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~~~~ ~~~~ 3M Medical Department Study: T-6316.8 BACK TO MAIN Analytical Report: FACT TOX-097 LRN-U2452 Study Title Oral (Stomach Tube) Developmental Toxicity Study of 2(N-Ethylperfluorooctanesulfonamido)-ethanolin Rabbits Analytical Laboratory Reporf Title Determination of the Presence and Concentration of Potassium Perfluorooctanesulfonate (PFOS) or another metabolite of 2(N-ethylperfluorooctanesulfonamido)-ethanol(N-EtFOSE) in Liver and Serum Specimens Data Requirement Not Applicable Author 3M Environmental Laboratory Study Completion Date At signing Performing Laboratory Liver and Serum, Extraction and Analyses 3M Environmental Laboratory Building 2-3E-09,935 Bush Avenue St. Paul, MN 55106 Project Identification 3M Medical Department Study: T-6316.8 Argus In-Life Study: 418-010 Analytical Report: FACT TOX-097 3M Laboratory Request No. U2452 Total Number of Pages 145 3M Environmental Laboratory Page 1 3M Medical Department Study: T-6316.8 BACK TO MAIN Analytical Report: FACT TOX-097 LRN-U2452 This page has been reserved for specific country requirements. 3M Environmental Laboratory Page 2 BACK TO MAIN 3M IMedical Department Study: T-6316.8 Analytical Report: FACT TOX-097 LRN-U2452 GLP Compliance Statement Analytical Laboratory Report Title: Determination of the Presence and Concentration of Potassium Perfluorooctanesulfonate(PFOS) or another metabolite of 2(N-ethylperfluorooctanesulfonamido)-ethanol(N-EtFOSE) in Liver and Serum Samples Study Identification Numbers: T-6316.8, FACT TOX-097, LRN-U2452 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 after teratological examination and shipment of specimens. The analytical study was considered to start at the receipt of these specimens for analysis. This resulted in having two separate study directors, one for each phase of the same study. However, since the technical performance of each phase was entirely separate, no effect is expected from this exception. 0 Stability not determined for test and control articles under all conditions of administration. 0 Characterizations of all analytical reference materials have not been completed at this time. An amendment to this report will be issued when characterizations have been completed for all reference materials with the exception of PFOSEA. A characterization of PFOSEA will not be completed as all the material was consumed during the analytical phase of the study. 0 There were no expiration dates on the reagentlsolutions bottle per 21 CFR Part 58.83. 0 The electronic data systems have not been validated and there is not an electronic audit trail of corrections currently available (21 CFR Part 58.130 (e)). Hardcopies of chromatograms will be Considered as the original raw data. (See next page for Signatures) 3M Environmental Laboratory Page 3 3M Medical Department Study: T-6316.8 BACK TO MAIN Analytical Report: FACT TOX-097 LRN42452 (GLPCompliance Statement Continued) A Mahin T. Case D.V.M., PhD., Study Director Date John L. Butenhoff Ph.D., Sponsor Representative Date b+)/O 1 - KristenJ. Hansen Ph.D. Principal Analytical lnvesfigafor Date 3M E3nvironrnental Laboratory Page 4 3M IWedical Department Study: T-6316.8 BACK TO MAIN Analytical Report: FACT TOX-097 LRN-U2452 GLP Study-Quality Assurance Statement Analytical Laboratory Report Title: Determinationof the Presence and Concentration of Potassium Perfluorooctanesulfonate(PFOS) or another metabolite of 2(Nethylperfluorooctanesu1fonamido)-ethanol (N-EtFOSE) in Liver and Serum Samples Study IdentificationNumbers: T-6316.8, FACT TOX-097, and LRN-U2452 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. I 01/22/01 -01/23/01 I Draftreport(2) I 01/23/01 I 01/23/01 QA U Reflesentative Date 3M Environmental Laboratory Page 5 ~~ BACK TO MAIN 3M Medical Department Study: T-6316.8 Analytical Report: FACT TOX-097 LRN-U2452 Table of Contents GLP Compliance Statement.............................................................................................. 3 GLP Study-Quality Assurance Statement ....................................................................... 5 Study Personnel and Contributors..................................................................................... 9 Introduction and Purpose .................................................................................................. 10 Test System ................................................................................................................. 10 Specimen Collection and Analysis ................................................................................ 11 Specimen Receipt and Maintenance................................................................................. 11 Chemical Characterizationof Analytical Reference MateriaVSubstance............................ 12 Method Summaries ........................................................................................................... 13 3M Environmental Laboratory....................................................................................... 13 Preparatory Methods ............................................................................................... 13 Analytical Methods ................................................................................................... 13 Analytical Equipment ............................................................................................... 14 Data Quality Objectives and Data Integrity........................................................................ 15 Data Summary, Analyses, and Results ............................................................................. 16 Summary of Quality Control Analyses Results.............................................................. 16 Statement of Data Quality ............................................................................................ 17 Summary of Sample Results ........................................................................................ 17 Statistical Methods and Calculations ................................................................................. 17 Statement of Conclusion ................................................................................................... 17 References......................................................................................................................... 17 Appendix A: Chemical Characterization of Test Material, Control Matrices ....................... 18 Chemical Characterizationof 2(N-Ethylperfluorooctanesulfonamido)-ethanol..............18 Characterizationof Control Matrices............................................................................. 18 Appendix B: Protocol, Amendments and Deviations.......................................................... 19 Appendix C: Extraction and Analytical Methods ................................................................ 42 FACT-M-1.O, "Extraction of Potassium Perfluorooctanesulfonateor Other Anionic FluorochemicalSurfactantsfrom Liver for Analysis Using HPLC-Electrospray/Mass Spectrometry," (8 pages) .................................................................................................. 43 FACT-M-3.0, "Extraction of Potassium Perfluorooctane!or Other Anionic Fluorochemical Compounds from Serum or Other Fluids for Analysis Using HPLC-Electrospray/Mass Spectrometry," (8 pages) .................................................................................................. 51 ETS-8-04.1, "Extraction of Potassium Perfluorooctanesulfonate or other Fuorochemical Compoundsfrom Serum for Analysis Using HPLC-Elec:trospray/Mass Spectrometry," (14 pages)......................................................................................................................... 59 ETS-8-06.0, "Extraction of Potasium Perfluorooctanesulfonate or other Fluorochemical Compoundsfrom Liver for Analysis Using HPLC-ElectrospraylMass Spectrometry," (14 pages)......................................................................................................................... 73 3M Environmental Laboratory Page 6 BACK TO MAIN 3M Medical Department Study: T-6316.8 Analytical Report: FACT TOX-097 LRN-U2452 FACT-M-2.0, "Analysis of Fluorochemicals in Liver Extracts Using HPLC- Electrospray/Mass Spectrometry,"(8 pages) ....................................................................87 FACT-M4.0, "Analysis of Fluorochemicals in Serum Extracts Using HPLC- Electrospray/MassSpectrometry,"(8 pages) ....................................................................95 ETS-8-05.1, "Analysis of Potassium Perfluorooctanesulfonate or other Fuorochemical Compounds in Serum Extracts Using HPLC-Electrospray/MassSpectrometry," (9 pages)........................................................................................................................... 103 ETS-8-07.0, "Analysis of Potassium Perfluorooctanesulfonateor other Fluorochernical Compounds in Liver Extracts Using HPLC-Electrospray/MassSpectrometry," ( I O pages)......................................................................................................................... 112 Appendix D: Data Summary Tables ................................................ ..................................122 Appendix E: Data Spreadsheets ....................................................................................... 128 Appendix F: Example Calculations....................................................................................140 Appendix G: Interim Certificate(s) of Analysis ................................................................... 141 Appendix H: Report Signature Page ................................................................................. 145 3M Ihvironmental Laboratory Page 7 BACK TO MAIN 3M Medical Department Study: T-6316.8 Analytical Report: FACT TOX-097 LRN-U2452 List of Tables Table 1. Dosage Levels. Concentration and Volumes for Argus Study #MI8-010.............10 Table 2. Characterizationof the Analytical Reference Materials/Substances in Study FACT-TOX-097.................................................................................................... 12 Table 3. Negative Ions Monitored..................................................................................... 15 Table 4. Characterizationof Test Substance in Study FACT-TOX-097............................. 18 Table 5. Characterizationof the Control Matrices Used for Liver and Sera Analyses in Study FACT-TOX-097.......................................................................................... 18 Table 6. Rabbit Sera FO PFOS, PFOSA and PFOSAA Data for FACT-TOX-097..............122 Table 7. Rabbit Sera FO N-EtFOSE and PFOSEA Data for FACT-TOX-097..................... 123 Table 8. Rabbit Liver FO PFOS, PFOSA and PFOSAA Data for FACT-TOX-097..............124 Table 9. Rabbit Liver FO N-EtFOSE and PFOSEA Data for FACT-TOX-097..................... 125 Table 10. FACT-TOX-097 Data Summary of Average Sera Concentration (pg/mL) and Standard Deviation (kSD) ................................................................................... 126 Table 11. FACT-TOX-097 Data Summary of Average L.iver Concentration (pg/g) and Standard Deviation (*SD) .................................................................................... 127 Table 12. Approximate LOQ Values Used in FACT-TOX-097........................................... 127 3M Environmental Laboratory Page 8 BACK TO MAIN 3M Medical Department Study: T-6316.8 Analytical Report: FACT TOX-097 LRN-U2452 Study Personnel and Contributors Study Director Marvin T. Case, D.V.M., Ph.D. 3M Corporate Toxicology 3M Center, Building 220-2E-02 St. Paul, MN 55133-3220 (651) 753-5180 Analytical Chemistry Laboratory Liver and Serum, Extraction and Analyses 3M Environmental Laboratory Kristen J. Hansen, Ph.D., Principal Analytical Investigator Sponsor 3M Corporate Toxicology 3M center, Building 220-2E-02 St. Paul, MN 55133-3220 John L. E3utenhoff, Ph.D., Sponsor Representative 3M Laboratory Contributing Personnel David R. Barnidge, Ph.D.* Lisa A. Clemen Kelly J. Dorweiler* Mark E. Ellefson Sarah A. Heimdal* Marlene M. Heying* Harold 0. Johnson *Contract lab professionalservice employees Kelly J. Kuehlwein* Glenn Langenburg* Sally A. Linda* Ian A. Smith* Thomas P. Wagner* Bob W. Wynne* Richard D. \/oungblom* Location of Archives All original raw data, protocol, and analytical report have been archived at the 3M Environmental Laboratory. The test substance 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 for a minimum of ten years. 3M Environmental Laboratory Page 9 BACK TO MAIN 3M Medical Department Study: T-6316.8 Introduction and Purpose Analytical Report: FACT TOX-097 LRN-U2452 The purpose of the analytical study is to provide semi-quantitative or qualitative determination of PFOS, PFOSA, PFOSAA, PFOSEA and N-EtFOSE in sera samples and liver samples collected from pregnant rabbits exposed orally to N-EtFOSE. The study was initiated on 18 September 1998. lest System The test system were timed-pregnant female New Zealand White [Hra: (NZW)SPF] Rabbits received from Covance Research Products Inc. The individual body weights of the female rabbits ranged from 2.9to 4.2kg;the rabbits were approximately five to six months of age at the time of study assignment (Argus Study ##418-010). Table 1 summarizes the number of female rabbits in each group. Group I consisted of control female rabbits that were administered 0.0 mg/kg/day in 2%)Tween@80 (vehicle). Group II through Group V female rabbits were administered 0.1, 1 .O,2.5,or 3.75mg of N-EtFOSE per kg of body weightlday in 2% Tween@80.Serum and liver samples were collected on day 21 of presumed gestation. Additionally, female rabbits were Caesarean-sectioned and pooled fetal tissue(s) taken from the rabbits. Details of the in-life phase of the study arie presented in the Argus Laboratory final report, "Oral (Stomach Tube) Developmental Toxicity Study of N-EtFOSE in Rabbits, M18010." Table 1. Dosage Levels, Concentration and Volumes for Argus Study W18-010 Assigned Rabbit Numbers *The test article will be considered 100% pure for the purpose of dosage calculations. 3M Environmental Laboratory Page 10 BACK TO MAIN 3M Medical Department Study: T-6316.8 Analytical Report: FACTTOX-097 LRN42452 Specimen Collection and Analysis In the analytical study reported here, 19 liver specimens, 19 sera specimens and 19 pooled fetal tissues were collected from 19 presumed pregnant female rabbits at the end of the in-life phase of Argus Study M18-010 and sent to the 3M Environmental Laboratory to be extracted and analyzed for perfluorooctanesulfonate(PFOS), perfluorooctanesulfonamide (PFOSA), perfluorooctanesulfonamido(ethy1)acetate(PFOSAA), N-ethyl perfluorooctanesulfonamidoethyl alcohol (N-EtFOSE), and perfluorooctanesulfonylethylamide (PFOSEA). Only non-quantitative screening data is provided for PFOSAA and N-EtFOSE determination in liver and sera samples. Blood specimens were centrifuged within one hour of collection. Serum was then harvested and stored in a freezer set to maintain specimens at -70" until shipped to the 3M Environmental Laboratory. Liver specimens collected from each animal were flash frozen in liquid nitrogen then stored in a freezer set to maintain specimens at -70C until shipped to the 3M Environmental Laboratory. Pooled fetal tissues (per litter, fetuses and placenta) were collected then stored in a freezer set to maintain specimens at -70C until shipped to the 3M Environmental Laboratory. (Note: Although fetal and placenta tissues were collected at the same time as liver and serum, results from these analyses will not be included in this report. A separate report may be issued for fetal tissue data.) Sera and liver samples were extracted beginning on 16 October 1998. Liver samples were homogenized prior to the extraction procedure. Sample extracts were analyzed beginning 17 October 1998 using high-pressure liquid chromatography-electrospray/tandem mass spectrometry (HPLC-ES/MS/MS) in the multiple reaction monitoring. Analytical details are included in this report. Specimen Receipt and Maintenance The 3M Environmental Laboratory received serum, liver, fetuses and placenta specimens for the in-life phase of this study FACT TOX-097 on 23 September 1998 from Argus Research Laboratories. All specimens were received frozen on dry ice and were immediately transferred to storage at -20C fl0"C. Control matrices used in liver and sera analyses performed during TOX-097 were obtained from commercial sources and are presented in Appendix A (see Table 5). Samples analyzed at the 3M Environmental Laboratory will be maintained for a maximurn period of 10 years and will be stored at the laboratory at -20C *lOC. 3M EInvironmental Laboratory Page 11 BACK TO MAIN 3M Medical Department Study: T-6316.8 Analytical Report: FACT TOX-097 LRN-U2452 Chemical Characterizationof Analytical Reference MateriaWubstance Purity I TBD~ TBD~ TBD~ 80.9% NA 3M E3vironmental Laboratory Page 12 BACK TO MAIN 3M Medical Department Study: T-6316.8 Method Summaries Analytical Report: FACT TOX-097 LRN-U2452 Following is a brief description of the methods used during this analytical study by the 3M Environmental Laboratory. Copies of the methods used in this study are located in Appendix C. As the present study progressed, more advanced methods evolved and earlier methods listed in the protocol were not used. It was determined that applying a 1/X weighting to the curve improved the method accuracy at the low end of the curve. The original data sets were reworked utilizing the improved practice. These changes only improved the effectiveness of the method. Amendments to the protocol were written to cover method changes. A copy of protocol amendments and method deviations are presented in Appendix B (Table 6) of this report. 3M Environmental Laboratory PREPARATORMYETHODS FACT-M-1.O, "Extractionof Potassium Pefluorooctanesulfonateor Other Anionic Fluorochemical Surfactantsfrom Liverfor Analysis Using HPLC-Electrospray/MassSpectrometry". 0 FACT-M-3.0, "Extractionof Potassium Perfluorooctanesulfonateor Other Anionic Fluorochemical Compounds from Serum or Other Fluids for Analysis Using HPLC-Electrospray/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 reconstitutedin 1.OrriL of methanol,and then filtered through a 3 mL disposable plastic syringe attached to a 0.2 vm nylon filter into glass autovials. 0 ETS-8-4.1, "Extraction of Potassium Pefluorooctanesulfonateor other FluorochemicalCompounds from Serum for Analysis using HPLC-Electrospray/MassSpectrometry," 0 ETS-8-6.0, "Extractionof Potassium Perfluorooctanesulfonateor Other FluorochemicalCompounds 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 methyl-tert-butyl-ether. The extract was transferred to a centrifuge tube and put onto a nitrogen evaporator until dry. Each extract was reconstitutedin 1.OrriL of methanoland passed through a 0.2 pm nylon filter, using a 3 mL disposable plastic syringe into glass autosampler vials. ANALYTICALMETHODS 0 FACT-M-2.0, "Analysis of Fluorochemicals in Liver Extracts Using HPLC-Electrospray/Mass Spectrometry" 0 FACT-M4.0, "Analysis of Fluorochemicals in Serum Extracts Using HPLC-Electrospray/Mass Spectrometry" 0 ETS-8-5.1, "Analysisof Potassium Perfluorooctanesulfonateor Other Fluorochemicalsin Serum Extracts Using HPLC-Electrospray/MassSpectrometry" 3M Environmental Laboratory Page 13 BACK TO MAIN 3M Medical Department Study: T-6316.8 Analytical Report: FACT TOX-097 LRN-U2452 0 ETS-8-7.0, "Analysis of Potassium Perfluorooctanesulfonateor 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 characteristicof a particularfluorochemical using HPLC/ES/MS/MS.For example, molecular ion 499, selected as the primary ion for PFOS (C8FI7SO3-)analysis,was fragmented to produce ion 99 (FS03-).The characteristicion 99 was monitoredfor quantitative analysis (Table 3). ANALYTICAELQUIPMENT 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 Chromatographsystem Analytical column: Keystone@BetasilTMCI8 2x100 mm (5 pin) Column temperature: Ambient Mobile phase components: Component A: 2mM ammonium acetate Component B: methanol Flow rate: 300 pL/min Injection volume: 10 pL Solvent Gradient: 13.5 minutes Time (minutes) %B 0.0 40% 8.5 90% 11.0 90% 12.0 40% 13.5 40% Mass Spectrometer;YMicromass@API/Mass Spectrometer Quattro I I m Software: Mass Lynx 3.4 Cone Voltage: 20-60 V Collision Gas Energy: 25-45 eV Mode: Electrospray Negative Source Block Temperature: 15OOC *lOC Electrode: Z-spray Analysis Type: Multiple Reaction Monitoring (MRM) Triple Quadrupole system 3M Einvironmental Laboratory Page 14 3M Medical Department Study: T-6316.8 Table 3. Negative Ions Monitored BACK TO MAIN Analytical Report: FACT TOX-097 LRN-U2452 I I I I THPFOS* ~~ ~ 427.0 ~~ ~~ 80.0 *Surrogate ** One or more product ions were used in the determination of PFOS. Deviations It should be noted that as the analytical phase of this study progressed, method parameters were evaluated to improve analyses. Earlier methods were used with deviations until amendments to the protocol were written. Deviations from the original protocol and methods are documented in the Appendix B. 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. ~~ Data Quality Objectives and Data Integrity The following data quality objectives (DQOs) were indicated in the protocol for this study: Linearity: The coefficient of determination (r2)equal to or greater than 0.980 Limits of Quantitation (LOQ): The LOQ is equal to the lowest acceptable standard in the calibration curve. Acceptable Precision: Precision is better than 30% for the method. Acceptable Spike Recoveries: 70-1 30% Demonstration of Specificity: Specificity to be demonstrated by chromatographic retention time and daughter ion characterization. 3M Environmental Laboratory Page 15 BACK TO MAIN 3M Medical Department Study: T-6316.8 Data Summary, Analyses, and Results Analytical Report: FACT TOX-097 LRN-U2452 Summary of Quality Control Analyses Results 0 Linearity: The coefficient of determination (rz)of the extracted standard curve was 20.980 except for the determination of PFOSAA. Acceptable curves could not be derived from the analysis of some data sets. All PFOSAA data should be regarded as non-qualitative screening data only. 0 Calibration Standards: Quantitation of the compounds was based on linear regression analysis (l/x weighted) of a single or of 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 the compounds was based on the response of one specific product ion using the multiple reaction monitoring mode of the instrument (see Appendix C, Analytical Methods). 0 Limits of Quantitation (LOQ): The LOQ is equal to the lowest acceptable standard in the calibration curve (defined as a standard within k30% of the theoretical value), and is at least two times the analyte peak area detected in the extraction blanks. 0 Blanks: All blanks were below the lower limit of quantitation for the compounds of interest, except as noted in Appendix B (see table 6). 0 Precision: Precision was not specifically determined within this study, but has been characterized to be better than *30% for this method. 0 Matrix Spikes: Matrix spikes and matrix spike duplicates were extracted with each set of samples and analyzed during analytical runs at the 3M Environmental Laboratory. Rabbit sera and liver from control animals were spiked prior to extraction. All target analytes were spiked approximatelt 250 ng/mL or 250 ng/g. Sera spikes for PFOS, PFOSA, PFOSAA, and PFOSEA were within &30% of expected values. Spike recoveries studies for N-EtFOSE were dramatically higher than expected, with an average recovery of 196%. The N-EtFOSE data shall be regarded as non-qualitative screening data only. Liver spikes for PFOS, PFOSA, PFOSAA, and PFOSEA were within &30% of expected values. Spike recoveries studies for N-EtFOSE were within *50%. The N-EtFOSE data shall be regarded as non-qualitative screening data only. 0 Surrogates: The surrogate (THPFOS) was added to all samples and standards prior to extraction. THPFOS was not used for quantitation, but was used to monitor for gross instrument failure. The surrogate response of each analytical run was monitored to determine that it did not vary more than *50% from the mean within each analytical run. No problems were observed with these data. 3M Ehvironmental Laboratory Page 16 BACK TO MAIN 3M Medical Department Study: T-6316.8 Analytical Report: FACT TOX-097 LRN-U2452 Statement of Data Quality It is not possible to verify true recovery of endogenous anailyte from tissues without radio labeled reference material. The only measurement of accuracy available at this time, matrix spike studies, indicates the data are quantitative to k30% or greater with the exception of PFOSAA and N-EtFOSE. The PFOSAA and N-EtFOSE data shall be regarded as non-qualitative screening data only. Summary of Sample Results Samples from Dosed Animals: In general, PFOS, PFOSA, PFOSAA and N-EtFOSE levels found in the sera and liver of the test animals increased with dose group. PFOSEA was not detected in sera or liver samples of dosed animals. 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 FACTTOX-097. Statement of Conclusion Under the conditions of the present studies, PFOS, PFOSA, PFOSAA and N-EtFOSE were observed in the sera and liver of rabbits dosed with the N-EitFOSE during the in-life phase of the study. References Argus In-life Final Report #MI8-010, "Oral (Stomach Tube) Developmental Toxicity Study of N-EtFOSE in Rabbits" 3M f:nvironmental Laboratory Page 17 BACK TO MAIN 3M Medical Department Study: T-6316.8 Analytical Report: FACT TOX-097 LRN-U2452 Appendix A: Chemical Characterization of Test Material, Control Matrices Chemical Characterizationof 2(N-Ethylperfluo~nesutfonamido~thanol CAS Number: 1691-99-2 Molecular Weight: 571.O Chemical Formula: CsFI7SO2N (CPHBC) H2CH20H Table 4. Characterization of Test Substance in Study FACT-TOX-097 I Test Substance I Chemical Name I Source I From Sponsor I Expiration Date 5/01I2000 Storage Conditions Ambient temperature Chemical Lot # FM-3929 (Mixture of Lots 30035,30037,30039) Physical Description Puritv 97.4% ~~ * The purity of the substances listedabovewas based only on NMR analyses.Subsequent chemical characterization is occurring and this anatykal report will be amended to indicate the purity of these substanceswhen a certilicateof analysis is issued. This information is from the iwlife protocol. Characterization of Control Matrices Table 5. Characterization of the Control Matrices Used for Liver and Sera Analyses in Study FACT-TOX-097 Control Matrix I Rabbit Liver Rabbit Sera Source Coming Hazelton Wisconsin Sigma I Emiration Date I 01/01/2010 I 01/01/2010 I Storage Conditions -20C * 1 0 T -20C *10"c Chemical Lot # Physical Description Rabbit Liver Rabbit Sera 3M Environmental Laboratory Page 18 BACK TO MAIN 3M Medical Department Study: T-6316.8 Appendix B: Protocol, Amendments and Deviations Analytical Report: FACT TOX-097 LRN-U2452 3M EInvironmental Laboratory Page 19 3M kledical Department Study: T-6316.8 BACK TO MAIN Analytical Report: FACT TOX-097 LRN-U2452 i a 3M ENVIRONM~NTLALBORATOR'Y - PROTOCOALNALYTICASLTUDY Oral (Stomach Tube) Developmental Toxicity Study of 2(N-Ethylperfluorooctanesulfonamido)-ethanolin Rabbits In-vivo study reference number: Argus M18-010 Study number: FACT-TOX-97 Test substance: 2(N-Ethylpeffluorooctanesulfon&do)-etha (N-EtFOSE-OH) Name ,and address of Sponsor: Marvin Case 3M Toxicology Services 3M Center Exact Copy of Original LAC, 91a d 98 'DcGu* Building 220-2E-02 St. Paul, MN 55144 Initial Date Yh.& ofip(L\ ppu'toh\ bw\d no./ Name and address of testing facility: bG cw;hd 3 c J &pab,ll k 3M Environmental Technology and Services oa$b. 935 Bush Avenue, Building 2-3E-09 Mt St. Paul, MN 55106 N(S1O1) Experimental start date: Expected termination date. Method numbers and revisions: FACT-M-1.0, Extraction of Potassium Perfluorooc.tauesulfonate or Other Anionic Surfactants from Liver for Analysis Using HPLC-ElectrosprayMass spectrometry . FACT-M-2.0A,nalysis of Fluorochemicals in Liver Extracts Using HPLCElectrospray/Mass Spectrometry FACT-M3.0, Extraction of Potassium Peffluorocxtanesulfonateor Other Anionic Surfactantsfrom Serum for Analysis Using HPLC-Electrospray/Mas Spectrometry FACT-M-4.0, Analysis of Fluorochemicals in Senirn Extracts Using HPLC- ElectrosprayMass Spectrometry Author: Lisa Clemen Kris Hansen Study Director 9/18 198 Date I&-, p5- Marvin Case Sponsor Representative 4X4L *' Date FACT-TOX-97, U2452 Argus #418-010 Page 1 of 5 3M Einvironmental Laboratory Page 20 3M Medical Department Study: T-6316.8 BACK TO MAIN Analytical Report: FACT TOX-097 LRN-U2452 ,j , 1.0 PURPOSE The analytical portion of this study is designed to evaluate levels of potassium perfluorooctanesulfonate(PFOS), or another metabolite of 2(N-ethylperfluorooctanesulfonamido)-ethanol (N-EtFOSE-OH) determined by the Study Director, in liver and serum samples of the test system when it is administered directly through a stomach tube. The in life portion of this study was conducted at Argus Research Laboratories. 2.0 REGULATORYCOMPLIANC~ This study is conducted in compliancewith the Food and Drug Administration Good Laboratory Practices regulation as stated in 21 CFR 58. Any exceptions will be noted in the final report. I. , 3.0 TESTMATERIALS 3.1 Test,control, and reference substancesand matrices 3.1.1 Analytical reference substatlce: Potassium perfluorooctanesulfonate(PFOS),lot # 217 3.1.2 Analytical reference substance mala& Rabbit liver and serum 3.13 Analytical control substance: None 3.1.4 Analytical control substancematrk Rabbit liver and serum 3.2 Sourceof materials 3.2.1 Analytical reference substance: 3M Specialty Chemical Division; traceability information will be included in the final i.eport , 3.3 3.4 32.2 Analytical reference substance matrix: Argus Research Laboratories; traceability information will be included in the final report 3.23 Analytical control matrix. 3.23.1 - Rabbit liver Argus Research IAmratories; traceability information will be included in the final report; or - Rabbit liver Covance Laboratories;traceability information will be included in the final report 3.23.2 Rat serum - Sigma Chemical Company; traceability information will be included in the final report Number of test and control samples. Liver samples for testing were received from 16 test and 3 control animals for the toxicokineticportion of the study. Liver samples for testing were received from 88 test and 22 control animals for the developmentalportion of the study. Serum samples will be tested at the discretion of the Study Director. Identification of test and control samples: Th,esamples are identified using the Argus Research Laboratories identifiers, which consist of a letter followed by the Argus project number, the animal number, the group designation, and the draw date. FACT-TOX-97, U2452 Argus #418-010 Page 2 of 5 3M Environmental Laboratory Page 21 3M Medical Department Study: T-6316.8 BACK TO MAIN Analytical Report: FACT TOX-097 LRN-U2452 , 3.5 3.6 3.7 3.8 3.9 i Purity and s t r e n b of materials: Characterizationbf the purity and identity of the reference material is the responsibility of the Sponsor. Stability of test material: Characterization of the stability of the test material is the responsibility of the Sponsor. Storage conditions for test materials: Test materials are stored at room temperature. Samples are stored at -20 f 10 OC. Disposition of test and/or control substances: Biological tissues and fluids are retained per GLP regulation. Safety precautions: Refer to the material safely data sheets of chemicals used. Wear appropriate laboratory attire, and follow adequate precautions for handling biological 'materials and preparing samples for analysis. 4.0 - EWEIUMENTAOLverview Tissues from animals dosed as desaibed in Argus Research Laboratories Protocol #418-010are received for analysis of fluorine compounds. Mated female rabbits were dosed on Day 7 of presumed gestation, with administration continuing through Day 20. At Day 21, serum and liver samples, as well as fetuses and placenta, were taken from rabbits in the toxicokinetic portion of the study. At Day 29 for the rabbits remaining in the study, samples of serum and liver were taken, as well as fetuses and placenta. At the discretion of the Study Director, a series of analytical tests will be performed on select tissues. Initially, all liver samples will be analyzed for PF:OS by Electrospray/mass spectrometry ( E S M S ) . On the basis of findings from these analyses, additional samples may be evaluated. If additional analysis is performed, a protocol amendment will be written to add the matrices and methods to the protocol. For analysis performed by the 3M Environmental Laboratory, the methods listed in the analytical methods section will be used. At the discretion of the StudyDirector, select analysis may be 1fwformed by a contract laboratory where competencehas been demonstrated, using validated analytical methods. If a contract laboratory is used, the methods and data provided to the Study . Director will be identified in the final report. 5.0 - EXPERIMENTALnalvtical Methods 5.1 For analysis performed by the 3M EnvironmentalLaboratory, the followingmethods will beused: ~ 5.1.1 FACT-M-1.0, Extraction of Potassium Peffluorooctanesulfonateor Other Anionic Surfactants from Liver for Analy!;is Using HPLC-ElectrospraylMas Spectrometry 5.1.2 FACT-M-2.0, Analysis of Fluorochemicds in Liver Extracts Using HPLCElectrosprayMass Spectrometry FACT-TOX-97,U2452 Arms #418-010 Page 3 of 5 3M Ehvironrnental Laboratory Page 22 3M Medical Department Study: T-6316.8 BACK TO MAIN Analytical Report: FACT TOX-097 LRN-U2452 j 5.1.3 FACT-M-3.0, Extraction of Potassium Peduorooctanesulfonate or Other Anionic Surfactants from Serum for Analysis Using HPLC-Electrospray/Mass Spectrometry 5.1.4 FACT-M-4.0, Analysis of Fluorochemicds in Serum Extracts Using HPLC- ElectrosprayNas 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 Data Reporting: For analysis performed by a contract laboratory, the contract laboratory, willprovide all data to the analyticalphase Study Director, and copies of the methods ' will be attached to the data. The contract laboratory and the data it provides will be identified in the data packet provided by the analytical phase Study Director to the Sponsor. 6.2 Data transformationsand analysis: Data will be reported as the concentration (weightlweight) of target analyte per tissue or sample, or of target analyte per unit of tissue or fluid. 6.3 Statistical analysis: Statistics used may include regression analysis of the serum concentrations over time, and standard deviations calculated for the concentrations within each dose group. If necessary, simple statistical tests, such as Student's t test, may be applied to evaluate statistical difference. 7.0 MAINTENANCEOFRAW DATAAND RECORDS 7.1 The following raw data and records will be retained in the study folder in the archives according to AMDT-S-8: I 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, Equipmcmt Procedures, and Methods FACT-TOX-97, U24.52 Argus #418-010 Page 4 of 5 3M Environmental Laboratory Page 23 3M Medical Department Study: T-6316.8 BACK TO MAIN Analytical Report: FACT TOX-097 LRN-U2452 1 i 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: I, 9.1.1 FACT-M-1.0, Extraction of Potassium Perfluorooctanesulfonateor Other Anionic Surfactants from Liver for Analysis Using HPLC-ElecttosprayMass Spectrometry 9.1.2 FACT-M-2.0, Analysis of Fluorochemicals in Liver Extracts Using HPLC- ElectrospraylMass Spectrometry 9.1.3 FACT-M3.0, Extraction of Potassium I?effluorooctanesulfonateor Other Anionic Surfactantsfrom Serum for Analysis Using HPLC-ElectrosprayMas Spectrometry 9.1.4 FACT-M-4.0, Analysis of Fluorochemicals in Serum Extracts Using HF'LCElectrosprayMass Spectrometry 9.2 If a contract analytical laboratory performs analpis, copies of the validated methods performed will be attached to the data packet provided to the Study Director. FACT-TOX-97, U2452 Argus #418-010 Page 5 of 5 3M Environmental Laboratory Page 24 3M Medical Department Study: T-6316.8 BACK TO MAIN Analytical Report: FACT TOX-097 LRN-U2452 Study Title Analytical Study Oral (StomachTube) Development Toxicity Study of 2(N-Ethylperfluorooctanesulfonamide:)-ethanolin Rabbits PROTOCOL AMENDMENT NO. 1 Amendment Date: . 18 February 2000 Performing Labora&ory 3M Environmental Technology & Safety Services 3M Environmental Laboratory 935 Bush Avenue St. Paul, MN 55106 Laboratory Project Identification ET&SS LRN-U2452 FACT TOX-097 Argus Study: 418-010 3M Medical Department Stud!,: T-63 16.8 3M Environmental Laboratory 3hM- at Laboratory Page 25 BACK TO MAIN 3M Medical Department Study: T-6316.8 Analytical Report: FACT TOX-097 LRN-U2452 PrOtOCOl LRN-U2452 Amendment Number 1 This amendment modifies the following portion@)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 effortto ensure compliancewith Good Laboratory Practice Standardsthat outline study personnel 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. Butenhoff, PbD., as of 18 February 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 responsibilitiesand workload are more evenly balanced. 3M Environmental Laboratory w- : Page 26 BACK TO MAIN 3M Medical Department Study: T-6316.8 Analytical Report: FACT TOX-097 LRN-U2452 PrOtOCOl LRN-U2452 Amendment Number f 3. PROTOCOL READS: Method numbers and revisions: FACT-M-1.O, Extraction of Potassium Perfluorooctanesulfonateor Other Anionic Surfactantsfrom Liver for Analysis Using HPLC-Electrosprayhlass Spectmmetry FACT-M-2.0, Analysis of Fluorochemicalsin Liver ExtractsUsing HPLC-Electrospray/ Mass Spectrometry FACT-M-3.O, Extraction of Potassium Perfluorooctanesulfonateor Other Anionic Surfactantsfrom Serum for Analysis Using HPLC-E1ectrosprayMa.w Spectrometry FACT-M-4.0, Analysis of Fluorochemicalsin Serum Extracts Using HPLC-Electrosprayhlass Spectrometry AMEND TO READ: Method numbers and revisions: ETS-8-6.0 "Extraction of Potassium Perfluorooctanesulfonateor Other FluorochemicalCompounds from Liver for Analysis Using HPLC-Electrosprayhlas Spectrometry" ETS-8-7.0 "Analysis of Potassium Perfluorooctanesulfonate or Other Fluorochemical Compounds in Liver Extracts Using HPLC-ElectrosprayIMassSpectrometry" ETS-8-4.1, "Extraction of Potassium Perfluorooctanesulfonateor Otlier FluorochemicalCompounds from Serum for Analysis Using HPLC-Electrosprayh4as Spectrometry" ETS-8-5.1, "Analysis of Potassium Perfluorooctanesulfonateor Other FluorochemicalCompounds in Serum Extracts HPLC-ElectrosprayMass Spectrometry" REASON: New methodologieswere implementedfollowingthe approval of the original protocol for FACT TOX-097. 3M Environmental Laboratory 3M Einvironmental Laboratory Page 27 BACK TO MAIN 3M Medical Department Study: T-6316.8 Analytical Report: FACT TOX-097 LRN-U2452 PrOtOCOl LRN-U2452 Amendment Number 1 4. PROTOCOL READS: 7.1 The followingraw data and records will be retained in the studyfolder in the archives according to AMDT-S-8: 7.1.1 7.1.2 7.1.3 7.1.4 7.1.5 Approved protocol and amendments Study correspondence Shipping records Raw data Electronic copies of data 7.2 Supportingrecordsto be retained separatelyfrom the study folder in the archives accordingto AMDT-S-8 will include at least the following: 7.2.1 7.2.2 7.2.3 7.2.4 7.2.5 Training records Calibration records Instrument maintenance logs StandardOperatingProcedures, EquipmentProcedures,and Methods Appropriate specimens AMENDTO READ: "The original data, or copies thereof,will be availableat the 3M Environmental Laboratory to facilitate audits of the study during its progress and before acceptanceof the final report. When the final report is completed,all originalpaper data, including: approved protocol and amendments, study correspondence,shipping records, raw data, approved final report, and electronic copies of data will be retained in the archivesof the 3M EnvironmentalLaboratory. All correspondingtraining records, calibration records, instrument maintenance logs, standard operating procedures, equipment procedures, and methods will be retained in the archives of the facility performing each analysis." REASON: To direct subcontract laboratoriesin the dispositionof the items listed above. 5. PROTOCOL READS: 3.1 Test, control, and referencesubstancesand matrices 3.1.2 .Analytical reference substance matrix: Rabbit liver and serum 3.1.4 Analytical control substance matrix: Rabbit liver and serum AMENDTO READ: 3.1 Test, control, and reference substancesand matrices 3.1.2 Analytical referencesubstancematrix: Rabbit liver, serum, and pooled fetal tissue(s) 3.1.4 Analyticalcontrol substancematrix: Rabbit liver, serum, and pooled fetal tissue(s) REASON: Analysis of fetal tissue for the target chemical andor its a!nalyteswas added to the scope of the study following tlie issuance of the original protocol. 3M Environmental Laboratoty 3M Environmental Laboratory Page 28 BACK TO MAIN 3M Medical Department Study: T-6316.8 Analytical Report: FACT TOX-097 LRN42452 P ~ O ~ OLCRON4~2452 Amendment Number I 6. PROTOCOL READS: The analytical portion of this study is designed to evaluate levels of potassium perfluoro- octanesulfonate(PFOS),or another meta%oliteof 2(N-Ethylpeffluorooctanesulfonamido)-ethanol(N- EtFOSE-OH)determined by the StudyDirector, in liver arid serum samples of the test system when it is administered directly through a stomach tube. AMENDTO READ: The analytical portion of this study is designed to evaluate levels of potassium peffluorooctanesulfonate(PFOS),or anothermetaboliteof 2(N-E~ylperfluorooctanesulfonamido)-ethanol(NEtFOSE-OH)determinedby the StudyDirector, in liver, serum, and fetal tissue(s) samples of the test system when it is administereddirectly through a stomachtube. REASON: Analysis of fetal tissue for the target chemical andlor its analytes was added to the scope of the study following the issuance of the original protocol. 7. PROTOCOL READS: 3.8 Disposition of test andlor control substances: Biological Tissues and fluids are retained per GLP regulation. AMENDTO READ: 3.8 Specimens will be maintained in the 3M EnvironmentalLaboratoryspecimen archives. All specimens sent to sub-contract laboratorieswill be returned to the 3M EnvironmentalLaboratory upon completion of analysisand submissionof the sub-contractlaboratory(s)final report. The specimenswill be returned with the followingdocumentation: the signed original chain of custody and records of storage conditionswhile at the sub-contractfacility. REASON: To direct subcontract laboratoriesin the disposition of the items listed above. 3M Environmental Laboratory 3M Einvironrnental Laboratory Page 29 BACK TO MAIN 3M Medical Department Study:T-6316.8 Analytical Report: FACT TOX-097 LRN-U2452 Protocol LRN-U2452 Amendment Number 1 8. PROTOCOL READS: 3.2.3 Analytical control matrix 3.2.3.1 Rabbit liver -Argus Research Laboratories;traceability informationwill be included in the final report; or Rabbit liver - CovanceLaboratories; traceabilityinformation will be included in the final report AMENDTO READ: 3.2.3 Analytical control matrix 3.2.3.1 Rabbit liver -CovanceLaboratories; traceabilityinformation will be included in the final report REASON: Argus ResearchLaboratorieswill be conductingthe in life portion of the study. 9. PROTOCOL READS: 3.2.3 Analyticalcontrol matrix 3.2.3.1 Rabbit liver-Covance Laboratories;traceabilityinformation will be included in the final report 3.2.3.2 Rat Serum - Sigma Chemical Company;traceability information will be included in the final report AMENDTO READ: 3.2.3 Analytical control matrix 3.2.3.1 Rabbit liver-Covance Laboratories;traceabilityinformation will be included in the final report 3.2.3.2 Rat serum - Sigma Chemical Company;traceability information will be included in the final report 3.2.3.3 Pooled fetal tissue(s) - traceabilityinformationwill be included in the final report REASON: Analysis of fetal tissue was added to the scope of the studyfollowingthe issuance of the original protocol. 3M Environmental Laboratory 3M Environmental Laboratory Page 30 3M Medical Department Study: T-6316.8 Amendment Approval BACK TO MAIN Analytical Report: FACT TOX-097 LRN-U2452 Protocol LRN42452 Amendment Number 1 /Ad\ Marvin i? Case, D.K M , Ph.D.,Outgoing Sponsor Representative A-ed Date John L. Butenhofl Ph.D., Incoming Sponsor Representative Date + KrisfenJ Hans& Ph.D., Outgoing Study Director &?L" Marvin T.Case, D.KM.,Ph.D., Incoming Study Director 2Lj-A~bh Date P, kJ Date 3M Environmental Laboratory 3M EnvironmentalLaboratory Page 31 3M Medical Department Study: T-6316.8 BACK TO MAIN Analytical Report: FACT TOX-097 LRN-U2452 Study Title Oral (StomachTube) DevelopmentalToxicity Study of ;!@I-Ethylperfluorooctanesu1fonamido)- ethanol in Rabbits; PROTOCOL AMENDMENT 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 Identification FACT TOX-097 ETBSS LFU'J-U2452 Argus Study: 418-010 3M Medical Department Study:T-6316.8 3M EnvironmentalLaboratory 3M Environmental Laboratory Page 32 BACK TO MAIN 3M Medical Department Study: T-6316.8 Analytical Report: FACT TOX-097 LRN-U2452 Protocol FACT TOX-097 Amendment No. 2 This amendment modifies the following portion(s) of the protocol: 1. PROTOCORLEADS: There is not a principal analytical investigator assigned for this study. AMENDTO READ: The role of principal analytical investigator for the study was assigned to Kristen J. Hansen, Ph.D. as of the signing of this amendment. REASON: The role of principal analytical investigator was assigned in an effort to ensure compliance with Good Laboratory Practice Standards that outline study peisonnel requirements. 3M EnvironmentalLaboratory 3M~i- .. Page 33 3M Medical Department Study: T-6316.8 BACK TO MAIN Amendment Approval Analytical Report: FACT TOX-097 .. LRN-U2452 Protocol FACT TOX-097 Amendment No. 2 Q&&Z k2zzeZgy John L.. Bhtenhofi Ph.D., Sponsor Re&entative SO /VQK il.aor@ Date Marvin T.Case, D.V.M., Ph.D., StudyDirector -A7 bud Date 3M EnvironmentalLaboratory 3MnviF Page 34 3M Medical Department Study: T-6316.8 3M Environmental Technology and Services PO Box 3333 I St. Ped. MN 55 133-333I 617778 6412. BACK TO MAIN Analytical Report: FACT TOX-097 LRN-U2452 Study Title Oral (Stomach Tube) Developmental Toxicity Study of Z,(N-Ethylperfluorooctanesulfonamido)- ethanol in Rabbits PROTOCOL AMENDMENT NO. 3 Amendment Date: January 23,2001 Performing Laboratory 3M Environmental Technology & Safety Services 3M EnvironmentalLaboratory 935 Bush Avenue St. Paul,MN 55106 Laboratory Project lderntification FACT TOX-097 ET&SS LRN-U2452 Argus Study: 418-010 3M Medical Department Study:T-6316.8 3M Environmental Labqmtoty 3wEF ~ Page 35 BACK TO MAIN 3M Medical Department Study: T-6316.8 Analytical Report: FACT TOX-097 LRN-U2452 PrOtOCOl FACT TOX-097 Amendment No. 3 This amendment modifies the following portion(s) of the protocol: 1. PROTOCOL READS: Sections 3.0 and 4.0 identify PFOS as the analytical reference substance. AMENDTO READ: To include the additionalanalytical reference substan.c.es, PFOSA,PFOSAA, PFOSEA, and N-EtFOSE. REASON: To identify all the compounds that was analyzedinthe analytical phase of the study. 2. CONSISTENCY: The test article 2(N-Ethylperfluorooctanesulfonamido)-etlolis given many different abbreviations in the protocol, study, raw data,and analytical report. Suchas EtFOSE, N-EtFOSE, EtFOSE-OH, and N-EtFOSE-OH. CLARIFICAnON: These different abbreviations are equivalent. 3M Environmental Laboratory 3M-E- Page 36 3M Medical Department Study: T-6316.8 BACK TO MAIN Analytical Report: FACT TOX-097 LRN-U2452 Amendment Approval PrOfOCOl FACT TOX-097 Amendment No. 3 John L. Butenhog Ph.D., Sponsor Representative 26 Date u7&/ Marvin ?.' erne,D.K M , PhD., Study Director ~~ Kristen J Hunsen, Ph.D., Principal fhalytical1nvestigatc)r Date 3M Environmental Laboratory 310Et~i- Page 37 BACK TO MAIN 3M Medical Department Study: T-6316.8 Analytical Report: FACT TOX-097 LRN-U2452 Record of Deviation 1. Identification . Study / Project No. FACT-TOX-097 Argus 418-010 ..-_ . ...- - - - I _ _ _ . - - - Deviation Type (Check one) CI SOP CI Method --------------I_.-- 0 Protocol other: 0 Equipment Procedure __ --I-------------_-- Doc6xNumber(s):%-cfr58.120 /1 (A) (11) ___I- I . - - - - - - - - - - - - Date(s) of occurrence: 9/18/98 /I. Description: Actual Procedureltxocess: ///. Actions Taken: -- (such as amendm-. ent issued, SOP revision, etc.) .-I--L ______I___ _T_h1is_d_e_v_i_at_io_n_1w_a_s_w_r_i-tt-e--n-.--I.-n-_th_e_f_u_tu_r_e_, _an_a_l_y_ti_ca_l_p_r_o_to1c_o_ls_w1_il_l _b_e_sIigned by the Sponsor . Representativebefore the S-t--u--d--y--Director. --I_ I _ - - - - - - - ---____-___ll_l_-_-.-- 1-1--------.--- - & + A &WIV. lmpact on Study/ ,Project 3M Envirohmentol Lnboratoly Form ETS-4-8.0 3bEft1- y u- Deviation No. 1 (assigned by Study Director or Project Lead at the end of study or project) 'Page 38 3M Medical Department Study: T-6316.8 BACK TO MAIN i Record of Deviation I. Identification Analytical Report: FACT TOX-097 LRN-U2452 1 . . 10/17/98,09/28/99 11. Description: Ill. Actions Taken: I I ~4Pi/oo on IV. Impact Study/ Project - ----_-________.I__ ---_--. __.__-- -.-.-I-_----_ I _ _ _. --____-- -A-l--t-houghno method was listed on 10/17/98,method FACT-M-2.0 was followed as_d--_e..t-ermined ----_-_I__ _ - _-_I_ - by parameters documented in the raw data. On 09/28/99 _--____,__._____I._I_ the extraction method_wa_s--w-.-ritte-.-.n in error analyticalmethod ETS-8-7.0 was used as determinedby parameters listed in ~ _____.____-____._______-_________11.__1..__.I-----._-----_-L--_--.--.---_-- the raw data. This me_t_hI_o_d-.is an improvement over FACT-M-2.0. No_a_dverse affect on these --__I___ ~ - - - - _ _ - _ I I _ _ _ data. ________.______-.---.--- -I___ C_I___.__._____l__ ~ _._I_ Authorized By (Study Director /Project Lead) 3M Environmental Laboratory Form ETS-4-8.0 Deviation No. (assigned by Sludy Director or Project Lead at the end of study or project) Page 39 BACK TO MAIN 3M Medical Department Study: T-6316.8 Analytical Report: FACT TOX-097 LRN-U2452 Record of Deviation I. Identification _-_I_._-___-I___-- Deviation Type (Check one) DTCGZGt NUber(S): --------- I_(---- _ - _ - _ _ _ _ _ _ _ l _ _ l _ l _ __.. -I 0 SOP 17 Method 0 Equipment Procedure XProtocol ---1 - FACT-TOX-097 0 Other: : Date(s) of - - - I - - - occurrence: 10/20/98, 10/23198,07/29/99 /I. Description: - A---cI-t-u-_a_l_P._ro_cedurdpr.o_c_e_s-s:I----I-------_---____. - I On 10/20/98 no method was listed, on 10123198 method FACT-M-4.1 was used, and on ---_.-I------ .--I__ _____ _ - I - - - _ _ 07/29/99 method ETS-8-7.0 (a liver method) was listed. 3M Environrnenid Laborntory Form ETS-4-8.0 3w- I Laboratory Y SLJ.3 D.re&: Ptwv Cn+cDeviation No. 7 2 (assignedby !Study Director or Project Lead at the end of study or PVOJoJect) Page 40 BACK TO MAIN 3M Medical Department Study: T-6316.8 Analytical Report: FACT TOX-097 LRN-U2452 Record of Deviation I. Identification Study / Project No. TOX0097 (-L- I-M--S--#-U-2.4.5-2-)- -- ---- -.-- ----- __ __ _ ---I-__ __-__.- --. Deviation type (Check one) 0 SOP X Method flEquipment Procedure I C1FYotocol _"- I - ) . - I-- --.- ---- --- C1 othe--.r.---: Document number FACT-M-2.1, ETS-8-5.1 and Date(s) of occurrence ~ ---l-lll._________ ETS-8-7.0 11125l98,9129199 and 7129199 /I. Description -R--e-q--u-i-r-ed procedurelprocess: Section 14.2.1:Solvent blanks, method blanks, and mat6~lankms ust be below the GLeSt---- .-sl_t.l_a.-_nI--d- ard on the calibration cu- rve. ~ --_- I _-_I- - Actual procedurelprocess: - -Occasionally, the first solvent blank injected for a runwas above the LOQ. _ I - - -____ - - - - - - - - - - - I - - - - - I _ - - - - - -.----I ___-_-~----- In each place a high solvent blank was analyzed, additional solvent blanks or method blanks were analyzed immediately following the high blank. This second injectionwas below the LOQ. Occassionally, the first injection of a run is high because it may immediately follow injection of a high standard from a previous run. For this reason,more than one blank is typically analyzed prior to the start of a calibration curve. These second and third, etc. blanks are below the LOQ and are more representative of the analytical conditions of the samples;the lls--t_u_d__yll_dl.a---t-a---_is---n---o---t--_a_d_v_e-_r_s_ely affected. kI -h I~llOlCI $,c,,ivf, ~~i\,n,,..,k,,h.it' ~U-dJkn~b:m c . C~ ast Deviation No. 't (assigned by Study Director or Project Lead at the end of study or project) Attachment A: Record of Deviation 3lbSE+ivimmental Laboratory ETS-4-8.0 Page 1 of 1 Page 41 3M lbledical Department Study: T-6316.8 Appendix C: Extraction and Analytical Methods BACK TO MAIN Analytical Report: FACT TOX-097 LRN-U2452 This appendix includes the following methods: FACT-M-1.O, "Extractionof Potassium Perfluorooctanesulfonateor Other Anionic FluorochemicalSurfactantsfrom Liver for Analysis Using HPLC-Electrospray/Mass Spectrometry," (8 pages) FACT-M3.0, "Extractionof Potassium Perfluorooctaneor Other Anionic Fluorochemical Compounds from Serum or Other Fluids for Analysis Using HPLC-Electrospray/Mass Spectrometry," (8 pages) ETS-8-04.1, "Extractionof Potassium Perfluorooctanesulfonateor other Fuorochemical Compoundsfrom Serum for Analysis Using HPLC-Electrospray/MassSpectrometry,''(14 pages) ETS-8-06.0, "Extractionof Potasium Petfluorooctanesulfonateor other Fluorochemical Compoundsfrom Liver for Analysis Using HPLC-Electrospray/MassSpectrometry,'' (14 pages) FACT-M-2.0, "Analysis of Fluorochemicals in Liver Extracts Using HPLC-Electrospray/Mass Spectrometry,''(8 pages) FACT-M-4.0, "Analysis of Fluorochemicals in Serum Extracts Using HPLC-Electrospray/Mass Spectrometry,'' (8 pages) ETS-8-05.1, "Analysisof PotassiumPerfluorooctanesulfonateor other Fuorochemical Compounds in Serum ExtractsUsing HPLC-Electrospray/MassSpectrometry,"(9 pages) ETS-8-07.0, "Analysisof Potassium Perfluorooctanesulfonateor other Fluorochemical Compounds in Liver Extracts Using HPLC-Electrospray/MassSpectrometry,"(10 pages) 3M E:nvironmentalLaboratory Page 42 3M Medical Department Study: T-6316.8 BACK TO MAIN Analytical Report: FACT TOX-097 LRWU2452 METHOD EXTRACTION OF POTASSIUMPERFLU0ROOCTA;NESU"ONATE OR OTlCIERANIONIC FLUOROCHEMICAL SURFACTANTS FROM LIVER FOR ANALYSIS USING HPLC-ELECTROSPRAYMASS SPECTROMETRY Method Number: FACT-M-1.O ' AU~IIO~:Lisa Clemen Approved By: AdoptionDate: 5/.26/F d Revision Date: b/,4 Group Leader S/zb /w Date 1.0 SCOPE AND APPLICATION I.lScope: This method is for the extraction of PotassiumPerfluorooctanesulfonate(PFOS)or other fluorochemid surfactantsh m liver. 1.2 Applicable Compounds: Fluorochemicalsurfactantsor other fluorinatedcompounds. 1.3 Matrices: Rabbit, rat, bovine, and monkey livers or other livers as designated in the validation report. Microsoft 7.0.1195 FACT-M-1.O Extraction of PFOS fiom Liver Page 1 of 8 3M EInvironrnentalLaboratory Page 43 3M Medical Department Study: T-6316.8 BACK TO MAIN Analytical Report: FACT TOX-097 LRN-U2452 2.0 SUMMAROYFMETHOD 2.1 This method describes how to extract potassium perfluorooctanesulfonate(PFOS) or other fluorochemicalsurfactan@fiom liver using ion pairing reagent and 5.0 m L s of ethyl acetate. An ion pairing reagent is added to each sample and partitioned into ethyl acetate. Four mLs of extract is removed to a centrifuge tube and put onto a nitrogen evaporator until dry. Each extract is reconstituted in 1.0 mL methanol then filtered through a 3 cc plastic syringe attached to a 0.2 pm filter into glass autovials. 3.0 DEFINITIONS 3.1 None. 4.0 WARNINGS AND CAUTIONS 4.1 Health and Safety Warnings: 4.1.1 Use universal precautions when handling d m a l livers, they may contain pathogens. , 5.0 INTERFERENCES 5.1 There are no known interferences at this time. 6.0 EQUIPMENT 6.1 The following equipment is used while carrying out this method. Equivalent equipment is acceptable. 6.1.1 Ultra-Turrax T25 Grinder for grinding liver samples 6.13 Vortex mixer, VWR, Vortex Genie 2 6.13 Centrifuge, Mistral 1000 or IEC 6.1.4 Shaker, Eberbach or VWR 6.1.5 Nitrogen Evaporator, Organomation 6.1.6 Balance 7.0 SUPPLIESAND MATERIALs 7.1 Gloves 7.2 Dissecthg scalpels 7.3 Eppendorf or disposable pipettes 7.4 Nalgene bottles, capable ofholding 250 mL and 1L 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 from Liver Page 2 of 8 3M Environmental Laboratory Page 44 3M Medical Department Study: T-6316.8 BACK TO MAIN Analytical Report: FACT TOX-097 LRN-U2452 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 from 3 separate vials. 8.0 REAGENTS AND STANDARDS 8.1 Reagents 8.1.1 Sodium Hydroxide (J.T 200 grams NaOH. Pour Baker into a 1o0r0e0qumivLableenatk),er(NcoaOntHai)ni1nOgN5.0w0eliigtehrsap(pLr)oMxiimllia-tQe1A water, mix until all solids are dissolved. Store in a 1 L nalgene bottle. 8.1.2 Sodium Hydroxide (J.T Baker or equivalent), (NaOHJ 1N. Dilute ION 1:10. Measure 10 mL of the 1ONNaOH solution into a 100mL volumetric flaskand dilute to volume using Milli-Qmwater. Store in a 125mL nalgene bottle. 8.1.3 Tetrabutylammonium hydrogen sulfate (Kodak or equivalent), (TBA) 0.5M: W e i e approximately 169 grams of TBA into a 1 L volumetric containing 500 L Milli-Q water. Adjust to pH 10using approximately 64mL 1ONNaOH and dilute to volume with Mi&-Q" 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 ensure pH = 10. Adjust as needed using 1N NaOH solution. 8.1.4 Sodium carbonatdSdum Bicarbonate Bifler (J.T. Baker or equivalent), (N%CO,/NaHCO,) 0.25M.Weigh approxhately 26.5 g of sodium carbonate (Na.&03)and 21.0 g of sodium bicarbonate (NaHCO,) into a 1 L volumetric flask and dilute to volume with Milli-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 frozen fiom testing laboratory. 8.1.9 Milli-Qm water, all water used in this method should be Milli-Qm 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 .O Extraction of PFOS from Liver Page 3 of 8 3M Environmental Laboratory Page 45 3M Medical Department Study: T-6316.8 BACK TO MAIN Analytical Report: FACT TOX-097 LRN-U2452 8.2.2 Weigh approximately 100 mg of PFOS into a 100 mL volumetric flask and record the actual weight. 8.2.3 Bring to volume with methanol for a stock standard of approximately 1000ppm (PdW. 8.2.4 Dilute the stock solution with methanol for a working standard 1 solution of approximately 50 ppm. 8.2.5 Dilute the stock solution with methanol for a working standard 2 solution of approx. 5.0 ppm. 8.2.6 Dilute the stock solution with methanol for a working standard 3 solution of approx. 0.50 ppm. 9.0 SAMPLEHANDLING 9.1 All livers are received'fiozenand must be kept frozenuntil the extractionis performed. 10.0 QUALITY CONTROL 10.1 Matrix Spikes 10.1.1 Prepare and analyze matrix spike and matrix spike duplicate samples to determine the accuracy of the extraction. 10.1.2 Prepare each spike using liver chosen by the analyst, usually a control liver. 10.13 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 . continuedlinearity of the initial calibrationcurve. 103.2 One check is preparedper group of ten samples. For example, if a sample set = 34, four checks are prepared and extracted. 10.23 Prepare each continuing calibration check from the same liver homogenate used to prep the initial curve. 10.2.4 The expected concentrationwill 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 rrL Nalgene bottle containing 200 m L s Milli-QTMwater. Grind to a homogeneous solution. 11.'1.2 If 40 g is not available, use appropriate amounts of liver and water in keeping with a 1 5 ratio. 11.1.3 See section 13.0 to calculate the actual density of liver. FACT-M-1 .O Extraction of PFOS from Liver Page 4 of 8 3M Environmental Laboratory Page 46 3M Medical Department Study: T-6316.8 BACK TO MAIN Analytical Report: FACT TOX-097 LRN-U2452 Working Standard (Approx. Conc.) 0.50 ppm 0.50 ppm 0.50 ppm 5.0 ppm 5.0 ppm 5.0 ppm 50 PPm PL Approx. final conc. of - PFOS in liver Blank 4 0.010 ppm H;: 20 0.050 ppm 4100 20 30 4 11.1.1 See section 13.0to calculate actualconcentrations of PFOS in calibration standards. 11.2 ExtrFt spiked liver homogenates following 12.14C12.24 of thismethod. Use these standards to establish each initial curve on the mass spectrometer. 12.0 PROCEDURES 12.1 Ob.tain frozen liver samples. In spent tissue, note that the liver has not been packaged with other tissues. 123 Cut approximately 1 g of liver using a dissecting scalpel. 12.3 Weigh the sample directly into a tared plastic sanipule vial. 12.4 Record the liver weight in the study notebook. 12.5 Label the sampule vial With the study number, weight, liver ID, date and analyst initials. 12.6 Add 2.5m L s 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 m L s waiter 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 I aye%?- 3bl Medical Department Study: T-6316.8 BACK TO MAIN Analytical Report: FACT TOX-097 LRN-U.2452 12.11Pipette 1 mL homogenate into a 15 rnL polypropylene centrikge tube. Label the centrifuge tube with the identical information as the sampule vial. (See Worksheet for documenting the remaining steps.) 12.12 Spike liver homogenates with the appropriate amount of PFOS standard as described in section 11.1 or Table 1. 12.13 Pipette two 1 mL aliquots of Milli-Qm water to centrifuge tubes. These will serve as instrument blanks. 12.14Add 1 mL 0.5 M TBA and 2 mL of the 0.25 M scdiurn carbona.te/sodiumbicarbonate buffer. 12.15 Using a volumetric pipette, add 5 mLs ethyl acetaite. 12.16 Cap each sample and put on the shaker for 20 minutes. 12.17 CentrZuge for 20 to 25 minutes, until layers are well separated. Set power on the centrifbge to approximately 3500 rpm. 12.18Remove 4 d s of organic layer, using a 5 mL gruluated glass pipette, to a clean 15 mL centrifuge tube. Label this fresh tube with the same informatiori as in 12.5. 12.19 Put each sample on the analytical nitrogen evapoiator until dry,a,pproximately 2 to 3 hours. . 12.20 Add 1.0 mL of methanol to each centrifuge tube using a graduated pipette., 12.21 Vortex mix for 30 seconds. 12.22 Attach a 0.2 pnnylon 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 withthe study number, animal number and gender, sample timepoint, matrix, final solvent, extraction date, and analyst(s) who performed the extraction. 12.24 Cap and hold for electrospray mass spectrometry analysis. 1225Completethe worksheetandtape to page ofstudy notebook. 13.0 DATAANALYSAISND CALCULATIONS 13.1 Calculations: 13.1.1 Calculate the density of liver (mg) in 1.0 i n L homogenate using the following equation: g of Liver x Average weight of ten 1m L aliquots (m& (g of Liver + g of Water) 3M Einvironmental Laboratory FACT-M-1 .O Extraction of PFOS from Liver Page 6 of 8 3M Medical Department Study: T-6316.8 BACK TO MAIN Analytical Report: FACT TOX-097 LRN-U2452 13.1.2 Calculate actual concentrations of PFOS in calibration standardsusing the following equation: pL of Standard x Concentration (pg /d,=!Final Concentration (pg/g or m a g ) mg Liver.! 1 mL 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 mL of Milli-Q water. 14.0 METHOPDERFORMANCE 14.1 The method detection limit is equal to half the lowest standard in the calibration curve. 15.0 POLLUTIONPREVENTION AND WAsTE MANAGEMENT 15.1 Sample waste is disposed in biohazard containers,flammable solveptwaste is disposed in high BTU containers,and used glass pipette wasti: is disposed in broken glass containers located in the laboratory. 16.0 RECORDS 16.1 Complete the extractionworksheet and tape into ihe study notebook. 17.0 TABLESD, IAGRAMSF,LOWCHARTS, AND VALIDATION DATA 17.1 The validation report associatedwith this method is FACT-M-11.0 & 2.0-V-1. 18.0 REFERENCES 18.1 AMDT-EP-22, "Routine Maintenance of Ultra-Turrax T-25" 19.0 AFFECTEDDOCUMENTS 19.1 FACT-M-2, ``Analysisof Liver Extracts for Fluorochemicalsusing HPLC-Electrospray Mass Spectrometry" 20.0 REVISIONS Revision Number. Reason For Revis& Revision 3M Environmental Laboratory FACT-M-1 .O Extraction of PFOS from Liver Page 7 of 8 - - - BACK TO MAIN 3M Medical Department Study: T-6316.8 I Study# I Extraction Worksheet for FACT-M-I Sample I PFOS I PFOSI Analytical Report: FACT TOX-097 LRN-U2452 PFOS I Date and I I I I 1 I 1 - Liver amount Temocmturc Vortex 30 SCC. Filter using a 3cc B-D syringewith a0.2umSRI filterinto a 1.5 mL autosamplevial - - MS/MSD/- Cont. Checks: Spiked uL of a ppm std ( ) for a final concentration of . ppm. MS/MSD used sample Cont. Checks used same homogenateas for std curve. FACT-M-1 .O Extraction of PFOS from Liver Page 8 of 8 3M E:nvironrnentalLaboratory 3M Medical Department Study: T-6316.8 3M ENVIRONMENTLAALBORATORY BACK TO MAIN Analytical Report: FACT TOX-097 LRN-U2452 EXTRACTIOONF POTASSmM PERl%uOROOCTANESULFONATE OR OTHERANIONIC FLUOROCHEMICSAULRFACTANTSFROM :SERUMFOR h7ALYSIS USING HPLC-ELECTROSPRAYLMASPSSECTRON~~TRY Method Number: FACT-M-3 .O Author: Lisa Clemen Adoption Date: I t x l q 8 Revision Date: Group Leader & ,&p Technical Reviewer ffz*/Gf Y Date YIzt198 Date 1.0 SCOPEANDAPPLICATION 1.1 Scope: This method is for the extraction of potassiim perfluorooctanesulfonate (PFOS) or other fluorochemical surfactants from serum. 1.2 Applicable Compounds: Fluorochemical surfactants or other fliiorinated compounds. 1.3 Matrices: Rabbit, rat, and bovine serum or other si:ra as designated in the validation report. Microsoft 7.0.1195 3M Ehvironrnental Laboratory FACT-M3.0 Extraction of PFOS from Serum Page 1 of 8 Page 51 3M IWedical Department Study: T-6316.8 BACK TO MAIN Analytical Report: FACT TOX-097 LRN-U2452 2.0 SUMMAROYFMETHOD 2.1 This method describes how to extract potassium perfluorooctane:sulfonate(PFOS)or other anionic fluorochemical surfactants from serum usirtg an ion pairing reagent and 5.0 mL of ethyl acetate. 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 arid 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 pm nylon filter into glass autovials. 3.0 DEFINITIONS 3.1 None. 4.0 WARNINGASND CAUTIONS 4.1 Health and Safety Wadngs: 4.1.1 Use universal precautions, especially laboratory coats, goggles, and gloves when handling animal serum, it may contain pathogens. 5.0 INTERFE~NCES 5.1 There are no known interferences at this time. 6.0 EOUIPMENT 6.1 The following equipment is used while canying out 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 V W R 6.1.4 Nitrogen evaporator, Organomation 6.1.5 Balance, (k 0.100 gm) 7.0 SUPPLIES AND MATERIALS 7.1 Gloves 7.2 Eppendorf or disposable pipettes 7 3 Nalgene bottles, capable of holding 250 mL and 1L 7.4 Glass, type A, volumetric flasks 7.5 40 mL glass I-CHEM vials 7.6 Polypropylene centrifuge tubes, 15mL 7.7 Labels 7.8 Syringes, capable of measuring 10 pL to 50 pL 7.9 Glass, type A, volumetric pipettes 7.10 Graduated pipettes FACT-M-3.O Extraction of PFOS from Serum Page 2 of 8 3M Environmental Laboratory Page 52 - 3M lvledical Department Study: T-6316.8 - BACK TO MAIN Analytical Report: FACT TOX-097 LRN-U2452 7.11 Electronic pipettor, Eppendorf or equivalent 7.12 Timer 7.13 Disposable plastic 3 cc syringes 7.14 Filters, nylon syringe fdters, 0.2 pm,25 mm 7.15 Crimp cap autovials Note: Prior to using glassware and bottles, rinse 3 times with methanol and 3 times with MilliQ" water. Rinse syringes a minimumof 9 times with methanol,,3 rinses from 3 separate Vials. 8.0 REAGENTS AND STANDARDS 8.1 Reagents 8.1.1 S o d i m hydroxide (J.T Baker or equivalent), (NaOH) 1 0 " weigh approximately 200 grams NaOH. Pour into a 1000mL beaker containing 500 liters Q Milli-Q"" water, mix until all solids are dissolved. Store in a 1 L Nalgene bottle. 8.1.2 Sodium hydroxide (J.T Baker or equivalent), (NaOH) 114. Dilute 1ON 1 : l O . Measure 10&I of 1ONNaOH solution into a 100 mL volumetric flask and dilute to volume using Milli-QTMwater. Store in a 125mLNalgene bottle. 8.13 %k Tetrabutylammonium hydrogen sulfate (Kodak or equivalent), (TBA) OSM: Wei approximately 169 grams of TBA into a 1L volumetric containing 500 L Milli-Q water. Adjust to pH 10using approximately 64mL of 1ON NaOH and dilute to volume withMilli-QTMwater. Add NaOH: slowly while adding the last mL of NaOH because the pH changes abruptly. Store in a 1 L Nalgene bottle. 8.1.3.1 TBA requires a check prior to each use to ensure pH = 10. Adjust as needed using 1N NaOH solution 8.1.4 Sodium carbonate/sodium bicarbonate buffer (J.T. Baker or equivalent), (Na+20,MaHC03) 0.25M.Weigh approximately 26.5 g of sodium carbonate (NqCO,) and 21.O g of sodium bicarbonate (NaHCO,) into a 1L volumetric flask and bring to volume with Milli-QTMwater. Store in a 1 L nalgene bottle. 8.1.5 PFOS (3MSpecialty Chemical Division).,molecularweight = 538. 8.1.6 Other fluorochemicals,as appropriate. 8.1.7 Ethyl Acetate, Omnisolv, glass distilled or.HPLC grade. 8.1.8 Methanol, Omnisolv, glass distilled or HPLC grade. 8i1.9 Serum, frozen liquid from Sigma. 8.1.10 Control serum received with each sampk: set. 8.1.11 Milli-QTMwater, all water used in this mlethodshould lbe Milli-Qm water and may be provided by a Milli-Q TOC Plus systcm. 3Nl Environmental Laboratory FACT-M3.O Extraction of PFOS liom Serum Page 3 of 8 3M Medical Department Study: T-6316.8 BACK TO MAIN Analytical Report: FACT TOX-097 LRN-U2452 8.2 Standards 8.2.1 Prepare PFOS standards for the standard curve. 8.2.2 Prepare other fluorochemical standards, as appropriate. 8.2.3 Weigh approximately 100 mg of PFOS into a 100 mL volumetric flask and record the actual weight. 8.2.4 Bring to volume with methanol for a stock standard of approximately 1000ppm (vg/mL). 8.2.5 Dilute the stock solution withmethanol for a working standard 1solution of approximately 50 ppm. 83.6 Dilute the stock solution withmethanol for a working standard 2 solution of approx. 5.0 ppm. 8.2.7 Dilute the stock solution with methanol for a working stzlndard 3 solution of approx. 0.50 ppm. . I 9.0 SAMPLHEANDLING 9.1 All sera are received frozen and must be kept frozen until the exlkaction is performed. 10.0 OUALITY CONTROL 10.1 Matrix Blanks and Method Blanks 10.1.1 Two 1.O mL aliquots of the serum are extracted following this procedure and used as matrix blanks. See section 11.1.2. 10.1.2 Two 1.0 mL aliquots of Milli-Q" water are extracted following this procedure and used as method blanks. 10.2 Matrix Spikes 10.2.1 Prepare and analyze matrix spike and matrix spike duplicate samples to determine the accuracy of the extraction. 10.2.2 Prepare each spike using serum chosen by the analyst, uually control serum received with each sample set. 10.2.3 Expected concentrations Will fall in the mid-range of the initial calibration curve. Additional spikes may be included and may fall in the low-range of the initial calibration curve. 103 ContinuingCalibration Checks 10.3.1 Prepare and analyze continuing calibration check samples to determine the continued linearity of the initial calibration curve. 10.3.2 One check is prepared per group of ten samples. For example, if a sample set = 34, four checks are prepared and extracted. 3M Erivironmental Laboratory FACT-M-3 .O Extraction of PFOS from Serum Page 4 of 8 -- 3M Medical Department Study: T-6316.8 BACK TO MAIN Analytical Report: FACT TOX-097 LRN-U2452 10.3.3 Prepare each continuing calibration check from the same serum used to prep the initial curve. 103.4' The expected concentration will fall within the mid-range of the initial calibration curve. 11.0 CALIBRATIAONNDSTANDARDIZATION 11.1 Prepare Serum Standards 11.1.1 Transfer 1 mL of s e m to a 15 mL centrifugetube. 11.1.2 If the majority of serum sample volumes ase less than 1.O mL, extract standards using serum volumes in the standards equal to the serum volumes in samples. Do not extract below 0.50 mL of serum. Record the serum volume on the extraction sheet. 11.13 Mix or shake between aliquots while preparing a total of sixteen aliquots of serum in 15 mL centrifuge tubes. . 11.1.4 Two 1 mL or appropriate aliquots serve as matrix blanks. Typically use the standard concentrations and spiking amourits listed in table 1to spike, in duplicate, two standard curves for a total of fourteen samples. 11.1.5 Refer to the validation report FACT-M-3.0-V-a1nd FACT-M4.0-V-1 which lists the working ranges for calibration curves. I 1 ' Approximate SpikingAmounts forStandards and Spikes Using 1.0 mL of Serum Working Standard (Approx. Conc.) - 0.500 pprn 20 5.00 ppm 5 5.00 ppm 10 5.00 ppm 20 50.0 ppm 5 50.0 ppm 10 50.0 ppm 15 I 11.1.4 See section 13.0to calcdate actual concentrations of PFOS in calibration standards. 11.2 Extract spiked serum standards following 12.6-12.16 of this method. Use these standards to establish each initial curve on the mass spectrometer. 3M E ivironrnental Laboratory FACT-M3.0 Extraction of PFOS from Serum Page 5 of 8 Page 5 5 3M Medical Department Study: T-6316.8 BACK TO MAIN Analytical Report: FACT TOX-097 LRN-U2452 12.0 PROCEDURES 12.1 Obtain frozen serum samples and allow to thaw. 12.2 Vortex mix for 15 seconds then remove 1.OmL or appropriatevolume to a 15 mL polypropylene centrifuge tube. 1 2 3 Return serum samplesto fieezer after extraction arnount has been removed. 12.4 Record the serum volume on the extraction worksheet. The final methanol volume will equal the initial serum volume. 12.5 Label the tube with the study number, serum ID, date and analyst initials. See attached worksheet for documentingthe remaining steps. 12.6 Spike serum with the appropriateamount of PFOS standard as described in section 11.1 or Table I for the calibration curve standards. Also spike matrix spikes and continuing calibration standards. 12.7 Vortex mix the standard curve samples, matrix spike samples, and continuing calibration samples for 15 seconds. 12.8 Add 1 mI, 0.5 M Tl3A and 2 mL of the 0.25 M sotiium carbonatdsodiumbicarbonate buffer. 12.9 Using a volumetric pipette, add 5 mL ethyl acetate. 12.10 Cap each sample and put on the shaker for 20 minutes. 12.11 Centrifuge for 20 to 25 minutes, until layers are wall separated, Set power on the centrifuge to approximately 3500 rpm. 12.12 Transfer 4 mL of organic layer, using a 5 mL graduated glass pipette, to a clean 15 mL centrifuge tube:Label this fresh tube with the same information as in 12.5. 12.13Put each sample on the analyticalnitrogen evaporator mtil dry,approximately2 to 3 hours. 12.14Add 1 .O mL or appropriate volume of methanol to each centrifugetube using a graduated pipette. (This volume equals the initial volume of serum used fixthe extraction.) 12.15Vortex mix for 30 seconds. 12.16Attach a 0.2 pnylon mesh filter to a 3 cc syringa and transfer the sample to this syringe. Filter into a 1.5 mL glass autovial. 12.17 Label the autovial with the study number, animal number and gender, sample timepoint, matrix, final solvent, extraction date, and analyst(;) who performed the extraction. 12.18 Cap and hold for HPLC-electrospray/massspectrometry analyds. Extracts may be stored at 4 O C until analysis. 12.19 Complete the extraction worksheet, attached to this document, ;md tape to page of study notebook. 3M Environmental Laboratory FACT-M-3 .O Extraction of PFOS from Serum Page 6 of 8 Page 56 3M Medical Department Study: T-6316.8 BACK TO MAIN Analytical Report: FACT TOX-097 LRN-U2452 13.0 DATAANALYSIASND CALCULATIONS 13.1 Calculations: 13.1.1 Calculate actual concentrations of PFOS,or other appropriate fluorochemical,in calibration standardsusing the following equation: mL of Standard x Concentration (UP / m k = Final Concentration (pg/mL) mL of Standard + Initial Serum Volume (mL) of PlFOS in Serum 14.0 METHODPERFORMANCE 14.1 The method detection limit is equalto halfthe lowest standard in the calibrationcurve. 15.0 POLLUTION PREVENTIONAND WASTE MANAGEMENT 15.1 Sample waste is disposed in biohazard containers, flammable solvent waste is disposed in high BTU containers, and used glasspipette waste is disposed in brokh glass containers located `inthe laboratory. 16.0 RECORDS 16.1 Complete the extractionworksheet attached to this method, and tape into the study notebook. 17.0 TABLESD,IAGRAMSF,LOWCHARTASN.D VALIDATION DATA 17.1 The validation report associatedwiththis method is FACT-M-3.0 & 4.0-V-1. 18.0 REFERENCES 18.1 None 19.0 AFFECTEDDOCUMENTS 19.i FACT-M-4, "AnstysisofSerum Extracts for Fluorochemicalsusing HPLC-Electrospray Mass Spectrometry" 20.0 REVISIONS Revision Number. Reason For Revis& Revision 3M EnvironmentalLaboratory FACT-M-3.O Extraction of PFOS from Serum Page 7 of 8 -fJ- BACK TO MAIN 3M Medical Department Study: T-6316.8 Analytical Report: FACT TOX-097 LRN-U2452 Extraction Worksheet for FACT-M-3 Study # Sample Number set # H,O Blank Serum Blank PFOS approx. 0.5 ppm actual ppm #W PFOS approx. 5 ppm actual ppm #W I 1 PFOS Date and approx. 50 ppm Initials for actual ppm Std. or #W Comments I I I I unt = Shake 20 min. Centrifuge20-25 min. Centrifugespeed Removea4 mL aliquotof oraaniclayer Put on NitroRenEvaporatorto dryness Evaporator#: Temperature: Add methanol Volume mL TN-A- Vortex 30 sec. Filter usinga 3cc B-D syrinaewith a 0.2umSRI filter into a 1.5 mL autosample vial - MS/MSD/--Cont. Checks:Spiked 2uL of a ppm sttf ( ) for a final concentrationof . ppm. MS/MSD used sample Cont. Checksused same serum as for std curve. FACT-M-3.0 Extraction of PFOS from Serum Page 8 of 8 3M Environmental Laboratory 3M Medical Department Study: T-6316.8 BACK TO MAIN Analytical Report: FACT TOX-097 LRN-U2452 3M ENVIRONMENTALLABORATORY METHOD EXTRACTION OF POTASSIUM PERF'LUOROOCTANESULI~ONATEOR O"BER FLUOROCHEMICALCOMPOUNDSFROM SERUM FOR ANALYSISUSING HPLCELECTROSPRAY/MSAP~SESCTROMETI~Y Method Number: ETS-8-4.1 Adoption Date: 03/01/99 Author: Lisa Clemen,Glenn Langenburg Revision Date: qla;llqj Approved By: M,h Technical Reviewer ojlad44 Date 1.0 SCOPE AND APPLICATION 1.1 Scope: This method is for the extraction of potassium perfluorooctanesulfonate(PFOS) or other fluorochemical compounds f?om serum. 1.2 Applicable compounds: Fluorochemical surfactants 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 E nvironrnental Laboratory ETS-8-4.1 Extractionof PFOS from Serum Page 1 of 14 Page 59 3M Medical Department Study: T-6316.8 BACK TO MAIN Analytical Report: FACT TOX-097 LRN-U2452 2.0 SUMMARYOF METHOD 2.1 This method describes the procedure for extracting potassium perfluorooctanesulfonate (PFOS)or other fluomchemical surfactants from serum, or other fluids, using an ion pairing reagent and methyl-teri-butyl ether (MtBE).In this method, seven fluorochemicalswere extracted: PFOS,PFOSA,PFOSAA, EtlFOSE-OH, PFOSEA, M556,and surrogate standard (see 3.0 Definitions). An ion patiring reagent is added to the sample and the analyte ion pair is partitioned into MtBE. The MtBE extract is removed and put onto a nitrogenevaporator 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: perfluorooctanesuEonate (anion of potassium salt) C,F, 7SO; 3.2 PFOSA perfluorooctane sulfonylamide C,F,,SO,W 3.3 PFOSAA: perfluorooctane sulfonylamido (ethy1)lacetatec8F17~~0,N(CH,CH,)CH,c4' 3.4 EtFOSE-OH. 2(N-ethylperfluorooctanesulfonanlido)-ethyl alcohol C8F1,SO~N(CHzCH&H~CH,OH 3.5 PFOSEA: perfluorooctane sulfonyl ethylamide C~8F,,S0,N(CHzCH3)H 3.6 M556:C8F,7S02N(H)(CHzCOOH) 3.7 Surrogate standard lH-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 EOUIPMENT 6.1 The following equipment is used while performing this method. Equivalent equipment is acceptable. 6.1.1 Vortex mixer, VWR, Vortex Genie 2 6.1.2 Centrifuge, Mistral 1000 or IEC 6.1.3 Shaker, Eberbach or VWR ETS-8-4.1 ExtractionofPFOS from Serum Page 2 of 14 3M Environmental Laboratory Page 60 3M Medical Department Study: T-6316.8 BACK TO MAIN Analytical Report: FACT TOX-097 LRN42452 6.1.4 Nitrogen evaporator, Organomation 6.1.5 Balance (* 0.100 g) 7.0 SUPPLIES AND MATERIALS 7.1 Gloves 7.2 Eppendorf or 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 Centrifhge tubes, polypropylene, 15 mL 7.7 Labels 7.8 Oxford Dispenser -3.O to 10.0 niL 7.9 Syringes, capable of measuring 5 pL to 50 pL 7.10 Graduated pipettes 7.11 Syringes, disposable plastic, 3 cc 7.12 Syringe filters, nylon, 0.2 pm, 25 mm 7.13 Timer 7.14 Crimp cap autovials and caps 7.15 Crimpers Note: Prior to using glasswareand bottles, rinse 3 timeswithmethancil and 3 times with Milli-QTMwater. Rinse syringes a minimum of9 timeswith methanol, 3 rinses h m 3 separate vials. 8.0 REAGENTS AND STANDARDS 8.1 Ty be pMeiIllri-eQa%enLwt garteardaenwdamteary, MbeilEpr-oQvTidNeodr equivalent; all water used in this by a Mlli-Q TOC P h d " system method s h o u l d 8.2 Sodiumhydroxide (NaOH), J.T Baker or equivalent 8.3 Tetrabutylammonium hydrogen 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 HPLC grade 8.7 Methanol, Omnisolv, glass distilled or HPLC wide 8.8 Serum or blood, eozen from supplier 8.9 Fluorochemical standards 8.9.1 PFOS (3MSpecialty Chemical Division), molecular weight = 538 8.9.2 PFOSA (3M Specialty Chemical Divisio:n), molecular weight = 499 ETS-8-4.1 ExtractionofPFOS from S a m Page 3 of 14 3M Environmental Laboratory Page 61 3M Medical Department Study: T-6316.8 BACK TO MAIN Analytical Report: FACT TOX-097 LRN-U2452 8.93 PFOSAA (3M Specialty Chemical Division), molecular weight = 585 8.9.4 EtFOSE-OH(3M Specialty Chemical Division), molecular weight = 570 8.9.5 PFOSEA (3M Specialty Chemical Division), molecular weight = 527 8.9.6 M556 (3M Special@Chemical Division),,molecular weight =557 8.9.7 Surrogate standard: 4-H, perfluorooctane sulfonic acid (1-HJ-H, 2-H,2-H CBF,,SO,H.)molecular weight = 428 8.9.8 Other fluorochemicals, as appropriate 8.10 Reagent preparation NOTE: When preparing largervolumes thanlisted 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-QWwater, mix until all solids are dissolved. Store in a 1L Nalgene bottle. 8.10.2 1N sodium hydroxide (NaOH): Dilute 110N NaOH 1:10. Measure 10mL of 10 N NaOH solution into a 100mL volumetric flask and dilute to volume using Milli-Qm water. Store in a 125 mL Nalgene bottle. 8.10.3 0.5 M tetrabutylammoniumhydrogen su1:fate(TBA): Mrei approximately 169g ofTBA into a 1L volumetric containing SO0 mL Milli-Ql+? water. Adjust to pH 10using approximately 44to 54 mL of 10N NaOH (While adding the last mL of ' NaOH, add slowly because the pH changes abruptly). Dilute to volume with Milli-Qm water, Store in a 1 L Nalgene bottle. 8.10.3.1 TBA requires a check prior to each use to ensure pH = 10. Adjust as ' needed using 1N NaOH solution. 8.10.4 0.25 M sodium carbonate/sodium bicarbonate buffer @~a+$O,/NaHCO,): Weigh approximately 26.5 g of sodium carbonate (NqCO,) and 21.0 g of sodium bicarbonate (NaHCO,) into a 1L voiumeixic flask and bring to volume with MilliQm water. Store in a 1 L Nalgene bottle. 8.11 Standards preparation 8.11.1 Prepare PFOS standards for the standard c m e . 8.11.2 Prepare other fluorochemical standards, as appropriate. Multicomponent fluorochemical standards are acceptable (for example, one working standard solution containing 1.00ppm PFOS,1.02 ppm PFOSA, 0.987 pprn 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 stock standard of ,approximately1000ppm (Pg/mL). 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 stidard 2 solution of approx. 5.0 ppm. ETS-8-4.1 ExtTacdon of PFOS from Serum Page 4 of 14 3M Erivironmental Laboratory Page 62 3M Medical Department Study: T-6316.8 BACK TO MAIN Analytical Report: FACT TOX-097 LRN-U2452 , 8.12 8.11.7 Dilute working standard 1 with rnethho:i for a working standard 3 solution of approx. 0.50 ppm. Surrogate stock standard preparation 8.12.1 Weigh approximately 50-60mg of surrogate standard l-H,l-H2,-H, 2-H, C8F,,S03Hinto a 50 mL volumetric flask and record the actual weight. 8.12.2 8.12.3 Bring to volume with methanol for a sunagate stock of approximately 1000-1200 PPm. Prepare a surrogateworking standard. Transfer approximately 1mL of surrogate stock to a 10 mL volumetric flask and bring to volume with methanol for a working standard of 100ppm. Record thLeactual volume transferred. 9.0 SAMPLHEANDLING 9.1 All samples are received b z e n and must be kept h z e n until the extraction is performed. 9.2 Allow samples to thaw to room temperature prior to extraction. 10.0 OUALITYCONTROL 10.1 Solvent Blanks, Method blanks and matrixblanks 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 Milli-Qm water following this procedure and use as method blanks. 10.1.3 Extract two 1.0mL aliquotsof 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 sample chosen by the analyst, usually the control matrix received with each sample set. 10.23 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 spike duplicate per 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 sanie matrix used to prepare the initial curve. 3M Environmental Laboratory ETS-8-4.1 Extractionof PFOS from Serum Page 5 of 14 Page 63 3M Medical Department Study: T-6316.8 BACK TO MAIN Analytical Report: FACT TOX-097 LRN-U2452 10.3.4 The expected concentrationswill fall wifln the mid-range of the initial calibration curve. Additional spikes may be included that fall in the Iow-range of the initial calibration curve. This is nece:;sary if the analyst must quantitate using only the low end of the calibration curve (for example, 5 ppb - 100ppb, rather than - 5 ppb 1000ppb). I l . 0 CALXBRATION AND STANDARDIZATION 11.1 Prepare matrix calibration standards 11.1.1 Transfer 1 mL of serum to a 15mL centr!fbgetube. 11.12 Ifmost sample volumes 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 fie extraction sheet. 11.1.3 While preparing a total of twenty aliquots in 15 mL centrifugetubes, mix or shake between aliquots. 11.1.4 Two 1 mL aliquots, or other appropdate volume, serve as matrix blanks. Typically use the standard concentrations and spiking mounts listed in Table 1, at the end of this section,to spike, in duplicate, two standardcurves, for a total of eighteen standards, two matrix blanks, and two method blanks. 11.1.5 Refer to validation report ETS43-4.0 & ETS-8-5.0-V-1, which lists the working ranges and the Linear Calibration Range (LCR) for calibration curves. 11.1.6 Use Attachment D as an aid in calculatingthe concentrations of the working standards. See Section 13.0 to calculate ectual concentrations of PFOS in calibration standards. - 11.2 To each standard, blank, or continuingcheck, add appropriatemount of surrogate working standard for the concentration to fall within the calibration curve range 5 ppb 1000 ppb. 1 1 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 ETS-8-4.1 Extraction of PFOS from Serum Page 6 of 14 Page 64 3M Medical Department Study: T-6316.8 BACK TO MAIN Analytical Report: FACT TOX-097 LRN-U2452 Working standard - (approx. conc.) 0.500 uum -- 5.OO ppm 5.00 ppm 5.00 ppm 50.0 ppm 50.0 ppm 50.0 ppm 50.0 ppm PL Approx. final conc. of - analyte in matrix Blank 10 0.005 rmm 5 10 20 5 10 15 20 12.0 PROCEDURE 12.1 Obtain h z e n samples and allow to thaw at room temperature lor in a lukewarm waterbath. 12.2 Vortex mix for 15seconds, then transfer 1.OmL or other appropriate volume to a 15 mL polypropylene centrifbge tube. 12.3 Return unused samples to fkeezer after extraction.amountshave been removed. 12.4 Record the initial volume on the extraction worksheet. 12.5 Label the tube with the study number, sample ID, date and analyst initials. See attached worksheet for documenting the remaining steps. 12.6 Spike all samples, including blanks and standardis, ready for 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 prepare 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. 1 12.9 Check to ensure the 0.5 M TBA reagent is at pH 10. If not, ad:iust accordingly. 12.10 To each sample, add 1 mL 0.5 M TBA and 2 mL, of 0.25M sodlium carbonatdsodium bicarbonate buffer. 12.11 Using an Oxford Dispenser, add 5 mL methyl-tert-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 3500 rpm, or until layers are well separated. m-8-4.1 Extraction of PFOS from Serum Page 7 of 14 3M Environmental Laboratory Page 65 3M Medical Department Study: T-6316.8 BACK TO MAIN Analytical Report: FACT TOX-097 LRN-U2452 12.14 Label a fresh 15 mL centrifuge tube with the same information as in 12.5. 12.15 Remove 4.0 mL of the organic layer to this clean 15 mL centrifuge tube. 12.16 Put each sample on the analytical nitrogen evaporator until dqr, approximately 1 to 2 hours. 12.17 Add 1.0 mL of methanol to each centrifuge tube using a graduated pipette. 12.18 Vortex mix for 30 seconds. 12.19 Attach a 0.2 pm nylon mesh filterto a 3 cc syringe and transferthe sample to this . syringe. Filter into a 1.5 mL glass autovial or low-volume autovial when necessary. 12.20 Label the autovial with the study number, animal number and gender, sample timepoint, matrix, final solvent, extraction date, and analysl:(s)perfomkg the 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 approprhte. 13.0 DATAANALYSIASND CALCULATIONS 13.1 Calculations 13.1.1 Calculate actual concentrations of PFOS,or other apphicable fluomchemical, in calibration standards using the following equation: mL of standard x concentration of standard (usr /mL) - mL of standard +mL of surrogate standard +initial matrix volume (mL) Final Concentration @g/mL,) of PFOS in matrix 14.0 METHODPERFORMANCE 14.1 The method detection limit (MDL) is analyte and matrix specific. Referto MDL report for specific MDL and limit of quantitation (LOQ) values (see ,4ttachmentsB 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 14of ETS-8-5.1for method performance criteria. 15.0 POLLUTION PREVENTION AND WASTE MANAGEMENT 15.1 Sample waste is disposed in biohazard containers, flammable solvent waste is disposed in high BTU containers, and used glass pipette waste is disposed in broken glass containers Iocated in the laboratory. ETS-8-4.1 Extraction of PFOS from Serum Page 8 of 14 3M Environmental Laboratory Page 66 3M Medical Department Study: T-6316.8 BACK TO MAIN Analytical Report: FACT TOX-097 LRN-U2452 16.0 RECORDS 16.1 Complete the extraction worksheet attached to tkis 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 ByMDULOQ values and summary 17.3 Attachment C, Calibration standard cancentratio n worksheet 18.0 REFERENCES 18.1 The validation report associated withthismethod is ETS-8-4.0& 5.0-V-1. 18.2 FACT-M-3.1, "Analysis of Senun or Other Fluid Extracts for Fluorochemicals using HPLC-ElectrosprayMass Spectrometry" 19.0 AFFECTEDOCUMENTS 19.1 ETS-8-5.1,"Analysis of Serum or Other Fluid Ektracts for Fluorochemicals using HPLC-Electrospray Mass Spectrometry" 20.0 REVISIONS Revision Number 1 Reason For Revis& Section12.21 Changedto includesamplestorage at room temperature. Section 12.13 Added the shaker speed. Section 12.17 Finalvolume is 1.0mL; not ardjusted for initial volumes less than 1.0 mL. Revision - Date 04/02/99 3M Environmental Laboratory ETS-8-4.1 Extraction of PFOS from. Serum Page 9 of 14 Page 67 3M Medical Department Study: T-6316.8 Extraction Worksheet ETS-8-4.1 Study #f Matrix Box # Way DateSpikedAnalyst ccv MS MSD Surrogate Std approx. pprn actual ppm # I FC-Mix approx. 0.5 Pm actual # BACK TO MAIN Analytical Report: FACT TOX-097 LRN-U2452 FC-Mix # Comments Vortex 30 sec. Filter using a 3cc B-Dsyringe with a 0.2um filter into a 1.5 mL autamD e vial Cont. Cal. Verifications used same matrix as for std curve. Attachment A ETS-8-4.1 Extraction of PFOS from Serum Page 10 of 14 Page 68 3 M Medical Department Study: T-6316.8 BACK TO MAIN Analytical Report: FACT TOX-097 LRN-U2452 Compound PFOS PFOSA PFOSAA EtFOSE-OH M556 PFOSEA MDL (ppb) 1.74 1.51 3.46 11.4 6.03 5.71 LOQ (ppb) 5.55 4.79 20.5 36.2 19.2 18.2 Linear Calibration Range (L,CR) Approximate concentrations to be used for preparing the - Standard Calibratian Curve 5 ppb 1000ppb - 5 ppb 1000ppb - 5 ppb 1000ppb - 5 ppb 1000ppb 5 ppb - 1000 ppb 5 ppb - 1000ppb Pleasesee LOQ Summary andMDL study in ETS-84.0Bt 5 .O-V-1for further information. Attachment B:MDYLOQ Summary 3MRttir?eftrnent&Laboratory ETS-8-4.1 Extraction of PFOS front Serum Page 11 of 14 Page 69 3M Medical Department Study: T-6316.8 BACK TO MAIN Analytical Report: FACT TOX-097 LRN-U2452 Rabbit Serum Full Range LOW Curie High curve 1/X Prepared range of standards (PPW W m L ) 0.995 - 978 4.94 - 248 97.8 - 978 0.995 - 978 Compound: PFOSA LCR from curve OPb) (ng/mL) 24.8 - 978 4.94 - 248 97.8 - 978 - 4.94 978 % Recovery 85-1 04 85-106 94-111 4.67- 11.0 5.34- 12.0 4.84-!9.80 4.60- 10.5 Full Range I LowCurve I 0.993 - 976 4.93 -97.6 24.8 - 976 4.93 - 976 I 4.93 -97.6 24.8 - 978 88-103 87-105 93-102 I ~~ 5.10-14.7 I 9.85-14.7 I 5.08-13.9 Rabbit Serum Full Range Low Curve High curve 1iX ~ Prepared range of standards 0W m L ) 0.991 - 974 4.92 - 247 49.2 - 974 0.991 - 974 LCR from curve 0 24.7 - 974 9.74 - 247 97.4 - 974 9.74 - 974 % Recovery 97-1Cl7 85-108 95-115 4.18-10.6 6.38-21.8 4.33-12.5 4.1 1-23.2 Attachment B: MDLnOQ Summary 3M Environmental Laboratory ETS-8-4.1 Extraction of PFOS from Serum Page 12 of 14 Page 70 3M Medical Department Study: T-6316.8 BACK TO MAIN Analytical Report: FACT TOX-097 LRN-U2452 Compoand: EtFOSE-OH Full Range I Low Curve I I Highcurve I Im I 0.993 - 976 4.93-97.6 49.3 -976 0.993-493 49.3 - 976 I 9.76-97.6 I I 97.6-976 I I 9.76-976 I 97-107 90-109 86-111 I 14.1-21.3 I I 11.5-19.61 I 11.1-:21.27 Prepared range Rabbit Serum of standards LCR from CurvC %R e c v T z r Range I FullRange 1 I I @pb) (ng/mL) 0.993-976 (PPb) (ndmL) 2423 -976 96106 I 10.1-16.2 High curve 1/X 49.3 * 976 0.993 - 976 Compound: M556 49.3 - 976 9.76 - 976 It 86-106 95-117 - 10.2-18.2 - 10.1-19.1 I I Preparedrange I LCRfrom 1 % R e c o ' r n 1 1 I ;:;; 1 1 :Curve 4.93 - 97.6 9.76-97.6 5.95-18.2 High curve 97.6 - 976 97.6 - 976 5.11-9.74 0.993 - 976 9.76 - 976 97-110 4.77-19.5 AttachmentB:MDULOQ Summary 3M Ehvironmental Laboratory ETS-8-4.1 Extractionof PFOS from Senun Page 13 of 14 Page 71 3M Medical Department Study: T-6316.8 BACK TO MAIN Analytical Report: FACT TOX-097 LRN-U2452 Ion Pair Standard Curves - Fluids; Prep date(s): Standard number: Analyte(s): Equipment number: Sample ma- Final solvent and TN: Blank fluidlidentifier: 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: Surrogate std approx 100 ppm: Actual concentrationsof standards in the FC mix Calculated concentrations of standards in the samplo matrix Bovine Human 5.00-1000 5.00-1 000 5 .OO-1 000 AttachmentC Ion Pair Standard Curves ETS-8-4.1 Extraction of PFOS from Serum 3M Environmental Laboratory Page 14 of 14 Page 72 3M Medical Department Study: T-6316.8 BACK TO MAIN Analytical Report: FACT TOX-097 LRN-U2452 3M ENVIRONMENTLAALBORATORY EXTRACTIONOF POTASsnrrclPERnUOROOCTANESULFONATE~ONA'l'EOR OTHER F'LUOROCHEMICALCOMPOUNDSFROM LIVERFOR ANALYSIS USING HPLCELECTROSPRAYMASS SPICCTROMETRY Method Number: ETS-8-6.0 Author: Lisa Clemen, Robert Wynne Approved By: Adoption Date: 071lL147 Rervision Date: IJk Laboratory Nkanaier U Group Leader Technical Reviewer Date ?/l+lff3 Date U?/,q (94 Date 1.0 SCOPEAND APPLICATION 1.1 Scope: This method is for the extraction of potassium perfluorooctanesulfonate (PFOS) or other fluorochemical compounds from liver. 1.2 Applicable Compounds: Fluorochemicalsurfactants or other fluorinated compounds. 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 1of 14 3M Environmental Laboratory Page 73 3M Medical Department Study: T-6316.8 BACK TO MAIN Analytical Report: FACT TOX-097 LRN-U2452 2.0 SUMMARYOF 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 (MtBE). In this method, seven fluorochemicals can be extracted: PFOS, PFOSA, PFOSAA, EtFOSE-OH,.P-FOSEA,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 transfim-edto a centrifugetube 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,,S03 3.2 PFOSA: perfluomoctane sulfonylamide C8F,,S02NH, 3.3 PFOSAA: perfluomoctane sulfonylamido (ethy1)acetate C8F,,SO,N(CH2CH3)CH2CO2 3.4 EtFOSE-OH: 2(N-ethylperfluorooctane su1fonamido)-ethyl alcoliol C,F,,SO~(CH,CH3)CH,C&OH 3.5 PFOSEA perfluorooctane sulfonyl ethylamide C,F,,S02N(CX2CJ33)H 3.6 M556:C,F,,SO,N(H)(CH,COOH) 3.7 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 interferences known at this t h e . 6.0 EOUIPMENT 6.1 The following equipment is used while performing this method. Equivalent equipment is acceptable. 6.1.1 6.1.2 6.1.3 6.1.4 U l t r a - T m T25 Grinder for grinding liver samples Vortex mixer, VWR,Vortex Genie 2 Centrifige, Mistral 1000 or IEC Shaker, Eberbach or VWR ETS-8-6.0 Extraction of PFOS from Liver Page 2 of 14 3M Environmental Laboratory Page 74 3M Medical Department Study: T-6316.8 BACK TO MAIN Analytical Report: FACT TOX-097 LRN-U2452 6.1.5 Nitrogen Evaporator, Organomation 6.1.6 Balance (sensitivity to 0.100 g) 7.0 SUPPLIES AND MATERIALS 7.1 Gloves 7.2 Dissecting scalpels 7.3 Eppendorf or disposable pipettes 7.4 Nalgene bottles, capable of holding 250 mL and 1 L 7.5 Volumetric flasks, glass, type A 7.6 I-CHEM vials, 40 mL glass 7.7 Plastic sampule vials, Wheaton, 6 mL (or apprOpriiitesize) 7.8 Centrifuge tubes, polypropylene, 15 mL 7.9 Labels 7.10 Oxford Dispensor - 3.Oto 10.0ml 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 times with methanol, 3 rinses @om3 separate Vials. 8.0 REAGENTASND STANDARDS 8.1 Type I reagent grade water, Milli-Qm or equivalimt; all water used in this method should be M.WQTMwater and be provided by a Milli-Q TOC PlusTusystem 8.2 Sodium hydroxide (NaOH), J.T Baker or equivalent 8;3 Tetrabutylammonium hydrogen sulfate(TBA), Kodak or equivalent 8.4 Sodium carbonate (NqCO,), 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 Eivironrnental Laboratory Page 75 3M Medical Department Study: T-6316.8 BACK TO M~~ AIN Analytical Report: FACT TOX-097 LRN-U2452 8.10.2 PFOSA (3M Specialty Chemical Division),molecular weight = 499 8.10.3 PFOSAA (3M Specialty Chemical Division), molecular weight = 585 8.10.4 EtFOSE-OH (3MSpecialty Chemical Division), molecular weight = 570 8.10.5 PFOSEA (3M Specialty Chemical Division), molecular weight = 527 8.10.6 M556 (3MSpecialtyChemical Division), molecular weight = 557 8.10.7 Surrogate standard: 4-H, perfluorooctane rmlfonic acid (1-H,l-H, 2-H, 2-H C,F,,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 surrogate preparation, adjust accordingly. 8.11.1 10N sodium hydroxide (NaOH): Weigh approximately200 g NaOH. Pourinto a 1000mLbeaker containing 500 mL Milli--Qmwater, 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 10N NaOH solution into a 100mL volumetric flask and dilute to volume using Milli-QTMwater. Storein a 125mL Nalgcme bottle. 8.11.3 0.5M tetrabutylammonium hydrogen sulfate (TBA): Weigh approximately 169 g of TBA into a 1L volumetric containing 500 mL Milli-Q"" water. Adjust to pH 10 using approximately 44to 54mL of 10N NaOH (While adding the last mL of NaOH, add slowly because the pH changes abruptly). Dilute to volume with Milli-QT" 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 solutiori. 8.11.4 0.25 M sodium carbonate/sodium bicarbonate buffer (Na&O,/NaHCO,): Weigh approximately 26.5 g of sodium carbonate!(Na$OJ and 21.0 g ofsodium bicarbonate(NaHCO,) into a 1 L volumetric flask and bring to volume with Milli- Qmwater. Store in a 1 L Nalgene bottle. 8.12 Standards preparation 8.12.1 Prepare PFOS standards for the standard curve. 8.12.2 Prepare other fluorochemical standards, as appropriate. Multicomponent fluorochemical standards are acceptable (jbr example, me working standard solution containing 1.OO ppm PFOS,1.02 ppm PFOSA, 0.987 ppm PFOSAA, and 1.10 ppm EtFOSE-OH.) 8.12.3 Weigh approximately 100mg of PFOS into a 100mL \volumetricflask and record the actual weight. 8.12.4 Bring to volume withmethanol for a stock standard of iipproximately 1000ppm (PglmL). 8.12.5 Dilute the stock solution with methanol for a working standard 1 solution of approximately 50 ppm. 3M Environmental Laboratoty ETS-8-6.0 Extraction of PFOS from. Liver Page 4 of 14 Page 76 3M Medical Department Study: T-6316.8 BACK TO MAIN Analytical Report: FACT TOX-097 LRN-U2452 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 surrogate standard 1-H,l-H, 2-H, 2-H, CIIF,$03Hinto a 50 mlvolumetric flask and record the actual weight. 8.13.2 8.13.3 Bring to volume withmethanol for a surrogate stock of approximately 1000-1200 PPm. Prepare a surrogateworking standard. Transfer approximately 1.0mi ofsurrogate stock to a 10ml volumetric flaskandbring to volume:with methanol for a working standard of 10-20 ppm. Recor(ithe actual volume transferred. 9.0 SAMPLHEANDLING 9.1 All samplesare received fiozen and must be kept frozenuntil the extraction is performed. 10.0 OUALIT~CONTROL 10.1 Matrix blanks and method blanks 10.1.1 An aliquot of 1.0 mL, methanol is used as a solvent blank. 10.1.2 Extract two 1.0mL aliquots of Milli-Qm water following this procedure and use as method blanks. 10.1.3 Extract two 1.0 mL aliquots of liver homogenate following this procedure and use as matrix blanks. Refer to 11.1.6. 10.2 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 chosen by the analyst, usually a control liver received with each sample set. 10.2.3 Expected concentrations Will fall in the mid-range of the initial calibration curve. Additional spikes may be included and may fall in the low-range of the initial calibration curve. 10.2.4 Prepare one matrix spike and matrix spike duplicate per 40 samples, with a minimum of 2 matrix spikes per batch. 10.3 Continuing calibration verifications 10.3.1 Prepare continuing calibration verification samples to ensure the accuracy of the initial calibration curve. 10.3.2 Prepare, at a minimum, one continuing Calibration verification sample per group of 10 samples. For example, if a sample set = 34, four verifications are prepared and extracted. 3M Ewironmental Laboratory ETS-8-6.0 Extraction of PFOS from Liver Page 5 of 14 Page??- 3M Medical Department Study: T-6316.8 BACK TO MAIN Analytical Report: FACT TOX-097 LRN42452 10.3.3 Prepare each continuing calibration verification from the same matrix used to prepare the initial curve. 10.3.4 The expected concentrations will fall within the mid-range of the initial calibration curve. Additional spikesmay be included thlat 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, 5 ppb - 100 ppb, rather than 5 ppb - 1000ppb). 11.0 CALIBRATIONAND STANDARDIZATION 11.1 Prepare matrix calibration standards 11.1.1 Weigh approximately 40 g of liver into a 250 mL Nalgene bottle containing 200 d s Milli-QTMwater. Grind to a homogeneous solution. 11.1.2 If 40 g is not available, use appropriate amountsof liver and water to ensure a 15 ratio. 11.1.3 Refer to 13.0 to calculate the actual density of liver homogenate and the concentration of solid livertissue dispersed.in 1.O mL of homogenate solution. 11.1.5 Add 1 mL of homogenate to a 15 mL centrifuge tube. Re-suspend solution by shakingbetween aliquots while preparing 2. total of eighteen 1 mL 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 Typicdly 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:&. 11.1.8 Refer to validation reports ETS8-6.0 and ETS-8-7.0-V-1 or Attachment B, 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 concentrations of the working standards. .Refer to 13.0 to calculate actual concentrations of PFOS in calibration standards. 11.2 To each working standard,blank, or continuing velification, add appropriateamount of surrogateworking standard for the concentration to fall within the calibration curve range 5 ppb - 1OOOppb. 3M Environmental Laboratory ETS-8-6.0 Extraction of PFOS from Liver Page 6 of 14 -- 3M Medical Department Study: T-6316.8 BACK TO MAIN Analytical Report: FACT TOX-097 LRN-U2452 11.3 Extract spiked liver homogenates following 12.14-l2.25 of this method. Use these standards to establish each initial curve on the mass spectrometer. Approximate Spiking r Calibration Standards Approx. final conc. of PFOS in liver Blank 0.005 ppm 0.010 ppm 0.025 pgm 0.050 pprn 0.100 ppm 0.250 ppm 0.500 ppm 0.750 ppm 1.OO ppm 12.0 PROCEDURE 12.1 Obtain frozen liver samples. 12.2 Cut approximately 1 g of liver using a dissectingsadpel. This part of the procedure is best performed quickly, not allowing the liver to thaw. . 12.3 Weigh the sample directly into a tared plastic samIx.de vial. 12.4 Record the liver weight in'the study 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 sample and grind for about 2 minutes, or until the sample is homogeneous. 12.8 Rinse the probe into the sample with 2.5 mLs water using a pipette. 12.9 Take the grinder apart and clean it with methanol after each sample. Refer to AMDT-EP- 22. 12.10 Cap the sample and vortex for 15 seconds. Label the sampule vial with the study number, weight, liver ID, date and analyst initials. 3M Environmental Laboratory ETS-8-6.0 Extractionof PFOS from Liver Page 7 of 14 Page 79 3M Medical Department Study: T-6316.8 BACK TO MAIN Analytical Report: FACT TOX-097 LRN-U2452 12.11 Pipette 1.0 mL, or other appropriate volume, of homogenate into1 a 15 mL polypropylene centrifuge tube. Label the centrifuge tube with the identical infonmation as the sampule vial. Refer to attached worksheet for documenting the remaining steps. 12.12 Pipette two 1mL aliquots of Milli-Q" 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 m a t h with the appropriate amount of standard as described in 11.1, or Table 1 of that section, for the calibration curve standards. Also prepare matrix spikes and continuing calibration standards. 12.15 Vortex mix the standard curve samples, matrix spike samples, and continuing calibration samples for 15 seconds. 12.16 Check to ensure 0.5 M TBA reagent is at pH 10. If not, adjust accordingly. 12.17 To each sample, add 1 mL 0.5 M TBA and 2 mL of the 0.25 M sodium carbonatdsodiurn bicarbonate buffer. 12.18 Using an Oxford Dispenser, add 5 mL methyl-tert-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 3508 rpm, or until layers are well separated. 12.21 Label a fresh 15 mL centrifuge tube with the same information as in 12.10. 12.22 Remove 4.0 mL of the organic layer to the fresh 1.5mL centrifuge tube. 12.23 Put each sample on the analytical nitrogen evaporator Until dry, approximately I to 2 hours. 12.24 Add 1.O mL to each centrifuge tube using a graduated pipette. 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 i;lumber and gender, sample timepoint, matrix, final solvent, extraction date, and analyst(@performing the extraction. 12.28 Cap and store extracts at room temperature or at approximately 4 "C until analysis. 12.29 Complete the extraction worksheet, attached to this document, and tape in study notebook or include in study binder, as appropriate. 3M Eivironmental Laboratoly ETS-8-6.0 . Extraction ofPFOS from Liver Page 8 of 14 Page 80 3M Medical Department Study: T-6316.8 BACK TO MAIN Analytical Report: FACT TOX-097 LRN-U2452 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 1.0 mZ, aliquots of homogenate. Average density (mg/mL) =Average mass (rnd of the aliauots 1.0 mL aliquot 13.1.2 Calculate the amount of liver (mg)per 1.0 i d homogenaie (or concentration of dispersed solid tissue per mL of homogenabe suspension)using the following equation: g of Liver x Average den&* of homogenate (mdmL1 (g of Liver + g of Water) * refer to 13.1.1 for details. 13.13 Calculate actual concentrations of PFOS arid other fluorcchemicals in calibration standards using the following equation: & of Standard x Concentration (DE /mL) =Final Concimtration (pg/g or mgkg) mg Liver/ 1mL homogenate* of PFOS in Liver *refer to 13.1.2 for details. 14.0 METHOD PERFORMANCE 14.1 The method detection limit (MDL) is analyte and i n a h specific. Refer to MDL report for specificMDL and limit of quantitation (LOQ)values (refer to Attachments B and C). 14.2 The followihg quality control samples are extracted with each batch of samples to evaluate the qualityofthe 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. 14.23 Continuing calibration verification samples to detennirte the continued accuracy of the initial calibration curve. 14.3 Refer to section 14 of ETS-8-7.0 for method perfclrmance criteria 15.0 POLLUTIOPNREVENTIOANND WASTEMANAGEMENT 15.1 Sample waste is disposed in biohazard containers, flammable solvent waste is disposed in high BTU containers, and used glass pipette waste:is disposed in broken glass containers located in the laboratory. 3M Environmental Laboratory ETS78-6.0 ExtractionofPFOS from Liver Page 9 of 14 Page 81 3M Medical Department Study: T-6316.8 BACK TO MAIN Analytical Report: FACT TOX-097 LRN-U2452 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 studybinder, as appropriate. 17.0 .TABLEDSI. AGRAMFSL.OWCHARATNSD. VALIDATIONDATA 17.1 Attachment A, Extraction worksheet 17.2 Attachment ByMDULOQ values and sumniary 17.3 Attachment C, Calibration standard calculation and concentration worksheet 18.0 REFERENCES 18.1 The validation report associated with this method is ETS-8-6.0 8i:7.0-V-1. 18.2 ANDT-EP-22, ``Routine Maintenance of Ultra-Turrax T-25" 18.3 FACT-M-1.1, "Exhction of PFOS or Other Anionic Fluorochmnical Surfactants fiom ' Liver for Analysis Using HF'LC-Electrospray/Mas Spectrometqr" 19.0 AFFECTEDDOCUMENTS 19.1 ETS-8-7.0, "Analysis of Liver Extracts for Fluomchemicals using HPLC-Electrospray Mass Spectrometry" 20.0 REVISIONS Revision Number. .. Reason For Revision - Revision Date 3M Environmental Laboratory ETS-8-6.0 Extraction of PFOS from Liver Page 10 of 14 Page 8 2 3M Medical Department Study: T-6316.8 Surrogate Std Date Spikedhalyst nnt r BACK TO MAIN Analytical Report: FACT TOX-097 LRN-U2452 I I I I r I K I Date & Initials AttachmentB:MDLLOQ Values 3M Environmental Laboratory ETS-8-6.0 Extraction of PFOS from Liver . .__.____ Page 11 of 16 n 3M Medical Department Study: T-6316.8 BACK TO MAIN Analytical Report: FACT TOX-097 LRN-U2452 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 78.3 345 262 108 Linear Calibration Range (LCR) Approximate concentrations to bt: used for preparing the Standard Calibration Curve 30ppb - 1200ppb 12ppb-1200ppb 30 ppb - 1200 ppb 60 ppb - 900 ppb* 60 ppb - 1200 ppb 30 ppb- 1200ppb Refer to LOQ Summary and MDL study in ETS-84.0 & 7.0-V-1for further information * EtFOSE-OH estimates only for MDL and LOQ. Did not meet criteria for validation. ComDound: PFOS Liver matrix Rabbit Attachment B: MDYLOQ Values 3M Environmental Laboratory ETS-8-6.0 Extraction of PFOS froin Liver Page 12 of 16 3M Medical Department Study: T-6316.8 BACK TO MAIN Analytical Report: FACT TOX-097 LRN-U2452 Compou id: M556 Liver Rabbit Attachment C Standard Calculations 3M Environmental Laboratory ETS-8-6.0 Extractionof PFOS from Liver Page 13 of 14 - - 3M Medical Department Study: T-6316.8 BACK TO MAIN Analytical Report: FACT TOX-097 LRN-U2452 Ion Pair Standard Curves - Tissue Prep date@): 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: Surrogate std approx. 100 ppm: Standard number: Equipment number:' Final solvent and TN: Blank livedidentifier: I I I I 1 1 E K c I I I Actual concentrations of standardsin the FC mix PFOS PFOSA PFOSAA EtFOSE PFOSEA Stdconc Stdconc Stdconc Stdconc Stdconc AI1 All Am'tspiked Density I00 surrogate I 0.005 Rabbit Bovine Attachment C: Standard Calculations 3M Environmental Laboratory ETS-8-6.0 Extraction of PFOS from Liver Page 14 of 14 Page 86 3M Medical Department Study: T-6316.8 BACK TO MAIN Analytical Report: FACT TOX-097 LRN-U2452 3M ENVIRONMENTLAALBORATORY METFIOD ANALYSIS OF FLUOROCHEMXCIANLIJSIVEREXTRACTUSSING HPLC-ELECTROSPRAYLMSAPESCS TROMETRY Method Number: FACT-M-2.0 Author: Lisa Clemen . Approved By: Adoption Date: 5/&+,[9# Revision Date: NIA Laboratory Manager Date Technical Reviewer 5h7/16 Date 1.0 SCOPE AND APPLICATION I.IScope: This method is for the analysis of extracts of liver or other tissues for fluorochemical surfactants using HPLC-electrospray/mas spectrometry. 1.2 Applicable Compounds: Potassium peduorooctanr:sulfonate,anionic fluorochemical surfactants, or other ionizable compounds. 1.3Matrices: Rabbit, rat, bovine, and m d e y livers or other livers as designated in the validation report. Word 7.0.1195 3M Environmental Laboratory FACT-M-2.0 Analysis of Liver Extract Using ES/MS Page 1 of 8 Page* 3M Medical Department Study: T-6316.8 BACK TO MAIN Analytical Report: FACT TOX-097 LRN-U2452 2.0 SUMMARY OF METHOD 2.1 This method describes the analysis of fluorochemical surfactants extracted from liver using HPLC-electrospray/mass spectrometry. The analysis is performed by monitoring a single ion characteristic of a particular fluorochemical, such as the potassium perfluorooctauesulfonate(PFOS)anion, M/Z= 499. Samples may also be screened to verify compound identification. 3.0' DEFINITIONS 3.1 None. 4.0 WARNINGS AND CAUTIONS 4.1 Health and Safety Warnings: I 4.1.1 Use caution with the voltage cable for the probe. When the voltage cable is plugged into the probe DO NOT TOUCH THE PROBE, there is risk of electrical shock. 4.2 Cautions: 4.2.1 Do not run solvent pumps above capacity of400 bar (5800 psi). If pressure goes over 400 bar, the HPl 100 will initiate automatic shutdown. 4.2.2 Do not runsolvent 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 Electrospray 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, rejiigerated liquid, regulated to approximately 100 psi. 7.1.2 HPLC column, specifics to be determined by the analyst. 7.1.3 Capped autovials or capped 15 r d centrifhge tubes. 8.0 REAGENTASNDSTANDARDS 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 88- 3M Medical Department Study: T-6316.8 BACK TO MAIN Analytical Report: FACT TOX-097 LRN-U2452 8.1.2 Milli-QTMwater, all water used in this methcd should be h.4illi-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 standards are prepared during the extraction procedure. See FACT-M-1. 9.0 SAMPLHEANDLING 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 lx 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 m e . 10.2 Matrix Spikes 10.2.1 Analyze a matrix spike and matrix spikd 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. 103 ContinuingCalibration Checks 103.1 Analyze a mid-range calibration standard after every tenth sample. If a significant change (rt 30%) in peak area occurs, relative to the initial standard curve, stop the run.Only those samples analyzed before thc last acceptable calibration standard will be used. The remaining samples must be reanalyzed. 10.3.2 See section 13 to calculate percent difference. 10.4 System Suitability 10.4.1 System suitability (e.g. peak area, retention time and peak shape, etc.) will be assessed for each run. 11.0 CALIBRATION AND STANDARDIZATION 11.1 Analyze the extracted liver standards prior to and hllowing 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 suitable s o h z e . 3M Environmental Laboratory FACT-M-2 .O Analysis of Liver Extract Using E S N S Page 3 of 8 -- 3M Medical Department Study: T-6316.8 BACK TO MAIN Analytical Report: FACT TOX-097 LRN-U2452 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. Sei:up a sample list. Assign a filename using letter-MO-DAY-last digit of year-sample number, assign a method (MS) for acquiring, and type in sample descriptions. 12.1.2 To create a method click on scan button in the Acquisition control panel and select SIR. Set Ionization Mode as appropriate and mass to 499 or other appropriate masses.. A scan is usually collected along with the SIRS. Save method. 12.13 Typically the sample list begins with the first set 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 tlo 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.l. 12.2.2 Set-up the HPllOO/autosampler at the following Conditions or at conditions the analyst considers appropriate for optimal re,sponse.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 12.2.23 Cycle time = 15 minutes 12.2.2.4 Solvent ramp = Time MeOH 0.00 min. 45% 55% Note: In this instrument configuration, the runmust be set up on the electrospray software with a "Waiting for inlet start" message before the "Start" button is pressed on the HP Workstation. 12.2.2.5 Press the "Start" button. 3M Environmental Laboratory FACT-M-2.0 Analysis of Liver Extract Using ES/MS Page 4 of 8 3M Medical Department Study: T-6316.8 BACK TO MAIN Analytical Report: FACT TOX-097 LRN-U2452 12.3 Instrument Sep-up 12.3.1 Refer to AMDT-EP-31 for more details. 1 2 3 3 Check the solvent level in reservoirs and refill if necessarr. 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 staixiless steel capillary. - 12.3.4 Set HPLC pump to "On". Set the flow to 10 500 uL/min.or as appropriate. Observe droplets coming out of the tip of the probe. Allow to equilibrate for approximately 10 minutes. 123.5 Turn on the nitrogen. A fine mist should be: expelled withno 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 literdhour - 12.3.63 ESI nebulizing gas 10-15 liters/hollr 123.6.3 LC constant flow mode flow rate 10 500 uL/min 12.3.6.4 Pressure -400bar (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 imtil it will not go any further. Connect the voltage cables to the probe. 123.8 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 spike percent recoveries using the following equation: % Recovery = Observed Result - Background Result x 100 Expected Result 13.1.2 Calculate percent difference using the follciwing equation: % Difference = Expected Conc. - Calculated &C Expected Conc. x 100 3M E:nvironmental Laboratory FACT-M-2.0 Analysis of Liver Extract IJsing ES/MS Page 5 of 8 -ragtrsl- 3M Medical Department Study: T-6316.8 BACK TO MAIN Analytical Report: FACT TOX-097 LRN-U2452 13.1.3 Calculate actual concentration of PFOS anion in total liver (mg): (ug PFOS anioncalc. h m std curve) I gofliverusedforanalysis 1OOOug/lmg I x Total mass of`liver(g) 14.0 METHOPDERFORMANCE 14.1 The method detection limit is equal to at least three times the baseline noise in the matrix blank. 14.2 The practical quantitation limit is equal to the lowest standard in the calibration curve. 15.0 POLLUTIONPREVENTIOANNDWASTMEANAGEMENT 15.1 Sample waste is disposedin biohazard containers, flammablesolvent 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 chromatogram should have the following information included either in the header or hand written on the chromatogram: study number, sample name, extraction date, and dilution factor (if applicable). 162 Plot calibration curve by linear regression and store in the study folder. 16.3 Print sample list fkom MassLynx and tape into the instrument runlog. 16.4 Print data integration suxnmary f h m MassLynx and tape into the instrument runlog. 16.5 Copy instrument runlog pages, including instrument parameters end sample results, and tape into appropriatestudy notebook. . 16.6 Summarizedata using suitable software and store in the study folder. 16.7 Back up electronic data to appropriate media. Record in study notebook the file name and location of backup electronic data. 17.0 TABLESD,IAGRAMFSL,OWCHARTANSD, VALIDATIODNATA 17.1 Attachment A: FACT-M-2 Data reporting spreadsheet 17.2 The validation report associatedwith 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 Ervironmental Laboratory FACT-M-2.0 Analysis of Liver Extract Using ES/MS - Page 6 of 8 3M Medical Department Study: T-6316.8 BACK TO MAIN Analytical Report: FACT TOX-097 LRN-U2452 19.0 AFFECTED DOCUMENTS 19.1 FACT-M-1.O, "Extraction of PotassiumPerfluorooctanesulfonatefrom Liver for Analysis Using KPLC-E1ectrospraylMas.s Spectrometry" 20.0 REVISIONS Revision Number. Reason For Revision - Revision Date 3M Environmental Laboratory FACT-M-2.0 Analysis of Liver Extract Using ESMS Page 7 of 8 ---- 3M Medical Department Study: T-6316.8 Laboratory Study # Study: Test Material: MatrWinal Solvent: MethodRevision: Analytical Equipment System Number: Instrument SoftwareNersion: Filename: R-Squared Value: Slope: Y Intercept: Date of ExtractiodAnalyst Date of AnalysidAnalyst: BACK TO MAIN Analytical Report: FACT TOX-097 LRN-U2452 Dilution Factor: Taken from the study folder. Final Cone. (ug/mL): Calculated by dividing the initial volume from the concentration 3M Environmental Laboratory FACT-M-2.0 Analysis ofLiver Extract Using ESMS Page 8 of 8 BACK TO MAIN 3M Medical Department Study: T-6316.8 Analytical Report: FACT TOX-097 ! 3M ENVIRONMENTLAALBORATORY I METHOD ANALYSIS OF FLUOROCEEMICALSIN SERUM EXTRACTUSSING HPLC-ELECTROSPRAYMASmSScTRonmmy Method Number: FACT-M-4.0 Author: Lisa Clemen Approved By: , e. Adoption Date: q/zt/q 8 Revision Date: ' NlA " Group Leader Technical Reviewer a Date /Y d 9 8 Date 1.0 SCOPEAND APPLICATION 1.1 Scope: This method is for the analysis of extracts of :ierumor tissue for fluorochemical surfactants using I-PLC-electrospray/mas spectrometry. 1.2 Applicable Compounds: Potassiumperfluorooctanesulfonate,anionic fluorochemical surfactants, or other ionizable compounds. 13 Matrices: Rabbit, rat,and bovine serum or other seraas designated.in the validation repqrt. Word 7.0.1195 FACT-M-4.0 Analysis of Serum Extract Using ESMS Page I of 8 Page 95 3M Medical Department Study: T-6316.8 BACK TO MAIN Analytical Report: FACT TOX-097 LRN-U2452 2.0 SUMMARYOF METHOD 2.1 This method describes the analysis of fluorochemicid surfactants extracted from serum using HPLC-electrospray/masspectrometry. The analysis is performed by monitoring a single ion characteristic of a particular fluorochemioal, such as the potassium perfluorooctanesulfonate(PFOS) anion, M/z= 499. Samples may also be screened to verify compound identification. 3.0 DEFINITIONS 3.1 None. 4.0 WARNINGS AND CAUTIONS 4.1 Health and Safety Warnings: 4.1.1 Use caution with the voltage cable for the probe. When the voltage cable is plugged into the probe DO NOT TOUCH THE PR013E,there is risk of electrical shock. 4.2 Cautions: 4.2.1 Do not run solventpumps above capacity of 400bar (5800 psi). If pressure goes over 400 bar, the HPl 100 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 instrurnentation that comes in contact with the sample or extract. 6.0 EOUIPMENT 6.1 Ekpipment listed below may be changed in order to optimize the system. 6.1.1 Micromass Electrospray Mass Spectrometer 6.1.2 HP 1100 low pulse solvent pumping system and autosampher. 7.0 SUPPLIESAND MATERIALS 7.1 Supplies 7.1.1 Nitrogen gas, refrigerated liquid, regulated to approximately 100psi. 7.1.2 HPLC column, specifics to be determined by the analyst. 7.13 Capped autovials or capped 15 mL centrifuga tubes. 8.0 REAGENTS AND STANDARDS 8.1 Reagents 8.1.1 Methanol, HPLC grade or equivalent. 3M Environmental Laboratory FACT-M-4.O Analysis of Serum Extract Using ESMS Page 2 of 8 Page 96 3M Medical Department Study: T-6316.8 BACK TO MAIN Analytical Report: FACT TOX-097 LRN-U2452 8.1.2 Milli-QW water, all water used in this method should be Edilli-Q" 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 serum blank, imd seven serum standards are prepared during the extraction procedure. See FACT-M-3. 9.0 SAMPLEHANDLING 9.1 Fresh serum standards are prepared with each analyriis. 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 at 4 O C until analysis can be performed. 10.0 Oufirry CONTROL 10.1 Matrix Blanks and Method 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 spike and matrix spike duplicate with eac,h 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. 10.2.3 See section 13 to calculate percent recovery. 10.3 Continuing Calibration Checks 10.3.1 Analyze a mid-range calibration standard &a every tenth sample. If a significant change (& 30%) in peak area occws, relative to the initial standard curve, stop the run.Only those samples analyzed before the ].atacceptable: calibration standard will be used. The remaining samples must be:reanalyzed. 10.3.2 See section 13 to calculate percent difference, 10.4 System Suitability 10.4.1 System suitability (e.g., peak area, retention time, peak shape, etc.) will be assessed for each m. 11.0 CALIBRATIOANND STANDARDIZATTON 11.1 Analyze the extracted serum standards prior to and following 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 suitable software. 3M Environmental Laboratory FACT-M-4.0 Analysis of Serum Extract Using ESNS Page 3 of 8 Page 97 3M Medical Department Study: T-6316.8 .. BACK TO MAIN Analytical Report: FACT TOX-097 LRN-U2452 11.2 The I? value for the data should be 0.98 or greater. Lower values may be acceptable at the discretion of the analyst. 11.3 If the curve does not meet requirements, perform routine maintenance or reextract the standard curve (if necessary) and reanalyze. Time MeOH 0.00 min. 45% 55% 7.5 min. 90% 10% 11.0min. 90% 10% 11.5 min. 45% 55% Note: In this instrument configuration, the mi must be set up on the electrospray software with a "Waitingfor inlet start" messa.gebefore the "Start"button is pressed on the HP Workstation. 12.2.2.5 Press the "Start" button. FACT-M-4.0 Analysis of Serum Extract Using ES/MS Page 4 of 8 3M Environmental Laboratory Page 98 3M Medical Department Study: T-6316.8 BACK TO MAIN Analytical Report: FACT TOX-097 LRN-U2452 12.3 Instrument Set-up 12.3.1 Refer to AMDT-EP-3 1 for more details. 12.3.2 Check the solvent level in reservoirs and refill if necessary. 12.33 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, disassemblethe probe and replace the stainlesssteel capillary. 123.4 Set HPLC pump to ''On''. Set the flowto 10 - 500 uL/min or as appropriate. Observe droplets coming out of the tip of the! probe. Allow to equilibratefor approximately 10 minutes. 123.5 T w 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 litendhour 12.3.6.2 ESI nebulizing gas 10-15 litershow 12.3.63 HPLC constant flow mode flow rate: 10- 500 pL/'min 12.3.6.4 Pressure <400 bar (This parameter is not set, it is a guide to ensure the HPLC is operating correctly.) 12.3.7 Carefully guide the probe into the opening. Insert probe until it will not go any further. Connect the voltage cables to the prabe. 12.3.8 Record tune parameters in the instrument log. 12.3.9 Using the cross-flow counter electrode in the ESMS source is recommended for the analysis of biological matrices. 12.3.1OClick on start button in the Acquisition Control Panel. Press the start button at top of sample list. Ensure start and end sample number includes dl samples to be analyd. ' 13.0 DATAANALYSIASND CALCULATIONS 13.1 Calculations: 13.1.4 Calculate matrix spike percent recoveries usingthe following equation: % Recovery = Observed Result - Background Result x 100 Expected Result 13.1.5 Calculate percent difference using the following equation: % Difference = ExDected Conc. - Calculated Conc. x 100 Expected Conc. 3M Eivironmental Laboratory FACT-M-4.0 Analysis of Serum Extract Using ES/MS Page 5 of 8 Page 99 3M Medical Department Study: T-6316.8 BACK TO MAIN Analytical Report: FACT TOX-097 LRN-U2452 13.1.6 Calculate actual concentration of PFOS, or other fluorochemical, anion in serum (PdmL): pn of PFO calc. from std. Curve x Dilution Factor x Final Volume (mL) Initial Volume of serum (mL) 14.0 METHOPDERFORMANCE 14.1 The method detectionlimit is equal to halfthe lowest standard in the calibration curve. 14.2 The practical quantitation limit is equal to the lowest standard in the calibration curve. 15.0 POLLUTION PREVENTIONAND WASTE MANAGEMENT 15.1 Sample extract waste and flammable solvent is disposed in high BTU containers, and glass pipette waste is disposed in broken glass containers located in the laboratory. 16.0 RECORDS 16.1 Store chromatograms in the study folder. Each chromatogram must have the following information included either in the header or hand written on the chromatogram: study number, sample name, extraction date, and dilution factor (if applicable). 16.2 Plot calibration curve by linear regression and store in the study folder. 16.3 Print sample list from MassLynx and tape into the imtmnent d o g . 16.4 Print data integration summary from MassLynx and tape into the instrument runlog. 16.5 Copy instrument runlog pages, including instrumen1 parameters and sample results, and tape into appropriate study notebook. 16.6 Summarize data using suitable software and store in the study folder. 16.7 Back up electronic data to appropriate medium. Record in study notebookthe file name and location of backup electronic data. 17.0 TABLESD. IAGRAMFSu,)WcEIARTS, AND VALIDATIONDATA 17.1 Attachment A. FACT-M-4 Data reporting spreadsheet 17.2 The validation report associated With this method is FACT-M-3.0 & 4.0-V-1. 18.0 REFERENCES 18.1 AMDT-EP-31, "Operation of VG Platform Electrospray Mass Spectrometer" 19.0 AFFECTEDDOCUMENTS 19.1 FACT-M-3.0, "Extraction of Fluorochemical Anions from Serum for Analysis Using HPLC-ElectrosprayMass Spectrometry" 3M Environmental Laboratory FACT-M-4.0 Analysis of Serum Extract Using ESMS Page 6 of 8 Page 100 3M Medical Department Study: T-6316.8 20.0 REVISIONS Revision Number. Reason For Revision BACK TO MAIN Analytical Report: FACT TOX-097 LRN-U2452 Revision - Date 3M E3wironmentaLl aboratory FACT-M-4.O Analysis of Serum Extract Using ESMS Page 7 of 8 Page 101 3M Medical Department Study: T-6316.8 Laboratory Study # Study: Test Material: MatridFinal Solvent: MethodRevision: Analytical Quipment System Number: Instrument SoftwareNersion: Filename: R-SquaredValue: Slope: Y Intercept: Date of Extraction/Analyst: Date of AnalysidAnaiyst: BACK TO MAIN Analytical Report: FACT TOX-097 LRN-U2452 GrouplDose: 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-4.0 Analysis of Senun Extract Using ESMS Page 8 of 8 Page 102 3M Medical Department Study: T-6316.8 BACK TO MAIN Analytical Report: FACT TOX-097 LRN-U2452 3M ENVIRONMENTLAALBORATORY METHOD ANALYSIS OF POTASSIUMPERFLUOROOCTANESULFONATE OR OTHER FLUOROCHEMICAILNSSERUM E:XTRACTS ZrSING HPLC-ELECTROSPRAYAWSAPESCSTROMETRY Method Number: ETS-8-5.1 Author: Lisa Clemen,Robert Wynne Approved By: 1 Laboratory Manager Group Leader c Technical Reviewer Adoption Date: 03/01/99 Revision Date: q / % f l VZC, Date 4/* /$v Date 0VI2L l 99 Date m-- 1.0 SCOPE AND APPLICATION 1.1 Scope: This method describes the analysis of serum extracts for fluarochemical surfactants using HPLC-electrospray/massspectrometry. 1.2 Applicable Compounds: Fluorochemical surfactantsor other fluorinated compounds, or other ionizable compounds. 1.3 Matrices: Rabbit, rat, bovine, monkey, and human senun, or other fluids as designated in the validation report. Word 6/95 ETS-8-5.1 Analysis ofSerum Extract Using ESNS Page 1 of 9 ratory Page 103 3M Medical Department Study: T-6316.8 BACK TO MAIN Analytical Report: FACT TOX-097 LRN-U2452 2.0 SUMMARY OF METHOD 2.1 This method describes the analysis of fluorochemical surfactants extracted from serum or other fluids, using HPLC-electrospray/mass spectrometry, or similar system as appropriate. The analysis is perfonned by monitoring a single ion characteristic of a particular fluorochemical, such as the perfluorooctanesulfonate (PFOS) anion, m/z= 499. Additionally, samples may be analyzed using a tandem mass spectrometer to fbrther verify the identity of a compound by detecting daughter ions of the parent ion. 3.0 DEFINITIONS 3.1 Atmospheric Pressure Ionization (API): The Micrornass Quattro 11 triple quadrupole systems allow for various methods of ionization by utilizing various sources, probes, and interfaces. These include but are not limited to: Elecmspray Ionization @SI), Atmospheric Pressure chemical Ionization (APcI), Thennospray, etc:. The ionization process in these techniques occurs at atmosphericpressure (Le., not under a vacuum). 3.2 Electrospray Ionization (ES,ESI): a method of ionization performed at atmospheric pressure, whereby ions in solution are transfmed to the gas phase via tiny charged droplets. These charged droplets are produced by the application of a strong electrical field. 3.3 Mass Spectrometry, Mass Spectrometer (MS), Tandem Mass Spectrometer (MS/MS): The MI Quattro 11triple quadrupole systems are equipped with quadrupolemass 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 @ I S M S ) for more specific frajpentation information. 3.4 Conventional vs. Zspray probe interface: The latest models of Micromass Quattro I1 triple quadrupole systems (post 1998)utilize a "Z-spray" conformation. The spray emitted from a probe is orthogonal to the cone aperture. In the conventional conformation it is aimed directly at the cone aperture, after passing through a tortuous pathway in the counter electrode. Though the configuration is different, the methods of operation, cleaning, and maintenance are the same. However, Z-spray components and conventional components are not compatible with one another, but only with similar :systems(i.e., :Z-spraycomponents are compatible with some other Z-spray systems, etc.) 3.5 Mass Lynx Software: System software designed for the specific opcxation of these Quattro 11triple quadrupole systems. Currently MassLynx has 'Windows95 and WindowsNT 4.0 versions. All versions are similar. For more details see the manuaI specific to the instrument (Micromass Quattro 11triple quadrupoleMassLynx or bdasslynx NT 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. 4.1.2 When handling samples or solventswear appropriateprotective gloves, eyewear, and clothing. ETS-8-5.1 Analysis of Serum Extract Using ESNS Page 2 of 9 3M -Ernvi Page 104 3 M Medical Department Study: T-6316.8 BACK TO MAIN Analytical Report: FACT TOX-097 LRN-U2452 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 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 instrumentation that comes in Icontact with the sample or extract. 6.0 EOUIPMENT 6.1 Equipment listed below may be modified in order to optimize the system. Document any modificationsin the raw data as method deviations. 6.1.1 Micromass Quattro II triple 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 7.0 SUPPLIES AND MATERIALS 7.1 Supplies 7.1.1 High purity grade nitrogen gas regulated to approximately 'LOO psi (House air system) 7.1.2 HPLC analytical column, specifics to be deteimined 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, 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 standards are prepared during the extraction procedure. See ETS-8-4.1. 9.0 SAMPLHEANDLING 9.1 Fresh matrix standards are prepared with each analysis. Extracted standards and samples are stored in capped autovialsor capped I5 mL centrihge tubesuntil analysis. ETS-8-5.1 Analysis of Serum Extract Using ESMS Page 3 of 9 Page 105 3M Medical Department Study: T-6316.8 BACK TO MAIN Analytical Report: FACT TOX-097 LRN-U2452 9.2 If analysis will be delayed, extracted standards and samples can be refrigerated at approximately4" C, or at room temperature, until analysis can be!performed. 10.0 QUALITY CONTROL 10.1 Solvent Blanks, Method Blanks and Matrix Blanks 10.1.1 Solvent blanks, method blanks and matrix blanks are prepared and analyzed with each batch to determine contamination or carryover, 10.1.2 Analyze a method blank and a matrix blank prior to each calibration curve. 10.2 Matrix Spikes 10.2.1 Matrix spikes are prepared and analyzed to determine the matrix effect on the . recovery efficiency. 10.2.2 Matrix spike duplicates are prepared and analyzed to measure the precision and the recovery for each analyte. 10.2.3 Analyze a matrix spike and matrix spike duplicate per forty samples, with a minimum of 2 spikes per batch. 10.2.4 Matrix spike and matrix spike duplicate concentrations will fall in the mid-range of the initial calibration curve. Additional spike:concentrations may fall in the lowrange of the initial calibration curve. 10.3 Continuing Calibration Verifications 10.3.1 Continuing calibration verifications are analyzed to verify the continued accuracy of the calibration curve. 10.3.2 Analyze a mid-range calibration standard after every tenth sample, with a minimum of one per batch. 11.0 CALIBRATIONAND STANDARDIZATION 11.1 Analyze the extracted matrix standards prior to and following each set of extracts. T h e average of two standard curves will be plotted by linear regression (y = my + b), weighted l/x,not forced through zero, using MassLyw or other suitable software. 11.2 If the curve does not meet requirements, perform rouline maintenance 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 fiJ1 range ofthe standard curve. Example: when attempting to quantitate approximate:ly 10 ppb of analyte, generate a calibration curve consisting of the standards from 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. ETS-8-5.1 Analysis of Serum Exnact Using E S N S Page 4 of 9 Page 106 3M Medical Department Study: T-6316.8 BACK TO MAIN Analytical Report: FACT TOX-097 LRN-U2452 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 Ionization Mode as appropriate and mass to 499 or other appropriate masses. A full scan is usually collected along with the SIRS. Save acquisition method. If MS/MS instruments are employed, additional product ion fragmentation information may be collected. See Micromass MassLynx GUIDE TO DATA ACQUISITION for additional information and MRM (Multiple Reaction Monitoring). 12.1.3 Typically the analytical batch run sequence begins with a set of extracted matrix standards and ends with a set of extracted ma.trix standards, 12.1.4 Samples are analyzed with a contmuing calibration check injected after every tenth sample. Solvent blanks should be analyzed priodically to monitor possible analyte carryover and are not considered samples but may be inclu'dedas 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 following conditionsor at conditions the analyst considers appropriate for optimal response. Record actual conditions in the instrument logbook 12.2.2.1 Sample size = 10 pL injection 12.2.2.2 Inject/sample = 1 12.2.2.3 Cycle time = 13.5 minutes 12.2.2.4 Solvent ramp = 0.00 min. 8.50 min. I 11.O min. I 90% I 12.0min. I 40% I 10% 60% I-- 12.2.2.5 Press the "Start"button. 12.3 Instrument Set-up 12.3.1 Refer to ETS-9-24.0for more details. 12.3.2 Check the solvent level in reservoirs and refill if necessary. ETS-8-5.1 Analysis of Serum Extract Using ESMS Page 5 of 9 Page 107 3M Medical Department Study: T-6316.8 BACK TO MAIN Analytical Report: FACT TOX-097 LRN-U2452 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 nojagged 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 uWmin 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 lWowing settings. These settings may change in order to optimize the response: 12.3.6.1 12.3.6.2 12.3.6.3 12.3.6.4 Drying gas 250-400liters/hour ESI nebulizing-gas10-15 literdhow HPLC constant flow mode, flow rate 10- 500 pLJmin Pressure 4 0 0 bar (This parameter is not set, it is a guide to ensure the HPLC is operating correctly.) 123.7 Carefully guide the probe into the opening. Insert probe uritil it will not go any further. Connect the voltage cables to 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 ESMS source is recommended for the analysis of biological matrices. 12.3.1OClick on start button in the Acquisition Control Pane1 (this may vary among MassLynx versions, see appropriateMassLynxUSERS GlJIDE). 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 using the followirtg equation: % Recovery = Observed Result - Backmound R& x 100 Expected Result 13.1.5 Calculate percent difference using the following equation: - % Difference = ExDected Conc. Calculated COIIII x 100 Expected Conc. 13.1.6 Calculate actual concentration of PFOS,or other fluorochenlical, in matrix (Clg/mL): {ng of PFOS calc. from std. Curve x Dilution Facto& x [Initial Volume of matrix (mL) + mL of`surroaate Standard) Final Volume (mL) 1 LIE 1000 ng ETS-8-5.1 Analysis of Serum Extract Using ESMS Page 6 of 9 Page 108 3M Medical Department Study: T-6316.8 BACK TO MAIN Analytical Report: FACT TOX-097 LRN-U2452 14.0 METHOPDERFORMANCE 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 Blainks 14.2.1 Solvent blanks, method blanks, and matrix ttlanks values are must be below the lowest standard in the calibration curve 14.3 Calibration Curves 14.3.1 The ? value for the calibration curve must be 0.980 or better. 14.4 Matrix Spikes 14.4.1 Matrix spike percent recoveries are must be within f 30% of thespiked concentration. 14.5 Continuing Caiibration Verifications 14.5.1 Continuing calibration verificationpercent recoveries must be k 30% of the spiked concentration. 14.6 If criteria listed in thismethod performance section isn't met, maintenance may be performed on the system and samples reanalyzed or (otheractions iiS determined by the analyst. Document all actions in the appropriate logbook. 14.7 If data are to be reported when performance criteria have not been met, the data must be footnoted on tables and discussed in the text of 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 hcated in the laboratory. 16.0 RECORDS 16.1 Each page generated for a study must have the following information included either in the header or hand Written on the page: study or project number, acquisition method, integration method, sample name, extraction date, dilution factor (if applicable), and analyst. 16.2 Print the tune page, sample list, and acquisition method from MassILynx to include in the appropriate study folder. Copy these pages and tape into the instruinent 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 method, and chromatograms, from MassLynx, and store in the study folder. ETS-8-5.1 Analysis ofSerumExtract Using E S M S Page 7 of 9 Page 109 3M Medical Department Study: T-6316.8 I' BACK TO MAIN Analytical Report: FACT TOX-097 LRN-U2452 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. Relcord in study :notebook the file name and location of backup electronic data. 17.0 TABLESD. IAGRAMFS.LOWCHARTASN,D VALIDATrONDATA 17.1 Attachment A: ETS-8-5.1 Data summary spreadsheet. 18.0 REFERENCES 18.1 FACT-M-4. I, "Extraction of Potassium Perffuorooctanesulfonate or Other Fluorochemical compounds from Senun for Analysis Using HPLC-I!lectrospray/hlassSpectrometry 18.2 ETS-9-24.0, "Operation and Maintenance of the Micromass Atmospheric Pressure Ionizationh4ass Spectrometer Quattro 11triple quadrupole Systems" 18.3 The validation report associated with this method is ETS-8-4.0 & 5.0-V-1. 19.0 AFFECTEDDOCUMENTS 19.1 ETS-8-4.1,`Zxtraction of Potassium Perfluorooctauesulfonateor Other Fluorochemical Compounds &om Serum for Analysis Using HPLC-I3lectrosprayb/iass Spectrometry" 20.0 REVISIONS Revision Number. 1 Reason For Revisioi! Section 6.1.2 Clarification of HPllOO system components. Section 11.1 Average of two curves, not standsud 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 fiom attachment B to A. - Revision Date 04/02/99 m ETS-8-5.1 Analysis of Serum Extract Using ESMS m Page 8 of9 Page 110 ~~ 3M Medical Department Study: T-6316.8 - BACK TO MAIN Analytical Report: FACT TOX-097 LRN-U2452 Laboratory Study # Study: Test Material: Matrix/Final Solvent: MethodIRevision: Analytical Equipment System Number: Instrument Softwarfleaion: Filename: R-Squared Value: Slope: Y Intercept Date of Extractiodhalyst: Date of AnalysidAnalyst: GrouplDose: Taken from the study folder. Sample#: Taken from the study folder. Concentration (ugh&): Taken from the MassLynx integration mmm;uy. Initial Volume (mL): Takenfrom the study folder. Dilution Factor: Taken from the study folder. Final Cone. (ug/mL): Calculated by dividing the initial volume from the concentration Attachment A: Summary Spreadsheet ETS-8-5.1 Analysis of Serum Extract Using ES/MS Page 9 of 9 Page 111 3M Medical Department Study: T-6316.8 BACK TO MAIN Analytical Report: FACT TOX-097 LRN-U2452 3M ENVIRONMENTLAALBORATORY ANALYSIS OF POTASSIUM PEIU?LUOROOCTANESULFONATE OR OTHER FLUOROCHEMICALSRy LIVEREXTRACTS USING HPLC-ELECI'ROSPRAY/MASS SPECTROMETRY Method Number: ETS-8-7.0 Author: LisaClemen, Glenn Langenburg Adoption Date: 0 ?/ 22 Revision Date: Nfl Approved By: m Group Leader 1A Technical Reviewer 31 14/33 Date 07& 7 Date 1.0 SCOPE AND APPLICATION 1.1 Scope: This method is for the analysis of liver extracts for fluorochemical surfactantsusing HPLC-electrospray/mass spectrometry. 1.2 Applicable Compounds:Fluorochemicalsurfactants or other fluorinated compounds, or other ionizable compounds. 1.3 Matrices: Rabbit, rat, bovine, monkey liver, or other tissues as designated in the validation report. Word 6/95 ETS-8-7.0 Analysis of Liver Extract Using ES/MS Page 1 of 10 v - Page 112 3M Medical Department Study: T-6316.8 BACK TO MAIN Analytical Report: FACT TOX-097 LRN-U2452 2.0 SUMMARY OFMETHOD 2.1 This method describes the analysis of fluorochemicalsurfactants extracted from liver using HPLC-electrospray/mass spectrometry, or similar system as appropriate. The analysis is performed by monitoring a single ion characteristic of a particular fluorochemical, such as the perfluorooctanesulfonate(PFOS)anion,m/z = 499. Additionally, samples may be analyzed using a tandem mass spectrometer to furlher verify the identity of a compound by detecting daughter ions of the selected parent ion. 3.0 DEFINITIONS 3.1 Atmospheric Pressure Ionization (MI):The Micromass Quattro 11triple quadrupole systems allow for variousmethods of ionization by utilizing v a r i o i ~sources, probes, and interfaces. These include but are not limited to: Elecb-ospray Ioniration @SI), Atmospheric Pressurechemical Ionization (APcI),Thermospray, atc. 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 iorlization performed at atmospheric pressure, whereby ions in solution are transferred to the gas phase via tiny charged droplets. These charged droplets are produced by the application of a strong electrical field. 3.3 Mass Spectrometry,Mass Spectrometer (MS),Tandem Mass Spectrometer (MS/MS): The API Quattro II triple quadrupole mass spectromc:tex is equipped with two quadrupole mass selectivedetectors and a collision cell. Ionsm:selectively discriminated by mass to charge ratio ( d z )and subsequently detected. A sinpJe MS may be employed for ion detectionor an ion may be selected in the first quadrupole, hgmented in the collision cell, and these fragmentsmay be analyzed in the second quadrupole. 3.4 Conventional vs. Z-spray probe interface: The latest models of Micromass Quattro I1 triple quadrupole (post 1998)utilize a "Z-spray" conformation. Tiespray 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 1:ortuouspathway in the counter electrode. Though the configuration is different, the methods of operation, cleaning, and maintenance are the same. However, Z-spray compaaents and con.ventional components are not compatiblewith one another, but only with similar systems (Le:. 2-spray components are compatiblewith other 2-spraysystems, etc.) 3.5 Mass Lynx Software: System software designed for the specific operation of these Quattro 11triple quadrupolesystems. Currently MassLynxhiis 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 IdassLynx or MassLynx NT 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 SO00 Volts. 3H- taboratory ETS-8-7.0 Analysis of LiverExtractUsing ESMS Page 2 of 10 Page 113 3M Medical Department Study: T-6316.8 BACK TO MAIN Analytical Report: FACT TOX-097 LRN-U2452 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 pressure of 400 bar (5800 psi). 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 interferenceswhen analyzing samples, Teflon shdl 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. Document any . modifications in the raw data as method deviations. 6.1.1 Micromass Quattro II triple quadrupole Mass Spectrometer equipped with an electrospray ionization source. 6.1.2 HP1100 low pulse solvent pumping systeni, solvent degiisser, column compartment, and autosampler 7.0 SUPPLIES AND MATERIALS 7.1 Supplies 7.1.1 High purity grade air regulated to approxiniately 100psi (houseair system) 7.1.2 HPLC analyticalcolumn, specifics to be determinedby the analyst and documented in the raw data 7.1.3 Capped autovials or capped 15 ml centrifuge tubes 8.0 REAGENTANSD STANDARDS 8.1 Reagents 8.1.1 Methanol, HPLC grade or equivalent 8.1.2 Milli-Qm water (ASTM type I), all water used in this method should be ATSM type I, or equivalent, and be provided by a Milli-Q TOC Plus system or other vendor 8.1.3 Ammonium acetate, reagent grade or equivalent 8.1.3.1 When preparing different amounts than those listed, adjust accordingly. 8.1.3.2 2.0 mM ammonium acetate solution: Weigh approximately 0.300 g ammonium acetate. Pour into a 2000 mL volumetric container containing 2000 mL Mil1i-Qm water, mix until all solids Ere dissolved. Store at room temperature. ETS-8-7.0 Analysis of Liver Extract Using ESMS Page 3 of 10 Page 114 3M Medical Department Study: T-6316.8 BACK TO MAIN Analytical Report: FACT TOX-097 LRN-U2452 8.2 Standards 8.2.1 Typically two method blanks, two matrix blanks, and eighteen matrix standards are prepared during the extraction procedure. Refer to ETS-8-6.0. 9.0 SAMPLHEANDLING 9.1 Fresh matrix standards are prepared with each analysis. Extracted standards and samples are stored in capped autovials or capped 15 ml centrifuge tubes until analySis. 9.2 If analysis will be delayed, extracted standards and samples 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 blanks, and matrix blanks are prepared and analyzed with each batch to determine contamination or oanyover. 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 rrnalyzed to measure the precision and the recovery for each d y t e . 10.23 Analyze a matrix spike and matrix spike duplicate per forty samplep. With a minimum of 2 spikes per batch. 10.2.4 Matrix spike and matrix spike duplicate concentrations will fall in the mid-range of the initial calibration curve. Additional spike concentrationsmay fall in the lowrange of the initial calibration curve. 10.3 Continuing CalibrationChecks 10.3.1 Continuing calibration verifications are analyzed to veri@ the continued accuracy of the calibration curve. 10.3.2 Analyze a mid-range calibration standard every tenth sample, with a minimumof 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 through the ongin, using MassLynx or other suitable somare. 11.2 If the curve does not meet requirements perform routine maintenance or reextract the standard curve (if necessary) and reanalyze. 3 r v l F- ETS-8-7.0 Analysis of Liver Extract Using E S N S Page 4 of 10 Page 115 3M Medical Department Study: T-6316.8 BACK TO MAIN Analytical Report: FACT TOX-097 LRN-U2452 11.3 For purposes ofaccuracy when quantitating low h e l s 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 approxiniately 10 ppb of analyte, generate a calibration curve 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 Acquisition Set up 12.1.1 Set up the sample list. 12.1.1.1 Assign a sample list filename using MO-DAY-Iast digit of yea-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 Reaction Monitoring). Set Ionization Mode as appropriate and mass to 499 or other appropriate masses. A full scan is usually collected along with the SIRS. Save acquisition method. If MS/MS instruments are employed, additional product ion hgmentation information may be collected. Refer to Micromass MassLynx GUIDETO DATA ACQUISITION for additional infomiation and h4R.M. 12.13 Typically the analytical batch run sequenct: begins and ends with a set of extracted matrix standards. 12.1.4 Samples are analyzed with a continuing ca.ibration 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 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 follclwingconditions or at conditions the analyst considers appropriatefor optimal n:sponse. 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 ETS-8-7.0 Analysis ofLiver Extract Using ESMS Page 5 of 10 Page 116 3M Medical Department Study: T-6316.8 BACK TO MAIN Analytical Report: FACT TOX-097 ' LRN-U2452 12.2.2.4 Solvent ramp conditions Ammoniuni acetate 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 II Triple Quadrupole Mass SpectrometerFittcd with an AtmosphericPressure Ionization Source," for more details. 12.3.2 Check the solvent level in reservoirs and refill if necessay. 123.3 Check the stainlesssteel capillary at the end of the probe. Use an eyepiece to check the tip. The tip should be flat with no jagged edges. Ifthe tip is found to be unsatisfactory, disassemble the probe and replace the stainless steel capillary. 123.4 Turn on the nitrogen. 123.5 Open the tune page. Clicks on operate to initiatesource block and desolvation heaters. 123.6 Open the Inlet Editor. 123.6.1 Set HPLC pump to "On" 12.3.6.2 Set the flow to 10 - 500Uymin 01'as appropriate 12.3.6.3 Observe droplets coming out ofthe tip ofthe probe. 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 12.3.6.4 Allow to equilibrate for approximately 10minutes. 123.7 The instrument uses these parameters at the: following settings. These settings may change in order to optimize the response: 12.3.7.1 Drying gas 250-400litersihour 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 of Liver ExtractUsing ESMS Page 6 of 10 3Fj)f`ivkmmmtal Laboratory Page 117 3M Medical Department Study: T-6316.8 BACK TO MAIN Analytical Report: FACT TOX-097 LRN-U2452 12.3.8 Print the tune page, with its parameters, arid store it in the study binder with a copy taped into the instrument log. 123.9 Click on start button in the Acquisition Control Pane1 (tllis may vary among MassLynx versions, refer to appropriate IvIassLynX User's Guide). Ensure start and end sample number includes all samples to be analyzed. 13.0 DATAANALYSIASND CALCULATIONS 13.1 Calculations: 13.1.4 Calculate matrix spikepercent recoveries using the following equation: % Recovery = - Observed Result Backmound Result x 100 Expected Result 13.1.5 Calculatepercent difference ushgthe folli~wingequation: - % Difference = Exuected Conc. Calculated (& Expected Conc. x 100 13.1.6 Calculate actual concentrations in matrix (pg/g): fna of PFOS calc. fromstd. Curve x DilutionFactor) [Initial Weipht of Liver (E) Final Volume (mL) x 1 UP 1000 ng 14.0 METHOPDERFORMANCE 14.1 Method Detection Limit (MDL)and Limit of Quantitation (LOQ) are method, andyte, and . matrix specific. Referto ETS-8-6.0, AttachmentB for a listing of current validatedMDL and LOQ values. 14.2 Solvent Blanks, Method Blanks and Matrix Blmks 14.2.1 Solventblanks, method blanks, and matrix.blanks must be below the lowest standard in the calibration curve. 143 CalibrationCurves 14.3.1 The 3 value for the calibration must be 0.980 or better. 14.4 Matrix Spikes 14.4.1 Matrix spikepercent recoveries must be within k 30%ofthe spiked concentration. 14.5 Continuing Calibration Verification 14.5.1 Continuing calibration verification percent recoveries must be within rt 30% ofthe 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-E- fatory ETS-8-7.0 Analysis of Liver Extract Using ESMS Page 7 of 10 Page 118 3 M Medical Department Study: T-6316.8 BACK TO MAIN Analytical Report: FACT TOX-097 LRN-U2452 14.7 If data are to be reported when performance cnteria have not been met, the data m u t 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 infomation included either in the header or hand written on the page: study or project number, acquisition method, integration method, sample name, extraction date, dilution factor (if applicable), and analyst. 16.2 Print the tune page, sample list, and acquisitionmethod from MnssLynxto 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/x7then print these graphs and store in the study folder. 16.4 Print data integration summary, integration method, and chromatograms from MassLynx and store in the study folder. 16.5 Summarize data using suitable software (Excel 5.W) and store in the study folder, refer to Attachment A for an example of a summary spreadsheet. 16.6 Back up electronic data to appropriate medium. Record in study notebook the file name and location of backup electronic data. 17.0 TABLESD, IAGRAMFSL. OWCHARTANSD, VALIDATION DATA 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 from Liver for Analysis Using HPLC-Electrospray/Mass Spectrometry" 18.2 ETS-9-24.0",Operation and Maintenance of the M!icromass Atmospheric Pressure Ionization/Mass Spectrometer Quattro II triple quadrupole Systems" 18.3 The validation report associated with this method is ETS-8-6.0CPC 7.0-V-1 19.0 AFFEC~EDDOCUMENTS 19.1 ETS-8-6.0",Extraction of Potassium Perfluorooctanesulfonateor Other Fluorochemical Compounds &om Liver or Fluid for Analysis Using HPLC-Electrospray/Mass Spectrometry" ETS-8-7.0 Analysis of Livcr.Extract Using ESMS Page 8 of 10 Page 119 3M Medical Department Study: T-6316.8 20.0 REVISIONS Revision Number BACK TO MAIN Analytical Report: FACT TOX-097 LRN-U2452. Reason For R e v i s h Revision - Date ETS-8-7.0 Analysis of Liver Extract Using ESlMS Page 9 of 10 Page 120 3M Medical Department Study: T-6316.8 BACK TO MAIN Analytical Report: FACT TOX-097 LRN-U2452 Laboratory Stud.y# Study: Test Material: MatrixFinal Solvent: MethodlRevision: Analytical Equipment System Number: Instrument SoftwareNersion: Filename: R-Squared Value: Slope: Y Intercept Date of ExtractiodAnalyst: Date of AnalysidAnalyst Sample#: Taken from the study folder. Concentration (ng/g): Taken from the MassLkx integration summary. InitIal Wt. (g): Taken from the study folder. Dilution Factor: Taken from the study folder. Final Cone. (uglg): Calculated by dividing the initial volume from the concentration 3M-nE Attachment A: Summary Spreadsheet ETS-8-7.0 Analysis of Liver Extract Using ESMS Page 10 of 10 Page 121 3M Medical Department Study: T-6316.8 Appendix D: Data Summary Tables BACK TO MAIN Analytical Report: FACT TOX-097 LRN-U2452 Table 6. Rabbit Sera FO PFOS, PFOSA and PFOSA4 Data for FACT-TOX-097 *Nonqualitative screening data only. It is not possible to verify true recovery of endogenous analyte from tissues without radio-labeled reference material. The only measurement of accuracy available at this, time, matrix spike studies, indicate that the data quantitative to 30% or greater. 3M Environmental Laboratory Page 122 3M Medical Department Study: T-6316.8 BACK TO MAIN Analytical Report: FACT TOX-097 LRN-U2452 Group Dose Sample # KEtFOSF rdmL PFOSEA IJglmL LOQ (0.00487) LOQ (0.00487) LOQ (0.00487) LOQ (0.00487) LOQ (0.00487) LOQ (0.00487) LOQ (0.00487) LOQ (0.00487) LOQ (0.00487) LOQ (0.00487) LOQ (0.00487) 2.5 mglkglday 0.5 mglmL Group 5 3.75 mglkglday 0.75 mglmL 8694F 8695F 8696F 8697F 8698F 8699F 8700F LOQ (0.00487) j i <LOQ(0.0216) <~0~;0.00487; I I <LOQ (0.0216) I <LOQf0.00487\ 0.0294 <LOQ (0.00487) 0.0279 <LOQ (0.00487) 0.0386 <LOQ (0.00487) cLOQ (0.0216) <LOQ (0.00487) 1 0.132 <-OQ (0.00487) *Non-qualitative screening data only. ~ ~ It is not possible to verify tnre recovery of endogenous analyte from tissues without radio-labeled reference material. The only measurement of accuracy atailable at this time, matrix spike studies, indicate that the data quantitative to 30% or greater. 3M Environmental Laboratory Page 123 3M Medical Department Study: T-6316.8 BACK TO MAIN Analytical Report: FACT TOX-097 LRN-U2452 Table 8. Rabbit Liver FO PFOS, PFOSA and PFOSPA Data for FACT-TOX-097 It is not possibleto verifyirue recoiery 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 quantitative to 30% or greater. 3M E~ivironmentaLl aboratory Page 124 3M Medical Department Study: T-6316.8 BACK TO MAIN Analytical Report: FACT TOX-097 LRN-U2452 Table 9. Rabbit Liver FO N-EtFOSE and PFOSEA D,atafor FACT-TOX-097 Group Dose 0.0 mglkglday 0.0 mglmL Group 2 0.1 mglkglday . 0.02 mglmL Group 3 1.O mglkglday 0.2 mglmL Group 4 2.5 mglkglday 0.5 rnglmL I Group 5 3.75 mglkglday 0.75 mnlmL 8683F 8684F 8685F 8686F 8687F 8688F 8689F 8690F 8691F 8692F 8693F 8694F 8695F 8696F 8697F 8698F 8700F P I I CLOQ (0.0525) CLOQ (0.0525) i I 4 cLOQ 10.0298) <LOQ (0.0298; cLOQ 10.0298) cLOQ (0.0525) CLOQ (0.0298) cLOQ (0.0525) CLOQ (0.0298) CLOQ (0.0525) cLOQ (0.0298) CLOQ (0.0525) cLOQ (0.0298) cLOQ (0.0525) cLOQ 10.0298) cLOQ (0.0525) 0.621 CLOQ (0.0525) cLOQ (0.0298) cLOQ 10.0298) 0.161 0.123 <LOQ (0.0298) CLOQ (0.0298) 0.140 0.417 0.477 93.5 30.8 ' 3 CLOQ10.0298) ,cLOQ (0.0298) ,:LOQ (0.0298) 1 *:LOQ (0.0298) 3M Eivironrnental Laboratory Page 125 3M Medical Department Study: T-6316.8 BACK TO MAIN Analytical Report: FACT TOX-097 LRN-U2452 Table 10. FACT-TOX-097 Data Summary of Average Sera Concenlration(pg/mL) and Standard Deviation ( S D ) i* N-EtFOSE* PFOSEA *Non-qualitative screening data only. It is not possible to verify true recovery of endogenous analyte from tissues without radio-labeled reference material. The only measurement of accuracy available at this time, matrix spike studies, indicate that the data quantitative to 30% or greater. NA = Not Applicable 3M Environmental Laboratory Page 126 3M Medical Department Study: T-6316.8 BACK TO MAIN Analytical Report: FACT TOX-097 LRN-U2452 Table 11. FACT-TOX-097 Data Summary of Average Liver Concentration (pglg) and Standard Deviation (ASD) *Non-qualitativescreening data only. It is not possible to verify true recovery of endogenous analyte from tissues without radio-labeled reference material. The only measurement of accuracy available at this time, matrix spike studies, indicatethat the data quantitative to 30% or greater. NA = Not Applicable Table 12. Approximate LOQ Values Used in FACT-TOX-097 I I I I Matrix I Compound 1 Loa I PFOS 0.0279 IJg/g 0.0298 1.1919 0.0458 pg/mL 0.00490 cig/mL - 1 I PFOSAA* 0.0124 .ud" mL 0.0216 py/mL I PFOSEA I 0.00487 Lia/mL I ~~ ~ ~~ *Nonqualitative screening data only. 3M Environmental Laboratory Page 127 3M Medical Department Study: T-6316.8 Appendix E: Data Spreadsheets BACK TO MAIN Analytical Report: FACT TOX-097 LRN-U2452 3M Environmental Laboratory Page 128 BACK TO MAIN F 2 a 0 n, 73 P, (0 (D A N W BACK TO MAIN W c PACT-TOX-097 Argmd416010 0 i!a(I, r UP, 3 b -0 P, (D (I, A W 0 BACK TO MAIN m22-. FACC-TOXm a ArgU#BMlO 3 22 4.nV- EL B mmtoQmIDs067afia DL U 1m18.m UI. UI. VI. in. in. iii P, a 1omm.n in%UI.UIO,in. VI. VI I-N inuVI.uloo,in. in. VI 2 1S- uim.in, mcqin. VI. in - logpuw inm. I M I ~ . in- VI. In. In 3 mmis.16 in. in. in. in. IR in v, a C u BACK TO MAIN 4 1 FACT-TOX497 (D ArgnrY418410 a E6. 8 e C a Y A w 10 BACK TO MAIN E E A A 0 w BACK TO MAIN 2 5aru FACT-TOX-097 Arg1~1?4lbolO 0 W p3 (0 (D 2 0 P BACK TO MAIN 5 PACT-Mx-097 a AguM 418410 ii` E FACT-M-LO -97 BACK TO MAIN z w I (aD 6' e, 9cnm FACTM-LO w9l FACT-TOX-097 ArgW 418-010 BACK TO MAIN P73 0 ;r FACT-TOX-097 A r g d 418-010 BACK TO MAIN FACT-M-20 Excd 91 s gn z-l FACT-TOX-097 Ar@as#418010 BACK TO MAIN NA NA PK NA Nh NA NA NA 0.161 NA 0.140 417 0.417 471 0.471 0.141 omn FAClM2.0 GxCl9l BACK TO MAIN 3M Medical Department Study: T-6316.8 Analytical Report: FACT TOX-097 LRN-U2452 Appendix F: Example Calculations Formula Used for Sera Analyses in Study FACT-TOX-097 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 3, Animal ID 8690F (PFOS) 519 ng/mL x 10 x 0.9275 x 1 mL x 1.0 pg 1.005 mL 1000 ng = 4,,79pg/mL 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-TOX-097 a AR (ng/g) x 3 curve sample x SC x DF x 1.0 pg = Reported concentration (pg/g) 1000 ng a curve is assumed to be: 1 g liver 5 mL H20 Calculation Used for Group 3, Animal ID 8690F (PFOS) 119 nglg x 1 g/5 mL x 0.9275 x 100 x 1.0 pg = 10.8 pg/g 1.0200 g/ 5mL 1000 ng AR- Analytical result from MassLynx summary a curve-Density of the liver standard curve, assumed to be lg liver/ 5 ml water a sample-Density of the liver sample (g sample/ 5 mL H2'0) SC-PFOS salt correction constant (0.9275) DF- Dilution factor 3M Environmental Laboratory Page 140 BACK TO MAIN 3M Medical Department Study: T-6316.8 ~~ ~~ Appendix G: Interim Certificate(s)of Analysis Analytical Report: FACT TOX-097 LRN42452 3M Environmental Laboratory Page 141 BACK TO MAIN 3M Medical Department Study: T-6316.8 - __ Analytical Report: FACT TOX-097 LRN-U2452 Centre Analyticarl Laboratories, Inc. 3048 Research Drive State College, PA 16801 Phone: (814)231-8032 Fax: (814)231-1253or (814)231-1580 INTERIM CERTIFICATE OF ANALYSIS Centre Analytical Laboratories COA Reference #: 023-022-1 3M Product: EtF0SE:-OH Test Control Reference #: SD-013 Purity: 88.9% 5. Nickel 6. Iron 1. <0.001 wt.lwt.% 2. <0.001 wt.iwt.% 3. <0.001 wt./wt.% 4. 0.002 wt./wt.% 3. Bromide 4. Nitrate 5. Nitrite 1. Carbon 2. Hydrogen 3. Nitrogen 4. Sulfur COA023-022-1 3M Environmental Laboratory 3. <0.040wt./wt.% 4. <0.009wt.iwt.% 5. e0.006wt./wt.% 6 . <0.007wt./wt.% 1. <0.1 wt.twt.% 1. Theoretical Value = 25.2% 2. TheoreticalValue = 1.75% 3. TheoreticalValue = 2.45% 4. Theoretical Value = 5.60% 1. 24.42 wt./wt.% 2. 1.78 wt.iwt.% 3. 2.72 wt.twt.% 4. 9.34 wt.iwt.% Page 1 of 3 Page 142 3M Medical Department Study: T-6316.8 BACK TO MAIN Analytical Report: FACT TOX-097 LRN-LJ2452 14) 231-8032 (814) 231-1253 Of (814) 231-1580 INTERTM CERTZFICATEOF ANALISIS Centre Analytical Laboratories COA Reference #: 023-022-1 3M Product: EtFOSE-(OR 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% + totd NMR impurities, 0.90% + GCMS impurities, 10.21 + POAA, 0.03%) Total impurity fkom all tests = 11.14% Purity = l o o % - 11.14%=88.9% z~~~ Trifluoroacetic acid rc4 HFBA Heptafluorobutyric acid NFPA Nonafluoropentanoic acid PFPA Pentafluoropropanoic acid 3Theoreticalvalue calculations based on the empirical formula, C I Z H ~ F ~ ~ N O ~ S (MW=571) COA023-022-1 3M Environmental Laboratory --- Page 2 of 3 Page 143 BACK TO MAIN 3NI Medical Department Study: T-6316.8 ' Analytical Report: FACT TOX-097 LRN-U2452 CEntrE Analytical Laboratories, Inc. 3048 Research Drive State College, PA 16801 Phone: (814) 231-8032 Fax: (814) 2311-1253or (814) 231-1580 pl INTERlM CERYTFICATE OF ANALYSIS Centre Analytical Laboratories COA R.eference#: (023-022-1 3M Product: EtFOSEXIH Test Control Reference #: SD-013 GC/MS PurityProfile This work was conducted under EPA Good Laboratory Practice Standards (40 CFR 160). Prepared By: Scientist ratories Centre halytical Laboratories COAO23-022-1 3M EnvironmentalLaboratory Page 3 of 3 Page 144 ~~ ' 3hrl Medical Department Study: T-6316.8 Appendix H: Report Signature Page BACK TO MAIN Analytical Report: FACT TOX-097 LRN-U2452 T L ..I. , D.V.M., Ph.D., Study Director TrA &/ Date ' John L. Butenhoff, Ph.D., Sponsor Representative , .. Date Kristen J. Hansen, Ph.D., PrincipalAnalytic81Investigator Date U-'d- ,n&/o, Date ..... a 3M Environmental Laboratory Page 145