Document mBXVkNno74X92D7n2prOJqXgO

SEHR _ 0800 _ 373 AR126-0962 Comte; yy By -_ COMPOSITE ANALYTICAL LABORATORY REPORT ON THE Quantitative Analysis of Fluorochemicals in Environmental Samples LE 2 22 = a8 4 2 _ ' REPORTLNRo.N--FWA2C4T7 9G1E,N-W022814,5G,EP WN-3012947,,E GEE0N0--01303,86GEN-033 ANALYTICAL STUDY INITIATION GENO021: 08/25/99 GENO024: 10/12/99 GENO30: 12/13/99 `GENO33: 03/14/00 A@RERpRaI-ToIrNReE 0008137992 S$ y& 3 >3 [NE = - 000003 ReportNo.Gan021, SGM EGn2va 4i,roGnamen nn0ta3l0,LGabaornat0o3ry3 TABLE OF CONTENTS Analytical Study Personnel and Contributors ---------- I, PuTersptosaned.Control ATCLa..n...r..--..--...--..--. ------------ --ac-------------------- Sample Collection and Analysis ---------------------- SAMPIE RECEIPt ANA MBIMENANCE .......cornsnnnimsssn3 ChePmiAcOaGlUCERMAErNA.E.T.Z.A.O.Noo. cceatm----------------------------------m--rs--s---------------- MethodPrSeupmarmaatroiryesa.n.d.ANGIVICAI MEIN-- OGS ...-- .........-- ..... ---- amm-- ----) rr Analytical EQUPMENT............... m------r" 5 o Data`SSuSumUmmMmaMararyIyYoo,ff QSAuMaaBmlIpiYtlSyeeSCR,OeNSAIuNOl.I.A..N.I.Y..S.E.S..R.OSRULOS.S..U..L..S.........orr........... e.-- oes sn-- s--nn : Data Qualty Objectivs....... so------------ 2 SB OfCOO MIUSIOE N inn mmo REOIONCES.....r " was AEC... ---------- 10 Report Signature..... ------------ LisT OF TABLES Table 1.DeSCOfHSApMPIHES,IBYOSHUNOY rrr mnt Table 2.ELProcurement Information for Reference Materialns int--he--Anal--ysi--s of--Environmental 3 Table 3. Ions Monitored in the Analyses of EXIFacs of GOUNGWGIer.......... -- Table 4. RangeOf LOGS fOrS81,BY SWAY. menses Table 5. RaonfLOgQSfeor Liver andOther TISSbUyESASY.,.. PropoContr 000004 rages Report No. Gen021, SGManEan, eGamn a30,LGabeornat0o3ry3 ANALYTICAL STUDY PERSONNEL AND CONTRIBUTORS Analytical Chemistry Laboratories 33MM EEnnvviirroonnmmeennttaall TLaebcohrnaotolroyg.y and Safety Services (ETASS) F2i3or.i0n0e Analytical Chemisiry Team (FACT) 9St3.5PBauuls,hMANve5n5u1e06 KHrairsolHdanJsoehnn,soPnh,.DA.nalPyAtiIcal Chemist LMiasrakA.ElClelfesmoen,n,AnAanlayltyitciaclalChCehmeimsitst Sponsor : mETass . BStu.ilPdaiunlg, 2M3N-0559133 Dale Bacon, Sponsor Representative 000005 Page 10110 Report. Gencar, GMoenonstSeonninLaSboonrisnady INTRODUCTION Purpose T3thuMedseptsuudraipreoessGeteiosnfs-uh0ei2ss1a,cmopGmelpne-sa0sc2i4at,ercGeetpneo-r0rt3so0L,moafpinrsdho,vGibednre.s0a,3s3m.uammAmlmlaarolyfsot,hfetahnsedamaapnmlaplehysitbiicinaaclnlsdu;adetOda.cInoJltolhheencsteeGdeifsory ocfonMdiucchtiegdantoStsautpepUonrivsetrusdiileyshdaessiugpnpeldlbdy Oarl. sGamepyles (0 3. These analyses have oan T3).hpetarrfguectroaoncaltyatneessuflorfonneyslaefmoidue s(tPuFdiOeSsA;weCrAeSpHer7f5l4u.c9r1o.o6c)t.anpeefsluulofronoaotceta(nPcFatOe;(CPAFSOA# o2f78P5O.A3A6-: `CASH 3625-26-1), and perfluorohexane sulfonate (PFHS; no CASH available). Deqvuuoaelnvi0itnatgthieaonnvala(yLrtiOicQea)lomwfelmrheoiduesi,stsvaoanmraielaybazlneea.dlyA(twisiclauhlmrdmeaastrpaeyqcutoaf0ltlhbyeootabhcjhseipceeivcveiedess,LsaOunGcdhi(bsayssusehpsee)c,iieam,nidisodfsuuee taond "t asptrtueadpyacroneludlmebcmtaeitror)nixiqsuppaoiksyeersoianmepdolei,ns.TsaMabomirpel2esopfpertceipiscqrudoaaplotlray.qcuToanhitetroyslaso,tbjeaednctddiavrteeascuoavnaedrlypaofr1satmbaeartsgeeerdsaonfnoarlrhyeesssufssoomf . Znatyical studies aro oulined ater in hs report Test and Control Article Tbapehpleroowtp,eritiantaeTr.taibcRllaeebsb1firtFoseraucaehl sssttuwudediyreesc,ocnthshoiessetcneodaiosfltvhaaerriciooulnstcotoilnssasiucsesteefdsroofbmervcaaabrubisioeusseprrsaepveaicnoiduessrsaaubncbdiiaevseerns,atveaesd icnodnitcoalteadrtviecrleyslwoewrleeveplrsovoifdeedndbyogtehneo3uMs EflnuvairroocnhmeemnitcaallsLaIbnohraetsoery.matrices, Samples of the oTbhtiasirneepdorftrdomoeDsr.noGteinsc.iude details or the collection ofthe est articles; these details should be Table 1.Descriopf Stamiploesn, byStudy |om | [--r---- rman CHaaocsSSooa,GooniSMepktarRoSveel| [FERRIER cSoma BamnSa oo Otnt Key| ri Ecerpaet .HHeTre SoGoaE| |,wnsmPt bien Comin Fae Sats Abstoss veeyr, =Comarartyok,Gut tSnESAtRgEuiD. || NPAMeTre t=Sp oinr Soeek|| S P CaptnFP ogCFopoTI eAGoi tn Trees Tk rece Lv a SosmcE oretGaSS roppgaoSton, Ne imtns ei eSon,SeT vron 600006 Pageza1t0 Reporte. Gen021,GSoMn0E2n4v,iGonemne0n30a,aGboarnst3o3ry3 Following analysis, extracts generated from these samples have been retained in cold storage. `Sample Collection and Analysis ``TTiesasmuebsyamKuprluenstwheacrheasluanbmKiatnendatnootfheMiEcnhviigraonnmSetnattealUnLiavbeorrsiattyo.ryD-eFtlauiosrionfetAhnealsyatmipcalleCrheecmeiipsttrayre documented on the chain of custody forms located in appendices of this ropor. SAMPLE RECEIPT AND MAINTENANCE (`@S/a2m6p/l9e9)s,wGeerne-r0e2c4ei(v1e0d/1i1n/t9h9)e,EGnevinr-o0n3m0en(t12a/l13L/a9b8)c,oladndorGferno-z0en33on(3t/h13e/0f0o)l.lowSianmgpdlaeterse:ceGiepnt,-021 fidoelndteirfsicaarteiolno,caanteddcihnaitnheof3Mcuasrtcohdiyveisn.formation are located in the study folder for each report; the . tThheeqsuaalmiptyleofexptrreapcatrsatviilolnbneomlaoinngtearinafefdorindscoplredssertvoartaigoen.at the 3M Environmental Laboratory until . CHEMICAL CHARACTERIZATION `PTrhoectuarregmetenatnadleyttaeislscohfartahceterreifzeerdenicnetshteasnadamrpdlsesusiendclfuodreaPnaFlOySs,isPaFreOSsAu,mmPaFrOiAze,dabnedloPwF.HS. Procurement re ERE| wens Table 2 Procurement Information for Reference Materials inthe Analysis of Environmental Samples -- Lor Nuseer Source Gonar: L2383, Gen026. 245; Gen-024 3M Specialty Chemicals `Fuclolmpclheetmeidcaatlhcihsartaicmtee.riUzpatoinoncostmupdlieetsi,oinncolfutdhinegsepusrtiutdyieasn,dastraebpoirlttyvidleltebremianracthiiovne,d hinavtehen3otMbeen Environmental Lab. METHOD SUMMARIES EFonlvliorwoinnmgenistaalbrLiaebfordaetsocrryi.ptiCoonpioefstohfetmheethacotdusalusmeedthdoudrisngustehidsfaonraltyhteiscealstsutduideysbayrethleoc3aMted in attachment H. N 60606007 Page3etio ReportNo.Gen-021,G3eMnE2n4vi,oGnemnen-t0a3l0,LaGbeonra.t0or3y3 PREPARANADATNALOYTRICYAL METHODS + AEnTaSl-y8s:i0s04u.s1in,g"EHxPtLrCa-cEtlioenctorfoPspFrOaSy MorasOsthSeprecFtlruoomreotcrhye"miwciatlh CsoommpeomuonddifsicfartoimonSs,erduesmcrfoirbed below. Because possible ttoheprmeaptarrieceesxtwrearcetesdosvtaarnidaabrlde caunrdvess.amApllleexstirzaectesxtwreermeeleyvalliumiatteedd, it was not versus iunneexatcrhacttiesdsusetsaandmaprlde cfurrovmese.aWchhesnpecsiaemptelsetesdizteo permitted, two provide some lmeavterlixofsepxitkreasctwieorneepfrfiecpiaernecyd determination. For some available vsoalmupmleesw,alsesesxttrhaacnte1dmaLccoofrsdianmgptloe twhaesmaevtahioladblwei.thFothretehxecseepstaimopnltehsa,t the. the final `volume of extraction solvent was adjusted to match the volume of the initial sample. `This method was used for the extraction of sera, plasma, and whole blood samples. . + UETsSi-n8g:H0P0L5C.-1E,l"eAcntarloysspirsaoyf MPaFsOsSSporecOttrhoemretFrlyu"orwoicthhesmoicmael mCoodimfpiocautniodnss,indSesecrruimbeEdxtbrealcotws. . Because possible the matrices weresovariable to prepare extracted standard caunrdvess.amAplll eexstirzaectesxtwreermeeleyvalliumiatteedd, it was not versus oufn1e.x2tr5actoteaddjsutsatndfoarrdthceurrveemso;vaals a result, all sample of 4/5 of the MTBE cfornocmentthreaetxitornacst.weTrheeafdajcutsotredibs y a factor unnecessary when an extracted curve is used for evaluation. HEPTLSC--8E:l0e0c6t,r"oAsnparlayysiMsaosfsPSFpOecStroromOetlhreyr" FwliuthorsoochmeemimcoadlifCicoamtpioonus,nddsesicnriLbievderbEexltorwa.cts using pBoescsaiubslee ttoheprmeaptarrieceesxtwrearcteesdosvtaarnidaabrlde caunrdvessa.mApllleexstirzaectesxtwreermeeleyvalliumaitteedd, viterwsauss.not iunneexatcrhacttiesdsusetsaandmaprlde curves. When sample size from each specie tested to permitted, two provide some matrix spikes were prepared level of extraction efficiency determination. tFhoersseosmaempslaemsp,lethse, less than available 1 g of sample mass of tissue (waasscaelxlterdacftoredinatchceormdeitnhgotdo)twheasmeatvhaoidla.ble. For `Samples of kidney, brain, egg, and muscle were extracted by this method. + AEnTaSl-y8s-i0s0u7s,in"gExHtrPaLcCt-iEolneocftrPoFsOprSayorMOatshserSpFelcutorroomcehtermyi"cawlitChosmopmoeunmdodsiffircoamtiLoinvse,rdfeosrcribed below. Because possible ttoheprmeaptarrieceesxtwrearcetesdosvtaarnidaabrlde caunrdvess.amAplll eexstirzaectesxtwreermeeleyvalliumiatteedd, it was not versus oufne1x.t2r5actoteaddjsutsatndfoarrdthceurrveemso;vaals oafr4e/su5lto,f atlhlesMamTpBlEe cfornocmentthreaetxitornacst.weTrheeadfjacutsotredibs y a factor unnecessary when an extracted curve is used for evaluation. dFeovreGleonp-me0n3t0,aanldl sGaemnp-l0e3s3doentlye:rmDiuneedttootchoenltaacikn ogfreeaxtceerstshatenst0.m0a1t5ergiailgfoorfmPeFtOhoSdwere. wsiulbhjercetstpoecatntoadtdhieti4on9a9l >PF9O9Strvaenrsiiftiicoantiaonndprtohcee4ss9.9 E>ac8h0 staramnpsliteiownas.sTahnealqyuzaendtisteaptaivreatreelsyults 600008 Pagedof10 Repro. Gen021,3GMemEnsvi.roGnmoennt0a3l0.LaCon458 o3b0t%a,intehde firdeonmtietaycohf tPrFanOsiStiwoansancaolnyfsiisrmweedre(sceoemRpeafreerd.encWeh1e).nTthhoessee rseasmupltlsesagwrheeerdettohweith identity of PFOS could not be confirmed are noted in the data tabi. PInFGHeSn-w0a2s1,connoduPcFtHeSd sbtaasneddarodnwraeassaovnaaibllaeblree.teInnttihoensteismaemapnldesa, kqnuaolwtnatPiFveHdSetterarnmsiintaitonio(n39of9 > 99). `ESnpveicirfoincmiennsttarlumLeanbtaalrcphairvaemse.ters are available in appendix IL of this report, stored in the 3M Anavyncat EquemenT For HPLC-Electrospray Tandem Mass Spectrometry: `LAinqauliydtiCcahlrocmoaltuomng:raph: Hewlett-Packard Series 1100 Liquid Chromatograph system Column 1txem3p0ermatmurCe:183B0edteagsrieleTMs C Cycle Time: 10 minutes : Mobil*e p"hCaosmepcoonmpeonnAte:nt2sm:M ammonium acetate Flow rateC:o3m0p0onpeLnmtin8: Methyl alcohol Injection volume: 10 pL Solvent Gradient: Time (min) %8 01 1100 55 95 [7]5 510 1 10 FMoarssDeStpeeccttiroonm:eter: Micromass API/Mass Spectrometer Quattro Ultima Triple Quadrapole ``AscyqsutiesmitoironMMiocdreo:maMssRMAP(Ire/fMearstso TSapbelcetr3o)meter Quattro Il Triple Quadrapole system SMoofdtew:areE:lecMtarsosspLryanyx"Neg3a.t3ive SSoouurrccee: Block Temperature: Z-spray 125-150C 000009 Pagesot 10 ReportNo.Gen21, SGMaEnn,GeemnGa30,LGaboornst0o5r3y Table3. ons Monitored ntheAnalysesofExtractsofGroundwater Er |_pros | ww0 [we| [es"Indi| cates the fwon uosedf| orquanttation Racetfuearltaonatlhyetiacnaallyetqicuailpmmeentthosdesttianngds euqsueidpimnetnhtelporgessefnotunsdtuidny.thTerhaeswedsaettaiongrsdmetaayilshaovnetvhaeried Snoolmeadwvheartsedluyrianfgfeacctttuhaelqdautaa ycololfectthieond.atHa.owEexvaecrt,sseltitgihntgvsadriuartiinognsallinpthhaeseisnsoifrudmaetnatcsoellteicntgisonwialre recorded and presented in the appendixofthis report, : DATA SUMMARY, ANALYSES, AND RESULTS Summary of Quality Control Analyses Results. + SbprrtaoacvnkidedateirandgbCueutsrteevarebsflt:eoTdvaheteractwohaeefslfii>ncei0ae.rn9rt6a2o.nfgdHeeitgaehprpmorifonpalrtoiiwaotcneu(rtvo)etfhpoeoriaanlttlas.1m/aXHiywgehhiagovhfetlebodewcecunurrvevexesclpuadinetdsto wmeorreedtehaacnti3v0a%t.edAicfctehpetacabllceudlaatteadwcaosnceevnatlruaattioend vvaerriseudsfarosmtatnhdeartdhecourertviecaclonctoanicneinntgraattiloenasbty 5cuprovinetss.usAeldatcotieovnasluaarteeaqcucaentpittaabtlieveadnadtaarweedroecaucmceenpttaebdlei.n specific data sets. All standard CoenvteirynufiivengsamCpalleisb.ratAicocnepVtearbilfiecdaattiaoniss:bOanckaeveerdagbey,coanliebrcaatliiobnractihoenckcsheqcukaniisiaantaeldyzteodbef.or wdiatthainis3b0r%ackofettehde tbhyeoarcecteipctaalbvlaelucea,ibervaatliuoantcehdeactksl,easatserveequriyretde.n samples. All quantitative Bloafnqkuasn:titEaxttiroanct(iLoOnGb)lfaonrkas wsepreceifciocmpanlailayntte.ifInnothtiasrgsettudayn,aleyxttreawctaisondebtleacntkesdwaebroevoefttehne hliimgihter tbhyadneflaouwltc,uralvlelpaoninktss.weBreecaduesteeramnianleydtetolebveelbseilnotwhethbellairmkitaorfequusaentdittaotdieontefromritnheethe LOQ, compounds of interest. Intonelyr.naInlteSmtaalndsatranddsa:rdInrteesmpaolnssteawndaasrdrerqeusiproendsteobweaws imlonhito5re0d%ionfGtehne-t0h3e0oralnidcaGlevna-lu0e3.3 If wsearmeplreesansahlyozweedd. afntihnetdeemvailasnttaInSdarredsproensspeonwsaesthcaotnfdiervmieadt,edthmeoarnealtyhtaend1a5t0a%w,atsherespaormtpelde,s but noted in the data table. 606010 Paget 10 Repro. Genc21 aGawnEazneo,meennoi%Go0n3. `Summary of Sample Results GENo21: PLiFoOnSfivwera,sHdaertbeocrteSdeailn iavtelre,asGtooznzle sIvaemrp,lMeinfrkovmetrh,eRfiovlelorwOitntgemraItvreirc.esT:urCtalelfIovemri.a Sea Cormorant blood, Oter blood, and Caspian Seal biood CPaFliOfSorAniwaaSsetaenLtiaotnivievleyr,idReintviefrieOdttienratlievears,tSoenaeOsttaemrpvleerf,roSmeathOetfeorllborwaiinng,maantdriOctetse:r blood + CPaFliOfAornwiaasSteeantLaitiovneliyveirdeantnidfiCeadsipniaatnlSeaesatlobnleoods.ample from the following matrices: CPaFliHfSornwiaasSteeantaLtiiovneliyveird,enGtiofzieid iivneart,lMeiasntkovneer,saRmipvleer Ofrtotmrtihveerf,olSleoawiOngttmaetriivceers.:Turtle fiver, Cormorant bood, Caspian Seal blood, and Otter blood. GENO: ": PARlObaStrowsasspldaestmeac,teAdlbiadelnrtoisfsiesderina,atColeramsotroannet spalmaspmlae,fCroormmtohreafnotlboiwoiondg,mHaetrriicnegs:Gull plasma, Herring Gull blood, Bald Eagle plasma, Loon ver, Albatross iver, Brown . Pelican iver, Albatross kidney, Cormorant yolk. and Gul yolk. `PCForOmSoArawntasbltoeondt,atBiavleldyEiadgenlteifpiledasimnaa,tLeoaosnt iovneer,sBarmopwlne Pfeolmictahneffivoelrl,oawinndgAmlabtartirceoss:s iver + `PCormoF wraansttO ebnltoaotdi,AvAellbyatirdeonstsifiiveedr,inCaotremaosrtanotneyolska,mapnlde Gfruolmytolhke. following matrices: + HPeFrHiSngwaGuslltepntlaatsimvealyaniddenBtailfdieEdaignlaet plleaasstmao,neLosoanmpIlveerf,rAolmbatthreosfsolvoewri,ngBmraotwrincePse:lican fiver, Albatross Kidney, Cormorant yolk, and Gul yolk. + GENO: + PblFooOdS, wPoalsardeBteeacrteidveirn,aMtilnekasitvoern,eNsoarmtphleemfFruorm Stehaelfiovlleorw,iMngapmatTruirctelse:iPvoel.aTruBneaariver, GCarrepe,nFFrrooggmluisvecrl,eC,hLiankoeokWhSiatlemfiosnhveegrg,s,LBarkoewWnhiTrloeuftshgigvse,rC,aBrrpowmnusTcrloeu,t Cihviern,ooWkh.ole `Salman muscle, Lake Whitefish muscle, and Brown Trout muscle. + PMiFnOkSvAerw.as tentatively dentiid in at least one sample from the following matrices: + PFOA was not tentatively identified in any sample analyzed. + PFHS was not tentatively identified in any sample analyze. + GENO: + BFSoeOlaplShliinwveavr,serCd,oetmSewocotrredadfniinsihvateLril,veeaBrs,ottToutnneNasoiasvmeepdrl,DeaofnlrdpohBimlntahicevkefrTo,aliGlloaewdnignGgeulsmlaDtIorvilecprehsi:n Mivierk, vSetrri,peBdaikal + PMiFnOkSlAivewr,asCotrenmtratainvtelyfidveer,ntainfddBoitntaletlNeaosstedonDeolspahmipnlveefr.rom the folowing matrices: 000011 Page7ol 10 Repo fe. Gen021, GSaMnE26n,Goann03,LGaosans0o3r%y + CPoFrOmAorwaanst itveenrt.atively identified in at least one sample from the following matrices: + lPivHerS, SwtraispetdenDtoaltpihveilnyfiidveenr,tiafniedd Sinwoardlfeiassht Loinveer.sample from the folowing matrices: Mink Appendices contain data summary tables. DATA QUALITY OBJECTIVES Ninotecgriirtcyuomfstthaencdeatsa.exiTshteeddadtuariqngutahye porbejseecnttivsetsu(dyDQthOast)wofoullldowheadvdeuraiffnegcttheed tphreesqeunatlatryeor indicated below. Lintheaanri0.t9y8:5 Twhileh caoeflfeiacsiten5taocftidveetepromiinntastuisoinng()a loifnetahre rsetgarnedsasridoncucruvrevewawistheq1u/axlwteoigohrtgirnegater + tIhnestcarluimberanttioQnuacnutrivtea(taicocnepLtiambilte1sQtLa)n:daTrhdeisIdGeLfiisneedqausaatosttahnedlaorwdeswtitahcince3p0ta%bloef tshteandard in " wthaesorentoitcsaplecviafliuce)a.llyASdettheirsmvianleude oirs snottatuesdeffuorl einvecroynssitduedry.ation of the sample data, the 10L . + cLailmiibtrsatoiofnQcuuarnvteit(adteifoinne(dLaOsG)a:sTtahnedaLrOdQwitishienqu3a0l%toofthtehelotwheesortetaicccaelptvaalbulee),staannddasrdainletahset tthweoatmiomeusntthoefasnaamlpytleepaevaaiklaabrleea fdoertaencatleydsiisn t(hpearetixcturlaacrtliyonforbasnakmsp.leTsheexLtrOaGctemdaayccvoarrdyidnugetoto vEaTrSi-o8u.s0i0s6s)uoerstaordealyi-sttoed-dianyTvaabrlieat4io(nsserian,tphleaasnmaal,ytaicnadl bslyosotde)m.anTdheTarbalneg5es(voferL,OkGiSdnfeoyr. muscle, egg, and brain). Table 4. Range of LOG for Sera, by Study [Aver |rooGo nene0s21ms.| o[co=rirsyom. | oocm[o=trsmppmt |t[rae | ow sont | oor pm. Goossen | 00208 ygimL__| 006240000858 pgm Tabl5e. RanofLgOGeS fo Liver and Other Tissues,bySty AvaLre [re [= Gen-030 [er PFOS Prosn | 00348pg oowmsus | o0c03o4r8suo9ufgo | 0.00606-0.g06y% |0.006096c-0.10699419 [rms| ww | coos | somos| uoomums 00359pig 0.180pg/g 00180007g1/9g _| 0.00718-0.07g1i8g NA = not applicae 600012 Pageot 10 Report Ho. Gen021, SGMenE2n4r,oGnemne0n3a0,LGaboonra0tory tiDsuspuleiscawteerleacrceepprtoadbulceibpleretcoiwsiitohnin(e1x5tr%action): Spikes conducted on samples of control Qu5a-l1i0tsyamCpolnetsr.oAlcRceesptpaobnlseeC:CAVcronetsipnounisneg wcaalsibwriatthiionn ve3r0if%icoaftitohne(tChCeoVr)etwiacaslavnaaluley.zeNdoevmeorrye than 10 samples were analyzed between acceptable CCV. + gSrpeiaktefvaarcicaebpilttayblinesrpeickoeverercioevse:riDesu.e Ftoorthaenynugmivbeenrmoaftdriifxfe(rsepnetcmiaetarnicdessuaena)l,yzsepdi,kethere was re3c0o%v)e;risepsikweitrheicno7v0er-i1e3s0b%etowfetehne 5e0xp-e1c5t0e%d cionndicceantterasteimoin-qiunadnitciattaetqiuvaendtiattaatifvoer tdhaattam(agtroioxd to (ugsoeoddffoor q2u5a0l%i)t.aiSvpeipkuerrpeocsoevseroinleys.oDutuseidteo osfatmhpilseralnimgietaitnidoincsa,tematthraitxssapmipkleestduadtiaesshwoeurlednboet cspoinkdeucsttueddiefosrsahlolumladtrbiececs.onFsiodrePreFdOfSoraqnuaallyisteast,ivseapmuprlpeosdeastaontlhya.tSiisnncoet nsoupipdoernttietdy by matrix valelriafniacaltyisoensetxhpaetrairmeennotts swueprpeorpteerdfobrymmeadtfroirxPsFpiOkAe,StPuFdHiSes,sahnodulPdFbOeScAo,nsfiodetrheedsetoapnarloyvtiedse, unconfirmed qualitative data only. " Usinteo tohfeIenxtterrancatlsSptoastn-deaxridrasc:tiToentraanhdydursoe-dpearsflaunorionotcetmaanlesstualnfdoanradtefo(rTsHaPmFpOlSe)s winaGsesnp-i0k3e0d :. aannadlyGteinca-l03s3o.unFdonresalsl sofamthpeledsatai.n tTheHsPeFsOtuSdileesv,elTsHtPhaFtOwSerleevdeeltsewremrienemdontiotboereddevtioavnetriffryotmhe `ceoxnpfeicttmeedd,vaalnuaeslbyylmevoerles wteharne 5re0po%rtweedrbeutreaarnealnyozteedd. IinftthheerdeesvuilatnttaTblHe.PFOS levels were + UEsSeMSofMSco)nfainrdmalatcokryofmaetvihaobdlse:alGtievmeantitvheefsoerlaenctailvyistiys,ofntohceoannfailrymtaitcaolrytomoeltuhsoedds(wHePrLeC-used. Detmiomnes(tmartacthieodntoosftsapnedcairfidcsittoy:wiStpheicnif3i%ci)tyawndasthdeemroenssptornasteoefdabty cleharsotomanteocghraarpahcitecrriestteinction product ion arising from cllisions of an analyte-specic parent ion. `mAastsriuxmisnpgiksepiskteudrieecsohvaervyesbteuedniesusfeodrmasaasnuiitnadbilceaitnodriocfatdiaotnaoqfuaelnidtoyg(esneoeuasboavnea)l.ytTehreecvoalviedriyt,y of ths assumption has not been verified by other techniques. STATEMENT OF CONCLUSION qUunadnetirttahteivecoanndailtyisoinssooffathseelpercetsieonntosftuedniveisr,otnhmeenptraelsemnaciecoefs.fluorochemicals was observed in the REFERENCES 1Q)ua"iAcacteipvteaDnecteeCrrmiitnearitaifoonroUflSturlatfroancyeluHrPeLaCH.eTrabincdideems MinaSsosirS;peLci,trLoYm.e;trCya:mpQbuealnlt,itaDtAiv;e aBenndnet, PK: Henion, J. Anal. Chem., 68 (19), 3397-3404, 1696 600013 Pagegal 10 Report No. Gen21,SGaMnE0n2v4t,oGnemann0t3l0,LaGboonrsGtoory ATTACHMENTS AttachmenAt: Gen-021 SeralPlasma/Blood Results AttachmenBt: Gen-021 Liver/Miscellaneous Results Attachment C: Gen-024 SeralPlasmalBlood Results Attachment D: Gen-024 Liver/Miscellaneous Results Attachment E: Gen-030 Sera/Plasma/Blood Results Attachment F: Gen-030 Liver/Miscellaneous Results Attachment G: Gen-033 Liver Results + Attachment H: Analytical Methods. " : Atatrachcihvems)e:ntAn|al-ytLic(aadldDiteitoanlals fbooruGnednd-o0c21u,meGnetn-a0v2ai4l,abGleen-in03t0he, 3andEGnevni-r0o3n3m.ental Lab REPORT SIGNATURE yA) He KastenJ. Hansen, Ph.D., Principal Analytical Investigator / Dale L. 8dcon, Spnsor Representative 5/10/00 Date 5 feo [nem D 000014 Paget0a1t0 -Farrer Lf=Lra i T--. =BEsTeo en o E SonRn. sacrament goores vis p J= onhris R EEEEEmom E T EAET sas [=7 = = om. o5 pe nTH . pLoei oondee : v,. Tfmoaiaems a aeoss BE ae [5%Ta erz | pgorLriinym ha orw.0er Eohnome]ss i A i I a PR & [<=[Ti f%oede[E] ESfprrnoaed Fa Ri EL eEm fEa Erdh nue a y a Sodia o a | 2 Lilien I 000015 win 2ES== =--oe-- ES=rrew rT me 2= * warns E fo mmtl 2=g-E BShuE o = EEErmeee TEE EE : E |a2== :: <oq(eoomm wo smeles e| Elel-- ss ma. -- 00co16 : nr ujsneeethyermob K=ieern BBBaeeaardh eBehin hcaip B000Samp Daa ac FACT.GEN2I otVio arefm Hi SEms usmemantine fe Sirniem sdUas i a ee ans 0 oEeeSSE seetocsetpotttaetsvtees > onlaaig tet je TSh oou Ph eepepesrae res 60C017 sePar stsms en a. SaEVitrewverin DBu uahbnieebie L000SameDaa ac FACT-GEN.02I G=t Vr om East wpmemes Sm p rfriepur eeirneya4s nin moreawns8hsse ane BpaeYt7e0e%rakpa vderttpra evot tcrten a = Hoaotbi mieteten fae TTfreotirreevpteeeeemmmrittioosie 600018 -) re i CE mE Cgia]z LE fan crea iiss Emin ELE FoouF oFio] mis NN ma LL me Fo] " Ega a mn Ra oa pTair A ems TFErhEme oas Fo] Er= asE 000019 4 = Se OESTCOPAVALAR ETT EIEEm EE Tees TT E Em Re I EP r E ma svar 000020 =a SLT estore aun oe E eb BE TT e EE TEE Ee Ter ToT Co [el]ELI = LLL = ELE me -- 000021 E-- a=r E=A I f. te --T= TLL Ee [Em [21 m . l=]e =[Llu] rT ----m ee Te PEt a TE Fn kn ons BS eavast ue os 000022 = = fro EE C m EEE fr---- fm-- BE rsssmemmmenne H=E BRR eam Bey Copy ayy Lae Le [TEL FEA T FFT Ta) [em T& 85 [ww| 8 T Tw || [emmsmegims,Tor Tow Tow om [or [| [esmmasn To |om |oe |;[we | | S eiE r. ESEaEme . Eo ! HEED E u om s m E meln E Ee a = = EEEErEReEE |i:Fi) Le HE m=eeE :ms NR [oomen Tow | Loon [oi [Semis =IPS frPrEEaiAEng]]Ea 3E=i> EEr EEooaiisss] 2&=5Zz EHBaaouasss Fi=H fPE EPeaaoe ]sss lpeHiiodiw = EER- EEmas =3| | pict Th iH H b Etio e e ses E b E Eae s e L a a oos. EE rgrTeors sn] P: mrEy || a - 600023 Emsam ug = pe-- a Sapam EE BESTCOPY AVAILABLE SamEbos: aa i es e ee r e e |2e |e [GE C Essieo8s or s aE B o r T n a CE EE T ReTe y E PhEEaEaEE eEsRs oEoEEEEEnnERR CEE EE ERsEs FIIEmEmmE ESFFErEErEesiEEy HE E = Es sE = 5. |= = HB EEE=EEF =E=I== mm= |.| i sti=eEen le EEhEiRmEe aEEmEs HEEEEE EEIIEIEEpREEEEEEEEEo. EEEESEEEEZRERE EEfEaEReEE EEEEmxR HEBREREREEEEE EZEEEERsR B oPEpptiioivgtmrisieoE y SEtIa=mmEm oo Td Cea hse omtama Eorme. eran ac somo a om oen Sfame=sys E SwaioEds mse E E E Es s s s So EEs ss 5 Sodus || aso Pettis 7 oN ete 006024 Erman Em n oi uy. rg v---- e---- a Sw . Copy,W : [asmsme =TwTw|& Tal ww i, [esaemmeis [| of Tow Tow Ton Tol a7 ZEaon CLE[bl r L 25 550 1 Le . EOB=EsEes iZTpeaiLynnz]ss EZobaorosnznd i oBoaasssr Soiaaairns 1. amanSaEsyys |. on wiz i: TEEell EELa Lmro IE rrr E [edae [rEe 2n 2]]| Ea r poonsacsamsan ence I 000025 Ee jrAenN. ooh E.ETolvoan bstoetabetiors frre pn BESTC0P)Y,AVAILABLE [mame 1 0 TwTw = oe | ow] CC [ Eeeem om[ow [ m =m [a 0[| Sodomons eens Ar aan Fe[Ems Temes TBToe liom | wn | E--E TER E1 E T ea pes esps RG | a pe meen meme Same me es Lo Lm[e wo [i Em]] | 600026 iA " BbE=B;= Dfe a rirtt ei fo racrcenan J fer-- ETFoEen e mimms s SSpSEoa mmitt | seiB2aeAin armaeR s B | Seema IDanmats pried BEsT,Copy,[7] e o e asm e mm Teentned pr E==BB IpIIneeein 2 Inn pihT TBsioe rdi sn a2B= InIenneaesememam B Inseam p o ISooe tns Tass BB=BE IfIoninsneeammaainnn Bb Soe o pIToosd edes Te T2o | SBoeem Ifcreinei aa. FH2ue Sama ams Sa preie) se Funiims SpTJooooemmtan FH Bes o a a a my a e 3 SA Temie na S r pTenE eee = pra Isetenem aan pad bina SEgeas sEa La nITTnceeseem Tg b Bosm n se HER EE ETE eo I me Er Tamm po, RT -Bcsmmrsseons mane E[ou e Spe SOinlBersAni Sens PE-- dfnemntsen pn rme = BE,STCop Sme mens A Mhe Suas s F BgE plameuS sssss Eas ||| SRSEuemaE mslii Ei=o77dn mantels || w Inemm adS :} . [mamma 8 Tol we 0 Tu] : [EemEdmRteT t W T Te| T a 0 T w Tu] ===== D ppniertea ttmy Hi a==== mme xeeemmmm a ===== STfermoraaaani A =BE e meam FSuiHsh un fSrpnhieu]mm Tee FinnHse ppmrrPeei ===== e JJTaasasmaieen Ta = Sse | ae a=lafi fa fIrrsoeen Tse te EeriFeEameere fmm D E oo e" -- n ---- ST ne tt hr, nA i Jr-- 006028 Eo "Bronte fi= a =m etyume E E S SaErn, rs racraman sa=dstenting SEEEEonEeEsn, EEpEEr,E 3EST aval ffErff riEtaeEe gns EHEEER ammii===e E=n a R iEDanas Tsin SENEED " [roisimens:| we| on | ew | aw |om [aw | om "| [o"s5s55i00m53p0 ialoln|WT 50T1eoeT [oo=|2W Ta[owlme PrEn==ihL fE===eS = = T mEe Eanrr mmst Ta pori oesmy afne==ih FTa I pr aons h tse E2e3) i= 2 semis]dE |" SFSTTrhhireaeeeeeen SEEIRES paEareaeenss Ems d f m HToee r e nis Een Zn a EEmEeE paamremmmss mdHEeeaaisss o=yaEaEy eEpJL msme PprrEEeoneeaE.s.. i as 000029 . po a ft eS EE===.tme E SSeeer f-- ome mete VEemH E EEEr2 EEsyEE, ERE R o Bmoon n en ||LL S S|oehe S yamn SRmEeEeeR|dS |eE S me BEST Copy ay AILABLE E E o Sh7en n im 7: " moL a| itesye| ||n r imemn eeet smeZ F ienR||E| E EmeSmReIs magEm | |mEeEmEe gSnElaoase sees|||| HMHmuaammEmmm SCDromloeeEeped |||mSEeemmmemms 2 a EeI , aA sEoSnFibiSm--p-nn | Solem eei asmn nt o ipmmooeeemts eem]er] mE Eamd Li Li i SpiEc. TLa Eireeer 000030 wprem air cr Dare emai foto Sample Data " MSUEracmr.ceonan nts =Tustin BEST Er, COPY AvAILARy Tm Scsmresn mus i} ] ] [esTammyTaras 8re] l= [ame[ol [a on [mas] || [Smmmemow|[oT w] .y: z22=Zz OO===EE a oa frerd 2 pd x = 2oa 1. a8 = Sogtonm|1aon . on 3i%lo | sotasto SmHoerEean = == ios [1 = si=en loa | eSooI nto)nm|Nace M w= = =ea EffEeeeeim] BaT=Eamnaa bs CE j= uscnerd i= diioma|I ace N b= aatoans, IR I PRNcneta WST wastes ton 0 DE E e aepmaS rin TrDotreoemseti 000031 MSU EsmamceraSle I" ween f=ot mentee Metter E EE E o t T E Diamar [EH= EEodE-- BESTCgpy gy, AlLABLE Esess [=1 EE a [a elm PesamrTamla8Toll a (Eool[El EL) ] | [lSoenmd s oam f Tow a we T| l ETa|e la| : of meBm= o i oLd a oamaen BOE TomlnL Smm2eeensn mm Tol| . E (|BZB mprmi CE oo `E ibatemm| aco ] = Sogtonm 1] o| y | ETE =n |=p NN moid = bi Le w=m | ls Ll: Ea 22d ll= | TE == =m|| |E EEEdeeee m= Ee ooooomaeoe Er S C ti SrE esE ET -- =] O= EE I EmFH mEN Ir ni SRL|IR EEE 600032 Eee CL w fr l Be BEST COPY Avg : IbidLE [a]fee esl 1) CEE Te] | 1 ==TFT.[EE] ool Ex[3[&| | a[Ee] |o] 000033 a Se STOAyOP Fe aE r = te mmla] 2[e ES [a =TE ETE a LE = TEE IEE oT] 000034 fe E Ea= AEEmr-- rie A nec A 7 XSEBREue-- mi an, eBe OPYAVAlLagyp' _ =Ee BEa-- ] Rml IaEH |E | mi v=m) IN | BBEEEuREERmEEE RER|| LmmmLi)mm ow | iBbEnm .LL oa inif=mo I T :Ea | E EE=Eir=EeE H2i Sii73ELE o= 7Eeai u5 TT em TSErTT T T T T e ET T CCE E TeeerrT re E T T e F Ea a=E 5DV,DSrreerE rerE e e F= ie=i i = iiA i3i]||| BmEmEieee rA r SI TEi= EE S2i oa ZZ= = =nan T a Bia EE cons 0003s zm fEe m snNrett bmet SEi En -- e--e EEia rElm, -- Allg BES]T00pya FBrEE a --m ----. ] L EEEE| wim, JwEm CBEE = RaEmE EaE||| o= aminmm I |, N oZEBhaeEmm I|, N . gAEmhELma || = amsemmmm nN ao= Eeall. ow LtemiesTwTel ow [2Tol wo =EET [CP A ommEierRs T TFoYlIIS Pwn | e <-~P Te oBPP| EEA ne [oemonedt| 8 Ton wn |2 Tw] [omerTo ToT we| To wm | | T[m= eeE EEEnEna R ||||| E = EseETH o N on LEa B=2 i=da|Ton a N ow | iiti obei LEEEEE 2BBE EE== a|||||E n EEuaaEEmEmmmR |, EBEE&ZP4iiiEos4sza |BE |, me i Em Ea . mano -- 6006036 so Frm Se BEST copyAyu pgyg TE TETET CCLEE E Ee e] l | EEE ECA i -- 000037 on _ = BESTCOPY Aviapy TCEEEETLEET : = Senses = = El[5] 8] . [.] meme |tm Tela|a | en |wn| TEE TEs EE me -- 000038 in Eee or -- a ER. o sucre oran wear a OPYAVAILABLE Err, HN EEe Ere n Eel gs | 17 EE El1, EL] EEE 3 2. =e [ome T8188 ToT aw [amlTw| . aa | fla= sls CEoeme Te aTs e] a CI I EE TET E S P PO S ESe EHPP PN (EhaseemesesTTTeoee oeT To o e T&8T] =--= FE TE E = T EmT e ] ed E a = 2 2 Tou -------- z= = re am | aoa Ed bo oe Sadie 00 rk me -- 000039 p. Hori Se ol meme PSTCOPY Apppig Eo. ri, 3. ov Ro on BASES Hee \ = P= =|LL [E= _L ] L] [e CEES r r a e e Ere re re ue -- 000040 -- i a me EE EnRiE, cramer Bmaata. E Lo a BESTCOPY AVAILABLE mFw- I= To EIT 2 i : i] 2 or = Bs i2d =hss ES one is = is = WU e------ pra EE pitied oeoe mm in En A Es 000041 cosmos a enenezens a E Ei S m pa Beis ELETm , s mmme w= Eai] a i ia3] a = FuEs] x on Ei]EL | naine == sintes 1 . E528a B28 EEmintnas ie - =To8n aim=ees Yuin So 00 = mE 8Bney aEaaEma Es i= oosn EFoE=Eoe]aS] E=EEmmea BE 2E]s = nn Sai meen ace HTEeE p Ee t HEEnE R EE n " mfi- d na fr a tos a ie piecta:an odammy poi E m amm ann a Em e pp brii t en fa a r E m ammyn x Srna 7800MR,UTR,CLIO MMLC ro 000042 a oe RLYAVAiLA --er eee 1 EB T=] Jls]. [=m Tm = Tw[m= [ow | C= TE Ea TT TE rE 000043 = Free Bz E = Ee sregy / (1 2 =[.1=81.1] 8[21[1288[] .] EET 000044 ~ CT BESTCOPYAVAILABLE TEETEE e E e ea e e ee eeLe 2 el Lo 000045 _ TT STCOPY ppp THEE Es eee re ede Tm1mI TmTm wn] Smee LE ow oaLETal=] 000046 3M ENVIRONMENTAL LABORATORY " . METHOD EXTRACTION OF POTASSIUM PERFLUOROOCTANESULFONATE OR OTHER FLUOROCHEMICAL COMPOUNDS FROM SERUMFOR ANALYSIS USING HPLC- ELECTROSPRAY/MASS SPECTROMETRY Method Number: ETS-8-4.1 `Adoption Date: 03/01/99 Author: Lisa Clemen, Glenn Langenburg. &, Approved By: Revision Date: Laboratory Manager Date Group Leader Date `Technical Reviewer Date LO_SCOPE AND APPLICATION 11 Scope: This method is for the extraction of potassium perfluorooctanesulfonate (PFOS) or other fluorochemical compounds from serum. 12 Applicable compounds: Fluorochemical surfactants or other fluorinated compounds. 13 Matrices: Rabbit, rat, bovine, monkey, and human serum or other fluids as designated in the validation report. Word 6195 ExtractionoEfTPSFeOaSlfrom Serum 000047 Page Lf 14 2.0 SUMMARY OF METHOD 2.1 This method describes the procedure for extracting potassium perfluorooctanesulfonate (paPiFrOinSg)roeragoetnhteranfdlumoertohcyhle-mfiecrat-lbsuutryflacettahnetrs(fMrtoBmE)s.eruImn,thoirs other fluids, using an ion method, seven fluorochemi c al s . `were extracted: PFOS, PFOSA, PFOSAA, EtFOSE-OH, PFOSEA, M556, and surrogate standard (see 3.0 Definitions). An ion pairing reagent is added to the sample and the anniatlryotgeenioenvappaoirraitsoprarutnititliodnrey.d inEtaocMhteBxEt.ractThiserMec(oBnsEtietxuttreadctinis1r.0emmoLveodf amnedthpauntolo,nttohean filtered through a 3 ce plastic syringe attached to a 0.2 pm nylon filter into glass autovials. 22 These sample extracts are analyzed following method ETS-8-5.1 or other appropriate `method. 3.0 DEFINITIONS - 3.1 PFOS: perfluorooctanesulfonate (anionof potassium salt) CyF1;SO5 " 32 PFOSA: perfluorooctane sulfonylamide CiF:SO;NH; : 33 PFOSAA: perfluorooctane sulfonylamido (ethyl)acetate CsF17SO;N(CH,CH:)CH,CO; "34 ECiFFO1SSE0-;ONH(:CH2:(NC-He;th)yClHpe,rCfHlu,oOrHooctane sulfonamido)-cthyl alcohol 3.5 PFOSEA: perfluorooctane sulfonyl ethylamide CyF1sSO:N(CH:CHy)H 3.6 M556: CsFiiSON(H)(CH.COOH) 3.7 Surrogate standard: 1H-1H-2H-2H perfluorooctane sulfonic acid 4W 0 ARNINGSANDCAUTION 4.1 Health and safety warnings 4.1.1 Use universal precautions, especially laboratory coats, goggles, and gloves when handling animal tissue, which may contain pathogens. S0 OIve0 meeme0 vces0000000000 5.1 There are no interferences known at this time. 6.0 EQUIPMENT 6.1 Tachceepftoalblleo.wing equipment is used while performing this method. 6.1.1 Vortex mixer, VWR, Vortex Genie 2 6.1.2 Centrifuge, Mistral 1000 or [EC 6.1.3 Shaker, Eberbach or VWR 6.1.4 Nitrogen evaporator, Organomation Equivalent equipment is Ext.ractionEofTSFO$S41from Serum Page20f14 000048 6.1.5 Balance (+ 0.100 g) 7.0 SUPPLIESANDMATERIALS 71 Gloves 7.2 Eppendororf disposable pipettes 7.3 Nalgene bottles, capableofholding 250 mL and 1 L 7.4 Volumetric flasks, glass, type A 7.5 1-CHEM vials, glass, 40 mL glass 7.6 Centrifuge tubes, polypropylene, 15 mL. 7.7 Labels 7.8 Oxford Dispenser ~3.0 to 10.0 mL 7.9 Syringes, capableofmeasuring 5 uL to 50 uL. 7.10 Graduated pipettes " 7.0L Syringes, disposable plastic, 3 cc . 712 Syringe filters, nylon, 0.2 pm, 25 mm 713 Timer 7.14 Crimp cap autovials and caps 7.15 Crimpers Note: Prior to using glassware and bottles, rinse 3 times with methanol and 3 times with Msielplaira-tQeTMvwiaatlser. Rinse syringes a minimumof 9 times with methanol, 3 rinses from 3 8.0 REAGENTS AND STANDARDS 81 TbeypMeilrle-QaTMgenwtagtreardaenwdatmeary, Mbeilplro-vQiTMdeodrbeyquaivMaillelnit-;QalTlOwCatePlrusuTMsedsiynsttheims method should 82 Sodium hydroxide (NaOH), J.T Baker or equivalent 83 Tetrabutylammonium hydrogen sulfate(TBA), Kodak or equivalent 84 Sodium carbonate (Na:COs), J.T. Baker or equivalent 8.5 Sodium bicarbonate (NaHCO3), IT. Baker or equivalent 86 Methyl-T-Butyl Ether, Omnisolv, glass distilled or HPLC grade 87 Methanol, Omisolv, glass distilled or HPLC grade 88 Serum or blood, frozen from supplier 89 Fluorochemical standards 8.9.1 PFOS (3M Specialty Chemical Division), molecular weight = 538 8.9.2 PFOSA (3M Specialty Chemical Division), molecular weight = 499 8.9.3 PFOSAA (3M Specialty Chemical Division), molecular weight = 585 Ex-tractionoEfTFSO8S41from Serum Page3of 14 60U049 8.9.4 E(FOSE-OH (3M Specialty Chemical Division), molecular weight = 570 8.9.5 PFOSEA (3M Specialty Chemical Division), molecular weight = 527 8.9.6 MSS6 (3M Specialty Chemical Division), molecular weight = 557 8.9.7 CSuarFriozgSaOtseHs)tamnodlaerdc:ula4r-Hw,eipgehrtfl=uo4r2o8octane sulfonic acid (1-H, 1-H, 2-H, 2-H 8.9.8 Other fluorochemicals, as appropriate 810 Reagent preparation NOTE: Wphreepnarparteiopna,riandgjulsatrgaecrcovrodliungmleys. than listed in reagent, standard, or surrogate 8.10.1 1100N00 smoLdibuemakheyrdrcoonxtiadieni(nNgaO5H0)0:mLWeiMiglhlaip-pQrTMoxwiamtaetre,lmyi2x0u0ntgilNaallOHso.lidPsouarreinto a. dissolved. Store ina | L Nalgene bottle. 8.102 1 N sodium hydroxide (NaOH): N NaOH solution into a 100 mL vDoilluumteet1r0icNflNaaskOaHnd1:d1i0l.uteMteoasvoulruem1e0umsiLngofM1il0li- B QTM water. Store in a 125mL Nalgene bottle. LE " 8",+103o0f.5TMBAteitnrtoabaut1 yLlavmomlourmieutrmihcycdornotgaeinnisnuglf5a0t0e (mTLBAM)i:lliW-eQi" ghwaapteprr.oxAidmjautsetlyto16p9Hg10 NusaiOnHg,apapdrdoxsilmoawtleylybe4c4autsoe5t4hempLoHfch1a0nNgesNaabOruHptl(yW)h.ilDeialudtdeintgo vthoelluamstemwiLthofMilli- QTM water. Storeina 1 L Nalgene bottle. 8.10.3.1 TBA requires needed using 1aNchNecakOpHrisoorluttoioena.ch use to ensure pH = 10. Adjust as 8.10.4 `0a.p2p5roMxismaotdeiluym26c.a5rbgoonfatseo/sdoiduimumcabribcoanrabtoena(tNeab:uCf0f5e)r (aNnda:2C1O.y0/gNaoHfCsOoyd)i:umWeigh bicarbonate (NaHCO) into a 1 L volumetric flask and bring to volume with Milli- QTM water. Storeina I L Nalgene bottle. 8.11 Standards preparation 8.11.1 Prepare PFOS standards for the standard curve. 8.11.2 Pflrueopraorceheotmhiecralflsutoarnodcahredmsiacraelasctcaenpdtaarbdlse, (afsorapepxraopmrpilaet,e.onMeuwlotrikcionmgposnteanntdard s1o.l1u0tipopnmcoEnFtaOiSnEin-gO1H..0)0 ppm PFOS, 1.02 ppm PFOSA, 0.987 ppm PFOSAA, and 8.11.3 tWheeiagchtuaaplpwreoixgihmtately 100 mgof PFOS into a 100 mL volumetric flask and record 8.11.4 Bring to volume with methanol for a stock standardofapproximately 1000 ppm (ng/mL). 8.11.5 Daiplpurtoexitmhaetsetloyck50soplpumt.ion with methanol for a working standard 1 solution of 8.11.6 aDpiplruotxe.wo5r.k0ipnpgmstandard 1 with methanol for a working standard 2 solution of Ex-tractionoEfTPSF$O4S1from Serum Page dof 14 000050 8.11.7 Dilute working standard approx. 0.50 ppm. 1 with methanol for aworking standard 3 solution of 812 Surrogate stock standard preparation 8.12.1 WCieFiyg:hSaOpspHroixnitomaate5l0ym5L0-v6o0lmumgeotfriscurflraosgkaatnedstraencdoarrddth1e-Ha,ct1u-aHl, w2e-iHg,ht2-H, 8.12.2 Bring to volume with methanol for a surrogate stockof approximately 1000-1200 ppm. 8.12.3 Psrteocpkarteoaas1u0rrmoLgatveolwuomrektirnicg fsltaasnkdaarndd. brTirnagnstfoervoalpuprmoexwiimtahtemleyth1amnoLlofforsuarrogate `working standard of 100 ppm. Record the actual volume transferred. 9.0 SAMPLE HANDLING 9.1 All samples are received frozen and must be kept frozen until the extraction is performed. 9.2 Allow samples to thaw to room temperature prior to extraction. % 10.0 QUALITY CONTROL , 10.1 Solvent Blanks, Method blanks and matrix blanks 10.1.1 An aliquot of 1.0 mL methanol is used as a solvent blank. 10.1.2 Eaxstmreactthotdwobla1.n0ksm. L aliquots of Milli-QTM water following this procedure and use 10.13 Emxattrraicxtbtlawnoks.1.0mSeLea1l1i.1q.u4o.tsof the serum following this procedure and use as 10.2 Matrix spikes 10.2.1 Prepare and analyze matrix spike the accuracyofthe extraction. and matrix spike duplicate samples to determine 102.2 Prerceepiavreedewaicthh sepaickhe using a sample sample set. chosen by the analyst, usually the control matrix 10.2.3 EAdxdpietcitoendalcospnickeenstrmaatiyonbsewiinlclfuadleldinatnhdemmaiyd-fraalnlgineotfhtehleowi-nritainalgecoalfitbhraetiionnitciaulrve. calibration curve. 10.2.4 P`rmeipnairmeumonoef2mamtartirxisxpsipkiekaensdpemratbraitxchs.pike duplicateper40 samples, with a 103 Continuing calibration checks 10.3.1 Pcarleipbarraeticononctuirnvue,ing calibration check samples to ensure the accuracy of the initial 103.2 Perxeapmaprlee,,aitf aa msaimnpilmeumse,to=ne34c,onftouirnucihnegcckhseacrkepperrepgarroeudpaonfd1e0xtsraamcptleeds.. For 10.3.3 Pthreepianirteialcaccuhrvce.ontinuing calibration check from the same matrix used to prepare Ex.tractionEofTFSOSaS1from Serum Page sof 14 600051 10.3.4 Tcuhrevee.xpAedcdtietdiocnoanlcesnptirkaetsiomnasywiblelifnalclluwidtehditnhatthefamlidi-nrtahnegleoowf-rtahnegienoitfitalhcaliinbirtaiatlion claolwibernatdioofntchuervec.aliTbhraitsioisn nceucrevsesa(rfoyriftexhaempalnea,ly5stpmpubs--t 1qu0a0ntpiptba,terautshienrgtohnalny the. 5 ppb ~ 1000 ppb) 11.0 CALIBRATION AND STANDARDIZATION 111 Prepare matrix calibration standards 11.11 Transfer 1 mL ofserum to a 15mLcentrifuge tube. 11.1.2Ivfomlousmtesseaqmupalletvootlhuemessamaprleelvesoslutmheasn.1.D0omLn,otexetxrtarcatctstleasnsdatrhdasn w0i.t5h0mmaLtroifxmatrix. Record each sample volume on the extraction sheet. 11.1.3 While preparing a between aliquots. totaloftwenty aliquots in 15 mL centrifuge tubes, mix or shake 11.1.4 uTsweoth1e mstLanadlairqudoctso,ncoerntortahteiroanpsparnopdrsipaitkeivnoglaummoeu,nstesrvleisatesdmiantrTiaxblbela1n,ksa.t Typically the end of " : tshtiasndsaercdtsi,on,twtoo spike, matrix ibnldaunpklsi,caatned, two two msettanhdoadrdblcaunrkvs.es, foar total of eighteen * 1115 RreafnegrestoanvdaltihdeatLiionneraerpCoarltiEbrTaSt-i8o-n4R.a0n&geE(TLSC-R8)-5fo0r-cVa-l1i,brawthiiocnhculrisvtess.the working 11.1.6 sUtsaendAatrtdsa.chmSeenetSDecatsioann1a3i.d0itnoccaallccuullaattiengactthuealcocnocnecnetnrtartaitoinosnosftofhPeFwOoSrkiinng calibration standards. 112 Twoorkeaicnhg ssttaannddaarrdd, fbolrantkh,e ocronccoennttirnautiinogncthoecfakl,l waidtdhianpptrhoepcrailaitberaatmioonunctuorvfesurrarnoggeat5e:ppb- 1000 ppb. 11.3 Extract spiked matrix standards to establish each initial curve on following the mass 12.6-12.16 of spectrometer. this method. Use these standards Ex.tractionEofTSPSR4OlS from Serum Page of 1s 000052 Table 1 Approximate spiking amounts for standards and spikes Using 1.0 mLofmatrix Working standard WL "Approx. final conc. of (approx. conc. analyte in matrix [00pm[10 1 | 00B0aSpkpm | | [00pm[20 |ooloppm [--sooppmTs1 oo2Sppm | | - 12.0PROCCEDURE 12.1 Obtain frozen samples and allow to thaw at room temperature or in a lukewarm waterbath. 12.2 Vpoolrytperxopmyilxenfoerce1n5tsriefcuognedst,ubte.hen transfer 1.0 mL or other appropriate volume to a 15 mL. 12.3 Return unused samples to freezer after extraction amounts have been removed. 12.4 Record the initial volume on the extraction worksheet. 12.5 `Lwaobreklsthheeettufbore dwoictuhmtehnetsitnugdythneurmebmeari,nisnagmpslteepsI.D, date and analyst initials. See attached 12.6 Spike all samples, including blanks and standards, ready for extraction withsurrogate standard as described in 11.2. 12.7 LSpiinktehaetacshecmtaiotnr,ixfowritthhetchaelaipbrpartoiporniactuervaemostuanntdaorfdsst.anAdlasrodparsepdaersecrmiabterdixinsp1i1k.1e,s oarndTable continuing calibration standards. 12.8 Vortex mix the standard curve samples, matrix spike samples, and continuing calibration samples for 15 seconds. 12.9 Check to ensure the 0.5 M TBA reagent is at pH 10. Ifnot, adjust accordingly. 12.10 To each sample, add 1 mL 0.5 MTBA and 2 mL of 0.25M sodium carbonate/sodium bicarbonate buffer. 12.11 Using an Oxford Dispenser, add 5 mL methyl-fert-butyl ether. 12.12 Cap each sample and put on the shaker at a settingof 300 rpm, for 20 minutes. 12.13 Centrifuge for 20 to 25 minutes ata setting of 3500 rpm, or until layers are well separated. Ex.tractionoEfrPsFsOaSlfrom Serum G0VOS3 Pagel 14 12.14 Label a fresh 15 mL centrifuge tube with the same information as in 12.5 12.15 Remove 4.0 mLofthe organic layer to this clean 15 mL centrifuge tube. 12.16 Put each sample on the analytical nitrogen evaporator until dry, approximately 1 to 2 hours. 12.17 Add 1.0 mLofmethanol to each centrifuge tube usinag graduated pipette. 12.18 Vortex mix for 30 seconds. 12.19 Attach 0.2 ym nylon mesh filter to a 3 cc syringe and transfer the sample to this syringe. Filter into a 1.5 mL glass autovial or low-volume autovial when necessary. 12.20 Label the autovial with the study number, animal number and gender, sample timepoint, `matrix, final solvent, extraction date, and analyst(s) performing the extraction. 12.21 Cap and store extracts at room temperature or at approximatel4y C until analysis. 12.22 Complete the extraction worksheet, attached to this document, and tape in the study notebook or include in study binder, as appropriate. " 13.0 DATA ANALYSISAND CALCULATIONS 13.1 Calculations ~ 13.1.1 CcaallicburlaattieonacsttuaanldacrodnsceunstirnagtitohnesfoofllPoFwOinSg,eoqruaottihone:r applicable fluorochemical, in mL of standard x concentrationofstandard (ug /m = `mL of standard + mL of surrogate standard + initial matrix volume (mL) Final Concentration (ug/mL)of PFOS in matrix 14.0 METHOD PERFORMANCE 14.1 The method detection limit (MDL) is analyte and matrix specific. Refer to MDLreport for specific MDL and limitofquantitation (LOQ) values (see Attachments B and). 14.2 Tthheeqfuoalliltoywoifntghqeualeixttyraccotnitornolansdamapnalleyssiasr.e extracted with each batchofsamples to evaluate 14.2.1 Method blanks and matrix blanks 14.2.2 Mpraetcriisxiosnpoifketahnedexmtartacrtiixons.pike duplicate samples to determine accuracy and 14.2.3 Continuing calibration check samples to determine the continued accuracyofthe initial calibration curve. 14.3 Refer to section 14 of ETS-8-5.1 for method performance criteria. 15.0 POLLUTION PREVENTION AND WASTE MANAGEMENT 15.1 Sample waste is disposed in biohazard containers, flammable solvent waste is disposed in `high BTU containers, and used glass pipette waste is disposed in broken glass containers located in the laboratory. Ext.raction oEfTPSF8O4S1from Serum Pagesof 14 000054 160 Recorns 16.1 Cnootmepbloeotke otrheinecxlturdaectiinonthweor3k-srhienesttautdtyabcihneddert,o at5hiaspmperotphroida,tea.nd tape in the study 17.0 ATTACHMENTS 17.1 Attachment A, Extraction worksheet 17.2 Attachment B, MDLILOQ values and summary 17.3 Attachment C, Calibration standard concentration worksheet 18.0 Rersrences 18.1 The validation report associated with this method is ETS-8-4.0 & 5.0-V-1. 182 HPFALCTC-ME-le3c.t1r,os"pArnaaylyMsaissosfSSpeecrturmoomretOrtyh"er Fluid Extracts or Fluorochemicals using i 19.0 AFFECTED DOCUMENTS 4 19.0 EETlSec-t8r-o5s.p1r,ay"MAanaslsysSipsecoftrSoemreturmy"or Other Fluid Extracts for Fluorochemicals using HPLC- 200 Revisions : RNeuvmisbieorn Reason For Revision ReDvaistieon 1" SSeeccttiioonn 1122..2113 AChdadnegdetdhetoshinackleurdsepseeadm.ple storage at room temperature. 04/02/99 tSheacnti1o.n01m2L..17 Final volume is 1.0 mL; not adjusted for intial volumes less Ex.tractionoEfsPsFOaSrfrom Serum Page gate 000055 4.12 When handling and clothing. samples or solvents wear appropriate protective gloves, eyewear, 42 Cautions: 4.2.1 Oprpeesrsautreetehxecesoeldvsen4t00pubamrp,sthbeelHoPw11a 0b0acwkilplreinsistuirateeoafut4o0m0abtairc (s5hu8t0d0owpsn).. Ifthe back 422 Do not run solvent pumps to dryness. 5.0 INTERFERENCES 5.1 Tstoormaigneiomrizaenyinptaerrtfoerfenicnesstwruhmeenntaantailoynztihnagtscaommpleess,inTceofnltoanctshwaillthnotthebesaumspeldefoorr esxatmrapclte. 6.0 EQUIPMENT * 6.1 Emoqduiifpimcaetnitonlsisitnedtbheelroawwmdaatyabaesmmoedtihfoiedddienvioartdioenrs.to optimize the system. Document any . 6.11 eMlieccrtoromsapsrsayQiuoantitzraotiIoIntrsiopulreceq.uadrupole Mass Spectrometer equippedwithan 6.1.2 HP1100 low pulse and autosampler solvent pumping system, solvent degasser, column compartment, 7.0 SUPPLIES AND MATERIALS 7.1 Supplies 7.1.1 High purity grade air regulated to approximately 100 psi (house air system) 7.12 HPLC analytical in the raw data column, specifics to be determined by the analyst and documented 7.13 Capped autovials or capped 15 ml centrifuge tubes 8.0 REAGENTS AND STANDARDS 81 Reagents 8.11 Methanol, HPLC grade or equivalent 8.12 M1,iolrle-qQuiTMvawlaetnte,r a(nAdSTbeMptryopveidIe,dalblywaatMeirlluis-eQdTiOn tChiPslmuestshyosdtesmhoourlodthbeerAvTenSdMortype 8.13 Ammonium acetate, reagent grade or equivalent 8.13.1 When preparing different amounts than those listed, adjust accordingly. 8.13.2 2.0 mM ammonium acetate solution: Weigh approximately 0.300 g a20m0m0onmiLumMialcletia-tQe.TM Pwaotuerr,inmtioxau2nt0i0l0amllLsolviodlsumareetrdiicsscoolvnetda.ineSrtcoornetaatinrionogm temperature. Analy.sisofLivEerTESxstraac0t Using ESMS. Page 3af10 000056 82 Standards 8.2.1 Typically prepared dtuwroinmgetthheoedxtbrlaacntkiso,ntpwroocmeadturriex. blanks, Refer taondETeSi-g8h-t6e.e0n.matrix standards are 9.0 SAMPLE HANDLING 9.1 Fresh matrix are stored in csatapnpdeadrdasutaorveiaplrsepoarrceadpwpietdh each 15 ml acnealnytsriisf.ugeExtturbaecsteundtisltaannadlayrsidss. and samples 9.2 Itfeamnpaelryastiusrew,illorberedferliagyereadt,edexattraacptperdoxsitmaantdealryds4anCd,suanmtiplleansamlyasyisbceasntboreepdeartforromoemd. 10.0 QuaurTy ConTroOL 10.1 Method Blanks and Matrix Blanks 10.1.1 cSaoclvhebnattbclhantkosd,emteertmhiondebcloanntkasm,iannadtimoantroirxcbarlraynokvsera.re prepared and analyzed with " 10.1.2 Analyze a method blank and a matrix blank prior to each calibration curve. + 102 Matrix Spikes 10.2.1 Mreactorviexrsypeifkfeisciaernecyp.repared and analyzed to determine the matrix effect on the 10.2.2 Matrix spike duplicates are recovery for each analyte. prepared and analyzed to measure the precision and the 10.2.3 Amnianliymzuemaoma2ftrsipxiskepsikpeearnbdatmcah.trix spike duplicate per forty samples. With a 10.2.4 Mthaetriinixtaslpickaeliabnradtmioantrciuxrvsep.ikAeddduiptliiocnaatlescpoinkceenctornacteinotnrsatwiiolnlsfamllaiynftahlelimnitdh-eralnogwe-of rangeofthe initial calibration curve. 10.3 Continuing Calibration Checks 103.1 Continuing calibration the calibration curve. verifications are analyzed to verify the continued accuracy of 103.2 Analyze a per batch. mid-range calibration standard every tenth sample, with a minimumof one 1.0 CALIBRATION AND STANDARDIZATION 11.1 TAnhaelayvzeertahgeeeoxftrtawcotesdtamnadtarridx csutravndeasrwdislprbieorpltoottaenddbfyolllionweairngreegarcehsssieotno(fys=ammpxle+bex)t,racts. weighted 1/x, not forced through the origin, using MassLynx or other suitable software 11.2 Isfttahnedacrudrcvuerdvoe(eisfnnoetcemseseatryr)eqaunidrermeeannatlsyzpee.rform routine maintenance or reextract the . ETSe0 AnalysisofLiver Extract Using ESMS. 000057 Page 4010 113 uFsoer tphuerplooswesenodfoafcctuhreaccayliwbhraetnioqnucanutrivteatriantghelrotwhalnevtehles ofuflalnralayntgeeo,fittmhaeystbaendnaercdescsuarrvye.to cEaxlaimbprlaet:ionwchurevneactotnesmipsttiinnggotfotqhueansttiatnatdearadpspfrorxoimm5atpeplby t1o0 1p0p0bopfpabnarlayttheer,tgheannertahteefuall rangeofthe curve (5 regression weighting opfpbhitgoh 1co0n0c0enpptbr)a.tioTnhisstawnidlalrdrse.duce inaccuracy attributed to linear 12.0 PROCEDURES 12.1 Acquisition Set up eer 12.1.1 Set up the sample list 12.1.1.1 lAeststiergnofathseamapllpehalbsettfistlaerntaimneg uwsiitnhgaMO-DAY-ast digitofyear-increasing 12.1.12 Assign a method (MS file) or acquiring 12.113 Assign an HPLC program (Inlet file) 12.1.1.4 Type in sample descriptions and vial position numbers : 12.12 sTpoecctrreoatmeetaermehtehaodidngclsicakndonsemleectthoSdIRin(StihnegAlceqTuoinsiRteicoonrcdoinntgr)olorpaMneRl Mthe(nMumlatsisple " * Reaction Monitoring). Set Ionization Mode as appropriate and mass to 499 or other appropriate masses. "A full scan is usually collected along with the SIRs. Save acquisition method. IfMS/MS instruments are employed, additional product ion fragmentation inforrnation may be collected. Refer to Micromass MassLynx GUIDE TO DATA ACQUISITION for additional information and MRM. 12.1.3 Typically the analytical batch run sequence begins and ends with a setofextracted matrix standards. 12.1.4 Samples are analyzed with a continuing calibration verification injected standard after every tenth sample. Solvent blanks should be analyzed periodically to monitor possible analyte carryover and are not considered samples but may be included as such. 122 Using the Autosampler 12.2.1 Set up sample tray according to the sample lst prepared in Section 12.1.1 12.2.2 Set-up analyst tchoensHiPd1e1r0s0a/papurtooprsiaamtpeleforratoptthiemaflolrleoswpionngsec.onRdeictoirodnsaocrtuaatlccoonnddiittiioonnsstihnethe instrument logbook: 12.2.2.1 Sample size = 10 uL injection 12.2.2.2 Inject/sample = 1 12.2.2.3 Cycle time = 9 minutes Analy.sisofLivEerTESx8tr7ac0t Using ESMS 00058 Page sof 10 12.2.2.4 Solvent ramp conditions Time 20mM Ammonium acetate [ooomin|[TM6a 0% 0%| [lOmin TT"7a%[60%| [[6455mmiinn| "ooss%[ | 5 5% %|| [Zomin 1""a | 60% | Loom --T| a0 6%%| 12.2.2.5 Press the "Start" button. 123 Instrument Set-up - 123.1 RTreifpelretQouEaTdSr-u9p-o2l4e.M0a,s"sOpSepreacttiroonmaetnedrMFaiitnttedenwaintcheoanftAhtmeoMsipchreroimcasPsreQsusuartet:ro II . Tonization Source," for more details. : 12.3.2 Check the solvent level in reservoirs and refilif necessary. hd 12.3.3 Check the tip. thTehsetatiinplsehssousltedebl ecafplialtlwairythatnothjeaegngdeodfedtghees.prIobfet.heUtsipe iasnfeoyuenpditeocebet.o check unsatisfactory, disassemble the probe and replace the stainless steel capillary. 12.3.4 Tum on the nitrogen 12.3.5 Open the heaters. tune page. Clicks on operate to initiate source block and desolvation 12.3.6 Open the Inlet Editor. 123.6.1 Set HPLC pump to "On" 12.3.6.2 Set the flow to 10 - 500 uL/min or as appropriate 12.3.6.3 Observe expelled droplets coming with no nitrogen outofthe tip ofthe leaking around the tpirpoobfet.heApfrionbee.misRteasdhjouusltd be the tipofthe probe ifno mist is observed 123.64 Allow to equilibrate for approximately 10 minutes. 123.7 Tchhaenginesitnruomrednetrustoesoptthiemsiezepatrhaemreetseprosnsaet:the following settings. These settings may 12:3.7.1 Drying gas 250-400 liters/hour 12.3.7.2 ESI nebulizing gas 10-15 liters/hour 12.3.7.3 HPLC constant flow mode flow rate 10 - 500 uL/min 12.3.7.4 Pressure HPLC is <op4e0r0atbianrg(cTohrirsecptalyr.a)meter is not set, it is a guide to ensure the 12.3.7.5 Source block temperature 150 12.3.7.6 Desolvation temperature 250 - ETS$70 AnalysisofLiver Extract Using ESMS. Pages of 10 000059 12.3.8 tPraipnetd tihnetotutnhee piangset,ruwmietnht its parameters, log. and store itin the study binder with a copy 12.3.9 CMlaiscskLoynnxstavretrsbiuotntso,nrienfetrhetoAacpqpuriospirtiioanteCoMnatsrsoLlyPnaxneUlse(rth'issGmuaidye)v.aryEnasmuornegstart and `end sample number includes all samples to be analyzed. 13.0 DATA ANALYSIS AND CALCULATIONS 13.1 Calculations: 13.1.4 Calculate matrix spike percent recoveries using the following equation % Recovery = OBbsearvecdReEksxupletgc-terd ReosultunResudlt x 100 13.15 Calculate pesent difference using the following equation: % Difference = ExpCoEnxe cp.e-ctc CealdcCutolnact.eedCodnc. x 100 k4 13.1.6 Calculate actual concentrations in matrix (g/g): " o (ng of PFOS calc. from std. Curvxe Dilution Factor) x Lug (Initial Weight of Liver (g) 1000 ng Final Volume (mL) 14.0 METHOD PERFORMANCE. 14.1 MmaettrhioxdspDeceitfeicc.tioRnefLeirmitto(EMTSD-L8)-6a.n0d,LAitmtiatocfhmQueannttiBtaftoiroanl(sLtOinQg)ofacruermreetnhtovda,liadnaaltyetdeM, DanLd and LOQ values. 14.2 Solvent Blanks, Method Blanks and Matrix Blanks 14.2.1 Sinoltvheenctalbilbarnaktsi,onmecutrhvoe.d blanks, and matrix blanksmust be below the lowest standard 143 Calibration Curves 14.3.1 The r* value for the calibration must be 0.980 or better. 14.4 Matrix Spikes 14.4.1 Matrix spike percent recoveries must be within + 30%ofthe spiked concentration. 145 Continuing Calibration Verification 14.5.1 sCpoinkteidnucionngcecnatlriabtriaotni.on verification percent recoveries must be within 30%of the 14.6 pIfcerriftoerrmieadliosntetdhienstyhsetmeemtahnoddspaemrpfloersmraenacnealseyczteidonoraroethneortamcetti,onmsaaisntdeentaenrcmeinmeadybybethe analyst. Document all actions in the appropriate logbook. Analy.sis ofLiveErTESxstr7ac0t Using ESMS G0VO60 Page Taf 10 14.7 fIfodoattnaotaerdeotno btaeblreespoarntdeddiwshcuesnsepderifnotrhmeatnecxetocfrittehreiarehpaorvte,not been met, the data must be 15.0 POLLUTION PREVENTION AND WASTE MANAGEMENT 15.1 Sample pipette extract waste is waste and disposed ifnlbarmomkaebnleglsaoslsvecnotnitaidniesrspolsoecdatiendhiinghthBeTlaUbocroanttoariyn.ers, and glass 16.0 RECORDS _ _ 16.1 Eheaacdherpaogrehgaennderwartietdtefnoornatshteudpyagme:uststhuadvyeotrheprfoojlelcotwniungmbienrf,oramcaqtuiiosnitiinocnlmudeetdhoedi,ther in the integration analyst. method, sample name, extraction date, dilution factor(ifapplicable), and 16.2 Parpipnrtoptrhieattuensetupdagyef,olsdaerm.pleColspty, atnhedseacpqaugiseistiaonndmteatpheoidntfortohmeMinasstsruLmyennttrounilnocgl.ude in the " 16.3 sPtloortetihnetchaelisbtruadtiyonfolcduerr.ve by linear regression, weighted 1/x, then print these graphs and 16.4. Print data integration summary, integration method, and chromatograms from MassLynx " and store in the study folder. 16.5 AStutmamcarhimzeentdaAtafuosrianngesxuiatmapblleesooffatwsaurmem(aErxcyeslp5r.e0a+d)shaenedt store in the study folder, refer to 16.6 Back up electronic data to appropriate medium. and locationofbackup electronic data. Record in study notebook the file name 17.0 TABLES, DIAGRAMS, FLOWCHARTS, AND VALIDATION DATA 17.1 Attachment A: ETS-8-7.0 Data summary spreadsheet 18.0 REFERENCES _ 18.1 FCAoCmTp-oMu-n2d.s1,fr"oEmxtLriavcetrifoonor fAnPaolytsaisssiUusminPgerHfPlLuCo-roEolcetcatnreossulpfroanya/tMeaosrsOStpheecrtFrloumoerroyc"hemical 18.2 EToTnSi-z9a-t2i4o.n0/,Ma"sOspeSrpaetcitornomaentderMaQiunattetnraonIcIeotrfiptlheequMaidcrruopmoalsesSAytsmteomssp"heric Pressure 183 The validation report associated with this method is ETS-8-6.0 & 7.0-V-1 19.0 AFFECTED DOCUMENTS 19.1 E`TCSo-m8p-6o.u0n,ds"EfxrtormacLtiivoenorforPFoltuaisdsfiourmAPnearlfylsuiosrUosoictnagnHesPuLlCf-oEnlaetectorroOstphreary/FMlausosrochemical Spectrometry" Analy.sisofLivEerTEsxstraac0t Using ESMS 000061 Page 8of 10 woRevSNs RNeuvimsbieorn 0000000000 ReasonForRevision ReDvaitsieon 5 An. aofLlivEeyrTExSts8ra0citUsisng ESIMS 000062 Page9o1f0 Laboratory Study # STeusdtyMaterial MMaettihxoFdiRneavlisSioolnvent IAnnasltyrtuimceanltSEoqfutiwpamreentVeSryssiotnm Number: . FRielSeqnuaamreed Value: SloIpnetercept DDaattee ooff AExntsryascitisoAnn/lAynsalty:st + " DGosre rop e [P SFacetoE r ] " GSiroopuep:/DToaskee:n fTaokmenlirneoarmrteghresstsuidoynfeoqludeart.ion `SCaomnpcelnet:ratTiaoknen(nrg/og)m:thTeaksetnyfrfoomldtehre MassLyn integration summary. IDniiltuitailoWneF.ac(2t)o:r:TaTkaeknenfrformotmhtehsetsutduydfyolfdoelrd.er Final Cone. (g/g): Calculated by dividing he initial volume rom the concentration Atzchment A: Summary Spreadsh`eAenatl.ysisof LivEerTESx$tr7ac0t Using ESMS . Page 100610 000063 SE 3MM EN NVIV RONMI ENTR AL LAO BORN ATTOOM RRYY ENT ~~A ~ L METHOD FLEUXOTRROACCTHIEOMNICOAFLPCOTOAMSPSOIUUNMDPSEFRRFOLMUOLRIOVOECRTFAONREASNUALLFYOSNIASTUESOINRGOHTPHLECRELECTROSPRAY/MASS SPECTROMETRY Method Number: ETS-8-6.0 | Author: Lisa Clemen, Robert Wynne Approved By: Adoption Date: Revision Date: Laboratory Manager Date Group Leader Date `Technical Reviewer Date 1.0 SCOPE AND APPLICATION 1.1 Scope: This method is for the extractionof potassium perfluorooctanesulfonate (PFOS) or other fluorochemical compounds from liver. 1.2 Applicable Compounds: Fluorochemical surfactants or other fluorinated compounds. 1.3 Matrices: Rabbit, rat, bovine, and monkey livers or other tissues as designated in the validation report. Word 6.095 Ex.tractionEoTf sPsOeSofrom Liver Page lof 14 000064 2.0 SUMMARY OF METHOD 2.1 (ThPiFsOSme)tohroodtdheesrcfrliuboesrotchheempircoacledsuurrfeacftoarnetxstfrarcotminlgivpero,taosrsoituhmerpetrifssluueosr,ooucstianngeasnulifoonnaptaeiring reagent and methyl-fert-butyl ether (MtBE). In this method, seven fluorochemicals can be esxttarnadcatredd.: APnFOiSon,pPaiFrOiSngA,reaPgFeOntSAisAa,ddEe{dFtOoStEh-eOsHa,mpPlFeOaSnEdAt,heMa5n5a6ly,teanidonspuarirrogisate partitioned into MtBE. The MIBE extract is transferred to a centrifuge tube and put onto a nitrogen evaporator until dry. Each extract is reconstituted in 1.0 mL methanol then filtered through a 3 cc plastic syringe attached to a 0.2 um nylon filter into glass autovials. 22 These sample extracts are analyzed following method ETS-8-7.0 or other appropriate methods. 3.0 DEFINITIONS 3.1 PFOS: perfluorooctanesulfonate (anion of potassium selt) CiFyrSOs tr 3.2 PFOSA: perfluorooctane sulfonylamide CsF1;SO:NH, + 33 PFOSAA: perfluorooctane sulfonylamido (ethyl)acetate CiFirSON(CH,CH:)CH:CO 3.4 ECtaFFO1S7ES-OO0H::N(2C(HN-;eCtHhsyl)pCeHr:flCuHoOrHooctane sulfonamido)-ethyl alcohol 3.5 PFOSEA: perfluorooctane sulfony ethylamide CaF17SO;N(CH;CHy)H 3.6 MSS6: CaF SON(H)(CH,COOH) 3.7 Surrogate standard: 1H-1H-2H-2H perfluorooctane sulforic acid 4.0 WARNINGS AND CAUTIONS 4.1 Health and Safety Warnings: 4.1.1 Uhasnedluinnigvearnsiamlaplreticsasuutei,onwsh,iceshpemcaiyallcyonltaabionraptaotrhyogceonast.s, goggles, and gloves when 5.0 INTERFERENCES 5.1 There are no interferences known at this time 6.0 EQUIPMENT 6.1 The following equipment is used while performing this method. Equivalent equipment is acceptable. 6.1.1 Ultra-Turrax T25 Grinder for grinding liver samples 6.1.2 Vortex mixer, VWR, Vortex Genie 2 6.1.3 Centrifuge, Mistral 1000 or IEC 6.1.4 Shaker, Eberbach or VWR. E.xtractionoEfsPsFOeS fiom Liver _ 00VO6S Page 20f 1s 6.15 Nitrogen Evaporator, Organomation 6.1.6 Balance (sensitivity to 0.100 g) 7S .0 UPPLIESANDMATERWAL 00S 0 7.1 Gloves 7.2 Dissecting scalpels 7.3 Eppendororf disposable pipettes 7.4 Nalgene bottles, capableofholding 250 mL and 1 L 7.5 Volumetric flasks, glass, type A 7.6 T-CHEM vials, 40 mL glass. 7.7 Plastic sampule vials, Wheaton, 6 mL (or appropriate size) 7.8 Centrifuge tubes, polypropylene, 15 mL 7.9 Labels 7.10 Oxford Dispensor ~3.0 to 10.0 ml " 7.11 Syringes, capableofmeasuring 5 uL 10 50 pL : 7.12 Graduated pipettes 7.13 Syringes, disposable plastic, 3 cc 7.14 Syringe filters, nylon, 0.2 um, 25 mm 7.15 Timer : 7.16 Crimp cap autovials and caps 7.17 Crimpers Note: Prior to using glassware and bottles, rinse 3 times with methanol and 3 times with Milli- va ias. water. Rinse syringes a minimum of9 times with methanol, 3 rinses from 3 separate 8.0 REAGENTS AND STANDARDS 8.1 Type reagent grade water, Mill-QTM or equivalent; all water used in this method should be Milli-Q water and be provided by a Milli-Q TOC PlusTM system 82 Sodium hydroxide (NaOH), J.T Baker or equivalent 83 Tetrabutylammorium hydrogen sulfate(TBA), Kodakor equivalent 84 Sodium carbonate (Na;COx), J.T. Baker or equivalent 8.5 Sodium bicarbonate (NaHCO), J.T. Baker or equivalent 8.6 Methyl-tert-butyl ether, Omnisolv, glass distilled or HPLC grade 8.7 Methanol, Omnisoly, glass distilled or HPLC grade 88 Liver, frozen from supplier 89 Dryice from supplier 8.10 Fluorochemical standards 8.10.1 PFOS (3M Specialty Chemical Division), molecular weight = 538 E. ximEoTfSFSF8O6S0fom Liver 0060 Page 3of 14 8.10.2 PFOSA (3M Specialty Chemical Division), molecular weight = 499 8.10.3 PFOSAA (3M Specialty Chemical Division), molecular weight = 585 8.10.4 E{FOSE-OH (3M Specialty Chemical Division), molecular weight = 570 8.10.5 PFOSEA (3M Specialty Chemical Division), molecular weight = 527 8.10.6 MSS6 (3M Specialty Chemical Division), molecular weight = 557 8.10.7 Surrogate standard: 4-H, perfluorooctane sulfonic acid (1-H, 1-H, 2-H, 2-H C4F;5SO5H) molecular weight = 428 8.10.8 Other fluorochemicals, as appropriate 811 Reagent preparation NOTE: When preparinglarger volumes than listed in reagent, standard, or surrogate preparation, adjust accordingly 8.11.1 10N sodium hydroxide (NaOH): Weigh approximately 200 g NaOH. Pour into a 1000 mL beaker containing 500 mL Mill-QTM water, mix untilal solids are dissolved. Store ina 1 L Nalgene bottle. 1 8.1"1.2101 NNs'oNdaiOumHhsyodlruotixoindein(tNoaaOH1)0:0 mDLilvutoelu1m0etNriNcaflOasHk a1:n1d0.dilMuetaestuorveol1u0mmeLusoifng * Milli-QTM water. Store in a 125mLNalgene bottle. 8.11.3 0o.5fTMBAteitnrtaobautIyLlavmomlounmieutmrihcycdornotgaeinnisnuglf5a0te0(mTLBA)Mi:lliW-eQi"ghwaaptperr.oxAidmjautsetlyto169 g PH 10 using approximately 44 to 54 mLof 10 N NaOH (While adding the last mL. ofNaOH, add slowly because the pH changes abruptly). Dilute to volume with Mill-QTM water. Store ina 1 L Nalgene bottle. 8.1.3.1 TBA requires a check prior to each use to ensure pH = 10. Adjust as, needed using 1 N NaOH solution. 8.11.4 0a.p2p5roMximsaotdeiluym2c6a.r5bgoonaftes/osdoiduimumcabribcoanrabtoena(tNeab:uCfOfse)r (Na;COy/NaHCO): Weigh and 21.0 gofsodium bicarbonate (NaHCO5) into a 1 L volumetric flask and bring to volume with Milli- QTM water. Storeina 1 L Nalgene bottle. 512 Standards preparation 8.12.1 Prepare PFOS standards for the standard curve. 8.12.2 Prepare other fluorochemical standards, as appropriate. Multicomponent. fluorochemical standards are acceptable (for example, oneworking standard solution containing 1.00 ppm PFOS, 1.02 ppm PFOSA, 0.987 ppm PFOSAA, and 1.10 ppm EtFOSE-OH.) 8.12.3 Weigh approximately 100 mg of PFOS into a 100 mL volumetric flask and record the actual weight. 8.12.4 Bring to volume with methanol for a stock standardof approximately 1000 ppm (ng/mL). 8.12.5 Dilute the stock solution with methanol for working standard 1 solution of approximately 50 ppm. E. ximEolTsEsOeSoonLive (00.0677 Page dof 14 8.12.6 Dilute the stock solution with methanol for a working standard 2 solution of approx. 5.0 ppm. 8.12.7 Dilute the stock solution with methanol for a working standard 3 solution of approx. 0.50 ppm. 813 Surrogate stock standard preparation 8.13.1 Weigh approximately 0-60 mg of surrogate standard 1-H,1-H, 2-H, 2-H, `CaF:5SOsH into a 50 ml volumetric flask and record the actual weight 8.13.2 Bring to volume with methanol for a surrogate stockof approximately 1000-1200 ppm. 8.13.3 Prsetpoacrketaosaur1r0ogmaltevowlourmkeitnrigcsftlaansdkaradn.d Tbrrianngsfteorvaoplpurmoxeiwmiatthelmyet1h.a0nmollofforsuarrogate working standard of 10-20 ppm. Record the actual volume transferred. 9.0 SAMPLE HANDLING " 9.1 All samples are received frozen and must be kept frozen unil the extraction is performed. : 10.0 OuALITY ConTROL "10.1 Matrix blanks and method blariks 10.1.1 An aliquot of 1.0 mL methanol is used as a solvent blank 10.1.2 Extract two 1.0 mL aliquots of Milli-QTM water following this procedure and use as method blanks. 10.1.3 Extract two 1.0 mL aliquotsof iver homogenate following this procedure and use: as matrix blanks. Refer to 11.16. 102 Matrix spikes 10.2.1 Prepare and analyze matrix spike and matrix spike duplicate samples to determine the accuracyofthe extraction 10.2.2 Prepare each spike using a sample chosen by the analyst, usually a control liver received with each sample set. 10.2.3 Expected concentrations will fallin the mid-rangeof the initial calibration curve. `Additional spikes may be included and may fal in the low-rangeofthe initial calibration curve, 10.2.4 Prepare one matrix spike and matrix spike duplicate per 40 samples, with a minimum of2 matrix spikes per batch. 10.3 Continuing calibration verifications 10.3.1 Prepare continuing calibration verification samples to ensure the accuracyofthe initial calibration curve. 10.3.2 Prepare, at a minimum, one continuing calibration verification sample per group of 10 samples. For example, ifa sample set = 34, four verifications are prepared and extracted. E.xtractionoEfTSPF8O6S0from Liver COVES Page Sof 14 10.3.3 Prepare each continuing calibration verification from the same matrix used to prepare the initial curve. 10.3.4 The expected concentrations will fall within the mid-rangeof the initial calibration curve. Additional spikes may be included that fall in the low-rangeofthe initial calibration curve. This is necessaryifthe analyst must quantitate using only the l1o0w00enppdob)f. the calibration curve (for example, 5 pp--b 100 ppb, rather than 5 ppb -- 11.0 CALIBRATION AND STANDARDIZATION 11.1 Prepare matrix calibration standards 11.1.1 Weigh approximately 40 gofliver into a 250mLNalgene bottle containing 200 `mLs Milli-QTM water. Grind to a homogeneous solution. 11.1.2 Irfat4io0. g is not available, use appropriate amounts of liver and water to ensure a 1:5 11.1.3 Refer to 13.0 to calculate the actual densityofliver homogenate and the " concentrationofsolid liver tissue dispersed in 1.0 mLof homogenate solution. 11.1.5 Add 1 mLof homogenate to a 15 mL centrifuge tube. Re-suspend solution by - shaking between aliquots while preparing a totalofeighteen 1 mL aliquots of homogeneous solution in 15 mL centrifuge tubes. 11.1.6 Two 1 mL aliquots, or other appropriate volume, serve as matrix blanks. 11.1.7 Typically use the standard concentrations and spiking amounts listed in Table 1, at the endofthis section, to spike, in duplicate, two standard curves, fora total of eighteen samples, two matrix blanks, and two method blanks. 11.1.8 Refer to validation reports ETS-8-6.0 and ETS-8-7.0-V-1 or Attachment B, `which lists the working ranges and the Linear Calibration Range (LCR) for calibration curves. 11.19 Use Attachment C as an aid in calculating the concentrationsofthe working ssttaannddaarrddss.. Refer to 13.0 to calculate actual concentrationsof PFOS in calibration 11.2. Tsuorreoagcahtewowrokriknigngstsatnadnadradr,dbfloarnkt,heorcocnocnetnitnruaitnigonvetroiffiacaltiwoint,hianddthaepcparloipbrriaattieonacmuoruvnetroanfge ppb -- 1000ppb. E.xtractonoEfTsFsOeSofrom Liver G0005y Page of 14 11.3 Extract spiked liver homogenates following 12.14-12.25ofthis method. Use these standards to establish each inital curve on the mass spectrometer. Table 1 Approximate Spiking Amounts for Calibration Standards Working Standard Wl "Approx. final conc. of (Approx. Con. PFOS in liver rT Bak | [oso|pp 10 m[ 0025pn [_o|s4o 0 | p01p 00ppmm| ' [ Soppm | 10 | 0250ppm of sopm 120 | 0500pm " [Soppm | 30 | 0.750 ppm (C_|S4 o | p1p 00ppmm| 12.0 PROCEDURE 12.1 Obtain frozen liver samples. 12.2 Cut approximately 1 gof liverusing a dissecting scalpel. This partofthe procedure is best `performed quickly, not allowing the liver to thaw. 12.3 Weigh the sample directly into a tared plastic sampule vial. 12.4 Record the liver weight in the study notebook. 12.5 Return unused liver portions to freezer. 12.6 Add 2.5 mLs ofwater to sampule vial. 12.7 Grind the sample. Put the grinder probe in the sample and grind for about 2 minutes, or until the sample is homogeneous. 12.8 Rinse the probe into the sample with 2.5 mLs water usinag pipette. 12.9 Take the grinder apart and clean it with methanol after each sample. Refer to AMDT-EP- 22. 12.10 Cap the sample and vortex for 15 seconds. Label the sampule vial with the study number, weight, liver ID, date and analyst initials. Ea xtractiono) fPFOS from Liver 060070 Page Tor 14 12.11 cPeinptertitfeug1e.0tumbeL.,Loarbeotlhtehreacpepnrtorpirfiuagteetvuobleuwmiet,hotfhehoimdeongteicnaaltienfinotromati1o5nmasLtphoelsyapmrpoupylleevnieal Refer to attached worksheet for documenting the remaining steps. 12.12 mPeitpehtotde btlwaonks1. mL aliquotsofMilli-QTM water to centrifuge tubes. These will serve as 12.13 sStpainkdearadllassadmepslcersi,biendclinudsiencgtibolnar1i1k.s2.and standards ready for extraction with surrogate 12.14ofSptihkaet esaeccthiomna,tfionr twhiethcatlhiebraaptpiroonpcruiartvee astmaonudanrtdos.f sAtlasnodaprrdepasardeesmcartirbiexdsipnik1e1s.1a,nodr Table 1 continuing calibration standards. 12.15`Vsoamrptleexs mfoirx 1t5hessetcaonnddasrd curve samples, matrix spike samples, and continuing calibration 12.16 Check to ensure 0.5 M TBA reagent is at pH 10. Ifnot, adjust accordingly. . 12.17bTiocaerabocnhastaembpulfef,eradd 1 mL 0.5 M TBA and 2 mLofthe 0.25 M sodium carbonate/sodium : 12.18 Using an Oxford Dispenser, add mL methyl-tert-butyl ether. . 12.19 Cap each sample and put on the shaker at a setting of 300 rpm, for 20 minutes. 12.20 Centrifuge for 20 to 25 minutes at a settingof 3500 rpm, or until layersarewell separated. 12.21 Label a fresh 15 mL centrifuge tube with the same information as in 12.10. 12.22 Remove 4.0 mLofthe organic layer to the fresh 15 mL centrifuge tube. 12.23 Put each sample on the analytical itrogen evaporator until dry, approximately 1 to 2 hours. 12.24 Add 1.0mLto each centrifuge tube using a graduated pipette. 12.25 Vortex mix for 30 seconds. 12.26 FAitlttearcihntao0a.21.u5mmnLylgolnasmseasuhtofviilatlerotrolao3w-cvooslyurmiengaeutaonvdiatlrawnhsfeenr ntehceesssaamrpyl.e to ths syringe. 12.27 mLaatbreixl,tfhienaalutsoovlivaelntw,ietxhtrtahcetsitounddyatneu,mabnedr,anaanliymsatl(sn)upmebrefroramnidnggetnhdeeerx,trsaactmipolne. timepoint, 12.28 Cap and store extracts at room temperature or at approximately 4 C until analysis 12.29oCroimnpclleutdee itnhesteuxdtyrabcitnidoenr,woarskasphpereotp,riaattet.ached to this document, and tape in study notebook E.xtractionEoTfSPF8O6S0from Liver 000071 Pages of 1s 13.0 DATAANALYSISAND CALCULATIONS 13.1 Calculations: 13.1.1 Cseaplacrualtaete1t.h0emaLvearlaigqeuodtesnosfithyoomfotgheenaltivee.r homogenatebyrecording each massoften Average density (mg/mL) = Avme ass(r mg)oa ftheg aliqe uots 1.0 mL aliquot 13.1.2 Cdailscpuelrasteed tsholeiadmtoisusnuteopferlimveLro(fmgh)ompoegre1n.a0tmeLsuhsopmenosgieonna)tuesi(nogr ctohnecfeonltlroawtiinogn of equation fLiver x * of homogenate (mg/mL) (8ofLiver + gofWater) *refer t0 13.1.1 for details. 13.1.3 Calculate actual concentrationsof POS and other fluorochemicals in calibration ~ standards using the following equation: uCl oofSntmangcdLairevdexrn/ 1tmLrhaomotgen(iuagto.emLn) =oFifnaPlFCOoSnceinntLriavteiron (ug/g or mg/kg) *refer t0 13.1.2 for details 14.0 METHOD PERFORMANCE 14.1 TspheecimfiectMhoDdLdeatnecdtliiomnitolifmitqu(aMntDiLt)atiiosnan(aLlyOtQe)avnadlumeastr(irxefseprectiofiAc.ttRaecfhemrentotsMBDLanrdepCo)r.t for 14.2 Tthheeqfuoalllitoywoifngthqeualeixttyraccotnitornolansdamapnlaelyssaisre extracted with each batchofsamples to evaluate 14.2.1 Method blanks and matrix blanks. 14.2.2 Matrix spike and matrix spike duplicate samples to determine accuracy and precisionofthe extraction. 14.2.3 Continuing calibration verification samples to determine the continued accuracy of the iniial calibration curve. 143 Refer to section 14of ETS-8-7.0 for method performance criteria 15.0 POLLUTION PREVENATNDIWAOSTNE MANAGEMENT 15.1 hSiagmhplBeTwUasctoentiasindeirssp,osaenddinubseidohgalzasasrdpicpoentttaeiwnearsst,efilsadmimsapbolseedsoilnvberntokweanstgelaisssdciosnptoasienderisn Tocated in the laboratory. Ext. ractionoEfTSPF8O6Sf0romLiver - 600072 Pager 14 160 REcorps ------ 16.1 oCromipnlcleutdeetihnetehxet3r-arcitnigonstwuodryksbhinedeetr,atatsaacphperdoptroitahties.method, and tape in the study notebook 17.0 TABLES. DIAGRAMS, FLOWCHARTS, AND VALIDATION DATA 17.1 Attachment A, Extraction worksheet 17.2 Attachment B, MDL/LOQ values and summary 17.3 Attachment C, Calibration standard calculation and concentration worksheet 18.0 REFERENCES 18.1 The validationreportassociated with this method is ETS-8-6.0 & 7.0-V-1. 182 AMDT-EP-22, "Routine MaintenanceofUltra-Turrax T-25" " 183 LFiAvCeTr-fMo-r1A.n1al,ys"iEsxtUrsaicntigoHnoPLfC-PEFlOeSctorrosOtphreary/AMnaisosniScpeFclturoormoecthremyi"cal Surfactants from 19.0 AFFECTED DOCUMENTS 19.1 LEiTvSe-r8-E7x.t0r,act"sAnuasliynsgiHsoPLfCP-oEtlaescstiruomspPrearfylMuoarsosocStpaencetsruolfmoentartye"or other Fluorochemicals in 20.0 REVISIONS Revision Revision Number. ReasonForRevision Date E.xtractionEoTf BSR8O6S0from Liver 600073 Page 10of 14 Study #_ Surrogate Std FC Mix Std FC Mix Std FC Mix Std `Comments Matrix |approx. ppm| approx. 0.5 ppm| approx. S ppm | approx. 50 ppm Box#, actual ppm| actual ppm | actual ppm | actual ppm WkDaDteaSypi_ked_/A_na_ly_st |#______\# (#0(#4 r eo p r r r Y ws [r T r r rr r r r r T r rr rr EE | r rr T r rr Tr ] rr SSSS ESTrEE r rr r rr rr rr rr rr rr rT 1 r > rr rrr --r ------ rrr rrT --rrr11 `Cont. Cal. Verifiucsadtthieosanmse matasrfoirtxh standardcurve. AtschmenAt: Extraction Worksheet.ExtracEotfTiSF8oO6Snf0rom Liver Page 110f14 000074 MDL/LOQ values for rabbit liver Compound| MDL | LOQ| Linear Calibration Range (LCR) (ppb) | (ppb)| Approximate concentrations to be used for preparing the Standard Calibration Curve PFOS | 845 [269[30ppb 1200 ppb PPFFOOSSAAA |35 | 10 1.1[1320 ppppb --b1122000ppp0pbb EtFOSE-OH|_108__|345 | 60 ppb -- 900 ppb* [Mss6 PFOSEA T78|22632 | |33 | 19 08 | 60 30 pp=b 1200 ppb ppb- 1200 ppb MinDeLac/hLoOfQthevasleumeastirnicraets, wboevrienee,xtarnadctmedonaknedyanlaivleyrzweedrweitnhotthsetartaibsbtiictallliyvedretceurrmviensedt.o deTtweormicnuerves reqeusipvoanlseensc,et.heRreefsoproen,stehseiirnMthDeLrata,nbdoLviOneQ, wainldl bmeonaksesuymleidvetrocbureveeqsuiwvearleenetqutiovtahloenste tvoaltuheesraasbbit . determined for the rabbit liver. [on ear Be | om mo | * *REe{tFfoOLSeEOr-QOHSuesmtmiamartyesanodnlyMfDoLrsMtDudLyianndELTOQS. -Di8d&-n7o6t.0m.-eVe0-t1fcroirtefruiratfhoerrivnalfiodramtaitoinon Compound: PFOS Liver | Prraempgaeroefd|| Raavnegregoef aLCwRfieom|| Rlaonwgsedof | LClRoFwsodm|| Rhaingghosodf | LOhiRghEeOtdW matrix| standards| curve ave ane ane uve. [me eam]ea |ee Compound: PFOSA Liver | Prraenpgaeroefd|| Raavngregof| LaCeRamfvoem|| Rlaonwgseodf | LOlRwfsodm|| Rhaingghosodf | LhOiRgEhSsH matsix| sandarss| curve ane are aire ave [un]S i B |Tn] Compound: PFOSAA Liver | Prrnegpaeroefd|| Raawnrgegoef matrix |62st)andaarmds || ee)curvent) LaOwRafrnoem|| Rlaonwgsodo 0) oxa) ne LlCoRwfsodm|| Rhaimgghewodf|| LhOiRgEhosm a2)r0e 6a2)ngemt) (o)curevpe ) Attachment B: MDLILOQ Values Ex.tractionoEfTSPF8O6S0from Liver 600O7S Page 120114 aoe|| 2 | Se Compound: EtFOSE-OH Prepared Rangeof LCR from Rangeof maT Eer : LCRfrom Rangeof LCRfrom F or | Emo ETrER Compound: PFOSEA Prepared. Rangeof LCR from Rangeof LCR from erRangeof LCR from cm] EEE Compound: M556 Liver |Prreapnagreeofd|Raanvegreaogef REE LCaRvefcruvoem||Ralnogwesodf LCRlofwrsdom| Rhaingghesodf LChRifgrhosm mss.oweT e es 000076 Ton Pair Standard Curves -- Tissue Prep date(s): Analyte(s): Sample matrix: Methodrevision: Targetanalyte(s): FFCCmmiixxssttddaappppr50r..o05o000pxpxppm.m:.: SFuCrrmoigxatsetdsatdpparpopxr.ox5.0.010p0pmp:pm: Standard number: Equipment number: Final solvent and TN: Blank liver/identifier: ASctPtdFucOaoSln|ec|oncSPeuFndtOcroSanAtci||onsPSotOfdsSctoAnaAc|n|dESaitIrdnFcOtdoShnsEce||FCPSFimdOicxSoEncA|| SiMdSco%nc | Sidconc |AmtIspiked| DenAsiity ug| ugnl | wpm | uml | ym | ugmi | ugm nL g : [0500T0500 [0500 [0500 "0.500 T0500 "0500| 050 | 050 | 0.50 |050|_| |0500 |_| 0002 000s | 0167] | 0167] ose [0500TT0o5s0e0[T0o.5s0o0 0500|os00|0500 || "0500 | 050 |0500 |__| ooo 0020 |oisr| | 0167| [050 T0500T0500 | 050 |050 | 0500 | [300T7500T7500 |"s00 | 500 | 500 | | 000 | 0167| | ooo | oie [500T7500 [500T7500 "500 "500 | | 500 500 | | 500 s00 | | 500 500 | | | 000 |0167 | | oo [500T7500 "500 "500 | s00 | 500| | 0004 | 0167| CaPFlRicnOaulSla||tedPcFFoiOnnaScleA|n|trFaPitFaiOlooSnosAnAoc|f|stEa(FniFdnOaaSlrEd||s iPnFFtiOhnSealEsA|a|mplMFeiSnmaSalt|rix Sweonc | SSurcroognaete|| AAmNt cnc | conc | ong | com | conc | come | ng | ngml | spiked [590 T7599 T7590 | s90 | 599 |se9 | | Pasteur busfaefT oo | 000s| IE 1 IE nr SFlolconc I [299T2900 [39% [soo so 1 20 | 299 | |500 | 599 | 99 seo | | | oso | | [898 eos mos | sos | wos | wes | | (nos Twos| 1198 | 1198 | ios | nos | | [LValb idated rang[es~- prapoprsoxim|atepcorncoenstra|tionsProsi |ewoseom| oar | PrOSEA| 2 Rat estimates anywembt mies ------ AtachmenCt: Standard CalculationsE. xtroEfTaSPF:cO6St0froimLoivner Page lof 14 600077? 2I 33M EN NVIV RONMI ENTAR L LAO BORRN AATTOOM RRYY E ~N ~T ~AL METHOD ANALYSIS FOLFUPOORTOACSHSEIMUIMCPAELRSFILNUSOERROUOCMTEAXNTERSAUCLTFSOUNSAITNEGOR OTHER HPLC-ELECTROSPRAY/MASS SPECTROMETRY Method Number: ETS-8-5. 1 I . Author: Lisa Clemen, Robert Wynne Approved By: `Adoption Date: 03/01/99 Revision Date: Laboratory Manager Date Group Leader Date Technical Reviewer Date 0_SCOPE AND APPLICATION 1.1 Scope: This method describes using HPLC-electrospray/mass the analysisofserum spectrometry. extractsfor fluorochemical surfactants 1.2 Applicable Compounds: Fluorochemical surfactants or other fluorinated `compounds, or other ionizable compounds. 1.3 vMaaltirdiacteiosn: reRpaobrbt.it, rat, bovine, monkey, and human serum, or other fluids as designated in the Word 695 Analys-isofSerEuTmsExetsratct Using ESMS. 000078 Page 10r9 2.0 SUMMARY OF METHOD 2.1 Tohtihesrmfeltuihdos,dudseisncgriHbPeLsCt-heeleancatlryossisporfayf/lmuaosrsocshpeemcitcraolmesturryf,acotranstismielxatrrascytsetdemfraosmapsperrouprmiaotre. `fTlhueoraoncahleymsiicsails,pseurcfhoramsetdhebypemrofnliutoorroioncgtaanessiungllfeoniaotnec(haPrFaOctSe)riasntiiocno, fma/zpa=rt4i9c9u.lar tAdhdeiitdieonntailtlyy,osfaamcpolmepsomuanydbbeyadneatleyczteidngusdianugghatteranidoenmsomfasthsesppaercetnrtomioent.er to further verify 3.0 DEFINITIONS m-- ce ---- 3.1 Atmospheric Pressure Ionization (API): The Micromass Quattro II triple quadrupole isnytsetrfeamcsesa.llTohwefsoer various include methodsofionization but are not limited to: by utilizing various sources, probes, and Electrospray Ionization (EST), Atmospheric Pressure chemical Ionization (APC), Thermospray, etc, The ionization process in these techniques occurs at atmospheric pressure (i.., not under vacuum). 32 Electrospray lonization (ES, ESI): a methodof ionization performed at atmospheric ": Tprheessseurceh,awrhgeerdedbryopiloentssianresoplrutoidouncaerdebtyratnhsefearprpeldictaotitohneogafas pshtarosnegveilaetcitrniyccalhafriegledd droplets. * 33 (MSM/aMsSs):SpeTchterAoPmeTtQruya,ttMraossIl Strpiepcletrqouamdertueprol(eMSs)y,stTeamsndareemeqMuaispspeSdpweictthrqoumaedtreurpole mass sseulbescetqiuveendteltyecdteotresc.tedT.onAs asriensgelleeMctSivemlayydibsecreimmipnlaotyeeddbyfomraisosn tdeotcehcatriogne orrataios(ermi/ezs)(aMnSd/MS) for more specific fragmentation information. 3.4 Conventional vs. Z-spray probe interface: The latest modelsof Micromass Quattro II triple quadrupole systems (post 1998) utilize a "Z-spray" conformation. The spray emitted from a probe is orthogonal to the cone aperture. In the conventional conformation it is aimed directly at the cone aperture, after passing through a tortuous pathway in the counter electrode. Though the configuration is different, the methodsofoperation, cleaning, and `maintenance are the same. However, Z-spray components and conventional components are not compatible with one another, but only with similar systems (i.., Z-spray components are compatible with some other Z-spray systems, etc.) 3.5 Mass Lynx Software: System software designed for the specific operationofthese Quattro II triple quadrupole systems. Currently MassLynx has Windows 95 and WindowsNT 4.0 versions. All versions are similar. For more details see the manual specific to the Giunisdter)u.ment (Micromass Quattro II triple quadrupole MassLynx or MassLynx NT User's 4.0 WARNANIDCNAUTGIOSNS 41 Health and Safety Warnings: 4.11 Use caution with the voltage cables for the probe. When engaged, the probe employs a voltageof approximately 5000 Volts. Analys.isofSerEumTsExstsraict Using ESMS C0007 Page 2069 4.1.2 Wanhdecnlohtahnindgl.ing samples or solvents wear appropriate protective gloves, eyewear, 4.2 Cautions: 4.2.1 Do not operate solvent pumps above capacity of400 bar (5800 psi) back pressure. If the back pressure exceeds 400 bar, the HP1100 will initiate automatic shutdown. 42.2 Do not run solvent pumps to dryness. 5.0 INTERFERENCES 5.1 To minimize interferences when analyzing samples, teflon should not be used for sample storage or any part of instrumentation that comes in contact with the sample or extract. 6.0 6.1 EQUIPMENT ER Equipment listed below may be modified inI order to optimiTzeEthe sA ystem. H Documeonyt-_an_yhr modifications in the raw data as method deviations. kK} 6.1.1 MelieccrtoromsapsrsayQuioantitzraotiIoTntrsiopulrecqeuadrupole Mass Spectrometer equipped with an 6.1.2 HP1100 low pulse solvent pumping system, solvent degasser, column compartment, and autosampler 7.0 SUPPLIES AND MATERIALS 7.1 Supplies 7.1.1 High purity grade nitrogen gas regulated to approximately 100 psi (House air system) 7.1.2 HPLC analytical column, specifics to be determined by the analyst and documented in the raw data. 7.1.3 Capped autovials or capped 15 mL centrifuge tubes 8.0 REAGENTS AND STANDARDS 81 Reagents 8.1.1 Methanol, HPLC grade or equivalent 8.1.2 Milli-QTM water, all water used in this method should be Milli-QTM water or equivalent, andmaybe provided by a Milli-Q TOC Plus system or other vendor 8.1.3 Ammonium acetate, reagent grade or equivalent 82 Standards 8.2.1 Typically two method blanks, two matrix blanks, and eighteen matrix standards are prepared during the extraction procedure. See ETS-8-4.1. 9.0 SAMPLE HANDLING Analys.is of SerEuTmSExstsra1ct Using ES/MS 600080 Page 30r9 9.1 Fresh matrix are stored in csatapnpdeadrdasutaorveiaplrsepoarrceadpwpietdh 1e5acmhLancaelynstirsi.fugEexttruabcetseudntsitlaanndaalrydsiss.and samples 9.2 aIfpapnraolxyismiastweillyl 4be Cd,eloaryeadt,reoxotrmactteemdpesrtaatnudraer,dsunatinldasnaamlypsliessccaannbbeepreerffroirgmeerda.ted at 10.0 QuaLITy ConTROL 10.1 Solvent Blanks, Method Blanks and Matrix Blanks 10.1.1 Solvent blanks, method blanks and matrix blanks are each batch to determine contamination or carryover. prepared and analyzed with 10.1.2 Analyze a method blank and a matrix blank prior to each calibration curve. 102 Matrix Spikes 10.2.1 Matrix spikes are prepared recovery efficiency. and analyzed to determine the matrix effect on the .T 10.2.2 Matrix spike duplicates are recovery for each analyte. prepared and analyzed to measure the precision and the . 102:3 `Amnianliymzuemaomaf2trsipxiksepsikpeeranbdatmcah.trix spike duplicate per forty samples, with a 10.2.4 tMhaetriinixtisalpickaeliabnradtimoantrciuxrvsep.ikAeddduiptliiocnaatlescpoinkceenctornacteinotnrsatwiiolnlsfamlaliynftahlelimnitdh-eralnogwe-of rangeofthe initial calibration curve. 10.3 Continuing Calibration Verifications 103.1 tChoentcianliubirnagticoanlicburravtei.on verifications are analyzed to verify the continued accuracy of 103.2 Analyze a mid-range of one per batch. calibration standard after every tenth sample, with a minimum 11.0 CALIBRATION AND STANDARDIZATION 11.1 aAvnearlaygzeeotfhteweoxtrsatcatneddarmdatcruirxvesstawnidlalrbdes pplroitotretdobaynldinfeoalrlroewgirnegsesiaocnh(syet=ofmeyxt+rabc)t,s.weiTghheted 1x, not forced through zero, using MassLynx or other suitable software. 11.2 Isfttahnedacrudrcvuerdvoee(sifnnoetcemseseatryr)eqaunidrermeeanntalsy,zep.erform routine maintenance or reextract the 113 uFsoer tphuerplooswesenodfoafccthueraccayliwbhraetnioqnucanutrivteatriantghelrotwhalnevtehlseoffulalnarlayntgee,ofitt hmaeystbaenndeacredscsuarrvye.to cEaxlaimbprlaeti:onwchurevneactotnesmipsttiinnggotfo tqhueansttiatnadtaeradpspfrroxoimmSatpeplby t1o0 1p0p0bopfpabnraaltyhteer,tgheannertahteefuall rangeofthe curve (5 ppb to regression weightingofhigh c1o0n0c0enptpbr)a.tioTnhisstawnidlalrdrse.duce inaccuracy attributed to linear Analys.isofSerEuTmsExatsraict Using ES/MS 0V081 Pagedof9 12.0 PROCEDURES 12.1 Acquisition Set up ese---------------- 12.1.1 Click on start button in the Acquisition Control Panel. Set up a sample list. Assigna filename using MO-DAY-last digitofyear-sample number, assign a method (MS) for acquiring, and type in sample descriptions. 12.1.2 To create a method click on scan button in the Acquisition control panel and select SIR (Single Ion Recording) or MRM. Set Ionization Mode as appropriate and mass 10 499 or other appropriate masses. Afull scan is usually collected along with the SIRs. Save product ion facrqaugimseinttiaotnimoentihnofdo.rmIaftiMoSn /mMaSy instruments be collected. arSeeeemMpilcoyreodm,asasddMiatisosnLalynx R`eGaUcItDiEonTMOoniDtAorTinAg)A.CQUISITION for additional information and MRM (Multiple 12.1.3 sTytpaincdaalrldys tahnedaennaldystiwciatlhbaatscehtorfuenxtsreaqucetnecdembaetgriinxsswtiantdharadss.etofextracted matrix 4 12.+".1.4 sSaammpplle.es Saroelvaennatlbylzaendkwsisthhoaulcdonbteinaunianlgyzceadlipberraitoiodniccahlleycktoinmjoercitteodrafptoesrsiebvleeryantaelnytthe . carryover and are not considered samples but may be included as such. 12.2 Using the Autosampler 12.2.1 Set up sample tray according to the sample list prepared in Section 12.1.1. 12.2.2 Set-up the HP1100/autosampler at the following conditions or at conditions the analyst considers appropriate for optimal response. Record actual conditions in the instrument logbook 12.2.2.1 Sample size = 10 pL injection 12.2.2.2 Inject/sample = 1 12.2.2.3 Cycle time = 13.5 minutes 12.2.2.4 Solvent ramp = Time MeOH 20mM Ammonium acetate [00min | a0% | 60% | [ilomn| 00% | 10% | [20min | a0%| 60% | 12.2.2.5 Press the "Start" button. 12.3 Instrument Set-up 12.3.1 Refer to ETS-9-24.0 for more details. 12.3.2 Check the solvent level in reservoirs and refillifnecessary. Analy's.isofSerEummsExetsract Using ne ESMS Page Sf 600082 12.3.3 Check the tip. thTehestatiinplsehssousltedeblecafplialtlwariythatntohjaegngdeodfetdhgees.proIbfet.heUtsipeiasnfoeuynepditeocebeto check unsatisfactory, disassemble the probe and replace the stainless steel capillary. 12.3.4 Set HPLC pump to "On". Set the flow to 10 - 500 uL/min or as appropriate. aOpbpsreorxviemadtreolplyet1s0 cmoimniutnegsoutofthe tipofthe probe. Allow to equilibrate for 12.3.5 Taurorunnodnthtehetinpiotfrtoghene. prAobef.ineRmeiasdtjusshtoutlhde btepeoxftpehlelepdrwoibtehifnononimtirsotgeisn olbesaekirnvged 12.3.6 The instrument uses these parameters at the following settings. These settings may change in order to optimize the response: 12.3.6.1 Drying gas 250-400 liters/hour 12.3.6.2 ESI nebulizing gas 10-15liters/hour 12.3.6.3 HPLC constant flow mode, flow rate 10 -- 500 uL/min 12.3.6.4 Pressure <400 bar (This parameter is not set, it is a guide to ensure the HPLC is operating correctly.) < 12.3.7 Carefully guide the probe into the opening. Insert probe until it will not go any . + further. Connect the voltage cables to the probe. 12.3.8 Print the tune page, with is parameters, and store it in the study binder with a copy taped into the instrument log. 12.3.9 Uansailnygsitsohefcbriooslso-gfilcoawl cmaoturnitceesr.electrode inthe ES/MS source is recommended for the 12.3.10Click on start button in MassLynx versions, see the Acquisition Control appropriate MassLynx Panel (this may vary among. USER'S GUIDE). Press the start button. Ensure start and end sample number includes all samples to be analyzed. 13.0 DATA ANALYSIS AND CALCULATIONS 13.1 Calculations: 13.1.4 Calculate matrix spike percent recoveries using the following equation % Recovery = OBbsearvecdReksulgt- rounResudlt x 100 Expected Result 13.15 Calculate percent difference using the following equation: % Difference = ExpCoEnxeepe.ctCcaeldcCutolnatce.edCodne. x 100 13.1.6 Calculate actual concentrationof PFOS, or other fluorochemical, in matrix (g/mL): (0g ofPFOS calc.fromstd.CurvexDilutionFactor) x Lug _ il Volume of +mLofSurre 1000ng. Final Volume (mL) `Analys.isofScrEuTmSExStsra)ct Using ESMS Pagesof9 000083 14.0 METHOD PERFORMANCE 14.1 mMaettrhioxdspDeceitfeicc.tioPnleLaismeitse(eMEDTLS)-8-a4n.d1L,imAitttoafcQhumaenntittaBt,ifoonra(LlOisQt)ingaorfe mceutrhreondt,vaanlaildyattee,dand MDL and LOQ values. 142 Solvent Blanks, Method Blanks, and Matrix Blanks 14.2.1 Sloolwveesnttsbtlaanndkasr,dmientthheodcablliabnrkast,ioanncdumravterix blanks values are must be below the 143 Calibration Curves 14.3.1 The r* value for the calibration curve mustbe 0.980 or better. 14.4 Matrix Spikes 14.4.1 Matrix spike percent recoveries are must be within + 30%ofthe spiked concentration. 14.5 Continuing Calibration Verifications " 145.1 Continuing calibration verification percent recoveries must be + 30%ofthe spiked : concentration. 146Ifcriteria listed in this method performance section isn't met, maintenance may be performed on the system and samples reanalyzed or other actions as determined by the analyst. Document all actions in the appropriate logbook. 14.7 Ifdata are to be reported when performance criteria have not been met, the data must be footnoted on tables and discussed in the textof the report. 15.0 POLLUTION PREVENTION AND WASTE MANAGEMENT 15.1 SpiapmepttleeweaxsttraectiswdaisstpeosaenddifnlbarmomkaebnleglsaoslsvceonnttiasidniesrspolsoecdatiendhiinghthBeTlaUbocroanttorayi.ners, and glass. 16.0 RECORDS _-- 16.1 Each page generated for a header or hand written on tshteudpyagmeu:ststhuadvyeotrheprfoojlelcotwniungmbienrf,oramcaqtuiiosnitiinocnlmuedetdhoedi,therin the integration analyst method, sample name, extraction date, dilution factor (if applicable), and 16.2 Print the tune page, sample list, and acquisition method from MassLynx to include in the appropriate study folder. Copy these pages and tape into the instrument runlog. 16.3 Plot the calibration curve by linear regression, weighted 1; then print these graphs and store in the study folder. 16.4 Print data integration summary, and store in the study folder. integration method, and chromatograms, from MassLynx, 16.5 AStutmamcarhimzeentdaAtfaoursainng esuxiatmapblleesooffatwsaurmem(aErxcyelsp5r.e0a)dsahnedets.tore in the study folder, see Analys.isofSerEuTmSExStSra1ct Using ESIMS 600084 Page Toro 16.6 Back up electronic data to appropriate medium. `and location ofbackup electronic data Record in study notebook the file name 17.0 TABLES, DIAGRAMS, FLOWCHARTS,AND VALIDATION DATA 17.1 Attachment A: ETS-8-5.1 Data summary spreadsheet. 18.0 REFERENCES 18.1 cFAoCmTp-oMu-n4d.s1f,ro"mExStrearcutmiofnoorfAPnoatlayssissiuUmsiPnegrfHlPuLoCr-oEolcetacntersouslpfroanayt/eMaosrsOtShpeerctFrloumoertorcyhemical 18.2 ETToSni-z9a-t2i4o.n0/,Ma"sOspeSrpaetcitornomaentderMaQiunattetnraonIcIetorfipltehequMaidcrruopmoalsesSAytsmtoesmsp"heric Pressure 18.3 The validation report-associated with this method is ETS-8-4.0 & 5.0-V-1. 19.0 AFFECTED DOCUMENTS " : 19.1 E`TCSo-m8p-o4.u1n,ds"EfxrtormacSteiornuomffPorotAansasliyusimsPUesrfilnugoHroPoLcCt-aEnleescultfroonsaptreaoyr/MOatshserSFpleucotrroocmheetmriyc"al "200 Revisions ---------- Revision Revision Numb1 er. Section 6.1.2 ClarificatioRneoafsHoPn1F1o0r0Resyvsitseimoncomponents. 04D/a02t/e99 pSleoctttiionng l1i1n.e1arAvreegrraegsesioofntawnod caudrdveeds,thneot1/sxtawnediagrhdtivanlgouefs,thaerecuursveed. for Section 12.2.2.4 Clarificationofsolvent ramp. Section 17.1 Changed from attachmentB to A. Analys.is ofSerEuTmSEx8tsra)ct Using ESMS. (0008s Page 8of9 Laboratory Study # STMtaeutsdryi:McaFtierniaallSolvent MAneatlhyotidcRaelvEiqsuoinp:ment System Number: FInisliernuammeent SoftwarrVersion: RSiSpqeuared Value: YDatIeneocfeEpxttracton/Analyst: DateofAnalysis/Analyst: [mr Joon |cog Troi miter T von] SGlroopuepr/DToaskee:n fTraokmennferaomrtehgeressusdioynfooqludaetrion CSoanmcpelnetr:atTiaokne(nufgrmoLmyt:heTsaukdeynoflrdoemrthe Massy integration summary DIiniltuitailoVnoFlaucmteo:(mTLa):keTnafkreonmftrhoemsttuedysftoulddyerfolder Final Cone. (ugmLy: Calultebyd dividing th intial volume rom th concenzation tachmenAt SummarySpreadsbost`Analy|sisofSerEuTmSEx5tr.a1ct Using ESAS Pagers 00086 3M ENVIRONMENTAL LABORATORY METHOD ANALYSIS OF POTASSIUM PERFLUOROOCTANESULFONATE OR OTHER FLUOROCHEMICALS IN LIVER EXTRACTS USING HPLC-ELECTROSPRAY/MASS SPECTROMETRY Method Number: ETS-8-7.0 Adoption Date: Author: Lisa Clemen, Glenn Langenburg Approved By; Revision Date: Laboratory Manager Date Group Leader Date `Technical Reviewer Date 1.0 SCOPE AND APPLICATION 1.1 HSPcLoCp-ee:leTchtirsomseprtahyo/dmaisssfosrptehcetraonmaeltyrsyi.sofliver extracts for fluorochemical surfactants using 1.2 Applicable Compounds: Fluorochemical surfactants or other fluorinated compounds, or other ionizable compounds. 1.3 rMeaptorrti.ces: Rabbit, at, bovine, monkey liver, or other tissues as designated in the validation Word695 Analy.sisof LivEessExstraoct Using EMS. Page laf 10 000087 2.0 SUMMARY OF METHOD 2.1 HThPiLsC-meeltehcotdrodsepsrcaryib/emsastshespaneacltyrsoimseotfrfyl,uoorroscihmielmaircaslysstuermfaacstaanptpsroepxrtiraatce.tedTfhreomanlailvyesrisusiisng ptheerfpoerrfmleudorboyocmtoannietsourlifnognaatsein(gPlFeOiSo)n cahnairoanc,temr/izst=ic4o99f.a pAadrdtiitciuolnaarlfllyu,osroacmhpelmeiscamla,ysubceh as daentaelcytziendgudsaiungghatetrainodnesmofmtahses ssepleeccttreodmpeatreernttoiofnu.rther verify the identity ofacompound by 3.0 DEFINITIONS 3.1 Atmospheric Pressure systems allow for various mIeotnihzoadtsioofnio(nAiPzTa)t:ioTnhebyMiutcirliozmiangssvaQruiaotutsrsoouIIrctersi,plperqoubaedsr,uapnodle Pinrteesrsfaucrees.cheTmhiecsael iInocnliuzdaetibount(aArePn)o,t Tlihmeirtmeodstpor:aEyl,ecettcr.ospTrhaey iIoonniizzaattiioonnp(rEoScIe)s,sAitnmtohsepsheeric techniques occurs at atmospheric pressure (i.e. not under 2 vacuum) " 32 Electrospray Ionization (ES, pressure, whereby ions in solution aErSeI)t:raansmfeertrheoddtoofthieongiazsatpihoanspeevrifaortimneydcahtaragtemdosdprhoeprlietcs. : These charged droplets are produced by the applicationofa strong electrical field. 33 (MSM/aMsSs):SpTehcterAoPmeItQruya,ttMraossII Strpiepcletrquoamdertuepro(leMSm)a,ssTaspnedcetrmomMeatsersiSspeeqcutirpopmedewteirthtwo qmuaasdsrtuopoclhearmgaesrsatsieole(cmt/izve) daentdecstuobrsseqaunedntalcyoldleitseicotnedc.ell.ATsoinnsglaerMe Sselmecatyivbeelyedmipslcroiymeidnaftoerdiboyn detection and these ofrraagnmeinotnsmmaayybebesealneacltyezdedinitnhtehefirsset cqounaddrquupaodlreu,poflrea;gmented in the collision cell, 3.4 triplCeoqnuvaednrtuipoonlael(vpso.stZ-1s9p98r)ayutpilriozbe ea interface: "Z-spray" The latest modelsofMicromass conformation. The spray emitted Quattro from a II pdirroebcetlyisaotrtthheogcoonnaelatpoertthuerce,onaetearpepratusrsei.ngItnhrtohuegchonavteonrttiuoonuasl pcaotnfhowramyaitniotnhetciosuanitemred melaeicnttroednea.nceThaoreugthhetshaemceo.nfiHgouwreavtieorn,iZs-dsipffrearyencto,mtphoenmeentthsodasnodfcoonpveernattiioonna,lclceoamnpinogn,enatnsd are ncootmpcaotmipbalteibwliethwoitthheornZe-sapnortahyers,ysbtuetmso,nleytcw.)ith similar systems (i.e. Z-spray components are 3.5 Mass Lynx Software: System software designed for the specific operation of these: 4Q.u0atvterrsoioInIst.ripAllelqvueardsriuopnoslaeresyssitmeimlasr.. CFuorrremnotrlyeMdeatsaislsLryenferhatsoWtihendmaonwusal95spaencdifWicitnodtohwesNT iGunisdter)u.ment (Micromass Quattro II triple quadrupole MassLynx or MassLynx NT User's 4.0 WARNINGS AND CAUTIONS 4.1 Health and Safety Warnings: 4.11 eUmspelcoayustiaovnowlittahgetohfe vaoplptraogxeimcaatbelelsy f5o0r0t0heVoplrtosb.e. When engaged, the probe Analys_isof LivEerTESxt8ra0ct Using ESMS Page20r10 600088 4.12 When handling and clothing. samples or solventswear appropriate protective gloves, eyewear, 42 Cautions: 4.2.1 pOrpeesrsautreetehxecesoeldvsen4t00pubamrp,stbheelHoPw11a 0b0acwkilplreinsistuirateeofau4t0om0atbiacr (s5h8u0t0dopswin). If the back 4.2.2 Do not run solvent pumps to dryness 5.0 INTERFERENCES 1 Tstoormaigneimorizaenyinptaerrtfoerfeinncsetsrwuhmeenntaatnailoynztihnagtscaommpleess,inTceofnltoanctshwaillthnotthbeesuasmepdlefoorresxatmrpalcte 6.0 Equipment " 6.1 E`mqoudiifpimceantitonlsistiendtbheelroawwmdaatyabaesmmoedtihfoiedddienvioartdioenrs.to optimize the system. Document any 6.1.1 Micromass Quattro II triple quadrupole Mass Spectrometer equipped with an * electrospray ionization source, 6.12 HP1100 low pulse and autosampler solvent pumping system, solvent degasser, column compartment, 7.0 SUPAPNDLMAITEREIASLS 7.1 Supplies 7.1.1 High purity grade air regulated to approximately 100 psi (house air system) 7.1.2 HinPtLheCraanwalydtaitcaal column, specifics to be determined by the analyst and documented 7.13 Capped autovials or capped 15 ml centrifuge tubes 8.0 REAGENTS AND STANDARDS 8.1 Reagents 8.1.1 Methanol, HPLC grade or equivalent 8.12 M1,iolrlieq-uQiTMvawlaetnet,r a(nAdSTbeMptryopveidI)e,dalblywaatMeirlulsie-Qd TiOn tChiPslmusetshyosdtesmhoourlodtbheerAvTenSdMortype 8.13 Ammonium acetate, reagent grade or equivalent 8.1.3.1 When preparing different amounts than those listed, adjust accordingly. 8.13.2 2.0 mM ammonium acetate solution: Weigh approximately 0.300 g ammonium acetate. Pour into a 2000 mL volumetric container containing 2te0m0p0ermatLurMei.lli-QTM water, mix until ll solids are dissolved. Store at room Anali|sisofLivEerTESx$tr7ac0t Using ESMS Page30f10 C0008Y 82 Standards 8.2.1 Typically two method blanks, two matrix blanks, and eighteen matrix standards are prepared during the extraction procedure. Refer to ETS-8-6.0. 9.0 SAMPLE HANDLING 9.1 Fresh matrixstandardsare prepared with each analysis. Extracted standards and samples. are stored in capped autovials or capped 15 ml centrifuge tubes until analysis 9.2 tIefmanpaelryastiusrewi,llorberedferilgaeyreadt,edexattraacptperdoxsitmaantdealryds4anCd,suanmtiplleansalmyasyisbceasntboerepderatforromoedm. 10.0 QUALITY CoNTROL 10.1 Method Blanks and Matrix Blanks 10.1.1 Solvent blanks, method blanks, and matrix blanks are prepared and analyzed with each batch to determine contamination or carryover. " 10.1.2 Analyze a method blank and a matrix blank prior to each calibration curve. 10.2 Matrix Spikes 10.2.1 Matrix spikes are prepared and analyzed to determine the matrix effect on the recovery efficiency... 10.2.2 Matrix spike duplicates are prepared and analyzed to measure the precision and the recovery for each analyte. 10.2.3 Analyze a matrix spike and matrix spike duplicate per forty samples. With a `minimum o2f spikes per batch. 10.2.4 Matrix spike and matrix spike duplicate concentrations will fal in the mid-range of the initial calibration curve. Additional spike concentrations may fall in the lowrangeofthe initial calibration curve. 10.3 Continuing Calibration Checks 10.3.1 Continuing calibration verifications are analyzed to verify the continued accuracy of the calibration curve. 10.3.2 Analyze a mid-range calibration standard every tenth sample, with a minimumofone: per batch. 11.0 CALIBRATION AND STANDARDIZATION 11.1 Analyze the extracted matrix standards prior to and following each setofsample extracts. `The averageoftwo standard curves willbe plotted by linear regression (y = mx +b), weighted 1/x, not forced through the origin, using MassLynx or other suitable software 11.2 Isfttahnedacrudrcvuerdvoe(eisfnnoetcemseseatryr)eqaunidrermeaennatlsyzpee.rform routine maintenance or reextract the Analy.sisof LivEerTESx8tr7ac0t Using ESMS Page 40710 6006090 11.3 uFsoertphuerploosweseonfdoafcctuheraccayliwbhraetnioqnucauntrivteatriantghelrotwhalenvtehlesoffulalnarlayntgeeo, fittmhaeystbaenndeacredscsuarrvye.to cEaxlaimbprlaet:ionwchurevneactotnesmipsttiinnggoftothqueansttiatantdearadpspfrorxoimm5atpeplby t1o0 1p0p0bopfpabnarlatyhteer,tgheannertahteefuall rangeofthe curve (5 ppb to regression weightingofhigh 1c0on0c0enpptbr)a.tioTnhisstawnidlalreddsuce inaccuracy attributed to linear 12.0 PROCEDURES 12.1 Acquisition Set up 12.1.1 Set up the sample fst. 12.1.1.1 lAeststiergonfathseamapllpehalbsettfistlaerntaimneguwsiitnhgaMO-DAY-last digitofyear-increasing. 12.1.1.2 Assign a method (MS file) for acquiring 12.113 Assign an HPLC program (Inlet file) 12.1.1.4 Type in sample descriptions and vial position numbers " : 12.1.2 sTpoecctrreoamteetaermheetahdoidngcsliacnkdonsemleectthoSdIRin(StihnegAlceqIuoinsiRteicoonrcdoinntgr)olorpaMneRl Mthe(nMumlatsisple +. Reaction Monitoring). Set Ionization Mode as appropriate and mass to 499 or * aoctqhueirsiatpipornopmreitahtoed.masIsfesM.S/AMfSullinssctarnumisenutssuaalrley ecmopllleocyteedd,aaldodnigtiwointahl tphreodSuIRcst.ioSnave GfrUaIgmDeEntaTtOioDn AinTfAormAaCtiQoUnImSaIyTbIeOcNolfloecrtaedd.ditRioenfaelritnofoMrimcartoimonasasndMaMsRsML.ynx 12.1.3 Typically the analytical matrix standards batch run sequence begins and ends with a setofextracted 12.1.4 aSfatmeprleevserayreteanntahlyszamepdlew.ithSo2lcvoennttibnulianngkscaslhioburladtibonevaenrailfyiczaetdiopneriinojdeicctaeldlysttaondmaornditor possible such. analyte carryover and are not considered samples but may be included as 12.2 Using the Autosampler 12.2.1 Set up sample tray according to the sample list prepared in Section 12.1.1 12.2.2 aSneatl-yuspttchoensHiPd1e1r0s0a/papurtoporsiaamtpelfeorraotptthiemaflolrleoswpionnsgec.onRdeictoirondsaocrtuaatlccoonnddiittiioonnsstihnethe instrument logbook: 12.2.2.1 Sample size = 10 uL injection 1222.2 Inject/sample = | 12.2.2.3 Cycle time = 9 minutes Analis_is ofLivEerTESx8tr7ac0t Using ESS Page sof 10 600091 12.2.2.4 Solvent ramp conditions Time MeOH 20mM Ammonium acetate [000min | a0% |" 60% | (omn|a0% | 60% | [45min 10 |55%%| [65min | 0% | 5% | Dom 1a0% | 60% | 12.2.2.5 Press the "Start" button. 12.3 Instrument Set-up _ 123.1 TRreifpelretQouEaTdSr-u9p-o2l4e.M0,as"sOpSepreacttiroonmeatnedrMFaiitnttedenwaintcheoanftAhtemoMsipchreorimcasPsreQsusautrtero II Tonization Source," for more details. 1 12.3.2 Check the solvent level in reservoirs and refillif necessary. " 12.3.3 Check the stainless steel capillary at the endofthe probe. Use an eyepiece to check tunhseattiip.sfaTcthoeryt,ipdsihsoauslsdembbelefltahtewiptrhobneoajnadggreedpleadcgeest.heIsftatihneletsisp sistefeolucnadpitlloarbye. 12.3.4 Tum on the nitrogen 12.3.5 Open the tune page. Clicks on operate to initiate source block and desolvation `heaters. 12.3.6 Open the Inlet Editor. 12.3.6.1 Set HPLC pump to "On" 12.3.6.2 Set the flow to 10- 500 uL/min or as appropriate 12.3.6.3 Observe droplets coming outofthe tipof the probe. A fine mist should be expelled with no nitrogen leaking around the tipofthe probe. Readjust the tipofthe probeif no mist is observed 12.3.6.4 Allow to equilibrate for approximately 10 minutes. 12.3.7 The instrument uses these parameters at the following settings. These settings may change in order to optimize the response: 12.3.7.1 Drying gas 250-400 liters/hour 12.3.7.2 ESI nebulizing gas 10-15 liters/hour 12.3.7.3 HPLC constant flow mode flow rate 10 ~ 500 L/min 12.3.7.4 PHrPeLssCuries <op4e0r0atbianrg(cTohrriesctplya.r)ameterisnot set, it is a guide to ensure the 12.3.7.5 Source block temperature 150 12.3.7.6 Desolation temperature 250 AnalisisofLivEerTExtSract Using ESMS Page of 10 000092 123.8 Print the tune page, with its parameters, and store it in the study binder with a copy taped into the instrument log. 123.9 Click on start buttonin the Acquisition Control Panel (this may vary among eMnadsssLaymnplxevnerusmibonesr,irnecfleurdteos aaplprsoapmrpilaetsetMoabsesLanyanlxyzUeds.er's Guide). Ensure start and 13.0 DATA ANALYSIS AND CALCULATIONS 13.1 Calculations: 13.14 Calculate matrix spike percent recoveries using the following equation: % Recovery = OB bsea rvec dReEk sxupleg tc-ter d Reo sultuRnesud lt x 100 13.15 Calculate percent difference using the following equation: % Difference = Exp ConExce p.e-cCtc aeldcCut olnact.ee dCod nc, x 100 " 13.1.6 Calculate actual concentrations in matrix (g/g): . ? (ng ofPFOScalc.fromstd, Curvxe Dilution Factor) x _Lug . (InitialWFieniaglhVtoolfuLmieve(rm(Lg)) 1000 ng. 14.0 METHOD PERFORMANCE 14.1 mMaettrhioxdspDeceitfeicc.tioRnefLeirmitto(EMTDS-L8)-a6.n0d,LAitmtitacohfmQeuannttiBtaftoiro2lni(stLiOnQgo)facruermreetnhtovda,liadnaatlyetdeM,DaLn.d and LOQ values. 14.2 Solvent Blanks, Method Blanks and Matrix Blanks 14.2.1 iSnotlvheenctalbilbarnaktsi,onmecutrhvoe.d blanks, and matrix blanks must be below the lowest standard 143 Calibration Curves 14.3.1 The r* value for the calibration must be 0.980 or better. 14.4 Matrix Spikes 14.4.1 Matrix spike percent recoveries must be within + 30%ofthe spiked concentration. 145 Continuing Calibration Verification 14.5.1 Continuing calibration verification percent recoveries must be within 30%ofthe spiked concentration. 14.6 Ifcriteria listed in the method performance section are not met, maintenance may be apnearlyfsotr.meDdoocnumtheentsyaslteamctainodnssianmptlheesaprperaonparliyazteedloorgboothoekr. actions as determined by the Analys_isofLivEeTrSExstnr0act Using ESS 000093 Page of 10 14.7 fIfodoattnaotaerdeotno tbaeblreespoarntdeddiwshceusnsepderinfotrhmeatnecxet ocrfittehreiarehpaovretnot been met, the data must be 15.0 POLLUTION PREVENTION AND WASTE MANAGEMENT 15.1 pSiapmepttleeweaxsttraectiswdaisstpeosaenddifnlbarmomkaebnleglsaoslsvceonnttiasidneirsspolsoecdatiendhiinghthBeTlaUbocroanttorayi.ners, and glass 16.0 RECORDS 16.1 hEeaacdherpaogrehgaennderwartietdtefn ooanr tshteudpyagme:uststhuadvyeotrheprfoojlelcotwninugmbienrf,oramcaqtuiiosnitiinocnlmuedetdhoedi,ther in the integration analyst. method, sample name, extraction date, dilution factor(ifapplicable), and 16.2 aPrpipnrtoptrhieattuensetpuadgyef,olsdaerm.pleColipsy, tahnedseacpqaugiseistiaonndmteatpheoidntfortohmeMiansstsrLuymennxttrounilnocgl.ude in the " 163 Psltoortetihnetchaelisbtruatdiyonfocludrerv.eby linear regression, weighted 1/x, then print these graphs and :" 16.4 Print data integration summary, and store in the study folder. integration method, and chromatograms from MassLynx 165 ASutmtmaacrhimzeentdaAtafuorsianng esuxiatmapblleesooffatwsauremm(aErxcyeslp5r.e0a+d)shaenedt store in the study folder, refer to 16.6 Back up electronic data to appropriate medium. and locationofbackup electronic data. Record in study notebook the file name 17.0 TABLES, DIAGRAMS, FLOWCHARTS, AND VALIDATION DATA 17.1 Attachment A: ETS-8-7.0 Data summary spreadsheet 18.0 REFERENCES 18.1 FCAoCmTp-oMu-n2d.s1,fr"oEmxtLriavcetrifoonrofAnPaoltysaisssiUusminPgerHfPlLuCor-oEolcetcatnreossulpfroanya/tMeaosrsOStpheecrtrFolmueotrrocyh"emical 18.2 ETToSni-z9a-t2i4o.n0/,Ma"sOspeSrpaetcitornomaentderMaQiunattetnraonIcIetorfitplheequMaidcrruopmoalsesSAytsmtoesmsp"heric Pressure 183 The validation report associated with this method is ETS-8-6.0 & 7.0-V-1 9.0 AFFECTED DOCUMENTS _ 19.1 ECToS-m8p-o6u.n0,ds"EfxrtormacLtiivoenroofrPFoltuaisdsfiourmAPnearlfylsuiosrUosocitnagneHsPuLlCf-oEnlaetcetorroOstphreary/FMlausosrochemical Spectrometry" Analys_isofLivEerTESx8tr7ac0t Using ESMS Pages of 10 0006094 20.0 Revisions ReNvuimsbieorn Reason For Revision --_ ReDvaitsieon AnalyS_isofLivEerTESxt$r7ac0t Using ESMS. Page 9of 10 600095 Laboratory Study # TseusdMoral: MMaesoidFRienrlsSoonlvent AInasattryuimceanltESqoufiwpamreenVteSnytsotenm Number: RFSiqnuaarned Vale SVionee: DfatmeoefBtxrhecwtiionv/Ainlyest: Broosu Conc5e:ntration | ATWe | DYiacoanr GSSahrroaoppuelephTesaskTe,askTeTrnuotferTnoomoethnertseepyselfayteorudero CTDooinicoelnnWtreFaatgcito:onr:(TsaTkgaek:nenoTfomkoemtnhfheesoosuytdheyFoMfladisytxgration sma: Final Con. (10: Cacuted by diving he iil solu romtheconnrion Atachment A; Summary SpeadshctAnalysis of LEriEsxvat0eUsring ESMS - * 000096N Page 00110 S3RM EERNVYIRROONNVMEENNTAALLLLAABBOORRAATTOORRYY ~~~ METHOD SULDFEOTNEYRLMAIMNIADTEIO(NPFOOFSPA)E,RFALNUDOPREOROFCLTUAONREOSOUCLTFAONNOAATTEE(P(FPOOSA)A,)PEINRWFLAUTOERROOBCYTLAINQUEIDSOLID EXTRACTION AND HIGSHP-EPCETRRFOOMREMTARNYCE(HLPILQUCI/DMCSH/RMOSM)ATOGRAPHY/TANDEM MASS Method Number: ETS-8-154.0 Adoption Date: Author: Kristen J. Hansen/Harold O. Johnson Approved By: William K. Reagen, Kent R. Lindstrom Revision Date: -- William K. Reagen, Laboratory Management -- Date Kristen J. Hansen, Ph.D. Group Leader Date Kent R: Lindstrom, Technical Reviewer Date MS Word 97 ETS8-1540 Page lof 17 DeterminationofPFOS, PEOSA, POAA in Wate by Liquid-Solid Extraction and LOMSMS 600097 110.0 ScSoCOrPeEaAvNpDAAePpPuLIiCcAaTImOeNy 000000000 1.1 TdehtiesrmmientahtoidonproofvPiedrefslcuoolrloeoccttiaonn,c esxutlrfaocntaitoen,(PaFnOdSa)n,alPyetricfalluoprroooccetdaunreeSsuflofrotnhyelamide (PFOSAY), and water samples. Perfluorooctanoate (POAA) in groundwater, surface water, and drinking 12 MTheitshomdesthtoodbewaPsropproespeadreadt a4c0cCorFdRinPgatrott1h3e6EoPrAPadrotc1u4m1e"nt(,se"eGRueifdeerliennecse a1n8.d1)F,oramndatisfor sbualsfeodnaitnep(arPtFOoSn)t,hePerrefplourotr"oMocettahnoedsouflfAonnayllyasmiisdefo(rPtFheOSDAe)t,eramnidnaPteirofnluoofrPoeorcftlaunooraotoectane (POAA) in Water" (see Reference 18.2). 2S 200 Sa UMMARYe OF METv HOD orMewos 21 Wfaacitleitry.saPmFpOlSes, aPrFeOcSoAll,ecatnedd PfrOoAmAa sairteeoefxtirnatcetreedstfraonmd 4sh0imppLedwactoelrdstaomapnleasnaulsyitincgalC, suosliindgpmheatsheaneoxlt.raScetpiaonra(tSioPnE,) icdaernttriifdigceast.ioTn,heancdommpeoausunrdesmaernetealrueteadccformopmltihseheC,d,bcyarhtirgihd-ge, performance liquid chromatography/tandem using multiple response monitoring (MRM). mass spectrometry (HPLC/MS/MS) analysis r`Tehsepocnosnecoefnttrhaetiqounoanfteiatacthionidieonntipfrieodduccoemdpobnyetnhtatiscommeapsouurnedd tboytcheomMpSarriensgptohneseMofSthe quantitation ion produced (extemal standard). by the same compound in an extracted calibration standard 3300 bDeEFIeNIeTIOnNS ~~ OOOOOO 3.1 Analytical Sample--A portion of an extracted Laboratory sample prepared for analysis. 32 CexatlriabcrtaetdiaocncSortdainngdatortdh--isAmseotlhuotdi.onTphreepcaarleidbrfatrioomn tshteanWdoarrkdisnogluStitoannsdaarrde u(sWeSd)taond calibrate the instrument response with respect to analyte concentration. 33 lDaubpolriactaotrey aSnadmpalnael(yzDeSd)s--epAarsaetpealryatweitahliiqdueonttiocafal psraomcpeldeu,retsa.keAnnailnytshiesoanfalDyStiscaclompared ptorotcheadtourfest,hebuftirsntoatlwiiqutohtsgaimvpelaemceolalseucrtieoonf, pthreesperrevcaitisoino,n aosrssotcoiraatgeedpwriotchedluarbeosr.atory 34 lFaibeolrdaBtolraynaknCdotnrteraoteldSaasmapslaemp(lFeBi)n--aTllypreespIewctast,erinpcllaucdeidnginexapsoasmuprleetocosnatmapilneirnginsitthee cdoentdeirtmiionnesi,fstteosrtagseu,bsptreasnecrevsaotrioonthaenrd all analytical interferences procedures. The are present in the purpose of the FB field environment. is to MSWord97 ETS81540 Page20r17 Determination ofPFOS, PFOSA, POA in Water by Liquid-Sold Extraction and LOMSMS 000098 3.5 FainedlpdlDaucpeldiucnadteer(iFdeDn)ti--caAl sciarmcpulmesctoalnlceecsteadndintdreuaptleidcaetxeacattltyhethseasmaemteimtehraosugthheoustamfipellde amnedasluarbeoroaftotrhye pprroecciesdiuornesa.ssAoncailaytseidswoifthFDsacmoplmepacroelldecttoiothna,tporfestehrevafitrisotnsaamndplsetogriavgees, aas well as with laboratory procedures. 3.6 oFfietlhdeMtaartgreitxanSapliyktees(aFreMaSd)de--dAisnatmhpelfeieclodlaltecttheedtiinmdeuopfliscaamteplteo wcohlilcechtikonn.owTnhequFanMtSities ssahmopulled.bTehsepiFkMedSatisaapnparloyxziemdatteolaysc5e0rt-a1i5n0i%foafnytmheaterxipxeecftfeedctasn,ailnytteerfceornecnecnetsr,aotriosntaibniltihtey issues may complicate the interpretation of the sample analysis. 37 qFuiaenltditSipeiskoefCtohenttraorgletSaanmapllyete(sFarSeCaSd)d--edAninatlhiequfoiteoldftatyptheeItwiamteoerfstoamwphilcehcoklnloecwtnion (at aanndapapnraolpyrzieadteexcaocntcleyntlriaketiaonsatmopbleedteotdeertmeirnmeidnebywthheethperrojaecltoslseoadf).anTahleytFeScCoSulids beextracted attributed to sample storage and/or shipment. 38 LquaabnotirtaiteosorfytChoenttarrogletSaanmapllytees(aLrCeSa)dd--eAdnianltihqeuoltaboofrattyoprey.I water to which Two levels are known included, one aatnotthheeLr OcoQnc(eanptprraotxi.on25tPogb/emdLe)t,erthmeinoetdhebryattahecpornocjeencttraletaido.noTfheapLpCroSx.is 1ex0t0r-a2ct5e0dPagn/dmL or aconnatlryozle,danexdawcthleythleikretahelalbaobroartaotroyrysiasmpclapeatboledeotfermmaikniengwhaectchuerrattehememaestuhroedmoelnotgsyaits tihne required method detection limit and higher. 39 Laboratory Sample--A portion ofa sample received from the field for testing. 3.10 mLeiamsiutroefdDaentdecrteipoonrt(eLd OwDit)h--9T9h%ecloonwfeisdtecnocnectehnattrathteioannoaflyatne caonnacleynttertahtaitocnains bgreeater than zero. The statistical LOD can be calculations. determined in several ways, including signal-to-noise ratio and 31 cLoinmcietntorfatQiuoannt(iUtLatOiQo)n t(haLtOcQa)n--bTehreelilaobwleysatcchoinecveendtrwaittihoinn(tLheLOspQe)ciofriehdiglhimeistts of precision and accuracy during routine operating conditions. cNoontcee:ntTrhaetiLonLOiQseilsegcteenderaaslltyheSl-o1w0esttimneosn-tzheerLoOsDt.anFdoarrdmianntyheancaallyitbersa,titohnecLurLveO.QHaonwaelvyetre, it `may be LLOQs naoremimnaatlrliyx-cdheopseenndewnitt.hin these stated guidelines to simplify data reporting. Sample 3.12 aMnaatlryitexsSapriekaed(deMdSi)n--tAhenlaabloirqautootryo.faThsaemMplSe,istoexwthriaccthedknaonwdnanqaulanytzietdieesxaocfttlayrgleitke a alnaabloyrtaitcoarlyrsesaumlptsl.eTthoedebtaecrkmgirnoeuwnhdectohnecrentthreastaiomnpsleofmathteriaxnacloynttersibiunttehsebsiaasmptloethmeatrix. must be determined in a separate background concentrations. aliquot and the measured valus in the MS corrected for 313 MsaemtphloedinBclluadninkg--eAxnpoasluiqrueottooaflltygpleasIswwaartee,r ethqautipismetnreta,tesdolevxeancttsl,yrleiakgeenatsl,abionrtaetronrayl MS standards, Word 97 and surrogates that are used with other ETS8-1540 laboratory samples. The method blank Page Jof17 Determinationof PFOS, PFOSA, POAA inWater by Liquid-Solid Extraction and LC/MS/MS (00099 is used to determineiftest environment, the reagents, substances or other or the apparatus. interferences are present in the laboratory 3.14 Museetdh1o0desDteatbelcisthioanLLOimDitva(luMeD.LT)heDesttaetirsmtiicnalaltyicoalnc--uOlnateeodfmsienviermaulmpraomcoeusnsetsoftahant may be azenraol.ytTehitshattecramnibseusmueaalsluyraesdsowciitahte9d9w%itchotnhfeidEePncAedtehfaitnitthieonreipnor4t0edCvFaRluPearistg1r3e6ater than Appendix B. 3.15 Sinatmenpdlede--toAresparmepsleentisthaesomrailglinpaolrtsioounrcceolmlaetcetreidalf.roma larger quantityofmaterial 3.16 pSrpeipkairnegtShteowcokrkSitnagndsatarndda(rSd.SS)--A solution prepared from stock standards used to 3.17 SIatboocrkatSotraynwdiatrhdan(SaSss)a--yAedcroenfceernetnrcaetecdomspolouutnidon.of a single analyte prepared in the 3.18 WfroormkSiSnsgaSntdandidlaurtded(aWsSne)e--dAedstooluptrieopnaoref scaelviebrraaltainoanlsyttaensdparredpsaarnedd oitnhtehrerleaqbuoirraetdory analyte solutions. 2W 400 iWARk NINM GS ANn D CAg UTIOs NS awoCaovions 41 Health and Safety Warnings 4.1.1 Tprheeciasceultyedaentdercmhirnoendi;chtoowxeicvietry,ocfatchhe sshtoanudladrbdes tfroerattheidsamseathpootdenhtaivael nheoatlbtehen hazard. 4.1.2 SUanmkpnloewcnonstaamipnleerss smhaoyulcdonbteaionpehniegdh cinonacehnotordatainodnshofanvdolleadtiwliethtogxliocvceosmtpoounds. prevent exposure. 4.1.3 cTuhrerelnatboarwaatroernyeisssorefsplooncsailbrleegufolratmiaoinnstraeignairndgi2ngsatfhee whoanrdklienngvoifrotnhmeecnhteamnidcaals suhsoeudlidnbtehiasvmaeitlhabolde. tAo arllefpeerresnocnenefilleinovfomlavteedriianltshaefseetyandaaltysaessh.eets (MSDS) 25)0 [IINvTeEeRrFeERReENNCCEBSS 5.1 During extraction and liquid-solid extraction analysis, devices. major potential contaminant sources are reagents and 52 uAlnldemratceornidailtsiounsseodfianntahleysainsalbyysersunshnailnlgbmeedtehmoodnsbtlarnaktse,d to be free from interferences 53 T`meafylocnausceonitnatienrifnegremnacteesraianldss(hc.ogu.ldcanpost, bweasuhsebdotdtulreis)ngcocnotllaeicntifolnu,osrtoocroagmep,ouexntdrsacwthioinc,hor analysis of the samples. MS Word 97 ETS81540 Pagedof17 Determination ofPFOS, PFOSA, POAA in Wate by Liuid-Solid Extraction snd LOMSMS(, 00.4. 00 6.0 EQUIPMENT, SUPPLIES, AND MATERIALS pNoetref:orBmraanncde nmaamyebse, saucphpileievresd,uasnidngpaarptpanruamtbuesrasnadrmeaftoerriialllusstortahteirvetphuarnptohsoesseosnpleyc.ifEiqeudihvearlee,ntbut rdeemsopnsotnrsatiiboinoolffittheyequilavbaolreanttorpye.rformance that meets the requirementsofthis method is the 61 Sampling Equipment 6.1.1 Sample collection screw cap. bottles--LDPE (e.g., NalgeneTM) narrow-mouth bottles with Note: 6.1.2 Do not use Coolers Teflon bortles or Teflon for sample shipment. lined caps. 6.13 Ice for sample shipment. 6.1.4 aBcotctolredsinmgustto tbheislomte-tcherotdi.fied to be free ofartifacts by running Method blanks 62 6L2a.b1oratBoarlyanEcqeu,iapnmaleynttica(lEx(tdriaspcltaiyonatalnedastAn0a.l0y0t0i1cga)l,)Mettler. 622 Vacuum pump, Bhi. 623 Visiprep vacuum manifold, Supelco. 62.4 62.5 SSeOpmLPadkisVpaocsabbelce (p1ogl)yp1rCo,pyclaretnreidcgeenstr(ipfaurgte#tWubAesT,0V3W6R7.95),Waters. 62.6 62.7 D1i5smpoLsadbislpeomsiacbrloeppiopeltytpersop(y5l0e-ne10ce0nLt,ri1fu0g0e-2t0ub0eusL,)V, WDRr.ummond, 628 6.2.9 HClyapsesrcAarpbipdertoteps-ianngduvaorlducmeotlruimcnfl(a4skmsm,)va(rpioaurs#t. 844017400), Keystone. 6.2.10 62.11 S1t2a5nmd-LalLoDnePEdronpa-rirnogwu-amroductahrtbroitdtglees,hoNladlegre,neK.eystone. 6.2.12 62.13 2HmPLLCclpeaurmHpP(LLCCIv0iAalDk)i,t (Schaitm#ad5z1u8.1-3400), Hewlett Packard. 6.2.14 6.2.15 LStCa/nMdaSr/dMlSabaenqduHipPmLenCts(ygsrtaedmusa,taesddceyslcirndiebresd,idnissepcotsiaobnle10t.u1b.es, etc), various. 63 Equipment Notes 63.1 Irnecorodmemretnodaevdoi(dtucboesn,tapmiipnetatteiso,n,cict.h)e useofdisposable labware is highly 63.2 vTieafllsoonrocraTpesflfoorn-tlhienHedPcLoCntaaiunteorssaomrpelqeurimpumsetntn,otinbceluudsiend.g Teflon-lined HPLC 63.3 tThyrpoeugIhwaatHeyrpuesrecdardburgiunagrdthceoslaummpnleusainndgsataHnPdaLrCd pexutmrpa.ctiTohnisshwoautledr biserfeifletrerreedd 634 0 as "filtered Itis necessary ttyopcehIewcaktetrh"e,sohlevreeanfttse(rmientthhainsorle)pofrotr the presence of contaminants (feosupnedcitaollbye PunOsAuAit)abblye LC/MS/MSprior for use. to use. Certain lot numbers have been 63.5 cUasleibdriastpioonsasbtlaendmaircdrsopainpdetmtaetsrioxr psippiektetse.s to aliquot standard solutions to make MS Word 97 ETS81540 Page sof 17 DeteminaionofPROS, PFOSA. POAA in Waterby LiquidSoid Exton snd LIMSIMS(3.104 7.0 REAGENTS AND STANDARDS Nmoatye:beSuapcphliieevresdaunsdincgatcahleomgincuamlsbeorbsiaairneedfofrriolmlusottrhaetrivseupppulripeorss.esDoonlyn.otEuqsueivalaelsesnterpgerrafdoeromfance chemical than those sed. 7.1 Chemicals 7.1.1 7.12 AMemtmhoannoilu(mMAecOeHt)at,e,HRPeLaCgegnrtadger,adJeT, BSaikgemra,-ACladtrailcohg,NCoa.taJlTo9g09N3o-.2A.-7330. 7.13 7.1.4 SWaotdeiru,mtTyhpieosI,ulpfraetpea,reRdeaigne-hnotusger.ade, JT Baker. 72 Standards 7.2.1 7.2.2 PPeortfalsusoiruomocptearnfelusourlofoocntyalnaemsiudlefo(nsaeteeA(tsteaecAhtmteancthAm,enFtigAu,reFi2g),ure 1). 72.3 Ammonium perfluorooctanoate (see Attachment A, Figure 3). 73 Reagent Preparation 73.1 250mg/mL sodium thiosulfate solution thiosulfate in 100mL reagent water. (Extraction)--Dissolve 25gof sodium 73.2 41001%.0mLewtihtahnroelag(eEnxttrwaatcetri.on)--Measure 400mL methanol and adjust the volume 73.3 a1c0et0amteManadmdmiossnoilvuemianc1e.t0aLtoefsorleuatgieonnt (wAatnearl.yDsiilsu)te--Wteheig1h070.m71M osofluatmiomnobnyiuam factor A of 50 to make the 2mM ammonium acetate solution used for mobile phase mNaoitnet:aiAnletde.mative volumes may be prepared as long as the ratios of the solvent to solute ratios are 7.4 Spiking 74.1 Stock Standard 100ug/mL each (SSS) PFOS, Preparation PFOSA, and POAA SSSs--Weigh out 10mg of paunraliyttyi9ca0l%st=a8n.da3r5dm(gcoCryrFe,c,tSe0d,f-o)rapnedrcdeinltutsealttoa1nd00pmurLitwyi--tih.cm.e,th1a0nmolgiCn,aF,1,0S00,mKL vfoorluemaecthriacnafllyatsek.. STtroarnesfseorluttoioans12in5amLreLfrDigPeEratbootrtalte.4Pr4e2paCrefaoasrepmaarxaitemusomlution 742 pIeprgio/dmLofm6ixmeodntShsSSf--roAmdtdhe1d.a0tmeoLfepraecphaorfattihoen1.00ug/mL SSSs (from 7.4.1) toa 743 01.010umgL/mvLolmumiexterdicSfSlSas--kAadnddb1ri0n.g0muLpotfotvhoelu1m.e0pwgi/tmhLm-emtihxaneodl.solution (from 744 70..40.12)ug10/maL1m0i0xmeLdvSoSluSm--etArdicdfl1a0s.k0amnLdofbrithneg 0u.p1tsogv/omlLu-mmeixweidthsomleutthiaonno(lf.rom 7.45 S7.t4o.r3a)gteoCao1nd0i0tmiLonvsol--uSmteotrreicalfllaSsSkSasnidnbarirnegfruigpertaotvorolinum1e2w5imtLh LmeDtPhaEnoblo,ttles at 442C foramaximum periodof 3 months from the date of preparation. MS Word 97 ETS81540 Page6of 17 Determination of PFOS, PFOSA, POAA in Waby tLiqueid-Srolid Extraction and LC/MS/MS 000102 75 CalibrationStandards 7.5.1 o1u0t01u0gm/gmoLfeaancahlyPtiFcOaSl,stPaFndOaSrAd,(caornrdecPteOdAfAorspteorcceknsttsaanltdaarndd spuorliutty)ioannsd--dWieluitgeh 1L0D1P0E0bmoLttlwei.thPrmeeptahraenaolseipnaara1t0e0smolLutvioolnumfeotrreiacchflaasnka.lyTtrea.nsSfteorrteosoalu1t2iSonmsL.in a refrigerator preparation. at 42C for a maximum period of6 months from the date of 752 (Ifprgo/mm7L.5W.1o)rtkoiang10S0tmaLndvoalrudm--etArdidc f1l.as0kmLanedacbhroifntg hupet1o0v0oilgu/mmeLwSitShsmoelutthiaonnosl. 753 0(f.rIopmg/7.m5L.2W)otroaki1n0g0SmtLanvdolaurmdet--rAicdfdlas1k0a.n0dmLborfintghuep1t.o0vgo/lmuLmemiwixtehdmseotlhuatniooln. 754 0(f.r0o1mug7./5m.L3)Wtoorak1i0n0gmSLtavnolduamredtr--icAdfldask10an.d0mbLroifntghuep t0o.1vuogl/ummLe wmiitxhedmestohlauntoilo.n 755 aStto4r4a2geCCofnodaritmioanxsi--mSutmorpeeralilodWSos3fimnoanrtehfsrifgerroamtotrhe(idnat1e2o5fmpLreLpDarPatEiobno.ttles) 7.56 Calibration filtered type Standard--Prepare I water according to a minimum of five the following table: calibration solutions in 0 _Conce0 ntratio0 n Vo0 lume o0 f Fina0 l Calib0 ration 0 Standar0 d 0 Final C0 oncent0 ration 0 of AofTWSS,upgg/mmlL., WWSS,upLL. 00) 0 VoVoulmumeem, lmLL. 0 CaClailibbrraattiioonnSSttaannddaarrdd,,PPgg//mmLL 0 0010 100 "0 2 0010 200 "0 50 0010 400 " 100 0.10 100 "0 250 010 200 "0 500 010 300 " 750" TT 010 400 a May be prepared to extend the ngs beyond SOP. 100 2 May be prepared to extend the ange beyond 750PmL. Nprootpeo:rtTihoensabosfsoollutuetevotlousmoelvsoenfttahree smtaainndtaaridnsedm.ay be varied by the analyst as long as the correct 75.7 Tidheentsictaalndtaortdhsealreabporroactoersysesdamtphlreosu.ghThteheeexxttrraacctteidoncopnrcoecnetdruarteio(nSeocfttihoenc9a.l0i)b,ration ssttaannddaarrdd idsureiqnugalthteo e8xXtrtahcetiionnitpiarlocceosnscentration, due to the concentration of the. 758 pSotloyrpargoepyCloenndeittiuboenssa--tSt4o4r2eaCl,l efxotraracmtaedxciamluibmrapteiroinosdotafndtawrodswienek1SsmfLr.om the dateofpreparation. MS Word 97 ETS8.1540 Page of 17 DeterminationofPFOS, PFOSA, POAA in Wate by Liquid-Solid Extraction and LOMSMS 000103 3P 800 RSASNoME AtMPe:PLS LSEaEmCCpOE lOLiLnLLgEReCqTuIiV pOmNe,ntAP,RiEnT cSlEuRdiVI nAgTaIuO tOoNmaAtN NiDc sSA aTmpOlReN rAsG,EmDust S be frT eeofTOefloR ~ n tuA bi~ ng,GgaskE ets, a`cnodmpootshietrepasratmspltheastomvaeyr tliemaechshinotuelrdfeurisngreafnrailgyetreasteidntpootlhyeprwoaptyelresnaempslaem.plAeutcoomntaatiincerssiamfpploesrssibtlhea.t `Sample bottles should not be rinsed before sample collection. 81 T(1a5pW1a0tCe)r--haOspsetanbitlhiezetdap(uasnudalallyloawbotuhtetswyostmeimnuttoefsl)u.sAhdujnutsilt tthheewfaltoewrttoeampbeoruatture. 500mL/min and collect samples from the flowing stream. 82 Ground Water--Purge sample directly from the the well ofstanding water pump or from the bailer. using a pumpor a bailer. Collect the 83 Surface Water--When sampling from `with water from a representative area. an open bodyofwater, fll the sample container 84 SinatmhpeldearDkecfhrloomrtihneattiimoeno--fAlclolslaemctpiloensusnthioluelxdtrbaectiicoend. oRrerseifdruiaglercahtleodraitne4s:h2oulCdabned kept rsaemdpulcee,d FbBy,aadnddinFgS2C0S0u(Lwhoicfah 2m5a0ymbge/pmlLacseoddiinuematchhiobsoutltflaetebesfoolruetiloenavtionegafcohrwtahteer sampling site.). 85 tHhoaltdtihnegthTrieme eco(mHpTou)n--dsReasurltsstaobflethfeorti1m4e/dsatyosraignewsattuedryosafmapllletsarwghetenantahleytseasmsphloesweadre dlaebcohrlaotroirnyatseadmapnledsstmoursetd abse deexstcrarcitbeedd wiintsheicnti1o4nd8a.y4s(sacnedatlhseoerxetfrearcetnscaen1a8l.y3z)e.dThweirtehfionr3e,0 days field ospfiskaemsptloeacvoolildecmtiisotn.epIrfetsheentHatTioenxocfetehdse 14 days, great care is sample concentration. used when evaluating 86 Field Blanks 86.1 Processa collected Field Blank Control Sample (FB) from the same general sample ite along with cach sample set at approximately the same (samples time). At tthyepelaIbwoartaetro,rys,eaplr,ioarndtosshaipmptlhee cFoBll1e0cttihoen,safimllpalisnagmspilteeacloonntgaiwnietrhwtihtehefmilptteyred 8.62 sWahmepnlesocodnituaimnetrhsi.osRuleftautrenisthaeddFeBdttootshaemplalbeosr,atuosreytwhietshatmhee pfirlolceeddsuarmepltoe bottles. preserve the FB. 87 Field Duplicates 8.7.1 sCaomlplleicnatg Fseite,lidflDeuspslitchaatne 1(0FDs)amfpolreesvearrye ctoelnl(ec1t0e)d.samples collected or per each 87.2 Separate FDs collected. must be collected for each type of water sample (ground, tap, etc.) 873 87.4 Collect the FD Preserve, store immediately and ship FD after using the the sample. same procedures as used for the samples. MS Word 97 ETS81540 Page of 17 DeterminationofPFOS, PFOSA, POAA in Water by Liguid-Solid Extraction and LOMS/MS 000104 88 Field Spike Control Sample (FSCS) 88.1 sAhiFpimeelndtS.piIfkemuClotnitprloelcoSoalmeprslear(eFuSsCeSd)tmousshtipbea psertepoafrseadmpfolrese,acchacshamcpoolleer must contain a FSCS. 882 Ashtitphteoltahbeorsaatmoprly,infgillsiatesaamlopnlge wciotnhtatihneeremwpitthy s1a0m0pmlLeocfonttyapieneIrwsataenrd.FSBeSa.l and 88.3 sWahmeenasmooduinutms tthoiotshuelFfaStCeSi.s added to samples, use the same procedure to add the 88.4 Seal and gently invert the FSCS to mix. procedures as used for the samples. Store and ship the FSCS using the same MS Word 97 ETS 1540 Page9or17 Determination ofPFOS, PFOSA, POAA in Water by Liquid-Solid Extraction and LOMS/MS 000105 9.0 EXTRACTION PROCEDURE 9.1 Extraction Scheme 9.1.1 Allow gently sinavmeprlteisngtotheequsialmipblraetebotttoler.oom temperature. Thoroughly mix samples by 912 QMeCasaunrdeM4at0rmiLxosfpsiakemspalserienqtuoiSreOdm,Lreppollaycperolipdyalenndemciexntwreilflu)g.e tubes (Spike the N(yoptiec:al*lySaSm0p-le1s50m%aoyfnseaemdptloebceonpcreenstcrrateieonne)d.to determine an appropriate matrix spike level 9.13 ConditiontheC, SPE by SmL filtered type | wcaatrterrid(g~e2sdr(o1pg/,se6c)m.L)Dboynpoatssleitncgo1lu0mmnL methanol run dry. followed Ndroytea:t aFnoyr tthiemefollowing steps, maintain a ~1droplsec flow rate. Do not allow the colum to run 9.14 9.15 WLoaasdhtwhietahna~lSytmiLca4l 0sa%mmpeltehoanntooltihnewCa,t,erS.PDEiscacratrrdidcgle.uatDei.scard eluate. 9.1.6 Eploultyeprwoiptyhl~enSemcLent1r0i0f%ugemettuhbeasn.olT.hiCoslilsetchteStmarLgoetfeelluutaitoen ifnrtaoctgiroand(ufaitneald v1o5lmuLme = SmL). 9.1.7 Analyzea portionofthe HPLC/MS/MS (Section 1t0a.r2g)e.t elution fraction eluent using negative clectrospray NFiontael:VSola.mp=leSsma.re concentrated by a factor ofeight during the extraction; Initial Vol = 40mL > 9.18 Smaamyplbeesstaorreesdtainblaeraetfrriogoemrattoermpater4at4u2reCfourntaitllaenaasltys2i4s.hours. Analytical samples 9.19 SbteannedcaersdsiazrayttioonstoafndCa,r,diSzPeEthce oC,luSPmEncso--lpuoImofnrsriencotvheerifeosllaorweionbgsmearvnende,ribtemfaoyre analyzing samples. 91.9.1" UPgs/emLa.stFaonldalrodwwtihtehexatnraacntailoyntesccohnecmeeniarsatoiuotnlibneetdwfereonm1s0t0ep0sa9n.d1.41010009.1.6, except, collect the eluate target elution fraction. fraction separately (approx. SmL), as well as the 9.192 After sep additional 9.1.6, collecta SmL of 100% post-elution methanol. fraction by, eluting with an 9.19.3 cAonnatlayiznse aallmithnrieemufrmaocftio8n5s%boyfHPthLeCr/eMspSe/cMtiSv.eIafnatlhyetetsa,rgiettmfaryacbtieon considered acceptable. 9.194 IofrtpheerwceansthagceonotfaiMnesOsiHgnsifhiocualndt bsetadnedcarreda(s>ed15%), ether the wash volume 919.5 eIlfutthieonpovsot-lculmuetisohnofurladctbieoninccornetaasiends.significant standard (>15%), the target MSWord 97 ETS8-1540 Page 100717 DemianofPEOS, ROSA, OAA in Water byLiquid:Sok Exacion and LOMSMS (0,690 101.0.00 ClNCAooALntLgeII:aBBsORRttAhAheTeTrIImeOiOntNNshtoANrdNuomDaenSiStTTesAAmNNaaDrDycAAbRRmeDDetIsI.ZeZATdAhTTaeIInOOodpNNet(hr(eaAAtcNNoqArAuLuLiYpYsmTTteIInoCCtAApaLLdiSSEsEcTToWUnnYPdd))id:ocamye5fevecryydomreentn oe inns 10.1 Establish the LC/MS/MS system and operating conditions equivalent to the following: Mass Spec: Micromass Quattro Ultima (Micromass) MInotderef:aceE:lecEtlreocstprroasyprNaeyga(tMiivcer,oMmualstsi)ple Response Monitoring (MRM) Harvard infusion pump (Harvard Instruments), for tuning Computer: COMPAQ Professional Workstation AP200 Software: Windows NT, MassLynx 3.3 HHPPLQCu:atHPewulmeptt Packard (HP) Series 1100 HHPP AVuatcousuammpDleegrasser roNoanttleim:naAy.HafPbtxueCnohIletmuepmmumnroHgbOeyipvvleeaerlnpvcehaaarsneddraoenpld-oiornrsgHuthParLdsCacmaaplpreviies(oKesysptoonte,0paarpt 4a4y01s7-e400E ) i as ched CHoPlLuCmnCoTleummpne:ratGuernee:si3s5C,C(Jones Chromatography), 2.1mm x 50mm, 4pm Injection Volume: 15uL Mobile Phase (A): 2mM Ammonium Acetate in filtered type I water (See 7.3.1) Mobile Phase (B): Methanol HPLC GradientProgram: min Time, `min 0.0 Phased Percent Mobile Phase A 60 Phase Percent Mobile Phase B 40 mimin FlowRate, `mL/min 03 04 60 40 03 1.0 10 90 0.3 7.0 10 90 03 75 0 100 03 9.0 0 100 04 9.5 60 40 04 13s 60 40 04 0 ee es 14.0 60 40 0.3 Not: Other HPLC gradients may be used slong as th method rieria are met 5 Ward? Ersasi0 -- Determination of PROS, PROSA, POAA in Watrby oLtig Soi Extraction and LCMSIMS 000107. CItomlauymnbsewnietchesdsiafrfeyrteontaddjiumsetnsthieonHsP(Lc.Cg. g2ra.d1imenmtxin3o0rmdemr)taonodptciomliuzmenisnsftrroummedniftfpereernftormance. `manufacturers (Keystone Betasil C,q etc.) may be used. Ions Monitored: Aualyte |Priimary lon Pr"oduct lon AAPpPpIrOoNxiImTaattee POAA | 4130 169.0 50 PFOS 499.0 99.0 52 PFOSA| 498.0 780 58 OfotrhePrFOprSo.duUcsteoiofntshmeasyugbgeesctheodsepnriamtatrhye idoinscirsetrieocnoomfmethnedeanda.lyRsett,eanlttihoonutgihmme/szma99yivsasruyggsleisgthetldy, oacncaepdtaayb-lteow-idtahyibnasains,andaelpyteincdailnrguno,natshelobnagtcahs othfemdorbiiftlecopnhtaisneuecstc.thDrroiufgthinthreeteennttiiroenatnialmyessisisand the standards are interspersed throughout the analytical run. 102 Tune File Parameters 102.1 Tinhsetrfuomlelnotwitnoginvsatlruuemseanrte. pArlosvo,idtehdesaes vaanlueexsammplaey. bAeccthuaalngveadlufersommatyimveartyo tfirmoem in order to optimize for greatest sensitivity Analyte | Dwell, sec POAA 02-04 PFOS 02-04 PFOSA 02-04 Collision Energy, eV 10-25 30-60 20-50 Cone, V. 20-30 50-80 30-60 Source Capillary Hexapole | Aperture | Hexapole 2 Source Block Temp. Desolvation Temp. Set 2.56-3.5kV 0sv 02v 08v 100-150C 250-400C MS Word 97 ETS8.1540 Page 120117 Determination of PFOS, PFOSA, POAA inWaterby Liquid-Solid Extraction and LC/MS/MS 600108 Analyzer LM Res| HM Res | Energy | Entrance Exit LM Res 2 HM Res 2 IEnergy 2 Multiplier Set 12.0-15.0V 12.0-15.0v 0.7v av w 1L0V 11.0v Lov 650V Gas Flows Cone Gas Desolvation Set 150L/hr 700L/hr Pressures Set eenSGasRCeSll heii 3.0-3mbar 1A111.00vANaALYu TICAvL QUm ALITeY COaNTRQ OL uaryCovmeor 111 Analytical results of the FB, FMS, FD, and FSCS should be `evaluated at the conclusion ofcotnhtreols/tduudpylitcoatheelspamipntleersprmeut stthebdearteapqouratleidtywoifthstahmeplseasmpdlatea.daAtan.alytical results for these MS Word 57 ETSs1540 Page 3087 Determination ofPEOS, PFOSA, POAA in Waterby Li;guid-Sold Extraction and LOMSIMS. 000109 1A 122.00 s ANALu YTICAv L PROm CEDURe E m0p0r 00oc0e00o00e 000e0 121 Sample Analysis 12.1.1 Setup analysis sample queue. 12.1.2 Irnejceocvtertyh,e scoanmteroalleitqcu.otin(tboetthweeeLnC/5-M2S5/yMLS)osfycstaecmh. standard, analytical sample, 12.13 All samples showing a response for on or more analytes above the response of the highest, active calibration curve level must be diluted andreanalyzed. 12.2 Calibration Curve 12.2.1 Starting with the standard of lowest concentration, inject the same size aliquot (between 10-25L)ofeach extracted calibration standard according to Section 12.1 and tabulate the response (peak height or area) versus the concentration in athnealsyttaendbayrd.linUeasrerleignreeasrssitoannwdiatrhd c1/uxrvweesigfohrtiqnugaontfipteataikonagreanevreartseudsfcoarleibarcahtion standard concentration. The correlation coefficient (r) for the calibration curves ampupsrtopbreia2t0e.s9t9e0ps(2m0u.s9t8b0e).tIafkceanlitboraadtjiuosnt rienssutlrtusmfeanllt oouptesriadteiotnheasnedlitmhietss,tatnhdeanrds reanalyzed. 122.2 122.3 tChueorrvetei--caTlhvealmueeasswuhreedn vcaulruvee fiosreevaaclhuactuerdovveeproiantrmaunsget abpeprwoiptrhiiant+e t3o0t%heodfata. aHcicgehptoarblleowcuprovientmsumsatycobnetadienacattilveaatsetdftioveaacchtiievvee thes curve criteria, points. but an Continuing Curve Verification (CCV)--Mid- and low-level calibrationchecks should be analyzed every 5-10 injections. The analyte level measured in the CCV should be within + 30% of theoretical values. If CCVs fall outsidoe fthis range, data collected subsequent to the last passing CCV should not be used. Only data collected between acceptable CCVs or the initial curve can beused. 1p133.00aDAmTA aANALAYSISvANDmCALvCULsATIsONS awCucuamoss 13.1 Cfaollcluolwaitneg tehqeuaatniaolny:tical sample (extract) concentration from the standard curve using the Extract Concentration, pg/mL=- {Peakaaroeaa-niintuecrceeptt)) (slope) 13.2 Calculate the percent recoveryof the FSCS using the following equation: FSCS % rec, =((CFEoSCnCcSS.cacodonndcee.d.,,PPP/gg/m/mmlLL))) 133 Calculate the percent recoveryof the MSs using the following equation: MS%rec, -- Scone. pg/mL ~SampleConc, PmL) | oo (Conc.added, Pg/mL) MS Ward En f---- Determination ofPFOS, PFOSA, POAA in Wair by Liuid-Soli Extraction and LCMSIMS 000110 1M144.00ETNMoEtHeT:HAOOnDyPDmEetRhPFoOdREpMerAfRNoCrmEFanPcAeORpAarMRaEmTeMtEeRrSsAt~ hatNareCnotEac~ hPieveAd mRust~ Abe cMon~ siEderTed~ inEtheRS edviaslcuuastsieodnoinfatnhye rdeaptao.rtNinognocfotnhfeordmataan.ce to any specified parameters must be described and 14.1 Lreignreeasrsiiotny--wiLtihne1a/rx swteaingdhatridngcourfvpeseafkoraqrueaantvietrastuisoncagleinbreartaitoendsftoarndeaarcdhcaonnacleyntterabtyiolni,neTahre correlation coefficient (r) for the calibration curves must be 20.990 (20.980). 142 wCiatlhiibnra+ti3o0n%CoufrtvheeoSrettaincadlavradlsu--esThwehemneacsuurrveedisvaelvuaelufaotrecdaocvhercuarvreanpgoeinatppmruosptribaete to atchecedpattaa.blHeicguhrvoer mluoswtpocionnttsaimnaaytlbeeasdtefaicvteivaactteidvetocuarcvheiepvoeintthse.se criteria, but an 143 CinjCecVtioPnes.rfTohremaannaclyet--eMliedvelanmdealsouwrleedveiln ctahleibCrCatVisonschhoeuclkdsbteowbietahnai+lnyz3e0d%eovferthye5or-e1t0ical CvaCluVes.shIofuCldCVnost fbaelluosuetds.iOdenloyf tdhaitsarcaonlglee,ctdeadtabecotlwleeecnteadcscuebptsaebqlueenCtCtoVthcealnasbtepuassesdi.ng 144 tLhiemipteaokfaDreetaoecftitohne (exLtOraDc)ti--oTnhbelanlkowtehsatt ccaalnibbreatmieoanssutraenddaartdawicotnhcaenpteraaktiaornegaraetatleerastth2aXn zero. 145 sLtiamnidtasrodfinQutahentciatlaibtriaotnio(nLcOurQv)e;--tThheepelaokwearreLaoOfQth(eLLLOLQO)Qismtuhsetlboeweasttlenaosnt-2zeXrothaacttoifve the extraction blank. By ofthe theoretical value. definition, the measured value of the LLOQ must be within 30% 14.6 Matrix Spikes--Matrix concentration. spike percent recoveries must be within + 30%of the spiked 147 SloowlevsetntnoBnl-aznekrso,aMcteitvheosdtaBnldaanrdksi,n athnedcMalaitbrraitxioBnlcaunrkves.--VMaatlruiexsbmluasnktsbaerebecloonswitdheered compliantif no test substance is detected above the LOD for that analyte. 148 sRpeipkreosduacnidbmialtirtiyx--Rsepipkreodduucpliibcialtiets.yoTfhteheMSm/etMhSoDd isshdoeuflidnbede breyptrhoedurceisbulltesotofwtihtehimnat2r0i%x. 149 UseofConfirmatory Methods--None 14.10 rDeetmeontnisotnrtaitmieo(nwoifthSipnec3i%foicfitsyt--aSnpdearcdi)fiacnidtythise dmeamsosnsstprecattreadl bryescphornosmeaotfougnriaqpuheicproduct fons generated from a characteristic primary ion. 14.11 Documentation 14.1.1I`fmcaryitbeeripaelrifsoterdmeind tohnistmheetshyosdtepmerafnodrsmaamnpcleessercteiaonnalayrzeedn,otormeott,hemraiacnttieonnasntcaeken 14.1.2Iafsdadettaearrmeinteodbebyretphoeratneadlywshte.nDopcerufmoernmtanacleaccrtiitoernisaihnatvhee naoptprboeperniamteell,otghbeoodka.ta must be footnoted on tables and discussed in the textofthe report, MSWord 97 ETS 81540 Page 150f17 Determination ofPFOS, PFOSA, POAA in Water by Liquid-Solid Extraction and LOMS/MS 600111 1125.00 TOPLOLLULUTTIIOONNPPRREEVVEENNTTIIOONNAANNDDWWAASSTTEEMMAANNAAGGEEMMEENNTT~~ 15.1 Sglaamspslpeipeextttreacwtaswtaesties dainsdcafrldaemdmianblbreoskoelnvegnltasiss dcoinstcaairndeerdsilnohciagthedBiTn Uthecolnatbaoirnaetrosr,y.and 118 60 kReEcCOoReDsSsom 16.1 Ethaechhepaadgeer goernhearnadt-ewdriftotreansotnudtyhemupasgteh:avsetutdhyeofrolprloojweicntgniunmfboerrm,ataicoqnuiisnictliuodnedm,etehitohde,r in integration analyst, method, sample name, extraction date, dilution factor (ifapplicable), and 162 Parpipnrtoptrhieattuensetupdaygef,olsdaerm.plCeolpiyst,tahnedseapcaqugiessitainodn method from MassLynx to tape into the instrument run include log, in the 163 Plot the calibration in the study folder. curves as described in this method, then print these graphs and store 164 Print data integration MassLynx, and store summary, integration in the study folder. method, and chromatograms, from 16.5 Summarize data using suitable software (MS Excel 97) and store in the study folder. 166 Banadckloucpateiloencotfrobnaicckduatpaetloecatprpornoipcrdiaattae. medium. Record in study notebook the file name A 17.0 AAtTaTcAwCHvMeErNTsS 17.1 Attachment A: Figures--Fluorochemical Compounds = 18.0 RXeErFeEmRewE<NCCqESSSooo 18.1 "U.GSu.idEenlviinreosnamnedntFaolrmPraottefcotriMoentAhgoednscyt,o bOeffPircoepoofseSdciaetn4ce0 aCnFdRTePacrhtno1l3o6goyrOPfafritce14o1f", Water, Washington, D.C. Draft 1996 182 "PeMreftlhuoordooofctAannaelyssuilsfofnoyrltahmeidDeet(ePrFmOiSnaAt)i,onanodfPPeerrfflluuoorrooooccttaannoeastuel(foPnOaAteA()PiFnOSW)a,ter, . IWnicc.krSteamteesCionlhleegaen,dPJe.nnFlsayhlevratnyi,aS,tJuadnyuaNruym2b0e00r.023-002, Centre Analytical Laboratories, 183 sVuallfiodnaattieon(PrFepOoSr)t,fPoerrtfhleu"orMoeotchtoandeosfuAlnfaolnyysliasmifdoer (thPeFODeStAe)r,miannadtiPoenrofflPuoerrofolcutoarnoooactetane (AnPaOlyAtAi)cailnLWaabtoerrat"o,riE.esW,iIcnkc.r,eSmteastienChoellaengde,J.PeFlnanhseyrltvya,nSiat,ud(yApNpurmobvaelrp0e2n3d-i0n0g2), Centre RT 1e9v.i0sifeion Rnevisions e Number, ReasonForRevision Revision Date MS Word 97 ETS81540 Page 160617 Determination ofPOS, PFOSA, POAA in Waterby Liquid-Solid Extraction and LC/MS/MS 600112 Figure 1: PFOS Chemical Name = Molecular ion = f "1 Perfluorooctanc sulfonate 499 (CF,S0,) PFOS Note: Standaarermaddse from the salt, potassium perfluorooctane sulfonate [CyF, SOK], mw 538. Figure 2: PFOSA Chemical Name Molecular fon = = Perfluorooctanesulfonylamide 498 (CF,,SONH,) ceFrp NH2 o PFOSA Figure 3: POAA Chemical Name = Molecular ion = Perfluorooctanoate 413(CF,C00) C7F45CO" POAA Note: Standards are made from the salt, ammonium perfluorooctanoate [C.FsCOONHL], mw 431 MS Word 97 Attachment A: ETS.5.1540 Page 170617 Determination of PROS, PFOSA, POAA in Water by Liquid-Solid Extraction and LC/MS/MS 000113 Compound-Specific, Quantitative Characterization of Organic Fluorochemicals in Biological Matrices Kristen J. Hansen; Lisa A. Clemen: Mark E. Ellefso; Harold O. Johnson (#Coresponding author email: G hansen mco, msphone 651-778-6018;fax651-778-6176) 3BuMilEdnivnigr2o-n3mEe.n0t9al Laboratory SPt.0.PaBulo,xM33N335151333331 Abstract Since the early 1980s, there has been a steady increase in the use of nonvolatile fluorinated organic compounds for a varietyof industrial applications. The industrial use ofthese relatively stable compounds has initiated debate over the fate of fluorochemicals in the environment and, ultimately, the bioavailabilityofthese compounds (1,2). Until recently, levelsoforganic fluorochemicals in biological matrices have been determined by non-chemical specific analytical methods such as total fluoride analysis (2-6). In this manuscript, we present a compound-specific method for the extractionof extremely low. levelsofseveral commercial organic fluorochemicals from sera and liver with `quantitative detection by negative ion electrospray tandem mass spectrometry. This technique represents a robust, previously undescribed approach to quantifying specific organic fluorochemicals in biological matrices. This method should prove useful in future studies designed to determine the levelsoforganic fluorochemnicals in humans and the environment. Results from a studyof 65 human sera samples purchased from biological supply companies and the detailsofthe analytical method for the quantitative analysis of specific organic fluorine containing compounds are described. 1 600114 Introduction `The unique chemical/physical properties of fluorine make fluorinated organic compounds useful for many commercial applications, and industrial production of these compounds has increased significantly since the early 1980s. Fluorinated organics are used as refrigerants, surfactants, and polymers and as components of pharmaceuticals, fire retardants, lubricants, and insecticides (1). Fluorochemical compounds that are not perfluorinated may be susceptible to partial chemical breakdown at functional group bonds (7). However, given the energyofthe carbon-fluorine bond, it is expected that `many organic fluorochemical compounds will be resistant to hydrolysis, photolysis, biodegradation, or metabolism (8). For example, even in the `high-energy environment of the stratosphere, the carbon-fluorine bonds in chlorofluorocarbons are exceptionally stable (9). In 1974, Guy et al. reported results for the determinationof organic fluorine levels in plasma from 106 individuals from five cities in the United States (2). These researchers demonstrated that although levelsofinorganic fluorine in human plasma could be correlated to fluoride levels in drinking water, organic fluorine levels showed no such correlation. Guy et al. showed that the organic fluorine levels measured from samples collected within a particular city were, basically, log normally distributed with few outliers. The average organic fluorine level in human plasma samples included in the study was reported to be 1.35 + 0.85 micromolar R-F (approximately 26 ppb organic fluorine). By concentrating the organic fluorine from 20 liters of plasma and performing nuclear magnetic resonance (NMR) analysis, Guy et al. postulated that the 600115 2 perfluorooctanoate anion (PFOA) or a structurally related compound may be the source ofthe organic fluorine. Further, they suggested that there may be three or more different componentsoforganic fluorine in plasma samples collected from the general population. Although the source of the organic fluorine in general population blood has been debated and never definitively determined, some have postulated that contamination of the environment with industrial fluorochemicals is the source of the organic fluorine compounds (4). Others suggest that the organic fluorine is likely to have a natural source (10). Despite the routeofexposure, researchers agree that such low levels oforganic fluorochemicals are unlikely to cause toxic effects (2,11,12). A large numberof studies in both humans and animals have been conducted to study the toxicity associated with PFOA. In these studies, when determinations ofthe PFOAlevels in tissues were necessary, a total organic fluorine method was employed or a study using radiolabeled material was designed, because easy, sensitive, compound-specific methods `have not been available (11-17). Historically, low-level detectionoffluorochemicals such as PFOA and perfluorooctanesulfonate (PFOS) has been limited to relatively insensitive or non-mass-specific detection methods, such as gas chromatography-flame ionization detection, gas chromatography-clectron capture detection and high performance liquid chromatography (HPLC)-ultraviolet detection (18-20). In the work presented here, a new method for the analysisofseveral low-level fluorinated organic compounds in sera and liver tissue is described. After initial extraction ofthe tissue with an ion-pairing reagent, extracts are analyzed with HPLC-negative ion electrospray tandem mass spectrometry (HPLC-ESMSMS). The ability to selecta unique 000116 : product ion upon fragmentation of the molecular fon provides a very selective analysis that is not as likely to be affected by biological interferences. Detection limits and the linear rangeofthe method were determined for four fluorinated organic compounds [PFOA, PFOS, perflucrooctanesulfonylamide (PFOSA), and perfluorhexancsulfonate (PFHS)] in both liver and sera by spiking each matrix with standard material and quantitatively recovering the compounds. Although detection limits can be improved by concentrating sample extracts, extractionof non-concentrated sera produced detection limits for all target analytes of 1-3 ppb. The method presented here has been used to quantitatively analyze four organic fluorochemicals in 65 human sera samples collected from several biological supply `companies in the United States. High-resolution time-of-flight mass spectrometry was used to confirm the identity of PFOS, PFOA, PFH, and PFOSA extracted from a single representative sera sample. Experimental Materials andMethods Rabbit and rat sera were purchased from Sigma (St. Louis, MO). HPLC-grade methyltert-butyl-ether (MTBE) and methanol were purchased from E.M. Science (Gibbstown, NJ); the tetra-butyl ammonium (TBA) hydrogen sulfate was purchased from Kodak (Rochester, NY); the pHof the TBA solution was adjusted with sodium hydroxide (J.T. Baker; Phillipsburg, NJ). Before use, water was purified with a Milli-Q system (Millipore; Bedford, MA). Human sera samples were purchased from the following biological supply companies: Sigma (St. Louis, MO); Golden West Biologicals 000117 4 (Temecila, CA); Biological Specialty Corporation (Colmar, PA); and Lampire Biological Laboratories (Pipersville, PA). New Zealand White [Hra:(NZW)SPF] rabbit liver was obtained from Covance Laboratories, Inc., in Madison, WI; Sprague Dawley rats were purchased from Harlan (Indianapolis, IN), and rat liver samples were harvested by 3M Toxicology personnel (St. Paul, MN). `The PFOS and PFOA used as standards and as matrix spikes were purchased from Fluka (Milwaukee, WI; standards of PFHS and PFOSA were made available from 3M Company (St. Paul, MN). The intemal standard, /1,1H,2H2H, perfluorooctane sulfonate (THPFOS), was purchased from ICN (Costa Mesa, CA), Extraction Procedure: One half mLofsera, 5 uLofintemal standard, 1 mL of 0.5 M TBA solution (adjusted to pH 10), and 2 mLof 0.25 M sodium carbonate buffer were added t0.a 15-mL polypropylene tube for extraction. Afier thorough mixing, $ mL of MTBE was added to the solution, and the mixture was shaken for 20 minutes. The organic and aqueous layers were separated by centrifugation, and an exact volume of MTBE (4.0 mL) was removed from the solution. The aqueous mixture was rinsed with MTBE and separated twice more; all rinses were combined in a second polypropylene tube. The solvent was allowed to evaporate under nitrogen before being reconstituted in 0.5 mL of methanol. The sample was vortex mixed for 30 seconds and passed through a 0.2 um nylon mesh filter into an autovial. Depending upon the species of test animal, the serum extract was typically either colorless or light yellow. 600118 E For the extraction of liver samples, a liver homogenate of 1 gramofliver to 5 mL of Milli-Q water was prepared. One mLofthe homogenate was added toa polypropylene tube, and the sample was extracted according to the procedure for sera (described above). Teflon or glass containers were avoided in this procedure; the former may cause analytical interferences, and the latter may bind the surfactants in an aqueous solution. Disposable polypropylene or plastic lab wear was used to minimize the possibility of sample contamination that can occur when glassware is reused. Any glassware used in the preparation of the reagents was thoroughly rinsed with methanol prior to use. To ensure that target analytes were not introduced to the matrix prior to extraction, blood collection supplies were extracted and analyzed. Blood bags were purchased from Baxter (Deerfield, IL), and five different typesofVacutainers (two labeled "gel and clot activator,"two labeled "K;EDTA, and one labeled "no activator") were purchased from Becton Dickinson (Franklin Lakes, NJ). Additional blood collection materials tested consistedof3-cc and 10-cc syringes, 19G1 1/2 Precision Guide sterile needles, multiple sample Vacutainer sterile needles, and Terumo winged infusion sets, all of which were obtained from Becton Dickinson. The inside surfacesofall blood collection supplies were exposed to methanol (from 0.5 mL to 80 mL, depending on the particular supply) for 1 hour, The extraction solvent was dried and reconstituted to exactly | mL of methanol. A second setofsamples was 00119 . spiked with analyte and extracted in exactly the same way as the first set to ensure that analyte could be recovered. Extraction blanks were prepared using Milli-Q water, and matrix blanks were prepared from rabbit or rat tissue spiked with THPFOS. Analyte separation was performed using a Hewlett-Packard HP1100 liquid chromatograph modified with low dead-volume internal tubing.Priorto the autosampler, a1 cm Hypercarb cartridge from Keystone (Bellefonte, PA) was added. Ten Ls of extract were injected onto a 50 x 2mm (5 um) Keystone Betasil Cys column with a 2 mM ammonium acetate/methanol mobile phase starting at 45% methanol. At a flow rate of 300 uL/minute, the gradient increased to 90% methanol before reverting to original conditions at 9 minutes. Column temperature was maintained at 25 C. For quantitative determination, the HPLC system was interfaced to a Micromass (Beverly, MA) Quattro I atmospheric pressure ionization tandem mass spectrometer operated in the electrospray negative mode. Instrumental parameters were optimized to transmit the [M-H] ion for all analytes. When possible, multiple daughter ions were `monitored, but quantitation was based on a single product ion. Refer to Table 2 foar summaryof transitions monitored. In all cases, the capillary was held between 1.6- 3.2 kV. For PFOA determination, the quantitation ion (m/z=169) corresponds to CsF; the product ion m/z=99 corresponds to 600120 ' FSOy for quantitative determination of PFOS. QuantitationofPFOSA occurs at m/z=78, corresponding to SO;N'; quantitationofPFHS occurs at m/z=80 (SO). In the ESMSMS system, the 499 Da. 80 Da. transition can provide a stronger signal than the 499 Da.> 99 Da. transitionofthe PFOS analysis. However, in the analysis of tissue samples collected from some speciesofanimals, an unidentified interferent was present in the 499 Da. 80 Da. transition. Although this interferent was rarely observed, to ensure complete selectivity, quantitation was based on the 499 Da. > 99 Da. transition. Exact mass determination was achieved by interfacing the chromatographic system to either a Micromass LCT; product ion spectra were collected with a Micromass Q-TOF. Both the LCT and Q-TOF are high-resolution time-of-flight mass spectrometers. An 800 ng/mL solutionofraffinose in 50/50 ACN/water was infused into the source at 20 wL/hars a lock mass (503.1612 Da). PFOS, PFOSA, and PFHS were measured ata cone voltage of 70 V; PFOA was measured ata 10-V cone voltage. For analysisofall analytes, the capillary was maintained at 3200 V. Results and Discussion Characterization of the Method A seriesof experiments, described in more detail below, was designed to characterize the analytical method. In general, all curves, extracted or unextracted, were plotted using linear regression, weighted 1/X. Tables 3 and 4 show the extraction efficiency, limit of detection, and linear range for the target analytes. 600121 * With the exception of PFOA, the extraction efficiency was determined by extracting and analyzing six replicate rat or rabbit sera samples spiked at approximately the following levels: 10 ng/mL, 50 ng/mL, 100 ng/mL, and 500 ng/mL. For PFOA, only the three higher levels were used for extraction efficiency calculations. Extraction efficiency in liver was determined by extracting samples spiked at 50 ng/g, 100 ng/g, and 500 ng/g. For both sera and liver, the extracted samples were evaluated versus the average curve produced by two unextracted solvent curves analyzed before and after the extracts. The extraction efficiency for PFHS and PFOSA from liver was determined to be significantly lower than those determined for the PFOS and POAA. However, because sample analysis is conducted using extracted curves, the relatively low recoveries should not affect the results. Table 3 shows the compiled average for all spike levels along with the standard deviation. For both sera and liver analyses, the internal standard was used for quantitative determination of PFOS and PFOA, only. `The limitofdetection was determined as per EPA Regulation 40 CFR part 136, Appendix B. For each analyte, seven low-level spikes were prepared and analyzed. Based on the standard deviation associated with the replicate analysis,a limitofdetection was calculated. This calculated limitofdetection was verified by analyzing a sample that was spiked at that level and extracted. G0v122 9? `The linear range was determined by analyzing duplicate curves extracted from each matrix over a wide range. Starting with the highest standard, points were removed from the curve until the correlation coefficient for the 1/x weighted fit was greater than 0.99. For the sera curves, all points except for the lowest standard level were evaluated to be. `within 20%ofthe expected value. For the standard curves extracted from liver, all points except the lowest point were within 30. Characterizationof Blanks Method blanks were prepared from Milli-Q water. Because analyte-free (less than 1 ng/mL) human sera matrix could not be located, surrogate matrix blanks were prepared from rabbit sera. Noneofthe analytes were detected in either setof blanks. Instrument blanks, consistingof HPLC-grade methanol, were analyzed after high-level-standard- curve points and after periodic calibration checks, to monitor potential carry-over. No carry-over was observed. Methanol extracts ofblood collection supplies were analyzed; noneof the target analytes were detected in these extracts. In addition, the Teflon cap linersofglass jars used for reagent storage were extracted with methanol. Low-levels of PFOS and PFOA were detected in someofthe extractsofthe Teflon liners. These materials were removed from the extraction procedure. Figure | compares the resultsof the `multiple response monitoring (MRM) analysis for PFOS in an extraction blank, in unspiked rabbit sera, and in unspiked,`commercially available human sera. 000123 0 Identification of Target Analytes `The retention times of the analytes extracted from human sera were matched to within 2%ofthe retention timeofstandard material spiked into and extracted from rabbit sera. MRM analysis was used for verification ofanalyte identity. For cach analyte except PFOSA, atleast two characteristic product ions were monitored, although quantitation was based on the response ofa single product ion. PFOSA was detected at such low. levels, only a single product ion could be monitored, even for qualitative purposes. In the. human sera samples, for all analytes except PFOSA, the relative abundances of two or `more product ions collected by MRM were confirmed to within 20%ofstandards as criteria for analyte verification (21) To further verify the identityofthe detected analytes, a 30-fold concentrated extraction of one sera sample was prepared. This concentrated extract was used for exact mass determinationofall four analytes using high-resolution time-of-flight mass spectrometry. The concentrationofthe detected analytes were confirmed to within ppm for al target analytes. Figure 2 shows the results of the PFOS and PFOSA high-resolution analysis. Additionally, using high-resolution time-of-flight mass spectrometry, full product ion spectra were collected for each analyte in the concentrated extract. The product ion spectra for the POS identified in human sera is shown in Figure 3. Quantitation of Target Analytes in Human Sera Quantitationof the analytes was based on comparisonof a single product ion peak area to the responseoftwo standard curves, weighted 1/X, bracketing each sample set. Mid-level calibration checks were analyzed every five to ten samples. Based on the precision 600124 n determined from repeat injections of the standard curves, results were considered quantitativeto + 30%. Quantitative results, presented as compound-specific average concentrations in sera are presented in Table S. In addition to the average analyte concentration, the concentrationoforganic fluorine represented by cach compound is presented. For example, by weight, PFOS is 63% fluorine; for samples reported here, the average PFOS concentration was determined to be 33 ng/mL of PFOS. This corresponds to about 22 ng/mLoforganic fluorine. Added together, the four specific fluorochemicals measured in this small setofsamples account for approximately 31 ngof organic fluorine per milliliter of sera. Within experimental error associated with cach technique, this value compares closely to the value obtained by Guy etal. (approx. 26 ng/mL) more than 20 years ago (2). Also in accordance with Guy etal., PFOA has specifically been identified in the sera samples, although not necessarily as the major component. For the 65 samples reported here, PFOS was present at the highest concentration. Each analyte measured was detected in every sample, with the following significant exceptions: PFOSA was not measured above the detection limit in 60of the 65 samples; PFHS was not detected in one sample. A combinationof extraction and analytical methods that do not require chemical derivitization, use small volumesofsamples, and are highly sensitive and mass specific were developed for the low-level analysisof several fluorinated organic compounds in sera and liver. Using these methods, samples of human sera collected from biological supply companies were analyzed for four separate fluorochemicals, PFOA, PFOS, PFHS, 600125 B and PFOSA. Taken together, these fluorochemicals accountfor 31 ng/mLoforganic fluorine in an examination of 65 human sera samples from biological supply companies, consistent with historical reportsoftotal organic fluorine studies. Although this study comprises a relatively small sample set, it does suggest the possibility ofamore complete characterization of the organic fluorine compounds present in human sera. Additionally, these compound-specific analyses should be paired `with a total organic fluorine analysis to determine what fraction of the total organic fluorine present is due to the four fluorochemicals quantified in this study. 600126 " Acknowledgements `The authors are grateful to Dr. Andrew Seacat and Deanna Luebker of 3M Toxicology for supplying rat liver for method development, and to Dr. George Moore for providing standard materials. Dr. Robert Voyksner is acknowledged for his thorough and timely reviewofthis work. 600127 14 References 1 EKnevyi,roBnD. .SciH.owTeeclhln,olR..D1.9,9a7n,d31C,ri2d4d4lc5,. C.S. Fluorinated Organics in the Biosphere. 2 PGluays,maW;S,PrTeavvaelesn,ceDRa.n,d aChnadraBcrteeyr,izWa.tSi.o,n.JrB.iOorcghaenmiicstFrlyuIonrvooclovimnpgouCnadrbsoinn-FHluumoarinne B`onnudmsb,erE;diptp.io1n1n7u-mXbXeXr; Publisher; Place of Publication; Year; Volume 3 T1a9v6e8s,,2D1.7,Ev1i05d1ence that there are Two FormsofFluoride in Human Serum. Nature 4 Taves, Nature D.R. Comparisonof 1971, 50, 783, `Organic' Fluoride in Human and Nonhuman Serum. S BCeolnitselne,tJo, faWnhdolHeagBelno,odD,.FS.erMuemt/hPoldasfmora,thaendDeOttehremrinBaitoiloongiocfalthSeaTmoptlaelsFAlnuaolr.ine Biochem. 1978, 87, 545. 6 FYoarmmasmootfoF,luGor,inYeosihnitWahkoel,eK.B,loSaotdo,ofT.H,uKmiamunraM,alTe.,Aannad.lABnidooc,heTm..Di1s9t8r9i,but1i8o2n, a37n1d. 7 7FlHuaorgienna,teDdIMFe;taBbeloilsiltee,sJb;yJoahGnassonC,hJr.oDm.;atVgernakpahtiecswHaerlliuu,mV.MiCcharroawcatvereizPaltaisomnaof DPeertfelucotroo--rocTtahneoaBtieo.trAannaslf.oBrimaotcihoenm.of191H8,11,H1,128H,,233H6-.Perfluorodecanol to 8 SOmragratn,ofBl.uEo;riTnaetlCohwe,mi1.sC.t,ryEdPsr.i;ncPipellensuamnPdreCsosm:mNeercwiaYlorAkp,pl1i9c9a4tions; Banks, R.E.; 9 Atmospheric Chemistry; Sons: New York, 1986. Finlayson-Pits, B.J, Pitts, LN. J. Eds; John Wiley & 10 SBecliiesnlcee, J.1O98r1g,an21i2c, F1l5u0o9r.ine in Human Serum: NaturalVersus Industrial Sources. 11 Gilliland, Enzymes, F.D., and Mandel, Lipoproteins, and C1hSo.leSseterruoml:PeArfSlutourdoyoocftaOncociucpAactiiodnaanlldyHEepxaptoisced Men Am. J. Ind. Med. 1996, 25, 560. 12 PGreirfffliutohr,oFo.cDt.a,noaantde.LoAnm.g,IJn.d.E.HyAgn.imAaslsoTco.xiJ.ci1t9y8S0t,u4d1i,es57w6ith Ammonium 000128 ee 13 TKoexninceold.y,ApJpRl..PahnadrmGaecroall.d,1L.9;85D,e8r1m,a3l48T.oxicity of Ammonium Perfluorooctanoate. 14 Kennedy, GL, of Ammonium Jr, Hall, G.T, Brittelli, M.R., and Chen, Perfluorooctanoate. Food Chem. Toxicol. H.C. Inhalation 1986, 24, 1325. Toxicity 15 HPaenrhfiljuaorrvoio,ctHa.n,oOatpehaEuxgcr,eRtiHo,n,ianntdheSiRnagte.rP,rLo.ceTehdeinSgexso-rfeltahteeSdoDciifefteyrfeonrce in Experimental Biology and Medicine 1982, 171, 50. 16 VTiesnsdueenDiHseturviebult,ioInP,,MeKtusalbiokliiss,mB,.1.,anVdaEnlimRianfaetligohneomf, PMe.rJf.l,uaonrdooPcettaenrosiocn,AcRi.dE.in Male and Female Rats. J. Biochem. Toxicol. 1991, 6, 83. 17 VExecnrdeetnioHnoeufvePle,rfJl.uPo,roDoacvtiasn,oiJc.WA.,ciIdI,iSnoMmamleersR,atRs.:, aInnhdibPiettoerrysoEnf,feRc.tE.o;fRenal TestosteroneJ.. Biochem. Toxicol. 1992, 7, 31. 18 OPehryfal,uoTr.i,naKtueddoC,arNb,o,xSyulziuckiA,ciEd.,sainndBiKoalwoagischailmSaa,mYp.l,eDsebteyrHmiignha-tpieonrfoofrmance Liquid Chromatography. J. Chromatogr. 1998, 720, | 19 ABecliidslien, BJ.l,oaonddaHnadgeOnt,heDr.FB.ioAlogMiectahloSdamfpolretsh.e ADneatle.rmBiinoacthieomn.o1f9P8e0r,fl1u0o1r,o3o6c9t.anoic 20 DYleitneernm,inMa.t,ioHanonhfijParFvOi,AH.a,s tPheeurBae,nzP.,ylREasmtoer,iOn.PQluaasnmtaitaantdivUerGinaes.CAhrcrho.maEtnovgirraopn.hic Contam. Toxicol. 1985, 14,713. 21 Lily Y.T., Ultratrace Campbell, D.A., Bennett, PX., and HPLC-Tandem Mass Spectrometry: Henion,J. Acceptance Criteria Quantitative and Qualitative for Determinationof Sulfonylurea Herbicides in Soil, Anal. Chem. 1996, 68, 3397. 600129 16 ER Orin [SCR IT CN JO Nia ray) PFOA 413 119,169%,219 | 2s 20 PFOS 99 50,99%, 130 0) as PFHS 399 80,99%, 130 0) as | [TrPwFeOrSoAs || 498 am w 78 w Te || as "Product ions were used for quantitation Table 2. Summary of Primary lons, Product lons, and ESMSMS Conditions 000130 1 RE PFOA PFOS PFOSA PFHS Ea [Crave RR DEVIATION 1019% 939% | 95:6% 857% [ DETECHON rea RANGE, EXTRACTED CORRKLATION COEFFICIENT 1.0 ppb 1.7 ppb 5-1000ppb | 0998 | 5-100 ppb 0.995 1.5 ppb 5-1000 ppb 0.998 20ppb | 5-1000 ppb 0.998 Table 3. Method in Sera. Characteristics for the (All concentrations are Analysisof Specific expressed as ng/g.) Organic Fluorochemicals 600131 " LTT Te Liner RANGE, CORRELATION SRE LL LL LR eT PFOA 87+12% 5.0 ppb 10-1000 0.989 PFOS 10013% 8.5 ppb 5-1000 0991 | PFOSA | s611% 35ppb_ | 5-100 ppb 0.995 | prs 71423% 20ppb | 5-1000 ppb 0.994 Tabled. Method in Liver. Characteristics for the Analysis of (All concentrations are expressed Specific as ng/g.) Organic Fluorochemicals 600132 ar CONCENTRATION IN eer CONCENTRATION IN CONCENTRATIOONF ORGANIC FLUORINE SSE Ra Ra fy PFOS 3321s 5-85 rfre e e 2 PFOA 663 1-13 46 PFHS 64%5% 1-13 37 PFOSA | 1803+ <12 11 * qSueavnetriatlatsiaonm,pltehserwefeorree,daevteerrmaigneecdotnocecnotnrtaatiinonthies target analyte estimated. below the limit of Table 5. Concentrations (ng/mL) of Various Organic Fluorochemicals in Human Sera 000133 2 Figure 2. High Resolution Analysis of PFOS and PFOSA 000134 2 Figure 3. Product lon Spectra for PFOS Endogenous in Human Sera 000135