Document r6qk7b4x3XX2XXyX8j26kRMkr

Norn Sissi ARQL6 -- 1210 suuyvorwssooon Report No. NWBROO-122 Quantitative Determinationof PFOS, PFOSA, PFOSAA, POAA, PFHS, M556 and M570 in Human Serum by LC/MS/MS Assay Revalidation Addendum Report Northwest Bioanalytical (NWB) ADivision of NWT Ine. 1121 East 3900 South Salt Lake City, UT 84124 PREPARED FOR: 3M Environmental Technology and Services 935 Bush Avenue St. Paul, MN 55133 mS 23D7 ST Cit = 85 = 89 2 AUTHOR: . Clrpnee O Lokarite Connie O. Sakashita, B.S., NWB Project Manager So = DATE: 1/7327 APPROVED FOR RELEASE BY: To a Patrick Bennett, M.S., M.B.A., NWB Laboratory Director DATE: _#"2-4/ CONTAIN NO GB! 050204 Page 1 Northwest Bioanalytical ReSptourdtyNNoo.. NNWWBBRS0000--102420 QUALITY ASSURANCE STATEMENT LABORATORY: `ANorDtivhiwseisotnBoifoNanWalTy,ticInacl. (NWE) S1a1l2t1LEaaksetC3i9ty0,0USTou8t4h124 SPONSOR: 933M5 EBnuvsihroAnvmeennuteal Technology Services StPaul, MN 55133 COMPOUNDS): NWB STUDY NUMBER: SPONSORSTUDYNUMBER: PFOS, PROSA, PFOSAA, BFHS, M536 aud M70 NWBS00040 Method Validation NWB STUDY TITLE: `LQOuManStIiMtiSv,e ADestsearymiVnaalitdiaotnoiofn PFOSandMetabolitesiaHuman Serum by Un"ToShn.eclmiFenDitcAhaolGdsotvouadldyiL.daabtHiooorwnasetvuoerdry,PrdNaeocsrtcitrchiewbeeRsdetgiBuniltoahatinsiaorlneyspftoiorctraliNsocnnooentldiuincnitccslaualdleLdapbrwooijrteahctitonsrtyihSatdcuecdfioeirsdiai(noTcnieoewif2at1hGCtLFhPRe.pruPedagruetllai5tn8e)ed,sotfhethe 4O`r2Od4Ei)CnDaTnPhcreeinffcooirlpllNeooswnoicnlfgiGniioncsoapdleLcLtaaibboonorsratwtoeorrryeyPSptreusrdcfiioecrsemoeandntbdhytehtSehaeJfaNeptWaynBoefsDeQrMAugUEsWp(eOrGrSdoOionPda.nLcaeboNroa.to2r1y, PraAcBtiNcoetiSftiacnadtairodn No. Inspection and Reporting Statement. I1n5sMpeactyi2on0D0a0te AhuaasleyotcfaSltuPdlayn Date Inspection ReportIssuedTo N1W5 BMaPyro2j0e0c0tManager \31NWMBaMya2n0a0g0ement 0175--1188SMeapy22000000 Re`ApsosarytVDarlaifdaUtRiaown Data 2108SMeapy22000000 2319SMeapy22000000 24302000 FiOnWaBlRROeOp-or1t08) (NWBR0O-108) 24 132.2000 311302000 1930 112001 05.07 Nov 2001 R(eNpWoBrRt0DOr-a1f2V2R)aw Data ReportDrafRaw Data 303012000 08 Nov 2001 3132000 30 Nov 2001 20 Nov 2001 OFWiBaaRlORCep-o1r2t 2) ONWBRG0-122) 20 Nov 2001 i: 30 Nov 2:001 c- Reportsto NWB Manaageeissmuedemonnthlty. . rAespocratnarcecaursaotnealbylyrebfeleecstttahbeliashwedd,attah.e methods and procedures described nd the results incorporated ito this ial S<Sheryel AgMat.hews, AAAS.Juttends NWB QAU Compliance Auditor 2 Mov 200), owe 030205 Page? Northwest Bioanalytical ReSptourdty NNoo,. NNWWBBRS0000-:102420 COMPLIANCE STATEMENT `The method validation study described in this addendum report is not included within the definitionof a GLP nonclinical regulated study. However, to the bestofour knowledge, the stability, extraction efficiency, and plasma abbreviated validation experiments were conducted in accordance with the guidelines ofthe U.S. FDA Good Laboratory Practice Regulations for Nonclinical Laboratory Studies (Titl2e1 CFR Part 48) and according to the methods and procedures described within this report. In addition, these experiments followed theguidelines ofthe OECD Principles of Good Laboratory Practice and the Japanese MHW GoodLaboratory Practice Standard Ordinance for Nonclinical Laboratory Studies on the SafetyofDrugs (Ordinance No. 21, PAB Notification No. 424.) `This addendum report also included results from research experiments conducted outside ofthe `validation study. These experiments `within section 7 ofthis report. were conducted according to the procedures described Serum sample results obtained using plasma curves for PEOSA, PFOSAA, POAA, PFHS, M556 and M570 did not meet NWB SOP requirements for validation. Any known circumstances that may have affected the quality or integrityofthe study or data are discussed within the report. This report represents an accurate recordofthe raw data. Conic O fOknatll Connie O. Sakashita, B.S. 'NWB Project Manager Date: 2077 GA EtroF---- Patrick K. Bennett, M.S, M.B.A. 'NWB Laboratory Director Date: _#-24-0, B 050206 Page Norwest Bioanalytical ReSptourdty NNoo.. NNWWBBRS0000--102420 TABLE OF CONTENTS SIONATURE PAGEcommunis QUALITY ASSURANCE STATEMENT .conrsmnsssssssssmsntsnssn2 COMPLIANCE STATEMENT... sss d TABLE OF CONcT ssENTS ssssss LIST OP TABLES... 1 INTRODUCTION... 13 2 METHmOmDmOUOG sssesmmmssmmssssmsninsilll 3. STABILITY EVALUATION ccna] 3.1. Freeze-Thaw and Room Temperature Matrix Stability .......crruersvscion|nT 222, SE tion SHI mmm ------------] FEN| J 3.4. LongTerm Matrix Stability in HUMAN SERUM... ssn19 4. EXTRACTION EFFICIENCY FROM HUMAN SERUM...) S. COMMENTS AND CONCLUSIONS... 21 6. ABBREVIATED VALIDA--THIUMOANN PLASMA ..c.oovvomrsrssnnn2s1 6.1. REDGE OF QUENIEON c.cccosrsssrssmmssensmesamsn 33 6.2. Lower Limit Of QUARKBHON mss ssssnsmssssssitssne 23 6.3. Intra-Assay Precision and ACCUICY wvmmmmmsnsssmsssnggnn 24 6.4. Proposed Acceptance Criteria for Human PaSTIA.....ovvvrnrnrsrirsnne28n 7. ADDITIONAL POST-VALIDATION EXPERIMENTS... srsnnn 25 7.1. 100 SUPPFESSION EXPERIMEN vss 2 7.2. Dilution Matrix EXPERMENtconsis 2 7.3. Serum and P1aSMaCOMPEISON errs 29 8. DATA MANAGEMENT ...ovccosmssmmsmssmssassnimmsmssssmssmmsndnl 8, REFERENCES ...ommmmmmsmmssmmmmmmmnssmsmmmnsnmmnndtl 10. DATA BBIBNTION smerny , 090207 Paget Northwest Biosmalytical ReSptourdtyNNoo.. NNWWBBRS0000-.102420 LIST OF TABLES Table 1. Summary of Calibration Curve Parameters for PFOS in Human Serum..........u.u.....32 Table 2. Summary of Calibration Curve Parameters for PFOSA in Human Serum.................32 Table 3. Summary ofCalibration Curve Parameters for PFOSAA in Human Serum................32 `Table 4. Summaryof Calibration Curve Parameters for POAA in Human Sequ ..........33 `Table 5. Summaryof Calibration Curve Parameters for PFHS in Human Setu...............33 `Table 6. Summary ofCalibration Curve Parameters for M556 in Human Serum............v......33 Table 7. SummaryofCalibration Curve Parameters for M570 in Human Serum.............v.v......33 `Table 8. Back-Calculated Concentrations ofCalibration Standards for PFOS in Table 9. Back-Calculated Concentrations of Calibration Standards for PFOSA in Table 10. Back-Calculated Concentrations ofCalibration Standards for PFOSAA in `Table 11. Back-Calculated Concentrationsof Calibration Standards for POAA in `Table 12. Back-Calculated Concentrationsof Calibration Standards for PFHS in `Table 13. Back-Calculated ConcentrationsofCalibration Standards for M556 in Table 14. Back-Calculated ConcentrationsofCalibration Standards for MS70in HUMAN SERUM corrosion36 `Table 15. Quality Control Samples for PFOS in HUMAN SEMUM ..vcvvvverversenrcnssssssssssssnnsn3T `Table 16. Quality Control Samples for PFOSA in Human Serum ...........ccuuuermuurmmssessssesssssonns 37 Table 17. Quality Control Samplesfor PFOSAA in Human Serum ...........oovcrvosrssssssssnnnn3T Pages 050208 Northwest Bioanalytical `Table 18. Quality Control Samples for POAA in HUMAN SERUM... ReSptourdtyNNoo,. NNWWBBRS00D0-.102420 3 `Table 19. Quality Control Samples for PFHS in Human Serum ......ouuwuvursmsssssssssssssssssisne3n8:. `Table 20. Quality Control Samples for M556 in HUMAN SEMUM .........ccovsevermsssssmsssssesssennn3en8:. `Table 21. Quality Control Samples for M570 in HUMAN SEMUM ....ccovevvreverrssnrsssssssesscsssssnens 38. Table 22. Freeze-Thaw and Room Temperature Matrix Stability for PFOS in Table 23. Freeze-Thaw and Room Temperature Matrix Stability for PFOSA in `Table 24. Freeze-Thaw Stability and Room Temperature Matrix for PFOSAA in `Table 25. Freeze-Thaw and Room Temperature Matrix Stability for POAA in `Table 26. Freeze-Thaw and Room Temperature Matrix Stability for PFHS in HUMAN SMUT. d `Table 27. Freeze-Thaw and Room Temperature Matrix Stability for M556 in `Table 28. Freeze-Thaw and Room Temperature Matrix Stability for M570 in `Table 29. Reinjection Stability for PFOS w......uvvuvmmmmmmssmsssssmsssssssssssssssssssssssgpassssssssssssd6 Table 30. Reinjection Stability for PFOSA v.rorcrcnrararcsvsnsmicnnnsssisssivnnndT = `Table 31. Reinjection Stability for PFOSAA........ccosmmmmmsmmmssmsssssssssssssssssssssssssssssssssssssnsid8. Table 32. Reinjection Stability for POAA .cccrcrcrnsnrnsmnsnnssnssnndd Table 33. Reinjection bility or PFHS vcore 50 `Table 34. Reinjection Stability for M556 .......uuuummmmsssmmssmmsssssssssssssssssssssssssssssssssssssssssssiSs|s Table 35. Reinjection StEity For MST0...cecnrararsnsnssssninssnsnssnssinisS2 050209 Pages Northwest Bioanalytical ReSptourdty NNoo.. NNWWBBRSO000-.102420 `Table 36. Room Temperature Extract Sbility or PEOS vss S3 Table 37. Room Temperature Extract Stability for PFOSA ovr 54 Table 38. Room Temperature Extract Stability for PROSAA vests55 `Table 39. Room Temperature Extract Stability for POAA mrss56 `Table 40. Room Temperature Extract Stability for PEHS vss ST Table 41. Room Temperature EXtract Stability for MSS6 rrr58 `Table 42. Room Temperature EXtract Stability fOr MST0 vermin59 `Table 43. Reduced Temperature (1 10 8 C) Extract Stability for PFOS....rrrercn6n0 `Table 44. Reduced Temperature (1 to 8 C) Extract Stability for PFOA... 61 `Table 45. Reduced Temperature (1 to 8 C) Extract Stability for PEOSAA rss62 `Table 46. Reduced Temperature (1 to 8 C) Extract Stability for POAA...crccvrrcerrn63 `Table 47. Reduced Temperature (1 10 8 C) Extract Stability for PEHS v.68 `Table 43. Reduced Temperature (1 10 8 C) Extract Stability for M56... 65 `Table 49. Reduced Temperature (1 t0 8 C) Extract Sability for MST0...rurrrcrrre66 `Table 50. Reduced Temperature (-20 C) Extract Stability for PROS... 67 `Table 51. Reduced Temperature (-20 C) Extract Stability for PFOSA crn 68 Table 52. Reduced Temperature (-20 C) Extract Stability for PFOSAA rrr69 `Table 53. Reduced Temperature (-20 C) Extract Stability for POAA woven 10 `Table 54: Reduced Temperature (-20 C) Extract Stability for PFHS.cvvcvvvvvvorrpvrvonrnTL `Table 55. Reduced Temperature (-20 C) EXtract Stability for MS56....vrsrncnT2 `Table 56. Reduced Temperature (-20 C) Extract Stability fOr M570. 3 Table 57. Long`Term Matrix Stability for PFOS in HUMAN SERUM vrs T Table 58. Long-Term Matrix Stability for PFOSA in HUMAN SERUM... 7 `Table 59. Long-Term Matrix Stability for PFOSAA in HUMAN Sum vrs 75 rege 030210 Northwest Bioanalytical ReSptourdtyNNoo.. NNWWBBRSO0O0-.102420 `Table 60. Long-Term Matrix Stability for POA in HUMAN Serum... 15 `Table 61. Long-Term Matrix Stability for PFHS in Human Serum rrr 16 Table 62. Long-Term Matrix Stability for M5S6 in HUMan Serum .cvvsssrevnrnne 76 `Table 63. Long-Term Matrix Stability for M570 in Human Serum ......ovvenemonranenT] `Table 64. PFOS Extraction Efficiency from Human Seru.......msnsunnsn 18 Table 65. PFOSA Extraction Efficiency om HUMAN SERUM...rvsvrsssrsnrn1) Table 66. PFOSAA Extraction Efficiency from HUMAN Sem... 80 `Table 67. POAA Extraction Efficiency from HUMAN SERUM v.81 `Table 68. PFHS Extraction Efficiency from HUmMaN SERUM... 82 `Table 69. M556 Extraction Efficiency om Human Serum... 83 `Table 70. M570 Extraction Efficiency from Human Serum... 34 `Table 71. Summaryof Calibration Curve Parameters for PFOS in Human PIasme..............85 `Table 72. SummaryofCalibration Curve Parameters for PFOSA in Human Pasma..............85 Table 73. SummaryofCalibration Curve Parameters for PFOSAA in Human Plasma............85 `Table 74. SummaryofCalibration Curve Parameters for POAA in Human PIasma ........86 `Table 75. SummaryofCalibration Curve Parameters for PFHS in Human Plasma...........86 Table 76. SummaryofCalibration Curve Parameters for MSS6 in Human Plasma................86 Table 77. SummaryofCalibration Curve Pazameters for MS7O in Human Pasa. ccc87 . Table 78. Back-Calculated Concentrations of Calibration Standards for PFOS in _ Table 79. Back-Calculated ConcentrationsofCalibration Standards for PFOSA in HHA PIRRs I ms `Table 80. Back-Calculated ConcentrationsofCalibration Standards for PFOSAA in FAIR PAIR mmmmmmmmm---------- 50211 Pages [oe ---- ReSpaoryt NNoo.. NNWWBBRSO0O0-014330 `Table 81. Back-Calculated Concentrations of Calibration Standards for POAA in HUMEN PlaScI oosTA scrsrsssssussssssssmsmmssssssssssssssssssess sass `Table 82. Back-Calculated Concentrations of Calibration Standards for PFHS in FIINR0 BLAST wuirmmrisuemmmmmmsssmmsssssimsnessmimsmmssmssmmmmssssmmpmorrimnndd `Table 83. Back-Calculated Concentrationsof Calibration Standards for M556 in `Table 84. Back-Calculated Concentrationsof Calibration StandardsforM570 in TEIRI PIASIA wirescesorormmsssormessmminemsenenisssesbimmsnssitssibiissimemmmssssmmmensimmmedly `Table 85. Lower Limit of Quantitation for PFOS in Human PI&SMA ......c.ccvevsersmsemsssenrseen|9 Table 86. Lower Limit of Quantitation for PFOSA in Human Plasma ...........cc.cusemmsmmrrenn92 Table 87. Lower LimitofQuantitation for PFOSAAin Human Plasma............oseemesresssrenn93. `Table 88. Lower Limit ofQuantitation for POAA in Human Plasma.........cu.uusemmssmesrn9e4s. Table 89. Lower Limit ofQuantitation for PFHS in Human PIasma ...........cwueesermsssssrenes 95. `Table 90. Lower Limitof Quantitation for M556 in Human Plasma ...........cccocuueverrrssssnerrensn96. `Table 91. Lower Limit of Quantitation for M570 in Human PIaSIMA ......c.cuevreseerresesssersisn9e7n Table 92. Intra-Assay Precision for PFOS Quality Controls in Human Plasma..........c.............98 `Table 93. Intra-Assay Precision for PFOSA Quality Controls in Human Plasma.............c.........99 Table 94. Intra-Assay Precision for PFOSAA Quality Controls in Human Plasma.................100 `Table 95. Intra-Assay Precision for POAA Quality Controls in Human PIaSma....perere lO]. _ `Table 96. Intra-Assay Precision for PFHS Quality Controls in Human Plasma........ spreverererererene] 02 Table 97. Intra-Assay Precision for M556 Quality Controls in Human Plasma..............c..cue1e0e3s. Table 98. Intra-Assay Precision for M570 Quality Controls in Human Plasma... 104 Table 99. Summary ofCalibration Curve Parameters for PFOS in Serum Diluted ' 050212 Page 9 Northwest Bioanalytical ReSptourdty NNoo.. NNWWBBRS00G0--102420 `Table 100. Summary of Calibration Curve Parameters for PFOSA in Serum Diluted WH BUST. 105 WA BUSEE sss `Table 101. Summary of Calibration Curve Parameters for PFOSAA in Serum Diluted Table 102. Summary of Calibration Curve Parameters for POAA in Serum Diluted WD BUT cts 105 106 WA BUST. 106 `Table 103. SummaryofCalibration Curve Parameters for PFHS in Serum Diluted WhBUcctv 106 Table 104. SummaryofCalibration Curve Parameters for M556 in Serum Diluted `Table 105. SummaryofCalibration Curve Parameters for M570 in Serum Diluted `Table 106. Back-Calculated ConcentrationsofCalibration Standards for PFOS in Serum `Table 107. Back-Calculated ConcentrationsofCalibration Standards for PFOSA in Serum DAU With BUTE. 108 `Table 108. Back-Calculated ConcentrationsofCalibration Standards for PFOSAA in Serum DHE With BURcorsets 109 Table 109. Back-Calculated Concentrationsof Calibration Standards for POAA in Serum Table 110. Back-Calculated ConcentrationsofCalibration Standards for PFHSinSerum 0 DAME With BUC c.sssssssssnsesssesvesn 109 DIVE With BUST... 109 Table 111. Back-Calculated Concentrationsof Calibration Standards for M556 in Serum DAIUEAWith BUTE... 10 `Table 112. Back-Calculated Concentrationsof Calibration Standards for M570 in Serum Table 113. PFOS Quality Controls Samples Prepared in Serum Diluted with BUffer ..........111 050213 Page 10 Northwest Bioanalytical ReSptourdty NNoo..NNWWBBRS0000-.102420 Table 114. PFOSA Quality Control Samples Prepared in Serum Diluted with Buffer ..........1 11 Table 115. PFOSAA Quality Control Samples Prepared in Serum Diluted with Buffer ........112 `Table 116. POAAQuality Control Samples Prepared in Serum Diluted with Buffer............112 `Table 117. PFHS Quality Control Samples Prepared in Serum Diluted with Buffer.............113 `Table 118. M556 Quality Control Samples Prepared in Serum Diluted with Buffer ...........113 `Table 119. M570 Quality Control Samples Prepared in Serum Diluted With BUFFer.............1 14 `Table 120. Summary of Calibration Curve Parameters for PFOS (Serum vs. Plasma) ..........115 Table 121. Summary of Calibration Curve Parameters for PFOSA (Serum vs. Plasma).........115 `Table 122. Summary of Calibration Curve Parameters for PFOSAA (Serum vs. Plasma).......1 15 `Table 123. Summaryof Calibration Curve Parameters for POAA (Serum vs. Plasma) .........116 `Table 124. Summaryof Calibration Curve Parameters for PHS (Serum vs. Plasma) ..........116 `Table 125. SummaryofCalibration Curve Parameters for MSS6 (Serum vs. Plasma)...........116 Table 126. SummaryofCalibration Curve Parameters for M570 (Serum vs. PIasma)...c.....117 Table 127. Back-Calculated ConcentrationsofCalibration Standards for PFOS. J EN `Table 128. Back-Calculated ConcentrationsofCalibration Standards for PFOSA (Sern v5, BUSES cummins1 Table 129. Back-Calculated Concentrationsof Calibration Standards for PFOSAA- (BOUE VS. PISS css 02 = Table 130. Back-Calculated ConcentrationsofCalibration Standards for POAA + LL LR EG:| `Table 131. Back-Calculated Concentrationsof Calibration Standards for PFHS (Berm is: EissiB mmm mst Table 132. Back-Calculated Concentrationsof Calibration Standards for M556 {Boras BSR assess 03 Page 11 030214 Northwest Bioanalytical ReSptourdtyNNoo.. NNWWBBRSO0O0-.102420 `Table 133. Back-Calculated ConcentrationsofCalibration Standards for M570 J `Table 134. PFOS Quality Controls (Serum vS. PISA)... 125 `Table 135. PFOSA Quality Controls (Serum VS. PIESTIA) ..ercrnscsnsnnn 27 `Table 136. PFOSAA Quality Controls (Serutn v5. PASTE)... 128 `Table 137. POAAQuality COBtrols (Serum v5. PIESIE).....erssmenn 129 `Table 138. PFHS Quality Controls (Serum v5. PLASMA) c.vvessnene131 `Table 139. M556 Quality Controls (Serum v5. PLASM). 132 `Table 140. M570 Quality Controls (Serum v5. PLASM). 133 `Table 141. Serum QC Results Based on PIaSIa CUIVES rns135 050215 Page 12 `Nortwest Bioanalytical ReSptourdty NNoo,. NNWWBBRSO0O0-.102420 Quantitative Determination of PFOS, PFOSA, PFOSAA, POAA, PFHS, M556 and M570 in Human Serum by LC/MS/MS Assay Revalidation Addendum Report 1 INTRODUCTION Northwest Bioanalytical (NWB) was contracted by 3M Environmental Technology and Services (3M) to modify and revalidate a liquid chromatography/tandem mass `spectrometry method forthe measurement of PFOS, PFOSA, PFOSAA, POAA, PFHS, M556 and M570 in human serum. The accuracy and precision results ofthe method revalidation were reported in NWB report NWBRO0-108 [9.1]. This report summarizes the results from the stability tests conducted as partofthe method revalidation forPFOS, PFOSA, PFOSAA, POAA, PFHS, M556 and M570 in human serum for 3M `Environmental Technologyand Services. The results from the following tests are also included: extraction efficiency from human serum; ion suppression experiment; and diluted matrix experiment. The suitabilityofthe method for use with plasma was determined by demonstrating intra-assay and LLOQ accuracy and precision and comparing serum and plasma results. Kris Hansen and James Lundberg at 3M Environmental Technology and Services served as the Study Monitors. The following isalist of NWB supervisory personnel involved in the completionofthis work: Connie O. Sakashita, B.S. (Current NWB Project Manager); Brad I. Coopersmith, Ph.D. (NWB Project Manager until October 28, 2000); Licong. - Jiang, Ph.D. (NWB Senior Scientist); Patrick Bennett, M.S., M.B.A. (NWBLaboratory Director). The following NWB laboratory personnel contributed to the completionofthis work: Suzanne Newman, B.S. (NWB Scientist). NWB SOPs were used in the conductofthis project and were available to project personnel in both electronic and hard copy formats. Date Study Initiated: _13-Apr-2000 Date Analyses Completed: 25-Apr-2001 090216 Page 13 Northwest Bioanalytical ReSptourdty NNoo.. NNWWBBRS0000-.102420 The method revalidation study described in this report is not included within the definition ofa GLP regulated nonclinical study. However, Northwest Bioanalytical conducts all studies within the guidelinesofthe US FDA Good Laboratory Practice Regulations for Nonclinical Laboratory Studies (Title 21 CFR Part 58), the OECD PrinciplesofGood Laboratory Practice and the Japanese MHW Good Laboratory Practice Standard Ordinance for Nonclinical Laboratory Studies on the Safety ofDrugs (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 Reference Materials andMatrices Analyte LotNumber PFOS (FC.95) 193 PFOSA 24 PFOSAA(FC-129) 617 POAA (FC-143) 25 PFHS* (used for serum) PFHS (used for plasma) Mss Ms70 S398182 SE036 NBLI83DOSZ11850626 THPFOS 59909 TT Reedw6200 Puriyy 100% 100% 38% 100% 100% 911% 9989% 99.75% 90% pom sohion ExDpiartaetion 1231200 12312010 1212000 12512010 12312010 Source Storage Conditions 3M TemRpeoroamture 3M Room Temperature 3M Room Temperature 3M Room Temperature 3M 20 LOI2010 3M 2c 12312010 3M: Room Temperature 12312010 3M Room Temperature 12312010 3M Room Temperature mene Reply 030217 Page 14 Northwest Bioanalytical ReSptourdty NNoo., NNWWBBSROO0G0-.102420 Matrix Lot Numbers TT HB ummsO enmOGw4BCSIOT Human plasma (Northern Chinese) C5186; C4929; C5517; C5200 The reference material purity for PFOS, PFOSA, PFHS and POAA was not available prior to the conductofthis study. Therefore, ll serum concentrations included in the report fo these analytes are based upon an assumed purity of 100%. After the revalidation was completed, 3M contracted with Centre Analytical Laboratories, Inc. in State College, Pennsylvania to determine the absolute concentration ofPFOS, POAAand PFHS in NW stock solutions used to prepare the analytical standards and controls used for this revalidation and subsequent analyses. All arrangements for purity determinations and transfers of NWB solutions to Centre Analytical Laboratories, Inc. were performed by 3M. Per agreement with the Sponsor, NWB quantitative results were not correctedforthe purity corrections. Therefore, based on the results obtained, the serum concentrations included in this report should be corrected according to the following table (except for the serum samples included in the serum versus plasma experiments, Section 7.3): Analyte PFOS POAA PFHS Principles of the Method Corection Factor 0836 0909 0855 : ~ . "The analytical method consistedof a liquid:liquid extraction procedure followed by evaporation and reconstitutionofthe extract residue with 30:70 (v/v) 20 mM ammonium acetate in water: 20 mM ammonium cetate in methanol. The samples were analyzed by liquid chromatography/tandem mass spectrometry using a PE Sciex API 3000. The instrument was operated in the multiple reaction monitoring (MRM) mode under 030218 Page 15 Northwest Bioanalytical ReSptourdty NNoo,. NNWWBBRS00O0-.102420 optimized conditions for PFOS, PFOSA, PFOSAA, POAA, PHS, M556 and M570 detection. A detailed descriptionofthe methodology is included in NWB report NWBROO-108 . . Acceptance Criteria for Human Serum Calibration Curve Each run wil include in duplicate calibration standards at six or more concentrations covering the lower to upper limitof quantitation. For all analytes except PFOSA, at least three-fourthsofthe calibration standards back-calculated concentrations must be within 15% (& 20% for LLOQ)oftheir individual target concentrations. For PFOSA, at least three-fourthsofthe calibration standards back-calculated concentrations must be within 20% ( 25% for LLOQ)oftheir individual target concentrations. A calibration standard will be considered a statistical outlierifthe back-calculated concentration is greater than two times the acceptance criteria for that standard. Lower LimitofQuantitation `The back-calculated concentrationsofat least oneofthe duplicate lowest points in the calibration curve must be within + 25%ofthe target concentration for PFOSA to qualify asthe LLOQ and within 20%ofthe target concentration to qualify as the LLOQ for all other analytes. [this criterion is not met, the next level is subjected totheSame test and the LLOQ raised accordingly. : oe Quality Control Samples Each analytical run will include low, medium and high QC samples in duplicate. The `measured concentrationsofatleast two-thirdsofall analytical QCs must be within + 20% of their target concentrations (+ 25% for PFOSA), and no two QCs at the same concentration can be outside the limit. If study samples require dilution, a dilution QC willbeanalyzed in triplicate for each dilution level (except for PFOSA and PFOSAA. 050219 Page 16 Northwest Bioanalytical ReSptourdty NNoo.. NNWWBBRS00O0--102420 `which should riot be diluted with control matrix). At least two dilution QC at each level `must be within + 20% of ts target concentrations in order to accept diluted study samples at that level. The dilution QC acceptance is independentofthe undiluted analytical QC acceptance. 3. STABILITY EVALUATION `The revalidated methods final acceptance criteria is used as a basis for evaluating fluorochemical stability results. Ifthe mean concentration obtained for stability test QC samples falls within 20% (& 25% for PFOSA) of the mean control QC concentrations, then stability is considered acceptable under the specified test conditions. Control QC `samples are defined as samples not subjected to test conditions. `The calibration curve parameters summary and individual calibration standard results for the serum stability runs can be foundin Tables 1-14. The analytical QC results for the stability runs can be found in Tables 15-21. 3.1. Freeze-Thaw and Room Temperature Matrix Stability The stability of PFOS, PFOSA, PFOSAA, POAA, and PFHS in human serum for three freeze-thaw cycles was demonstrated and reported under the original validation study NWBS98-092, NWB report NWBR99-005 [9.2]. Therefore, only freeze-thaw stability for M556 and MS70 was needed. Acceptable results for the three freezethaw cycle stability test was obtained on Run 4 for M556 and M570 (data not included). However, since some analyzed subject samples subsequentlyunderwentmore than three freeze-thaw cycles, the stability ofPFOS, PFOSA, PFOSAA, POAA, PFHS, M556 and M570 was evaluated after seven freeze-thaw cycles. All analytes demonstrated acceptable stability when compared against controls that underwent one: freeze-thaw cycle (Tables 22-28). `During the seven freeze-thaw cycle test, samples were left at room temperature for up 106.75 hour periods between each freeze cycle. Since the freeze-thaw samples Page 17 090220 Northwest Bioanalytical RSeptourdtyNNoo.. NNWWBBRSO0O0--102420 demonstrated acceptable stability, the stabilityofserum samples left at room temperature for up to 6.75 hours is also demonstrated. 32. Reinjection Stability `The quantitative reproducibilityofstandards and controls afer reinjection was determined for PFOS, PFOSA, PFOSAA, POAA, PFHS, M556, and M570. The samples were extracted and analyzed on June 30, 2000 (Run 15 - data shown in NWBROO-108). These samples were reinjected on July 3, 2000 after storage at room. temperature (Run 23). The results from Run 23 indicate reinjection stability when compared with data obtained from the original injection [Run 15] (Tables 29 35). 33. Extract Storage Stability Extract stability was evaluated at different storage temperatures and for various lengthsoftime. The evaluations were performed using QC lots prepared on June 27, 2000 for all analytes and conditions. Although the same QC lots (prepared on June 27,2000) were not used for PFOSA and PFOSAA run acceptance determination, they still provided acontrol value for stability evaluations. 33.1. Room Temperature Extract Stability, The extract stability ofPFOS, PFOSA, PFOSAA, POAA, PHS, M556 and M570 was determined for low and high serum QC extracts stored at room temperature for seven days prior to analysis. These test QC samples were compared against serum control samples that were analyzed immediately following extraction. `The extracted test QC samples demonstrated acceptable stability after seven days at normal room temperature (Tables 36 - 42). 3.3.2. Reduced Temperature (1 to 8C) Extract Stabili The extract stability ofPFOS, PFOSA, PFOSAA, POAA, PHS, M556 and MST0 was determined for low and high serum QC extracts stored at 1 to 8C for four Page 18 050221 Norwest Bioanalytical ReSptourdtyNNoo.. NNWWBBRS00O0--102420 days prior to analysis. These test QC samples were compared against serum control samples analyzed immediately following extraction. The extracted test QC samples demonstrated acceptable stability after four days at 108C (Tables 43-49). 333. Reduced Temperature (-20 C) Extrect Stability `The extract stability of PFOS, PFOSA, PFOSAA, POAA, PFHS, M556, and MS70 was determined forlowandhigh serum QC extracts stored at -20 C for five dayspriorto analysis. These test QC samples were compared against serum control samples analyzed immediately following extraction. `The extracted test QC samples demonstrated acceptable stability afer five days at 20C (Tables 50-56). 34. LongTerm Matrix Stability in Human Serum `The long-term matrix stability ofPFOS, POAA, PFHS, M556 and M570 in human serum was determined by storing QC samples for approximately 55 days at -20 C. The long-term matrix stability ofPFOSA and PFOSAA in human serum was determined by storing QC samples for approximately 42 days at -20 C. The mean test QC concentrations were compared against theoretical. The test QC samples demonstrated acceptable stability after approximately 42 days for PFOSA & PFOSAA or 55 days for other analytes, at -20 C (Tables 57-63). - 4. EXTRACTION EFFICIENCY FROM HUMAN SERUM : `The extraction efficiencies ofPFOS, PFOSA, PFOSAA, POAA, PFHS, M556 and MS70 were determined by comparing the area ratios obtainedforthe following three cases: 1. Both the analyte and internal standard added following the extraction (postextract). 090222 Page 19 Northwest Bioanalytical ReSptourdty NNoo.. NNWWBBRSO000--102420 2. The analyte added to serum prior to extraction and the internal standard added following extraction (pre-extract analyte). 3. The intemal standard added to serum prior to extraction and the analyte added following the extraction (pre-extract intemal standard). The extraction efficiencies were then determined by the area ratio of the pre-extract samples to the post-extract samples. The extraction efficiency experiments were performed at both low and high concentrations to determine that there was no concentration bias. The total amountofanalyte in the sample prior to extraction (pre-extract) is equal to the amountofanalyte added plus the persistent amount in the blak matrix. The amount of analyte in the post-extract solution is the amountofanalyte added plus the amount of analyte recovered (extracted) from the blank matrix. Therefore, the following formula was used to determine the extraction efficiency for PFOS, PFOSAA, POAA, PFHS, M556 and M570: ((Spk + Per)* EE)/ (Sp+k (Per * EE)= Rpre / Rpost; where SPepti==tthheepaemrsoiusnttenotfaamnoaulnytteoafadndaeldyte in the blank matrix EE =th extraction efficiency Rinotreern=altshtearnadtairoodfadadneadlyaefltienteexmtraalcsttiaonndard determined forth analyte added prior snd Rstpaonsdtar=dtahddreadtoaofefrsneaxlayctteiionntemal siandard determined forth analyte and intemal PFOS PFOSA PFOSAA Mean extraction efficiency (%) 37.3 69.6 627 :. a POAA PFHS Mss6 `Mean extractionefficiency(%) 15.6 153 39.1 Ms70 Mean extraction efficiency (%) 57.1 The results are shown in Tables 64 ~ 70. Page20 THPFOS 599 030223 Northwest Bioanalyical ReSptourdtyNNoo,. NNWWBBRS00O0--102420 5. COMMENTS AND CONCLUSIONS Per agreement with the Sponsor, the regressions were not recalculated based upon the updated purity information for PFOS, POAA and PFHS. Some differences might occur due to the effectof roundingifthe regressions were performed with the purity corrected concentrations, but these differences would have a negligible effect on the overall interpretationofthe revalidation results. PFOS, PFOSA, PFOSAA, POAA, PFHS, M556 and MS70 in human serum appear to be stable for seven freeze-thaw cycles and demonstrated matrix stability for up to 6.75 hours at room temperature. PFOS, PFOSA, PFOSAA, POAA, PFHS, MSS6 and MS70 exhibit extract stability for seven daysat room temperature, four days at 1 to 8 C, and five days 2t-20C. The analytes also demonstrated matrix stability for approximately 5S days (for PFOS, POAA, PFHS, M556 and M570) and 42 days (for PFOSA and PFOSAA) at 20C in human serum. 6. ABBREVIATED VALIDATIO-N HUMAN PLASMA The choiceofmatrix for calibration curve preparation is dependent upon the desired LLOQ. 3M supplied NWB with human plasma identifiedas"Norther Chinese" plasma. (referred to as Chinese plasma or CP). This matrix has very low levels of fluorochemicals and will be used for studies with a desired LLOQof < 5.00 ppb. The following sections summarize the accuracy and precisionofthe fluorochemicals in human plasma and the quantitative accuorfsaecruym sample results obtained against plasma calibration curves using the revalidated serum method. i . The absolute concentrationsofPFOS, PFHS and POAA in the stock solution used for plasma analyses were determined by Centre Analytical Laboratories, Inc. in State College, Pennsylvania. Based upon the results, the concentrationsofplasma standard and quality control samples were corrected using the following factors: . 090224 Page2! Northwest Bioanalytical ReSptourdtyNNoo.. NNWWBBRSO0O0-.102420. Analyte PFOS POAA PFHS Correction Factor 0.821 0.961 1.043 All plasma experiment concentrations included in this report reflect adjustments based `upon the correction factors above. The serum target concentrations for these experiments have also been adjusted based upon the correction factors (listed on page 15ofreport). `The tests for the abbreviated human plasma validation were conducted after the serum revalidation was completed. They were performed outsideofthe revalidation or in `sample analysis studies conducted after the serum revalidation. However, all applicable NWB SOPs were followed. Persistent Levels of Analytes in Chinese Plasma `The persistent levels of the analytes in Chinese plasma varied in each tube of plasma tested. Therefore, several tubesofplasma were pooled and the amountsof persistent analytes were determined. Previous determinationofpersistent levels was achieved by `comparison with rabbit serum curves. For the experiments included in this report, the persistent levels were determined as follows: Known amountsofthe analytes were added to Chinese plasma in the range of 1 to SO or 1 10 100 ppb.The standards were then analyzed according to the revalidated method on at least two separate occasions. A linear regression was performed using the spiked analyte concentration as the y-value and the ratio of the analyte area to internal standard area as the x-value (Microsoft Excel 97 formulas). The mean resultant y-intercept determined from multiple runs was then used as an estimateofthe persistent level ofanalyte ifit was greater than 0.2 ppb. rg 030225 NorthwestBioanalytical 6.1. Rangeof Quantitation ReSptourdtyNNoo.. NNWWBBSR0O0--102420 `The target calibrator concentrations were approximately 1.00, 2.50, 10.0, 25.0, 50.0, 100, 250, 400 and 500 ppb for all analytes except M5S6. The target calibrator concentrations for M556 were approximately 2.50, 10.0, 25.0, 50.0, 100, 250, 400 and 500 ppb. The final curve range can vary based upon the persistent levelsofthe analyte in the human plasma pool used. For the validation study,thecalibration curve ranges were as follows: PFOS PFOSA PFOSAA POAA PFHS Mss6 M570 39410414 ppb 10010500 ppb 16010 501 ppb 19210481 ppb 1.36 10 523 ppb 25010 500 ppb 1.000 500 ppb `The calibration curve parameter summaries and individual calibration standard results for the plasma runs can be found in Tables 71 - 84. 62. Lower Limit of Quantitation `The precision at the Lower Limit ofQuantitation (LLOQ)ofthe assay in human plasma was determined by using six 100-L aliquots ofthe pooled Chinese plasma fortified with PFOS, PFOSA, PFOSAA, POAA, PFHS, MSS6, and M570 at concentrations equal to the LLOQ. The LLOQ concentrationwasdependent upon the ~~ persistent levels found in the plasma. . Lower Limit ofQuantitation Precision (4CV) Accuracy (%Bies) PFOS 3.94ppb 54 1.5 PFOSA 1.00ppb 10.1 10 PFOSAA 1.60pp> 57 25 POAA 1.92 ppb 44 89 Page 23 090. fs Northwest Bioanalytical Lower Limitof Quantitation Precision (%CV) Accuracy (%Bias) PFHS 136 ppb 99 -S.1 The results are shown in Tables 85 - 91. 63. Intra-Assay Precision and Accuracy M536 2.50ppb 57 20 ReSptourdtyNNoo.. NNWWBBRSO000--102420 M570 1.00 ppb 52 30 `The precision and accuracyofthe LC/MS/MS method for PFOS, PFOSA, PFOSAA, POAA, PFHS, M556, and M570 in human plasma were deterbmyainanlyzeindg three levelsof quality control samples in replicates of five on a single run. Precision (%CV) Accuracy (Theoretical) Accuracy (%Bias) PFOS Diaassay 15028 1012101123 1210123 PFOSA Itaassy 181057 104010 1163 400163 PFOSAA Inimassay 26158 102610 1117 26117 POAA Intraassey 08t058 103.410 1062 341062 Precision (4CV) Accuracy (Theoretical) Accuracy (Bias) PFHS Intassay L7t03.0 957101060 431060 `The results are shown in Tables 92 ~98. M56 Intassay 471076 98.710 1113 -13t0113 M70 Intra-assay 19156 101310 118.0 1310180 64. Proposed Acceptance Criteria for Human Plasma ? Calibration Curve Each run will include in duplicate calibration standards at six or more concentrations covering the lower to upper limitofquantitation. For all analytes except PFOSA and PFOSAA, at least three-fourths of the calibration standard's back-calculated concentrations must be within 15% (+ 20% for LLOQ)oftheir individual target Page 24 030227 Northwest Bioanalytical ReSptourdtyNNoo.. NNWWBBRSO0O0-.102420 concentrations. For PFOSA and PFOSAA,atleast three-fourthsofthe calibration standard's back-calculated concentrations must be within 209% (+ 25% for LLOQ) of their individual target concentrations. A calibration standard will be considered a statistical outlierifthe back-calculated concentration is greater than two times the acceptance criteria for that standard. Lower Limit ofQuantitation `The back-calculated concentrationsofat least one ofthe duplicate lowest points in the calibration curve must be within 25% of the target concentration for PFOSA and PFOSAA to qualify as the LLOQ and within 20%of the target concentration to qualify as the LLOQ forall other analytes.Ifthis criterion is not met, the next level is `subjected to the same test and the LLOQ raised accordingly. Quality Control Samples Each analytical run will include low, medium and high QC samples in duplicate. The measured concentrations of at least two-thirdsofall analytical QCs must be within 20%oftheir target concentrations (+ 25% for PFOSA and PFOSAA), and ro two QCs at the same concentration can be outside the limit. Based upon the results, the method is suitable for use with human plasma. 7. ADDITIONAL POST-REVALIDATION EXPERIMENTS `Additional experiments were conducted subsequent to completion of the revalidation to further elucidate the applicabilityofthe revalidated method to sample analysis. These tests were performed outside of any revalidation or sample analysis studies. 7.1. Ion Suppression Experiment During analysis ofmedical surveillance serum samples, it was noted that the internal standard response for the study samples differed from the standards and quality controls. POAA and the intemal standard (THPFOS) have very similar retention Pages 050228 Northwest Bioanalytical ReSptourdtyNNoo.. NNWWBBRSO000--102420 times. Therefore, THPFOS and POAA are not chromatographically resolved in extracted samples containing high concentrations of POAA. This co-elution can suppress the intemal standard response and introduce a (high) bias in the measured concentration ofanalytes when compared against the standard curve. The suppression of the intemal standard signal is proportional to the concentration of POAA in the `sample and dependent on the LC resolutionofthe two compounds. An experiment was performed to evaluate the magnitudeofthis effect. Low control samples were fortified with various (high) levels of POAA. The test samples were analyzed in triplicate in asinglerun (PFOSMD-41). The mean concentration of the test samples were then compared against the meanofthe analytical low control samples. A summary ofthe results is shown in the following table: brosprosd rosidprs wis M0Bare Control Mean Conc. (ppb) 51.8 4.05 6.83 8.04 541 6.87 108857 FOANaddedams0pey % change from control mean 182% 152% 180% 180% 216% 207% -75% TPoARwddmomm0Opes % change from control mean 28% 39% 52% 29% 52% 56% 43% TroMnasdwmmpn % change fromconol mean 16% 21% 30% 16% 36% 34% 32% TROANwddwsze ApS % change from control mean 2% -11% -5% G1%eW 8% E -8% u+1m1% `The experiment verified that very high levels of POAA resulted in a lowerresponse for the intemal standard, which then results in a falsely elevated concentrationforthe othe6r analytes. No significant bias is introduced when the extracted concentration of POAA is within the revalidated rangeofthe analytical method (approximately 500 ppb). However, some samples can contain POAA at concentrations above the revalidated range ofthe analytical method, and will likely introduce abias in the reported concentrations of PFOS, PFHS, PFOSA, PFOSAA, M556, or M570 when the concentrationofthese analytes are determined prior to sample dilution. The amount ofbias could range up to approximately 56%. However, the amount of rt 030229 Northwest Bioanalytical ReSptourdtyNNoo.. NNWWBBRSO000--102420 PFOS, PFHS, PFOSA, PFOSAA, M556, or M570 impurities in the POAA reference material is not known. This prevents accurate determinationofthe % bias. It should be noted that since the amountofion suppression is dependent upon the amount of `extracted POAA, no concentration bias is expectedforan analyte result determined from a pre-diluted sample wherein the extracted POAA concentration s less than approximately 500 ppb. This bias will not significantly effect the interpretationofthe analytical results. 7.2. Dilution Matrix Experiment The amountof Chinese plasma supplied by 3M is limited. Therefore, it cannot be `used on a routine basis for dilution. Human serum (control serum) is used to dilute test samples as needed to obtain measurable concentrations within the range of the calibration curve. The measured concentrations of fluorochemicals (FC) obtained for diluted samples must then be corrected for persistent levels of the analytes in the control serum. Experiments were performed to determine whethear substitute`matrix that did not contain persistent levels of FC analytes could be used when performing dilutions. Previous experiments had shown that samples diluted with buffer insteaod f serum did not accurately quantitate against a typical serum curve (data not reported). This indicatedthatthe matrix to buffer ratioofthe `sample affected the extraction recovery. For the experiments described in this report, QC samples were diluted with buffer. `The calibration curve was prepared in the same buffer to matrix ratioasthe QC samples to determineif this was a viable method for diluting samples. A different oT dilution ratio was prepared to determine if samples could be quantitated from a curve prepared in a matrix to buffer ratio that was different from the QC samples (e.g. if QC samples diluted with matrixby a factorof50 (2% matrix in buffer) could be quantitated against a curve prepared in 10% matrix in buffer). For the experiments described below, ammonium acetate buffer was used to dilute the matrix for the QCs and calibration curves. Page27 050230 Norwest Bioanalytical ReSptourdtyNNoo.. NNWWBBRS0000-014220 Calibration Curve and QC Preparation The experiment (PFOSMD run 39) used a curve prepared in 10% serum in buffer, and thee levels of QC samples diluted with buffer bya factorof 10 (10% matrix to buffer ratio). The QC concentrations prior to dilution were 150, 400, and 4000 ppb plus the aplesrosidsitleuntteldev1e:l50fowrietahcbhufafnearl.yte. QC samples at 4000 ppb (plus persistent level) were `The calibration curve parameter summaries and individual calibration standardresults for the runs can be foundin Tables 99 -- 112. The quality control sample results can be found in Tables 113 - 119. Results for 10% Serum in Buffer (PFFOSMD Run 39) `The mean accuracy (% theoretical) for all analytes at all QC concentration levels diluted with buffer by a factorof 10 (the same serum to buffer ratio as the curve) ranged from 94.1% to 114.3%. The intra-assay precision (%CV)forall analytes were less than or equal to 5.6% for each QC concentration. `The mean accuracy (% theoretical) for the QC samples diluted with buffer by a factor of 50 ranged from 56.5% to 84.8%. The inter-assay precision (%CV) for all analytes `were less than or equal to 4.4%. All analytes were biased low, and all analytes except for POAA and PFHS were not within +20%ofthe target concentrations. QC samples met the acceptance criteriaof 20% of target concentration (425% for PFOSAA) when diluted with bufferif quantitated against a curve prepared in the same serum to buffer ratio. QC samples diluted with buffer at a different ratio than - the calibration curve did not meet the acceptance criteia for al analytes, It appears that quantitating samples diluted with buffer against a curve prepared in the same serum to buffer rato is a viable way minimizing the corrections needed due to persistent levels of analytes in the dilution matrix. However, since mostanalytes cannot be accurately quantitated against a calibration curve that is prepared in a serum/buffer ratio different from thatof the diluted samples, a. separate curve with the same buffer to matrix ratio would need to be prepared for each dilution factor needed. Page2s 030231 Northwest Bioanalytical 73. Serum and Plasma Comparison ReSptourdtyNNoo.. NNWWBBRS0000--102420 Because the choiceofcalibration curve and quality control matrix is dependent upon. the desired LLOQ, it is not always possible to match the subject sample matrix with thatofthe calibration curve and quality contral matrix. For instance, itis necessary to use Chinese plasma to prepare the calibration standards and quality control samples when analyzing serum samples where a LLOQof < 5.00 ppb is desired. Therefore, to evaluate if, and to what extent, the useof plasma calibration standards affects the quantitative analysis of fluorochemicals in serum, a comparisonof quantitative results obtained from plasma curves versus serum curves was performed. Calibration curves were prepared in both human serum and Chinese plasma, and were analyzed along with serum and Chinese plasma quality control samples in replicates of four. The results for the calibration and quality control samples are shown in Tables 120 ~ 140. `The confidence interval around the serum QC mean was determined for serum samples quantitated against the plasma curve using Microsoft Excel 97. Alpha (o) was set t0 0.05 foar 95% confidence level. The confidence interval around the mean `was compared to the analytical acceptance rangeofthe QC which is 20% (+ 25% for PFOSA and PFOSAA)ofthe target concentration. The summary resultsofthe experiment are shown in Table 141. `The 10/27/00 serum curve for M70 had a raised LLOQof5.20 ppb. Data for PFOSA and PFOSAA analyzed on 10/27/00 and data for PHS and M356 analyzed on 4/25/01 are not used because either the serum or plasma calibration curveorthe ~~ QC samples did not meet the acceptance criteria. One high QC replicat analyzed on 10/27/00 was not included in the statistical calculations becauseofan extraction error. `Theconfidence intervals around the mean serum QC results for POS are within the acceptance criteria at all levels. -- 030232 Northwest Bioanalytical ReSptourdty NNoo.. NNWWBBRS0000-.102420 The confidence intervals around the mean serum QC results for POSA are greater than the acceptance raonf g25e% deviation. The confidence intervals ranged up to 78% deviation from target. The confidence intervals around the mean serum medium QC result for PFOSAA was 33.9% deviation which isgreaterthan the plasma acceptance rangeof25% deviation. The confidence interval for the PFOSAA low and high QC samples are within the acceptance criteria. `The confidence intervals around the mean serum QC results for POAA and PFHS are greater than the acceptance rangeof20% deviation. The confidence intervals ranged up 10 27.4% deviation from target for POAA and up to 37.6% deviation from target for PFHS. `The confidence interval around the mean serum low QC results for M556 was 26.9% deviation. This is greater than the acceptance range of209 deviation and may be due alow value obtained for one replicate. The confidence intervals for the M556 medium and high QC samples are within the acceptance criteria. The confidence intervals around the mean serum medium QC result for M570 was 21.9% deviation which is slightly greater than the plasma acceptance range of 20% deviation. The confidence interval for the M570 low and high QC samples are within the acceptance criteria. CONCLUSION an Serum sample results obtained using plasma curves for PFOSA, PFOSA, POAA, PFHS, M556, and M570 did not meet NWB SOP requirements for validation. Results mayvaryon average up 0 26% from QC results obtained using human serum calibration curves at some concentration levels for PFHS, PEOSAA, POAA, MSS6, and M570. For PFOSA, results may vary on average up to 43% from QC results obtained using human serum calibration curves. However, when a lower limit of Page30 030233 Northwest Bioanalytical ReSptourdtyNNoo.. NNWWBBRSO000-.102420 `quantitation less than 5 ppb is required by the Sponsor, it will be necessary to use: plasma curves. 8. DATA MANAGEMENT PFOS, PFOSA, PFOSAA, POAA, PFHS, M556, M570, and THPFOS chromatographic peaks were integrated using PE Sciex MecQuan software (version 1.6) with a smooth factorof one. Quantitation was based upon quadratic regression analysis ofcalibration curves (weighted 1/x?) using the area ratio vs. concentration calculated by the Watson DMLIMS software (versions 6.1.1.03 and 6.11.04). 9. REFERENCES [9.1] C. Sakashita. "Quantitative Determination of PFOS, PFOSA, PFOSAA, POAA, : PFHS, M556 and M70 in Human Serum by LC/MS/MS." Assay Revalidation Report. NWB Study NWBS00-040. NWB Report NWBR0O-108. January 24, 2001. [9:2] D. Vollmer. "Quantitative Determinationof PFOS, PFOSA, PFOSAA, N-MeFOSE-OH, N-EtFOSE-OH, POAA and PFHS in Human Serum by LC/MS/MS." Assay Validation Report. NWB Study NWBS98.082. NWB Report NWBR99-005. May 13, 1999. 10. DATA RETENTION `Theraw data and final report for this study wilbestointrheeNWdB Archive, 1121 East 3900 South, Salt Lake City, UT 84124 per regulations and contract agreement. 3M ~~ = Environmental Technology and Services will be notified concerning final disposition of records at completionofcontract obligations. Page 31 090234 rs ustrnom ne Table 1. Summary of Calibration Curve Parameters for PFOS in Human Serum `Quadratic weighted x'. All concentarraeetxpiroesnsesd as ppb. `Run Date [zisimeo Run A Number c LLOQ| ULOQ is Toso[cams[amen] osm | sa A, B, and C are coefficients used to define the calibration curve. 57] Table 2. SummaryofCalibration Curve Parameters for PFOSA in Human Serum Quadratic weighted Lx". All concentrations are expressed as ppb. Run A Number Cc R-Squared| LLOQ|ULOQ sw Table 3. Summary of Calibration Curve Parameters for PFOSAA in Human Serum Quadratic weighted 1/x%. All `concentarreaextpireossnesd asppb. [roeTam,| *[=][rswealmoa[uon [ous | 15Toromes[ome [om]ams|eon | | 030235 Table 4. Summary of Calibration Curve Parameters for POAA in Human Serum Quadratic weighted 1/x'. All concentrations are `expressed as ppb. Run A C |RSquired| LLOQ [ULOQ Tee i Table 5. Summary of Calibration Curve `Parameters for PFHS in Human Serum Quadratic weighted 1/x'. All concentrations are `expressed as ppb. [[ezssumeammoon||vTooswoiolr [woomnrems[oouarrs]|aanms|55155 || sis|] `Table 6. `Summary of Calibration Curve Parameters for M556 in Human Serum Tx Quadratic weighted 1x'. EEE All concentrations are expressed as ppb. pr [[Eeuasvss aon||e v ooocmooe onme[[sooonmeoe [oemsoms ||a"saossns ||33317|| ir|| Table 7. SummaryofCalibration Curve Parameters for M570 in Human Serum Quadratic weighted x', All concentrationsare expreseads ppb. ) Run A < R-Squared|LLOQ ULOQ [Es o0 isomers [oso[sors]"sos |sr| Page 33 030236 Nm FR Table 8. Back-Calculated son Concentrations of Calibration Standards for PFOS in Human Quadratic Ta weighted 1x'. All wT concentrations. are expressed Toas ppb. Tr Tor Tom [Cowrsai|| T[[wswourro[sssweoowsls7a7al[mooraa]| onJa[soamn oasrTor]] T Tos [T os[ms [our ow [oe TarTr Table 9. Back-Calculated eum Concentrations ofCalibration Standards for PFOSA in Human `Quadratic weighted 1/%. All concentrations are expressedss ppb. [CT eommoas |[No 5um5ber wowor[[n 22 [|{iooeew[[[a30vl o] re7ae]o]1[e[[|e 5a]eaarso]]s0]] [ToC wle om [mi srT ]om owlsn om] Table 10. Back-Calculated Concentrations of Calibration Standards for PFOSAA in Human Serum Quadratic weighted 1. All concentrationsareexpressed 2 ppb. . . Run |: 405 [[CCaomamsm|T[T N5umbTeTr owsnw [[oo7non[[e1n0esa[om8s[[ws5e7o[m|10a3l[n2 s| [3w0 [T[owotr]]] [T Teulsw welmlsam m[alonm] Page 34 030237 Table 11. Se mn Back-Calculated ConcentrationsofCalibration Standards for POAA in Human Quadratic weighted 1/x'. All concentrations are `expressed as ppb. [enim Zion | | Nn umber ssw[aonn [sos[oo |1s2e[[2m0e8 | 09 ana [[soao[oe3ao na os |Ta Tan o5n]] a| ITewTaw [wa [ms[sso ow [oon] Table 12. Back-Calculated Concentrations of Calibration Standards for PFHS in`Human `Quadratic weighted 1x". All concentrations are. expressed as ppb. [[eC oransmesoT [| a[[|zsswa[w[aswmw[[|wira[[[zmne7[enisswoors [m2 eTaml[6]7]| 1TwTem[wi[fosTo ooTar Tor] Table 13. Back-Calculated coum `Concentrations of Calibration Standards for M556 inHuman `Quadratic weighted 1/x'. All concentratioanrse expressaesd ppb. . ot - ow [oem Run 430 252 [402 502 [[ >TTn eeew][euoa f[waes[aewsas lfo[[ammoree [wiTfwaer]]] ToT {ma fos [wsTne] i. 030238 Table 14. Back-Calculated Concentrations of Calibration Standards for M570 in Human Serum Quadratic weighted 1/x. All concentrations are. `expressed as ppb. we Run 7.10 29.6 [oT wnsaiisor||s xso[aewr[[i oons[|[awr7s[[[0ome 2wrfs5o3eoTwiioooT[maan swfTarnFoowr|]] I Toole [wefonsTosTao Toe] Page 36 030239 metas Cer Alsosrs, `Table 15. Quality Control Samples for PFOS in Human Serum Run Date CEE i CE 2I EZ ETI ETE) Low QC| %Dif. {Medium QC| %Dif. 51.1 ppb 197 ppb High QC| %Dif. 446 ppb "Table tots Gres, emesis seeps, 16. Quality Control `Samples for PFOSA in Human Serum Run | Low QC| %Dif. [Medium QC] %Dif.| High QC| %Dif. Number| 4.00 ppb 150 ppb 400 ppb. [erm]won [ow] Jam] um] Table tice 17. Quality Go. tems gt, Control Samples for PFOSAA in Human Serum oT amas ronan aT aT [[envsmms e]o] 5[[ vn[o aor] s 00JTsas aoi]]"mss] TJion]] 030240 ess sisson etn Gt. Alsen Table 18. Quality Control Samples for POAA in nts Human Serum Run | Low QC |%Dif. Medium QC|%Dif| High QC| %Dir [[Eemroanl| 55[|vre J3u0s]] 00[J2a6a] maw 7To)d] iotepicn rs. Amen cts Table 19. Quality Control Samples for PFHS in Human Serum Run | Low QC| %Dif [Medium QC| %Dif| High QC | %if. ferns] 5[ss{is |ier [rer] so Tar] ioticn ss. cs Table 20. Quality Control Samples for M556 in enn, Human Serum [[ovmamsoa]] 5 ||sswn[{ae0s]wwa[rw]a] swemrT[e5o2]] Table 21. Quality Control Samples for M570 in Human Serum. Me of a replin cate QCs reported. All concenarterexparetssiedaosnppsb. * - Run Low QC| %Dif. (Medium QC| %Dif.| High QC| %Dif. [rrsma] 55 | svown[Lwore]] weJ[aawu]] woT[e3wm]] Number| 8.60 ppb 155 ppb 405 ppb Page 33 030241 Northwest Bioanalytical ReSptourdty NNoo.. NNWWBBRS00O0--102420 Table 22. Freeze-Thaw and Room Temperature Matrix Stability for PFOS in Human Serum All concentrations ar expressed as ppb. RunRNuunmDbaetre 21-Au1g8-2000 After 7 Low Qc Freeze-Thaw cycles 445613 440 MesDa.n 4L15431 Control % cv (ng/mL) 3.1 67 Mean % Dev. from Control 3.0 After 7MFEreDeIzeU-MThQawC cycles 115780 164 MesDa.n 610640 Control (a%g/mCLv) 13.770 Mean % Dev. from Control 35 After 7 FHrIeeGzHe-QTChaw cycles| 336796 ,: on 361 Mean sD. 369 7.51 Control (n%g/mcLv) 32708 Mean % Dev. from Cantrol 24 NOTE: Samples Cwoenrterollef=amt rooeomftaenamapleytrniactaulrQe Cfosraumpp0le6s7a5nahloyuzredpenritohdessabemtewereunneach freeze cycle Page39 03 0242 Northwest Bioanalytical ReSptourdty NNoo.. NNWWBBRSO0O0-.102420 `Table 23. Freeze-Thaw and Room Temperature Matrix Stability for PFOSA in Human Serum Allconcentratiaornes expresseasd ppb. Run Number Run Date 18 21-Aug-2000 After 7 Low Qc Freeze-Thaw cycles 373 353 3.8 MesDa.n 03..15382 Control (n%g/mCLy) 3.7 438 Mean% Dev. from Control 17.8 After MEDIUM QC 7Freese-Thaw cycles 108 126 125 Mean sD. 120 10.1 Control % Cv (ng/mL) 8.4 150 Mean% Dev. from Control 200 After HIGH QC 7 Freeze:Thaw cycles . 348 361 . 350 MesDa.n 353 7.00 Control % cy (ng/mL) 20 395 Mean% Dev. from Control 10.6 NOTE: Samples Cwoenrterollef=amteoaonmofatneamlpyetriactaulrefQoCrusapmp0le6s.7a5nahloyuzredpeirnitohdessbaemtewereunneach freezs cycle Page 40 030243 Northwest Bioanalytical RepStourdytNNoo.. NNWWBBRS00O0--102420 Table 24. Freeze-Thaw Stability and Room Temperature Matrix for PFOSAA in Human Serum All concenarteerxparetssiedoaspnpsb. RunRuNnumDbaetre 21-Au1gs-2000 After 7 FrLeoezwe-QThcaw cycles 9.08 9.85 9.46 Mean 9.46 sD. 0.385 Control (n%g/mcLv) a1 9.95 Mean% Dev. from Control "9 After MEDIUM QC 7 Freeze-Thaw eyeles 114 147 147 Mean sD. 136 19.1 wey Control (ng/mL) 114600 Mean % Dev. from Control "15.0 After HIGH QC 7Freeze-Thaw epeles| 383 403 . oe 391 : Mean sD. 1309.21 % Cv Control (ng/mL) 26 a3 Mean% Dev. from Control 1 NOTE: SamplesCwoenrterollef=am t rooeomftaeanmapleyrtnaitcaulrQe Cfosaumpptloe6s.a75nhaloyuzrepdeirnitodhse sbaemtewereunneach freeze cycle: Pagel - 650244 Norwest Biosnaytsl RepSourdtyNNoo.. NNWWBBRSOIDD-102420 Table 25. Freeze-Thaw and Room Temperature Matrix Stability for POAA in Human Serum All concentmionsaecxpresed 5 ys. RuaRNuunmDbaetre 21-Au1gs-2000 After 7FLroeweToshcae. cycles 55.8036 525 Msepn. cy 05..0381 is Mean % DeCvo.ntfrroolm nCgonitmrLo)l 272 After 7MFErDeeIsUe-MThqawccpetes 11591 I Mesab.n 215512 Mean % DeCvo.ntfrroolm (agimcLy) Control 1749 13 After 7 ruerecGeH.Tohca cycles 35361249 MesaD.n 7s5n1 Mean % DeCvo.ntfrraolm ng"icmLy) Control 52102 00 ,: oT NOTE: SamplesCeatneoeftmeraonoomfeanmapleytriacarleQCfrsupss6.s75hloepderiinotdhessenesrenn exh ze cele rages 030245 re Northest Bioanalytical ReSptourdtyNNoo.. NNWWBBRSO0D0-.102420 Table 26. Freeze-Thaw and Room Temperature Matrix Stability for PFHS in Human Serum All concenatrerexparetssiedossnppsb, Run Number, Run Date 18 21-Aug-2000 Low Qc After 7Freeze-Thaw cycles 6.05 627 7.12 Mean sD. 06..54635 % Cy 87 Mean% DeCvo.ntfrroolm (Cnogn/tmroLl) 6.86 55 After MEDIUM QC 7Freeze-Thaw cycles 165 157 163 Mean 162 sD. % Cv 416 2.6 Mean %DevC.onftrrooml (ag/mL) Control 164 12 After HIGH QC 7Freeze-Thaw eyeles| * 397 a 405 : MesaDn. 406 10.1 Control (n%g/mcLv) 2.5 403 Mean % Dev. from Control 0.7 NOTE: Samples`werCe lo=eamtnefraontotomfratneamlpoyetrialctaulrQefCorsuapmptloe6s.7a5nhaloyuzrepderinitohdessbaemtewereunne.achfreeze cycle. Pgs 030248 ---------- Northwest Bioanalytical RepSoturdty NNoo.. NNWWBBRS0000--102420 `Table 27. Freeze-Thaw and Room Temperature Matrix Stability for M556 in Human Serum All concenatre rexparetssiedoasnppsb. RunRuNnumDbaetre 18 21-Aug-2000 LOW QC After 7Freese-Thaw cycles 495 5.07 5.09 Mean 5.04 sD. 0.0757 Control (n%g/mcLv) Is 542 Mean% Dev. from Control 70 After 7MFErDeeIzUe-MThQaCw cycles 128 140 135 Mean 134 sD. 6.03 Control (n%g/mcLv) 4s 141 Mean% Dev. from Control 50 After 7FrHeIeGzeH-TQhCaw cytes |. 359 : an 374 : Mean 368 sD. 8.14 Control (n%g/mcLv) 22 367 Mean% Dev. from Control 03 NOTE: SamplesCwoenrterollef=amteraonomoftaenmpaelryattuirceQafClorusapmptloe6s7a5nahloyuzredpenritohdessbeatwmereeunneachfreezecycle Page dd 090247 Northwest Bioanalytical ReSptourdtyNNoo.. NNWWBBRS0000-.102420 Table 28. Freeze-Thaw and Room Temperature Matrix Stability for M570 in Human Serum All concentrationsareexpresseads ppb. RunRuNnumDbaetre 21-Au1g3-2000 After 7 FrLeoezwe-QThcaw cycles 7.68 885716 Mespa.n 532 0.565 Control (n%g/mcLv) 6 502 Mean % Dev. from Control 37 After 7MFErDeeIzUeM-TQhCaw cycles 115522 163 MesDa.n 0.155727 Control (n"g/cmLy) 04 149 Mean % Dev. from Control a1 After 7FrHeIezGeH:TQhCaw cycles| - e Loe a 337110 : Mean sD. 366 839 Control (n%g/mcLy) 32836 Mean % Dev. from Control 52 NOTE: SamplesCwoenrterollef=amt ooemoftaa enmapleytrniactaulreQfCosuapmptle6s.7a5nhaolyuzrepdernitohdessabemtewereunneach fieeze cycle Pages 030248 Norwest Bioanalytical `Table 29. Reinjection Stability for PFOS All concentrations are expressed 1s ppb. RuarNuanmtbeer auto Mesabn. Contra (awme)y Mesn 4 Dv. rom Contrl MeSba.n Cour agwienty) Mesa % De. rom Comet M5ebs.a Coote (awienty) Mesa % De.tromCone (Apes 2.5RLeDepOiWnQeooCon.m Ten awo0r a500 pri a20 2"uw MReepiIjUciMoQnC Apr25Doe oa Temp) 6in m8n in a8m nFy a Rwejaesctioocn 4p 2.5ys RoamTemp mx PmY at nisms i a ReSptourdtyNNoo., NNWWBBRSO000--102420 3 : oe . Control = mean ofanalytical QC samples analyzed in run 15 (original injection) Posed 050243 Northwest Bioanalytical Table 30. Reinjection Stability for PFOSA All concentrations are expressed as ppb. RunRNuonrDbaetre Mseoa.n Contrl (gw/emLy) Mean % Dev from Control Masaon. Contra (rwiemy) Mean % Dev from Control Mesaon. Contr (awmely) Mean% Dev. from Control gutz2000 RLejoecwtaiocn Apr2.5 Days RoomTemp) 226 226865 268 o2omm 23m0 a2 M`ERDenIjUctMioQnC Afr 25 Days Room Temp) 0122 9su9s 105 a10a0 "103 29 RHiInGHcoQnC 2.5Days Room Tem ums 3fn0 so 25576 03 pr} ReSptourdtyNNoo.. NNWWBBRS0000--102420 . a : `Control = meanofanalytical QC samples analyzed i run 1S (orginal injection) Page 47 030250 oo . Northwest Bioanalytical `Table 31. Reinjection Stability for POSAA All concentratiaornes expreasssppebd. RunRNuunmDbaetre duin2000 RLeionwjcqiocn er2.5 Days Roam Tey 7u5m8 718s1 2 Mesaon. 0222136 Control (ngc)y 5395 Me%aDenv from Const is MREeDnjIcUtMioQnC 4p 25Deys RoomTemp m10n9 1304 uo Mseoa.n snto Contrl (nwiemy) 332 Mean % Dev. from Control a7 Rneincjhcioocn Afr 25Days RoomTemp aws nFl 2s Mseoa.n 1a082 Control (g/mcLy) 035 Mean% Dev from Contest wo ReSptourdty NNoo.. NNWWBBRS0000--102420 : " 2 i : Contro=l meanofsnalytical QC samples analyzed in run 15 original infection) 090251 Page ds _ `Northwest Bioanalytical `Table 32. Reinjection Stability for POAA All concentrations are expressed as ppb. ReSptourdtyNNoo.. NNWWBBRSO0O0--102420 RunRNuonmDbaevre 3aun00 RLejoecwiaon Up 2.5 Days BoumT srse p53r1 sa MesaDn. o5uas Control (nsgiemLv) 110s Me%aDenv. from Control " MREeDnjIcUtMioQnC Ug 2.5 Days Roum 119520 115664 ww Messoa. 418500 Control (agimcLd) 3114 Mean% Dev from Control RmeijGecHiQoCn 4ter 2.5 Days RoamTo oasn ko3r 1w6 sMoa.n o1o2 Contra (nginctyy I33 Mean % Dev from Control . tea : Conieol= meanofanalytical QC samples analyzed in run 13 (original injection) Page 49 030252 Northwest Bioanalytical Table 33. Reinjection Stability for PFHS All concentrations are expressed as ppb. ReSptourdtyNNoo.. NNWWBBRSO0D0--102420 RunRNuunmbDeart.e duin2000 RLeionwecaiocn (Uper 2.5 Days Room Tem 6830 s59a8 su Mesaon. 021965 Contrl (wnegy) 251 Mean% Dev, from Control o MREeDnjIeUcMioQnC dpe 2.5 Days Boom Temp) 11556 115569 1 Mseon. 21551 Contes (nwg/emLy) 11568 Mean % Dev. rom Conteal a Rnenijccioocn fir 2.5Days Room Temp) ""6 aolw ps Mesoa.n a14 Contr (rwgiemly) par Mean% Dev. from Contest "0 ::, . . `Control = meanof analytical QC samples analyzed in run 15 (original injection) 030253 Page 50 `Northwest Bioanalytical RSeptourdtyNNoo.. NNWWBBRSO000--102420 Table 34. Reinjection Stability for M556 All concentrations are expressed 55 ppb. RunRuNnumDbaetre sukn000 RLenojwcaiocn (4p 2.5DaysRoomTemp) ssna s5i2s9 53 Man so. ossm Contr (nwpeny) saet Mean% Dev. from Control as MREeDjeIcUtMioQnC per2.5 Days Room Te Wuew 0w 6 Measno. iusn Contra (nng/emlv) wisw Me%aDenv. from Control a4 RmejieuctiQocn 72.5Days RoamTemp) " mES ` a EFia -: 0 Mesao.n nmz Contra (nng/emlv) 2362 Mea%n Dev. from Control 38 Control = meanof analytical QC samples analyzed in run 1S (original injection) Page st 050254 Northwest Bioanalytical Table 35. Reinjection Stability for M570 All concentrations are expressed as ppb. ReSptourdtyNNoo.. NNWWBBSR000--102420 RunRNuanbDeart.e auk1000 RLeoinweacocn (4p 2.5 Days Room Temp) 13 90 21 Mesaon. 0a3212 Contr (angeinv) 155 Mesn % Dev. from Contra a3 MREeDnjIeUctMioQnC Ape 2.5 Days Room Temp) uuso 1w6w us Mseoan. 9u2s Contea (nwg/emly) wisw Me%aDenv. from Conteal "01 Rmeenjncqiocn er 15 Days RoomTemp) i1u +. 30 : ws Mseon. 933%1 Control (amle)v o24 Mean % Dev, fram Conteal a1 Control = mean ofanalytical QC samples analyzed in rn 13 (original injection) 0902535 Pages2 Northwest Bioanalytical ReSptourdtyNNoo.. NNWWBBRS00G0--102420 `Table 36. Room Temperature Extract Stability for PFOS All concentrations are expressed s ppb. Run Number 18 RunDate 21-Aug-2000 LOW QC After 7Days 517 46.0 462 Mean 48.0 SD. 323 % CV 61 Control (ng/mL) 46.7 Mean % Dev. from Control 28 HIGH QC After 7Days 3m 402 397 Mean 301 SD. 155 % CV 40 Control (ng/mL) 3m Mean % Dev. from Control 34 . : ot `Control= meanofQC samples analyzed in the same run immediately following extraction Pages 090258 Northwest Bioanalytical ReSptourdty NNoo.. NNWWBBRS0000--102420 `Table 37. Room Temperature ExtractStability for PFOSA All concentrations are expressed as ppb. Run Number 18 Run Date 21-Aug-2000 LOW QC Afier 7 Days 348 334 330 Mean 337 SD. 00945 % Cv 28 Control (ng/mL) 3.13 Mean% Dev. from Control 77 HIGH QC After 7Days 337 362 334 Mean 344 sD. 154 % CV 45 . Control (ng/mL) 322 Mean % Dev. from Control 68 . or Control = meanofQC samples analyzed in the same run immediately following extraction Page 54 090257 Norwest Biosmalycal ReSptourdtyNNoo.. NNWWBERSOIDD-014202 `Table 38. Room Temperature Extract Stability for PFOSAA All concentrations sre expressed ss ppb. `Run Number Run Date 18 21-Aug-2000 Low QC After 7 Day 82 9923 Mean S.D. % CV Control (ng/mL) Mean % Dev. from Control 8.89 0.607 68 8.02 10.8 AfHtIerG7HDQayCs 332317 343 Mean 334 S.D. 114 % CV 34 . Control (ng/mL) 297 Mean % Dev. from Control 125 co Control = mean ofQCsamplesanalyzed in the same run immediately following extraction poss 00258 Northwest Bioanalytical ReSptourdty NNoo.. NNWWBBRSO0O0-.102420 Table 39. Room Temperature Extract Stability for POAA All concentration are expressed as ppb. Run Number 18 Run Date 21-Aug-2000 LOW QC After 7Days 692 7.98 8.15 Mean 7.68 SD. 0.667 % Cv 87 Control (ng/mL) 7.82 Mean % Dev. from Control 13 HIGH QC After 7Days 360 395 378 Mean 378 sD. 17.5 % CV 46 . Control (ng/mL) 372 Mean % Dev. from Control 16 ot Control = meanofQC samples analyzed inthe same run immediately folowing extraction riget 030253 Northwest Bioanalytical StudyNo. NWBS00-040 Report No. NWBR0O-122 `Table 40. Room Temperature Extract Stability for PFHS All concentrations are expressedas ppb. Run Number 18 Run Date 21-Aug-2000 LOW QC After 7 Days 631 621 590 Mean 614 SD. 0214 % CV 3s Control (ng/mL) 686 Mean% Dev. from Control 105 HIGH QC After 7 Day 39 405 a2 Mean 404 sD. 8.02 % Cv 20 Control (ng/mL) 403 Mean% Dev. from Control 02 - co os `Control =meaofQnC samples analyzedinthesame run immeditely lowing extraction Page 7 030260 Northwest Bioanalytical Study No. NWBS00-040 Report No. NWBR00-122 `Table 41. Room Temperature Extract Stability for M556 All concentrations are expressed a ppb. Run Number 18 RunDate 21-Aug-2000 Low QC After 7Days 5.13 565 5.50 Mean 5.43 sD. 0268 % CV 49 Control (ng/mL) sa Mean% Dev. from Control 02 HIGH QC After 7Days 373 376 387 Mean 379 SD. 737 % Cy 19 Control (ng/mL) 367 Mean % Dev. from Control 33 oe Control = meoafQnC samples analyzed inthe same run immediately following extraction Page 58 050261 Northwest Bioanalytical ReSptourdty NNoo.. NNWWBBRS00O0--102420 `Table 42. Room Temperature Extract Stability for M570 All concentrations acs expressed a5 pb. Run Number 18 Run Date 21-Aug-2000 Low QC After 7Days 336 9.13 874 Mean 891 sD. 0.200 % CV 22 Control (ng/mL) 8.02 Mean% Dev. from Control 11 HIGH QC After 7 Days 409 429 423 Mean 420 SD. 103 % CV 2.5 . Control (ng/mL) 386 Mean % Dev. from Control 88 co oe Control = meanofQC samples analyzed inthe same run immediately following extraction Page 59 090262 Northwest Bioanalytical ReSptourdtyNNoo., NNWWBBRS00O0--102420 Table 43. Reduced Temperature (1 to 8 C) Extract Stability for PFOS Allconcentrations are expresasspepbd. Run Number Run Date 18 21-Aug-2000 LOW QC Af1tte4or8DayCs 49.3 45.6 4438 Mean 46.6 S.D. 240 % CV 52 Control (ng/mL) 46.7 Mean % Dev. from Control 0.2 HIGH QC Aft1etro48DaCys 391 385 377 - Mean 384 S.D. 7.02 % CV 18 Control (ng/mL) 378 Mean % Dev. from Control 16 or oC . Control = mean ofQC samples analyzed nthe same un immediately following extraction J. 050263 Northwest Bioanalytical ReSptourdtyNNoo.. NNWWBBRS00O0--102420 `Table 44. Reduced Temperature (1 to 8 C) Extract Stability for PFOSA All concentrations are expressed as pb. Run Number 18 RunDate 21-Aug-2000 Low QC 1to8C After 4 Days 32 279 286 Mean 2.96 SD. 0231 % CV 78 Control (ng/mL) 313 Mean % Dev. from Control 54 HIGH QC 1to8C After 4 Day 350 297 304 Mean 317 SD. 2838 % CV 91 Control (ng/mL) 32 Mean % Dev. from Control 16 . 2a o `Control=meanofQC samples analyzed in the same run immediatly following extraction Page 61 030264 Northwest Bioanalytical RepSoturdtyNNoo.. NNWWBBRS00O0--102420 `Table 45. Reduced Temperature (1 to 8 C) Extract Stability for PFOSAA All concentrations are expresassppebd. Run Number 18 RunDate 21-Aug-2000 LOW QC 1togC After 4 Days 778 8.55 8.32 Mean 8.22 SD. 0395 % CV 43 Control (ng/mL) 802 Mean % Dev. from Control 25 HIGH QC 1to8C After 4 Days 312 306 3 Mean 310 sD. 321 % CV 10 Control (ng/mL) 297 Mean % Dev. from Control 44 - } al o- . Control = meanofQC samples analyze in the same ru immediately following extraction rage 090265 Northwest Bioanalytical RSeptourdtyNNoo.. NNWWBBRSO000--102420 `Table 46. Reduced Temperature (1 to 8 C) Extract Stability for POAA All concentrations ae expressed as ppb. Run Number 18 RunDate 21-Aug-2000 LOW QC 1t08C After 4 Days 778 875 839 Mean 831 SD. 0490 % CY 59 Control (ng/mL) 782 Mean% Dey. from Control 63 HIGH QC 1t08C After 4 Days 381 394 385 Mean 387 . SD. 6.66 % Cv 17 co - Control (ng/mL) 372 . Mean % Dev. from Control 40 Control = meanofQC samples analyzed in the sume run immediately following extraction Pages 090265 Northwest Bioanalytical ReSptourdtyNNoo.. NNWWBBRSO0O0--102420 Table 47. Reduced Temperature (1 to 8 C) Extract Stability for PFHS. All concentrations ar expressed ss ppb. Run Number 18 RunDate 21-Aug-2000 Low QC 1to8C After 4 Da 6.76 634 7.74 Mean 695 sD. ong % Cv 103 Control (ng/mL) 636 Mean % Dev. from Control 13 HIGH QC 1to8C After 4 Days 400 41 423 Mean au SD. 115 % CV 28 Control (ng/mL) Mean % Dev. from Control 403 20 . : or : (Control = mean ofQC samples analyzed inthe same run immediatly following extraction Page 64 030267 Northwest Bioanalytical ReSptourdtyNNoo.. NNWWBBRS00O0-.102420 Table 48. Reduced Temperature (1 to 8 C) Extract Stability for M556 All concentrationsareexpressed as ppb. `Run Number 18 RunDate 21-Aug-2000 Low QC 1to8C After 4 Days 547 657 682 Mean 629 SD. 0718 % Cv 1.4 Control (ng/mL) 542 Mean % Dev. from Control 161 HIGH QC 1to8C After 4 Days 398 404 403 Mean 402 SD. 321 % Cv 08 Control (ng/mL) 367 Mean% Dev. from Control 95 : Ct - . Control = meen ofQC samples analyzed inthe same run immedistely folowing extraction Pages 050268 Northwest Bioanalytical Study No. NWBS00.040 Report No. NWBR0O-122 Table 49. Reduced Temperature (1 to 8 C) Extract Stability for M570 All concentrations are expressed as ppb Run Number RunDate 18 21-Aug-2000 Low Qc 1to8C Afier 4 Days 7.96 7.90 921 Mean 8.36 SD. 0.740 % CV 89 Control (ng/mL) 8.02 Mean % Dev. from Control 42 HIGH QC 1togC After 4 Days 396 394 402 Mean 397 . SD. 4.16 % CV 10 Control (ng/mL) 386 .: Mean % Dev. from Control 28 `Control = meanofQC samples analyzed in the same run immediately following extraction Page 66 050269 Northwest Bioanalytical Study No. NWBS00.040 ReportNo. NWBR0O-122 Table 50. Reduced Temperature (-20 C) Extract Stability for PFOS All concentrations are expressed as ppb. Run Number 18 Run Date' 21-Aug-2000 Low QC 20C After 5 Days 412 45.5 409 Mean 445 sD. 326 % CV 73 Control (ngml) 46.7 Mean % Dev. from Control 4.7 HIGH QC 20C After 5Days 376 373 365 Mean 371 SD. 569 % CV 15 Control (ng/mL) ~~ 378 Mean % Dev. from Control ~~ 1.9 I. et o . Control = meanofQC samples analyzed in the same run immediatly following extraction Pages? 050270 Northwest Bioanalytical ReSptourdtyNNoo.. NNWWBBRS00O0--102420 `Table 51. Reduced Temperature (-20 C) Extract Stability for PFOSA All concentrations are expresseda ppb. Run Number 18 RunDate 21-Aug-2000 Low QC 20C After 5 Days 295 3.16 314 Mean 3.08 sD. 0.116 % CV 38 Control (ng/mL) 313 Mean % Dev. from Control 16 HIGH QC 20C After 5 Days 312 312 296 Mean 307 . SD. 9.24 %CV 3.0 co o Control (ng/mL) 322 Mean% Dev. from Control "7 ' Control = meanofQC samples analyzed in the same run immedistely following extraction 030271 Page 68 Northwest Bioanalytical ReSptourdtyNNoo.. NNWWBBRS00O0-.102420 Table 52. Reduced Temperature (-20 C) Extract Stability for PFOSAA. All concentrations are expressed s ppb. Run Number 18 Run Date 21-Aug-2000 LOW QC 20C After $ Days 7.52 832 7.89 Mean 791 SD. 0400 % CV 5.1 Control (ng/mL) 8.02 Mean % Dev. from Control 14 HIGH QC 20C After 5 Days 204 306 287 Mean 296 SD. 9561 % CV 32 Control (ng/mL) Mean % Dev. from Control 297 03 . Bi - : Control = meanofQC samples analyzed in the same run immedisely following extraction 090272 Page 69 Northwest Bioanalytical RSeptourdtyNNoo.. NNWWBBRS00O0-.102420 Table 53. Reduced Temperature (-20 C) Extract Stability for POAA All concentrations are expressed as pb. Run Number 18 Run Date 21-Aug-2000 Low QC 20C After 5 Days 7.84 102 6.85 Mean 830 SD. 172 %Ccv 207 Control (ng/mL) 7.82 Mean % Dev. from Control 61 HIGH QC 20C After 5 Days 351 373 386 Mean 370 . SD. 1.7 % CV 48 Control (ng/mL) 32 co > oC Mean% Dev. from Control 05 Control = mean ofQC samples analyzedi the same run immediately following extraction Page 70 030273 Northwest Bioanalytical ReSptourdtyNNoo.. NNWWBBRSO0O0--102420 Table 54. Reduced Temperature (-20 C) Extract Stability for PFHS All concentrations ase expressed as ppb. Run Number 18 Run Date 21-Aug-2000 LOW QC 20C After 5 Days 6.68 6.06 7.70 Mean 681 SD. 0828 %CV 122 Control (ng/ml) 6.86 Mean % Dev. from Control ~~ -0.7 HIGH QC 20C After 5Days. 403 407 a7 Mean 409 sD. 721 % CV 18 Control (ng/ml) 403 : - Mean % Dev. from Control 15 `Control = meanofQC samples anlyzed in the same run immediately following extraction 090274 Page 71 Northwest Bioanalytical ReSptourdtyNNoo.. NNWWBBRSO0O0-.102420 Table 55. Reduced Temperature (-20 C) Extract Stability for M56 All concentrations are expresseads pb. Run Number 18 Run Date 21-Aug-2000 Low QC 20C After 5Days 451 5.69 597 Mean 539 sD. 0775 %CY 144 Control (ng/mL) 5.42 Mean % Dev. from Control ~~ -0.6 HIGH QC 20C After 5 Days 358 373 378 Mean 370 " SD. 104 % CV 28 - Control (ng/ml) Mean% Dev. from Control 367 08 ' Conteol = meanofQC samples analyzed in the same run immediately following extraction Page 72 030275 Northwest Bioanalytical ReSptourdtyNNoo.. NNWWBBRSO0O0-.102420 `Table 56. Reduced Temperature (-20 C) Extract Stability for M570 All concentrations are expresseda pb. Run Number 18 Run Date 21-Aug-2000 LOW QC 20C After 5 Da 843 8.52 7.94 Mean 830 sD. 0312 wey 38 Control (ng/ml) ~~ 8.02 Mean % Dev. from Control ~~ 3.5 HIGH QC 20C After 5 Days 366 388 369 Mean 374 sD. 119 %Cy 32 Control (ng/mL) ~~ 386 Mean % Dev. from Control ~~ -3.1 y : "0 : `Control = meanofQC samples analyzed inthe same run immediately following extraction 030275 Pages Soong eyeowEsn030 Table 57. Long-Term Matrix ---- Stability for PFOS in Human Serum vRunn | LEoywQeC || MeMdieummeQC|| HHiagkhaQcC T T Ts =T T m mw w] ] T T TwT o T Tw o ow |] o ml TwowT T | oms| awen]] woeml se T| nor[ |=]| CW 515T 15 Table 58. Long-Term Matrix Stability for PFOSA in Human Serum TA o Tar [emma| mma Number| 4.00 ppb 150 ppb. 400 ppb T memo] 5T |w T | ma|mow | T TweonT [w wmw | ] wi] TT wo wmwol Toow Twwm awr]] wweele Tosa 00 ] |1] CW 15T 15] 050277 -- o-- rn `Table 59. Long-Term Matrix eerie Stability for PFOSAA in Human Serum woe Run | ove Low QC [Metimac Medium QC Tamrac High QC GC omme| 5T[soueT | Tw0T | aw| TT To ow wa a] e so T Tow |w am w0a|] m we[ l ou sT [1m | 50 | CT 151 voaATml|coTpnrveeepre[[hsMeithimmeca| emaace Table 60. Long-Term Matrix Stability for POAA in Human Serum C ome [5Tn wT Tm w |e ]| 55 Days 55 Days. 55 Days Tm ws] TT ww esol Toww |wmw w | n] e | [wseell TToo sT [as a[a5 r] C1 TT 55 1] es 050278 emo Table 61. Al ceieocrsne s5 Long-Term Matrix Stability for PFHS in Human Serum EE Run Low QC ETMedium QC ITS High QC aC anT ] 0Teom [w waw e | T To o w ww w es wes| o om To w |w em w |w me | e me ise [[weeese s | a 15 51 5 | Table 62. Long-TAercmeMmaotrrmiioxnSstavbsilityefsordM5556 in Human Serum wm] TEdTams ||[mMemae ||| TmmeEes C ame| eeweT w | wm w ws | T TweT w Twwe swee] TT Tw |w| msol seeenT | w ew | mwse|] meell Tw we[Teosr| wr|] w ETT T Ts | 050279 meses nFeUms,e `Table 63. Long-Term Matrix Stability for M570 in Human Serum wo Tec [Mame Hae Number 8.60 ppb 155 ppb 405 ppb f C r T Tw w 55 Daya T sT |ow 55wDays.|] 5w5wDmaiys ] T T wew T [ooww wwem] Tew we] vseoll Toem |w 5% w we]| veewlT er |w 59||a1]| C5T TT] -- 090280 Northwest Bioanalytical ReSptourdtyNNoo.. NNWWBBRSO0D0--102420 `Table 64. PFOS Extraction Efficiency from Human Serum Run22 "Tow Concentration = #00 ng/mL 1.AssTiymaenod fStpiekrienxgstion Mean Rati0o.A0m6l7v9e/lS 0.0683 00735 0.0699 RatioIS1/8A7malvte 146 136 143 2Asay prio and 5ahrciction 0.0656 0.0605 0.0649 Mean 0.0637 3.15 priorsodAnalyteafer ection 071s ons 0305 Mean 0769 Adjusted mean extraction efficiency = 44.4% Mean extraction efficiency for the internal standard = 538% High Concentration = 400 ng/mL. Timeof Spiking RatioAmlvilS 1Anite30d15 serctction 165 155 16s Mean 162 RatiolS/Analvte 0.605 0.643 0.607 0618 2 Ansepirsd1Saterection o0s2 0538 0518 Mean 0.526 3.1prioradAnseser extcion Mean = 006 . | aa 00379 00401: 00392 Adjusted mean extraction efficienc=y 30.1% `Mean extraction efficiency for the internal standard = 634% Overallmean extraction ficiencyfor analyte = 37.3% Overall mean extraction efficiencyfor internalstandard = 5.86% * Due t0 rounding, calculations based on displayed values may difer rom actual. 030281 Page 78 Northwest Bioanalytical ReSptourdty NNoo.. NNWWBBRS0000--102420 Table 65. PFOSA Extraction Efficiency from Human Serum R22 Tow Concentration = S00 mgnL 1. ATnimenedo1f5Sspiekeinxgci Rati0o0A2m7i5velS 000022124 Pr-- Ratio [3A63mie 5pr0 27 2Antec 1 cision o00o14n7 I-- oi 2.15 pricsndAssi secsion 247 222855 Mean 239 Mean extraction efficiency for the internal standse=d 5.60% High Concentration = 400ng/mL. 1. Av sterxe ction 225487 Mean 2Z6T5 0387 003:7075 L 0390 2 Ase prs 5 aicxntion 180 1178s Mean Tar . 3.1a5 stestrction Mean 00002233 + se 0G0o2m333 Mean extraction efficiency for the analyt=e 70.4% Overall meOavnereaxltlrmacetaionneexftfriacciteinocnyfeofrfiinciteenrcnyaflorstaannadlarydt==e 659..769%% * Due to rounding, calculations based on displayed values may differ from acral. 030282 Page Norwest Bioanalytical ReSptourdtyNNoo.. NNWWBBRSO0O0--102420. Table 66. PFOSAA Extraction Efficiency from Human Serum Ran22 "Tow Concentration = F00 gin 1AnTin0eo1fShoeirkeinxgci Rati0o0A2n4a6lveS 0o02s57 Mean 0.0287 Ratio[0SA6nalvte 32900 05 2Asiepsd 5 srction 0000128s6 Mean 0000119945 3.15 pricsndAva srcsion 227415 Mean 225588 Mean extraction`Aefdfjiucsietnecdymefoarntehxetirnatcetrinoanlesftfaincdiaerndc==y 6623.76%% High Concentration = 400ng/mL. 1 A`Timn neodf5Sat ptierkine gction Ratio1A5m0aiveS L1a 56 Mean 150 Ratio0I6S/6A5nalvte 00663913 0666 2Aas pri ad1sfxtion 0055692 Mean 00904167 ; 3.15 rirsndAnsyscnion Mean 0000431094 <= 0000:40513: `Mean extractionAedfjfuscteendcymfeoarntehxetirnatcetrinoanlesftfiacnedncayr=d= 66.21.47%% Overallmean extractionefficiencyfor analyte 62.7% Overall mean extraction efffioricnteirnaelstnancdaryd= 626% * D0uroundeing, calculations based on displayed valuesmay ifroe mactual. 050283 Page 0 Northwest Bioanalytical ReSptourdtyNNoo.. NNWWBBRSO0O0--102420 Table 67. POAA Extraction Efficiency from Human Serum Rin22 Tow Concentration = #00 gil 1. Anite nd 15 erxin 0000223981 334473 Mean 00.0032092% 3E1Q 2Anepd 5a xin 000000973640 Mean 0o.o0t090017 3.1 priorsnd Anish ncn 210530 205 Mean 700 Mean extractionAdefjfuicsiteencdymefoarntehxetirnatcetrinoanlesftfaincideanrc==dy 51.68.33%% High Concentration = 400 ng/mL 1. Tyiemenod1f5Speirkixngsi Ratio2A6m1ivelS 225m9 Mean 26 Rati0o3I8S/3Aalve 00336876 3 2Aas pri 1d1 atercron 00338757 Mean 00338832 = 3.15 piesndArarcnn Mean 0000222s7 - ae 000223451 Mean extractionAedfjfuisctieendcymefoarntehxetirnatcetrinoanlesftfaicnideanrcdy == 61.4331%% Overallmean extraction effcincyforanalyte = 15.6% Overall mean extraction fficencyfor internalstandard = 6.10% * Dtou rounde ing, caeultion bse on displayed values may differ from aca, 030284 Pages! Northwest Bioanalytical ReSptourdtyNNoo.. NNWWBBRSO0O0--102420 `Table 68. PFHS Extraction Efficiency from Human Serum Rn22 "Low Concentration = 4.00 ng/mL 1.ArTtimsendo1fS Ssohieksixnsgion Rusi0o.A03m6i7velS 0061 Mean 0000336656 RatolSAnsivie 2773 T7i3 [RO -- 000000787780 000878 Mean "0.00882 3.1rind Arsaticnction 11762 Mean 6I Mean extractionAedfjfcuesnctyed mefoarntheextirnatcetrinoanlesftfaicnideanrcdy == 61.61.73%% High Concentration = 400 ng/mL. 1. Anemd 5st ttn 33429 00238064 Mean 335485 05227900 2Aasbtepriors 1 erction o0a5t1s4 Mean 0"0449s5 - 2.15 prosod Asstser xcion Mean ooiso! "- 0o1os? + Mean extractionAedfjfuisctieendcymefoarntehxetirnatcetrinoanlesftfacnednacryd == 61.44.83%% Overall mean extraction ffciencyfor analyte = 15.3% Overall mean extraction ffcencyorinternalstandard = 6.33% + Due o rounding,calculationsbased on displayed values may dife from scnal. Page 82 050285 Norwest Bioanalytical ReSptourdty NNoo.. NNWWBBSR000-.102420 Table 69. M556 Extraction Efficiency from Human Serum Rin22 "Tov Concentration = 400mgnL 1. Anndi5artxeo 00..0000778560 1127 Mean 000.000788120 1238 2Aste rirad1 terxsion o0.o000s362615 Mean 00.0000331608 2.15pr anAn heree xci 765148 Mean 608 MeaAndjeuxtsrtaecdtimoenaenffeixctriaecntfciooyrtnheefafincliyent=c=ey 4367..81%% High Concentration = 400 ng/mL. 1. A`nTeoif5Saimkriexngion Rati[oAImalvelS 00785386 Mean 0754 Ratio [1S2/7Amivie 112220 126 2Asiesd1 ste ccc 00333276 Mean 00330232 = 5.1 pri ndArm tercrcien Mean o00r85s0+ -- o0o7p7 * Mea`nAdejxutsrtaecdtimoneaefnfiecxiterncsyeftoorntheefafnicaileynt=c=ey 4400..64%% Overall mean extractionefficiency for analyte = 39.1% Overall mean extraction efficiencyfor internal standard = 5.52% * Due o rounding, calulations baonsdisepladyed values may differ fom actu, 090288 Pages Northwest Bioanalytical ReSptourdtyNNoo,. NNWWBBRS00O0-.102420 Table 70. M570 Extraction Efficiency from Human Serum Rn22 "Tow Concentration = T007g/mE 1. Avie 15ae xtctin 0000333137 33101s Mean 0000333258 30095 2Asstpio nd1 erxin 000022404 Mean 000022346 3.15 prior esi he xacion 152 717s Mean 5) Mean extractionAedfjfuiscteendcymefoarntehxetirnatcetrinoanlesftfacnednacryd == 5847.4%% High Concentration = 400 nginL. 1.AnTiennedo1f5Saoikxingin Ratio2A2m0lvelS 221365 Mean 220 Ratio0[4VA5n8alvie 00446236 0a 2Astpir 1 erxin 113347 Mean 113 32 = 3.15 pra Ani ter xin Mean 00002240 "a 000221s + Mean extractionAedfjfuiscteendcymefoarntehxetirnatcetrinoanlesftfaicnideanrcdy == 5692.77%% Overall mean extraction efficiencyfor analyte = 57.1% Overall mean extraction efciencyforinternal standard = 607% * Due to rounding, caleulations based on splayed values may differ fom acu. Page 84 050287 [-- ReSptourdtyNNoo.. NNWWBBRSO0O0--102420 Table 71. Summary of Calibration Curve Parameters for PFOS in Human Plasma Quadratic weighted 1. All concentrations ae expresaspepd. 12:0ct:2000| S000912_| -0.000007| 0.030663| -0.005620| 0.9986 27-0ct-2000|PFOSMD--40| 0.000000| 0.028132| 0.010586| 09931 |39 |414 4 | ALB,andCare coefficientusd tdein the calibration curve. Table 72. Summaryof Calibration Curve Parameters for PFOSA in Human Plasma Quadratic weighted Ux'. All concentrraeetxpiroesnsesdss ppb. 12-0012000|_S000912_| -0.000011| 0.073810| 0006421| 09970 |10 |s00 0_| 27-Oct-2000| PFOSMD-40| 0.000001 | 0.052188| 0.010966| 09912 ALB,andCarecoefficientsusedtodeintecalibration curve. Table 73. Summary of Calibration Curve Parameters for PFOSAiAn Human Plasma Quadratic weighted 1s' Allconcensrte rexparetssiedoasnpbs. Run A C |R-Squarea|LLOGQ[ULOQ Number 12-0ct-2000| S00091-2 | 0.000003| 0.032601 09988 _|1.|6so10_| 27-0ct-2000|PFOSMD-40| 0.000005| 0.021209|0.002450| 09873 |[6 | 50 01| A.B, and Care cosfints ued 0 define the calibration curve Pagess 030258 Northwest Biosalyial ReSptourdtyNNoo.. NNWWBBRSO0O0-.102420 Table 74. Summary of Calibration Curve Parameters for POAA in Buman Plasma Quadratic weighted 1. All concentrations are expresasspepbd. [= roman| Som009e12 || 000=000| 0| 0* 017245|0. 0010[72|m09o985]|1i.|9h4i82 1i| 27-0w2000|PFOSMD-40] 0.000002 | 0.014346 | 0002192| 0918 |15 | as2 i| A,B, 20d Carecoefientsuse tdefine he calibration ve. Table 75. Summary of Calibration Curve Parameters for PFHSin Human Plasma Quad weighed 1. All concenratonsserxpresse3d pb. RunDate | Run A C |RSquirea|LLOQ|ULOQ | Number 12-0200| S00091-2_| -0.00|00.002058518| 0.000970| 0.9983 27-00-2000|PFOSMD-40| -0.000001| 0.025534| 0011247| 0.9953 AB nd Carecoefiietsusd 0definethe calibrationcurve. Table 76. Summary of Calibration Curve Parameters for M556 in Human Plasma Quadratic weighted 1. All concentration ae expressedas ppb Run A Number C |ReSquared |LLOQ|ULOQ - Tr0ma00| 000912 | 000002|0018315| 000sset| _osvsa_| 25 |so0 o| SLow202040|| 0.000001 [0018373|0.001923| 09972 ALB, 20d Care coefensused to define hecalibration curve. Pages 050289 Northwest Bioanalytical ReSptourdtyNNoo.. NNWWBBRS00O0--102420 Table 77. Summaryof Calibration Curve Parameters for M570 in Human Plasma Quadratic weighted 1/x". All concentrations are: expressed as ppb. 12-00-2000 | S000912 | 0.000006| 0.048518| 0.005367| 09978 [100|50| 27-0ct-2000| PFOSMD-40| 0.000001| 0.034031| 0.011681| 0.9910[100 [500| AB, and Care coccienusted to definethecalibrationcurve. 050290 Pages? `Table 78. Back-Calculated Concentrations of Calibration Standards for PFOS in Human Plasma Quadratic weighted 1/x'. All concentrations are expressed as ppb. [| oRoanene T|rasnNoamoboe[a5l5m4s[s5o1ur71wn03zf35m504]lmt2s3a[[ws5sa7T|movuol5sa[[r3e]]] 1 oii rowTo7s2o[ls1er[l1u3]o10u4r4s5[s1u1s [o2m T[sa3rar]| Table 79. Back-Calculated Concentrationsof Calibration Standards for PFOSA in Human Plasma Quadratic weighted 1/x'. All concentrations are expresasspepbd. [amonzpane[|rasnwoaonmber| 1o20r[2z5oo0]0w0a35510[0ia[siaosTs[so0]) Tolan on m]sos]wa[ossao] `Table 80. Back-Calculated Concentrations of Calibration Standards for PFOSAA in `Human Plasma Quadratic weighted 1/x'. All concentrations are. expressed as ppb. 2. |rsI uarznoenT|rusnwSouombnTe[riv3se0lso55n11[02m]o1sso0[s7m32s55s36[[ss0ou3ao[]2or[[5aossnl[wo5er]s]]] [ rowT romTaol1o65s0[]o1s7[2n1s3]5m1m10ss a9l]au]] 090281 meso p-- e orn Table 81. Back-Calculated Concentrations of Calibration Standards for POAA in Human Plasma Quadratic weighted 1/x". All concentrations are expressed as ppb. [rroemmeoT|owroamaer[1am2o 3v3a6rwwss a3so[[nao o7nsa[aan ss oorm]| 1 Tiseonwi[sassofm weer] T Dwpnlwlsiawmml]] Table 82. Back-Calculated Concentrations of Calibration Standards for PFHS in Human Plasma Quadratic weighted 1/x'. All concentrations are expressed as ppb. EoTem iose[d1de27awlswossda[eell m [oln] T ce|FronTilao[u27s 1T w0us[]a0s]ssvalo1s7[a2w[tens[oe0]] [[[lonuslas]sulmlzolmle] Table 83. Back-Calculated Concentrations of Calibration Standards for M556 in Human Plasma Quadratic weighted 1x. All concentrationsar expresseads ppb. "- Eouln| sowiae [azw d 10]75[7 [1e 1[2 a sI eam] 3 aewp[w0lu[s2lo0rf3w0[e20l[om5w[o029]] CTTewpmlusloslmmwlmle] 030282 SO o-- e Table 84. Back-Calculated Concentrations of Calibration Standards for M570 in`Human Plasma [Foe Joe sr5) `Quadratic weighted 1/x". All concentrations are expressed as ppb. owzon| SowsTioTow[n[s1w5[[ou[1 me[s3n0o]w[m2 m[[wswm0]lm1|] [1 Ton[ms[wlmlwslms mmm] 090293 -- E-- u `Table 85. Lower Minne Limit of Quantitation for PFOS in HumanPlasma ---- [[oBoewme [[roorsewmo]li(o3.94,nppb)]| T Tw a] ]| I m-- w-- ] I o-- T T ] CT [eo mmaw Ts] ] CT] 090294 Sa Table 86. Lower Limit of Quantitation for PFOSA in Human Plasma `Run Number LLOQ (1.00 ppb) Tw TT T i w] ] T To w w] ] TT ow esolow]| EE el wn ST] a 090285 RR am Table 87. Lower min ss rt Limit of Quantitation for PFOSAA in `Human Plasma `Run Number LLOQ (1.60 ppb) Tw we T T a T ] ww] I so ow | E-- e E -- w I malTa i 030238 enim fot mst Table 88. =tense Lower Limit of Quantitation for POAA in (1.92 ppb) Human Plasma C TT w] ow] T T w T ] w T --T wT ] I ew] [e ieresa w] ou] C wela T] ] 000297 [Fe eg `Table 89. Lower Limit of Quantitation for PFHS in Human Plasma (1.36 ppb) [TT ww [To we [TT =e | [Te [ T [de] Tm [sol Tom| [e[ meomica|e T1ov 5 s || [ [m 1 a] l si 090298 Sun T ties sa tEoa, Table 90. Lower Limit of Quantitation for M556 in Human Plasma Number (2.50 ppb) T Tw w] ] T Tw w] ] T 1 e] voelTow]]| [e fal l o0]] 1 mal ] 30] 050299 -- s-- hut Snes Table 91. Lower Amis Limitof Quantitation for M570 in `Human Plasma Run Number row LLOQ (1.00 ppb) TT T ow] e solT wo] w| [me ea]w] mo] C m T w ]| 000300 ae vase Table 92. Intra-Assay Atoms se pete Precision for PFOS Quality Controls in Human Plasma Run Number| Low QC |Medium QC| High QC 6.40 ppb 126 ppb 332 ppb. T TTw oT [m Tw w5] ] TT Tow w ww e] ] e e sol TomsT T |a awmm ||iew] e] C mel T Tm|eT Tm eit mee set mre 050361 te mn et mss Table 93. Intra-Assay Precision for PFOSA Quality Controls in Human Plasma Run Number| Low QC| Medium QC| 4.00 ppb 150 ppb High QC 400 ppb TTTTeom ww w| C T ToeoT [aw| ww vel Te|wwe el ToTo uw] eeT TeT n1w 5 , 090302 apes Table 94. Intra-Assay Precision for PFOSAA Quality Controls in Human Plasma All concentrations are expressed as ppb. [[oreoo[Tsoommer|][ 4so.r60oppb[m |w15e1pa pb||Sa4] 01kp]pb| C TToeeoT w owe ] T T TTwoT T www e[] | e T so T e TomT| oT e wem awwo| [me ea w7 |e ne |w ea] C smal T TwT21 |e]] * Sample deactivated due to unacceptable intemal standard response. 0350303 Table 95. Intra-Assay JN Precision for POAA Quality Controls in Human Plasma C T oone | SowTsTTw |ooe ewnT |w w w we w e w ewwm|] Run Date [Run Number| Low QC 481ppb |Medium QC| | 145ppb | High QC 385ppb vseol TTomno |w on |w an] Tel |ee Te] C Tee T Te 1 a ] uw] ements ra tre 0950304 ape msn Table 96. Intra-Assay Precision for PFHS Quality Controls in Human Plasma Run Number| Low QC | Medium QC| 4.48 ppb 156 ppb High QC 417 ppb. T T TT Tewa T w e w we] T Ts o w wwe ] voell TToown[m aww|e] w] [ficeal] Tewos|a ois |] a] C val TeT T[ s | 5a]] 090305 `Table 97. Intra-Assay Precision for M556 Quality Controls in Human Plasma Run Date Low QC 4.00 ppb Medium QC| High QC 150 ppb 400 ppb. T TTe eT Tw w e ee Te eeTe w e we ew ww oe ow| w T eT o1w FE erate Page 103 030308 Table 98. Intra-Assay rest Precision for M570 Quality Controls in Human Plasma Run Number| Low QC [Medium QC| High QC 4.00 ppb | 150 ppb 400 ppb T TewswT Twwo [wi] T TT Tonw [ow w [owm] seom TToew[w0w |e]| [meseal [Tooo[wsre[uwvos]] mel we er | F TER Page 104 930307 Northwest Bioanalytical ReSptourdtyNNoo.. NNWWBBRS0000-.102420 Table 99. Summary of Calibration Curve Parameters for PFOS in Serum Diluted with Buffer Quadraic weighted I', All concenraions se expressed as ppb 23-0ct:2000|PFOSMD-39 [0.000001[ 0.009444 [0.004315|09|8 5305 | 54 04| ALB, 200 C are coeficients ued to define te calibration curve. `Table 100. Summary of Calibration Curve Parameters for PFOSA in Serum Diluted with Buffer Quadratic weighted I, All concentrations se expressed as pb. 23-0ct:2000| PFOSMD-39| 0000000 [ 0014752 [ 0.002265| 09946 |1.|0500 0| A,B, 204 C are confit used to define the calibration ure. Table 101. Summary of Calibration Curve Parameters for PFOSAA in Serum Diluted with Buffer Quadraic weighted 1. All conceraions ar expressed a ppb. NRuumbner A c LLoQ[uLOQ] - 23-0ct-2000 |PFOSMD-39 0.009880[ 0004993| 09960 | 00|500| ALB, 304 C are cosfcint used to dein the calibration curve. Page 105 090308 -- ReSptourdtyNNoo., NNWWBBRSO0000.-014202 Table 102. Summary of Calibration Curve Parameters for POAA in Serum Diluted with Buffer Quadratic weighed 1c. All concentratioanrse expressedas pb [oo imme | [ ] [oe] Hoje 23-0ct-2000|PFOSMD-39 |-0.000001| 0.011351|0.000137] 0.9967 ALB, 2d Cae cosficients ustodefdine thecalibration curve. `Table 103. Summary of Calibration Curve Parameters for BFHS in SerumDiluted with Buffer Quadratic weighted 1c. All concentrations ae expressed 2 ppb. RunDate | Run A Number Cc [ReSquarea[LLOQ[ULOQ 23-0ct-2000|PFOSMD-39| -0.000003| 0.018220| 0.007453 | 0.9980 AB 0Cdare costicients usedtodeinthecalibration curv. `Table 104. Summaryof Calibration Curve Parameters for M556 in Serum Diluted with Buffer EP la Quadratic weighted Uc. All concentrations re expresed s gob. 23-0ct-2000|PFOSMD-39| 0.000000| 0.008729 [0.000146] 09936 |1.|05000| A,B, 20d C are coefficients use to define the calibration curve. 050309 Page 106 Norwest Bioanalytical ReSptourdtyNNoo.. NNWWBBRS0DG0-014202 `Table 105. Summary of Calibration Curve Parameters for M570 in Serum Diluted with Buffer Quadratic weighted 1%, Allconcentratiaornes expressaeds ppb. 23-0ct:2000| PFOSMD-39| 0.000000 [0.015628[0.005711] 09969 |1.|05000| ALB, 2dC acr oefcuiseedtnodteisnthecalibration curve. Page 107 030310 `Table 106. Back-Calculated Concentrations of Calibration Standards for PFOS in Serum Diluted with Buffer Quadratic weighted 1/+%. All concentrations are expressedas ppb. [moewmneT[rroam[n7 aN1[ov43o[2m5963][55r43 2wore[0ors oone[[s15]] [[swlmlwslmilwsslloiwn] Table 107. Back-Calculated Concentrations of Calibration Standards for PFOSA in Serum Diluted with Buffer Quadratic weighted 1/x'. All concentrations are expressedas ppb. Eceln e[reow|1l1[o22[d5 [d72s[5E02[dova dl[Eoeo[5 CTDolsonmmole] Table 108. Back-Calculated Concentrations of Calibration Standards for PFOSAA in Serum Diluted with Buffer Quadratic weighted 1/x'. All concentrations are expressed as ppb. [soumowneT[mrSoumwbesr|172[25500[037 |350[s0w0 iosv[[520 on[[50]] [1Doofaaloalm wel fmlmlo] 090311 `Table 109. Back-Calculated Concentrations of Calibration Standards for POAA in Serum Diluted with Buffer Quadratic weighted 1/x". All concentrations are expressed as ppb. [Foewma|rSoamsir 1502 332702[[11007s[3527[70s7 3o5t[2asa[a1 [[|| 1Dsbulm(mlmlwlmlele] . Table 110. Back-Calculated Concentrations of Calibration Standards for PFHS in Serum Diluted with Buffer Quadratic weighted 1/x'. All concentrations are expressed as ppb. ErEam romes a10)[o20lw)t[i32R6E3EE[E1E ED T Toles lT olmlsolmlmlw]m] "Table 111. Back-Calculated Concentrations ofCalibration Standards for M556 in Serum Diluted with Buffer Quadratic weighted 1/x'. All concentrationsareexpresseads ppb. IEroman row[2a8[l 106 2d 5[3 10ad[E25c E[Eo)s C1Dwbslwleulmmle] 050312 Ror Table 112. Back-Calculated Concentrations of Calibration Standards for M570 in Serum Diluted with Buffer i es a EE CO ll Quadratic weighted Lx'. All concentrations are expressed as ppb. [ oman1 rossT|oowr[2m5[[s03w3m[72e5 n1s uoslm[s1l[e5s _-- 030313 orld S-- e Table 113. PFOS Quality Controls Samples Prepared in Serum Diluted with Buffer [ 1 FSroovsT T ovses||o w o PmrrgT T t|[o o swmwmat[|o e[oawmwmwwem|ooomwwwwmo]]|] DF=10 DF=10 DF=10 DF=50 smol T Tw wT Tw o[mwew || wee | [emite]d Tw iors a oa | mw a| a ] C aw eTT o asTe| w] e] ien er `Table 114. Toronto vages| oman| omg PFOSA Quality Control Samples Prepared in Serum Diluted with Buffer Pros T Tw eTTo w | oewe || || smol TTewsTTawe[worw | awmea ] [emiea]l Tea0 55s|| wws ||5sss | mal ToTw [| es] raten me i 090314 rns re vaso `Table 115. PFOSAA Quality Control Samples Prepared in Serum Diluted withBuffer Run Number| 153 ng/mL | 403 ng/mL| 4000 ng/mL| 4000 ng/mL 1 romT p] w DFvw=10|TDW nF=10T|owDwFw=10 || wwDF=we50|] w TwTw oT|oonn || omwe]] s elw o wew [m wemw || mmwo]] emeil] 0 5Tw00 m5o|| o5s]] woe 5TnTw |ou] J, `Table 116. POAA Quality Control Samples Prepared in Serum Diluted with Buffer T rob] 16 T|w wmw | emwo[|owwo]| TwTw ew | oe] e so Tew w owes|weeu ||wwe]] e mesial] 5tose[[T woo|woso[|wwoe || el To[oo[so[wa| 650315 big Table 117. PFHS Quality Control Samples Prepared in Serum Diluted with Buffer Run Number| 154 ng/mL | 404 ng/mL | 4000 ng/mL| 4000 ngimL 1 roTs oiw |a ow ||wwooT| wwo]| T Te wTeee emae aww] Te Sewl TTw oTT 5 a e[[ueawse| [wwnee]] mematd] TewsT[our|eser | was] CTT 1] `Table 118. M556 Quality Control Samples Prepared in SerumDiluted with Buffer om [ownTs] wtw T |w a | wawe |woew |] T Te wT Tw w | ewwe |wweo] e wsol T Te wTT owwoom |w aew w] e | [Femmiw] ToiorTnmis[iewsm wroo]] ert tere pets 050316 mn ae Table 119. M570 Quality Control Samples Prepared in Serum Diluted `with Buffer Run Number| 157 ng/mL| 407 ng/mL| 4010 ng/mL| 4010 ng/mL C C T TTw o e[[ [T 1 oonwoe eooeoo] wo]] IemllT T Ts w w esY n eT |e w] a Cowmeds] [ {ow5s |n to n | orsa |r]] 0950317 `Table 120. Summary of Calibration Curve Parameters for PFOS (Serum vs.Plasma) To] : Quadratic weighted 1/x'. All concentrations are expressed as, ppb. Number Toate ro] sewn[91003 [cori te ee Table 121. Summary of Calibration Curve Parameters for PFOSA (Serum vs. Plasma) `Quadratic weighted 1/x". All concentrations are expressed as ppb. Run Matrix A c LLOQ Te `Table 122. Summary of Calibration Curve Parameters for PFOSAA (Serum vs.Plasma) wo Tre Tr] Quadratic weighted /x'. All concentrations are expressed Treg as ppb. Number A 030318 Table 123. Summary of Calibration Curve Parameters for POAA (Serum vs. Plasma) `Quadratic weighted 1/x". All concentrations are `expressaesd ppb. Run Matrix A c R-Squared| LLOQ| ULOQ [[aEttoeprm|| 555T[E sroomn[oo0mmm [sooorrsre[ooreson|oomsamr||"voovas||s]r] Table 124. Summary of Calibration Curve Parameters for PFHS (Serum vs. Plasma) Quadratic weighted 1/x. All concentrations are expreasssppebd. [[zoo2m rT| rrooowmtasno[[Foanmues] 4[3c[wsmara]iioocuog] [roam [ sro |sun [ors [ome [ now |_aoo | 335 | or] Table 125. Summary of Calibration Curve Parameters for M556 (Serum vs. Plasma) Quadratic weighted 1/x'. All concentrations are expressed as ppb. Run Date Run A Cc R-Squared|LLOQ| ULOQ 7Pr0o8sia s] seves oooco r[[o0w0e1ni0a[aoma| ow[3I50 50| : 090319 soon -- Table 126. Summary of Calibration Curve Parameters for M570 (Serum vs.Plasma) = : TremTioag Quadratic weighted Ux. All concentrations are expressed as ppb. [zai | a5 [eum[oom ovsas[a asm omsi |50 | | 090320 Norwest Bioanalytical ReSptourdtyNNoo.. NNWWBBSR000-:102420 (Serum vs. Plasma) `Table 127. Back-Caleulated Concentrationsof Calibration Standards for PFOS `Quadratic weighed 1x'. All concentrations are expresssesd ppb. [unDate [27-0cv2000 runNumber[atari [394 prOSMD-4o| lesa[572 [517[113[236 518 |113 | 19:6 42[554] 209 a5 [32.1[214 | 32 357 [ase 30 |RlLunDate[|RunNumberT[Tatsi[s365o|55f0[4s43 e565r[[s72sl.o7f|1ou2n0s|s]35l[337a0 aae [R[ unDate [RunNumber MeriT[s3o28s[T4w51 o[l1w0s7e25r0[m55]ssao[2s08]a31s[aas1) [| 2saocao[ ot| 25 [pismaT[e27n0e5o50[u91 65s[1aa[s2s[3s03o |190]z 324[] 32 [RunDate |RunNumber|Mari [365 550|443|568[777120|245|370[450 [L 25ae20ni[26 [serumT[o56e5l[5w5T o5l[4w4i2[s54e7me1a]|v126o[2s34 3w6o3[wa0w]] Page 18 \ 090321 tsimm wr sos `Table 128. Back-Calculated Concentrations of|Calibration Standards for PFOSA (Serum vs. Plasma) [ron BreTr a n womieie 130 Quadratic weighted 1/x". All concentrations are. 330 00 expressed 0 as ppb 0 . 5 ao] C aeraT | 1 T TTToioTet]l1e5a8[l9ww5e [il2m56sswd0.s1us1li0 [m]]lvwmiolrsai]]| 090322 Table 129. Back-Calculated Concentrations of Calibration Standards for PFOSAA (Serum vs.Plasma) (oC Ewraen mT o5smQuaedrratimciweeigh]mTtede1/1x'.s25A0lhl[[c3oan2c5enlt0ra[ts03io2n3s7la[r2ew`2e2xlp7res0m0sed7las]ppwsb. rlT[mslan3ls1]]| [T oBanwaa nT 2o6mer|isouTne[5s23l00[o[55n7901l[u11a32l[[m2328o92[l|55s027sl11u50 [|a25lT[ae1s7]]| ea rms ~ 090323 tre ST `Table 130. Back-Calculated Concentrations of Calibration Standards for POAA (Serum vs. Plasma) [[e27r0i02e0 Traorsees[ saen][117552 Quadratic weighted 1/x". All concentrationsareexpresseasd ppb. T[3301311005[[225208[o7a[[r1t[[2 o35 [a0] [[oT Frrobszae{roSsumvnesr|sew[no|7ow5s2 [[[36T 7n81]1l250s[227s25t0 |[033[3m2 [mo0aLt]]| [[C Eodmean|r2asme[woweeTn[|o13w5s[[o32T 0n05[[11w00[31[[m32s002w3[ |a[o[e3 o]ss[u2lsT5losuwrn]]| [[C oEswete|n2a6mesaenTn||ovow5 [o5aT aw180[[11n5560a[3f205s43[52s10e48|[s3s75o[os2neu[[sssos]]]ws7]]| T TeetsbmT ulmfminslwlmlmle] 000324 Northwest Bioanalytical peStudy No. vsme NWBS00-040 Table 131. Back-Calculated Concentrations of Calibration Standards for PFHS (Serum vs. Plasma) [o27m00e00T|rsanrsoombeern 115527T227 201078[2342[[44s1o1[[75[s5[9]] Quadratic weighted 1/x". All concentrationsareexpressedas ppb. [1m1e saTo3r 9s[h57w1a 5l20u15d7s m[msimvse [svus ]l [TTTFwesTnlustmslmeorlmolulm| 0950325 Stage FT Table 132. Back-Calculated Concentrations of Calibration. Standards for M556 (Serum vs. Plasma) [r2T o7.b00e00TR$7QT 0ua9adr0a.tmi4c 0woeighrtiedse1T/nx'.o[[A2l3sl65co1hnce0wn1tr0al8t7iom2n2s3sare5.l`ex25par2es0s5sedo1a0s lppb,mT5lw5Tlsrel]|] [ewbe{RomNomea[30113 [265[13 1 ror 1] TT Toolwolmiaslwlm wa] 090328 me men Table 133. Back-Calculated Concentrations of Calibration Standards for M570 (Serum vs. Plasma) [[eL 27n-0e0T 20Ro0m5[N0aQ-umad4rJatic waeiogehmtsed[1/mx01'.90A90ll[DcoT 3noce7ntlr[atmi5oni5s amr2ea .`1e]xp2rme[sss3eda[s6nps pb1os. lTTomolso umrlm01]]] [[D2C :0o02o00r370S5o-m4er|stT uamT[r|2e357s50][15o35m02[1n3s[a22e7sT 7[o1s27s0oi1o0 m73insseaf]] |[I zosnnpebaeom[Ro2mN5ame|1aaswTm e[w110010l[[52a10[10l0500s]v5a0n[]50]02w|8moslTdaainsmmsnm]s]1] [[oT eneermo]aSamer[sa ownT[o|o3w7vl0[[s1sT 50l31n30s53[27s1a7o3l20n37[i1i3ra aa[iTsom]]| 050327 `Table 134. PR. PFOS Quality Controls (Serum vs.Plasma) T(Plasma) (Plasma) (Plasma) T [C romT mommolT ow|w[Tawe]| T r T TeT w T 7 e m r eT mw eell TTTes omeTT eaww a ] 15TT EEE [T Fos rromT ou|e0T |Tie|5e| rT TTeeT Te wTe ] eollTTwee TweTen] C mal T Te a15ae]] T= 050328 rm a-- e ssn `Table 134, bere PFOS Quality Controls (Serum vs. Plasma) Continued T m Toneo s e [mT ee [eme ESE r 1 Te T a eww] ECTOE sa T r (Serum) I I e T e E T Cr -- E r e a e m T-- roru a e r e-- re a nTe ] ] -- aa]r 39.3 ppb. (Serum) 161 ppb (Serum) 370 ppb. _ 090329 Northwest Bioanalytical ) ne noo Study No. NWBS00-040 Table 135. ens PFOSA Quality ss Controls (Serum vs. Plasma) al EEE [T BownT ]5Twoes[w0T[m|] T TTT Two T w wwa]] ve 1 l 1|wo a] T T TTonws e w [oww] ] T oe T Two[w ] o] (Serum) (Serum) (Serum) [C ErmT ]wT o4n.0o 0 ppb[e o150 ppbT.Ta40w0 op]pb] TTews w sn a] ] r ewT 1w7e 1 s Tal o Toa [|sa r | worr]] C A RANn T + 1. 050550 eresBri oto sss `Table 136. rr PFOSAA Quality Controls (Serum vs. Plasma) TEon9S Tictmac[EHhSoc [T Ew | 5Toow [ooww [mww]| Tw Ton mo wTw]] lesEE eso t Teat s[ow ] | ue] oeea TTww[mos ww]r] TT] far2 [gmac [aac [C EmmT T Toe n[wwo 7.20 ppb 153 ppb. | wo|] 403 ppb Tw ToT w ow]] e rr wTwT w] e sl TowT a0[orwn]] TE T tT T eare ] a . 0350331 rears Swier dsuseo Table 137. Acosmseisotns POAA Quality Controls (Serum vs. Plasma) TE [Ee EEE CTTTwowww[o]w] T r rT 111] s om o Tw 5 om w w] [eeall 5[n sswa ] 15151] (Serum) (Serum) (Serum) [FC rowam [rosiTeoornT [[wm e Tmw] TTa w w ww w] ] r mlw T e 1] esoll TTosm[oowsT|oai]] CET A eT A157. 050332 Noes a Table 137. POAA Quality Controls (Serum vs. Plasma) Continued (Plasma) (Plasma) (Plasma) TE [Erm]5Teswn [w 0 |w w] 4.32 ppb. 145 ppb. 385 ppb. r TTeeT we ewer eoll rel Towsa w 5 a e ur] o me T l Ta] e | EE | [T Een]5e Tw0T| ew [oowmwe ] ToeT Tw e ow e rel To m ww e mw] C on rT TTw 1 0 a. ] - 050333 meni s-- re meres Table 138. PFHS Quality Controls (Serum vs. Plasma) ErSae | mec [aac T T TT e eT e e e TT T T a oe T ] (row amma] so | 6.41 ppb we132 ppb | 50|] 345 ppb. E T Te a =R Te eTee]] EI E EO ToI NEowTIo-- re n-- ] e T i Te . Te] Ea i. 050331 -- rnteo Nasms Table 139. J M556 Quality Controls (Serum vs.Plasma) I EE T TTooeT wwow[a]] rTTT EEE --ol--Tow ww] [eewlm Tea ww e [a]] aTTT] (Serum) (Serum) (Serum) [FC owam [rowsT|o5s.30moT pp|b 1w51mppb. o| 4ew01mp]p|b To Toos ww e o]n r ml TwT w7 a] e ol T Tow[oosao]n] T ealT RETT w[] 5Te. ] . 090335 SN Te Table 140. At cmins eps 5. M570 Quality Controls (Serum vs. Plasma) EE (Plasma) (Plasma) (Plasma) [Fon[mToaow|w ow n T[=w]]| --rr TwTe Te Ee e wo Towe [we[as] e oom l Tos[awe re[wsse]] 15TT LowQC|MediumQC| High QC FonT[ o T moox|n w e|ow]nw]| To Tee w we [[wwee]] --oe 1Ter[w 1 w eollT Toe w[eow [wr] we] T ee e nTieT ---- ar T51. . 0950338 ers reson See `Table 140. on eps M570 Quality Controls (Serum vs. Plasma) Continued [= aS| ns Mine | noc ETmoam]T 5[oe w [ow w | wr]| T TeTm owT [e ww |] wo] Tr om To1 T [m1 w]| T Tel TToe ws [ en[om r]c a em TwT [wm | |e]] EEEEEE [T Emm 5TToweT [[oww|w]| T TTw eT wwe e Tw]] r om Twr ws |e T ol e TTowww [al ww| ] ae | Ta T t] s . ei 050337 Norwest Bioanalytical RepStourdtyNNoo.. NNWWBBRSO0O0--102420 Table 141. Serum QC Results Based on Plasma Curves Sple D PFOS PFOSA PFOSAA POAA PFHS MsSS MST (ppb) LLooww ((aallyyzzeedd 110022770000)) 441.12 3 (ppb)* (ppb)* (ppb) 0W8U (7pp6b)* (sppeb)* (6pp9b%). am asl es LoLoww ((aallyyzzeedd 11002277/0000)) 442.113 nois o9m2 ssees;oe LLooww((uawnaallyyzeedd44225500)) 446157 6S4074 a6s63 9993 7sa7l4 LLooww((aumalliyzeedd442285001)) 446770 668785 8a8n3 111330 889249 amde.adenv.+ 2w6e o6s2 8Lo1l L1o0r9 0870676 044319 07920% udcevve+ l6o09%% 3ST%i% 113264%% 194%% 29%% o98m% sLaLsww 957S%ecccopnlfaindceencreanrgaeng*e 43141110044857.24 53.0405110075..1020 7$.0413 21008..0808 763 to 114 10216117 51310 7.69 7326882 41206438 3871047 53610808 66010757 MMeedd ((aanlaylyzzeedd 110022770000)) 116532 11m36 117800 113469 175s5 MMeedd ((aannaallyyzzeedd 11002277/0000)) 116653 1174 11662 113397 1166810 MMeedd ((asnnaallyysaeedd4422550001)) 118359 w0ms 201902 116640 11898 MMeedd ((aanlaylyzzeedd4422550000)) 119919 w wow w 11608 119911 Tadamean + I18s1 626116 26041 81m3 J1a0l 134%0 11735 siwdeevne L63%% d3oMw S3l2o%w 149%5% 2A0s0k% 4%aw s7o%n 95S%ecccopnufaindceenrcaenrgeange* 127921100119930 2[0D43t1o0211875 1T91561100210951 1161541to0117777 1160361t0o 115788 11342ttoo1118416 1T62521100 1186%5 HHiigghh a(annaallyyzzeedd 1002277/10000)) 336740 prwr 5a0 363491 a38s4 `High(analyzed 1027/00) 374 459. a2 366 20 High(analyzed 4/25/01) 396 493 451 453 433 `HHiigghh ((annaallyyzpeedd44/2255/001)) 439096 S50O9 44607 447760 a45n0... - High (analyzed 42550) 825 S60 s06 an 5 amdeadenv.+ a39s0 35067 2a3w a1s02 2a6x0 83758 3a6s0 Saedev SSs6 e58w% 1S7I% d3a3%w m6lw% 1204%% m82w% 957S%ccccopnufaindceencaenrgeange 3674ttoo 440374T90201t0o S5060 13_02.411006459007% 4 TDttoo944G4713T 96t1oe 0A4T5T5 3T42511100346ST9 4T1z1 1t0o446054 TT `*NDOueTtEo:Oronulnyddinagt,acfalrcoumlaatcicoenpstaablseeruodnns diinscplluadyeeddivnatlhueesTambaey (disfcfeerreFpoormtactemxa)l.. (Data biained on 10127100fromrunPFOSMD-40. Data obtained on4/25/01 romrun S00040-25.) 050338 Page 135