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3M Medical Department Study: T-6316.7 BACK TO MAIN Analytical Report: FACT TOX-098 LRN-U2402 Study Title Oral (Gavage) Developmental Toxicity Study of 2(N-Ethylperfluorooctanesulfonamido)-ethanol in Rats Analytical Laboratory Report Title Determination of the Concentration of PFOS, PFOSA, PFOSAA, and EtFOSE-OH in the Sera and Liver of Crl:CDBR VAF/Plus Rats Exposed to N-EtFOSE-OH Data Requirement Not Applicable Author 3M Environmental Laboratory Study Completion Date At signing Performing Laboratories Liver and Serum Analyses 3M Environmental Laboratory Building 2-3E-09, 935 Bush Avenue St. Paul, MN 55106 Project Identification 3M Medical Department Study: T-6316.7 Argus In-Life Study: 418-011 Analytical Report: FACT TOX-098 3M Laboratory Request No. U2402 Total Num ber o f Pagres 151 3M Environmental Laboratory Page 1 3M Medical Department Study: T-6316.7 BACK TO MAIN Analytical Report: FACT TOX-098 LRN-U2402 This page has been reserved for specific country requirements. 3M Environmental Laboratory Page 2 3M Medical Department Study: T-6316.7 BACK TO MAIN Analytical Report: FACT TOX-098 LRN-U2402 GLP Compliance Statement Analytical Laboratory Report Title: Determination of the Concentration of PFOS, PFOSA, PFOSAA, and EtFOSE-OH iri the Sera and Liver of Crl:CDBR VAF/Plus Rats Exposed to N-EtFOSE-OH Study Identification Numbers: T-6316.7, FACT TOX-098, LRN-U2402 This study was conducted in compliance with United States Food and Drug Administration (FDA) Good Laboratory Practice (GLP) Regulations 21 CFR Part 58, with the exceptions in the bulleted list below. Exceptions to GLP compliance: There were two study directors in this study. This study was designed as two separate studies. The in-life phase was considered to end upon weaning of F2 pups and shipment of analytical specimens. The analytical study was considered to start at the receipt of these specimens for analysis. This resulted in having two separate study directors, one for each phase of the same study. However, since the technical performance of each phase was entirely separate, no effect is expected from this exception. No expiration date on reagents/solutions labels. Sample storage stability was not determined. Some analytical reference materials have not been completely characterized. QAU did not perform an in-phase inspection during the study. u-v. / La/_ Marvin T. Case, D.V.M., Ph.D., Study Director b F ith Date Ch-o f X . * '/? /' John Butenhoff, Ph.D., Sponsor Representative o f , ?<*/ Date u*-- ... ... F<M> o )j 7XTDS Kristen J. Hansen, Ph.D., Principal Analytical Investigator Date 3M Environmental Laboratory Page 3 3M Medical Department Study: T-6316.7 BACK TO MAIN Analytical Report: FACT TOX-098 LRN-U2402 GLP Study-- Quality Assurance Statement Analytical Laboratory Report Title: Determination of the Concentration of PFOS, PFOSA, PFOSAA, and EtFOSE-OH in the Sera and Liver of Crl:CDBR VAF/Plus Rats Exposed to N-EtFOSE-OH Study Identification Numbers: T-6316.7, FACT TOX-098, LRN-U2402 This study has been inspected 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 Phase Date Reported to Management Study Director 11/06/00-1/08/00 Data 11/08/00 11/08/00 12/13/00-12/15/00 Draft report 12/15/00 12/15/00 12/21/00-12/22/00 Draft report 12/22/00 12/22/00 QAU Representative /% lc> \ Date 3M Environmental Laboratory Page 4 3M Medical Department Study: T-6316.7 BACK TO MAIN Analytical Report: FACT TOX-098 LRN-U2402 Table of Contents GLP Compliance Statement.................................................................................................3 GLP Study--Quality Assurance Statement......................................................................... 4 Study Personnel and Contributors....................................................................................... 8 Introduction and Purpose.....................................................................................................9 Test System.....................................................................................................................9 Specimen Collection and Analysis.................................................................................. 9 Specimen Receipt and Maintenance................................................................................... 10 Chemical Characterization of the Reference Standards..................................................... 10 Method Summaries.............................................................................................................. 11 3M Environmental Laboratory......................................................................................... 11 Preparatory Methods..................................................................................................11 Analytical Methods......................................................................................................11 Analytical Equipment..................................................................................................12 Data Quality Objectives and Data Integrity..........................................................................13 Data Summary, Analyses, and Results............................................................................... 13 Summary of Quality Control Analyses Results................................................................13 Statement of Data Quality...............................................................................................14 Summary of Sample Results.......................................................................................... 14 Statistical Methods and Calculations................................................................................... 15 Statement of Conclusion......................................................................................................15 Appendix A: Chemical Characterization and Control Matrices........................................... 16 Appendix B: Protocol, Amendments, and Deviations.......................................................... 17 Appendix C: Extraction and Analytical Methods................................................................. 37 FACT-M-1.0, Extraction of Potassium Perfluorooctanesulfonate or Other Anionic Fluorochemical Surfactants from Liver for Analysis Using HPLC-Electrospray/Mass Spectrometry, (8 pages).......................................................................................................38 FACT-M-3.1, Extraction of Potassium Perfluorooctane or Other Anionic Fluorochemical Compounds from Serum or Other Fluids for Analysis Using HPLC-Electrospray/Mass Spectrometry, (17 pages).................................................................................................... 46 ETS-8-4.1, Extraction of Potassium Perfluorooctanesulfonate or Other Fluorochemical Compounds from Serum for Analysis Using HPLC-Electrospray/Mass Spectrometry, (14 pages)............................................................................................................................. 63 ETS-8-6.0, Extraction of Potassium Perfluorooctanesulfonate or Other Fluorochemical Compounds from Liver for Analysis Using HPLC-Electrospray/Mass Spectrometry", (14 pages)............................................................................................................................. 77 FACT-M-2.0, Analysis of Fluorochemicals in Liver Extracts Using HPLCElectrospray/Mass Spectrometry, (8 pages)........................................................................ 91 3M Environmental Laboratory Page 5 3M Medical Department Study: T-6316.7 BACK TO MAIN Analytical Report: FACT TOX-098 LRN-U2402 FACT-M-4.1, Analysis of Potassium Perfluorooctanesulfonate or Other Fluorochemicals in Serum or Other Fluid Extracts Using HPLC-Electrospray/Mass Spectrometry, (9 pages)...............................................................................................................................99 ETS-8-5.1, Analysis of Potassium Perfluorooctanesulfonate or Other Fluorochemicals in Serum Extracts Using HPLC-Electrospray/Mass Spectrometry, (11 pages)..................108 ETS-8-7.0, Analysis of Potassium Perfluorooctanesulfonate or Other Fluorochemicals in Liver Extracts Using HPLC-Electrospray/Mass Spectrometry, (12 pages).....................119 Appendix D: Data Summary Tables..................................................................................... 131 Appendix E: Data Spreadsheets.......................................................................................... 135 Appendix F: Example Calculations.......................................................................................140 Appendix G: Interim Certificates of Analysis........................................................................ 141 Appendix H: Report Signature Page....................................................................................151 3M Environmental Laboratory Page 6 3M Medical Department Study. T-6316.7 BACK TO MAIN Analytical Report: FACT TOX-098 LRN-U2402 List of Tables Table 1. Test System Population Demographics and Dosage Levels for Study (4 1 8-0 1 1 )................................................................................................................. 9 Table 2. Characterization of the Analytical Reference Standards in Study FACT TOX-098................................................................................................................. 10 Table 3. Negative Ions Monitored in 3M Laboratory Analyses.......................................... 13 Table 4. Determinations of the LOQ in the Analyses of Sera and Liver Extracts.............. 14 Table 5. Characterization of Test Article in Study FACT TOX-098......................................16 Table 6. Characterization of the Control Matrices Used for Liver and Sera Analyses in Study FACT TOX-098......................................................................................... 16 Table 7. Reported Fluorochemical Levels in Sera Analyses in Study FACT TOX-098...... 132 Table 8. Reported Fluorochemical Levels in Liver Analyses in Study FACT TOX-098...... 133 Table 9. Average Concentration of Fluorochemical Levels in Sera Analyses in Study FACT TOX-098........................................................................................................134 Table 10. Average Concentration of Fluorochemical Levels in Liver Analyses in Study FACT TOX-098........................................................................................................134 3M Environmental Laboratory Page 7 3M Medical Department Study: T-6316.7 BACK TO MAIN Analytical Report: FACT TOX-098 LRN-U2402 Study Personnel and Contributors Study Director Marvin T. Case, D.V.M., Ph.D. 3M Corporate Toxicology - Medical Department 3M Center, Building 220-2E-02 St. Paul, MN, 55144-1000 Sponsor John L. Butenhoff, Ph.D. 3M Corporate Toxicology - Medical Department 3M Center, Building 220-2E-02 St. Paul, MN, 55144-1000 Analytical Chemistry Laboratories Liver and Serum Analyses 3M Environmental Laboratory Kristen J. Hansen Ph.D., Principal Analytical Investigator 3M Lab Contributing Personnel Lisa A. Clemen Kelly J. Dorweiler* Mark E. Ellefson Sarah A. Heimdal* Marlene M. Heying* Harold O. Johnson Kelly J. Kuehlwein* 'Contract lab professional service employees Sally A. Linda* Joseph C. Pilon* Ian A. Smith* Kathleen M. Stock* Bob W. Wynne* Richard D. Youngblom* Location of Archives All original raw data, protocol, and analytical report have been archived at the 3M Environmental Laboratory. The test article and analytical reference standard reserve samples, as well as the specimens pertaining to the analytical phase of this study are archived at the 3M Environmental Laboratory. . 3M Environmental Laboratory Page 8 3M Medical Department Study: T-6316.7 BACK TO MAIN Analytical Report: FACT TOX-098 LRN-U2402 Introduction and Purpose The purpose of the analytical study is to quantify levels of PFOS, PFOSA, PFOSAA, and EtFOSE-OH in sera samples and liver samples collected from rats exposed to N-EtFOSE-OH. This study was initiated on 30 September 1998. Test System Nineteen presumed pregnant female rats were assigned to each of five dosage groups (Groups 1 through V). Table 1 outlines the dosage levels and the number of rats per group designated for collection of analytical samples for Argus In-life study 418-011. The test system species and strain selected was the Crl:CDBR VAF/Plus (Sprague-Dawley) rat received from Charles River Laboratories, Inc., permanently identified using a Monel self piercing ear tag. Table 1. Test System Population Demographics and Dosage Levels for Study (418-011) Dosage Group I II III IV V Dosage (mgfkg/day) 0 1 5 10 20 Number of Rats 3 5 3 3 5 Specimen Collection and Analysis Sample specimens were collected from Argus (study 418-011) and sent to the 3M Environmental Laboratory for analysis. Liver, sera, fetal, and placental specimens were collected from female rats on day 18 of presumed gestation. Although fetal and placenta specimens were collected, results from these analyses will not be included in this report. A separate report may be issued for fetal tissue data. The number and type of specimens collected for analyses in the analytical phase of this study are presented below. Specimens Collected from Study Groups I through V: Serum Specimens-- 19 specimens Liver Specimens-- 19 specimens Fetuses-- 19 specimens Placentas-- 19 specimens Blood specimens were centrifuged after collection. Serum was then harvested and immediately frozen on dry ice and maintained frozen at -70C until shipped to the 3M Environmental Laboratory. Liver specimens collected from each animal were excised, weighed, and a sample section (lateral lobe) was frozen and retained at -70C until shipped to the 3M Environmental Laboratory. Fetuses and placenta were pooled per litter and retained frozen at-70C until shipment to the 3M Environmental Laboratory. The specimens were shipped to the 3M Environmental Laboratory frozen and on dry ice. 3M Environmental Laboratory Page 9 3M Medical Department Study: T-6316.7 BACK TO MAIN Analytical Report: FACT TOX-098 LRN-U2402 Sera and liver samples were extracted beginning on 30 September 1998 using an ion pairing reagent and either ethyl acetate or methyl-terf-butyl ether (MtBE). Liver samples were homogenized prior to the extraction procedure. Sample extracts were analyzed using highpressure liquid chromatography-electrospray/tandem mass spectrometry (HPLC-ESMSMS) by multiple reaction monitoring. PFOS, PFOSA, PFOSAA, and EtFOSE-OH levels were quantitated by external calibration. Analytical details are included in this report. Specimen Receipt and Maintenance The 3M Environmental Laboratory received serum, liver, fetus, and placenta specimens collected at the end of the in-life phase of Argus study 418-011 on 9-15-98 and 9-18-98 from Argus. All specimens were received frozen on dry ice and were immediately transferred to storage at -20C 10C. Control matrices used in liver and sera analyses performed during TOX-098 were obtained from commercial sources and are presented in Appendix A. Samples analyzed at the 3M Environmental Laboratory will be maintained for a period of 10 years and will be stored at the laboratory at -20C 10C. Chemical Characterization of the Reference Standards Chemical characterization information on the analytical reference standards used in this study is presented in the tabular form below. Table 2. Characterization of the Analytical Reference Standards in Study FACT TOX-098 Refe rence Standard / Form ula Acronym Source Potassium Perfluorooctanesulfonate C8F17 SO3-K+ KPFOSa 3M 3M N-Ethyl Perfluorooctanesulfbnamido ethyl alcohol C 8F i7 S 0 2N(C2H5)CH2C H 2 0 H EtFOSE-OH 3M Perfluorooctanesulfonylam ido(ethyl)acetate C 8F 7S02N(CH 2C H3)C H2C 0 0 - N a Perfluorooctanesulfonylam ido(ethyl)acetate C 8F i7S 0 2N (C H 2C H 3 )C H 2 C 0 0 - h PFOSAA 3M 3M Perfluorooctanesulfbnylam ide C8F |7S 0 2N H 2 PFOSA 3M 1H, 1H, 2H, 2HTetrahydroperfluorooctanesulfonic acid C8H 4F i3 S 0 3H THPFOS ICN aPFOS--Perfluorooctane (C8F17SO3-) "Assumed 100% until Certificate of Analysis is completed. CNA--not applicable. This lot is exhausted and cannot be characterized. TBD--to be determined Expiration Date 20 10 0 1 /0 1 /2 0 1 0 2 0 10 2 0 10 20 10 20 10 2 0 10 Storage C o n d itio n s Ambient temperature Ambient temperature Ambient temperature Ambient temperature Ambient temperature Ambient temperature Ambient temperature Chemical Lot Number 193 171 936 Physical Description White crystals Light colored powder Amber waxy solid 617 Yellow to amber liquid NB 112999-99 Tan waxy solid L2353 Amber brown waxy solid 59909 Brown powder P u r it y 1' NAC 86.4% 88.9% TBD TBD TBD TBD 3M Environmental Laboratory Page 10 3M Medical Department Study: T-6316.7 BACK TO MAIN Analytical Report: FACT TOX-098 LRN-U2402 Method Summaries Following is a brief description of the methods used during this analytical study by the 3M Environmental Laboratory. Detailed descriptions of the methods used in this study are located in Appendix C. Data collected prior to November 1999 was reworked in 2000 to accommodate improvements in data reduction methods. Both the original and "reworked" data are archived; reworked data is presented in the final results. The improved methods are documented in the form of method modifications. 3M Environmental Laboratory Preparatory Methods FACT-M-1.0, "Extraction of Potassium Perfluorooctanesulfonate or Other Anionic Fluorochemical Surfactants from Liver for Analysis Using HPLC-Electrospray/Mass Spectrometry". FACT-M-3.1, "Extraction of Potassium Perfluorooctanesulfonate or Other Anionic Fluorochemical Compounds from Serum or Other Fluids for Analysis Using HPLCElectrospray/Mass Spectrometry". An ion-pairing reagent was added to the sample and the analyte ion pair was partitioned into ethyl acetate. A portion of the ethyl acetate was transferred to a centrifuge tube and put onto a nitrogen evaporator until dry. Each extract was reconstituted in 1.0 mL of methanol, and then filtered through a 3 cc plastic syringe attached to a 0.2 pm nylon filter into glass autovials. ETS-8-4.1, "Extraction of Potassium Perfluorooctanesulfonate or Other Fluorochemical Compounds from Serum for Analysis Using HPLC-Electrospray/Mass Spectrometry" ETS-8-6.0, "Extraction of Potassium Perfluorooctane-sulfonate or Other Fluorochemical Compounds from Liver for Analysis Using HPLC-Electrospray/Mass Spectrometry" An ion-pairing reagent was added to the sample and the analyte ion pair was partitioned into MtBE. The MtBE extract was transferred to a centrifuge tube and put onto a nitrogen evaporator until dry. Each extract was reconstituted in 1.0 mL of methanol, and then filtered through a 3 cc plastic syringe attached to a 0.2 pm nylon filter into glass autovials. Analytical Methods % FACT-M-2.0, "Analysis of Fluorochemicals in Liver Extracts Using HPLC-Electrospray/Mass Spectrometry" FACT-M-4.1, "Analysis of Potassium Perfluorooctanesulfonate or Other Fluorochemicals in Serum or Other Fluid Extracts Using HPLC-Electrospray/Mass Spectrometry" ETS-8-5.1, "Analysis of Potassium Perfluorooctanesulfonate or Other Fluorochemicals in Serum Extracts Using HPLC-Electrospray/Mass Spectrometry" 3M Environmental Laboratory Page 11 3M Medical Department Study: T-6316.7 BACK TO MAIN Analytical Report: FACT TOX-098 LRN-U2402 ETS-8-7.0, "Analysis of Potassium PerfluorooctanesuHonate or Other Fluorochemicals in Liver Extracts Using HPLC-Electrospray/Mass Spectrometry" The analyses were performed by monitoring one or more product ions selected from a single primary ion characteristic of a particular fluorochemical using HPLC/ES/MS/MS. For example, molecular ion 499, selected as the primary ion for PFOS (C8F17SO;r) analysis, was fragmented to produce ion 99 (FS03-). The characteristic ion 99 was monitored in the samples and was evaluated versus one or two 1/X weighted, extracted standard curves. Analytical Equipment The actual analytical equipment settings used in the present analytical phase of this study varied slightly during actual data collection. The following is representative of the settings used during the analytical phase of this study. Liquid Chromatograph: Hewlett-Packard Series 1100 Liquid Chromatograph system Analytical column: Keystone BetasilTM C 2x50 mm (5 pm) Column temperature: Ambient Mobile phase components: Component A: 2mM ammonium acetate Component B: methanol Flow rate: 300 pL/min Injection volume: 10 pL Solvent Gradient: 13.5 minutes (minutes) %B 0.0 40% 8.5 90% 11.0 90% 12.0 40% 13.5 40% Mass Spectrometer: Micromass API/Mass Spectrometer Quattro IITMTriple Q uadruple system Software: Mass LynxTM 3.1, 3.3, and 3.4 Cone Voltage: 30-60 V Collision Gas Energy: 25-45 eV Mode: Electrospray Negative Source Block Temperature: 150C 10C Electrode: Z-spray Analysis Type: Multiple Reaction Monitoring (MRM) 3M Environmental Laboratory Page 12 3M Medical Department Study: T-6316.7 BACK TO MAIN Analytical Report: FACT TOX-098 LRN-U2402 Table 3. Negative Ions Monitored in 3M Laboratory Analyses Target Analyte Primary Ion (amu) Product Ion (amu) PFOS 499.0 80.0, 99.0, 130.0 PFOSA 498.0 78.0 PFOSAA 584.0 83.0, 169.0 EtFOSE-OH 630.0 59.0 THPFOS 427.0 80.0 Data Quality Objectives and Data Integrity The following data quality objectives (DQOs) were indicated in the method performance section of ETS-8-5.1, Analysis of Potassium Perfluorooctanesulfonate or Other Fluorochemicals in Serum Extracts Using HPLC-Electrospray/Mass Spectrometry and ETS-8-7.0, Analysis of Perfluorooctanesulfonate or Other Fluorochemicals in Liver Extracts Using HPLCElectrospray/Mass Spectrometry: Linearity: The coefficient of determination (r2) equal to or greater than 0.980 Acceptable Spike Recoveries: 70-130% Data Summary, Analyses, and Results Data quality objectives for the analytical phase of this study outlined in the 3M Environmental Laboratory Methods ETS-8-5.1 and ETS-8-7.0 (see Appendix C) were met with the exceptions noted in this report. Summary of Quality Control Analyses Results Linearity: The coefficient of determination (r2) of the standard curve was >0.980. Calibration Standards: Quantitation of the target analytes was based on linear regression analysis 1/x weighted of an opening extracted curve or two extracted matrix curves bracketing each group of samples. High or low points on the curve may have been deactivated to provide a better linear fit over the curve range most appropriate to the data. Low curve points with peak areas less than two times that of the extraction blanks were deactivated to disqualify a data range that may have been significantly affected by background levels of the analyte. Occasionally, a single mid-range curve point that was an obvious outlier may have been deactivated. Quantitation of each analyte was based on the response of one or more specific product ion(s) using the multiple response-monitoring mode of the instrument (see Appendix C, Analytical Methods). Limits o f Quantitation (LOQ): The LOQ is equal to the lowest acceptable standard in the calibration curve (defined as a standard within 30% of the theoretical value), and is at least two times the analyte peak area detected in the extraction blanks. 3M Environmental Laboratory Page 13 3M Medical Department Study: T-6316.7 BACK TO MAIN Analytical Report: FACT TOX-098 LRN-U2402 Table 4. Determinations of the LOQ in the Analyses of Sera and Liver Extracts A n a ly te PFOS PFOSA PFOSAA EtFOSE-OH Values are approximate LOQ--Limit of Quantitation Sera Method LOQ (p g /m L )* 0.025 0.005 0.025 0.010 Liver M ethod LOQ (M9/g)* 0.060 0.120 0.060 0.060 Blanks: All blanks were below the lower limit of quantitation for the quantitative analysis of compounds of interest. Although the matrix blanks were clean, some liver data for G1-G3 should be considered qualitative, as these samples may have been affected by background levels of the analyte found in the method blanks; specific data points affected are noted in the results table. To simplify analyses that were complicated by endogenous levels of fluorochemicals in unexposed rat sera and liver, rabbit sera and liver were selected as a suitable surrogate matrices. Precision: Precision was not specifically determined within this study, but has been characterized to be better than 30% for this method. Matrix Spikes: Matrix spikes and matrix spike duplicates were extracted with each set of sera and liver samples and analyzed during analytical runs at the 3M Environmental Laboratory. Rat sera and liver from control animals were spiked prior to extraction. All target analytes were spiked at approximately 250 ng/mL or 250 ng/g. Sera matrix spikes for PFOSAA and EtFOSE-OH were within 30% of the theoretical concentration. One matrix spike for PFOS and one for PFOSA were outside of this range (152% and 149%, respectively). The average spike recovery for PFOS in sera was 137% and for PFOSA it was 126%. Matrix spikes prepared in liver (PFOS, PFOSA, PFOSAA, and EtFOSE-OH) were compliant within 30% for all analytes. Surrogates: The surrogate (THPFOS) was added to all samples and standards. THPFOS was not used for quantitation, but was used to monitor for gross instrument failure. Statem ent of Data Quality It is not possible to verify true recovery of endogenous analyte from tissues without radio-labeled reference material. The only measurement of accuracy available at this time, matrix spike studies, indicate that the data are quantitative to 40%. Summary of Sample Results Some PFOS results (those obtained using lot # 171) have been corrected for purity of the analytical reference material. Uncorrected results are noted in the data tables. Samples from Control Animals: Low levels of PFOS were detected in the liver of the control animals. These levels were significantly lower than those found in the low dose test animals. 3M Environmental Laboratory Page 14 3M Medical Department Study: T-6316.7 BACK TO MAIN Analytical Report: FACT TOX-098 LRN-U2402 Samples from Dosed Animals: In general, levels of the target analytes present in the sera and liver of the test animals increased with dose group. Detailed sample data tables are presented in Appendices D and E. Statistical Methods and Calculations Statistical methods were limited to the calculation of means and standard deviations. See Appendix F for example calculations used to generate the liver and serum sample data in FACT TOX-098. Statement of Conclusion Under the conditions of the oral development toxicity of N-EtFOSE, PFOS, PFOSA, PFOSAA, and EtFOSE-OH were observed in the sera and liver of pregnant rats dosed with N-EtFOSE-OH during the in-life phase of the study. 3M Environmental Laboratory Page 15 3M Medical Department Study: T-6316.7 BACK TO MAIN Analytical Report: FACT TOX-098 LRN-U2402 Appendix A: Chemical Characterization and Control Matrices Table 5. Characterization of Test Article in Study FACT TOX-098 Test Article Chemical Name Source Expiration Date Storage Conditions Chemical Lot # Physical Description Purify N-EtFOSE-OH 2(N-Ethylperf!uorooctanesulfonamido)-ethanol 3M 5/2000 Ambient temperature FM-3929 (30035, 30037, 30039) Waxy solid 97.4% Table 6. Characterization of the Control Matrices Used for Liver and Sera Analyses in Study FACT TOX-098 Control Matrix Rat Serum TN-A-2001 Rabbit Serum TN-A-2382 Rabbit Liver TN-A-0809 Rabbit Liver TN-A-0810 Source Expiration Date Storage Conditions Chemical Lot # Physical Description Sigma 2010 -20C 10C 17H9306 Rat Serum Sigma 2010 -20C 10C 118H8418 Rabbit Serum CHW 2010 -20C 10C F00012 Rabbit Liver CHW 2010 -20C 10C F00013 Rabbit Liver 3M Environmental Laboratory Page 16 3M Medical Department Study: T-6316.7 BACK TO MAIN Analytical Report: FACT TOX-098 LRN-U2402 Appendix B: Protocol, Amendments, and Deviations 3M Environmental Laboratory Page 17 3M Medical Department Study: T-6316.7 BACK TO MAIN Analytical Report: FACT TOX-098 LRN-U2402 3M Environmental Laboratory Pr o to c o l - A n alytic al Study Oral (Gavage) Developmental Toxicity Study of 2(N-Ethylperfluorooctanesulfonamido)-ethanol in Rats In-vivo study reference number: Argus 418-011 Study number: FACT-TOX-098 Test substance: 2(N-Ethylperfluorooctanesulfonamido)-ethanol (N-EtFOSE-OH) Name and address of Sponsor: Marvin Case 3M Toxicology Services 3M Center Building 220-2E-02 St. Paul, MN 55144 Name and address o f testing facility: 3M Environmental Technology and Services 935 Bush Avenue, Building 2-3E-09 St. Paul, MN 55106 Sponsor approval date: Experimental start date: October 9,1998 Expected termination date: July 16, 1999 Method numbers and revisions: . FACT-M-1.0, Extraction of Potassium Perfluorooctar esulfonate or Other Anionic Surfactants from Liver for Analysis Using HPLC-Electrospray/Mass Spectrometry . ? FACT-M-2.0, Analysis of Fluorochemicals in Liver Extracts Using HPLCElectrospray/Mass Spectrometry FACT-M-3.1, Extraction o f Potassium Perfluorooctamesulfonate or Other Anionic Surfactants from Serum or Other Fluid for Analysis Using HPLCElectrospray/Mass Spectrometry FACT-M -4.I, Analysis o f Fluorochemicals in Serum or Other Fluid Extracts Using HPLCElectrospray/Mass Spectrometry Author: Lisa Clemen ihL'U*____ Kris Hansen Study Director FACT-TOX-098, U2402 Argus #418-011 Page 1 of 5 `W g g Date Marvin Case Sponsor Representative Date 3M Environmental Laboratory Page 18 3M Medical Department Study: T-6316.7 BACK TO MAIN Analytical Report: FACT TOX-098 LRN-U2402 1.0 Purpose__________________________________________________________ ;________ The analytical portion of this toxicity study is designed to evaluate the levels o f potassium perfluorooctane sulfonate (PFOS), or another metabolite of 2(Nethylperfluorooctanesulfonamido)-ethanol (N-EtFOSE-OH) designated by the study director, in the livers of the test system, or other tissues and fluids as necessary. The in life portion of this study was conducted at Argus Research Laboratories (Argus 418-011). 2.0 Regulatory Compliance____________________________ ;________________________ This study will be conducted in compliance with the Food and Drug Administration Good Laboratory Practices regulation as stated in 21 CFR 58. Any exceptions will be noted in the final report 3.0 Test Materials___________________;______________________________ :____________ 3.1 Test, control, and reference substances and matrices 3.1.1 Analytical reference substance: Potassium perfluorooctanesulfonate (PFOS), lot #217 3.1.2 Analytical reference substance matrix: Fat liver, serum, and whole blood 3.1.3 Analytical control substance: None 3.1.4 Analytical control substance matrix: Rat liver, serum, and whole blood 3.2 Source of materials 3.2.1 Analytical reference substance: 3M Specialty Chemical Division; traceability information will be included in the final report 3.2.2 Analytical reference substance matrix: Argus Research Laboratories; traceability information w ill be included in the final report 3.2.3 Analytical control matrix: 3.2.3.1 Rat liver - Argus Research Laboratories; traceability information will be included in the final report; or Rabbit liver - Covance Laboratories; traceability information will be included in the final report. 3.2.3.2 Rat serum - Sigma Chemical Company; traceability information will be included in the final report. 3.2.3.3 Rat whole b lo o d -3 M Toxicology; traceability information will be included in the final report. 3.3 Number of test and control samples. Liver and serum samples will be received for testing from 16 test and 3 control animals for the toxicokinetic portion of the study. Liver and serum samples for testing will be received from 100 test and 25 control FACT-TOX-098, U2402 Argus #418-011 Page 2 of 5 3M Environmental Laboratory Page 19 3M Medical Department Study: T-6316.7 BACK TO MAIN Analytical Report: FACT TOX-098 LRN-U2402 animals for the developmental portion of the study. Fetus, placenta, or other samples will be tested at the discretion of the Study Director. 3.4 Identification o f test and control samples: The samples are identified using the Argus Research Laboratories identifiers, which consist of the Argus project number, the anim al number, the group designation, and the draw date. 3.5 Purity and strength o f materials: Characterization of the purity and identity of the reference material is the responsibility of the Sponsor. 3.6 Stability of test material: Characterization of the stability of the test material is the responsibility of the Sponsor. 3.7 Storage conditions for test materials: Test materials are stored at room temperature. Samples are stored at -20 10 C. 3.8 Disposition o f test and/or control substances: Biological tissues and fluids are retained per GLP regulation. 3.9 Safety precautions: Refer to the material safety data sheets of chemicals used. Wear appropriate laboratory attire, and follow adequate precautions for handling biological materials and preparing samples for analysis. 4.0 Experimental - Overview_____________________________________________________ Tissues from animals dosed as described in Argus Research Laboratories Protocol #418-011 will be received for analysis of fluorochemicals. Mated female rats were dosed on Day 6 of presumed gestation, with administration continuing through Day 17. At Day 18, serum and liver samples, as well as fetus and placenta samples, were taken from rats in the toxicokinetic portion of the study. A t Day 20 for the rats remaining in the study, samples of serum and liver were taken, as well as fetus and placenta. Dosage samples will be provided from Argus Research Laboratories for concentration level confirmation. These samples will not be extracted and analyzed according to GLP regulations. The data collected will be provided to the Sponsor as an attachment to the data package. At the discretion of the Study Director, a series of analyticd tests will be performed on select tissues. Initially, all liver and serum samples will be analyzed, using the methods listed in section 5.0, for PFOS by Electrospray/mass spectrometry (ES/MS). On the basis of findings from these analyses, additional samples may be evaluated. If additional analysis is performed, a protocol amendment will be written to add the matrices and methods to th protocol. At the discretion of the Study Director, select analysis may be performed by a contract laboratory where competence has been demonstrated, using validated analytical methods. If a contract laboratory is used, this protocol will be amended to include the required information. The methods, data, and contract laboratory will be identified in the data package provided to die Sponsor. FACT-TOX-098, U2402 Argus #418-011 Page 3 o f 5 3M Environmental Laboratory Page 20 3M Medical Department Study: T-6316.7 BACK TO MAIN Analytical Report: FACT TOX-098 LRN-U2402 5.0 Experimental - Analytical Methods______________________________ ^______________ 5.1 For analysis performed by the 3M Environmental laboratory, the following methods will be used: 5.1.1 FACT-M-1.0, Extraction of Potassium Perfluorooctanesulfonate or Other Anionic Surfactants from Liver for Analysis Using HPLC-Electrospray/Mass Spectrometry 5.1.2 FACT-M-2.0, Analysis of Fluorochemicals in Liver Extracts Using HPLCElectrospray/Mass Spectrometry 5.1.3 FACT-M-3.1, Extraction of Potassium Perfluorooctanesulfonate or Other Anionic Surfactants from Serum or Other Fluid for Analysis Using HPLCElectrospray/Mass Spectrometry 5.1.4 FACT-M-4.1, Analysis of Fluorochemicals in Serum or Other Fluid Extracts . Using HPLC-Electrospray/Mass Spectrometry 5.2 If analysis is performed at a contract analytical laboratory, copies of the validated methods will be included in the data packet provided to the Study Director. 6.0 Data Analysis_______________________________________________________________ 6.1 Data reporting: For analysis performed by a contract laboratory, the contract laboratory will provide all data to the analytical phase Study Director, and copies of the methods will be attached to the data. The contract laboratory and the data it provides will be identified in the data packet provided by the analytical phase Study Director to the Sponsor. 6.2 Data transformations and analysis: Data will be reported as the concentration (weight/weight) of the target analyte per tissue or s:ample, or of the target analyte per unit of tissue or fluid. 6.3 Statistical analysis: Statistics used may include regression analysis o f the serum concentrations over time, and standard deviations calculated for the concentrations within each dose group. If necessary, simple statistical tests, such as Student's t test, may be applied to evaluate statistical difference. 7.0 Maintenance of Raw Data and Records______ _________________________________ 7.1 The following raw data and records will be retained in th study folder in the archives according to AMDT-S-8: 7.1.1 Approved protocol and amendments 7.1.2 Study correspondence 7.1.3 Shipping records 7.1.4 Raw data 7.1.5 Electronic copies of data FACT-TOX-098, U2402 Argus #418-011 Page 4 o f 5 . 3M Environmental Laboratory Page 21 3M Medical Department Study: T-6316.7 BACK TO MAIN Analytical Report: FACT TOX-098 LRN-U2402 7.2 Supporting records to be retained separately from the study folder in the archives according to AMDT-S-8 will include at least the following: 7.2.1 Training records 7.2.2 Calibration records 7.2.3 Instrument maintenance logs 7.2.4 Standard Operating Procedures, Equipment Procedures, and Methods 7.2.5 Appropriate specimens 8.0 References______'______ ^__________________________________________________ 8.1 3M Environmental Laboratory Quality System Chapters 1, 5 and 6 8.2 Other applicable 3M Environmental Laboratory Quality System Standard Operating Procedures 9.0 Attachments___________^____________________________________ ;______ '_______ 9.1 Copies o f the following validated 3M Environmental Laboratory methods are attached for information purposes: 9.1.2 FACT-M-1.0, Extraction of Potassium Perfluorooctanesulfonate or Other Anionic Surfactants from Liver for Analysis Using HPLC-Electrospray/Mass Spectrometry 9.1.2 FACT-M-2.0, Analysis ofFluorochemicals in Liver Extracts Using HPLCElectrospray/Mass Spectrometry 9.1.3 FACT-M-3.1, Extraction of Potassium Perfluorooctanesulfonate or Other Anionic Surfactants from Serum or Other Fluid for Analysis Using HPLCElectrospray/Mass Spectrometry . 9.1.4 FACT-M -4.1, Analysis o f Fluorochemicals in Serum or Other Fluid Extracts Using HPLC-Electrospray/Mass Spectrometry 9.2 Argus protocol 418r011 FACT-TOX-098, U2402 Argus #418-011 Page 5 o f 5 3M Environmental Laboratory Page 22 3M Medical Department Study: T-6316.7 BACK TO MAIN Analytical Report: FACTTOX-098 LRN-U2402 Study Tide Oral (Gavage) Developmental Toxicity Study of 2(N-Ethylperfluorooctanesulfonamido)-ethanol in Rats PROTOCOL AMENDMENT NO. 1 Amendment Date: 22 February 2000 Performing Laboratory 3M Environmental Technology & Safety Services 3M Environmental Laboratory 935 Bush Avenue St. Paul, MN 55106 Laboratory Project Identification FACT TOX-098 ET&SS-- U2402 Argus Study: 418-011 . 3M Medical Department Study: T-6316.7 3M Environmental Laboratory 3M Environmental Laboratory Page 23 3M Medical Department Study: T-6316.7 BACK TO MAIN Analytical Report: FACT TOX-098 LRN-U2402 Protocol LRN-U2402 Amendment Number 1 This amendment modifies the following portion(s) of the protocol: 1. Protocol reads: The study director for the present study was identified in the protocol, as Kristen J. Hansen, Ph.D. Amend to read: The role of study director for the present study was reassigned to Marvin T. Case, D.V.M., Ph.D., as of the signing o f this amendment. Reason: The role of study director was reassigned in an effort to ensure compliance with Good Laboratory Practice Standards that outline study personnel requirements (refer to 21 CFR Part 58). 2. Protocol reads: The sponsor for the present study was identified as M aran T. Case, D.V.M., Ph.D. Amend to read: The role of sponsor for the present study was reassigned to John L. Butenhoff, Ph.D., as of 20 January 2000. Reason: To ensure that the study director does not also carry the duties of study sponsor, the sponsor role was reassigned. In this manner, personnel responsibilities and workload are more evenly balanced. 3. Protocol reads: 3.1 Test, control, and reference substances and matrices 3.1.2 Analytical reference substance matrix: Rat liver, serum, and whole blood 3.1.4 Analytical control substance matrix: Rat liver, serum, and whole blood Amend to read: 3.1 Test, control, and reference substances and matrices 3.1.2 Analytical reference substance matrix: Rat liver, serum, pooled fetal tissue(s), and whole blood 3.1.4 Analytical control substance matrix: Rat liver, serum, pooled, fetal tissue(s), and whole blood . Reason: Analysis o f fetal tissue for the target chemical and/or ils analytes was added to the scope of the study following the issuance of the original protocol. 3 M Environmental Laboratory 3M Environmental Laboratory Page 24 BACK TO MAIN 3M Medical Department Study: T-6316.7 Analytical Report: FACT TOX-098 LRN-U2402 Protocol LRN-U2402 Amendment Number 1 4. Protocol reads: 7.1 The following raw data and records will be retained in the study folder in the archives according to AMDT-S-8: 7.1.1 Approved protocol and amendments 7.1.2 Study correspondence 7.1.3 Shipping records 7.1.4 Raw data 7.1.5 Electronic copies of data 7.2 Supporting records to be retained separately from the study folder in the archives according to AMDT-S-8 will include at least the following: 7.2.1 Training records 7.2.2 Calibration records 7.2.3 Instrument maintenance logs 7.2.4 Standard Operating Procedures, Equipment Procedures, and Methods 7.2.5 Appropriate specimens Amend to read: "The original data, or copies thereof, will be available at the 3M Environmental Laboratory to facilitate audits of the study during its progress and before acceptance of the final report. When the final report is completed, all original paper data, including: approved protocol and amendments, study correspondence, shipping records, raw data, approved final report, and electronic copies of data will be retained in the archives of the 3M Environmental Laboratory. All corresponding training records, calibration records, instrument maintenance logs, standard operating procedures, equipment procedures, and methods will be retained in the archives of the facility performing each analysis." Reason: To direct subcontract laboratories in the disposition of ihe items listed above. 3M Environmental Laboratory 3M Environmental Laboratory Page 25 3M Medical Department Study: T-6316.7 BACK TO MAIN Analytical Report: FACT TOX-098 LRN-U2402 Protocol LRN-U2402 Amendment Number 1 5. Protocol reads: 3.8 Disposition of test and/or control substances: Biological Tissues and fluids are retained per GLP regulation. Amend to read: 3.8 Specimens will be maintained in the 3M Environmental Laboratory specimen archives. All specimens sent to sub-contract laboratories will be returned to the 3M Environ mental Laboratory upon completion o f analysis and submission of the sub-contract laboratory(s) final report. The specimens will be returned with the following documentation: the signed original chain of custody and records of storage conditions while at the sub-contract facility. Reason: To define in detail the appropriate disposition of specimens analyzed at subcontract laboratories. 6. Protocol reads: Method numbers and revisions: FACT-M-1.0, Extraction of Potassium Perfluorooctanesulfonate or Other Anionic Surfactants from Liver for Analysis Using HPLC-Electrospray/Mass Spectrometry. FACT-M-2.0, Analysis of Fluorochemicals in Liver Detracts Using HPLC-Electrospray/ Mass Spectrometry FACT-M-3.1, Extraction of Potassium Perfluorooctanesulfonate or Other Anionic Surfactants from Serum or Other Fluid for Analysis Using HPLC-Electrospray/ Mass Spectrometry . FACT-M-4.1, Analysis o f Fluorochemicals in Serum or Other Fluid Extracts Using HPLC- Electrospray/Mass Spectrometry Amend to read: Method numbers and revisions: ETS-8-6.0 "Extraction of Potassium Perfluorooctanesulfonate or Other Fluorochemical Compounds from Liver for Analysis Using HPLC-Electrospray/Mass Spectrometry" ETS-8-7.0 "Analysis of Potassium Perfluorooctanesulfonate or Other Fluorochemical Compounds in Liver Extracts Using HPLC-Electrospray/Mass Spectrometry" ETS-8-4.1, "Extraction of Potassium Perfluorooctanesulfonate or Other Fluorochemical Compounds from Serum for Analysis Using HPLC-Electrospray Mass Spectrometry" ETS-8-5.1, "Analysis of Potassium Perfluorooctanesulfonate or Other Fluorochemical Compounds in Serum Extracts HPLC-Electrospray Mass Spectrometry" Reason: New methodologies were implemented following the approval of the original protocol for FACT Tox-098. 3M Environmental Laboratory 3M Environmental Laboratory Page 26 3M Mendicai Department Study: T-6316.7 Amendment Approval BACK TO MAIN Analytical Report: FACT TOX-098 LRN-U2402 Protocol LRN-U2402 Amendment Number 1 ___ _ * ___________ ____________________________________________________________________________ ____________________________ John L. Butenhoff, PhD., Sponsor Representative Date Kristen J. Hansen, PhD., Outgoing Study Director <2 Date - 74100 Marvin T. Case, D.V.M., PhD., Incoming Study Director / tfXrJ'VtA Q--g-) Date 3M Environmental Laboratory 3M Environmental Laboratory Page 27 3M Medical Department Study: T-6316.7 BACK TO MAIN Analytical Report: FACT TOX-098 LRN-U2402 Study Title Oral (Gavage) Developmental Toxicity Study of 2(N-Ethylperfluorooctanesulfonamido)-ethanol in Rats PROTOCOL AMENDMENT NO. 2 Amendment Date: November 21,2000 Performing Laboratory 3M Environmental Technology & Safety Services 3M Environmental Laboratory 935 Bush Avenue St. Paul, MN 55106 Laboratory Project identification FACT TOX-098 ET&SS LRN-U2402 Argus Study: 418-011 3M Medical Department Study: T-6316.7 3M Environmental Laboratory 3M Environmental Laboratory Page 28 3M Medical Department Study: T-6316.7 BACK TO MAIN Analytical Report: FACT TOX-098 LRN-U2402 Protocol FACT TOX-098 Amendment No. 2 This amendment modifies the following portion(s) of the protocol: 1. Protocol reads: There is not a principal analytical investigator assigned for this study. Amend to read: The role of principal analytical investigator for the study was assigned to Kristen J. Hansen, Ph.D. as of the signing of this amendment. Reason: The role of principal analytical investigator was assigned in an effort to ensure compliance with Good Laboratory Practice Standards that outline study personnel requirements. . 3M Environmental Laboratory 3M Environmental Laboratory Page 29 3M Medical Department Study: T-6316.7 BACK TO MAIN Analytical Report: FACT TOX-098 LRN-U2402 Amendment Approval Protocol FACT TOX-098 Amendment No. 2 ___ Z JohnL. Butenhoff, Ph.D., Sponsor Representative Y Date TQm . Marvin f . Case, D.V.M., Ph.D., Study Director ^A c Date Q jytrtj 3M Environmental Laboratory 3M Environmental Laboratory Page 30 3M Medical Department Study: T-6316.7 BACK TO MAIN Analytical Report: FACT TOX-098 LRN-U2402 Study Title Oral (Gavage) Developmental Toxicity Study of 2(N-Ethylperfluorooctanesulfonamido)-ethanol in Rats PROTOCOL AMENDMENT NO. 3 Amendment Date: November 21,2000 Performing Laboratory 3M Environmental Technology & Safety Services 3M Environmental Laboratory 935 Bush Avenue St. Paul, MN 55106 Laboratory Project Identification FACT TOX-098 ET&SS LRN-U2402 Argus Study: 418-011 3M Medical Department Study: T-6316.7 . 3M Environmental Laboratory 3M Environmental Laboratory Page 31 BACK TO MAIN 3M Mcidical Department Study: T-6316.7 Analytical Report: FACT TOX-098 LRN-U2402 Protocol FACT TOX-098 Amendment No. 3 This amendment modifies the following portion(s) of the protocol: 1. Protocol reads: 1.0 Purpose: The analytical portion of this toxicity study is designed to evaluate the levels of potassium perfluorooctane sulfonate (PFOS), or another metabolite of 2(N-ethylperfluorooctanesulfonamido)-ethanol (N-EtFOSE-OH) designated by the study director in the livers of the test system, or other tissues and fluids as necessary. A mend to read: 1.0 Purpose: The analytical portion of this toxicity study iis designed to evaluate the levels of potassium perfluorooctanesulfonate (PFOS), N-ethyl periluorooctanesulfonamido ethyl alcohol (EtFOSE-OH), perfluorooctanesulfonylamido(ethyl)acetate (PFOSAA), and perfluorooctanesulfonylamide (PFOSA) in the livers of the test systems, or other tissues and fluids as necessary. Perfluorooctanesulfonylethylamide (PFOSEA) and M556 will be monitored but not used for GLP purposes and will not be part of this study. Reason: Specific target analytes are known. 2. Protocol reads: 3.1.1 Analytical reference substance: Potassium perfluorooctanesulfonate (PFOS), lot #217. Amend to read: 3.1.1 Analytical reference substances: Potassium perfluorooctanesulfonate (PFOS), N-ethyl periluorooctanesulfonamido ethyl alcohol (EtFOSE-OH), perfluorooctanesulfonylamido(ethyl)acetate (PFOSAA), and perfluorooctanesulfonylamide (PFOSA). Reason: Include the additional reference substances used. 3 M Environmental Laboratory 3M Environmental Laboratory Page 32 3M Meidical Department Study: T-6316.7 BACK TO MAIN Analytical Report: FACT TOX-098 LRN-U2402 Amendment Approval Protocol FACT TOX-098 Amendment No. 3 John L. Butenhoff, Ph.D., Sponsor Representative jy f/S SO Date TOoof Marvin T. Case, D.V.M., PhD., Study Director /X, Date 3 M Environmental Laboratory 3M Environmental Laboratory Page 33 3M Medical Department Study: T-6316.7 BACK TO MAIN Analytical Report: FACT TOX-098 LRN-U2402 Record of Deviation Study / Project No. .Identification TOX0098 (LIMS #U2402) Deviation type (Check one) SOP X Method Equipment Procedure Protocol Other: Document number FACT-M-2.1 Date(s) of occurrence 10/02/98 and 10/06/98 !`i-- II. Description Required procedure/process: Section 11.1.: .. ..The average o f two standard curves will be plotted by linear regression, not forced through zero,... Actual procedure/process: Data was originally analyzed as described by the method. However, after the analysis was complete, it was determined that applying a 1/X weighting: to the curve dramatically improved method accuracy at the low end of the curve. The original data sets were reworked utilizing the improved practice. :&W?' -i ' III. ACtionsTaker,1 (such as amendment issud/SOP revision, etc.) ` '"' l - Deviation written. Method revalidated utilizing improved practices. Original and reworked data are included with the raw data. The reworked data is reported in the final results. Recorded by Date tuzo/cn The reworked data has a higher degree of accuracy than the original data. There is no adverse impact on the study. khh n / t o / 07) Authorized by T ( L ^ 2i)Mbr Tobr. BvitX Date D,'rtcU: AW Co, Deviation No /(aaescsiingnnwedl bkvy SCthuI.dtvy DDiirr^ercft/ovrr ro\rr DPrmoij'ec/11t Lead at tbe end o f study or project) Attachment A: Record of Deviation 3M Environmental Laboratory ETS-4-8.0 lofi P a g e 'T o iZ . V f k-h UlZ/eO Page 34 3M Medical Department Study: T-6316.7 BACK TO MAIN Analytical Report: FACT TOX-098 LRN-U2402 Record of Deviation I. Identification Study/Project No. FACT-TOX-098 Argus 418-011 Deviation Type (Check one) SOP X Method Equipment Procedure Protocol Other: Document Numbers): TS-8-5.1 Date(s) o f occurrence: 09/28/99 and 10/02/99 II. Description: Required Procedure/process: 14.4.1 Matrix spike recoveries must be within +/-30% o f the spiked concentration. Actual Procedure/process: One matrix spike in sera showed a higher recovery for PFOS (152%, average 137%) And PFOSA (149%; average 126%). : ~~ ill. Actions Taken: (such as amendment issued, SOP revision, etc.) This deviation was written. The stated accuracy of these data will be changed in the final report to reflect these recoveries. ~ Recorded By Date 13-/Iw/o IV. Impact on Study / Project This deviation will have no adverse affect on these data. toll IIINIOO Authorized By (Study Director / Project Lead) fe d * b& Z' (/a Date sponsor RtprfrytniaFli^T)in BuftAhoff 3M Environmental Laboratory Form ETS-4-8.0 -Sliuiu Dire-dor'. Kori- Ca.sc. 0 Deviation No. (assigned by Study Director or Project Lead at the end o f study o r project) 3M Environmental Laboratory Page 35 3M Medical Department Study: T-6316.7 BACK TO MAIN Analytical Report: FACT TOX-098 LRN-U2402 Record of Deviation I. Identification Study/Project No. FACT-TOX-098~ Argus 418-011 Deviation Type (Check one) SOP X Method Equipment Procedure Protocol Other: Document Num bers): ETS-8-7.0 Date(s) of occurrence : Entire study II. Description: Required Procedure/process: Section 13.1.6 describes the calculations that should be used to convert extract concentration to matrix concentration. Actual Procedure/process: In order to accommodate purity information, the actual calculation used varied somewhat from that written in section 13.1.6. Two additional factors, salt correction and standard purity, were added. The first accommodates the mass difference between the analytical standard (C8F17S03K) and the target analyte (C8F17S03-), while: the second addresses the purity o f the analytical reference material, determined after the study was completed. III. Actions Taken: (such as amendment issued, SOP revision, etc.) This deviation was written. Recorded By Date tjh WwloX) IV. Impact on Study / Project The updated calculations accommodate new information and are an improvement. No adverse affect on the study. _____ ___________________________ ^ __. i%lxo/tn> _____ Authorized By (Study Director / Project Lead) [Date Sponsor fUprt-Unia-hrt.- Toll ftuWytiiof-C 3M Environmental Laboratory Form ETS-4-8.0 Dm&W- Mftrv G&1*. 4 iD eviation N o. (assigned by Study Director or Project Lead at the end o f study or project) 3M Environmental Laboratory Page 36 3M Medical Department Study: T-6316.7 BACK TO MAIN Analytical Report: FACT TOX-098 LRN-U2402 Appendix C: Extraction and Analytical Methods This appendix includes the following methods: Preparatory Methods FACT-M-1.0, Extraction of Potassium Perfluorooctanesulfonate or Other Anionic Fluorochemical Surfactants from Liver for Analysis Using HPLC-Electrospray/Mass Spectrometry, (8 pages) FACT-M-3.1, Extraction of Potassium Perfluorooctane or Other Anionic Fluorochemical Compounds from Serum or Other Fluids for Analysis Using HPLC- Electrospray/Mass Spectrometry, (17 pages) ETS-8-4.1, Extraction of Potassium Perfluorooctanesulfonate or Other Fluorochemical Compounds from Serum for Analysis Using HPLC-Electrospray/Mass Spectrometry, (14 pages) ETS-8-6.0, Extraction of Potassium Perfluorooctanesulfonate or Other Fluorochemical Compounds from Liver for Analysis Using HPLC-Electrospray/Mass Spectrometry", (14 pages) Analytical Methods . FACT-M-2.0, Analysis of Fluorochemicals in Liver Extracts Using HPLC-Electrospray/Mass Spectrometry, (8 pages) FACT-M-4.1, Analysis of Potassium Perfluorooctanesulfonate or Other Fluorochemicals in Serum or Other Fluid Extracts Using HPLC-Electrospray/Mass Spectrometry, (9 pages) ETS-8-5.1, Analysis of Potassium Perfluorooctanesulfonate or Other Fluorochemicals in Serum Extracts Using HPLC-Electrospray/Mass Spectrometry, (11 pages) ETS-8-7.0, Analysis of Potassium Perfluorooctanesulfonate or Other Fluorochemicals in Liver Extracts Using HPLC-Electrospray/Mass Spectrometry, (12 pages) 3M Environmental Laboratory Page 37 3M Medical Department Study: T-6316.7 BACK TO MAIN Analytical Report: FACTTOX-098 LRN-U2402 3M Environmental Laboratory M ethod E x tr a c tio n o f P o ta ssiu m perflu o ro o cta n esu lfo n a te o r O t h e r An io n ic F l u o r o c h e m ic a l Su rfa cta nts fr o m L iv e r fo r An alysis U sing H P L C -E lectrospray /M ass Spectr o m etr y M ethod N um ber: FACT-M-1.0 Author: Lisa Clemen Approved By: 's H I Laboratory Manager Group Leader nl*+ H Technical Reviewer ------` Adoption Date: ` f a h j ' i Revision Date: lijj\ -------------------. Date ,5/Z-U Date s h ih ? Date 1.0 Scope and Application 1.1 Scope: This method is for the extraction of Potassium Perfluorooctanesulfonate (PFOS) or other fluorochemical surfactants from liver. 1.2 Applicable Com pounds: Fluorochemical surfactants or other fluorinated compounds. 1.3 M atrices: Rabbit, rat, bovine, and monkey livers or other livers as designated in the validation report M icrosoft7.0.1/95 FACT-M-1.0 Extraction of PFOS fr om Liver Pagel of8 3M Environmental Laboratory Page 38 3M Medical Department Study: T-6316.7 BACK TO MAIN Analytical Report: FACTTOX-098 LRN-U2402 2.0 Summary of Method________________;_________________________________________ 2.1 This method describes how to extract potassium perfluorooctanesulfonate (PFOS) or other fluorochemical surfactants from liver using ion pairing reagent and 5.0 mLs o f ethyl acetate. An ion pairing reagent is added to each sample and partitioned into ethyl acetate. Four mLs of extract is removed to a centrifuge tube and put onto a nitrogen evaporator until dry. Each extract is reconstituted in 1.0 mL methanol then filtered through a 3 cc plastic syringe attached to a 0.2 pm filter into glass autovials. 3.0 Definitions_____________________ ________________ i___________________________ 3.1 None. 4.0 Warnings and Cautions__________ ' 4.1 Health and Safety Warnings: ______________________ ^___________ . 4.1.1 Use universal precautions when handling animal livers, they may contain pathogens. . 5.0 Interferences________________________ ._______________________________________ 5.1 There are no known interferences at this time. 6.0 E quipment______________________________________________________________ ' 6.1 The following equipment is used while carrying cut this method Equivalent equipment is acceptable. 6.1.1 Ultra-Turrax T25 Grinder for grinding liver samples 6.1.2 Vortex mixer, VWR, Vortex Genie 2 6.1.3 Centrifuge, Mistral 1000 or IEC 6.1.4 Shaker, Eberbach or VWR . 6.1.5 Nitrogen. Evaporator, Organomation 6.1.6 Balance 7.0 Supplies and Materials ______________________________________________ __ 7.1 Gloves 7.2 Dissecting scalpels 7.3 Eppendorf or disposable pipettes 7.4 Nalgene bottles, capable o f holding 250 mL and 1 L 7.5 Glass, type A, volumetric flasks 7.6 40 mL glass I-CHEM vials 7.7 Plastic sampule vials, Wheaton, 6 mL 7.8 Polypropylene centrifuge tubes, 15 mL 7.9 Labels FACT-M-1.0 Extraction of PFOS from Liver Page 2 of 8 3M Environmental Laboratory Page 39 3M Medical Department Study: T-6316.7 BACK TO MAIN Analytical Report: FACT TOX-098 LRN-U2402 7.10 Syringes, capable of measuring 10 p.L to 50 pL 7.11 Glass, type A, volumetric pipettes 7.12 Graduated pipettes 7.13 Electronic pipettor, Eppendorf or equivalent 7.14 Timer 7.15 Disposable plastic 3 cc syringes 7.16 Filters, nylon syringe filters, 0.2 pm, 25 mm . 7.17 Crimp cap autovials Note: Prior to using glassware and bottles, rinse 3 times with methanol and 3 times with MilliQTM water. Rinse syringes a minimum of 9 times with methanol, 3 rinses from 3 separate vials. 8.0 Reagents and Standards_____________ .______________._________________________ 8.1 Reagents 8.1.1 Sodium Hydroxide (J.T Baker or equivalent), (NaOH) 10N: weigh approximately 200 grams NaOH. Pour into a 1000 mL beaker containing 500 liters (L) Milli-QTM water, mix until all solids are dissolved. Store in a 1 L nalgene bottle. 8.1.2 Sodium Hydroxide (J.T Baker or equivalent), (NaOH) IN. Dilute 1ON 1:10. Measure 10 mL o f the 10N NaOH solution into a 100 mL volumetric flask and dilute to volume using Milli-QTM water. S tore in a 125 inL nalgene bottle. 8.1.3 Tetrabutylammonium hydrogen sulfate (Kodak or equivalent), (TBA) 0.5M: Weigh approximately 169 grams of TBA into a 1 L volumetric containing 500 L Milli-QTM ' water. Adjust to pH 10 using approximatel y 64 mL 10N NaOH and dilute to volume with Milli-QTM water. Add NaOH slowly while adding the last 1 mL of NaOH because the pH changes abruptly. Store in a 1 L nalgene bottle. 8.13.1 TBA requires a check prior to each use to ensure pH = 10. Adjust as needed using IN NaOH solution. 8 .1 .4 Sodium carbonate/Sodium Bicarbonate Buffer (J.T. Baker or equivalent), (Na2C03/NaHC03) 0.25M: Weigh approximately 26.5 g of sodium carbonate (NajC03) and 21.0 g of sodium bicarbonate (NaHC03) into a 1 L volumetric flask and dilute to volume with Milli-QTM water. Store in a 1 L nalgene bottle. 8.1.5 PFOS (3M Specialty Chemical Division), molecular weight = 538. 8.1.6 Ethyl Acetate, Omnisolv, glass distilled or HPLC grade. 8.1.7 Methanol, Omnisolv, glass distilled or HPLC grade. 8.1.8 Liver and control liver, received frozen from testing laboratory. 8.1.9 Milli-QTM water, all water used in this melhod should be Milli-QTM water and may be provided by a Milli-Q TOC Plus system. 8.2 Standards 8.2.1 Prepare PFOS standards for the standard curve. FACT-M-1.0 Extraction of PFOS from Liver Page 3 of 8 3M Environmental Laboratory Page 40 3M Medical Department Study: T-6316.7 BACK TO MAIN Analytical Report: FACT TOX-098 LRN-U2402 8.2.2 Weigh approximately 100 mg of PFOS into a 100 mL volumetric flask and record the actual weight. 8.2.3 Bring to volume with methanol for a stock standard of approximately 1000 ppm (fig/mL). 8.2.4 Dilute the stock solution with methanol for a working standard 1 solution of approximately 50 ppm. 8.2.5 Dilute the stock solution with methanol for a working standard 2 solution of approx. 5.0 ppm. 8.2.6 Dilute the stock solution with methanol for a working standard 3 solution o f approx. 0.50 ppm. 9.0 Sample H andling___________ ;_______________________________ _________________ 9.1 All livers are received frozen and must be kept frozen until the extraction is performed. 10.0 Quality Co n t r o l ____________________________ ;____________ ;_______________ 10.1 Matrix Spikes 10.1.1 Prepare and analyze matrix spike and matrix spike duplicate samples to determine the accuracy of the extraction. 10.1.2 Prepare each spike using liver chosen by the analyst, usually a control liver. 10.1.3 Expected concentrations will fall in the mid-range o f the initial calibration curve. 10.2 Continuing Calibration Checks 10.2.1 Prepare and analyze continuing calibration check samples to determine the continued linearity of the initial calibration curve. 10.2.2 One check is prepared per group of ten samples. For example, if a sample set = 34, four checks are prepared and extracted. 10.2.3 Prepare each continuing calibration check from the same liver homogenate used to prep the initial curve. 10.2.4 The expected concentration will fall within the mid-range of the initial calibration curve. 11.0 Calibration and Standardization_____________ ;___________________________ __ 11.1 Prepare Liver Homogenate to Use for Standards 11.1.1 Weigh approximately 40 g of liver into a 250 mL Nalgene bottle containing 200 mLs Milli-QTM water. Grind to a homogeneous solution. 11.1.2 If 40 g is not available, use appropriate amounts of liver and water in keeping with a 1:5 ratio. 11.1.3 See section 13.0 to calculate the actual density of liver. FACT-M-1.0 Extraction of PFOS from Liver Page 4 of 8 3M Environmental Laboratory Page 41 3M Medical Department Study: T-6316.7 BACK TO MAIN Analytical Report: FACT TOX-098 LRN-U2402 11.1.4 Add 1 mL of homogeneous solution to a 15 mL centrifuge tube. Re-suspend homogeneous solution by shaking between aliquots while preparing a total of sixteen 1 mL aliquots of homogeneous solution in 15 mL centrifuge tubes. 11.1.5 Two 1 mL aliquots serve as matrix blanks. Use the standard concentrations and spiking amounts listed in table 1 to spike, in duplicate, two standard curves for a total of fourteen samples. Table 1 Approximate Spiking Amounts for Calibration Standards Working Standard (Approx. Cone.) - 0.50 ppm 0.50 ppm 0.50 ppm 5.0 ppm 5.0 ppm 5.0 ppm 50 ppm pL Approx, final cone, of PFOS in liver - Blank 4 0.010 ppm 20 0.050 ppm 40 0.100 ppm 10 0.250 ppm 20 0.500 ppm 30 0.750 ppm 4 1.000 ppm 11.1.1 See section 13.0 to calculate actual concentrations o f PFOS in calibration standards. 11.2 Extract spiked liver homogenates following 12.14-12.24 o f this method. Use these standards to establish each initial curve on the mass spectrometer. 12.0 Procedures________________________ ^________________________________________ 12.1 Obtain frozen liver samples. In spent tissue, note that the liver has not been packaged with other tissues. 12.2 Cut approximately 1 g of liver using a dissecting scalpel. 12.3 Weigh the sample directly into a tared plastic sampule vial. 12.4 Record the liver weight in the study notebook. 12.5 Label the sampule vial with the study number, weight, liver ID, date and analyst initials. 12.6 Add 2.5 mLs of water to sampule vial. 12.7 Grind the sample. Put the grinder probe in the sample and grind for about 2 minutes, or until the sample is homogeneous. 12.8 Rinse the probe into the sample with 2.5 mLs water using a pipette. 12.9 Take the grinder apart and clean it with methanol after each sample. Follow AMDT-EP-22. 12.10 Cap the sample and vortex for 15 seconds. FACT-M-1.0 Extraction of PFOS from Liver Page 5 of 8 3M Environmental Laboratory Page 42 3M Medical Department Study: T-6316.7 BACK TO MAIN Analytical Report: FACT TOX-098 LRN-U2402 12.11 Pipette 1 mL homogenate into a 15 mL polypropylene centrifuge tube. Label the centrifuge tube with the identical information as the sampule vial. (See Worksheet for documenting the remaining steps.) 12.12 Spike liver homogenates with the appropriate amount o f PFOS standard as described in section 11.1 or Table 1. 12.13 Pipette two 1 mL aliquots o f Milli-QTM water to centrifuge tubes. These will serve as instrument blanks. 12.14 Add 1 mL 0.5 M TBA and 2 mL of the 0.25 M sodium carbonate/sodium bicarbonate buffer. 12.15 Using a volumetric pipette, add 5 mLs ethyl acetate. 12.16 Cap each sample and put on the shaker for 20 minutes. 12.17 Centrifuge for 20 to 25 minutes, until layers are well separated. Set power on the centrifuge to approximately 3500 rpm. 12.18Remove 4 mLs of organic layer, using a 5 mL graduated glass pipette, to a clean 15 mL centrifuge tube. Label this fresh tube with the same information as in 12.5. 12.19 Put each sample on the analytical nitrogen evaporator until dry, approximately 2 to 3 hours. ' 12.20 Add 1.0 mL o f methanol to each centrifuge tube using a graduated pipette. 12.21 Vortex mix for 30 seconds. 12.22 Attach a 0.2 pm nylon mesh filter to a 3 cc syringe and transfer the sample to this syringe. Filter into a 1.5 mL glass autovial. 12.23 Label the autovial with the study number, animal number and gender, sample timepoint, matrix, final solvent, extraction date, and analyses) who performed the extraction. 12.24 Cap and hold for electrospray mass spectrometry analysis. 12.25 Complete the worksheet and tape to page o f study notebook. 13.0 Data Analysis and Ca l c u l a t i o n s ___________ ._________;_____________ 13.1 Calculations: 13.1.1 Calculate the density of liver (mg) in 1.0 mL homogenate using the following equation: g o f Liver x Average weight of ten 1 mL aliquots (mg) (g of Liver + g of Water) 3M Environmental Laboratory FACT-M-1.0 Extraction of PFOS from Liver Page 6 of 8 Page 43 3M Medical Department Study: T-6316.7 BACK TO MAIN Analytical Report: FACT TOX-098 LRN-U2402 13.1.2 Calculate actual concentrations ofPFOS in calibration standards using the following equation: uL o f Standard x Concentration Q-ig /mL) = Final Concentration (pg/g or mg/kg) mg Liver /1 mL homogenate ofPFOS in Liver *Average weight o f liver in solution as determined in 13.1.1, by weighing ten 1 mL homogenates o f approximately 40 mg liver in 200 mL o f Milli-Q water. 14.0 Method Performance____________________________ _________________________ 14.1 The method detection limit is equal to half the lowest standard in the calibration curve. 15.0 Pollution Prevention and Waste Management_____________________________ 15.1 Sample waste is disposed in biohazard containers, flammable solvent waste is disposed in high BTU containers, and used glass pipette waste is disposed in broken glass containers located in the laboratory. 16.0 R ecords____________________________________________________'__________________ 16.1 Complete the extraction worksheet and tape into the study notebook. 17.0 Tables, Diagrams, Flowcharts, and Validation Data____________^___________ 17.1 The validation report associated with this method is FACT-M-1.0 & 2.0-V-l. 18.0 References_____________________________________________ ;______________ 18.1 AMDT-EP-22, "Routine Maintenance o f Ultra-Turrax T-25" 19.0 Affected Documents_________ ' .__________________________________' 19.1 FACT-M-2, "Analysis o f Liver Extracts for Fluorochemicals using HPLC-Electrospray Mass Spectrometry" 20.0 Revisions Revision Number. _______________ ;_________ Reason For Revi sion Revision Date 3M Environmental Laboratory FACT-M-1.0 Extraction ofPFOS from Liver Page 7 of 8 Page 44 3M Medical Department Study: T-6316.7 BACK TO MAIN Analytical Report: FACTTOX-098 LRN-U2402 Extraction Worksheet for FACT-M-1 Study # Sam p le Number set# PFOS approx. 0.5 ppm actual ppm PFOS approx. 5 ppm actual ppm PFOS approx. 50 ppm actual ppm D ate and Initials for Std. - - 1 H ,0 Blank Liver Blank #W - -. ! > '- #W - #W ' - - - - .- .- - -- - . - - -- -- -- -r -. - - - - - - .- -- -- .- - -- - - . 1Study numi ier where the original workshcet is-located,____ Blank Liver Homogenate: Std # Liver Extraction Method : Liver amount - e Date & Initials _ Vortex 15 sec. Pioette 1 mL of Liver Solution . Pipette 1mL of tO.S M TBA, pH 10. Std. # Pipette 2 mL of 0.25 Na?COV0.25M NaHCOt Buffer Std.# Pipette 5 mL of Ethyl Acetate TN-A- Shake 20 min. _______________________________________________________________ ---------- Centrifuge 20-25 min. Centrifuge Speed . ... Remove a 4 mL aliauot of organic layer Put on Nitrogen Evaporator to drvness Evaporator Temperature . . ... Add 1.0 mL of Methanol TN-A- Vortex 30 sec. ...._______ ________________________________________________________ :________ ------------------ Filter using a 3ccB-D syringe with a 0.2um SRI filter into a 1.5 mL autosample vial MS/MSD/___Cont. Checks: Spiked_____ uL of a _____ ppm std (______________) for a final concentration of ppm. MS/MSD used sample______ ;_______ . Cont Checks used same homogenate as for std curve. FACT-M-1.0 Extraction of PFOS from Liver Page 8 of 8 3M Environmental Laboratory Page 45 3M Medical Department Study: T-6316.7 BACK TO MAIN Analytical Report: FACT TOX-098 LRN-U2402 3M Environmental Laboratory M ethod E x tr a c tio n o f P o ta ssiu m P erflu o ro o cta n esu lfo n a te o r O t h e r F l u o r o c h e m ic a l c o m po u n d s fr o m Seru m o r O t h e r F luid f o r Analysis U s in g H P L C - E l e c t r o s p r a y /XIAss Sp e c t r o m e t r y M ethod N um ber: FACT-M-3.1 Adoption Date: 04/22/98 Author: Lisa Clemen, Glenn Langenburg Revision Date: to Approved By: k j^ ~ Laboratory Manager /o/ / / r v Date Group Leader ` *1/- 2 - ti f / Date A rlwb&K Technical Reviewer ' k s f a s Date 1.0 Scope and Application 1.1 Scope: This method is for the extraction of potassium perfluorooctanesulfonate (PFOS) or other iluorochemical compounds from serum or other fluid. 1.2 Applicable compounds: Fluorochemical surfactants or other fluorinated compounds. 1.3 M atrices: Rabbit, rat, bovine, and monkey serum, rat whole blood, and rat milk curd. ' Word 6/95 3M Environmental Laboratory FACT-M-3.1 Extraction of PFOS from Serum and Other Fluids Page 1 of 17 Page 46 3M Medical Department Study: T-6316.7 BACK TO MAIN Analytical Report: FACT TOX-098 LRN-U2402 2.0 Summary of Method_______________ ________________________________ 2.1 This method describes the procedure for extracting potassium perfluorooctanesulfonate (PFOS) or other fluorochemicals from serum, blood, or milk curd using an ion pairing reagent and 5.0 ml o f ethyl acetate. In this method, seven fluorochemicals were extracted: PFOS, PFOSA, PFOSAA, EtFOSE-OH, POAA, PFOSEA, and FC-807 monoester (see 3.0 Definitions). An ion pairing reagent is added to the sample and the analyte ion pair is partitioned into ethyl acetate. Four ml o f extract are removed and put onto a nitrogen evaporator until dry. Each extract is reconstituted in 1.0 ml o f methanol, then filtered through a 3 cc plastic syringe attached to a 0.2 pm nylon filter into glass autovials. 3.0 Definitions__________________________________________________ .____________ 3.1 PFOS: perfluorooctanesulfonate (anion o f potassium salt) C,,F]l7S 0 3" 3.2 PFOSA: perfluorooctane sulfonylamide C8F l7S 0 2NH2 3 3 PFOSAA: perfluorooctane sulfonylamido (ethyl)acetate C,,Fl7S 02N(CH2CH3)CH2C 0 2' 3.4 EtFOSE-OH: 2(N-ethylperfluorooctane sulfonamido)-ethyl alcohol C8F 17S 0 2N(CH2CH3)CH2CH20 H 3.5 POAA: perfluorooctanoate (anion o f ammonium salt) C ^ C O O ' . 3.6 PFOSEA: perfluorooctane sulfonyl ethylamide C,,F17S 0 2N(CH2CH3)H 3.7 FC-807 monoester C8F17S 0 2N(CH2CH3)CH2CH20 -P 0 3H) 3.8 Surrogate standard: 1H-1H-2H-2H perfluorooctane sulfonic acid 4.0 Warnings and Cautions____________________________________________ 4.1 H ealth an d safety warnings 4.1.1 Use universal precautions, especially laboratory coats, goggles, and gloves when handling animal tissue, which may contain pathogens. 5.0 Interferences___________________________________________________________ 5.1 There are no known interferences at this time. 6.0 Equipment_______________________________________________________________ 6.1 The following equipment is used while performing; this method. Equivalent equipment is acceptable. 6.1.1 Vortex mixer, VWR, Vortex Genie 2 6.1.2 Centrifuge, Mistral 1000 or IEC 6.1.3 Shaker, Eberbach or VWR 6.1.4 Nitrogen evaporator, Organomation 6.1.5 Balance ( 0.100 g) FACT-M-3.1 Extraction of PFOS from Serum or Other Fluid Page 2 of 17 3M Environmental Laboratory Page 47 3M Medical Department Study: T-6316.7 BACK TO MAIN Analytical Report: FACT TOX-098 LRN-U2402 7.0 Supplies and Materials 7.1 Gloves 7.2 Eppendorf or disposable pipettes 7.3 Electronic pipettor, Eppendorf or equivalent 7.4 Graduated pipettes 7.5 Nalgene bottles, capable o f holding 250 mL and 1 L . 7.6 Volumetric flasks, glass, type A 7.7 Volumetric pipets, glass, type A 7.8 I-CHEM vials, glass, 40 mL glass 7.9 Crimp cap autovials 7.10 Centrifuge tubes, polypropylene, 15 mL 7.11 Labels 7.12 Syringes, capable o f measuring 5 pL to 50 pL 7.13 Syringes, disposable plastic, 3 cc 7.14 Syringe filters, nylon, 0.2 pm, 25 mm 7.15 Timer Note: Prior to using glassware and bottles, rinse 3 times with methanol and 3 times with Milli-QTM water. Rinse syringes a minimum o f 9 times with methanol, 3 rinses from 3 separate vials. 8.0 Reagents and Standards___________;_______________________________________ 8.1 Type I reagent grade water, Milli-QTM or quivalait; all water used in this method should be Milli-QTM water and may be provided by a Milli-Q TOC PlusTM system 8.2 Sodium hydroxide (NaOH), J.T Baker or equivalent 8.3 Tetrabutylammonium hydrogen sulfate(TBA), Kodak or equivalent 8.4 Sodium carbonate (Na2C03), J.T. Baker or equivalent 8.5 Sodium bicarbonate (NaHC03), J.T. Baker or equivalent 8.6 Ethyl acetate, Omnisolv, glass distilled or HPLC grade 8.7 Methanol, Omnisolv, glass distilled or HPLC grade 8.8 Serum or blood, frozen from supplier 8.9 Control matrix or blank matrix for purpose of standards, QC checks, blanks, etc. 8.10 Fluorochemical standards 8.10.1 PFOS (3M Specialty Chemical Division), molecular weight = 538 8.10.2 PFOSA (3M Specialty Chemical Division), molecular weight = 499 FACT-M-3.1 Extraction of PFOS from Seram or Other Fluid Page 3 of 17 3M Environmental Laboratory Page 48 3M Medical Department Study: T-6316.7 BACK TO MAIN Analytical Report: FACT TOX-098 LRN-U2402 8.10.3 PFOSAA (3M Specialty Chemical Division), molecular weight = 585 8.10.4 EtFOSE-OH (3M Specialty Chemical Dmsion), molecular weight = 571 8.10.5 POAA (3M Specialty Chemical Division), molecular weight = 431 8.10.6 PFOSEA (3M Specialty Chemical Division), molecular weight = 527 8.10.7 FC-807 monoester (3M Specialty Chemical Division). FC-807 is a mixture of triester, diester, and monoester fluorochemical components. The monoester molecular weight = 650 8.10.8 Surrogate standard: 4-H, perfluorooctane sulfonic acid (1-H.l-H, 2-H, 2-H C8F 13S 0 3H) molecular weight = 428 8.10.9 Other fluorochemicals, as appropriate 8.11 Reagent preparation 8.11.1 10 N sodium hydroxide (NaOH): Weigh approximately 200 g NaOH. Pour into a 1000 mL beaker containing 500 mL Milli-QTM water, mix until all solids are dissolved. Store in a 1 L Nalgene bottle. 8.11.2 1 N sodium hydroxide (NaOH): Dilute 10 N NaOH 1:10. Measure 10 mL o f 10 N NaOH solution into a 100 mL volumetric flask and dilute to volume using Milli-QTM water. Store in a 125 mL Nalgene bottle. 8.11.3 0.5 M tetrabutylammonium hydrogen sulfate (TBA): Weigh approximately 169 g o f TBA into a 1 L volumetric containing 500 mL Milli-QTM water. Adjust to pH changes abruptly. Store in a 1 L Nalgene bottle. 8.11.3.1 TBA requires a check prior to each use to ensure pH = 10. Adjust as needed using 1 N NaOH solutio n. 8.11.4 0.25 M sodium carbonate/sodium bicarbonate buffer (Na2C 0 3/NaHC03): Weigh approximately 26.5 g of sodium carbonate (NajC03) and 21.0 g o f sodium bicarbonate (NaHC03) into a 1 L volumetric flask and bring to volume with MilliQTM water. Store in a 1 L Nalgene bottle. 8.12 Standards preparation 8.12.1 Prepare PFOS standards for the standard curve. 8.12.2 Prepare other fluorochemical standards, as appropriate. Multicomponent fluorochemical standards are acceptable (for example, one working standard solution containing 1.00 ppm PFOS, 1.02 ppm PFOSA, 0.987 ppm PFOSAA, and 1.10 ppm EtFOSE-OH.) . 8.12.3 Weigh approximately 100 mg of PFOS into a 100 ml volumetric flask and record the actual weight. . 8.12.4 Bring to volume with methanol for a stock standard o f approximately 1000 ppm (jig/ml). 8.12.5 Dilute the stock solution with methanol for a working standard 1 solution of approximately 50 ppm. FACT-M-3.1 Extraction of PFOS from Serum or Other Fluid Page 4 o f 17 3M Environmental Laboratory Page 49 3M Medical Department Study: T-6316.7 BACK TO MAIN Analytical Report: FACT TOX-098 LRN-U2402 8.12.6 Dilute the stock solution with methanol for a working standard 2 solution o f approx. 5.0 ppm. 8.12.7 Dilute the stock solution with methanol for a working standard 3 solution of approx. 0.50 ppm. 8.13 Surrogate stock standard preparation 8.13.1 Weigh approximately 50-60 mg o f surrogate standard l-H .l-H , 2-H, 2-H, C8F 13S 03H into a 50 ml volumetric flask and record the actual weight. 8.13.2 Bring to volume with methanol for a surrogate stock of approximately 1000-1200 ppm. 8.13.3 Prepare a surrogate working standard. Transfer approximately 0.5 ml o f surrogate stock to a 50 ml volumetric flask and bring to volume with methanol for a working standard o f 10-20 ppm. Record the actual volume transferred. 9.0 Sample Handling _____________ ;_____________________ :______________ 9.1 All samples are received frozen and must be kept frozen until the extraction is performed. 10.0 Quality Control__________- ______________________________ ;_________ 10.1 Matrix blanks and method blanks 10.1.1 Extract two 1.0 mL aliquots o f the appropriate matrix (serum or blood, with blood samples diluted 1:1 wifla Milli-QTM water) following this procedure and use as matrix blanks. See 11.1.4. 10.1.2 Extract two 1.0 ml aliquots o f Milli-QTM water following this procedure and use as method blanks. 10.2 Matrix spikes 10.2.1 Prepare and analyze matrix spike and matrix spike duplicate samples to determine the accuracy of the extraction. 10.2.2 Prepare each spike using a sample chosen by the analyst, usually the control matrix received with each sample set. 10.2.3 Expected concentrations will fall in the mid-range o f the initial calibration curve. Additional spikes may be included and may fall in the low-range o f the initial calibration curve. 10.2.4 Prepare one matrix spike and matrix spike duplicate per 40 samples, with a minimum o f 2 matrix spikes per batch. 10.3 Continuing calibration checks 10.3.1 Prepare and analyze continuing calibration check samples to ensure the accuracy of the initial calibration curve. If the percent difference between the initial curve and the continuing check differ by >30%, re-analyze samples analyzed after the last acceptable check. 10.3.2 Prepare one check per group of ten samples. For example, if a sample set = 34, prepare and extract four checks. FACT-M-3.1. Extraction of PFOS from Serum, or Other Fluid Page 5 of 17 3M Environmental Laboratory Page 50 3M Medical Department Study: T-6316.7 BACK TO MAIN A nalytical Report: FAC T TO X -098 LR N -U 2 4 0 2 10.3.3 Prepare each continuing calibration check from the same matrix used to prepare the initial curve. 10.3.4 The expected concentration will fall within the mid-range o f the initial calibration curve. Additional spikes may be included that fall in the low-range o f the initial calibration curve. This is necessary if the analyst must quantitate using only the low end of the calibration curve (for example, 5 ppb - 100 ppb, rather than 5 ppb -1 0 0 0 ppb). 11.0 Calibration and Standardization_______________________ ____________________ 11.1 P repare m atrix calibration standards Note: Blood coagulates in air; therefore, minimize air contact until dilution. At this point, add TBA and buffer to each centrifuge tube as in step 12.9, then add 1.0 mL o f the diluted matrix sample to each tube. 11.1.1 Transfer 1 mL of serum or 1 mL of blood (blood is diluted 1:1 with Milli-QTM water) to a 15 mL centrifuge tube. The blood is similar in composition to milk curd and can be used in place of milk curd for standard curves when extracting that matrix. 11.1.2 I f most sample volumes are less than 1.0 mL, extract standards with matrix volumes equal to the sample volumes. Do not extract below 0.50 mL o f matrix. Record the sample volume on the extraction sheet. 11.1.3 While preparing a total of twenty aliquots; in 15 ml centrifuge tubes, mix or shake between aliquots. 11.1.4 Two 1 mL aliquots, or other appropriate volume, serve as matrix blanks. Typically use the standard concentrations and spiking amounts listed in Table 1, at die end o f this section, to spike, in duplicate, two standard curves, for a total of eighteen standards and two matrix blanks. 11.1.5 Refer to validation reports FACT-M-3.1-V-l and FACT-M-4.1-V-1, which list the working ranges and the Linear Calibration Range (LCR) for calibration curves. 11.1.6 Use Attachment D as an aid in calculating the concentrations o f the working standards. See Section 13.0 to calculate actual concentrations o f PFOS in calibration standards. 11.2 To each standard, blank, or QC check, add appropriate amount o f surrogate working standard for the concentration to fall within the calibration curve range 5 ppb -1000 ppb. 11.3 Extract spiked matrix standards following 12.6-12.16 o f this method. Use these standards . to establish each initial curve on the mass spectrometer. 3M Environmental Laboratory FACT-M-3.1 Extraction of PFOS from Serum or Other Fluid Page 6 o f 17 Page 51 3M Medical Department Study: T-6316.7 BACK TO MAIN A nalytical Report: FACT TO X -098 L R N -U 2 40 2 Table 1 Approximate spiking amounts for standards and spikes using 1,0 ml of m atrix Working standard (approx, cone.) pL Approx, final cone, of analyte in matrix - - Blank 0.500 ppm 10 0.005 ppm 0.500 ppm 20 0.010 ppm 5.00 ppm 5 0.025 ppm 5.00 ppm 10 0.050 ppm 5.00 ppm 20 0.100 ppm 50.0 ppm 5 0.250 ppm 50.0 ppm 10 0.500 ppm 50.0 ppm 15 0.750 ppm 50.0 ppm 20 1.00 ppm Table 2 Approximate spiking amounts for standards and spikes using 0.5 ml of m atrix Working standard M-L Approx, final cone, o f (approx, cone.) analyte in matrix - - Blank 0.500 ppm 5 0.005 ppm 0.500 ppm 10 0.010 ppm 5.00 ppm 2.5 0.025 ppm 5.00 ppm 5 0.050 ppm 5.00 ppm 10 0.100 ppm 50.0 ppm 2.5 0.250 ppm 50.0 ppm 5 0.500 ppm 50.0 ppm 7.5 0.750 ppm 50.0 ppm 10 1.00 ppm 12.0 Procedure______________________________________________________________ _ 12.1 Obtain frozen samples and allow to thaw. . 12.2 Vortex mix for 15 seconds, then transfer 1.0 mL or other appropriate volume to a 15 mL polypropylene centrifuge tube. For blood samples, remove 0.5 mL and dilute to 1.0 mL with Milli-QTM water. As soon after diluting as possible, pipet diluted blood into TBAbuffer mixture shown in step 12.9 and mix well. 12.3 Return samples to freezer after extraction amount has been removed. 3M Environmental Laboratory FACT-M-3.1 Extraction of PFOS from Serum or Other Fluid Page 7 of 17 Page 52 3M Medical Department Study: T-6316.7 BACK TO MAIN Analytical Report: FACT TO X-098 L R N -U 2 40 2 12.4 Record the volume on the extraction worksheet. The final methanol volume equals the volume transferred from the sample. For example, if 0.5 mL is removed for a blood sample, the final methanol volume will equal 0.5 mL. 12.5 Label the tube with the study number, sample ID, date and analyst initials. See attached worksheet for documenting the remaining steps. . 12.6 Spike each matrix with the appropriate amount o f standard as described in 11.1 or Table 1 or 2 in that section for the calibration curve standards. Also prepare matrix spikes and continuing calibration standards. 12.7 Spike all samples, including blanks and standards, ready for extraction with surrogate standard as described in 11.2. 12.8 Vortex mix the standard curve samples, matrix spike samples, and continuing calibration samples for 15 seconds. 12.9 To each sample, add 1 mL 0.5 M TBA and 2 mL o f 0.25 M sodium carbonate/sodium bicarbonate buffer. 12.10 Using a volumetric pipette, add 5 mL ethyl acetate. 12.11 Cap each sample and put on the shaker for 20 minutes. 12.12 Centrifuge for 20 to 25 minutes at approximately 3500 rpm, until layers are well separated. 12.13 Transfer 4 mL o f organic layer, using a 5 mL graduated glass pipette, to a clean 15 mL centrifuge tube. Label this fresh tube with the same information as in 12.5. 12.14 Put each sample on the analytical nitrogen evaporator until dry, approximately 2 to 3 hours. 12.15 Add 1.0 mL or other appropriate volume of methanol to each centrifuge tube using a graduated pipette. Methanol volume to add equals the initial volume of sample used for the extraction. 12.16 Vortex mix for 30 seconds. . 12.17 Attach a 0.2 pm nylon mesh filter to a 3 cc syringe and transfer the sample to this syringe. Filter into a 1.5 mL glass autovial or low-volume autovial when necessary. 12.18 Label the autovial with the study number, animal number and gender, sample timepoint, matrix, final solvent, extraction date, and analyst(s) performing the extraction. 12.19 Cap and store extracts at approximately 4 C until analysis. 12.20 Complete the extraction worksheet, attached to tliis document, and tape in the study notebook or include in study binder, as appropriate. 3M Environmental Laboratory FACT-M-3.1 Extraction of PFOS from Serum or Other Fluid Page 8 of 17 Page 53 3M Medical Department Study: T-6316.7 BACK TO MAIN A nalytical Report: FAC T TO X -098 L R N -U 2 40 2 13.0 Data Analysis and Calculations______________________ _______________ _______ 13.1 Calculations 13.1.1 Calculate actual concentrations o f PFOS, or other applicable fluorochemical, in calibration standards using the following equation: mL of standard x concentration o f standard (ug AnT.i_________________ = mL o f standard + mL o f surrogate standard + initial matrix volume (mL) Final Concentration (pg/mL) o f PFOS in matrix 14.0 Method Performance_______________________________________________________ 14.1 The method detection limit (MDL) is analyte and matrix specific. Refer to MDL report for specific MDL and limit of quantitation (LOQ|) values (see Attachments B and C). 14.2 The following quality control samples are extracted with each batch o f samples to ensure the quality o f the extraction and analysis. 14.2.1 Method blanks and matrix blanks 14.2.2 Matrix spike and matrix spike duplicate samples to determine accuracy and precision of the extraction 14.2.3 Continuing calibration check samples to determine the continued accuracy of the initial calibration curve 15.0 Pollution Prevention and Waste Management_______________ ,______ __ 15.1 Sample waste is disposed in biohazard containers, flammable solvent waste is disposed in high BTU containers, and used glass pipette was te is disposed in broken glass containers located in the laboratory. 16.0 Records__________________ ____________________ ;_________________________ . 16.1 Complete the extraction worksheet attached to this method, and tape in the study notebook or include in study 3-ring binder, as appropriate. 17.0 Attachments_________ __________________________________ 17.1 Attachment A, Extraction worksheet 17.2 Attachment B, MDL/LOQ values 17.3 Attachment C, LOQ Summary 17.4 Attachment D, Calibration standard concentration worksheet ________ 18.0 References_________________________________________________ ;___________ ___ 18.1 The validation reports associated with this method are FACT-M-3.1 & 4.1-V -l. FACT-M-3.1 Extraction of PFOS from Serum or Other Fluid Page 9 of 17 3M Environmental Laboratory Page 54 3M Medical Department Study: T-6316.7 BACK TO MAIN A nalytical Report: FACT TO X -098 L R N -U 2 40 2 19.0 Affected Documents______________________________ ;___________________ 19.1 FACT-M-4.1, "Analysis o f Serum or Other Fluid Extracts for Fluorochemicals using HPLC-Electrospray Mass Spectrometry" 20.0 REVISIONS_____________ _____________ ___________________________ Revision Number 1 Reason For Revision Validation of method to include 7 fluorochemicals, an additional matrix, new API/MS(MS) systems, monkey serum cross validation, improvements to ion pairing extraction, MDL study, updates in record keeping and storing policies, etc. Revision Date 07/01/98 3M Environmental Laboratory FACT-M-3.1 Extraction of PFOS from Serum or Other Fluid Page 10 of 17 Page 55 3M Meidical Department Study: T-6316.7 BACK TO MAIN Analytical Report: FACT TO X -098 L R N -U 2 40 2 S tu d y # Sam p le Number .1 set# H ,0 Blank Blank FC-M ix . approx. 0.5 ppm actual ppm #W - FC-M ix approx. 5 ppm actual ppm #W - - FC-M ix approx. 50 ppm actual ppm #W * D ate and Initials for Std. or C om m en ts - -. '- - - - .- -- - - .- - -' - -. - - '- -- - -- -- -- - -. -- -- -- - - -- - - -- - -' 1Study number where the original worksheet is located.--------------- Blank Std # amount = mL Serum Extraction Method Vortex 15 sec. :- Date & Initials Pinette Matrix Volume ml Pipette 1 ml of 0.5 M TBA, pH 10. Std. # Pipette 2 ml of 0.25 Na2COV0.25M NaHCOt buffer Std. # Pipette 5 ml of ethyl acetate TN-A- Shake 20 min. Centrifuge 20-25 min. Centrifiiae speed: Remove a 4 ml aliauot of organic laver Put on Nitrogen Evaporator to drvness Evaporator #: Temperature: Add methanol Volume ml TN-A- Vortex 30 sec. Filter using a 3cc B-D syringe with a 0.2um SRI filter into a 1.5 ml autosample vial___________________________ MS/MSD/___Cont. Checks: Spiked_____ uL of a _____ ppm std (______________) for a final concentration of ________ ppm. MS/MSD used sample _______ __. Cont. Checks used same matrix as for std curve. Surrogate Standard: Spiked____ _ uL of a _____ ppm std (_____________ ) to all samples, standards, and blanks Attachment A: Extraction worksheet FACT-M-3.1 Extraction ofPFOS from Serum or Other Fluid Page 11 of 17 3M Environmental Laboratory Page 56 3M Medical Department Study: T-6316.7 BACK TO MAIN A nalytical Report: FAC T TO X -098 LR N -U 2 4 0 2 MDL/LOQ values for Rabbit Seram : Compound MDL LOQ Linear Calibration Range (LCR) (ppb) (ppb) Approximate concentrations to be used for preparing the Standard Calibration Curve PFOS 1.38 4.39 5 ppb -1 0 0 0 ppb PFOSA 2.23 7.09 lO ppb-lO O O ppb PFOSAA 2.84 9.04 10 ppb -1 0 0 0 ppb EtFOSE-OH 3.90 12.4 15 ppb -1 0 0 0 ppb POAA 4.31 13.7 15 p p b - 7 5 0 ppb PFOSEA 1.09 3.48 25 ppb -1 0 0 0 ppb Monoester 149 248 MDL and LOQ are estimates only. No valid MDL was determinable from MDL study. Any quantitation performed for monoester will be an estimate only. Please refer to FACT-M-3.1 & 4.1-V-l for specifics. MDL/LOQ values for R at Serum: Compound MDL LOQ Linear Calibration Range (LCR) (PPb) (PPb) Approximate concentrations to be used for preparing the Standard Calibration Curve PFOS 1.27 4.04 10 ppb -1 0 0 0 ppb PFOSA 2.14 6.81 25 ppb -1 0 0 0 ppb PFOSAA 2.32 7.38 10 ppb - 1000 ppb EtFOSE-OH 3.25 10.3 50 ppb - 1000 ppb POAA 1.20 3.81 5 ppb -1 0 0 0 ppb PFOSEA 1.84. 5.86 10 ppb -1 0 0 0 ppb Monoester 149 248 MDL and LOQ are estimates only. No valid MDL was determinable from MDL study. Any quantitation performed for monoester will be an estimate only. Please refer to FACT-M-3.1 &4.1-V-1 for specifics. MDL/LOQ values for Bovine Serum: Compound MDL LO Q Linear Calibration Range (LCR) (ppb) (PPb) Approximate concentrations to be used for preparing the Standard Calibration Curve PFOS 2.11 6.70 25 ppb-lOOOppb PFOSA 5.04 16.0 25 ppb -1 0 0 0 ppb PFOSAA 2.34 7.45 260 ppb-1 0 0 0 ppb EtFOSE-OH 11.3 35.8 50 ppb-lOOOppb POAA 4.64 14.8 15 ppb -1 0 0 0 ppb PFOSEA 3.71 11.8 15 ppb - 1000 ppb Monoester 149 248 MDL and LOQ are estimates only. No valid MDL was determinable from MDL study. Any quantitation performed for monoester will be an estimate only. Please refer to FACT-M-3.1 & 4.1-V-l for specifics. N o data is available for MDL or LOQ in Monkey Serum. U se validated Linear Calibration Range instead. . Please see Attachment C (LOQ Summary) and MDL study in. FACT-M-3.1 & 4 .1-V -l for specifics. Attachment A: Extraction worksheet FACT-M-3.1 Extraction of PFOS from Serum or Other Fluid Page 12 of 17 3M Environmental Laboratory Page 57 3M Mtidical Department Study: T-6316.7 BACK TO MAIN A nalytical Report: F A C T T O X -0 9 8 L R N -U 2 40 2 M DL/LOQ values for IVlonkey Serum: Compound MDL LOQ Linear Calibration Range (LCR) (PPb) (PPb) Approximate concentrations to be used for preparing the Standard Calibration Curve PFOS 1.38 4.39 MDL and LOQ are estimates only. No valid MDL was determinable from MDL study. Any quantitation performed for PFOS will be an estimate only. Please refer to FACT-M-3.1 & 4.1-V-l for specifics. PFOSA 2.23 7.09 MDL and LOQ are estimates only. No valid MDL was determinable from MDL study. Any quantitation performed for PFOSA will be an estimate only. Please refer to FACT-M-3.1 & 4.1-V-l for specifics. PFOSAA 2.84 9.04 MDL and LOQ are estimates only. No valid MDL was determinable from MDL study. Any quantitation performed for PFOSAA will be an estimate only. Please refer to FACT-M-3.1 & 4.1-V-l for specifics. EtFOSE-OH 3.90 12.4 MDL and LOQ are estimates only. No valid MDL was determinable from MDL study. Any quantitation performed for EtFOSE-OH will be an estimate only. Please refer to FACT-M-3.1 & 4.1-V-l for specifics. POAA 4.31 13.7 MDL and LOQ are estimates only. No valid MDL was determinable from MDL study. Any quantitationperformed for POAA will be an estimate only. Please refer to FACT-M-3.1 & 4.1-V-l for specifics. PFOSEA 1.09 3.48 MDL and LOQ are estimates only. No valid MDL was determinable from MDL study. Any quantitation performed for PFOSEA-OH will be an estimate only. Please refer to FACT-M-3.1 & 4.1-V-l for specifics. Monoester 149 248 . MDL and LOQ are estimates only. No valid MDL was determinable from MDL study. Any quantitation performed for EtFOSE-OH will be an estimate only. Please refer to FACT-M-3.1 & 4.1-V-l for specifics. MDL/LOQ vaines for R at Whole Blood: Com pound M DL LOQ Linear Calibration ]Range (LCR) (PPb) (PPb) Approximate Concentrations to be used for preparing the Standard Calibration Curve PFOS 1.25 3.96 5 p p b - lOOOppb PFOSA 1.77 5.65 10 ppb -1 0 0 0 ppb PFOSAA 17.3 55.0 55 ppb - 1000 ppb EtFO SE-O H 7.89 . 25.1 MDL and LOQ are estimates only. No valid MDL was determinable from MDL study. Any quantitation performed for EtFOSE-OH will be an estimate only. Please refer to FACT-M-3.1 & 4.1-V-l for specifics. POAA 4.73 15.1 15 ppb - lOOOppb PFOSEA 24.2 77.1 80 ppb-lOOOppb M onoester 58.0 185 MDL and LOQ are estimates only. No valid MDL was determinable from MDL study. Any quantitation performed for monoester will be an estimate only. Please refer to FACT-M-3.1 & 4.1-V-l for specifics. Please see Attachment C (LOQ Summary) and MDL study in FACT-M-3.1 & 4.1-V-l for specifics. Attachment A: Extraction worksheet FACT-M-3.1 Extraction of PFOS from Serum or Other Fluid 3M Environmental Laboratory Page 13 o f 17 Page 58 3M Medical Department Study: T-6316.7 BACK TO MAIN A nalytical Report: FAC T TO X -098 L R N -U 2 40 2 Ion Pairing Extraction of Fluorochemicals from Serum and Analysis by API/MS(MS) Summary Table: Limits o f Quantitation Compound PFOS Matrix Rabbit Bovine Rat Monkey MDL 1.38 ppb 2.11 ppb 1.27 ppb n/d LOQ 4.39 ppb 6.70 ppb 4.0-1 ppb n/d Approximate linear range2 Low std High std 5 ppb 1000 ppb 25 ppb 1000 ppb 10 ppb 1000 ppb 25 ppb 1000 ppb PFOSA Rabbit Bovine Rat 2.23 ppb 5.04 ppb 2.14 ppb 7.09 ppb 16.0 ppb 6.81 ppb 10 ppb 25 ppb 25 ppb 1000 ppb 1000 ppb 1000 ppb PFOSAA Monkey Rabbit Bovine Rat Monkey n/d 2.84 ppb 2.34 ppb 2.32 ppb n/d n/d 9.04 ppb 7.4:5 ppb 7.38 ppb n/d 25 ppb 10 ppb 263 ppb 10 ppb 25 ppb 1000 ppb 1000 ppb 1000 ppb 1000 ppb 1000 ppb EtFOSE-OH Rabbit Bovine ' 3.90 ppb 113 ppb 12.4 ppb 35.8 ppb 15 ppb 50 ppb 1000 ppb 1000 ppb Rat 3.25 ppb 10.3 ppb 50 ppb 1000 ppb Monkey n/d n/d 10 ppb 1000 ppb POAA Rabbit 4.31 ppb 113.7 ppb 15 ppb 750 ppb Bovine Rat 4.64 ppb 1.20 ppb 14.8 ppb 3.81 ppb 5 ppb 5 ppb 1000 ppb 1000 ppb Monkey n/d n/d 5 ppb 1000 ppb PFOSEA Rabbit 1.03 ppb 3.48 ppb 25 ppb 1000 ppb Bovine 3.71 ppb 11.8 ppb 5 ppb 1000 ppb . Rat 1.84 ppb 5.86 ppb 10 ppb 1000 ppb Monkey n/d n/d 5 ppb 1000 ppb Monoester1 Rabbit 149 ppb 474.0 ppb 250 ppb 1000 ppb Bovine 149 ppb 474.0 ppb 250 ppb 1000 ppb Rat 149 ppb 474.0 ppb 250 ppb . 1000 ppb Monkey n/d n/d 100 ppb 1000 ppb 1. Values for monoester axe estimates only. 2. Highest standard (approx. 1500 ppb) was excluded from final LCR and upper LOQ values due to poor R & R values and excessive weighting o f the calibration curve. Compound: PFOS Serum Prepared Range of Range of Range of -l^jLCRfrom'; matrix range o f standards (ppbXng/mL) average curve (ppbXng/mL) l i low std curve ^r1! high std curve .. '-'ic u r v e t > (PPb)(ng/rnL) l i l (P P b )(n g /m L ) (ppbXng/mL) . Rabbit Bovine 4.93 - 1450 4.93 - 1450 4.93 - 1450 ;49:3U:100(>4 49.3 - 97.6 97.6 - 1450 97.6; -1 0 0 0 S ilf io -i*: >*- 4.93 - 1450 4.93 - 248 ; 24,8t,'248. 97.6 - 1450 `97.6 -1100 Rat Monkey 4.93 - 1450 4.93 - 1450 4.93 - 976 .24.8 r 976 ' j-.CJ ~ 4.93 - 1450 24,8-OOO: 4.93 -248 24.8 -493 ! 9.76-248 : 97.6 - 1450 . 24.8 -493 97.6 - 1450 248 - T00 97.6 1000 Attachment C: LOQ Summary FACT-M-3.1 Extraction of PFOS from Serum or Other Fluid 3M Environmental Laboratory Page 14 of 17 Page 59 3M Medical Department Study: T-6316.7 BACK TO MAIN Analytical Report: FACT TO X-098 L R N -U 2 40 2 Compound: PFOSA Serum matrix Prepared range of standards (ppb)(ng/mL) Rabbit 5.00 -1470 Range of average curve (ppbXng/mL) 5.00-1470 LCRfrom ave curve (ppbXng/mL) 9.89-1000 Range of low stcl curve (Ppb)(ng/mL) 5.00-2:51 LCRfrom low std curve (ppbXng/mI.5 Range of high std curve (ppbXng/mL) n/a 98.9 - 1470 LCRfrom high std curve . (ppbXng/mL) 98.9-1000 Bovine Rat 5.00 -1470 5.00- 1470 5.00- 1470 ':n:vi25EA0<k 5.00-98.9 5.00-1470 9.89-500 : L:?; 9 8 .9-1470 9 8 .9 -1 0 0 0 98 .9 -1 4 7 0 :9?.9 -1 0 0 0 Monkey . 5.00 -1470 5.00 -1470 Ijfcgstgawij'fiwl ii^ 25.1 - 500 2 5 lf e o a 98.9-1470 _n/a \r :; Compound: PFOSAA Serum Prepared matrix range of standards (ppbXngAnL) Range o f average curve (ppbXng/mL) LCR.fromj i IJ.^v^ turves j'- (:!${ ' (ppbXng/mL) I R" 8e o f I g P oe i low stcl | | | | g oe p curve (ppbXng/niL) < 6 W 9 Range of high std curve (ppbXng/mL) tiEGR'from. :-,lnglistd^ ^.(ppV^rtg/mL)!1^ Rabbit 5 .2 0 -1 5 4 0 5 .2 0-1540 d`Oft|oOO~ 5-2 0 - " 3 1 1 1 1 104 - 1540 r',263 --100 Bovine 5.2 0 -1 5 4 0 5.20 - 1540 IQOL , M - a * # S # I 104-1540 Rat Monkey 5.20-1540 5.20-1540 m s m 5.20-1540 5.20-1540 | | S | l f a S 5.20-263 104-1540 ' 263-1000. ^ :-i^'arv 'r'->i i;:::y 5 .2 0 -2 5 3 T2$ai3^3?| 1 0 4-1540 ,'_2>63 4' lOOO i Compound: EtFOSE-OH Serum Prepared Range o f ^LRfront,^ matrix range of average standards curve (ppbXng/mL) (ppbXng/mL) i l i ^ b W w w l Range,,f low std curve (ppbXng/mL) |tefilo^Sta|5 Cf{ppiwM K|ii: Range of curve (ppbXng/mL) ^ ''( p b k f fg /n it)* Rabbit Bovine 4.94 -1450 4.94-1450 4.94 -1450 4.94 - 1450 4.94 - 248 4.94-248 m m 97.8 -1450 i^^'h/vKtfr; WSii-.i'W- 97.8 - 1450 :|<248-pHQ0 Aa*dtei*C Rat Monkey 4.94 -1450 4.94- 1450 4.94,-1450 4.94-1450 4.94-248 H i s i l i f m u ^ jtoO O ^ i t 4.94-248 >;-'`"Vj!r: .:?."*J1.TYv: :*,v9;!.78_-'248 : 97.8 -1450 : Attachment C: LOQ Summary FACT-M-3.1 Extraction of PFOS from Serum or Other Fluid 3M Environmental Laboratory Page 15 of 17 Page 60 3M Medical Department Study: T-6316.7 BACK TO MAIN Analytical Report: FACT TOX-098 LRN-U2402 Compound: POAA Serum Prepared matrix range of standards (ppbXng/mL) Rabbit 5.01 - 1480 Bovine 5.01 - 1480 Rat 5.01 - 1480 Monkey 5.01 - 1480 Range of LCR from average aye curve curve .-Vl.'.-f: (ppb)(ng/mL) (ppbXng/mL) Range o f low std curve (ppbXng/inL) LCR from low std curve {ppbXnR/fnLY Range o f high std curve (ppbXng/mL) 5 .1 3-1510 *2 5 .8-1000 5 .1 3 -2 5 8 102-1510 SIISI!--+ <&->* -ir 5 .1 3 -1510 JSiK:if0di2si4iSl'-0i0d0iiy; 5-13- 258 102-1510 5.13 - 1510 iSmMoOEl '.stm&.. 5.13-102 .5113-102. 102-1510 5-'3 - 151 i l 5 .1 3 -1 0 2 ;|Sl)2if 102-1510 LCR from high std curve (ppbXng/mL) , n/a 258-1000 102 -1 0 0 0 2*58-1900 Compound: PFOSEA Serum matrix Prepared range of standards (ppbXng/mL) Rabbit 5.13-1510 Bovine 5.13-1510 Range of l|EaEIBSiPi# average ^ave;eurye;,, low std curve curve (ppb)(ng/mL) ifppV^g'nii,')" (ppbXng/rnL) lM X m ittft 5.13-1510 kkH oO ! 5.13-258 lilil mSSMM 5.13-1510 `'idjt'Wpf 5.13 -2 5 8 | | f | | | | | | | | Range of ; LCR;from-i: high std :4:-i^isti||i ^ (ppbXng/mL) Ilfw Wf C^ nSfelBi 102 - 1510 Y-m-Fn.>:"t 102 - 1510 ;;S8'.1000j: Rat Monkey 5.13-1510 5.13 -1510 5.13-1510 j5l 3 ^ilOOtj 5.13 - 1 0 2 I I i l l i l 5.13 - 1510 . 'lO2;v'^0;j 5 .1 3 -1 0 2 102 - 1510 Mdii-*Yi2r & r*|r^t..1J,; 102 - 1510 ...i/ Compound: Monoester Serum matrix Prepared range of standards (ppbXng/mL) Range o f iJLCKfipn g average !iwfeSurv>;j curve j;S (ppbXng/rrtL) Rabbit 4.94- 1450 9 .7 8 -9 7 8 Bovine 4.94 -1450 9 7 .8 -1 4 5 0 Rat 4.94 -1450 2 4 8 -1 4 5 0 Monkey 4.94 -1450 49.4 - 1450 f97.;8;-;:'l0i In general, the chromatography for the monoester was very poor (broad peaks, high baseline). Curves for monoester in rabbit and bovine were unacceptable. Any quantitation performed with the monoester is only an estimate and should not be used for reliable, accurate data reporting. Attachment C: LOQ Summary FACT-M-3.1 Extraction of PFOS from Serum or Other Fluid 3M Environmental Laboratory Page 16 of 17 Page 61 3M Medical Department Study: T-6316.7 BACK TO MAIN Analytical Report: FACT TOX-098 LRN-U2402 Ion Pair Standard Curves - Fluids Prep date(s): Standard number: Analyte(s): Equipment number: Sample matrix: Final solvent and TN: Blank fluid/identifier: Method/revision: Target analyte(s): FC mix std approx. 0.500 ppm: W398-641 FC mix std approx. 5.00 ppm: W398-640 FC mix std approx. 50.0 ppm: W398-639 Surrogate std approx. 17.71 ppm: W398-605 Actual concentrations of standards in the FC mix PFOS Std cone PFOSA Std cone PFOSAA EtFOSEOH Std cone Std cone POAA Std cone PFOSEA Monoeste r Std cone Std cone ug/mL ug/mL ug/mL ug/mL ug/mL ug'mL ug/mL ' 0.500 0.507 0.532 0.501 0.509 0.521 0.501 0.500 0.507 0.532 0.501 0.509 0.521 0.501 5.00 5.07 5.32 5.01 5.09 5.21 5.01 5.00 5.07 5.32 5.01 5.09 5.21 5.01 5.00 5.07 5 3 2 5.01 5.09 5.21 5.01 50.0 50.1 53.2 50.1 50.9 52.1 50.1 50.0 50.1 53.2 50.1 50.9 52.1 50.1 50.0 50.1 53.2 50.1 50.9 52.1 50.1 50.0 50.1 53.2 50.1 50.9 52.1 50.1 Calculated concentrations oi'standards in the sample matrix PFOS Final cone ng/mL 4.93 9.76 24.8 49.3 97.6 248 493 735 976 PFOSA Final cone ng/mL PFOSAA Final cone ng/mL EtFOSE POAA PFOSEA Monoester Final cone Final cone Final cone Std cone ng/mL ng/mL ng/mL ng/mL 5.00 5.24 4.94 5.01 5.13 4.94 9.89 10.4 9.78 9.93 10.2 9.78 25.1 26.3 24.8 25.2 25.8 24.8 50.0 52.4 49.4 50.1 51.3 49.4 98.9 104 97.8 99.3 102 . 97.8 251 263 248 252 258 248 500 524 494 501 ' 513 494 746 782 737 749 7(56 737 989 1038 978 993 1017 978 AU All Am't spiked mL 0.010 0.020 0.005 0.010 0.020 0.005 0.010 0.015 0.020 Final Volume mL 1.015 1.025 1.010 1.015 1.025 1.010 1.015 1.020 1.025 Surrogate Std cone ng/mL . 2.64 AH Am't spiked (mL) 0.005 Surrogate Final cone ng/mL 81.0 Validated ranges - approximate concentrations Sera PFOS PFOSA PFOSAA Rabbit 5-1000 ppb 10-1000 ppb 10-1000 ppb Bovine 25-1000 ppb 25-1000 ppb 263-1000 ppb Rat 10-1000 ppb 25-1000 ppb 10-1000 ppb Monkey Estimates only. Use values for Rabbit EtFOSE-OH 10-1000 ppb 5-1000 ppb 50-500 ppb POAA 10-750 ppb 5-1000 ppb 5-1000 ppb PFOSEA 25-1000 ppb 5-1000 ppb 5-1000 ppb Attachment D: Ion Pair Standard Curves FACT-M-3.1 Extraction of PFOS from Serum or Other Fluid 3M Environmental Laboratory Page 17 o f 17 Page 62 3M Medical Department Study: T-6316.7 BACK TO MAIN Analytical Report: FACT TOX-098 LRN-U2402 3M Environmental Laboratory M ethod E x tr a c t io n o f P otassium P erflu o ro o cta n esu lfo n a te o r O t h e r F l u o r o c h e m ic a l c o m p o u n d s f r o m Se r u m f o r A n a l y sis U s in g HPLC- E lectrospray/M ass Spectrom etry M ethod N um ber: TS-8-4.1 Adoption Date: 03/01/99 Author: Lisa Clemen, Glenn Langenburg Revision Date: 4 /5 Approved By: 01 j Laboratory Manager ---- . Date U t-- Group Leader y/tL /S d Date A Technical Reviewer oifaU w Date 1.0 Scope and Application 1.1 Scope: This method is for the extraction of potassium perfluorooctanesulfonate (PFOS) or other fluorochemical compounds from serum. 1.2 Applicable compounds: Fluorochemical surfactants or other fluorinated compounds. 1.3 M atrices: Rabbit, rat, bovine, monkey, and human serum or other fluids as designated in the validation report. Word 6/95 3M Environmental Laboratory ETS-8-4.1 Extraction o f PFOS from Serum Page 1 o f 14 Page 63 3M Medical Department Study: T-6316.7 BACK TO MAIN Analytical Report: FACT TOX-098 LRN-U2402 2.0 SUMMARY OF METHOP_________________ ;_________ .________________________________________ 2.1 This method describes the procedure for extracting potassium perfluorooctanesulfonate (PFOS) or other fluorochemical surfactants from serum, or other fluids, using an ion pairing reagent and methyl-terf-butyl ether (MtBE). In this method, seven fluorochemicals were extracted: PFOS, PFOSA, PFOSAA, EtFOSE-OH, PFQSEA, M556, and surrogate standard (see 3.0 Definitions). An ion pairing reagent is added to the sample and the analyte ion pair is partitioned into MtBE. The MtBE extract is removed and put onto a nitrogen evaporator until dry. Each extract is reconstituted in 1.0 mL o f methanol, then filtered through a 3 cc plastic syringe attached to a 0.2 jam nylon filter into glass autovials. 2.2 These sample extracts are analyzed following method ETS-8-5.1 or other appropriate methods. 3.0 Definitions_________ __ ;_________________________________________._________________ 3.1 PFOS: perfluorooctanesulfonate (anion o f potassium salt) C,F17S 03' 3.2 PFOSA: perfluorooctane sulfonylamide C8F 17S 02NH2 3.3 PFOSAA: perfluorooctane sulfonylamido (ethyl)acetate C8F17S 0 2N(CH2CH3)CH2C 0 2` 3.4 EtFOSE-OH: 2(N-ethylperfluorooctane sulfonamido)-ethyl alcohol C8F 17S 0 2N(CH2CH3)CH2CH20 H 3.5 PFOSEA: perfluorooctane sulfonyl ethylamide C8F17S 02N(CH2CH3)H 3.6 M556: C8F 17S 0 2N(H)(CH2C 00H ) 3.7 Surrogate standard: 1H-1H-2H-2H perfluorooctane sulfonic acid 4.0 W arnings and Ca u t i o n s _________________________________________________ 4.1 Health and safety warnings 4.1.1 Use universal precautions, especially laboratory coats, goggles, and gloves when handling animal tissue, which may contain pathogens. 5.0 Interferences__________________________________________________________________ _ 5.1 There are no interferences known at this time. 6.0 E quipment________________________________________________________ ___ 6.1 The following equipment is used while performing this method. Equivalent equipment is acceptable. 6.1.1 Vortex mixer, VWR, Vortex Genie 2 6.1.2 Centrifuge, Mistral 1000 or IEC 6.1.3 Shaker, Eberbach or VWR ETS-8-4.1 Extraction of PFOS from Serum Page 2 of 14 3M Environmental Laboratory Page 64 3M Medical Department Study: T-6316.7 BACK TO MAIN Analytical Report: FACT TOX-098 LRN-U2402 6.1.4 Nitrogen evaporator, Organomation 6.1.5 Balance ( 0.100 g) 7.0 S upplies and Materials_________________ ;________________________________________ 7.1 Gloves 7.2 Eppendorf or disposable pipettes 7.3 Nalgene bottles, capable o f holding 250 mL and 1 L 7.4 Volumetric flasks, glass, type A 7.5 I-CHEM vials, glass, 40 mL glass 7.6 Centrifuge tubes, polypropylene, 15 mL 7.7 Labels 7.8 Oxford Dispenser - 3.0 to 10.0 mL 7.9 Syringes, capable of measuring 5 pL to 50 pL 7.10 Graduated pipettes 7.11 Syringes, disposable plastic, 3 cc 7.12 Syringe filters, nylon, 0.2 pm, 25 mm 7.13 Timer 7.14 Crimp cap autovials and caps 7.15 Crimpers Note: Prior to using glassware and bottles, rinse 3 times with methanol and 3 times with Milli-QTM water. Rinse syringes a minimum o f 9 times with methanol, 3 rinses from 3 separate vials. 8.0 Reagents and Standards________________________ ;____________________________ ; 8.1 Type I reagent grade water, Milli-QTM or equivalent; all water used in this method should be Milli-QTM water and may be provided by a Milli-Q TOC PlusTM system 8.2 Sodium hydroxide (NaOH), J.T Baker or equivalent 8.3 Tetiabutylammonium hydrogen sulfate(TBA), Kodak or equivalent 8.4 Sodium carbonate (NajCOj), J.T. Baker or equivalent 8.5 Sodium bicarbonate (NaHC03), J.T. Baker or equivalent 8.6 Methyl-T-Butyl Ether, Omnisolv, glass distilled or HPLC grade 8.7 Methanol, Omnisolv, glass distilled or HPLC grade 8.8 Serum or blood, frozen from supplier 8.9 Fluorochemical standards 8.9.1 PFOS (3M Specialty Chemical Division), molecular weight = 538 8.9.2 PFOSA (3M Specialty Chemical Division), molecular weight = 499 ETS-8-4.1 Extraction of PFOS from Serum Page 3 of 14 3M Environmental Laboratory Page 65 3M Medical Department Study: T-6316.7 BACK TO MAIN Analytical Report: FACT TOX-098 LRN-U2402 8.93 PFOSAA (3M Specialty Chemical Division), molecular weight = 585 8.9.4 EtFOSE-OH (3M Specialty Chemical Division), molecular weight = 570 8.9.5 PFOSEA (3M Specialty Chemical Division), molecular weight = 527 8.9.6 M 556(3M Specialty Chemical Division), molecular weight = 557 8.9.7 Surrogate standard: 4-H, perfluorooctane sulfonic acid (1-H,1-H, 2-H, 2-H C8F13S 0 3H) molecular weight = 428 8.9.8 Other fluorochemicals, as appropriate 8.10 Reagent preparation NOTE: When preparing larger volumes than listed in reagent, standard, or surrogate preparation, adjust accordingly. 8.10.1 IO N sodium hydroxide (NaOH): Weigh approximately 200 g NaOH. Pour into a 1000 mL beaker containing 500 mL Milli-QTM water, mix until all solids are dissolved. Store in a 1 L Nalgene bottle. 8.10.2 1 N sodium hydroxide (NaOH): Dilute 10 N NaOH 1:10. Measure 10 mL o f 10 N NaOH solution into a 100 mL volumetric flask and dilute to volume using Milli-QTM water. Store in a 125 mL Nalgene bottle. 8.10.3 0.5 M tetrabutylammonium hydrogen sulfate (TBA): Weigh approximately 169 g o f TBA into a 1 L volumetric containing 500 mL Milli-QTM water. Adjust to pH 10 using approximately 44 to 54 mL of 10 N NaOH (While adding the last mL of ' NaOH, add slowly because the pH change abmptly). Dilute to volume with Milli-QTM water. Store in a 1 L Nalgene bottle. 8.10.3.1 TBA requires a check prior to each use to ensure pH = 10. Adjust as needed using 1 N NaOH solution. 8.10.4 0.25 M sodium carbonate/sodium bicarbonate buffer (NajCO/NaHCOj): Weigh approximately 26.5 g o f sodium carbonate (NajC03) and 21.0 g of sodium bicarbonate (NaHCOj) into a 1 L volumetric flask and bring to volume with MilliQTM water. Store in a 1 L Nalgene bottle. 8.11 Standards preparation ' 8.11.1 Prepare PFOS standards for the standard curve. 8.11.2 Prepare other fluorochemical standards, as appropriate. Multicomponent fluorochemical standards are acceptable (for example, one working standard solution containing 1.00 ppm PFOS, 1.02: ppm PFOSA, 0.987 ppm PFOSAA, and 1.10 ppm EtFOSE-OH.) 8.11.3 Weigh approximately 100 mg of PFOS into a 100 mL volumetric flask and record the actual weight. 8.11.4 Bring to volume with methanol for a stock standard o f approximately 1000 ppm (pg/mL). 8.11.5 Dilute the stock solution with methanol for a working standard 1 solution o f approximately 50 ppm. 8.11.6 Dilute working standard 1 with methanol, for a working standard 2 solution of approx. 5.0 ppm. ETS-8-4.1 Extraction of PFOS from Serum Page 4 of 14 3M Environmental Laboratory Page 66 3M Medical Department Study: T-6316.7 BACK TO MAIN Analytical Report: FACT TOX-098 LRN-U2402 8.11.7 Dilute working standard 1 with methanol for a working standard 3 solution o f approx. 0.50 ppm. 8.12 Surrogate stock standard preparation 8.12.1 Weigh approximately 50-60 mg of surrogate standard 1-H,1-H, 2-H, 2-H, CaF |3S 0 3H into a 50 mL volumetric flask: and record the actual weight. 8.12.2 Bring to volume with methanol for a surrogate stock o f approximately 1000-1200 ppm. 8.12.3 Prepare a surrogate working standard. Transfer approximately 1 mL o f surrogate stock to a 10 mL volumetric flask and bring to volume with methanol for a working standard o f 100 ppm. Record the actual volume transferred. 9.0 Sample Handling___________ ;_______________________________ i_____________________ 9.1 All samples are received frozen and must be kept frozen until the extraction is performed. 9.2 Allow samples to thaw to room temperature prior to extraction. 10.0 Quality Control______________________ ;_______________ .________________________ 10.1 Solvent Blanks, M ethod blanks and m atrix blanks 10.1.1 An aliquot o f 1.0 mL methanol is used as; a solvent blank. 10.1.2 Extract two 1.0 mL aliquots o f Milli-QTM water following this procedure and use as method blanks. 10.1.3 Extract two 1.0 mL aliquots o f the serum following this procedure and use as matrix blanks. See 11.1.4. 10.2 M atrix spikes 10.2.1 Prepare and analyze matrix spike and matrix spike duplicate samples to determine the accuracy of the extraction. 10.2.2 Prepare each spike using a sample chosen by the analyst, usually the control matrix received with each sample set. 10.2.3 Expected concentrations will fall in the mid-range of tire initial calibration curve. Additional spikes may be included and may fall in the low-range o f the initial calibration curve. 10.2.4 Prepare one matrix spike and matrix spike duplicate per 40 samples, with a minimum o f 2 matrix spikes per batch. 10.3 Continuing calibration checks 10.3.1 Prepare continuing calibration check samples to ensure the accuracy o f the initial calibration curve. 10.3.2 Prepare, at a minimum, one continuing check per group of 10 samples. For example, if a sample set = 34, four checks are prepared and extracted. 10.3.3 Prepare each continuing calibration check from the same matrix used to prepare the initial curve. ETS-8-4.1 Extraction of PFOS from Serum Page 5 of 14 3M Environmental Laboratory Page 67 3M Medical Department Study: T-6316.7 BACK TO MAIN Analytical Report: FACTTOX-098 LRN-U2402 10.3.4 The expected concentrations will fall witliin the mid-range of the initial calibration curve. Additional spikes may be included that fall in the low-range of the initial calibration curve. This is necessary if the analyst must quantitate using only the low end o f the calibration curve (for example, 5 ppb - 100 ppb, rather than 5 ppb - 1000 ppb). 11.0 Calibration and Standardization_________ ____________________;________________ 11.1 Prepare matrix calibration standards 11.1.1 Transfer 1 mL o f serum to a 15 mL centrifuge tube. 11.1.2 If most sample volumes are less than 1.0 mL, extract standards with matrix volumes equal to the sample volumes. Do not extract less than 0.50 mL of matrix. Record each sample volume on the extraction sheet 11.1.3 While preparing a total o f twenty aliquot, in 15 mL centrifuge tubes, m ix or shake between aliquots. 11.1.4 Two 1 mL aliquots, or other appropriate volume, serve as matrix blanks. Typically use the standard concentrations and spiking amounts listed in Table 1, at the end o f this section, to spike, in duplicate, two standard curves, for a total of eighteen standards, two matrix blanks, and two method blanks. 11.1.5 Refer to validation report ETS-8-4.0 & ETS-8-5.0-V-1, which lists the working ranges and the Linear Calibration Range (LCR) for calibration curves. 11.1.6 Use Attachment D as an aid in calculating the concentrations of the working standards. See Section 13.0 to calculate actual concentrations of PFOS in calibration standards. 11.2 To each standard, blank, or continuing check, add appropriate amount o f surrogate working standard for the concentration to fall within the calibration curve range 5 ppb 1000 ppb. 113 Extract spiked matrix standards following 12.6-12.16 o f this method. Use these standards to establish each initial curve on the mass spectrometer. 3M Environmental Laboratory ETS-8-4.1 Extraction of PFOS from Serum Page 6 of 14 Page 68 3M Medical Department Study: T-6316.7 BACK TO MAIN Analytical Report: FACT TOX-098 LRN-U2402 Table 1 Approximate spiking amounts for standards and spikes Using 1.0 mL of m atrix Working standard p.L Approx, final cone, of (approx, cone.) analyte in matrix - - Blank 0.500 ppm 10 0.005 ppm 0.500ppm 20 0.010 ppm 5.00 ppm 5 0.025 ppm 5.00 ppm 10 . 0.050 ppm 5.00 ppm . 20 0.100 ppm 50.0 ppm 5 0.250 ppm 50.0 ppm 10 0.500 ppm 50.0 ppm 15 0.750 ppm 50.0 ppm 20 1.00 ppm 12.0 Procedure______________________ _________ ________________________ ____________ _ 12.1 Obtain frozen samples and allow to thaw at room temperature or in a lukewarm waterbath. 12.2 Vortex mix for 15 seconds, then transfer 1.0 mL or other appropriate volume to a 15 mL polypropylene centrifuge tube. 12.3 Return unused samples to freezer after extraction, amounts have been removed. 12.4 Record the initial volume on the extraction worksheet. 12.5 Label the tube with the study number, sample ID, date and analyst initials. See attached worksheet for documenting the remaining steps. 12.6 Spike all samples, including blanks and standards, ready for extraction with surrogate standard as described in 11.2. 12.7 Spike each matrix with the appropriate amount o f standard as described in 11.1, or Table 1 in that section, for the calibration curve standards. Also prepare matrix spikes and continuing calibration standards. 1 12.8 Vortex mix the standard curve samples, matrix spike samples, and continuing calibration samples for 15 seconds. 12.9 Check to ensure the 0.5 M TBA reagent is at pH 10. If not, adjust accordingly. 12.10 To each sample, add 1 mL 0.5 M TBA and 2 mL of 0.25M sodium carbonate/sodium bicarbonate buffer. 12.11 Using an Oxford Dispenser, add 5 mL methyl-feri-butyl ether. 12.12 Cap each sample and put on the shaker at a setting of 300 rpm, for 20 minutes. 12.13 Centrifuge for 20 to 25 minutes at a setting o f 3:500 rpm, or until layers are well separated. ETS-8-4.1 Extraction of PFOS from Serum Page 7 of 14 3M Environmental Laboratory Page 69 3M Medical Department Study: T-6316.7 BACK TO MAIN Analytical Report: FACT TOX-098 LRN-U2402 12.14 Label a fresh 15 mL centrifuge tube with the same information as in 12.5. 12.15 Remove 4.0 mL of the organic layer to this clean 15 mL centrifuge tube. 12.16 Put each sample on the analytical nitrogen evaporator until dry, approximately 1 to 2 hours. 12.17 Add 1.0 mL of methanol to each centrifuge tube using a graduated pipette. 12.18 Vortex mix for 30 seconds. 12.19 Attach a 0.2 pm nylon mesh filter to a 3 cc syringe and transfer the sample to this . syringe. Filter into a 1.5 mL glass autovial or low-volume autovial when necessary. 12.20 Label the autovial with the study number, animal number and gender, sample thnepoint, matrix, final solvent, extraction date, and analyses) performing the extraction. 12.21 Cap and store extracts at room temperature or at approximately 4 C until analysis. 12.22 Complete the extraction worksheet, attached to this document, and tape in the study notebook or include in study binder, as appropriiite. 13.0 Data Analysis and Calculations____________________________________________ 13.1 Calculations 13.1.1 Calculate actual concentrations of PFOS, or other applicable fluorochemical, in calibration standards using the following equation: mL o f standard x concentration of standard fug /m L i__________;________ = . mL o f standard + mL of surrogate standard + initial matrix volume (mL) Final Concentration (pg/mL) o f PFOS in matrix 14.0 Method Performance___________________ ;___________________________________ 14.1 The method detection limit (MDL) is analyte and matrix specific. Refer to MDL report for specific MDL and limit of quantitation (LOQ) values (see Attachments B and C). 14.2 The following quality control samples are extracted with each batch of samples to evaluate the quality o f the extraction and analysis. 14.2.1 Method blanks and matrix blanks. . 14.2.2 Matrix spike and matrix spike duplicate samples to determine accuracy and precision o f the extraction. 14.2.3 Continuing calibration check samples to determine the continued accuracy o f the initial calibration curve. 14.3 Refer to section 14 of ETS-8-5.1 for method performance criteria. 15.0 Pollution Prevention and Waste Management________________________ 15.1 Sample waste is disposed in biohazard containers, flammable solvent waste is disposed in high BTU containers, and used glass pipette waste is disposed in broken glass containers located in the laboratory. ETS-8-4.1 Extraction of PFOS from Serum Page 8 of 14 3M Environmental Laboratory Page 70 3M Medical Department Study: T-6316.7 BACK TO MAIN Analytical Report: FACTTOX-098 LRN-U2402 1 6 -0 RECO RD S________________ ;__________ ;_________________________________________________ ^________ 16.1 Complete the extraction worksheet attached to this method, and tape in the study notebook or include in the 3-ring study binder, as appropriate. 17.0 attachm ents___________________________________________________________________ 17.1 Attachment A, Extraction worksheet 17.2 Attachment B, MDL/LOQ values and summary 17.3 Attachment C, Calibration standard concentration worksheet 18.0 References_____________________________________________________________________ 18.1 The validation report associated with this method is ETS-8-4.0 & 5.0-V -l. 18.2 FACT-M-3.1, "Analysis of Serum or Other Fluid. Extracts for Fluorochemicals using HPLC-Electrospray Mass Spectrometry" 19.0 affected D ocuments________ ;__________________________________________________ 19.1 ETS-8-5.1, "Analysis o f Serum or Other Fluid Extracts for Fluorochemicals using HPLC-Electrospray Mass Spectrometry" 20.0 R evisions ________________:____________________ !____________________________ Revision Mumher 1 Reason For Revision Section 12.21 Changed to include sample storage at room temperature. Section 12.13 Added the shaker speed. Section 12.17 Final volume is 1.0 mL; not adjusted for initial volumes less than 1.0 mL. Revision Date 04/02/99 3M Environmental Laboratory . ETS-8-4.1 Extraction of PFOS from Serum Page 9 o f 14 Page 71 BACK TO MAIN 3M Medical Department Study: T-6316.7 Analytical Report: FACT TOX-098 LRN-U2402 Extraction Worksheet ETS-8-4.1 Studv# Matrix Box# Wk/Dav DateSpiked/Analyst CCV MS MSD Surrogate Std approx, ppm actual ppm # FC-Mix approx. 0.5 pm actual ppm # FC-Mix approx. 5 ppm actual ppm # FC-Mix approx. 50 ppm actual ppm # Comments ---- --- -' ------- - - '- Blank Std# amount- Serum Extraction Method : Vortex 15 sec. Pioette Matrix ' Volume mL Pipette 1 mL of 0.5 M TBA, pH 10. pH - Std. # Pipette 2 mL of 0.25 Na2COV0.25M NaHCOi buffer Std. # Dispense 5 mL of methyl-t-butyl ether TN-A- Shake 20 min. Shaker speed: Centrifuee 20-25 min. Centrifuee speed: Remove a 4 mL aliauot of oreanic laver Put on Nitroeen Evaporator to drvness Temperature: Add methanol Volume mL TN-A- Vortex 30 sec. Filter usine a 3cc B-D svrinee with a 0.2um filter into a 1.5 mL autosamcle vial Cont Cal. Verifications used same matrix as for std curve. - - - .- - - - - mL Date & Initials Attachment A 3M Environmental Laboratory ETS-8-4.1 Extraction of PFOS from Serum Page 10 of 14 Page 72 3M Medical Department Study: T-6316.7 BACK TO MAIN Analytical Report: FACT TOX-098 LRN-U2402 MDL/LOQ values for rabbit serum Compound MDL LOQ Linear Calibration Range (LCR) (PPb) (PPb) Approximate concentrations to be used for preparing the Standard Calibration Curve PFOS 1.74 5.55 5p p b -1 0 0 0 p p b PFOSA 1.51 4.79 5 ppb -1000 ppb PFOSAA 3.46 20.5 5 p p b - 1000 ppb EtFOSE-OH 11.4 36.2 5 p p b -1000 ppb M556 6.03 19.2 5 ppb -1000 ppb PFOSEA 5.71 18.2 5 p p b -1000 ppb MDL/LOQ values in rat, bovine, monkey, and human serum, and monkey plasma were not statistically determined. Two curves in each of these matrices were extracted and analyzed with the rabbit serum curves to determine equivalence. Responses in the rat, bovine, monkey, and human were equivalent to the rabbit responses, therefore, their MDL and LOQ will be the same values as determined in rabbit serum. Please see LOQ Summary and MDL study in ETS-8-4.0 & 5,0-V-l for further information. Attachment B: MDL/LOQ Summary ETS-8-4.1 Extraction of PFOS from Serum 3M Environmental Laboratory Page 11 of 14 Page 73 3M Medical Department Study: T-6316.7 BACK TO MAIN Analytical Report: FACT TOX-098 LRN-U2402 Com pound: PFOS Prepared range Rabbit Serum o f standards (ppb) (ng/mL) Full Range Low Curve High curve 1/X 0.995 - 978 4.94 - 248 97.8 - 978 0.995 - 978 LCR from curve (pph) (ng/mL) 24.8 - 978 4.94-248 97.8-978 4.94 - 978 % Recovery Range 83-103 85-104 85-106 94-111 Compound: PFOSA Prepared range Rabbit Serum o f standards (ppb) (ng/mL) Full Range Low Curve High curve 1/X 0.993 - 976 4.93 - 97.6 24.8-976 0.993 - 976 LCR from curve (PPb) (ng/mL) 4.93-976 % Recovery Range 88-103 4.93 - 97.6 . 87-105 24.8-978 93-102 4.93-976 94-103 Compound: PFOSAA Prepared range Rabbit Serum o f standards (ppb) (ng/mL) Full Range Low Curve High curve 1/x 0.991 - 974 4.92 - 247 49.2-974 0.991 - 974 LCR from curve (PPb) (ng/mL) 24.7 - 974 9.74-247 97.4 - 974 9.74-974 % Recovery Range 81-111 97-107 85-108 95-115 RSD Range 4.67-11.0 5.34-12.0 4.84-9.80 4.60-10.5 RSD Range 5.10-14.7 9.85-14.7 5.08-13.9 5.10-14.5 RSD Range 4.18-10.6 6.38-21.8 4.33-12.5 4.11-23.2 Attachment B: MDL/LOQ Summary ETS-8-4.1 Extraction of PFOS from Serum 3M Environmental Laboratory Page 12 o f 14 Page 74 3M Medical Department Study: T-6316.7 BACK TO MAIN Analytical Report: FACT TOX-098 LRN-U2402 Compound: EtFOSE-OH Prepared range Rabbit Serum o f standards (ppb) (ng/mL) Full Range Low Curve High curve 1/X 0.993 - 976 4.93-97.6 49.3 - 976 0.993-493 LCR from curve (ppb) (ng/mL) 49.3 -976 9.76-97.6 97.6 - 976 9.76 - 976 % Recovery Range 77-110 97-107 90-109 86-111 Compound: PFOSEA Prepared range Rabbit Serum o f standards (ppb) (ng/mL) Full Range Low Curve High curve 1/X 0.993-976 4.93 - 248 49.3 - 976 0.993 - 976 LCR from curve (PPb) (ng/mL) 24.8 - 976 9.76 - 248 49.3 - 976 9.76 - 976 % Recovery Range 96-105 91-113 86-106 95-117 Compound: M556 Prepared range Rabbit Serum o f standards (PPb) (ng/mL) Full Range Low Curve High curve 1/X 0.993 - 976 4.93 - 97.6 97.6 - 976 0.993 - 976 LCR from curve (ppb) (ng/mL) 24.8 - 976 9.76-97.6 97.6 - 976 9.76-976 % Recovery Range 88-106 100-105 81-111 97-110 RSD Range 11.2-25.5 14.1-21.3 11.5-19.6 11.1-21.2 RSD Range 10.1-16.2 11.8-19.5 10.2-18.2 10.1-19.1 RSD Range 4.82-17.9 5.95-18.2 5.11-9.74 4.77-19.5 Attachment B: MDL/LOQ Summary ETS-8-4.1 Extraction of PFOS from Serum 3M Environmental Laboratory Page 13 of 14 Page 75 3M Medical Department Study: T-6316.7 BACK TO MAIN Analytical Report: FACT TOX-098 LRN-U2402 Ion Pair Standard Curves - Fluids Prep date(s): Standard number: Analyte(s): Equipment number: Sample matrix: Final solvent and TN: Blank fluid/identifier: Method/revision: Target analyte(s): FC mix std approx. 0.500 ppm: FC mix std approx. 5.00 ppm: FC mix std approx. 50.0 ppm: Surrogate std approx. 100 ppm: Actual concentrations of standards in the FC mix PFOS PFOSA PFOSAA EtFOSE PFOSEA Std cone Std cone Std cone Std cone Std cone ng/mT. ug/mL ug/mL ug/mL ug/mL 0.500 0.507 0.532 0.501 0.521 0.500 0.507 0.532 0.501 0.521 5.00 5.07 5.32 5.01 5.21 5.00 5.07 5.32 . 5.01 5.21 5.00 5.07 5.32 5.01 5.21 50.0 50.1 53.2 50.1 52.1 50.0 50.1 53.2 50.1 52.1 50.0 50.1 53.2 50.1 52.1 50.0 50.1 53.2 50.1 52.1 M556 Std cone ug/mL 0.501 0.501 5.01 5.01 5.01 50.1 50.1 50.1 50.1 AU Am't spiked mL 0.010 0.020 0.005 0.010 0.020 0.005 0.010 0.015 0.020 All Final voi mL 1.015 1.025 1.010 1.015 1.025 1.010 1.015 1.020 1.025 Calculated concentrations o f standards in the sample! matrix PFOS PFOSA PFOSAA EtFOSE PFOSEA M556 Surrogate All Final cone Final cone Final cone Final cone Final cone Final cone Std cone Amt spiked ng/mL____ ng/roL_______ ng/mL____ ng/mL ng/mL n;ptnL ng/mL_______ mL 4.93 5.00 5.24 4.94 5.01 5.13 100 9.76 9.89 10.4 9.78 9.93 10.2 24.8 25.1 26.3 24.8 25.2 25.8 Surrogate 49.3 50.0 52.4 . 49.4 50.1 51.3 Final cone 97.6 98.9 104 97.8 99.3 102 ng/mL 248 251 263 248 252 258 500 493 500 524 494 501 513 735 746 782 737 749 766 976 989 1038 978 993 1017 Validated ranges - approximate concentrations Serum PFOS PFOSA PFOSAA EtFOSE-OH PFOSEA M556 Rabbit 5.00-1000 I 5.00-1000 | 5.00-1000 | 5.00-1000 | 5.00-1000 | 5.00-1000 Bovine Estimates only. Use values for rabbit . Rat Estimates only. Use values for rabbit Monkey & Plasma Estimates only. Use values for rabbit. Human Estimates only. Use values for rabbit. Attachment C: Ion Pair Standard Curves ETS-8-4.1 . Extraction of PFOS from Serum 3M Environmental Laboratory Page 14 of 14 Page 76 3M Medical Department Study: T-6316.7 BACK TO MAIN Analytical Report: FACT TOX-098 LRN-U2402 3M Environmental Laboratory M ethod E x tr a c t io n o f P o ta ssiu m P erflu o ro o cta n esu lfo n a te o r o t h e r F l u o r o c h em ic a l C om pounds fr o m L iv er fo r Analysis usin g H P L C - E lectrospray/M ass Spectrom etry M ethod N um ber: ETS-8-6.0 Author: Lisa Clemen, Robert Wynne Approved By: yiTw- Laboratory Manager u fa it* , Group Leader ----------- (Htx. A Technical Reviewer .. Adoption Date: Revision Date: " V 2- V h <? Date Date 0 ? /ii/< H Date ... 1.0 Scope and Application 1.1 Scope: This method is for the extraction of potassium perfluorooctanesulfonate (PFOS) or other fluorochemical compounds from liver. 1.2 Applicable Compounds: Fluorochemical surfactants or other fluorinated compounds. 1.3 M atrices: Rabbit, rat, bovine, and monkey livers or other tissues as designated in the validation report. Word 6.0/95 ETS-8-6.0 Extraction of PFOS from Liver Page 1 of 14 3M Environmental Laboratory Page 77 3M Me dical Department Study: T-6316.7 BACK TO MAIN Analytical Report: FACT TOX-098 LRN-U2402 2.0 Summary of Method________________________________________________________ ' 2.1 This method describes the procedure for extracting potassium perfluorooctanesulfonate (PFOS) or other fluorochemical surfactants from liver, or other tissues, using an ion pairing reagent and methyl-ieri-butyl ether (MtBE). In this method, seven fluorochemicals can be extracted: PFOS, PFOSA, PFOSAA, EtFOSE-OH,.PFOSEA, M556, and surrogate standard. An ion pairing reagent is added to the sample and the analyte ion pair is partitioned into MtBE. The MtBE extract is transferred to a centrifuge tube and put onto a nitrogen evaporator until dry. Each extract is reconstituted in 1.0 mL methanol then filtered through a 3 cc plastic syringe attached to a 0.2 pm nylon filter into glass autovials. 2.2 These sample extracts are analyzed following method ETS-8-7.0 or other appropriate methods. 3.0 D efinitions______________________________________________________________________ 3.1 PFOS: perfluorooctanesulfonate (anion of potassium salt) CgF17S 0 3 3.2 PFOSA: perfluorooctane sulfonylamide C8FI7S 0 2NH2 3.3 PFOSAA: perfluorooctane sulfonylaxnido (ethyl)acetate C8F17S 0 2N(CH2CH3)CH2C 0 2 3.4 EtFOSE-OH: 2(N-ethylperfluorooctane sulfonamido)-ethyl alcohol C8F nS 0 2N(CH2CH3)CH2CH2OH 3.5 PFOSEA: perfluorooctane sulfonyl ethylamide C8F17S 0 2N(CH2GH3)H 3.6 M556: C,F,7S 0 2N(H)(CH2C 00H ) 3.7 Surrogate standard: 1H-1H-2H-2H perfluorooctane: sulfonic acid 4.0 W arnings and Cautions_________________________________1 _ ________________ ' 4.1 H ealth and Safety W arnings: 4.1.1 Use universal precautions, especially laboratory coats, goggles, and gloves when handling animal tissue, which may contain pathogens. . 5.0 Interferences__________________________________________________________________ 5.1 There are no interferences known at this time. ' 6.0 Equipment________________________ ;_________________'___________________ ;_________ 6.1 The following equipment is used while performing this method. Equivalent equipment is acceptable. 6.1.1 6.1.2 6.1.3 6.1.4 Ultra-Turrax T25 Grinder for grinding liver samples Vortex mixer, VWR, Vortex Genie 2 Centrifuge, Mistral 1000 or IEC Shaker, Eberbach or VWR ETS-8-6.0 Extraction of PFOS from Liver Page 2 of 14 3M Environmental Laboratory Page 78 3M Me dical Department Study: T-6316.7 BACK TO MAIN Analytical Report: FACTTOX-098 LRN-U2402 6.1.5 Nitrogen Evaporator, Organomation 6.1.6 Balance (sensitivity to 0.100 g) 7.0 Supplies and Materials____________________________________________________ 7.1 Gloves 7.2 Dissecting scalpels 7.3 Eppendorf or disposable pipettes 7.4 Nalgene bottles, capable of holding 250 mL and 1 L 7.5 Volumetric flasks, glass, type A . 7.6 I-CHEM vials, 40 mL glass . 7.7 Plastic sampule vials, Wheaton, 6 mL (or appropriate size) 7.8 Centrifuge tubes, polypropylene, 15 mL 7.9 Labels 7.10 Oxford D isp en so r-3.0 to 10.0 ml 7.11 Syringes, capable o f measuring 5 pL to 50 pL 7.12 Graduated pipettes 7.13 Syringes, disposable plastic, 3 cc 7.14 Syringe filters, nylon, 0.2 pm, 25 mm 7.15 Timer 7.16 Crimp cap autovials and caps 7.17 Crimpers Note: Prior to using glassware and bottles, rinse 3 times with methanol and 3 times with MilliQTM water. Rinse syringes a minimum o f 9 times with methanol, 3 rinses from 3 separate ' vials. 8.0 Reagents and Standards________________________________ ________________ '___ 8.1 Type I reagent grade water, Milli-QTM or equival ent; all water used in this method should be Milli-QTM water and be provided by a Milli-Q TOC PlusTM system 8.2 Sodium hydroxide (NaOH), J.T Baker or equivalent 8.3 Tetrabutylammonium hydrogen sulfate(TBA), Kodak or equivalent 8.4 Sodium carbonate (Na^COj), J.T. Baker or equivalent 8.5 Sodium bicarbonate (NaHC03), J.T. Baker or equivalent 8.6 Methyl-te/7-butyl ether, Omnisolv, glass distilled or HPLC grade 8.7 Methanol, Omnisolv, glass distilled or HPLC grade 8.8 Liver, frozen from supplier 8.9 Dry ice from supplier 8.10 Fluorochem ical standards 8.10.1 PFOS(3M Specialty Chemical Division), molecular weight = 538 . ETS-8-6.0 Extraction of PFOS from Liver Page 3 of 14 3M Environmental Laboratory 3M Medical Department Study: T-6316.7 BACK TO MAIN Analytical Report: FACT TOX-098 LRN-U2402 8.10.2 PFOSA (3M Specialty Chemical Division), molecular weight = 499 , 8.10.3 PFOSAA (3M Specialty Chemical Division), molecular weight = 585 8.10.4 EtFOSE-OH (3M Specialty Chemical Division), molecular weight = 570 8.10.5 PFOSEA (3M Specialty Chemical Division), molecular weight = 527 8.10.6 M556 (3M Specialty Chemical Division), molecular weight = 557 8.10.7 Surrogate standard: 4-H, perfluorooctane sulfonic acid (l-H .l-H , 2-H, 2-H C8F,3S 0 3H) molecular weight = 428 8.10.8 Other fluorochemicals, as appropriate 8.11 Reagent preparation N O TE: When preparing larger volumes than listed in reagent, standard, or surrogate preparation, adjust accordingly. 8.11.1 10 N sodium hydroxide (NaOH): Weigh approximately 200 g NaOH. Pour into a 1000 mL beaker containing 500 mL Milli-QTM water, mix until all solids are . dissolved. Store in a 1 L Nalgene bottle. 8.11.2 I N sodium hydroxide (NaOH): Dilute 10 N NaOH 1:10. Measure 10 mL o f 10 N NaOH solution into a 100 mL volum etric flask and dilute to volume using Milli-QTM water. Store in a 125 mL Nalgene bottle. 8.11.3 0.5 M tetrabutylammonium hydrogen sulfate (TBA): Weigh approximately 169 g of TBA into a 1 L volumetric containing 500 mL Milli-QTM water. Adjust to pH 10 using approximately 44 to 54 mL of 10 N NaOH (While adding the last mL o f NaOH, add slowly because the pH changes abruptly). Dilute to volume with Milli-QTM water. Store in a 1 L Nalgene bottle. 8.11.3.1 TBA requires a check prior to each use to ensure pH = 10. Adjust as needed using 1 N NaOH solution. 8.11.4 0.25 M sodium carbonate/sodium bicarbonate buffer (NajC03/NaHC03): Weigh approximately 26.5 g o f sodium carbonate (NajCO,,) and 21.0 g of sodium bicarbonate (NaHC03) into a 1 L volumetric flask and bring to volume with Milli- QTM water. Store in a 1 L Nalgene bottle. 8.12 Standards preparation 8.12.1 Prepare PFOS standards for the standard curve. 8.12.2 Prepare other fluorochemical standards, as appropriate. Multicomponent fluorochemical standards are acceptable (for example, one working standard solution containing 1.00 ppm PFOS, 1.02 ppm PFOSA, 0.987 ppm PFOSAA, and 1.10 ppm EtFOSE-OH.) . 8.12.3 Weigh approximately 100 mg of PFOS into a 100 mL volumetric flask and record the actual weight. 8.12.4 Bring to volume with methanol for a stock standard of approximately 1000 ppm (pg/mL). 8.12.5 Dilute the stock solution with methanol for a working standard 1 solution of approximately 50 ppm. ETS-8-6.0 Extraction of PFOS from Liver Page 4 of 14 3M Environmental Laboratory Page 80 3M Medical Department Study: T-6316.7 BACK TO MAIN Analytical Report: FACT TOX-098 LRN-U2402 8.12.6 Dilute the stock solution with methanol for a working standard 2 solution of approx. 5.0 ppm. 8.12.7 Dilute the stock solution with methanol for a working standard 3 solution of approx. 0.50 ppm. 8.13 Surrogate stock standard preparation 8.13.1 Weigh approximately 50-60 mg o f surrogate standard 1-H.l-H, 2-H, 2-H, CgF,3S 0 3H into a 50 ml volumetric flask and record the actual weight. 8.13.2 Bring to volume with methanol for a surrogate stock o f approximately 1000-1200 ppm. 8.13.3 Prepare a surrogate working standard. Transfer approximately 1.0 ml o f surrogate stock to a 10 ml volumetric flask and bring to volume with methanol for a working standard of 10-20 ppm. Record the actual volume transferred. 9.0 Sample H andling________________________________________________________________ 9.1 All samples are received frozen and must be kept frozen until the extraction is performed. 10.0 Quality C ontrol______________________________________________________________ 10.1 M atrix blanks and m ethod blanks . 10.1.1 An aliquot o f 1.0 mL methanol is used as a solvent blank. 10.1.2 Extract two 1.0 mL aliquots of Milli-QTM water following this procedure and use as method blanks. 10.1.3 Extract two 1.0 mL aliquots of liver homogenate following this procedure and use as matrix blanks. Refer to 11.1.6. - 10.2 M atrix spikes 10.2.1 Prepare and analyze matrix spike and matrix spike duplicate samples to determine the accuracy of the extraction. 10.2.2 Prepare each spike using a sample chosen by the analyst, usually a control liver received with each sample set 10.2.3 Expected concentrations will fall in the mid-range of the initial calibration curve. Additional spikes may be included and may fall in the low-range o f the initial calibration curve. 10.2.4 Prepare one matrix spike and matrix spiks duplicate per 40 samples, with a minimum of 2 matrix spikes per batch. 10.3 Continuing calibration verifications 10.3.1 Prepare continuing calibration verification samples to ensure the accuracy o f the initial calibration curve. 10.3.2 Prepare, at a minimum, one continuing calibration verification sample per group o f 10 samples. For example, if a sample set - 34, four verifications are prepared and extracted. ' ETS-8-6.0 Extraction of PFOS from Liver Page 5 of 14 3M Environmental Laboratory Page 81 3M Medical Department Study: T-6316.7 BACK TO MAIN Analytical Report: FACT TOX-098 LRN-U2402 10.3.3 Prepare each continuing calibration verification from the same matrix used to prepare the initial curve. 10.3.4 The expected concentrations will fall within the mid-range of the initial calibration curve. Additional spikes may be included that fall in the low-range of the initial calibration curve. This is necessary if the analyst must quantitate using only the low end o f the calibration curve (for example, 5 ppb - 100 ppb, rather than 5 ppb - 1000 ppb). 11.0 Calibration and Standardization_______________________________ ^________ 11.1 P repare m atrix calibration standards ' 11.1.1 Weigh approximately 40 g o f liver into a 250 mL Nalgene bottle containing 200 mLs Milli-QTM water. Grind to a homogeneous solution. 11.1.2 If 40 g is not available, use appropriate amounts of liver and water to ensure a 1:5 ratio. 11.13 Refer to 13.0 to calculate the actual density of liver homogenate and the concentration o f solid liver tissue dispersed in 1.0 mL of homogenate solution. 11.13 Add 1 mL of homogenate to a 15 mL centrifuge tube. Re-suspend solution by shaking between aliquots while preparing a total o f eighteen 1 mL aliquots of homogeneous solution in 15 mL centrifuge tubes. 11.1.6 Two 1 mL aliquots, or other appropriate volume, serve as matrix blanks. 11.1.7 Typically use the standard concentrations and spiking amounts listed in Table 1, at the end o f this section, to spike, in duplicate, two standard curves, for a total o f eighteen samples, two matrix blanks, and two method blanks. 11.1.8 Refer to validation reports ETS-8-6.0 and ETS-8-7.0-V-1 or A ttachm ent B, which lists the working ranges and the Linear Calibration Range (LCR) for calibration curves. 11.1.9 U s e A ttachm ent C as an aid in calculating the concentrations o f the w orking standards. Refer to 13.0 to calculate actual concentrations o f PFOS in calibration standards. 11.2 To each working standard, blank, or continuing verification, add appropriate amount o f surrogate working standard for the concentration to fall within the calibration curve range 5 ppb - lOOOppb. 3M Environmental Laboratory ETS-8-6.0 Extraction of PFOS from Liver Page 6 of 14 Page 82 3M Medical Department Study: T-6316.7 BACK TO MAIN Analytical Report: FACT TOX-098 LRN-U2402 11.3 Extract spiked liver homogenates following 12.14-12.25 o f this method. Use these standards to establish each initial curve on the mass spectrometer. Table 1 Approximate Spiking Amounts for Calibration Standards Working Standard (Approx. Cone.) 0.50 ppm 0.50 ppm 0.50 ppm 0.50 ppm 0.50 ppm 5.0 ppm 5.0 ppm 5.0 ppm 50 ppm '2 4 10 20 40 10 20 30 4 Approx, final cone, o f PFOS in liver Blank 0.005 ppm 0.010 ppm 0.025 ppm 0.050 ppm . 0.100 ppm 0.250 ppm 0.500 ppm 0.750 ppm 1.00 ppm 12.0 Procedure_____________________________________________________________________ 12.1 Obtain frozen liver samples. 12.2 Cut approximately 1 g of liver using a dissecting scalpel. This part of the procedure is best performed quickly, not allowing the liver to thaw. 123 Weigh the sample directly into a tared plastic sampule vial. 12.4 Record the liver weight in'the study notebook. 12.5 Return unused liver portions to freezer. 12.6 Add 2.5 mLs o f water to sampule vial. 12.7 Grind the sample. Put the grinder probe in the sample and grind for about 2 minutes, or until the sample is homogeneous. 12.8 Rinse the probe into the sample with 2.5 mLs water using a pipette. 12.9 Take the grinder apart and clean it with methanol after each sample. Refer to AMDT-EP- 22. 12.10 Cap the sample and vortex for 15 seconds. Label the sampule vial with the study number, weight, liver ID, date and analyst initials. 3M Environmental Laboratory ETS-8-6.0 Extraction of PFOS from Liver Page 7 of 14 Page 83 3M Medical Department Study: T-6316.7 BACK TO MAIN Analytical Report: FACTTOX-098 LRN-U2402 12.11 Pipette 1.0 mL, or other appropriate volume, of homogenate into a 15 mL polypropylene centrifuge tube. Label the centrifuge tube with the identical information as the sampule vial. Refer to attached worksheet for documenting the remaining steps. 12.12 Pipette two 1 mL aliquots o f Milli-QTM water to centrifuge tubes. These will serve as method blanks. 12.13 Spike all samples, including blanks and standards ready for extraction with surrogate standard as described in section 11.2. 12.14 Spike each matirx with the appropriate amount of standard as described in 11.1, or Table 1 of that section, for the calibration curve standards. Also prepare matrix spikes and continuing calibration standards. . 12.15 Vortex mix the standard curve samples, matrix spike samples, and continuing calibration samples for 15 seconds. 12.16 Check to ensure 0.5 M TBA reagent is at pH 10. If not, adjust accordingly. 12.17 To each sample, add 1 mL 0.5 M TBA and 2 mL of the 0.25 M sodium caibonate/sodium bicarbonate buffer. 12.18 Using an Oxford Dispenser, add 5 mL methyl-terf-butyl ether. 12.19 Cap each sample and put on the shaker at a setting of 300 ipm, for 20 minutes. 12.20 Centrifuge for 20 to 25 minutes at a setting o f 3500 rpm, or until layers are well separated. 12.21 Label a fresh 15 mL centrifuge tube with the same information as in 12.10. 12.22 Remove 4.0 mL of the organic layer to the fresh 1:5 mL centrifuge tube. 12.23 Put each sample on the analytical nitrogen evaporator until dry, approximately 1 to 2 hours. 12.24 Add l .0 mL to each centrifuge tube using a graduated pipette. 12.25 Vortex mix for 30 seconds. 12.26 Attach a 0.2 p.m nylon mesh filter to a 3 cc syringe and transfer the sample to this syringe. Filter into a 1.5 mL glass autovial or low-volume autovial when necessary. 12.27 Label the autovial with the study number, animal number and gender, sample timepoint, matrix, final solvent, extraction date, and analyst(s) performing the extraction. 12.28 Cap and store extracts at room temperature or at approximately 4 C until analysis. 12.29 Complete the extraction worksheet, attached to this document, and tape in study notebook or include in study binder, as appropriate. 3M Environmental Laboratory ETS-8-6.0 Extraction of PFOS from Liver Page 8 of 14 Page 84 3M Medical Department Study: T-6316.7 BACK TO MAIN Analytical Report: FACT TOX-098 LRN-U2402 13.0 Data Analysis and Calculations____________________________________________ 13.1 Calculations: 13.1.1 Calculate the average density of the liver homogenate by recording each mass of ten separate 1.0 mL aliquots of homogenate. Average density (mg/mL) = Average mass (me) o f the aliquots 1.0 mL aliquot 13.1.2 Calculate the amount of liver (mg) per 1.0 rnL homogenate (or concentration of dispersed solid tissue per mL of homogenate suspension) using the following equation: g of Liver x Average density* o f homogenate (mg/mL) (g of Liver + g o f Water) * refer to 13.1.1 for details. 13.1.3 Calculate actual concentrations o f PFOS and other fluorochemicals in calibration standards using the following equation: uL o f Standard x Concentration (ug /mL) = Final Concentration (pg/g or mg/kg) mg Liver / 1 mL homogenate* of PFOS in Liver refer to 13.1.2 for details. 14.0 Method Performance__________________________________________________________ 14.1 The method detection limit (MDL) is analyte and matrix specific. Refer to MDL report for specific MDL and limit o f quantitation (LOQ) values (refer to Attachments B and C). 14.2 The following quality control samples are extracted with each batch o f samples to evaluate the quality o f the extraction and analysis. 14.2.1 Method blanks and matrix blanks. 14.2.2 Matrix spike and matrix spike duplicate samples to determine accuracy and . precision of the extraction. 14.2.3 Continuing calibration verification samples to determine the continued accuracy of the initial calibration curve. 14.3 Refer to section 14 o f ETS-8-7.0 for method performance criteria. 15.0 Pollution Prevention and W aste Management________________________________ 15.1 Sample waste is disposed in biohazard containers, flammable solvent waste is disposed in high BTU containers, and used glass pipette waste is disposed in broken glass containers located in the laboratory. 3M Environmental Laboratory ETS-8-6.0 Extraction of PFOS from Liver Page 9 of 14 Page 85 3M Medical Department Study: T-6316.7 BACK TO MAIN Analytical Report: FACT TOX-098 LRN-U2402 16.Q Records__________________________________________________ ;________ 16.1 Complete the extraction worksheet attached to this method, and tape in the study notebook or include in the 3-ring study binder, as appropriate. 17.0 Tables. Diagrams. Flowcharts, and Validation Data_______________________ 17.1 Attachment A, Extraction worksheet 17.2 Attachment B, MDL/LOQ values and summary 17.3 Attachment C, Calibration standard calculation and concentration worksheet 18.0 References____________________________________________ ,__________________ 18.1 The validation report associated with this method is ETS-8-6.0 & 7.0-V-l. 18.2 AMDT-EP-22, "Routine Maintenance of Ultra-Timax T-25" 18.3 FACT-M-1.1, "Extraction o f PFOS or Other Anionic Fluorochemical Surfactants from Liver for Analysis Using HPLC-Electrospray/Mass Spectrometry" 19.0 Affected Documents____________________________________________________ 19.1 ETS-8-7.0, "Analysis of Liver Extracts for Fluoroohemicals using HPLC-Electrospray Mass Spectrometry" 20.0 Revisions Revision Number. Reason For Revision Revision Date 3M Environmental Laboratory ETS-8-6.0 Extraction of PFOS from Liver Page 10 of 14 Page 86 3M Medical Department Study: T-6316.7 BACK TO MAIN Analytical Report: FACT TOX-098 LRN-U2402 Studv # Matrix Box # Wk/Dav Date Spiked/Analyst CCV MS MSD Surrogate Std approx, ppm actual ppm # FC Mix Std approx. 0.5 ppm actual ppm # FC Mix Std approx. 5 ppm actual ppm # I FC Mix Std approx. 50 ppm actual ppm # Comments Blank_______________Liver Homogenate: Std# Liver Extraction Method - - .-- : -. '- - - Liver amount = - - - - - - - - - g Date & Initials Spike surrogate and Standard mix. Vortex IS sec.___________________________________________ _______________________________ Pipette 1 mL of Liver Solution_______________________________ _______________________________________________ _ _ Pipette 1 m L of fO.S M TBA,pH 10. p H - Std. # _________________________ Pipette 2mLof0.2SNa2CO3/0.25MNaHCQt Buffer Std.# Dispense 5ml of Methyl-t-Butyl Ether _____________________TN-A- ___________________________ - Shake 20 mig^ -- ---- = Centrifuge 20-25 min. __________ Shaker Speed Centrifuge Speed ~ Remove a 4 mL aliquot of organic layer __ Put on Nitrogen Evaporator to dryness______________________ Evaporator Temperature_________ Add 1.0 mL of Methanol______________________________ TN-A- . ________________________ _______ Vortex 30 sec. -- Filter using a 3cc B-D syringe with a 0.2um SRI filter into autosample vial_______________________________________________________ Cont. Cal. Verifications used the same matrix as for the standard curve. Attachment B: MDL/LOQ Values . ETS-8-6.0 Extraction of PFOS from Liver Page 11 of 16 3M Environmental Laboratory Page 87 3M Medical Department Study: T-6316.7 BACK TO MAIN Analytical Report: FACT TOX-098 LRN-U2402 MDL/LOQ values for rabbit liver Compound MDL LOQ Linear Calibration R ange (LCR) (PPb) (PPb) Approximate concentrations to be used for preparing the Standard Calibration Curve PFOS 8.45 26.9 30 ppb - 1200 ppb PFOSA 3.50 11.1 1 2 p p b - 1200 ppb PFOSAA 24.6 78.3 30 ppb - 1200 ppb EtFOSE-OH 108 345 60 ppb - 900 ppb* M556 82.3 262 60 ppb -1 2 0 0 ppb PFOSEA 33.9 108 30 ppb- 1200 ppb MDL/LOQ values in rat, bovine, and monkey liver were not statistically determined. Two curves in each of these matrices were extracted and analj-zed with the rabbit liver curves to determine equivalence. Responses in the rat, bovine, and monkey liver curves were equivalent to the rabbit responses, therefore, their MDL and LOQ will be assumed to be equivalent to those values as determined for the rabbit liver. Refer to LOQ Summary and MDL study in ETS-8-6.0 & 7.0-V-l for further information * EtFOSE-OH estimates only for MDL and LOQ. Did not meet criteria for validation. Com pound: PFOS___________________________________ ________________ Liver matrix Prepared Range of range of average standards 1 curve (ppb) (ng/mL) (ppb) (ng/mL) LCR frothy,, Range of vave'CiMe^: low std .i yV curve (ppb)'(ng/mLK (ppb) (ng/mL) LRTroni: i. Tow std: . crve; : (P pbj(rigfoiL ). Range of high std curve (ppb) (ng/mL) LCR from: high,std curve . (ppb) (n/ml.) : Rabbit 6.19 -1237 12 -1200 i 2Vri 6 -3 0 0 ' 2^;3QK\ 6 0 - 1200 . ]6Q r 1200 : Compound: PFOSA Prepared Liver range of matrix standards (ppb) (ng/mL) Rabbit 6.19-1237 Range of average curve (ppb) (ng/mL) 12 - 1200 LGftftomv .avecuiye'i.: . (ppb):(/mLJ: 12Vl200> Range of low sti curve (ppb) (ng/mL) 12 - 300 'iLGRftorn: i : :loW:St,.; --rcutve,:?. '(PPb) (ng/mL) : 12-300 Range of high std curve (ppb) (ng/mL) 6 0 - 1200 .^DCR from; : :.hi|hstd:' ' v curvet b - ._(ppb).(ng/mL):: '60 - 1200 : Compound: PFOSAA Prepared Range of Liver range of average matrix standards curve (ppb) (ng/mL) (ppb) (ng/mL) Rabbit 6.16-1232 12 - 1200 LCR,from, ave curve (ppb) (ng/mL) 30-1200 Range of low s(d curve (ppb) (ng'mL) 30 - 900 LCR from Range of low std . : high std curve curve (ppb) (ng/mL) (ppb) (ng/mL) 60 - 900 . N/A LCR from high std curve (ppb) (ng/mL) ' N/A Attachment B: MDL/LOQ Values ETS-8-6.0 Extraction of PFOS from Liver 3M Environmental Laboratory Page 12 of 16 Page 88 3M Medical Department Study: T-6316.7 BACK TO MAIN Analytical Report: FACT TOX-098 LRN-U2402 Compound: EtFOSE-OH Prepared Range of Liver matrix range of standards average curve (ppb) (ng/raL) (ppb) (ng/mL) Rabbit 6.17-1235 31-900 LCR from ave curve (ppb) (ng/mL) 31-900 Range of low std curve (ppb) (ng/mL) N/A LCR from low std curve (ppb) (ng/mL) N/A Range of high std curve (ppb) (ng/mL) N/A LCR from high std curve (ppb) (ng/mL) N/A Compound: PFOSEA Prepared Range of Liver range of average matrix standards curve (ppb) (ng/mL) (ppb) (ng/mL) Rabbit 6.17-1235 31 - 1200 LCR from Range of ave.curve low std curve (ppb).: (ng/mL) (ppb) (ng/riL) 31 - 1200; N/A LCR from low std curve (PPb) (ng/mL) Range of high std curve (ppb) (ng/mL) N /A . N/A LCR from high std curve. ' ' (ppb) (ng/mL) N/A ' Compound: M556 Prepared Liver range of matrix standards (ppb) (ng/mL) Rabbit 6.17-1235 Range of average curve (ppb) (ng/mL) 31-1200 .LCRiftoni' ave.curye.; (ppbJiCng/mL)., Range of low std curve (ppb) (ng/mL) LCRfir*iom . :; Range of lowstd :' high std curve'' curve .(ppb) ;(hg/mL) (ppb) (ng/mL) LCR from high std. - curve (ppb) (ng/inL) 6 0 -1 2 0 0 :- N/A N/A . N/A ' r Attachment C: Standard Calculations ETS-8-6.0 Extraction of PFOS from Liver 3M Environmental Laboratory Page 13 of 14 Page 89 3M Medical Department Study: T-6316.7 BACK TO MAIN Analytical Report: FACT TOX-098 LRN-U2402 Ion Pair Standard Curves - Tissue Prep date(s): Analyte(s): Sample matrix: Method/revision: Target analyte(s): FC mix std approx. 0.500 ppm: FC mix std approx. 5.00 ppm: FC mix std approx. 50.0 ppm: Surrogate std approx. 100 ppm: Standard number: Equipment number: Final solven t and TN: Blank liver/identifier: Actual concentrations of standards in the FC mix PFOS PFOSA PFOSAA EtFOSE PFOSEA Std cone Std cone Std cone Std cone Std cone ug/mL ug/mL ug/mL ug/mL ug/mL 0.500 0.500 0.500 0.500 0.500 0.500 0.500 0.500 0.500 0.500 0.500 0.500 0.500 0.500 0.500 0.500 0.500 0.500 0.500 0.500 0.500 0.500 0.500 0.500 0.500 5.00 5.00 5.00 5.00 5.00 5.00 5.00 5.00 5.00 5.00 5.00 5.00 5.00 5.00 5.00 50.0 50.0 50.0 50.0 50.0 O MI56 Std cone ug/mL 0.500 0.500 0.500 0.500 5.00 5.00 5.00 50.0 Std cone ug/mL All Ain't spiked mL 0.002 0.004 0.010 0.020 0.040 0.010 0.020 0.030 0.004 All Density B 0.167 0.167 0.167 0.167 0.167 0.167 0.167 0.167 0.167 Calculated concentrations of standards in the sample m ain PFOS PFOSA PFOSAA EtFOSE PFOSEA M556 Final Final Final cone Final Final Final cone cone ng/g cone cone cone ng/g ng/g ng/g ng/g ng/g 5.99 5.99 5.99 5.99 5.99 5.99 12.0 12.0 12.0 12.0 12.0 12.0 29.9 29.9 29.9 29.9 29.9 29.9 59.9 59.9 59.9 59.9 59.9 59.9 120 120 120 120 120 120 299 299 299 299 299 299 599 599 599 599 599 599 898 898 898 898 898 898 1198 1198 1198 1198 1198 L198 Std cone ng/g Surrogate Std cone ng/mL too Surrogate Final cone ng/mL 0.500 All Am't spiked mL 0:005 Validated ranges - approximate concentrations Liver PFOS PFOSA PFOSAA Rabbit . 5-1000 ppb 5-1000 ppb 5-1000 ppb Bovine ' Estimates only, use rabbit values. Rat Estimates only, use rabbit values. Monkey Estimates only, use rabbit values. EtFOSE-OH 5-1000 ppb POAA 5-1000 ppb PFOSEA 5-1000 ppb Attachment C: Standard Calculations ETS-8-6.0 Extraction of PFOS from Liver Page 14 of 14 3M Environmental Laboratory Page 90 3M Medical Department Study: T-6316.7 BACK TO MAIN Analytical Report: FACT TOX-098 LRN-U2402 3M Environmental Laboratory M ethod Analysis o f F l u o r o c h em ic a ls in L iv e r E xtracts U sin g H P L C -E lectrospray/M ass Spectrom etry M ethod Num ber: FACT-M-2.0 Adoption Date: Author: Lisa Clemen Revision Date: A Approved By: AH Laboratory Manager --______ ^heA y Date ---f//W.^.v. .U..?..'.--------- --- -- ' Group Leader Date Technical Reviewer Date 1.0 Scope and Application 1.1 Scope: This method is for the analysis o f extracts of liver or other tissues for fluorochemical surfactants using HPLC-electrospray/mass spectrometry. 1.2 Applicable Compounds: Potassium perfluorooctanesulfonate, anionic fluorochemical surfactants, or other ionizable compounds. 1.3 M atrices: Rabbit, rat, bovine, and monkey livers or other livers as designated in the validation report. Word 7.0.1/95 FACT-M-2.0 Analysis of Liver Extract Losing ES/MS Page 1 of 8 3M Environmental Laboratory Page 91 3M Medical Department Study: T-6316.7 BACK TO MAIN Analytical Report: FAC T TO X -098 LR N -U 2 40 2 2.0 Summary of Method___________________________________________ '________"" 2.1 This method describes the analysis o f fluorochemical surfactants extracted from liver using HPLC-electrospray/mass spectrometry. The analysis: is performed by monitoring a single ion characteristic of a particular fluorochemical, such as the potassium perfluorooctanesulfonate (PFOS) anion, M/Z= 499. Samples may also be screened to verify compound identification. 3.0 Definitions__________________________________________________________________ 3.1 None. 4.0 Warnings and Cautions___________________________________ ____________________ 4.1 Health and Safety Warnings: 4.1.1 Use caution with the voltage cable for the probe. When the voltage cable is plugged into the probe DO NOT TOUCH THE PROBE, there is risk of electrical shock. 4.2 Cautions: 4.2.1 Do not run solvent pumps above capacity of 400 bar (5800 psi). If pressure goes over 400 bar, the HP 1100 will initiate automatic shutdown. 4.2.2 Do not run solvent pumps to dryness. 5.0 I n t e r f e r e n c e s ________________________________________________________________ ;_________ 5.1 Teflon should not be used for sample storage or any part of instrumentation that comes in contact with the sample or extract. 6.0 E q u ip m e n t _______________________________________________________________________________ 6.1 Equipment listed below may be changed in order to optimize the system. 6.1.1 Micromass Electrospray Mass Spectrometer 6.1.2 HP 1100 low pulse solvent pumping system and autosampler. 7.0 Supplies and Materials________ ;________________ ;_______________ _____________ 7.1 Supplies 7.1.1 Nitrogen gas, refrigerated liquid, regulated 1o approximately 100 psi. 7.1.2 HPLC column, specifics to be determined by the analyst. 7.1.3 Capped autovials or capped 15 mL centrifuge tubes. 8.0 Reagents and Standards________________________________________________ _____ 8.1 Reagents 8.1.1 Methanol, HPLC grade or equivalent. Word 7.0.1/95 FACT-M-2.0 Analysis of Liver Extract Using ES/MS Page 2 of 8 3M Environmental Laboratory Page 92 3M Medical Department Study: T-6316.7 BACK TO MAIN Analytical Report: FACT TO X-098 L R N -U 2 40 2 8.1.2 Milli-QTM water, all water used in this method should be Milli-QTM water and may be provided by a Milli-Q TOC Plus system. 8.1.3 Ammonium acetate, HPLC grade or equivalent. 8.2 Standards 8.2.1 Typically one H20 blank, one liver blank, and seven liver standards are prepared during the extraction procedure. SeeFACT-M-1. 9.0 S a m p l e H a n d l in g _____________________ ^__________________________________________________ 9.1 Fresh liver standards are prepared with each analysis. Extracted standards and samples are stored in capped autovials or capped 15 mL centrifuge tubes until analysis. 9.2 If analysis will be delayed, extracted standards and samples may be refrigerated until analysis can be performed. 10.0 Q u a l it y C o n t r o l _________________________________________________________ ^____________ 10.1 Matrix Blanks and Method Blanks 10.1.1 Analyze a method blank and matrix blank prior to each calibration curve. 10.2 Matrix Spikes 10.2.1 Analyze a matrix spike and matrix spike duplicate with each analysis. 10.2.2 Expected concentrations will fall in the mid-range of the initial calibration curve. Additional spike concentrations may fall in the low-range o f the initial calibration curve. 10.2.3 See section 13 to calculate percent recovery. 10.3 Continuing Calibration Checks 103.1 Analyze a mid-range calibration standard after every tenth sample. If a significant change ( 30%) in peak area occurs, relative to the initial standard curve, stop the run. Only those samples analyzed before the last acceptable calibration standard will be used. The remaining samples must be reanalyzed. 10.3.2 See section 13 to calculate percent difference. 10.4 System Suitability 10.4.1 System suitability (e.g. peak area, retention time and peak shape, etc.) will be assessed for each run. 11.0 C a l ib r a t io n a n d St a n d a r d iz a t io n ______________;_____________________________________ 11.1 Analyze the extracted liver standards prior to and following each set of extracts. The mean of two standard values, at each standard concentrati on, will be plotted by linear regression for the calibration curve using MassLynx or other suitable software. FACT-M-2.0 Analysis of Liver Extract Using ES/MS Page 3 of 8 3M Environmental Laboratory Page 93 3M Medical Department Study: T-6316.7 BACK TO MAIN Analytical Report: FAC T TO X -098 LR N -U 2 4 0 2 11.2 The r2value for the data should be 0.98 or greater. Lower values may be acceptable at the discretion o f the analyst. 11.3 If the curve does not meet requirements, perform routine maintenance or reextract the standard curve (if necessary) and reanalyze. 12.0 P r o c e d u r e s _________________________________________ '__________________________________ 12.1 Acquisition Set up 12.1.1 Click on start button in the Acquisition Control Panel. Set up a sample list. Assign a filename using letter-MO-DAY-last digit of year-sample number, assign a method (MS) for acquiring, and type in sample descriptions. 12.1.2 To create a method click on scan button in the Acquisition control panel and select SIR. Set Ionization Mode as appropriate and mass to 499 or other appropriate masses.. A scan is usually collected along with the SIRs. Save method. 12.13 Typically the sample list begins with the first set o f liver standards and ends with the second set of standards. 12.1.4 Samples are analyzed with a continuing calibration check injected after every tenth sample. Solvent blanks should be analyzed periodically to monitor possible analyte carryover and are not considered samples but may be included as such. 12.2 Using the Autosampler 12.2.1 Set up sample tray according to the sample list prepared in section 12.1.1. 12.2.2 Set-up the HP 1100/autosampler at the follo'ving conditions or at conditions the analyst considers appropriate for optimal response. Record actual conditions in the instrument logbook: 12.2.2.1 Sample size = 10 pL injection with a sample wash 12.2.2.2 Inject/sample = 1 12.2.2.3 Cycle time = 15 minutes 12.2.2.4 Solvent ramp = Time MeOH 0.00 min. 7.5 min. 11.0 min. 11.5 min. 45% 90% 90% 45% 2.0 mM Ammonium acetate 55% 10% 10% 55% Note: In this instrument configuration, the run must be set up on the electrospray software with a "Waiting for inlet start" message before the "Start" button is pressed on the HP Workstation. 12.2.2.5 Press the "Start" button. FACT-M-2.0 Analysis of Liver Extract Using ES/MS Page 4 of 8 3M Environmental Laboratory Page 94 3M Medical Department Study: T-6316.7 BACK TO MAIN A nalytical Report: FAC T TO X -098 LR N -U 2 4 0 2 12.3 Instrum ent Sep-up ' 12.3.1 Refer to AMDT-EP-31 for more details. 123.2 Check the solvent level in reservoirs and refill if necessary. 123.3 Check the stainless steel capillary at the end of the probe. Use an eye piece to check the tip. The tip should be flat with no jagged edges. If the tip is found to be unsatisfactory, disassemble the probe and replace the stainless steel capillary. 12.3.4 Set HPLC pump to "On". Set the flow to 10 - 500 uL/min or as appropriate. Observe droplets coming out of the tip of the probe. Allow to equilibrate for approximately 10 minutes. 123.5 Turn on the nitrogen. A fine mist should be expelled with no nitrogen leaking around the tip of the probe. 12.3.6 The instrument uses these parameters at the following settings. These settings may change in order to optimize the response: 123.6.1 Drying gas 250-400 liters/hour 123.6.2 ESI nebulizing gas 10-15 liters/hour 123.6.3 LC constant flow mode flow rate 10 - 500 uL/min 123.6.4 Pressure <400 bar (This parameter is not set, it is a guide to ensure the instrument is operating correctly.) 123.7 Carefully guide the probe into the opening. Insert probe until it will not go any further. Connect the voltage cables to the probe. 123.8 Record tune parameters in the instrument log. 123.9 Using the cross-flow counter electrode in the ES/MS source is recommended for the analysis of biological matrices. 123.10 Click on start button in the Acquisition Control Panel. Press the start button at top of sample list. Ensure start and end sample number includes all samples to be analyzed. 13.0 Data Analysis and Calculations__________________________________ 13.1 Calculations: 13.1.1 Calculate matrix spike percent recoveries using the following equation: % Recovery" Observed Result - Background. Result x 100 Expected Result 13.1.2 Calculate percent difference using the following equation: % Difference " Expected Cone. - Calculated Clone, x 100 Expected Cone. 3M Environmental Laboratory FACT-M-2.0 Analysis of Liver Extract Using ES/MS Page 5 of 8 Page 95 3M Medical Department Study: T-6316.7 BACK TO MAIN A nalytical Report: FACT T O X -098 L R N -U 2 40 2 13.1.3 Calculate actual concentration o f PFOS anion in total liver (mg): ug PFOS anion calc, from std curve < g of liver used for analysis > x Total mass o f liver (g) 1000 u g /1mg 14.0 M e t h o d P e r fo r m a n c e __________________________________________________ ;_____________ 14.1 The method detection limit is equal to at least three times the baseline noise in the matrix blank. 14.2 The practical quantitation limit is equal to the lowest standard in the calibration curve. 15.0 P o l l u t io n P r e v e n t io n a n d W a st e M a n a g e m e n t ___________________________________ 15.1 Sample waste is disposed in biohazard containers, flammable solvent waste is disposed in high BTU containers, and glass pipette waste is disposed in broken glass containers. All containers are located in the laboratory. 16.0 R e c o r d s _______________________ .________________________________________________________ 16.1 Store chromatograms in the study folder. Each chromatogram should have the following information included either in the header or hand written on the chromatogram: study number, sample name, extraction date, and dilution factor (if applicable). 16.2 Plot calibration curve by linear regression and store in the study folder. 16.3 Print sample list from MassLynx and tape into the instrument runlog. 16.4 Print data integration summary from MassLynx and tape into the instrument runlog. 16.5 Copy instrument runlog pages, including instrument parameters and sample results, and tape into appropriate study notebook. 16.6 Summarize data using suitable software and store in the study folder. 16.7 Back up electronic data to appropriate media. Record in study notebook the file name and location of backup electronic data. 17.0 T a b l e s , D ia g r a m s , F l o w c h a r t s, a n d V a l id a t io n D a t a _________________________ __ 17.1 Attachment A: FACT-M-2 Data reporting spreadsheet 17.2 The validation report associated with this method is FACT-M-1.0 & 2.0-V-l. 18.0 R e f e r e n c e s _____________________ ;_____________________________________ '_______________ 18.1 AMDT-EP-31, "Operation of VG Platform Electrospray Mass Spectrometer" 3M Environmental Laboratory FACT-M-2.0 Analysis of Liver Extract Using ES/MS Page 6 of 8 Page 96 3M Medical Department Study: T-6316.7 BACK TO MAIN Analytical Report: FACT TO X-098 L R N -U 2 40 2 19.0 Affected Documents____________________________________________________" 19.1 FACT-M-1.0, "Extraction of Potassium Perfluorooctanesulfonate from Liver for Analysis Using HPLC-Electrospray/Mass Spectrometry" 20.0 Revisions Revision Number. Reason For Revision Revision Date 3M Environmental Laboratory FACT-M-2.0 Analysis of Liver Extract Using ES/MS Page 7 of 8 Page 97 3M Medical Department Study: T-6316.7 BACK TO MAIN Analytical Report: F A C T T O X -098 L R N -U 2 40 2 Laboratory Study # Study; Test Material: Matrix/Final Solvent: Method/Revision: Analytical Equipment System Number Instrument Software/Version: Filename: R-Squared Value: Slope: Y Intercept: Date o f Extraction/Analyst: Date o f Analysis/Analyst: Group Dose Sample# Concentration ug/mL Initial VoL mL Dilution Factor Final Cone. ug/mL Slope: Taken from linear regression equation. Group/Dose: Taken from the study folder. Sample#: Taken from the study folder. Concentration (ug/mL): Taken from the MassLynx integration summary. Initial Volume (mL): Taken from the study folder. Dilution Factor: Taken from the study folder. Final Cone. (ug/mL): Calculated by dividing the initial volume from the concentration 3M Environmental Laboratory FACT-M-2.0 Analysis of Liver Extract Using ES/MS Page 8 of 8 Page 98 3M Medical Department Study: T-6316.7 BACK TO MAIN Analytical Report: FAC T TO X -098 L R N -U 2 40 2 3M Environmental Laboratory M ethod An a ly sis o f P o ta ssiu m P erflu o ro o cta n esu lfo n a te o r O t h e r F lu o ro ch em ica ls in Seru m o r O th ef:F luid E x tra cts U sin g H P L C -E lectrospray/M ass Spectro m etry M ethod Num ber: FACT-M-4.1 Author: Lisa Clemen, Glenn Langenburg Approved By: Laboratory Manager (a/ja. Group Leader ----- --* A__ fibma*.__________________ Technical Reviewer Adoption Date: 4/22/98 Revision Date: /*/, t j s r Date Vt-lHjl Date stl9a Date 1.0 Scope and Application____________________ ;___________________________________ 1.1 Scope: This method is for the analysis of extracts from serum or blood for fluorochemical surfactants using HPLC-electrospray/mass spectrometry. 1.2 Applicable Compounds: Fluorochemical surfactants or other fluorinated compounds, or other ionizable compounds. 1.3 M atrices: Rabbit, rat, bovine, or monkey serum and rat whole blood or milk curd. Word 6.0.1/95 FACT-M-4.1 Analysis of Serum or Fluid Extract Using ES/MS Page 1 of 9 3M Environmental Laboratory Page 99 3M Medical Department Study: T-6316.7 BACK TO MAIN Analytical Report: FACT TO X-098 L R N -U 2 40 2 2.0 Sum m ary of M ethod _____________ ;___________________________ :______ " 2.1 This method describes the analysis o f fluorochemical surfactants extracted from serum, whole blood, or milk curd using HPLC-electrospray/mass spectrometry, or similar system as appropriate. The analysis is performed by monitoring a single ion characteristic o f a particular fluorochemical, such as the potassium perfluorooctanesulfonate (PFOS) anion, M/Z= 499. Samples may also be analyzed using an .API/MS/MS system to further verify compound identification. 3.0 De f i n i t i o n s __________________________________________________' .___________ 3.1 Atm ospheric Pressure Ionization (API): The Micromass platform systems allow for various methods o f ionization by utilizing various sources, probes, and interfaces. These include but are not limited to: Electrospray Ionization (ESI), Atmospheric Pressure chemical Ionization (APcI), Thermospray, etc. The ionization process in these techniques occurs at atmospheric pressure (i.e. not under a vacuum). 3.2 Electrospray Ionization (ES, ESI): a method of ioniz ation performed at atmospheric pressure, whereby ionization occurs through the production o f tiny charged droplets in a strong electrical field. 3.3 M ass Spectrom etry, Mass Spectrometer (MS), Tanilem Mass Spectrom eter (MS/MS): The API platforms are equipped with quadrupole mass selective detectors. Ions are selectively discriminated by mass to charge ratio (m/z) and subsequently detected. A single MS may be employed for ion detection or a series (MS/MS) for more specific fragmentation information. 3.4 Conventional vs. Z-spray probe interface: The latest models o f Micromass platform systems (post 1998) utilize a "Z-spray" conformation. The spray emitted from a probe is orthogonal to the cone aperture, hi the conventional conformation it is aimed directly at the cone aperture, after passing through a tortuous pathway in the counter electrode. Though the configuration is different, the methods of operation, cleaning, and maintenance are the same. However, Z-spray components and conventional components are not compatible with one another, but only with similar systems (i.e. Z-spray components are compatible with other Zspray systems, etc.) 3.5 M ass Lynx Software: System software designed for the specific operation of these platform systems. Currently MassLynx has Windows 95 and WindowsNT 3.1 versions. All versions are similar. For more details see the manual specific to the instrument (Micromass Platform II or Quattro II MassLynx or MassLynx NT USER'S GUIDE). 4.0 W arnings and Cautions________________________________________________________ 4.1 H ealth and Safety W arnings: 4.1.1 Use caution with the voltage cables for the probe. The probe employs a voltage of approximately 5000 Volts. 4.1.2 When handling samples or solvents wear appropriate protective gloves, eyewear, and clothing. Word 6.0.1/95 FACT-M-4.1 Analysis of Serum or Fluid Extract Using ES/MS Page 2 of 9 3M Medical Department Study: T-6316.7 BACK TO MAIN Analytical Report: FAC T TO X -098 L R N -U 2 40 2 4.2 Cautions: 4.2.1 Do not operate solvent pumps above capacity of 400 bar (5800 psi) back pressure. If the back pressure exceeds 400 bar, the HP 1100 will initiate automatic shutdown. 4.2.2 Do not run solvent pumps to dryness. 5.0 Interferences_____________________________________________________________ 5.1 To minimize interferences when analyzing samples for perfluorooctanoate(POAA), teflon should not be used for sample storage or any part o f instrumentation that comes in contact with the sample or extract. 6.0 Equipment__________________________________________________________________ 6.1 Equipment listed below may be modified in order to optimize the system. 6.1.1 Micromass Electrospray Mass Spectrometer . 6.1.2 HP1100 low pulse solvent pumping system iind autosampler 7.0 Supplies anp Materials_____________________________________________________ 7.1 Supplies 7.1.1 High purity grade nitrogen gas regulated to approximately 100 psi 7.1.2 HPLC analytical column, specifics to be determined by the analyst 7.1.3 Capped autovials or capped 15 ml centrifuge: tubes 8.0 Reagents and St a n d a r d s _______________________ __________________________ 8.1 Reagents 8.1.1 Methanol, HPLC grade or equivalent 8.1.2 Milli-QTM water, all water used in this method should be Milli-QTM water and may be provided by a Milli-Q TOC Plus system 8.1.3 Ammonium acetate, reagent grade or equivalent 8.2 Standards 8.2.1 Typically one method blank, one matrix blank, and ten matrix standards are prepared during the extraction procedure. SeeFACT-M-3.1. 9.0 Sample Handling_________________ |_________ ________________________________ 9.1 Fresh matrix standards are prepared with each analysis. Extracted standards and samples are stored in capped autovials or capped 15 ml centrifuge tubes until analysis. 9.2 If analysis will be delayed, extracted standards and samples can be refrigerated at approximately 4 C until analysis can be performed. FACT-M-4.1 Analysis of Serum or Fluid Extract Using ES/MS Page 3 of 9 3M Environmental Laboratory Page 101 3M Medical Department Study: T-6316.7 BACK TO MAIN A nalytical Report: FAC T TO X -098 L R N -U 2 40 2 10.0 Quality Control_______________________ 10.1 M ethod Blanks and M atrix Blanks .__________________________________ 10.1.1 Analyze a method blank and a matrix blank prior to each calibration curve. 10.2 M atrix Spikes 10.2.1 Analyze a matrix spike and matrix spike dup licate per forty samples. With a minimum o f 2 spikes per batch. 10.2.2 Expected spike concentrations will fall in the: mid-range o f the initial calibration curve. Additional spike concentrations may fall in the low-range o f the initial calibration curve. 10.2.3 See Section 13 to calculate percent recovery. 10.3 Continuing Calibration Checks 10.3.1 Analyze a mid-range calibration standard after every tenth sample. If a significant change ( 30%) in peak area occurs, relative to the initial standard curve, stop the run. Only those samples analyzed before the last acceptable calibration standard will be used. The remaining samples must be reanalyzed. 10.3.2 See Section 13 to calculate percent difference. 11.0 Calibration and Standardization____________ ;_________________________________ 11.1 Analyze the extracted matrix standards prior to and following each set o f extracts. The mean of two standard values, at each standard concentration, will be plotted by linear regression (r^for the calibration curve using MassL;mx or other suitable software. 11.2 The r2value for the data should be 0.980 or greater. Lower values may be acceptable at the discretion of the analyst and documented approval o f the Project Lead. 11.3 If the curve does not meet requirements, perform routine maintenance or reextract the standard curve (if necessary) and reanalyze. 11.4 For purposes of accuracy when quantitating low levels o f analyte, it may be necessary to use the low end of the calibration curve rather than the full range o f the standard curve. Example: when attempting to quantitate approximately 10 ppb o f analyte, generate a calibration curve consisting of die standards from 5 ppb to 100 ppb rather than the full range of the curve (5 ppb to 1000 ppb). This will reduce inaccuracy attributed to linear regression weighting o f high concentration standards. 12.0 Procedures____________________________________________________________________ 12.1 Acquisition Set up 1 12.1.1 Click on start button in the Acquisition Conlxol Panel. Set up a sample list. Assign a filename using letter-MO-DAY-last digit o f year-sample number, assign a method (MS) for acquiring, and type in sample descriptions. FACT-M-4.1 Analysis of Serum or Fluid Extract Using ES/MS Page 4 of 9 3M Environmental Laboratory Page 102 3M Medical Department Study: T-6316.7 BACK TO MAIN Analytical Report: FACT TO X -098 L R N -U 2 40 2 12.1.2 To create a method click on scan button in the Acquisition control panel and select SIR (Single Ion Recording). Set Ionization Mode as appropriate and mass to 499 or other appropriate masses. A full scan is usually collected along with the SIRs. Save acquisition method. If MS/MS instruments are employed, additional product ion fragmentation information may be collected. See Micromass MassLynx GUIDE TO DATA ACQUISITION for additional information and MRM (Multiple Reaction Monitoring). 12.1.3 Typically the analytical batch run sequence begins with a set of extracted matrix standards and ends with a set o f extracted nuitrix standards. 12.1.4 Samples are analyzed with a continuing calibration check injected after every tenth sample. Solvent blanks should be analyzed periodically to monitor possible analyte carryover and are not considered samples but may be included as such. 12.2 Using the Antosampler 12.2.1 Set up sample tray according to the sample list prepared in Section 12.1.1. 12.2.2 Set-up the HP1100/autosampler at the following conditions or at conditions the analyst considers appropriate for optimal response. Record actual conditions in the instrument logbook: 12.2.2.1 Sample size = 10 pL injection with a sample wash 12.2.2.2 Inject/sample = 1 12.2.2.3 Cycle time = 15 minutes 12.2.2.4 Solvent ramp = Time MeOH 0.00 min. 7.5 min. 11.0 min. 11.5 min. 45% 90% 90% 45% 2.0 mM Ammonium acetate 55% 10% 10% 55% Note: In this instrument configuration, the run must be set up on the electrospray software with a "Waiting for inlet start" message before the "Start" button is pressed on the HP Workstation. 12.2.2.5 Press the "Start" button. 12.3 Instrument Set-up 12.3.1 Refer to FACT-EP-3.0 for more details. 12.3.2 Check the solvent level in reservoirs and refi ll if necessary. 12.3.3 Check the stainless steel capillary at the end o f the probe. Use an eyepiece to check the tip. The tip should be flat with no jagged edges. If the tip is found to be unsatisfactory, disassemble the probe and replace the stainless steel capillary. FACT-M-4.1 Analysis of Serum or Fluid Extract Using ES/MS Page 5 of 9 3M Environmental Laboratory Page 103 3M Medical Department Study: T-6316.7 BACK TO MAIN Analytical Report: FACT T O X -098 L R N -U 2 40 2 12.3.4 Set HPLC pump to "On". Set the flow to 10 500 uL/min or as appropriate. Observe droplets coming out o f the tip o f the probe. Allow to equilibrate for approximately 10 minutes. 12.3.5 Turn on the nitrogen. A fine mist should be expelled with no nitrogen leaking around the tip of the probe. 12.3.6 The instrument uses these parameters at the following settings. These settings may change in order to optimize the response: 12.3.6.1 Drying gas 250-400 liters/hour 12.3.6.2 ESI nebulizing gas 10-15 liters/hour 12.3.6.3 HPLC constant flow mode flow rate: 10 - 500 pL/min 12.3.6.4 Pressure <400 bar (This parameter is not set, it is a guide to ensure the HPLC is operating correctly.) 12.3.7 Carefully guide the probe into the opening. Insert probe until it will not go any further. Connect the voltage cables to the probe. 12.3.8 Record tune parameters in the instrument log;. 12.3.9 Using the cross-flow counter electrode in the ES/MS source is recommended for the analysis of biological matrices. 12.3.10Click on start button in the Acquisition Control Panel (this may vary among MassLynx versions, see appropriate MassLynx USER'S GUIDE). Press the start button at top of sample list. Ensure start and end sample number includes all samples to be analyzed. 13.0 D ata Analysis and Calculations_________________;_____________________________ 13.1 Calculations: 13.1.4 Calculate matrix spike percent recoveries using the following equation: % Recovery = Observed Result - Background Result x 100 Expected Result 13.1.5 Calculate percent difference using the following equation: % Difference = Expected Cone. - Calculated Cone, x 100 Expected Cone. 13.1.6 Calculate actual concentration of PFOS, or ether fluorochemical, in matrix (pg/ml): fag o f PFOS calc, from std. Curve x Dilution Factor) x 1 tig /Initial Volume of matrix (mil + ml o f Surrogate Standard! 1000 ng Final Volume (mL) 14.0 M ethod Performance___________ '______________________________________________ 14.1 Method Detection Limit (MDL) and Limit of Quantitation (LOQ) are method, analyte, and matrix specific. Please see FACT-M-3.1, Attachm ent A for a listing o f current validated MDL and LOQ values. FACT-M-4.1 Analysis of Serum or Fluid Extract Using ES/MS Page 6 of 9 3M Environmental Laboratory Page 104 3M Medical Department Study: T-6316.7 BACK TO MAIN Analytical Report: FACT TO X-098 L R N -U 2 40 2 14.2 Method Blanks and Matrix Blanks 14.2.1 Method blanks and matrix blanks will be analyzed with each sample set for possible contamination or carryover. Values are expected to fall below the lowest standard in the calibration curve. 14.3 MatrixSpikes 14.3.1 Matrix spikes are analyzed with each sample set and the percent recoveries are expected to fall within 30% of the spiked concentration. 14.4 Continuing Calibration Checks 14.4.1 Continuing calibration checks are analyzed at a minimum of after every 10 samples with each sample set. The percent recoveries are expected to fall within 30% of the spiked concentration. 14.5 If any criteria listed in the method performance section isn't met, maintenance may be performed on the system and samples reanalyzed or other actions as determined by the analyst. All actions will be documented in the instrument runlog, the maintenance log, or on the summary sheet with the sample results. . 15.0 P ollution P revention and Waste Management_____________________ :__________ 15.1 Sample extract waste and flammable solvent is disposed in high BTU containers, and glass pipette waste is disposed in broken glass containers located in the laboratory. 16.0 Records_______________ ;___________________________________________ ________ 16.1 Store chromatograms in the study or project folder. Each chromatogram must have the following information included either in the header or hand written on the chromatogram: study or project number, acquisition method, integration method, sample name, extraction date, dilution factor (if applicable), and analyst. 16.2 Plot calibration curve by linear regression and store in the study folder. 16.3 Print sample list from MassLynx and tape into the instrument runlog. 16.4 Print data integration summary from MassLynx and tape into the instrument runlog. 16.5 Copy instrument runlog pages, including instrument parameters and sample results, and store in appropriate study folder. 16.6 Summarize data using suitable software and store in the study folder. 16.7 Back up electronic data to appropriate medium. Record in study notebook the file name and location of backup electronic data. 17.0 T ables. Diagrams. Flowcharts, and Validation Data_______________ ;_________ 17.1 Attachment A: FACT-M-4.1 Data reporting spreadsheet 17.2 The validation report associated with this method is FACT-M-3.1 & 4.1-V-l. FACT-M-4.1 Analysis of Serum or Fluid Extract Using ES/MS Page 7 of 9 3M Environmental Laboratory Page 105 3M Medical Department Study: T-6316.7 BACK TO MAIN Analytical Report: FACT TO X-098 L R N -U 2 40 2 18.0 R eferences______________________________________________________________ ' ' .. 18.1 FACT-EP-3.0, "Operation and Maintenance of the Micromass Atmospheric Pressure Ionization/Mass Spectrometer Platform Systems" 19.0 Affected Documents___________________________!_________ ^______________;_______ 19.1 FACT-M-3.1, "Extraction of Potassium Perfluorooctanesulfonate or Other Fluorochemical Compounds from Serum or Fluid for Analysis Using HPLC-Electrospray/Mass Spectrometry" 20.0 Revisions____________________________________________________ ~__________________ Revision NnmTw 1 Reason For Revision Validation o f method to include 7fluorochemicals addition o f whole blood matrix, surrogate standard, new API/MS(MS) systems, monkey sera cross validation, MDL study, updates in record keeping and storing policies, etc. Revision Date 07/01/98 3M Environmental Laboratory FACT-M-4.1 Analysis of Serum or Fluid Extract Using ES/MS Page 8 of 9 Page 106 3M Medical Department Study: T-6316.7 Attachment A BACK TO MAIN Analytical Report: FACT TO X-098 LR N -U 2 4 0 2 Laboratory Study # Study: Test Material: Matrix/Final Solvent Method/Revision: Analytical Equipment System Number: Instrument Software/Version: Filename: R-Squared Value: Slope: Y Intercept: Date o f Extraction/Analyst: Date o f Analysis/Analyst: Group Dose Sample# Concentration ug/mL Initial Voi. mL Dilution Factor Final Cone. ug/mL Slope: Taken from linear regression equation. . Group/Dose: Taken from the study folder. Sample#: Taken from the study folder. Concentration (ug/mL): Taken from the MassLyrx integration summary. Initial Volume (mL): Taken from the study folder. ' Dilution Factor: Taken from the study folder. Final Cone. (ug/mL): Calculated by dividing the initial volume from the concentration 3M Environmental Laboratory FACT-M-4.0 Analysis of Serum or Fluid Extract Using ES/MS Page 9 of 9 Page 107 3M Medical Department Study: T-6316.7 BACK TO MAIN Analytical Report: FACT TO X-098 L R N -U 2 40 2 3M Environmental Laboratory M ethod A n alysis o f P ota ssiu m P erflu o ro o cta n esu lfo n a te o r O t h e r F l u o r o c h em ic a ls in Serum E x tra cts U sin g H P L C -E lectrospray /M ass Spectro m etry M ethod N um ber: ETS-8-5.1 Author. Lisa Clemen, Robert Wynne Approved By: 9-1 Laboratory Manager Group Leader 'f'J z* -------------- Technical Reviewer Adoption Date: 03/01/99 Revision Date: ^ _ - Date 7 /2 4 / ? Date oh/ m I m Date 1.0 Scope and Application_________;_________________________________________________ 1.1 Scope: This method describes the analysis o f serum extracts for fluorochemical surfactants using HPLC-electrospray/mass spectrometry. 1.2 Applicable Compounds: Fluorochemical surfacfcints or other fluorinated compounds, or other ionizable compounds. 1.3 M atrices: Rabbit, rat, bovine, monkey, and human serum, or other fluids as designated in the validation report. Word 6/95 ETS-8-5.1 Analysis of Serum Extract: Using ES/MS Page 1 of 9 3M Environmental Laboratory Page 108 3M Medical Department Study: T-6316.7 BACK TO MAIN Analytical Report: FACT TO X-098 L R N -U 2 40 2 2.0 Summary of M ethod_____________________________________________________ ;_______ 2.1 This method describes the analysis of fluorochemieal surfactants extracted from serum or . other fluids, using HPLC-electrospray/mass spectrometry, or similar system as appropriate. The analysis is performed by monitoring a single ion characteristic of a particular fluorochemieal, such as the perfluorooctanesulfonate (PFOS) anion, m/z=499. Additionally, samples may be analyzed using a tandem mass spectrometer to further verify the identity o f a compound by detecting daughter ions of the parent ion. 3.0 D efinitions__________________________:_________________;__________________________ 3.1 Atm ospheric Pressure Ionization (API): The Micromass Quattro II triple quadrupole systems allow for various methods of ionization by utilizing various sources, probes, and interfaces. These include but are not limited to: Elec trospray Ionization (ESI), Atmospheric Pressure chemical Ionization (APcI), Thermospray, etc. The ionization process in these techniques occurs at atmospheric pressure (i.e., not under a vacuum). 3.2 Electrospray Ionization (ES, ESI): a method o f ionization performed at atmospheric pressure, whereby ions in solution are transferred to 1he gas phase via tiny charged droplets. These charged droplets are produced by the application of a strong.electrical field. 3.3 M ass Spectrometry, Mass Spectrometer (MS), Tandem Mass Spectrometer (MS/MS): The API Quattro 13 triple quadrupole systems are equipped with quadrupole mass selective detectors. Ions are selectively discriminated by mass: to charge ratio (m/z) and subsequently detected. A single MS may be employed for ion detection or a series (MS/MS) for more specific fragmentation information. 3.4 Conventional vs. Z-spray probe interface: The latest models of Micromass Quattro II triple quadrupole systems (post 1998) utilize a "Z-spray" conformation. The spray emitted from a probe is orthogonal to the cone aperture. In the conventional conformation it is aimed directly at the cone aperture, after passing through a tortuous pathway in the counter electrode. Though the configuration is different, the methods o f operation, cleaning, and maintenance are the same. However, Z-spray components and conventional components are not compatible with one another, but only with similar systems (i.e., Z-spray components are compatible with some other Z-spray systems, etc.) 3.5 M ass Lynx Software: System software designed for the specific operation o f these Quattro II triple quadrupole systems. Currently MassLynx has Windows 95 and WindowsNT 4.0 versions. All versions are similar. For more details see the manual specific to the instrument (Micromass Quattro n triple quadrupole MassLynx or MassLynx NT User's Guide). 4.0 W arnings and Cautions______________________ ;_______________________________ _ 4.1 H ealth and Safety W arnings: . 4.1.1 Use caution with the voltage cables for the probe. When engaged, the probe employs a voltage o f approximately 5000 Volts. 4.1.2 When handling samples or solvents wear appropriate protective gloves, eyewear, and clothing. ETS-8-5.1 Analysis of Serum Extract Using ES/MS . Page 2 of 9 3M Environmental Laboratory Page 109 3M Medical Department Study: T-6316.7 BACK TO MAIN Analytical Report: FACT TO X -098 L R N -U 2 40 2 4.2 Cautions: 4.2.1 Do not operate solvent pumps above capaci ty o f 400 bar (5800 psi) back pressure. If the back pressure exceeds 400 bar, the HP1100 will initiate automatic shutdown. 4.2.2 Do not run solvent pumps to dryness. 5 .0 I N T E R F E R E N C E S _____________________________________________________________________ 5.1 To minimize interferences when analyzing samples, teflon should not be used for sample storage or any part of instrumentation that comes in contact with the sample or extract. 6.0 Equipment_______________________________________ ,_______________________________ 6.1 Equipment listed below may be modified in order to optimize the system. Document any modifications in the raw data as method deviations:. 6.1.1 6.1.2 Micromass Quattro II triple quadrupole Msiss Spectrometer equipped with an electrospray ionization source HP1100 low pulse solvent pumping system, solvent degasser, column compartment, and autosampler 7.0 Supplies and M aterials________________________________ ;_________________________ 7.1 Supplies 7.1.1 High purity grade nitrogen gas regulated to approximately 100 psi (House air system) 7.1.2 HPLC analytical column, specifics to be determined by the analyst and documented in the raw data. 7.1.3 Capped autovials or capped 15 mL centrifuge tubes 8.0 Reagents and Standards_________________ _________________ :__________ ;__________ 8.1 Reagents 8.1.1 Methanol, HPLC grade or equivalent 8.1.2 Milli-QTM water, all water used in this method should be Milli-QTM water or equivalent, and may be provided by a Milli-Q TOC Plus system or other vendor 8.1.3 Ammonium acetate, reagent grade or equivalent 8.2 S tandards - 8.2.1 Typically two method blanks, two matrix blanks, and eighteen matrix standards are prepared during the extraction procedure. See ETS-8-4.1. 9.0 Sample H andling_________________________________________________ ;___ __________ _ 9.1 Fresh matrix standards are prepared with each an alysis. Extracted standards and samples are stored in capped autovials or capped 15 mL centrifuge tubes until analysis. ETS-8-5.1 Analysis of Serum Extract Using ES/MS Page 3 of 9 3M Environmental Laboratory Page 110 3M Medical Department Study: T-6316.7 BACK TO MAIN A nalytical R eport: FAC T TO X -098 L R N -U 2 40 2 9.2 If analysis will be delayed, extracted standards and samples can be refrigerated at approximately 4 C, or at room temperature, until analysis can be performed. 10.0 Quality C ontrol_______________________ ,___________________ ;___________________ 10.1 Solvent Blanks, Method Blanks and Matrix Blanks 10.1.1 Solvent blanks, method blanks and matrix blanks are prepared and analyzed with each batch to determine contamination or carryover. 10.1.2 Analyze a method blank and a matrix blank prior to each calibration curve. 10.2 Matrix Spikes 10.2.1 Matrix spikes are prepared and analyzed to determine the matrix effect on the recovery efficiency. 10.2.2 Matrix spike duplicates are prepared and analyzed to measure the precision and the recovery for each analyte. 10.2.3 Analyze a matrix spike and matrix spike duplicate per forty samples, with a minimum o f 2 spikes per batch. 10.2.4 Matrix spike and matrix spike duplicate concentrations will fall in the mid-range of the initial calibration curve. Additional spike concentrations may fall in the lowrange o f the initial calibration crave. 10.3 Continuing Calibration Verifications ' 10.3.1 Continuing calibration verifications are analyzed to verify the continued accuracy o f the calibration curve. 10.3.2 Analyze a mid-range calibration standard sifter every tenth sample, with a minimum o f one per batch. 11.0 Calibration and Standardization____________ ;________________________________ 11.1 Analyze the extracted matrix standards prior to and following each set o f extracts. The average of two standard curves will be plotted by linear regression (y = my + b), weighted 1/x, not forced through zero, using MassLynx or other suitable software. 11.2 If the crave does not meet requirements, perform routine maintenance or reextract the standard curve (if necessary) and reanalyze. 11.3 For purposes o f accuracy when quantitating low levels of analyte, it may be necessary to use the low end o f the calibration crave rather than the full range o f the standard curve. Example: when attempting to quantitate approximately 10 ppb o f analyte, generate a calibration curve consisting of the standards from. 5 ppb to 100 ppb rather than the full range o f the curve (5 ppb to 1000 ppb). This will reduce inaccuracy attributed to linear regression weighting of high concentration standards. 3M Environmental Laboratory ETS-8-5.1 Analysis of Serum Extract Using ES/MS Page 4 of 9 Page 111 3M Medical Department Study: T-6316.7 BACK TO MAIN A nalytical Report: FACT TO X-098 L R N -U 2 40 2 1 2 . 0 P r o c e d u r e s _________________________________________ ;____________________________ ;_________ 12.1 Acquisition Set up 12.1.1 Click on start button in the Acquisition Control Panel. Set up a sample list. Assign a filename using MO-DAY-last digit o f yetir-sample number, assign a method (MS) for acquiring, and type in sample descriptions. 12.1.2 To create a method click on scan button in the Acquisition control panel and select SIR (Single Ion Recording) or MRM. Set Ionization Mode as appropriate and mass to 499 or other appropriate masses. A full scan is usually collected along with the SIRs. Save acquisition method. I f MS/MS instruments are employed, additional product ion fragmentation information may be collected. See Micromass MassLynx GUIDE TO DATA ACQUISITION for additional information and MRM (Multiple Reaction Monitoring). 12.1.3 Typically the analytical batch run sequence begins with a set o f extracted matrix standards and ends with a set o f extracted matrix standards. 12.1.4 Samples are analyzed with a continuing calibration check injected after every tenth sample. Solvent blanks Should be analyzed periodically to monitor possible analyte carryover and are not considered samples but may be included as such. 12.2 Using the Autosampler 12.2.1 Set up sample tray according to the sample list prepared in Section 12.1.1. 12.2.2 Set-up the HP1100/autosampler at the following conditions or at conditions the analyst considers appropriate for optimal response. Record actual conditions in the instrument logbook: 12.2.2.1 Sample size = 10 p.L injection 12.2.2.2 Inject/sample =1 12.2.2.3 Cycle time = 13.5 minutes 12.2.2.4 Solvent ramp = Time 0.00 min. 8.50 min. 11.0 min. 12.0 min. MeOH 40% 90% 90% 40% 2.0 mM Ammonium acetate 60% 10% 10% 60% 12.2.2.5 Press the "Start" button. 12.3 In stru m en t Set-up 12.3.1 Refer to ETS-9-24.0 for more details. 12.3.2 Check the solvent level in reservoirs and refill if necessary. 3M Environmental Laboratory ETS-8-5.1 Analysis of Scrum Extract Using ES/MS Page 5 o f 9 Page 112 3M Medical Department Study: T-6316.7 BACK TO MAIN Analytical Report: FACT TO X-098 L R N -U 2 40 2 12.3.3 Check the stainless steel capillary at the end o f the probe. Use an eyepiece to check the tip. The tip should be flat with no jagged edges. If the tip is found to be unsatisfactory, disassemble the probe and replace the stainless steel capillary. 12.3.4 Set HPLC pump to "On". Set the flow to 10 - 500 uL/min or as appropriate. Observe droplets coming out of the tip of the probe. Allow to equilibrate for approximately 10 minutes. 12.3.5 Turn on the nitrogen. A fine mist should be expelled with no nitrogen leaking around the tip o f the probe. Readjust the tip of the probe if no mist is observed. 12.3.6 The instrument uses these parameters at the following settings. These settings may change in order to optimize the response: 12.3.6.1 Drying gas 250-400 liters/hour 12.3.6.2 ESI nebulizing gas 10-15 liters/hour 12.3.63 HPLC constant flow mode, flow late 10 - 500 pL/min 12.3.6.4 Pressure <400 bar (This parameter is not set, it is a guide to ensure the HPLC is operating correctly.) 12.3.7 Carefully guide the probe into the opening. Insert probe until it will not go any further. Connect the voltage cables to the pirobe. 12.3.8 Print the time page, with its parameters, and store it in the study binder with a copy taped into the instrument log. 12.3.9 Using the cross-flow counter electrode in the ES/MS source is recommended for the analysis of biological matrices. 12.3.10Click on start button in the Acquisition Control Panel (this may vary among MassLynx versions, see appropriate MassLynx USER'S GUIDE). Press the start button. Ensure start and end sample numb er includes all samples to be analyzed. 13.0 D ata Analysis and Calculations______________________________________________ 13.1 C alculations: 13.1.4 Calculate matrix spike percent recoveries using the following equation: % Recovery = Observed Result - Background Result x 100 Expected Result 13.1.5 Calculate percent difference using the following equation: . % Difference" Expected Cone. - Calculated Cone, x 100 Expected Cone. 13.1.6 Calculate actual concentration of PFOS, or other fluorochemical, in matrix (pg/mL): (he o f PFOS calc, from std. Curve x Dilution Factor) x 1 ue /Initial Volume o f matrix CmLl + iriL o f Surrogate Standard! 1000 ng Final Volume (mL) ETS-8-5.1 Analysis of Serum Extract Using ES/MS Page 6 of 9 3M Environmental Laboratory Page 113 3M Medical Department Study: T-6316.7 BACK TO MAIN Analytical Report: FACT TOX-098 LRN-U2402 14.0 M e t h o d P e r f o r m a n c e ___________________________ ___________________________________ ;________ 14.1 Method Detection Limit (MDL) and Limit o f Quantitation (LOQ) are method, analyte, and matrix specific. Please see ETS-8-4.1, Attachment B, for a listing of current validated MDL and LOQ values. 14.2 Solvent Blanks, Method Blanks, and Matrix Blanks 14.2.1 Solvent blanks, method blanks, and matrix blanks values are must be below the lowest standard in the calibration curve 14.3 Calibration Curves 14.3.1 The r2value for the calibration curve must be 0.980 or better. 14.4 Matrix Spikes 14.4.1 Matrix spike percent recoveries are must be within 30% o f the spiked concentration. 14.5 Continuing Calibration Verifications 14.5.1 Continuing calibration verification percent recoveries must be 30% o f the spiked concentration. 14.6 If criteria listed in this method performance section isn't met, maintenance may be performed on the system and samples reanalyzed or other actions as determined by the analyst. Document all actions in the appropriate logbook. 14.7 If data are to be reported when performance criteria have not been met, the data must be footnoted on tables and discussed in the text of this report. 1 5 . 0 P o l l u t i o n P r e v e n t i o n a n d W a s t e M a n a g e m e n t _______________________;________________ 15.1 Sample extract waste and flammable solvent is disposed in high BTU containers, and glass pipette waste is disposed in broken glass containers located in the laboratory. 16.0 Records__________________ ;_________ ;_________________________________ 16.1 Each page generated for a study must have the following information included either in the header or hand written on the page: study or project number, acquisition method, integration method, sample name, extraction date, dilution factor (if applicable), and analyst. 16.2 Print the tune page, sample list, and acquisition method from MassLynx to include in the appropriate study folder. Copy these pages and tape into the instrument runlog. 16.3 Plot the calibration curve by linear regression, weighted 1/x, then print these graphs and store in the study folder. 16.4 Print data integration summary, integration method, and chromatograms, from MassLynx, and store in the study folder. 3M Environmental Laboratory ETS-8-5.1 Analysis of Serum Extract Using ES/MS Page 7 of 9 Page 114 3M Medical Department Study: T-6316.7 BACK TO MAIN Analytical Report: FAC T TO X -098 L R N -U 2 40 2 16.5 Summarize data using suitable software (Excel 5.0) and store in the study folder, see A ttachm ent A for an example of a summary spreadsheet. 16.6 Back up electronic data to appropriate medium. Record in study notebook the file name and location o f backup electronic data. 17.0 T ables. Diagrams. Flowcharts, and V alidation D ata__________________________ 17.1 Attachment A: ETS-8-5.1 Data summary spreadsheet 18.0 References__________;____________ ;___________________________________________ 18.1 FACT-M-4.1, "Extraction o f Potassium Perfluorooctanesulfonate or Other Fluorochemical compounds from Serum for Analysis Using HPLC-Electrospray/Mass Spectrometry 18.2 ETS-9-24.0, "Operation and Maintenance o f the Micromass Atmospheric Pressure Ionization/Mass Spectrometer Quattro II triple quadrupole Systems" 18.3 The validation report associated with this method is ETS-8-4.0 & 5.0-V-l. 19.0 A ffected D ocuments_________________________________________ :_________________ 19.1 ETS-8-4.1, "Extraction o f Potassium Perfluorooctanesulfonate or Other Fluorochemical Compounds from Serum for Analysis Using HPLC-Electrospray/Mass Spectrometry" 20.0 Revisions_____________________________________________________________ Revision Number. 1 Reason For Revision Section 6.1.2 Clarification of HP1100 system components. Section 11.1 Average o f two curves, not standard values, are used for plotting linear regression and added the 1/x weighting of the curve. Section 12.2.2.4 Clarification o f solvent ramp. Section 17.1 Changed from attachment B to A. Revision Date 04/02/99 3M Environmental Laboratory ETS-8-5.1 Analysis of Serum Extract Using ES/MS Page 8 of 9 Page 115 3M Medical Department Study: T-6316.7 BACK TO MAIN Analytical Report: FACT TO X-098 L R N -U 2 40 2 Laboratory Study # Study: Test Material: Matrix/Final Solvent Method/Revision: Analytical Equipment System Number: Instrument Software/Version: Filename: R-Squared Value: Slope: Y Intercept Date o f Extraction/Analyst: Date o f Analysis/Analyst: Group Dose Sample# Concentration ug/mL Initial Vol. mL Dilution Factor Final Cone. ug/mL Slope: Taken from linear regression equation. Group/Dose: Taken from the study folder. Sample#: Taken from the study folder. Concentration (ug/mL): Taken from the MassLynx integration summary. Initial Volume (mL): Taken from the study folder. Dilution Factor: Taken from the study folder. Final Cone. (ug/mL): Calculated by dividing the initial volume from the concentration Attachment A: Summary Spreadsheet ETS-8-5.1 Analysis of Serum Extract Using ES/MS 3M Environmental Laboratory Page 9 of 9 Page 116 3M Medical Department Study: T-6316.7 BACK TO MAIN Analytical Report: FACT TO X-098 L R N -U 2 40 2 Study #: FACT-TOX-098 3M Environmental Lab -- Method Modification Method: ETS-8-5.1 "Analysis o f Potassium Perflucrooctanesulfonate or Other Fluorochemicals in Sera Extracts Using HPLC-Electrospray/Mass Spectrometry" Section modified: Effective date of modifications: 10.3.2,14.5.1, add sections 14.3.2-14.3.6 April 26,1999 Section 10.3.2___________'_______:____________________________________________ Method reads: 10.3.2 Analyze a mid-range calibration standard after every tenth sample, with a minimum of one per batch. Modify method to read: 10.3.2 Analyze a mid-range calibration standard at least after every ten samples, with a minimum o f one per batch. Section 14.5.1_______________________________________________________________ Method reads: 14.5.1 Continuing calibration verification percent recoveries must be within 30% of die spiked concentration. Modify method to read: 14.5.1 At least one continuing calibration verification per ten samples must show a percent recovery within +/-30% of die spiked concentration. Section 14.3.2____________ ;_________________________________________________ Method reads: NA Modify method to read: 14.3.2 The second (bracketing) calibration curve may be deactivated if instrumental drift affects the data. The first curve and acceptable calibration checks shall bracket usable data. 3M Environmental Laboratory Page 117 3M M edical D epartm ent Study: T-6316.7 BACK TO MAIN Analytical Report: F A C T T O X -09 8 L R N -U 2 40 2 Study #: FACT-TOX-098 Section 14.3.3_______ :_______________________________________________________ ` Method reads: . NA Modify method to read: 14.3.3 Calibration standards with peak areas less than 2 times the curve matrix blank should be deactivated to disqualify a data range that may be affected by background levels o f the analyte. Section 14.3.4 Method reads: ______________________ ;_______ ;_________ ;_______________ . NA Modify method to read: . . 14.3.4 Low or high curve points may be deactivated to optimize a linear range appropriate to the data. Section 14.3.5_______ ;_____________________ _________________________;____________ Method reads: NA Modify method to read: 14.3.5 A curve point may be deactivated if it deviates more than 30% from the theoretical value when the curve is evaluated over a linear range appropriate to the data. Section 14.3.6 Method reads: ___________________ _____________________________________ NA Modify method to read: 14.3.6 A valid calibration curve must contain at least 5 active points. - " jjz ----- U lrt/od Signature of PAI and date Signature of Sponsor and date Signature o f Study Director and date l?A<_ 3M Environmental Laboratory Page 118 3M Medical Department Study: T-6316.7 BACK TO MAIN A nalytical Report: FAC T TO X -098 L R N -U 2 40 2 3M Environmental Laboratory M ethod A nalysis o f P o ta ssiu m P erflu o ro o cta n esijlfo n a te o r O t h e r F lu o r o c h e m ic a l s in L iv e r E x tra cts U sing H P L C -E lectrospray/M ass Spectrom etry M ethod Num ber: ETS-8-7.0 Author: Lisa Clemen, Glenn Langenburg Approved By: IM Laboratory Manager Adoption Date: 0 *lX ^ Revision Date: 7A Date Group Leader Date Technical Reviewer Date 1.0 Scope and Application________ :_________________________________________________ 1.1 Scope: This method is for the analysis of liver extents for fluorochemical surfactants using HPLC-electrospray/mass spectrometry. 1.2 Applicable Compounds: Fluorochemical surfactants or other fluorinated compounds, or other ionizable compounds. 1.3 M atrices: Rabbit, rat, bovine, monkey liver, or other tissues as designated in the validation report. Word 6/95 ETS-8-7.0 Analysis of Liver Extract Using ES/MS Page 1 of 10 3M Environmental Laboratory Page 119 3M Medical Department Study: T-6316.7 A nalytical Report: FACT TO X -098 L R N -U 2 40 2 2.0 S u m m a r y of M ethod_____________________._____ __________________ ______ 2.1 This method describes the analysis of fluorochemical surfactants extracted from liver using HPLC-electrospray/mass spectrometry, or similar system as appropriate. The analysis is performed by monitoring a single ion characteristic of a particular fluorochemical, such as the perfluorooctanesulfonate (PFOS) anion, m/z = 499. Additionally, samples may be analyzed using a tandem mass spectrometer to furlher verify the identity o f a compound by detecting daughter ions o f the selected parent ion. 3.0 Definitions________________________ '______________ :_____________________ 3.1 Atm ospheric Pressure Ionization (API): The Micromass Quattro II triple quadrupole systems allow for various methods o f ionization by utilizing various sources, probes, and interfaces. These include but are not limited to: Electrospray Ionization (ESI), Atmospheric Pressure chemical Ionization (APcI), Thermospray, etc. The ionization process in these techniques occurs at atmospheric pressure (i.e. not under a vacuum). 3.2 Electrospray Ionization (ES, ESI): a method of ionization performed at atmospheric pressure, whereby ions in solution are transferred to the gas phase via tiny charged droplets. These charged droplets are produced by the application o f a strong electrical field. 3.3 Mass Spectrometry, Mass Spectrometer (MS), Tandem Mass Spectrometer (MS/MS): The API Quattro II triple quadrupole mass spectrometer is equipped with two quadrupole mass selective detectors and a collision cell. Ions are selectively discriminated by mass to charge ratio (m/z) and subsequently detected. A single MS may be employed for ion detection or an ion may be selected in the first quadrupole, fragmented in die collision cell, and these fragments may be analyzed in the second quadrupole. 3.4 Conventional vs. Z-spray probe interface: The latest models o f Micromass Quattro II triple quadrupole (post 1998) utilize a "Z-spray" conformation. The spray emitted from a probe is orthogonal to the cone aperture. In the conventional conformation it is aimed directly at the cone aperture, after passing through a tortuous pathway in the counter electrode. Though the configuration is different, the methods o f operation, cleaning, and maintenance are the same. However, Z-spray components and conventional components are not compatible with one another, but only with similar systems (i.e. Z-spray components are compatible with other Z-spray systems, etc.) 3.5 Mass Lynx Software: System software designed for the specific operation o f these Quattro II triple quadrupole systems. Currently MassLynx has Windows 95 and WindowsNT 4.0 versions. All versions are similar. For more details refer to the manual specific to the instrument (Micromass Quattro 13triple quadrupole MassLynx or MassLynx NT User's Guide). 4.0 W a r n i n g s a n d C a u t i o n s _____________________________ 4.1 H ealth and Safety W arnings: ___________________ 4.1.1 Use caution with the voltage cables for the probe. When engaged, the probe employs a voltage of approximately 5000 Volts. ETS-8-7.0 Analysis of Liver Extract Using ES/MS Page 2 of 10 3M Medical Department Study: T-6316.7 BACK TO MAIN A nalytical Report: FAC T TO X -098 L R N -U 2 40 2 4.1.2 When handling samples or solvents wear appropriate protective gloves, eyewear, and clothing. 4.2 Cautions: 4.2.1 Operate the solvent pumps below aback pressure o f 400 bar (5800 psi). If the back pressure exceeds 400 bar, the HP 1100 will initiate automatic shutdown. 4.2.2 Do not run solvent pumps to dryness. 5.0 I nterferences__________________________ ______________________________ 5.1 To minimize interferences when analyzing samples, Teflon shall not be used for sample storage or any part of instrumentation that comes in contact with the sample or extract. 6.0 Equipment_____________________________________________________________;_________ 6.1 Equipment listed below may be modified in order to optimize the system. Document any modifications in the raw data as method deviations. 6.1.1 6.1.2 Micromass Quattro II triple quadrupole Miiss Spectrometer equipped with an electrospray ionization source. HP 1100 low pulse solvent pumping system, solvent degasser, column compartment, and autosampler 7.0 Supplies and M aterials________________ ,____________ ____________________________ 7.1 Supplies 7.1.1 High purity grade air regulated to approximately 100 psi (house air system) 7.1.2 HPLC analytical column, specifics to be determined by the analyst and documented in the raw data 7.1.3 Capped autovials or capped 15 ml centrifuge tubes 8.0 Reagents and Standards________________________________________________________ 8.1 Reagents 8.1.1 Methanol, HPLC grade or equivalent . 8.1.2 Milli-QTM water (ASTM type I), all water used in this method should be ATSM type I, or equivalent, and be provided by a Milli-Q TOC Plus system or other vendor 8.1.3 Ammonium acetate, reagent grade or equivalent 8.1.3.1 When preparing different amounts than those listed, adjust accordingly. 8.1.3.2 2.0 mM ammonium acetate solution: Weigh approximately 0.300 g ammonium acetate. Pour into a 2000 mL volumetric container containing 2000 mL Milli-QTM water, mix until all solids are dissolved. Store at room temperature. . ETS-8-7.0 Analysis of Liver Extract Using ES/MS Page 3 of 10 3M Environmental Laboratory Page 121 3M Medical Department Study: T-6316.7 BACK TO MAIN Analytical Report: FACT TOX-098 LRN-U2402 8.2 Standards 8.2.1 Typically two method blanks, two matrix blanks, and eighteen matrix standards are prepared during the extraction procedure. Refer to ETS-8-6.0. 9.0 S a m p l e H a n d l i n g ________ ,______________________________________________________________________ 9.1 Fresh matrix standards are prepared with each analysis. Extracted standards and samples are stored in capped autovials or capped 15 ml centrifuge tubes until analysis. 9.2 I f analysis will be delayed, extracted standards and samples may be stored at room temperature, or refrigerated at approximately 4 C, until analysis can be performed. 10.0 Q u a l i t y C o n t r o l _________________________________ ,_______________________ 10.1 M ethod Blanks and M atrix Blanks '___________ 10.1.1 Solvent blanks, method blanks, and matrix blanks are prepared and analyzed with each batch to determine contamination or carryover. 10.1.2 Analyze a method blank and a matrix blank prior to each calibration curve. 10.2 M atrixSpikes 10.2.1 Matrix spikes are prepared and analyzed to determine the matrix effect on the recovery efficiency. 10.2.2 Matrix spike duplicates are prepared and analyzed to measure the precision and the recovery for each analyte. 10.23 Analyze a matrix spike and matrix spike duplicate per forty sample^. With a minimum o f 2 spikes per batch. 10.2.4 Matrix spike and matrix spike duplicate concentrations will fall in the mid-range of the initial calibration curve. Additional spike concentrations may fall in the lowrange o f the initial calibration curve. 10.3 C ontinuing C alibration Checks 10.3.1 Continuing calibration verifications are analyzed to verify the continued accuracy of the calibration curve. 10.3.2 Analyze a mid-range calibration standard every tenth sample, with a minimum of one per batch. 11.0 'C a l i b r a t i o n a n d S t a n d a r d i z a t i o n _________________________________________________ 11.1 Analyze the extracted matrix standards prior to and following each set of sample extracts. The average o f two standard curves will be plotted by linear regression (y = mx + b), weighted 1/x, not forced through the origin, using MassLynx or other suitable software. 11.2 If the curve does not meet requirements perform routine maintenance or reextract the standard curve (if necessary) and reanalyze. 3M Environmental Laboratory ETS-8-7.0 Analysis of Liver Extract Using ES/MS Page 4 of 10 Page 122 3M Medical Department Study: T-6316.7 BACK TO MAIN A nalytical Report: F A C T T O X -0 9 8 L R N -U 2 40 2 11.3 For purposes of accuracy when quantitating low levels o f analyte, it may be necessary to use the low end of the calibration curve rather than the full range of the standard curve. Example: when attempting to quantitate approximately 10 ppb o f analyte, generate a calibration curve consisting of the standards from 5 ppb to 100 ppb rather than the full range of the curve (5 ppb to 1000 ppb). This will reduce inaccuracy attributed to linear regression weighting o f high concentration standards. 12.0 P rocedures_______________________ ;____________________________________________ 12.1 Acquisition Set up 12.1.1 Set up the sample list. 12.1.1.1 Assign a sample list filename using MO-DAY-last digit o f year-increasing letter of the alphabet starting with a 12.1.1.2 Assign a method (MS file) for acquiring 12.1.1.3 Assign an HPLC program (Inlet file) 12.1.1.4 Type in sample descriptions and vial position numbers 12.1.2 To create a method click on method in the Acquisition control panel then mass spectrometer headings and select SIR (Single Ion Recording) or MRM (Multiple Reaction Monitoring). Set Ionization Mode as appropriate and mass to 499 or other appropriate masses. A full scan is usually collected along with the SIRs. Save acquisition method. If MS/MS instruments are employed, additional product ion fragmentation information may be collected. Refer to Micromass MassLynx GUIDE TO DATA ACQUISITION for additional information and MRM. 12.1.3 Typically the analytical batch run sequence begins and ends with a set of extracted matrix standards. 12.1.4 Samples are analyzed with a continuing calibration verification injected standard after every tenth sample. Solvent blanks should be analyzed periodically to monitor possible analyte carryover and are not considered samples but may be included as such. 12.2 U sing the A utosam pler . 12.2.1 Set up sample tray according to the sample list prepared in Section 12.1.1. 12.2.2 Set-up the HP1100/autosampler at the following conditions or at conditions the analyst considers appropriate for optimal response. Record actual conditions in the instrument logbook: 12.2.2.1 Sample size = 10 pL injection 12.2.2.2 Inject/sample = 1 12.2.2.3 Cycle time = 9 minutes . 3M Environmental Laboratory ETS-8-7.0 Analysis of Liver Extract Using ES/MS Page 5 of 10 Page 123 3M Medical Department Study: T-6316.7 BACK TO MAIN Analytical Report: FACT TO X-098 L R N -U 2 40 2 12.2.2.4 Solvent ramp conditions Time MeOH 0.00 min. 1.0 min. 4.5 min. 6.5 min. 7.0 min. 9.0 mi. 40% 40% 95% 95% 40% 40% 2.0 mM Ammonium acetate 60% 60% 5% 5% 60% 60% 12.2.2.5 Press the "Start" button. 12.3 Instrument Set-up 12.3.1 Refer to ETS-9-24.0, "Operation and Maintenance o f the Micromass Quattro II Triple Quadrupole Mass Spectrometer Fitted with an Atmospheric Pressure Ionization Source," for more details. 12.3.2 Check the solvent level in reservoirs and refill if necessary. 123.3 Check the stainless steel capillary at the end of the probe. Use an eyepiece to check the tip. The tip should be flat with no jagged edges. If the tip is found to be unsatisfactory, disassemble the probe and replace the stainless steel capillary. 123.4 Turn on the nitrogen. 12.3.5 Open the tune page. Clicks on operate to initiate source block and desolvation heaters. 123.6 Open the Inlet Editor. 123.6.1 Set HPLC pump to "On" 123.6.2 Set the flow to 10 - 500 uL/min or as appropriate 12.3.6.3 Observe droplets coming out of the tip of the probe. A fine mist should be expelled with no nitrogen leaking around the tip of the probe. Readjust the tip o f the probe if no mist is observed 12.3.6.4 Allow to equilibrate for approximately 10 minutes. 123.7 The instrument uses these parameters at ths following settings. These settings may change in order to optimize the response: 123.7.1 Drying gas 250-400 liters/hour 12.3.7.2 ESI nebulizing gas 10-15 liters/hour 1 2 3 .7 3 HPLC constant flow mode flow rate 10 - 500 jiL/min 12.3.7.4 Pressure <400 bar (This parameter is not set, it is a guide to ensure the HPLC is operating correctly.) 123.7.5 Source block temperature 150 123.7.6 Desolvation temperature 250 ETS-8-7.0 Analysis of Liver Extract Using ES/MS Page 6 of 10 3M Environmental Laboratory Page 124 3M Medical Department Study: T-6316.7 BACK TO MAIN Analytical Report: FACT TO X-098 L R N -U 2 40 2 12.3.8 Print the time page, with its parameters, and store it in the study binder with a copy taped into the instrument log. 12.3.9 Click on start button in the Acquisition Control Panel (this may vary among MassLynx versions, refer to appropriate MassLynx User's Guide). Ensure start and end sample number includes all samples to be analyzed. 13.0 Data Analysis and Calculations___________________________ ___________________ 13.1 Calculations: 13.1.4 Calculate matrix spike percent recoveries using the following equation: % Recovery = Observed Result - Background Result x 100 Expected Result ' 13.1.5 Calculate percent difference using the following equation: % Difference = Expected Cone. - Calculated Cone, x 100 Expected Cone. 13.1.6 Calculate actual concentrations in matrix (p,g/g): fag of PFOS calc, from std. Curve x Dilution Factor! x 1 pg (Initial Weight of Liver (et 1000 ng Final Volume (mL) 14.0 Method Performance_________________________________________________________ 14.1 Method Detection Limit (MDL) and Limit o f QuEintitation (LOQ) are method, analyte, and . matrix specific. Refer to ETS-8-6.0, Attachm ent B for a listing o f current validated MDL and LOQ values. 14.2 Solvent Blanks, M ethod Blanks and M atrix Blanks 14.2.1 Solvent blanks, method blanks, and matrix blanks must be below the lowest standard in the calibration curve. . 14.3 C alibration Curves 14.3.1 The r2 value for the calibration must be 0.980 or better. 14.4 M atrix Spikes 14.4.1 Matrix spike percent recoveries must be within 30% of the spiked concentration. 14.5 Continuing Calibration Verification 14.5.1 Continuing calibration verification percent recoveries must be within 30% o f the . spiked concentration. 14.6 If criteria listed in the method performance section are not met, maintenance may be performed on the system and samples reanalyzed or other actions as determined by the analyst. Document all actions in the appropriate logbook. 3M Environmental Laboratory ETS-8-7.0 Analysis of Liver Extract Using ES/MS Page 7 of 10 Page 125 3M Medical Department Study: T-6316.7 BACK TO MAIN Analytical Report: FACT TO X-098 LR N -U 2 4 0 2 14.7 If data are to be reported when performance criteria have not been met, the data must be footnoted on tables and discussed in the text of the: report. 15.0 P ollution Prevention and W aste M anagement_________________________________ 15.1 Sample extract waste and flammable solvent is disposed in high BTU containers, and glass pipette waste is disposed in broken glass containers located in the laboratory. 16.0 R ecords______ _________________ ;_________;_______________'______ ;__________ 16.1 Each page generated for a study must have the following information included either in the header or hand written on the page: study or project number, acquisition method, integration method, sample name, extraction date, dilution factor (if applicable), and analyst. 16.2 Print the tune page, sample list, and acquisition method from MassLynx to include in the appropriate study folder. Copy these pages and tape into the instrument runlog. 16.3 Plot the calibration curve by linear regression, weighted 1/x, then print these graphs and store in the study folder. 16.4 Print data integration summary, integration method, and chromatograms from MassLynx and store in the study folder. 16.5 Summarize data using suitable software (Excel 5.0+) and store in the study folder, refer to A ttachm ent A for an example o f a summary spreadsheet. 16.6 Back up electronic data to appropriate medium. Record in study notebook the file name and location o f backup electronic data. 17.0 T ables. Diagrams. Flowcharts, and Validation Data_________________________ 17.1 Attachment A: ETS-8-7.0 Data summary spreadsheet 1 8 .0 References_________;__________________ _______________________________ 18.1 FACT-M-2.1, "Extraction of Potassium Perfluorooctanesulfonate or Other Fluorochemical Compounds from Liver for Analysis Using HPLC-Electrospray/Mass Spectrometry" 18.2 ETS-9-24.0, "Operation and Maintenance o f the Micromass Atmospheric Pressure Ionization/Mass Spectrometer Quattro II triple qu:idrupole Systems" 18.3 The validation report associated with this method is ETS-8-6.0 & 7.0-V-l 19.0 A ffected D ocuments_________________________________________ ________________ 19.1 ETS-8-6.0, "Extraction of Potassium Perfluorooctanesulfonate or Other Fluorochemical Compounds from Liver or Fluid for Analysis Using HPLC-Electrospray/Mass Spectrometry" ETS-8-7.0 , Analysis of Liver Exttact Using ES/MS Page 8 of 10 3M Medical Department Study: T-6316.7 20.0 Revisions Revision Number BACK TO MAIN Analytical Report: FACT TO X -098 L R N -U 2 40 2 Reason For Revision Revision Date 3M Environmental Laboratory ETS-8-7.0 Analysis of Liver Extract Using ES/MS Page 9 of 10 Page 127 3M Medical Department Study: T-6316.7 BACK TO MAIN A nalytical Report: F A C T T O X -09 8 L R N -U 2 40 2 Laboratory Study # Study: Test Material: Matrix/Final Solvent: Method/Revision: Analytical Equipment System Number Instrument Software/Version: Filename: R-Squared Value: Slope: Y Intercept: Date of Extraction/Analyst: ' Date of Analysis/Analyst G roup Dose Sample# C oncentration ng/e in itial W t. g D ilu tio n F actor Final C one, ugfe Group/Dose: Taken from the study folder. Sample#: Taken from the study folder. Concentration (ng/g): Taken from the MassLynx integration summary. Initial Wt. (g): Taken from the study folder. Dilution Factor: Taken from the study folder. Final Cone, (ug/g): Calculated by dividing the initial volume from the concentration Attachment A: Summary Spreadsheet ETS-8-7.0 Analysis of Liver Extract Using ES/MS 3M Environmental Laboratory Page 10 of 10 Page 128 3M Medical Department Study: T-6316.7 BACK TO MAIN Analytical Report: FACT TOX-098 LRN-U2402 Study #: FACT-TOX-098 3M Environmental Lab --Method Modification Method: ETS-8-7.0 "Analysis o f Potassium Perfluorooctanesulfonate or Other Fluorochemicals in Liver Extracts Using ]HPLC-Electrospray/Mass Spectrometry" Section modified: Effective date o f modifications: 10.3.2,14.5.1, add sections 14.3.2-14.3.6 July 22,1999 Section 10.3.2______ : _______________________________________________________ Method reads: 10.3.2 Analyze a mid-range calibration standard after every tenth sample, with a minimum of one per batch. Modify method to read: 10.3.2 Analyze a mid-range calibration standard at least after every ten samples, with a minimum o f one per batch. Section 14.5.1____________'_______________ ;____________________________ __________ Method reads: 14.5.1 Continuing calibration verification percent recoveries must be within 30% of the spiked concentration. Modify method to read: 14.5.1 At least one continuing calibration verification per ten samples must show a percent recovery within +/-30% of the spiked concentration. Section 14.3.2 ___________________ _____________________________________________ Method reads: NA Modify method to read: ' 14.3.2 The second (bracketing) calibration curve may be deactivated if instrumental drift affects the data. The first curve and acceptable calibration checks shall bracket usable data. 3M Environmental Laboratory Page 129 BACK TO MAIN 3M Medical Department Study: T-6316.7 Analytical Report: FACT TOX-098 LRN-U2402 Study #: FACT-TOX-098 Section 1 4 .3 .3 ____________________________________;_____________________ ' Method reads: NA Modify method to read: 14.3.3 Calibration standards with peak areas less than 2 times the curve matrix blank should be deactivated to disqualify a data range that may be affected by background levels of the analyte. Section 14.3.4_____________________________ _________________________________ Method reads: NA . Modify method to read: . 14.3.4 Low or high curve points may be deactivated to optimize a linear range appropriate to the data. Section 14.3.5______________________________________________________________ Method reads: NA Modify method to read: '. 14.3.5 A curve point may be deactivated if it deviates more than 30% from the theoretical value when the curve is evaluated over a linear range appropriate to the data. Section 14.3.6 ________________________ ____________________________________ Method reads: NA Modify method to read: 14.3.6 A v alid calibration curve m ust contain at least 5 active points. H z------ Signature of PAI and date 7. Signature of Sponsor and date U/^/irO /J ^ , rd Signature of Study Director and date J, ? Z 3M Environmental Laboratory Page 130 3M Medical Department Study: T-6316.7 Appendix D: Data Sum m ary Tables BACK TO MAIN Analytical Report: FACT TOX-098 LRN-U2402 3M Environmental Laboratory Page 131 3M Medical Department Study: T-6316.7 BACK TO MAIN Analytical Report: FACT TOX-098 LRN-U2402 Table 7. Reported Fluorochemical Levels in Sera Analyses in Study FACT TOX-098 Dosage Group Specimen ID PFOS (pg/mL) PFOSA (pg/ml.)a PFOSAA (pg/mL)a EtFOSE-OH (pg/mL)a Group 1 (Control) 0 mg/kg/day 12573F 12574F 12575F <LOQ (0.0248)3 <LOQ (0.0248)3 <LOQ (0.0248)a <LOQ (0.005) <LOQ (0.0263) <LOQ (0 005) <LOQ (0.0263) <LOQ (0.005) <LOQ (0.0263) <LOQ (0.0098) <LOQ (0.0098) <LOQ (0.0098) Group 2 1 mg/kg/day 12576F 12577F 12578F 12579F 3.35a 3.28a 2.70a 3.69a 0.0893 0.0478 0.0292* 0.0786 5.08 3.72 2.18 6.01 0.00706 0.00397 0.00794 <LOQ (0.00493) 12580F 4.01a 0.0788 6.65 0.00848 Group 3 5 mg/kg/day 12581F 12582F 12583F 28.5a 24.7a 16.2a 0.500 0.308* 0.375* 56.1 48.0 14.4 0.0159 0.0121 0.0181 Group 4 10 mg/kg/day 12584F 12585F 12586F 32.0a 38.0a 34.5a 0.918 0.888 0.924 31.2 39.1 20.9 0.0406 0.0356 0.0352 12587F m i6 1.72 48.3 0.0800 Group 5 20 mg/kg/day 12588F 12589F 12590F 731e 60.9b 755 1.27 1.27 1.89 53.6 33.7 53.6 0.0661 0.0886 0.0762 12591F 47.7b 1.08 26.5 0.105 LOO--Limit of Quantitation aResults have not been corrected for the purity of the analytical reference material. ""Results have been corrected for the purity of the analytical reference material. "Tentative value, diluted extracts were below valid linear range of calibration curve. It is not possible to verify true recovery of endogenous analyte from tissues without radio-labeled reference material. The only measurement of accuracy available at this time, matrix spike studies, indicate that the data are quantitative^ to 40%. 3M Environmental Laboratory Page 132 3M Medical Department Study: T-6316.7 BACK TO MAIN Analytical Report: FACT TOX-098 LRN-U2402 Table 8. Reported Fluorochemical Levels in Liver Analyses in Study FACT TOX-098 Dosage Group Specimen ID PFOS (Mg/g)a PFOSA (Mg/g)a PFOSAA (Mg/g)a EtFOSE-OH (Mg/g)a Group 1 (Control) 0 mg/kg/day 12573F 12574F 12575F 0.0994 0.0253 0.137 <LOQ (0.120) <LOQ (0.120) 0.148 <LOQ (0.063) <LOQ (0.063) <LOQ (0.063) <LOQ (0.0593) <LOQ (0.0593)* <LOQ (0.0593)* Group 2 1 mg/kg/day 12576F 12577F 12578F 12579F 9.68 7.60 6.34 8.58 1.52 1.25 1.13 1.39 7.73 5.49 3.93 8.81 <LOQ (0.0593)* 0.292* 0.0774* 0.166* 12580F 7.00 1.24 7.95 0.132* Group 3 5 mg/kg/day 12581F 12582F 12583F 38.9 38.7 33.1 5.65 4.50 3.54 58.6 53.4 21.3 1.56 0.803 0.854 Group 4 10 mg/kg/day 12584F 12585F 12586F 75.9 85.8 76.4 7.33 6.07 6.67 40.8 58.4 26.8 6.96 5.26 6.40 12587F 145 9.18 96.1 15.3 Group 5 20 mg/kg/day 12588F 12589F 12590F 209 167 121 8.23 6.51 5.90 154 76.1 103 16.8 16.5 10.4 12591F 114 6.96 97.0 12.5 LOQ--Limit of Quantitation "Resuits have not been corrected for the purity of the analytical reference material. ` Data are qualitative only, matrix blank was unusually high and a suitable calibration curve (r2>0.98) could not be determined from this analysis. It is not possible to verify tore recovery of endogenous analyte from tissues without radio-labeled reference material. The only measurement of accuracy available at this time, matrix spike studies, indicate that the data are quantitative to 40%. 3M Environmental Laboratory Page 133 3M Medical Department Study: T-6316.7 BACK TO MAIN Analytical Report: FACT TOX-098 LRN-U2402 Table 9. Average Concentration of Fluorochemical Levels in Sera Analyses in Study FACT TOX-098 Dosage Group PFOS (pg/mL) PFOSA (pg/mL)a PFOSAA (pg/mL)a EtFOSE-OH (pg/mL)a Group 1 (Control) 0.0 mg/kg/day <LOQa <LOQ <LOQ <LOQ Group 2 1 mg/kg/day 3.40a 0.0647 4.73 0.00686 1 Anomaly Group 3 5 mg/kg/day 23.1a 0.395 39.5 0.0154 Group 4 10.0 mg/kg/day 34.8a 0.910 30.4 0.0371 Group 5 20.0 mg/kg/day 67.4b 1.44 43.1 0.0833 LOQ--Limit of Quantitation a Results have not been corrected for the purity of the analytical reference material. bResult has been corrected for purity of the analytical reference material. It is not possible to verify true recovery of endogenous analyte from tissues without radio-labeled reference material. The only measurement of accuracy available at this time, matrix spike studies, indicate that the data are quantitative to 40%. Table 10. Average Concentration of Fluorochemical Levels in Liver Analyses in Study FACT TOX-098 Dosage Group PFOS (pg/g)a PFOSA (pg/g)a PFOSAA (Mg/g)a EtFOSE-OH (Mg/g)a Group 1 (Control) 0.0 mg/kg/day 0.0873 0.148 <LOQ <LOQ Group 2 1 mg/kg/day Group 3 5 mg/kg/day Group 4 10.0 mg/kg/day 7.84 36.9 79.4 1.31 4.56 6.69 6.78 44.4 42.0 0.167* 1.07 6.21 Group 5 20.0 mg/kg/day 151 7.36 105 14.3 LOQ--Limit of Quantitation aResults have not been corrected for the purity of the analytical reference material. * Qualitative only It is not possible to verify true recovery of endogenous analyte from tissues without radio-labeled reference material. The only measurement of accuracy available at this time, matrix spike studies, indicate that the data are quantitative to 40%. 3M Environmental Laboratory Page 134 3M Medical Department Study: T-6316.7 Appendix E: Data Spreadsheets BACK TO MAIN Analytical Report: FACT TOX-098 LRN-U2402 3M Environmental Laboratory Page 135 3M Medical Department Study: T-6316.7 Analytical Report: FACT TOX-098 LRN-U2402 BACK TO MAIN i ! 3M Environmental Laboratory r i I sa ! iB *1 1*!2 \ 5 t* 1 h Page 136 3M Medical Department Study: T-6316.7 Analytical Report: FACT TOX-098 LRN-U2402 8ES^SSS|. ,s M| < 2 i =1 Cf < y p i s H i 1 ? ? ! j 23 SE i o litfiif t oI - |s| i U IH II id I 4* tet H I f i li i ! ! m1 lilis < C | l | H U S I i n ISSISI: 3335 33155 FACT-TOX-098 A rgus# 418-011 8SII-. 5 SI'- m ul u l l l h i l l l l i i J l i l i iffffi i ' i ` z< ss 15 13 S3 I1 I=ffe I,I.I ill ? i * f 1 J i ii ill |i*s g i l K l i t 55313 S 3533 83S33 H ? | I III I t t H I1IS SIS S S i ISSs S5SS8 I I I 18gS88 8 Ijiu i l h Hi iiiiii lili] H i II, 5 s 3 If I I m l u p i lallsj n 2 * 3! i i 11 2 $ it i } t j t | l u 3M Environmental Laboratory 9 1 5 2 Is 31 Page 137 3M Medical Department Study: T-6316.7 Analytical Report: FACT TOX-098 LRN-U2402 BACK TO MAIN 3M Environmental Laboratory ni Si Page 138 3M Medical Department Study: T-6316.7 Analytical Report: FACT TOX-098 LRN-U2402 BACK TO MAIN 33 X 0 ! 3M Environmental Laboratory 3J s5iJ Si Page 139 3M Medical Department Study: T-6316.7 BACK TO MAIN Analytical Report: FACT TOX-098 LRN-U2402 Appendix F: Example Calculations Formula Used for Sera Analyses in Study FACT TOX-098 AR (ng/mL) x DF x SC x FV (mL) x 1.0 pg = Reported Concentration (pg/mL) EV (mL) 1000 ng Calculation Used for Group 2, Animal ID 12576F 363 ng/mL x 10 x 0.9275 x 1 mL x 1.0 pg = 3.35 pg/mL 1.005 mL lOOOng AR-- Analytical result from MassLynx summary DF-- Dilution factor SC-- PFOS salt correction constant (0.9275) FV-- Final extract volume (1.0 mL unless otherwise noted) EV-- Volume of sera extracted Formula Used for Liver Analyses in Study FACT TOX-098 AR (ng/g) x d curve(1) x SC x DF x 1.0 pg = Reported Concentration (pg/g) d sample 1000 ng (1) d curve is assumed to be: 1 g liver 5 mL H2O Calculation Used for Group 2, Animal ID 12576F 527 ng/g x 1 g/ 5 mL x 0.9275 x 20 x 1.0 pg = 9.68 pg/g 1.0101 g/5mL lOOOng AR-- Analytical result from MassLynx summary d curve--Density of the liver standard curve, assumed to be lg liver/ 5 ml water 3 sample--Density of the liver sample (g sample/ 5 mL H20 ) SC-- PFOS salt correction constant (0.9275) DF-- Dilution factor 3M Environmental Laboratory Page 140 3M Medical Department Study: T-6316.7 Appendix G: Interim Certificates of Analysis BACK TO MAIN Analytical Report: FACT TOX-098 LRN-U2402 3M Environmental Laboratory Page 141 3M Medical Department Study: T-6316.7 BACK TO MAIN Analytical Report: FACTTOX-098 LRN-U2402 Centre Analytical Laboratories, Inc. 3048 Research Drive Phone: (814) 231-8032 State College, PA 16801 F:ax: (814) 231-1253 or (814) 231-1580 INTERIM CERTIFICATE OF ANALYSIS Revision 1(9/7/00) C entre Analytical Laboratories COA Reference #: 023-018B 3M Product: PFO S,Lotl71 Reference #: SD-009 ____________________ Purity: 86.4% Test Name Specifications Purity* Result 86.4% Appearance Identification NMR Metals (ICP/MS) 1. Calcium 2. Magnesium 3. Sodium 4. Potassium2 5. Nickel 6. Iron 7. Manganese Total % Impurity (NMR) Total % Impurity (LC/MS) Total % Impurity (GC/MS) Related Compounds - . POAA Residual Solvents (TGA) Purity by DSC Inorganic Anions (IC) 1. Chloride 2. Fluoride 3. Bromide 4. Nitrate 5. Nitrite 6. Phosphate 7. Sulfate4 Organic Acids5(IC) 1. TFA 2. PFPA 3. HFBA 4. NFPA Elemental Analysis": 1. Carbon 2. Hydrogen 3. Nitrogen 4. Sulfur 5. Fluorine . White Crystalline Powder ' , :r. . '* ' : .. ' ' ' ... ; . . . ' '' ' ' ; " ' ---- - - ...... ' ' ' - ' - .... - '. ' 1. Theoretical Value = 17.8% 2. Theoretical Value = 0% 3. Theoretical Value = 0% 4. Theoretical Value = 5.95% 5. Theoretical Value = 60% Conforms Positive 1. 0.017 wt./wt.% 2. 0.007 wt./wt.% 3. 1.355 wt./wt.% 4. 6.552 wt./wt.% 5. 0.003 wt/wt.% 6. 0.004 wt./wt.% 7. <0.001 wt./wt.% 1.00 wt./wt.% 10.60 wt./wt.% None Detected 0.30 wt/wt.% None Detected Not Applicable* 1. <0.015 wt/wt.% 2. 0.27 wt/wt.% 3. <0.040 wt/wt.% 4. <0.009 wt/wt.% 5. <0.006 wt/wt.% 6. <0.007 wt/wt.% 7. 8.82 wt/wt.% 1. <0.1 wt/wt.% 2. <0.1 wt/wt.% 3. <0.1 wt/wt.% 4. <0.25 wt/wt.% 1. 12.08 wt/wt.% 2. 0.794 wt/wt.% 3. 1.61 wt/wt.% 4. 10.1 wt/wt.% 5. 50.4 wt/wt.% COA023-018B Page 1 o f 3 3M Environmental Laboratory Page 142 3M Medical Department Study: T-6316.7 BACK TO MAIN Analytical Report: FACT TOX-098 LRN-U2402 Centre Analytical Laboratories, Inc. k 3048 Research Drive State College, PA 16801 ^ Phone: (814) 231-8032 Fax: (814) 231-1253 or (814) 231-1580 INTERIM CERTIFICATE OF ANALYSIS Centre Analytical Laboratories COA Reference #: 023-018B Date o f Last Analysis: 08/31/00 Expiration Date: 08/31/01 Storage Conditions: Frozen <-10C Re-assessment Date: 08/31/01 'Purity = 100% - (sum of metal impurities, 1.39% +LC/MS impurities, 10.60%+Inorganic Fluoride, 0.27%+NMR impurities, 1.00%+ POAA, 0.30%) Total impurity from all tests = 13.56% Purity = 100% -13.56% = 86.4% 2Potassium is expected in this salt form and is therefore not considered an impurity. 3Purity by DSC is generally not applicable to materials o f low purity. No endotherm was observed for this sample. 4Sulfur in the sample appears to be converted to SO4 and hence detected using the inorganic anion method conditions. The anion result agrees well with the sulfur determination in the elemental analysis, lending confidence to this interpretation. Based on the results, the SO4 is not considered an impurity. 5TFA HFBA NFPA PFPA Trifluoroacetic acid Heptafluorobutyric acid Nonofluoropentanoic acid Pentafluoropropanoic acid 6Theoretical value calculations based on the empirical formula, CgFi7S03TC+ (MW=538) This work was conducted under EPA Good Laboratory Practice Standards (40 CFR 160). COA023-018B 3M Environmental Laboratory Page 2 o f 3 Page 143 3M Medical Department Study: T-6316.7 BACK TO MAIN Analytical Report: FACT TOX-098 LRN-U2402 Centre Analytical Laboratories, Inc. 3048 Research Drive Phone: (814) 231-8032 State College, PA 16801 Fax: (814) 231-1253 or (814) 231-1580 INTERIM CERTIFICATE OF ANALYSIS Centre Analytical Laboratories COA Reference #: 023-018B LC/MS Purity Profile: Im p u rity C4 C5 C6 C7 Total wt/wt. % 1.03 1.56 6.38 1.63 10.60 Note: The C4 and C6 values were calculated using the C4 and C6 standard calibration curves, respectively. The C5 value was calculated using the average response factors from the C4 and C6 standard curves. Likewise, the C7 value was calculated using the average response factors from the C6 and C8 standard curves. Prepared By: f <y*> Davitl S. Bell Date Srietftist, Ce^Ltr^^pfilytical Laboratories Reviewed By: L/t/y Ah ? /vU Date Laboratory Manager, Centre Analytical Laboratories COA023-018B 3M Environmental Laboratory Page 3 o f3 Page 144 3M Medical Department Study: T-6316.7 BACK TO MAIN Analytical Report: FACT TOX-098 LRN-U2402 Centre Analytical Laboratories, Inc. 3048 Research Drive State College, PA 16801 Phone: (814) 231-8032 Fax: (814) 231-1253 or (814) 231-1580 o INTERIM CERTIFICATE OF ANALYSIS Centre Analytical Laboratories COA Reference #: 023-022-1 3M Product: EtFOSE-OH Test Control Reference #: SD-013 Purity: 88.9% Purity1 Test Name Specifications Result 88.9% Appearance Identification Yellow-white, waxy solid -y:v "-P". Conforms NMR Metals (ICP/MS) V;', ; . :) : ; - ' . 'j i". . Positive 1. Calcium 2. Magnesium 3. Sodium 4. Potassium 5. Nickel 6. Iron 7. Manganese Total % Impurity (NMR) '" ; ' 7 :! ; : ' :; ' ' .. . - '. , ' '' ' .1 . 1. <0.001 wt./wt.% 2. <0.001 wt./wt.% 3. <0.001 wt./wt.% 4. 0.002 wt./wt.% 5. <0.001 wt.Avt.% 6. <0.001 wt./wt.% 7. <0.001 wt.Avt.% 0.90 wt./wt.% Total % Impurity (LC/MS) None Quantified r Total % Impurity (GC/MS) Related Compounds - POAA Residual Solvents (TGA) . : .! ; .: '* : 10.21 wt.Avt.% 0.03 wt.Avt.% None Detected Purity by DSC Inorganic Anions (IC) - 87.6 wt.Avt.%. 1. Chloride 2. Fluoride 3. Bromide 4. Nitrate 5. Nitrite 6. Phosphate 7. Sulfate Organic Acids'* (IC) : ': * . .1 . ... ......... 1. <0.015 wt.Avt.% 2. <0.005 wt.Avt.% 3. <0.040 wt.Avt.% 4. <0.009 wt.Avt.% 5. <0.006 wt.Avt.% 6. <0.007 wt.Avt.% 7. <0.040 wt.Avt.% 1. TFA 2. PFPA 3. HFBA 4. NFPA Elemental Analysis'*: 1. <0.1 wt.Avt.% 2. <0.1 wt.Avt.% 3. <0.1 wt.Avt.% 4. <0.25 wt.Avt.% 1. Carbon 2. Hydrogen 3. Nitrogen 4. Sulfur 5. Fluorine 1. Theoretical Value = 25.2% 2. Theoretical Value = 1.75% 3. Theoretical Value = 2.45% 4. Theoretical Value = 5.60% 5. Theoretical Value = 56.6% 1. 24.42 wt.Avt.% 2. 1.78 wt.Avt.% 3. 2.72 wt.Avt.% 4. 9.34 wt.Avt.% 5. 58.4 wt.Avt.% COA023-022-1 3M Environmental Laboratory Page 1 of 3 Page 145 3M Medical Department Study: T-6316.7 BACK TO MAIN Analytical Report: FACT TOX-098 LRN-U2402 Centre Analytical Laboratories, Inc. 3048 Research Drive Phone: (814) 231-8032 State College, PA 16801 Fax: (814) 231-1253 or (814) 231-1580 INTERIM CERTIFICATE OF ANALYSIS Centre Analytical Laboratories COA Reference #: 023-022-1 3M Product: EtFOSE-OH Test Control Reference #: SID-013 Date of Last Analysis: 11/26/00 Expiration Date: 11/26/01 Storage Conditions: <-10 C Re-assessment Date: 11/26/01 'Purity = 100% - (total metal impurities, 0.002% + total NMR impurities, 0.90% + GC/MS impurities, 10.21 + POAA, 0.03%) Total impurity from all tests = 11.14% Purity = 100% -11.14% = 88.9% 2TFA Trifluoroacetic acid n- HFBA Heptafluorobutyric acid NFPA Nonailuoropentanoic acid PFPA Pentafluoropropanoic acid 3Theoretical value calculations based on the empirical formula, C12H10F17NO3S (MW=571) COA023-022-1 3M Environmental Laboratory Page 2 of 3 Page 146 3M Medical Department Study: T-6316.7 BACK TO MAIN Analytical Report: FACTTOX-098 LRN-U2402 Centre Analytical Laboratories, Inc. 3048 Research Drive Phone: (814) 231-8032 State College, PA 16801 Fax: (814) 231-1253 or (814) 231-1580 INTERIM CERTIFICATE OF ANALYSIS Centre Analytical Laboratories COA Reference #: 023-022-1 3M Product: EtFOSE-OH Test Control Reference#: SD-013 GC/MS Purity Profile Peak# 1 2 3 4 5 6 7 8 9 10 11 12 13 Total Retention Time (min) 6.163 8.011 8.206 9.065 9.844 13.93 14.238 15.130 15.52 15.941 16.379 16.801 17.222 - Identity Unknown Unknown Unknown Unknown Unknown Unknown Unknown C2 C3 C4 C5 C6 C7 - % Impurity 0.12 0.23 0.51 0.21 0.34 0.62 0.11 0.11 1.11 1.55 1.07 3.30 0.93 10.21 This work was conducted under EPA Good Laboratory Practice Standards (40 CFR 160). Prepared By: 22^ rfetftd S. Bell Scientist Centre Analyrifcai Laboratories Reviewed By: ft) /ULfctU ) /John Flaherty / / Laboratory Manager Centre Analytical Laboratories C' COA023-022-1 3M Environmental Laboratory Date //k M Date Page 3 o f3 Page 147 3M Medical Department Study: T-6316.7 BACK TO MAIN Analytical Report: FACT TOX-098 LRN-U2402 / --s i Centre Analytical Laboratories, Inc. 3048 Research Drive Phone: (814) 231-8032 S tate C ollege, PA 16801 Fax: (814) 231-1253 o r (814) 231-1580 INTERIM CERTIFICATE OF ANALYSIS C entre Analytical Laboratories COA Reference #: 023-022-2 3M Product: EtFOSE-OH Test Control Reference #: TCR-00017-52 Purity: 97.4% Test Name Purity1 Appearance Identification NMR Metals (ICP/MS) 1. Calcium 2. Magnesium 3. Sodium 4. Potassium 5. Nickel 6. Iron 7. Manganese Total % Impurity (NMR) Total % Impurity (LC/MS) Total % Impurity (GC/MS) Related Compounds - POAA Residual Solvents (TGA) Purity by DSC Inorganic Anions (IC) 1. Chloride 2. Fluoride 3. Bromide 4. Nitrate 5. Nitrite 6. Phosphate 7. Sulfate . Organic Acids'1(IC) 1. TFA 2. PFPA 3. HFBA 4. NFPA Elemental Analysis1: 1. Carbon 2. Hydrogen 3. Nitrogen 4. Sulfur 5. Fluorine Specifications ;r ! . ! . vi-" .i-i f ;? v. i , ;: Yellow-white, waxy solid Result 97.4% Conforms ; . : ' v Positive " 1. <0.001 wt./wt.% 2. <0.001 wt./wt.% 3. <0.001 wt./wt.% 4. <0.001 wt./wt.% 5. <0.001 wt./wt.% 6. <0.001 wt./wt.% 7. <0.001 wt./wt.% 1.26 wt./wt.% None Quantified 1.29 wt./wt.% . 0.10 wt./wt.% None Detected . ' ' ` 90.3 wt./wt.%. 1. <0.015 wt./wt.% 2. <0.005 wt./wt.% 3. <0.040 wt./wt.% 4. <0.009 wt./wt.% 5. <0.006 wt./wt.% 6. <0.007 wrt./wt.% 7. <0.154 wt./wt.% 1. <0.1 wt./wt.% 2. <0.1 wt./wt.% 3. <0.1 wt./wt.% ' ' ' , 4. <0.25 wt./wt.% 1. Theoretical Value = 25.2% 2. Theoretical Value = 1.75% 3. Theoretical Value = 2.45% 4. Theoretical Value = 5.60% 5. Theoretical Value = 56.6% 1. 25.04 wt./wt.% 2. 1.69 wt./wt.% 3. 2.61 wrt./wt.% 4. 8.88 wt./wt.% 5. 56.8 wt./wt.% COA023-022-2 3M Environmental Laboratory Page 1 of 3 Page 148 3M Medical Department Study: T-6316.7 BACK TO MAIN Analytical Report: FACT TOX-098 LRN-U2402 Centre Analytical Laboratories, Inc. 3048 R esearch Drive Phone: (814) 231-8032 S tate C ollege, PA 16801 Fax: (814) 231-1253 o r (814) 231 -1580 INTERIM CERTIFICATE OF ANALYSIS Centre Analytical Laboratories COA Reference #: 023-022-2 3M Product: EtFOSE-OH Test Control Reference#: TCR-00017-52 Date of Last Analysis: 11/26/00 Expiration Date: 11/26/01 Storage Conditions: <-10 C Re-assessment Date: 11/26/01 'Purity = 100% - (total NMR impurities, 1.26% + GC/MS impurities, 1.29 + POAA, 0.10%) Total impurity from all tests ==2.65% Purity = 100% - 2.65% = 97.4% TFA HFBA NFPA PFPA Trifluoroacetic acid Heptafluorobutyric acid Nonafluoropentanoic acid Pentafluoropropanoic acid 3Theoretical value calculations based on the empirical formula, C12H10F17NO3S (MW=571) COA023-022-2 3M Environmental Laboratory Page 2 o f 3 Page 149 3M Medical Department Study: T-6316.7 BACK TO MAIN Analytical Report: FACT TOX-098 LRN-U2402 Centre Analytical Laboratories, Inc. L 3048 Research Drive State College, PA 16801 ^ Phone: (814) 231 -8032 Fax: (814) 231 -1253 or (814) 231 -1580 c INTERIM CERTIFICATE OF ANALYSIS Centre Analytical Laboratories COA Reference #: 023-022-2 3M Product: EtFOSE-OH Test Control Reference #: TCR-00017-52 GC/MS Purity Profile Peak# 1 2 Total Retention Time (min) 13.934 17.307 - Identity PFOSDEA C7 - % Impurity 0.36 0.93 1.29 This work was conducted under EPA Good Laboratory Practice Standards (40 CFR 160). Prepared By: fete Hd S. Bell Scientist Centre Anal' oratories Reviewed By: t John Flaherty Laboratory Manager Centre Analytical Laboratories COA023-022-2 3M Environmental Laboratory Date Date Page 3 o f 3 Page 150 3M Medical Department Study: T-6316.7 Appendix H: Report Signature Page BACK TO MAIN Analytical Report: FACT TOX-098 LRN-U2402 T " GiiL& t^ Marvin T. Case, D.V.M., Ph.D., Study Director %*o Date ^ John L. Butenhoff, Ph.D., Sponsor Representative f -S z & t? ' Date Q Z'/ol/of Kristen . Hansen, Ph.D., Principal Analytical Investigator Date William K. Reagen, Laboratory Manager Date 3M Environmental Laboratory Page 151