Document 85Z3QMYYpy9VMbgnrR45kaKZd

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-Ethylperfluorooctanesulfonamidc~)-ethanoinl Rats Analytical Laboratory Report Title Determination of the Concentration of PFOS, PFOSA, PFOSAA, and EtFOSE-OH in the Sera and Liver of Crl:CD@BRVAF/PIUS@Rats Exposed to PJ-EtFOSE-OH Data Requiremeni! Not Applicable Aufhor 3M Environmental Laboratory Study Completion D#ate At signing Performing Laboratories Liver and Serum Analyses 3M Environmental Laboratory Building 2-3E-09,935 Bush Avenue St. Paul, MN 55106 Project Identificaticm 3M Medical Department Study: T-6316.7 Argus In-Life Study: 418-011 Analytical Report: FACT TCIX-098 3M Laboratory Request No. U2402 Total Number of Pages 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 {countryrequirements. 3M Erivironmental 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 in the Sera arid Liver of Crl:CD@BR VAF/PIUS@Rats Exposed to N-RFOSE-OH Study IdentificationNumbers: 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: 0 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 performanceof 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, 0 Some analytical reference materials have not been completely characterized. QAU did not perform an in-phase inspectionduring the study. -6- 9 l L W t . A f Marvin T. Case, D.V.M., Ph.D., Study Director 6 FA / Date John f. Butenhoff, Ph.D., Sponi%r Representative Ffiy 06 ~ LZd/ Date Kristen J. Hansen, Ph.D., Principal Analytical lnvesfigator F i b 01, m / Date 3M Environmental Laboratory Page 3 3M Medical DepartmentStudy: 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 Cr1:CD"BR VAF/PIUS@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 - 11/06/00 1/08/00 Phase Data Date Reported to Management Study Director 11/08!00 11/08/00 12/13/00- 12/15/00 - 12/21/00 12/22/00 Draft report Draft report 12/15/00 12/22f00 12/15/00 12/22/00 /&/.* QA U Representative 2 / 2 / 0I 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 EnvironmentalLaboratory....................................................................................... 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.O, Extraction of Potassium Perf1uorooctane:sulfonateor 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, Extractionof Potassium Perfluorooctanesulfonateor Other Fluorochemical Compounds from Serum for Analysis Using HPLC-Elelctrospray/MassSpectrometry, (14 pages)......................................................................................................................... 63 ETS-8-6.0, Extraction of Potassium Perfluorooctanesulfonate or Other Fluorochemical Compounds from Liver for Analysis Using HPLC-ElectrospraylMass Spectrometry", (14 pages)......................................................................................................................... 77 FACT-M-2.0, Analysis of Fluorochemicals in Liver Extracts Using HPLC- Electrospray/MassSpectrometry, (8 pages)...................................................................... 91 3M Erivironmental 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/MassSpectrometry. (9 pages)........................................................................................................................... 99 ETS-8-5.1, Analysis of Potassium Perfluorooctanesulfonate or Other Fluorochemicals in Serum Extracts Using HPLC-Electrospray/MassSpectrometry. (1 1 pages) ..................108 ETS-8-7.0, Analysis of Potassium Perfluorooctanesulfonateor Other Fluorochemicals in Liver Extracts Using HPLC-Electrospray/MassSpectrometry. (1Z! 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 Erivironmental 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 (418-011) ............................................................................................................. 9 Table 2. Characterizationof the Analytical Reference Standards in Study FACT TOX-098 ............................................................................................................... 10 Table 3. Negative Ions Monitored in 3M Laboratory Arialyses .......................................... 13 Table 4. Determinationsof the LOQ in the Analyses of Sera and Liver Extracts..............14 Table 5. Characterizationof Test Article in Study FACT' TOX-098 .................................... 16 Table 6. Characterizationof 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 Levlels 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 0. Johnson Kelly J. Kuehlwein* *Contract lab professionalservice employees Sally A. Lintla* Joseph C. F'ilon* Ian A. Smith* Kathleen M. Stock* Bob W. Wyinne* Richard D. 'r'oungblom* 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 EnvironmentalLaboratory 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 nurnber 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:CD@BRVAF/PIUS@(Sprague-Dawley) rat received from Charles River Laboratories, Inc., permanently identified using a Monel@selfpiercing ear tag. Dosage Group I II Ill IV V Dosage (mglkglday) I 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-I 9 specimens Liver Specimens-I9 specimens Fetuses-I 9 specimens Placentas-I 9 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 shlipped 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-tert-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 SI-15-98and !3-18-98 from Argus. All specimens were received frozen on dry ice and were immediately transferred to storage at -20C *IO"C. 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 k1O"C. Chemical Characterizationof the Referencle Standards Chemical characterizationinformation on the analytical reference standards used in this study is presented in the tabular form below. Table 2. Characterization of the Analytical ReferenceStandards in Study FACT TOX-098 Reference Standard I Formula Acronym Source Expiration Date Potassium Perfluorooctanesulfonate CaF17SO3-K+ 3M KPFOS~ 3M N-Ethyl Perfluorooctanesulfonamidoethyl alcohol EtFOSE-OH 3M C~F.I~SO~N(C~H~)CH~CH~OH Perfluorooctanesulfonylarnido(ethy1)acetate 3M CE,F&OZN(CH~CH~)CHZCOO-N~ PFOSAA Perfluorooctanesulfonylarnido(ethy1)acetate 3M C ~ F , I ~ S O ~ N ( C H ~ C H ~ ) C HH~ C O O - Perfluorooctanesulfonylarnide CaF 17SozNHz PFOSA 3M IH, IH, 2H, 2HTetr.3hydropetfluorooctanesulfonic acid THPFOS ICN CaH4Fi3S03H 2010 01/01/2010 2010 2010 2010 2010 2010 bAssurned100%untilCertificateof Analysis is completed. 'NA-not applicable. This lot is exhausted and cannot be characterized. TBD-to be determined 3M Erivironmental 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 during1 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 Envimnmental Laboratory PREPARATORMYETHODS FACT-M-I.O, "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.OmL 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 Compoundsfrom Serum for Analysis Using HPLC-Electrospray/MassSpectrometry" ETS-8-6.0, "Extraction of Potassium Perfluorooctane-sulfonate or Other Fluorochemical Compounds from Liver for Analysis Using HPLC-Electrospray/MassSpectrometry" 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.OmL of methanol, and then filtered through a 3 cc plastic syringe attached to a 0.2 pm nylon filter into glass autovials. ANALMICAL METHODS FACT-M-2.0, "Analysis of Fluorochemicals in Liver Extracts Using HPLC-Electrospray/Mass Spectrometry" 0 FACT-M-4.1, "Analysis of Potassium Perfluorooctaneslulfonate or Other Fluorochemicalsin Serum or Other Fluid Extracts Using HPLC-Electrospray/MassSpectrometry" 0 ETS-8-5.1, "Analysis of Potassium Perfluorooctanesulfonateor Other Fluorochemicalsin 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 Perfluorooctanesullonateor Other Fluorochemicalsin Liver Extracts Using HPLC-Electrospray/MassSpectrometry" The analyses were performed by monitoring one or more product ions selected from a single primary ion characteristic of a particular fluorochemicall using HPLC/ES/MS/MS. For example, molecular ion 499, selected as the primary ion for PFClS (CsF17SO:,-)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@BetasilTMCis 2x50 mm (5 pi) Column temperature: Ambient Mobile phase components: Component A: 2mM ammonium acetate Component B: methanol Flow rate: 300 vL/min Injection volume: 10 pL Solvent Gradient: 13.5 minutes Time (minutes) %B 0.0 40% 8.5 90% 11.0 90% 12.0 40% 13.5 40% Mass Spectrometer: Micromass@API/Mass Spectrometer Quattro IllMTriple Quadrupole system Software: Mass Lynxn 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 *1O"C Electrode: Z-spray Analysis Type: Multiple Reaction Monitoring (MRM) 3M Ertvironmental Laboratory Page 12 3M Medical Department Study: T-6316.7 BACK TO MAIN Analytical Report: FACT TOX-098 LRN-U2402 PFOS I PFOSA 1 PFOSAA I EtFOSE-OH I THPFOS I 499.0 498.0 584.0 630.0 427.0 I I 80.0, 99.0, 130.0 ~ I 78.0 I I 83.0, 169.0 I 59.0 I 80.0 I 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 Perfluorooctanesulforiate or Other Fluorochemicals in Serum Extracts Using HPLC-Electrospray/MassSpectrometry and ETS-8-7.0, Analysis of Perfluorooctanesulfonateor Other Fluorochemicals in Liver Extracts Using HPLCElectrospray/Mass Spectrometry: 0 Linearity: The coefficient of determination (rz)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 10.980. Calibration Standards: Quantitationof the target analytes was based on linear regression analysis l / 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 of Quantitation (LOQ): The LOQ is equal to the lowest acc.eptablestandard 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 EnvironmentalLaboratory Page 13 3M Medical Department Study: T-6316.7 BACK TO MAIN Analytical Report: FACT TOX-098 LRN-U2402 Analyte PFOS PFOSA PFOSAA EtFOSE-OH Values are approximate LOQ--Lirnit of Quantitation Sera Method LOQ (WmLP 0.025 0.005 0.025 0.010 ~~ Liver Method LOQ (P9W 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 GI-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: Precisionwas 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, arid 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. Statement 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 anldControl Matrices Table 5. Characterization of Test Article in !study FACT TOX-098 I Te!it Article 1 - Chemical Name N-EtFOSE-OH I source I 3M I Storage Conditions Ambient temperature I Physical Description I Purity I Waxy solid 97.4% I 1 - 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-A2382 Rabbit Liver TN-A-0809 Rabbit Liver TN-A-0810 Source Sigma Sigma Storage Conditions -20C *10"C -20C * I 0C -20C *I0C Chemical Lot # 17H9306 118H8418 FOOOl2 Physical Description I Rat Serum I Rabbit Serum I Rabbit Liver 1 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 Madical Department Study: T-6316.7 BACK TO MAIN Analytical Report: FACT TOX-098 LRN-U2402 3M ENVIRONMENTALLABORATORY PROTOCOL- ANALYTICASLTUDY Oral (Gavage) Developmental Toxicity Study of 2(N-Ethylperfluorooctanesulfonamido)ethanoX 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 of testing facility: 3M Environmental Technology and Services 935 Bush Avenue, Building 2-3E-019 St. Paul, MN 55106 Sponsor approval date: Experimental s t a r t date: October 9,1998 Expected termination date: July 16,1999 Method numbers and revisions: FACT-M-1.0, Extraction of Potassium Pduorooctanesulfonate or Other Anionic . Surfactants from Liver for Analysis Using HPLC-EkctrosprayMass Spectrometry < FACT-M-2.0, Analysis of FluorocheqicalsioLiver Eutracts Using :HpLC- Electrospray/Mass Spectrometry FACT-M-3.1, Extractionof Potassium Perfluorooctaniesulfonateor Other Anionic Surfactants from Serum or Other Fluid for Analysis lJsing HPLCElectrosprayhhss Spectrometry FACT-M-4.1, Analysis of Fluorochemicals in Serum or Other Fluid Extracts Using HPLCElectrosprayMass Spectrometry Author: Lisa Clemen Study Director FACT-TOX-098, U2402 A r p #418-011 Page 1of 5 3M EnvironmentalLaboratory Sponsor Representative 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 of potassium perfluorooctane sulfonate(PPOS), or another metaboliteof 2(Nethylperfluorooctanesulfonamid0)-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 Rtsearch Laboriittories (Argus 4 18-01 1). 2.0 REGULATORYCOMPLIANCE This study will be conducted in complianceWith the Food and Drug Administration Good Laboratory Practicesregulation as stated in 21 CFR 58. Any exceptionswillbe noted in the final report. 3.0 TESTMATEM 3.1 Test,control, and reference substances and matrices 3.1.1 Analyticalreference substance: Potassium perfluorooctmesulfonate(PPOS), lot # 217 3.1.2 Analyticalreference substancematrix: Rat liver, serum, and whole blood ' 3.13 Analytical control substance: None 3.1.4 Analyticalcontrol substancematrix: Rat liver, serum, and whole blood 3.2 Source of materials 3.2.1 Analyticalreference substance: 3M Specalty Chemical Division; traceability informationwill be included in the finalreport 3.2.2 Analytical reference substance matrix: Argus Research Laboratories; traceabilityinfomationwill be included in the final report 3.23 Analytical control matrix: 3.2.3.1 Rat liver -Argus ResearchLaboratories; traceahility informationwill be included in the final report; or Rabbit liver - Covance Laboratories;traceabilityinformation will be included in the final report. 3.23.2 Rat serum - Sigma Chemical Company;traceabilityinformationwill be included in the final report. 3.2.3.3 Rat whole blood -3M Toxicology; traceability informationwill be included in the final report. 3.3 Number of test and control samples. Liver and serum sampleswill 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 Argas #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 developmentalportion of the study. Fetus,placenta, or other samples will be tested at the discretion of the Study Director. 3.4 Identification of test and control samples: The samples are idmtifiedusing the Argus Research Laboratories identifiers,which consistof the Argus project number, the animal number, the group designation, and the draw date. 3.5 Purity and strength of materials: Characterizaticeof the purity and identity of the reference material is the responsibility of the Sponsor. 3.6 Stability of test material: Characterizationof the stability of the test materialis the responsibility of the Sponsor. 3.7 Storage conditions for test materials: Test materials are stored at room temperature. Samples are stored at -20 f 10 "C. 3.8 Dispositionof test and/or control substances: Biological tissues and fluids are retained per GLP regulation. 3.9 Safety precautions: Refer to the material safety dita sheets of chemicals used. Wear appropriate laboratory attire, and follow adequate precautions for handling biological materials and preparing samples for analysis. 4.0 - EXPERIMEN~O~Lverview Tissues from animals dosed as described in Argus Research Laboratoria Protocol #418-011will 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. At Day 20 for the rats remainingin the study.,samples of s t m and liver were taken, as well as fetus and placenta. Dosage samples willbe provided from Argus Research Laboratories for concentrationlevel confirmation. These samples will not be extracted and analyzed according to GLP regulations. The data collectedwill be providedto the Sponsor as an attachmentto the datapackage. At the discretion of the Study Director, a series of andytical tests will be performed on select tissues. Initially, all liver and serum samples will be analy:zed, using the methods listed in section 5.0, for PFOS by Electrospray/massspectrometry(WMS). On rhe basis of findings from these analyses, additionalsamples may be evaluated. If additionalanalysis is performed, a protocol amendment will be written to add the matrices and methods to the protocol. At the discretion of the Study Director, select analysis may be performed by a contract laboratorywhere competencehas been demonstrated,using validated anidytical methods. If a contract laboratory is used, this protocol willbe amendedtr3 include the required information, The methods, data, and contract laboratory will be identifiedin the data package provided to the Sponsor. FACT-TOX-098, U2402 A r e s #418-011 Page 3 of 5 3M Environmental Laboratory Page 20 3 M Medical Department Study: T-6316.7 BACK TO MAIN Analytical Report: FACT TOX-098 LRN-U2402 5.0 EXPERIMENTA-LAnalyticalMethods 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 Perfluorooctanesulfonateor Other Anionic Surfactants from Liver for Analysis Using HPLCI-Electrospray/Mass Spectrometry 5.1.2 FACT-M-2.0, Analysis of Fluorochemicals in Liver Extracts Using HPLCElectrosprayMass Spectrometry 5.13 FACT-M-3.1, Extraction of Potassium Perfluorooctanesulfonateor Other Anionic Surfactants from Serum or Other Fluid for Analysis Using HPLC- ElectrosprayMass Spectrometry 5.1.4 FACT-M-4.1, Analysis of Fluorochemical,sin Serum or other Fluid Extracts Using HPLC-ElectrwprayMass 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 DATAANALYSIS 6.1 Data reporting: For analysis perfomed by a conbract 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 datapacket provided by the analytical phase Study Director to the Sponsor. 6.2 Data transformations and analysis: Data will be reported as the concentration (weightlweight) of the target analyte per tissue or simple, or of tlle targe.t analyte per unit of tissue or fluid. 6.3 Statisticalanalysis: Statistics used may include regression analysis of the s e r u m concentrations over time, and standard deviations calculated for the concentrations within ,each dose group. If necessary, simple statistical teats, such as Student's t test, may be applied to evaluate statistical difference. 7.0 MAINTENANCE OF RAWDATAAND RECORDS 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 PACT-TOX-098, U2402 Argus M18-011 Page 4 of5 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 foIlowing: 7.2.1 Training records 7.2.2 Calibration records 7.23 Instrumentmaintenance 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 of the following validated 3M Environmental Laboratory methods are attached for information purposes: 9.1.2 FACT-M-1.0, Extractionof Potassium Perfluorooctanesulfonateor Other Anionic Surfactants from Liver for Analysis Using HPLC-Electrosprayhfass Spectrometry 9.1.2 FACT-M-2.0, Analysis of Fluorochemicals in Liver Extracts Using HPLC- Electrosprayhiass Spectrometry 9.1.3 FACT-M-3.1, Extraction of Potassium Perfluorooctanesulfonateor Other Anionic Surfactants fiom Serum or Other Fluid for Analysis Using HPLCBlectrosprayhiass Spectrometry 9.1.4 FACT-M-4.1, Analysis of Fluorochemicals in S e r u m or Other Fluid Extracts Using HPLC-ElectrosprayMass Spectrometry 9.2 Argus protocol 418-01 1 FACT-TOX-098, U2402 Argus #418-011 Page 5 of 5 3M Environmental Laboratory Page 22 3M Medical Department Study: T-6316.7 BACK TO MAIN Analytical Report: FACT TOX-098 LRN-U2402 Study Titie Oral (Gavage) DevelopmentalToxicity Study of2(N-Ethylperfluorooc~mesulfonamido)-ethanol in Rats PROTOCOLAMENDMENT NO. I Amendment Date: 22 February 2000 Performing Laboratory 3M Environmental Technology & 13afety Services 3M EnvironmentalLaboratory 935 Bush Avenue St. Paul, MN 55106 LaboraforyProject ldentficafion 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 I 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. AMENDTO 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 of this amendment. REASON: The role of study director was reassigned in an effort to ensure compliance with Good Laboratory Practice Standardsthat outline study persortnel requirements (refer to 21 CFR Part 58). 2. PROTOCOL READS: The sponsor for the present study was identified as Marvin T. Case, D.V.M., Ph.D. AMENDTO READ: The role of sponsor for the present study was reassigned to John L. Eiutenhoff, PbD., 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 substancematrix: Rat Iik er, serum, and whole blood 3.1.4Analytical control substancematrix: Rat liver, serum, and whole blood AMENDTO READ: 3.1 Test, control, and reference substances and matrices 3.1.2 Analytical reference substancema& Rat liver,serum, pooled fetal tissue(s), and whole blood 3.1.4Analytical control substancematrix: Rat liver, serum, pooled, fetal tissue(s), and whole blood REASON: Analysis of fetal tissue for the target chemical andor ils analytes was added to the scope of the study following the issuance of the original protocol. 3M Environmental Labohfory 3M EnvironmentalLaboratory Page 24 BACK TO MAIN 3M Medical Department Study: T-6316.7 Analytical Report: FACT TOX-098 LRN-U2402 PrOtOCOl LRN42402 Amendment Number I 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 AMENDTO 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 tiefore acceptance of the finalreport. When the final report is completed, all original paper dlta, 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 correspondingtrainingrecords, calibrationrecords, instrument maintenance logs, standard operating procedures, equipment procedures, and methlods will be retained in the archives of the facility performing each analysis." REASON: To direct subcontract laboratories in the disposition of The items listed above. 3 M 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. AMENDTO 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 Environmental Laboratory upon completion of analysis and submission of the sub-contract laboratory(s) finaI report. The specimenswill 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 specinens analyzed at subcontract laboratories. 6. PROTOCOL READS: Method numbers and revisions: FACT-M- 1.O,Extraction of Potassium Perfluorooctani:sulfonate or Other Anionic Surfactants from Liver for Analysis Using HF'LC-ElectrosprayMass Spectrometry. FACT-M-2.0, Analysis of Fluorochemicalsin Liver E:ctractsUsing WLC-Electrospray/ Mass Spectrometry FACT-M3.1 ,Extraction of Potassium Perfluorooctanizsulfonateor Other Anionic Surfactantsfrom Serumor Other Fluid for Analysis Using HPLC-Electrospray/ Mass Spectrometry FACT-M-4.1, Analysis of Fluorochemicals in Sen& or Other Fluid Extracts Using HPLCElectrospray/Mass Spectrometry AMENDTO READ: Method numbers and revisions: ETS-8-6.0 "Extraction of Potassium Perfluorooctanesulfonateor Olher Fluorochemical Compounds from Liver for Analysis Using HPLC-Electrospray/Mass Spectrometry" ETS-8-7.0 "Analysis ofPotassium Perfluorooctanesulifonate or Other Fluorochemical Compounds in Liver Extracts Using HPLC-ElectrosprayhfassSpectrometry" ETS-8-4.1,"Extraction of Potassium Perfluorooctanesulfonateor Other Fluorochemical Compounds from Serum for Analysis Using HPLC-Electrospray Mass Spectrometry" ETS-8-5.1,"Analysis of Potassium Perfluorooctanesulfonateor Other Fluorochemical Compounds in Serum Extracts HF'LC-ElectrosprayM,ssSpectrometry" REASON: New methodologies were implementedfollowingthe ,approvalof the original protocol for FACT Tox-098. 3M Environmental Laboratory 3M EnvironmentalLaboratory Page 26 3M Medical Department Study: T-6316.7 Amendment Approval BACK TO MAIN Analytical Report: FACT TOX-098 LRN-U2402 P ~ O C O LI RKU2402 Amendment Number I John L. Butenhofl Ph.D., Sponsor Representative Date Kristen J.Hansen, Ph.D., Outgoing Study Director Date T i h' Mahin T. Case, D.KM,Ph.D., Incoming Study Director /I?!=!!= Date 3 M 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) DevelopmentalToxicity Study of 2(N-Ethylprfluorooctstnesulfonamido)-ethanol in Rats PROTOCOLAMENDMENIT NO.2 Amendment Date: November 2 1,2000 Performing Laboratory 3M Environmental Technology & Safety Services 3M Environmental Laboratory 935 Bush Avenue St.Paul, MN 55106 Laboratory Project Idenljfication FACT TOX-098 ET&SS LRN-U240:!. 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 PrC~tOCOlFACT TOX-098. Amendment No. 2 This amendment modifiesthe following portion(s) of the protocol: 1. PROTOCOL READS: There is not a principal analyticalinvestigatorassigned for this study. AMENDTO READ: The role of principal analyticalinvestigatorfor the study was assignedto Kristen J. Hansen, Ph.D. as of the signing of this amendment. REASON: The role of principalanalyticalinvestigatorwas assigned in an effort to ensure compliancewith 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 &e q A 2 . y I John L. ButenhoB Ph.D., Sponsor gepresentative AEL Date &4 &;7-L J Marvin 1: Case, D.V.M., Ph.D., Study Director :L&C Date k j 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-Ethylperfluoro~tanesulfonamido)-ethanol in Rats PROTOCOL AMENDMENT NO. 3 Amendment Date: November 21,2000 Performing Labofiatory 3M Environmental Technology dk Safety Services 3M Environmental Latomtory 935 Bush Avenue St. Paul, MN 55106 Laboratory Project ldeirltification FACT TOX-098 ET&SS LRN-U2402 Argus Study: 418-011 3M Medical Department Study:T-6316.7 . 3M Environmental Laboretory 3M Environmental Laboratory Page 31 3M Medical Department Study: T-6316.7 BACK TO MAIN 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.OPurpose: 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. AMENDTO READ: 1.OPurpose: The analytical portion of this toxicity study is designed to evaluate the levels of potassium perfluorooctanesulfonate (PFOS), N-ethyl perfluorooctanesulfonamido ethyl alcohol (FAFOSE-OH),perfluorooctanesulfonylamido(ethy1)acetzi (PFOSAA), and perfluorooctanesulfonylamide (PFOSA) in the livers of the test systems, or other tissues and fluids as necessary. Perfluorooctanesulfonylethylamide (I'FOSEA) 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. PROTOCOLREAD,S: 3.1.1 Analytical referencesubstance: Potassium perfluorooctanesulfonate(PFOS),lot a 1 7 . AMENDTO READ: 3.1.1 Analytical reference substances: Potassiumpeffluorooctanesulforiate (PFOS),N-ethyl perfluorooctanesulfonamidoethyl alcohol (EtFOSE-OH), peffluorooctanesulfonylamido(ethy1)acetate (PFOSAA), and perfluorooctanesulfonylamide (PFOSA). REASON: Include the additional reference substances used. 3M Environmental Laboratory . .- Page 32 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. 3 John L: Butenhofi Ph.D., Sponsor Representative M /o Date ZOO? Marvin T. Case, D.V.M., PlrD., Study Director I Q L %/ Date 3M 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. I TOXOO98 (LIMSW2.402) Deviation type (Check one) 0 SOP OProtocol Document number FACT-M-2.1 X Method 0Equipment Procedure B other: 1 Datel.s,) of occurrence and 10106198 // . Required procedurelprocess: .. Section 11.1.: ..The average of two standard curves will be plotted by linear regression, not .. forced through zero, Actual procedurdprocess : Data was originally analyzed as described by the method. However, a.ftrthe analysis was complete, it was determined that applying a 1lX weighting to the curve dramatically improved method accuracy at the low end of the curve. The original data sets were reworked utilizing the _im-proved practice. Deviation written. Method revalidated utilizing improved practices. Original and reworked data are included with the raw data. The reworked data is reported in the iind results. Recorded by - -.______---I --- ---___ - The reworked data has a higher degree of accuracy thanthe original data, There is no adverse impact on the study. ~____---__- I _ _ - - - @-Lgw@- A 4 4 5?+4 %$' D,&; 1 inow C a c ~ Deviation No. (assigned by Stuiy Director or Roject Lead at the end of study or project) Attachment A: Record of Deviation 3M Environmental Laboratory ETS-4-8.0 1 off Page- DG li'h l!lW/ao Page 34 3M Medical Department Study: T-6316.7 Record of Deviaition 1. Identification BACK TO MAIN Analytical Report: FACT TOX-098 LRN-U2402 i 09/28/99 and 10/02/99 11. Description: Required Proceddprocess: - - I _ _ - - - - -------......- .-I-_.--_____I___________ 14.4.1 Matrix spike recoveries must be within +/-30% of the spiked concentration. - Actual Procedurdprocess: --- - --_I__. - I _ - - - - - - I - - - - - - - - - - OneI ma_ trix spik_ e in ser- a showed-__aI------.--hI--igher recovery for PPOS (152%--, -a_v_erag_e_1_3_7_%_)________.I__ And PFOSA (149Yiavera~e126%). Ill. Actions Taken: (such as amendment issued, SOP revision, etc.) _--- - - _ _ ~ _ _ - - - _ - - - I - This deviation was written.--T_h-e--s-ta-t~e-d--a-c-c-u_ra_c_y_of these data will be changed in the fina-l report to reflect these recoveries. --_--------------- _ . - - - - - I _ - -_l___-__ -- Authorized By (StudyDirector /Project Lead) /I --__---- TDate F o ET~S-4-8.0 (assigned by Study Director or Project Lead at the end of study or project) 3M EnvironmentalLaboratory Page 35 3M Medical Department Study: T-6316.7 BACK TO MAIN Analytical Report: FACT TOX-098 LRN-U2402 Record of Deviation 1. identification Deviation Type (Check one) B&GGZNUmber(S): 0 SOP 0Prot0c01 - ----- ---__-_.-.___-._l__l__I___ X Method 0Equipment Procedure 0 other: --__- ETS-8-7.0 _--- ----; Date(s) Iof occurrence: 1 Entire study I/. Description: ___________ - Required Proceddprocess: ____-- _____--------_- -l_l___ ---- - Section 13.1.6 describes the calculations that should be usedto convert extract concentrationto matrix concentration. Actual Procedurdprocess: - - - I - - - _ - _I I-___-.--.____ In order to accommodate purity information, the actual calculation used varied somewhat fiom that written in section 13.1.6. Two additional factors, sal1 correction and standard purity, were added. The first accommodates the mass difference betw1:en the analytical standard (C8F17S03K) and the target analyte (CSF17S03-), while: the second addresses the purity of the analytical reference material. determined after the study was completed. ~ Recorded By _- - IDate . I-._ I__-____ I @I /UW/UU I N.Impact on Study/ Project The updated calculations accommodate new information and are an improvement. No adverse affect on the study. k J L . : - 'Z'Ld/pD _____________~-----_--____*------I_ --- * I____-_.____-I-___._ ____ - Authorized By (StudyDirector /Project Lead) TDate 3M EnvironmentalLaboratory Page 36 3M Medical Department Study: T-6316.7 BACK TO MAIN Analytical Report: FACT TOX-098 LRN-U2402 ~~ Appendix C: Extraction and Analytical Metlhods This appendix includes the following methods: Preparatory Methods FACT-M-1.O, Extractionof Potassium Perfluorooctanesulfonateor Other Anionic FluorochemicalSurfactantsfrom Liver for Analysis Using HPLC-Electrospray/Mass Spectrometry, (8 pages) FACT-M-3.1, Extractionof Potassium Perfluorooctaneor Other Anionic Fluorochemical Compounds from Serum or Other Fluids for Analysis Using HPLC-Electrospray/Mass Spectrometry, (17 pages) ETS-8-4.1, Extractionof Potassium Perfluorooctanesulfonateor Other Fluorochemical Compoundsfrom Serum for Analysis Using HPLC-Electrospray/MassSpectrometry, (14 pages) ETS-8-6.0,Extractionof Potassium Perfluorooctanesulfonateor Other Fluorochemical Compoundsfrom Liver for Analysis Using HPLC-Electrospray/MassSpectrometry",(14 pages) Analytical Methods FACT-M-2.0,Analysis of Fluorochemicalsin Liver ExtractsUsing HPLC-Electrospray/Mass Spectrometry, (8 pages) FACT-M-4.1, Analysis of Potassium Perfluorooctanesulfonateor Other Fluorochemicalsin Serum or Other Fluid ExtractsUsing HPLC-Electrospray/MassSpectrometry, (9 pages) ETS-8-5.1, Analysis of Potassium Perfluorooctanesulfonateor Other Fluorochemicalsin Serum Extracts Using HPLC-Electrospray/MassSpectrometry, (11 pages) ETS-8-7.0, Analysis of PotassiumPerfluorooctanesulfonateor Other Fluorochemicalsin Liver Extracts Using HPLC-Electrospray/MassSpectrometry, (12 pages) 3M Environmental Laboratory Page 37 3M Medical Department Study: T-6316.7 BACK TO MAIN Analytical Report: FACT TOX-098 LRN-U2402 3M ENVIRONMENTLAALBORATORY METHOD EXTRACTIOOFNPOTASSIUMPERFLUOROOCTANESULFONATEOR OTHER ANIONIC kUOROCHEMlcAL SURFACTANTSFROM LIVERFOR ANALYSIS USING HPLC-ELECTROSPRAYSWPE~CTROMETRY Method Number: FACT-M-1.0' Author': Lisa Clemen Approved By: Adoption Date: . 5 / 2 L / $ f RevisionDate: ~ / , 4 Group Leader r L T&hnical Reviewer 3/2b /4Q Date 512319 d Date 1.0 SCOPE AND APPLICATION 1.IScope: This method is for the extraction of Potassium Perfluorooctanesulfonate (PFOS) or other fluorochemical surfactants from liver. 1.2 Applicable Compounds: Fluorochemical surfactants or other fluorinated compounds. 1.3 Matrices: Rabbit, rat, bovine, and monkey livers or other livers i1s designated in the validation report. Microsoft 7.0.1195 FACT-M-1.O Extractionof PFOS fhrn Liver Page 1 of 8 3M Environmental Laboratory Page 38 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 how to extract potassium perfluorooctanesulfonate(PFOS)or other fluorochemicalsurfactimpfrom liver using ion pairing reagent and 5.0 mLs of ethyl acetate. An ion pairing reagent is added to each simple and partitioned into ethyl acetate, Four mLs of extract is removed to a centrifige tube and put onto a nitrogen evaporator until dry. Each extract is reconstituted in 1.O mL methanol then filtered through a 3 cc plastic syringe attachedto a 0.2 pfilter into glass autovials. 3.0 DEFINITIONS 3.1 None. 4.0 WARNINGS AND CAUTIONS 4.1 Health and Safety Warnings: 4.1.1 Use universal precautions when handling rinimal livers, they may contain pathogens. 5.0 INTERFERENCES 5.1 There are no h o r n interferencesat this time. 6.0 EQUIPMENT 6.1 The following equipment is used while carrying clut this method. Equivalent equipment is acceptable. 6.1.1 Ultra-Turrax 'I25 Grinder forgrinding liver samples 6.12 Vortex mixer, VWR,Vortex Genie2 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 of 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 .O Extraction of PFOS fiom 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 pL 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 Milli- Qm water. Rinse syringes a minimum of 9 times with methanol, 3 rinses fiom 3 separate vials. 8.0 REAGENTS AND STANDARDS 8.1 Reagents' 8.1.1 S20o0di&umramHsyNdraoOxHid.eP(oJu.rTBinatokear 1o0r0e0qumivLableenatk)e, r(NcoaOntHai)ni1nOgN5.0w0eliigtehrsap(Lpr)oMxiimllia-tQel& water, mix until all solids are dissolved. Store in a 1L rialgene bottle. 8.1.2 Sodium Hydroxide (J.T Baker or equivalent), (NaOH) 1". Dilute 1ON 1:10. Measure 10 mL of the 1ONNaOH solution into a 100mL volumetric flaskand dilute to volume using Milli-Qm water. Storein a 125ILnalgene bottle. 8.1.3 Tetrabutylammonium hydrogen sulfate (Kodak or equivalent), (TBA) 0.5M. W e i g approximately 169 grams of TBA into a 1 L volumetric containing 500 L Milli-Q water. Adjust to pH 10 using approlrimately 64mL 10NNaOH and dilute to volume with Milli-Qm water. Add NaOH slowly while adding the last 1mL 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 e m pH = 10. Adjust as needed using 1N NaOH solution. 8.1.4 Sodium CarbonatdSodiumBicarbonate Buffer (J.T.B&er or equivalent), (NqC03/NaHC03)0.25M:Weigh approximately 26.5 g of sodium carbonate (Na&O,) and 21.0 g of sodium bicarbonate (NaHCO,) into a 1 L volumetric flask and dilute to volume withMlli-Qm water. Store in a 1L 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 fiozen from testing laboratory. 8.1.9 Milli-Qm water, all water used in this melhod should be Milli-Qw 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 .GI 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 100mg of PFOS into a 100mL volumetric flask and record the actual weight. 8.2.3 Bring to volume with methanol for a stock standard of approximately 1000ppm (Pg/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 pprn. 8.2.6 Dilute the stock solution with methanol for a working standard3 solution of approx. 0.50 ppm. 9.0 SAMPLEHANDLING 9.1 All livers are received'fiozen and must be kept fiozen until the extraction is performed. 10.0 QUALITCYONTROL 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, w d l y a control liver. 10.13 Expected concentrations will fall in the mid-range of 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. 103.2 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 CALIBRATIOANND 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-QTMwater. Grind to a homogerteous solution. 11.1.2 If 40 g is not available, use appropriate aniounts of liveir 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.O 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 centrifugetube. Re-suspend homogeneous solution by shaking between aliquots while preparing a total of sixteen 1 mL aliquots of homogeneous solution in 15 mL centrifugetubes. 11.1.5 Two 1mL aliquots serve as matrix blanks. Use the standard concentrationsand spiking amounts listed in table 1 to spike, in duplicate, AVO standard curves for a total of fourteen samples. Table 1 Approximate Spiking Amounts for Callibration Standards I 1 WorkingStandard I PL I Approx.final-1 - (Approx. Conc.) - 0.50 ppm 4 I 0.50 DIXII a0.50 Dum I 1 20 1 40 I 0.100 uDm 5.0 ppm 20 11.1.1 See section 13.0 to calculate actual Concentrations of PFOS in calibration standards. 11.2 Extract spiked liver homogenates following 12.14-12.24 of this method. Use these standards to establisheach 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 d y s t 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. 3M Environmental Laboratory FACT-M-1.0 Extraction of PFOS from Liver Page 5 of 8 - Page 42 3M Medical Department Study: T-6316.7 BACK TO MAIN Analytical Report: FACT TOX-098 LRN-U2402 12.11Pipette 1 mL homogenate into a 15 mL po1ypropy:lenecentrihge tube, Label the centrifuge tube with the identical information as the sampuIe vial. (See Worksheet for documenting the remaining steps.) 12.12 Spike liver homogenates with the appropriate amount of PFOS standard as described in section 11.I or Table 1. 12.13 Pipette two 1mL aliquots of Milli-Q" water to centrifuge tubes. These will serve as instrument blanks. 12.14 Add 1mL 0.5 M TBA and 2 mL of the 0.25 M sociium carbonatdsodiumbicarbonate buffer. 12.15 Using a volumetric pipette, add 5 m L s ethyl acetate. 12.16 Cap each sample and put on the shaker for 20 minutes. 12.17 Centrifbge 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 centrifugetube. Label this fresh tube withthe same information as in 12.5. 12.19 Put each sample on the analytical nitrogen evaponttor until dry,approximately 2 to 3 hours. 1230Add 1.O mL of methanol to each centrifugetube using a gradmiled pipette., 12.21 Vortex mix for 30 seconds. 12.22 Attach a 0.2 pmnylon mesh filter to a 3 cc syringe and transfer the sample to this syringe. Filter into a 1.5 mL glass autovial. 12.23Label the autovial with the study number, animal ,numberand gender, sample timepoint, matrix, final solvent, extraction date, and analyst(li) who performed the extraction. 12.24 Cap andhold for electrospray mass spectrometry i d y s i s . 1235Complete the worksheet and tape to page of study notebook. 13.0 DATAANALYSIS AND CALCULATIONS 13.1 Calculations: 13.1.1 Calculate the density of liver (mg) in 1.O rLhomogenate using the following equation: R of Liver x Average weight of ten 1mL aliquots (mi9 (g of Liver + g of Water) 3M Environmental Laboratory FACT-M-1 .O 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 of PFOS in calibration standards using the following equation: pL of Standard x Concentration(WE/mL) = Final Concentration (pg/g or mg/kg) mg Liver'/ 1mL homogenate of PFOS in Liver *Averageweight of liver in solution as determinedin 13.1.1, by weighing ten 1 mL homogenates of approximately 40 mg; liver in 200 inL of Milli-Q water. 14.0 METHODPERFORMANCE 14.1 The method detection limit is equaltq halfthe lowest standard in the calibration curve. 15.0 POLLUTIONPREVENTION AND W A m E MANAGEM'ENT 15.1 Samplewaste is disposed in biohazard containers,flammablesolvent waste is disposed in high BTU containers, and used glass pipette waste is disposed in broken glass containers located in the laboratory. 16.0 RECORDS 16.1 Complete the extraction worksheet and tape into the study notebook. M. DATA TI 17.1 The validation report associatedwith thismethod is FACT-M-1.0 & 2.0-V-1. 18.0 REFERENCES 18.1 AMDT-EP-22, "RoutineMaintenanceof U l t r a - T m T-25" 19.0 AFFECTEDDOCUMENTS 19.1 FACT-M-2, "Analysisof Liver Extractsfor Fluorochemicalsusing HPLC-Electrospray Mass Spectrometry'' - 20.0 REVISIONS Revision Number. Reason For R e v i a - Revision Date 3M Environmental Laboratory FACT-M-1 .O Extraction of PFOS from Liver Page 7 of 8 ... Page 44 3M Medical Department Study: T-6316.7 BACK TO MAIN Analytical Report: FACT TOX-098 LRN-U2402 Extraction Worksheet for FACT-M-I Study # Sample Number PFOS set # #W #U' - H,O Blank Liver Blank - PFOS Date and ppm Initials #W - r I I - Blank ~~ ~ the is located. LiverHomopmate: S t d 6 ~ ~ Liver ExtractionMetbod L i v a I - amount K Date & Initials ppm. MS/MSD used sample . Cont. Checks used 3for a final concentrationof same homogenate as for std curve. FACT-M-1 .O Extraction of PFOS fiom 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 ENVIRONMENTLAALBORATORY METHOD EXTRACTIONOF POTASSIUM~ " E R ~ U O R O < ~ A N E S O N AORT OETHER FLUOROCHEMICALCOMPOUNDSFROM SERUMOR OTHER kW FOR ANALYSIS USING HPLC-ELECTROSPRAYD'IASS SPECTROMETRY Method Number: FACT-M-3.1 Adoption Date: 04/22/98 Author Lisa Clemen,Glenn Langenburg Revision Date: 10 1 a i I c18 Lab0 toryManager Date Technical Reviewer 9/28/98 Dite 1.0 SCOPE AND &PLICATION 1.1 Scope: This method is for the extraction of potassiumperfluorooctanesulfonate (PFOS) or other fluorochemical compounds from serum or other fluid. 1.2 Applicable compounds: Fluorochemical surfac:tants or other fluorinated compounds. 1.3 Matrices: Rabbit, rat, bovine, and monkey serum, rat whole blood, and rat milk curd. Word 6/95 3M Environmental Laboratory FACT-M-3.1 Extraction ofPFOS fromS e m rtnd 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 ffuorochemicals from serum, blood, or milk curd using an ion pairing reagent and 5.0 ml of 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 of extract are removed and put onto a nitrogen evaporator until dry. Each extract is reconstituted in 1.Oml of methanol, then filtered through a 3 cc plastic syringe attached to a 0.2 pni nylon filter into glass autovials. 3.0 DEFKNITIONS 3.1 PFOS:perfluorooctanesulfonate(anion of potassium salt) C,F,,,SO; 3.2 PFOSA: perfluomoctane sulfonylamide C$,,SObNH, 3.3 PFOSAA: perfluorooctane sulfonylamido (ethy1)acetate C,F,,SO,N(CH,CH,)CH,CO~ 3.4 EtFOSE-OH.2(N-ethylperfluomoctane sulfonanlido>ethyl alcohol c8F17s02N(C%CH3)C%C0H 3.5 POAA: perfluorooctanoate (anion of ammoniumsalt) C,Fl5CO0' 3.6 PFOSEA perfluomoctane sulfonyl ethylamide C!,F,,SO,N(CH,CH,)H 3.7 FC-807monoester C$,7S0,N(CH2CH,)~CH,0-P03H) 3.8 Surrogate standard: 1H-lH-2H-2H perfluorooctane sulfonic acid 4.0 WARNINGS AND CAUTIONS 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 knowninterferences at this time. 6.0 E~WPMENT 6.1 The f o l b v h g equipment is used while performing;thismethod. Equivalent equipment is acceptable. 6.1.1 Vortex mixer, VWR, Vortex Genie 2 6.1.2 Centrifbge, Mistral 1000 or IEC 6.1.3 Shaker, Eberbach or V W R 6.1.4 Nitrogen evaporator, Organomation 6.1.5 Balance (rt 0.100 g) FACT-M3.1 Extractionof PFOS from Serum or OtherFluid 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 disposablepipettes 7.3 Electronic pipettor, Eppendorf or equivalent 7.4 Graduated pipettes 7.5 Nalgene bottles, capable of 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 Centrifhge tubes, polypropylene, 15 mL 7.1 1 Labels 7.12 Syringes, capable of measuring 5 pL to 50 a . 7.13 Syringes, disposable plastic, 3 cc 7.14 Syringe filters, nylon, 0.2 pm, 25 mm 7.15 Timer Note: Prior to using glasswareand bottles, rinse 3 times With methanol and 3 times with Milli-Q" water. Rinse syringes a minimum of 9 times withmethanol, 3 rinses from 3 separate vials. 8.0 RJIAGENTS AND STANDARDS 8.1 Type I reagent grade water, Milli-Q" or equivalm%all water used in thismethod should be Milli-Qm water and may be provided by a Millli-Q TOC PlusTMsystem 8.2 Sodium hydroxide (NaOH), J.T Baker or equivalent 8.3 Tetrabutylainmoniumhydrogen sulfate(TBA),Kodak or equivalent 8.4 Sodium carbonate (N+C03), J.T.Baker or equivalent 8.5 Sodium bicarbonate (NaJXO,), 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 SpecialtyChemical Division), molecular weight = 538 8.10.2 PFOSA (3M Specialty Chemical Division), molecular weight = 499 FACT-M-3. I Extraction of PFOS fromSerum lor 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 (3MSpecialty Chemical Division),molecular weight =585 8.10.4 EtFOSE-OH(3M Specialty Chemical Division), molecular weight =571 8.10.5 POAA (3M SpecialtyChemical Division), molecular weight =431 8.10.6 PFOSEA (3M Specialty Chemical Division), molecular weight = 527 8.10.7 FC-807monoester (3MSpecialtyChemical Division). FC-807is a mixture of triester, diester, and monoester fluorocheinical components. The monoester molecular weight = 650 8.10.8 Surrogate standard 4-H, perfluorooctane sulfonic acid (1-H,l-H, 2-H, 2-H C$,,SO,H) molecular weight = 428 8.10.9 Other fluorochemicals,as appropriate 8.11 Reagent preparation 8.11.1 10 N sodium hydroxide WaOH): Weigh iipproximately 200 g NaOH. Pour into a 1000mL beaker containing 500 mL Milli-QTMwater, mix until all solids are dissolved. Storein a 1L Nalgene bottle. 8.11.2 1N sodium hydroxide (NaOH): Dilute 10N NaOH 1:lO. Measure 10 mL of 10N NaOH solutioninto a 100mL volumetric flaskand dilute to volume using MiX-Qm water. Store in a 125 mL Nalgene bottle. 8.11.3 0.5 M tetrabutyl&onium hydrogen sulhte (TBA): Weigh approximately 169 g of TBA into a 1 L volumetric containing 500 mL Milli-Qm water. Adjust to pH k 10usin approximately 44to 54 mL of 10N NaOH and dilute to volume with Milli-Q water. While adding the last m L of NaOH, add slowly because the pH changes abruptly. Store in a 1 L Nalgene bottle. 8.11.3.1 TBArequires a checkprior to each use to ensure pH = 10. Adjust as needed using 1 N NaOH solution. 8.11.4 0.25M sodium carbonate/sodiumbicarbonate buffer (Na$O,/NaHCO,): Weigh approximately26.5 g of sodium carbonate (Na$O,) and 21.O g of sodium bicarbonate (NaHCO,) int6 a 1L vo1umeik-k flask and bring to v. o.lume with MilliQmwater. Store in a 1L Nalgenebottle. 8.12 Standardspreparation 8.12.1 Prepare PFOS standards for the standard Ixrve. 8.12.2 Prepare other fluorochemicalstandards, 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.) 8.123 Weigh approximately100mg of PFOS into a 100ml volumetric flask and record the actual weight. 8.12.4 Bring to volume with methanol for a stock standard of approximately 1000ppm (PfYmU- 8.12.5 Dilute the stock solution with methanol fix a working standard 1 solution of approximately 50 ppm. - -- 3M Environmental Laboratory FACT-M3.1 Extraction of PFOS fiom Serum or Other Fluid Page 4 of 17 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 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 of surrogate standard 1-H,l-H, 2-H, 2-H, CsF,,S03Hinto a 50 mlvolumetric flaskand record the actual weight. 8.13.2 Bring to volume with methanol for a surrogate stock of approximately 1000-1200ppm. 8.133 Prepare a s m g a t e working standard. `Transferapproximately 0.5 ml of surrogate stock to a 50 ml volumetric flask and bring to volume with methanol for a working standard of 10-20 ppm. Record the actual volume transferred. 9.0 SAMPLHEANDLING 9.1 All samples are received h z e n and must be kept h z e nuntil !he extraction is performed. 10.0 OUALrrY CONTROL 10.1 Matrix blanks and method blanks 10.1.1 Extract two 1.0 mL aliquots of the appro])riatematrix (senunor blood, with blood samples diluted 1:1 withMilli-Qm water) followingthisprocedure and use as matrix blanks. See 11.1.4. 10.1.2 Extract two 1.0 ml aliquotsof MillGQ" water followixig 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 Prepate each spike using a sample chosen by the analyst, usually the control matrix received with each sample set. 10.2.3 Expected concentrationswill fall in themid-range ofthe initial calibration curve. Additional spikes may be included and may fall in the low-range of the initial calibration curve. 10.2.4 Prepare one matrix spike and matrix spike duplicate per 40 samples, with a minimumof 2 matrix spikes per batch. 10.3 Continuing calibration checks 10.3.1 Prepare and analyze continuing calibration check samples to ensure the accuracy ofthe initial calibration curve. If the peri:ent differencebetween the initial curve and the continuing check differ by >30%, re-analyze simples 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. - ___ -. 3M Environmental Laboratory FACT-M-3.1, Extractionof PFOSfrom Serum.or Other Fluid Page 5 of 17 Page 50 3M Medical Department Study: T-6316.7 BACK TO MAIN Analytical Report: FACT TOX-098 LRN-U2402 10.3.3 Prepare each continuingcalibration check: h m the same matrix used to prepare the initialcurve. 10.3.4 The expected concentrationwill fall Within the mid-range of the initial calibration curve. Additional spikes may be included that fall in b e low-range of the initial caliiration c w e . This is necessary if the analyst must quantitate using only the low end of the calibration curve (for example, 5 ppb - 100ppb, rather than 5 ppb - 1000ppb). 11.0 CALIBRATTONAND STANDARDIZATION 11.1 Prepare matrix calibration standards Note: Blood coagulates in air, therefore, minimize air contact until dilution. At this point, add TBA and buffer to each centrifugetube as in step 12.9, then add 1.0 mL of the diluted matrix sample to each tube. 11.1.1 Transfer1 mL of serum or 1mL ofblood (blood is diluted 1:l with Milli-Qm water)to a 15mL centrifugetube. The b loodis similar in compositionto milk curd and can be used in place of milk curd for standard curves when extracting that matrix. 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 below 0.50 mL of matrix. Record the sample volume on the extraction sheet. 11.1.3 While preparing a total of twenty aliquotsin 15mlcea%rifuge tubes, mix or shake between aliquots. 11.1.4 Two 1mL, aliquots,or other appropriate770lume, serve as matrix blanks. Typically use the standard concentrations and spiking amounts listed in Table 1, at the end of 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 reportsFACT-M-3.1-V-1and 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 aidincalculatingthe concentrations ofthe working standards. See Section 13.0 to calculateactual concentrationsof PFOS in calibration standards. 11.2 To each standard, blank, or QC check, add appropriate amount of surrogateworking standard for the concentrationto fall withinthe calibrationcurverange 5 ppb -1000 ppb. 11.3 Extract spiked matrix standards following 12.6-12.16 of this method. Use these standards , to establish each initial curve on the mass spectrometer. 3M Environmental Laboratory FACT-M3.1 Extraction of PFOS from Serum or Other Fluid Page 6 of 17 Page 51 3M Medical Department Study: T-6316.7 BACK TO MAIN Analytical Report: FACT TOX-098 LRN-U2402 Approximate spiking amounts for standards and spikes 12.0 PROCEDURE 12.1 Obtain f?ozen samples and allow to thaw. 12.2 Vortex mix for 15 seconds, thentransfer 1.0 mL or other appropriatevolume to a 15 mL polypropylenecentrihge tube. For blood samples,remove 0.5 mL and dilute to 1.OmL withMilli-QTMwater. 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 fieezer after extraction amount has been removed. I - -___ 3M EnvironmentalLaboratory FACT-M3.1 Extractionof PFOS from Serum or Other Fluid Page 7 of 17 Page 52 3M Medical DepartmentStudy: T-6316.7 BACK TO MAIN Analytical Report: FACT TOX-098 LRN-U2402 12.4 Record the volume on the extractionworksheet. The finalmethanol volume equals the volume transferredfrom 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 appropriateamount of standard as described in 11.1or 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 exhaction 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 of 0.25 M sodium carbonatelsodium bicarbonate bufer. 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 of organic layer, using a 5 mL graduated glass pipette, to a clean 15 xnL centrifugetube. Label this fkesh tube with the sarne informationas in 12.5. 12.14 Put each sample on the analytical nitrogenevaporator until dry,approximately 2 to 3 hours. 12.15 Add 1.0 mL, or other appropriate volume of methanol to each centrifige tube using a graduatedpipette. Methanolvolume to add equalsthe 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 syrin$e and transfer the sample to this syringe. Filter into a 1.5 mL glass autovial or lowvolume autovial when necessary. 12.18 Label the autovial with the study number, animal number and gender, sample timepoint, matrix, finalsolvent, extraction date, and analystl[s)performing the extraction. 12.19 Cap and storeextracts at approximately4 "Cuntil analysis. 12.20 Completethe extractionworksheet, attached to this document, and tape in the study notebook or include in studybinder, as appropriate. 3M Environmental Laboratory FAm-M3.1 Extraction of PFOS from Serum or Other Fluid Page 8 of 17 Page 53 3M Medical Department Study: T-6316.7 BACK TO MAIN Analytical Report: FACT TOX-098 LRN-U2402 13.0 DATAANALYSIASND CALCULATIONS 13.1 Calculations 13.1.1 Calculate actual concentrationsof PFOS,or other applicable fluorochemicd, in calibration standards using the following equation: mL, of standard x concentration of standard fua /mL) = - mL of standard+mLof surrogate standard +initialmatrix volume (d) Final Concentration(pg/mL) of PFOS in matrix 14.0 METHODPERFORMm a 14.1 The method detection limit (MDL) is analyte and matrix specific. Refer to MDL report for specific MDL and limit of quatitation (LOQvalues (see Attachments B and C). 14.2 The following quality control samples are extracted with each batch of samples to ensure the qualityof the extraction and analysis. 14.2.1 Method blanks and matrix blanks 14.2.2 Matrix spike and matrix spike duplicatesamples to detemine accuracy and precision of the extraction 14.23 Continuing calibration check samples to determine the continued accuracy of the initial calibration curve 15.0 POLLUTIONPREVENTIONAND WASTMEANAGEM~ENT 15.1 Samplewaste is disposed in biohazard containers, flammable solventwaste is disposed in high BTU containers, and used glass pipette waste is disposed in broken glass containers located in the laboratory. 16.0 RECORDS 16.1 Complete the extractionworksheet 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, h4DrJLOQ values 17.3 Attachment C, LOQ Summary 17.4 Attachment D, Calibration standard concentration worksheet 18.0 REFERENCES 18.1 The validation reports associatedwith this methad are FACT-RI-3,l & 4.1-V-1. ~ 3M EnvironmentalLaboratoly FACT-M-3.1 Extraction of PFOS from Serum or Other Fluid Page 9 of 17 Page 54 ~ ~- 3M Medical Department Study: T-6316.7 BACK TO MAIN Analytical Report: FACT TOX-098 LRN-U2402 1 19.1 FACT-M-4.1,"Analysis of Serum or OtherFluid Extracts for Fluorochemicalsusing HPLC-Electrospray Mass Spectrometry" 20.0 REVISIONS Revision Number 1 Reason For Revi:;& Validation of method to include 7 fluorochrmicals,an additionalmatrix, new API/MS(MS) systems, monkey senun cross validation, improvementsto ion pairing extraction,MIIL study, updates in record keeping and storing policies, etc. Revision Date 07/01/98 -. 3M Environmental Laboratory FACT-M3.1 Extraction ofPFOS from Serum or Other Fluid __ Page 10 of 17 Page 55 BACK TO MAIN 3M Medical Department Study: T-6316.7 Analytical Report: FACT TOX-098 LRN-U2402 [ Study# I Sample Number set # HZO Blank Blank I FC-Mk I approx. 0.5 ppm actual PPm #w approx. 5 ppm ;ctuaI: ppm 1 FC-Mix approx. 50 ppm ycd ppm 1I Dateand I Initials for Std.or Comments I I I I I I I I - I amount = I 1 I mL Date & Initials - ppm. MS/MSD used sample Surrogate Standard Spiked uL of a . Cont.Checks used same matrix as for std curve. ppm std ( ) to all samples, standards, and blanks Attachment A: Extractionworksheet FACT-M-3.1 Extraction of PFOS fromSerum or OtherFluid Page 1 1 of 17 .. -_ 3M Environmental Laboratory Page 56 3M Medical Department Study: T-6316.7 MDL/LOQ values for Rabbit Serum: BACK TO MAIN Analytical Report: FACT TOX-098 LRN-U2402 jntrations to be used for preparing the ates only. No validMDL was determinable fkom ationperfiimed for monoester wiU be an Pto FACT-M-3 . I & 4.1-V-1 for specifics. lMDLdLOQ values for Rat Serum: rogunate conceiitrations to be used for preparing the i t a only. No valid MDL was determinablefkom ttionperformed for monoester will be an r to FACT-M-3.1 & 4.1-V-1 for specifics. MDL/LOQ values for Bovine Serum: roximate concentrationsto be used for preparing the ites only. No valid MDL was determinable from Ition perfonned for monoester will be an r to FACT-M-3.1 & 4.1-V-1 for specifics. No data is available for MDL or LOQ in Monkey Serum. Use validated Linear Calibration Range instead. Please see Attachment C (LOQSummary)and MDL study in. FACT-M3.1 & 4.1-V-1 for specifics. Attachment A: Extraction worksheet FACT-M-3.1 Extraction ofPFOS from Serum or Other Fluid - 3M Environmental Laboratory Page 12 of 17 Page 57 3M Medical Department Study: T-6316.7 BACK TO MAIN Analytical Report: FACT TOX-098 LRN-U2402 MDLLOQ values for Monkey Serum: Compound MDL LOQ Linear Calibration .Range (LCR) PFOS (ppb) 1 1.38 I (ppb) Approximate conceintrations to be used for p- re-parin-g the Standard Calibration Curve I 4.39 I MDL andLOQ are estimatesonly. 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-1 for specifics. PFOSA PFOSAA 2.23 I 12.84 I I 7-09 MDL and LOQ are estimatesonly. No valid MDL was determinable from MDL study. Any quantitationperformed for PFOSA willbe an estimate only. Please refer to FACT-Mi3.1 & 4.1-V-1 for specifics. I 9-04 I 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-1 for specifics. EtFOSE-OH 3.90 12.4 MDL and LOQ BIZ estimates only. No valid MDL was determinable fkom MDL study. Any quantitationperformedfor EtFOSE-OH will be an estimateonly. Please refixto FACT-M-3.1 & 4.1-V-1 for specifics. POAA 4.3 1 PFOSEA I 1.09 13.7 I 3.48 MDL and LOQ arc estimates only. No valid MDL was determinable from I MDL study. Any quantitatimpexformedforPOAA will be an estimate only. Please refer to FACT-M-3.1 & 4.1-V-1 for specifics. I MDL and LOQ are estimates only. No valid MDL was determinable from MDL study. Any quantitationperformed for PFOSEA-OH willbe an estimate only. Please refm to FACT-M-3.1& 4.1-V-1 for specifics. only. No valid MDL was determinable from MDL study. Any quantitation perfomed for EtFOSE-OH will be an estirnate only. Please refi:r to FACT-M-3.1& 4.1-V-1 for specifics. rMDL/LOQ values for Rat Whole Blood: Compound 1 MDL [ LOQ I Linear Calibration ]Range ( L C R ) P roximate Concentrations to be used for preparing the ites only. No valid MDL was determinable fium antihtion performed for EtFOSE-OH will be an r to FACT-M-3.1 8c 4.1-V-1 for specifics. stes only. No valid MDL was determinable from ation performed for monoesterwillbe an :r to FACT-M-3.1& 4.1-V-1 for specifics. Please see Attachment C (LOQ Summary)and M D L study in FACT-M-3.1 & 4.1-V-1 for specifics. Attachment A ExtTaction worksheet FACT-M3.1 Extraction of PFOS from Serum or Other Fluid .._ . - 3M Environmental Laboratory Page 13 of 17 Page 58 3M Medical Department Study: T-6316.7 BACK TO MAIN Analytical Report: FACT TOX-098 LRN-U2402 Ion Pairing ExtractionofFluomchemicalsfrom Senun and Analysis by APIIMSWS) Summary Table: Limits ofQuantitation Compound PFOS Matrix Rabbit MDL 1.38 ppb Bovine Rat Monkey 2.1 1ppb 1.27 ppb dd PFOSA Rabbit Bovine 2.23 ppb 5.04 ppb PFOSAA Rat Monkey Rabbit 2.14 ppb dd 2.84 ppb Bovine Rat Monkey 2.34 ppb 2.32 ppb dd EtFOSE-OH Rabbit Bovine . 3.90 ppb 113 ppb POAA Rat . Monkey Rabbit 3.25 ppb dd 4.31 ppb PFOSEA Bovine Rat Monkey Rabbit Bovine Rat Monkey 4.64 ppb 1.20 ppb dd 1.03 ppb 3.71 ppb 1.84 ppb dd Monoester' Rabbit 149 ppb Bovine 149 ppb Rat 149 ppb Monkey dd 1. Values for monoesterarc estimatesonly. Compound PFOS LOQ 4.39 ppb 6.70 ppb 4.01 ppb xdd 7.00 ppb 16.0 ppb 6.8 1 ppb rdd 9.04 ppb 7.45 ppb 7.313 ppb rid 12.4 ppb 35.13 ppb 10.3 ppb dd 113.7 ppb 14.8 ppb 3.8 L ppb rdd 3.43 ppb 11.8 ppb 5.86 ppb rdd 474.0 ppb 474.0 ppb 474.0 ppb rdd Low std 5 PPb 25 ppb 10 PPb 25 PPb 10 PPb 25 PPb 25 ppb 25 ppb 10 ppb 263 ppb 10 ppb 25 ppb lSppb 50 PPb 50 PPb 10 ppb 15 PPb 5 PPb 5 PPb 5 PPb 25 ppb 5 PPb 10 ppb 5 PPb 250 ppb 250 ppb 250 ppb 100 ppb High std 1000 ppb 1000 ppb 1000 ppb 1000 ppb 1000 ppb 1000 ppb 1000 ppb 1000 ppb 1000 ppb 1000 ppb 1000 ppb 1000 ppb 1000 ppb 1000 ppb 1000 ppb 1OOOppb 750 ppb PPb 1000 ppb 1000 ppb 1000 ppb 1000 ppb 1000 ppb 1000 ppb 1000 ppb 1000 ppb 1000 ppb 1000 ppb Attachment C: LOQ Summary FACT-M3.1 ExtractionofPFOS from Serum or Other Fluid -3M Environmental Laboratoty Page 14 of 17 Page 59 3M Medical Department Study: T-6316.7 Compound.PFOSA BACK TO MAIN Analytical Report: FACT TOX-098 LRN-U2402 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 Compound P O L 4 BACK TO MAIN Analytical Report: FACT TOX-098 LRN-U2402 ComDound PFOSEA Cornpow Rabbit 4.94- 1450 I 9.78-978 4.94 - 1450 I 49.4 - \ - 7 1450 In general, he chromatographyfor the monoester was very1 poor (broad peaks, high baseline). Curves for monoester in rabbit and bovine were unacceptable. Any quantitationperformed with the monoester is only an estimate and should not be used for reliable, accurate data reporting. Attachment C:LOQ Summary - 3M Environmental Laboratory FACT-M-3.1 Extraction of PFOS fiom Serum or Other Fluid 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@): Standard number: Analytets): Equipmenl number: Sample matrix. Final solveint and 'I": Blank fluidlidentifier: Methodrevision: Target analyte(s): FC mix std approx. 0.500 ppm: W398-641 FC m k 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 concentrationsof standards in the FC mix )SEA Monoes ikedml. Volume Validated ranges - approximate concentrations Bovine Rat Monkey 25-1000 ppb 10-1000 ppb Estimates only. 25-1000 ppb 25-1000 ppb Use values for 263-1000 ppb 10-1000 ppb Rabbit EtFOSEOH 10-1000 ppb 5-1000 ppb 50-500 ppb ITl 10-750 ppb 25-1000 pb 5-1000 ppb 5-1000 ppb 5-1000 ppb 5-1000 ppb Attachment D: Ion Pair Standard Curves FACT-M3.1 Extraction of PFOS from Serum or Other Fluid 3M Environmental Laboratory Page 17 of 17 Page 62 3M Medical DepartmentStudy: T-6316.7 BACK TO MAIN Analytical Report: FACT TOX-098 LRN-U2402 3M ENVIRONMENTALLABORATORY EXTRACTION OF POTASSIUMPERFLUOROOCTANESULFONATEOR OTHER FLUOROCHEMICALCOMPOUNDS FROM SERUM FOR ANALYSIS USING HPLC- ELECTROSPRAYIRISAPS~S:CTROMETRY Method Number: ETS-8-4.1 Adoption Date: 03/01/99 Author: Lisa Clemen, Glenn Langenburg Revision Date: w 7 m Approved By: Laboratory Manager Date Group Leader Technical Reviewer Date QY/2b/q 4 Date 1.0 SCOPE AND APPLICATION 1.1 Scope: This method is for the extraction of potassium perfluorooctanesulfonate (PFOS) or other fluorochemical compounds h m serum. 1.2 Applicable compounds: Fluorochemical surfacRants or other fluorinated compounds. 1.3 Matrices: Rabbit rat, bovine, monkey, and hunian serum or other fluids as designated in the validation report. Word 6/95 3M EnvironmentalLaboratory ETS-8-4.1 Extraction ofPFOS from Serum Page 1 of 14 Page 63 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 fluorochemical surfactants from serum, or other fluids, using an ion pairing reagent and methyl-teri-butyl ether (M@E). In this method, seven fluorochemicalswere extracted. PFOS, PFOSA, PFOSAA, EtFOSE-OH,PFOSEA, M556,and surrogate standard (see 3.0 Definitiorrs). An ion pairing reagent is added to the sample and the analyte ion pair is partitioned into MBE. ` h e MtBE extract is removed and put onto a nitrogen evaporator until dry. Each extract is reconstituted in 1.O mL of methanol, then filtered through a 3 cc plastic syringe attached to a 0.2 p 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 of potassium salt) C,F,,SO; 3.2 PFOSA. perfluorooctane sulfonylamide C$,,SO,NIE, 3.3 P F O S U perfluorooctane sulfonylamido (ethy [)acetateC$,,S02N(CH,CH,)CH~CO; 3.4 , EEOSE-OH: 2(N-ethylperfluorooctanesulfonamido)-ethyl C$ ,SO,N(CH,CH,)CH,CH,OH alcohol 3.5 PFOSEA. perfluorooctane sulfonyl ethylamide C,F,,SO,N(CH,CH,)H 3.6 M556:C8F,,SO,N(H)(CH,COOH) 3.7 Surrogate standard lH-lH-2H-2H perfluorooclme sulfonic sicid 4.0 WARNINGS AND CAUTIONS 4.1 Health and safety warnings 4.1.1 Use Universalprecautions, especially laboratory coats, goggles, and gloveswhen 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 perfommg 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 (k 0.100 g) 7.0 SUPPLIES AND MATERIALS 7.1 Gloves 7.2 Eppendarfor disposable pipettes 7.3 Nalgene bottles, capable of holding 250 mL and 1L 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 ofmeasuring 5 pL to 50 pL 7.10 Graduated pipettes 7.11 Syringes, disposable plastic, 3 cc 7.12 Syringe filters, nylon, 0.2 p,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 Mil1i-Qm water. Rinse syringes a minimum of 9 times with methanol, 3 rinses fiom 3 separate vials. 8.0 REAGENTS AND STANDARDS 8.1 Type I reagent grade water, Milli-Qm or equivalent;dl water used in this methodshould be Milli-Qm water and may be provided by a Milli-Q TOC Phism system 8.2 Sodiumhydroxide (NaOH), J.T Baker or equivalent 8.3 Tetxabutylammoniumhydrogen sulfate(TBA), Kodak or equivalent 8.4 Sodium carbonate (Na&O,), J.T.Baker or equivalent 8.5 Sodium bicarbonate (NaHCO,), J.T.Baker or equivalent 8.6 Methyl-T-Butyl Ether, Omnisolv, glass distilled or KPLC grade 8.7 Methanol, Omnisolv, glass distilled or HPLC wide 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 Extractionof 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(3MSpecialty Chemical Division), molecular weight = 570 8.9.5 PFOSEA (3M Specialty Chemical Division), molecular weight = 527 8.9.6 M556 (3M Specialty Chemical Division), molecular weight = 557 8.9.7 Surrogate standard: 4-H, perfluorooctane sulfonic acid Q-HJ-H, 2-H, 2-H CBF13S03Hm)olecular 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 10 N sodium hydroxide (NaOH): Weigh approximately 200 g NaOH. Pour into a 1000mL beaker containing 500mL Milli-Qm water, mix until all solids are dissolved. Store in a 1L Nalgene bottle. 8.10.2 1N sodium hydroxide (NaOH): Dilute 10N NaOH 1:10. Measure 10mL of 10 N NaOH solution into a 100 mL volumebfc flask and dilute to volume using Milli-QTMwater. Store in a 125 mL Nalgae bottle. 8.10.3 ' 0.5 M tetrabutylammonium hydrogen sulfate (TBA): Weigh approximately 169 g of TBA into a 1L volumetric containing 500mLMilli-QWwater. Adjust to pH 10using approximately 44to 54 mL of 10N NaOH (While adding the last mL of NaOH, add slowly because the pH changcs abruptly). Dilute to volume with Milli-Qm water. Store in a 1L Nalgene hottle. 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 carbonatdsodium bicarbonate buffer (Na&O3/NaJ4CO,): Weigh approximately 26.5 g of sodium carbonata (NhCO,) and 21.0 g of sodium bicarbonate (NaHCO,) into a 1L volumetric flask and bring to volume with MilliQm water. Store in a 1L 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 standardsare acceptable (for example, m e 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 100mg of PFOS into a 100mL volumetric flask and record the actual weight. 8.11.4 Bring to volume with methanol for a stoc8kstandard of approximately 1000ppm WmL). 8.11.5 Dilute the stock solution with methanol for a working :standard 1solution of approximately 50 ppm. 8.11.6 Dilute working standard 1 with methanol for a working st&dard 2 solution of approx. 5.0 ppm. ETS-8-4.1 Extractionof 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 , 8.12 8.11.7 Dilute working standard 1with methanol for a working standard 3 solution of approx. 0.50 ppm. Surrogate stock standard preparation 8.12.1 Weigh approximately50-60mg of surrogate standard 1.-H,1-H, 2-H, 2-H, C$,,S03H into a 50 mL volumetric flask and record the actual weight. 8.12.2 8.12.3 Bring to volume with methanol for a surrogate stock of approximately 1000-1200 PPm. Prepare a surmgateworking standard. Transfer approximately 1mL of surrogate stock to a 10mL volumetric flask and bring to volume withmethanol for a working standard of 100ppm. Record the actual volume transferred. 9.0 SAMPLEHANDLING 9.1 All samples are received fi-ozenand must be kept h z e n until the extractionis performed. 9.2 Allow samples to thaw to room temperature prior to extraction. 10.0 OUALITYCONTROL 10.1 Solvent Blanks, Method blanks and matrix blanks 10.1.1 An aliquot of 1.0 mL methanol is used as a solvent blank. 10.1.2 Extract two 1.0 mL aliquots of MilG-QT" water following this procedure and use as method blanks. 10.1.3 Extract two 1.0 mL aliquots of the serum following this procedure and use as matrix blanks. See 11.1.4. 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 simple chosen by the analyst, usually the control matrix received with each sample set. 10.2.3 Expected concentrationswill fall in the mid-range of the initial calibration curve. Additional spikes may be included and may fall in the low-range of the initial calibration curve. 10.2.4 Prepare one matrix spike and matrix spik.e duplicate pea 40 samples, with a minimum of 2 matrix spikes per batch. 10.3 Continuing calibration checks 10.3.1 Prepare continuing calibration check samples to ensure the accuracy of 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 ofPFOS frorn Serum Page 5 of 14 3M Environmental Laboratory Page 67 3M Medical Department Study: T-6316.7 BACK TO MAIN Analytical Report: FACT TOX-098 LRN-U2402 10.3.4 The expected concentrationswill 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 anidyst must quantitate using only the low end of the calibrationcurve ([forexample, 5 ppb - 100ppb, rather than 5 ppb - 1000 ppb). 11.o CALIBRATlONAND STANDARDIZATION 11.1 Prepare matrix calibration standards 11.1.1 Transfer 1mL of serum to a 15mT,centrifugetube. 11.1.2 Ifmost samplevolumes are less than 1.0 mL, extract standardswith 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 of twenty aliquots in 15mI,centrifugetubes, mix or shake between aliquots. 11.1.4 Two 1mL aliquots, or other appropri'ate volume, serve as matrix blanks. Typically use the standardconcentratiomi and spiking amounts listed in Table 1, at the end of this section, to spike, in duplicate, two standard curves, for a total of eighteen standards, two matrix blanks, arid two method blanks. 11.1.5 Refer to validation report ETS-8-4.0 & ETS-8-5.0-V-I, which lists the working ranges and the Linear CalibrationRange (LCR)for calibrationc w e s . 11.1.6 Use AttachmentD as an aid in calculatingthe concentrations of the working standards. See Section13.0 to calculate actual concentrations of PFOS in calibration standards. 11.2 To each standard, blank, or continuingcheck, add appropriateamount of surrogate working standard for the concentrationto fall within the calibrationcurve range 5 ppb - 1000 ppb. 113 Extract spiked matrix standards following 12.6-112.16 of this method. Use these standards to establish each initial curve on the mass spectrometer. 3M Environmental Laboratory ETS-8-4.1 Extraction of PFOS frwn Serum Page 6 of 14 Page 68 3M Medical Department Study: T-6316.7 BACK TO MAIN Analytical Report: FACT TOX-098 LRN-U2402 Approximate spiking amounts for standards and spikes Using 1.0 mL of matrix Working standard Approx. final conc. of 12.0 PROCEDURE 12.1 Obtain frozen samples and allow to thaw at rooni temperature or in a lukewarm waterbath. 12.2 Vortex mix for 15seconds, then transfer 1.O mL or other appropriate volume to a 15mL 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 d y s t initials. See attached worksheet for documenting the remaining steps. 12.6 Spike all samples,includingblanks and s,tandard.s, ready far extraction with surrogate standard as described in 11.2. 12.7 Spike each matrix with the appropriate amount of standard as described in 11.1, or Table 1in that section, for the calibration curve standards. Also prqme matrix spikes and continuing calibration standards. I 12.8 Vortex mix the standard curve samples, matrix spike samples, and continuing calibration samples for 15 seconds. I 12.9 Check to ensure the 0.5 M TBA reagent is at pH 10. If not, atljust accordingly. 12.10 To each sample, add 1mL 0.5 M TBA and 2ml, of 0.25Msodium carbonatelsodium bicarbonate buffer. 12.11 Using an Oxford Dispenser, add 5 mL methyl-ttrt-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 of 3.500rpm,or until layers are well separated. ETS-8-4.1 ExbactionofPFOS from Serum Page 7 of 14 3M EnvironmentalLaboratory Page 69 3M Medical Department Study: T-6316.7 BACK TO MAIN Analytical Report: FACT TOX-098 LRN-U2402 12.14 Label a fiesh 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 15mL centrifuge tube. 12.16 Put each sample on the analytical nitrogenevaporator until dry, approximately 1to 2 hours. 12.17 Add 1.OmL of methanol to each centrifige 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. FiIter into a 1.5 mL glass autovial or lo w-volume autovial when necessary. 12.20 Label the autovial with the study number, animal number and gender, sample timepoint, matrix, final solvent, extraction date, and analysf(s)performingthe extraction. 12.21 Cap and store extracts at room temperature or at approximately4 "Cuntil analysis. 12.22 Complete the extraction worksheet, attached to this document, and tape in the study notebook or include in study binder, as appropriate. 13.0 DATAANALYSIASND CALCULATIONS 13.1 Calculations 13.1.1 Calculate actual concentrations of PFOS,or other applicable fluorochemical, in calibration standards using the following equation: mL of standard x concentration of stanciard (UP /I&) - mL of standard + mL of surrogate standard + initial ma.trix volume (a) Final Concentration (pg/mL) of PFOS in matrix 14.0 METHODPERFORMANCE 14.1 The method detection limit (MDL) is d y t e and matrix specific. Refer to MDL report for specific MDL and l ~of qtuantitation (LOC!) values (see AttachmentsB and C). 14.2 The following quality control samples are extracted with each batch of samples to evaluate the quality of 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. 14.3 Refer to section 14 of ETS-8-5.1 for method perfcirmance 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 fioni Serum Page 8 of 14 3M Environmental Laboratory Page 70 3M Modical Department Study: T-6316.7 BACK TO MAIN Analytical Report: FACT TOX-098 LRN-U2402 16.0 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 ATTACHMENTS 17.1 Attachment A, Extraction worksheet 17.2 Attachment B, MDULOQ values and summary 17.3 Attachment C, Calibration standard cmcentration worksheet 18.0 REFERENCES 18.1 The validation report associated with this method is ETS-84.0 & 5.0-V-1. 18.2 FACT-M-3.1, "Analysis of Serumor Other Fluid.Extracts for Fluorochemicals using HPLC-Electrospray Mass Spectrometry" 19.0 AFFECTEDDOCUMENTS 19.1 ETS-8-5.1,"Analysis of Serum or OtherFluid Extracts for Flutorochemicalsusing HPLC-Electrospray Mass Spectrometry" 20.0 REVISIONS Revision Number 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 Finalvolume is 1.0 mL; not adjustedfor initial volumes less than 1.0 mL. - Revision Date 04/02/99 3M EnvironmentalLaboratory ETS-8-4.1 Extractionof PFOS from Serum Page 9 of 14 Page 71 3M Medical Department Study: T-6316.7 Extraction Worksheet EXS-84.1 F Study # Surrogate Std DatcSpiked/Analyst MSD -+---I BACK TO MAIN Analytical Report: FACT TOX-098 LRN-U2402 FC-Mix approx. 50 ppm actual ppm Comments +- I I I I *- I I I I --I amount = I mL I I Date & Initials mL Std. # Std. # TN-A- speed speed rature: TN-A- Vortex 30 sec. Filterusing a3cc B-D svnnge with a0.2ttrnfilterinto a 1.5 mL autosamplevial Cont. Cal. Verifications used same matrix as for std CUNC. Attachment A 3M Environmental Laboratory ETS-84.1 Extraction of PFOS from Serum Page 10 of 14 Page 72 3M Medical Department Study: T-6316.7 MDLLOQ values for rabbit serum BACK TO MAIN Analytical Report: FACT TOX-098 LRN-U2402 e used for preparing the asrna were not statistically determined. Two curves in each of these matrices were extracted and analyzed with the rabbit serum curvesto determineequivalence. Responsesin the rat, bovine,monkey, and human were equivalentto the rabbit responses, therefore, theirMDL and LOQ will be the same values as determinedin rabbit serum. Please see LOQ Summary andMDL atudy in ETS-84.0 & 5.0-V-1for further information. Attachment B: MDULOQ Summary .. 3M Environmental Laboratory ETS-8-4.1 Extraction of PFOS from Serum Page 11of 14 Page 73 3M Medical Department Study: T-6316.7 BACK TO MAIN Analytical Report: FACT TOX-098 LRN-U2402 Rabbit Serum Full Range LOW Curve High curve 1/X Prepared range of standards (PPW (nglmL) - 0.995 978 4.94 - 248 97.8- 978 - 0.995 978 Compound: PFOSA I Full Range Low Curve High curve 1/X - 0.993 976 - 4.93 97.6 24.8 - 976 0.993 - 976 LCR from CUNe (PPb) (ng/mL) 24.8 - 978 - 4.94 248 97.8 - 978 - 4.94 978 % Recovery 83-108 85-104 85-1015 94-111 curve (PPb) (ng/mL) - 4.93 976 4.93 - 97.6 24.8 - 978 - 4.93 976 Rang: 88-10f' 87-105 93-102 94-103 4.67-1 1.0 5.34-12.0 4.84-9.80 4.60-1 0.5 5.10-14.7 9.85-14.7 5.08-13.9 5.10-14.5 RabbitS m Full Range . Lowcurve High curve 1/X Preparedrange of standards 0 (ng/mL) 0.991 - 974 4.92 - 247 - 49.2 974 0.991 - 974 LCRfiom curve 0 (ndW - 24.7 974 9.74 - 247 - 97.4 974 - 9.74 974 %Recovery 97-107 85-108 95-115 4.18-10.6 6.38-'21.8 4.33-12.5 4.1 1-23.2 AttachmentB:h4DLnOQ Summary ETS-8-4.1 ExtractionofPFOS from Serum 3M Environmental Laboratory Page 12 of 14 Page 74 3M Medical Department Study: T-6316.7 Prepared range LCR from % RabbitSerum of standards curve (ppb) (ng/mL) (PPb) (ng/mL) Full Range 0.993 - 976 49.3 - 976 BACK TO MAIN Analytical Report: FACT TOX-098 LRN-U2402 Rabbit Serum Prepared range of standards (PPW (ndmL) FullRange 1 0.993-976 1 Low Cwve I 4.93-248 I High curve 49.3 - 976 1/X - 0.993 976 Rabbit Serum Full Range Prepared range of standards (PPW (ng/mL) 0.993 - 976 LCR from CWO (PPW (ndmL) 24A - 976 9.76-248 49.3 - 976 9.76 - 976 LCR from curve (ppb) (ng/mL) 24.8 - 976 % Recoirery -."-- I, 96-106 I 91-1113 ~- 1 10.1-16.2 -- I 11.8-19.5 10.2-1 8.2 % Recovery 88-106 4.82- 17.9 0.993 - 976 - 9.76 976 4.77-19.5 Attachment B: h4DULOQ Summary ETS-84.1 ExtractionofPFOS fiom Senun 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@): Standard number: Analyte(s): Equipment number: , Sample ma- Final solverit and TN: BIank fluidr'identifler: Methodlrevision: Target analyte(s): FC mix std approx. 0.500 ppm: FC mix std approx. 5.00 ppm: FC mix std approx. 50.0 ppm: Surrogatestd approx 100ppm: Actual concentrationsof standardsin the FC mix PFOS PFOSA PFOSAA EtFOSE PFOSEA Std conc Std conc Stdconc Stdconc Std conc Std conc Calculated concentrations of standards in the sample!matrix PFOS PFOSA PFOSAA EtFOSE PFOSEA M556 Surop1Final conc Final conc Final conc Final conc Final COnc Fhd conc Std MIIC Am't apiked Snrrogate Final conc 513 766 1017 Bovine Human Attachment C Ion Pair Standard Curves ETS-84.1 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 ENVIRONMENTLAALBORATORY EXTRACTION OF POTASSIUM PERFLUOROOCTANESULFONATEOR OTHER FLUOROCXEMICAL COMPOUNDS FROM LIVERFOR ANALYSIS USING HPLCELECTROSPRAYMASPSSE:CTROMETRY Method Number: ETS-8-6.0 Author: Lisa Clemen,Robert Wynne Approved By: Ad.option Date: RevisionDate: $k Moratory I&anaier U Date Technjcal Reviewer o.t/,q 199 Date 1.0 SCOPEAND APPLICATION 1.1 Scope: Thismethod is for the extractionof potassium perfluorooctanesulfonate(PFOS) or other fluorochemicalcompounds from liver. 1.2 ApplicableCompounds: Fluorochemical surfactants or other fluorinatedcompounds. 1.3 Matrices: Rabbit, rat, bovine, and monkey livers or other tissues as designated in the validation report. Word 6.0/95 ETS-8-6.0 Extractionof PFOS from Liver . Page 1 of 14 3M Environmental Laboratory Page 77 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 fluorochemical surfactants from liver, or other tissues, using an ion pairing reagent and methyl-tert-butyl ether @BE). 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 malyte ion pair is partitioned into MtBE. The MtBE extract is transferred to a c e n ~ f u gteube and put onto a nitrogen evaporator until dry. Each extract is reconstituted in 1.O mL methanol then filtered through a 3 cc plastic syringe attached to a 0.2 pmnylon filter into glass autovials. 2.2 These sample extracts are analyzed following method ETS-8-7.0 or other appropriate methods. 3.0 DEFINITIONS 3.1 PFOS: perfluorooctanesulfonate(anion of potassium salt) C,F,,SO, 3.2 PFOSA: perfluorooctane sulfonylamide C,F,,SO,NH, 3.3 P F O S U perfluorooctane sulfonylamido (ethy1)acetate C,F17S0,N(CH,CH,)CHzCOz 3.4 , EtFOSE-OH: 2(N-ethylperfluorooctane su1fonamiclo)-ethyl alcohol C,F 7SO~(CH,CH,)CH,C-&OH 3.5 PFOSEA perfluorooctane sulfonyl ethylamide C8~,7S0,N(CHzCX13)H 3.6 M556:C~17SOzN(H)(CH2COOH) 3.7 Surrogatestandard: 1H-lH-2H-2H perfluorooctane: sulfonic acid 4.0 WARNINGS AND CAUTIONS 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 EQUIPMENT 6.1 The following equipment is used while performhls this method. Equivalent equipment is ,acceptable. 6.1.1 Ultra-Tunax 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 ETS-8-6.0 Extractionof PFOS from Liver Page 2 of 14 3M EnvironmentalLaboratory Page 78 3M Medical Department Study: T-6316.7 BACK TO MAIN Analytical Report: FACT TOX-098 LRN-U2402 6.1.5 Nitrogen Evaporator, Organomation 6.1.6 Balance (sensitivity to 0.100 g) 7.0 SUPPLIESAND MATERIALS 7.1 Gloves 7.2 Dissecting scalpels 7.3 Eppendorf or disposable pipettes 7.4 Nalgene bottles, capable of holding 250 mL and 1I, 7.5 Volumetric flasks, glass, type A 7.6 I-CHEM vials, 40 mL glass 7.7 Plastic sarnpule vials, Wheaton, 6mL (or approprisrte size) 7.8 Centrifuge tubes, polypropylene, 15 rnL 7.9 Labels 7.10 Oxford Dispensor- 3.0 to 10.0 ml 7.11 Syringes, capable of 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 Milli- Qm water. Rinse syringes a minimUmof 9 timeswith methanol, 3 rinses from 3 separate Vials. 8.0 REAGENTASND STANDARDS 8.1 Type I reagentgrade water, Milli-Qm or equival'ent;all water used in this method should be M U Q Wwater and be providedby a Milli-Q TOC Plus- system 8.2 Sodium hydroxide (NaOH), J.TBaker or equivalent 8.3 Tetrabutylammonium hydrogen sulfate('TBA), K odak or equivalent 8.4 Sodium carbonate (N%CO,),J.T.Baker or equivalent 8.5 Sodium bicarbonate (NaHCO,), J.T.Baker or equivalent 8.6 Methyl-tert-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 Fluorochemical 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 Page 79 3M Medical Department Study: T-6316.7 BACK TO MAIN Analytical Report: FACT TOX-098 LRN-U2402 8.10.2 PFOSA (3M SpecialtyChemical 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, pduorooctane sulfonic acid (I-H,l-H, 2-H, 2-H CBF,,SO,H) molecular weight = 428 8.10.8 Other fluorochemicals, as appropriate 8.11 Reagent preparation NOTE: When preparing larger volumes than listed in reagent, standard, or m g a t e preparation, adjust accordingly. 8.11.1 10N sodium hydroxide (NaOH): Weigh a]pproximately200 g NaOH. Pour into a ' 1000mL beaker containing 500 mLMilli-Q" water, mix until all solids are dissolved. Storein a 1L Nalgene bottle. 8.11.2 1 N sodium hydroxide (NaOH): Dilute 10N NaOH 1:lO. Measure 10mL of 10 N NaOH solution into a 100 mL volumetric flask and dilute to volume using Milli-Q" water. Store in a 125 mL Nalgeme bottle. 8.11.3 0.5 M tetrabutylammonim hydrogen sulfate W A ) : Weigh approximately 169 g of TBA into a 1L volumetric containing 5100mL Milli-QTMwater. Adjust to pH 10using approximately 44 to 54 mL of 10N NaOH (While adding the last mL of NaOH, add slowly because the pH changes abruptly). Dilute to volume with Milli-Q" water. Storein a 1L Nalgene bottle. 8.11.3.1 TBA requires a check prior to each use to ensure pH = 10, Adjust as needed using 1N NaOH solution. 8.11.4 0.25 M sodium carbonatelsodium bicarbonate buffer (Na$O,/NaHCO,): Weigh approximately 26.5 g of sodium carbonate (Na$OJ and 21.O g of sodium bicarbonate(NaHCO,) into a 1L volumetric flask and hring to volume with MilliQm water. Store in a 1 L Nalgenebottle. 8.12 Standards preparation 8.12.1 Prepare PFOS standards for the standard curve. 8.12.2 Prepare other fluorochemical standards, as appropriate. Multicomponent fluorochemical standards are acceptable (or example, one working standard solution containing 1.00 ppm PFOS, 1.02 ppm PFOSA, 0.987 pprn PFOSAA, and 1.10 ppm EtFOSE-OH.) 8.12.3 Weigh approximately 100 mg of PFOS into a 100 mL volumetric flask and record the actual weight. 8.12.4 Bring to volume With methanol for a stock standard of approximately 1000ppm (CIdmL). 8.12.5 Dilute the stock solution with methanol for a working standard 1 solution of approximately 50 ppm. ETS-8-6.0 Extraction of PFOS from Liver Page 4 of 14 3M Environmental Laboratory Page 80 3M Medical Department Study: T-6316.7 BACK TO MAIN Analytical Report: FACT TOX-098 LRN-U2402 8.12.6 Dilute the stock solution with methanol for a working standard 2 solution of approx. 5 .O ppm. 8.12.7 Dilute the stock solution with methanol for a working standard 3 solution of approx. 0.50 ppm. 8.13 Surrogate stock standard preparation 8.13.1 Weigh approximately 50-60mg of m g i i t t e standard I-H,l-H, 2-H,2-H, C$,,SO,H into a 50 ml volumetric flask and record the actual weight. 8.13.2 8.13.3 Bring to volume with methanol for a surrclgate stock of approximately 1000-1200 PPm. Prepare a surrogate working standard. Transfer approximately 1.0 ml of surrogate stock to a 10mlvolumetric flask and bring to volume with methanol for a working standard of 10-20 ppm. Record the actual volume transferred. 9.0 SAMPLHEANDLING 9.1 All samples are received fiozen and must be kept frozen until the extraction is performed. 10.0 OUALITYCONTROL 10.1 Matrix blanks and method blanks 10.1.1 An aliquot of 1.O mL,methanol is used as a solvent blank. 10.1.2 Extract two 1.0 mL aliquots of Milli-Qm water following this procedure and use as method blanks. 10.1.3 Extract two 1.0mL aliquots of liverhomogenate following this procedure and use as matrix blanks. Refer to 11.1.6. 10.2 Matrix spikes 10.2.1 Prepare and analyze matrix spikeand matrix spikeduplicate samples to determine the accuracy of the extraction. 10.2.2 Prepare each spike using a sample choseni by the anaiyst, 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 spikesmay be included and may fall in the low-range of 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 calibrationverifications 10.3.1 Prepare continuing calibration verification samples to r:nsure the accuracy of the initial calibration curve. 103.2 Prepare, at a minimum, one continuing calibration verificationsample per group of 10 samples. For example, if a sample set = 34, four verifications are prepared and extracted. 3M Environmental Laboratory ETS-8-6.0 Extractionof PFOS from Liver Page 5 of 14 - __ Page 81 3M Medical Department Study: T-6316.7 BACK TO MAIN Analytical Report: FACT TOX-098 LRN-U2402 103.3 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 spikesmay be included that fall in the low-range of - the initial calibration curve. This is necessary if the analyst must quantitateusing only the low end of the calibration curve (For example, S ppb - 100ppb, rather than 5 ppb 1000 ppb). 11.0 CALIBRATIONAND STAN~~RDIZATION 11.1 Prepare matrix calibration standards 11.1.1 Weigh approximately 40 g of liver into a 250 mL Nalgene bottle containing 200 m L s Mils-QTMwater. 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 of solid liver tissue dispersed in 1.O mL of homogenate solution. 11.15 Add 1 mL of homogenateto a 15 dcentrifuge tube. Re-suspend solution by shakingbetween aliquots whileprepaxing a total of eighteen 1mL aliquotsof 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 of this section, to spike, in duplicate, two standard curves, for a total of eighteen samples, two matrix blanks, and two method bl;mks. 11.1.8 Refer to validation reports ETSS6.0 and ETS-8-7.0-V-1 or Attachment By which lists the working ranges and the Linear Calibration Range (LCR) for calibration curves. 11.1.9 Use Attachment C as an aid in calculating the concentrationsofthe working standards. .Referto 13.0to calculate actual concentrations of PFOS in calibration standards. 11.2 To each working standard, blank, or continuing velification, add appropriate amount of surrogateworking standard for the concentration to fall within the calibration curve range 5 ppb - 1OOOppb. 3M Environmental Laboratory ETS-8-6.0 ExtractionofPFOS ko:n 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 12.14-12.25of this method. Use these standards to establish each initial cuwe on the mass spectrometer. 1 Table 1 Approximate Spiking Amounts for Calibration Standards PFOS in liver .L 0.50 ppm ~ 0.50 pprn 0.50 ppm 5.0 ppm 5.0 ppm 5.0 ppm 50 PPm 10 20 40 10 20 30 4 1.OO pprn 12.0 PROCEDURE 12.1 Obtain frozen liver samples. 12.2 Cut approximately 1 g of liver using a dissecting soalpel. This part of the procedure is best performed quickly, not allowing the liver to thaw. . 123 Weigh the sample directly into a tared plastic samyle vial. 12.4 gecord the liver weight in'thestudy notebook. 12.5 Return unused liver portions to freezer. 12.6 Add 2.5 m L s of water to sampule vial. 12.7 Grind the sample. Put the grinder probe in the sarriple and grind for about 2 minutes, or until the sample is homogeneous. 12.8 Rinse the probe into the sample with 2.5 mLswatzr using a pipette. 12.9 Take the grinder apart and clean it with methanol after each sample. Refer to AMDT-EP22. 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 ofPFOS from Liver Page 7 of 14 Page 83 3M Medical Department Study: T-6316.7 BACK TO MAIN Analytical Report: FACT TOX-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 infomation as the sampule vial. Refer to attached worksheet for documenting the remaining steps. 12.12 Pipette two 1mL aliquots of Milli-Qm 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. Ifnot, adjust accordingly. 12.17 To each sample, add 1mL 0.5 M TBA and 2 mL of the 0.25 M r;odiumcarbonatdsodium bicarbonate buffer. 12.18 Using an Oxford Dispenser, add 5 mL methyl-ted-butyl ether. 12.19 Cap each sample and put on the shaker at a setting of 300 rpm, for 20 minutes. 12.20 Centrifuge for 20 to 25 minutes at a setting of 3500rpm, or until layers are well separated. 12.21 Label a fresh 15mL centrifuge tube with the same information as in 12.10. 12.22 Remove 4.0 mL of the organic layer to the fresh 1.5mL centrifuge tube. 12.23 Put each sample on the analytical nitrogen evaporator until dry, approximately 1to 2 hours. 12.24 Add 1.O mL to each centrifuge tube using a graduiitedpipette. 12.25 Vortex mix for 30 seconds. 12.26 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. 12.27 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, imd 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 DATAANALYSIASND CALCULATIONS 13.1 Calculations: 13.1.1 Calculate the average density of the liver homogenate by recording each mass of ten separate 1.OmL aliquots of homogenate. Average density (mg/mL) = Average mass (mdof the a l i m 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 densitv* of homogenate (mdmL') (g of Liver + g of Water) * refer to 13.1.1 for details. 13.1.3 Calculate actual concentrations of PFOS and other fluorochemicals in calibration standards using the following equation: pL of Standard x Concentration (UE/n1L1=Final Concentration (pglg or mg/kg) mg Liver/ I mL homogenate* of PFOS in Liver *referto 13.1.2 for details. 14.0 METHOD PERFORMANCE 14.1 The method detectionlimit (MDL)is d y t e and matrix specific. Refer to h4DL report for specific MDL and limit of quantitation (LOQ)valnes (refer to AttachmentsB and C). 14.2 The followkg quality control samples are extractedwith each batch of samples to evaluate the quality ofthe extraction and analysis. 14.2.1 Method blanks and matrix blanks. 14.2.2 Matrix spike and matrix spike duplicate samples to detc:rmine accuracy and . precision of the extraction. 14.23 Continuing calibration verification samples to determiriethe continued accuracy of the initial calibration curve. 14.3 Refer to section 14 of ETS-8-7.0 for method perfcmance criteria. ~ I E N T 15.1 Sample waste is disposed in biohazard containers, flammable solvent waste is disposed in high BTU containers, and used glass pipette wastc: is disposed in broken glass containers located in the laboratory. 3M Environmental Laboratory ETS-8-6.0 ExtractionofPFOS 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 16.0 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 TABLEDS,IAGRAMFSL, OWCHARATNSD. VALIDATION DATA 17.1 Attachment A, Extraction worksheet 17.2 Attachment B, MDWLOQ values and sumniary 17.3 Attachment C, Calibration standard calculation and concentrationworksheet 18.0 REFERENCES 18.1 The validation report associated with this method is ETS-8-6.0 & 7.0-V-1. 18.2 AMDT-EP-22, ``Routine Maintenance of Ultra-Tulrax T-25" 18.3 FACT-M-1.1, "Extraction of PFOS or Other Anionic Fluorochemical Surfactants from ' Liver for Analysis Using HPLC-Electrospray/Mass Spectrometry" 19.0 AFFECTEDDOCUMENTS 19.1 ETS-8-7.0, "Analysis of Liver Extracts for Fluorochemicals usuig HPLC-Electrospray Mass Spectrometry" 20.0 REVISIONS Revision Number. Reason For Revision Revision Date 3M Environmental Laboratory ETS-84.0 Extraction of PFOS from Liver Page 10 of 14 - Page 86 ~~ 3M Medical Department Study: T-6316.7 Date SDiked/halYst BACK TO MAIN I Analytical Report: FACT TOX-098 LRN-U2402 - FC Mix Std actual ppm FC Mix Std approx. 50 ppm actual ppm # Comments I I I I - - I Date & Initials Attachment B: MDLLOQ Values 3M Environmental Laboratory ETS-8-6.0 Extraction of PFOS froin Liver Page 1 1 of 16 ~~ Page 87 3M Medical Department Study: T-6316.7 BACK TO MAIN Analytical Report: FACT TOX-098 LRN-U2402 Compound PFOS PFOSA PFOSAA EtFOSE-OH M556 PFOSEA MDL (ppb) 8.45 3.50 24.6 108 82.3 33.9 LOQ (ppb) 26.9 11.1 78.3 i 345 262 I 108 Linear Calibration Range (LCR) Approximate concentrationsto be used for preparing the Standard Calibratioii Curve 30 ppb - 1200 ppb - 12ppb 1200ppb 30 ppb - 1200ppb 60 ppb - 900 pPb* i 60pib-1206ppb I 3 0 P ~ b 1- 2001~1b ~ M D U O Q values in rat, bovine, and monkey liver were not statisticallydetermined. Two curves in each of these matriceswere extracted and analyzedwith 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 equivalentto those values as determined for the rabbit liver. Refer to LOQ Summary and MDL studyin ETS-8-6.0 & 7.0-V-1for finther information * EtFOSE-OH estimates only for MDL and LOQ. Did not meet criteria for validation. I Liver range of average ::; I- curve we highstd . ,'.hi@ Rabbit 6.19 - 1237 12 - 1200 l2--'1200..3 12 - 300 12-300 60- 1200 -'60 1200 Attachment B: MDULOQ Values 3M Environmental Laboratory ETS-8-6.0 Extractionof PFOS from Liver 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 I I Prepared 1 Rangeof LCRfrom 1 RangeoF L C R h m I Rangeof LCRfrom Attachment C Standard Calculations ETS-8-6.0 Extractionof PFOS fro,mLiver 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: Methodhevision: Target analyte(s): FC mix std approx. 0.500 ppm: FC mix std approx, 5.00 ppm: FC mix std approx. 50.0 ppm: Surrogatestd approx. 100ppm: Standard number: Equipment immber:' Final solvent and TN: Blank liver/identifier: Actual concentrations of standardsin the FC mix Calculatedconcentrationsof standards in the sample!matrix * PFOS PFOSA PFOSAA EtFOSE PFOSEA Final Find Finalconc Final Final conc conc ndg conc conc c onc 299 599 898 1198 - Validated ranges approximate concentrations Rabbit Bovine 100 Surrogate Final conc nglmL 0.500 spiked mL 0.005 Attachment C: Standard Calculations ETS-8-6.0 Extraction of PFOS from Liver 3M Environmental Laboratory Page 14 of 14 Page 90 3M Medical Department Study: T-6316.7 BACK TO MAIN Analytical Report: FACT TOX-098 LRN-U2402 3M ENVIRONMENTLAALBORATORY METHOD ANALYSIS OF FLUOROCHEMICIANLLSJVER EXTRACTUSING HPLC-ELECXROSPRAY/MASSPSECTROME:TRY Method Number: FACT-M-2.0 Author: Lisa Clemen Approved By: &w - Laboratory Manager Adoption Date: 5/J& 21 Revision Date: li, Date &SA h Technical Reviewer 5-/a7/sa Date 1.0 SCOPEAND APPLICATION 1.1 Scope: This method is for the analysis of extracts of liver or other tissues for fluorochemical surfactants using HPLC-electrospray/mas spectromt:try. 1.2 Applicable Compounds: Potassium perfluorooctanesulfonate, anionic fluorochemical surfactants, or other ionizable compounds. 1.3 Matrices: Rabbit, rat, bovine, and mobkey livers or other livers a13 designated in the validation report. Word 7.0.1195 FACT-M-2.0 Analysis of Liver Extract Using ESMS Page 1 of 8 3M Environmental Laboratory Page 91 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 analysis of fluorochemicad 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 WARNINGSAND 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 PROIBE, 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 400bar, the HP1100 will initiate automatic shutdown. 4.2.2 Do not run solvent pumps to dryness. 5.0 INTERFERENCES 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 EQUIPMENT 6.1 Equipment listed below may be changed in order to optimize the system. 6.1.1 Micromass Electrospmy Mass Spectrometer 6.1.2 HP1100 low pulse solvent pumping system and autosampler. 7.0 SUPPLIES AND MATERIALS 7.1 Supplies 7.1.1 Nitrogen gas, refrigerated liquid, regulated l o approximately 100psi. 7.1.2 HPLC column, specifics to be determined by the analyst. 7.1.3 Capped autovials or capped 15 mL centrihge tubes. 8.0 REAGENTASND 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 ESMS Page 2 of 8 3M Environmental Laboratory Page 92 3M Medical Department Study: T-6316.7 BACK TO MAIN Analytical Report: FACT TOX-098 LRN-U2402 8.1.2 Milli-QTMwater, all water used in this metho,dshould be Milli-Qm 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 H,O blank, one livd blank, and seven liver hqandards are prepared during the extraction procedure. See FACT-M-l . 9.0 SAMPLHE ANDLING 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 refiigerated until analysis can be performed. 10.0 QUALITYCONTROL 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 of the initial calibration curve. 10.23 See section 13 to calculate percent recovery. 10.3 Continuing CalibrationChecks 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 he 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 CALIBRATIOANND STANDARDIZATION 11.1 Analyze the extracted liver standards prior to and following each set of extracts. The mean of two standard values, at each standard concentratj.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: FACT TOX-098 LRN-U2402 11.2 The ? value for the data should be 0.98 or greater. Lower values may be acceptable at the discretionof 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 PROCEDURES 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 firzit set of 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 butt 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.l. 12.2.2 Set-up the HPl lOO/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 Time MeOH 0.00 min. 45% 55% 7.5 min. 90% 10% 11.0 min. 90% 10% 11.5 min. 45% 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 "Startyybutton is pressed on the HP Workstation. 12.2.2.5 Press the "Start" button. FACT-M-2.0 Analysis of Liver Extract Using ESMS Page 4 of 8 3M Environmental Laboratory Page 94 3M Medical Department Study: T-6316.7 BACK TO MAIN Analytical Report: FACT TOX-098 LRN-U2402 12.3 Instrument Sep-up 12.3.1 Refer to AMDT-EP-31 for more details. 1233 Check the solvent level in reservoirs and refill if necessary. 12.3.3 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 thr: probe. Allow to equilibratefor 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: 12.3.6.1 Drying gas 250-400 literslhour - 12.3.6.2 ESI nebulizing gas 10-15 litedhour 123.6.3 LC constant flow mode flow rate 10 500 uL/min 12.3.6.4 Pressure 4 0 0bar (This parameter is not set, it is a guide to ensure the instrument 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 inthe instrument log. 123.9 Using the cross-flow counter electrode in the ESMS 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 DATAANALYSIS AND CALCULATIONS 13.1 Calculations: - 13.1.1 Calculate matrix spikepercent recoveries using the following equation: % Recovery = Observed Result Background Result x 100 Expected Result 1.3.1.2 Calculate percent differenceusing the following equation: YODifference = Expected Conc. - Calculated.@C Expected Conc. x 100 3M Environmental Laboratory FACT-M-2.0 Analysis of Liver Extract Using ESMS Page 5 of 8 Page 95 3M Medical Department Study: T-6316.7 BACK TO MAIN Analytical Report: FACT TOX-098 LRN-U2402 13.1.3 Calculate actual concentration of PFOS anion in total liver (mg): 14.0 METHODPERFORMANCE 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 1OWe:jtstandard in the calibration curve. 15.0 POLLU'MON PREVENnON AND WAsTE MANAGEMENT 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 RECORDS 16.1 Store chromatograms in the study folder. Each chmmatogratn should have the following informationincluded either in the header or hand written on the chromatogram: study number, sample name, extraction date, and dilution factor (if applicable). 16.2 Plot calibrationcurve by linear regression and store in the study folder. 16.3 Print sample list from MassLynx and tape into the ilnstrument 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 Suwnarize data using suitable s o h a r e 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 TABLESD,IAGRAMS, BLOWCHARTS, AND VALIDATION DATA 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-1. 18.0 REFERENCES 18.1 AMDT-EP-3 1,"Operation of VG Platform Electrospray Mass Spectrometer" 3M Environmental Laboratory FACT-M-2.0 Analysis of Liver Extract Using EWMS Page 6 of 8 Page 96 3M Medical Department Study: T-6316.7 BACK TO MAIN Analytical Report: FACT TOX-098 LRN-U2402 19.0 AFFECTEDDOCUMENTS 19.1 FACT-M-1.O, "Extraction of Potassium Peffluorooctanesulfonatefrom Liver for Analysis Using HPLC-Electrosprayhfass Spectrometry" 20.0 REVISlONS Revision Number. Reason For Revision - Revision Date 3M Environmental Laboratory FACT-M-2.0 Analysis of Liver Extract Using ESMS Page 7 of 8 Page 97 3M Medical Department Study: T-6316.7 Laboratory Study # Study: Test Material: Matrix/Final Solvent: MethodRevision: Analytical Equipment System Number: Instmment SoftwareNersion: Filename: R-SquaredValue: Slope: Y Intercept Date of Extraction/Analyst Date of AnalysidAnalyst: BACK TO MAIN Analytical Report: FACT TOX-098 LRN-U2402 Slope: Taken from linear regressionequation. GroupDose: 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 Conc. (ug/mL): Calculated by dividing the initial volume from the concentration 3M Environmental Laboratory FACT-M-2.0 Analysis of Liver Extract IJsing ESMS Page 8 of 8 Page 98 3M Medical Department Study: T-6316.7 BACK TO MAIN Analytical Report: FACT TOX-098 LRN-U2402 3M ENVIRONMENTLAALBORATORY METHOD ANALYSIS OF POTASSIUM ~ R F L U O R O 0 C T d ~ S U L F O N A TOER OTHER ~UOROCHEMICALSIN SERUMOR OTHERF.LUIDEXTRACTUSSING HPLC-EIXCTROSPRAYMASSPSECTROMETRY Method Number: FACT-M-4.1 Author: Lisa Clemen, Glenn Langenburg Approved By: p/72& Laboratory Manager &LA I$. Group Leader Adoption Date: 4/22/98 Revision Date: I O-I -9 Date 9/29/49 Date 1.o SCOPEAND APPLICATION 1.1 Scope: This methodis for the analysis of extractsfiom serum or blood for fluorochemical surfactantsusing HPLC-electrospray/masspectrometry. 1.2 Applicable Compounds: Fluorochemical surfactants or other fluorinatedcompounds,or other ionizable compounds. 1.3 Matrices: Rabbit, rat, bovine, or monkey serum and rat whole blood or milk curd. Word 6.0.1195 FACT-M-4.1 Analysis of Serum or Fluid Extract Using ESMS Page 1 of 9 3M Environmental Laboratoly Page 99 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 analysis of fluorochemical surfactants extracted from serum, whole blood, or milk curd using HFLC-electrospray/massspectrometry,or similar system as appropriate. The analysis is performed by monitoring a single ion characteristic of a particular fluorochemical, such as the potassium pedluorooctanesulfonate(PFOS) anion, WZ= 499. Samplesmay also be analyzed using an .4pI/MS/MSsystem to further verify compound identification. 3.0 DEFINITIONS 3.1 Atmospheric Pressure Ionization (MI): The Micronlassplatform systems allow for various methods of 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 ionizationprocess in these techniques occurs at atmospheric pressure (i.e. not under a vacuum). 3.2 Electrospray Ionization (ES, ESI): a method of ionizationperformed at atmospheric pressure, whereby ionizationoccurs through the production of tiny charged droplets in a strong electrical field. 3.3 Mass Spectrometry, Mass Spectrometer(M),Tandem Mass Spectrometer( M S M S ) : The A P I platforms are equippedwith quadrupole mass selectivedetectors. Ionsare selectivelydiscriminatedby mass to charge ratio ( d z ) and subsequently detected. A single MS may be employed for ion detection or a series (MS/MS) for more specific fiagmentation information. 3.4 Conventional vs. %spray probe interface: The latest models of Micromass platform systems (post 1998) utilize a "2-spi-a~c'o~nformation. The spray emitted fiom 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 of operation, cleaning, and maintenance are the same. However, Z-spray components and conventional components are not compatible with one another, but only with similarsystems (i.e. Z-spray Components are compatiblewith other Z - spray systems, etc.) 3.5 Mass Lynx Software: System software designed for the specific operationof 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 11or Quattro 11MassLynx or MassLynx NT USER'S GUIDE). 4.0 WARNINGASND CAUTIONS 4.1 Health and Safety Warnings: 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 6.0.1195 FACT-M4.1 Analysis of Serum or Fluid Extract Using ES/MS Page 2 of 9 3M Environmental Laboratory Page 100 3M Medical DepartmentStudy: T-6316.7 BACK TO MAIN Analytical Report: FACT TOX-098 LRN-U2402 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 1100will initiate automatic shutdown. 4.2.2 Do not run solvent pumps to dryness. 5.0 INTERFERENCES 5.1 To minimize interferencesw h q analyzing samples for perfluorooctanoate(POAA), teflon should not be used for sample storage or any part of instrumentation that comes in contact with the sample or extract. 6.0 EOUIPMENT 6.1 Equipment listed below may be modified in order to optimize the system. 6.1.1 Micromass Electrospray Mass Spectrometer . 6.1.2 HPllOO low pulse solvent pumping system and autosampler 7.0 SUPPLIES AM)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 dettmnined by the analyst 7.1.3 Capped autovials or capped 15ml centrifuge tubes 8.0 REAGENTSAND STANDARDS 8.1 Reagents 8.1.1 Methanol, HPLC grade or equivalent 8.1.2 Milli-Qm water, all water used in this methosd should be Mila-Q"" 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 blarlk, and ten matrix standards are prepared during the extraction procedure. See FACT-M-3.1. 9.0 SAMPLEHANDLING 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 ;samplescan be refrigerated at approximately 4 O C until analysis can be performed. FACT-M-4.1 Analysis of Serum or Fluid Extract Using ES/MS 'Page3 o f 9 3M EnvironmentalLaboratory Page 101 3 M Medical Department Study: T-6316.7 BACK TO MAIN Analytical Report: FACT TOX-098 LRN-U2402 10.0 OUALITY CONTROL 10.1 Method Blanks and Matrix Blanks 10.1.1 Analyze a method blank and a matrix blank prior to each calibration curve. 10.2 Matrix Spikes 10.2.1 Analyze a matrix spikeand matrix spike duplicate per forty samples. With a minimm of 2 spikes per batch. 10.2.2 Expected spike concentrations will fall in the mid-range of the initial calibration curve. Additional spike concentrationsmay fall in the low-range of 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 standardcurve, 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 CALIBRATIOANND STANDARDIZATION 11.1 Analyze the extracted matrix standards prior to and rollowing each set of extracts. The mean of two standard values, at each standard concentration, will be plotted by linear regression (?)for the calibration curve using MassLynx or other slfitable software. 11.2 The 2value for the data should be 0.980 or greater. Lower values may be acceptable at the discretion of the analyst and documented approval of the Project Lead. 11.3 If the curve does not meet requirements,performroutine maintenance or reextract the standard curve (if necessary) and reanalyze. 11.4 For purposes of accuracy when quantitating low levssls of analyte, it may be necessary to use the low end of the calibration curve rather thanthe full range of the standard curve. Example: when attempting to quantitate approximately 10ppb of'analyte, generate a calibration curve consisting of the standards h m 5 ppb to 100ppb rather than the full range of the curve (5 ppb to 1000ppb). This will reduce inaccuracy attributed to linear regression weighting of high concentration standards. 12.0 PROCEDURES 12.1 Acquisition Set up 12.1.1 Click on start button in the Acquisition Con1;rolPanel. 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. FACT-M-4.1 Analysis of Serum or Fluid Extract Using ESMS Page 4 of 9 3 M Environmental Laboratory Page 102 3M Medical DepartmentStudy: T-6316.7 I BACK TO MAIN Analytical Report: FACT TOX-098 LRN-U2402 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 appropriatemasses. A fill scan is usually collected along with the SIRs. Save acquisition method. If MSMS instruments are employed, additionalproduct ion fragmentation information may be collected. See Micromass MassLynx GUIDE TO DATA ACQUISITION for additional information and MRM (Multiple Reaction Monitoring). 12.1.3 Typicallythe analyticalbatch runsequenceklegins with a set of extractedma&x standards and ends with a set of extracted matrix standards. 12.1.4 Samples are analyzed with a continuing calihration 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 HP1lOO/autosamplerat the followingconditions 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 Injecdsample = 1 12.2.2.3 Cycle time = 15 minutes 12.2.2.4 Solvent ramp = 1 1 1 0.00min. 17.5min. 11.Omin. 11.5 min. 45% 55% Note: In this instrument configuration, the run must be set up on the electrospray software with a "Waitingfor 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.11if necessary. 12.3.3 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. FACT-M-4.1 Analysis of Serum or Fluid Extract Using ESMS Page 5 of 9 3M Environmental Laboratory Page 103 3M Medical DepartmentStudy: T-6316.7 BACK TO MAIN Analytical Report: FACT TOX-098 LRN-U2402 - 12.3.4 Set HPLC pump to ``On''S.et the flow to 10 500 Wmin 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 cxpelled 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 litendhour 12.3.6.2 ESI nebulizing gas 10-15 liters/how 12.3.6.3 HPLC constant flow mode flow rate: 10 - 500 pL/min 12.3.6.4 Pressure 4 0 0bar (Thisparameter is not set, it is a guide to ensure the HPLC is operating correctly.) 12.3.7 Carefully guide the probe into the opening. Insertprobe until it will not go any fkther. 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. 12.3.1OClick 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 startand end sample number includes all samples to be analyzed. 13.0 DATAANALYSIASND CALCULATIONS 13.1 Calculations: 13.1.4 Calculate matrix spike percent recoveries using the following equation: % Recovery = Observed Result - Backmound Result x 100 Expected Result 13.1.5 Calculate percent difference using the following equation: % Difference = Expected Conc. - Calculated Cog& x 100 Expected Conc. 13.1.6 Calculate actual concentration of PFOS, or other fluorochemical, in matrix (pg/ml): [ne of PFOS calc. from std. Curve x Dilution FaqM x 1 ug: [Initial Volume of matrix (ml) + ml oPSurrogate Standard) 1000 ng Final Volume (mL) 14.0 METHODPJCRFORMANCE 14.1 Method Detection Limit (MDL) and Limit of Quantitation (LOW are method, analyte, and matrix specific. Please see FACT-M3.1, Attachment A for a listing of current validated MDL and LOQ values. FACT-M-4.1 Analysis of Serum or Fluid Extract Using E S N S Page 6 of 9 3M Environmental Laboratory Page 104 3M Medical DepartmentStudy: T-6316.7 BACK TO MAIN Analytical Report: FACT TOX-098 LRN-U2402 14.2 Method Blanks and Matrix Blanks 14.2.1 Method blanks and matrix blanks will be andlyzed with each sample set for possible contamination or carryover. Values are expected to fall below the lowest standard in the calibration curve. 14.3 Matrix Spikes 14.3.1 Matrix spikes are analyzed with each sample set and the percent recoveries are expected to fall within f 30% of the spiked concentration. 14.4 Continuing Calibration Checks 14.4.1 Continuing calibration checks are analyzed a: a minimum of after every 10 samples with each sample set. The percent recoveries are expected to fall within f 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 80tha~ct~ions 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 POLLUTION PREVENTION 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 chromatogramsin 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, acquisitionmethod, 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 h m 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 TABLESD.IAGRAMFSL,OWCHARTASN,D VALIDATLONDATA 17.1 Attachment A. FACT-M4.1 Data reporting spreadsheet 17.2 The validation report associatedwith this method is FACT-M-3.1 & 4.1-V-1. FACT-M-4.1 Analysis of Serumor Fluid Extract Using ESMS Page 7 of 9 3M EnvironmentalLaboratory Page 105 -- 3M Medical Department Study: T-6316.7 BACK TO MAIN Analytical Report: FACT TOX-098 LRN42402 18.0 REFERENCES 18.1 FACT-EP3.0, "Operation and Maintenance of the Micromass Atmospheric Pressure IonizationlMass Spectrometer Platform Systems" 19.0 AFFECTEDDOCUMENTS 19.1 FACT-M3.1, ``Extraction ofPotassiumPerfluorooctanesulfonate or Other Fluorochemical Compounds from Serum or Fluid for Analysis Using HPLC-ElectrospraylMass Spectrometry" 20.0 REVISIONS Revision Number. Reason For Revision 1 Validationof method to include 7fluorocheml:calsaddition of whole blood matrix,surrogatestandard, new API/MS(MS) systems, monkey sera cross validation,MDL study, updates in ,recordkeeping and storing policies, etc. - Revision Date 07/01/98 3M Environmental Laboratory FACT-M-4.1 Analysis of Serum or Fluid Extract Using ESMS Page 8 of 9 Page 106 3M Medical Department Study: T-6316.7 Attachment A Laboratory Study ib Study: Test Material: M a M i n a l Solvent: MethodlRevision: Analytical Equipment System Number: Instrumat SoffwareNersion: Filename: R-Squared Value: Slope: Y Intercept: Date of ExtractiodAnalyst: Date of AnalysidAnalyst BACK TO MAIN Analytical Report: FACT TOX-098 LRN-U2402 GrouplDose: Taken from the study folder. Sample#: Taken h m the study folder. Concentration (uglmL): Taken from the MassLynx integration suinmary. Initial Volume (mL): Taken Erom the study folder. Dilution Factor: Taken from the study folder. Final Conc. (ug/mL): Calculated by dividing the initial volume frcm the c6ncentration 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 TOX-098 LRN-U2402 3M ENVIRONMENTLAALBORATORY ANALYSIS OF POTASSIUM ~RnUOROOC~TANESULFONATOER OTHER FLUOROCFJEMICALSINSERUMEXTRACTUSSING HPLC-ELECTROSPRAY/MAS!S SPECTROMETRY Method Number: ETS-8-5.1 Author Lisa Clemen, Robert Wynne Approved By: Laboratory Manager Group Leader qylLc Technical Reviewer Adoption Date: 03/01/99 Revision Date: qlabfl W2C, Date 4 / 2 4 /y9 Date dq 0y12 4 Date 1.0 SCOPE AND APPLICATION 1.1 Scope: This method describes the analysis of serum extracts for fluorochemicalsurfactants using HPLC-electrospray/mas spectrometry. 1.2 Applicabie Compounds: Fluorochemical surfactants or other fluorinated compounds, or other ionizable compounds. 1.3 Matrices: Rabbit, rat, bovine, monkey, and humm serum, or other fluids as designated in the validation report. Word 6/95 ETS-8-5.1 Analysis of Serum Extract:Using ESMS Page 1 of 9 3M Environmental Laboratory Page 108 3M Medical Department Study: T-6316.7 BACK TO MAIN Analytical Report: FACT TOX-098 LRN-U2402 2.0 SUMMARY OF METHOD 2.1 Thismethod describes the analysis of fluomchemical 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 fluorochemical, such as the perfluorooctanesulfonate(PFOS)anion, dz=499. Additionally, samples may be analyzed using a tandem mass spectrometerto furtherverify the identity of a compound by detecting daughter ions of the parent ion. 3.0 DEFIMTIONS 3.1 Atmospheric Pressure Ionization (API): The Micromass Quattro IItriple quadrupole systems allow for various methods of 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 Ihe gas phase via tiny charged droplets. These charged droplets are produced by the application of a strong,electricalfield.. 3.3 Mass Spectrometry, Mass Spectrometer(MS), Tandem Mass Spectrometer( M S M S ) : The API Quattro II triple quadrupole systems are equipped with quadrupole mass selective detectors. Ions are selectively discriminated by mass to charge ratio ( d z )and subsequently detected. A single MS may be employed for ion detection or a series ( M S / M S ) for more specific hgmentation information. 3.4 Conventional vs. Zspray probe interface: The latest models of Micromass Quattro 11 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, a.ferpassing 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 (is., Z-spraycomponentsare compatible with some other Z-spray systems, etc.) 3.5 Mass Lynx Software: System software designed fcir the specific operation of 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 IItriple quadrupoleMassLynx or MassLynxNT User'sGuide). 4.0 WARNINGS AND CAUTIONS 4.1 Health and Safety Warnings: 4.1.1 Use caution with the voltage cables for the probe. When engaged, the probe employs a voltage of 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 of9 3M Environmental Laboratory Page 109 3M Medical Department Study: T-6316.7 BACK TO MAIN Analytical Report: FACT TOX-098 LRN-U2402 4.2 Cautions: 4.2.1 Do not operate solvent pumps above capacj.ty of 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 INTERFERENCES 5.1 To minimize interferences when analyzing samples, teflon should not be used for sample storage or any part of idrumentation that comes i n contact withthe sample or extract. 6.0 EOIJIPMENT 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 MicromassQuathp IItriple quadrupole Mass Spectrometer equipped with an electrospray ionization source 6.1.2 Hpl 100 low pulse solvent pumping system, solvent degasser, column compartment, and autosampler 7 r 7.1 Supplies 7.1.1 High purity grade nitrogen gas regulated to approximately 100psi (House air system) 7.1.2 HPLC analytical column, specifics to be dtzterminedby the analyst and documented in the raw data. 7.1.3 Capped autovials or capped 15 dcentrifiigetubes 8.0 REAGENTS AND STANDARDS 8.1 Reagents 8.1.1 Methanol, HPLC grade or equivalent 8.1.2 Mlli-Q" water, all water used in this method should be:Milli-Qm 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 Standards 8.2.1 Typically two method blanks, two matrix blanks, and eighteen matrix standardsare prepared during the extraction procedure. See ETS-8-4.1. 9.0 SAMPLEHANDLING 9.1 Fresh matrix standards are prepared with each analysis. Extracted standards and samples are stored in capped autovials or capped 15 d centrifbge tubes until analysis. ETS-8-5.1 Analysis of Serum Extract IJsing ESMS Page 3 of 9 3M Environmental Laboratory Page 110 3M Medical Department Study: T-6316.7 BACK TO MAIN Analytical Report: FACT TOX-098 LRN-U2402 9.2 If analysis will be delayed, extracted standards and samples can be refiigerated at approximately 4" C, or at room temperature, until analysis can be performed. 10.0 OUALITYCONTROL 10.1 Solvent Blanks, Method Blanks and Matrix Blanks 10.1.1 Solvent blanks, method blanks and matrix lblanks are prepared and analyzed with each batch to determine contaminationor carryover. 10.1.2 Analyze a method blank and a matrix blanlc prior to each calibration curve. 10.2 Matrix Spikes 10.2.1 Matrix spikes are prepared and analyzedto 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 duplicateper forty samples,with a minimum of 2 spikes per batch. 10.2.4 Matrix spike and matrix spike duplicatecancentrationswill fall in the mid-range of the initial calibration curve. Additional spike concentrationsmay fall in the lowrange of the initial calibration curve. 10.3 C o n t h i n g Calibration Verifications i0.3.1 Continuingcalibrationverificationsare analyzedto veri@ the conhued accuracy of the calibration curve. 10.3.2 Analyze a mid-range calibration standard after every tenth sample, with a minimum of one per batch. 11.0 CALIBRATIONAND STANDARDIZATION 11.1 Analyze the extractedmatrix standards prior to and followingeach set ofextracts. The average of two standard curves will be plotted by linear regression (y = my f b), weighted l/x, not forced through zero,using MassLynx or other suitable software. 11.2 Ifthe curve does not meet requirements, perform routinemaintenance or reextract the standard curve (if necessary) and reanalyze. 11.3 For purposes of accuracy when quantitating low levels of analyte, it may be necessary to use the low end of the calibration curve rather than the full range of the standard curve. Example: when attemptingto quantitateapproximately 10ppb of analyte, generate a calibration curve consisting of the standards h m 5 ppb to 100ppb rather than the full range of the curve (5 ppb to 1000ppb). This will reduce inaccilracy attributedto linear regression weighting of high concentration standards. 3M Environmental Laboratory ETS-8-5.1 Analysis of Serum Extract 1Jsing ESlMS Page 4 of9 Page 111 3 M Medical Department Study: T-6316.7 BACK TO MAIN Analytical Report: FACT TOX-098 LRN-U2402 12.0 PROCEDURES 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 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 (Single Ion Recording) or MRM. Set 1:onizationMode as appropriate and mass to 499 or other appropriate masses. A full !%carist usually collected along with the SIRS.Save acquisition method. IfMS/MS instruments are employed, additional product ion fkagmentation informationmay be collected. See Micromass MassLyrur GUIDE TO DATA ACQUISITI[ON for additional information and MRM (Multiple Reaction Monitoring). 12.1.3 Typically the analytical batch runsequence:begins with a set of extracted matrix standards and ends with a set of extracted matrix standards. 12.1.4 Samples are analyzed with a conthuhg 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 HPl lOO/autosampler at the following conditions ox at conditions the analyst considers appropriate for optimal rixponse. Record actual conditions in the instrument logbook 12.23.1 Sample size = 10 pL injection 12.2.2.2 Injectlsample = 1 12.2.23 Cycle time = 13.5 minutes 12.2.2.4 Solvent ramp = I Time I MeOH1-0.2( Ammonium acetate pZ-pKjK-1 8.50 min. 12.0 min. 12.2.2.5 Press the "Start"button. 12.3 Instrument 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. 3 M Environmental Laboratory ETS-8-5.1 Analysis of SerumExtractUsing E S N S Page 5 of 9 Page 112 3M Medical Department Study: T-6316.7 BACK TO MAIN Analytical Report: FACT TOX-098 LRN-U2402 12.3.3 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. - 12.3.4 Set HPLC pump to "On".Set the flow to 1D 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 of 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 literdhow - 12.3.6.2 ESI nebulizing gas 10-15liters/hciur 12.3.63 HPLC constant flow mode, flow late 10 500 pL/rnin 123.6.4 Pressure 4 0 0bar (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 M e r . Connect the voltage cablesto the probe. 12.3.8 Print the tune 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 E S N S source is recommended for the analysis of biological matrices. 12.3.1OClick on start button in the Acquisition Control Panel (this may vary among MassLynx versions, see appropriate MassLynx USERS GUIDE). Press the start button. Ensure start and end sample number includes all samples to be analyzed. 13.0 DATAANALYSIASND CALCULATIONS 13.1 Calculations: 13.1.4 Calculate matrix spike percent recoveries ilsing the following equation: % Recovery = Observed Result - Backmund Result x 100 Expected Result 13.1.5 Calculate percent difference using the folliDwing equation: . - % Difference = ExDected Conc. Calculated- 1 Expected Conc. x 100 13.1.6 Calculate actual concentration of PFOS,or other fluorochemical, in matrix (vg/mL): (ng of PFOS calc. from std. Curve x Dilution Factor1 x 1 LLP (Initial Volume of matrix (mL) +mL of Surrogate Standard) 1000 ng Final Volume (d;) ETS-8-5.1 Analysis of Serum Extract IJsing ESlMS Page 6 of 9 3M Environmental Laboratoly Page 113 3M Medical Department Study: T-6316.7 BACK TO MAIN Analytical Report: FACT TOX-098 LRN-U2402 14.0 METHODPERFORMANCE 14.1 Method Detection Limit (MDL) and Limit of 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 3 value for the calibration curve must lie 0.980 or better. 14.4 Matrix Spikes 14.4.1 Matrix spike percent recoveries are must be Within f 30% of the spiked concentration. 14.5 Continuing Calibration Verifications 14.5.1 Continuing calibrationverificationpercent recoveries must be f 30% of the spiked concentration. 14.6 If criteria fisted in this method performance section isn't met,maintenance may be performed on the system and samples reanalyzed or other actions as determinedby 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 the report. 15.0 POLLUTION PREVENTION 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 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, acquisitionmethod, 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 l/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 S e m Extract Ilsing ESMS Page 7 of9 Page 114 3M Medical Department Study: T-6316.7 BACK TO MAIN Analytical Report: FACT TOX-098 LRN-U2402 16.5 Summarize data using suitable software (Excel 5.0:) and store in the study folder, see Attachment A for an example of a summary spreadsheet. 16.6 Back up electronic data to appropriate medium. Record in study notebook the filename and location of backup electronic data. 17.0 TABLESD. IAGRAMSFL.OWCHARTSA,ND VALIDATION DATA 17.1 Attachment A: ETS-8-5.1 Data summary spreadslneet. 18.0 REFFXENCES 18.1 FACT-M-4.1, "Extraction of Potassium Peduorooctanesulfonate or Other Fluorochemical compounds fkom Serum for Analysis Using HPLC-ElectrosprayMass Spectrometry 18.2 ETS-9-24.0, "Operation and Maintenance of the M:icromass Atmospheric Pressure IonizationlMass Spectrometer Quattro II triple quadrupole Systems" 18.3 The validation report associated withthis method is ETS-8-4.0 & 5.0-V-1. 19.0 AFFECTEDOCUMENTS 19.1 ETS-8-4.1, "Extraction of PotassiumPerfluorooctanesulfonateor Other Fluorochemical Compounds from Serum for Analysis Using HPLC-Electrosprayhfass Spectrometry" 20.0 REVISIONS Revision Number. 1 Reason For Revislion Section 6.1.2 Clarification of H P l l O O systemcomponents. Section 11.1 Average of two curves, not standard values, are .used for plotting linear regression and added the l/x weighting of the curve. Section 12.2.2.4 Clarification of solvent ramp. Section 17.1 Changed from attachment B to A. Revision 04/02/99 3M EnvironmentalLaboratory ETS-8-5.1 Analysis of Serum Extract Using ESlMS Page 8 of 9 Page 115 3M Medical Department Study: T-6316.7 Laboratory Study # BACK TO MAIN Analytical Report: FACT TOX-098 LRN-U2402 Group Dose Sample# Concentration ug/mL Initial Vol. mL Dilution Factor Final Conc. UglmL Attachment A, Summary Spreadsheet ETS-8-5.1 Analysis of Serum Extract lJsing ES/MS 3M Environmental Laboratoty Page 9 of 9 Page 116 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-8-5.1 "Analysis of Potassium Perfluolrooctanesulfonate or Other Fluorochemicalsin Sera Extracts Using IHPLC-Electsospray/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 caliition standard after every teath 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 minimumofone per batch. Section 14.5.1 Method reads: 14.5.1 Continuing calibrationverification percentrecoveries must be within k 30% of the spiked concentration. Modify method to read 14.5.1 At least one continuing caliition verificationper ten samplesmust show a percent recovery within +/-30% of the spiked concentration. Section 14.3.2 Method reads: NA Modifl method to read: 14.3.2 The second (bracketing) calibration curve may be deactivated if instmnientaldrift affectsthe data. The fmt curve and acceptable calibration checks shall bracket usable data. 3M Environmental Laboratory Page 117 3M Medical Department Study: T-6316.7 BACK TO MAIN Analytical Report: FACT TOX-098 LRN-U2402 Study #: FACT-TOX-098 Section 14.3.3 Method reads: NA Modify method to read: 14.3.3 Calibration standards withpeak areas less than 2 times the c w e matrix blank should be deactivated to disqualify a data range that may be affectlsd by background levels of the adyte. Section 14.3.4 Method reads: NA Modify method to read: 14.3.4 Low or high curve points may be deactivatedto optimize a linear range appropriateto the data. Section 14.3.5 Method reads: NA Modi@ method to read: 14.3.5 A curve point may be deactivated if it deviatesmore th;m 30% from the theoreticalvalue when the c w e is evaluated over a linearrange appropriateto the data. Section 14.3.6 Method reads: NA Modify method to read: 14.3.6 A valid calibration c w e must contain at least 5 active points. &-. /f- + Signature of PAI and date lI//$/d Sigrfature of Sponsor and date I /Tb.&= ZL7- G ,A& Si&itdre of Study Director and date L3A.4 k,' 3M Environmental Laboratory Page 118 3M Medical Department Study: T-6316.7 BACK TO MAIN Analytical Report: FACT TOX-098 LRN-U2402 3M ENVIRONMENTLAALBORATORY METHOD ANALYSIS OF POTASSIUM PERnuoROOC"ANEsULF0NATE OR OTHER F'LUOROCBEMICALS IN LIVEREXTRACTS USING HPLC-ELECI'ROSPRAYMASS SPECTROMETRY Method Number: ETS-8-7.0 Author: LisaClemen, Glenn Langenburg Approved By: Adoption Date: 0 7/21141 Revision Date: flf? Group Leader Date c Technical Reviewer Date 1.0 SCOPE AND APPLICATION 1.1 Scope: This method is for the analysis of liver extracts for fluorochemical surfactants using HPLC-electrospray/mass spectrometry. 1.2 Applicable Compounds: Fluorochemical surfactants or other fluorinated compounds, or other ionizable compounds. 1.3 Matrices: Rabbit, rat, bovine, monkey liver, or othta 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 BACK TO MAIN Analytical Report: FACT TOX-098 LRN-U2402 2.0 SUMMARY OF METHOD 2.1 This method describes the analysis of fluorochemical surfactants extracted from liver using WLC-electrospraylmasspectrometry, 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 spectrometerto furlherverify the identity of a compound by detecting daughter ions of the selected parent ion. 3.0 DEFINITIONS 3.1 Atmospheric Pressure Ionization (-1): The Micromass Q u a m 11triple quadrupole systems allow for various methods of ionization by utilizing various sources, probes, and interfaces. These include but are not limited to: 33lec:krospray Ioniiation (EST), Atmospheric Pressure chemical Ionization (APoI), Thermospray, etc. The ionization process in these techniques occurs at atmospheric pressure (i.e. not wader a vacuum). 3.2 Electrospray Ionization (ES, ESI):a method of ionization performed at atmospheric pressure, whereby ions in solution are transferredto the gas phase via tiny charged droplets. These charged droplets are produced by the application of a strong electrical field. 3.3 Mass SpectrometryyMass Spectrometer (MS), Tandem Mass Spectrometer ( M S M S ) : The AJ?I Quattro II triple quadrupole mass spectrometer is equipped with two quadrupole mass selective detectors and a collision cell. Ions an: selectively discriminated by mass to charge ratio (mlz) and subsequently detected. A single MS may be employed for ion detection or an ion may be selected in the first quadrupole, fragmented in the collision cell, and these hgments may be analyzed in the second quadrupole. 3.4 Conventional vs. Z-spray probe interface: The latest models ofMicromassQuattro 11 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 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 s . Z-spray components are compatiblewith other Z-spray systems,etc.) 3.5 Mass Lynx Software: System software designed for the specific operation of these Quattro I1 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 II triple quadrupole MassLynx or MassLynxNT User's Guide). 4.0 WARNINGS AND CAUTIONS 4.1 Health and Safety Warnings: 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 ExtractUsing ESMS Page 2 of 10 3M Environmental Laboratory Page 120 3M Medical Department Study: T-6316.7 BACK TO MAIN Analytical Report: FACT TOX-098 LRN-U2402 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 a back p~.essureof 400 bar (5800 psi). If the back pressure exceeds 400 bar, the H P l l O O will initiate automatic shutdown. 4.2.2 Do not run solvent pumps to dryness. * 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 EOUI'PMENT 6.1 Equipment listedbelow may be modified inorder to optimize the system. Document any modifications in the raw data as method deviationii. 6.1.1 Micromass Quattro 11triple quadrupole MiEs Spectrometerequipped with an electrospray ionization source. 6.1.2 HPl 100low pulse solvent pumping systenl,solvent degmser, column compartment, and autosampler 7.0 SUPPLIEASND MATERIALS 7.1 Supplies 7.1.1 High purity grade air regulated to approxkaately 100psi (house air system) 7.1.2 HPLC analyticalcolumn, specifics to be d1:termined 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-Q" water (ASTM type I), all water iised 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 volunietric container containing 2000 mLMilli-QR"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 l~lanksa,nd eighteen matrix standards are prepared during the extraction procedure. Refer to ETS-8-6.0. 9.0 SAMPLHEANDLING 9.1 Fresh matrix standards are prepared With each analysis. Extracted standards and samples are stored in capped autovialsor capped 15 ml centrifugetubes until analysis. 9.2 If 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 OUALITYCONTROL 10.1 Method Blanks and Matrix Blanks 10.1.1 Solvent blanks, method b l e s , 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 tcl 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 d y t e . 10.23 Analyze a matrix spike and matrix spike duplicateper f o e samplep. With a minimum of 2 spikes per batch. 10.2.4 Matrix spike and matrix spike duplicate ccmenbtionswill fall in the mid-range of the initial calibration curve. Additional spike concentrations may fall in the lowrange ofthe initial calibration curve. 10.3 Continuing CalibrationChecks :10.3.1 Continuing calibration verifications are analyzedto veri@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 CALIBRATION AND STANDARDIZATION 11.1 Analyze the extracted matrix standards prior to and following each set of sample extracts. The average of two standard curves will be plotted by linear regression (y =mx +b), weighted l/x,not forced throughthe origin, using MassLynx or other suitable software. 11.2 If the curve does not meet requirementsperform routine maintenance or reextract the standard curve (if necessary) and reanalyze. - -. 3M Environmental Laboratory ETS-8-7.0 Analysis of Liver Extract U:ring ESMS Page 4 of 10 Page 122 3M Medical Department Study: T-6316.7 BACK TO MAIN Analytical Report: FACT TOX-098 LRN-U2402 11.3 For purposes of accuracy when quantitating low lervels of 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 of analyte, generate a calibrationcurve consisting of the standards from5 ppb to 100ppb rather than the full range of the curve (5 ppb to 1000ppb). This will reduce inaccuracy attributed to linear regression weighting of high concentration standards. 12.0 PROCEDURES 12.1 AcquisitionSet up 12.1.1 Set up the sample list. 12.1.1.1 Assign a sample list filename using MO-DAY-last digit of year-increasing letter of the alphabet starting with a 12.1.1.2 Assign a method (MSfile) 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 ReactionMonitoring). Set IonizationMode as appropriate and mass to 499 or other appropriate masses. A full scan is usually collected along with the SIRS. Save acquisition method. If MSMS 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 Typicallythe analytical batch runsequencebegins and endswith 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 Using t h e Autosampler 12.2.1 Set up sample tray according to the samph: list prepared in Section 12.1.1. 12.2.2 Set-up the HPllOO/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 hject/sample = 1 12.2.2.3 Cycle time = 9 minutes - __ 3M Environmental Laboratory ETS-8-7.0 Analysis of Liver Extract Using ESlMS Page 5 of 10 Page 123 3M Medical Department Study: T-6316.7 BACK TO MAIN Analytical Report: FACT TOX-098 LRN-U2402 12.2.2.4 Solvent ramp conditions FFl Time 4.5 min. 95% 6.5 min. 1 7.0 min. 40% 19.omi. I 40% I 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 of the Micromass Quattro 11 Triple Quadrupole Mass SpectrometerFitted with an AtmosphericPressure 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 shouldbe flat with no jagged edges. Ifthe tip is found to be unsatisfactory, disassemble the probe and Ieplace the stainless steel capillary. 123.4 Turn on the nitrogen, 12.3.5 Open the tune page. Clicks on operate to initiatesource block and desolvation heaters. 123.6 Open the Inlet Editor. 12.3.6.1 12.3.6.2 12.3.6.3 12.3.6.4 - Set HPLC pump to "On" Set the flow to 10 500 Uymino:r as appropriate Observedroplets coming out of the tip of theprobe. A fine m i s t shou 1 be expelled with no nitrogen leaking around the tip of the probe. Readjust the tip of the probe if no mist is observed 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: 12.3.7.1 Drying gas 250-400litershour 12.3.7.2 ESI nebulizing gas 10-15 literdhour 123.7.3 HPLC constant flow mode flow rate 10-500 pUmin 12.3.7.4 Pressure <400 bar (This parameter is not set, it is a guide to ensure the HPLC is operating correctly.) 12.3.7.5 Source block temperature 150" 12.3.7.6 Desolvation temperature 250' ETS-8-7.0 Analysis ofLiver Extract Using ESMS Page 6 of 10 3M Environmental Laboratoty Page 124 3M Medical Department Study: T-6316.7 BACK TO MAIN Analytical Report: FACT TOX-098 LRN-U2402 12.3.8 Print the tune 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 DATAANALYSAISND CALCULATIONS 13.1 Calculations: 13.1.4 Calculatematrix spike percent recoveries ilsing the followingequation: % Recovery = - ObservedResult BackmoundResult x 100 Expected Result 13.1.5 Calculatepercent differenceusing the following equation: - % Difference= Exuected Conc. Calculated Cone. x 100 Expected Conc. 13.1.6 Calculate actual concentrations inmatrix (pg/g): Ing of PFOS calc. h m std. Curve x Dilution Factor) (Initial Weisht of Liver Final Volume (mL) x 1up 1000 ng 14.0 METHOPDERFORMANCE 14.1 Method Detection Limit (MDL)and Limit of Qumtitation (LOQ)are method, analyte, and . matrix specific. Refer to ETS-8-6.0, Attachmenit B for a listing of current validated MDL and LOQ values. . 14.2 Solvent Blanks, Method Blanks and Matrix B1:anks 14.2.1 Solvent blanks, method blanks, and matrix blanks must be below the lowest standard in the calibration curve. 1 4 3 Calibration Curves 14.3.1 The 1.2 value for the calibrationmust be 0.980 or better. 14.4 Matrix Spikes 14.4.1 Matrix spike percent recoveries must be within i:30% ofthe spiked concentration. 14.5 Continuing Calibration Verification 14.5.1 Continuing calibration verification percent recoveries must be within k 30% of 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 ESMS Page 7 of 10 Page 125 3M Medical Department Study: T-6316.7 BACK TO MAIN Analytical Report: FACT TOX-098 LRN-U2402 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 POLLUTION PREVENTION AND WASTE MANACEMIENT 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 studymust have the folllowing information included either in the header or hand written on the page: study or project number, acquisition method, integrationmethod, sample name, extraction date, dilution factor (if applicable), and analyst. 16.2 Print the tune page, sample list, and acquisition method fiom MassLynx to include in the appropriatestudy folder. Copy these pages and tape into the instrument runlog. 16.3 Plot the calibrationcurve by linear regression, weighted l/x,then print these graphs and store in the study folder. 16.4 Print data integration summary, integration methold, and chromatogramsh m 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 Attachment A for an example of a summary SpRiidSheet. 16.6 Back up electronic data to appropriate medium. Record in study notebook the file name and location of backup electronic data. 17.0 TABLESD,IAGRAMFSL.OWCHARATNSD, VALIDATIONDATA 17.1 Attachment A ETS-8-7.0 Data summary spreadsheet 18.0. REFERENCES 18.1 FACT-M-2.,1"Extraction of PotassiumPerfluorooctanesulfonateor Other Fluorochemical Compounds fiom Liver for Analysis Using HPLC-Electrospray/Mass Spectrometry" 18.2 ETS-9-24.0, "Operation and Maintenance of the hlicromassAtmospheric Pressure Tonization/Mass Spectrometer Quattro II triple qu:&pole Systemsy' 18.3 The validationreport associated with this method is ETS-8-6.0 & 7.0-V-1 19.0 AFFECI'EDDOCUMENTS 19.1 ETS-8-6.0,"Extraction of Potassium Perfluorooct;mesulfonateor Other Fluorochemical Compounds fkom Liver or Fluid for Analysis Using HPLC-ElectrosprayMass Spectrometry" -- ___ 3M Environmental Laboratory ETS-8-7.0 Analysis of Livcr.ExtractU!iing E S h S Page 8 of 10 Page 126 3M Medical Department Study: T-6316.7 20.0 REYISIONS Revision Number BACK TO MAIN Analytical Report: FACT TOX-098 LRN-U2402 Reason For R e v i s h Revision - - .. 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 Analytical Report: FACT TOX-098 LRN-U2402 Laboratory Study # Study: Test Material: MatrixlFinal Solvent: MethodRevision: Analytical Equipment System Number Instrument SofhvareNersion: Filename: R-Squared Value: Slope: Y Intercept: Date of ExtractiodAnalyst: Date of Analysis/Analyst Croup/Dose: Taken from the study folder. Sample#: Taken from the study folder. Concentration (nglg): Taken from the MassLyk integrationsummary. Initial Wt. (g): Taken from the study folder. Dilution Factor: Taken from the study folder. Final Conc. (ug/g): Calculatedby dividingthe initial volume from the concentration Attachment A. Summary Spreadsheet ETS-8-7.0 Analysisof Liver Extract UsJng ESMS .. 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 of Potassium Perfluo-rooctanesulfonateor Other Fluorochemicals in Liver Extracts Using !KPLC-Electrospray/Mass Spectrometry" Section modified: Effective date of modifications: 10.3.2, 14.5.1, atld sections 143.2-14.3.6 July 22, 1999 Section 10.3.2 Method reads: 10.3.2 Analyze a mid-mge calibration standardaftereverytenth sample, with a minimumofone per batch. Modify method to read: 10.3.2 Analyze a mid-range calibrationstandardat least after (:very ten samples, with a minimumof one per batch. Section 14.5.1 Method reads: 14.5.1 Continuingcalibrationverification percent recoveries must be within f 30Y0of the spiked concentration. Modi@ method to read: 14.5,l At least one continuing calibrationverification per ten riamplesmust 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 instruniental drift affects the data. The first curve and acceptable calibration checks shall bracket usable diita. 3M Environmental Laboratory .._ _ Page 129 3M Medical Department Study: T-6316.7 BACK TO MAIN Analytical Report: FACT TOX-098 LRN-U2402 Study # FACT-TOX-098 Section 14.33 Method reads: NA Modify method to read: 14.3.3 Calibrationstandardswith peak areas less than2 times the curve matrix shouldbe deactivatedto disqualify a data range that may be affecttd by background levels of the analyte. Section 14.3.4 Method reads: NA Modify method to read: 14.3.4 Low or high curvepoints 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 mom than 30% from the theoreticalvalue 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 c a h i t i o n curve must contaki at least 5 activepoints. Signature of PAI and date Il/L.G/1b-d I' Signature of Sponsor and date A Signahre of Study Director and date A 7 . A ? c Aek 3M Environmental Laboratory Page 130 3M Medical Department Study: T-6316.7 Appendix D: Data Summary 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 I I I I I 1 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 Group 1 (Control) 0 mglkglday Group 2 1 mgkglday Group 3 5 mgkglday Group 4 10 mgkglday Group 5 20 mgkglday Specimen ID 12573F 12574F 12575F 12576F 12577F 12578F 12579F 12580F 12581F 12582F 12583F 12584F 12585F 12586F 12587F 12588F 12589F 12590F PFOS (ClS19)" 0.0994 0.0253 0.137 9.68 7.60 6.34 8.58 7.00 38.9 38.7 33.1 75.9 85.8 76.4 145 209 167 121 PFOSA PFOSAA EtFOSE-OH <LOQ <LOQ (0.120) 0.148 1.52 1.25 1.13 1.39 I.24 5.65 4.50 3.54 7.33 6.07 6.67 9.18 6.51 5.90 <LOQ (0.063) <LOQ (0.063) 7.73 5.49 3.93 8.81 7.95 58.6 53.4 21.3 40.8 58.4 26.8 96.1 <LOQ (0.0593)* <LOQ (0.0593)* <LOQ (0.0593)* 0.292* 0.0774* 0.166* 0.1 32* 1.56 0.803 0.854 6.96 5.26 6.40 15.3 10.4 L W i r n i tof Quantitation `Results havenot beencorrected for the purityof the analyticalreferencematerial. `Data are qualitativeonly, matrixblankwas unusually highand a suitablecalibrationtwve (P >0.98)couldnot bedetermined from this analysis. It is not possibleto v e mtrue recovery of endogenousanalytefrom tissueswithout radioqabeledreferencematerial. Theonly measurement of accuracy available at this time, matrixspike studies, indicatethat the data are quantitativeto *40%. 3M Environmental Laboratory Page 133 3M Medical Department Study: T-6316.7 BACK TO MAIN Analytical Report: FACT TOX-098 LRN-U2402 PFOSAA (IJglmL)" <LOQ 4.73 39.5 30.4 43.1 EtFOSE-OH (PSlmL)" r TOX-098 <LOQ 0.00686 1 Anomaly 0.0154 0.0371 0.0833 PFOSAA (Pglg)" <LOQ 6.78 44.4 42.0 105 EtFOSE-OH (PSkI)" T TOX-098 <LOQ 0.167* 1.07 6.21 14.3 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 BACK TO MAIN W 3 | = | t BACK TO MAIN i f [ry mee ------ i { Ee Bn ELE Em E EFaEe sEm a E mrm ereTEe a TETEs ree ; EEE Ete ETtte EE E rrersE tE L e t Te R Er rE r e=eC e a r EEEEEE LETTEIsl.1. , i| . -- 3 : 1 { _ sotk i BACK TO MAIN z t| IE = =T = ELE F re || FE Sime SE i Ee - EE ee s eepbhrisiraP teRre PtPeErPeSR eT) [ CE E=reearEEEEnE EseLelr.rT sreE E 0 rerE eoTt L]| CEE EREEEEEL TEETTTEE CT. LiJ | - -i BACK TO MAIN P-0 0 3 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 1000 ng AR- Analytical result from MassLynx summary DF- Dilution factor SC-PFOS salt correction constant (0.9275) FV-Final extract volume (1 .O mL unless otherwise noted) EV-Volume of sera extracted Formula Used for Liver Analyses in Study FACT TQX-098 a AR (ng/g) x 3 curve ('Ix SC x DF x 1.0 pg = Reported Concentration (pg/g) sample 1000 ng (I) a curve is assumed to be: 1 g liver 5 mL H20 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 = ,9.68pglg I .0101 g/ 5mL 1000 ng AR- Analytical result from MassLynx summary a curve-Density of the liver standard curve, assumed to 'be l g liver/ 5 ml water a 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: FACT TOX-098 LRN-U2402 CEntrE Analytical Laboratorks, Inc. /1 3048 Research Drive Phone: (814) 231-8032 State College, PA 16801 Fax: (814) 231-1253 or (814) 231-1580 INTERIM CERTIFICATE 0F A N a Y S I S Revision 1(9/7/00) Centre Analytical Laboratories COA Reference #: 023-018B 3M Product: PFOS, Lot 171 Reference #: SD-009 Purity: 86.4% 3. Sodium 4. PotassiumZ 5. Nickel 6. Iron ,- 1. 0.017 wt.lwt.% 2. 0.007 wt.lwt.% 3. 1.355 wt./wt.% 4. 6.552 wt.lwt.% 5. 0.003wt./wt.% 6. 0.004wt.fwt.% 3. Bromide 4. Nitrate 5 . Nitrite I. <0.015 wt.lwt.% 2. 0.27 wt.lwt.% 3. c0.040wt./wt.% 4. 0.009wt.lwt.% 5. <0.006wt.lwt.% /A. COAO23-018B - -. .. 3M Environmental Laboratory 1. 12.08 wt.lwt.% 2. 0.794 wt.lwt.% 3. 1.G1 wt.lwt.% 4. 10.1 wt.lwt.% Page 1 of 3 Page 142 ~~ 3M Medical Department Study: T-6316.7 BACK TO MAIN Analytical Report: FACT TOX-098 LRN-U2402 Centre Analytical Laboratorks, Inc. 3048 Research Drive State College, PA 16801 FI Phone: (814) 231-8032 Fax: (814) 231-'1253or (814) 231-1580 INTERIM CERTIFICATE OF ANALYSIS Centre Analytical Laboratories COA Reference #: 023-018B Date of Last Analysis: 08/31/00 Expiration Date: OW3 1/01 Storage Conditions: Frozen I-10C Re-assessment Date: 08/31/01 'Purity = 100%- (sum of metal impuritds, 9 i +LC/TdS impurities, 10.60%+InorganicFluoride, 0.27%+NMRimpurities, 1.OO%+POAA, 0.30%) Total impurity from all tests = 13.56% Purity = 100% - 13.56% = 86.4% 2Potassiumis expected in this salt form and is therefore riot considered an impurity. 3pUrityby DSC is generallynot applicable to materials of low purity. No endotherm was p observedfor this sample. 4Sulfbrin the sample appears to be converted to SO4and 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. 'TFA WA NFPA PFPA Trifluoroacetic acid Heptafluorobutync acid Nonofluoropentanoic acid Pentafluoropropanoic acid %'heoretical value calculations based on the empirical formula, C B F ~ ~ S O(~MXW=+538) This work was conducted under EPA Good Laboratory Practice Stmdiuds (40 CFR 160). COA023-018B 3M Environmental Laboratory Page 2 of 3 Page 143 BACK TO MAIN 3 M Medical Department Study:T-6316.7 Analytical Report: FACT TOX-098 LRN-U2402 a\Centre Analytical Laboratories, Inc. 3048 Research Drive Phone: (814)231-8032 State College, PA 16801 f'ax: (814)231-1253or (814)231-1580 INTERIM CERTIFICATE OF ANALYSIS Centre Analytical Laboratories COA Reference #: 023-018B LCMS purity Profile: Impurity WLiwt. Yo I c5 I 1.56 I C6 c7 Total Note: The C4 and C6 values were calculated using the (24and 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. /A,&&- Prepared By: - Dayd S. Bell tal Laboratories Date Laboratory Manager, Centre Analytical Laboratories COA023-018B 3 M Environmental Laboratory Page 3 of 3 Page 144 3M Medical Department Study: T-6316.7 BACK TO MAIN Analytical Report: FACT TOX-098 LRN-U2402 CEntre Analyticall Laboratories, Inc. 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 #: 023-022-1 3M Product: EtFOSE-OH Test Control Reference #: SD-013 Purity: 88.9% 3. <0.001 wt.lwt.% 4. 0.002 wt.lwt.% 5. <0.001 wt.lwt.% 6. <0.001 wt.lwt.% 1. Chloride 2. Fluoride 3. Bromide 4. Nitrate 5. Nitrite 1. <0.015 wt.lwt.% 2. <0.005 wt.lwt.% 3. <0.040 wt.lwt.% 4. <0.009 wt.lwt.% 1. Carbon 2. Hydrogen 3. Nitrogen 4. Sulfur j 5. Fluorine fl COA023-022-1 3M Environmental Laboratory 1. Theoretical Vellue = 25.2% 2. Theoretical Value = 1.75% 3. Theoretical Value = 2.45% I 1 4. TheoreticalValue = 5.60% I 5. Theoretical Value = 56.6% 1. 24.42 wt.lwt.% 2. 1.78wt.lwt.% 3. 2.72 wt.lwt.% 4. 9.34 wt.lwt.% 5. 58.4 wt.lwt.% Page I of 3 Page 145 BACK TO MAIN 3M Medical Department Study: T-6316.7 Analytical Report: FACT TOX-098 LRN-U2402 a\CEntrE Analytical Laboratories, Inc. 3048 Research Drive State College, PA 16801 ILL4 Phone: (814) 231-8032 Fax: (814) 231-1253 or (814) 231-1580 I INTERIM CERTIFICATE OF ANALYLYIS Centre AnalyticalLaboratoriesCOA Reterence #: 023-022-1 3M Product: EtFOSE-OH Test Control Reference #: SD-013 Date of Last Analysis: 11/26/00 Expiration Date: 11/26/01 Storage Conditions: <-lo "C Re-assessment Date: 11/26/01 'purity = 100% - (total metal impurities,0.002% + total NMR impurities,0.90% + G U M S impurities, 10.21 + POAA, 0.03%) Total impurity fiom all tests = 11.14% PUrity = 100% - 11.14% = 88.9% 2TFA Trifluoroacetic acid f- HFBA Heptafluorobutyric acid NFPA Nonafluoropentanoic acid PFPA Pentafluoropropanoic acid 3Theoreticalvalue calculationsbased on the empirical fcmnula, C ~ ~ H I O F ~ ~ N O ~ S (MW=571) COAO23-022-1 - .. 3M EnvironmentalLaboratory Page 2 of 3 Page 146 3M Medical Department Study: T-6316.7 BACK TO MAIN Analytical Report: FACT TOX-098 LRN-U2402 7 ii 3048 Research Drive Phone: (814) 231-8032 State Colleae. PA 16801 ax: (814) 231-1253 or (874j 231-1580 INTERIM CERTIFICATE OF ANALYSIS Centre Analytical Laboratories COA Reference #: 023-022-1 3M Product: EtFOSE-OH Test Control Reference #: SD-013 GCMS Purity Profile Peak # 1 2 3 L 4 5 6 7 8 9 10 11 12 13 Total I RetentionTime I I (min) 6.163 I 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 c4 C5 C6 c7 I %x@ajq 1.07 3.30 10.21 This work was conducted under EPA Good Laboratory Practice Standards (40 CFR 160). Prepared By: Scientist ratories Centre Analytical Laboratories COAO23-022-1 3M EnvironmentalLaboratory D.ate Page 3 of3 Page 147 BACK TO MAIN 3M Medical Department Study: T-6316.7 Analytical Report: FACT TOX-098 LRN-U2402 a\Centre Analytical Laboratories. Inc. 3048 Research Drive State College, PA 16801 Phone: (814) 231-8032 Fax: (814) 231-'1253 or (814) 231-1580 INTERIM CERTIFICATE OFANALYSIS Centre Analytical Laboratories COA Reference #: 023-022-2 3M Product: EtFOSE-OH Test Control Reference #: TCR-00017-52 Purity: 97.4% Test Name I Soecificatims Result 2. Magnesium 3. Sodium 4. Potassium 5. Nickel 6. Iron 1. Chloride 2. Fluoride 3. Bromide 2. PFPA 3. HFBA 3. Nitrogen 4. Sulfur 5. Fluorine 1. Theoretical Value = 25.2% 2. Theoretical Value = 1.75% 3. Theoretical Value = 2.45% 4. Theoretical Vilue = 5.60% 5. Theoretical Value= 56.6% Conforms Positive 1. <0.001 wt.lwt.% 2. <0.001 wt./wt.% 3. <0.001 wt.lwt.% 4. <0.001 wt.lwt.% 5. <0.001 wt./wt.% 6. <0.01)1 wt.lwt.% 7. <0.001 wt.lwt?h 1.26 wi./wi.% None Quantified 1.29 wt.lwt.% 0.10 wt./wt.% None Detected 90.3 wt./wt.%. 1. <0.015 wt./wt.% 2. <0.005 wt.lwt.% 3. <0.040 wt./wt.% 4. <0.009 wt.lwt.% 5 . <0.006wt./wt.% 6. <0.007 wt.lwi.% 7. <0.154 wt./wt.% 1. <0.1 wt./wt?h 2. CO.1 wt./wt.% 3. <0.1 wt.lwt.% 4. <0.25 wt./wt.% 1. 25.04 wt./wt.% 2. 1.69 wt./wt.% 3. 2.61 wt./wt.% 4. 8.88 wt./wt.% 5. 56.8 wt.lwt.% 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 L 3048 Research Drive State College, PA 16801 Phone: (814) 231-8032 Fax: (814) 231-1253 or (814)231-1580 INTENM CERTIFICATE 0 . FANALYSIS Centre Analytical Laboratories COA Reference #: 023-022-2 3M Product: EtFOSE-OH Test Control Reference #: TCFL-00017-52 Date of Last Analysis: 11/26/00 ExpirationDate: 11/26/01 Storage Conditions: <-lo "C Re-assessmentDate: 11/26/01 'Purity = 100%- (totalNMR impurities, 1.26%+ GCMS impurities, 1.29 + POAA, 0.10%) Total impurity from all tests ==2.65% Purity = 100%- 2.65% = 97.4% *TFA Tnfluoroacetic acid P HFBA Heptafluorobutyric acid NFPA Nonafluoropentanoic acid PFPA Pentafluoropropanoic acid 3Theoreticalvalue calculationsbased on the empirical fixmula, C12Hn-,F17NO3S (MW=571) COA023-022-2 3M Environmental Laboratory Page 2 of 3 Page 149 3M Medical Department Study: T-6316.7 BACK TO MAIN Analytical Report: FACT TOX-098 LRN-U2402 L 3048 Research Drive State College, FA 16801 Phone: (814) 231-8032 Fax: (814) 231-1253 or (814) 231-1580 INTERIM CERTIFICATE0.FANALYSIS Centre Analytical Laboratories COA Reference #: 023-022-2 3M Product: EtFOSE-OH Test Control Reference #: TCEI-00017-52 G C / M S Purity Profile Retention Time 17.307 Total 1.29 This work was conducted under EPA Good Laboratory Practice Standards (40 CFR 160). Prepared By: Scientist '/Sohn~laherty Laboratory Manager Centre Analytical Laboratories COA023-022-2 3M EnvironmentalLaboratory /r/ddd Date Page 3 of 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 re- Makin T. Case, D.V.M.,Ph.D., Study Director lF2* m/ Date d John L. Butenhoff, Ph.D., Sponsor Representative 6 F&Z. Zeal Date az/oz/o 1 Kristen 5. Hanseh, Ph.D., Principal Analytical InvestlQator Date *E L' / William K. Reagen, Laboratory Manager Date 3M Environmental Laboratory Page 151