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Northwest Bioanalytical a Study No. NWBS99-117 Report No. NWBR99-114 Quantitative Determination of PFOS, PFOSA, PFOSAA, PFHS, POAA, M556 and M570 in Human Serum by LC/MS/MS Sample Analysis Report for Protocol EPI-PEuro Northwest Bioanalytical (NWB) A Division of NWT Inc. 1121 East 3900 South Salt Lake City, UT 84124 PREPARED FOR: 3M Environmental 935 Bush Avenue Technology and Services (3M) St. Paul, M-N 55133 AUTHOR: j David Vollmer, Ph.D., NWB Project Manager APPROVED FOR L E BY: Rodge, _. 1 ultz, Ph.D oratory Director Page 1 Northwest Bioanalytical QUALITY ASSURANCE STATEMENT Study No. NWBS99-117 Report No. NWBR99-114 LABORATORY: Northwest Bioanalytical (NWB) A Division NWT, Inc. 1121 East 3900 South Salt Lake City, UT 84124 SPONSOR: 3M Environmental Technology Services (3M) 935 Bush Avenue St. Paul, MN 55133 COMPOUND(S): NWB STUDY NUMBER: PFOS, PFOSA, PFOSAA, PFHS, POAA, M556 and M570 NWBS99-117 SPONSOR STUDY NUMBER: EPI-PEuro NWB STUDY TITLE: Extraction and Analysis of PFOS and other Fluorochemicals in Human Serum by HPLC/Electrospray Mass Spectrometa:y The clinical sample analysis study described in this report is not included within the definition of a GLP regulated nonclinical study. However, Northwest Bioanalytical conducts all studies within the guidelines of the U.S. FDA Good Laboratory Practice Regulations for Nonclinical Laboratory Studies (Title 21 CFR Part 58), the OECD Principles of Good Laboratory Practice and the Japanese MHW Good Laboratory Practice Standard Ordinance for Nonclinical Laboratory Studies on the Safety of Drugs (Ordinance No. 21, PAB Notification No. 424.) The following inspections were performed by the NWB QAU per SOP. Inspection and Reporting Statement. Inspection Date 27-30 Aug 1999 03 Sep 1999 08 Sep 1999 17-22 Sep 1999 01 Dec 1999 09 Dec 1999 Phase of Study Sample Receipt Analytical Plan Draft Analytical Plan Sample Analysis Report Draft/Raw Data Final Report Date Inspection NWB Project Manager 30 Aug 1999 03 Sep 1999 08 Sep 1999 22 Sep 1999 02 Dec 1999 09 Dec 1999 Report Issued To *NWB Management 31 Aug 1999 30 Sep 1999 30 Sep 1999 30 Sep 1999 30 Dec 1999 30 Dec 1999 *Reports to NWB Management are issued monthly. As can reasonably be established, the methods and procedures described and the results incorporated into this final report accurately reflect the raw data. --"" Quality Assurance Complian S_._list Page 2 Date / NorthwestBioanalytical StudyNo. NWBS99-117 ReportNo. NWBR99-114 Compliance Statement The clinical sample analysis study described within this report is not included within the definition of a GLP regulated nonclinical study set forth in Title 21 of the U.S. Code of Federal Regulations Part 58. However, to the best of our knowledge, this project was conducted in accordance with the guidelines of the U.S. FDA Good Laboratory Practice Regulations for Nonclinical Laboratory Studies (Title 21 CFR Part 58) and according to the methods and procedures described within this report. In addition, the study followed the guidelines of the OECD Principles of Good Laboratory Practice and the Japanese MHW Good Laboratory Practice Standard Ordinance for Nonclinical Laboratory Studies on the Safety of Drugs (Ordinance No. 21, PAB Notification No. 424.) There were no known incidents that affected the quality or integrity of the project or data reported. This report represents an accurate record of the raw data. David Vollmer, Ph.D. NWB Project Manager Rod L._FoI_, Ph.D_ NWB Laboratory Director Date Date le-_/_//_ ,e Page3 i Northwest Bioanalytical StudyNo. NWBS99-117 Report No. NWBR99-114 TABLE OF CONTENTS SIGNATURE PAGE ........................................................................................................................ 1 QUALITY ASSURANCE STATEMENT ...................................................................................... 2 COMPLIANCE STATEMENT ....................................................................................................... 3 TABLE OF CONTENTS ................................................................................................................. 4 LIST OF TABLES ........................................................................................................................... 4 1. INTRODUCTION .................................................................................................................... 6 2. METHODOLOGY ................................................................................................................... 7 3. SAMPLE ANALYSIS ............................................................................................................. 9 4. RESULTS AND DISCUSSION ............................................................................................ 10 5. REFERENCES ...................................................................................................................... 10 LIST OF TABLES Table 1. Calibration Curve Summary for PFOS in Human Serum ............................................... 12 Table 2. Calibration Curve Summary for PFOSA in Human Serum ............................................ 12 Table 3. Calibration Curve Summary for PFOSAA in Human Serum ......................................... 12 Table 4. Calibration Curve Summary for PFHS in Human Serum... ............................................. 13 Table 5. Calibration Curve Summary for POAA in Human Serum .............................................. 13 Table 6. Calibration Curve Summary for M556 in Human Serum ............................................... 13 Table 7. Calibration Curve Summary for M570 in Human Serum ............................................... 14 Table 8. Back-Calculated Concentrations of Calibration Standards for PFOS in Human Serum .............................................................. _.................................. 15 Table 9. Back-Calculated Concentrations of Calibration Standards for PFOSA in Human Serum .............................................................................................. 15 Page 4 Northwest Bioanalytical Study No. NWBS99-117 Report No. NWBR99-114 Table 10. Back-Calculated Concentrations of Calibration Standards for PFOSAA in Human Serum ........................................................................................... 16 Table 11. Back-Calculated Concentrations of Calibration Standards for PFHS in Human Serum ................................................................................................. 16 Table 12. Back-Calculated Concentrations of Calibration Standards for POAA in Human Serum ................................................................................................ 17 Table 13. Back-Calculated Concentrations of Calibration Standards for M556 in Human Serum ................................................................................................. 17 Table 14. Back-Calculated Concentrations of Calibration Standards for M570 in Human Serum ................................................................................................. 18 Table 15. Analytical QC Summary for PFOS in Human Serum .................................................. 19 Table 16. Analytical QC Summary for PFOSA in Human Serum ................................................ 19 Table 17. Analytical QC Summary for PFOSAA in Human Serum ............................................. 20 Table 18. Analytical QC Summary for PFHS in Human Serum .................................................. 20 Table 19. Analytical QC Summary for POAA in Human Serum ................................................. 21 Table 20. Analytical QC Summary for M556 in Human Serum .................................................. 21 Table 21. Analytical QC Summary for M570 in Human Serum .................................................. 22 Table 22. Study Sample Concentrations ....................................................................................... 23 Page 5 Northwest Bioanalytical Study No. NWBS99-117 Report No. NWBR99-114 Quantitative Determination of PFOS, PFOSA, PFOSAA, PFHS, POAA, M556 and M570 in Human Serum by LC/MS/MS Sample Analysis for Protocol EPI-PEuro 1. INTRODUCTION This report summarizes the analytical results from the quantitation ofPFOS, PFOSA, PFOSAA, PFHS, POAA, M556 and M570 in human serum samples for 3M Environmental Technology and Services in support of Protocol EPI-PEuro [5.1 ]. The validated LC/MS/MS method provided a lower limit of quantitation (LLOQ) of 31.4 (PFOS), 1.00 (PFOSA), 1.45 (PFOSAA), 2.61 (PFHS) and 5.27 (POAA) ppb. All analytes exhibited a linear response to approximately 500 ppb with the exception of PFOSAA, which had an upper limit of quantitation Cu-LOQ) of 270 ppb [5.2]. The nonvalidated method for the quantitation for M556 and M570 in human serum provided a lower limit of quantitation (LLOQ) of 1.00 and 5.00 ppb, respectively, and exhibited a linear response to 500 ppb for both analytes. The testing facility was 3M Corporate Occupational Medicine (Bldg. 220-3W-05, St. Paul, MN 55144-1000), and Jean Burris at the facility served as the Principal Investigator. The following is a list of NW-B supervisory personnel involved in the completion of this work: David Vollmer, Ph.D. (NWB Project Manager); Rodger L. Foltz, Ph.D. (NWB Laboratory Director). NWB SOPs were used in the conduct of this study and were available to study personnel in electronic and/or hard copy formats. Date Study Initiated: September 7, 1999 Date Analyses Completed: September 20, 1999 _J The clinical study described in this report is not included within the'definition of a GLP J regulated nonclinical study. However, Northwest Bioanalytical conducts all studies within the guidelines of the U.S. FDA Good Laboratory Practice Regulations for Page 6 Northwest Bioanalytical Study No. NWBS99-117 Report No. NWBR99-114 Nonclinical Laboratory Studies (Title 21 CFR Part 58), the OECD Principles of Good Laboratory Practice and the Japanese MHW Good Laboratory Practice Standard Ordinance for Nonclinical Laboratory Studies on the Safety of Drugs (Ordinance No. 21, PAB Notification No. 424). Any changes to or deviations from the original protocol (Analytical Plan) were documented through approved memos and are retained within the raw data. protocol amendments or deviation 2. METHODOLOGY The validated assay used for this study is Meth98082.00 Bioanalytical report NWBR99-005 [5.2]. and was reported in Northwest Samples for this study were received at NWB on the following date: Receipt Date August 27, 1999 Referenee Material Number of Samples Received 18 Storage Condition (except during analysis) - 20C Analyte Lot Number Purity Expiration Date Source Storage Conditions *PFOS (FC-95) 193 PFOSA *PFOSAA (FC-129) PFHS 214 617 $398-182 100% 100% 53.8% 100% 12/31/2010 12/31/2010 12/31/2010 12/31/2010 3M Room Temperature (dry) 3M Room Temperature 3M Room Temperature 3M - 20C *POAA (FC 143) M556 245 100% NB 113047-80 99.89% 12/31/2010 12/31/2010 3M Room Temperature (dry) 3M Room Temperature M570 118506-26 99.75% 12/31/2010 3M Room Temperature SDS (sodium 17H0459 99% dodecylsulfate) 1/16/2001 Sigma Room Temperature * Analyte names used throughout this report do not include their FC identifier; for example, PFOS is synonymous with PFOS (FC-95) Page 7 Northwest Bioanalytical Study No. NWBS99-117 Report No. NWBR99-114 Nine calibration standards were prepared fresh daily by adding the appropriate spiking standard solution into 0.200 mL of human serum. The final calibration standard concentrations in human serum for each analyte were as follows: Analyte PFOS PFOSA PFOSAA PFHS POAA M556 M570 Calibrd tion Standard Concentrations (ppb) 31.4, 35. 40.4, 55.4, 80.4, 130, 280, 430, 530 1.00, 5.(), 10.0, 25.0, 50.0, 100, 250, 400, 500 1.45, 3._ 3, 6.29, 14.4, 27.8, 54.7, 135,216, 270 2.6_)11.6, k. 26.6, 51.6, 102, 252, 402, 502 5.27"9727, 14.3, 29.3, 54.3, 104, 254, 404, 504 1.00, 5.00, 10.0, 25.0, 50.0, 100, 250, 400, 500 1.00, 5.00, 10.0, 25.0, 50.0, 100, 250, 400, 500 Analytical QCs were prepared in human serum on September 13, 1999 and frozen in a -20C freezer. Analytical QCs at the following levels were assayed in duplicate in each analytical run: Analyte " PFOS PFOSA PFOSA.A PFHS POAA M556 M570 QC Concentrations (ppb) Low (50.4) and High (380) Low (20.0) and High (350) Low (11.7) and High (189) Low (21.6) and High (352) Low (24.3) and High (354) Low (20.0) and High (350) Low (20.0) and High (350) After the addition of HPLC-grade water and sodium dodecyl sulfate (SDS, internal standard) to 0.200 mL of human serum, the serum mixture is made basic with the addition of 0.5 M tetrabutylammonium hydrogen sulfate (TBA, pH 10) and 0.25 M carbonate buffer. This mixture is then extracted with methyl-tert-butyl ether. After sufficient mixing, the sample is centrifuged. The organic layer is transferred via pipette into a clean test tube. The organic layer is then evaporated to dryness and the sample is Page 8 Northwest Bioanalytical Study No. NWBS99-117 Report No. NWBR99-114 reconstituted into 2 mM ammonium acetate water:methanol (50:50 v/v). The extracts are then analyzed by liquid chromatography/tandem mass spectrometry using negative-ion electrospray ionization and multiple reaction monitoring. 3. SAMPLE ANALYSIS The prepared samples, calibration standards and QCs were injected into the PE Sciex API 3000 LC/MS/MS system in a systematic order. Chromatographic peak integration=was performed using MacQuan software (version 1.6). Quadratic regression analysis (weighted l/x) all analytes was performed using the peak area ratio vs. concentration utilizing Watson DMLIMS software (version 5.4.10.2). 3.1. Acceptance Criteria For an analytical run to be accepted, it must have met the acceptance criteria listed below which are consistent with regulatory and industry recommendations. Calibration Curve Each run will include duplicate calibration standards at 7 or more concentrations covering the lower to upper limit of quantitation. At least two-thirds of the calibration standard's back-calculated concentrations must be within + 15% (+ 20% for LLOQ) of their individual target concentrations. If a calibration standard is not within the acceptance criteria, it is deactivated. This process starts from the highest calibration standard down to the lowest until all the active standards are within the acceptance criteria. Lower Limit of Quantitation The back-calculated concentrations of at least one of the duplicate lowest points in the calibration curve must be within + 20% of the target concentration to qualify as the LLOQ. If this criterion is not met, the next level is subjected to the same test and the LLOQ raised accordingly. Page 9 NorthwestBioanalytical Quality Control Samples StudyNo. NWBS99-117 ReportNo. NWBR99-114 Each analytical run will include low and high QC samples in triplicate. The measured concentrations of at least two-thirds of all analytical QCs must be within _+20% of their target concentrations. If study samples require dilution, a dilution QC is analyzed in duplicate for each dilution level. At least one dilution QC at each level must be within _+20% in order to accept study sample results at that dilution level. The dilution QC acceptance was independent of the undiluted analytical QC acceptance. 4. RESULTS AND DISCUSSION Each accepted run met the acceptance criteria set for the calibration curve points, for the lower limit of quantitation (LLOQ) and for the analytical quality control (QC) samples. Run PFOS PFOSA PFOSAA PFHS ID Result Result Result Result POAA Result M556 Result M570 Result Comments 1 accepted accepted accepted accepted accepted accepted accepted There were no known circumstances that may have affected the quality or integrity of the data. In conclusion, based on the quality control and calibration curve data, it is our opinion that the data submitted to 3M Environmental Technology and Services for PFOS, PFOSA, PFOSAA, PFHS, POAA, M556 and M570 are accurate for all of the EPI-PEuro study samples. Please note that the results for analytes M556 and M570 should be considered unvalidated data. 5. REFERENCES 5.1. 3M Protocol EPI-PEuro, "Analysis of Fluorochemicals in Poo!ed Sera from Europe," August 24, 1999. Page 10 NorthwestBioanalytical StudyNo. NWBS99-117 ReportNo. NWBPO9-114 5.2. D. Vollmer, "Quantitative Determination ofPFOS, PFOSA, PFOSAA, N-MeFOSEOH, N-EtFOSE-OH, POAA and PFHS in Human Serum by LC/MS/MS," NWB Study Number NW-BS98-082, NWB Report Number NW'BR99-005, May 13, 1999. The raw data and final report for this study will be stored in the NWB Archives, 1121 East 3900 South, Salt Lake City, UT 84124 per regulations and contract agreement. After submission of the final report to the Sponsor, remaining study samples will be stored under required conditions until confirmation of Sample Disposition/Return Authorization is received from the Sponsor. 3M Environmental Technology and Services and 3M Corporate Occupational Medicine will be notified concerning final disposition of records at completion of contract obligations. Page 11 Northwest Bioanalytical Study No. NWBS99-117 Report No. NWBR99-114 Table 1. Calibration Curve Summary for PFOS in Human Serum Quadratic weighted 1/x. All concentrations are expressed as ppb. Run Date 17-Sep-1999 Run Number 1 A B C R-Squared LLOQ ULOQ -0.000003 0.007758 0.060028 0.9961 31.4 530 Mean n -0.000003 1 0.007758 1 0.060028 1 0.9961 1 *A, B and C are coefficients used to define the quadratic curve. Table 2. Calibration Curve Summary for PFOSA in Human Serum Quadratic weighted 1/x. All concentrations are expressed as ppb. Run Date 17-Sep-1999 Run Number 1 A B C R-Squared LLOQ ULOQ -0.000007 0.016542 0.006140 0.9960 1.00 500 Mean n -0.000007 1 0.016542 1 0.006140 1 0.9960 1 *A, B .andC are coefficients used to define the quadratic curve. Table 3. Calibration Curve Summary for PFOSAA in Human Serum Quadratic weighted 1/x. All concentrations are expressed as ppb. Run Date 17-Sep-1999 Run Number 1 A B C R-Squared LLOQ ULOQ -0.000002 0.001907 0.002064 0.9971 1.45 270 Mean n -0.000002 1 0.001907 1 0.002064 1 0.9971 1_ *A, B and C are coefficients used to define the quadratic curve. Page 12 Northwest Bioanalytical Study No. NWBS99-117 Report No. NWBR99-114 Table 4. Calibration Curve Summary for PFHS in Human Serum Quadratic weighted 1/x. All concentrations are expressed as ppb. Run Date 17-Sep-1999 Run Number 1 A B C R-Squared LLOQ ULOQ -0.000006 0.012069 0.031955 0.9980 6.61 502 Mean n -0.000006 1 0.012069 1 0.031955 1 0.9980 1 * A, B and C are coefficients used to define the quadratic curve. Table 5. Calibration Curve Summary for POAA in Human Serum Quadratic weighted 1/x. All concentrations are expressed as ppb. Run Date 17-Sep-1999 Run Number 1 A B C R-Squared LLOQ ULOQ -0.000006 0.014672 0.018212 0.9977 5.27 504 Mean n -0.000006 1 0.014672 1 0.018212 1 0.9977 1 * A, B and C are coefficients used to define the quadratic curve. Table 6. Calibration Curve Summary for M556 in Human Serum Quadratic weighted 1/x. All concentrations are expressed as ppb. Run Date Run Number A B C R-Squared LLOQ ULOQ 17-Sep-1999 1 0.000000 0.000556 0.001320 0.9977 1.00 500 Mean n 0.000000 1 0.000556 1 0.001320 1 0.9977 1, * A, B and C are coefficients used to define the quadratic curve. Page 13 Northwest Bioanalytical Study No. NWBS99-117 Report No. NWBR99-114 Table 7. Calibration Curve Summary for M570 in Human Serum Quadratic weighted 1/x. All concentrations are expressed as ppb. Run Date 17-Sep-1999 Run Number 1 A B C R-Squared LLOQ ULOQ -0.000001 0.002742 0.015241 0.9984 5.00 500 Mean n -0.000001 1 0.002742 1 0.015241 1 0.9984 1 * A, B and C are coefficients used to define the quadratic curve. Page 14 Northwest Bioanalytical Study No. NWBS99-117 Report No. NWBPO9-114 Table 8. Back-Calculated Concentrations of Calibration Human Serum Standards Quadratic weighted 1/x. All concentrations are expressed as ppb. for PFOS in Run Date Run Number 31.4 35.4 40.4 55.4 80.4 130 280 430 530 17-Sep-1999 1 29.7 34.4 41.4 57.2 88.2 126 266 444 490 27.5 35.9 42.2 56.2 84.3 130 278 417 581 Mean S.D.] %CV %Bias nl 28.6 35.2 41.8 56.7 86.3 128 272 431 536 1.56 1.06 0.566 0.707 2.76 2.83 8.49 19.1 64.3 5.5 3.0 1.4 1.2 3.2 2.2 3.1 4.4 12.0 -8.9 -0.6 3.5 2.3 7.3 -1.5 -2.9 0.2 1.1 2 2 2 2 2 2 2 2 2 Table 9. Back-Calculated Concentrations of Calibration Human Serum Standards for PFOSA in Quadraticweighted 1/x. All concentrationsare expressed as ppb. Run Date Run Number 1.00 5.00 10.0 25.0 50.0 100 250 400 500 17-Sep-1999 1 0.958 *7.04 10.3 *28.8 55.5 99.2 241 438 451 0.852 "6.51 11.3 *30.0 51.2 88.5 259 377 542 Mean 0.905 10.8 53.4 93.9 250 408 497 S.D. 0.0750 0.707 3.04 7.57 12.7 43.1 64.3 %CV 8.3 6.5 5.7 8.1 5.1 10.6 12.9 %Bias _ -9.5 8.0 6.8 -6.1 0.0 2.0 -0.6 n 2 2 2 2_ 2 2 2 p- * Sample deactivated as an outlier (> +15% theoretical for non-LLOQ; > +20% theoretical for LLOQ ) Page 15 Northwest Bioanalytical Study No. NWB$99-117 i Report No. NWBR99-114 Table 10. Back-Calculated Concentrations Human of Calibration Serum Standards for PFOSAA in Quadratic weighted 1/x. All concentrations are expressed as ppb. Run Date 17-Sep-1999 Run Number 1 1.45 3.60 1.16 3.60 *0.720 3.53 6.29 14.4 27.8 54.7 135 216 270 6.37 14.7 30.9 53.5 125 217 251 6.41 14.9 30.6 54.9 133 214 300 Mean S.D. %CV %Bias n 1.16 N.C. N.C. -20.0 1 3.57 0.0495 1.4 -0.8 2 6.39 0.0283 0.4 1.6 2 14.8 0.141 1.0 2.8 2 30.8 0.212 0.7 10.8 2 54.2 129 216 276 0.990 5.66 2.12 34.6 1.8 4.4 1.0 12.5 -0.9 -4.4 0.0 2.2 2 2 2 2 * Sample deactivated as an outlier (> _+15%theoretical for non-LLOQ; > +_20%theoretical for LLOQ ) N.C. = not calculable Table 11. Back-Calculated Concentrations of Calibration Standards for PFHS in Human Serum Quadratic weighted 1/x. All concentrations are expressed as ppb. Run Date Run Number 2.61 6.61 11.6 26.6 51.6 102 252 402 502 17-Sep-1999 1 *0.735 5.57 10.9 27.3 58.3 101 232 404 491 "0.571 6.57 11.7 27.7 56.4 102 243 412 523 Mean S.D. %CV %Bias 6.07 11.3 27.5 57.4 102 238 408 507 0.707 0.566 0.283 1.34 0.707 7.78 5.66 22.6 11.6 5.0 1.0 2.3 0.7 3.3 1.4 4.5 -8.2 -2.6 3.4 11.2 0.0 -5.6 1.5 1.0 k. n 2 2 2 2 *2 2 2 2 * Sample deactivated as an outlier (> +15% theoretical for non-LLOQ; > +20% theoretical for LLOQ ) Page 16 Northwest Bioanalytical Study No. NWBS99-117 Report No. NWBR99-114 Table 12. Back-Calculated Concentrations of Calibration Human Serum Standards for POAA in Quadratic weighted 1/x. All concentrations are expressed as ppb. Run Date Run Number 5.27 9.27 14.3 29.3 54.3 104 254 404 504 17-Sep-1999 1 4.41 9.29 14.7 30.1 60.2 99.9 235 403 480 4.35 9.85 15.0 31.4 59.3 103 246 421 533 Mean S.D. %CV %Bias n 4.38 9.57 14.9 30.8 59.8 101 241 412 507 0.0424 0.396 0.212 0.919 0.636 2.19 7.78 12.7 37.5 1.0 4.1 1.4 3.0 1.1 2.2 3.2 3.1 7.4 -16.9 3.2 4.2 5.1 10.1 -2.9 -5.1 2.0 0.6 2 2 2 2 2 2 2 2 2 Table 13. Back-Calculated Concentrations of Calibration Standards for M556 in Human Serum Quadratic weighted 1/x. All concentrations are expressed as ppb. Run Date Run Number 1.00 5.00 10.0 25.0 50.0 100 250 400 500 17-Sep-1999 1 1.03 4.49 "7.17 21.3 50.5 103 239 394 467 *<LLOQ *2.56 10.1 27.7 54.9 101 248 401 544 Mean S.D. %CV %Bias n 1.03 N.C. N.C. 3.0 1 4.49 N.C. N.C. -10.2 1 10.1 24.5 52.7 102 244 398 506 N.C. 4.53 3.11 1.41 6.36 4.95 54.4 N.C. 18.5 5.9 1.4 2.6 1.2 10.8 1.0 -2.0 5.4 2.0 -2.4 -0.5 1.2 1 2 2 2 2 2 2 * Sample deactivated as an outlier (> +15% theoretical for non-LLOQ; > +20% tfieoretical for LLOQ ) N.C. = not calculable Page 17 NorthwestBioanalytical StudyNo.NWBS99-117 ReportNo. NWBR99-114 Table 14. Back-Calculated Concentrations of Calibration Standards for M570 in Human Serum Quadratiwc eighted1/x. Allconcentrationasreexpressedasppb. Run Date Run Number 1.00 5.00 10.0 25.0 50.0 100 250 400 500 17-Sep-1999 1 *<LLOQ 4.82 10.4 27.9 "61.7 102 240 425 485 *<LLOQ 4.17 9.69 26.3 54.1 96.9 242 386 517 Mean S.D. %CV %Bias n 4.50 10.0 27.1 54.1 0.460 0.502 1.13 N.C. 10.2 5.0 4.2 N.C. -10.0 0.0 8.4 8.2 2 2 2 1 99.5 241 406 501 3.61 1.41 27.6 22.6 3.6 0.6 6.8 4.5 -0.5 -3.6 1.5 0.2 2 2 2 2 * Sampledeactivatedasan outlier > +15%theoreticalfor non-LLOQ>; +20%theoreticalfor LLOQ) N.C. = notcalculable Page 18 Northwest Bioanalytical Study No. NWBS99-117 Report No. NWBR99-114 Table 15. Analytical QC Summary for PFOS in Human Serum All concentrations are expressed as ppb. Run Date Run Number 17-Sep-1999 1 Low QC 50.4 ppb 49.9 49.4 53.5 High QC 380 ppb 393 322 369 Mean S.D. %CV %Theoretical %Bias n 50.9 361 2.24 36.1 4.4 10.0 101.0 95.0 1.0 -5.0 3 3 Table 16. Analytical QC Summary for PFOSA in Human Serum All concentrations are expressed as ppb. Run Date Run Number 17-Sep- 1999 1 Low QC 20.0 ppb 18.0 20.0 18.5 High QC 350 ppb 302 *264 280 Mean S.D. %CV %Theoretical %Bias n 18.8 1.04 5.5 94.0 -6.0 3 * > +20%theoretical 282 19.1 6.8 80.6 - 19.4 3 3 Page 19 Northwest Bioanalytical Study No. NWBS99-117 Report No. NW'BR99-114 Table 17. Analytical QC Summary for PFOSAA in Human Serum All concentrations are expressed as ppb. Run Date Run Number 17-Sep- 1999 1 Low QC 11.7 ppb 11.0 10.1 11.0 High QC 189 ppb 186 156 184 Mean S.D. %CV %Theoretical %Bias n 10.7 175 0.520 16.8 4.9 9.6 91.5 92.6 -8.5 -7.4 3 3 Table 18. Analytical QC Summary for PFHS in Human Serum All concentrations are expressed as ppb. Run Date Run Number 17-Sep-1999 1 Low QC 21.6 ppb 21.7 20.9 22.6 High QC 352 ppb 374 313 356 Mean S.D. %CV %Theoretical %Bias n 21.7 348 0.850 31.3 3.9 9.0 100.5 98.9, 0.5 - 1.1 3 3 Page 20 Northwest Bioanalytical Study No. NWBS99-117 Report No. NWBR99-114 Table 19. Analytical QC Summary for POAA in Human Serum All concentrationsareexpressedas ppb. Run Date 17-Sep-1999 Run Number I 1 Low QC 24.3 ppb 23.5 22.9 25.6 High QC 354 ppb 362 311 357 Mean S.D. %CV %Theoretical %Bias n 24.0 343 1.42 28.1 5.9 8.2 98.8 96.9 - 1.2 -3.1 3 3 Table 20. Analytical QC Summary for M556 in Human Serum All concentrationsare expressedas ppb. Run Date Run Number 17-Sep-1999 1 Low QC 20.0 ppb 16.7 "15.6 18.5 High QC 350 ppb 290 281 352 Mean S.D. %CV %Theoretical %Bias n 16.9 1.46 8.6 84.5 -15.5 3 * > +20%theoretical 308 38.7 12.6 88.0 -1213 3 - Page 21 NorthwestBioanalytical StudyNo. NWBS99-117 ReportNo. NWBR99-114 Table 21. Analytical QC Summary for M570 in Human Serum All concentrationasreexpressedasppb. Run Date Run Number 17-Sep-1999 1 Low QC 20.0 ppb 19.7 18.2 19.2 High QC 350 ppb 340 280 342 Mean S.D. %CV %Theoretical %Bias n 19.0 321 0.764 35.2 4.0 11.0 95.0 91.7 -5.0 -8.3 3 3 Page 22 r-- _ == dd ZZ _ O' 0 '_ 0 '_ O' 0,' 0" O' 0 ,'_ 0 'I 0" 0 'I O' O' O' O' O' * _- S o o o o o o o o 9 o o o o o o o V VVV VVV VVVVV VV VVV 99 9_o o999 99oo oo oo V VV VVVV VVVVV VV VV