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250i7p ORIGINAL ~~ pr226- 095/ 3M Medical Deparment Study:T-6265.7 -_ LaboratRoerpy oRretquNeos.t FNAuCmbTeTrOUXz023705 ANALYTICAL LABORATORY REPORT FROMTHE 26-Week Capsule Toxicity Study with Perfluorooctanesulfonic Acid Potassium Salt (T-6295) in Cynomolgus Monkeys oNTHE Determination of the Presence and Concentration of Perfluorooctanesulfonate (PFOS) in Liver and Serum Samples Project Identification 3M Medical Department Study: T-6295.7 Covance In-Life Study: #6329-223 Analytical Study: FACT TOX-030 3M Laboratory Request No. U2279 Study Completion Date Atsigning Total NumobfPeagres 233 53 T Sn F 2 3MEnvironmental Laboratory Contz: NC CBI 10 Paget 3m Medical Department Study: T-6255.7 SM Medal Deparment Sy. 762057 G_L--P COMPLIANCE STATEMENT LaboratorRyepoRretq.ueNsot. NFuACmTbeTro-xU-s05330 oratorRyeRpoersti, NAaCbTeTro0x5023703 Sty The: APDeneLtifaaeumtrioaanrnaLdsoaSonboercsfnntnnoeerSsyPuaReteeApaesdnsnt fcPoaam hsCeso2nScit5ee(n15tC5a5po8s)ualeerTCorooascmeatmSoeunsciVwoiinmssyrona) Sty detain uber: FACT TOX.030, T6265,Covance #6329:223 oahLraiwbsoodrs osftoatylnaPdrssascy acaiocmnedeu(eycGetLaoePrdsr).iScTnhacoonmapdlraaanr4ct0elCwpihRnhasne7caIeno7rm5See2ittweisshEnnsovrogiorSxSoHiWnampLLeooinpsotwnaPaosrvpoervtsieotAcrgeeissonra God Ad Sconewth SM ETESS Senders Opa Pocodses Exceptions to 6LP compiance + TTTTroeierseansnwueeteriepdcruaisnsshcsachnwgyarswacseoScenotpnarsraairtddeeyrsosyts.ndtdTcioss,eaogsyr rwoaoesoffis5hn5eishnshdnsaSpvaeohowofoenpbnsoaoertmtcossyni.s, rHoweevcerrnocmei ceacpioc.l paroranc of sac ras wos eho septs mys +* TcLhoOaenndbaSufocMetrweTadoOciPKncra.sascciiocncosor,stsdaSoanotaeatwdwesairerenneo4nt0UrtneChicFeoRRrsdoeS1gda6tou2erswscitoEhrrrruneycrstCcecodomreeexnpaacplttsartnvoasoticonreaoeynnteAqgsTebuinyoscityhsoerCGoyLnesPwsdl. be mtocoanenndlcaeemsneaitsraanprdornnhcciosocminoeocdhegnonesStp,fon1sshore"yAsA.rmyihstocrre!mwocaenruo.er.nc,oawcinsvoeearobnCoyomwomehprcnlooooGmLelryeClointecs, Ceaminasons wets nl cried Si Dre duns PY) Sen Alon 2. Butiniqy SS oe 19/00 6 seer zecr me Enna Laborry 3M Environmental Laboratory 1n Pag2e BoE 2 sopra v7 onbTeset TerR ror ete GLP STUDY--QUALITY ASSURANCE STATEMENT Syme E B AA mpTs Enon e e ctpeCTooanynrn PSeotretra ieeyeo 1 eorttyy7eo etSenopsyeerone brevet | ore | om Ta s romTe| e ya C " QAURepreskitative Coster Date ry 3M Environmental Laboratory " ons EageT 3m Medical Department Study: T-6295.7 laboratorRyepoRretqueNso.t NFuAmCbTerTOUXz-s0s3s0 3M Medial DopartmentStay: 762657 LaboratoRreypRoeratuNesot FNuArCmToeTro0X2a23709 _ STUDY PERSONNEL AND CONTRIBUTORS 3AS0tnMucedrdyoiMDwa.ilSreeDcaetcpoaartr,mPenrt. S3PtMOPBCaeounx,te3Mr3,N2B2u50l5k1i3n3g3222200-26-02 (51) 575.3161 3`3SSMLMpPToCaonoussn.ctoroMr,oNgByu5Si5d1ei3rn3gv.29e02s2.02-M6e.d0i3cal Deparment JonL Buterhof, Ph.SponsorRepresentative LiA3vnMaelErynavtniidrcoaSnlomeCrnhutmeamlAirTnseatcyhrsnyeoslLoagbyoarnadtSoarfyetyServices (IM ETASS) 23Fi3Ms2oEi0nnv0eirAonnamieynttcaall CLahbaosrtaT(3teMaomLro(byF)ACT) S5t35PBauu.nMANve5n3u1e06 Kisten J. Hansen, Ph.D. PrincipalAnataInvestigator COomntirdRibuBtaimngcgPeersonnel Usa A Camen HaKertdy JOK.uJeorhtnseonn KMaerklJ. DEolneufesioenr Swat Estes SMeariaAey!DL.inLdvangston JosepCh Pion BSaorroanAA.GHraommenaz Cans. Hewtt Sacant RSiFonst Aehaovo Marne M. Heyig Bon, wyne C3I3on0vd1aineKciTenesLsmatbaiannrgsBtoLounaleebsvo,arrnadct.ory Madison, Wi 53704-2505 PeteJr.Thomond, Ph.D. iL PhasoStuyDirector IM EnvironmentalLaboratory Pages 3M Environmental Laboratory 3 Bat 4 3m Medical Department Study: T-5295.7 3MMedicalDepartmentSy: T2657 TABLE oF CoNTENTS - LaboratorRyepoRretqueNos.t NFuAmCbTerT-o0x3-307300 LabReoRpeocrruteseatNFuAtmCbToeTrOUrX3.30y7380 - GLPCarmpiancesstement LPStucy--Qualty AssuranceSatement . . 2 5 Stay PeransdCoonntoes BL oe s . s Test System... EE s SPOTROCHA. SpecimenCollectionandAnalysis. wt Dose ConfmatnAnayses } - :7 8 MeanrdMetls... Chemical Charactrzason } } .: . s MethodSummaries AnaycalEqupmen... } --. -- s 5 DOS... DataQuaityObjectivesand Daa gry BE . 0 Dota Summary,Anases,andResults, Summary ofQualty Coto AnasesRess } we . tt Summary of Sample Ress Sttstcal Methods and Catuiatons } a 2 Statementof Concusion Ustof Atacrmens..... } " 2 tAatacchhmmeenn8ttA:: PCroontorclolMaantdtDeevCihartaiocntSruzmamtairnya.ndDose Confimaton Arases n AAtacthmenatD:cDExattrhaacStuimommnaanredyAaTianybitsc.atl eCtod:s...} 8) 1s Attaacchhmmeenntt. DEaxtaamSpperCoaacdushaeosnss..............__ 18 zs AAtatchmaenHtc:RnehpeormtmSCoenaattnefotfcAratbsePage. 2 . m WMEnvronmenta Laboratory 3M Environmental Laboratory si Pages ats 3m Medical Department Study: T-6295.7 3MMedical Department Study: T62857 FE -- INTRODUCTION AND PURPOSE laboratorRyepoRretqueNos.t NFuAmCbTerT-OUX2-207350 LaboratorRyegReoqruteNso: FNAumCbTeTrO-XU.3023709 CsF1------ i --0 o Perfiucrooctanesutfonate (PFOS) (CAS Number: 2750.39:3 Chemical Formula: CFO; Molecuar Weight: 498.98 oPTrhRaelOypSud(ropsCoesdFewoSifht)hpeeianrnuavoleryrtoiaoccnatldapsnheeasrsuuemfoosfnpthcoicsaismctedundpsycooialsetocsdtueemtdesdmauntriin(ne1g6th2he6e9ps)rteusdeynocfeCaynndorcmocnicgeuntsrmatoiornsofe Test System YPIrohouednutgcetsats,sdyInsoc.teamedusnlptte,ceaindedsuawsneidinggshtaercadoinlsapserplareogcxt,eaAdtewhlaeys3it-hne5atCkiygonnoofmlegautsmernor,ikteey fCyonmomCoolvoaunscemoRnekseeayrscwhere csTtowunedtnyr.tolyF-Ootyuwnroogmrmaooulipegsuasnofdmtoe2ns2tkfeaeynmsiamltaehlasCtywdnedormneooelsgetuacsbeimnsoehrektdheeaycescsowtredsriuenbgsuts0aenddceoa.ssatoghle lrteeevscelessnyGsdrthoemuepnet+chucevonapalrseeisneenntof T`Daw6oes2rmae9ga5oedpmoaifeunnrldiaknscGgttraootrsofeeudbponddCgyhyealwraewatiicignthnecir0asd.pia0sc3yusio()emswhaod5iotcshaeaty))a0d.um1ia5nti(seitdedrdewodisoeh)t.fceatnoGdsre0oui7np5g4e(lhaagnthmnadctoasspes)uGlrmeogsum(esssep2ee.5cT,iaaballneyd.|4oifr Table 1. Dosage and Group Characteristics of Test System in Stuy T-6265.7 aL | DOSAGE LEVEL [bosace rao) fo [mr e Tome |meen[me ToC [o[ommmo erwonm s||[t itrmm ee om ]aoo a n[em e|| 48animalwerr ince e inn-- e Bassin ser colecianbut4 sims were assigned frseaiment IMEnvironmental Laboratory 3M Environmental Laboratory 5 Pages sage 6 sn Medical Department seudy: T-6295.7 adc Depament Susy T2957 LaboracorRyepRoretquewos.t NFoAwCbTerToxp-i0es30 [RSRepp,iFpATOC]0T30 SmAaoHaltemoLasrnntaaranoybsipsms(vwva0eAodvnwee htma.mssiTiecsoe moareoosotBe2]0rwoeveeks. SrSsoeciarwosr chtcetcoes? HFaFeo3sumosaarctkonhreisnoirceeceofldvaermay5v7GeGs0ronuCugrsresw1i.4w5eevasnryoenvreesrspsrhdt.tnhesocrrsessnoecssoe:nSpeSgdorbupCsoaitp, nTas2 Seer2000 ecovey ares ri1SteotpseehaacSanirrhmctGLceeifalcnEseaaIlCcrFotpaOlohclSaeEsocecfPtHeLiooOponSoaAmsnPdtaOA,rAnpteailPaeynoHrrsOe0isSnsEosAPsrsSP.BrSiowPermOssrScoepmeewecnsose)vh,fSoenrpmsst ievlescpeewserreressen Tieoercreeusroetsnages0cv5oe0emcprelay0 es 2c00deoninHnr CenyoseaLconoeo(eSsee miT n SSSBopotreeacsi iipFcnaeosslceoesooicromhoemsocnmsmoeonanag8as5oarsttSpiOinCesnsathltonshhBsaF5opfe0ossBPuieoKeenThomspesnewemrnna hoesftoe deonzs SSig2pa0di)c reamrysLiatfsewzrneacncoy on0cLptaapycre fo8 rg r er USrsa aSn eravsralsei hsomoaegs eexceesr 1si5ganexpeicncossget Ssremtrioprtet aerp(!GE) CEeSntSonA)Araabliiceaoaairestsh PvsOSpeonv ws aoa re ir using high-pressure liquidchromatographyelecirosprayitandem mass spectrometry (HPLCSSrpoeoctiS mSepnsaCcoa ilmlaanctei o$5r0oamnSctuty GrSoesup1btrabugh rsough 22559, CSSpeeerrcaiSmpSenespcCiamoecnms2l5poa4lmpanethceecoaRn-ecmoscorva1tr e.r5yn0Grss ouapcfaoorm p22e+7r1si28 t30(780v0a:py) --- SPECIMEN RECEIPT e oSerAopmTg waew 19r9rcbeee csaeedeoLomrhGrod2vs0a.n SLeiarrrassstFoa sopsrosdcearltyn.oignrgevesiSfephaseof i sc, CCoamndamalaSmtosrauessorsoivneroree3seosssr1a0eArrrssanprwomoosdtcrTasogtTAO)XM3o 0eweroTsee esform Ermer Laat 3M Environmental Laboratory mn Page oyu 5m Medical Department study: T-6295.7 LaboratorRyepoRretquevos.t NEuAmcbTerToxs-ie0n30 odesDeparmentsuc izes nsoReRpernotFmACoTnOvRoG 2SDD1oo)ss5eT0chCGooudnnoftsgierancmsoadrtriamaolonegrAsnanwaanalotryefsevpesrsutrtaycentlseae cmincgesrs+em ome(1e 393 1452 9)ccStC2n%gon oDLosrecEnonattmOartanewaspaaemrenfnooForrsimaacnchsetfdoroosces,reSoS13e5d11S0ssanS140a9- y HesaPPrceaLapiCarrneeEdgdQ(csastopNpGnrsoScoxiiaTomaanhntdnealayphrHaSos0mva0oee0r,eg1sniTs5gheaetpnadaev4r.w0aa0amuegsdn/vgn)esedbvymesarpiasykiynsgstehseendosseoe snolsuo tnieonn arnd tho en dBiSlL uting an2d02 _ MATERIALS AND METHODS STChaelmy2icrasalenChsSanrlamctatetroinzaegtirornrScastnratoioodn of nest sustacncee wSeinDnee hase Tabl2e.GharactrtzatonofTest ardAnaly! Rfrenc SubstancesinSudACTT0030 ET I Ce r= [Em ovencm een KPFOS Possum. |sevKPeFimOSePontnascsi,urm o|| Snare] THPROS Ea | [mmo 7wan | eae [ww [omen|To TmTaw RResse5shameesofvee aScel ekrioc sinsirceevsionf5n07 Ln are21sS or 10 a2FMe0omt5lh1iao2gd5n.SruDmoeetmtttanordieoressrsteiitoonafnrroefstaemstaenrooefcpssesseyitrnogospseytrnaeofnnisss oy SvChrreeraensaypssttcofoercncbmyoearamnao0sfs4oteemscnCsroawrordroteeSseatsoSocowaoodneToi hserermemocer re inanese e tBE(e oPs SmeeEoeRSt.e A a Enimmentatatoy 5 Environmental Laboratory I~ Pages es 3n Medical Department Study: T-6295.7 3M Medial Department Stay: T2657 laboratorRyepoRretqueNso.tNuFAmCbTerTo-Uxz-s0r3s0 LaboratorRyepRoerctuNesot NFaAmCbTeTr02023705 Proparatory Mathods: + ErToSm8-L60i,foSrvxAnvaeolcytsroisnoufsPiontgHasPsLiCumSPecerosrprafyihuiosss Srpoecoctetort.harFnoreocshamuiclaCoomapotundes L2riTverHa3s(a0mPnnapdlneteFodsssaaewemxceOpareelcenetSdthaeiorndmgdouesgiehuenebngeiaazadnnendoadinntpwauaaltropenag.trAowaonxantlisatucprtooaagtneinopnferevoeacadpecodhrunarhoteoo.mrVAoIugnBelEon.caTnytope.woweEvaaxcshcsseaopxsrceakucrnrswtswiass deicsopnosable lians1.s0yiLnogfemelolGraanslsaantcassasemdplrvoeiursgh a 6.2 um ion11 cagee + ECTSo841m, EpfxrtormocSieorunuomfnPoortdaAsnssisyusmsPUesrinfguHcPrLCo.oEcetcatneossupoorMrnoteasrtyseSFpiercoocohmeernryic:at 2TeShce0reooannMss-BipEateaiunxigemtdnwrere1rpaeag.wesa0cnlpsLtiwrkoaeeefsmdmaovesvdihetdadhenTacdnlHdaPtpnhOudtSposanaasnomsdepdeixahrtnroodaguctetgenehd ss.ai0nv.na2ygai1aepmnaornynnappootildwnrnreygs.eapkxttarirhatcnitsoignov5nespecrcoot.coeesduhre. 3posable lastesyinge lo gas aucsamplervis Anatyical Methods: + EETxStr.a7ct.s0U,si"nAgnsHyPsLCoEflPcottoassspirumaPyieasrsoSpreckoomoetncy"taonroethserFuucfroochneramctasei Liver + EETxSa8c:5t.1U,si"ng HAPLnEolaefcPyootsapssrsaisyuimiPaesrsuaSrpoeoccvaonmeesruyo"nate or Over Fsochemcailn Serum PTomhoerralnaieilioooyinnscoec9nmshl4w9da9(er9rraFa,ceSitepOerlr)eseTryochfetf5cepbhohyadomrraotpnrrmiiitrmaoearurciyndoogeotonnran5oese9rhowiFeascmOmoSir.mveci(apnaCgrloon1cu45ci70ooLrn)oCscsEorsSnaMoreesNtaS,ldi.weeorFasoocrmnfaatasapmieeinmegdee.nc to Analytical Equipment cPTuhhraeinsgaecotafucattlusaalndsaauyttcaccaaeectciuopnm.eTnhtesftlionwgns useedpirnehsapnreisaeentoafna1lsytiscael ehs vseoefdehGius rsytcy vearciedti)gnty AnCLaoiyqutumcindalCtehcmrpooemruaatt:uargeKr:eayAgsmhtbsoineHneetwBeetttsPa"cCkaar2dx5S0ermiems 1(130m0)Lind Ghomatogeasn system NobCClooemmpppohonaesnenectnoA5tm:pmomeniteannatcmlmonium acotinawatteer SFIolelocvwtenaottneG:vro0aud0meenu:tm1100n,m1utes WM Enionmental Laboratory 3M Environmental Laboratory 8 Pages ed 3m Medical Department Study: T-6205.1 LazoracorRyepoRretqueNso.t NFuAmCbTerTo9x-70s30 OM Modal Onprent Say Ta2657 [SrResso t FACT Tb oxo F5IiSn1ic6rtaa10sat00o4n95sf5f8o4o5r 5o1v0mrmi4itnmsines iRekanio10048%oro3v5rm0snnes ass Spectrometer tomas CSoofntwaavroe:taMgansssLoyrnxTM 3.2 APIMss Spacers Qt I Tile Guang system SSoCooadrtcetii:oaonBBscGlesakupTEponyrneopre:Nrs4oa0rg-ea601500% 10C Aras Te, aka Resco nin (MF) Tabla. NoognsWoankorvedin FACT T0X030 [oemeer acs T prs oar | rover onan | rosDovetncviiadaatvdbieooarnremscwoaraonnaatDbaovyaisosnasnEmtaircnmhernagaofswietssosstceycspwnrdiogcrbeoesrdsmsr omnaosdeprn eomnsrwsoeee Rescsoo Taba 1) TDhATtoAwQinUgALdaItaTcYuOsBtJcEiCocTiIveVsE(S A0NDwa0DrAeT0ActIsNoTEaGRhITrYocfor i + Linearity: The coefficient of determination () equal toor greater than 0.98 + + 9uL 7p6l1i c5aotpm fulAQuci eacnatit ptaabts iTlohenaPL(raOcLGiiIoOTnc:hGuerMte)cth: nsodoDaewtsetcrtisoonosLipmiite(MoDsLc)wofonnr sP2F0O%S ais: 12 ppb for sem ++ CS3lokcnaAtcoacatponiaMbaifnoRedcseo:vedroireeos:,mm70i-1o3s0o%nnpeama9ysoaonydssibngacnntrsry mets + Donammaasns sspioorfc Sapeictoo cynO:nSpes aaccrctsobnodamsiaed oycromatororionnmce SM Envionmans ars 3M Environmental Laboratory 1019 Page 10 eageTro 20 Medical Department Seudy: T-6295.7 LaboratorRyepoRretquexos.t NeAacmresTouxr-e03s0 SM Maat DepartSuc Tez057 _-- LoranReRpeorqtsFmAoCmTTooxs0o3n0 DATA SUMMARY, ANALYSES, AND RESULTS DTaOtXaGc5i0t(ysseectascohvBey avertyiraof isscvienes eboS31 3 rotFACT SummaryofQuality Control Analyses Results +* SoSLCianaielsamerlis-tayCU:snTxThocSSeptatc1anadso0adraedrssf:rdfoQh1furideactreSerSitm5iieon9aotnffiunotnvim(aavrop)efsethrce sHetoanmssnede earwmdeesceunonrvesSeewoaS mns 2e0e .9t85,eroe IsoiOornP aCbollsnaweanGtueOeoalnocochasfaaisdoeownnpFisasorpSavgaaecycnt kegeasrarossgeumtareeassvnge1oeHchoovSpceroaosrnenppmseyteo tfreep eceessy ei 0 eedee 0 s oe + lLomilmieirttesroofnaaQCpuraaientGaoailorona(cn3O.Cs2:osetTheeeeLnh0cGse oecSamotooSeooseso es? ceo tas ssn1reatno v altos00 fnman corssnp cee Table. Dotaminsionof POSLOGI 0X30Anayees + SeBlanyaks:asArrshsoros trosblcoaws ncsaiotonm uoatgis ocnoteor ecosmmse ofoies.ITo + r DuplciatsRacepanbi Precision: sion wosctortyays NSISDnwo + ocMaattiiroSnhoceosnnMhcaeteerhsteitsnsasFnSemeastouiElspDiSTopiycE0ropsa weeerrcrce orvieevsn rochtsettsho0rmi s + SSepimreslaAekcScoowkpaeabplaeRseprcoaeuasrdieers0e:4.SWioeenrrmsovcewnresnws504FofossptecBsie essmrsne senets t5USs3eoo0ofS%lumfroogsatsreasomhoeeofnscurnogea t0kehTeHe ePsFS)nr vasestres ek eose searnisen esSarcsETHEPlESSOS SUEnmans asestory 3M Environmental Laboratory \ Page see 3m Medical Department Study: T-s295.7 LaboratorRyepoRretqueNos.t NFuAmCbTerToux3-903300 SM Medal Deparment Sy T2657 LaraResRoortaNso.t FnAoCmT Tovxion2g0 u Assumia ng sp0ia k3e0o%c,oTvhrevtouiddesoomfsausmsoeionndroakofeendnogennouys aonee eecoa. data ae +`SuSahamnemmnpaClaroe.ynsrfofoamSnaiCmmopntlTrehoelRsAeensiuvmlsatslsewLioswSevesoofPFclOvSweearersfPenscs ecee nesn a nd ro + fSaleanermaespnllhecnseacsrenboaemltswDeaeodesnwiaimgtadrlAGeenoiFnsmOdarSseo:ml.enlveigFseOPaRSrO,eStlPlaeRerOssiSeweeseaplrssontfsuGanebrnsodpemnsapcrsesamdoovensrneeofoahertfest Goalsrested Araceae dE E STATISTICAL METHODS Ae ND CALCULATIONS e fSoatesxiacmapllme ceactothoenrossiudsiasd01tGeecna0rcoledaofatmnedaasnnos ansostranedasrdtsdevaptoensn. SsooAtachment STE ATEMTENT OF CsONeCLe USIOe N e TPPURenFOdrOSeSS.r ehPveaOeclSooenmferdGverienoloioulnpssrseio2aeef.s3p,rraensv3e1snetcsaooidrrnlao.asHt.otawTaepvnsheerCCasronofscoishsrf4ocs(coy0.mh,d|GrProFoviO)nSmrewasalrssseehnlostnoaom ilnynr spcpsosorasrpanness DTooOvtiXs5u3na0et(ryaecabAmtjaeocshcmifvaoanvrtneGBsryeoarss4mnotmwpeihaa,solfeisxstoy ucoteesd cioaLtasoo or FACT mLIIsESmTEeOEFrRAeTEeTEeACeHeMmEeNTeSmeeeeemsemememeerereeseeeeesme + + AAtaacchhmmaannBAtt: GProotoncolMaantdixOCvriaaScuemztmiaornynd Doss Contato Anas + + AAttaacchhmmoonnttC:: EDaxtvaacStuomnmaanrdyATnabaleysa etna + + tAtaacchhmmeonnttE:: EDxaatampSlreeCaadsuhteitosns + + AAttaacchhmmoonntt :G: nRoesromnCSngtrcaaretoPoagneayses SM Envhonmant Laboratory 3M Zavironmental Laboratory 21 Page 2 23012 31 Medical Department Study: T-6295.7 Mod Oepremnt Suey T2557 LaboratorRyepoRretquevso.t NFaacmteThoex-a03d0 aReBt epntsh FACmo TeT0030 ATTACHMENAT (CONTROL MATRIX CHARACTERIZATION AND DoSE CONFIRMATION ANALYSES Tile 1. CharcutcatonofhCon Fao Scr MatsUndoLoe adSrAnsonSty PACHT0050 | Roa Senn | owe Semin | owersexoe | owerSen | [[Sowee SimaCremicas | SigmaCrema | LampeSongs | Sere Some = ET LE a ETrT a [PSrvoay |mcs | Rotstinn|| rseeyrmiees{ ||sosarn r| ry | [ome Commarimion| |Comvarier| retanir Somoi | Lo TT " 2 Wn rrt e aEmionmantaborty 2M Environmental Laboratory 2 -- raat" am Medical Department Study: T-s295.7 SMMecicl DeparmentStuy: 2957 LaboratorRyepoRretqueNso.t NFuAmCbTerTo0x3-90390 Lobes RReeespoFmACbrTaTrotoxa,n Taio A2% Lactose Doe ertcaton (PRoOrSStu)y 2822342159 eomey|| Mvaosm3e eoaweee)|eveeoesmra oepiron [A2 E FCoetom[E 500|Esz2 om0| zso0000E |"ari22s|IIwes[s-- rr s3 n] ealwn EE L C E C 00 00Z 3 3TO CW E C || (AScehaUsS CoownwcianaatonrScralsarSLcohreeaeaie oa snSv.ideeo pe oe. inese 100 ] Table A.LSaeS cvteorseoD| ooener|atim|oarne(PFO[Sm[-a--eHreaerse|Spmik[eeesea)feeor[|Saeyac6ao3n20T225o42159 on fom| om| G55 | 635 | 5 FE[ooeEteoTn|ooE --sar TairsE sT|wesn%|EoawCwweo [|ovass|[T ovor2 |eameT s vee eT vi2| wr] CF| oosteonT||Teno$o06n[T|T e--osro[TA [7ovasen|ST|otaama0en1[|[ os0wm0[|rwomr ||o wae wus|T]T esereTTw]| SMEnsonmentl sors 3 Environmental Laboratory 23 Pager seen_ am Medical Department Study: T-6295.7 3MMetal DeparStoym:Te:6n26t57 APRTOTTAOCCHOMLEANNBTD DEVIATION SUMMARY RaboratorSyeveRresqueNso.tNuFmAbCeTr ToUxr-aoy3e0 ReportNo FACTToxa30 Laboratory Request Number-U2279 Table 1DeviationSummary or FACTT0X.030 TR [ TE veoermeaer1owes oE rccoumence | RT T TA SErT [Er|[ereenve --------| im tadby foearwpRaian sig pnd FOGPession 2s spaced in heproteus. Tomswren Secon Ee C ee @ gvuartiseadmvaetsheivewas ot 73 vi TE 322005995.,342717/19896,,431211319990,,43/12249%6,, 17195. 5/1999.52299,6999. 6/1499. `tNhoevpergecaitsiivoeniamnpdaaccteounrathceysotfuadnyal--y1siixs.weighted curvesimproved 25039 3153091 |SNoeooaeorsmsorocasoFeeir n awro 19%,5322040%0.,14022679050, 121/00, |ehnesacariobcronattionnucehdeacckcsuriascufyofifcitehnetaannadityhseesc.aTthaeqQuCaiptryowviidneodlbbye.. EET S onCR e e v | rad CTETS70 | E10S2E785TOV RR Te 5I4,V,in| arosveesmosonme Pe ee sya aor [EERE| semen [mem oie e s Sor ras E re dl Ie |v indtis rep 7540 Nori TossSess pet358 ovron cys ORE EE nt Roose GC va mr sarst mie Hndoa or Tecre sinsver sins CC or ore on E R Eem g QSLdn | J ve.e Jramaes.3v2ymsvwdueeyivsossh.,|| eSSLeErrcosS6o73wscr aaimsesrla resseas no0d.5Smi.wPOeorneeHxToketracted. WM EmionmentalLaboratory 3M Environmental Laboratory 24 Paget rage Ts 3m Medical EDepamrtmrent TSetkumldeyy: T-s2R95O.7 BB smn 1aboracorRyepPoRretrqueoNsoE.ttAGhFoTAeTCcTOroNT3ol3nx5u-0i3s0 Study Title 26-Week Capsule Toxicity Study with Perfluorooctane Sulfonic Acid Cynomolgus Potassium Monkeys Salt (T-6295) in PROTOCOL LisAauCtlheomern . JanuarDyat2e5,: 1999 3M Enviro3nMPmeeEnrntfvaoilrroTmneimcnehgnntoLalalobgLoyarb&aotrSaotarofyrtyy Services St9.3P5auBlu,sMhNAve5n5u1e06 Laboratory Project Identification FACT-TOX-030 SM Environmental Laboratory 3M Environmental Laboratory "as Page toro 2a0e Ts 3m Medical Department Study: T-6255.7 laboratorRyepoRPrreoeqtouceoNlsoFt.ANCFuATmCTbTOeXrT0OU3X0z-s0r3s0 Study Identification 26-Week Capsule Toxicity Study with Perfluorooctane Sulfonic Acid Potassium Salt (T-6295) in Cynomolgus Monkeys Test Material P(eTr6f2l9u5or)ooctane sulfonic acid potassium salt Sponsor 3M Center, Building 220.2E-02 3M Toxicology Services - Medical Department St. Paul, MN 55144-1000 Sponsor Representative A3nMdrToexwicSeoalcoagty, SPehr.vDi.ces `Telephone: 612-575-3161 Facsimile: 612-733-1773 Study Director Kristen Hansen, Ph.D. S3BeuMrivlEidcnievnsgir2o-n3m6e-n0t9al Technology andSafety SS17T8-6018 Study Location(s) In vivo Testing Facility Analytical Testing Laboratory `M3Ca3od0vi1asnoKcnie,nLsWamibasonrcaotBnoosruiilneesv,$a3Irn7dc0. 4 3BuMilEdnivnigr2o.n3mEe.n0t9al Laboratory S9t35PBauuls,hMANven5u1e06 Proposed Study Timetable Study Initiation Date Study Completion Date January 25, 1999 January 25, 2000 SM Environmental Laboratory 3M Environmental Laboratory Nab Pagezots Pres 3m Medical Department Study: T-6295.7 laboratorRyepPoRrorettqocuoelNsoF.tACNTF.uAmCTbTOXe0rT3O00X3-307300 1. Sruov cTwyennotmyo-lsgiuxswmeoenkkecyasp.sule toxicity study with potassium perfluorooctane sulfonic acid (T-6295) in 2. Purpose a(TnhPaiFlsOyzSaen)da.liyntTitchhaeellisinvtleuirdfyaenipdsodsreeisroiungmonfeodtfhtcioysdnseottmueodrlymgiwunasesmlcoeovnenkldesuycostf.epdoAtdaatdsiCstoiivouanmanplceertifLslasubuooersroaootrcotrfailneuesis,dusslmtfuaodnyyat#bee6329- 223, 3. REGULATORY COMPLIANCE `ATghiesnsctyuGdyoowidllLabbeocroantdoruyctPerdacitnicaecscoSrtdaandnacredsw,it4h0tCheFURni7t9e2d. SwtiatthetshEenevxicreopntmieonntatlhaPraontaelctyisoenof cthoendtuecsttemda,tearnidalims itxhteurreespfoonrsicboinlcietnytroaftitohne,Spsoolnusboirl,ity, homogeneity, and stability will not be 4. QuALITY Assurance SsTtthuaednyd3acMrodnsEdnauvncid,rowdniamttehan,3taaMlndELnafvibnioarrloanrtemoperonyrttQautlaoLldaiebttoyerArasmtsiounrreyaScntocamenpUldniaiartndcweOiplwleirrtaehtviGineogwoPdtrhoLecaepbdrouorrtaeotsco.orlyaPnrdacatuidciet 5. TestMaTeRAL 5:1 Refer to Covance Laboratory protocol fo study #6329-223, 6. CONTROL MATRICES 6.1 nIudemnbteirfsicwailtliobne Mroecnokredeydliivnetrhaenrdaswedrautma aanndd/oirncrlaubdbeidt liinvetrheanfdinsaerruemp,ertraceability 6.2 Source Covance Research and/or Sigma Chemical 66..43 PPuhryistiycaanldDeSstcarbiiplittiyonNoMtoanpkpleiycalbilveer and serum and/or rabbit liver and serum 6.5 Storage Conditions Frozen at -20 C %10Cor-55C+10C 6.6 lRoensgearsvteh following eMaqtuarliixty Aofpotrhetiporneopfartahteioconntarfoflormdasterviaxluwaitlliobne, rbeuttainnoetd the effective date of the final test rule if applicable). lionntgheer atrhcahnivteesn fyoerarass 6.7 uDriisnep,osaintdibolnoodM)atmraiycebsewdiilslpboseerdetaafienredQpAeUr GveLrPifirceagtuiloant.ion. Certain matrices (feces, WMEnvironmental Laboratory 3M Environmental Laboratory w2a7 Page dots rageTs 3m Medical Department Scudy: T-6295.7 laboratorRyepoRretqueNso.tNuFAmCbTerTOUXs-s0r3s0 Protocol #FACT-Tox.020 6:8 plSraaebfpoearrtaityonrgPyrsaoaicmpralu,etsainfodonrfsaonlRalleoyfsweirsa.dteoqMuaStDeSprefocraucthieomniscafolrshuasnedd.linWgebairolaopgpircoaplrmiaatteerials and 7. RerEReNceMATemIAL 7.1 (Iedqeunitviafliecnattlioosn) Potassium perfluorooctancsulfonste (PROS). ot #s 171, 215, or 217 7.2 Source 3M Specialty Chemicals 7.3 Physical Description Whit powder 7.4. Purity and Stability Responsibility of the Sponsor 7.5 Storage Conditions Room temperanure 7.6 hbReaensreettreanivnyeeedaMraasstefloorlnilgaolawsiAntghreetshqeeuraelvfieftesycatomifpvetlhedeafptrreoeopmfacrtaahtcihofinbnaaatlfcfhtoerotdfsrPuelFveaOl(Suiafutaispoepndl,iicbnaubtthlines)ostluodnygewril 7.7 EDnivsiproosnimteinotanl ULnaubsoreadtorrefyearnendcewimlaltbeeridailscwairldebde longer affords evaluation wrheeainntehde fqouraulisteyboyf tphreep3aMration no 7.8. plSraaebfpoeraarttiyonrgPyrsteaicmpealu,etsainfodonrfsaonlRalleoyfsweirsa.dteoqMuaStDeSprefocraucthieomniscaflorshuasnedd.linWgebairolaopgpircoaplrmiaatteerials and 8. TesrSvsrem GiCnryonCuoopvmsaonl2cg.eu3,spramonotdnoc4koelrye#sc6ew3iev2er9de-2tu2hs3ee.tdaesGtrtsohuubpsttIeasnctcosenytdsrtoaellmya,nfaionmrad2lw6sewdrieedekmnsao,itnarteaccieoninevcdeenattnhrdeatdeisootsnessdoubfasst0da.e0n9sc.ecr0i.b5e,d woafenrddea2ta.a.0llmTogww/ekodg/aadtnailyme,aaslrtseasepae1cc3thiwvefeerlkyo.,m wGRhreiofcuehprsmtoa1,yC3o,bveaanenxcdte4enpwdreeortdeo,cdroelesci#og6vn3ear3ty9e-dpa2e2ri3ordfeocarofttveearrbycuelasansiapmtraieolsnesnoatfnatdion treatment 9. SPECIMENAND SAMPLE RecEIPT ssTppheeeccii3mmMeennEssnvwoiifrltolhnbemefeponlatlcaoklweiLdnagobnobrodadrtyyorciyseswuifleolrsrsahenicdpepiMivunegi.hdosmforgoemnetihteyndsiacmapileeds pfooindtossienatnhaelyssaidsy.andAll WM Environment Laboratory 3M Environmental Laboratory 2 Page oto rapt 3n Medical beparement Study: T-295.7 LaboratorA y PmeqruecotACmcToT-- mooanala0is Body tissue/fluid Expected # of Serum - all animals 7 days prior to treatment specimens 616 from main. Lveoryettcww oonweekn s during esiment |2-- S4usdsdionalfrom Urine and feces ~ recovery animals Day zeroofrecovery 6,30, and 90 (with a potential 180)| 24 24 urine. feces doef means [Civer--allanimals | After terminationof the sta [a TToottaal nnumubemroofbfcteeostnrean)imaanlis:me3:2 12 aSpppelciicmabelnesSsteanntdtaord3MOpeErnavtiirnognmPernotceadluLraebso.ratories will be received and tracked according to 10. P10R.E1PARFAATCTO-RMY-M1.E0T,HOExDtSacion of Potassium PerucroctansuOotnhearAtneioonirc Fluorochemical Spectrometry Surfactant from Liver for Analysis Using HPLC-Electrospray/Mass 10.2 FACT-M-3.1, Extraction of Potassium Perfluorooctanesulfonate or Other FEllueocrtroocshpermaiyc/aMlasCsomSppeocutnrdomseftrryom Serum or Other Fluid for Analysis Using HPLC- 10.3.1 tpudresewipthmameethrsodysaodttoesoha rthyese ise shovesus,an amendment his protocol will be written. Any deviations from these methods will be documented and 11. ANALYTICAL METHODS 11.7 Elecrosprayhias Speen FACT-M-2.0, AnalysisofFluorochemicals in Liver Extracts Using HPLC71.2 Specwometry FFlAuCoTr-oMc-he4m.i1c,alAsnailnySsiesruofm PoortOatshseirumFlPueirdflEuxotrroaoctcstaUnseisnulgfHonPaLtCe-EorleOctthreorspray/Mass 71.3 Idrfoatneoacldyotliwcwiailltlhmbeemtehworsditsteyont.heArntyhadnethvoisae tliisotednmsaboevseearme useed, awnsialmoerhndcmoeoent tbo thies aEnvironment assy 34 Environmental Laboratory 2029 Pagesors aged 3m Medical Department Study: T-5295.7 laboratorRyepRoretqueNos.t NFuAmCbTerT-OUX2-207350 Protocol #FACT.TOX.030 12.DATA QuaLITY OssEcTIVES Tinhdeicnautomrbsearreofinscpliukedse/dduinpltihceataensa,lyutiscaolfmseutrhroogdast.es,Inaandddiitnifoonr,mtahteiofnololnowoitnhgercrdiatteariqauwailliltybe met: 12.1 Linearity #2098 12.2 Limits ofdete/cquatntiitaotinon 12.2.1 Method Detection Limit (MDL) for PFOS a) Sem: I2ppb b) Liver: 15ppb 12.2.2 cParlaicbtricaatlioQnucaunrtivteation Limit (PQL) ~ Equal to the lowest standard in the 12.3 Duplicate acceptable precision < 30% for the method 12.4 Spike acceptable recoveries 70% - 130% 72.5 cUosnfeiromfatcoornyfmiertmhaotdo.rIyfmaecotnhfoirdmsatIonrdyetmeertmhiondatiessuasmepd,leasnwaimllenbdemreen-atnatolytzheisd using a protocol will be written. 12.6 dDaeumgohtnesrtiroantcihaornaoctfersipzeatciiofn.icity Chromatographic retention time, mass spectral 13.5U8-CONTRACTED ANALYSIS . 73.1 LAlalboarnaatloyriseess,aBsudieludiilnegd2i-n36t-hi0s9,pr9o3t5ocBoluswhillAvbeenpueer,fSotr.mPeadula,t M3MNE5n5v1i0r6o,nmental 73.2 Alanboaramteonridemseontthetrotthhaisnptrhoeto3cMolEwnivlilrboenmwernitttaelnLiafbaonraatloyrsye.s are performed at 14.STATISTICAL ANALYSIS dAevsecrraigbeesd and standard below: deviations will be calculated. The statistical methods that will be used are 14.1 (Dwaetiaghttrwaenisgfhtoromrawteiioghnts'vaonld)ofanPaElOySsiosr Dmaettaabwoilliltebeperreptoirsstueed oars ftlhueidconcentration 14.2 cSotnacteinsttriactailonasnaovleyrsitismeS,taatnidstisctsanudsaerddmdaevyiaitnicolnusdecarlecgurleastseidonfoarnathleysciosnocefntrations `wmiatyhibneeaapcphlideodsetogervoaulpu.aItfe snaetciessiscarayl,disfifmeprleneces.tatistical tests, such as Students t test, 9M Environmental Laboratory. 3M Environmental Laboratory 2 30 Page6of9 rags ai sm Medical Deparement Study: T-6295.7 laboratorRyePproRoretotqcuoeNlos.4tFANCFTua:mcTbtOeR.r70o03x07-907350 15. Reponr wAilclepnocrltudoef,thbeutrensoutltbseoflitmhietesdtutd,ytwhilfoblelopwrienpga,rewdhbeyn3apMplEincvaiblreonmental Laboratory. The report 15.1. 75.2. NDaatmees uapnodnawddhriecshs tohfetshteufdayciwlaitsy ipneirifaotremdiangntchomsptluedyted 75.3 LAabsotraatteomrenytPorafcctoicmeplSitaanncdearbdys the Study Directo addressing any exceptions to Good 75.4 iOnbjheectoirviegsinaanldpprrotoocceodlures as sated inthe approved protocol, including any changes 75.5 isThthereeSntgpetoshtn,sspouurbrsittya.ncaendidceonmtipfoisciattiioonnboyrnoatmhee,acphpermoipcriaaltaebcshtararcatcstenruismtbiecsr orpcroodveidneudmbbye.r, 15.6 aStdambiinliitsycaantidotnheifsporluobviildietyd obfythhetSesptosnusbosrtances under the conditions of 75.7 15.8 AA ddeessccrriippttiioonn ooff tthhee emsetthsoydssteumsetdo conduc the es(s) 75.9. oAfdtehsecdriapation of any circumstances that may have affected the qualityo the integrity 75.10 sTuhpeernvaimsoeryofpethresoSntnuedlyiDnivraelcvteodrianntdhtehstnudaymes of other scientists, professionals, and 15.11 cdAaotndace,lsaucssruiipomtnmisoandrroyafwtanhnedfrtarolamnystsfrioesramnaoatflityohsneess,ancaallyctuilcaatliocnhse,moistorpyerdaattiao,nasnpderfsoaremmeednotn otfhehe 7755..1732 TiStnhavetoilsstviiegcdnaelidnmaethntedhosddtaustdeyud,sreiefdppotprltesivcoaaflbuclaaetcehtohfetdhaea,iindfiavpipdluiaclasbclieentists or othr professionals 1755..1145 iATnhsseptealctotceiamoteninsotnarnwedhpeaarusdeeirtdsabwwyedtraheaemQaaundadel,itthayenAdfsintshaulrradenapcoterstUoanfeian1l0yisbfteiinnsgdoitnrhgeesddraetpoerttheadt1s0ttuhdeyStudy Director and Management acihmtaeinnsgdemnseewcneitslswabirelymctaloedmaeralkiyeitdcheoenrtrfieofcyrtmtihooenfspaaonrrtoaamdfdetinhtedimoiennnattloirasesfpiuoneradtl btryhepatohrietSbatefuiendrgyiatDimhraeesncdtbeoerde.,nTtahceecerpetaesdo,nsthfeor the amendment, and willb signed by the Study Director. aM EnvironmentalLaboratory 3M Environmental Laboratory 223) Page rors sate i Medica) Dopassaens seudy: 5-sass.s SasoracorySpeegs BvpEL 16.LOCATION OF RAW DATA, (RECORDS, AND FiNAL REPORT Sui of hey Go r Original data, or copies thereof, will be available at 3M Environmental Laboratory to facilitate onhivecsomopftSeE,E1 8 omFoatsdbi orpleoeripnsrpsser m ee e le regulation, and as established by 3M Environmental Laboratory Standard Operating Procedures. Fc 16.1 sTthuedyfloplrloojewcitngarrcahwivdeastaacacnodrdriencgortdos3wMillEnbveirreotnamienendtailn tLhaebosrtautdoyrfyolSdtearndinartdheOperating 76.1.1 Approved protocol and amendments 111666...111.342 SRahnidnsg ors Study correspondence 76.1.5 Approved final report (original signed copy) 16.2 TF1o6e.rn1(.il6oetwEilnecogtrsornpicbaciop6niesno1fEddatanwilobensiLndoseepaSreroime sy older 11662212 CTaatirnocnrecsorts 162.0 16.2.3 Sans Opera Bsus, Instrument maintenance logs Een ries od hods 17. SPECIMEN RETENTION Specimens will be maintained in specified by regulation or as long the as laboratory the quality specimen archives of the preparation for a periodoftime as affords evaluation, but not longer than ten years following the effective dateofthe final test rule (if applicable), and as established by 3M Environmental Laboratory Standard Operating Procedures. 18. PROTOCOL AMENDMENATNSD DEVIATIONS DPePliaInaonEerd Dn ceahnoatnnghii.eedsAS1rw0pyitohShreetoprtsocteochnolaswile1olbde.ninrthAee rfloreFmnmofdoowfrietesn abmeendmnentgs sSiegned 2by the Stu dy oH Ennmeras amos 3 Eavirommensal Laboratory Rags Famous aT: 3m Medical Department Study: T-6295.7 laboratorRyePproRoretotqcuoleNsoSt.FACNFTu-ATmCObTXe.rT0-3O00X2-207350 19. ATTACHMENTS 19.1 AttachmenAt Preparatory and analytical methods 20. SIGNATURES Andrew Seacat, Ph.D. Spona sor Representative Z, DaeZ Valorfe -- Kristen Hansen, Ph.D., 3M Environmental Laboratory Study Director 1/27/78 Date 3M Environmental Laboratory 3M Environmental Laboratory 2423 Pagesar9 rie 3m Medical Department Study: T-6295.7 laboratorRyepoRretqueNso.t NFuAmCbTerT-OUXz-207350 GLP Study Protocol Amendment Study Number: FACT Tox-010 Covenes, 4324-223 StudyTitle: A uk Capunly Torcily Shay with, #05 in Cypomgas Mankegs Study Director: Kris Hansen A_m--e_n--dment Date: 03]aseg Amendment Number: | This amendment modifies the following portion of the protocol: Sechion 10 Fupurchg Hebuds and Sechinn 11 Arslgheal Matheds These sechoms lab FACT MAL and Farm as fhe serum exhachon and analyheal methads. The methods hare bun wpdaled to 75: 5.40 osneraumE15ex8ha:c5h0o.ns Thaened wapdnoldeedsmethod will be wed fe he remaining Approved by: T A Y Sudy Director Date 3M Environmental Laboratory 2s 3y per 3m Medical Department Study: T-5285.7 LaboratorRyepoRretqueNso.t NFuAmCpTerT-ouxz-eca3s0 26-Week Capsule Toxicity Study Title Study with Perfluorooctane Sulfonic Acid Potassium Salt (PFOS, T-6295) in Cynomolgus Monkeys PROTOCOL AMENDMENT NO. 2 Ame"Anprdilm5e,nt19D9a6te: 3M EnviroPnemrenftoarlmTiencghLnoalboogrya&toSarfyety Services 3M Environmental Laboratory 935 Bush Avenue St. Paul, MN 55106 Laboratory Project Identification ET&SS FACT-TOX-030 LIRN U2279 WM Environmental Laboratory 3M Environmental Laboratory 2635 rages am Medical Department Study: T-6285.7 LaboratorRyepRoertqueNso.t NFuAmCbTerTogxr-0a3s0 Protocol FA"CaTm-enTdOmXe.n0t302 This amendment modifies the following portion(s) of theprotocol: extaction and analytical methods. + oe dain 1. PROTOCOL READS: In amendment 1, section10Preparatory Methods and Section 11 Analytical Methods were updated to ETS-8-4.0 and ETS-8-5.0 as the revised serum AMEND 70 READ: These methods were revised on 4/26/99 to ETS-8-4.1 and ETS-8-5.1 `which will be used for all future analyses. `RwEeAigShOtiNn:g fTohreinmiteitahl ocdursvewse.re revised for clarification and to include linear regression, 1/x Amendment Approval LiSed acat Pah, SponsA oo r Represd entative h 7/B3at0e,r foto flo -- Kris J. Hansen Ph.D., Study Director WMEnvironmental Laboratory 34 Environmental Laboratory 79 3 2/7/35 Date Page TT 2m Medical Department Study: T-6255.7 LaboratorRyepomretgNeo.t MFaoczeaTroxt-083200 Study Title 26-Week Capsule Toxicity Study with Perfluorooctane Sulfonic Acid Potassium Salt (PFOS, T-6295) in Cynomolgus Monkeys PROTOCOL AMENDMENT NO. 3 `Amendment Date: June 03, 1999 Performing Laboratory 3M Enviromen55t5ecvgee& oo Seis 3M Environmental Laboratory St. Paul, MN 55106 Laboratory Project Identification [ET&SS FACT-TOX-030 LIRN U2279 F------ 20 Environmental Laboratory % 230% 20 Medical Department seu: 1-6295.7 LaborasoltyaPpRoresgeuseNoc.Ft AMpaeCctmTbT0oO%ex-0ro0s10 verano This amendment modifies the following portion(s) of the protocol: 1. PROTOCOL READS: Section 10 Preparatory Methods and Section 11 Analytical Methods list FACT-M-1.0 and FACT-M-2.0 as the liver extraction and analytical methods. AME TON READ D: These methodswere revised on 06/03/99 to FACT-M-1.1 and FACT-M2.1 which will be used for all future analyses. a REASON: The methods were revised for clarification and to expand on the listof target Amendment Approval ( India J Rue? Andrew Seacat Ph.D., Sponsor Representative /3(19 Date. 4 4 4s Kris J. Hansen Ph.D., Study Director Menino 3M Environmental Laboratory 7195 Date 38 wagts 3m Medical Department Study: T-6295.7 laboratorRyepoRretqueNos.t NFuAmCbTerT-O0X2-207350 Study Title Sulfo2ni6c-WAeceidk PCoatpassuslieuTmoSxailctit(yT-S6t2u9d5y)wiintChyPneorfmloulogruoosctMaonnekeys PROTOCOL AMENDMENT NO. 4 AmeApnridlm2e5n,2t0D0a0te: 3M EnvironPmeerntfaolrTmeicnhgnoLlaobgoyr&atSoarfyety Services 3M En9vi3r5oBnumesnhtaAlveLnaubeoratory St.Paul, MN 55106 LaboratorFyAPCrTojTeocxt-i0d3e0ntification ET&SS LRN-U2279 3MEnvironmentalLaboratory 3M Environmental Laboratory Page TG 3m Medical Department Study: T-8295.7 aboratoTryReeporBeqtueesteNo. SFENAuDCmTb8erTs50X7-0730 Amendment Number 4 This amendment modifies the following portion(s)of the protocol: AMEND 10 READ: 1. TPhReOTsOpCoOnLsoRrEAfoDrSt:he present study was identified as Andrew Seacat, Ph.D. The role Ph.D. as of of sponsor the date for of the present study was reassigned signature approval of this protocol to John L.Butenhoff, amendment. Reason: `Tsopoennssourrreoltehawtatsheresatsusdiygndierde.ctIonrtdhiosemsannontera,lsopecrasrornyntehle rdeustpioenssiobfilsittuiedsy asnpdonsor, the workload are more evenly balanced. 2. PROTOCOL READS: Oannalpytaigceal2pohfasteheopfrtothoecoslt,udKyr.iPseHtaenrsTehnomifs oirdedntiisfiaeldsoasidtehnetifsiteuddyasdiarescttuodryfodrirtehcetor, but for the in-life phase of the study (see Covance Laboratories Protocol6329-223). AMEND 10 READ: TOKrhniosmpHfaaognresd2ewnoiflwtibhleepipedrreofnttooircfomile,tdhAienndtduhrteieefwsinSoaelfartcehapeotrpwrtiilanlcsibpteahleidapenrnaitlniycftiiipecadallaisin-ntivihefesetsiintgvauetdsoyrt,idgiaaretncodtroPare,steofr the date of signature approval of this protocol amendment. Reason: The original study the study and one design for the identified analytical two study directors; phase of the study. one The for the role of in-fife study phase of director has been reassigned in Practice Standards that aountleifnfeortsttuodeynpseurrseoncnoemlplrieaqnucireewmietnhtsG.ood Laboratory 3. PRoTocoL reaps: 10. Preparatory Methods: 10.1 FACT-M-1.1, "Extraction of Potassium Perfluorooctanesutfonateor Other Fluorochemical Compounds from Liver for Analysis Using HPLC- AMEND TOERlEeAcDt:rospray/Mass Specirometry* 10. Preparatory Methods: 10.1 ETS-8-6.0, "Extraction of Potassium Perfluorooctanesulfonate or Fluorochemical Compounds from Liver for Analysis Using HPLC- Other Electrospray/Mass Spectrometry" Reason: Tmehtehyml-ettehrto-dbuwtaylsertehveirse(dVItBoEi)ncilnusdteeaedxtorfacettihoynlsaocfetoattheerintitshseueexttyrpacetsioanndprtohceesuss.e of 4. PROTOCOL READS: 11. Analytical Methods: aM Environment Laboratory 3M Environmental Laboratory 3b W rage ST 5m Medical Department Study: T-6295.7 LaboratorRyepoRretqueNso.t NFuAmCmTerTonxa-a03r0 Protocol LAN-U2279 Amendment Number 4 AMEND TO READ: 11.1 EFleAcCtTr-oMs-p2r.a1y,/M"aAsnsa.lSypseicstorfoFmleutorryo"chemicals in Liver Extracts Using HPLC- 11. Analytical Methods: 11.1 ETS-8-7.0, "Analysis of Potassium Perfluorooctanesulfonate or Other Fluorochemical Compounds in Liver Extracts. Using HPLC- Reason: Electrospray/Mass Spectrometry" 5. ProtocoL reaps: The and amertehdoudcewdacsycrleevitsiemde tforionmcl1u3d.e5 cmuirnvuetsespltoott9e.d0 bmyinluitneesa.r regression weighted 1/x 8. Test System AMEND To READ: aRneifmearltso etahcehCofrvoamncGeropurpotsoc1o,l3,#6a3n2d94-2w2e3refordetsaibgulnaartepdreassenrteactoivoenroyfadnaitmaa.lTswaond were allowed at least a 13 week, which may be extended, recovery period after cessation of treatment. 8. Test System A tabular presentation of the test system data will be included in the final report for this 3, and p 4 roject were (FACT designa Tox-030). Four ted as recovery animals animals each and (two/gender) from were observed for Groups a recove 1, ry period after cessation of treatment until study cut-off on March 7.2000 (Week 80). The observation and analysis of the recovery group ll animals beyond the cut-off date of this study will be reported in a new long-term recovery study. Reason: Additional animals were assigned to the recovery group following approval of the 6. ProTocoL reads: prreomtaoicnoilnfgoranFiAmCalTsTforxo-m03r0e.coAvenreywgrsotuupdyllwi(llmirdep-odrotsethgeroluopn)g.-term recovery of the Liver-al animals: 44 9. ESxppeeccitmeedn#aonfdSSpaemcipmleenRse:ceipt: [Sample Receipt Table] `Serum-all animals: 616 from main study, 24 additional from recovery Urine and feces-recovery animals: 24 urine, 24 feces Collected: Urine and feces-recovery animals: Day 0, 6, 30, 90 (potential 180) Total number of test animals: 32 aM Envinmenta Laborstry 3M Environmental Laboratory 4 Je 3m Medical Department Study: T-s355.7 LaboratoryRYeBpoRretgbtuosNcsoo.tt LNFAeACrRTmCaT2roo5xr-3z07e3e0 Amendment Number 4 AMEND TO READ: 9. #SpofecSipmeceinmaennds:Sample Recelpt: [Sample Receipt Table] SLievreurm--a~lallalnainmiamlasl;s:4251(e6igfhrtosmpmeaciinmeSntusdyv,ia2b8i0opasdydiftrioonmalrescpoevceriymegnrsoufpr)omrecovery Total number of test animals: 32 Total number of animals: 48 (12 animals in control group) Note: 4 animals were not assigned were collected. Specimens of body to a study group. tissues and fluids Day 0 other serum specimens than seru specimens will be collected and received from Covance m and liver Laboratories(6329-223, Study T-6295.7). Reason: gAprdrodoiuttopic.oonSlapfleoracniFimAmeCanlTscTowloelxer-ce0t3ia0osn.sfiTighgneuerdersetcosohtvoheewrynrepacerorevieotrdhyrhogaursgohbupethefenoleelnxodwtiaonnfgdtaehpdepfrsootrvutadhlyeo3rf/e0tc7ho/ev0e0,ry Amendment Approval ZL 2 CZorip Toh Butenhofl, PD. Spoor Represemais 9fomre Dae ( mdi 2 W . Soca Andrew Seacat, Ph.D. Incoming Study Director Y(2? [2000 Date KristeJn.eHeansen, PhD. Owgorng Sud Dirsior To--at2e - 200 Dale Bazan, Library Manager STh/eY rv aM Erviormontat Laboratory 3M Environmental Laboratory v2 ser 3m Medical Department Study: T-6295.7 laboratorRyepoRretqueNos.t NFuAmCbTerT-OuXz-2073s0 26-Week Capsule Toxicity Study with PerfSltuuordoyocTtiatnlee Sulfonic Acid Potassium Salt (T-6295) in Cynomolgus Monkeys PROTOCOL AMENDMENT NO. 5 AmAeugnudsmte1n4,t20D0a0te: 3M EnvironPmeenrtfaolrTmeicnhgnoLlaobgoyra&tSoarfeyty Services 3M En9v3i5roBnumesnhtAalveLnaubeoratory St. Paul, MN 55106 LaboraEtTo&rSySPrFoAjCeTc-tTIOdXe-nt0i3f0ication CovLaInMcSe ##60322297-9223 3M Environmental Laboratory 3M Environmental Laboratory age In Medical Deparcment stacy: 1-6295.7 taboraoly seqFuroeatnaotcmoelNn1d0mo5e0n-- 3tp0s e038 Tia sandman nls hollowing soriont) of heros 1. ApPdoRdtOiatTsisOoiCnuaOmlLpteiRrsEfsulAeuDsoSro:orocf2lt.uainPdesUsRumlPafOyoSnbaEet:aenT(ahlPiyOszSead)n.aliyntitchael lsivteurdyanids dseesriugmnoefdcytonodemtoelrgmuinse`mloenvkeelysso.f REASON: Feces saps vec ddd afer the inl pret was wis AMENDTO READ: Add that select feces samples will be analyzed for PFOS at Centre. 2. EPnRvOrToOnCmOenLiaRlEALDaSr: nSeTrUDY Locarions(race 2): Analytical Testing Laboratory: 3M sREiASeONs:eASnH EyvioafnfmeceenseseLsosibgneod to Cee, in sition to the asses of ater AMEND TO READ: 3M (Centre), 3048 Research Environmental Laboratory and Drive, State: College, PA 16801 Centre Analytical LaboratoriesInc. 3. APNRDOT1O2.CODLATRAEAQDUSA:LISTEYCOTBIJOENCSTI10V.ESPREPARATORY METHODS, 11. ANALYTICAL METHODS PAoNmEoNgGen7z0ziRoEnAD:seyAdidmmtoeithneiseysaions the mos ume version of "Deemiation of 0F0luMo-r0o2c3h-e0m0i3c,alwiRtehsiadr.ueLs OiQn Mionnkfeecye/sRaoft 1F0ecnegs/g.byThLeC/mMeSt/hMoSd,"wilClenitnrceorpmoertahteodannuimnibtearl SRREoeSgOceNrr:se,Tto1sssSpeeiwty LshteCoovmamliisddimteesteeaddnditfnogtbehesuspesderrfvrmsocnstsessonamneaslosmons2wilth analytical limits. NOTE: LC/MS/MS is an abbreviation for "liquid chromatography/mass 4. PROTOCOL Metros READS: SECTION 10,PREPARATORY METHODS AND 11. 3M ANALYTICAL oAMENoD 7t0 Read: Chane to spiy hat the mst cunt version of te methods ise mReEtAhSoOdNs:shToouldspbeeciufsyedthdautritnhge tmhoesctouarpspeorofprtihaetestuvdeyr.sion of the preparatory and analytical Erman tabs 34 Savi ronmental Laboratory rss 3m Medical Department Study: T-6295.7 laboratorRyepoRretqueNos.t NFuAmCbTerTO0X3-307300 ProtAomcoelnTdOmXe-n0t305 S. iPsRtOheTOPrCiOncLipRaElAADnSa:lyTtihcealamInevnedsetidgaPtroortofcoorlth(eAmenetnirdemsetnudty.#4) states that Kris J. Hansen, Ph.D. CAeMnEtrNeDfoTrOfRecEeAsDa:naAldysdesE.naksha Wickremesinhe, Ph.D. as the PrincipaBleLan-ltfyeh,Iinvaerstigator at REASON: To specify the PAI at Centreforfeces analysis. aafmfie 6. RPeRpOoTrOtCsOL READS: SECTION 16. Locamon oF Raw Dara, Recoros AND FiNaL EAnMvEiNroDnmTeOntRaElADLa:boArdatdortiheast, Cteongtertehewriwlitfhorcwoapridesaolforaipgpirnoalprsitautdey-fsapceilciitfyi-cspreaciwfidcatraawto d3atMa rdaaeptcpaol,ridcpsar.bolteoctool thainsdsatnuadlyy.tiCceanltrreepowritllinmatihnetaCienntarecoaprycohifvtesh,easapwpellilcabalse alsltuodryi-gsipneacliffaiccilriatwy REASON: Centre, To specify the archival requirement for the portion of the data developed by 7. PROTOCOL READS: SECTION 17. SPECIMEN RETENTION AMEND TO director, all READ: Add that after the analytical report feces specimens of this study will be retum on feces is signed by the study These specimens may then be discarded by written direcetdiotnoo3fMtheEnstvuidryondmireencttaolrL.aSbpoercaitmoreyn.s daoinfraeslcyettroiucrmal arnedpourrtinefomr atyheal3soMbeEndviisrcoarndmeedntbayl wrLiatbtoernatdoirryectainoanlyosfetsheisstsuidgyneddirebcytorthaefstterutdhye Reason: vTeorisfpieccaitfiyontihse chonasnidderleoidfncaolgmlpblieotleogical fluids, and to define when the quality assurance 34 Environmental Lasaratory 3M Environmental Laboratory rege om Medina, Depgsiment Shiga R395, 7, TEE pan om amonia *BobroarcaonroyR'SPROeEgyeNot.s ENApGhTtTsobwooo a8 | i. LAB 2-36-09 + 614 231 1mm co | CEI : : ProtAocmoelnTdomxe-n0t2s0 AmendmentApproval | i! SET 7 "Gpocsor's :: =. Seacat, 7D, Shady . : Ii A. IS-- i Ph.D, PrincipalIn-fifeInv i 3 i\ :! : od i ~ ' :: 14 fasme ee ;i 2 Die ; oo : Date : 'I ii : ji5 3M Environmental Laboratory ii $Y PagessiA 3m Medical Department Study: T-6295.7 1aboratorRyepoRretqueNos.t NFuAmCbTerT-O0X2-207350 Study Title 26-Week Capsule Toxicity Study with Perfluorooctane Sulfonic Acid Potassium Salt (T-6295) in Cynomolgus Monkeys PROTOCOL AMENDMENT NO. 6 Amendment Date: September 11, 2000 3M Enviro3nMPmeeErnntfvaoilrromTneimcnehgnntoLalalobgLoyarba&otraoSatrfoyertyy Services . 935 Bush Avenue St. Paul, MN 55106 Laboratory Project Identification ETC&oSvaSnFceAC#6T32T9O-X223030 LIMS #U2279 a Environmental Lasaratory 3M Environmental Laboratory age SE 3m Medical Department Study: T-s295.7 laboratorRyepoRretqueNso.t NFuAmCbTerTo0x3-303350 Protocol TOX 030 Amendment 6 This amendment modifies the following portion(s) of the rotacol: 1. PROTOCOL READS: c8o.nTcEenStTratSiYoSnTsoEfM:0.G0r2,ou0p.5s, 2, 3, and and 4 received 2.0 mg/kg/day, trheespteecsttisvueblys.tance daily for 26 `weeks, at AMEND TO READ: 0G.r1o5u,p2snd2,03.,75anmdgl4krge/cdeaiyv.ed the test substance daily for 26 weeks, at concentrations 00.03, Reason: Ttheestacstuubasltdaonsceesodofsetesstwesruebscthaanncegetdhatafweerrpergoitvoecno.l was written. The Covance protocol reflects PThReOTamOeCnOdLeRdEpArDotSo:col (Amendment Number 4, section 2. states: On page 2ofthe protocol, taaAhmneedtnphrerdeimpnwerciniSnpteca.ailcpaaantlawilniy-ltlliicfbaeeliinivndevseestntiigtgaaittoaforsri,atseaohdnefdtshPteeutdedyradtTieorheofcmtsofirgo,nrKadrtiwusrielHaabnpepsredonevawnlitloleftpdheir1sfotprhrmeottfohicenoslduetiseassof AMEND TO READ: Peter Thomford will remain as the Covance Study Director for the purposeofissuing the Covance final report for the in-life phase of thestudy. StRhteeuadasynoidnmia:rlecetxopresrhiipmewnataslnpohtasreelwinaqsuicsohmepdlebtyeCdobveafnocreeAfmorenthdemienn-ltifNeupmhbaseero4fwtahee sstoudsy,, since Amendment Approval pln 7 Ef John L Butenhoff. PH.D.. Sponsor Representative ryfe Date AndrewCSeoacnatc,lPihDi.nStuJdy Drees Se seDaoe WM Environmental Laboratory 3M Savironmental Laboratory asl 3m MeddwadMepamsment Seuss 5628517, !0 HiBoratorRyepRoretqueNso.t FACT ToX-030 Numbdbas ses Study Title ProtoAcmolenTdOmXe-n0t3?0 26-week Capsule Toxicit(yTS6t2u9d5y)wiinthCyPnecofmloulogruoosctMaonnekSeuylsfonic Acid Potassium Salt Protocol Amendment No. 7 Amendment Date: September 14, 2000" 3M EnvirPoenmrefntoarlmTiecnhgnoLloagbyoarndatSaofrelyy Services : 3M En9v3iz5oBnumsenhtAalveLnaubeoratory St Paul, MN 55106 LaboratEoTr&ySSPrFoAjCeTc-tTOIXd-e0n3t0ification `CoLvIanMcSe ##6U322297.9223 ! | i | 3M Environmental Laboratory TTT TR 3m, MedRABBeparememit tpoxi StOUNTARY 7 yp 884'91; BBratoRreypRoertqueNsot. NFuAmCbTeoT.0X3-2027330 ou Amendment Approval A John L. Butenhoff, Ph.D., Sponsor'sRepresentative 7s Date `Seacat, Ph.D., Study Director pz Date i|: i i Bl " 3m Medical Department Study: T-6295.7 SMMedicalDepartment Suey: T2957 ATTACHMENCT EXTRACTION AND ANALYTICAL METHODS LaboratorRyepRoertqueNso.t NFaAmCbTerTouxs-0e3s0 LasorsteRyeRsooqriuNeos. FACmToToorxi0n3g0 SW EmionmortlLaborsory 3M Environmental Laboratory Pagect Based 3m Medical Department Study: T-6295.7 laboratorRyepRoretqueNsot. NFuAmCbTerT-OUXs-3013e0 3M ENVIRONMENTAL LABORATORY . METHOD FLEUXOTRROACCHTEIMONICOAFLPCOOTMASPSOIUUNMDPSEFRRFOLMUOLRIOVOECRTFAONREASNUALLFYOSNIASTUESOIRNGOHTHPERL.C ELECTROSPRAY/MASS SPECTROMETRY Method Number: ETS-8-60 Adoption Date: 0 114g Author: Lisa Clemen, Robert Wyme Approved By: IN Toe Laboratory Manager Lit toe Group Leader Revision Date: Nit ar fog Date 2/119 Dac Techxnicbail RBeovmiaenw,er Daitleiils 0 Score Ap ArrLicATION 1-1 oStchoepre:fluTohrioschmeemtihcoald cisomfoprotuhnedesxtfrracotmiolnivoerf.potassium perfluorooctanesulfonate (PFOS) or 1.2 Applicable Compounds: Fluorochemical surfactants or other fluorinated compounds. 1.3 vMaaltirdiacteiso:n rReapbobrtit, rat, bovine, and monkey livers or other tissues as designated in the Word 6.095 ExvacionEofrPSFsO.S60fiom Liver 3M Environmental Laboratory Page tori Page dd 3m Medical Department Study: T-6295.7 laboratorRyepRoretqueNso.t NFuAmCbTerT-OUX2-207350 2.0 SUMMARY oF MeTHOD 21 Tr(ehPaiRgsOenSmt)etaohnrododtmhedeterhsycf/ru-ifoberersoic-thbheuemtpiyrlcoaecltehdseuurrrf(eaVcfItoBarEnet)xs.trrIaonctmtihnilgsivpmeoer,ttahosorsdoi,tushmerpveeicnsfslufuelesar,oouorscoitcnahgneeastnunliifoconeanaolpinsaeirbieng pseaxtrtatrniadtcaitroednd.e:dAPniFnOtioSon,MpIPaBiEErO.iSngAT,rheePagFMeOIntSBAiEAea,xdtdEreaIdcFtOtioSstEth-reaOnsHsa,fmeprPrlFeedOaStnoEdAa,tcheeMnt5arn5ia6fl,uygtaeentjduobnseupraarinordgaptuet onto a fniilttreorgedenthervoapuogrhaato3rcucntpillasdtriy.c sEyraicnhgeexattrtaaccthiesdrteoco2n0s.t2ituutmedniynlo1n.0fmillt.emientohagnloalssthaeunt.ovials 22 Tmhetehsoedss.ample extracts are analyzed following method ETS-3-7.0 or other appropriate 3.0 Derrvmions 33.21 PPFFOOSS:A:peprefrlfulourooroocotcatnaenseulsfuolnfaotney(laanmiiodneofCyFp,o,tSaOss,iNuHm,sali) C.F,SO, 33 POSAA: perfluorooctane sulfonylamido (cihyl)acetateC.F.SO,N(CH.CH,)CH,CO, 34 CE.FFO,SSEO-NO(HC:H.A(CNH-,ct)hCylHp,eCrfHl,u0orHaoctane sulfonamido)-cthy! alcohol 33.56 PMFSSO6S:EAC:Fp,eSrfOl.uNor(oHo)c(taCnHe,sCuOlfOoHny)l ethylamideC,F,SON(CH,CH,) 3.7 Surrogate standard: 1H-1H-2H-2H perfluorooctane sulfonic acid 4.0 WARNINGS AND CAUTIONS 4.1 Health and Safety Warnings: 411 hUasnedluinnigvearnsailmaplretciasustuie,onwsh,iecshpemcaiayllcyonltaabionraptaotrhyogceonatss., goggles, and gloves when SO 51 I vveme There earmeennocienster f er en ce s known at t hi s time. 00000000 S60 .O1 FoTuh0 eewfoelwlo0 rwing eq0 uipmen0 t is use0 d while0 perform0 ing thi0 s method0 . Equi0 valent e0 quipme0 nt is acceptable. 6.1.1 6.1.2 VUolrirtae-xTumrirxaexr,TV25WRGr,inVdoerrtefxorGgerniinedi2ng liver samples 6.1.3 Centrifuge, Mistral 1000 or [EC 6.1.4 Shaker, Eberbach or VWR ExtractioEnToSf.P8O.S60from Liver 3M Environmental Laboratory Page 2014 Pages HMs53 3m Medical Department Study: T-6295.7 laboratorRyepoRretqueNso.t NFuAmCbTerT-OUX2-207390 6.1.5 Nitrogen Evaporator, Organomation 6.1.6 Balance (sensitivity 0.0.10 g) 7.0 SUPPLIES AND MATERIALS 71 Gloves 7.2 Dissectingscalpels 73 74 NEalgpenepbootetrldeisns,pcodsaabolpepoairfphebotfltledsieng 250 mL and 1 L 75 Volumetric flasks, glass, type A 7.6 1-CHEM vials, 40 mL glass 7.7 7.8 CPleansttriicfusgaemptuublees;vipaolsl,ypWrhoepaytloenne,,61m5LmL(.or appropriate size) 79 Labels 7.10 OxfordDispensor ~3.0to 10.0 ml 7.11 Syringes, capableofmeasuring 5 uL to 50 uL. 7.12 Graduated pipettes T.13 Syringes, disposable plastic, 3 cc 7:14 715 Syringe Timer filter, nylon, 0.2 um, 25 mm 7.16 Crimp cap autovials and caps 7.17 Crimpers Note: PQvriia7olrs.wtaotuesri.ngRignlsaessswyarriengaensdabomtitlneis,murimnsoe f93 tiimmeesswwiitthhmmeetthhaannooll,an3dri3nsteismefsrowmit3h sMeiplalria-te 8801 RerTcypeev|trseaagevnot Sgrtaadeowaatmers, Milli-QTM be Milli-QTM water and be providedby oar eMqiulilvia-lQenTtO; CallPwlautseTMr suyssetdeimn this method should 82 Sodium hydroxide (NaOH), LT Baker or equivalent 83 Tetrabutylammonium hydrogen sulfate(TBA), Kodak or equivalent 84 Sodium carbonate (N,CO,), 1.T. Baker or equivalent 85 Sodium bicarbonate (NaHCO)LT,.Bakeror equivalent 86 Methyl-tert-butyl ether, Omnisolv, gla distilled or HPLC grade 87 Methanol, Omnisolv, glass distilled or HPLC grade 88 Liver, frozen from supplier 89 Dry ice from supplier 810 Fluorochemical standards 810.1 PFOS (3M Specialy Chemical Division), molecular weight = 538 3M Environmental Laboratory ExuactioEnTofSP8E.OS60fiom Liver qs 59 PageSorts Pagede 3m Medical Department Study: T-6295.7 laboratorRyepoRretqueNso.t NFuAmCbTerT-OUX3-307340 8.10.2 8.10.3 PPFFOOSSAAA(3(M3MSpSepceicaiallytyChCehmeimciaclalDiDviivsiisoinc)n,),momloelceuclualrarwewiegihgtht= =495985 8.104 8.10.5 PEFFOOSSEEA-O(H3M(S3pMecSipaelctiyalCthyeCmhiecmalicDailviDsiivoins)i,onm)o,lemcoulleacrulwaerigwhetig=ht52=7$70 8.10.6 M556 (3M Specialty Chemical Division), molecular weight = 557 8.10.7 CSu\rFr,ogSaOteH)stamnodlaercd:ula4r-Hw,epiegrhftlu=o4ro2o8ctane sulfonic acid (1-H,1-H, 2-H, 2H 8.10.8 Other Auorochemicals, as appropriate 811 Reagent preparation pNrOepTaEra:tioWnh,eandjupsrtepaacrcionrgdilnagrlgyer volumes than listed in reagent, standard, or sumogate B11 d11i00s0s0olsmvoiedd..ibueSmatkoheryredrcioonnxatiad|ienL(inNNgaalOSgH0e)0n:emWLbeoiMrgel.hla-pQprTMoxwiamtaetre,lmyi2x0u0ntgilNaalOHsa,ldPsouarreinto a 811.2 M11 i0NlNls'-oNdQaiTMQumwHathseyord.lruoiSxotinodreien(tioNnaaaOH11)02:05mmDLLi.lvuNotalelugm1ee0ntNeribNcoatfllOea.Hsk a1:n1d0.dilMuetaestuorveo1l0ummeLusoifng 8:11:3 PO0f.H5TM1B0Atuseiitnnrtgosba2uptp| yLrloaxvmiommloautnmeielutyrmi4hc4ycdtoronot5ga4einnmiLsnuglo5ff0t0e10(mTNLBANM)ai:lOlWH-eQi(TMWghhiwalapeteparr,dodxAiidnmjgauttsehtleytloa1s6t9mLg. MofilNlaiO-HQ, waadtders.loSwtloyrbeeicnaau1seLthNeaplHgecnheabnogtelseabruptly). Dilute to volume with 8113.1 nTeBeAdedreuqusiirnegs|aNheNcakOpHrisoorluttoioena.ch use to ensure pH = 10. Adjust as 8.11.4 ba0i.pc2pa5rrobMxoinmsaaottdeeil(uyNma2c6Ha.Cr5bOoo)nafitsneot/odsaiodu1imuLmcavbroiblcauanmraebttorenia(ctNeal,baCkuO,ar)nda(nNbdari,2n1Cg.O0t/.ogNvaooHflsCuoOmd,ei)uw:imtWheiMgilhi- Q water. Store ina | L Nalgene boule. 812 Standards preparation 8.12.1 Prepare PFOS standards for the standard curve. 812.2 sPfolrlueuoptraiorocenhoectmohinectraalifnlsiutnoagrnod1ca.hr0ed0msipacpramel asPctFcaeOnpdSat,radb1sl.,e0a2(sfpoarppempxroaPpmFrpiOlaSet,Ae.,on0Me.u9lw8to7irkcpiopnmmgpoPsntFeanOntdSaArAd, and 1.10 ppm EEFOSE-OH.) 8.12.3 Wtheeiagchtuaaplpwreoixgihmtately 100 mg of PFOS into 100 mL volumetric flask and record 8.12.4 Bring to (ng/mL). volume with methanol foa sock standardofapproximately 1000 ppm 8.12.8 aDiplpurtoexitmhaetsetloyck50sopluptmion with methanol for a working standard 1 solution of 34 Envizonmenta Zasoratory ExncionoEfTsPsO.S60fiom Liver Pagedof 4 saget7 3m Medical Department Study: T-6295.7 laboratorRyepoRretqueNos.t NFuAmCbTerT-oyxz-207350 812.6 Dapiplruotxe.th5e.0stpopckmsolution with methanol fora working standard 2 solution of 8.12.7 aDiplpurtoex.th0e.5s0topcpkms.olution with methanol fora working standard 3 solution of 813 Surrogate stock standard preparation 813.1 WeCiFg1hSaOpp,rHoxiinmtaotaelSyO5m0i-6v0olmugmeotfriscufmloagsaktaensdtraencdoarrdd t1h-eH,a1c-tuHa,l2w-eHi,gh2t..H, 8132 Bring to ppm. volume with methanolfor a surrogate stock ofapproximately 1000-1200 813.3 Prswetoporacrkkein(a0gsasutr1ar0nodgmaalrtdevoowflourm1ke0it-nr2gi0csptpfalmna.dsakrRaden.cdoTbrrrdainntsghfeetoracavtpouplarluomvxeoimlwauitmtehelmytrea1t.0nhsafnemroirleodff.osru3rrogate 9.0 SAMPLE HANDLING S11 All samples are received frozen and must be kept frozen until the extraction fs performed 10.0 QuALITY ConTror 10.1 Matrix blanks and method blanks 10.1.1 An aliquot of 1.0 mL methanol is used as a solvent blank. 10.1.2 Eaxstmreactthotdwobla1n.0ksm. L aliquotsofMilli-QTM water following this procedure and use 10.1.3 aEsxmtraatcrtixtwbola1n.k0s.mLReafleirqutoots11o.1f.6l.iver homogenate following this procedure and use 10.2 Matrix spikes 10.2.1 tPhreepaacrceuraancdyoanfaltyhezeexmtartarctiixons.pike and matrix spike duplicate samples to determine 10.22 rPerceepiavreedcwaicthhscpaickhe ussaimnpglea sseatmple chosen by the analyst, usually a control liver 1023 cEAadxldpiiebtrciatotenidaonlcocsnuprcivekene.tsramtaiyonbsewiilnlclfuadleldinatnhdemmaiydfa-llrianontfhtgehelowi-nirtainagceaolifbtrhaetiionnitciualrve, 10.2.4 mPrienpiarmeumonoef2matmraitxrisxpiskpeikaensdpmeartbraitxcshpike duplicate per 40 samples, with a 10.3 Continuing calibration verifications 10.3.1 Pinrietpiaalrceacliobnrtaitniuoinncgucravlei.bration verification samples to ensure the accuracyofthe 10.3:2 Poarfned1pa0erxsetar,macpatlteeds..minFiormuemx,amopnlee,coinfatisnauimnpglecasleitbr=at3i4o,nfvoeurrifviecraitfiiocnatsiaomnsplaerpperregpraoruepd 3M Environmental Laboratory ExtractioEnToSf 8P.O6S0from Liver Pagesof14 Pages 3m Medical Department Study: T-6295.7 laboratorRyepoRretqueNsot. NFuAmCbTerT-OUX2-207330 10.3.3 pPrreeppaarreetehaechinciotnatlicnuurivneg calibration verification from the same matrix used to 10.3.4 tcThaheleiibenrixattpiaielocntcaeclduirbcvroaetn.icoenAndtcdruiarttvioeo.nnaslTwhsiiplslikifesasllnmewciyetshsbianeryitihnfeclmtuihdede-darntaahnlagytseotfflmltuhsiettqihuneiatnlitaoilwt-artaenugseinogf tohnalny 5tphepblo~w e1n0d0o0fptphb)e. calibration curve (for example, 5 ppb ~ 100 ppb, rather 1.0 CALIBRATION AND STANDARDIZATION 11.1 Prepare matrix calibration standards HLL mWeLisgMhialplpir-oQxTMimwaatteelr.y 4Gr0ignodftloivaehroimnotogean2e5o0usmLsolNutailogn,ene bottle containing 200 11.1.2 rat4io0. gisnot available, use appropriate amountsof iver and water to ensurea 1:5 11.1.3 cRoenfceernt10ra1t3i.o0ntoofcsaollciudlaitveetrhteiascuteuadlisdpeenrssietdyoifnl1i.v0ermhLoomfogheonmaotgeenaantdetshoelution. TLLS hsAhodamdkoig1negmnLbeoeotufwsehesonomloaultgiieqonunaotitnsew1th5oimaleL15pcrmeenpLtarrciiefnnugtgreaitftouotgeaelsotufbee.igRhet-eseuns|pemnLd saolliuqtuiootns obfy 11.1.6 Two 1 mL aliquots, or other appropriate volume, serve as matrix blanks 11.1.7 Tethiyegphietcneadelonlyfsauhmsipeslteshsee,ctstitowanon,dmatarotdsrpicioxknebc,leanintnkrsda,utpialoinncdsattaewn,odtmswpeoitkshitonadgndbaalmraodnukcnsutrsvelsi,stfeodrain tToatablleofI, at 11.18 csRaeelhfiiebcrrhattloiisovtnaslctiuhdreavtweiosornkrienpgorrtasnEgTesS-a8n-d6.th0eaLnidneEaTrSC-a8l-i7br.a0t-iVo-n1RoarngAett(aLcChRm)enftorB, 11.1.9 sUtsaendaArtdts.achRmeefenrt tCo standards. a1s3.a0ntaoicdalicnuclaaltceulacattuianlg ctohnecceonntcreanttiroantsoiofnsoPFfOStheiwnocrakliibnrgation 11.2. TPsuoPmoe~gaca1ht0e0w0owpropkrbik.nigngstsatnadnadradr,dbfloarntk,heocrocnocnetnitnruaitnigonve0riffialclatwiiotnh,i3ndtdheacpaplriobprraitaitoenacmuoruvnetraonfge $ ExtacioEnTofSP8F.OS60from Liver 3M Environmental Laboratory Page Goris Pagers 3m Medical Department Study: T-s295.7 laboratorRyepoRretqueNso.t NFuAmCbTerT-OUX3-307390 11.3 sEtxatnrdacatrdsspitokeedstlaibvleirshhoemaocgheinnaitteals cfuorlvleowoinngth1e 2ma.s1s4s-pe1co2tfr.otm2hei5tsemrethod. Use these Table1 Approximate Spiking Amounts for Calibration Standards Working Standard (Approx. Conc.) Wl Approx. final conc. of PFOSin liver 00pm| | Gos : 030 ppm 0.50 ppm. [aT ootopm 10 [| 0025ppm | 00.500 pppmm [8020 | 00.100500ppppmm [ [SSo0pmemm[73%10 [Som TT 00.s2o50oppppmm 0.750 pom. Som7% 100ppm 120 Procepure 12.1 Obuain frozen liver samples, : 122 Cupteraforpmedpquicrkly|,ognootfxallilvieorwuisnmigntghaaedliisvtesrecoteitnhgaslwc,alypel. Thispart of the procedureisbest 12:3 Weigh the sample directly into a tared plastic sampule via. 12.4 Record the liver weight in the study notebook. 125 Retum unused liver portions to freezer. 126 Add 2.5 mLs of water to sampule vil, 12.7 Gruintnidl tthhee ssaammpplle.e iPsuhtotmhoeggerniendoeursprobe inthe sample and grind for about 2 minutes, or 12.8 Rinse the prose ino the sample with 2.5 mLs water usinga pipette 12.9 Ta22k.e the grinder apart and clean it with methanol afer each sample, Refer to AMDT-EP- 12.10 Cweaipghtth,e lsiavmerplIeD,adnadtevoarntdexanfaolys1t5 sineictioanlds.s. Label the sampule vial with the study number, ExuaEcTotfSiP.RoO6nS0from Liver 3M Environmental Laboratory Page 7of1s 39e50 3m Medical Department Study: T-6295.7 laboratorRyepRoretqueNso.t NFuAmCbTerT-oUXz-z0r3s0 12:11 vciPeainlp.tertiRtfeeufge1e.r0t1um0beLa.,ttLoaacrbhoeetldhewtrhoerakcpsepnhrtoerpeirtfiuafgtoere tdvuoobcleuummweei,tnhtoiftnhhgeoitmdhoeengtreiencmaaaltieniinnfntogorsmataetp1is5onmaLs ptohelysparmoppuylleene 12.12`Pmieptehtotde btlwaonk|s.mL aliquotsofMill-QTM water to centrifuge tubes. These will serve as 12.13 sStpainkdearadllassadmepslcersi,beidncilnudseicntgiobnla1n1k.s2.and standards ready for extraction with surrogate 12.14ofSptihkaet eseaccthiomna,tiorrx twhietchatlihberaaptpiroonpcruiravtee continuing calibration standards. satanmdarodos.fustAalnnsdoatprrdepaasrdeesmcartirbiexdspinik1e1s.1a,nodr Table | 12.15sVaomrptleexsmfiorx 1t5hesesctoannddsa.rd curve samples, matrix spike samples, and continuing calibration 12.16 Check to ensure 0.5 M TBA reagent is at pH 10. If not, adjust accordingly. 12:17bTiocacrabocnhastaembpulfefe,r.add | mL 0.5 M TBA and 2. mLofthe 0.25 M sodium carbonate/sodium 12.18 Using an Oxford Dispenser, ad S mL methyl-tert-buty] ether. 12.19 Cap cach sample and put on the shaker at a seting of 300 rpm, for 20 minutes, 12:20 Centrifuge for 20 10 25 minutesat a settingof 3500 rpm, or unil layers are well 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 hPouurts.cach sample on the analytical nitrogen evaporator until dry, approximately 1 102 12.24 Add 1.0 mL 10 each centrifuge tube using a graduated pipette. 12.25 Vortex mix for 30 seconds. 12:26 FAitltiearchinato0a.21u.5mmnLylgolnasmseasuhtofviilalleo0r lao3w-cvcoslyurmiengaeutaonvdiatlrawnhsfeenr tnheecessasmaprlye o this syringe. 12.27mLaatbriexl,tfhienaalutsoovlivaelntw,itexhttrhacetsitonuddyatneu,mabnedr,anaanliymsai(ls)nupmebrfeorramnidnggtehnedeerx,trsaacmtipolne timepoint, 12.28 Cap and store extcacts at room temperature ot at approximately 4 C unil analysis 12.29oCroimnpclleudteeitnhesteuxdtyrabcitnidoenr,woarsksaphpereotp,riaatttea.ched to this document, and tape in study notebook ExvactioEnTofSP8R.O6S0fom Liver 3M Environmental Laboratory Pagesoris rage tl 3m Medical Department Study: T-6295.7 LaboratorRyepoRretqueNos.t NuAmcbrerTosKa-0i3n0 10 DATA AnaLYSIS AND CatcuaTions 13.1 C1a3.l1c.u1latCteianolnessle:paatreattehe1av0ermagae dleniioqoffouhreoogivetenrishcomogenate by recordin cach mass of Averagedensity (mg/mL)= Average mass (ng ofthe alquos 1.0 mL aliquot 13.1.2 Cdiaslpceurlsaetde sthleidamiosusntuopfelrivmeLr (ofmhgo)mpaegre1n.0smeLsuhsopmeongseinoantewi(norscon Foon centration of of Liver x A(v8oer rvagee drens+ity*ooffWhaomtogenate (mg/mL) * refer to 13.1.1 for details. 13.1.3 Cstaalncdualradtse wacitnuaglhceoncTeonltorwaitinognssofuPnFOS and other fluorochemicals incalibration dof Standard x Concenation(ug mL = Fina] Concentration (us or mk) mg Liver/ | mL homogenate of PFOS in Liver "refer to 13.1.2 for details. 140 Merion Pensomasc 14.1 1432 TshTphheeeecqiumffaoiellctilthMoyowDdionLfdgetatqhenueacdeltiilxtoiaymniccltioionmfoitqntruo(aalnMntsdDiLtsaa)tyiimssoanan.pa(elLyOeltQxe)aaecnvtdaselumdeastwri(itrxhefaseprcecthiofibAcat. ttahRcoehffmeserantmtopslMeBD.sLatnodreevpCo)a.rttutfoer 142.1 142.2 MpMreaetcthiisoidosnbpolifkatenkhasnadenxmdaamcittosnpibklaendks, opsalmpleis tocdettermisne acurscy and 14:23 oCfotnhtieniuinngiccaalliibbrraattiioonnvceurirfviecation smpis to determine the continued accuracy 14.3 Refer to section 14 of ETS-8-7.0 for method performance criteria. 155:.10 PgSOahLmpLlUBeTTIwUOaNsctonPetRiaesinvdeiersxsp,ToIsiOeddN locsed inthe labors. AiunsNebDdioWlhaaazssatreid pcMoeantesaiawncearessnt,gOsarGmsmpaobslceds1olvbernotkewnassos dsiosmpaosseedrs ExvcionEorfPsaOsSofom Liver 3M Environmental Laboratory - sears Page 3m Medical Department Study: T-6295.7 laboratorRyepoRretqueNos.t NFuAmCbTerT-OUX2-207390 16.0 RECORDS 16.1. oCromipnclleutdeetihnetehxet3r-acrtiinognstwuodryksbhienedetr,atatsacahpepdrotporitahties.method, and tape in the study notebook 17.0 TABLES, DIAGRAMS, FLOWCHARTS, AND VALIDATION DATA 17.1 Attachment A, Extraction worksheet 17.2 Atachment B, MDL/LOQ values and summary 173 Atachment C, Calibration standard calculation and concentration worksheet B18O.1ReTeheemvaelnidcateiosn report associated with this me0tho0d 0is E0TS0-8-06.00&07.0-V-1 18.2 AMDT-EP-22, "Routine MaintenanceofUltra-Turrax T-25" 18.3 LFiAvCeTr-fMo-r1A.n1a,ly"sEixstrUascitnigonHPoLfCP-FEOleSctorroOstphrearyA/nMiaosnsicSpFelcutorroomcehtermyi"cal Surfactants from 19.0 AFFECTED DOCUMENTS 19.1 EMTaSs-s8S7p.e0c,tr"oAmneatlryys"is of Liver Extracts for Fluorochemicals using HPLC-Electrospray 200 Revisions ReNvuimsbieorn, Reason For Revision ReDvaitseion ExwacioEnTofSF3O6S0from Liver Page 100f14 3m Medical Department Study: T-6295.7 laboratorRyepoRretqueNso.t NFuAmCbTerT-OUX3-207350 Study7. MaBtorckix Surrogate Si@ FC Mix 5S FC Mix Std FCMixStd | Comments a|apcpuraolx. ppppmm|| aapcpwrsolx. 0.5 ppppmm|| aspcpuraolx. 5ppppmm || aappcrosx. SOppppmm Webs |gPm gee em --Dat-- e Spikeed/sAownwaolyT so t [ p o r p 1 r r r r r r ] r Fr s oa Ie m r---- r F ---- B ---- f E r -- e-- e--r -- ee -- ] r r rr r t ---- r -- r t r T r ] 1 r r r eel ir r p meem eee----1 rr II I TI --1 1 T Mfir obmdmyesEET Cpe Talo Sopen r : ri E T S -- --E1 S[ r E Tori E op -- e aeee n -- ly [Remove tmClommdentor Rew eve y] DonCoeno(PisCrieoeeV=erBsSionm:eseweds bSeomeSmHHaenp Tor he d Save Anschment B: MDLILOQ Vales ExeEoafrPscOssStofirmoLivne 3M Environmental Laboratory Page 1116 Perl 3m Medical Department Study: T-6295.7 laboratorRyepRoretqueNsot. NFuAmCbTerT-OUXz-307390 [MCDoLm/pLoOuQn|dvaluMesDfLor|rabLbiOtQl|iveLrinear Calibration Range (LCR) ] (Pb) | (ppb)|Approximate concetnobetusredafortpriepaorinngtshe | PFOS | 845 | 69||3S0tapnpdbard12C0al0ipbprbation Curve | 1 [PPFFOOSSAAA T73[52406 ||I7L8.I3 || 3102ppppbb=1122000ppp0pbb. ECFOSE-OH| M556 108 _| 823 | 236425 || 6600 ppppbb-- 9102000ppppbbTM 1 PFOSEA 3391 108 | 30ppb- 1200ppb cMuDrvLe/sLinOeQacvhaolfuetshiensreamta,tbroivciense,waenrde mexotnrkacetyedliavnedr waenraleynzoetdswtiattihsttihcealrlaybbdiettelrimvienrecdu.rveTswtoo tdheeterrambibintereeqsupiovnasleesn,cet.herReefosrpeo,ntsheesiriMn tDhLe raatn,dboLvOinQe,wialnldbmeoanskseuymeldivetro bcuerevqeusiwvaelreenteqtuoitvhaolseent to values as determined for the rabbit iver. A *RefEeGrFtOoSLEO-QOHSeusmtmimaartyesaonndlyMfDorLMsDtuLdyainndELTOS-Q5.-6.D0id&no7t.0m-eVe-t1 efioerrfiuartfhoerr vianlfiodramtaitoinon r Compound:PrPepFarOeSd| Rargear Loe | ongeol | vege [aCvRfeiocm|| Rlaonwgseds [CRlofwrsodm || Whagnhgeeodl LChRghfsodm marx t| osuonndardospt||oo)cuorvgemts on cum | ocuorvempm) ocounrtvoepnts| mycurpveris_ py cgurvte s f Compound:PrPepFarOeSd A| Rangeol | mLiavme L|| esayonnitgmetls||| opw)ceionnegsy Rabe [619-1297 [12-200 i Compound:PrPepFarOeSd A|A Rongeol | mLaern || smumnsdaerodrs|| wcveunege Lootog | on rgmts LaCeRcfnoem|| Rlaonwgsedor om cont| om cmuients 12-200 | 12-30 [CRiiom| Rargeolr aves| laomwaed co)oprmts | eos toms TOlRowTsodm|| Rmarnagedr utyaorens || eo atmients 12-300 CCRTom| Famgeor alomwesd || hagmtaed omy opts | commits LhCiRgThosm oapme m|| LCR Form agmhoew mts AtschrenBt:MDLILOQ Values ExwacioEnToSf.F8O.S60from Liver 3M Environmental Laboratory Page 12016 Page-ss" 3m Medical Department Study: T-6295.7 laboratorRyepoRretqueNos.tNuFAmCbTerTo0x3-507350 I | Covmepou|nd:ForEeiteFrdOS||E-ORwHaerngseael mars|orsunltapcmts|| oo"cupeets Rabbit | 617-1235| 31.90 [mCeRafnoms|| Tomgeor lows om) gmt | mysremts 3i.s00 | na Compound:FiPeFpuOeSd |EA Loo | meal | Twanegeer TwoeRafmoem|| Roanwgesor max | (0 saongiay||oncunmt sm cps|onanepnts | Rabbit 617-1235 |- 31-1200 31-1200 NA [CR om dgolwesed | || Khaprgvereodr and TCR gTaREd | omy mms| eomts snes| a Na Na | ToORwnTsom|| ame | Tgaemeds de TmCeT| me nents| sotsp NA NA NA T Covmepou|nd:PaMnp5ge5ar6|| Raavmegnegset ma | osoanmiamrdss || ry courves [Rave [617-1235| 31-1200 wCeCaRfnoem|| Flaomiaedst ave [oRwsodm|| Rmoemwpesr ame | ldme pnts| ot mts fs | mms 60-1200 Nia CoCnaR dmetats NA Avschmen C: Sindand Caeusions ExacionEofrPsRsOcSofom Lier 34 Environmental Laboratory Page 30r14 2asest Laboratory Request Number use Ton Pair Standard Curves - Tissue S APrneapttdaaes: FESitqnauanlisdpamreodntnvuaamembnbednre:rTt:N: ToMentghoedsaeneavlsyion: . Blak ienfdentter FFFCC memiixmsstdd apappepprrreoanx.n.0S$5h3o00r0ppoom: Surrogace sd approx. 108por: AcFtFuOalSc|oPnROcS|eRnoOftsStaArn|daarEdtIsFnOtiShEoe|FFnCrmOsiSsAT FI AT wnt| gmt" | Soni | Sp | Sper | See|Srtee | amp [055010500 "0560 {003.05000JOS00T0500 | 0500 |0500 |0500 || oor 0.560 |0500|0500 | | ood | oie 0.167 [0350T05o 00To 0500|0o 50 |r 0500 |o 0300| ToooonteaOir6e7r| [300 jx 1500 | 500 S00 1 S00 1 S00 || 0001000010o1e6r7 ] [ C5S00055000 550000 | | 5000|1 550000|] 55000| | ] 0To.w0e3r0 To0.l1e6r7 Calculated concentrations of standards in the sample matrix Dome | ame hs | om | ame oan | Cnt EON Ro me e no | i260 | wo [3E95N 59 560 | 399 | 599 | 505 {| mateo [3%Tso2TJ s1 5% J 1 se se 1 SEE EEE T ws J eeY s TeVwJ wVsT || L ETrorn Cam--m-- [The Rabr bi TT5P.1R00O0 Sppb_|T31P0r0O0SpAg5| | 5P1R0O0S0AppAb |EWOSEOR | | 3-100 ppb | FORA 35-1000 pp PFOSEX 13-1000 pot ExsoafPRcOSfiomoLins 34 Environmental Laboratory ames Seles 3m Medical Department Study: T-6295.7 laboratorRyepRoerqtueNos.t NFuAmCbTerT-O0X7-307350 3M ENVIRONMENTAL LABORATORY MeTHOD ANALYSIS OFFLUPOORTOACSHSEIMUMICPAELRSFLINUOLIRVOEORCTEAXNTERSAUCLTFSOUNSAITNEGOR OTHER HPLC-ELECTROSPRAY/MASS SPECTROMETRY Method Number: ETS-87.0 Adoption Date: 0 [22.19% Author: Lisa Clemen, Glenn Langenburg. Approved By: a 7 1p Taboratsry Manager JAN Group Leader On 4 Cree Technical Reviewer Revision Date: Ni 2/5) Dae 3/14/39 Dae othifyy Dae Loscoreavpaerncamos 000000000 1.1 HSPcLoCp-ee:leTchtirsomspertahyo/dmaisssfosrptehcetraonmaeltyrsiys.ofliver extracts for fluorochemical surfactants using. 1.2 oAtphpelricioanbilzeabCloemcpoomupnodusnd:sFluorochemical surfactants or other fluorinated `compounds, or 1-3 Mreaptorritc.es: Rabbit, rat, bovine, monkey livoreotrhe,r tissues as designated in the validation Word 695 Ansyss ofLiveErrsE8x.a7c0t Using ESAS 3 Environmental Laboratory Page 1or10 sae 3m Medical Department Study: T-6295.7 laboratorRyepoRretqueNos.t NFuAmCbTerT-OUXz-207350 2.0 SUMMARY oF MeTHOD 2:1 pTHhePirLsfCo-mreemlteehdcotbdryodsmepsrocarnyi/biemsatstoshearsspaiienncangltyrlgsoeimsejtoornfyc,fhuaocrrarcsaticemhrieilsmatiriccsayolstfaseumrpfaaarcsttiaacpnuptlrsaorperxfitlarutaoecr.toecdThehfmeriocamanlal,liyvsseuricsuhssi2nsg dtaehnteaelpcyetzriefndlguudosariounoggchtt2aentrcasonandlsefomofnmattaheses(sPseFpleOeccStt)erdoamnpeiatornee,rnttmo/iozfnu.=rth49e9r.verAidfdyittihoenaildleyn,itsyamopfiacscommapyouben.d by 3.0 Deenvimions 3.1 siAnyttsmetroefsampcsehsae.lrliTochwePfsroeresivsnaucrlriuoeduesIobmnuitzeaatrietonnooth(fAliioPomn)ii:tzdeaTdthistoeon:MEbilycercutotrimloaisszpisrnagQyuvIaaortniriozouastIisootnruir(pcElSeeIsq)u,parAdotrbmueposo,slpaehnedric Ptreecshsnuirqeuecshoecmciucrasl altonaitzmaotsipohne(rAiPcclp)r,esTshuererm(oisc.prnaoyt,uentdc.erTahveaicounuimz)ation process in these 32 pTErlheeescssteurrcoeh,sapwrrghaeeydrldbornyoipzilaoetntissoiannre(sEoplSru,otdiEuoScnIe)ad:rebatymraetnhtsehfaoeprdprloeidfctiaootnitiohzneoatgfiaosanpsphiearrosfneogrvemilaeedcttirantiycaaculhmafoirsegpledh.derdirocplets, 3.3 TmMahasesssAsPSeIpleeQccuttiarvtoetmrdeoettIreycrt,oirpMslaeasqnsudaSadprceuocpltlorilsoeimomenatcseeslrl.s(pMeTSco)tn,rsoTamraeetnesdereliemscteMiqvauesilpsypdeSidpsecwcriittmrhiontmawetoteedqruba(ydMrmSua/pcMosSlte)o: cdaehntaderctgtheieosrnaetoiforraa(gnmm/eizon)ntasmnmadaysyubbebseesqeaulneeacnlttyelzdyeiddneittnehcetthefedir.sstecAqounasddirnqguulpeaodlMre,uSpfomrlaaeygmbeenteemdpilnoytheedcfoolrliisoinon cell, 3.4 tpCrroiopvbleeenqtiusiaoodrrntuahpologlovesn.a(lZpo-tssotptrh1ae9y9c8op)nreuoibalepezrietnutareer".fZa-cIsenp:rtahTyeh"cecoonlnvafetoensrttmiamotnoiadolenl.csoonTffhoMerimscaprtrioaomynaesimsiistQeusdaimtferrdoomIfa demilareiecnctttreloydneaa.ntctTehheaorcueognthheetashpeaermcteou.nrfei,Hgoauwrtaaetvrieoprna,sissZi-dnsigpffrteahryreonctuo,gmhtphaoentmeoerntttuhsoouadsnsdpoafcotophnewvraeanyttiiiononnt,ahleclcceooaumnnpitnoegnr,enatnds arc cnootmpcaotmipbalteibwliethwiotthheornZe-sapnrotahyesr,ysbtuetmso,nleytcw.)ith similar systems (i.e. Z-spray components are 3.5 MveartssriisopnlLse.yqnuAxaldlSrouvfpeotrlwseaiornsesy:satrSeeymsss.itmeiClmuarrs.roefntFtwolaryremModaresessidLgeyntenaidxlfshoerrseftehWreitsnopdetochiwefsimca9on5pueaarnladtsiWpoeincoinffdicotwhteossNethTQeu4a.t0tro Giunisdter)u.ment (Micromass Quattro I triple quadrupole MassLynxorMassLynx NT User's 41 Health and Safety Warnings: 41.1 Uemspelcoayustiaonvowlittahgethoefvaoplptraogxeicmaabtleelsyf5o0r0t0heVoplrtosb.e. When engaged, the probe Analysis of Live1r 5Ex3a7c0t Using ESAS 3M Environmental Laboratory Page 20110 Be 3m Medical Department Study: T-6295.7 laboratorRyepoRretqueNso.t NFuAmCbTerTOuXr-a0t3e0 41.2 aWnhdeCnlohtahnidnlging samples or solvents wear appropriate protective gloves, eyewear, 42 Cautions: 42.1 Oprpeesrsautree tehxeceseoldvsen4t00pubmarp,sthbeelHoPw11a0b0acwkilplriensistuiraeteoafu4t0o0mabtairc (sSh8u0t0dapsnin).y If the back 4.2.2 Do not run solvent pumps to dryness, 50 InTeRrEmeNces $1 To minimize intecferences when analyzing samples, Teflon shall not be used for sample storage or any part of instrumentation that comes in contact with the sample or extract. 60 Fournier _ 61 mEoqduiifpimcaetnitonlsistiendtbheelroawwmdaatya baes mmoedtihfoideddeivniaotridoenrs.to optimize the system. Document any 6.1.1 61.2 ccHMolPiemcLcprtIaroOromtOsampslresaonywtQ,uipoauantlnitsdzreaostsiuoIo1lonvstersanimuptlpreplcuqeeumrapdirnugposlyestMeams,ss Spectrometer equipped wi olvent degasser, column th an 7.0 Supeuies ano MATERIALS 71 Supplies 7.11 High purity gradeai regulated to approximately 100 ps (house air system) 7.1.2 iHnPtLheCraanwaldyattiscal column, specifics to be determined by the analyst and documented 7.13 Capped awovials or capped 15 ml centrifuge tubes 0 REAGENT AxD STANDARDS 81 Reagents 8.1.1 Methanol, HPLC grade or equivalent 8.1.2 svMeipnledloi1r-oQrTMewqauitvearle(nAt,STanMd tbyepeprIo,vaildl ewdatbeyr&usMeidliln QthiTsOmCetPhloods sshyosutledmabe AaTtShM 8.13 Ammonium acetate, reagentgradeor equivalent 813.1 When preparing diferent amounts than those listed, adjust accordingly. taemmmpoernaituurmescetate, Pour into a 2000 ml. solumeurc coments coring 8.1.3.2 2.0 mM ammoniumacetatesolution: `Weigh approximately 0.300 g. 2000 mL Milli-QTM water, mix until all solids are dissolved. Store at room Asis of Lvas E)xe Using ESAS 3M Eavironmental Laboratory Page 30ri0 a) 3m Medical Departmen: Study: T-6295.7 laboratorRyepoRretqueNso.t NFuAmCbTesT-OUXz-2073s0 82 Standards 82.1 pTryeppiacraeldlydutrwionmgetthheoedxtbrlaacntkiso,n wproocemdautrrei.x bRleafnekrs,10anEdTSe-i8gh.t6e.e0n matrix standards are 9.0 SaweLe HanbLING 9:1 aFrreesshtomraetdriinxcsatpapnedadradustoarveiaplrseoprarceadpwpietdh1c5amchiacneanltyrsiifsu.geExtturbaecstaendtsltaannadlayrsdiss.and samples 92 tIfeamnpaelryastiusrew,ilolrbreefdreilgaeyreatde,deaxttraapcptreodxsitmaantdealryd4saCn,d usnatmilplaensalmysaiysbceasntobreepderaftorrmoeodm. 10.0 Quatity ConTroL, 10.1 Method Blanks'and Matrix Blanks 10.1.1 Seoalcvhebnattbclhantoksd,etmeertmhionde bcloanntkasm,ianantdimoantorrixcabrlraynokvsera.re prepared and analyzed with 10.2 10.12 Analyze a method blank and amatrix blank Matrix Spikes prier to each calibration curve. 10.2.1 rMeactorviexryspeifkfeisciaernecyp.repared and analyzed to determine the matrix effect on the. 10.2.2 rMeactorviexrsypfiokreedaucphliacnaatleystea.re prepared and analyzed to measure the precision and the 10.23 mAnianliymzuemaomaf2trsipxiskpeiskpeearnbdatmcah,trix spike duplicate per forty samples. With a 10.2.4 rtMhaaentgiienxiotifsalptihckeaeliianbintrdiaatlmiaocntarlciiubxrrvasetp.iioknAedcdduuirpvtleii,ocnaatlescpoinkceenctornacteinotnrsatwiiolnlsfmalayi'n ftahle imnidt-heralnogwe. of 10.3 Continuing Calibration Checks 10.3.1 oCfontthiencuailnigbrcaatliiobnrcautrivoen.verifications are analyzed to verify the continued accuracy 10.3.2 Aonnealpyezrebaatcmhi.d-range calibration standard every tenth sample, with a minimum of 11.0 CALIBRATION AND STANDARDIZATION 111 wTAehniaeglhaytvzeeerdatgh1eexo,efxnttorwtaocftoesrdtcaemndadttarhridrxocusugtrahvnetdshaerwdoisrlilpgrbiineo,rpultsooittnaegnddMbufysolslliLonyewanirngroecrgarocethshsesireotnosuf(rytsa=ablmmepulseo=fetbxwyta,rraec.ts 11:2 sItfatnhdeacrudrcvuerdvoee(sIfnnoetcmeesseatryr)eqaunidrermeeanntaslypzeer.form routine maintenance of reextract the Analysis ofLiveTrSE.x8a.c70t Using ESIMS 3M Environmental Laboratory Page dof 0 Ps sm Medical Department Study: T-s295.7 LaboratorRyepoRretqueNsot. NFuamcbterzouxr-o033e0 11.3. uFosreputhreplooswesenodfoafcctuhreaccayliwbhraetnioqnuacnutrivteatriantghelrotwhalnevtehlesoffulalnraalyntgeeo,fitt hmeaystbaendnaercdescsuarrvye to cEaxlaimbrpaltei:onwchurevneactotnesmipsttiinngg otfotqhueanstiatnadtaeradpsprfooxmimatpeplby t1o0 1p0p0bopfbanaaltyhtee,hgaeyneeratefa rreagnrgeessoifotnhweeciugrhvtein(5gopfphbitgoh1c0o0n0cepnptbr)a.tiTohnisstawnidlalrdrse.duce inaccuracy attributed to linear 1122..10 PARcOquCiEsDiUtRiEonS.Se-tup 12.14 Set up he sample is 12.1.1.1 {Aestseirgonfathseamapllpehalbiestfsitlaetnianmgewuistihng MO-DAY-last digiot fyear-increasing 12.1.1.2 12.1.1.3 Assign Assign a method an HPLC (MS file) for acquiring program (Inlet file) 12:12 T1o2.1r.e1.t4eTaypmeetihnsoadmcpllicekdaenscrmieptthioonds ianthdevAiaclqupiossiittiioonn numbers contol panel then mass Spectrometer headings aRpeparcotpiroinatMeonmiatsosreisn.g).A and fSueltl select SIR (Single lon Recording) or MRM (Multiple slocnainzaistiuosnuaMloydecolalseacptperdotproinagte4atnhd mtaessSTtRo.49S9aovreother facrqaugimseinttiaotnimoentihnofdo.rmaIftiMoSn/mMaSybiencsotlrluemcetnetds.arRceefmeprl(o0yMeid,craodmdiatsisonMalopwrorduyct ion GUIDE TO DATA ACQUISITION for additional information and MRM. . 12.1.3mTaypircalsltyantdhaeradnsalytical batch run sequence begins and ends with a set of extracted 12:14 imaSnotacenmlriputledoeevrdserpsryossetsseiuancbnthlah,elsyaaznmepadllew.yitchSaoralrcvyoeonnvtteirbnlauainnndkgsrcesalhniobortuactoioonensiavdneearrliefydcsaestdaimoppneliernsjiebcoottdedassybiynbdeard 122 Using the Autosampler 12:20 Set up sample tray secocing 1 the sample st prepared in Section 12.1.1 12.22 Sianneastlt-yrusuptmtechnoetnsHliPod1ge1hr0os0na/kpapurtoopsraiamtpelefroaotptihemaflolrleoswpionngsec.onRdeictoiorndssouraatlccoonnddiittiioonnsstnhethe 12.221 Samplesize = 10 ul injetion 122.22 njcysampl=e 1 12.223 Cycle ime =9 minutes Arty of LiveersEaxreo Using E53 3M Environmental Laboratory ot 70 Pesaro Pages 3m Medical Department Study: T-6295.7 laboratorRyepoRretqueNso.t NFuAmCbTerTotx3-90330 12.2.4 Solvent ramp conditions T| ime om Ammon2i0ummMacetate . [Lomin SaSSmminn 177405 160% | | o0w5%| [se CT 7---- om 60% 12.2.2 Press the "Start" buton. 123 Instrument Setup 12.3.1 TTRorenifipezlraettQioouEnaTdSSro-uu9pr-co2el4,e."0M,faosr"sOmpoSerpreeactdtieroconamlae.ntderMFaiitnttedenwaintcheaonfAtthmeoMsipcherroimcasPsreQsusautrtero II 12.32 Check the solvent level in reservoirs and refl if necessary. 12.3.3 iuCnhhseeatctiipk.sftahTcehtoesrtyai,ipndlsiehssosausslsedeemlbbeclaeNpaittlhlewaiprtryhoabtneothajenadegngrdeedpolefadtcgheeest.hpro1sb1teah.ienlUetssipsefsascnoeeuycenappitiegcfeano. check 12.3.4Tum on thnitvogen. 12:35 Ohepateenrs.the tune page. Clicks on operate to inate source block snd desolvation 12.3.6 Open the Inlet Editor 1122.33..66..12 SSeett HthPeLfClopwutomp1t0-o "SO00n"ulmioas ppropriate 12.3.6.3 etOhxbepseetlrplveeoddfrwtoihptelhpenrtooscbieotmirionngoegnmoiluestatoksifntoghbeasretorpuvonefddtthhepiroboef.iheAfpirnoebme.isRteasthyouusltd be 12.3.6.4 Allow to equilibrate for spproximately 10 mints. 12.37 cThhaenginesitruomrednetr utoseosptthiemsiezepahreamreetseprosnasteth folowing stings. These settings may 12.3.7.1 123.7:2 DErSyIinnegbuglaiszi2n5g0-g4a0s01l0i-t1er5slhitoeursr hour 12.3.7.3 12.3:7.4 HPrPesLsCurceo<ns4t0a0ntbalro(wThmiosdpearfalmoewterraei HPLC is operating comely) 10 - 500 wLmin not se, it i guide o ensure the 12.375 Source block temperature 150 123.76 Desolation temperature 250 AlyseLiveerrEsxaurs0t Using ESAS 34 Environmental Laboratory Fase gario Pager 3m Medical Department Study: T-6295.7 laboratorRyepoRretqueNso.t NFuAmCbTerT-OUXz-207350 12.3.8 tPraipnetdtihnetotutnhee piangset,ruwmietnhttlsog,parameters, and store it in the study binder with a copy 123.9 eMCnlaidscsskLaoymnpnlxsetvanenursbmiubotentsro,nirinencfleturhdeteosAcaapqlplurisosaptmripioalnteesCMotonatsbresoLlaynPnaaxlnyeUzlseed(rth'issGmuaidye)v,aryEnasmuornegstart and 13.0 DATA ANALAYNDSCIALSCULATIONS 13.1 Calculations: 13.1.4 Calculate mateix spike percent recoveries using the following equation % Recovery = Observed REexspuelc-tteBdacReksgurlotund Result x 100 13.15 Calculate percent difference using the following equation: %Differenc=e ExpCoEnxeep,e-ctCcaeldcCutolnaet.eedCodne, x 100 13.1.6 Calculate actual concentrations in matrix (ug/g): (2 of PFOS cal(cI.nitfiralomWsetdi,ghCturovfe x Dillion Liver (0) Factor) x ug 1000 ng. Final Volume (mL) 14.0 METHOD PERFORMANCE. 14.1 aMmnaetdtrhiLoxOdsQpDeecvitafelicuct.eisoRneLfiemritto(EMTSD-L8)-6a.n0d,LAitmtiatochfmQeunatntBitafotrioanl(iLsiOnQg)ofarceurmreetnhtovda,liadnaatleytde,MDanLd 14.2 Solvent Blanks, Method Blanks and Matrix Blanks 142.1 sStoalnvdeanrtdbianntkhse,cmaleitbhroatdiobnlacnukrsve,.and matrix blanks must be below the lowest 143 Calibration Curves 14.3.1 The valuefor the calibration must be 0.980 or beter. 14.4 Matrix Spikes 145 C14o.n4t.i1nuMiantgriCxalspiibkreatpieornceVnetrirfeciocvaetriioens must be withia + 30%of the spiked concentration. 14.5.1 sCpoinkteidnucionngcecnatlriabtriaotni.on verification percent recoveries must be within = 30%of the 14:6 Iapfnearrlifytsoter.rmieaDdloiocsntuemtdheiennsttyhaseltlmeamecttahinoodndsspaienmrptflhoeersamprapenraconeparslieyacztteeidolnooragorbetohnoeorktacmteito,nmsaaisntdeenlaenmcmeinmeadybybethe Ansys ofLivEerTSE.8t7s0 Using SIMS 3M Environmental Laboratory Page Tarl0 Pager 3m Medical Department Study: T-625.7 LaboratorRyepoRretqueNso.t NFuAmCbTerT-O0X2-307350 14.7 Iffodoattnaotaerdeotno btaeblreespoarntdeddiwshceusnsepderifnotrhmeantecxetocrfittereiarhepvoer not been met, the data must be 50 15:1 PpSiOapLmeptLtlUeeTwIeaOxisNrtaPciRtsEwdVaiEsstNpeoTsIaeOnddNifnAlNbarDmomWkaeAbnSlegTlEsaosslMvAceonNntAtaiGsiEndeMirsEspNolsoTecadtiend hiingth hBlTaUborcaotnotrayi.ners, and gloos 60 Reconos 16.1. hEineatacedhgerrpaatoigroenhgamenendtehrwoardtie,tdesnfaomorpanletshtneuadpmyaeg,meu:esxtstrthauacdvtyeiootnrhdepartfoeoj,leldcoiwntiuinmogbneinrff,aocratmocraq(utiiisofinat3iiponlniecmuaedbdeiiedo).dei.atnhedr in he analyst 16:2 aPrpipnrtoptrhieatteunsetpuadgyef,olsdaerm.plCeolpsty, tahnedseacpqauigssitainond mtzeptehoindtofrtohemiMnasisrsuLmeynnt rtuonliongc.lude in the 16.3 sPtloortetihnetchaelisbtruadtyionfocluderrve by linea regression,; weighted 1, then print these graphs and 16.4. aPrnidntsodrateaiinntthegrsattiuodny sfuolmdmera.ry, integration method, and chromatograms from MassLynx 165 `SAucmtmaacrhimzeentdaAtafsoianrngesxuaimtapbllee osoffatwsaurmem(aErxyceslpr5e.a0d4s)heaentd. store in the study folder, refer (0 16.6 aBnadckloucpateiloenoctfronbiacckduatpaetloecatprpornoipcrdiaatte.medium. Record in study notebook the file name 2.0 17.1 TAAaBcLhESm.enDtIAAG:RAEMTSS,-8F.7L.O0WEDHaAtRaTsSu,mmAaNrDyVsApLreIaDdAsThIeeOtN DaTA 18.0 Reerrences 18.1 FCAoCmTp-oMu-n2d.s1,fr"oEmxtLriavcetriofnoorfAnPaoltyassissiUusmiPnegrfHlPuLorCo.oEclteacntcrsoaslpfroanyatMeaosrsOtShpeerctFrloumoerrorcyhemical 18.2 EToTnSi-z9a-t2i4o.n0/,Ma"sOspeSrpaetcitornomaentdeMraQiunatretnoanIceriopfltehequMaidcrrupoomlaessSyAsttmeomsep:heric Pressure 183 The validation report associated with this method is ETS-$-6.0& 7.0-V-1 1A 189.01 Ee CToSm-8p:oe 6u.n0,ds"c Efxrtoramcr Ltiivoenroe ofrPFoltaip sdsifuormD APnearlfylsuo iosroUoscitc nangeHsuPlw LfCo-nEalteew cotrroOste hperrayF/ln Muaorsoecht ermicas l Spectrometry" Analysis of LiveerrExsar io ESAIS 3M Environmental Laboratory Fase sar10 Ege . 3m Medical Department Study: T-6295.7 laboratorRyepoRretqueNso.t NFuAmCbTerTo0x3-30730 200 Revisions Revision J Revision AnofLiEyverrEsxi7ac0t Using ESS 34 Environmental Laboratory Page 90110 Paget" 3m Medical Department Study: T-6295.7 laboratorRyepoRretqueNsot. NFuAmCTbeTrO-Xz-307390 STueesy:Material MMeatihxoFdiRneavlisSioolnv:ers. AInnastlryuimceanlt ESqoufiwpamreenVteSrsyisotnem Number FRielSeqnuarmeed Valu: SDlaotmpeea:rofcEexpstctioniAnaiysr Duc ofAmlysiAnalst Laboratory Study # SGarmopuplieD:o5sTeL:akweTLonakrenoJ mfothme stthuedryafoyldefZroldeor TT] `DniCloiuntlcieonWnterF.aatc(it)oo:nr:(TnaTgka)ek:nenfTofarmkoemtnhterheosmsatyuhdeyfoMflodlaedesrr.sty negraon summary. Final Conc ug: Caeutdbydividing the nis volume from he cancentation AuachmenAt: Summary Spreadsheet Analysis of LEivrersEax7t0 Using ESAS 3M Environmental Laboratory Page 10010 2agas7TM 3m Medical Department study: T-6295.7 laboratorRyepRoerqtueNos.t NFuAmCbTerTO(X2-307350 3M ENVIRONMENTAL LABORATORY MeTHOD FLEUXOTRROACCHETMIIOCNOAF.LPCOOTMAPSOSUIUNMDSPEFRRFOLMUSOEROROUCMTFAONRESANUALLFYOSNIASTEUSOIRNGOTHHPELRC- ELECTROSPRAY/MASS SPECTROMETRY Method Number: ETS-8-4.1 Adoption Date: 03/01/99 Author: Lisa Clemen, Glenn Langenburg. Revision Date: 4/27/93 Approved By: 1) 11 Yo Laboratory Manager Vp ode fo Group Leader l Techa nicaf l Reviewer "fir /ss Da 4/24/99 Dae gDatu e e 0 Score AnD AppLiCATION L1 Sorcoopteh:er Tfhliuosrmocehtehmmoidcails fcoormtphoeuenxdtrsacftrioomn osfpmotuassium perfluorooctanesulfonate (PFOS) 12 Applicable compounds: Fluorochemical surfactantso other fucrinated compounds 13 Mthaetrviacleidsa:tiRoanbrbeipto,rtrat, bovine, `monkey, and human serum or other fluids as designated in Ward 495 3M Environmental Laboratory Exvacion oEfTPSRO.S from Serum 37 Page tof 14 PagesT 3m Medical Department Srudy: T-6295.7 LaboratorRyepoRretqueNso.t NFuAmCbTerT-OiX3-s0g3e0 20 SUMMARY OF METHOD 2.1 This method describes the procedure for extracting potassium perfluorooctanesulfonate p(aPiFrOinSg)roeragoetnhteranfldumoertohcyhle-mtiecratl-bsuutryflacettahnetrs(fVrIoBmE)s.eruImn,hoirs omtehtehrofdl,uisdse,veuns.ing an ion Mfl5u6or,ocahnedmiscuarlrsogwaetreesetxatnrdaactredd:(scPeF3O.0 SD,efPinFiOtSiAon,s)P.FOASnAAi,onEptaiFrOiSngE-rOeaHg,enPtFiOs SaEddAe,d to fmrtihelLetmesoroavfimenmptdeoltaeghnlaadannsopdslu,attuhttoeohnveatinnoaalflasiy.lnttieetrireoodngtephnarieorvuaigsphopr2aar3ttioctrciuopnnlteaidsltiirncyt.soyMrEiBanEgc.eh aetxTttharcaehcteMdiB(0rEeace0o.x2 ntrsajtcuutmticnsdylionn1.0 22 mTehtehsoedss.ample extracts are analyzed following method ETS-8-5.1 or otherappropriate 20 DesviTIons _ -- 3.1 PFOS: perfluorooctanesulfonate (anionofpotassium salt) C,F SO; 32 PFOSA: perfluorooctane sulfonylamide CyFSO,NH, 33 PFOSAA: perfluorooctane sulfonylamido (ethyl)acetateC.F, SO,N(CH,CH,)CH,CO; 34 CEtFF,O,SSEO-,ONH(:CH2(,NC-He,th)yClHpe,rCfHl,uo0rHooctane `sulfonamido)-ethy] alcohol 35 PFOSEA: perfluorooctane sulfonyl ethylamideC,F, SO,N(CH,C)H 3.6 M556: C,F,SO,N(H)(CH,COOH) 37 Surrogate standard: 1H-1H-2H-2H perfluorooctane sulfonic acid 4.0 WARNINGS AND CauTiONS 41 Health and safety warnings 4.1.1 hUasnedluinnivgearnsailmaplretciasustuei,onwsh,iecshpemcaiayllcyonltaabionraptaotrhyogceonatss., goggles, and gloves when i 5.1 There are no interferences known at this time. 60 61 Equipment The lollowing equipment is used -- while performing this method. Equivalent equipment is acceptable. 66..11..21 CVeonrttreixfumgiex,erM,isVtrWaRl ,10V0o0rtoerx[GEeCnie 2 6.1.3 Shaker, Eberbach or VWR. ExtscioEnoTfSFOS fom Serum 3 Environmental Laboratory % 77 Page 20f1s vases 3m Medical Department Study: T-s255.7 Report No. FACT Tox-030 laboratory Request Number-U2279 6.1.4 615 NBiatloangceen(e+v0a1p0o0ra), Organommaion 10 7 Sureuies avoMassie Cloves 72 73 NEalgpenepbootetrledsni,scpdosaabopepoairpfebhtotflledsieng 250 mL and | L 74 Volumetric flasks, glass, type A 7.5 I-CHEM vials, glass, 40 mL glass 7.6 Centrifuge tubes, polypropylene, 15 mL 7.7 Labels 77.98 710 SOyrxifnogreds,Dicsappeanbsleero--f3m.ea0s0ur1i0.n0gm5LK.L to Graduated pipes 0 pL. 77.1112 SSyyrriinnggees,ideissp,osnayblloenp,la0s2tiuc,m,3c2c5 mm 3 Timer 744 15 Crimp cap Crimpers utovaindlcasps Note: Prior to using glassware and bottles, rinse 3 times with `methanol and 3 times with 0 Reasgeepnatsr vAniDnSTANDARDS Milli-QTM water. Rinse syringes a minimum of9 times with methanol, 3 rinses from 3 8.1 8:2 ST5o2ydpiMeuImIhr-yeGadgreonxtwiagdtreaed(aeNnawdaOtmKe)ar,,ybMIeiTlpBlraio-eQvrTMioodrreeoedqyquuii3vvMaalleennLtt; alTl owaCteBrrausssedninetthiss method should 8834 TSeotdriaubmutcayrlbaomnmaotnei(uNm&,`hCyOd)r,og.eTn.suBlafkaetre(oTrBeAq)u,iKvaoldeantk or equivalent 85 Sodium bicarbonate (NaHCO), J.T. Baker or equivalent 8.6 Methyl-T-Butyl Ether, Omnisolv, glass distilled or HPLC grade 8.7 Methanol, Omnisolv, glass distilled or HPLCgrade 8589 FSleuraruomcohrembilocoadl,sftraonzednarfdrosmsupplier 8.9.1 89:2 PPFFOOSSA(3(M3MSpSepceiaclitaylyChCehmeimciaclalDiDviivsiisoonn,),momloelceuclualrarwewieghitgh= -534699 ExvacinerofsPRaOaSsom Sern Poe are 3M Environmental Laboratory 0% 7# Pags-70 3m Medical Department Study: T-6295.7 laboratorRyepRoretqueNos.t NFuAmCbTerT-OUX2-307330 89.3 8.9.4 PFOSAA (3M Specialty Chemical Division), molecular weight = 535 EWFOSE-OH (3M Specialty Chemical Division), molecular weight = 570 89.5 8.9.6 PFOSEA (3M Specialty Chemical Division), molecular weight = 527 MSS6 (3M Specialty Chemical Division), molecular weight = 557 8.9.7 CS.urFr,o,gSaOt,eHs)tamnodlaercdu:l4a-rHw,eipgerhftlu=o4ro2o8ctane sulfonic acid(1.H, 1-H, 2-H, 2.1 8.9.8 Other fluorochemicals, as appropriate 810 Reagent preparation NOTE: Wprheepanraptrieopna,riadnjgusltaragcecovrodlinugmleys. than listed in reagent, standard, or surrogate 8.10.1 d11i0s00sNo0lvsmeoLdd.ibueSmatkoheryredcrioonnxatiad1ienLi(nNNgaalO5gH0e0)n:emWLbeotitMlgieh.llaip-pQrTMoxwiamtaert,elmyi2x0u0ntgilNaalOHso.liPdosuarre.into a 8.10.2 MN| iNNlalsiOod-HiQusmwoalthueytrid.ornoSxntitodoree2(iN1na0aO0H1m)25:LmvDoLilluuNmtaeeltg1re0incNeflbNoatsatklOea.Hnd1:d1i0l.uteMetoasvuorleum1e0umsLingof 10 8103 O01f05TuMsBitAnegtiaantpbopuartoyx|liLamamvtmoeollunymie4ut4mritcohy5cdo4rnomtgaLeinnoifnsug1l0f5a0Nt0eNm(TaLBOAMH)i:l(lWW-heQiTMilgehawadaptdpeirrn.ogxAitdhmjeautlseatslty10m1tp6.i9logf MNialOlHi,-QaTMddwastlero.wlyStboerceauisnea t1heLpNHalcgheannegebsolaberuptly). Dilute to volume with 8103.1 nTeBeAderdequusiirnegs1acNheNcakOpHrisoorltuotieoanch use to ensure pH = 10, Adjust as 810.4 b0ai.pc2pa5rrbMooxnsiaotmdeait(ueNmlacyHaCr26.5bOgoo)nfaitnset/oosdaoiduIimuLmcavbroiblcuoamnreabttorenia(ctNef2l,bauCsfOkf,ea)rnda(nNbdai,n2C1g.O0t,oNgoavfoHlCsuOom,de)i:wuimtWheiMgilhli QTM water. Store ina | L Nalgene bottle. 811 Standards preparation 8.11.1 Prepare PFOS standards for the standard curve, 8.11.2 sPforuleouprtaoircoenheocmtoihnectraalifnlsiutnoagrnod1.c0ahpredmspiacmrael asPctFcaOenpdSat,radb1sl.,e02a(sfpoarppempxroaPpmFrpiOlaSet,Ae.o,n0Me.uw9lo5tr7ikcpiopnmmgposPntFeanOntdSaArAd, and 1.10 ppm E(FOSE-OH.) 8.11.3 tWheeiagchtuaalppwreoixgihmtately 100 mgof PFOS into a 100 mL volumetric flask and record 8.11.4 Bring to volume with methanol for a stock standard of approximately 1000 ppm (ng/mL). 8.11.5 aDpiplurtoexitmhaetsetloyck50soplpumt.ion with methanol for a working standard 1 solution of 8.11.6 aDpiplruotxe.wo5.r0kipnpgm.standard | with methanol for a working standard 2 solution of Extraction EofTSPF8O4S1from Serum 3M Environmental Laboratory Pagedoris eager 3m Medical Department Study: T-6295.7 laboratorRyepoRretqueNos.tNuFAmCbTerTo0x2-20735c 8.11.7 aDpiplruotex.w0o.r5k0inpgpms.tandard | with methanol for working standard 3 solution of 812 Surrogate stock standard preparation 8.12.1 `WCeiiFg,h)SaOp,pHrionxtiomaat5e0lmy L50-v6o0lummgeotrficsufrlrasokgaatnedsrteacnodradrdth1e-Ha,c1tuHa,l w2e.iHg,ht2,.H, 8.12.2 pBprimn.g to volume with methanolforasurrogate stockofapproximately 1000-1200 8123 sPtroecpkar10e.2 a s1u0rrmoLgatveolwuomrektirnigc sftlaasnkdaarndd. bTrrianngsftoervaoplpurmoexiwmiatthemleyt|hmanL!offosru1r,rogate `workingstandardof 100ppm. Record the actual volume transferred. S0sameeMaownse 0000000000000 91 92 All samples are Allow samples received frozen to thaw 10 room and must be temperature kept prior frozen until the to extraction. extraction is performed. 10.0 QuaiTy 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 Extract two 1.0 mL aliquots of Milli-QTM water following this procedure and use. 10.13 as method blanks. mEaxttrraicxtbtlwanoks1..0 SmeLe 1a1l.i1q4u,otsof the serum following tis procedure and use as 10.2 Matrix spikes 102.1 tPhreepaacrceuraancdyoanfatlhyezeexmtartarcitxiosnp.ike and matrix spike duplicate samples to determine 10.2:2 Prepare cach spike usingasample chosen by the analyst, usually the control `matrix received with each sample set. 10.2.3 cAEadxldpiiebtrciatotenidaolncocsnupcrievkene.tsramtaiyonbsewiilnlclfualdledinatnhde mmaidy-rfaalnlgien otfhethleowin-irtaianlgceoalfibtrhaetiionnitciualrve. 10.24 Prepare one mati spike and matrix spike minimum of2 matrix spikes per batch. duplicate per 40 samples, with a 103 Continuing calibration checks 103.1. Prepare continuing calibration check samples to ensure the accuracy of the initial calibration curve. 103.2 ePxreapmaprlee,,iatfaamsianmipmluems,eto=ne34c,onftoiunrucihnegcckhseacrke pperrepgarroeudpaonfd1e0xtsraamcptleeds.. For 103.3 tPhreepianrietaelaccuhrvceo.ntinuing calibration check from the same matrix used to prepare. ExracionoEf1PR0O4S1from Serum 3M Environmental Laboratory Page Sof is PageTT 3m Medical Department Study: T-6235.7 laboratorRyepoRretqueNso.t NFuAmCbTerT-OUXz-207350 10.3.4 Tctahhleeiibneriaxttpiiaelocntcaecludirbvcero.antcieoAnndtdrciauttriivoeon.naslTwhsipilliskfieassllnmewacieytshsibanertyihnieclmtuihddee-darnatahnlagytseotfafmltuihsnettqihnueiatlinaotlwi-areanugseiongf othnalny the low endofthe calibration curve (for example, 5 ppb - 100 ppb, rather 5 ppb - 1000 ppb). 11.0 CALIBRATION AND STANDARDIZATION ILI Prepare matrix calibration standards 11.11 Transfer | mLofserum to a 15 mL centrifuge tube. 11.1.2 mIvafotmlroiuxsm.tesRseaecmqupoalrledtvoeoatlchuhemsseaasmmapprlleeelvvesoosllutumhmeaesn.o1n.D0tomheLn,eoxtterexxattrcratacictotnstlseahsenesdtat,rhdasn w0i.t5h0 mmaLtrioxf 11.1.3 Wbehtiwleeepnraelpiaqruiontgs.a totalof twenty aliquots in.15 mL centrifuge tubes, mix or shake 11.1.4 TTatywptoihcea1elmnldyLuoasfleitthqhiuesotssste,actnoirdoano,rtdhteocrosanppcipekern,otiprranitaidtuopenlsviacoalntudem,sep,tiwksoienrsgvteaanamdsoaumrndattcrsuirxlviebsslte,adnkfison.rTatbolteal16,7 eighteen standards, two matrix blanks, and two method blanks. TLLS rRaenfgeerstoanvdaltihdeatLiionnearerpCoarltiEbTraSt-i8o-n4R.a0n&geE(TLS-C8R-)5.fo0r-Vc-al1i,brwahtiiocnhcluirsvtesst,he working 1116 sUtsaendAatrdtsa.chSmeeentSeDctaisonan13a.i0d calibration standards. itno ccaallccuullaatteinagcttuhaelccoonncceennttrraattiioonnssofotfhPeOwSorkining 11:2 Twoorekaicnhgssttaannddaarrdd, fbolrantkh,e ocronccoennttirnautiinogncthoefcakl,l waidtdhianpptrhoeprcialaitberaatmioounncturfvesruarnroggeat5eppb - 1000 ppb. 113 E10xtersatcatblsipsihkeeadcmhatinriitxialstcaunrdvaerdosnftohlelmowaisnsgs1p2e.c6t-r1o2m.et1e6ro.f this method. Use these standards Excraction EofTPSF8O4S1from Serum 3 Environmental Laboratory Page Gor 14 Pagers" Sn Medical Department study: T-5295.7 LaboratorRyeporretqueNos.t NFoACsTmeTroxn-o0a3s0 Approximate spiking Table amounts 1 for standards and spikes Woprkisng Fan Fo a Using 1.0 mLof matrix v SS30p0pmpm o 2o 00r 0C02p 5] ppm SSopopmm [T7o] Bo0LsT0pm 00pm [5 $0 pom ozopm3 | 5730pom $0 0m 5 Tom L0 opeoce0 pure 0000000 12.1 Veto Obtain frozen samples and allow to thaw at room temperature or in a lukewarm 122 pVortoexlmixeforo1m5asfeocognsdsi, nthen easter 1.0 mLo othe pproprst volume 0:3 15 ml. 123 Retum unused samples to fczer ae extraction amounts ha been removed, 112254 RLaebceolrdthtehewhineitwiailtvhotlhuemsetuodnytnhuemebxetrr,acstiaonmwpor1kDs,hdeaette snd anys nls. See attached 126 SwShopirinkdksahnledtetassafmdopriedeossc,ueimnecnl1 tuidninggtbhea:nrkesmaainndinsgnsdtaepnsd.s, ready or cxracion wth sumogate 12:7 SCopinintkiehntaciancsgheccmaoatnte,riafonwroinhthestachmaeilpbnrrgoasptroniactuervaemaonudnstnodfes.tanNdoarodpaescpduensscrmibeedhins1o1.1s, oosoTable 128 SVaomrptleexsmiToxr t1h5essetcaonnddasrd curve samples, mate sike samples, and continuing calibration 1122..190. SCThoeeaccatkcohtnosaemnbpsluier,eetadhde| mL0.5 M T0.B5AMrTeaBgeAntanisda2t pmHL 1o0f. If0no.t5,saoddjiusutmacccoomrdoinntgleyo.adiom 1122.1121 CUasipnegacahnsOaxmfpolred aDinsdpepnusteorn, atdhdshamkLermaetthysl-etetrrt-obifutny0]g0etrhemr., or 20minutes 12.13 -- Centrifuge for 20 to 25 minutes atasettoif3n50g0 rpm, or until layers are well Euracoefir7so0an4arhom Serum nar tores 3M Environmental Laboratory Paget 3m Medical Department Study: T-6295.7 laboratorRyepRoerqtueNsot.NuFAmCbTerTo0X3-30336 12.14 Label a fresh 15 mL centrifuge tube with the same information as in 12.5, 1122..1165 hPRoueutrmseo.avceh 4s.a0mpmlLeofonththee oarngaalyntiiccallayneirttroogtehnisecvlaepaonra1t5ormnLiclendtrryi,fuagpeprtuobxei.mately | 0.2 12.17 Add LO mLofmethanol to each centifuge ube using a graduated pipet 12.18 Vortex mix for 30 seconds. 12.22 Complete the extraction worksheet, atached to this document, and spe in he study 12.19 12.20 1221" Attach a 0.2 um nylon mesh syringe. Filter into a 1.5 mL filter glass to a 3 cc autovial syringe and transfer the or low-volume autovial sample to this. when necessary. Label the autovial with the study number, matrix, final solvent, extraction date, and animal number and gender, sample analysi(s) performing the extraction. timepoint, Cap and store extracts at room temperature or at approximately 4 C until analysis. notebook or include in study binder, asappropriate. 13.0 DATA AvaLysIS AND CALCULATIONS 131 Calculations 13.1.1 cCaallicburlaattieonacsttuaanldacrodnsceunstirnagtithonsfoofllPoRwOiSng,oequoatthieornapplicable flsorochemical, in nLof standard concentrationof standard (er - mL ofstandard + mLofsurrogate standard + initial matrix volume (mL) Final Concentration (g/mL) of PFOS in matrix 140MevwooPemeomwance 14.1 The method detection limit (MDL) is 0000000000000 analyte and matrix specific. Refer to MDL report 142 TfohresfpeoclilfoiwcinMgDqLualaintdy cliomnittooflqusaanmtpilteastairoene(xtLrOaQct)edvawliutehs e(asecch AbtattcahcohfmesnatmsplBesatnod C). 1ev4a.l2u1ateMetthehoqudalbiltaynokfstahnedemxattrraictxibolnaanknsd.analysis. 142.2 pMraetcriisxisopniokfetahnedexmtartarcitxiosnpike duplicate samples to determine accuracy and 14.2.3 Cinointitailncuailnigbrcaatliiobnractuirvoen.check samples to determine the continued accuracyof the 14.3 Refer to section 14 of ETS-8-5.1 for method performance criteria. 15.0 151 POLLUTION PREVENTION AND WASTE MANAGEMENT. ShiagmhplBeTwUasctonetiasindeirssp,osaendd iunsbeidoghlaazsasrpdipceotntteaiwnearsst, filadmismpaobsleeds1omlvbernotkewnasgtleasfss cdiosnpaomseedrsin located inthe laboratory. Exeacion oEfrPsOaStfrom Sear 3M Environmental Laboratory Page saris rages 3m Medical Department Study: T-6295.7 laboratorRyepoRretqueNso.t NFuAmCbTerT-OUX2-207330 16.0 Recoros 16.1 nCootmepbloeotkeotrheinecxlturdaectiinonthweo3r-krsihnegesttautdtyabcihneddert,o tahisapmpertophroida,tea.nd tape in the study 17.0 ATTACHMENTS 171 Attachment A, Extraction worksheet 17.2 AttachmenBt,MDL/LOQ values and summary 17.3 Attachment C, Calibration standard concentration worksheet 180 18.1 Reegmences Thevalidation feport a s s ociated w it h 00000 this method is ETS-8-4.0 0000 & 5.0-V-1. 18.2 HFPALCCT--EMl-e3c.t1r,os"pArnaaylyMsaissosfSpSeecrturmomoertrOyt"her Fluid Extracts for Fluorochemicals using 19.0 19.1 AFFECTED Documents ETS-8-5.1, "Analysis of Serum or Other Fluid see ExtractsforFluorochemicals using HPLC-Electrospray Mass Spectrometry" 20.0 Revisions RNeuvmisbieorn Reason For Revision ReDvaistieon 1 SSeeccttiioonn 1122.2113 CAhdadnegdetdhetoshiankcelurdsepeseadm.ple storage at room temperature. 04/02/09 Sleescstitohnan121..017mLF.inal volume is 1.0 mL; not adjusted for initia volumes ExtacionoEfTFOSS fiom Serum 3M Environmental Laboratory Pagedoris Eames 3m Medical Department Study: T-6295.7 LaboratorRyepoRretqueNos.t NFuAmCbTerT-O0X2-207350 Extraction Worksheet ETS 8.4.1 SMarT Soc {[Saiproege scl spppaomm||| aapcparFoox w0.E5ppomm|| psperFonnE. Rpppmm|| aspeprFnonO30Npopemm [Commer _ W_ D. DateSpikedAnsys:r | 2 2. '. r || = _ r r _ r r r r _ t E r r e r r SoA 1 r _ 1 ---- r ro r r I T r I SSS E T E ArS Sy 7 r . eS EX S E tr EE E S y -- -- r T r T r T r T r r FS r r RS,WSsWa ---- SS -- Vom [re [Esmee SloreoA s copg CS foSmcmhbentme movesls saws Sees Ere Cont. Cal Veriicatons sed same max for 2d on 3M EnAvsiarcohmnemnetnAtal Laboratory ExticionoEf1F5OS41from Serum Page 10013 PageTT 3m Medical Department Study: T-6295.7 LaboratorRyepoRretquewso.t NFAaCmTeTNo-00330 MDL/LOQ values for rabbit serum `Compound MDL LOQ|LinearCalibrationRange (LCR) 1 (ppb) | (ppb)| Approximate concentrations to be used for preparing the | [[FPOFSOS[A|T1751 5||54.759[3[Spstpoaobnbd--a1r10d00C00apl0pibb0ration Curve } [[E[ NF5O5SFE-OF1[61O310..6S46T11|2A3506727R|15s[opsroobbp-hTT1oo00o000n3pp5mo0y | [PtvMFeDerOLmsSiLnE0OeRdQG1.taeTr7lumioenc1escuq1suslbi5eonvncican2eceh.,omfRaetsnhpssyoptn,spersi8-hTcaoeormwsp,ipseebco,vuinpe,3ind rmbtnysa Fsm oswSo re eoer hbiedaenbi responses neton, the ML endLO will ie mom ee ete Sale Please se LOQ Summary and MDL say in ETS-840 & 50.1 fr fsther norton. M EnvAiarcohnmeenntaMlBLLLaObGoSrautmoryy Excion ePsOaSsfomSen I5 rgnsiorse -- 3n Medical Department Study: T-6295.7 laboratorRyepoRretqueNso.t NFuAmCbTerT-OUXz-207350 CRaobmbpiotuSnedr:umP|FOPrSoefpasraenddsarndsge|| [CcRurfvreom | %RRaesnogveery | RTanDge } pb) (ngiml)| (ppb) | | (ng/mL) [[s0iwworeTe n[oo m||omn [van]| RaCbobmiptouSnerdu:mP|FPOrSoefApsatraenddaarndgse|| LCcRurfvreom | %RoRaensgoveery | RSanDge BY) (gm)| Gb) (ng/mL) = [mLoowrCaurnve vn 493-976 won || mo] 985-147 Compound: PFOPSreApAaedrange| LCRfom | % Recover|y RSD Rabbit Serum| of standards curve. Range | Range (ppb) (ng/mL) (ppb) (ng/mL) ieee |r Higheve | 02.9% | onaom | sam | esis 3M EnvAitraocnhmmeennBtt:aMlDLILLaObQoSruartorryy ExracioEnoTfSPFdO.S01from Serum Page 20114 see 3m Medical Department Study: T-6295.7 laboratorRyepoRretqueNos.t NFuAmCbTerT-O(X2-207330 Compound: EFPOrSepEa-reOdHrange| LCR fiom | %Recovery| Rabbit Serum| ofstandards | curve Range RRaSngDe | pb) (ng/mL)| (ppt) | (ng/mL) | | Full Range | 0993-976 m0 | nas | Compound: PFPOrSeEpaAredrange| Rabbit Serum| ofsundards | LCcRurovem | %ReRsaongveery pd) (ng/mL)| (gp/pmb)L) |Full Range | 0953-976 10.1162 [ere | re| non| ew |ee] Compound: MssP6reparedrange|LCR fom | % Recover|y Rabbit Serum | of standards curve Rage | RRaSngDe | Gob) wml)| (nogo/tm)L) || [arene Ux | | wee owen |Tore | aan | ow| |omon[e|mamo s| tachmenBt:MDLALOQ Summary ExicionEoTfSP.O4S1fom Serum 3M Environmental Laboratory Page 130014 Paetl 3m Medical Department Study: T-6295.7 laboratorRyepoRretqueNso.t NFuAmCbTerT-oUx3-3c7. PAnraelpydtaet(es(:s): Ton Pair StandSatranddaCrudrnvuemsbe-r:Fluids Equipment number: Sample matrix: Methodrevision: BFilnaanlksMoulivde/nitdeanntdifTieNr:: TFaCrgmeitxansatldyatpepsr)o:x. 0.500 ppm: SFFuCCrrmmoiigxxattsetddstaadpppparrpoopxxr..ox55.0.0p.01p0p0mpp:mpm: ASPdcFctOounSse--concSPecFnoOtrnSaAetionsPSEoufOcsoStnAasAndarESdscWoOinnSteEhe FPSCiFdmOciSoxEneA SMueSosnc | AAmTi Fioiurvol 05m00L uT0g5m07l T05u3gm7l | osr [oEeC CEL sogerl C ugml L | pikedmt 1me LX 0CY 2 2 I A 0STTs SSa e ss er s T o $5o [ T oo on TT Aao eY [0o00 geeSoi ST y T os o TSTn o| e oaott a] e ra CFaiFlRccOooSunTleatwedFFcrnRoOnoScnKentraFtoFriRgaoOnnoSslnAoRef stFanngoEdeOalSgrEds iTFnmRtOaghSemEosRamprlaSegoSemSnantrixSSeaIerrrOe Amispiked aH n Isem T TE nT w ey en sumege|e1ATal | I h eo TS a e Te a y e Rane | im = LS T | es----i ) 5698 038 S798 iorT| Validated ranges - approximate concentrations FrVeerke,Bov&inpFe riem||EEsmmmtsesoorulyby UiUii avlieeeTffrorobers man me ns Cavsey Sooo] 500 00 | ARachment C: fon Par Standar Curves ExmacionoEfPTEGSS from Serum 3M Environmental Laboratory Page aor 16 pager 3m Medical Department Study: T-6295.7 laboratorRyepoRretqueNos.t NFuAmCbTerT-OUX2-30735c 3M ENVIRONMENTAL LABORATORY METHOD ANALYSISFQLFUPOORTOACSHSEIMUIMCPAELRSFILNUSOERROUOMCTEAXNTERSAUCLTFSOUNSAITNEGOR OTHER HPLC-ELECTROSPRAY/MASS SPECTROMETRY Method Number: ETS-8-5.1 Adoption Date: 03/01/99 Author: Lisa Clemen, Robert Wynne : Revision Date: 4/2/71 0g Approved By: E Laboratory Mana7ger1) e 0 e "/z2e, Dae Coote fh Group Leader Aad Chime Technical Reviewer S/26195 Date oyDaftaefpr 0 SCOPE AND APPLICATION 1.1 Scope: This method describes the analoyfsseriusm using HPLC-electrospray/mass`spectrometry. extracts for fluorochemical surfactants 1:2 Applicable Compounds: Fluorochemical surfactants or other fluorinated compounds, or other ionizable compounds. 1.3 tMhaetrvialciedsa:tiRoanbrbeipto,rtrat, bovine, monkey, and human serum, or other fluids as. designated in Word 695 Analysis ofSeruE1ms.Ex8t.r5a1ct Using ESIMS 3M Environmental Laboratory Page toro sages 3m Medical Department Study: T-6295.7 laboratorRyepoRretqueNso.t NFuACmTbeTrO-Xy-s02370 2Z .1 TtO hhiesrmfs eltuihdos,du udseisncgrm iHbPesLtChw eclaencatla ryosissproafm yf/lmuaosrsv oscpheec0 mtio rcoaml0 etsurr ryf,0aoct0aM sni0tmsi0elxatre 0rsayc0tset0dw emf0ro0mo s0er0umn 0or0 ffTalhdueiotraionocanhlaeylmsliiycs,ailss,apmsepurlcfehosrammseatdhyebbypeemraofnnlaiultoyrozoreoidcntguasnasieinsgnuglaflotenaaintodene(cmhPamFraOascSts)ersaipnseitciotcn,roommfeaztpeearr4tt9io9ca.fusulraatrphperropriate. the identity ofa compound by detecting daughter ionsofthe parent jon verity 33-.10 sADytesmtsoeismipsmhaeilrolinocswPfroresvsaurrioeuIsomneiztahtoidosno(fiAoPnYi)z:aTthie oenMbiycruotimlaiszisngQuvaartiro I e rple quadrupole tPierntceehsrnsfiuaqrcueesec.sheoTcmhcieucsraesl iaftnocnalituzmdaoetsibpouhnte(raAirPceclnp)roe,tslTsihumreiertm(eodisctp.or:anEoylt,eucenttcrd.oesrTparhaevyailcoounnuiiomzzua)ast.tiisooonnurp(crEeosSc,Des,psrAoitbnmeots.hseparhneedric 3:2 TEprhleeesscseturrceoh,sapwrrhgaeeydrledobrnyoipzflaoetntsisoainnre(sEoplSru,otdEiuoScnIe)ad:rabytmretahnteshfoaepdrprolefidci0aotniitozhneatogiafoasnspptherarosfneogrvemileedcttirantiycaactlhmaforisgipedhderdircoplets 3:3 dTMehatesecsAtoPSrIps.eQcutTaortnotsmreoatrIerfyst,erliMpelcaetsiqsvuealSdypreduicpstocrlroeimmseiytnsaettreemd(sMbSay)r,emaeTsqasunitpdopecemhdawMrigateshsrqatSuiapode(rcumtpzro)olmeaentmdearssus(bMsseSelq/eucMetSni)vte:ly sdepteeccitfeidc.frAagmsienngtlaetiMoSn imnafoyrmbaeteiomnployed for ion detection or a series (MS/MS) for more 34 fiCiroponlmveeaqnputaridoorbnueaplioslvoesr.stZyh-ossgtpoenrmaasly(tppoorstothbe1e9c9oi8nnt)eeuraifplaeirctzeue:reaT."hZeInslpatrthaeesyt"comncovodenenfltosiromoanftailMoicnco.rnofTmohramesasstpiQruoaanytiterm1oistIatIiemded medliaericentctrtoeldynea.antctTehheaorcueognthehetahspeaemrcetou.nrfei,Hgouawfrtaeetvrieporna,sis2i-dnsigpffrteahryreonctuo,gmhtphaoentmeoernttthusooudassnodpafctoohnpwveaerynattiiinoonnt,ahleleccaoonumniptnoegnr,enantds are cnootmpcaotmipbalteibwliethwistohmoenoethaenrotZh-esrp,rbauytsoynsltyemwsi,thets.i)milar systems (1c. Z-spray components are 3:5 Mv1eartssriispolnLesy.qnuAxaldlSrouvfpetorwlsaieorsnesy:satrSeeymsss.itmeiClmuarrs.roefntFtwolaryremModaresessiLdgeyntenadixlfshoasrscteWhteihsnepdemocaiwnfsuica9lo5pseaprneacdtiifWoiicnntodofotthwheessiNenTsQtu4ra.ut0mternot (Micromass Quatro II tiple quadrupole MassLym or MassLynx NT User's Guide) 4.0 WARNINGS AND CAUTIONS 41 Health and Safety Warnings: 11 eUmspelcoayustiaonvowlittahgetohfe vaoplptraogxeicmaabtleelsyf5o0r0t0heVporlosb.e. When engaged, the probe 1:2 aWnhdecnlohtahnidnlging samples or solvents wear appropriate protective gloves, eyewear, Analysis ofSerEumTExStract Using ESS Page 2079 3m Medical Department Study: T-6295.7 laboratorRyepoRretqueNos.t NFuAmCbTerT-OUX2-207350 42 Cautions: 4.21 Do not operate solvent pumps above capacityof 400 bar (5800 psi) back pressure. Ifthe back pressure exceeds 400 bar, the HP1100 will initiate automatic shutdown. 422 Do not run solvent pumps to dryness 50 InerrereNces 51 Tstoormaigneiomrizaenyinptaerrtfoefrienncsetsruwmheentnaatniaolnytzhiantgcsoammeplsesin,ctoenftlaocntshwiotuhldthneotsabmepulseeodrfeoxrtsraacmtple 60 Boutpment 6.1 Equipment listed below may be modified in order to optimize the system. `modifications inthe raw data as method deviations. Document any 6.1.1 Micromass Quattro II triple quadrupole Mass Spectrometer equipped with an 6.1.2 eHcloPem1cpt1ar0rot0smpleraonywt,ipouanlnisdzeatasiuootlnovsesnaotmuprplcueemrping system, solvent degasser, column 2.0 SUPPLIES AND MATERIALS. 7.1 Supplies 7.1.1 Hsyisgthemp)urity grade nitrogen gas regulated to approximately 100 psi (House air 7.1.2 iHnPtLheCraanwaldyatiac.al column, specifics to be determined by the analyst and documented 7.13 Capped autovials or capped 15 mL centrifuge tbes 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, and may be provided by aMilli-Q TOC Plus system or other vendor 81.3 Ammonium acetate, reagent grade or equivalent 82 Standards 8.2.1 Tpyrpeipcaarleldydtuwrionmgetthheoedxtbrlaacntkiso,ntprwoocemdautrrei.x bSleaenkEs,TSa-n8d.4e.i1ghteen matrix standards are 9.0 SAMPLE HANDLING _ 9.1 aFrreesshtomraetdriinx csatpapndeadrdaustaorveiaplrseporarceadpwpietdh1c5acmhLancaelnytsirsi.fugEextturbaecsteudnsltaanndaalrydssisand samples Anlyis ofSerEuTmsEexsa1r sing ESS 3M Environmental Laboratory Page 309 raged 3m Medical Department Study: T-5295.7 laboratorRyepoRresqueNso.t NFuAmCbTerTOXv-i0a3g0e 9.2 Ifaapnparolxyismiastweillyl 4beCd,eloayaetd,roexotmratcetmepdersattaunrdea,rdusnaindasnalmyp&liescacnanbbeepreesfrfiogemrcated at 100 QuaLITY Control 10.1 Solvent Blanks, Method Blanks and Matrix Blanks 10.1.1Seoxlcvhenbatibclhantkosd,etmeertmhionde bcloanntkasmiannadtimoantorixcbalramnykosvearre prepared and analyzed with 10.1.2 Analyze a method blank and amatrix 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 10.2.2 rMeactorviexrsy pefkficdiuepnlciyc.ates are prepared and analyzed to measure the precision and he 10.2.3 recovery Analyze for each a maiix analyte. spike and matrix spike duplicate per forty samples, wih a 10.2.4 tM`hmaeitinniiitmiusapmloickfaeli2abnrsdaptimikoeanstcpiuerrvsepb.aitkcAehd.dduiptliiocnaatlespcoinkceenctornacteinotnsatwiiolnsfmlaiyntfahlle imnidt-eraJnogne.of rangeof the intial calibration curve. 103 Continuing Calibration Verifications 10.3.1 oCfonttheincuailnbgractaliiobnrcautrivoen.verifications sre analyzed to verify the continued accuracy 10.3.2 Aonfaolnytzpeear bmaitdc-hrange calibration standard after every tenth sample, with a minimum LC1O1.1aAnualymze tmhe eaxtramctedomatxrixastansdardos prSiortto aand fsolloowingseacehseotofzextaractsm. Tohe x average oftwo standard curves will be plotted by linear regression (y =my +b), weighted 11:2 Is1tf/xat,nhdenaocrtudrfcvouerrcdveoede(tshfnrnooetucgmehsesezaetrryor),eqauusniidrnecgmeaMnantssd,slLpeyernfxororm other su routine itable software, maintenance of ret ract he 11.3 uEFsxoearmtpphulerep:oowsewsehneodfnaoacftctuehremapcciaylniwgbhrtaeotniqoqunuaacnnuttriivtaeactriaantpghpelrroohxwiamlneavtteehllesoyffl1al0nrapalpynbtgeeo,foafitntmahiaeyytia,bnesdonaenncdoerssseuamryeto calibration curve consistingof the standards from 5 ppb to 100 ppb rather than the full rreagnrgeesosfiotnheweciugrhvtein(5gopfphbitgoh1c0o0n0cepnptbr)a.tTiohnisstwaindlalrdrse.duce inaccuracy atiributed to linear Ansys of ScEumsEaxe Cio ESS 34 Environmental Laboratory Page sor Pagesr am Medical Department Study: T-6295.7 LaboratorRyepoRretqueNso.t NFoamcbterTooxi-s030 122.10 PAcRqOuCiEsiDtUiRoEnSSetup 12.1.1 C2lfiiclkenoanmestaurstibnugtMtoOn-DinAYth-elAacsqtuidsiigtiitoofnyCeoanrt-rsolamPpalneel.nuSmebteurp, a sample list Assign assign a method (MS) for acquiring, and type in sample descriptions. 12.1.2 TS10Io.R4c9r(9eSaiotnregloaetmh[erotnhpRpoerdcoocprlridciiknaglo)nmoasrscsaMenRsb.Mut.AtofnuSelitln IctohaneinzAac1tqiuwoiansliMtloiyodneScaoosntcarpopalropnparnicgaltaewnadonndsemleacsts SIRs. Save `product ion acquisition method. If MS/MS fragmentation information may instruments be collected. are employed, additional SeeMicromass MMaRssMLy(nVxulGpUlIeDREeaTcOtioDnAMToAnitAoCrQinUgI)SITION for additional information and 12.13 TstayndpardistachnedaaneanlldysltiwciyatlhbetaoufetnasescqueedhncembaegkinSswaindtahvset ofextracted matrix 12.1.4 Samples sample. are anal Solvent ybzleandkwsitshhoaulcdonbteinaunianlgyzceadlipberratiioodniccahlelycktoinmjoencittedo after every r possible a tenth nalyte 122 Using tchaerAryuotvoesraamnpdlaerre not considered samples but may be included as such. 12:21 12:22 SSsneeattl-uyupspttschaoemnpsHliPdeear1s0y0apapacrusotoporrstiaiamntpgeleofrtoahtotephtsieammafplollreleosswpitonngpserc.eopRnaecdceoirtidniSosoecncautastilocncoo1nn2dd.1it.io1onnsshe the insirument logbook 1122.22.22.21 Snjaemcpulseasmipzlee==101 LL injection 1122.22..22.43 CSoylcvleenttiamem=p -13.5 minutes Time MeOH [oT0S0Ommiin]n[ww50% Ammo2n.i0ummMcette [0oo%m (20Ommiin[ 90%a || o19 0o% n]| 123 Instrum1e2n.2t.2S.e5tPurpess the "Start" button. 1122.:33.21 CRehfeecrkttohEeTsSo-l9v-en2t4.l0eveflorinmoerservdeotarisls.and refi necessary Ary of SrEamreExsp:os Using ES 3M Environmental Laboratory - ge sof rad 3m Medical Department Study: T-6295.7 laboratorRyepoRretqueNsot. NFuAmCbTerT-OUXz-2073s0 12.3.3 tCuhnheseacttiikp.sftahTcehtosertyat,iipndlsiehssosausslstdeeemlbbelcaefplitatlhlewaiprtyrhoabtneotahjenadgegnrededpolefadctgeehset.hperoLsbteta.hineltUeisspseissainefeoelyucenapdpiitelolcaebreyt.o check 12.3.4 OSebtseHrPvLeCdroppulmetpstcoo"mOinn"g. oSuettotfhtehfeltoiwp approximately 10 minutes. otof1t0he- p5r0ob0e.uL/Amlilnowor1a0seqaupiplriobprraitaetef.or 12.3.5 aTruomunodttthtehetinpiotfrtogheen.prAobefi.neRmeiasdtjusshtoutlhed tbiepoefxptehlelepdrowbiethifnnoonmiitsrtogiesnolbesearkviendg. 12.3.6 cThhaenginestinruomrednetrutsoeosptthiemsiezepatrhaemreetseprosnsaet:the following setings. These settings may 1236.1 Drying gas 250-400 liters/hour 12.362 ESI nebulizing gas 10-15litershour 12.3.6.3 HPLC constant flow mode, flow rate 10 - 500 wLimin 12.3.6.4 PHrPeLssCuries <op4e0r0atbianrg(cTohmiesctplayr,a)meter is not set, it is a guide to ensure the. 12.3.7 Cfuarrtehferu.llCyognuniedcettthheepvroolbteagientcoatbhleesopteontihneg.proIbnes.ert probe until it wil not go any 12.3.8 tPraipnetdtihnetottnhee piangset,ruwmietnhtiltosg.parameters, and store it in the study binder with a copy 12.3.9 tUhseinangatlhyesicsroofsbsi-oflloogwiccoaulnmtaetrreilceecst.rode in the ES/MS source is recommended for 12.3.10bCMulatitsocnsk.L"oynnExsntsavruetrresbiusottnaosr,nt asienncdtahepenpdArcosqpaurimispaitlteeiMonanusmCsboLenyrinoixlncUPlaSundEeeRls'a(SltlhiGssUamImDapElye)s.vatroyPrbaeesmasonntahgleyzsteadrt 3.0 DATA ANALYSIS AND CALCULATIONS 13.1 Calculations: 13.14 Calculate matrix spike percent recoveries using the following equation: % Recovery = Observed Result = und Result x 100 Expected Result 13.1.5 Calculate percent difference using the following equation: %Differenc=e EC xpea cteldCocEnxepu .e-ctled a Conct, e Condc. x 100 13.1.6 Calculate actual concentrationofPFOS, or other fluorochemical, in matrix (ng/mL): (08 of PFOS calc, from (niial Volumeofmarx std. (mL) Curve x + mLof Dilution Factor) x Surrogate Standard) lug 1000 ng Final Volume (mL) Analysis of SEerTumS8Ex5tr1act Using ESMS 3M Environmental Laboratory < Page 609 rogers am Medical Department Study: T-6295.7 laboratorRyepoRretqueNso.t MEeAmCbTerTo0x3-9033s0 140 MeTuop PeRroRvance 14.1 mMaettrhioxdspDeectiefcictioPnleLaismeitsc(eMEDTLS)-5a.n4d.1L,imAitotafchQmuaennttitBa,tioorn (LaOtQi)ngaroefmseotmhoedn, wc analyte, and MDL and LOQ values, 14.2 Solvent Blanks, Method Blanks, and Matrix Blanks 14.2.1 Sololwveesnttsbtlaanndkasr,d minethheodcablliabnraktsi,onanvd meatrix blanks values are must be below the 143 Calibration Curves 144 M14a.t3r.i1xTShpeikesva_lue for the calibration curve must be 0.980 of better. 14.4.1 Matrixspikepercent recoveriesaremustbewithin 30% ofthe spiked 145 ContinucionngceCnatlriabtiroant.ion Verifications 14.5.1 cCoonncteinnturiantgiocanlibration verification percent recoveries must be + 30%of thespiked 14.6 analyst. Documental aeiiontnne sppropre logbook pIfecrrfioterrmieadliosntetdhienstyhsitsemmeatnhdodsapmeprlfeosrmraenacnealsyezcetdioonr iostnh'termeactt,imoansinatsedneatnecremimnaeyd be by the 14.7 1f6odoattnaotaedro0n abebireespoarntdeddiwshceusnsepderifnohrmeantcxeteoifehreahepaovre.not been me, the daa must be 15.0 15.1 PSPiOapLmepLtlUeeTwIeaOxtNsrtaPctiRsEwdVaisEstNpeoTsIneOddNifnAlNbarDmomkWaeAbnSlelTaEssosMlvaceonntnatiGasiednvieserpNoosTcesdiemnd hrighheBbTUocroonanimers, and gins l0 6160.1ReEac0 cohrppags0 e ~ gener0 a~ ted fo0 ra study0 must h0 ave the0 Followi0 ng infor0 mation0 include0 d ether0 im he header or hand written on the page: study or project number, acquisition method, anaiya integration method, sample name, extraction date, dilution facto(r if applicable), and 16:2 Pintth tune page, sample ist, and acquisition method from MassLyn to include in the appropriate study folder. Copy these pages and tape into the instrument runlog. 16.3 Pstloortetihne tchaelisburadtyiofnocludrevre by linear regression, `weighted 1/x, then print these graphs and 16.4 Print data integration and store in the study summary, folder. integration method, and chromatograms, from MassLynx, Ary ofSeEmrEasva Using ESS 3M Environmental Laboratory Page 0r0 rages 3m Medical Department Study: T-6295.7 laboratorRyepoRretqueNso.t NFuAmCbTerT-OUXz-207330 165 ASutmtmaacrhimzeentdaAtfaoursainngesuixtablaeosofmfatwpsaurmlm(aEerxycelsp5r.e0a)dsahnedetstore in the study folder, se 16.6 aBnadckloucpateiloencotfrobnaicckduaptaetloecatprpornoipcridaattea medium. Record instudy 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 Rererences 18.1 FcAoCmTp-oMu-n4d.s1f,ro"mExSterarcutmiofnoorfAnPaoltyassissiuUmsiPnegrfHlPuLorCo-oEclteacntersuolsfpornaayt/eMoarssOtShpeercFtlruoomreotcrhyermical 18.2 TEoTnSi-z9a-t2i4o.n0/,Ma"sOspeSrpaetcitornomaentdeMraQiunattetnraonIcIetorfiptlehequMaidcrruopomlaessSyAsttmeomssp"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 ECToSm-8p-o4u.n1,ds"EfxrtoramcStieornuomfPfoortaAnsasliyusmisPeUrsfilnugorHoPoLctCa-nEelseucltfroonastperaoyr/OMtahsesrSFpleucotrroocmheetmriyc"al woRevsiovs ReNvuimsbeiro,n ReasonFor Revision 1 SSeeccttiioonn 61.11..12AvClearraigfiecoaftitownoofcuHrPv1e1s,00nsotysstteanmdacrodmvpaolnueens,tsa.re used for pSleoctttiionng 1li2n.e2a.r2.r4egCrlaersisfiiocnatainodnoafdsdoeldvethnet 1r/axmpw.eighting of the curve. Section 17.1 Changed from attachment B to A. ReDvaitsieon 04/02/99 Analysis ofSerEuTmSE8xtsra1ct Using ESMS 3M Environmental Laboratory 47 Pages of9 Pagses 3m Medical Department Study: T-6295.7 laboratorRyepoRretqueNso.t NFuAmCbTerT00x2-207350 Laboratory Study # Tsnedxyh.aeral: MMeatvrodiRnevaiiSoonl:vent: + InAsntlryuimecnatESqaufivpamreenVtcSsyitone:m Number: FRielSeqnuanmreed Vale FSplooe -- DDaatteooffEAxntsyasciisoannAalryasleys FCrolup| iSamplleh| Canceemliaoln AVAL Fiar Cone SGorpoeurpDToaskee:n fTaokmennfeormghseisoynfqer.o SCToainmcplelnetsVr:oaltTuiamokene((nmsfLymo:m):TckeeTsanukefdonrfrolodhmeerhseuMdafslyre negation srry. DFiinlaltoCnonFa.ct(ors:pmTLa)kenaflomlhte sbyydivfiodlidnegr he nisl volume fom the conccasion AvschineAn:SummarySpreadshestAnotfSrsaEmrsEaxsic Using ESMS 3M Environmental Laboratory 8 9g Page 901s Cd 3m Medical Department Study: T-6295.7 laboratorRyepoRretqueNso.t NFuAmCbTerT-OUXz-2073s0 = L 3M EN NVIV RONMI ENTR ALLAO BORN AATTOOM RRYY E~N ~T ~ AL MeTHOD FLEUXOTRROACCTHIEOMNI-COAFLPCOOTMAPSOSIUUNMDSPEFRRFOLMUSOREOROUCMTFAONRESAUNLALFYOSNIASTEUSOIRNGOTHHPELRC- ELECTROSPRAY/MASS SPECTROMETRY Method Number: ETS-84.0 Adoption Date: 3/,/}) Author: Lisa Clemen, Glenn Langenburg Approved By: J > 7/3 Laboratory Manager' Yidow Hn Grdup Leader Yur Technical Reviewer Revision Date: 3/4/54 Date 3/1/99 Date ill Date 10 scoreawpApeyicamon 0000000000000 L1 oSrcoopteh:er Tfhliusormoechtehmoidcails fcoormtphoeuenxtdrsacftrioomn osfe|rpuomt.assium perfluorooctanesulfonate (PFOS) 12 Applicable compounds: Fluorochemical surfactants or other fluorinated compounds. 13 tMhaetrviacliedsa:tioRnabrbeipto,rtra, bovine, monkey, and human serum or other fluids as designated in Word 97 $8.1 ExtacioEnTofSP8R.O4S0from Serum 3 Environmental Laboratory ay Page or13 pages 5m Medical Department study: T-6295.7 LaboratorRyepoRretqueNso.t NFaamcbterT-oUxs-50s3s0 2.0 SUMMARY OF METHOD. 2.1 FThOisSm)eothoodthdeerscurcirbeoschtehemipcraocledsuurreffaoctraenxttrfarcotminegrpuortsaswsiinugm npeorfnluopraoroicntganscesnalsfoornaatned mase5xnti.taa0trlmnoaydgcLtaeteroenddof:e(nvsPmacpeFeptiOoh3rrSy.al,0st-DotPepreFfraiatOnn-StitbAtultit,odoyrnlPyns.)Fe.tOihESneAaroAcn(AhMM,iBetoxBnEEEttp.)raF.aiOcrTSihiInEneg-rtOMerhicIeHsaoS,gnEmesePniettFnhxuOiotasSdeca,EddtAsnd,eeivsd1Me.o5nt0ro5fmm6tlioh,uveooaersfndoadecmsahnpuceilrdmenripoceagaolnla,dstoetmwhheyoerea afiulotveiraedlst.hrough a 3 cc plastic syringe attached to.a 0.2 um nylon filter into glass 3.0 DEFINITIONS 3.1 PFOS: perfluorooctanesulfonate (anion of potassium salt) CsF1nSOy 32 PFOSA: perfluorooctane sulfonylamide CyFy7SO;NH; 34. EFOSE-OH: 20%thylperfuoroctan sulfonamido-ehyl alcohol 3.3 PFOSAA: perflucrooctane sulfonylamido (thyl)acetateCaF17SO:N(CH:CH3)CH;CO CsF17SO;N(CH,CH;)CH:CH,OH 3.5 PFOSEA: perfluorooctane sulfonyl ethylamide CyF ,SON(CH:CHy)H 33.76 SMu5m5o6g:atCeyFst1a;ndSaOrdN:(H1)H(-C1HH,.C2HO.O2H8)peflucrooctane sulfonic acid 40 41 WaHreanlethsaandnnsaCfaetvyriwoanrnsings 4111 hUasneduinnigvearsnailmraeicstsiuo,nsw,hiscphecmiaayllcyolnaabonraptuorhyogceonss, goggles, and gloves when 50 InreRreRexces 5.1 There are no interferences known at this time. 60 Eouevex 6.1 Tachceepfoilalbolwing equipment is used while performing this method. Equivalent equipment is 61.1 6.12 CVeonrtiefxugmei,xerM,isViWalR,10Vo0rot0rexEGCenie 2 6:13 Shaker, Ekerbach or VR 6.14 615 Nirogenevaporator, Balance (01008) Organormation Suvscoen orfRsOaS0fom Serum 3M Environmental Laboratory Sw [ Pagser 3m Medical Department Study: T-6295.7 laboratorRyepoRretqueNos.t NFuAmCbTerT-O0X2-207390 7.0 SUPPLIES AND MATERIALS 71 Gloves 72 Eppoerdinspodsabolepirpetftes 7.3 Nalgene bottles, capable of holding 250mL and 1 L 74 Volumetric flasks, glass, type A 7.5 L-CHEM vials, glass, 40 mL glass 7.6 Centrifuge tubes, polypropylene, 1S mL 77 Labels 7.8 Oxford Dispense--r 3.010 10.0 ml 79 Syringes, capable of measuring 5 pL to SO UL 7.10 Graduated pipettes 7.41 Syringes, disposable plastic,3 cc: 7.12 Syringe fikers, nylon, 0.2 um, 25 mm 713 Timer 7.14 Crimp cap autovials and caps 7.5 Crimpers Note: PrMiiolrlti0-uQsTMinwgatgerl.assRwianrseeasnydribnogtetlsesa,miinnsiem3umtimoefs9 with methanol and 3 times with times with methanol, 3 rinses from 3 separate vials, 8.0 REAGENTS AND STANDARDS 81 TbeyMpielliL-rQe"agentwagtreardeanwdatemra,yMbielplr-oQvTMidoerdebqyuiavaMlielnlti;-QallTwOaCtePrluusTMsd isnysthtiesmmethod should 82 Sodium hydroxide (NaOH), J.T Baker or equivalent 83 Tetrabutylammonium hydrogen sulfate(TBA), Kodak or equivalent 84 Sodium carbonate (Na:COy), 1.T. Baorkequievalrent 8.5 Sodium bicarbonate (NaHCOy), J.T. Baker or equivalent 86 Methyl-T-Butyl Ether, Omnisolv, glass distilled or HPLC grade 87 Methanol, Omnisolv, 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 Specialy Chemical Division), molecular weight = 499 8.93 PFOSAA (3M Specialty Chemical Division), molecular weight = S85 8.9.4 EWFOSE-OH (3M Specialty Chemical Division), molecular weight = 570 Excacion EofTPSF8O4S0from Serum 3M Environmental Laboratory xR /0/ Page af 13 Page 3m Medical Department Study: T-6295.7 laboratorRyepoRretqueNos.t NFuAmCbTerT-OUXz-207330 8.9.5 PFOSEA (3M Specialty Chemical Division), molecular weight = 527 8.9.6 MS56 (3M Specialty Chemical Division), molecular weight = 557 89.7 CSuarFr1o:gSaOt3eHs)tamnodlaercd:ul4a-rH,wepiegrhftlu=or4o2o8ctane sulfonic acid (1-H,1-H, 2-H, 2-H 8.9.8 Other fluorochemicals, as appropriate 810 Reagent preparation 810.11d1i00sNs0o0lvsmeodLd.ibueSmatkoheryredcrioonnxatiadIienLi(nNNgaalOSgH0e)0n:emWLbeoiMliegl.hlia-pQp"rTMoxwiamtaetre,lmyi2x00untgilNaalOl Hs.oliPdosuarreno a 8.102 N1 NNasoOdHiusmolhuytidornoxiindtoea(N1a0O0H)m:L vDoilluumteetr1i0cNflNaaskOaHnd1:d1i0l.uteMetoasvuorleum1e0 Milli-QM water. Store in a 125mL Nalgene bole. umsLingof 10 8103 o0f.5TMBAteitnrtoabaut|yLlavmomlounmeiturmichycdornotgaeinnisnuglf5a0t0e m(TLBAM)i:lliW-eQi"gh waaptperro.xAimdajtueslyto1p69H N1a0OuHsi.ngadadppsrlooxwilmyatbeelcyau4s4e1t0he54pHmLchoafng1e0sNabNruaptOlHy).(WhDiillueteadtdoivnogltuhmeelawsittmhi. of Milli-QTM water. Store ina | L Nalgene bottle. 8.10.31 TneBeAderdequusiirnegs|aNchNecakOpHrisoorluttoicona.ch use to ensure pH = 10. Adjust as 8.10.4 0a.p2p5roMximsaotdeiluym2c6a.r5bgonoaftseo/sdoiduimucmabribcoanrabtoena(tNeabCuOff,e)r a(nNda:2C1.O0YgNaoHfCsOoyd)i:umWeigh QbicTMarwbaotneart.e S(tNoareHCinOa)|iLntoNaalgI eLnevoblourtmlee.tric flask and bring to volume with Mili- 811 Standards preparation 8.11.1 Prepare PFOS standards for the standard curve 8.11.2 PMsoruleouprtaoircoehneocmtohinectraalifnlsiutnoagrnod1ca.hr0ed0msipcaparmel asPctFcaOenpdSat,radb1sl.,e0a2(sfpoarppempxroaPpmFrpiOlaeSt,eA..on0Me.u9lw8to7irckpoipmnmpgoPsntFeanOntSdaArAd. and 1.10 ppm EFOSE-OH.) 8.11.3 tWheeiagchtuaaplpwreoixgihmt.ately 100 mg of PFOS intao 100 ml volumetric flask and record 8.11.4 Bring (0 volume with methanol for a stock standard of approximately 1000 ppm (ug/ml). 8.11.5 Daiplpurtoexitmhaetsetloyck50soplpumt.ion with methanol for a working standard | solution of 8.116 aDpiplruotxe.wo5.r0kipnpgm.standard | with methanol for a working standard 2 solution of 8.11.7 aDpiplruotxe.wo0r.5k0inpgpms.tandard | with methanol for a working standard 3 solution of ExiscioEnTofSP8R.O4S0from Sra 3M Environmental Laboratory * 102 Pa or1s ome 3m Medical Department Study: T-6285.7 laboratorRyepoRretqueNso.t NFuAmCbTe:T-oUx3-207350 812 Surrogate stock standard preparation 8.12.1 WCoeFi1gxhSaOp:pHroixnitmoaate5l0ym5l0-v6ol0ummegtroifcsfulrarsokgaatnedsrteacnodradrdth1e-aHc,tua1l-H,w2e-igHh,t,2-H, 8.12.2 Bring to volume with methanol foar surrogate stock of approximately 1000-1200 ppm. 8.123 sPtroecpkar0e.a s1u0rrmolgavtoeluwmoertkriincgastsakndaarndd. brTirnagnstfoevroalpupmreoxiimhatemleyth|amnoiloffosru3rrogate `working standard of 100 ppm. Record the actual volume transferred, 100 QuaLITy Control, 9.0 SAMPLE HANDLING -- --_-- 9.1 All samples arereccived frozen and must be kept frozen until the extraction is performed. 10.1 Method blanks and matrix blanks 10.1.1 Easxtmreactthotdwobl1a.n0ksm.l aliquots of Milli-QTM water following this procedure and use 10.12 mExtaraxctbtlwanoks1..0SmeLc 1al1i1q.u4ots of the serum following this procedure and use as 102 1M0a.t2r.1ixtPshrpeeipakacercseuraancdyaonfal(yhezetrmaactrtiixons,pike and mati spike duplicate samples to determine 10.22 mPraepiarereeacecihvesdpiwkietuhsciancghassaammpplleescehosen by the analyst, usually the control 10.2.3 cEAaxldpiebdrcatoteidnoncocpnuicrkevenetsramtaiyonbsewiilnlclfuldedi atnhde mmaidy-r3a1nge ohftehlowi1i3lngecaolfibiraesimoniclurve 10.2.4 Prepare one malix spike and mati spike duplicate per 40 samples, with a `minimum of2 matrix spikes per batch. 10.3 1C0o.n3t.i1nuaoPifnrtnedphghaerceeainlciaiotnbnadrtlaiatcnnaiuloliinynbgzrcaehctehiccoeokncnsktciudrnivufeif.nergIbcaytl>ihbe3r0paet%ir,ocnernect-hacemcfakflyrszeaenmcpselaembspeltteowseeaennnsaultryheezteihdeiaiafc)ecrucrtuahrecvye fast acceptable check 103.2 sPertepar34e,ofnoeurccohneticnkusinagechperecpkapreerd garnoduepxotfr1a0cs.amples. For example,if a sample 103.3 iPhreenpiatcalcaccuhrvceontinuing calibration check from th same mir used to prepare 10.3.4 cTuhrevee"xpAedcdtietdiocnoanlcesnptirkaetsiomnasyfablel wiintchliundetdhethmaida-rlanigethoef othwe rinaintigael coaflitbhreatiinonit Bunion EofrPsFeO4S0fom Seu 3M Environmental Laboratory /03 Paseo 13 PagSEe 3m Medical Department Study: T-6295.7 laboratorRyepoRretqueNos.t NFuAmCbTerT-OUX2-2073s0 c5laolpwipbbernatdio1onf00ct0uhrepvpcea.bl)iTbrhaitsioins nceucrevse(sfaroyriefxtahmepalnea.ly5stpmpu--bst 1q0u0anptpibt,aterautsheirngthoannly the ILL Prepare matrix calibration standards ILL Transfer | mLofserum toa 15mLcentrifuge tube. 11.1.2 Imvfaotmlrouixsm.tessRaeemqcupoalrledtvoeoatlchuhemsseaasmmapprlleeelevvsoosllutumhmaeens.o1n.D0tomheLm,eoxtetexrxtatrcratacictotnstlseahsnesdeattrhdasn w0i.t5h0 mmaLtrioxf 11.1.3 Wbehtiwleeepnraelpiaqruiontgs.a total of twenty aliquots in 15 mi centrifuge tubes. mixor shake 11.1.4 TtThywepoiecna1dlmloyLfutsaheilsitsqheeucotsittooasnrn,,daotrtodhsepcrioknaecp,eprniotnprdrauitpailtoiencsavtoaeln,udtmswep,oikssietnragvnedaaamrsodumcnauttrrsviexlsi,sbtlfeadnokirs&n.tToatballeof1, at eighteen standards, two matrix blanks, and two method blanks. 11.1 Rraenfgeers0anvdaltihdeatLiionncarrepCoarltiEbrTaSt-io8n4.Ra0n&geE(TLSC-8R-)5.fo0r-Vca-l1i,brwahtiiocnhcluirsvtsesthe working 11.16 sUtsaendaArtdtsa.chSmeeentSeDcatsionan13a.id0 tion ccaallccuullaattienagcttuhaelccoonncceennttrraattiioonnssoofftPheFwOoSrkining calibration standards. 11.2 wT1oo0r0ek0aicpnphgb.ssttaannddaarrdd, fbolrantkh,e coornccoennttirnautiinogn cthoefcakl,l waidtdhianpptrhoeprcailaitberaatmioounncturovfesurrarnoggeat8epp-b 11.3 0Extersatcatblsipsihkeeadchmatinriitxialstcaunrdvaerdosnftohlelomwaisnsgs1p2e.c6t-r1om2e.t1e6r.of this method. Use these standards %S /0y Exacion oEfTPSF.O8S40from Serum 3M Environmental Laboratory Page6or 13 wr PagetE 3m Medical Department Study: T-6295.7 laboratorRyepoRretqueNso.t NFuAmCbTerT-O0X3-207350 Table 1 Approximate spiking amounts for standards andspikes. Working suandard Using 1.0 mWlLof mat|rixApprox. final conc. of (approx. conc.) analyte in matrix [0500ppm | 20 0.010 ppm [50pm | 20 50.0 ppm S 0.100 ppm [0250ppm| _-- 500mm 130 | ss 00pm] 120 Procepure 12.1 Obain frozen samples and allow fo thaw, 122 Vortex mix for 15 seconds, then transfer 1.0 mL or other appropriate volume to a 15 mL. 12.3 polypropylene centrifuge tube. Return samples to freezer after extraction amount has been removed. 124. Record the volume on the extraction worksheet. 12.5 wLaobreklshtehettufobredwoictuhmtehnetsitnugdythneurmebmeari,nisnagmpslteepsI.D, date and analyst initials. See attached 126 SStpainkdearaldl assadmepslcersibiendclinud1i1n.g2.blanks and standards, ready orextraction with surrogate 12.7 ScpLoiinnkteitnhueatiancsgheccmtaialotinrb.irxaftowiriottnhhesttcahaneldiaabrpdrpsar.toiporniactuervaemsotuanndtarodfs.staAnldasrodparsepdaersecrmiabierdixinsp1i1.k1e,s oarndTable 128 sVaomrpelxesmfioxr t1h5e ssetcaonnddasrd curve samples, matrix spike samples, and continuing calibration 129 Tbiocacrabcohnastaempblufef,era.dd 1 mL 0.5 M TBA and 2 mLof 0 25M sodium carbonate/sodium 12.10 12.11 Using an Cap each Oxford sample Dispenser, and put on add the 5 mL. shaker methyl-rert-buty] for 20 minutes ether. 12.12 Cseepnatrraitfeudge for 20 to 25 minutes at approximately 3500 rpm. until layers are well 12.13 Rtuebmeowvieth4.t0hemsLamoef tihnefoorrmgaatniiocnlaasyeirn t1o2.a5c.lean 15 mL centrifuge tube. Label this fresh ExvacioEn TofPSR8O4S0from Serum 3 Environmental Laboratory Se jo5 Pagar Fase 3m Medical Department Study: T-6295.7 laboratorRyepoRretqueNso.t NFuAmCbTerTOUX2-207350 12.14 hPouutres.ach sample on the analytical nitrogen evaporator until dry, approximately | to 2 12.15 gArdaddu1a.t0edmLpipoerttoet.heMreatphparnooprliavtoeluvmoelutmoeadofd the extraction. meequtahlasnotlhetoineiataclh vceonlturmiefuogfe staubmepluesienegda for Note: 1216 If the initial volume is less than 0.500mL th Vortex mix for 30 seconds. final methanol volume will equal 1.0 mL. 1217. AFitlttaecrhin2t.o0.a2 1u.5mmnLylgolnasmsesauhtofviiltaelr toro.lo3wc-cvoslyurimnegeauatnodvitarlanwshfeerntnheecessasmaprly.e to this syringe. 12:18. mLaatbreilx,thfeinaaultosvoilavelnwti,tehxttrhaectsitoundydantuem,baenrd,anaanliymsatl(sn)upmebrefroramnidnggetnhdeere,xtsraacmtpiloen.timepoirt, 12.19 Cap and store extracts at approximatel4y C until analysis. 12.20 Cnootmepbloeotkeotrheinecxlturdaectiinonstwuodryksbhinedeetr,,aatstaacphperdoporiathties document, and tape in the study 13.0 DATA ANALYSIS AND CALCULATI 13.1 Calculations 13.1.1 CcaallicburlaattieonacsttuaanldacrodnsceunstirnagtitohnesfoofllPoFwiOn,g eoqruaotthieorn applicable fluorochemical, in mLmLofosftasntadnadradrd+ xmcLonocfensturrartoigaotneosftsatnadnadradr+d (ug /mL) initial matrix volume (mL) = Final Concentration (ug/L) of POS in matrix 14.0 METHOD PERFORMAN 14.1 TfohrespmeectihfiocdMdeDtLectainodn lliimmiitt o(fMqDuLan)tiitsaatniaolnyt(eLOanQd) mvaaltueexs s(pseeceifAict.tacRehfmeernttosMBDaLndreCpoyrt 14.2 Tevhaelufaotleltohweinqguaqluiatlyitoyfctohnetreoxltrsaactmipolnesanadreaenaxltyrsaicst.ed with cach batch of samples to 14.2.1 Method blanks and matrix blanks. 14.2.2 Mpraetcriisxiosnpiokfetahendexmtraatcrtiixons.pike duplicate samples to determine accuracy and 14.2.3 Cinointitailncuailnigbrcaatliiobnracturivoen.check samples to determine the continued accuracyof the 5.0 POLLUTIONPREVENTION AND WASTE MANAGEMENT 15.1 hSloiacgmahptleBdeTiwUnastchtoenetilaaibndoerirasstp.oorasy.endd iunsebdioghlaazsasrpdipceotntteaiwnaesrst,e fisladmismpaobsleedsionlvbernotkewnasgtleasiss cdoinstpaoisneedrsin ExcacioEn TofSPO8.S40from Serum 3M Environmental Laboratory a 00 Pager 13 PPaaygeTT 3m Medical Department Study: T-6295.7 laboratorRyepoRretqueNos.t NFuAmCTbeTrO-XU-aCs3e0 160 Recorps 161 nCootmepbloeotkeotrheinecxlturdaectiinonthweor3-krsihnegesttautdtyabcihneddert,o tahsisapmperotphroida,te.and tape i the scudy Doamamens 17.1 Auachment A. Extraction worksheet 0000000000 17.2 Auachment B, MDL/LOQ values and summary 17.3 Autachment C, Calibration standard concentration worksheet Bopemmses 18:1 The validation report associated with this me0 thod0is 0ET0S-08.04.00&050.00-v.01,000 19.0 ArrECTED Documents 191 HETPSL-C8:-E5l.e0c,tr"oAsnparlayysiMsaosfsSSepreuctmroormeOttrhye"r Fluid Extracts for Fluorochemicalsusing WReoviRsieovn gos Number 0000000000000 ReasonForRevision Revision Date Exrcion oEfTPSR8O4S0from Serum. 3M Environmental Laboratory a 07 Pagegor 1 PagevT 2m Medical Department Study: T-6295.7 LaboratorRyepoarqtueNsot. MFaAnCpTerToxa-d0i3l0 Se TSS i ow = Extraction Worksheet ETS-8-4.0 pail Bll nT | ee, rr-- r rr rr r i 1 etr7 ee} _--t rr TYr r r tt r -- r _, rr T --r--t-- rrr r tt t t ed rr r e fr mm oe r ee eee} er ee r r t e r meevnc] i eee PS re Sy | --t--free} r r r r r Tr r 1 T 1 T I reeem rr ee| eee eee} rt rr 1 a or = Bskeddmn E om AIS we E e n | wm Ereisraostoom Sne 0 Snvizonmental Laboratory 4 "ps pe 00013 Page x15 3m Medical Department Study: T-6295.7 laboratorRyepRoretqueNsot. NFuAmCbTerT-O0X2-207330 [MCoDmLp/oLuOnQd|valuMesDfLor rabbit serum [ LOQ [Linear Callbratin Range (LCR) | L (ppb) | | (ppb) | Approximate concentrations Standard Calibration Curve to be usedforpreparing the PROS PROSA 476 289 | | 91.5129 1[55ppppbb -1100000ppp0pbb 1 [PFOSAA [EFOSE-OH 12.94 5.33 41915 [5 ppb- 1000 ppb ppb-100pp0b [[MP5r5o6sEA T71121.16] | 3450.42[155oppobb-- 11000000ppppbb. 1 cdMueDrtveLerIsmLiinOneQeeaqcvuahilvouafelsetnhicenes.reat.hRrbeeosevpiomnanets,reimscoeinsnktewheyer,earane,xdtbhroauvcimtnaeend,aansneddraumnmoanlwkyezereyed mwwoeitrtehstetaqhtueiircvaaabllbelinyst ds1eerftuohmercmsueardbvessTroo s fesponses. therefore, their MDL andLOQwillbe the same values a determined in rabbi serum PinlfeoarsmeasteieonA.ttachment C (LOQ Summary) and MDL study in ETS-8-4.0 & 5 0-V-1 for further fon Pairing Extraction ofSFluumomroacrhyemTiacballes:fLriomimtSseorfuQmuaanntditAantailoynsis by APUMS(MS) [eros PRs IT Al Tam 10Q 152 ATeTy Low std I 251 High std. 1002 seo FEA ErOSEOH" | AAIIT The ar en aieTa 1 csooo] ] 1000 [PrOSER MM3D6L 2nd LOG we ai esimates Ths only for EFFOT SE OTF A 402 CX 250. 1 1000 250 I 1000 Auachmen8t:MDLLOQ ExteactioEn TofSBR8O.S40from Serum 3M Savironmental Laboratory 100 109 Page 11013 age Tor sm Medical Department Seudy: T-295.7 LaboratorRyepoRretqueNso.t NFoacmtero-xs-i0e3ss T Compouna:aprpoess | Rome I serum | ey rangeof |e average [roo [100-1002 [100-1000 rears | Compound: PFOSA ~ | Sem | | rnangeeo,f | eeaverage [rom [130-100 100-10 Cosmcpuomu||nd:nFmPeFepOasySeldAA|| Rwo|emenpesr [Roi [10-550 [100-038 r[| Csoampnou|n|d:sPfEonSpreE,s.||| RSSopoTer Rte 100-100 | 100-1000 Csormpuooum|ndp| :eaPornoo,satEn|| o[crrheemr wove | 100-100 [ 100-10 | Coosmopmmou|nd:aP|saspeeused ||[| RSereseeor wove | 100100 | 100-100 Ro| Toor Teo Tear ave curve Gree low sid lowsid | hee highsid nmhighsid || 251-|130010.220 So1|-3003-1000 Rn| eer ave curve. re Towstd Towstd | hae highstd. 10-100| 510.100 30-100| 50-108 To wn highsid mn an | Teeoen|| Toooarerdo LoEeR|| e ahade T ovnn 250-598 | 430-30 omni | mom ns RWoRn|IRTTerreee TE ERT| Enaee TeaR 00.100| 500-100 100-10 | 300. 1000 sor o EeeReT|| Toe ee e TewR|| e bMody o ownae | 250-100| 500-250 st0-50| 10.108 sora CW ERT|W ToereertTeREwR|| |rteee Cemero || 350-100 | 500-100 sm. 10| 1m. som oe. om Ansan5:40LLOQ ExssEfnFsOiaSioooSn 3M Environmental Laboratory To 110 J. PageTOZ 5m Medical Departmen Study: T-5295.7 LaboratorRyepRoretquewso.t MFAaCpTesToRx-S030 PSAraemnppladeamtta:rt: Ton Pair StandFESaqiirunndildpsaCmoreuvdnertnnvuseoomsnrbde-brTF:l:uids TMaertgheosdarmeavrseons Blank Ridndenditin FFFCCCmmmiiiexx tistddd aasppppeooerreeen.s0SS30p000eoegmnms Surrsotdgapapreox 100 pos SAscFitrnuoOalSlncoS"nalaFccOeSontFrSoSRfahOsgotSttaAninAdoarndSHssSOhoSnouEtrw"he FPSCFemOSiSenxeK sSVeeEeTw||aAAmb TrmTei -0300 |-9300 _ 050710532 TTT0S0l T0521 sor 03~i0 "057 32 TI 0s0 sa1 0501 | |. 0010 0020 7 101s 7} 1025 Cf 390 300 SoS R s07 sa TS0 sa L To 301 TTS isa so 0.005. 0010 L010, 1015 300 1-300 SOUTTsnTTTsTalSesl or T7S0.1TT Ts32 TT Sor [sai Tso I) 1.025, 000s oi" 00g THe |--300. "so. TT Ts3g 1300 S00 ssa OL sa S00 Tso) Ts S00 i es 0010 0015" iots 71.020, TEGS RO Calculated concentrations of standards itn he sample matrix TS PrEacSTTe oSr eAE FFa riomee e aBS nOSe efpE e moamsm eit {Tn|En Tiasl]| 923823 3S0e0T TT TIS eTTTaTem TeG no TSr E Fianaglictonc: { 28 9% CaTe Te 7 S00 TTTUSTTane --_2 a oe 3OL1 m TSIY TC S00 i a 25 [nee mdT9006 Validated ranges pprosimate concentrations e [Serum| pros PFOSA EE ae L E e ee] RMa onkeytTEqimatesonly_Usevalfourreabbsit Human OSAA___EFOSE-OH___PFOSEA M556. 250-1000 3 Abner. on Po Snot Caves Euacion ETP0S8 homSer 3 Snvironmenzal Laboratory oe uy ge 101 Pagers 3m Medical Department Study: T-6295.7 laboratorRyepoRretqueNso.t NFuAmCbTerT-oUx3-307350 S3EM ENNVVIIRROONNMENTALLLABORATORRYY~~~ METHOD ANALYSISOFFLUPOORTOACSHSEIMUIMCPAELRSFILNUSOERROUOMCTEAXNTERSAUCLTFSONUSAITNEG.OR OTHER HPLC-ELECTROSPRAY/MASS SPECTROMETRY Method Number: ETS-8.5.0 Author: LisaClemen, Robert Wynne Approved By: | Lo /Se Laboratory Manbger J Group Leader Af Corse Techical Reviewer Adoption Date: >/, jj; Revision Date: IVAS, Dae 24/9 Dac 2/1]%; Dae 10 Score anp ArrLicATION 1.1 Suscionpge:HPTLhCi-selmeectthroosdpirsafyo/rmathses asnpaelcytsirsomoefterxytracts from serum for fluorochemical surfactants 1:2 oAtphpelriicoanbilzeabCloemcpoompuonudnsd:s.Fluorochemical surfactants or other fluorinated compounds, or 1:3 Mthaetrviacliedsa:tiRoanbrbeipto,rtat, bovine, monkey, and human secu, or other fluids a designated in Word 9758.1 Analysis ofSerEumTsExat5ra0s Using ESMS 3M Environmental Laboratory 3 12 Page 1019 Page TOT 3m Medical Department Study: T-6295.7 laboratorRyepoRretqueNso.t NFuAmCbTerT-OUX3-2073s0 202:1suuwTshwiinsagmmHePtvLhCoo-derldMeecsetcrrmoibsoepsroatyh/emaansaslysspiescotfrofmleotrroy,choermisicmaillasrursfyasc0ttea0nmt0sase0xatp0rpra0ocptr0eida0fter0.om0Tsh0eer0a0, aSfnalaumlopyrlsoiecssheimmsiapcyearlaf,lossroumcbehed aabsnyatlmhyeozpenodtiuasstisnoigaurmasniipneAngrPlfgHleuEoiSorno/oMcchtSaar/naMecstSuelrsifysotsnitacetemo(ftaoPEpfuOarrStt)hiecarunlivaoernri,fym/z= 499. compound identification. 03.D1 eAvatermiowosumpshmoeertsihcsodPsreosfsiuorneizIaotniioznabtyiounti(lAizPi)ng:vTahrie0 oMusic0 srouorm0 caesss,0pQruoa0btetsr,0 oaIn0 dsyisntt0eermfsa0caesl.l0owTh0 feosre0 iTaontncmilozusadptehieobrnuit(cAaPrpcerlen)so,stuTrihemei(rit.me.odsnptorot:ayuE,lnedccttecar.rosTvpharecauyiulomon)in.ziaztaitoinonpr(EoScIe)s,s Aitn mtohsepsehetreicchnPirqeusessuroecccuhresmiatcal 32 spEtrlreesocsntugrroeel,sepcwrthraeiycraellobnfyiieizldoa.ntiizoanti(oEnS,ocEcSuIr)s:tahrmoeutghhotdheofprioodnuiczattiioonnopfetrifnoyrcmheadrgatedatdmroosplpehtesriicn a 3.3 TsMehalesecsAtiSPvpeelQycutdaritostcmrreoitmIriyns,aytsMetadesmbssyaSmrpaeesecsqtutroiopmcpheeatdregrwei(trVhaStqi)ou,a(Tdmrazun)pdoalenemdsmMuabassssessqeuSlepencettciltvyreoddemeteteetccetterodr.s(.MASl/oSMniSsn)gal:ree MinSformmaatyiobne.employed for ion detection or a series (MS/MS) for more specific fragmentation 3.4 Csoryotsnhtvoeegmnostnia(ploonstatolt1hv9se9.8cZ)o-nusetpiarlpiaezyretpaurrZeo.bseIpnrinattyhee"rcfcoaocnnevf:eonrtmTiahoteniaolnla.tceostnTfhmooerdmseaplrtsaiyoonfemiMititictsreadoimmfaersodsmdQi2urpeactrttolrbyoeaIiIsthe ccHooonnwfeeivgaeuprrear,ttuiZro-ens,pirasfadtyiefrcfoepramespnsoti,nnetgnhetthsmreaotunhgdhocdaosnotvoferntotupioeournsaatpliaoctnoh,mwcpaloyenaienninnttghs,e aacrnoedunnmtoaetircneotlmeepcnatartnoicdbeel.earwTeihttohheuogsnhaemet.he oatnhoetrheZr',sbpurtayonslyystweimtsh,seitmc.i)lar systems (i.c. Z-spray components are compatible with some 3.5 IMvearsssysisotnLesmysna.rxeCSsuiormfirlteawnr.at"lrye:FMoarSsymssoLtryeenmxdsetohafaitslwsWarsieeneddteohsweisgmna9en5duaafnolrdstWpheiecnisfdpieocciwtfosicNthToep4ei.rn0asttvireournsmieoonfntst.(hMesiAelclrQoumaatstrso `Quatro IT MassLynx or MassLynx NT USER'S GUIDE). 4.0 WARNINGS AND CAUTIONS 41 Health and Safety Warnings: 41.1 aUpsperocxaiutmiaotnelwyit5h00th0eVvooltlst.age cables for the probe. The probe employs a voltage of 41.2 aWnhdecnlohtahnidnlging samples or solvents wear appropriate protective gloves, eyewear, Analysis of SerEuTmSEx8t5ra0ct Using ESAMS 3 Environmental Laboratory oY 1/3 Pase2019 Pagex05" 3m Medical Department Study: T-6295.7 laboratorRyepoRretqueNos.t NFuAmCbTerT-O0X2-207350 4.2 Cautions: 42.1 Do not operate solvent pumps above capacity of 400 bar (S800 psi) back pressure. 422 If the back Do not run pressure exceeds 400 bar, solvent pumps to dryness. the HP1100 wil initiate automaticshutdown, 5.0 INTERFERENCES 51 Tstoormaigneiomriazneyinptaretrfoefreinncsetsruwmheenntaatniaolnytzhiantgcsoammeplseisn,ctoenftlaocntshwiotuhldthneotsabmepulseoedrfeoxrtrsaacmtp.le 6.0 EQUIPMENT 6.1 Equipment listed below may be modified in order to optimize the system, 6.11 Micromass Electrospray Mass Spectrometer 6.1.2 HP1100 low pulse solvent pumping system and autosampler 2.0 7.1 SUPPLIES AND Supplies MATERIALS 7.1.1 Hsyisgthemp)urity grade nitrogen gas regulated to approximately 100 psi (House air 7:12 7.1.3 HPLC analytical column, specifics to be determined Capped autovials or capped 15micentrifuge tubes by the analyst 8.0 REAGENTS AND STANDARDS 81 Reagents 8.1.1 Methanol, HPLC grade or equivalent 8.12 eMqiulilvail-eQnTMt,waatnedr,maalyl wbaetperrouvsieddedinbythaisMmieltlih-oQdTsOhoCulPdlubse sMyisltleim-oQrTMotwhaetrerveonrdor 8.1.3 Ammonium acetate, reagent grade or equivalent 8.2 Standards 82.1 Tpyrpeipcaarleldydtuwroinmgetthheoedxtbrlaacntkiso,n tpwroocemdautrrei.x bSleaenkEs,TSa-n8d4e.i0g.hteen matrix standards are 9.0 SawPLE HanpLING 9-1 aFrreesshtomraetdriinxcsatpapnedadradustoarveiaplrseporarceadpwpietdh1e5acmhlacneanltyrsiisf.ugeExtturbaecstuedndstaanndaalrydsissand samples 92 aIfppanraolxyismiastweillyl 4bedCeluantyield,aneaxltyrsaicstecdanstbaenpdearrdfsoramnedd.samples can be refrigerated at Analysis of SerEuTmSE8xi5s0t Using ESMS 3M Environmental Laboratory ToS- /14 Page dats Page-T0 3m Medical Department Study: T-6295.7 laboratorRyepoRretqueNso.t NFuAmCbTerT-oUx2-307350 10.0 QuaLITY CONTROL 10.1 MethodBlanks 10.1.1 Analyze and Matrix Blanks a method blank and a matrix blank prior to each calibration curve. 10.2 Matrix Spikes 10.2.1 mAinniamulmaymoafzt2riesxpiskpeiskpeerabnadtmcahtrix spike duplicate per forty samples. Witha 10.2.2 Eccuaxrlpvieeb.cratteAiddodnsipctiuikroevneac.lonscpeinkteractoinocnesntwrialtliaolnls imnatyhefamlildi-nrtahnegeloowf-trhaengienitoifal(hcealmibrpation 10.23 See Section 13 to calculate percent recovery. 10.3 Continuing Calibration Checks 103.1 cAhnaanlgyeze(xa 3m0id%-)rainngpeeacakliabrreaatoiocncusrtsa,nrdealradtaifvettroevtheeryinitteanlthStsaamnpdlaer.d cIuraves.igsntiofpictahnet run. Only those samples analyzed before the last acceptable calibration standard. 10.32 will See be used. Section The 13 10 remaining calculate p samples ercent di must bereanalyzed. fference. 110 CALIBANDRSTAANDTARDIIZOATINON 111 rmAenegaarlenyszsoiefotnthw(eofsextotarrnadctathreeddcavmlaailtburreiasxs,isoattnaecnaducarhrvdesstuapsrniidnoagrrdMtoacosansncLdeynftnorxlaltooirwointn.hgewerialcsluhbietsaepbtolloefttseeoxdfttrbawycatrsle,eaTrhe 11.2 Tdihsecretivoanluoefftohretahnealdyasttaasnhdouslpdprboeva0l.9o8f0othregrPeraotjeerc.t LLeaodw:er values may be acceptable at the TL3 Isftatnhdeacrudrcvuerdvoee(sifnnoetcmeesseatryr)eqaunidrermeeanntasl,yspeerform routine maintenance or reextract the 114. FuEsoxeramtpphuelrepl:ooswewsehneodfnaoacftcttuheremapcctayilniwgbhrteaotniqoqunauncatnuittraivtaeiranatgphpelrrootwxhialmneavtteehleslyofuf1l0lanrpaaplnybgteeo,foifantmahlaeyytset,baesnencnerecdreasccsuearrey.to crraealgnirgbeersasotifiootnnhewceucirugvrhevteicno(n5gsopisphtbiingtgoho1cf0o0tnh0ceepnspttbr)aa.ntdiaTorhndissstfawrnidolamlrd5rse.pdupcbetoin1i0c0euprpibcyraanthreirbuttheadn ttohelifnuelalr 12.0 Procepures 12.1 AcquisiSteitounp 12.1.1 Cfolfriicalkceqnouanimrseinaugrs,iabnngudtMoOtny-pDienAiYtnh.es-aAImcapqtlueidsidigetisitcoornifpCyroeonantrsr.-oslamPpanleel.nuSmebteru,p aasssaimgnplaemlest.hoAds(siMgSn) AliofSerEumTsxs5s0 Using ESAS 3M Environmental Laboratory 10% 1/5 Page 4019 Pagero7 3m Medical Department Study: T-6295.7 laboratorRyepoRretqueNso.t NFuAmCbTerT-OUX2-207350 12.1.2 TS10Io.R4e9r(9eSaiotnregloaethmfeeortnhaRpopedrcoocprlridiciakntgoe)nmoasrscsaMensR.bMu.tAionSfuelitlnIstochnaeinzAaictqiuuoisnsuiaMtloilyodnecocaloslnetacrptopelrdoppaarlnioealntgeawnaidntdhsemtlheacests pMSrIaRossds.uLcytSnaixvoneGafUcrIqaugDimsEeintTtiaOotniDomnAetiThnoAfdo.ArmCIafQtiUMoISnS/mIMaTSyIOibenNsctforolrulmeaecdntdetidst.iaorSneaeleemiMpnlifocoyrremodamt,aisoasdndiatnidonal MRM (Multiple Reaction Monitoring). 1213 Tstyapnidcaarldlsy athnedaennadlystiwciatlhbaatscethofrunexsterqaucteendcemabtergiixnsstwaintdharadss.etof extracted matrix 12.1.4 scSaaarmmrppylloeev.serSaoraelnvdaennaartleybnzloeatdnkcwsointsshhiodauelcrdoendbtesianaumnipanllgeyzsceabdluitbpremartaiioyodnibcceahlielnycckltouidnmejodenciatsteodsruafcpthoe.srsiebvleeryantaelnytthe 12.2. Using the Autosampler 122.1 Set up sample tray according to the sample list prepared in Section 12.1.1. 12.22 Saneatl-yuspttchoensHiPd1e1r0s0a/papurtoopsriaamtpeleforraotptthiernfalolrleoswpionngsec.onRdeictoirondsaoctruaatlccoonnddiittiioonnsstihen.the instrument logbook: 12.221 Sample size = 10 iL injection with a sample wash 12222 Injectsample = | 1222.3 Cycle time = 13.5 minutes 12.224 Solvent ramp = Time MeOH 20mM 000min_| __a0% Ammonium acetate 60% 7.5 min Hiomin T0o % i 0 o 10m% n|| (iSmin TT s0% |60%] 1222.5 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 refill if necessary. 12.3.3 tChheetcipk. thTehestatiipnlsehsosusltdeeblecafpliatllwairtyhatnothjeagegneddoefdtghees.proIbfet.heUtispeisanfoeuynepditeocebeto check unsatisfactory, disassemble the probe and replace the stainless sel capillary. 123.4 SOebtseHrPveLCdrpopulmetpstcoo"mOinn"g.oSuettotfhethfeltoiwptoof 1th0e-pSr0ob0e.uL/Amlilnoowrtaoseqaupiplriobprraitaete.for approximately 10 minutes AnslysisofSeErTumSE8xt.r5ac0t Using ESS 3M Zavironmental Laboratory +1 0 Page 50r9 Fageror 3m Medical Department Study: T-6295.7 laboratorRyepRoerqtueNos.t NFuAmCbTerT-OUX2-207390 1235 Taruomundonthtehetiptcoofgtehne.prAobef.ine mist should be expelled with no nitrogen leaking 12:36 cThhaenginestinruomrednetru(s0eosptthiemsiezepatrhaemreetseprosnsaet:the following settings. These settings may 12.3.6.1 Drying gas 250-400 liersrhour 123.6.2 ESI nebulizing gas 10-15 litershour 12.3.6.3 123.64 HPLC constant flow mode flow rate Pressure <400 bar (This parameter is 10 - 500 uLimin not set, it is a guide to ensure the HPLC is operating correctly.) 12.3.7 Cfaurretfheurl.lyCgounniedcettthheepvroolbteagientcoabtlheesopteontihneg.proIbnseert probe until it will not go any. 123.8 Record tune parameters in the instrument log, 12.39 Uthseinangalthyesicsroofssb-ifolloowgiccoaulnmtaetrriecleesc.trode in the ESMS source is recommended for 12:3.10MbCulatistcsoknL.oynnExsntsvauerrresbiusottntasro,tnsaienncdtahepenpdArcospqarumiiapstileteiMnoanusmCsboLnetyrrnoixlncUPlSaundEeeRls'a(SltlhiGssUaImmDpaEly)e.sva1r0Pyrbaeesmsaontnahlgeyzsetda.rt 13.0 DATA ANALYSIS AND CaLcuLATIONS 13.1 Calculations: 13.1.4 Calculate matrix spike percent recoveries using the following equation %Recovery= Observed Result - Background Result x 100 Expected Result 13.15 Calculate percent difference using the following equation %Difference = Expected CoEncx.pectedCaClcounlca,ted Cone, x 100 13.16 Calculate actual concentration of PFOS, or other fluorochermical, in matrix (ug/mi: (Ini(t0igaloVfoPlEuOmSe coaflcm.atfrrioxm(msit)d, C+umrlveofx_SuDrirlougtiaotne FSatcatnord)ard)x | ug 1000 ng Final Volume (mL) 4.0 METHOD PERFORMANCE 14.1. MmMaeDttrLhioxadnspdDeecLtifeOiccQt.iovnPalluLeeiassm.eitse(cMEDTLS)-8a-n4d.0L,imAitttoafcQhumaenntittaBt,ifoonr(aLlOisQt)inagroefmceutrhroendt, vaanlaildyatet.edand Analysis ofSerEumTSEx8tr5a0ct Using ESS 3M Environmental Laboratory ro% 117 Page 60f9 Page-ToT 3m Medical Department Srudy: T-6295.7 laboratorRyepoRerqtueNsot. NFaAmCbTerT-OUXs-s07360 14.2 Method Blanks and Matrix Blanks 14.2.1 SpMotesatsnhidboalrdedbcilonantnthkaesmicaannladitbimroaanttirooirnxccbaulrrayvnoekvserw.illVbaeluaensalayrzeeedxwpietchteedactho oslalmpelle osethfeorlowest 14.3 Matrix Spikes 143.1 eMxaptercitxesdptiokeasllarweiatnhainly+ze3d0w%itohfetahcehsspiakmepdlecosnectenatnrdatthieonpercent recoveries are 14.4 Continuing Calibration Checks 14.4.1 twChioetnhtsipeniaukciehndgscaocmnapcleilbnertarsteatit.oinoTnc.hheecpkesrcaernetarneacloyvzeerdieast aaremiexnpiemcutmedotfoafftaellrewviethriyn 103s0am%ploefs. 14:5 Iapfneaarlnfyyostr.cmreiAtdelrloinaaitlhiisotesndyiswtiletlmhebaenmdedtoshcaoumdmpelpneetsrefrdoeraimnnaatlnhycezeensdseiocrrtiuoomtnehnirstn'artcutmienotng,s amtsahiecnemtteeenranmnicsneeedmsabyyotbghee, or on the summary sheet with the sample resuls. 15.0 POLLUTION PREVENTION AND WASTE ManaGEMENT. 15.1 pSiapmepttleeweaxsttreacitswdaisstpeosaenddifnlbarmomkaebnleglsasoslvceonnttaisindeirsspolsoecadtiend hiingthheBsTbUorcaotnotraiyners, andglass 160 16.1. Recoros Store chromatograimns the studyorproject folder. Each chromatm ogrammmust-- have -- the dsfatotuledl,yowdoiiflnupgtriioonjnefcoftramcntauotrmiboenfri,anppcallciuqcudaiebsdlieet)ii,tohnaernmdeitnahntoahdle,yhsietnatdeegrraotriohnamnedtwhroidt,tesnaomnpltehencahmreo.meaxttogeraimo:n 16:2 16.3 Plot calibration curve by linear regression and stor in the sudy folder Print sample list from MassLynx and tap int the instrument unlog 16.4. 165 sCPtrooiprneyt idinanasaptprirunomtpeergnirtaattrieuonsnltosugudympamfgaoelrsd,yerifnrcolmudMiangssiLnystnrxumaenndt tpaapreamnettoertsheanidnsstraammpelnetruensullotsg, and 16.6 Summarize data using suitable software and store in the study folder. 16.7 aBnadckloucpateiloenctorfbonaiccdkuapaetloecatprpornoipcrdiaattae medium, Record in stdy notebook the ile name 17.0 17.1 TAAtBtLaEcSh,menDtIABG:RAEMTSS,-8-F5L.O0WCDHaAtaRrTeSp,ortAiNnDg VALIDATION spreadsheet, DATA 172 The validation report associated with this method is ETS-8-40 & 5.0-V-1 AnalysisofSeru1m5E4.x5c0 Using ESAS 3M Environmental Laboratory 4 1& Page 7ofs PageTT0 3m Medical Department Study: T-6295.7 laboratorRyepRoretqueNos.t NFuAmCbTerT-OuX2-207350 18.0 REFERENCES 18.1 EoTnSi-z9a-t2i4o.n0/,Ma"sOspeSrpaetcitornomaentderMaQiunatternoanIcfeSyosfttehmesM"icromass Atmospheric Pressare 19.0 AFFECTEDDocuments 19.1 E`TCSo-m8p-4o.u0n,ds"EfxrtormacSteiornumoffPoortAansasliyusmisPeUrsfilnugorHoPoLcCt-anEelseucltfroonsatperaoyr/OMtahsesrSFpleucotrrocohmeemtircyal 200 Revisions ReNvuimsbieorn, Reason For Revision Revision Dae Analysisof SEeTaSm.E8x.i5s0t Using ESM 3M Environmental Laboratory me~ 1/5 Page Bory PagerIT 3m Medical Department Study: T-6295.7 laboratorRyepoRretqueNos.t NFuAmCbTerT-O0X3-307360 AttachmenAt TsewsatyMaris: MMaettioxdFRienvailsiSocne:nt InAsviarluymiecnatl ESqoufitpamreentVeSryssioenm Number: RFiSlqeunaarmeed Valu S iipneercep DaDtaeeooffAExnracvtioin/Alna:lysi: Laboratory Study # S| Garomuonl/eD:asTea:keTnakfeonm5 fotmo sthtedi suoddeorder s IDCnioilntuciteainoVtnroFalatucitmooenr:((mwLTyog.k)en:TofcoTnamkrtehoemrsohumedtshfteouMlddaesrfsolLdyern nterion summary Final Cone. (vemL1; Calculated by didn the iil volume rom the concenrion Anlysis of SerEumtEsx50aUcsitng ESAS 3M Environmental Laboratory (20 Fase 9019 BaseTZ 3m Medical Department Study: T-625.7 laboratorRyepoRretqueNos.t NFuAmCbTerTO0X2-307350 S 3M ENVIRE ONMENTAR LLLAABBOORRAATI TOORRYY ~A ~ L MeTrOD EXTRAFCLTUIOORNOOCFHEPOMTIACSASLISUUMRPFEARCFTLAUNOTRSOFORCOTMANLEISVUELRFFOONRATAENAOLRYSOITSHUESRINAGNIONIC HPLC-ELECTROSPRAY/MASS SPECTROMETRY Method Number: FACT-M-1.0 Adoption Date: 5/2. [7 Author: Lisa Clemen Approved By: al) Laboratory Manager frida th Group Leader Sie A Cima Technical Reviewer Revision Date: 1/4 Sle /e Date sig Dac s/22/av Date 1.0 Score Anb ArpuicaTioN 1.1 Scope: This method is for the extraction of other fluorochemical surfactants from liver. Potassium Perfluorooctanesulfonate (PFOS) or 1.2 Applicable Compounds: Fluorochemical surfactants or other fluorinated compounds. 1.3 vMaaltirdiacteison: reRpaobrbti.t, rat, bovine, and monkey livers or other livers as designated in the Microsoft 7.0.1/95 FACT-M-1.0 Extraction ofPFOS from Liver 34 Environmental Laboratory 1am Page 1 of8 BseeTr 3m Medical Department Study: T-6295.7 laboratorRyepRoerqtueNos.t NFuAmCbTerT-OUX2-207350 2.0 SUMMARY OF METHOD 2.1 aATchleiutsoatremo.echtehAmonidciadolensspcuarriifrbaiecnstgahnrtoeswagfetronotemxitlsiravadecrtdeupsdoittnaogsesiaiocunhmpsapaiermrifpnllgueorraeonaodgcetpnaatnrcatsinutdilof5no.en0damtieLnts(oPtoFfhOyeS!t)hyaoc]retoatteh.er upFnlotauisltridcmryLs.ysroiEfnaegcxehtarteatxcatctrhaicestdrs1e0mr.oe3cv0oe.n2dstutiotmuatfceiedlntetirnrin1ft.u0ogemglLtausbmseeatauhntadonvpoiulatltsohnetnoafilnteirterdogtehnroeuvgahposra3tcocr 2390DDeeerivneiomioonns 31 None 444..110 HWeAHeaRaNllIttNhhGaaSnndAdNSSaDaffCeetAtyyUWTWIaaOrrNnniSinngsg:s -------- 41.1 Upastehougneinvse.rsal precautions when handling animal livers, they may contain 50 INTERFERENCES SI There are no known interferences at this time. 60 Equipment 6:1 Tachceepftaobllleo.wing equipment is used while carrying ou this method. Equivalent equipment is 6.0.1 6.1.2 Ulra-Turrax T25 Grinder for grinding liver samples Vortex mixer, VWR, Vortex Genie 2 6.13 Centrifuge, Mistral 1000 or [EC 6.04 Shaker, Eberbach or VWR 6.15 Nitrogen Evaporator, Organomation 6.16 Balance I7S 7.1O0 Su uGPlpoLvIer EsS Au ND MAt TERIe ALSsaxoM0 av 0emnas 7.2 Dissecting scalpels 7.3 Eppendoorrf disposable pipettes 74 Nalgene botles, capableofholding 250 mL and 1 L 75 Glass, type A, volumetric flasks 7.6 40 mL glass CHEM vials 7.7 7:8 Plastic sampule vials, Wheaton, 6 mL, Polypropylene centrifuge tubes, 1S mL 79 Labels ExtractioFnAoCfTP-FMO-S1.f0rom Liver 3M Environmental Laboratory H3 122 Page2of8 page sa 3m Medical Department Study: 7-6295.7 laboratorRyepoRretqueNos.t NFuAmCbTerT-OUX2-207350 7.10 Syringes, capofamebaslurieng 10 ul to 50 uL. 7.11 Glass,type A, volumetric pipettes 7.12 Graduated pipers 7.13 Electronic pipenor, Eppendorofr equivalent 7.14 Timer 7.15 Disposable plastic 3 ce syringes 7.16 7.17 Filters, nylon syringe filters, 0.2 ym, 25 Crimp cap autovials mm Note: PQriPor wtaoteurs.ingRignlsaessswyarrienagnedasbmoitensi,murimnosef39 vials. ttiimmeess wwiitthh mmeetthhaannooll,an3dri3nsteismefsrowmit3h sMeiplalria-te S88O.10 RReERAAecaGegEenNntTtsSsaAnNoDSStTaAsNoDaArRDsSs 0000000000000 8:14 2wSa0ot0degir,ruammmiHsxydNurnatoOixlHiad.lelPs(ooIulrTidisBnaatroeeardi1os0rs0oe0lqvumeidLv.albeSentao)kr,eer(icNnoAanOtHa| i)Lni1nn0agNl:5ge0wn0eeilbigothtetrslaep(.pL)roMxiilmlait-eQly 8.1.2 SdMieoladustieuurm1e0Hv1yo0dlmruoLmxeoidfuesti(hn1egT1Mi0BlNaklNeira-QOoTMrHweaqstuoeilrvu.atlieoSnntto)ir,net(oiNnaaa1O01H02)5mImLN.LvonDlaiullmugetetenrei1bc0oNtftlla1es:k10.and 8:13 Twaaeptpuerarob.xuiAtmdyajtulesaltmym0o16np9iHugm1r0ahmuyssdironofggTaeBpnpAsruolixfniattmoea2t(eK|loLyd6av4kolmourmLeetq1rui0icNvcaNolnaetOna,Hin(iaTnngBdA5d)0i0l0u.tL5eMMt:iolWlie-iQgh NvoalOuHmebweictahusMeilthlei-pQHTMcwhaatnegr.esAadbrdupNtlay.OHStsolroewliynawh|iLlenaadldgienngetbhoeullaest | mL. of 8.1.3.1 TneBeAderdequusiirnegsIaNchNeacOkHprisoorluttoioen.ach use to ensure pH = 10. Adjust as 8.1.4 ((SNNoaad,,iCCuOOm,/)cNaaGrnHbdoCn2Oa1,t.e)0/gS0oo.fd2si5uoMm:diBWuiecmiagrbbhiocanaparptberonoBaxutifemfa(etrNe(la1y.HT2.C6O.B5)agkieonrftosoorade|qiLuuimvvoacllauermnbe)ot,nraitceflask and dilute to volume with Milli-QTM water. Store ina | L nalgene bottle. 8.1.5 PFOS (3M Specialty Chemical Division), molecular weight = 538 8.16 Ethyl Acetate, Omnisolv, glass distilled or HPLC grade 8.1.7 Methanol, Omnisolv, glass distilled or HPLC grade. 8.1.8 Liver and control liver, received frozen from testing laboratory. 8.1.9 bMeilplrio-vQidTMewdatbeyr,a aMlillwlait-eQrTuOseCdPilnutshissysmteemt.hod should be Milli-QTM water and may 82 Standards 82.1 Prepare PFOS standards for the standard curve, FACT-M-10 ExtracoftPiFoOnS from Liver Page 3 of8 3M Environmental Laboratory 4 /23 Pagers 3m Medical Department Study: T-6295.7 laboratorRyepoRretqueNos.t NFuAmCbTerT-OUXs-2073s0 8.2.2 tWheeiagchtuaalppwreoixgihmta.tely 100 mg of PFOS into a 100 mL volumetric flask and record 8:23 B(rgi/nmgL)to.volume with methanolfo a stock standardofapproximately 1000 ppm 8.2.4 aDpilpurtoexitmhaetsetloyck50soplputmi.on with methanolfor aworking standard 1 solution of 8.2.5 Dapiplruotxe.th5e.0stpopckmsolution with methanol for a working standard 2 solution of 8.2.6 aDpiplruotxe.th0e.5s0topcpkm.solution with methanol for a working standard 3 solution of 9.0 SavpLe HanoLing 91 Alllivers are received frozen andmustbe kept frozen until th extraction s performed. 10.0 QuaLITY Control 10.1 Matrix Spikes 10.1.1 tPhreepaacrceuraancdyoanfatlhyezeexmtartarctiixons.pike and matrix spike duplicate samples to determine 10.12 Prepare each spike using liver chosen by the analyst, usually a control liver. 10.13 Expected concentrations will fall in the mid-rangeofthe initial calibration curve. 102 Continuing Calibration Checks 10.2.1 cPornetpianrueeadnldinaenaarliytyzoefctohnetiinnuiitinaglccaalliibbrraattiioonncchuervcek.samples to determine the 1022 Ofonuer cchheecckksisarpereppraerpeadrepderangdroeuxtproafctetde.n samples. For example, if a sample set = 34, 10.2.3 pPrreeppatrheeeiancithalcocnutrivneu.ing calibration check from the same liver homogenate used to 10.2.4 cTuhrevee.xpected concentration wil fall within the mid-range ofth initial calibration A11L11.O01 CCPAArULeBIpaBRrRAeATTLIIiOvNerAAHNoDmSSoTTgAAeNNnDaAtReDtIoZAUsTeIOfONoNr Standards 11.1.1 mWLeisgMhialplpir-oQxTMimwaatteerl.y 4Gr0ignodftoivaehroimnotgoean2e5o0usmLsolNutailogn.ene bottle containing 200 11.12 211:7540 rgaitsionot available, use appropriate amounts of iver and water in keeping with 11.13 See section 13.0 t0 calculate the actual densityof iver. ExtractioFnAoCfTP-FMO-S1f0rom Liver 3M Environmental Laboratory +s /24 Page dof Page re Sm Medical Department Study: T-6295.7 LaboratorRyepoRretqueNso.t hFaacmterTroax-o0i3d0 11.1.4 Add I mLof homogeneous solution to a 15 mL centrifuge tube.Re-suspend hsioxmtoeegnen| emoLusasloilquutoitoson fbyhosmhoagkienngeboeutswesoelnutailoinquionts15whmiLlecpernetprairfiungge a total tubes. of 11.1.5 Two 1 mL aliquots serve as matrix blanks. Use the standard concentrations and `spiking total of amounts listed in fourteen samples. table 1 to spike, in duplicate, two standard curves fora Working Sande WT Ro arena - Approximate Spiking AmoTuanbltes 1 for Calibration Standards (Approx. Conc.) PFOS in liver o S0S00pppmm |h 3400 ] 00X0os00ppmm [[5S0tmemnT Tm T 70520500 ppoprm IT 0mm-- 150 0570s00ppomm 1LL1 See section 13.0 to calculate actual concentrationsofPFOS in calibration standards. 11.2 Extract spiked liver homogenates following 12.14-12.24of this method. standards to establish each initial curve on the mass spectrometer. Use these 12.0 12.1 PcOrbttoracienapruT o,ngeesn vrT samples. Tspeent sso, mre fh Tv smo or poked oi 12:2 Cut approximately 1 g of liver using adissecting scalpel 12.3 Weigh the sample directly into a tared plastic sampule vial. 12.4 Record the liver weight in the `study notebook. 12.5 Label the sampule vial with the study number, weight, liver ID, date and analyst initials. 1122.76 GArdidnd2.t5hemsLasmpolfe.waPtuetrtthoesgarmipnudlerepviraol.be in he sample and grind for shout 2 mimes or 12.8 uRnitnislethtehesparmopblee nistohotmhoegseanmepolues.with 2.5 ms water usinga pipet 12.9 Take the grinder apart and clean it with `methanol after each sample. Follow AMDT-EP-22, 12.10Cap the sample and vortex for 15 seconds, FACT-M-1.0 Extractionof PFOS from Liver 3M Zavironmental Laboratory +H )25 Page Sof8 age ser 3m Medical Department Study: T-6295.7 laboratorRyepoRretqueNos.t NFuAmCbTerT-OUK2-207340 12.11 ttPhiuepbeertewtmeiat|ihnmtiLhn.eghisdotemenpotsig.ce)anlaitnefoirnmtoataio1n5amLthpeoslaymppruolpeylveinale. c(eSneteriWfuogrekstuhbeee.tLfaobredlohceumceenntciinfguge 12.12Sspeicktieonli1v1e.r1hoormoTgaeblneat|es with the appropriate amount of PFOS standard as described in 12.13 iPinpsettrtuemetnwtob|lamnLksaliquots ofMilli-QTM water to centrifuge tubes. These will serve as 12.14bAudffder.| mL 0.5 M TBA and 2 mLofthe 0.25 M sodium carbonate/sodium bicarbonate. 12.15 Using a volumetric pipette, add 5 mLs ethyl acetate. 12.16 Cap cach sample and put on the shaker for 20 minutes. 12.17Cteonatprpirfouxgiemaftorel2y0 31500205 rmpimn.utes, until layers are well separated. Set power on the. centrifuge 12.18cReenmtroivfeuge4 tmuLbes. oLfaboerlgatnhiics lfaryeesrh,tuusbiengwiath tmhLe sgarmaeduiantfeodrgmlaatsisonpiapsetitne,12t.o5,aclean 15 mL. 12.19hPouutrse.ach sample on the analytical nitrogen evaporator until dry, approximately 2 to 3 12:20 Add 1.0 mLof methanol to each centrifuge tube using a graduated pipette. 12.21 Vortex mix for 30 seconds. 12.22FAitlttaecrhin2t0o.2 1y.5mmnLylgolnasmsesauhtofviilatle.r 10a3 cc syringe and transfer the sample to this syringe. 12.23 Lmaatbreilx,thfeinaaultosvoilvaelntw,itehxttrhaectsitoundydantue,mbaenrd,aannailymsatl(sn)uwmhboerpearnfdogremneddert,hesaexmtprlaectitoinm,epoint, 12.24Cap and hold for electrospray mass spectrometry analysis. 12.25 Complete the worksheet and tape to pageofstudy notebook. 1D11333..010aDCaArlTcAualAaNtiAAoLnYsvS:ISaANDLCAyLCUsLATiIOsNS awpCatcotamons 13.1.1 eCqaulactuiloant:e the densityofliver (mg) in 1.0 mL homogenate using the following Bof Liver x A(veorfagLeivweerig+hgtoofftWeante|rm)L aliquots (me) ExtractioFnoAfCTP-FMO-SLfOrom Liver 3M Environmental Laboratory 3/20 Page6 ors aeTH 3m Medical Department Study: T-6295.7 laboratorRyepoRretqueNso.t NFuAmCbTerT-OUX3-307390 13.1.2 Cfaollcluolwaitneg aecqtuuaatliocnoncentrationsofPFOS in calibration standards using the LofSta`mngdaLridvexr'CoIncmeLnthraotmioognen(autge/mL) =OFfinPalFOCoSnciennLtirvaetrion (ug/g or mg/kg) m"ALvehroamgoegewneaitgehstooffalpipvreoxinimsaoltuetliyon40asmdgetleirvmeirniend2i0n01m3.L1o.1f, by weighing ten Milli-Q water. | 14.0 METHOD PERFORMANCE 14.1 The method detection limit is equal tohalfthe lowest standard.in the calibration curve. 15.0 POLLUTION PREVENTION AND WASTE MANAGEMENT 15.1 Sample waste is disposed in biohazard containers, flammable solvent waste is disposed in `high BTU located in containers, and the laboratory. used glass pipette waste : is. disposed in broken glass containers 161166...110CRoCoemmcpploleretptesetthhee eexxtvraaccitiioonnwwoorrkksshheeestsanddtaspe iTnetoTteheo study noe tebook 17.0 TABLES, DIAGRAMS, FLOWCHARTS, AND VALIDATION DATA 17.1 The validation report associated with this method is FACT-M-1.0 & 2.0-V-1 18.0 REFERENCES 18.1 AMDT-EP-22, "Routine MaintenanceofUlra-Turrax 1-25 1e 11999...4100 FAFAFACFCTETC--TMME--D22,,D"O"ACAnnUaaMllp EyyssNiiTssSooffLLivheerBExrtreacnts foe r Fluorochemicals usinge HPLC-Electrospra Mass Spectrometry" y WRO20 e0vR0iesviRoei0 nvsiisioon0 ss 000000000Re0 vision Number. Reason For Revision Date FACT-M-1.0 ExtractionofPFOS from Liver 3M Environmental Laboratory +H )27 Page 7of8 Page-TTS 3m Medical Department Study: 7-8295.7 LasoratorRyepoRretqueNso.t naacmtesoNx-oci3d0 Extraction Worksheet for FACT-M-1 Narer Study # Sample i set# Eee {sopPcFO03S ppm | sprPeFOpSom|prePsFOgS n|Date and aetael ppm| actual ppm| awcwwal ppm|for Sug, -- T --rr Tr r F z r---- r Trt T rr r rr F r tr e] } r TY rt -- r rr r F rrr 0 _tr r ] -- rr rr T rr r e r m r ete eetemer} _--_---- a =r7 Romie ey EE 1 Comme EEa-- Ee--DSH a nEl TT oTmTen FACT-M-1.0 TM Environmental Laboratory Ne 128 Extraction of PFOS from Liver aor Page 8 of8 rage 120 3m Medical Department Study: T-6295.7 laboratorRyepoRretqueNso.t NFuAmCbTerT-OUXs-207390 S3MSENSVIRROONNMMEENNTTAALLLLAABBOORRAATTOORRYY ~~~ MeTHOD ANALYSHIPSLOCF-FELLUEOCRTORCOHSEPMRIACYA/LMSASINSLSIPVEECRTERXOTMREATCRTYS USING Method Number: FACT-M-2.0 Author: Lisa Clemen Adoption Date: 5/3.,/27 Revision Date: 10j4 Approved By: BD J` p Laboratory Manager Verto Hoo Group Leader Aa A Chin Technical Reviewer Sets Dae Sets y Bae 5/23/92 Date 11.01SSccooppee:TAhNiDs AmPeptLhIoCdATis IfOoNrtemootsoFemo aTTo ep 1.1 sSucrofapcet:antTshiussimnegtHhPoLdC-iselfoerctthreosapnralayys/imsaofssexstpreaccttrsomoeftrlyi.ver or other tissues for fluorochemical 1.2 Asuprpflaictcaanbtlse, oCroomtphoeruniodnsi:zabPloetcaosmspiouumnpdesr.fluorooctanesulfonate, anionic fluorochemical 1.3 Mvaaltirdiacteiso:n rRepaobrbtit, rat, bovine, and monkey livers or other livers as designated in the Word 7.0.1/95 FACT-M-2.0 AnalysisofLiver Extract Using ES/MS 3M Environmental Laboratory 12a /29 Page 1 of8 Paget 3m Medical Department Study: T-6295.7 laboratorRyepoRretqueNso.t NFuACmTbeTrO-XU-202370 2.0 Sumwary oF MeTHoD 21 fHToPhnLiCsc-hmsaerlatechcttoerrdiosdstepisrccaroyifaibemsapsatsrhtesipcaeunlacaltryrsofimlseutoorrfyo.fclhTuehomeriocacanhlae,lmysisucicashliass5uprtefarhcftoparonmtteasdsesbxiyaumcmtoenditofroimngivesrinwgloeng vpeerrilfuyorcooomcptoanuensdulifdoenntaitfeic(aPtiFoOnS.) anion, M/Z= 499. Samples may also be screened to 3.0 DermiTions 3.1 None. 40 WARNINGS AND CAUTIONS 41 Health and Safety Warnings: 41.1 iUnsteo tchaeutpiroonbweiDthOthNeOvToltTaOgeUcCabHleTfHoEr tPhRe OprBoEbe,. tWhehreenitrhieskvoolfetlaegcetrciacballe sihepel:ugged 42 Cautions: 42.1 `Dooverno4t00rubnars,oltvheenHtPp1u1m0p0swaiblolvienictaiaptaeciatuytoofma4ti0c0sbhaurt(d5o8w0n0.ps). Ifpressure goes 42.2 Do not run solvent pumps to dryness. a 50 InTeRreReNces $1 cToenftlaocntswhiotuhldthneostabmeplueseodrfeoxrtrsaactm.ple storage or any part of instrumentation frat comes in 6.66.110 EEqEqquuuiiipppmmmeeennntttlliisstteeddbbeelloowwmmaayybbee cshhaanmgeeddhinoorrdeeretoeoptrimize the system 6.1.1 61.2 MHiPc1r1o0m0aslsowElpeucltsreossporlvaeynMtapsusmpSipencgtsryosmetteemrand autosampler 77u.1n0 SsSwuUpppplPlieisAPsNDLMAITEREIASLS 71.1 7.1.2 HNiPtLroCgecnolguamsn,resfpreicgiefriactsedtolibqeuidde,treergmuilnateeddbtyo approximately the analyst. 100 psi 7.1.3 Capped autovials or capped 15 mL centrifuge tubes. 388.100RReERaAecaGegEenNntTtsSsaAvNoDSStTaAnNoDAaRmDoSs 0000000000000 8.1.1 Methanol, HPLC grade or equivalent. Word 7.0.1/95 FACT-M20 Analysis of Liver Extract Using ESIMS 3M Environmental Laboratory +2130 Page20f8 Page TID 3m Medical Department Study: T-6295.7 laboratorRyepRoretqueNsot. NFuAmCbTerT-oUxa-2073s0 812 Mbeilprloiv-iQdewdatbeyr,a aMlillwlia-tQeTuOseCdPilnutshissysmteetm,hod should be Mil-QTM water and may 82 S8.t1a.n3darAdmsmonium acetate, HPLC grade or equivalent. 821 dTuyrpiicnagltlhyeoenxetrHacOtiobnlapnrko,ceodnuerel.iveSreeblFanAkC,Ta-nMd.s1even liver standards are prepared 9.0 SamPLE HANDLING 9:1 Fstroersehdliinvecrasptpaenddaarudtsovairaclsproerpcaarepdpewdit1h5emaLc.hcaennatlrysiifsu.ge Etxutbreasctunetdilstaannaldyasridss and samples are 92 `Iafnaanlaylsyissicsanwible bpeerdfeolramyeedd., extracted standards and samples may be refrigerated until 10.0 QuawiTy ConrroL 10.1 Matrix Blanks and Method Blanks 10.2 M1a0.t1r.i1xASnpailkyezse a methodblankand matrix blank prior to each calibration curve. 10.2.1: Analyze a mateix spike and matrix spike duplicate with each analysis 10.2.2 cAEudxrdpvieetc.itoendalcosnpcieknetrcaotnicoennstrwaitlilofnasllmianythfealmliidn-trhaengloeow-fratnhgeeionftiatlhecailniibtraalticoanlicburravtei,on 10.2.3 See section 13 tocalculate percent recovery. 103 Continuing Calibration Checks 10.3.1 crAuhnnaa.nlgOyenzle(y=amt3ih0od%s-e)rasiannmgppeelacekaslaiarbnreaaaltoyiczocneudsrtbsae,nfrdoearlreadttiahvfeetetlraosettvhaeecrycienipttteianalbthlsetsaacnamdlpailbrerd.atcIiuforanvess.itgsantniodfapirctdahnet will be used. The remaining samples must be reanalyzed, 10.32 See section 13 to calculate percent difference. 10.4 System Suitability 10.4.1 aSsyssetsesmedsufiotrabeialcihtyru(ne..g. peak area, retention time and peak shape, etc.) will be T11L11.01 ACAnAnaaLllIyyBzzReeAtTthhIeeOeeNxxttArraNaccDtteSeddTlAliiNvveDerArsRsttDaaInnZddaAarrTddIssOpNrsryiootrosoangd Ffoorllowing each setof extracts. of two standard values, at cach standard concentration, will be plotted by l The mean for the calibration curve using MassLynx or other suitable software inear regression Analysis of LiFvAerCETx-trMac2t0Using ES/MS 3M Environmental Laboratory +22 1/3) Page 3 of8 Pages75 3m Medical Department Study: T-6295.7 laboratorRyepoRretqueNsot. NFuAmCbTerT-O0X2-207355 11.2 The value discretionof for the the data analyst. should be 0.98 or greater. Lower values may be acceptable at the 11.3 sItftahnedacrudrcvuerdvoee(sifnneoctemseseatryr)eqaunidrermeeanntasl,yspee.rform routine maintenance or reextract the 12.0 12.1 PROCEDURES Acquisition St up 12.1.1 Click on star buton in the Acquisition Control Panel. Set up sample lst. Assign a(MfSi)lefnoarmeacuqsuiinrignlg,etatnedr-tMyOp-eDiAn Ys-almasptledidgeits.corfiptyieoanrs.-sample number, assign a method 12.1.2 To create a method SIR. Setlonization cMliocdkeonasscapapnrbouptrtiaotneinantdh e Acquis massto i4t9i9onocroonttrhoelr panel approp and s riate. e l e ct masses. A scan isusually collected along with the SIRs. Save method. 12.1.3 Tthyepisceaclolnydtsheetsoafmpstlaendliasrtdbsegins with the first setofliver standards 2nd ends with 12.1.4 Samples sample. aSroelvaennatlybzleadnkwsisthhoaulcdonbteinaunianlgy zceadlipberraitoidoinccahlleycktoinmjoencitteodrafptoesrseibvleeryantaelnytthe 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 list prepared in section 12.1.1 12.2.2 Saneatl-yuspttchoensHiPd1e1r0s0a/papurtoopsriaamtpeleforraotptthiemaflolrleoswpionnsge.conRdeictoirodnsaocrtuaatlccoonnddiittiioonnsstihne the instrument logbook: 12.2.2.1 Sample size = 10 uL injection with a sample wash 12.2.2.2 Injecvsample = 1 12.2.2.3 Cycle time = 15 minutes 12.2.2.4 Solvent ramp = Time MeOH 20mM Ammonium acetate 00min | 45% 1 55% | [[C7O5mmiinn T7[ 75900%%| to to%w|| Note: In this instrument configuration, the run must be set up on the electrospray psroefstsweadreow n thi eaH"WPt aWiotrh iknsgtaftoironin.let stan" message before the "Stat" button is 12.2.2. Press the "Start" button. FACT-M-2.0 Analysis of Liver Extract Using ES/MS 3M Environmental Laboratory 123 132 Pa4gofe8 PageTIE 3m Medical Department Study: T-6295.7 laboratorRyepoRretqueNso.tNuFAmCbTerTo0x3-5073s0 12.3 Instrument Sep-up 12.3.1 Refer to AMDT-EP-31 for more details. 12.3.2 Check the solvent level in reservoirs and refill if.`necessary. 12.3.3 utCnhhseeatctiipk.sftahTcehtoesrtyatii,pndlseihsssoausslstdeeemblbelcaefpliattlhlewaiprtyrhonabtoetahjenadgegnrededpolfetadchgeeest.hpeIrofsbteta.hienUltesispesiasstnfeeoeluynecdappiitloelcbaereyt.o check 12.3.4 aOSepbtpsreHorPxviLemCadrtopeplulymetp1s0tcmooi"mnOiutnne.gs.oSuettoftthehefltoiwpotfo1t0he- p5r0o0beu.L/Amlilnoowrtaoseqaupiplriobprraitaetef.or 12.3.5 aTruomunodnthtehetinpitorfogtehne.prAobef.ine mist should be. expelled with no nitrogen leaking 123.6. Drying gas 250-400litershour 12.3.6 cThhaenginesitnruomrednetr utsoeosptthiemsiezepatrhaermeestpeornssaet:the following settings. These settings`may 12.3.6.2 ESI nebulizing gas 10-15 liters/hour 112233..66.34 PiLrnCesstcsrouanrmseetna<tn4ti0s0folpboeawrram(toTihdnigescfpolarorrweacmrtealttyee)r10is-no5t00seutLi/tm5in guide to ensure the 12.3.7 Carefully guide the probe into the opening. Insert further. Connect the voltage cables to the probe. probe until it will not go any 12.3.8 Record tne parameters in the instrumentlog. 12.3.9 Using the cross-flow counter electrode in the the analysis of biological matrices. ES/MS source is. recommended for 12.3.10a0nCpaloliyfczkesdoa.nmpslteartlsbtu.ttEonnsuinrethseaArcqaunidsietnidonsCaomnptlreolnuPmanbeel.r iPnrcelsasdetshealstasratmbpultetson10atbe 111333..110CDaCaAllTccAoullAaaNttiAooLnnYssS:IsS AND CALCULATIONS 13.1.1 Calculate % Recovery = matrix spike Observed percent Result - recoveries using the Background Result following x 100 equation: Expected Result 13.12 Calculate percent difference using the following equation: % Difference = Expected CoEnxcp.ec-tCeadlcCuolnact.ed Conc. x 100 FACT-M-2.0 Analysis of Liver Extract Using ES/MS 3M Environmental Laboratory +o 133 Page Sof8 _BageTIS . 3m Medical Department Study: T-6295.7 laboratorRyepoRretqueNso.t NFuAmCbTerT-O0X2-207390 13.13 Calculate actual concentration of PFOS anion in totalliver (me) (errsmince fromstd curve' L oflivo er uesedd tforr aane anlysis J Totalmassoffiver 3) 1000 ug/1mg 14.0 METHOD PERFORMANCE 14.1 The method blank. detection limit is equal to at least three times the baseline noise in the matrix 142 The practical quantitationlimit is equal to the lowest standard in the calibration curve, 15.0 POLLUTION PREVENTION AND WASTE MANAGEMENT 151 Sample waste is disposed in biohazard containers, flammable solvent waste is disposed in high BTU containers containers, are located and glass pipette waste in the laboratory. is disposed in broken glass containers. All lO0 106R0 eRceco0 ornopss 00000000 16.1 Sitnofroermcahtrioonmaitnocglruademds eiinthtehre isnttuhdy hfeoladdeerr. oErahcahndchwrroitmtaetnoognratmheshcohurlodmahtaovgeratmh:e fosltluodywing number, sample name, extraction date, and dilution factor(ifapplicable). 16.2 Plot calibration curve by linear regression and store in the study folder. 16.3 Print sample lst from MassLynxand tape into the instrument runlog 16.4 Print data integration summary from MassLyn and tape into the instrument runlog. 16:5 tCaoppeyinitnosatprpurmoepnrtiartuenlsotgudpyagneost,eibnocolkuding instrament parameters and sample results, and 16.6 Summarize data using suitable software and store in the study folder. 16.7 lBoaccaktiuopn oeflebcatrcoknuipc edlaetcatrtoonaipcpdraotpariate media. Record in study notebook the file name and. 17.0 TABLES, DIAGRAMS, FLOWCHARTS, AND VALIDATION DATA 17.1 Attachment A: FACT-M-2 Data reporting spreadsheet 17.2 The validation report associated with this method is FACT-M-1.0 & 2. 0-V-1. Wo0 18R.0erREsFm0 EeREnNcCeESs00000000 18.1 AMDT-EP-31, "Operation of VG Platform Electrospray Mass Spectrometer" FACTM.20 Analysisof Liver Extract Using ES/MS 3M Environmental Laboratory 12s. /34 Page 60f8 Page--tre-- 3m Medical Department Study: T-6295.7 laboratorRyepoRretqueNos.t NFuAmCbTerT-OUX2-207350 19.0 AFFECTED DocumexTs 19:1 FUsAiCnTg-MH-PL1C.-0E,le"Ecxttrroascptrioany/oMfaPsostaSspseciturmoPmeertfrlyu"orooctanesulfonate from Liver for Analysis 200 Revisions ReNvuimsbieorn - Reason For Revision ReDvaitseion Analysis of LiFvAeCrTEx-tMr2ac.t0Using ESMS 3M Environmental Laboratory rel 135 Page 7018 Page-TIT 3m Medical Department Study: T-6295.7 laboratorRyepoRretqueNso.t NFuAmCbTerT-OUX2-207350 Laboratory Study # TSetsutdMyateria MMaetnroidcRFeinvailsoSonl:vent ~ AInnasliyutmiceanltSEoqfutiwpamreenVterSsyisotne:m Numbers FRielSeqnuasmreed Value: Siionptee:rcep DDaateeooffEAxntaryascitsonA/nAanlaylsytt: Concentration Dilation Final Conc. _ Group/D1 ese: T-- aken from the sudy folder BN | BN ] S`Caomnpcleenst:ratTiaokne(nufgrmoLmyt:heTsaukdeyn oflodmerthe MssLyn Initial Volume (mL): Taken rom he study folder. integration summary. DFiilnuatliCoonncF.act(ourg:/LT)a:kenCaflciuolattheedsbtyddyivfiodldienrg. the ntl volume from the concentration AnaloyfsLiiFvAesrCETx-trMac2t0Using ESMS 3M Environmental Laboratory 3 134 Page 8of _PageTIT Im Medical Department Study: 6295.7 LaboratorRyepoRretqueNso.t NFeAsCpTerToxa-o03n0 3M ENVIRC DNMENTAL LABC DRATORY METHOD EXTRACTIOONF POTASSIUM PERFLUOROOCTANESULFONATE OR OTHER FLUOROCHEMICAL COMPOUNDS FROM LIVER FOR ANALYSISUsiNG HPLC-ELECTROSPRAY/MASS SPECTROMETRY Method Number: FACT-M-1.1 Adoption Date: 0526/98 Authors Lis CleGlemnneLangnenb,urg Approved By: , Caba|na)Ma[nagerdr Revision Date: 06319 , </3/59 Bic 2L2elaedver DLi/c11a 99 AZlin AiRevtiewieanr gBatw e ASR AeA 1.1 Scope: This method is for the extraction of potassium perfluorooctanesulfonate (PFOS) or other fluorochemical compounds from liver. prow 1.2 Applicable Compounds: Fluorochemical surfactants or other fluorinated compounds. 1.3 Matrices: Rabbit, rat, bovine, and monkey liverorother liver as designated in the validation Meatans Excc-- oif$o0n8 fom Lvs 3M Environmental Laboratory t2% 37 Page o13 Page 120 20 Suvsaey os Merion Pationed nt lil Sete. Four ml ofexes re remitans ohPI 2.1 T(hPiFsOSm)etohroodthdeerscfrliuboersocthheempircoaclesdufrreomfolriv`eerxthroacmtoinggenpaottaesussiiunmgpaenrfilounorpoaiorcitnagnerseualgfeonntataend 5.0 mlofethyl acetate. In this method, seven fluorochemicals were extracted: PFOS, DPeFfiOnSitAi,onPs)F.OSAAnA,ionEtpFaiOrSiEng-OrHea,gePnOt AisAa,ddPeFdOStoEtAh,e and FC-807 monoester sample and the analyte (see 3.0 ion pair is `evaporator until dry. Each extract is reconstituted in 1.0 mlofmethanol, then filtered through a 3 ce plastic syringe attached to a0.2 um nylon filter into glass autovials. 30 Desnumons 3.1 PFOS: perfluorooctanesulfonate (anionofpotassium salt) C,F,,SO," 3.2 PFOSA: perfluorooctane sulfonylamide CF,;SO,NH, 3.3 PFOSAA: perfluorooctane sulfonylamido(ethylacetate.CF ,SO,N(CH,CH,)CH,CO; 3.4 EtFOSE-OH: 2(N-ethylperfluorooctane sulfonamido)-ethyl alcohol C4F;SO,N(CH,CH,)CH,CH,0H 3:5 POAA: perfluorooctanoate (anionofammonium salt) C.F,,COO' 3.6 PFOSEA: perfluorooctane sulfonyl ethylamide C,F,,SO,N(CH,CH,)H 3.7 FC-807 monoester C,F,SO,N(CH,CH,)CH,CH,0-PO,H) 3.8 Surrogate standard 1H,1H,2H,2H perfluorooctane sulfonic acid 40 WagaspCeaurisons Randing amie sve ep cont oman 50 tereRemmacrs 5.1 There are no known interferences at this time. 60 6.1 Fou Tphefoellowing equipmentsused wil sayingout his mend. Equivalent cosipmento 66.11..21 UVolritnexTumirxaerw,iYtWhRT.2,5VogrrindeGrenaiaec3imenforgrinding ispersingemusifying 6.1.3 Centrifuge, Mistral 1000 or [EC 6G6.111.5.6.4 SBNhaialtkarenorcg,ee,nE(beevar0pb1oar0ca0ht5oor)r,VOrWgRanomaion 3M Environmental Laboratory 124. 138 _Pagai3e-- 3m Medical Department Study: T-6295.7 laboratorRyepoRretqueNso.t NFuAmCbTerT-OUX2-207330 7.0 SUPPLIES AND MATERIALS 7.1 Gloves 72 Eppendoorfr disposable pipettes 73 7.4 Nalgene bottles, capable of holding 250 ml and 1 L Wheaton 6 ml Plastic Sampule Vials 75 Glass, type A, volumetric flasks 7.6 40 ml glass I-CHEM vials 7.7 Polypropylene centrifuge tubes, 15 mi 78 Labels 7.9 Syringes, capable ofmeasuring 5 kL to 50 pL 7.10 Glass, type A, volumetric pipettes 7.11 Graduated pipettes 7.12 Electronic pipettor, Eppendorofr equivalent 7.13 Timer 7.14 Disposable plastic 3 cc syringes 7.15 Filters, nylon syringe filters, 0.2 um, 25 mm 7.16 Crimp cap autovials Note:QvPiwarliaso,tretor.usiRnigngsleassysrwianrgeeasnadmbionttilme,umrionfs93ettiimmeesswwiitthhmmeetthahnaonlo,l 3anrdin3setsifmresomwi3tsheMpairlaltie 8.0 REAGENTS AND STANDARDS 8.1 sAhSouTlMd bTeypMiellIir-eQ"agwenattgerradaenwdamteary, Mbeilplrio-vQiTMdedorbeyquaiMvaillelnit-;QaTllOwCatPelruussTMedsiynsttehmi,s method 82 Sodium hydroxide (NaOH), J.T Baker or equivalent 83 Tetrabutylammonium hydrogen sulfate (TBA), Kodak or equivalent 84 Sodium carbonate (N2,C0,), 1. Baker or equivalent 85 Sodium bicarbonate (NaHCO), J.T. Baker or equivalent 86 Ethyl acetate, Omnisolv, glass distilledorHPLC grade 87 Methanol, Ormnisolv, glass distilled or HPLC grade 88 Liver tissue, frozen from supplier 8.9 Control matrix or blank matrix for standards, QC checks, blanks, etc 8.10 Fluorochemical standards 8.10.1 PFOS (3M Specialty Chemical Division), molecular weight = 538 8.10.2 PFOSA (3M Specialty Chemical Division), molecular weight = 499 8.10.3 PFOSAA (3M Specialty Chemical Division), molecular weight = 585 3M Environmental Laboratory ExtacionFAoCfTPMF.LO.S1from Lives 130 /35 Page3of15 Basen 3m Medical Department Study: T-6295.7 laboratorRyepoRretqueNos.t NFuAmCbTerT-OUX2-207390 8.10.4 EXFOSE-OH (3M Specialty Chemical Division), molecular weight = 571 8.10.5 POAA (3M Specialty Chemical Division), molecular weight = 431 8.10.6 PFOSEA (3M Specialty Chemical Division), molecular weight = 527 8.10.7 `tFrmCioe-ls8etec0ru7,ladmrioewnsetoeiergs,httaenr=d(6m35oM0noSepsetceiarlftlyuCohreomchiecmailcaDlivciosimopno)n.entFsC.-80T7heism2onmoiexstutreer of 8.10.8 SCu.rFro,gSaOteH)Stamnodlaercdu:la4r-Hw,epiegrfh=ltuo4r2o8octans sulfonic acid (1-H, 1-H, 2.H,2-H 8.10.9 Other fluorocheamsaippcraoplrisat,e 811 8R1e1a.g1ent10pNrsepoadriautimonhydroxide (NAOH): Weigh spprosimatly 2006 NaOH, Pour iio d1i0s0so0lvmeid.beaSkteorrecoinntaai1nLinNga5l0g0enmelbMoilllei.-Q" water, mix until al sods are 811.2 wN1aatNeOrs.HodsSiotuloumrteihoyinndraiontx1oi2d5aem1(l0N0NamaOlHlg)ve:onleuDmbieolrtutrtlieec1f0lNasNk aanOdHdi1l:u1t0e. toMevaosluurmee 1u0simnlg Moifl1l0iN-QTM 8:11:3 0PgrH5aMm1s0otufseiaTnbgBuAatpypilnratooxmimamo1artLieulvymol4hu4ymdetrtoro5ig4cenmcolsnutolaffit1nei0n(NgTNB5A0a)0O:mHiWaeMniidgllhdii-laQapTMtperotwoxaivtmeoarl.tuemAledyjw1uis6tt9h.to PMiHlclhia-nQgTMeswaatberru.ptlWyh.ilSetaodrediinngat|heLlNaastlgfeewnemibosttloef. NaOH, add slowly because the 8.1.3.1 nTeBeAdedreuqusiirnegs IaNchNeacOk Hprisoorluttoioena.ch use to ensure pH = 10. Adjust as 811.4 ba0ip.cp2ar5robMxoiSnmaoatdteeil(uyNma2cH6a.Cr5Ob,goon)aftisneot/odsaoidu1imucLmavrbobilcouanmraebttoerni(acNteef.lbaCusfOkf,ea)rnd(aNnbdar,i21Cn.0gOg,toNaovHofClsOuo,md)ei:uwimWtheiMgilhliQTM water. Store ina | L nalgene bottle. 812 Standards 8.12.1 Prepare PFOS standardsforthe standard curve. 8.122 cPforlneutpoaarironecihonetgmhie1cr.a0lf0lsputoparnmodcaPhreFdmsOiSca,arel1a.sc0tc2aenppdtaparmdbsl,PeaF(scO.aSgp.Apr,oonp0er.i9wa8ot7rek.pipnMmgulsPttFaiOncdSoamArpdAo,snoealnunttdio1n.10 ppm EXFOSE-OH.) 8.12.3 Wtheeiagchtuaalppwreoixgihtm.ately 100 mg of PFOS into a 100 ml volumetric flask and record 8.124 B(rgi/nmg)t.o volume with methanol for a stock standardof approximately 1000 ppm 8.12.5 aDiplpurtoexitmhaetsetloyck50sopluptmi.on with methanol for a working standard 1 solution of ExtactioFnAoCfTPEMO.SLLfrom Liver 3M Environmental Laboratory 5+ 140 Page dof 15 ageirr 3m Medical Department Study: T-s295.7 laboratorRyepRoertqueNos.t NFuAmCbTerTOUXz-s07350 8.12.6 aDiplpurtoex.th5e.0stpopcmk.solution with methanol fo2r `working standard 2 solution of 8.12.7 Dapiplruotxe.th0e.5s0topcpkms.olution with methanol for a working standard 3 solution of 8.13 Surrogate stock standard preparation 8.13.1 actual weight. sPtraenpdaarreds1s-uH,rr1o-gHa,te2-stHo,c2k-Hs,taCn,daFr,d,.SOW,eHiignhtoaapp5r0oxmilmavtoelluyme5t0r-i6c0fmlagskoafsnudrrreocgoartdethe 8.13.2 Bproimn.g to volume with methanolfor asurrogate stockofapproximately 1000-1200 8.13.3 Prepare a sumogate working sandard. Transfer approximately 0.5 ml of surrogate ssttoacnkdatrod&of5010m-l20voplpumm.etrRieccoflradskthaendacbtruialngvtooluvmoelutmraenswfietrhremd.ethanol for a working 814. Liver homogenate preparation - Niiostes:. TPhreefvoelnltowiisnsguperforcoemduthraewwiinlgl:bkeemeupcshtoeraesdieorntioc peurnfoirmexwciitsihnfgroazpeonrtliivoenrof 8.14.1 Weigh 40 gofblank or control liver into a 250 ml Nalgene bottle containing 100 mls Milli-QTM water. Record the actual weightof liverand total volumeof water ugsreidn.derGr(ihnigdhtshpeeleidvefroirnatpaporofxiniemlaytdeilsype3rmsiednuhtoemsoogreunnattile swuiftfhicainenUtllytra-Turrax T25 homogenized). bring the total Rinse grinder with an voloufwmateer added additional to 200 mi. 100 mlof MilliQTM water, to 8.14.2 oTlnioqdueobtraesloarnmfcien.tehetThhheoemcooagnvceeernnaatgtreeadtoeinosntioaftryeotdfhpetohblleyaspnerkoapllyiilqveeunroethsotumisboegdsee,tneaarntdmei,wneteridagnahsnfdeeartchtheennli1tq.hu0eomrl concentration (gof liver/mlof homogenate) can be calculated as follows: 8.14.3 {lgrams (g) of liver] + [grams (g)ofwater]} 8.14.3 Prepare sample livers as described in 8.3.1, but weigh out 1 gofliver, homogenize wUistehW2h.5eamtloonf6MmilllpilaQsTMtiwcastearm,paunlde vriianlsse owritahpparnooptrhiearte2.r5ecmepltoafcleM.ilRliinQsTMe wgartienrd.er aRupneipctrooarpfdrtiearaltleevwleeyriiygnhsctalsumdpnildnegwvsiottluhduymweanstuemurbseeadrn.,d st(ahDemonpl1w0ei1tIhpDe,mrelftiohvraenmrowl8e..3i.g2Lhfa,obredalttheve,iasalasnmdpalnealfysetr. homogenates). ExvscionFAofCTFAOLSLLfrom Liver 34 Environmental Laboratory 52 14 Page Saris Pages 3m Medical Department Study: T-6295.7 laboratorRyepRoretqueNos.t NFuAmCbTerT-0UX2-307350 9.0 SAMPLE HanpLING 9-1 Alllivers are received frozen and must be kept frozen until the extraction is performed. Lo 10.1 oMuatwrmixvbCloanvksmaonrd method blanks 10.1.1 Efxotlrlaocwtinigwtohi1s.p0romcleadluirqueotasndofutsheeaisvmeartrhioxmoblgaennksa.teS(epereSpeacrteidonin181..11.43.1-2) 10.1.2 Emxettrhaocdt tblwaonk1s..0 ml aliquotsofMill-QTM water following this procedure and use as 10.2 Matrix spikes 102.1 Pthreepaacrceuraancdyoafntalhyezeexmtartarcitixosn.pike and marix spike duplicate samples to determine 10.2.2 Prerceepiavreedewaicthhscpaickhe suasimnpglelisveetr.chosen by the analyst, usualy the control iver 10.2.3 EAdxdpietcitoendalcosnpcieknetsramtaiyonbsefianlcilnudtehde calibration curve, maindd-mraanygefoalf itnhethieniltioawl-caanligberaotfiotnhecumrvael 10.2.4 mPrienpiarmeumoonefm2atmraitxrisxpiskpeikaensdpoenrebamtacthrix spike duplicate per 40 samples, with a. 10.3 Continuing calibration checks 103.1 Ptthhreeepicanorintetailannucdailnaignbarclahyteizcoekncdcoiunfrtfvieer.nubiyIng>h3cea0l%pi,berrarcteeianontnadlciyhfzefecerkseansmcapemlpbelesetsawnetaeolneynztsehuderiaefntitethraeltahcceucru1vrseatccyngof acceptable check 10.3.2 Pprreeppaarree aannde ecxhtercakctpfeorugrrcohuepcoksf.ten samples. For example, ifa sample set = 34, 10.3.3 uPsreedpatroeperaecpharceontthiniuniintigalcacluirbvreation check from the same blank liver homogenate 10.3.4 Tccuahrlevieb.erxatpAiedocndtiectduirocvneoa.nlceTsnhptiirksaetisisomnnaescyfeasblseawirinyitchlfiutndhetedheatnhmaaltiysfdtal-mliurnsatothnfqetugalhneotwi-itrnaaittneagluescoiafntlgihboernaltiiyniotntihael plbo)w.end ofthe calibration curve (e.g. 10 ppb ~ 100 ppb, rather than 10 ppb. 1000 11.0 CALIBRATION AND STANDARDIZATION ILI Prepare liver homogenate standards 1.1.1 Tmriancsenfterrif|umgle taulbieqsu.otsofblank/control liver homogenate prepared in 8.14.1-2 to 15 3 Environmental Laboratory ExacionFoAfCPTFMO.S1Lfrom Liver 33 192 Page gor 1s Pager3d 3m Medical department study: T-625.7 LazoratorRyepoRretqueNsot. mFaamctpeTrooxi-r0o3s0 11.1.2 hohFomevoagevhnooalmtuoemgevesonolafutmseeasmpcRleqecaoilrvdetrthhheosmusoagmmeppnlaetvevsosormerloinmiBmteoo,mreooxstornearcatcsettancdslaornds waith ner 1113 tWuhbiels,e opeoarrinSgkaelbofewinesnosloq ofer homogenate in 15 ml centifge 11.14 Tscnttudaionodtna1wr)mdlomc,ooansrpcnieaknept,lproamtpairdosnutsepasniadq,usopttiksoi,nsgaeranvmoduaanstmscaulitessite,dblifnanrTkasb,slaeTsy1pC(iathctaehleruesnnedotfOhtFtehiss 111 hReefewroortkhien vaanlgideastiaonndriepnoretss FCAalCrTa-tMio-nL1RVa-g1e a(nLdekF)ACoTr-Mn-n2e.r4a.nYe-s1bawhiiec?h i 11.1.6 soUtsoaenrdmAartidtsoa.nchSsmeeaenntdSuDeadcssonan1a3i.d01i0n clea sto conspire calculating the concentrations of theworking 11.2 113 ETsSxttoatanrnedadaacacrtrhddsssift1okarnedetdashrteladicb,vloeinbrslchhaeonnaktm,rohagoterimnoQaniCttleochsfcaeilucalknrw,dviaatrodhdnisdntfatopehplerpoocwpaairlniigabtrae1t2ai6omn-o1ucun2rt1v6oeforfsaunhrgiresog1ma0ettephpwobdo,r~kUi1ns0ge00tphpbe. TET WAoprpkrionxgimSaatnedSapikUisnignAgm1o0unRmtSisoffoLriSvtearnT dardsT and SpT ikes | - (Approx. Conc.) PFOS in liver O05W0g0pprm 050m || [T30 o| o0Go0o06pnm0p] O0S000ppmem |Td oT oom3omppmm | 05000ppmp [4 iTC5az000005p3mppmm | S50mwm |6 Tsopm 20 12.1 POrbtoacienofurnozeesn iver samples and harmog=edenseiriabesd 8.107. 12.2 Vertex mix homogenste for 15 seconds, then transfer 0mo ther appropri volume to 215 ml polypropylene centrifuge tube. 12:3 Retum liver homogenate samples to freezer after extraction amount has been removed. ScionFoaFcrHanLyho Liver 3M Environmental Laboratory 34 143 PageTors Pagesrs 3m Medical Department Study: T-6295.7 laboratorRyepoRretqueNso.t NPuAmCbTerT-O0X2-207390 12:4 vtRreoaclnousfrmederrtwehidellfloiervqeueraxHtlroatmchoteigoiennni,taitatlheehnvootmhloeugmfeiennaaoltnertevhceoonlesuxtmtiert.aucttiFiooonnrmeweoxtrahkmaspnhloeele,tv.iolITuhmmeiefoeifqnauhlaolmmseot1gheoannn-aotle is 12.5 wLoarbkeslhteheettfuobredwoictuhmtehenstitnugdythneurmebmeari,nilnivgerstIeDps,.date, and analyst initial. See attached 126 dASelpssicokreipbrbeelpdaanirknelSmieavcettrriihoxonms1op1gi.ke1ensoartaTnedaacblolineqtuiontisuniwtnihgtathcastlehicebtriaaoptnpirofonoprrstithaaentdecaaarldmisob.uranttioonfcsutravnedasrtdanadsards, 12.7 sStpainkdearaldlassamdpelsecsr,ibiendclinudSiencgtibolnan1k1s.2a,nd standards, ready for extraction with surrogate 12.8 Vsaomrptleexsmifoxr t1h5essetcaonnddasr.d curve samples, matrix spike samples, and continuing calibration 129 Tbiocacrabcohnastaempblueff,era.dd 1 ml 0.5 M TBA and2 mlof the 0.25 M sodium carbonate/socium 12.10Using a volumetric pipette, add 5 mi ethyl acetate. 12.11 Cap each sample and put on the shaker for 20 minutes. 12.12 Centrifuge for 2010 25 minutes at approximately 3500 rpm, until layers are well separated. 12.13 Tcernatnrsiffeurge4 tmulbe.ofLoarbgealnitchilsafyerre,shustiunbge waithmlthgersadaumaeteidnfgolramsastpiiopnetatse,into12a.5c,lean 15 ml 12:14 hPoutusc.ach sample on the analytical nitrogen evaporator until dry, approximately 2 to 3 12.15peAixdpterdtatcet1.i.o0nMm.eltohranaoplprovporliuamtee veoqulaulmsetohfe mineitthiaalnvoolltuomceacofhlicvenetrrihfoumgoegteunbaetuesuisngedafgorratdhueated 12.16 Vortex mix for 30 seconds. 12:17 AFitlttaecrhinto0.2a 1u.5mmnlylgolnassmeasuthovfiilatler(toor alo3wc-ovoslyruimnegeauatnovdiatlranwshfeerntnheecessasmapryl)e.to this syringe. 12.18 Lmaatbreilx,thfeinaaultosvoilavelnwti,tehxttrhaectsitoundydantue,mbaenrd,aannaliymsatl(sn)uwmhbeorpearnfdogremneddert,hesaexmtprlaecttiiomnepoint, 12.19Cap and store extracts at approximately 4 C until analysis. 12:20 nCootmepbloeotke otrheinecxlturdaectiinonstwuodryksbhienedetr,,aatstascphperdoptroiathties document, and tape to pageofstudy 13.0 DATA ANALYSIS AND CALCULATIONS 13.1 Calculations: 13.1.1 cCaallicburlaattieonacsttuaanldacrodnsceunstirnagtitohnesofofllPoFwOiSng,oerquoatthioenr:appropriate luorochemical, in ExmactioFnAofCTPMO.SL1from Liver 3M Environmental Laboratory 38 144 Page8of 15 Pager 3m Medical Department Study: T-6295.7 laboratorRyepRoretqueNso.t NFuAmCbTerT-OUXz-207350 2`C]ooncfeSnttraantdiaonrdofxBCloannkceLnitvaertiHoonmoofgeSntaatneda(rgd/m()ug(/smcle)8.143) __ Final Concentration (ug/g) of PFOS in Liver See Attachment Dfor a sample form to calculate the concentrations of standards. 14.0 METHOD PenroRMANCE 14.1 TspheecimfiecthMoDdLdetaencdtliiomnitloifmiqtua(nDtiLta)tiiosnan(aLlyOtQe)anvadlmuaeisr(isxeespAectitfiacc.hmReenftesrBtoaMnDdLC)report for 142 tThheeqfuaolliltoywoifntghqeuaelxittryacctoinotrnaolndsaamnpallyessisa,re extracted with each batchof samples to evaluate 14.2.1 Method blanks and matrix blanks 14.22 pMraetcriisxiosnpoifkethaendexmtartarcitxiosnpike duplicate samples to determine accuracy and, 14.2.3 iCnointtailncuailnigbrcaatliiobnracturivoen.check samples: to determine the continued accuracyofthe 15.0 POLLUTION PREVENTION AND WASTE MANAGEMENT 15.1 ShlioacgmahptlBedeTiwUnastchtoenetliaasibndoeirrasstp,oorsaye.ndd iunsebdioghlaazsasrpdicpeotntteaiwnaesrst,e fisladmimspaobsleedsionlvbernotkewnasgtleasiss dcoinstpaoisneedrsin 16.0 16.1. Reconps Complete the extraction worksheet atached to ths method, emt and tape into the srudy notsbook or include into study binder, as appropriste. 20 Arracuments 17.1 Auachment A, Extraction worksheet 17.2 Atachment B, MDL/LOQ values 17.3 Attachment C, LOQ summary 17.4. Atiachment D, Calibration standard concentration worksheet 18.0 Rererexces 18.1 The validation reports associated with this method are FACT-M-1.1 and 2.1-V-1. 19.0 AFFECTED DocumEnTs 19.1 FMAaCsTs-SMp-e2c.t1ro,me"tArnya"lysisofLiver Extract for Fluorochemicals using HPLC-Electrospray. ExuacioFnAofPCRTOASLLfom Liver 3M Environmental Laboratory rol 145 Page sof 1s Pagar 3m Medical Department Study: T-6295.7 laboratorRyepoRretqueNos.t NFuACmTbeTrO-X0-202370 Womewwoss Revision 0000 Numb1er, sVyasltiedmast,iomnoonfkmeeytlhiovedr1c0roisnRscelvauasdleoidn7aFftloiuoronR,roeicvmhipesrmiioocvnaelmse,ntnsewtiAoPnIpMaSiIriMnSg) eexttcraction, MDL study, updates in record keeping andstoring polices ReDvaitseion 08101198 3M Environmental Laboratory ExuacioFnoAfCFTOASLIfrom Liver += 194 Page 100715 PageTIE 3m Medical Department Study: T-6295.7 laboratorRyepRoerqtueNso.tNuFAmCbTerTouxz-s073s0 BSoMtuxad#ty#e.s ToonacStui.rarlogateppSoptdmm. w, | afacFptuCaplMri0xo5Spxpipdp.mm|| Aw, pacrFtoCuanlM.ixpSpoodmm aw, || aapcFtruoCanl.Mi2x0Sppiodmm Aw Comments. -- f As nalysuDate ie mpy | T 0 r r T R r tr r -- --r1 r r Tt-- r r 1 ] ar r ---------- A r BO Re--1 ET B r e ------ r t r y r tr r t ]Re r r r: E r t EE r p ----B--]1 rt r Ri re saa. -- Fost IE noT03i5 E Kozo 0 BN SCO See Siz mmr | AGS meters Votoms = TNA a CEF MsSJMeSm E D_Cov MntSMCSa heDs cTkuresSdpiiSkhmedTg hossat m ConeaCChace raTnorsesFr] oat eoohncemvamn or AsschimenAt: Exaction worshess ExeFaAcoCCiTPoFOnSfom Liver Page 11 of15 34 Environmental Laboratory % 147 Page--TT5 3m Medical Department Study: T-6295.7 laboratorRyepoRretqueNso.tNFuAmCbTerTO0X3-303300 MDLILOQ values for Rabbit Liver: | Compound | MDL (ppb) | [LOQ (ppb) ||LSAitpnapenardroaxrCidamlaCitabelriaCbtorinaoctneinoRtnaraCntugireovn(esLCtoRb)eusedforpreparing the 1 PFOS PPFFOOSSAAR [55711.8 37.4138 ppb -- 10p0p0b. 6.06 19.3 20 pp--b 1200 ppb i FEtOFROASE-OH T87_| 190ppb = 18p0p0b ([VPoFnOoeSsEtRer n16v2|Sv17|[6o2oppsbtTeav00opdpp iama oe be ames CMoDmLp/oLuOnQd|valuMesDfLor|RatLLOivQe|r:Linear Calibration Range (CCR) PFOS (Pb) | (ppb)| SAtpapnrdoaxridmaCtaleiCbraotinon cCureventotberusaedftoriproepnarisng the || 78.7 | 62ppb -- 120pp0b. | PFOSA [| FPEOFFOAOSLSAEA-OR |wvv. n/v_| 62 p-p12b 00 ppb LE MDL/LOQ values for Monkey Liver: [Compound| | MDL pb) | | LOQ| pb)| LAipnperaorxiCamlaitberaCtoinocnenRtarnagteio(nLsCtRo)be Standard Calibration Curve usedforpreparing the ] | [T BPrFoOSsA | 274 [s2o8ppppbb---t1a02ov 0ppp0mb. | PROSAR | wv [EWFOSE-OR wv |v |55pb 1200p [PPFOOASAEA | wnvv. | w/vv_| | 112200 ppppb b-- 112200p0ppy0b. v= NocUtnotnviaclleindft.urratUthipeoronnasnaaflnoyraslciyaszliiibnsrgcatotihmoepnidciauetrd,v,evauplsrueeeptadhrieadtLinoContR. ptaossdettheercmriinteeritahesrtanfogretohfisScahamraicaterization, AnachmenBt: MDLILOQ Values 3M Savironmental Laboratory ExcacionFoAfCTP:EMO-S11from Lier 18 4g Page 12 0f 15 _Esseti 3m Medical Department Study: T-6295.7 laboratorRyepoRretqueNos.t NFuAmCbTerT-OUX2-207350 [I 5 CoSmumnoinmaa|ryTvaablcesLimitsvoofcQuan|Ttit1a0t0ion | Approsimte Linear Range righ Standard Ee T ! T | Standard | Bovine OE | wd- sodcmnedT 60pps 100g TT [okey [FFOSA | Rai ora? Sem | 107 6% | 3| Tome | Tosh ho Bove CC ET okey 1 T7470 Toms | Troon 2=3 FROSAR a Rabb S377b | Tops T8005 Tonk Tor ww op Tm g2 z EFGSEOR [Hodkey Rat Tw 57 ovTiops Tig > z okTaery ww wwvi] TT TToeps Soph TToomgess i007 33 = PORK | Rabi | Trp | Tse | Tom Tews 0n Bovine wd VioRwakey ||vwv w5C d "E Tm20ophpb |T 12T20000ppprb a @ PROSER | Rabon ToT Si itn Te [FR ewBosvie yne w wwd a Twwd T_3T00p5p5b5 Tope | T20007p5pb5. Toe TBowT e )0) Tw = eT=nDcireCwmesgeiSseosowdnhdessrrd avv e avawaife R|epCwrei&RCas| arroaedm rebss FiaoCg sO-- hR So| ra | tl fro Ga or rls ed et esGETo SOTO |determination. C[Coommpound|" rr| Rraose Ep Rar I ER 1 J eo THE 1 [Matrix sRuanodgaeroels|| v"Ceonee [[SRRGRRRG]E L`oCwonse.|"|BLoiwSeiar.| i"phSeu. PGES eso oPooe) |Gree) HMR [Rss {575-0[an Gooveue be GroNys 555 vrs me romeo ro oe [gkaw ee] B{ eCnCkNer 5 CESENEC9 L B|sEv- E s0 =2e 37 ] P Wa wwTiames]| ArachmenCt:LOQ summary ExacionFAofCTPFMO.S11fom Liver Page 130715 3M Environmental Laboratory TO yg PageTaT sn Medical Departmen: Seay: T-295.7 LasoracorRyeRperquoes.t Naecwtberzoax-r03s0 I I Be `Compound' PFOSA | I -- pane | Ramet| vee |Sgducl| Lowse | Lous] is |mu en Standards Curve DACUNGRE] Curve |. CubvetC] Curve | id Q-3E |[r Eene SRT e T [os r feea ik ie vva er EEae 3 rosa | Zz Prepared |Taree of CRETE ; CRT] TE] T CEr RN Eme eE r d.Eo C e |GnaE on Bl) [eoag en 2 a [Tier Prepared ECE CRE --TER Standards nal Stim Sn C BSaorovinney S] [SCGT SRTRIT |||M p eE e0R p S E 7w R wne|] u e e hT vhgpaei]rg| [Compound POAA A [f[ [ormee sI Seor o [ [eesoTs nE aE v vo e soawew r 0-1 oohgn] `T Cpoamrpeou|nde mat PFOSEA ro ed i= T ay .- 1 SG Se Jaw] [Bovine TSTT0060Candi awe ae JE -- ExFaoPcROtSnsomsc 0 Environmental Laboratory -- ge 140015 Pagesee 3m Medical Department Study: T-6295.7 LaboratorRyepoRretqueNso.t NFuAmCbTerT-o(-2023705 m mmapt: RWeyWLr Ton Pair StandardT Curves--Tise suesre Reva R [agereie n RCacomaTo T slatef a IN T ow T or T IFFCCSSiSidSaAppproo5D0S0T200 3W36O10O05 -- i [SerpeSuApprox. les wows I :i: ActuaClPornoCsson|ci SeofnFSFtCtaOonSndrAaerad|sitntihSPeoFroCOnnSMAseiAx | E4wOCSoEe| SFoaRn I ProSER T Si Ewer 3 mCooEre C CC EwJe| . eTe 5> e : ] 3 |T SpTSiokooeordgooceonree Esoso ser CE Tos To1m Toi wson DTowooTie SY: SS + SS or oFiraSoiHobsi oTorr STCoomaE i{atoelr| ooo ote CFTroSCCoreom--epFPriROsCSotonf_eSrFaPrRTCOoonSe|STR FBNiwOSConeE FFeOaRr FRFOoSEeA| Dono fSmsgm : Ew =e o Te owsm Ts Te w ssr: e e HSie e Conee, sAo] mee T3S -- --m-- o 1 [ree2eTe wi1e]Te = nETTLaSa --mSfie r]cwe I CT me ] I a i-- ri=sr [Te] 1 -- C--o ] [tVae lnidateRd eaReaT nges--60fA+p11pr20ro00ox00i5sm55a85t_e[[T33C00Fon1co2e2n00str00as3t5i50o5nJs[] 18P02r:01o5s-0t00508|]1TE53r00.o1as800E0o0onb0[400-p11o280r000g55k] E 50F0-r1-o7s10e20a09505|]] | CL Fonkey 1760 13005[90-2002 Y [C 130 H 200]E0E1200ToenGE M13050E040a4)g00 CcAoatnalccietbnmraetenotsnDawonrdahect 3M Environmental Laboratory ExtocioFnaAfCPTRAOSLIfom Liver re 5 157 Page 15 of 15 SaeeTIT 3m Medical Department Study: T-6295.7 laboratorRyepoRretqueNos.t NFuAmCbTerT-O0X2-207350 3M ENVIRONMENTAL LABORATORY METHOD ANALYSIS OF FLUOROCHEMICALS IN LIVER EXTRACTS USING HPLC-ELECTROSPRAY/MASS SPECTROMETRY Method Number: FACT-M-2.1 Author: Lisa Clemen Approved By: D fe TCaboratory Manager GroupALeafdenr Hon Te(chsniacal4Revi(edwimeer. Adoption Date: 05/26/98 Revision Date: 06[03112 </3/55 Dae DLa/te1/55 LDaie o L0_ SCoPE AND APPLICATION 1.1 sSucrofapcet:antTshiussimnegtHhPoLdC-iselfeorcttrhoesapnraalyys/imsaosfsexstpreacctrtosmoeftlriyv.er or other tissues for fluorochemical 1.2 sAuprpflaicctaabnlt,e oCroomtphoeruinodnisz:ablPeotcaosmspiouumnpdesrfivoroostanesalfonate, anionic fluorochemical 1.3 vMaaltirdiacteison: rRepaobrbtit, rat, bovine, and monkey livers or other livers as designated in the Word 70.95 AnalysisofLiFvAeCrTEMxac1t Using SIMS 3M Environmental Laboratory 3/52 Page 1019 Page rer 3m Medical Department Study: T-6295.7 laboratorRyepoRretqueNos.t NFuAmCbTerT-OUXz-2027350 2201 suHThPmiLwsC-ameegltevhcootdrrodMsepesrctaryiw/boemsaostshespaencatlryosmiestorfy.flTuhoreoa0cnhae0lmyis0cias0liss0upref0racf0toarn0mtes0dexb0tyra0mcotn0eidt0forrionmglaivseirnugsleing ipeornfclhuaarraocotcetriasnteiscuolffoanaptaert(iPcuFlOaSr)luaoniroonc,heMm/icZa=l,49s9u.chSaasmptlheespomtaayssailusmo verify compound identification. be screened to 3.0 DeriniTions 3.1 `AvtamrioosupshmeertihcodPsroefssiuorneizIaotniioznabtyiounti(lAizPi)ng:vTahrieoMusicsrouormcaesss, pprloabtefso,rmansdysitnetmersfaaclelso.wTfhoerse iTonncilzuadteibonut(aPreeln)o,tTlhiemrimtoedsptro:ayE,leecttc.rosTphreayiIoonniziaztaitoinonpr(oEScIe)s,s AitnmtohsepsheetreicchnPirqeusessuroecccuhresmiatcal atmospheric pressure (i.. not unadvaceuurm) 3.2 pErleescsturreo,spwrhaeyrelobnyizioantiizoanti(oEnS,ocEcSuIr)s:tahrmoeutghhotdheofprioodnuiczattiioonnopfetrifnoyrcmheadragtedatdmroospplheetrsici:n a strong electrical field. 3.3 TMhaessAPSIpepcltartofmoertmsrya,reMeaqsusipSppeedcwtirtohmqeutaedrr(uMpSol)e, mTaasnsdseemlecMtaisvesdeStpeectcotrrs.omeltoenrs a(rMeS/MS): sMeSlecmtaivyelbyedeismcprliomyiendatefodrbiyonmdaestsecttoicohnoarrgaesreartiieos(m(/M2S)/aMnSd)sufbosremqoureentslpyecdieftiecctferd.agmAentsiantgiloen information. 3.4 Csyosntveemnsti(oponsatl1v9s9.8Z)-ustpirliazye par"oZb-espirnatye"rfcaocnef:orTmahteiolna.testTmhoedseplrsaoyfemMiitctreodmfarsosmpalaptrfoobremis corotnheogapoenratlurteo,tahfetceopnaesaspienrgtutrher.ou[gnhtahetocrotnuvoeunstpioantahlwcaoynifnortmheatcioounntietriselaeicmtreodded.ireTthloyuaghthtehe cHoonwfeivgeurra,tiZo-nspirsadyifcfoermepnot,netnhetsmeatnhdocdosonvfenotpieornaatliocno,mcploenaneinntgs, aarnednmoaticnotmepnaatnicbelearweitthheosnaeme. asnportahyesr,ysbtuetmso,nleytc.w)ith similar systems (i.e. Z-spray components are compatible with other Z- 3.5 MsyasstesmsL.ynCxurSroefnttwlayrMea:ssSy_sLtyenmhsaosfWtwianrdeodwessi9g5neadndforWithnedsopwecsiNfiTc 3o.p1ervaetrisoionnso.f thAelslevpelrastifoonsrm aIlreorsiQmuialtart.roFIoIrMmaosrseLdyetnaiolrs MscaestshLeymnaxnNuaTlUspSeEcRif'iSc tGoUtIhDeEi)n.strument (Micromass Platform 4.0 WARNINGS AND CAUTIONS. 4.1 Health and Safety Warnings: 4.1.1 aUpsperocaxuitmiaotnelwyit5h0t0h0eVvoollttsage cables for the probe. The probe employs a voltage of 4.1.2 aWnhdecnlohtahinndgl.ing samples orsolvents wear appropriate protective gloves, eyewear, Word 70.195 Analysis of LFivAerCTEx:trMa2ct1Using ESIMS. 3M Environmental Laboratory Ex] 153 Page 2019 _Page las 3m Medical Department Study: T-6295.7 laboratorRyepoRretqueNso.t NFuAmCbTerT-OUX2-207350 42 Cautions: 4.2.1 oDvoerno4t00rubnaxs,oltvheentHPpLu1m0p0swaiblovienictaiaptaeciatuytoofma4ti0c0 sbhaurt(do8w0n0. psi). Ipressure goes 4.22 Do not run solvent pumps to dryness. 5.0 InTEReERENCES. 5.1 Tshooumlidninmoitzbeeiunsteedrffeorrenscaemspwlheesntoarnaagleyozrinagnsyapmaprltoesffionrspterurmfelnutoartoioocnttahnaotacteo(mPeOsAAi)n,cotnetfalocnt with the sample or extract. 60 OEoueweNr 6.1 Equipmentlisted below may be modified in order to optimize the system. 6.1.1 Micromass Electrospray Mass Spectrometer 6.1.2 HP1100 low pulse solvent pumping system and autosampler. 7.0 SUPPLIES AND MATERIALS 7.1 Supplies 7.11 High purity grade nitrogen gas regulated to approximately 100 psi, 7.1.2 HPLC column, specifics to be determined by the analyst. 7.1.3 Capped autovials or capped 15 mL centrifuge tubes. 8.0 REAGENTS AND STANDARDS 81 Reagents 8.11 Methanol, HPLC grade or equivalent. 812 MAiSlTlMi,-QTTMywpaeteI rwaatnedr,mMaiylblei-pQrTMovwiadteedr,bayllawMaitlelri-uQseTdOiCn tPhliussmesytshtoemd.should be 8.1.3 Ammonium acetate, reagent grade or equivalent. 82 Standards 82.1 Tpyrpeipcaarleldydounreinmgetthheoedxtbrlaacntki,ononpreocmeadturriex.blSaenke,FaAnCdTt-eMn-m1a.tr1ix standards are 92.0 SAMPLE HanpLinG 9.1 Farceesshtomraetdriinxcsatpapndeadrdausatroveiaplrsepoarrceadpwpietdh1e5acmhLancaelnytsriisf.ugEextturbacetseudntsitlanadnaalrydssisand samples 9.2 Itfeamnpaelryastiusrewiolrl rbeefrdieglearyaetde,d aetxt4racCteudntsitlanadnaalrydssisancdansabmeppleersfomramyed.be stored at room Analysis ofLiFveArCTEx:trMa2ct1Using ESMS 3M Environmental Laboratory 154 Page 3of9 Page rie 3m Medical Department Study: T-6295.7 laboratorRyepoRretqueNso.t NFuAmCbTerT-OUX2-207330 10 100.01 QMuataru0 irx yBlCaookvsr0 aonrd Meth0 od Blanks 00000 10.1.1 Analyze a method blank and matrix blank prior to each calibration curve 102 Matrix Spikes 10.2.1 mAnianliymzuemaomaf2trispxiskpeiskpeearnbdatmcaht.rix spike duplicate with each analysis. With a 10.2.2 EAdxdpietcitoendalcosnpcieknetrcaotnicoennstrwaitlilonfasllmianythfaellmiidn-trhaenlgoeowf-trahnegeionifttiahlecanliicbarlatciaolnicburravtei.on curve. 10.2.3 See section 13 10 calculate percent recovery. 10.3 Continuing Calfbration Checks 103.1 cAhnaanlgyeze(=a3mi0d%-)rainngpeecaakliabrreaatoicocnusrtsa,nrdealradtiavfetelroetvheeriynitteinatlhstsaanmdpalred.cIufraves,igsntiofpictahnet wruinl.l bOenluysedt.hosTehseamrpelmeasinainnaglsyzaemdplbeesfomruestthebelarsetaancacleypzetda.ble calibration standard 10.3.2 See section 13 to calculate percent difference. 1.0 CALIBRATION AND STANDARDIZATION 11.1 aAvnearlaygzeeofthetweoxtsrtaacntdeadrmdacturrivxesstawinldlarbdespprliootrttedobaynldinfeoalrlorewgirnegsseiaocnh(syet=ofmeyxtr+acb)t,s.notThfeorced through zero, using MassLynx or other suitable software. 11.2. sIftatnhdeacrudrcvuerdvoe(eisfnnoetcemseseatryr)eqaunidrermeeanntasl,yzpee.rform routine maintenance or reextract the 11.3 uFsoertphuerploosweseonfdofactchueraccayliwbhraetnioqnuacnutrivteatriantghelrotwhalnevtehles foufllanraalnygteeo,fitt hmeaystbaendnaercdescsuarrvey.to cEaxlaimbprlaet:ionwchuervneactotnesmipsttiinngg otofqtuhaentsittaantdearadpsprforxoimma5tpeplyb t1o0 1p0p0bopfpbanraaltyhteer,tgheannetrhaetefuall rreagnrgeessoifotnhweeciugrhvtein(gSopfpbhitgoh1c0o0n0cepnptbr)a.tiTohnisstawnildlarrdesd.uce inaccuracy attributed to linear 12.0 Procepures 12.1 Acquisition Set up 12.1.1 aClfiiclkenoanmsetaurstibnugtMtoOn-DinAYth-elAacsqtudiisgiittioonfyCeoantre-oslamPpanleel.nuSmebteurp, aasssaimgpnlaemliestt.hoAdss(iMgSn) for acquiring, and type in sample descriptions, 12.1.2 STIoRcr(eSaitneglaemfeotnhRoedcocrlidcikngo)n osrcaMnRbMut.tonSeitn ltohneiAzactqiuoinsiMtoiodnec3osntarpoplrpoapnreilataenadndsemleacsts S0IR4s9.9 oSravoethaecrquaipspirtoiporniamteethmoasds.es.If MAS/fuMllSsciannstirsuumseunatlslyarceolelmepcltoeydeadl,onagddwiititohnatlhe production fragmentation information may be collected. See Micromass. Analysis ofLiFvAerCETxMtr2a.ct1Using ESMS 3M Environmental Laboratory te 155 Page dof9 pager 3m Medical Department Study: T-6295.7 laboratorRyepoRretqueNos.t NFuAmCbTerT-OUX2-207390 `MMRasMsL(yMnuxltGiUplIeDREeaTctOioDnAMTonAitAorCiQngU)I.SITION for additonal information and 12.1.3 Tthyepisceaclolnydthseetosfamsptlaendlasrtdsb.egins with the first set of liver standards and ends with 12.1.4 Ssaammppllee,s Saorelvaennatlyblzaendkwsitshhoaulcdonbteinaunianlgyzcealdipberratiioodniccahlleycktoinmjoencittedorafptoesrseibvleeryantaelnytthe camyover and are not considered samples but may be included as such. 12.2 Using the Autosampler 12.2.1 Set up sample ray according to the sample list prepared in section 12.1.1 12.2.2 Saneatl-yuspttchoensHiPd1e1r0s0a/papurtoopsraiamtpelefroraoptthiemaflolrleoswpionngsec.onRdeictoiorndsaocrtuaatlccoonnddiittiioonnsstihnethe instrumen logbook: 12.2:2.1 Sample size = 10 pL injection with a sample wash 12.2.2.2 Injectsample = | . 12.2.2.3 Cycletime = 13.5 minutes 12.2.2.4 Solvent ramp = Time MeOH | 20mM 000min_| 40% | Ammon5i0u%m acetate SOmin_| 90% [TLOmin | 90% | 10% 10% 20min | a0% | 50% 12.2.2.5 Press the "Start" button. 123 Yostrument Sep-up 12.3.1 Refer to ETS-9-24.0 for more details 12.3.2 Check the solvent level in reservoirs and refiifnecessary. 12.3.3 Cthheetcipk. thTehsetatiinplsehssousltdeeblecalpaitllwairtyhatnothjeaegnsdedofetdhgees.prIofbteh.eUtspeiasnfoeuynedtpioebceeto check unsatisfactory, disassemble the probe and replace the stainless steel capillary. 12.3.4 SOebtseHrPveLCdroppulmetpstcoo"mOinn"g. oSuettoftthehefltoiwp tooft1h0e-p5ro0b0e.uL/Amlilnowortoaseqaupiplriobprraitaetef.or approximately 10 minutes 12.3.5 Taruomunodnthtehetinpitorfogtehne. prAobef.ineRemaidsjtussthotuhledbtiepofetxpheellperdowbieithfnnoo nmiitsrtog1sencblseearkviendg 123.6 Tchhaenignestinruomrednetrutsoeosptthiemsiezepatrhaemreetseprosnsaet:the following setings. These settings may `Analysis ofLiFvAerCTExMtr-a2ct1Using ESMS Page 5 09 3M Environmental Laboratory 3 156 Page TEE 3m Medical Department Study: T-6295.7 laboratorRyepoRretqueNso.t NFuAmCbTerT-OUXz-20735 12.3.6.1 Drying gas 250-400 itershour 123.62 ESI nebulizing gas 10-15 liters/our 123.63 LC constant flow mode flow rate 10 - 500 uL/min 12.3.6.4 Pirnesstsruurmeen<t4i00opbearra(tTihnigscporarreacmtelyt.e)r is not set, itis guide to ensure the 12.3.7 Cfuarrtehfeurl.lCyognuniedcettthheepvroolbteagientcoabtlheesotpoentihneg.proIbnes.ert probe until it will not go any 12.3.8 Ptraipnetdtihnetotutnhee piangset,ruwmietnhtiltosgp.arameters,and store it in the study binder with a copy 12.3.9 Uthseianngaltyhseicsroofsbsi-oflloogwiccoaulntmaetrreilceesc.trode in the ES/MS source is recommended for 123.10toCploifcksaomnpslteartlibstu. ttEonnsiunrethsetaAtcqaunidsietnidonsCaomnptlreolnuPmanbeel.r iPnrcelsusdetshealsltasratmbpultetsontoatbe analyzed. 3.0 DATA ANALYSIS AND CALCULATIONS. 13.1 Calculations: 13.1.1 Calculate matrix spike percent recoveries using the following equation: % Recovery = Observed Result - Background Result x 100 Expected Result 13.1.2 Calculate percent difference using the following equation: % Difference = Expected CoEnxcp.ec-tCeadlcCuolnact.ed Cone. x 100 13.1.3 Calculate actual concentrationofPFOS anion in total liver (me): (ma Sadan) SBO ofliv1E e0r0u0suegdf/oE Irmagn,alN ysis xTotal massiveor(fp) 14.0 METHOD PERFORMANCE 14.1 MmaettrhioxdspDeecitfeicct.ioPnlLeiamseitse(eMEDTLS)-8a-n1d.1L,imAitttoafcQhumaenntittBat,ifoonr (aLlOisQt)ingaroefcmuertrheondt,vaanlaildyattee,dand MDL and LOG values. 141 142 SolventBlanks, Method Blanks, and Matrix Blanks 14.1.1 Sloolwveesnttstbalnandkasr,d minetthheodcablliabnrkast,ioanncdurmvaet.rix blanks values are must be below: the 142 Calibration Curves 14.21 The value for the calibration curve must be 0.980 or better Analysisof LFivAerCTExMt-ra2ct1Using ESMS 34 Environmental Laboratory & 157 Page 609 pestis 3m Medical Department Study: T-6295.7 laboratorRyepoRretqueNsot. NFuAmCbTerT-OUX2-207350 143 Matrix Spikes 14.3.1 Mcoantcreinxtrsaptiikoenpercent recoveries are must be within = 30oft%he spiked 14.4 Continuing Calibration Verifications 14.4.1 cCoonncteinnturiatnigocna.libration verification percent recoveries must be + 30% of te spiked 14.5 paIfnecarrlfiytsoter.rmieaDdoloicsnutemtdheeinnsttyhasilstemamecttaihnodondssaipnmeprtlhfeeosramprapenraconpearlsiyeazctetediolonorgibosotnoh'kte.rmeasc,timonasinatsedneatnecremimnaeyd bbey the 14.6 1ffoodtantoataerdeo1n0 btaebrleespoarntdeddiwshceunsspeedrifnotrhmeanecxetcorfitthereiarehpaovrte.not been met, the data must be 15. POLLUTION PREVENTION AND WASTE MANAGEMENT 15.1 chSoiangmthpaliBneTerwUsascatorenetliasoicndaeitrsespd,osaiennddthigenlalbsaisboohpriaapzteoatrrtyde.cwoansttaeiniesrsd,isfploasmemdaibnlebrsooklevnengtlawsassctoentiasidniesrsp.oseAdllin 160 16.1 Reconos Each page generated forastudy must have the following erate information included either sree in the ahinenataledygersrattoironhamnedthwordi,tesnamopnlethneapmaeg,e:extsrtaucdtyioonr dpartoej,ecdtilnuutmiobnerf,acatcoqru(iisaiptpiolnicmaebtlheo,d,and 16.2 aPrpipnrtoptrhieatteunseupdaygef,olsdaemrp.leColpsty, tahnedseacpqaugiesistaionndmteaptehoindtofrthoemiMnasstsraLmyennxt rtaonilnocg.lude in the 16:3 sPtloortetihnetchaelisbtruadtyionfolcduerrv,e by linear regression, weighted 1/x, then print these graphs and 16.4. aPnrdinstdoarteaiinnttheegrsattiuodnysfuolmdmearry, integration method, and chromatograms, from MassLyns, 165 AStutmamcahrmieznetdAaafoursianngesxuaimtapbllee osofftwsaurmem(aErxyceslp5r.e0a)dsahnedetstore inthe study folder, sce 16.6 aBnadckloucpateiloenctorfbonaicckduaptaetloecatprpornoipcridaattae.medium. Record in study notcbook the ile name 12.0 TABLES, DIAGRAMS, FLOWCHARTS, AND VALIDATION DATA 17.1 Attachment A: FACT-M-2.1 Data reporting spreadsheet Analysis of LFivAeCrTE:xtMr2a.ct1Using ESIMS 3M Environmental Laboratory q 15% Page 7079 Pag50e 3m Medical Department Study: T-6295.7 laboratorRyepoRretqueNso.t NFuAmCbTerT-OuX2-207350 180 Rerewesces 18.1 `FcAoCmTp-oMu-nLd.s1f,ro"EmxStrearcutmiofnoorfAnPaoltyassissiuUmsiPnegrfHlPuLorCo-oEclteacntersuolsfpornaayt/eMoarssOtShpeerctFlruoomreotcrhyemical 18.2 TEoTnSi-z9a-t2i4o.n0/,Ma"sOspeSrpaetcitornomaentderMaQiunattetnraonIcetorfitplheequMaidcrruopomlaessSyAsttmeomssp"heric Pressure 183 The validation report associated with this method is FACT-M-L1-V & 2.1.V-1 19.0 AFFECTED DOCUMENTS 19.1 UFsAiCnTg-MH-PLL.C-1E,l"eEcxttrroascptrioanyo/fMaPsostaSspseicutmroPmeertfrlyu"orooctanesulfonste from Liver for Analysis 200 Revisions _ ReNvuimsbieorn, Reason For Revision 1 SSeeccttiioonn 61.11..12AvCelarraigfiecoaftiownoofcHurPv1e1s,00notsyssttaenmdacrodmvpaolnueesn,tsre used for pSleoctttiionng 1l2i.n2e.a2r.r4egCrleasrsiifoinc.ationofsolvent ramp. Section 17.1 Changed from tachment B to A. ReDvaistieon 03/04/99 AnaloyfsLiiFvAesrCTEMxt.r2ac.t1Using ESIMS 3 Environmental Laboratory S65 159 Page 809 _Paga-3sT-- am Medical Department Study: T-6285.7 laboratorRyepoRretqueNso.t NFuACmTbeTro-XU-z0z370 Laboratory Study # sTeusa:Merial MMeatrhsoudFRieavilsSoanv,es|: * Asvramlyeinets SEoqfutiwpamreenVteSrsyiomn Number FRelSnqavmree:dValo: YStenpeer: DaDatceooffEAcuascsiioAnAlnya:st Cran Conceaton | TRAVEL For Cone. SGrioouppe/DToraee: rTaokwenJ fom he sy oSdero ISCnoaitnmicpalelneVr:oaltTuoamknee((ngmf/Lom:Lm)Th:aekkesneyfnrooomdhmee.hseyMsolsdy.o negation summary. DFiinlaultiCoannFea.ct(ogr/:mLT)a:kenCraocmttie sbyydivoilddienrg.he nin volume fom ie concenation AttachmenAt:Data Sheet Anat ofLiFvAerCETxMt2r0Uasicntg ESAS 3M Snvizonmental Laboratory rst 160 Page 909 Pager 3m Medical Department Study: T-6295.7 laboratorRyepoRretqueNso.t NFuAmCbTerT-OUX2-207350 3M ENVIRONMENTAL LABORATORY METHOD FLUEOXRTORCAHCETMIIOCNAOLFCPOOMTPAOSUSNIDUMS PFERROFMLSUEORROUOMCOTRANOETSHUELRFOFNLUAITDEFOORROATNHALEYRSIS USING HPLC-ELECTROSPRAY/MASS SPECTROMETRY Method Number: FACT-M-3.1 Adoption Date: 04/22/98 Author: Lisa Clemen, Glenn Langenburg Revision Date: 10 [o1(8 Approved By: 1y /7 /ho Labordiory Manager ' ahe Dae Vck he Group Leader ise A (hirer. Technical Reviewer 1/25/98 Date 9/23/92 Date 0 SCOPE AND APPLICATION 1.1 Sorcoopteh:er Tfhliusormoecthehmoidcails fcoormtphoeuenxtdrsacftrioonm osfeproutmasorsioutmheprefrlufidl. uorooctanfoensautel (PFOS) 12 Applicable compounds: Fluorochemical surfactants or other fluorinated compounds. 1.3 Matrices: Rabbit, rat, bovine, and monkey serum, rat whole blood, and rat milk curd. Word 695 Extraction of PFOSFAfComTAS3e1m and Other Fics 3 Environmental Laboratory = Jel Page tart? efi 3m Medical Department Study: T-6295.7 laboratorRyepoRretqueNso.t NFuAmCbTerT-OUXz-207350 2.0 SUMMARY OF METHOD 21 T(hPiFsOSme)tohroodthdeerscfrliuboersocthheempircoaclesdufrreomfosreerxutmr,acbtlionogd,pootramsislikumcupredrfulsuionrgooacntainoensupalifroinnagte rexetargaecntteda:ndPF5O.0S,mlPoFfOSetAh,ylPaFceOtaStAe.A,InEXthFiOs SmEe-thOoHd,, PsOevAeAn,flPuFoOroScEhAem,icaanlds FwCe-r8e07 `anmaolnyotcestieorn(psaeier3i.s0pDaertfiitniiotnieodnsi)n.toAenthyilonacpeatiartien.g rFeoaugrenmtliosfaedxdterdacttoatrheerseammopvleedaannddtphuet othnetno afinltietrreodgtehnreovuagphoraa3tocrcupnltaisltidrcy.syrEiancgehaetxttarcahcteds(0reaco0n.s2tuitmutendylionn1.f0iltmelroifntmoegtlhaasnsol, autovials. opemvmoss 00000000 3.1 PFOS: perfluorocctanesulfonate (anionofpotassium salt) C,F SO; 32 PFOSA: perfluorooctane sulfonylamide CF,,SONH, 33 PFOSAA: perfluorooctane sulfonylamido (ethyDacetateC.F,SO,N(CH,CH,)CH,CO; 3.4 ECX.FFOSSEO-NO(HC:H2,(CNH-e,t)hCylHp,eCrfHl,uo0rHooctane sulfonamido)-cthyl alcohol 35 POAA: perfluorooctanoate (anionofammonium salt) C,F,,COO" 3.6 PFOSEA: perfluorooctane sulfonyl ethylamide C,F.,SO,N(CH,CHJH, 37 FC-807 monoester C,F,,SO;N(CH,CH,JCH,CH,0-PO,H) 38 Surrogate standard: 1H-1H-2H-2H perfluorooctane sulfonic acid 4.0 WARNINGS AND CAUTIONS 41 Health and safety warnings 4.1.1 Uhasnedluniinvgerasnailmaplretciasustuie,onwsh,iecshpemcaiayllcyonltaabionraptaotrhyogceonatss., goggles, and gloves when 50 InterrereNces ---- eee 5.1 There are no known interferences at this time. 6.0 EQuipmENT ee emsi-------- 6.1 Tachceepftoalblloewing equipment is used while performing this method. Equivalent equipment is 6.11 Vortex mixer, VWR, Vortex Genie2 6.1.2 Centrifuge, Mistral 1000 or [EC 6.1.3 Shaker, Eberbachor VWR 6.1.4 Nitrogen evaporator, Organomation 6.1.5 Balance (0.1005) ExtractionofPOFSACrToMm3S.e1rum or Ot Fluid 3M Environmental Laboratory 53 Jol Page 20017 sages 3m Medical Department Study: T-6295.7 laboratorRyepoRretqueNos.t NFuAmCbTerT-OUX2-207350 7.0 SUPPLIES AND MATERIALS 71 Gloves 72 Eppendorofr disposable pipettes 7.3 Electronic pipettor, Eppendorf or equivalent 74 Graduated pipettes 75 Nalgene bottles, capableofholding 250 mL and 1 L 7.6 Volumetric flasks, glass, type A , 7.7 Volumetric pipets, glass, typAe 78 I-CHEM vials,glass, 40 mL glass 7.9 Crimp cap autovials 7.10 Centrifuge tubes, polypropylene, 15 mL 711 Labels : 712 Syringes, capableof measuring 5 uL to 50 uL 7.03 Syringes, disposable plastic, 3 cc 7.14 Syringe filters, nylon, 0.2 ym, 25 mm 7.15 Timer Note: Prior to using glassware Milli-QTM water. Ririse asnyrdibnogtetsleas,mriinnsiem3utmimoefswititmhesmewtihtahnmoelthaanndol3,t3imreisnsweisthfrom 3 separate vials, 80REAGENTSANDSTANDARDS 81 TbeyMpiellir-eQaTMgenwtagtreardaenwdatmear,ybMeilplr-oQv"iTMdeodrbeyquiavMalielnlti;-QllTOwaCtePrluusTMsed siynshtiesm method should 82 Sodium hydroxide (NaOH), J.T Baker or equivalent 83 Tetrabutylammonium hydrogen sulfate(TBA), Kodak or equivalent 84 Sodium carbonate (N3,CO,), J.T. Baker or equivalent 85 Sodium bicarbonate (NGHCO,), J.T. Baker or equivalent 86 Ethyl acetate, Omnisoly, glass distilled or HPLC grade 87 Methanol, Omnisoly, glass distilled or HPLC grade 88 Serum or blood, frozen from supplier 89 Control matrix or blank matrix for purpose of standards, QC checks, blanks, etc 810 Fluorochemical standards 8.10.1 PFOS (3M Specialty Chemical Division), molecular weight = 538 8.102 PFOSA (3M Specialty Chemical Division), molecular weight = 499 Exuaction of PFOFSACfTr:oMm:3S.er1um or Other Fluid 34 Environmental Laboratory 4 163 Page 30017 _BageTSS 3m Medical Department Study: T-6295.7 laboratorRyepoRretqueNos.t NFuAmCbTerT-O0X2-207350 8.10.3 8.10.4 8.10.5 8.10.6 8.10.7 PFOSAA (3M Specialty Chemical Division), molecular weight = 585 EXFOSE-OH (3M Specialty Chemical Division), molecular weight = 571 POAA (3M Specialty Chemical Division), molecular weight = 431 PFOSEA (3M Specialty Chemical Division), molecular weight = 527 FtmroCil-se8tc0eu7,ladmrioenwsoeteiergs,htatenr=d(6m35oM0noSepsetceiarlftlyuCohreomcihecmailcaDlivciosimopno)n.enFtsC.-80T7heismaonmioxetsutreerof 8.10.8 CSu,rFr,oSgaOt,eHs)tamnodlaercd:ula4r-Hw,epiegrhftlu=o4r2o8o.ctane sulfonic acid (1-H, 1-H, 2-H,2-H 8.10.9 Other fluorochemicals, as appropriate 811 Reagent preparation 8.11.1 10Nsodium hydroxide (NaOH): Weigh approximately 200 8 NaOH. Pour into a 1000 mL beaker containing 500 mL Milli-QTM water, mix until all solids are dissolved. Store ina 1 L Nalgene bottle. 8.11.2 1 N sodium hydroxide (NaOH): Dilute 10 N NaOH 1:10. Measure 10 mL of M1il0NliN-aQTMOHwatseorl.utiSotnoriention a a 100 125 mL mL vNoallugmeenteribcotftllaes.k and dilute to volumeusing. 8 .11. 3 0.5 M tetrabutylammonium h of1T0 uBsiAngianptopraox1iLmavtoelluyme4tritco ydrogen sulfate c5o4nmtaLinoifng1050N0 (TBA): Weigh approximately 169 NmaLOMHilalnid-dQi"luwtaetetor.voAdljuumset wtiotphH. g cMhialnlgie-sQTMabrwuapttelr.y. WShtiolree aidndin1gLthNeallgasetnmeLboottflNe aOH, add slowly because the pH 8113.1 TBA requachiecrkperisor t cach use to ensure pH = 10, Adjust as needed using | N NaOH solution. 812 Standards preparation 8.11.4 0.25 M sodium carbonate/sodium bicarbonate buffer (N2,CO,/NaHCO,): Weigh approximately 26.5 g ofsodium carbonate (N2,CO,) and 21.0 gofsodium bQiTMcarwbaotnera.te S(tNoraeHiCnOa)1 iLntNoaalg1 eLnevoboltutmlee.tric flask and bring to volume with Milli- 88..1122..12 PPfrlreuepoparaorrceehoPetmFhiOecrSalfslstutaoanrndodacarhrdedsmsifacorareltahsetacsntdacanrdedasr,pd(afcstourravapcepx.rboapmrlpilaeete,.onMeulwtoirckionmgposnteanntdard solution containing 1.00 ppm PFOS, 1.02 ppm PFOSA, 0.987 ppm PEOSAA, and 1.10 ppm E{FOSE-OH.) 8.12.3 Wtheeiagchraalppwreoixgihmtately 100 mg of PFOS into & 100 ml volumetric flask and record 8.12.4 BGraiinmg).to volume with methanol for a stock standardofapproximately 1000 ppm 8.12.5 Daiplpurtoexitmhaetsetloyck50soplpumt.ion with methanol for a working standard 1 solution of ExracionofPROFSAfComTS;e1rum or Otter Fad 3M Environmental Laboratory 15 Jey Page of 17 Bases am Medical Department Study: T-6295.7 laboratorRyepoRretqueNos.t NFuAmCbTerT-OUX2-207350 8.12.6 aDpiplruotxe. 50 ppm. the stock solution with methanol for a working standard 2 solution of 8.12.7 Dilute `approx. th0e.5s0t opcpkms.olution wit h methanol foar working standard 3 solution of 8.13 Surrogate stock standard preparation 8.13.1 Weigh approximately 50-60 mgofsurrogate CiF,,SO,H into a 50 ml volumetric flask and standard 1-H,1-H, 2-H, 2.H, record the actual weigh. 8.132 B1r0i0n0g-1020v0olpupmme. with methanol fora surrogate stockof approximately 8133 Prepare a surrogate sumogate stock to a working standard. 50 ml volumetric Transfer flask and approximately 0.5 ml of bring to volume with methanol for a working standard of 10-20 ppm. Record the actual volume transferred. 20 SAuPLE HanpLNG 91 All samplesare received frozen and must be kept frozen until the extraction is performed. 00 QuaLITY CorrroL 10.1 1M0a.t1r.1ixEbxltarnacktstawnod1m.0etmhLodalbilguasntkssofth appropriate matsx (serumo blood, with blood msaatmrpilxesbldainlkust.edS1e:e1 1w1i.t1h4M.illi-QTM water) following this procedure and use as. 10.1.2 Extract two 1.0 ml aliquotsof Milli-QTM water following thisprocedure and use as 102 Matrix`smpeitkheosd blanks. 10.2.1 tPhreepcaureraancdyaonfatlhyezeexmtartacitxiosnp.ike and matrix spike duplicate samples o determine 10.22 mParterpiaxrerceaccihvesdpiwkietuh esachaissaanmmppgllee sct.hosen by the analyst, usually the contro! 10.2.3 EAdxdpietcitoendalcosnpcieknetsramtaiyonbsewiilnlclauldeidnatnhde calibration curve mmiady-rfaalnlgneotfhtehlowin-irtaanlgceaolfibrheatiionnitciualrve 10.2.4 Prepare one matrix spike and matrix spike duplicate per 40 samples, with a 103 Continu`imnignicmaulmiboraf2tiomnatcrhiexckspsikes per batch. 10.3.1 aPornfetdphteahreiecnoiantnatdliancnuaalilinbygrzaecthicoeoncnktciudnriuvfief.negIfbctyahl>ieb3r0pae%tri,coenrnect-hadenicafklfyeszraeemnspcalemebpseltteowseeaennsnaultrhyeezteihdneiatafaclecrucrutarhcevye last acceptable check. 10.3.2 Prepare one check per groupof ten samples. For example,if a sample set = 34, prepare and extract four checks. Excacion of BOFSAfrComTSumo Other Fd 3M Environmental Laboratory 15% 165 Page sort? _pasersT 3m Medical Department Study: T-6295.7 laboratorRyepoRretqueNos.t NFuAmCbTerT-OUX2-20733 10.3.3 Pthreepianriteiaclaccuhrvceo.ntinuing calibration check from the same matrix used to prepare: 10.3.4 Tcuhrevee.xpAedcdtietdiocnoanlcesnptirkaetsiomnawyilblefianlcwliutdheidnthtahte fmaild-irnatnhgeeloofwt-haenginetoiafl tchaeliibnriatitailon claolwibernadtioonf ctuhrevec.aliTbrhaitsioins nceucrevses(afroyrifetxhaempalnea,ly5stppmbus~t1q0u0anptpibt,atreatuhseirngthoannly the 5 ppb.- 1000 ppb). 1.0 CALIBRATION AND STANDARDIZATION 111 Prepare matrix calibration standards Note: Bploionotd, caodadguTlBatAeasnidnbauirf;ftehrerteofeoraec,hmciennitmriifzuegeaitrucboentaascitnusntteipl d1i2l.u9t,iotnh.enAatddthi1s.0 mL. ofthe diluted matrix sample to each tube. 11.1.1 Twartaenrs)fetro |2m15LomfLsecrenutmroifruge1 mtuLboe.fTbhleoobdlo(boldoiosdsiimsidlialruitendc1o:m1pwoistihtiMoinltloi-miQlTMk urd and can that matrix. be used in placeofmilk curd for standard curves when extracting 11.1.2 Ivfomlousmtessaemqupalletvootlhuemseasmaprleelvesoslutmheans.1.D0omLn,oteexxttrraaccttsbteanldoawrd0s.5w0itmhLmoaftrmiaxtrix. Record the sample volume on the extraction sheet 11.1.3 bWehtiwleeepnreapliaqruiontgs.a total oftwenty aliquois in 15 mi centrifuge tubes, mix or shake 11.1.4 TTywpoica|lmlyLusaleiqtuhoetss,taondaortdhecroanpcpernotprraitaitoensvoalnudmsep,iksienrgveaamsoumanttrsixlibstleadnkisn.Table I, aetigthhteeeenndsotfatnhdiarsdsseactnidont,wtoomsaptirkie,x ibnladnukplsicate, two standard curves, for a total of 11.15 tRheefewrortokivnalgirdaatnigoensraenpdortthseFLAiCnTea-rMC-a3l.i1br-aVt-i1onaRnadngFeAC(TL-CMR-)4.fo1r-cVa-l1ib,rawthiiocnh list curves. 11.1.6 UstsaendAartdtsa.chSmeeentSDectaisonan13si.d0 i0n ccaallccuullaattienagcttuhaelccoonncceennttrraattiioonnsosfofthPeFwOoSrkiinng calibration standards. 11.2 Tstoancdaacrhdfsotrantdhaerdc,obnlcaennkt,raotrioQnCtochfealclkw,iatdhidnatphperocparliiabtreataiomnoucnurtovfesruarnrgoeg$atpepbwo-r1k0i0n0gppb. 11.3 1E0xtersatcatblsipsihkeeadcmhatinriitxialstcaunrdvaerodns ftohlelmowaisnsgs1p2e.c6t-r1o2me.t1e6ro.fthis method. Use these standards Extraction of PFOFSACfTr:omM3S.er1um or Other Fld 3M Environmental Laboratory 153 Ib Page sof 17 pages 3m Medical Department Study: T-6295.7 laboratorRyepoRretqueNso.t NFuAmCbTerT-OUX2-207390 Table 1 Approximate spiking amounts for standards and spikes using 1.0 mi of matrix (approx. conc.) analyte in matrix [o1 stopm[- To |1 0Btaspmem || [So[ opo pms|t 5 2o 0 | po0p m0lsoppmm m || 50pm |0 oo0s0pem | [sooppm [5 | 0250pm | Approximate spiking amToaubnltes for standards and spikes using 0.5 ml of matrix (approx. conc) analyte in matrix -- TT sm | [os|t1o 0 po5op 0t2oppeom mm | Soopp5m| 000mm | [ [ SS 00o p3e T0 5Om |p o00.a1s000m pppemm | [Soomm[5 0s00pm | [0 |0 70 | p T00m ppm | 120 proceoure 12.1 Obtain frozen samples and allow to thaw. 122 Vortex mix for 15 seconds, then transfer 1.0 mL or other appropriate volume to a 15 mL polypropylene centrifuge tube. For blood samples, remove 0.5 mL and dilute to 1.0 mL `with Milli-QTM water. As soon after diluting as possible, pipet diluted blood into TBA`buffer mixture shown in step 12.9andmix well. 123 Rerum samples to freezer afer extraction amount has been removed. Extacionaf FOFS AfrCamTSerum or Othe Fluid 3M Environmental Laboratory 15% 767 Page T0017 PagesT 3m Medical Department Study: T-6295.7 laboratorRyepoRretqueNos.t NFuAmCbTerT-OUX2-207350 124 Record volume the volume transferred on the extraction from the sample. worksheet. The final methanol volume equals For example,if 0.5 mL is removed for a blood the sample, the final methanol volume will equal 0.5 mL. 12.5 Label the tube with the study number, sample ID, date and analyst initials. See attached worksheet for documenting the remaining steps. 12.6 Spike Lor 2 each matrix with the appropriate in that section for the calibration acmurovuentsotafnsdatradnsd.arAdlassodeprsecprairbeedmaitnri1x1.s1piokreTsaabnlde continuing calibration standards. 12.7 Spike all samples, including blanks and standards, ready for extraction with surrogate. standardasdescribed in 11.2. 12.8 Vortex mix the standard curve samples, matrix spike samples, and continuing calibration samples for 15 seconds. 12.9 To each samplZ, add | mL 0.5 M TBA and 2 mL of 0.25 M sodium carbonate/sodium `bicarbonate buffer. 12.10 Usinag volumetric pipette, add mL ethyl acetate. 12.11 Cap each sample and put on the shaker for 20 minutes. 12.12 Cseepnatrraitfeud.ge for 20 to 25 minutes at approximately 3500 rpm, until layers are well 12.13 Transfe4r mLof organic layer, using a 5 mL graduated glass pipette, (0 a lean 15 mL. centrifuge tube. Label this fresh tube with the same information as in 12.5. 12.14. Putcach sample on the analytical nitrogen evaporator until dry, approximately 2 to 3 hours. 1215 gArdaddu1a.t0emd Lpipoerttoe.theMreatphparnooplriavtoeluvmeoto laodfdumeeqtumhaalnseotlhtoinietaicalh vceonlturmiefoufgesatumbpeluesiunsged for the extraction. 12.16 Vortex mix for 30 seconds. 12.17 Asyurtiancghe.20F.i2ltuerminntyolaonLmSemshL fgillatserstaoutao3vicael soyrrliongwe-vaonldutmreanasufteorvtihael swahmepnlenetcoestshairsy. 12.18 Label the autovial with the study number, animal numberand gender, sample timepoint, 12.19 `matrix, final solvent, extraction date, and Cap and store extracts at approximatel4y analyst(s) performing C until analysis. the extraction. 12.20 Cnootmepbloeotkeotrheinecxlturdaectiinonstwuodryksbhienedetr,,a3tstaacphperdoptroitahties document, and tape in the study ExtractionofPOFSACfToMm S3erum or Other Fluid 3M Environmental Laboratory TY 168 Pages ory? paseo Sm Medical Department Study: T-6295.7 LaboratorRyepoRresqueNso.t NFuAmCbTerTOUXs-s073s0 5.0 DATA ANALYSIS AND CALCULATIONS 13.1 Calculations 13.1.1 CcaallicburlaartieonacsttuaanldacrodnsceunstirnagtitohnesofofllPoFwOiSn,g coaruottihoern applicable fluorochemical, in FLmLoofsftsatnadnadradrd+ xmLc.onocfensrauimoongaotefstansdtaanrddar+d(mugaJl maT vole (i) - Final Concentration (g/mL)of PFOS in matrix 140MevHoDPERFORMANCE 0000000000000 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 The following quality control samples are extracted with each batcoh f samples to ensure the qualityof the extraction and analysis. 14.2.1 Method blanks and matrix blanks 14.2.2 Matrix spike and matrix spike duplicate samples to determine accuracy and 14.2.3 pCinroientcitiailsnicuoainlonigfbrctaahtlieionreaxctturiraovcneticohneck samtopdelteremisn the continued accuracy ofthe 150 POLLUTION PREVENTION AND WASTE MANAGEMENT 15.1 ShloiacgmahptleBdeTiwUnastchtoentilsaaibndoerirasstp,oorsayen,dd iunsbeidoghlaazsasrpdicpeotntteaiwnaesrst,efisladmimspaobsleedsionlvbernotkweansgtleasiss dciosntpaoisneedrsin 60 Reconps 16.1 Complete the extraction worksheet attached to this method, and tape in the study notebook or include in study 3-ring binder, as appropriate. 7.0 ATTACHMENTS 170 AtaAc,Ehxtacmtioenwornkshteet 17.2 Attachment B, MDL/LOQ values 17.3 Attachment C, LOQ Summary 17.4 Attachment D, Calibration standard concentration worksheet 5.0 Reeenences 18.1 The validation reports associated with this method are FACT-M-3.1 & 4.1-V-1. Excacion ofPFOFSaocmtSserr ar Ofer Fld 3M Environmental Laboratory Ho Je) Pageooriz EagerTEL 3n Medical Department Study: T-6295.7 laboratorRyepoRretqueNso.t NFuAmCbTerT-OUX2-207350 1199.01 arFeAcCTr-eMp-4D.o1,cy"mAnealsyssisofSerum or Other Fluid Extr0act0s 0for0Flu0or0och0em0ica0ls0us0ing00 HPLC-Electrospray Mass Spectrometry" R00 e0viRseivo0 nsioN0 s 000000000Re0 vision Numb1 er VnaelwidAatPiU-oMnSof(MmeSt)hosdysttoemiRsne,calmusodonenk7eFfyolrusRoererovucimhsecimrioocnsaslsv,alaindaatdidoint,ional matrix, 07/D0a1t/e98 ikemepprionvgemaenndtsstotroiinognpoplaiicriiensg, eextct.raction, MDL study, updates in record ExtractionofPROFSACfTr:omM3S.er1um or Othe Fluid 3M Environmental Laboratory Het 170 Page 10017 _Bagetrezr 3m Medical Department scudy: T-6295.7 LaboratorRyepoRretqueNso.t NFuacmtbeTroUx3-907350 Study # Sample `Number FC-Mix FC-Mix FC-Mix | Dateand approx. 0.5 ppm | approx. ppm| approx. 50 ppm| Initials for seus wawc,hal pom | Payctu pom| sacwat pom| CoSmhmeonrts --T rrHmw mm r Tp------ Fe eo e ee e tee meeseteoy)reece] ee r eeeer m oe r eee]etme) fe eee mmee elt F I r mm-- ~] meee-- ee] r r r r r r T r T r r r r r r Lr r r r r 7 ee -- Ee ---- r -- T -------- ep -- } r r r r p r Ir N rr r r r r r r T r 1] r EEBM a Soemnpbmoboesvhomeehesnp we bead e o {SemExtactonMethod ___ Bie gi -- premeieee -- (BpeneMuox _--_ Wowme ---- pigimaan | wm | ET | [Comfoeed0 mn Comfgespees | (mm J FEERrRErCo Colsy Sad I ArTamA s. sani. nd eT ens [EP ------Eriof FnOFSafcoTmaSaea1m Other Fd 3 Environmental Laboratory Ket 177 Page? pagers 3m Medical Department Study: T-6295.7 laboratorRyepoRretqueNso.t NFuAmCbTerT-O0X2-207350 MDL/LOQ values for Rabbit Serum: Compound | MDL (ppb) [PROS |T38 [PEOSAR [285- 439 [Sppb-tovopep | | LoQ LinearCalibrRaangtei(LoCRn) | (ppb)| Approximate concentrations to be usedforpreparing the | | Standard Calibration Curve | [9.05 [10pm --Todopp | Monoester | 149 | 248 || | MsMDDtLLmssttduodnyLl.yO.AQnPsyeeqssuasnrteiaetrritoaonoiPpAeeyCrfToNrMome3Vda1floar&mW4oOn.To1c.ws1atsrfewoirrlmbesipecainfnicse rom | MDLILOQ values for Rat Serum: `Compound |MDL LOQ[LinearCalibration Range (LCR) | (ppb) | (ppb)|SAtpapnrdoaxridmaCtaelicbornacteinotnraCtuiornvse tobeusedforpreparing the [PFOS [2[7 403 10 ppb 1000355 ] Monoester | 149 | |248 | DMDLLssnudd.LOAQnsyeausainmtaiatteisoonlpyeroNmoedvaloird MmoBnLeewissreweirlibneaabnie om csimae only. Plese tefe 0 FACT-M.1 & &1-v1 for specifics CMoDmLp/oLuOnQd|valMueDsLfor|BovLiOneQ|SerLuimn:ear Calibration Range (LCR) [[PFFFOOSSA (ppb) (ppb)| AStpapnrdoaxridmaCtaleibcroantcieonntrCautriovnes to be used for preparing the [1750014 [[167600||sZs5ppppob-=t1o00000ppepep | [PPOEOASAER Tags [iss |Isppo--t000ppp Monoester ] 248 | MMDDLL astnddyL.OQAanryeqeusataitmiaatetsononpley.rfoNromevdalfiod MmoDnLecwassedweitlerominaanble from Ninostdeaatda is available for MDL or LOQ in Monkey Serum. Use validated Linear Calibration Range Plesse see Attachment: C (LOQ Summary) and MDL study in FACT-M-31 & 4.1-V-1 for specifics. AvschmenAt;Exacion worksheeEtxacionofFOFSAfCoTm MSe1am or Otter Fluid 3M Environmental Laboratory Fo 122 Page 12017 BaggTed 3m Medical Department Study: T-6295.7 laboratorRyepoRretqueNso.t NFuAmCbTerT-O0X2-207350 MDL/LOQ values for Monkey Serum: Compound [MDL | LOQ| Linear Calibration Range (LCR) 1 | (Pb) || (ppb)| SAtpapnrdoaxridmaCtaelicbornacteinotnraCtuirovnes to be used for preparing the || FFOSA 138 [223 4.39 | oMMnlDDy.LL saPtlnueddayLs.eOeAQenayrrqeoueasFnttAiiCmsaTtt-eiMso.no3npl.ey1.rf&oNromev4da.1lf-iVod.r1MPfFoDrOLSwswapeiscilfdbileectearnmtuniambaiteefrom | [7:09 | MDL and LrO eestiQ mates only. No vid MDL vs Geermirsbie om PFOSAA |288 | oMnlDy.L sPlyea.se dreaeyrqto FiACTn-MLp3r1t&om$e1d-Vo1r1PfoOrSspAecwilfilc an cate | | 904 | MDL nd LOQur ctmates oly No valid MDLne determinable For MDL study. Ay auactiaion performed ox PEOSA will br a ssn, EFOSE-OH| 390 ]|{ 124 |MMonDDlyLL. snPtludedayLs.OerQeAafneryretqouxatFniAsmCatTtae-isoMn-o3pl.ey1rf&oNrom4.ev1da-lVfio-dr1fMEoIDrFsLOpSewEcia.ftiOccFse.ewrmiimbeabalne Fors | POAA TT [137 |MesDtiLmantedonlLyO.QPslresestmealteetsooFlAy.CTNMo-v3al1id&MDL&.1.wV-a1sfdoeresrpmeciinfsibcsle For MDL study. Any quaniaton performed for POAA will be am etimte | | ony. Pease refe0r FACT-ML3.1 & .1-V- for specifics | PFOSEA 1.09 | |I 3.48 | | cMMtDDiLLmtsaetnudodnyLl.yO.AQnPayeraeqsueesatrniiemaaftieearosnoFnplAeyrC.fToN-romMe.vda1loird8 MPFDO4L.S1.wEV-aA1s-fOdoreFtsepwreimcliilnfbaiebclsaen from. | Monoester | 149 | 238 ||MMDDLLsdnyd.LOQAssyegsusiamanieorlpye. sNtoovaoirMEDFOLSwEaOdtetwoimmenssbn rom | estimate only. Please refer fo FACT-M-3.1 & 4.1-V-1 for specifics. | MDL/LOQ values for Rat Whole Blood: `Compound |MDL LOQ | Linear Calibration Range (LCR) (ppb) | (PPD) | Approximate Concentrations to be used for preparing the [PPFFOOSSA [1251596 Standard Calibration [5 pp-b 1000 ppb Curve 565 [t0ppo-t000ppy ] [PFOSAA [173 [550 [55 ppb-- 1000ppb EFOSE-OH || 789 | | 25.| 1 | e0 MsDtLima2mraodyn,LlyO.RQePalyreqewuseenstrtieamfaeitroetsnopoTnelAyr.CtoTNmoMe-vda.iloid&EM$DOL+Swas.ffdotersswepriimitbnsea.bni rom PPOFAOASEA | 244723 [1511550ppppbb--- 110000p0ppp0bb. Monoester | 580 | 185 | MDL ndCOQare ctmarcs only No valid MBL wis eerminable om 1| } cMsDiLmasetuodnyl.y.AnPlyesqusaenrteifteartitoonPpAeCrTfMo-r3m.e1d for&m$on1oe.s1tefrowrislplebciefiacns. Please see tachment C (LOQ Summary) and MDL study in FACT-M-3.1 & 4 1.V-1 fo specifics. AtachmenAt: Extraction worksheeEtxvacionofPOFSAfrComTSerum or Other lid 3M Environmental Laboratory Het 173 Page 130r17 pagazes om um ng mee o Ion Pairing Extraction of Fluorochemicals from Serum and Analysis bAy PIMS(MS) Bovine 21th 70m 259mb woos | | SE Ra|r rrrme eRdil rWdReS8oBn)n EE100ee0p ER ER) EB | Es Bovine Iw Tom Seo | 1000p rid Monk wd wd |__100ppb 1000ppb | `Compound: PFOS omer ra Seg [[oEmet[vorn[avonon][mrsmm[[rreonnrv] [Teron[ecn orono a[ronr 01 Enviromental Laboratory M5179 eases06-- 3m Medical deparcaent study: T-265.7 LaboratorRyepomratqetcot. HFoArCsTesTobx-a0n3e0 SeiCompound: FPeFOeSsA | Tomer To| Re TCT | Re Io | Saeunt|| eis mens | irseomn|| wMiwn msiee | [raw [500-1070 [500-160 sso-t0m| 500-251 wa [89-1670 89-1000 Soro 300-100 asim| some wn | moi samme [[f5n00-107| 500-10 _soaiom| om sor m1 0 |se3-170 sma Von| 500-107| 500-10 swam | 51s ma [mee | STom`Compound: | PSFmnrOIeSgeeeAtA|||.| Tioartr es ToSmooeen||| wTor iveer)n e iTonwotT'e||||oTpSaenr ToOmRT i Ra [0-110| 530-1500 to| 530-200 T0420| Tor 160 3 io To [sme [sms ww| sm Toa| ne 1am EE EE EE EE Compound: EtFOSE-OH So Ramnwg | esr (osbogmL)| (spblingiml) soe |430|-av1100 [oonveem|soin Von| 434-100| avium Cot (ope Re oary u || e ra T (ppbXngiml) (pobXn|g/(pmpbbd)ngiml) _(ppbegmb|) sna tuo | 4342 e342 8 v0 ro wm|ovm me [ose maw| wien mam [me ee ion 708 omrr OeFi 54 Environmental Laboratory Hor 175 Fagerer Laboratoly request Number 57500 [[CmSomoopou|nd :ca PePoaOnpAreAss||| oegnee wis Coomoen|| compen | FoeeeiaerlooeowRnSTs'||||Rewoeh iree-- e di Rabbit Bovine 501-1480| 513-1510 25.8-1000| 5.13-258 | 501-1 s-1s10 a-ia0| 513m wa [102-1510 533-2| errs wa moi | Monkey SO1-1480| 513-1510 102-1000| 513-102 513-102 | 102-1510 258-1000 | So | Compound: oaePFOewtSE||A| eps | Twowpo TeOmR| Ren eese w CeRwT|| E vEee TaO Sn ovutiom ns |) envpet) oe| os sm _ ony [Rabi [some [5[35--1155000 | m[5-031-1i5100 2ws8aor0i00[|35S3ia-a2s8 wa sm | [m10i2-e10 70 woe w | [=| So ] Snes | Compound: Monoester Tarr ore | EE Ravbic | 494-1450 | LT 578-978 oa wa aren aren 7am J[Ta votres,|aBsEme| rsaT:16e B3t0R ie tonVmSgompeosy(rnod sns,itgh), JR 3 Environmental Laboratory acta HA /7 rp ssarin a 3m Medical Department Study: T-6295.7 laboratorRyepoRretqueNos.t NFuAmCbTerT-OUX2-207350 Ton Pair Standard Curves - Fluids Prep date(s): AnSaalmyptlee(sm)a:trix: Methodseevision: TFaCrmgeitxsantadlyatep(psr)o:x. 03500 ppm: FFCCmmiixx sstddaapppprrooxx.. 55.00.00 pppmm:: Surrogate sta approx. 1771 ppm: `Standard number: FEiqnuailpsmoelnvetntnuamnbdeTrN:: Blank fuid/identifer: WWIiRs64e0 WW339988..660359 AcPtRuOaSl concFeFnOtrSaAtionsFEofOSstAaRndarEdWsOnSEt-he FCPmOiAx PROSEA Momwesis AT A Swcolne Swdcmolmc Ssupcoenlc oScxone wel Swcmonle Sudgcmonhc Scorne upml spAimetdml VFealolme [T0o330w0T00530077[T0o3s37 T|u0m3o0r1 | 0s3w%|o0msar[|eossoo || oaobm o pr tooniss|] [SS0w0 [ssoorr[[sswm[Tssoo [[ssemo gsamr[ssoorrT|omoosl | ttoorls ] I S00 sor S32 | sor |sJ eTsai[sor] Coo1m 1 tos | [[CC3e0o0 [sSeer[[s5ss[ [s0 or1 [sSs[|ssiarr]ssroi || oooo t1o05|] CoCamlcoulaItedScoornce[ntr5ati2ons o|f sstaondtard|s fat5he[sasmplaermat|rixsor |oo 1 1055] TFFaiOlS FPFcOSK FFRiOSmAX AEmWoOSnE fFlORaAn PPFuOlSEmA MSaaomme SSuopae AmAiT k mL ON E 1S OY EO [376 T7588 | lod | 57% | 993 | joa | vu | T E3sTas00S a6 Toa | asa | ass | Ta S00 S13 | 208 #04 | Surrogate | Famalcon|c O76 [TORY loa ors 993 |tz| 978 | ngmi EC NN JO N+J J 1os A} 7735 TsTioe 1S TeTR Ts ae 77 fo--iy 5 81 Validatedranges - approximate concentrations [Sfe --rmTTT[5[-T1P0F0OSppb |T10P-F10O0S0pAp_b|_1P0R-1O0S00ApApb__|E1W0O-1S0E00-pOpNb _ | 10F-O75A0pAp_|_3P5-F1O0S00EpApb|| [ [FRonakt ey [Est1i0m-a1t0e0s0opnplyb||Us2e5v:a1l0v00epspfbor|[R1a0-b1b000ppb ||--50---501 0pob | 5-1000ppn |[ 51000| 0p0 | Atachment fon Pac Standard CuErxvressctionofPFOFSAfCoTmSerum or Other lid 3M Environmental Laboratory D177 Page 170017 Paget Laboratory Taausel Senr Wa ORG 3M ENVIRONMENTAL ABORATORY WEDS HT, we METHOD ANALYSIS OF POTASSIUM PERFLUOROOCTANESULFONATE OR OTHER FLUOROCHEMICALS IN SERUM OR OTHER FLUID EXTRACTS USING 'HPLC-ELECTROSPRAY/MASS SPECTROMETRY Method Number: FACT-M-4.1 Author: Lisa Clemen, Glenn Langenburg Approved By: D [95 Toboatory Marager Yh b. - Group Leader i So hi nie. `Adoption Date: 4/22/98 Revision Date: 1011-15 Asp Bue 9/21/98 Date 42D4a)e a 1L.01 SsSucctoaopcert:eanTvthspiwsiAmnregetuhHiocPdaLitCio-ornshlechanoastyptmrsaaosfyesxsapcetemsofmreoym.sero blood for aoroahamical 1.2 Applicable Compounds: Fluorochemical surfactants or other fluorinated compounds, or other ionizable compounds. 1.3 Matrices: Rabbit, rat, bovine, or monkey serum and rat whole blood or milk curd. Werds0.195 oy ofSeuFacFtidvaEsc Using ESAS 4 Envixonmental Laboratory +4 17 ---- rages 3m Medical Department Study: T-6295.7 laboratorRyepoRretqueNso.t NFuAmCbTerT-OUX2-2073s0 20S 2.1 ummaRvoFMeTMOD This method describes the analysis of f luo ro ch em 000000000 ical surfactants extracted from serum, 2`swhaoplperobplroioadt,e.orTmhielkacnaulrydsiussiinsgpeHrPfLoCr-meldecbtyromsopnriatyo/rmiansgsasspiencgtlreoimoentrcyh,aroarctseirmiisltairc soyfsatem Mpa/rtzi=cu4l9a9r.flSuaormopclheesnimcaaly,asluscohbaesatnhaelpyozetdasussiiunmg paenrfAlPuoUrMooSc/taMnSessulyfsotneamteto(PfuFrOtShe)r avneiroinf,y compound ideitification. M3p.1eAtemvosmphoersicsPressure Ionization (API): The Micromass platform sy0st0em0s 0al0lo0w 0fo0r 000 `ivnacrliuodues bmuetthaoredsnootfliiomniitzeadtitoo:n EblyecuttirloiszpirnagyvIaorniiozuastsioounr(cEeSsI,),prAotbemso,spahnedriinctePrrfaecsessu.reTchheesmeical aTtonmiozsapthieorni(cApPre)s,suTrheer(im.o.spnrotayu, entc.advTahceeuuimorn)i.zation process in these techniques occurs at 3.2 pErleescsturreo,spwrhaeyreIobnyizioantiizoanti(oEnS,ocEcSuIr)s:tahrmoeutghhotdheofprioodnuizcattiioonnopfetrifnoyrcmheadragteadtdmroospplhetesriicn a strong electrical field 3.3 `MTahsesAPSIpepcltartofmoermtsrya,reMeaqsusipSppeedcwtirtohmqeutaedrr(uMpSol)e, mTaasnsdseemlecMtaivsesdeStpeectcotrrs,omeltoensr a(rMeS/MS): MseSlecmtaivyelbyedeismcprliomyiendatefodrbiyonmdaestsecttoiconhaorrge sraetriioes((mMzS)/aMnSd)sufobrsemqoureentslpyecdieftiecctfedr.agAmentsiantgiloen information. 3.4 sCyosntveemnsti(oponsatl1V9s9.8Z)-ustpilriazye apr"oZb-espirnatye"rfcaocnef:orTmahteiolna.testTmhe sopradoyfeMmeiitctrleodmfasrsosmpalpartofobremis corotnheoagpoenratlurteo,tahteecropnaesaspienrgtutrher.ouIgnhtahetocrotnuvoeunstipoantahlwcaoynifnorthmeatcioounntiet riselaeicmterodddei.recTtolyugaththtehe Hcoonwfeivgeurra,tiZo-nspirsadyifcfoermepnot,netnhetsmeatnhdocdosnovfeonpteiroantailocno,mcploenaeninntgs, aarnednmoaticnotmepnaatnicbelearweitthheosnaeme. sanportahyers,ysbtuetmso,nletyc.)with similar systems (i.. Z-spray components are compatible with other Z- 3.5 sMyasstesmsL.ynCxurSroefnttwlayrMe:asSsyLsytenmhsaosftWwianrdeodwessi9g5neadndforWithnedsopewcsifNiTc 3o.p1ervaetrisoiononsf. thAelslevpelrastifoonrsm laroersiQmuilaatrt.roFIofrMmaosrseLydentxaiolrs MseaestshLeymnaxnNuaTlUsSpeEcRif'icStGoUtIheDEin)strument (Micromass Platform 4.0 WARNINGS AND CAUTIONS 4.1 Health and Safety Warnings: 4.1.1 aUpsperocaxuitmiaotnelwyit5h00th0eVvoolltst.age cables for the probe. The probe employs a voltage of 4.1.2 aWnhdecnlohtahinndgl,ing samples orsolvents wear appropriate protective gloves, eyewear, Word 60.195 Analysis ofSeruoFmArCFTlMu.i4d.E1xtract Using ESMS 3M Environmental Laboratory +70 /79 Page20r9 amet 3m Medical Department Study: T-6295.7 laboratorRyepoRretqueNso.t NFuAmCbTerT-OUXz-2073s0 42 Cautions: 4.2.1 DIfothneotbaocpkerparteesssoulrveeenxtcpeuedmsps40a0bobavre, ctahpeaHciPt1y1o0f04w0i0llbairni(ti5a8t0e0auptsio)mabtaiccksphruetsdsouwren.. 42.2 Do not run solvent pumps to dryness. 5.0 INTERFERENCES 5.1 Tshooumlidninmoitzbeeiunsteerdfeforrenscaemspwlheesntoarnaagleyzoirnagnsyapmaprlteosfifnosrtpreurmfelnutoartoioocnttahnaotacroe(mPeOsAAi),cotnetfalocnt `with the sample or extract. S0 6o.1pouEqeu0 iwpemesnrt li0 sted below0 may be m0 odified i0 n order to 0 optimize 0 the system.00 6.1.1 Micromass Electrospray Mass Spectrometer 6.1.2 HPL100 low pulse solvent pumping system and autosampler 7.0 SUPPLIES AND MATERIALS 71 Supplies 7.11 High purity grade nitrogen gas regulated to approximately 100 psi 7.1.2 HPLC analytical column, specificsto be determined by the analyst 7.13 Capped autovials or capped 15 mi centrifuge tubes 8.801 RERAeGaEgNenTtSsANDSTANDARDS ~~~ 8.1.1 Methanol, HPLC grade or equivalent 8.1.2 bMeilprloiv-iQdewdatbeyr,a aMlillwlait-eQrTuOseCdiPnlutshissymsteetmhod should be Milli-QTM water and may 8.1.3 Ammonium acetate, reagentgradeor equivalent 82 Standards 82.1 Tpryeppiacraeldlydounrienmgetthheoedxtbrlaacntki,ononperomcaetdruirxe.blSaneke,FaAnCdTt-eMn-m3at.r1ix standards are 9.0 SAMPLE HANDLING. 9.1 arFreessthormeatdriinxcsatpapndeadrdaustaorveiaplrsepoarrceadpwpietdh1e5acmhlacneanltysriisf.ugeExtturbaecsteundtisltaannadlayrsdiss.and samples 92 aIpfapnraolxyismiastweillyl4beCdeulnaiyielda,neaxlytsriasctceadnsbtaenpdearrfdosramendd.samples can be refrigerated at AnalysisofSeruFomArCFTluMi.d 4Ex1tract Using ESMS 3M Environmental Laboratory 190 Page 309 maser 3m Medical Department Study: T-6295.7 laboratorRyepoRretqueNos.t NFuAmCbTerT-OUXz-207350 10.0 QuaLITY ConTROL 10.1 Method Blanks and Matrix Blanks 10.1.1 Analamyetzhoed blank aanmatdrix blank prior to each calibration curve. 102 Matrix Spikes 10.2.1 Amnianliymzuemaomfat2risxpiskpeiskpeearnbdatmcaht.rix spike duplicate per forty samples. With a 10.2.2 cEuxrpvee.cteAdddsiptiikoencaolnscpenitkreactoinocnesntwrilaltioalnsimnatyhefmalildi-nrtahnegeloofw-trhaengieniotfatl hcaliinbirtaiatlion calibration curve. 10.2.3 See Section 13 to calculate percent recovery. 10.3 Continuing Calibration Checks 10.3.1 Achnaanlgyeze(&am3i0d%-)rainngpeeacakliabrreaatioocncusrtsa,nrdealradtiavfettroetvheeriyntiteanlthsisaanmdpalred. cIurfaves,igsntiofpictahnet rwuinl.l bOenluysetdh.osTehseamrpelmeasinainnaglyszaemdplbeesfomruestthebelarsetaancacleyzpetda.ble calibration standard 10.3.2 See Section 13 to calculate percent difference. 11.0 CALIBRATION AND STANDARDIZATION ILI mAneaalnyzoefttwhoe esxttarnadcatredd vmaaltuceisx, sattacnadcahrdsstaprnidoarrdtocoanncdenftorlaltoiwoinn,gwielalcbhespelootfteexdtrbayctlsi.neTarhe regression (for the calibration curve using MassLynx or other suitable software. 112 Tdihsecr#etvioanluoeftfhoretahnealdyastta sahnodudlodcbumeen0t.9e8d0 oarppgrreoavtaerl.ofLtohweePrrovjaelcutesLemaad.y beacceptable at the 113 Istfatnhdeacrudrcvuerdvoe(eisfnnoetcemseseatryr)eqaunidrermeeanntasl,yzpee.rform routine maintenance of reexiract the 11.4. Fusoertphuerploosweseonfdaocftcuhreaccayliwbhreatnioqnuacnutrivteatriantgheorwthalnevtelhsoffulalnarlayntgeeo,fittmhaeystbaendnaercdescsuravrey.to cEaxlaimbprlaet:ionwchuervneactotnesmipsttiinngg toofqtuhaensttitaantdearadpsprforxoimma5tpelpyb t1o0 1p0p0bopfpbanraaltyhteer,tgheannertahetefuall rreagnrgeesosfiotnheweciugrhvtei(n5gopfphbi0gh1c0o0n0cepnptbr)a.tiTohnisstwanidlalrdrse.duce inaccuracy atibuied to linear 12.0 ProcEDURES 12.1 Acquisition Set up 12.1.1 Calfiiclkenoanmsetaurstibnugtlteontiienrt-hMeO-ADcqAuYi-sliatsitondiCgoitntorfolyePaarne-ls.amSpelteunpumabsearm,palsesiigsnt.aAmsestihgond (MS) for acquiring, and type in sample descriptions. Analysis ofSerumFoArCPTiMd.AE1xact Using ESS Page dor 3M Environmental Laboratory +E /f/ Eager 3m Medical Department Study: T-6295.7 laboratorRyepRoretqueNos.t NFuAmCbTerT-0UX2-207330 12.1.2 STIoRcr(eSaitneglaemIeotnhRoedcocrldiicnkgo)n. scan Set IbountitzoantiionntMheoAdcequaissiatpipornopcroinattreolanpdanmealsasntdos4e9le9ctor oStahveer aacpqpuriospirtiiaotnemmeatshsoeds.. AI MfuSll/sMcSaniinssutsruuamlelnytcsolalreecetmepdloayleondg, with the SIRS. additional product GonUIfDraEgmTeOntDatAioTnAinAfCorQmUaItiSoInTmIaOyNbfeorcoaldldeictteido.nalSienfoMrimcartoimoansasndMaMsRsLMyn(xMultiple Reaction Monitoring). 12.1.3 Tsytpaincdaarldlsy atnhed aennadlsytwiciatlhabsaetcthorfuenxtsreaqcuteendcmeabtergiixnsstwaintdahradss.etof extracted matrix 12.1.4 Ssaammppllee.s Saorelvaennatlybzleadnkwsisthhoacuolndtbienaunianlgyzcaeldibpreartiioodniccahlelycktoinmjoencittedorafptoesrseibvleeryantaelnytthe carryover and are not considered samples but may be included as such. 12.2 Using the Autosampler 12.2.1 Set up sample tray accorditnog the sample lit prepared in Section 12.1.1. 12.2.2 aSneatl-yusptthcoenHsPi1de1r0s0/aappurtoopsraiamtpelefroraotptthiemaflolrleoswpionngsec.onRdeictoiorndsaocrtuaatlccoonnddiittiioonnsstihnethe instrument logbook: 12.2.2.1 Sample size = 10 kL injection with a sample wash 122.22 Injecusample = 1 12.2.2.3 Cycle time = 15 minutes 12.2.2.4 Solvent ramp = Time [0.00min 20mm Ammonium acetate [45% 5% [0min[71 00 i0% %| Note:`wIinththais"iWnasittriunmgenftorcionnlfeitgsutraartti"omn,estshaegreunbemfuosrte tbhees"eSttuaprto"nbutthteocnlicstprroesspsreadyosnofttwhaere. HP Workstation. 122.25 Press the "Start" button. 123 Instrument Set-up 123.1 Refer to FACT-EP-3.0 for more details. 12.3.2 Check the solvent level in reservoirs and refillifnecessary. 12.3.3 tChheetcipk. thTehsetatiinplsehssousltdeeblecafpliatllwairtyahtntohjeaegngedodfetdghees.prIofbet.heUtsipeisanfoeyuenpditeocebeto check unsatisfactory, disassemble the probe and replace the stainless steel capillary. Analysis ofSerumFoArCFTlu:idM4Ex1act sing ESS. Page Sof RO -- 3/62 ee Sn Medical Department Study: T-6295.7 LaboratorRyepoRretquewos.t NFeAmCbTesT-0K0-7033s0 12.3.4 OSebtseHrPvLedCroppluemtps ctoom"iOnng.ouStetofthteefltopw toof t1h0e-pr5o0b0e.uLA/lmlionwor10ascaqpuprmopirbiaeten.otr `approximately 10 minutes. 12.3.5 Tum on the nitrogen. around the tipof the A fine probe. mist should be expelled with no nitrogen leaking 1236.1 Drygisi25n0-4g00 ershour 12.3.6 The instrument uses these parameters at the following settings. These settings may. changein order to optimize the response: 12.3.6.2 ESI nebulizing gas 10-15 liters/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 2 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 12:38 Rfuercthoerrd. Ctuonnenepcatcatmheeevrosltiangheecabilnessratometnhte plrogo.be. 12:39 Using he cross-flow counter lectrode inthe ESMS source i recommendedfo the analysisofbiological matrices. butionat tp ofsampl14e. Eneure statand end sample number incloaes ai 12.3.10Click on start button in the Acquisition Control MassLynx versions, see appropriate MassLynx Panel (this may vary among USER'S GUIDE). Press the start samples to be analyzed. 12.0 13.1 DCAalTcAulAaNtAioLnYsS:IS AND Cat coLaions 13.1.4 Calulate matix spike percent recoveries usin th following equation - % Recovery = Observed REexspuelc-teBdaRceksgurlotund Result x 100 13.1.5 Calculate percent difference using the following equation: % Difference = Exp ConEcxe .po-sCc eadlcCut olantee edCod ne, x 100 13.1.6 Calculatie(ialc0tu0aVloPlcFouOnmSceeonctflrema.tairoinoosmfmsPdF+OCSu,mrlovreoofxtShDueirolgfulatuitooernoSFcnahcedtmaoirrc)atl), ixn matriLuxg (ug/ml): 00075 Final Volume (mL) 140 Merion Pencommance VDL 228 L0G vals Facts Paesors 14.1 `MmeattrhioxdspDeectieficct.ioPnlLeiamseitse(eMDFALC)Ta-nMd-3L.im1i,t AotftQaucanhtmietnattiAonf(oLr OaQl)istairnegmoeftchuordr,enatnavlayltied,ataendd 3 Environmental Laboratory 83 Boge TH 3m Medical Department Study: T-6295.7 laboratorRyepoRretqueNso.t NFuAmCbTerT-OuX2-207350 142 Method Blanks and Matrix Blanks 14.2.1 psMotesatsnhidbaolrdedbcilonanntthkaesmciaannladitbimroaatntriooirnxccbaulrrarvnyeok.vserw.illVbaeluaensalayrzeeedxwpietchteedactho fsaalmpbleeloswettfhoerlowest 14.3 Matrix Spikes 14.3.1 `Meaxtpreicxtesdptiokefsalalrweiathnianly+ze3d0w%iothftehaechspsiakmepdlecosnectenatnrdattihoenp.ercent recoveries are 144 Continuing Calibration Checks 14.4.1 wCiotnhtienaucihngsacmalpilberasteti.oncThheecpkesracreentarneacloyvzeerdieast aaremiexnpiemcutemdoftoafftalelrweivtehriyn 1&03s0a%mpolfes the spiked concentration. 145 pIfearnfyorcmreitderoina tlhisetsedysitnetmheamndetshaomdplpeesrfroeramnaalnyczeesdeocrtiootnheirsn'atctmieotn,smaasidnetteenramnicneedmabyy tbhee aonnaltyhset.suAmlmlaarcytisohnesewtilwlitbhetdhoecsuammepnlteedresiunlttsi.e instrument runlog, the maintenance log, or 15.0 POLLUTION PREVENTION AND WASTE MANAGEMENT 15.1 pSiapmepttleeweaxsttreacitswdaisstpeosaenddifnlbarmomkaebnleglsaosslvceonnttasindeirsspolsoecdatiendhiingthheBTlaUborcaotnotrayi.ners, and glass 60 Recorns 16.1. Sftoolrleowcihnrgoimnaftoorgmrataimosniinntchleudsetdudeyitohrerprionjtehcet hfoeladdere.r oErahcahndchwrroimttaetnogornatmhemucshtrohmaavteogtrheam: sdattued,ydoirluptrioojnecftacntuormbiefr,apaplciqcuaibsliet)i,onanmdetahnoadl,ysti.ntegration method, sample name, extraction 162 Plot calibration curve by linear regression and store in the study folder. 163 Print sample list from MassLynx and tape into the instrument runlog, 16.4. Print data integration summary from MassLynx and tape into the instrument runlog 16:5 Cstoopreyiinnasptprruompernitarteunsltougdypafgoelsde,r.including instrument parameters and sample results, and 16.6 Summarize data using suitable software and store in the study folder. 167 Banadckloucpateiloenctorfonbiacckdautpaetloecatprpornoipcrdiaattae.medium. Recordinstudy notebook the file name 17.0 TABLES, DIAGRAMS, FLOWCHARTS, AND VALIDATION DATA 17.0 Attachment A: FACT-M-4.1 Data reporting spreadsheet 17.2 The validation report associated with this method is FACT-M-3.1 & 4.1-V-1. ---- 5/8 AnalysisofSerumFoArCTF:uMi4d.E1xtract Using ESMS Page 70f9 psa am Medical Department Study: T-6295.7 laboratorRyepoRresqueNsot. NFuAmCbTerTOUX-r03s0 180 RepsRnces 18.1. ToFnAiCzTa-tEiPo-n/3M.a0s,s"OSppeercattrioomnetaenrdPMlaaitnftoremnaSnycsetoefmtshe Micromass Atmospheric Presmare 190. AseECTED DocMENTs 19.1 FCSApoCemTcpt-orMou-mn3ed.ts1r,yf""oEmxtrSaecrtuiomnoorfFPoutasfsoiruAmnaPleyrsfiusorUosaicntganHcPsLuCl.fEonlaeicotr rOtohserpMrFaaasaysroshemical RmN0 euovmimbsVUievo0 rngoVsa0 lsidation-0 ofmeth0 od to Ri0 necalusdoen0 7FforroRcehv0 eimsiiocnal0 s addit0 ion of0 whole 0Re0 Dvaitseion sbelroaodcrmoasisriva.lisduartrioogna,teMsDtLansdtaurdd,. nuepwda4tPeIiMnSr(ecHoSr)dytsetcepimnsg. amnodnisetsing Goenios policies, oe Avayis of SennFFacaTdaaEnxec Using ESS 3M Environmental Laboratory Fo fs Fagesors _Bager7T-- 3m Medical Department Study: T-628A5v.a7chmenAt LaboratorRyepoRretqueNos.t NFAmCsTerTo0x3-903300 Laboratory Study # TsNhnatasreFraotSven McreaubrmoaednReESvqouoepnmmeenvtesSipoanm Nob: Fiemme: So vi De SaomamteEes ieiusi: [or [eCrommenr ter | T[RoeV| Deer | FTeCe S C SuptT r , TI Sne or e estyafrr CoDoinwtcteonntVroFatteiosen,((mTroikemnTafkoTeeneoennafoem htetMysasoeye gation sry. 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Avarage PFOS Concarratons inSani Doge Grou naWeshom Sy FACTT0030 RacerGros) [[SeToraoormsT Totimrroitio| s|Goowvnat|crus| v|croowismaGtoopn||crtoprani| mreore]| [eEdr[[dmoenrecies | SSomm TT owomomn[o v5[ tse wa ny] ] Er es [A mE n [[RSeomT emrarnosio| t| oomoomwr |17 oooomm [eCwnEeI |7 esw iw s] ]| VoA kls |E ASoamapnro |Totomsn ToomieoT asTw vBm re er [Bf [worroion | sms [wom ios wis [mT] EP a e s ee [nso, mos Sebmien [Pomeroiea vow smn 1ogomurmi13[TsiwsTswiewsi] Wowk 57 "Average PROShome 0.0327 cous. wz 323 |70 | 16 | [[ Cr memoe an Ts own ve Twi Tw Tw ET TN [Eo P[m oSEeommesPrmoSieam --ooSommom T|ooeoaoomsn|o3r55P|mef ssee | ase raa r [Fovoenrs [meioTsim oo 50T0055] [So |b oom o|or wes | os am] ms ve WM EmionmenalLaboratory 3M Environmental taboratory 12 191 Pages Pagers 3m Medical Department Study: T-6295.7 LaboratorRyepoRretqueNos.t NFuAmCbTerTO0X3-307360 3M Medical apartment Sc: T2657 LaboratorRyegReecruteos,t FNAumCbTeTrOUXz.20730 [Taabloe0s-3.[PFO0S Am[ounotsyRep[ortoedoinSarTuombyIndmeidAounaiel(B[aseolinye [Weeik 26) oe [| FP C roeseeTonTe oa|a ooIo[e 0WTC e CooW Ts wnT Teeeofnos e ooerr]| oE e mmrrr eoinsoenTTTaaecoeoTeceneaTTTroooeTTooonwnW TeoT oosnneE T|| o ieonT r mcia ote ssos ma aaser oooooe|]||| ee e e teee tateebanhem fe o fe he a E eC asE e -- aw ore aT sr s C 0et ea] Pome os aa oF mer oee ow rer t aeoTert s wTro e ea rr r e a]| frE eeE TooenTB ow TowTaw owTe ae]T --P tw--am] er Fe TeToTToe TwTmTwTeerTerTeTrTo] FP emfea eb ae re e ee te tee ee a Ce[ica ssT TR nee]| e I a eEe C 0ee F e J eie a 2 = | eo EC TJ CE tm | E E C e Ce wmJ ra Ce 0T E 0 7 eeLeaLo || E e BEe rr T T 7 E rant Je a e a a| ae Bt eT CT 0 e Jtm J0 0 IMEninmental Laboratory 3 Environmental Laboratory 19% /52 Pagans a 3m Medical Department Study: T-6295.7 3 Metal Dartment Study: 6205.7 laboratorRyepoRretqueNso.t NFuAmCbTerT-OUXz-207350 LaboratRoreypoRretquNeos.t FNuAmCbTerT-OUXz02%750 TableD4. 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PFOS ConcanratonsnLivebyDessgeGroup [E =n |r Zea]|mee| FIT 7 Co TT iver | [[CroFoassmmeTf enaem f=] FFossosseossrT Te s a si-- -- e m 5-- ----m ] --o-- u----]] foFoe Ty Peset=a r % [m[ seaT n Fr -- -- e m Ls r r -- --5e 7---- | 4 [FT r STm] FS s [C [eI CsmT a T Too>me imy o -- a 1-- Se r o --] -- I a2 [[[[emmmsssssee[T T Somome ese T m go iw rs--a ------r a --] 5er-- S I es | Soi bogey| mseS-------- r7r] = a8 ([[ FSI romwr nige iooo es p_gp_t --------e ----r n --------------| [rom mo mr w a --] ET 0To --m -- o-- r] [oF|om moeey |v| [mse[mm | El 2 TC i A -- E>-- J | LO Ls Tn oe rrGn eSTT WM Environmental Laboratory 3% Environmental laboratory fo 55 Pages page TET 3m Medical Department Study: T-6295.7 3M MedicalDepartmentStudy: T-6205.7 ATTACHMENT E DATA SPREADSHEETS laboratorRyepoRretqueNos.t NFuAmCbTerT-OUX2-207330 LaboratorRyeRpeoqrtueNso.t FNAuCmbTeTrO-XU.202370 -- IMEnvironmental Laboratory 3M Environmental Laboratory 18% /7 Paget _Bagese-- 30 Medical Department =Bima Study:= nTm-e52CScotSmroIanaDnt LLaabboorraatorRyepRoesquewos.t haocmterTooixa-t0a3s0 EE=B =EELEee B[ = E=E=on EEFeyr.y. oEiEyE So E ns [= TE[maE ] [orTemTE==rzEe ToTo B=g= = 2 a] . 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E 2 EEmEm ne ET |TweT e Ee TET e eT TT T] om ToTe To oe | | om To ee mem TeL w T ee [mm TwTw a r owee[Lmor oem |w =|w w T w e 3m Environmental Laboratory mom 2 2/6 _pagezus 3m Medical Department Study: T-6C2%T%n ocx t LaboratorRyepSoretqueNco.t NFoAsCpTerTOiX-o03s0 =Borers Frrd -- en bE ee ESEIiSinEeeoEnEram EEE EEEnnrimaas,a L[e iiwsT[o8r7 To2 rT To T or | [om Fem TorTo Toe | To 7Ts T gr [Co TwTw mmsToTwTT god A, ee -- Bo 2/7 Sm Medical Department Study: T-62C%hrcheroanm:e LaboratorReypomrte.geNo.t LFaAICT0%To;r0-30300 =Trin' ELeernner rman EE-mm Eaf [fEE rr EEmc E E EsrYmmi.m rr [=F [| TE (mam | EE] [oso 8TwTo] | | EL CE P CoE e E TE eT) ) eT BE I em I ETI E r eeT LBy--TE 1n, re n] je semar---- So S-- 34 Environmental Laboratory -- "x 2/8 Page2rr-- Sm Medical Department Study: T-62a 9actr: orem LaboratorRyepnoretsmNco.i SFAACCIT 1T0o1x--002300 iH.oran oAeI Asoo Ene pata f=Eoxri SSEeIrr RE Ffateon Eau,nsmuow mar, =f le [EL | h0 eme eSe S l Ie a TeT-- aEe oSeE [ ee E T E Te aT EERE Ea EE CR, Hii 24 2/5 3m Medical Department Study: T-62%cT Tox-as0 Ea-- Pr[i ComGi a Laboratory Request frre pred Report No. Pam Eoe un Eooe nam mBaers fotos [=[Tee(Em et =] TT CE ee IHH CE a E m r Tar Ce Em Ta ET m-- ia SFAoCmTerTOrX-l03)0 2 220 3m Medical Department Study: Coa T-629AcTToX-a30 LaboratorRyepoRretqueNos.t hFoAvCpTerTOhX-i0l3l0 EE=ovens oeKerere tasen s EEEmm EEEenme gBeE GiE--E--- S bEE era mai ns. I |Z EN I= sie |wT T a erhsTala mm |= em TeTe LT Pareto |rF eEE-- T Tr Tao mms TEERR E e R eE co im 3M Environmental Laboratory -- #22] vr PageaTi 3m Medical Department Study: T-629%actroxas e apRImEDetsabsoraacoty Semen: Homer isis meen X rr Era = Report No. FACT TOX-030 SEEEEEihErcee.ri ET ns [= [TET (Ea = Ee e ooBE a T aSea e [E ae Ei T a 2 HH w-- m TERRIER er vn, me ct Mes ot ed sbtt LC 01370 3M Environmental Laboratory Page2rs" ee BIE 0 FOE, 1on rnp EeSener ctr E a E Sane oe = fre Tow a rn a EET [rm me TT To Tm] [om [orTa To] IEE JI | Ywee m[eomw[Ta H Tm BeTm 8 Le ToTw1 = [25 To1 =| w= wm TEEeR eue 3m Ehvironmental Laboratory moe 24 223 RagezTE 3m Medical Department i abRSARG) Study: T-6295ack Toxam Report No. r------ pri FACT TOX-030 Fir sit EE Pritts e Trr T= FeTei] | IEEETETea [om To Te Te we] 2 [T Tonee | a | a. a wen | mm Fi Fy a[me] E ES E TT [lo 3m Evironmental Laboratory em 2 229 _eaga iar-- 3m Medical Department Study: T-629C5rxmFcreo@xansii laboratorRyepRoretqueNso.t NFuAmCTbeTrOoXi-a0a3a0 fo -- Rt SEE Ef mmm [aTT eA EECE [i Co om a 2a TaaTar ooTT LE 5] au Eivironmental Laboratory -- Zr 225 pag Bre . BEL porn FEEL, TOTTO ee E == HEaee [ome Te are LEE essas ToT ae [Ema TeT= CT] [emTe |=FE oT] ae TTi I I I~ | os [= Pe wT= 1To] eH I J | 3 Eivironmental Laboratory mene 2H 226 page Bry 31 Medical Department Seudy: T-625fCadrkmrotnaSn LaboratorRyepoRretqueNos.t MFoAsCpTerToaxr-e0s30 I Sim Een TEE Fea] e EE S e E oTE] Te]- E LEe aSC H a I : = | 3m Environmental Laboratory ZH 227 Page320 Rea bi bm Ere.a EE soesm fo I ot [aed i= = o emsvas | Eee mn |0| Se oo]. eeee = 1Im] ee ToToT= . I HO | [[a emeT3 e1 T =|. r Ta a] 31 Environmental Laboratory Tm Ut 228 eagaior 3m Medical Department Scud: T-6295,7 FACTTOXA% laboratorRyegoRreequeNso.tNuFAmCbTerT0o-3093sy0 es Eipg------ i ene ar eemvee fow ntFeri AeSt pe ve wn Sins aie in saw A Se oe wa se SE nO Er [Co esTe Ten] [[ Coe eeae [(ooiprrE |ee VR T Ta e Po e ) PO sonra E frEanr E on onE F C TE oE ee n E Een] rs J Snip rA roe | T-LE i[ r1ma, ] fHaimnmg E CroE setd FER EI ee Ren mesons g ot Et MD.eT a eeGe e Oen R rms ow Eiiromental laboratory TI229 eager  5it3 8 i 1511 i i EH] i : Pou i} 5 ]#2325 Pos ag fa] FH : gst Ef 2 Fie 81 JH5 eddeeddddaga 2255%1 ggiifnt ] FoRaTmHI EBESE feefsiiltl if y e piiireld , nff3 ge a IHi N23] FLHhE2 3 iH Hi g Hii it EE i E2222Esz2d25s318o12l5s alu 2s 2ie I ind EE EEE ET E yoo Tig i dil ! is 3 EERE HFA ipo Ela HE 3 iJ}] ACE 2 22 230 cil 3m Medical Department Study: T-6295.7 Laboratory megunct 0S nae iz 3 i i : 22 EET ET Po Ee i. $SEFEEES5ASB5T ls EEsN3EsEiEcEiEslSedhplesh ols | IHrpEisRiiDss [isegeesensess , =: pi 3 firsts] :3g !s: ESS EEIaEERHEa REEaaaezCd l iollaeaakkyss fCg afl EEE ERe FEE Is giz EEE 5 = L ifin s 2 J aEb3aElf:iE 3 ERiEjEiEIsEiEtIeEI iibhidopiSs Per fRE 5ff Asiied: Bsiif EEFEH 3M Enviromental Laboratory 277.23) i: eagetis am Medical Departmen: Scudy: T-6295.7 SM Medial Deparment Sucy: 62957 AmEtXAaMcPhLmE eCAnLtCFULATIONS LaboratorRyepoRerqtueNso.tNuFmACbTerT00%3-207390 LaboratRoepRoertdNeo.s FuAmCbTerTosxa0r3e0 FormuiaUsed for Sera Analyses in Study FACT TOX.030 AR (g/mL) DEX SC FVVn(ml)) x 01000gmg_= pg/mL. xPC = Reported Conc ug) Calculation Used for Group 3, Week 1, Animal ID 105505M 287ngimL x 1009275 x OImSE x T1o0npgg = 5.32 ugimL x 0.864 =4.60 pg/mL. ADFR----DAinlaultyitoincacl orersult from MassLynx summary PC--PFOS purity comestion factor (86.4%) 'SC--PFOS salt correction constant (0.9275) FV--Final extract volume (1.0 mL unless otherwise noted) [EV--Volumeofsera extracted FAorRmu(lgaf)UseddefuorLi(v1)erxASnaClXysDeFs*in_LS0tuugd.y F=AgCiTgTxOPXC.=43[0PROS] spe ug) Same 000mg TM cuisr assuv medte obe: SHH 1 gliver Calculation Used for Group 3, Week 27, Animal ID 105510M SHnge x0ly6SmLS_ x09275x 100% W1o0nugg =488 uy x084622u4gly 2 sample--Density ofhe lver sample (1g ample 3 mL. 1,0) AR-- Analytical result from MassLynx summary 9 curve--Densityofthe liver standard curve, assumed to be 1g liver/ S ml water 'SC--PFOS salt comection constant (0.9275) DPCF----PDFiOluStipounriftayctcoorrrection factor (86.4%) WMEnronmentt atorsory 3M Environmental Laboratory Paget pre 3m Medical Department Study: T-6295.7 WMMedicalDepartment Sucy:T:62057 A--T_T-- ACHMENGT INTERIM CERTIFICATE OF ANALYSES laboratorRyepoRretqueNso.t NFuACmTbeTrO-X0-202370 Repent No. FACT Toxa30 TT LaboratoryRequest Number-U2279 WMEnvironmental Laborscry 3M Environmental Laboratory 224 233 Page Gt Pagea?e INTERIM CERTIFICATE OF ANALYSIS Revision 1(9/7/00) Centre Analytical Laboratories COA Reference #:023-0184 3M Product: PFOS,Lot 217 Referenc#e: SD-018 fone TTT PuriStyp: e86t.o9%n f Fhppearnce 1 oe [rou| Metal P/MS) Wii Crysalline Powder | Conforms 33a foopoee. 3} peSnitmeertasl 4. Potassium' 4. 6.849 wriwt% To5IEEmoym 3 iSerms [Foals mmpuiyORY[ym rr a (LEMS) nl [[RPesuidyubalySDoSlCves (TGA)7| organicAnions (IC) Z&iIrfNITiegee. 7._Sulfate* [Te IEiT oI S= feamloso `Organic Acids (IC) er, NNootnApDpelieciabelde | 5T bJ Spaamnn %: SShormvmxx 7b.i g ahtinmi 8.76 wtiwt% Ln TDI OooRRDTomeemommmEaaeminvevRhIieeegemreoln || ||r1))oa aiiesss mes HE onillI rt COA023-018A M Environmental Laboratory 22s0a 239 Exaca tCopyofOCraiglilnasl sage Page 1of3 3m Medical Department Study: T-6295.7 laboratorRyepoRretqueNos.t NFuAmCbTerT-OUXz-207390 CenItNreTAEnaRlyItiMcalCLEabRoTraItoFriIeCs CAOTAEROefFerAenNceA#L: Y02S3I-0S18A Date of Last Analysis: 08/31/00 Expiration Date: 08/31/01 Storage Conditions: Frozen <-10C Re-assessment Date: 08/31/01 F"lPuuorriitdye=, 01.050%9%-+(NsuMmRofimmpeutraitlieism,pu1r.i9ti3e%s+,or1g.a4n5i%c+aLciCd/iMmSpuriimtpiuersi,ti0es.,388.%4+1P%O+AnAo,rganic 033%) ToPtuarliitmypu=ri1t0y0f%ro-m1al3l t.es0t=s9=8%61.39.%09% "Potassium is expectedinthis salt form and is therefore not considered animpurity. oPbusreirtvyebdyfoDrStChisissagmepnlerea.lly not applicable to materialsof low purity. No endotherm was di"Sneuotlrefgruamrniinicantatinhoienosnianmmtephlteeheoaldpepmceeoannrtdsailttioaonnbasel.ycsoTinshv,eelraetnneiddoinntgorecSsouOnlftiaadngerdnecheeesntwcoeelthldieswtiietnchtteertdphreuestsaiutlnifgountr.he Based on the results, the SO is not considered an impurity. TFA NHFFPBAA PFPA THreipftlaufolruooacreotbiuctyarciicdacid NPeonutoafflluuoorrooppernotpaannooiicc aacciidd "Theoretical value calculations based on the empirical formula, CiF S05" (MW=538) `This work was conducted under EPA Good Laboratory Practice Standards (40 CFR. 160). coauzs018a 3M Environmental Laboratory 235 Page20r3 PageTie 2m Medical Department Study: 7-6295.7 laboratorRyepoRretqueNso.t wFuAmCpTerT-oUxz-i073s0 INTERIM CERTIFICATE OF ANALYSIS Centre Analytical Laboratories COA Reference #: 023-0184 LC/MS Purity Profile: mopo ------r wwm e 0 oo r r Fr a a ] Cw ma Note: The C4 curves, respect and C6 values ively. The CS were value calculated using the C4 and C6 was calculated using the avera st ge andard ca response libration factors from the C4 and C6 standard curves. average response factors from the C6 Likewise, the C7 value was and C8 standard curves. calculated using the Prepared By: David S. Bell Date Scientist, Centre Analytical Laboratories Reviewed By: ee John Flaherty Date Laboratory Manager, Centre Analytical Laboratories conomaien 3M Environmental Laboratory 230 Pasaona pags755 3m Medical Department Study: T-6295.7 laboratorRyepoRretqueNso.t NFuAmCbTerT-OUX3-307350 INTERIM CERTIFICATE OFANALYSIS Revision 1(9/7/00) Centre Analytical Laboratories COA Reference #: 023-0188 3MProduct: PFOS, Lot 171 Reference #: SD-009 Purity: 86.4% . fF Fu same 7 [Apdeenaimcnaieoen | Speffatons | 7 R=ew itsCyl Powder |Conforms ER1 CCPatcum 2. Magnesium 2L 0000017w7wimas% 3. Sodhum 5 LISSwins 45&.. NPLiooctnkaeslsium' 45.. 006.005005432wwwiiirihwt%% 7._Manganese 7._<0.001wt./wt.% | ToT: Tmt TOS GoRaehledpaComypou| n--d ee5 FOAL [[TRPreuorsigdyianbilycSDAonSlivCoensrs(ITC)GA)| [N | o NonAeDpeptelcitecd]] 2. CFuloorirdee 5. Bromide 2L 0O01zsmwamn 5. 0040 wi 55. NNiitee 6. Phosphate 55 00.00060wmnm 0007 win O| 7rga7n. sAEScuialdfsateICY 2 FRA 7.882 wt/wi% 2LL Dtlwmwnn || [Elem3e. ntaHnlFwABraaAlyeie 3. <00215wwtwiw% | 21. 5. HCayrdbroongen Niwogen 21 5 TThhTeeoohrreeettiioccaarllVVVeaaallltuvieeei=-c1007%a%.8l% 2L 5 0n9omiwn Le wim o5._sFluuorine 45. TThheeoorreettiiccaallVVaalluee =- 509%5% 55. 10S014wweinn coA023-0188 3M Environmental Laboratory `Bact Copyof Original TH Data 226 237 Page Lof3 ET 3m Medical Department Study: T-6295.7 laboratorRyepRoretqueNos.t NFuAmCbTerT-OUX2-207350 CenItNreTAEnaRlyItiMcalCLEabRoTraItoFriIeCs CAOTAEROefFerAenNceA#LY02S3I-0S18B Date ofLast Analysis: 08/31/00 Expiration Date: 08/31/01 Storage Conditions: Frozen <-10C Re-assessment Date: 08/31/01 "1P0u.r6i0ty%+=In1o0r0ga%n-ic(sFulumoorifdmee,ta0l.2im7pu%r+itNiMesR, i1m.p3ur9i%ti+eLs,C/1.M0S0%im*puPrOitAiAes,, 0.30%) "Totalimpurityfrom all tess = 13.56% Purity = 100% - 13.56% = 86.4% "Potassium is expectedinthis saltformand is therefore not considered an impurity. o*PbusreirtvyebdyfDorStChsissagmenpelrea.lly not applicable to materialsof low purity. No endotherm was i"Snuolrfguarniicn atnhieosnammepltehoadppceoanrdsittioonbse.coTnhveeratneidontoreSsOul4t aangdreheesnwceelldewtietchtetdheussiunlfgutrhe odenttehremirneasutlitosn, tinhtehSeOexleimsennottacloannsaildyesries,d laennidmipnugrictoyn.fidence to this interpretation. Based THFFABA NFPA PFPA TrHiefplaufolruooarcoebtuitcyarciicdacid PNeonntoafflluuoorrooppernotpaannooiicc aacciidd "Theoretical value calculations based on the empirical formula, CyF;SOyK" (MW=538) `This work was conducted under EPA Good Laboratory Practice Standards (40 CFR 160). coauzs.0isB 34 Envirroonnmmeennttaall LaLbaobroarta:tory 2 235 Page 2013 aase 5 3m Medical Department Study: T-6295.7 laboratorRyepoRretqueNos.t NFuAmCbTerT-OUX2-207330 CenItNreTAEnaRlyItiMcalCLEabRoTraItoFriIeCs ACOTAEROefFerAenNceA#L: Y02S3I-0S188 LOMS Purity Profile: [ Mwpariy TT wem% es ie ese eT ew ie | CC wr ee Ncuortvee:s,TrheespCec4tiavnedlyC.6TvhaeluCeSs wvereawcaallscuclaalutceudlauetseidngutshiengC4theanadveCr6agsetarnedsaprodnscealfiabcrtaotrison afvreormatgheerCespoannsdeCf6acsttoarsndrarodmcutrhveesC.6 LainkdeCwi8ses,tatnhdearCd7cvuarvleuse.was calculated using the Prepared By: DScaivenitdisst.,BCeelnltre Analytical Laboratories Dae Reviewed By: John Flaherty Dae _ Laboratory Manager, Centre Analytical Laboratories coamsosn 3M Environmental Laboratory 230 235 Page30f3 ser