Document 6BEjpjJMO6LQLxZM3BXbD4G3m

NorthwesiBioamlytcs R226 - 1209 ReSptourdtyNNoo..NNWWBBRS000..014080 Quantitative Determination of PFOS, PFOSA, PFOSAA, POAA, PFHS, M556 and M570 in Human Serum by LC/MS/MS Assay Revalidation Report ) Northwest Bioanalytical (NWB) A Division of NWT Inc. Sal1t12L1akEeasCtit3y9,00UTSo8u4t1h24 = Sgz o2u0 33 PREPARED FOR: 3M Environmental Technology and Services (3M) 935 Bush Avenue St. PaMu N5l 51, 33 AUTHOR: lone DLbeetX. Connie O. Sakashita, B.S., NWB Project Manager + i`. DATE: 2d/os APPROVEDFOR RELEASE BY: LETS] fon F-- Patrick Bennett, MS., M.B.A., NWB Laboratory Director DATE: _ 4/26, CONTAIN NO CRI 000101 Paget NorthwestBioanalytical QUALITYASSURANCE STATEMENT Rene WAL Study No.NWBS00-040 . LABORATORY: NorthwestBioanalytical (NWB) A Division Of NWT, Inc. 1121 East 3900 South Salt Lake City, UT 84124 SPONSOR: 3M 935 Environmental Bush Avenue Technology andServices (3M) St. Paul, MN 55133 COMPOUNDS): NWB STUDYNUMBER: PFOS, PFOSA, PFOSAA, POAA, PFHS, M556 and M570 NWBS00-040 SPONSOR STUDY NUMBER: NA, Assay Validation - NWB STUDY TITLE: QuantitativeDetermination of PFOS, PFOSA, PFOSAA, POAA, PFHS, M556 and M570 LeMsms, Assay Validation in Human Serumby The assay validation study described regulated nonclinicalstudy.Howev in this report is not included within the definition ofa GLP guidelinesof the Studies (Title 21 U. S. FDA Good Learb,oNroartotrhywPersatctBiicoeaRneaglyutliactailoncsonfdourNcotnsclalilnstudies within the = Japanese CFR. MHWGood Part 58), the OECD Laboratory Practice Principles Standa of Good Laboratory icalLaboratory Practice andthe SintsupdeicetsioonnstwheerSeafpeetryfoorfmDerdugbsy (tOherdNinWaBnceQNAoU. per SOP. rd Ordinance for NonclinicalLaboratory 2 1, PAB Notification No. 424) Thefollowing Inspection and Reporting Statement, T Dat nspesion Rens sued To ect ERRE Mads 5h Si 51 15 Sern, Foot Ropar Bis 07 - 18 -18 SMeapy22000000 of Study ARespsoarytVDalriadfatRiaonw Data NWBProjectManager 18 20 MSeapy22000000 NWBManagement 31 29 SMeapy22000000 oa Seri "Reports to NWB `Management are issuedmonthly. o As caa n reasoniantbolythbies feisntaalblriesphoerdt,atchceurmaettehloydrsefalencdt ptrhoecreadwurdeastsd,escribed and the ` results Y,. A2) (. Wl AD, 0 ShaundelPercey, BS, P Quality Assurance Compfiage)Specialist - Date rae 000102 SR FT, COMPLIANCE STATEMENT "The methodvalidation study described in this `GLP nonclinical regulated study. However report is not included within the definition ofa RceognudluacttieodnsinfaorccNoorndcalnicniecwailtLhatbhoeragtuoirdyelSitnue,dsitoeoftsth(hTeeitbUle.esSt2. 1oFfDouArkGnooowdleLdagbeo,rtahtiosrystPuradcytwicaes methods and procedures described within this report. In CFR Part 58) and addition, the study according fol to the guidelinesofthe OECD Principles Laboratory Practice Stan of Good Laboratory Practice and the lowed the Japanese MHW Good Drugs (Ordinance No. affected the quality or dard Ordinance forNonclinical Laboratory Studies on the 21, PAB Notification No. 424) Any known circumstances Safety that of integrity ofthe study or data are discussed. within the may have represents an accurate recordofthe raw data. report. This report Connie O. Sakashita, B.S, NWB Project`Manager rE a Patrick Bennett, M.S., MBA. NWBLaboratoryDirector DATE: fate, 000103 Northwest Bioanalytical ReSptourdtyNNoo.. NNWWBBRS0000-.100480 TABLE OF CONTENTS SIGNATURE PAGE bss nl QUALITY ASSURANCE STATEMENT coco 2 COMPLIANCE STATEMENT... TABLE OF CONTENTS c..csssssssmssmmsmssssssmmemsssssssn son dh LI OFTSABLTES sss IST OF FIGURES...] 1 INTRODUCTION... onsen 8 2 VALDATION SUMMARY. 10 I2.1. a Persistent Levels ofARIES in MAIFI...vcnrsessrscssromomsseser on 10 23. PrEadCAIGUSA Ic ONoronas 12 24 EXURCHON EMCIENCY corres 1S 25. SbIity EVBIUGUON crm sms 17 3: DATAMANAGEMENT cc. msssssssssmmssmssnsnsnnss 17 4: COMANM DCOE NCLN USIOT NS..S . 17 41. Proposed Sample Analysis Acceptance Criteria......uronrmromsnree 18 6. DATARETENTION coors19 7 ANALYTICAL METHOD w.cocovrssssissssensmomssssonso ots 67 = 7:1. Reference Materialsand Mes... 72. Chemicals and EQUIMENt cco oo 69 70 73. Reagents, Calibration Standard and Quality Control (QC) SOlutoRS v.72 74. PreparationofValidation Quality Control SAMPLES... 77 7:3. 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. PrepofaCalirbraatiotnStaindaordsn... 80 Pages 000104 Northwest Bioanalytical Study No. NWBS00.040 Report No. NWBR00-108 i1 7:9: LOMMSIMS CONGIIONS rors 83 710. QUENON cms APPENDIX Avs sesso 84 96 APPENDIX Bsns sss. +100 LIST OF TABLES Table 1. SumomfCaalibrratiyon Curve Parameters for PFOS ..cc.oasmres....20 Table 2. SumomfCaalibrratyion Curve Parametersfor PFOSA...c...ovosermr....20 Table 3. SumomfCaalibrratiyon Curve Parameters for PFOSAA vv... 21 Table 4. SumomfCaalibrratyion Curve Parameters for POAA crores... 21 Table 5. Summary of Calibration Curve Parameters for PEHS wc...22 Table 6. Sum ofCm alibra atior n.Cuy rve Parameters for MSS6 nr... 22 Table 7. SumomfCaalibrratyion Curve Parametersfor MS70..ce.rorororosorer..23 Table 8. Back-Calculated Concentrations of Calibration Standards for POS ...................24 Table 9. Back-Calculated ConcentrationsofCalibration Standards for PFOSA................... 24 Table 10. Back-Calculated ConcentrationsofCalibration Standards for PFOSAA...............25 Table 11. Back-Caleulated ConcentrationsofCalibration Standards for POAA...............25 Table 12. Back-Calculated ConcentrationsofCalibration Standards for PFHS smn Table 13. Back-Calculated Concentrationsof Calibration Standardsfor M56. grrr 2. Table 14. Back-Calculated Concentrationsof Calibration Standards for M50... 27 Table 15. Intra-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 Accuracyfor POAA Quality Control Sarmples.........34 Table 19. Intra-Assay Precision and Accuracy for PFHS Quality Control Samples ............ 36 Pages 000105 Northwest Bioanalytical Study No. NWBS00.040 Report No. NWBR00-108 `Table 20. Table 21. Table 22. Intra-Assay Precision and Accuracy for M556 Quality Control Samples ..............38 Intra-Assay Precision and Accuracy for M570 Quality Control Samples..................40 Inter-Assay Precision for PFOS Quality Control Samples .................. reser 82 Table 23. Inter-Assay Precision for PFOSA Quality Control Samples ............... ress 83 Table 24. Intr-Assay Precision for PFOSAA Quality Control Samples... th Table 25. Inter-Assay Precision for POAA Quality Control Samples............... omens 8S Table 26. Inter-Assay Precision for PFHS Quality Control SAMPLES rrr 46. Table 27. Inter-Assay Precision for MSS6 Quality Control Samples ron...47 `Table 28. Inter-Assay Precision for M570 Quality Control SAMPLES covers 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..................... EE T-- 1 | Table 32. PFOSA DilutionQuality Control Samples................... Table 3. PFOSAA Dilution Quality CORTON SADIE... ssn 32 53, Table 34. POAA DilutionQualityControl Samples...................... ss ---------- 34 Table 35. PFHS Dilution Quality Control SAmples.........v....... rns SS `Table 36. M56 Dilution Quality Control Samples........................ wrens ----Sl Table 37. M70 Dilution Quality Control Samples... Co -- Table 38. PFOSA. Specific Dilution Quality Controls.......ovveverreee. EE Tt 1. Table 39. PFOSAA Specific Dilution Quality Controls.......ov...... wens Table 40. PFOS Extraction EFCIENCY corre oessssrnessssssesss ses Ta ------ Table 41. PFOSA Extraction EFfICIONCY vvroerorrsrer sess wens G1 Table 42. PFOSAA Extraction EFCIENCY covers ssesssensnn ET -------1 Table 43. POAA EXIraction EFCIency oom... smsmssiSE Pages 000106 NorthwestBioanalytical ReSptourdty NNoo.. NNWWBBRS0000--100480 Table 44. PFHS Extraction Efficiency........cuummmmnnne ------------------------------ Gd. Table 45. M556 Extraction EfCIENCY.cvenerrrsssesersssssnsnsne 65 Table 46. M570 Extraction EFCIENCY.covvorrrrssreesesersssessn ES '- LIST OF FIGURES Figure 1. Representative Calibration Curve for PFOS ......ccervrrre. rss 83 Figure 2. Representative Calibration Curve or PEOSA.vevvccvr gS Figure 3. Representative Calibration Curve or PFOSAA cra... 86 Figure 4. Representative Calibration Curve for POAA ver... SR." Figure 5. Representative Calibration Curvefor PFHS ....ccoccercrrrronnse rrr 87 Figure 6. Representative Calibration CUIVE FOF M556 ....ocorerveesrrn ress 87 Figure 7. Representative Calibration Curve for M570 .....ocovvvvrererrrnnn EE -- Figure 8. Standard (57.1 ng/mL) Chromatogram for PEOS .................. emmy Figur9e. Standard (10.1 ng/mL)Chromatogram for PFOSA..... 7...SSR Figure 10. Standard (15.0 ng/mL) Chromatogram for PFOSAA................. rss 91 Figure 11. Standard (14.8 ng/mL) Chromatogram for POAA......... sien 92 Figure 12. Standard (12.2 ng/mL) Chromatogram for PFHS re --------------93 Figure 13. Standard (11.8 ng/mL) Chromatogram for M556 .................. ssn 9 Figure 14. Standard(14.6 ng/mL) Chromatogram forLLC2IRR--GEG--G--------LY + did Ap pdires # = Taser ise Jp Lo PFOSK + PROSEA Lal dwe Study 00-002 QC Sep Soler tug Lond Lo Calriishion of pecistnd Fonds an diluted bia cern (atosgites) 000107 Page? Northwest Bioanalytical ReSptourdtyNNoo.. NNWWBBRS0000-.100480 Quantitative Determination of PFOS, PFOSA, PFOSAA, POAA, PFHS, M556 and M570 in Human Serum by LC/MS/MS Assay Revalidation Report I. INTRODUCTION Northwest Bioanalytical (NW) was contracted by 3M Environmental Technology and Services to develop and validatae liquid chromatography/tandem mass spectrometry method for the measurementof perfluorooctanesulfonate (PFOS), perfluorooctanesulfonamide (PFOSA), N-ethyl perfluorooctanesulfonamidoacetate (PFOSAA), perfluorooctanoate (POAA), perfluorohexanesulfonate (PFES), perfluorooctanesulfonamidoacetate (M556) and N-methyl perfluorooctanesulfonemidoacetate (M570) in human serum. The reference material purityfor PFOS, PFOSA, PFHS and POAA was not available tprhieosre taonatlhyetecsonadruecbtaosfetdhiusposntuadny.asTsheurmeefdorpeu,riatly coofnc1e0n0t%r.ations includedinthe report for After the validation was completed, 3M contracted with Centre Analytical Laboratories, Inc. in State College, Pennsylvania to determine the absolute concentration of PEOS, 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 inthis report should be corrected according to he following table: Ce = -- "AnaFlyFte ~~CoComrercecitoinonFFaaccttoorr : PFOS 0336 POAA 0.909 _-- PFHS 0s.8s55 000108 Pages Northwest Bioanalytical ReSptourdtyNNoo,. NNWWBBRS000D-.100480 This report summarizes the accuracy, precision and the Northern Chinese plasma extraction efficiency results rom the validationof the method fo the quantiation of PFOS, PFOSA, PFOSAA, POAA, PEHS, M556 aad M70 in human serum for 30 Environmental Technology and Services. Stability results wil be reported in separate addendum report. Kis Hansen ai 31 Environmental Technology and Services served as the Study Monitor. The following is a list of NWB supervisory personnel involved in the Ccooomppelrestmiiotnoh,ftPhhi.sD.wo(rNk:WBConSenniieorO.ScSiaeknatsihsti)t;a,LiB.cSo.ng(JNiaWnBg,PPrho.jDe.ct(MNanWaBgeSre)n;ioBrrad I PShc.iDe.nti(sN);WPBatTreicchknBiecnanletDti,reMc.toSr.),.M.B.A. (NW Laboratory Director); Rodger Foltz, NWB SOPs were used in the condouftchits 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 0a GLP regulated nonclinical study. However, Northwest Bioanalytical conducts all studies within the guidelines of the U.S. FDA Good Laboratory Practice Regulations for NLoanbcolriantiocraylPLraabctoircaetoarnydStthuedJieaspa(nTeisleeM21HCWFRGoPoardt L5a8b),ortahteoOryEPCraDctPirciencSitpalnedsaorfd Good OPrAdBinNaontciefifcoartiNoonncNloi.ni4c2a4)l.LaAbonryatcohrayngSetusdtioesorodnetvhieatSioans fforfeomDrttuhgeysor(iOgirndailnparnocteocNool. 21, (Analytical Plan) were documented through approved protocol amendments or deviation memos and are retained within the aw data. : es Principles of the Method : The newly developed method is a modificationofa previous method developed under study NWBS98.082 and reported in NWB report NWBR99-005 (5.1). The new method was developed to provide improved accuracy, precision and ruggedness using less sample volume, and to add MSS6 and M570 to the method. 030109 Pages Northwest Bioanalytical ReSptourdty NNoo.. NNWWBBRS0000-.100480 The analytical method consistedof a liquid:liquid extraction procedure followed by evaporation and reconstitution of the extract residue with 30:70 20 mM ammonium acetate in water: 20 mM ammonium acetate in methanol (v/v). The samples were analyzed by liquid chromatography/tandem mass spectrometry using a PE Sciex API 3000. The instrument was operated in the multiple reaction monitoring (MRM) mode under optimized conditions for PFOS, PFOSA, PFOSAA, POAA, PFHS, M56 and MS70 detection. 2. VALIDATION SUMMARY Three separate analytical runs were used in the determinationoflinearity, precision, and accuracy. An additional run was used to determine intra-assay precision and accuracy for PFOSA and PFOSAA. The extraction efficiencies for all analytes and the internal standard 1H, 1H,2H,2H-perfluorooctane sulfonic acid (THPFOS) were also determined. 21. Persistent Levels of Analytes in Matrix Because PFOS, PFOSAA, POAA, PFHS, M556 and M570 demonstrate measurable levels in control humaii serum, a procedure to account for these persistent levels is needed for this method validation. `The sponsor provided NWB with blank matrix (Norther Chinese human plasma). `This matrix was tested and demonstrated no quantifiable concentrations for anyofthe analytes. In order to determine the persistent levelsofthese analytes in the lot of human serum used for the validation, a partial calibration curve was prepared with the blank Norther 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 ofthe 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 amountofanalyte. For example,ifthe target concentration at the lower Page 10 000110 Northwest Biowalytcal ReSptourdtyNNoo.. NNWWBBRS00G0--100480 limit ofquantitation was 1.00 ng/mL and the persistent levelofthe analyte in human serum was 2.00 ng'mL, the target concentrationofthe 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 levelsof the analytes in matrix must be performed whenever a new lot of human serum is used during the course of a study. 22. Range of Quantitation Each analytical run included calibration standards in duplicate at nine different concentrations (cight for M556), a minimumofsix quality control samples (QCs) (three levels in replicatesoftwo), two serum blanks and two 0-ng/mL QCs (serum blank with internal standard). `The target calibrator concentrations were approximately 1.00, 2.50, 10.0, 25.0, 50.0, 100, 250, 400 and 500 ng/mL for all analytes. Each analyte has a different final curve range based upon the persistent levels of the analyte in the human serum used. For the validation study, the calibrator concentrations were as follows: PFOS 48.1, 49.6, 57.1,72.1, 97.1, 147, 297, 447, 547 PFOSA 1.00, 2.51, 10.1, 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 oT MsS6 430,118,268, 51.8, 102, 252, 402, 502 : Ms70 5.60, 7.10, 14.6, 29.6, 54.6, 105, 255, 405, 505 000111 Page 11 Northwest Bioanalytical ReSptourdtyNNoo.. NNWWBBRS0000--100480 Range Mean Coefficient _of--Determination PFOS 48110547 ng/mL 0.9925 (Table 1) PFOSA 10010502 ng/mL 0.9899 T (TablF e 2) PFOSAA 60010505 ng/mL 0.9968 T(TTablRe 3)) POAA 5.761 505 ng/mL 0.9922 (T(Tsabblleed4y) -_-- _Ra--nge Mean Coefficient of D_et--ermination PFHS 31510502 ng/mL 0.9954 (TableS) M556 43010502 ng/mL 09935 (TTabEle R6) PM57P0 5.600505 ng/mL 0.9967 T(TaT ble 7D ) The results for the individual calibration standards can be found in Tables -- 14, 23. 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 levelsof the analytes in the human serum used for preparation. For the validation study, the quality control concentrations were as follows: PFOS Low (ng/mL) st Medium (ng/mL) 197 High (ng/mL) 446 Dilution (ng/mL) 4460 .- PFOSA 400 150 400 4000 - 2a PFOSAA 9.00 155 05 050 * POAA EE 154 403 4030 PFHS 615 152 02 4020 M336 580 152 402 020 M570 8.60 155 405 4050 000112 Page 12 Northwest Bioanalytical Study No. NWBS00-040 Report No. NWBR00-108 23.1. Precision and Accuracy for PFOS, PFOSA, PFOSAA, POAA, PFHS, Mss6 and M570 Quality Controls The precision and accuroafcthye LC/MS/MS method for the quantitation of PFOS, PFOSA, PFOSAA, POAA, PFHS, M556 and M570 in human serum were determined by analyzing three levelsofquality controls in replicates of five on three separate days. The intra-assay precision (%CV) for PFOS, POAA, PEHS, M556 and M570 were less than or equal to 9.5% for each undiluted QC concentration. The mean accuracy (%Theoretical) for all levelsofundiluted quality controls ranged fom 84.8% to 111.2% (Tables 15 and 18 = 21). The intra-assay precision (%CV) for PFOSA and PFOSAA were less than or equal to 19.3% for each undiluted QC concentration. The mean accuracy (%Theoretical) for all levelsofundiluted QCs ranged from 68.7% to 91.7% (Tables 16 -- 17). `The inter-assay precision (%CV) for PFOS, POAA, PFHS, M556 and M570 i. were less than or equal to 9.7% for each undiluted QC concentration. The mean accuracy (%Theoretical) for all levels ofundiluted QCs ranged from 87.4% 0 108.5% (Tables 22 and 25 - 28). The inter-assay precision (%CV) for PFOSA and PFOSAA were less than or equal to 14.0% for each undiluted QC concentration. The mean accuracy (%Theoretical) for all levels of undiluted QCs ranged from 73.1% to 88.9% (Tables 23 24), For diluted QCs, the inter-assay precision (%CV) for PFOS, POAABFHS, = MS56 and M570 were less than or equal t0 6.6%. The mean accuracy (%Theoretical) for the diluted QCs ranged from 92.8% to 113.9% (Tables 31 and 34 - 37). The inter-assay precision (%CV) for PFOSA and PFOSAA. were less than or equal to 9.7% for each diluted QC. The mean accuracy (Theoretical) for the diluted QCs ranged from 79.0% to 81.3% (Tables 32 3). 050113 Page 13 Norwest Bioanalytical Study No, NWBS00.040 Report No. NWBR00-108 The slightly high negative bias for the calculated concentrationsof the PFOSA. and PFOSAA QC samples was a resultofthe 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 contentof spiked serum samples. For this preparation, evaporation was required to obtain appropriate concentrations. However, becauseofthe high volatilityofPFOSA 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 asolution that was rot evaporated (Tables A. ~ A.2.). 232. Precision and Accuracyfor PFOSA and PFOSAA Specific Quality Controls Becauseofthe bias demonstrated for PFOSA and PFOSAA calculated concentrations from the original QCs prepared as described in section 23.1, a new setofQCs containing Sly 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 al levelsofundiluted QCs ranged from 72.9% to 107.7% (Tables 29- 30) - The PFOSAA LLOQ calibration standards for the intra-assay Run 17 demonstrated unacceptable accuracies resulting in both replicates being rejected and a raised LLoOf7.Q50 ng/mL for the run. The low QCs for PFOSAA also demonstrated variability greater than 20%. This resulted in a. negative bias (72.9%oftheoretical) for the low PFOSAA QC. 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 resultofwithin-run Page 14 000114 Northwest Bioanalytical ReSptourdtyNNoo.. NNWWBBRS0000-.100480 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 deviationofthe calculated concentrations from theoretical concentrations for these QC diluted with control matrix were both greater than 20%. This indicatesthat 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 accuracyofthe PFOSA and PFOSAA specific QCs was demonstrated with the analytical QC performance during sample . analysis study NWBS00-062 (medical surveillance). The results can be found" in Appendix A, Tables A.1. and A.2. These tables only contain analyses for reported PFOSA and PFOSAA sample data, and do not include runs that did not meet the acceptance criteria for PFOSA or PFOSAA. -- For the PFOSA and PFOSA specific QCs, the inter-assay precision (%CV) was less than or equal to 11.4% for each undiluted QC concentration. The `mean accuracy (%Theoretical) for all levels of undiluted QCs ranged from ~~ 98.5% to 107.7%. # 24. Extraction Efficiency `The extraction efficiencies of PFOS, PFOSA, PFOSAA, POAA, PFHS, M556 and M570 were determined using Northern Chinese human plasma by comparing the area ratios obtained for the following three cases: 000115 Page 1s Northwest Bioanalytical ReSptourdty NNoo.. NNWWBBRS0000-.100480 1. eBxottrhactt)h.e analyte and internal standard added following the extraction(post2. Tfohleloawnianlgyteextardadcetdiotno(pplrae-semxatrparcitorantaolyetxet)r.action and the intemal standard added 3. Tfohleloiwnitnegmatlhesteaxntdraacrtdioandd(epdret-oexptlraacstmainptrerinoarltosteaxntdraradc)t.ion and the analyte added The extraction efficiencies were then determined by the area ratioofthe pre-extract samples tg 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%), MS70 (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 intemal standard are very low. This low recovery is a result ofthe 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 rangeofpolarities for the analytes prevented the useof acidic pH during the extraction and prevented the use ofan effective SPE extraction for allofthe analytes. While the recovery is low, the intra andinter-assay precision and accuracy values demonstrated good reproducibility. The lowrecovery should not affect the assay performance. `The Chinese plasma containing very low persistent levelsof most analytes was used to estimate the extraction recovery ofthe analytes from matrix. This plasma was used. - 0 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. 000116 Page 16 Northwest Bioanalytical 25. Stability Evaluation ReSptourdtyNNoo.. NNWWBBRS0000-.100480 The stability of PFOS, PFOSA, PFOSAA, POAA, PFHS, M556 maintained under various storage conditions wil be documented and in 2 M570 separate NWB report. 3. DATA MANAGEMENT iPnFtOeSgr,atPeFdOuSsAin,g PPFEOSScAieAx,MPaOcAQAu,anPFsoHfSt,waMr5e5(6v,erMsi5o7n01.c6h)rwoimtahtoagsrmapohoitchpfeaacktsoroafr1e , Quantitation is based upon quadratic regression analysis ofcalibration curves (weighted Ux') using the area ratio vs. concentration calculated by the Watson DMLIMS software (version 6.1.1.04). 4. COMMENTS AND`CONCLUSIONS Per agreement updated purity with the Sponsor, the regressions were not information for PFOS, POAA and PFHS. recalculated based upon Some differences might the occur dueto the effectof roundingifthe regressions were performed with the purity corrected concentrations, but these differences would have a negligible effect on the overall interpretation ofthe validation results. oOfnolnyeToHfPthFeOoSriwgainsalusiendteransaalnstianntdeamradls,stNa-nEdtar-dFObeScEa-uOsHeo,fwhciocnchegranvseovaenratcheteastpeecfiofnicaistay product. - The volatility ofPFOSA and PFOSAA is an important aspect t be cautiousofduring the evaporation step ofthe assay. An evaporation step was used for the initial preparation of QC samples to obtain an adequate concentrationof the analytes. This setdemonstrated acceptable precision, but slightly high negative bias for PFOSA and PFOSAA. A separate set of QCs containing only PFOSA and PFOSAA were prepared without using an evaporation step. The second set (PFOSA and PFOSAA specific QCs) demonstrated Page 17 000117 Northwest Bioanalytical ReSptourdty NNoo.. NNWWBBRS0000--100480 both acceptable precision and accuracy. Asa result, two setsof intra-assay and interassay precision and accuracy are shown in the report. The method described in this report has been validated for the determinationof PFOS, PFOSA, PFOSAA, POAA, PFHS, MSS6 and M570 in human serum. However, study samples with experimentally calculated PFOSA or PEOSAA concentrations above the ULOQ should rot be diluted with control matrix. Any known circumstances that may have affected the quality or integrityofthe data are discussed in this report. 4.1. Proposed Sample Analysis Acceptance Criteria Calibration Curve Each run wil include in duplicate calibration standards at six or more concentrations covering the lower to upper limit of quantitation. For all analytes except PFOSA, at least three-fourthsofthe calibration standard's back-calculated concentrations must be within 15% (& 20% for LLOQ)oftheir individual target concentrations. For PFOSA, at least three-fourthsofthe calibration standards back-calculated concentrations must be within + 20% (+ 25% for LLOQ)oftheir individual target concentrations.A calibration standard will be considered a statistical outlier if the back-calculated concentration is greater than two times the acceptance criteria for that standard, Lower Limit ofQuantitation `The back-calculated concentrationsofat least one of the duplicate lowest pints 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 qualifyas the LLOQ for all other analytes. Ifthis criterion is not met, the next level is subjected to the same test and the LLOQ raised accordingly. 030118 Page 18 Northwest Bioanalytical ReSptourdtyNNoo.. NNWWBBRS0000--100480 Quality Control Samples Each analytical run will include low, medium and high QC samples in duplicate. The measured concentrationsofat least two-thirds ofall 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.Ifstudy samples require dilution, a dilution QC will be analyzed in triplicate for each dilution level (except for PFOSA and PFOSAA which should not be diluted with control matrix). At least two dilution QC at each level must be within 20% of it target concentrations inorder to accept diluted study samples at that level. The dilution QC acceptance is independentofthe undiluted analytical QC acceptance. 5. REFERENCES (5.1) D. Vollmer, "Quantitative Determination of PFOS, PFOSA, PFOSAA, NMEFOSE-OH, N-EtFOSE-OH, POAA and PFHS in Human Serum by LC/MS/MS," NWB study NWBS98-082, NWB report NWBR99-005, May 13, 1999. 6. DATARETENTION 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 completionofcontract obligations. : Page 19 000119 Norwest Bioanalytical ReSptourdtyNNoo.. NNWWBBRS0000-.100480 Table 1. Summaryof Calibration Curve Parameters for PFOS Quadsatic weighed Ux. Allconcentrationsare expressed 3 nymL. alI 29-Jun-2000 I I I ogres 0.000009[ 0.035473 | 0.368276| 0.9877 |48|.se71| 30-Jun-2000 -0.000001| 0.018768 09949 |as |st a7| 02-Jul-2000|16| -0.000005| 0.028829| 0235310| 0.9948 [ Mem| T-oo0000s[00276%0 0250778] 09925 ||| CB ECT CE L 5T5T 5515 1 177 A,B and C sre cosflicints used to define the quadratic curve, Table 2. SummaryofCalibration Curve Parameters for PFOSA Quadratic weighted 1, All concentrations re expressed 8 agin. Run A Number Cc [rSquirea[LLOQ[ULOQ 29un2000| 13 |-0000030 [0.093650 |0.023501| 09761 |1.|0so0 z| 30-Jun-2000|15| -0.000012| 0.051196| 0.004685 02-7u12000|16| 0.000020 | 0.088242 [ 0.025293| 09960 |1.|0soz0| [ Mean| 0000021 [0077709 [0017826| 09899 |- || e [wss [eo[ua |] C= T51 TT5 1 1T T5 7] A.B 4ndCarecoefTiiens useto deinethe quadratic curve, Page20 000120 Norwest Bioanalytical RSeptourdty NNoo., NNWWBERS0O00-.100450 Table 3. Summaryof Calibration Curve Parameters for PFOSAA Quads weighted 1. All concenttion ee expressed a ng. El I i ed 25un-| 210300| 0.000003| 0023347| 0.035783| 0.9969 |6.|0500 5 | 30-Jun| -2105 00| 0.000000| 0013890| 0020748 | 0.9971 |6.|0500 s| 027u12000|16| 0.000001 | 0.023426| -0033118| 09964 |60 | 50 05 | [ [MS emT] o|-u00o00t00r1| 0T0o20t22o1|0o0v2o98w8a5s]ooosores ||| ||| TsTsTsTsTT] A.B 2d C ace coeficints use to define the quadratic curve Table 4. Summaryof Calibration Curve Parameters for POAA Quadratic weighted 1, All concentrations are expresseasd sgn. RuaDate | Run A Cc [R-SquareaLLOQ[ULOQ Number 29-1un-2000 0.000002| 0.018721| 0.026984| 09847 30-7u|n210500| 0.000000| 0012512 [0.009407| 0.9958 02Jul2000|__16___| -0.000002| 0.014490|0.015595| 09962 [Mea] [oo0o000 [oors2er|ooirao| 0922 || |. TT el LowT sT[ossT[osoTT1 ]| ALB and Cace coeficiems usedto defn the quad cure Page21 ee CL 000121 Norwest Biosralyial RepSoturdtyNNoo.. NNWWBBRSO0O0--100480 Table 5. Summaryof Calibration Curve Parameters for PFHS Quadratic weighted Lx', All concentrations are expressed as ng/mL. Run A Cc [Resquared| LLOQ |ULOQ 29-Jun-2000 Number 13 0.000006| 0.023682| 0021811 | 09947 |31 |s5 oz| 30-Jun-2000 0.000005| 0.016787| 0.011507 02Jul2000|1 __ 6| 0000009|0.021344|0.020627| 09968 S etTweT LmT oeT TwT ||]| Band Csr coefficients used 0 defn the quadratic curve. "Table 6. Summaryof Calibration Curve Parameters for M556 Quadratic weighted 1/x'. All concentrations are expresseadsg/mL. 29Jun2000| 13| 0000001 [0.005000 0004952| 09881 300-277un--22000000|__16 | 00..000000000000[| 00..000032889812||00..000000754488|| 00997459 [4|3s00 2| [so| Toooo0ot [0001050 | 0.002487| 00049 [I S e T leeT r[wssTs s[|1| A.B 32Cdar coelcint used fin theuadsatccurve 090122 Page 22 Nortwen Biosralytcal ReSptourdtyNNoo.. NNWWBBRSO0OD-.100480 Table 7. Summaryof Calibration Curve Parameters for M570 Quadra weighted /, All concentrations ar expressed as g/mL 297un2000 | 13 |-0000002| 0.006621| 0.007115| 09962 30-J|un15-2| 00.00000001| 0.003911| -0005406| 0.9972 |56 | 50 05| 02312000 |__16 | 0.000000| 0.006433| -0.009231| 09967 |56 |s00 s| So] ooo [oman [omoons [somos ||| [ Men| [0000001 |000s6ss|-00072s1| 0s967 ||| [we [ooo [aes[266[or|[| Co TsTs TT 1] A.B 30Cadre coefficients used to detfheqiuadnrateic curve. 000123 Pages Norwest Bioanal pnyoowsehne s Oi77,ResupdkyNeo NNWWBBRSOIO0-1.0080 Table 8. Back-Calculated Concentrationsof Calibration Standards for PFOS [Comme[mew os[eal onTis [0 on) Quads weighed Ux. Cae SAllhcongcrernaio3rne expriessedjau ngfi ci 4d? ore| al or] ni | 73 | 2S| zed| sen - [ovmooo| 5 Jas[sai[368[ur[GEO] 15s|so ao[sus | 757 [TT Tess sso]us]as[as[sw] [o[ mmaoe[ [Ts5o[lwesw[essssese[nl rfomaanluwsra[[oooasr|]saano[[ss]nu|] [oviwaooo[16 [sie |s05 [eos |7as[ous | ustJawTass[om | [1 Tealwaslssmalo] us]om [wo[sw] -- T rTrr r7TT] vem [wis[soo[sama [oar [ur[307[wes | se | [sol Tamura[sm eor[emfow ea]ma) wovl [sew] Tso [os[se[soles[aa[arJarJar) [foes [ielaslaaesloTsTooos) Co [elelelelelelslels] Table 9. Back-Calculated Concentrations of Calibration Standards for PFOSA a iI a 2ld 2EE w Quadiatc weighted Uc, S AllT coE ncaeeexnpraesasedtasongn/msl. oz [mmm | 5 | wo [ae | wos| [cop] tor[m0| am |wr| [T [ao[ni[E98w 7oso ase[am [sor] [o[ omaooo[1T 5 |too[|zo 2ea[|oomo[[a2s7o a[[asnoea][oonussJ|aar mss[Jaooor[[asru|| [osmaooo| ts |oss|ze| is|26a[ne[om [ass[oonTse]. voz[om[wr[wilw[mw [eo [su] C rrT Trrrr] [vel io [ae [os[mews[en[am[aor[am] [sol ow [oo am [svar[aso[ura[wo [ass [ev [os[e072[nsfteo]as[eras[sal [wC m]o[ [oeol [ae2]e3f0 [eleeolsealeslso[snlass]] Page2s 090124 Naive Bsc Pnrieysos.foBdurSan4d0 `Table 10. Back-Calculated Concentrations of Calibration Standards for PFOSAA hd Quadraticweighted 1'. All concenarteerxpraestsedisongnmsl. _ 7%: =p Of og i3uT Feed, ster grid Tpsictey. sR (C omommT | 5T[sssosot [|77aa [|wwaaaa[2 030]fewgrseys[]ivoiso:[|azurs[[awmn[isuer]] [oC rn | 5 |sT w[|7as[| 74s[[m001] 2s 3[[tmoos[[oomn[aamms [ao|e| [C Comma | 1s T[osuos[|noea[|uv1 osn[[3m0aa]]snsss[[oisw[[oams [[on3s[Tnomre]] [I won| Tsoi [7aT| 5T T 0[m0T[531[orT[5sTJooTn o7e| [[seovl To5w3[[o3a1r[[#o6m[[3195]|2501[[eesm[6a3a7[n0is[i5s1e]] C [oo wl Teils[eal[0s0l[oea]ls9[5se3esl[e07l[aea]] Sampledst ni ins espe ie). Rhor dedin amy sts, Table 11. Back-Calculated ConcentrationsofCalibration Standards for POAA Il ad Ea ADEF EI dd Quadraticweigchtead 1/d5'. Ahllac]qnceL ngations arse exEpsra[seApsg/mslA Lepd h 5. |zEee (mC emo1 | T[e5n1[[6o1g9n||n1aa[[4 a0a7[]) Gsogeeoens[tou/[] oae[[7 a7s[[/| s0u|] (o[ man[5T [sas[[voams||tts woo [[3a075]| sssaas[[tonoae r[aomso[[a0[|saisr]] [[ Coma [v6aoi[C53u6g[l7am0[|t1s5e2[[3320aa]]sseoro[[iooss[|aasss0[|ssmosa[ssmmi| C onl Ts [ar[w1 T r[30T 0|saT a|ioT r[3aI]so1e[so1r| [sowel TToosso[[owras[[oowr[[2a010][aa00[[s|ssna]]tueaala73]] C [mw TTosa T|oerl[eoo olr[ealr]s0l7e[l10e]leso[l2as]e00]] Sarl dsc1i2oeed(3 es he epsen), Rel mrad samets. peas 000125 SO RSuatotsBtSIOiO0 Table 12. Back-Calculated Concentrations of Calibration Standards for PFHS Quadraicweighted 1, All concentratiaonesexpressedsgl, 3c. 47 13a Ea T3Y re in "ase wa | 1 [Croemrmeo|remem [[o3n|Tsee[[iaes[{vw2e J[oonoeJ[wsr [oanm[[ato[arm C somoT [15 T[easwa[Tsemen[wwra{[aoon1T[wenss[[osoa[woLoaou[[vosn| [C armorT [we T[ssear[[oaon[wuos[[aases[[ssuona[[oiwoe [aasu [[oosnLoewar| DTTTsT u Tosrs[uTs[aT ss[soa[T wr[aw [T ow[0] e sol Tow Teomws[a oe s [oaw[[2e0ew [wew[er[ssa]| [ ed T TwTToeT[aa ooToos[[osa[oas llouo[[ualeasa a Telelelsielelolole] Table 13. Back-Calculated Concentrations of Calibration Standards for M556 `Quadratic ER weighted 1x'. All concNeEntSrSatiAoanrLes eAxpressed as 4 ng/mL. 3e9ra2l-33097 [roSmnee [[ memorieh s[iugJ[eoega ][oCg[dreJ[oTr me[[oe|Jwowr| CC ommon 5[TaaamTTuosmT [aoollwors[[0w[onm[[oowe[ooro]] [C emma | teTaeamwnT[TuuuseT [TsaasoT|oo0uesT[Tioosoai[[[aaeam[[[3aoomns[[[0o|]e -c T Tr rTTTT] eo.l Taoiarlw[ows[Taom[awnTeosseo[asal[uus[a]is| a | TTael[aesT[eelossT[ealnssT[oorl[eas]] ae ame gei et et my 000126 FE iyo msc Report No, NWBR0O-108 Table 14. Back-Calculated ConcentrationsofCalibration Standards for M570 Qi LY,MSTnL 00 5 |e [mCC oornoenT T[mwoTTswsioJ[4JonanJ|w4 ise[{vooese[A[ote9zsn|n[l[owevraJT]aows[[aossw[[50a]| E(eEmo|EeTTTesEeo[TT oeww[[Evwrs[DEo5rEsoraALeon[E oa [TEoan[Ee|] e TsTuoeT e[[e wT oesT ewT wLne lraTwee]] [seall wel TTToowaeTl[oowor[[[ooourn{[loraree[]aasv[T[oanone l[oonuiilToeolaer]]]| m oTu n eeleelsstelFoAseTeAleTeTeeTs] ra 000127 mers pioees Table 15. Intra-Assay Precision and Accuracy for PFOS Quality Control Samples All concentrations are expressed as ng/mL. wind ro | [En 2 [mCmoT5 |e esss[Tw wow[[|owee=m ]|] Number [51.1 ng/mL| 197ng/mL 446 ng/mL. TTe es[Twwss | [ow]] : -- 1I [ T Mesan o]l-- [e uw T|[ewsm l m[| |amso s]|| C[ aes [m mr |T m os wa ] ] roe|EE " C ome TeweT |owwm [|ewnm]] Number [51.1 ng/mL| 197 ng/mL_| 446 ng/mL TTeess[ Tww[men]] --rr Te1 we|e] T meseonle T [mel [[wws ewsm e |||wawii]|] C Tsa Ts 5] 030128 -- oqnoemsmeonosn `Table 15. Intra-Assay Precision and Accuracy for PFOS Quality Control Samples (continued) All concentrations are expressed as ng/mL. Run Low QC | Medium QC| High QC Number [51.1 ng/mL| 197 ng/mL | 446 ng/mL TTTT T Tee ww w wm T TTeowT w ww w] ] m ol w Tw w ew |w ai] mmeal] Tome|awes |5we] CT 1515] 000129 Comers S cooe rs Table 16. Intra-Assay Precision and Accuracy for PFOSA Quality Control Samples All concentarreaetxpiroesnsesd as ng. 207 0o 4.00 ng/mL| 150 ng/mL| 400 ng/mL T T TTe eT T T wwwo] w] T TTe TosTw w[m wm] T em TowTwT w] ] sesllyTs owT| eew [|owce]| TsTs = oRemaR]5 m] ve | ee|dg| vam | cw | om | 4.00 ng/mL| 150 ng/mL| 400 ng/mL T Tee T eTree[ooww]] aeer[[rwws[|aeww em w 11T w Tw]m] se] {wow [or|ws| C5T Ts] 000130 aot 0d Page 30 4 esedtl Tote 2 msn PD Table 16. Intra-Assay Precision and Accuracy for PFOSA Quality Control Samples (continued) Run Number| Low QC [Medium QC| High QC 4.00 ng/mL| 150 ng/mL 400 ng/mL T T TTa oT T orww aa] n] T Tee e wm |e e e sol Too0nT [wamw| ] ee Demmwia]l [C7o5o wwoa | ] er | CT TT5s T=] - 000131 Norwest Bioanalytical ReSptourdty NNoo,. NNWWBBRSO0G0--100480 Table 17. Intra-Assay Precision and Accuracy for PFOSAA Quality Control Samples All concentrations ae expressed s ngimL. 100% Los Run Number| LowQC | Medium QC| High QC 9.00 ng/mL| 155 ng/mL | 40S ng/mL T Te e rwms eeea C TTew eT w =oe |oe w] ewsoew ll T Tw oem |a m aw e |w m) o] WToeoweica| l| 06 "800 |ar TsTs Run Number| Low QC | Medium QC| High QC 9.00 ng/mL| 155ng/mL| 405 ng/mL T C Tomm T mrwe eeew] T T ToemT |rnww oeww el Ts ww] Ce ita] o wr|[7es s [e| s] Lo TT 5 ae +> 220% theoretical rao MTN CE 3 07 Page32 E327rd000132 emir nFeT soe (continued) Table 17. Intra-Assay Precision and Accuracy for PFOSAA `Quality ControlSamples Run Number| Low QC Medium QC 9.00 ng/mL| 155 ng/mL. High QC 405 ng/mL T TT Te on[ohre we ueoe aw] Te uw [oo] el Te ww [eelrT|sw5] 1 a [Cio[se] C5T 15] * > 20% theoretical or 000133 Report No. NWBR0O-108 "Table 18. Intra-Assay Precision and Accuracy for POAA Quality Control Samples All concentrations are expresseasd ng/mL. L2: 5.79 EEAEAE EA Gama no | ow| wo | ae] Number| 8.74 ng/mL| 154 ng/mL | 403 ng/mL T C T ToewT T w [see] ] TTe e[e we e] T ml TwT [ww] soell essw [n oow |w] e] CTsss Dom1 a]Nu5mbTer|| 8o.w 74 nng/m|[ L| 15w4e neg/mL||w4w03 ne] g/m|L T T Tw eT w wew [w] e | rT TowT TwT ow] e sol TosweT [[eeoeT|aoew C mmm] T 97T[wsss T[ose]] 000134 Northwest Bioanalytical ReSptourdtyNNoo. NNWWBBRS0000--100480 Table 18. Intra-Assay Precision and Accuracy for POAA Quality Control Samples (continued) All concentrations ae expresed ss g/mL. Run | LowQC | Medium QC| HighQC Number| 8.74 ng/mL| 154 ng/mL| 403 ng/mL ouaeeo | 16 |ms[se|e | [TT es [ uwT [ wm |] [Te |w [e 3s| [TT en | wT s [ es | [T Twe [aw | [ven [oo | we [395 | [sol Tom |se [ us | Theoretical] | 905 | 929 98.0 CT Tss [5| 000135 Page 3s I rseeasmssn Table 19. Intra-Assay Precision and Accuracy for PFHS Quality Control Samples All concentrations are expressed as ng/mL. Loos md Run Low QC | Medium QC Number| 6.15 ng/mL| 152 ng/mL| High QC 402 ng/mL T TTeorwT Twno) we] C TToewT [wwae] ] Seoml Towww [ew | ar] [ase imr 071| 3 ws |awo]o] Run Low QC | Medium QC Number| 6.150g/mL| 152 ng/mL | High QC 402 ng/mL T C TToonwT T [ow w w a] ] C TTeownT [wewe] ] esoll TToea [aaws |ise]] C1 Ts5 15] 000136 meant rsee mmssmn Table 19. Intra-Assay Precision and Accuracy for PFHS Quality Control Samples (continued) Run Date NRuruinber |6L.1o5 wngQ/CmL| | M1e5d2inugm/mQLC| 4H0i2gnhgQ/mCL TTToeeenw [[wowwoTaow] ]] To ow] SeeollloTTTosew [m a0m w [u] r] CTTTe] 090137 Table 20. Intra-Assay Precision and Accuracy for M556 Quality Control Samples en a All concentrations are expressed as ng/mL. woes 4.0 Number [5.80 ng/mL| 152 ng/mL | 402 ng/mL T w T T w eew we Treee wwem owee e T e T sel Tow ww Te Bg| [eT miao ] T | 8 |s ors v wz [T ton]1T|e snT |w w |w wo | Number |5.80 ng/mL| 152 ng/mL| 402 ng/mL TT s w e www e C T TaT T woTw 1. e emsso]lw | oomer[|e wower||w unes] CT Tss Tv] 000138 emesis pceomme Table 20. Intra-Assay Precision and Accuracy for M556 Quality Control Samples (continued) Run Number Low QC | Medium QC |5.80 ng/mL| 152 ng/mL High QC 402 ng/mL. C TT TeennT ooww [|ewno]] T T ToaT e [owwe [|ewrm] el Tw ww] Te we el e|w] oT Ts 5] 030139 meron SI orte rms Table 21. Intra-Assay Precision and Accuracy for M570 Quality Control Samples Allconcenaions reexpressed sng, 0297 262 Run Number| 8.L6o0 wngQ/CmL| | M1e5d5inugm/QmLC_|| High QC 405 ng/mL C T TTw eT T T Te wT a e C TTeweT T Tw w owes CT Tss Ts win Run Number| Low QC | Medium QC| 8.60 ng/mL| 155 ng/mL | High QC 405 ng/mL T T Tn mT T Tw w|oe w T TeT ww Te sol Tw om[w sm T|ueo Pe ome] l | 3s wee ee| |w| o| 030140 Cet Ste T `Table 21. Intra-Assay Precision and Accuracy for M570 Quality Control Samples (continued) Run Low QC | Medium QC| High QC Number| 8.60 ng/mL| 155 ng/mL | 405 ng/mL T TToenT Twww [w] i] T TTs eT [w we wa] ] mo ww el Tes re 090141 mers p-- e mene Table 22. Inter-Assay Precision for PFOS Quality Control Samples RdEAE Ad [ T m 5T[weom T [ wo] w m | Number |51.1 ng/mL| 197 ng/mL | 446 ng/mL T C TTeesT T Tw wwwm | T TwT Tw T TT T Te eT T TmwTwwm] T ATIeT TOwT wm [mT am|eT|e wzT w | 0 |w wm] T TTeerT Twwe Twwo] --TTT woToT m a] e sw owos w w | w] in [eI rmalI 1IoiTs |ET1E [mETaT] C TvT TsTs] I~ 030142 Report No. NWBR00-108 Table 23. Inter-Assay Precision for PFOSA Quality Control Samples [Er rgFB| All concentrations are expressed as ng/mL. 4.00 ng/mL| 150 ng/mL| 400 ng/mL C T TToowwT T www m Ta] w] TToew w wm m] ] oT man Te awT e | ew o [w] e] T Tee wwea[nwr] (EmmT|T eTw emra [e os |] ow] T T TsT w Twwn |s]] T Te TowT w we |w we ]] -- -- mesil] w0e0 mosw | ] es] a aw] m5T oT[aw15mi]] J. 000143 Cosmin -- te vasa Table 24. Inter-Assay Precision for PFOSAA Quality Control Samples pe [5075 |ieoe] om | a | mowo |me[wol | om | Number |9.00 ng/mL| 155 ng/mL| 405 ng/mL C TTowoT w owe |o]] T C Teew T [aw| m ow [oC mmaion| 5T|mveT w | ua |w ow] T T TmT e rwwe |ewse] [ezow| T 5|ear[wweas]] T TTeeenT w rw |w ew T TToesT owwe |oowe -- -- eoll TToee [|sesn [|sewe]s.] ... Comeerssl] m9|aas5 || mwae]] C TI wo Tw 5] 030144 ren Roopume `Table 25. Inter-Assay Precision for POAA Quality Control Samples [Faf mT w] uoT|wooeT [ wo | aa]| 8.74 ng/mL| 154 ng/mL 403 ng/mL T T TowmT Tw w e wm] TT TonTw wm] C T TToowwT T T www] wm] TTT owT Tww Twwm]] FT ozmamo| 6T|oossT T| w0[|ow ]| T Teee T Tw w awmw]] TT ToeT Tw T we esoll Toemw |w wn w wa] PoCem=s] I5Cs |sETi| mCT0] SO Tel TvasTw > 5a0]] 090145 -- pJen--s `Table 26. Inter-Assay Precision for PFHS Quality Control Samples [|Fe J All concentrations are expressed as ng/mL. T maw] voT|eowoT | wves |awe]| Number| 6.15 ng/mL| 152 ng/mL 402 ng/mL TTeer[w on ea]] GT erman| 5[onw | 0we |aw] n] T TToewmT w www ] T TTT TeewT e we w ew | [mT am|5T|eonoT |[ww o w wi]| : T TT TToewsT T Tww = [| | T TTT e Tw T we | e slw Tow [[wmwew]i] e mei] l | oaT| woe ss|moesl ] mel Tsse[e5sTues]] Page 46 030148 Snes Table 27. Inter-Assay Precision for M556 Quality Control Samples liPAE 580 ng/mL| 152ng/mL| 402 ng/mL C Tw ewT [e we w [w] e] T Tesw T Tw wTowe] DT imon| 5T[oemT Tw w ow e| Teew TTww owwe ] [emmm| w | eseT| www [wwir]] T TTeernT Twwe w we] T Tsee [ TewTw e mw 1T wT w] sol Tew ow wo e e mm e toesT[r oe oa r |] oeoar]] To Ts[ou] 000147 mes S wero m `Table 28. Inter-Assay Precision for M570 Quality Control Samples EGr L a T mmo 5T| o0T | wws T |e| Number| 8.60 ng/mL| 155 ng/mL| 405 ng/mL Te ow Twes Towe ET E EE ATe TCwNTEwT 1 TToeeTT[wwwew]e] ToneTewo Toww Fomo1r| aT| w 30T | oww T|wwm]| T Tne wwee Twwe] T 1 w TeT-- w [Mewsaeo]-- e om ow s | se] eTl wT[asso Tuar]] Page ds 000148 a `Table 29. Intra-Assay Precision and Accuracy for PFOSA Specific Quality Controls [Com wn m Too me]a E w [A oawgs |crnwmhn|| T T TTooeT T eToww [mwem T T r T TeeT T T w we |w e| T so TTeewe [ | bw e | w wa] T me o a[wl n ese |[wui|] TT ot Th togrfini r pt ddp ertn. Quadratic weighted 1/x. i[ems e[ | r (wow o [s3omo[[or men[|rowws reasf|uivoo] an]|uoomn|) CT or mcs r rirer e . 1] [oiC snmozoT n|[rrrToowm0n[T2a2ss[[[ioooarr[|[muias[[[sssoese[a[ooosa[[[o3|nJvonm[[551] 38]. T w ET ITa T TEasT [E 30[EaT TeE[aEr T e[EoCnI[EneTEaAr]E]O a [alalilalelel:ls1:] 050149 Northwest Bioanalytical Study No. NWBS00-040 Report No. NWBR0O-108 `Table 30. Intra-Assay Precision and Accuracy for PFOSAA Specific Quality Coatrols [ov ome]fo dB) [Cowan[0[70[ we Tse | [n Ts wTm| [ [wes1 [ wooTos| [Ts Twe Tse | [ [en7 e[wm [ 6| 1[1 - [veal 1 ess[ wo [sn | [sof 1 we[ 15s [ es | [wo [ms [ os | so| [meosict] | mo |wa| ois | C=[+T 5s T5| pe `Quadratic weighted Ux'. cc 11 T 1TT1 C= TT Tv oT 7 1 i meoe d dl [ez[7Toms[san[wa 32s[esa]vonJas [ai [5| [TTTsofenulwws[e mi]s[] ws] Tr rr1 rrr 1] [sem[ess]usol[aueso[amsJoem |ssie) [wow T-Torleolou3ss]la0afure] Co [-T-l202722(a2Tz2]z] Page 50 000150 mero -- pte m--s Table 31. PFOS Dilution Quality `Coantrol Samples Run Number| 4Di4l6u0tinogn/QmCL es | im T I CT eTs e|| Tee wm ew | Tmeemews] | | 2 | Co Tw] Page SL 000151 my -- ReNp o.No WBROrO-1t08. "Table 32. PFOSA Dilution Quality Control Samples Rl +1 4000 ng/mL To TTT w wn] ] Tw T] w T I w] I TTTEewT| C E TE TEwE ] T Tw e ] T TT T ] well Ts] | eeam lai eso] s]| : CTs] 000152 emesmin worsen `Table 33. PFOSAA Dilution Quality Control Samples Rl G3 [ Tm T5m T|40wm 5w 0 nog/|mL T I --T CT EE Te TT e Te]e TTo TTw e E CT ETeE w] T I Ie] T 1 TTw I I ---- Caiea m0 a] m] 0 | : CT5] 000153 eres nmi S-- te Tum Table 34. POAA Dilution Quality Control Samples All concentrations are expressed as ng/mL. SF 4030 ng/mL. T I Iw] TTw T T w] o [oTm T T 5an w ] e] T Tw w o] ] [aT|T Tma |e on n | T [e ew n T Tw eo o T eT w sl Tw| remoo]a] as] oo 000154 Norwest Biosalyical RStuedyNNopo..NNoWWBBRSr0000..t100480 Table 35. PFHS Dilution Quality Control Samples Al concenttions re espresed as nginL. Run Date | Run Number| Di4l0u2t0inogn/QmCL TTTe Te] 30-Jun-2000 4980 |. T Teew] ] 0202000 |16| 4700 T Te e I -- I -- a Cm m]auw]s] oT] +> 220%theoretical 000155 Page 55 . Norwest Bownalyical RStuedyNNapo.. NNoWWBBRSr0000--t100480 `Table 36. M556 Dilution Quality Control Samples All concentations ee expressed ss nya. Run Number] D4i0l2u0tinogn/QmCL 2992000 13| 4330 I-- Som I I -- 0202000 [16| am [Tee] [Tw] [ww [weal Teno ImalE------ Pagess 090156 [ET S-- RStuedyNNopo..NNoWWBBRSrO0O0-.t100480 Table 37. MS70 Dilution Quality Control Samples All concentrations se expressed 3 ngmL. Te] Run Date |Run Number] D4i0l5u0tinogn/QmCL 250un2000 | 13 | 4710 Tew soneao| 1s | seo | Tw 080 02-Jul-2000 270 Tw E elT m] m I e --l wr| 090157 ~ Pages? Northwest Bioanalytical ReSptourdty NNoo.. NNWWBBRS0000.-100480 Table 38. PFOSA Specific Dilution Quality Controls All concentrations are expressed as ng/mL. Run Date |Run Number| Dilution QC 4000 ng/mL 16-1ul-2000 +2630 eee l s Tw ] [veealmaina] 0] 3 | 15] = +> 225% theoretical 080158 Page ss Northwest Bowssytcal ReSptourdty NNoo.. NNWWBBRSOOO0--10008 Table 39. PFOSAA Specific Dilution Quality Controls All conceaatiosnes expressaed ng. Run Date | Run Number| D4i0l5u0tinogn/QmLC. 16701-2000 "2560 I ---- e I =--e iT ma T ne] [---- - 030159 Page Northwest Bioanalytical Study No. NWBS00.040 Report No. NWBROO-108 Table 40. PFOS Extraction Efficiency Ren? To CoAT ire Ma mig Laer iStrem pr - : 3.1pirsodA trxin =5 on Sa . on tn io nst eiecyeboeo ssnebtsts 5145035%0. elmCorton = 10f8o Agrc Re magn male rsstecmon an 2. r tA an ehConran gn ordr . 1 Ao Tim atss abine cin tiwirnts BaFpEiose en 5 2 yerar stration oan 2.18 rs etcxcon aon Mes cto ice hin 5 Crm rst cn otrnb S81 Page 60 030160 Northwest Bioanalytical ReSptourdtyNNoo., NNWWBBRSO0X0--100480 Table 41. PFOSA Extraction Efficiency Run? CTT r E Dosn a bEsos tists apr cin asons -- | sm amecm uis : nt Mean o eis 65 10 tnCortean 188gmt Can pags mae 2 ec oncom poya ie ptrcn am ns ts resi 63% isont 84rgnL rt Cfrrt Ee borras esro a Mon Br ia 2 per timc am 25rd Aestcmn re son ee Sa se contee n arcdne shordtoaisne 4340%% PO icresnteetsrccooominnnEser A41% _-- 090161 Northwest Bioanalytical Study No. NWBS00-040 Report No. NWBROO-108 Table 42. PFOSAA Extraction Efficiency Run? To Coeemraon = TTAR r Im Sn estou oe 2.15prandAna aterxncion i - 158 Medium Concnraion = 109 gifonrAna.bne 1.Ansem1de15 she extn Baoioondmmsbvels stan a5 2Asa rior 20d1S sexction oase Raagan fr i 3.15 porsndAneafer enion ome os ertos ey brew+ 144%. High Cnconredn= 400 gform drat Asr ct als ba 2Ami rior 115s xnion os 2.18rirsnAnayaexmcion Moon Moss curacon ficincy or he aly = OvralmcOuvnecrxertcmieoannefixcuiecnocynfoocresnrcylfosasndnaaryde== oom Ea TSK L7043%5 Page 62 . 090162 Northwest Bioanalytical fo R`eSptourdtyNNoo..NNWWBBSR0O0L-100480 Table 43. POAA Extraction Efficiency LL fun? Has sega . air tr in cam = en S008 = re. 3.15 prior ndAnalyte aterexvaciion w3m% ston c Mss cco s ese is 331520% etn Corin 80pm snr . rEvR. mear s mss7ase soir tr in ocoinee 3.8m re rio oam Mw = gett i130 at Cor og fp Lr ai a IH] os sep 8 ion ox 15 cit Astro min en ses --a_ c-- ics e--cte-- io Ormecnstre esdsrrctrtmsa . pr HE 1645 180% I 000163 Northwest Bioanalytical ReSptourdty NNoo,. NNWWBBRSO0O0--100480 Table 44. PFHS Extraction Efficiency Rin? LE S ras R Saben mGsone mangees 2 Ampere 15 xcs aon - | seae miesrtrcmson Po ts cnSes ncceto toieda e rei sus 033%% etmConnon 180g Art TR eggs age 2 Aste ren5 arcmon aus [3.15 sd resctsrcieon a Se stotyrrdt tas 15% HhComrise= 8fg rrr ni 1 alssear ton mrT4o s beaaala i aes 2 Aste re 15 er xin an 3.15 ir0 Aatere ction cans OotmOnrstotmeonepcotyoopr rndorr eieds 7115 Pag6es 090164 Northwest Bossalyial ReSpuodrtyNNoo.. NNWWEBRS0000-.100680 Table 45. M556 Extraction Efficiency TTRunI ? rndCE otseR n en bShm smsase Amir s cmon isam Sen [EE -- i nSr et ie tst s tmCorin tng 1. Ani nd 5 20er4 ax 370 >Ame recon or id aps tse ss tC tapisr [TeO matar BoIiasst bsoSna e os Sawenrcn sa nSe earcy eimies iao OrvstnrCr pi ints. hk pases 090165 Northwest Bioanalytical RStuedyNNopo.. NNoWWBBRSr00O0--t100480 "Table 46. M570 Extraction Efficiency Run? TT TR Is a an Sr 2 Aye rad 5 trxin pe i ao _ Le] sani termine we Son cabSoe icnosneihne esrmoarlhoaerse G13T8% etm Cncinren= 109ormArgle re gps aie 3.18 pirsdAr hrxin RS aoms mariceorses PE -- 1AiTioeagsearicsscion osaLwmdans en a00n Amierhd rxin om Saiias HIi 2.15rr aAmster con an Stn nce cnbt eres 614% resOsrsemsmeFceoncos nrondrnsbirrte= P13N8% Page66 030168 `Northwest Bioanalytical ReSptourdtyNNoo.. NNWWBBSR0000.-014008. 7 ANALYTICAL METHOD Principles of the Method The analytical method consisted of a liquid:liquid extraction procedure followed by `evaporation and reconstitution ofthe 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 oC instrument was operated in the multiple reaction monitoring (MRM) mode under optimized conditions for PFOS, PFOSA, PFOSAA, POAA, PFHS, M556 and M570 detection. CHEMICAL STRUCTURES aq F FP FR I FC Cc OOnCc ONT|N, F 2 F Foo PFOSA MW=499.0 (M-H]=498.0 I BON Fr One OFea OFea [TN 2 2 2 Il 20 OH S$ PFOSAA MW=585.0 [M-H]=584.0 BONeOeBem n5e po on 2 2 2 0 Page? POAA ) MW=4140 [M-H}=413.0 030167 west Bioazalyia Norwest Blossayial Fa Ba % FC. C. C. AO SF:e NFT NFeB 9 I ~S~ou 20Il StudyNo, NWBS00.040 Report No. NWBR00-108 PEOS [MMW-=H4]9=949.89.9 9 : FFI PFHS i. . BO SON ON To MW=399.9 . ff fo MH]=398.9 0 Fs Fa Fy I FON ON ON ON A F FE Rg H on o M556 NWosses MH]=S55.9 i F EB FON Oe SNe ON A> o | CH, G8 M570 MW=571.0 a}(-H]=570.0 st 090168 Northwest Bioanalytical ReSptourdtyNNao.. NNWWBBRSO000-.100480 re EAA a TO a E Ne ONT NEN--T 3 F F Hog vs 71. Reference Materials and Matrices LT Analyte LotNumber Purity ExDpairtaetion Source CoSntdoirtaigoens J PFOS (FC-95) 3 00% A10S0 ER3MS TempReoroamture PFOSA 26. 00% 12300 3M TempReoroamture PEOSAA(FC-129) 617 S38% 12312010 3M TempReoroamture POAA (FC-143) 2s 100% LALO 3M TempReroaotmure* PFS? M536 M570 THPFOS sI8-152 100% NBIL3O)ST- 99.89% 11850626 975% so09 0% Rede me 12312000 3M 123L2010 3M 12312000 3M LAL20W0 3M el Kd. 20C TemRpeoroamnure TempReoroamture TempReoroamture The reference material purity for PFOS, PFOSA, PFHS and POAA was not available prior to the conduct ofthis study. Therefore, all concentrations included in the report for these analytes are based upon an assumed purity of 100%. Prior to the completionofthis final report, 3M contracted with Centre Analytical Laboratories, Inc. in State College, Pennsylvania to determine the absolute concentration of PFOS, POAA and PFHS in the stock standard solution prepared. using seven reference materials (PFOS, PFOSA, PFOSAA, POAA, PFHS, MSS6 and Pages _ 090 169 Northwest Bioanalytical ReSptourdtyNNoo.. NNWWBBRS0000-.100480 MS70) at a target concentration of 5000 ppb. Absolute determinationofthe concentration of PFOSA in the stock standard solutions cannot be made until a full purity determination of these reference materials is completed. Based on the results obtained, the concentrations included in this report should be corrected according to the following table: . Analyte . PFOS . POAA Correction Factor 0.836 0.909 PFHS : 03855 Matrix Human serum Human 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) ethanol] Methanol, HPLC-grade Methyl-Tert-Butyl Ether (MTBE), HPLC-grade Water, HPLC-grade Name Autosampler: PE Series 200 Balance: Meter Toledo MTS Equipment/Supplies 1 Source Perkin Elmer, Norwalk, CT HMiegthltesrt-oTwonl,edNoJ, Inc., rg 030170 Northwest Bioanalytical ReSptourdtyNNoo.. NNWWBBRSO000--100480 Name Centrifuge: Beckman GS-6R Equipment/Supplies Source Beckman Instruments, Fullerton, CA Evaporator: Turbo Vap LV, Model 43750 | Zymark Corp., Hopkinton, i MA `Hematology/Chemistry Mixer, Model 346 Fisher Scientific, Pittsburgh, PA HPLC Chromatographic column: Genesis Lightning, | Jones Chromatography, Cis, 4 pm, 2 x 50 mm. | Lakewood, CA oe Liquid Chromatograph: Hewlett Packard 1100 `Agilent (Hewlett Packard), . Palo Alto, CA. Liquid Chromatograph: PE Sciex Series 200 Perkin Elmer, Norwalk, CT wLiitqhuiCdLC-h1ro0mAaDtopgurmappha:nSdhCiTmOad-z1u0ASCcLo-l1u0mAn coovnetnroller || `Shimadzu, Columbia, MD Mass Spectrometer: Perkin Elmer Sciex API3000 | PE Sciex, Concord, Ontario Multi-tube Vortexer : | VWR Scientific Products, | Bridgeport, NJ Pipentes: Eppendorf Repeater Pipette | BWersitnbkumrayn, INnYstruments, Inc., Pipettes: Finnpipette: Digital 1-5 mL | Fisher Scientific, Pittsburgh, PA Pipettes: Rainin EDP Digital 100-1000uL TM | Rainin Instrument Co., | Wobum, MA Pipettes: Volumes: Rainin M adjustable 20-50, 10-100, 100-1000 ul __ || WRoaibnuinm,InsMtArument Co., Pipettes: Rainin RL Rainin Instrument Co., Volumes: adjustable 5-20, 20-100, 40-200, 200-1000 | Woburn, MA --ul--e--e ------------------------------------ `Sonicator, Branson [ Branson, Danbury, CT Sonicator, Fisher | Fisher Scientific, Fair Lawn, | NI Vortex: Fisher Genie 2 i---- `Water Pro Plus eee | Fisher Scientific, Fair Lawn, ceNgSe ---------------- | Labeonco, Co., Kansas City, | MO Page 71 0 301 "1 Norwest Bioanalytical ReSptourdtyNNoo.. NNWWBBRS0000-.100480 73. Reagents, Calibration Standard and Quality Control (QC) Solutions The calibrator, quality control and stock solution preparations listed below serve as a general guideline for auaining the targeted concentrations. Due to the fact that PFHS was received at NWB as a solution, both the calibration standard and quality control stocks are from the same solution and are not from separate weighings of reference material as per NWB SOP LABOP003. However, separate aliquots were used for calibration standard and QC stock solution preparation. Reagents All reagent solutions are stored at room temperature unless otherwise noted. + 50m ammonium acetate in water (unadjusted: pH~6.9) Fill a 1-L volumetric flask approximatelyhalffull with water. Weigh out 3.85 g ofammonium acetate and transfer to the flask. Use a stir plate to mix and fill to volume with water. + 20mMammonium acetate in water (unadjusted:pH ~6.9) Filla 1-L volumetric flask approximately half full with water. Weigh out 1.54 g ofammonium acetate and transfer to the flask. Use a str plate to mix and fill to volume with water. + 20mM ammonium acetate in methanol Prepare as described above, substituting methanol for water. + 30:70 20 mM ammonium acetate in water (unadjusted: pH~6.9): 20 mM. ammonium acetate in methanol (v4) + 50:50 water:metha(nvo4l) 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. Page 2 030172 Northwest Biosralyical ReSprourdtyNNoo.. NNWWBBRS0000--100480 + PFOS Stock Standard (0.05904 mg/mL) Weigh 1.476 mg of PFOS and add to a 25-mL volumetric flask. Fill to volume with methanol, mix by inverting 5 times and sonicating for approximately 10 minutes. . PFOSA Stock Standard (0.07652 mg/mL) Weigh 1.913 mg ofPFOSA and add to a 25-mL volumetric flask. Fill to volume with methanol, mix by inverting 5 times and sonicating for approximately 10 er +. minutes. + 'PFOSAA Stock Standard (0.02264 mg/mL) Weigh 1.052 mg ofPFOSAA 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 ofPOAA and add to a 25-mL volumetric flask. Fill to volume with methanol, mix by inverting 5 timesand sonicating for approximately 10 minutes. . M556 Stock Standard (0.04579 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. + M570 Stock Standard (0.04796 mg/mL) Weigh 1.202 mg ofM570 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 Standar9d (5000 ng/mLfor PFOS, PFOSAA, POAA. PFHS, M556and ` M570; 5020 ng/mLfor PFOSA) Add 2.117 mLofPFOS Stock Standard (0.05904 mg/mL), 1.639 mL of PFOSA PageT3 030173 Northwest Biosralyial RSeptourdty NNoo.. NNWWEBRSO0O0--100480 Stock Standard (0.07652 mg/mL), 5.521 mL of PFOSAAStock Standard (0.02264 mg/mL), 3.232 mL of POAA StockStandard (0.03868 mg/mL), 20.16 WL of PFHS (6200 ppm), 2.73 mL of M556 Stock Standard (0.04579 mg/mL) and 2.606 mL of M570 Stock Standard (0.04796 mg/mL) to a 25-mL volumetric flask. Fill to volume with 50:50 water:methanol and mix by inverting 10 times and sonicating for approximately 10 minutes. . Spiing Standard 8 (4000 ng/mL for PFOS, PFOSAA, POAA, PFHS, M556, 0 * "M570; 4020 ng/mLfor PFOSA) `Add 8.00 mL of Spiking Standard 9 to 2 16 x 100 mm polypropylene tube and add 2.00 mLof 50:50 water:methanol. Mix by inverting 20 times. . Spiking Standard 7 (2500 ng/mLfor PFOS, PFOSAA, POAA, PFHS, M556,M570; 2510 ng/mL for PFOSA) `Add 5.00 mLof Spiking Standard 9 to a 16 x 100 mm polypropylene tube and add 5.00 mLof 50:50 water:methanol. Mix by inverting 20 times. . Spiking Standard 6 (1000 ng/mLfor PFOS, PFOSA, PFOSAA, POAA, PFHS, M556 and M570) Add 2.00 mL ofSpiking Standard 9 to 2 16 x 100 mm polypropylene tube and add. 8,00 mLof 50:50 water:methanol. Mix by inverting 20 times. + Spiking Standard 5 (S00 ng/mL.for PFOS, PFOSAA, POA, PFHS, M556, M570; 503ng/mL for PFOSA) Add 1.25 mLofSpiking Standard 8 to a 16 x 100 mm polypropylene tube and add 8.75 mL of50:50 water:methanol. Mix by inverting 20 times. . Spiking Standard 4 (250 ng/mLfor PFOS, PFOSAA, POAA, PFHS, M556, M570; 251 ng/mL for PFOSA) Add 0.500 mLofSpiking Standard 9 to a 16 x 100 mm polypropylene tube and add 9.50 mLof 50:50 water:methanol. Mix by inverting 20 times. 090174 Pages Northwest Bioanalytical ReSptourdty NNoo,, NNWWBBRS0000-.100480 + Spiking Standard 3 (100 ng/mLfor PFOS. PFOSAA. POAA, PFHS, M555, M570: 101 ng/mLfor PFOSA) Add 0.250 mLofSpiking Standard 8 to a 16 x 100 mm polypropylene tube and 2dd 9.75 mL of 50:50 water:methanol. Mix by inverting 20 times. + Spiking Standard 2 (25.0 ng/mL.for PFOS. PFOSAA, POAA, PFS, M556, MS70; 25.1 ng/mLfor PFOSA) "Add 0.100 mLofSpiking Standard 7 to a 16 x 100 mm polypropylene tube and or "3dd 9.90mL of 50:50 water:methanol. Mix by inverting 20 times. + Spiking Standard 1 (10.0 ng/mLfor POS, PFOSA, PFOSAA, POAA, PFHS, M556 and M570) Add 0.100 mLof Spiking Standard 6 to a 16 x 100 mm polypropylene tube and 2dd 9.90 mLof 50:50 watermethanol. Mix by inverting 20 times. internal Standard Solutions All internal standard solutions are transferred to polypropylene containers and stored ina-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:methanolandmix by inverting times and sonicating approximately 10 minutes. + Working Internal Standard (400 ng/mL. THPFOS) Add 413 pLof the THPFOS Stock Solution (0.04342 mg/mL) to a S0-mL volumetric flask and fil to volume with 50:50 water:methanol. Mix by inverting 510 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 2-20C freezer. Pages -- 030175 Norwest Bioanalycical ReSptourdtyNNoo.. NNWWBBRS0000--100480 + PFOS QCStock (0.07732 mg/mL) Weigh 1.933 mgof PFOS and add to a 25-mL volumetric flask. Fill to volume with methanol, mix by inverting $ times and sonicating for approximately 10 minutes. + PFOSA QC Stock (0.04876 mg/mL) Weigh 1.219 mg of PFOSA and add to a 25-mL volumetric flask. Fill to volume with methanol, mix by inverting $ times and sonicating for approximately 10 - thinutes. + PFOSAA QC Stock (0.02920 mg/mL) - Weigh 1.357 mg ofPFOSAA and add to a 25-mL volumetric flask. Fill to volume with methanol, mix by inverting times and sonicating for approximately 10 minutes. + POAA OC Stock (0.04228 mg/mL) `Weigh 1.057 mg of POAA and add to a25-mL volumetric flask. Fill to volume . with methanol, mix by inverting $ times and sonicating for approximately 10 `minutes. + PFHS QC Stock (1000ppm) Aliquot 807,Lof PHS stock solution (6200 ppm) to a 5-mL volumetric flask. Fill to volume with methanol and mix by inversion. + M556 OC Stock (0.05230 mg/mL) Weigh 1.309 mg of M556 and add to 2 25-mL volumetric flask. Fill to volume with methanol, mix by inverting $ times and sonicating for approximately 10 minutes. + M570 QC Stack (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 $ times and sonicating for approximately 10 `minutes. Page 76 000176 Northwest Bioanalytical ReSptourdty NNoo,. NNWWBBRS0000--100480 + 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.L. nitrogen. Reconstitute with 131 uL ofmethanol. . PFOSH Concentrated OC Solution (1,000,000 ng/mL) Evaporate 2.05 mL ofPFOSA (0.04876 mg/mL) at 20 C and below 5 P.S.L nitrogen. Reconstitute with 100 uL of methanol. .e . PFOSAA Concentrated QC Solution (1,000,000 ng/mL) Evaporate 4.42 mL of PFOSAA (0.02920 mg/mL) at 20C and below 5 PSL. nitrogen. Reconstitute with 129 uLof methanol. . POAA Concentrated OC Solution (996,000 ng/mL) Evaporate 2.59 mL ofPOAA (0.04228 mg/mL) at 20C and below 5 P.S.L nitrogen. Reconstitute with 110 uLofmethanol. . PFHS Concentrated QC Solution No PFHS concentrated solution was prepared. + M556 Concentrated OC Solution (1,000,000 ng/mL) " Evaporate 2.18 mL ofM556 (0.05230 mg/mL) at 20 C and below S P.SL nitrogen. Reconstitute with 114 uL of methanol. + M570 Concentrated OC Solution (1,000,000 ng/mL) Evaporate 2.09 mL ofM570 (0.05474 mg/mL) at 20 C and below 5 P.S.L nitrogen. Reconstitute with 114.4 uL ofmethanol. 7.4. Preparationof Validation Quality Control Samples All quality control target concentrations will be corrected for the persistent levels of the analytes in human serum. 090177 Page 77 . Northwest Bioanalytcsl ReSptourdty NNoo.. NNWWBBRSO000--100480 Dilution Quality Control (3990 ng/mL PFOS, 4000 ng/mL PFOSA, 4000 ng/ml PEOSAA, 3980 ng mL POAA, 4000 ng/mL PFHS, 4000 ng/mL M556 and 400: ng/mL M570) Transfer 100 uLof each Concentrated QC Solution (997,000 ng/mL PFOS, 1,000,000 ng/mL PFOSA, 1,000,000 ng/mL PFOSAA, 996,000 ng/mL POAA, 1,000,000 ng/mL M556, and 1,000,000 ng/mL M570) and 100 kL of the 1000 ppm PFHS QC Stock into'a 25-mL volumetric flask. Fill to volume with human serum and mix by op inversion. Sonicate for approximately 10 minutes and equilibrate for approximately 10 minutes. `High Quality Control (399 ng/mL PFOS, 400 ng/mL PFOSA, 400 ng/mL PFOSAA, 398 ng/mL POAA., , 400 ng/mL PFHS, 400 ng/mL M556 and 400 ng/mL M570) Transfer 5.00 mL ofthe Dilution Quality Control into a 50-mL volumetric flask. Fill to volume with human serum and mix by inversion. Sonicate for approximately 10 minutes and equilibrate for approximately 10 minutes. Medium Quality Control (150 ng/mL PFOS, 150 ng/mL PFOSA, 150 ng/mL PEOSAA, 149 ng/mL POAA, 150 ng/mL PFHS, 150 ng/mL M556 and 150 ng/mL M570) Transfer 1.875 mLof the Dilution Quality Control into a 50-mL volumetric flask. Fill to volume with human serum and mix by inversion. Sonicate for approximately 10 minutes and equilibrate for approximately 10 minutes. Low Quality Control (3.99 ng/mL PFOS, 4.00 ng/mL PFOSA, 4.00 ng/ mL PFOSAA, 3.98 ng/mL POAA, 4.00 ng/mL PFHS, 4.00 ng/mL M556 and 4.00 ng/mL M570) Transfer 0.500 mLof the High Quatity 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 ofQC Samples After preparation, place aliquots of the low, medium, high and dilution QC pools into cryogenic vials, and store in a -20C freezer. 030178 Pages Northwest Bioanalytical ReSptourdtyNNoo.. NNWWBBRS0000-.100480 75. Preparation of PFOSA and PFOSAA Specific Validation Quality Control Samples Quality Control Solutions Allqualitycontrol solutions are transferred to polypropylene containers and stored in 2-20 C freezer. + PFOSA OC Stock (0.2166 mg/mL) `Weigh 5.414 mg of PFOSA and add to a 25-mL volumetric flask. Fill to volume `with methanol, mix by inverting S times and sonicating for approximately 10 minutes. + PFOSAA QC Stock (0.2322 mg/mL) `Weigh 10.790 mgof PFOSAA and add to a 25-mL volumetric flask. Fill to volume with methanol, mix by inverting times and sonicating for approximately 10 minutes. Preparation of Quality Control Samples All quality control target concentrations will be corrected for the persistent levels of the analytes in human serum. Dilution Quality Control (4000 ng/mL PFOSA and PEOSAA) Transfer 0.462 mLof the PFOSA QC Stock Solution (0.2/66 mg/mL) and 0.431 mL. ofthe PFOSAA QC Stock Solution (0.2322 mg/mL ) into a 25-mL volumetric flask. Fill to volume with human serumandmix by inverting approximately 10 times. Sonicate for 5 minutes and equilibrate for approximately minutes. High Quality Control (400 ng/mL PFOSA and PFOSAA) Transfer 2.50 mLof the Dilution Quality Control into a 25-mL volumetric flask. Fill 10 volume with human serum and mix by inverting approximately 10 times. Sonicate for 5 minutes and equilibrate for approximately 5 minutes. Pages 030179 NorwestBioanalytical SRtepuodryt NNoo..NNWWBBRS0000-.100480 Medium Quality Control (150 ng/mL PFOSA and PFOSAA) Transfer 0.938 mL of the Dilution Quality Control into a 25-mL volumetric flask. Fill to volume with human serum and mix by inverting approximately 10 times. Sonicate for 5 minutes and equilibrate for approximately 5 minutes. Low Quality Control (4.00 ng/mL PFOSA and PFOSAA `Transfer 0.250 mLof the High Quality Control into a 25-mL volumetric flask. Fill to volime with human serum and mix by inverting approximately 10 times. Sonicate - for minutes and equilibrate for approximately 5 minutes. : Storage ofOC Samples After preparation, place aliquotsofthe 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 solutionsofthe analytes can be used to prepare calibration standards and quality control samples, the evaporationofstock 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 dayofeach run by adding 100 pL blank human serum and 400 Lof 50 mM ammonium acetate in water (unadjusted: pH ~6.9) to 13 x 100 mm polypropylene tubes. After abrief vortex mixing, spike 10.0 HLofthe appropriate spiking solution into the tubes as shown in the table below. [wo wo | wno | sw | `SNtuanmdbaerrd| ConcSeonlturtaitoino(nnogf/mSLp)iking | VoSpliukmiengof || BlVaonlkuHmuemoafn| ConceFnitnralation [CCTT woowme owoo|w ow |e mi] PFOS3,1SP5F6O,SMASAT,O/PPOFAOSAPFHS, | SoWluLt)ion | Serum (iL)| (sgl) Page 80 030180 Northwest Bioanalytical ReSptourdtyNNoo.. NNWWBBRSO000--100480 SNtuamnbdaerrd| ConcSeonlturtaitoino(nnogf/mSpLi)king | VSpoilluimnegof|| BlVaonlkuHmuemoafn| ConceFnitnraaltion PROSN.iPseR,OSEATAD,)POPAFAO,SPAFHS, | Soolutyion | Serum(uL) | (ng/mL) [ wwo [1wwoo |[| w w ow ||ww us || C oT T wwse Twwo| Tw wo [owowwi]| mos [ee | wm [wan] LoTw [wo |w o | rw | * The target calibration curve range is 1.00 ng/mL to 500 ng/mL. Each analyte has a different final curve range based upon the persistent levelsofthe analyte in the human serum used. 7.8. Sample Preparation Calibration Curve Samples (prepare in duplicate) as indicated above Quality Control Samples (prepare in duplicate) Aliquot 100 uLofeach of the Low, Medium andHighcontrols into separate 13 x 100 `mm polypropylene tubes. If needed, prepare a dilution control (in triplicate) according to the following formula: for DF=X, aliquot (0.500 mL/X) ofdilution control into [0.500mL -- (0.500 mL/X)]ofblank human serum. Aliquot 100 pL of the prepared dilution control into separate 13 x 100 mm polypropylene tubes. Blank Control Samples (prepare in duplicate) Aliquot 100 uLofblank human serum into separate 13 x 100 mm polypropylene tubes and label as QCO. Aliquot 100 pL ofblank human serum into separate 13 x 100 mm polypropylene tubes and label as BLANK. as 030181 Norwest Bioanalytical Study Samples ReSptourdtyNNoo. NNWWBBRSO0O0--100480 Transfer 100-4L aliquotsof each study sample into appropriately labeled 13 x 100 cum polypropylene tubes. [f necessary, dilute study samples in the same manner as the Dilution QC. Extraction Procedure 1. Add 400 uLof 50 mM ammonium acetate in water (unadjusted: pH ~6.9) to cach sample except calibrators and vortex mix briefly. 2. Add 50.0 iL of Working Intemal 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-ert-butyl ether to each tube, cap and vortex mix for approximately 30 seconds. 5. Rotate samples for approximately 10 minutes 6. Centrifuge samples at 3000 rpm for approximately 10 minutes. 7. Transfer the top MTBE layer into clean 13 x 100 mm polypropylene tubes using a transfer pipette. 8. Evaporate the organic layer to dryness in a TurboVap set at 25C under gentle nitrogen stream (setting <5 psi) for approximately 40 minutes. 9. Reconstitute in 100 uLof 30:70 20 mM ammonium acetate in water (unadjusted: PH ~6.9): 20 mM ammonium acetate in methanol and vortex mix for approximately 15 seconds. 10. Transfer samples to autosampler vials and centrifuge at 3000 rpm for approximately 2 minutes 090182 Pages Northwest Bioanalytical 79. LC/MS/MS Conditions ReSptourdtyNNoo.. NNWWBBRSO0O0-.100480 LC Conditions HPLC Column Mobile Phases L Conditions Genesis Lightning Cys, 4 pum, 2450 mm A: 20 mM ammonium acetate in water (unadjusted: pH ~6.9) B: 20 mM ammonium acetate in methanol Gradient - LEN Time (min.) %B . 0 50 1 -s0 7 97.5 9 97.5 91 50 n end Flow Rate 300 WL/minute: Post Column Addition | 50 uL/minute of2-(2-methoylethoxy) ethanol Column Temperature ~~ 40C Injection Volume 2-204 MS Conditions Post Column Split Source Source Temp. Ionization Mode Analysis type None TurbolonSprayTM (flow rate L/min.) 400C Negative lon Multiple reaction monitoring (MRM) PFOS PFOSA Transitions monitored (0.3) 499 > 80 498578 Dwell Time (ms) 70 70 Collision Energy (eV) 80 65 pages 00183 Northwest Bioanalytical ! PFOSAA POAA PFHS M56 M570 THPFOS Transitions monitored (03 384-419 4135169 39980 556 > 498 570 > 419 427-407 ReSptourdtyNNoo.. NNWWEBRS0000-.100480 Dwell Time (ms) 70 70 0 400 0 0 Collision. Energy (eV) 29 2 J a a a `Theprepared standards and QCs were injected into the instrument LC/MS/MS system ina 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. Severalofthe analytes possess iosmers. The chromatographic conditionsofthis method can achieve baseline separationofthese isomers. Because the goslof this method i to determine th 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/x) using the area ratio vs. concentration by the `Watson DMLIMS software (version 6.1.1.04) 030184 Page s Norwest Biosalyial ReSptourdtyNNoo.. NNWWBBRSO0O0--100680 Figure 1. Representative Calibration Curve for PFOS eg aIdtPSp RSEIAS rS e 1 Pg . IilT | TT 5 oA . i : | 3pse------reereeeeeeeppeemreeeed]| ime Figure 2. Representative Calibration Curve for PFOSA ER nt | 2 wih | Seint 030185 Pages Norwest Bianca ReSptourdtyNNoo., NNWWBBRSOI0D..100480 Figure 3. Representative Calibration Curve for PFOSAA. l ERe i _ h Nomina Conc. (ng) Figure 4. Representative Calibration Curve for POAA Tees QueLi a0 Jdi I bt h . " : NosiauConc. (agal) Pages 090188 Northwest Bioanalytical Se Study No. sess NWBS00-040 Figure 5. Representative Calibration Curve for PFHS -- FoRE-- "I Ld | z | i mista - " Figure 6. Representative Calibration Curve for M556 e At Fe, e Fl ; | ` " ema * | | - " 090187 Northwest Bioanalytical ReSptourdty NNoo..NNWWBBRS0QO0--100480 Figure 7. Representative Calibration Curve for M570 Ra e HRr Gii a : LL "1 al - - [---- 090188 Page88 - Norwest Bioanalytical RStuedyNNopo.. NNoWWBBRSrO000--t100480 Figure 8. Standard (57.1 ng/mL) Chromatogram for PFOS fEomesoes -- ota ren S = a isS 98 52 r m rrme a = e8] E = t C-- r a-- -- ], . E C~ E Emm-- a a, . [Gec=-= fe Be BW NW Her [o--r---- ---- ] r = rE = - Pe. n m sen Em8]. . emt = E Ea m -- , C-- r E EE w] w GEES BE EEE) 090189 Page89 Northwest Bioarayical RepSotrudty NNoo..NNWWBBRSO000..100480 Figure 9. Standard (10.1 ng/mL) Chromatogram for PFOSA EE ne ET oG ormgeT ann m eFmraR ia]I bs I [Ra E E ea a vr iasa r]]TM] = Gee--el, ERIE ARE ARE E-- E -- -- m B m = en = ae233 eEe s 2 Pam]= ar] E r = a-- l r L E tho ea BA Na AR a a we i: 12 TE EB Wh BF har Pageso 090190 NostuwestBiosalyial RepSoturdty NNoo,.NNWWBBRSO000-.100480 Figure 10. Standard (15.0 ng/mL) Chromatogram for PFOSAA E wSm oarm e ay) m z amrmm t e. d a1 mmmemesrs .% pE E e r -- n r| EE e--r -- Eee SA CTTETE TS, 5 BW Gu = m= r) i Sa m mae etn0 rt" 12 mpemomteses % = [megsr =. e = Ea m e ieee BA CTURTE Wh th 8 Bh slr Pages 090191 Norwest Bioanalytical RStuedyNNopo,. NNoWWBBRSr0000-.t100480 Figure 11. Standard (14.8 ng/niL) Chromatogram for POAA BiE Ei--n_-- Emena) P= O me% mE mn tn _-- wemmememmnesamsst Sov i i . I : EEa -- =Ee-- ---- -- ---- . ) w EGi=meey G--IAr | TRH Bh ih Th Fr Sear EE -- Em) mem m= mrme ry -- Co-- a mmmaE em=se o2s2 i ---- b or eesio ng TM] Ef E -- Ee ms m-- l --. l he EE TTR WB th BP fhm Pages 090192 extenSot Nutysaobaa Figure 12. Standard (12.2 ng/mL) Chromatogram for PFHS H>TEmee--rw E= F ZC= SEEEEEEE ESGwe Em N en [row----------1.TM] mm E m --rE e E e GEE BE fed WW BW Sewar = pT STmehT EneT fe mEn EE8] tT Em wee = [ B E E ee /e i e mEa 1TM me EEE BE ENE NEN RE EY rs 050193 ne Figure 13. Standard (11.8 ng/mL) Chromatogram for M556 H= == eer En E EIE2EEEE0EeT SF E3EkJ4 EE fren -- = a e LLaee-- e aeus Ts EA ., Seo HW Th AWN --= m= = En= TBTSREEiEEEEEYRtRS4 ee Gor -- o a - e fer e emeTM'| ey =H NeoE WW BOW Sor Paget 09019. 4 Norte: iowa RepSoturdty NNoo.. NNWWBBSRI00-.100480 Figure 14. Standard (14.6 ng/mL) Chromatogram for M570 E --ree eoe = E smmaen y % amen wmaFwneEeioogg ---- ae -= eEa e -- f ema -- mr a ---- a d Eoc=--F- EN fim a TR Be BOW flr ws = SR zmaEmb = abEs Z 23 me fm, = rm fi rE E E -- = -- at a m a -- e = s-- e 1TM| c=: TE Te th BF fer Pagess 090195 Report No. NWBR0O-108 APPENDIX A The following quality control results are from sample analysis study NWBS00-062. Table A.1. Inter -Assay Precision and Accuracy for PFOSA Quality Control Samples : 4.00 ng/mL| 150 ng/mL | 400 ng/mL [C om | mTooweT [woie] ]| C imam | 5ToawT |uwmww]m] [oman]rT ae e[wwwwe] ] e T rs T[m weaT w |mw ]w m 5 GC as] 5T[ oowmT e wwe ] C TeT ww [C eran 5TTooawwmT w [w wew w | ] e| [mC sm] 5ToomnT w | w [] | C TowT mw] I ET NV 090195 men Jeeronysmsonsnn Table A.1. Inter -Assay Precision and Accuracy for PFOSA Quality Control Samples (continued) [Fe fer y FR e All concentrations are expressed as ng/mL. Fommaon] aT|o4e.00wnng/m[[L|wo150weng/mL|| 4a00wnrsg/m]L [C Emm meTeweT Te w w ow T FrT[oi awwT |[a oew |[s wwmm ]| ST ermon T| oowwT T [wwwe]e] I= --1 [oremwsieca]] [mwse T | me7T|eoses]| C eal T To wa [ w5w] as] : 000197 Table A.2. Inter -Assay Precision and Accuracy for PFOSAA Quality Control Samples [Fo J]Jog, er | 9.00 ng/mL 155 ng/mL. 405 ng/mL. DC ow| 5ToweT [eise |e]| ee foramI]TT Te os |m|w ae | [mC mm Tw neT T[wem a| [T emg TwT w |w ew ] C TeT Tw em [C ama]5To eT mw |w ws] T TwT Tw we] [C msm]5TewwsT T|e e w ew | [C emsmom|5Tw0T |wwe me] w [C ram]5Tw osT e |wm w ] C TomT Tw a] 050198 ere nmi coro Table A.2. Inter -Assay Precision and Accuracy for PFOSAA Quality Control Samples (continued) Run Number| Low QC [Medium QC| High QC 9.00 ng/mL| 155ng/mL| 405 ng/mL Tew we C ToeT Tw |or To ww | C TeeT Tw[we] swom [Twe o ||w ws[w un | [meae]l | wore |mwwe| toss| ve T|m we|e] mC 090199 Northwest Bioanalytical ReSptourdtyNNoo.. NNWWBBRSO000-.100480 APPENDIX B Calculation of Persistent Levelsof Analvtes in Diluted Human Serum Samples Because there are persistent levelsof the analytes in human serum, diluted samples must account for the amountofanalyte added from the sample and the amountofanalyte added from the human serum matrix. The following section describes equations used to correct for the amount of analyte added from the human serum matrix used to dilute samples. " "Thie total volume (Vr) of a diluted sample is defined as the volume aliquoted fromtheoriginal sample (Vs) plus the volume ofmatrix (Va) added. The dilution factor (DF) is defined as the total volume divided by the volume aliquoted from the original sample. Vp=Vs+ Vy DF=V1/Vs Example Vr =0.1 mL sample + 0.4 mLcontrol matrix Vr=05 The amountofanalyte A in a diluted sample is equal to the volume aliquoted from the original sample times the concentrationofanalyte in the sample (Cs) plus the volumeof control matrix added times the concentrationofanalyte in the control matrix (Cy). The concentration ofanalyte in the sample (Cs) is determined during sample analyses. The concentrationofanalyte in the control matrix (Cy) is determined experimentally for each lotofcontrol matrix as described in Section 2.1. Equation 1. Total Amount of analyte = Vs*Cs + Vy*Cyt Example Total Amooufannalytte = [0.1 mL sample * 100 ng/mL}+ [0.4 mL control matrix * 10 ng/mL] Total Amountofanalyte = 14 ng. 090200 Page 100 Northwest Bioanalytical Amooufannalytte from sampl=e 10 ng ReSptourdty NNoo.. NNWWBBRS0000-.100480 ; Amountof analyte from control matrix = 4 ng `The concentrationof the analyte (Cy) in the diluted sample is equal to the sum of the following: 1) the ratioof the volumeof sample (Vs) to the total volumeof the diluted sample (V-) times the `concentration in the sample (Cs), and 2) the ratioofthe volumeof control matrix in the diluted sample (Vi) to the total volume of the diluted sample(Vr) times the concentration in the control matrix (Cv). (This is the concentration of analyte in the diluted samplepriorto any adjustment - for the actual dilution.) `The equation for the above is: Equation 2. Ca=(VS/Vi)*Cs + (Vs/VD)* Cut . Example Cp =[(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 volumeof control matrix Vi with (V1-Vs) into this equation produces: Equation 3. Ca= (VIVD)*Cs + (VV) V)*Cu or Ca=(Vs/VD)*Cs + (1-Vs/VDC)u*t Example Ca ={(0.1 mL/0.5mL)*100 ng/mL] + [(1-(0.1 mL/0.5 mL))*10 ng/mL] Ca=20 ng+/8nm g/mLL. Ca=28 ng/mL. Inserting 1/DF for the term V/V yields the final equation for Ca: 090z1 . Page 101 Northwest Biowalyscal Equation 4. ReSptourdtyNNoo.. NNWWBBRS00O0-.100480 Ca =(I/DF)*Cs + (1-1/DF)*Cxn Example Ca = [(1/5)*100 ng/mL] + [(11/5))*10 ng/mL] Cu= 28 ng/ml The concentration determined by the Watson LIMS system (Cw) for any sample is: " Equation 5. or Cw=DF*Cs Example Cw=5*28ngmL Cw = 140 ng/mL (This is the concentrationofanalyte after adjusting for the actual dilution.) Substituting the final equation for C, (Equation 4.) into the equation for Cy gives: Equation 6. * - Cw = DF *[(1/DF)*Cs + (1-1/DF)*Cyi] Exsmple Cu=5* (U5) *100 ngimL) + (1415)* 10 ng/mL) Cw =5* [20 ng/+mL8 ng/mL] Cw = 140 ng/mL. Solving this last equation for Cs produces the equation for correction due to persistent levels which is used to adjust theoretical calibration and QC concentrations. Equation 7. Cs = Cw- (DF-1)*Cu `Example Cs = 140 ng/mL -- (5-1) * 10 ng/mL] 050202 Paget Northwest Bioanalytical Cs= 140 ng/-m4L0 ng/mL Cs= 100 ng/mL. ReSptourdtyNNoo.. NNWWBBRSO0D0--100480 Page 103 050203