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TITLE
HodofAoki fieDerm ofPectin cd (PFOA) Se
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AutHoR
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`EmilyDecker
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DATEISSUED
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January8, 2004
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`Haskell \Baboratoryafposor HiBealathxse o& Ens viroomental Sciences.
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ExypaReseach
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BM008276Revi 2
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MANAGEMENTAPPROVAL
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sMhaaknKeeamdy fz Dus
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Doron SponsorRepresentative
Exygea Research
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`TABLE OF CONTENTS
L SUMMARY eons
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3. CHEMICALSANDSUPPLIES ..cecossssssmmessmmeensnoeonon]
F3.E3. EQUJANID SUPPPLIMEScEecvNerTcmresmesems-- essesseno on 8
3F 5. PREPARATION OFSTANDARDS An NDFORTIFICATIONSOLUTIONS corfc--e--cr--r--o--r----1
33..55.21..SFTOROTCIKFSIOCALTUITOINOSNOcLrUrTsIsOsNsS.s.s.sc smsmo eeoso eesr nor s os
D 3.53.CALIBRATION STANDARDS ..T cerenrrnsmresmmeroore 10
4A2L..
PSLOAWDMIAPGRLAMEcPreRtOcrccCmeEosSrSoImeNnGemossommosio s er
10 10
43.SAMPLEPREPARATION recesses
45. Q4.U5A.N1.TLITCA/TMISO/NMc SSysteo mand Oe pezatineg eCoendeitsioenssvmmsoeemweoneorornroen111
44..5532..CTAULNIEBFRIALTEIOPNARPARMOECTEEDRSU.R.E.Sc __._.o .....____s ___.....m ..... co omms 1122
46. A4C5C4E.PTSAANMPCLEECRAINTAERLIYA.S.I.S. cee mommsmssesoonoseoees 1134
5.
44.87..TPIEMRERORREMQUAINRCEEDCFROIRTAENRAILAY.S.I.S...r momo en or ers1134. CALCULATIONS .o.eeesecemsmmmmsosmsesoes 15
6. SAFcmeETYcensoreens 13.
Ey Research |
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LIST OFTABLES
Table RecoveryofPFOAfrom FortificationsInMonkey SED......ceo.ecvnen 16
LISTOF FIGURES
FFiigpuarne21..RPCaFplOirAbeawtaionihcuvrvCnegffoourLoP.oFfmOAaioCfontCroelrMoroMcoekskSepySurr ec Sempov leese1l7f
FigurR e 3. RepresC entativeChioma stogamofa.10pp Sr tafonr PFdOAafa nCrodz
Figor4e. ReiptrhesPeFntOaAtiavedChwriotmhaBtogLsam.ofoCfonStCrBoFlMOoAnr keySeerumFe ortifir edat10Ep0pt20
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1 SUMMARY `sTehrisumr,eport details amethodof analysisforperfluorooctancicacid (PFOA)in
Toechericl fom of PFOA vs sein me `PQFuaOntAifiicsateixotnroafctPeFdOfAriosmacscoempmlisbheyd bpryoltieqiunipdrcehcirpoimtaattioongirnapshcyethoanmnitdaielsems,
`spectrometry(LC/MS/MS)analyisusing selectedreactionmonitoring (SRM).
sem. `Thelowerlimitofquantitation (LLOQ)forthismethodis10ppbforPOAin Quantificationis performed. singextractedcalibrationstandardscontainingan
s"aThniismaethdovdiwiaosndfeovePloRpOeNdusingmonnkyecyasenrum.1T,h1e0ondvoperyceent rrecove]ry& 6.9%(Table T). A1.toRperpesrcenstcattaitvievceaclhbrroimoantocgarrvaemsfofrorPFPOFAOAi.mionnmkoetykseeyrusmerisumhaotwe.sihnoFviegsure Figures 2to 4.
.
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2. sergo e---------------- EXPERIMENTALCOMPOUNDS
BCumcodNewne := opm homa
FF
F
Hc Core = ompum1265
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3. CHEMICALSANDSUPPLIES
31. CCw aenels e cage "Methanol(MOE)
Acetonitrile(ACN)
Comalil ErTll artoenalAIOi `AmmoniumAcetate
HPLC
HPLC Reagent
EMSceocs
BM Science: Sigma-Aldrich
JT90932
AX0145-1 A-1330
32. STANDARDS
a ad Sl (PFOA)
Tae Fay
Se
Products.
2afTQ H TMTDeR e perfluoroctancicacid, NA
9635
DuPont.
Resi
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33.CETQrUIaePMEswNT AeNDSuEpepeLIyES Sow Sohcritceitg es
STpoat --
CDFiErrNepoicecsGOgAL 1002000)
arson
DreamFnaysy
Sraintteontfyopanrteeit oir KNepueme
ES SRoni Sne ew( ndiHLrn, 2mLclearHPLCvial kit (cat# 5181-3400) vAaivoH:iPFaotnis
eeeyEs ikomei m ny beei frd petiri timorn
1Noe Irr como, woopicava gy
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5
oHeWPFrLsIei CrvecialosFfrItm oNrA(tuSmihbeetee Hssac,PpknLiepdCCed sasu,oe teotsced sa.m)p.lcecrmeuqstiT mnpootibne,musceed,iy PPE oi Eof
54CFTlihetm ynareestehnrdacsnetmxsnpsarmosve
htsitstsoe wgidewieon r ome
24.Scumone
2 mn wewien is peur by ping
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Nyete:T0hp.e1t 5d8ofammmongr io umacetati eandodiissoelo viongcn iindc1 cLsod ,f,OehonntiStateye Wsatet, n
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35.PREPARATIONOFSTANDARDSAND FORTIFICATIONSOLUTIONS Analyt1.iscCaearbsubtmraanaidnaodrndsSsrt1aenuvdsaedrdd1fs0sc--atTlhhieesenetpseutrtdphoeasrreedsss:psonospeorefptahreeddetiecctoonrtrwolimdoinnkseyy
2. `Lacanoabnloyorscainst.aortyoCnosnctoamsosSppoinkdeisn--gTthoetsheefoLrtLiOfiQcattidons1a0exLuLsuOalQlyoprdeapaerwedesdt. 3. M1iancdteoitnxetSropmliimnkeeosnankaelTyyhsteiesceaulorr.etciofvceartyi.onsLareaprebparceoodnbtryroslpsiapkiiknegtsaortoeotprrhepyaeld seafmfpelceosfstthksnaomwpnlecmonactenitxroatniosnnsa.lMyiactalarescpoivkeecsy.aresso 19 evaluatth `Thesolutevolumesofthesiadardsmayb viedbyth analystsslong sth mcopoammyrebcoetepprpiirorfpenpaoporahtsrdrioseoserndsra1eydows.fdsoadonlngu.tae5tNtoohsteoolPvarelensptoaratarhetaimtoaanidndtniadtiincoeondns.c)eAnclttroeartnniaoctneesbormncsecnsitearoaryt.rnioyns1s
P3r.o5.p1.eSrtioncdikvsiodluuatlisotnock solionsof ~ 100 gl. ofPFOA and "C-
.
EPpxuFrtcOiihtgAyes)rbtaonycdwkeds1iiol2gluhtueiiCn1o0gn1o1u06a0tmCa~L113w0i5imtmhsgmlaeoLtlfDheaPafnnEoaoblleyot1arimceaaaslx1)s0it0a.1nsdtaorvpdboee(lsc.uosomridreooencefst1deidpfencoassr.3
romthedatof preparation.
23.51.P2sr0.eF0pooarrteislfL.iocftafoitttniioaohfneiSvncooalPliuFioutnOimoAetsnsrtsoaeilndtaailrkodn.odfes1c0r0ibpediLn 3f.o5r.1PtFoOA1b00ymLilwting bod`PimrleLupwtaiirnsgh 1aaf0comerttLifiioccfattfihnoenvPstoCa-lnPduFamrOdeAostflfrulaii1sock0n.pgd/esmclr,ifbeodrfn"3C5P.F1O10A1b0y9
P0de.rs0oc0pr5eipbge/dimfLno.c3oi.f5f2iP.cCa2-i2oPdnFsO0a.A5nbmtyaLdiolofufttihneLg "O1pC0-0pPmF.lOAoffsootrlhuPerFiPOoFnAd.OecAsosenssoiinnoidnsogs @ P3r5o2p1s0f1o0c0lfmiLcawiiotnhsaacnctdoendiloefn0.31vpo/lumm.etfcfPkFOAcontsining e. dPeG 3isz5cp2ro ai1eb1e0m dif1no0a 30nm.c5Lt2iCwoiRnnthsdOst0aN.tn5dbmarrLdcodofifntii0nen0ag1vLopglO/ummmll.etosfsfoofrltuaPotsFiPoOFnAOdceAossncnbliendogin 005pg/mL.ofPC PFOAby diluting 0.1mL.ofthe FRO sonics
orp omc
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d3e5s2crbi1b0edi1n003.a5..2 wi4t0h4a0c.t5omniLtdolfethine a"voCl-uPmFeOAsollusthio.n describedin
bSotetpoleeasat)lisloftno2raiiCfcra0iwo6insctfhaontrdiamaredmsiatoxliutmnieuocnmespsiesnrraiyrotedofomifgae1kraytenoeaiwfsnrto1am2n5td-hamerLtdLotsDsnPogEf
hestock sohuion.
Ti3hd.e5en3tc.iacCaallllirtbocraottnihoesntaSsnatdmaapnrlddesasar.drs pTrhoecefsosrtitfhicraotiuognhtofhetxhet.rasctuinondparyocepdeupree,;
`exactionsdoneaccording thefolowing ble:
"ConMicxeOdF
RoSroliuctaitoinon
VFoorltmeeat(ilo)n VCaonmmosli `Sample(mL)
EGaumro Calibentic
Sunda!
Ao0o-- 1 -- 050 --oT0-- 11r 02
--0 0l0011_ 210 a ooil o2m100
4. METHOD
T4dheLsecrPiBrpotoiwowndDoifuaegoarcnahmasoepf.themetiodis given below,followedby
| a dotaled
Measure 0.1mL.soyfMss ameplp etF(luoshwiinDgioadign isrdpgaosmnaeblemiceopipete)
Piapeii ncine
LOM iaussi
42.SunProcesses Nosstibmepallelporwoecdetsoincoimspnleeetdlyedtfhoarws,ewriumdseam,plesr.ooHmotweems,pefvrzae,n Santisamsples
EomResees
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43.SunPrzmancron s`tsoamrpeldexsemfiugsetcbaetetdhsohroouuglhdlaylsmoibxeedablelfoowreedtbeoienqguislaimbrpalteedtfoarroexotmrtacetmipone.rate. All
5a.cEp aoTcchobrar etcnhocfopsy naempepl(egesieexthiraohcstdedl(Ntybpbiscaxlw lym3d0torixnledscsop)uriansl t(ionsctludeaHtlreast
CNai seciom`paica ayettrooe pFooal 1e ivetdy 3 rtoooonm
Pr 1. Mesos0 mk.of
\ le to 2a.rentcmt
oct
2 3
(wcC v hiotdhmcaniaos lnyrto eawsxneeafdlesdes,i tncliosncelvoiedi,hae nnndvaoarmte,x ~1S02sedaconrdos). 0005
er
4. AnalyzesamplesusingelectrosprayLC/MS/MS.
45. QUANTITATION
45.1. LC/MS/MSSystemandOperatingConditions
Instrument:
PE SCIEXAPI 4000 BiomolecularMassAnalyzer
SCIEXTurbolonSprayLiquid IntrodactionInterface
HPSCLouCEmppugmen CPfDoArvSeometiPAtCroemomkptzaaxn)S1e31i106s0 Quatpamp GI311A
pin py isees og Ble BNo4t0eL:7T40w0o) w4 ox t10amcmhiynpetrceaarfbedrrotph-inigpuasndvcaerterisdogesB(Koeystone,Lt#
and/orHPLC system.
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NICHonMojPbleiLcuoltCmieConPbnPoThlaeaVmussiopmelen(urA:lma)5te:u:,re:Gc23Se0ynMh.eCsiA esCy(Tm ones ACho creomsatteor gnrOapShyi o)l,2y.1Wm amtmerx 30 mum,dy.
w3 ou%4 %B He03mali
37357s o00 111@00%00 5
00a03s33
5u o @ ,00533
pmeayrbienoeccsay adjust theHILC gradicat inonder tooptimize instrument
Tap
Appeosie
POA Nepive
UCPFOA Negative
ga
"23min
415-5370
~2.8min.
i2hbe1els0eern1piphotenehdcmcile.hesDmdcioyntothivuaiyn,lgiyohoonunogihdstaeyeoai-stcybeeapsia,pdeewliepaenp onthebs th of
ivi ot: aaka LCIMSIMSsyste maybo usedonce demonstratedtobo
452. TineFlePramas
1 os com CO a) osA anTsom s0oo3leeutdL miofanosrotfsi hpePepFcorOiwzoAnocn(teeet1seoT0i.htmCusinoeendutfowreietnheiaallnkyyobnyesipndfduufsogiengeagp~mh1pga. s2a2d0a
ng a4r3Ie3s,atvCedalt25teartcaunaiemnfPeirldeo.cTewhdiitrsetsun5e4le)iotfhecnaucsehdcduulrbienngtroonutinaeennaldysst. he
Eames
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S Ea ES O T . i pe sss toaa uesfor quan, nt Po Liverwandard cuherve ar
ES E SEeE,nTE RT EE EE hE `statisticaloutlierbyusing
outliertest (e.g.
lest),
T SErESorrS ea : 209925 (Z20.985). IFcalibrationresultsfalloutsidetheselimits, then
<TT2 -- a Injectthesamealiquot (5iL) of each standard,sample,
, control,etc.
mSE EESmeEkmSE z e EA ter sampleset. In eithercas,calibration standardsmustbethe firstandJay ES EI rnc tT Ta E E n rR s `withinthestandardcurve range. ---- e Fortificationrecoveries falingwithin8510 115% (80to120% forlevelsat _--r e S -- rr e t co e---- 5 Samplesinwhich00peaks sredeta i.signal ntrio<3.1) he
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t(i1m0epspbbutwaherreelLesLsOtQhan=th10eplpobw)ewsitlclobnecreenptarratteidoan os f<1th0epcpabl.ibration standards
meene 4.6.ACCEPTANCECRITERIA
on Thefollowing criteriamustbemet toensure the presenceofPFOA and UC-
1. ToFnRoO: sutamts oaonst3ih0owstooe r lgloirne pes4t 13a
2.
An"yC-sPFOeA:
pesto mtd lak 2daughterionat370smufr
om aparentof
tbe tes
415amu
1dowe
thn
tfohledLlLowOeQr.thAanntyhaenLalLyOtQep.resentinthe reagent blankmustbe atleast5-
3B1eto5kn%rdw(nfaRu0ai21e0aioaonctr(ootiironccosonfsbooLpoLmltaetp losnfig.ySopnon
ieeenvp315. ibe
cioitte eptaol egelBOd ee ee
4 CpSnEyici3bae5ctsoofifchsadleinawnfyaosueasofbessesttsilsei eeolrrdeb7y mitr g8 1x1
BCU cnet mpm 0 to11n0 47 Peroncarn 5. TreeTesvaea e pe umentste i, T2he0com9e(la79t2io02n9c8o95e)f.fiIcifecnatlib)raftoiorncarleisburlatstifoanlcouruvseisdegtehneesraetLeidmimtsu,sttheben
Thiem lovine igtomcofctesrssthbeephrwtorned myeasytttt,b,eor
cdii4 nnS`ternsteeiofapn Foi
aenit 2rSiioned1L0Q
BogenResch
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R`SuemcondsCeriotefrisotna:ndardsof iv ormorsconcentration levels,spamming a ange i`esngttarterehrtiisenaoignnoaantlsyomswriiebste.,hlsGoaewcpntoelhereftLcoLiaeOaytcQboaulefipbedrn1ae0lttityoheznecnehtudiwigrihoeetnshots(rce2gon)andccoaehnnf,dtsaraamtllioeonmslptaee0vn9enl8eot5so.bbGesirne3celuwndoced. 43. TomRaqTOuRAENiLEYEDS IpsAibsoocereatdtoaofrrsy3ic5nsoapompllrespsio(ke1sx,eaaidg8ah3tmoc0ounssatabomlypt,lae1m)emscpaalenbrxseyocnto.aknTeenhH,eoL1uOlagMbhoSrtaMthoSry.osopniaksa,on2s. (standards30035sample) willak spp 9ho:
5. CALCULATIONS
5.pUbesyaektBaqrAeuasa)oaunsyi1nsgfotofhtcewaalsrcteualpnardteaortgdhaecmsa.rovem(elionefasrraogyrosssfioounnpdariamdetenrs,)gbenaesreatoend oe Eautio1n;
Analytefound(ppb)=(AssisPeak realocaksca) iteresg)x1Sconc. (ng/mL)
b.EFqourastaimopnle2s0fcoratlicfuieldatweitthehpkrnoewan recovery. amountsofanalyteprior toextraction,use
ReEcqouvaetriyo2(n:)=
Analytefound(pp"bA)m=ovun3t.AAnnaslyytsefaodduerd (ty incontrol(ppb), x 100%
6. SAFETY
ThIahangebneodrnaiatntnaosglryyscbsteotafnusodshrao,ersudlpsede0rcyeoarmrdleitaanhsggeesmt,tasht,oseirdnimgcaleldotisvhnaeogfsde.wtDoyrodUkasitsabne.gsIuhinneiefvtueosrmsfeaoolbrpaopedlrdslecspbutaeatsnniododanwrsocdsaweathkneesedg : Prowohean biandoingnsecsu.
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Table L RecoveryofPFOAfromFortificationsIn Monkey Serum
Bp nm bm td Ried Skee T EomEmeRisaitc Lcaomovnnweoen sE Gmowe llbs 0%=
amansAtAoDmpBypova x3%
SummaryofPOARecover 10 ppbection okey eran
Be me bee ae ied de
mTmmy miay GR LuemEeoGTs ne meane = i 2 %
mama sSoAuDEoben
5=
Suey oPFOARecaro 100pp Bottini nMokeSor
T Gr miTnRal men r oes w
~~
ma ecosup ws me 8
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seAmnSTEAANBDAARDDEDEVVIAATRION: B2e
ovina cvmace: "
OVERALLKATIESona pA 2
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000169
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Figur2e. Representative Chromatogramof a ControlMonkey Serum
`Samplefor PFOA with ~ 5ng/mL of "C-PFOA
Bl; EpE----------
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5
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Figure3. RepresentativeChromatogramof a PRO inConnMonkeySer ih
EE-
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Figur4e.RepresentativeChromatogram of Control MonkeySerum Fortified at 10ppbwith PFOA andwith ~ 5ng/mLofC-
PFOA
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