Document M4mKEVRmKny3qLO1G9Geq83xM

| Northwest Bioanalyical RSetpuodrytNNoo..NNWWBBRSS999--111136 Quantitative Determination of PFOS, PFOSA, PFOSAA, PFHS, POAA, M556 and M570 in Human . Serum by LC/MS/MS Sample Analysis Report for Protocol EPI-Swed Northwest Bioanalytical (NWB) A Division of NWT Inc. 1121East 3900 South Salt Lake City, UT 84124 PREPARED FOR: 3M Environmental Technology and Services (3M) 935 Bush Avenue St. Paul, MN' 55133 g == oBnE SS = 5% 2 AUTHOR: David Vollmer, Ph.D., NWB Project Manager APPROVED FO) FE BY: 77 RodgefL. Foltz, Ph.D., NWB Laboratory Director Pagel pate: 5% 4 DATE: ae vo Lo QUALITY ASSURANCE STATEMENT Report No.NWBR99-113 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 NWBS99-116 `SPONSOR STUDY NUMBER: EPI-Swed NWB STUDY TITLE: `Extraction and Analysis of PFOS and other Fluorochemicals in Human Serum by HPLC/Electrospray Mass Spectrometry `The clinical sample analysis study described in this repiosrnott included within the definitionof a GLP regulated nonclinical study. However, Northwest Bioanalytical conducts all studies within the guidelines ofthe U.S. FDA Good Laboratory Practice Regulations for Nonclinical Laboratory Studies (Title 21 CFR. Part 58), the OECD PrinciplesofGood Laboratory Practice and the Japanese MHW Good Laboratory Practice Standard Ordinance for Nonclinical Laboratory Studies on the Safety ofDrugs (Ordinance No. 21, PAB Notification No. 424.) The following inspections were performedbythe NWB QAU per SOP. Inspection and Reporting Statement. Inspection Date 0278-S3e0pA1u9g991999 17Sep 1999 01 Dec 1999 09 Dec 1999 PhaseofStudy ASnaamlpylteicRaelcPeliapnt Sample Analysis Report DraftRaw Data Final Report NW ProjDaetcetMIansnpaecgteiron Rep*oNrtWBIsMasnuageemdentTo 0380SAeupg 11999999 3310SAeupg11999999 17Sep 1999 30 Sep 1999 02 Dec 1999 30 Dec 1999 09 Dec 1999 30 Dec 1999 *Reports to NWB Management are issued monthly. Northwest Bioanalytical RSetpuodryt NNoo,. NNWWBBSR3S99--111163 Compliance Statement d`Tehfeinciltiinoincaol fasaGmpLlPe raengaulylsaitsedstnuondcyldineisccarlibsetdudwyitsheitnftohritshrienpToirttleis2n1ootfitnhceluUd.eSd. wCitohdienotfhe Federal Regulations Part 58. However, to the best ofour knowledge, this project was conducted in accordance with the guidelinesof the U.S. FDA Good Laboratory Practice Regulations for Nonclinical Laboratory Studies (Title 21 CFR Part58)and according to the g`umiedtehloidnseasonfd ptrhoecOedEuCreDs Pdreisnccriipbleeds woiftGhionotdhiLsarbeoproartto.ryInPraadcdtiiticoena,ntdhethsetuJdaypafnoelsleowMedHtWhe "Good Laboratory Practice Standard Ordinance for Nonclinical Laboratory Studies on the iSnacfiedteynotsf tDhrautgasff(eOcrtdeidntahnecequNaoli.ty21o,r PinAtBegrNiottyiofifctahteiopnroNjoe.ct4o2r4.d)atTahreerpeorwteedr.e Tnhoiksnroepwonrt represents an accurate record of the raw data. | oN NDaWviBd ProjWeiclmterM,aPnha.gDe.r Rodger L. Foltz, PhD, NWB Laboratory Director fy [5 Date 12/1/v2 Date ' `Northwest Bioanalytical Study No. NWBS99-116 Report No. NWBR99-113 TABLE OF CONTENTS SIGNATURE PAGE...hmmm QUALITYASSURANCE STATEMENT ccc 2 COMPLIANCE STATEMENT... ssid TAOFB CONLTENETS covered LI OFTSABLT ES eo monly 1. BITROOUCTION mmm 2 METHODOLOS ummm 3. SAMPLEANALYSE commas nm---------- 4. REAS NDDU ISCL USTT IONS ...mmmmmmmmmmmmmmnsmmmmmnondd 5. BERERBHCES mmm LI OF TSABLTES Table 1. Calibration Curve Summary for PFOS in Human Serum.......owvvsens 12 Table 2. Calibration Curve Summary for PFOinHSUMAAN SERUM... 12 Table 3.Calibration CurveSummaryfor PFOSAAin HUMAN Semm ...........rrnre 12. Table 4. Calibration Curve Summary for PFHSin HUMAN Serum... 13 Table 5. Calibration Curve Summary for POAA in HUMAN SErUM vermis 13 Tabl6e. CalibrationCurveSummary for MSS6 in HUMAN SERUM... 13 Tabl7e.Calibration Curve Summaryfor M570 in HUMAN SEU. ...evvvvvrnins 14 Table 8. Back-Calculated ConcentrationsofCalibration Standards for PROS intHUMAN SEM... ssn 1S Table 9. Back-Calculated Concentrationsof Calibration Standards for PFOSAin HumanSerur............ sm ----------tg Paged CT . etme Bowen ReSptourdtyNNoo.. NNWWBBRS9999-.111136 `Table 10. Back-Calculated ConcentrationsofCalibration Standards for PFOSAA its FUND SEIU cc. nese 16 Table 11. Back-Calculated Concentrations of Calibration Standards for PFHSin HUMAN SCRUM... sss 16 Table 12. Back-Calculated Concentrationsof Calibration Standards for POAA in HUD SUL. ssn]1 Table 13. Back-Calculated ConcentrationsofCalibration Standards for Table 14. Back-Calculated ConcentrationsofCalibration Standards for MST0 in HUIAR SEIU. 18 Table 15. Analytical QC Summary for PFOS in HUMEN SMU crn 19 Table 16. Analytical QC Summary for PFOSAin HUMAN SERUM... 19 `Table 17. Analytical QC Summary for PFOSAA in HUMAN SETUIM.....vvrrvvrrrn 20 `Table 18. Analytical QC SummfoarPrFHyS int HUMAN SERUM rvs 20 Table 19. Analytical QC Summaryfor POAA in HUME Ser vrs 21 Table 20. Analytical QC Summary for MSS6 in HUME SNM v.21 Table 21. Analytical QC Summaryfor MS70 in Human Sem vse 22 Table 22. Study Sample CONCERTAONS srs: 23 Pages Northwest Bioanalytical ReSptourdty NNoo.. NNWWBBRSS399--111136 Quantitative Determination of PFOS, PFOSA, PFOSAA, PFHS, POAA, M556 and M570 in Human Serum by LC/MS/MS `Sample Analysis for Protocol EPI-Swed 1. INTRODUCTION . `This report summarizes the analytical results rom the quantitation ofPFOS, PFOSA, PFOSAA, PFHS, POAA, M556 and MS70 in human serum samples for 3M `Environmental Technology and Services in supportofProtocol EPI-Swed [5.1]. The validated LC/MS/MS method provided a lower limit of quantitation (LLOQ)of 31.4 (PFOS), 1.00 (PFOSA), 1.45 (PFOSAAY), 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 (ULOQ) of 270 ppb [5.2]. The non- validated method for the quantitation for M556 and M570 in human serum provided a lower limit ofquantitation (LLOQ) of 1.00 and 5.00 ppb, respectively, and exhibited a linear response to 500 ppb for both analytes. `The testingfacility was 3M Corporate Occupational Medicine (Bldg. 220-3W-05, St. Paul, MN 55144-1000),and JH. Mandel, M.D.,a the facility servedasthe Principal Investigator. The following is a list of NWB supervisory personnel involved in the completionofthis work: David Vollmer, Ph.D. (NWB Project Manager); RodgerL. Foltz, Ph.D. (NWB Laboratory Director). NWB SOPs were used in the conduct ofthis 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 "The clinical 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 guidelinesofthe U.S. FDA Good Laboratory Practice Regulations for Pages Norwest Bioasalytcal ReSptourdtyNNoo. NNWWEBRSS9S9--111136 Nonclinical Laboratory Studies (Title 21 CFR Part 58), the OECD Principles of Good Laboratory Practice and the Japanese MEW Good Laboratory Practice Standard Ordinance for Nonclinical Laboratory Studies on the SafetyofDrugs (Ordinance No. 21, PAB Notification No. 424). Any changes to or deviations from the original protocol (Analytical Plan) were documented through approved protocol amendments or deviation `memos and are retained within the raw data. 2. METHODOLOGY `The validated assay used for this study is Meth98082.00 and was reported in Northwest Bioanalytical report NWBR99-005 [5.2]. Samples for this study were received at NWB on the following date: Receipt Date August 27, 1999 Reference Material NumberofSamples Received 39 Storage Condition (except during analysis) -20C Analyte Lot Number Purity Expiration Date Source Storage Conditions *PFOS (FC-95) 193 100% 12312010 3M Room Temperature (ry) PFOSA 214 100% 12/31/2010 ES `Room Temperature *PFOSAA 67 538% 12612010 3M RoomTemperature (FC-129) PFHS S9B182 100% 12512000 3M ~20c (PFCo1A4L3) 245 100% 12312010 3M Room Temperature a Mss6 113N04B7-80 9989% 12312010 3M RoomTemperature M570 11850626 975% 12312010 3M, Room Temperature dSodDeScyl(soudlfiautme) 17HO459 99% 1162001 Sigmp Room Temperature TT sAynnoanlymaoumsewsitwhoPeEOrSau(gFhCo.n9t5)is report do mot inde heir FC HeniherTo example, PROS 5 Page Northwest Bioanalytical ReSptourdtyNNoo.. NNWWBBRS9999-.111136 Nine calibration standards were prepared fresh daily by adding the appropriate spiking standard solution into 0.200 mL.ofhuman serum. The final calibration standard concentrations in human serum for each analyte were as follows: Analyte PFOS PFOSA . PFOSAA PFHS POAA M556 M570 Calibration Standard Concentrations (ppb) 314,35.4,40.4, 55.4, 80.4, 130, 280, 430, 530 1.00, 5.00, 10.0, 25.0, 50.0, 100, 250, 400, 500 145,3.60,6.29, 14.4, 27.8, 54.7, 135,216,270 261,6.61,11.6,26.6, 51.6, 102, 252, 402, 502 527,9.27,143,29.3, 543, 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 cach analytical run: Analyte PFOS PFOSA PFOSAA PFHS POAA M556 M570 OC 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 mLof human serum, the serum mixture is made basic with the addition of 0.5 M tetrabutylammonium hydrogen sulfate (TBA, pHL10) and 0.25 M carbonate buffer. This mixtureis then extracted with methyl-tert`butyl ether. After sufficient mixing, the sample is centrifuged. The organic layer is transferred via pipette into aclean test tube. The organic layer is then evaporated to dryness and the sample is Pages Northwest Bioanalytical SRtepuodryt NNoo..NNWWBBSR999-S111163 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 1/x) for all analytes was performed using the `peak area ratio vs. concentration utilizing Watson DMLIMS software (version 5.4.10.2) 31. 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 lowerto upper limitofquantitation. At least two-thirdsofthe calibration standard's back-calculated concentrations must be within + 15% (+20% for LLOQ)oftheir 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 arewithinthe acceptance criteria. Lower Limitof Quantitation `The back-calculated concentrations of at least oneofthe duplicate lowest points in the calibration curve must be within + 20%ofthe target concentration to qualify as the LLOQ. Ifthis criterion is not met, the next level is subjected to the same test and the LLOQ raised accordingly. Pages Northwest Bioanalytical Quality Control Samples ReSptourdtyNNoo.. NNWWEBRS9999..111136 Each analytical run will include low and high QC samples in triplicate. The measured concentrationsofat least two-thirdsofall analytical QCs must be within 20% of their target concentrations. Ifstudy samples require dilution, a dilution QC is analyzed in duplicate for each dilution level. At least one dilution QC at cach level `must be within 20% in order to accept study sample results at that dilution level. "The dilution QC acceptance was independentofthe undiluted analytical QC : acceptance, 4. RESULTS AND DISCUSSION Each accepted run met the acceptance criteria st for the calibration curve points, for the lower limit of quantitation (LLOQ) and for the analytical quality control (QC) samples. RIuDn PRResOulSt FReFsOuSltA FRFesOulStAA RPesFuSlt FROesAulAt MRSesSu6lt RMessuilt Comment T accepted accepted sccepied accepted accepied accepted seeped|FSAE 2 accepted accepted accepted accepted accepted accepted accepied PSAR = Pre-study assay evauation There were no known circumstances that may have affected the quality or integrity of the data. Inconclusion, 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 allofthe EPLSwed study samples. Please note that the results for analytes M556 and M570 should be considered unvalidated data. 5. REFERENCES . 5.1. 3M Protocol EPL-Swed, "AnalysisofFluorochemicals in Individual Sera from Sweden," August 24, 1999. Page 10 Northwest Biowalytical ReSptourdtyNNoo.. NNWWBBSR9S9-111136 52. D. Vollmer, "Quantitative DeterminationofPFOS, PFOSA, PFOSAA, N-MeFOSEOH, N-EtFOSE-OH, POAA and PFHS in Human Serum byLC/MS/MS,"NWB `Study Number NWBS98-082, NWB Report Number NWBR99-005, 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 submissionofthe final report to the Sponsor, remaining study samples will be stored under required conditions until confirmationof Sample Disposition/Retum Authorization : is received from the Sponsor. 3M Environmental Technology and Services and 3M Corporate Occupational Medicine will be notified concerning final dispositionofrecords at completionof contract obligations. Page lt tn roor vmoms Table 1. iin stom Calibration Curve Summary for metons PFOS in Human Serum [[resoo]s]aNumgber[[o| +ws][o*rn [ owas [|oowmnearl|i5i1ao|a5i0on] r i T LowT es[woT w [vasT e |omT|T || CA tT eet ner emTT `Table 2. Calibration Curve `Summary for PFOSA in Human Serum al cs `Quadratic weighted 1/x. All concentrations are expressed as ppb. [som]Numbe1rTow1 voi |[oIma|T wer| oT v 5 | CT Tomo [ousT [oweb |omerT ||T| ptr ere eon Table 3. Cmte. Calibration Curve AtoSummary for et, PFOSAA in Human Serum alINumber IE I I es viW l TrTT owomn wos[T T ooie|om . [| BS -- ' Northwest Bioanalytical Study No. NWBS99-116 `Table J 4. Calibration Curve Summary for PFHS in Human Serum [| iesm95]|s> Numgber |[-oo]mos[vo *[[worse[|roswsna|]zoora|]wro|g CSen]T[T woos [owe[oT res own|T || arT eT s mret teatmor Table sett Abe 5. Calibration Curve Summary fornto POAA in Human Serum ||| Number *[] [renaloa]wod) Siem T [ovotT ms oom[mT nomT a |||| r mitert ie tere Table eter seme 6. Calibration Curve Summary for M556 in Human Serum [esaols]= Nu2mber[Ioom oI mnesI[s ovos |om l |i | | Tr | or s T zT T Trm eot m na er ieeee] os spn pwer Table cmt sr 3s 7. Calibration Curve Summary for ment M570 in Human Serum mse Run A C R-Squared| LLOQ os Number ow a E steve en vei aw pe m `Table 8. Back-Calculated Concentrationsof Calibration Standards for PFOS in ti ae--g [[rimeDsuoe|un3Number[] w35n+[355040[Ta2na 556534[Ta2na[|1130 3o30s[s0o [351] + [r wel TameeT[mrvoen[Twrr sarsaor[Teeaar sm[[er so5aor [55 seovl TInnee [nwe[eai0][oas[w3a0[feus[mnee sr[s]o C [me TTeos[0a[oo taaiolTaorl[aasiles a[]a2a]] US. `Table 9. Back-Calculated Concentrations of Calibration Standards for PFOSA in Cmte. mt Human Serum st [iRaoI mps]ru3 nNomsTovaow0e[l[5oe0o0[[[1oe0vn0a[[32a55a0an[[s3st100ss[]atvoo n[r235a80s[rstoo[55r0]| v re Tn Taon|T vo on |zeT n[im [ova | s sw] [[iseo [Juwies[[wwee[[wwec[aaam[oooniissss[[nnec[srio]os6]] Cm o Tel [la0[eo[taaoiaas efase]i1aa[55t]3]0] : ' Northwest Bioanalytical re Study No. mee NWBS99-116 "Table 10. Back-Calculated Concentrationsof Calibration Standards for PFOSAA in [S eon Cie int [Some]ve [cos[i[so Human Serum tn 7 [r ieSemion| 3T|wir [svwoes or s[weoar x|220r sr 0[[ssuesr a s5ersoorss|| [ (sowelal T[vioT w[sxweewe[loaamioanff[o5aeaf[|woavw nee[as0ofsr ovTeTonrlonv]r| C [i saao st[waat[a20f[asa teartaaslee] eo 58 emeoanet ee Table 11. Back-Calculated Concentrationsof Calibration Standards for PFHS in [iormC]mateapoe[ss [ tn | `Human Serum e r CC raln Tr [ors oesT r[[nmmsri[aa ur s[ooser ronr [[3om]| [ (oseo mIInno eolwsweael[oaoo aw|oasmsa[[oaomr es[[rrraaetl[leemr] [e[5eort56]]] T rb 5a TteeemfaterT)aTe] I sen -- "Table 12. Back-Calculated Concentrationsof Calibration Standards for POAA in `Human Serum [oSmeemo_m3][a7waT|sao[o3s]5525[ooee]2 [5 7] Towle vanseses[uo aw [os as] (ees nv o TTTnNeesl T[wweee[[aom ieaotn[s1[eo2ers[[7nT7oe0rom9wee5inn]] Cro m ToTol[aaT[aarl[eas toaz[taaloTsa[lsasie]] Sabet i3eee [ene me] J [sl[ar] Coot er `Table 13. Back-Calculated `Concentrations of Calibration Standards for M556 in `Human Serum osm3][|eoma[l3][owmiea]semso]]snoso[[osnia[ [av [o5 o 1]| w sveonl [o"e oen[[[swwaeeo[voeem[[iaoers|sofoe ursse[[ooovooaoa w2oa3ra ]i77s1|]] C mal T Ta[TsafaartaaeetTaas erT[aatr ise Srp ---------- re Co Northwest Bioanalytical ems Study No. NWBS99-116 Coit. Aton ete Table 14. Back-Calculated Concentrations of Calibration Standards for M570 in Human Serum [[iotwee|Tra3 nmbr]1000J]ssos |110600[|73051055070i[swn[5o0e[a55 1|| E vel ToHnS nin wA os[om aa[ous osA[ses| ew os wo[on[oe [50 | [s sreadl oveaTfeal smroe[naweaeJoosmsoa[[ae5se as[snsee[oawosos[eoansa||] C"So ette meT as55etee n0[0 a Saea [rarT0a)ls] em Table 15. Analytical QC TOS Summary for PFOS in Human Serum T C Cain E T we owwe]] Run Date |Run Number| Low QC 50.4 ppb High QC 380 ppb svoell ww |awem ] [ [eo ewellrTia we oer| a |aioe ] n]s] CTT Table 16. emoar PrO% Analytical QC Summary for PFOSA in Human Serum Run Number Low QC 20.0 ppb High QC 350 ppb. A I esollT aw we [e erleile soeseos T|iw s]i] C To T mee Table 17. Aro----r---- Analytical QC `Summary for PFOSAiAn Human Serum Run Number| LowQC 11.7 ppb High QC 189 ppb ww ew a t e rl e | a nsa | m] i] W To T Table 18. I . Analytical QC Summary for PFHS in Human Serum Run Date |Run Number| Low QC High QC 21.6 ppb. 352 ppb. T TT m ow vseo m w|a e wel a a C ewT ww : ' Northwest Bioanalytical Study No.NWBS99-116 Report No. NWBR99-113 `Table 19. JA, Analytical QC Summary for POAA in Human Serum IEE REEe F ETET 24.3 ppb. 354 ppb. r wT a me ow vseela we E[rE eo laTa0e T |aT 0] 1 Table 20. eam septa Analytical QC Summary for M556 in Human Serum [[eFsee Tomwm2o0.r0epepib|||e35eo0 p]npb]| T T e T wee ewee e s vel s e er ae a ET E me T T ae1a] emesis mons Table 21. arent Analytical QC Summary for M570 in Human Serum Run Date |Run Number| Low QC | High QC 20.0 ppb. 350 ppb. : I 1 E TosY we] elm we ala a CT TI Te J. zz i EE] 1 1, i |2i2iz|2 SLE 3 282122 SE 2 2 sz 2 22g Ee oss ees HEEHEEEEEEEERERREEERE AEE na ene SSE EE ee g(g|g g g/3/3/8 HEEER J 3 HEHE HHL EHLE EE |2|5/5|z(2 2 2525] os|<|g o|z| 2s5] 3S EEE ee ne Z HEN zlg|g|1gls 212g] Il: SiE d EL EREEEEEREEERERARARARR iz i S HEBEEHE EHaERnEeEE REnEReOR z Pf 0A 2ic: i i 3i | 0 : lg2izizisic)s2/|s25)|5g| 555s 33 EE EE see PEEE as