Table 6. Liquid ChromatographyGradient Program................................................................................ 12 Table 7. MassTransitions.......................................................................................................................... 12 Table 8. Lab ControlSpike Results........................................................................................................... 14 Table 9. Sample and QC Results.............................................................................................................. 16 Table 10. Analytical Method Uncertainty.................................................................................................. 16 Page 6 of 119 STUDYINFORMATION 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 05-0210; 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 Experimental Initiation: March 1, 2006 Interim Experimental Completion: August 27, 2006 Interim Study Completion: Date of interim report signing Location of Archives All original raw data, protocol, and analytical report have been archived at the 3M Environmental Laboratory according to 40 CFR Part 792. The test substance and analytical reference standard reserve samples are archived at the 3M Environmental Laboratory according to 40 CFR Part 792. Page 7of I19 3M ENVIRONMENTAL LABORATORY PROJECT 05-0210; 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 PerfluorinatedAcids, Alcohols, Amides, and Sulfonates in Water by Solid Phase Extractions and High Performance Liquid Chromatography/Mass Spectrometry" in accordance with Exygen Research Protocol POOOl 131 (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 [I,2 "C] was not included as a surrogate. This is a deviation from Protocol POOOl130 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"' 3M ENVIRONMENTAL LABORATORY PROJECT05-0210; INTERIM REPORT#20 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 resultsare as follows: PFBS 100% f 17%, PFHS 100% f 15%, and PFOS 100% f 22%, based on control chart data of laboratory control samples. All resultsand 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% f 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% f 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% f 38% due to the low field matrix spike recovery. Additionaldiscussion is provided on the Data Summary and Discussionsection of the report. (5) The analytical method uncertainty for PFBS on this sample is 100% f 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 analyticalmethod uncertainty for PFBS on this sample is 100%f 40% due to the low field matrix spike recovery. Additionaldiscussion is provided on the Data Summary and Discussionsection of the report. Page9of119 TESTSAMPLES 3 M ENVIRONMENTALLABORATORY PROJECT05-0210; INTERIMREPORT#20 Sample Description Abbreviation DPWS WMEL SWTP MSTP FWTP TVATP PW FW 0 DB LS HS Definition Decatur Potable Water System West MorganlEast Lawrence Water Treatment Plant, Lawrence County, AL Sheffield Water Treatment Plant, SheffieldAL Muscle Shoals Treatment Plant, Muscle Shoals,AL FlorenceWater Treatment Plant, Florence,AL Tennessee Valley Authority Treatment Plant, Muscle Shoals, AL Potable Water Finished Water Matrix Sample Duplicate Low Spike High Spike REFERENCSEUBSTANCES 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 Identifier (''Source Expiration Date Storaae Conditions Chemical Lot Number TCR Number Physical Description Purity (*)Solubility C4FsSOiK' CAS # 29420-49-3 3M 1/17/2007 Frozen 2 TCR-281 (99131-039) White Powder 97.3% 54,400 ppm CsFi3SOiK' CAS # 3871-99-6 3M 10/18/2006 Frozen NB# 120067-69 TCR-83 (SE036) White Powder 98.6% No informationavailable CaF1+Oj.K* CAS # 2795-39-3 3M 8/31/2006 Frozen 171 TCR-696 White powder 86.4% 680 ppm Page 10 of 119 3M ENVIRONMENTALLABORATORY PROJECT 05-0210; INTERIM REPORT #20 METHODSUMMARIES 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 I `"Bottle Description Nominal Fina Volume Collected (mu Final Concentration(ng/mL) I I PFBS PFHS PFOS Sample 450 NA NA NA Sample Dup 450 NA NA NA I I 1 I I Low Field Matrix Spike (LS) 450 High Field Matrix Spike (HS) 450 0.0499 0.499 0.0502 0.502 0.0496 0.496 (1) The sample descriptionwas 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/MassSpectrometry". 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 05-0210; INTERIM REPORT #20 Table 5. Instrument Parameters Instrument Name Liquid Chromatograph Guard column Analytical column Injection Volume Mass Spectrometer Electrode Ion Source Polarity Software ETSGinger Agilent 1100 Betasil C18 (2.1 mm X 100 mm), 5 pp Betasil C18 (2.1 mm X 100 mm), 5 pn 5 pL Applied Biosystems API 5000 2-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 I.o 11.0 13.5 14.0 17.0 Flow Rate (Wmin) 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 8 (Methanol) 40.0 40.0 90.0 90.0 40.0 40.0 Table 7. Mass Transitions Analyte ("PFBS ("PFHS "'PFOS Mass Transition QW3 299180 299199 3991130 399199 399180 4991130 499199 499180 resultant TIC was used for quantitation Dwell Time (msec) 150 150 150 150 150 150 150 150 Page 12 of I19 3M ENVIRONMENTALLABORATORY PROJECT 05-0210; INTERIM REPORT #20 ANALYTICARLESULTS 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, l / 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 100k25% (100*30% for the LOQ standard). The correlation coefficient was greater than 0.999 for all analytes. Limit of Quantitation (LOQ) The LOQ for this analysis, as defined in ETS-8-154.1, is the lowest non-zero calibration standard in the curve in which the area counts are 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.O 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 3M ENVIRONMENTALLABORATORY PROJECT 05-0210; 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"'. PFBS - I I I PFHS I Conc. Conc. Conc. LCS-060825-1 0.0999 0.109 109 0.0999 0.103 LCS-060825-2 0.0999 0.108 108 0.0999 0.104 LCS-060825-3 0.0999 0.111 111 LCS-0608254 0.999 1.oo 100 0.0999 0.107 105 0.999 LCS-060825-5 0.999 1.09 109 0.999 1.07 107 LCS-060825-6 0.999 1.14 114 - 0.999 1.15 116 Average 1 %Recoverv f %RSD 109% f 4.3% 110% f 3.3% : (1) Table displaysroundedvaluesfor all concentration and percent recoveryvalues (3 significantfigurt 106%f4.6% ) and %RSD (2 significantfigures) using EPA rounding rules. Reportedvalues may vary slightly from the raw data. Sample Duplicates Because a field sample duplicate (separate container) was collected at each sampling location, duplicate/replicate extractions of a given sample were not performed. Overall method precisionwas 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 100i30% 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. DATASUMMARAYND DISCUSSION 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 05-0210:INTERIM REPORT#PO 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 PFHSwere ~0.0250ng/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 ~0.0250ng/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 ~0.0250ng/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 ~0.0250ng/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 ~0.0250ng/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 ~0.0250ng/mL. The analytical method uncertainty for PFBS on this sample is 100%*40%. Page 15of I19 3M ENVIRONMENTALLABORATORY PROJECT 05-0210; lNTERlM REPORT #20 Table 9. Sample and QC Results ('). Location #1 DPWS 'W FW03 1- ~~~ - PEES_.._-- I PFHS PFOS 3M LlMS ID Description Conc. (ng/mL) %Recovery E05-0210-94967 DPWS PW FW03 0 060301 E05-0210-94968 DPWS PW FW03 DB 060301 E05-0210-94969 DPWS PW FW03 LS 060301 I E05-0210-94970 DPWS PW FW03 HS 060301 Average Sample Concentration (ng/mL) f %RPD'*' <0.0250 NA ~0.0250 NA 0.0612 123 0.626 126 <00250 1 Location #2 WMEL PW FW03 I~~ ~ PFBS Conc. (ng/mL) %Recovery <0.0250 NA <0.0250 NA 0.0622 124 I -- 0.617 123 . - e0.0250 -~ - _ PFHS Conc. (ng/mL) %Recovery ~0.0250 NA <0.0250 NA 0.0496 99.4 0.493 98.8 <0.0250 PFOS 3M LlMS ID Description 3M LlMS ID I Description Avera e Sam le Concentration n mL f %RPD@' Cone. (ng/mL) %Recovery 1Conc.(ng/mL) %Re: ~ ~0.0250 <0.0250 0.0650 131'4' 0.661 133"' <0.025d4) Conc. (ng/mL) %Recovery <0.0250 NA <O 0250 0 0801 0 666 - ~ - NA 159'3' 133'3' ~ <0.0- 250'3' ~~- ----_ __ PFHS - Cone. (ng/mL) %Recovery Conc. (ng/mL) %Recovery 0.102 NA 0.105 NA 0.159 110 0.590 97.5 0.104 f 2.8% PFOS Conc. (ng/mL) %Recovery Page 16 of I19 3M ENVIRONMENTALLABORATORY PROJECT 050210; INTERIM REPORT #20 ~Loca_tion _#4 TV_ATP PWFWO3 PFBS 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 - E A T P PW FW03 HS 060301 ~ Av- erage Sample Concentration (ng/mL) %RPdz' ~~~ Conc. (ng/mL) %Recovery <O 0250 NA <O 0250 NA 0.0687 138`5' - 0 620 125 <0.025d5' PFHS __ Conc. (ng/mL) %Recovery <0.0250 NA e0 0250 NA 0.0626 125 0 627 125 c0.0250 PFOS Conc. (ng/mL) %Recovery 0.0441 NA 0.0410 NA 0 0921 99.4 0 512 94.1 0.0425 f 7.1% 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) 5 %RP@Y .. ... -... Conc. (ng/mL) %Recovery e0 0250 NA <O 0250 NA 0 0737 148"' 0 642 ~ 129 ~ 0.0250 " ___~ PFBS 3M LIMS ID Description Conc. (ng/mL) %Recovery <0.0250 NA <0.0250 0.0696 NA 140"' 0.632 128 <0.025c1~ <0.0250 0.0628 0.649 <0.0250 _____~ - - PFHS ~ ~- ~~ Conc. (ng/mL) %Recovery <0.0250 NA <0.0250 NA 0.0636 127 0.629 125 <0.0250 %Recove 0.0448 0.0432 0.0977 108 0.547 101 0.0440 f: 3.7% ~ . ~ PFOS Conc. (ng/mL) %Recovery 0.0448 NA 0.0428 NA 0.0962 105 0.542 108 0.0438 f 4.4% Page 17of I19 3M ENVIRONMENTALLABORATORY PROJECT 050210; INTERIM REPORT #20 Trip Blank -~ -~ -~ ~ PFBS PFHS PFOS 3M LlMS ID E05-0210-94964 E05-0210-94965 E05-0210-94966 Description DPWS PW TRIP 0 060301 DPWS PW TRIP LS 060301 DPWS PW TRIP HS 060301 Conc. (ng/mL) c 0.0250 0.0507 0.599 %Recovery NA 102 121. - - Conc. (ng/mL) C0.0250 0.0533 0.603 %Recovery NA 106 120 Conc. (ng/mL) < 0.0250 0.0422 0.478 %Recovery NA 84 5 95 8 (1) The analyticalmethod uncertainties associatedwith the reported results are as follows: PFBS 100% f 17%, PFHS 100% f 15%, and PFOS 100% f 22%, based on control chart data of laboratory control samples. All results and averages are presentedwith three significantfigures, %RPD to two significant figures. Sample concentrations, averages, and %RPD values may vary slightly from the raw data. (2) Not applicable. %RPD values not determinedwhen concentrationsfor the sample andlor 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 dup!icate resultsfor PFHS were <O.0250ng!mL. The analytical method uncertaintyfor 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 resultsfor PFBS were <0.0250 ng/mL. The analytical method uncertaintyfor 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 ~0.0250nglmL, and the low field matrix spike recovery of 120% was within the acceptance criteria,the sample is accurate to the stated method uncertaintyfor PFHS. (5) Sample locationTVATP 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 resultsfor PFBS were ~0.0250nglmL. The analytical method uncertaintyfor 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 resultsfor PFBS were <0.0250 ng/mL. The analytical method uncertaintyfor 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 resultsfor PFBS were ~0.0250nglmL. The analytical method uncertaintyfor PFBS on this sample is 100%*40%. Page 18 of I 1 9 3 M ENVIRONMENTAL LABORATORY PROJECT05-0210; INTERIM REPORT#20 STATISTICAL METHODASND CALCULATIONS Statistical methods used to interpret sample results include averages and standard deviations. The Analyst software program calculated sample concentrations using resultant analyte peak areas and the established quadratic, Ilx 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 LCSlSurrogate Percent Recovery = Calculated Concentration oo% Spike Concentration Sample Spike Recovery = (Spiked Sample Concentration - Average Concentration : Field Sample & Field Sample Dup.) 100% Spike Concentraton % RSD (Relative Standard Deviation) = standard deviation of replicates 100% ~ replicate average % RPD (Relative Percent Difference) = Absolute difference between sample duplicates 100% average sample concentration Determination of Analytical Uncertainty Analytical uncertainty is based on historicalQC 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. Pagel9of119 Table I O . 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 ENVIRONMENTALLABORATORY PROJECT 05-0210; INTERIM REPORT #20 Method Uncertainty 100% f 17% 100% f 15% 100% f 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: 100+5.2%; PFHS: 100+9.3%, 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. "StatisticalTreatment 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. LISTOF ATTACHMENTS Attachment A: Selected Chromatograms and Calibration Curves Attachment B: Extraction and Analytical Methods Attachment C: Protocol and Protocol Amendments Attachment D: Protocol andlor Method Deviations Page 20 of I I9 3M ENVIRONMENTAL LABORATORY PROJECT 05-0210; INTERIM REPORT #20 SIGNATURPEAGE We certify that this report is a true and complete representation of the data for this study: . - -X I 1 Jaisimha Kesari, P.E., DEE, Susan Wolf, Study Director 3M Senior Chemist Date I-29-U Date William K. Reagen, Ph.D., 3M Environmental Laboratory Manager Date Sponsor Representative / date Page 21 of 119 3M ENVIRONMENTAL LABORATORY PROJECT 05-0210; INTERIM REPORT #20 ATTACHMENAT: SAMPLE CHROMATOGRAMSAND CALIBRATION CURVES Page22of 119 *Ginger AGO1330509 ample Index: ample Type: oncentration: a l c u l a t e d Conc: cq. Date: cq. Time: 17 Standard 0.9390 0.9871 8/27/2006 3 : 4 8 : 4 0 PH ng/mL ng/mL odified: T Window: x p e c t e d RT: s e R e l a t i v e RT: Yes 30.0 sec 4 . 8 6 rain No n t . Type: Manual e t e n t i o n Time: 4.882 mi" rea: 1109323 counts eight: 82100. cps t a r t Time: 4.40 rain nd Time: 5.35 min 8% 80.4 7&4 78.4 74e4 7% 7004 6&4 8604 8464 6204 60.4 58.4 54.4 5 2.4 50.4 4w 48.4 44e4 8 42.4 5 1 40.4 - C 58.4 3w 34e4 3 2.4 30.4 2&4 2w 24e4 2 2e4 2004 18.4 18.4 14 1% 1W 8woC mc 4OOoE Moot OC Printing Time: 1:48:47 PM sample Index: sampleType: concentration: calculated Conc: A C ~ . Date: Acq. Tine: 17 Standard 1.000 1.009 8/27/2006 3 : 4 8 : 4 0 PH ng/mL ng/mL Modified: Yes RT window: 30.0 sec Expected RT: 8.67 min u s e R e l a t i v e RT: No I n t . Type: Manual Retention Time: 8.675 min Area: 1675489 counts Height: 262000. c p s S t a r t Time: 8.26 min End Time: 9.07 mi" 2.6e5 2.6e5 2.505 2.95 2.405 2.405 2.305 2.305 2.2e5 2.2e5 2.le5 2.1e5 2.Oe5 2.Oe5 1 .%5 1. a 5 1.8.35 1.&5 1.705 1.7e5 1.6e5 1.Be5 1.95 1.95 1.405 8 1.- @. 1.3e5 d 1.395 1.2e5 1.295 l.le5 l.le5 1.Oe5 9.504 9.W 834 8.oe4 7.w 7.w 8.M 6.W 5.w 5.M 4.M 4.w 3.- 3.w 2.w 2.w 1.- 1.004 5000.E OS 323 8.41 35 % Results Name: g060827a.rdb . .. I oncentration: 0.9990 ng/mL a l c u l a t e d Conc: 1.006 ng/mL cq. Date: 8/27/2006 c q . Time: 3 : 4 8 : 4 0 PH - l o d i f i e d : NO roc. Algorithm: IntelliQuan HQII o i s e Percentage: 50 a s e . Sub. Window: 1.00 min .eeapko-rSt pLl iatr.g eFs at cPt oear :k: 5No lin. Peak Height: 0.00 c p s lin. Peak width: 0.00 s e c moothing Width: 0 points .T Window: 30.0 sec : x p e c t e d RT: 10.4 lain lse R e l a t i v e RT: No n t . Type: Base To Base e t e n t i o n Time: 10.312 min ,rea: 1411930 counts leight: 181000. cps . t a r t Time: 9.72 min :nd Time: 10.7 min #a - 1IM REPORT 1-5 18e5 1.7e5 1.7e5 1.c5 1.Be5 195 1.5e5 145 1.4e5 135 135 1.2e5 1.2e5 1.1.5 l.le5 1.005 8 9.W i5 9.W - d 8.W 8.W 7.W 7.w 6.504 6.W 5.w 5.0.4- 10.13 4.W- 4.0.4 - 3.584 - 3.oe4- 2.W- 2.0.4 - 1.W- 1.0.4- 50oo.O - Page 1 of 1 *Ginger AGO1330509 - smpb Nm:"g060827so24' SapleID:W 4 6 0 6 2 5 1 " fae:'g060827a.rrin Peak Nnw: p a . Mpu(es): 199.w98.0mu.299.0m0.0 mu' carment:"MemodBlank-1" I\nnomon: implc Index: 24 ample Type: Unknown mcentration: N/A i l c u l a t e d Conc: < 0 245 :q. Date: 8/27/2006 :q. Time: 5:54:01 Pn 240 Jdified: roc. Algorithm: NO IntelliQuan - IQA i n . Peak Height: 0.00 cps i n . Peak Width: 0.00 sec noothing Width: r Window: 0 30.0 points sec x p e c t e d RT: 4.86 lain se R e l a t i v e RT: No n t . Type: e t e n t i o n Time: rea: eight: t a r t Time: nd Time: Valley 4.861 min L119 c o u n t s 113. cps 4 . 6 5 mi" 4.95 mi" 235 230 225 220 215 42 0 5.: 6. ample Index: ample Type: oncentcation: alculated Conc: cq. Date: cq. Time: 24 Unknown N/A < 0 8/27/2006 5 : 5 4 : 0 1 PM odified: Yea T window: 30.0 sec x p e c t e d RT: 8.67 min s e R e l a t i v e RT: No n t . Type: e t e n t i o n Time: rea: eight: t a r t Time: nd Time: Manual 8.648 min 2115 counts 332. c p s 8.58 min 8.74 min 1 360350- 340 - 530- 320 - 310- m- 175 /j 170 165 180 ~ 155 130 125 80 75 70 65 80 55 50 45 40 33 30 25 20 15 10 5 Printing Time: 1:49:50 PM Results Name: g060827a.rdb a l c u l a t e d Conc: ,cq. Date: c q . Time: < 0 8/27/2006 5:54:01 Pn lodified: 'roc. A l g o r i t h m : I n NO tell i Q u a n - MQII loire Percentage: 50 Lase. Sub. Window: 1.00 lain 'eak-Split. Factor: 5 : = p o r t L a r g e s t Peak: No lin. Peak Height: 0.00 cps lin. Peak Width: 0.00 s e c moothing Width: 0 points :T Window: 30.0 sec : x p e c t e d RT: 10.4 min I r e R e l a t i v e RT: No n t . Type: Letention Time: mea: [eight: : t a r t Time: :nd Time: Base To Base 10.349 min 3005 counts 644. cps 10.3 min 10.5 min 760 - 740 - 720 700- 680 - 880640620- 600 580 580 - 540 520500480460440 ~ 420- 400 - 380360340 - 320 - 300280280240P O200180180140120100- 80- Page 1 of 1 Tm. min *Ginger AGO1330509 imple Index: imple Type: mcentration: i l c u l a t e d Conc: :q. D a t e : :q. Time: 38 Qc 0.9990 ng/mL 1.002 ng/mL 8/27/2006 1 0 : 0 4 : 4 6 PM Ddified: Yes I Window: 30.0 sec x p e c t e d RT: 4.86 min s e R e l a t i v e RT: No nt. Type: manual e t e n t i o n Time: 4.843 min rea: 1733925 counts eight: 82900. c p s t a r t Time: 4.30 min nd Time: 5.37 mrn 8.W 7.8.4 7.8.4 744 7.2.4 7.094 6.8.4 &Be4 8.4e4 8.2.4 6.094 5.894 5.8.4 5.404 5.2.4 5.004 4.w 4.w 4.M 8 4.204 - 4.0.4 3.894 3.w 3.m 3.284 3.w 2.m 2.m 2.& 2.2w 2.w 1.W 1.w 1 .& 1 .% 1.h WW.1 6000.1 4WO.l 2ooo.1 0.1 Printing Time: 1:50:42 PM 4.7R ample Index: ample Type: oncentration: a l c u l a t e d Conc: cq. Date: cq. Time: 38 QC 1.000 1.049 8/27/2006 10:04:46 ng/mL ng/mL PM odiiicd: Yea T Window: 30.0 aec xpected RT: 8.67 min se R e l a t i v e RT: No n t . TYPe: Manual etention Time: 8.667 min rea: 1741795 counts eight: 269000. c p s t a r t Time: 8.23 min nd Tine: 9.09 min 2.66 295 2.95 2.495 2.495 2.3.5 2.M 2.295 2.295 2.105 2195 2.0~5 2.Oa5 1.985 1.985 1.Be5 1.805 1.7e5 1.795 1.6~5 1.805 1.95 1.585 1.495 8 1.495 @ 1.3.5 a C 1.3.5 1.2e5 1.2e5 1.195 1.195 1.Oa5 9.504 9.0.4 8.504 8.M 7.504 7.M 8.W 6.0.4 5.594 5.0.4 4.594 4.w 3.594 3.064 2.5.4 2.w 1.5.4 1.w 5wOs a( Results Name: g060827a.rdb ample TI n dY P ~ R O J l?0Z50210:, INTE-RIM REPORT #2 oncentration: 0.9990 ng/mL a l c u l a t e d Conc: 1.024 ng/mL cq. Date: 8/27/2006 cq. Time: 1 0 : 0 4 : 4 6 PH lodifi e d : 'roc. a l g o r i t h m : No IntelliQuan - MQII loise Percentage: 50 a s e . Sub. Window: 1.00 min 'eak-Split. Factor: 5 !:::i::: . e p o r t L a r g e s t Peak: ~~~~~: N:o::: moothing Width: 0 points .T Window: 30.0 sec : x p e c t e d RT: 10.4 min Ise R e l a t i v e RT: No n t . Type: Base T o Base f e t e n t i o n Time: 10.372 min ,rea: 1437256 counts [eight: 180000. cps , t a r t Time: 9.72 min :nd Time : 10.8 min 1.7e5 1.795 1.605 1.661,s1.951.4a5- 1.&5 - 1.3e5- 1.3e5- 1 1.2e5 i 1.2e5 - 1.195- l.le5- l.Oe5- 9.594 r8: 9 . w - .D f 8.w; 8.oe4- 7.594- 7.w- 6.M- 8.084 - 5.w5.w- 10.12 4.- - 4.w- 3.594- 3.w- 2.w- 2.oe4- 1.W- 1.0.4- 5ooo.O- ?age 1 of 1 2 474 495 I1 *Ginger AGO1330509 - Sample Name: "g060627soSl' Sa@n ID: YO50210-94941" File:" ~ 7 a . w w Psak N m : 'p(bs' Mur(es):'299.w99.0 anlu.299.0180.0mu' canInNl1:M L p w m0 0803M' Annotation: ample Index: 51 ample Type: Unknown 4.04 oncentration: N/A 61W2 a l c u l a t e d Conc: 0.05961 ng/mL cq. D a t e : 8/28/2006 6M)o- cq. Time: 1:57:46 M 5900- odified: Yes T Window: 30.0 sec x p e c t e d RT: 4.86 min s e R e l a t i v e RT: No 5800- 5700 - 5800- n t . Type: Manual e t e n t i o n Time: 4.838 min rea: 120505 counts eight: 6000. c p s t a r t Time: 4.40 min nd Time : 5.19 min 5500540053005200- 5100- Moo- 4900- 4800- 4700 - 4600- 4500- 4400- 4300- 4200- 4100- 4m- 3900- 3ww)- 3700- 3800- 3500- 3400- 33003200- ! 3100: .i. -' 2900; 2800- 2m- 26M)- 2500- 2a- 2m- 2200- 2100- 2000- 1900- 1800- 1700- 1800- 1500- 1400- 1300- 1200- 1100- 1000- 800- 800- 700- 600- 500- am- 200- 100 I I ........................... O3:4 3:s 3.8 4.0 4.2 4.4 4.6 4.8 SO 5 2 5.4 5.8 5.8 6.0 6.2 147 156 164 173 162 190 199 207 216 225 233 242 250 259 288 ample Index: ample Type: Incentration: ~ i c u l a t e dC o n c : :q. Date: cq. Time: 51 Unknown N/A 0.01718 8/28/2006 1 : 5 7 : 4 6 AM ng/mL - o d i f i e d : Yes roc. Algorithm: IntelliQuan oise Percentage: 50 MQII a s e . Sub. Window: 1.00 min eak-split. Factor: 5 e p o r t L a r g e s t Peak: NO i n . Peak H e i g h t : 0.00 cps i n . Peak Width: 0.00 sec moothing Width: 0 points T Window: 30.0 aec x p e c t e d RT: 8.67 min se R e l a t i v e RT: No n t . Type: etention Time: rea: eight: t a r t Time: nd Time: Base To Base 8.652 min 43387 counts 6590. cps 8.26 min 8.84 rain BBw 8500 E400 6300 8Mo 6100 6wo 5900 5800 5700 5800 5500 5400 5300 5Mo 5100 Moo 4900 4800 4700 4500 4400 4300 4200 4100 4m 3900 3800 3700 3800 B 3500 3400 g33w - c 22 3200 3100 3oM) 2900 2800 2700 2800 2500 2400 2m 2m 21 00 2m lgw 1600 17W 1600 15LX 14w 13OC 1Mo 11w 10M 900 800700600500400300200- 0' I. 7.5 323 P r i n t i n g T i m e : 1:51:28 PM 8.41 : 6.0 8.5 9.0 345 368 388 T i ,mil R e s u l t s Name: g 0 6 0 8 2 7 a . r d b :% :;:pROJ&@ 05-0210; INTERIM REPORT #;4 oncentration: a l c u l a t e d Conc: 0.1023 ng/mL c q . Date: Cq. Time: 8/28/2006 1 : 5 7 : 4 6 AM 1.704 - lodi f i e d : No 'roc. A l g o r i t h m : I n t e l l i Q u a n bise Percentage: 50 MQII a s c . Sub. Window: 1.00 min leak-Split. Factor: 5 ;:. e p o r t ;;g;;: L a r g e s t Peak: N;:;O ,moothing Width: IT Window: 0 30.0 points sec X p e c t e d RT: 10.4 min Ise R e l a t i v e RT: NO n t . Type: Base TO B a s e I e t e n t i o n Time: 10.349 mi" mea: 154062 counts [eight: 17400. cps l t a r t Time: 9.12 lain :nd Time: 10.6 min 1.704 1.694 1.694 1.W 1.Ad 1.484 1.394 1 ,394 1.204 1.204 1.1.4 1.104 1 .oe4 95W.O 8 gow.0 E.i. 6500.0 MMo.0 7500.0 7000.0 6500.0 6wo.O 5500.0 5000.0 4500.0 4WO.O 3500.0 3000.0 2500.0 m.0 1500.0 9.5 10.0 409 431 Page 1 of 1 1m.c 500.c 0.c P-L? rvJ/""" 152.5 14714.0 1419.55 b 3M ENVIRONMENTAL LABORATORY PROJECT E05-0210; INTERIM REPORT #20 ATTACHMENBT: EXTRACTIOANND ANALYTICAMLETHODS Page 27 of 119 3M ENVIRONMENTALLABORATORY PROJECT E05-0210; INTERIM REPORT #20 3M Environmental Laboratmy Method Determinationof PerfluorinatedAcids, Alcohols, Amides, and Sulfonates In Water By Solid Phase Extraction and High Performance Liquid Chromatography/Mass Spectrometry Method Number: ETSa8-154.I Adoption Date: 28 Apr 2000 Revision Date: 5 May, 2003 Effective Date: 5 May, 2003 Approved By: -- William K. Reagen Manager os+I-/o3 Date ETS-8-154.1 Page 1 of 17 Determinationof PerfluorinatedCompounds in Water Using SPE and LC/MS. Page 28 of 119 3M ENVIRONMENTAL LABORATORY PROJECT E05-0270; INTERIM REPORT #20 1 Scope and Application This method was validated for the collection, extraction, and analytical procedure for the determinationof Perfluorooctanesulfonate(PFOS), Perfluorooctanesulfonylamide (FOSA), and Perfluorooctanoate(PFOA) in groundwater,surfacewater, and drinking water samples. This method may also be applied to the determinationof other perfluorinatedacids, alcohols, amides, and sulfonates in similar matrices, as long as the defined QC elements are satisfied and with the understandingthat the method is not validatedfor compounds outside the scope of the original protocol This method is based in part on the report "Method of Analysis for the Determination of Perfluorooctanesulfonate (PFOS), Perfluorooctanesulfonylamide(PFOSA), and Perfluorooctanoate(POAA) in Water" (see Section 17),as developed and validated by Exygen Research (formerly Centre Analytical Laboratories, Inc.). 2 Method Summary Water samples are collected from a site of interest and shipped cold to an analytical facility. Perfluorinated acids, alcohols, amides, and sulfonates are extracted from 40mL water samples using C18 solid phase extraction (SPE) cartridges. The compounds are eluted from the C18 cartridge, using methanol. Separation, identification,and measurementare accomplished by highperformance liquid chromatography/tandem mass spectrometry (HPLCMSNS) analysis. Highperformance liquid chromatography/massspectrometry(HPLCMS) may be used if the defined QC 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 MS response of the quantitation ion produced by the same compound in an extracted calibration standard (external standard). 3 Definitions 3.1 Analytical Sample A portion of an extracted Laboratory Sample prepared for analysis. 3.2 Calibration Standard A solution preparedfrom the Working Standard(WS) 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 laboratorythat is extracted and analyzed separatelywith identical procedures.Analysis of DSs comparedto that of the first aliquot give a measure of the precisionassociatedwith laboratory procedures,but not with sample collection, preservation, or storage procedures. 3.4 Field Blank Control Sample (FB) ASTM Type Iwater placed in a sample container in the laboratoryand 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 Determinationof PerfluorinatedCompounds in Water Using SPE and LC/MS. Page 29 of 179 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 "fillto this level" line on the outside of the sample container. The FMS should be spiked at approximately50-150% 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 FMS 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 Iwater to which known quantities of the target analytes are added in the field at the time of sample collection (at an appropriateconcentrationto 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 ASTM Type Iwater 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 concentrationof approx. 100-250 pg/mL or another concentrationto be determined by the project lead. The LCS is extracted and analyzed exactly like a laboratorysample 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 may be determined in several ways, includingsignal-to-noiseratio and statistical calculations. 3.11 Limit of Quantitation (LOQ) The LOQ for a dataset is the lowest concentration(LLOQ) or highest concentration (ULOQ) 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 matrixdependent. 3.12 Matrix Spike (MS) ETS-8-154.1 Page 3 of 17 Determinationof PerfluorinatedCompounds in Water Using SPE and LC/MS. Page 30 of 719 3M ENVIRONMENTAL LABORATORY PROJECT EO5-0210; 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 concentrationsof the analytes in the sample matrix must be determined in a separate aliquot and the measured values in the MS correctedfor backgroundconcentrations. 3.13 Method Blank An aliquot of ASTM Type Iwater that is treated exactly like a laboratorysample including exposure to all glassware, equipment, solvents, and reagents that are used with other laboratory samples. The method blank is used to determine if test substances or other interferences are present in the laboratoryenvironment,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 40 CFR 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 laboratoryfrom 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 concentrationsof volatile toxic compounds. Sample containers should be opened in a hood and handled with gloves to prevent exposure. The laboratory is responsiblefor maintaininga safe work environmentand a current awareness of local regulations regardingthe handlingof the chemicals used in this method. A reference file of material safety data sheets (MSDS) should be available to all personnel involved in these analyses. 4.2 Cautions None 5 Interferences ETS-8-154.1 Page 4 of 17 Determinationof PerfluorinatedCompounds 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 demonstratedto be free from interferencesunder 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 micropipettesor pipettesto aliquot standard solutions is recommendedto 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 demonstrationof equivalent performancethat meets the requirementsof this method is the responsibilityof the laboratoryperformingthe analysis. 6.1 Instrumentation Balance,analytical (display at least 0.0001g), Mettler HPLC/MS/MSor HPLC/MS system, as described in Section IO. 6.2 Supplies and Materials. Sample collection bottles-LDPE (e.g., NalgeneTMn)arrow-mouthbottles 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 Vac 6cc (Ig) tC18 cartridges (part# WAT 036795),Waters. 50mL disposable polypropylenecentrifugetubes, WVR. 15mL disposable polypropylenecentrifugetubes, WVR. Disposablemicropipettes(50-1 OOpL, 100-ZOOpL), Drummond. Class A pipettes and volumetric flasks, various. Hypercarbdrop-in guard column (4mm) (part# 844017400), Keystone. Stand-alone drop-in guard cartridge holder, Keystone. 125mL LDPE narrow-mouth bottles, Nalgene. 2mL clear HPLC vial kit (cat # 5181-3400), AgilenVHewlett Packard. Standard lab equipment (graduated cylinders, disposable tubes, etc.), various. 7 Reaaents and Standards Note: Suppliers and catalog numbers are for illustrative purposes only. Equivalent performance may be achieved using chemicals obtainedfrom other suppliers. Do not use a lesser grade of chemical than those listed. ETS-8-154.1 Page 5 of 17 Determinationof PetfluorinatedCompounds in Water Using SPE and LC/MS. Page 32 of 119 3M ENVIRONMENTAL LABORATORY PROJECT E05-0210; INTERIM REPORT #20 7.1 Chemicals Methanol (MeOH), HPLC grade, JT Baker, Catalog No. JT9093-2. Ammonium Acetate, Reagentgrade, Sigma-Aldrich,Catalog No. A-7330. ASTM Type I Water, prepared in-house. Sodium Thiosulfate, Reagent grade, JT Baker. 7.2 Standards Potassium perfluorooctanesulfonate Pemuorooctanesulfonylamide Ammonium peffluorooctanoate Others as required. 7.3 Reagent Preparation 250mg/mL sodium thiosulfate solution-Dissolve259 of sodium thiosulfate in 100mL reagent water. 40% methanolwash solution - Measure400mL methanol and adjust volume to 1.OL with reagent water. 1OOmM ammonium acetate solution (Analysis)-Weigh 7.71g of ammonium acetate and dissolve in 1.OL of reagentwater. Dilutethe 1OOmM solution by a factor of 50 to make the 2mM ammonium acetate solution used for mobile phase A. Note: Alternative volumes may be prepared as long as the ratios of the solvent to solute ratios are maintained. 7.4 Spiking Stock Standard (SSS) Preparation The following standard preparation procedure 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. IOOpglmLeach PFOS, PFOSA, and POAA SSSs-Weigh out 10mg of analytical standard (correctedfor percent salt and purity-i.e., 10 mg CBFl7SO3K purity 90% = 8.35mg C8F17SOc)and dilute to 100mL with methanol in a 100mL volumetric flask. Transfer to a 125mL LDPE bottle or other suitable container. Prepare a separatesolutionfor each analyte. Solutions may be stored in a refrigerator at 4"*2"C for a maximum periodof 6 months from the date of preparation. 1pg/mL mixedSSS-Add 1.OmLeach of the 1OOpg/mL SSSs (from 7.4.1) to a 1OOmL volumetric flask and bring up to volume with methanol. 0.1 pglmL mixed SSS-Add 1O.OmL of the 1.Opg/mL-mixed solution (from 7.4.2) to a 1OOmL volumetric flask and bring up to volume with methanol. 0.01pglmL mixed SSS-Add 10.0mL of the O.Ipg/mL-mixedsolution (from 7.4.3) to a 100mL volumetric flask and bring up to volume with methanol. Storage Conditions-Store all SSSs in a refrigeratorat 4"*2"C for a maximum period of 6 months from the date of preparation. 7.5 CalibrationStandards 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 Determinationof PerfluorinatedCompounds in Water Using SPE and LC/MS. Page33ofl19 3M ENVIRONMENTAL LABORATORY PROJECT E05-0210; INTERIM REPORT #20 100pglmL each PFOS, PFOSA, and P O A A stock standard solutions-Weigh out 1Omg of analytical standard (corrected for percent salt and purity) and dilute to 1OOmLwith methanolin a 100mL volumetric flask. Transfer to a 125mL LDPE bottle or other suitable container. Prepare a separate solution for each analyte. Store solutions in a refrigerator at 4"?2"C for a maximum period of 6 months from the date of Ipreparation. Ipg/mL Working Standard-Add 'I.OmL each of the IOOpg/mL SS solutions (from 7.5.1)to a 100mL volumetric flask and bring up to volume with methanol. 0.1pglmL Working Standard -Add 10.0mLof the l.O~ig/mLmixed solution (from 7.5.2)to a 100mL volumetric flask and bring up to volume with methanol. 0.01pglmL Working Standard -Add 10.0mL of the O.lpg/mL mixedsolution (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 4"&2"Cfor 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: 0.0 0 40 0.010 100 40 0.010 200 40 0.01 0 400 40 0.10 100 40 0.10 200 40 0.10 300 40 0.10 400 40 1.o 100 40 1.o 400 40 1.o 1000 40 0 25 50 100 250 500 750 1000 2500 10000 25000 The standards are processed through the extraction procedure (Section 1l ) , 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 4"&2"C,for a maximum period of two weeks from the date of preparation 8 Sample Collection and Haridling 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 over time should use refrigerated polypropylene sample containers if possible. Sample bottles should not be rinsed before sarnple 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 05-0210; 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 performedin 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 Water: Open the tap and allow the system to flush untilthe water temperature(15"kIOOC) has stabilized (usually about two minutes). Adjust the flow to about 5OOmUmin and collect samples from the flowing stream. Ground Water: Purge the well of standing water using ai 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. Sample Dechlorination: All samples should be iced or refrigerated at 4"*2"C 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 Time (HT): Results of the timektorage study of all target analytes showed that the three compounds are stable for 14 days in 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 analyzedwithin 30 days of sample collection. If the HT exceeds 14 days, great care is used when evaluatingfield 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 preservethe FB. 8.2 Field Duplicates Collect a Field Duplicate (FD) for every ten ( I O ) samples collected or per each sampling set, 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 saniples, 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 Perfluorinat'ed Compounds in Water Using SPE and LC/MS. Page35ofll9 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 proceduresas used for the samples Provide information on sample collection, preservation, shipment and storage. List applicable holding times. Include sample stability and extract storage requirements. Referencethe 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 unspikedsample 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 employedshould 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 personnelto 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 controVduplicate 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 blanksshould return to 4 0 % 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 kill within & 25% of expected values. If the matrix spikes fail, evaluate the lab control spikes. If the L.CSare within acceptancecriteria there may be matrix issues in the samples,. Disciiss 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 I19 3M ENVIRONMENTAL LABORATORY PROJECT E05-0210; INTERIM REPORT #20 Matrix spike duplicates are prepared periodically to measurethe precision associatedwith 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-rangeof 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 miid-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 preparedat a minimum of 2 levels and in duplicate or triplicate. Recoveryof these samples should be within f 25% of expected values, and the RPD (or RSD) be 5 15%. If recoveries fall outside these limits the samples should be addressed in the final report. 10 Calibration and Standardization 10.1 Instrument Setup Note: In this example, a MicroMass UltimaTMLiquid Chromatography Tandem Mass Spectrometer (LC/MS/MS) is used. Other brands of LC/MS/MSsas 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 illustrativepurposes only. Equivalent performance may be achieved using apparatus aiqd 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. Establishthe LC/MS/MSsystem and operating conditions equivalent to the following: Mass Spec: MicromassUltima (Micromass) Interface: Electrospray (Micromass:l Mode: Electrospray Negative, Multiple Response Monitoring (MRM) Harvard infusion pump (Harvard Instruments), for tuning 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: 1Omm Hypercarb drop-in guard cartridge (Keystone, part # 844017-400) may be attached on-line after the purge valvle and before the sample injector port to trai any residue contaminants that may be in the mobile phase and/or HPLC system. HPLC Column: Genesis C8 (JonesChromatography),2.lmm x 50mm, 4pm ETS-8-154.1 Page 10 of 17 Determination of Perfluorinated Compounds in Water Using SPE and .C/MS. Page 37 of 119 3M ENVIRONMENTAL LABORATORY PROJECT E05-0210; INTERIM REPORT #20 Column Temperature: 35C Injection Volume: 15vL Mobile Phase (A): 2mM Ammonium Acetate in ASTM Type I water (See 7.3.1) Mobile Phase (B): Methanol 60 40 0.3 I 0.4 60 1.o 10 40 0.3 90 0.3 7.0 10 90 0.3 7.5 0 100 0.3 9.0 0 100 0.4 9.5 60 40 0.4 13.5 60 40 0.4 1 14.0 60 40 0.3 Note: Other HPLC: gradients may be used as long as the mettiod criteria are met. It may be necessary to adjust the HPLC gradient in order to optimize instrument performance. Columns with different dimensions (e.g. 2.1mm x 30mm) and columns from different manufacturers(KeystoneBetasil C18 etc.) may be used. Ions Monitored: I PFOA PFOS FOSA 41 3 499 498 169 99 78 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 I ;;I=: ::lo: 1 sensitivity. 0.2-0.4 0.2-0.4 FOSA 0.2-0.4 20-50 30-60 ETS-8-154.1 Page 11 of 17 Determination of Perfluorinated Compounds in Water Using SPE and LCIMS. Page 38 of 119 3M ENVIRONMENTAL LABORATORY PROJECT E05-0210; INTERIM REPORT #20 CapiI lary Hexapole 1 Aperture 1 Hexapole 2 Source Block Temp. Desolvation TemD. 2.6-3.5kV 0.5V 0.2v 0.8V 100-1 50C 250-400 "C LM Res 1 HM Res 1 IEnergy 1 Entrance Exit LM Res 2 HM Res 2 IEnergy 2 Multidier Cone Gas 12.5-1 5.0V 12.5-15.OV 0.7v -2v 1v 11.ov 11.ov 1 .ov 650V 15OL/hr I Gas Cell 3.Oe-3m bar I ETS-8-154.1 Page 12 of 17 Determination of Perfluorinated Compounds in Water Using SPE and LC/MS. Page 39 of I19 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 9 the standard curve should be plotted using a linearfit, wei hted l / x or unweighted. However, the standard curve may also be plotted by quadraticfit (y = a + bx + c), weighted l / x or unweighted, using suitable software. The calibration curves may include but should not be forced through zero. The mathematical method used to calculate the calibration curve should be applied consistently throughout a study. Any change should be thoroughly 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 inaccuracyattributedto linear regressionweighting 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 appropriateto the data or becausethey did not meet the predeterminedacceptance 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 requirementso f f 25% (and k 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 (CCV) are analyzed to venfy the accuracy of the calibration curve. Analyze a mid-range calibration standard, one of the same 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. Calibrationverification injections must be within G 5 % to be considered acceptable. The calibration curve and the last passing CCV will then bracket acceptable samples. Multiple CCV 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. The system suitability injections must have area counts with an RSD of 55% and a retention time RSD of S2% when evaluated independently. 11 Procedures 11.I Extraction Scheme Allow samples to equilibrate to room temperature, Thoroughly mix samples by gently inverting the sample bottle. Measure40mL of sample into 50mL polypropylenecentrifugetubes (Spike the Matrix spikes as required*, replace lid and mix well). Note: * Samples may need to be prescreenedto determine an appropriate matrix spike level (typically 50-1 50% of sample concentration). Alternativelythe samples could be spiked at more than one level, allowing for the inappropriatespike level to be eliminated. ETS-8-154.1 Page 13 of 17 Determinationof PeifluorinatedCompounds in Water Using SPE and LC/MS. Page40of 119 3M ENVIRONMENTAL LABORATORY PROJECT EO5-0210; INTERIM REPORT #20 Condition the C18 SPE cartridges (1g, 6mL) by passing approximately 1OmL methanol followed by approximately 50mL ASTM Type I water (flow rate approximately 2 droplsec). Do not let column run dry. Note: For the following steps, maintain a -Idrop/sec flow rate. Do not allow the column to run dry at any time. Load the analytical sample onto the C18 SPE cartridge. Discard eluate. Ten mL of the 40% methanol in water wash mixture is passed through the C18 SPE cartridge to rinse away potential interferences and then discarded. This step must be omitted if perfluorinated compounds with chain lengths less than C8 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 approximately4.5 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/MS/MS or HPLC/MS. + Note: Samples are concentratedby a factor of eight during the extraction; InitialVol = 40mL Final Vol. = 5mL. Samples are stable at room temperaturefor at least 24 hours. Analytical samples may be stored in a refrigerator at 4"&2"C until analysis. Standardization of C18 SPE columns-If poor recoveriesare observed, it may be necessaryto standardize the C18 SPE columns in the following manner before analyzing samples. Use a standard with an analyte concentrationbetween 1000and 4000 pg/mL. Repeatthe extraction scheme from the beginning up through the eluting with -5mL 100% methanol. After the eluting with -5mL 100% methanol step, collect an additional post-elution fraction by eluting with an additional 5mL of 100% methanol. Analyze both fractions by HPLC/MS/MS 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 (> 5%I), either the wash volume or percentage of MeOH should be decreased. If the post-elutionfraction contains significantstandard (> 5%)I, the target elution volume should be increased. 11.2 Sample Analysis Set up analysis sample queue. Inject the same volume (between 5-25vL) 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 produceacceptable results (e.g. poor MS recoveries)dilute the original sample and re-extract. Calculatethe analytical sample (extract)concentrationfrom the standard cuwe using the following equation: ETS-8-154.1 Page 14 of 17 Determinationof PerfluorinatedCompounds in Water Using SPE and LC/MS. Page41 of119 3M ENVIRONMENTAL LABORATORY PROJECT EO5-0210; INTERIM REPORT #20 Extract Concentration, pg/mL = /Peak area - intercept) (slope) Calculate the percent recovery of the FSCS using the following equation: FSCS % rec. = [FSCS conc., Dcl/mL) x 100 (Conc. added, pg/mL) Calculate the percent recovery of the MSs using the following equation: MS % rec. = (MS conc., - pg/mL Sample conc., pg/mL)x 100 (Conc. added, pg/mL) 13 Method Performance Note: Any method performanceparametersthat are not achieved must be considered in the evaluation of the data. Nonconformanceto any specified parameters must be described and discussed in any reporting of the data. If criteria listed in this method performancesection 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 reportedwhen performancecriteria 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 55% and a retention time RSD of 52% when evaluated independently. 13.2 Quantitation CalibrationCurve: The coefficientof determination(?)value for the calibration curve must be greater than or equal to 0.990. Each point in the curve must be within e 5 % of the theoretical concentrationwith the exception of the LLOQ, which may be within *30%. Demonstrationof Specificity:Specificity is demonstratedby chromatographicretention time (within 3% of standard) and the mass spectral response of unique ions. 13.3 Sensitivity Solvent Blanks and Method Blanks: Solvent and method blank area counts must be < 50% that of the lowest standard used in the calibration curve. Limits of Quantitation (LOQ): The lower LOQ (LLOQ) is the lowest non-zero active standard in the calibration curve; the peak area of the LLOQ must be at least 2X that 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 Determinationof PerfluorinatedCompounds in Water Using SPE and LC/MS. Page42of119 3M ENVIRONMENTAL LABORATORY PROJECT E05-0210; INTERIM REPORT #20 13.4 Accuracy CCV Performance:Calibrationverification injectionsmust be within S?5%to be considered acceptable. The calibration curve and the last passing CCV will then bracket acceptable samples. Multiple CCV levels may be used. Matrix Spikes: Matrix spike percent recoveries must be within f 25% of the spiked concentration. If matrix effects are suspected, evaluate the LCS results to determine if a matrix 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 Reproducibility: Reproducibilityof the method is defined by the results of duplicate or triplicate analysis of samples. A RPD or RSD of S 15% will be considered acceptable. System Suitability: The system suitability injections must have area counts with an RSD of S5% and a retention time RSD of S2% 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 Determinationof Perfluorooctanesulfonate (PFOS), Perfluorooctane sulfonylamide(PFOSA), and Perfluorooctanoate(POAA) in Water", E. Wickremesinhe and J. Flaherty,Study Number 023402,Centre Analytical Laboratories,Inc., State College, Pennsylvania,January 2000. Validation report for the "Methodof Analysis for the Determinationof Perfluorooctanesulfonate (PFOS), Perfluorooctanesulfonylamide(PFOSA), and Perfluorooctanoate(POAA) in Water", E. Wickremesinheand J. Flaherty,Study Number 023402,CentreAnalytical Laboratories, Inc., State College, Pennsylvania. ETS-8-154.1 Page 16 of 17 Determinationof PerfluorinatedCompounds in Water Using SPE and LC/MS. Page43of 119 3M ENVIRONMENTAL LABORATORY PROJECT E05-0210; INTERIM REPORT #20 18 Affected Documents None. 19 Revisions Revision Number 1 Revision Number Updated to the new format. Changed Title. Section 1: States the validationof 3 analytes, removes reference to PA document that's no longer applicable. Section 2: Provided for the extraction of more than the 3 validated analytes, allows the use of a LC/MS system, not only the LS/MS/MS previously mentioned. Section 3: Revised definitions for field matrix spike, field control spike, LLOQ, method blank, and MOL. Section 5: Reworded the interferences, added recommendationto use disposable pipettes. Section 6: Recategorized and pared down. Section 7: Changed storage time to 6 months. Added more calibration points to the table. Section 8:Added statement addressing labeling requirements and spiking procedures. Expanded section 8.8. Section 9: New Section Section 10: Changed some of the parameters in the tables. Allowed for use of different instrumentation. Added information from section 12 of previous version, extensivelyrevised. Section 11 (section 9 in previous version): Clarificationof wash step, stated exact volume of eluate is 5 mL, revised standardizationprocess, removed requirement to use LC/MS/MS. Section 12 (section 13 in previous version: no changes Section 13 (section 14 in previous version): Extensively rewritten. Section 14 (section 15 in previous version): no changes Section 75 (section 76 in previous version): Minor changes to recording requirements. Section 16 (section 17 in previous version): Removed attachment. Section 17 (section 18in previous version).' Removed reference to PA document that no longer applied to this SOP. Section 18: New section. Revision Date 7 ETS-8-154.1 Page 17 of 17 Determinationof PerfluorinatedCompounds in Water Using SPE and LC/MS. Page 44 of 119 3M ENVIRONMENTAL LABORATORY PROJECT E05-0210; INTERIM REPORT #20 ATTACHMENTc: PROTOCOLAND PROTOCOLAMENDMENTS Page45of 119 3M ENVIRONMENTAL LABORATORY PROJECT 050210; INTERIM REPORT #20 Exygen Protocol Number: PO001 131 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 LCIMSIMS for the 3M Decatur Monitoring Program Exygen Protocol Number: PO001 131 Perfoming; Laboratory: Exygen Research 3058 Research Drive State College, PA 16801 Phone: (814) 272-1039 I..- Sponsor Representative: Michael A. Santoro Director of Regulatory Affairs 3M Building 0236-01-B- 10 St. Paul, MN 55144 Phone: (651) 733-6374 ---_ P q y I Cij Page 46 of 119 3M ENVIRONMENTAL LABORATORY PROJECT E05-0210; INTERIM REPORT #20 Exygen Protocol Number: PO001 13 1 DISTRIBUTION: 1) Jaisimha Kesari, Study Director, Weston Solutions 2) John M. Flaherty, Principal Investigator, Exygen Research 3) Michael A. Santoro, SponsorRepresentative, 3M Company 4) Exygen Research Quality Assurance Unit c Page 2 of 65 Page47of 119 3M ENVIRONMENTAL LABORATORY PROJECT 05-0210; INTERIM REPORT #20 Exygen Protocol Number: PO00 1 131 PROTOCOL APPROVAL Study Title: Analysis of Perfluorobutanesulfonate(PFBS), Perfluorohexanesulfonate (PFHS), and Perfluorooctanesulfonate(PFOS) in Water, Soil, Sediment, Fish, Clams, Vegetation, Small Mammal Livers and Small Mammal Serum Using LC/MS/MS for the 3M Decatur Monitoring Program Exygen Protocol Number: PO001131 APPROVALS Weston Solutions Michael A. Sfitoro, Sponsor Representative 3M Comparfy John M. Flaherty, ?rincipal Investigator / Exygen Research Exygen R e s e w r, Facility Management -* . /$I& ,flAh Date Date Ewgen Research Puge 3 q j 7 . Page 48 of 1 19 3M ENVIRONMENTAL LABORATORY PROJECT EO5-0210; INTERIM REPORT #20 Exygen Protocol Number: PO00 1131 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 ......................................................... Y SAMPLE PROCUREMENT, RECEIPT AND RETENTION ......................................................................... 8 SAMPLE IDENTIFICATION .......................................................................................................................... 9 ANALYTICAL PROCEDURE SUMMARY................................................................................................... 9 VERIFICATION OF ANALYTICAL PROCEDURE..................................................................................... - 9 METHOD FOR CONTROL OF BIAS .........................................................................................................1.1... STATISTICAL METHODS ............................................................................................................................1. 1 GLP STATEMENT .......................................................................................................................................... 11 REPORT ........................................................................................................................................................... 1 1 SAFETY AND HEALTH ................................................................................................................................. 12 AMENDMENTS TO PROTOCOL .................................................................................................................. 1 3 DATA RECORDKEEPING ............................................................................................................................ 13 QUALITY ASSURANCE ................................................................................................................................ 14 RETENTION OF DATA AND ARCHIVING ................................................................................................. 14 APPENDIX I, ANALYTICAL METHODS..................................................................................................... 15 P q e 4 of65 Page 49 of I19 .. 3M ENVIRONMENTAL LABORATORY PROJECT E050210; INTERIM REPORT #20 Exygen Protocol Number: PO001 13 1 INTRODUCTION The purpose of this study is to perform analysis for perfluorobutanesulfonate (PFBS), perfluorohexanesulfonate (PFHS) and perfluorooctanesulfoiiate (PFOS) in water, soil, sediment, fish, clams, vegetation, small mammal livers and small mammal serum using LC/MS/MS for the 3M Decatur Monitoring Program. The study will be audited for compliance with EPA TSCA Good Laboratory Practice Standards 40 CFR 792 by the Quality Assurance Unit of Exygen Research. TEST MATERIALS The test materials are perfluorobutanesulfonate (PFBS), perfluorohexanesulfonate (PFHS) and perfluorooctanesulfonate (PFOS) and are all supplied by 3M. PFBS .- Chemical Name: Perfluorobutanesulfonate Molecular Weight: 338 supplied as the potassium salt (C4F9S03-K') Lot Number: 101 Purity: 96.7% Transitions Monitored: 299 -+ 99 Structure: F F PFHS Chemical Name: Perfluorohexanesulfonate Molecular Weight: 438 supplied as the potassium salt ( C ~ F I ~ S O ~ K ' ) Lot Number: SE036 Purity: 98.6% Transitions Monitored: 399 3 80 Structure: F F F Page 5 qf25 Page 50 of 119 3M ENVIRONMENTAL LABORATORY PROJECT E05-0210; INTERIM REPORT #20 Exygen Protocol Number: PO00 1 13 I c PFOS Chemical Name: Perfluorooctanesulfonate Molecular Weight: 538 supplied as the potassium salt (CgFi,SO,-K+) Lot Number: 217 Purity: 86.9% Transitions Monitored: 499 + 99 Structure: FFFF 3 OBJECTIVE The purpose of this study is to perform analysis for perfluorobutanesulfonate (PFBS), perfluorohexanesulfonate (PFHS) and perfluorooctanesulfonate (PFOS) in water, soil, sediment, fish, clams, vegetation, small mammal livers and small mammal serum for the 3M Decatur Monitoring Program using the current versions of the following Exygen analytical methods: VOOOl780: VOOO 1781: VOOOl782: V0001783: V0001784: VOOOl785: V0001786: "Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Water by LC/MS/MS" "Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Soil by LC/MS/MS" "Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Sediment by LC/MS/MS" "Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Fish and Clams by LC/MS/MS" "Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Vegetation by LC/MS/MS" "Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Small Mammal Liver by LC/MS/MS" "Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Small Mammal Serum by LC/MS/MS" TESTING FACILITY Exygen Research 3058 Research Drive State College, PA 16801 Phone: (814) 272-1039 Page 6of 65 Page 51 of 119 3M ENVIRONMENTAL LABORATORY PROJECT 05-0210; INTERIM REPORT #20 Exygen Protocol Number: PO00113 1 ry STUDY DIRECTOR Jaisimha Kesari P.E., DEE Weston Solutions, Inc. 1400 Weston Way West Chester, PA 19380 Phone: (610) 701-3761 Fax: (610) 701-7401 j .kesari@westonsolutions.com SPONSOR REPRESENTATIVE Michael A. Santoro 3M Company Director of RegulatoryAffairs 3M Building 0236-01-B-10 St. Paul, MN 55144 Phone: (651) 733-6374 - PRINCIPAL INVESTIGATOR John M. Flaherty Exygen Research 3058 Research Drive State College, PA 16801 Phone: (814) 272-1039 john. flaherty@exygen.corn PROPOSED EXPERIMENTAL START AND TERMINATION DATES It is proposed that the analytical portion of this study be conducted from October 01, 2004 to December 31, 2005. The actual experimental start and termination dates will be included in the final report. Puge 7 oj'6S Page 52 of 119 3M ENVIRONMENTAL LABORATORY PROJECT 05-0210; INTERIM REPORT #20 Exygen Protocol Number: PO001 131 14 IDENTIFICATION AND JUSTIFICATION OF THE TEST SYSTEM The following are the test systems for this study: Water (groundwater and surface water) Soil . Sediment Fish Clams Vegetation 0 Small Mammal Liver Small Mammal Serum The samples will be collected by Weston Solutions. The control samples will be purchased and prepared by the testing facility. Purchase and processing details for the control samples will be included in the final report associated with this study. The test systems were chosen to access the environmental impact of PFBS. PFHS and PFOS in the Decatur, Alabama area. SAMPLE PROCUREMENT, RECEIPT AND RETENTION Water, soil, sediment, fish, clam, vegetation, small mammal liver and small mammal serum samples will be received at Exygen directly from Weston Solutions. The details of sample procurement for this study are outlined in the 3M work plan entitled "Phase 2 Work Plan for Sampling Environmental Media." The number and types of samples collected will vary depending availability in the field. The total number of samples received and analyzed for each matrix will be documented in the final report associated with this study. Water, soil, and sediment samples will be used as received without further processing at Exygen. These samples will be stored refrigerated at 2OC-8"C. Fish, clam, vegetation and small mammal liver samples will be processed according to the appropriate analytical method (see Appendix I). These samples will be stored frozen at I -10C. Small mammal whole blood samples will be centrifuged in the field at the time of collection and the serum fraction will be used for the study. Small mammal serum will be stored frozen at 5 -10C. The receipt and processing of the samples will be documented in the final report and raw data associated with the study. Page 8 o j 6 5 Page 53 of I 19 3M ENVIRONMENTAL LABORATORY PROJECT 05-0210; INTERIM REPORT #20 Exygen Protocol Number: PO001 131 SAMPLE IDENTIFICATION Prior to analysis, each sample will be assigned a laboratory sample reference number. The reference number will be unique and will distinguish each laboratory sample that is processed throughout the analytical procedure. Chromatographic data will be identified by the laboratory sample reference number. Sample storage conditions and locations will be documented throughout the study. ANALYTICAL PROCEDURE SUMMARY References: VOOOl780: VOOOl781: VOOOl782: VOOOl783: VOOOl784: VOOOl785: VOOOl786: "Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Water by LC/MS/MS" "Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Soil by LC/MS/MS" "Method o f Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Sediment by LC/MS/MS" "Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Fish and Clams by LC/MS/MS" "Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Vegetation by LCMSMS" "Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Small Mammal Liver by LC/MS/MS" "Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Small Mammal Serum by LC/MS/MS" The above methods use analytical conditions capable of separating the isomers of PFBS, PFHS and PFOS. The final report will include the isomers summed into total PFBS, total PFHS, and total PFOS found. VERIFICATION OF ANALYTICAL PROCEDURE A laboratory control sample will be used for the preparation of fortified control samples. The test substance will be made into solutions as per the method, and added to the matrices via a micropipette. For water sampling, Exygen will supply one bottle per sample collected. The .- bottles will be 500 mL precleaned Sci/Spec Premier wide mouth HDPE bottles. These bottles have been routinely used for fluorochemical sample Page 54 of 119 3M ENVIRONMENTAL LABORATORY PROJECT 05-0210; INTERIM REPORT #20 Exygen Protocol Number: PO00 1 13 1 collection at the testing facility and have been shown to be fiee of PFBS, PFHS and PFOS. Samples will be added to each container to a volumetric fill line at 200 mL. A field duplicate, a low field spike and a high field spike of each sample will be collected. The low and high field spike bottles will contain PFBS, PFHS and PFOS as well as perfluorooctanoic acid (PFOA) and 1.2-13C perfluorooctanoic acid (I3C PFOA). PFOA and I3C PFOA are included in the solutions used to spike the samples. The results for PFOA and I3C PFOA will not be reported in this study. Exygen will supply one field blank (control water) and two field blank spikes (control water fortified with PFBS, PFHS and PFOS at a low and high level) for every twenty samples collected. At the testing facility, each water sample (excluding field duplicates and field spikes) will be extracted in duplicate and will also be fortified at a low and high concentration with PFBS, PFHS and PFOS and processed through the described procedure to determine method accuracy and to check for bias. For soil, sediment, clams, and vegetation, Exygen will supply one 500 mL precleaned Sci/Spec Premier wide mouth HDPE bottle per sample collected or a zip-seal bag. All containershags used for sample collection will be shipped to the sample location. Samples will be added to each container or bag in the field. At the testing facility, each sample will be extracted in duplicate and will also be fortified at a known concentration with PFBS, PFHS and PFOS at both a low and high level and processed through the described procedure to determine method accuracy and to check for bias. For small mammal liver, Exygen will supply a 50 mL polypropylene centrifuge tube. For small mammal serum, Exygen will supply a collection kit for each sample containing serum separator tubes (red top), vacutainers, needle holders and needles, transfer pipettes, and polypropylene tubes. At the testing facility, each liver and serum sample will be extracted in duplicate and will also be fortified at a known concentration with PFBS, PFHS and PFOS at both a low and high level and processed through the described procedure to determine method accuracy and to check for bias. Low and hi& stikine levels for each matrix are defined below: Matrix Low Spiking Level High Spiking Level Water 500 ng/L 5000 ng/L ,- Page IO of65 Page 55 of 119 --- 3M ENVIRONMENTAL LABORATORY PROJECT EO5-0210; INTERIM REPORT #20 Exygen Protocol Number: PO001131 Recoveries are anticipated to be between 70% and 130% of the fortified levels; however, the exact precision and accuracy will be determined by the analysis of the quality control samples described above. A statement of accuracy will be included in the final report. METHOD FOR CONTROL OF BIAS Control of bias will be addressed by taking representative sub-samples from a homogeneous mixture of each matrix from untreated control samples, and by analyzing at least two levels of fortifications. STATISTICAL METHODS Statistics will be limited to those specified in the subject methods and to the calculation of average recoveries, as applicable. GLP STATEMENT All aspects of this study shall be performed and reported in compliance with EPA TSCA Good Laboratory Practice Standards 40 CFR 792. The filial report or data package (supplied to the Sponsor) shall contain a statement that the study was conducted in compliance with current and applicable GLP standards and will outline any deviations in the study from those standards. This statement will be signed by the Study Director and Sponsor Representative. REPORT A final report will be prepared by the principal investigator or their designee at the conclusion of the study. The report will include, but will not be limited to, the following: 0 The name and address of the Study Director, Sponsor Representative, and of the testing facility. A statement of GLP compliance (any related documentation, such as chain-of-custody records, must be in the study records). Page 56 of 1 19 3M ENVIRONMENTAL LABORATORY PROJECT E05-0210; INTERIM REPORT #20 Exygen Protocol Number: PO00 1 13 1 0 The signed and dated statement by the Exygen Research Quality Assurance Unit regarding dates of study inspections and dates findings were reported to the Study Director and Management. 0 A description of the exact analytical conditions employed in the study. If the subject method was followed exactly, it is necessary to include only a copy of the analytical method. Any modifications to this method will be incorporated into the report. If the method is photo-reduced, the project number and page number must be included on each page. 0 Descriptionof the instrumentationused and operating conditions. 0 All results fiom all sets analyzed. Control and fortified samples will be identified and the data table will include sample number and fortification level. 0 Representative chromatograms for each analyte in each matrix, including chromatogramsof a standard and a control sample, and a chromatogram at a fortification level. The location of the analyte peaks will be clearly identified in all chromatograms. All circumstances that may have affected the quality or integrity of the data will be documented in the report. 0 Locations where raw data and the final report are to be archived. 0 Additions or corrections to the final report shall be in the form of an amendment signed by the Study Director. The amendment shall clearly identify that part of the report that is being altered and the reasons for the alterations. The amendment will be signed and dated by the Study Director and the SponsorRepresentative. All applicable requirements for reporting of study results as per 40 CFR 792.185. SAFETY AND HEALTH 0 Laboratorypersonnel will practice good sanitation and health habits. 0 Every reasonable precaution shall be taken to prevent inadvertent exposure of personnel and the environment to the test or reference substance(s). Page 12 of 65 Page 57 of f 79 3M ENVIRONMENTAL LABORATORY PROJECT E05-0210; INTERIM REPORT #20 Exygen Protocol Number: PO001131 AMENDMENTS TO PROTOCOL All significant changes to the analytical protocol outlined here will be expressed in writing, signed and dated by the Study Director and Sponsor Representative. Amendments usually will be issued prior to initiation of study plan change. However, when a change is required without sufficient time for the issue of a written amendment, that change may be effected verbally with supporting documentation signed and dated by the Study Director and followed with a written amendment as soon as possible. In this case, the effective date of the written amendment will be the date of the documented change. Copies of the signed amendments will be appended to all distributed study plan copies. The original amendment will be maintained with the original study plan. Any deviations from the study plan or from the analytical method as provided will be documented and reported promptly to the Sponsor Representative. DATA RECORD KEEPING Records to be maintained include the following (as appropriate): Sample tracking sheet@) Sample receipt records, storage history, and chains of custody History and preparation of standards (stock, fortification, calibration) Description of any modifications to the method Instrument run sheets, bench-sheets or logs Analytical data tables All chromatographic and instrumental conditions Sample extraction and analysis dates A complete listing of study personnel, signatures and initials Chronological presentation of all study correspondence Any other documentation necessary for the reconstruction of the study Chromatograms-All chromatograms will contain the following: Sample identification, injection date, arrow or other indication of the area of interest, and injection number corresponding to the run. Additionally, fortifications will include the amount of analyte added and the sample number of the sample that was fortified. ..-. Analytical standard chromatograms will additionally include the concentration (e.g., pg/mL). Page 58 of I I 9 3M ENVIRONMENTAL LABORATORY PROJECT 05-0210; INTERIM REPORT #20 Exygen Protocol Number: PO00113 1 .0 As part of the documentationthe following sheets will be included in each analytical set: a run sheet listing the samples to be run in the set, and an instrument conditions sheet describing the instrument type and operating conditions. QUALITY ASSURANCE The QA Unit of Exygen Research will inspect the study at intervals adequate to assure compliance with GLP's, and will report the findings of audits to the Study Director, Exygen Management, and the Sponsor Representative. RETENTION OF DATA AND ARCHIVING All hard copy raw data, including, but not limited to, the original chromatograms, worksheets, correspondence, and results shall be included with the data package submitted to the Study Director. These will be archived with the original study plan, amendments, final report, and all pertinent information fiom the Sponsor. The testing facility shall keep all electronic raw data and any instrument, equipment, and storage logs for the period of time specified in 40 CFR 792.195. An exact copy of the materials submitted to the study director will also be kept at Exygen Research. Exygen will obtain permission from the study director before discarding or returning samples. Page I 4 o f 6 j Page 59 of 779 . 3M ENVIRONMENTAL LABORATORY PROJECT 05-0210; INTERIM REPORT #20 Exygen Protocol Number: PO001 131 APPENDIX I ANALYTICAL METHODS VOOOl780: "Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Water by LCMSMS" VOOOl781: "Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Soil by LC/MS/MS" VOOOl782: "Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Sediment by LC/MS/MS" VOOOl783: "Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Fish and Clams by LC/MS/MS" '- VOOOl784: "Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Vegetation by LCMS/MS" VOOOl785: "Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Small Mammal Liver by LC/MS/MS" VOOOl786: "Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Small Mammal Serum by LC/MS/MS" c .. Page 15 of65 Page 60 of 119 .I .- 3M ENVIRONMENTAL LABORATORY PROJECT EO5-0210; INTERIM REPORT #20 Exygen Protocol Number: PO001 13 1 ANALYTICAL METHOD Method Number: VOOOl780 Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA)in Water by LC/MS/MS Analytical TestingFacility: Exygen Research 3058 Research Drive State College, PA 16801 Approved By: Paul Connolly Technical Leader, LC-MS,Exygen Research ' Vice President,Operations,ExygenResearch --- Total Pages: 7 Page 16 of 65 Page 61 of 119 3M ENVIRONMENTAL LABORATORY PROJECT E05-0210; INTERIM REPORT #20 Exygen Protocol Number: PO00 1 131 Exygcn Research Method Number VOOO 1780 1 i ANALYTICAL.METHOD Method of Analysis for the Determinationof Perfluorooctanoic Acid (PFOA) in Water by LC/MS/MS 1.o 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 SpectrometricDetector (LC/MS/MS)in water. 2.0 Safety 2.1 Always observe safe laboratorypractices. 2.2 Consult the appropriateMSDS 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 4.3 Ammonium Acetate A.C.S. Reagent Grade 4.4 PerfluorooctanoicAcid - 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 5-100 pL connected to a tandem Mass Spectrometer (LC/MS/MS). 5.2 A device to collcct raw data for peak integration and quantitation. 5.3 Analytical balance capable of reading to O.OOOO1 g. 5.4 50 mL disposablepolypropylenecentrifuge tubes. 5.5 15 mL disposablepolypropylenecentrifuge tubes. 5.6 Disposable micropipets(50- IOOuL, 100-2OOuL). 5.7 125-mL LDPE narrow-mouthbottles. 5.8 2 mL clear HPLC vial kit. 5.9 Disposable pipettes. 5.10 Autopipenes (100-10oO pL and 10-100 pL), with disposable tips. 5.1 1 Waters Sep Pak Vac 6 cc (lg) tC18 SPE cartridges. Page J 7 of65 Page 62 of 119 3M ENVIRONMENTAL LABORATORY PROJECT EO5-0210; INTERIM REPORT #20 Exygen Protocol Number: PO001 131 Exygen Research Method Number VOOO 1780 I 1 AhALYTICAL ,METHOD Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Water by LC/MS/MS 5.12 SPE vacuum manifold. 5.13 Centrifbge 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. 511 (PM: 82505-052130) 6.2 Temperature: 3OoC 6.3 Mobile Phase (A) : 2 mM Ammonium Acetate in Water 6.4 Mobile Phase (B) : Methanol 6.5 Gradient Program: Time (min) %A 0.0 65 1 .o 65 8.0 25 20.0 25 22.5 65 Flow Rate (mUmin) 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 FL). 6.7 Quantitation: Peak Area - external standard calibration curve. 6.8 Run Time: 23 minutes. The above conditions an intended as a guide and may be changed in order to optimize the HPLC system. 7.0 MSMS 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 IO 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 y of ammonium acetate to 1000 mL of water. Alternate volumes may be prepared. - Page -- 3 -. - 01' Page 18 of 65 Page 63 of I19 3M ENVIRONMENTAL LABORATORY PROJECT E05-0210; INTERIM REPORT #20 Exygen Protocol Number: PO00113I Exwen Research Method Number VOOOl780 I ANALYTICAL METHOD I 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 pB/mL of PFOA by weighing 10 nis of analytical standard (corrected for punty) and dilute to 100 rnL with methanol in a 125-mLLDPE bottle. 9.1.2 A 10 pg/mL fortification solution of PFOA is prepared by bringing 10 mL of the 100 &nL 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 p@mL solution to a final volume of 100 with methanol i n a 125 mL LDPE bottle. 9.1.4 A 0.1 pg/mL fortification solution of PFOA is prepared by bringing I O mL of the 1.0 pg/mL solution to a final volume of 100 with methanol in a 12s 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 pB/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 h m the date of preparation. 9.2 Standard Calibration Solutions 9.2.1 LC/MS/MS calibration standards are prepared in HPLC water. The calibration standards are processed through the extraction procedure, identical to samples. 9.2.2 The following is a typical example: additional concentrations may be prepared as needed. Final Concentration Fortification Volume of Concentrationof Calibration of Fortification Volume Fortified Control Calibration Standard ID Solution (ppb) (pL) Sample (mL) Standard(ppt)' (example) 0 0 40 0 XCmmddyy -0 10 100 40 10 200 40 10 400 40 25 XCmmddyy-1 50 XCmmddyy -2 100 XCmmdd yy -3 100 100 40 250 XCmmddyy -4 IO0 200 40 500 XCmmddyy-5 100 400 40 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 19 of 65 Page 64 of I 19 3M ENVIRONMENTAL LABORATORY PROJECT 050210; INTERIM REPORT #20 Exygen Protocol Number: PO001 131 1 ' ExygenResearch Method Number VOOOl780 ANALYTICAL METHOD Method of Analysis for the Determinationof Pertluorooctanoic Acid (PFOA)in Water by LCMSMS 9.2.3 A zero standard solution (reagent blank) wt be preDare4 with each set of standards extracted. 9.2.4 Store all extracted calibration standards in 15-mL polypropylene tubes at 2OC to 6OC, up to two weeks. 9.2.5 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) arid two rugcnl 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 assuranceplan for this project. 11.O Sample Extraction 11.1 Measure 40 mL of sample or a portion of sample diluted to 40 mL with water into 50 mL polypropylenecentrifuge tubes (fortify as needed, replace lid arid mix well). 11.2 Condition the Clg SPE cartridges (1 g, 6 rnL) by passing 10 rnL methanol followedby 5 mL of HPLC water (- 2 dropkc). Do not let column run dry 11.3 Load sample on conditionedCISSPE cartridge. Discard eluate. ,- 11.4 Elute with -5 mL 100% methanol. Collect 5 mL of eluate into graduated 15mL polypropylenecentrifugetubes (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 Standardsof PFOA correspondingto at least five or more concentration levels must be included in an analytical set. 12.3 An entire set of extracted caljbratjon standards must be included at the beginning and at the end of a sample set. Extracted standards must hc interspersedbetween every 5-10 samples. As an alternative. an entire set o f extracted calibration standards may be injected at the beginning of a sei followed by extracted calibration standards interspersed every 5- 10 samples (to account for a second set of extracted standards). In either case, extracted calibrationstandardsmust 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 20 01'65 Page 65 of 119 3M ENVIRONMENTAL LABORATORY PROJECT E05-0210; INTERIM REPORT #20 Exygen Protocol Number: PO001131 Exygen Research Method Number VOOO I780 I 1 Ah'ALYTICALMETHOD 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 thai exceed standard responses should be fiuther diluted and reanalyzed. 13.0 Acceptance Criteria 13.1 Chromatogram must show a peak of a daughter ion at 369 a m u from a parent of 413 amu. The 413 amu parent corresponds to the PFOA anion. while the. daughter ion (369m u ) 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 coeficicnt (R) for calibration curves generated must be 20.992 (I 20.9'85). 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 f 4 % within an analytical run. Ifretention time drift exceeds this limit within an analytical run then the set must be reanalyzed. 14.0 Calculations 14.1 Use the following equation to calculate 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 - intercent)- x D F slope DF = factor by which the final volume was diluted, if necessary. Page 6 01' Page 21 of 65 Page 66 of 1 19 c. 3M ENVIRONMENTAL LABORATORY PROJECT E05-0210; INTERIM REPORT #20 Exygen Protocol Number: PO00 1 131 Exygen Rcoeuch Method Number VOW I780 1 i AULYTICAL METHOD Method of Analysis for the Determinationof PerfluorooctanoicAcid (PFOA) in Water by LC/MS/MS 14.2 For samples fortified with known amounts of PFOA prior to extraction. usc the following equation to calculate the percent recovery. Recovery (%) = I - (n#L)l totalanalytefound (ne/L) analytefound in control oo analyteadded (ng/L) Page 7 o f 7 Page 22 01'65 Page 67 of 1 19 3M ENVIRONMENTAL LABORATORY PROJECT E050210; INTERIM REPORT #20 Exygen Protocol Number: PO001 131 ANALYTICAL METHOD Method Number: V000178 1 Method of Analysisfor the Determinationof Perfluorooctanoic Acid (PFOA)in Soil by LC/MS/MS Analytical TestingFacility: Exygen Research 3058 Research Drive State College, PA 16801 Approved By: Paul Connolly \ Technical Leader, LC-MS,Exygen Research I o Ju, 4 Date /Vice Presidek, Operations,Exygen Research c Total Pages: 7 Page 23 of65 Page 68 of 119 . I 3M ENVIRONMENTAL LABORATORY PROJECT 050210; INTERIM REPORT #20 Exygen Protocol Number: PO001131 .- Exvnen Rerearch Method Number VOOO I78 I ANALYTICAL METHOD 1 1 Method of Analysis for the Determinationof Perfluorooctanoic Acid (PFOA) in Soil by LCiMSMS 1.o 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 SpectrometricDetector (LC/MS/MS)in soil. 2.0 Safety 2.1 Always observe safe laboratorypractices. 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 samplesmust be thoroughlymixed before being sampled for extraction. 3.5 Sample collection procedures will be specified in the sampling plan for this project. 4.0 Reagents and Standards - 4.1 Water HPLC grade - 4.2 Methanol HPLC ~ o d e - 4.3 AmmoniumAcetate A.C.S. Reagent Grade 4.4 PerfluorooctanoicAcid - Sigma-Aldrich 5.0 Instrument and Equipment 5.1 A high performance liquid chromatograph capable of pumping up to I 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 disposablepolypropylenecentrifuge tubes. 5.5 15 mL disposablepolypropylenecentrihge tubes. 5.6 Disposablemicropipets(50- 1OOuL. 100-2OOuL). 5.7 125-mL LDPE narrow-mouthbottles. 5.8 2 m L clear HPLC vial kit. 5.9 Disposablepipettes. 5.10 Autopipettes(100-1OOO pL and 10-100 pL), with disposable tips. 5.11 Waters Sep Pak Vac 6 cc (lg) tC18 SPEcartridges. 5.12 SPE vacuum manifold. 5.13 Ultrasonicbath. Page 24 oj'6S Page 69 of 119 3M ENVIRONMENTAL LABORATORY PROJECT E05-0210: INTERIM REPORT #20 Exygen Protocol Number: PO00113 1 Exvnen Research Method Number VOOO 178 I J ANALYTICAL METHOD Method of Analysis for the Detemination of Perfluorooctanoic Acid CPFOA) in Soil by LCMSMS 5.14 Wnst-action shaker. 5.15 Centrifuge capable of spinning 50 mL polypropylene tubes at 5000 rprn. 6.0 Chromatographic System 6.1 Analytical Column: Fluophase RP (Keystone Scientific), 2.1 mm x 50 mm. S i t (PM: 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: Iirdm& !&A 0.0 65 1 .o 65 8 .o 25 20.0 25 22.5 65 a 35 35 75 75 35 Flow Rate [mumid 0.3 0.3 0.3 0.3 0.3 - 6.6 Injection Volume: 15 pL (can be increased to as much as 50 pL). 6.7 Quantitation: Peak Area - extcmal standard calibration curve. 6.8 Run Time: 23 minutes. The above conditions am intended 88 a guide and may be changed in order to optimize the HFW system. 7.0 MSMS System 7.1 Mode: Electrospray Negative MRM mode, monitoring 413 + 369 miz 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 15 of6S Page 70 of 1I 9 3M ENVIRONMENTAL LABORATORY PROJECT E05-0210; INTERIM REPORT #20 Exygen Protocol Number: PO001131 Exygen Research Method Number VOOO 178 I I 1 ~~ ANALYTICAL METHOD Method of Analysis for the Determinationof PerfluorooctanoicAcid (PFOA) in Soil by LC/MS/MS 9.0 Standard Preparation 9.1 Standard Stock/FortificationSolution 9.1.1 Prepare a stock solution of -100 pg/mL of PFOA by weighing 10 my 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 I O mL of the 100 &mL solution to a final volume of 100 with nieth:inol in a 125 mL LDPE bottle. 9.I .3 A 1.0 p@mL fortification solution of PFOA is prepared by bringing I O mL of the 10 p@mL solution to a final volume of 100 with mrrhanol 111 a 125 mL LDPE bottle. 9.1.4 A 0.1 p@mLfortificationsolution of PFOA is prepared by bringing I O mL of $he 1.0 pe/mL solution to a final volume of 100 with methanol in a 125 mL LDPE bottle. 9.1.5 A 0.01 pg/mL fortificationsolution of PFOA is prepared by bringing 10 mL of the 0.1 pg/mL solution to a final volume of 100 with methanolin a 125mL LDPE bottle. 9.1.6 The stock and fortificationsolutions are to be stored in a refrigeratora1 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 HPLC water. Thc calibration standards are processed through the extraction procedure, identical to samples. 9.2.2 The followingis a typical example: additional concentrations may be prepared as needed. Final Concentration o_ f F..o.rtification Solution (ppb) 0 Fortification Volume of Volume Fortified Control (k L) Sample (mL) 0 40 Concentration of Calibration Standard (ppt)* 0 Calibration Standard ID (example!__ XCmmddy y-O 10 100 40 10 200 40 25 XCmmddyy-I 50 XCmmddyy-2 10 400 40 100 XCmmddyy-3 100 100 40 250 XCmmddy y -4 100 200 40 500 XCmmddyy-5 100 400 40 1000 XCmmddyy -6 The extractedconcentration of the calibrationstandard is equal to 8x its initial concentration, due to the concentrationof the standardduring the extraction(SPE). XC = extracted calibrationstandard. Page 4 o f 7 Page 26 4 ' 6 5 Page 71 of 119 - - 3M ENVRONMENTAL LABORATORY PROJECT 05-0210; INTERIM REPORT #20 Exygen Protocol Number: PO001131 I I ExygenResearch Method Number VOOO I78I ANALYTICAL METHOD Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA)in Soil by LCIMSMS 9.2.3 A zero standard solution (reagent blank) must be preDared with each set of standards extracted. 9.2.4 Store all extracted calibration standards in 15-mL polypropylene lubes at 2OC to 6OC, up to two weeks. 9.2.5 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.O 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 -1 5 minutes. 11.3 Transfer the tubes to an ultrasonic bath and sonicate for -15 minutes. 11.4 Bring the volume up to 40 m L with water in the 50 mL polypropylene centrifuge tube. 11.5 Centrifuge for -10 minutes at -3000 rpm. 11.6 Condition the CI: SPE cartridges (1 g, 6 mL) by passing 10 mL methanol followed by 5 mL of HPLC water (- 2 dropkc). Do not let column run dry 11.7 Load (decant) the sample on the conditioned C I ESPE cartridge. Discard eluate. 11.8 Elute with -5 mL 100% methanol. Collect 5 mL of eluate into grdduated I5 mL polypropylene centrifuge tubes (final volume = 5 mL). 11.9 Analyze samples using electrospray LC/MSiMS. 12.0 Chromatography 12.1 Inject the same amount of each standard, sample and fortified sample into the LCIMSIMS 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 a1 [he beginning and at the end of a sample set. Extracted standards must be interspersedbetween every 5-10 samples. As an alternative. an entire set o f Page 5 of7 Poge 27 Cfns Page 720f 119 3M ENVIRONMENTAL LABORATORY PROJECT E05-0210; INTERIM REPORT #20 Exygen Protocol Number:PO001131 Exygcn Research Method Number VOW I78I I I ANALYTICALMETHOD 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 sei followed by extracted calibration standards interspersed every 5- IO 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 I/x weighting of peak area versus calibration standard concentration using MassLynx 3.3 (or equivalcniI 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 fiom a parent of 413 m u . 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 70-130o/u 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 70130% should be evaluated by the analyst to determine if re-extraction is warranted. 13.4 Any calibration standard found to be a statistical outlier by using the Huge Error Test, may be excluded from the calculation of the calibration 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 niust be 20.992 (R' 20.985). If calibration results fall outside these limits, ilicii appropriate steps must be taken to adjust instrument operation, and thc standards or the relevant set of samples should be reanalyzed. 13.6 Retention times between standards and samples must not drift more that1 i 4 % within an analytical run. If retention time drift exceeds this limit within an analytical runthen the set must be reanalyzed. Page 6 of 7 -.. .- Page 28 of65 Page 73 of 119 3M ENVIRONMENTAL LABORATORY PROJECT E05-0210; INTERIM REPORT #20 Exygen Protocol Number: PO001 13 1 c Exvnen Research Method Number VOOO I78I ~ AYALYTICAL METHOD I Method of Analysis for the Determinationof Perfluorooctanoic Acid (PFOA)in Soil by LC/MS/MS 14.0 Calculations 14.1 Use the following equation to calculate the amount of PFOA found (in n g L , based on peak area) using the standard curve (linear regression parameters) generatedby the Mass Lynx s o h a r e program: PFOA found (n@) =p e a k area - interceDQ 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 followingequationto calculate the percent recovery. Recovery (%) * I total analytefound (ng/L) - ~ a l y tfeoundin control (ng/L)] analyteadded (ng/L) 14.3 Use the following equation to convert the amount of PFOA found in ng/L to - ng43 (PPb). PFOA found (ppb) ( P F O ~ t r a c t e (0d.04LU sample weight (5 g) 14.4 Use the following equation to calculate the amount of PFOA found in pph based on dry weight. PFOA found (ppb) dry weight = PFOA found (ppb) x [100%/ total solids(%)] Page 7 of 7 _a- Page 74 of 119 3M ENVIRONMENTAL LABORATORY PROJECT 05-0210; INTERIM REPORT #20 Exygen Protocol Number: PO001131 e- - ANALYTICAL METHOD Method Number: V0001782 Method of Analysis for the Determinltion of Perflucrrooctanoic Acid (PFOA) in Sediment by LC/MS/MS Analytical TestingFacility: Exygen Research 3058 Research Drive State College, PA 16801 Approved By: Paul Connolly Technical Leader, LC-MS. Exygm Research 1 Vice President,Operations,ExygenResearch L Date TotalPages: 7 Page 30 of 65 Page 75 of 119 3M ENVIRONMENTAL LABORATORY PROJECT E05-0210;INTERIM REPORT #20 Exygen Protocol Number: PO001 13 1 Exygcn Rereucb Method Number VOOO I782 1 ANALYTICAL METHOD Method of Analysis for the Determinationof Perfluorooctanoic Acid (PFOA)in Sediment by LCIMSIMS -- 1.o 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 SpectrometricDetector(LC/MS/MS) in sediment. 2.0 Safety 2.1 Always observe safe laboratorypractices. 2.2 Consultthe appropriateMSDS 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 Methi11101 HPLC @e 4.3 Acetic Acid - Reagent grade - 4.4 Ammonium Acetate A.C.S. Reagent Grade 4.5 PerfluorooctanoicAcid - Sigma-Aldnch 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 connectedto a tandem Mass Spectrometer (LC/MS/MS). 5.2 A device to collect raw data for peak integration and quantitation. 5.3 Analyticalbalance capable of reading to 0.00001g. 5.4 50 mL disposablepolypropylenecentrifuge tubes. 5.5 15 mL disposablepolypropylenecentrifuge tubes. 5.6 Disposable micropipets (50-100uL, 100-2OOuL). 5.7 125-mL LDPE narrow-mouth bottles. 5.8 2 mL clear HPLC vial kit. 5.9 Disposablepipettes. 5.10 Autopipettes(100-lo00 pL and 10-100 kL), with disposable tips. 5.11 Waters Sep Pak Vac 6 cc (le)tC18 SPE cartridges. 5.12 SPE vacuum manifold. Page 31 of65 Page 76 of 119 3M ENVIRONMENTAL LABORATORY PROJECT E05-0210; INTERIM REPORT #20 Exygen Protocol Number: PO00 1131 ExygcnR e r u c h Method Number VOOO I782 L ANALYTICAL METHOD T I Method of Analysis for the Determinationof Perfluorooctanoic Acid (PFOA)in Sedimenr h~ LCIMSIMS 5.13 Vortexer. 5.14 Wrist-actionshaker. 5.15 Centrifugecapable of spinning 50 mL polypropylene tubes at 3000 rpm. 6.0 ChromatographicSystem 6.1 AnalyticalColumn:FluophaseRp (Keystone Scientific), 2.1 m m x 50 mm.Sp (P/N: 82505-052130) 6.2 Temperature: 3OoC 6.3 Mobile Phase (A) : 2 mM Ammonium Acetate in Water 6.4 Mobile Phase (B): Methanol 6.5 Gradient Program: Time (&) 0.0 1 .o 8.0 20.0 22.5 %A 65 65 25 25 65 Flow Rate {mllrniq) 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 an intended as a guide and may be changed in order to optimize the HPLC system. 7.0 MSIMS System 7.1 Mode: ElcctrosprayNegativeMRM mode, monitoring 413 -+ 369 d 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 Phasc 8.1.1 2 mM ammonium acetate in water is prepared by adding 0.154 8 o f ammoniumacetate to 1000mL of water. Page 3 o f 7 Page 32 of65 - Page 7 1 4 f MQ---- 3M ENVIRONMENTAL LABORATORY PROJECT 050210; INTERIM REPORT #20 Exygen Protocol Number: PO00 1 I3 I Exygcn Remuch Method Number VOOOl782 ~~ ANALYTICAL METHOD I Method of Analysis for the Determination of PerfluorooctanoicAcid (PFOA) in Sedirneni h! LCMSMS 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 StocldFortificationSolution 9.1.1 Prepare a stock solution of -100 pB/mL of PFOA by weighing 10 nig of analytical standard (comcted for purity) and dilute to 100 rnL with methanol in a 125-mL LDPE bottle. 9.1.2 A 10 pg/mL fortification solution of PFOA is prepared by bringing I O mL of the 100 F@mLsolution to a final volume of 100 with methanol in a 125 mL LDPE bottle. 9.1.3 A 1.0 pg/mL fortification solutionof PFOA is prepared by bringing I O mL of the 10 pB/mL solutionto a final volume of 100 with methanol in a I25 mL LDPE bottle. 9.1.4 A 0.1 pg/mL fortificationsolution of PFOA is prepared by bringin8 I O mL of the 1.O&mL solutionto a final volume of lo0 with methanol in a 125 mL LDPE bottle. 9.1S A 0.01 pB/mL fortificationsolution of PFOA is prepared by bringing 10 mL of the 0.1 &mL solution to a final volume of 100 with methanol in a 125mL LDPE bottle. 9.1.6 The stock and fortificationsolutions are to be stored in a refrigerator at approximately 4C and arc stable for a maximum period of 6 months from the date of preparation. 9.2 Standard CalibrationSolutions 9.2.1 LCMSMS calibrationstandards arc prepared in methanol via dilution of the 0.1 pg/mL fortificationsolution. 9.2.2 The following is a typical example: additional concentrations may he prepared as needed. Concentration Final of Fortification Volume Diluted to Concentration Solution (nglmL) (mL) (mL) (ng/mL) 100 10 100 10.0 100 5 100 5 .O 100 2 100 2.0 10 10 100 1 .o 5 IO 100 0.5 2 IO 100 0.2 Page 4 of 7 Page 33 of65 Page 78 of 119 3M ENVIRONMENTAL LABORATORY PROJECT E05-0210; INTERIM REPORT #20 Exygen Protocol Number: PO001 131 1 I ExygcnRelciuch Method Number VOOO1782 ANALYTICAL METHOD Method of Analysis for the Determinationof Perfluorooctanoic Acid (PFOA)in Sediment by LC/MS/MS 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 vcrify 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.O Sample Extraction 11.1 11.2 11.3 11.4 11.5 11.6 11.7 11.8 11.9 11.10 11.11 11.12 11.13 Weigh 5 g of sample into 50 mL polypropylene centritbge tubes (fortif) as needed, replace lid and mix well). Add 35 mL of 1% acetic acid, cap, vortex and shake on a wrist action shaker for 4 0 minutes. Centrifugethe tubes at -3000 rpm for -20 minutes. Condition the Cia 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 Load (decant) the sample on the conditioned CII W E cartridge. Discard eluate. 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. Centrifugethe tubes at -3000 rpm for -20 minutes. Decant the methanol onto the same SPE cartridge. Collect the eluate. Wash the column with 4 mL of methanol. Collect the eluate and add it to the eluate collected in step 11.8. Condition a second CISSPE c h d g e (1 g. 6 mL) by passing IO mL methanol followed by 20 mL of HPLC water (- 2 drop/sec). Do not let column run dry Add the methanol to -200 mL of water and load on the second conditioncd SPE cartridge. Elute with -5 mL 100% methanol. Collect 5 mL of eluate into graduated I5 mL polypropylene centrifuge tubes (final volume = 5 mL). Analyze samples using tlcctrospray L C M S M S . Page 5 of 7 Page 34 of 65 Page 79 of 119 .c. c 3M ENVIRONMENTAL LABORATORY PROJECT EO5-0210; INTERIM REPORT #20 Exygen Protocol Number: PO001 13 I Exygen Research Metbod Number VOOOl782 I ANALYTICAL METHOD ! Method of 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 Standardsof PFOA correspondingto at least five or more concentration levels must be included in an analytical set. 12.3 An entire set of extracted calibration standards must be included 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 lasr injection in a sample set. 12.4 Use linear standard curve0 for quantitation. Linear standard curves arc generated for the analyte by linear regression using l/x weighting of peak area versus calibration standard concentration using MassLynx 3.3 (or equivalent) software system. 12.5 Sample response should not e x c d 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 a m u from a parent of 413 amu. The 413 m u parent corresponds to the PFOA anion, while rhc daughter ion (369 m u ) represents the lossof 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 samplemust be obtained and the entire set must be rc-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 70130% should be evaluated by the analyst to determine if re-extraction is warranted. 13.4 Any calibration standard found to be a statistical outlier by using the Huge Error Test, may be excluded from the calculation of the calibration curvc. However, the total number of extracted calibration standards that could be excluded must not exceed 20% of the total number of extracted standards injcctcd. 13.5 The correlation coefficient (R) for calibration curves generated must he 20.992 (R220.985). If calibration results fall outside these limits, then appropriate steps must be taken to adjust instrument operation, and the standardsor the relevant set of samplesshould be reanalyzed. Page 6 of 7 Puge 35 of65 Page 80 of I I9 3M ENVIRONMENTAL LABORATORY PROJECT E05-0210; INTERIM REPORT #20 Exygen Protocol Number: PO001 131 ExygcnRsrcuch Method Number VOOOl782 1 I ANALYTICAL METHOD Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA)in Sediment by LC/MS/MS - - --- 13.6 Retention times between standards and samples must not drift more than f 4 % within an analytical run. If retention time drift exceeds this limit within an analyticalrunthen the set must be reanalyzed. 14.0 Calculations 14.1 Use the followingequation 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 interceDt1 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 ("A)= - [total analytefound (ng/mL) analytefound in control (ng/mL)l oo analyte added (ng/mL) 14.3 Use the following equation to convat the amount of PFOA found in ng/mL lo - nele @PW PFOA found@pb) -p d &Ll x final volume (5 mL)J sample weight (5 g) 14.4 Use the following equation (if necessary) to calculate the amount of PFOA - found in ppb based on dry weight. PFOA found @pb) dry weight PFOA found (ppb) x [ 1 0 0 % 1 total solids(u/)] Page 36 4 6 . 5 Page 81 of 119 _- 3M ENVIRONMENTAL LABORATORY PROJECT E05-0210; INTERIM REPORT #20 Exygen Protocol Number: PO001 13 1 ANALYTICAL METHOD Method Number: VOOO1783 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: Technical Le&er, LC-MS,ExygenResearch /Vice Presideh,Operations,Exygen Research 4??r Date TotalPages: 8 L Pcige 37 01'65 Page 82 d 1IQ-. 3M ENVIRONMENTAL LABORATORY PROJECT EO5-0210; INTERIM REPORT #20 Exygen Protocol Number:PO001131 Exygen Research Method Number VOOO I783 1 1 APiALYfICALMETHOD Method of Analysis for the Determinationof Perfluomctanoic Acid (PFOA)in Fish and Clams by LC/MS/MS - 1.o 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 SpectrometricDetector(LC/MS/MS) in fish and clams. 2.0 Safety 2.1 Always observe safe laboratorypractices. 2.2 Consult the appropriateMSDS 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 (120-400mesh) Reagent grade 4.4 Methanol- HPLC grade - 4.5 Silica gel (60-200 mesh) Reagent grade 4.6 Florid (60-100 mesh)- Reagent grade - 4.7 SupercleanLC-NHz Reagent grade 4.8 I-OctanOl- HPLC gade - 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 PerfluorooctanoicAcid Sigma-Aldrich 5 .O Instmment and Equipment 5.1 A high ~ C I ~ O ~ M lCiqCuid chromatograph capable of pumping up to 1 solvents quipped 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 Analyticalbalance capable of reading to 0.00001 g. Page 2 of s Piigt. 36: o/ 65 Page83of119 - 3M ENVIRONMENTAL LABORATORY PROJECT E05-0210; INTERIM REPORT #20 Exygen Protocol Number: PO00 1131 Exygen Research Method Number VOoO1783 I 1 ANALYTICAL METHOD Method of Analysis for the Determination of PerfluorooctanoicAcid (PFOA) in Fish and Clams by LCiMSiMS 5.4 Rotary evaporator. 5.5 Tissumizer. 5.6 125 mL pear-shaped flash. 5.7 50 mL disposablepolypropylene centrifugetubes. 5.8 15 mL disposablepolypropylene centrifugetubes. 5.9 Disposable micropipets (SO- 1OOuL, 100-2OOuL). 5.10 125-mLLDPE nanow-mouth bottles. 5.11 2 mL clear HPLC vial kit. 5.12 Disposablepipettes. 5.13 Autopipettes (100-lo00 pL and 10-100pL), with disposable tips. 5.14 SPE tubes (2OmL)(Supelcocat. no.NO57177). 5.15 Wrist action shaker. 5.16 Centrifbgecapableof spinning50 mL polypropylenetubes at 2000 rpm. 6.0 Chromatographic System 6.1 Analytical Column: Fluophase RP (Keystone Scientific). 2.1 mm x 50 mm, 511 (PM: 82505-052130) 6.2 Temperature: 3OoC 6.3 Mobile Phase (A) : 2 mM Ammonium Acetate in Water 6.4 MobilePhase (B) : Methanol 6.5 Gradient Program: lhhid 0.0 1 .o 8.0 20.0 22.5 %A 65 65 25 25 65 Flow Rate %Bo 35 0.3 35 0.3 75 0.3 75 0.3 35 0.3 6.6 6.7 6.8 - - injection Volume: 15 pL (can be increased to as much as 50 pL). Quantitation: Peak Area external standard calibration curve. Run Time: 23 minutes. The above conditionsarc intended as a guide and may be changed in order to optimize the HPLC system. Page 3 of 8 Page 39 qf65 Page 84 Ofr 149 3M ENVIRONMENTAL LABORATORY PROJECT 050210; INTERIM REPORT #20 Exygen Protocol Number:PO001131 .- Exygen Rerurch Method Number VOOO1783 I I ~~ ANALYTICAL METHOD Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Fish and Clamsby LC/MS/MS 7.0 MSMS System 7.1 Mode: Electrospray Negative MRh4 mode, monitoring 413 + 369 m/zfor 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.I54 8 of ammonium acetate to 1000mL of water. 8.2 Extraction Solutions 8.2.1 2% k o r b i c acid in methanol is prepared by dissolving 2 g of ascorbic acid in 100mL of merhanol. 8.2.2 30% Dimethyldichlorosilane in toluene is prepared by bringing 3 ml.. of dimethyldichlorosilane to a final volume of IO mL with toluene. Altemate volumes may be prepared. 9.0 Standard Preparation 9.1 Standard StwWortification Solution 9.1.1 9.1.2 9.1.3 9.1.4 9.1.5 Prepare B stock solution of -1 00 p g h L of PFOA by weighing IO m g of analytical standard (corrected for punty) and dilute to 100 niL with methanol in a 125-mLLDPE bottle. A 1.O pg/mL fortification solution of PFOA is prepared by bringing I mL of the 100 p@mL solution to a final volume of 100 with methanol in a 125mL LDPE bottle. A 0.1 &mL fortification solution of PFOA is prepared by bringing I O mL of the 1.0 pB/mL solution to a final volume of 100 with methanol in a 125 mL LDPE bottle. A 0.01 pB/mL fortification solution of PFOA is prepared by bringing 10 mL of the 0.1 p&mL solution to a final volume of 100 w i t h 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 monlhs h m the date of preparation. Page 4 01'8 Puge 40 of65 Page 85 of 119 3M ENVIRONMENTAL LABORATORY PROJECT E05-0210; INTERIM REPORT #20 Exygen Protocol Number: PO00113 1 c Exyeen Research Method Number VOOO 1783 L I ANALITICAL bUTlIOD Method of Analysis for the Determinationof Perfluorooctanoic Acid (PFOA) in Fish and Clams by LCMSMS -- 9.2 Standard CalibrationSolutions 9.2.1 LCMSMS calibration standards are prepared in methanol via dilution of the 1.O pg/mL fortificationsolution. 9.2.2 The following is a typical example: additional concentrations may be prepared as needed. Concentration Final of Fortification Solution (~g/mL) 1 .o 1 .o 1 .O Volume (mL) 5.0 2.5 1 .o Diluted to (d) 100 100 100 Concentration (jlg//mL) 0.05 0.025 0.01 0.05 10 100 0.005 0.025 10 100 0.0025 0.1 10 100 0.001 0.005 10 100 0.0005 9.2.3 Store all calibration standards in 125-mL LDPE narrow-mouth bottles at 2C to 6OC, up to six months. 9.2.4 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.O Sample Extraction 11.1 Weigh 5 g of frozen sample into SO mL polypropylene centrifuge tubes (fortify as needed, replace lid and mix well). 11.2 Add 30 mL of acetonitrileand shake on a wrist action shaker for -1 5 minutes. 11.3 Place the tubes in a freezer for -I hour. 1 1.4 Pack and condition the SPE tubes and silanizc 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-NHz. Condition the columns with 20 mL of methanol. then 20 mL of acetonitde. Discard all washes. Do not allow the column to dry. 11.6 Silanize the 125 mL pear-shaped flasks by rinsing with the 30Y0 dimethyldichlorosilanein toluene solution. Rinse the flask with toluene once, followed by methanol (three times). Dry the flasks completely before use. either by aitdrying or with a stream of nitrogen. Page 4 I of65 -.--. Page 864f 11%- 3M ENVIRONMENTAL LABORATORY PROJECT EO5-0210; INTERIM REPORT #20 Exygen Protocol Number: PO001131 Exygen Research Method Number VOOO I783 L 1 ANAL.\TICAL METHOD Method of Analysis for the Determination of PerfluorooctanoicAcid (PFOA)in Flsh and Clams by LC/MS/MS _. 11.7 11.8 11.9 11.10 1 1.1 1 11.12 11.13 11.14 11.15 11.16 1 1.17 1 1.18 Centrifuge the 50 mL polypropylene tubes containing sample at -2000 rpm for -10 minutes. Decant the extract on to a conditionedSPE column fitted inside the mouth of' the pear-shaped flask. Collect the eluate in the 125 mL silanized pear-shape flask. Add 10 mL of acetonitrile to the sample in the 50 mL centrifuge tube. Homogenize the frozen fat phase using a tissumizer for -30 seconds and rinsc the tissumizerwith -10 mL ofacetonitrileinto the tube. Shake the sample again for -10 minutes on a wnst-action shaker. - Place the tubes in a freezerfor 1 hour more. Centrihge the 50 mL polypropylene tubes containing sample at -2000 rpni -for 10 minutes. Decant the extract onto the same SPE column. Collect the eluate into the same pear-shaped flask and combine with the eluent from the initial extraction. Pass 20 mL of acetonitrilethrough the SPE column and combine the eluate in the same pear-shaped flask. Add 3-4 drops of I-octanol to the extract in the pear-shaped flask and evaporateat reduced pressureusing a rotary evaporator (at e 40C). Make the final volume, by adding 2 rnL of 2% ascorbic acid in methanol to the pear-shaped flask and swirl to middissolve. Transfer the extractsto HPLC vials using disposable pipets. Analyze samples using electrosprayLC/MS/MS. 12.0 Chromatography 12.1 Inject the same amount of each standard, sample and fortified sample into the LC/MS/MS system. A calibration standard must precede and follow all analyzed samples. 12.2 Standardsof PFOA conesponding to at least five or more concentration levels must be included in an analytical set. 12.3 An entire set of calibrationstandardsmust be included at the beginning and 31 the end of a sample set. Standards must be interspersed between every 5-10 samples. As an alternative, an entire set of calibration standards m y 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 CUNCS for quaniitation. Linear standard curves are generated for the analyte by linear regressionusing I/x weighting of peak area versus calibration standard concentrationusing MassLynx 3.3 (or equivalent) software system. -. . .__ . Page 6 o f 8 Page 42 o j 6 5 Page 870f 119 - - 3M ENVIRONMENTAL LABORATORY PROJECT E05-0210; INTERIM REPORT #20 Exygen Protocol Number: PO001 13 1 ,.- Exygen Research Method Number VOW1783 1 I ANALYTICALMETHOD 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 thai 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 m u . The 413 amu parent corresponds to the PFOA anion, while the daughter ion (369 m u ) 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 calibralion curve. However, the total number of calibration standards that could be excluded must not exceed 20% of the total number of standardsinjected. 13.5 The correlation coefficient (R) for calibration curves generated must be 20.992 (R' 20.985). If calibration results fall outside these limits, then appropriate steps must be taken to adjust instrument operation, and the standardsor the relevant set of samples should be reanalyzed. 13.6 Retention times between standards and samples must not drift more than f 4 % within an analyticalrun. If retention time dritt exceeds this limit within an analytical run then the set must be reanalyzed. 14.0 Calculations 14.1 Use the followingequation 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 - interceDt) slope 14.2 Use the followingequation to convert the amount of PFOA found in ng/mL to no/e @PW. PFOA found (ppb) =[PFOAfound W L )x fmal volume (mL1 x DF] sample weight (g) DF = factor by which the final volume was diluted, if necessary. Page 7 of 8 Page 43 of 65 -- -- Page 88 of 3-1-9--. 3M ENVIRONMENTAL LABORATORY PROJECT E05-0210; INTERIM REPORT #20 Exygen Protocol Number: PO001 131 "- Exygen Rcscuch Method Number VGW I 78.7 I I A X G Y T 1 C . U AlE'I'HOD Method of Analysis for the Determinationof PerfluorooctanoicAcid (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 calculatethe percent recovery. Recovery (Yo) I - total analytefound (nglg) analytefound in control (ng/g)l oo analyteadded (ng/g) Page 8 or X Page 44 of 65 3M ENVIRONMENTAL LABORATORY PROJECT E05-0210; INTERIM REPORT #20 Exygen Protocol Number: PO001 131 ANALYTICAL METHOD Method Number: VOOOl784 Method of Analysis for the Determlnatlonof 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,ExygenResearch 'Vice President,Operations,Exygen Research Total Pages: 7 --.-- Page 45 of65 Page 90 of 119 - 3M ENVIRONMENTAL LABORATORY PROJECT 05-0210; INTERIM REPORT #20 Exygen Protocol Number: PO001131 Exygen Research Method Number VOOO I '84 E ."riALYTlCAL METHOD iL Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA)in Vegetation by LCIMSIMS 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 SpectrometricDetector (LCIMSMS)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 Rquirement 3.1 At least 20 g of test sample for extraction. 3.2 Samplesshould be processed before extraction. Place the frozen sample in a food processor and homogenize with dry ice. Place the samples in containers and leave 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 Reageiits and Standards 4.1 Water - HPLC grade 4.2 Acetonitrile- HPLC grade 4.3 Carbon (1 20-400 mesh) - Reagent grade 4.4 Methanol - HPLC grade 4.5 Silica gel (60-200 mesh) - Reagent grade 4.6 Florisil(60-lo0mesh)- Reagent grade 4.7 Superclean LC-MI2 - Reagent grade - 4.8 1-0ctanol- 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 perfonnance liquid chromatograph capable of pumping up to 2 solvents equipped with a variable volume injector capable of injecting S-ZO(1 )tLconnected to a tandem Mass Spectrometer(LCIMSMS). 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 46 o j 6 5 Page 91 of 119 3M ENVIRONMENTAL LABORATORY PROJECT 05-0210; INTERIM REPORT #20 Exygen Protocol Number: PO001 13 1 Exygcn Research Method Number VOW I784 1 I ANALYTICAL METHOD Method of Analysis for the Determination of PerfluorooctanoicAcid (PFOA) in Vegetation by LC/MS/MS 5.4 Rotary evaporator. 5.5 125 mL pear-shaped flasks. 5.6 50 mL disposablepolypropylenecentrifugehibes. 5.7 15 mL disposablepolypropylenecentrifugetubes. 5.8 Disposable micropipets(~O-~OOU1L0,0-2OOuL). 5.9 125-mL LDPE narrow-mouth bottles. 5.10 2 mL clear HPLC vial kit. 5.1'1 Disposable pipettes. 5.12 Autopipetta (100-1000 pL and 10-100JIL),with disposable tips. 5.13 SPE tubes (2OmL)(Supelcocat. no. N057177). 5.14 Wrist action shaker. 5.15 Centrifugecapableof spinning50 mL polypropylenetubes at 2000 rpm. 6.0 Chromatographic System 6.1 Analytical Column: Fluopha~eRP (Keystone Scientific).2.1 mm x 50 mm, 511 (PM: 82505-052130) 6.2 Temperature: 3OoC 6.3 Mobile Phase (A) : 2 mM Ammonium Acetate in Water 6.4 Mobile Phase (B): Methanol 6.5 Gradient Program: u Flow Rate fmUmin) 0.0 65 35 0.3 1 .o 65 35 0.3 8.0 25 75 0.3 20.0 25 75 0.3 22.5 65 35 0.3 6.6 6.7 6.8 - - Injection Volume: IS pL (can be increasedto as much as 50 pL). Quantitation: Peak Area external standard calibration curve. Run Time: 23 minutes. The above conditionsare intended as a guide and may be changed in order to optimizethe HPLC system. 7.0 MS/MSSystem 7.1 Mode: Electrospray Negative MRM mode, monitoring413 -+ 369 m/z for PFOA. Page 3 01'7 Page 4 7 oj'4S - - -?age-82Bf-l41.9------ 3M ENVIRONMENTAL LABORATORY PROJECT EO5-0210; INTERIM REPORT #20 Exygen Protocol Number: PO00113 1 c Exygen Research Method Number VOOO I784 I I ANALYTICAL JIETHOD Method of Analysis for the Determinationof Perfluorooctanoic Acid (PFOA) in Vegetation by LC/MS/MS --- The above conditionsare intendedas a guide and may be changed in order to optimize the MSMS system. 8.0 Preparationof Solutions 8.1 Mobile Phase 8.1.1 2 mM ammonium acetate in water is prepared by adding 0.154 g o f ammoniumacetate to 1000 mL ofwater. 8.2 Extraction Solutions 8.2.1 2% ascorbic acid in methanol is prepared by dissolving 2 g of ascorbic acid in 100 mL of methanol. 8.2.2 30% Dimethyldichlorosilae in toluene is prepared by bringing 3 m L of dimethyldichlorosilaeto a final volume of 10 mL with toluene. Alternatevolumes may be prepared. 9.0 Standard Preparation 9.1 StandardStocklFortificationSolution 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 r n l with methanol in a 125-mLLDPE bottle. 9.1.2 A 1.0 pg/mL fonification solution of PFOA is prepared by bringing I mL of the 100 pg/mL solution to a final volume of 100 with methanol in a 125 mL LDPE bottle. 9.1.3 A 0.1 p g h L fortificationsolution of PFOA is prepared by bringing I O 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 A 0.01 pg/mL fortification solution of PFOA is prepared by bringing 10 mL of the 0.1 pglmL solution to a final volume of 100 with methanol in a 125 mL LDPE bottle. 9.1.5 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 StandardCalibrationSolutions 9.2.1 LC/MS/MS calibration standards are prepared in methanol via dilution of the 1.O p@mL fortificationsolution. Page 48 oj6S Page 93 of 119 3M ENVIRONMENTAL LABORATORY PROJECT E05-0210; INTERIM REPORT #20 Exygen Protocol Number: PO001 13 I Exvncn Research Method Numbcr VOOO I784 ~~ ~ AhikLYI K A L IkiE'THOD Method of Analysis for the Determinationof Perfluorooctanoic Acid (PFOA) in Vegetation by LC/MS/MS 9.2.2 The following is a typical example: additional concentrations may be prepared as needed. Concentration Final of Fohfication Volume Diluted to Concentration Solution (pe/mL) (mL) 1 .o 5.0 1 .o 2.5 1 .o 1 .O (Id) 100 100 100 (NdmL) 0.05 0.025 0.01 0.05 10 100 0.005 0.025 10 100 0.0025 0.1 10 100 0.00 1 0.005 IO 100 0.0005 9.2.3 Store all calibration standards in 125-mLLDPE 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 hvo untreated controls fortified a1 known concentrations(lab control spike) to verify procedural recovery for the batch. 10.2 Requirements for field and laboratory duplicates and spikes will be specilied in the quality assuranceplan for this project. 11.O 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 -1 5 minutes. 11.3 Centrifuge the 50 mL polypropylene tubes containing sample at -2000 rpm for -1 0 minutes. 11.4 Pack and condition the SPE tubes and silanize the pear-shaped flasks. 11.5 Pack the 20 mL SPE tubes in sequence with 2 g florisil, 2 g silica gel. 2 g carbon, and 1 g LC-NH2. Condition the columns with 20 mL of methanol, then 20 mL of acetonitrile. Discard all washes. Do not allow the column to dry. 11.6 Silanize the 125 mL pear-shaped flasks by nnsing with the 30% dimethyldichlorosilaein toluene solution. Rinse the flaskwith toluene oncc, followed by methanol (three times). Dry the flasks completely before use. either by air-dryingor with a stream of nitrogen. 11.7 Decant the extract on to a conditioned SPE column fitted inside the mouth 01` the pear-shaped flask. Collect the eluate in the 125 mL silanized pear-shape flask. Page 49 of65 Page 94 of 119 . - 3M ENVIRONMENTAL LABORATORY PROJECT EO5-0210; INTERIM REPORT #20 Exygen Protocol Number: PO001 13 1 c 1 ExygcnResearch -' a: Method Number VOW I 784 Ah#i.Y'rIc:'hL \lS*l'HOD Method of Analysis for the Determinationof Perfluorooctanoic Acid (PFOA)in Vegetation by LC/MS/MS 11.8 11.9 11.10 11.1 1 11.12 11.13 11.14 11.15 11.16 Add 20 mL of acetonitrileto the sample in the 50 mL centrifuge tube. Shake the sample again for -10 minutes on a wrist-action shaker. Centrifuge the 50 mL polypropylene tubes containing sample at -2000 rpm for -5 minutes. Decant the extract onto the same SPE column. Collect the eluate into the same pear-shaped flask and combine with the eluent from the initial extraction. Repeat steps 11.8 through 11.11 again. Add 3 4 drops of l-octanol to the extract in the pear-shaped flask and evaporate at reduced pressure using a rotary evaporator (at < 4OOC). Make the final volume, by adding 2 mL of 2% ascorbic acid in methanol to the pear-shaped flask and swirl to mixldissolve. Transfer the extracts to HPLC vials using disposable pipets. Analyze samples using electrospray LC/MS/MS. 12.0 Chromatography 12.1 Inject the same amount of each standard, sample and fortified sample into thc LCMS/MS system. A calibration standard must precede and follow all analyzed samples. 12.2 Standardsof PFOA correspondingto at least five or more concentration lewls 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 bc 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 sccond set of extracted standards). In either case. extracted calibrationstandardsmust 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 of peak area versus calibration standard concentration using MassLynx 3.3 (or equivalent1 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 ACCC~WCrCiteria 13.1 Chromatogrammust show a peak of a daughter ion at 369 amu from a parent of 413 m u . The 413 amu parent corresponds to the PFOA anion, while the daughter ion (369 amu) represents the loss of carbon dioxide. Page 6 01'7 Page SO of 65 Page 95 of 1f9---- 3M ENVIRONMENTAL LABORATORY PROJECT 05-0210; INTERIM REPORT #20 Exygen Protocol Number: PO001 131 Exygen Research MethodNumber V0001784 1 I 4-Wn AYALYTlCAL METHOD 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. 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% 01` their known values. If a control spike falls outside the acceptable limits, the entire set of samplesshould be re-extracted. 13.4 Any calibration standard found to be a statistical outlier by using the Huge Error Test, may be excluded fiom 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 20.992 (R220.985). 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 f 4 %within an analytical run. If retention time driR 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 cuwc (linear regression parameters) generated by the Mass Lynx software program: - PFOA found (ng/mL)=p e a k area intercept) slope 14.2 Use the following equation to convert the amount of PFOA found in ng/mL to nul? @Pb)* PFOA found @pb)=lpFOA found C&L) x fina1volume (mL) x DF] sample weight (g) DF = factor by which the final volume was diluted. if necessary. 14.3 For samples fortified with known amounts of PFOA prior to extraction, use the following equation to calculate the percent recovery. Recovery (%) = total analytefound (ng/g) - analytefound in control (ng/g)] ,oo analyteadded (ng/g) Page 51 of65 Page 96 of:1 - L 3M ENVIRONMENTAL LABORATORY PROJECT 050210; INTERIM REPORT #20 Exygen Protocol Number: PO001 13 1 .c ANALYTICAL METHOD Method Number: V0o01785 Method of Analysis for the Determinationof Perfluorooctanoic Acid (PFOA) in Small Mammal Liver by LC/MS/MS Analytical Testing Facility: Exygen Research 3058 Research Drive State College. PA 16801 7-1, u Approved By: Paul Connollv Technical hider, LC-MS,Exygen Research ra\zafoll Date /.A! Date . -- Total Pages: 7 Page 52 qf6.5 . - ._ __ Page 97 of 1I 9 ---- 3M ENVIRONMENTAL LABORATORY PROJECT EO5-0210; INTERIM REPORT #20 Exygen Protocol Number: PO001131 .- Exygen Research Method Number V0001785 I I Ahi.LYTICtU METHOD Method of Analysis for the Determination ofPerfluorooctanoic Acid (PFOA) in Small Mammal Liver by LC/MS/MS - 1.o scope This method is to be mploycd for the isolation and quantitation of perfluorooctanoic acid by High Performance Liquid Chromatography coupled to a tandem Mass Spectrometric Detector (LCMShlS) in small mammal liver. 2.0 Safety 2.1 Always observe safe laboratory practices. 2.2 Consult the appropriate MSDS before handling any chemical for proper safely precautions. 3.0 Sample Requirement 3.1 At least 5 g of test sample for extraction. 3.2 Samples should be processed before extraction. Place the frozen sample in il 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 AmmoniumAcetate A.C.S. Reagent Grade - 4.5 Perfluorooctanoic Acid Sigma-Aldrich 5.O instrument and Equipment 5.1 A high performance liquid chromatograph capable of pumping up to 7 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,100-2OOuL). 5.7 125-mL LDPE narrow-mouth bottles. 5.8 2 mL clear HPLC vial kit. Page 2 of' .- - - . ._ - . Page 53 of65 Page 9 8 & - # L 3M ENVIRONMENTAL LABORATORY PROJECT E05-0270; INTERIM REPORT #20 Exygen Protocol Number: PO001131 Exvnen Research Method Number VOOO1785 - ~~ AKALYTICAL METHOD 1 Method of Analysis for the Determination of PerfluorooctanoicAcid (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 Stp Pak Vac 6 cc (le) tC18 SPE cartridges. 5.12 SPE vacuum manifold. 5.13 Tissuemizer. 5.14 Wrist-action shaker. 5.15 Centrifuge capable of spinning 15 mL polypropylenetubes at 3000 rpm. 6.0 Chromatographic System 6.1 Analytical Column: Fluophase RP (Keystone Scientific),2.1 mm x 50 mm,511 (PM: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: Timelmin) %A 0.0 65 1 .o 65 8.0 25 20.0 25 22.5 65 Flow Rate lmUminl 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 6.8 - Quantitation: Peak Area - external standard calibration curve. Run Time: 23 minutes. The above conditionsam intended as a guide and may be changed in order 10 optimizethe HPLC system. 7.0 MSMS System 7.1 Mode: Electrospray NegativeMRM mode, monitoring 413 --+ 369 d z for PFOA. The above conditions are intended 85 a guide and may be changed in order 10 optimize the MSMS system. Page 54 4 ' 6 5 Page 99 of I19 -- 3M ENVIRONMENTAL LABORATORY PROJECT EO5-0210; INTERIM REPORT #20 Exygen Protocol Number: PO001131 1 i ExygenResearch .--- .4N:+ZYTICAL METHOD Method Number VOOO1785 Method of Analysis for the Determination of Perfluorooctanoic 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 o f ammonium acetate to 1000mL of water. Alternate volumes may be prepared. 9.0 Standard Preparation 9.1 Standard StocWFortificationSolution 9.1.1 Prepare a stock solution of -100 pg/mL of PFOA by weighing 10 mg of analytical standard (corrected for punty) and dilute to 100 mL with methanol in a 125-mL LDPE bottle. 9.1.2 A 1.0 pB/mL fortification solution of PFOA is prepared by bringing I mL of the 100 &mL solution to a final volume of 100 with methanol in a 125 mL LDPE bottle. 9.1.3 A 0.1 pdmL fortification solution of PFOA is prepared by bringing 10 mL of the 1.0 pdmL solution to a final volume of 100 with methanolin a 125 mL LDPE bottle. 9.1.4 The stock and fortification solutions are to be stored in a refrigerator at approximately 4 O C 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 &mL fortification solution. 9.2.2 The following is a typical example: additional concentrations may be prepared as needed. Concentration Final of Fortification Volume Diluted to Concenbation Solution (ng/mL) (mL) (mL) (ng/mL) 100 5.0 100 5 .O 100 2.0 100 2 .o 100 1 .o 100 I .o 5.0 10 100 0.5 2.0 10 100 0.2 1.0 10 100 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. Page 4 of 7 ,r- 1 -_- . - Page 55 qf6S Page 100 of 119 ----- 3M ENVIRONMENTAL LABORATORY PROJECT 05-0210; INTERIM REPORT #20 Exygen Protocol Number: PO001131 Exygcn Rereuch Method Number VOOO I785 L A3.UYTICAL METHOD J I Method of Analysis for the Determinationof Perfluorooctanoic Acid (PFOA) in Small Mammal Liver by LCMSMS 10.0 Batch Set Up 10.1 Each batch of samples extracted (typically 20 or less) must include at leas1 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 assuranceplan for this project. I 1.O Sample Extraction 11.1 11.2 11.3 11.4 11.5 11.6 11.7 11.8 11.9 11.10 11.11 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 r measureddependingon the sample size available for use. Add water to the sample for a final volume of 10 mL. Homogenizesample using a tissuemizer for -1 minute. Transfer 1 mL of the sample using a disposable pipette into a 15 m L disposable centrifuge tube. Add 5 mL of acetonitrileand shake for -20 minutes on a wrist-action shaker. Centrifugethe tubes at -3000 rpm for -5 minutes. Decant the supernatant into a 50 mL disposable centrifuge tube and add 35 mL of water. Condition the cl8 SPE cartridges (1 g. 6 mL) by passing 10 mL methanol followed by 5 mL of HPLC water {- 2 drop/sec). Do not let column run dry Load the sample on conditioned CISSPE cartridge. Discard eluate. Elute with -2 mL of methanol. Collect 2 mL of eluate into a graduated 15 mL polypropylenecentrifuge tube (final volume = 2 mL). 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 - I O samples. As an alternative, an entire set of calibration standards may hc injected at the beginning of a set followed by calibration standards interspersedevery 5-10 samples (to account for a second set of standards). In either case, calibration standards must be the first and last injection in il sample set. 12.4 Use linear standard c u r m for quantitation. Linear standard curves are generated for the analyteby linear regression using l l x weighting of peak area ,"- Piige 56 of 6j Page 101 of 119---- 3M ENVIRONMENTAL LABORATORY PROJECT EO5-0210; INTERIM REPORT #20 Exygen Protocol Number: PO001 13 1 Exygea Research Method Number V0001785 I ANALYrICriL METHOD I Method of Analysis for the Determinationof Perfluorooctanoic Acid (PFOA) in Small Mammal Liver by LC/MS/MS versus calibration standard concentration using MassLynx 3.3 (or equivalenl) software system. 12.5 Sample response should not exceed standard responses. Any samples thai exceed standard responses should be hrther diluted and reanalyzed. 13.0 AcceptanceCriteria 13.1 Chromatogrammust show a peak of a daughter ion at 369 m u from a parent of 413 m u . The 413 amu parent corresponds to the PFOA anion. while the daughter ion (369 m u ) represents the loss of carbon dioxide. 13.2 Method blanks must not contain PFOA at levels greater than the LOQ. If il blank contains PFOA at levels greater than 10 ndg, then a new blank sample must be obtained and the entire set must be re-extracted. 13.3 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 70130% should be evaluated by the analyst to determine if re-extraction is warranted. 13.4 Any calibration standard found to be a statistical outlier by using the Huge Error Test, may be excluded &om 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 cocfllcicnt (R)for calibration curves generated ntust be 20.992 (R' 20.985). If calibration results fall outside these limits. then appropriate steps must be taken to adjust instrument operation, and the standardsor the relevant set of samples should be reanalyzed. 13.6 Retention times between standards and samples must not drift more than f 4 % within an analytical run. Ifretention time drift exceeds this limit within an analytical run then the set must be reanalyzed. 14.0 Calculations 14.1 Usc the followingequation to calculate the amount of PFOA found (in n@niL. based on peak area) using the standard curve (linear regression parameters) generatedby the Mass Lynx softwareprogram: - PFOA found (ng/mL)* ( P e a k a intercentl x DF x aliquot factor slope DF = factor by which the final volume was diluted, if necessary. Aliquot factor = 10 Page 6 of 7 ..- _-. - __ - Pnge 57 of 65 --ri-t.8foftf4--------- 3M ENVIRONMENTAL LABORATORY PROJECT 05-0210; INTERIM REPORT #20 Exygen Protocol Number:PO001 131 Exypcn Research Method Number VOOO1765 1 i ANALY i Ic'AL AlE'THOD Method of Analysis 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 (%) = - ltotal analyte found(ng/mL) analyte found in control (ng/mL)L oo analyteadded (ng/mL) 14.3 Use the following equation to convert the amount of PFOA found in n g h L io ng/g @Pb)* PFOA found (ppb)=IPFOA fourd.b@mL) x final volume (mLu sample weight (8) Page 7 of 7 .- Page 58 of 65 Page 103of119 -I - - - 3M ENVIRONMENTAL LABORATORY PROJECT 05-0210; INTERIM REPORT #20 Exygen Protocol Number: PO001131 ANALYTICAL METHOD Method Number: VOOOl786 Method of Analyrls for the Determination of Perfluorooctanolc Acid (PFOA)in Srnull Mammal Serum by LC/MS/MS Analytical TestingFacility: Exygen Research 3058 Research Drive State College, PA 16801 Approved By: Paul Connolly 1 Technical Leader, LC-MS,ExygenResearch / Vice President,Operations,ExygenResearch TotalPages: 7 c Page 59 cf65 -Page 104 of 119 ------ - 3M ENVIRONMENTAL LABORATORY PROJECT EO5-0210; INTERIM REPORT #20 Exygen Protocol Number: PO001131 Exygen Research Method Number VOW I786 L I ANAL'I'TICAL hlETHOD Method of Analysis for the Determinationof Perfluorooctanoic Acid (PFOA)in Small Mammal Serum by LC/MS/MS 1.o Scope This method is to be employed for the isolation and quantitation of perfluorooctanoic acid by High Performance Liquid Chromatography coupled to a tandem Mass SpectrometricDetector(LCIMSIMS) in small mammal serum. 2.0 Safety 2.1 Always observe safe laboratorypractices. 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 strum 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 Pade 4.3 Acetonitrile HPLC grade 4.4 Ammonium Acetate- A.C.S. Reagent Grade 4.5 PerfluorooctanoicAcid - Sigma-Aldrich 5.0 Instrumentand Equipment 5.1 A high performance liquid chromatograph capable of pumping up LO 1 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 Analyticalbalance capable of reading to 0.00001 g. 5.4 50 mL disposablepolypropylenecentrihge tubes. 5.5 15 mL disposablepolypropylenecentrifbge tubes. 5.6 Disposablemicropipets(50-1OOuL, 100-2OOuL). 5.7 125-mLLDPE narrow-mouthbottles. 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. 5.1 1 Waters Scp Pak Vac 6 cc (le)tCl8 SPE cartridges. 5.12 SPE vacuum manifold. 5.13 Vortexer. 3M ENVIRONMENTAL LABORATORY PROJECT EO5-0210; INTERIM REPORT #20 Exygen Protocol Number: PO001 13 1 Exypcn Research Method Number VOOO I786 I .AI\~ALY*I'~C'AMLETHOD Method of Analysis for the Determination of PerfluorooctanoicAcid (PFOA)in Small Mammal Serum by LCIMS/MS --- 5.14 Wrist-action shaker. 5.15 Centrifugecapableof spinning 15 mL polypropylenetubes at 3000 rpm. 6.0 Chromatographic System 6.1 Analytical Column: Fluophase RP (Keystone Scientific), 2.1 mm x 50 mm. 511 (PM:82505-052130) 6.2 Temperature: 30C 6.3 Mobile Phase (A) : 2 mM Ammonjum Acetate in Water 6.4 Mobile Phase (B): Methanol 6.5 Gradient Program: Time 0.0 1.o 8.0 20.0 22.5 Flow Rate %LA %B lmYmin) 65 35 0.3 65 35 0.3 25 75 0.3 25 75 0.3 65 35 0.3 - 6.6 Injection Volume: 15 pL (can be increasedto as much as 50 pL). 6.7 Quantitation: Peak Area - external standard calibration curve. 6.8 Run Time: 23 minutes. The above conditionsare intended as a guide and may be changed in order IO optimizethe HPLC system. 7.0 MSMS 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 lo 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 acetateto 1000 mL of water. Alternate volumes may be prepared. Page 106of 119 .-.- 3M ENVIRONMENTAL LABORATORY PROJECT EO5-0210; INTERIM REPORT #20 Exygen Protocol Number: PO001 13I h Exygen Research Method Number VOOO1786 I I ANALYTICAL !METHOD Method of Analysis for the Determination of PerfluorooctanoicAcid (PFOA) in Sniall Mammal Serum by LCIMSIMS ~ 9.0 Standard Preparation 9.1 Standard StocWFortificationSolution 9.1.1 Prepare a stock solution of -100 p#mL of PFOA by weighing I i)iiig of analytical standard (corrected for punty) and dilute to 100 m L H Ill) methanol in a 125-mL LDPE bottle. 9.1.2 A 1.0 p#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 p#mL fortificationsolutionof PFOA is prepared by bringing I O mL ofthe 1.0 p#mL solution to a final volume of 100 with methanol in a 125 mL LDPEbottle. 9.1.4 The stock and fortificationsolutionsare to be stored in a refrigeratorat approximately 4OC and are stable for a maximum period of 6 months !?om the date of preparation. 9.2 Standard Calibration Solutions 9.2.1 LC/MS/MS calibrationstandardsare prepared in methanol via dilution of the 0.1 pg/mL fortificationsolution. 9.2.2 The following is a typical example: additional concentrations may be prepared as needed. Concentration Final of Fortification Volumc Diluted to Concentration Solution (ng/mL) (mL) (mL) (ng/mL) 100 5 .O 100 5.0 100 2.0 100 2.0 100 1 .o 100 1 .o 5.0 10 100 0.5 2.0 10 100 0.2 1 .o 10 100 0.1 9.2.3 Store all calibrationstandards in 125-mL LDPE narrow-mouthbottles at 2 O C to 6OC, up to six months. 9.2.4 Alternate volumes and concentrationsof standards may bc 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 knowti concentrations(lab controlspike) to verify procedural recovery for the batch. 10.2 Requirements for field and laboratory duplicates and spikes will be specified in the qualityassuranceplan for this project. Page 4 01' 7 *- . . -.--- I- Page 62of 65 --Pagel o r n - " 3M ENVIRONMENTAL LABORATORY PROJECT E05-0210;INTERIM REPORT #20 Exygen Protocol Number: PO00 1131 Exygen Research Method Number VOOOl786 1 ANtcLYTICAL METHOD Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Small Mammal Serum by LC/MS/MS - 11.O Sample Extraction 11.1 11.2 11.3 11.4 11.5 11.6 11.7 11.8 11.9 11.10 11.11 Measure 1 mL of sample into a 50 mL polypropylene centrifuge tubes (fortify as needed, replace lid and mix well). Note that alternate volumes of serum may be measured dependingon the sample size available for use. Add water to the sample for a final volume of 20 mL. Cap tightly Vortex for -1 minute. Transfer 1 mL of the sample using a disposable pipette into a 15 mL disposable centrifuge tube. Add 5 mL of acetonitrileand shake for -20 minutes on a wrist-action shaker. Centrifugethe tubes at -3000rpm for -5 minutes. Decant the supernatant into a 50 mL disposable centrifuge tube and add 35 mL of water. Condition the CISSPE cartridges (1 g, 6 mL) by passing 10 rnL methanol followed by 5 mL of HPLC water (- 2 drophec). Do not let column nm dry Load the sample on conditioned CISSPE cartridge. Discard eluate. Elute with -2 mL of methanol. Collect 2 mL of eluate into a graduated 15mL polypropyleneccntrihge tube (finalvolume = 2 mL). Analyze samples using electrospray LCiMSiMS. 12.0 Chromatography 12.1 Inject the same amount of each standard, sample and fortified sample into thc LC/MS/MS system. A calibration standard must precede and follow all analyzed samples. 12.2 Standards of PFOA correspondingto 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 - I O samples. As an alternative, an entire set of calibration standards may be injected at the beginning of a set followed by calibration siandards 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 curyes for quantitation. Linear standard curves are generated for the analyte by linear regression using I/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 responsesshould be m e r diluted and reanalyzed. - ..__^__. - P ~ g 6e3 OJ 45 -egap 3M ENVIRONMENTAL LABORATORY PROJECT EO5-0210; INTERIM REPORT #20 Exygen Protocol Number: PO001131 Exygen Research Method Number W O O I7116 1 1 ANALYTICALGETHOD Method ofAnalysis for the Determination ofPerfluorooctanoic 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 [tic. daughter ion (369 m u ) represents the loss ofcarbon 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 valuee. If a control spike falls outside the acceptable limits, the entire set of samples should be re-extracted. Any matrix spike outside 70130% should be evaluated by the analyst to determine if re-extraction is warranted. 13.4 Any calibration standard found to be a statistical outlier by using the Huge Error Test, may be excluded from the calculation of the calibration 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 20.992 (R220.985). If calibration results fall outside these limits. ~hrn appropriate steps must be taken to adjust instrument operation, and [tic' standards or the relevant set of samples should be reanalyzed. 13.6 Retention times between standards and samples must nor drift more thltii f 4 % within an analytical run. If retention time driA exceeds this limit withill c 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 - interceDt) x DF x aliquot factor slope DF = factor by which the final volume was diluted, if necessary. Aliquot factor = 20 14.2 For samples fortified with known amounts of PFOA prior to extraction. use the following equation to calculate the percent recovery. Recovery (%) = 1total analyte found(ng/mL) - analyte found in control (ng/mL)L analyte added (ng/mL) oo Page 6 of 7 .- Page 64 of 4S Page 109 of 119- 3M ENVIRONMENTAL LABORATORY PROJECT 05-0210; INTERIM REPORT #20 Exygen Protocol Number: PO001 13 1 IExygenRerearch *--* A i i u m x L METHOD Method Number VOOO I786 1 Method of Analysis for the Determinationof PerfluorooctanoicAcid (PFOA)in Small Mammal Serum by LC/MS/MS -- 14.3 Use the following equation to convert the amount of PFOA found in n u n i l to PPb. PFOA found @pb)=lpFOA found IndmL) x final volume ImL)] sample volume (mL) Page 65 01'65 Page 110 of 1-19-- -. * MAY. 30. 2006 12:2lPM . SHEM EHSR 236 78 3M ENVIRON LAB 260 5 b5.I fa3 'lme 05/26 '06 1 3 5 4 NE0-. 0 903- 71 casP,,,* 4 ... , ."... *. 05/26 '06 1 3 5 4 N!-. 0.9.3-71 06P. 5 " . .. ..#I.. GLP A.oracol Amendment BEREAN . Michael A. TIES 260 5 05/26 106 13:55 ND. 9371072. Protocol ExygenPoorsSHAERR 0210 AmendmontApproval , 3M `Sponsor Representative S fos fs neil POS Be Wiliam K. Reagen, 3M Tk Environmental Laboratory Manager Date Jaisimha Kesar, Weston Solutions, `Study Director Date one B0s20 6 wn) GLP Protocol Amendment Baie Protocol Exygen PO007 737;3M Sfudy Number EO5-0270 3MENVIRONM- T Y PROJECT 05-0210; INTERIM R m B O Study Titie ANALYSISOF PERFLUOROBUTANESULFONATE(PFBS), PERFLUOROHf3ANESULFONATE (PFHS), AND PERFLUOROOCTANESULFONATE(PFOS) fN WATERS, OIL, SEDIMENT, FISH, CLAMS, VEGETATION, SMALL MAMMALIVERAND SMALL MAMMASLERUM USING LC/MS/MS FOR THE 3M DECATUR MONlTORtNG PROGRAM PROTOCOL AMENDMENT NO. 9 Amendment Date: July 7,2006 Performing Laboratory 3M Environmental, Health, and Safety Operations 3M Environmental Laboratory 935 Bush Avenue St, Paul, MN 55106 Laboratory Project IdenfMcatian E050210 EOS-02 IO GLP Protocol Amendment #9 Page 1of 3 Page 114 of 119 Protocol xygen PO007 3'33'3;M Study Number EO5-QZlO 44T9FTP9 3M ENVIRONMENTA PROJECT E05-0210; INTERIM R P RT #2 This amendment modifies the following portion($) of the protocol: The following modification to ETS 8-154.1 will be incorporated into the study: (1) Section 11.Iof the methodstatesthat the columnwill not beallowed 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. Once all the sample has loaded onto the cartridge, isolatethe cartridgefrom thevacuum and wait until ali sampleson the vacuum manifoldhave been loaded. Discardeluate. Open manifold valves and pulla vacuum on the SPE cartridgesfor approximately3 minutes to removeas much residualwater fmm the SPE cartridge. A new revision of ETS 8-154 will incorporatethe modification listedabove. If a new version of ETS 8154 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, methads, etc.) that occur during the course of this investigation. E05-02IO GLP Protocol Amendment #9 Page 2 of 3 Page 115of 119 Amendment Approval MichaelA. Shtoro, 3M Sponsor Representative Date 7A3AL 49 r 1c 3M Environmental Laboratory Manager Date Jaisjmha !@sa@ Weston Solufrbns, Study Director Date Eo5-0210 GLJ Protocol Amendment #9 Page 3 of3 Page 116of 119 ATTACHMENDT: METHODDEVIATIONS 3M ENVIRONMENTAL LABORATORY PROJECT E05-0210; INTERIM REPORT #20 Page 117of 179 3M Confidential 3M ENVIRONMENTAL LABORATORY RECORDOF NONCONFORMAN/ Ct#%E@?f@#flo;lNTERIM (3) E73 8-154.3 makes no mention of sample surrogate spikes; however, all samples will be spiked with a radiolabeled surrogate.PFOA [l,2 %I. The cor?centrationof the surrogate spike may vary ~ e or?the (~ ~ 0 ~~ev1en~t, b~u~t t~ypi~cal0~sa~mples a~re spike~d with a ~g o ~c oi~ c~e n ~af a~toifo ~ 0.05nglmL. Surrogate spike recoveries will be ~ o ~ u ~ ean~d dtisecu(ss~ed it? the final report. Actuai procedurelprocess: PFOA[I ,2 ''C] was not included as a surrogate spike in any of the samplelsarnple duplicates, or field matrix spikes collected for this project. Surrogate recoveries from the other water projects and previous interim reports for E05-0209indicate that there are ~ o ~ u b i l ~ ~ / s t aibsis~uietsywith 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 recaverics. Until this situation is fully understood and a method modification has been incorporatedto eliminate or circumvent the issue, PFOAII,2 I3C]will not be added as surroaate. - I _ _ _ _ -- ___ IN ~ c t i o n sm e n Jsuch as amendment issued, SOP revision, efc ) Corrective Action ( ~ 1Yes 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 fur the given matrix. Non-inclusion of the surrogate spike has no impact on the quality and integrity of the data presented here. Actions: LJ Halting of Work U Client Notrficatton 1zi Work Recall U ~ ~ ~ h h ~o~f Rdeiponrtg El Other Nan-i?c!usion of the surrogate will be addressed in the final report. *I I Date: -__---I_______ _- - - - I _ - I __ __--___ ~ Pk A-i ~ _ _ _ _ ~_ _ _ - ~ tV. Authorization to Resume Work _W- -lw-r-t?hatfrng q f w w k o'_ccurredr,esumptior; of work must first b ~afppmved by TechnicalManagement __- I 1 1 1 Technical Manager Approval: , j\/ ma;d)r"(*wd*lM-ip. Date: if- ===---- __ pJAi- ' -__ . _ _ _ _ _ _ _ _ _ - ~ _ _ _ _ _ - "'I *-....---7 Deviation NO. (assignedby Study Director or Project Lead at the end of study or projecf,) Protocol Deviation Page 1 of 1 Page 118of 119 3M Confidential RECORDOF NONCONFORMAN/ CDWAETK~MENTAL LABORATORY 1 1. ldentification Study / Project No. Date@)of Occurrence: Document Number: Em-0210 Interim RepoM20 February 20,2006 Exygen Protol #PO001131 Deviation type (Check one) 0 SOP E7 Protocol Amendment ##3(3M) 0 Equipment Procedure Method 0 GPO El Other: 11. 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 [7,2r3C], The concentration of the surrogate spike may vary depending on the collection event, but typical samples are spiked wit!?a nominal concentrationof 0.05 ng/mL. Surrogate spike recoveries will be documented and discussed in the final report. Actual procedurelprocess: PFOA[I,2'k]was not included as a surrogate spike in any of the sample/sample duplicates, or field matrix spikes collected for this project. Surrogate recoveriesfrom the other water projects and previous interim reports for E05-0210 indicate that there are solubility/stability issues with this surrogate in water. The 3M Environmental l a b has started an official investigation (which is not part of EO5-0210)to determine the root cause for the low recoveries. Until this situation is fully understoodand a method modificationhas been incorporated to eliminate or circumvent the issue, PFOA[I ,213Cw]ill not be added as surrogate. J//. Actions Taken (such as amendment issued, SOP revision, etc.) CorrectiveAction (ayes 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 i has no impact on the quality and integrity of the data presented here. i: Actions: 0 Halting of Work El Client Notification U Work Recall Cl Withholding of Report @OIther: Non-inclusionof the surrogate will be addressed in the final report. Is): (if halting of work or work recall are indicated): ot3h / Q k Date: +G/06 Date: li IV. Aufhurizatbn to Resume Work Where halting of work occurred, resumption of work must first be approved by Technical Management b Technical Manager Approval: Date: Protocol Deviation (assigned by Study Director or Project Lead at the end of study orpmject) Page 779of 719 Page 1 of 1