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lAbOMtDry ComposiRteport AnalyticaRleportsofData for FluorochemicalAnalysisinHuman Sera LIEMS No. 1623 TestingLaboratory 3M EnvironmentaTlechnology& SafetyServices 3M EnvironmentalLaboratory FluorineAnalyticaClhemistryTeam (FACT) 2-3E-09 935 Bush Avenue, St.PauL MN 55106 Laboratory Cont"t KrisHansen,Ph.D. Bldg.2-3E-09 P.O.Box 3331 St.Paul,MN 55133-3331 Phone: (651)778-6018 FAX: (651)778-6176 Requesters LarryZobel,JeffMandel,Gemy Olsen 3M MedicalDepartment Bldg.220 St.Paul,MN page I 3MDnIMONMENTAL LABORA7'ORY ..................... no compositereportmciudestentechnicarleporttshatwere issuedtothe3M MedicalDepmunat btween March 2,1998and April24,1998.Each mpm do=ncits theresultosfanalysiosfhuman 9= mniples collectefdromvarious==. Inmost am, eachtechnicarleporitncludemsformatlonaboutthedateof ddiveryofthedatatotherequesterI.aterpretatiomftheresultisstheresponsibiliotfythe3M Medical Departrnerd- Althoughrigorousqualitcyontrolmeasuresand 3M EnvironmentalLaboratoryStandardOpwating Proceduresand methodswere followedwhen possiblet,heaoqmmttonoftlusdatawas notn==* coum-tedaccordingtoapplicablGeood LaboratoryPracticesD.ata was collectetdomad m=mbate needsto helpd@ar@ thepotentiaflorfunmn healthconcernsi;twas notalwayspossiblteofollowlaboratory proceduresthatwere inplaceatthefim. Data was wdnic@ mvL-@@ by 3M Ew&oumemW Lab technical st4 butwas notrewev@vdby theQualityAssuranceUmt inpl= at3Ni Appendix A includetsheanalyticanlzdwds thatwere followedinthecollectioonfthedatam44xfftindgxw technicarleports. ETS-8-4.1ExtractioonfPotassiumPcrflwroocLmesWfonateorodia FluorocbemicalQmmgpdg Serum forArahsis UsingHPLC-Elecftospray/h4ass withthefollowingmeptlon: ethylacetate was added totheaqueousexuw insteaotfiMME. ETS-&5.1 AnalysisofPotassiumPeffluorow==Mnate orotherFluorochemicaClormxxmds inSenun ExtractsUsingIHPLC&LeqMEMA4ass . Data kduded inthePhaseI[andPhase M reports was collecteudsingESHE, whiledataforsubsequentphaseswere collecteudsingESMSIZ. Detailcsanbe foundineachWJmical reporti,ncludedinAppendicesB ftmgh K ChromalogaphicdataforeachUdinicalreporitsmdived atthe3M En*mnenW Lab. KrisHan-wA Ph.D.,P&icipalAnalyticaklxestiptor Date Appendix A: AnalyticaMlethods Appendix B: Phase2 Appendix C: Phase3 Appendix D: Phase4 Appendix E:Phase 5 Appendix F: Phase6 Appendix G: Phase7 Appendix H: Phase8 Appendix 1:Phase 9,partI Appendix J:Phase 9,part2 AppendixK: Summary ofPOAA Imb forPhases2-9 Page 2 3M ENviRoNwNTAL LABORATORY METHOD ExTRAcTiON OF POTASSIUM PERFLUOROOCTANESULFONATE FLUOROCHEMICAL COMPOUNDS FROM SERUM FOR ANALYSIS ELECTROSPRAY/MASS SPECTROMETRY OR OTHER USING HFLC- Method Number: ETS-84.1 Adoption Date: 03/01/99 Author: Lisa Clemen, Glenn Langenburg Revision Date: Approved By: LaboratoryManager Date Group Leader Date TechnicalReviewer Date 1.0 SCOPE Am APPLicATioN 1.1 Scope: This method isforthe extractioonf potassiumperfluorooctanesulfona(tPeFOS) or otherfluorochemicaclompounds from serum. 1.2 Applicable compounds: Fluorochemicalsurfactantosr otherfluorinatecdompounds. 1.3 Matrices: Rabbit,rat,bovine,monkey, and human serum or otherfluidsas designatedin thevalidationreport. Word 6/95 ETS-9-4.1 Extractionof PFOS from Senun Page I of 14 2.0 SUMMARY OF METHOD 2.1 This method describetsheprocedureforextractinpgotassiumperfluorooctanesulfonate (PFOS) or otherfluorochen@caslurfactantfsrom serun-o4r otherfluidsu,singan ion pairingreagentand methyl-tert-buteytlher(MtBE). Inthismethod,sevenfluorochemicals were extractedP:FOS, PFOSA, PFOSAA, ETFOSE-OI-L PFOSEA, M556, and surrogate standard(see3.0 Definitions)A.n ionpairingreagentisadded to thesample and the analyteionpairispartitioneidntoMTBE. The MTBE extractisremoved and put onto a nitrogenevaporatoruntildry.Each extractisreconstitutiend1.0mL of methanol,then filteretdhrougha 3 cc plastiscyringeattachedto a 0.2limnylonfilteirntoglassautovials. 2.2 These sample extractasreanalyzedfollowingmethod ETS-8-5.1 or otherappropriate method. 3.0 DEnNrrioNs 3.1 PFOS: perfluorooctanesulfona(taenionofpotassiumsaltC)sFl7S% 3.2 PFOSA: perfluorooctanseulfonylamidCegFl,7SO2NH2 3.3 PFO SAA: perfluorooctanseulfonylamido(ethyl)acetaCtieF 17SO2N(CH2CH3)CH2CO2 3.4 ETFOSE-OH: 2(N-ethylperfluorooctasnuelfonaniido)-etahlyclohol CSF17SO2N(CH2CH3)CH2CH20H 3.5 PFOSEA: perfluorooctanseulfonyelthylamideC8Fl7SO2N(CH2CH3)H .3.6 M556: Cl@F17SO2N(H)(CH2COOH) 3.7 Surrogatestandard:IH- IH-2H-2H perfluorooctanseulfoniaccid 4.0 WARNINGS AND CAUTIONS 4.1 Healthand safetywarnings 4.1.1 Use universaplrecautionse,specialllyaboratorcyoats,goggles,and gloveswhen handlinganimaltissuew,hich may containpathogens. 5.0 INTERFTRENCFs 5.1 There areno interferenckensown atthistime. 6.0EouipmzNT 6.1 The followingequipmentisusedwhileperformingthismethod. Equivalentequipmentis acceptable. 6.1.1 Vortexmixer,VWII, VortexGenie2 6.1.2 CentrifugeM,stral 1000orEEC 6.1.3 Shaker,EberbachorVWR 6.1.4 NitrogenevaporatorO,rganomation ETS-8-4.1 ExtractionofPFOS kom Senun Page 2 of 14 6.1.5 Balance(0.100g) 7.0 SUPPLIES AND MATERJALS 7.1 Gloves 7.2 Eppendorf or disposablepipettes 7.3 Nalgene bottles,capableof holding250 mL and I L 7.4 Volumetric flasks,glass,type A 7.5 I-CHEM vials,glass,40 niL glass 7.6 Centrifugetubes,polypropylene,15 mL 7.7 Labels 7.8 Oxford Dispenser -,-3.0to 10.0 mL 7.9 Syringes,capable of measuring 5 pL to 50 pL 7.10 Graduated pipettes 7.11 Syringes,disposableplastic3, cc 7.12 Syringefiltersn,ylon,0.2 gm, 25 mm 7.13 Timer 7.14 Crimp cap autovialsand caps 7.15 Crimpers Note: Priorto using glasswareand bottlesr,inse3 times with methanol and 3 times with NM-QTMwater. Rinse syringesa minimum of 9 times with methanol,3 rinsesfrom 3 separate vials. 8.0 REAGENTS AM STANDARDS 8.1 Type I re!y4ntgrade water,NM-QTM or equivalent;allwater used inthismethod should be Mflli-Q water and may be providedby a MM-Q TOC Pluim system 8.2 Sodium hydroxide (NAOM, J.T Baker or equivalent 8.3 Tetrabutylammorium hydrogen sulfate(TBA),Kodak or equivalent 8.4 Sodium carbonate(Na2C03), J.T.Baker or equivalent 8.5 Sodium bicarbonate(NaHC03), J.T.Baker or equivalent 8,6 Methyl-T-Butyl Ether,Omnisolv, glassdistilleodr BPLC grade 8.7 Methanol, Omnisolv, glassdistilleodr EPLC 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),molecularweight = 499 8.9.3 PFOSAA (3M SpecialtyChemical Division),molecularweight = 585 ETS-8-4.1 ExtmctioonfPFOS fromSenun Page3 of14 8.9.4 ETFOSE-OH (3M SpecialtCyhemicalDivision)m,olecularweight= 570 8.9.5 PFOSEA (3M SpecialtCyhemicalDivision)m,olecularweight= 527 8.9.6 M556 (3M SpecialtCyhemicalDivision)m,olecularweight= 557 9.9.7 Surrogatestandard4:-H, perfluorooctanseulfoniaccid(I-K I-I-L2-K 2-H CsFl3SO3H) molecularweight= 428 8.9.8 Other fluorochemicalsa,s appropriate 8.10 Reagent preparation NOTE: When preparinglargervolumes thanlisteidnreagent,standardo,r surrogate preparationa,djustaccordingly. 8.10.1 10 N sodium hydroxide(NaOIi):Weigh approximately200 g NAOH. Pour intoa 1000 mL beakercontainin5g00 mL NM-i:fm water,mix untilallsolidsare dissolved.Storeina IL Nalgenebottle. 8.10.2 1 N sodium hydroxide(NaOH):. Dilute10 N NAOH 1:10. Measure 10 mL of 10 N NAOH solutioinntoa 100 mL volumetricflaskand dilutteo volume usingMUiQTm water. Storeina 125 mL Nalgene bottle. .8.10.30.5M tetrabutylammoniuhmydrogen sulfat(eTBA):Weigh approximately169 g ofTBA intoa I L volumetriccontainin5g00 mL MI-Q" water.AdjusttopH 10 usmg approximately44 to 54 mL of 10 N NAOH (WhileaddingthelastmL of NAOK add slowlybecausethepH changesabruptly)D.ilutetovolume withNMwater. Storeina I L Nalgenebottle. 8.10.3.1TBA requiresa check priorto eachuseto ensurepH =.10. Adjustas neededusing I N NAOH solution. 8.10.4 0.25 M sodium carbonate/sodiumbicarbonatebuffer(Na2C03/NaHC03): Weigh approximately26.5g of sodium carbonate(Na2C03) and 21..O-ogf sodium bicarbonate(NaHC03) intoa I L volumetricflaskand bring'to-volumweithNEE-@. QTm water. Storeina 1 L Nalgene bottle. 8.11 Standards preparation' 8.11.1 PreparePFOS standardsforthestandardcurve. 8.11.2Prepareotherfluorochemicasltandardsa,sappropriateM.ulticomponent fluorochemicasltandardsareacceptable(forexample,one working standard solutioncontaining1.00ppm PFOS, 1.02ppm PFOSA, 0.987ppm PFOSAA, and 1.10 ppm EtFOSE-OH.) 8.11.3Weigh appro)dmately100 mg of PFO S intoa 100 mL volumetricflaskand record the actualweight. 8.11.4Bring to volume withmethanolfora stockstandardof approximately1000 ppm (@L9/ML). 8.11.5 Dilutethe stocksolutionwithmethanol fora working standardI solutionof approximately50 ppm. 8.11.6 Diluteworking standardIwithmethanolfora workingstandard2 solutionof approx.5.0 ppm. ETS-84.1 ExtractioonfPFOS from Senun Page 4 of14 8.11.7DiluteworkingstandardI withmethanolfora workingstandard3 solutionof approx.0.50ppm. 8.12 Surrogate stockstandard preparation 8.12.1 Weigh approximatel5y0-60 mg ofsurrogatestandardI-K I-K 2-K 2-K CgFl3SO3H intoa 50 mL volumetrifclaskand recordtheactualweight. 8.12.2 Bringto volume withmethanolfora surrogatestockof approximately1000-1200 PPM. 8.12.3Preparea surrogateworking standard.TransferapproximatelyI mL of surrogate stockto a 10 mL volumetricflaskand bringto volume withmethanolfora Working standardof 100 ppm. Record theactualvolume transferred. 9.0 SAWLF, HANDL]ING 9.1 AU samplesarereceivedfrozenand must be kept frozenuntilthe extractioinsperformed. 9.2 Allow samplesto thaw to room temperaturepriorto extraction. 10.0 OuALrry CoNTRoL 10.1 SolventBlanks,Method blanks and matrix blanks 10.1.1An aliquotof 1.0mL methanolisused as a solventblank. 10.1.2Extracttwo 1.0mL aliquotosfl@ffi-Ql"waterfollowingthisprocedureand use as method blanks. 10.1.3 Extracttwo 1.0mL aliquotosftheserum followingthisprocedureand use a's matrixblanks.See 11.1.4. 10.2 Matrixspikes 10.2.1 Prepareand analyzematrixspikeand matrixspikeduplicatesamplesto determine theaccuracyof theextraction. 10.2.2 Prepareeach spikeusinga sample chosenby theanalystu,suallythecontrolmatrix receivedwitheach sample set. 10.2.3 Expected concentrationwsillfalilnthemid-rangeoftheinitiaclalibratiocnurve. Additionalspikesmay be includedand may fallinthelow-rangeoftheinitial calibratiocnurve. 10.2.4 Prepareone matrixspikeand matrixspikedupecateper 40 samples,with a minimum of2 matrixspikesperbatch. 10.3 Continuingcalibratiocnhecks 10.3.1Preparecontinuingcalibratiocnheck samplesto ensuretheaccuracyoftheinitial calibratiocnurve. 10.3.2 Prepare,ata minimum, one continuingcheck pergroup of IO'samples.For example,ifa sampleset= 34,fourchecksarepreparedand extracted. 10.3.3 Prepareeach continuingcalibratiocnheck from thesame matrixused to prepare theiniticaulrve. ETS-8-4.1 ExtractioonfPFOS from Sennn Page 5 of14 10.3.4 The expectedconcentrationwsillfallwithinthemid-rangeof theinitiaclalibration curve.Additionalspikesmay be includedthatfallinthelow-rangeof theinitial calibratiocnurve.Thisisnecessaryiftheanalystmust quantitatuesingonly the low end ofthecalibraticounrve(forexample,5 ppb - 100 ppb,ratherthan 5 ppb - 1000 ppb). 11.0 CALIBRATION AND STANDARDIZATION 11.1 Preparematrixcalibratiosntandards 11.1.1TransfeIrmL ofserumtoa 15 mL centrifutguebe. 11.1.2 Ifmost samplevolumes arelessthan 1.0mL, extractstandardswith matrix volumes equalto thesamplevolumes. Do not extractlessthan0.50mL of matrix. Record each sample volume on theextractionsheet. 11.1.3 While prepa#nga totaloftwenty aliquotisn15 mL centrifugteubes,mix or shake between aliq@uots. 11.1.4 Two I mL aliquotso,r otherappropriatveolume,serveas matrixblanks.Typically use thestandardconcentrationasnd spikingamounts listedinTable 1,attheend of thissectiont,o spike,induplicatet,wo standardcurves,fora totalof eighteen standardst,wo matrixblanks,and two method blanks. 11.1.5Referto validatiorneportETS-8-4.0 & ETS-8-5.0-V-1,which listtsheworking rangesand theLinearCalibratioRnange (LCR) forcalibratiocnurves. 11.1.6Use AttachmentD as an aidincalculatintgheconcentrationosf theworldng standards.See Section13.0to calculataectualconcentrationosfPFOS'in calibratiosntandards. 11.2 To each standardb,lank,or continuingcheck,add appropriataemount of surrogate working standardfortheconcentratiotno falwlithinthecalibration.cuir-avnege 5 ppb 1000 ppb. 11.3 Extractspikedmatrixstandardsfollowing12.6-12.16ofthismethod. Use thesestandards to establisehach initia6l@@e on the mass spectrometer. ETS-8-4.1 Extractionof PFOS from Senun Page 6 of 14 Table 1 Approximate spikingamounts forstandardsand spikes Usi g 1.0ML of matrix Working standard (approxc.onc.) ILL Approx.finaclonc.of analyteinmatrix - - Blank 0.500 ppm 10 0.005 ppm 0.500 ppm 20 0.010 ppm 5.00 ppm 5 0.025 ppm 5.00 ppm 10 0.050 ppm 5.00ppm 20 0.100 ppm 50.0pprn 5 0.250ppm 50.0ppm 10 0.500ppm 50.0ppm 15 0.750 ppm 50.0ppm 20 1.00ppm 12.0 PROCEDURE 12.1 Obtainfrozensamplesand allowto thaw atroom temperatureor in a lukewarm waterbath. 12.2 Vortexmix for15 seconds,thentransfe1r.0mL or otherappropriatveolume to a 15 niL polypropylenecentrifugteube. 12.3 Return unused samplesto freezerafterextractioanmounts have been removed. 12.4 Record theinitiavlolume on theextractiownorksheet. 12.5 Label thetubewith thestudynumber,sampleBD, dateand analystinitialsS.e.e attached worksheet fordocumentingthe remainingsteps. 12.6 Spikeallsamples,includinbglanksand standardsr,eadyforextractiownith surrogate standardas describedin11.2. 12.7 Spike each matrixwiththeappropriateamount of standardas describedin11.1,or Table 1 inthatsectionf,orthecalibratiocnurvestandards.Also preparematrixspikesand continuingcalibratiosntandards. 12.8 Vortex mix thestandardcurvesamples,matrixspikesamples,and continuingcalibration samplesfor 15 seconds. 12.9 Check to ensurethe0.5 M TBA reagentisatpH 10. Ifnot,adjustaccordingly. 12.10 To each sample,add I mL 0.5M TBA and 2 mL of 0.25M sodium carbonatelsodium bicarbonatebuffer. 12.11 Using an Oxford Dispenser,add 5 mL methyl-tert-buteytlher. 12.12 Cap each sample and put on theshakerata settingof 300 rpm, for20 minutes. 12.13 Centrifugefor20 to25 minutesata settinogf3500 rpm, or untilayersarewellseparated. ETS-8-4.1 ExtractioonfPFOS from Serum Page 7 of 14 12.14Labelafres1h5mL centriftuugbeewiththesameinformataisoinn12.5. 12.15Remove4.0mL oftheorganilcayetrothicslea1n5mL centriftuugbee. 12.16 Put each sampleon theanalyticanlitrogenevaporatoruntildry,approximatelyI to 2 hours. 12.17 Add 1.0mL of methanolto eachcentrifugteubeusinga graduatedpipette. 12.18 Vortex mix for30 seconds. 12.19 Attach a 0.2 gm nylonmesh filtetro a 3 cc syringeand transfetrhesample to thissyringe. Filterintoa 1.5mL glassautoviaolr low-volume autoviawlhen necessary. 12.20 Label theautovialwith thestudynumber,animalnumber and gender,sampletimepoint, matrix,finalsolvente,xtractiodnate,and analyst(sp)erformingtheextraction. 12.21 Cap and storeextractastroom temperatureor atapproximatel4y 'C untilanalysis. 12.22 Complete theextra@@onworksheet,attachedtothisdocument,and tapeinthestudy notebook or includeinstudybinder,asappropriate. 13.0 DATA ANALYSIS ANID CALCULATIONS 13.1 'Calculations 13.1.1 CalculateactualconcentrationosfPFOS, or otherapplicablfeluorocherinicianl, calibratiosntandardsusingthefollowingequation: mL of standardx concentratioonf standard(gg LLIQ mL of standard+ mL of surtogatestandard+ initimaaltrixvolume (mL) FinalConcentratio(n4mL) ofPFOS inmatrix 14.0 MIETHOD PFMORMANCE 14.1 The method detectiolnimit(MDL) isanalyteand matrixspeciflcR.efertoMDL report forspecifiNcML and ffiniotfquantitati(oLnOQ) values(seeAttachments B and C). 14.2 The followingqualitycontrolsamplesareextractewditheachbatch of samplesto evaluate thequalityoftheextractioannd analysis. 14.2.1Method blanksand matrixblanks. 14.2.2Matrix spikeand matrixspikeduplicatseamplesto determineaccuracyand precisioonf theextraction. 14.2.3 Continuingcalibratiocnheck samplesto determinethecontinuedaccuracyofthe initiaclalibratiocnurve. 14.3 Referto section14 of ETS-8-5.1formethod performancecriteria. 15.0 POLLUTION PREVENTION AND WASTE MANAGEMZNT 15.1 Sample waste isdisposedinbiohazardcontainersf,lammablesolventwaste isdisposedin highBTU containersa,nd used glasspipettewaste isdisposedinbroken glasscontainers locatedinthelaboratory. ETS-8-4.1 ExtractioofnPFOS fromSenun Page8 of14 16.0RFcoRDs 16.1 Complete theextractiownorksheetattachedto thismethod,and tapeinthe study notebook or includeinthe 3-ringstudybinder,as appropriate. 17.0 ATTAcHmzNTs 17.1 Attachment A, Extractionworksheet 17.2 Attachment B, L@MLJLOQ valuesand summary 17.3 Attachment C, Calibrationstandardconcentrationworksheet 18.0 REFIZRENCES 18.1 The validationrepoit associatedwith thismethod isETS-8-4.0 & S.O-V-l. 18.2 FACT-M-3. 1,"Analysis of Senun or Other Fluid Extractsfor Fluorochemicals using IHPLC-ElectrosprayMass Spectrometry" 19.0 AFncrED DocummNTs 19.1 ETS-8-5. 1, "Analysisof Serum or Other FluidExtractsforFluorochemicalsusingBPLCElectrosprayMass Spectrometry" 20.0 RFvisioNs Revision Number I Reason For Revision Section 12.21 Changed to includesample storage atroom temperature. Section 12.13 Added the shaker speed. Section 12.17 Finalvolume is1.0mL; not adjustedforinitiavlolumes less than 1.0mL. Revision Date 04/02/99 ETS-8-4.1 ExtractionofPFOS from Senun Page 9 of 14 3M ENvmoNwNTAL LABORATORY METHOD ANALYSIS OF POTASSIUM PERFLUOROOCTANESULFONATE OR OTHER IFLUOROCHEAUCALS IN SERUM ExTRAcTs USING HPLC-ELECTROSPRAY/MASS SPECTROMEETRY Method Number: ETS-8-5.1 Author: LisaClemen,RobertWynne Approved By: Adoption Date: 03/01/99 RevisionDate: LaboratoryManager Date Group Leader Date TechnicalReviewer Date l.b SCOPE AND APPLICATION 1.1Scope: Thismethod describesthe analysisof serum extractsforfluorochemicalsurfactants usingBPLC-electrospray/massspectrometry. 1.2Applicable Compounds: Fluorochemicalsurfactantosr otherIluorinatecdompounds, or otherionizablecompounds. 1.3 Matrices: Rabbit,rat,bovine,monkey, and human serum,or otherfluidsas designatedinthe validatiorneport. Word 6/95 ETS-8-5.1 AnalysisofSenun ExtractUsing ES/A4S Page Iof9 2.0 SUMMARY OF METHOD 2.1 Thismethod describetsheanalysisof fluorochemicaslurfactantesxtractedfrom serum or otherfluidsu,singBPLC-electrospray/massspectrometryo,r similasrystemas appropriate. The analysiissperformedby monitoringa singleioncharacteristoifca particular fluorochemicals,uchas theperfluorooctanesulfona(tPeFOS) anion,nVz--499. Additionallys,amplesmay be analyzedusinga tandem mass spectrometerto furtherverify theidentitoyf a compound by detectingdaughterionsoftheparention. 3.0 DEFUGTIONS 3.1 Atmospheric PressureIonization(API):The Mcromass QuattroI[t[riplqeuadrupole systemsallowforvariousmethods of ionizatiobny utilizivnagrioussources,probes,and interfacesT.heseincludebut arenot limitedto:ElectrospraIyonizatio(nESI),Atmospheric PressurechemicalIonizatio(nAPcl),Thermospray,etc.The ionizatiopnrocessinthese techniquesoccursatatmosphericpressure(i.e.n,otunder a vacuum). 3.2 ElectrosprayIonization(ES, ESI): a method ofionizatiopnerformedatatmospheric pressurew,hereby ionsinsolutionaretransferretdothegasphase viatinychargeddroplets. These chargeddropletsareproduced by theapplicatioonfa strongelectricaflield. 3.3 Mass Spectrometry, Mass Spectromete r (MS), Tandem Mass Spectrometer (MS/MS): The API QuattroH triplequadrupolesystemsare equippedwith quadrupole mass selectivdeetectors.Ionsareselectiveldyiscriminatebdy mass to chargeratio(nVz)and subsequentldyetected.A singleMS may be employed foriondetectionor a serie(sMS/MS) formore specififcragmentatioinnformation. 3.4 Conventional vs.Z-spray probe interface:The latesmtodels ofMcromass Quattro R triplqeuadrupolesystems(post1998)utilizae"Z-spray"conformation.T.he sprayemitted from a probe isorthogonalto thecone aperture.Intheconventionaclonfor'mationitisaimed directlaytthecone aperturea,fterpassingthrougha tortuouspathway inthecounter electrode.Though theconfiguratioinsdifferenth,emethods ofoperation,cleaninga,nd maintenancearethesame. However, Z-spraycomponents and conventionalcomponents are notcompatiblewithone another,butonlywithsimilasrystems(i.e.Z,-spraycomponents are compatiblewithsome otherZ-spraysystems,etc.) 3.5 Mass Lynx Software: System softwaredesignedforthespecifiocperationofthese QuattroIItriplqeuadrupolesystems.CurrentlyMassLynx hasWindows 95 and WindowsNT 4.0versions.AllversionsaresimilarF.or more detailsseethemanual specifitco the instnunen(tMcromass QuattroIItriplqeuadrupoleMassLynx orMassLynx NT User's Guide). 4.0 WARNINGS AND CAUTIONS 4.1 Healthand SafetyWarnings: 4.1.1 Use cautiowniththevoltagecablesfortheprobe.When engaged,theprobe employs a voltageof approximately5000 Volts. ETS-8-5.1 Analysisof SeniinExtractUsingES/MS Page 2 of 9 4.1.2 When handlingsamplesor solventswear appropriatperotectivgeloves,eyewear, and clothing. 4.2 Cautions: 4.2.1 Do not operatesolventpumps above capacityof 400 bar (5800 psi)back pressure. iftheback pressureexceeds400 bar,theBPI 100 willinitiataeutomaticshutdown. 4.2.2 Do not runsolventpumps todryness. 5.0 INTERFERENCES 5.1, To minimizeinterferencwehsen analyzinsgamplest,eflonshouldnotbe usedforsample storageor any partof instrumentatiotnhatcomes incontactwiththesampleor extract. 6.0 EOLTIPMENT 6.1 Equipment listebdelow may be modifiedinorderto optimizethesystem.Document any modificationisntheraw data as method deviations. 6.1.1 NEcromass QuattroH triplqeuadrupoleMass Spectrometerequippedwith an electrospraiyonizatiosnource 6.1.2 BPI 100 low pulsesolventpumping system,solventdegasser,column compartment, and autosainpler 7.0 SUPPLrEs AND MATERIALS 7.1 Supplies 7.1.1 High puritygradenitrogengas regulatetdo approximately100 psi(House air system) 7.1.2 BPLC analyticaclolumn, specifictso be determinedby the an@dy@tand documented intheraw data. 7.1.3 Capped autovialosr capped 15 mL centrifugteubes 8.0 REAGIENTS AND STAMARDS 8.1 Reagents 8.1.1 Methanol,BPLC gradeorequivalent 8.1.2 NM-Q"z" water,allwater used inthismethod shouldbe N&M-Qlm water or equivalenta,nd may be provided by a MM-Q TOC Plus system or othervendor 8.1.3 Anunonium acetate,reagentgrade or equivalent 8.2 Standards 8.2.1 Typicalltywo methodblankst,wo matrixblanksa,nd eighteemnatrixstandardasre prepared duringthe extractionprocedure. See ETS-8-4.1. 9.0 SAMIPLE HANDLING ETS-8-5.1 AnalysisofSerum ExtractUsing ES/MS Page 3 of 9 9.1 Fresh matrixstandardsarepreparedwith each analysisE.xtractedstandardsand samples arestoredincapped autovialosr capped IS mL centrifugteubesuntilanalysis. 9.2 Ifanalysiwsillbe delayed,extractedstandardsand samplescan be refrigerateadt approximately4' C, oratroom temperatureu,ntilanalysicsan be performed. 10.0 OUALrry CONTROL 10.1 Solvent Blanks, Method Blanks and Matrix Blanks 10.1.1 Solventblanks,method blanksand matrixblanksareprepared and analyzedw.ith each batchto determinecontaminationor carryover. 10.1.2 Analyzea method blankand a matrixblankpriorto each calibratiocnurve. 10.2 Matrix Spikes 10.2.1Matrixspikes'arpereparedand analyzedto determinethematrixeffecton the recoveryefficiency. 10.2.2 Matrixspikeduplicateasrepreparedand analyzedto measure theprecisionand the recoveryforeach analyte. 10.2.3 Analyzea matrixspikeand matrixspikeduplicatpeerfortysamples,with a minimum of2 spikesperbatch. 10.2.4 Matrixspikeand matrixspikeduplicatceoncentrationwsillfallinthemid-rangeof theiniticaallibraticounrve.Additionalspikeconcentrationmsay falilnthelowrangeof theinitiaclalibratiocnurve. 10.3 Continuing CalibrationVerifications 10.3.1 Continuing calibratiovnerificatioanrse analyzedto verifythe continuedaccuracy of the calibratiocnurve. 10.3.2Analyzea mid-rangecalibratiosntandardaftereverytenthsample,witha minimum of one perbatch. 11.0 CALIBRATION AND STANDMWMATION 11.1 Analyzetheextractemdatrixstandardspriorto and followingeach setof extracts.The averageoftwo standardcurveswillbe plottedby linearegressio(ny= my + b),weighted I/x,not forcedthroughzero,usingMass]Lynxor othersuitablseoftware. 11.2 Ifthecurvedoes notmeet requirementsp,erformroutinemaintenanceor reextracthe standardcurve (ifnecessary)and reanalyze. 11.3 For purposesof accuracywhen quantitatinlgow levelsofanalyte,itmay be necessaryto usethelow end of thecalibratiocnurveratherthanthefiffrlange ofthe standardcurve. Example: when attemptingto quantitataepproximately10 ppb of analyteg,eneratea calibratiocnurveconsistinogf thestandardsfrom 5 ppb to 100 ppb ratherthanthefall rangeof thecurve(5ppb to 1000 ppb). Thiswillreduceinaccuracyattributetdo linear regressiowneightingof highconcentratiosntandards. ETS-8-5.1 Analysisof Senun ExtractUsing ES/MS Page 4 of 9 12.0PRocEDuREs 12.1AcquisitiSoentup 12.1.1Clickon starbtuttonintheAcquisitioCnontrolPanel.Setup a sample Est.Assign a filenameusingMO-DAY-last digitofyear-samplenumber,assigna method (MS) foracquiringa,nd typeinsunple descriptions. 12.1.2 To createa method clickon scanbuttonin theAcquisitiocnontrolpaneland select SIR (SingleIon Recording)orNIRM. Se'tIonizatioMnode as appropriat'eandmass to 499 or otherappropriatmeasses.A fullscanisusuallycollecteadlongvvitthhe SIRS. Save acquisitiomnethod. IfMS[MS instrumentasreemployed,additional productionfragmentatioinnformatiomnay be collectedS.ee Mcromass MassLynx GUI]DE TO DATA ACQLJISITION foradditionailnformatioannd MRM (Multiple ReactionMoiitoring). 12.1.3 Typicallytheanalyticablatchrun sequencebeginswith a setof extractedmatrix standardsand ends with a setof extractedmatrixstandards. 12.1.4 Samples areanalyzedwith a continuingcalibratiocnheck injectedaftereverytenth sample. Solventblanksshouldbe analyzedperiodicaltloy monitorpossibleanalyte carryoverand arenotconsideredsamplesbutmay be includedas such. 12.2 Using theAutosampler 12.2.1 Setup sampletrayaccordingtothesample fisptreparedinSection12.1.1. 12.2.2 Set-uptheBPI I00/autosampleratthefollowingconditionsor atconditionsthe analystconsidersappropriateforoptimalresponse.Record actualconditionsinthe instrumentlogbook: 12.2.2.1 Sample size= 10 gL injection 12.2.2.2Inject/sainp=le1 12.2.2.3 Cycletime= 13.5minutes 12.2.2.4Solventramp = Time 0.00 min. 8.50 min. 11.0min. 12.0min. MEOH 40% 90% 90% 40% 2.0mM Ammonium acetate 60% 10% 10% 60% 12.2.2.5Pressthe"Start"button. 12.3 InstrumentSet-up 12.3.1 RefertoETS-9-24.0formore details. 12.3.2Check thesolventlevelinreservoirasnd refiilflnecessary. ETS-8-5.1 Analysisof Serum ExtractUsing ES/MS Page 5 of 9 12.3.3 Check thestainlesssteelcapillarayttheend oftheprobe. Use an eyepieceto check thetip.The tipshouldbe flatwithno jaggededges.Ifthetipisfoundto be unsatisfactordyi,sassembletheprobe and replacethestainlesssteelcapillary. 12.3.4 SetBPLC pump to "On". Settheflow to 10 - 500 uL/niinor as appropriate. Observedropletscoming out ofthetipoftheprobe.Allow to equilibratfeor approximately10 minutes. 12.3.5 Turn on thenitrogen.A fineiiissthouldbe expelledwithno nitrogenleaking around thetipoftheprobe. Readjustthetipof theprobe ifno mistisobserved. 12.3.6 The instrumentusestheseparametersatthefollowingsettingsT.hese settingsmay change inorderto optimizetheresponse: 12.3.6.1Dryinggas250400 fiters/hour 12.3.6.2ESI nebulizinggas 10-15fiters/hour I'@. 12.3.6.3 BPLC constant flow mode, flow rate 10 - 500 Wmin 12.3.6.4 Pressure <400 bar (This parameter is not set, it is a guide to ensure the BPLC isoperatincgorrectly.) 12.3.7 Carefullgyuidetheprobeintotheopening.Insertprobeuntilitwillnotgo any furtherC.onnectthevoltagecablesto theprobe. 12.3.8 Printthetunepage,withitsparametersa,nd storeitinthestudybinderwitha copy tapedintotheinstrumentlog. 12.3.9 Using thecross-flowcounterelectrodientheES/MS sourceisrecommeride@ forthe analysiosf biologicamlatrices. 12.3.10CHckon starbtuttonintheAcquisitioCnontrolPanel(thismay varyamong MassLynx versionss,eeappropriatMeassLynx USEWS GUIDG). Pressthestart button.Ensure startand end saznplenumber includesallsample'sto be analyzed. 13.0 DATA ANALysis AND CA'LCULATIONS 13.1 Calculations: 13.1.4 Calculatematrixspikepercentrecoverieussingthefollowingequation: % Recovery Observed Result-Backyround Resultx 100 Expected Result 13.1.5Calculatepercentdifferencuesingthefollowingequation: % Difference EKpectedConc.-CalculateCdonc. x 100 Expected Conc. 13.1.6CalculateactualconcentratioonfPFOS, or otherfluorochernicailn,matrix(gg/mL): (ngof PFOS calc.from std.Curve x DilutionFactorj x I LLg anitiaVlolume ofmatrix(mL) + mL ofSur@ogateStandard) 1000 ng FinalVolume (mL) ETS-8-5.1 AnalysisofSenm ExtracUtsingES/MS Page 6 of9 14.0 METHOD PERFORMANCE 14.1 Method DetectionLimit(MDL) and Limitof Quahtitatio(nLOQ) aremethod,analytea,nd matrixspecificP.leaseseeETS-8-4.1,Attachment B, fora listinogf currentvalidated MDL and LOQ values. 14.2 Solvent Blanks, Method Blanks, and Matrix Blanks 14.2.1 Solventblanks,method blanks,and matrixblanksvaluesare must be below the lowest standardinthe calibratiocnurve 14.3 CalibrationCurves 14.3.1The r2 valueforthecalibratiocnurvemust be 0.980or better. 14.4 Matrix Spikes 14.4.1 Matrix spikepercentrecoveriesaremust be within 300/oof ihe spiked concentration. 14.5 Continuing CalibrationVerifications 14.5.1 Continuingcalibratiovnedficationpercentrecoveriesmust be 30% of the spiked concentration. 14.6 Ifcriterilaistedinthismethod performancesectionisn'tmet, maintenancemay be performed on the system and samplesreanalyzedor otheractionsas determinedby the analyst.Document allactionsinthe appropriatelogbook. 14.7 Ifdataare tobe reportedwhen performance criterihaave not been met, the datamust be footnotedon tablesand discussedinthe textofthe report. 15.0 POLLUTION PREVENTION AND WASTE MANAGEMMNT 15.1 Sample extractwaste and flammable solventisdisposedinhighBTU containersa,nd glass pipettewaste isdisposedinbroken glasscontainerslocatedinthe laboratory. 16.0 REcoRD@ 16.1 Each page generatedfora studymust have the followinginformationincludedeitherinthe headeror hand writtenon the page: studyor projectnumber, acquisitiomnethod, integratiomnethod, sample name, extractiodnate,dilutiofnactor(ifapplicable)a,nd analyst. 16.2 Printthe tune page,sample lista,nd acquisitiomnethod from MassLynx to includeinthe appropriatestudyfolder.Copy thesepages and tapeintothe instrumentrunlog. 16.3 Plotthe calibratiocnurve by linearregressionw,eighted I/x,thenprintthesegraphsand storeinthe studyfolder. 16.4 Printdata integratiosnummary, integratiomnethod, and chromatograms, from MassLynx, and storein the studyfolder. 16.5 Summarize datausingsuitablesoftware(Excel5.0)and storeinthe studyfolder,see Attachment A foran example ofa summary spreadsheet. ETS-8-5.1 Analysisof Senun ExtractUsing ES/N4S Page 7 of 9 16.6 Back up electronidcatato appropriatmeedium. Record instudynotebook thefilename and locationofbackup electronidcata. 17.0 TABLES, DiAGRAms, FLOWCHARTS, AND VALIODATioN DATA 17.1 AttachmentA: ETS-8-5.1 Data summary spreadsheet. 18.0 RFFERFNCES 18.1 FACT-M-4. compounds 1,"Extractionof Potassium Perfluorooctanesulfonatoer Other Fluorochemical from Serum forAnalysisUsing EPLC-Electrospray/NWs Spectrometry 18.2 ETS-9-24.0, "Operation and Maintenance of the Mcromass Atmospheric Pressure Ionization/MassSpectrometer Quattro H triplequadrupole Systerr&' 18.3 The validationreport-associatedwith thismethod isETS-8-4.0 & 5.0-V-l. 19.0 Amc7m DocumNTs 19.1 ETS-8-4. 1, "Extractionof Potassium Perfluorooetanesulfonatoer Other Fluorochemical Compounds from Serum forAnalysisUsing BPLC-Electrospray/i\4assSpectrometry" 20.0 REvisioNs Revision Number. I Reason For Revision Section 6.1.2 Clarificatioonf IHP 1100 system components. Section 11.1 Average of two curves, not standardvalues,are used for plottinglinearregressionand added the I/xweighting of the curve. Section 12.2.2.4Clarificatioonf solventramp. Section 17.1 Changed from attachment B to A. Revision 04/02/99 ETS-8-5.1 Analysisof Senun ExtractUsing ES/MS Page 8 of9 LaboratoryStudy Study: TestMaterial: Matfix/FinaSlolvent: Method/Revision: AnalyticalEquipment System Number: InstrumentSoftwareNersion: Filename: R-Squared Value: Slope: Y Intercept: Date ofExtraction/Analyst: Date ofAnalysis/Analyst: Group Dose Sample# Concentration mg/mT, InitiaVloL mL Dilution Factor FinalConc. uatmt, S@lope:Taken from linearregressionequation. Group/Dose: Taken from thestudyfolder. Sample#: Taken from thestudyfolder. Concentration(ugfmL): Taken from theMassLynx integratiosnummary. InitiaVlolume (mL): Taken ftom thestudyfolder. DilutionFactor: Taken from thestudyfolder. FinalConc. (ug/mL): Calculatebdy dividingtheinitiavlolume from theconcentration Attachment A: Summary Spreadsheet ETS-8-5.1 Analysisof Senun ExtractUsing ES/MS Page 9 of 9 Fluorine Analytical Chemistry Team Contact Ki.Han9cm 8-6019 Detersitago6n ofPFOS inseraby ElectrosprayMass Spectrometry(ESMS) Quantitation: LimitofDetectio(nLOD): LimitofQuantitatio(nLOQ) Range of C&Bration Curve: 1.0ppb 1.0ppb I.Oppb- 100ppb Spechscity: RetentionTime: moiam9mion. 7.35 499 amu Curve CorrelatioCnoefficient 0.999 PHASE Let 1006 1007 1008 S009 solo soil W012 W013 W014 IEL-An*sisofpooledsem US 1!993 Avemp cone. St& % Rowyery PFOS (Dpb)@ Dev. 44 2 -27* 73 44 6 -14* 35* 43 2 65* 37* 26 8 82 92 28 7 -6* 27* 45 2 100 108 54 13 78 92 50 5 90 102 41 6 106 92 lrange ofAverage Concmbudon ofPFOS IinLot Smorram: Avemge eme LigL PFOS (ODb)@ RM W015 41 3 W016 47 8 W017 42 3 - fiffa6mppb to54 ppb % Remvery m 120 112 51* 92 -45* 65* ResultsofQuantitatioonfMethod Blanks Concentmdon link Medwd Blank B20-1 PFOS (pub) <LOD Method Blank MO-2 <LOD MeffiodBlank,um-1 (1) 7 Method Blani@u.m-1 (2) Method Blank,mm-1 (3) Method Blank.3cm-2 (1) <LOD -<LOD 4.OD SamvW QCI QC2 QC3 QC4 QC5 [PFOSI rowyered 49 49 49-.50 53 [PFOSI emnew 49 49 49 49 49 % rewyery 100 100 100 102 108 Sy stem Reprodua*bilityand PrecisionChecks producib spampk uclb 31;00(67-23((22)) 9_3 S009-3(2) WO 12.3(2) W015-3 (2) W017-3 (2) PFOS (pob) 45 I1 54 33 37 L% 0.7% 0.40A 0.4% 1.80/0 0.8% Prock1w.. Am* 1007-1(1) 1007-1(2) 1007-1(3) 1007-1(4) 1007-1(5) Avemge Std.Dev. RSD PFOS (npb) 47 55 49 47 49 49 I 211/o 'Reportedvaluesare theaverages concentratioPnFOS ofsampleseximctedinMplicate. 2QC sampki are extractsof blankseraspikedwithPFOS standar&. 3Reproducibilitsyamples are repeatinjectionosf lotsera extractsp,erformedafter every 15 imn plei 4Precision.samplesare 5 consecutiveinjectionsofthe same lotsera extract *QuestionableData. Sou= of ailotmm=tly undeAmiinecL 3M Envkommental Lab 5nlgg PHASE.U rtuorineAnalyticalChemistryTeam in seraby ElectrosprayMass Spectrometry (ESMS) Quantitd&@ LimitofDetectio(nLOD) LimitofQuantitatio(nLOQ) Range ofCalibratioCnurve: 10 ppb 1.0ppb loppb -100ppb Specificity: RetentionI"une: MolecularIon: 6.90min. 413 amu' 369 amu@ Curve CorrelatioCnoefficien(tr@: 0.991 Comm: K.J.Hansm 8-60191 Phase U: Analysisof pooled sem Avenige con Std. i,ot POAA (D]Db Dev. 1006 <LOD 0 1007 <,OD 1 1009 <LOD 0 S009 <LOD 1 solo <.OD 0 soil <WD 0 W012 <LOD 1 W013 <LOD 0 W014 <,OD 0 (POAA) % Recovery MS MSD 56* 96 53* 79 90 74 65* 68* 45* 570 71 67* 92 94 100 100 98 95 Range ofAverage ConcentrationofPOAA inLot Sera.- Avcmge conc Std. _LA POAA (IDub? Dev. W015 <LDD 0 W016 <LOD 0 W017 <.OD 0 BOOI <IOD 0 B002 <LDD 0 B003 -CLOD 0 B004 <,OD 0 B005 <DD 0 allvahm wm belowtheLODIOQ % Recovery MS MD 100 101 83 .97 83 97 98 89 100 100 108 110 60* 94 94 89 (10ppb) Resultsof QuantitationofMethod Blinks Slmk Method Blank,B20-1 Method Bbu3k H20-2 Method Blank,scra-I(1) MethodBlank,stra-1(2) MethodBlank sem-1(3) Method Blank sera-2 (1) concentration POAA (IDD-W <LOD <.OD <LOD <LOD -<LOD <.OD Samew QCI QC2 QC3 QC4 QC5 [POAA] recovemd 94 91 91 .92 9.3 [JPOAA) emmcted 98 95 95 96. 97 % recoym 98 95@ 95 96 97 System Reproducibilityand PrecisionChecks Reproducibilty': Samiple 1006-3(2) S009-3(2) W012-3 (2) W015-3 (2) W017-3 (2) B003-3(2) POAA (area) 7700 3100 6500 4700 4900 3100 RSD 0.9% 7.00/o o.oo/o 0.0% 5.801* 2.00/o Sample1007-1(1) 1007-1(2) 1007-1(3) 1007-1(4) 1007-1(5) Avmge Std.Dev. RSD Peak 5$w 5600 6300 6300 6300 6100 300 5% 'IonnLIz-413usedforpurposes ofquantitation. 21onmlz-369 usedforpurposes ofverficatioonf compound identity. 'Reportedvaluesare theaveragesconcentratioPnOAA ofsmples c&wted intnplicate 4Qc samplesare extractsof blankseraspikedwithPOAA standards. sreproducibilitsyamples are repeatInjectionosf lotsera extractsp,erfonnedafterevery15 samples. 6Precisiosnamples are 5 consecutiveinjectionosfthesaw lotseraextract. *QuestionableData. Source of errorau=tly undetermined. 3M EnvironmentalLab 5nlgg PHASE 11(POAA) Y-1.Hanwn Phase III 3/W8 3M EnvironmentalLab Quantitative Analysis of PFOS in Ind*m*dual's Sera Determination of PFOS in sera by Electrospray Mass Spectrometry (ESMS) Quandtation: Spedfidty: LimitofDctwdon (LOD) 1.0ppb Retudon Time: 7.35min. LimitofQuandtodon (LOQ) 1.0ppb MolecularIon: 499 amu Range ofCabbradw Curve: l.Oppb- 100ppb CurveCorrelatioCn4efficie(nrt@: I ResultsofQuantitationoflnd*m*duaSleraSamples Avenkgecow. Lot PFOS (opb) 198 58 298 41 398 46 498 32 598 67 698 S3 798 42 898 49 % RSD 41* 14 2 N-k 0 7 N-k 35* Let 1798 1898 im 2099 2198 2298 2398 2498 998 39 11 2598 1098 54 5 2698 1198 60 11 2798 1299 37 1 2899 1399 45 NA 2998 1498 31 12 3098 1598 96 2 3199 1699 74 1 venkgceonc. PFOS (vpb) % RSD 46 15 56 10 70 3 40 2 49 9 60 13 72 1 35 20 41 33* 53 11 57 15 33 N-k 65 7 67 50* 33 11 N.A- - No 0/" calculatedas therewu only enough smplc forI mabsis. QuantitatiAvnealysiosfPFOS and POAA inInd*m*dualS'esra DeterminatioonfPFOS/POAA inseraby lelectrosprMaayn Quantitation: LimiotfDetectio(nLOD): LimitofQumdtadOn (LOQ): Rmp ofCalibradonCurve: CoffelatioCnoefficien(tr@: Spedrakr. 1.0ppbtlo ppb 1.0ppb/10 ppb RdenticeT'mc: bblecwwim I.Oppb- 100ppb/10 ppb -100ppb 0.999/0.999 Spectrometz7(ZSMS) 7.35minJ6.9miil 499 awd4l3 Lwu.369 a= Preliminary Results 198 2F 2199181 398 498 598 698 798 899 999 1099 1198 1298 1398 1498 1598 1698 of Ouantitation of Indhid%W Som Avlg.cone. Std. S(lo Dev. 46 nd. Avg. cosm 'FOAA (o <LOQ 35 nd. 45 nd. 32 nd. .167 nd. 53 4 42 nd. 49 17 37 ncL :LOQ Is 10 <.OQ <ZQ gLOQ <.OQ -GLOQ 52 tLdL CLOQ 39 nd. 37 nd. CLOQ 4.OQ 45 nd. 28 nd. 96 3 CLOQ <.OQ 10 73 tLd. .4LOQ Samples St4L RM ME zLd. 1798 nd. 1999 3 1999 nd. 2098 zLd. 2198 n.& 2298 nd. 2398 nd. 2498 nd. 2598 nd. 2698 ndL 2798 iLdL 28"0 nd. 2998 zLd. 3098 1 3198 nd. Avemp mm PFOS (nimb4 41 52 71 40 45 54 71 23 30 47 51 30 41 41 36 Std. 2m nd. zLd. nd. 1 ;LdL zLd. zLd. nd. zLd. 1 nd. iLd. zLd. nd. iLd. Avg. am& POAA (io CLOQ <LOQ -<LOQ <.OQ CLOQ 4.OQ 12 <.OQ -4,OQ <.DQ <.OQ LOQ <IOQ LOQ <LOQ St4L um nd. iLd. zLd.' iLd. iLd. n.& nd. nd. nd. nd. zLd. iLd. nd. nd. nd. Resultsof Quantitaflonof Method Blanbs llmk IPFOSI(pub) iS7B Blank.Eao-l .4LOD Method BILuk,Sao-1 (1) 4 MethodBlock=%-I (2) -4LOD IPOALAI (DUM Method Blxmk.MO-1 -4.OD MethodBlock=*-I (1) -4LOD IMetiodBlockum-1 (2) 4.OD System ReproducibilityChecks produdbw: sm2h 0198-2(2) o1lr0o9t9.2(2) 2098-1(2) 2698-1(2) PFOS (Dpb) 33 20 40 47 an 4.3% 3.6% 1.9% 3.0% s QCI QC2 QC3 Rios] recovend 99% 93% 90% VPOAA) .-.recovered 93% 87% 82% POAA (area) 28W 0.0% 13M 0 .0% 1400 0.0% 2300. 6.1% 'R*portovdalumwvfivm a iftleawlysb mdow a MA dev.isbuffeated. indon cam. 2 alliquotsof somple wo a amal>wd od evoogd. 2QC =MpeS We c&ac& ofblankmm ipikedwithPPOS4:'OAA amdar& 'Repro&cibifitmymples arerepeatinleefiomoflotjorac*wta pwfonmd evoy 15 so**& KJ. Hansen Phne III 3M EwArannuntalLAb Quantitative Analysis of PFOS in Individual's Sera Determination of PFOS in sem Quandtadon: Limit of Detectkm (LOD) LimitofQwmdtwm OX)Q) Rmp ofCabUatkm Curve. CurveCorrelatioCnacffidwt(r@: by Electrospray Man Spectrometry (ESMS) specindly: 1.0ppb 1.0ppb Remdon Time: MolocularIon: 7.35min. 4992ml I.Oppb- lOOpIpb I ResultsofQwmtitation ofIndividuidSera Samples Awrw came. Iqt PFOS (opb) % 3M 198 58 410 299 41 14 399 46 2 498 32 NJL 598 67 0 6" 53 7 798 42 Nk 898 49 35* "B 39 11 1098 54 5. 1199 60 11 1298 37 1 1393 45 N.A. 1498 31 12 1599 96 2 1 1698 74 1 Lot 1798 1899 1"3 2099 2198 2299 2399 2498 2598 2698 2798 2899 2998 3098 3198 verw cow. PFOS (nob) 46 56 70 40 48 60 72 35 41 53 57 33 65 67 33 % RSD is 10 3 2 9 13 1 20 33* 11 15 Nk 7 50* 11 Nfi- - No VJW calcubod as that was onlymougbisamplefor1 =absil 4/9/98 FluorineAnalyticalChemistry Team Determination of PFOS in sera by Electrospray Mass Spectrometry (ESMS) Quintitation: LimitofDewdon (LOD): LimitofQuantitado(nLM Rwp ofCahlwationCurve: Specffldty: 1.0ppb RetentionTime: 1.0ppb MolecularIon: I.Oppb- 100ppb 7.35min. 499 amu CurveCorrelatioCn4cffwicn(tr): I PHASE IUL-Analysisof individualserasamples,1993 US Averageconc. Lot PFOS (npb) % RSD 198 59 41* 298 41 14 398 46 2 499 32 N.A. 598 67 0 698 53 7 798 42 N.A. 898 49 35* 998 39 11 1098 54 5 1198 60 11 1298 37 1 Aymp cone. L&L PFOS (pub) % RSD 1798 46 15 1898 56 10 1999 70 3 2098 40 2 2198 49 9 2298 60 13 2398 72 1 2499 35 20 2598 41 33* 2698 53 11 2793 57 15 2898 33 N.A. 1399 45 N.A. 1498 31 12 .1598 96 2 2998 65 7 3098 67 50* 3198 33 11 1698 74 1 N.A. = No 0/" mlmbod as them wu onlyewugh sampleforI anabsis- contga. 8-6018 3M Ew&onmnud IAb 5nl9g PHASEM 3M EnvironmentalLaboratory-FluorineAnalyticaClhemistry Team KrisHamn - Sr.AnalyticaClhemist Muorine AnalyticaClmmisuy Team Building2-3E-09 612-778-6018 Phaie IV: PFOS analysis of US human sera samples collected in the 1970s Slimmau: Twelve frozenserasamplesofvariousvolumeswem suppliedtotheEnvLro=cntal Lab by jeff Mandel (3M Medical).One mL orno more thanone halfofthevolume ofeachsamplewas extracted usingan ion-pairmgreagentlle ewacts wereanalyzedqmumvcly fi)PrFOS by EEPLC-ESHO. Analyteidentificatwiaosnvcdfwd by comparisonofmoleculairon-EPLC retentiofnim oftheexuacted analyteand standardmataw. Sampleswerequantitativelvyaluateadgainsat VwWUy pmpuid extractecdurve. The twelvesamplescontainedan averageconcenusfim of27 ppb PFOS =nzin from 2-48 ppb. Ile PFOS levelwsese sigmfi=* lowerthaninthe31 amples collectefdtom individualisno2/98and =bbited significantglmyatervariabiliitnyPFOS levelsthanthosesame samples.The percent abuddanceofthebranchedchainisomerwas 37.3+/-4.90/oc,omparedto33.7+/-70/ionthe1998 samples In standardsolutionosfPFOS, thebranchedchainaccountsfor21.7+/-3.1% ofthetotalconcmftadm A detailedsummary oftheresultissincludecl The identitoyfPFOS inseveralrandomlyselectesdamplesinthisphasewas Tumed using EPLC-Esmsms. In allcasestheanalyteoxbbiteda cbaracwasdcretentiofnim, a characteristic primaryion,and fivecharacteristsiecondaryions. Experimental=unwary: SonplepreparationI:on-palyinegxtraction Ina pH controllendvironmentan ion-pamngreagenttcuabutyalmmomum (IBA),isusedtD ==a theanalytferom thematnx Ilm cabowc reagentwlectn* targetasmomc compounds,likePFOS and POAA. SubsequenttotheformationoftheTBA-anion pair,theambu istransferretdoa non-polm organicsolven(tethywletaw).dmd. and reconstitutiendmcftwl forhG analysis. BPLC. CharacteristriectentioanmesforPFOS InBPLC, an aliquootfthecKft2aisinjecteadnd passeddumigh a column. Based on theaffiwtyoftheandyteforthestationary-phaisnethecolumn relativteotheliquidmobile- phasepassingthroughthecolumn,theanatytcisretainefdora chamuensticamount ofum. For ekample,ina standardsolutionP,FOS may clutoat9.1minutes.Patentiontimesbetweena standard PFOS solutioannd theanalyteodmcted from seraintlusanalysiwserematched towithin1% on the BPLC system. ESCDS. Detectionand monitoringofthemolecularion AnalysisofPFOS standardisndicatetsha theprimaryioncb=Mrisfic ofPFOS isn* - 499 amu, correspondingtothemaw offt miionicmufamnt (CO17S%-)- ThisionWas monitoredWICC&dY tomaximize sensitivitAy.scanofm/z-100 to1210 (negativoenly)was alsocohecte& HPLCEY-MSAWS. Detectionand monitoringofcharacteristsieccondaryions ES-MSMS isverysimilartoESMS, exceptthatitadds-anadditionadlimensionofcertainttyo compound identificatioAns.inESMS, achamcteristiiconisselectedA.fterselectiotnh,eES-MSMS chamcteritmheionfiutkbrysmsithiintaparwtithhighenau pL AsamWt oftheuusb*nit secondairoynifcragmen(tdsaughtieornsd)w,wtuisfiocfthemoleculaem,amaw anddcmcm& Forexamplef,orPFOS analysiiso,n499isselectaesdthecharacteripsrtiimcariyoilThisionis smashed intootherionssuchasgo -g-u(correspondintgoS031, 99 -u (correspondintgoFS%), 130 Amu (concvonding toCF2SO3), 180 i-mu (C2F4S%). and 230 amu (C@F&S%). Each ofthen m=ndary ftagmentsis&Aectcd atthedc=wr. Qualitycontrolmmmary: Due tosample lumtationss,erasampleswon notodmcted induplicateFor thissome remn, matrixspdm couldnotbe evaluatedforthesamples. Tinsteatdwo samplesof pooledserapurchasedftom Sigma were used forduplicatmeatrixspilmanalysis.hfcdmd Wanks wac analyzedbeforeand dber each sample toensurecompleteisolatioonfthe sample. A mid-lad qualitycontrolsample was analyzed afterthesixthserasample. The fourpowt extractedcurvewas analyzedbeforeand aft thesm3@pla. Quantitatioonf thesamples isbasedon the linea regmisionequationdaived hom theaverageofthetwo bracketingcurvm SpecifiQcC parameterswe availabloen theWoded resulttsalk butmmental specwwj: HPLC system HewlettPa&ard Series1100LiquidChromatogfifm,nh- Column:Kcystme BetasilCis 2 x 100 mm 5 pm particlseize Flow rate: 300 pumin. SolventA: 2.0mM Ammonhm Acetat SolventB: Methanol SolventGradient 45 to 90 O/oBin 9.50 mins. Hold at90 V*B for3.50mins. Remm to45 0/aliBn 1.50mins. Hold at45 O/oBfor3.5 injectiovnolume: 10 ;LL Injection/ssample:I EJechvspray nuissspectrometer Mcromass PlafformH API Mm Spectrometer MassLynx 2.1 Softwarr. Cone volu4m: -20v,-60v Mode: clecuospraynegative Sourcetempuature: 115 OC. Analy=r vacuum pre==: 0.000043mbar, 0.000079mbar Ions:369, 413, 499 Elecb-ode:cross-flow FluorineAnalytical Chemistry Team Samples Received: 3/6/99 Samples Extracted: 3/9/98 Samples Analyzed: 3/11-3/12/98 Analysisby: CalibratioCnurve Ransc 1?ofCaUbratim Curvc LOQALODI: PHASE IV: Analysis of historic samples, 197"Os Conc. of PFOS b+B40 P07-SE13 13 PG3-SEll 32 98SE774 30 98SE117 24 PG25 40 PGS 29 Sample IDD CRAH 0003 3"1-0089-01 3"14)044-01 Will0002 Do= 0001 80-52-0514-01 COUC. OfPFOS (pob) 48 15 39 2 44 25 Average(ppb) 28 Std.Dev. 9 %RSD 32 KJHmun 77"018 ESM Ippb- 49ppb O.M 1 ppb QC Results QC Check- CalibratiCohnadc (QCI) bo NW % Recovery 1000/0 105% 105% Branched Isomer Abundance Percentages % Abund-n of % Abundanceof Branched Isomers Branched Isomers Sample Pool SUndakd Nfaterial IndividualSsampled in 1998 3M CMW GrovePlantWorkers IndividualSsampled in 1970's ,PooledSem Samples le) 220/o 34% 45% 370/o 31% (St&DCV.) 3% 5% 4% 5% 5% n (Msmoled inpool) n/a 31 3 12 I -LOD/LOQ - LimitofDetwdon/Limit ofQuantitation 3M EnvironmentaIlab 5nlgs PHASE rV Pi@17090 phmrv FUS Is"& 3149101 hf$OH blmk 31 4nM gx&aow bmm - 3149M SWaaWbu@@blmk 3:115 me= blmk 3 49s,6 M@OH blmk 3149817 P074813 3149911 ?074813 3149119 P074813 31499211MOgbbak 3149921 P034811 31499= P034211 3149rM P034811 3149624 USCH blmk 3149M 9L4M"4 3149926 99SB774 3149W 99313"4 31 M*CH blmk 314grJg9MI17 3149= 9=117 314MI 98= 17 31 980117 31 Mo=bbok 31499M PWS 3149835 P025 3149M P025 314@ Us= bbmk 3149M Skd4-1.4" 3149639 3td4-1.4U 3149M Me= blmk 3149941 POS 314994 POS 3149M POS 3149944 POS 3149945 Me= blmk 3149946 GRABDO3 3149M GRAND03 3149M GRARD03 3149M Me= Wmk 31499" 80-01-0089-01 3149M W41-00041 3149LI28"1-008"1 3149953 M*OHbLmk 314995420-01-0""1 3149955 3"14044-01 3149Li68"14)044-01 3149M 30-41-004"1 31491= MOCH Wmk 3149959 S& 4-1.4LO ppb 3149M Me= bimk 3149Ul WiD 0002 3149= VIM OOM 3149M VIM 0002 3149864 WM OOM 31~ WM 0002 3149M MoM blwk 3149M DMWOMI 314ma DEUM 0001 49gd9 DEUM 0001 31.49lr7MOe= bimt 3149971 80-n45l4-01 3149M 80-524514-01 3149M 30-524514-01 3149974)@@bimk 3149675 kivOHblmk 3149976 B20 bimic-I 31498" B20 blmk.2 3149M M*ORbhwk 3149979 PoolsdL%wl 31 POOW sgw2 314MI MoOHWmk 3149M )M 19.9ppb 3149M MSD 19.9ppb 3l49U4 MoOHblmk 3149US )6WH bL=k 3149486 Exwootedb@ me 3149U? Ro@b..@Wmk bl-k blmk pfasippbMY04" OLDw. 3117 0.0 3490 CLO 34a 0.0 3$47 0.0 3756 ao 27470 14.0 27393 13.9 26140 13.2 13.7 0.3 3 36M 0.0 -S7153 32.2 So" 3" 3016 31J 31.7 0.4 1 3d26 0.0 $25M 29.3 30755 2L3 31951 29.0 2L9 0.6 2 3476 0.0 4"% 25.0 4S761 25.2 25.2 44= 24.3 24.9 0.4 2 32ZI 010 96619 3&4 94M 53.3 gum SC7 553 2 3241 0.0 79= 45.9 7d$41 443 3186 0.0 54= 3Q3 ism 30.9 54670 30.7 5380 30.2 su Q3 I 2907 mo 77394 44A 73104 42.0 1 45.1 4" 1.7 4 3458 0.0 2M7 I&I 27410 13.9 -27137 13.8 13.2 0.1 1 35" OLO 72475 41.6 713d3 4OL9 omi 70013 39.6 40.1 4&5 0.9 2 33si 0.0 7W2 453 35M ao 75M 1.8 7395 1.6 7= ii 7$D 1.8 73d2 IA 1.7 0.1 6 3w ao 75M 431 1 425 76"5 44.0 43.3 u 2 34M 0.0 41094 22.3 40551 22.0 4ON7 E.9 22.1 0.2 1 39= OLO m 0.0 4799 0.1 468 0.0 395 0.0 3"32 29.9 35" 313 30.5 1.0 3 4035 mo sow 31.6 to" 49-9 su 1.2 2 3729 0.0 3177 0.0 4019 0.0 4ID13 0.0 &N-(QC) QC "MM7 (%) .6 94 .0 91 .7 n %dw. ao -3.0 spft @vwy(%) 10&0 97.0 7@-- Andy-d Chow-y TSANwirowunwl,ab Eb@ 778-Ml 3M EnvironmentaLlaboratoryF-luorinAenalyticCahlemishNTeani KrisHansen-Sr.AnalyticCahlemist FluorineAnalyticalChemistryTeam Building2-3E-09 612-778-6018 Phase V and VI: QuantitativeanalysisofPFOS inhistorihcuman serasamples and inhuman serasamples from ruralNorthern China ,Rilinmary: Two setsof samples,thefim with 12 sampla inplastviical(sZ-seatn)dthesecondwith5 samplesinglassvials(M-M), wm suppliedtotheEwAmnmental Lab by JeffU@mM (3M Mefficala)n March 20, 1998. For bothsok samplevolunkevariedfmm 0.2to1.0niLofhun"n =2 A volume between0.4- 1.0mL ofeachsamplewas exuactedusingan ion-oft reapm Sixunmi inthe7,.sa didnotcontainenoughvolumefororbacdm intheseca twosampla (e,&ZZA6 andZZAS) wac combinedand cxuactedasone=moo. lU cwactswereanalyzeqduanutatn* for - mffonaw(PFOS)by high-pmmm liquidMIUDgWby-de=oWq mm Vwuomefty (EEFW-ESM. Anabu idenflficatwiaosnvafflobdy ofnwlecwu ion-EPLCretmdondm oftheextracted analyteand standardmatuw. Sampleswerequantitativelvyaluateadgainsta speciallpyrqpare4frm@ pointextractecdam (zA2- 0.999).Eachsamplecc= wasanalyzetdwim Calibraticolnimb analyzedevery5 samples,wem within6% ofexpectedvaluel The identitoyfPFOS inseverarlandomlyselectesdamplesinUm phase(M-M only)was confirmedusing13PLC-ESMSNO. In allcasestheanabu exhibiteadcharacterisztaitcentiotnime,a characterisptriicmaryion,andfivedmderistic secondariyons. Ristopiscamples Ile sem samplesintheM-set,collectferdom donorsintheUS inthe1960s,had an average PFOS concentrationf33.4ppb;thesamplesrangedinIPFOSconcentratifornom Il..tt5o9.4. Foreignsamples ne 9 samplesintheZ-set(includin3gcompositesamples)wen couacwd f3romdonorsinrural China and weredeterminedtohavea much lowerconcentratiofnPFOS thanany him2n seradataset analyzedpreviouslyS.ixsamplesdidnotcontainPFOS abovethe &Awdon limitofthisanalytical method(Ippb).M@e remaining3 samplesevidencePdFOS concenuationlseathan5 pyb,thelimitof quantiwon. With thewwmdon ofwe human =a samplecollectiendthe1980s(Will0002),thenm theonlybi,m-nsamplesdeterminetdocontailnessthan10ppbPFOS. Experimentalsummarr. SwnplepreparafioIno:n-paipinegxtraction Ina pH controllewdamnment.an ion-pdnngreagentwtrabutyalmmonium adfiftCMA), is 'usedtoexU= theanabu fromthematrixAmomc compoun(klikethecauomc=agent selwuvely targetPsFOS andpeawrow=oate (POAA). SubsequenttotheformatioonftheIBA-anionpair,the anabu is=Ld@ffed toa non-polaorrganicsolven(tethyalcetated)n,e4 andn=nstium:d inmethanol forMS analysis. HPLC CharacteristriectentiohnmesforPFos InBPLC, an aliquootftheextracitsinjecteadndpassedthrougha chromatographiccolumm Based on theafrimtyoftheanwyteforthestationary-phiansethecolumnrelativteotheliquimdobil-.Phase Passingthroughthecolumn,theanalytiesretainefdora characterisatmiocunt oftime.For example,ina standardsolutionP,FOS may cluteat10.5minutel Rdentiontin= betweena standard 05/07/98 PFOS solutionand theanalyteextractedfrom serainthisanalysiwsere niatchadtowithin 1% on the HPLC system. ESlAa. Detection and monitoring of the mokcular ion Analysis of PFOS standards indicatesthatthe primary ion characteristicof PFOS is m/z - 499 ami@ corresponding tothe mass of the anionicmufactant(CsFl7SO3-).This ion was monitored selectively to maximize sensitivityA. scan ofm/z-100 to 1210 (negativeonly)was alsocobected. HPLC ES-MSIMS. Detection and monitoring ofcharacteristic secondary ions ES-MSMS is very sitnila to ESMS, except that itadds an additional dimension ofcmiaintyto compound identificationA.s inESNM, a characteristiocnissdacte& Afterselectiont,he ES-MSNE charactchm the ionfurtherby smashing itapartwith high-emgy ga& As a resultof the smashin& secondary ionic fragments (daughter ions),characwmc of the molecule, am created and deUM& For example, for PFOS analysis,ion 499 isselectedas the characteristicprimary ion. lliis ion is smashed into other ions such as 80 -gmu (convsponding to S03), 99 stirn(ucorresponding to Fsoi), 130 amu (coffevonding to CF2SO3), 180 qmu (C,2F4SI%), and 230 amu (C3F&%%). Each afthm secondary ftagments isdetected at the doADoor. Quality control summary: Due to sample hunt3uomL% sera samples were not exuacted in duplicate,with I exception. For this same reason, matrix spikes could not be evaluated for the samples. Each sample exuad was analyzed in duplicate. One sample, M001278 contained enough material for two exuacdm& Tk presented rmft for thissample are based on duplicate analysis of each oftbe two extractions. NL-thanol blanks were analyzed penodicauy to ensure complete isolationof the sample. A nud-level (24 ppb) quality control lewas analyzed every 5 samplel Because human sera without PFOS is not available,sampim of sem purchased from Sigma were pre-cKuact4 spflmd with a standard concentration ofPFOS, and n-carwted. This pm-odmcted sera isunderstood to be very similn to the sample matrix This fin pomt extracted curve was analyzed before and afterthe samples. QuantLtation of the samples isbased on tborinea mgmssion equation derived from the average of the two bracketing curves (r"2 0.999). Specific QC parameters uc available on the appended resultstable LimitofQuantitation 7U current analyticalmethod has be= used to evaluate a large range ofFFOS levels odmcted fim sem ft method isnot optimized for pr=*SC low-lcvd (< 5 ppb) quantitaflon,but instcaifor samples in the range of 20-60 ppb. Cnven the conccm=ons chosen for Ons optimization of the standard mm, while them is a sl-itmcal ddkrt= for samples evaluated from 5-100 ppb, them is no sudLsUW difference between samples quantitated from 1-5 ppb. The quantitationdoes not become MdWcany defendable unth 5 ppb; thus thisvalue represents the limit ofquantitadon for this method. Samples designated <LOQ am estimated to contain 1-5 ppb PFOS. Nfurther accuracy and precision in quantitatton of the low-kvcl Z-4 samples (samples lessthan 5 ppb) isrequired, a sp=W low-level quantitation system will be required Limit ofdetection I'helimitofdetectiofnorthisanalyticmaelthodisbasedon analysrtecognitioonfthedistinct peaksbapereswungfromPFOS aiwygs. PFOS =dud mataialand PFOS exuactodfrom scm analyzed by the conditions reported hem, show new basdin resolutionof I brandied and I 'in= PFOS isomer. lle apex of each peak in the BPLC-U= o=im ata spedfic, reproducible retention fim Ew the ratioof the branched peak to the linm peak ishtwy consisftt down to the low s=dard. 1.0 ppb. Thm samples that a) had PFOS levelsevaluated at lessthan the limitof quantitationand b) evidenced the distinctpeak shape, were designated < LOQ. Samples that a) have PFOS levels evaluated at less than the limit of quantitation and that b) do not reflectthe unique PFOS peak shape, were designated < LOD. The= samples are estituted to contain from 0-1 ppb PFOS. 05/07/98 2 Instnunestalspecirwi: HPLC system Hcwkn Padeard Series1100 Liquid Column:Kcimne Betod Cis 2 x 100 mm 5 limpardclesize Flow rate: 300 gLAnin. SolventA: 2.0mM Ammonium Acetm SolventB: Nlethanol SolventGradient 45 to 90 %B in9.50minl Hold at90 %B for3.50minl Returntik45%B in1.50minl BDid at45 %B for3.5minl injecdmvob=: 10 ILL Injecdm / nm&: 2 meefts'prayM= SWcftmeter bficromm Pb&rm I[A[M ItfawSpwftomam, "CMW M&%sLy= 2.4Software Cone volbigm:--20v;-6ft Mode: clocamprayneptm Source tmnperature: 115 OC. Analrw va=m pmmm: 0.000043mbar, o.oooo7gmbor Ions: 369,413, 499,427 Electrode:cmss-fiow 05/07/98 3 FluorineAnalytical Chemistry Team XJHwmn 77"018 Samples Received: Samples Extracted: Samples Analyzed: 3/6/99 3/9/98 3/11- 3/12/98 Analysisby: CalibrationCurve Range: ?of CalibratioCnurve: LODL.- LOQ2: ESMS ippb.-49ppb 0.998 I pi)b 5 pyb - PHASE V and VI: Analysisofforeign(ruralNorthern China) and historiscerasamples(1960) Conc. ofPFOS Sample M ZZAI <DD ZZA2 <DD ZZA3 ZZA4 <IOQ ZZA5 <M ZZA6 and & <,OD ZZA7 <,OD ZZA9 and 10 ZZAL I and 12 <.OQ <,OD Smple IID mDO1205 M001278 M01202 NMMI MD01208 Cw- ofPFOS 0 24.4+/-0.4 48.4+/-1.5 23.2+1-0.2 11.8+/-O.l 59.4+/-O.l QC Results QC ChecILCahbmtionCheck (QC I) CaMradon Check(QC2) % Recovery 1040/o 1060/o Branched Isomer Abundance Smple Pool StandardNiaterial WMduals Sampledin1998 3M CoftapGrovePlantWorkers Individual@sa@ed in1970's ,PooledSeraSamples Percentages % Abundanceof BranchedIsomers (Averaw) 220/o 34% 45% 370/9 31% % Abundanceof BranchedLomen (StdD.ev.) 3% 5% 4% 5% 5% I -LOD = LimitofDet=don 2-LOQ - LimitofQuantitation a sampledInpool) nia 31 3 12 11 3M EnvironmentalLab 5nl9g PHASE V &W VI Quantify COMPOUOD SU@CY ASPOrt P"e \WJXLYM 3 M\ 9 Last mo"tied: sun mar 22 14:33:39 It" Method: e: \.USLTNX\PRUBB. M\MTMCB\PM Last mo"fiod: &at Mae 21 19:19!42 1996 saw a "St@ C: ","J,, O"M"\012" 9 Job Code: Printed: Sun Mar 22 14:52:37 1998 Camqxnind 1: IPMS t N@ 1 03219801 2 03219402 Type AnalYto Anslyto 3 03219803 4 03219804 5 03219005 Analyto Standard Standard 6 03219806 Standard 7 03219807 S 03219006 9 03219809 Standard Standard Standard 10 03219810 Analyto 11 03219011 Analyto 12 03219812 Analyto 13 03219013 Analyto 14 03219014 Analyto 15 03219815 Analyto 16 03219816 Anglyto 17 03219827 Analyto If 03219011 Analyto 19 03219019 AnslYto 20 03219820 Analyto 21 03219821 Anslyto 22 03219822 Amlyto 23 03219923 OC 24 03219824 Malyt* 25 03219025 Analyto 26 03219826 Analyto 27 03229927 Analyto 28 03219826 Analyto 2: 0321::2: A-alyto Analyt. 3 0321 3 31 03219031 AnalYte 32 03219632 Analytt 33 03219633 Analyto 3: 0321::3: Analyte 3 0321 3 36 03219836 AoCnalyto 37 03219837 Analyt* 38 03219838 Analyto 39 03219839 Analyto 40 03219440 Analyto 41 03219041 Analyto 42 03219$42 Analyto 43 03219043 Analyto 44 03219144 Analyto 45 03219845 Analyto 46 03219846 Analyto 7 0321::47 AAnnaallyyttoo :8 0321 49 49 03219849 Analyto 30 03219850 Analyto 51 03219851 Standard 52 03219052 Standard 33 03219033 Standard 54 03219054 Standard 55 03219655 Standard 56 03219056 Standard ample Text NoW Blank U20 blank-I Sera blank-I 0.996 PPb re mi 4.90 PPb FC Mix 24.8 ppb FC Mix 49.0 ppb FC Nix 96.2 ppb FC Nix 142 ppb TC Mix M*(M Blank M001203 M001205 12) N001218-1 M001278-1 (2) M001278-2 M001270-2 (2) M01202 U01202 (2) M001221 U001221 (2)"@-' W001208 U001200 (2) Cal check, 24.4 ppb NoW blank ZZAI ZZAI (2) ZZA2 ZZA2 9TA3 IZ13 (2) J21 ZZM ZZR4 42) ZZIL5 ZZAS (2) Cal check, 24.0 ppb MGM ZZAS ZZh6 and 1 and 1*(2) ZZR'Y ZZA7 (2) ZIA9 and 20 ZZA9 and 10 (2) ZZRll and 12 ZZAII and 12 (21 AZ26-1 AZ26-1 (2) NoW blank K*OR blank 320 blank-I Sera blank-I 0.996 ppb IC Mix 4.90 ppb YC Kix 24.8 ppb TC Xix 49.0 ppb TE Nix 96.2 ppb FC Mix 142 ppb FC Xix IPFW PMS As" 4292 3239 2913 sots 10464 41137 74118 142*7$7 193769 3257 39673 380" 74992 79499 71794 72374 37934 37494 21224 23.018 $0087 $9709 41304 2021 3310 3397 3277 3300 5598 $721 7108 7038 3046 3340 41633 2900 4883 5071 4181 4207 6631 #4$0 5342 5262 44210 44259 2803 2744 3397 3223 Sall 10482 40948 75059 243153 203166 com. 0.26 0.00 0.00 0.76 4.53 25.72 49.51 95.94 131.18 0.00 24.71 24.15 49.11 50.16 46.90 47.45 23.32 23.20 11.94 11.02 St.S4 39.34 2S.94 0.00 0.00 0.00 0.00 0.40 1.17 1.25 2.21 2.17 0.80 0.99 25.20 0.00 0.67 0.80 0.19 0.20 1.08 1.78 0.99 0.93 27.25 27.18 0.00 0.00 0.00 0.00 0.81 4.54 25.39 49.16 96.21 137.66 wev news bb bb m -23 M -8 M 4 mm -1 M 0m -8 NU bb m NK m PK m mm m m mm m m m 4m bb m m mm m m xx mm m m mm 6 34K bb m m m Nk mm m m m NK mm mm m bb w -19 m -7 M 3M 0m 0m -3 NNX t Quantity ciaLbiratLon Report calibration, L"t =&tied: ftirttod* Ct\oo&SZMM\PNAUS.M\CURV=S\pm am bar 22 14:49..38 logo fta mr 22 14:52:38 It" @on-"undIname:PFOS Corretafiocnoefficienrt-: 0.999912,rl\2- 0.999823 ,Calibraflcounrve:1447.275720 0 x + 3910.409399 Responsetype:ExternalStd,Area lcurvetype:Linear,Origin:Exclude,Weighting:Null,Axis tms: None 2.O9e5- ftes I Response- x r 0- one' 3M EnvironmentalLaboratory-FluorineAnalyticaClhemistry Teani KrisHansen -Sr.AnalyticaClhemist FluorineAnalyticaClhemistryTeam Building2-3E-09 612-778-6018 ]?hueV and VI: QuantitativaenalysiosfPFOS inhistorihcuman serasamples and inhuman serasamplesfrom ruralNorthernChina Sunnuary: Two setsofsamples,thefLm with 12 mVIes inplastivcials(Z,.M)and thesecondwith5 samplesin glassvWs (M-ed),were affhad tothe rwmenftlLabby JeffMandel (3M h4adimo on March 20,1998. ForbothsM umple volumevariedfrom0.2to1.0mL ofhum2" am A volume between0.4- 1.0mL ofciaisamplewas extracteudsingan ion@ ream= Sixamples intheZeed didnotcontainenoughvohm foradracdon;indme cum twosampla (e,&ZZA6 and ZZAS) vim fm binedandckuactedwoneunq&. Tk odmcts wereanaly2Wquandbdvdy forpeifilinnnff,@tand mffomw (PFOS)by hghvemm hqtud mmtogWby-clec@@ mm Vocametry (EPLC-P-SM. Anabft identificatiwoans vatud by comparisonofmoleadu ion-HPLC retentiodnm oftheomacted ambft and standardMatM2' Smpla wm quammv* evguftd aga= a Vmd@y pqmmd, frv- pointumctw curve(irA-20."9). Each sampleacum wasanab-adtwice.Cahimadonchecks anabmed evray5 samplm werewithin6% ofeq)ecievdaluel The ulentitoyfPFOS inwvmal randomlyseladedmupla inthisphase(M-M only)was confmned usingEPLC-ES&MO. Inallca theanalyteexhfl)itaecdharacteristriectentiotnime, characteristpircimaryion,and Bm r wns. historicsamples Tli.-serasamples in theM-sek collectefdmm donm intheUS in ft 1960s,bad an average PFOS concentratioonf33.4ppb;thesamplesrangedinPFOS concentratiofnrom II..tto59.4. Foreign samples The 9 samplesintheZ-W (indmung 3 =nposite samples)were coU=W from donorsinrural China and were determinedtohave a much lowerconcentratioonfPFOS thanany hlim2n seradataset anab7,edpreviouslySixamples didnotcontainPFOS abovethedftmon lumt ofdus analytical method (1pi)b)T.he remainin3g sampla awenced PFOS ednm=dm I= than5 ppk thelimitof quantitagoilWith thecwmdon ofoneInim2nma samplecollecteidndw 1980s(Will0002),tlmm an theonlyhil-2nsampla determinedtocontainI= than10ppb PFOS. entalxununary.- S@vnplepreparationI:on-pairinegxtracdon In a pH Controlle=dvironmn4 an ion-pdringrogmt =rabutylammonium mdfat(eMA), is used tocxum theanabu from thematnx Amomc conip=W Wre thecationirceagent,wl=dvely targetPsFOS and pefflwrooc=oate(POAA). SW=quent tothefimnationofthe7BA-mon pai4the anabu isMmsfeffedtoa non-polarorganicsoh=t (ethyalmtm), dn4 andn=nmuftd inmdmd forNM analysis. HPLC. Characterisdrcetenhonhmesfor PFOS @ InBPLC, an aliqwtoftheext= isinjecteadndpassedthrougha chromatographiccolumil Based on theafftnitoyftheanalyteforthestationary-phaiwnthecolumn mWM totheliquidmobile. phasepassingthroughthecolumn.thea=lyw isretainefdora characterisatmiocunt ofdm For example,ina standardsolutionP,FOS may cluteat10.5inini . Reftntiontimesbetweena standard 05/07/98 PFOS solutionand the analyteextractedfrom serainthisanalysiswere matched towithin 1% on the BPLC systeal E&MS. Detection and monitoring of the molecular ion Analysis of PFOS standards indicates thattheprimaryioncharacteristoifcPFOS isM/z - 4" amu .,correspondintgothemassoftheanioniscurfacmt(CIF17SO3-).Thisionwas monitoredselecdvely toma)e@ sensitivitAy.scanofm/z-100 to 1210(negativoenly)was alsocouacte& HPLC F.S-MSIMS.-Dewdon and monitoringofcharacteristsiecondwy ions ES-MSMS isverysimOI2toESNC, exceptthatitad& an additionadlimensionofcutainty*to compound identificatiAosn.inESW, a characterisitoincisselectedA.fterselectiont.heES-B(SM characterimtheionfurthebry smashingitapartwithhigh-c=U ga& As a resulotf thesmashing secondaryionicfragments(daughterions)c,harwm= ofthemolowle,arecreatedand debwte& For example,forPFOS a=bi* ion499 isselecteadsthecharacterisptriicmaryim Thisionis mnnr.lheidntootherionsa# n 80 jimu(correspon&mtoS%I, 99 amu (COIJL 22--tDFS03 130 amu (correspondintog CF2SO3), 130 -Qmu (C2F4SO3), and 230 amu (C3F&S%). Each ofthesesecondary ftpients isdcwctedattheddector. QuWft controlmmmary: Due tosamplelimitationsse,rasampleswerem extracteidnduplicatew,ithI exccpdm For this== rema, matrixsp&w couldnotbe evaluatefdorthesamplesE.ach sampleam= was amb=d induplicateO.ne sample,MOO 1278 containedenough matmialfortwo exuacdon& Mm presentedresults forthissample arebasedon duplicataenalysiosfeachofthetwo extractionsN.(ethanolblanks wei analyzedperiodicalltyoensurecompleteisolatioonfthesample.A nud-lcvd(24ppb) quaht3rcontrol sample was anabrzodcvciy5 sampk& BecauseIhilim2snerawithoutPFOS isnotavailables,amplespf pooledserapurchasedf3romSigma wen pre-exbwted,sp&od witha sumdard concentratioonfPFOS, iimd re-mmacte& Thisprt-uuactedseraisunderstoodtobe vay simil-t4othesamplematrix Thisfmc pointcxbww curvewas analyzedbeforeand afterthesamples.Quanti=on ofthesamplesisbasedon therinea regressioenquationderivedfrom theaverageofthetwobracketingcurves(rA2*='0.999). SpecificQC parametersareavailabloen theappended resulttsable. Limitofquantitafion The currentanalyticamlethod hasbe= usedtD evaluatea bW mp'of PFOS levelsextracted from wxa; themethod isnotopdndzed forpre*= low-kvd (< 5 ppb)quantitadon,butinsteadfor samplesintherange of20-60ppb. Given theconcentrationcshosenfx)trhisoptimizatioonfthe sbmdard curve,whilethem isa statistidciaflfcrc= fDrsamplesevaluatefdimm 5-100ppb,then isno statistical diffm= betweensamplesquanbuttedfrom 1-5ppb. The quantitatidoonesnotbecome MdWcaBy defendab]until5 ppb;thusthisvaluerepreantsthelimitofqwmtitation:btrhismethodl -qnmpks desizo2ted<LOQ areestimatedtocontain1-5ppb PFOS. Nfiutha accuracyand precisionin tatioonfthelow-kvclZ,-sasamples(sampleslea than5 ppb)isrequireda,specialow-level quantitatiosnystemwillbe reqmrc& Limitofdetection I'hehaut ofdetectiofnordus analyficamlethm isblsedon arwystrecogiiitioofnthedisd peak shaperesultinfg3romPFOS angyns. PFOS standardm2tenn'and PFOS exuactodfrom sera analyzedby theconditionsreportedhere,show nearbaselinreesolutioonf Ibranched and I lines PFOS isomer Ile apex ofeach peak intheMC-u= o=us ata specifirce,prod=ble rdendon dm oW & ratioofthebranchedpeak tothe'*no peak ishirlyconsistendtown tothelow smda4 1.0ppb.Those samplesthata) had PFOS levelesvaluatedatI= thanthelimitofquantitatiaond b) evidencedthe distincpteak shape,were designated< LOQ. Samples,thata)havePFOS ImU evaluatedatlessthan the limitofquantitatioannd thatb)do not reflecttheuniquePFOS peak shape,were designated< LOD. These samplesareestimatedtocontainfrom 0-1 ppb PFOS. 05/07/98 2 instmmentaispecirwc BPLC system Howlen Padcard Series1100 Liquid Column:Krimm Betug Cis 2 x 100 mm 5 pm pardde size Flow rate: 300 gumin. SolventA: 2.0mM Ammonium Acmo SolventB: Methanol SolventGradient 45 to 90 O/oBin9.50minl Ekid at90 %B for3.50minl ReturntD_45%B in 1.50minl EUd aC4S %B for3.5minl injadm vobm. 10AL Injecdm /sanvk:2 F-Jecbwpaymm specftmtzr bfi=mass Platform]IAPI Man SpecUcmeW, -ChicL- MassLynx 2.4 Software Cone voltalp:.-20v-;60v Mode: clecaospmynegatm source 115 OC Aulpw vacuum press=: 0.000043mbar,0.000079mbor Ions: 369,413,499,427 Electrode:cros*41ow 05/07/98 3 FluorineAnalytical Chemistry Team r@JHanwn Samples Received: 3/6/99 Samples Extracted: 3/9/98 Samples Analyzed: 3/11-3/12/98 Analysisbr. CalibrathmCurve Range: 1?ofCalibrationCor-m LOD': LOQ2: ESM lppb- 49ppb O."s 1 plpb 5 ppb PHASE V and VI: Analysisofforeign(ruralNorthern China) and historicserasamples (1960) Conc. ofPFOS Sample ED ZZA6 ZZA2 ZZA3 27A4 ZZAS and 8 <DD <,OD <LOQ <A)Q <,OD <,OD ZZA7 <A)D ZZA9 and 10 ZZAII and 12 <A)Q <.OD Smple ED mOO1205 IMDMO1270 127085 bo 2I12 COW. ofPFOS (pob) 24.4+/-0.4 48.4+/-I.S 23.2+/-0.2 lh=1203 59.4+1-0.1 QC Results QC ChedLCalibratioCnhwk (QCl) CaM=tkm Cho* (QC2) % BwAn" 104'Yo 106% Bmnched Isomer Abundance Samle Pad SUndard Matmial hdhidualsSampled in 1999 3M C4op Gwn PlantWorken IndividukSampM in1970's 1POoledSera Samples Percentages % Abmdazm of % Abmdana of BrancbW Immm Brawbed Immm (Averap) (St4DLev.) 22% 3% 340/o 5% 45% 4% 37'Yo 5% 1 31% 1 5% 1 a (0@obd Inpoold nla 31 3 12 I -LOD - LimitofDewdon 2-LOQ - LimitofQuantitation 3M EwAmnmentd lAb 5nl" PEULSE V Ed VI Quantity ConvowA 9@ry FAmpart Sasple List: Lost modified: moth",. Last modified: Job Co": C: Sun Mar 22 14.33:59 lose C: %whosLymx\pnuu. nw\mms\pm Set War 21 19:19:42 1998 Printed: Sun Mae 22 14:52:37 Ito$ Compound 1: PM 0 m@ TY" 1 03219801 Analyto 2 03219002 An&lyto 3 03219403 Analyto 3219004 Standard 4s 00321980S Standard 0 03219806 Standard 7 03219807 Standard 8 03219608 Standard 9 03219809 Standard 10 03219810 Amlyto 11 03219811 Malyto 12 03219012 Malyto 13 03219913 Analyt* 14 03219914 Analyto 15 03219813 Analyt* 16 03219816 Analyto 17 03219917 Analyto 18 03219818 Analyto 19 03219919 Anslyto 20 03219620 Analyto 21 03219021 Analyto 22 03219822 Amlyto 23 :321:923 Qe 24 321 024 Analyto 23 03219825 Analyto 26 03219826 Auslyto 27 03219427 An&lyto 28 03215120 Analyto 29 03219829 Analyto 30 03219430 Analyto 31 03219431 Analyto 32 03219832 Analyto 33 03219833 Anslyto 34 03219034 Analyto 31 03219835 OC 36 03219036 Analyto 37 03219837 Analyto 38 03219834 Analyto 39 03219839 Analyto 40 03219040 Analyto 41 03219841 Analyt* 42 03219042 Analyt* 43 03219943 Analyte 44 03219944 Analyto 45 03219845 Analyto 46 03219940 Analyto 47 03219847 Analyto 1 03219840 Analyto :9 03219049 Analyto 50 03219050 Analyto 51 03219851 Standard 52 03219132 Standard S3 03219853 Standard 5345 0321:::: Standard 0321 Standard 36 03219956 Standard Semple Tmt N*M blank H20 blank-I Set& blank-I 0.996 ppb FC xix 4.90 ppb fC Kix 24.1 ppb fC lUx 49.0 ppb IC KLm %.2 ppb FC kix 142 ppb rC Mix MOCK Blank M001203 MOO1205 (2) MOO1278-1 U001278-1 121 MOO2271-2 =01271-2 (2) U01202 *01202 (2) HOO1221 HOO1221 (2j,@, W0120t "-' =41206 121 Caa cbeek, 24.9 ppb PIM no= blank ZZRI ZLU t2) ZZA2 IZA2 (2) --A' ZZA3 (2) ZZh4 Z7A4 (2) ZZAS ZZA3 (2) C" check, 24*$ ppb PrW NoW ZZAG =4 9 ZZKG and $-(2) Z7A7 ZL%7 (2) ZZRS and 10 &Zht and 10 (2) ZZLII and 12 ZZPUI wW 12 (2) AZ26-1 AZ26-1 (2) HOOK blank No= blank 22o blank-I Sera blazLk-I 0.996 ppb ]rCKix 4.90 ppb fC Kiz 24.8 ppb PC KLx 49.0 ppb IC Kix 26.2 ppb FC KLx 142 ppb rC KU Area 4252 3239 2963 5013 104" 41137 74119 142707 193746 3287 39973 30848 74992 764" 71794 72378 37934 37494 21224 itoll 9M7 99709 41304 2221 3310 3397 3277 3300 3396 5721 7104 7054 soff 3340 4L$33 2900 4843 3072 4101 4207 9931 6488 S342 5241 44210 44250 2803 2744 3397 3223 sogi 10482 40946 7SOSS 143233 203144 cona. umv nave 0.29 bb 0.0 bb 0.00 m 0.76 -23 M 4.53 -8 NK 23.72 4m 46.31 -1 M 93.96 0m 131.18 -9 MM 0.00 bb 24.71 mm 24.15 m 49.11 m so.is m 49.90 m 47.43 NK 23.31 m 23.20 m 11.96 m 11.92 m 39.54 m St.34 m 25.64 4 m C." bb 0.00 m 0.00 m 0.00 m 0." m 1.17 m 1.23 m 2.21 m 2.17 m 0.80 m 0. m 24.20 4 m 0.00 bb 0.67 m 0.80 m 0.19 m 0.20 m 1.08 m 1.79 m 0." m 0.93 m 27.85 m 27.88 m 0.00 m 0.00 m 0.00 bb 0.00 m 0.81 -19 M 4.34 -7 M 23.39 3 M 49.10 0 m 96.21 0 M 137. -3 OW - 1444% guatley calLbv*LLOO ammm emalbntlent Lost ao"fiods ftletod: fte Nw 22 14.49-.S@ 1999 am "W 22 14:32:301"9 ConipounIdnum: PFOS CorreWon coefficienrt- 0.999912,rft2- 0.999823 Calibradoncurve:1447-2757200 x + 3910.409399 ResponserM: ExUmal Std,Area Curve rM: Linew,Origin:Exclude,WeiShting:Null,Axis tram:None 2.O9c5- Responw- x x c 3M EnvironmentalLaboratory-FluorineAnalyticaClhemistry Team KrisHamn -Sr.ArWydcW Chemist FluorineAnalyticalChemistryTeam Building2-3E-09 612-778-6018 Phase VIOI: US Geographical Distribution Study - PFOS levels in human sera Slimmm: Fifty-fivseamples ofcoldhuman serain pwdc cenbiffigueiba were suppliedtothe EnvironmentalLab by JeffMandd (3M Mefficalo)n Mamh 19 and Mu& 20, 1998. The samplm collectefdrom sitesaroundtheUnitedSLite4containedsevew mTs ofsa2 One mL ahquob ofsua viae renwved from each sampleand ekbuted vnthan ion-pamg ragod, nearlyallofft sampw were pir-a@'- induplicatel.U ftm ckuam wem analyzedquanumvely for mlfmft (PFOS)by highprasm liquiddmmatogWby-el@@ == Vecftmetry(HPLC-ESW). AnW* identificaflwoans verifiebdy comparisonofmolecularion-EPLC =tendon dm oftheacuictedanabu and standardmaterial.Samplesvm qmntltum* evaluatedapum a spemauy prepar4 five-pomt muzcted curve(rA2= 0.996).A matrixspikecahlrationcheck,analyzedinthemiddle oftheanalysis sequence,was within1% of expectedvalues. The sem samples in thegeographicdistributiosntudyhad an averagePFOS odwmtmtion of 28 ppb; the samplesranged in PFOS conmnbadon from 9 ppb to59 ppb. The percentabundance ofthe branched chainisomer was 41 +/-40/e.In standardsolutionosfPFOS, thebranched chainaccountsfor 21.7+/-3.1%ofthetotalconcenuadorl A detaileodnimaiy oftheresultissattached The identitoyfPFOS inseveralrandomly selectesdamplesinthisphase W-M o*) was confmned usingHPLC-ESMSNO. In allca theanalyteodubited a cbaractmuticretentionhuac.a, characteristpircimaryion,and fivecharacteristsieccondaryions. Experimental summary. SamplepreparaftonI:on-pairingixtraction Ina pH controlleednvironmentan ion-panng reagent=,abutyl ammonium sulfateCIBA), is usedtocxvw theanalytefrom thematrix Anionicconipminds,Mm PFOS and peawrooc=oate (FoAA), arewiwtn* targetebdy thecatiomcreagent subwquw totheformatlonoftheTBA-emon pair,thenabft istransferretdoa non-polaorrganicsolvent(ethyalce=), dn4 and m=Wbdcd in methanol forIHPLC-ESKA analysis. HPLC CharacterisheretentionhmesforPFOS In EPLC, an aliquotof theextractisinjectedand passedthrougha column. Based on the affinitoyf theanalyteforthestationary-phasienthe column relativteo the liquidmobile- phase passingthrough thecolumn, the analyteis retainedfora characteristaimcount of fim. IFOR example,ina standardsolutionP,FOS may aluteat 10.5minutes.Retentiond= between a standard PFOS solutionand the analyteextractefdrom serain thisanalysiswere niawliedto within 1% on the EPLC system ESMS. Detectionand monitoringofthemokcular ion Analysisof PFOS standardsindicatetshattheprimaryion characteristoifcPFOS ism/z - 499 amt% correspondingtothe mass of theanionicnufactant(CsFl7SO3-). This ionwas monitored selwdvcly tomaximize sensitivitAy.scanofm/z-100 to1210 (negativoenly)was alsocollected. 05/07/98 1 HPLC ES-M&MS.-Detwdonandmonitorionfcgharactersiesctoincdairoyns ES-MSMS isverysimiltaorESMS,exceptthaittaddsanadditiodniamlensiofncertaitnoty compound identificatioAns. in ESMS, a characteristiiocnisselectedA.fterselectiont,he ES-MSMS characterizetsheionfurtherby smashing itapartwith high-energygas. As a resultof the-rm24thing secondaryionicfragments(daughterions),characteristoifcthemolecule,arecreatedand deuztc& For example,fbrPFOS analysisi,on499 isselectedas thecharacteristpircimary ion. 1-nision is smashed intootherionssuch as 80 amu (correspondintgo S03), 99 amu (correspondingto FS03), 130 amu (correspondingto CF2SO@), 190 amu (U4SO3), and 230 2mu (C3F6SO31. Each of theseseoon&ry fiagments isdetectedatthedewmr. Qualitycontrolmmmary: In most cases theserasampleswere extractedin duplicatef;orthosesamples thatwere,standard deviationvaluesareincludedinthetableofr=WtL Nb= spdm werepreparedforseveralsampla too. Ifavalable,thematrixspikerecoverieasreincludedintheresulttsable.A nud-kvel (49ppb)quality controlsample was analyzed,rwmry forthematrixspikewas &-Uwmmed tD be 1010/e. Be== h-man sera-%ithouPtFOS isnota@@@le, samplesofpoolednm purchasedfrom Sigma were pro-exuwtmt spikedwitha standardconcentratioonfPFOS, and re-c@ Thispre-axu=W seraisunderstoodto be veryc*mila tothesamplematrix Thisfivepointexuactedcurvewas analyzedbeforemW dta the samples. Quantitatioonfthesamplesisbasedon thelinm regressioenquationdaived from theavmp ofthetwo bracketingcurves(rA2- 0.996).SpecifiQcC parametersareavailabloen theappended msdts table. Limit of Quanittation The currentanalyticamlethod has been used to evaluatea largerangeof PFOS levels=Uacted from sera;the method isnot optimizedforpreciselow-level(< 5 ppb) quantitationb,ut insteadfor . samples in the range of 20-60 ppb. Given the concentrationschosen forthisoptimization of the standard mm while thereis a smmcal diffeam forsamples evaluated from 5-100 ppb, them is no sbbswd differencebetween samples quantitatedfrom 1-5 ppb IU quantitabon does not become mtmUcaUy defensibleuntil5 ppb, thus tlnsvalue representsthe limitof quand=on forthismethod. -Sampla d@edgnated <.OQ are estimated to contain 1-5 ppb PFOS. Iffurtheraccuracy and pro@mon in quantitationof the low levelZ-wt samples (samples lessthan 5 ppb) is requiria a speciallow-lmd quantitationsystem will be required. Limit of Detection The linutof detectionfor thisanalyticalmethod isbased on analystrecognitionof the distinct peak shape rowtmg from PFOS analysis.PFOS standard materialand PFOS extractedftom sera analyzebdy the conditions reportedhere,show near baselineresolutionof I branched and I linearPFOS isomer The apex of each peak in the HPLC-U= occurs ata specificm,yroducfl)lcretentionum and the ratioof the branched peak to the linearpeak isfairlycondsw down to the low standard, 1.0 ppb. lUm samples thata) had PFOS Imb evaluatedat lea than the limitof quantitationand b) evidenced the distinctpeak shape, were designated< LOQ. Samples thata) have PFOS levelsevaluated at lessth4n the limitof quantitationand thatb) do not refled theunique PFOS peak shape,were desipated < LOD. These samples are esdmted to containfrom 0-1 ppb PFOS. Instnimental specifics: HPLC system Hewlett Packard Series1100 Liquid Chromatograph ColuzLn:Kcystone BetasilCis 2 x 100 mm 5 @mm particle size 05/07/98 2 Flow rate: Solvent A: 300 IAUmin. 2.0mM Ammonium Aettgo SolventB: Methanol SolventGradient: 45 to 90 O/oBin 9.50mins. Hold at90 'YeBfor3.30minl Return to45 YeB in 1.50minl Hold at45 %B for3.5mins. Injocdonvohme: 10 pL Injwdons /sample: I Electrospramyass spec&ometer mcroman PlatformiiAPI Mm SpwUomeW, -ChicL- ManLynx 2.4 Saftwm Cone voltnw: .60v .Mode: clewupny neptive sou= wmpuviu@ 115-C. Analym vacamm presmu: 0.000043mbar,0.000079mBa I=: 499,427 Electrode:amss4low 05/07/98 3 3M Eavi=vncnWLAb FluorineAna"cal Chemistry Team S@pin Reedved: Smapin Ezbm&M: Sompks Aambwd: 3/19/98 3/19M 3120-3/25/99 Ansirb brCW[brsdm C)uve Rmp: 3@ofCmubradm Cwve: LOD': LOO': PHASE VII Results: US geographical distribution study - PFOS Ineb in human sen [P" St& sm*m @ (ppb) Dev- AKOI 21 0.4 AK02 31 1.2 AK03 25 NA AK04 19 NA AZO26 28 2.5 AZO27 37 6.4 AZMS 47 5.9 CA029 23 4.1 CA030 23 0.7 CA031 26 0.0 DE016 24 4.5 DE017 32 2.2 DEOIS 21 0.1 DED19 21 NA DBO20 23 0.3 IA013 40 0.1 IA014 29 4.2 TAOIS 28 3.0 LA47 49 7.4 IA048 39 1.9 LA049 so 7.9 bOO3 31 NA M3D4 is NA M105 22 0.9 bUO6 31 1.1 M007 24 4.9 moos 31 6.6 M009 35 0.6 msso 35 5.3 QC Results QC C3wdr. IC4h-WSdOnChWk(QCI) % Recovery 101% Branched Isomer Abundance Percentages Somph Pool StandardMataial I"vkkmkb Sampled in 1"8 3M Cottap Grove PlantWorksm 1"9 k&vw"b SagAW in 1970s and SW PooledSam S=Wles from 1997-98 FOcciP Sam &=pit$. 1994 wW 1994 hAvWuak S=Vled inlate1950t US Coop2phied Su* Samples,iggs % Abundsnce of Branched homen (Av 22% 34% 45% 370/o 31% na 39% 41% I - LA)D - Lbnk of Dc"on 2 - LA)Q - LimitofQuantitafion m I Wb) mssi 56 MS52 27 bff4l 26 Mr42 24 Mr43 is NE21 20 NE22 12 NE23 27 NE24 9 NEZS 19 moio 27 NIOII 20 N1012 24 NV44 28 NV4S 25 NV46 X7 SB38 13 SM9 14 SBD40 16 SCM2 56 SCO33 47 SCO34 52 IX035 32 TX036 26 T=7 29 WY53 35 WYS4 13 WY55 20 % Abm@ Brmwl" of l@ (ML Da.) 3% 5% 4% 5% 5% na 4% 4% a(#MPM) nta 31 3 12 11 na 5 55 Dw. S.1 9.5 4.1 3.5 3.2 1.3 1.0 6.7 2.0 0.8 5.9 0.8 2.0 0.8 0.1 6.6 0.2 0.7 0.6 1.8 2.8 2.1 11.2 1.2 3.6-.-0.7 0.4 IJ 5/7/98 778-60io ESLO Ippb to248 ppb 0.9% I ppb 5.ppb PHASEVU Quantify C=Wound Sumary Report BeAmple List; LAst modified: Method: Lost Modified: Job Code: C;\NUSLYMX\PHUZI -VW\BAWWM\PXMZI Thu Apr 09 09:39:18 It" C: \MUSLYMX\PKAUI. PMD\MTMB\VM Thu Apr 09 10:00:36 is*@ Printed: Thu Apr 09 10:11:30 1990 Compound 1: PTOS N@ Type 1 04079901 Analyto 2 04079$02 Analyto 3 0407::03 Analyto 4 0407 04 Analyto 5 04079605 Analyt* 6 04079806 Standard 7 04079007 Standard 8 04079408 Standard 9 04079009 Standard 10 04079810 Standard 11 04079811 Standard 12 04079912 Standard 13 04079813 Standard 14 04079814 Standard 15 04079615 Standard 16 0:07::16 Standard 17 0 oll 17 Standard 10 04079118 Anslyto 19 04079419 Analyto 20 04079820 Analyt* 21 04079821 Analyto 22 04079422 Analyto 23 00 ::7::23 Analyto 24 7 24 Analyto 25 04079825 Analyto 26 04079826 Artalyto 27 04079027 Analyto 21 04079128 Analyto 2: 04:7::209 OC 3 04 7 3 Analyto 31 04079831 Anslyto 32 04079132 Analytt 33 04079633 Analyto 34 04079834 Analyt* 35 0407::3S Analyto 36 0407 36 Analyt* 37 0407::37 Analyto 30 0407 38 Analyto 39 04079839 Analyto 40 04079040 Analyto 41 04079041 OC 42 04079642 Analyto 43 04079943 AA&lyto 44 04079044 Analyto 45 04079945 Analyt* 46 04079046 Analyto 47 04079847 Analyto 48 04079849 Analyt* 49 04079849 Anslyto 50 04079850 Analyto 51 04079051 Analyto 52 04079852 Analyto 53 04079OS3 QC 34 04079954 Analyto 55 04079855 Analyto 56 04079856 AA&Lyto 57 04079857 Analyto 54 04079858 Analyto 59 04079859 Analyto 60 04079060 ArL&lyto 61 04079861 Analyto 62 04079642 Analyto 63 04079663 Analyto 64 04079064 Analyto GS 04079665 Analyto 66 04079946 Analyto 67 04079847 Analyt* 04:7:::: Analyto :39 04 7 Analyte 70 04079870 Analyto 71 04079871 Analyt* 72 04079872 )Lnalyto 73. 04079873 Analyto 74 04079874 Analyto 75 04079075 Analyto 76 04079076 Analyto 77 04079877 Analyto 78 04079870 Analyto 79 04079979 Analyto 10 04079800 Anslyto 81 04079@el Analyto gmwle Text Hoch Blank Water blank inj 1 Water blank inj 2 KS Blank inj 1 KS Blank inj 2 0.996 ppb inj-l 0.996 ppb inj-2 4.90 ppb inj-l 4.90 ppb Lnj-2 24.8 ppbinj-l 24.0 ppb inj-2 49.0 ppb inj-l 49.0 ppb inj-2 96.2 ppb inj-l 96.2 ppb ial-2 142 ppbLnj-2 142 ppbinj-l meah blank St-11 RS04078 inj 1 39-11 US04079 lal 2 SE-12 U304079 inj 1 SZ-12 US04079 inj 2 SZ-13 K$04078 inj 1 SZ-13 9304070 inj 2 SZ-14 NS04076 inj 1 SZ-14 MS04078 Lnj 2 SZ-15 US04078 Lnj 1 SX-15 3304070 inj 2 24.8 ppb cal check aboob blank 3F,-16 M504078 inj 1 SE-16 RS040'18inj 2 SE-17 M304078 Lnj 1 SE-17 R504079 Lal 2 St-18 U504078 inj 1 SE-19 KS04078 inj 2 SE-19 U504070 inj 1 SZ-19 8304076 inj 2 39-20 MS04071 inj 1 SE-20 KS*4078 inj 2 24.Sppb cal check mah blank SE-1 RS04070 lnj 1 SE-1 H304070 Lnj 2 SE-2 MS04076 inj 1 SE-2 H304078 inj 2 SE-3 KS04070 Lnj 1 SE-3 KS04079 inj 2 SE-4 K304078 inj 1 SZ-4 KS04078 inj 2 SE-5 NS04079 inj 1 SZ-5 NS04074 inj 2 24.8 ppb cal check "ch blank SZ-6 M304078 inj 1 SE-6 NS04076 lnj 2 SE-7 H304072 inj 1* SE-7 US04075 inj 2 SE-0 NS04079 Lnj 1 39-8 U3040-19 Lnj 2 39-9 K$04078 inj 1 St-D U504079 Lnj 2 SIC-10 R$04071 inj 1 $Z-10 R504076 Lnj 2 moh blank Water blank lnj 1 Water blank Inj 2 Sera blank Lnj 1 S*ra blank Luj 2 0.996 ppb lal-I 0.996 ppb iml-2 4.90 ppb inj-l 4.90 ppb Lal-2 24.4 ppbinj-l 24.1 ppbinj-2 49.0 ppb inj-l 49.0 ppb inj-Z 96.2 ppb inj-l 96.2 ppb inj-2 142 ppbinj-l 142 ppbxnj-2 a? 9.910 10.471 10.479 10.487 10.489 20.411 10.430 10.423 10.415 10.405 10.411 10.419 10.373 10.421 10.418 10.434 10.425 10.496 10.503 10.401 10.405 10.478 10.481 10.485 10.486 10.4$5 10.479 10.477 10.365 10.402 20.486 20.48S 10.424 10.479 10.487 10.477 10.437 10.420 10.411 10.43S 10.358 10.438 10.353 10.357 10.352 10.340 10.470 10.467 10.336 10.337 10.434 10.403 10.309 10.388 10.305 10.291 10.410 10.470 10.463 10.4ss 10.336 10.343 10.461 10.404 10.406 10.402 10.407 10.390 10.404 20.336 10.334 10.339 10.340 10.340 10.340 10.340 10.344 10.339 10.342 10.346 10.342 Area 5$375 3243 4594 5092 4829 5970 S750 11454 illes 41237 42564 73645 74906 132595 127206 192332 196237 4489 ssso 9246 7869 7340 9139 9473 7496 7116 7746 7431 12549 3709 7723 7611 3624 3633 5265 5330 4707 4569 3712 4203 12009 3757 9377 9822 9759 9610 10125 10474 0107 7985 9294 9300 11515 4037 8249 7947 10301 17578 7772 7839 022S 9132 20521 20053 4367 4524 4379 4342 4493 5875 5794 11191 11296 40768 41257 68332 66993 116061 115447 162113 165349 Cma. IDOV flags 36.04 bb 0.00 bb 0.00 bb 0.00 bb 0.00 bb 0.00 -100 m 0.00 -100 m 3.29 -33 NK 3.09 -37 MN 25.35 2 MN 25.59 3M 50.83 4m 50.29 3m 93.01 -3 M $9.09 -7 M 137.26 -3 bbX 140.15 -1 bbX 0.00 bb 1.37 NK 1.66 m 0." m 0.24 m 0.00 m 0.00 m 0.34 m 0.00 m 0.54 m 0.31 m 4.10 -93 MM 0.00 XK 0.53 im 0.44 m 0.00 m 0.00 m 0.00 m 0.00 m 0.00 m 0.00 m 0.00 bb 0.00 bb 3.70 -85 m 0.00 m 1.90 m 2.08 m 2.04 m 1.92 m 2.31 m 2.56 m 0.81 m 0.72 m 1.69 m 1.70 m 3.34 -$? M 0.00 bb 0.92 0..69 -m m 1 57 m 7.83 m 0.36 m 0.41 m 0.90 m 1..57 bb 10 ol m 9.66 m 0.00 m 0.00 bb 0.00 NK 0.00 m 0.00 bb 0.00 -100 bb 0.00 -100 bb 3.10 -37 bb 3.17 -35 bb 25.00 1 bb 25.36 2 bb 45.42 -7 M 43.91 -6 m 80.77 -16 M 00.64 -16 M 114.11 -19 M 117.42 -17 M fton I 2.91. LL;t: I C:\N"SLTIMVMAM -MNAN@%0322" I"% -." Ld U" U" as L2:st.34 IS" N.U.dj C:\WAALIM\pm"u -pm\@\DM& 07 14,09,39 toss 14- j4p&07 14,23,32 IS" PC" S..pL* I- 0032-Igool ATOY"LYYt.-. A n:l, 3 03239403 AnslYts 3239404 Standard M"@ US.c* lb@llomokk'-lI K20 0.996 PPb FC MIX 16 0.0332233:::: :1t1..1dgrdd 4.*41.: PpPp-b IrCc -IlnuLx: 1 .0,111:::7St.:"4Ld :::20 IPb IC 03223990: St@l",g 3 n"rd It 323::110 Analyto 10 .323 1 An lyt: 12 03239#12 AN:IY% Pl- VC KL142 ppb FC KLS NOCW 916&k ::J::l 12 13 0323::13 A. lYt: 4 0313 14 An:@, I16S 03239415 Analyt. S338 M Hwu IU-k S331-1 323::16 A^:Iy . L7 0323 17 An lytte is o@23::,I: An lyt 19 032 A.:lyt: 20 0323::zo An lyt 21 0323 21 An:lyt: 22 0323::2 23 032 Analyt: 22 A, 24 0323982 43 A..Iyrtt. 2: 003 31826 An.lyt. 2 12239024 A"Iyto 27 0323::27 Amlyt: 2: 032233,02291 Amlyt 23 Amlyto 3310 00332233::3301 AAnnaallyyttoo 0323,432 3,1 A"IytA 33 0323 :33 A"Iyt 3831-2 5540-1 5940-2 mr4l-l mr4 t Wr4l:m2 Me= ILLmk 'a4 PC 42:21 Mr43-1 PC43-2 OV44 I MV44:, OV44-M N*CS U." 3rV45-1 IrVdS-2 WV46-1 34 0313: 34 A"Iyt: 3: 032 3983S A"Iyt. 3 o3z39s34 Analyto MV46-2 LA47 I LA47:2 31 :3233::37 An&1 yto 30 2 34 Am lyt. 39 03239830 Ablyto 40 02239440 A"Iyto LA47-M mm St@k IA4::l 1^4 2 41 03239241 42 03239042 43 03231943 44 03239944 3 03231845 Anal A.'Ii-t. AMlyt* A"Iyt: A..Iyt LMS@2 NSWI 19250@2 msso-ms :4 03239$46 A"Iyte U&ak 47 03230147 A" Yt. 41 03230049 Am 1 Yto - mxsl-l 923 41 03231941 AD41yt* ns 50 03231430 Aalyt. "352-2 03239@Sl A..lyto 52 0323::3S2 Analyto 0323 53 3 Amlyto 54 033223::554 A"lyto W"3-1 WV$3-2 WYSI-= MOCO Blank 3 4'.0 M& KLX 0323965: OADCalyto M.Cm Blank 57 0323::37 )"Iyt VT3::l 5* 0323 54 .115 2 59 03239451 A"Iyte WY35:1 03239SGO A"Iyt. vns 2 61 0323:::l A-lyt62 0323 2 Analyto AX03:1 AR04 I 63 03239063 Am iyt 64 03221964 Analyt: IU03:2 KZ04 2 6S 03239165 An.lyt M104-SKINaak 66 03239666 An.lyt: ".= I 03ZJ:::7 A"Iyt. :6: a0323 1 A-lyto MDOO-1 "009-2 32 70 03233::7 71 03231671 Analyt A-lyt: A"Iyt. :JOU:L J012 z 1"15-1 72 00323::732 A-1 Yt: 73 323 7 An"Yt 7I4S 03239974 A-1 9975 Analyytt: 76 77 00322233 ::7774 Aaalrte Aaalyts 7: 00332323::7: A-IY" 7 7 A"Lyt: IMIS-I2Vaal 1 :2 Pm"eItLD 2 DWZC@-I D9020-2 NMI-M No= Blank 40 032398$0 01 02231681 2 03231682 :3 03239943 Anal A"Iyrtt. A"Iyt: Analyt 94 03231614 Anal 15 03239865 An.lylt: $6 0323,006 Analyt 67 03231807 A..Iyt: 323:::: A"Iyt NNz"o223,:l2 MCOZ ::1 uto. 2 3-1 :.800.25-2 AZOI::L AZ.. 2 CA031:1 :I01 t-0.323 AR&Iyt: CA031 2 0123:::0 Analyt: 2 .031223 1 Anslyt 3:::l Amirts 3CO34-1 SCO34-2 Txo3s-l 93 03213.8943 1, lyt An:lyt: 23982$ An.lyt. :3123:::,dAMn:Iyt* 91 0323 t. TX 13 TX0033:.1 NOW 6 Blank ?X*3 -1 TX036-2 14 03239498 Anallyyto 1: 012,1::Oll.Antlyt, to 032 An Lyt. 1 0 23::Gl An:ly : TX037-1 TX037-2 SCO32-K$ woom3a a 102 0323 02 An lytt 103 0323::03 Anslyto SCO3 -11 SCO23-2 104 03 3,90054 Alelyt: 105 032 A..Iyt M.. 51 A: S.C. liL..-1 0323:::f Axalyt. M20 bl"k-I 1.0,1 0323 7 Stan" rd 0.996 ppb rc mix 1:0 00111:::: .3,t.:n".,l 1-1: ppb IC KIR 1@to 0 3322331910 St.an" @d III 032399LI at& rd 111 03239912 2ta."Cd 494:0 ppb rC KL 96.2 "pptb 7rcc mit: 142 PP6 FC KLa 1. 9.921 4., :::2I's 9.737 9.742 ::171 a I.uo ::711 99..6422@01 t.:21 9.6312 9::2 1 9.414 ::122 622 S.617 9.677 I.il: 9.92 9. .:,1: S...622 77 9.419 S,43 9. I..l 9.621 7 :::'#a :::Is 16 1."0 9.11, 1.123 9.832 ::1.31 :33: 2214 0430 1"94 352146 4 197 11: 4@ 2.4 2422 223 1164 1:1 1 34 I,.: 2159 2078 214 l17o0s,s 1a1.34"7 211,.l 22 IIS3 23 @:1 3.5 3"4 269 24: 2 0 @4 2. 212, 24., 1514 last ll:,l 1.:20 9..5,377 .$$: 22670 2127 lIL2 3 213:33 @::,Is5,-7, 101..25207 1.354 ::161 354 2111 114'315: 1210 13.:7 1a 11 ::SSS" $.67: :: ,,,,7, ::-,I, 1.S" 9.362 ::I:l 53 24:: 2269 2240 301 274 24774 121* "7 7 22224"0 273 &o.094 ::'1'131 ::,531 62 9.55 1.5s: ::3,:: ::1"171 1:13 1: 22 .2 I0s:: 1743 1164 2:01 .3 2 171: 1720 ::* 9.3:5 2674 :9.:6'7S5:'3 '22*4.:& ::33 SS 217,1 171 ::I'll 9.557 7"6 1ze:i ::'3814 ::::1 ::,171 1.':7 2111 178 400 9..55565, 21476 0.5" ::,3" 795: 15390 2,z:l .3 3 COW, O*V 0.:0@: 3.73 23.25 31.20 101.63 140.74 .:** U 6. 17 0.37 4.200 3.48 '3 :7 ::It 02 5.21 0.00 44..70 @L3 3: 334S 43: IL4 70 5 31 0: bb 11 bb 2 bb 4 bb 1 bbX -1 bbx bbbl bb bb m bb bb bb M bb bb bb NK I.:, M ..2. hb 9.12 7.23 m C." bb 52 bb 7:33 16 6 57 3 32 bb 3:74 ':' 16 7. bMb 5. 13 S.'32 bb 4::: 53 .74 bMb 40 410..4*40 -3 bb 2.:Io bb ,I:Iol lb 3.1 lb .7 bb :..:3.1".1 Ilb S0.S0O0 bbl' bb 7 it bb 6: 53 bb 1,:71 1 07 22. .1.'3'7 7.34 m 4: :4 bb 2 62 1:.:6 bb 2. 1.1 16 4:::3 1 ..71 4:11 bb 4.1. I'1.1: bl 47.16 bb S.17 bb 03..0700 M M 4. 173 M ..@2 7.75 bb *:t"o bb :0..49 bb ....370, "b 0.26 -74 2,3:,3: !: 11 46.96 1::7' 63 -4 bb -9 IM $VAA 0-7 L::3 67 23 19" PC*A Conipound I nwm: POAA Correlatiocnoefficienrt-: 0.997996,r^2- 0.995995 Calilradoncurve:319-335230 * x + 393.755503 Response type:ExternalStd,Area Curve type:Linear,Origin:Exclude,Weighting:Null,Axis transN:one 4.56o4- Response- x x 4d.'O' I .0 ibb.'o -T- 1,7o7-'- Conc 14 .0 3M EnvironmentalLaboratory-FluorineAn KrisHan= -Sr.AnalyticaClhmist FluorineAnalyticaClhem6try Team Building2-3E-09 612-'778-6019 Quantitative analysis of PFOS in hbtoric human serilsamples from Sweden 'eam Two groupsoftenmm samplesinplastivcialswei supplietdotheEwAronmental Lab by kff NL-M&L (3M Medical)on March 20,1998.The two groups(sampla SE-1 toSE-10 and SEI 1-SE-20) repre*nttwo dlcaw ww penods.Both groupsofumpla onpuod inSweden and inallcans,sample volume variedfmm 0.5to1,0mL of taim2n.am An exactvolum% usualltyheanti=samplewas =uactod usingan ion-paW4 reagent.The =MM wa analyzedquantiftti%f*orPeffluOrOOCtIm milfougeCPFOS)by highpmmm liquidmmatopMby4e=ospmy massq=UomctrY (EMP-ESM. Analyftidentificatwiaosn vedftedby comparisonofmolecularion-HPLC reftndm time oftheexhacted anabu and standarmdgmg. Sampleswerequantift&* ewuated againsat VedWly preparedf.ourpointcxuactedmm (rA2- 0.992).Each sampleactradwas nab7od twice.Cah-brafm chocks6 analyzedevery5 samplm werebetween W106% ofexpectedvalues. Descriptionofmnples groups.SE-1 toSE-10 and SE-1I to20 Sem samplesSFl throughSE-10 were coUacW from Swedishdonorsmore recentlythan samplesSE-11throughSE-20. The avemp PFOS concentratioin ffifcirsgtroupswas determinedtobe 1.3ppb;them were threesamplesbelow thelimitofdetechollThe wmd setofsamplesbad an.aveiap concentratioonf 2.0ppb. Intwo ofthesesamples,PFOS conccnusdm was determinedtorboet 1I:=4:mtih@abntloe thedetectiolnimitofthemedw& Allsampla containePdFOS atlea than5 ppb. Itwas deftmm theisimm ratiofdmc low levelamplel icxperimentaslmmarjr Samplepreparation:.Ion-paeixrtirnagction CMA), is Ina pH controlle=dWronment, an ion.-pairirnegagentf,tmbuty@ammonium sumft usedtomtr= theangytefromthematnx Am=c wmpowW4 blwPFOS and peffluomwmoate (POAA), am sdecdvtlytargetebdy thecationirceagenlSubsequentotheftmufm oftheTBA-anion pair,ft andyw isftandwed toa nw-polarorpnic solven(tethyalcetat)d.rie4and r=zmdtuted in methanolforMS anakysil HPLC.- characteristircetentiotnimesforPFOS jacw and passedthrougha &romatograpbicCOIUMIL InEPLC, an aliquootfthe=U-Ad is in inthecolumn mbwm totheliquidmb*L- Based on theaT2nityoftheanalyteforthestadonary-phase phasepassin throughthecolumn,theanalyteis mWnW. fora cbamcw&& amount ofdm. For example,ina standardsohitionP,FOS May cluteat10 5 mmML Rctention bm Wtwwn a smndud PFOS solutionand theanalytextractefdrom serainWs analysiwsere matched towithin1% on the BPLC rjstm EZ&. Detectioannd monitorinogfthemoleculaIron ArWysisofPFOS standardisndicattehsatheprimaryioncharacteriosftPiFcOS ismlz 4" amu, correspondingtothemassoftheanionmiubcctant(Wl7So3-)- ThiisonWasmonitorseedlectively tomaximize sensitivitAy.scanofm/z-100 to1210 (nepdvi only)was alsoCOUcctc& In those=MPICB 04/20/98 thatwat determinedtocontainPFOS at identityof the anabft usingIRPLC-ESMSHO 1'rnagreaterthan10 ppb,isposuble0 vaffythe to MOWtOr fourchwAcWrWc fragmcn daughterion& Qualitycontrolsummm7: Due toumple limitationsse,rasampleswere notwdmcted induplicateF.or thissame reason, matrixspflm couldnotbe evaluatedforthesamplel Each sample =Uact was analyzed in&Vhcatr. bfetbanobllankswei analyzedperiodicaltloyensm complft isolatio(nifthesample.Two mid4md (2.98ppb and 4.95ppb)qualitcyontrolsampleswere analyzedbetweenevay 5 samplel Bacmisehuman mm witboutPFOS isnotavailables,amplesofpooledserapurchasedfrom Sigma we= pre-exbwft4 spiked@itha standardconcentrationfPFOS, and m-=ftacte& Thispm-=UwW sem isunderstoodto be vm similato the samplematnx. lbs fourpomt odmcted curvewas analyzedbeforeand afterthe samples.Quantitatioonfthenmples isbasedon thelinearegmssionequationderivedf3romtheavmp Ofthetwo bracketincgurves(zA2- 0.992).SpecifiQcC pamme= am availabloen thegpended results table. Limitofquanatation Thm low iavd sampleswac analyzedagainsta vemauy prqmcd low kvd =ve, As a rerdti6 confide= limitcsalculodaroundft =m valuesindicataequandflabldeiffc=m bctwm samples evaluatedat0 and 1 ppb. For thisanalysist,helimitofquwdtadon and thelimitofdetect*mare Limitofdetecdon The limitofdetecuonforthisanalyticamletbod isbasedon analystrecogmtlonofthedadnct peak shaperesultmgfiromPFOS analysisP.FOS ==lard ma=d and PFOS extmctedfrom sera amb7ed by theconditionrseportedhem show nearbasd resolutioOnf Ibmn'cbed and I li PFM iso=. The apexofeachpeakin theHPLC-U= occursata Wcific,reprod=Uc retentiodnm gndthe ratiofthebrmdzd peaktothelineapreak ishirlyconsistendtown to thelow s=dard, 1.0ppb.Those thata)have PFOS levelesvaluatedatlessthanthelimitofquantitadonand thatb) do notnfiect the PFOS peak shape,were designatebd.dl.O)dm &=don limit.Them samplesareestimated tocontainlessthan 1 ppb PFOS. 2 04/20/99 Instnunentalspecifics:. HPLC system HewlettPadmrd Series1100Liquid Column:KcystoneBeUsil Cis 2 x 100 mm 5 @= particlsehe Flow rate: SolventA. 300 gjkiin. 2.0mM Ammonium Acetate Soh=t B: Methanol SolventGradient 45 to 90 O/oBin 9.50 minl Hold at90 O/Z br 3.50mins. Returnto45 %B in 1.50m!nL Hold at45 %B for3.5minl Injectiovnohuw-. 10 JAL InjectionIsumple-. 2 Ekchvspray mass spech-ometer bficromassPhdorm n API Nfm Specftometer".ChW MassLynx 2.4Saftwuc Cone volukm: -20v,-60v Mode: elccuwray neptive Source temperature: 115 OC. Analyzervacuum pre==: 0.000043mbar, O.OWM Ions: 369,413,499,427 Mccuvde: cross-flow mbar Elec&osprayM&?JS mcromam QuattroItAPI MRA, -NbddW MassLynx 3.1Software Cone voltkges:-60v ConWon ps enew. 40 Mode: elecuDsPraYnqp*m Sourcewmpcratm: 115 *C. Anab= vacuum pressures0:.000043mbar, 0.000079 ud3ar Primary Ion:499 Daughter ions:90,99, 130, 180,230 Mectmde: Zesom= 04/20/98 3 FluorineAnalytical Chemistry Team SamplesReceived: Samples Extracted: Samples Analyzed: 4/6/98 4n4logIgg 4/094/13/98 Anabmb by. camrsuon CurveRange: iAofCalibratioCnurve: LOW: IAW: Study of PFOS levelsin historicalSwedish human wxa smples Sample ID SE-1 SE-2 SE-3 SE-4 SE-5 SE-6 SE-7 SE-9 SE-9. SE-10 2 VPP+++FF(/1/O_L__I__S0L@00ID..II00@ b1LS0).'9 1.2++1/_-0O..1 l 1.55+++///_-0O,1l 1.0+/-0.L 1.0+/-O.l bdd.1 1.22+/-0.0 2.8+/-0.0 b.c-dLL ! 21.066+/-0.9 ab.d.L Sample ID SE-11 SE-12 SE-13 SE-14 SE-15 SE-16 SE-17 SE-19 SE-19 SE-20 VPFOSI .=b 4.1+1-0.2 2.9+/-0.0 2.0+/-Ol 2.8+1-0.0 2.9+/-0.0 2.7+1-0.2 1.2+/-0.0 1.2+/-0.0 bd.L bd.L 778-6018 EShg lpybto 10 ppb 0.992 I ppb I ppb QC Results QC Chedc CalibratioCnheck 2.98ppb CalibraticCmheck 4.95ppb CalibratioCnheck, 2.98ppb Cahb@on Check,4.95ppb CalibratioCnheck, 2.98 ppb CalibmtionCheck, 4.95ppb Reavery 106% 100% 100% 96% 89% 990/0 Brandied Isomer Abundanci Percentages Sample Pool SUndard MAWW IndividualSsampled in 1998 3M CottageGrm PlantWatkas, 1998 IndividualsSampled in 1970s and 803 Pooled Sem Samples fmm 1997-98 ForeignSem SampIM 1994 and 1994 IndividualSsampled inlate1950s US Geogmphical StudySamplm 1998 %Abundancoof BranciwdImmm %Abuadmceof Brandiedboomn .%22CL22% 400A 45% 37% 31% na 39% 41% --(&&D".) 3% n/a 5% 31 4% 3 5% 12 5% 11 na na 4% 5 4% 55 I -LOD - LimitofDetection 2 -LOQ - LimitofQuantitadon Pop I Shooti filenumber 4099807 4099808 4099809 4099810 4099811 4099812 4099813 4099818 4099819 4099820 4099821 4099822 4099823 4099824 4099825 4099826 4099827 4099828 4099829 4099830 40998Sl 4099832 4099833 4099834 4099835 4099836 4099837 4099838 4099839 4099840 4099841 4099842 4099843 4099844 4099845 4099846 4099847. 4W9848 4099849 4099850 4099851 4099852 4099853 4099854 4099855 4099856 4099857 4099858 4099859 4099860 sampledescriptionpeak area 0.998pp@ W 1 4654 2.98ppb inj1 sm 2.98ppb W 2 8247 4.95ppb inj1 11239 4.95ppb W 2 11381 9.8ppb inj1 17949 9.8ppb inj2 17582 Meoh blank 4466 SE- 11 HS04078 ini1 9659 SE- II HS04078 inj2 9265 SE- 12 HS04078 ini1 7886 SE- 12 HS04078 ini2 7794 SE- 13 HS04078 inj1 6507 SE. 13 HS04078 inj2 6735 SE 14 H.SO407,8'?n*j1 7684 SE-14 HS04079 inj2 7674 SE- 15 HS04078 ini1 7808 SE- 15 HS04078 inj2 7803 Meoh blank 4133 2.98 ppb calcheck 8173 4.95ppb calcheck 10721 Meoh blank 8844 SE- 16 HS04078 ini1 7766 SE- 16 HS04078 ini2 7411 SE-17 HS04078 inj1 5539 SE- 17 HS04078 ini2 5450 SE- 18 HS04078 ini1 5380 SE- 18 HS04078 ini2 5459 SE- 19 HS04078 ini1 4579 SE-19 HS04078 inj2 4335 SE-20 HS04078 ini1 4018 SE-20 HS04078 ini2 3971. Meoh blank 3569- 2.98 ppb calcheck 7932 4.95 ppb calcheck 10483 Meoh blank S377 SE. 1 HS04078 ini1 6330 SE- I HS04078 inj2 m SE-2 HS04078 ini1 5773 SE-2 HS04078 inj2 6878 SE-3 HS04078 inj1 5197 SE-3 HS04078 inj2 5114 SE-4 HSD4078 ini1 5337 SE-4 HS04078 ini2 5127 SE-5 HS04078 ini1 4807 SE-5 HS04078 inj2 4937 Meoh blank 8416 2.98 ppb calcheck 7479 4.95 ppb calcheck 9904 Meoh blank 3219 [analytel 0.6 3.1 3.2 5.4 5.5 10.2 9.9 0.5 [PFOS]avg 4.2 3.9 t1 3.0 2.9 2.9 2.0 2.1 2.0 2.8 2.8 2.8 2.9 2.9 2.9 0.3 3.2 5.0 0.0 2.9 2.6 2.7 1.3 1.2 1.2 1.2 1.2 1.2 0.6 0.4 0.5 0.2 0.1 0.2 -0.1 3.0 t8 -0.3 1.1 1.2 1.2 1.4 1.5 1.5 1.0 1.0 1.0 1.1 1.0 1.0 0.7 0.8 0.8 -0.3 2.7 4.4 -0.4 St. Dev. 0.2 0.0 O..l 0.0 0.0 %Rec, QC o 106 100 0.2 0.0 0.0 0.1 0.0 100 96 0.1 0.1 0.0 0.1 0.1 89 88 Page I Shooti 4099861 SE-6 HS04078 inj1 5421 1.2 4099862 SE-6 HS04078 W 2 5480 1.2 1.2 0.0 4099863 SE-7 HS04078 W 1 7673 2.8 4099864 SE-7 HS04078 inj2 7729 2.8 2.8 0.0 W39865 SE-8 HS04078 inj1 4556 0.6 4099866 SE-8 HS04078 inj2 4682 0.7 0.6 0.1 4099867 SE-9 HS04078 inj1 6497 10 4099M SE-9 HS04078 inj2 8183 3.2 2.6 0.9 4099869 SE-10 HS04078 W 1 4312 0.4 4099870 SE-10 HS04P78 inj2 4710 0.7 0.5 0.2 4099871 Mooh blank 1555 0.0 4099872 water blankinj1 3813 0.0 4099873 water blankinj2 3891 0.1 4099874 serablank inj1 4273 0.4 4099875 seraWank inj2 4498 0.5 4099876 0.998ppb i,N-1 5199 1.0 4099877 0.998ppb inj2 5054 0.9 ,4099878 2.98ppb inj1 7712 2.8 4099879 2.98ppb inj2 7792 2.9 4099880 4.95ppb iai1 10297 C7 4099881 4.95ppb inj2 10556 4.9 4099882 9.80ppb inj1 16807 9.4 4099883 9.80ppb inj2 16888 9.4 Page 2 3M EnvironmentalLaboratory-FluorineAnalyticalChemistry Team Yds Hansen - Sr.AnalyticaClhemist FluorineAnalyticaClhemistryTeam Building2-3E-09 612-778-6019 Quantitative analysis of PFOS in historic human sera samples from the University of Minnesota Summary: Ten samplesweresuppliedtotheEnvironmentalLab by JeffMan&l (3M Medical)on April14, 1998. The tensampleswere partofa group ofsamplesthat,afteruuaction,were pre-screenefdorPFOS levelsT.hese tensampleswere classifiaesd"low-lever(<10ppb);dW arenumbered 1,2A, 3A, 4,5, 6A, 7A, 9,9A,and IOA. Two-one mL portionosfthesampleswen extracteudsingan iontaidn rea t The === wereanab7jedquantLMaWy forpeifluorooctaanmemge CPFOS) usinghigh. pressureliquid hmmatog6i@by-elwftosprmaayss spwmmcay (EPLC-ESbffi).Ambft MaWfication was verifiebdy compuison@ofmolecularion-HPLC refttiontimeofthec&wW a=bU and standard material.Sampleswem quandta&* evaluateadgainsta speciallpyrepu4 five@-poiawdractedcurve (rA2 = 0.987).Cahbratiduchecks,analyzedevery5 samples,we= between80-1086/o9fexpectedvalue& Descfipiffofnswnples.In alltenofthesepre-screenesdampIM PFOS concentratiownas determinedtobe lessthanthc* detectiolnimitof the method, 1 ppb. Experimentalwammary: SwnplepreparationI.on-pairinegxtraction In a pH controlleednvironmenta,n ioxl-paidnrgeagenttetrabutaymlmonium Mil (TJBA)i,s used tow&= theanalytefrom thematrix Amomc compounds,likePFOS and pmguomocmnoate (POAA), areselectiveMlpyted by thecationirceagent Subsequenttotheformationafthe7BA-anion pair,theanalyteiswanderredtoa non-polarorganicsolvent(ethyalce=), dnecland reconstltutiend methanolforMS analysis. HPLC Charactenshc retentiohnmesfor PFOS InBPLC, an allquotoftheuwda *5injecteadnd passedthrougha coiunm Basedon theafonityoftheanabftforthestagonary-pbaisnethecolumnrelativteo theliquidmobffo.- phasepawing throughthecolumn,theanabu is retainefdora cbaracmmc amount ofwm. For example,ina standardsolutionP,FOS may clutoat10.5minutm Retentiontimesbetweena standafd PFOS solutioannd theanalytexuactedfrom serainthisanalysiwsere matcw towithin1% an the EPLC systenl F.SIZ. Detectionand monito?ingofthemolecularion AnalysisofPFOS standardisndicatetshattheprimaryioncbaracwas* ofPFOS ism/z - 499 amu, correspondintgothemass oftheanionicsurhctant(CsFl7SO3-)T.hisionwas monitoredselectively tonmxhniw sensitivitAy.scanofm/z--100to 1210(negatneonly)was alsocollected. Qualitycontrolnummary: Scm sampleswereextracteidnduplicateM.ethanolblankswereanalyzedperiodicaltloyenmm completeisolatioonfthesaxnpleand two mid-leve(l2.98ppb and 4.95ppb)qualitcyontrolmunpleswere analyzedbetweenevery5 samples.Becausehuman sem withoutPPOS ism availables,ampla of pooledserapurchasedfrom Sigma were pm-moacted, spikedwitha standardconoonuadon ofPFOS, and re-extracte&This pre-exuwtedseraisunderstoodtobe verysomila tothesample matrix Thisfive 04/22/98 1 pointexuactedcurvewas analyzedbeforeand afterft samples.Quantitadonofthemmi)l isbasedon thelinea regressioenquadonderivedfrom thenwap oftbetwo bmdmft curves(zA2- 0.9M. SpecificQC parametersareavailabloen theappendedresulttsome. LimitofQuanhtahonILimit ofdetection These low level=Wles wen analyzedapmst a q=auy preparedlow levelcurve As a results, confide= limitcsalmwod aroundthecurvevaluesindoca aquandfablediffmwmbdweensainples evaluatedat0 and I ppb. Forthis=&isi4 thelimitofqundbtbm andthelimitofdetectioanre equivalent- speciries: C system HewlettPadmrd Series110014Wd Column:Y.eystonBeetud Cis 2 x 100 mm Flow rate: Solventk. SolventB: 5,pm particlsein 360 PL/Mi& 2.0mM Ammonhm Nfethan.01 Acetate SolventGradienL 45 to90 O/oBin 9.50minl Hold at90 O/Z hr 3.50mins. PetUM tD45 %B in 1.50mins. Hold at45 */&Bfor3.5mins. Injectiovnolume: 10 pL Injection/ssample:2 Electrospraymass spectrometer Mcromass PlatfbrmH API Mass Spwftometer,-OW MassLynx 2.4Software Cone voltaga:-20v,-60v Mode: elccftvrayncpdm Sourcetemperature: 115 OC. Analyzervacuum pressures0:.000043mbar, 0.000079mbar Ions:369,413,499 Electrode:cross-flow 04/22/99 2 FluorineAnalytical ChemistryTeam Samples Received: Sampla Extracted: Samples Analyzed: 4/14/98 4/15/98 4/16-4/21/98 Analysisby: CalibratioCnurve Range: ?of CalibntionCurm LOD': Analysis of PFOS in historicsamples of human sera (U of Al) Sample IDD I 2A 3A 4 5 <Lo @IFV<PIFAO)S] <LLLO40obD)@D ,If < LODS' < LOD < LODD < LODD < LODD Simple M 6A 7A 9 9A 10A IPFOS] (pob) I < LOD < LOD < LOD <LOD < LOD K-fHanwn 77"018 E,%iS lppb tD 24.9ppb 0.997 1 ppb I ppb QC Results QC Check-. CahbrationCheck (QC1 at2.98ppb) CaMration Check (QC2 at4.95ppb) % Recovm 30% 108% I -LOD - LimitofDetection 2 -LOQ - LimitofQuantitation 4n2AS 3M EnvironmentalLaboratory-FluorineAnalyticalChemistry Team KrisHamn - Sr.Analydol Chen2W FluorineAnalyticgCkmWq Team Budding 2-3E-09 612-778-6018 Phase 2C4 Part 2: PFOS levels in individual's human sera samples, a blind repeatability experiment Summary: Fifteensamples of hitn-qnserain plasticentrifugetubeswen suppliedtotheEnvironmental Lib by Jeffb&ndel (3M Nledicalo)n April15,1998. The Aftacm=mples werepartofa gmup ofminples tb4 afterextractionw,erepro-screenafdorPFOS levels.These fiftm sampleswere classifieads"andlevel"(>10 ppb and <100 ppb);theyarenumbered IA, 2B, 2C@ 4A, 5A, 5B, 6B, 7B, &A, 9B, 9C, IOB, 11, 12,and 13. sampic ofse@Afrom dim don= have be= anab-zedpreviouslbyy theFAcr. one mL ahquotsofserawere removedfrom eachample and extractewdft an wn-pamng ragmt; allofthe samples were extractedin duplicate. Ilicseracxtwm were aiwyzed quantl=vely forp i-ifnirnooctaneswfonate (JPFOS)byhigh pressurehqtudchromatograpby-ekcummy mm V=Uometty (HPLC-LUB). Anabft identificatim was verifiedby comparisonof molecularion-EPLC rctmtlontime of theextractedanalyteand standard m@itmisal.Samples were quantitativeelvyaluatedagainsta Wedally prepar4 five-poimextractedcurve (rA2 = 0.996).MaWx sp&c calibratiocnhwjcs,anab7Ad inthemiddle ofthe analysisequence,wo within100/.ofexpectedvalues. The presenceofPFOS in sevezw ofthesamples (IA tbmugh 6B) was verifieudsingEPLCESMMS. This techniqueprovidesan additionadlegme ofcertainttyo theanalytoidentdcationby.' specificalmloynitoringftginmts daughterions(m/z-80,99,130,190,230)characteristoifcthePFOS primaryion(m/z- 499). The PFOS valuesin thisblindrepro&u*ilityanalysisme within11% ofvaluescollected previously.Specificrebmltasreattachedtothisreporl Experimentalmmmarr. Smple prepffadon:.Ion-,wifi=ntgraction la a pH controllednvironmenta,n ion-pairinrgeagenta,usbutylammonium mdfiftCMA), is usedtowm-ad themmbft from thematrixAmomc compundi6 Ww PFOS and paflumowmoate (POAA), areselocawy targetebdy thecationirceagent Subsoqmw tothehrnmdon oftheIBA-anion poar,the analyteisuuns&rred toa non-polarorganicsolvent(aftlacetate)d,rnd, and reconstitutemd methanol forEPLC-ESMS analysis. HPLC. Characte)4sfircetenfiodnmesfor PFOS In HPLC, an aliquotofthe aft= isinjectedand passeddvmgh a cdhbxmomato column. Based on the atmiityoftheanalyteforthestadonary-phasien de coW= relativteotheliquidmobilpphase passingthrough thecolumn,theanalyten retuned fDra characteristaimcount of Wa. For example, in a standardsolutionP,FOS may cluteat 10.5minutm Rdention timesbetween a standard PFOS solutionand theanalyteextractefdrom serain thisanabsiswem matched to within1% on the EPLC systeul 05/07/98 ESAC. DetectioanndmonitorionfgthemokcularIon AnalysiosfPFOS standaridnsdicattehsattheprimaryioncharacteriosftPiFcOS ism/z- 499 amu,corresponditnogthemassoftheanionicMUhM=nt (C4Fl7So3-).Thisionwas monitoresdelectively tomaximize sensitivitAy.scanofm/z-100 to 1210 (negativeonly)was alsocollected. ES&YSMS.- Confinnafionofanalyteidenfification sevem UMPICS indus setwen analyzedby ESUSO tovm* theidentitoyfthePFOS analyte ion.ES.NlcJv% isverysimil2toE,9W, exceptthatitadds an additionadlmmdon ofcertainttyo con4ound identificatioAms inFSM, a compound specifiicm isselectedAlfterwlectiox@theselected ,onischwwwnzed ffirtbebry smashmg itapartwith highc=U gas-As a resultotfhesmashing Ionic fragments,characteristoifcthemolecule,arecreatedand detecte& For example,forPFOS analysisi,on499 issdww asthecompound sped& primaryim This ionissma bed intootherionssuch as90 amu (conwqwnding toS03), 99 amu (cmil11 " g toFS%), 130amu (oDntVondingtoCF2SO3), 190 skmu (C2F4S%l and 230,umu(C3F&SO3')I.fPFOS ispresent inthe samples,each ofthesesecondary&Agments isddeaed atthe detecw. Qualitycontrolmmmary! Each serasunple was extractedin duplicates,tandarddcvmon vahm areincludedin thetable ofresultsT.wo mid-ILwd(25jo and 49 ppb) qualitycontrolumple wm analyzedbetween everyfmc samples;recoveryforthe= matrixsp&e analysiwsen detumined tobe within10'Yofcwud values. Because human sm %i&M "OS isnotavail&le,=,moles ofpwW sm pmrband km Sig= wm pm--e=@ spdmd witha standardconcenuabon ofPFOS, and re-odmcte& This pre-ccuactedserais understoodtobe verysirniltaothemunple matdx This fivepointexuactedmm was analyzedbefi= and afterthe samples.Quantitatioonf the samples isbased an the li regressionequationderivedfrom theavemge of thetwo bracketingcurves(e2 - 0.996).SpecificQC parametersare availablean the appended resultstable. Limit of Quantitation As thesesamples we= pre-screened,itwas detanninedthatopdmizing instrumentaldetection hnuts was not necessaryforacmwaw analysisofthese"imd-kvw samples. As a resultt,he limitof quantitatiownas 10 ppb. All sampleswere quantitatewdellabove thislevel Instnunentalspecirics: HPLC system HewlettPackard Senes 1100laquid Column:Y.eystonBeemsflCis 2 x 100 mm 5 pm particlseize Flow rate: SolventA: 300 ltT,/min 2.0mM Ammonium Ace= SolventB: methool SolventGradient: 45 to 90 O/oBin 9.50mins. Hold at90 O/oBfor3.50 mins. Rctum to45 O/oBin 1.50 mins. Hold at45 0/0 for3.5mins. Injectiovnolume: 10 ;LL Injection/ssample: I Electrospraymass spectrometer bhcromass PlatformR API Mass Spe=meter. MassLynx 2.4 Software -Chick- 05/07/98 2 cone voltqw: -60V Mode: decbwray neptivc Sourcetemperature: 115 OC. Analy= vacuum pressurm:0.000043mbar, 0.000079mbar IDns:499.413,369 Electmde:crws-Ilow EJec&wpray AME hncrmm QuaMro H API Ntm Spectrometeurb,bddw MassLynx 3.1Software Cone voltna: -60v CoUWon ps enerw.45 V Mode: deWoWM neptin SourceUmpuature: 115 OC. An*= vacmim pmaum: 0.000043mbar, 0.000079mbar Pdmuy I= 4" Daugbier ions:W;199, 130,180,230 Electmde: Z-optay,. 05/07/99 3 inuorineAnalyticaClhemistryTeam conw. XJ. Hamen 8-6018 SamplesReceived: Samples Extracted: Samples Analyzed: 4/14/98 4/15/98 4/16-4/22/98 LOD': LOQI: Analysisbr. CalibratioCnum Rmg iAofCalibratioCnum10 ppb 10ppb ESMS 10ppb to96 ppb . 0.996 PHASE M part2: Blind repeatabiliteyxperiment Somnvolmeme@110 1A 213 2C 4A -SX SB 6B 7B SA VPFOS] (pob) 28+/-0.3 50+/-3 38+/-0.2 47+/-0.4 62+/-l 54+/-2 44+/-4 54+/-4 78+/-S ReportW Previoud 28 41 37"" 45 S4 49 42 4S 67 VPFOS] +Ob;ES9 9B 33V44+/-l 9c 477P+F/-9 10B 51+/-l 11 44+/-4 12 79 +I-4 13 70+/-S 14 79+/-26 16 37+/-2 17 80+/-14 Rqmrbd =gL 30 39 41 na. -n-.a. na. na. 32 73 QC Results QC ChedLCalibratioCnhock, 24.9 ppb CalibratioCnheck, 49 ppb CalibratioCnheck, 24.9 ppb CalibratioCnheck, 49 ppb CalibratiOownk, U.9 "b Cdibmdon Cho& 49 Mb % Recovery 96% 100% 92% 95% 90% 98% I -LOD - LimitofDetection 2 -LOQ - LimitofQuantitafion 3M EnvironmentalLab 3/7/98 PHASE ICK part2 3M EnvironmentaLlaboratoryF-luorinAenalyticCahlemistrT-evam KsisPranm -Sr.AnalydcdChernist FluorineAwlydol Ckmisuy Team IRiltildi2n-g3E-09 612-778-6018 Preliminaryresults:Analysisof POAA in human sera samples Summary: To date,datafrom nine phases of a su* offluorochemicianlhcinnan-sehmas been collected. PHASE I:Semi-quantitativhermdgadon ofPFOS in sampla ofpooled Ihilimsskeimn PHASE U: QuantitativeanalysisofPFOS and POAA in samples ofpooled human US sm samplc4 1997-1998 PHASE 13L-Quantiu&c analysisof PFOS 1998 PHASE TV Quantimm analysisofPFOS and POAA in himumd in sampla of Wdhidual's som US sem samplek 1970s-80s PHASE V.'QuantitaCm analysisof PFOS-M@ l@6@ US am samples, 1960s PHASF, VI: QuantiutiveanalysisofPFOS in samplesof sua fiam indi@ in rural northem China, 1995-1997 PHASE VU: CieogVhicd disftflmdon:a quantitativaenalysisof PFOS in seracollected around theUnited Suitt*,1998 PHASE VM: Quanti=w analysisof PFOS in butmud samplesftm SwedM specific date not suppliedto EnvirwTngntnl Lab PHASE M part 1:Quandtafm analysisof PFOS in historicaUlS samples, 1948-1951 PHASE M part2:PFOS levelsin indhidual'shuman serasampla; Blind RepeataWHty yveriment no analysisof perfluorooc=enffonate (PFOS) has be= theprimary focusin dww phases; quantitativdeataforpeinwmoctonoate (POAA) has alsobe= collectefdc)rseveralphasesof ft 0* Due totime constWnM we have not devoted the same attentiontothePOAA as we ba@v to the PFOS analysis The followingreportdescribesobsmvauons ofPOAA levelsin some of dw umples analyzed as partof thisongomg study Reportsdeudmg thePFOS analysislistedabovehave been issuedtoJeff Mandel inft 3M Medical Deputment LOQ forPOAA is10 ppb. PHASE 11-OuantitattyetwalysisofPFOS and POAA Number of sampleswith POAA > LOQ - 0 Number of samples with POAA > LOD - 4 Total munber ofsamples - 12 Comnmu: InLam lesofyooled human PHA SE IffOuantitattvaenalys7sgL PFOS and POAA Number ofsampleswithPOAA > LOQ - 4 Total number ofsamples - 31 Comments: Data has not been ammmed cardbuy inLiE@le_sofindividual'ssera PHASE IV- QLantitghve anal@gl ofPFOS inhistoricaslera LeL/es Number of wimples vvithPOAA > LOQ - 0 Total number of samples - 10 Comments: Data has not be= preciselyquantiuded 1970s-80s 04/24/98 PFL4SEV OuantitatainvayelsisofPFOSInhistoriscearlammle_.s,1960s NumberofsmiplewsithPOAA > LOQ - 0 Number ofsampleswithPOAA > LOD = 6 Totalnumber ofsamples- 6 Comments: Data hasnotbeenpreciselqyuantibod PHASE P7 OuantitativaenalymsofPFOS in1WR_le__sof-le-fraom indtvi&alsinrural northernChina Number ofsampleswithPOAA > LOQ - 0 Number ofsampleswithPOAA > IA)D - 5 Totalnumber ofsamples= 9 Comments: Data has notbeen preciselqyuantibued PHASE P71-Geographicaldistdbutiona: guantitafivaenahvisofPFOS collecteadround theUnitedStates Number of,samplawidiPOAA > LOQ - 2 (1at22 ppb, I at12pyb) Number ofsampleswithPOAA > LOD - 20 Totalnumber of ..p.,la 55 Comments. Data hainotbeenan on&gy insera PHASE VUl OuantitadavnvahsiOsLEM InhisoricsamlnvlefsromSweden Numberof=Wlw withPOAA > I.OQ- 0 Number ofsamplw withPOAA > LOD - 0 Totalnumber ofsainpla- 20 Comments: Data has notbeen preciselqyuandtacd PHASE IX gga 1:Qu_antitaitaynealysisofPFOS InhistoricaUlS mW-I-e-s1,948-19s] Number ofsampleswithPOAA > IA)Q = 0 Totalnumber ofsamples- 20 Comments: Data has notbeen preciselqyuantitged Experimentalsommarr. SamplepreparationI:on-pairing'e2&action In a pE contmuedenviromncmta,n !on-pairinrgeagentwuabutylammom*unk m-1 CIBA),is usedtoamracttheawlytefromthematnx Ammw wmpunds, Mm PFOS and (POAA), arewlecdvdy Wgded by thecadanicreagenl Subwqtumttothehonadon oftheTRA-anion pair,theanalyteisunnsfayedtoa non-polarorgamc sowm (ethyalcotgo)&,14 and reconsbuftdin methanolforNE analysi& HPLC. Chffwterisfircetentiohnmesfor POAA InBPLC, an aliquootftheckum n mjectedand passedOmagh a 00hunn. Based on theaffiwtyoftheanabu forthesudonary-phaninthecohnnn rdada totheHquidnwbile- phasepasung throughthecohmn, theanabu is remniedfora characunstimcmot oftune For CULMplelina standardsolufimPOAA may elutoat10.5minutes.Rdmdon timesbetweena sumdard POAA sohitm and theanalytocxbactedfromseram thisan*m weremached towithin1% on the HPLC qftm E&2& DetectioanndmonitonngofthemoleculaIron AnalysiosfPOAA stanaaridn-dcicatdeisaitheprimariyondkwacterisotfiPcOAA ismiz 413 amu, correspondintgothemass oftheanionicmubctant (C,7FsCOr).This ionwas monitoredwlecdvely toma2ritnisemidvity.Frap=tation ofthePOAA anioncanbe in&xw; ion369,cha=ctaistiocfthe 04/24/98 2 dmftxylation productr,esultsB.oth ionswere monitoredinmostofthesu* phaseslisteadbove,A smn ofm/z--100to 1210 (negativeonly)was alsocollected. EMDSUS.- Confirmationof analyteidentification Severalsamples inthissetwere analyzedby FSNLSI@M to verifythe identityof thePFOS anabu ion.ES-NL%S isvery sumlar to ESNS, exceptthatitadds an addition&dimension of certaintyto compatmd identificatioArslinESNO, a compound spedficion issclectc&Afterscl=don, theselected ioniscbaracwdzed hwther by smashing itapartwith high energygas. As a r=Wt ofthesmasbinz ionic ftapients,characteristoifcthe molecule,arecreatedand dcu=te& For example,for PFOS analysisi,on 499 isselectedasthecompound spwfficprimary ioillids ionissmashed intootherionssuchas 80 2mu (correspondingtoS031, 99 airnu(coffespon(Ungto FS03), 130 amu (correspondintgo CF2SO3), 180 skmu (C2F4SO31, and 230 2mu (C3F6S%). IfPFOS ispresent inthesamplm eachofthesesecondaryftp=U isdmcw atthe&ft=r. Qualitycontromln=mt: When possibles,eraumples w= cxuactedinduplicataend analyzedindupl=ft. Oftenthis was notpossg)lca,ssamplesnze and/orum were lumted. Methawl blanimwm analyzedperiodically toensurecompleteLwlationofthen@aple.)&d-levclqu-W* gonuol =Wles w= analyzedpuiodWaUy ihmgh each analyticasleque=, typicalleyveryfivesankplm tomoidtorbubument sbbffityB.e== bitirnsaenravatboutPFOS isnotavailables,amaplaofpooledserapurchasedfrom Sig= were proocuact4 spflwdvhtha standardconcentratioonfPFOS, and re-odracted.Thispre-exuacted8= b understoodtobe verysimil2tothesample matrix In allcasesPFOS was evaluatedvmw an ckuact* for,mostphasesofthissb*, POAA was alsospflwdand extractetdoform a sbuward curve In then cases quantitadonofthesimplesisbasedon thelinea mgmsdon equationderivedfrm theavmp of thetwo curvesbracketingthe samples. Limitof Quantitaflon The currentanabtkml method has been used to evaluatea UM mp of PFOS Imb axuwftd from =a, themethod isnotoptimizedforprocim I(yw-kvd(< 5 ppb)quantimfim but insteadA)r sampleswithPFOS concentrationisnthermp of20-60ppb. A speciallow4evelcurveh.as be= for prcclw quantitatievvealuatioonfPFOS kvds betwm 1-10ppb. As thePOAA levels observedinnon-plantworka wra areverylow,in futm analysm POAA willbe.evaluateadgain a special low-lcvtl curvr, sim,12 to thatpreparedforPFOS. Samples designated<.OQ containlessthan 10 ppb POAA. Iffiutheraccuracyand precisionin quantitadonofthesm nmplas (sampleska dm 10 ppb) isrequireds,ampleswillbe re-evaluated versusa speciallow-levelquantitativceurve, Limitofdetecfion The hnut of d&Amon forthisanalyticamlethod n based on analystrempition of the distinct peak shape resultingfrom POAA analysis.POAA stuxiardnu&jw and POAA extractedfrom nm analyzedby theconditionsreportedhoM show nearb@wji resolutiodn Ibranched and I lin POAA isomer.The al= ofmch peak in theHPLC4r= o=rs ata qmdfic,reprod=ble rdmft dm Unlikc,PFOS, and theratiofthebranchedpeak tothelin peak isnot necessarilcyonsistent particularlaytlow level& POAA, when present,seem tobe presentatmich low levelsthataccurate on ofthesmallerpeak (thebrandied isomer)isverydifficult. Qu&Wicadons: Tiusdatawillbe consideredprelindnaryuntiltheanalyt cxbactedftm thesesampks can be venfiedasPOAA usingLC-MShg whmques and untilunuumental senutivitcyan be ophnuzed tD MM clearlyidentiftyheangytc responw. The method used ADrtheanalysiosfdme ampla hasbeen validatebdy LC-MSNM previouslybut wn&=on ofPOAA forspeaficsamplesinthiseight-phan 0* has notbe= pedormed. 04/24/99 3 InstnamentalSPCMCI: BPLC system HewlettPadmrd Serie1s100Uquid Column:KcysWne BdasilCis 2 x 100 mm 5 pm parficlseize Flow rate: 300 itTImin SolventA. 2.0mM Ammonhm Solv=tB: 14ethanol SolventCTrAffient 45 to 90 'Y*Bin 9.50 mins. Hold at90 O/oBfor3.30minl Returnto45 'YeBin 1.50 Hold at45 %B for3.5minl Injectiovnolume:,10 )iL Injecdm /nmpla: 2 Acebft -Elecbwpay massVectrometer M=mam Plodom H APIMass SpecUomeM, -CWW MassLynx 2.4Software Cone volUM: -20v,-&)v Mo&: elc=Mrq m-ga&c So= temperamm: 115 OC Analym vacuum pressures0:.000043mbar, 0.000079mBa Ions:369,413;499,427 ElecaWe: crawflow EJec&osprayMSMS bfimmass Quawo H API Mass SpecuonitteIr&,ddine MassLynx 3.1Software Cone voltwo: -20v Collisiopns enaw. 45 V Mo&: clechvspranyega&e Sourceten4)erAUm: 115 OC Aralyzervacuum pi mm: 0.000043mbar, 0.000079mbar Pdm" I=- 413 Daugbter iom: 119,169,369 Mactrode: Z-spray z 04/24/98 4