Document NG0wMewERG4xRbLM27YBgBN2R
Corl)orateHealth Physics Corporate OccupationalMedicine Corporate Product Responsibility
Corporate Toxicology 3M MedicalDepartment
3M Center,220-2E-02 PO Box 33220 St.Paul,MN 55133-3220 651733 1110
DeteriminationofSerum EliminationHalf-LivesofAmmonium Perfluorooctanoate, PerfluorooctaneSulfonicAcid,Perfluoroheaxanesulfonicacidand TotalOrganic Fluorinein Decatur Chemical PlantRetirees
The objectivoefthisstudyisquantitatehehuman serum eliminatiohnalflifeof perfluorooctanesulfon(aPtFeOS),perfluoroo.ctano(aPtFeOA) , perfluoroohexanesulfon(aPtFeOS) andtotalorganicfluorin(eTOF). Blood willbe collectebdiannuallyfrom approximately25 retiree3dM Decaturemployeesfora period of 5 years.Upon collectioannd analysisof thethirdsample (baseline6,months and then 12 months) and thereaftera,n interimdrafthalf-lifaenalysisreportwillbe prepared.
3M Company Page I of 19
PROTOCOL
Epidemiology,220-3W-05 Medical Department 3M Company SL Paul,NIN 55144
Date: October14,1998
Title:DETERMINATION PERFLUOROOCTANOATE, PERFLUOROBEXANE DECATUR CBFMCAL
OF SERUM ELIMINATION HALF-LIVES OF AMMONIUM
PERFLUOROOCTANE
SULFONIC ACED,
SULFONIC ACID AND TOTAL ORGANIC FLUORINE IN 3M
PLANT RETWEM.
Study StartDate:
EstimatedDate of FinalReport:
IRB ApprovalDate:
ProtocolNumber # 0007 IRB Approval# 98095 Exempt XX Expedited
June,2004
14 October 1998
PrincipaIlnvestigator.
JeanM. BurrisRN, NTH
Co-investigators:
Geary Olsen,DVM, PhD' John C. Schumpert MD Cathy Simpson, RN2 JeffreyMandel,MD, MPH'
Study Director-:
JeffreyMandel,MD, NTH'
Study Sponsor:
Medical Department 3M Company 220-3W-05 SaintPaul,NfN 55144
1
OccupationalMedicine,MedicalDepartment,3M Company, 220-3W-05, St.Paul,MN
55114
2. 3M DecaturSpecialtyAdhesivesand ChemicalsPlant,P.O.Box 2206,Decatur,AL
35609-2206
3M Company Page 2 of 19
ABSTRACT 3M manufactures products which containchemical compounds, eitheras intentionalcomponents or residualimpurities,thathave as a parentmolecule, perfluorooctanesulfonylfluoride(POSF). These compounds may be expected to transform metabolically,to an undetermined degree,to PFOS as an end-stage metabolite.Other compounds manufactured by 3M include ammonium perfluorooctanoate(PFOA) and perfluorohexanesulfonicacid (PFHS). These molecules entera number of product applications(e.g.,surfactantsf,ood packaging additives,polymers).
The biologicaleliminationratesof fluorochemicalshave been studiedin rats,pregnant rats, female rabbitsand hamsters. Biologicalhalf-livehsave been calculatedfrom the studiesin rats and dogs. In addition,the half-lifoef serum totaiorganicfluorinein a fluorochemicalworker was estimatedusing serialcollectionsof serum totalorganicfluorine.These studiesrevealthat PFOS and PFOA may persistin the body forprolonged periods,and with continuingexposure, accumulate over time in biologicalsystems.To date,theactualeliminationhalf-lifoef perfluorooctanesulfonate(PFOS), ammonium perfluorooctanoate(PFOA), perfluorohexane sulfonicacid(PFHS), and totalorganicfluorine(TOF) inhuman serum isnot completely understood. The objectivesof thisstudy are to quantitatetheeliminationhalflifein human serum of PFOA, PFOS, PFHS, and TOF. These half-liveswillbe calculatedfrom declinesin serum fluorochemicallevelsin 3M Decatur Chemical PlantRetirees.
INTRODUCTION
3M Company Page 3 of 19
3M manufacturesproductswhich containchemicalcompounds, eitheras intentionaclomponents
or residualimpuritiest,hathave asa parentmolecule,perfluorooctanseulfonylfluoride(POSF).
These chemicalsinclude:perfluorooctanseulfonate(PFOS),N-ethyl
perfluorooctanesulfonamidNe-,ethylperfluorooctanesulfonamiedtohanol,N-methyl
perfluorooctanesulfonamideothanoland chemicalsderivedfrom it,and themixtureof mono-, di-
and tri(N-ethylperfluorooctanseulfonamidoethylp]hosphates.There may be otherprecursor-isn
theworkplace,includingperfluorohexanesulfonfylluorid(ePHSF). These moleculesentera
number of productapplication(se.g.s,urfactantfso,odpackagingadditivesp,olymers).These compounds may be expectedtotransformmetabolicallyt,oan undetermineddegree,toPFOS
(andPFHS) asan end-stagemetabolite.Potassiumperfluorooctanseulfonat(eCgFl70SO2K) is itselfa,surfactanutsed as a wettingand foaming agentinindustriaalnd commercial processes. (Olsen,etal.,1998)
Subchronicstudiesin ratsand primatessuggesttheremay be a potentiaflorcumulativetoxicity withPFOS over timewith theprimaryeffectrelatedtometabolicwasting..-Thesemoleculeshave longeliminatiohnalflivesinbiologicaslystems.Althoughthemechanism oftoxicitiysnotfully understood,toxicitmyay be due toan effecton peroxisomeproliferatiofna,ttyacidmetabolism, membrane functionp,roteinsynthesiasnd/ormitochondrialbioenergetic(sO.lsen,etal.,1998)
The biologicaelliminatiornatesof fluorochemicalhsave been studiedin ratsp,regnantrats, femalerabbitsand hamsters.Biologicalhalf-livehsave been calculatefdrom thestudiesinrats and dogs. In additiont,he half-lioffeserum totalorganicfluoriniena fluorochemicawlorker
3M Company Page 4 of 19
was estimatedusingseriaclollectionosfserum totaolrganicfluorineT.hese studiersevealthat
PFOS and PFOA may persistin thebody forprolonged periods,and with continuing exposure, accumulate over time in biologicalsystems. To date,the actualeliminationhalf-lifoef perfluorooctanesulfonate(PFOS), ammonium perfluorooctanoate(PFOA), perfluorohexane sulfonicacid(PFHS), and totalorganicfluorine(TOF) inhuman serum isnot completely understood.
The objectivesof thisstudy areto quantitatetheeliminationhalflifein human serum of PFOA, PFOS, PFHS, and TOF; thesehalfliveswillbe calculatedfrom declinesin serum fluorochemical levelsin 3M Decatur Chemical PlantRetirees.This informationis necessaryin order to:1) plan the most appropriateperiodicityof medical surveillanceand 2) guide medical decisionmaking of workers from fluorocheznicalproductionfacilitiewshen those workers' serum fluorochemical levelsare determined to be unsafelyelevated.A benefitof thisresearchwil.1be to understand the naturalhistoryof fluorochemical excretion.
LITERATURE
REVEEW
AMMONIUM
PERFLUOROOCTANOATE
(PFOA or FC- 143)
Excretion ratesof PFOA have been observed in mts, and found to be differentby gender and
route of excretion.Following singlei.v.doses of 14C-FC-143 (carbonylcarbon labeled)in rats,
Johnson et al,found thatfemales excretedvirtuallyallthe administered 14C within I day. Urinaryexcretionformales was about 50% ofthe dose by day 6 and 83% by day 36. Fecal '4C
excretionforfemales was 1.5% by 3 days and formales was 5.4% by 36 days. (Johnson,et al.,
1980) Rapid urinaryexcretionof 14C followingoraldoses of 14C_FC-
3M Company Page 5 of 19
143 was also shown to
occur in pregnant rats.(Johnson,etal.,1983)
Excretionratesvary by species studied.Excretionof radiolabeledPFOA was studiedin four speciesby Dupont in 1988. Excretion as a percentage of administereddose 120 hours after dosing was in the following order;female rat,male and female rabbitand male hamster (> 99%); female hamster (60%); male rat(39%); male and female n-@ce(21%). (Dupont, 1988) Of note, theadministereddose and routesof exposure and excretionwere not specifiedin thisstudy report.
Rats and dogs respond to phannacologic interventionsthatchange PFOA excretionrate.In male
ratsadministered singlei.v.doses
of
14
C-FC-143
(carbonylcarbon labeled)cholestyramine(4%
w/w in feed)increasedcumulative 15 day fecal14C:excretio9n.8-foldversuscontrols..Total '4C:l
excretion(fecesplus urine)was alsoenhanced, although lessdramatically(84.3% of dose vs.
71.8% forcontrols).(Johnson,et a].,1980a; Johnson JD, et al.,1984) There isno difference
between the renalclearances of '4C in male and female dogs eitherbeforeor afterprobenecid.
Probenecid significantlryeduces PFOA clearancein dogs. Glomerular filtratiornatesof PFOA
were simflarinratsand dogs. (Hanhijarvie,tal.,1988)
Elimination half-lifheas been calculatedunder numerous conditionsin ratsand dogs. The results show the same differentiaelliminationratein ratsby gender (i.e.f,emale > male), as well as halflifedifferencesby routeof exposure and species.Following a singleoraldose of 14C_FC-143 (carbonylcarbon labeled)in male rats,theplasma half-lifweas 4.8 days.(Johnson,et a].,1979)
Infemaleratsadministeredoralor i.v.dosesof 14C_FC_
3M Company Page6of19
143 (carbonylcarbon labeled),over 90%
of theadministereddose was recovered in theurine withinthe first12 hours.The whole body
eliminationhalf-lifoef PFOA in male and female ratswas 15 days and lessthan one day,
respectivelyf,ollowinga single4-mg/kg intraperineadlose. (Vanden Heuval, etal.,1991)
(Johnson,et al.,1983; Johnson, et al.,1980) The half-lifoef PFOA in the liverwas 60 hours for
females and 2 10 hours for male rats.(Ylinen,et al.,1990) The decreased excretionrate(i.e.,
increasedeliminationhalf-lifei)n males isseen acrossat leasttwo species:rats(asabove) and
dogs. The plasma half-lifoef PFOA was longer in male dogs (473 to 541 hours)than in females (202 to 305 hours).(Hanhijarvie,tal.,1988)
The eliminationhalf-lifaeppears to be similarin male ratsexposed to eitherinhalationalor dermal exposure. Following repeatedinhalationexposures to FC-143 over a two-week period, blood organicfluoridelevelsin male ratsshowed a half-lifoef fiveto seven days. (Kennedy, et al.,1986) A blood half-lifoef fiveto seven days was seen followingrepeatedderznalexposures in male rats.(Kennedy, 1985)
A calculationof the eliminationhalf-lifoef an organic fluorinespecieshas been reportedin the human. The half-lifoef totalserum organicfluorinein a fluorochemicalworker who was removed fr-omfurtherexposure was greaterthan 18 months. This worker had a blood organic fluorinelevelof 40 partsper millionover the periodof one year. The worker'sorganicfluorine levelrose to 70 partsper millionforno apparentreason. The worker was then removed from the fluorochemicalproductionarea and hisblood and urinesamples were periodicallcyhecked for organic fluorineand PFOA over an 18-month period. A totalof 18 months transpiredbeforethe
3M Company Page 7 of 19
worker'sbloodorganicfluorinleevelhad returnedtoitsbaselineof40 partspermillion.(Ubel, etal.1980) Otherestimatesof serum fluorochemicalhalf-livehsave been calculateidntheone thousandday range.T This anecdotalaccountdoes notactualltyracktheserum PFOA level, butrathertheserum totalorganicfluorin(eTOF) level.Itwas found throughempiricalmeans that90% oftheblood organicfluorineexistedasPFOA. (Ubel,etal.1980)
PERFLUOROOCTANE
SULFONIC ACED (PFOS orFC-95)
Perfluorooctanseulfonicacidappearsto persisitn biologicaslystems,beingexcretedmore slowlythanPFOA. Singlei.v.doses(mean 4.2mg/kg) of 14C-FC-95 and 0.9% NACL were
administeredto male rats.By 89 daysafterdosing,30.2% of theadministered14C had been
excretedintheurineand 12.6% had been excretedin thefeces.(Johnson,etal.,1979)
Pharrnacologicinterventiondso appearto increasetheexcretoryrateof PFOS. -Fecaland totalexcretionof 14C were markedly increasedinmale ratsadministeredCholestyrainin(eabout2.7 gnvkg per day)and theirdietfollowingsinglei.v.dosesof 14C-FC-95.The resultsuggestthat therewas significanetnterohepaticcirculatioonfFC-95. (Johnson,etal.,1980a;Johnson,etal., 1984)
Much lessisknown concerningeliminatiohnalf-lioffePFOS thanPFOA; only one calculation
of 14C
of theelin-@dnatihoanlf-lifoef PFOS has been attempted.The plasma eliminationhalf-life followingsingleoraladministratioonf 14C-FC-95(mean dose4.2 mgtkg) to male ratswas 7.5 days. (Johnson,eta].1979a)
T Mandel,]. Personalcommunication.
3M Company Page 8 of 19
RESEARCH METHODS
3M Company Page 9 of 19
The overallresearchdesign isprospectiveinnature,obtainingmultipleserialblood samples from retireetshroughoutthecourseof a five-yeapreriod.We willtrackthedeclineinseveralserum
fluorochemicalevelsin humans. The healthoutcomes thatwillbe documented inthisstudyare
thehalf-lidfeterminationosfserum ammonium perfluorooctanoa(tPeFOA), perfluorooctane
sulfoniaccid(PFOS),perfluorohexanseulfoniaccid(PFHS),and serum totalorganicfluorine (TOF).
ELgh-performanceliquidchromatographymass spectrometry/masspectrometrywillbe utilized toanalyzeallserum samples. The accuracyand reliabiliotfythisdevicewillbe thestateof the artatthetime ofanalysis.Periodicallsyp,litsamplesfrom one ortwo subjectwsillbe utilizetdo assessreliabiliotfytheanalysis.
Only individualwsho have retirefdrom 3M Decaturchemicalplantfrom theIstof January1995 throughthe IstofJanuary 1998,willbe includedinthestudy.Using inforznatiorneceivedfrom theHuman ResourcesDepartment of 3M Decatur,34 individualwsere identifietdo have retired duringthistimeperiod.Of the34 individualisdentifie7d,have previouslbyeen enrolledinthe fluorochemicamledicalsurveillancperogram. Four of theseindividualpsarticipateidn the medicalsurveillancperogram in 1994 butnot 1997;3 individualpsarticipateidn 1997 only.We willattempttoenrollall34 retireeisntothestudy.Futureretireewsillnotbe enrolled. Serum fluorochemicalevelswillbe drawn everysixmonths. Allserum fluorochemicallevels willbe drawn withina one-month timeframe(i.e.w,ithinone month ofMarch and September).
3M Company Page 10 of 19
Retirr.ewsillbe recruitetdo thestudythroughfirsta,n introductorlyettearnd second,a phone conversationfrom theprinciplienvestigator hisdesignee.Subsequentto thefollow-up telephoneconversationand iftheretireieswillingtoenterthestudy,a writteninformedconsent form (AppendixA) and a medicalquestionnair(eAppendix)3)willbe senttotheretirefeorhis review.Once theretir-eseignstheconsentform,completesthequestionnairea,nd retums the documents totheprincipleinvestigatohre,willbe enteredintothestudyand scheduledforblood collectionP.articipatiointhestudyisvoluntaryand retireemsay drop out atany time.
Blood collectiownillbe scheduledbiannuallyi,nApriland October(tonotconflicwtithHoliday and summer vacationtravel)L.ettersannouncingbloodcollectiodnatesand timeswillbe sent outto each studysubjectone month priortoeach bloodcollectiomnonth. Retireeswillupdate theirmedicalquestionnairaeteach bloodcollectionA.llbloodcollectionwsillbe.performed underthesupervisioonfthe3M Decaturplantnurse,Cathy Simpson,RN. Two red-toppedtubes--@ willbe obtainedfrom each retireet;he3M OccupationalMedicine Servicemethodology for handlingand shippingof fluorochemicalbloodsampleswillbe appliedto allblood samples. Samples willbe analyzedby the3M EnvironmentalLaboratoryor itsdesignatedcontract laboratoryR.etireeswillreceivewrittenreportsoftheirsenun fluorochemicalevelsfollowing each blood collectiocnycle.
Two methods ofcalculatintgheserum fluorochemicahlalflifewillbe utilizedI.nthefirst method, a one compartment model.willbe assumed and theformula
qt= qo
3M Company Page II of 19 willbe utilizedto estimatethe halflifeof eliminationforPFOS, PFOA, PFHS, and TOF. The terms q, and qo referto the serum concentrationsattime t and time 0, tisthe elapsedtime;tl.is what we seek to solve.
The second method will be used to verifythe first.Italsoassumes a one compartment model. Each retiree'fsluorochemical levelswill@begraphed on a log-linearscaleas serum fluorochemicalvs.elapsed time from to(inmonths) and enteredintoa database. Statistical analysiswillbe perforxnedto determine thebest-fimtodel thatdescribesthe slope of the loglinearline.(Appendix C)
The slopeof the log-linearIineisrelatedto the eliminationconstant(-kl)through the equation
Slope= -k,1(2.303)
Once the eliminationconstant iscalculatedt,he halflifecan be calculatedusing therelationship
t@6 0.6931k-el
EliminatiohnalflivefsorTOF, PFOS, PFHS, andPFOA willbedeterznine(dB.eneze,tal.1985, Medinsky,etal.1996.)
DISCUSSION Therearea numberoflimitatioansssociatweidththestudyi,ncluding:
1.The lackofpre-studsyerumfluorochemiclaelvelisnthemajoritoyfretireemsa,kingit
impossibletodevelopbaselinelevelsforthemajorityof thestudygroup. The tO valueswill thereforeneed to be theirfirsftluorochemicallevelpost-retirement.
3M Compan'y Page 12 of 19 2. Previous studieshave perfor7nedserum totalorganic fluorinelevelsand not serum testing specificforserum PFOS or PFOA; comparabilitywith previous work isthereby compromised. 3. Retireeson medications such as colestipolor cholestyramine,which can artificialilnycrease the rateof eliminationof fluorochemicalsfrom the blood willartificialilnycreasethe eliminationin unpredictableways, therebycausing an underestimationof theeliminationhalf lifein theentirestudy population. 4. Retireesmay unknowingly have medical conditionsthatwilldecreasetheirtoxicant eliminationrateunpredictably(e.g.r,enalfailurec,ongestiveheartfaflurec,holelithiasiest,c.), thereby causing an inaccurateestimationof theeliminationhalflifein theentirestudy population. For thesereasons,the study willoccur over the protractedperiodof fiveyears insteadof a shorter time period (i.e.t,hreeyears). As the only human data plottingtotalorganicfluorinesuggest-f: @ -. plasma eliminationhalf-lifoef 18 months, a threeyear studywould allow forverificatioonf that estimatesincethreeyearswould be equal to two half-lives.A five-yearstudy,therefore,should includeover threehalf-livesof elimination,and make itpossibleto use severalyears'data to calculatetheeliminationhalflife.
The overallresultscollectedin thisstudy may be used in publicationsor in publicpresentations. Retireenames or otherindividualdata willnot be revealedin any publicationor other documentations intended forpublicexamination. Individualresultswillbe communicated only to ther-etireeI.ndividualresultswillbe consideredconfidentiailnformationand willnot be disclosedto anyone outside of the 3M Medical Department without the retiree'wsrittenconsent.
3M Company Page 13 of 19
Qualityassurancewillbe performedpertheguidelinessetforthinthestandardoperating procedureforthecreationr,eview,and approvalof3M epidemiologystudyprotocols.
Archivingofstudymaterialsu,pon completionof thestudy,willbe performedperthestandard operatinpgrocedureforthecreationa,uditingr,eviewand approvalof 3M epidemiologyfinal reports.
ne studywillbe communicated totheretireemsa,nagement,and thetechnicaclommunity. Communicationstotheretireewsillbe intheform ofa lettewrh,ich statetsheretireev'asrious bloodfluorochemicallevels.Theseletterwsillbe sentoutfollowingeachblooddraw. Connnunicationtomanagement willbe intheform ofa writtendocument which willundergo thereviewprocessas setforthin thestandardoperatingprocedureforthecreation@a,uditing, reviewand approvalof 3M epidemiologyfinarleports.The communicationtothetechnical community willbe intheform ofa scientifpiacper,which willbe submittedtoa relevanpteer reviewedscientifpiucblication.
REFERENCES
3M Company Page 14 of 19
1. Olsen GW, BurrisJM, Mandel JH, and LR Zobel.An epidemiologicinvestigationfclinical chemistriesh,ematology and hormones in relatiotno serum levelsof perfluorooctane sulfonatein male fluorochemicalproductionemployees. J Occup EnvironMed 1998;
2. Johnson JD and Ober RE. Extentand routeof excretionand tissuedistributiofntotal
carbon-14inmale and femaleratsaftera singleIV dose of FC-143-14C . RikerLaboratories, Inc.St.,Paul,NIN; 1980.
3. Johnson JD and Conard GJ. Extentand routeofexcretionof totalcarbon-14inpregnantrats aftera singleoraldose of ammonium 14C-perfluorooctanoatRei.ker LaboratoriesI,nc.,St. Paul,MN; 1983.
4. Vanden Heuval J,KuslikisB, Van Refelghem M, and PetersonR. Tissuedistribution, metabolism,and eliminationof perfluorooctanoiacid.J Biochem Toxicol1991;6:83-92.
5. DuPont. Ammonium perfluorooctanoatTeo.xicityHazard Informationfrom MSDS; 1988.
6. Johnson JD and Ober RE. Enhanced eliminatioonf FC-95-14C and FC-143- 14C inratswith cholestyraminetreatment.RikerLaboratoriesI,nc.,St.Paul,MN, 1980a.
7. JohnsonJD, Gibson SJ,and Ober RE. Cholestyramine-enhancefdecaleliminatioonf carbon14 inratsafteradministratioonf ammonium [14C]perfluorooctanoaotre potassium['t]perfluorooctanesuIfonatFeu.ndAppi Toxicol1984;4:972-976.
8. CniffitFhD and Long JE. Animal toxicitsytudieswith ammonium perfluorooctanoatAem. Ind Hyg Assoc J 1980;41:576-583.
9. HanhijarviH, YlinenM, Haaranen T and NevaWnen T. A proposedspeciesdifferencienthe renalexcretionof perfluorooctanoiaccidin thebeagledog and therat.In:Beynen AC and SolleveldHA, eds.New DevelopmentsinBiosciences:77ieiIrmplicationsfoLraborotory Animal Science.MartinusNijhoff,Dordrecht,1988.
10.GillilanFdD. Fluorocarbonsand human health:studiesinan occupationaclohort.Ph.D. DissertatioUn., of Minnesota,NfinneapolisM,N; 1992.
11.Johnson JD and Ober RE. Absorptionof FC-143-14C inratsaftera singleoraldose. Riker LaboratoriesI,nc.,St.Paul,MN; 1979.
12.Johnson JD and Ober RE. Extentand routeof excretionof totalcarbon-14pregnantratsafter a singleoraldoseof ammonium 14-C perfluorooctanoatRei.kerLaboratoriesI,nc.,St.Paul, M; 1983.
3M Company Page 15 of 19
13.Kennedy GL, Jr.,HallGT, BtittelMlRi, Bames JR, and Chen HC. Inhalationatloxicitoyf ammonium perfluorooctanoatFeu.nd Chent Toxicol1986;24:1325-1329.
14.Kennedy GL, Jr.Dermal toxicitoyfammonium perfluorooctanoatTeo.xicolAppl Pliamiacol 1985;81:348-355.
15.YlinenM, Kojo A, HanhijarviH, and PeuraP. Dispositioonfperfluorooctanoiacidinthe rataftersingleand subchronicadministrationB.ullEnvironContam Toxicol1987;44:46-53.
16.Ubel FA, Sorenson SD, and Roach DE. Healthstatusof plantworkersexposed to fluorochemicalsa,preliminaryreport.Am Ind Hyg Assoc J 1980;41:584-589.
17.Johnson JD, Gibson SJ,and Ober RE. Extentand routeof excretionand tissuedistribution of totalcarbon-14inratsaftera singlei.v.dose of FC-95-'4C.RikerLaboratoriesI,nc.,St. Paul,MN; 1979.
18.Johnson JD and Ober RE. AbsorptionofFC-95-14C aftera singleoraldose.Riker LaboratoriesI,nc.,St.Paul,NW; 1979a.
19.BenetIZ and SheinerLJ3.PhannacokineticsT:he dynamics of drug absorptionD.istribution, and eliminationI.n:Goodman Gilman A, Goodman LS, RallTW, and Murad F, ed. Goodman and Gilman's The PharmacologicBasisof Theapeutics,SeventhEdition.Macmillan PublishingCompany, New York, 1985.
20.Medinsky MA and Klaasen CD. ToxicokeneticsI.n:Carasettand Doull'sToxicology:The BasicScienceofPoisons,FifthEdition.KlaasenCD, Amdur MO, and DoullJ,editors.New York,McGraw-Hill,1996.
Appendix A
3M Company Page 16 of 19
CONSENT FORM FOR COLLECTION OF BLOOD FOR SERUM
FLUOROCHEMICAL
LEVEL HALF-LIFE DETERMINATION
INTRODUCTION You and yourcolleagueswere involvedinfluorochemicalproductionat3M Decaturpriorto your retirementY.ou areinvitedtoparticipatiena researchstudybeingconductedby 3M OccupationalMedicine ServicesregardingfluorochemicalsY.our participatiwoonuld involve donatingone red-toppedtesttubeof bloodapproximatelyeverysixmonths forthenextfive years.Your blood willonly be testedfortheamount and kindoffluorochemicalistcontainsi;t willnot be used totestforany othersubstancesordisease.Pleasereview thisconsentform carefullaynd be sureyour questionsareanswered beforeyou make a decisionto participateT.he plantnursewillbe availablteoprovideyou withadditionailnformatioantthetimeofyour medicalsurveillancbelood donation.
PURPOSE OF STUDY The purposeof thestudy isto determinehow much and what typesof fluorochemicalsare eliminatedfrom thehuman body overtime.
STLTDY PROCEDURES Your bloodwillbe drawn with one needlestickand requiretwo testtubes.This willoccurabout everysixmonths forthenext fiveyears.A lettewrillbe senttoyou inadvanceinformingyou of theproperdate,time,and placetoreportto forblood collection.
POTENTLKL RISKS/BENEFITS The onlydiscomfortyou may feelisfrom theneedlestick.You may alsohave some temporary redness/bruisingtswelliinntghisareaafterblood collection."
BENEFITS There willbe no directbenefitfrom yourparticipatiionnthisstudy.However, theinformation gainedfrom thisstudywillfurtherhelpus understandhuman exposuresto fluorochernicals. Your individuarlesultasswell astheoverallresultwsillbe communicated toyou only.Your individuarlesultswillbe consideredconfidentiailnformationand willnotbe disclosedto anyone outsidethe3M MedicalDepartment withoutyour writtenconsent.
COMPENSATION Ifyou sufferinjuryor a medicalconditionthatappearstobe theresultofparticipatiinngthis study,you willbe referredtoanotherhealthcareprofessionaaltno costtoyou. In theeventof a researchrelatedinjuryc,ompensationwillbe determinedon a caseby casebasisby 3M.
3M Company Page 17 of 19
CONFIDENTIALITY The overallresultcsollectedinthisstudymay be used in publicationosr publicpresentation. Your name willnotbe revealedinany publicatioonr otherdocuments intendedforpublication examination.Your individuarlesultwsillbe communicated toyou only.Your individuarlesults willbe consideredconfidentiailnformatioannd willnotbe disclosedtoanyone outsidethe3M MedicalDepartment withoutyourwrittenconsent.
SUBJECT RIGHTS/AVAI]LAB=Y OF INFORMATION Ifyou have any questionsabout thestudynow, or latero,r intheeventof a researchrelatedinjury oremergency,contactDr. JeffreyMandel (651-733-8670)orJeanBurtis,R.N. (651-737-7867). For answerstoquestionsabout yourrightsinregardtothisresearchy,ou may contactDr.Larry Zobel,Chair,3M InstitutionRaelview Board at651-733-5181.
VOLUNTARY PARTICE?ATION AND WM-EDRAWAL Participatiionthisstudyisvoluntary.Refusaltoparticipawtiellinvolveno penaltyoflossof benefitstowhich you areotherwiseentitledY.ou arefreetowithdraw from thestudyatany time forany reason.The investigatomray stopyour participatiionthisstudyshoulditbe detemiined thatcontinuedparticipatiomnay be detrimentatioyour health.Followingeach serni-annual bloodcollectiony,ou willreceivea check for$50.00as compensationforyour time and effort.
SUBJECT CONSENT By signingtheconsentform,IcertiftyhatIam atleast18 yearsold.IconfirmthatIhave read. thisconsentform,and thatI have been givenadequateopportunittyo ask any questionsI may have aboutthisconsentform or thestudy.IalsoconfirmthatIunderstandthescopeof my participatiion thisstudy,and thatallof my questionshave been answered tomy satisfactio.In. am signingthisconsentform voluntafilayn,d Idesireto participaitnethestudy.Iunderstand thatIwillreceivea copy of thissignedconsentform. I understandthatIwillreceivea check for $50.00immediatelyfollowingeachserni-annuaclollectioonfmy blood.
Signature
Date
PrintedName
Witness
Ai)i)endiBx
MEDICAL HISTORY OUESTIONNAIRE
3M Company Page 18 of 19
Pleaselisthenames of theALL themedicationsyou takeatleastonce a day.
Pleasecircletheconditionsyou now sufferfrom or have sufferedfr-omin thelastthreeyears.
Kidney Failure Gall Bladder Disease Hepatitis/Jaundice CongestiveHeart Failure LiverDisease InflammatoryBowel Disease Crohn'sDisease PerniciousAnen-da
3M Company Page 19 of 19
Aipi)endiCx
Example ofa log-lineaprlotthatwillbe usedtoestimatetheserum fluorochemicaellimination halflifeinDecaturretirees.
20- - - - -- I
Slope -k4d2=
TIM
3M ENVIRONMENTAL LABOPATORY
METHOD
EXTRACTION FLUOROCHENUCAL
OF POTASSIUM PERFLUOROOCTANESULFONATE
COMPOUNDS FROM SERUM FOR ANALYSIS
ELECTROSPRAY/MASS
SPECTROMETRY
OR OTHER USING HPLC-
Method Number: ETS-8-4.1
Adoption Date: 03/01/99
Author: LisaClemen, Glenn Langenburg
RevisionDate: q/.)7/99
Approved By:
I,)...LaboratoryManager
lKtd4, &--
Group Leader
d@- A
TechnicalReviewer
7 Date
qlztlgg
Date
QYLIIIqq -
Date
1.0 SCOPE ANID APPLICATION
1.1 Scope: This method isfortheextractioonfpotassiumperfluorooctanesulfona(tPeFOS) or otherfluorocheinicaclompounds from senun.
1.2 Applicable compounds: Fluorochemical surfactantsor other fluorinatedcompounds.
1.3 Matrices: Rabbit,rat,bovine,monkey, and human serum or otherfluidsas designatedin thevalidationreport.
Word 6/95
ETS-8-4.1 Extractionof PFOS from Serum
Page I of 14
2.0SUMMARY OF METHOD 2.1 Thismethod describestheprocedureforextractinpgotassiumperfluorooctanesulfonate
(PFOS) orotherfluorochemicaslurfactantfsrom serum,orotherfluidsu,singan ion pairingreagentand methyl-tert-buteytlher(MtBE). Inthismethod, seven fluorochemicalwsere extractedP:FOS, PFOSA, PFOSAA, ETFOSE-OH, PFOSEA, M556, and surrogatestandard(see3.0Definitions)A.nion pairingreagentisadded to thesample and theanalyteionpairispartitioneidntoMTBE. The MTBE extractis removed and put onto anitrogenevaporatoruntildry.Each extractisreconstituteidn 1.0 mL of methanol,thenfilteretdhrougha 3 cc plastiscyringeattachedto a 0.2Am nylon filteirntoglassautovials. 2.2 These sample extractsareanalyzedfollowingmethod ETS-8-5.1 or otherappropriate methods.
3.0 DEFINITIONS 3.1 PFOS: perfluorooctanesulfonat(eanionof potassium salt)C.F,7SO33.2 . PFOSA: perfluorooctanseulfonylamideC,F,,SO2NH2 3.3 PFOSAA: perfluorooctanesulfonylamido(ethyl)acetatC,eFl7SO2N(CH2CH,)CH2CO23.4 ETFOSE-OH: 2(N-ethylperfluorooctasnuelfonamido)-ethaylcohol
C,F,:7SO2N(CH2CH3)CH2CH20H 3.5 PFOSEA: perfluorooctanseulfonylethylamideCsF,7SO2N(CH2CH3)H 3.6 M556: CgFl7SO2N(H)(CH2COOH) 3.7 Surrogatestandard:IH-IH-2H-2H perfluorooctanseulfoniaccid
4.0 WARNINGS AND CAUTIONS 4.1 Health and safetywarnings
4.1.1 Use universalprecautions,especiallylaboratorycoats,goggles,and gloveswhen handlinganimal tissuew,hich may containpathogens.
5.0 INTERFERENCES 5.1 There areno interferenceksnown atthistime.
6.0 EOUIPMENT 6.1 The followingequipmentisused whileperformingthismethod. Equivalentequipmentis
acceptable. 6.1.1 Vortexmixer,VWP, VortexGenie2 6.1.2 CentrifugeM,istral1000 orIEC 6.1.3 Shaker,EberbachorVWR
ETS-84.1 Extractionof PFOS from Senim
Page 2 of 14
6.1.4 NitrogenevaporatorO,rganomation 6.1.5 Balance(0.100g)
7.0 SUPPLIES AND MATERIALS 7.1 Gloves 7.2 Eppendorf or disposablepipettes 7.3 Nalgene bottlesc,apableof holding250 mL and 1 L 7.4 Volumetric flasks,glass,typeA 7.5 I-CHEM vials,glass,40 mL glass 7.6 Centrifugetubes,polypropylene,15 mL 7.7 Labels 7.8 Oxford Dispenser- 3.0to 10.0mL 7.9 Syringes,capableof measuring 5 RL to 50 liL 7.10 Graduated pipettes 7.11 Syringes,disposableplastic3, cc 7.12 Syringe filternsy,lon,0.2lim,25 mm 7.13 Timer 7.14 Crimp cap autovialsand caps 7.15 Crimpers Note: Priortousing glasswareand bottlesr,inse3 timeswith methanol and 3 timeswith
water. Rinse syringesa minimum of 9 timeswith methanol,3 rinsesfrom 3 separatevials.
8.0 REAGENTS AND STANDARDS 8.1 Type I reagentgrade water,Milli-QTmorequivalenta;llwater used inthismethod should
be Milli-Q' water and may be providedby a l@filliT-OQC PluJ' system 8.2 Sodium hydroxide(NaOH), J.TBaker or equivalent 8.3 Tetrabutylammonium hydrogen sulfate(TBA)K,odak or equivalent 8.4 Sodium carbonate(NaCO,), J.T.Baker or equivalent 8.5 Sodium bicarbonate(NaHC03), J.T.Baker or equivalent 8.6 Methyl-T-Butyl Ether,Omnisolv,glassdistilleodrBIPLC grade 8.7 Methanol, Onmisolv, glassdistilleodr BPLC grade 8.8 Serum or blood,frozenfrom supplier 8.9 Fluorochemical standards
8.9.1 PFOS (3M SpecialtyChemical Division),molecularweight = 538 8.9.2 PFOSA (3M SpecialtyChemical Division)m,olecularweight= 499
ETS-8-4.1 ExtractionofPFOS from Serum
Page 3 of 14
8.10 8.11
8.9.3 PFOSAA (3M SpecialtyChemical Division)m,olecularweight = 585
8.9.4 ETFOSE-OH (3M SpecialtyChemical Division)m,olecularweight= 570
8.9.5 PFOSEA (3M SpecialtyChemical Division)m,olecularweight = 527
8.9.6 M556 (3M SpecialtyChemicalDivision)m,olecularweight= 557
8.9.7 Surrogatestandard:4-H, perfluorooctanseulfonicacid(I-H,I-H, 2-H, 2-H C,F,3So3H) molecularweight= 428
8.9.8 Other fluorochemicalsa,s appropriate
Reagent preparation
NOTE: When preparinglargervolumes thanlisteidnreagent,standard,or surrogate preparation,adjustaccordingly.
8.10.1 10 N sodium hydroxide (NaOH): Weigh approximately 200 g NAOH. Pour into a 1000 mL beakercontaining500 mL Milli-Q' water,mix untilallsolidsare dissolved.Storeina 1 L Nalgene bottle.
8.10.2 1 N sodium hydroxide(NaOH): Dilute10 N NAOH 1:10. Measure 10 mL of 10 N NAOH solutionintoa 100 mL volumetricflaskand dilutetovolume using Milli-Q' water. Storeina 125 niL Nalgene bottle.
8.10.3 0.5M tetrabutylanunoniuhmydrogen sulfat(eTBA): Weigh approximately169 g ofTBA intoa 1 L volumetriccontaining500 mL Milli-(@'water.Adjust topH IQ usingapproximately44 to54 mL of 10 N NAOH (While addingthelastmL of NAOH, add slowlybecausethepH changes abruptly).Diluteto volume with Milli-Q' water. Storeina 1 L Nalgene bottle.
8.10.3.1TBA requiresa checkpriortoeach use toensurepH = 10. Adjustas needed using 1 N NAOH solution.
8.10.4 0.25 M sodium carbonate/sodiumbicarbonatebuffer(NaC03/NaHC03): Weigh approximately26.5 g ofsodium carbonate(NaCO,) and 21.0 g of sodium bicarbonate(NaHC03) intoa 1 L volumetricflaskand bringtovolume withMilliQ' water. Storein a 1 L Nalgene bottle.
Standards preparation
8.11.1 PreparePFOS standardsforthestandardcurve.
8.11.2 Prepareotherfluorochemicalstandards,asappropriate.Multicomponent fluorochemicalstandardsareacceptable(forexample, one working standard solutioncontaining1.00ppm PFOS, 1.02ppm PFOSA, 0.987 ppm PFOSAA, and 1.10 ppm EtFOSE-OH.)
8.11.3 Weigh approximately100 mg ofPFOS intoa 100 mL volumetricflaskand record the actualweight.
8.11.4 Bring to volume with methanol fora stockstandardof approximately1000 ppm (AgIML).
8.11.5 Dilutethestocksolutionwith methanol fora working standard1 solutionof approximately50 ppm.
8.11.6 Diluteworking standardI with methanol fora working standard2 solutionof approx.5.0 ppm.
ETS-8-4.1 ExtractioonfPFOS from Serum
Page 4 of 14
8.11.7Diluteworking standard1 withmethanolfora working standard3 solutionof approx.0.50 ppm.
8.12 Surrogatestockstandardpreparation 8.12.1 Weigh approximately50-60mg of surrogatestandard1-H,1-H,2-H, 2-H, C,F,3So3H intoa 50 mL volumetricflaskand recordtheactualweight. 8.12.2 Bringto volume withmethanolfora surrogatestockof approximately1000-1200 PPM. 8.12.3 Preparea surrogateworking standard.Transferapproximately1 mL of surrogate stockto a 10 mL volumetricflaskand bringto volume with methanol fora working standardof 100 ppm. Record theactualvolume transferred.
9.0 SAMPLE HANDLING 9.1 All samples arereceivedfrozenand must be keptfrozenuntiltheextractioinsperformed. 9.2 Allow samplesto thaw toroom temperaturepriorto extraction.
10.0 OUALITY CONTROL
10.1 SolventBlanks,Method blanksand matrixblanks 10.1.1An aliquootf1.0mL methanoilsusedasa solvenbtlank. 10.1.2Extracttwo 1.0mL aliquotosfMilli-Q' waterfollowingthisprocedureand use asmethod blanks. 10.1.3Extracttwo 1.0mL aliquotosfthesenun followingthisprocedureand use as matrixblanks.See 11.1.4.
10.2 Matrix spikes 10.2.1Prepareand analyzematrixspikeand matrixspikeduplicatesamples todetermine theaccuracyof theextraction. 10.2.2 Prepareeach spikeusinga sample chosen by theanalyst,usuallythecontrol matrixreceivedwitheach sample set. 10.2.3 Expected concentrationwsillfallinthemid-rangeof theinitiaclalibratiocnurve. Additionalspikesmay be includedand may fallinthelow-rangeof theinitial calibratiocnurve. 10.2.4 Prepareone matrixspikeand matrixspikeduplicatpeer 40 samples,with a minimum of 2 matrixspikesperbatch.
10.3 Continuingcalibratiocnhecks 10.3.1 Preparecontinuingcalibratiocnheck samplesto ensuretheaccuracyof theinitial calibratiocnurve. 10.3.2 Prepare,ata minimum, one continuingcheck pergroup of 10 samples.For example, ifa sample set= 34,fourchecks arepreparedand extracted. 10.3.3 Prepareeach continuingcalibratiocnheck from thesame matrixused to prepare theinitiaclurve.
ETS-84.1 ExtractioonfPFOS from Serum
Page 5 of 14
10.3.4 The expectedconcentrationwsillfallwithinthemid-rangeoftheinitial calibratiocnurve.Additionalspikesmay be includedthatfallinthelow-rangeof theinitiaclalibratiocnurve.Thisisnecessaryiftheanalystmust quantitatuesing onlythe low end ofthecalibraticounrve(forexample,5 ppb - 100 ppb,rather than 5 ppb - 1000ppb)
11.0 CALIBRATION
AND STANDARDIZATION
11.1 Prepare matrix calibrationstandards
11.1.1 Transfer 1 mL of serum to a 15 mL centrifugetube.
11.1.2 Ifmost sample volumes arelessthan 1.0niL,extractstandardswithmatrix volumes equaltothesamplevolumes. Do notextractlessthan0.50mL of matrix.Record each sample volume on theextractiosnheet.
11.1.3 VAiilepreparinga totaolf twentyaliquotisn 15 mL centrifugteubes,niixor shake between aliquots.
11.1.4 Two I mL aliquotso,r otherappropriatveolume,serveasmatrixblanks. Typicallyuse thestandardconcentrationasnd spikingamounts listedinTable 1, attheend of thissectiont,ospike,induplicatet,wo standardcurves,fora totalof eighteenstandardst,wo matrixblanks,and two method blanks.
11.1.5Refer to validatiorneportETS-84.0 & ETS-8-5.0-V-1,which listtsheworking rangesand theLinearCalibratioRnange (LCR) forcalibratiocnurves.
11.1.6Use AttachmentD as an aidincalculatintgheconcentrationosf theworking standards.See Section13.0to calculataectualconcentrationosfPFOS in calibratiosntandards.
11.2 To each standardb,lank,or continuingcheck,add appropriataemount of surrogate working standardfortheconcentratiotnofallwithinthecalibratiocnurverange5 ppb -
1000 ppb.
11.3 Extractspikedmatrix standardsfollowing12.6-12.16ofthismethod. Use thesestandards to establisehach initiaclurveon themass spectrometer.
ETS-8-4.1 ExtractioonfPFOS from Serum
Page 6 of 14
Table 1
Approximate spikingamounts forstandardsand spikes
Using 1.0mL of matrix
Working standard
@iL
Approx.finalconc.of
(approx.conc.)
analyteinmatrix
-
-
Blank
0.500 ppm
10
0.005ppm
0.500 ppm
20
0.010ppm
5.00 ppm
5
0.025ppm
5.00 ppm
10
0.050ppm
5.00 ppm
20
0.100 ppm
50.0 ppm
5
0.250 ppm
50.0 ppm
10
0.500 ppm
50.0 ppm
15
0.750 ppm
F-50.0 ppm
20
1.00ppm
12.0 PROCEDURE
12.1 Obtain frozensamples and allowtothaw atroom temperatureor ina lukewarm waterbath.
12.2 Vortex mix for15 seconds,thentransfe1r.0mL orotherappropriatveolume to a 15 mL polypropylenecentrifugteube.
12.3 Return unused samplesto freezerafterextractioanmounts have been removed.
12.4 Record theinitiavlolume on theextractiownorksheet.
12.5 Label thetubewith thestudynumber, sample ID,dateand analystinitialsS.ee attached worksheetfordocumenting theremainingsteps.
12.6 Spike allsamples,includingblanksand standardsr,eady forextractiownith surrogate standardasdescribedin11.2.
12.7 Spike each matrixwith theappropriataemount ofstandardas describedin11.1,or Table I inthatsection,forthecalibratiocnurve standards.Also preparematrixspikesand continuingcalibratiosntandards.
12.8 Vortex mix thestandardcurve samples,matrixspikesamples,and continuingcalibration samples for15 seconds.
12.9 Check to ensurethe0.5M TBA reagentisatpH 10. Ifnot,adjustaccordingly.
12.10 To each sample,add I mL 0.5M TBA and 2 mL of 0.25M sodium carbonate/sodium bicarbonatebuffer.
12.11 Using an Oxford Dispenser,add 5 niL methyl-tert-buteytlher.
12.12 Cap each sample and puton theshakerata settinogf 300 rpm, for20 minutes.
12.13 Centrifugefor20 to25 minutesata settingof 3500 rpm, or untillayersarewell separated.
ETS-8-4.1 ExtractioonfPFOS from Serum
Page 7 of 14
12.14 Label a fresh15 mL centrifugteubewiththesame informationas in 12.5. 12.15 Remove 4.0mL of theorganiclayertothisclean15 mL centrifugteube. 12.16 Put each sample on theanalyticanlitrogenevaporatoruntildry,approximatelyI to2
hours. 12.17 Add 1.0mL of methanol toeach centrifugteubeusinga graduatedpipette. 12.18 Vortex mix for30 seconds. 12.19 Attacha 0.2gm nylon mesh filtetroa 3 cc syringeand transfetrhesample tothis
syringe.Filteirntoa 1.5niL glassautovialor low-volume autovialwhen necessary. 12.20 Labeltheautovialwith thestudynumber,animalnumber and gender,sample timepoint,
matrix,finalsolvente,xtractiodnate,and analyst(sp)erformingtheextraction. 12.21 Cap and storeextractsatroom temperatureor atapproximately4 'C untilanalysis. 12.22 Complete theextractiownorksheet,attachedtothisdocument,and tapein thestudy
notebook or includeinstudybinder,as appropriate.
13.0 DATA ANALYSIS AND CALCULATIONS 13.1 Calculations
13.1.1 CalculateactualconcentrationosfPFOS, or otherapplicablefluorochemicali,n calibratiosntandardsusingthefollowingequation: mL of standardx concentratioonf standard(alz/mL) mL ofstandard+ mL ofsurrogatsetandard+ initiamlatrixvolume (mL)
FinalConcentratio(ngghnl)ofPFOS inmatrix
14.0 METHOD PERFORMANCE 14.1 The method detectionlimit(MDL) isanalyteand matrixspecificR.efertoNML report
forspecifiMcDL and limitofquantitatio(nLOQ) values(seeAttachments B and C). 14.2 The followingqualitycontrolsamplesareextractewdith eachbatchof samplesto
evaluatethequalityof theextractioannd analysis. 14.2.1 Method blanksand matrixblanks. 14.2.2 Matrix spikeand matrixspikeduplicatesamplestodetermineaccuracyand
precisionoftheextraction. 14.2.3 Continuingcalibratiocnheck samplestodeterminethecontinuedaccuracyofthe
initiaclalibratiocnurve. 14.3 Refertosection14 of ETS-8-5.1formethod performancecriteria.
15.0 POLLUTION PREVENTION AND WASTE MANAGEMENT
15.1 Sample waste isdisposedinbiohazardcontainersf,lammable solventwaste isdisposedin highBTU containersa,nd used glasspipettewaste isdisposedin broken glasscontainers locatedinthelaboratory.
ETS-8-4.1 ExtractioonfPFOS from Serum
Page 8 of 14
16.0RECORDS
16.1 Complete theextractiownorksheetattachedto thismethod, and tapein thestudy notebook or includein the3-ringstudybinder,as appropriate.
17.0 ATTACHMENTS 17.1 AttachmentA, Extractionworksheet 17.2 AttachmentB, MDL/LOQ valuesand summary 17.3 Attachment C, Calibratiosntandardconcentratiownorksheet
18.0 REFERENCES
18.1 The validatiorneportassociatewdiththismethodisETS-84.0 & 5.0-V-1.
18.2 FACT-M-3. 1,"AnalysiosfSeniinorOtherFluidExtractfsorFluorochemicalusing BPLC-ElectrosprayMass Spectrometiy'
19.0 AFFECTED DOCUMENTS
19.1 ETS-8-5.1, "Analysisof Serum or OtherFluidExtractsforFluorochemicalsusing BPLC-ElectrosprayMass Spectrometry"
20.0 REviSIONS
Revision Number
I
Reason For Revision Section12.21Changed toincludesample storageatroom temperature. Section12.13Added theshakerspeed. Section12.17Finalvolume is1.0mL; notadjustedforinitiavlolumes lessthan 1.0mL.
Revision Date
04/02/99
ETS-84.1 Extractionof PFOS from SenLm
Page 9 of 14
ExtractionWorksheet ETS-8-4.1
Study
SurrogateStd
Matrix
approx. ppm
Box #
actual ppm
Wk/Day_
DateSpiked/Analyst
ccv
ms_
MSD
FC-Mix approx.0.5pm actual ppm
FC-Mix approx.5 ppm actual pptn
FC-Mix approx.50 ppm actual PPM
Comments
Blank
Std
amount
Serum ExtractioMnethod
Vortex15 sec.
PipettMeatrix
Volume
ML
PipettIemL of 0.5M TBA, pH 10.pH
Std.#
Pipett2emL of0.25Na2CO3/0.25M NaHC03 buffer
Std.#
Dispense5 mL ofmethyl-t-buteytlher
TN-A-
Shake20 min. Centrifug2e0-25min.
Shakerspeed: Centrifugespeed:
Remove a 4 mL aliquootforganiclayer
Puton NitrogenEvaporatOTtodryness
Temperature:
Add methanol
Volume
mL
TN-A-
Vortex30 sec.
Filteursinga 3ccB-D syringewitha 0.2gm filtienrtoa 1.5mL autosamplveial Cont.Cal.Verificatiounsedsame matrixasforstdcurve.
AttachmentA
ETS-84.1 ExtractioonfPFOS from Serum
ML Date& Initials
Page 10 of 14
MDL/LOQ valuesforrabbisterum
Compound MDL LOQ LinearCalibratiRoannge(LCR)
(ppb) (ppb) Approximatceoncentratitoonbseusedforpreparintghe
StandarCdalibratiCounrve
PFOS
1.74 5.55 5 ppb - 1000 ppb
PFOSA
1.51 4.79 5 ppb - 1000 ppb
PFOSAA
3.46 20.5 5 ppb - 1000 ppb
ETFOSE-OH
11.4 36.2 5 ppb - 1000 ppb
M556
6.03 19.2 5 ppb - 1000 ppb
IPFOSEA
5.71 1 18.2 15 ppb - 1000 ppb
MDL/LOQ valuesinrat,bovine,monkey,and human senun,and monkey plasma were notstatistically
determined.Two curvesineachofthesematriceswere extracteadnd analyzedwiththerabbitserum
curvestodetermineequivalence.Responsesinthera@bovine,monkey, and human were equivalento
therabbitresponses,thereforet,heirMDL and LOQ willbe thesame valuesasdeterminedinrabbit
serum.
PleaseseeLOQ Summary and MDL studyinETS-8-4.0& 5.0-V-1forfurtheirnformation.
AttachmentB: MDL/LOQ Sununary
ETS-8-4.1
ExtractioonfPFOS from Serum
Page IIof 14
Compound: PFOS Prepared range
RabbitSerum of standards (ppb)(ng/mL)
FullRange
0.995-978
LCR from curve (ppb)
(ng/mL)
24.8-978
% Recovery Range
83-108
Low Curve
4.94-248
4.94-248
85-104
High cur-ve
97.8-978
97.8-978
85-106
I/X
0.995-978
4.94-978
94-111
I
I
I
Compound: PFOSA Prepared range
RabbitSenun of standards (ppb)(ng/mL)
LCR from curve (ppb)
(ng/mL)
% Recovery Range
FullRange
0.993-976
4.93-976
88-103
Low Curve
4.93-97.6
4.93-97.6
87-105
High curve
24.8-976
24.8-978
93-102
I/X
0.993-976
4.93-976
94-103
Compound: PFOSAA Preparedrange
RabbitSerum of standards (ppb)(ng/mL)
FullRange
0.991-974
LCR from curve (ppb)
(ng/mL)
24.7-974
Low Curve
4.92-247
9.74-247
High curve
49.2-974
97.4-974
1/X
0.991-974
9.74-974
% Recovery Range
81-111@ 97-107 85-108 95-115
RSD Range
4.67-11.0 5.34-12.0 4.84-9.80 4.60-10.5
I
RSD Range
5.10-14.7 9.85-14.7 5.08-13.9 5.10-14.5
RSD Range
4.18-10.6 6.38-21.8 4.33-12.5 4.11-23.2
AttachmentB: MDL/LOQ Sununary
ETS-8-4.1 ExtractioonfPFOS from Senim
Page 12 of 14
Compound: ETFOSE-OH Preparedrange
Rabbit Serum of standards (ppb)(ng/mL)
FullRange
0.993-976
LCR from curve (ppb)
(ng/mL)
49.3-976
Low Curve
4.93-97.6
9.76-97.6
High curve
49.3-976
97.6-976
1/X
0.993-493
9.76-976
% Recovery Range
77-110 97-107 90-109 86-111
Compound: PFOSEA Prepared range
RabbitSenun of standards (ppb)(ng/mL)
FullRange Low Curve High curve I/X
0.993-976 4.93-248 49.3-976 0.993-976
LCR from curve (ppb)
(ng/mL) 24.8-976
9.76-248
49.3-976
9.76-976
% Recovery Range
96-106 91-110 86-106 95-117
Compound: M556 Preparedrange
RabbitSerum of standards (ppb)(nghnl)
FullRange
0.993-976
LCR from curve (ppb)
(ng/mL)
24.8-976
Low Curve
4.93-97.6
9.76-97.6
High curve
97.6-976
97.6-976
1/X
0.993-976
9.76-976
% Recovery Range
88-106 100-105 81-111 97-110
RSD Range
11.2-25.5 14.1-21.3 11.5-19.6 11.1-21.2
RSD Range
10.1-16.2 11.8-19.5 10.2-18.2 10.1-19.1
RSD Range
4.82-17.9 5.95-18.2 5.11-9.74 4.77-19.5
AttachmentB: NIDL/LOQ Summary
ETS-84.1 ExtractioonfPFOS from Serum
Page 13of 14
Ion PairStandard Curves- Fluids
Prep date(s):
Standard number:
Analyte(s):
Equipment number:
Sample matrix:
Finalsolventand TN:
Blank fluid/identifier:
Method/revision:
Target analyte(s):
FC mix std approx. 0.500 ppm:
FC mix std approx. 5.00ppm:
FC mix std approx. 50.0ppm:
Surrogate stdapprox. 100 ppm:
Actual concentrationsof standardsin theFC mix
PFOS
PFOSA
PFOSAA
ETFOSE PFOSEA
Std conc Std conc Std conc Std conc Std conc
ughnl 0.500
ug/mL
ug/mL
0.507 1 0.532
ug/mL 0.501
ug/mL 0.521
0.500
0.507
0.532
0.501
0.521
5.00
5.07
5.32
5.01
5.21
5.00
5.07
5.32
5.01
5.21
5.00
5.07
5.32
5.01
5.21
50.0
50.1
53.2
50.1
52.1
50.0
50.1
53.2
50.1
52.1
50.0
50.1
53.2
50.1
52.1
50.0
50.1
53.2
50.1
52.1
M556 Std conc ug/mL 0.501 0.501
5.01 5.01 5.01 50.1 50.1 50.1 50.1
All ArWt spikedmL 0.010 0.020 0.005 0.010 0.020 0.005 0.010 0.015 0.020
All Finalvol
ML 1.015 1.025 1.010 1.015 1.025 1.010 1.015 1.020 1.025
Calculated concentrationsofstandards in the sample matrix
PFOS Finalconc
ng/mL 4.93 9.76 24.8 49.3 97.6 248 493 1 735
PFOSA Finalconc
nghnl 5.00 9.89
25.1 50.0
98.9 251 50 746
PFOSAA Finalconc
ng/mL 5.24 10.4 26.3 52.4 104
263 524
782
ETFOSE Finalconc
ng/mL 4.94 9.78 24.8 49.4
97.8 248 494
737
PFOSEA Finalconc
ng/mL 5.01 9.93 25.2 50.1 99.3 252 501 749
M556 Finalconc
ng/mL 5.13 10.2 25.8 51.3 102 258 513 766
Surrogate Std conc ng/mL
100
Surrogate Finalconc
ng/mL 500
976
989
1038
978
993
1017-
All Ariftspiked
ML 0.005
Validatedranges - approximate concentrations
Serum Rabbit Bovine
PFOS
PFOSA
PFOSAA
5.00-1000 1 5.0-100
1 5.0-100
Estimatesonly.Use valuesforrabbit.
Rat
Estimatesonly.Use valuesforrabbit.
Monkey & Plasma Estimatesonly.Use valuesforrabbit.
Human
I Estimatesonly.Use valuesforrabbit.
ETFOSE-OH 1 5.0-100
PFOSEA 5.0 -10 0
M556 5.0 -10 0
Attachment C: Ion PairStandardCurves
ETS-84.1
Extractioonf PFOS from Serum
Page 14 of 14
3M ENVIRONMENTAL
LABORATORY
METHOD
ANALYSIS
OF POTASSIUM PERFLUOROOCTANESULFONATE
OR OTHER
FLUOROCIIEAUCALS IN SERUM EXNIACTS USING
HPLC-ELECTROSPRAY/MASS
SPECTROMETRY
Method Number: ETS-8-5.1
Author: LisaClemen, RobertWynne Approved By: LaboratoryManager
Adoption Date: 03/01/99 RevisionDate: q
e-,/ 2,c@l
Group Leader
-.Y;.;A.,
TechnicalReviewer
Date
oqL;2&hg
Date
1.0 SCOPE AND APPLICATION
1.1Scope: This method describesthe analysisof serum extractfsorfluorochemicalsurfactants using IPLC-electrospray/mass spectrometry.
1.2Applicable Compounds: Fluorochemicalsurfactantosr otherfluorinatecdompounds, or otherionizablecompounds.
1.3Matrices: Rabbit,rat,bovine,monkey, and human serum, or otherfluidsas designatedin thevalidationreport.
Word 6/95
ETS-8-5.1 Analysisof Serum ExtractUsing ES/MS
Page I of 9
2.0SUM MARV OF METHOD
2.1 Thismethod describestheanalysisof fluorochemicaslurfactantesxtractedfrom serum or otherfluidsu,sing HPLC-electrospray/masspectrometryo,r similarsystem as appropriate. The analysisisperformedby monitoringa singleioncharacteristoifca particular fluorochemicals,uch as theperfluorooctanesulfon(aPtFeOS) anion,m/z--499. Additionallys,amples may be analyzedusinga tandem mass spectrometerto furtherverify theidentitoyf a compound by detectindgaughterionsof theparention.
3.0 DEFINITIONS
3.1Atmospheric Pressure Ionization(API):The Micromass QuattroH triplqeuadrupole systemsallow forvariousmethods ofionizatiobny utilizinvgarioussources,probes,and interfacesT.hese includebutarenot limitedto:ElectrospraIyonizatio(nESI),Atmospheric PressurechemicalIonizatio(nAPcI),Tbermospray,etc.The ionizatiopnrocessinthese techniquesoccursatatmosphericpressure(i.e.n,otundera vacuum).
3.2ElectrosprayIonization(ES,ESI): a-methodof ionizatiopnerformedatatmospheric pressurew,hereby ionsinsolutionaretransferretdothegas phase viatinychargeddroplets. These chargeddropletsareproducedby theapplicatioonf a strongelectricfaileld.
3.3Mass Spectrometry,Mass Spectrometer(MS), Tandem Mass Spectrometer (MS/MS): The API QuattroH triplqeuadrupolesystemsareequippedwith quadrupolemass selective detectorsI.onsareselectiveldyiscriminatebdy mass tochargeratio(m/z)and subsequently detected.A singleMS may be employed foriondetectioonra series(MS/MS) formore specififcragmentationinformation.
3.4Conventionalvs.Z-spray probe interface:The latesmtodels ofNficromassQuattroH triplqeuadrupolesystems(post1998)utilizae"Z-spray"conformation.The sprayemitted from a probeisorthogonaltothecone aperture.In theconventionalconformationitisaimed directlaytthecone aperturea,fterpassingthrougha tortuouspathway inthecounter electrodeT.hough theconfiguratioinsdifferenth,emethods ofoperationc,leaning,and maintenancearethe same. However, Z-spraycomponents and conventionalcomponents are not compatiblewith one another,butonlywithsimilarsystems(i.eZ.-,spraycomponents are compatiblewith some otherZ-spraysystems,etc.)
3.5Mass Lynx Software: System softwaredesignedforthespecifiocperationoftheseQuattro IItIriplqeuadrupolesystems.CurrentlyMassLynx has Windows 95 and WindowsNT 4.0 versions.All versionsaresimilar.For more detailseethemanual specifitcotheinstrument (Micromass QuattroIItIriplqeuadrupoleMassLynx orMassLynx NT User'sGuide).
4.0 WARNINGS
AND CAUTIONS
4.1 Health and SafetyWarnings:
4.1.1 Use cautionwith thevoltagecablesfortheprobe.When engaged,theprobe employs a voltageofapproximately5000 Volts.
4.1.2 When handlingsamplesor solventswear appropriatperotectivgeloves,eyewear, and clothing.
ETS-8-5.1 Analysisof Semm ExtractUsing ES/NIS
Page 2 of 9
4.2 Cautions: 4.2.1 Do notoperatesolventpumps above capacityof400 bar (5800psi)back pressure. Iftheback pressureexceeds400 bar,theHP 1100 willinitiataeutomaticshutdown. 4.2.2 Do not run solventpumps todryness.
5.0 INTERFERENCES 5.1 To minimize interferencwehsen analyzingsamples,teflonshouldnotbe used forsample
storageor any partof instrumentatiotnhatcomes incontactwith thesample or extract.
6.0 EouiPMENT 6.1 Equipment listebdelow maybe modifiedinordertooptimizethesystem.Documentany
modificationsintheraw dataasmethod deviations. 6.1.1 Micromass Quattro11triplqeuadrupoleMass Spectrometerequippedwith an
electrospraiyonizatiosnource 6.1.2 HP1100 low pulsesolventpumping system,solventdegasser,column
compartment,and autosainpler
7.0 SUPPLIES AND MATERIALS 7.1 Supplies
7.1.1 High puritygradenitrogengasregulatedto approximately100 psi(House air system)
7.1.2 BPLC analyticaclolumn,specifictsobe determinedby theanalystand documented intheraw data.
7.1.3 Capped autovialosr capped 15 mL centrifugteubes
8.0 REAGENTS AND STANDARDS 8.1 Reagents
8.1.1'Methanol,B?LC gradeorequivalent 8.1.2 Nfilli-Q*wmater,allwaterusedinthismethod shouldbe Milli-Q' wateror
equivalenta,nd may be providedby a Milli-QTOC Plussystemor othervendor 8.1.3 Ammonium acetater,eagentgradeor equivalent
8.2 Standards 8.2.1 Typicallytwo method blanks,two matrixblanks,and eighteenmatrixstandardsare preparedduringtheextractiopnrocedure.See ETS-84.1.
9.0 SAMPLE HANDLING 9.1 Fresh matrixstandardsarepreparedwith each analysis.Extractedstandardsand samples
arestoredincapped autovialosr capped 15 mL centriftitguebesuntilanalysis.
ETS-8-5.1 AnalysisofSenun ExtracUtsingES/MS
Page 3 of9
9.2 Ifanalysiswillbe delayed,extractesdtandardsand samples can be reftigerateadt approximately4' C, or atroom temperatureu,ntilanalysiscan be performed.
10.0 OUALITY CONTROL 10.1 SolventBlanks,Method Blanks and Matrix Blanks
10.1.1 Solventblanks,method blanksand matrixblanksarepreparedand analyzedwith eachbatch todeterminecontaminationorcarryover.
10.1.2Analyze a method blankand a matrixblankpriortoeach calibratiocnurve.
10.2 Matrix Spikes
10.2.1Matrix spikesarepreparedand analyzedto determinethematrixeffecton the recoveryefficiency.
10.2.2 Matrix spikeduplicateasrepreparedand analyzedtomeasure theprecisionand the recoveryforeach analyte.
10.2.3 Analyze a matrixspikeand matrixspikeduplicatpeerfortysamples,witha minimum of 2 spikesperbatch.
10.2.4Matrixspikeand matrixspikeduplicatceoncentrationwsillfallinthemid-rangeof theinitiaclalibraticounrve.Additionalspikeconcentrationmsay fallinthelowrangeof theinitiaclalibratiocnurve.
10.3 Continuing CalibrationVerifications
10.3.1 Continuingcalibratiovnerificatioanrseanalyzedtoverifythecontinuedaccuracy of thecalibratiocnurve.
10.3.2Analyze a mid-rangecalibratiosntandardaftereverytenthsample,with a minimum of one perbatch.
11.0 CALIBRATION
AND STANDARDIZATION
11.1 Analyze theextractemdatrixstandardspriorto and followingeach setof extractsT.he averageof two standardcurveswiU be plottedby linearegressio(ny = my + b),weighted 1/x,not forcedthroughzero,usingMassLynx or othersuitablseoftware.
11.2 Ifthecurve doesnotmeet requirementsp,erformroutinemaintenanceorreextracthe standardcurve (ifnecessary)and reanalyze.
11.3 For purposesof accuracywhen quantitatinlgow levelsof analytei,tmay be necessaryto use thelow end of thecalibratiocnurveratherthanthefullrangeof thestandardcurve. Example: when attemptingto quantitataepproximately10 ppb of analyteg,eneratea calibratiocnurveconsistingofthestandardsfrom 5 ppb to 100 ppb ratherthanthefull rangeofthecurve(5ppb to1000 ppb).Thiswillreduceinaccuracyattributetdolinear regressionweightingof highconcentratiosntandards.
ETS-8-5.1 Analysis of Serum ExtractUsing ES/MS
Page 4 of 9
12.0PROCEDURES
12.1 AcquisitionSetup
12.1.1 Clickon startbuttonintheAcquisitionControlPanel.Setup a sample list.Assign a filenameusingMO-DAY-last digitof year-samplenumber, assigna method (MS) foracquiring,and typeinsample descriptions.
12.1.2 To createa method clickon scanbuttoninthe Acquisitioncontrolpanel and select SIR (SingleIon Recording)orMRM. SetIonizatioMnode as appropriateand mass to499 or otherappropriatmeasses. A fullscanisusuallycollectedalong with the SIRS. Save acquisitiomnethod. IfMS/MS instrumentsareemployed, additional production fragmentationinformationmay be collected.See Micromass MassLynx GUIDE TO DATA ACQLTISITION foradditionailnformationand MRM (MultipleReactionMonitoring).
12.1.3Typicallytheanalyticablatchrun sequencebeginswith a setof extractedmatrix standardsand ends with a setofextractedmatrix standards.
12.1.4.Samples areanalyzedwith a contiri4incgalibratiocnheck injectedafterevery tenth sample. Solventblanksshouldbe analyzedperiodicalltyomonitor possibleanalyte carryoverand arenot consideredsamples but may be includedas such.
12.2 Using the Autosampler
12.2.1 Set up sample trayaccordingtothe sample listprepared inSection12.1.1.
12.2.2 Set-up theBP 1100/autosampleratthe followingconditionsor atconditionsthe analystconsidersappropriateforoptimalresponse.Record actualconditionsin the
instrumentlogbook:
12.2.2.1Sample size= 10 @tLinjection
12.2.2.2Inject/sampl=e 1
12.2.2.3Cycle time = 13.5minutes
12.2.2.4Solventramp =
Time
0.00 min. 8.50 niin. 11.0 min. 12.0niin.
MEOH
40% 90% 90% 40%
2.0mM Ammonium acetate
60% 10% 10% 60%
12.2.2.5Press the"Start"button. 12.3 Instrument Set-up
12.3.1 Refer to ETS-9-24.0 formore details. 12.3.2 Check the solventlevelinreservoirasnd refililfnecessary.
ETS-8-5.1 AnalysisofSenun ExtracUtsingES/MS
Page 5 of9
12.3.3 Check the stainlesssteelcapillaraytthe end ofthe probe. Use an eyepiecetocheck thetip.The tipshouldbe flatwith no jaggededges. Ifthetipisfound tobe unsatisfactoryd,isassembletheprobe and replacethestainlessteelcapillary.
12.3.4 Set HPLC pump to "On". Set the flow to 10 - 500 uL/min or as appropriate. Observe dropletscoming outofthetipof theprobe. Allow toequilibratfeor approximately 10 minutes.
12.3.5 Turn on thenitrogen.A finemistshouldbe expelledwith no nitrogenleaking around the tipof theprobe. Readjustthe tipof theprobe ifno mist isobserved.
12.3.6 The instrumentusestheseparametersatthefollowingsettings.These settingsmay change in ordertooptimizethe response:
12.3.6.1Drying gas 250-400 liters/hour 12.3.6.2ESI nebulizinggas 10-15 liters/hour 12.3.6.3BPLC constantflow mode, flow rate10 - 500 JiUmin 12.3.6.4Pressure<400 bar (Tbisparameterisnot set,itisa guide to ensurethe
B?LC isoperatingcorrectly.)
12.3.7 Carefullyguidetheprobe intotheopening. Insertprobe untilitwillnot go any further.Connect thevoltagecablestotheprobe.
12.3.8 Printthe tune page,with itsparameters,and storeitin thestudy binder with a copy taped intothe instrumentlog.
12.3.9 Using the cross-flowcounterelectrodeintheES/MS sourceisrecommended for theanalysisofbiologicamlatrices. -
12.3.10CIickon startbuttonintheAcquisitionControlPanel (thismay vary among MassLynx versions,see appropriatMeassLynx USER'S GUIDE). Pressthe start button. Ensure startand end sample number includesallsamples tobe analyzed.
13.0 DATA ANALYSIS AND CALCULATIONS 13.1 Calculations:
13.1.4 Calculatematrixspikepercentrecoveriesusing the followingequation:
% Recovery
Observed Result-Background Result x 100 Expected Result
13.1.5 Calculatepercentdifferenceusing the followingequation:
% Difference
Expected Conc. - CalculatedConc. x 100 Expected Conc.
13.1.6 Calculateactualconcentratioonf PFOS, orotherfluorochemicali,nmatrix (gg/ML):
(ng of PFOS calc.from std.Curve x DilutionFactor) x 1 Mg (InitiVaollume ofmatrix(mL) + mL of SurrogateStandard) 1000 ng
FinalVolume (mL)
ETS-8-5.1 AnalysisofSerum ExtractUsing ES/MS
Page 6 of 9
14.0 METHOD PERFORMANCE 14.1 Method DetectionLimit(MDL) and LimitofQuantitatio(nLOQ) aremethod,analytea,nd
matrixspecificP.leaseseeETS-84.1, Attachment B, fora listinogfcurrentvalidated MDL and LOQ values.
14.2 SolventBlanks, Method Blanks,and Matrix Blanks
14.2.1 Solventblanks,method blanks,and matrixblanksvaluesaremust be below the loweststandardinthecalibratiocnurve
14.3 CalibrationCurves
14.3.1The r'valueforthecalibraticounrvemust be 0.980or better.
14.4 Matrix Spikes
14.4.1Matrix spikepercentrecoverieasremust be within 30% of thespiked concentration.
14.5 Continuing CalibrationVerirications
14.5.1 Continuingcalibratiovnerificatipoenrcentrecoveriemsust be 30% of thespiked concentration.
14.6 Ifcriterilaisteidn thismethod performancesectionisn'mtet, maintenancemay be performed on thesystem and samplesreanalyzedor otheractionsas determinedby the analyst.Document allactionsintheappropriatleogbook.
14.7 Ifdataareto be reportedwhen performancecriterihaave not been met, thedatamust be footnotedon tablesand discussedinthetextof thereport.
15.0 POLLUTION PREVENTION AND WASTE MANAGEMENT
15.1 Sample extwt waste and fiammablesolventisdisposedinhighBTU containersa,nd glass pipettewaste isdisposedinbroken glasscontainerlsocatedinthelaboratory.
16.0 REcoRDs 16.1 Each page generatedfora studymust have thefollowinginforxnatioinncludedeitherin the
headeror hand writtenon thepage: studyor projectnumber, acquisitiomnethod, integratiomnethod, samplename, extractiodnate,dilutiofnactor(ifapphcable),and analyst.
16.2 Printthetunepage,samplelista,nd acquisitiomnethod from MassLynx to includeinthe appropriatestudyfolder.Copy thesepages and tapeintotheinstrumentrunlog.
16.3 Plotthecalibratiocnurveby linearegressionw,eighted 1/x,thenprintthesegraphsand storeinthe studyfolder.
16.4 Printdataintegratiosnummary, integratiomnethod, and chromatograms,from MassLynx, and storeinthestudyfolder.
ET'S-8-5.I Analysisof Serum ExtractUsing ES/MS
Page 7 of 9
16.5 Summarize datausingsuitablseoftware(Excel5.0)and storeinthestudyfolder,see Attachment A foran example of a summary spreadsheet.
16.6 Back up electronidcatatoappropriatmeedium. Record in studynotebook thefilename and locationof backup electronidcata.
17.0 TABLES,,DIAGRAms, FLOWCHARTS, AND VALIDATION DATA 17.1 AttachmentA: ETS-8-5.1 Data summary spreadsheet.
18.0 REFERENCES 18.1 FACT-M-4. 1,"ExtractionofPotassiumPerfluorooctanesulfonaotreOtherFluorochemical
compounds from SeniinforAnalysisUsing BPLC-Electrospray/MassSpectrometry
18.2 ETS-9-24.0,"Operationand MaintenanceoftheMicromass Atmospheric Pressure Ionization/MassSpectrometerQuattroI[t[riplqeuadrupoleSystems"
18.3 The validationreportassociatedwith thismethod isETS-84.0 & 5.0-V-1.
19.0 AFFECTED DOCUMENTS
19.1 ETS-84. 1,"Extractionof PotassiumPerfluorooctanesulfonoarteOtherFluorochemical Compounds from Serum forAnalysisUsing BPLC-Electrospray/MassSpectrometry"
20.0 REvisioNs
Revision Number.
I
Reason For Revision Section6.1.2ClarificatiofnBPI 100 systemcomponents. Section11.1 Average of two curves,notstandardvalues,areused for plottinglinearegressioannd added theI/xweightingof thecurve. Section12.2.2.4Clarificatioofnsolventramp. Section17.1Changed from attachmentB toA.
Revision Date
04/02/99
ETS-8-5.1 AnalysisofSenun ExtracUtsingES/MS
Page 8 of9
Laboratory Study
Study: TestMaterial: Mabix/FinalSolvent: Method/Revision: AnalyticalEquipment System Number: InstnunentSoftwareNersion: Filename: R-Squared Value: Slope: Y Intercept: Date of Extraction/Analyst: Date of Analysis/Analyst
Group Dose
Sample#
Concentration uginil
InitiaVlol. ML
Dilution Factor
Final Conc.
Slope: Taken from linearegressionequation. Group/Dose: Taken from thestudyfolder. Sample#: Taken from the studyfolder. Concentration(ug/mL): Taken from theMassLynx integratiosnummary. InitiaVlolume (niL):Taken from thestudyfolder. DilutionFactor: Taken from thestudyfolder. FinalConc. (ug/mL): Calculatedby dividintgheinitiavlolume from theconcentration
AttachmentA: Sun=ary Spreadsheet
ETS-8-5.1
Analysisof Serum ExtractUsing ES/MS
Page 9 of 9
3M ENVIRONMENTAL
LABORATORY
METHOD
EXTRACTION FLUOROCREWCAL
OF POTASSIUM PERFLUOROOCTANESULFONATE
CompouNDs FRom LIVER FOR ANALYSIS
ELECTROSPRAY/MASS
SPECTROMXTRY
OR OTHER USING HPLC-
Method Number: ETS-8-6.0
Author: LisaClemen, RobertWynne Approved By: LaboratoryManager
AdoptionDate: 031-17-1.gLI
RevisionDate:
2,
Date
AA
Group Leader
1-iv,A
TechnicalReviewer
Date
07/lqlqti Date
1.0 SCOPE AND APPLICATION
1.1Scope: This method isfortheextractioonfpotassiumperfluorooctanesulfona(tPeFOS) or otherfluorochemicalcompounds from liver.
1.2 Applicable Compounds: Fluorochemicalsurfactantosrotherfluorinatecdompounds.
1.3 Matrices: Rabbit,rat,bovine,and monkey liversorothertissuesas designatedin the validatiorneport.
Word 6.0/95
ETS-8-6.0 Extractionof PFOS from Liver
Page 1 of 14
2.0 SUMMARY OF METHOD 2.1 This method describestheprocedureforextractinpgotassium perfluorooctanesulfonate
(PFOS) or otherfluorochemicalsurfactantfsrom livero,r othertissuesu,singan ionpairing reagentand methyl-tert-butyelther(MtBE). In thismethod, seven fluorochemicalscan be extracted:PFOS, PFOSA, PFOSAA, ETFOSE-OH, PFOSEA, M556, and surrogate standard.An ion pairingreagentisadded tothesample and the analyteionpairis partitioned into MTBE. The MTBE extract is transferred to a centrifuge tube and put onto a
nitrogen evaporator untildry. Each extractisreconstitutedin 1.0 mL methanol then filteredthrough a 3 cc plasticsyringe attachedto a 0.2 gm nylon filterinto glassautovials.
2.2 These sample extractsare analyzed following method ETS-8-7.0 or other appropriate methods.
3.0 DEFINITIONS
3.1 PFOS: perfluorooctanesulfonate (anion of potassium salt)C,F,7SO3
3.2 PFOSA: perfluorooctane sulfonylamide C,F,7SO2NH2
3.3 PFOSAA: perfluorooctane sulfonylamido (ethyl)acetatCesF,7SO2N(CH2CH3)CH2CO2 3.4 ETFOSE-OH: 2(N-ethylperfluorooctanesulfonamido)-ethylalcohol
C,F,7So2N(CH2CH3)CH2CH20H
3.5 PFOSEA: perfluorooetane sulfonylethylamide C8F,7SO2N(CH2CH3)H
3.6 M556: CBF17So2N(H)(CH2COOH)
3.7 Surrogate standard: IH- 1H-2H-2H perfluorooctane sulfonicacid
4.0 WARNINGS AND CAUTIONS 4.1 Health and Safety Warnings:
4.1.1 Use universal precautions, especiallylaboratory coats,goggles, and gloves when handling animal tissue,which may contain pathogens.
5.0 INTERFERENCES 5.1 There are no interferencesknown
at thistime.
6.0 EOUIPMENT 6.1 The following equipment isused while performing thismethod. Equivalent equipment is
acceptable.
6.1.1 Ultra-Turrax T25 Grinder for grinding liversamples 6.1.2 Vortex mixer, VWI;@ Vortex Genie 2 6.1.3 Centrifuge,Mistral 1000 orIEC 6.1.4 Shaker, Eberbach or VV@R
ETS-8-6.0
Extraction ofPFOS from Liver
Page 2 of 14
6.1.5 NitrogenEvaporatorO,rganomation 6.1.6 Balance(sensitivittoy0.100g)
7.0 SUPPLIES AND MATERIALS 7.1 Gloves 7.2 Dissectingscalpels 7.3 Eppendorfor disposablepipettes 7.4 Nalgene bottlesc,apableof holding250 mL and 1 L 7.5 Volumetricflasksg,lass,typeA 7.6 I-CHEM vials4,0 mL glass 7.7 Plasticsampule vialsW,heaton, 6 mL (orappropriatseize) 7.8 Centrifugetubes,polypropylene,15 mL 7.9 Labels 7.10 Oxford Dispensor- 3.0to10.0ml 7.11 Syringes,capableof measuring5 gL to 50 gL 7.12 Graduatedpipettes 7.13 Syringes,disposableplastic3,cc 7.14 Syringefilternsy,lon,0.2lim,25 mm 7.15 Timer 7.16 Crimp cap autovialsand caps 7.17 Crimpers Note: Priortousingglasswareand bottlesr,inse3 timeswithmethanoland 3 timeswithMilli-
Q' water.Rinse syringesa minimum of 9 timeswithmethanol,3 rinsesfrom 3 separate vials.
8.0 REAGENTS AND STANDARDS 8.1 Type I reagentgrade water,MiRi-QTm or equivalenta;llwaterused inthismethod should
be Milli-Q' water and be providedby a Milli-QTOC PluiTmsystem 8.2 Sodium hydroxide(NaOH), J.TBaker orequivalent 8.3 Tetrabutylammoni= hydrogensulfate(TBA)K,odak orequivalent 8.4 Sodium carbonate(Na2CO3),J.T.Baker orequivalent 8.5 Sodium bicarbonate(NaHC03), J.T.Baker orequivalent 8.6 Methyl-tert-buteytlher,Omnisolv,glassdistilleodrBPLC grade
8.7 Methanol,Oinnisolv,glassdistilleodrBPLC grade 8.8 Liver,frozenfrom su@plier 8.9 Dry icefrom supplier
8.10 Fluorochemicalstandards 8.10.1PFOS (3M SpecialtCyhemicalDivision)m,olecularweight= 538
ETS-8-6.0 ExtractionfPFOS fromLiver
Page 3 of 14
8.10.2PFOSA (3M SpecialtCyhemicalDivision)m,olecularweight= 499
8.10.3 PFOSAA (3M SpecialtCyhemicalDivision)m,olecularweight= 585
8.10.4 ETFOSE-OH (3M SpecialtCyhemicalDivision)m,olecularweight= 570
8.10.5 PFOSEA (3M SpecialtCyhemicalDivision)m,olecularweight= 527
8.10.6 M556 (3M SpecialtCyhemicalDivision)m,olecularweight= 557
8.10.7 Surrogatestandard4:-H,perfluorooctanseulfonicacid(1-H,1-H,2-H, 2-H C,F13SO3H) molecularweight= 428
8.10.8 Other fluorochemicalsa,s appropriate
8.11 Reagent preparation
NOTE: When preparinglargervolumes thanlisteidnreagent,standard,or surrogate preparationa,djustaccordingly.
8.11.1 10 N sodium hydroxide(NaOH): Weigh approximately200 g NAOH. Pour intoa 1000 mL beakercontainin5g00 niL Milli-Q' water,mix untilallsolidsare dissolved.Storeina 1 L Nalgene bottle.
8.11.2 1 N sodium hydroxide(NaOH): Dilute10 N NAOH 1:10. Measure 10 mL of 10 N NAOH solutionintoa 100 mL volumetricflaskand dilutetovolume using Milli-Q' water.Storeina 125 mL Nalgenebottle.
8.11.3 0.5M tetrabutylammoniumhydrogensulfat(eTBA): Weigh approximately169 g ofTBA intoa 1 L volumetricontainin5g00 mL Milli-Q"Mwater.Adjustto pH 10 usingapproximatel4y4 to54 mL of 10N NAOH (WhileaddingthelastmL of NAOH, add slowlybecausethepH changesabruptly)D.iluteto volume with Milli-Q' water.Storeina 1 L Nalgenebottle.
8.11.3.1TBA requireascheckpriortoeachusetoensurepH = 10. Adjustas needed using I N NAOH solution.
8.11.40.25M sodium carbonate/sodiubmicarbonatbeuffer(NaCO,/NaHCO,): Weigh approximately26.5g of sodium carbonate(Na2C03)and 21.0g of sodium bicarbonat(eNaHC03)into a 1 L volumetricflaskand bringtovolume withNElliQ' water.Storeina 1 L Nalgenebottle.
8.12 Standards preparation
8.12.1PreparePFOS standardsforthestandardcurve.
8.12.2 Prepareotherfluorochemicasltandardsa,s appropriateM.ulticomponent fluorochemicalstandardsareacceptable(forexample,one working standard solutioncontaining1.00ppm PFOS, 1.02ppm PFOSA, 0.987ppm PFOSAA, and 1.10 ppm EtFOSE-OH.)
8.12.3 Weigh approximately100 mg of PFOS intoa 100 niL volumetricflaskand record theactualweight.
8.12.4 Bring to volume withmethanolfora stockstandardof approximately1000 ppm (gg/ML).
8.12.5 Dilutethestocksolutiownithmethanolfora workingstandardI solutionof approximately50 ppm.
ETS-8-6.0 ExtractioonfPFOS fromLiver
Page 4 of 14
8.12.6Dilutethestocksolutiownithmethanolforaworking standard2 solutioonf approx.5.0ppm.
8.12.7Dilutethestocksolutiownithmethanolfora working standard3 solutionof approx.0.50 ppm.
8.13 Surrogatestockstandardpreparation 8.13.1Weigh approximately50-60mg of surrogatsetandard1-H,I-H,2-H,2-H, C,F13So3H intoa 50 ml volumetricflaskand recordtheactualweight. 8.13.2Bring tovolume withmethanolfora surrogatsetockof approximately1000-1200 PPM8.13.3 Preparea surrogateworking standard.Transferapproximately1.0ml of surrogate stockto a 10 ml volumetricflaskand bringtovolume with methanol fora working standardof 10-20ppm. Record theactualvolume tr=ferred.
9.0 SAMPLE HANDLING 9.1 All samplesarereceivedfrozenand must be keptfrozenuntiltheextractioinsperformed.
10.0 OUALITY CONTROL
10.1 Matrix blanksand method blanks
10.1.1 An aliquotof 1.0mL methanolisused asa solventblank.
10.1.2Extracttwo 1.0niL ahquotsofl@filli-Qw'aterfollowingthisprocedureand use asmethod blanks.
10.1.3Extracttwo 1.0mL aliquotosfliverhomogenate followingthisprocedureand use asmatrixblanks.Referto11.1.6.
10.2 Matrix spikes
10.2.1 Prepareand analyzematrixspikeand matrixspikeduplicatesamples todetermine theaccuracyof theextraction.
10.2.2 Prepareeach spikeusinga sample chosenby theanalyst,usuallya controlliver receivedwith each sample set.
10.2.3 Expectedconcentrationwsillfallinthemid-rangeof theinitiaclalibratiocnurve. Additionalspikesmay be includedand may fallinthelow-rangeoftheinitial calibratiocnurve.
10.2.4 Prepareone matrixspikeand matrixspikeduplicatpeer40 samples,with a minimum of 2 matrixspikesperbatch.
10.3 Continuing calibrationverifications
10.3.1 Preparecontinuingcalibratiovnerificatisoanmplestoensuretheaccuracyof the initiaclalibratiocnurve.
10.3.2Prepare,ata minimum, one continuingcalibrativoenrificatisoanmpleper group of 10 samples. For example,ifa sample set= 34,fourverificatioanrseprepared and extracted.
ETS-8-6.0 ExtractionfPFOS fromLiver
Page 5 of 14
10.3.3 Prepareeach continuingcalibratiovnerificatifornom thesame matrixusedto preparetheinitiaclurve.
10.3.4 The expectedconcentrationwsillfallwithinthemid-rangeof theinitial calibratiocnurve.Additionaslpikesmay be includedthatfallinthelow-rangeof theinitiaclalibratiocnurve.Thisisnecessaryiftheanalystmust quantitatuesing onlythe low end of thecalibratiocnurve(forexample,5 ppb - 100 ppb,father than5 ppb - 1000 ppb).
11.0 CALIBRATION AND STANDARDIZATION
11.1 Preparematrixcalibratisotnandards
11.1.1Weigh approximately40 g ofliverintoa 250 mL Nalgene bottlecontaining200
mLs Milli-QwTamter.Grindtoa homogeneous solution.
11.1.2 If40 g isnot availableu,se appropriataemounts of liverand watertoensurea 1:5 ratio.
11.1.3 Refer to 13.0to calculatteheactualdensityof liverhomogenate and the concentratioonf solidlivertissuedispersedin 1.0mL of homogenate solution.
11.1.5 Add I mL of homogenate toa 15 mL centrifugteube.Re-suspendsolutionby shakingbetween aliquotwshilepreparinga totalof eighteenImL aliquotsof homogeneous solutionin15 mL centrifugteubes.
11.1.6 Two 1 mL aliquotso,r otherappropriatveolume,serveasmatrixblanks.
11.1.7 Typicallyuse thestandardconcentrationasnd spikingamounts listedinTable 1,at theend of thissectiont,o spike,induplicatet,wo standardcurves,fora totalof eighteensamples,two matrixblanks,and two method blanks.
11.1.8 Refer tovalidationreportsETS-8-6.0 and ETS-8-7.0-V-1 or Attachment B, which listtsheworking rangesand theLinearCalibratioRnange (LCR) for calibratiocnurves.
11.1.9 Use Attachment C as an aidincalculatintgheconcentrationosftheworking standards.Referto 13.0tocalculataectualconcentrationosf PFOS in calibration standards.
11.2 To eachworking standardb,lank,or continuingverificatioand,d appropriataemount of surrogateworking standardfortheconcentratiotno fallwithinthecalibratiocnurverange 5 ppb -1000ppb.
ETS-8-6.0 ExtractionfPFOS fromLiver
Page 6 of 14
11.3 Extractspikedliverhomogenates following12.14-12-25of thismethod.Use these standardsto establisehach initiaclurveon themass spectrometer.
Table 1 Approximate SpikingAmounts forCalibrationStandards
Working Standard (Approx.Conc.)
0.50ppm 0.50ppm 0.50ppm 0.50ppm 0.50ppm 5.0ppm 5.0ppm 5.0ppm 50 ppm
[LI
Approx.finalconc.of
PFOS inliver
-
Blank
2
0.005ppm
4
0.010ppm
10
0.025ppm
20
0.050ppm
40
0.100 ppm
10
0.250ppm
20
0.500ppm
30
0.750
4
1.00ppm
12.0 PROCEDURE 12.1 Obtainfrozenliversamples. 12.2 Cut approximately1 g of liverusinga dissectinsgcalpel.Thispartof theprocedureisbest
performedquickly,notallowingthelivertothaw.
12.3 Weigh thesample directliyntoa taredplastiscampule vial.
12.4 Record theliverweight inthestudynotebook.
12.5 Returnunused liverportionstofreezer.
12.6 Add 2.5mLs of waterto sampulevial. 12.7 Grind thesample.Put thegrinderprobeinthesample and grindforabout2 minutes,or
untilthesample ishomogeneous.
12.8 Rinse theprobe intothe samplewith 2.5mLs waterusinga pipette.
12.9 Take thegrinderapartand cleanitwithmethanolaftereach sample.Referto AMDT-EP22.
12.10 Cap thesample and vortexfor15 seconds.Label thesampule vialwiththestudynumber, weight,liverID, dateand analystinitials.
ETS-8-6.0 ExtractioonfPFOS fromLiver
Page 7 of 14
12.11 Pipette1.0mL, or otherappropriatveolume, of homogenate intoa 15 mL polypropylene centrifugetube.Label the centrifugetubewith theidenticailnformationas the sampule vial.Refer to attachedworksheet fordocumenting the remaining steps.
12.12 Pipettetwo 1 mL aliquotsofMilli-Q' watertocentrifugteubes.These willserveas method blanks.
12.13 Spike allsamples,includingblanksand standardsready forextractionwith surrogate standardas describedinsection11.2.
12.14 Spike each matirx with the appropriateamount of standardas describedin 11.1,or Table I of thatsection,forthe calibratiocnurve standards.Also preparematrixspikesand continuingcalibratiosntandards.
12.15 Vortex mix the standardcurve samples,matrixspikesamples,and continuingcalibration samples for15 seconds.
12.16 Check to ensure 0.5 M TBA reagentisatpH 10. Ifnot,adjustaccordingly.
12.17 To each sample,add 1 niL 0.5M TBA and 2 mL ofthe0.25 M sodium carbonate/sodium bicarbonatebuffer.
12.18 Using an Oxford Dispenser,add 5 mL methyl-tert-buteytlher.
12.19 Cap each sample and put on the shakerata settingof 300 rpm, for20 minutes.
12.20 Centrifugefor20 to25 minutes ata settingof3500 rpm, or untillayersarewell separated.
12.21 Label a fresh15 mL centrifugetubewith thesame informationasin 12.10.
12.22 Remove 4.0 mL of theorganiclayerto thefresh15 niL centrifugetube.
12.23 Put each sample on the analyticanlitrogenevaporatoruntildry,approximately I to2 hours.
12.24 Add 1.0mL to each centrifugetube using a graduatedpipette.
12.25 Vortex mix for30 seconds.
12.26 Attach a 0.2 4m nylon mesh filtetro a 3 cc syringeand transferthe sample to thissyringe. Filterintoa 1.5mL glassautovialor low-volume autovialwhen necessary.
12.27 Label the autovialwith thestudynumber, animal nwnber and gender,sample timepoint, matrix,finalsolvent,extractiodnate,and analyst(sp)erformingthe extraction.
12.28 Cap and storeextractsatroom temperatureor atapproximately4 'C untilanalysis.
12.29 Complete the extractionworksheet,attachedtothisdocument, and tapeinstudy notebook or includein studybinder,as appropriate.
ETS-8-6.0 ExtractioonfPFOS from Liver
Page 8 of 14
13.0DATA ANAL'VSIS AND CALCULATIONS 13.1 Calculations:
13.1.1 Calculatethe averagedensityofthe liverhomogenate by recordingeach mass of ten separate1.0mL aliquotsofhomogenate.
Average density(mg/mL) = Average mass (mg) of thealiguots
1.0mL aliquot
13.1.2 Calculatetheamount of liver(mg) per 1.0mL homogenate (orconcentratioonf dispersedsolidtissueper mL of homogenate suspension)usingthe following equation:
g of Liver x Average density*of homogenate (m-g/mL) (g ofLiver+.g ofWater)
referto13.1.1fordetails.
13.1.3 CalculateactualconcentrationosfPFOS and otherfluorochemicalisncalibration standardsusing thefollowingequation:
aL of Standardx Concentration(ag /n-iL=) FinalConcentration(pLg/ogr mg/kg)
mg Liver/1 niL homogenate*
of PFOS inLiver
*referto 13.1.2fordetails.
14.0 METHOD
PERFORMANCE
14.1 The method detectionlimit(NIIDL)isanalyteand matrixspecific.RefertoMDL reportfor specificMDL and limitofquantitatio(nLOQ) values(refertoAttachments B and C).
14.2 The followingqualitycontrolsamplesareextractedwitheachbatchof samplesto evaluate the qualityof the extractionand analysis.
14.2.1 Method blanks and matrixblanks.
14.2.2 Matrix spikeand matrixspikeduplicatesamples todetermineaccuracyand precisionof theextraction.
14.2.3 Continuing calibratiovnerificatiosnamples todeterminethe continuedaccuracy of theinitiaclalibratiocnurve.
14.3 Refer to section14 of ETS-8-7.0 formethod performancecriteria.
15.0 POLLUTION
PREVENTION
AND WASTE MANAGEMENT
15.1 Sample waste isdisposedinbiohazardcontainersf,lammable solventwaste isdisposedin high BTU containers,and used glasspipettewaste isdisposedinbroken glasscontainers locatedin thelaboratory.
ETS-8-6.0 ExtractioonfPFOS from Liver
Page 9 of 14
16.0RECORDS
16.1 Complete theextractiownorksheetattachedtothismethod, and tapeinthestudynotebook or includeinthe 3-ringstudybinder,as appropriate.
17.0 TABLES. DIAGRAms, FLOWCHARTS, AND VALIDATION DATA 17.1 AttachmentA, Extractionworksheet 17.2 AttachmentB, MDL/LOQ valuesand sununary 17.3 Attachment C, Calibratiosntandardcalculatioannd concentratiownorksheet
18.0 PEFERENCES 18.1 The validatiorneportassociatewdiththismethod isETS-8-6.0 & 7.0-V-1.
18.2 AMDT-EP-22, "RoutineMaintenanceofUltra-Tun-aTx-25"
18.3 FACT-M- 1.1,"ExtractionofPFOS or OtherAnionicFluorochemicalSurfactantfsrom LiverforAnalysisUsing BFLC-Electrospray/MassSpectrometry"
19.0 AFFECTED DOCUMENTS
19.1 ETS-8-7.0,"AnalysisofLiverExtractfsorFluorochemicalussingIiPLC-Electrospray Mass Spectrometry"
20.0 REvisIONS
Revision Number.
Reason For Revision
Revision Date
ETS-8-6.0 Extractionof PFOS from Liver
Page 10 of 14
Study Matrix Box # Wk/Day_
Date Spiked/Analyst ccv ms MSD
SurrogateStd approx. ppm actual ppm
FC Mix Std approx.0.5ppm actual PPM
FC Mix Std 'approx.5 ppm actual PPM
FC Mix Std approx. 50 ppm actual PPM
Comments
Blank
LiverHomogenate: Std
Liveramount
LiverExtractioMnethod
Spikesurro-gaatned Standardmix,Vortex15 sec.
PipettIemL ofLiverSolution
PipettIemL oftO.5M TBA, pH 10. pH
Std.
Pipett2emL of0.25Na2CO3/0.25M NaHC03 Buffer
Std.
Dispense5ml of Methyl-t-ButEytlher
TN-A-
Shake 20 min.
ShakerSpeed
CentrifuLy2e0-25min.
CentrifuiSzveeed
Remove a 4 mL aliquootforganiclayer
Puton NitrogenEvaporatotrodryness
EvaporatoTremperature
Add 1.0mL ofMethanol
TN-A-
Vortex30 sec.
I Filteursinga 3ce B-D syringewitha 0.2@LmSRI fiiteitn-toautosamplevial Cont.Cal.Verificationused thesame matrixasforthestandardcurve.
AttachmentB: MDL/LOQ Values
ETS-8-6.0 ExtractioonfPFOS from Liver
g Date& Initials
Page II of 16
:L, 7'7
MDL/LOQ valuesforrabbiltiver
Compound
MDL LOQ LinearCalibratiRoannge(LCR) (ppb) (ppb) Approximatceoncentratitoonbseusedforpreparintghe
StandarCdalibratiCounrve
PFOS
8.45 26.9 30 ppb - 1200 ppb
PFOSA
3.50 11.1 12 ppb - 1200 ppb
PFOSAA
24.6 78.3 30 ppb - 1200ppb
ETFOSE-OH
108 345 60 ppb - 900 ppb*
M556
82.3 262 60 ppb - 1200 ppb
PFOSEA
33.9 108 130 ppb- 1200 ppb
MDL/LOQ valuesinrat,bovine,and monkey liverwere notstatisticadleltyermined.Two curvesineach of thesematriceswere extracteadnd analyzedwiththerabbitlivercurvesto determineequivalence.Responsesintherat,bovine,and monkey livercurveswere equivalento therabbitresponses,thereforet,heirNML and LOQ willbe assumed tobe equivalentothose valuesas determinedfortherabbitliver.
RefertoLOQ Summary andMDL studyinETS-8-6.0& 7.0-V-1forftutheirnformation * ETFOSE-OH estimateosnlyforMDL andLOQ. Did notmeet criterifaorvalidation.
Compound: PFOS
Prepared Range of LCR from Range of :LCR from. Range of
Liver rangeof matrix standards
average curve
ave curve.-.: low std curve
@:.@l6@i, vid:: highstd ,.cuvre..:,,, curve
(ppb)(ng/ffiL)(ppb)(ng/-L) (pOb) (ng/
(ppb)(ng/-L) (ppb (ligihLi) (ppb)(ng/mL)
Rabbit 6.19-1237 12 - 1200 12 1200: 6 300 12 -:@00 60-1200
LCR from high @ts.d:
(0@b)
60 - 1200..-.;.
Compound:
Liver matrix
Rabbit
PFOSA
Prepared rangeof
standards
(ppb)(ngImL)
Range of LCR fibm average ave:curve curve
(ppb)(ng/TnL) (ppb)(ng/mL)
Range of low std curve
(ppb)(ng/mL)
L.CR froni.i;Range of LCR froi@n.
low std highstd curve::t; curve
'.hiis4jij': curve.@@..
.(ppb):,(nit-fi(ipLp)b)(ngirnl)
6.19-1237 12-1200 12 l@200.1 12-300 .12-.300 60-1200 60 1200
Compound: PFOSAA
Prepared Liver rangeof matrix standards
@ (ppb)(ng/mL)
Range of average curve
(ppb)(ng/mL)
LCR from ave curve
(ppb)(ng/mL)
Range of low std curve
(ppb)(ng/mL)
LCR from low std curve
(ppb)(ng/mL)
Range of high std
curve
(ppb)(ng/mL)
LCR from high std curve (Ppb)(ng/mL)
Rabbit 6.16- 1232 12-1200 30-1200
30-900
60-900
N/A
N/A
1
AttachmentB: MDL/LOQ Values
ETS-8-6.0 Extractioonf PFOS from Liver
Page 12 of 16
Compound: ETFOSE-OH
Prepared Range of LCR from Range of
Liver rangeof
average ave curve low std
matrix
standards
curve
curve
(ppb)(ng/mL) (ppb)(ng/-L) (ppb)(ng/-L) (ppb)(ng/mL)
LCR from Range of
low std
high std
curve
curve
(ppb)(ng/-L) (ppb)(ng/mL)
LCR from high std curve
(ppb)(ng/mL)
Rabbit 6.17- 1235 1 31 -900
31-900
N/A
N/A
N/A
N/A
Compound: PFOSEA
Prepared Liver rangeof matrix standards
(ppb)(ng/-L)
Range of average curve
(ppb)(ng/mL)
LCR from Range of ave curve low std
curve
(Ppb)(ng/-L) (ppb)(ng/mL)
LCR from low std curve
(ppb)(ng/mL)
Range of highstd curve
(ppb)(ng/mL)
LCR from highstd curve
(ppb)(ng/mL)
Rabbit 6.17-1235 31 - 1200 .31 - 1200@.. N/A
N/A
N/A
N/A
Compound: M556
Prepared Range of LCR from Range of
Liver rangeof
average ave,.curve, low std
matdx
standards
curve
curve
(ppb)(ng/-L) (ppb)(ng/InL) (ppb)--(ng/rnL)(ppb)(ng/-L
Rabbit 6.17-1235 31-1200
N/A
LCR from Range of
low std
highstd
.curve,@. curve
(pob)@'(nghnL) (ppb)(ng/mL)
LCR front... .highstd
curve,,.
(Vpb)(.nerfi4y
;N/A
N/A
N/A
AttachmentC: StandardCalculations
ETS-8-6.0
Extractioonf PFOS from Liver
Page 13 of 14
Ion PairStandard Curves - Tissue
Prep date(s): Analyte(s): Sample matrix:
Method/revision: Target analyte(s): FC mix std approx. 0.500 ppm: FC mix std approx. 5.00ppm: FC mix std approx. 50.0ppm: Surrogate stdapprox. 100 ppm:
Standard number: Equipment number: Finalsolventand TN: Blank liver/identifier:
Actual concentrationsof standardsin theFC mix
PFOS
PFOSA
PFOSAA ETFOSE PFOSEA
Stdconc Std conc ug/mL ug/niL
Std conc ug/n2L
Stdconc Stdconc ug/raL ug/mL
0.500
0.500
0.500
0.500
0.500
0.500
0.500
0.500
0.500
0.500
0.500
0.500
0.500
0.500
0.500
0.500
0.500
0.500
0.500
0.500
0.500
0.500
0.500
0.500
0.500
5.00
5.00
5.00
5.00
5.00
5.00
5.00
5.00
5.00
5.00
5.00
5.00
5.00
5.00 1 5.00
50.0
50.0 1 50.0
50.0
50.0
M556 Std conc ug/mL
0.500 0.500 0.500 0.500 0.500 5.00 5.00 5.00 50.0
Std conc ug/mL
AU An@t spiked
ML 0.002 0.004 0.010 0,020 0.040 0.010 0.020 0.030 0.004
AU Density
9 0.167 0.167 0.167 0.167 0.167 0.167 0.167 0.167 0.167
Calculated concentrationsof standards in the sample mafaix
PFOS
PFOSA PFOSAA ETFOSE PFOSEA
M556
Final
Final Finalconc Final Final
Final Stdconc
conc
conc
ng/g
conc
conc
conc
ng/g
ng/g
ng/g
ng/g
ng/g
nglg
5.99
5.99
5.99
5.99
5.99
5.99
12.0
12.0
12.0
12.0
12.0
12.0
29.9
29.9
29.9
29.9
29.9
29.9
59.9
59.9
59.9
59.9
59.9
59.9
120
120
120
120
120
120
299
299
299
299
299
299
599
599
599
599
599
599
898
898
898
898
898
898
1198
1198
1198
1198
1198
1198
Surrogate Std conc ng/mL
100
Surrogate Finalconc
ng/mL 0.500
All Ardt spiked ML 0.005
Validatedranges - approxim e concentrations
Liver Rabbit Bovine
PFOS
PFOSA
PFOSAA
5-1000ppb 5-1000ppb 5-1000ppb
Estimatesonly,usembbitvalues.
Rat
Estimatesonly,usembbitvalues.
Monkey
Estimatesonly,userabbitvalues.
ETFOSE-OH 5-1000ppb
P@ 5-1000ppb
PFOSEA 5-1000ppb
Attachment C: StandardCalculations
ETS-8-6.0
Extractionof PFOS from Liver
Page 14 of 14
3M ENVIRONMENTAL
LABORATORY
METHOD
ANALYSIS
OF POTASSIUM PERFLUOROOCTANESULFONATE
OR OTHER
FLUOROCHENUCALS
IN LRVER EXTRACTS USING
HPLC-ELEcTRospRAY/MAss
SpEcTRomzTRY
Method Number: ETS-8-7.0
Author: LisaClemen, Glenn Langenburg Approved By:
Laborat'o"rNyfanager A -/Z.W.W@-
GroupLeader
(Ii/)As- C)P*rit-
TechnicalReviewer
AdoptionDate: RevisionDate:
? Date -7/ 1115@9 Date Date
1.0 SCOPE AND APPLICATION
1.1 Scope: This method isforthe analysisof liverextractfsorfluorochemicalsurfactantussing BPLC-electrospray/mass spectrometry.
1.2 Applicable Compounds: Fluorochemicalsurfactantosr otherfluorinatecdompounds, or otherionizablecompounds.
1.3 Matrices: Rabbit,rat,bovine,monkey livero,r othertissuesasdesignatedinthe validation report.
Word 6/95
ETS-8-7.0 Analysisof LiverExtractUsing ES/MS
Page 1 of 10
2.0 SUMMARYOFMETHOD
2.1 This method describestheanalysiosf fluorochemicaslurfactantesxtractedfrom liverusing HPLC-electrospray/massspectrometryo,r similarsystem asappropriate.The analysisis performed by monitoringa singleioncharacteristoifca particulafrluorochemicals,uch as theperfluorooctanesulfon(aPtFeOS) anion,m/z = 499. Additionallys,amplesmay be analyzedusinga tandem mass spectrometetro furthevrerifytheidentitoyf a compound by detectingdaughter ionsof the selectedparention.
3.0 DEFINITIONS 3.1Atmospheric Pressure Ionization(API):The Micromass QuattroH triplequadrupole
systemsallow forvariousmethods of ionizatiobny utilizinvgarioussources,probes,and interfacesT.hese includebutarenotlimitedto:ElectrospraIyonizatio(nESI),Atmospheric PressurechemicalIonizatio(nAPcI),Thermospmy, etc.The ionizatiopnrocessinthese techniquesoccursatatmosphericpressure(i.en.ot undera vacuum).
3.2ElectrosprayIonization(ES,ESI): a method of ionizatiopnerformedatatmospheric pressure,whereby ions insolutionaretransferretdothegasphase viatinychargeddroplets. These chargeddropletsareproducedby theapplicatioonfa strongelectricaflield.
3.3Mass Spectrometry,Mass Spectrometer(MS), Tandem Mass Spectrometer (MS/MS): The API Quattro][Itriplqeuadrupolemass spectrometerisequippedwithtwo quadrupole mass selectivdeetectorsand a collisiocnell.Ionsareselectiveldyiscriminatebdy mass to chargeratio(m/z)and subsequentldyetected.A singleMS may be employed forion detectionor an ionmay be selecteidnthefirsqtuadrupole,fragmentedinthecollisiocnell, and thesefragmentsmay be analyzedinthesecond quadrupole.
3.4Conventional vs.Z-spray probe interface:The latesmtodels ofMicromass QuattroH triplqeuadrupole(post1998)utilizae"Z-spray"conformation.The sprayemittedfrom a probe isorthogonalto thecone aperture.In theconventionaclonformationitisaimed directlaytthecone aperturea,fterpassingthrougha tortuouspathway inthecounter electrode.Though theconfiguratioinsdifferenth,emethods of operationc,leaning,and maintenancearethe same. However, Z-spraycomponents and conventionalcomponents are not compatiblewith one another,butonlywithsimilarsystems(i.eZ.-spraycomponents are compatiblewith otherZ-spraysystems,etc.)
3.5Mass Lynx Software: System softwaredesignedforthespecifiocperationoftheseQuattro ][tIriplqeuadrupolesystems.CurrentlyMassLynx has Windows 95 and WindowsNT 4.0 versions.All versionsaresimilar.For more detailrsefertothemanual specifitcothe instrument(Micromass QuattroH triplqeuadrupoleMassLynx orMassLynx NT User's Guide).
4.0 WARNINGS AND CAUTIONS
4.1 Healthand SafetyWarnings:
4.1.1 Use cautionwiththevoltagecablesfortheprobe.When engaged,theprobe employs a voltageof approximately5000 Volts.
ETS-8-7.0 Analysisof LiverExtractUsing ESIMS
Page 2 of 10
4.1.2 When handling samplesor solventswear appropriateprotectivegloves,eyewear, and clothing.
4.2 Cautions:
4.2.1 Operatethesolventpumps below aback pressureof 400 bar (5800 psi).Iftheback pressureexceeds 400 bar,theHP 1100 willinitiataeutomaticshutdown.
4.2.2 Do not ran solventpumps to dryness.
5.0 INTERFERENCES
5.1 To minimize interferencewshen analyzingsamples,Teflonshallnot be used forsample storageor any partofinstrumentatiotnhatcomes incontactwith the sample or extract.
6.0 EouIPMENT
6.1 Equipment listedbelow may be modified inordertooptimizethe system.Document any modificationsintheraw dataasmethod deviations.
6.1.1 6.1.2
Micromass QuattroR triplequadrupoleMass Spectrometerequipped with an electrosprayionizationsource.
HP 1100 low pulse solventpumping system,solventdegasser,column compartment and autosampler
7.0 SUPPLIES AND MATERIALS
7.1 Supplies 7.1.1 High puritygradeairregulatedto approximately100 psi (houseairsystem) 7.1.2 BPLC analyticaclolumn, specifictsobe determinedby the analystand documented intheraw data 7.1.3 Capped autovialsorcapped 15 ml centrifugetubes
8.0 REAGENTS AND STANDARDS 8.1 Reagents
8.1.1 Methanol, BPLC gradeorequivalent
8.1.2 Milli-QTmwater (ASTM type1),allwaterused inthismethod shouldbe ATSM type I,or equivalenta,nd be providedby a Milli-QTOC Plus system or other vendor
8.1.3 Ammonium acetate,reagentgrade or equivalent
8.1.3.1 When preparingdifferentamounts thanthose listeda,djustaccordingly.
8.1.3.2 2.0 mM ammonium acetatesolution:Weigh approximately0.300 g ammonium acetateP.our intoa 2000 mL volumetriccontainercontaining 2000 mL Milli-Q' water,mix untilallsolidsaredissolved.Storeatroom temperature.
ETS-8-7.0 Analysisof LiverExtractUsing ES/MS
Page 3 of 10
8.2 Standards
8.2.1 Typicallytwo method blanks,two matrixblanks,and eighteenmatrixstandardsare preparedduring theextractiopnrocedure.RefertoETS-8-6.0.
9.0 SAMPLE HANDLING
9.1 Fresh matrix standardsarepreparedwith each analysis.Extractedstandardsand samples
are stored in capped autovialsor capped 15 ml centrifugetubes untilanalysis.
9.2 Ifanalysis will be delayed, extractedstandards and samples may be stored atroom temperature, or refrigeratedat approximately 4' C, untilanalysiscan be performed.
10.0 OUALITY CONTROL 10.1 Method Blanks and Matrix Blanks
10.1.1 Solvent blanks,method blanks,and matrix blanks are prepared and analyzed with each batch to determine contamination or carryover.
10.1.2 Analyze a method blank and a matrix blank prior to each calibrationcurve.
10.2 Matrix Spikes
10.2.1 Matrix spikes are prepared and analyzed to determine the matrix effecton the recovery efficiency.
10.2.2 Matrix spike duplicates are prepared and analyzed to measure the precision and the recovery for each analyte.
10.2.3 Analyze a matrix spike and matrix spike duplicateper forty samplep. With a minimum of 2 spikes per batch.
10.2.4 Matrix spike and matrix spike duplicateconcentrationswill fallin the mid-range of the initialcalibrationcurve. Additional spike concentrationsmay fallin the lowrange of the initialcalibrationcurve.
10.3 Continuing Calibration Checks
10.3.1 Continuing calibrationverificationsare analyzed to verify the continued accuracy of the calibrationcurve.
10.3.2 Analyze a mid-range calibrationstandard every tenth sample, with a minimum of one per batch.
11.0 CALIBRATION AND STANDARDIZATION
11.1
Analyze the extractedmatrix standardspriorto and following each setof sample extracts. The average of two standard curves will be plottedby linearregression(y = mx + b), weighted I/x,not forced through the origin,using MassLynx or other suitablesoftware.
11.2 Ifthe curve does not meet requirements perform routinemaintenance or reextractthe standard curve (ifnecessary) and reanalyze.
ETS-8-7.0 Analysisof LiverExtractUsing ES/MS
Page 4 of 10
11.3 For purposes of accuracywhen quantitatinlgow levelsof analyte,itmay be necessaryto use the low end of the calibratiocnurveratherthanthe fullrange of the standardcurve. Example: when attemptingtoquantitataepproximately10 ppb of analyte,generatea calibratiocnurve consistingof thestandardsfrom 5 ppb to 100 ppb ratherthan the full rangeof thecurve (5 ppb to 1000 ppb). This willreduceinaccuracyattributetdo linear regession weighting ofhigh concentratiosntandards.
12.0 PROCEDURES 12.1 AcquisitionSet up
12.1.1 Set up the sample list.
12.1.1.1Assign a sample listfilenameusingMO-DAY-last digitof year-increasing letteorf the alphabetstartinwgith a
12.1.1.2Assign a method (MS filef)oracquiring 12.1.1.3Assign an BEPLC program (Inletfile) 12.1.1.4Type in sample descriptionasnd vialpositionnumbers
12.1.2 To createa method clickon method in theAcquisitioncontrolpanel then mass spectrometerheadingsand selectSIR (SingleIon Recording)or NIRM (Multiple Reaction Monitoring).Set IonizatioMnode as appropriataend mass to499 or other appropriatemasses. A fullscanisusuallycollectedalongwith the SIRS. Save acquisitiomnethod. IfMS/MS instrumentsare employed, additionaplroduction fragmentationinformationmay be collected.Referto Micromass MassLynx GUIDE TO DATA ACQUISITION foradditionailnformationand MRM.
12.1.3 Typicallythe analyticablatchrun sequence beginsand ends with a setof extracted matrixstandards.
12.1.4 Samples are analyzedwith a continuingcalibratiovnerificatioinniectedstandard afterevery tenthsample. Solventblanksshouldbe analyzedperiodicalltyo monitor possibleanalytecarryoverand arenot consideredsamples but may be included as such.
12.2 Using the Autosampler
12.2.1 Set up sample trayaccordingtothe sample listpreparedin Section12.1.1.
12.2.2 Set-up the BPI 100/autosampleratthe followingconditionsor atconditionsthe analystconsidersappropriateforoptimalresponse.Record actualconditionsinthe instrumentlogbook:
12.2.2.1Sample size= 10 @LLinjection
12.2.2.2Inject/sample= 1
12.2.2.3Cycle time= 9 minutes
ETS-8-7.0 AnalysisofLiverExtractUsing ES/MS
Page 5 of 10
12.2.2.4Solvent ramp conditions
Time
MEOH
0.00 min.
40%
1.0min.
40%
4.5min.
95%
6.5min.
95%
7.0min.
40%
9.0mi.
40%
2.0 mM Ammonium acetate
60% 60% 5% 5% 60% 60%
12.2.2.5Press the"Start"button.
12.3 Instrument Set-up
12.3.1 Refer to ETS-9-24.0, "Operationand Maintenance of theMicromass QuattroII TripleQuadrupole Mass SpectrometerFittedwith an Atmospheric Pressure IonizationSource,"formore details.
12.3.2 Check the solventlevelinreservoirasnd refililfnecessary.
12.3.3 Check the stainlesssteelcapillaraytthe end of theprobe. Use an eyepieceto check thetip.The tipshouldbe flatwith no jagged edges.Ifthetipisfound to be unsatisfactoryd,isassembletheprobe and replacethestainlessteelcapillary.
12.3.4 Tum on the nitrogen.
12.3.5 Open the tune page. Clickson operateto initiatseourceblock and desolvation heaters.
12.3.6 Open the InletEditor.
12.3.6.1Set BPLC pump to"On" 12.3.6.2Set the flow to 10 - 500 uL/min or asappropriate 12.3.6.3Observe dropletscoming out of thetipofthe probe. A fineraistshould be
expelledwithno nitrogenleakingaround thetipof theprobe. Readjust the tipof theprobe ifno mist isobserved 12.3.6.4Allow toequilibratfeorapproximately10 minutes.
12.3.7 The instrumentusestheseparametersatthe followingsettings.These settingsmay change in orderto optimizethe response:
12.3.7.1Drying gas 250-400 liters/hour 12.3.7.2ESI nebulizinggas 10-15 liters/hour 12.3.7.3BPLC constantflow mode flow rate10 - 500 @iL/min 12.3.7.4Pressure<400 bar (Thisparameterisnot set,itisa guide to ensurethe
HPLC isoperatingcorrectly.)
12.3.7.5Source block temperature150'
12.3.7.6Desolvationtemperature250'
ETS-8-7.0 Analysisof LiverExtractUsing ES/MS
Page 6 of 10
12.3.8 Printthe tunepage,with itsparameters,and storeitinthestudybinderwith a copy taped intothe instrumentlog.
12.3.9Clickon startbuttonintheAcquisitionControlPanel (thismay vary among MassLynx versions,refertoappropriateMassLynx User'sGuide). Ensure startand end sample number includesallsamples tobe analyzed.
13.0 DATA ANALYSIS AND CALCULATIONS 13.1 Calculations:
13.1.4 Calculatematrix spikepercentrecoveriesusingthe followingequation:
% Recovery
Observed Result- BackUound Result x 100 Expected Result
13.1.5 Calculatepercentdifferencuesingthefollowingequation:
% Difference
ExRected Conc. - CalculatedConc. x 100 Expected Conc.
13.1.6 Calculateactualconcentrationisnmatrix (@ig/g):
(naof PFOS calc.from std.Curve x DilutionFactor) x (InitiWaelight of Liver(2) FinalVolume (mL)
I L!g 1000 ng
14.0 METHOD PERFORMANCE 14.1 Method DetectionLimit(MDL) and Limitof Quantitatio(nLOQ) aremethod, analytea,nd
matrixspecificR.eferto ETS-8-6.0,Attachment B fora listinogf currentvalidatedMDL and LOQ values.
14.2 Solvent Blanks, Method Blanks and Matrix Blanks
14.2.1 Solvent blanks,method blanks,and matrixblanksmust be below the lowest" standardinthe calibratiocnurve.
14.3 CalibrationCurves
14.3.1 The r'value forthecalibratiomnust be 0.980 orbetter.
14.4 Matrix Spikes
14.4.1 Matrix spikepercentrecoveriesmust be within 30% ofthe spiked concentration.
14.5 Continuing CalibrationVeriflcation
14.5.1 Continuing calibratiovnerificatiopnercentrecoveriesmust be within 30% of the spikedconcentration.
14.6 Ifcriterilaistedin themethod performance sectionarenot met, maintenance may be performed on the system and samples reanalyzedor otheractionsas determined by the analyst.Document allactionsinthe appropriatelogbook.
ETS-8-7.0 Analysisof LiverExtractUsing ES/MS
Page 7 of 10
14.7 Ifdataareto be reportedwhen perfon-nancceriterihaave not been met, thedatamust be footnotedon tablesand discussedinthetextof thereport.
15.0 POLLUTION PREVENTION AND WASTE MANAGEMENT
15.1 Sample extractwaste and flammablesolventisdisposedinhigh BTU containersa,nd glass pipettewaste isdisposedinbroken glas'csontainerlsocatedin thelaboratory.
16.0 RECORDS
16.1 Each page generatedfora studymust have thefollowinginformationincludedeitherin the headeror hand writtenon thepage: studyor projectnumber, acquisitiomnethod, integratiomnethod, samplename, extractiodnate,dilutiofnactor(ifapplicable)a,nd analyst.
16.2 Printthetunepage,samplelista,nd acquisitiomnethod from MassLynx toincludein the appropriatsetudy folder.Copy thesepages and tapeintotheinstrumentranlog.
16.3 Plotthecalibratiocnurveby linearegressionw,eighted I/x,thenprintthesegraphsand storeinthestudyfolder.
16.4 Printdataintegratiosnummary, integratiomnethod,and chromatograinsfrom MassLynx and storeinthestudyfolder-.
16.5 Summarize datausingsuitablseoftware(Excel5.0+)and storein thestudyfolderr,eferto Attachment A foran example of a summary spreadsheet.
16.6 Back up electronidcatato appropriatmeedium. Record instudynotebook thefilename and locationof backup electronidcata.
17.0 TABLES. DIAGRAms, FLOWCHARTS,
AND VALIDATION DATA
17.1 AttachmentA: ETS-8-7.0 Data summary spreadsheet
18.0 REFERENCES 18.1 FACT-M-2. 1,"ExtractioonfPotassiumPerfluomoctanesulfonaotreOtherFluorochemical
Compounds from LiverforAnalysisUsing BPLC-Electrospray/MassSpectrometry"
18.2 ETS-9-24.0,"Operationand Maintenanceof theMicromass Atmospheric Pressure Ionization/MassSpectrometerQuattroIItriplqeuadrupoleSystems"
18.3 The validationreportassociatedwith thismethod isETS-8-6.0 & 7.0-V-1
19.0 AFFECTED DOCUMENTS
19.1 ETS-8-6.0,"ExtractioonfPotassiumPerfluorooctanesulfonoartOetherFluorochemical Compounds from LiverorFluidforAnalysisUsing HPLC-Electrospray/Mass Spectrometry"
ETS-8-7.0 AnalysisofLiverExtracUtsingES/MS
Page 8 of 10
20.0REVISIONS
Revision Number
Reason For Revision
Revision Date
ETS-8-7.0 AnalysisofLiverExtractUsingES/MS
Page 9 of 10
Laboratory Study
Study: Test Material: Matrix/FinaSlolvent: Method/Revision: AnalyticaElquipment System NumbC@T: InstrumentSoftwaTeNersion: Filename: R-SquaTed Value: Slope: Y Intercept: Date of ExtTaction/Analyst: Date ofAnalysis/Analyst:
Liroup Dose
Sample#
Concentration ng/g
lnitial.Wt. 9
Dilution Factor
FinalCone. ug/g
Slope: en from linearegressionequation. Group/Dose: Taken from thestudyfolder. Sample#: Taken from the studyfolder. Concentration(ng/g):Taken ftom theMassLynx integratiosnummary. InitiaWlt. (g):Taken from thestudyfolder. DilutionFactor: Taken from thestudyfolder. FinalConc. (ug/g):Calculatedby dividingtheinitiavlolume ftom theconcentration
Attachment A: Summary Spreadsheet
ETS-8-7.0
AnalysisofLiverExtractUsing ES/MS
Page 10 of 10