Document QJKVw5j6q0VaRRweYXMNeRmX5

ARRRG _ 31404 --~ 8 9 i - 000075 | J lmD || | || i ARBKAG - 3404 METHOD VALIDATION REPORT TITLE: DATE: MAemtmhoondiVuamliPdeartfilounofroorocthteanQouaatneti(tAaPtFiOo)n,of (MPeFasOuAr,e0d.0a5s ttohe10Peprgf/lmuLo.roCoocntcaennotartaetiAonnioRnange), in Human Serum by Turbo Ton Spray LC/MS/MS ' 12 July 2002 REPORT: 02120VDJA_DUDOC Far owe] AUTHORS: David J. Anderson, M.S. Kimberly L. Norwood, B.A. , JKeantnhiyeJLo. RPaolmmneery,,B.MS..PS. PREPARED FOR: 1A5dvCiaotnheBirowSocoidenRceosa,dInc. Ithaca, NY 14850 DuPoenritmCeenntatlral RSetastieonarch and Development di ui 1985 PNAUGMEBSE:R OF 54 `CatipanySenlized.DossnotcontalnTSCA CB 000076 .? PD | I~ | | | i | 8 AUTHORS: TITLE: KDiamvbiedr1l.yAnL.deNrosrowno,oMd.,S.B.A. KaJetahniyeJLo. PRaolmmaecry,,B.MSP.S, `PeMreftlhuoodrVoaolcitdaantoiaotnef(oAPtFhOe)Q,uMaentaistuarteiodnoaftAhem. monium S`PCpeorrnfaclyeunoLtrrOoaoMtciStoan/nMcRaaStnegAen),ioinnH(oPmFaOAn,S0e0r5umob1y0fugu/mel.fou ABSTRACT bAysAndsviitovneBisocSlceiteinvcee,sa,cIonuer.th,aacned,rNeepwrYodourctkoanqaulaynttiictaalteeatmdowns developed hpuemrfalniosreorouctmaszacmaptele(sA.PFPOF),OAmeansdsiesdinsttemhalpsetarndfarod(r1)owoerceaitnsioaloannt(eoPdfFnOotAmo) in threuomregnasncircumaysearfmrploemsca(2c5hs1a)mbpylewlaisqueivda-plioqruaitdeedxtadcytinoen spr,orceecdounrset.itAutneadlainqduot of (alyLzeCdbyfIirnMothoeSnnesgar/tyivMeliioSndmoc)dhreo.mTahteosgsrspahyydfaendmemomanssssatpleosrwteraromlteitmeeiy odf ufoamn0t.0i5agtomfi.0o.0n05 p10gpgh/ufLom.rPfoRLcOPA.F.OAT.heTcheaoleifcuibrvoecfrrdaneiagteenwtnattsiiqosunaodr(a2n)tiocf hocalibration carveswere209950 or PFOA. OPr0ec5is,io3n, aannddac8cugra/cmy.qPuaFlOiAty.cTonhteroilnt(eQCa)nsdaimnptleer-sawsesraeyprperceipasrieodna(tCcVo)nrceenstirstions : inlteearalsaseadyfcrcomuQrcCys(aRmEp)lecslrealgteeddfrroomm Q2.C07s1u0p7l.e7s0%rafnogrePdFOroAm. T-h12e.f3r0a5.s0nd%. forPFOA. sTohrsrgeesufulotspfr0P6F5OAhouwrese rreopormodutciebmlepuepron.rTinheecetxitornacotfpironepraerceovdesruymopflePsFaOlAr and theISfrom humanserum were 81.8 md909%,tespecively. Themat offct [ cnsovoun puoc 000077 | onthe oniatofiPFoOnA andtheIS espetively. wasanenhancemeonft1.62and 8.075%, ' || | | rerracse cronpose 000078 TLE: Report Number: SIGNATURE PAGE MPeertfhloudomVoaclitdaantoinoenf(oArPtFhOe),QuManetaistuarteiodnaoftAhemmonium CLPoenOrcfMelnuStorrMaotoScitoannRcaantgeeA)n,iionnH(uPmFaOnAS,e0r.0u5mbtoy1T0 upgb/mlLo.n Spray 02120VDJA_DUDOC Reportedby: f Bh ) David. Ms. ResearchScientist Iyuiez. Dus Authorizedor 2 | Yh fu / D Releasoby: iclE.Malvans,B.3. Director of Bioanalytical Science: agli | . Seomortorin: Ph 4), Th dor. Marylh. Kaiser,PLD hd `DuPontCorporateCenter for Analytical Sciences Pastors voi busoc, 000079 | QAU STATEMENT A Pt thmmem. t tttmst `BioSciences, Inc. (Advion). Rt = -- [Dataview 6r7yi u)e 007 20Jn 2007 Basedon theinspectionsand thedatareviewed,thisreport is acompletoand [=x Amedro, B.S. Date `Quality Assurance Auditor wo -- 000080  D i TABLE OFCONTENTS SIGNATURE PAGE QUSTATEMENT css sss LIST OF TABLES vss LIST OFFIGURES vss 8 Le INTRODUCTION cvs hse 10. 23. SAMPLE PREPANADEXRTRAACTITON IPROCOEDUNRE ................. 2.4. HUMANSERUMVALIDATDAITOA N weve 1] AL 25.1.Intra:adEMEr-ASSRY PICCISION.... vores 12 2:52. Tatra 0d IN ASS2YACCURCY ress 2:53.Sensitivity: LowerLimit ofQuantita(tLiLQo)n v..cvcccuvvuro 12 13 2:55.CIOMAOGTAPHICCHITYOVER csr 13 2.56. AccanduPrecrisioanOfDcIONy... 246. STAOFB PFOIA INL HUMIANTY SERUM. 14 soso 14 27. REPROOFDREIUNECCTINIGPRB EPARIED LSAIMT PLYES... 14 28. 2.9. EMXATTRRAICXTEIFOFNERCETCOONVIEROYNIZAOvTFmPIeFrOmOmNAseAmNsDssSsHe-sHsDssFsNsAsImNmHmsUeMsAeNsms so 4. 3. REAS NDDU ISCL USSIT ON S cocoons 16. 3.1. ASSAYEVALRUESUALTSTc IONco 1 3.1L.Inte:anc leeASSayPROGSION. co. 312. traanc Tler-AACSE$IaY corse 1] 17 Pegssotse @ia0voin punoc 000081 | S13. Callaion Cuvier 17 & B3.L1S.4.. SUNY co Sensitivity:Lower Limit of Quantita(tLiLQo)n ........ 316. CHROMMOGIPE CATING SS-- 181 Ee te 3332..REXTREACTIPON RRECOOVOFERRYEDNJc ECUTINGCEXTIRAc CTBEDSALvtiYso s.. 1 15 54. MATROX ERECTON NIZATION OF PFOA ADSHLD A mrs be > 10. APPENDIAX:QUANTIOTFAAMTMOINOIUNM P C PE ER RF ELLUC UO KOAR NRGO E(OA,AO NAPIOFOOCE N)C,(T aMTTEOAAAASS,N U0RN.oO E05DOTmA AoA SAe1TT0L pHTEoErEn.o rs son poo 000082 | LISTOF TABLES TTalbele21:: AVCconrtaeSaaRmnlpudlnePrsDecEifirsSoimopTninhfOehneesVP.aFl.iOd.aAtcioAcnsRcsysin.oman SeuoforrQcuoay_2271 abl3e: AcCcuaracly abndrrcuiionooofnmtThSherPteFeOaVA nAasdalyainltasnann Sceuef.r .. 23 Table 4 CallanCarve Paraeesfo PEOA inHumanSera fo Thr Table: LowerLiitofQunaion Tabl6e: LoverLimitofQuaistion of of PFOA i Human Sra... PFOAandSelvinSi Separt Los 25 | Tabl7e: SAacmcpulmeosyfaonmdPOrnieoVnaolfithdoaRPntF.OiA.oA.nssy inHamanSeu ionQC 7 Table: SRteofrnaegcetiionnRReepcrosdiciiboinlySlofaQiCnSaomrp6l3eHoCuorsnisRionugPnFTOAeAmfpteer...28 | TTaabbllee1:0:EExxtrtactoionnRReeccoovveerryyoofftPheOtAfaolm SHtuanmdaarnd, SLEDFSNAe, fuo.r .Hu.man 39 D Tlee112:MMaatitllettonnootaiioonn ooffPEONDEiNA nmS S 3 | [-- cmonpuso: 000053 | LIST OF FIGURES Figure 1: Full-ScanTurboTon SpayMassSpectus Of PFOA...33 Figur2e: Pull-ScanProdMususScpecttruimOFon Figur3e Full-ScanTurbo fon SprayMass Spectrumof PFOA... 34 HDF... 35 Figure 4: Ful-Scan Product lon Mass Spocteum of HLHDENA..........._ 35 Figur5e: SStealencdtaerddRienaCcotnitornoMloBnliitnokriHnugtCthSreorummatEXoTogifcr.aP.Fm.O.sA.caondntehe Interal 37 Figure 6 SSetlaencdtaerddRienac4tHiuonmaMonsSiteoruinEgxCthrracotmCaotnotgarianmisnogfnPteFraOlaStAatndhaerndtOenrlmyal Figure 7: SSetlaencdtaerddfRreaocmtiaoSntMaonsdiatrdai|ngHCuhmraonmaSterougmraExmtsorafctPF0O.0A5apngdmth)e Internal 39 Figure 8: `SSetlaencdtaerddRferaocmtiaoSntaMentdiatrodi9nHguCmlarnomSaeirougmrEaxmtsroafctP(F1O0Apadnth Interncaoe. 40 |D | | Paesarss avonpuso: 000084  1. INTRODUCTION "deTehnpmuirnpaotsieonofoftshisoepnrtii 0pedfeusocrroiobctahmeomsteeth(AoPvFaOl)i,damteioansuorredthse qthueanti pmeertahoordovoacltiadnatoinotnewseironto(vPaFOlAd)t,e ianshuupmaen psrerpuamrsiaomnplperso.cTedhureo,bjdeectteirvemsionftthhee loiwteironl,imdietotefrgmiuneacnoivie(ytLsoLndQn)mraotuitifnietanaalnydsios,pdreomviodnessierletctsivcee,uarcccyuorafte ad rmeaprsosdpueccitblreomqeuatnrtyi(tLatCi/vMeSr/uMiS)l.byThaibs owofrkn wsarsycoindiudctecdhrinotsocgsroarphdyalwnaicneden Advion BioSciences I. (Adio) SandrdOpertingProcodus (SPP. 2 EXPERIMENTAL 21. CummaipcMATaERrIAsLS Penadecalfaorooccando moni sit (oth 4212071) ws tained from ~9 `9SigEma-Abldriech,xSt.aLouids, MoO.cc`iTdheOaiFn-telHrDnaEslNsAt,aoNndoarrLdo(to1hS)a,nvilaoble)nw' aas onano frfooumnSd ig in mAap-peAolddirxiAc.Sht,. Louis, MO. Adetailed listofchemeindmcatearilalssis | cionotcrols(dQwCo)rskaimnpglseoslfutoioPnsFOeAdwerepprreeppaarreetheaadreastcriiobendciunrAvpepsaenndiqxuAa.l | 22 LOMSIMS NsTRBENTATION he uidchromatography:spectrometry systemconsistedoftwoLC-10ADvp . || pCuolmupmsb(iSah,iMmDa)dz,ya,CPeoslkuimnbEiiam,eMrDS)e,raen2SC0L0-s1t0oAsvmpplpru(pPcronitrnolBliere(rSChoirmpa,da, | Norwalk,CT), Betas Cocolumn2x501mm, anpielsize, "CThhreommaytopgersaplh.yKUeStAonIen,, BLeaklelwoonocd,, PCAO)),,aamdo4dSeC7I9E7X0 cAoPlIu3m0n0h0emtarssones | -- caravan puso 000085 | & ctor (plied ioSems, Conca,Ona. Adet iof instrumentationandinstrumentconditions isfound inAppendixA. 25. SAMPLE PRAPARATION A EXTRACTION PROCEDURE WFoerrecapchrseupl1idne,bpe intAp2p5e1l Al.quoTshofcalbesiconleorenccuorvne stamiplnes Wer005,01,025,05,1,2,5,75,ad 10 pgfocP.FOA. HuomnancsenQoCcs11po5l,,n0ws4erppemrPdFiOAdfoo cQC,f0h0%,linQCo3,smyci, || caoasncodeentntarialteidoinneAxpcpeeenddiinxgAh.eQcCalAibr(aSti0o-ngc/umrLvedialuntigo,n aQnCd)wwasasspsraeypeadresdiantgaa 10-od. | 24. HUMAN SERUM VALIDATIONDATA ls J Th ta weecolt ings aonmori RM)to rey LCIintMhenSegat/iv fMonmoSde. Pak rca wretgrteby heSt a roam Mb avcasQupse,ovra ersioniLnG,gn recsiedingon810cMoarienrohcomppruetsecra.cBfPtROnte | cointolnsegnrd bocrkpoduiendseobitnsAcniponp, esneA(udA1si40x)b,lesFM ollo oewnitu wnweegsr nsve ote ls nd plod 0hAdio ieee i.(hi le ss, | W+weieghteid61(.1n050)3g(udhrebasoi.Pnsawpaipeant,o,mPeAd).Ailn somscdf atptess idhoevnxlcopatdionfroofQmWCLaLoQ d0 Sce 97lwisth,cweidcehciwrespcdeofornieyoown Tsoumdiang,lCaocsn.cnMtottioo,nCdVatvcoince,ntniRoSnvmales ws ltd wl and Reiwts Homa sr liso restoredin Adio Ntebonk0212, roundedtothreesignificantfiguresinExcelpriortoreporting.`Thedataforthe str p---- 000086 @ AClolncsclnerautiioonnsswoefrPFOaAsidn QotChseapmpelseswrceaeraetitoeomfinPeFdOAbyfitnhvneseparclicstitondr,om the calibration curve. 25 AssavEvaoamon 250. Intra and Tter-Asay Precision Then andners poisonofthemethod was sesbydetermininhge Q`dcCeoteefenfivmceiilneenatdonbdfyveadxririvaeitdsiiinongngt(thChVs)etoiabsdteravsdeedvpiinrstctiheoetnabanyagly.tshiTse.mhoeefiaQntCcossamnaplycepse.r(eTnhceifsCoicwVaoarwsnas exunp.rTehsesinatsert-haesCsaVyopfretchiesriopnlwacstexapnarleysssesafsahQeCCVloevfeltdhiovneralslimngelsemvaolfidtahteion QinCdi1v,idQuCal2re0pldicQatCecwonecerntsrantiodnsoifnethpelQiCcstamspiloef.s fIrnties vialihdoean mtieonm, s.sy CV was als report orcairostandards a levels. | 252 atra andnerAwayAccursey | Theraad nessucofthemethodwassebsydestermeinindg he brelylevaetsli,vdeieivrirdotirn(gtttRhEee)nicoobimsintearblveydcithneotnhonema iananlna vfuool emf,ty QahCedssmatemi hapnelescsc.xornTcehnseaiRtnEowtnhaefsrodreeatlecrghmeiQ.nCe.d | pserclenstageo.f QTChsainmtpenlsedeurinsgcnscicnyglweavsalcidrateisons.LthtREsaofysheomayaepliste. xparsheReEoftsheosvrmeanoftheindividu replicate concmtationf tsehapelnQidcaCarsideassmotpfaslxel.sflvoTlrnst,hareievoa,litdeatnriuonesn.QsC1R,EQwCs2,lanod QreCp3orwteerd eoraessaatyehdtion n -- avonppoc 000057 | H j-D | | | || 253. Sensitivity: Lower Limit ofQuantitation (LLQ) aAhsuampanisnerrpulmicQtCeswfaisxr.epThreeLLaQcaosncdeeirtactionneoby0o.b05taginnin,gPthOo Abacakn. called concentrations L-- fom hse QC semples. Th mean concent, CY, nd 254 Seteeiviy iTnhtescrcealccetfrioovmftthhyeebaisoslaoygiwcasldmeatetrimiwneidkibyrLunOs,MfSoMrtSi,fieTdocmoonneibflor human rum samples wer assayed with ll experiments exceptrecoveryodmati fs. iJahad1d5it)iwone,rixexrterpalcitceadtie 1eachsoafmtphleet(wcoondtirfoflrbenltaokthoufncosnetoulssearmupmlaesadrfioretdhe standard carvesndQCsamples. Thesezerosunpleswereuf ebro subtractionduetthe presence of POA i thecontol serum ot (Section A. 14.0), v"eTotmpolneodfiollosPpFeOcAitfhmtLQoaerdiaenpcee, stixosfeppaatfsofbGmpn ser copnrceepnatrreadtfioonmesvcehslont.dwerealyzedslongwith contol blank andze sample 255. Chromatographic Carryover "Th camyover ofaltefomth jection ofon sample othe wasassessedin : 1orhmatbtyanldai5nresoponnsoe: binsat xhhighstndd (STD) mfg reas amv puso 000088 | 256. Acearacyand PrecisionofDilation @ Theaccuracyad precision ofdition inthearly of PEOAinhumanserumwas sss. QC samples (50 pgm)wer analyzed at 10-6 dition replicates ofsin. ThemeanconcnttioCnY,adREwrecalle 26. STABIOLFIPRTOYA I HUMAN SERUM halyseis siallobifePaFlOAsinasbndurerseaposdrterirntihneg11sa1m0p0lensgtocragoe,nprcartstrioin raonadnnge \ human serum method validation." 27. REPRODUCOIFBREIINLIEICTTINYG PREPARED SAMPLES Tihnoveespteibdy iuncofciribileeyonfepxtrracetevd ainoruecsonssiatdtidndsseraunmdsQamCplseasmpwlaess that thaedQbCensasmoprleedsawfer nsjcctonsastosopmrotceesmpersaatmeplfoerst6a5bioliwtys.inTrheceoCnVsianionRnEdof ? solution. AllQCsampleswer aseyedi epics of si. 28 Exmaacrion Recovery bTuhtetdhedettaeyainnoetfdfexiotmraoctthionosnercexcpoevceirmyenwtasssieballvpedrftoorbmeedaisncRcuunrst,6maanidnl7,y douemtportonceidguhreearfttheanceoxepcetcitoend faobs1o 0alciqouovteresmsovned(fr1o0%orgaasniom yeerxprecntegd coenxctreancttriaotni.onibetswuesnpcthtedstahleesssapuikedfbtehfolorrgxtdritfoeenn(crees-(<1xt0r-afctldai)nndthe oaomnplliensasrptiykseedcafitnrdcetxetcstotrornes(ppoonssets-,xthaiscpt rinothcoesercexcpoeviermieenstws,ecroupilseedd.wiTthth 1c0x-p6cimteontcwaos emppefeeoitnhReasn10asitlwuhetic,h tAhepocsteoxtrantcht sciaomnpclenenstrwagetrieonldsoifeyhde | Page thotss onovoupuboc 000089 | & rVsdispeobtyxhetotnsaamprlyes.woldowbe uch ose xchat nd has pheortcoxmtirnagtsonpactoewvioefPgrROh(y1Atfoesmaonsuoshwerse-ixn ipnMaccxuen y c`samiplescwhLiccrShvwevrS orptrehpaprsedtatE thte noumisna,l" cTonhceentrwatsions--s 0i--.15p--, 3s--, an--nd--8oganl.firn PROrordterminaion. Thheexktrsa e isso(PeARo)ftphe oxm amanpstenrwth e slst8pit, c 1SH5L3HDEtNrAa)ssted inadevpstkofdve.pootsis txiteioon fnoeoySC, ItOoRPNFAOA ws ictovienrgy dsermatint, PTAhR xyhipnstcovery POAkRRaecdoverrysws dettm by percentage. . ||| > | | Ths itn me FRA fn mc ms 29. sna MATRIXEFFECTON IONIZATION OFPFOAAND9H-HDIvFHUNMAAN by comparingthepeakareaofsamples (0.15, 3,and pg/mL.forPFOA) spiked in bsularnknehumfeaaontssetireeu)pm.oeTxtthrcoacmtt(apoipste-cfxtsrcaectv)adwsiitvdhietnchgemtpieneakdaertteybahof aenamunexthrpt oacektetdptekc p[ec--an--and--crielnta genea. Five plwesmst RT sSinedeitrh5mpigsbFycSHoDmPpNeAnfteecptkionofweitkhhpeneosfeontet f-- arsonue 000090 | @ ; | | pnreepxatreatiendespollutiiocn (oarftetievssei.e). Th post-extact and nest solo spl were 3. RESULTS AND DISCUSSION A qunitaive satya procedure sag ro onsprayLOMSMS in he negative ieoqnumieondeewntassdoervtehleodpeetdertmoimnaeteitotnhoefhiPgFhOsAensiinthivuimtya,nsesleercutmiv.ityA,ainsdtroefprtohdeucibility aldarustiireoscon inTabl|e Tahee =ul4l13cawnhiacoisafotnisburtyedmfaotsshpdeepsrotonaoftedPFmOolAeschuolewe(Fditghuoreb1g), Tphekloan roduc omsccmof PFOA showed the ba pes producto 169 (gure 2, Tehmetullca=n4h5owhoicnhsisraytmasus spehcetdraerofsoEnLteIDdFNlAseholwe(dFihgesa3s, 1 es llcan produto 381 Figare 4 specif HLHDFNA shoved these pis proto "1T5hienfohlulmoawinnsgcreusmpec(toivmei[nMa:H] ionswereusedindatssequisitonfor PFOAandthe Proa SHEENA wm a3os 169 wads381 aTehse SRhMiocnhrFowimgatnog5r.amSsRfMrocm arhprersenoafivmomcoantroetlprbesosenstaghtiavnrecaosnateolumhaexpstraact. sFeriugmsex.trBaocttchoFnitgauinrieng aonnldyt6hsehiontwerthnealpsrteansdnacredo(fzePrFoOsaAmipnleth)e,ecroenstheowanmina `serum. ractiosa [------ 000091 | Figures 7and 8are SRMchromatogramsfom represenativeextracts om HD calibration standards 1 (LLQ) and 9 (ULQY, respectively. IL ASSAYEVALUATION RESULTS SLL Intra and nter-Asay Precision Table 2presents theprecisiondataforthe PFOAQCsamplesfom th three QvaCli,daQtiCo2n,uannsd.QTCh3e cinotnrcaenatardatiinotnerl-evaeslssa.ypTraebcliesionpr(eCsVe)ntwstahsep<r7e.c7i0s%iofnordaPtEaOfAorat eslibestionstandard.TheCV forcalibration sandsedswas 10.0%forPFOA. "Thess datindicateacceptable OfPFOA in human serum, itz nd inter-assayprecision fothe determination Ontulbey.fiAvlesor,erepsulltsifoocfoQalUsyIwfeivreersuecpcelssifcuolafltyQpeCrs3epianreRduinnR5 uwenr3edcuaeltlaebdrodkuecnoeast. probable preparation cor. Thisi in compliance with Advion SOPs & 312. Intra snd Tnter-AssayAccuracy "vTaalbildeat2iponrorusnnst.stThheeaicnctuar-aacnyddattaefaorsathce cPFuOArQaCsc(aREmy)plreasngfeod frtohmet12h.3 0 R9E.0f0o%cfaolriPbrFaOtiAo.n sTtaabnldear3dsprraesnegnetdsftrhoeamcc3u.r9a9ctydoa6ta9f2o%rcfaolriPbrFaOtiAonisthanudaradss,erThuem. "Toes datindicateacceptable OfPFOA inhumansecu. ita and inter-assay sccuracyfor the detcmminaton 313. CallirationCarveFit "The calibration curves wee itby weighted (1c)quadraticregression. cCoefificiaentctsuorfvideesttoaetirsmntiicns aasrti(ohn) weorienT2wa0.b9ln9e540. for PFOA in humzn serum, The Pa 7034 avonpunoc | 000092 & 504. Sent:LoweLinkofQuantitation (LO) `GTeabmloesn5ss tadh ttahteo 0r.e0s5uw lptgsfas r.omsthaeQcCepattatbhleeLLLLQQfolrevPelFOfAo.P`FTOhAe.expTehreimCeYntwas 295%, ad the RE wis 9.73% for he LLQQC 315. Soetivity Tb he syr washtie gohloysnsln eicnieveoffd oPrFPOFAOaAa .hNeo1chirsomatcoonptspicsnteprfnlces were analyzed(Fie 5.Thecontolblanksdi control human seam. howthopresence of PRAfn he hsuebtrsaeclteictoinv(iTtyabbleet)we.enThmeaprriecitssionpr(oCdVu)cefdosthee PpOaAsraemspulsesfweasb7ac1k8graonund 521%fortheLLOQ andULQsamples spectively.Thecomesposncudminwags. 0.133and 11.8% fortheLLQandULQsamples. | 316 CuromsograpicCams seArcuhmrcoonmtoaltbolagnkasepRslilwoawsinobhsiegrhvseadndahredsr.Tehneirnesipoensfo wPeFrOAomitphaehrubmtlao.n ahyoooftshwacsoegntloibblsfo PjFsOtA.bCoatnyhovesftoarntdhaordctuerrvneasl.sTaidsiandwaiscahtte | insignificant, 317. Assarscy aud PreckionofDilation y `4T2he5re%s4ultstfhreoRmEQvCa4lsuamwpalsesd1ilu0tefd.1o0-6PfoFlO%dAa.rTehsohoswnditns Tdaebmleo7n.sThesCcVewas rests forthe dituionofsamples. Pastner rr 000093 L 32. RoRn en c Beacrao ssoiss oS "TSThlesr.esul0tTs5oofbrCo eirntejxtreaecstpecdarnetdtrecticonsntritugtthedysnnamtpls tesoconatapinning PFOA after cE A stesnoE ation strn---- wie TJ 2i2e. cDomcnnoosnP tiaocfoRnOyAsd-- , DA-- fr -- rn ROA mis HEF | i a : Tertt ht tas re een 7708 34. MATRIX EFFECTONIONIZATIOFN PFOAAND 9H-HDFNAIvHUMAN SERUM ` A 1S aednTt apy trent toon T-- eehnsern nsoirgoavAvriToinoSnOY DOCU 2, lune tsmb | forspikingQCsamplesmustbe<5%ofthetotalvolume. QC4(dilutionQC)was | preparedby spiking avolumeofstocksolutionthatwas 10%of thetotalvolumeso | J------ { thatnewstocksolution didnot avetobeprepared. Thisdeviationdidnotaffectthe Thrwr melonSderotion man aonsee 000094  5 DATARETRIEVAL TihntehdeiAtdavoirotnhearmchaivme srsvalidationsse ored i Advion Notebook02120and & concuusions pTrhoeveanrbiboonssepnrasyoE,CIsMlSMiSvasesarypr,ocenddurreefproorduPcFiOblAei.nIhsuimgahnsierta y ianlblouwms elselrumibnadsperdoond2u5-c4 tsqamupalenst.o of PEOA down tv \of 0.05 gin, J| . 7. REFERENCE 1. QAudavniiomnioRneofpAtmNmoc0n2i0u86mVPDeIcAi_nrDoUaDcOaCn,onMteet(hPoFdO)Va,liMdatuionrfoor thsehe FPuerrafSleuaomrboycTcAunttioaoTnno(oPSaFpOtrAae,yL1OtMo1S0I0MnSg,/m0L Cboncoeaetr,ationRange)in [-- avinpsec 000095 > + mamas Tar Vda a2T|rtaronn |Rt oeres | sa 2 || omrrm|| emr2m || gees P05| + | | nrsinne || e r. ry m ||t! pietostsh, 10-May-02 | Acc sdPurecirsiona;Selc ectiy vity| | Accepted | | i -- mos 00009 | @ | "Table 2: Accuracy and Precision of the PFOA Asis nHa umayn Serum for Quality Control Samples from Three Validation Runs IE PFOAConcentration (g/mL) T 0.147 273 aT CV (%) RE(%) 733 268 626 561 -123 261 Mean 5 0.157 277 Ta 0.169 296 8 0.162 288 EQB Overall Mean 054 276 Es RCEV ==([S(MDe/aMne-aNn)o*10m0 iNonminaal]l1)00 78 Gluuyy  P| pepzlezlapas {: |! ; | Ebb f sifessaas LL p|bpeeedhe | Pak = eREE an] i2 I CL=Enp mk | : silsalifas | lo cle & IS = = 5 HI EH | 3 EEEEEEgeng] E Ig "of gga . 52 3 g 00098 L& "Table 4: "CTahlrieberaVtaiolniCduravtReiuPonansrameters for PFOA in Human Serum from TanDie x CT] Coothcemior mbes Detemiaati(o2)n Far TTR| 0360080| 0006 |09950 BMay02 "000249| 0317953| 0000595| 09955 10:May-02 0008130| 0289836| 0000240| 09951 View |[00073| omen 59557 TASTER TCweigheTdY | ---- carovoi puso 000099 L "Table 5: Lower Li ofQm uanti itatt ion of PFOA in Human Serum wat, PFOA Conc. (g/mL) asQCLLQ oro ss0.0562 onsto =0.0532 ' CV (%) 295 T RRmE(d%)eE mi 9.73 a0 | 9 | | Page25054 02120VD1A_DUDOC. 0001.00 | o Table6: LowerLimitofQuantitation of PFOA and Selectivity inSix SeparateLots of Human Serum =Runs TT2248635 "PFOA Concentration LQ :(g/mL) ULO 0.0544 06 L. CV5 == (esoNomis (SD/Mean)*100 mie 100 . | | || ne ign BD 000101 I & | Table: AccuanrdPareccisiyonofthe PFOAAss in Human Serum Dilution QC Samples from One Validation Run PFOA Cone. (gl) Runt 50 QC4(1'in 10dilution) ws 7 43 #9 29 ) Vea 746.0 CV (4) 425 Tm RE (%) -10.6 CV =(SD/Mean)*100 | ~& ruemase cvspoo 600102 I @ | | L | { | Tabled: AfRetienrjeSstiton RoiepnrRroedcuocanisbtiigltiutyteioofnQSCaluStaimopnlfeosr6C5onHtoauinrisngatPRFoOoAm Temperature IE el "PFOA Concentration (4g) qct QC3 [QCA(Iidni10tion) TRm1 0.147 273 B47 0 oss | 25s | 3s oe | 256 | 754 o1s6 | 25 | as os | 267 | 237 ois | 268 | 75 Mean | 01 | 26 | 7m oven | 73 | 268 | eas REG) | ser | 23 | 2a Rand | 0150 | 275 | 810 woshous| 06 | 288 | sar EC a1 53 "9, 29 460 aT 42s 106 6 a2 ous | 2m | 1m 458 ua | 265 | 76 60 ous | 257 | 7s0 463 Mea | 010 | 2m | 78 Wx owen | sn | as | ex 205 REC) | 661 | oa | a 221 C= GDM 100 RE = (MeanNominal)Nomioal}*100 Puezsorss a2novoinpusoc 000103 L Tabeds Extraction Recovery ofPOAfro amanSern ORC GD oRsu2n)ge | a01a Post-Exiract | ois oottss | | ois | ooonuiin RiEG) | oSTms | o14m9 vclz I 321 295 340 338 , View S5o0 | foemd Sie woo | s5sm 7105 ~~ 8 pg/mL 777450 7 | faT7i1ds5 7m . RVor ToraiH TCoopnta sT Rewer DeteremSienoaterion| (|))| oo0o.z9n9z3ss0 || om0.eo98m7t8 | ws RPioBtE(iMxaena-WNMcaarxtot pkmaldoiAwidBFsPOR FiatoosrOoSnAsi Recover=y(Slcopmere/Slopeemar)a*10s0 | Page290134 120VDIA_DUDOC 000104 | & D Table 10: ExtractionRecoveryofthe Internal Standard, 9H.HLDENA,from HumanSeram PeakAreaRatio(IS/PFOA) Recovery ow5 sul | View | TH | OA oases | oasen ours | oss odes | oso omens | oamss 0a | 0mm | soe reBrot Mate sedwis SE FIDTNA rir Satin Post tact =MatixspikedwithSELEIDRNAate xtacton CV =(SD/Mean)*100 %Recovery = (Meantre cane Melina crac) 100 | | [er avo ousoc 600105 L Tle MaetoEO o mS | sar [riaJwe D) 0.15 pg/mL o| i | 19835 18856 ow | 20597 18333 | Mean 20183 19180 523 Qc2 3pg/ml. 426002 418407 399353 408972 | | o--e Mean 179 406010 389 ' Dow ; cs TA | 7 8pg/mL 747001 794112 Ss o araeoa s_| || soonz me OverallMean = 162 p Si e erYemen MCaVti=x(ESfDf/eMceta=n)(*M1e00ats ut~Mts32.YMen55100, ns npn ~ 000106 | | i| roars debs tiss l eSerdem ve]FE sndl Noomw Mean 341696 8.07 " Post-=EMatxritxsrpikaedcwitth 9H-HaftDereF xtrN actiAon ! NeatSpike =Solution of 9H-HDFNAwithoutmatrix CV = (SD/Mean)*100 Co`MwahterrixanEefgfaetci=tve[n(uMmebreorinnEdxiceacte~siMoentstupaprsepsasci)oMn aendaa npspoasici]tie1ve0::0, --. ns 9 000107 | 9. FIGURES : Pret: FlkScnTurk on SreyMas SpectumofPROS -- RET 9% " [0 " g" ] Pow iw a &w D u w " . ! . | | kf LE RS - S. ras avon occ 000108 L Figure 2: Full-Scan Product Ion Mass Spectrum of PFOA emmmosssaTCD 000 sion . . |e-n : || 1= . i. | "0 ~D - . ; , Ew-a sn FR 000109 i || || ~ Figured: Full-Scan Turbo Ion Spray Mass Spectrumof 9H-HDFNA Spectrfom FHLADRNAQ1-10OR e " wo us Less oe Pjo o I ie w 2 . w . 1 ; . i wm wm ns 0 [-- anonpune 000110 | | ~~ | | | |i Figured: Full-Scan Product lon Mass Spectrumof 9H-HDFNA. Spc"tunfo HLHDENAMSIMS PROD 10OR v wn 0 gn ]a io iw 2] u 16 s 63 100 RK jt 20 331 300 wi 400 sstes an ) . 500 rnsiars cazovoi usoc 000111 |@ bs > | ! Figures: SIentleercntaetd SRetaacntdieonn MionnCiotnotrrionlgBCahsrkomHaatromgramSsroafmPFEOxAeand the wenn 0 stn = w gw ii -- , Tonite an. oan a wo 2 z > Time,min TT Control Blank, Rua 1, Injection 7 msn [r-- 000112 @ - Figure 6: SIenlteecrtneadl RSetaacntdiaornd naman Monitoring. CShrroammatEoxgtrraacmtsCoofPnFtOiAnand the | | -- . oe .. : 2 1 m-ssonssion [Tr ~ ..;-2 E Zero Sample, Run 1, Injection 1 rene H--- 000113 N@ Figur7e: SelectedReaction Monitoring ion: ChromatofoPgFOrA aandmtshe (0.05 pg/mL) I i . v | e: w"0 1 la i essmnms amma i, ston |D .0 | || Y i) Sit nbn ron A 600114 .9 Figure8: InSteelrecntaeldSRteaanctdiaorndMfornoimlorSitnagnCdharrodm9aHtougmraanmsSeofruPm EFxtarOaucdttAhe (10 pg/mL) ama130-2 160,70 Sttesan || w " . i || i Tinea i 415033803,SADA 225000 Ig i D wo " | 60 | o . PF) Tetia 5 Sun5,Rduna1, cjectdion 16 [-- town suse: 000115 | FR -- @ PPEREFLRIOFROLOCTUANOOARTEO(AANPOIFOO)NC,(MT5E0AA,SU0RN0E5DO7A0ST1TH0EEm. sCmaOLoNws CRRANGNE)IINHUMRANKSERTUMBIYTuOgsN0 ATHORS: DaE Kvibdsae.yAenR Nonnwem5s3..0 Taawzorconta , 9 RAGSDD Mrmr COMICScosmos 83 RR8500. PP ups r ofQhtryi CSuondmaSn Sret paetnrose1y A10N0 LPteLsopfBiioSontrns.ss gayy rn anonoes BD ve.oi6 | L | | | ALO CHEMICAL STRUCTURES) MY+ To--o1c--cie Ammann Perotoociancale (4PFO)Cy 5NO;Monoisotpi Mass = 431.0 oI ` 0--C--CrFys. PerfiuoroocAntiaonn(oPaFOtAe) CoF150; MonolsMoatsso=p1i3.c0 vod_cxsoracrn SHeradocaranaAcnaGHnHOeFiNAc)Co Monoiclok Mo=s440s. reas aasowih usec 000117 | ThAi2.s0 254 SPECIMENS) tf rs. Hi sm slfosn sorsdat-20C. A30 ASSAY PRINCIPLE (PeSsHuLorwHeorsDioexotEcnra(cPttNeFdOaAAo)smah)tae,tns 5s,e9rFhuemxsaadmeplceasu(o2r5oinLo)auosionlgefaciidc. evmupiodrexstoratctoidonynpreocseducreo.nAsnisdli,unotdotfhtenhsoyrendabyyetrorf socnspamrpylleiwqauisd chromatopranpuhssypeactnrodmeetrmy (LC/inMtheSnega/tivMeioSnmo)de. A40 COMPOUNDS | Pentpeatsdoreoocciaanhosoes,irAdoamaPmocLnFtoituahOms4na2)lc1t2(i0:d7c1o, 1o0v0%apurimty,o09n58i2 sl ctor, L Sig Aldih,St.Losi,MO. . SE NGiFGa0n_:_ 4131.00620s 09% | SHCHDENA: "Mo. NoLot# avail, 100% uriassumed, ign Alc, St. Lois Aso cummcaLs - Aolfchcheemmiiccaalls aybesubsti withtaof32eile mamctrerand rode Tetabuslemmonium Bytropen Sulfate. HPLC GrCaatd13e604,7, 1.7. Baker Phillipsburg, NJ Sodium Carbonate,Anzhydrous HPLCGrade, Catff 32503-27-8, Aldrich, Milwaukee, WI (Calif 3604-01,J.T. Baker, Phillipsburg, NJ [-- crnpuso 000118 | |@ i 1 i} ) & i ace Sodium Bicarbonate AmmoniumAcetate Ammonium Hydroxide Water Motanol Jr-- HumanSerum (CCat 13509-001,t0.iT. Ba4kecr,oP,nhi,llipsburg, NI Muskegon, MI Cath24.019:2, Aldrich,Milwaukee,Wi CNathAXI3EM0Sci3enc-e, 1Gib3sto,wn, CMautskhe36g5o-n4,,MBI ur&tikcaonk, CMutshk2e3g0o-4n,MBI ucJnkoon, CMtushkeBgon0, Mi1Bur5dick-&I4ko,n, BiologicalSpecialty Corpration,Colm, PA A60 MATERIALS AND EQUIPMENT MassSpectrometer SDyutteaAmcqisiion HPLC Pumps LCPumpConoller Atosampler EPLCColmn SCIEXAPL 3000atmopsrepssheienatrioen {ander ripe quadnpolemass spectrometerequipped `with TurbolonSprayTM interface, AppliedBioSystems, Concard, Ontario AaLPdI4SSaMtuaanpdecarCDdoSnvoAtLfrtDoiwla,rve,1B4M, canQu3uoa1wd4ye,s1MM.6ia,liLwCace2uonye14, Gis, Applied Byte, Consors Ot ShimadzuLC-IOAD vp pumps, Shima Co, `Columbia, MD ShSiCL10mAvp,aSmdad aCo,Coal,MD PekinEimerSes 20, Pein ElmerCop,Norwalk, cr CeBeltlkal8o5nC2in0,,5o551Ap0m1,pTaroticc siy,pe2s5s0Kmonn,e, -- coinpuso 000119 & | ColumnMor MLaokdeewlo9od7,0,CfOons ChromatograUpShAy Ic, HaPruvmaprd11Syringe HarvardApparatus nc,SouthNts,MA P5o6l-yWperlaplyeoass Cat 267006, Beckman Instruments, ne, Fulton, CA SPoclayopwr-oapyplpeende Visls 1S6C.52861%081mm,Cath60.541,Sarto,Tn,Noto, FSoclryepwrcoappypiendo Vis 16557mm,Cath60542,Sarstedt, ns, Newton, SC | SP SiNGliicdctonnniibzseeds aLSpmLa,,CaCtAMH9-48541558,PhenixResearchProducts, ConniugeTubes Scniie Bdge 0 BPaobtptrsopyeae B1r7d5mgelpoNra,lg1e0ne, Cath 16120.953, VWR Sic | i wm ~ PBooltyptrsopylene B1rLidNgaelpogretn,e,1Cat 16120962, VWR Scientific, |! TEavbapooVrasoprss C[at471000, mark Intranets, Hopkin,MA | CReemtiitgomgised | Malt ube Vortexer Cat SSB6-115, VWR Scientific, WestChester, PA MechanicalShaker Cat6000,BorbachCo,An Arbor,MT !| Watson LoaubaorPahtaosroyCorfpo,rmHataioncMhaenTahgSeysmteemnv t 6.0.3, Othergralaborstonygsevar so supp were used, [-- cumvinpuso 600120 L |o | | 1 ! 3 AT0 PREPARATIONOFCALIBRATION STANDARDS Mixall solutionswell. Unle ambient temperature before ss otherwisenoted,storesolutions use. Unless otherwise noted, prepa at re 4 Candbring fresh solutions to every three months or as ncdod. ATL PreparationofAnalytical Standard Solutions Standard StockSolation,PFOA (500 g/mL): Weigh spproximately 2 mg pentadecaacidfamlmounioumsralotansdtcrantsfaertn81o6.5ix c57 pgolayplrospoyllueineovnifal.eDciolnuetcewtiitohnfaoppsraolptricattoevr.olVumoeroaffmoetohvaenomlitoouytieeyld a500- A72 PreparationofStandard Curve PrepareStandards 1through 9byditionofStandardStandard tockSolutiono he apprpreopparrinetdeisntanSd-armdLivntoolcuomnetrolfsekr.umacSctaonrddairndg1ttohtohuegfohllSowtingaabnlead,veSpatraornpdrader9d in 1.5m siliconizedpolypropylene microtbes. i Aualytical Standard Dilution Table: StaNnod.ard FinGaelCmo)nc. SaUluteion SoLuoiton jCioSnetrmol Tom 9 0 SdSeck 100 Addseramio VolSume 687 755 ssDO9 315 Sl1msark 0s 250 250 03 2 sTD9 100 00 . +53 015 025 SkTdD sTD7 5500 45500 25 475 00s5 0o5s 21 0001s tSoDsS 250 4o5s0 o0s5 Po tsarse voi pus0c 000121 | & | | ! ; ~ A80 PREOPFINATERR NALSATANT DARI DSOLOUTIN ONS aMimxbailltstoelmupieornastwuerleble.fUonrleeussseo.thUesrlweissse ontheedr,wisteonrostoedl,utpionsr4resChaspolubtariionnsgto everythremonsoras needed. ternalStandard Stock Slaton, IH HIDFNA (S00uginL: Weigh epoximatly 2mg SH HDENAandtransfer. 16.5 x 57polypropyien vil, Dilute with appropriate foroneminut volume ofmethatonyoiekld a 00-ug/inL souton. Vortex oIfnfttrneaallSSttaannddaarrddWSotrokciknSoglSuotliuotnionnt,oSa2H5mIL lAass5"An"vLol:umeTtrribcsfltaesk2.50ut. ctornatasitni1n0gaappSr0o-xmiLmapteolyy2p0mr.oofcpweayttieirf,gcDeitlnuubtsee:tovolwiuthmweee, Vortexsad x90 Emones cova ssa ASL PrepoafSreruamQtCiSamoplnes PrSeoplauctia5A0o0Lns.gnP.reQpCarSetQoCcskaSmolpulteisoin nian t5h-e1a.mveolmuamneatcriecafltahsekSbtadnidlautridoSntoofchk e Q(ComStaodcikfSeorleunttono,ttQCh3n,oruQseCd20nptroeappaprreotxhimsattaenldyar3dmLcu.rvoef)acoandtroilthiunmgatnoserum volumeacctoothre ldowiingnabgle: QCF Aube Cone. Gynt) Sowdonts Ve por Souton DietaFat Volum (ul) or oa 5 5 Wok sek 50 30 5 5 oz 5 a ais ws 5 os 5 ocue aos w1 s rare o20v08poo 000122 | | I D | | | 2"T0eaCns.eNro0t.e2t:mhLatatlhiequQotCsoLfLeQacwhasQCoinnt-otmiimcerpoceepnatrsiaftugoenatnbdewsaasnndosttosrteorferdoz,en a. A100 PREPARATION OFOTHER SOLUTIONS HMPiLxCllgrsaolduetiboontstlweedl.wDiafofserqetnutivvaoelleunmtesshooftulhd bseoluusteidonwsheinrehviesrsweactteiornimacayllbeed for. preparedif ocessary. Unlessotherwis ote, treat room temperate. Prepare fish solutions every three months or as needed, unlessotherwisenoted. 1075gMofTtetsebuttylaammboniuahmydtHryodgryeongselunlfSiautlefianmt4e,0mpmiLf.o1w0a:tneDriisias8l1eug7p5mrmox.imicly polypropylene bot. Adjust the pH to 10with 10 M and 1 M sodium hydroride. Transfeeto 2 100-mL class"A"volumetric fas anddilute to the mackwith water. "Transfert04 175-mL.polypropyblotetnlee. `w1i0thMwSaotdeirianma H1y0d0r-omxLidcel:asDsi"lAu"tevaolupmetipcflrask4.o0Mgixxsaodniidutmhmroaanittosat1efo7t5ehlmemlr,ayrk polypropylene botde. 1atMerSoidniau1m00H-ymdLrocxliadse:"AD"ivloultueme1t0rmiLc fl1a0skM. sMoidxiuamnhdytdrroxaidnetotso:t1fh7e5me-amrrLk,with Cn SteBnhst `polypropylene bole. ca2r1b-oLnpaoltyeparnodpy2l1egnesgordaiduumatbeidccbyolniandteorainndpdirlutoetxo tihemm5a0ar0ktmwiletwhalwteayrt,e.TMriaxnsafnedtro transfe0r 1.polypropylene bot, 1M AmmoniumAcetate: Disolve 7.7 ammonium cettetothemarkwithwater pionlayp1r0o0pmylle.ncelabsostt"lAe"volumetfrlaiskc. Mix and trantosa1f75e-mrL cssarst cova puooc 000123  ||. ~| D | || || mtAmmanAcetate ALS Lo Manionssit prim i90t0hmvLawta.teMriixn and carton 1 panos bo a 1000-mL polypropylene graduatedcylinderand dilute to volume 10:50Methanol:mt Arman Act: Combine 100m. ett ad T90o0nsmtLi2romaMLaLmpmoolnyiruempyalcaetsatbeoitnea 1000-mL.`polypropylenegraduated cylinder. 9[ 0:10Meth320:2mYAmmL oniaAste: Cabiv n300ml.tat a Tans 1.pobpropycas bot w19r4Aiso1l0o0mHcyarso"sdAevDeitt t3.omkL..sMoisninoiod t1os0m0aat0ith 001%Armonia rcwith wate H2y5d0rmoL.scdleosD"Ai"so25moL.1%maaskm. oMissaymaotronn 250 hs gente, J NE 80:20 Acetonitrile:0.01% Ammonium Hydroxide: Combine 800mLacetonitrile ~-D wn mnusec 600124 | A1.1T1o0 LIQUpIlDo-LdIQUsID EXTRACTION PROCEDURE 2. Prepacthe standards3soutlined inSeconA72. 3. sLtaabnedlar16,QCx ,10co1nmtrmolsbarnekw,caanpdpzeedrpooslaympprloep.yleSntantduabredss,fcoarrerxycovhesracmoplreo, alnaalnyez,eadnidn eeeplcitciavteisycoofnitxr.olSibxanrekpslaiceataenaelryzseadmipnldeusplriecaptree.paQrCesaieachof 1ahndthwuomadinffsreernutm0QsCsofamcopnlterso.lseu: usd orthehumanserumsapard cures 4. Aliquot 25kL tho spproprist ofeachstandard,QC, ube. zerosample, contol blssk, adsample ! cto 5. Add25 uL.of tealStandardWorkingSolution (5ign SELHDENA) oa | | tubes exceptfo blak. thecontrolblanksamples. Add25 wL ofwater to eachcoatrol | 6. 7. AAdAd 21mmLL0oFf002.55MMtsoediutmcrabaonbteu.h2y5tdrMoysgoeldnisuualmfabtmie,cspmbHo1ors0tnetotcoiaccahuchtumbbee.. ~ 85.. SAtdadk3emonLcetihpyrloacclestatle0rcfaocrh2t0bmei.tesst di pc. 10. Cntt320i0pmffor2g0muetes } 11. Taster 300-4 aliquot ofth ganiclaye 0 6-vel lock. 12. Bvaporat th utetodrynessundeironat 35 Cforsgproximately 30mousat amaximumpressuref0p. 15.Ror6e0seccondtosh. Adnrdiedds4e0x0trptacLtspiiinltewtodstuetpess;Aadnddvo4r0te0xuLors60 stendsao.ndviores - 14.Apply cove lm1 the96-vellbk. ~B Pesrst canonpunoc 000125 I I. D | | | | i y ~D HPA1L2C0 CTYoPnICtAeL INSTRUMENT CONDITIONS Mobile Phase (gradient) aA:ce1s0:90 methanol:2 mM ammonium 5: 90:10 methanol mM amisoniar acetate Mobil Phase Gradient Condions: @ 0s 23 30 3s 500 Flow Rate Autosampler AAuvttoossaammpplleeer NTeesmtplereaWtaurseh njction Volume HPLC Cola A`uTyopsicsamlIpnilteiaRlCuonlTuimmnPeressure: HE , sup 100% B too xp stop 300 unin PE Serics 200 (with 50-pL stainlesssteel oop) Ambicat h8y0:i2r0oiadce eon0.01%ammonia 106150 Belasi Ci, 5pmpatilsize, 2mm x Somm 4733bmari(nvuarsiable) SpHrEoaDsENA ntHl 2235 cesASAEne Pasi ase caavoinpuso 600126 & [| 3D | || | i `MassSpectrometerConditions: CurtinGas Nebulizer Gas Collison Gas TubolonSprayTM Temperature AuxiliaryGas SelectedReston Monitoring: UHP Nitrogen UHPNiogen UHP Nitrogen 30C UHPNitrogenat 8L/min SPHrLOHADENA Tonizasion Mods IonSprayVoltage: DeclusteringPotential 5 son ine CollisionEnergy PauseTime mnlizz==44451>3381619 220000mmss Negaivelon 3000 V ov Some 255eV 2s Calibrateth nas is of the instrumentbyinfusionofRhodspexmasscalibration siolnutfionuastoaiff4lo0o.nwr1 tgefof.1m0iuxledmsio.luOtpitonimoifzePFthOeAseansnidtOivFityHoDfFtNheAinsstt1r0umLenit unsing i808Bowof 19 WLminofmilephaseusingthe inlgradiatconditon (Ba4l5%heeliugehtnitnB)b.oMtah sinsgspeMcStraumnpdeMaSk/wiMdStmhossdheosu.ldbeapproximately0.6amuat cOalnibrcattioofncsatliabnrdaatridonswtaasnidnjaercttewd antheeetndoaftehaechagmilnyntiincglarao. nAddsitoofnal contolbikandzero samples maybesed 0 mito possiblecamyovesor rss crave uo 000127 | | L |2 | | | | : ~B pcloancteadmibnaotrioen.exTchhsitanrdeaprldiccautrevoefcach background sbicaction 10 sample re PAlulmpposlayntderterpaliacoerdowtiytlhnei(tPeTrFsEt)ilcoenstasitniengoxmaPtEerEiKa.ls TahesreismdoovneedtroormomthoneLC hes ems sspotential sores ofPOA. A130 SYSTEM SUITABILITY FsSpyosntsmssuiotfatbhileit0y.0c5ritger/imaL.ePrFeOeAstacballiisbheeuidodnusrtianngdtahredvsailihdautmioannbsaesamu,p the The nsdtiesmecsuiiotnabsislirtysmaamdpeloetooxthseamspelemapnrtiostaseapmrpelpeaarneadloyrscaancdhsbjtcytteo rhae, stldlicniseouilad b.eTchoepmeapkssha0pehantdobestcernvieodn times in forth anyan he validation,Tho tonal gal 4(0p0opnceihnood)eorettohpr0o.c0e5edgw/imth.ssaymptleemysusiitsab,lysamplesshou be isi of ermal CAlteioceaLsceuLnTtIiOrNs dnp ar sof 5. Tepe 05 orth analyte nso dividedbyth peakaafethecomping regression. Background Subtraction procedure: 1. uBsacek(g0ropruenpdarseubtthraecsttiaonnd2a2r7d0csuarmvpelsesrepirdeepnatriefiewdittshZhEeRoOn,tHroSlTDu,ssadstheo u ot prideepnatirfeidedwiaslhZtEhROo_HnQlC.hiTmharnesreeaplnilcaotessadre1p0lacreed pberfotrhee eOaCchssatmpalnedme are, 2PARfOtA/lrieI,S)tfhoedmatthaesisp2rocessasmepdilensMianceQsuano,fnthdettheimcoenstossenr oo(ecn ruses anominpupoc 000128 | | || | i] jc 2 --_-- Yiceladls lwaomnexa eareGaraomitsh.e dotthaectoheeMiccoQfiuvaerraienatsnitson b(lCeY)(ofDAamTefain)tTohi rTeestEexchladnat2a0a%eshaevneud tlersrovanliuaelslsye(rZeEmRovOedFal)n, tanhdemaheasdoiosepcyoaflthuettg dsocupmentratioons.haowidngtshe heanearcarattio,si incinlth uanaldytiecaldyun 3 ShenappldicnaabdleQmnCeastanmaplrseesr.atBiaockcgersoutnadstuebtdracftrioomntc herwlsenarrieponoershte in {he EdaitcalinithneMfaocuQsuainrgessuilte(DrAtTh mean res to,ndsu wing fe). ThExce duamrsage cdrlogceoumcaeltntaoantndiotinh.eaSbalUcykg1r)o,u nsdubaraacterddadts,oithipensclyurdeodsi f,the sahlyyinhgae m hebckgrou (DAT fle i ndsubtracteddtarcops othooriinlMacQu Excel withthe background subiccted ec tio in t he ests ie coplalcofeutihomDA1nT5fAler."ThceoElxucmenlinstharDoAfT"plaest,eTvhialmulesgsei, Anpvreago dcaaotbmaelca"te0bsa"acvikesgdcrnoceulerncedcosnuiibcaitlhcliay(en_daaSlUryBa.rDraetaocofolrutmhaefoWractosnotrnoLlIbMlSraeg,rTehsseioEnx.ceTlhe Theesbadckagtrwouinldl gspubetrcitnetdhedhartd(cSoUyBAp.TrDifAnloTu)tlbeuo)tmanorWtiuptprslioonsntd,eIdMtoS(WatsonLiv, InformaPetskiAovens):. ent tThehielelserevenrinltehseaLrIeMpFlIacLeEdSiditretchteorpyp.opeite der or thentti mon oin(5f1.i4o) rmaactainaodncMmaiancadgQenumdea(nnt.sg1.r6.eTwhidnagtSaCaIreEXfsoromfattwteadrei aptphleiAcdatviioonnsSaambpolrerCyool ytem (Watson, v1.03). eo sonccauionscab ial factor, prs 8 APFObymulling thePEOAvey Pastorse vonpusoc 000129