Document QXyGoq1MDbRBrwqGMm9Y9oaBE

Northwest Bioanalytical Study No. NWBS00-040 Report No. NWBR00-122 Quantitative Determination of PFOS, PFOSA, PFOSAA, POAA, PFHS, M556 and M570 in Human Serum by LC/MS/MS Assay Revalidation Addendum Report Northwest Bioanalytical (NWB) A Division of NWT Inc. 1121 East 3900 South Salt Lake City, UT 84124 PREPARED FOR: 3M Environmental Technology and Services 935 Bush Avenue St. Paul, MN 55133 AUTHOR: / (q('-,3,_d:9/c,._ L tz-yE-_'_zL Connie O. Sakashita, B.S., NWB Project Manager APPROVEDF_ P3,ELEASEBY: /X'-'__,_b r _,_..-p_.,_--- Patrick Bennett, M.S., M.B.A., NWB LaboratoryDirector DATE: II'qVC' DATE: _" 2 ,..-/ Page l Northwest Bioanalytical Study No. NWBS00-040 Report No. NWBR00-122 QUALITY ASSURANCE STATEMENT LABORATORY: SPONSOR: COMPOUND(S): NWB STUDY NUMBER: SPONSOR STUDY NUMBER: Northwest Bioanalytical (NWB) A Division of NWT, Inc. 1121 East 3900 South Salt Lake City, UT 84124 3M Environmental Technology 935 Bush Avenue St Paul, MN 55133 Services PFOS, PFOSA, PFOSAA, PFHS, M556 and M570 NWBS00-040 Method Validation NWB STUDY TITLE: Quantitative Determination of PFOS and Metabolites in Human Serum by LC/MS/MS, Assay Validation The method validation study descn_oed in this report is not included within the definition of a GLP regulated nonclinical study. However, Northwest Bioanalytical conducts all projects in accordance with the guidelines of the U.S. FDA Good Laboratory Practice Regulations for Nonclinical Laboratory Studies (Title 21 CFR Part 58), the OECD Principles of Good Laboratory Practice and the Japanese MHW Good Laboratory Practice Standard Ordinance for Nonclinical Laboratory Studies on the Safety of Drugs (Ordinance No. 21, PAB Notification No. 424.) The following inspections were performed by the NWB QAU per SOP. Inspection and Reporting Statement. Inspection Date 15 May 2000 15-18 May 2000 07-18 Sep 2000 24 Jan 2000 19-30 Iu12001 05-07 Nov 2001 20 Nov 2001 Phase of Study Analytical Plan Assay Validation Report Draft/Raw Data (NWBR00-108) Final Report ('NWBR00-108) Report Drain/Raw Data (NWBR00-122) Report Drain/Raw Data (NWBR00-122) Final Report (NWBR00-122) Date Inspection Report Issued To NWB Project Manager iNWB Management 15 May 2000 31 May 2000 18 May 2000 31 May 2000 20 Sep 2000 29 Sep 2000 24 Jan 2000 30 Ju12001 31 Jan 2000 31 Ju12001 08 Nov 2001 20 Nov 2001 30 Nov 2001 .:': 30 Nov 2001 : :' ":" IReports to NWB Management are issued monthly. As can reasonably be established, the methods and procedures described and the results incorporated into this final report accurately reflect the raw data. Sheryl A. Mathews, A.A.S. Date NWB QAU Compliance Auditor Page 2 Northwest Bioanalytical Study No. NWBS00-040 Report No. NWBR00-122 COMPLIANCE STATEMENT The method validation study described in this addendum report is not included within the definition of a GLP nonclinical regulated study. However, to the best of our knowledge, the stability, extraction efficiency, and plasma abbreviated validation experiments were conducted in accordance with the guidelines of the U.S. FDA Good Laboratory Practice Regulations for Nonclinical Laboratory Studies (Title 21 CFR Part 48) and according to the methods and procedures described within this report. In addition, these experiments followed the guidelines of the OECD Principles of Good Laboratory Practice and the Japanese MHW Good Laboratory Practice Standard Ordinance for Nonclinical Laboratory Studies on the Safety of Drugs (Ordinance No. 21, PAB Notification No. 424.) This addendum report also included results fi:omresearch experiments conducted outside of the validation study. These experiments were conducted according to the procedures described within section 7 of this report. Serum sample results obtained using plasma curves for PFOSA, PFOSAA, POAA, PFHS, M556 and M570 did not meet NWB SOP requirements for validation. Any known circumstances that may have affected the quality or integrity of the study or data are discussed within the report. This report represents an accurate record of the raw data. :u-j,'L,:-_:.,_//_,-..,__z_.._S Connie0.Sakash_/Bt.aS,. NWB ProjecMtanager Date: /7-D:-)':: Patrick K. Bennett,M.S., M.B.A. o;" : NWB Laboratory Director Page 3 NorthwestBioanalytical StudyNo. NWBS00-040 Report No. NWBR00-122 TABLE OF CONTENTS SIGNATURE PAGE ........................................................................................................................ 1 QUALITY ASSURANCE STATEMENT ...................................................................................... 2 COMPLIANCE STATEMENT ....................................................................................................... 3 TABLE OF CONTENTS ................................................................................................................. 4 LIST OF TABLES ........................................................................................................................... 5 1. INTRODUCTION ..... ............................................................................................................. 13 2. METHODOLOGY ....................................................................................... _......................... 14 3. STABILITY EVALUATION ................................................................................................ 17 3.1. Freeze-Thaw and Room Temperature Matrix Stability ..................................................... ! 7 3.2. Reinjection Stability ........................................................................................................... 18 3.3. Extract Storage Stability .......................................................... .......................................... 18 3.4. Long-Term Matrix Stability in Human Serum ................................................................... 19 4. EXTRACTION EFFICIENCY FROM HUMAN SERUM ................................................... 19 5. COMMENTS AND CONCLUSIONS .................................................................................. 21 6. ABBREVIATED VALIDATION - HUMAN PLASMA ...................................................... 21 6.1. Range of Quantitation ........................................................................................................ 23 6.2. Lower Limit of Quantitation .............................................................................................. 23 6.3. Intra-Assay Precision and Accuracy ............................................................. ..... ................ 24 6.4. Proposed Acceptance Criteria for Human Plasma ............................................................. 24 7. ADDITIONAL POST-VALIDATION EXPERIMENTS ...................................................... 25 7.1. Ion Suppression Experiment .............................................................................................. 25 7.2. Dilution Matrix Experiment ............................................................................................... 27 7.3. Serum and Plasma Comparison ......................................................................................... 29 8. DATA MANAGEMENT ............................................ _.......................................................... 31 9. REFERENCES ........................................................ .............................................................. 31 10. DATA RETENTION ............................................................................................................. 31 J Page4 NorthwestBioanalytical LIST OF TABLES StudyNo. NWBS00-040 ReportNo. NWBR00-122 Table I. Summary of Calibration Curve Parameters for PFOS in Human Serum ........................ 32 Table 2. Summary of Calibration Curve Parameters for PFOSA in Human Serum ..................... 32 Table 3. Summary of Calibration Curve Parameters for PFOSAA in Human Serum .................. 32 Table 4. Summary of Calibration Curve Parameters for POAA in Human Serum ...................... 33 Table 5. Summary of Calibration Curve Parameters for PFHS in Human Serum ........................ 33 Table 6. Summary of Calibration Curve Parameters for M556 in Human Serum ........................ 33 Table 7. Summary of Calibration Curve Parameters for M570 in Human Serum ........................ 33 Table 8. Back-Calculated Concentrations of Calibration Standards for PFOS in Human Serum ...................................................................................... _...................... 34 Table 9. Back-Calculated Concentrations of Calibration Standards for PFOSA in Human Serum ............................................................................................................. 34 Table 10. Back-Calculated Concentrations of Calibration Standards for PFOSAA in Human Serum ............................................................................................................. 34 Table 11. Back-Calculated Concentrations of Calibration Standards for POAA in Human Serum ................. ............................................................................................ 35 Table 12. Back-Calculated Concentrations of Calibration Standards for PFHS in Human Serum ............................................................................................................. 35 o;" ; Table 13. Back-Calculated Concentrations of Calibration Standards for M556 in ." , :" ,.it. 4 Human Serum ................................................................ ......................... '......... _......... 35 Table 14. Back-Calculated Concentrations of Calibration Standards for M570 in Human Serum ................................. _........................................................................... 36 Table 15. Quality Control Samples for PFOS in Human Serum .................................................. 37 Table 16. Quality Control Samples for PFOSA in Human Serum ............................................... 37 Table 17. Quality Control Samples for PFOSAA in Human Serum ............................................ 37 Page5 NorthwestBioanalytical StudyNo. NWBS00-040 ReportNo. NWBR00-122 Table 18. Quality Control Samples for POAA in Human Serum ................................................. 38 Table 19. Quality Control Samples for PFHS in Human Serum .................................................. 38 Table 20. Quality Control Samples for M556 in Human Serum .................................................. 38 Table 21. Quality Control Samples for M570 in Human Serum .................................................. 38 Table 22. Freeze-Thaw and Room Temperature Matrix Stability for PFOS in Human Serum ............................................................................................................. 39 Table 23. Freeze-Thaw and Room Temperature Matrix Stability for PFOSA in Human Serum ............................................................................................................. 40 Table 24. Freeze-Thaw Stability and Room Temperature Matrix for PFOSA.A in Human Serum ............................................................................................................. 41 Table 25. Freeze-Thaw and Room Temperature Matrix Stability for POAA in Human Serum ............................................................................................................. 42 Table 26. Freeze-Thaw and Room Temperature Matrix Stability for PFHS in Human Serum ............................................................................................................. 43 Table 27. Freeze-Thaw and Room Temperature Matrix Stability for M556 in Human Serum ............................................................................................................. 44 Table 28. Freeze-Thaw and Room Temperature Matrix Stability for M570 in Human Serum .................................................................................................. ........... 45 Table 29. Reinjection Stability for PFOS ................................................................. ..... ............... 46 Table 30. Reinjection Stability for PFOSA ..................................... ......................... -.......:..-......... 4"7_' Table 3 I. Reinjection Stability for PFOSAA ..................................... ........................ ._.................. 48 Table 32. Reinjection Stability for POAA .................................................................................... 49 Table 33. Reinjection Stability for PFHS ..................................................................................... 50 Table 34. Reinjection Stability for M556 ..................................................................................... 51 Table 35. Reinjection Stability for M570 ..................................................................................... 52 Page6 Northwest Bioanalytical Study No. NWBS00-040 Report No. NWBR00-122 Table 36. Room Temperature Extract Stability for PFOS ....................................................... 53 Table 37. Room Temperature Extract Stability for PFOSA ............................................. .54 Table 38. Room Temperature Extract Stability for PFOSAA ............................................. .55 Table 39. Room Temperature Extract Stability for POAA ............................................... 56 Table 40. Room Temperature Extract Stability for PFHS ................................................ .57 Table 41. Room Temperature Extract Stability for M556 .................................................... 58 Table 42. Room Temperature Extract Stability for M570 ......................................................... .59 Table 43. Reduced Temperature (1 to 8 C) Extract Stability for PFOS ............ .. ...................... .60 Table 44. Reduced Temperature (1 to 8 C) Extract Stability for PFOSA ................................... 61 Table 45. Reduced Temperature (1 to 8 C) Extract Stability for PFOSAA .............................. .62 Table 46. Reduced Temperature (1 to 8 C) Extract Stability for POAA .................................... .63 Table 47. Reduced Temperature (1 to 8 0C) Extract Stability for PFHS ...................................... 64 Table 48. Reduced Temperature (I to 8 C) Extract Stability for M556 .................................... .65 Table 49. Reduced Temperature (1 to 8 C) Extract Stability for M570 ..................................... .66 Table 50. Reduced Temperature (-20 C) Extract Stability for PFOS. ...................................... .67 Table 51. Reduced Temperature (-20 C) Extract Stability for PFOSA ...................................... .68 Table 52. Reduced Temperature (-20 C) Extract Stability for PFOSAA ................................... 69 Table 53. Reduced Temperature (-20 C) Extract Stability for POAA ................. _ii.'............ ...70 Table 54: Reduced Temperature (-20 C) Extract Stability for PFHS..........................................71 Table 55. Reduced Temperature (-20 C) Extract Stability for M556... ................ " ............... .72 Table 56. Reduced Temperature (-20 C) Fxtract Stability for M570... ..................................... ..73 Table 57. Long-Term Matrix Stability for PFOS in Human Serum ........................................ .74 Table 58. Long-Term Matrix Stability for PFOSA in Human Serum ................ ..... ................. .74 Table 59. Long-Term Matrix Stability for PFOSAA in Human Serum ............................... .75 Page 7 Northwest Bioanalytical Study No. NWBS00-040 Report No. NWBR00-122 Table 60. Long-Term Matrix Stability for POAA in Human Serum ............................................75 Table 61. Long-Term Matrix Stability for PFHS in Human Serum .............................................76 Table 62. Long-Term Matrix Stability for M556 in Human Serum .............................................76 Table 63. Long-Term Matrix Stability for M570 in Human Serum .............................................77 Table 64. PFOS Extraction Efficiency from Human Serum.........................................................78 Table 65. PFOSA Extraction Efficiency from Human Serum ......................................................79 Table 66. PFOSAA Extraction Efficiency from Human Serum ...................................................80 Table 67. POAA Extraction Efficiency from Human Serum ...............................:.......................81 Table 68. PFHS Extraction Efficiency from Human Serum .........................................................82 Table 69. M556 Extraction Efficiency from Human Serum.........................................................83 Table 70. M570 Extraction Efficiency from Human Serum .........................................................84 Table 71. Summary of Calibration Curve Parameters for PFOS in Human Plasma.....................85 Table 72. Summary of Calibration Curve Parameters for PFOSA in Human Plasma ..................85 Table 73. Summary of Calibration Curve Parameters for PFOSAA in Human Plasma ...............85 Table 74. Summary of Calibration Curve Parameters for POAA in Human Plasma ...................86 Table 75. Summary of Calibration Curve Parameters for PFHS in Human Plasma ..................... 86 Table 76. Summary of Calibration Curve Parameters for M556 in Human Plasma.....................86 .:,.. Table 77. Summary of Calibration Curve Parameters for M570 in Human Plasma.....................87 Table 78. Back-Calculated Concentrations of Calibration Standards for PFOS in Human Plasma ............................................................................................................ 88 Table 79. Back-Calculated Concentrations of Calibration Standards for PFOSA in Human Plasma ............................................................................................................ 88 Table 80. Back-Calculated Concentrations of Calibration Standards for PFOSAA in Human Plasma ............................................................................................................ 88 Page 8 Northwest Bioanalytical Study No. NWBS00-040 Report No. NWBR00-122 Table 81. Back-Calculated Concentrations of Calibration Standards for POAA in Human Plasma ............................................................................................................89 Table 82. Back-Calculated Concentrations of Calibration Standards for PFHS in Human Plasma ............................................................................................................89 Table 83. Back-Calculated Concentrations of Calibration Standards for M556 in Human Plasma ............................................................................................................89 Table 84. Back-Calculated Concentrations of Calibration Standards for M570 in Human Plasma ............................................................................................................90 Table 85. Lower Limit ofQuantitation for PFOS in Human Plasma ...........................................91 Table 86. Lower Limit of Quantitation for PFOSA in Human Plasma ........................................92 Table 87. Lower Limit of Quantitation for PFOSAA in Human Plasma......................................93 Table 88. Lower Limit of Quantitation for POAA in Human Plasma ..........................................94 Table 89. Lower Limit of Quantitation for PFHS in Human Plasma ...........................................95 Table 90. Lower Limit of Quantitation for M556 in Human Plasma ...........................................96 Table 91. Lower Limit of Quantitation for M570 in Human Plasma ...........................................97 Table 92. Intra-Assay Precision for PFOS Quality Controls in Human Plasma ...........................98 Table 93. Intra-Assay Precision for t;FOSA Quality Controls in Human Plasma ........................99 Table 94. Intra-Assay Precision for PFOSAA Quality Controls in Human Plasma ..............._...100 Table 95. Intra-AssayPrecision for POAA Quality Controls in Human Plasma........................I01 " : " . .". .o Table 96. Intra-Assay Precision for PFHS Quality Controls in Human Plasma.........................102 Table 97. Intra-Assay Precision for M556 Quality Controls in Human Plasma ......................... 103 Table 98. Intra-Assay Precision for M570 Quality Controls in Human Plasma .........................104 Table 99. Summary of Calibration Curve Parameters for PFOS in Serum Diluted with Buffer ................................................................................................................ 105 Page 9 NorthwestBioanalytical StudyNo. NWBS00-040 ReportNo. NWBR00-122 Table 100. Summary of Calibration Curve Parameters for PFOSA in Serum Diluted with Buffer ................................................................................................................ 105 Table 101. Summary of Calibration Curve Parameters for PFOSAA in Serum Diluted with Buffer ................................................................................................................ 105 Table 102. Summary of Calibration Curve Parameters for POAA in Serum Diluted with Buffer ................................................................................................................ 106 Table 103. Summary of Calibration Curve Parameters for PFHS in Serum Diluted with Buffer ................................................................................................................ 106 Table 104. Summary of Calibration Curve Parameters for M556 in Serum Diluted with Buffer ................................................................................................................ 106 Table 105. Summary of Calibration Curve Parameters for M570 in Serum Diluted with Buffer ................................................................................................................ 107 Table 106. Back-Calculated Concentrations of Calibration Standards for PFOS in Serum Diluted with Buffer ................................................................................................... 108 Table 107. Back-Calculated Concentrations of Calibration Standards for PFOSA in Serum Diluted with Buffer ................................................................................................... 108 Table 108. Back-Calculated Concentrations of Calibration Standards for PFOSAA. in Serum Diluted With Buffer ................................................................................................... 108 Table 109. Back-Calculated Concentrations of Calibration Standards for POAA in Serum Diluted with Buffer ................................................................................. ".:._.............. 109 ;l'able 110. Back-Calculated Concentrations of Calibration Standards for PFHS in'Serum a Diluted with Buffer ................................................................................................... 109 Table 111. Back-Calculated Concentrations of Calibration Standards for M556 in Serum Diluted with Buffer ............. _..................................................................................... 109 Table 112. Back-Calculated Concentrations of Calibration Standards for M570 in Serum Diluted with Buffer ................................................................................................... 110 Table 113. PFOS Quality Controls Samples Prepared in Serum Diluted with Buffer .................. Page I0 Northwest Bioanalytical Study No. NWBS00-040 Report No. NWBR00-122 Table 114. PFOSA Quality Control Samples Prepared in Serum Diluted with Buffer .............. 111 Table 115. PFOSAA Quality Control Samples Prepared in Serum Diluted with Buffer ........... 112 Table 116. POAA Quality 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 ................. 114 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) ........ 115 Table 123. Summary of Calibration Curve Parameters for POAA (Serum vs. Plasma) ............ 116 Table 124. Summary of Calibration Curve Parameters for PFHS (Serum vs. Plasma) .............. 116 Table 125. Summary of Calibration Curve Parameters for M556 (Serum vs. Plasma) .............. 116 Table 126. Summary of Calibration Curve Parameters for M570 (Serum vs. Plasma) .............. 117 Table 127. Back-Calculated Concentrations of Calibration Standards for PFOS (Serum vs. Plasma) ................................................................................................... i 18 Table 128. Back-Calculated Concentrations of Calibration Standards for PFOSA (Serum vs. Plasma) ................................................................................................... 119 Table 129. Back-Calculated Concentrations of Calibration Standards for PFOSAA: ... (Serum vs. Plasma) .................................................................................... .....:.......... 120-- - Table 130. Back-Calculated Concentrations of Calibration Standards for POAA (Serum vs. Plasma) ................................................................................................... 121 Table 131. Back-Calculated Concentrations of Calibration Standards for PFHS (Serum vs. Plasma) ................................................................................................... 122 Table 132. Back-Calculated Concentrations of Calibration Standards for M556 (Serum vs. Plasma) ................................................................................................... 123 Page 11 Northwest Bioanalytical Study No. NWBS00-040 Report No. NWBR00-122 Table 133. Back-Calculated Concentrations of Calibration Standards for M570 (Serum vs. Plasma) ...................................................................................................124 Table 134. PFOS Quality Controls (Serum vs. Plasma) .............................................................. 125 Table 135. PFOSA Quality Controls (Serum vs. Plasma) ...........................................................127 Table 136. PFOSAA Quality Controls (Serum vs. Plasma) ........................................................ 128 Table 137. POAA Quality Controls (Serum vs. Plasma) .............................................................129 Table 138. PFHS Quality Controls (Serum vs. Plasma) ..............................................................131 Table 139. M556 Quality Controls (Serum vs. Plasma) ..................................i..........................132 Table 140. M570 Quality Controls (Serum vs. Plasma) .............................................................133 Table 141. Serum QC Results Based on Plasma Curves ............................................................135 : ; : .. . ";..,"..o Page 12 Northwest Bioanalytical Study No. NWBS00-040 Report No. NWBR00-122 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 for the measurement ofPFOS, PFOSA, PFOSAA, pOAA, PFHS, M556 and M570 in human serum. The accuracy and precision results of the method revalidation were reported in NWB report NWBR00-108 [9.I]. This report summarizes the results from the stability tests conducted as part of the method revalidation for PFOS, PFOSA, PFOSAA, POAA, PFHS, M556 and M570 in human serum for 3M Environmental Technology and Services. The results from the following tests are also included: extraction efficiency from human serum; ion suppression experiment; and diluted matrix experiment. The suitability of the 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 is a list of NWB supervisory personnel involved in the completion of this work: Connie O. Sakashita, B.S. (Current NWB Project Manager); Brad I. Coopersmith, Ph.D. (NWB Project Manager until October 28, 2000); Lico.ng . ... Jiang, Ph.D. (NWB Senior Scientist); Patrick Bennett, M.S., M.B.A. (NWB Laboratory Director). The following NWB laboratory personnel contributed to the completion of this work: Suzanne Newman, B.S. (NWB Scientist). NWB SOPs were used in the conduct of this 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 Page 13 Northwest Bioanalytical Study No. NWBS00-040 Report No. NWBR00-122 The method revalidation study described in this report is not included within the definition of a GLP regulated nonclinical study. However, Northwest Bioanalytical conducts all studies within the guidelines of the US FDA Good Laboratory Practice Regulations for Nonclinical Laboratory Studies (Title 21 CFR Part 58), the OECD Principles of Good Laboratory Practice and the Japanese MHW Good Laboratory Practice Standard Ordinance for Nonclinical Laboratory Studies on the Safety of Drugs (Ordinance No. 21, PAB Notification No. 424). Any changes to or deviations from the original protocol (Analytical Plan) were documented through approved protocol amendments or deviation memos and are retained within the raw data. 2. METHODOLOGY Reference Materials and Matrices Analyte PFOS (FC-95) PFOSA Lot Number Purity 193 100% 214 100% Expiration Date 12/31/2010 12/31/2010 Source 3M 3M PFOSAA (FC-129) 617 53.8% 12/31/2010 3M POAA (FC- 143) 245 100% 12/31/2010 3M PFHS* $398-182 100% 12/31/2010 3M (used for serum) PFHS SE-036 91.1% 1/01/2010 3M (used for plasma) M556 NBl13047- 99.89% 12/31/2010 3M 8D M570 118506-26 99.75% 12/31/2010 3M THPFOS 59909 90% 12/31/2010 3M *Received as a 6,200 ppm solution in methanol. **Kept dry. Storage Conditions Room Temperature Room Temperature Room Temperature Room Temperature** -20C .: .: -20C : " -" Room Temperature Room Temperature Room Temperature Page 14 NorthwestBioanalytical StudyNo. NWBS00-040 ReportNo. NWBR00-122 Matrix Human serum Human plasma (Northern Chinese) Lot Numbers BC30399-4; BC061900-1 C5186; C4929; C5517; C5200 The reference material purity for PFOS, PFOSA, PFHS and POAA was not available t prior to the conduct of this study. Therefore, all serum concentrations included in the report for 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 of PFOS, POAA and PFHS in NWB 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 corrected for the 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 Correction Factor 0.836 0.909 o: -. 0.855 Principles of the Method The analytical method consisted of a liquid:liquid extraction procedure followed by evaporation and reconstitution of the extract residue with 30:70 (v/v) 20 mM ammonium acetate in water: 20 mM ammonium acetate in methanol. The samples were analyzed by liquid chromatography/tandem mass spectrometry using a PE Sciex AP13000. The instrument was operated in the multiple reaction monitoring (MRM) mode under Page 15 NorthwestBioanalytical StudyNo. NWBS00-040 ReportNo. NWBR00-122 optimized conditions for PFOS, PFOSA, PFOSAA, POAA, PFHS, M556 and M570 detection. A detailed description of the methodology is included in NWB report NWBR00-108 [9.1]. Acceptance Criteria for Human Serum Calibration Curve Each run will include in duplicate calibration standards at six or more concentrations covering the lower to upper limit ofquantitation. For all analytes except PFOSA, at least three-fourths of the calibration standard's back-calculated concentrations must be within + 15% (+ 20% for LLOQ) of their individual target concentrations. For PFOSA, at least three-fourths of the calibration standard's back-calculated concentrations must be within + 20% (+ 25% for LLOQ) of their individual target concentrations. A calibration standard will be considered a statistical outlier if the back-calculated concentration is greater than two times the acceptance criteria for that standard. Lower Limit of Quantitation The back-calculated concentrations of at least one of the duplicate lowest points in the calibration curve must be within + 25% of the target concentration for PFOSA to qualify as the LLOQ and within _+20% of the target concentration to qualify as the LLOQ for all other analytes. If this criterion is not met, the next level is-subjected to the "s_-netest 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-thirds of all 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 will be analyzed in triplicate for each dilution level (except for PFOSA and PFOSAA '" Page 16 Northwest Bioanalytical Study No. NWBS00-040 Report No. NWBR00-122 which should not be diluted with control matrix). At least two dilution QC at each level must be within _ 20% of its target concentrations in order to accept diluted study samples at that level. The dilution QC acceptance is independent of the undiluted analytical QC acceptance. 3. STABILITY EVALUATION The revalidated method's final acceptance criteria is used as a basis for evaluating fluorochemical stability results. If the 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 found in 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 ofPFOS, 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 M570 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 subsequently underwentmore 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 to 6.75 hour periods between each freeze cycle. Since the freeze-thaw samples Page 17 Northwest Bioanalytical Study No. NWBS00-040 Report No. NWBR00-122 demonstrated acceptable stability, the stability of serum samples left at room temperature for up to 6.75 hours is also demonstrated. 3.2. Reinjection Stability The quantitative reproducibility of standards and controls after 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 NWBR00-108). 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). 3.3. Extract Storage Stability Extract stability was evaluated at different storage temperatures and for various lengths of time. 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 a control value for stability evaluations. 3.3.1. Room Temperature Extract Stability The extract stability ofPFOS, PFOSA, PFOSAA, POAA, PFHS, 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 eg_:action. 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 Stability The extract stability ofPFOS, PFOSA, PFOSAA, POAA, PFHS, M556 and M570 was determined for low and high serum QC extracts stored at 1 to 8C for four Page 18 Northwest Bioanalytical Study No. NWBS00-040 Report No. NWBR00-122 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 1 to 8 C (Tables 43 - 49). 3.3.3. Reduced Temperature (-20 C) ExtractStability The extract stability ofPFOS, PFOSA, PFOSAA, POAA, PFHS, M556, and M570 was determined for low and high serum QC extracts stored at -20 C for five 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 five days at -20 C (Tables 50 - 56). 3.4. Long-Term 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 of PFOSA 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 approximat.e!y42 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 M570 were determined by comparing the area ratios obtained for the following three cases: 1. Both the analyte and internal standard added following the extraction (postextract). Page 19 Northwest Bioanalytical Study No. NWBS00-040 Report No. NWBR00-122 2. The analyte added to serum prior to extraction and the internal standard added following extraction (pre-extract analyte). 3. The internal standard added to serum prior to extraction and the analyte added following the extraction (pre-extract internal 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 amount ofanalyte in the sample prior to extraction (pre-extract) is equal to the amount of analyte added plus the persistent amount in the blank matrix. The amount of analyte in the post-extract solution is the amount of analyte 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 arid M570: ((Spk + Per)* EE)/(Spk + (Per * EE)) = Rpre / Rpost; where Spk = the amount of analyte added Per = the persistent amount of analyte in the blank matrix EE = the extraction efficiency Rpre -- the ratio of analyte/intemal standard determined for the analyte added prior and internal standard added after extraction Rpost = the ratio of analyte/internal standard determined for the analyte and internal standard added after extraction Mean extraction efficiency (%) PFOS 37.3 PFOSA 69.6 PFO.$ .AA 62.7 ; . " .- _ Mean extraction efficiency (%) POAA 15.6 PFHS 15.3 M556 39.1 Mean extraction efficiency (%) M570 57.1 The results are shown in Tables 64 - 70. THPFOS 5.99 Page 20 NorthwestBioanalytical 5. COMMENTS AND CONCLUSIONS StudyNo. NWBS00-040 ReportNo. NWBR00-122 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 effect of rounding if the regressions were performed with the purity corrected concentrations, but these differences would have a negligible effect on the overall interpretation of the revalidation results. PFOS, PFOSA, PFOSAA, POAA, PFHS, M556 and M570 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, M556 and M570 exhibit extract stability for seven days at room temperature, four days at 1 to 8 C, and five days at -20 C. The analytes also demonstrated matrix stability for approximately 55 days (for PFOS, POAA, PFHS, M556 and M570) and 42 days (for PFOSA and PFOSAA) at -20 C in human serum. 6. ABBREVIATED VALIDATION - I:IUMAN PLASMA The choice of matrix for calibration curve preparation is dependent upon the desired LLOQ. 3M supplied NWB with human plasma identified as "Northem 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 LLOQ of< 5.00 ppb. The following sections summarize the accuracy and precision of the fluorochemicals in human plasma and the quantitative accuracy of serum sample results obtain,e:d. against plasma calibration curves using the revalidated serum method. The absolute concentrations ofPFOS, 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 concentrations of plasma standard and quality control samples were corrected using the following factors: Page21 Northwest Bioanalytical Study No. NWBS00-040 Report No. NWBR.00-122 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 15 of report). The tests for the abbreviated human plasma validation were conducted after the serum revalidation was completed. They were performed outside of the 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 tubes of plasma were pooled and the amounts of persistent analytes were determined. Previous determination of persistent levels was achieved by comparison with rabbit serum curves. For the experiments included in this report, the persistent levels were determined as follows: Known amounts of the analytes were added to Chinese plasma in the range of 1 to 50 or 1 to 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 starldard area as the x-value (Microsoft Excel 97 formulas). The mean resultant y-intercept determined from multiple runs was then used as an estimate of the persistent level of analyte if it was greater than 0.2 ppb. Page 22 NorthwestBioanalytical 6.1. Range of Quantitation StudyNo. NWBS00-040 ReportNo. NWBR00-122 The target calibrator concentrations were approximately 1.00,2.50, I0.0, 25.0, 50.0, i00, 250, 400 and 500 ppb for all analytes except M556. The target calibrator concentrations for M556 were approximately 2.50, I0.0, 25.0, 50.0, I00, 250, 400 and 500 ppb. The final curve range can vary based upon the persistent levels of the analyte in the human plasma pool used. For the validation study, the calibration curve ranges were as follows: PFOS PFOSA PFOSAA POAA PFHS M556 M570 3.94 to 414 ppb 1.00 to 500 ppb l.d0 to 501 ppb 1.92 to 481 ppb 1.36 to 523 ppb 2,50 to 500 ppb 1.00 to 500 ppb The calibration curve parameter summaries and individual calibration standard results for the plasma runs can be found in Tables 71 - 84. 6.2. Lower Limit of Quantitation The precision at the Lower Limit of Quantitation (LLOQ) of the assay in human plasma was determined by using six 100-_L aliquots of the pooled Chinese plasma fortified with PFOS, PFOSA, PFOSAA, POAA, PFHS, M556, and M570. at concentrations equal to the LLOQ. The LLOQ concentration was dependent upon the,= persistent levels found in the plasma. Lower Limit of Quantitation Precision (%CV) Accuracy (%Bias) PFOS 3.94 ppb 5.4 -1.5 PFOSA 1.00 ppb 10.1 1.0 PFOSAA 1.60 ppb 5.7 -2.5 POAA. 1.92 ppb 4.4 -8.9 Page23 NorthwestBioanalytical Lower Limit of Quantitation Precision (%CV) Accuracy (%Bias) PFHS 1.36 ppb 9.9 -5. I The results are shown in Tables 85 - 91. M556 2.50 ppb 5.7 -2.0 StudyNo. NWBS00-040 ReportNo. NWBR00-122 M570 1.00 ppb 5.2 3.0 6.3. Intra-Assay Precision and Aeeuracy The precision and accuracy of the LC/MS/MS method for PFOS, PFOSA, PFOSAA, POAA, PFHS, M556, and M570 in human plasma were determined by analyzing three levels of quality control samples in replicates of five on a single run. Precision (%CV) Accuracy (%Theoretical) Accuracy (%Bias) PFOS Intra-assay 1.5 to 2.8 101.2 to 112.3 1.2 to 12.3 PFOSA Intra-assay 1.8 to 5.7 104.0 to 116.3 4.0 to 16.3 PFOSAA Intra-assay 2.6 to 5.8 102.6 to i 11.7 2.6 to 11.7 POAA lntra-assay 0.8 to 5.8 103.4 to 106.2 3.4 to 6.2 Precision (%CV) Accuracy (%Theoretical) Accuracy (%Bias) PFHS Intra-assay 1.7 to 3.1 95.7 to 106.0 -4.3 to 6.0 M556 I.rttra-assay 4.7 to 7.6 98.7 to 111.3 -1.3 to 11.3 M570 Intra-assay 1.9 to 5.6 101.3 to 118.0 1.3 to 18.0 The results are shown in Tables 92 - 98. 6.4. 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 limit ofquantitation. 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) of their individual target Page24 NorthwestBioanalytical StudyNo. NWBS00-040 ReportNo. NWBR00-122 concentrations. For PFOSA and PFOSAA, at least three-fourths of the calibration standard's back-calculated concentrations must be within _+20% (_+25% for LLOQ) of their individual target concentrations. A calibration standard will be considered a statistical outlier if the back-calculated concentration is greater than two times the acceptance criteria for that standard. Lower Limit of Quantitation The back-calculated concentrations of at least one of the 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 for all other analytes. If this 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-thirds of all analytical QCs must be within + 20% of their target concentrations (+ 25% for PFOSA and PFOSAA), and no two QCs at thesame concentration can be outside the limit. Based upon the results, the method is suitable for use with human plasma. 7. ADDITIONAL POST-REVALIDATION EXPERIMENTS .o: _ ; Additional experiments were conducted subsequent to completion of the reyalida_.ion to : =-_ further elucidate the applicability of the revalidated method to sample analysis. These tests were performed outside of any revalidation or sample analysis studies. 7.1. Ion Suppression Experiment During analysis of medical 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 internal standard (THPFOS) have very similar retention Page25 NorthwestBioanalytical StudyNo. NWBS00-040 ReportNo. NWBK00-122 times. Therefore, THPFOS and POAA are not chromatographically resolved in extracted samples containing high concentrations of POAA. This co-elution can suppress the internal standard response and introduce a (high) bias in the measured concentration of analytes when compared against the standard curve. The suppression of the internal standard signal is proportional tOthe concentration of POAA in the sample and dependent on the LC resolution of the two compounds. An experiment was performed to evaluate the magnitude of this effect. Low control samples were fortified with various (high) levels of POAA. The test samples were analyzed in trip.licate in a single run (PFOSMD-41). The mean concentration of the test samples were then compared against the mean of the analytical low control samples. A summary of the results is shown in the following table: PFOS ControlMean Cone. (ppb) 51.8 POAA added at --59200 ppb % change from control mean 182% POAA added at-11800 ppb % change from control mean 28% POAA added at ---5920ppb % change from controlmean 16% POAA added at -592 ppb % change from control mean -2% PFOSA PFOSAA PFHS 4.05 6.83 8.04 152% 180% 180% 39% 52% 29% 21% 30% 16% -11% -5% -7% M556 5.41 216% 52% 36% -8% 11/I570 IS Area 6.87 108857 207% -75% 56% -43% 34% -32% -8% +1 i% The experiment verified that very high levels of POAA resulted in a lower response for the internal standard, which then results in a falsel3/elevated concentration for the other 6 analytes. No significant bias is introduced when the extracted concen_ation of POAA is within the revalidated range of the analytical method (approXimately 500 ppb). However, some samples can contain POAA at concentrations above the revalidated range of the analytical method, and will likely introduce a bias in the reported concentrations of PFOS, PFHS, PFOSA, PFOSAA, M556, or M570 when the concentration of these analytes are determined prior to sample dilution. The amount of bias could range up to approximately 56%. However, the amount of J Page26 Northwest Bioanalytical Study No. NWBS00-040 Report No. NWBR00-122 PFOS, PFHS, PFOSA, PFOSAA, M556, or M570 impurities in the POAA reference material is not known. This prevents accurate determination of the % bias. It should be noted that since the amount of ion suppression is dependent upon the amount of extracted POAA, no concentration bias is expected for an analyte result determined from a pre-diluted sample wherein the extracted POAA concentration is less than approximately 500 ppb. This bias will not significantly effect the interpretation of the analytical results. 7.2. Dilution Matrkx Experiment The amount of 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 whether a substitute matrix that did not contain persistent levels ofFC analytes could be used when performing dilutions. Previous experiments had shown that samples diluted with buffer instead of serum did not accurately quantitate against a typical serum curve (data not reported). This indicated that the matrix to buffer ratio of the 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 ratio _'he QC samples to determine if this was a viable method for diluting samples. A different " '_ " 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 matrix by a factor of 50 (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. Page 27 NorthwestBioanalytical StudyNo. NWBS00-040 ReportNo. NWBR00-122 Calibration Curve and QC Preparation The experiment (PFOSMD run 39) used a curve prepared in 10% serum in buffer, and three levels of QC samples diluted with buffer by a factor of 10 (10% matrix to buffer ratio). The QC concentrations prior to dilution were 150, 400, and 4000 ppb plus the persistent level for each analyte. QC samples at 4000 ppb (plus persistent level) were also diluted 1:50 with buffer. The calibration curve parameter summaries and individual calibration standard results for the runs can be found in Tables 99 - 112. The quality control sample results can be found in Tables 113 - 119. Results for 10% Serum in Buffer (PFOSMD Run 39) The mean accuracy (% theoretical) for all analytes at all QC concentration levels diluted with buffer by a factor of 10 (the same serum to buffer ratio as the curve) ranged from 94.1% to 114.3%. The intra-assay precision (%CV) for all 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%of the target concentrations. QC samples met the acceptance criteria of_+ 20% of target concentration (+_25%for PFOSAA) when diluted with buffer if quantitated against a curve prep_e.d 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 criteria for all analytes. It appears that quantitating samples diluted with buffer against a curve prepared in the same serum to buffer ratio is a viable way minimizing the corrections needed due to persistent levels Of analytes in the dilution matrix. However, since most analytes cannot be accurately quantitated against a calibration curve that is prepared in a serum/buffer ratio different from that of the diluted samples, a separate curve with the same buffer to matrix ratio would need to be prepared for each dilution factor needed. Page28 NorthwestBioanalytical 7.3. Serum and Plasma Comparison StudyNo. NWBS00-040 ReportNo. NWBR00-122 Because the choice of calibration curve and quality control matrix is dependent upon the desired LLOQ, it is not always possible to match the subject sample matrix with that of the calibration curve and quality control matrix. For instance, it is necessary to use Chinese plasma to prepare the calibration standards and quality control samples when analyzing serum samples where a LLOQ of< 5.00 ppb is desired. Therefore, to evaluate if, and to what extent, the use of plasma calibration standards affects the quantitative analysis of fluorochemicals in serum, a comparison of 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 Microsoi_@ Excel 97. Alpha (a) was set to 0.05 for a 95% confidence level. The confidence interval around the mean was compared to the analytical acceptance range of the QC which is _ 20% (__2_5% for PFOSA and PFOSAA) of the target concentration. experiment are shown in Table 141. The summary results of the The i0/27/00 serum curve for M570 had a raised LLOQ of 5.20 ppb. Data for ,.t . PFOSA and PFOSA.A. analyzed on 10/27/00 and data for PFHS and M556 analyzed " ,4 :. .m on 4/25/01 are not used because either the serum or plasma calibration _urve br the QC samples did not meet the acceptance criteria. One h!gh QC replicat_ analyzed on 10/27/00 was not included in the statistical calculations because of an extraction error. The confidence intervals around the mean serum QC results for PFOS are within the acceptance criteria at all levels. Page29 Northwest Bioanalytical Study No. NWBS00-040 Report No. NWBR00-122 The confidence intervals around the mean serum QC results for PFOSA are greater than the acceptance range of+_.25% 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 is greater than the plasma acceptance range of+_25% 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 range of+_20% deviation. The confidence intervals ranged up to 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 of+_20% deviation and may be due a low 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 o; ; : : ,: . Serum sample results obtained using plasma curves for PFOSA, PFOSA?A, POAA, PFHS, M556, and M570 did not meet NWB SOP requirements for validation. Results may vary on average up to 26% from QC results obtained using human serum calibration curves at some concentration levels for PFHS, PFOSAA, POAA, M556, 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 Page 30 NorthwestBioanalytical StudyNo. NWBS00-040 ReportNo. NWBR00-122 quantitation less than 5 ppb is required by the Sponsor, it will be necessary to use plasma curves. 8. DATA MANAGElVlENT PFOS, PFOSA, PFOSAA, POAA, PFHS, M556, M570, and THPFOS chromatographic peaks were integrated using PE Sciex MacQuan software (version 1.6) with a smooth factor of one. Quantitation was based upon quadratic regression analysis of calibration curves (weighted 1/Xz) using the area ratio vs. concentration calculated by the Watson DMLIMS software (versions 6.1.1.03 and 6.1.1.04). 9. REFERENCES [9.1.] C. Sakashita. "Quantitative Determination ofPFOS, PFOSA, PFOSAA, POAA, PFHS, M556 and M570 in Human Serum by LC/MS/MS." Assay Revalidation Report. NWB StudyNWBS00-040. NWB Report NWBR00-108. January 24, 2001. [9.2.] D. Vollmer. "Quantitative Determination ofPFOS, 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 NWBR_99-005. May 13, 1999. 10. DATA RETENTION The raw data and final report for this study will be stored in the NWB Archi;ce, 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 completion of contract obligations. Page 31 Northwest Bioanalytical Study No. NWBS00-040 Report No. NWBR00-122 Table 1. Summary of Calibration Curve Parameters for PFOS in Human Serum Quadratic weighted 1/x2. All concentrationsare expressed as ppb. Run Date 03-Jul-2000 21-Aug-2000 Run A B C R-Squared Number 23 -0.000004 0.0i9854 0.112640 0.9862 18 0.000001 0.028989 0.257857 0.9849 A, B, and C arecoefficients used to define the calibration curve. LLOQ 48.1 48.1 ULOQ 547 547 Table 2. Summary of Calibration Curve Parameters for PFOSA in Human Serum Quadratic weighted 1/x2. All concentrations areexpressed as ppb. Run Date 03-Jul-2000 21-Aug-2000 Run A B C R-Squared LLOQ Number 23 -0.000017 0.054058 0.006149 0.9952 1.00 18 0.000005 0.082782 0.019970 0.9952 1.00 A, B, and C are coefficients used to define the calibration curve. ULOQ 502 502 Table 3. Summary of Calibration Curve Parameters for PFOSAA in Human Serum Quadratic weighted 1/x2. All concentrationsare expressed as ppb. Run Date 03-Jul-2000 21-Aug-2000 Run Number 23 A B C R-Squared LLOQ ULOQ -0.000002 0.013656 -0.020557 0.9903 6.00 505 18 0.000005 0.025511 -0.020269 0.9978 A, B, and C are coefficients used to define the calibration curve. 6.00.,. 505 : :. :- ..-.. Page 32 Northwest Bioanalytical Study No. NWBS00-040 Report No. NWBR00-122 Table 4. Summary of Calibration Curve Parameters for POAA in Human Serum Quadratic weighted I/x2. All concentrations are expressed as ppb. RunDate 03-Jui-2000 21-Aug-2000 Run A B C R-Squared LLOQ ULOQ Number 23 -0.000002 0.013683 0.010580 0.9979 5.76 505 18 0.000003 0.013704 0.017950 0.9966 5.76 505 A, B, and C are coefficients used to define the calibration curve. Table 5. Summary of Calibration Curve Parameters for PFHS in Human Serum Quadratic weighted l/x 2. All concentrations are expressed as ppb. RunDate 03-Iul-2000 21-Aug-2000 Run Number 23 A B C R-Squared LLOQ -0.000006 0.018323 0.012882 0.9971 3.15 18 -0.000001 0.022486 0.017181 0.9970 3.15 A, B, and C are coefficients used to define the calibration curve. ULOQ 502 502 Table 6. Summary of Calibration Curve Parameters for M556 in Human Serum Quadratic weighted l/x2. All concentrations are expressed as ppb. RunDate 03-Jul-2000 21-Aug-2000 Run A B C IR-Squared LLOQ Number 23 0.000000 0.002714 0.000585 0.9925 4.30 18 0.000001 0.011156 0.005643 0.9958 4.30 A, B, and C are coefficients used to def'me the calibration curve. ULOQ 502 502 Table 7. Summary of Calibration Curve Parameters for M570 in Humail Quadratic weighted l/x". All concentrations are expressed as ppb. Serum . " _. --. .o" RunDate 03-Jul-2000 21-Aug-2000 Run Number 23 A B C R-Squared LLOQ 0.000000 0.003818 -0.004160 0.9963 5.60 18 0.000005 0.036404 -0.017720 0.9977 5.60 A, B, and C are coefficients used to define the calibration curve. ULOQ 505 505 Page 33 Northwest Bioanalytical Table 8. Back-Calculated Concentrations of Calibration Serum Standards Study No. NWBS00-040 Report No. NWBR00-122 for PFOS in Human Quadratic weighted l/x 2. All concentrations are expressed as ppb. Run Date 03-Jul-2000 21-Aug-2000 Run Number 23 18 48.1 49.6 57.1 72.1 97.1 147 297 447 547 50.7 52.1 64.0 77.3 98.4 158 309 472 564 45.8 45.6 53.4 68.2 86.6 138 298 435 508 51.7 54.3 60.3 80.5 101 152 304 489 566 44.1 45.6 50.5 70.5 91.1 139 286 417 521 Table 9. Back-Calculated Concentrations of Calibration Serum Standards for PFOSA in Human Quadratic weighted 1/x2. All concentrations are expressed as ppb. RunDate 03-Jul-2000 21-Aug-2000 Run Number 23 18 1.00 2.51 10.1 25.1 50.3 100 251 402 502 1.10 2.54 10.6 27.3 51.6 103 259 419 534 0.918 2.38 9.36 24.6 46.2 95.4 265 398 436 1.01 2.73 10.4 27.9 52.7 106 253 416 530 0.990 2.30 9.62 25.1 44.7 95.8 229 378 502 Table 10. Back-Calculated Concentrations of Calibration Standards for PFOSAA in Human Serum Quadratic weighted 1/x2. All concentrations are expressed as ppb. : RunDate 03-Jul-2000 21-Aug-2000 Run Number 23 18 6.00 7.50 15.0 30.0 55.0 105 255 i_ 405 505 6.74 7.73 16.6 31.3 57.6 111 278 446 524 5.49 6.93 13.6 27.3 52.0 99.6 249 382 451 5.83 7.71 14.5 29.8 53.7 99.3 239 397 499 6.13 7.50 14.4 32.2 57.0 108 268 414 513 Page 34 Northwest Bioanalytical Table 11. Back-Calculated Smdy No. NWBS00-040 Report No. NWBR00-122 Concentrations of Calibration Serum Standards for POAA in Human Quadratic weighted I/x2. All concentrations are expressed as ppb. . Run Date Run 5.76 7.26 14.8 29.8 54.8 105 255 405 505 Number 03-Jul-2000 23 5.93 7.11 15.2 30.6 52.0 109 265 406 524 5.74 6.93 15.6 29.4 53.1 99.6 261 404 474 21-Aug-2000 18 5.98 7.19 13.9 28.2 52.8 102 256 401 481 6.06 6.58 15.2 32.5 55.2 108 270 412 511 Table 12. Back-Calculated Concentrations of Calibration Serum Standards for PFHS in Human Quadratic weighted 1/x2. All concentrations are expressed as ppb. Run Date 03-Jul-2000 Run Number 23 21-Aug-2000 18 3.15 4.65 12.2 27.2 52.2 102 252 402 502 3.13 4.45 12.8 27.7 49.3 103 256 405 515 3.20 4.55 13.7 26.3 49.1 99.7 253 402 487 2.85 4.72 11.8 28.1 48.8 98.3 245 408 497 3.17 5.23 12.1 27.9 51.9 105 258 404 502 Table 13. Back-Calculated Concentrations of Calibration Serum Standards for M556 in Human .:_ Quadratic weighted [Ix2. All concentrations are expressed as ppb. : " '=" " Run Date 03-Jul-2000 21-Aug-2000 Run Number 23 18 4.30 11.8 26.8 51.8 102 252 _402 502 4.44 12.6 28.9 53.2 113 274 434 528 4.18 11.I 23.8 47.5 95.1 247 381 448 4.36 I 1.0 25.5 49.0 101 234 391 469 4.35 11.8 27.2 55.4 106 284 415 516 Page 35 Northwest Bioanalytical Study No. NWBS00-040 Report No. NWBR00-122 Table 14. Baek-Caleulated Concentrations of Calibration Standards for M570 in Human Serum Quadratic weighted I/x2. All concentrationsare expressed as ppb. Run Date 03-J'ul-2000 21-Aug-2000 Run Number 23 18 5.60 7.10 14.6 29.6 54.6 105 255 405 505 6.16 6.90 15.4 29.8 54.0 107 273 414 510 5.54 6.43 14.3 28.7 52.6 104 265 404 464 5.11 7.35 13.7 30.2 53.3 101 249 401 503 5.84 7.43 14.4 31.1 54.5 106 269 406 505 ; ; Page 36 Northwest Bioanalytical Study No. NWBS00-040 ReportNo. NWBR00-122 Table 15. Quality control Samples for PFOS in Human Serum Mean of replicate QCs reported. All concentrations are expressed as ppb. Run Date 03-Jul-2000 Run Number 23 2 l-Aug-2000 18 Low QC 51.1 ppb 47.7 46.7 %Dif. -6.65 Medium QC 197 ppb 168 %D/f. -14.7 -8.61 170 -13.7 High QC 446ppb 382 378 %Dif. -14.3 -15.2 Table 16. Quality Control Samples for PFOSA in Human Serum Mean of replicate QCs reported. All concentrations are expressed as ppb., Run Date 03-Jui-2000 21-Aug-2000 Run Number 23 18 Low QC 4.00 ppb 2.73 4.34 %D/f. Medium QC %D/f. 150 ppb -31.8 100 -33.3 8.50 150 0.00 High QC 400 ppb 286 395 %Dif. -28.5 -1.25 Table 17. Quality Control Samples for PFOSAA in Human Serum Mean of replicate QCs reported. All concentrations are expressed as ppb. Run Date 03-Jul-2000 21-Aug-2000 Run Number 23 18 Low QC 9.00 ppb 7.73 9.95 %Dif. Medium QC %Dif. 155 ppb -14.1 110 -29.0 10.6 160 3.23 High QC 405 ppb 288 413 %Dif. -28.9 1.98 ,:_ z " _'. ,;.". _ Page 37 Northwest Bioanalytical Study No. NWBS00-040 Report No. NWBR00-122 Table 18. Quality Control Samples for POAA in Human Serum Mean of replicate QCs reported. All concentrations are expressed as ppb. Run Date 03-Jul-2000 Run Number 23 2 l-Aug-2000 18 Low QC 8.74 ppb 8.45 7.82 %Dif. Medium QC %Dif. 154 ppb -3.32 150 -2. 60 -10.5 149 -3.25 High QC 403 ppb 400 372 %Dif. -0. 744 -7.69 Table 19. Quality Control Samples for PFHS in Human Serum Mean of replicate QCs reported. All concentrations are expressed as ppb. Run Date 03-Jul-2000 21-Aug-2000 Run Number 23 18 Low QC 6.15 ppb 6.24 6.86 %Dif. Medium QC %D/f. 152 ppb 1.46 157 3.29 11.5 164 7.89 High QC 402 ppb 427 403 %Dif. 6.22 0.249 Table 20. Quality Control Samples for M556 in Human Serum Mean of replicateQCs reported. All concentrationsare expressedas ppb. Run Date 03-Jul-2000 2 l-Aug-2000 Run Number 23 18 Low QC 5.80 ppb 5.52 5.42 %D/f. -4.83 Medium QC %D/f. 152 ppb 145 -4.61 High QC 402 ppb 381 -6.55 141 -7.24 367 %D/f. -5.22 -8.71 Table 21. Quality Control Samples for M570 in Human Serum.:': Mean of replicateQCs reported. All concentrationsare expressedas ppb. Run Date 03-Jul-2000 2l-Aug-2000 Run Number 23 18 Low QC 8.60 ppb 8.20 8.02 %D_ -4.65 Medium QC %Dif. 155 ppb 146 -5.81 -6.74 149 ' -3.87 High QC 405 ppb 390 386 %Dif. -3.70 -4.69 Page 38 Northwest Bioanalytieal Study No. NWBS00-040 Report No. NWBR00-122 Table 22. Freeze-Thaw and Room Temperature Matrkx Stability for PFOS in Human Serum All concentrations are expressed as ppb. Run Number Run Date 18 21-Aug-2000 LOW QC After 7 Freeze-Thaw 46.8 45.1 44.0" cycles Mean 45.3 S.D. 1.41 % CV 3.1 Control (ng/m L) 46.7 Mean % Dev. from Control -3.0 ....After MEDIUM QC 7 Freeze-Thaw cycles 170 158 164 Mean 164 S.D. 6.00 % CV 3.7 Control (ng/mL) 170 Mean % Dev. from Control -3.5 HIGH QC After 7 Freeze-Thaw cycles ":". 369 376 361 Mean 369 S.D. 7.51 % CV 2.0 Control(ng/mL) 378 Mean % Dev. from Control -2.4 Control = mean of analytical QC samples analyzed in the same run NOTE: Samples were left at room temperature for up to 6.75 hour periods beo,veen each freeze cycle Page 39 Northwest Bioanalytical Study No. NWBS00-040 Report No. NWBR00-122 Table 23. Freeze-Thaw and Room Temperature Matrix Stability for PFOSA in Human Serum All concentrations are expressed as ppb. Run Number Run Date 18 21-Aug-2000 LOW qc After 7 Freeze-Thaw 3.73 3.53 3.48 cycles Mean Mean S.D. % CV Control (ng/mL) % Dev. from Control 3.58 0.132 3.7 4.34 -17.5 MEDIUM QC After 7 Freeze-Thaw cycles 108 126 125 Mean Mean S.D. % CV Control (ng/mL) % Dev. from Control 120 10.1 8.4 150 -20.0 HIGH QC o:'-. After 7 Freeze:Thaw cycles :. _,.'._ 348 361 350 Mean Mean S.D. % CV Control (ng/mL) % Dev. from Control 353 7.00 2.0 395 -10.6 Control = mean of analytical QC samples analyzed in the same run NOTE: Samples were left at room temperature for up to 6.75 hour periods between each freeze cycle Page 40 Northwest Bioanalytical Study No. NWBS00-040 Report No. NWBR00-122 Table 24. Freeze-Thaw Stability and Room Temperature Matrix for PFOSAA in Human Serum All concentrations are expressed as ppb. Run Number Run Date 18 21-Aug-2000 LOW QC After 7 Freeze-Thaw 9.08 9.85 9.46 Mean Mean S.D. % CV Control (ng/mL) % Dev. from Control 9.46 0.385 4.1 9.95 -4.9 cycles MEDIUM QC After 7 Freeze-Thaw cycles :: 114 147 147 Mean Mean S.D. % CV Control (ng/m L) % Dev. from Control 136 19.1 14.0 160 -15.0 HIGH QC After 7 Freeze-Thaw 383. 403 391 cycles .:_ : :_: - Mean 392 S.D. 10.1 L % CV 2.6 Control (ng/m L) 413 IVlean % Dev. from Control -5.1 Control = mean of analytical QC samples analyzed in the same run NOTE: Samples were left at room temperature for up to 6.75 hour periods between each freeze cycle Page 41 Northwest Bioanalytical Study No. NWBS00-040 Report No. NWBR00-122 Table 25. Freeze-Thaw and Room Temperature Human Serum Matrix Stability for POAA in All concentrations are expressed as ppb. Run Number Run Date 18 21-Aug-2000 LOW Qc After 7 Freeze-Thaw 8.06 8.83 8.25 cycles Mean Control % Dev. from Mean S.D. % CV (ng/mL) Control 8.38 0.401 4.8 7.82 7.2 MEDIUM QC After 7 Freeze-Thaw cycles 151 149 154 Mean 151 S.D. 2.52 % CV 1.7 Control (ng/m L) , 149 Mean % Dev. from Control 1.3 HIGH QC After 7 Freeze-Thaw 364' 379 372 cycles Mean 372 S .D. 7.51 % CV 2.0 Control (ng/m L) 372 Mean % Dev. from Control 0.0 ,:_: : _ _.._ Control = mean of analytical QC samples analyzed in the same run NOTE: Samples were left at room temperature for up to 6.75 hour periods between each fi:eeze cycle Page 42 Northwest Bioanalytical Study No. NWBS00-040 Report No. NWBR00-122 Table 26. Freeze-Thaw and Room Temperature Human Serum Matrix Stability for PFHS in All concentrations are expressed as ppb. Run Number Run Date 18 21-Aug-2000 LOW QC After 7 Freeze-Thaw 6.05 6.27 7.12 cycles Mean Mean S.D. % CV Control (ng/mL) % Dev. from Control 6.48 0.565 8.7 6.86 -5.5 MEDIUM QC After 7 Freeze-TlJaw cycles 165 157 163 Mean 162 S.D. 4.16 % CV 2.6 Control (ng/mL) 164 Mean % Dev. from Control -1.2 HIGH QC After 7 Freeze.Thaw 397 417 4O5 cycles Mean Mean S.D. % CV Control (ng/m L) % Dev. from Control 406 10.1 ' 2.5 403 0.7 Control = mean of analytical QC samples analyzed in the same run NOTE: Samples were left at room temperature for up to 6.75 hour periods between each freeze cycle Page 43 Northwest Bioanalytical Study No. NWBS00-040 Report No. NWBR00-122 Table 27. Freeze-Thaw and Room Temperature Human Serum Matrix Stability for M556 in All concentrations are expressed as ppb. Run Number Run Date 18 21-Aug-2000 LOW QC After 7 Freeze-Thaw 4.95 5.07 5.09 cycles Mean Mean S.D. % CV Control (ng/m L) % Dev. from Control 5.04 0.0757 1.5 5.42 -7.0 MEDIUM QC After 7 Freeze-Thaw cycles 128 140 135 Mean 134 S.D. 6.03 % CV 4.5 Control (ng/mL) 141 Mean % Dev. from Control -5.0 HIGH QC After 7 Freeze-Thaw 359. 372 374 cycles Mean 368 S.D. 8.14 % CV 2.2 Control (ng/m L) 367 Mean % Dev. from Control 0.3 ,:_, "" :'-.- Control = mean of analytical QC samples analyzed in the same run NOTE: Samples were left at room temperature for up to 6.75 hour periods between each freeze cycle Page 44 Northwest Bioanalytical Study No. NWBS00-040 Report No. NWBR00-122 Table 28. Freeze-Thaw and Room Temperature Matrix Stability for M570 in Human Serum All concentrations are expressed as ppb. Run Number Run Date 18 21-Aug-2000 LOW QC After 7 Freeze-Thaw 7.68 8.76 8.51 cycles Mean Control % Dev. from Mean S .D. % CV (ng/m L) Control 8.32 0.565 6.8 8.02 3.7 MEDIUM QC After 7 Freeze-Thaw cycles 142 142 143 Mean Mean S.D. % CV Control (ng/m L) e/. Dev. from Control 142 O.577 0.4 149 -4.7 HIGH QC After 7 Freeze-.Th aw cycles ":_". 356 371 370 M ean 366 S.D. 8.39 % CV 2.3 Control(ng/mL) 386 Mean % Dev. from Control -5.2 i Control = mean of analytical QC samples analyzed in the same run NOTE: Samples were left at room temperature for up to 6.75 hour periods between each freeze cycle Page 45 Northwest Bioanalytical Table 29. Reinjection Stability for PFOS All concentrations are expressed as ppb. Run Number Run Date 23 3-Jul-2000 LOWQC Rei::jeetion (After 2.5 Days Room Ten:_.) 49.1 48.0 50.0 46.1 45.1 Mean 47.7 S.D. 2.04 % CV 4.3 Control (ag/mL) 47.2 Mean % Dev. from Control 1.1 MEDIUM QC Reinjection (After 2.5 Days Room Temp. 164 172 170 164 170 Mean S.D. % CV Control (ng/mL) Mean % Dev. from Control i 68 3.74 2.2 173 -2.9 HIGH QC Reinjection (After 2.5 Days Room Temp.) 391 369 368 372 411 Mean 382 S.D. 18.6 % CV 4.9 Control (ng/mL) 393 Heart % Dev. from Control -2.8 Study No. NWBS00-040 Report No. NWBR00-122 ':': :" l. "- " Control= meanofanalyticaiOCsamplesanalyzedin run 15(originalinjection) Page 46 Northwest Bioanalytical Table 30. Reinjeetion Stability for PFOSA All concentrations are expressed as ppb. Run Number Run Date 23 3-Jul-2000 LOW QC Reinjection (After 2.5 Days Room Temp.) 2.79 2.69 2.85 2.66 2.68 Mean S.D. % CV Control (ng/mL) Mean % Dev. from Control 2.73 0.0820 3.0 2.82 -3.2 MEDIUM QC Reinjection (After 2.5 Days Room Temp.) [02 Z02 97.9 94.4 105 Mean $.D. % CV Control (ngtmL) Mean % Dev. from Control I00 4.14 4.1 103 -2.9 HIGH QC Reinjection (After 2.5 Days Room Temp.) 275 273 290 284 307 Mean S.D. % CV Control(ng/mL) Mean % Dev.fromControl 286 13.7 4.8 300 -4.7 Study No. NWBS00-040 Report No. NWBR00-122 s: _.. : : _;.. Control = mean of analytical QC samples analyzed in run 15 (original injection) Page 47 Northwest Bioanalytical Table 31. Reinjeetion Stability for PFOSAA All concentrations are expressed as ppb. Study No. NWBS00-040 Report No. NWBR.00-122 Run Number Run Date 23 3-Jul-2000 LOW QC Reh_jection (After 2.5 Days Room Temp.) 7.78 7.58 8.15 7.41 7.74 Mean $.D. % CV Control (ng/mL) IVlean% Dev. from Control 7.73 0.276 3.6 7.59 1.8 MEDIUM QC Xeinjection (After 2.5 Days Room Temp.) 109 112 113 104 110 Mean !I0 S.D. 3.51 % CV 3.2 Control (ng/mL) 113 Mean % Dev. from Control -2.7 HIGH QC Rehzjectlon (After 2.5 Days Room Temp.) 287 285 284 278 305 Mean 288 S.D. 10.2 % CV 3.5 Control (ng/mL) 30 I lVlean% Dev. from Control -4.3 .:. : : : :... Control = mean of analytical QC samples analyzed in run 15 (original injection) Page 48 Northwest Bioanalytical Table 32. Reinjection Stability for POAA All concentrationsareexpressed as ppb. Run Number Run Date 23 3-Jul-2000 LOW QC Reinjection (After 2.5 Days Room Temp.) 8.68 8.43 8.31 8.41 8.41 Mean S.D. % CV Control (ng/mL) Mean % Dev. from Control 8.45 0.138 1.6 8.10 4.3 MEDIUM QC Reinjection (After 2.5 Days Room Temp.) 150 152 156 144 147 Mean !50 S.D. 4.60 % CV 3.1 Control (ng/mL) 144 Mean % Dev. from Control 4.2 HIGH QC Reinjection (After 2.5 Days Room Temp.) 417 408 386 388 401 Mean ];00 S.D. 13.2 % CV 3.3 Control (ng/mL) 384 Mean % Dev. from Control 4.2 Study No. NWBS00-040 Report No. NWBR00-122 ,:_: :. " .=.. Control= mean ofanalytical QC samplesanalyzed in run15 (original injection) Page 49 Northwest Bioanal_ical Table 33. Reinjection Stability for PFHS All concentrations are expressed as ppb. Run Number Run Date 23 3-Jul-2000 LOW QC Reinjection (After 2.5 Days Room Temp.) 6.30 6.32 6.48 5.98 6.11 Mean S.D. % CV Control (ng/mL) Mean % Dev. from Control 6.24 0.195 3.1 6.22 0.3 MEDIUM QC Relnjection (After 2.5 Days Room Temp. 153 156 156 159 159 Mean 157 S.D. 2.51 % CV 1.6 Control (ng/mL) 158 Mean % Dev. from Control -0.6 HIGH QC Reinjection (After 2.5 Days Room Temp.) 441 416 421 409 450 lVlean 427 S.D. 17.4 % CV 4.1 Control (ng/mL) 445 Mean % Dev. from Control -4.0 Study No. NWBS00-040 Report No. NWBR00-122 .:_.. :. "_. ,... - Control = mean of analytical QC samples analyzed in run 15 (original injection) Page 50 Northwest Bioanalytical Table 34. Reinjeetion Stability for M556 All concentrations are expressed as ppb. Run Number Run Date 23 3-Jul-2000 LOW QC Reinjection (After 2.5 Days Room Temp.) 5.78 5.73 5.45 5.29 5.33 lVlean S.D. % CV Control (ng/mL) Mean % Dev. from Control 5.52 0.227 4.1 5.61 -I.6 MEDIUM QC Reinjection .(After 2.5 Days Room Temp.) 144 147 147 , 143 146 /Vlean 145 S.D. ! .82 % CV !.3 Control (ng/mL) 147 IVlean% Dev. from Control -1.4 nIGH QC RMnjection (After 2.5 Days Room Temp.) 388 373 381 367 398 Mean 38 I S.D. 12.2 % CV 3.2 Control (n_mL) 396 /Vlean % Dev. from Control -3.8 Study No. NWBS00-040 Report No. NWBR00-122 : ; ..:. , Control = mean of analytical QC samples analyzed in run 15 (original injection) Page 51 Northwest Bioanalytical Table 35. Reinjeetion Stability for M570 All concentrations arc expressed as ppb. Run Number Run Date 23 3-Jul-2000 LOWQc Reinjection (After 2.5 Days Room Temp.) 8.31 8.36 8.52 8.10 7.71 Mean S.D. % CV Controi (ng/m L) Mean % Dev. from Control 8.20 0.312 3.8 8.3 l -1.3 MEDIUM QC Reinjection (After 2.5 Days Room Temp.) 145 149 146 144 145 Mean S.D. % CV Control (ng/mL) Mean % Dev. from Control 146 1.92 1.3 147 -0.7 HIGH QC Reinjection (After 2.5 Days Room Temp.) 391 384 399 380 405 IVlean 390 S.D. 9.51 % CV 2.4 Control (ng/mL) 401 Mean % Dev. from Control -2.7 StudyNo. NWBS00-040 Report No. NWBR00-122 :. : .: .. Control = mean of analytical QC samples analyzed in run 15(original injection) Page 52 Northwest Bioanalytical Study No. NWBS00-040 Report No. NWBR00-122 Table 36. Room Temperature Extract Stability for PFOS All concentrations are expressed as ppb. Run Number Run Date Mean S.D. % CV Control (ng/mL) Mean % Dev. from Control Mean S.D. % CV Control (ng/mL) Mean % Dev. from Control 18 21-Aug-2000 LOWQc After 7Days 51.7 46.0 46.2 48.0 3.23 6.7 46.7 2.8 HIGH QC After 7 Days 373 402 397 391 15.5 4.0 378 3.4 - : " :..._...-. Control -- mean of QC samples analyzed in the same run immediately following extraction Page 53 Northwest Bioanalytical Study No. NWBS00-040 Report No. NWBP-.00-122 Table 37. Room Temperature Extract Stability for PFOSA All concentrations are expressed as ppb. Run Number Run Date 18 21-Aug-2000 LOW QC After 7 Days 3.48 3.34 3.30 Mean Mean S.D. % CV Control (n_mL) % Dev. from Control 3.37 0.0945 2.8 3.13 7.7 HIGH QC After 7 Days 337 362 334 Mean 344 S.D. 15.4 % CV 4.5 j:t_ Control (ng/mL) 322 Mean % Dev. from Control 6.8 g Control = mean of QC samples analyzed in the same run immediately following extraction Page 54 NorthwestBioanalytical StudyNo. NWBS00-040 Report No. NWBR00-122 Table 38. Room Temperature Extract Stability for PFOSAA All concentrationsare expressedas ppb. Run Number 18 Run Date 21-Aug-2000 LOW QC After 7 Days 8.2 9.2 9.3 Mean Mean S.D. % CV Control (ng/mL) % Dev. from Control 8.89 0.607 6.8 8.02 10.8 HIGH QC After 7 Days 321 337 343 Mean 334 S.D. 11.4 % CV 3.4 Control (ng/mL) 297 Mean % Dev. from Control 12;5 : "" "_" i.d-. Control_-meanof QCsamplesa"nalyzedinthesame runimmediatelyfollowingextraction Page55 Northwest Bioanalytical Study No. NWBS00-040 Report No. NWBR00-122 Table 39. Room Temperature Extract Stability for POAA All concentrations are expressed as ppb. Run Number Run Date 18 21-Aug-2000 LOW QC After 7 Days 6.92 7.98 8.15 Mean Mean S.D. % CV Control (ng/mL) % Dev. from Control 7.68 0.667 8.7 7.82 -1.8 HIGH QC After 7 Days 360 395 378 Mean 378 S.D. 17.5 % CV 4.6 Control (ng/mL) 372 Mean % Dev. from Control 1.6 . "_- .,,.. Control -- mean of QC samples analyzed in the same run immediately following extraction Page 56 NorthwestBioanalytical StudyNo. NWBS00-040 ReportNo. NWBR00-122 Table 40. Room Temperature Extract Stability for PFHS Allconcentrationsare expressedasppb. Run Number Run Date 18 21-Aug-2000 LOW QC After 7 Days 6.31 6.21 5.90 Mean Mean S.D. % CV Control (ng/mL) % Dev. from Control 6.14 0.214 3.5 6.86 -10.5 mGrl QC After 7 Days 396 405 412 Mean 404 S.D. 8.02 % CV 2.0 Control (ng/mL) 403 Mean % Dev. from Control 0.2 ] 1 Control= mean of QC samplesanalyzedin thesamerun immediately following extraction Page57 NorthwestBioanalytical StudyNo.NWBS00-040 ReportNo.NWBR00-122 Table 41. Room Temperature Extract Stability for IV1556 All concentrationsareexpressedasppb. Run Number Run Date 18 21-Aug-2000 LOW QC After 7 Days 5.13 5.65 5.50 Mean Mean S.D. % CV Control (ng/mL) % Dev. from Control 5.43 0.268 4.9 5.42 0.2 HIGH QC After 7 Days 373 376 387 Mean 379 S.D. 7.37 % CV 1.9 Control (ng/mL) 36_/ Mean % Dev. from Control 3.3 o: : , _'. ,_ Control=mean of QCsamplesanalyzedin the samerun immediatelyfollowingextraction Page58 NorthwestBioanalytical StudyNo. NWBS00-040 ReportNo. NWBR00-122 Table 42. Room Temperature Extract Stability for M570 All concentrationsare expressedasppb. Run Number Run Date 18 21-Aug-2000 LOW QC After 7Days 8.86 9.13 8.74 Mean S.D. % CV Control (ng/mL) Mean % Dev. from Control 8.91 0.200 2.2 8.02 11.1 HIGH QC After 7 Days 409 429 423 Mean 420 S.D. 10.3 % CV 2.5 Control (ng/mL) 386 Mean % Dev. from Control 8.8 Control--mean of QCsamplesanalyzedin thesamerun immediatelyfollowingextraction Page 59 NorthwestBioanalytical StudyNo. NWBS00-040 ReportNo. NWBR00-122 Table 43. Reduced Temperature (1 to 8 C) Extract Stability for PFOS All concentrationsare expressedasppb. Run Number Run Date 18 21-Aug-2000 LOW Qc 1 to 8C After 4 Days 49.3 45.6 44.8 Mean 46.6 S.D. 2.40 % CV 5.2 Control (ng/mL) 46.7 Mean % Dev. from Control -0.2 HIGH QC 1 to 8C After 4 Days 391 385 377 - Mean 384 o;" : S.D. 7.02 % CV 1.8 : ;. "_'..2..o Control (ng/mL) 378 Mean % Dev. from Control 1.6 Control= meanof QCsamplesanalyzedin the samerun immediatelyfollowingextraction Page 60 NorthwestBioanalytical StudyNo. NWBS00-040 ReportNo. NWBR00-122 Table 44. Reduced Temperature (1 to 8 C) Extract Stability for PFOSA All concentrationsare expressedasppb. Run Number Run Date 18 21-Aug-2000 LOW QC I to 8C After 4 Days 3.22 2.79 2.86 Mean S.D. % CV Control (n_mL) Mean % Dev. from Control 2.96 0.231 7.8 3.13 -5.4 HIGH QC 1 to 8C After 4 Days 350 297 304 Mean S.D. % CV Control (n_mL) Mean % Dry. from Control 317 28.8 9.1 322 -1.6 ,: : :. . , i _" ..;-. Control= meanof QC samplesanalyzedin thesame run immediatelyfollowingextraction Page61 NorthwestBioanalytical StudyNo. NWBS00-040 ReportNo. NWBR00-122 Table 45. Reduced Temperature (1 to 8 C) Extract Stability for PFOSAA All concentrationsare expressedas ppb. Run Number Run Date Mean S.D. % CV Control (ng/mL) Mean % Dev. from Control Mean Mean S.D'. % CV Control (ng/mL) % Dev. from Control 18 21-Aug-2000 LOWQC 1 to 8C After 4 Days 7.78 8.55 8.32 8.22 0.395 4.8 8.02 2.5 HIGH QC I to 8C After 4 Days 312 306 311 310 3.21 1.0 297 4.4 ..: :. , :'..o , Control= meanof QCsamplesanalyzedin the samerun immediatelyfollowingextraction Page 62 NorthwestBioanalytical StudyNo. NWBS00-040 ReportNo. NWBR00-122 Table 46. Reduced Temperature (1 to 8 C) Extract Stability for POAA All concentrationsare expressedasppb. Run Number Run Date Mean S.D. % CV Control (ng/mL) Mean % Dev. from Control Mean S.D. % CV Control (ng/mL) Mean % Dev. from Control 18 21-Aug-2000 LOW QC I to 8C After 4 Days 7.78 8.75 8.39 8.31 0.490 5.9 7.82 6.3 HIGH QC I to 8C After 4 Days 381 394 385 387 6.66 1.7 372 4.0 ' :. , " .-. _.,/-. .. Control-- meanof QC samplesanalyzedin thesamerun immediatelyfollowingextraction Page63 NorthwestBioanalytical StudyNo. NWBS00-040 ReportNo. NWBR00-122 Table 47. Reduced Temperature (1 to 8 C) Extract Stability for PFHS All concentrationsareexpressedas ppb; Run Number Run Date Mean Mean S.D. % CV Control (ng/mL) % Dev. from Control Mean S.D. % CV Control (ng/mL) Mean % Dev. from Control 18 21-Aug-2000 LOW QC 1 to 8C After 4 Days 6.76 6.34 7.74 6.95 0.718 10.3 6.86 1.3 HIGH QC 1 to 8C After 4 Days 400 411 423 411 11.5 2.8 " 403 2.0 .:_ . .".._ . Control=meanof QCsamplesanalyzedin the samerunimmediatelyfollowingextraction Page 64 Northwest Bioanalytical Study No. NWBS00-040 Report No. NWBR00-122 Table 48. Reduced Temperature (1 to 8 C) Extract Stability for M556 All concentrations are expressed as ppb. Run Number Run Date Mean Mean S.D. % CV Control (ng/mL) % Dev. from Control Mean Mean S.D. % CV Control (ng/mL) % Dev. from Control 18 21-Aug-2000 LOW QC 1 to 8C After 4 Days 5.47 6.57 6.82 6.29 0.718 11.4 5.42 16.1 HIGH QC 1 to 8C After 4 Days 398 404 403 402 3.9.1 0.8 367 9.5 _. , ': _:-. _ Control = mean of QC samples analyzed in the same mn immediately following extraction Page 65 NorthwestBioanalytical StudyNo. NWBS00-040 ReportNo. NWBR00-122 Table 49. Reduced Temperature (1 to 8 C) Extract Stability for M570 Allconcentrationsareexpressedas ppb. Run Number Run Date Mean S.D. % CV Control (ng/mL) Mean % Dev. from Control Mean S.D. % CV Control (ng/mL) Mean % Dev. from Control 18 21-Aug-2000 LOW QC I to 8C After 4 Days 7.96 .7.90 9.21 8.36 0.740 8.9 8.02 4.2 HIGH QC I to 8C After 4 Days 396 394 402 397 4.16 1.0 386 2.8 :. ' _- ,_.... Control= meanof QCsamplesanalyzedinthe samerun immediatelyfollowingextraction Page66 NorthwestBioanalytical SmdyNo. NWBS00-040 ReportNo. NWBR00-122 Table 50. Reduced Temperature (-20 C) Extract Stability for PFOS Allconcentrationsare expressedas ppb. Run Number 18 Run Date 21-Aug-2000 LOW QC -20oC After 5 Days 47.2 45.5 40.9 Mean 44.5 S.D. 3.26 % CV 7.3 Control (n_mL) 46.7 Mean % Dev. from Control -4.7 HIGH QC -20oC After 5 Days 376 373 365 Mean 371 S.D. 5.69 % CV 1.5 Control (ng/mL) 378 Mean % Dev. from Control -1.9 i Control= mean ofQC samplesanalyzedin the same runimmediatelyfollowingextraction Page67 NorthwestBioanalytical StudyNo. NWBS00-040 ReportNo. NWBR00-122 Table 51. Reduced Temperature (-20 C) Extract Stability for PFOSA All concentrationsareexpressedasppb. Run Number Run Date 18 21-Aug-2000 LOW QC -20oc After 5 Days 2.95 3.16 3.14 Mean Mean S.D. % CV Control (ng/mL) % Dev. from Control 3.08 0.116 3.8 3.13 -1.6 HIGH QC -20C After 5 Days 312 312 296 Mean 307 .:_: S.D. 9.24 % CV 3.0 Control (ng/mL) 322 . Mean % Dev. from Control -4.7 Control= meanof QC samplesanalyzedin thesamerunimmediatelyfollowingextraction Page68 Northwest Bioanalytical Study No. NWBS00-040 Report No. NWBR00-122 Table 52. Reduced Temperature (-20 C) Extraet Stability for PFOSAA All concentrations are expressed as ppb. Run Number Run Date 18 21-Aug-2000 LOW Qc -20C After 5 Days 7.52 8.32 7.89 Mean S.D. % CV Control (n_mL) Mean % Dev. from Control 7.91 0.40O 5.1 8.02 -1.4 HIGH QC -20oC After 5 Days 294 306 287 Mean 296 S.D. 9.61 % CV 3.2 Control (ng/mL) 297 Mean % Dev. from Control -0.3 :. ""-., _.-. Control = mean of QC samples analyzed in the same run immediately following extraction Page 69 NorthwestBioanalytical StudyNo. NWBS00-040 ReportNo. NWBR00-122 Table 53. Reduced Temperature (-20 C) Extract Stability for POAA All concentrationsare expressedasppb. Run Number Run Date 18 21-Aug-2000 LOW QC -20C After 5 Days 7.84 10.2 6.85 Mean 8.30 S.D. 1.72 % CV 20.7 Control (n_mL) 7.82 Mean % Dev. from Control 6.1 HIGH QC -20oC After 5 Days 351 373 386 Mean S.D. % CV Control (ng/mL) Mean % Dev. from Control 370 1_/.7 4.8 372 -0.5 Control= meanof QCsamplesanalyzedinthe samerun immediatelyfollowingextraction Page70 NorthwestBioanalytical StudyNo. NWBS00-040 ReportNo. NWBR00-122 Table 54. Reduced Temperature (-20 C) Extract Stability for PFHS All concentrationsareexpressedas ppb. Run Number Run Date 18 21-Aug-2000 LOW QC -20oC After 5 Days 6.68 6.06 7.70 Mean S.D. % CV Control (ng/mL) Mean % Dev. from Control 6.81 0.828 12.2 6.86 -0.7 HIGH QC -20oC After 5 Days 403 407 417 Mean 409 .:,: S.D. 7_21 % CV 1.8 Control (ng/mL) 403 Mean % Dev. from Control 1.5 Control= meanof QCsamplesanalyzedin the same run immediatelyfollowingextraction Page71 NorthwestBioanalytical StudyNo. NWBS00-040 ReportNo. NWBR00-122 Table 55. Reduced Temperature (-20 C) Extract Stability for M556 All concentrationsare expressedasppb. Run Number Run Date 18 21-Aug-2000 LOW QC -20oc After $ Days 4.51 5.69 5.97 Mean Mean S.D. % CV Control (ng/mL) % Dev. from Control 5.39 0.775 14.4 5.42 -0.6 mGn Qc -20C After 5 Days 358 373 378 Mean 370 S.D. 10.4 %CV 2.8 Control (ng/mL) 367 Mean % Dev. from Control 0.8 "" :'_'" Control= meanof QCsamplesanalyzedin the samerun immediatelyfollowingextraction Page72 NorthwestBioanalytical StudyNo. NWBS00-040 ReportNo. NWBR00-122 Table 56. Reduced Temperature (-20 C) Extract Stability for M570 All concentrationsare expressedasppb. Run Number Run Date 18 21-Aug-2000 LOW QC -20oc After 5 Days 8.43 8.52 7.94 Mean Mean S.D. % CV Control (ng/mL) % Dev. from Control 8.30 0.3 I2 3.8 8.02 3.5 HIGH QC -20C After 5 Days 366 388 369 Mean S.D. % CV Control (ng/mL) Mean % Dev. from Control 374 1"1.9 3.2 386 -3.1 Control= meanof QC samplesanalyzedin thesamerun immediatelyfollowingextraction Page73 Northwest Bioanalyfical Study No. NWBS00-040 Report No. NWBR00-122 Table 57. Long-Term Matrix Stability for PFOS in Human Serum All concentrations are expressed as ppb. Run Date 21-Aug-2000 Run Number 18 Low QC 51.1 ppb 55 Days 52.2 50.1 42.3 43.8 45.0 Medium QC 197 ppb 55 Days 175 171 174 I66 165 High QC 446 ppb 55 Days 381 391 361 371 388 Mean SD %CV %Bias N 46.7 170 378 4.26 4.55 12.4 9.1 2.7 3.3 -8.6 -13.7 -15.2 5 5 5 Table 58. Long-Term Matrix Stability for PFOSA in Human Serum All concentrations are expressed as ppb. Run Date 21-Aug-2000 Run Number 18 Low QC 4.00 ppb 42 Days 4.28 4.56 4.40 4.12 4.32 Medium QC 150 ppb 42 Days 155 158 154. 142 143 High QC 400 ppb 42 Days 376 420 381 ':': 397 .. 400 : ,: .- Mean SD %CV %Bias N 4.34 150 395 0.161 7.37 17.4 3.7 4.9 4.4 8.5 0.0 -1.3 5 5 5 Page 74 Northwest Bioanalytical Study No. NWBS00-040 Report No. NWBR00-122 Table 59. Long-Term Matrix Stability for PFOSAA in Human Serum All concentrations are expressed as ppb. Run Date 21-Aug-2000 Run Number 18 Low QC 9.00 ppb 42 Days 9.64 9.11 10.5 10.2 10.3 Medium QC 155 ppb 42 Days 157 158 164 159 160 High QC 405 ppb 42 Days 403 429 406 413 416 Mean SD %CVi %Bias N 9.95 160 413 0.568 2.70 10.2 5.7 1.7 2.5 10.6 3.2 2.0 5 5 5 Table 60. Long-Term MatrLx Stability for POAA in Human Serum All concentrations are expressed as ppb. Run Date 21-Aug-2000 Run Number 18 ,., Low QC 8.74 ppb 55 Days 7.74 7.54 7.72 8.05 8.03 Medium QC 154 ppb 55 Days 145 138 155 151 157 High QC 403 ppb 55 Days 364 380 354 .:': 371 . 389 J Mean SD %CV %Bias N 7.82 149 372 0.219 7.76 13.6 2.8 5.2 3.7 -10.5 -3.2 -7.7 5 5 5 Page 75 Northwest Bioanalytical Study No. NWB$00-040 Report No. NWBR00- !22 Table 61. Long-Term Matrix Stability for PFHS in Human Serum All concentrations are expressed as ppb. Run Date Run Number 2 l-Aug-2000 18 Low QC 6.15 ppb 55 Days 7.01 7.06 7.10 6.57 6.56 Medium QC 152 ppb 55 Days 163 158 169 161 168 High QC 402 ppb 55 Days 387 414 375 404 435 Me_ SD %CV] %Bias N 6.86 164 403 0.271 4.66 23.4 4.0 2.8 5.8 11.5 7.9 0.2 5 5 5 Table 62. Long-Term Matrix Stability for M556 in Human Serum All concentrations are expressed as ppb. Run Date 2l-Aug-2000 Run Number 18 Low QC 5.80 ppb 55 Days 6.07 5.67 *4.60 4.97 5.79 Medium QC 152 ppb 55 Days 141 137 149 137 143 High QC 402 ppb 55 Days 355 382 340': 36.2: : . 394 l' Mean 5.42 141 367 SD 0.612 4.98 21.5 I %CV 11.3 3.5 5.9 %Bias -6.6 -7.2 -8.7 N 5 5 5 * > :i:20% deviation from theoretical : ,_- ,. Page 76 Northwest Bioanalytical Study No. NWBS00-040 Report No. NWBR00-122 Table 63. Long-Term Matrix Stability for M570 in Human Serum All concentrations are expressed as ppb. Run Date 2 l-Aug-2000 Run Number 18 Low QC 8.60 ppb 55 Days 7.93 8.07 7.87 8.04 8.20 Medium QC 155 ppb - 55 Days 147 147 158 144 150 High QC 405 ppb 55 Days 373 391 373 384 408 Mean SD %CV %Bias N 8.02 149 386 0.128 5.36 14.6 1.6 3.6 3.8 -6.7 -3.9 -4.7 5 5 5 Page 77 Northwest Bioanalytical Study No. NWBS00-040 Report No. NWBR00-122 Table 64. PFOS Extraction Efficiency from Human Serum Run 22 Low Concentration = 4.00 ng/mL Time of Spiking !. Analyte and IS after extraction Mean Ratio AnalyteflS 0.0679 0.0683 0.0735 0.0699 Ratio IS/Analyte 14.7 14.6 13.6 14.3 2. Analyte prior and IS after extraction Mean 0.0656 0.0605 0.0649 0.0637 3. IS prior and Analyte after extraction Mean 0.775 0.726 0.805 0.769 Adjusted mean extraction efficiency = 44.4% Mean extraction efficiency for the internal standard = 5.38% High Concentration = 400 ng/mL T.irne of Spiking 1. Analyte and IS after extraction Mean Ratio Analyte/IS 1.65 1.55 1.65 1.62 Ratio IS/Analyte 0.605 0.643 0.607 0.618 2. Analyte prior and IS afc_ extraction Mean 0.522 0.538 0.518 0.526 3. IS prior and Analyte after extraction Mean 0.0396 : 0.0379 0.0401 : 0.0392 : -" -- Adjusted mean extraction efficiency = 30.1% Mean extraction efficiency for the internal standard = 6.34% Overall mean e,,ctraction efjTciency for analyte ffi 37.3% Overall mean extraction efficiency for internal standardffi 5.86% * Due to rounding, calculations based on displayed values may differ from actual. Page 78 Northwest Bioanalytical Study No. NWBS00-040 Report No. NWBR00-122 Table 65. PFOSA Extraction Efficiency from Human Serum Run 22 Low Concentration = 4. O0 ng/mL .Time of Spikin_ I. AnalyteandIS aRerextraction Mean Ratio Anal,fte/IS 0.0275 0.0222 0.0214 0.0237 2. Analytepriorand ISafterextraction Mean 0.0I 7 l 0.0147 0.0172 0.0163 Ratio IS/Analvte 36.3 45.0 46.8 42.7 3. 1SpriorandAnalyteafterextraction 2.47 2.25 2.45 Mean 2.39 Mean extraction efficiency for the internal standard -- 5.60% High Concentration = 400 ng/mL Time of Spiking 1.AnalyteandIS afterextraction Mean Ratio Analvte/IS 2.58 2.47 2.65 2.57 Ratio IS/Analvte 0.387 0.405 0.377 0.390 2. AnalytcpriorandISafterextraction 1.80 1.87 1.75 Mean 1.81 ,: _. 3. lSpriorandAnalytcafterextraction Mean 0.0243 : 0.0223 0.0233. : 0.0233 Mean extraction efficiency for the analyte = 70.4% Overall mean extraction efficiency for analyte = 69.6% Overall mean extraction efficiency for internal standard = 5. 79% * Due to rounding, calculations based on displayed values may differ from actual. "_ .,i'.. Page 79 Northwest Bioanalytical Study No. NWBS00-040 Report No. NWBR00-122 Table 66. PFOSAA Extraction Effieieney from Human Serum Run 22 Low Concentration = 4.00 ng/mL Time of Spiking 1. Analyte and 1Safter extraction Mean Ratio Analyte/IS 0.0246 0.0257 0.0238 0.0247 Ratio IS/Anal_e 40.6 39.0 42.0 40.5 2. Analyte prior and IS after extraction Mean 0.0206 0.0186 0.0194 0.0195 3. IS prior and Analyte after extraction Mean 2.71 2.45 2.58 2.58 Adjusted mean extraction efficiency = 62.6% Mean extraction efficiency for the internal standard = 6.37% High Concentration = 400 ng/mL Time of Spiking 1. Aaalyt and IS after extraction Mean Ratio Analyte/IS 1.50 1.44 1.56 1.50 Ratio IS/Analyte 0.665 0.693 0.641 0.666 2. Analytc prior and I$ after extraction Mean 3. IS prior and Analyte aRer extraction Mean 0.962 0.959 0.917 0.946 ,:,: 0.0419 : 0.0394 0.0413 0.0409 Adjusted mean extraction efficiency = 62.7% Mean extraction efficiency for the internal standard = 6.14% Overall mean extraction efficiency for analyte = 62. 7% Overall mean extraction efficiency for internal standard = 6.26% : -" - * Due to rounding, calculations based on displayed values may differ from actual. Page 80 Northwest Bioanalytical Study No. NWBS00-040 Report No. NWBR00-122 Table 67. POAA Extraction Efficiency from Human Serum Run 22 Low Concentration = 4. 00 ng/mL Time of Spiking I. AnalyteandIS afterextraction Mean Ratio Analyte/IS 0.0288 0.0291 0.0302 0.0294 Ratio IS/Analyte 34.7 34.3 33.1 34.0 2. AnalytepriorandIS afterextraction Mean 0.00934 0.00760 0.0101 0.00901 3. ISpriorandAnalyteafter extraction 2.02 1.93 2.05 Mean 2.00 Adjusted mean extraction efficiency -- 16.8"/, Mean extraction efficiency for the internal standard -- 5.88/, High Concentration "_-400 ng/mL Time of Spiking I.AnalyteandISafterextraction Mean Ratio Anal_e/IS 2.61 2.59 2.72 2.64 Ratio IS/Analyte 0.383 0.386 0.367 0.379 2. AanlytepriorandISafterextraction 0.377 0.385 0.383 Mean 0.382 .:'.. 3. Is priorandAnalyteafterextraction Mean 0.0248 0.0227 0.0241 -" 0.0239 Adjusted mean extraction efficiency = 14.3"/o Mean extraetion efficiency for the internal standard = 6.31% Overall mean extraction effieiency for analyte = 15. 6% Overall mean _vtraction efdqciency for internal standard = 6.10% * Due to rounding, calculations based on displayed values may differ from actual. Page 81 Northwest Bioanalytical Study No. NWBS00-040 Report No. NWBR00-122 Table 68. PFHS Extraction Efficiency from Human Serum Run 22 Low Concentration = 4.00 ng/mL Time of S_iking 1.AnalyteandIS afterextraction Mean Ratio Analyte/IS 0.0367 0.0361 0.0366 0.0365 Ratio IS/Analyte 27.3 27.7 27.3 27.4 2. Ana/ytepriorandISafterextraction Mean 0.00870 0.00778 0.00878 0.00842 3. ISpriorandAnalytealterextraction 1.72 1.63 1.71 Mean 1.69 Adjusted mean extraction efficiency = 16.3% Mean extraction efficiency for the internal standard = 6.17% High Concentration = 400 ng/mL Time of Spiking I. Analytand ISal_erextraction Mean 2. Anal_e priorand IS alterextraction Mean 3. IS priorandAnalytafterextraction Ratio Analyte/IS 3.49 3.29 3.58 3.45 0.486 0.514 0.485 0.495 Mean Ratio IS/Analyte 0.286 0.304 0.279 0.290 ,:_.. 0.0192 0.0181 0.0192 " 0.0188 Adjusted mean extraction efficiency ffi 14.3% Mean extraction efficiency for the internal standard = 6.48% Overall mean extraction efficiency for analyte = 15.3% Overall mean extraction effidency for internal standard = 6.33% :" _:'" " * Due to rounding, calculations based on displayed values may differ from actual. Page 82 Northwest Bioanalytical Study No. NWBS00-040 Report No. NWBR00-122 Table 69. MSS6 Extraction Efficiency from Human Serum Run 22 Low Concentration = 4.00 ng/mL Time of Spiking i. AnalyteandIS afterextraction Mean Ratio Analyte/IS 0.00786 0.00750 0.00810 0.00782 Ratio IS/Analyte 127 133 123 128 2. Analytepriorand IS afterextraction Mean 0.00421 0.00366 0.00310 0.00366 3. ISpriorand Analyteafterextraction 7.54 6.18 6.78 Mean 6.83 Mean extraction efficiency for the analyte = 46.8% Adjusted mean extraction efficiency = 37.7*/, High Concentration = 400 ng/mL Time of Spiking 1.Analyteand IS after extraction Mean 2. Analytepriorand IS afterextraction Mean Ratio Analyte/IS 0.788 0.758 0.836 0.794 0.337 0.326 0.303 0.322 Ratio IS/Analyte 1.27 1.32 1.20 1.26 .:', 3. ISpriorand Anal_e after extraction Mean 0.0753 0.0690 0.0709 0.0717 : "- Mean extraction efficiency for the analyte = 40.6% Adjusted mean extraction efficiency = 40.4% Overall mean extraction efficiency for analyte = 39.1% Overall mean extraction efficiency for internal standard = 5.52% : "" " * Due to rounding, calculations based on displayed values may differ from actual. Page 83 Northwest Bioanalytical Study No. NWBS00-040 Report No. NWBR00-122 Table 70. IVI570 Extraction Efficiency Run 22 from Human Serum Low Concentration = 4. O0 ng/mL Time of Spiking I. Analyte and IS after extraction Mean Ratio AnalyteflS 0.0333 0.0317 0.0335 0.0328 Ratio IS/Analyte 30.1 31.5 29.9 30.5 2. Analyte prior and IS after extraction Mean 0.0244 0.0220 0.0244 0.0236 3. IS prior and Analytc after extraction Mean 1.92 1.72 1.74 1.79 Adjusted mean extraction efficiency ffi 54.4*/, Mean extraction efficiency for the internal standard = 5.87% High Concentration = 400 ng/mI, Time of Spiking I. Analyte and IS after extraction Mean Ratio Analyte/IS 2.20 2.16 2.35 2.24 Ratio IS/Analyte 0.454 0.463 0.426 0.448 2. Analyte priorand IS after extraction Mean 3. IS prior and Analyte after extraction Mean 1.34 1.37 1.32 1.34 .: .. 0.0294 0.0270 0.0280 " 0.0281 Adjusted mean extraction efficiency = 59.7% Mean extraction efficiency for the internal standard = 6.27% Overall mean extraction effieiency for analyte= 57.1% Overall mean extraction efficiency for internal standard = 6.07% i * Due to rounding, calculations based on displayed values may differ from actual. : "z:" Page 84 Northwest Bioanaly'tical Study No. NWBS00-040 Report No. NWBR00-122 Table 71. Summary of Calibration Curve Parameters for PFOS in Human Plasma Quadratic weighted l/x 2. All concentrations are expressed as ppb. Run Date Run Number 12-Oct-2000 27-Oct-2000 S00091-2 PFOSMD-40 A -0.000007 0.000000 B C R-Squared 0.030663 0.028132 -0_005620 0.010586 0.9986 0.9931 LLOQ ULOQ 3.94 414 3.94 414 A, B, and C are coefficients used to define the calibration curve. Table 72. Summary of Calibration Curve Parameters for PFOSA in Human Plasma Quadratic weighted 1/x2. All concentrations are expressed as ppb. RunDate Run Number 12-Oct-2000 27-Oct-2000 S00091-2 PFOSMD-40 A -0.000011 0.000001 B C R-Squared 0.073810 0.052188 0.006421 0.010966 0.9970 0.9912 LLOQ ULOQ 1.00 500 1.00 500 A, B, and C are coefficients used to define the calibration curve. Table 73. Summary of Calibration Curve Parameters for PFOSAA in Human Plasma Quadratic weighted 1/x2. All concentrations are expressed as ppb. Run Date 12-Oct-2000 27-0ct-2000 Run Number S00091-2 PFOSMD-40 A -0.000003 0.000005 B C R-Squared 0.032601 0.021209 -0.006279 -0.002450 0.9988 0.9873 L.t,OQ 1.60 {.60 ULOQ : 501 501 A, B, and C are coefficients used to define the calibration curve. Page 85 Northwest Bioanalytical Study No. NWBS00-040 Report No. NWBR.00-122 Table 74. Summary of Calibration Curve Parameters for POAA in Human Plasma Quadratic weighted I/x 2. All concentrations are expressed as ppb. Run Date 12-Oct-2000 27-Oct-2000 Run Number S00091-2 PFOSMD-40 A 0.000000 0.000002 B 0.017245 0.014346 C R-Squared 0.001072 0.002192 0.9985 0.9918 LLOQ 1.92 1.92 ULOQ 481 481 A, B, and C are coefficients used to define the calibration curve. Table 75. Summary of Calibration Curve Parameters for PFHS in Human Plasma Quadratic weighted l/x 2. All concentrations are expressed as ppb. Run Date 12-Oct-2000 27-Oct-2000 Run Number S00091-2 PFOSMD-40 A -0.000005 -0.000001 B C R-Squared 0.025818 0.025534 -0.000970 0.011247 0.9983 0.9953 LLOQ ULOQ 1.36 523 1.36 523 A, B, and C are coefficients used to define the calibration curve. Table 76. Summary of Calibration Curve Parameters for M556 in Human Plasma Quadratic weighted l/x 2. All concentrations are expressed as ppb. Run Date 12-Oct-2000 31-Oct-2000 Run Number S00091-2 24 A -0.000002 -0.000001 B 0.018315 0.018373 C 0.005561 0.001923 R-Squared LI_()Q ULOQ " :" - 0.9954 2.,50 500 0.9972 2.50 500 A, B, and C are coefficients used to define the calibration curve. Page 86 Northwest Bioanalytical Study No. NWBS00-040 Report No. NWBR00-122 Table 77. Summary of Calibration Curve Parameters for M570 in Human Plasma Quadratic weighted 1/x2. All concentrations are expressed as ppb. RunDate Run Number A B C R-Squared LLOQ ULOQ 12-Oct-2000 S00091-2 27-Oct-2000 PFOSMD-40 -0.000006 0.000001 0.048518 0.034031 0:005367 0.011681 0.9978 0.9910 1.00 500 1.00 500 A, B, and C are coefficients used to define the calibration curve. Page 87 NorthwestBioanalytical StudyNo. NWBS00-040 Report No. NWBR00-122 Table 78. Back-Calculated Concentrations of Calibration Plasma Standards for PFOS in Human QuadraticweightedL/x2. Allconcentrationsare expressedas ppb. Run Date Run Number 3.94 5.17 11.3 23.6 44.2 85.4 209 332 414 12-Oct-2000 S00091-2 27-Oct-2000 PFOSMD-40 4.00 5.01 11.2 25.0 42.9 83.7 203 335 390 3.86 5.35 11.1 24.3 43.5 85.3 209 350 424 3.72 5.18 11.3 19.4 45.5 82.1 214 337 369 3.90 5.67 11.7 24.1 45.9 84.4 221 367 407 Table 79. Back-Calculated Concentrations of Calibration Standards for PFOSA in Human Plasma Quadraticweighted1/x2. All concentrationsareexpressedasppb. Run Date Run Number 1.00 2.50 10.0 25.0 50.0 100 250 400 500 12-Oct-2000 S00091-2 27-Oct-2000 PFOSMD-40 1.04 2.60 10.4 25.1 49.1 95.9 252 380 462 0.941 2.53 8.93 25.7 50.5 102 255 444 508 0.961 2.59 9.81 20.2 53.4 103 259 356 443 0.999 2.71 10.4 22.1 52.3 101 267 458 515 Table 80. Back-Calculated Concentrations of Calibration Standards for PFOSAA in Human Plasma .,. - Quadraticweightedl/x2. All concentrationsare expressedas ppb. -. ": ._..-- Run Date Run Number 1.60 3.10 10.6 25.6 50.6 101 251 401 501 12-Oct-2000 S00091-2 27-Oct-2000 PFOSMD-40 1.65 3.12 10.7 25.3 50.5 98.2 255 393 469 1.54 3.10 10.4 26.3 51.0 101 245 432 513 1.66 3.50 11.7 21.3 53.1 104 299 406 440 1.47 2.96 10.8 23.5 47.3 90.3 261 441 475 Page88 Northwest Bioanalytical Study No. NWBS00-040 Report No. NWBI_00-122 Table 81. Back-Calculated Concentrations of Calibration Plasma Standards for POAA in Human Quadratic weighted 1/x2. All concentrations are expressed as ppb. Run Date IRunNumber 1.92 3.36 10.6 25.0 49.0 97.1 241 385 481 12-Oct-2000 S00091-2 27-Oct-2000 PFOSMD-40 2.00 3.24 10.5 26.1 49.3 97.5 241 386 477 1.96 3.11 10.1 25.2 52.1 95.0 239 392 477 1.73 3.01 10.5 22.9 49.7 99.1 250 367 439 2.30 3.12 10.9 25.1 50.7 96.7 262 422 474 Table 82. Back-Calculated Concentrations of Calibration Plasma Standards for PFHS in Human Quadratic weighted l]x 2. All concentrations are expressed as ppb. Run Date Run Number 1.36 2.92 10.7 26.5 52.6 104 262 418 523 12-Oct-2000 S00091-2 27-Oct-2000 PFOSMD-40 1.28 2.74 10.6 27.5 50.2 104 253 441 518 1.44 3.08 10.8 26.9 53.2 104 259 413 524 1.52 2.72 10.6 24.2 54.6 107 259 466 489 1.28 2.78 10.4 28.4 54.1 102 270 425 491 Table 83. Back-Calculated Concentrations of Calibration Standards for M556 in Human Plasma Quadratic weighted l/x 2. All concentrations are expressed as ppb. :. ::- Run Date Run Number 2.50 10.0 25.0 50.0 100 250 400; 500 12-Oct-2000 31-Oct-2000 S00091-2 24 2.48 11.3 27.9 49.7 101 259 398 521 2.47 9.43 24.5 43.7 96.7 245 384 499 2.33 9.67 24.3 51.9 95.2 255 388 499 2.70 9.81 25.8 49.3 104 249 438 470 Page 89 NorthwestBioanalytical StudyNo. NWBS00-040 ReportNo. NWBR00-122 Table 84. Back-Calculated Concentrations of Calibration Standards for M570 in Human Plasma Quadraticweightedl/x:. All concentrationsare expressedasppb. Run Date Run Number 1.00 2.50 10.0 25.0 50.0 100 250 400 500 12-Oct-2000 27-Oct-2000 S00091-2 PFOSMD-40 0.991 1.00 0.993 0.951 2.57 10.2 25.0 50.8 97.0 250 380 451 2.48 9.46 25.4 50.3 101 244 448 528 2.71 10.6 21.2 53.6 104 284 401 444 2.65 10.4 20.9 48.6 93.5 254 443 481 Page90 Northwest Bioanalytical Study No. NWBS00-040 Report No. NWBP.00-122 Table 85. Lower Limit of Quantitation for PFOS in Human Plasma All concentrations are expressed as ppb. Run Date Run Number 27-Oct-2000 PFOS MD 40 LLOQ (3.94 ppb) 3.98 3.51 3.89 4.04 4.06 3.77 Mean S.D. 3.88 0.208 %CV 5.4 %Theoretical 98.5 %Bias -1.5 N 6 Page 91 Northwest Bioanalytical Study No. NWBS00-040 Report No. NWBR.00-122 Table 86. Lower Limit of Quantitation for PFOSA in Human Plasma All concentrations are expressed as ppb. Run Date Run Number 27-Oct-2000 PFOS MD 40 LLOQ (1.00 ppb) 1.08 1.07 1.11 0.900 1.01 0.865 Mean S.D. %CV %Theoretical %Bias N 1.01 0.102 10.1 101.0 1.0 6 Page 92 Northwest Bioanalytical Study No. NWBS00-040 Report No. NWBR00-122 Table 87. Lower Limit of Quantitation for PFOSAA in Human Plasma All concentrations are expressed as ppb. Run Date Run Number 27-Oct-2000 PFOS MD 40 LLOQ (1.60 ppb) 1.63 1.58 1.54 1.39 1.63 1.57 Mean S.D. %CV %Theoretical %Bias N 1.56 0.0889 5.7 97.5 -2.5 6 I Page 93 Northwest Bioanalytical Study No. NWBS00-040 Report No. NWBR00-122 Table 88. Lower Limit of Quantitation for POAA in Human Plasma All concentrationsare expressed as ppb. Run Date Run Number 27-Oct-2000 PFOS MD 40 LLOQ (1.92 ppb) 1.72 1.84 1.80 1.74 1.79 1.62 Mean S.D. %CV %Theoretical %Bias N 1.75 0.0776 4.4 91.1 -8.9 6 j:. Page 94 Northwest Bioanalytical Study No. NWBS00-040 Report No. NWBR.00-122 Table 89. Lower Limit of Quantitation for PFHS in Human Plasma All concentrations are expressed as ppb. Run Date Run Number 27-Oet-2000 PFOS MD 40 LLOQ (1.36 ppb) 1.23 1.29 1.36 1.35 "1.06 1.42 Mean 1.29 S.D. 0.128 %CV 9.9 %Theoretical 94.9 %Bias -5.1 N 6 *> + 20% deviation from theoretical m; : Page 95 Northwest Bioanalytical Study No. NWBS00-040 Report No. NWBR00-122 Table 90. Lower Limit of Quantitation for M556 in Human Plasma All concentrations are expressed as ppb. Run Date 31-Oct-2000 Run Number 24 LLOQ (2.50 ppb) 2.44 2.25 2.50 2.65 2.50 2.34 Mean S.D. %CV %Theoretical %Bias N 2.45 0.139 5.7 98.0 -2.0 6 Page 96 Northwest Bioanalytical Study No. NWBS00-040 Report No. NWBR00-122 Table 91. Lower Limit of Quantitation for M570 in Human Plasma All concentrations are expressed as ppb. Run Date Run Number 27-Oct-2000 PFOS MD 40 LLOQ (1.00 ppb) 1.14 1.02 1.01 1.01 1.01 0.997 Mean S.D. %CV %Theoretical %Bias N 1.03 0.0538 5.2 103.0 3.0 6 Page 97 Northwest Bioanalytical Study No. NWBS00-040 Report No. NWBR00-122 Table 92. Intra-Assay Precision for PFOS Quality Controls in Human Plasma All concentrations are expressed as ppb. Run Date Run Number 12-Oct-2000 S00091-2 Low QC 6.40 ppb 7.33 7.38 *7.89 7.07 6.98 Medium QC 126 ppb 132 129 138 132 137 High QC 332 ppb 343 331 334 334 340 Mean S.D. %CV %Theoretical %Bias n 7.19 0.195 2.7 112.3 12.3 4 134 3.78 2.8 106.3 6.3 5 336 4.93 1.5 101.2 1.2 5 * Sample deactivated due to unacceptable internal standard response. : _. g "":" . ...."- Page 98 Northwest Bioanalytical Study No. NWBS00-040 Report No. NWBR00-122 Table 93. Intra-Assay Precision for PFOSA Quality Controls in Human Plasma All concentrations are expressed as ppb. Run Date Run Number 12-Oct-2000 S00091-2 Low QC 4.00 ppb 4.69 4.73 *4.67 4.53 4.66 Medium QC 150 ppb 148 157 172 166 163 High QC 400 ppb 423 409 425 414 409 Mean S.D. %CV %Theoretical %Bias n 4.65 0.0866 1.9 116.3 16.3 4 161 9.15 5.7 107.3 7.3 5 416 7.62 1.8 104.0 4.0 5 * Sample deactivated due to unacceptable internal standard response. , Page 99 Northwest Bioanalytical Study No. NWBS00-040 Report No. NWBR00-122 Table 94. Intra-Assay Precision for PFOSAA Quality Controls in Human Plasma All concentrations are expressed as ppb. Run Date Run Number 12-Oct-2000 S00091-2 Low QC 4.60 ppb 5.20 5.50 *7.43 4.96 4.89 Medium QC 151 ppb 146 148 169 156 155 High QC 401 ppb 411 423 431 404 425 Mean S.D. %CV %Theoretical %Bias n 5.14 0.276 5.4 111.7 11.7 4 155 9.04 5.8 102.6 2.6 5 419 11.0 2.6 104.5 4.5 5 * Sample deactivated due to unacceptable internal standard response. Page 100 Northwest Bioanalyfical Study No. NWBS00-040 Report No. NWBR00-122 Table 95. Intra-Assay Precision for POAA Quality Controls in Human Plasma All concentrations are expressed as ppb. Run Date Run Number 12-Oct-2000 S00091-2 Low QC 4.81 ppb 5.33 5.17 *7.82 4.86 4.69 Medium QC 145 ppb 144 157 163 155 152 High QC 385 ppb 400 395 399 394 401 Mean S.D. %CV %Theoretical %Bias n 5.01 0.290 5.8 104.2 4.2 4 154 6.98 4.5 106.2 6.2 5 398 3.11 0.8 103.4 3.4 5 * Sample deactivated due to unacceptable internal standard response. ,.; Page 101 NorthwestBioanalytical StudyNo. NWBS00-040 ReportNo. NWBR00-122 Table 96. Intra-Assay Precision for PFHS Quality Controls in Human Plasma All concentrationsare expressedasppb. Run Date Run Number 12-Oct-2000 S00091-2 Low QC 4.48 ppb 4.91 4.80 "5.4I 4.72 4.58 Medium QC 156 ppb 162 158 162 150 158 High QC 417 ppb 406 388 399 403 401 Mean S.D. %CV %Theoretical %Bias n 4.75 158 399 0.139 4.90 6.88 2.9 3.1 1.7 106.0 101.3 95.7 6.0 1.3 -4.3 4 5 5 * Sampledeactivateddueto unacceptableinternalstandardresponse. R; _ Page 102 NorthwestBioanalytieal StudyNo. NWBS00-040 ReportNo. NWBR00-122 Table 97. Intra-Assay Precision for M556 Quality Controls in Human Plasma All concentrationsare expressedas ppb. Run Date Run Number 12-Oct-2000 S00091-2 Low QC 4.00 ppb 4.65 4.35 *4.76 4.14 3.89 Medium QC 150 ppb 147 140 150 146 159 High QC 400 ppb 441 **492 459 403 431 Mean S.D. %CV %Theoretical %Bias n 4.26 0.322 7.6 106.5 6.5 4 148 6.95 4.7 98.7 " -1.3 5 445 33.1 7.4 111.3 I 1.3 5 * Sampledeactivateddue to unacceptableinternalstandardresponse. **> +__20d%eviationfromtheoretical g Page 103 Q Northwest Bioanalytical S tudy No. NWBS00-040 Report No. NWBR00-122 Table 98. Intra-Assay Precision for M570 Quality Controls in Human Plasma All concentrations are expressed as ppb. Run Date Run Number 12-Oct-2000 S00091-2 Low QC 4.00 ppb **4.82 4.75 *5.89 4.56 4.74 Medium QC 150 ppb 146 161 170 165 158 High QC 400 ppb 407 407 414 393 403 Mean S.D. %CV %Theoretical %Bias n 4.72 0.111 2.4 118.0 18.0 4 160 9.03 5.6 106.7 6.7 5 405 7.69 1.9 101.3 1.3 5 * Sample deactivated due to unacceptable internal standard response. ** > +_20% deviation from theoretical 0..'.. : " _" ;.;". _ Page 104 NorthwestBioanalytical StudyNo. NWBS00-040 ReportNo. NWBR00-122 Table 99. Summary of Calibration Curve Parameters Buffer for PFOS in Serum Diluted with Quadraticweightedl/x:. All concentrationsare expressedas ppb. Run Date [Run Number A B C R-Squared 23-Oct-2000 PFOSMD-39 -0.000001 0.009444 -0.004315 0.9954 LLOQ ULOQ 5.30 504 A, B, and C are coefficientsused to definethe calibrationcurve. Table 100. Summary of Calibration Curve Parameters for PFOSA in Serum Diluted with Buffer Quadraticweighted1/x2. Allconcentrationasreexpressedas ppb. Run Date Run Number A 23-Oct-2000 PFOSMD-39 0.000000 B C R-Squared LLOQ ULOQ 0.014752 0.002265 0.9946 1.00 500 A, B,and Care coefficientsusedto definethe calibrationcurve. Table 101. Summary of Calibration Curve Parameters for PFOSAA in Serum Diluted with Buffer Run Date 23-Oct-2000 Quadraticweightedl/x2. Allconcentrationsare expressedas ppb. Run Number PFOSMD-39 A 0.000002 B 0.009880 C .R-Squared 0.004993 0.9960 m: : L,LOQ: ULOQ - .. {.00 500 A, B, and C are coefficientsused to definethecalibrationcurve. Page 105 Q Northwest Bioanalytical Study No. NWBS00-040 Report No. NWBR00-122 Table 102. Summary of Calibration Curve Parameters Buffer for POAA in Serum Diluted with Quadratic weighted l/x _-. All concentrations are expressed as ppb. Run Date 23-Oct-2000 Run Number PFOSMD-39 A -0.000001 B 0.011351 C R-Squared -0.000137 0.9967 LLOQ ULOQ 1.70 501 A, B, and C are coefficients used to define the calibration curve. Table 103. Summary of Calibration Curve Parameters for PFHS in Serum Diluted with Buffer Quadratic weighted l/x 2. All concentrations are expressed as ppb. Run Date 23-Oct-2000 Run A B C R-Squared Number PFOSMD-39 -0.000003 0.018220 0.007453 0.9980 i, A, B, and C are coefficients used to define the calibration curve. LLOQ ULOQ 1.00 500 Table 104. Summary of Calibration Curve Parameters for M556 in Serum Diluted with Buffer QuadratwiecighteId/2x.Allconcentratairoeenxspressaespdpb. .:'.. Run Date 23-Oct-2000 Run Number PFOSMD-39 A 0.000000 B C R-Squared LLOQ uLoQ : 0.008729 -0.000146 0.9936 1.00 500 A, B, and C are coefficients used to define the calibration curve. Page 106 Northwest Bioanalytical Study No. NWBS00-040 Report No. NWBR.00-122 Table 105. Summary of Calibration Curve Parameters Buffer for M570 in Serum Diluted with Quadratic weighted l/x 2. All concentrations are expressed as ppb. Run Date RunNumber 23-Oct-2000 PFOSMD-39 A 0.000000 B C R-Squared LLOQ ULOQ 0.015628 0.005711 0.9969 1.00 500 A, B, and C are coefficients used to define the ealibration curve. Page 107 Northwest BioanalyticaI Study No. NWBS00-040 Report No. NWBR00-122 Table 106. Back-Calculated Concentrations of Calibration Diluted with Buffer Standards for PFOS in Serum Quadratic weighted 1/x2. All concentrations are expressed as ppb. Run Date Run Number 5.30 6.80 14.3 29.3 54.3 104 254 404 504 23-Oet-2000 PFOSMD-39 5.32 6.18 14.0 25.6 52.3 5.45 7.23 14.8 30.1 54.6 102 264 410 476 113 265 417 488 Table 107. Back-Calculated Concentrations of Calibration Standards for PFOSA in Serum Diluted with Buffer Quadratic weighted 1/x2. All concentrations are expressed as ppb. Run Date Run Number 1.00 2.50 10.0 25.0 50.0 100 250 400 500 23-Oct-2000 PFOSMD-39 1.03 2.21 9.29 22.6 50.2 98.2 265 410 455 1.03 2.45 I0.1 25.8 50.6 112 271 423 468 Table 108. Back-Calculated Concentrations of Calibration Standards for PFOSAA in Serum Diluted with Buffer Quadratic weighted l/x 2. All concentrations are expressed as ppb. Run Date !RunNumberi 1.00 2.50 10.0 25.0 50.0 100 250 400 500 23-Oct-2000 PFOSMD-39 1.12 2.50 9.73 23.6 50.0 104 267 .408 485 0.901 2.41 9.41 24.1 50.6 111 254 399 .479 ::.... Page 108 Northwest Bioanalytical Study No. NWBS00-040 Report No. NWBR00-122 Table 109. Back-Calculated Concentrations of Calibration Diluted with Buffer Standards for POAA in Serum Quadratic weighted l/x 2. All concentrations are expressed as ppb. Run Date Run Number 1.70 3.20 10.7 25.7 50.7 101 254 401 501 23-Oct-2000 PFOSMD-39 1.56 3.22 10.4 24.7 50.9 99.8 260 401 458 1.88 3.10 10.6 25.3 50.8 109 263 432 491 Table 110. Back-Calculated Concentrations of Calibration Standards for PFHS in Serum Diluted with Buffer Quadratic weighted I/x 2. All concentrations are expressed as ppb. Run Date Run Number 1.00 2.50 10.0 25.0 50.0 100 250 400 500 23-Oct-2000 PFOSMD-39 1.01 2.40 I0.I 24.2 49.6 99.3 254 424 450 1.01 2.53 I0.0 24.3 49.0 I06 260 433 473 Table 111. Back-Calculated Concentrations of Calibration Standards for M556 in Serum Diluted with Buffer QuadratiwceightedI/x2.Allconcentratioanrseexpresseadsppb. Run Date Run Number 2.50 10.0 25.0 50.0 100 250 400 500 23-Oct-2000 PFOSMD-39 2.68 I0.6 22.9 51.3 109 2.36 9.33 22.9 48.6 i02 283 432 505 242 379 45i "" ,,;. Page 109 Northwest Bioanalytical Study No. NWBS00-040 Report No. NWBR00-122 Table 112. Back-Calculated Concentrations of Calibration Diluted with Buffer Standards for M570 in Serum Quadratic weighted l/x z. All concentrations are expressed as ppb. Run Date Run Number 1.00 2.50 10.0 25.0 50.0 100 250 400 500 23-Oct-2000 PFOSMD-39 1.07 2.48 9.93 22.5 51.6 0.964 2.34 9.59 24.6 50.8 103 268 408 489 110 254 393 476 o; ; Page 1I0 Northwest Bioanalytical Study No. NWBS00-040 Report No. NWBR00-122 Table 113. PFOS Quality Controls Samples Prepared in Serum Diluted with Buffer Run Date 23-Oct-2000 Run Number PFOSMD-3 9 193 ng/mL DF=10 204 219 205 202 443 ng/mL DF=10 464 423 451 477 4040 ng/mL DF=10 4240 4420 4320 4320 4040 ng/mL DF=50 *3180 *3020 *3150 "3100 Mean S.D. %CV %Theoretical %Bias n 208 7.77 3.7 107.8 7.8 4 454 23.1 5.1 102.5 2.5 4 * >+_20%deviation from theoretical 4330 73.7 1.7 107.2 7.2 4 3110 69.9 2.2 77.0 -23.0 4 Table 114. PFOSA Quality Control Samples Prepared in Serum Diluted with Buffer Run Date Run Number 23-Oct-2000 PFOSMD-39 150 n_mL DF=10 147 147 145 136 400 ng/mL DF=10 388 360 370 409 4000 n_mL DF=10 4020 3850 3980 3960 4000 ng/mL DF=50 *2380 *2240 "2210 "2210 Mean S.D. %CV %Theoretical %Bias n 144 382 5.25 21.5 3.6 5.6 96.0 95.5 -4.0 -4.5 4 4 * >+20*/odeviation from theoretical 3950 72.7 1.8 98.8 -1.3 4 o 2260 =81.2 3.6 56.5 -43.5 4 Page 111 Northwest Bioanalytical Study No. NWBS00-040 Report No. NWBR00-122 Table 115. PFOSAA Quality Control Samples Prepared in Serum Diluted with Buffer Run Date 23-Oct-2000 Run Number PFOSMD-39 153 ng/mL DF=10 144 146 142 142 403 ng/mL DF=10 405 379 389 417 4000 ng/mL DF=10 3920 4100 3970 4010 4000 ng/mL DF=50 *2750 *2560 *2620 *2550 Mean S.D. %CV %Theoretical %Bias n 144 398 1.91 16.8 1.3 4.2 94.1 98.8 -5.9 -1.2 4 4 * >+_20%deviation from theoretical 4000 76.2 1.9 100.0 0.0 4 2620 92.0 3.5 65.5 -34.5 4 Table 116. POAA Quality Control Samples Prepared in Serum Diluted with Buffer Run Date Run Number 23-Oct-2000 PFOSMD-39 157 n_mL DF=10 163 174 164 166 407 ng/mL DF=10 410 387 418 411 4010 ng/mL DF=10 4230 4190 4280 4140 4010 n_mL DF=50 3610 3310 3330 ': !3350 Mean S.D. %CV %Theoretical %Bias n: 167 4.99 3.0 106.4 6.4 4 407 13.5 3.3 100.0 0.0 4 4210 59.4 1.4 105.0 5.0 4 :3400 141 4.1 84.8 -15.2 4 Page 112 Northwest Bioanalytical Study No. NWBS00-040 Report No. NWBR00-122 Table 117. PFHS Quality Control Samples Prepared in Serum Diluted with Buffer Run Date Run Number 23-Oct-2000 PFOSMD-39 154 ng/mL DF=10 157 163 161 166 404 ng/mL DF=10 421 389 416 419 4000 n_mL DF=10 4200 4210 4330 4300 4000 ng)mL DF=50 3490 3300 3410 3260 Mean S.D. %CV %Theoretical %Bias n 162 3.77 2.3 105.2 5.2 4 411 15.0 3.6 101.7 1.7 4 4260 64.8 1.5 106.5 6.5 4 3370 105 3.1 84.3 -15.8 4 Table 118. M556 Quality Control Samples Prepared in Serum Diluted with Buffer Run Date 23-Oct-2000 Run Number PFOSMD-39 151 ng/mL DF=10 156 157 145 148 401 ng/mL DF=10 421 385 403 417 4000 ng/mL DF=10 4480 4600 4710 4490 Mean S.D. %CV %Theoretical %Bias n 152 5.92 3.9 100.7 0.7 4 407 16.3 4.0 101.5 1.5 4 * >+_.20%deviation from theoretical 4570 108 2.4 114.3 14.3 4 4000 nffmL DF=50 *2980 *2740 *2780 *27 I0 o; : . 2800_ _ 122 4.4 70.0 -30.0 4 " =" - Page 113 Northwest Bioanalytical Study No. NWBS00-040 Report No. NWBR00-122 Table 119. M570 Quality Control Samples Prepared in Serum Diluted with Buffer Run Date 23-Oct-2000 Run Number . 157 ng/mL DF=10 PFOSMD-39 159 167 158 160 407 ng/mL DF=10 430 401 408 441 4010 ng/mL DF=10 4240 4390 4290 4330 4010 ng/mL DF=50 "2910 *2730 *2700 *2680 Mean S.D. %CV %Theoretical %Bias n 161 4.08 2.5 102.5 2.5 4 420 18.7 4.5 103.2 3.2 4 * >+_20%deviation from theoretical 4310 63.4 1.5 107.5 7.5 4 2760 105 3.8 68.8 -31.2 4 ,:'. Page 114 Northwest Bioanalytical Study No. NWBS00-040 Report No. NWBR00-122 Table 120. Summary of Calibration Curve Parameters for PFOS (Serum vs. Plasma) Quadratic weighted l/x 2. All concentrations are expressed as ppb. Run Date 27-Oct-2000 27-Oet-2000 25-Apr-2001 25-Apr-2001 Run Number PFOSMD-40 PFOSMD-46 Matrix Plasma Serum A 0.000000 -0.000003 B 0.028132 0.031081 C R-Squared LLOQ ULOQ 0.010586 0.084427 0.9931 0.9506 3.94 414 36.8 454 25 Plasma -0.000003 0.048192 0.045435 0.9841 26 Serum -0.000020 0.057680 -0.301033 0.9934 A, B, and C are coefficients used to define the calibration curve. 3.28 413 36.8 454 Table 121. Summary of Calibration Curve Parameters for PFOSA (Serum vs. Plasma) Quadratic weighted 1/x2. All concentrations are expressed as ppb. Run Date 25-Apt-2001 25-Apr-2001 Run Matrix A B C R-Squared Number 25 Plasma -0.000005 0.120662 0.028023 0.9887 26 Serum -0.000032 0.166445 0.051786 0.9873 A, B, and C are coefficients used to define the calibration curve. LLOQ 1.00 1.00 ULOQ 500 502 Table 122. Summary of Calibration Curve Parameters for PFOSAA (Serum vs. Plasma) Quadratic weighted l/x 2. All concentrations are expressed as ppb. Run Date 25-Apt-2001 25-Apt-2001 Run Number ,,1 25 Matrix A B C R-Squared LLOQ ULOQ Plasma 0.000009 0.033608 0.035470 0.9938 .:'1:.30 500 26 Serum 0.000007 0.044621 -0.040847 0.9947 4.20. 503 - A, B, and C are coefficients used to define the calibration curve. a Page 115 Northwest Bioanalytical Study No. NWBS00-040 Report No. NWBR00o122 Table 123. Summary of Calibration Curve Parameters for POAA (Serum vs. Plasma) Quadratic weighted l/x _. All concentrations are expressed as ppb. RunDate 27-Oct-2000 27-Oct-2000 25-Apr-2001 25-Apr-2001 Run Number PFOSMD-40 PFOSMD-46 Matrix Plasma Serum A 0.000002 0.000003 B C R-Squared LLOQ ULOQ 0.014346 0.016839 0.002192 0.003246 0.9918 0.9894 1.92 481 6.82 461 25 Plasma -0.000003 0.011215 0.005552 0.9929 26 Serum 0.000001 0.011414 0.000898 0.9931 A, B, and C are coefficients used to define the calibration curve. 1.44 481 6.82 461 Table 124. Summary of Calibration Curve Parameters for PFI:IS (Serum vs. Plasma) Quadratic weighted l/x 2. All concentrations are expressed as ppb. Run Date Run Number MatrLx A B C R-Squared LLOQ ULOQ 27-Oct-2000 27-Oct-2000 PFOSMD-40 Plasma -0.000001 0.025534 0.011247 0.9953 PFOSMD-46 Serum -0.000012 0.033256 0.018733 0.9890 A, B, and C are coefficients used to define the calibration curve. 1.36 523 3.85 431 Table 125. Summary of Calibration Curve Parameters for M556 (Serum vs. Plasma) Quadraticweighted I/x2. All concentrationsare expressedas ppb. RunDate 27-Oct-2000 27-Oct-2000 Run Matrix A B C JR-Squared LLOQ Number PFOSMD-40 Plasma 0.000002 0.016888 0.007835 0.9949 ": 2.50 PFOSMD-46 Serum 0.000005 0.014650 0.008960 0.9903 '3.8_3: A, B, and C are coefficients used to define the calibration curve. " ULOQ 500 501" Page 116 NorthwestBioanalytical Study No. NWBS00-040 Report No. NWBR00-122 Table 126. Summary of Calibration Curve Parameters for M570 (Serum vs. Plasma ) Quadratic weighted I/x2. All concentrations are expressed as ppb. RunDate 27-Oct-2000 27-Oct-2000 25-Apr-200I 25-Apr-2001 Run Number PFOSMD-40 PFOSMD-46 Matrix Plasma Serum A 0.000001 0.000003 B 0.034031 0.034699 C R-Squared 0.011681 0.028660 0.9910 0.9919 25 Plasma 0.000009 0.039312 0.045599 0.9964 26 Serum 0.000002 0.046817 0.021413 0.9931 A, B, and C are coefficients used to define the calibration curve. LLOQ 1.00 5.20 1.00 3.70 ULOQ 500 503 500 503 o; ; Page I 17 Northwest Bioanalytical StudyNo. NWBS00-040 Report No. NWBR00-122 Table 127. Baek-Caleulated Concentrations of Calibration Standards for PFOS (Serum vs. Plasma) Quadratic weighted 1/x2. All concentrations are expressed as ppb. Run Date 27-Oct-2000 Run Date 27-Oct-2000 Run Date 25-Apr-2001 Run Date 25-Apt-2001 Run Number PFOSMD-40 Run Number PFOSMD-46 Run Number 25 Run Number 26 Matrix Plasma Matrix Serum Matrix Plasma Matrix Serum 3.94 5.17 11.3 23.6 44.2 85.4 209 332 414 3.72 5.18 11.3 19.4 45.5 82.1 214 337 369 3.90 5.67 11.7 24.1 45.9 84.4 221 367 407 36.8 38.0 44.3 56.8 77.7 120 245 370 454 34.0 31.7 37.3 49.3 70.7 114 247 374 392 50.5 39.0 45.8 60.7 78.7 125 282 418 436 3.28 4.52 10.7 23.0 43.5 84.6 208 331 413 2.70 _3.90 9.65 21.4 42.2 80.3 190 324 392 4.15 4.63 11.3 25.0 47.6 90.3 217 336 441 36.8 38.0 44.3 56.8 77.7 120 245 370 454 38.3 35.5 44.2 54.7 71.1 124 234 363 420 37.4 39.0 46.1 56.1 75.9 128 256 400 459 = Page 118 NorthwestBioanalytical StudyNo. NWBS00-040 ReportNo. NWBR00-122 Table 128. Back-Calculated Concentrations of Calibration Standards for PFOSA (Serum vs. Plasma) QuadraticweightedI/x2. Allconcentrationsareexpressedas ppb. Run Date Run Number Matrix 1.00 2.50 10.0 25.0 50.0 100 250 400 500 25-Apr-2001 25 Plasma 0.935 2.52 7.89 23.0 45.4 102 223 387 477 *1.77 2.99 10.3 27.3 52.5 113 254 402 546 25-Apr-2001 26 Serum 0.951 1.96 9.62 25.6 48.1 109 240 403 422 1.18 2.21 10.7 24.3 52.4 111 279 450 491 * Sampledeactivatedas anoutlier o; *. Page 119 Northwest Bioanalytieal Study No. NWBS00-040 Report No. NWBR00-122 Table 129. Back-Calculated Concentrations of Calibration (Serum vs. Plasma) Standards for PFOSAA Quadratic weighted l/x 2. All concentrations are expressed as ppb. Run Date 25-Apr-2001 Run Number 25 Run Date 25-Apro2001 Run Number 26 Matrix Plasma Matrix Serum 1.30 2.80 10.3 25.3 50.3 100 250 400 500 1.28 2.65 8.27 24.7 50.7 102 253 399 480 *2.23 3.22 9.79 24.9 54.4 110 257 391 509 4.20 5.70 13.2 28.2 53.2 103 253 403 503 4.27 5.31 12.8 26.9 50.7 119 259 391 477 4.38 5.78 13.6 25.0 51.9 111 260 407 511 * Sample deactivated as an outlier _.. Page 120 NorthwestBioanalytical StudyNo. NWBS00-040 ReportNo. NWBR00-122 Table 130. Back-Calculated Concentrations of Calibration (Serum vs. Plasma) Standards for POAA Quadraticweighted1/x2. Allconcentrationsareexpressedasppb. Run Date 27-Oct-2000 Run Number PFOSMD-40 Matrix Plasma Run Date 27-Oct-2000 Run Number PFOSMD-46 Matrix Serum Run Date 25-Apr-2001 Run Number 25 Matrix Plasma Run Date 25-Apr-2001 Run Number 26 Matrix Serum 1.92 1.73 2.30 6.82 7.32 "18.3 1.44 1.35 "2.14 6.82 7.98 6.23 3.36 10.6 25.0 49.0 97.1 241 385 481 3.01 10.5 22.9 49.7 99.1 250 367 439 3.12 10.9 25.1 50.7 96.7 262 422 474 8.18 15.0 28.6 51.4 97.3 234 370 461 6.86 13.9 27.2 51.9 105 252 399 421 9.23 14.9 29.4 46.2 94.9 246 392 424 2.88 10.1 24.5 48.5 96.6 241 385 481 2.65 10.5 24.1 43.4 91.1 225 396 470 3.35 11.2 26.6 46.5 90.9 241 405 498 8.18 15.0 28.6 51.4 97.3 234 370 461 8.28 15.6 28.8 50.2 103 242 383 457 7.23 15.0 26.9 48.0 100 235 378 433 * Sampledeactivatedas an outlier Page 121 Northwest Bioanalytical S tudy No. NWBS00-040 Report No. NWBR00-122 Table 131. Back-Calculated Concentrations of Calibration (Serum vs. Plasma) Standards for PFHS Quadratic weighted 1/x2. All concentrations are expressed as ppb. Run Date 27-Oct-2000 Run Date 27-Oct-2000 Run Number PFOSMD-40 Run Number PFOSMD-46 Matrix Plasma Matrix Serum 1.36 2.92 10.7 26.5 52.6 104 262 418 523 1.52 "_ 2.72 10.6 24.2 54.6 107 259 466 489 1.28 2.78 10.4 28.4 54.1 102 270 425 491 3.85 5.13 11.5 24.4 45.7 88.9 217 345 431 4.08 4.36 11.2 21.8 47.5 89.1 235 371 363 "15.4 5.80 10.6 23.5 49.0 89.7 230 388 389 * Sample deactivated as an outlier o_ Page 122 Northwest Bioanalytical Study No. NWBS00-040 Report No. NWBR00-122 Table 132. Back-Calculated Cohcentrations of Calibration Standards (Serum vs. Plasma) Quadratic weighted I/x2. All concentrations are expressed as ppb. for M556 Run Date 27-Oct-2000 Run Date 27-Oct-2000 Run Number PFOSMD-40 Run Number PFOSMD-46 Matrix 2.50 10.0 25.0 50.0 100 250 400 500 Plasma 2.64 I0.7 23.4 52.9 I01 276 396 456 Matrix 2.35 9.95 23.3 48.3 94.1 260 440 481 =., 3.80 11.3 26.3 51.3 101 251 401 501 Serum 3.89 II.I 25.3 53.7 116 280 441 480 3.91 10.0 25.7 42.9 102 258 396 450 _ ; ; :. _ ,4"" Page 123 Northwest Bioanalytical Study No. NWBS00-040 Report No. NWBR00-122 Table 133. Back-Calculated Concentrations of Calibration (Serum vs. Plasma) Standards for M570 Quadratic weighted l/x 2. All concentrations are expressed as ppb. Run Date 27-Oct-2000 Run Date 27-Oct-2000 Run Date 25-Apr-2001 Run Date 25-Apr-2001 Run Number PFOSMD-40 Run Number PFOSMD-46 Run Number 25 I Run Number 26 Matrix Plasma Matrix Serum Matrix Plasma Matrix Serum 1.00 2.50 10.0 25.0 50.0 100 250 400 500 0.993 2.71 10.6 21.2 53.6 104 284 401 444 0.951 2.65 10.4 20.9 48.6 93.5 254 443 481 3.70 5.20 12.7 27.7 52.7 103 253 403 503 *5.54 4.92 11.8 27.4 51.0 116 277 432 464 "16.4 5.75 12.4 26.9 45.5 106 271 404 468 1.00 2.50 10.0 25.0 50.0 100 250 400 500 1.01 2.43 8.50 24.6 52.8 105 246 420 483 "1.90 2.66 9.29 25.4 51.9 108 247 387 504 3.70 5.20 12.7 27.7 52.7 103 253 403 503 4.07 4.60 12.5 27.4 50.3 118 262 395 478 3.94 4.60 13.3 26.0 49.7 109 264 405 502 * Sample deactivated as an outlier Page 124 Northwest Bioanalytical Study No. NWBS00-040 Report No. NWBR00-122 Table 134. PFOS Quality Controls (Serum vs. Plasma) All concentrations are expressed as ppb. Run Date Run Number Low QC Medium QC High QC _ (Plasma) (Plasma) (Plasma) 6.40 ppb 126 ppb 332 ppb 27-Oct-2000 PFOSMD-40 6.44 115 316 5.99 136 326 5.91 118 298 Mean SD %CV %Bias n Run Date Run Number 27-Oct-2000 PFOSMD-46 6.11 0.286 4.7 -4.5 3 Low QC (Serum) 39.3 ppb 35.0 35.6 35.5 35.8 123 11.4 9.3 -2.4 3 Medium QC (Serum) 161 ppb 164 151 149 152 313 14.2 4.5 -5.7 3 High QC (Serum) 370 ppb 336 "199 343 346 Mean SD %CV %Bias n 35.5 154 342 0.340 6.78 5.13 1.0 4.4 1.5 -9.7 .4.3 -7.6 4 4 3 * Sample deactivated due to a sample preparation error. ** > +_20%deviation from theoretical '; ": : =" " Page 125 Northwest Bioanalytical Study No. NWBS00-040 Report No. NWBR00-122 r Table 134. PFOS Quality Controls (Serum vs. Plasma) Continued All concentrations are expressed as ppb. Run Date 25 -Apr-2001 Run Number 25 ' Low QC (Plasma) 5.75 ppb 5.44 5.62 5.59 5.90 Medium QC (Plasma) - 126 ppb 143 139 136 132 High QC (Plasma) 331 ppb 333 325 347 333 Mean SD %CV %Bias 5.64 138 335 0.192 4.65 9.15 3.4 3.4 2.7 -I .9 9.5 1.2 ;' n 4 4 4 Run Date Run Number Low QC (Serum) 39.3 ppb Medium QC (Serum) 161 ppb High QC (Serum) 370 ppb 25-Apt-2001 26 41.3 174 377 45.4 182 388 45.8 172 408 45.6 169 381 Mean SD %CV %B ias n 44.5 174 2.16 5.56 4.9 3.2 13.2 8.1 4 4 * > +_20% deviation fi'om theoretical 389 13.8 ': ': 3.5 : 5.1 : 4 Page 126 Northwest Bioanalytical Study No. NWBS00-040 Report No. NWBR00-122 Table 135. PFOSA Quality Controls (Serum vs. Plasma) All concentrations are expressed as ppb. Run Date 25-Apr-2001 Run Number 25 Low QC (Plasma) 4.00 ppb 4.06 3.99 4.49 4.73 Medium QC (Plasma) 150 ppb 158 175 170 159 High QC (Plasma) 400 ppb 395 412 438 424 Mean SD %CV %Bias n Run Date Run Number 25-Apr-2001 26 4.32 0.353 8.2 8.0 4 Low QC (Serum) 4.00 ppb 3.52 4.75 4.55 **4.85 166 8.35 5.0 10.7 4 Medium QC (Serum) 150 ppb 153 163 155 151 417 18.2 4.4 4.3 4 High QC (Serum) 400 ppb 390 431 417 377 Mean SD %CV %Bias n 4.42 156 404 0.611 5.26 24.6 13.8 3.4 6.1 10.5 4.0 1.0 4 4 4 * Sample deactivated due to a sample preparation error. ** > +9_0%deviation from theoretical ":': " Page 127 Northwest Bioanalytical Study No. NWBS00-040 Report No. NWBR00-122 Table 136. PFOSAA Quality Controls (Serum vs. Plasma) All concentrations are expressed as ppb. Run Date Run Number 25-Apr-2001 25 Low QC (Plasma) 4.30 ppb 3.59 3.85 3.74 4.96 Medium QC (Plasma) 150 ppb ** 187 175 172 169 High QC (Plasma) 400 ppb 429 412 440 429 Mean SD %CV %Bias n Run Date Run Number 25-Apr-2001 26 4.04 0.626 15.5 -6.0 4 Low QC (Serum) 7.20 ppb 6.74 8.11 8.29 8.37 176 7.89 4.5 17.3 4 Medium QC (Serum) 153 ppb 150 162 159 157 428 11.6 2.7 7.0 4 High QC (Serum) 403 ppb 363 409 396 376 Mean SD %CV %Bias n 7.88 157 386 0.766 5.10 20.5 9.7 3.2 5.3 9.4 2.6 -4.2 4 4 4 Sample deactivated due to a sample preparation error. * > +_20%deviation from theoretical ":' " g : "" - Page 128 Northwest Bioanalytical Study No. NWBS00-040 Report No. NWBR00-122 Table 137. POAA Quality Controls (Serum vs. Plasma) All concentrations are expressed as ppb. Run Date Run Number 27-Oct-2000 PFOSMD-40 Low QC (Plasma) 4.81 ppb 4.57 4.31 4.52 Medium QC (Plasma) .145 ppb 146 167 147 High QC (Plasma) 385 ppb 373 419 393 Mean SD /oCV %Bias n Run Date Run Number 27-Oct-2000 PFOSMD-46 4.47 0.138 3.1 -7. I 3 Low QC (Serum) 9.54 ppb 9.42 9.17 8.53 9.62 153 395 11.8 23.1 7.7 5.8 5.5 2.6 3 3 Medium QC (Serum) 143 ppb 158 High QC (Serum) 370 ppb 373 146 I *253 148 375 150 388 Mean SD %CV %Bias n 9.19 151 379 0.474 5.26 8.14 5.2 3.5 2.1 -3.7 5.6 2.4 4 4 3 Sample deactivated due to a sample preparation error. * > +_20%deviation from theoretical ': "" :--= .... Page 129 Northwest Bioanalytical Study No. NWBS00-040 Report No. NWBR.00-122 Table 137. POAA Quality Controls (Serum vs. Plasma) Continued All concentrations are expressed as ppb. Run Date 25-Apt-2001 Run Number 25 Low QC (Plasma) 4.32 ppb 5.13 4.47 4.35 4.44 Medium QC (Plasma) . 145 ppb 143 145 145 137 High QC ('Plasma) 385 ppb 394 383 412 404 Mean SD %CV %Bias n Run Date Run Number 25-Apt-2001 26 4.60 0.359 7.8 6.5 4 Low QC (Serum) 9.54 ppb 10. I 11.4 *'13.2 9.82 143 3.79 2.7 -1.4 4 Medium QC (Serum) 143 ppb 157 152 149 152 398 12.6 3.2 3.4 4 High QC (Serum) 370 ppb 374 389 385 391 Mean SD %CV */,Bias i n 11.1 153 1.54 3.32 13.9 2.2 16.4 7.0 4 4 ** > +_20% deviation from theoretical 385 7.59 2.0 "" " 4.1 : " '- - 4 g Page 130 Northwest Bioanalytieal Study No. NWBS00-040 Report No. NWBR00-122 Table 138. PFHS Quality Controls (Serum vs. Plasma) All concentrations axe expressed as ppb. Run Date Run Number 27-Oct-2000 PFOSMD-40 Low QC (Plasma) 4.48 ppb 4.62 4.35 4.39 Medium QC -(Plasma) 156 ppb 157 173 151 High QC (Plasma) 417 ppb 397 407 384 Mean SD %CV %Bias n Run Date Run Number 27-Oct-2000 PFOSMD-46 4.45 0.146 3.3 -0.7 3 Low QC (Serum) 6.41 ppb 5.65 5.70 6.87 5.71 160 11.4 7.1 2.6 3 Medium QC (Serum) 132 ppb 144 136 129 135 396 11.5 2.9 -5.0 3 High QC (Serum) 345 ppb 343 "221 395 367 Mean SD %CV %Bias n 5.98 136 368 0.592 6.16 26.0 9.9 4.5 7.1 -6.7 3.0 6.7 4 4 3 Sample deactivated due to a sample preparation error. * > .+.20%deviation from theoretical ._' : : _- _ Page 131 Northwest Bioanalytical Study No. NWBS00-040 Report No. NWBR00-122 Table 139. M556 Quality Controls (Serum vs. Plasma) All concentrations are expressed as ppb. Run Date 27-Oct-2000 Run Number PFOSMD-40 Low QC (Plasma) 4.00 ppb 3.73 3.45 3.34 Medium QC (Plasma) 150 ppb 133 168 139 High QC (Plasma) 400 ppb 389 420 387 Mean SD %CV %Bias ' n 3.51 147 399 0.201 18.7 18.5 5.7 12.7 4.6 -12.3 -2.0 -0.3 3 3 3 Run Date Run Number 27-Oct-2000 PFOSMD-46 Low QC (Serum) 5.30 ppb 5.03 5.23 *'4.15 5.01 Medium QC (Serum) 151 ppb 166 152 152 155 High QC (Serum) 401 ppb 383 **243 371 388 Mean SD %CV */,Bias n 4.86 156 0.480 6.65 9.9 4.3 -8.3 3.3 4 4 ** > +_20%deviation from theoretical 381 8.74 2.3 """ -5.0 : 3 : '--" " Page 132 Northwest Bioanalytical Study No. NWBS00-040 Report No. NWBR00-122 Table 140. M570 Quality Controls (Serum vs. Plasma) All concentrations are expressed as ppb. Run Date 27-0ct-2000 Run Number PFOSMD-40 Low QC (Plasma) 4.00 ppb 3.95 3.54 3.38 Medium QC (Plasma) 150 ppb 133 161 136 High QC (Plasma) 400 ppb 379 403 360 Mean SD %CV %Bias n Run Date Run Number 27-Oct-2000 PFOSMD-46 3.62 0.294 8. I -9.5 3 Low QC (Serum) 6.70 ppb 6.36 6.35 **5.33 6.39 143 15.4 I 0.8 -4.7 3 Medium QC (Serum) 153 ppb 169 150 156 155 381 21.5 5.6 -4.8 3 High QC (Serum) 403 ppb 397 *227 367 400 Mean SD %CV %Bias n 6.11 158 388 0.519 8.10 18.2 8.5 5.1 4.7 -8.8 3.3 -3.7 4 4 3 * Sample deactivated due to a sample preparation error. ** > +90% deviation from theoretical ': " : .:" Page 133 Northwest Bioanalytical Study No. NWBS00-040 Report No. NWBR00-122 Table 140. M570 Quality Controls (Serum vs. Plasma) Continued All concentrations are expressed as ppb. Run Date 25-Apt-2001 Run Number 25 Low QC (Plasma) 4.00 ppb 3.24 3.49 3.58 4.05 Medium QC (Plasma) . 150 ppb 180 175 164 171 High QC (Plasma) 400 ppb 420 417 440 432 Mean SD %CV %Bias n Run Date Run Number 25-Apr-2001 26 3.59 0.339 9.4 -10.3 4 Low QC (Serum) 6.70 ppb 5.90 7.49 7.03 8.03 173 6.76 3.9 15.3 4 Medium QC (Serum) 153 ppb 166 167 167 161 427 10.7 2.5 6.8 4 High QC (Serum) 403 ppb 392 445 437 409 Mean SD %CV %Bias n 7. I 1 165 0.906 2.87 12.7 1.7 6.1 7.8 4 4 * > +__20%deviation from theoretical 421 24.6 5.8 ":" " 4.5 : 4 : '= " Page 134 Northwest Bioanalytical "_, Study No. NWBS00-040 Report No. NWBR00-122 i Table 141. Serum QC Results Based on Plasma Curves Sample ID PFOS (ppb) Low (analyzed 10/27100) 41.2 Low (analyzed 10/27/00) 41.8 Low (analyzed 10/27/00) 41.8 Low (analyzed 10/27/00) 42.1 Low (analyzed 4/25/01) 41.7 Low (analyzed 4/25/01) 46.5 Low (analyzed 4/25/01) 46.7 Low (analyzed 4/25/01) 47.0 mean * std. dev. * %CV * %dev * 43.6 2.61 6.0% 10.9% acceptance range * 31.4to47.2 95*,/, confidence range * 41.8 to 45.4 PFOSA (ppb)* 5.04 6.47 6.75 6.88 6.29 0.847 13.5% 57.1% 3.00to5.00 5.45 to 7.12 PFOSAA (ppb)* 6.68 8.5 8.72 8.83 8.18 1.01 12.4% 13.6% 5.40to9.00 7.48 to 8.88 POAA (ppb) PFHS ,(ppb)* 11.1 7.64 10.8 7.71 10.I 9.22 I 1,4 7.72 9.62 9.93 11.3 13.0 10.9 1.07 9.9% 14.3% 8.07 0.766 9.5% 25.9% 7.63to 11.4 5.13to7.69 10.2 to 11.7 7.32 to 8.82 M556 (ppb)* 4.44 4.61 3.67 4.42 4.29 0.419 9.8% -19.2% 4.24to6.36 3.87 to 4.7 M570 (ppb) 6.99 6.98 5.93 7.02 6.41 7.74 8.29 8.94 7.29 0.986 13.5% 8.8% 5.36to8.04 6.60 to 7.97 Meal Med Med Med Med Med Med Med (analyzed (analyzed (analyzed (analyzed (analyzed (analyzed (analyzed (analyzed 10/27/00) 10/27/00) 10/27/00) 10/27/00) 4/25/01) 4/25/01) 4/25/01) 4/25/01) mean * std. dev. * %CV * %dev * acceptance range* 95% confidence range* 182 168 165 168 185 189 191 199 181 12.5 6.9% 12.3% 129 to 193 172 to 190 205 207 210 220 211 6.66 3.2% 40.3% 113to188 204 to 217 192 200 203 207 201 6.4 3.2% 31.0% 115to191 196 to 205 186 172 174 177 160 164 164 170 171 8.37 4.9% 19.5% 114to172 165 to 177 180 170 162 169 170 7.41 4.4% 29.0% 106 to 158 163 to 178 149 136 137 139 140 5.97 4.3% -7.1% 121 to 181 134 to 146 175 155 161 160 184 190 191 191 176 15.3 8.7% 15.0% 122 to 184 165 to 186 High (analyzed 10/27/00) High (analyzed 10/27/00) High (analyzed 10/27/00) High (analyzed 4/25/01) High (analyzed 4/25/01) High (analyzed 4/25/01) High (analyzed 4/25/01) mean * std. dev. * %CV * %dev * 364 370 374 396 399 406 424 390 21.8 5.6% 5.5% 493 509 543 560 526 30.7 5.8% 31.6% 451 467 490 506 479 24.3 5.1% 18.7% 441 443 459 453 470 476 478 460 15.2 3.3% 24.3% 400 452 424 425 26.0 6.1% 23.3% 361 349 366 -. ; 359 8.74 2.4% -10.6% 416 384 420 433 450 ,,-. 478 486 438 36.0 8.2% 8.7% acceptance range* 95%eonfideneerange* 296 to 444 374 to 407 300 to 500 492 to 561 302 to 504 460 to 497 296 to 444 449 to 471 276 to 414 396 to 455 321to481 349 to 369 322 to 484 411to465 Due to rounding, calculations based on displayed values may differ from actual. NOTE: Only data from acceptable runs included in the Table (see report text). (Data obtained on 10/27/00 from run PFOSMD-40. Data obtained on 4/25/01 from run S00040-25.) Page 135