Document 062xm2V3287mpR628wgXN4YvV

Northwest Bioanalytical Study No. NWBS99-116 Report No. NWBR99-113 Quantitative Determination of PFOS, PFOSA, PFOSAA, PFHS, POAA, M556 and M570 in Human Serum by LC/MS/MS Sample Analysis Report for Protocol EPI-Swed Northwest Bioanalytical (NWB) A Division of NWT Inc. 1121 East 3900 South Salt Lake City, UT 84124 PREPARED FOR: 3M Environmental Technology and Services (3M) 935 Bush Avenue St. Paul, MN 55133 AUTHOR: David Vollmer, Ph.D., NWB Project Manager Laboratory Director Page 1 DATE: DATE: Northwest Bioanalytical QUALITY ASSURANCE STATEMENT Study No. NWBS99-116 Report No. NWBR99-113 LABORATORY: NAS11aoD2lrt1tiLhvEwaiskaeiseostntC3BNi9ti0yoW0,aUnTSa,TolIyun8tteih4c.1a2l 4(NWB) SPONSOR: 39S3Mt.5PEBanuuvls,ihrMoAnNvme5en5nu1tea3l3Technology Services (3M) COMPOUND(S): PFOS, PFOSA, PFOSAA, PFHS, POAA, M556 and M570 NWB STUDY NUMBER: NWBS99-116 SPONSOR STUDY NUMBER: EPI-Swed NWB STUD, Y TITLE: HExutmraacntioSneraunmdbAynHalPysLisC/oEflPecFtOroSsparnadyoMthaesrsFSlpueocrotrcohmeemtriycals in rPTP2oe1rafhga,retucthPltce5iAalc8tiUBeen),d.iSScNthna.taoloeFntnsDOidafcaimAElcirnCadpGitlDciOeooarnoaPldndsrNiaitnLnuloaycda.nsiyb4pci.ose2ler4Hsafst.otu)ooordwrfTNyyeGhovdPeoneerocrasfd,locciNlrtnLliioicboacewerbatdRliohnrLiwegnagateiobtusnhroltsaiyrsBptaiPeriotoeocrnaptarsicynootfarniSotclsytreiustwNidanceinoaeordnlsetcctioplhnoineencnrlitdfJuchoauaderpclematSdLsneaaweadfbleslibeottyshyrMtaioutnthHodfteriDhWeyNesrSuWwdGgteuisoBftdhio(inOiedQnistrLAidto(haTiUnnbeiaotopglnrefeuacarit2edo1SGeNrOylCLionPPF.e.Rs Inspection and Reporting Statement. I20001n18977s-pDDSS3eeee0ecppcctAi11o11u999n9g9999D99991a9t9e9 FPARSSihaaennmmapaasollppeyrllRtteeoiDecfRAparSleonatcPrafutetlld/yiaRypsntaisw Data N0300128097WADDSSBeeeeuppccgP11r111oD99999j99999ea9999c9tet MInsapneacgteiorn Re33333*p10000NorAWDDSSteeueeIBppsccgsMu1111199e999ad99999n99999Tagoement ^Reports to NWB Management are issued monthly. tAhisc_afinnarleraespoonratbalyccbueraetsetlaybrleisfhleecdt,tthheermawethdoadtas. and procedures described and the results incorporated into SQhuaaulintdyeAl sPseurrcaenyc,eBC.So.m, Mpii.;T Date Page 2 Northwest Bioanalytical Study No. NWBS99-116 Report No. NWBR99-113 Compliance Statement The clinical sample analysis study described within this report is not included within the definition of a GLP regulated nonclinical study set forth in Title 21 of the U.S. Code of Federal Regulations Part 58. However, to the best of our knowledge, this project was conducted in accordance with the guidelines of the U.S. FDA Good Laboratory Practice RmeegthuoladtsioannsdfporroNceodnucrliensicdaelscLraibbeodrawtoirtyhiSntuthdiisesre(pToirttl.e 2In1 aCdFdRitioPna,rtth5e8)staunddyafcoclloorwdiendgtthoethe guidelines of the OECD Principles of Good Laboratory Practice and the Japanese MHW Good Laboratory Practice Standard Ordinance for Nonclinical Laboratory Studies on the Safety of Drugs (Ordinance No. 21, PAB Notification No. 424.) There were no known rinepcirdeesenntststhaant aacfcfeucrtaetde rtheceoqrudaoliftythoerrianwtedgarittay. of the project or data reported. This report NWB Project Manager NWB Laboratory Director Page 3 / Northwest Bioanalytical TABLE OF CONTENTS Study No. NWBS99-116 Report No. NWBR99-113 SIGNATURE PAGE.... ........................................................................................................................... 1 QUALITY ASSURANCE STATEMENT........................................................................................... 2 COMPLIANCE STATEMENT..............................................................................................................3 TABLE OF CONTENTS........................................................................................................................ 4 LIST OF TABLES................................................................................................................................... 4 1. INTRODUCTION............................................................................................................................6 2. METHODOLOGY.......................................................................................................................... 7 3. SAMPLE ANALYSIS....................................................................................................................9 4. RESULTS AND DISCUSSION.................................................................................................. 10 5. REFERENCES.............................................................................................................................. 10 LIST OF TABLES Table 1. Calibration Curve Summary for PFOS in Human Serum.................................................12 Table 2. Calibration Curve Summary for PFOSA in Human Serum..............................................12 Table 3. Calibration Curve Summary for PFOSAA in Human Serum............................................12 Table 4. Calibration Curve Summary for PFHS in Human Serum................................................... 13 Table 5. Calibration Curve Summary for POAA in Human Serum................................................. 13 Table 6. Calibration Curve Summary for M556 in Human Serum...................................................13 Table 7. Calibration Curve Summary for M570 in Human Serum...................................................14 Table 8. Back-Calculated Concentrations of Calibration Standards for PFOS in Human Serum................................................................ i.....................................15 Table 9: Back-Calculated Concentrations of Calibration Standards for PFOSA in Human Serum.....................................................................................................15 Page 4 Northwest Biqanalytical Study No. NWBS99-116 Report No. NWBR99-113 Table 10. Back-Calculated Concentrations of Calibration Standards for PFOSAA in Human Serum.....................................................................:..........................16 Table 11. Back-Calculated Concentrations of Calibration Standards for PFHS in Human Serum...................................................................................................... 16 Table 12. Back-Calculated Concentrations of Calibration Standards for POAA in Human Serum...................................................................................................... 17 Table 13. Back-Calculated Concentrations of Calibration Standards for M556 in Human Serum....................................................................................................... 17 Table 14. Back-Calculated Concentrations of Calibration Standards for M570 in Human Serum....................................................................................................... 18 Table 15. Analytical QC Summary for PFOS in Human Serum....................................................19 Table 16. Analytical QC Summary for PFOSA in Human Serum..................................................19 Table 17. Analytical QC Summary for PFOSAA in Human Serum..............................................20 Table 18. Analytical QC Summary for PFHS in Human Serum....................................................20 Table 19. Analytical QC Summary for POAA in Human Serum.................................................. 21 Table 20. Analytical QC Summary for M556 in Human Serum................................................... 21 Table 21. Analytical QC Summary for M570 in Human Serum....................................................22 Table 22. Study Sample Concentrations.............................................................................................23 Page 5 Northwest Bioanalytcal Study No. NWBS99-116 Report No. NWBR99-113 Quantitative DetearnmdinMat5i7o0n ionfHPFumOSan, PSFeOruSmA,bPyFLOCS/AMAS,/MPFSHS, POAA, M556 Sample Analysis for Protocol EPI-Swed 1. INTRODUCTION This report summarizes the analytical results from the quantitation of PFOS, PFOSA, PFOSAA, PFHS, POAA, M556 and M570 in human serum samples for 3M Environmental Technology and Services in support of Protocol EPI-Swed [5.1]. The validated LC/MS/MS method provided a lower limit of quantitation (LLOQ) of 31.4 (PFOS), 1.00 (PFOSA), 1.45 (PFOSAA), 2.61 (PFHS) and 5.27 (POAA) ppb. All analytes exhibited a linear response to approximately 500 ppb with the exception of PFOSAA, which had an upper limit of quantitation (ULOQ) of 270 ppb [5.2]. The nonvalidated method for the quantitation for M556 and M570 in human serum provided a lower limit of quantitation (LLOQ) of 1.00 and 5.00 ppb, respectively, and exhibited a linear response to 500 ppb for both analytes. The testing facility was 3M Corporate Occupational Medicine (Bldg. 220-3W-05, St. Paul, MN 55144-1000), and J.H. Mandel, M.D., at the facility served as the Principal Investigator. The following is a list ofNWB supervisory personnel involved in the completion of this work: David Vollmer, Ph.D. (NWB Project Manager); Rodger L. Foltz, Ph.D. (NWB Laboratory Director). NWB SOPs were used in the conduct of this study and were available to study personnel in electronic and/or hard copy formats. Date Study Initiated: September 7, 1999 Date Analyses Completed: September 20, 1999 The clinical study described in this report is not included within the definition of a GLP regulated nonclinical study. However, Northwest Bioanalytical conducts all studies within the guidelines of the U.S. FDA Good Laboratory Practice Regulations for Page 6 Northwest Bioanalytical Study No. NWBS99-116 Report No. NWBR99-113 Nonclinical Laboratory Studies (Title 21 CFR Part 58), the OECD Principles of Good Laboratory Practice and the Japanese MHW Good Laboratory Practice Standard Ordinance for Nonclinical Laboratory Studies on the Safety of Drugs (Ordinance No. 21, PAB Notification No. 424). Any changes to or deviations from the original protocol (Analytical Plan) were documented through approved protocol amendments or deviation memos and are retained within the raw data. 2. METHODOLOGY The validated assay used for this study is Meth98082.00 and was reported in Northwest Bioanalytical report NWBR99-005 [5.2]. Samples for this study were received at NWB on the following date: Receipt Date August 27, 1999 Reference Material Number o fSamples Received 39 Storage Condition (except during analysis) - 20C Analyte Lot Number Purity Expiration Date Source Storage Conditions *PFOS (FC-95) 193 100% 12/31/2010 3M Room T(edmryp)erature PFOSA *(PFFCO-1S2A9A) PFHS (*FPCO1A4A3) M556 214 100% 617 53.8% S398-182 100% 245 100% 113N04B7-80 99.89% 12/31/2010 12/31/2010 12/31/2010 12/31/2010 12/31/2010 3M Room Temperature 3M Room Temperature 3M - 20C 3M Room T(demryp)erature 3M Room Temperature M570 118506-26 99.75% dSoDdeScy(slsoudlifuamte) 17H0459 99% 12/31/2010 1/16/2001 3M , Room Temperature Sigma Room Temperature * Analyte names used throughout this report do not include their FC identifier; for example, PFOS is synonymous with PFOS (FC-95) Page 7 Northwest Bioanalytical Study No. NWBS99-116 Report No. NWBR99-113 Nine calibration standards were prepared fresh daily by adding the appropriate spiking standard solution into 0.200 mL of human serum. The final calibration standard concentrations in human serum for each analyte were as follows: Analyte PFOS PFOSA PFOSAA PFHS POAA M556 M570 Calibration Standard Concentrations (ppb) 31.4, 35.4, 40.4, 55.4, 80.4, 130, 280, 430, 530 1.00, 5.00, 10.0, 25.0, 50.0, 100, 250, 400, 500 1.45, 3.60, 6.29, 14.4, 27.8, 54.7, 135, 216, 270 2.61, 6.61, 11.6, 26.6, 51.6, 102, 252, 402, 502 5.27, 9.27,14.3, 29.3, 54.3, 104, 254, 404, 504 1.00, 5.00,10.0, 25.0, 50.0, 100,250,400, 500 1.00, 5.00, 10.0, 25.0, 50.0, 100, 250, 400, 500 Analytical QCs were prepared in human serum on September 13, 1999 and frozen in a -20C freezer. Analytical QCs at the following levels were assayed in duplicate in each analytical run: Analyte PFOS PFOSA PFOSAA PFHS POAA M556 M570 QC Concentrations (ppb) Low (50.4) and High (380) Low (20.0) and High (350) Low (11.7) and High (189) Low (21.6) and High (352) Low (24.3) and High (354) Low (20.0) and High (350) Low (20.0) and High (350) After the addition of HPLC-grade water and sodium dodecyl sulfate (SDS, internal standard) to 0.200 mL of human serum, the serum mixture is made basic with the addition of 0.5 M tetrabutylammonium hydrogen sulfate (TBA, pH. 10) and 0.25 M carbonate buffer. This mixture is then extracted with methyl-tert-butyl ether. After sufficient mixing, the sample is centrifuged. The organic layer is transferred via pipette into a clean test tube. The organic layer is then evaporated to dryness and the sample is Page 8 Northwest Bioanalytical Study No. NWBS99-116 Report No. NWBR99-113 reconstituted into 2 mM ammonium acetate watermethanol (50:50 v/v). The extracts are then analyzed by liquid chromatography/tandem mass spectrometry using negative-ion electrospray ionization and multiple reaction monitoring. 3. SAMPLE ANALYSIS The prepared samples, calibration standards and QCs were injected into the PE Sciex API 3000 LC/MS/MS system in a systematic order. Chromatographic peak integration was performed using MacQuan software (version 1.6). Quadratic regression analysis (weighted 1/x) for all analytes was performed using the peak area ratio vs. concentration utilizing Watson DMLIMS software (version 5.4.10.2). 3.1. Acceptance Criteria For an analytical run to be accepted, it must have met the acceptance criteria listed below which are consistent with regulatory and industry recommendations. Calibration Curve Each run will include duplicate calibration standards at 7 or more concentrations covering the lower to upper limit of quantitation. At least two-thirds of the calibration standard's back-calculated concentrations must be within 15% ( 20% for LLOQ) of their individual target concentrations. If a calibration standard is not within the acceptance criteria, it is deactivated. This process starts from the highest calibration standard down to the lowest until all the active standards are within the acceptance criteria. Lower Limit of Quantitation The back-calculated concentrations of at least one of the duplicate lowest points in the calibration curve must be within 20% of the target concentration to qualify as the LLOQ. If this criterion is not met, the next level is subjected to the same test and the LLOQ raised accordingly. Page 9 Northwest Bioanalytical Quality Control Samples Study No. NWBS99-116 Repon No. NWBR99-113 Each analytical run will include low and high QC samples in triplicate. The measured concentrations of at least two-thirds of all analytical QCs must be within 20% of their target concentrations. If study samples require dilution, a dilution QC is analyzed in duplicate for each dilution level. At least one dilution QC at each level must be within 20% in order to accept study sample results at that dilution level. The dilution QC acceptance was independent of the undiluted analytical QC acceptance. 4. RESULTS AND DISCUSSION Each accepted run met the acceptance criteria set for the calibration curve points, for the lower limit of quantitation (LLOQ) and for the analytical quality control (QC) samples. Run PFOS ED Result PFOSA PFOSAA PFHS Result Result Result POAA Result MS56 Result M570 Comments Result 1 accepted accepted accepted accepted accepted accepted accepted PSAE 2 accepted accepted accepted accepted accepted accepted accepted PSAE = Pre-study assay evaluation There were no known circumstances that may have affected the quality or integrity of the data. In conclusion, based on the quality control and calibration curve data, it is our opinion that the data submitted to 3M Environmental Technology and Services for PFOS, PFOSA, PFOSAA, PFHS, POAA, M556 and M570 are accurate for all of the EPI-Swed study samples. Please note that the results for analytes M556 and M570 should be considered unvalidated data. 5. REFERENCES 5.1. 3M Protocol EPI-Swed, "Analysis of Fluorochemicals in Individual Sera from Sweden," August 24, 1999. Page 10 Northwest Bioanalytical Study No. NWBS99-116 Report No. NWBR99-113 5.2. D. Vollmer, "Quantitative Determination of PFOS, PFOSA, PFOSAA, N-MeFOSEOH, N-EtFOSE-OH, POAA and PFHS in Human Serum by LC/MS/MS," NWB Study Number NWBS98-082, NWB Report Number NWBR99-005, May 13, 1999. The raw data and final report for this study will be stored in the NWB Archives, 1121 East 3900 South, Salt Lake City, UT 84124 per regulations and contract agreement. After submission of the final report to the Sponsor, remaining study samples will be stored under required conditions until confirmation of Sample Disposition/Retum Authorization is received from the Sponsor. 3M Environmental Technology and Services and 3M Corporate Occupational Medicine will be notified concerning final disposition of records at completion of contract obligations. Page 11 Northwest Bioanalytical Study No. NWBS99-116 Report No. NWBR99-113 Table 1. Calibration Curve Summary for PFOS in Human Serum Quadratic weighted 1/x. All concentrations are expressed as ppb. Run Date Run Number A B C R-Squared LLOQ LLOQ 16-Sep-1999 2 -0.000003 0.006330 0.056522 0.9964 31.4 530 Mean -0.000003 0.006330 0.056522 0.9964 n 1 11 1 * A, B and C are coefficients used to define the quadratic curve. Table 2. Calibration Curve Summary for PFOSA in Human Serum Quadratic weighted 1/x. All concentrations are expressed as ppb. Run Date Run A B C R-Squared LLOQ LLOQ Number 16-Sep-1999 2 . -0.000009 0.015340 0.006786 0.9967 1.00 500 Mean -0.000009 0.015340 0.006786 0.9967 n 1 11 1 * A, B and C are coefficients used to define the quadratic curve. Table 3. Calibration Curve Summary for PFOSAA in Human Serum Quadratic weighted 1/x. Ail concentrations are expressed as ppb. Run Date Run Number A B C R-Squared LLOQ ULOQ 16-Sep-1999 2 -0.000002 0.001756 0.001162 0.9952 1.45 270 Mean -0.000002 0.001756 0.001162 0.9952 n 1 1 1 1r ' * A, B and C are coefficients used to define the quadratic curve. Page 12 Northwest Bioanalytical Study No. NWBS99-116 Repon No. NWBR99-113 Table 4. Calibration Curve Summary for PFHS in Human Serum Quadratic weighted 1/x. All concentrations are expressed as ppb. Run Date Run Number A B C R-Squared LLOQ LOQ 16-Sep-1999 2 -0.000005 0.009627 0.017026 0.9970 2.61 502 Mean -0.000005 0.009627 0.017026 0.9970 n 1 11 1 * A, B and C are coefficients used to define the quadratic curve. Table 5. Calibration Curve Summary for POAA in Human Serum Quadratic weighted 1/x. All concentrations are expressed as ppb. Run Date NuRmunber A B C R-Squared LLOQ ULOQ 16-Sep-1999 2 -0.000005 0.011221 0.022280 0.9961 5.27 504 Mean -0.000005 0.011221 0.022280 0.9961 n 1 11 1 * A, B and C are coefficients used to define the quadratic curve. Table 6. Calibration Curve Summary for M556 in Human Serum Quadratic weighted 1/x. All concentrations are expressed as ppb. Run Date NuRmunber A B c R-Squared LLOQ ULOQ 16-Sep-1999 2 0.000000 0.000362 0.001145 0.9977 1.00 500 Mean n 0.000000 0.000362 0.001145 111 0.9V9/77 * A, B and C are coefficients used to define the quadratic curve. Page 13 Northwest Bioanalytical Study No. NWBS99-116 Report No. NWBR99-113 Table 7. Calibration Curve Summary for M570 in Human Serum Quadratic weighted 1/x. All concentrations are expressed as ppb. Run Date Run Number A B C R-Squared LLOQ ULOQ 16-Sep-1999 ' 2 -0.000002 0.002831 0.017415 0.9953 5.00 500 Mean -0.000002 0.002831 0.017415 0.9953 n 111 1 * A, B and C are coefficients used to define the quadratic curve. Page 14 Northwest Bioanalytical Study No. NWBS99-116 Report No. NWBR99-113 Table 8. Back-Calculated Concentrations of Calibration Standards for PFOS in Human Serum Quadratic weighted 1/x. All concentrations are expressed as ppb. Run Date Run Number 31.4 35.4 40.4 55.4 80.4 130 280 430 530 16-Sep-1999 2 *42.2 36.0 42.6 56.5 83.2 119 305 397 534 28.6 *26.3 40.8 56.1 78.7 131 *340 419 . 552 . Mean S.D. %CV %Bias n 28.6 36.0 41.7 56.3 81.0 125 305 408 543 N.C. N.C. 1.27 0.283 3.18 8.49 N.C. 15.6 12.7 - N.C. N.C. 3.0 0.5 3.9 6.8 N.C. 3.8 2.3 -8.9 1.7 3.2 1.6 0.7 -3.8 8.9 -5.1 2.5 1 1 2 2 22 122 * Sample deactivated as an outlier (> 15% theoretical for non-LLOQ; > 20% theoretical for LLOQ) N.C. = not calculable Table 9. Back-Calculated Concentrations of Calibration Standards for PFOSA in Human Serum Quadratic weighted 1/x. All concentrations are expressed as ppb. Run Date Run Number 1.00 5.00 10.0 25.0 50.0 100 250 400 500 16-Sep-1999 2 1.04 *7.31 t--* A tb-- `o 25.3 51.5 87.0 253 354 494 0.809 4.90 10.8 26.7 51.5 109 *348 419 535 Mean 0.925 4.90 10.8 26.0 51.5 98.0 253 387 515 S.D. 0.163 N.C. N.C. 0.990 0.00 15.6 N.C. 46.0 29.0 %CV 17.6 N.C. N.C. 3.8 0.0 15.9 N.C. 11.9 5.6 %Bias -7.5 -2.0 8.0 4.0 3.0 - 2.0 1.2 -3.3 3.0 n 2 1 1 2 22 122 * Sample deactivated as an outlier (> 15% theoretical for non-LLOQ; > 20% theoretical for LLOQ) N.C. = not calculable Page 15 Northwest Bioanalytical Study No. NWBS99-116 Report No. NWBR99-113 Table 10. Back-Calculated Concentrations of Calibration Standards for PFOSAA in Human Serum Quadratic weighted 1/x. All concentrations are expressed as ppb. Run Date Run Number 1.45 3.60 6.29 14.4 27.8 54.7 135 216 270 16-Sep-1999 2 1.28 *4.35 6.76 15.1 28.2 48.6 135 185 284 1.36 3.16 7.06 16.0 28.9 51.5 154 210 285 Mean 1.32 3.16 6.91 15.6 28.6 50.1 145 198 285 S.D. 0.0566 N.C. 0.212 0.636 0.495 2.05 13.4 17.7 0.707 %CV 4.3 N.C. 3.1 4.1 1.7 4.1 9.2 8.9 0.2 %Bias -9.0 -12.2 9.9 8.3 2.9 -8.4 7.4 -8.3 5.6 n 2 1 2 2 2 222 2 * Sample deactivated as an outlier (> 15% theoretical for non-LLOQ; > 20% theoretical for LLOQ) N.C. = not calculable Table 11. Back-Calculated Concentrations of Calibration Standards for PFHS in Human Serum Quadratic weighted 1/x. All concentrations are expressed as ppb. Run Date Run Number 2.61 6.61 11.6 26.6 51.6 102 252 402 502 16-Sep-1999 2 2.33 6.97 11.7 27.4 51.3 89.6 257 368 501 *1.44 *5.13 11.5 28.8 52.3 99.8 281 391 528 Mean 2.33 6.97 11.6 28.1 51.8 94.7 269 380 515 S.D. N.C. N.C. 0.141 0.990 0.707 7.21 17.0 16.3 19.1 %CV N.C. N.C. 1.2 3.5 1.4 7.6 6.3 4.3 3.7 %Bias -10.7 5.4 0.0 5.6 0.4 -7.2 6.7 -5.5 2.6 n 1 1 2 2 2 /2 2 2 2 * Sample deactivated as an outlier (> 15% theoretical for non-LLOQ; > 20% theoretical for LLOQ) N.C. = not calculable Page 16 Northwest Bioanalytical Study No. NWBS99-11 Report No. NWBR99-113 Table 12. Back-Calculated Concentrations of Calibration Standards for POAA in Human Serum Quadratic weighted 1/x. All concentrations are expressed as ppb. Run Date Run Number 5.27 9.27 14.3 29.3 54.3 104 254 404 504 16-Sep-1999 2 5.27 8.96 14.0 29.9 53.9 90.6 260 367 507 *3.38 *6.93 14.4 32.6 54.8 100 287 399 519 Mean 5.27 8.96 14.2 31.3 54.4 95.3 274 383 513 S.D. N.C. N.C. 0.283 1.91 0.636 6.65 19.1 22.6 8.49 % cv N.C. N.C. 2.0 6.1 1.2 7.0 7.0 5.9 1.7 %Bias 0.0 -3.3 -0.7 6.8 0.2 -8.4 7.9 -5.2 1.8 n 1 1 2 2 2 2222 * Sample deactivated as an outlier (> 15% theoretical for non-LLOQ; > 20% theoretical for LLOQ ) N.C. = not calculable Table 13. Back-Calculated Concentrations of Calibration Standards for M556 in Human Serum Quadratic weighted 1/x. All concentrations are expressed as ppb. Run Date Run Number 1.00 5.00 10.0 25.0 50.0 100 250 400 500 16-Sep-1999 2 0.973 5.26 *8.16 25.0 50.0 95T 244 375 513 *<LLOQ *1.84 9.53 26.5 50.5 95.1 281 393 502 Mean 0.973 5.26 9.53 25.8 50.3 95.1 263 384 508 S.D. N.C. N.C. N.C. 1.06 0.354 0.00 26.2 12.7 7.78 %CV N.C. N.C. N.C. 4.1 0.7 0.0 10.0 3.3 1.5 %Bias -2.7 5.2 -4.7 3.2 0.6 -4.9 5.2 -4.0 1.6 n 1 1 1 2 2 22 22 * Sample deactivated as an outlier (> 15% theoretical for non-LLOQ; > 20% theoretical for LLOQ) N.C. = not calculable Page 17 Northwest Bioanalytical Study No. NWBS99-116 Report No. NWBR99-113 Table 14. Back-Calculated Concentrations of Calibration Standards for M570 in Human Serum Quadratic weighted 1/x. All concentrations are expressed as ppb. Run Date Run Number 1.00 5.00 10.0 25.0 50.0 100 250 400 500 16-Sep-1999 2 *<LLOQ 4.05 10.0 26.1 53.7 89.6 244 350 504 *<LLOQ *1.78 10.9 *29.1 54.3 96.6 283 386 558 Mean 4.05 10.5 26.1 54.0 93.1 264 368 531 S.D. N.C. 0.636 N.C. 0.424 4.95 27.6 25.5 38.2 %CV N.C. 6.1 N.C. 0.8 5.3 10.5 6.9 7.2 %Bias -19.0 5.0 4.4 8.0 -6.9 5.6 -8.0 6.2 n 1 2 1 2 2222 * Sample deactivated as an outlier (> 15% theoretical for non-LLOQ; > 20% theoretical for LLOQ) N.C. = not calculable Page 18 Northwest Bioanalytical Study No. NWBS99-116 Report No. NWBR99-113 Table 15. Analytical QC Summary for PFOS in Human Serum All concentrations are expressed as ppb. Run Date Run Number 16-Sep-1999 2 Low QC 50.4 ppb 53.2 55.4 55.9 High QC 380 ppb 432 416 387 Mean S.D. %CV %Theoretical %Bias n 54.8 412 1.44 22.8 2.6 5.5 108.7 108.4 8.7 8.4 33 Table 16. Analytical QC Summary for PFOSA in Human Serum All concentrations are expressed as ppb. Run Date Run Number 16-Sep-1999 2 Low QC 20.0 ppb 23.6 19.3 20.1 High QC 350 ppb *448 385 405 Mean S.D. %CV %Theoretical %Bias n 21.0 2.29 . 10.9 105.0 5.0 3 * > 20%theoretical 413 32.2 7.8 118.0 183/).0 Page 19 Northwest Bioanalytical Study No. NWBS99-116 Report No. NWBR99-113 Table 17. Analytical QC Summary for PFOSAA in Human Serum All concentrations are expressed as ppb. Run Date Run Number 16-Sep-1999 2 Low QC 11.7 ppb 13.3 *14.4 *14.1 H18ig9hpQpbC 218 221 194 Mean S.D. %CV %Theoretical %Bias n 13.9 211 0.569 14.8 4.1 7.0 118.8 111.6 18.8 11.6 33 * >20% theoretical Table 18. Analytical QC Summary for PFHS in Human Serum All concentrations are expressed as ppb. Run Date Run Number 16-Sep-1999 2 Low QC 21.6 ppb 21.8 22.7 23.8 H35ig2hpQpbC 384 381 330 Mean S.D. %CV "/(/Theoretical %Bias n 22.8 365 1.00 30.3 4.4 8.3 105.6 103.7 5.6 3.7 33 Page 20 Northwest Bioanalytical Study No. NWBS99-116 Report No. NWBR99-113 Table 19. Analytical QC Summary for POAA in Human Serum All concentrations are expressed as ppb. Run Date Run Number 16-Sep-1999 2 Low QC 24.3 ppb 23.1 24.3 25.6 High QC 354 ppb 369 381 333 Mean S.D. %CV %Theoretical %Bias n 24.3 361 1.25 25.0 5.1 6.9 100.0 102.0 0.0 2.0 33 Table 20. Analytical QC Summary for M556 in Human Serum All concentrations are expressed as ppb. Run Date Run Number 16-Sep-1999 ' 2 Low QC 20.0 ppb 16.6 19.0 23.8 High QC 350 ppb 374 360 312 Mean S.D. %CV "/Theoretical %Bias n 19.8 349 3.67 32.5 18.5 9.3 99.0 99?! - 1.0 -0.3 33 Page 21 Northwest Bioanalytical Study No. NWBS99-116 Report No. NWBR99-113 . Table 21. Analytical QC Summary for M570 in Human Serum - All concentrations are expressed as ppb. Run Date Run Number 16-Sep-1999 2 Low QC 20.0 ppb 22.4 *24.5 22.5 High QC 350 ppb 392 382 355 Mean S.D. %CV "/(Theoretical %Bias n 23.1 1.18 5.1 115.5 15.5 3 * > 20%theoretical 376 19.1 5.1 107.4 7.4 3 Page 22 Northwest Bioanalytical Sample ID 9506 9507 9508 9509 9510 9511 9512 9513 9514 9515 9516 9517 9518 9519 9520 9521 9522 9523 9524 9525 9526 PFOS < L L O Q (3 1.4) < L L O Q (3 1.4) < L L O Q (3 1.4) < L L O Q (3 1.4) < L L O Q (3 1.4) < L L O Q (3 1.4) < L L O Q (3 1.4) <LLOQ(31.4) < L L O Q (3 1.4) <LLOQ(31.4) < L L O Q (3 1.4) < L L O Q (3 1.4) <LLOQ(31.4) 32.1 31.8 <LLOQ(31.4) <LLOQ(31 4) <LLOQ(31.4) 33.1 < L L O Q (3 1.4) <LLOQ(31.4) PFOSA <LLOQ(1.00) <LLOQ(1.00) <LLOQ(1.00) <LLOQ(1.00) <LLOQ(1.00) <LLOQ(1.00) <LLOQ(1.00) <LLOQ(1.00) <LLOQ(1.00) <LLOQ(1.00) <LLOQ(1.00) <LLOQ(1.00) <LLOQ(1.00) <LLOQ(1.00) <LLOQ(1.00) <LLOQ(1.00) <LLOQ(l .00) <LLOQ(1.00) <LLOQ(1.00) . <LLOQ(1.00) <LLOQ(1.00) Table 22. Study Sample Concentrations All concentrations are expressed as ppb. PFOSAA PFHS POAA <LLOQ(1.45) <LLOQ(1.45) <LLOQ(1.45) <LLOQ(1.45) <LLOQ(l .45) <LLOQ( 1.45) <LLOQ(l .45) <LLOQ(1.45) <LLOQ(1.45) <LLOQ(1.45) <LLOQ(1.45) <LLOQ(1.45) <LLOQ(1.45) <LLOQ(l .45) <LLOQ(1.45) <LLOQ(1.45) <LLOQ(1.45) <LLOQ(1.45) <LLOQ(1.45) <LLOQ(1.45) <LLOQ(1.45) <LLOQ(2.61) <LLOQ(2.61) <LLOQ(2.61) <LLOQ(2.61) <LLOQ(2.61) <LLOQ(2.61) <LLOQ(2.61) <LLOQ(2.61) <LLOQ(2.61) <LLOQ(2.61) <LLOQ(2.61) <LLOQ(2.61) <LLOQ(2.61) <LLOQ(2.61) <LLOQ(2.61) <LLOQ(2.61) <LLOQ(2.61) <LLOQ(2.61) <LLOQ(2.61) <LLOQ(2.61) <LLOQ(2.61) <LLOQ(5.27) <LLOQ(5.27) <LLOQ(5.27) <LLOQ(5.27) <LLOQ(5.27) <LLOQ(5.27) <LLOQ(5.27) <LLOQ(5.27) <LLOQ(5.27) <LLOQ(5.27) <LLOQ(5.27) <LLOQ(5.27) <LLOQ(5.27) <LLOQ(5.27) <LLOQ(5.27) <LLOQ(5.27) <LLOQ(5.27) <LLOQ(5.27) 6.43 <LLOQ(5.27) <LLOQ(5.27) ( ) value in parentheses is the LLOQ Page 23 Study No. NWBS99-116 Report No. NWBR99-113 M556 <LLOQ(1.00) <LLOQ(1.00) <LLOQ(1.00) <LLOQ(1.00) <LLOQ(1.00) <LLOQ(1.00) <LLOQ(1.00) <LLOQ(1.00) <LLOQ(1.00) iLLOQ(l.OO) <LLOQ(1.00) <LLOQ(1.00) <LLOQ(1.00) <LLOQ(1.00) <LLOQ(1.00) <LLOQ(1.00) <LLOQ(1.00) <LLOQ(1.00) <LLOQ(l .00) <LLOQ(1.00) <LLOQ(l .00) M570 <LLOQ(5.00) <LLOQ(5.00) <LLOQ(5.00) <LLOQ(5.00) <LLOQ(5.00) <LLOQ(5.00) <LLOQ(5 00) <LLOQ(5.00) <LLOQ(5.00) <LLOQ(5.00) <LLOQ(5.00) <LLOQ(5.00) <LLOQ(5.00) <LLOQ(5.00) <LLOQ(5.00) <LLOQ(5.00) <LLOQ(5.00) <LLOQ(5.00) <LLOQ(5.00) <LLOQ(5.00) <LLOQ(5.00) Northwest Bioanalytical Sample ID PFOS 9527 <LLOQ(31.4) 9528 <LLOQ(31.4) 9529 <LLOQ(31.4) 9530 43.3 9531 <LLOQ(31.4) 9532 <LLOQ(31.4) 9533 45.9 9534 58.5 9535 37.6 9536 <LLOQ(31.4) 9537 <LLOQ(31.4) 9538 85.4 9539 72.9 9540 <LLOQ(31.4) 9541 31.6 9542 56.1 9543 <LLOQ(31.4) 9544 <LLOQ(31.4) Table 22. Study Sample Concentrations (concentrations) All concentrations are expressed as ppb. PFOSA <LLOQ(1.00) <LLOQ(1.00) <LLOQ(1.00) <LLOQ(1.00) <LLOQ(1.00) <LLOQ(l .00) <LLOQ(1.00) <LLOQ(1.00) <LLOQ(1.00) <LLOQ(l .00) <LLOQ(l .00) <LLOQ(1.00) <LLOQ(1.00) <LLOQ(1.00) <LLOQ(1.00) <LLOQ(1.00) <LLOQ(1.00) <LLOQ(1.00) PFOSAA i PFHS POAA <LLOQ(l .45) <LLOQ(l .45) <LLOQ(1.45) 1.72 1.57 <LLOQ(1.45) 5.75 <LLOQ(1.45) ' <LLOQ(l .45) <LLOQ(1.45) <LLOQ(l .45) <LLOQ(1.45) <LLOQ(1.45) <LLOQ(l .45) <LLOQ(l .45) <LLOQ(1.45) <LLOQ(1.45) <LLOQ(1.45) <LLOQ(2.61) <LLOQ(2.61) <LLOQ(2.61) <LLOQ(2.61) <LLOQ(2.61) <LLOQ(2.61) <LLOQ(2.61) <LLOQ(2.61) <IXOQ(2.61) <LLOQ(2.61) <LLOQ(2.61) <LLOQ(2.61) <LLOQ(2.61) <LLOQ(2.61) <LLOQ(2.61) <LLOQ(2.61) <LLOQ(2.61) <LLOQ(2.61) <LLOQ(5.27) <LLOQ(5.27) 6.10 7.03 <LLOQ(5.27) <LLOQ(5.27) 5.41 <LLOQ(5.27) 6.15 <LLOQ(5.27) 6.12 12.4 7.42 <LLOQ(5.27) <LLOQ(5.27) <LLOQ(5.27) ; <LLOQ(5.27) <LLOQ(5.27) ( ) value in parentheses is the LLOQ Study No. NWBS99-116 Report No. NWBR99-113 M 556 <LLOQ(1.00) <LLOQ(1.00) <LLOQ(l .00) <LLOQ(1.00) <LLOQ(1.00) <LLOQ(1.00) 2.92 <LLOQ(1.00) <LLOQ(l .00) <LLOQ(1.00) <LLOQ(1.00) cLLOQ(l.OO) <LLOQ(1.00) <LLOQ(1.00) <LLOQ(l .00) <LLOQ(1.00) <LLOQ(1.00) <LLOQ(1.00) M 570 <LLOQ(5.00) <LLOQ(5.00) <LLOQ(5.00) <LLOQ(5.00) <LLOQ(5.00) <LLOQ(5.00) <LLOQ(5.00) <LLOQ(5.00) <LLOQ(5.00) <LLOQ(5.00) <LLOQ(5.00) <LLOQ(5.00) <LLOQ(5.00) <LLOQ(5.00) <LLOQ(5.00) <LLOQ(5.00) <LLOQ(5.00) <LLOQ(5.00) Page 24