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Northwest Bioanalytical Study No. NWBSOO-040 Report No. NWBR00-108 Quantitative Determination of p r o s , PFOSA, PFOSAA, POAA, PFHS, M556 and M570 in Human Serum by LC/MS/MS Assay Revalidation Report Northwest Bioanalytical (NWB) A Division of NWT Ine. 1121 East 3900 South Salt Lake City, UT 84124 PREPARED FOR: 3M Environmental Technology and Services (3M) 935 Bush Avenue St. Paul, MN 55133 AUTHOR: ' ^ doTULAJ 0 ;_______________ DATE: t f a ' l l o f Connie O. Sakashita, B.S., NWB Project Manager APPROVED FOR RELEASE BY: -- _______________ _______ DATE: Patrick Bennett, M.S., M.B.A., NWB Laboratory Director //* -> /< > r Paee 1 Northwest Bioanalytical QUALITY ASSURANCE STATEMENT Study No. NWBS0O-040 Report No. NWBR00-108 LABORATORY: Northwest Bioanalytical (NWB) A Division of NWT, Ine. 1121 East 3900 South Salt Lake City, UT 84124 . SPONSOR: 3M Environmental Technology and Services (3M) 935 Bush Avenue St. Paul, MN 55133 COMPOUND(S): PFOS, PFOSA, PFOSAA, POAA, PFHS, M556 and M570 NWB STUDY NUMBER: NWBS00-040 SPONSOR STUDY NUMBER: NA, Assay Validation NWB STUDY TITLE: Quantitative Determination of PFOS, PFOSA, PFOSAA, POAA, PFHS, M556 and M570 in Human Serum by LC/MS/MS, Assay Validation The assay validation study described in this report is not included within the definition of a GLP regulated nonclinical study. However, Northwest Bioanalytical conducts all studies within the guidelines of the U.S. FDA Good Laboratory Practice Regulations for 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 0 7 -1 8 Sep 2000 24 Jan 2001 Phase of Study Analytical Plan Assay Validation Report Draft/Raw Data Final Report Date Inspection Report Issued To NWB Project Manager *NWB Management 15 May 2000 31 May 2000 18 May 2000 31 May 2000 20 Sep 2000 . 29 Sep 2000: . 24 Jan 2001 31 Jan 2001 *Reports to NWB Management are issued monthly. As can reasonably be established, the methods and procedures described and the results orated into this final report accurately reflect the raw data. Shaundel Percey, B.S., NLTrrASCP'' Quality Assurance CompkanceiSpecialist Page 2 Northwest Bioanalytical Study No. NWBSOO-040 Report No. NWBROO-108 COMPLIANCE STATEMENT The method validation study described in this report is not included within the definition of a GLP nonclinical regulated study. However, to the best of our knowledge, this study was conducted in accordance with the guidelines of the U.S. FDA Good Laboratory Practice Regulations for Nonclinical Laboratory Studies (Title 21 CFR Part 58) and according to the methods and procedures described within this report. In addition, the study 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.) 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. (JfTttou Connie O. Sakashita, B.S. NWB Project Manager , _______________________ DATE: ' 1/6-c/ / Patrick Bennett, M.S., M.B.A. NWB Laboratory Director -- ____________ __________ DATE: /A v A , Page 3 Northwest Bioanalytical Study No. NWBSOO-040 Report No. NWBR00-108 TABLE OF CONTENTS SIGNATURE PAGE............................ 1 QUALITY ASSURANCE STATEMENT........................... 2 COMPLIANCE STATEMENT......................................................................................................... 3 TABLE OF CONTENTS..................................................................... 4 LIST OF TABLES................................................................................ 5 LIST OF FIGURES............................................................................................... 7 1. INTRODUCTION........................................................................................ 8 2. VALIDATION SUMMARY..................................................................................................... 10 2.1. Persistent Levels of Analytes in Matrix..............................................................................10 2.2. Range of Quantitation............................................ 11 2.3. Precision and Accuracy...................................................................................................... 12 2.4. Extraction Efficiency.......................................................................................................... 15 2.5. Stability Evaluation.............................................................................................................17 3. DATA MANAGEMENT............................................ 17 4. COMMENTS AND CONCLUSIONS...................................................................................... 17 4.1. Proposed Sample Analysis Acceptance Criteria.................................................................18 5. REFERENCES...............................................................................................:................... .......19 6. DATA RETENTION......................................................................................... ...................... 19 7. ANALYTICAL METHOD..................................... .:6T - 7.1. Reference Materials and Matrices.................................................................................... 69 7.2. Chemicals and Equipment.................................................................................................. 70 7.3. Reagents, Calibration Standard and Quality Control (QC) Solutions..............................72 7.4. Preparation of Validation Quality Control Samples..........................................................77 7.5. Preparation of PFOSA and PFOSAA Specific Validation Quality Control Samples 79 7.6. Recommended Calibration Standard and Quality Control Preparation for Analysis..... 80 7.7. Preparation of Calibration Standards........................................................... 80 Page 4 Northwest Bioanalytical Study No. NWBSOO-040 Report No. NWBROO-108 7.8. Sample Preparation..............................................................................................................81 7.9. LC/MS/MS Conditions....................................................................................................... 83 7.10. Quantitation...................................................................................................................... 84 APPENDIX A ................................................................................................................................... 96 APPENDIX B ..... ............................................................. !............................................................ 100 LIST OF TABLES Table l . Summary of Calibration Curve Parameters for PFOS....................................................20 Table 2. Summary of Calibration Curve Parameters for PFOSA.................................................20 Table 3. Summary of Calibration Curve Parameters for PFOSAA............................................. 21 Table 4. Summary of Calibration Curve Parameters for POAA...................................................21 Table 5. Summary of Calibration Curve Parameters for PFHS................................................... 22 Table 6. Summary of Calibration Curve Parameters for M556....................................................22 Table 7. Summary o f Calibration Curve Parameters for M570....................................................23 Table 8. Back-Calculated Concentrations of Calibration Standardsfor PFO S............................ 24 Table 9. Back-Calculated Concentrations of Calibration Standardsfor PFOSA.......................... 24 Table 10. Back-Calculated Concentrations of Calibration Standards for PFOSAA....................25 Table 11. Back-Calculated Concentrations of Calibration Standards for POAA........................ 25 Table 12. Back-Calculated Concentrations of Calibration Standards for PFHS......................... 26 Table 13. Back-Calculated Concentrations of Calibration Standards for M556 .......... .:2,6- Table 14. Back-Calculated Concentrations of Calibration Standards for M570 .......................27 Table 15. Intra-Assay Precisionand Accuracy for PFOS Quality Control Samples.....................28 Table 16. Intra-Assay Precisionand Accuracy for PFOSA Quality Control Samples............. .....30 Table 17. Intra-Assay PrecisionandAccuracy for PFOSAA Quality Control Samples................32 Table 18. Intra-Assay PrecisionandAccuracy for POAA Quality Control Samples....................34 Table 19. Intra-Assay PrecisionandAccuracy for PFHS Quality Control Samples.....................36 Page 5 Northwest Bioanalytical Study No. NWBS00-040 Report No. NWBR00-108 Table 20. Intra-Assay Precision and Accuracy for M556 Quality Control Samples....................38 Table 21. Intra-Assay Precision and Accuracy for M570 Quality Control Samples....................40 Table 22. Inter-Assay Precision for PFOS Quality Control Samples........................................... 42 Table 23. Inter-Assay Precision for PFOSA Quality Control Samples........................................ 43 t Table 24. Inter-Assay Precision for PFOSAA Quality Control Samples...................................... 44 Table 25. Inter-Assay Precision for POAA Quality Control Samples.......................................... 45 Table 26. Inter-Assay Precision for PFHS Quality Control Samples........................................... 46 Table 27. Inter-Assay Precision for M556 Quality Control Samples.................. .......47 Table 28. Inter-Assay Precision for M570 Quality Control Samples........................................... 48 Table 29. Intra-Assay Precision and Accuracy for PFOSA Specific Quality Controls................49 Table 30. Intra-Assay Precision and Accuracy for PFOSAA Specific Quality Controls..............50 Table 31. PFOS Dilution Quality Control Samples...................... 51 Table 32. PFOSA Dilution Quality Control Samples........................................................ 52 Table 33. PFOSAA Dilution Quality Control Samples ................................................................. 53 Table 34. POAA Dilution Quality Control Samples......................................................................54 Table 35. PFHS Dilution Quality Control Samples.......................................................................55 Table 36. M556 Dilution Quality Control Samples.......................................................... 56 Table 37. M570 Dilution Quality Control Samples...................... i...............................................57 Table 38. PFOSA Specific Dilution Quality Controls.................................................................. 58 Table 39. PFOSAA Specific Dilution Quality Controls............................................................... 59 Table 40. PFOS Extraction Efficiency................ ..........................................................................60 Table 41. PFOSA Extraction Efficiency.................... ...................................................................61 Table 42. PFOSAA Extraction Efficiency.....................................................................................62 Table 43. POAA Extraction Efficiency.........................................................................................63 Page 6 Northwest Bioanalytical Study No. NWBS00-040 Report No. NWBR00-108 Table 44. PFHS Extraction Efficiency.................................. 64 Table 45. M556 Extraction Efficiency..........................................................................................65 Table 46. M570 Extraction Efficiency..........................................................................................66 LIST OF FIGURES Figure 1. Representative Calibration Curve for PFO S................................................................. 85 Figure 2. Representative Calibration Curve for PFOSA............................................................... 85 Figure 3. Representative Calibration Curve for PFOSAA.............................................................86 Figure 4. Representative Calibration Curve for POAA................................-.................................86 Figure 5. Representative Calibration Curve for PFHS................................................................. 87 Figure 6. Representative Calibration Curv for M 556................................................................. 87 Figure 7. Representative Calibration Curve for M 570................................................................. 88 Figure 8. Standard (57.1 ng/mL) Chromatogram for PFO S......................................................... 89 Figure 9. Standard (10.1 ng/mL) Chromatogram for PFOSA........................................ 90 Figure 10. Standard (15.0 ng/mL) Chromatogram for PFOSAA................................... 91 Figure 11. Standard (14.8 ng/mL) Chromatogram for POAA.......................................................92 Figure 12. Standard (12.2 ng/mL) Chromatogram for PFHS........................... 93 Figure 13. Standard (11.8 ng/mL) Chromatogram for M 556...... 94 Figure 14. Standard (14.6 ng/mL) Chromatogram for M 570......... ........................ :....................95 f-f -- P)h l if P Fosa / PF Of4/1 C- T ' "" " KJ * v w ^t s o-, a ( SL/i, - / Page 7 Northwest Bioanalytical Study No. NWBSOO-040 Report No. NWBROO-108 Quantitative Determination of PFOS, PFOSA, PFOSAA, POAA, PFHS, M556 and M570 in Human Serum by LC/MS/MS Assay Revalidation Report 1. INTRODUCTION Northwest Bioanalytical (NWB) was contracted by 3M Environmental Technology and Services to develop and validate a liquid chromatography/tandem mass spectrometry method for the measurement of perfluorooctanesulfonate (PFOS), perfluorooctanesulfonamide (PFOSA), N-ethyl perfluorooctanesulfonamidoacetate (PFOSAA), perfluorooctanoate (POAA), perfluorohexanesulfonate (PFHS), perfluorooctanesulfonamidoacetate (M556) and N-methyl perfluorooctanesulfonamidoacetate (M570) in human serum. The reference material purity for PFOS, PFOSA, PFHS and POAA was not available prior to the conduct of this study. Therefore, all concentrations included in the report for these analytes are based upon an assumed purity of 100%. After the validation was completed, 3M contracted with Centre Analytical Laboratories, Inc. in State College, Pennsylvania to determine the absolute concentration of PFOS, POAA and PFHS in the stock solutions used to prepare the analytical standards and controls used for this validation and subsequent analyses. Based on the results obtained, the concentrations included in this report should be corrected according to -the following table: ' ; Analyte PFOS POAA PFHS Correction Factor 0.836 0.909 0.855 Page 8 Northwest Bioanalytical Study No. NWBSOO-O40 Report No. NWBROO-108 This report summarizes the accuracy, precision and the Northern Chinese plasma extraction efficiency results from the validation of the method for the quantitation of PFOS, PFOSA, PFOSAA, POAA, PFHS, M556 and M570 in human serum for 3M Environmental Technology and Services. Stability results will be reported in a separate addendum report. Kris Hansen at 3M Environmental Technology and Services served as the Study Monitor. The following is a list of NWB supervisory personnel involved in the completion of this work: Connie O. Sakashita, B.S. (NWB Project Manager); Brad I. Coopersmith, Ph.D. (NWB Senior Scientist); Licong Jiang, Ph.D. (NWB Senior Scientist); Patrick Bennett, M.S., M.B.A. (NWB Laboratory Director); Rodger Foltz, Ph.D. (NWB Technical Director). 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: April 13, 2000 Date Analyses Completed: July 16, 2000 The method validation study described in this report is not included within the definition of a GLP regulated nonclinical study. However, Northwest Bioanalytical conducts all studies within the guidelines of the U.S. FDA Good Laboratory Practice Regulations for 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. ' :: " Principles of the Method . The newly developed method is a modification of a previous method developed under study NWBS98-082 and reported in NWB report NWBR99-005 [5.1]. The new method was developed to provide improved accuracy, precision and ruggedness using less sample volume, and to add M556 and M570 to the method. Page 9 Northwest Bioanalytical Study No. NWBSOO-040 Report No. NWBROO-108 The analytical method consisted of a liquid:liquid extraction procedure followed by evaporation and reconstitution of the extract residue with 30:70 20 mM ammonium acetate in water: 20 mM ammonium acetate in methanol (v/v). The samples were analyzed by liquid chromatography/tandem mass spectrometry using a PE Sciex API 3000. The instrument was operated in the multiple reaction monitoring (MRM) mode under optimized conditions for PFOS, PFOSA, PFOSAA, POAA, PFHS, M556 and M570 detection. 2. VALIDATION SUMMARY Three separate analytical runs were used in the determination of linearity, precision, and accuracy. An additional run was used to determine intra-assay precision and accuracy for PFOSA and PFOSAA. The extraction efficiencies for all analytes and the internal standard lH,lH,2H,2H-perfluorooctane sulfonic acid (THPFOS) were also determined. 2.1. Persistent Levels of Analytes in Matrix Because PFOS, PFOSAA, POAA, PFHS, M556 and M570 demonstrate measurable levels in control human serum, a procedure to account for these persistent levels is needed for this method validation. The sponsor provided NWB with blank matrix (Northern Chinese human plasma). This matrix was tested and demonstrated no quantifiable concentrations for any of the analytes. In order to determine the persistent levels of these analytes in the lot of human serum used for the validation, a partial calibration curve was prepared with the blank Northern Chinese human plasma and extracted along with blank human serum samples. The human serum samples were quantitated against the Northern Chinese human plasma calibration curve to determine the persistent levels of the analytes in the lot of human serum being tested. Once the persistent level of each analyte was determined, the target concentrations for each calibration standard and quality control sample were adjusted to account for the persistent amount of analyte. For example, if the target concentration at the lower Page 10 Northwest Bioanalytical Study No. NWBS00-040 Report No. NWBROO-108 limit of quantitation was 1.00 ng/mL and the persistent level of the analyte in human serum was 2.00 ng/mL, the target concentration of the LLOQ was adjusted to 3.00 ng/mL. Appendix B outlines the procedure used to calculate persistent levels of analytes in diluted human serum samples. This procedure to account for persistent levels of the analytes in matrix must be performed whenever a new lot of human serum is used during the course of a study. Range of Quantitation Each analytical run included calibration standards in duplicate at nine different concentrations (eight for M556), a minimum of six quality control samples (QCs) (three levels in replicates of two), two serum blanks and two 0-ng/mL QCs (serum blank with internal standard). The target calibrator concentrations were approximately 1.00,2.50, 10.0, 25.0, 50.0, 100,250, 400 and 500 ng/mL for all analytes. Each analyte has a different final curve range based upon the persistent levels of the analyte in the human serum used. For the validation study, the calibrator concentrations were as follows: PFOS PFOSA PFOSAA POAA PFHS M556 M570 48.1, 49.6, 57.1, 72.1, 97.1, 147, 297, 447, 547 1.00, 2.51, 10.1, 25.1, 50.3, 100, 251, 402, 502 6.00, 7.50, 15.0, 30.0, 55.0, 105, 255, 405, 505 5.76, 7.26, 14.8, 29.8, 54.8, 105, 255,405,.505 3.15, 4.65, 12.2, 27.2, 52.2, 102, 252, 402, 502 4.30, 11.8, 26.8, 51.8, 102, 252, 402, 502 5.60, 7.10, 14.6, 29.6, 54.6, 105, 255, 405, 505 Page 11 Northwest Bioanalytical Study No. NWBS00-040 Report No. NWBR00-108 Range Mean Coefficient of Determination PFOS 48.1 to 547 ng/mL 0.9925 (Table 1) PFOSA 1.00 to 502 ng/mL 0.9899 (Table 2) PFOSAA 6.00 to 505 ng/mL 0.9968 (Table 3) POAA 5.76 to 505 ng/mL 0.9922 (Table 4) Range Mean Coefficient of Determination PFHS 3.15 to 502 ng/mL 0.9954 (Table 5) M556 4.30 to 502 ng/mL 0.9935 (Table 6) M570 5.60 to 505 ng/mL .9967 (Table 7) The results for the individual calibration standards can be found in Tables 8 -1 4 . 2.3. Precision and Accuracy The target concentrations for the quality control samples were 4.00, 150, 400 and 4000 ng/mL for all analytes. All quality control target concentrations were corrected for the persistent levels of the analytes in the human serum used for preparation. For the validation study, the quality control concentrations were as follows: PFOS PFOSA PFOSAA POAA PFHS M556 M370 Low (ng/mL) 51.1 4.00 9.00 8.74 6.15 5.80 8.60 Medium (ng/mL) 197 150 155 154 152 152 155 High (ng/mL) 446 400 405 403 402 402 405 Dilution (ng/mL) 4460 . 4000 4050 4030 4020 4020 4050 Page 12 Northwest Bioanalytical Study No. NWBSOO-040 Report No. NWBROO-108 2.3.1. Precision and Accuracy for PFOS. PFQSA. PFQSAA. POAA. PFHS, M556 and M570 Quality Controls The precision and accuracy of the LC/MS/MS method for the quantitation of PFOS, PFOSA, PFOSAA, POAA, PFHS, M556 and M570 in human serum were determined by analyzing three levels of quality controls in replicates of Five on three separate days. The intra-assay precision (%CV) for PFOS, POAA, PFHS, M556 and M570 were less than or equal to 9.5% for each undiluted QC concentration. The mean accuracy (%Theoretical) for all levels of undiluted quality controls ranged from 84.8% to 111.2% (Tables 15 and 18-21). The intra-assay precision (%CV) for PFOSA and PFOSAA were less than or equal to 19.8% for each undiluted QC concentration. The mean accuracy (%Theoretical) for all levels of undiluted QCs ranged from 68.7% to 91.7% (Tables 16 - 17). The inter-assay precision (%CV) for PFOS, POAA, PFHS, M556 and M570 were less than or equal to 9.7% for each undiluted QC concentration. The mean accuracy (%Theoretical) for all levels of undiluted QCs ranged from 87.4% to 108.5% (Tables 22 and 25 - 28). The inter-assay precision (%CV) for PFOSA and PFOSAA were less than or equal to 14.0% for each undiluted QC concentration. The mean accuracy (%Theoretical) for all levels of undiluted QCs ranged from 73.1% to 88.9% (Tables 23 - 24). For diluted QCs, the inter-assay precision (%CV) for PFOS, POAA,:PFHS, M556 and M570 were less than or equal to 6.6%. The mean accuracy (%TheoreticaI) for the diluted QCs ranged from 92.8% to 113.9% (Tables 31. and 34 - 37). The inter-assay precision (%CV) for PFOSA and PFOSAA were less than or equal to 9.7% for each diluted QC. The mean accuracy (%Theoretical) for the diluted QCs ranged from 79.0% to 81.3% (Tables 32 - j j ). Page 13 Northwest Bioanalytical Study No. NWBSOO-04O Report No. NWBROO-108 The slightly high negative bias for the calculated concentrations of the PFOSA and PFOSAA QC samples was a result of the QC preparation procedure. A stock solution containing all the analytes was prepared and evaporated to dryness in order to obtain a concentration high enough for the QC concentration range. This methodology is commonly used when stock solution concentrations are too dilute to obtain the targeted concentration or to reduce the organic content of spiked serum samples. For this preparation, evaporation was required to obtain appropriate concentrations. However, because of the high volatility of PFOSA and PFOSAA, approximately 25% of these analytes evaporated from the solution. The quality control samples from this preparation were biased approximately 25% lower (Tables 23 - 25) than those prepared from a solution that was not evaporated (Tables A.l. - A.2.). 2.3.2. Precision and Accuracy for PFOSA and PFOSAA Specific Quality Controls Because of the bias demonstrated for PFOSA and PFOSAA calculated concentrations from the original QCs prepared as described in section 2.3.1, a new set of QCs containing'only PFOSA and PFOSAA were prepared using concentrated solutions that did not require evaporation. For the PFOSA and PFOSA specific QCs, the intra-assay precision (%CV) for PFOSA and PFOSAA was less than or equal to 23.8% for each undiluted QC concentration. The mean accuracy (%Theoretical) for all levels of undiluted QCs ranged from 72.9% to 107.7% (Tables 29 --30). - The PFOSAA LLOQ calibration standards for the intra-assay Run 17 demonstrated unacceptable accuracies resulting in both replicates being rejected and a raised LLOQ of 7.50 ng/mL for the run. The low QCs for PFOSAA also demonstrated variability greater than 20%. This resulted in a negative bias (72.9% of theoretical) for the low PFOSAA QCs. The mid-level and high-level QCs demonstrated acceptable precision and accuracy. Because this bias only appears at the Low QC level and is a result of within-run Page 14 Northwest Bioanalytical Study No. NWBS0O-040 Report No. NWBROO-108 variability, study sample analysis acceptance criteria will be maintained at 20% precision and accuracy for QCs. For the PFOSA and PFOSAA specific QCs containing PFOSA or PFOSAA concentrations above the ULOQ, the intra-assay precision (%CV) for PFOSA and PFOSAA was 19.8% and 20.9%, respectively. The mean accuracy (%Theoretical) for diluted PFOSA and PFOSAA specific QCs were 78.3% and 78.0%, respectively (Tables 38 - 39). The deviation of the calculated concentrations from theoretical concentrations for these QCs diluted with control matrix were both greater than 20%. This indicates that samples with experimentally calculated PFOSA or PFOSAA concentrations above the ULOQ should not be diluted with control matrix. However, the levels of PFOSA and PFOSAA in study samples are expected to be within the range of the calibration curve. The inter-assay precision and accuracy of the PFOSA and PFOSAA specific QCs was demonstrated with the analytical QC performance during sample analysis study NWBS00-062 (medical surveillance). The results can be found"' in Appendix A, Tables A .l. and A.2. These tables only contain analyses for reported PFOSA and PFOSAA sample data, and do not include runs that did . not meet the acceptance criteria for PFOSA or PFOSAA. i' For the PFOSA and PFOSA specific QCs, the inter-assay precision (%CV) was less than or equal to 11.4% for each undiluted QC concentration. The mean accuracy (%Theoretical) for all levels of undiluted QCs rangedTrom 98.5% to 107.7%. : 2.4. Extraction Efficiency The extraction efficiencies of PFOS, PFOSA, PFOSAA, POAA, PFHS, M556 and M570 were determined using Northern Chinese human plasma by comparing the area ratios obtained for the following three cases: Page 15 Northwest Bioanalytical Study No. NWBS00-040 Report No. NWBR00-108 1. Both the analyte and internal standard added following the extraction (post extract). 2. The analyte added to plasma prior to extraction and the internal standard added following extraction (pre-extract analyte). 3. The internal standard added to plasma 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, medium and high concentrations to determine that there was no concentration bias. The mean extraction efficiencies were: PFOS (42.1%), PFOSA (65.3%), PFOSAA (73.4%), POAA (18.0%), PFHS (19.7%), M556 (43.9%), M570 (66.9%). The overall mean extraction efficiency for the internal standard THPFOS was 7.86% (Tables 40 - 46). ' The recovery for many of the analytes and for the internal standard are very low. This low recovery is a result of the following factors: a neutral pH was required to minimize any matrix effects that were observed from the previous extraction method which used a basic pH and, the wide range of polarities for the analytes prevented the use of acidic pH during the extraction and prevented the use of an effective SPE extraction for all o f the analytes. While the recovery is low, the intra and inter-assay precision and accuracy values demonstrated good reproducibility. The low recovery should not affect the assay performance. The Chinese plasma containing very low persistent levels of most analytes was used to estimate the extraction recovery of the analytes from matrix. This plasma.was used.to provide the most accurate results possible at the lower concentrations. The recovery from general population control serum will be evaluated and reported in an addendum report. Page 16 Northwest Bioanalytical 2.5. Stability Evaluation Study No. NWBSOO-040 Report No. NWBR00-108 The stability of PFOS, PFOSA, PFOSAA, POAA, PFHS, M556 and M570 maintained under various storage conditions will be documented in a separate NWB report. 3. DATA MANAGEMENT PFOS, PFOSA, PFOSAA, POAA, PFHS, M556, M570 chromatographic peaks are integrated using PE Sciex MacQuan software (version 1.6) with a smooth factor of 1. Quantitation is based upon quadratic regression analysis of calibration curves (weighted 1/x2) using the area ratio vs. concentration calculated by the Watson DMLIMS software (version 6.1.1.04). 4. COMMENTS AND CONCLUSIONS Per agreement with the Sponsor, the regressions were not recalculated based upon the updated purity information for PFOS, POAA and PFHS. Some differences might occur duelo 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 validation results. Only THPFOS was used as an internal standard because of concerns over the specificity of one of the original internal standards, N-Et-FOSE-OH, which gave an acetate ion as a product. . The volatility of PFOSA and PFOSAA is an important aspect to be cautious of during the evaporation step of the assay. An evaporation step was used for the initial preparation of QC samples to obtain an adequate concentration of the analytes. This set demonstrated acceptable precision, but slightly high negative bias for PFOSA and PFOSAA. A separate set of QCs containing only PFOSA and PFOSAA were prepared without using an evaporation step. The second set (PFOSA and PFOSAA specific QCs) demonstrated Page 17 Northwest Bioanalytical Study No. NWBSOO-040 Report No. NWBROO-108 both acceptable precision and accuracy. As a result, two sets of intra-assay and inter assay precision and accuracy are shown in the report. The method described in this report has been validated for the determination of PFOS, PFOSA, PFOSAA, POAA, PFHS, M556 and M570 in human serum. However, study samples with experimentally calculated PFOSA or PFOSAA concentrations above the ULOQ should not be diluted with control matrix. Any known circumstances that may have affected the quality or integrity of the data are discussed in this report. 4.1. Proposed Sample Analysis Acceptance Criteria Calibration Curve ' Each run will include in duplicate calibration standards at six or more concentrations covering the lower to upper limit of quantitation. For all analytes except PFOSA, at least thxee-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 o f Quantitation The back-calculated concentrations of at least one of the duplicate lowest points in the' calibration curve must be within 25% o f 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 same test and the LLOQ raised accordingly. Page 18 Northwest Bioanalytical Quality Control Samples Study No. NWBSOO-040 Report No. NWBROO-108 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 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 o f the undiluted analytical QC acceptance. 5. REFERENCES [5.1] D. Voilmer, "Quantitative Determination of PFOS, PFOSA, PFOSAA, NMeFOSE-OH, N-EtFOSE-OH, POAA and PFHS in Human Serum by LC/MS/MS," NWB study NWBS98-082, NWB report NWBR99-005, May 13, 1999. 6. DATA RETENTION The raw data and final report for this study will be stored in the NWB Archives, 1121 East 3900 South, Salt Lake City, UT 84124 per regulations and contract agreement. 3M Environmental Technology and Services will be notified concerning final disposition of records at completion of contract obligations. Page 19 Northwest Bioanalytical Study No. NWBS00-040 Report No. NWBROO-108 Table 1. Summary of Calibration Curve Parameters for PFOS Quadratic weighted l/x:. All concentrations are expressed as ng'mL. Run Date 29-Jun-2000 30-Jun-2000 02-Jul-2000 Run Number 13 15 16 A B C R-Squared LLOQ ULOQ -0.000009 0.035473 0.368276 0.9877 48.1 547 -0.000001 0.018768 0.148749 0.9949 48.1 547 -0.000005 0.028829 0.235310 0.9948 48.1 547 Mean S.D. %CV n -0.000005 0.027690 0.250778 0.000004 0.008411 0.110578 -80.0 30.4 44.1 3 33 0.9925 0.0041 0.4 3 A, B and C are coefficients used to define the quadratic curve. Table 2. Summary of Calibration Curve Parameters for PFOSA Quadratic weighted 1/x2. All concentrations are expressed as ng/mL. Run Date 29-Jun-2000 30-Jun-2000 02-Jul-2000 Run Number 13 15 16 Mean S.D. %CV n A B C R-Squared LLOQ ULOQ -0.000030 0.093690 0.023501 -0.000012 0.051196 0.004685 -0.000020 0.088242 0.025293 0.9761 0.9975 0.9960 -0.000021 0.000009 -42.9 3 0.077709 0.023122 29.8 3 0.017826 0.011416 64.0 3 0.9899 0.0119 1.2 3 1.00 1.00 1.00 '' : 502 502 502 A, B and C are coefficients used to define the quadratic curve. Page 20 Northwest Bioanalytical Study No. NWBS00-040 Report No. NWBR00-108 Table 3. Summary of Calibration Curve Parameters for PFOSAA Quadratic weighted 1/x2. All concentrations are expressed as ng/mL. Run Date 29-Jun-2000 30-Jun-2000 02-Jul-2000 Run Number 13 15 16 A B C R-Squared LLOQ ULOQ -0.000003 0.023347 -0:035788 0.9969 6.00 505 0.000000 0.013890 -0.020748 0.9971 6.00 505 0.000001 0.023426 -0.033118 0.9964 6.00 505 Mean S.D. %CV n -0.000001 0.020221 -0.029885 0.000002 0.005483 0.008024 -200.0 27.1 -26.8 333 0.9968 0.0004 0.0 3 A, B and C are coefficients used to define the quadratic curve. Table 4. Sum m ary of Calibration Curve Parameters for POAA Quadratic weighted 1/x2. All concentrations are expressed as ng/mL. Run Date 29-Jun-2000 30-Jun-2000 02-Jul-2000 Run Number 13 15 16 Mean S.D. %CV n A B C R-Squared LLOQ ULOQ 0.000002 0.018721 0.026984 0.9847 5.76 505 0.000000 0.012512 0.009407 0.9958 5.76 505 -0.000002 0.014490 0.015595 0.9962 5.76 505 0.000000 0.015241 0.017329 0.9922 : . 0.000002 0.003172 0.008916 0.0065 - 20.8 51.5 0.7 3 33 3 A, B and C are coefficients used to define the quadratic curve. Page 21 Northwest Bioanalytical Study No. NWBS0O-040 Report No. NWBROO-108 Table 5. Summary of Calibration Curve Parameters for PFHS Quadratic weighted l.'.x:. All concentrations are expressed as ng/mL. Run Date 29-Jun-2000 30-Jun-2000 02-Jul-2000 Run Number 13 15 16 A B C R-Squared LLOQ ULOQ -0.000006 0.023682 0.021811 0.9947 3.15 502 -0.000005 0.016787 0.011507 0.9946 3.15 502 -0.000009 0.021344 0.020627 0.9968 3.15 502 Mean S.D. %CV n -0.000007 0.020604 0.017982 0.Q00002 0.003507 0.005638 -28.6 17.0 31.4 33 0.9954 0.0012 0.1 3 A, B and C are coefficients used to define the quadratic curve. Table 6. Summary of Calibration Curve Parameters for M556 Quadratic weighted 1/x2. All concentrations are expressed as ng/mL. Run Date Run Number A B C R-Squared LLOQ ULOQ 29-Jun-2000 13 0.000001 0.005000 0.004952 0.9881 4.30 502 30-Jun-2000 15 0.000000 0.002882 0.000748 0.9975 4.30 502 02-Jul-2000 16 0.000000 0.003891 0.000548 0.9949 4.30 502 Mean S.D. %CV n 0.000000 0.003924 0.002083 0.000001 0.001059 0.002487 - 27.0 119.4 3 -> 3 0.9935 0.0049 0.5 3 A, B and C are coefficients used to define the quadratic curve. *: Page 22 Northwest Bioanalytical Study No. NWBSOO-040 Report No. NWBR00-108 Table 7. Summary of Calibration Curve Parameters for M570 Quadratic weighted l/x:. All concentrations are expressed as ns/mL. Run Date 29-Jun-2000 30-Jun-2000 02-Jul-2000 Run Number 13 15 16 A B C R-Squared LLOQ ULOQ -0.000002 0.006621 -0.007115 0.9962 5.60 505 -0.000001 0.003911 -0.005406 0.9972 5.60 505 0.000000 0.006433 -0.009231 0.9967 5.60 505 Mean S.D. %CV n -0.000001 0.005655 -0.007251 0.000001 0.001513 0.001916 -100.0 26.8 -26.4 333 0.9967 0.0005 0.1 3 A, B and C are coefficients used to define the quadratic curve. Page 23 Northwest Bioanalytical h ^Jjj >' i f Study No. NWBSOO-040 Report No. NWBR00-1Q8 Table 8. Back-Calculated Concentrations of Calibration Standards for PFOS Run Date 29-Jun-2000 30-Jun-2000 02-Jul-2000 Quadratic weighted l/x:. All concentrations are expressed as ng/mL. __________ 5/7.y -9.^ /,/rri u'i- 'jug Run Number 13 48.1 y in 44.9 49.6 57.1 72.1 97.1 147 i l 2 * S i t ^ 1 ?3 . & / - y i / Ov 52.1 56.6 71.7 (81.1J 153 297 2, 302 46.5 51.5 58.5 80.8 94.6 146 316 15 46.5 49.8 58.6 72.7 92.2 147 321 50.3 48.3 58.4 71.2 98.9 135 306 16 51.6 50.5 60.3 74.9 94.3 151 299 45.2 47.6 54.3 71.2 94.9 148 298 447 l.to 449 445 438 471 415 449 547 ^0 515 560 541 518 571 547 Mean S.D. %cv %Bias n 47.5 50.0 57.8 73.8 92.7 147 307 445 542 2.78 1.77 2.07 3.72 6.07 6.28 9.47 18.2 22.3 5.9 3.5 3.6 5.0 6.5 4.3 3.1 4.1 4.1 -1.2 0.8 1.2 2.4 -4.5 0.0 3.4 -0.4 -0.9 66666666 6 Table 9. Back-Calculated Concentrations of Calibration Standards for PFOSA Quadratic weighted 1/x*. Allconcentrations are expressed as ng/mL. Run Date Run Number 29-Jun-2000 13 30-JIU1-2000 15 02-Jul-2000 16 I-00/ 1.00 1.03 1.03 0.957 0.952 1.04 2.51 -V; ..^/ 2.44 2.23 2.46 2.68 2.48 2.57 10.1 l y 25.1 50.3 c' f 100y 251 402 <1 2 502 y 10.6 25.7 104 280 420 483 vyf " 11.1 45.7 95.0 256 379 507 9.90 25.7 48.6 98.5 255 401 518 9.71 25.7 50.4 94.8 277 . ; 418 447 11.5 26.2 47.6 99.0 244 . 391 . 506 : 9.79 22.7 48.7 107 237 431 501 Mean S.D. % cv %Bias n 1.00 2.48 10.4 26.6 45.3 99.7 258 407 494 0.0388 0.150 0.753 3.54 7.27 4.90 17.3 19.7 25.6 3.9 6.0 7.2 13.3 16.0 4.9 6.7 4.8 5.2 0.0 -1.2 3.0 6.0 -9.9 -0.3 2.8 1.2 -1.6 6 6 6 6666 66 \.) Page 24 Northwest Bioanalytical Study No. NWBSOO-040 Report No. NWBR00-108 Table 10. Back-Calculated Concentrations of Calibration Standards for PFOSAA Quadratic weighted l 'x:. All concentrations are expressed as ng mL. ,, >r7 ' T~ : ; f Run Date Run Number 6.00 7.50 15.0 30.0 55.0 105 ' 255 405 505 f.t S 29-Jun-2000 13 5.90 7.74 14.4 31.3 n * 3 J J P n o 262 412 476 5.92 7.85 14.1 30.8 49.4 106 266 400 512 30-Jun-2000 13 6.00 7.82 14.9 29.0 53.3 105 271 403 494 5.58 7.81 15.7 30.1 53.3 99.9 271 426 475 02-Jul-2000 16 5.92 7.22 15.8 28.2 49.8 102 255 392 501 6.15 7.65 14.8 30.2 55.8 119 260 413 504 Mean S.D. % cv %Bias n 5.91 7.68 15.0 29.9 52.3 107 264 408 494 0.187 0.237 0.683 1.15 2.69 6.83 6.37 11.9 15.2 3.2 3.1 4.6 3.8 5.1 6.4 2.4 2.9 3.1 -1.5 2.4 0.0 -0.3 -4.9 1.9 3.5 0.7 -2.2 6 6 6 6 5 6666 * Sample deactivated as an outlier (> 2 times the acceptance criteria). Result not included in summary statistics. Table 11. Back-Calculated Concentrations of Calibration Standards for POAA Quadratic weighted l/x:. All concentrations are expressed as ng/mL. , n Run Date 29-Jun-2000 30-Jun-2000 02-Jul-2000 Run Number 5.76 7.26 13 5.15 <M2> 6.41 6.41 15 6.26 7.24 5.67 6.80 16 5.36 7.09 Z.o5 ( ' u .s ) 7.74 14.8 29.8 54.8 / 105 255 405 505 j // 14.6 30.9 fb T ) 104 246 377 450 13.4 25.3 56.6 116 287 423 541 14.0 29.5 54.4 108 280 390 517 15.0 29.7 55.2 97.6 276 406 466 15.2 28.2 50.7 103 250: 385 532 15.9 32.2 54.9 106 255 ' 394 521 Mean S.D. . % cv %Bias n 5.77 7.31 14.7 29.3 54.4 106 266, 396 505 0.550 0.754 0.891 2.38 2.20 6.12 17.4 16.4 37.3 9.5 10.3 6.1 8.1 4.0 5.8 6.5 4.1 7.4 0.2 0.7 -0.7 -1.7 -0.7 1.0 4.3 -2.2 0.0 5 6 6 6 5 6666 !Sample deactivated as an outlier (> 2 times the acceptance criteria). Result not included in summary statistics. Northwest Bioanalytical Study No. NWBSOO-040 Report No. NWB ROO-108 Table 12. Back-Calculated Concentrations of Calibration Standards for PFHS Run Date 29-Jun-2000 30-Jun-2000 02-Jul-2000 Quadratic weiehted l.'x:. All concentrations are expressed as ng/mL. ; ice-', ^ ? ' ~ r ' >- - -i 4 - V - -3> / 0 t V/V7 !I 2?>", 3- !' V y - ; i n7 ` 1 u o ?i Q & z *! J~ ~ rni Run Number 3.15 4.65 12.2 / / 27.2 52.2 102 252 402 502 // / // 13 3.28 5.17 11.8 28.0 55.3 107 252 377 448 2.85 4.59 11.7 27.3 48.8 97.6 250 414 574 15 2.96 5.08 13.2 26.2 47.6 101 264 395 515 3.00 4.70 13.8 26.6 50.4 94.6 255 415 484 16 3.21 4.70 12.9 28.4 50.0 101 244 382 545 3.12 4.26 13.5 26.5 50.3 99.7 249 399 509 Mean S.D. %cv %B ias n 3.07 4.75 12.8 27.2 50.4 10d 252 397 513 0.162 0.333 0.880 0.887 2.63 4.14 6.77 15.8 44.3 5.3 7.0 6.9 3.3 5.2 4.1 2.7 4.0 8.6 -2.5 2.2 4.9 0.0 -3.4 -2.0 0.0 -1.2 2.2 6 6 6 6 6 6 6 6 6 Table 13. Back-Calculated Concentrations of Calibration Standards for M556 Run Date 29-Jun-2000 30-Jun-2000 02-Jul-2000 Run Number 13 15 16 430 ./ 5.11 3.78 4.25 4.39 3.78 4.74 11.8 11.3 11.6 11.7 11.5 13.0 3*2' ."nr 26.8 51.8 y 30.6 (*33.3) 24.0 49.7 27.4 49.8 26.1 51.1 25.6 47.5 26.9 53.6 102 / 111 101 107 95.1 98.2 110 252 y 266 272 273 266 250 254 402 ./ 396 395 403 412 379 408 502 y 468 514 499 466 513 506 Mean S.D. %cv %Bias n 4.34 11.5 26.8 50.3 104 264 399 494 0.527 0.996 2.22 2.23 6.56 9.46 11.8 21.9 12.1 8.7 8.3 4.4 6.3 3.6 3.0 4.4 0.9 -2.5 0.0 -2.9 2.0 4.8 -0.7 -1.6 6 6 6 5 6666 * Sample deactivated as an outlier (> 2 times the acceptance criteria). Result not included in summary statistics. Page 26 Northwest Bioanalytical Study No. NWBS00-040 Report No. NWBROO-108 Table 14. Back-Calculated Concentrations of Calibration Standards for M570 Run Date 29-Jun-2000 30-Jun-2000 02-Jul-2000 Quadratic weighted 1/'r. All concentrations are e:cpressed asng/mL. (o' 2.2 " U x /a. / ! , 3'U wo'* Run Number 5.60 / s o 14.6 29.6 54.6 105 255 405 / >3% y 13 5.50 7.08 13.7 31.6 & 107 264 418 5.47 7.61 14.6 30.5 50.5 98.9 269 388 s?/ 505 y 466 530 15 5.26 7.11 14.8 29.1 55.2 101 266 408 498 6.00 6.94 14.5 30.7 54.7 97.0 272 422 474 16 5.67 6.55 15.7 27.5 49.8 108 255 394 509 5.82 6.98 14.9 29.5 56.3 113 251 401 513 Mean S.D. %cv %Bias n 5.62 0.266 4.7 0.4 6 7.05 0.342 4,9 -0.7 6 14.7 0.648 4.4 0.7 6 29.8 53.3 1.44 2.94 4.8 5.5 0.7 -2.4 65 104 263 405 .498 6.16 8.18 13.4 24.4 5.9 3.1 3.3 4.9 -1.0 3.1 0.0 -1.4 6666 * Sample deactivated as an outlier (> 2 times the acceptance criteria). Result not included in summary statistics. Page 27 Northwest Bioanalytical Study No. NWBS00-040 Report No. NWBR00-108 Table 15. Intra-Assay Precision and Accuracy for PFOS Quality Control Samples 9 - S1 All concentrations are expressed as ng/mL. Run Date 29-Jun-2000 Run Low QC Medium QC High QC Number 51.1 ng/mL 197 ng/mL 446 ng/mL 13 45.9 191 392 44.8 177 403 47.6 183 429 48.8 175 383 48.6 179 377 Mean S.D. %cv %Theoretical n Run Date 30-Jun-2000 Run Number 15 47.1 181 1.74 6.32 ' 3.7 3.5 92.2 91.9 55 Low QC Medium QC 51.1 ng/mL 197 ng/mL 48.8 173 46.7 174 47.8 175 45.8 166 47.1 176 397 20.5 5.2 89.0 5 High QC 446 ng/mL 382 395 402 382 406 Mean S.D. %cv %Theoretical n 47.2 173 393 ' 1.13 3.96 11.1 ; 2.4 2.3 2.8 92.4 87.8 88.1 55 5 Page 28 Northwest Bioanalytical Study No. NWBS00-040 Report No. NWBR00-108 Table 15. Intra-Assay Precision and Accuracy for PFOS Quality Control Samples (continued) All concentrations are expressed as ng/mL. Run Date 02-Jul-2000 Run Low QC Medium QC High QC Number 51.1 ng/mL 197 ng/mL 446 ng/mL 16 45.2 170 380 48.7 160 393 46.4 166 385 47.7 169 387 44.0 172 .361 Mean S.D. %cv "/Theoretical n 46.4 ' 1.88 4.1 90.8 5 167 4.67 2.8 84.8 5 381 12.2 3.2 85.4 5 Page 29 Northwest Bioanalytical Study No. NWBS0O-040 Report No. NWBROO-108 Table 16. Intra-Assay Precision and Accuracy for PFOSA Quality Control Samples All concentrations are expressed as ng/mL. LLO 0 ' a O Run Date Run Number Low QC 4.00 ng/mL 29-Jun-2000 13 3.43 3.21 3.39 3.58 3.67 iMedium QC 150 ng/mL 133 122 128 129 139 ' High QC 400 ng/mL 325 345 357 322 342 Mean 3.46 S.D. %cv y 0.178 5.1 "/(Theoretical 86.5 n5 Run Date Run Number Low QC 4.00 ng/mL 30-Jun-2000 15 * 2.88 * 2.76 * 2.89 * 2.78 * 2.77 130 6.30 4.8 86.7 5 Medium QC 150 ng/mL * 100 * 105 * 103 * 97.8 * 110 338 14.6 4 3 --=? S ' 84.5 V---- 5 High QC 400 ng/mL *278 *291 318 * 297 314 Mean S.D. %cv / %Theoretical n 2.82 103 300 : : ' 0.0635 4.71 16.5; 2.3____ ____ 4.6------' -- -5:5------- <70.5 68.7 75.0 5 - 5 .... ........ 5. _..... - * > 25% theoretical r o<- Ov" /0 >` H Page 30 1 :A Northwest Bioanalytical Study No. NWBS0O-040 Report No. NWBR00-108 Table 16. Intra-Assay Precision and Accuracy for PFOSA Quality Control Samples (continued) All concentrations are expressed as ng/mL. Run Date 02-M-2000 Run Number Low QC 4.00 ng/mL 16 * 2.58 *2.96 3.50 3.94 *2.51 Medium QC 150 ng/mL * 105 * 102 * 103 * 102 * 112 High QC 400 ng/mL * 289 311 315 304 305 Mean S.D. %CV %Theoretical n 3.10 ' 0.613 ? 77.5 5 105 305 4.21 9.91 4.0 70.0 76.3 5 - ...... - 5 ..... * > 25% theoretical Page 31 Northwest Bioanalytical Study No. NWBSOO-040 Report No. NWBROO-108 Table 17. Intra-Assay Precision and Accuracy for PFOSAA Quality Control Samples All concentrations are expressed as ng/mL. 1.0 0 - L . 0 0 Run Date Run Number Lo>v QC 9.00 ng/mL 29-Jun-2000 13 7.98 7.43 ' 8.05 8.81 8.46 Medium QC 155 ng/mL 129 * 120 * 123 * 121 127 High QC 405 ng/mL * 272 * 303 *317 *260 *286 Mean 8.15 S.D. 0.522 %CV ' 6.4 %Theoretical 90.6 n5 Run Date Run Number Low QC 9.00 ng/mL 30-Jun-2000 15 7.65 7.76 7.73 7.29 7.52 124 3.87 3.1 ' 80.0 5 Medium QC 155 ng/mL * 112 * 117 * 116 * 104 * 117 288 23.0 8.0 71.1 . 5 High QC 405 ng/mL *285 * 300 *315 *290 *313 Mean S.D. %cv y %Theoretical n 7.59 0.192 2.5 ; 84.3 5 ' * > 20% theoretical 113 5.54 4.9 72.9 5 301' ' 13.4* .... 4.5 __... 74.3 5 Northwest Bioanalytical Study No. NWBS00-040 Report No. NWBR00-I08 Table 17. Intra-Assay Precision and Accuracy for PFOSAA Quality Control Samples (continued) All concentrations are expressed as ng/mL. Run Date 02-M-2000 Run Number Low QC 9.00 ng/mL 16 7.37 8.11 8.47 9.67 7.63 Medium QC 155 ng/mL * 117 * 107 * 111 * 114 * 116 High QC 405 ng/mL * 287 * 309 * 301 *312 *291 Mean S.D. %CV %Theoretical n v 8.25 ' 0.900 10.9 91.7 5 113 4.06 3.6 72.9 5 * > 20% theoretical 300 10.9 3.6 ~ ~ 7 4 .1 ^ " 5..... " ~ Page 33 Northwest Bioanalytical Study No. NWBS00-040 Report No. NWBR00-108 Table 18. Intra-Assay Precision and Accuracy for POAA Quality Control Samples All concentrations are expressed as ng/mL. Run Date 29-Jun-2000 Run Number 13 Low QC 8.74 ng/mL 7.74 8.83 9.28 10.1 9.01 Medium QC 154 ng/mL 157 156 154 153 163 High QC 403 ng/mL 404 401 434 422 377 Mean S.D. %cv %Theoretical n Run Date Run Number 30-Jun-2000 15 8.99 0.852 9.5 102.9 5 Low QC 8.74 ng/mL 8.79 8.00 8.26 7.59 7.88 157 3.91 2.5 101.9 5 Medium QC 154 ng/mL 144 153 147 136 139 408 21.8 5.3 101.2 5 High QC 403 ng/mL 374 389 382 376 398 Mean S.D. %cv ./ %Theoretical n 8.10 0.453 5.6 92.7 5 144 6.69 4.6 93.5 5 384 ' 9.86 4 2.6 95.3 5 Page 34 Northwest Bioanalytical Study No. NWBSOO-040 Report No. NWBROO-108 Table 18. Intra-Assay Precision and Accuracy for POAA Quality Control Samples (continued) All concentrations are expressed as ng/mL. Run Date Run Number O2-J11I-2OOO 16 Low QC 8.74 ng/mL 7.15 8.15 8.42 8.72 7.12 Medium QC 154 ng/mL 150 136 140 148 141 High QC 403 ng/mL 401 393 398 405 .376 Mean S.D. %cv y %Theoretical n 7.91 0.737 9.3 90.5 5 143 5.83 . ' 4.1 92.9 5 395 11.3 2.9 98.0 5 Page 35 Northwest Bioanalytical Study No. NWBS00-040 Report No. NWBROO-108 Table 19. Intra-Assay Precision and Accuracy for PFHS Quality Control Samples All concentrations are expressed as ng/mL. Run Date 29-Jun-2000 Run Low QC Medium QC Number 6.15 ng/mL 152 ng/mL 13 7.03 ' 175 6.44 166 6.77 171 6.89 158 7.05 165 High QC 402 ng/mL 454 431 468 434 424 Mean 6.84 167 S.D. 0.249 6.44 %cv / 3.6 3.9 "/(Theoretical 111.2 109.9 n 55 Run Date Run Low QC Medium QC Num ber 6.15-ng/mL 152 ng/mL 30-Jun-2000 15 6.53 163 6.22 157 6.06 158 6.00 149 6.31 164 442 18.2 4.1 110.0 5 High QC 402 ng/mL 429 455 463 429 ' 447 Mean S.D. %CV -/ %Theoretical n 6.22 0.211 3.4 101.1 5 158 5.97 3.8 103.9 5 445 ' 15.3 : 3.4 110.7 5 Page 36 Northwest Bioanalytical Study No. NWBS0O-040 Report No. NWBROO-108 Table 19. Intra-Assay Precision and Accuracy for PFHS Quality Control Samples (continued) All concentrations are expressed as ng/mL. Run Date 02-Jul-2000 Run Low QC Medium QC Number 6.15 ng/mL 152 ng/mL 16 6.43 154 6.52 150 6.23 152 6.86 153 5.93 151 High QC 402 ng/mL 408 441 422 430" 409 Mean S.D. %cv %Theoretical n y 6.39 0.345 5.4 103.9 5 152 1.58 1.0 100.0 5 422 14.1 3.3 105.0 5 Page 37 Northwest Bioanalytical Study No. NWBSOO-040 Report No. NWBROO-108 Table 20. Intra-Assay Precision and Accuracy for M556 Quality Control Samples AH concentrations are expressed as ng/mL. U . O ^ - 'I.'IO Run Date 29-Jun-2000 Run Number 13 Low QC 5.80 ng/mL ** 8.98 6.34 Medium QC 152 ng/mL 165 159 ** 7.98 161 6.55 163 5.86 170 High QC 402 ng/mL 427 429 439 413 366 Mean S.D. %CV %Theoretical n 6.25 0.354 5.7 107.8 3 164 4.22 2.6 107.9 5 Run Date Run Low QC Medium QC Number 5.80 ng/mL 152 ng/mL 30-Jun-2000 15 5.74 143 5.89 147 5.46 152 5.33 139 5.61 153 415 28.8 6.9 103.2 5 High QC 402 ng/mL 380 395 408 390 407 Mean S.D. %CV y %Theoretical n 5.61 0.221 3.9 96.7 5 147 5.93 4.0 96.7 5 396 ` 11.8 ' 3.0 98.5 5 ** Sample deactivated due to injector carryover (not included in summary statistics) Page 38 Northwest Bioanalytical Study No. NWBS00-040 Report No. NWBROO-108 Table 20. Intra-Assay Precision and Accuracy for M556 Quality Control Samples (continued) All concentrations are expressed as ng/mL. Run Date 02-Jul-2000 Run Low QC Medium QC Number 5.80 ng/mL 152 ng/mL 16 6.23 149 6.17 137 6.17 149 6.33 152 5.61 150 High QC 402 ng/mL 391 418 390 417 .378 Mean S.D. %CV %Theoretical n 6.10 0.283 4.6 105.2 5 147 5.94 4.0 96.7 5 399 17.8 4.5 99.3 5 Page 39 Northwest Bioanalytical Study No. NWBSOO-040 Report No. NWBROO-108 Table 21. Intra-Assay Precision and Accuracy for M570 Quality Control Samples All concentrations are expressed as ng/mL. Run Date 29-Jun-2000 Run Low QC Number 8.60 ng/mL 13 8.23 8.07 8.34 8.87 8.04 Medium QC 155 ng/mL 168 155 164 153 170 High QC 405 ng/mL 393 403 424 378 377 Mean 8.31 S.D. %cv S 0.336 4.0 %Theoretical 96.6 n5 Run Date Run Low QC Num ber 8.60 ng/mL 30-Jun-2000 15 8.17 9.09 8.72 7.71 7.88 162 7.65 4.7 104.5 5 Medium QC 155 ng/mL 151 149 148 138 150 395 19.5 4.9 97.5 5 High QC 405 ng/mL 396 396 412 388 413 Mean S.D. %cv ./ %Theoretical n 8.31 0.579 7.0 96.6 5 147 . 5.26 3.6 94.8 5 401 ' 11.0 * 2.7 99.0 5 Page 40 Northwest Bioanalytical Study No. NWBS0O-040 Report No. NWBROO-108 Table 21. Intra-Assay Precision and Accuracy for M570 Quality Control Samples (continued) All concentrations are expressed as ng/mL. . Run Date 02-M-2000 Run Low QC Num ber 8.60 ng/mL 16 8.67 8.49 9.73 9.69 7.95 Medium QC 155 ng/mL 148 137 148 145 151 High QC 405 ng/mL 382 391 384 412 368 Mean S.D. %CV %Theoretical n 8.91 0.780 8.8 103.6 5 146 5.36 3.7 94.2 5 387 16.1 4.2 95.6 5 Page 41 Northwest Bioanalytical Study No. NWBS0O-040 Report No. NWBROO-108 Table 22. Inter-Assay Precision for PFOS Quality Control Samples All concentrations are expressed as ng/mL. Run Date 29-Jun-2000 30-Jun-2000 02-Jul-2000 Run Low QC Medium QC High QC Number 51.1 ng/mL 197 ng/mL 446 ng/mL 13 45.9 191 392 44.8 177 403 47.6 183 429 48.8 175 383 48.6 179 377 15 48.8 173 .382 46.7 174 395 47.8 175 402 45.8 166 382 47.1 176 406 16 45.2 170 380 48.7 160 393 46.4 166 385 47.7 169 387 44.0 172 361 . Mean S.D. %CV %Theoretical %Bias n 46.9 174 390 1.55 7.46 15.7 3.3 4.3 . 4.0 . 91.8 88.3 87.4 . -8.2 -11.7 -12.6 15 15 15 Page 42 Northwest Bioanalytical Study No. NWBSOO-040 Report No. NWBR00-108 Table 23. Inter-Assay Precision for PFOSA Quality Control Samples All concentrations are expressed as ng/mL. Run Date Run Number Low QC 4.00 ng/mL 29-Jun-2000 13 3.43 3.21 3.39 3.58 3.67 30-Jun-2000 15 *2.88 *2.76 *2.89 *2.78 *2.77 02-Jul-2000 16 *2.58 *2.96 3.50 3.94 *2.51 Medium QC 150 ng/mL 133 122 128 129 139 *100 *105 *103 *97.8 *110 *105 *102 *103 *102 *112 High QC 400 ng/mL 325 345 357 322 342 *278 ' *291 318 *297 314 *289 311 315 304 305 Mean S.D. %cv %TheoreticaI %Bias n 3.12 0.437 14.0 78.0 -22.0 15 113 13.7 12.1 75.3 -24.7 15 * > 25% theoretical 314 21.9. 7.0 . . 78.5 -21.5* 15 Page 43 Northwest Bioanalytical Study No. NWBS00-040 Report No. NWBROO-108 Table 24. Inter-Assay Precision for PFOSAA Quality Control Samples All concentrations are expressed as ng/mL. Run Date 29-Jun-2000 30-Jun-2000 02-Jul-2000 Run Low QC Medium QC High QC Number 9.00 ng/mL 155 ng/mL 405 ng/mL 13 7.98 129 *272 7.43 *120 *303 8.05 *123 *317 8.81 *121 *260 8.46 127 *286 15 7.65 *112 *285 7.76 *117 *300 7.73 *116 *315 7.29 *104 *290 7.52 *117 *313 16 7.37 *117 *287 8.11 *107 *309 8.47 *111 *301 9.67 *114 *312 7.63 *116 *29i Mean S.D. %CV %Theoretical %Bias n 8.00 0.640 8.0 88.9 -11.1 15 117 6.79 5.8 . 75.5 -24.5 15 * > 20% theoretical 296 1.6.6 >!: 5.6 . 73.1 -26.9 15 Page 44 O OO Northwest Bioanalytical Study No. NWBSOO-i Report No. NWBROO- Table 25. Inter-Assay Precision for POAA Quality Control Samples All concentrations are expressed as ng/mL. Run Date 29-Jun-2000 30-Jun-2000 02-Jul-2000 Run Number 13 15 16 Low QC 8.74 ng/mL 7.74 8.83 9.28 10.1 9.01 8.79 8.00 8.26 7.59 7.88 7.15 8.15 8.42 8.72 7.12 Medium QC 154 ng/mL 157 156 154 153 163 144 153 ' 147 136 139 150 136 140 148 141 High QC 403 ng/mL 404 401 434 422 377 .374 389 382 376 398 401 393 398 405 376 Mean S.D. %cv %Theoretical %Bias n 8.34 0.812 9.7 95.4 -4.6 15 148 8.27 5.6 . 96.1 -3.9 15 395 17.4 4.4 . ; 98.0 . -2.0 15 Page 45 Northwest BLoanalytical Study No. NWBS00-040 Report No. NWBROO-108 Table 26. Inter-Assay Precision for PFHS Quality Control Samples All concentrations are expressed as ng/mL. Run Date 29-Jun-2000 30-Jun-2000 02-Jul-2000 Run Low QC Medium QC Number 6.15 ng/mL 152 ng/mL 13 7.03 175 6.44 166 6.77 171 6.89 158 7.05 165 15 6.53 . 163 6.22 157 6.06 158 6.00 149 6.31 164 16 6.43 154 6.52 150 6.23 152 6.86 153 5.93 151 High QC 402 ng/mL 454 431 468 434 424 429 455 463 429 447 408 441 422 430 409 Mean S.D. %CV %Theoretical %Bias n 6.48 0.368 5.7 105.4 5.4 15 159 7.96 5.0 104.6 4.6 15 436 l 8-1 .... 4.2 , 108.5 8.5 * 15 Page 46 Northwest Bioanalytical Study No. NWBS0O-040 Report No. NWBROO-108 Table 27. Inter-Assay Precision for M556 Quality Control Samples All concentrations are expressed as ng/mL. Run Date Run Number Low QC 5.80 ng/mL 29-Jun~2000 13 **8.98 6.34 **7.98 6.55 5.86 30-Jun-2000 15 5.74 5.89 5.46 5.33 * 5.61 02-Jul-2000 16 6.23 6.17 "" 6.17 6.33 5.61 Medium QC 152 ng/mL 165 159 161 163 170 143 147 152 139 153 149 137 149 152 150 High QC 402 ng/mL 427 429 439 413 366 . 380 395 408 390 407 391 418 390 417 378 Mean S.D. %cv 5.95 153 403 0.381 9.49 21.0... 6.4 6.2. 5.2 ; ; %Theoretical %Bias n 102.6 2.6 13 100.7 0.7 15 100.2 0.2 15 ** Sample deactivated due to injector carryover (not included in summary statistics) Northwest Bioanalytical Study No. NWBSOO-040 Report No. NWBROO-108 Table 28. Inter-Assay Precision for M570 Quality Control Samples All concentrations are expressed as ng/mL. Run Date 29-Jun-2000 30-Jun-2000 02-Jul-2000 Run Low QC Medium QC Number 8.60 ng/mL 155 ng/mL 13 8.23 168 8.07 155 8.34 164 8.87 153 8.04 170 15 8.17 151 9.09 149 8.72 148 7.71 138 7.88 150 16 8.67 148 8.49 137 9.73 148 9.69 145 7.95 ` 151 High QC 405 ng/mL 393 403 424 378 377 396 396 412 388 413 382 391 384 412 368 Mean S.D. %cv %Theoretical %Bias n 8.51 0.621 7.3 99.0 - 1.0 15 152 9.51 6.3 98.1 -1.9 15 394 15.8 4.0 ' 97.3 ' -2.7 * 15 Page 48 Northwest Bioanalytical Study No. NWBS0O-040 Report No. NWBR00-108 Table 29. Intra-Assay Precision and Accuracy for PFOSA Specific Quality Controls All concentrations are expressed as ng'mL. Run Date 16-Jul-2000 Run Number Low QC Medium QC High QC (4.00 ng/mL) (150 ng/mL) (400 ng/mL) 17 3.55 145 322 3.58 135 324 3.84 171 326 4.01 149 330 3.46 144 341 Mean S.D. %CV %Theoretical n 3.69 0.229 6.2 92.3 5 149 . 329 13.4 7.54 9.0 2.3 99.3 82.3 5 ,5 Note: The following calibration curve and standard statistics are included for analytical run review, (not included in overall validation summary statistics) Quadratic weighted l/x:. Run Date Run Number A B C R-Squared LLOQ ULOQ 16-Jul-2000 17 0.000002 0.020014 0.006490 0.9985 1.00 502 n 1 1 .1 1 A, B and C are coefficients used to define the quadratic curve. Run Date Run Number 1.00 2.51 10.1 25.1 50.3 100 251 402 .502 16-Jul-2000 17 0.998 2,48 10.1 24.5 52.6 96.2 244 39p 531 0.980 2.67 10.4 24.2 50.4 104 239 399 505 Mean %Bias n 0.989 2.58 10.3 24.4 51.5 100 242 395 518 1 bo -1.1 2.8 2.0 2.4 0.0 -3.6 -1.7 3.2 2 2 2 2 22222 Page 49 Northwest Bioanalytical Study No. NWBSOO-040 Report No. NWBR00-108 Table 30. Intra-Assay Precision and Accuracy for PFOSAA Specific Quality Controls All concentrations are expressed as ng/mL. Run Date 16-Jul-2000 Run Number 17 Low QC (9.00 ng/mL) 7.30 *4.93 *5.65 8.36 **21.4 o(J\ # Medium QC (155 ng/mL) 161 176 159 151 High QC (405 ng/mL) 368 382 395 365 346 Mean S.D. %CV %Theoretical n 6.56 167 371 1.56 15.5 18.5 23.8 9.3 5.0 72.9 107.7 91.6 4 55 * > 20% theoretical ** Deactivated as an outlier (not included in summary statistics) Note: The following calibration curve and standard statistics are included for analytical run review, (not included in overall validation summary statistics) Quadratic weighted 1/x2. Run Date Run Number A B C R-Squared LLOQ ULOQ 16-Jul-2000 17 0.000001 0.005669 0.008710 0.9764 7.50 505 n Run Date 111 1 A, B and C are coefficients used to define the quadratic curve. Run Number 6.00 7.50 15.0 30.0 55.0 105 255 405 505 16-Jul-2000 . 17 **8.89 8.60 *17.8 32.5 *65.3 107 287 413 559 **3.30 6.49 *12.1 *25.4 47.6 104 231 372 465 Mean %Bias n - 7.55 15.0 29.0 56.5 106 259 393 512 - 0.7 0.0 -3.3 2.7 1.0 1.6 -3.0 1.4 - 22222222 * > 15% theoretical ** Sample deactivated as an outlier (> 2 times the acceptance criteria). Result not included in summary statistics. Page 50 O OO Northwest Bioanalytical Study No. NWBSOO-i Report No. NWBR00- Table 31. PFOS Dilution Quality Control Samples All concentrations are expressed as ng/mL. Run Date Run Number Dilution QC 4460 ng/mL 29-Jun-2000 13 ' 4310 4170 4420 3900 4110 30-Jun-2000 15 4490 3990 4180 4050 . 4400 02-Jul-2000 16 4160 4160 -4020 3790 3970 Mean S.D. %CV %Theoretical %Bias n 4140 199 4.8 92.8 -7.2 15 Page 51 Northwest Bioanalytical Study No. NWBSOO-040 Report No. NWBR00-108 Table 32. PFOSA Dilution Quality Control Samples All concentrations are expressed as ng/mL. Run Date Run Number Dilution QC 4000 ng/mL 29-Jun-2000 13 3720 3570 3720 3380 3690 30-Jun-2000 15 . 3020 *2890 3100 *2750 *2860 02-Jul-2000 16 3270 3130 3130 3210 3240 . Mean S.D. %cv %Theoretical %Bias n * > 25% theoretical 3250 315 9.781.3 -18.8 15 Page 52 Northwest Bioanalytical Study No. NWBS00-040 Report No. NWBROO-108 Table 33. PFOSAA Dilution Quality Control Samples All concentrations are expressed as ng/mL. Run Date Run Number Dilution QC 4050 ng/mL 29-Jun-2000 13 3490 3280 3270 *3170 3390 30-Jun-2000 15 . 3420 *3030 3240 *3060 *3120 02-Jul-2000 16 *3220 *3110 *3210 *2960 *3000 Mean S.D. %CV %Theoretical %Bias n 3200 157 4.9 79.0 -21.0 15 * > 20% theoretical Page 53 Northwest Bioanalytical Study No. NWBSOO-040 Report No. NWBROO-108 Table 34. POAA Dilution Quality Control Samples All concentrations are expressed as ng/mL. Run Date Run Number Dilution QC 4030 ng/mL 29-Jun-2000 13 4220 3820 4250 4040 4000 30-Jun-2000 15 4550 3850 4010 4000 4020 02-Jul-2000 16 4170 4090 .... 4230 3910 4040 Mean S.D. %CV %TheoreticaI %Bias n 4080 184 4.5 . 101.2 1.2 15 Page 54 Northwest Bioanalytical Study No. NWBS00-040 Report No. NWBROO-108 Table 35. PFHS Dilution Quality Control Samples All concentrations are expressed as ng/tnL. Run Date Run Number Dilution QC 4020 ng/mL 29-Jun-2000 13 4830 4430 4530 4150 * 30-Jun-2000 15 4190 *4980 4500 4550 4490 4790 02-Jul-2000 16 4700 *5030 4550 4260 4760 Mean S.D. %cv %Theoretical %Bias n * > 20% theoretical 4580 267 5.8. 113.9 13.9 15 Page 55 O 00 Northwest Bioanalytical Study No. NWBSOO-I Report No. NWBR00- Table 36. M556 Dilution Quality Control Samples All concentrations are expressed as ng/mL. Run Date Run Number Dilution QC 4020 ng/mL 29-Jun-2000 13 4330 4050 4260 3940 4000 30-Jun-2000 15 4490 3980 4130 4000 4170 02-Jul-2000 16 4270 3990 4240 3890 3980 Mean S.D. %CV %Theoretical %Bias n 4110 172 4.2 102.2 2.2 15 Page 56 Northwest Bioanalytical Study No. NWBS00-040 Report No. NWBR00-108 Table 37. M570 Dilution Quality Control Samples All concentrations are expressed as ng/mL. Run Date Run Number Dilution QC 4050 ng/mL 29-Jun-2000 13 4710 4590 4550 4100 4490 30-Jun-2000 15 4610 3980 4080 *' 4080 4280 02-Jul-2000 16 . 4270 3950 4120 3890 3960 Mean S.D. %CV %Theoretical %Bias n 4240 278 6.6 104.7 4.7 15 Page 57 Northwest Bioanalytical Study No. NWBSOO-040 Report No. NWBROO-108 Table 38. PFOSA Specific Dilution Quality Controls All concentrations are expressed as ng/mL. Run Date Run Number Dilution QC 4000 ng/mL 16-Jul-2000 17 *2630 *2570 3110 3250 4110 Mean S.D. %CV %Theoretical %Bias n * > 25% theoretical 3130 620 19.8 78.3 -21.8 5 Page 58 Northwest Bioanalytical Study No. NWBS00-04O Report No. NWBR00-108 Table 39. PFOSAA Specific Dilution Quality Controls All concentrations are expressed as ng/mL. Run Date Run Number Dilution QC .4050 ng/mL 16-JuI-2000 17 *2560 *2650 *2900 3640 4070 Mean S.D. %CV %Theoretical %Bias n * > 20% theoretical 3160 661 20.9 78.0 -22.0 5 Page 59 O 00 Northwest Bioanalytical Study No. NWBSOO-i Report No. NWBROO- Table 40. PFOS Extraction Efficiency Run 7 Low Concentration * 2.S0 ng/mL fo r Analyte Tijne of Spikin# 1. Analyte and IS after extraction Mean Ratio Analvte/IS 0.00936 0.0I0I 0.0103 0.00992 Ratio IS'Analvte 107 99.2 97.5 101 2. Analyte prior and IS after extraction Mean . 0.00532 0.00568 0.00517 0.00539 3. IS prior and Analyte after extraction Mean 6.94 7.51 8.42 7.62 Mean extraction efficiency for the analyte Mean extraction efficiency for the internal standard * 54.3% 7.54% ' Medium Concentration " 100 ng/mL fo r Analyte Time o f Spiking 1. Analyte and IS after extraction Mean Ratio A nalvtdS 0.326 0.297 0.312 0.312 Ratio IS/Analvte 3.07 3.37 3.21 3.22 2. Analyte prior and IS after extraction Mean 0.117 0.121 0.113 0.117 3. IS prior and Analyte after extraction Mean 0.263 0.269 0.280 0.271 Mean extraction efficiency for the analyte " Mean extraction efficiency for the internal standard * 37.5% 8.42V. High Concentration 400 ng/mL fo r Analyte Time o f Spiking 1. Analyte and IS after extraction Mean Ratio Analvte/IS 1.17 1.25 1.21 1.21 Ratio IS/Analvte 0.855 0.797 0.828 0.827 2. Analyte prior and IS after extraction Mean 0.435 0J98 0.418 0.417 3. IS prior and Analyte after extraction Mean 0.0692 0.0652 0.0668 0.0671 Mean extracdon eftlciency for the analyte * Mean extracdon efflciencv for the interna! standard " 34.5% 8.11% Overol! mean extraction ejflciencyfo r analyte OvaraU mean extraeran efficiencyfo r inurnal standard - 42.1% 102% Page 60 Northwest Bioanalytical Study No. NWBS00-040 Report No. NWBR00-108 Table 41. PFOSA Extraction Efficiency Run 7 Low Concentration m 2,50 ng/mL fo r Analyte Time of Spiking I. Analyte and IS after extraction Ratio Analvte.*lS . 0.00603 0.006 U 0.00587 Mean 0 .0 0 6 0 0 Ratio [S/Analvte 166 164 170 167 2. Analyte prior and IS after extraction Mean 0.0037S 0.00434 0.00383 0.00397 3. IS prior and Analyte after extraction Mean 11.4 11.2 13.4 12.0 Mean extraction efficiency for the analyte * Mean extraction efficiency for the internal standard * 661.'/. 7.19% . Medium Concentration ** J 00 ng/mL fo r Analyte T im eofS pikim t - 1. Analyte and IS after extraction Mean Ratio A nalvte/IS 0.240 0.203 0.2U 0.219 Ratio iS/A nalvte 4.16 4.92 4.68 4.59 2. Analyte prior and IS after extraction Mean 0.148 0.128 0.152 0.143 3. IS prior and Analyte after extraction Mean 0.331 0.357 0.358 0.349 Mean extraction efficiency for the analyte * Mean extraction efficiency for the internal standard * 6 5 1 '/. 7.60% High Concentration " 400 ntfm L fo r Analyte T iiPfii?f.SB ikiniL . 1. Analyte and IS after extraction Mean Ratio AnalvtcflS 0.950 0.901 0.973 0.941 f o f o IS/A nalyte 1.05 1.11 1.03 1.06 2. Analyte prior and IS after extraction Mean 0.595 0.574 0.648 0.606 3. IS prior and Analyte after extraction Mean 0.0880 0.0778 0.0817 0.0825 Mean extraction efficiency for the analyte * 64.4V* Mean extraction efficiency for the Internal standard ______ 7,78*/ Overall mean extraction efficiency fo r analyte m Overall mean extraction efficiencyfo r internal standard 45,3% 7.52 % Page 61 Northwest Bioanalytical Study No. NWBS0O-040 Report No. NWBR00-108 Table 42. PFOSAA Extraction Efficiency Run 7 Low Concentration * 2.50 ng/mL fo r Analyte Time of Spikimr l. Analyte and IS after extraction Mean Ratio Analvte/IS 0.00526 0.00589 0.00559 0.00558 Ratio IS/Analvte 190 170 179 130 2. Analyte prior and (S after extraction Mean 0.00401 0.00427 0.00399 0.00409 3. IS prior and Analyte after extraction ' Mean 12.8 14.1 15.6 14.2 Mean extraction efficiency for the analyte * Mean extraction efficiency for the internal standard 73.3% 7.89% Medium Concentration m 100 ng/mL fo r Analyte Time of Spiking I. Analyte and IS after extraction Mean Ratio Analvte/IS 0.373 0.325 0.342 0.347 Ratio IS/Analvte 2.68 3.08 2.93 2.90 2. Analyte prior and IS after extraction Mean 0.254 0.270 0.249 0.258 3. IS prior and .Analyte after extraction Mean 0.234 0.238 0.248 0.240 M ean extraction efficiency for the analyte * Mean extraction efficiency for the internal standard 74.4% 8.28/ High Concentration * 400 ng/mL fo r Analyte Time o f Spiking 1. Analyte and IS after extraction Mean Ratio Analvte/IS 0.864 0.900 0.389 0.884 Ratio IS'Analvte 1.16 Ml 1.12 1.13 2. Analyte prior and IS after extraction Mean 0.642 0.624 0.658 0.641 3. IS prior and Analyte after extraction Mean 0.0944 0.0878 0.0885 0.0902 Mean extraction efficiency for the analyte Mean extraction efficiency for the internal itandard " 72.5% 7.98% . Overall mean extraction efficiencyfo r analyte m Overall mean extraction efficiencyfo r internal standard 73.4% 1.05% Page 62 Northwest Bioanalytical Study No. NWBS0O-O4O Report No. NWBR00-108 Table 43. POAA Extraction Efficiency Run 7 Low Concentration 2.50 ng/mL fo r Analyte Time ofSoiktnK I. Analyte and IS after extraction Mean Ratio Analvte/IS 0.00921 0.00982 0.0105 0.00934 Ratio IS/Analvte 109 102 95.0 102 2. Analyte prior and IS after extraction Mean 0.00209 0.00213 0.00224 0.00215 3. IS prior and Analyte after extraction Mean Mean extraction efficiency for the analyte Mean extraction efficiency for the internal standard 21.8% 7.30% 7.02 7.35 7.97 7.45 Medium Concentration * 100 ng/mL fo r Analyte Time of Soikintr ' 1. Analyte and IS after extraction Mean Ratio Analvte/IS 0.358 0.313 0.336 0.337 Ratio IS/Analvte 2.79 3.15 2.98 2.97 2. Analyte prior and IS after extraction Mean 0.0526 0.0570 0.0516 0.0537 3. IS prior and Analyte after extraction Mean * 0.227 0.237 0.248 0.237 Mean extracdoo efficiency for the analyte Mean extracdoo efficiency for the Internal standard * 15.9% 7.98% High Concentration * 400 ng/mL fo r Analyte Time q f Spiking , l. Analyte and IS after extraction Mean Ratio Analvte/IS 1.19 1.31 1.29 1.26 Ratio IS/Analvte 0.841 0.762 0.775 0.793 2. Analyte prior and IS after extraction Mean 0.205 0.201 0.216 0.207 3. IS prior and Analyte after extraction Mean 0.0633 0.0595 0.0627 0.0620 Mean extraction efficiency for the analyte Mean extracdoo efficiency for the internal standard 16.4% 7.82% Overall mean extraction efficiencyfo r analyte Overall mean extraction efficiencyfo r internal standard m 110% 7.70% Page 63 Northwest Bioanalytical Study No. NWBSOO-040 Report No. NWBR00-108 Table 44. PFHS Extraction Efficiency Run 7 Lo*v Concentration * 2.50 ng/mL fo r Analyte Tin ol'Soikinjr t. Analyte and IS after extraction Mean Ratio Analvte/IS 0.0133 O.OI63 0.0169 0.0165 Ratio ISAnalvte 63.2 59.6 59.3 60.7 2. Analyte prior and IS after extraction Mean 0.00371 0.00384 0.00399 0.00385 3. IS prior and Analyte after extraction Mean 4.43 4.47 5.27 4.72 Mean extraction efficiency for the analyte * Mean extraction efficiency for the internal standard * 23.3% 7.78% . Medium Concentration m 100 ng/mL fo r Analyte Time of Soficins l. Analyte and IS after extraction Mean Ratio AnalvteilS 0.632 0.567 0.598 0.599 Ratio fS/Analvte 1.53 1.76 1.67 1.67 2. Analyte prior and IS after extraction Mean 0.105 0.113 0.108 0.109 3. IS prior and Analyte after extraction Mean 0.134 0.146 0.146 0.142 Meaa extraction efficiency for the analyte Mean extraction efficiency for the internal standard * 18.2% 8.50% High Concentration 400 ng/mL fo r Analyte . T'TP*? 1. Analyte and IS after extraction Baiaj\MlYWflS.. 2.07 2.25 2.15 Mean 2.16 Ratio IS/Analvte 0.484 0.444 0.465 0.464 2. Analyte prior and IS after extraction , Mean 0.387 0.363 0.385 0.378 3. IS prior and Analyte after extraction Mean 0.0394 0.0360 0.0365 0.0373 Mean extraction efficiency for the analyte * Mean extraction efficiency for the internal standard * 17.5% 8.04% Overall mean extraction efficiencyfo r analyte " Overall mean extraction efficiencyfa r internal standard * 19.7% !% Page 64 Northwest Bioanalytical Study No. NWBSOO-040 Report No. NWBR00-108 Table 45. M556 Extraction Efficiency Run 7 Low Concentration " 2.50 ng/mL for Analyte Tirr* ofSoikinit 1. Analyte and IS after extraction Mean Ratio AnalvtC'IS 0.00635 0.00659 0.00684 0.00659 Ratio IS/Anatvie 157 152 146 152 2. Analyte prior and IS after extraction Mean 0.00293 0.00299 0.00279 0.00290 3. IS prior and Analyte alter extraction Mean Mean extraction efficiency for the analyte " Mean extraction efficiency for the internal standard 44.0% 7.43% 10.7 10.7 12.4 11.3 Medium Concentration = 100 ng/mL fo r Analyte Time of Spiking 1. Anai'.te and IS after extraction ' * Mean Ratio Analvte/TS 0.270 0.240 0.252 0.254 Ratio IS/Analvte 3.70 4.17 3.97 3.95 2. Analyte prior and IS after extraction Mean 0.106 0.124 o.m 0.114 3. IS prior and Analyte after extraction Mean 0.309 0.320 0.325 0.318 Mean extraction efficiency for the analyte Mean extraction efficiency for the Internal standard 44.9% 8.05% High Concentration 400 ng/mL fo r Analyte Time o f Snikinz I. Analyte and IS after extraction Mean Ratio AnalvtcflS 1.02 1.07 1.05 1.05 Ratio IS/Analvte 0.984 0.934 0.953 0.957 2. Analyte prior and IS after extraction Mean - 3. IS prior and Analyte after extraction Mean 0.444 0.441 0.463 0.449 0.0800 0.0744 0.0765 0.0770 Mean extraction efficiency for the analyte " Mean extraction efficiency for the internal standard " 42.8% 8.05% Overall mean extraction efficiencyfo r analyte m Overall mean extraction efficiencyfo r internal standard 43.9% 7.34% Page 65 Northwest Bioanalytical Study No. NWBSOO-040 Report No. NW BR00-108 Table 46. M570 Extraction Efficiency Run 7 Low Concentration m 2.S0 ng/mL fo r Analyte Time o f Spikim: 1. Analyte and IS after extraction Mean Ratio Analvte/lS 0.00470 0.00487 0.00530 0.00496 Ratio (S Analvte 213 205 189 202 2. Analyte prior and IS after extraction Mean 0.00317 0.00355 0.00320 0.00331 3. IS prior and Analyte after extraction Mean 14.4 14.0 15.8 14.7 Mean extraction efficiency for the analyte m Mean extraction efficiency for the internal standard * 66.7% 7.28% . Medium Concentration / OOng/m L fo r A naiyte ' Time o f Snilcina 1. Analyte and IS after extraction Mean Ratio A n a W I S 0.217 0.191 0.204 0.204 Ratio IS/Analvte 4.61 5.24 4.90 4.92 2. Analyte prior and IS after extraction Mean 3. IS prior and Analyte after extraction Mean 0.130 0.143 0.136 0.136 .... 0.391 0.396 0.422 0.403 Mean extraction efficiency for the analyte Mean extraction efficiency for the internal standard * 66.7% 8.19% High Concentration * 400 ng/mLfo r Analyte TinK.qfSnifcing ' 1. Analyte and IS after extraction Mean 2. Analyte prior and IS after extraction Mean Ratio AnalytcfIS . 0.772 0.831 0.796 0.800 0.533 0.521 0.563 0.539 Ratio IS/Analvte 1.29 1.20 1.26 1.25 2. IS prior and Analyte after extraction Mean 0.102 0.0970 0.0984 0.0991 Mean extraction efficiency for the analyte * Mean extraction efficiency for the internal standard 67.4% 7.93% Overall mean extraction efficiency for analyte m Overall mean extraction efficiencyfo r internal standard " ti.9% t.SOS Page 66 Northwest Bioanalytical Study No. NWBSOO-040 Report No. NWBR00-108 7. ANALYTICAL METHOD 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 API 3000. The ' instrument was operated in the multiple reaction monitoring (MRM) mode under optimized conditions for PFOS, PFOSA, PFOSAA, POAA, PFHS, M556 and M570 detection. CHEMICAL STRUCTURES .0 f2 f2 f2 II F,C f2 f2 f2 f2 PFOSA MW=499.0 [M-H]=498.0 F3C c F2 PFOSAA MW=585.0 [M-H]=584.0 POAA MW=414.0 [M-H]=413.0 O Page 67 Northwest Bioanalytical f 3c Study No. NWBS00-040 Report No. NWBROO-108 PFOS M[MW-H=]4=9499.98.9 f2 F3C c> c F2 PFHS M[MW-H=]3=9399.98.9 D Fv F- F2 1 f3c . / C . F2 Fi F, /1 F, o H ,O H O M556 M[MW-H=]5=5565.95.9 M570 M[MW-H=]5=7517.00.0 Page 68 Northwest Bioanalytical Study No. NWBS00-040 Report No. NWBR00-108 F, F, h2 f3c C, C. Cs C' C C' f 2 F2 F2 I THPFOS MW=428 7.1. Reference Materials and Matrices Analyte Lot Number Purity Expiration Date Source Storage Conditions PFOS (FC-95) 193 100% 12/31/2010 3M PFOSA 214 . 100% 12/31/2010 3M PFOSAA (FC-129) " 617 53.8% 12/31/2010 3M POAA (FC-143) 245 100% 12/31/2010 3M PFHS* M556 M570 S398-182 NB1130478D 118506-26 100% 99.89% 99.75% 12/31/2010 12/31/2010 12/31/2010 3M 3M 3M .THPFOS 59909 90% 12/31/2010 3M * Received as a 6,200 ppm solution in methanol. ** Kept dry. Room Temperature Room Temperature Room Temperature Room Temperature** -20C Room Temperature Room Temperature Room Temperature The reference material purity for PFOS, PFOSA, PFHS and POAA was not available prior to the conduct of this study. Therefore, all concentrations included in the report for these analytes are based upon an assumed purity of 100%. Prior to the completion of this final report, 3M contracted with Centre Analytical Laboratories, Inc. in State College, Pennsylvania to determine the absolute concentration of PFOS, POAA and PFHS in the stock standard solution prepared using seven reference materials (PFOS, PFOSA, PFOSAA, POAA, PFHS, M556 and Page 69 Northwest Bioanalytical Study No. NWBS0O-040 Report No. NWBROO-108 M570) at a target concentration of 5000 ppb. Absolute determination of the concentration of PFOSA in the stock standard solutions cannot be made until a full purity determination of these reference materials is completed. . Based on the results obtained, the concentrations included in this report should be corrected according to the following table: Analyte PFOS POAA PFHS Correction Factor 0.836 0.909 ' 0.855 Matrix Human serum Human plasma (Northern Chinese) Lot Numbers BC30399-4 C5186; C4929; C5517; C5200 Chemicals and Equipment Chemicals Ammonium acetate, 99.9% Di (ethylene glycol) methyl ether [may be written as 2-(2-methoxyethoxy) ethanol] Methanol, HPLC-grade Methyl-Terr-Butyl Ether (MTBE), HPLC-grade Water, HPLC-grade Name Autosampler: PE Series 200 Balance: Mettler Toledo MT5 Equipment/Supplies Source Perkin Elmer, Norwalk, CT Mettler-Toledo, Inc., Hightstown, NJ Page 70 Northwest Bioanalytical Study No. NWBSOO-040 Report No. NWBR00-108 Equipment/Supplies Name Source Centrifuge: Beckman GS-6R Beckman Instruments, : Fullerton, CA Evaporator: Turbo Vap LV, Model 43750 i Zymark Corp., Hopkinton, | MA Hematology/Chemistry Mixer, Model 346 HPLC Chromatographie column: Genesis Lightning, Cis, 4 pm, 2 x 50 mm Liquid Chromatograph: Hewlett Packard 1100 j Fisher Scientific, Pittsburgh, PA Jones Chromatography, Lakewood, CA Agilent (Hewlett Packard), Palo Alto, CA Liquid Chromatograph: PE Sciex Series 200 Perkin Elmer, Norwalk, CT Liquid Chromatograph: Shimadzu SCL-10A controller Shimadzu, Columbia, MD with CL-10AD pump and CTO-10A column oven Mass Spectrometer: Perkin Elmer Sciex API 3000 PE Sciex, Concord, Ontario Multi-tube Vortexer VWR Scientific Products, Bridgeport, NJ Pipettes: Eppendorf Repeater Pipette Brinkman Instruments, Inc., Westbury, NY Pipettes: Finnpipette: Digital 1-5 mL Fisher Scientific, Pittsburgh, PA Pipettes: Rainin EDP Digital lOO-lOOOpL TM Rainin Instrument Co., Wobum, MA Pipettes: Rainin M Volumes: adjustable 20-50, 10-100, 100-1000 pL Rainin Instrument Co., Wobum, MA Pipettes: Rainin RL Rainin Instrument Co., Volumes: adjustable 5-20, 20-100,40-200, 200-1000 Wobum, MA pL . Sonicator, Branson Branson, Danbury, CT Sonicator, Fisher Fisher Scientific, Fair Lawn, NJ - Vortex: Fisher Genie 2 Fisher Scientific, Fair Lawn, NJ Water Pro Plus Labconco, Co., Kansas City, MO Page 71 Northwest Bioanalytical Study No. NWBSOO-040 Report No. NWBROO-108 7.3. Reagents, Calibration Standard and Quality Control (QC) Solutions The calibrator, quality control and stock solution preparations listed below serve as a general guideline for attaining the targeted concentrations. Due to the fact that PFHS was received at NWB as a solution, both the calibration standard and quality control stocks are from the same solution and are not from separate weighings of reference material as per NWB SOP LABOP003. However, separate aliquots were used for calibration standard and QC stock solution preparation. Reagents All reagent solutions are stored at room temperature unless otherwise noted. 50 mM ammonium acetate in water (unadjusted: pH ~6.9) Fill a 1-L volumetric flask approximately half full with water. Weigh out 3.85 g of ammonium acetate and transfer to the flask. Use a stir plate to mix and fill to volume with water. . 20 mM ammonium acetate in water (unadjusted: pH ~6.9) Fill a 1-L volumetric flask approximately half full with water. Weigh out 1.54 g of ammonium acetate and transfer to the flask. Use a stir plate to mix and fill to volume with water. 20 mM ammonium acetate in methanol Prepare as described above, substituting methanol for water. . 30:70 20 mM ammonium acetate in water (unadjusted: pH ~6.9): 20 mM ammonium acetate in methanol (v/v) . 50:50 water:methanol (v/v) ' Calibration Standard Solutions All calibration standard solutions are transferred to polypropylene containers and stored in a -2 0 C freezer. The PFHS reference material was provided as a 6200 ppm solution. Page 72 Northwest Bioanalytical Study No. NWBSOO-040 Report No. NWBR00-108 PFOS Stock Standard (0.05904 mg/mL) Weigh 1.476 mg of PFOS and add to a 25-mL volumetric flask. Fill to volume with methanol, mix by inverting 5 times and sonicating for approximately 10 minutes. . PFOSA Stock Standard (0.07652 mg/mL) Weigh 1.913 mg of PFOSA and add to a 25-mL volumetric flask. Fill to volume with methanol, mix by inverting 5 times and sonicating for approximately 10 : . minutes. PFOSAA Stock Standard (0.02264 mg/mL) Weigh 1.052 mg of PFOSAA and add to a 25-mL volumetric flask. Fill to volume with methanol, mix by inverting 5 times and sonicating for approximately 10 minutes. POAA Stock Standard (0.03868 mg/mL) Weigh 0.967 mg of POAA and add to a 25-mL volumetric flask. Fill to volume with methanol, mix by inverting 5 times and sonicating for approximately 10 minutes. ; M556 Stock Standard (0.04579 mg/mL) Weigh 1.146 mg of M556 and add to a 25-mL volumetric flask. Fill to volume with methanol, mix by inverting 5 times and sonicating for approximately 10 minutes. M570 Stock Standard (0.04796 mg/mL) Weigh 1.202 mg of M570 and add to a 25-mL volumetric flask. Fill to volume with methanol, mix by inverting 5 times and sonicating for approximately 10 minutes. . Spiking Standard 9 (5000 ng/mLfor PFOS, PFOSAA, POAA, PFHS, M556 and M5 70; 5020 ng/mLfor PFOSA) Add 2.117 mL of PFOS Stock Standard (0.05904 mg/mL), 1.639 mL of PFOSA Page 73 Northwest Bioanalytical Study No. NWBSOO-040 Report No. NWBR00-108 Stock Standard (0.07652 mg/mL), 5.521 mL of PFOSAA Stock Standard (0.02264 mg/mL), 3.232 mL of POAA Stock Standard (0.03868 mg/mL), 20.16 |iL of PFHS (6200 ppm), 2.73 mL of M556 Stock Standard (0.04579 mg/mL) and 2.606 mL of M570 Stock Standard (0.04796 mg/mL) to a 25-mL volumetric flask. Fill to volume with 50:50 water:methanol and mix by inverting 10 times and sonicating for approximately 10 minutes. Spiking Standard 8 (4000 ng/mLfor PFOS, PFOSAA, POAA, PFHS, M556, : ' M570; 4020 ng/mLfor PFOSA) Add 8.00 mL of Spiking Standard 9 to a 16 x 100 mm polypropylene tube and add 2.00 mL of 50:50 watenmethanol. Mix by inverting 20 times. . Spiking Standard 7 (2500 ng/mLfor PFOS, PFOSAA, POAA, PFHS, M556.M570; 2510 ng/mLfor PFOSA) Add 5.00 mL of Spiking Standard 9 to a 16 x 100 mm polypropylene tube and add 5.00 mL of 50:50 watenmethanol. Mix by inverting 20 times. . Spiking Standard 6 (1000 ng/mLfor PFOS, PFOSA, PFOSAA, POAA, PFHS, M556 and M570) Add 2.00 mL of Spiking Standard 9 to a 16 x 100 mm polypropylene tube and add 8.00 mL of 50:50 watenmethanol. Mix by inverting 20 times. . Spiking Standard 5 (500 ng/mLfor PFOS, PFOSAA, POAA, PFHS, M556, M570; SOingtmL for PFOSA) Add 1.25 mL of Spiking Standard 8 to a 16 x 100 mm polypropylene tube and add 8.75 mL of 50:50 watenmethanol. Mix by inverting 20 times. . Spiking Standard 4 (250 ng/mLfor PFOS, PFOSAA, POAA, PFHS, M556, M570; 251 ng/mLfor PFOSA) Add 0.500 mL of Spiking Standard 9 to a 16 x 100 mm polypropylene tube and add 9.50 mL of 50:50 watenmethanol. Mix by inverting 20 times. Page 74 Northwest Bioanalytical Study No. NWBSOO-040 Report No. NWBR00-108 Spiking Standard 3 (100 ng/mLfor PFOS, PFOSAA, POAA, PFHS, M556, M570; 10i ng/mLfor PFOSA) ' Add 0.250 mL of Spiking Standard 8 to a 16 x 100 mm polypropylene tube and add 9.75 mL of 50:50 watenmethanol. Mix by inverting 20 times. Spiking Standard 2 (25.0 ng/mLfor PFOS. PFOSAA, POAA, PFHS, M556, M570; 25.1 ng/mLfor PFOSA) Add 0.100 mL of Spiking Standard 7 to a 16 x 100 mm polypropylene tube and ' ' add 9.90 mL of 50:50 watenmethanol. Mix by inverting 20 times. . Spiking Standard 1 (10.0 ng/mLfor PFOS, PFOSA, PFOSAA, POAA, PFHS, M556 and M570) Add 0.100 mL of Spiking Standard 6 to a 16 x 100 mm polypropylene tube and add 9.90 mL of 50:50 watenmethanol. Mix by inverting 20 times. Internal Standard Solutions All internal standard solutions are transferred to polypropylene containers and stored in a -20 C freezer. THPFOS Stock Solution (0.04842 mg/mL) Weigh 0.2690 mg of THPFOS and add to a 5-mL volumetric flask. Fill to volume with 50:50 watenmethanol and mix by inverting 5 times and sonicating approximately 10 minutes. Working Internal Standard (400 ng/mL THPFOS) Add 413 pL of the THPFOS Stock Solution (0.04842 mg/mL) to a 50-mL volumetric flask and fill to volume with 50:50 watenmethanol. Mix by inverting 5 to 10 times and sonicating approximately 5 to 10 minutes. Quality Control (OC) Solutions All quality control solutions are transferred to polypropylene containers and stored in a -20 C freezer. Page 75 Northwest Bioanalytical Study No. NWBS00-040 Report No. NWBR00-108 . PFOS QC Stock (0.07732 mg/mL) Weigh 1.933 mg of PFOS and add to a 25-mL volumetric flask. Fill to volume with methanol, mix by inverting 5 times and sonicating for approximately 10 minutes. . PFOSA QC Stock (0.04876 mg/mL) - Weigh 1.219 mg of PFOSA and add to a 25-mL volumetric flask. Fill to volume With methanol, mix by inverting 5 times and sonicating for approximately 10 ' -minutes. . PFOSAA QC Stock (0.02920 mg/mL) ' Weigh 1.357 mg of PFOSAA and add to a 25-mL volumetric flask. Fill to volume with methanol, mix by inverting 5 times and sonicating for approximately 10 minutes. ' . POAA QC Stock (0.04228 mg/mL) Weigh 1.057 mg of POAA and add to a 25-mL volumetric flask. Fill to volume with methanol, mix by inverting 5 times and sonicating for approximately 10 minutes. . PFHS QC Stock (1000 ppm) Aliquot 807/xL of PFHS stock solution (6200 ppm) to a 5-mL volumetric flask. Fill to volume with methanol and mix by inversion. . M 556QC Stock (0.05230 mg/mL) Weigh 1.309 mg of M556 and add to a 25-mL volumetric flask. Fill to volume with methanol, mix by inverting 5 times and sonicating for approximately 10 minutes. . M570 QC Stock (0.05474 mg/mL) Weigh 1.372 mg of M570 and add to a 25-mL volumetric flask. Fill to volume with methanol, mix by inverting 5 times and sonicating for approximately 10 minutes. Page 76 Northwest Bioanalytical Study No. NWBSOO-040 Report No. NWBROO-108 PFOS Concentrated QC Solution (997,000 ng/mL) Evaporate 1.69 mL of PFOS (0.07732 mg/mL) at 20 C and below 5 P.S.I. nitrogen. Reconstitute with 131 fiL of methanol. PFOSA Concentrated QC Solution (1,000,000 ng/mL) Evaporate 2.05 mL of PFOSA (0.04876 mg/mL) at 20 C and below 5 P.S.I. nitrogen. Reconstitute with 100 iL of methanol. . PFOSAA Concentrated QC Solution (1,000,000 ng/mL) .^Evaporate 4.42 mL of PFOSAA (0.02920 mg/mL) at 20 C and below 5 P.S.I. nitrogen. Reconstitute with 129 /L of methanol. . POAA Concentrated QC Solution (996,000 ng/mL) Evaporate 2.59 mL of POAA (0.04228 mg/mL) at 20 C and below 5 P.S.I. nitrogen. Reconstitute with 110 ptL of methanol. . PFHS Concentrated QC Solution No PFHS concentrated solution was prepared. . M556 Concentrated QC Solution (1,000,000 ng/mL) Evaporate 2.18 mL of M556 (0.05230 mg/mL) at 20 C and below 5 P.S.I. nitrogen. Reconstitute with 114 fiL of methanol. . M570 Concentrated QC Solution (1,000,000 ng/mL) . Evaporate 2.09 mL of M570 (0.05474 mg/mL) at 20 C and below 5 P.S.I. nitrogen. Reconstitute with 114.4 /iL of methanol. 7.4. Preparation of Validation Quality Control Samples ' All quality control target concentrations will be corrected for the persistent levels of the analytes in human serum. . Page 77 Northwest Bioanalytical Study No. NWBSOO-040 Report No. NWBR00-108 Dilution Quality Control 0990 ng/mL PFOS. 4000 ng/mL PFOSA. 4000 ng/mL PFOSAA, 3980 na'mL POAA. 4000 ng/mL PFHS. 4000 ng/mL M556 and 4000 ng/mL M570) Transfer 100 pL of each Concentrated QC Solution (997,000 ng/mL PFOS, 1,000,000 ng/mL PFOSA, 1,000,000 ng/mL PFOSAA, 996,000 ng/mL POAA, 1,000,000 ng/mL M556, and 1,000,000 ng/mL M570) and 100 juL of the 1000 ppm PFHS QC Stock intefa 25-mL volumetric flask. Fill to volume with human serum and mix by .inversion. Sonicate for approximately 10 minutes and equilibrate for approximately 10-minutes. ' High Quality Control (399 ng/mL PFOS, 400 ng/mL PFOSA. 400 ng/mL PFOSAA. 398 ng/mL PO AA.. 400 ng/mL PFHS. 400 ng/mL M556 and 400 ng/mL M5701 Transfer 5.00 mL of the Dilution Quality Control into a 50-mL volumetric flask. Fill to volume with human serum and mix by inversion. Sonicate for approximately 10 minutes and equilibrate for approximately 10 minutes. Medium Quality Control (150 ng/mL PFOS. 150 ng/mL PFOSA. 150 ng/mL PFOSAA. 149 ng/mL POAA. 150 ng/mL PFHS. 150 ng/mL M556 and 150 ng/mL M5701 Transfer 1.875 mL of the Dilution Quality Control into a 50-mL volumetric flask. Fill to volume with human serum and mix by inversion. Sonicate for approximately 10 minutes and equilibrate for approximately 10 minutes. Low Quality Control f3.99 ng/mL PFOS. 4.00 ng/mL PFOSA. 4.00 ng/ mL PFOSAA. 3.98 ng/mL POAA. 4,00 ng/mL PFHS. 4.00 ng/mL M556 and 4,00 ng/mL M570) Transfer 0.500 mL o f the High Quality Control into a 50-mL volumetric flask. Fill to volume with human serum and mix by inversion. Sonicate for approximately 10 minutes and equilibrate for approximately 10 minutes. Storage o f QC Samples After preparation, place aliquots of the low, medium, high and dilution QC pools into cryogenic vials, and store in a -20C freezer. Page 78 Northwest Bioanalytical Study No. NWBS00-040 Report No. NWBR00-108 7.5. Preparation of PFOSA and PFOSAA Specific Validation Quality Control Samples Quality Control COO Solutions All quality control solutions are transferred to polypropylene containers and stored in a -20 C freezer. PFOSA QC Stock (0.2166 mg/mL) Weigh 5.414 mg of PFOSA and add to a 25-mL volumetric flask. Fill to volume with methanol, mix by inverting 5 times and sonicating for approximately 10 minutes. ' PFOSAA QC Stock (0.2322 mg/mL) Weigh 10.790 mg of PFOSAA and add to a 25-mL volumetric flask. Fill to volume with methanol, mix by inverting 5 times and sonicating for approximately 10 minutes. Preparation of Quality Control Samples All quality control target concentrations will be corrected for the persistent levels of the analytes in human serum. Dilution Quality Control 4000 ng/mL PFOSA and PFOSAA1 Transfer 0.462 mL of the PFOSA QC Stock Solution (0.2166 mg/mL) and 0.431 mL of the PFOSAA QC Stock Solution (0.2322 mg/mL ) into a 25-mL volumetric flask. Fill to volume with human serum and mix by inverting approximately 10 times. Sonicate for 5 minutes and equilibrate for approximately 5 minutes. High Quality Control (400 ng/mL PFOSA and PFOSAAl Transfer 2.50 mL of the Dilution Quality Control into a 25-mL volumetric flask. Fill to volume with human serum and mix by inverting approximately 10 times. Sonicate for 5 minutes and equilibrate for approximately 5 minutes. . Page 79 Northwest Bioanalytica! Study No. NWBS00-040 Report No. NWBR00-108 Medium Quality Control (T50 ng/mL PFOSA and PFOSAA) Transfer 0.938 mL of the Dilution Quality Control into a 25-mL volumetric flask. Fill to volume with human serum and mix by inverting approximately 10 times. Sonicate for 5 minutes and equilibrate for approximately 5 minutes. Low Quality Control (4.00 ng/mL PFOSA and PFOSAA) Transfer 0.250 mL of the High Quality Control into a 25-mL volumetric flask. Fill to volume with human serum and mix by inverting approximately 10 times. Sonicate ` for 5'minutes and equilibrate for approximately 5 minutes. Storage o f QC Samples - After preparation, place aliquots of the low, medium, high and dilution QC pools into cryogenic vials, and store in a -20C freezer. 7.6. Recommended Calibration Standard and Quality Control Preparation for Analysis While concentrated solutions of the analytes can be used to prepare calibration standards and quality control samples, the evaporation of stock solutions to prepare concentrated stock solutions is not recommended for PFOSA and PFOSAA. 7.7. Preparation of Calibration Standards The calibration curve is prepared on the day of each run by adding 100 pL blank human serum and 400 pL of 50 mM ammonium acetate in water (unadjusted: pH -6.9) to 13 x 100 mm polypropylene tubes. After a brief vortex mixing, spike 10.0 pL of the appropriate spiking solution into the tubes as shown in the table below. Standard Number 9 8 7 Concentration of Spiking Solution (ng/mL) PFOS, PFOSAA, POAA, PFHS, M556, M570 / PFOSA 5000 / 5020 4000 / 4020 2500/2510 Volume of Spiking Solution (PL) 10.0 10.0 10.0 Volume of Blank Human Serum (pL) Final Concentration* (ng/mL) 100 500/502 100 400 / 402 100 250/251 Page 80 Northwest Bioanalytical Study No. NWBS00-O40 Report No. NWBROO-108 Standard Number 6 5 4 3 _2 1 Concentration of Spiking Solution (ng/mL) PFOS. PFOSAA, POAA, PFHS, M 5 5 6 , M 5 7 0 / PFOSA 1000 500/503 250/251 100/101 25.0/25.1 10.0 Volume of Spiking Solution (pL) 10.0 10.0 10.0 10.0 10.0 10.0 Volume of Blank Human Serum (pL) Final Concentration* (ng/mL) 100 IQO 100 50.0/50.3 100 25.0/25.1 100 10.0/10.1 100 2.50/2.51 100 1.00 * The target calibration curve range is 1.00 ng/mL to 500 ng/mL. Each analyte has a different final curve range based upon the persistent levels of the analyte in the human serum used. 7.8. Sample Preparation Calibration Curve Samples (prepare in duplicate') as indicated above Quality Control Samples (prepare in duplicate) Aliquot 100 pL of each of the Low, Medium and High controls into separate 13 x 100 mm polypropylene tubes. If needed, prepare a dilution control (in triplicate) according to the following formula: for DF=X, aliquot (0.500 mL/X) of dilution control into [0.500 mL --(0.500 mL/X)] of blank human serum. Aliquot 100 pL of the prepared dilution control into separate 13 x 100 mm polypropylene tubes. Blank Control Samples (prepare in duplicate) Aliquot 100 pL of blank human serum into separate 13 x 100 mm polypropylene tubes and label as QC0. Aliquot 100 pL of blank human serum into separate 13 x 100 mm polypropylene tubes and label as BLANK. Page 81 Northwest Bioanalytica Study Samples Study No. NWBSOO-040 Report No. NWBR00-108 Transfer 100-jiL aliquots of each study sample into appropriately labeled 13 x 100 mm polypropylene tubes. If necessary, dilute study samples in the same manner as the Dilution QC. Extraction Procedure 1. Add 400 pL of 50 mM ammonium acetate in water (unadjusted: pH ~6.9) to each sample except calibrators and vortex mix briefly. 2. Add 50.0 pL o f Working Internal Standard to each sample (except Blanks) and vortex mix for approximately 30 seconds. 3. Centrifuge samples at 3000 rpm for approximately 2 minutes. 4. Add 3 mL of methyl-teri-butyl ether to each tube, cap and vortex mix for approximately 30 seconds. 5. Rotate samples for approximately 10 minutes. 6. Centrifuge samples at 3000 rpm for approximately 10 minutes. 7. Transfer the top MTBE layer into clean 13 x 100 mm polypropylene tubes using a transfer pipette. 8. Evaporate the organic layer to dryness in a TurboVap set at 25C under gentle nitrogen stream (setting <5 psi) for approximately 40 minutes. 9. Reconstitute in 100 jiL of 30:70 20 mM ammonium acetate in water (unadjusted: pH ~6.9): 20 mM ammonium acetate in methanol and vortex mix for approximately 15 seconds. 10. Transfer samples to autosampler vials and centrifuge at 3000 rpm for approximately 2 minutes Page 82 Northwest Bioanalytical 7.9. LC/MS/MS Conditions Study No. NWBS0O-040 Report No. NWBR00-108 LC Conditions HPLC Column Mobile Phases LC Conditions Genesis Lightning Cis, 4 pm, 2x50 mm A: 20 mM ammonium acetate in water (unadjusted: pH ~6.9) B: 20 mM ammonium acetate in methanol Gradient Time (min.) %B 'i 0 50 1 50 7 97.5 9 97.5 9.1 50 11 end Flow Rate 300 pL/minute Post Column Addition 1 50 pL/minute of 2-(2-methoylethoxy) ethanol Column Temperature 40C Injection Volume 2 - 20 pL MS Conditions Post Column Split Source Source Temp. Ionization Mode Analysis type None TurboIonSprayTM (flow rate 8 L/min.) 400 C Negative Ion Multiple reaction monitoring (MRM) PFOS PFOSA Transitions monitored (0.3) 499 -> 80 498 78 Dwell Time (ms) 70 70 Collision Energy (eV) 80 65 Page 83 Northwest Bioanalytical Study No. NWBS00-040 Report No. NWBR00-108 PFOSAA POAA PFHS M556 M570 THPFOS Transitions monitored (0.3) 584 -4- 419 413 ->169 399 -> 80 556 -> 498 570 ->419 427 -> 407 Dwell Time (ms) 70 70 70 400 70 70 Collision Energy (eV) 29 25 70 40 42 42 The prepared standards and QCs were injected into the instrument LC/MS/MS system in a systematic order. . 7.10. Quantitation PFOS, PFOSA, PFOSAA, POAA, PFHS, M556, M570 and THPFOS chromatographic peaks are integrated using PE Sciex MacQuan software (version 1.6) with a smooth factor of 1. Several of the analytes possess iosmers. The chromatographic conditions of this method can achieve baseline separation of these isomers. Because the goal of this method is to determine the total amount for each analyte, all isomer peaks are integrated together and the peak areas are treated as a single peak by MacQuan. Quantitation is based upon quadratic regression analysis of calibration curves (weighted 1/x2) using the area ratio vs. concentration by the Watson DML1MS software (version 6.1.1.04) Page 84 Northwest Bioanalytical Study No. NWBS00-040 Report No. NWBR00-108 Figure 1. Representative Calibration Curve for PFOS Analytical Run 13 analyzed on 29-Jun-2000 Calibration Standards for PFOS (ng/mll Regression Method QUADRATIC Weighting Factor l/X**2 Quadratic Limit * 1930 Instrument Response 0 100 200 300 400 . 500 ` Nominal Cone. (ngftnL) Figure 2. Representative Calibration Curve for PFOSA Analytical Run 13 analyzed on 29-Jun-2000 Calibration Standards for PFOSA (ng/mT) Regression Method - QUADRATIC - Weighting Factor " l/X**Z Quadratic Limit 1560 600 ln Atraillen! R p o n s e Page 85 Northwest Bioanalytical Study No. NWBS00-040 Report No.NWBROO-108 Figure 3. Representative Calibration Curve for PFOSAA A nalytical R un 13 analyzed on 2 9 -Ju n -2 0 0 0 C alibration S tan d a rd s for PFO SA A ( n g m l) R ec essio n M ethod * Q U A D R A TIC W eighting Factor 1/X**2 Q uadratic Limit 3880 Inurnment Ropn Figure 4. Representative Calibration Curve for POAA Analytical Run 13 analyzed on 29-Jun2000 Calibration Standards for POAA (ng/ml) Regression Method QUADRATIC - Weighting Factor * ]/X**2 Quadratic Limitm-4810 Inurnment Region: Page 86 Northwest Bioanalytical Study No. NWBS0O-040 Report No. NWBR00-108 Figure 5. Representative Calibration Curve for PFHS Analytical Run 13 analyzed on 29>Jun*2000 Calibration Standards for PFHS Ing-ml) Recession Method - QUADRATIC - Weighting Factor * i X**2 Quadratic Limit - 1870 Insmiiiioii Response Figure 6. Representative Calibration Curve for M556 A n aly tical R un 13 analyzed on 2 9 -Ju n -2 0 0 0 C alib ratio n S ta n d a rd s for M S56 (ng/m l) Regression M ethod QUADRATIC W eighting Factor UX**2 Quadratic Limit * -1 7 1 0 Inanim o Response Page 87 Northwest Bioanalytical Study No. NWBS00-040 Report No. NWBR00-108 Figure 7. Representative Calibration Curve for M570 A nalytical R un 13 analyzed on 29-Jun*2000 C alibration S tan d ard s for M 570 (ng/m l) Regression M ethod - QUADRATIC * W eighting Factor - l/X * * Q uadratic Limit 1770 Instrument Region* Page 88 Northwest Bioanalytical Study No. NWBS00-040 Report No. NWBROO-108 Figure 8. Standard (57.1 ng/mL) Chromatogram for PFOS internal Standard' TH PFO S U se Area Absolute Retention Time Eipeeted RT 5 35 Current Method N oise Three. Quant Three. 10.0 2.0 too 2.0 Mm. width 5 5 20 20M utt Width 100 100Base. Width 20 20RT Win. (seca) 1 1Smooth "`CZ'jTOZ ~"<33 30CG62 Std J : No Comineo! > ,. .u n 29. 2000 : -D A M 3 S hi 1 penod 1oc 1: 6 59 M R M . 446 scans io 4 9 9 .0>6 0 .0 so- Area.. 602815 Hoqht 63160 Start Time End Time intearation Width Retention Time intec ration Tvoe m+MMa 5:05 6:03 0:58.1 5:35 A - BB OE3 70SO SO40 30 20- 10 . 20 43 SS SI04-- i-- i-- i-- 1:14 ,- 121 181 2:26 3:39 ITKPFQS u se as interna! Standard Exoected RT 4:56 I Current Method Noise Three. 50.0 50.0 Quant Three. 5.0 5.0 Min. Width 12 12 Mutt Width 10 10 Baee. Width 100 100 RT Win. (eecs) 20 20 Smooth t 1 00c207003 7 0 03 S 00062 SU13 1 1 No Comment intensity : 63187 cps 277 J ....................................... 241 301 3S1 421 Scan-- 4:52 6:04 7:17 S :3 0 Time|-{ Page 89 Northwest Bioanalytical Study No. NWBS00-040 Report No. NWBROO-108 Figure 9. Standard (10.1 ng/mL) Chromatogram for PFOSA Ip f o s a Internal Standard: T H P F O S U se Area Absolute Retention Time Expected R T 6:18 Current Method Noise Three. 200 20.0 Q uant Thres. 4.0 4.0 Min. Width 53 M u ll Width 12 12 Base. Width 100 100 RT Win. (sees) 20 20 Smooth 1 1 006207003 ?003 300062 S l d i ? Thu. Jun 29. 2000 2 tO AM 9 59 m 1 penod 1: 6:59 M R M , 446 scans 498.0>78.0 Area 261240 Hetqht 31061 S tart Time End Time Integration Width Retention Time Integration Type rtTikH a 5:50 6:36 0:48.4 6:18 A B0 he No Comment in tffu ity : 31176 eps IT B P FO S use as Internat Standard Expected RT 4:56 I Current Method Noise Thres. 50.0 50.0 Q uant Thres. 5.0 5.0 Min. Width 12 12 MuK. Width 10 10 Base. Width 100 100 R T Win. (secs) 20 20 Smooth 1 1 006207003 7 003 S 00062 Std3 t 1 Thu. Jun 29. 2000 2 10 A M 6.59 >n i p e rcd l: 6:59 M RM . 446 scans 427.0->407.0 Area 256706 HetQM 40943 Start Time End Time Integration Width Retention Time infeorabon Type 4:47 5:09 0:21.8 4:57 A B8 No Comment intensity : 41002 cps Page 90 Northwest Bioanalytical Study No. NWBSOO-040 Report No. NWBR00-108 Figure 10. Standard (15.0 ng/mL) Chromatogram for PFOSAA IPFOSAA Internal Standard T H P F O S Use Area Assolute Retention Time Expected R T 6:32 Current Method N oise Thres. 12.0 12 0 Q uant Three. 4 0 4 0 Min. Width 6 6 Mult. Width 15 15 Base. Width 100 100 RT Win. (secs) 20 20 Smooth 1 1 ' U 7 0 O 3 7 riQ3 3 0 3lfl3 ! 1 Thu. Juti 29. 2000 2 10 AM 6 59 ui 1 penod 1: 8:59 M R M , 446 scans 564.0->419.t Area 76838 Height 11102 Start Time End Time intearation Width Retention Time Intearabn Tvoe 6:10 6:44 0:33.9 6:32 A B8 No Comment intensity : 11172 cp* ITHPFOS u m as Internai Standard Expected R T 4:50 Current Method Noise T V e s. 50.0 50.0 Quant Three. 5.0 5.0 Mm. Width 12 12 M u lt Width 10 to Base. Width too too R T Win. (secs) 20 20 Smooth t 1 0011207003 7 0 03 S0CQ62 3ui3 1 t Thu. Jim 29. 20C0 2:10 A M 6.59 in 1 penod 1: 8:SB M RM . 448 scans 427.0->407.0 Area Heiaht 256706 40943 Start Time End Time Integration Width Retention Time Integration Tvoe ussy 4:47 5:09 0:21.8 4:57 A SB No Comment intensity : 4 1002 cp* Page 91 Northwest Bioanalytical Study No. NWBSOO-040 Report No. NWBR00-108 Figure 11. Standard (14.8 ng/mL) Chromatogram for POAA internal Standard. r H P F C S U se Area Aosoiute Retention Time Expected RT 4 59 Current Method Noise Thres. 20 0 20 0 Q uart Thres. 20 2.0 . Wm Width toMutt w idth tooBase. Width 6 100 8 10 R T Win. (secs) 20 20 Smooth 1 1 5 M 2970 3 7 C03 SJC 0 6 2 St-33 t i Tini. Jun 29.2000 2 :0 AM 3 59 M oenod 1: 8:59 M R M , 448 s can s 413.0->169.1 Area 77412 Heiqht 8757 Start Time End Time lotearaben w idth Retention Time integration Type 111+MHa 4:34 5:18 0:44.8 5:00 A - 08 SHI No Comment intensity : 878S eps (t h p f o s ~~ u se a s Internal Stand ard Expected RT 4:58 I Current Method Noise Thres. 50.0 50.0 Q uant Thres. 5.0 $.0 Min. Width 12 12 Mutt. Width 10 10 Base. Width 100 100 R T Win. (secs) 20 20 Smooth 1 1 306207003 7 0 03 S 00062 Std3 1 I Thu. Jim 29. 2000 2.10 A M 8.59 m t penod t: 8:59 M R M . 448 scan s 427Q-407.Q Area 258706 H eio h t 40943 Start Time End Time integration Width Retention Time i t l i w aIntegrabon Tvoe 4:47 5:09 0:21.8 4:37 H QA BB No Comment intensity: 41 0 0 2 eps Page 92 Northwest Bioanalytical Study No. NWBS00-040 Report No. NWBR00-108 Figure 12. Standard (12.2 ng/mL) Chromatogram for PFHS internat Standard. T N P F O S Use Area Absolute Retention Time Expected R T 4 17 Current Method Noise Thres. 200 20 0 Q uant Thres. 2.0 2.0 Min. Width 55 MuH, Width 10 10 Base. Width 100 100 R T Win. (secs) 20 20 Smooth 1 1 >V.t2t:?003 7 X3 SOOM2 SlOd 1 Thu. Jun 29. 2000 2 10 AM 3 59 ui 1 oenod 1: 8:59 M R M . 448 s can s 399.0>79.9 Area 77378 Weight 9782 S tir i Time End Time integration W idth Retention Tim e Integration Tvoe 3:57 4:30 0:32.7 4:18 A B8 Hi H hbI No Continent intensity : 9818 cps ITHPFQS use as Internai Standard Expected R T 4:58 Noise Three. Quant Three. Min. Width Mutt Width Base. Width R T Win. (secs) Smooth Method 50.0 3.0 5.0 12 12 ___ 10. 10 100 100 1 120 20 X>207003 7 003 S0062 Std3 1 I Thu. Jun 29. 2000 2:10 AM d 59 in i penod t: 8:59 M R M . 448 scans 427.0-407.0 Area 258706 Height 40943 Start Time End Time Integration Width Retention Tim e [tl4hHaIntegration Tvoe 4:47 5:09 0:21.8 4:57 E QA - S B No Comment intensity : 41002 eps Page 93 Northwest Bioanalytical Study No. NWBS00-040 Report No. NWBR00-108 Figure 13. Standard (11.8 ng/mL) Chromatogram for M556 Im SSS internal Standard. TH PFO S Use Area Absolute Retention Time Expected RT 6.04 Current Method Noise Thres. Quant Three. Min. Width Mutt. Width Base. Width 12.0 S4.0 12 4 00 5 to too 10 100 1RT Win. (sacs) Smooth 20 1 20 n(i2>7W\ 70C3 SQ0G62 S OJ 1 t No Comment Thu. Juu 29. 2000 2 10 AM 9 59 in i senod t: 8:59 M RM . 446 scans 1oc io 556.(^*498.0 so- Area 15635 Heioht 1227 Start Time End Time Intear?tion Width Retention Time Integration Tyoe HHnn& 5:45 6:33 0:484 6:02 A . SB .70 60 30 40 30 20- 10- 0 11 61 I :14 299 12122:2116 .` 161368:319 T --224T41--4 4:52 360:014 intensity : 1243 cps 376:117 462:310 STcmanel--a ITNPFOS u m as internai Standard Expected AT 4:58 NoiMI Current Method Three. 50.0 Quant Three. Mm. w idth Mu. Width 5.0 12 __ 10 5.0 12 10 8 a. Width 100 100 RT Win. (sacs) Smooth 120 20 1 006207C03 7 003 S0G062 Std3 1 t Thu. Jun 29. 2000 2:10 AM 6.59 m * period 1: 6:59 MRM. 448 scans 427.0->407.0 Area 256706 Heioht 40043 Start Tune End Time Intearation Width Retention Time Integrabon Tvoe [t I4 l*HI(SI 4:47 5:09 0:21.8 4:57 A 0B No Comment intensity : 41002 cps Page 94 Northwest Bioanalytical Study No. NWBS0O-O4O Report No. NWBROO-108 Figure 14. Standard (14.6 ng/mL) Chromatogram for M570 internal Standard: T H P P O S Use Area Absolute Retention Time Expected RT 6.18 Current Method Noise Three. Quant Three. 12.0 20 12.0 2.0 Min. Width 53 Mult, w idth 10 10 Base. Width too 100 RT Win. (sees) 20 20 Smooth 1 1 V.C'J?50 7 003 SCf.062 Sld3 * Thu. ,, um 29. 2000 2 10 A M 3 53 'M t period 1: 8.59 M RM . 446 scan s 570 0^419.1 Area 21349 Height 2928 Stan rune End Time Integration Width 6:02 6:28 0:26.6 Retention Time Integration Type 6:19 A BB im m us H U No Comment intensity : 2958 eps ITHPFQS use as Internai Standard Expected RT 4:58 ] Current Method N o is e T h re s . 50.0 50.0 Quant Three. 5.0 5.0 Min. Width 12 12 Mutt. Width 10 10 B ase . Width 100 100 R T Win. (secs) 20 20 Smooth 1 1 006207003 7 003 S00062 Std3 1 t Thu. Jun 29. 2000 2:10 AM 8 59 m 1 penod t: 8:59 M R M . 446 s can s 427.0->407.0 Area 256706 Height 40943 Start Time End r*ne integration Width Retention Time Integration Tvoe 4:47 5:09 0:21.8 4:57 A-BB 630 No Comment intensity : 41002 cps Page 95 Northwest Bioanalytical Study No. NWBS0O-O4O Report No. NWBROO-108 APPENDIX A The following quality control results are from sample analysis study NWBS00-062. Table A.l. Inter -Assay Precision and Accuracy for PFOSA Quality Control Samples All concentrations are expressed as ng/mL. Run Date Run Number Low QC 4.00 ng/mL . 22Jul-2000 . 21 3.95 *5.18 25-M-2000 22 4.14 3.50 31-Jui-2000 23 3.68 4.35 01-Aug-2000 24 4.14 3.84 02-Aug-2000 26 4.51 3.96 09-Aug-2000 29 3.70 4.9 12-Aug-2000 . 31 4.17 3.95 14-Aug-2000 32 3.92 3.86 15-Aug-2000 33 4.98 3.81 16-Aug-2000 36 4.20 3.33 17-Aug-2000 37 4.56 3.43 Medium QC 150 ng/mL 158 153 171 ' 152 151 141 150 144 169 161 141 134 148 148 142 147 156 157 144 126 149 121 High QC 400 ng/mL 461 364 394 379 345 356 395 359 378 406 * 390 364 382 346 319 364 403 413 409 382 384 470 * >25% theoretical Page 96 Northwest Bioanalytical Study No. NWBS00-040 Report No. NWBROO-108 Table A.l. Inter -Assay Precision and Accuracy for PFOSA Quality Control Samples (continued) All concentrations are expressed as ng/mL. Run Date Run Number Low QC 4.00 ng/mL 24-Aug-2000 42 4.57 ; 3.06 26-Aug-2000 43 4.61 4.02 28-Aug-2000 44 3.97 3.87 30-Aug-2000 46 3.85 ' 3.84 Medium QC 150 ng/mL 148 136 162 161 142 147 137 145 High QC 400 ng/mL 471 425 403 388 409 ' 392 381 474 Mean S.D. %CV %Theoretical %Bias n 4.03 0.458 11.4 100.8 0.8 30 148 11.3 7.6 98.7 -1.3 30 394 37.8 9.6 98.5 -1.5 30 Page 97 Northwest Bioanalytical Study No. NWBSOO-040 Report No. NWBR00-108 Table A.2. Inter -Assay Precision and Accuracy for PFOSAA Quality Control Samples All concentrations are expressed as ng/mL. Run Date Run Number 25-Jul-2000 22 31-Jul-2000 ; 01-Aug-2000 23 24 02-Aug-2000 26 04-Aug-2000 09-Aug-2000 27 29 12-Aug-2000 31 14-Aug-2000 32 15-Aug-2000 33 16-Aug-2000 36 17-Aug-2000 37 Low QC 9.00 ng/mL 8.50 7.33 10.2 10.5 10.5 10.4 *11.4 9.24 ' *11.3 8.37 8.59 9.46 10.2 10.2 8.46 8.89 *10.9 9.51 9.40 8.70 10.5 9.74 Medium QC 155 ng/mL 147 142 *115 170 156 161 177 171 153 155 152 152 149 168 140 142 160 161 151 163 158 157 * >20% theoretical High QC 405 ng/mL 359 431 426 396 416 '444 452 434 430 424 393 399 395 455 370 391 439 449 438 449 409 421 Page 98 Northwest Bioanalytical Study No. NWBS0O-040 Report No. NWBROO-108 Table A.2. Inter -Assay Precision and Accuracy for PFOSAA Quality Control Samples (continued) All concentrations are expressed as ng/mL. Run Date Run Number Low QC 9.00 ng/mL 24-Aug-2000 ; 42 9.83 8.00 26-Aug-2000 43 *11.0 10.4 28-Aug-2000 44 ` 9.71 10.4 30-Aug-2000 46 9.30 ' 9.65 Medium QC 155 ng/mL 144 137 169 167 155 159 152 165 High QC 405 ng/mL 461 475 424 438 436 447 445 *518 Mean S.D. %CV %Theoretical %Bias n 9.69 1.01 10.4 107.7 7.7 30 * >20% theoretical 155 12.5 8.1 100.0 0.0 30 429 31.7 7.4 105.9 5.9 30 Page 99 Northwest Bioanalytical Study No. NWBS0O-O4O Report No. NWBR00-108 APPENDIX B Calculation of Persistent Levels of Analvtes in Diluted Human Serum Samples Because there are persistent levels of the analytes in human serum, diluted samples must account for the amount of analyte added from the sample and the amount of analyte added from the human serum matrix. The following section describes equations used to correct for the amount of analyte added from the human serum matrix used to dilute samples. The total volume (VT) of a diluted sample is defined as the volume aliquoted from the original sample (Vs) plus the volume o f matrix (Vm) added. The dilution factor (DF) is defined as the total volume divided by the volume aliquoted from the original sample. ' VT= VS+ VM DF = Vt / V S Example VT= 0.1 mL sample + 0.4 mL control matrix VT= 0.5 The amount of analyte A in a diluted sample is equal to the volume aliquoted from the original sample times the concentration of analyte in the sample (Cs) plus the volume of control matrix added times the concentration of analyte in the control matrix (Cm)- The concentration of analyte in the sample (Cs) is determined during sample analyses. The concentration of analyte in the control matrix (Cm) is determined experimentally for each lot of control matrix as described in Section 2.1. Equation 1. Total Amount of analyte = Vs*Cs + Vm*Cm Example Total Amount of analyte = [0.1 mL sample * 100 ng/mL]+ [0.4 mL control matrix * 10 ng/mL] Total Amount of analyte = 14 ng Page 100 Northwest Bioanalytical Study No. NWBS00-040 Report No. NWBR00-108 Amount of analyte from sample = 10 ng Amount of analyte from control matrix = 4 ng The concentration of the analyte (Ca) in the diluted sample is equal to the sum of the following: 1) the ratio of the volume of sample (Vs) to the total volume of the diluted sample (V-r) times the concentration in the sample (Cs), and 2) the ratio of the volume of control matrix in the diluted sample (Vm) to the total volume of the diluted sample(V-r) times the concentration in the control matrix (Cm)- (This is the concentration of analyte in the diluted sample prior to any adjustment for the actual dilution.) The equation for the above is: . Equation 2. CA= (Vs/Vt)*Cs + (Vm/Vt)* Cm Example CA= [(0.1 mL/0.5 mL) * 100 ng/mL] + [(0.4 mL/0.5 mL) * 10 ng/mL] CA= 28 ng/mL (28 ng/mL = 14 ng total amount calculated above/0.5 mL total volume) Replacing the volume of control matrix Vmwith (Vf-Vs) into this equation produces: Equation 3. CA= (Vs/VT)*CS+ ((Vt-Vs)/Vt)*Cm or CA= (Vs/VT)*CS+ (1-Vs/VT)*CM Example . CA= [(0.1 mL/0.5 mL)*100 ng/mL] + [(l-(0.1 mL/0.5 mL))*10 ng/mL] CA= 20 ng/mL + 8 ng/mL CA= 28 ng/mL Inserting 1/DF for the term Vs/VTyields the final equation for CA: Page 101 Northwest Bioanalytical Study No. NWBSOO-040 Report No. NWBR00-108 Equation 4. Ca = (1/DF)*Cs + (1-1/DF)*Cm Example CA= [(1/5)* 100 ng/mL] + [(1-(1/5))*10 ng/mL] CA= 28 ng/mL The concentration determined by the Watson LEMS system (Cw) for any sample is: Equation 5. Cw = D F * C a Example Cw = 5 * 28 ng/mL Cw= 140 ng/mL (This is the concentration of analyte after adjusting for the actual dilution.) Substituting the final equation for CA(Equation 4.) into the equation for Cw gives: Equation 6. Cw = DF *[(1/DF)*CS+ (1-1/DF)*CM1 Example Cw = 5 * [((1/5) *100 ng/mL) + (((l-(l/5)) * 10 ng/mL)] Cw = 5* [20 ng/mL + 8 ng/mL] Cw = 140 ng/mL Solving this last equation for Cs produces the equation for correction due to persistent levels which is used to adjust theoretical calibration and QC concentrations. Equation 7. Cs = Cw -(DF-1)*Cm Example Cs = 140 ng/mL - [(5-1) * 10 ng/mL] Page 102 Northwest Bioanalytical Cs = 140 ng/mL - 40 ng/mL Cs = 100 ng/mL Study No. NWBS00-040 Report No. NWBR00-108 Page 103