Document 8Dmwa1vkYj4EJwLz52YyRBZm

mi 3M 3.V1 C.eneral Ottices 3M Center St. Paul. MN 55144-1000 612 733 1110 A&Z26-l050qOtr geHQ.oqoi -0373 September 5, 2001 D ocum ent Processing Center (7407) O ffice o f Toxic Substances U .S. Environmental Protection A gency 401 M Street, SW W ashington, DC 20460 Attn: T SC A Section 8(e) Coordinator % (( L_ Dear Section 8(e) D ocket Coordinator: Re: T SC A 8(e) Supplemental N otice on Sulfonate-based Fluorochem icals W ith this letter, 3M is providing final reports and other supplemental inform ation related to previous T SC A Section 8(e) notifications. M any o f the enclosed item s are analytical reports providing blood serum and liver levels o f test materials for w hich the in-life report referring to adm inistered d oses has already been subm itted to the 8(e) docket. In other cases w here the 8(e) notification consisted o f preliminary data, w e are subm itting a final study report. A ll o f the enclosed item s are already in E PA 's possession and available in T SC A D ocket A R -226. W e believe, how ever, that placing these item s in the 8(e) docket m ay allow for m ore convenient access to inform ation directly related to previous 8(e) notifications by 3M. The table b elow lists the enclosed item s and references the study or data w hich already has been the subject o f an 8(e) notification by 3M: Attached Submission Related Study/Data Already Filed Under 8(e) 1. Amended Analytical Study, 2(N-Ethylperfluorooctane sulfonamido)-ethanol in Two Generation Rat Reproduction, Determination o f the Presence and Concentration o f PFOS, M 556, PFOSAA, and PFOSA in the Liver and PFOS, M 556, PFOSAA, PFOSA and EtFOSE-OH in the Sera o f C rfC D BR VAF/Plus Rats Exposed to EtFOSE-OH, 3M Reference N o. T-6316.5, Analytical Report TOX-013, LRN-U2095, June 11,2001. Combined Oral (Gavage) Fertility, Developmental and Perinatal/Postnatal Reproduction Toxicity Study o f NEtFOSE in Rats, 3M Reference No. T6316.5, June 30, 1999, full report submitted February 15, 2000 to supplement earlier filing Iz '-8 'Hi 82 83S \m Contain NO CBI TSCA Section 8(e) Docket Coordinator Page 2 Attached Submission Related Study/Data Already Filed Under 8(e) 2. Analytical Laboratory Report, Determination o f the Presence and Concentration o f Potassium Perfluorooctanesulfonate (CAS Number: 2759-39-3) in the Serum and Liver o f Sprague-Dawley Rats Exposed to PFOS via Gavage, Laboratory Report No. U2006, Requestor Project No. 3M TOX 6295.9, October 27, 1999. 3. Report Amendment 1, Combined Oral (Gavage) Fertility, Developmental and Perinatal/Postnatal Reproduction Toxicity Study o f PFOS in Rats, Argus Research Laboratories, Inc., Protocol 418-008, Sponsor's Study No. 6295.9, April 13, 2000. Combined Oral (Gavage) Fertility, Developmental and Perinatal/Postnatal Reproduction Toxicity Study o f PFOS in Rats, Argus Research Laboratories, Inc., Sponsor's Study No. 6295.9, June 10, 1999, full report submitted February 15, 2000 supplementing earlier filing 4. Analytical Report, Determination o f the Presence and Concentration o f Perfluorooctanesulfonate, Perfluorooctanesulfonylamide, M556, and M 570 in the Liver and Sera Samples, 3M Environmental Laboratory Ref. No. U2636, TOX-028, February 23, 2001 13-Week Dietary Study o f N-Methyl Perfluorooctanesulfonamido Ethanol (N-MeFOSE) in Rats, 3M Ref. No. T6314.1, Covance Study No. 6329-225, dated June 30, 2000, Section 8(e) filing July 24, 2000 5. Analytical Laboratory Report, Determination o f the Concentration o f PFOS, PFOSA, PFOSAA, and EtFOSEOH in the Sera and Liver o f Crl.CDBR VAF/Plus Rats Exposed to N-EtFOSE, 3M Environmental Laboratory Report No. TOX-098, Laboratory Request No. U2402, 3M Ref. N o. T-6316.7, February 6, 2001. Final Report, Oral (Gavage) Developmental Toxicity Study o f 2(NEthylperfluorooctanesulfonamido)ethanol in Rats, 3M Reference No. T6316.7, December 17, 1998, submitted to Section 8(e) docket per letter o f August 21, 2000 6. Analytical Laboratory Report on the Determination o f the Presence and Concentration o f Potassium Perfluorooctanesulfonate (PFOS) or another metabolite o f 2(N-ethylperfluorooctanesulfonamido)-ethanol (NEtFOSE) in Liver and Serum Specimens, 3M Environmental Laboratory Report No. TOX-097, Laboratory Request No. U2452, 3M Ref. No. T-6316.8, February 8, 2001 Final Report, Oral (Stomach Tube) Developmental Toxicity Study o f NEtFOSE in Rabbits, 3M Reference No. T-6316.8, January 11, 1999, submitted to Section 8(e) docket per letter o f August 21, 2000 7. Final Report, Alexander, B., Mortality Studies o f Workers Employed at the 3M Decatur Facility, University o f Minnesota, April 26, 2001. Preliminary data submitted to Section 8(e) docket in letter o f December 15, 2000 TSCA Section 8(e) Docket Coordinator Page 3 . Attached Submission Related Study/Data Already Filed Under 8(e) 8. Final Report, Acute Oral Toxicity Screen with T-3290CoC in Albino Rats, Safety Evaluation Laboratory, Riker Laboratories, Inc., Project No. 0882AR0362, 3M Reference N o. T-3290 (40 % K+PFOSAA in 3 % EtOH, 17 % IPA and 40 % H20, L-6778, F-6873, Lot 501), November 5, 1982 [Bibliography entry in Docket AR-226, final report was to be moved to TSCA 8(e) docket] Acute Oral Toxicity Screen with T3290CoC in Albino Rats, Safety Evaluation Laboratory, Riker Laboratories, Inc., Project No. 0882AR0362, 3M Reference No. T3290 (40 % K+PFOSAA in 3 % EtOH, 17 % IPA and 40 % H20, L-6778, F6873, Lot 501), November 5, 1982, submitted to Section 8(e) docket in August 21, 2000 self-audit letter (which erroneously refers to rabbits rather than rats) 9. Giesy, J.P., and K. Kannan, Accumulation o f Perfluorooctanesulfonate and Related Fluorochemicals in Fish Tissue, Michigan State University, June 20, 2001. Preliminary data submitted to Section 8(e) docket May 26, 1999 10. G iesy, J.P., and K. Kannan, Accumulation o f Perfluorooctanesulfonate and Related Fluorochemicals in Mink and River Otters, Michigan State University, June 20, 2001. 11. G iesy, J.P., and K. Kannan, Perfluorooctanesulfonate and Related Fluorochemicals in Oyster, Crassostrea Virginica, From the G ulf o f M exico and Chesapeake Bay, Michigan State University, June 20,2001. 12. G iesy, J.P. and K. Kannan, Perfluorooctanesulfonate and Related Fluorochemicals in Fish-Eating Water Birds, M ichigan State University, June 2 0 ,2 0 0 1 . 13. G iesy, J.P. and K. Kannan, Accumulation o f Perfluorooctanesulfonate and Related Fluorochemicals in Marine Mammals, Michigan State University, June 20, 2001. If you have any questions about this subm ission, please contact m e at (651)737-4795. Sincerely, AA G eoijan Adams Manager, 3M Corporate Product Responsibility Enclosures 3M Medical Department Study: T-6316.8 Analytical Report: F A C T TO X -097 LRN-U2452 flR Study Title Oral (Stomach Tube) Developmental Toxicity Study of 2(N-Ethylperfluorooctanesulfonamido)-ethanol in Rabbits 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 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 -3 E -0 9 ,935 Bush Avenue St. Paul, M N 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 0 1c,o"vi `"O o .3 o EPA-OTS 000811830L a ilfi3 D L 3M Environmental Laboratory 4 3M M edical D epartm ent Study: T-6316.8 BACK TO MAIN Analytical Report: FA C T TO X -097 L R N -U 2 4 52 This page has been reserved for specific country requirements. 3M Environmental Laboratory Page 2 BACK TO MAIN 3M Medical 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: 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. Stability not determined for test and control articles under all conditions of administration. 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. There were no expiration dates on the reagent/solutions bottle per 21 CFR Part 58.83. 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 M edical D epartm ent Study: T -6316.8 BACK TO MAIN A nalytical R eport: FA C T TO X -097 L R N -U 2 4 5 2 (GLP Com pliance Statem ent Continued) t 71aaak. ____________ 7/57 w Marvin t7 Case D.V.M., Ph.D., Study Director * Date 1 _________________Q Z - / o John L. Butenhoff Ph.D., Sponsor Representative Date Kristen J. Hansen Ph,D. Principal Analytical Investigator Date 3M Environmental Laboratory Page 4 3M Medical Department Study: T-6316.8 BACK TO MAIN Analytical Report: FACT TOX-097 LRN-U2452 GLP Study-- Quality Assurance Statement Analytical Laboratory Report Title: Determination of the Presence and Concentration of Potassium Perfluorooctanesulfonate (PFOS) or another metabolite of 2(Nethylperfluorooctanesulfonamido)-ethanol (N-EtFOSE) in Liver and Serum Samples Study Identification Numbers: 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. Inspection Dates Phase Date Reported to Management Study Director 09/23/98 Sample receipt 11/21/98 11/21/98 10/30/00-11/01/00 Data 11/02/00 11/02/00 01/02/01 - 01/05/01 Draft report (1) 01/05/01 01/05/01 01/22/01 -01/23/01 Draft report (2) 01/23/01 01/23/01 QAU Representative Z Q ? /o \ 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 Characterization of Analytical Reference Material/Substance............................ 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 Characterization of 2(N-Ethylperfluorooctanesulfonamido)-ethanol............. 18 Characterization of Control Matrices...............................................................................18 Appendix B: Protocol, Amendments and Deviations...........................................................19 Appendix C: Extraction and Analytical Methods................................................................. 42 FACT-M-1.0, "Extraction of Potassium Perfluorooctanesulfonate or Other Anionic Fluorochemical Surfactants from 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 Compounds from Serum for Analysis Using HPLC-Electrospray/Mass Spectrometry," (14 pages)............................................................................................................................ 59 ETS-8-06.0, "Extraction of Potasium Perfluorooctanesulfonate or other Fluorochemical Compounds from Liver for Analysis Using HPLC-Electrospray/Mass Spectrometry," (14 pages)............................................................................................................................ 73 3M Environmental Laboratory Page 6 BACK TO MAIN 3M Medical Department Study: T-6316.8 Analytical Report: FACT TOX-097 L R N -U 2 4 5 2 FACT-M-2.0, " Analysis of Fluorochemicals in Liver Extracts Using HPLCElectrospray/Mass Spectrometry," (8 pages)...................................................................... 87 FACT-M-4.0, "Analysis of Fluorochemicals in Serum Extracts Using HPLCElectrospray/Mass Spectrometry," (8 pages)...................................................................... 95 ETS-8-05.1, "Analysis of Potassium Perfluorooctanesulfonate or other Fuorochemical Compounds in Serum Extracts Using HPLC-Electrospray/Mass Spectrometry," (9 pages)............................................................................................................................... 103 ETS-8-07.0, "Analysis of Potassium Perfluorooctanesulfonate or other Fluorochemical Compounds in Liver Extracts Using HPLC-Electrospray/Mass Spectrometry," (10 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 Environmental 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 #418-010............ 10 Table 2. Characterization of the Analytical Reference Materials/Substances in Study FACT-TOX-097........................................................................................................ 12 Table 3. Negative Ions Monitored........................................................................................ 15 Table 4. Characterization of Test Substance inStudy FACT-TOX-097..............................18 Table 5. Characterization of the Control Matrices Used for Liver and Sera Analyses in Study FACT-TOX-097..............................................................................................18 Table 6. Rabbit Sera F0 PFOS, PFOSA and PFOSAA Data for FACT-TOX-097...... 122 Table 7. Rabbit Sera F0 N-EtFOSE and PFOSEA Data for FACT-TOX-097..............123 Table 8. Rabbit Liver F0 PFOS, PFOSA and PFOSAA Data for FACT-TOX-097...... 124 Table 9. Rabbit Liver F0 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 (SD)...................................................................................... 126 Table 11. FACT-TOX-097 Data Summary of Average Liver Concentration (|jg/g) and Standard Deviation (SD)....................................................................................... 127 Table 12. Approximate LOQ Values Used inFACT-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. Butenhoff, 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 O. Johnson 'C ontract lab professional service employees Kelly J. Kuehlwein* Glenn Langenburg* Sally A. Linda* Ian A. Smith* Thomas P. Wagner* Bob W. Wynne* Richard D. Youngblom* Location of Archives All original raw data, protocol, and analytical report have been archived at the 3M Environmental Laboratory. The test 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. T e s t 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 2.9 to 4.2 kg; 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 0.1,1.0, 2.5, or 3.75 mg of N-EtFOSE per kg of body weight/day 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 are presented in the Argus Laboratory final report, "Oral (Stomach Tube) Developmental Toxicity Study of N-EtFOSE in Rabbits, #418010." Table 1. Dosage Levels, Concentration and Volumes for Argus Study #418-010 Dosage Group Number Dosage* Concentration Dosage of (mg/kg/day) (mg/mL) Volume Rabbits (ml_/kg) I 3 0 (Vehicle) 0 5 II 5 III 3 0.1 1.0 0.02 5 0.2 5 IV 3 2.5 0.5 5 V 5 3.75 0.75 5 *The test article will be considered 100% pure for the purpose of dosage calculations. Assigned Rabbit Numbers 8682F - 8684F 8685F - 8689F 8690F - 8692F 8693F - 8695F 8696F - 8700F 3M Environmental Laboratory Page 10 BACK TO MAIN 3M Medical Department Study: T-6316.8 A nalytical Report: F A C T T O X -0 9 7 L R N -U 2 4 5 2 S pecim en C o llectio n and A nalysis 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 #418-010 and sent to the 3M Environmental Laboratory to be extracted and analyzed for perfluorooctanesulfonate (PFOS), perfluorooctanesulfonamide (PFOSA), perfluorooctanesulfonamido(ethyl)acetate (PFOSAA), N-ethyl perfluorooctanesulfonamido ethyl 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 10C. 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 maximum period of 10 years and will be stored at the laboratory at -20C 10C. 3M [Environmental Laboratory Page 11 BACK TO MAIN 3M Medical Department Study: T-6316.8 Analytical Report: FACT TOX-097 LRN-U2452 Chemical Characterization of Analytical Reference Material/Substance Table 2. Characterization of the Analytical Reference Materials/Substances in Study FACT-TOX-097 Substance KPFOSa,c PFOSA PFOSAA N-EtFOSE PFOSEAc Source 3M Specialty 3 M IC P /P C P 3M IC P /P C P 3M Chemicals Division Division 3M Formula Expiration Date C 8F 17S O 3-K + 01/01/2010 C 8F 17S O 2N H 2 /01 01/2010 C bFitS O jN (C H tCH-,) CH2CO O -H 01/01/2010 CeF17S 02N (C2H5) C H 2C H 2OH 0 1/0 1/20 10 C bFwS O jN H C j h5 TBD Storage Conditions Chemical Lot Number Physical Description Purity Ambient temperature 193 W hite powder T B D 1' Ambient temperature Ambient temperature L2353 617 Amber to brown waxy sol id TBDb Yellow to amber liquid TBDb Ambient temperature 936 Am ber waxy solid 88.9% Ambient temperature TN-A-1885 Am ber waxy solid NA TBD - To be determined a The target analyte is PFOS (Perfluorooctanesulfonate), C8F17SO3b The purity o f the substance listed above was based only on NMR analyses. Subsequent chemical characteristics are occurring and this analytical report will be amended to indicate the purity of these substances when a certificate o f analysis is issued. c Purity will not be determined due to lack o f material. See compliance statement. Note: All reference materials that were used during the course o f the study were stored at ambient temperature and were frozen when moved to storage. 3M [Environmental 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 Environm ental Laboratory Preparatory Methods FACT-M-1.0, "Extraction of Potassium Perfluorooctanesulfonate or Other Anionic Fluorochemical Surfactants from Liver for Analysis Using HPLC-Electrospray/Mass Spectrometry". FACT-M-3.0, "Extraction of Potassium Perfluorooctanesulfonate or 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 reconstituted in 1.0 rnL of methanol, and then filtered through a 3 mL disposable plastic syringe attached to a 0.2 pm nylon filter into glass autovials. ETS-8-4.1, "Extraction of Potassium Perfluorooctanesulfonate or other Fluorochemical Compounds from Serum for Analysis using HPLC-Electrospray/Mass Spectrometry," ETS-8-6.0, "Extraction of Potassium Perfluorooctanesulfonate or Other Fluorochemical Compounds from Liver for Analysis Using HPLC-Electrospray/Mass Spectrometry" An ion-pairing reagent was added to the sample and the analyte ion pair was partitioned into methyl-terf-butyl-ether. The extract was transferred to a centrifuge tube and put onto a nitrogen evaporator until dry. Each extract was reconstituted in 1.0 mL of methanol and passed through a 0.2 pm nylon filter, using a 3 mL disposable plastic syringe into glass autosampler vials. Analytical Methods FACT-M-2.0, "Analysis of Fluorochemicals in Liver Extracts Using HPLC-Electrospray/Mass Spectrometry" FACT-M-4.0, "Analysis of Fluorochemicals in Serum Extracts Using HPLC-Electrospray/Mass Spectrometry" ETS-8-5.1, "Analysis of Potassium Perfluorooctanesulfonate or Other Fluorochemicals in Serum Extracts Using HPLC-Electrospray/Mass Spectrometry" 3M [Environmental Laboratory Page 13 BACK TO MAIN 3M Medical Department Study: T-6316.8 Analytical Report: FACT TOX-097 LRN-U2452 ETS-8-7.0, "Analysis of Potassium Perfluorooctanesulfonate or other Fluorochemicals in Liver Extracts Using HPLC-Electrospray/Mass Spectrometry" The analyses were performed by monitoring one or more product ions selected from a single primary ion characteristic of a particular fluorochemical using HPLC/ES/MS/MS. For example, molecular ion 499, selected as the primary ion for PFOS (C8F17S 03-) analysis, was fragmented to produce ion 99 (FS03-). The characteristic ion 99 was monitored for quantitative analysis (Table 3). Analytical Equipment The actual analytical equipment settings used in the present analytical phase of this study varied slightly during actual data collection. The following is representative of the settings used during the analytical phase of this study. Liquid Chromatograph: Hewlett-Packard Series 1100 Liquid Chromatograph system Analytical column: Keystone BetasilTM Ci82x100 mm (5 pm) Column temperature: Ambient Mobile phase components: Component A: 2mM ammonium acetate Component B: methanol Flow rate: 300 pL/min Injection volume: 10 pL Solvent Gradient: 13.5 minutes Time (minutes) %B 0.0 40% 8.5 90% 11.0 90% 12.0 40% 13.5 40% M ass S p e c tro m e te r: Micromass A PI/M ass Spectrometer Quattro IITM Triple Quadrupole system Software: Mass LynxTM 3.4 Cone Voltage: 20-60 V Collision Gas Energy: 25-45 eV Mode: Electrospray Negative Source Block Temperature: 150C 10C Electrode: Z-spray Analysis Type: Multiple Reaction Monitoring (MRM) 3M [Environmental Laboratory Page 14 BACK TO MAIN 3M Medical Department Study: T-6316.8 Analytical Report: FACTTOX-097 LRN-U2452 Table 3. Negative Ions Monitored Target Analyte Primary Ion (AMU) Product Ion (am u) PFOS** 499.0 80.0, 99.0, 130.0 PFOSA 498.0 78.0 PFOSAA 584.0 83.0, 169.0 N-EtFOSE PFOSEA 630.0 526.0 59.0 65.0,119.0 D eviations THPFOS* 427.0 80.0 Surrogate ** One or more product ions were used in the determination of PFOS. 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 Lim its o f 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: 70-130% 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 Sum m ary o f Q u ality Control Analyses Results Linearity: The coefficient of determination (r2) of the extracted standard curve was >0.980 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. Calibration Standards: Quantitation of the compounds was based on linear regression analysis (1/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). Limits o f Quantitation (LOQ): The LOQ is equal to the lowest acceptable standard in the calibration curve (defined as a standard within 30% of the theoretical value), and is at least two times the analyte peak area detected in the extraction blanks. Blanks: All blanks were below the lower limit of quantitation for the compounds of interest, except as noted in Appendix B (see table 6). Precision: Precision was not specifically determined within this study, but has been characterized to be better than 30% for this method. Matrix Spikes: Matrix spikes and matrix spike duplicates were extracted with each set of 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. 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 [Environmental Laboratory Page 16 BACK TO MAIN 3M Medical Department Study: T-6316.8 Analytical Report: FAC T TO X -097 L R N -U 2 4 5 2 S tatem en t o f D ata Q uality 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, indicates the data are quantitative to 30% 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. Sum m ary o f S am ple R esults 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 #418-010, "Oral (Stomach Tube) Developmental Toxicity Study of N-EtFOSE in Rabbits" 3M [Environmental 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 C hem ical C h aracterizatio n o f 2(N -Ethylperfluorooctanesulfonam ido)-ethanol CAS Number: 1691-99-2 Molecular Weight: 571.0 Chemical Formula: C8Fi7S 02N (C2H5) CH2CH20H Table 4. Characterization of Test Substance in Study FACT-TOX-097 Test Substance Chemical Name Source N-EtFOSE 2(N-Ethylperfluorooctanesulfonam ido)-ethanol From Sponsor Expiration Date 5/01/2000 Storage Conditions Am bient temperature Chemical Lot # F M -3929 (Mixture of Lots 30035, 30037, 30039) Physical Description W axy solid Purity 97.4% * The purity of the substances listed above was based only on NMR analyses. Subsequent chemical characterization is occurring and this analytical report will be amended to indicate the purity of these substances when a certificate of analysis is issued. This information is from the in-life protocol. C haracterization o f Control M atrices Table 5. Characterization of the Control Matrices Used for Liver and Sera Analyses in Study FACT-TOX-097 Control Matrix Rabbit Liver Rabbit Sera Source Expiration Date Storage Conditions Chemical Lot # Physical Description C om ing Hazelton W isconsin 0 1 /0 1 /2 0 1 0 -20C 10C F00013 Rabbit Liver S ig m a 0 1 /0 1 /2 0 1 0 -20C 10C 96H4639 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 [Environmental Laboratory Page 19 3M Medical Department Study: T-6316.8 BACK TO MAIN Analytical Report: FACT TO X -097 L R N -U 2 4 5 2 3M Environmental Laboratory ________ .. Protocol - Analytical Study Oral (Stomach Tube) Developmental Toxicity Study of 2(N-Ethylperfluorooctanesulfonamido)-ethanol in Rabbits In-vivo study reference number: Argus #418-010 Study number: FACT-TOX-97 T est substance: 2(N-Ethylperfluorooctanesulfonamido)-ethanol (N-EtFOSE-OH) Name and address of Sponsor: Marvin Case 3M Toxicology Services 3M Center Building 220-2E-02 St. Paul, MN 55144 Exact Copy of Original Initial -* D a te Name and address o f testing facility: 3M Environmental Technology and Services 0n; 935 Bush Avenue, Building 2-3E-09 St. Paul, MN 55106 Experimental start date: Expected termination date: . Method numbers and revisions: FACT-M -1.0, Extraction of Potassium Perfluorooctanesulfonate or Other Anionic Surfactants from Liver for Analysis Using HPLC-Electrospray/Mass Spectrometry FACT-M -2.0, Analysis of Fluorochemicals in Liver Extracts Using HPLCElectrospray/Mass Spectrometry FACT-M -3.0, Extraction of Potassium Perfluorooctanesulfonate or Other Anionic Surfactants from Serum for Analysis Using HPLC-Electrospray/Mass Spectrometry FACT-M -4.0, Analysis of Fluorochemicals in Serum Extracts Using HPLCElectrospray/Mass Spectrometry Author: Lisa Clemen Kris Hansen Study Director FACT-TOX-97, U2452 Argus #418-010 Page 1 of 5 3M Einvironmental Laboratory Date Marvin Case Sponsor Representative Date Page 20 3M Medical Department Study: T-6316.8 BACK TO MAIN Analytical Report: FACT TOX-097 LRN-U2452 i' 1.0 Purpose_______ L_____________________________ |____________________________ __ The analytical portion of this study is designed to evaluate levels o f potassium perfluorooctanesulfonate (PFOS), or another metabolite of 2(N-ethylperfluorooctanesulfonamido)-ethanol (N-EtFOSE-OH) determined by the Study Director, in liver and serum samples o f the test system when it is administered directly through a stomach tube. The in life portion o f this study was conducted at Argus Research Laboratories. 2.0 Regulatory Compliance_________________________________________ . This study is conducted in compliance with the Food and Drug Administration Good Laboratory Practices regulation as stated in 21 CFR 58. Any exceptions will be noted in the final report. i, 3.0 Test Materials___________ ;__________________________________________________ 3.1 Test, control, and reference substances and matrices 3.1.1 Analytical reference substance: Potassium perfluorooctanesulfonate (PFOS), lot #217 3.1.2 Analytical reference substance matrix: Rabbit liver and serum . 3.1.3 Analytical control substance: None 3.1.4 Analytical control substance matrix: Rabbit liver and serum 3.2 Source o f materials 3.2.1 Analytical reference substance: 3M Specialty Chemical Division; traceability information will be included in the final report 3.2.2 Analytical reference substance matrix: Argus Research Laboratories; traceability information will be included in the final report 3 .2 3 Analytical control matrix: 3 .2 3 .1 Rabbit liver - Argus Research Laboratories; traceability information will be included in the final report; or Rabbit liver - Covance Laboratories; traceability information will be included in the final report 3 .2 3 .2 Rat serum - Sigma Chemical Company; traceability information will be included in the final report 3.3 Num ber o f test and control samples. Liver samples for testing were received from 16 test and 3 control animals for the toxicokinetic portion o f the study. Liver samples for testing were received from 88 test and 22 control animals for the developmental portion o f the study. Serum samples will be tested at the discretion of the Study Director. 3.4 Identification o f test and control samples: The samples are identified using the Argus Research Laboratories identifiers, which consist of 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 Einvironmental Laboratory Page 21 3M Medical Department Study: T-6316.8 BACK TO MAIN Analytical Report: FACT TO X-097 L R N -U 2 4 5 2 . 3.5 3.6 3.7 3.8 3.9 !' Parity and strength o f materials: Characterization of the purity and identity o f the reference material is the responsibility of the Sponsor. ` Stability o f 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 -2 0 10 C. Disposition o f test and/or control substances: Biological tissues and fluids are retained per GUP regulation. Safety precautions: Refer to the material safety data sheets o f chemicals used. Wear appropriate laboratory attire, and follow adequate precautions for handling biological 'materials and preparing samples for analysis. 4.0 Experimental - Overview_______________________________________________________ Tissues from animals dosed as described in Argus Research Laboratories Protocol #418-010 are received for analysis o f fluorine compounds. Mated female rabbits were dosed on Day 7 o f 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 PFOS by Electrospray/mass spectrometry (ES/MS). On the basis of findings from these analyses, additional samples may be evaluated. If additional analysis is performed, a protocol amendment will be written to add the matrices and methods to 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 Study Director, select analysis may be >performed by a contract laboratory where competence has been demonstrated, using validated analytical methods. If a contract laboratory is used, the methods and data provided to the Study Director will be identified in the final report. 5.0 Experimental - Analytical Methods______________________________________ _ 5.1 For analysis performed by the 3M Environmental Laboratory, the following methods will be used: 5.1.1 FACT-M-1.0, Extraction o f Potassium Perfluorooctanesulfonate or Other Anionic Surfactants from Liver for Analysis Using HPLC-Electrospray/Mass Spectrometry 5.1.2 FACT-M-2.0, Analysis ofFluorochemicals in Liver Extracts Using HPLCElectrospray/Mass Spectrometry FACT-TOX-97, U2452 Argus #418-010 Page 3 of 5 3M Einvironmental 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 Perfluorooctanesulfonate or Other Anionic Surfactants from Serum for Analysis Using HPLC-Electrospray/Mass Spectrometry 5.1.4 FACT-M-4.0, Analysis of Fluorochemicals in Seram Extracts Using HPLCElectrospray/Mass Spectrometry 5.2 If analysis is performed at a contract analytical laboratory, copies o f the validated methods will be included in the data packet provided to the Study Director. 6.0 Data Analysis___________________________ ;____________________________________ 6.1 , Data Reporting: For analysis performed by a contract laboratory, the contract laboratory will provide all data to the analytical phase Study Director, and copies o f the methods ' will be attached to the data. The contract laboratory and the data it provides will be identified in the data packet provided by the analytical phase Study Director to the Sponsor. 6.2 Data transformations and analysis: Data will be reported as the concentration (weight/weight) of target analyte per tissue or sample, or of target analyte per unit o f tissue or fluid. 6.3 Statistical analysis: Statistics used may include regression analysis o f the serum concentrations over time, and standard deviations calculated for the concentrations within each dose group. If necessary, simple statistical tests, such as Student's t test, may be applied to evaluate statistical difference. 7.0 Maintenance of Raw Data and Records_____________________________ ;_________ 7.1 The following raw data and records will be retained in the study folder in the archives according to AMDT-S-8: 7.1.1 Approved protocol and amendments 7.1.2 Study correspondence 7.1.3 Shipping records 7.1.4 Raw data 7.1.5 Electronic copies of data 7.2 Supporting records to be retained separately from the study folder in the archives according to AMDT-S-8 will include at least the following: 7.2.1 Training records . 7.2.2 Calibration records 7.2.3 Instrument maintenance logs 7.2.4 Standard Operating Procedures, Equipment Procedures, and Methods FACT-TOX-97, U2452 Argus #418-010 Page 4 of 5 3M Environmental Laboratory Page 23 3M Medical Department Study: T-6316.8 BACK TO MAIN Analytical Report: F A C T T O X -0 9 7 L R N -U 2 4 5 2 / 12.5 Appropriate specimens. ; ' r ' 8.0 References___________________ ;_______ ;________________ ____________ ______ . 8.1 3M Environmental Laboratory Quality System Chapters 1 ,5 and 6 8.2 Other applicable 3M Environmental Laboratory Quality System Standard Operating Procedures 9.0 Attachments___________________ ;____________________________________,_________ 9.1 Copies of the following validated 3M Environmental Laboratory methods are attached for information purposes: . 9.1.1 FACT-M -1.0, Extraction o f Potassium Perfluorooctanesulfonate or Other ^ Anionic Surfactants from Liver for Analysis Using HPLC-Electrospray/Mass Spectrometry 9.1.2 FACT-M -2.0, Analysis o f Fluorochemicals in Liver Extracts Using HPLCElectrospray/Mass Spectrometry 9.1.3 FACT-M -3.0, Extraction of Potassium Perfluorooctanesulfonate or Other Anionic Surfactants from Serum for Analysis Using HPLC-Electrospray/Mass Spectrometry 9.1.4 FACT-M -4.0, Analysis o f Fluorochemicals in Serum Extracts Using HPLCElectrospray/Mass Spectrometry 9.2 If a contract analytical laboratory performs analysis, copies o f 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 TO X-097 L R N -U 2 4 5 2 Study Title Analytical Study Oral (Stomach Tube) Development Toxicity Study o f 2(N-Ethylperfluorooctanesulfonamide)-ethanol in Rabbits PROTOCOL AMENDMENT NO. 1 Amendment Date: 18 February 2000 Performing Laboratory 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 Study: T-6316.8 3M Environmental Laboratory Laboratory Page 25 BACK TO MAIN 3M Medical Department Study: T-6316.8 Analytical Report: FACT TO X-097 L R N -U 2 4 5 2 Protocol LRN-U2452 Amendment Number 1 This amendment modifies the following portion(s) of the protocol: 1. Protocolreads: The study director for the present study was identified in the protocol as Kristen J. Hansen, Ph.D. Amend to read: The role of study director for the present study was reassigned to Marvin T. Case, D.V.M., Ph.D., as of the signing of this amendment. Reason: The role of study director was reassigned in an effort to ensure compliance with Good Laboratory Practice Standards that outline study personnel requirements (refer to 21 CFR Part 58). 2. Protocol reads: The sponsor for the present study was identified as Marvin T. Case, D.V.M., Ph.D. Amend to read: The role of sponsor for the present study was reassigned to John L. Butenhoff, Ph.D., as of 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 responsibilities and workload are more evenly balanced. 3M Environmental Laboratory BACK TO MAIN 3M Medical Department Study. T-6316.8 Analytical Report: FACT TOX-097 LRN-U2452 Protocol LRN-U2452 Amendment Number 1 3. Protocol reads: Method numbers and revisions: FACT-M-1.0, Extraction of Potassium Perfluorooctanesulfonate or Other Anionic Surfactants from Liver for Analysis Using HPLC-Electrospray/Mass Spectrometry FACT-M-2.0, Analysis of Fluorochemicals in Liver Extracts Using HPLC-Electrospray/ Mass Spectrometry FACT-M-3.0, Extraction of Potassium Perfluorooctanesulfonate or Other Anionic Surfactants from Serum for Analysis Using HPLC-Electrospray/Mass Spectrometry FACT-M-4.0, Analysis of Fluorochemicals in Serum Extracts Using HPLC-Electrospray/Mass Spectrometry Amend to read: Method numbers and revisions: ETS-8-6.0 "Extraction of Potassium Perfluorooctanesulfonate or Other Fluorochemical Compounds from Liver for Analysis Using HPLC-Electrospray/Mass Spectrometry" ETS-8-7.0 "Analysis of Potassium Perfluorooctanesulfonate or Other Fluorochemical Compounds in Liver Extracts Using HPLC-Electrospray/Mass Spectrometry" ETS-8-4.1, "Extraction of Potassium Perfluorooctanesulfonate or Other Fluorochemical Compounds from Serum for Analysis Using HPLC-Electrospray/Mass Spectrometry" ETS-8-5.1, "Analysis of Potassium Perfluorooctanesulfonate or Other Fluorochemical Compounds in Serum Extracts HPLC-Electrospray/Mass Spectrometry" Reason: New methodologies were implemented following the approval of the original protocol for FACT Tox-097. 3M Environmental Laboratory 3M Environmental Laboratory Page 27 BACK TO MAIN 3M Medical Department Study: T-6316.8 Analytical Report: FACT TO X-097 L R N -U 2 4 5 2 Protocol LRN-U2452 Amendment Number 1 4. Protocol reads: 7.1 The following raw data and records will be retained in the study folder in the archives according to AMDT-S-8: 7.1.1 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 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 7.2.2 7.2.3 7.2.4 7.2.5 Training records Calibration records Instrument maintenance logs Standard Operating Procedures, Equipment Procedures, and Methods Appropriate specimens Amend to read: `The original data, or copies thereof, will be available at the 3M Environmental Laboratory to facilitate audits of the study during its progress and before acceptance of the final report. When the final report is completed, all original paper data, including: approved protocol and amendments, study correspondence, shipping records, raw data, approved final report, and electronic copies o f data will be retained in the archives of the 3M Environmental Laboratory. All corresponding training records, calibration records, instrument maintenance logs, standard operating procedures, equipment procedures, and methods will be retained in the archives o f the facility performing each analysis." Reason: To direct subcontract laboratories in the disposition o f the items listed above. 5. Protocol reads: 3.1 Test, control, and reference substances and matrices 3.1.2 Analytical reference substance matrix: Rabbit liver and serum 3.1.4 Analytical control substance matrix: Rabbit liver and serum Amend to read: 3.1 Test, control, and reference substances and matrices 3.1.2 Analytical reference substance matrix: Rabbit liver, serum, and pooled fetal tissue(s) 3.1.4 Analytical control substance matrix: Rabbit liver, serum, and pooled fetal tissue(s) Reason: Analysis of fetal tissue for the target chemical and/or its analytes was added to the scope of the study following the issuance of the original protocol. 3M Environmental Laboratory 3M Environmental Laboratory Page 28 BACK TO MAIN 3M Medical Department Study: T-6316.8 Analytical Report: FACT TO X-097 L R N -U 2 4 52 Protocol LRN-U2452 Amendment Number 1 6. Protocol reads: The analytical portion of this study is designed to evaluate levels of potassium perfluorooctanesulfonate (PFOS), or another metabolite of 2(N-Ethylperfluorooctanesulfonamido)-ethanol (NEtFOSE-OH) determined by the Study Director, in liver and serum samples of the test system when it is administered directly through a stomach tube. Amend to read: The analytical portion of this study is designed to evaluate levels of potassium perfluorooctanesulfonate (PFOS), or another metabolite of 2(N-Ethylperfluorooctanesulfonamido)-ethanol (NEtFOSE-OH) determined by the Study Director, in liver, serum, and fetal tissue(s) samples of the test system when it is administered directly through a stomach tube. Reason: Analysis of fetal tissue for the target chemical and/or its analytes was added to the scope of the study following the issuance o f the original protocol. 7. Protocol reads: 3.8 Disposition o f test and/or control substances: Biological Tissues and fluids are retained per GLP regulation. . Amend to read: 3.8 Specimens will be maintained in the 3M Environmental Laboratory specimen archives. All specimens sent to sub-contract laboratories will be returned to the 3M Environmental Laboratory upon completion of analysis and submission of the sub-contract laboratory(s) final report. The specimens will be returned with the following documentation: the signed original chain of custody and records of storage conditions while at the sub-contract facility. Reason: To direct subcontract laboratories in the disposition of the items listed above. 3M Environmental Laboratory 3M Environmental Laboratory Page 29 BACK TO MAIN 3M Medical Department Study: T-6316.8 Analytical Report: FACT TO X-097 L R N -U 2 4 5 2 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 information will be included in the final report; or Rabbit liver - Covance Laboratories; traceability information will be included in the final report Amend to read: 3.2.3 Analytical control matrix 3.2.3.1 Rabbit liver - Covance Laboratories; traceability information will be included in the final report Reason: Argus Research Laboratories will be conducting the in life portion of the study. 9 . Protocol reads: 3.2.3 Analytical control matrix 3.2.3.1 Rabbit liver - Covance Laboratories; traceability information will be included in the final report 3.2.3.2 Rat Serum --Sigma Chemical Company; traceability information will be included in the final report Amend to read: 3.2.3 Analytical control matrix 3.2.3.1 Rabbit liver - Covance Laboratories; traceability information will be included in the final report 3.2.3.2 Rat serum - Sigma Chemical Company; traceability information will be included in the final report 3.2.3.3 Pooled fetal tissue(s) - traceability information will be included in the final report Reason: Analysis o f fetal tissue was added to the scope o f the study following the 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 TO X -097 L R N -U 2 4 5 2 Protocol LRN-U2452 Amendment Number 1 /flO lU s XU*e) Marvin T. Case, D. V.M., Ph.D., Outgoing Sponsor Representative Date John L. Butenhoff, Ph.D., Incoming Sponsor Representative ____f i b - f i > - ____ _______________________ Kristen J. Hansen, PhD., Outgoing Study Director Date Date Marvin T. Case, D. V.M., PhD., Incoming Study Director Date 3M Environmental Laboratory 3M Environmental Laboratory Page 31 3M Medical Department Study: T-6316.8 BACK TO MAIN Analytical Report: FACT TO X -097 L R N -U 2 4 52 Study Title Oral (Stomach Tube) Developmental Toxicity Study o f 2(N-Ethylperfluorooctanesulfonamido)- ethanol in Rabbits PROTOCOL AMENDMENT NO. 2 Amendment Date: November 21, 2000 Performing Laboratory 3M Environmental Technology & Safety Services 3M Environmental Laboratory 935 Bush Avenue St. Paul, MN 55106 Laboratory Project Identification FACT TOX-097 ET&SS LRN-U2452 Argus Study: 418-010 3M Medical Department Study: T-6316.8 3M Environmental Laboratory 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. Protocol reads: There is not a principal analytical investigator assigned for this study. Amend to read: The role of principal analytical investigator for the study was assigned to Kristen J. Hansen, Ph.D. as o f the signing of this amendment. Reason: The role o f principal analytical investigator was assigned in an effort to ensure com pliance with Good Laboratory Practice Standards that outline study personnel requirements. 3M Environmental Laboratory 3M M edical D epartm ent Study: T-6316.8 BACK TO MAIN Analytical Report: FACT TO X-097 L R N -U 2 4 52 Amendment Approval Protocol FACT TOX-097 Amendment No. 2 John L. Butenhoff, Ph.D., Sponsor Representative Date &4A Marvin T. Case, D. V.M., Ph.D., Study Director /Cl A -,____ Date 3M Environmental Laboratory 3M Medical Department Study: T-6316.8 3M Environmental Technology and Services PO Box 33331 St. Paul. MN 55133-3331 612 778 6442 BACK TO MAIN Analytical Report: FACT TOX-097 LRN-U2452 Study title Oral (Stomach Tube) Developmental Toxicity Study o f 2(N-Ethylperfluorooctanesulfonamido)- ethanol in Rabbits PROTOCOL AMENDMENT NO. 3 Amendment Date: January 23,2001 Performing Laboratory 3M Environmental Technology & Safety Services 3M Environmental Laboratory 935 Bush Avenue: St. Paul, MN 55106 Laboratory Project Identification FACT TOX-097 ET&SS LRN-U2452 Argus Study: 418-010 3M Medical Department Study: T-6316.8 3M Environmental Laboratory 3Me nvironmental Laboratory 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. Amend to read: To include the additional analytical reference substances, PFOSA, PFOSAA, PFOSEA, and N-EtFOSE. . Reason: To identify all the compounds that was analyzed in the analytical phase o f the study. 2. Consistency: The test article 2(N-Ethylperfluorooctanesulfonamido)-ethanol is given many different abbreviations in the protocol, study, raw data, and analytical report. Such as EtFOSE, N-EtFOSE, EtFOSE-OH, and N-EtFOSE-OH. Clarification: These different abbreviations are equivalent. 3M Environmental Laboratory 3M'Environmental Laboratory Page 36 3M M edical D epartm ent Study: T-6316.8 BACK TO MAIN Analytical Report: F A C T T O X -097 L R N -U 2 4 5 2 Amendment Approval Protocol FACT TOX-097 Amendment No. 3 John L Butenhoff, PhD., Sponsor Representative Z- arrtyv i Date afcfr:.. Marvin T. Case, D. V.M., PhD., Study Director Date Kristen J. Hansen, PhD., Principal Analytical Investigator l fi -Hl O\ Date 3M Environmental Laboratory 3 M E iiviruriiiibrmal LaUui atu i y Page 37 BACK TO MAIN 3M Medical Department Study: T-6316.8 Analytical Report: FACT TO X -097 L R N -U 2 4 5 2 Record of Deviation I. Identification Study/P roject No. FACT-TOX-097 Argus 418-010 Deviation Type (Check one) SOP Method Equipment Procedure Protocol 1^ Other: Document Number(s): 21 cfr 58.120 (A) (11) Date(s) o f occurrence: 9/18/98 II. Description: Required Procedure/process: The Sponsor Representative approval o f the analytical protocol is required prior to the Study Director's approval. Actual Procedure/process: The Study Director approved the analytical protocol before the Sponsor Representative. III. Actions Taken: (such as amendment issued, SOP revision, etc.) This deviation w as written. In the future, analytical protocols w ill be signed by the Sponsor Representative before the Study Director. Recorded By A C ioW\Ja~ IV. Impact on Study / Project This deviation w ill not adversely affect the outcome o f this study. Date a l m i Ou ty , IXIHIGD Authorized By (Study Director / Project Lead) I Date Spense^ fttpre.ientalu/t.: ' Te'hn BulenhcV3M Environmental Laboratory Form ETS-4-8.0 ih-- / Qfi- Director - Mcuv Cast. Deviation No. i (assigned by Study Director or Project Lead at the end of study or project) 3M Environmental Laboratory Page 38 BACK TO MAIN 3M M edical D epartm ent Study: T-6316.8 Analytical Report: FACT TO X-097 L R N -U 2 4 52 Record of Deviation 1. Identification Study/Project No. FACT-TOX-097 Argus 418-010 Deviation Type (Check one) SOP Method Equipment Procedure ^ P r o to co l Other: Document Number(s): FACT-TOX-097 I Date(s) o f occurrence: [ 10/17/98,09/28/99 II. Description: Required Procedure/process: The protocol states the liver analytical method to follow for this study is FACT-M -2.0. Actual Procedure/process:___ _______________ ______ On 10/17/98 no method was listed and on 09/28/99 method ETS-8-6.0 (the extraction method) was listed. '~ III. Actions Taken: ______________ '_______ (such as amendment issued, SOP revision, etc.) This deviation was written. ' Recorded. By Date IV. Impact on Study / Project ^/w /o O Although no method was listed on 10/17/98, method FACT-M-2.0 was followed as determined by parameters documented in the raw data. On 09/28/99 the extraction method was written in error - analytical method ETS-8-7.0 was used as determined by parameters listed in the raw data. This method is an improvement over FACT-M -2.0. No adverse affect on these data. A n ( M / r J Authorized By (Study Director /Project Lead) D ate spense- Repre.Rnkvk.t.'. Toko (baleninof P 3M Environmental Laboratory Form ETS-4-8.0 Stink* D^dci'^r. IM.rv Cent, Deviation No. 2 (assigned by Study Director or Project Lead at the end of study or project) 3 M ~gnvliu i ii i lu i Hal L ab u iatui y Page 39 BACK TO MAIN 3M M edical D epartm ent Study: T-6316.8 Analytical Report: FACT TO X -097 L R N -U 2 4 52 Record of Deviation I. Identification Study/Project N o. FACT-TOX-097 Argus 418-010 Deviation Type (Check one) SOP Method Equipment Procedure ^ P r o to c o l Other: Document Number(s): FCT-TOX-097 Date(s) o f occurrence: 10/20/98, 10/23/98, 07/29/99 II. Description: Required Procedure/process: The protocol states the sera analytical method to follow for this study is FACT-M -4.0. Actual Procedure/process: On 10/20/98 no method was listed, on 10/23/98 method FACT-M-4.1 was used, and on 07/29/99 method ETS-8-7.0 (a liver method) was listed. III. Actions Taken: _________' _______ (such as amendment issued, SOP revision, etc.) This deviation was written. Recorded By Date 0o IV. Impact on Study / Project Although no method was listed on 10/20/98, method FACT-M-4.0 was followed as determined by parameters documented in the raw data. The method FACT-M-4.1, used on 10/23/98 is an imporvement over FACT-M -4.0. Method ETS-8-7.0 was recorded in error. Method ETS-8-5.1 was used as determined by parameters listed in the raw data. ETS-8-5.1 is an improvement over FACT-M-4.0;there isn o jdverse affect on these data. ^ 't (XjifV Authorized By (Study Director / Project Lead) Date 2- ^ `jpo.-ijLr L.'pr0-jmLih . To CuiihtA'-V 3M Environmental Laboratory Form ETS-4-8.0 I w w , T(f^ i i P u . n * Deviation No. n (assigned by Study Director or Project Lead at the end of study or project) Laboratory Page 40 BACK TO MAIN 3M M edical D epartm ent Study: T-6316.8 Analytical Report: FACT TO X -097 L R N -U 2 4 52 Record of Deviation I. Identification Study / Project No. TOXOQ97 (LIMS #U2452) Deviation type SOP X Method Equipment Procedure (Check one) Protocol Other: Document number FACT-M-2.1, ETS-8-5.1 and ETS-8-7.0 Date(s) of occurrence 11/25/98, 9/29/99 and 7/29/99 II. Description Required procedure/process: Section 14.2.1: Solvent blanks, method blanks, and matrix.blanks must be below the low est standard on the calibration curve. Actual procedure/process: Occasionally, the first solvent blank injected for a run was above the LOQ. : j IU. zActions.Taken' :, (sch as amendment issued, SOI1revision,; etc.) , Deviation written. Recorded by Date (UXotcr IV. irtpacton Studf Project i-(compieteti by Study Director o fPtjct.Lead) In each place a high solvent blank was analyzed, additional solvent blanks or method blanks were analyzed immediately following the high blank. This second injection was below the LOQ. O ccassionally, the first injection o f a run is high because it may immediately follow injection o f a high standard from a previous run. For this reason, more than one blank is typically analyzed prior to the start o f a calibration curve. These second and third, etc. blanks are below the LOQ and are more representative o f the analytical conditions o f the samples; the study data is not adversely affected. Vfk II llofCO Authorized by -Sp-n.iv." At? -['A/ / Ccu Deviation No. (assigned by Study Director or Project Lead at the end of study or project) Attachment A: Record of Deviation 3M-Erflvtroftmental Laboratory ETS-4-8.0 Page 1 of 1 Page 41 3M Medical Department Study: T-6316.8 Appendix C: Extraction and Analytical Methods BACK TO MAIN A nalytical Report: FA C T T O X -097 L R N -U 2 4 52 This appendix includes the following methods: FACT-M-1.0, "Extraction of Potassium Perfluorooctanesulfonate or Other Anionic Fluorochemical Surfactants from Liver for Analysis Using HPLC-Electrospray/Mass Spectrometry," (8 pages) FACT-M-3.0, "Extraction of Potassium Perfluorooctane or Other Anionic Fluorochemical Compounds from Serum or Other Fluids for Analysis Using HPLC-Electrospray/Mass Spectrometry," (8 pages) ETS-8-04.1, "Extraction of Potassium Perfluorooctanesulfonate or other Fuorochemical Compounds from Serum for Analysis Using HPLC-Electrospray/Mass Spectrometry," (14 pages) ETS-8-06.0, "Extraction of Potasium Perfluorooctanesulfonate or other Fluorochemical Compounds from Liver for Analysis Using HPLC-Electrospray/Mass Spectrometry," (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, "Analysis of Potassium Perfluorooctanesulfonate or other Fuorochemical Compounds in Serum Extracts Using HPLC-Electrospray/Mass Spectrometry," (9 pages) ETS-8-07.0, "Analysis of Potassium Perfluorooctanesulfonate or other Fluorochemical Compounds in Liver Extracts Using HPLC-Electrospray/Mass Spectrometry," (10 pages) 3M Environmental Laboratory Page 42 3M Medical Department Study: T-6316.8 BACK TO MAIN Analytical Report: FACT TO X-097 L R N -U 2 4 52 3M Environmental Laboratory Method Extraction of Potassium perfluorooctanesulfonate or Other Anionic Fluorochemical Surfactants from Liver for Analysis Using HPLC-Electrospray/Mass Spectrometry M ethod N um ber: FACT-M -1.0 Author: Lisa Clemen - Approved By: . 0 1s f ' i Laboratory Manager -- ---- --I--w-- h1--l--s--t-*-------l--i------------------"--------------------- -------- Group Leader - nil* H C ita to Technical Reviewer A doption Date: 5'2h<ii R evision Date: 5 y 4 c /7 Date 5 /z -t/ / 4 f Date 5 /2 7 /4 F Date 1.0 S c o p e a n d A p p l i c a t i o n ____________________________ ;____________._________________________________ 1.1 Scope: This method is for the extraction o f Potassium Perfluorooctanesulfonate (PFOS) or other fluorochem ical surfactants from liver. 1.2 A pplicable Com pounds: Fluorochemical surfactants or other fluorinated compounds. 1.3 M atrices: Rabbit, rat, bovine, and monkey livers or other livers as designated in the validation report Microsoft 7.0.1/95 3M [Environmental Laboratory FACT-M-1.0 Extraction o f PFOS from Liver Page 1 of 8 Page 43 3M Medical Department Study: T-6316.8 BACK TO MAIN Analytical Report: FACT TO X -097 L R N -U 2 4 5 2 2.0 S u m m a r y o f M e t h o d ________________ ;_____________ _____________________________________ 2.1 This method describes how to extract potassium perfluorooctanesulfonate (PFOS) or other fluorochemical surfactants from liver using ion pairing reagent and 5.0 mLs o f ethyl acetate. An ion pairing reagent is added to each sample and partitioned into ethyl acetate. Four mLs o f 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 D e f i n i t i o n s __________________________ :____________________________________________________ 3.1 None. 4.0 W a r n i n g s a n d C a u t i o n s ______________ ;_______________'___________________________ 4.1 Health and Safety Warnings: 4.1.1 U se universal precautions when handling animal livers, they may contain pathogens. . . 5.0 I n t e r f e r e n c e s ________________________________ .____________________________________________________ 5.1 There are no known interferences at this time. 6.0 E q u i p m e n t ________________________________________________________________________________ ' 6.1 The follow ing equipment is used w hile carrying out this method. Equivalent equipment is acceptable. 6.1.1 Ultra-Turrax T25 Grinder for grinding liver samples 6.1.2 Vortex mixer, VW R, Vortex Genie 2 6.1.3 Centrifuge, Mistral 1000 or IEC 6.1.4 Shaker, Eberbach or VWR . 6.1.5 Nitrogen Evaporator, Organomation 6.1.6 Balance 7.0 S u p p l i e s a n d M a t e r i a l s ________ ;____________________________________________________ _ 7.1 G loves 7.2 D issecting scalpels 7.3 Eppendorf or disposable pipettes 7.4 N algene bottles, capable o f holding 250 mL and 1 L 7.5 G lass, type A , volumetric flasks 7.6 40 mL glass I-CHEM vials 7.7 Plastic sampule vials, Wheaton, 6 mL 7.8 Polypropylene centrifuge tubes, 15 mL 7.9 Labels FACT-M-1.0 Extraction o f 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 TO X-097 L R N -U 2 4 5 2 7.10 Syringes, capable o f 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 N ote: Prior to using glassware and bottles, rinse 3 times with methanol and 3 time; with M illi- QTM water. Rinse syringes a minimum o f 9 times with methanol, 3 rinses from 3 separate vials. 8.0 R e a g e n t s a n d S t a n d a r d s 8.1 R eagents ____________ .__________________ ._____________ '____________________ 8.1.1 Sodium Hydroxide (J.T Baker or equivalent), (NaOH) 10N: w eigh approximately 200 grams NaOH. Pour into a 1000 mL beaker containing 500 liters (L) M illi-QTM water, m ix until all solids are dissolved. Store in a 1 L nalgene bottle. 8.1.2 Sodium Hydroxide (J.T Baker or equivalent), (NaOH) IN . Dilute 1ON 1:10. Measure 10 mL o f the 10N NaOH solution into a 100 mL volumetric flask and dilute to volum e using Milli-QTM water. Store in a 125 mL nalgene bottle. 8.1.3 Tetrabutylammonium hydrogen sulfate (Kodak or equivalent), (TBA) 0.5M: W eigh approximately 169 grams o f TBA into a 1 L volumetric containing 500 L M illi-QTM ' water. Adjust to pH 10 using approximately 64 mL 10N NaOH and dilute to volum e with M illi-QTM water. Add NaOH slow ly while adding the last 1 mL o f NaOH because the pH changes abruptly. Store i n a l L nalgene bottle. 8 .1 3 .1 TBA requires a check prior to each use to ensure pH = 10. Adjust as needed using IN NaOH solution. 8.1.4 Sodium carboriate/Sodium Bicarbonate Buffer (J.T. Baker or equivalent), (Na^COj/NaHC03) 0.25M: W eigh approximately 26.5 g o f sodium carbonate (NasCOj) and 21.0 g o f sodium bicarbonate (NaHCO,) into a 1 L volumetric flask and dilute to volume with Milli-QTM water. Store in a 1 L nalgene bottle. 8.1.5 PFOS (3M Specialty Chemical D ivision), molecular weight = 538. 8.1.6 Ethyl Acetate, Omnisolv, glass distilled or HPLC grade. 8.1.7 M ethanol, Omnisolv, glass distilled or HPLC grade. 8.1.8 Liver and control liver, received frozen from testing laboratory. 8.1.9 Milli-QTM water, all water used in this method should be Milli-QTM water and may be provided by a M illi-Q TOC Plus system . 8.2 Standards 8.2.1 Prepare PFOS standards for the standard curve. FACT-M-1.0 Extraction of PFOS from Liver Page 3 of 8 3M Environmental Laboratory Page 45 3M Medical Department Study: T-6316.8 BACK TO MAIN Analytical Report: FACT TO X-097 L R N -U 2 4 5 2 8.2.2 W eigh approximately 100 mg o f PFOS into a 100 mL volumetric flask and record the actual weight. 8.2.3 Bring to volum e with methanol for a stock standard o f approximately 1000 ppm (p g/m L ). 8.2.4 D ilute the stock solution with methanol for a working standard 1 solution o f approximately 50 ppm. 8.2.5 Dilute the stock solution with methanol for a working standard 2 solution o f approx. 5.0 ppm. 8.2.6 Dilute the stock solution with methanol for a working standard 3 solution o f approx. 0.50 ppm. 9.0 S a m p l e H a n d l i n g _______________ ;____________________________________ _________________ 9.1 A ll livers are received frozen and must be kept frozen until the extraction is performed. 10.0 Q u a l i t y C o n t r o l _____________________________________ ;________________ ;____________________ 10.1 Matrix Spikes 10.1.1 Prepare and analyze matrix spike and matrix spike duplicate samples to determine the accuracy o f the extraction. 10.1.2 Prepare each spike using liver chosen by the analyst, usually a control liver. 1 0 .1 3 Expected concentrations w ill fall in the mid-range o f the initial calibration curve. 10.2 Continuing Calibration Checks 10.2.1 Prepare and analyze continuing calibration check samples to determine the continued linearity o f the initial calibration curve. 1 0 3 .2 One check is prepared per group o f ten samples. For example, if a sample set = 34, four checks are prepared and extracted. 1 0 .2 3 Prepare each continuing calibration check from the same liver homogenate used to prep the initial curve. 10.2.4 The expected concentration w ill fall within the mid-range o f the initial calibration curve. 11.0 C a l i b r a t i o n a n d S t a n d a r d i z a t i o n _________________ ;________________________________________ 11.1 Prepare Liver Homogenate to Use for Standards 11.1.1 W eigh approximately 40 g o f liver into a 250 mL N algene bottle containing 200 mLs Milli-QTM water. Grind to a homogeneous solution. 11.1.2 If 40 g is not available, use appropriate amounts o f liver and water in keeping with a 1:5 ratio. 1 1 .1 3 See section 13.0 to calculate the actual density o f liver. FACT-M-1.0 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 TO X-097 L R N -U 2 4 5 2 11.1.4 Add 1 mL o f homogeneous solution to a 15 mL centrifuge tube. Re-suspend homogeneous solution by shaking between aliquots while preparing a total o f sixteen 1 mL aliquots o f homogeneous solution in 15 m l. centrifuge tubes. 11.1.5 Two 1 mL aliquots serve as matrix blanks. Use the standard concentrations and spiking amounts listed in table 1 to spike, in duplicate, two standard curves for a total o f fourteen samples. T able 1 A pproxim ate Spiking Am ounts for C alibration Standards Working Standard (Approx. Cone.) 0.50 ppm 0.50 ppm 0.50 ppm 5.0 ppm 5.0 ppm 5.0 ppm 50 ppm (iL Approx, final cone, o f PFOS in liver - Blank 4 0.010 ppm 20 0.050 ppm 40 0.100 ppm 10 0.250 ppm 20 0.500 ppm 30 0.750 ppm 4 1.000 ppm 11.1.1 See section 13.0 to calculate actual concentrations o f PFOS in calibration standards. 11.2 Extract spiked liver homogenates following 12.141-12.24 o f this method. U se these standards to establish each initial curve on the mass spectrometer. 12.0 P r o c e d u r e s _________________________________ |_____________________________________________________ 12.1 Obtain frozen liver samples. In spent tissue, note that the liver has not been packaged with other tissues. 12.2 Cut approximately 1 g o f liver using a dissecting scalpel. 12.3 W eigh the sample directly into a tared plastic sampule vial. 12.4 Record the liver weight in the study notebook. 12.5 Label the sampule vial with the study number, weight, liver ID, date and analyst initials. 12.6 Add 2.5 mLs o f water to sampule vial. 12.7 Grind the sample. Put the grinder probe in the sample and grind for about 2 minutes, or until the sample is homogeneous. 12.8 Rinse the probe into the sample with 2.5 mLs waiter using a pipette. 12.9 Take the grinder apart and clean it with methanol after each sample. Follow AM DT-EP-22. 12.10 Cap the sample and vortex for 15 seconds. 3M Environmental Laboratory FA C T -M -1.0 Extraction o f PFOS from Liver Page 5 o f 8 Page-47- 3M Medical Department Study: T-6316.8 BACK TO MAIN Analytical Report: FACT TO X-097 L R N -U 2 4 5 2 12.11 Pipette 1 mL homogenate into a 15 mL polypropylene centrifuge tube. Label the centrifuge tube with the identical information as the sampule vial. (See Worksheet for documenting the remaining steps.) 12.12 Spike liver homogenates with the appropriate amount o f PFOS standard as described in section 11.1 or Table 1. 12.13 Pipette two 1 mL aliquots o f Milli-QTM water to centrifuge tubes. These w ill serve as instrument blanks. 12.14 Add 1 mL 0.5 M TBA and 2 mL o f the 0.25 M sodium carbonate/sodium bicarbonate buffer. 12.15 U sing a volumetric pipette, add 5 mLs ethyl acetate. 12.16 Cap each sample and put on the shaker for 20 minutes. 12.17 Centrifuge for 20 to 25 minutes, until layers are w ell separated. Set power on the centrifuge to approximately 3500 rpm. 12.18 Remove 4 mLs o f organic layer, using a 5 mL graduated glass pipette, to a clean 15 mL centrifuge tube. Label this fresh tube with the same information as in 12.5. 12.19 Put each sample on the analytical nitrogen evaporator until dry, approximately 2 to 3 hours. 12.20 Add 1.0 mL o f methanol to each centrifuge tube using a graduated pipette. 12.21 Vortex m ix for 30 seconds. 12.22 Attach a 0.2 pm nylon mesh filter to a 3 cc syring;e and transfer the sample to this syringe. Filter into a 1.5 mL glass autovial. 12.23 Label the autovial with the study number, animal number and gender, sample timepoint, matrix, final solvent, extraction date, and analyse s) who performed the extraction. 12.24 Cap and hold for electrospray mass spectrometry analysis. 12.25 Complete the worksheet and tape to page o f study notebook. 13.0 D a t a A n a l y s i s a n d C a l c u l a t i o n s ________________________.__________ ;_________________ 13.1 C alculations: 13.1.1 Calculate the density o f liver (mg) in 1.0 mL homogenate using the follow ing equation: g o f Liver x Average weight o f ten 1 mL aliquots (mg) (g o f L iver+ g o f Water) 3M E-nvironmental Laboratory FACT-M-1.0 Extraction of PFOS from Liver Page 6 of 8 Page 48 3M Medical Department Study: T-6316.8 BACK TO MAIN * Analytical Report: FACT TO X -097 L R N -U 2 4 5 2 13.1.2 Calculate actual concentrations o f PFOS in calibration standards using the follow ing equation: uL o f Standard x Concentration (ug /mL'i = Final Concentration (pg/g or mg/kg) mg Liver /1 mL homogenate o f PFOS in Liver *Average w eight o f liver in solution as determined in 13.1.1, by weighing ten 1 mL homogenates o f approximately 40 mg liver in 200 mL o f M illi-Q water. 14.0 M e t h o d P e r f o r m a n c e __________________________________________________ '____________________ 14.1 The method detection lim it is equal to h alf the low est standard in the calibration curve. 15.0 P o l l u t i o n P r e v e n t i o n a n d W a s t e M a n a g e m e n t _______ _____________________ :________ 15.1 Sample waste is disposed in biohazard containers, flammable solvent waste is disposed in high BTU containers, and used glass pipette waste is disposed in broken glass containers located in the laboratory. 16.0 R e c o r d s ___________________________________________________ '__________________ 16.1 Complete the extraction worksheet and tape into the study notebook. 17.0 T a b l e s , D i a g r a m s , F l o w c h a r t s , a n d V a l i d a t i o n D a t a ________________^_______________ 17.1 The validation report associated with this method is FACT-M -1.0 & 2 .0 -V -l. 18.0 R e f e r e n c e s _________________________ ________________________________________________________ 18.1 AM DT-EP-22, "Routine Maintenance o f Ultra-Turrax T-25" 19.0 A f f e c t e d D o c u m e n t s__________ ' -._____________________________________ ________ 19.1 FACT-M -2, "Analysis o f Liver Extracts for Fluorochemicals using HPLC-Electrospray M ass Spectrometry" 20.0 Rvisions R evision Number. _________________ ;__________ Reason For Revision R ev isio n Date 3M Environmental Laboratory FACT-M-1.0 Extraction of PFOS from Liver Page 7 o f 8 Page 49 BACK TO MAIN 3M Medical Department Study: T-6316.8 Extraction Worksheet for FACT-M-1 Analytical Report: FACT TO X-097 L R N -U 2 4 5 2 Study# S am ple PFOS PFOS PFOS Date and .- 1 Number set# H,0 Blank Liver Blank approx. 0.5 ppm actual ppm #W - approx. 5 ppm actual ppm #W - approx. 50 ppm actual ppm #W - - Initials for Std. - e - '- - - - .- .- - - - - . - - - - -- '- .- -- - .- - - - . 1Study numb er where the original worksheet is located. Rlank Liver Homogenate: Std.#- Liver amount- g Liver Extraction Method : Date & Initials Vortex 15 sec. Pioette 1mL ofLiver Solution . Pipette 1mL of tO.S M TBA, pH 10. Std. # Pipette2 mL of0.25Na?COV0.25MNaHCO^ Buffer Std.# Pipette 5 mL of Ethyl Acetate TN-A- Shake 20 min. Centrifuge 20-25 min. Centrifuge Speed . Remove a 4 mL aliauot of organic laver Put on Nitrogen Evaoorator to drvness Evaoorator Temperature Add 1.0 mL of Methanol TN-A- Vortex 30 sec. Filter using a 3cc B-D syringe with a 0.2um SRI filter into a 1.5 mL autosample vial MS/MSD/__ Cont. Checks: Spiked_____uL of a _____ppm std (____________ ) for a final concentration of _______ ppm. MS/MSD used sample______ :______ . Cont Checks used same homogenate as for std curve. FACT-M-1.0 Extraction of PFOS from Liver Page 8 of 8 3M Environmental Laboratory 3M Medical Department Study: T-6316.8 BACK TO MAIN Analytical Report: FACT TO X-097 L R N -U 2 4 5 2 3M Environmental Laboratory M ethod E x t r a c t io n o f P o t a ssiu m per flu o r o o c t a n e su lfo n a te o r o t h e r A n io n ic F l u o r o c h e m ic a l su r fa c t a n t s fr o m Ser u m fo r A n a l y sis U sin g H P L C -E le c t r o spr a y /M a ss Spe c t r o m e t r y M ethod Num ber: FACT-M -3.0 Author: Lisa Clemen . Approved By: ) Laboratory Manager -- --s _ _ lb * ----------- Group Leader A di/m /.v Technical Reviewer JA doption D ate: ^ tZ `l 3 R evision Date: vji^M D ate D ate n lu ln D ate 1.0 S c o p e a n d A p p l i c a t i o n 1.1 Scope: This method is for the extraction o f potassium perfluorooctanesulfonate (PFOS) or other fluorochemical surfactants from serum. 1.2 A pplicable Com pounds: Fluorochemical surfactants or other fluorinated compounds. 1.3 M atrices: Rabbit, rat, and bovine serum or other sera as designated in the validation report. Microsoft 7.0.1/95 3M Environmental Laboratory FACT-M-3.0 Extraction of PFOS from Serum Page 1 of 8 Page 51 3M Medical Department Study: T-6316.8 BACK TO MAIN Analytical Report: FAC T TO X -097 L R N -U 2 4 52 2.0 S u m m a r y o f M e t h o d ______________________________________ ;_____________________________ 2.1 This method describes how to extract potassium perfluorooctanesulfonate (PFOS) or other anionic fluorochemical surfactants from serum using an ion pairing reagent and 5.0 mL o f ethyl acetate. An ion pairing reagent is added to the sample and the analyte ion pair is partitioned into ethyl acetate. Four mL o f extract are removed and put onto a nitrogen evaporator until dry. Each extract is reconstituted in 1.0 mL o f methanol, then filtered through a 3 cc plastic syringe attached to a 0.2 pm nylon filter into glass autovials. 3.0 D e f i n i t i o n s _____________________________!__________________ ;_________________ 3.1 None. 4.0 W a r n i n g s a n d C a u t i o n s ___________ ;_________ ,________________________________;__________ ;---------4.1 Health and Safety Warnings: 4.1.1 U se universal precautions, especially laboratory coats, goggles, and gloves when h a n d lin g animal serum, it may contain pathogens. 5 .0 I n t e r f e r e n c e s __________________ ___________________________________ _______________________________ 5.1 There are no known interferences at this time. 6.0 E q u i p m e n t _____________________ . _________________ _________________________ 6.1 The follow ing equipment is used w hile carrying out this method. Equivalent equipment is acceptable. 6.1.1 6 .1 .2 6 .1 .3 6 .1 .4 6 .1 .5 Vortex mixer, VWR, Vortex Genie 2 Centrifuge, Mistral 1000 or IEC Shaker, Eberbach or VWR Nitrogen evaporator, Organomation Balance, ( 0.100 gm) .. 7.0 S u p p l i e s a n d M a t e r i a l s ______________________ ______________ _________________________________ 7.1 G loves 7.2 Eppendorf or disposable pipettes 7 3 N algene bottles, capable o f holding 250 mL and 1 L 7.4 G lass, type A, volumetric flasks 7.5 40 mL glass I-CHEM vials 7.6 Polypropylene centrifuge tubes, 15 mL 7.7 Labels 7.8 Syringes, capable o f measuring 10 pL to 50 pL 7.9 G lass, type A , volumetric pipettes 7.10 Graduated pipettes FACT-M-3.0 Extraction of PFOS from Serum Page 2 of 8 3M Environmental Laboratory Page 52 3M M edical D epartm ent Study: T -6316.8 BACK TO MAIN Analytical Report: FAC T TO X-097 L R N -U 2 4 5 2 7.11 Electronic pipettor, Eppendorf or equivalent 7.12 Timer 7.13 Disposable plastic 3 cc syringes 7.14 Filters, nylon syringe filters, 0.2 pm, 25 mm 7.15 Crimp cap autovials N ote: Prior to using glassware and bottles, rinse 3 times with methanol and 3 tim es with M illi- QTM water. Rinse syringes a minimum o f 9 tim es v/ith methanol, 3 rinses from 3 separate vials. 8.0 R e a g e n t s a n d S t a n d a r d s _____________________________________ ;_______________________ 8.1 R eagents 8.1.1 Sodium hydroxide (J.T Baker or equivalent), (NaOH) 1ON: w eigh approximately 200 grams NaOH. Pour into a 1000 mL beaker containing 500 liters (L) M illi-QTM water, m ix until all solids are dissolved. Store in a 1 L N algene bottle. 8.1i2 Sodium hydroxide (J.T Baker or equivalent), (NaOH) IN . D ilute 1ON 1:10. Measure 10 mL o f 10N NaOH solution into a 100 mL volumetric flask and dilute to volum e using Milli-QTM water. Store in a 125 mL Nalgene bottle. 8 .1 .3 Tetrabutylammonium hydrogen sulfate (Kodak or equivalent), (TBA) 0.5M: W eigh approximately 169 grams o f TBA into a 1 L volumetric containing 500 L M illi-QTM water. Adjust to pH 10 using approximate ly 64 mL o f 1ON NaOH and dilute to volum e w ith Milli-QTM water. Add NaOH slow ly while adding the last mL o f 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 IN NaOH solution, 8 .1 .4 Sodium carbonate/sodium bicarbonate buffer (J.T. Baker or equivalent), (N ajC 03/N aH C 03) 0.25M: W eigh approximately 26.5 g o f sodium carbonate (NajCOj) and 21.0 g o f sodium bicarbonate (NaH C03) into a 1 L volumetric flask and bring to volum e with Milli-QTM water. Store in a 1 L nalgene bottle. 8.1.5 PFOS (3M Specialty Chemical Division)., molecular weight = 538. 8.1.6 Other fluorochemicals, as appropriate. 8.1.7 Ethyl Acetate, Omnisolv, glass distilled or HPLC grade. 8.1.8 M ethanol, Omnisolv, glass distilled or HPLC grade. 8.1.9 Serum, frozen liquid from Sigma. 8.1.10 Control serum received with each sample set. 8.1.11 Milli-QTM water, all water used in this method should be Milli-QTM water and may be provided by a M illi-Q TOC Plus system. 3M Environmental Laboratory FACT-M-3.0 Extraction of PFOS from Serum Page 3 o f 8 Page 53 3M Medical Department Study: T-6316.8 BACK TO MAIN Analytical Report: FACT TO X -097 L R N -U 2 4 5 2 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 W eigh approximately 100 mg o f PFOS into a 100 mL volumetric flask and record the actual weight. 8.2.4 Bring to volum e with methanol for a stock standard o f approximately 1000 ppm (pg/m L ). 8.2.5 Dilute the stock solution with methanol for a working standard 1 solution o f approximately 50 ppm. 8.2.6 Dilute the stock solution with methanol for a working standard 2 solution o f approx. 5.0 ppm. 8.2.7 Dilute the stock solution with methanol for a working standard 3 solution o f approx. 0.50 ppm. 9.0 S a m p l e H a n d l i n g ______________________________ __________________________________________ ' 9.1 A ll sera are received frozen and must be kept frozen until the extraction is performed. 10.0 Q u a l i t y C o n t r o l _______________ ____________________________ ;________________________ 10.1 Matrix Blanks and Method Blanks 10.1.1 Two 1.0 mL aliquots o f the serum are extracted follow ing this procedure and used as matrix blanks. See section 11.1.2. 10.1.2 Two 1.0 mL aliquots o f Milli-QTM water are extracted follow ing 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 o f the extraction. 10.2.2 Prepare each spike using serum chosen by the analyst, usually control serum received with each sample set. 10.2.3 Expected concentrations w ill fall in the mid-range o f the initial calibration curve. Additional spikes may be included and may fall in the low-range o f the initial calibration curve. 103 Continuing Calibration Checks 10.3.1 Prepare and analyze continuing calibration check samples to determine the continued linearity o f the initial calibration curve. 10.3.2 One check is prepared per group o f ten samples. For example, if a sample set = 34, four checks are prepared and extracted. 3M Environmental Laboratory FACT-M-3.0 Extraction of PFOS from Serum Page 4 o f 8 Page 54- 3M Medical Department Study: T-6316.8 BACK TO MAIN Analytical Report: FACT TO X-097 L R N -U 2 4 5 2 10.3.3 Prepare each continuing calibration check from the same serum used to prep the initial curve. 10.3.4 The expected concentration w ill fall within the mid-range o f the initial calibration curve. 11.0 C a l i b r a t i o n a n d S t a n d a r d i z a t i o n _________ |__________________________________ ;______________ 11.1 Prepare Serum Standards 11.1.1 Transfer 1 mL o f serum to a 15 mL centrifuge tube. 11.1.2 If the majority o f serum sample volumes are less than 1.0 mL, extract standards using serum volum es in the standards equal to the serum volum es in samples. Do not extract below 0.50 mL o f serum. Record the serum volum e on the extraction sheet. 1 1 .1 3 M ix or shake between aliquots while preparing a total o f sixteen aliquots o f 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 amounts listed in table 1 to spike, in duplicate, two standard curves for a total o f fourteen samples. 11.1.5 Refer to the validation report FACT-M-3.0-V-1 andFA CT-M -4.0-V-l which lists the working ranges for calibration curves. Table 1 A pproxim ate Spiking A m ounts for Standards and Spikes U sing 1.0 mL o f Serum Working Standard (Approx. Cone.) - 0.500 ppm 5.00 ppm 5.00 ppm 5.00 ppm 50.0 ppm 50.0 ppm 50.0 ppm PL Approx, final cone, o f PFOS in serum - B lank 20 0.010 ppm 5 0.025 ppm 10 0.050 ppm 20 0.100 ppm 5 0.250 ppm 10 0.500 ppm 15 ! 0.750 ppm 11.1.4 See section 13.0 to calculate actual concentrations o f PFOS in calibration standards. 11.2 Extract spiked serum standards following 12.6-12.16 o f this method. Use these standards to establish each initial curve on the mass spectrometer. 3M Environmental Laboratory FACT-M-3.0 Extraction of PFOS from Serum Page 5 of 8 Page 55- 3M Medical Department Study: T-6316.8 BACK TO MAIN Analytical Report: FACT TO X -097 L R N -U 2 4 5 2 12.0 P r o c e d u r e s ________________________________________ :_______________________________________________ 12.1 Obtain frozen serum samples and allow to thaw. 12.2 Vortex mix for 15 seconds then remove 1.0 mL or appropriate volum e to a 15 mL polypropylene centrifuge tube. 1 2 3 Return serum samples to freezer after extraction amount has been removed. 12.4 Record the serum volum e on the extraction worksheet. The final methanol volum e w ill equal the initial serum volum e. ' 12.5 Label the tube with the study number, serum ID, date and analyst initials. See attached worksheet for documenting the remaining steps. 12.6 Spike serum with the appropriate amount o f 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 mL 0.5 M TBA and 2 mL o f the 0.25 M sodium carbonate/sodium bicarbonate 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 w ell separated. Set power on the centrifuge to approximately 3500 rpm. 12.12 Transfer 4 mL o f organic layer, using a 5 mL graduated glass pipette, to a clean 15 mL centrifuge tube; Label this fresh tube with the same information as in 12.5. 12.13 Put each sample on the analytical nitrogen evaporator until dry, approximately 2 to 3 hours. 12.14 A dd 1.0 m L or appropriate volume o f methanol to each centrifuge tube using a graduated pipette. (This volum e equals the initial volume of serum used for the extraction.) 12.15 Vortex mix for 30 seconds. 12.16 Attach a 0.2 pm nylon mesh filter to a 3 cc syringe and transfer the sample to this syringe. Filter into a 1.5 mL glass autovial. 12.17 Label the autovial with the study number, animal number and gender, sample timepoint, matrix, final solvent, extraction date, and analyst(s) who performed the extraction. 12.18 Cap and hold for HPLC-electrospray/mass spectrometry analysis. Extracts may be stored at 4 C until analysis. 12.19 Complete the extraction worksheet, attached to this document, and tape to page o f study notebook. 3M Environmental Laboratory FACT-M-3.0 Extraction o f PFOS from Serum Page 6 of 8 Page5& 3M Medical Department Study: T-6316.8 BACK TO MAIN Analytical Report: FACT TO X-097 L R N -U 2 4 5 2 1 3 .0 D a t a A n a l y s i s a n d C a l c u l a t i o n s _________________________________ :______________________ 13.1 Calculations: 13.1.1 Calculate actual concentrations o f PFOS, or other appropriate fluorochem ical, in calibration standards using die following equation: . mL o f Standard x Concentration fne /mLl = Final Concentration (pg/mL) mL o f Standard + Initial Serum Volume (mL) o f PFOS in Serum 1 4 .0 M e t h o d P e r f o r m a n c e ________________________________________________________________________ 14.1 The method detection lim it is equal to half the low est standard in the calibration curve. 1 5 .0 P o l l u t io n P r e v e n t io n a n d W a st e M a n a g e m e n t ____________________________ 15.1 Sample waste is disposed in biohazard containers, flammable solvent waste is disposed in high BTU containers, and used glass pipette waste is disposed in broken glass containers located in the laboratory. 1 6 .0 R e c o r d s _________________________ ;___________________________________________________________ 16.1 Complete the extraction worksheet attached to this; method, and tape into the study notebook. 1 7 .0 T a m f s - D i a g r a m s . F l o w c h a r t s , a n d V a l i d a t i o n D a t a _______________________________ 17.1 The validation report associated with this method is FACT-M -3.0 & 4 .0 -V -l. 1 8 .0 R e f e r e n c e s ________________________________________ :________ ;________________________________ 18.1 None 1 9 .0 A f f e c t e d D o c u m e n t s ___________________:__________________ _j ___________________ :_______ 19.1 FACT-M -4, "Analysis o f Serum Extracts for Fluorochemicals using HPLG-Electrospray M ass Spectrometry" 20.0 R e v i s i o n s R ev isio n Number. Reason For Revision R evision D ate 3M Environmental Laboratory FACT-M-3.0 Extraction of PFOS from Serum Page 7 of 8 ...Page-57- BACK TO MAIN 3M Medical Department Study: T-6316.8 Analytical Report: FACT TO X-097 L R N -U 2 4 52 Extraction Worksheet for FACT-M-3 Study# 1 Sample Number set # H ,0 Blank Serum Blank PFOS approx. 0.5 ppm actual ppm #W - PFOS approx. 5 ppm actual ppm #W - PFOS approx. 50 ppm actual ppm #W -' - Date and Initials for Std. or Comments - -' - -- - - - - --- * -' - - - ' - - -- - - - - --- --- -. - - - .- - --- --- - - - - - .- - .- - - .- - - -. --- 1Studv numtter where the original worksheet is located.____ Blank Scrum Std # Serum amount ** Serum Extraction Method : R Date & Initials Vortex 15 sec. PiDette Serum Volume mL Pipette 1mL of 0.5 M TBA, pH 10. Std.# Pipette 2 mL of 0.25 Na?CO"?/0.25MNaHCOt buffer Std. # Pipette 5 mL of ethyl acetate TN-A- Shake 20 min. Centrifuge 20-25 min. Centrifuge speed: Remove a 4 mL aliauot of organic laver Put on Nitrogen Evaporator to dryness Evaporator #: Temperature: Add methanol Volume mL TN-A- Vortex 30 sec. Filter using a 3cc B-D svringe with a 0.2um SRI filter into a 1.5 mL autosample vial MS/MSD/___Cont. Checks: Spiked ___ uL of a _____ ppm std (_____________) for a final concentration of ________ppm. MS/MSD used sample______________. Cont Checks used same serum as for std curve. FACT-M-3.0 Extraction o f PFOS from Serum Page 8 of 8 3M Environmental Laboratory Page 58 3M Medical Department Study: T-6316.8 BACK TO MAIN Analytical Report: F A C T T O X -097 L R N -U 2 4 52 3M Environmental Laboratory M ethod E x t r a c t io n o f P o t a ssiu m P e r flu o r o o c t a n e su lfo n a te o r O t h e r F l u o r o c h e m ic a l c o m p o u n d s f r o m S e r u m f o r A n a l y sis U s in g HPLC- E l e c t r o spr a y /M a ss Spe c t r o m e t r y M ethod Num ber: ETS-8-4.1 Adoption Date: 03/01/99 Author: L isa Clemen, Glenn Langenburg R evision Date: Approved By: Q f ; S --- Laboratory Manager j j z .-------- Group Leader Technical Reviewer Date Date otf/W w Date 1.0 S c o p e a n d A p p l i c a t i o n 1.1 Scope: This method is for the extraction o f potassium perfluorooctanesulfonate (PFOS) or other fluorochem ical compounds from serum. 1.2 A pplicable com pounds: Fluorochemical surfactants or other fluorinated compounds. 1.3 M atrices: Rabbit, rat, bovine, monkey, and human serum or other fluids as designated in the validation report. Word 6/95 3M Environmental Laboratory ETS-8-4.1 Extraction of PFOS from Serum Pagel o f 14 Page 59 3M Medical Department Study: T-6316.8 BACK TO MAIN Analytical Report: FACT TO X -097 L R N -U 2 4 5 2 2.0 s u m m a r y o f M e t h o d _____________________________'__________________ __ ________________________ 2.1 This method describes the procedure for extracting potassium perfluorooctanesulfonate (PFOS) or other fluorochemical surfactants from serum, or other fluids, using an ion pairing reagent and methyl-tert-butyl ether (MtBE). In this method, seven fluorochemicals were extracted: PFOS, PFOSA, PFOSAA, EtFOSE-OH, PFOSEA, M 556, and surrogate standard (see 3.0 Definitions). An ion pairing reagent is added to the sample and the analyte ion pair is partitioned into MtBE. The MtBE extract is removed and put onto a nitrogen evaporator until dry. Each extract is reconstituted in 1.0 mL o f methanol, then filtered through a 3 cc plastic syringe attached to a 0.2 pm nylon filter into glass autovials. 2.2 These sample extracts are analyzed follow ing method ETS-8-5.1 or other appropriate m ethods. 3.0 D e f i n i t i o n s _______________________________ ____________________ ;_____________________ 3.1 PFOS: perfluorooctanesulfonate (anion o f potassium salt) C,F,7S 0 3` 3.2 PFOSA: perfluorooctane sulfonylamide C8F17S 0 2NH2 3.3 PFOSAA: perfluorooctane sulfonylamido (ethyl)acetate C8F17S 0 2N(CH2CH3)CH2C 0 2' 3.4 EtFOSE-OH: 2(N-ethylperfluorooctane sulfonamido)-ethyl alcohol C8F 17S 0 2N(CH2CH3)CH2CH20H . 3.5 PFOSEA: perfluorooctane sulfonyl ethylamide C8F17S 0 2N(CH2CH3)H 3.6 M 556: C8F17S 0 2N(H)(CH2C 0 0 H ) 3.7 Surrogate standard: 1H-1H-2H-2H perfluorooctane sulfonic acid 4.0 W a r n i n g s a n d C a u t i o n s __________________________________________________________ 4.1 Health and safety warnings 4.1.1 U se universal precautions, especially laboratory coats, goggles, and gloves when handling animal tissue, which may contain pathogens. 5.0 I n t e r f e r e n c e s _________________________________________________________________________ '________ 5.1 There are no interferences known at this time. 6.0 E q u i p m e n t __________________________________'_____________________________________________ 6.1 The follow ing 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 3M Environmental Laboratory ETS-8-4.1 Extraction of PFOS from Serum Page 2 of 14 Page 60 3M Medical Department Study: T-6316.8 BACK TO MAIN Analytical Report: F A C T T O X -0 9 7 L R N -U 2 4 5 2 6.1.4 Nitrogen evaporator, Organomation 6.1.5 Balance ( 0.100 g) 7 .0 S u p p l i e s a n d M a t e r i a l s ______________________;________ 7.1 G loves 7.2 Eppendorfor disposable pipettes 7.3 Nalgene bottles, capable o f holding 250 mL and 1 L 7.4 Volumetric flasks, glass, type A 7.5 I-CHEM vials, glass, 40 mL glass 7.6 Centrifuge tubes, polypropylene, 15 mL 7.7 Labels 7.8 Oxford Dispenser - 3.0 to 10.0 mL 7.9 Syringes, capable o f 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 N ote: Prior to using glassware and bottles, rinse 3 times with methanol and 3 tim es with M illi-QTM water. Rinse syringes a minimum o f 9 times with methanol, 3 rinses from 3 separate vials. 8.0 R e a g e n t s a n d S t a n d a r d s ________________________________ ,____________________________________ 8.1 Type I reagent grade water, Milli-QTM or equivalent; all water used in this method should be M illi-QTM water and may be provided by a M illi-Q TOC PlusTM system 8.2 Sodium hydroxide (NaOH), J.T Baker or equival ent 8.3 Tetrabutylammonium hydrogen sulfate(TBA), Kodak or equivalent 8.4 Sodium carbonate (N ajC 03), J.T. Baker or equivalent 8.5 Sodium bicarbonate (NaHCOj), J.T. Baker or equivalent 8.6 M ethyl-T-Butyl Ether, Omnisolv, glass distilled or HPLC grade 8.7 M ethanol, Omnisolv, glass distilled or HPLC grade 8.8 Serum or blood, frozen from supplier 8.9 Fluorochemical standards 8.9.1 PFOS (3M Specialty Chemical D ivision), molecular weight = 538 8.9.2 PFOSA (3M Specialty Chemical Division), molecular weight = 499 ETS-8-4.1 Extraction of PFOS from. Serum Page 3 of 14 3M Environmental Laboratory Page 61 3M M edical D epartm ent Study: T -6316.8 . BACK TO MAIN Analytical Report: FACT TO X -097 L R N -U 2 4 5 2 8.9.3 PFOSAA (3M Specialty Chemical D ivision), molecular weight = 585 8.9.4 EtFOSE-OH (3M Specialty Chemical D i'/ision), molecular w eight = 570 8.9.5 PFOSEA (3M Specialty Chemical D ivision), molecular weight = 527 8.9.6 M 556(3M Specialty Chemical Division)., molecular w eight = 557 8.9.7 Surrogate standard: 4-H, perfluorooctane sulfonic acid (1-H .l-H , 2-H, 2-H C8F13S 0 3H) molecular weight = 428 8.9.8 Other fluorochem icals, as appropriate 8.10 Reagent preparation NOTE: W hen preparing larger volumes than listed in reagent, standard, or surrogate preparation, adjust accordingly. 8.10.1 IO N sodium hydroxide (NaOH): W eigh approximately 200 g NaOH. Pour into a 1000 mL beaker containing 500 mL Milli-QTM water, m ix until all solids are dissolved. Store in a 1 L Nalgene bottle. 8.10.2 1 N sodium hydroxide (NaOH): Dilute 1 0 N NaOH 1:10. Measure 10 mL o f 10 N NaOH solution into a 100 mL volumetric flask and dilute to volum e using M illi-QTM water. Store in a 125 mL Nalgene bottle. 8.10.3 0.5 M tetrabutylammonium hydrogen sulfate (TBA): W eigh approximately 169 g o f TBA into a 1 L volumetric containing 500 mL Milli-QTM water. Adjust to pH 10 using approximately 44 to 54 mL o f 10 N NaOH (W hile adding the last mL o f ' NaOH, add slow ly because the pH changes abruptly). D ilute to volum e with Milli-QTM water. Store in a 1 L Nalgene bottle. 8.10.3.1 TBA requires a check prior to each use to ensure pH = 10. Adjust as needed using 1 N NaOH solution. 8.10.4 0.25 M sodium carbonate/sodium bicarbonate buffer (N ajC 03/N aH C 03): W eigh approximately 26.5 g o f sodium carbonate (NajCOj) and 2 1.0 g o f sodium bicarbonate (NaHCO,) into a 1 L volumetric flask and bring to volum e w ith M illiQTM water. Store in a 1 L Nalgene bottle. 8.11 Standards preparation 8.11.1 Prepare PFOS standards for the standard curve. 8.11.2 Prepare other fluorochemical standards, as appropriate. Multicomponent fluorochemical standards are acceptable (for example, one working standard solution containing 1.00 ppm PFOS, 1.02 ppm PFOSA, 0.987 ppm PFOSAA, and 1.10 ppm EtFOSE-OH.) 8.11.3 W eigh approximately 100 mg o f PFOS into a 100 mL volumetric flask and record the actual weight. 8.11.4 Bring to volum e with methanol for a stock standard o f approximately 1000 ppm (p g/m L ). 8.11.5 D ilute the stock solution with methanol for a working standard 1 solution o f approximately 50 ppm. 8.11.6 D ilute working standard 1 with methanol for a working standard 2 solution o f approx. 5.0 ppm. ETS-8-4.1 Extraction of PFOS from Scrum Page 4 of 14 3M Environmental Laboratory Page 62 3M Medical Department Study: T-6316.8 BACK TO MAIN Analytical Report: FACT TO X -097 L R N -U 2 4 5 2 8.11.7 Dilute working standard 1 with methanol for a working standard 3 solution o f approx. 0.50 ppm. 8.12 Surrogate stock standard preparation 8.12.1 W eigh approximately 50-60 mg o f surrogate standard l-H ,l-H , 2-H , 2-H , C8F13S 0 3H into a 50 mL volumetric flask and record the actual weight. 8.12.2 Bring to volum e w ith methanol for a surrogate stock o f approximately 1000-1200 ppm. 8.12.3 Prepare a surrogate working standard. Transfer approximately 1 mL o f surrogate stock to a 10 mL volumetric flask and bring to volume with methanol for a working standard o f 100 ppm. Record the actual volum e transferred. 9.0 S a m p l e H a n d l i n g ______________ ;_______________________________________ '___________________________ 9.1 A ll samples are received frozen and must be kept frozen until the extraction is performed. 9.2 A llow samples to thaw to room temperature prior to extraction. 10.0 Q u a l i t y C o n t r o l ____________________________ ;_______________ _________________________ 10.1 Solvent B lanks, M ethod blanks and m atrix blanks 10.1.1 An aliquot o f 1.0 mL methanol is used as: a solvent blank. 10.1.2 Extract two 1.0 mL aliquots o f Milli-QTM water following this procedure and use as method blanks. 10.1.3 Extract two 1.0 mL aliquots o f the serum following this procedure and use as matrix blanks. See 11.1.4. 10.2 M atrix spikes 10.2.1 Prepare and analyze matrix spike and matrix spike duplicate samples to determine the accuracy o f the extraction. 10.2.2 Prepare each spike using a sample chosen by th analyst, usually the control matrix received with each sample set. 10.2.3 Expected concentrations w ill fall in the mid-range o f the initial calibration curve. Additional spikes may be included and may fall in the low-range o f the initial calibration curve. 10.2.4 Prepare one matrix spike and matrix spike duplicate per 40 samples, with a minimum o f 2 matrix spikes per batcli. 10.3 C ontinuing calibration checks 10.3.1 Prepare continuing calibration check samples to ensure the accuracy o f the initial calibration curve. 10.3.2 Prepare, at a minimum, one continuing check per group o f 10 samples. For example, if a sample set = 34, four checks are prepared and extracted. 10.3.3 Prepare each continuing calibration check from the same matrix used to prepare the initial curve. 3M Environmental Laboratory ETS-8-4.1 Extraction of PFOS from Serum Page 5 of 14 Page 63 3M Medical Department Study: T-6316.8 BACK TO MAIN Analytical Report: FACT TO X-097 L R N -U 2 4 5 2 10.3.4 The expected concentrations w ill fall within the mid-range o f the initial calibration curve. Additional spikes may be included that fall in the low-range o f the initial calibration curve. This is necessary if the analyst must quantitate using only the low end o f the calibration curve (for example, 5 ppb - 100 ppb, rather than 5 ppb -1 0 0 0 ppb). 11.0 C a l i b r a t i o n a n d S t a n d a r d i z a t i o n _____________;________________________;_____________________ 11.1 Prepare matrix calibration standards 11.1.1 Transfer 1 mL o f serum to a 15 mL centrifuge tube. 11.1.2 I f m ost sample volum es are less than 1.0 mL, extract standards with matrix volum es equal to the sample volum es. Do not extract less than 0.50 mL o f . matrix. Record each sample volum e on tie extraction sheet. 11.1.3 W hile preparing a total o f twenty aliquots; in 15 mL centrifuge tubes, m ix or shake between aliquots. 11.1.4 Two 1 mL aliquots, or other appropriate volume, serve as matrix blanks. Typically use the standard concentrations and spiking amounts listed in Table 1, at the end o f this section, to spike, in duplicate, two standard curves, for a total o f eighteen standards, two matrix blanks, and two method blanks. 11.1.5 Refer to validation report ETS-8-4.0 & 1T S-8-5.0-V -1, which lists the working ranges and the Linear Calibration Range (LCR) for calibration curves. 11.1.6 U se Attachment D as an aid in calculating the concentrations o f the working standards. See Section 13.0 to calculate Eictual concentrations o f PFOS in calibration standards. 11.2 To each standard, blank, or continuing check, add appropriate amount o f surrogate working standard for the concentration to fall within the calibration curve range 5 ppb 1000 ppb. 1 1 3 Extract spiked matrix standards following 12.6-12.16 o f this method. U se 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 TO X-097 LR N -U 2 4 52 Table 1 Approxim ate spiking am ounts for standards and spikes U sing 1.0 mL o f m atrix Working standard (approx, cone.) - pL Approx, final cone, o f analyte in matrix - Blank 0.500 ppm 10 0.005 ppm 0.500ppm 20 0.010 ppm 5.00 ppm 5 0.025 ppm 5.00 ppm 10 . 0.050 ppm 5.00 ppm 50.0 ppm . 20 5 0.100 ppm 0.250 ppm 50.0 ppm 10 0.500 ppm 50.0 ppm 15 0.750 ppm 50.0 ppm 20 1.00 ppm 12.0 P r o c e d u r e _______________________________________ _________ ;_____________________ __________________ 12.1 Obtain frozen samples and allow to thaw at room temperature or in a lukewarm waterbath. 12.2 Vortex m ix for 15 seconds, then transfer 1.0 mL or other appropriate volum e to a 15 mL polypropylene centrifuge tube. 12.3 Return unused samples to freezer after extraction ,amounts have been removed. 12.4 Record the initial volum e on the extraction worksheet. 12.5 Label the tube w ith the study number, sample ID, date and analyst initials. See attached worksheet for documenting the remaining steps. 12.6 Spike all samples, including blanks and standards, ready for extraction with surrogate standard as described in 11.2. 12.7 Spike each matrix with the appropriate amount o f standard as described in 11.1, or T able 1 in that section, for the calibration curve standards. Also prepare matrix spikes and continuing calibration standards. 1 12.8 Vortex m ix the standard curve samples, matrix spike samples, and continuing calibration samples for 15 seconds. 12.9 Check to ensure the 0.5 M TBA reagent is at pH 10. If not, adjust accordingly. 12.10 To each sample, add 1 mL 0.5 M TBA and 2 mL o f 0.25M sodium carbonate/sodium bicarbonate buffer. 12.11 Using an Oxford Dispenser, add 5 mL methyl-te/7-butyl ether. 12.12 Cap each sample and put on the shaker at a setting o f 300 rpm, for 20 minutes. 12.13 Centrifuge for 20 to 25 minutes at a setting o f 3500 rpm, or until layers are w ell separated. ETS-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: FAC T TO X -097 L R N -U 2 4 52 12.14 Label a fresh 15 raL centrifuge tube with the same information as in 12.5. 12.15 Rem ove 4.0 mL o f the organic layer to this clean 15 mL centrifuge tube. 12.16 Put each sample on the analytical nitrogen evaporator until dry, approximately 1 to 2 hours. " 12.17 Add 1.0 mL o f methanol to each centrifuge tube using a graduated pipette. 12.18 Vortex mix for 30 seconds. 12.19 Attach a 0.2 pm nylon mesh filter to a 3 cc syringe and transfer the sample to this . syringe. Filter into a 1.5 mL glass autovial or low-volum e autovial when necessary. 12.20 Label the autovial with the study number, animal number and gender, sample tim epoint, matrix, final solvent, extraction date, and analyst(s) performing the extraction. 12.21 Cap and store extracts at room temperature or at approximately 4 C until analysis. 12.22 Complete the extraction worksheet, attached to this document, and tape in the study notebook or include in study binder, as appropriate. 13.0 Data Analysis and Calculations_____________________________________ 13.1 C alculations 13.1.1 Calculate actual concentrations o f PFOS, or other applicable fluorochem ical, in calibration standards using the follow ing equation: mL o f standard x concentration o f standard fug /mTT__________ '________= . mL o f standard + mL o f surrogate standard + initial matrix volum e (mL) Final Concentration (pg/mL) o f PFOS in matrix 14.0 Method Performance____________________L__________________________________ 14.1 The method detection lim it (MDL) is analyte and matrix specific. Refer to MDL report for specific MDL and lim it o f quantitation (LOQ) values (see A ttachm ents B and C). 14.2 The follow ing quality control samples are extracted with each batch o f samples to evaluate the quality o f the extraction and analysis. 14.2.1 Method blanks and matrix blanks. . 14.2.2 Matrix spike and matrix spike duplicate samples to determine accuracy and precision o f the extraction. 14.2.3 Continuing calibration check samples to determine the continued accuracy o f the initial calibration curve. 14.3 Refer to section 14 o f ETS-8-5.1 for method performance criteria. 15.0 POLLUTION PREVENTION ANDWASTE MANAGEMENT________________________ 15.1 Sample waste is disposed in biohazard containers, flammable solvent waste is disposed in high BTU containers, and used glass pipette waste is disposed in broken glass containers located in the laboratory. ETS-8-4.1 Extraction of PFOS from Serum Page 8 of 14 3M Environmental Laboratory Page 66 3M Medical Department Study: T-6316.8 BACK TO MAIN Analytical Report: FACT TO X -097 L R N -U 2 4 5 2 16.0 R e c o r d s __________________;___________ _____________________________________________________ _ 16.1 Complete the extraction worksheet attached to this method, and tape in the study notebook or include in the 3-ring study binder, at appropriate. 17.0 ATTACHMENTS________________________________________ |_________________________ 17.1 Attachment A, Extraction w oiksheet 17.2 Attachment B , MDL/LOQ values and summary 17.3 Attachment C, Calibration standard concentratio nworksheet 18.0 R e f e r e n c e s __________________________________________________________________________________ 18.1 The validation report associated with this method is ETS-8-4.0 & 5 .0 -V -l. 18.2 FACT-M -3.1, "Analysis o f Serum or Other Fluid Extracts for Fluorochemicals using HPLC-Electrospray M ass Spectrometry" 19.0 A f f e c t e d D o c u m e n t s ____________________________________________________________________ 19.1 ETS-8-5.1, "Analysis o f Serum or Other Fluid Extracts for Fluorochemicals using HPLC-Electrospray M ass Spectrometry" 20.0 Revisions R evision Number 1 Reason For Revision Section 12.21 Changed to include sample storage at room temperature. Section 12.13 Added the shaker speed. Section 12.17 Final volume is 1.0 mL; not adjusted for initial volum es less than 1.0 mL. R ev isio n D ate 04/02/99 3M Environmental Laboratory . ETS-8-4.1 Extraction of PFOS iron:. Serum Page 9 o f 14 Page 67 BACK TO MAIN 3M Medical Department Study: T-6316.8 Analytical Report: FACT TO X-097 L R N -U 2 4 5 2 Extraction W orksheet ETS-8-4.1 Studv # Matrix Box# Wk/Dav DateSpiked/Analyst CCV MS MSD Surrogate Std approx, ppm actual ppm # FC-Mix approx. 0.5 pm actual ppm # FC-Mix approx. 5 ppm actual ppm # FC-Mix approx. 50 ppm actual ppm # Comments Blank --- - -- -- - -' ---- -- -- -- - - - 'Std# amount- Serum Extraction Method : V ortex 15 sec. Pioette Matrix Volume mL Pipette 1mL of 0.5 M TBA, pH 10. pH - Std. # Pipette 2 mL of 0.25 Na?COy0.25M NaHCOi buffer Std. # Dispense 5 mL of methyl-t-butyl ether TN-A- Shake 20 min. Shaker speed: Centrifuge 20-25 min. Centrifuge speed: Remove a 4 mL aliauot oforganic laver Put on Nitrogen Evaporator to drvness Temperature: Add methanol Volume mL TN-A- Vortex 30 sec. Filter using a 3cc B-D svringe with a 0.2um filter into a 1.5 mL autosamolevial Cont Cal. Verifications used same matrix as for std curve. . - - - .- - - - - mL Date & Initials .. Attachment A ETS-8-4.1 Extraction of PFOS from Serum Page 10 of 14 3M Medical Department Study: T-6316.8 BACK TO MAIN Analytical Report: FACT TO X -097 L R N -U 2 4 52 M DL/LO Q values for rabbit serum Com pound M DL LOQ Linear C alibration Range (LCR) (PPb) (PPb) Approxim ate concentrations to be used for preparing the Standard C alibration Curve PFOS 1.74 5.55 5p p b -1 0 0 0 p p b PFOSA 1.51 4.79 5 ppb -1 0 0 0 ppb PFOSAA 3.46 20.5 5 p p b - 1000 ppb EtFOSE-OH 11.4 36.2 5 p pb -1 0 0 0 ppb M 556 6.03 19.2 5 ppb -1 0 0 0 ppb PFOSEA 5.71 18.2 5 ppb -1 0 0 0 ppb MDL/LOQ values in rat, bovine, monkey, and human serum, and monkey plasma were not statistically determined. Two curves in each o f these matrices were extracted and analyzed with the rabbit serum curves to determine equivalence. Responses in the rat, bovine, monkey, and human were equivalent to the rabbitresponses, therefore, their MDL and LOQ will be the same values as determined in rabbit serum. Please see LOQ Summary and MDL study in ETS-8-4.0 & 5.0-V-l for further information. Attachment B: MDL/LOQ Summary ETS-8-4.1 Extraction of PFOS from Serum 3M Environmental-Laboratory Page 11 of 14 Page 69 3M M edical D epartm ent Study: T -6316.8 BACK TO MAIN Analytical Report: FAC T TO X-097 L R N -U 2 4 5 2 Compound: PFOS Prepared range Rabbit Serum of standards (ppb) (ng/mL) Full Range Low Curve High curve 1/X 0.995 - 978 4.94 - 248 97.8 - 978 0.995 - 978 Compound: PFOSA Prepared range Rabbit Serum of standards (ppb) (ng/mL) Full Range Low Curve High curve 1/X . 0.993 - 976 4.93 - 97.6 24.8 - 976 0.993 - 976 Compound: PFOSAA Prepared range Rabbit Serum of standards (ppb) (ng/mL) Full Range Low Curve High curve !/X 0.991 - 974 4.92 - 247 49.2 - 974 0.991 - 974 LCR from curve (PPb) (ng/mL) 24.8 - 978 4.94 - 248 97.8 - 978 4.94 - 978 % Recovery Range 83-108 85-104 85-106 94-111 LCR from curve (ppb) (ng/mL) 4.93 - 976 % Recovery Range 88-103 4.93 - 97.6 . 87-105 24.8-978 93-102 4.93-976 94-103 LCR from curve (PPb) (ng/mL) 24.7-974 9.74 - 247 97.4 - 974 9.74-974 % Recovery Range 81-111 97-107 85-108 95-115 RSD Range 4.67-11.0 5.34-12.0 4.84-9.80 4.60-10.5 RSD Range 5.10-14.7 9.85-14.7 5.08-13.9 5.10-14.5 RSD Range 4.18-10.6 6.38-21.8 4.33-12.5 4.11-23.2 Attachment B: MDL/LOQ Summary ETS-8-4.1 Extraction of PFOS from Serum 3M Environmental Laboratory Page 12 of 14 Page 70 3M Medical Department Study: T-6316.8 BACK TO MAIN Analytical Report: FACT TO X-097 LRN-U2452 Compound: EtFQSE-QH Prepared range Rabbit Serum of standards (PPb) (ng/mL) Full Range Low Curve High curve 1/X 0.993 - 976 4.93-97.6 49.3 - 976 0.993 - 493 LCR from curve (PPb) (ng/mL) 49.3 -976 9.76-97.6 97.6 - 976 9.76 - 976 % Recovery Range 77-110 97-107 90-109 86-111 Compound: PFOSEA Prepared range Rabbit Serum o f standards (ppb) (ng/mL) Full Range Low Curve High curve 1/X 0.993 - 976 4.93 - 248 49.3 - 976 0.993 - 976 LCR from curve (PPb) (ng/mL) 24,8 - 976 9.76 - 248 49.3 - 976 9.76 - 976 % Recovery Range 96-106 91-110 86-106 95-117 Compound: M556 Prepared range Rabbit Serum o f standards (ppb) (ng/mL) Full Range Low Curve High curve 1/X 0.993-976 4.93 - 97.6 97.6 - 976 0.993 - 976 LCR from curve (PPb) (ng/mL) 24.8 - 976 9.76-97.6 97.6 - 976 9.76 - 976 % Recovery Range 88-106 100-105 81-111 97-110 RSD Range 11.2-25.5 14.1-21.3 11.5-19.6 11.1-21.2 RSD Range 10.1-16.2 11.8-19.5 10.2-18.2 10.1-19.1 RSD Range 4.82-17.9 5.95-18.2 5.11-9.74 4.77-19.5 Attachment B: MDL/LOQ Summaiy ETS-8-4.1 Extraction of PFOS from Serum 3M Environmental Laboratory Page 13 of 14 Page 71 3M Medical Department Study: T-6316.8 BACK TO MAIN Analytical Report: FACT TO X -097 L R N -U 2 4 5 2 Ion Pair Standard Curves - Fluids Prep date(s): Standard number: Analyte(s): Equipment number: Sample matrix: Final solvent and TN: Blank fluid/identifier: M ethod/revision: Target analyte(s): FC mix std approx. 0.500 ppm: FC mix std approx. 5.00 ppm: FC mix std approx. 50.0 ppm: Surrogate std approx. 100 ppm: Actual concentrations of standards in the FC mix PFOS PFOSA PFOSAA EtFOSE PFOSEA Std cone Std cone Std cone Std cone Std cone ug/xnL ug/mL ug/mL ug/mL ug/mL 0.500 0.507 0.532 0.501 0.521 0.500 0.507 0.532 0.501 0.521 5.00 5.07 5.32 5.01 5.21 5.00 5.07 5.32 . 5.01 5.21 5.00 5.07 5.32 5.01 5.21 50.0 50.1 53.2 50.1 52.1 50.0 50.1 53.2 50.1 52.1 50.0 50.1 53.2 50.1 52.1 50.0 50.1 53.2 50.1 52.1 MB56 Std cone ug/mL 0.501 0.501 5.01 5.01 5.01 50.1 50.1 50.1 50.1 AU Amt spiked mL 0.010 0.020 0.005 0.010 0.020 0.005 0.010 0.015 0.020 AU Final vol mL 1.015 1.025 1.010 1.015 1.025 1.010 1.015 1.020 1.025 Calculated concentrations of standards in the sample matrix PFOS PFOSA PFOSAA EtFOSE PFOSEA M556 Surrogate Final cone Final cone Final cone Final cone Final cone Final cone Std cone ng/mL ng/mL ng/mL ng/mL ng/mL ng/mL ng/mL 4.93 5.00 5.24 4.94 5.01 5.13 100 9.76 9.89 10.4 9.78 9.93 10.2 24.8 25.1 26.3 24.8 25.2 25.8 Surrogate 49.3 50.0 52.4 . 49.4 50.1 51.3 Final cone 97.6 98.9 104 97.8 99.3 102 ng/mL 248 251 263 248 252 258 500 493 500 524 494 501 513 735 746 782 737 749 766 976 989 1038 978 993 1017 AU Amt spiked mL 0.005 Validated ranges - approximate concentrations Serum PFOS PFOSA PFOSAA EtFOSE-OH PFOSEA M556 Rabbit 5.00-1000 | 5.00-1000 I 5.00-1000 | 5.00-1000 | 5.00-1000 i 5.00-1000 Bovine Estimates only. Use values for rabbit . Rat Estimates only. Use values forrabbit Monkey &Plasma Estimates only. Use values for rabbit Human Estimates only. Use values for rabbit Attachment C: Ion Pair Standard Curves ETS-8-4.1 Extraction of PFOS from Serum 3M Environmental Laboratory Page 14 o f 14 Page 72 3M Medical Department Study: T-6316.8 I BACK TO MAIN Analytical Report: FAC T TO X-097 L R N -U 2 4 52 3M Environmental Laboratory M ethod E x t r a c t io n o f P o t a ssiu m P er flu o r o o c ta n esu lfo n a te o r o t h er F l u o r o c h e m ic a l C o m p o u n d s f r o m L iv e r f o r A n a l y s is u s in g HPLC- E l e c t r o spr a y /M a ss Sp e c t r o m e t r y M ethod Num ber: ETS-8-6.0 Author: Lisa Clemen, Robert Wynne Approved By: 'V t A w -- Laboratory Manager u / / **--------- Group Leader ( A .dWtu ' Technical Reviewer Adoption Date: R evision Date: "V ^ Ditte h Date o i/w h 't D ate 1.0 Scope and Application 1.1 Scope: This method is for the extraction o f potassium perfluorooctanesulfonate (PFOS) or other fluorochem ical compounds from liver. 1.2 A pplicable Com pounds: Fluorochemical surfactants or other fluorinated compounds. 1.3 M atrices: Rabbit, rat, bovine, and monkey livers or other tissues as designated in the validation report. Word 6.0/95 ETS-8-6.0 Extraction of PFOS from Liver Page 1 of 14 . 3M Environmental Laboratory Page 73 3M Medical Department Study: T-6316.8 BACK TO MAIN Analytical Report: FACT TO X-097 L R N -U 2 4 5 2 2.0 S u m m a r y o f M e t h o d ____________________ ;___________________________________________________ ' 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-/eri-butyl ether (MtBE). In this method, seven fluorochemicals can be extracted: PFOS, PFOSA, PFOSAA, EtFOSE-OH,.PFOSEA, M 556, and surrogate standard. An ion pairing reagent is added to the sample and the analyte ion pair is partitioned into MtBE. The MtBE extract is transferred to a centrifuge tube and put onto a nitrogen evaporator until dry. Each extract is reconstituted in 1.0 mL methanol then filtered through a 3 cc plastic syringe attached to a 0.2 pm nylon filter into glass autovials. 2.2 These sample extracts are analyzed following method ETS-8-7.0 or other appropriate m ethods. 3 .0 D e f i n i t i o n s _________________________________________________________________________ ;_______________ 3.1 PFOS: perfluorooctanesulfonate (anion o f potassium salt) C8F17S 0 3 3.2 PFOSA: perfluorooctane sulfonylamide C8F17S 0 2NH2 3.3 PFOSAA: perfluorooctane sulfonylamido (ethyl)acetate C,,F,7S 0 2N (CH2CH3)CH2C 0 2 3.4 EtFOSE-OH: 2(N-ethylperfluorooctane sulfonamido)-ethyl alcohol C,F17S 0 2N(CH2CH3)CH2CH20H 3.5 PFOSEA: perfluorooctane sulfonyl ethylamide C8FnS 0 2N(CH2CH3)H 3.6 M 556: C8F i7S 0 2N(H)(CH2C 00H ) 3.7 Surrogate standard: 1H-1H-2H-2H perfluorooctane sulfonic acid 4.0 W a r n i n g s a n d C a u t i o n s _____________________________________________ ;_____________________ ' 4.1 Health and Safety Warnings: 4.1.1 U se universal precautions, especially laboratory coats, goggles, and gloves when handling animal tissue, which may contain pathogens. . 5.0 Interferences_________________________ 5.1 There are no interferences known at this time. 6.0 E q u i p m e n t ___________________________________________________________________________ ;___________ 6.1 The follow ing equipment is used while performing this method. Equivalent equipment is acceptable. 6.1.1 6.1.2 6.1.3 6.1.4 Ultra-Turrax T25 Grinder for grinding liver samples Vortex mixer, VWR, Vortex Genie 2 Centrifuge, Mistral 1000 or IEC Shaker, Eberbach or VWR ETS-8-6.0 Extraction of PFOS from Liver Page 2 of 14 3M Environmental Laboratory Page 74 3M Medical Department Study: T-6316.8 BACK TO MAIN Analytical Report: FACT TO X -097 L R N -U 2 4 52 6.1.5 Nitrogen Evaporator, Organomation 6.1.6 Balance (sensitivity to 0.100 g) 7.0 S u p p l i e s a n d M a t e r i a l s _________________________________________________________________________ 7.1 G loves 7.2 D issecting scalpels 7.3 Eppendorf or disposable pipettes 7.4 Nalgene bottles, capable o f holding 250 mL and 1 L 7.5 Volumetric flasks, glass, type A . 7.6 I-CHEM vials, 40 mL glass 7.7 Plastic sampule vials, Wheaton, 6 mL (or appropriate size) 7.8 Centrifuge tubes, polypropylene, 15 mL 7.9 Labels 7.10 Oxford Dispensor - 3.0 to 10.0 ml 7.11 Syringes, capable o f measuring 5 pL to 50 pL 7.12 Graduated pipettes 7.13 Syringes, disposable plastic, 3 cc 7.14 Syringe filters, nylon, 0.2 pm, 25 mm 7.15 Timer 7.16 Crimp cap autovials and caps 7.17 Crimpers Note: Prior to using glassware and bottles, rinse 3 times with methanol and 3 tim es with M illiQTM water. Rinse syringes a minimum o f 9 times with methanol, 3 rinses from 3 separate vials. 8.0 Reagents and Standards 8.1 Type I reagent grade water, Milli-QTM or equivalent; all water used in this m ethod should be M illi-QTM water and be provided by a M illi-Q TOC PlusTM system 8.2 Sodium hydroxide (NaOH), J.T Baker or equivalent 8.3 Tetrabutylammonium hydrogen sulfate(TBA), Kodak or equivalent 8.4 Sodium carbonate (N a^ O j), J.T. Baker or equivalent 8.5 Sodium bicarbonate (NaH C03), J.T. Baker or equivalent 8.6 M ethyl-iert-butyl ether, Omnisolv, glass distilled or HPLC grade 8.7 M ethanol, Omnisolv, glass distilled or HPLC grade 8.8 Liver, frozen from supplier 8.9 Dry ice from supplier 8.10 Fluorochem ical standards 8.10.1 PFOS (3M Specialty Chemical D ivision), molecular weight = 538 ETS-8-6.0 Extraction of PFOS from Liver Page 3 of 14 3M Environmental Laboratory Page 75 3M Medical Department Study: T-6316.8 BACK TO MAIN Analytical Report: FACT TO X -097 L R N -U 2 4 5 2 8.10.2 PFOSA (3M Specialty Chemical D ivision), molecular weight = 499 8.10.3 PFOSAA (3M Specialty Chemical D ivision), molecular weight = 585 8.10.4 EtFOSE-OH(3M Specialty Chemical D ivision), molecular weight = 570 8.10.5 PFOSEA (3M Specialty Chemical D ivision), molecular weight = 527 8.10.6 M 556 (3M Specialty Chemical D ivision), molecular weight = 557 8.10.7 Surrogate standard: 4-H, perfluorooctane sulfonic acid (1-H .l-H , 2-H, 2-H C8F13S 0 3H) molecular weight = 428 8.10.8 Other fluorochemicals, as appropriate 8.11 R eagent preparation NO TE: When preparing larger volum es than listed in reagent, standard, or surrogate preparation, adjust accordingly. 8.11.1 10 N sodium hydroxide (NaOH): W eigh approximately 200 g NaOH. Pour into a 1000 mL beaker containing 500 mL Milli-QTM water, m ix until all solids are . dissolved. Store in a 1 L Nalgene bottle. 8.11.2 1 N sodium hydroxide (NaOH): Dilute 10 N NaOH 1:10. Measure 10 mL o f 1 0 N NaOH solution into a 100 mL volumetric flask and dilute to volum e using M illi-QTM water. Store in a 125 mL Nalgene bottle. 8.11.3 0.5 M tetrabutylammonium hydrogen sulfate (TBA): W eigh approximately 169 g o f TBA into a 1 L volumetric containing 500 mL Milli-QTM water. Adjust to pH 10 using approximately 44 to 54 mL o f 10 N NaOH (While adding the last mL o f NaOH, add slow ly because the pH changes abruptly). Dilute to volum e with M illi-QTM water. Store in a 1 L Nalgene bottle. 8.11.3.1 TBA requires a check prior to each use to ensure pH = 10. Adjust as needed using 1 N NaOH solution. 8.11.4 0.25 M sodium carbonate/sodium bicarbonate buffer (Na^COj/NaHCOj): W eigh approximately 26.5 g o f sodium carbonate; (Na^COj) and 21.0 g o f sodium bicarbonate (NaH C03) into a 1 L volumetric flask and bring to volum e with M illiQTM water. Store in a 1 L Nalgene bottle. 8.12 Standards preparation 8.12.1 Prepare PFOS standards for the standard curve. 8.12.2 Prepare other fluorochemical standards, as appropriate. Multicomponent fluorochemical standards are acceptable (for example, one working standard solution containing 1.00 ppm PFOS, 1.02 ppm PFOSA, 0.987 ppm PFOSAA, and 1.10 ppm EtFOSE-OH.) . 8.12.3 W eigh approximately 100 mg o f PFOS into a 100 mL volumetric flask and record the actual weight. 8.12.4 Bring to volume with methanol for a stock standard o f approximately 1000 ppm (p g/m L ). 8.12.5 D ilute the stock solution with methanol for a working standard 1 solution o f approximately 50 ppm. 3M Environmental Laboratory 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: FAC T TO X-097 L R N -U 2 4 5 2 8.12.6 Dilute the stock solution with methanol for a working standard 2 solution o f approx. 5.0 ppm. 8.12.7 Dilute the stock solution with methanol for a working s tandard 3 solution o f approx. 0.50 ppm. 8.13 Surrogate stock standard preparation 8.13.1 W eigh approximately 50-60 mg o f surrogate standard l-H .l-H , 2-H , 2-H, C,F13S 0 3H into a 50 m l volumetric flask and record the actual weight. 8.13.2 Bring to volume with methanol for a surrogate stock o f approximately 1000-1200 ppm. 8.13.3 Prepare a surrogate working standard. Transfer approximately 1.0 m l o f surrogate stock to a 10 m l volumetric flask and bring to volum e with methanol for a working standard o f 10-20 ppm. Record the actual volum e transferred. 9.0 S a m p l e H a n d l i n g _________________________________________________________________________________ 9.1 A ll samples are received frozen and must be kept frozen until the extraction is performed. 10.0 Q u a l i t y C o n t r o l _____________________ :_________________________________________ 10.1 Matrix blanks and method blanks . 10.1.1 An aliquot o f 1.0 mL methanol is used as a solvent blank. 10.1.2 Extract two 1.0 mL aliquots o f Milli-QTM water following this procedure and use as method blanks. 10.1.3 Extract two 1.0 mL aliquots o f 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 spike and matrix spike duplicate samples to determine the accuracy o f the extraction. 10.2.2 Prepare each spike using a sample chosen, by the analyst, usually a control liver received w ith each sample se t 10.Z.3 Expected concentrations w ill fall in the mid-range o f the initial calibration curve. Additional spikes may be included and may fall in the low-range o f the initial calibration curve. 10.2.4 Prepare one matrix spike and matrix spike duplicate per 40 samples, with a minimum o f 2 matrix spikes per batch. 10.3 Continuing calibration verifications 10.3.1 Prepare continuing calibration verification samples to ensure the accuracy o f the initial calibration curve. 10.3.2 Prepare, at a minimum, one continuing calibration verification sample per group o f 10 samples. For example, if a sample set - 34, four verifications are prepared and extracted. ' 3M Environmental Laboratory ETS-8-6.0 Extraction of PFOS from Liver Page 5 of 14 Page-77- 3M Medical Department Study: T-6316.8 BACK TO MAIN Analytical Report: FACT TO X-097 L R N -U 2 4 5 2 10.3.3 Prepare each continuing calibration verification from the same matrix used to prepare the initial curve. 10.3.4 The expected concentrations w ill fall with in the mid-range o f the initial calibration curve. Additional spikes may be included that fall in the low-range o f the initial calibration curve. This is necessary if the analyst must quantitate using only the low end o f the calibration curve (for example, 5 ppb - 100 ppb, rather than 5 p p b - 1000 ppb). 1 1 . 0 C a l i b r a t i o n a n d S t a n d a r d i z a t i o n _______________________________________ ^___________ 11.1 Prepare matrix calibration standards ' 11.1.1 W eigh approximately 40 g o f liver into a 250 mL Nalgene bottle containing 200 mLs Milli-QTM water. Grind to a homogeneous solution. 11.1.2 If 40 g is not available, use appropriate amounts o f liver and water to ensure a 1:5 ratio. 11.1.3 Refer to 13.0 to calculate the actual density o f liver homogenate and the concentration o f solid liver tissue dispersed, in 1.0 mL o f homogenate solution. 11.1.5 Add 1 mL o f homogenate to a 15 mL centrifuge tube. Re-suspend solution by shaking between aliquots while preparing z. total o f eighteen 1 mL aliquots o f homogeneous solution in 15 mL centrifuge tubes. 11.1.6 Two 1 mL aliquots, or other appropriate volume, serve as matrix blanks. 11.1.7 Typically use the standard concentrations and spiking amounts listed in Table 1, at the end o f this section, to spike, in duplicate, two standard curves, for a total o f eighteen samples, two matrix blanks, and tsvo method blanks. 11.1.8 Refer to validation reports ETS-8-6.0 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 U se Attachment C as an aid in calculating; the concentrations o f the working standards. Refer to 13.0 to calculate actual, concentrations o f PFOS in calibration standards. 11.2 To each working standard, blank, or continuing verification, add appropriate amount o f surrogate working standard for the concentration to fall within the calibration curve range 5 ppb - lOOOppb. 3M Environmental Laboratory ETS-8-6.0 Extraction o f PFOS from Liver Page 6 of 14 Page 78 3M Medical Department Study: T-6316.8 BACK TO MAIN Analytical Report: FAC T TO X -097 L R N -U 2 4 52 11.3 Extract spiked liver homogenates following 12.14-12.25 o f this method. U se these standards to establish each initial curve on the mass spectrometer. T able 1 Approxim ate Spiking Am ounts for Calibration Standards Working Standard (Approx. Cone.) - 0.50 ppm 0.50 ppm 0.50 ppm 0.50 ppm 0.50 ppm 5.0 ppm 5.0 ppm 5.0 ppm 50 ppm Ml '2 4 10 20 40 10 20 30 4 Approx, final cone, o f PFOS in liver Blank 0.005 ppm 0.010 ppm 0.025 ppm 0.050 ppm 0.100 ppm 0.250 ppm 0.500 ppm 0.750 ppm 1.00 ppm 12.0 P r o c e d u r e _______________________________________________________________________________________ 12.1 Obtain frozen liver samples. 12.2 Cut approximately 1 g o f liver using a dissecting scalpel. This part o f the procedure is best p erform ed q u ick ly, n o t a llo w in g th e liver to thaw . 12.3 W eigh the sample directly into a tared plastic sampule vial. 12.4 Record the liver weight in'the study notebook. 12.5 R e tu r n u n u s e d liv e r p o r tio n s to fr e e z e r . 12.6 Add 2.5 mLs o f water to sampule vial. 12.7 Grind the sample. Put the grinder probe in the sample and grind for about 2 minutes, or until the sample is homogeneous. 12.8 Rinse the probe into the sample with 2.5 mLs water using a pipette. 12.9 Take the grinder apart and clean it with methanol after each sample. Refer to AMDT-EP- 22. 12.10 Cap the sample and vortex for 15 seconds. Label the sampule vial with the study number, weight, liver ID, date and analyst initials. 3M Environmental Laboratory ETS-8-6.0 Extraction of PFOS from Liver Page 7 of 14 Page 79 3M Medical Department Study: T-6316.8 BACK TO MAIN Analytical Report: FAC T TO X-097 L R N -U 2 4 5 2 12.11 Pipette 1.0 mL, or other appropriate volume, o f homogenate into a 15 mL polypropylene centrifuge tube. Label the centrifuge tube with the identical information as the sampule vial. Refer to attached worksheet for documenting the remaining steps. 12.12 Pipette two 1 mL aliquots o f Milli-QTM water to centrifuge tubes. These w ill serve as method blanks. 12.13 Spike all samples, including blanks and standards ready for extraction with surrogate standard as described in section 11.2. 12.14 Spike each matirx with the appropriate amount o f standard as described in 11.1, or Table 1 o f that section, for the calibration curve standards. A lso prepare matrix spikes and continuing calibration standards. . 12.15 Vortex m ix 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 o f the 0.25 M sodium carbonate/sodium bicarbonate buffer. 12.18 U sing an Oxford Dispenser, add 5 mL methyl-teri-butyl ether. 12.19 Cap each sample and put on the shaker at a setting o f 300 rpm, for 20 minutes. 12.20 Centrifuge for 20 to 25 minutes at a setting o f 3500 rpm, or until layers are w ell separated. 12.21 Label a fresh 15 mL centrifuge tube with the same information sis in 12.10. 12.22 Rem ove 4.0 mL o f the organic layer to the fresh 1:5 mL centrifuge tube. 12.23 Put each sample on the analytical nitrogen evaporator until dry, approximately 1 to 2 hours. 12.24 Add 1.0 mL to each centrifuge tube using a graduated pipette. 12.25 Vortex m ix for 30 seconds. 12.26 Attach a 0.2 pm nylon mesh filter to a 3 cc syringe and transfer the sample to this syringe. Filter into a 1.5 mL glass autovial or low-volume autovial when necessary. 12.27 Label the autovial with the study number, animal number and gender, sample timepoint, matrix, final solvent, extraction date, and analyst(s) performing the extraction. 12.28 Cap and store extracts at room temperature or at approximately 4 C until analysis. 12.29 Complete the extraction worksheet, attached to thi s document, and tape in study notebook or include in study binder, as appropriate. 3M Environmental Laboratory ETS-8-6.0 Extraction of PFOS from Liver Page 8 of 14 Page 80 3M Medical Department Study: T-6316.8 BACK TO MAIN Analytical Report: FACT TO X -097 L R N -U 2 4 5 2 13.0 Data Analysis and Calculations__________________________________________ 13.1 C alculations: 13.1.1 Calculate the average density o f the liver homogenate by recording each mass o f ten separate 1.0 mL aliquots o f homogenate. Average density (mg/mL) = Average mass (mg) o f the aliquots 1.0 mL aliquot 13.1.2 Calculate the amount o f liver (mg) per 1.0 mL homogenate (or concentration o f dispersed solid tissue per mL o f homogenate suspension) using the follow ing equation: g o f Liver x Average density* o f homogenate (Tng/mTl (g o f Liver + g o f Water) * refer to 13.1.1 for details. 13.1.3 Calculate actual concentrations o f PFOS and other fluorochemicals in calibration standards using the following equation: U.L o f Standard x Concentration fug AnTT = Final Concentration (pg/g or m g/kg) mg L iver/ 1 mL homogenate* o f PFOS in Liver refer to 13.1.2 for details. 14.0 M e t h o d P e r f o r m a n c e _________________________________________________________________________ 14.1 The method detection lim it (MDL) is analyte and matrix specific. Refer to MDL report for specific MDL and lim it o f quantitation (LOQ) values (refer to Attachm ents B and C). 14.2 The follow ing quality control samples are extracted with each batch o f samples to evaluate the quality o f the extraction and analysis. 14.2.1 Method blanks and matrix blanks. 14.2.2 Matrix spike and matrix spike duplicate samples to determine accuracy and . precision o f the extraction. 14.2.3 Continuing calibration verification samples to determine the continued accuracy o f the initial calibration curve. 14.3 Refer to section 14 o f ETS-8-7.0 for method performance criteria. 15.0 P o l l u t i o n P r e v e n t i o n a n d W a s t e M a n a g e m e n t _____________________________________ ___ 15.1 Sample waste is disposed in biohazard containers, flammable solvent waste is disposed in high BTU containers, and used glass pipette waste: is disposed in broken glass containers located in the laboratory. 3M Environmental Laboratory ETS.-8-6.0 Extraction of PFOS from Liver Page 9 of 14 Page 81 3M Medical Department Study: T-6316.8 BACK TO MAIN Analytical Report: FAC T TO X-097 L R N -U 2 4 52 1 6 .0 R e c o r d s ______________________________________________________________________ ;___________ 16.1 Complete the extraction worksheet attached to this method, and tape in the study notebook or include in the 3-ring study binder, as appropriate. 17.0 T a b l e s . D i a g r a m s . F l o w c h a r t s , a n d V a l i d a t i o n D a t a ________________________________ 17.1 Attachment A, Extraction worksheet 17.2 Attachment B , MDL/LOQ values and summary 17.3 Attachment C, Calibration standard calculation and concentration worksheet 1 8 .0 R e f e r e n c e s ___________________________________________________________ ;__________________________ 18.1 The validation report associated with this method is ETS-8-6.0 & 7 .0-V -l. 18.2 AM DT-EP-22, "Routine Maintenance o f Ultra-Tirrrax T-25" 18.3 FACT-M -1.1, "Extraction o f PFOS or Other Anionic Fluorochemical Surfactants from Liver for Analysis Using HPLC-Electrospray/Mass; Spectrometry" 19.0 A f f e c t e d D o c u m e n t s _________________________________________________________________________ 19.1 ETS-8-7.0, "Analysis o f Liver Extracts for Fluorochemicals using HPLC-Electrospray M ass Spectrometry" 20.0 Revisions R evision Number. Reason For Revision R evision D ate 3M Environmental Laboratory ETS-8-6.0 Extraction of PFOS from Liver Page 10 of 14 Page 82- 3M Medical Department Study: T-6316.8 BACK TO MAIN Analytical Report: FACT TO X-097 L R N -U 2 4 5 2 Studv # ' Matrix Box # W k/D av Date Spiked/Analyst CCV MS MSD Surrogate Std approx, ppm actual ppm # FC M ix Std approx. 0.5 ppm actual ppm # FCM ix Std approx. 5 ppm actual ppm # FC M ix Std approx. 50 ppm actual ppm # Comments Blank Liver Homoeenate: Std# Liver Extraction Method .-.- : 1 Liver amount" -. - - SDikesurrogate and Standard mix. Vortex 15 sec. PiDette 1mL of Liver Solution Pipette 1mL of t0.5 M TBA, pH 10. pH - Std. # PiDette 2 mL of 0.25 NapCOt/0.25M NaHCOt Buffer Std. # Dispense 5ml of Methyl-t-Butyl Ether TN-A- Shake 20 min. Centrifuse 20-25 min. Remove a 4 mL aliquot of organic laver Put on Nitrosen Evaporator to drvness Shaker Speed Centrifutte Speed Evaporator Temperature Add 1.0 mL of Methanol TN-A- . Vortex 30 sec. Filter using a 3cc B-D syringe with a 0.2um SRI filter into autosample vial Cont. Cal. Verifications used the same matrix as for the standard curve. Attachment B: MDL/LOQ Values 3M Environmental Laboratory . ETS-8-6.0 Extraction of PFOS from Liver -` .e Date & Initials , .. .... __ . ... Page 11 of 16 Faye 83 3M Medical Department Study: T-6316.8 BACK TO MAIN Analytical Report: FACT TO X-097 L R N -U 2 4 5 2 M DL/LOQ values for rabbit liver Com pound MDL LOQ Linear C alibration Range (LCR) (PPb) (PPb) A pproxim ate concen trations to be used for preparing the Standard Calibration Curve PFOS 8.45 26.9 30 ppb - 1200 ppb PFOSA 3.50 11.1 12 ppb - 1200 ppb PFOSAA 24.6 78.3 30 p p b - 1200 ppb EtFOSE-OH 108 345 60 ppb - 900 ppb* M 556 82.3 262 60 ppb - 1200 ppb PFOSEA 33.9 108 30 ppb- 1200 ppb MDL/LOQ values in rat, bovine, and monkey liver were not statistically determined. Two curves in each o f these matrices were extracted and anah/zed with the rabbit liver curves to determine equivalence. Responses in the rat, bovine, and monkey liver curves were equivalent to the rabbit responses, therefore, their MDL and LOQ w ill be assumed to be equivalent to those values as determined for the rabbit liver. Refer to LOQ Summary and MDL study in ETS-8-6.0 & 7.0-V-l for further information * EtFOSE-OH estimates only for MDL and LOQ. Did not meet criteria for validation. Com pound: PFOS______________________________________________________ Liver matrix Prepared range of standards (ppb) (ng/mL) Range of average curve (ppb) (ng/mL) LCR from.* avefcurverJ j.) 'I.',;'' !.I V v (ppb) (ng/mL)-- Range of low std curve (ppb) (ng/mL) . ;LGK3roEir;: Range of low;sdd . high std . cupe'; ; - / curve : (ppb)(rig/riL). (ppb) (ng/mL) LCR from high std ; - curve (ppb) (ng/niL); Rabbit 6.19-1237 12 - 1200 12 - 1200/- 6-300 2 ' t 300/ l. 60- 1200 j ;6r 120a Compound: PFOSA Prepared Liver range of matrix standards (ppb) (ng/mL) Rabbit 6.19 -1237 Range of average curve (ppb) (ng/mL) 12 - 1200 LC3t`ftbni. .ave curve-; (ppb) (ng/mL) 12V1200 Range of low std curve (ppb) (ng/mL) 12 - 3C0 ^LGR/frorn. Range of : .low:htd.-i : high std curve (ptib) (lig/rrl.) ; (ppb) (ng/mL) ! 12-300 i 60 - 1200 -LCR from; .hh std . curve;^ ;n' (ppb) (ng/mL)- '60 - 1200 : Compound: PFOSAA Prepared Range of Liver m atrix range of average standards curve (ppb) (ng/mL) (ppb) (ng/mL) Rabbit 6.16- 1232 12-1200 LCR,from ave curve (ppb) (ng/mL) Range of low std curve: (ppb) (ng/mL) 30-1200 ; 30 - 900 LCR fro m Range of lo w std i : high std curve curve (ppb) (ng/mL) (ppb) (ng/mL) 60-900 N/A LCR from, high std curve (ppb) (ng/mL) 'N/A - Attachment B: MDL/LOQ Values 3M Environmental Laboratory ETS-8-6.0 Extraction of PFOS from Liver Page 12 of 16 Page 84 3M Medical Department Study: T-6316.8 BACK TO MAIN Analytical Report: FACT TO X -097 L R N -U 2 4 52 Com pound: EtFOSE-OH Prepared Range of Liver matrix range of average standards curve (ppb) (ng/mL) (ppb) (ng/mL) Rabbit 6.17 - 1235 31-900 LCR from ave curve (ppb) (ng/mL) 31-900 Range cif low stcl curve (ppb) (ng/mL) N/A LCR from low std curve (ppb) (ng/mL) N/A Range of high std curve (ppb) (ng/mL) N/A LCR from high std curve (ppb) (ng/mL) N/A Compound: PFOSEA Prepared Range of Liver range of average matrix standards curve (ppb) (ng/mL) (ppb) (ng/mL) Rabbit 6.17 -1235 31 - 1200 LCR from Range of ave.curve low std curve (ppb). (ng/mL) (ppb) (ng/mL) 31-1200- N/A LCR from low std . curve (ppb) (ng/mL) Range of high std curve (ppb) (ng/mL) N/A- ' N/A LCR from high std curve. " (ppb) (ng/mL) . ' N/A. ' Compound: M556 Prepared Liver range of matrix standards (ppb) (ng/mL) Rabbit 6.17-1235 Range of average curve (ppb) (ng/mL) 31 - 1200 LCRifroni' ave cur: i (ppb) (ng/mL). Range of low std curve (ppb) (ng/:mL) LCRfrom : Range of LCR from lowstd :' high std highstd '/ curve/ ; curve n : curve: (Ppb) (ng/mL) (ppb) (ng/mL) (ppb) (ng/mL) 60rl20:.`- N/A V' n / C ' ' N/A . N/A ' : Attachment C: Standard Calculations ETS-8-6.0 Extraction of PFOS from Liver 3M Environmental Laboratory Page 13 of 14 Page-05 3M Medical Department Study: T-6316.8 BACK TO MAIN Analytical Report: FACT TO X-097 L R N -U 2 4 52 Ion Pair Standard Curves - Tissue Prep date(s): Analyte(s): Sample matrix: M ethod/revision: Target analyte(s): FC mix std approx. 0.500 ppm: FC mix std approx. 5.00 ppm: FC mix std approx. 50.0 ppm: Surrogate std approx. 100 ppm: Standard number: Equipment number: Final solvent and TN: Blank liver/identifier: Actual concentrations of standards in the FC mix PFOS PFOSA PFOSAA EtFOSE PFOSEA Std cone Std cone Std cone Std cone Std cone ug/mL ug/mL ug/mL ug/mL ug/mL 0.500 0.500 0.500 0.500 0.500 0.500 0.500 0.500 0.500 0.500 0.500 0.500 0.500 0.500 0.500 0.500 0.500 0.500 0.500 0.500 0.500 0.500 0.500 0.500 0.500 5.00 5.00 5.00 5.00 5.00 5.00 5.00 5.00 5.00 5.00 5.00 5.00 5.00 5.00 5.00 50.0 50.0 50.0 50.0 50.0 M`556 Std cone ug/mL 0.500 0.500 0.500 0.500 0.500 5.00 5.00 5.00 50.0 Std cone ug/mL All All Ain't spiked Density mL 0.002 g 0.167 0.004 0.167 0.010 0.167 0.020 0.167 0.040 0.167 0.010 . 0.167 0.020 0.167 0.030 0.167 0.004 0.167 C alculated concentrations o f standards in the sam ple m atr : PFOS Final cone PFOSA PFOSAA EtFOSE PFOSEA Final Final cone Final Final cone ng/g cone cone 1M556 Final cone Std cone ng/g ng/g ng/g ng/g ng/g ng/g 5.99 5.99 5.99 5.99 5.99 5.99 12.0 12.0 12.0 12.0 12.0 12.0 29.9 29.9 29.9 29.9 29.9 29.9 59.9 59.9 59.9 59.9 59.9 59.9 120 120 120 120 120 120 299 299 299 299 299 299 599 599 599 599 599 599 898 898 898 898 898 898 1198 1198 1198 1198 1198 1198 Surrogate Std cone ng/mL 100 Surrogate Final cone ng/mL 0.500 All Ain't spiked mL 0.005 Validated ranges --approximate concentrations Liver PFOS PFOSA PFOSAA Rabbit . 5-1000 ppb 5-1000 ppb 5-1000 ppb Bovine ' Estimates only, use rabbit values. Rat Estimates only, use rabbit values. Monkey Estimates only, use rabbit values. EtFOSE-OH 5-1000 ppb POAA 5-1000 ppb PFOSEA 5-1000 ppb Attachment C: Standard Calculations ETS-8-6.0 Extraction of PFOS from Liver 3M Environmental Laboratory Page 14 of 14 Page 80 3M Medical Department Study: T-6316.8 BACK TO MAIN Analytical Report: FACT TO X-097 L R N -U 2 4 5 2 3M Environmental Laboratory M ethod A n a l y sis o f F l u o r o c h e m ic a l s in L iv e r E x tr a c t s U sin g H P L C -E l e c t r o spr a y /M a ss Spe c t r o m e t r y M ethod Num ber: FACT-M-2.0 Author: Lisa Clemen . Approved By: yi Laboratory Manager j/url^ Group Leader -------------- A CJlmu. Technical Reviewer Adoption Date: Revision Date: $ \/\ Date Date f'Jaihir Date 1.0 Scope and Appucation_________ ;_______________ :_________________ ;____________ __ 1.1 Scope: This method is for the analysis o f extracts of liver or other tissues for fluorochemical surfactants using HPLC-electrospray/mass spectrometry. 1.2 A pplicable Com pounds: Potassium perfluorooctanesulfonate, anionic fluorochemical surfactants, or other ionizable compounds. 1.3 M atrices: Rabbit, rat, bovine, and monkey livers or other livers as designated in the validation report. Word 7.0.1/95 3M Environmental Laboratory FACT-M-2.0 Analysis of Liver Extract Using ES/MS Pagel o f8 Page-87- 3M Medical Department Study: T-6316.8 BACK TO MAIN Analytical Report: F A C T T O X -097 L R N -U 2 4 5 2 2.0 Summary of Method______________________________________________________ "" 2.1 This method describes the analysis o f fluorochemical surfactants extracted from liver using HPLC-electrospray/mass spectrometry. The analysis is performed by monitoring a single ion characteristic o f a particular fluorochemical, such as the potassium perfluorooctanesulfonate (PFOS) anion, M/Z= 499. Samples may also be screened to verify compound identification. 3.0 Definitions______________________________________________________________________ 3.1 N one. 4.0 W arnings and Cautions_____________________________________ ______________ 4.1 H ealth and Safety W arnings: 4.1.1 U se 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 o f electrical shock. 4.2 Cautions: 4.2.1 Do not run solvent pumps above capacity o f 400 bar (5800 psi). If pressure goes over 400 bar, the HP 1100 w ill initiate automatic shutdown. 4.2.2 Do not rim solvent pumps to dryness. 5.0 Interferences__________________ ______________________________________ ,_________ 5.1 Teflon should not be used for sample storage or any part o f instrumentation that com es in contact with the sample or extract. 6.0 E quipment_________________________ _____________________________________________ 6.1 Equipment listed below may be changed in order to optimize the system. 6.1.1 Micromass Electrospray Mass Spectrometer 6.1.2 HP 1100 low pulse solvent pumping system and autosampler. 7.0 Supplies and M aterials__________________________;_______________________________ 7.1 Supplies 7.1.1 Nitrogen gas, refrigerated 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 mL centrifuge tubes. 8.0 Reagents and Standards_______;_________________________________________________ 8.1 R eagents 8.1.1 Methanol, HPLC grade or equivalent. Word 7.0.1/95 FACT-M-2.0 Analysis of Liver Extract Using ES/MS Page 2 of 8 3M Environmental Laboratory Page 88- 3M Medical Department Study: T-6316.8 BACK TO MAIN Analytical Report: FACT TO X-097 L R N -U 2 4 5 2 8.1.2 Milli-QTM water, all water used in this method should be Milli-QTM water and may be provided by a M illi-Q TOC Plus system. 8.1.3 Ammonium acetate, HPLC grade or equivalent. 8.2 Standards 8.2.1 Typically one H20 blank, one liver blank, and seven liver standards are prepared during the extraction procedure. See FACT-M-1. 9.0 Sample H andling____________________'_______________________________________ ' 9.1 Fresh liver standards are prepared with each analysis. Extracted standards and samples are stored in capped autovials or capped 15 mL centrifuge tubes until analysis. 9.2 I f analysis w ill be delayed, extracted standards and samples may be refrigerated until analysis can be performed. 10.0 Quality Control_______ .___________________________________________ ^___________ 10.1 M atrix Blanks and M ethod Blanks 10.1.1 Analyze a method blank and matrix blank prior to each calibration curve. 10.2 M atrix Spikes 10.2.1 Analyze a matrix spike and matrix spike duplicate with each analysis. 10.2.2 Expected concentrations w ill fall in the mid-range o f the initial calibration curve. Additional spike concentrations may fall in the low-range o f the initial calibration curve. 10.2.3 See section 13 to calculate percent recovery. 1 0 3 C ontinuing C alibration Checks 1 0 3 .1 Analyze a mid-range calibration standard after every tenth sample. If a significant change ( 30%) in peak area occurs, relative to the initial standard curve, stop the run. Only those samples analyzed before the last acceptable calibration standard w ill 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 peal: shape, etc.) w ill be assessed for each run. 11.0 Calibration and Standardization__________________________________________ ___ 11.1 Analyze the extracted liver standards prior to and following each set o f extracts. The mean o f two standard values, at each standard concentration, w ill be plotted by linear regression for the calibration curve using MassLynx or other suitable software. 3M Environmental Laboratory FACT-M-2.0 Analysis of Liver Extract Using ES/MS Page 3 of 8 3M Medical Department Study: T-6316.8 BACK TO MAIN Analytical Report: FACT TO X -097 L R N -U 2 4 5 2 11.2 The r2 value for the data should be 0.98 or greater. Lower values may be acceptable at the discretion o f the analyst. 11.3 If the curve does not meet requirements, perform routine maintenance or reextract the standard curve (if necessary) and reanalyze. 12.0 P rocedures_____________________________________ '_______________________________ 12.1 A cquisition Set up 12.1.1 Click on start button in the Acquisition Control Panel. Set up a sample list. A ssign a filename using letter-MO-DAY-last digit o f year-sample number, assign a method (M S) 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. 12.1.3 Typically the sample list begins with the first set o f liver standards and ends with the second set o f standards. 12.1.4 Samples are analyzed with a continuing calibration check injected after every tenth sample. Solvent blanks should be analyzed periodically to monitor possible analyte carryover and are not considered samples but may be included as such. 12.2 U sing the Autosam pler 12.2.1 Set up sample tray according to the sample list prepared in section 12.1.1. 12.2.2 Set-up the HP1100/autosampler at the following conditions or at conditions the analyst considers appropriate for optimal response. Record actual conditions in the instrument logbook: 12.2.2.1 Sample size = 10 pL injection with a sample wash 12.2.2.2 Inject/sample = 1 1 2 .2 .2 3 Cycle time = 15 minutes 1 2 3 .2 .4 Solvent ramp = Time MeOH 0.00 min. 7.5 min. 11.0 min. 11.5 min. 45% 90% 90% 45% 2.0 mM Ammonium acetate 55% 10% 10% 55% Note: In this instrument configuration, the run must be set up on the electrospray software with a "Waiting for inlet start" message before the "Start" button is pressed on the HP Workstation. 12.2.2.5 Press the "Start" button. 3M Environmental Laboratory FACT-M-2.0 Analysis of Liver Extract Using ES/MS Page 4 o f 8 Paye 90 3M Medical Department Study: T-6316.8 BACK TO MAIN Analytical Report: FACT TO X -097 L R N -U 2 4 5 2 12.3 Instrum ent Sep-up ' 12.3.1 Refer to AM DT-EP-31 for more details. 1 2 3 .2 Check the solvent level in reservoirs and refill if necessary. 12.3.3 Check the stainless steel capillary at the end o f the probe. U se an eye piece to check the tip. The tip should be flat with no jagged edges. If the tip is found to be unsatisfactory, disassemble the probe and replace the stainless steel capillary. 12.3.4 Set HPLC pump to "On". Set the flow to 10 - 500 uL/min or as appropriate. Observe droplets coming out o f the tip o f the probe. A llow to equilibrate for approximately 10 minutes. 1 2 3 .5 Turn on the nitrogen. A fine mist should be expelled with no nitrogen leaking around the tip o f the probe. 12.3.6 The instrument uses these parameters at the following settings. These settings may change in order to optimize the response: 12.3.6.1 Drying gas 250-400 liters/hour 1 2 3 .6 .2 ESI nebulizing gas 10-15 liters/hour 1 2 3 .6 .3 LC constant flow mode flow rate 10 - 500 uL/min 1 2 3 .6 .4 Pressure <400 bar (This parameter is not set, it is a guide to ensure the instrument is operating correctly.) 12.3.7 Carefully guide the probe into the opening. Insert probe until it w ill not go any further. Connect the voltage cables to the probe. 1 2 3 .8 Record tune parameters in the instrument log. 1 2 3 .9 U sing the cross-flow counter electrode in the ES/MS source is recommended for the analysis o f biological matrices. 1 2 3 .1 0 Click on start button in the Acquisition Control Panel. Press the start button at top o f sample list. Ensure start and end sample number includes all samples to be analyzed. 13.0 Data Analysis and Calculations______________________________________________ 13.1 Calculations: 13.1.1 Calculate matrix spike percent recoveries using the following equation: % Recovery = Observed Result - Background Result x 100 Expected Result 13.1.2 Calculate percent difference using the following equation: % Difference = Expected Cone. - Calculated Cone, x 100 Expected Cone. 3M Environmental Laboratory FACT-M-2.0 Analysis of Liver Extract Using ES/MS Page 5 of 8 Page-91 3M Medical Department Study: T-6316.8 BACK TO MAIN Analytical Report: FAC T TO X -097 L R N -U 2 4 5 2 13.1.3 Calculate actual concentration o f PFOS anion in total liver (mg): ' ug PFOS anion calc, from std curve' ^ g o f liver used for analysis j 1000 u g /1 mg x Total mass o f liver (g) 14.0 M eth o d P erform ance_____________________________________________ ;____________ 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 lim it is equal to the lowe st standard in the calibration curve. 15.0 P o llu tio n P revention and W aste M anagem ent________________________________ 15.1 Sample waste is disposed in biohazard containers, flammable solvent waste is disposed in high BTU containers, and glass pipette waste is disposed in broken glass containers. A ll containers are located in the laboratory. 16.0 R ecords_____________________._______________________________________ ;__________ 16.1 Store chromatograms in the study folder. Each chromatogram should have the follow ing information included either in the header or hand written on the chromatogram: study number, sample name, extraction date, and dilution factor (if applicable). 16.2 Plot calibration curve by linear regression and store in the study folder. 16.3 Print sample list from MassLynx and tape into the instrument runlog. 16.4 Print data integration summary from MassLynx and tape into the instrument runlog. 16.5 Copy instrument runlog pages, including instrument parameters and sample results, and tape into appropriate study notebook. 16.6 Summarize datausing 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 o f backup electronic data. 17.0 T a b l e s , D i a g r a m s , F l o w c h a r t s , a n d V a l i d a t i o n D a t a _________________ 17.1 Attachment A: FACT-M-2 Data reporting spreadsheet 17.2 The validation report associated with this method is FACT-M-1.0 & 2.0-V -l. 18.0 R e f e r e n c e s ______________________________________________________________________ 18.1 AM DT-EP-31, "Operation o f VG Platform Electrospray Mass Spectrometer" 3M Environmental Laboratory FACT-M-2.0 Analysis of Liver Extract Using ES/MS Page 6 of 8 Page 92 3M Medical Department Study: T-6316.8 BACK TO MAIN Analytical Report: FACT TO X-097 L R N -U 2 4 52 19.0 Affec ted Documents___________________________________________________ 19.1 FACT-M -1.0, "Extraction o f Potassium Perfluorooctanesulfonate from Liver for Analysis U sing HPLC-Electrospray/Mass Spectrometry" 20.0 Revisions R evision Number. Reason For Revision R evision D ate 3M Environmental Laboratory FACT-M-2.0 Analysis of Liver Extract Using ES/MS Page 7 of 8 Page-93 3M Medical Department Study: T-6316.8 BACK TO MAIN Analytical Report: FACT TO X -097 L R N -U 2 4 5 2 Laboratory Study # Study: Test Material: Matrix/Final Solvent: Method/Revision: Analytical Equipment System Number Instrument Software/Version: Filename: R-Squared Value: Slope: Y Intercept: Date of Extraction/Analyst Date of Analysis/Analyst: Group Dose Sample# Concentration ug/mL Initial VoL mL Dilution Factor Final Cone. ug/mL Slope: Taken from linear regression equation. Group/Dose: Taken from the study folder. Sample#: Taken from die study folder. Concentration (ug/mL): Taken from the MassLynx integration summary. Initial Volume (mL): Taken from the study folder. Dilution Factor: Taken from the study folder. Final Cone. (ug/mL): Calculated by dividing the initial volume from the concentration 3M Environmental Laboratory FACT-M-2.0 Analysis of Liver Extract Using ES/MS Page 8 o f 8 Page 94 3M Medical Department Study: T-6316.8 BACK TO MAIN Analytical Report: FACT TO X -097 l|R N -U 2452 3M Environmental Laboratory Method Analysis of Fluorochemicals in Serum Extracts Using HPLC-Electrospray/Mass Spectrometry M ethod Num ber: FACT-M -4.0 A doption Date: Hj l l j $ Author: Lisa Clemen R evision Date: fjjft \ Approved By: , - j%,_________;______ j / W 9 f Laboratory Manager Date Group Leader Technical Reviewer Date hJj h H s Date 1.0 S c o p e a n d A p p l i c a t i o n _________________________________________________________ ;________________ 1.1 Scope: This method is for the analysis o f extracts o f serum or tissue for fluorochemical surfactants using HPLC-electrospray/mass spectrometry. 1.2 A pplicable Com pounds: Potassium perfluorooctanesulfonate, anionic fluorochem ical surfactants, or other ionizable compounds. 1 3 M atrices: Rabbit, rat, and bovine serum or other sera as designated in the validation report. Word 7.0.1/95 3M Environmental Launratury FACT-M -4.0 Analysis o f Serum Extract U sing ES/MS Page 1 o f 8 Page 95 3M Medical Department Study: T-6316.8 BACK TO MAIN Analytical Report: FACT TO X -097 L R N -U 2 4 52 2.0 S u m m a r y o f M e t h o d ___________________________________________________________________ ;_________ 2.1 This method describes the analysis o f fluorochemical surfactants extracted from serum using HPLC-electrospray/mass spectrometry. The analysis is performed by monitoring a single ion characteristic o f a particular fluorochemical, such as the potassium perfluorooctanesulfonate (PFOS) anion, M/Z= 499. Samples may also be screened to verify compound identification. 3 .0 D e f i n i t i o n s ________________________'_________________________________________________________________ 3.1 N one. . 4.0 W a r n i n g s a n d C a u t i o n s _________________________________________________________________________ 4.1 Health and Safety Warnings: 4.1.1 U se 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 o f electrical shock. 4.2 Cautions: 4.2.1 Do not run solvent pumps above capacity o f 400 bar (5800 psi). I f pressure goes over 400 bar, the HP 1100 w ill initiate automatic shutdown. 4.2.2 Do not run solvent pumps to dryness. 5.0 I n t e r f e r e n c e s ____________________________________________________________________________________ 5.1 Teflon should not be used for sample storage or any part o f instrumentation that com es in contact with the sample or extract. 6.0 E q u i p m e n t _____________________________;____________________________________________________________ 6.1 Equipment listed below may be changed in order to optimize the system. 6.1.1 Micromass Electrospray Mass Spectrometer 6.1.2 HP 1100 low pulse solvent pumping system and autosampler. 7.0 S u p p l i e s a n d M a t e r i a l s ________________________________________________________________________ 7.1 Supplies 7.1.1 Nitrogen gas, refrigerated 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 mL centrifuge tubes. 8.0 R e a g e n t s a n d S t a n d a r d s ______________________________________________________________________ 8.1 Reagents 8.1.1 M ethanol, HPLC grade or equivalent. 3M Environmental Laboratory FACT-M-4.0 Analysis of Serum Extract Using ES/MS Page 2 of 8 Page 96 3M Medical Department Study: T-6316.8 BACK TO MAIN Analytical Report: FACT TO X -097 L R N -U 2 4 5 2 8.1.2 Milli-QTM water, all water used in this method should be Milli-QTM water and may be provided by a M illi-Q TOC Plus system. 8.1.3 Ammonium acetate, HPLC grade or equivalent. 8.2 Standards 8.2.1 Typically one H20 blank, one serum blank, md seven serum standards are prepared during the extraction procedure. See FACT-M-3. . 9 . 0 S a m p l e H a n d l i n g _________________________________________________________________________________ 9.1 Fresh serum standards are prepared with each analysis. Extracted standards and samples are stored in capped autovials or capped 15 mL centrifuge tubes until analysis. 9.2 I f analysis w ill be delayed, extracted standards and samples may be refrigerated at 4 C until analysis can be performed. 10.0 Q u a l i t y C o n t r o l _________ ;____________________________________________________________________ 10.1 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 each analysis. . 10.2.2 Expected concentrations w ill fall in the mid-range o f the initial calibration curve. Additional spike concentrations may fall in the low-range o f the initial calibration curve. 10.2.3 See section 13 to calculate percent recovery. 10.3 Continuing Calibration Checks 10.3.1 Analyze a mid-range calibration standard after every tenth sample. If a significant change ( 30%) in peak area occurs, relative to the initial standard curve, stop the run. Only those samples analyzed before the last acceptable calibration standard w ill 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.) w ill be assessed for each run. 1 1 .0 C a l i b r a t i o n a n d S t a n d a r d i z a t i o n _________________________________________________________ 11.1 Analyze the extracted serum standards prior to and following each set o f extracts. The mean o f two standard values, at each standard concentration, w ill 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 ES/MS Page 3 of 8 Page 97 3M M edical D epartm ent Study: T-6316.8 BACK TO MAIN Analytical Report: FACT TO X -097 L R N -U 2 4 52 11.2 The r2 value for the data should be 0.98 or greater. Lower values may be acceptable at the discretion o f the analyst. 11.3 If the curve does not m eet requirements, perform routine maintenance or reextract the standard curve (if necessary) and reanalyze. 12.0 P r o c e d u r e s _______________________________________________________________________________________ 12.1 A cquisition Set up 12.1.1 Click on start button in the Acquisition Control Panel. Set up a sample list. Assign a filenam e using letter-MO-DAY-last digit o f year-sample number, assign a method (M S) 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). Set Ionization Mode as appropriate and mass to 499 or other appropriate m asses. A scan is usually collected along with the SIRs. Save method. 12.1.3 Typically the sample list begins with the first set o f serum standards and ends with the second set o f standards. 12.1.4 Samples are analyzed with a continuing calibration check injected after every tenth sample. Solvent blanks should be analyzed periodically to monitor possible analyte carryover and are not considered samples buit may be included as such. 12.2 U sing the A utosam pler 12.2.1 Set up sample tray according to the sample list prepared in section 12.1.1. 12.2.2 Set-up the HP1100/autosampler at the following conditions or at conditions the analyst considers appropriate for optimal response. Record actual conditions in the instrument logbook: 12.2.2.1 Sample size = 10 pL injection with a sample wash 12.2.2.2 Inject/sample = 1 12.2.2.3 Cycle time = 15 minutes 12.2.2.4 Solvent ramp = Time 0.00 min. 7.5 min. 11.0 min. 11.5 min. MeOH 45% 90% 90% 45% 2.0 mM Ammonium acetate 55% 10% 10% 55% N ote: In this instrument configuration, the run must be set up on the electrospray software with a "Waiting for inlet start" message before the "Start" button is pressed on the HP Workstation. 12.2.2.5 Press the "Start" button. FACT-M-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 TO X -097 L R N -U 2 4 5 2 12.3 Instrument Set-up 12.3.1 Refer to AM DT-EP-31 for more details. 12.3.2 Check the solvent level in reservoirs and refill if necessary. 12.3.3 Check the stainless steel capillary at the end o f the probe. U se an eye piece to check the tip. The tip should be flat with no jagged edges. Ifth e tip isfo u n d to b e unsatisfactory, disassemble the probe and replace the stainless steel capillary. 12.3.4 Set HPLC pump to "On". Set the flow to 10 - 500 uL/min or as appropriate. Observe droplets coming out o f the tip o f the: probe. A llow to equilibrate for approximately 10 minutes. 1 2 3 .5 Turn on the nitrogen. A fine mist should be expelled with no nitrogen leaking around the tip o f the probe. 12.3.6 The instrument uses these parameters at the following settings. These settings may change in order to optimize the response: 12.3.6.1 Drying gas 250-400 liters/hour 12.3.6.2 ESI nebulizing gas 10-15 liters/hour 1 2 .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 w ill not go any further. Connect the voltage cables to the probe. 1 2 3 .8 Record tune parameters in the instrument log. 1 2 3 .9 Using the cross-flow counter electrode in the ES/MS source is recommended for the analysis o f biological matrices. 12.3.10C lick on start button in the Acquisition Control Panel. Press the start button at top o f sample list. Ensure start and end sample number includes all samples to be analyzed. 1 3 .0 D a t a A n a l y s i s a n d C a l c u l a t i o n s __________________________________________ ________________ 13.1 Calculations: 13.1.4 Calculate matrix spike percent recoveries using the follow ing equation: % Recovery = Observed Result - Background Result x 100 Expected Result 13.1.5 Calculate percent difference using the following equation: % Difference = Expected Cone. - Calculated Cone, x 100 Expected Cone. 3M Environmental 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: F A C T T O X -097 L R N -U 2 4 5 2 13.1.6 Calculate actual concentration o f PFOS, or ether fluorochemical, anion in serum (pg/m L ): qg o f PFO calc, from std. Curve x Dilution Factor x Final Volume (mL) Initial Volume o f serum (mL) 1 4 .0 M e t h o d P e r f o r m a n c e _______________________________________________ ;_________________________ 14.1 The method detection lim it is equal to half the lowest standard in the calibration curve. 14.2 The practical quantitation lim it is equal to the lowest standard in the calibration curve. 1 5 .0 P o l l u t i o n P r e v e n t i o n a n d W a s t e M a n a g e m e n t _________________________________________ 15.1 Sample extract waste and flammable solvent is dispased in high BTU containers, and glass pipette waste is disposed in broken glass containers located in the laboratory. 1 6 .0 R e c o r d s ________________'______________________________________________________________ ;___________ 16.1 Store chromatograms in the study folder. Each chromatogram must have the follow ing information included either in the header or hand written on the chromatogram: study number, sample name, extraction date, and dilution factor (if applicable). 16.2 Plot calibration curve by linear regression and store in the study folder. 16.3 Print sample list from MassLynx and tape into the instrument runiog. 16.4 Print data integration summary from MassLynx and tape into the instrument runiog. 16.5 Copy instrument runiog pages, including instrument parameters and sample results, and tape into appropriate study notebook. 16.6 Summarize data using suitable software and store in the study folder. 16.7 Back up electronic data to appropriate medium. Record in study notebook the file name and location o f backup electronic data. 1 7 .0 Tables. Diagrams. Flowcharts, and Validation Data________________________ 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 -l. 18.0 References________________________________________________________________ 18.1 AM DT-EP-31, "Operation o f VG Platform Electrospray Mass Spectrometer" 1 9 .0 A f f e c t e d D o c u m e n t s _________________________________________________________________________ 19.1 FACT-M -3.0, "Extraction o f Fluorochemical Anions from Serum for Analysis Using HPLC-Electrospray/Mass Spectrometry" 3M Environmental Laboratory FACT-M-4.0 Analysis of Serum Extract Using ES/MS Page 6 of 8 Page 100 BACK TO MAIN 3M M edical D epartm ent Study: T-6316.8 * Analytical Report: F A C T T O X -0 9 7 L R N -U 2 4 5 2 ' 20.0 Revisions___________________________________________________________________ R ev isio n Number. Reason For Revision R ev isio n Date 3M Environm ental Laboratory FA C T -M -4.0 Analysis o f Serum Extract Using ES/MS Page 7 o f 8 P age 101 3M Medical Department Study: T-6316.8 BACK TO MAIN Analytical Report: FACT TO X -097 L R N -U 2 4 5 2 Laboratory Study # Study: Test Material: Matrix/Final Solvent: Method/Revision: Analytical Equipment System Number: Instrument Software/Version: Filename: R-Squared Value: Slope: Y Intercept: Date of Extraction/Analyst: Date of Analysis/Analyst: Group Dose Sample# Concentration ug/mL Initial Vol. mL Dilution Factor Final Cone. ug/mL Slope: Taken from linear regression equation. Group/Dose: Taken from the study folder. Sample#: Taken from the study folder. Concentration (ug/mL): Taken from the MassLynx integration summary. Initial Volume (mL): Taken from the study folder. Dilution Factor: Taken from the study folder. Final Cone. (ug/mL): Calculated by dividing the initial volume from the concentration 3M Environmental Laboratory FACT-M-4.0 Analysis of Serum Extract Using ES/MS Page 8 of 8 Page 102 3M Medical Department Study: T-6316.8 BACK TO MAIN Analytical Report: FAC T TO X-097 L R N -U 2 4 5 2 3M Environmental Laboratory Method Analysis of Potassium Perfluorooctanesulfonate or Other Fluorochemicals in Serum Extracts Using HPLC-Electrospray/Mass Spectrometry M ethod N um ber: ETS-8-5.1 Author: Lisa Clemen, Robert Wynne Approved By: Laboratory Manager A doption D ate: 03/01/99 R evision D ate: `i f V ^/ D ate Group Leader A _________________________________ Technical Reviewer D ate Date 1 . 0 S c o p e a n d A p p l i c a t i o n _________________________________________________________________________ _ 1.1 Scope: This method describes the analysis o f serum extracts for fluorochemical surfactants using HPLC-electrospray/mass spectrometry. 1.2 A pplicable Com pounds: Fluorochemical surfactants or other fluorinated compounds, or other ionizable compounds. 1.3 M atrices: Rabbit, rat, bovine, monkey, and human serum, or other fluids as designated in the validation report. Word 6/95 ETS-8-5.1 Analysis of Serum Extract Using ES/MS Page 1 of 9 3M Page 103 3M Medical Department Study: T-6316.8 BACK TO MAIN Analytical Report: FAC T TO X -097 L R N -U 2 4 5 2 2 .0 Summary o f M eth o d ______ _________________________ ________________ .______ 2 .1 This method describes the analysis o f fluorochemical surfactants extracted from serum or other fluids, using HPLC-electrospray/mass spectrometry, or sim ilar system as appropriate. The analysis is performed by monitoring a single ion characteristic o f a particular fluorochem ical, such as the perfluorooctanesulfonate (PFOS) anion, m /z= 499. Additionally, samples may be analyzed using a tandem mass spectrometer to further verify the identity o f a compound by detecting daughter ions o f the parent ion. 3 .0 D efin itio ns____________________________ ;_________________________________________ 3 .1 Atmospheric Pressure Ionization (API): The Micromass Quattro II triple quadrupole system s allow for various methods o f ionization by utilizing various sources, probes, and interfaces. These include but are not limited to: Electrospray Ionization (ESI), Atmospheric Pressure chem ical Ionization (APcI), Thermospray, etc:. The ionization process in these techniques occurs at atmospheric pressure (i.e., not under a vacuum). 3 .2 Electrospray Ionization (ES, ESI): a method o f ionization performed at atmospheric pressure, whereby ions in solution are transferred to the gas phase via tiny charged droplets. These charged droplets are produced by the application o f a strong electrical field. 3 .3 Mass Spectrometry, Mass Spectrometer (MS), Tandem Mass Spectrometer (MS/MS): The API Quattro II triple quadrupole systems are equipped with quadrupole m ass selective detectors. Ions are selectively discriminated by mass to charge ratio (m /z) and subsequently detected. A single MS may be employed for ion detection or a series (M S/M S) for more specific fragmentation information. 3 .4 Conventional vs. Z-spray probe interface: The latest models o f Micromass Quattro II triple quadrupole system s (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 cotifiguration is different, the methods o f operation, cleaning, and maintenance are the same. However, Z-spray components and conventional components are not com patible with one another, but only with similar systems (i.e., Z-spray components are compatible with some other Z-spray systems, etc.) 3 .5 M ass L ynx Softw are: System software designed for the specific operation o f these Quattro II triple quadrupole system s. Currently MassLynx has Windows 95 and W indowsNT 4.0 versions. A ll versions are similar. For more details see the manual specific to the instrument (M icromass Quattro II triple quadrupole MassLynx or MassLynx NT U ser's Guide). 4.0 Warnings and Cautions____________________________________________________ 4.1 H ealth and Safety W arnings: 4.1.1 U se caution with the voltage cables for the probe. When engaged, the probe em ploys a voltage o f approximately 5000 Volts. 4.1.2 When handling samples or solvents wear appropriate protective gloves, eyewear, and clothing. ETS-8-5.1 Analysis of Serum Extract Using ES/MS Page 2 of9 3M Page 104 3M Medical Department Study: T-6316.8 BACK TO MAIN Analytical Report: FAC T TO X-097 L R N -U 2 4 5 2 4.2 Cautions: 4.2.1 Do not operate solvent pumps above capacity o f 400 bar (5800 psi) back pressure. If the back pressure exceeds 400 bar, the HP 1100 w ill initiate automatic shutdown. 4.2.2 Do not run solvent pumps to dryness. 5.0 INTERFERENCES____________________________________________________________ .__________ 5.1 To m inim ize interferences when analyzing samples, teflon should not be used for sample storage or any part o f instrumentation that com es in contact w ith the sample or extract. 6.0 Equipment ____________________________________________________________ _ 6.1 Equipment listed below may be modified in order to optimize the system . Document any m odifications in the raw data as method deviations. 6.1.1 6.1.2 M icromass Quattro n triple quadrupole Mass Spectrometer equipped with an electrospray ionization source H P1100 low pulse solvent pumping system , solvent degasser, column compartment, and autosampler 7.0 Supplies and Ma t e r i a l s ___________________ :________________________ 7.1 Supplies 7.1.1 High purity grade nitrogen gas regulated to approximately 100 psi (H ouse air system ) 7.1.2 HPLC analytical column, specifics to be determined by the analyst and documented in the raw data. 7.1.3 Capped autovials or capped 15 mL centrifuge tubes 8.0 Reagents and Standards______________________________ ____________________ 8.1 Reagents 8.1.1 Methanol, HPLC grade or equivalent 8.1.2 M illi-QTM water, all water used in this method should be Milli-QTM water or equivalent, and may be provided by a M illi-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 Sample Handling____________________ ;__________________________ ______________ 9.1 Fresh matrix standards are prepared with each analysis. Extracted standards and samples are stored in capped autovials or capped 15 mL centrifuge tubes until analysis. 3M EnvironmeiUdl Laboratory ETS-8-5.1 Analysis of Serum Extract Using ES/MS Page 3 o f 9 Page 105 3M Medical Department Study: T-6316.8 BACK TO MAIN Analytical Report: F A C T T O X -097 L R N -U 2 4 52 9.2 I f analysis w ill be delayed, extracted standards and samples can be refrigerated at approximately 4 C, or at room temperature, until analysis can be performed. 1 0 .0 Q u a l i t y C o n t r o l __________ ;______________________________________________________________ 10.1 Solvent B lanks, M ethod Blanks and M atrix Blan ks 10.1.1 Solvent blanks, method blanks and matrix blanks are prepared and analyzed with each batch to determine contamination or carryover. 10.1.2 Analyze a method blank and a matrix blank prior to each calibration curve. 10.2 M atrix 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 fo rt/ samples, w ith a minimum o f 2 spikes per batch. 10.2.4 Matrix spike and matrix spike duplicate concentrations w ill fall in the mid-range o f the initial calibration curve. Additional spike concentrations may fall in the low range o f the initial calibration curve. 10.3 C ontinuing C alibration V erifications 10.3.1 Continuing calibration verifications are analyzed to verify the continued accuracy o f the calibration curve. ' 10.3.2 Analyze a mid-range calibration standard after every tenth sample, with a minimum o f one per batch. 1 1 . 0 C a l i b r a t i o n a n d S t a n d a r d i z a t i o n __________________________________________________________ 11.1 A nalyze the extracted matrix standards prior to and following each set o f extracts. The average o f two standard curves w ill be plotted by linear regression (y = my + b), weighted 1/x, not forced through zero, using MassLynx or other suitable software. 11.2 If the curve does not meet requirements, perform rouline maintenance or reextract the standard curve (if necessary) and reanalyze. 11.3 For purposes o f accuracy when quantitating low levels o f analyte, it may be necessary to use the low end o f the calibration curve rather than the full range o f the standard curve. Example: when attempting to quantitate approximately 10 ppb o f analyte, generate a calibration curve consisting o f file standards from 5 p pb to 100 ppb rather than the full range o f the curve (5 ppb to 1000 ppb). This w ill reduce inaccuracy attributed to linear regression weighting o f high concentration standards. 3M'EnvirunTiientat Laboratory ETS-8-5.1 Analysis of Serum Extract Using ES/MS Page 4 of 9 Page 106 3M Medical Department Study: T-6316.8 BACK TO MAIN Analytical Report: FACT TO X-097 L R N -U 2 4 52 12.0 P rocedures ________________________ ;_____________________________________ _ 12.1 A cquisition Set up 12.1.1 Click on start button in the Acquisition Conlrol Panel. Set up a sample list. A ssign a filenam e using MO-DAY-last digit o f year-sample number, assign a method (M S) 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 em ployed, additional product ion fragmentation information may be collected. See Micromass MassLynx GUIDE TO DATA ACQUISITION for additional information and MRM (M ultiple Reaction Monitoring). 12.1.3 Typically the analytical batch run sequence begins with a set o f extracted matrix standards and ends with a set o f extracted matrix standards. 12.1.4 Samples aire analyzed with a continuing calibration check injected after every tenth sample. Solvent blanks should be analyzed periodically to monitor possible analyte carryover and are not considered samples but may be included as such. 12.2 U sing the A utosam pler 12.2.1 Set up sample tray according to the sample list prepared in Section 12.1.1. 12.2.2 Set-up the HP1100/autosampler at the follow ing conditions or at conditions the analyst considers appropriate for optimal response. Record actual conditions in the instrument logbook: 12.2.2.1 Sample size = 10 pL injection 12.2.2.2 Inject/sample = 1 12.2.2.3 Cycle time = 13.5 minutes 12.2.2.4 Solvent ramp = Time 0.00 min. 8.50 min. 11.0 min. 12.0 min. MeOH 40% 90% 90% 40% 2.0 nM .Ammonium acetate 60% 10% 10% 60% 12.2.2.5 Press the "Start" button. 12.3 Instrum ent Set-up 12.3.1 Refer to E T S-9-24.0 for more details. 12.3.2 Check the solvent level in reservoirs and refill if necessary. 3M ~E rivIroTii i lei ita l Ldlroiato iy ETS-8-5.1 Analysis of Serum Extract Using ES/MS Page 5 of 9 Page 107 3M Medical Department Study: T-6316.8 BACK TO MAIN Analytical Report: FACT TO X-097 L R N -U 2 4 52 12.3.3 Check the stainless steel capillary at the end o f the probe. Use an eyepiece to check the tip. The tip should be flat with no jagged edges. If the tip is found to be unsatisfactory, disassemble the probe and replace the stainless steel capillary. 12.3.4 Set HPLC pump to "On". Set the flow to 10 - 500 uL/min or as appropriate. Observe droplets coming out o f the tip o f the probe. Allow to equilibrate for approximately 10 minutes. 12.3.5 Turn on the nitrogen. A fine mist should be expelled with no nitrogen leaking around the tip o f the probe. Readjust the tip o f the probe if no m ist is observed. 12.3.6 The instrument uses these parameters at the following settings. These settings may change in order to optimize the response: 12.3.6.1 Drying gas 250-400 liters/hour 12.3.6.2 ESI nebulizing gas 10-15 liters/hour 12.3.6.3 HPLC constant flow mode, flow rate 10 - 500 pL/min 12.3.6.4 Pressure <400 bar (This parameter is not set, it is a guide to ensure the HPLC is operating correctly.) 12.3.7 Carefully guide the probe into the opening. Insert probe until it w ill 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 w ith a copy taped into the instrument log. 12.3.9 U sing the cross-flow counter electrode in the ES/MS source is recommended for the analysis o f biological matrices. 12.3.10C lick on start button in the Acquisition Control Panel (this may vary among M assLynx versions, see appropriate M assLyrx USER'S GUIDE). Press the start button. Ensure start and end sample number includes all samples to be analyzed. 13.0 Data Analysis and Calculations_______________ __________________________ 13.1 Calculations: 13.1.4 Calculate matrix spike percent recoveries using the following equation: % Recovery = Observed Result - Background Result x 100 Expected Result 13.1.5 Calculate percent difference using the following equation: % D ifference = Expected Cone. - Calculated Cone, x 100 Expected Cone. 13.1.6 Calculate actual concentration o f PFOS, or other fluorochemical, in matrix (p g/m L ): (ng o f PFOS calc, from std. Curve x Dilution Factor-) x 1 ua /Initial Volume o f matrix (m D + mL o f Surrogate Standard! 1000 ng Final Volume (mL) ETS-8-5.1 Analysis of Serum Extract Using ES/MS Page 6 of 9 3M~Eiiviiuiinibiital Laboratory Page 108 3M Medical Department Study: T-6316.8 BACK TO MAIN Analytical Report: FACT TO X -097 L R N -U 2 4 5 2 14.0 M ethod P e r f o r m a n c e ____________________________ _______________________ 14.1 Method Detection Limit (MDL) and Limit o f Quantitation (LOQ) are method, analyte, and matrix specific. Please see ETS-8-4.1, Attachm ent B, for a listing o f current validated MDL and LOQ values. 14.2 Solvent Blanks, Method Blanks, and Matrix Blanks 14.2.1 Solvent blanks, method blanks, and matrix blanks values are must be below the low est standard in the calibration curve 14.3 Calibration Curves 14.3.1 The r2 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 30% o f the spiked concentration. 14.5 Continuing Calibration Verifications 14.5.1 Continuing calibration verification percent recoveries must be 30% o f the spiked concentration. 14.6 I f criteria listed in this method performance section isn't met, maintenance may be performed on the system and samples reanalyzed or other actions as determined by the analyst. Document all actions in the appropriate logbook. 14.7 I f data are to be reported when performance criteria have not been met, the data must be footnoted on tables and discussed in the text o f the report. 15.0 Pollution Prevention and Waste Ma n a g e m e n t ___________:_______________ 15.1 Sample extract waste and flammable solvent is disposed in high BTU containers, and glass pipette waste is disposed in broken glass containers located in the laboratory. 1 6 .0 R e c o r d s _____________________________________________________________________________ 16.1 Each page generated for a study must have the following information included either in the header or hand written on the page: study or project number, acquisition method, integration method, sample name, extraction date, dilution factor (if applicable), and analyst. 16.2 Print the tune page, sample list, and acquisition method from MassLynx to include in the appropriate study folder. Copy these pages and tape into the instrument runlog. 16.3 Plot the calibration curve by linear regression, weighted 1/x, then print these graphs and store in the study folder. 16.4 Print data integration summary, integration method, and chromatograms, from M assLynx, and store in the study folder. ETS-8-5.1 Analysts o f Serum Extract Using ES/MS Page 7 of 9 3M Medical Department Study: T-6316.8 BACK TO MAIN Analytical Report: FAC T TO X-097 L R N -U 2 4 5 2 16.5 Summarize data using suitable software (Excel 5.0) and store in the study folder, see Attachment A for an example o f a summary spreadsheet. 16.6 Back up electronic data to appropriate medium. Record in study notebook the file name and location o f backup electronic data. 17.0 Tables. Diagrams. Flowcharts, and Validation Data 17.1 Attachment A: ETS-8-5.1 Data summary spreadsheet. _________________ 18.0 References____________________________;_____________________________________ 18.1 FACT-M -4.1, "Extraction o f Potassium Perfluorooctanesulfonate or Other Fluorochemical compounds from Serum for Analysis Using HPLC-Electrospray/Mass Spectrometry 18.2 E TS-9-24.0, "Operation and Maintenance o f the Micromass Atmospheric Pressure Ionization/M ass Spectrometer Quattro II triple quadrapole System s" 18.3 The validation report associated with this method is ETS-8-4.0 & 5 .0 -V -l. 19.0 Affected Documents___________________________________________________________ 19.1 ETS-8-4.1, "Extraction o f Potassium Perfluorooctanesulfonate or Other Fluorochemical Compounds from Serum for Analysis Using HPLC-Electrospray/Mass Spectrometry" 20.0 R evisions____________ _________________________________________________________ _ R evision Numher. 1 Reason For Revision Section 6.1.2 Clarification o fH P l 100 system components. Section 11.1 Average o f two curves, not standtird values, are used for plotting linear regression and added the 1/x weighting o f the curve. Section 12.2.2.4 Clarification o f solvent ramp. Section 17.1 Changed from attachment B to A. R ev isio n Date 04/02/99 3OTEnvironmental Laboratoiy ETS-8-5.1 Analysis of Serum Extract Using ES/MS Page 8 of 9 Page 110 3M Medical Department Study: T-6316.8 BACK TO MAIN Analytical Report: FACT TO X-097 L R N -U 2 4 52 Laboratory Study # Study: Test Material: Matrix/Final Solvent: Method/Revision: Analytical Equipment System Number: Instrument Software/Version: Filename: R-Squared Value: Slope: Y Intercept: Date of Extraction/Analyst: Date of Analysis/Analyst Group Dose Sample# Concentration ug/mL Initial Vol. mL Dilution Factor Final Cone. ug/mL Slope: Taken from linear regression equation. Group/Dose: Taken from the study folder. Sample#: Taken from the study folder. Concentration (ug/mL): Taken from the MassLynx integration summitry. Initial Volume (mL): Taken from the study folder. Dilution Factor: Taken from the study folder. Final Cone. (ug/mL): Calculated by dividing the initial volume from the concentration Attachment A: Summary Spreadsheet ETS-8-5.1 Analysis of Serum Extract Using ES/MS , 3M E n y tremmei'il'crt't a b o rate n y Page 9 of 9 Page 111 3M Medical Department Study: T-6316.8 BACK TO MAIN Analytical Report: FACT TO X -097 L R N -U 2 4 5 2 3M Environmental Laboratory Method Analysis of Potassium Perfluorooctanesulfonate or Other Fluorochemicals in Liver Extracts Using HPLC-Electrospray/Mass Spectrometry M ethod Num ber: ETS-8-7.0 Author: Lisa Clemen, Glenn Langenburg Approved By: Adoption Date: R evision Date: N * . ______ Laboratory Manager Date Group Leader -- - ____________ _________________ ___________ W H f i a Date CL,ra^ Technical Reviewer Date 1 .0 S c o p e a n d A p p l i c a t i o n ______________________________________________________________ 1.1 Scope: This method is for the analysis o f liver extracts for fluorochemical surfactants using HPLC-electrospray/mass spectrometry. 1.2 A pplicable Com pounds: Fluorochemical surfactants or other fluorinated compounds, or other ionizable compounds. 1.3 M atrices: Rabbit, rat, bovine, monkey liver, or other tissues as designated in the validation report. Word 6/95 ETS-8-7.0 Analysis of Liver Extract Using ES/MS Page 1 of 10 3M Enviiunmental Laboratory Page 112 3M Medical Department Study: T-6316.8 BACK TO MAIN Analytical Report: FACT T O X -097 L R N -U 2 4 5 2 2 . 0 S u m m a r y o f M e t h o d _________________________________________ ___________________________________ 2.1 This method describes the analysis o f fluorochemical surfactants extracted from liver using HPLC-electrospray/mass spectrometry, or similar system as appropriate. The analysis is performed by monitoring a single ion characteristic o f a particular fluorochemical, such as the perfluorooctanesulfonate (PFOS) anion, m /z = 499. Additionally, samples may be analyzed using a tandem mass spectrometer to fufliier verify the identity o f a compound by detecting daughter ions o f the selected parent ion. 3 .0 Defin itio n s____________________________ '________________ :________________________ 3.1 A tm ospheric Pressure Ionization (API): The Micromass Quattro II triple quadrupole system s allow for various methods o f ionization by utilizing various sources, probes, and interfaces. These include but are not limited to: Electrospray Ionization (ESI), Atmospheric Pressure chem ical Ionization (APcI), Thermospray, etc. The ionization process in these techniques occurs at atmospheric pressure (i.e. not under a vacuum). 3.2 E lectrospray Ionization (ES, ESI): a method o f iomzation performed at atmospheric pressure, whereby ions in solution are transferred to the gas phase via tiny charged droplets. These charged droplets are produced by the application o f a strong electrical field. 3.3 M ass Spectrom etry, M ass Spectrom eter (M S), Tandem M ass Spectrom eter (M S/M S): The API Quattro 13 triple quadrupole mass spectrometer is equipped with two quadrupole mass selective detectors and a collision cell. Ions an: selectively discriminated by mass to charge ratio (m /z) and subsequently detected. A single MS may be employed for ion detection or an ion may be selected in the first quadrupole, fragmented in file collision cell, and these fragments may be analyzed in the second quadrupole. 3.4 C onventional vs. Z-spray probe interface: The latest models o f Micromass Quattro II triple quadrupole (post 1998) utilize a "Z-spray" conformation. The spray emitted from a probe is orthogonal to the cone aperture. In the conventional conformation it is aimed directly at the cone aperture, after passing through a tortuous pathway in the counter electrode. Though the configuration is different, the methods o f operation, cleaning, and maintenance are the same. However, Z-spray components and conventional components are not com patible w ith one another, but only with similar systems (i.e. Z-spray components are com patible w ith other Z-spray system s, etc.) 3.5 M ass L ynx Softw are: System software designed for the specific operation o f these Quattro II triple quadrupole system s. Currently MassLynx has W indows 95 and WindowsNT 4.0 versions. A ll versions are similar. For more details refer to the manual specific to the instrument (M icromass Quattro 13triple quadrupole MassLynx or MassLynx NT User's Guide). 4 . 0 W a r n i n g s a n d C a u t i o n s _____________________________ - 4.1 H ealth and Safety W arnings: . ____________________ 4.1.1 U se caution with the voltage cables for the probe. When engaged, the probe em ploys a voltage o f approximately 5000 Volts. ETS-8-7.0 Analysis of Liver Extract Using ES/MS Page 2 of 10 3M Medical Department Study: T-6316.8 BACK TO MAIN Analytical Report: FACT TO X-097 L R N -U 2 4 5 2 4.1.2 When handling samples or solvents wear appropriate protective gloves, eyewear, and clothing. '' 4.2 Cantions: 4.2.1 Operate the solvent pumps below a back pressure o f 400 bar (5800 psi). I f the back pressure exceeds 400 bar, the H P1100 w ill initiate automatic shutdown. 4.2.2 Do not run solvent pumps to dryness. 5.0 I n terferen ces___________________________________________________________________ 5.1 To m inim ize interferences when analyzing samples, Teflon shall not be used for sample storage or any part o f instrumentation that com es in contact with the sample or extract. 6.0 E q u i p m e n t ____________________________________________________________________________________ 6.1 Equipment listed below may be modified in order to optimize the system. Document any m odifications in the raw data as method deviations. 6.1.1 6.1.2 Micromass Quattro II triple quadrupole M:iss Spectrometer equipped with an electrospray ionization source. HP 1100 low pulse solvent pumping system , solvent degasser, column compartment, and autosampler 7.0 Su pplies and M aterials_______________ ;__________________ ;_______________________ 7.1 Supplies 7.1.1 High purity grade air regulated to approximately 100 psi (house air system ) 7.1.2 HPLC analytical column, specifics to be determined by the analyst and documented in the raw data 7.1.3 Capped autovials or capped 15 ml centrifuge tubes 8 . 0 R e a g e n t s a n d S t a n d a r d s __________________________________________________________________ 8.1 R eagents 8.1.1 M ethanol, HPLC grade or equivalent . 8.1.2 Milli-QTM water (ASTM type I), all water used in this method should be ATSM type I, or equivalent, and be provided by a M illi-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: W eigh approximately 0.300 g ammonium acetate. Pour into a 2000 mL volumetric container containing 2000 mL Milli-QTM water, mix until all solids are dissolved. Store at room temperature. ETS-8-7.0 Analysis of Liver Extract Using ES/MS Page 3 of 10 3M Eiiviiumnental Laboratory Page 114 3M Medical Department Study: T-6316.8 BACK TO MAIN Analytical Report: FACT TO X-097 L R N -U 2 4 52 8.2 Standards 8.2.1 Typically two method blanks, two matrix blanks, and eighteen matrix standards are prepared during the extraction procedure. Refer to ETS-8-6.0. 9 .0 S a m p l e H a n d l i n g ________________________ ________________________________________________________ 9.1 Fresh matrix standards are prepared with each analysis. Extracted standards and samples are stored in capped autovials or capped 15 m l centrifuge tubes until analysis. 9.2 I f analysis w ill be delayed, extracted standards and samples may be stored at room temperature, or refrigerated at approximately 4 C, until analysis can be performed. 1 Q .0 Q u a l i t y C o n t r o l ___________________________________________________________________ ;___________ 10.1 M ethod Blanks and M atrix Blanks . 10.1.1 Solvent blanks, method blanks, and matrix blanks are prepared and analyzed with each batch to determine contamination or carryover. 10.1.2 Analyze a method blank and a matrix blank prior to each calibration curve. 10.2 M atrix 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. 1 0 .2 3 Analyze a matrix spike and matrix spike duplicate per forty sample^. With a minimum o f 2 spikes per batch. 10.2.4 Matrix spike and matrix spike duplicate concentrations w ill fall in the mid-range o f the initial calibration curve. Additional spike concentrations may fall in the low range o f the initial calibration curve. 10.3 C ontin u in g C alib ratio n C hecks 10.3.1 Continuing calibration verifications are analyzed to verily the continued accuracy o f the calibration curve. 10.3.2 Analyze a mid-range calibration standard every tenth sample, with a minimum o f one per batch. 11.0 C a l i b r a t i o n a n d S t a n d a r d i z a t i o n __________________________________________________ 11.1 Analyze the extracted matrix standards prior to and following each set o f sample extracts. The average o f two standard curves w ill be plotted by linear regression (y = mx + b), weighted 1/x, not forced through the origin, using MassLynx or other suitable software. 11.2 If the curve does not meet requirements perform routine maintenance or reextract the standard curve (if necessary) and reanalyze. ETS-8-7.0 Analysis of Liver Extract Using ES/MS Page 4 of 10 3M Medical Department Study: T-6316.8 BACK TO MAIN Analytical Report: FAC T TO X -097 L R N -U 2 4 5 2 11.3 For purposes o f accuracy when quantitating low levels o f analyte, it may be necessary to use the low end o f the calibration curve rather than the full range o f the standard curve. Example: when attempting to quantitate approximately 10 ppb o f analyte, generate a calibration curve consisting o f die standards from 5 ppb to 100 ppb rather than the full range o f the curve (5 ppb to 1000 ppb). This w ill reduce inaccuracy attributed to linear regression weighting o f high concentration standards. 12.0 P rocedures_____________'__________ ________'_____________________________________ 12.1 A cquisition S et up 12.1.1 Set up the sample list. 12.1.1.1 Assign a sample list filename using MO-DAY-last digit o f year-increasing letter o f the alphabet starting with a 12.1.1.2 A ssign a method (MS file) for acquiring 12.1.1.3 A ssign 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 (Sinjjle Ion Recording) or MRM (M ultiple Reaction M onitoring). Set Ionization M ode as appropriate and mass to 499 or other appropriate masses. A full scan is usually collected d ong with the SIRs. Save acquisition method. If MS/MS instruments are employed, additional product ion fragmentation information may be collected. Refer to Micromass MassLynx GUIDE TO DATA ACQUISITION for additional information and MRM. 12.1.3 Typically the analytical batch run sequence begins and ends with a set o f extracted matrix standards. 12.1.4 Samples are analyzed with a continuing calibration verification injected standard after every tenth sample. Solvent blanks should be analyzed periodically to monitor possible analyte carryover and are not considered samples but may be included as such. 12.2 U sing th e A utosam pler 12.2.1 Set up sample tray according to the sample list prepared in Section 12.1.1. 12.2.2 Set-up the HP1100/autosampler at the following conditions or at conditions the analyst considers appropriate for optimal response. Record actual conditions in the instrument logbook: 12.2.2.1 Sample size = 10 pL injection 12.2.2.2 Inject/sample = 1 ' 12.2.2.3 Cycle time = 9 minutes . ETS-8-7.0 Analysis of Liver Extract Using ES/MS Page 5 of 10 3M Medical Department Study: T-6316.8 BACK TO MAIN Analytical Report: FACT TO X-097 ' LRN-U2452 12.2.2.4 Solvent ramp conditions T im e MeOH 0.00 min. 1.0 min. 4.5 min. 6.5 min. 7.0 min. 9.0 mi. 40% 40% 95% 95% 40% 40% 2.0 mM Ammonium acetate 60% 60% 5%, 5% 60% 60% 12.2.2.5 Press the "Start" button. 12.3 Instrum ent Set-up 12.3.1 Refer to E TS-9-24.0, "Operation and Maintenance o f the Micromass Quattro II Triple Quadrupole M ass Spectrometer Fitted w ith an Atmospheric Pressure Ionization Source," for more details. 12.3.2 Check the solvent level in reservoirs and refill if necessary. 12.3.3 Check the stainless steel capillary at the end o f the probe. U se an eyepiece to check the tip. The tip should be flat with no jagged edges. If the tip is found to be unsatisfactory, disassemble the probe and replace the stainless steel capillary. 12.3.4 Turn on the nitrogen. 12.3.5 Open the tune page. Clicks on operate to initiate source block and desolvation heaters. 12.3.6 Open the Inlet Editor. 12.3.6.1 Set HPLC pump to "On" 12.3.6.2 Set the flow to 10 - 500 uL/min or as appropriate 12.3.6.3 Observe droplets coming out o f the tip o f the probe. A fine m ist should be expelled with no nitrogen leaking around the tip o f the probe. Readjust the tip o f the probe if no mist is observed 12.3.6.4 Allow to equilibrate for approximately 10 minutes. 12.3.7 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 250-400 liters/hour 12.3.7.2 ESI nebulizing gas 10-15 liters/hour 1 2 3 .7 .3 HPLC constant flow mode flow rate 1 0 -5 0 0 pL/min 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 Extract Using ES/MS Page 6 of 10 3M Civironmerttal Laboratory Page 117 3M Medical Department Study: T-6316.8 BACK TO MAIN Analytical Report: FACT TO X -097 L R N -U 2 4 5 2 12.3.8 Print the time page, with its parameters, and store it in the study binder with a copy taped into the instrument log. 12.3.9 Click on start button in the Acquisition Control Panel (this may vary among M assLynx versions, refer to appropriate MassLynx User's Guide). Ensure start and end sample number includes all samples to be analyzed. 13.0 Data Analysis and Calculations___________________________ ___________________ 13.1 C alculations: 13.1.4 Calculate matrix spike percent recoveries using the following equation: % Recovery = Observed Result - Background Result x 100 Expected Result ' 13.1.5 Calculate percent difference using the following equation: % Difference = Expected Cone. - Calculated Clone, x 100 Expected Cone. 13.1.6 Calculate actual concentrations in matrix (pig/g): fag o f PFQS calc, from std. Curve x Dilution Factor! (Initial W eight o f Liver (gl Final Volume (mL) x 1 p.g 1000 ng 1 4 . 0 M e t h o d P e r f o r m a n c e _________________________________________________________________________ 14.1 Method Detection Limit (MDL) and Limit o f Quantitation (LOQ) are method, analyte, and . matrix specific. Refer to E TS-8-6.0, A ttachm ent B for a listing o f current validated MDL and LOQ values. 1 4 .2 Solvent B lanks, M ethod Blanks and M atrix Blanks 14.2.1 Solvent blanks, method blanks, and matrix: blanks must be below the low est standard in the calibration curve. . 14.3 C alibration Carves 14.3.1 The i3 value for the calibration must be 0.980 or better. 14.4 M atrix Spikes 14.4.1 Matrix spike percent recoveries must be within 30% o f the spiked concentration. 14.5 C ontinuing C alibration V erification 14.5.1 Continuing calibration verification percent recoveries must be within 30% o f the . spiked concentration. 14.6 If criteria listed in the method performance section are not met, maintenance may be performed on the system and samples reanalyzed or other actions as determined by the analyst. Document all actions in die appropriate logbook. ETS-8-7,0 Analysis o f Liver Extract Using ES/MS Page 7 o f 10 3M Medical Department Study: T-6316.8 BACK TO MAIN Analytical Report: FACT TO X-097 L R N -U 2 4 52 14.7 I f data are to be reported when performance criteri a have not been met, the data must be footnoted on tables and discussed in the text o f the report. 15.0 P o llu tio n P revention and W aste M anagem ent________________________________ 15.1 Sample extract waste and flammable solvent is disposed in high BTU containers, and glass pipette waste is disposed in broken glass containers located in the laboratory. 16.0 R ecords______ :_______________________ ;_________;_______________' __________ 16.1 Each page generated for a study must have the follow ing inform ation included either in the header or hand written on the page: study or project number, acquisition method, integration method, sample name, extraction date, dilution factor (if applicable), and analyst. 16.2 Print the tune page, sample list, and acquisition method from MassLynx to include in the appropriate study folder. Copy these pages and tape into the instrument runlog. 16.3 Plot the calibration curve by linear regression, weighted 1/x, then print these graphs and store in the study folder. 16.4 Print data integration summary, integration method, and chromatograms from MassLynx and store in the study folder. 16.5 Summarize data using suitable software (Excel 5.0+) and store in the study folder, refer to A ttachm ent A for an example o f a summary spreadsheet. 16.6 Back up electronic data to appropriate medium. Record in study notebook the file name and location o f backup electronic data. 17.0 T a b les. Dia g ram s. F low charts, and Validation Data_________________________ 17.1 Attachment A: ETS-8-7.0 Data summary spreadsheet 1 8 .0 Re fer en c es______________ ____________________________________________________ _ 18.1 FACT-M -2.1, "Extraction o f Potassium Perfluorooctanesulfonate or Other Fluorochemical Compounds from Liver for Analysis Using HPLC-Electrospray/Mass Spectrometry" 18.2 E TS-9-24.0, "Operation and Maintenance o f the Micromass Atmospheric Pressure Ionization/M ass Spectrometer Quattro II triple quadrupole Systems" 18.3 The validation report associated with this method is E TS-8-6.0 & 7.0-V -l 19.0 Affec ted Documents__________________________________________ ________________ 19.1 ETS-8-6.0, "Extraction o f Potassium Perfluorooctanesulfonate or Other Fluorochemical Compounds from Liver or Fluid for Analysis U sing HPLC-Electrospray/Mass Spectrometry" ETS-8-7.0 , Analysis of LiverExtract Using ES/MS Page 8 of 10 3M Medical Department Study: T-6316.8 20.0 REVISIONS R ev isio n Number BACK TO MAIN Analytical Report: FACT TO X -097 L R N -U 2 4 5 2 Reason For Revision R ev isio n Date ETS-8-7.0 Analysis of Liver Extract Using ES/MS Page 9 of 10 Page 120 3M Medical Department Study: T-6316.8 BACK TO MAIN Analytical Report: FAC T TO X -097 L R N -U 2 4 5 2 Laboratory Study # Study: Test Material: Matrix/Final Solvent: Method/Revision: Analytical Equipment System Number: Instrument Software/Version: Filename: R-Squared Value: Slope: Y Intercept Date of Extraction/Analyst: ' Date of Analysis/Analyst G roup Dose Sam ple# C oncentration ng/g initial W t. g D ilu tio n F actor Final Cone. ug/g Group/Dose: Taken from the study folder. Sample#: Taken from the study folder. Concentration (ng/g): Taken from the MassLynx integration summary. Initial Wt. (g): Taken from the study folder. Dilution Factor: Taken from the study folder. Final Cone, (ug/g): Calculated by dividing the initial volume from the concentration Attachment A: Summary Spreadsheet ETS-8-7.0 Analysis ofLiver Extract Using ES/MS 3M Envi ronm entaM -abui aim y Page 10 of 10 P age 121 3M Medical Department Study: T-6316.8 Appendix D: Data Summary Tables BACK TO MAIN A nalytical Report: F A C T T O X -0 9 7 L R N -U 2 4 5 2 Table 6. Rabbit Sera FO PFOS, PFOSA and PFOSA* Data for FACT-TOX-097 Group Dose Sample # PFOS pg/mL PFOSA pg/mL PFOSAA* pg/mL Group 1 0.0 mg/kg/day 0.0 mg/mL 8682F 8683F 8684F <LOQ (0.0458) <LOQ (0.0458) <LOQ (0.0458) <LOQ (0.00490) -LOQ (0.00490) =LOQ (0.00490) <LOQ (0.0124) <LOQ (0.0124) <LOQ (0.0124) Group 2 0.1 mg/kg/day 0.02 mg/mL 8685F 8686F 8687F 8688F 8689F 0.484 0.919 0.839 0.622 0.806 0.0155 0.00696 0.0154 0.0132 0.00988 0.975 1.00 1.15 0.660 1.01 Group 3 1.0 mg/kg/day 0.2 mg/mL 8690F 8691F 8692F 4.79 3.66 8.66 0.0954 0.0561 0.242 9.19 6.67 11.6 Group 4 2.5 mg/kg/day 0.5 mg/mL Group 5 3.75 mg/kg/day 0.75 mg/mL 8693F 8694F 8695F 8696F 8697F 8698F 8699F 24.4 14.1 9.88 22.6 21.5 18.8 19.5 0.378 0.433 0.460 0.489 0.592 1.01 0.450 27.6 20.7 22.7 44.3 59.8 54.1 45.6 8700F 14.8 1.41 45.4 *Non-qualitative screening data only. It is not possible to verify true recovery o f endogenous analyte from tissues without radio-labeled reference material. The only measurement o f accuracy available at this, time, m atrix 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: FAC T TO X -097 L R N -U 2 4 5 2 Table 7. Rabbit Sera FO N-EtFOSE and PFOSEA Data for FACT-TOX-097 Group Dose Sample # N-EtFOSP pg/mL PFOSEA pg/mL Group 1 0.0 mg/kg/day 0.0 mg/mL 8682F 8683F 8684F <LOQ (0.0216) <LOQ (0.0216) <LOQ (0.0216) <LOQ (0.00487) <:LOQ (0.00487) <LOQ (0.00487) Group 2 0.1 mg/kg/day 0.02 mg/mL 8685F 8686F 8687F 8688F <LOQ (0.0216) <LOQ (0.0216) <LOQ (0.0216) <LOQ (0.0216) <LOQ (0.00487) <LOQ (0.00487) <LOQ (0.00487) <LOQ (0.00487) 8689F <LOQ (0.0216) <LOQ (0.00487) Group 3 1.0 mg/kg/day 0.2 mg/mL 8690F 8691F 8692F <LOQ (0.0216) 0.0368 <LOQ (0.0216) <LOQ (0.00487) <LOQ (0.00487) <LOQ (0.00487) Group 4 2.5 mg/kg/day 0.5 mg/mL 8693F 8694F 8695F <LOQ (0.0216) <LOQ (0.0216) <LOQ (0.0216) <LOQ (0.00487) <LOQ (0.00487) <LOQ (0.00487) Group 5 3.75 mg/kg/day 0.75 mg/mL 8696F 8697F 8698F 8699F 0.0294 0.0279 0.0386 <LOQ (0.0216) <LOQ (0.00487) <LOQ (0.00487) <LOQ (0.00487) <LOQ (0.00487) 8700F 0.132 <_OQ (0.00487) *Non-qualitative screening data only. It is not possible to verify true recovery o f 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 123 3M Medical Department Study: T-6316.8 BACK TO MAIN A nalytical R eport: FA C T T O X -097 L R N -U 2 4 52 Table 8. Rabbit Liver FO PFOS, PFOSA and PFOS/A Data for FACT-TOX-097 Group Dose Sample # PFOS M9/g PFOSA M3/g PFOSAA* pg/g Group 1 0.0 mg/kg/day 0.0 mg/mL 8682F 8683F 8684F 0.0519 0.0346 <LOQ (0.0279) <LOQ (0.0120) <LOQ (0.0120) 0.0120 0.0458 0.119 0.0680 8685F 1.83 0.147 5.32 Group 2 8686F 1.58 0.0947 6.32 0.02 mg/mL 8687F 1.64 0.156 5.74 8688F 1.41 0.0663 4.47 8689F 1.44 0.100 3.91 Group 3 1.0 mg/kg/day 0.2 mg/mL 8690F 8691F 8692F 10.8 7.89 13.4 1.01 1.04 1.98 36.2 25.0 37.5 Group 4 2.5 mg/kg/day 0.5 mg/mL 8693F 8694F 8695F 24.3 22.2 26.0 6.85 4.27 4.61 65.4 67.5 90.9 Group 5 3.75 mg/kg/day 0.75 mg/mL 8696F 8697F 8698F 8699F 37.5 48.5 33.2 32.1 7.83 8.65 11.9 5.61 123 259 148 88.5 8700F 25.2 18.3 133 *Non-qualitative screening data only. It Is not possible to verify true recovery o f endogenous analyte from tissues without radio-labeled reference material. The only measurement o f accuracy available at this time, m atrix spike studies, indicate that the data quantitative to 30% or greater. 3M Environmental Laboratory Page 124 3M Medical Department Study: T-6316.8 BACK TO MAIN Analytical Report: FAC T TO X -097 L R N -U 2 4 52 Table 9. Rabbit Liver FO N-EtFOSE and PFOSEA Data for FACT-TOX-097 Group Dose Sample # N-EtFOSE* Mg/g PFOSEA pg/g Group 1 0.0 mg/kg/day 0.0 mg/mL 8682F 8683F 8684F <LOQ (0.0525) <LOQ (0.0525) <LOQ (0.0525) <LOQ (0.0298) <LOQ (0.0298) <LOQ (0.0298) Group 2 0.1 mg/kg/day 0.02 mg/mL 8685F 8686F 8687F 8688F <LOQ (0.0525) <LOQ (0.0525) <LOQ (0.0525) <LOQ (0.0525) <LOQ (0.0298) <LOQ (0.0298) <LOQ (0.0298) <LOQ (0.0298) 8689F <LOQ (0.0525) <LOQ (0.0298) Group 3 1.0 mg/kg/day 0.2 mg/mL 8690F 8691F 8692F <LOQ (0.0525) 0.621 <LOQ (0.0525) <LOQ (0.0298) <LOQ (0.0298) <LOQ (0.0298) Group 4 2.5 mg/kg/day 0.5 mg/mL 8693F 8694F 8695F 0.161 0.123 0.140 <LOQ (0.0298) <LOQ (0.0298) <LOQ (0.0298) Group 5 3.75 mg/kg/day 0.75 mg/mL 8696F 8697F 8698F 8699F 0.417 0.477 93.5 0.235 <LOQ (0.0298) <LOQ (0.0298) <LOQ (0.0298) < O Q (0.0298) 8700F 30.8 <LOQ (0.0298) *Non-qualitative screening data only. It is not possible to verify true recovery o f 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 125 3M Medical Department Study: T-6316.8 BACK TO MAIN A nalytical R eport: FA C T T O X -097 L R N -U 2 4 5 2 Table 10. FACT-TOX-097 Data Summary of Average Sera Concent ration (pg/mL) and Standard Deviation (SD) Dosage Group PFOS Average SD PFOSA Average SD PFOSAA* Average SD N-EtFOSE* Average SD PFOSEA Average +SD Group 1 0.0 mg/kg/day 0.0 mg/mL Group 2 0.1 m g/kg/day 0.02 mg/mL Group 3 1.0 mg/kg/day 0.2 mg/mL <LOQ NA (n=3) 0.734 +0 177 (n=5) 5.70 2.62 (n=3) <LOQ NA (n=3) 0.0128 0.00277 (n=5) 0.131 0.0977 (n=3) <LOQ NA (n=3) 0.959 0.180 (n=5) 9.15 2.46 (n=3) <LOQ NA (n=3) <LOQ NA (n=5) 0.0267 0.00875 (n=3) <LOQ NA (n=3) <LOQ NA (n=5) <LOQ NA (n=3) Group 4 2.5 mg/kg/day 0.5 mg/mL 16.1 7.47 (n=3) 0.423 0.0497 (n=3) 23.7 3.51 (n=3) <LOQ NA (n=3) <LOQ NA (n=3) Group 5 3.75 mg/kg/day 0.75 mg/mL 19.4 2.98 (n-5) 0.790 0.411 (n=5) 49.8 6.82 (n=5) 0.0499 0.0464 (n=3) <LOQ NA (n=5) *Non-qualitative screening data only. It is not possible to verity 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 A nalytical R eport: F A C T T O X -0 9 7 L R N -U 2 4 52 Table 11. FACT-TOX-097 Data Summary of Average Liver Concentration (|jg/g) and Standard Deviation (SD) Dosage Group PFOS Average SD PFOSA Average SD PFOSAA* Average SD N-EtFOSE* Average SD PFOSEA Average SD Group 1 0.0 mg/kg/day 0.0 mg/mL 0.0381 0.0124 (n=3) <LOQ NA (n=3) 0.0776 0.0375 (n=3) <LOQ NA (n=3) <LOQ NA (n=3) Group 2 0.1 mg/kg/day 0.02 mg/mL 1.58 0 171 (n=5) 0.113 0.0376 (n=5) 5.15 0.968 (n=5) <LOQ NA (n=5) <LOQ NA (n=5) Group 3 1.0 m g/kg/day 0.2 mg/mL 10.7 2.78 (n=3) 1.34 0.550 (n=3) 32.9 6.86 (n=3) 0.242 0.328 (n=3) <LOQ NA (n=3) Group 4 2.5 mg/kg/day 0.5 mg/mL 24.1 1 92 (n=3) 5.24 1.40 (n=3) 74.6 14.1 (n=3) 0.141 0.0187 (n=3) <LOQ NA (n=3) Group 5 3.75 mg/kg/day 0.75 mg/mL 35.3 8.60 (n=5) 10.5 4.93 (n=5) 150 64.8 (n=5) 25.1 40.4 (n=5) <LOQ NA (n=5) *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 Table 12. A pproxim ate LOQ Values Used in FACT-TOX-097 Matrix Liver Sera Compound PFOS PFOSA PFOSAA* N-EtFOSE* PFOSEA PFOS PFOSA PFOSAA* N-EtFOSE* PFOSEA LOQ 0.0279 |jg/g 0.0120 (jg/g 0.0324 |jg/g 0.0525 jjg/g 0.0298 |jg/g 0.0458 pg/mL 0.00490 pg/mL 0.0124 pg/mL 0.0216 pg/mL 0.00487 pg/mL *Non-qualitative 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 3M Medical Department Study: T-6316.8 i f * s Sdi Si i | si 1 mI ! 1 "1 ? 1I 8 ! < i|l l 8Ii In8I8l l 1II |e. S f i ! I l l l 8SISSSaSs h m f I? o I* l i l i i n 531 r n s m n ? . 3353*332 o < C 1i 1l 2i - ![ji! !J! II iiiif if ii H lH ili 11 ij i S| 23 23 21 ; : i 13! 81 m e 1a. 1 S- s f? 5 'i 1 i l I l l II 1 3 i ti |di|8.ImI I 11 ti 88 8 t i l l i l i 25 SS=S=18 1 1 . U f a i n 532S2 58!!3535331 iMl sl 21 i | I n i " JI J i i i l i l J J J j j l j j j f hi f i l md e e a Uo o 111111111 sEeEdBoBd6#& n IS - It li i 1 Ji i 1 ll!i iiil * f\ i l l i l i fi SS i} i i ji j i. li % !4 : l Ml ,1 f 6M * i M f H jl if! fiiit il si a 3M Environmental Laboratory Analytical Report: FACT TO X-097 LRN-U2452 I IS Page 129 BACK TO MAIN 3M Medical Department Study: T-6316.8 > i{i * a u a| tt ai ill I I i E 1 I 1 I il ? j E e I i j f l II |ifilfl 1 Iil! 1 99119 I s ! p 1S !! H | ! a eeee i l l e ie nH i i\ ? m ? 1 m I H i S S l i l i l l a i 351 3 = 3 3 : ili-- o 111s s a a s s ssssa [ | ! H i ! U *o 3o $o d3 5o #o Id dl i o o d l i i l l S S3 !i3t i ;2 a ! S3 5 J ? ,I IV I 5 i 5e 1 1 I I 13a f 51 i j i l l I I I I rl 1 i3 3 I S=l l III25 55 = h- rRari *> 2 78 9 9 * 9 | 1 l ?$ n? 2 l u i * i i 5 J 1 1 1 3 = 5 3 * S S 5 3 * 3 Ss s 2 2 1 I I* * 5 Ili- n -f n _ li a l Il li lIlIlIljils fjHS ijSo S i 33 99S9SI 22 3 ili ati 6g i sa 2f f 3 2 i ii i a 11 i i l i l i l l l l i l l l | ii 11 ity J} if j l 1 21 jj fS i8 ff iS 3 | ! if! 1 3? s.a isii If *I II Il I 3M Environmental Laboratory Analytical Report: FACT TOX-097 LRN-U2452 is ll Page 130 3M Medical Department Study: T-6316.8 4 if i i z li i i H E I\ l I? I I < H * !a *e 1 l se i i e1 1 % 1 ? 1 i mo I- m 1I 111; i n i m u * ; i 11! 11 i i i l l i l i i i M i i i i i i o m m mmi mi < lism 5 s.;!-s' O8o88o H i m i s ii m i i i i i i i --- -- ... - ---- i l l I i i i I J i i i i l i i i i i i i i 5 I] ifI j FACT-TOX-097 ArgutiM l&410 4 i i X m iii i l i ii t Si i i 1j ! H i i i i i Si i i i i`3 j1 H I* Hi ii Hi HiHH itii H i*1l3lSI* i.......... 7, 2l i l t l liilfiIifliiif2liI 1 il-H i i i H i i i a m i l I If IllfJ t1 18 I I 11 111 m m i i i i m u i i l I !il i Hi iilJi I il jh i a St !i j & if! lf| ij H o' xi *! IS 1 IS I 3M Environmental Laboratory Analytical Report: FACT TOX-097 LRN-U2452 25 II Page 131 3M Medical Department Study: T-6316.8 I a*i I!I) mmmli1i1 l a iiiie1S!lsis1mn1n1ii1!?mmml uluu 3111H!?!1l1?Im2mmgas-1g1tiSisSlSs?p1S-a1| Io" ,i| * 3 ssi 5- 5* S 3 5? o2 ill Jif|l 11 aS 33, i?m1 e III?l323 3 se 1 i?a Pe 5e ISIIlSIi5 SS3 M o; n' 5 SRSsis i] lili 53 I l i 3 5 5 2 5 2 5 = 5 3 = s 3 2 2 i 22 i2 1 i lo * }| Si 111111 S iCCi t ! 1 I1 III a 3 ci s ci i lili li filPlllipIII s s B8888 lili 15 g I l i 1 1I P i l ! tS Sf U- t* ;- II?? III 3S33S liiiliil, i d l i l i SS 111 2 5 S 5 3 3SS15 a! SS 53 ! 1i 22 3 ]2 * 1 1 1 iI !I iIt1 I1 -JIiPn imri1nii1ini 5S5 3a523ii3 11* i f , .1 % S 13 m lili as ? is 11 4 p ri>???? m i5 !II I J f i ! | 1)1 lis cSas m sas?,*s ^ jUiiilii . i n i S 3 * l H h- , I III HS 5 II 1Ni!1 l i l i i lil illll l< .!1 S si! $ f! 11iili,!lili!* 1 S I\ * |1 3 ill i l i l l W i i r 3 i 1 f] r~, t j!! i IJ i 3M Environmental Laboratory Analytical Report: FACT TOX-097 LRN-U2452 Page 132 3M Medical Department Study: T-6316.8 < o I* o < C Analytical Report: FACT TOX-097 LRN-U2452 3M Environmental Laboratory I5 El Page 133 3M Medical Department Study: T-6316.8 Analytical Report: FACT TOX-097 LRN-U2452 FACT-TOX-097 A rgus# 418-010 3M~Ervironmental-Laboratory I p S3-3 Page 134 3M Medical Department Study: T-6316.8 Analytical Report: FACTTOX-097 LRN-U2452 I? o<4 3M Medical Department Study: T-6316.8 Analytical Report: FACT TOX-097 LRN-U2452 55 gg Is FACT-M-10 3WI1 i*romji'imental Laboratory Page 136 3M Medical Department Study: T-6316.8 Analytical Report: FACT TOX-097 LRN-U2452 g3s TOX-0974ivct010-3E.xli 3M Dh v to i ii nerital Laboratory- J 6 r? P a g e 137 3M M edical D epartm ent Study: T-6316.8 sill! 3M E i'iv ii u n iiie iila l L a b o ra to ry ....................................... BACK TO MAIN FACT-TOX-097 Argus# 418-010 Sbxfy: Product Numb(Tc*t Subitanee): Matrix: Metfad/Reviaiou; A uahttcd Equipment Syitan Number liu taa n en t Softw w W em on: D aterfE x tretico /A w ly it: D te etf A oaly aif /A iu Jy at: Date of Data ReduoW A naty*: S u a p ie D ata A rftu 418-010, Oral (Stomach Tube) Dck)pnett Twucity Sbufy ofN-fUFOSE in Rabbits E tF O S E -O H (T -6 3 1 6 .5 ) Rabbit Liver FCT-M-1.0 f t FACT-M-2.0 - fa ta r iwgriioo. w agtntd 1At Amelia 062498 tod Madeline 041098 Ma*sLynx3.1 0/ 109* s a h /j c p \ o m m , i ] a $ m ,0 9 / z v 9 9 h o wa s 1023/98, hqj/m m h Fiknwne: See Aitachdart* R-Sqaired Value: See Attachments Sfepc Sec Attachments Y-lteocept: See Attacbajccti R A B B IT L IV E R P I -------------8 5 5 ------------ Dew Method BBt Matrix Blk QC*500ppb G ite jl O.Owcfctfdty 0.0 m #nL 0.1 m i W 0.02 mgfaL C iw fJ l.O m ptftiay 0.2 m*taL 2.5 rag/*^tfiy 0.5 n ^ u L Sam p le# H20B3k-l H 20B U -2 Rabb# Liver B it-1 Rabbit Lm rBflc-2 8682F-MSD 86S3F KS4F 868F 8687F 8688F 8689F 869IF 692P 8694F 8695F B55------- -- Cafe. Case. .. i d -------4.37 9.99 0.00 0.00 554 563 515 34.6 21.0 CaacsatrattM aTPFOS *% . < O Q (0.0279 V t ) <LOO (0.0279 xAt <LOQ (0.0279 oj/g) < 0 0 (0.0279 V ) 93% 95% 0.0519 05346 < O Q (0.0279 V x ) 1 . 1-5 1.64 1.41 1.44 p ro s <LOQ (0.0279 V ) < 0 0 (0.0279 V c ) 94% 0.0381 158 78*9 13445 22155 25987 7.89 13.4 245 22J 260 10.7 24.1 3.75 tnpftj/day 0 .7 5 m ffaL 8697F 8698F B699F 8700F 48502 33213 32139 25193 485 33.2 32.1 25J 35.3 SUL Dev. M & iM SD W D NA NA 2% 32.4 0.0124 10-8 0.171 26.0 2.78 7.95 1.92 24J 860 H Sa a Cale. Ceue. _!#* 000 0.00 0.00 0.00 695 795 455 119 68.0 5314 6318 5742 4474 3906 36175 25003 37474 65389 67485 90855 123403 259440 147948 88494 132664 afffO SA A V xar% *ae. < O Q (00324 V |) < 00(0.0324 V a l < O Q (0.0324 V i ) <LOO <0.0324 V ) 117% 134% 0.0458 0.119 0.0680 5.32 6.32 5.74 4.47 3.91 36 3 15.0 37.5 65.4 67.5 909 123 259 148 886 133 Vina PFOSAA Vk < 0 0 (0.0324 V a ) < 00(0.0324 ufo) 125% 0.0776 5.15 32.9 74.6 150 Connection FactorsnotapplicabletoM S/M SD Q CdU t The carve r2 far PTOSAA, and EtFOSE-OII did not meet criteri*, tb o e d m and MS56 data (which w n calculated m trg ihc PFOSAA curve) will be coosdeied quiikMivc data. LAC 12/13/00 Origiiial PFOSJTOSAA, EtPOSO LOQs (30.1 ng/g.) updated lo reflaet caiwSwoftcaori&nnatiiOI/JOitlL LAC 01/3001 D ue Entered/Anatpr IO/27/X) LAC DateVeriSayAaaJyst: 1Q/308MKJH SM. te r . M S/M 8DBPB NA NA 13% 48J 0 .0 3 7 5 188 0.968 20.8 686 19.0 14.1 43.1 64.8 .6 6 * I ti -- m s-- Cafe. Came. -0.0906 -0.0906 NA NA 0.701 0.485 0.108 0.00885 0.293 7.24 6.96 122 24.8 6.02 22.3 23.7 45.7 40.4 17.9 49.6 r w r a n l f tin atM 5S6 <LOQ (0.0602 ag/f) Mm M556 S5B----------- ---------- SUL t e r . <LOQ (0.0602 V i ) NS < o q (00602 V i ) <LOQ (DD602 V g ) < O Q (0.0602 V k) < O Q (00602 V |) < O Q <0.0602 V i ) <LOQ (0,0602 ug/|) NA NA < 0 Q (0.0602 V i ) < O Q (0.0602 V i ) < O Q (0.0602 V 8 ) < O Q (0.0602 V B ) <UX ^0.0602 Uflty <LOQ (0.0602 u*/(0 < O Q (0.0602 V l> <LOQ (0.0602 V l ) < O Q (0.0602 V l ) <00(0.0602 V a l < O Q (0 0602 V ) NA NA NA NA Analytical Report: FAC T TO X -097 L R N -U 2 4 5 2 Page 138 FACT-M-2.0 Excel 37 T O X-097-lh'crO 10- 3.Jd s 2/5/2001 9--26AM 3M Medical Department Study: T-6316.8 < o I* o < CO 12 H5 Analytical Report: FACT TOX-097 LRN-U2452 F A C T - M -2 .0 3M'Er'VlroninBiual LabcrrartOTy Page 139 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 A R (ng/mL) x DF x SC x FV (mL) x 1.0 jag = R eported C oncentration (pg/m L) 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 = 4.,79 pg/m L 1.005 mL 1000 ng AR-- Analytical result from MassLynx summary DF-- Dilution factor SC-- PFOS salt correction constant (0.9275) FV-- Final extract volume (1.0 mL unless otherwise noted) EV-- Volume o f sera extracted Formula Used for Liver Analyses in Study FACT-TOX-097 AR (ng/g) x 9 cu rv e(1) x SC x DF x 1.0 pg = Reported C oncentration (pg/g) 9 sample 1000 ng (1) 9 curve is assumed to be: 1 g liver 5 mL H2 O Calculation Used for Group 3, Animal ID 8690F (PFOS) 119 ng/g x 1 g/ 5 mL x 0.9275 x 100 x 1.0 pg = 10.8 pg/g 1.0200 g/5m L 1000 ng AR-- Analytical result from MassLynx summary 9 curve-- Density o f the liver standard curve, assumed to be lg liver/ 5 ml water 9 sample-- Density o f the liver sample (g sample/ 5 mL H2 O) SC-- PFOS salt correction constant (0.9275) DF-- Dilution factor 3M Environmental Laboratory Page 140 3M M edical D epartm ent Study: T -6316.8 Appendix G: Interim Certificate(s) of Analysis BACK TO MAIN A nalytical R eport: F A C T T O X -0 9 7 L R N -U 2 4 5 2 3M Environmental Laboratory Page 141 BACK TO MAIN 3M Medical Department Study: T-6316.8 Analytical Report: FACT TO X-097 L R N -U 2 4 5 2 Centre Analytical Laboratories, Inc. k 3048 Research Drive State College, PA 16801 ^ Phone: (814) 231-8032 Fax: (814) 231-1253 or (814) 231-1580 IN T E R IM C E R T IF IC A T E O F A N A L Y S IS Centre Analytical Laboratories COA Reference #: 023-022-1 3M Product: EtFOSE-OH Test Control Reference #: SD-013 Purity: 88.9% Purity1 Test Name Appearance Identification NMR Metals (ICP/MS) 1. Calcium 2. Magnesium 3. Sodium 4. Potassium 5. Nickel 6 . Iron 7. Manganese Total % Impurity (NMR) Total % Impurity (LC/MS) Total % Impurity (GC/MS) Related Compounds - POAA Residual Solvents (TGA) Purity by DSC Inorganic Anions (IC) 1. Chloride 2. Fluoride 3. Bromide 4. Nitrate 5. Nitrite 6 . Phosphate 7. Sulfate Organic Acids'* (IC) l. TFA 2. PFPA 3. HFBA 4. NFPA ' Elemental Analysis'1: 1. Carbon 2. Hydrogen 3. Nitrogen 4. Sulfur 5. Fluorine Specifications ; 1 it _ | Yellow-white, waxy solid . ' "1 1 ' ; -;J = si..' ' ' 7 ': J- I'":' - . . -; ' 1' 1. Theoretical Value - 25.2% 2. Theoretical Value = 1.75% 3. Theoretical Value = 2.45% 4. Theoretical Value = 5.60% 5. Theoretical Value = 56.6% Result 88.9% Conforms Positive 1 . <0 .0 0 1 wt./wt.% 2 . <0 .0 0 1 wt./wt.% 3. <0.001 wt./wt.% 4. 0.002 wt./wt.% 5. <0.001 wt./wt.% 6 . <0 .0 0 1 wt./wt.% 7. <0.001 wt./wt.% 0.90 wt./wt.% None Quantified 1 0 .2 1 wt./wt.% 0.03 wt./wt.% None Detected 87.6 wt./wt.%. 1. <0.015 wt./wt.% 2. <0.005 wt./wt.% 3. <0.040 wt./wt.% 4. <0.009 wt./wt.% 5. <0.006 wt./wt.% 6 . <0.007 wt./wt.% 7. <0.040 wt./wt.% 1 . <0 .1 wt./wt.% 2 . <0 .1 wt./wt.% 3. <0 .1 wt./wt.% 4. <0.25 wt./wt.% 1. 24.42 wt./wt.% 2. 1.78wt./wt.% 3. 2.72 wt./wt.% 4. 9.34 wt./wt.% 5. 58.4wt./wt.% COA023-022-1 3M Environmental Laboratory Page 1 o f 3 Page 142 BACK TO MAIN 3M Medical Department Study: T-6316.8 Analytical Report: FACT TO X-097 LRN-U2452 Centre Analytical Laboratories, Inc. 3048 Research Drive State College, PA 16801 Phone: (814) 231-8032 Fax: (814) 231-1253 or (814) 231-1580 IN T E R IM C E R T IF IC A T E O F A N A L Y S IS Centre Analytical Laboratories COA Reference #: 023-022-1 3M Product: EtFOSE-OH Test Control Reference #: iSD-013 Date o f Last Analysis: 11/26/00 Expiration Date: 11/26/01 Storage Conditions: <-10 C Re-assessment Date: 11/26/01 'Purity = 100% - (total metal impurities, 0.002% + total NMR impurities, 0.90% + GC/MS impurities, 10.21 + POAA, 0.03%) Total impurity from all tests = 11.14% Purity = 100% -11.14% = 88.9% 2TFA HFBA NFPA PFPA Trifluoroacetic acid Heptafluorobutyric acid Nonafluoropentanoic acid Pentafluoropropanoic acid 3Theoretical value calculations based on the empirical formula, C12H10F17NO3S (MW=571) COA023-022-1 3M Environmental Laboratory Page 2 o f 3 Page 143 BACK TO MAIN 3M Medical Department Study: T-6316.8 Analytical R eport: FACT TO X -097 L R N -U 2 4 52 Centre Analytical Laboratories, Inc. 3048 Research Drive State College, PA 16801 Phone: (814) 231-8032 Fax: (814) 231-1253 or (814) 231-1580 IN T E R IM C E R T IF IC A T E O F A N A L Y S IS Centre Analytical Laboratories COA Reference #: 023-022-1 3M Product: EtFOSE-OH Test Control Reference #: SD-013 GC/MS Purity Profile Peak# 1 2 3 4 5 6 7 8 9 10 11 12 13 Total Retention Time (min) 6.163 8.011 8.206 9.065 9.844 13.93 14.238 15.130 15.52 15.941 16.379 16.801 17.222 - Identity Unknown Unknown Unknown Unknown Unknown Unknown Unknown C2 C3 C4 C5 C6 C7 - % Impurity 0.12 0.23 0.51 0.21 0.34 0.62 0.11 0.11 1.11 1.55 1.07 3.30 0.93 10.21 This work was conducted under EPA Good Laboratory Practice Standards (40 CFR 160). Prepared By: id S. Bell Scientist iratories Reviewed By: John Flaherty Laboratory Manager Centre Analytical Laboratories COA023-022-1 3M Environmental Laboratory Date Date Page 3 of 3 Page 144 ' 3M M edical D epartm ent Study: T-6316.8 Appendix H: Report Signature Page BACK TO MAIN Analytical Report: FAC T TO X -097 LR N -U 2 4 52 Marvin T. Case, D.V.M., Ph.D., Study Director ' Date ' 7John L. Butenhoff, Ph.D,, Sponsor Representative <>/ V / V / Date ------------ - Kristen J. Hansen, Ph.D., PrincipalAnalytical Investigator D2JO &} f)l Date . William K. Reagen, Ph.D., Laboratory Manager O J j& y o ; Date 3M Environmental Laboratory Page 145