Document begL548RMB3j6ba3V7pBG0q0

"E en - XY8RESEARCH en Res - ? oe - Analytical Report - 3M Environmental Technology and Safety Services - Analysis of Perfluorooctanesulfonate in Mallard and Quail Egg Yolk Exygen Report No. 023-070 Testing Laboratory - Exygen Research 3058 Research Drive State College, PA 16801 Requester William K. Reagen, Ph.D. 3M Environmental Technology and Safety Services a BuPiOldiBnogx23-333E0-109 St. Paul, MN 55133-3331 - s0 s een rBiiveon, usa )- pace1065 X R wR0zEsyY A210 CRLL0S _--_-- - - - WinsS ediT eienEEHSER SERLEMR e eEN ry to ree - qReusaul0t9s9ayroolrkseamppolesfo. Ahleoatnoapoirstsedoifslahel PpReOuoSrcoooncttaainneedsiunfosnaclhe o(fPtRoOSt)h in fmaalciirodnsaonfd - Geogngdiycotkedinvaecrcyorldoawncdeownstthytlhpoapnraoytiecnal(VpLhDaLs)e.prpohtooscovlin,veanndnAptoiuctheliimne.nt This sty was i Tame Mae - oTfhlassaampmlpolseowge-rnoisnufbomrimatteidoinn plraesstiecnbtaegdsinAbtoalcdhmweinhtmAagic markerand pedprin. Acopy . TThwaelsvaemmpallelsawredargogyshopkpseadmfprlozeesnaonndwryacoqvuiaaFloadgogryoEkxsparmepslses(wFeedrEex)r,eceTihvoetdwoan 0m8a1l2a,r yok samples worecombinedandhomogenized 0 ane sample. Thesamewas Gand fo the tFwoeuemaallalrdyoaklsbaummpelnes.andTheshsealmpmleemsbwrearndatsoeadpsfr.ozeannfdofmourmeGuaofd aacumaaunr aandnaSenesl - omteimsbrsatnudeys.ampleswero aso fecavedonOS'0102. Thesesamwperionstanalazspeardt . 3 Methods - AnalayndtPriepcaraatolry =~ EERE . 3.1 3S1a.m1pTloetalPrPeFpOaSraCtoinotnent aA 1.d0 1g0sdathmapselaempwdlaes.uTshedosfoarmtphleewxatsacptloancepdroacnacuwrro.saTcwtaonnysihvaokemrilfiodrer1s5omfimentshaannoldtwheerno - 0co5ngtoifucgaeardbio-nw2a00s0acpdmedf.orT-he10smaimnpelse.weAss{hveakmielnitearnalaolotwoefdhfoe ssia.mEpslechwassamapkleonwaansd fredandanahzedby LMSMSolctospray. 3.1.2 PFOS Content Yolk Fractions - Mg5S0O,gsa1 mmpMlepwhaesnyuismaetdhfaonetshuelofnryafcuioorniadtoen(.PMTSeFn)m.ialntder2soufM lseuopeoptnin cwoenrteaianicndged0t5o7thMe - coSanmtplueg.eThuobse.Tahemsaapmspsllheawkeaesncforni2ugMeidtaets20e0n,d0h0a0nx hoatS4ufso 2p4whooausrnts.asnTshicaesnneadmp1l0ea waanssthreancsieopnar(aHtOePd)into 2 segments: he very ow densiy ipoproten (VLDL) and the hgh - T1hmeMVLPDMLSwFa,sadnisds5oluveMdlieuapseopltunt.ioTnhcaoHnOtFavia2ns0irmnesMgusTpreisn-dHeCdI,i1520smolMuiNoanCclo,n0a2rimnMg 0E4D5TA, . 2M0G0S.0O0,0 x1 gmaitd PCMSf,or 1a6ndho2us. TlehuepeVptLinD..wEaosinthVeLnDLisasnodvHedOFin f2raocktiionosnwecronetaciennitngiu2g0emd bat TTihseHCHLO,F1w80asmsMsoNlaCvle,d0n2 m2 MSoElDoTnA,c1onmnMinPgSF0.,45anMd MSGJSMO.lau1pamptin aPnMdSFs,toraenddat24p.hi race2065 6n1ung _--mm-- - - " Jweucp.epinand hentwovolumesofcodwater asaddedcropsandthenstoredovemightat am`lTMh4eCHP.DMFSTFsh,uepaeHnmDdatF2apn4rteMciiplpiootuvapetoelp(iipinhn)oswavnaidtsndt)ehawcnaandstdieediadnsdwdeshioicenoalddwsvowilatuethtecirdoolnadncwdoantsteairiraneindndg0osv.te4ronrMieMgdhogtveaSmtiO4g,h1Ct hTohueraipnodvidailsisnoalnveddpihnoassvoiltutwieornceotnteaninriencgov1eMreNdabCyl,ce5nmtuMgaTtHoSnHaCt, 1|0m60M0 PHxASgEa,ta4nCd 2foph1 eupeptn. Alofthe resultingpreciitates(-1.0g)from each fractionwereusedforthe extraction - pmpliranouccteeeddsuo.ren.AawvTrwoesmntitaylc-lfteiorvnesahmiialukloiettreorffsotho1fes5mametimhnpaulnteoelwsaawsentrdaektaehdnedanencddteo0.t5nhgeotsfacmaraptlreib-.o2nfw0T0ahu0seogasmacmefepodlrde,~wTah1se0 - samplowas shakenthen allowed 0si. Eachsamplewas itered andanalyzedby LOMSMS electrospray. p, - 3.2 Sample Analysis by LC/MS/MS In High Prossuro Liquid Chvomatagraphy (HPLC), an aiuot of extract is jected and passed - tcshthraaotruiaognchatrrayilpsihtqaausimedo-uipnhnattshoeecfhctroiolmmeu.amtnoFg1orla8opwahiincg1c2HoPhlLeuCmGnsu.eipBdearmasoteibodinlo,enmptahhasseseas,fpfetichnteirtoaynmoaefllytrthyeepairnoarvleiytcateiesnfeaodrrftoarhpea - arndaacgcumraetaenmnodtadneetsecdftoer,da.nTalhyazilnogncawhiadreacrtaengreiosfoofrghaeniccomcpoomupnodunidso.bsMervoedalndaarqeucainoiuntiazsteed,d `against standards. Each sample was analyzed in duplicate. - An HP1000 systom ntriaced t a Micromass Quatro Utima system was used0 analyze the 5sa0m2p.l1e mexmtrxactsu.mcAolugmrandwieanstuseeutdioonrsetpharroautgihon.a Jones Chrcmatography Genesis C-8 The folowing gradient was performed: - MMoobbilooPPrraassea((A8)): 2MmetMhaAnol mmAcoetatneinTiypau|Wamter > 0T0ime "A xw8 04 & 2 7100 1100 % - 5705 o0 11000 91540 6 - Thefollowingparameterswereusedforoperationofthe mass spectrometer: . TFToanimzamtieonwHod Sewmg Ehcospray FoTlraanrstiyions Moritored 4N5og9a5t9v9e . paceaces onLnsT -------- - - CDryim ng GaT s (2empomwe70UminC_-- a HRiaialnanssaGERA R RSE B e NUTR SE e Ra i - 41 Calibration Aa6n-aplotianltcsaebqruaetnicoon cfourrPvFeOwSa.sTahnacbavleberdalaltotnhpeobinetgsiwreinrga,ptrvepoaurgehdofuo,r aPnFdOaStatt0e.e1n0d2o,f0t5e, . aG1o0ct,her2mp0io,nnaa.ntodn5Us.(i70n)gnwge/nrmeeLad.ertTeehgrremeisinsenidso.ntruwAmcieahnltb1rreaswlpoeonincgsuhervieenirhgssae,usStichpee,cco.nncfieeenrt=oraeptp0ito.atn9wn90da.Csopiuoftiiecdfnootrf - Fontrhtehrearwedsauttsa rinepAotratcedhmheernot,Cc.alibration crtria wera mel. The calibration curvesaroincluded - 42 SSuurrrorgoatgeastpeiksesaranot a componentof teanayicalmethod. - 4.3 Laboratory Control Spikes - Laobfotrhaetaonrayycioensirtlospcoinkiorslinotghgeyaonkalystaicmapllseest.weLraobporroaplaoryecdbonyiraldisnpigkoasknroownUsceodnc1e0ntarsasteisosn hmeotchaotdaaicscruerjaeccyt.edT.hFeolratbhoerreersuclosnetpoorstpiekdehsermos,ttshahoSwpirkeecsowveerrieeswbietnwetehne7a0cc-e1p3t0a%bloer - range. 44 MaMtaitxisxpikSepsiwkeersanotincludedwihtisstudy. - 45 Sample Related Comments Oupicato nections were performed or all sample anaes. 5 Data Summary - PplaetasspeosrebeilAotnta(cnhmie)nftorBfPoRrOSa.detailedlisting oftheanalyticalresults. Resultsarereportedin - 6 Data/Sample Retention sSlacmtprloenscardoatdaisispoasrecdhoifveodnoenmornottihoavfaeblre tmheedrieapoarntds ihsasrudecdoupnylersespootrhtesrwariesesslpoerceidiend.caAtla foEkndvierrosnmmeantialniltbabayinoEtnxfyiegededn.0dHaayrsdcporpiyrctoaheisdisspoorsealdfoofrhaarcopiymduamta of veyears, SHA . raceaoes chee ------------------------------------------ - J - - - TE71ReAtlaachCrmanoefCuns t - 72 AtachmentB:AnataResuts STR a ay 73 AnaCciRhawmAnaejcanlDatta - 74 AttachmenDt: Study Personnel - 75 AtachmentE: AnataPhaseProcol - 8' Signatures BE i Ss - VRDecker,Pincpal esigaor Toesjo2_ - ar Abs - oN.Flory,vie Frasers Tae a pacesors nant ------------ . |B " lB " H 3F2E5E5S] Lj5k |B - 8 Ik le ' 15 " il 3 - : H32HH . " ' 1|1 pl : 3 i | 3 ia & Gnas _--mm . } SERIE EERIE EEEEE EEEEL EI - Hl ` Sl an BEE EERE EERE EEE REE * = Ella lial, HEE o he Re g [2 2lE2[E3]E3 33 3 diFel : gl 5 . E28 2H 3d 3 3 i \ " oe - 1 EREEEE gg Al) J11111111111111118 h zz [5 zz ZEEE EE Z= HERE g [@ BEEREE 3 3 a - 3 8 3 5 > GAL _--_--m--mm - : 3 (i SEEEEERREERE ER 3 is alalalaiaiaiatartdiasiasatsians - fe - E; EE EERE EER . 3 EER 3 iE . . lo B|EhRiuEsEatneas 3 EOE B naslsialndebo a alll ot alhelddats 8| 83F8 - 5 EH s E 3 2 : Se \ . repr FREER JHE EEE le EEE 8 &$ ' SElgE fs fe 5 2 " . carn _-- " BERT .. PEBiLas: ` EEE i i: tlle . Ele5 [EECiEtan QI 33 fi . F2 ae i: ` ` HEE . c 98d S1152 2 [5 [8 H 5B ! ) nN <Q 3 z $ NL:G snow `CONTRASCMTELNAVBIORROANTMOERNYTAWLOLRAKBOORRDAETROBRYYSAMPLE of2 Project BOLT ContractLabs): EXYGEN a RDeeapuaersmeern:t:Ro0b2i1d8ea0, RSitoe ScuRrhe(:0e002l.03lE.0e9) ProjectNumber PrCoomjpelcettLieosn:DaRte:esR.eRfiljdelnn Phone Numbers 65178-7065 PDraojeeRcetcDeesicvreidp:ti1on0:47N2.00B1obwhiteReproduction(Eggs) Email Address: obidess@mnm.com - swous 5/9/0--2 Comments: NOTE: MISCSERY = roti searaion to be conduct by Eygen Research, , rMBsOaea mLpcIleNrGeeseecSea1m9p2el0e0d0mDatee _eSa1em0poloeemaDieesBcrOeipAtiYnoonkesesmmmmereeree MIAwSiCu_sSCEesRcVeee AMinsac tSearvMiceetshodeee _ CMoimsponSeenrtvisces ree ATan3a0Oh20i0D2ueDate ee EL AminiCote GDA Antica Mttog l0semai 2HYok Componenn . AnDaueDuie o MA SCSERY MiscServices Misc Services ree Ta302R002 ee BOLI o Aono 610001 100m Ansa Method 234HYok Compan AnasDueDate MI ISC_SERY Mise Services Mise. ServistimeTsa02T002R a aBsiOnNCotIeS AGnt2i0Method 10somaiC3on6pHanYionsk A"nDueote MISC_SERV earMie Service es t Mise Services rese 3020a 02 rc: Ancsinoi Cmotes A1n32s0e0l1Methed 1000maiC2o91mYpolsk AnasDuc te . MB ICSERV Mis Services . MiseServices S 8302002 BLISS . AuiniCode a61m00l1Mtg 100omaiC2o3n1p1oYnonlsk Anas~DueDe MISC_SERV Mic Services MiscServices an02002 L AnEsDnLiTsCSoOtNeST aRns0c0nManet 10soC2o3mm3p1aoVnoeilnks AnasDuc ie E MISCSEe RV Mie Services Misc Services ee SsnO 02002 ee * w AA nMM EIaSnCs _CSoEtRaeV AOMniNsaeASnNervMiecetshod10vemaCiMosimsiovSreeA rekvisces ee Aa30u20t0>D2uoe ste BOLI a dousce 6n2i1s20i0n1Meted10oomaiC2o2n61pYaomlakn AntsDue se MISC_SERV Mic Services Mis Services eee sa0m0m nnty son 3MENVIRONMENTAL LABORATORY 2002 CONTRACTLABORATORY WORK ORDER BYSAMPLE Project F011379 cont) Contract Lab(s): EXYGEN a 3MsSaamnplke -- EoLmae SaSmampplleedd DDaattee_ SSaammpplleeDDeesscriigpttieonn 6212000 10ai.0 226)0Alwummen - AusiaitCote -- MISC SERV Ans Misc ServMiecehsog ere MCT oimsp.re Saeervaie nces oO A6v s3a0l20m e0D2osDats E0L137931942 6212001 1050m a. 226) Shell Membrane: @ Ane --Ms_ISC_CSoEnReV AMnisse SaervMiecetsog CMoimspc.anSaee arsvices 0Ae2n0e 0a2DuisDast0eoJ 01137931943 212001 105om i 230)Yolk Mr fI--wS_sCi--_nSiECReYi. AMniaseh,iSerMviecteshod CoMimspc.onSeee rnvtices GA50nm 2a00l2DyuesDiatse eas 202001 105mai 230 Albumen o AnMStCiSCEoRneY AMinsc.sSaevMioeetshod CMoismc.mSuernvices Ansani0n2i0s0DueDate E0L1379.31946 AnsinisCode --w_is--c_serv E0L1379.31067 f=ume 6212000 1050m 81.2301Shell Membrane Aus Mobod Componens Misc. Services Misc. Services 202000 1050mai. 232 Yolk AnPsRiOvSicbay MEeSthMod CPomrpoosnent AnataDueDate 6302002 Aneal0n0is0DueDate Eolas PR Ans Cote E0LL1379:31950 = ApnraoissCote 6212000 1000m si. 232 Albumen APnROsS bay MESaMhSon Copmrpoosncag. 202001 1000ma. 232 Shell Membrane AuPRiOcSsbyMEaStMtSed Cpornopusneon A3n0a2i0s0D2ueDate AnnaoismDueDate E011379:31951 7 APnRsOiSniaCode 6202000 100om al 234 Yolk APnRtOiScbayMEeStMhSod Copmrpousnensy 6A302n00D2aueDlate Eas 6202001 100oma. 234) Albumen = Lo AnPsoivsisCode F AnPaRlOtSicbs ylMEeStMhoSd Cpormopsanens E0L1379.31954 . Amimecow 6212001 10 00m i. 234. Shell Membrane AnsaMthog Componcn pros. PROS by ESMS rros 6A0n2e0a0wl2DuysDlatse AnnauisaDusDate - . . anions5 _am--m W WeE e | LTT TIT o`sBE EE El a e FE Lf Heri ! . NLSy ] H-- HHH 3I 3 [=e]J Fee tH e |||5 | 3 Sy[(W]| [ey] EEE 318 | ||| Se s"Hiii;1 hesitate |3|) | ) Hh SPER ; $ FbPeiOllEl | JE L m fl ilR sil ABBTlmlO|E T eeEE EE a RL _-- [LI TIT FE WC HEl E RER A e|E: L$ . HEE HH [16| - o|||=NSaE I ser| 7 9 [SINS] [wd FE] 2 8 sll] "3 aid "| Shs JES iis N o | [oon TT] N | C{n= RT RET NNRL ER] CC An STII "111 [fhe -3iHfE| ATT TT hToogs]| re[[] 1]| Ne 13 i ]: 4 HT i1 i|n: ESR { git] | In } Sali "8 Eels oF HE [nen | EE SRINRERR iT Someo reTT RSE Chl --e--ee------------------------------------ spinon - Project: E01-1378. CONTRASCMTELNAVBIORROANTMOERNYTWALORLKABOORRDAETROBRYYSAMPLE Lof2 Contract Labs): EXYGEN @ DReeqpuaersmteern:t:Ro0b2id1e8au0, RSioteSocuRrhc(e0:0e02l-03lE-0e9) PDraotjeeRcetcNeuimvebde:r:1042001 Project Description: Mallard Reporduction (Eggs) `PCroomjpelcettLieoandD:atRea:chelRle ~ Robiceau EPmhaoinleANdudmrbesesr:: r6r51o7b7i81d0e6s5u@mcnom. - spo S[3|[ 07 Comments: NOTE: MISCSERY = Procn separation condcied by Exygen Research. nN FE3oMorSaaimpileT6sN..S.a2mp0.l0ed1.D.ateW_ iS0ia0m0palmelaiDess2cr.i3HptYioo0nlk MjInSsCiv_eSCEoRteV AuMissie.tiSeedrMveitshod eee_ CMiosmep.oSneernvnices memeATnaaslVyos0iR0sDz.ueDate * poLmsien 61132001 10 pom ai. 23 HYo2 lk. a AeMnIseSiChSCEoRdYe eeAnMgiisyet.icSselrvMiecteshoed ee CMoimspeo.nSeenrtvesictes E0L137831640 132000 1000m ai 234H Yok rsicrmeorrssae SsAnEn0zaE0l0D2vuesDiatse. errs a AnimiCode Ansivticsl Method Component AnaivsisDoeDate. -- MISC_SERV. MaMiesc.dmSerivikcess Misc. e Services G6307e 2002 o EUTIG AnantCoge 6132001 100mai 23HYo6lk Ans Motos Comprass AmaDlueiDaate MISC_SEReV eeeae Misc. Services eeMisc.e ServiceseiiivrimsmaraeneT &n02E 002 _ernenmn . EO1-1378-31654 D . AnMsIlE SvnCiSsCEoRdVe,. EoL17831655 . MAIE nSsiCn_iE SCEoRdeV . EoLTaIess AnaisCode MISC_SERV 6132001 10 pom 4.3. 212Yo9lk AnMiasiet.ieSaelrvMiectehsod CoMmispeo.nSeenrtvisces 132001 10a0i m2311 Yolk eae eBAn0Ia0lE0DyyesDiatse rm reeAnMailsxe.ticSaelrvMiecteshod eeCMoimsep.onSeernviyceserstensAnnB02aE0iW0D2Lvy_esDiaetsemirm: 2001 10ai0,23o31Ymolk AualticalMethod MMisee. Seervices Compneny e Misc. See rvices AsaluisDueDate 6502002 " E01-1378-31657 Anaivsis Code | bMiIcSCSERVSO E0L1378:31694 PR MISC_SERV 6/1372001 10 vom ai. 23 1Yo5 lk Analvtical Method Componeny AnalysisDueDate. Mise. SerovsicesOOOMi. sc. ServicesA..6. 302002SS 202001 1050m 2.2301 Yolk AMniss,eSelrvMiceetsod CoMmipso.nSeernvsices eae An6s3i0n2s00D2uc ate odnrl ---------------------------------------------- smi CONTRASCMTELNAVBIORROANTMOERNYTWALORLKABOORRDAETROBRYYSAMPLE 20f2 Project: 011378 cont) Contract Labs): EXYGEN 3MESoSarammnppmleies S2Soa0mmp2pl0le0edd0DDaattee S_e1Sm0a0pmopllmeeaD.Dee2ssc3cr0ei)pitApioltnbnumen - AustCote a = MISC_SERV AnsonMethod MMeiseS. nSerhvicees Componeny Misc. Services AssisDueDate G63e 02002 E01-1378.31697 6212000 1050m 81.230. Shell Membrane AustCog --M_iscsERY Aus Method Compona MMise c Servn ices e Mise.e Services h A6n0a2e i0s0D2usDate E01-1378.31698 6212001 100om ai.2321Yolk e M--AIm_aS--iCn_isSCEoRneV AnMaiiscv.tiScelrMviectehsod CoMimscp.oSsee ervrisces Anena0i20s0D2ueDate * Eonomies 202001 100pmai 232) Albumen _ AnMsSCaSCEodReV (MAnMaeilsy,etiSctearlvMiectehsod WCoMmispe eonSeenrvtisces G6A5e 02n00D2iueDlate E01-137831701 202001 10pom a. 232.) Shell Membrane o AM--IuSn_Cm_--sSEC--RoY AMniscs.oSnervMiecteshod CMoimsep.rSseervniyces Aesa0i0is0Do ueDate e o EET Aus Cote 6202000 105omai 2341 Yolk Autti Mohod Compra . Ausini DusDate pros PROS by ESMS PROS 6502002 roma APnRsOSaCate 202000 100om ai 234) Albumen APnOsS bay EMeStMhSod CPoRmOpSara A6n0a2i0s02DucDate E0L-1378:31705 . AubniCo 212001 10 bom a. 231She4ll Membrane AnslMethod Compencas AssisDueDate e F--R_O-- h PROS bye ESMS Pros e 6302002 . EoLLTE3ING 6212001 10pom 8.2361 Yolk Anp--r_so--saCog APRuOStby EMSeMtSod CoPmrpoosnent As6a3ei020m0D2ucDate Eolas 6212000 1000ma. 236 Albumen , AP--uR_sO--StCote APnRsOeS byeMEeStMhSod Componcas Pros AssiDuneDiatte een020m02 EOL1378.31709 AvsiatCote 6212001 1000m a. 236 Shell Membrane Asati Method Compsncas. AnsysDueDate eros PROS by ESMS ros 602002 . ; N . n15. -_----- SSNS FEO TT ; se re Hn EY - . PoCR reRed NH Hr o8 sPeE EPt EE:| : = oei JIE N led EEE | | Te LY LE [ow TTT il 4 iHTT Sybal] a1] Ail Eason ; y "Ei - BRS i | Meddmmmeeisd [Ny 1 [1] Jl EE [11 HR] Fil a EE HE Se : fs ii EE Hess 1 Le 0 rel _--mnmn--mwrm--m--m----m-m-mmm - - [Tel TT TTT] | Aif E E elE HHH E - a C [13E ] R 5I] |HHH Ei az Fre (SLY CT ki [FEE. qlBaa0Lad) = =e e I | .3 Te TTT EN alDE ATE Csi lee]| [121] . :i on ||| BP le | IE 3 F5il | JM H d CEE 138 Aaiazage | LH 8 Bal FREE i| _-- `E en - XY8RESEARCH Fonfos . - Sample "Condition Upon Receipt" Form - Protocol # o MA. - Exygen Study # __ 022 --070 D470 . Date & Time Received __ &-/-02 _/ 245 - Conditionof Samples __ 2 Dry zo - 9 - Temporary Storage Location __~ Initial&s Date Lu & 02 - - Waybill# _F2vs pep sop (red fi) . Comments: Spfivrday ded coo did tf buwe rldy gumber, SK free --_-- -- ww - A Q:WORDIprcepsrs-oocgiemsSdasompin SampieConditononRscepztdoc Go. n on GAL _-- )| -- ET Fay i Fao EET . HLi y |iiCl ( SEa E E Gh LSE at 14 Bb innide ` BR ER TEE Li . i ge ES Bn) Se : wil 21d ely 3 cl ise .- E BeAaNr | Eg 1a | i| [88B1SCNS = ` al ! 3 - 183 | 4 38% FOB 2 a= a= Pdliad,d1e05s0 i15= ZSahnogolJehAni= = BE Pi FEE Ron. a 4 CAlsrs -_-- ip - Pl En i23 i " ] gis RE . HEE i Eh 1B i . I| - : a | Ny QNW g chan? -- - ` : - a - - for tly LM5 : EEA :Lo ee oe seenee! us WETS 4 3 ste coe, | wThHee an eccse Price ge can 0 CHANGE. '. ai soe vd ny esas | AsCuonsSWUoEu1'HvsUaRlcu4use5snesEvrueosrevitnasy cus |i } WAVE THE POUER 10 SAVE NORE i TER1S3 AEN sec EXACT COPY oF OF . 5.02 la o towe6520 nan -- : Exygen FrecPhreovReensReRaErechS.EARCH - - SinuCmommbairnoeyfdPMFaOllSarRdosEigdgusYoFlokuSnadmp(plpebs) ree in Sponsor Analyte Found Fort. Lovel Recovery - n7aa((RReeaaggeienonttCCoonnttrrooll)) (ppNba) na (ppb- ) - (5%) . - nnaa((RReeaaggeennttCCoonnttrrooll)) nNOa -: :- nnaa((MMaattrriixxCCoonnttrrooll))* nNao -- - nnaa((MMaattrriixxCCoonnttrrooll} nnaa .- - - nnaa ((LLCCSS)) 1108 101000 1n0m 8 a nnaa ((LLCCSS)) 101003 101000 110003 "dup 5523360000 s2600 -: ,p N = Dup* 52500 . : DNuQpl=iRceasutletIinsjeacbtoiovne the detection limit of - 0.5 ppb but below the quantitation imi of 10 ppb. compositoef amples E01-137B31EI, 31659, 31640, 3164, 31654, 3165, 1658, 31657, 21694, 2688 31702, 31708 - . ROE BX Tear, un CFrE)S Cha -_-- } Exyogren ProvenRess. - ID - na (Reagent Control) - na (Matrix Control)* id ey b) (%) NO - . NO . . . . } Co ree m-th iaee I - EXxYy8gten a - RESEARCH . 0 ob) (%) - na (Reagent Control) ND - - - na (Matrix Control)-VLDL* 143 - . ceren GNLBIL ------------------------------------------------------ " " Exygen o S3t0a5t8e RCeolsleeagrec,hPDAri1v6e801 TF:: ((881144)) 227321--11508309 RESEARCH - STUDY PERSONNEL LOG : PROTOCOL __NA EXYGEN STUDY _02%-0%0 - : . Nn 11] ~~ 1 [| : RN lL] . SN SN 1 |] . NL 1 . ie, I7 - rN] TN] : rm ~_1 1 .I 1] 1 Ne] [>] . 1 June 28, 2001/1 o GnABIT - - - ANALYTICAL PHASE PROTOCOL - ANALYTICAL PHASE TITLE EXTMRAALCLTAIROND AOFNDPOQTUAASISLIEUGMGPEYROFLLKUFOORROOACNATLAYNSEISSULUSFIONNGATHEPLFCR-OM ELECTROSPRAY/MASS SPECTROMETRY . SPONSOR 3M EnvironmentaBluiTledcihnngo2lo3gEy-0an9d Safety Services St. PauPlO, MBoNx5353133331-3331 DATA REQUIREMENTS Analytical Method Requirements PERFORMING LABORATORY - Exy"g16e5n8 RReesseeaarrcchh (DErxiyvgeen) . StaPtheoCnoell8e1g4e-,2P72A-110638901 - . Beygen Research Page 10639 COB0Z - - Title: EMXATLRLAACRTIDOANNODFQPUOATIALSESIGUGMYPOELRKFFLOURORAONOACLTYSAINSESUUSLIFNGONHAPTLCE-FROM ELECTROSPRAY/MASS SPECTROMETRY TABLE OF CONTENTS - Page TABLE OF CONTENTS... 2 - 1. PURPOSE. nd " 2 REFERENCEMATERIAL...... nd 3. SPONSOR nnd - 4 TESTING FACILITY /PERFORMING LABORATORY .....coch - 56. PSRAOMPPOLSEEPDREOCXEPSESRIINMGE,NS TALTTIMO AEN-DR FIDREANATMIEFIG .CA.T.E IO.N.c.o. corrnd 7 ANALYTICAL METHOD ccc 8 EXPERIMENTALDESIGN.......... nS 9. RECORDS. SR mn 10. DATAANDREPORTccc JE ---- in . 12. PROTOCOL APPROVAL FE 7 APPENDIX. ANALYTICAL METHODS .....c.co Exvgen Research Page 20639 Coa . - Tit EXTMRAALCLTAIROND OAFNDPOQTUAASISLIEUGMGPEYROFLLKUFOORROOACNTALAYNSEISSUULSFIONNGATHPELFCR-OM - ELECTROSPRAY/MASS SPECTROMETRY 1. PURPOSE `The purpose of this stud is to analyze mallard and quail egg yolk samples and - their fractions for residues of perfluorooctanesulfonate (PFOS) using methods entitled, "Lipovitellin, Phosvitin, and Very Low Density Lipoprotein (VLDL) Isolation from Chicken and Japanese Quail Egg Yolk" and "Determination of Perfluorooctanesulfonate in Egg Membrane, Albumen and Yolk by LC/MS/MS." . Both methods can be found in the Appendix. 2. REFERENCE MATERIAL - `The following analytical standard will be used: a Chemical name and structureofthe compound ijs presented below. i - PFOS Chemical Name: ~~ Perfluorooctanesulfonate - IUPAC Name: 1-Octanesulfonic acid. 1,1,2,2,3,3,4,4.5.5,6,6,7,7888heptadecafluoro-, potassium salt CAS Number. 2795-393 Molecular Weight: 499 (CiF17805TM) - carpe . o Nobottaei:neTdheis. nepuetrrfalluomrooolcetcaunleesualfnodnatsetanpdoatradssfiruomm which the PFOS salt [CsFi7SOSK], (anion) is molecular weight 538. A record of test and reference substance receipt, storage conditions, and a record. ofuse will be maintained at Exygen. Forms documenting chain-of-custody and shipping records for trackingofthe test substances will be included as partofthe raw, data package. Exygen Research . Page 3of 39 CGRLLI " uAlnliqsuteanldaabredls/otresntumsubbesrtaonncetsheacnodntaaniyneprreopracrreodsss-orleufteiornesncmeudsttobtheeicdoennttaiifnieerd.with a HAZARDINFORMATION A current MSDS for the chemical(s) used in this study will be maintained at the " testing facility 3. SPONSOR . 3M Environmental Technology and Safety Services Building 2-38-09 PO Box 33331 : " St. Paul,MN 55133-3331 . `BiSlplonRseoargReenpresentative:: 4 TESTING FACI/PELRFIORTMIYNG LABORATORY . E3x0y5g8eRnesReeasrecahrcDhrive State College, PA 16801 . PErmoijleyctR.MaDneacgkeerr:, Exygen 5. PROPOSED EXPERIMENTAL TIME-FRAME ` AAnnaallyyttiiccaall Start Date Termination Date June 3, 2002 June 21, 2002 . Report Issued July 31, 2002 6. SAMPLE PROCESSING, STORAGE AND IDENTIFICATION . All mallard egg yolk samples received will be combined into sample using a blender. The same process will also be used for aolneofcotmhepoqsuiatiel egg yolk samples. - Each sample will be assigned a unique sample identification number at Exygen, . which will be wil be stored used for tracking and identification in a temperature-monitored freezer, of the samples. maintained at < The samples -10 C, except wsahmepnlersewmiollvebde kfeoprt eixstorlaacttediofnroamndtheantaelsytssisubsatsandceescdruirbiendg sitnortahgee. method. The : dSoacmupmleentreedc.eiptAlalndsamsptloreasgeanldocaantiyonreasunldtincgondsiatmipolnes edxutrrianctgs twhiellstbuedyidweinltlifibeed . wcointthaianeurnioqrucerolsabse-lreofrerseanmcepdle10ntuhmebecro.ntaiSnuecrh identification will be either on the Exygen Research - Page 4 of 39 GALLS _--_--m - 7. ANALYTICAL METHOD `The composite mallard/quail egg yolk samples will be extracted and analyzed first - according to the analytical method tiled "Determination of Perfluorooctanesulfonate in Egg Membrane, Albumen and Yolk by LC/MS/MS" 10 determine the total amountof PFOS in each sample. Then the mallarc/quail composite yolk samples will be separated into three fractions using the method - "Lipovitellin, Phosvitin, and Very Low Density Lipoprotein (VLDL) Isolation from Chicken and Japanese Quail Egg Yolk." Each fraction will then be extracted and analyzed according to the method "Determination of - Perfluorooctanesulfonate in Egg Membrane, Albumen and Yolk by LC/MS/MS". 8. EXPERIMENTAL DESIGN Samples obtained by 3M Environmental will be shipped to Exygen Research for analysis. Samples will be extracted and analyzed at Exygen according to method, - "Determination of Perfluorooctanesulfonate in Egg Membrane, Albumen and `Yolk by LC/MS/MS." - Methods to control bias will include assay of untreated control samples, forification of untreated control samples to obtain recovery data, and replicate . analysis of fortified samples to provide an indication of reproducibility. Fortification will be made to the matrix prior to extraction. `The average recovery and relative standard deviation ofthe fortified samples will be calculated. Ifnecessary, apply a standard test for outliers. If an outlier exists, . tfhoeunnditamndaystbanedearxdclduedveidatfiornomfotrheeascthatmiasttirciaxl wainlallbyseics.alcuAllastoe,d.the average residue 9. RECORDS Records to be maintained include, but are not limited the following (as . ap1p. roSparmipaltee)t:rackingsheet(s) - 32.. HSiasmtpolrey arencdeipprtepraercaotridosn,osftosrtaagnedahirsdtsor(ys,toacnkd, cfohratiinfsiocafticouns,tcoadliybration) 4 Descriptionof any modifications to the method S. Instrument run sheets, bench-sheets or logs - 6. Analytical data tables 7. All chromatographic and instrumental conditions. ! 9.. ASacmopmlpeleextteralcsttiionngoafndstaundaylypseisrsdoantneesl, signatures and initials 10. Chronological presentationofall study correspondence - 11. Any other data necessary for the reconstruction of the study All chromatograms will contain the following: " a Sample identification, date, arrow or other indication of the area of interest, and injection number corresponding to the run. - Exygen Research Page Sof39 Gas " b. Additionally, fortifications will include the fortification level of the analyte. - c. Acnoanlcyetnitcraaltionst(aen.gd.a,rdpg/mclh,ronmga/tmoLg,rpapmbs, will additionally ppt, tc.) include the Each data set will contain information on temperatures, flow rates, column - parameters, gases, instrument parameters, andinstrumenttype, etc. 10. DATA AND REPORT . 1. All raw data and the original signed protocol will be maintained in the study file. This data includes the laboratory notebooks, analytical standard solution preparation, sample chain Of custody sheets, sample work sheets, - chromatograms, calibration curves, andanyother appropriate data generated. 2. A report will be issued by Exygen at the completionofthe study according to the Sponsor's specifications. The report contents should include, but not limited to 1. Objectives and procedures stated in the protocol - 2 Analytical and statistical methods used 3. Reference materials identified by name, lot purity, and other characteristics 4. Nameofperforming laboratory and analytical start and termination dates 5. Tables containing all applicable data 6. All chromatographic and instrumental conditions 7. A complete listingof Exygen study personnel 11. Costs `The total cost for performanceofthe study will be $30,000.00. . Exygen Research . Page 6 of39 [tk SER _--mnmmmm--m - - 12. PROTOCOL APPROVAL - Project Manager, Exygen . . Scientist . Sponsor, 3M . . . BJ ifRee agenre -- . . . 5Jz pe Da ate fb ox - Exygen Research - Page 7039 Lan _-- . ' . " " . . . . APPENDIX: ANALYTICAL METHODS A. "DeterminationofPerfluorooctanesulfonate in Egg Membrane, Albumen and Yolk by LC/MS/MS" B. "Lipovitellin, Phosvitin, and Very Low Density Lipoprotein (VLDL) Isolation from Chicken and Japanese Quail Egg Yolk" . Exygen Research - ' Page 8 of 39 GAs --_ =, Lo mmE : DeterofmPeiclonr:oaosatneislLfooonnMieSinMESggMembAbrinaanndiYeolk,by * | * :i ElySuAffUeTrHanOdJRoShn: Fiery :: }{ i . : DATEISSUED . Co mbay2aon . sowor 3M EnvironmentalLaboratory | i . . BuPlOlBinogr2333383015 . St.MPNa551u33l33,31 ii . : " PERFORMINGLABORATORY Centre Ana ly3t0i4c8alRLeisbeoarrtcohrDiresi,v In. e Cente) `State College, PA 16801 .: ' . - .| | i CENTRESTUDYNUMBER .: 023-015 `CENTREMETHODNUMBER .. oomozs015 `TOTAL NUMBEROFPAGES 28 + t : . ! i| . i Exygen Research . Page9039 61s _ - \ " (CentreMethodNo: 00M-0Z3-015 k " : . MANAGEMENT APPROVAL . . ".wAiatpheGco4o0dCLaRbor7a1t9o2r,ymPreatcthicesd.evoewleavpoeern,ti howterekewasdin0cboncfoonrmdaewniictnehcoamppplicibslcee |i - standardgperaingprocedures ndgeneral GLPregulations. t "! ! . ii | " i | . . i| i . 2: Sauer Hh Date P`rCiennctiepaAlnaIlnyvteiscdagaLoarbortries .ne. . . chm hh LaobnorFaoarbyMyanager Dae Cente Analytical Labortoribces. " | `RichardA Grbac, PnHD. etrD-ioen + . PCreenstirdeenAtnalytical Labordories nc. = Nala -- LfJ DSee nsornRepreseiaive ble Envitonments Libortory | . i. : : i{ t . "i i;) | - Vo )i | . . a . Coerp ste Sy43015 Exygen Research er Page 100139 CALs . " Sl! " . : | | . : ! . . ;i |{i .| - 11 | i[ vo 1 . w . Cent:reMethod No: 00M-0Z3-015 . `TABLE OF CONTENTS | : + + TITLEPAGEcancers mermmimmemssessmessssmemmemees | t MANAGEMENTAPPROVALrorsmmmenmss i TABLE OF CONTENTS rere emer LIST OFTABLES rerererremeermieemreeeer LIST OF FIGUREScert immmessminoeeed 1 SUMMARYcerececrmmemeeeeemrmememe | 2. EXPERIMENTALCOMPOUNDSworecrmreommerrecoesmsmmsesmsiemnines] t NE 4 COS reer eer eee eee] i 3F F2o. Sr Tr ANDAe ReDSr..-- . -- o ---- S-- -- m-- -- --m-- -- -- s ------ ] --|| $333 t P S opr o S eT e T T mI mmesmme, nrros) 8. METHODerenca e10. AJEEm F Smrec-- on-- C m --I -- N r --I ---- mreI emrereI rrrchemI tamanel] +" i 04.E ANAmLYSISBStoYtLsOIBMpSooaInMee taSPtadvrorGraeo imrm in Ge om dinsm be ae p s n iii I] i oA s!Pamaa mmm r mr emgeoomssmesmme s--a--mm--e i ES -- ------TTTTTTTTTT TABLES cie merss 18 FIGURESworsening2 [--------T Pw | Exygen Research Page 110739 "" GALE . : + Centre MeNto:0h0M-o123.d015 : v. LIST OFTABLES - : TABLEL SUMMARY OFRECOFVO; RPEFOSRIN. QIUAELESGG .. ; - TABLET: SUMMARYOFRECOVERIESFORPFOSINMALLARDEGG f PB TABLE I SUMMARYOFRECOVERIESFORFFOSIN QUAILEGG ABLE IV: SUMMARYOFRECOVERIESFORPFOSIN MALLARDEGG "TABLEV: SUMMARY OFRECOFOV RPE FOSR INQI UAIE LES GG ; i " i "TABLE VI:SUMMARY OFRECOVERIESFORPFOS INMALLARDEGG i - "i :. : { |. - : i; i Con epi List. ty 0S Exygen Research -i Page 12013) 613d _----,--,-- - " Centre Method No: 00M-023-015 ~ - Lok LIST OF FIGURES 3: | FigurIe: RepresentativeChromstogram of 8QualEggMembrane COR... 23 i PM Figw2eRepresentativeCororiogamof a alldEggMembraneCon...23 ' ? " Figur3e: Representative Chroralogram of Qu]Egg YolkCOntol cer 26. } Figur4e. RepresentativeChroiogam of 8 MallEgagYroldk Con)... 24 `Figore 5: Representative Chromatogram of 8 Qual Egg ATbumen Control. 25 " Figur : Regsenaive h:romiogan of Malin Egg Albuns Conol: cc25 i . `Figur7e: RepresentativeChromatogram of aQuelEggMembraneControl Forifiedat b ", . ; Figur&eRepresentativeChroraatogyamof Mallsrd Egg Membrane CoT ntrolFore tified at ii Figures: eFrerseptoaiiveChiofmssQuailEegodgYoglkCaonmt Forifiead : Figure 10: RepresentativeChromatogzamof aMallardEggYolkControlFortifiedst oo Ph . i || Figure 11:RF epresentatE iveChromatogramorf ae QuailrEggeAlbmumemn Co--ntr--ol--Fortifieadt | +*+ 1 - |. Figute 12: Ra epresentative Choromamtogram of aMallardEggn Albumen Con niolFotficd st. ~ | - i To { . Cone Atae Sy HS Exygen Research " res | Page 130139 60s - . ii :: Ce.ntre Method No: 00MAZ0IS . . : 1 SUMMARY : cy . Tpfheoifrusmoudrlooaacocfuttmnhecensnatldaieloantlssiscgi(8vPeFmnOeStn)hSoeicdntoieogfnga2mneafmlbyisrisasnnfet,ohraoldtb.huemernesniddualyodlke.ermTihneacthoenmicoafl | i f . . .i i . ePebcafrmlnueoner2oos2cadtmyaponlelesskl,sfaotnhmaepemleset,heaxanaoc0l.t5eedoxfafrcotcmaeirssbopcnahsmsiseatddtxdreodwuigt6hhamanemteshmalbniorqlaupn(eMoeffitOlhHte)er..meFFtoohrarneeogglsg | | SapmeecrtomQaueayn(iLscOaMiSoInMoSf)PaRnOalSysiissaicncosemlepciledibeyslahitqeiuinddmcohnriotomraitnogg,ra(pShRyMl)u.nders ass GFE oovriotpimse enmte.thodetehe oLOsQhais,10angc/gh(putmsp4eraybiolkliocn)o. l"Tshspisebasaetdinogn astmudoiess . t LF . i . .| - 1 + a . ) . i ConteAoiLibri Sty #23015 Exygen Research . Pes | . Page 140139 adn _ . . ! .i .| . || " i| PB A i .| ii| i i1 . . - . : . CentreMethod No: 00M.023.015 : : 2. EXPERIMENTAL COMPOUNDS ThemolculastrctreofPFOSisgivenbelow: i eros |: i Molecularweigh: 499 CiFnSOY) i o : CanCdTee IChUePmAiCcanl mNaem1e-:OstanPcearlflloutriococtiadne1su1l2fo2n3a3te44,55,6617.883- P case: "hepade7c1af9l5uo3r5o-3potassium salt : : Note: Toomnfseplecmfooloeocuclteanacnsd usftaonndaatrdpoftoasmswiuhmicsahltP(ECO.SF (aSniOoKn])is(mdoilevceuldar i| weigh $30). . 3. CHEMICALSAND SUPPLIES . 31. Cimacas { CChaermbiocnal Methanol (MeOH) G1r2a0d4e00 HPLC. SSuopuerleeso 7C2o10e0 . ! (VWR)LT.Baker TT90932 1! . AmmoniumAces Ser Regen Tel C Aldich m Chemical . 111475 |{ TypeTwier = cocamsvit,minimom of1667 Ma 3c 5C, foma } LibeoncaTM waerpr workstation, based on ASTM classification) : 32. STANDARDS Era RetCerTene Number FOS } Amul TOROX S EmLavboIiMratoorny, mSt. ePaunl,aMlN unreAny Liboraric Sy #023015 Puse? i Exygen Research . Page 15 of 39 CLL NY (Ceatre Method No: 00M-023-015 - 3.3. EQUIAPNM DSEUPPNLITES ' --BBaalla_ nnecee,,_51 ppll_ aaccee so_ apllyotai_ dcailng__E. QURM SMMOeeE tetRerrN ET tb . MWianitBtescatdoBn eshearkesr BloSpBeuclPrlodues i! . SBMieanalccehdnoiispptocuseganbeilf1eu2pg0oel1ypropylene ene bes vEVwCRR : M`Sitcarion-ecsesneiefubgeesbtse(scat #11075) Bio SpVee RProducts ol : `D1i5s.p0os0adbilseppoispaebtlcesp,otleysptrtoupbyelseneec.centifgo bes VWwRR i . i: 362`3-Wmh3La5Et2pmpa)enaTMdAonroftTMomipcTMroFicletnetrr(icfua#gtet6u8b0e9-s10(2ca2c)h 22- VVWWRR . i!| ii S2tamndcalredaarbHcPLqvieikCtpsestrA# p1e8i1p.n3e4s0t0a)nd 2 | > L`CIvMSoMSl ftlansudksH,mPgrLae Cduayt tsetdecrmylsiindec rs, etc.) As decribed " .. - slat i . Note 1. Iingohrderrectoomamveoniddedcocntoammi.nianti,on,tubtehse, pseetofsdiicpo.ssle labware bod | i 2. H"PTeLflConvoiratceafplso,n-slhionuelddconngtsbaieneursseodr.equipment, including tefloThn-lined y . {|| . 4. ScteoiensntafnmeicneaosnstasruybiyaosLcnuhOetMcakdSbltIehMeSosrobwlevefe.onrtes(umsee.thaCnealr)ifnorlotthenpurmbeesresncheavoef oId : 5. Use disposable icropipees or pets 0 aliquot sandrd solutions : . 6. mBaaenudtihwvoahdle.ennptreHmpoarweiengrl,stamhnadeayrsdbsee.asnodufbscasamirpbltoeens fffoorrroemhxtorsSaucetpiesolpnce.coifiisedstirnontgliy i reommended. i . i . i| 34. 1S.50oLSaucrmitmoMnsm| LoofASnTsMsipsesuoIlwuattmieo.nS;toDrisisnoalvnes3.p5prof olamapmaobiroitorme : Pos 2. 2 rmodom tamepmepnieurmaftosruucpreteoo|lyueoun.: Dilute 40 FL. of he 50 mM . A `rmmonium sett scion na lerofASTM ye Twat, for mobile ". ConeAnyi Libre a.Sud 0015 Pit . `Exygen Research . Page160f 39 Gnas ----------------------------ereeree et re ee - - " i| -v - | iii | ' i - - Ny - : : CeatreMethodNo:0M:023.015 : . ophlayseeAs.. Strensn appropri lsbord at oomtemperafo up Note: Thevolumesshown ce providedfo guidance;airmasv; evolumed. | . maybe prepared. . i; 3.5. PREPARATION OF STOCK, FORTIFICATION, AND CALIBRATION i `SOLUTIONS . : . Arenusanpltoyntesieecdaolfssattmhipenlddeaesrsdisnorrorwedpeerrdeapianrdeehtdeefrnomriyntew.noapluyrptocslesr.eTcohveeryy!aarnedutsoecdatollfaorttifhy Tphreoposratliyosntsmoafysvoalye tthoestoblsvoelnuttesvrolummaentsionfetdh. stTahnedasrodlsu1isonlsoncgea3dthbeecloorwrewcet bi Evenssim cxampls aliemaiveconccauatonsmaybeprepa f cde. { | 3.5.1. Stock Solution ! `Pnraeypairaetstsoacnkdsarodl(cuoofmtePiFtOfooSnapterc1e0n0t n sl). gAbdyu/ swteifgim nhailnvgL ooluutme101.10m00gmoLf. b ov1fi2t56hmmoLe.ntLthDhaPEnrobmiontshed)1a0ie0naoLf.rpevrfeoiplgauermaretttirooirnc. tas.C toSt6-oCrfothris!asmiacxkimsoulmipoenriGond : 3&.5.L2.OuFogrt/iLfiFcoartiifoincaStoilonutSioolnustion - Fipete 1.0mLofthe 100pgasock omleaiaonn.ito a 100 mL volumetric sk. Bring up to, volume wit b.-0.1ui.Esuifcaton Scluian ~ Pipe 100mLofthe' LOpg/ml florutimfiecawtiiohn sotluhtiaono..into & 100-mL volumetric flsk and bring up c. 0v{0oo1rliuAmcega/wimioltn,hFomsraeiitfsihocoantlimonoSo8lut1i0o0n1~. Pvipoeltutmeer1i0c.0 fmlaLskofsntdhen0.g1 p0gp/mL1.0 ' HE! i$ i : Store. all fortification sands solutions (in 125-mL. LDPE bates) in a refigratorat 2Cto 6Cfor 8maximumperiod of 6months fromthedate : ofpreparation. : 3.53. Caltraion Standards 0Pr1eppapremsixanLdC0I0M1SIpMSy.cliorriafiiocnaisoannsdrudissi.n methanolvi ctionofthe + | CentreAnalyticalLaboraories, nc.Stady #023-015. Page. I : Exygen Research Page 170139 GaLsT - " . , `Centre Method No: 00M.423-015 : -| : Tehspis 4a opedtypical exearmpele; additonal concentrationsons mamay be propaed -ws ee - - Tnital Conaoe1.i (ug/ml) Volums 2e (wl)o Diluw ted1t0o (ml) Final Co0o0n0oe8. (ug/ml) ii! .: oool 51 1100 + ool 0.005 i . 0o0o1t 01s 11000 00..00600015 - i, eSftoirgeesalolrcsatli2braCti0on6stCafnodrarodmasosliutmiuonms pneriod1o2f5mlm.onLtDhsPErobmotchdeesd)atinofa preparation. . 4. METHOD } . { | 4.1. FLOW DIAGRAM i Tdehsecrifpltoiwonodfiacgarcahmseopf. the method is given below, followed by a depuy siled : . i | | } : Weigh 1 of ati 1fomembrane)Frynett : _ Ext winmeoH cite tn ptmente) } . Fil|er Ditufiion LOMSAMS alia .ty . Ii oo! = 42. SAMPLE PROCESSING : i -; "Aolfltseamepxleesciaoen.ceivedfrozenadwil bekeptfrozen olow -10 C)unc ime -{ 43. SMPLEEXTRACTION NOTE: All egg sample riatices were separated prior to amval at Cee. -. - Cn Anite Libros.Sy $023.01 Exygen Research . Page 10 . i Page 18039 CALs - . (CentreMethod No: 00M-023-018 : 43.1 Egg Membrane . - & Allowsample: tothaw. : . i - . Weeheiiwghoef0i1G+r0ma0e05rt9eoof0fos0a0bm0np3cl,ehF1o0rty2a.EmppeapcecodmeorsteteTMm.eRecord i! ! .: i -i f| -. i. ii | - | 1 - .CAoRhdmnode1gtcmeaLneoflwMitewchOiabHnseaaednwsbidteethhmrrreiasettt<aieenplneoissgsots3enenl2obwe(0a20d2s50,.0eo0plpsEapceeonslddmto3inrgahttetlywseen:'hd e,11 Ging wit WiatmssTM AncopTM 10 ls cosy 4 Make a 5X dilution by transferi0n.1g moffL le to HPLC viel snd i ding0.4mLofmethanol usingdisposablemicropipetes (100-200pL), + Soreefto xin a ppnienty FCC | forfuture re-dilotion or re-injection. ; 432. Egg YolkandAlbumen . [R-- : : b Weigh10 g (005)of maple to + 0m posablepoyiopiene cuenonrtvareitfneu.dge ctobnetr.olRescaomprldestheat wtehiigshtpoi1n0t tfhoer ndeaerteesrtii0n0a0i0on1g.of mFeotrhtfoyd | Co i Ad 25 ml of MOH, ee 10 ight, and she on risa I `sfhora15kmineuters. Centrifugetubesat ~ 2,000rpmfor ~ 10minutes. ! . & TCriorbmatatpsbheoent,e300m3gLofbeoxnt.oepo 154.mUL digswpodslaol epyooy ,opkso |! |i . || Lientothaetu1b0e-sms),itfdoisrpo~s2sbmlienustyersi,ncgaerefbualrrleyltracnosnfneerc~te2d.0mtoL+ofWthheastammapnleTM. =`cAnoeitoopnTMoor1r0rfsaileigr,oF8inler samploetnp to twolHPLCyvi2as, o1ne0fo6rionrjetctiron : . - Cee Amiri, Sy403905 Expgen Research Fett i Page 190139 [LEER"Y --_-- " " - 3 " -1 | ; .i !| .| . ii !i . - . mr : te CentreMethodNo:00M-023.015 : . 44. ANALYSISBY LC/MS/MS Ce 4.4.1. LCI2MS/MS SyasndtOpereatimng Conditions (Turbolonsprey) ; . MassSpec: PE SCIEXAPI3000BiomolMeascsAunllyazerr b Ineaces | HSaCrIvEX Tiunrbfoluosn iSporanyLiquipud; nrcuctonIerface i | Compu' PowerMacinohG3 Sofa WPiEndSocwseNATmm LL pL: HeleHnPPacQkuaartdGPFu)mpS.eries 1100 HHPP VAaucousuemmDpelgrasser HP ColumnOven >` :i i CHoPlLuCmCoTleummpa:: G3e5neCsis, foes Chromat2o.1gmamgx $h0myn),4,. Lo MojbeictlieoPnhVaasle:(A): 120a4ML AmmoniumAcettifASTM pe Iwater MoFboiwlReaPeh:ase(3): M03itmhUnnoiln. :: : . Tiimee 2a4v 10 o EYwio io Tety 4 fiii!o a" o ' 3{0nsmtiuanmy)meabnnedpnccfoeolrcummeannssefe.1omCaoddilfouftmenrsntthwemiaHhnPfdLisCfcfuegerrrsasdmie(enKntesyiisntoononsredBe(re5a.fol2o.Cpptmiemoicz.ne) ii can be ued,povidedequivalentchromsogrphy s Gbned. tommoon: . Approx.imate . APmROiS.e rMeotgee Toms4io9n9Mo9niond RecnionETEi}ne(min) bOanchdofemadlyepbass, etc.enon nsmy vr ig depewndieong te ConeAnLabyor inc.Sy 4023915 nen2 ; Exygen Research Page 200139 CALE _-- " . . CenteMethod No: 0M423015 : - i + 442. Esample Tune File Parameters . . ! . 1 fTrhoemfnollsowinng uvtaolmuiensesrreunmpetrno.viAdleds,ashaenseevxaalmupelsem.ayAcbteuaclhvnagleuedsfmoamyivmarey _ time nordertooptimizeforratssensitiv. i : "| | i| .! : Pos . ! . .i } - . "Tlhuetmioansofpethcerosomcekersiosluttuionnedinsimendghan0cl..5ThLe BsFolOuStsiolnaiinin,fpusreedpa(rweidnvgia& ! "cToTMncsoimspottfio4n0n1%g) 1me0thaLn/olmi1n0id6nt0o% 82m0Mmammmionnsieaasmcoef.mTobhielnepyhtaesse `rhee niansltlruymtenet ids ftoectdh,eptahreopntiamniazedd tpahreanmeftuenrsdfaortshaevperdodasuc8t"itounn.eOin"c.e b"Teirsepneeseldadfsnetcheetnuareydo ernisunrgerpouttinme nsaeyis.t:The ingprocedumraey 1 t CSolnotsroplrsay : OR.Orfce . "06S0a10t00 i RQNOG-QFoucaudsRoRidnOgffset . QU.Interquad lens 2110000 53 + STR.SOuLbbQieusRedOfict 1903 IRQO2Qtueardq2uardo2d olfefnsset. 28400 3 TROaSS:uQbbuiaeds3 rod ffct DF.CEM Deflecon Plate. 150600 3000 :|vd (CEM:ChanelElectron Mulipier 2000 ii GNeabusliFzleorGwsas sImt |i CCaullmiisnonGaGass 3" i! TIS Temperature asec . i| i : 4.4I.3n.ceCaltihbersaatmioenvoPlroucmeed(ubreersween 101020 BL) ofeach calibestion stanrd . {prepinaMreOeRd)into the LCMSIMS. b. sUtsaendalirndecaur,rveUsx aweegiegnhteerdicsufnodrucradchcusrtvebsyfloirneqauranetgirfiecsaiteionn.usinLigntehaer Cin Anyi Labrie oc.Sud 023013 3 | Exygen Research . Page210f39 GHABST --_ w - =A w . -. i} i:i i *i .i i .i i - . :i | : 1i - ' Centre Methoa No: 00M23-015 5a0p%p,rboapsreidosonfwiatsrecaalycusleamte.d cAonncyencralaitbiroanti,omnusutnbdaeresxcflluidnegdfooutmstidh+e . Cm alibbraa etieoxnccy luurdvee.d mHowevnsoer,extt cheeetdota3l0r%ofuthmeotfaobiabreandieuon mosftasbntdaarneddsatrrdss injected i } , 1 . T2h0e99c2o5ne(l2a0t9on85c)o.effiIcfiecnltf(r)tifoon<raelsiulbtrsaifolnlcouurtvseisdegtehersefdmsmu,stthbeen ;! . slpperovpriisnioctsfcspsasmepslteshmuosttubaeklernea1dn0aalbydzejed. instrumentopgpemion eration, andthe 44:I4n. oSamthpelseaAmnealvylsuimseusedfort1hecalibration senda (between' 10. 25of )cach sample, orfication, control tc. ntothe LOMSIMS. i b. Staannadanradysacolrmsestpondingtoalessix concenrtionsruebeincludedin |{ . Tnanjdcatradnsemteirssspetrosfesdtaanbdouatrdes(v3ey)5-a1t0hesabmepgliensi.ngAolftshuerpuen nindjeicntfioencst i . ustb backedbystandardinjections (seSection 4.5). & Eaacghensettcoonfstaomlp(lmeestahnoadlbylzaendk()o,otoneeAxSceTeMd2T5y)pmes1twaitnecrlbulanatk,eastleoanse. i oCnonecmenattraiticoonsntnodl,anrde(twhoomugahxt.hcpornotcoedlusreamtpovleersiffyrrieecodverya.known ` All implsmostbeanalyzedwithduplicate injections. :i J ---- fortifaic10a,5t0ianod2n5s0 ng/g(ppb)fortheanalyte. yo {. Trhoemctohenscuendnadotcfurravacehtsbaiamspoelden,ofnrtihcetpieoank,accoanocf,heec.aniadleyteirmninaeld i asnpodnsaersojfetchteerdesdcuurienfgou4nsdei.n hTehseamspelnesdeat.rIefspnoencseessaruys,tdilburtaectkheet Samples and analyz0 ivspoense withtsthanidcunrve range. i & SFaomrpilieaiwoilnlsbethaitncblruadecdewittheh hcighhesstarmepslieduseete.xpe1ctreedsiidnueesacwhrecfaotuendd ouubsaicdqeuctnheasepleimitsse, a0dcdittaiolnials tfhoartdmfectahioodnsrecwoivlelribese ariencalvuadeidbleinfo3r nc ani of retatconcentexrceaeditngithoosnesn rejsamples. i `CentreAcaiyealLabonte,Ic.Sey #023015. Page14 i Exygen Research * Page 220139 coat _ " } 4 ner remem i ems . : . (CentreMethod No: 00M.023-015 : " . h.aFtorhiefi1ct0iQonlreevcoevlerieRsecvoivners6b0e0tw1e3e0n%7a0res1 3e0%pweftoacfacoetpftcaifeoonsg - : . i. rsfSaoearmmtpriplflieeecsast.tiinonnsmwahstitlciehvgehlnisoopgnreoafecr atahrsaenedk.tihcedtLOeaQfd,ualtFltamhileaurnecdotoymsmpeooefnsdttihhneegsaeiacfrmilte i|+ prneeeneiorneTtodiwemetesstctiadlnltdbaehreoiprolerlsepdorne1pd3oNirntDgeda(na8aoltNydQec(tioced)na.onnStaiammpellse.ostaewilehss . - :i | | . Background levels of sy found in contr samples that `cnomedspocndotocvialucesobenlowetchoeveLreOsQ. butare scl quantifiable, willbe iW xseandp,easgwelesmmiekptesnstSoecoe l ig oewesse rpttrdt atyedo2gC eyeCe !|| ReTcaobvleersiLeVsLfrom methoddeve"lopmentforallmatics anbe f: ound in i - . 4.5.PERFORMANCECRITERIA . . RE : . | i . " i | .i - ibo m"CeTohpeoecfdaollllyowwihngentwwoincgrtdriifafrtesntinbs:eummeettabetfoorne ethekienitiGalananatlyhseisSoifdsamiplehs, { FesiitmCartietderLioOnQ- I(n1j0ecptpabstLaOndQaridseoqluuitviaolnenotnt10he&LsCa/nMdaSi/dMsSoclourtiroenspofo0nd.i2ngngfmtLot)he + | "ndchain &signalto noiseTao Of atleast9:1relativetotheesgeblaansk. If ptuhisrsceriste.rion cannot be met; optimize and: . change .instument operating | i `SecondCricon -nee st of anc ein rom crbelow heLOQ, up ] othe highest concentration level. Geneacraliabrtatieoncurve forthe analyatned. : fo`lorbittahieneada lmwineiaersreagOnrendscssieonihnwieisrthsrpeaecreoiedof.fihciaesnbtoefdnetderme"inamtioon (nr')sasomfparltesleiastde0y.9b,8e5 i . Cente Analyst Laboratories I. Sly #023015 Exygen Research , Page 15 i Page 230139 COLT - : . CentreMethod No:00M-023-015 : / Cad pn rept | . wf - 46. TOMEREQUREDFOR ANALYSIS . A ttof14 samplescanbe ken trough thecxincrionpect ; . i 7 LTE pei sssma hou. . !! 5. CALCULATIONS . 5.1 ANALYTEFOUND: i . Calculatetheamountofanalyte found (in ng,basedonpeakwre) sing the andandcurvegenerated bythe MscQuansoftwareprogram usingEquation | Equation 1: PI . Analy found (ag/nL) =suakacsao.gien-trest) | . 52 ConRpesDoUECNoNCERNTNRATTION Determine he compen sceconceniaion ing Equation FC ---- a b . EqA uati.on: oe ford go xDPxEY uy g:o3 . - : Note:ng/g =ppb. v i : 5.3 PERCENTRECOVERY : : iCeusiitreepexreontns,cooveny Eforksiamnpl.es foiled with know, mous of i Sows k Recoey (= (opto veWngaespgatoonincn 10 - ` Cre iiri.St 4515 ExygenReseach rts Page 40139 ns " : "Centr Method No: 00M-023.015 . .: ~ 6. SAFETY : ! . . + eNThoaerdrmteaealrlbonreotraiunlrusyspufraeltcahdaaazoatnrdshs esahstsooflocdiabateleldicaWiagte.hnttshibsefmoertehpode. oThe anatlhystmsthnould |hit! t .i " ie ! . . . ) : . " . bo :i " CI | . - : Sa | { | 1 CureAmbLitmcrinn Sy #025015 Exygen Research i Pt? | Page250139 CALS - ie - - : - - i * i -i -! . .i | oC . Centre MethodNo:00M-023-015 : . . i ` .! : . . ' . . . oo . oo : f i . ; . { a ) i `| . : i I - Coe ApLisa,Sy 42015 ExygenResearch --t Page260739 nL . " am . Cavemenowners . Table: Summaryof Recoveres forPFOSInQual EggMembrane _ - Sure Fo Low pr .: - Tm o a vaer eEltnsnk Eo0Eeee 0"e e. $| mo8maspBnak wom spe lIo "u5 | }i . pc0r0o0o3s7m9i8meSasptk yC P15Y00 . 52 s2 . . !i . pwomwsrostmsesinEkrda 2=50 wo | .i comm spt STR2ATDEVEIAGTIEON: EBn3 . ti: [ "RELATIVESTANDARD DEVIATION: 3 i .| - i "TableI: SummaryofRecoveriesfor PROS(nMallardEggMembrane . P|o Sage" TOES Feort wLoevel Recovery ww . ot i BkD o wm : . i i `000w3c8oo02mnMitactrispxpeBelnask C wo N44A i|A i woe ws i || . 0003n8i0n2 SsppkeD. 0x siz spk 95% y 1i - wwiomrsppeea 00 no i1 coon get x EGE i -i RELATIVEsSTTAaNDDAARRDDDDEEVVIIAATTIIOONN:: H .i NA= NotAgpicble - . [EV -------- " Exygen Research " 19 i Page270f39 GH - : : (CentreMeNto:0h0M-0o23.d015 + TableIDl:Summary of Recoverles forPEOSInQuailEggYolk - _ CSE upeA Feo Lw0ov Aha7 gey ! - 0a7s8MMair Bmkk8 o wash 0 awe 5 ;i! - aassssssuac Cw a* { ossen Cow, n wcoausssseere worms so s0o , = 5w5o ii cmoeuss eutn 200 M : . :| STeDADDEVEIAGTOEN. 03 |} RELATIVESTANDARDDEVIATION: n i . TablIV: Sumary ofRecoverefor PROStn MallardEggYolk i . _--oT H SumpB " Reawo o 7 oo=r i| 0000007398viiBBk oo : cours ~an H5 i i cccooommsmsspypeece ww iivod . |! wwoaasmsatee 30 uxww RETo Ei - wworaege b=y ERAGE 5a> iii : RELATIVESSTTAANNDDAARRDDDDEEVVIIAATTIIOONN: 5: ii NNoA tAgpl= icitle :. . ConteAneLabi, Sty 4023013 Exygen Research P20 oC Page28013) GRLELD . _-- . . CentreMethod No: 00M-023-015 "'. TableV: SummaryofRecoveries forPFOS InQuailEggAlbumen - | seve Fo Lewd hacomey --t HE e a Bn 00796Marc BaD o0. ww a 00037w9o6MeasaBank C o 1 " aoosmsssspce 110 nFI . waoomessspn 00 556s .. wonsomessika 05 onomsspn Lm 5a0 @ asso 20 AEE 00] RELATIVESSTTAANNDDAARRDD DDEEVVIIAATTIIOONN:: 7i i | i} { ii ; " . i i . - -i i "Table VI: SummaryofRecoveriesorPFOS In Mallard EggAlbumen supe O _--RMa A Foentwe Rents = 0000080000MMaaiBBaarkk o an aoosni0sspeA 0 - wwoolonsspkc woos . 510. 55 anwsooimosaskno Sw0 w>5n Bag osmsnt 0 AEG IE] `RELATIVESSTTAANNDDAARRDD DDEEVVIIAATTIIOONN:: 7: NA =Nat Applicable. i : ]5 co L' o]i | | i ; . . ConteAnyi Libraries.Sy E3015 Exygen Research Peat | Page 29039 crane _-- . Cometic . . . . : ' . . 3 . . ; '| FIGURES i . . ] .} , -| CH ood Co } ] : . CentreAnalyticalLaboratories.lc.Stody #023-015. Exygen Reseach * Page 22 ! Page00639 Goon -_--_-- . - 0 u . " " - . . : -i i : * Centre MethodNo:00M-023.015 E "Figure 1:Representative Chromatogramof a QuailEggMembraneControl E| E ( EEE L ku qv : ' . } !|ii [asEa i [~PE ' ! EBEE L yEd | PR 17 EEEheER3E.1 : .i IfiEee "--dE3E : EE : !!! Figure2: RepresentativeChromatogramof aMallardEggMembraneControl : - * ; TE a, i CEE n : oo BSEE hH5a: 5e 7 . ka |: Co EEEE :| e E (EErEee 5T g LT s TS=T T T - ! ! * GenteAnsysLaborore, oe.Susy #023015 Pl \ Baygen Research re ! Page 310039 ents -_-- . ee . . Contre MethodNo: toM.az3.015 . Po - Fi. gure3: RepresentativeChromatogramof a QuallEgeYolk Control . . BfEm E . ' Eeaei : it |' - " Er5Ee.jo) a i l A ' BSEdE 2# ul . ] . |i " i 2 . *Figur4e:RepresentativeChromatogram of aMallard Egg YolkControl ": 7 . me LT | TEE, - ' COEEE . [AY i CEBERERHO : 1 | [Fs . - Fr HE Liem om 'od } : i : " . CentreAnalyticalLaboratories,Inc.Study #023-015 Bxygen Research Page 24 P-age 32039 ors _-- . . " . . "vo |1 hb . vl - cy | co I Contre MeNto chontoazsadts : ++. ifures::RepresentativeChromatogr ofaQualEgg AlbumenCoro. . RN wo i - "e | mFRErE"a Lh di. Lu o -- f i Eee ] EEE : C: i BIRF J ' i i HBoite HmewI, BEE, T rT LTTrEeTevwaw : r: ii Figure 6:RepresentativeChromatogransof &Mallard Egg Albumen Control Ui : = di. : . :} EE Lal { Eo E oll : 4 oH !i te [BT HS w er LL rePl i : CentreAnalytica Laborasocie, nc. Stady # 023.015 " Exygen Research . Page 25 ! Page 33 of39 Cav tan 5 _--_-- ee .: " " . . . - - 0|! : "od : . (CentreMethodNo: 00M-023-015 : Figure 7: Rr epresentativi e Chromatogram of aQuail Egg Membrane Control . . i. t is . " FE" oh, : i! Efe 7 7 BE fx EN EEE EF : f Be HE Lert ! :+ `Figure 8: Representative Chromatogramof aMalEglgMa embrrad ne Control Fortified at 10.0 ng/g(ppb) - me ESEE a :: . - :. s "| = Bi hotg rem pp , " : . - CentreAsalyticalLaboratories,Inc. Swudy # 023015 Expgen Research - Page 26 ! Page 340139 Godin EE EE -- . W " . "' " . wo " " " Po o --- Core MetodNooumomass | "Figure: Representative ChromatofoaQguarilaEgmg Yolk Control Fortifiedat 10.0 ng/g(ppb) : : : EEETE ud . ' EEEeE 7) BB 4x i ELE BF - . } ! . v od Hr WE = ; Firs 10 Hepresotatve ChromatogrE amof aMallard Egg YolkControl : . Fortifiedat 10.0 ng/g(ppb) .: Ba ma EEE Bum oh, 7 BE oid EfEeEi i. : | ol ! sn i: C- rs AiLibros Sui 4 015 . . Exygen Research rat? ! Page350139 6oLsY" _--mnmm--m--m-- . " " . . : . ! " Cli i : *CentreMethod No: 00M-023.015- : Figure 11: RepresentativeChromatogeamof a Quail EggAlbumenControl `Fortifiedat 10.0ng/g(ppb) - ELL {| a E - i - BEC dR, 1 Co E Enea1 =Jd _. : ' wi | EEE 47 wn i erm wal PR ! Ptr1: bRepbreietetCaroyppaf MalEggAlbaresCor , ESmE a oo B= SE hai HIT. EE: - 1 so _ at i + . CentreAnalyticalLaboratories,Ine.Study #023-015 " " ExygenResearch Page 28 ! Page 360139 60s ny --_-------- pun " : WEI I . ' ._ froUmpoCultlhin,iaPnhodcsJvaitpkna,neaensieGVnUerSy LoEGwE DeYDnsity LipToHApTrotein (VCooLk DLB)aAIs-o4la4t:i8on6 96 [1960] and Stfai er al., JAC 265:882.888 (1950]) . " - (Mey, 1994) . i: 1) Breraekmoopveenaedghge,risnegpaarlabtuemeynol.k, and roll yolk on a moist paper towel to : " 2) Puncture yolk membrane and let yolk drain nto a graduated cylinder. A 3) DiluMteM1gvSoOlu,me1 omfMagpgheynoyllkmweitthahn2esvuolflounmyeisfioufor8isdoelu(tPiMoSnFc)o,ntaanidni2nguM0.67 leupeptin " 4) Covseervecryallintdiemrest.ightly with ParafiimM and mix well by gently inverting i u SI Using a 20 gauge needle and 12 cc syrings, transfer the suspension to i aBmeocuknmtanofpoyloylaklplromeeprarQeudicakn-dSeroatlorswabveasiab(lteu)b.e size will depend on ' . 6) Seal tubes, place into appropriata rotor, and centrifuge at 200,000 x g at 4+C or 26 hours. nm 0 . 7) Foldlioawignrgamcaanttriifguhgta]:tioAn,,a4flianyelrasyewirllofbeyeelvliodwengtelin(VtLhDeLt)u:bBe,s(scelsear jr ' pLcaoallyloeerrtlseastBstashoneldubtoCitontc;oonmsC;t,iDt8,utv8eisftclhuoefufsyhiygyehellldloeownwssisutoyslpufterinaoscnitiogonrna(d(ViHLnDDgFL)t)o. a firm A= a]| . a(nadryl)ayleorwAdaannsdityhefrascutsiopne.nded material in D constitute the A. i|: 8) Usi1n0gpaof2t0hegatuugbeanaeneddlweitahnddr1aw2 lcacyesryrDinagss,wemlalkaestswevoerpaulncmtiulrteesrisn the ! - below the leva of layer A until layer 8 s reached. ) Wit`hYe3llsocwaluppeple,rcupatlltehteotfutbheeatbuobvee(tVhLeDLl)evesl tohfetnhererdaimsaslanlviendg ilniquaids.oluTthieon mcoMntaPiMnSinFg, 2a0ndm5M uTMrisl-eHuCpeIpt(ipnH. 8). 150 mM The lowar NaCl, pallet 0.2 mM EDTA, (high dansity 1 :. rfreascutsiopne,ndHeOdF;in,La3yesrolCutisohnocuokndtabienignegnt0l.y4w5aMshMegdSwOisth,, 1anmdMthPeMnSF, " Exygen Research . Page 370f39 cry _--_--m " " : " . aGenndt2it4uMgedleautpe2p0ti0n,,00B0otxhgVaLtD4L aCndfoHrO1F6 shcauutsi.ans are then 1 01 . F ollowing cantifugation redissolved In 8 minim of al the VLDL souton, amount of 20 mM the ur pe Tris-HCI (pH lle 8), (VLDL) ls 150 mM |: ' ' 4sNpratMeCo)plc.a,krsa0soto.dil2iuotnumimoMisnaznE1ioD0dewT0A08c,ox6m,1pdlmlei0MtueodaPt.MefSidTnF,ah,leacanlondondwscet5aro'nrputeMaadtllaoaetnu4(poeLpafCtv1.ienrF,AClMf)lilotpffehraheede(rHV0.LeE4D5L t i ' Tau0bde2is4fMrsltourpea-gdtiisns.avTehdeInn,atswooluvtoiolnumoefs0.o4f5coMldM"gapSuOr,e"1wmatiePaMrSoF, added 1 the HOF solution dropwise, wih sing, at & C (thus, sMtgaSndOigngc(ownictehnoturtatsitoninig)naow40.C15oMveir.nTighhat.solution is then lft ; " 11) On`{5td0h0ehsfatoelpltoo1w2it]nh,getmbhoeronnilrnsgs.oofiavtyehaetlhlfeoiwcpkrg.eelcDaitaiicanatonautsn(parnedcsiopsiiettvaieto)enhsohefou0sl.ud4pebMrenste t ; "aMenG-dpSulOarts,"stw1aanmtdeirovdPerMroSmpFgw,hitsae(nwd(itt2uhusouMtsosllueitunipeogpnt)iisns,dt i4AedCdd. o1nOan0.vt2ehlMufmMaetGooSfwiOcno)gld i " mD1uo0tr6fn,oir0n0gc,0onxtthageianptirneg4ci1piCMtaftNoea(1Cs,hhoo6usrvmiaiinndTirtsihese-cHroCevIsau[ripeHndg7b.py8)lc,oe1ntimsiifdduigsPaeAimSeoEne,sat3 4M leupeptin, and sodium sds added to a inal cancoruatan of " `mM (Le., dilute stock solution 1000 x). Aliquot and store at 4 C. . 12) Thd1el*t0s,au0dp5ow1miatthMagt"Sw(Oo0.v.)1o5ldurMmoepMswigosSfeOcto;ldhss-saai.spttuiernpeg"1a1wt)a4sthCo.u(lfadinnbadecoodnewcceatnnttrea0dt,ion | ' S3ptr5aonc8dp0to4avtesCri(fgiohprto1v(ihwaoiltuihrnoauitnsdhsuiersnisnogf)elceaodtva4nreCbd.ubtytTehceaconrntetsraufiiuniignnaggown1 haMte1N0a6C,l0030 | HasodAluotTiroansddH1aC0dI00t(opxaH.f7i.An81af.icn1oanlmcpeMrnottrPeaMitnSiFco,onnoc3fennd1trm2atMuiMo(n.leoe.fu,p8edp-tr1i0en,msatgnodcmksofda ium ] desired. . Exygen Research . i Page 380139 onan _--mmmmm-- . . Lirovirerin, Prasumio, &8 vist isecamend : Rom. E66 York (Mas, 114) - EGE --= Yok. | . . . DILUTE: 1:2 fo.67MMaloy Li| . CEM@. 2000 xy Ru2the tC | " Vp LD CAG evDendy b3r A10h )@aC-- HBG(FasahcohyeBred @ deg <p bid ge Corlmegn: BEDf ORIy EGE ' hy 5 oBuATHT SuSuipouinTANT HOF Cpt) =Nil} ! Pret (ovum); tec . 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