Document Lo426yjpJn3NnDLVjLL36exyq

Northwcst_ioanal_ical StudyNo. NWBS00-040 ReportNo. NWBR00-108 Quantitative Determination of PFOS, PFOSA, PFOSAA, POAA, PFHS, M556 and M570 in Human Serum by LC/MS/MS Assay Revalidation Report J Northwest Bioanalytical (N'WB) A Division of NWT Inc. 1121 East 3900 South Salt Lake City, UT 84124 PREP._ED FOR: 3M Environmental Technology and Services (3M) 935 Bush Avenue St. Paul, MN 55133 AUTHOR: ConnieO. Sakashita, B.S., NWB Project Manager APPROVED FOR RELEASE BY: __" PatrickBennett,M.S., M.B.A., NWB LaboratoryDirector "" : "" " DATE: ///Z'r/O( Pa_eI NorthwestBioanalytical QUALITY ASSURANCE STATEMENT Study No.NWBS00-040 Report No. N'WBR00-108 LABOR.ATORY: SPONSOR: COMPOUND(S): NWB STUDY NUMBER: Northwest Bioanalytical (NWB) A Division of NWT, Inc. 1121 East 3900 South Salt Lake City, UT 84124 3M Environmental Technology 935 Bush Avenue St. Paul, MN 55133 and Services (3M) PFOS, PFOSA, PFOSAA, POAA, PFHS, M556 and M570 NWBS00-040 . SPONSOR STUDY NUMBER: NA, Assay Validation N_VB 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 descril3ed 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 07 - 18 Sep 2000 24/an 2001 Phase of Study Analytical Plan Assay Validation Report Draft/Raw Data Final Report *Reportsto NWB Managementareissued monthlyl Date Inspection Report Issued To NWB Project Manager *NWB Management 15 May 2000 31 May 2000 18 May 2000 31 Ma_>:2000 20 Sep 2000 " . 29 Sep 2000:. , " ,.c.- 24/an 2001 31 Jail 2001 As can reasonably be established, the methods and procedures described and the results )_orated into thi_.._ report accurately reflect the raw data. ( " "/'/'7 Shaundel Perc@, B.'_., _ Quality Assurance Comp'Kai)._Specialist /I" Dat_,/ Page2 NorthwestBioamlytica| COMPLIANCE STATEMENT Study No.NWBS00-040 ReportNo. NWBR00-108 The method validation study described in this report is not included within the definition of a GLP nonclinical regulated study. However, to the best o four knowledge, this study was conducted in accordance with the guidelines oft.he U.S. FDA Good Laboratory Practice Regulations for Nonclinical Laboratory Studies (Title 2 i 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. / Connie O. Sakashita, B.S. NWB Project Manager Patrick Bennett, M.S., M.B.A. NWB Laboratory Director .... DATE: ,:t.. ," . " ] -..U'- ,o Page3 NorthwesBt ioanalytical StudyNo.NWBS00-040 ReportNo.NWBR00-108 TABLE OF CONTENTS SIGNATURE PAGE....... ................................................... 1 QUALITY ASSURANCE STATEMENT ...................................................................................... 2 COMPLtANCE STATEMENT ....................................................................................................... 3 TABLE OF CONTENTS .................................................................... i............................................ 4 LIST OF TABLES .............................................................................. ............................................. 5 LIST OF FIGURES.......................................................................................................................... 7 1. INTRODUCTION ..................................................................................... ................................ 8 2. V._LIDATION S_Y ................................................................................................... 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 ........................................... i............................................................ 17 4. COMMENTS AND CONCLUSIONS .................................................................................... 17 4.1. Proposed Sample Analysis Acceptance Criteria ............................................................... 18 5. REFERENCES ............................................................................................. ....................,......19 6. DATA RETENTION ........................................................................................ ,:....................19 7. ,_N'ALY'TICALMETHOD .................................... _............................................ :....".............167 " 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 Page4 NorthwestBioanalytical StudyNo. NWBS00-040 ReportNo. NWBR00-I08 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 of Calibration Curve Parameters for M570 .................................................... 23 Table 8. Back-Calculated Concentrations of Calibration Standards for PFOS ............................ 24 Table 9. Back-Calculated Concentrations of' Calibration Standards for PFOSA .......................... 24 Table i 0. Back-Calculated Concentrations of Calibration Standards for PFOSA.A ........ . ............ 25 Table l 1. 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 ....:...... :............ ::26- Table 14. Back-Calculated Concentrations of Calibration Standards for M570 ..... _................... 27 Table 15. I.ntra-Assay Precision and Accuracy for PFOS Quality Control Samples .................... 28 Table 16. Intra-Assay Precision and Accuracy for PFOSA Quality Control Samples .................. 30 Table 17. Intra-Assay Precision and Accuracy for PFOSAA Quality Control Samples ............... 32 Table 18. Intra-Assay Precision and Accuracy for POA.A Quality Control Samples ................... 34 Table 19. Intra-Assay Precision and Accuracy for PFHS Quality Control Samples .................... 36 Page 5 NorthwestBioanalytical StudyNo. NWBS00-040 ReportNo. NWBR00-108 Table 20. Intra-Assay Precision and Accuracy for M556 Quality Control Samples .................... 38 Table 21. [ntra-Assay Precision and Accuracy for M570 Quality Control Samples .................... 40 Table 22. Inter-Assay Precision for PFOS Quality Control Samples ............................... i........... 42 Table 23. Inter-Assay Precision for PFOSA Quality Control Samples ........................................ 43 Table 24. Inter-Assay Precision for PFOSA.A Quality Control Samples ...................................... 44 Table 251 Inter-Assay Precision for POA.A 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. POA.A Dilution Quality Control Samples ..................................................................... 54 Table 35. PFHS Dilution Quality Control Samples ...................................................................... 55 Table 36. M556 Dilution Quality Control Samples ................................... i..................... -............ 56 .:I: Table 37. M570 Dilution Quality Control Samples ....................... _.............................................. 57 Table 38. PFOSA Specific Dilution Quality Controls ............................................... . .................. 58 Table 39. PFOSAA Specific Dilution Quality Controls ............................................................... 59 Table 40. PFOS Extraction Efficiency ................. ......................................................................... 60 Table 4 i. PFOSA Extraction Efficiency ..................... .................................................................. 61 Table 42. PFOSAA. Extraction Efficiency .................................................................................... 62 Table 43. POA.A Extraction Efficiency ........................................................................................ 63 Page6 NorthwesBt ioanalytical StudyNo. NWBS00-040 ReportNo. NWBR00-108 Table 44. PFHS Extraction Efficiency ............................................................................. ............. 64 Table 45. M556 Extraction Efficiency .......................................................................................... 65 Table 46. M570 Extraction Efficiency .......................................................................................... 66 LIST OF FIGURES Fig_'e l. Representative Calibration Curve for PFOS ................................................................. 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 Curve for M556 ................................................................. 87 Figure 7. Representative Calibration Curve for M570 ................................................................. 88 Figure 8. Standard (57.1 ng/mL) Chromatogram for PFOS ......................................................... 89 Figure 9. Standard (10.1 ng/mL) Chromatogram for PFOSA ....... ::'............................... .............. 90 Figure I0. Standard (15.0 n_mL) Chromatogram for PFOSAA ................................... ............... 91 Figure 11. Standard (14.8 ng/mL) Chromatogram for POA.A ...................................................... 92 Figure 12. Standard (12.2 ng/m.L) Chromatogram for PFHS ............................ ........................... 93 Figure 13. Standard (11.8 ng/mL) Chromatogram for M556 .................................. _.';................. 94 " "- :._. Figure 14. Standard (14.6 ng/mL) Chromatogram for M570 .......... . ....................... 2._.... _............. 95 _, . _ ,--_,_.t _.<..,._ p,)_ _r.>_ I_?oSi_ _ _ o;_i_ QC. _.._I-.".>/_.. ". " 6 '" '6.. <. ) Page7 NorthwestBioanalytical Study No. NWBS00-040 ReportNo. NWBR00-108 Quantitative Determination of PFOS, PFOSA, PFOSAA, POAA, PFHS, M556 and M570 in Human Serum by LC_IS_IS Assay Revalidation Report 1. INTRODUCTION Northwest Bioanalytical (NWB) was contracted by 3M Environmental Technolog7 and Services to develop and validate a liquid chromatography/tandem mass spectrometry method for the measurement ofperfluorooctanesulfonate (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. NWBS00-040 Report No. NWBR00-108 This report summarizes the accuracy, precision and the Northem Chinese plasma extraction efficiency results from the validation of the method for the quantitati0n 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 ori_nal protocol (Analytical Plan) were documented through approved protocol amendments'or deviation memos and are retained within the raw data. _" " " , .;" ,d Principles of the Method The newly developed method is a modification of a previous method developed under study N'W'BS98-082 and reported in NWB report NW'BR99-005 [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. N'WBS00-040 Report No. NWBR00-108 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 A.PI 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 PFOSA.A. The extraction efficiencies for all analytes aridthe internal standard 1H, 1H,2H,2H-perfluorooctane sulfonic acid (THPFOS) ,,,,'erealso determined. 2.1. Persistent Levels of Analytes in Matrix Because PFOS, PFOSAA., POAA, PFHS, M556 and M570 demonstrate measurable levels in control hum;/.h"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 determinethe persistent levels of these analytes in the lot of human serum used for the validation, a pa.rtial calibration curve was prepped with th.e 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 I0 Northwest Bioanalytical Study No. NWBS00-040 Report No. NWBR00-108 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. 2.2. 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, I0.0, 25.0, 50.0, I00, 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 48.1, 49.6, 57.1,72.1, 97.1,147, 297, 447, 547 PFOSA 1.00, 2.51, 10.I, 25.1, 50.3, 100, 251,402, 502 PFOSAA 6.00, 7.50, 15.0, 30.0, 55.0, 105, 255,405, 505 POAA 5.76, 7.26, 14.8, 29.8, 54.8, 105, 255,405, 505 .: _: PFHS 3.15, 4.65, 12.2, 27.2, 52.2, 102, 252,402, 502 a lVI556 4.30, 11.8, 26.8, 51.8, 102, 252, 402, 502 M570 5.60, 7.10, 14.6, 29.6, 54.6, 105, 255,405,505 Page I 1 NorthwestBioanalytical .... StudyNo. N'WBS00-040 ReportNo. NWBR00-108 Range Mean Coefficient of Determination PFOS 48. l to 547 n_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 - 14. 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 Low (ng/m.L) 51.1 Medium (ng/m.L) 197 High (ng/mL) 446 Dilution (ng/rnL) 4460 .: PFOSA 4.00 150 400 4000 " " '" PFOSA.A 9.00 155 405 4050 ; POAA 8.74 154 403 4030 PFHS 6.15 152 402 4020 M556 5.80 152 402 4020 M570 8.60 155 405 4050 Page 12 Northwest Bioanalytical Study No. NWBS00-040 Report No. NWBR00-108 2.3.1. Precision and Accuracy for PFOS, PFOSA, PFOSAA, POAA, PFHS, M556 and M570 Quality Controls The precision and accuracy of the LCEvIS/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 accurac(y%Theoreticaflo)ralllevelosfundiluteqdualitcyontrols rangedfrom 84.8%to111.2%(Tables15and 18- 2 i).The intra-assay precisio(n%CV) forPFOSA andPFOSAA werelessthanorequalto19.8% foreachundiluteQdC concentratioTnh.e mean accurac(y%Theoreticaflo)r alllevelosfundiluteQdCs rangedfrom68.7% to91.7% (Tables16- 17). The inter-assparyecisio(n%CV) forPFOS, POAA, PFHS, M556 and M570 werelessthanorequalto9.7% foreachundiluteQdC concentratioTnh.e mean accuracy(%Theoreticaflo)ralllevelosfundiluteQdCs rangedfrom 87.4% to108.5% (Table2s2 and 25- 28).The inter-assparyecisio(n%CV) forPFOSA and PFOSAA werelessthanorequalto14.0%foreachundiluted QC concentratioTnh.e mean accurac(yTheoreticaflo)ralllevelosf undiluteQdCs rangedfi'om73.I% to88.9%(Tables23 - 24). FordiluteQdCs, theinter-assparyecisio(n%CV) forPFOS, POA.A,:PFHS, :-_-M556 and M570 werelessthanorequalto6.6%.The mean accgracy (%Theoreticaflo)rthediluteQdCs rangedfrom92.8%to 113.9%(Tables31 and 34 - 37).The inter-assparyecisio(n%CV) forPFOSA andPFOSAA werelessthanorequalto9.7% foreachdiluteQdC. The mean accuracy (%Theoreticaflo)rthediluteQdCs rangedfrom79.0%to81.3% (Table3s2 33). Page 13 NorthweBsitoanalytical StudyNo.NWBS00-040 ReporNto.NWBR00-108 2.3.2. 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. 1. - A.2.). .. Precision and Accuracy for PFOSA and PFOSAA Specific Quality Controls Because of the bias demonstrated for PFOSA and PFOSAA. calculated concentrations from the ori_nal QCs prepared as described in section 2.3.1, a new set of QCs containingtSialy 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 PFOSA.A LLOQ calibratisotnandardfsortheintra-assRauyn 17 demonstrated unacceptable accuracies resulting in both replicates being rejected and a raised LLOQ of 7.50ng/mL for the run. The low QCs for PFOSA.A 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 NorthwesBt ioanalytical StudyNo.NWBS00-040 ReportNo.NWBR00-108 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 _'om theoretical concentrations for these QCs diluted with control matrix were both greaterthan 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 oft_he PFOSA and PFOSAA specific QCs was demonstrated with the analytical QC performance during sample " analysis study NWBS00-062 (medical surveillance). The results can be fotmff'" in Appendix A, Tables A.1. and A.2. These tables only contain analyses for _'_| reported PFOSA and PFOSA.A sample data, and do not include runs that did t not meet the acceptance criteria for PFOSA or PFOSA.A. ....._ji For the PFOSA and PFOSA specific QCs, the i.nter-assayprecision (%CV) , was less than or equal to 11.4% for each undiluted QC concenffafion. The mean accuracy (%Theoretical) for all levels of tmdiluted QCs rkngeclTrom 98.5% to 107.7%. 2.4. Extraction Efficiency The extraction efficiencies ofPFOS, PFOSA, PFOSAA, POA.A, PFHS, M556 and M570 were determined using Northern Chinese human plasma by comparing the area ratios obtained for the following three cases: Page 15 NorthwestBioanalyticai Study No. NWBS00-040 ReportNo. NWBR00-108 1. Both the analyte and intemal standard added following the extraction (postextract). 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 of 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 anal,yt.es was Used to estimate the extraction recovery of the analytes from matrix. This p.lasma: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 NorthwestBioanalytical 2.5. Stability Evaluation StudyNo. NV_3S00-040 ReportNo. NV_R00-108 The stability ofPFOS, PFOSA, PFOSA.A, 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 soft'ware (version 1.6) with a smooth factor of I. Quantitation is based upon quadratic regression analysis of calibration curves (weighted l/x 2) using the area ratio vs. concentration calculated by the Watson DMLIMS sofb,vare (version 6. I. 1.04). 4. COMMENTS .-MNDCONCLUSIi)NS 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 dualothe effect of rounding if the regressions were performed with the purity corrected concentrations, but these differences would have a negliNble 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. Art 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 PFOSA.A. A separate set of QCs containing only PFOSA and PFOSAA were prepared without using an evaporation step. The second set (PFOSA and PFOSA.A specific QCs) demonstrated Page 17 NorthweBsitoanalytical StudyNo.NV/BS00-040 ReporNto.NWBR00-108 both acceptable precision and accuracy. As a result, two sets of intra-assay and interassay precision and accuracy are shown in the report. The method described in this report has been validated for the determination of PFOS, PFOSA, PFOSA.A, POAA., PFHS, M556 and M570 in human serum. However, study samples with experimentally calculated PFOSA or PFOSA.A 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 ofquantitation. For all analytes except PFOSA, at least three-fourths of tSe calibration standard's back-calculated concentrations must be within -4-15% (+ 20% for LLOQ) of their individual target concentrations. For PFOSA, at least three-fourths of the calibration standard's back-calculated concentrations must be within + 20% (+ 25% for LLOQ) of their individual target concentrations. A calibration standard will be considered a statistical outlier if the back-calculated concentration is greater than two times the acceptance criteria for that standard. Lower Limit of Quantitation .:_.. The back-calculated concentrations of at least one of the duplicate lowest 15oints in the calibration curve must be within _+25% of the target concentration for PFOSA to qualify as the LLOQ and within + 20% of the target concentration to qualify as the LLOQ for all other analytes. If this criterion is not met, the next level is subjected to the same test and the LLOQ raised accordingly. Page 18 NorthweBsitoanalytical Quality Control Samples StudNyo.NWBS00-040 ReporNto.NWBR00-108 Each analHical 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% o fits target concentrations in order to accept diluted study samples at that level. The dilution QC acceptance is independent of the undiluted analytical QC acceptance. 5. REFERENCES [5.1] D. Vollmer, "Quantitative Determination of PFOS, PFOSA, PYOSAA, NMeFOSE-OH, N-EtFOSE-OH, POAA and PFHS in Human Serum by LC/MS/MS," NW'B study NWBS98-082, NWB report NW'BR99-005, 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. _, . " :". ;.-_,". Page19 Northwest Bio_alytical Study No. NWBS00-040 Report No. NWBR00-108 Table 1. Summary of Calibration Curve Parameters for PFOS Quadratic weighted I/x'-. All concentrations are expressed as n_'mL. Run Date 29-Jun-2000 30-Jun-2000 02-Jul-2000 Run Number i3 15 16 A BI C R-Squared LLOQ ULOQ -0.000009 -0.000001 -0.000005 0.035473 0.018768 0.028829 0.368276 0.148749 0.235310 0.9877 0.9949 0.9948 48.1 547 48.1 547 48.1 547 Mean S.D. %CV n -0.000005 0.000004 -80.0 3 0.027690 0.008411 30.4 I. 3 0.250778 0.110578 44. I 3 0.9925 0.0041 0.4 3 A, B and C are coefficients used to defLnethe quadratic curve. Table 2. Summary of Calibration Curve Parameters for PFOSA Quadratic weighted l/x 2. All concentrations are expressed as n#mL. Run Date 29-Jun-2000 30-J'un-2000 02-J'ul-2000 Run Number 13 15 16 A B C R-Squared LLOQ ULOQ -0.000030 -0.000012 -0.000020 0.093690 0.051196 0.088242 0.023501 0.004685 0.025293 0.9761 0.9975 0.9960 1.00 502 1.00 502 1.00 502 Mean S.D. %CV nl -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 A, B and C are coefficients used to define the quadratic curve. : : ' .... Page 20 -'. e* Northwest Bioanalytical Study No. NWBS00-040 Report No. NWBR00-108 Table 3. Summary of Calibration Curve Parameters for PFOSAA Quadratic weighted I/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 -0.000003 0.000000 0.000001 0.023347 0.013890 0.023426 -0:035788 -0.020748 -0.033118 0.9969 0.9971 0.9964 6.00 505 6.00 505 6.00 505 Mean S.D. %CV n -0.000001 0.000002 -200.0 3 0.020221 0.005483 27.1 3 -0.029885 0.008024 -26.8 3 0.9968 0.0004 0.0 3 A, B and C are coefficients used to def'me the quadratic curve. Table 4. Summary of Calibration Curve Parameters for POAA Quadratic weighted 1/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 0.000002 0.000000 -0.000002 0.018721 0.012512 0.014490 0.026984 0.009407 0.015595 0.9847 0.9958 0.9962 5.76 505 5.76 505 5.76 505 Mean S.D. %CV n 0.000000 0.000002 3 0.015241 0.003172 20.8 3 0.017329 0.008916 51.5 3 0.9922 0.0065 0.7 3 A, B and C are coefficients used to define the quadratic curve. :. Page 21 ... NorthwestBioanalytical StudyNo. NWBS00-040 Report No. NW'BR00-108 Table 5. Summary of Calibration Curve Parameters for PFI-IS Quadraticweighted l;x:. Allconcentrationsare expressedas ng,;mL. Run Date 29-3un-2000 R ,,. 30-3un-2000 02-Jul-2000 Run Number 13 15 16 A B C R-Squared LLOQ ULOQ -0.000006 -0.000005 -0.000009 0.023682 0.016787 0.021344 0.0218 l 1 0.011507 0.020627 0.9947 0.9946 0.9968 3.15 502 3.15 502 3.t5 502 Mean S.D. %CV n -0.000007 0.000002 -28.6 3 0.020604 0.003507 17.0 3 0.017982 0.005638 31.4 3 0.9954 0.0012 0.1 3 A, B and C are coefficientsused to define thequadraticcurve. Table 6. Summary of Calibration Curve Parameters for M556 QuadraticweightedI/x". Allconcentrationsare expressedas ng/mL. Run Date Run Number A B C R-Squared LLOQ ULOQ 29-3un-2000 30-3un-2000 02-Jul-2000 13 0.000001 0.005000 0.004952 0.9881 4.30 502 15 0.000000 0.002882 0.000748 0.9975 4.30 502 16 0.000000 0.003891 0.000548 0.9949 4.30 502 Mean S.D. %CV rt 0.000000 0,00000l 3 0.003924 0.001059 27.0 3 0.002083 0.002487 119.4 3 0.9935 0.0049 0.5 3 A, B and C are coefficientsusedto definethe quadraticcurve. . :_ Page 22 Northwest Bioanalytical Study No. NWBS00-040 Report No. NWBR00-108 Table 7. Summary of Calibration Curve Parameters for MS70 Quadratic weighted I/x". All concentrations are expressed as ng/mL. Run Date 29-Iun-2000 30-J'un-2000 02-Jul-2000 Run Number 13 15 16 A B C R-Squared LLOQ ULOQ -0.000002 -0.000001 0.000000 0.006621 0.003911 0.006433 -0.007l 15 -0.005406 -0.009231 0.9962 0.9972 0.9967 5.60 505 5.60 505 5.60 505 Mean S,D. %CV n -0.000001 0.000001 -100.0 3 0.005655 0.001513 26.8 3 -0.007251 0.001916 -26.4 3 0,9967 0.0005 0.1 3 A, B and C are coe_cients _ed to define _e qua_atic curve. o;:: ,e Page 23 Northwest Bioanalytical ,L :.:.'2":p_.;" <"Z ="):! :" S r 0_.," f :J"" f" _lq-")"4._' I Study No. NWBS00-040 Report No. NWBR00-108 Table 8. Back-Calculated Concentrations of Calibration Standards for PFOS ! Quadratic weighted l,x'. All concentrations are expressed as ng/mL. Run Date 29-Iun-2000 30-ltm-2000 02-Iul-2000 Run Number 13 15 16 48.1 ,3q,_ 44.9 46.5 46.5 50.3 51.6 45.2 49.6 "f_,2, 52.1 51.5 49.8 48.3 50.5 47.6 57.1 72.1 =7.., ',..". 56.6 71.7 58.5 80.8 58.6 72.7 58.4 71.2 60.3 74.9 54.3 71.2 97.1 147 23 .,,_'_,_."- koL._j! 153 94.6 146 92.2 147 98.9 135 94.3 151 94.9 148 297 _,' _ 302 316 321 306 299 298 447 _e'o 449 445 438 47l 415 449 547 ..._..:._ 515 Z[. '7' , ; 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. I 4.1 4.1 - 1.2 0.8 1.2 2.4 -4.5 0,0 3.4 -0.4 -0.9 6 6 6 6 6 6 6 6 6 Table 9. Back-Calculated Concentrations of Calibration Standards for PFOSA .... Quadratic weighted I/x z. All:concentrations are expressed as n_mL. L" ./. G _'_ _'_ '" ,7' Run Date 29-Iun-2000 Run Number 13 1.00/ 1.00 2.51 c,*', " 2.44 10.1 25.1 50.3 10011 251 402 502 t, _,-' _.,_ _. 10.6 25.7 _ 104 280 420 483 30-Jun-2000 1.03 2.23 1I. 1 _ 45.7 95.0 256 379 507 15 1.03 2.46 9.90 25.7 43.6 98.5 255 401 518 0.957 2.68 9.71 25.7 50.4 94.8 277 : ..418 447 02-Jul-2000 16 0.952 2.48 11.5 26.2 47.6 99.0 244 ,39:1. 506" _ 1.04 2.57 9.79 22.7 48.7 107 237 431 501 Me_ S.D. %CV %Bias n 1.00 0.0388 3.9 0.0 6 2.48 0.150 6.0 -1.2 6 10.4 0.753 7.2 3.0 6 26.6 45.3 99.7 258 3.54 7.27 4.90 " 17.3 13.3 16.0 4.9 6.7 6.0 -9.9 -0.3 2.8 6 6 6 6 407 494 19.7 25.6 4.8 5.2 1.2 -1.6 6 6 Page 24 .. Northwest Bioanalytical Study No. NWBS00-040 Report No. NWBR00-108 Table I0, Back=Calculated Concentrations of Calibration Standards for PFOSAA Run Date 29-Jun-2000 30-/un-2000 02-Iul-2000 Quadratic I Run Number weighted I'x'. G. g 6.00 All concentrations :, _ 'L,q f.7. " ;it'I:' 7.50 15.0 are expressed as, ntt,.mL. . _ . _ ' - "J-._., .'.', _ 3._ : ,Z_,'.:?".!,.:,- _"#_. t 30.0 55.0 105 255 _- : _ " -..-.,,, ' 405 -".,:)'. " S_" 505 / 13 5.90 7.74 14.4 31.3 t-,___L._ 110 262 412 476 5.92 7.85 14.1 30.8 49.4 106 266 400 512 15 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 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 6 6 6 6 Sample deactivated as an outlier (> 9j times the acceptance criteria). Result not included in summary statistics. Table 11. Back-Calculated Concentrations of Calibration Standards for POAA Run Date m IN29_2000 Quadratic weighted 1/xZ'/,_-&_c'olncentrations are )ressed as_mL ....... -= , Run Number 13 5.76 7.26 I!_,% 5.15 14.8 29.8 54.8 " / _,_ ,"_ 14.6 30.9 105 255 405 505 J '/ / _" I04 246 377 450 30-Iun-2000 02-Jul-2000 15 6.26 7.24 5.67 6.80 16 5.36 7.09 /,0_" _11.8_ 7.74 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 15.9 32.2 54,9 103 25(5:: 385 532 106 255" 394 52i Mean S.D. %CV %Bias n 5.77 7.31 14.7 29.3 54.4 106 266, 396 505 0.550 I 0.754 0.891 2.38 2.20 6.12 17.4 16.4 37.3 i 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 6 6 6 6 Sample deactivated as an outlier (> 2 times the acceptance criteria). Result not included in summary, statistics. Page 25 NorthwestBioanalytical StudyNo. NWBS00-040 ReportNo. NWBR00- I08 Table 12. Back-Calculated Concentrations of Calibration Standards for PFHS Run Date 29d'tm-2000 30-lun-2000 02-Jul-2000 Quadratic weighted_ l,'x'. All concentrations, are ,,.r L, 4_ [ /'V7 Run Number 3.15 : 4.65 / 12.2 27.2 - i ._;u, - I 3 _'_-."t (-4 ? - 52.2 102 252 402 502 "/ J / ; ," 13 3.28 5.17 11.8 28.0 55.3 107 252 377 448 2.85 4.59 I t.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 0.162 5.3 -2.5 .6 4.75 0.333 7.0 2.2 6 12.8 0.880 6.9 4.9 6 27.2 50.4 100 252 0.887 2.63 4.14 6.77 3.3 5.2 4.1 2.7 0.0 -3.4 -2.0 0.0 I 6 I6 6 6 397 513 15.8 44.3 4.0 8.6 - 1.2 2.2 6 6 Table 13. Back-Calculated Concentrations of Calibration Standards for M556 Quadratic weig..h..t..ed _,1/_/x'. A'll/0c"o-ncenI tria-t_io2ns a!re _ressed as ng/mL. _ 3 _2" i --.r:" "" Run Date 30-Jun-2000 02-J'ul-2000 Run Number 15 16 4.30 .)! 1.8 26.8 51.8 102 252 402 502 111 266 396 468 ] 24.049.-, 514 4.25 11.6 27.4 49.8 107 273 403 499 4.39 11.7 26.1 51.1 95.1 266 412 466 3.78 11.5 25.6 47.5 98.2 250 379 513 4.74 13.0 26.9 53.6 110 254 408 506 ",:.... Mean S.D. %CV %Bias n 4.34 1.5 26.8 50.3 104 264 3_9 494 0.527 0.996 2.22 2.23 6.56 9.46 11.8 21.9 / 12.1 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 6 6 6 6' * Sample deactivated as an outlier (> 2 times the acceptance criteria). Result not included in summary statistics. Page 26 NorthwestBioanalytical StudyNo. NWBS00-040 ReportNo. NWBR00-108 Table14.Back-CalculatCeodncentratioonfsCalibratiSotnandardfsorM570 Run Date QuadraticwexghtedI/x'.Allconcentrations Run :Number 5.60 7.10 14.6 29.6 54.6 ...f. _.-'- _'_'-_ _,l i ! 105 255 405 505 29-1un-2000 13 30-Jun-2000 15 02-Jul-2000 16 5.50 5.47 5.26 6.00 5.67 5.82 7.08 7.61 7.11 6.94 6.55 6.98 13.7 31.6 _ 107 264 418 466 14.6 30.5 50.5 98.9 269 388 530 14.8 29.1 55.2 I01 266 408 498 14.5 30.7 54.7 97.0 272 422 474 15.7 27.5 49.8 108 255 394 509 14.9 29.5 56.3 I13 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 6 5 104 263 405 498 6.16 8.18 13.4 24.4 5.9 3.1 3.3 4.9 -1.0 3.1 0.0 -1.4 6 6 6 6 * Sample deactivated as a.u outlier (> 2 times the acceptance criteria). Result not included in summary statistics. . " _ ;_-. Page 27 Northwest Bioanalyfical Study No. NWBS00-040 ReportNo. NWBR00-I08 Table 15. Intra-Assay Precision and Accuracy for PFOS Quality All concentrations are expressed as ng/mL. Control Samples :-:.o5) : 9 _', ) Run Date 29-Jun-2000 Run Number 13 Low QC 51.1 ng/mL 45.9 44.8 47.6 48.8 48.6 Medium QC 197 ng/mL 191 177 183 175 179 High QC 446 ng/mL 392 403 429 383 377 Mean S.D. %CVI %Theoretical i n Run Date .... 30-Jun-2000 Rnn Number 15 47.1 1.74 " 3.7 92.2 5 Low QC 51.1 ng/mL 48.8 46.7 47.8 45.8 47. I 181 6.32 3.5 91.9 5 Medium QC 197 ng/mIJ 173 174 175 166 176 397 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 5 5 5 Page 28 NorthwestBioanalytical StudyNo. NWBS00-040 ReportNo. NWBR00-108 Table 15. Intra-Assay Precision and Accuracy for PFOS Quality Control Samples (continued) All concentrationsare expressedas ng/mL. Run Date 02-Jul-2000 Run Number 16 Low QC 51.1 ng/mL 45.2 48.7 46.4 47.7 44.0 Medium QC 197 ng/mL 170 160 166 169 172 High QC 446 ng/mL 380 393 385 387 .361 Mean S.D. %CV I %Theoretical nl 46.4 167 38l 1.88 4.67 12.2 4.1 2.8 3.2 90.8 84.8 85.4 5 5 5 Page29 o. NorthwestBioanalytical Study No. NWBS00-040 ReportNo. NWBR00-108 Table 16. lntra-Assay Precision and Accuracy for PFOSA Quality Control Samples .:_.LD_:.-) , .:Jo All concentrationsare expressedas ng/mL. Run Date 29-3un-2000 Run Number 13 Low QC 4.00 ng/mL 3.43 3.21 3.39 3.58 3.67 Medium QC 150 ng/mL 133 122 128 129 139 High QC 400 n_mL 325 345 357 322 342 Mean S.D. %CV ,/ %Theoretical n Run Date Run Number 30-Jun-2000 15 3.46 0.178 5. l 86.5 5 Low QC 4.00 ng/mL * 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 _, : .... 84.5 "---5 High QC 400 ng/mL * 278 * 291 318 * 297 314 Mean S.D. %CV / %Theoretical n .... 2.82 0.0635 2.3 ,_70.5 5 . 103 4.71 _ ,i . 300 16.5' " _ . ,:d', .......... 4,6 ................. -5_5_ ---_ 68.7 75.0 '-_1 .,. ...... .. ....5....................... 5............. "> +25% theoretical _'/o",''"_(:"",:.":_.-.0'"r", l''<J ;,,-- U Page 30 ") :' "L.e_ NorthwestBioanalytical "StudyNo. NWBS00-040 ReportNo. NWBR00-I08 Table 16. Intra-Assay Precision and Accuracy for PFOSA Quality Control Samples (continued) All concentrationsare expressedas ng/mL. Run Date 02-Jul-2000 Run Number 16 Low QC 4.00 ng/mL * 2.58 2.96 3.50 3.94 2.51 Medium QC 150 ng/mL * 105 * 102 * 103 * 102 * 112 High QC 400 ng/m.L * 289 311 315 304 305 Mean S.D. %CV %Theoretical n 3.10 105 305 0.613 4.21 9.91 _ _ 77.5 5 4.0 _____..3_,__.__.__ 70.0 76.3 " 5 .........5............".......... > +_25%theoretical e:_.: Page3 l NorthwestBioanalytical StudyNo. NWBS00-040 ReportNo. NWBR00-108 Table 17. Intra-Assay Precision and Accuracy for PFOSAA Quality Control Samples Allconcentrationsareexpressedas ng,.'mL. Run Date 29-Jun-2000 ' Run Number 13 Low QC 9.00 ng/mL 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 S.D. %CV -" %Theoretical n Run Date Run Number .... 30-Jun-2000 15 8.15 0.522 6.4 90.6 5 Low QC 9.00ng/mL 7.65 7.76 7.73 7.29 7.52 124 3.87 3.1 : 80.0 5 Medium QC 155ng/m.L * 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 .. %Theoretical n 7.59 0.192 2.5 .., < 84.3 5 113 5.54 4.9... :-( 72.9 5 > +_.20%theoretical 301' 13.4: 4.5 74.3 5 '" _, .,)_" "'"- . _. r _,_.._' :_ Page32 NorthwestBioanalytical StudyNo. NWBS00-040 ReportNo. NWBR00-108 Table 17. Intra-Assay Precision and Accuracy for PFOSAA Quality Control Samples (continued) All concentrationsare expressedas ng/mL. Run Date 02-Jul-2000 Run Number 16 Low QC 9.00 ng/mL 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 8.25 113 0.900 4.06 10.9 3.6 91.7 \..-72.9 5 5 * > __.20%theoretical 300 10.9 3.6 74.1 .......... 5 ....... w: _.. Page 33 r. NorthwestBioanalytical Study No. NWBS00-040 ReportNo. NWBR00-108 Table 18. Intra-Assay ==, Run Date 29-Jun-2000 Precision and Accuracy for POAA Quality Control Samples All concentrationsare expressedas ng/mL. .7._ t _/-'2)`5.5 _J 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 30-Jun-2000 Run Number 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 i57 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 2.6 95.3 5 :. _.. " _".,.:.". Page34 NorthwestBioanalyticsl StudyNo. NWBS00-040 ReportNo. NWBR00-I08 Table 18. Intra-Assay Precision and Accuracy for POAA Quality Control Samples (continued) Allconcentrationsare expressedas ng/mL. Run Date 02-Jul-2000 Run Number 16 Low QC 8.74 n_mL 7.15 8.15 8.42 8.72 7.12 Medium QC 154 n_mL 150 136 140 148 141 High QC 403 ng/mL 401 I 393 398 405 376 Mean S.D. %CV ./ %Theoretical n 7.91 143 395 0.737 5.83 11.3 9.3 4.1 2.9 90.5 92.9 98.0 p, 5 5 5 ::; Page35 NorthwestBioanalyfical StudyNo. NWBS00-040 ReportNo. NWBR00-108 Table 19. Intra-Assay Precis/on" and Accuracy for PFHS Quality Control iI Allconcentratairoeenxspressaesdnffml.. Samples I , .t '_2_ Run Date 29-Jun-2000 Run Number 13 Low QC 6.15 ng/mL 7.03 6.44 6.77 6.89 7.05 Medium QC 152 ng/mL 175 166 171 158 165 High QC 402 n_mL 454 431 468 434 424 Mean S.D. %CV / %Theoretical n Run Date Run Number 30-Jun-2000 15 6.84 0.249 3.6 l l 1.2 5 Low QC 6.15-ng/mL 6.53 6.22 6.06 6.00 6.31 167 6.44 3.9 109.9 5 Medium QC 152 ng/mL 163 157 158 149 164 442 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 "_.._,.7-_ Page 36 NorthwestBioanalytical StudyNo. NWBS00-040 ReportNo. NWBR00-108 Table 19. lntra-Assay Precision and Accuracy for PFHS Quality Control Samples (continued) Allconcentrationsare expressedas ng/mL. Run Date 02-Jul-2000 Run Number 16 Low QC 6.15 ng/mL 6.43 6.52 6.23 6.86 5.93 Medium QC 152 n_mL 154 150 152 153 151 High QC 402 ng/mL 408 44l 422 430" 409 Mean 6.39 152 422 S.D. 0.345 1.58 t4.1 %CV / 5.4 1.0 3.3 %Theoretical 103.9 100.0 105.0 ' n 5 5 5 .... g Page 37 NorthwestBioanalytical StudyNo. NWBS00-040 Report No. NWBR00-108 Table 20. Intra-Assay Precision and Accuracy for M556 Quality Control Samples Allconcentrationsare expressedas ngjmL. L: :.9_ "= LI, "_'3 Run Date 29-Jun-2000 Run Number l3 Low QC 5.80 ng/mL ** 8.98 6.34 ** 7.98 6.55 5.86 Medium QC 152 n_mL 165 159 16l 163 170 High QC 402 ng/mL 427 429 439 413 366 Mean 6.25 164 415 S.D. 0.354 4.22 28.8 %CV ,1 5.7 2.6 6.9 %Theoretic al 107.8 107.9 103.2 : n 3 5 5 Run Date 30-Jun-2000 Run Number 15 Low QC 5.80 ng/mL 5.74 5.89 Medium QC 152 ng/mL 143 147 High QC 402 ng/mL 380 395 5.46 152 408 5.33 139 390 5.61 153 407 Mean S.D. %CV "/ %Theoretical n 5.61 147 396 0.221 5.93 11.8 3.9 4.0 3.0 96.7 96.7 98.5 5 5 5 ** Sampledeactivateddue to injectorcarryover(not includedin summarystatistics) Page 38 ... NorthweBsitoanalytical StudNyo.NWBS00-040 Rcl_oNro_.NWBR00-108 Table20. Intra-AssayPrecisioannd Accuracy forM556 QualityC.ontrolSamples (continued) Allconcentratairocenxspressaesdn_rnL. Run Date 02-Jul-2000 I- Run Number 16 Low QC 5.80 ng/mL 6.23 6.17 6. I7 6.33 5.61 Medium QC 152 ng/mL 149 137 149 152 150 High QC 402 ng/mL 391 418 390 417 .378 Mean S.D. 6.10 147 399 ," ,. 0.283 5.94 17.8 %CV J 4.6 4.0 4.5 Theoretical 105.2 96.7 99.3 n ..o. 5 5 5 Page39 o,. NorthwestBioanalytical Study No.NWBS00-040 ReportNo. NWBR00-108 Table 21. Intra-Assay Precision and Accuracy for M570 Quality Control Samples All concentrationsare expressedas ng/mL. Run Date 29-Jun-2000 Run Number 13 Low QC 8.60 n_mL 8.23 8.07 8.34 8.87 8.04 Medium QC 155 ng/mL 168 155 164 153 170 High QC 405 n_mL 393 403 424 378 377 Mean S.D. %CV l %Theoretical n 8.31 162 395 0.336 7.65 19.5 4.0 4.7 4.9 96.6 104.5 97.5 5 5 5 Run Date 30-Jun-2000 Run Number 15 Low QC 8.60 n_mL 8.17 9.09 8.72 7.71 7.88 Medium QC High QC 155 n_mL 405 ng/mL 'i ....... 151 396 149 396 148 412 138 388 150 413 ,o h Mean S.D. %CV ,/ %Theoretical i n 8.31 147 0.579 5.26 7.0 3.6 96.6 94.8 5 5 - 40 I 11.0 2.7 99.0 5 " 3. ,:.;-. Page 40 .. NorthwestBioanalytical StudyNo. NWBS00-040 Rq_ortNo. NWBR00-108 Table 21. lntra-Assay Precision and Accuracy for M570 Quality Control Samples (continued) All concentrationsare expressedas ng/mL. Run Date Run Number Low QC 8.60 ng/mL Medium QC 155 n_mL High QC 405 ng/mL 02-3ui-2000 16 8.67 148 382 t" 8.49 137 391 9.73 148 384 9.69 145 412 == 7.95 151 368 Mean S.D. %CV %Theoretical n 8.91 146 387 0.780 5.36 16.1 8.8 3.7 4.2 103.6 94.2 95.6 5 5 5 a.,_.. Page41 NorthwestBioanalytical StudyNo. NW'BS00-040 ReportNo. NWBR00-I08 Table 22. Inter-Assay Precision for PFOS Quality Control Samples All concentrationasre expressedasngimL. Run Date 29-Jun-2000 30-Jun-2000 02-Jul-2000 Run Number 13 15 16 Low QC 51.1 ag/mL 45.9 44.8 47.6 48.8 48.6 48.8 46.7 47.8 45.8 47.1 45.2 48.7 46.4 47.7 44.0 Medium QC 197 ng/mL 191 177 183 175 179 173 174 175 166 176 170 160 166 169 172 High QC 446 ag/mL 392 403 429 383 377 .382 395 402 382 406 380 393 385 387 361 Mean S.D. %CV %Theoretical %Bias n 46.9 174 1.55 7.46 3.3 4.3 91.8 88.3 -8.2 -11.7 15 15 390 15.7 ...: 4.0 , : 87.4 g -12.6 15 : ._-. .. Page42 NorthwestBioanalytical Study No. NWBS00-040 ReportNo. NW'BR00-108 Table 23. Inter-Assay Precision for PFOSA Quality Control Samples Allconcentrationsare expressedas ng/mL. Run Date 29-Jun-2000 Run Number - 13 30-Jun-2000 15 02-Jul-2000 16 .... Low QC 4.00 ng/mL 3.43 3.21 3.39 3.58 3.67 *2.88 2.76 2.89 2.78 2.77 *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 1211 75.3 -- -24.7 15 * > __.25%theoretical 314 21.9 7.0 _ : ::,,., 78.5 $ -21.5 15 Page43 NorthwestBioanal_ical Study No. NWBS00-040 Repog No. NWBR00-108 Table 24. Inter-Assay Precision for PFOSAA Quality Control Samples Allconcentrationsare expressedas n_mL. Run Date 29-1un-2000 30-Jun-2000 02-Jul-2000 Run Number 13 15 16 Low QC 9.00 n_mL 7.98 7.43 8.05 8.81 8.46 7.65 7.76 7.73 7.29 7.52 7.37 8.11 8.47 9.67 7.63 Medium QC 155 ng/mL 129 *120 "123 "121 127 i112 *117 "116 "104 "117 "117 "107 *Iii "114 "116 High QC 405 ng/mL *272 *303 "317 *260 *286 *285 *300 "315 *290 "313 *287 *309 "30_ "312 "291 Mean S.D. %CV %Theoretical %Bias n 8.00 0.640 8.0 88.9 -1 I. I 15 117 6.79 5.8 75.5 -24.5 15 * > +_.20%theoretical 296 16.6 5.6 73.1 -26.9 15 .. .:._.... Page44 NorthweBsitoanalytical StudyNo.NWBS00-040 ReporNto.NWBR00-108 Table25. Inter-AssaPyrecisiofnorPOAA QualityControlSamples Allconcentratairocenxspressaesdng/mL. Run Date 29-Jun-2000 30-lun-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 n_ml, 404 40l 434 422 377 374 389 382 376 398 401 393 398 405 376 Mean S.D. %CV %Theoretical %Bias n 8.34 148 0.812 8.27 9.7 5.6 95.4 96.1 -4.6 -3.9 15 15 395 17.4 ...... 4.4 _ .. 98.0 -2.0 15 : .:=-.- Page45 NorthwestBioanalyfical StudyNo. NWBS00-040 ReportNo. NWBR00-108 Table 26. Inter-Assay Precision for PFHS Quality Control Samples All concentrationsare expressedas ng/mL. Run Date 29-Jun-2000 Run Number 13 Low QC 6.I5 ng/mL 7.03 Medium QC 152 ng/mL 175 High QC 402 ng/mL 454 6.44 166 431 6.77 171 468 6.89 158 434 7.05 165 424 30-Jun-2000 15 6.53 163 429 6.22 157 455 6.06 158 463 6.00 149 429 6.31 164 447 i 02-Jul-2000 16 6.43 154 408 6.52 150 441 .... 6.23 152 422 6.86 153 430 5.93 151 409 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 18.1 o.; . 4.2 _,.. : . "".':._" 108.5 8.5 15 Page 46 Northwest Bioanaly_ical Study No. HWBS00-040 ReporNt o.NWBR00-I08 Table 27. Inter-Assay Precision for M556 Quality Control Samples All concentrations are expressed as ng/mL. Run Date 29-Jun-2000 30-Jun-2000 02-Jul-2000 Run Number Low QC 5.80 nglmL 13 *'8.98 6.34 **7.98 6.55 5.86 15 5.74 5.89 5.46 5.33 5.61 16 6.23 i 6.17 o4". 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 %Theoretical %Bias n 5.95 0.381 6.4 102.6 2.6 13 153 9.49 6.2. 100.7 0.7 15 403 21.0.;.. 5.2 : 100.2; , 0.2 15 * Sample deactivated due to injector carryover (not included in summary statistics) :-,=... Page 47 Nor_west Bioanal_al StudyNo. NWB$0-040 ReportNo. NWBR00-I08 Table 28. Inter-Assay Precision for M570 Quality Control Samples All oacentrationsareexpressedas ng/mL. Run Date Run Number Low QC 8.60 ng/mL Medium QC 155 ag/mL High QC 405 n_mL 29-Jun-2000 13 8.23 168 393 8.07 155 403 8.34 164 424 8.87 153 378 8.04 170 377 30-.l'un-2000 15 8.17 151 396 9.09 149 396 8.72 148 412 7.71 138 388 7.88 150 413 02-Jul-2000 16 8.67 148 382 8.49 137 391 9.73 148 384 .... 9.69 145 412 7.95 151 368 Mean S.D. %CV %Theoretical %Bias n 8.51 152 0.621 ,., 9.51 7.3 6.3 99.0 98.1 -1.0 -1.9 15 15 394 15.8 o; ' 4.0 97.3 -2.7 15 Page48 Northwest Bioanalyfical Study No, NWBS00-040 Report No. NWBR00-108 TaMe 29. Intra-Assay Precision and Accuracy for PFOSA Specific Quality Controls All concentrationsare expressed as ng/mL. Run Date 16-.Iu[-2000 Run Number 17 Low QC Medium QC High QC (4.00nedmL) (1SOnedmL) (400uedmL) 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 (not included in overall validation summary statistics) Quadratic weighted I/x". run review. RunDate 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 I 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 39,0 531 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 - 1.1 2.8 2,0 -2.8 2.4 0.0 -3.6 - 1.7 3.2 2 2 2 2 2 2 2 2 2 Page 49 Northwest Bioanalytical Study No. NWBS00-040 Report No. NWBK00-108 Table 30. Intra-Assay Precision and Accuracy for PFOSAA, Specific Quality Controls All concentrations are expressed as n_mL. Run Date 16-Jul-2000 Run Number 17 [" Low QC tMedium QC High QC (9.00nedmL) (15Sng/mL) (40Sng/mL) 7.30 161 368 *4.93 176 382 "5.65 * 190 395 8.36 159 365 **21.4 151 346 Mean 6.56 167 371 . S.D. %CV %Theoretical n 1.56 15.5 .18.5 23.8 9.3 5.0 72.9 107.7 91.6 4 5 5 * > +_290%theoretical ** Deactivated as an outlier (not included in summary statistics) Note: The following .... calibration curve and standard statistics are included for analytical (not included in overall validation summary statistics) Quadratic weighted 1/xz. run review. Run Date Run Number A B C I R-Squared LLOQ ULOQ , 16-Iu1-2000 17 0.000001 0.005669 0.008710 0.9764 7.50 505 n Run Date 1 1 1 I 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 - 2 2 2 2 2 2 2 2 * > 15% theoretical ** Sample deactivated as an outlier (> 2 times the acceptance criteria). Result not included in summary statistics. Page 50 NorthwestBioanalytical StudyNo. NWBS00-040 ReportNo. NWBR00-108 Table 31. PFOS Dilution Quality Control Samples All concentrationsareexpressedas ng/mL. Run Date 29-Jun-2000 30-Jun-2000 02-Jul-2000 Run Number 13 15 16 Dilution QC 4460 ng/mL 4310 4170 4420 3900 4110 4490 3990 4180 4050 4400 4160 4160 --4020 3790 3970 Mean S.D. %CV %Theoretical %Bias n 4140 199 4.8 , .,, 92.8 -7.2 15 : ,7 . " 3 ,_.,,.;-.._ Page 51 NorthweBsitoanalytical StudNyo.NWBS00-040 ReporNto.NW'BR00-108 Table32. PFOSA DilutioQnualityControlSamples All concentrationsare expressedas ng/mL. Run Date 29-Jun-2000 30-Jun-2000 02-Jui-2000 Run Number 13 15 16 Dilution QC 4000 n_mL 3720 3570 3720 3380 3690 3020 *2890 3100 *2750 *2860 3270 3130 3130 3210 3240 Mean 3250 S.D. 315 %CV 9.7 %Theoretical %Bias 81.3 -18.8 n 15 " > ._+25%theoretical .: :. '" :-.-'- - Page52 NorthweBsitoanalytical StudNyo.NWBS00-040 Rc'poN_o.N'WBR00-108 Table33. PFOSA.A DilutioQnualityControlSamples Allconcen_atiaorneesxpressaesdn_mL. Run Date 29-Ju_-2000 Run Number 13 DilutioQnC 4050 ng/m.L 3490 3280 3270 "3170 3390 30-Jim-2000 15 3420 *3030 3240 *3060 "3120 , 02-Jul-2000 16 *3220 "3110 "3210 *2960 *3000 Mean S.D. %CV %Theoretical %Bias n * > +_290t%heoretical 3200 157 4.9 79.0 -21.0 15 ,: .., _: g :-..-:- - Page 53 NorthwestBioanalytical StudyNo. NWBS00-040 ReportNo. NWBR00-108 Table 34. POAA Dilution Quality Control Samples All concentrationsare expressedas ns/mL. Run Date 29-Jun-2000 Run Number 13 Dilution QC 4030 ng/mL 4220 3820 4250 4040 4000 30-]'un-2000 15 4550 3850 4010 4000 4020 02-3"ul-2000 16 4170 4090 .... 4230 3910 4040 Mean S.D. %CV %Theoretical %Bias n 4080 184 4.5 101.2 1.2 15 .:. _ :. : :_. _ Page54 .. . NorthwesBtioar_lytical StudyNo.NWBS00-040 ReporNt o. NW'BR00-108 Table 35. PFHS Dilution 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 Dilution QC 4020 ng/mL 4830 4430 4530 4150 4190 *4980 4500 4550 4490 4790 4700 *5030 4550 4260 4760 Mean S.D. %CV %Theoretical %Bias n * > +_.20%theoretical 4580 267 5.8. 113.9 l 3.9 15 .::.. -. ..=-- ,g Page55 NorthweBsitoanalytical StudNyo.NWBS00-040 ReporNto.NWBR00-108 Table36. M556 DilutionQualityControlSamples Allconcentratairoeenxspressaesdng/mL. Run Date 29-Jun-2000 30-.lun-2000 02-J'ul-2000 Run Number 13 15 16 DilutionQC 4020 ng/mL 4330 4050 4260 3940 4000 4490 3980 4130 4000 4170 4270 3990 4240 3890 3980 Mean S.D. %CV %Theoretical %Bias n 4110 172 4.2 102.2 2.2 15 .:.: : .. g : ..--.. Page 56 NorthwesBtioanalyt/cal StudyNo.NWBS00.040 ReportNo.NWBR00.108 Table 37. M570 DilutionQualityControl Samples All concentrations are expressed as ng/mL. Run Date 29-Jun-2000 30-Jun-2000 02Jul-2000 Run Number 13 I5. 16 , Dilution QC 4050 ng/mL 4710 4590 4550 4100 4490 4610 3980 4080 4080 4280 4270 3950 4120 3890 3960 Mean S.D. %CV %Theoretical %Bias n 4240 278 6.6 . 104.7 4.7 15 .: '. _ : . :.: - Page57 NorthwesBtioanalytical StudyNo.NWBS00-040 ReportNo.NWBR00-10S Table 38. PFOSA Specific Dilution Quality Controls Allconcentrationsare expressedas ng/mL. Run Date 16-Jul-2000 Run Number 17 Dilution QC 4000 ng/mL *2630 *2570 3110 3250 4110 Mean 3130 S.D. 620 %CV 19.8 %Theoretical 78.3 r %Bias -21.8 n 5 .... * > +_25%theoretical m: _.. Page58 NorthwestBiomalytical StudyNo, NWBS00-040 R_>ortNo. NWBR00-108 Table 39. PFOSAA Specific Dilution Quality Controls Allconcentrationas reexpressedas ng/mL. Run Date 16-Jul-2000 Run Number 17 Dilution QC 4050 nglmL *2560 *2650 *2900 3640 4070 Mean 3160 S.D. 661 %CV 20.9 i %Theoretical 78.0 %Bias -22.0 n 5 .q-, * > -+20% theoretical Page 59 Northwest Bioanalytical Study No. NWBS00-040 Report No. NWBR00-108 Table 40. PFOS Extraction Efficiency Run 7 Law Concentration " 2.50 ng/mLfor Analyte Time of Spiking I. Analyte and IS after extr'_ction Mean P,_"0 Analvt_IS 0.00936 0,0101 0.0103 0.00992 Ratio IS/AnaIyte 107 99.2 97.5 I01 2. Amlyte prior and IS after extraction : Mean 0.00532 0.00568 0.00517 0.005.]9 3. IS prior and Analy_ after extraction 6.94 7.51 8.42 Mean 7.62 Mean extraction efficiency for the analyte = Mean ran'action efficiency for the internal standard = 54.3% 7.54% ._edtum Cancentral_on _ IOO ng/mL for Anal)'te "_!me 0fSoiking I. Aaal v_ and IS at_r exwaction Mean Ratio Analvtc, IS 0.326 0.297 0.312 0.312 Rado tS_'Analyte 3.07 3.37 3.21 3.22 2. Aaaly_ prior and IS after ex_cdon Mean 0.! ! 7 0,121 0.113 O.117 3, IS prior and Analyte after extraction Mean 0.263 0.269 0.280 0.271 Mean extraction efficiency for the analyte = i_,|ean extraction efflcien,:_ for the internal standard _ H_* C#=snwat/o= - 400 ng/m for.4a_.rt 37.5% 8.42e/o T_me 0fSoikinf I. Analyte and IS after e,xmtcdoa Mean _,ati0 Amly_$ 1.17 1.25 1.21 1.21 Ratio I$/Analvte 0.$55 0.797 0.828 0.827 2. Analyte prior and IS after exwaction Mean 0.435 0.398 0.418 0.417 3. IS prior and Anal,,._ al_r ext,.edna Mean 0.0692 0,0652 0.0668 0.067 I Mean extraction efficiency far the attalyte ,, Mean extraction efficiency for the internal standard ", O_ra//_tna exmlction e2T/c/sn_fir aaa/.v_" O_arr_l meam e._m,acrlan eff_iency fnr mttrnal st_da_d " 34.5% 8.1 I% 4_ I &02 _ Page 60 NorthwesBtioanaly_ical StudyNo.NWBS00-040 ReportNo.N'W'BR00I-08 " =" . ; :: _ : ; :, Table 41. PFOSA Extraction Efficiency Run 7 La_ Concentration" 2.50 ng/mL for Analyte Time of Soikin_ I. Analyte and IS aRnr extraction Mean Ratio.Analvtc/I$ 0.00603 0.00611 0.00587 0.00600 Ra_io[S/Analvte 166 164 170 167 2. Analyte prior and IS aRcr can-action ,Wean 0,00375 0,00434 0,00383 0.00397 3. IS priorand Anal vU_a_rexlraction Mean 11.4 I 1.2 t3.4 12.0 Mean extraction efficiency for the aaalyte '! Mean extraction efficiency for the Internal standard = 66_'/. 7.19% ._edium Concentration ** l OOnglmL foe Anal)_ Time of Soikinz - I..A.nai_e and IS aRcr exff'_ction : Mean Rado Analv_e/IS 0.240 0.203 0.214 0.219 Ra6o [S/Analvte 4.16 4.92 4.68 4.59 2..4a2aly_ prior and IS att_r extraction Mean 3. [S prior _nd Anal.v_ aRer extraction O.148 0.128 0,152 O.143 Mean 0,331 0.357 0.358 0.349 Mean extraction eflklency for the analyte = Mean extraction efficiency for the internal standard = &_J% 7.60% High Concentrm#.on " 4OOn_/mL for AnaJy_ Time of _ikinsl 1.' Analyze and IS alter extraction Mean Ratio Analvt/lS 0.950 0.901 0.973 0.941 Ratio IS/Ann|yen 1.05 1.11 1.03 1.06 2. Analy_ prior and IS afterxn'action Mean 0.595 0.574 0.648 0.606 J, IS prior and Anal_e aRnr extraction Mean 0.0880 0,0778 0.0817 0.0825 Mean extraction efflcie_'7 for the analyte ,, Mean extraction efficiency fortheInternal standard _ Oyera_ mean _racdan effic_ncT fae _ncdyre 01,er,*ll menn _acdon e_flci#ncy foe intlenM_n[andard- 64.4% 7.78% L$.J_ 7.$2% Page 61 Northwest Bioanalytical Study No. NWBS00-040 Report No. NWBR00-108 Table 42. PFOSAA Extraction Efficiency Run 7 Low Concentration = 2.JO ng/mL fne Analj_e Time of Soikin_ 1. Analyse and IS aRer extraction Mean Ratio Analyse/IS 0,00526 0.00589 " 0.00559 0.00.558 Ratio !_Analvte 190 170 179 180 2. Analyt= prior and l$ after extraction Mean 0.00401 0.00427 0.00399 0.00409 3. IS prior and Ana|yt after extraction 12.8 14.1 g:o 15,6 Mean 14.2 .'*lean extraction efficiency far the analyte =. Mean extraction effitlencv for the internal standard == 73.3% 7.89% Medium Concentration - lO0 ng/mL for Analyse Time of Soikin_ I. Analyse and IS aRer extraction Mean Ratio Analvte/lS 0.373 0.325 0.342 0.347 Ratio IS/Analyse 2.68 3.08 2.93 2.90 2...t.naIFte prior and IS after extraction -. ". Mean 0.254 0.270 0,249 0.2.58 3. IS prior and ._l_c after extraction , Mean 0.234 0._8 0.248 0.240 Mean extraction efficiency for the analyte = ,Mean extraction efficiency for the internal standard = 74.4% 8.28*/. High Concenwatton = 400 ng/mL for Analy_ Time of Soikin 1. AnalySe and IS aRer extraction A/can Ratio Analvte._S 0.S64 0.900 0.889 0.S84 Ratio IS/Analyse 1.16 1.11 1,12 I, 13 2. Analyte prior and IS aR=r eswaction Mean 0.642 0.624 0.6.58 O.64I 3. IS prior and Anal.v_ a_r extraction Mean 0.09_ 0.0878 0.0885 0.0902 Mean extraction efficiency for the analyse = Mean extraction efficienc_ far the internal standard = O_r_l _,_an _rtraction ,ffkitno.l'ot inJlyte ", Overall meam e._acC_n efficiency far iattrnai standard - 72.$% 7.98% 73. J & 05 Page62 Northwest Bioaaalytical Study No. NWBS00-040 Report No. NWBE00-108 Table 43. POAA Extraction Efficiency Run 7 Low Concentration = _JO ng/mL far .4nalyte Time QSo/kin_ I. Anaiyt and IS after extraction Mean Ratio Anaivt_/IS 0.00921 0.00982 0,0105 0.00984 Ratio {S/Angevin 109 102 95.0 102 2. Analyte prior and IS after extraction 0.00209 0.00213 " 0.00224 Mean 0.00215 - t 3. IS prior and Analyze after extraction 7.02 7.35 " _ :' 7.97 Mean 7.4 $ Mean extraction efficiency for the analyte = Mean extraction efficlencv for the internal standard = 21.$% 7.30% ,$fediam Concentration = lOO ng/mL for Analyre Time of Spiking " I, Anal_e and iS at_r extraction Mean RatioAnaiv'te/[$ 0.358 0.3t8 0.336 0.337 Ratio{S/Analvte 2.79 3.i5 2.98 2.97 2. Analyze prior and IS after ext_ction 0.0526 0.0570 0.0516 Mean 0.0537 .... 3. IS prior and Analyze after extraction Mean 0.227 0.237 0.248 0.237 Mean extraction efficiency for the analyte Mean extraction efl'lclencv for the Internal standard = 15.9% 7.98*/, Ili_glt CancenO_an - ,tOOnAp/roLleArnal3_ Time of Sot'k[nsE !. Amlyte and IS ai_er exU'_tion Mean RatioAnalvte/lS 1.19 1.31 1.29 1.26 Ratio lS/Analyte 0.841 0.762 0.775 0.793 2. Anal vle priorand IS after extnction Mean 0.205 0.201 0.216 0.207 3. IS priorand Analyteafterextraction Mean 0.0638 0.0595 0.0627 0.0620 Mean extraction efficiency for the analyt Mean extraction efficiency for the internal standard = Overall mean t.vwat'_n e.Olci_ncy fer analyt " Overall mtnn e._ractton e_/_i_ncy fer internasltanderd " 16.4% 7.82% l& O_ 7.70_ Page 63 NorthwesBtioanalytical StudyNo.NWBS00-040 ReportNo.NWBR00-108 - ..z' .. r :; : . :, Table 44. PFHS Extraction Efficiency Run 7 l.a_, Concentration - 2.50 ng/mL for .4nalyte Time ol'5piking I..Anal_ and IS aRar e._r'action ,Wean Ratio ,AnalvtedS 0.0158 0.0168 0.0169 0.0165 R:,do [.,_,Analv_e 63.2 $9.6 59.3 60.7 2. Analyte prior and IS aRcr extraction Mean 0.00371 0.00384 0.00399 0.00385 3. IS prior and/naiyte after extraction 4.43 4.47 5.27 ,Wean 4.72 ,Mean extraction efficiency for ti_ aaalyte = Mean e:ffracfloa efficiency, for the internal se_adard =. 23.3% 7.78% ,$[edium Concentration = 100 ng/mL for rtnalyte Time of So_ine I. Ana|yte and IS after extraction Mean Rado Anal_e_IS 0,632 0.$67 0.598 0.599 Ratio[S/Analvte 1.$_ 1.76 1.67 1.67 2. Aaalyte prior and iS at_er extraction Mean O.I05 0.II3 0.I08 O.109 3. iS prior and Analyte after extraction _ Mean 0.134 0.146 0.I-_ O.142 Mean extraction efficiency for the analyte =, Mean extraction efficient 7 for the internal standard = IS.Z% 8..0% High Concentration- 400 ng/mL forAnal_ Time of So_in_ !. A_lyte and IS after exaction Mean Ratio Analvte/IS 2.07 2.25 2.15 2.16 P,igio [S/Amhne 0.484 0,4.44 0,465 0,464 2. Analyu: prior and IS after extraction Mean 0.387 0.363 0.385 0.378 3. IS prior and .-_naly*.eaRer eaWaction Mean 0.0394 0,0360 0.0365 0,0373 Mean extration emcteacy for the analyte = Mean extraction efficiency for the Internal standard = Overtll Overall mean e,rtracrion efficiency for _tdyte mean _tractian efficiency far internal $'atndard 1" 17.5% 8.04% It 7_ & 11 Page 64 Northwest Bioaaalytical Study No. NWBS00-040 ReportNo. NWBRO0-108 - .:' ._. : : " :" Table 45. M556 Extraction Efficiency Run 7 Lov_ Concentration " _-.fO ng/mL for .4nalyte Tirr,_fSoikin_ I.Analyteand IS a_er extraction Mean RatioAnalvtedS 0.00635 0.00659 0.00684 0.00659 RatlolS/Analvte 157 l$2 146 152 2. Analy_e prior and IS a_er ex_'action Mean 0.00293 0.00299 0.00279 0.00290 3. IS prior and -_l,v_ a_er extraction 10.7 10.7 12.4 Mean l 1.3 Mean extractioenfficiencfyor theanalyteMean ezn"lction efficlencv for the internal standard - .I..I.0% 7.43% Medium Concentration_' lOO ng/mLforAnalyce Time of Soikin_ 1. Anai'._ and :S a_cr exmacdon Mean Ratio Analv_e/IS 0.270 0.240 0.252 0.254 Ratio [S/Analv_e 3.70 4.[7 3.97 3.95 2. Analy_e prior and IS after exn'acfion Mean 0.106 0.124 0.111 0.| 14 3. IS prior and .-_al.vu: aP_erextraction Mean 0.309 0.320 0,325 0.318 Mean extraction efficiency for the aaalyte = ,Mean extraction efficiency forthe internal standard = 44.9% 8.05% High Com#ntratton - 400 mg/mL for AnnJJrt_ Time of Soikim' I. Anal3ae and IS after extraction _ 1.02 1.07 1.05 Mean 1.05 0.984 0.934 0.953 0.957 2. Analyte prior and _S a_er ex_action Mean 0.4.44 0.4-;1 0.463 0.4,4.9 3. IS prior andAnalyteai_:r extraction Mean 0.0800 0.0744 0.0765 0.0770 Mean extraction efficiency for the analy_ Mean eat'action emclencv for the Internal standard - 42.11"/. 1.05% Overtll mean .xa.acriom e.Oicien,:yfor ,,Mlvw Overall meam _waet_am effiiancy far _ngtrmaJ standard - 4J. 9_, 7, _4 Page 65 NorthwestBioanalytical StudyNo. NWBS00-040 ReportNo. NWBR00-108 Table46. M570 Extraction Efficiency Run 7 Law Concentration" 2.50ng/mL for .4naiyte Time or'Soikinz I,Anaiyteand IS aRer exaction ' ?-lean RatioAnal_hedlS 0.00470 0.00487 0.00530 0.00496 RatiofS,'An-',Ivte 213 205 X89 202 2. Analyte prior and IS after extraction : Mean 0.00317 0.00355 0.0G320 0._331 - "3"' . - ;: ; :" 3. IS priorand Analy_ afterextnccion Mean 14.4 14.0 15.8 14,7 Mean extraction efllciency forthe analyte Mean extraction efflciencv for the in|ernal sundzrd = 66.7"/. 7.28% .$fedtum Concentration- lOO n_/mL forAnatjrte Time of Soikin__ I.AnaIyteand 1S aRcr extraction Mean RaEo Analv_e/IS 0.217 0.191 0.204 0.204 RatioIS/Analvie 4.6[ 5.24 4.90 4.92 ( 2..-_naly_e prior and 1S after extraction 0.130 0.143 0.136 Mean 0.136 3. IS priorand Analyteafter extraction Mean 0.391 0.396 0.422 0,403 Mean extraction efiqcieocy for the analyte = Mean extraction efficiency for the intern_'tl standard = 66.7% g,19% High Concentration -- 400 nf,/mJ for Ancs_Fte Time of Sn_in_ I.Analy_ and IS ai_r exlxactina Mean _ 0.772 0.831 0.796 0,800 Ratio[S/Analy_q 1.29 1.20 1,26 1.25 2. Analyte prior and IS after exuaction ?.lean 3. [SprioarndAnavi_aefter extraction Mean 0.533 0.._21 0.563 0.539 0.102 0.0970 0.098-_ 0.0991 Mean extraction efnciency for else znalyte Mean extraction emciencv for the internal standard t O_r_! mean ex_al_n efficienc)" fnr enalyte m Overallme._n e._:rrac6nn efficiency far intern "t stmxdnrd m 6"/.4"/s 7.93"/* _(t.9_ 7,BO_ Page 66 NorthweBsitoanalytical StudNyo".NWBSO0-040 ReporNto.NW'BR00-108 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 insti'ument was operated.in the multiple reaction monitoring (MRM) mode under optimized conditions for PFOS, PFOSA, PFOSAA, POAA, PFHS, M556 and MS70 detection. CHEMICAL STRUCTURES 0 F2 F2 F2 F3C_c/C_c/C_c/C_c/ F2 F2 F2 _ NI-12 F2 O PFOSA MAY=499.0 [M-H]=498"0 OI| F3C_c/C_.c/C_../C_._ _ _ / SI[_ tN/_._]I/OH II F2 F_ F2 F_ O _ _ \ PMF-O7:S=A5A85.0 [M-n]=584.0 Fx Fz F_ F_ _ F3C_c/C_c/.C_c/C_oH Fz IIit! O POA.A MW--414.0 [M-H]-----413.0 Page 67 Northwest Bioanalytical F_ F3C _ c/C_ F. F, c/C_ F, F2 _/cC_ ["_ O [[ c/S_oIHI _O Study No. NWBS00-040 Report No. NWBR00-108 PFOS Mw_99 9 [M-H]-=498.9 O : F2 F2 I[ F3C_c/C_c/C_c _ _O H "._:o F2 F2 F2 O PFHS MW--399.9 O F2 F2 F2 I[ F3C_cF2/C_c/CF2_c./CF_2 C II N F2/sO_ H _OH O M556 MW=SS69 [M-H]=555.9 O F2 F2 F2 11 F3C.c/C.cF/C2.c"/C--c/i_ F2 F2 F2 O. N[/'_O CH 3 0 H M570 M[MW-H=]5=7517"00.0 Page 68 Northwest Bioanalytical Study No. NWBS00-040 Report No, NWBR00-108 F2 F3C_cJC_cJC_cJC_cJ F2 F2 F2 F2 O [-[2 :_OH H2 O THPFOS MW=428 7.1. w ..-, . Reference Materials and Matrices ";:" Analyte Lot Number Purity Expiration Date Source Storage Conditions PFOS CFC-95) PFOSA 193 100% 12/31/2010 3M 214 . 100% 12/31/2010 3M PFOSAA (FC-129) 617 53.8% 12/31/2010 3M POA.A (FC- 143) 245 100% 12/31/2010 3M PFHS* M556 M570 $398-I 82 100% 12/31/2010 3M NB 113047- 99.89% 12/31/20 t0 3M 8D 118506-26 99.75% 12/31/2010 3M .THPFOS 59909 90% 12/31/2010 3M * Received as a 6,200 ppm solution in methanol. ** Kept dry. 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, PFOSA.A, POAA, PFHS, M556 and Page 69 ... NorthwestBioanalytical StudyNo. N'WBS00-040 ReportNo. NWBR00-I08 M570) at a target concentration of 5000 ppb. Absolute determination of the concentration of FFOSA 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 : _:. POA.A PFHS Correction Factor 0.836 0.909 0.855 Matrix Human serum H.uman plasma ('Northern Chinese) Lot Numbers BC30399-4 C5186; C4929; C5517; C5200 7.2. Chemicals and Equipment Chemicals Ammonium acetate, 99.9% Di (ethylene glycol) methyl ether [may be written as 2-(2-methoxyethoxy) Methanol, HPLC-grade Methyl-Tert-Butyl Ether (MTBE), HPLC-grade Water, H_PLC-grade ethanol] Name Autosampler: PE Series 200 Balance: Merrier Toledo MT5 EquipmentSupplies Source Perkin Elmer, Norwalk, CT Mettler-Toledo, Inc., Hightstown, NJ Page 70 - Northwest Bioanalytica[ Study No. NWBS00-040 Report No. NWBR00-108 Name EquipmentSupplies Source Centrifuge: Beckman GS-6R Beckman instruments, FuIlerton, CA Evaporator: Turbo Vap LV, Model 43750 Zymark Corp., Hopkinton, MA Hematology/Chemistry Mixer, Model 346 Fisher Scientific, Pittsburgh, PA HPLC Chromatographic column: Genesis Lightning, Jones Chromatography, Cis, 4 lam, 2 x 50 mm Lakewood, CA " :': : : Lkiuid Chromatograph: Hewlett Packard 1100 :_:. Agilent (Hewlett Packard), Palo Alto, CA Liquid Chromatograph: PE Sciex Series 200 Perkin Elmer, Norwalk, CT Liquid Chromatograph: Shimadzu SCL-10A controller with CL-10AD pump and CTO-10A column oven Shimadzu, Columbia, MD 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: Rain[n EDP Distal 100-10001.tL rM Pipettes: Rainin M Volumes: adjustable 20-50, 10-100, 100-1000 p.L Pipettes) Rainin ILL Volumes: adjustable 5-20, 20-100, 40-200, 200-1000 gL Sonicator, Branson Sonicator, Fisher Rainin Instrument Co., Wobum_ MA Rainin Instrument Co., Woburn, MA Rainin Instrument Co., Woburn, MA Branson, Danbury, CT Fisher Scientific, Fair Lawn, NJ Vortex: Fisher Genie 2 Fisher Scientific, Fair Lawn, NJ Water Pro Plus Labconco, Co., Kansas City, MO Page 71 NorthwestBioanalytica[ StudyNo. NWBS00-040 ReportNo. N'WBR00-108 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 N%VBas 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. Z?_ , " ,* , Reagents All reagent solutions are stored at room temperature unless otherwise noted. 50 mM ammonium acetate in water (unadjusted: pH-6.9) Fill a I-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 mMammonium 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 -20 C freezer. The PFHS reference material was provided as a 6200 ppm solution. .. Page72 NorthwestBioanalytical StudyNo. N'WBS00-040 Report No. NWBR00=108 PFOS Stock Standard ((9.05904 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 ((9.07652 mg/rnL) 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 ((9.02264 mg/mL) Weigh 1.052 mg of PFOSA.A 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 POA.A and add to a 25-mL volumetric flask. Fill to volume with methanol, mix by inverting 5 times and sonicating for approximately I0 .o-, minutes. I M556 Stock Standard ((9.04579 mg/mL) Weigh 1.146 mg ofM556 and add to a 25-mL volumetric flask. Fill to volume with methanol, mix by inverting 5 times and sonicating for approximately 10 minutes. M5 70 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/mL for PFOSA) Add 2.117 mL of PFOS Stock Standard (0.05904 mg/mL), 1.639 mL of PFOSA Page73 NorthwestBioana[y_ica[ Study No. NWBS00-040 ReportNo. NWBR00-I08 Stock Standard (0.07652 m_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 p.L of PFHS (6200 ppm), 2.73 mL ofM556 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 l0 minutes. Spiking Standard 8 (4000 ng/mLfor PFOS, PFOSAA, POAA, PFHS, M556, : ' M570; 4020 ng/m.Lfor PFOSA) "'Add 8.00 mL of Spiking Standard 9 to a 16 x I00 mm polypropylene tube and add 2.00 mL of 50:50 water:methanol. Mix by inverting 20 times. Spiking Standard 7 (2500 ng/mLfor PFOS, PFOSAA, POAA, PFHS, M556,M5 70,"2510 ng/mL for 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 water:methanol. Mix by inverting 20 times. Spiking Standard 6 (1000 ng/mL for PFOS, PFOSA, PFOSAA, POd_l, PFHS, :_,[556and M5 70) Add 2.00 mL of Spiking Standard 9 to a 16 x 100 mm polypropylene tube and add 8.00 mL of 50:50 water:methanol. Mix by inverting 20 times. Spikzlng Standard 5 (500 ng/mLfor PFOS, PFOSAA, POAA, PFHS, M556, M570; 503ng/mL 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 water:methanol. Mix by inverting 20 times. Spiking Standard 4 (250 ng/mL for PFOS, PFOSAA, POAA, PFHS, M556, M570; 251 ng/mL for 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 water:methanol. Mix by inverting20 times. Page74 NorthwestBioanaIyticaI StudyNo. NWBS00-040 Repor_No. NWBR00-108 Spiking Standard 3 (100 ng/mLfor PFOS, PFOSAA, POAA, PFHS, M556, ,1,[570."lO/ ng/mL for PFOS,4) Add 0.250 mL of Spiking Standard 8 to a 16 x 100 mm pol_20ropylene tube and add 9.75 mL of 50:50 water:methanol. Mix by inverting 20 times. ": : Spiking Standard 2 (25.0 ng/mL for PFOS, PFOSAA, POAA, PFHS, M556, M570; 25.1 ng/mL for PFOSA) Add 0.100 mL of Spiking Standard 7 to a 16 x 100 mm polypropylene _ : _add 9.90 mL of 50:50 water:methanol. Mix by inverting 20 times. :_:. tube and Spiking Standard 1 (10.0 ng/mL for PFOS, PFOSA, PFOSA_A, POAA, PFHS, M556 and M570) Add 0.100 rnL of Spiking Standard 6 to a 16 x 100 mm polypropylene tube and add 9.90 mL of 50:50 water:methanol. 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 water:methanol and mix by inverting 5 times and sonicating approximately 10 minutes. Worldng Internal Standard (400 ng/mL THPFOS) Add 413/.tL of the THPFOS Stock Solution (0.04842 m_mL) to a 50-m.L volumetric flask and fill to volume with 50:50 water:methanol. Mix by inverting 5 to 10 times and sonicating approximately 5 to 10 minutes. Quality Control (QC) Solutions All quality control solutions are transferred to polypropylene containers and stored in a-20 *C freezer. Page75 NorthwestBioanalytical StudyNo. NWBS00-040 ReportNo. 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 I0 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 %ith methanol, mix by inverting 5 times and sonicating for approximately 10 - -, _: .minutes. ".;:w PFOSAA QC Stock (0.02920 rng/rnL) Weigh 1.357 mg of PFOSAA and add to a 25-mL volume_c flask. Fill to volume with methanol, mix by inverting 5 times and sonicating for approximately l 0 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 (1000ppm) Aliquot 807#L of PFHS stock solution (6200 ppm) to a 5-mL volumetric flask. Fill to volume with methanol and mix by inversion. M556 QC Stock (0.05230 mg/mL) Weigh 1.309 mg ofM556 and add to a 25-mL volumetric flask. Fill to volume with methanol, mix by' inverting 5 times and sonicating for approximately I 0 minutes. 34570 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 NorthwestBioanalytical StudyNo. NWBS00-0.40 ReportNo. NWBR00-I08 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 #L 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 #L ofmethanoI. - -, : :, 'PFOSAA Concentrated QC Solution (I, 000, 000 ng/mL) ._:,Evaporate 4.42 mL ofPFOSA.A (0.02920 mg/mL) at 20 *C and below 5 P.S.I. nitrogen. Reconstitute with 129 ]zL of methanol. POAA Concentrated QC Solution (996, 000 ng/mL) Evaporate 2.59 mL of PO._. (0.04228 mg/mL) at 20 C and below 5 P.S.I. nitrogen. Reconstitute with 110 #L 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 ofM556 (0.05230 mg/mL) at 20 *C and below 5 P.S.L nitrogen. Reconstitute with 114 #L 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 1 I4.4 p,L 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 NorthwestBioanalytical Study No. NWBS00-040 ReportNo. NWBR00-108 - .:-, Dilution Quality Control (3990 n_mL PFOS, 4000 n._mL PFOSA, 4000 ng/mL PFOSAA, 3980 n_mL POAA, 4000 n_'rnL PFHS, 4000 n._mL M556 and 4000 n_rnL M570) Transfer 100 #L of each Concentrated QC Solution (997,000 ng/mL PFOS, 1,000,000 ng/mL PFOSA, 1,000,000 n_mL PFOSA.A, 996,000 n#mL POAA, 1,000,000 ng/mL M556, and 1,000,000 nffmL M570) and 100 _L of the 1000 ppm PFHS QC Stock into_a 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. Hich Ouality Control (399 ng/mL PFOS,400 n_mL PFOSA, 400 ng/mL PFOSAA, 398 nodmL POAA.. 400 n_mL PFHS, 400 na/mL M556 and 400 n._mL M570) 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 (15.0 n_mL PFOS, 150 n_mL PFOSA, 150 ng/mL PFOSAA, 149 n._mL POAA, 150 n_mL PF.HS, 150 n_mL M556 and 150 n_mL .... M570) 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 10minutes and equilibrate for approximately 10 minutes. Low Ouality Control (3.99 n._/mL PFOS, 4.00 n._mL PFOSA, 4.00 n_ mL PFOSAA, 3.98 n_mL POAA. 4.00 ng/mL PFHS, 4.00 ng/mL M556 and 4.00 n_mL M570) Transfer 0.500 mL of 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 of 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 Speeifie Validation Quality Control Samples Oualitv Control (QC) 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 : .:a "withmethanol, 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 sorLicatingfor 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 PFOSAA) Transfer 0.462 mL of the PFOSA QC Stock Solution (0.2166 mg/mL) and 0.431 mL of the PFOSA.AQC 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 Oualitv Control(400 ng/mL PFOSA and PFOSA.A) Transfer 2.50 mL of the Dilution Quality Control into a 25--mLvolumetric flask. Fill to votume with human serum and mix by inverting approximately 10 times. Soaicate for 5 minutes and equilibrate for approximately 5 minutes. Page 79 Northwest Bioanalyfical Study No. NWBSO0-040 Report No. NWBR00-108 Medium Quality Control (150 ng/mL PFOSA and PFOSA.A) 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 n_mL PFOSA and PFOSA.A) 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 of QC Samples Afterpreparation, 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 ILLblank human serum and 400 gL 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 gL 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, PFOSA.A, POAA, PFHS, M556, M570 / PFOSA 5000/ 5020 4000 / 4020 2500 / 2510 Volume of Spiking Solution (ILL) lo.o 10.0 I0.0 Volume of BiankHuman Serum (gL) loo 100 I00 Final Concentration* (ng/mL) soo/502 400 / 402 250 / 251 Page 80 ... Northwest Bioanalytical Study No. NWBS00-040 Report No. NWBR00--108 Standard Number 6 5 4 3 2 1 Concentration of Spiking Solution (ng/mL) PFOS. PFOSAA, POAA, PFHS, M556, M570 / PlrOSA 1000 500 / 503 250 / 251 100/I01 25.0 / 25.1 I0.0 Volume of Spiking Solution (IzL) 10.0 10.0 I0.0 I0.0 10.0 I0.0 Volume of Blank Human Serum (_L) Final Concentration* (ng/mL) t00 t0,0 100 50.0 / 50.3 100 25.0/25.1 I00 10.0/10.1 I00 2.50 / 2.51 100 E00 * The target calibration curve range is 1.00 n_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 CalibrationCurveSan_ples(preparein duplicate) as indicated above Quality ControlSamples (preparein duplicate) ++o, Aliquot 100 p.Lof each of the Low, Medium and High controls into separate 13x 100 mm polypropylene tubes. If'needed, prepare a dilution control (in triplicate) according to the following formula: for DF=X, aliquot (0.500 miD() of dilution control into [0.500 mL - (0.500 mL/X)] of blank human serum. Aliquot 100 I.tL of the prepared dilution control into separate 13 x 100 mm polypropylene tubes. Blank Control Samples (prepare in duplicate) Aliquot 100 _.L of blank human serum into separate 13 x 100 mm poiypropylene tubes and label as QC0. Aliquot 100 _L of blank human serum into separate 13 x 100 mm polypropylene tubes and label as BLANK. Page 81 +.. NorthwestBioanalytical Study Samples StudyNo. NWBS00-040 ReportNo. NWBR00-108 Transfer 100-/aL 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 _L of 50 mM ammonium acetate in water (unadjusted: pH _6.9) to each . sample except calibrators and vortex mix briefly. 2. Add 50.0 _L of 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-tert-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 _<5psi) for approximately 40 minutes. 9. Reconstitute in 100 _.L of 30:70 20 mM ammonium acetate in water (unadjusted: pH -6.9): 20 m.M 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 Page82 NorthwestBioanalytical 7.9. LC/MS/MS Conditions StudyNo. NWBS00-040 ReportNo. NWBR00-108 " _"' :. LC Conditions HPLC Column Mobile Phases LC Conditions Genesis Lightning CIs, 4/am, 2x50 mm A: 20 mM ammonium acetate in water (unadjusted: pH -6.9) B: 20 mM ammonium acetate in methanol Gradient :: '_:" Time (min.) %B 0 50 1 50 7 97.5 9 97.5 9.1 50 11 end Flow Rate 300 gL/minute Post Column Addition I 50 p.L/minute of 2-(2-methoylethoxy) ethanol Column Temperature 40C Injection Volume 2 - 20 p.L MS Conditions Post Column Split Source Source Temp. Ionization Mode Analysis type None TurbolonSpray rM (flow rate 8 L/rain.) 400 C Negative Ion Multiple reaction monitoring (M1LM) PFOS PFOSA Transitions monitored (+0.3), 499 --_ 80 498 _ 78 Dwell Time (ms) 70 70 Collision Energy(eV) 80 65 Page 83 NorthwestBioanalytical - PFOSAA Transitions monitored (+_0.3) 584 --).419 POA.A. 413 --> 169 PFHS 399 _ 80 M556 556 --_ 498 M570 570 -_ 419 THPFOS 427 -_ 407 StudyNo. NWBS00-040 ReportNo. NWBR00-108 Dwell Time (ms) 70 70 70 400 70 70 Collision Energy (eV) 29 25 70 40 42 42 The,prepared standa/-ds 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 DMLIMS software (version 6. I. 1.04) Page84 o. Northwest Bioanalytical Study No. NWBS00-040 Report No. NWBR00-108 [$ 16- 14- 12I . ..:..-,.._ IO- :; Figure 1. Representative CalibrationCurve for PFOS Analytical Run [.1analyzedon 29-$un-2000CalibrationStandards['orPFOS (ng/mll Rcocssion Method =QUADRATIC- Wci_htini Factor- [iX'2 QuadraticLimit- 1930 ] 'ta _T o 100 200 3o0 40o 500 600 Nominal Conc.(ns/mL) Figure 2. Representative Calibration Curve for PFOSA -., A_aly_cal Ran 13 aaa.ly'ztdon 29-Jua-2000CallbradoaStandardsfor PFOSA (ab'm-q Rz_esslon Method - QUADRATIC - Weightlng Factor - l/X*2 Quadratic Limit - 1560 40 30 _ 2o I0 $ 0 0 I I00 200 300 400 $00 600 Nominal Cone.(as/''/) Page 85 Northwest Bioanai)cicaI Figure 3. Representative Study No. NWBS00.040 Report No. NWBR00-108 Calibration Curve for PFOSAA I" . i I0"_ I _1r i i['12,,- 1 0-'_ 0 amaly_ica|Run I.t analyzedon 29-Jun-2000 Calibration Slanda.rdsfor PFOSA.-_(n;'ml) RckTessionMethod -QUADRATIC- Wei_t/ng Factor- it.'<": Quad_tic L_| = .1880 - , i 100 200 300 400 500 600 Nominal Cont. (n_'mL) Figure 4. Representative Calibration Curve for POAA '12 I Aaaly6cal Ram 13 analyzed on zg-Jua.Z000 Ca_br'a6ou Standards for POAA (ng/m_) R_ression Me,hod - QUADRATIC - Weigh6ag Facior - 1/3("2 Quadrate Limit - -48 ;0 t A '11 0_ ' 0 I O0 , "* ZOO 300 400 NominalCont. (nV'ml..) ' _00 600 Pagc 86 NorthwestBioanalytical StudyNo. NWBS00-040 ReportNo. NW'BR00- I08 Figure 5. Representative Calibration Curve for PFHS Axlal_,_cRauln 13 ;malyzeodn 29-Jun-2OOC0alibratiSolnandardsforPFHS (ng,,ml) _c_cssion Method - QUADRATIC - Weighting Factor - I X"2 Quadrat/c Lim/t - 1870 12 IO 6 ..... "i 4 0, , , I00 200 ." 300 400 500 600 Nora/hal Cooc. _nf/mL) 3.0 2.$ 2.0 i i--= '1..0$i Figure 6. Representative Calibration Curve for M556 Analytical Pare 13 analyzed on 29-1un-2000 Calibration Standards for M556 (nWml) Regx'_sioMncdtod - QUADRATIC - Weighting Factor - I.'X"2 Qu._h-_c Lim/t --I 710 0.0 0 1 " b _ _ I00 200 300 400 SO0 600 Nomlaal Conc. (ag/mL) Page 87 NorthwesBtioanalytical StudyNo.NWBS00-040 ReportNo.NWBR00-108 Figure 7. Representative Calibration Curve for M570 .knaly_ical Run I3 aaall,'zedon 29.1un-2000 Cafibratioa Slaadatds for MS'/0 (ntvmi) RecessionMethod = QUADRATIC - Wcighdntt Factor = I_.'X"2 Quadratic Limit - | 770 L0 2J 2.0 -! I.$ LO OJ 0.0 , , . t00 200 :}00 400 500 600 Nominal Cone.(nWmL) Page 88 Northwest Bioanalytical Study No. NWBS00-040 R_ort No. NWBR00-108 Figure 8. Standard (57.1 ng/mL) Chromatogram for PFOS " "-_"-._ . p=cs tnlemal SlSt_Std" TNPFOS Use Ares AbsO_ule Reten,JOl Time r-:oected RT 5 35 I Cun'en! N_se Thres, [10.01 Method 10.0 Quanl Thrlul. _ 2.0 M_. Wid_ _ 5 Mull W_lth _ 20 Base Width _ -- 100 RT Win. (See4} _ Smaom I 20 11 t "_:'J/C_3 ? _3 S_G62 $id3 T_,;..;u, 2g, 20X) " '0 ,_M ,_ _-_ .I ! l_'.nrxI 1: a'sg MRM. 440 Stalls : NO CGtmne.I I00T0" " " ' 499._.>E0.0 90" Area., 602815 70" He_ht 63160 Start Tone End 1",me 5:05 60 - 0:03 II_l_l'ifJOl Wk_h Relel-lflo_ "rime Inte_fial Type 0:_1.1 _0" 5:35 A BO 40- intensitq : r:_ 187 cps 277 10- 0" 20 45 _ St 1:14 116 ' 1'_t 2:26 160 187211 ' ' 1_1 3:-39 / J 241 4:52 _313 ",_31 6:04 342 372 36,1 7:17 426 ' 4_1 ':_,u_ S:30 Time _F l_p_os I Cufre_ Memcd NO_eTh_I _01 5o.o a,t Inteenai StaJ1l_d _ ThteL __ 5.0 Mi_. W'idll 1121 12 ''*" MulL Width 1101 10 Ezoected lit 4:58 Base. WId_ _ RT wi_. (se_) _ s_othI 100 _l 20 ,._d_.07003 7 003 500062 St(J3 : No Cornmeal T_u. Jun 29. 2000 2:10 AM ._g,n 1 per*xl '00 1:8:59 MRM, 4445 m '90 427.0->407.0 Aru" 25_70e _m 4o_3 Start T1eml E_ Tgne 4:47 60 fl:O_ Inlo_ralIo_ _ R_lm_n Time Inle_raboI1 Type 0:21.8 _0 4:57 A - B8 40 _ntensitQ : 4|002 1_ 245 0 42 " 154 204 299 1:14 ._." 3.3'9 4:52 6:04 371 7:17 420 8:30 Time[ Page 89 Northwest Bioanaiytical Study No. NWBS00-040 Report No. NWBR00-108 Figure 9. Standard (10.1 ng/mL) Chromatogram for PFOSA _ .2', ./ p_OSA I Internal $1ancland:THPFOS Use Alea Absolute RelenlJot_"J_me [[xDeted RT 6:16 Cun'ent m Nodse Thre|. [ 20.0_ Quant Thres. _ Min. Width 151 Mull. Wldlh 1121 Base. WIdIJ_ _ wzn. (s_) _ Sm_ [ 1] 20.0 4.0 5 12 100 20 I 006_07003 ? _a33300(;62 Srd._ ! ; NO Comment Th6u5.9JtmJllI 2p9_. O2d0_0 2:0 '.V,I 1: _:_;gMP.M. 446 _ml ! I 00 " 498.0->78,0 _, Area Hel(Jht 261240 31081 Siart:'rJme f=nd T'_e I_lle_r_{_ll _ Relenuon l"_me IntecJrat_n Type 70" 5:50 _. 6:34 0:48.4 - _. iS:18 A * BO 40, _nt_itIl : 31 t "/6 cp$ 312 20- ;0 _,_ 29 ,6-;_';l 99 t_1131 158 t1_94i 219 22446T 27"JMZol _ z__7: 1:14 2:26 3:39 4:_2 6.'04 7:17 4'104_t s_ ;J:30 T_ei: [THPFOS USeas Inltm%ll Standaf_ Expected RT 4:M' I i Cum_ Memod C_Jarlt"l'hr_. _ 5.0 Min. Wk:l_l 1121 12 Mull V_ _ 10 8a._,e. Width _ 100 Smooth( 11 1 C06207003 7 003 S000_2 SKI3 1 No C_nmenl ;'_u, Jun 29.2000 2.10 AM 24S I: 8:._) MRM. 446 scans 427.0J,407.0 2567O6 Start Tmle _nd Time Irde_l_0_ Widm R_ Teme Inle_rat/_n T_ 4:47 5:09 0.'21,8 4:57 A B8 _O 70. 60, _0' , 40' _tensit_/: 4 ! 002 cps -'0' '0" | 42 1_4 204 ._ __L. 299 371 420 I :14 2:_._ 3:39 a':52 _;:04 7t7 _) :3CI Tk_l: Page 90 Northwest Bioanalytical Study No. N'WBS00-040 Report No. NWBR00- I 0g Figure 10. Standard (15.0 ng/mL) Chromatogram for PFOSAA [PFOSAA Internal 51anOa_ THPFO$ Use Area AOsok_teRetention"/';me FJDected RT 6:32 1 No=se Thtel. JCun'ont Method _ 12.0 ] 12 O Cluan| Thtll. _ Mitt. Wklth _ Mull IAqII/I _ BaSil, VVlat_ _ RT W_. (le_) _ Smoo_ I 40 .8 15 100 20 It I 7.('_?fJTqq3 7 _03 S0(:Cd2 $113 1 No Ccmwent Thu..,;u_t29, ._,l] ._ 10 _,_.1 ,qSO.I I penod t00' 1:8".'59 MRM. 446 SCans 30 584.0->419.1 Area He_jht 76838 11102 Si, it _'=me End "rime Int_m_ W_ 70. 6:10 60' 8:_ 0:33.9 _0, _Integration Ty_e A - 99 40. Intensi_ : I 1172 P-,pS 324 tO. _,1 1:14 121 2:25 181 3:39 ITHPFOS use as Internal Standa."d *. *,ExpecIe0 RT 4:58 I Cummt Memod Quant _ _ 5.0 MluUlBl _WWiIdctlhOt _ 9a..*e. wkim _ RT W_ (secs) _ Smoom [ 1120 100 20 Ij PA.'_207C_3 7 003 SOC_0_2Stu3 t NOComment The Jim 2J, _ d.59 _nI _ 2:10 AM I 0Cl- 427.0->407.0 Ama 25_70_ Helcjlat 40_43 Start _me En_ Tune Relentio_t T_me Intecjralion Type! 4:47 5:00 4:57 A. =tO ?O60- J,_l- 241 4:5_. j 7T,0I 6:04 24_ 3,_t 7:17 4_! ' g_ ._-S:30 Tk_ >. _t*nsit_ : 4 t 002 ps 20, I0- 0 ' 42' _'i ' "_t 1_4 _ 20'4 '2,_" '_299 '_ 3"71 ',_420 " Page 91 NorthwesBtioanalytical StudyNo.N'WBS00-040 ReportNo.NW'BK00-108 Figure 11. Standard (14.8 ng/mL) Chromatogram for POAA ..:, [PC'_'_ mtema Standard. _'I_PFCS USe Area ADSOlUISRSI_'II_On '_mo Eso_ecl RT 4 59 Currenl Method I No.sTshree._ Q,za_t Thrs_. _ . Min. Widl/1 [ Moll WIcIUI _ 20o 2.0 6] 6 10 Bass. WkRh _ RT W'm. ($ecJI) _ Smoo=i 100 20 tI I -:.'C_I'_79U:_ ? C0"Z S;JC'062Sta3 I NO Comment TIll. Jun .,9. 2000 2 I_ AM 248 59 m_! penoO [00" I: 8:';g MRM, 446 scans 413.0->169.1 :: ' Area 77412 H.eKJh 8757 St31 "nine End "nine Inte_i'21J_cIn ReCeption Time Integration Ty_ 4:34 5:18 0:44.8 5:(X) A BB '.:_0- 80" .70 60 50, /i 4G_ tntensit_ : 8785 cps 20 I0 34 G,3 84 122 154 2t_ ,-- . , , i ,-,_ 307 _;9 _;'/G 409 _ - ..... 1:14 2:26 3:37 4:52 604 7:17 8:.30 Ttm,_t_ ITHPFOs Cuce_ Memol I No_T_.. I----'_--I 50.0 Min. Wld_ _ 12 Expected RT 4:58 Base. Width _ Smoo_I 100 sI '_)6207L_03 7 003 500_2 StY3 I NOComment Thu. j_n 29. ZOO0 2.10 AM 245 d..c.cJ,nI 0enz_d 100. t: 8:5g MRM. 446 scans 427.0->407.0 '_0, _0" ,Am.1 2567O6 70. HeXer '4o943 Star_ Tkne "t'_ne 4:47 _0" 5:09 Inte<jfalio_ _ Rete_bon Tone .!.,,,eqra,o."r,,_t 0:21.8 _0' 4:57 . =,e _0- k_tensftu : 4100'2 cps 10 0 42 61 121 124 181 2_4 _ t I :14 ..2 3;3_ 4;$2 _2:3_0<1) G:04 3_31"71 7:17 442201 8:30 Se,anr---T_meI': Page 92 Nonhwcst Bioanalyticat Study No. NWBS00-040 Report No. NWBR00-108 Figure 12. Standard (12.2 ng/mL) Chromatogram for PFHS l No_seThres. Currefll MelflOd [ 20.01 200 Irllemdll SlarlClar_. Tk4PFOS Use Arei Absolute RSletltlo,n Tm_e E,,IOeCtlKIRT 4 17 Quant Threl. _ 2.0 Mm. WtdUI 151 5 Mull Wid_ 11OI 10 Bale. WIdU__ RT Win, {sect) _ Smoom{ 100 20 11 .1 L'_._7003 "P_,(_'tSrJ_ i_i]2 ; No Camment Thor. JUJI2(_. -_00_ "* _k"Al M 213 3 50 .I I OenOU :00" 1:8:50 k4/_d. 446 sc_Inll 399.0->79.9 Area 77378 Hr _h|. $h'ln Tone End Time 9782 _Fa.on W_ RetemUonTime mte(j_Uon T_ 3:57 4:30 o:.:_.7 4:18 A - B8 i _" 7060. 15040- intensit_ : 981 8 cps ' 13 33 _.3 a ;, ! :14 j I ! 0 I _59 ,_, : ,_, ._ 274 323 3G7 :_, 3o, z_, 2:-?.6 3:39 4.52 6:04 7;17 425 4:, _. r-"'- _:3Q Tcnel*. l-r+.+pr-os use al Into'ha Standard EXl_ RT 4:_ I ._._r Cun'_! MeO_od tool m,o Quar4 Thnm. Min. w_m Mull Wk:l_ P'-----'--1_ 1121 1101 Base. _ _ RT W+m.(_ca) 12Ol Smoo__ Ij 5.0 12 10 100 20 t C0fi207003 7 003 500062 SIU3 I NO Comment _,. J_n ._j. 2000 2:10 AM 245 dGOm _ penoa 00 1: 8:51) MRM. ,M6 scan_ 427.0-)407.0 25a70e He,hi _3 Stan _ 4:47 End T_ 5:09 Inte_lr_b_l _ Retlmtk0n "_ mle<j_on T-,I_ 0:21.8 4:57 A - 8B [*l+!"'l""l_ _ "tO 60. _0, 40 _o :0" 10" O 4'2 , .14. + 1_4 ._:_.G 204 3.3._ . ,,..J 4 52 2'99 +:04 _ntens_t_ : 41002 CpS 371 7:'_7 420 +:30 Page 93 Northwest Bioanalytical Study No. NWBS00-0.40 Report No. NWBR00-108 Figure 13. Standard (11.8 ng/mL) Chromatogram for M556 "2 ' 1. _58 Inlerrla Slannatl. THPFCIS Use Area ADSQ_UIIRetent_n Time E_Dected RT 6.04 J C_yent MeU_od Nc,se "rares. I T2.0 I 12 0 Quan[ _. _ Mitt. WIdUl _ Mu#. WIdlh _ Base. WidU__ RT Win. (sect) _ smoom I 40 5J 5 10 100 20 11 1 O0d=_700"_ ? _C'_ S0tlG6_ S:(_:_I 1 rhu. Ju. 2g. 2000 2 10 AM a 5g ._ 1 _ f : 100 t: 8:';9 MRM. 446 $MIa _j. NO Conlment 5_._->498.0 :: Area Hex_.t 15835 1227 ; Stdrt Trne i End Twe4 5:45 e:33 SO. ,70' 60. Reran,on "r',a,e 8:02 Intonation T,/pe A - 88 40, 2<)9 _ttnsit_ : 1243 cps i Inte_rat_on WIdlh 0:48,4 '_50, 72 61 121 19! 1:14 2:26 3:39 ITHPFOS usa as Intecnai Standa;'d C4Jmm I/,eelod I "il_m. [ _.0] . 80.0 Quart! Thn_ _ 5.0 F.xl_ RT 4:58 Mln. Wldm _ Bm_'. _ _ RT Win. (sacs) _ sn'_mt 12 100 11 20 (_6207C'03 7 003 SOC,062$tu3 t "rhu. j_ 29. 2000 2:10 AM P4oComrmmt 1:8:59 MRM. 448 scans _. 427.0->407.0 _0" 256706 70" HeKJh 40943 Stact Tmrm End "l"._e 4:47 60" 8:09 Inte(J/31bonWidth Retention "r_me 0:21.8 _0" 4:57 In_e(Jtabo_ T_/pa A - B8 40" 241 4.'52 _ 7._01 6;04 :3_1 ?:17 3 421 G:30 ,' S4mnf..-Tim_l >. intensit_ : 4|002 ps 24_ 4a _,'_' 1:14 _s_ 2o4 J a ' _ ' ,_ "2_,_ 2:.,?.6 3::39 4:_2 _ *-_ 6:04 _?_ 42o ' _;_ ' 4_ "s_,_f--. 7;17 _:30 T_m_[: Page 94 Northwest BioanaTytical Figure I4. Standard (14.6 ng/mL) Chromatogram Study No. NWBS00-040 Report No. NWBR00- I08 for M570 (U570 I Nc.se Thte|. Curren! 112.01 Metf_od 12.0 tnlemsl SI;incJarfJ:THPFOS Use ._.rea AbS_ute R4ieflbon Time Quan! Thres. __ Mm. WI_USi MuR. Wld_ _ 2.0 5J 5 10 E_pected RT 6.$8 Base. Wi(l_ _ 100 RT Win. (sect) _ 20 smooIm I I $ ,'_._:_?._rJ3 7 003 SC.Go'_SId._ i No Commefll T_u..;u, _9. 2Q(X)2: I 0 ,._'.4 8 .S_.I ! p_1o,Jr 100. 1:8.59 MRM, 448 scans 570.0->419.1 .':<3. Area 21348 ?O- Start Tm_e End Time 8:02 _0- 6:28 InmE_uon Widm Reclmli(_ "l-.'na Inte_raUon T_)e 0:.28.8 50. 6:lg A - BB 40- :313 intensih : 2958 cp8 20. 10- I_ o.;. 46 87 6t 1:14 110136 121 2:..26 $71 213 257282r_ a_ 181 3:39 241 4_.?. lOt 6;04 3693?5 361 7:17 423 421 _r-'- S:_;O Time( _. i LT_pr<xs I c_, _.Xl:)_ctReTcl 4:M W'Jdh_1121 12 Mull. Width _ 10 _ __ Wkn. (_) _ sm_mI 100 20 _I i 006207003 7 003 500062 Std3 t NOC_m_$ _u. ,'tin29. _000 2:t0 AM 245 59 _nI pen_Id 00 1:8:';9 MI_vl. 448 scans _1 427.0o407.0 2_70_ 4o_ Sla_ '_ ' End '1",me Inte(_ra_on _ Reten60n T_'_e ,)n,eq,='_r,tl_ 4:47 5:09 0:.21.8 4:$'7 A - so 70 60 _0. -_0. inttnsit_ : 41 002 cpS 10' ! 0 42 , '$_I t54 ',_i 20'4 '_4.J __. " 'z29d9_ ' '_ 3;71 '.4-2_0I 's=..r-- 1:14 ._ :-""_ 3:39 4:52 _:04 7:t7 0;30 T_ne( ; Page 95 ... Northwest Bioanalytical Study No. NWBS00-040 Report No. NWBK00-108 APPENDIX A The following quality control results are from sample analysis study NWBS00-062. Table A.I. Inter -Assay Precision and Accuracy for PFOSA Quality Control Samples All concentrations are expressed as ng/mL. Run Date _: 22-,Jul-2000 25-,1ul-2000 31-Jul-2000 Run Number 21 22 23 Low QC 4.00 ng/mL 3.95 "5.18 4.14 3.50 3.68 4.35 Medium QC 150 ng/mL 158 153 171 152 151 141 High QC 400 ng/mL 461 364 394 379 345 356 01-Aug-2000 24 4.14 150 395 i 3.84 144 359 02-Aug-2000 26 4.51 169 378 3.96 161 406 " 09-Aug-2000 29 3.70 141 390 4.69 134 364 12-Aug-2000 31 4.17 148 382 3.95 148 346 14-Aug-2000 32 3.92 142 319 3.86 147 364 15-Aug-2000 33 4.98 156 403 3.81 157 413 16-Aug-2000 36 4.20 144 409 3.33 126 382 17-Aug-2000 37 4.56 149 384 3.43 121 470 * >+_25%theoretical Page 96 NorthwestBioanalytical StudyNo. NWBS00-040 ReportNo. NWBR00-108 Table A.1. Inter -Assay Precision and Accuracy for PFOSA Quality Control Samples (continued) Allconcentrationsare expressedasng/mL. Run Date 24_Aug-2000 . Run Number 42 Low QC 4.00 ng/mL 4.57 3.06 Medium QC 150 ng/mL 148 136 High QC 400 ng/mL 471 425 26-Aug-2000 43 4.61 162 403 .. 4.02 161 388 28-Aug-2000 44 3.97 142 409 3.87 147 392 30-Aug-2000 46 3.85 137 381 / 3.84 145 474 Mean S.D. %CV %Theoretical %Bias n 4.03 148 394 0.458 11.3 37.8 11.4 7.6 9.6 100.8 98.7 98.5 0.8 -1.3 -1.5 30 30 30 Page 97 NorthwestBioanalytical StudyNo. NWBS00-040 ReportNo. NWBR00-108 Table A.2. Inter-Assay Precision and Accuracy for PFOSAA Quality Control Samples Allconcentrationas reexpressedas n_'mL. Run Date Run Number 25-.fui-2000 22 Low QC 9.00 ng/mL 8.50 7.33 Medium QC 155 ng/mL 147 142 High QC 405 ng/mL 359 431 31-Jul-2000 23 .. 01-Aug-2000 24 10.2 * 115 426 10.5 170 396 10.5 156 416 10.4 161 444 02-Aug-2000 26 * 11.4 177 452 9.24 171 434 04-Aug-2000 27 * 11.3 153 430 8.37 155 424 : 09-Aug-2000 29 8.59 152 393 9.46 152 399 -" 12-Aug-2000 31 10.2 149 395 10.2 168 455 14-Aug-2000 32 8.46 140 370 8.89 142 391 15-Aug-2000 33 * 10.9 160 439 9.51 161 449 16-Aug-2000 36 9.40 151 438 8.70 163 449 17-Aug-2000 37 10.5 158 409 9.74 157 421 >+..20%theoretical Page98 NorthwestBioanalytical StudyNo. NWBS00-040 ReportNo. NWBR00-108 Table A.2. Inter -Assay Precision and Accuracy for PFOSA.A Quality Control Samples (continued) Allconcentrationsare expressedas ng/mL. Run Date 24-Aug-2000 : Run Number 42 Low QC 9.00 ng/mL Medium QC 155 ng/mL 9.83 144 8.00 ' 137 High QC 405 ng/mL 461 475 . :_ 26-Aug-2000 . 43 *11.0 169 424 .._:. 10.4 167 438 28-Aug-2000 44 9.71 155 436 10.4 159 447 30-Aug-2000 46 9.30 " 9.65 152 445 165 "518 Mean S.D. %CV %Theoretical %B'ias n 9.69 155 1.01 12.5 10.4 .... 8.1 107.7 100.0 7.7 0.0 30 30 * >+..20%theoretical 429 31.7 7.4 105.9 5.9 30 Page99 -. Nor',hwesBt ioanaly_ica! StudyNo. NWBS00-040 ReportNo. NW'BR00I-08 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 io dilute samples. The total volume (VT) of a diluted sample is defined as the volume aliquoted from the original sample (Vs) plus the volume of matrix 0/M) 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 / Vs 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 ofanalyte 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*C.,,I Example Total Amount of analyte = [0.l mL sample * I00 ng/mL]+ [0.4 mL control matrix * 10 ng/rnL] Total Amount of analyte = 14 ng Page I00 NorthwesBtioanalytical StudyNo.N'WBS00-040 ReportNo.NWBR00-108 Amount ofanalytcfromsample= 10ng Amount ofanalytfcromcontromlatrix= 4 ng The concentratioonftheanalyte(CA)inthedilutedsample isequaltothesum of thefollowing: l)theratioofthevolume ofsample (Vs)tothetotalvolume ofthedilutedsample (VT) timesthe concentratioinnthesample (Cs),and 2)theratioof thevolume ofcontrolmatrixinthediluted sample (VM) tothetotalvolume ofthedilutedsamplc(VT)tirncstheconcentratioinnthecontrol matrix(CM). ('l'hiisstheconcentratioonfanalyteinthedilutedsample priortoany adjustment - -fortheacfualdilution.) :.:. The equationfortheabove is: Equation 2. CA = (Vs/V+)*Cs + (V,,dVT)* C,,,I 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 VM with Cv'r-Vs) into this equation produces: Equation 3. CA= CCs/Vr)*Cs+ (CCrVs)rV'r)*CM or CA = (VS/YT)*Cs + (1-VsfVT)*CM Example C., = [(0.i mL/0.5 mL)*100 ng/mL] + [(1-(0.l mL/0.5 mL))*10 ng/mL] CA = 20 ng/rnL+ 8 ng/mL Cx = 28 ng/mL Inserting 1/DF for the term VS/VT yields the final equation for CA: -. Page l 01 Northwest Bioanalytical Study No. NWBS00-040 Report No. NWBR00-108 Equation 4. CA = (I/DF)*Cs + (I-I/DI_*C._t Example CA = [(I15)* IO0 ng/mL]+ [(I-(IlS))* I0 ng/mL] CA = 28 ng/rnL The concentration determined by the Watson LIMS system (Cw) for any sample is: " quatio g. : :* Cw = DF * CA Example Cw=5 * 28 ng/mL ,*' Cw = 140 ng/mL (This is the concentration ofanalyte after adjusting for the actual dilution.) Substituting the final equation for CA (Equation .*. Equation 6. 4.) into the equation for Cw gives: Cw -- DF *[(1/DF)*Cs + (1-1/D10*CM] Example Cw = 5 * [((1/5) *100 n_mL) + (((1-(i/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 which is used to adjust theoretical calibration and QC concentrations. due to persistent levels Equation 7. Cs = Cw - (DF-1)*CM 'Example Cs = 140ng/mL - [(5-I*) lOng/mL] Page102 .. Northwest Bioanalytical Cs = 140 ng/mL - 40 ng/mL Cs = I00 ng/mL Study No. NWBS00-040 Report No. N'WBR00-!08 Page 103