Document KRVyaVEON05pnKK1MMkq0OEjx

AOVANCEO BIOANALY71CAL SERVICES, INC. TWE-Z-IlnlAI,Y49 ANALYTICAL REPORT K0iv 1 9 NIO, TITLE: QUALITATIVE INVESTIGATION OF THE IN VITRO METABOLISM OF T-62929 T-62939 T-6294 AN]) T-6295 BY RAT AND HUMAN HEPATO- CYTES USING ION SPRAY LC/MS AND LC/MS/MS DATE: November 12, 1996 REPORT: 96ADEM01.3M AUTHORS: Daniel E. Mulvana, B.S. Jack Henion, Ph.D. PREPARED FOR: NO. OF PAGES: 3M Medical Department, Toxicology Services, St. Paul, MN 55133 45 15 Catherwood Road a Ithaca, New York 14850 n (607) 266-0665 o Fax (607) 266-0749 2 ADVANCED SIGNATURE BIOANALYTICAL PAGE SERVICES Title: QUALITATIVE INVESTIGATION OF THE IN VITRO METABOLISM OF T6292,T-6293,T-6294 AND T-6295 BY RAT AND HUMAN HEPATOCYTES USING ION SPRAY LC/MS AND LC/MS/MS Reported by: DanielE. Mulvana,B.S. Date SeniorResearchScientist Reviewed and Approved by: Mary Ringwood, BQ Date QualityAssuranceManager Authorized for Release by: 1157@ Jack Henion,Ph.D.(@'q Date LaboratoryDirector Advanced BioAnalyticaSlervices AOVANCED BICANALYTICAL SERVICES, INC. REPORT: 96ADEMOI.3M QUALITATIVE METABOLISM AND HUMAN LC/MS/MS 3 INVESTIGATION OF THE IN VITRO OF T-6292,.T-6293, T-6294 AND T-6295 BY RAT HEPATOCYTES USING ION SPRAY LC/MS AND ABSTRACT The in vitrometabolism of T-6292, T-6293, T-6294 and T-6295 was investigated usinga sensitivaend highlyspecifiacnalyticamlethod developedby Advanced BioAnalyticaSlervicesI,nc.,IthacaN,Y. Thismethod involvesa simpledirect injectioLnC/MS analysisof 'processedhepatocyteincubate. Siliconized polyethylenteransfepripetsand polypropyleneautosamplervialswere used forall samples.LCIMS andLC/MS/MS experimentwsere performedinthenegativeion mode usingan ionsprayinterfaceC.hromatographywas performedon a 2 rinnx 100mm BetasilODS column usinggradienetlutiownitheitheraqueous ammonium acetatweithmethanolor waterwith methanol.Using a directinjectiomnethod, ratherthanone usingextractioenn,ableddetectioonf a wide varietyof metabolites ina singlienjectioInn.cludedinthisreportaremetabolismdatafrom individuarlat and human samples.Severalmetabolitewsere foundintheratand human samples and differencewsere observedbetweenthespecies. AOVANCEO BIOANALYTICAL SERVICES, INC. REPORT: 96ADFMOI.3M 4 TABLE OF CONTENTS SIGNATURE PAGE 2 ABSTRACT PAGE 3 1- INTRODUCTION 7 1.1 Introduction 7 1.2 Study Objective 7 2. EXPEREQENTAL 7 2.1 Chemicals and Materials 7 2.2 LC/MS Instrumentation 7 2.3 StandardSolutions 8 2.4 HPLC Eluent Preparation 8 2.5 Sample Preparation 8 2.6 Sample Analysis 9 2.7 Data Handling 10 3. RESULTS AND DISCUSSION 11 3.1 Mass SpectrometriRcesultsforTestArticles 11 3.2 Metabolism by Rat Hepatocytes .11 3.3 Metabolism by Human Hepatocytes 15 4. CONCLUSIONS 16 5. REFERENCES 16 FIGURES Figure 1. Structureosf thestudytestarticles. 17 Figure2. Negativeionizatiomnass spectraobtainedfrom thetestarticleTs-6293 ([M- H]-= 650.1),T-6294 ([M-H]-= 526.1)and T-6295 ([M-H]-= 498.9). 18 Figure3. Production spectraobtainedfrom the deprotonatetdestarticleTs-6293, T- 6294, and T-6295. 19 Figure4. Proposed CID fragmentatiomnechanism forT-6293. 20 Figure5. Proposed CID fragmentatiomnechanism forT-6294. 21 Figure6. Proposed CID fragmentatiomnechanism forT-6295. 22 ACVANCEO BIOANALYTICAL SERVICES, INC. REPORT: %ADEMOI.3M 5 Ficru7r.e Totalion and extractedfragmention chromatogramsforT-6292 obtained from Rat ControlSample 13. 23 Figure8. Totalion and extractedfragmention chromatogramsfor T-6292 obtained from Rat 1,0-Hr sample. 24 Figure9. Extractedion chromatogramsof fragmentpeaks for T-6292 homologous seriesinRat 1,0-Hr sample. 25 Figure 10a. Proposed structuroef parentcompound forT-6292 homologous seriesfrom Figures8 and 11. 26 Figure 10b. Proposed structuroef glucuronidefor homologous seriesfrom Figures 12 and 13. 26 Figure 11. Totalion and extractefdragmention chromatograms for T-6292 obtained from Rat 1,6-Hr sample. 27 Figure12. Extractedion chromatogramsof putativeglucuronidepeaks for T-6292 homologous seriesinRat 1,6-Hr sample. 28 Figure 13. Extractedion chromatogramsof putativeglucuronidepeaks for T-6292 homologous seriesinRat 1,0-Hr sample. 29 Figure 14. Proposedstructureosfthesulfonamideand carboxylicacidmetabolitesforT- 6292 from Rat 1. 30 Figure 15. Extractedion chromatogramsof sulfonamide(nVz 498) and carboxylicacid (nVz 584) metabolitefsorT-6292 from Rat 1,0-Hr (top)and 6-Hr (bottom) samples. 31 Figure16. Production scanforcarboxyliaccidmetabolitoef T-6292 in Rat 1, 6-Hr sample and proposedfragmentstructures. 32 Figure17. Total ion and extractedparent(nVz 650) and proposed metaboliteion chromatograms forT-6293 obtainedfrom Rat ControlSample 13. 33 Figure 18. Totalion and extractepdarent(M/z 650) and proposed metaboliteion chromatograms forT-6293 obtainedfrom Rat 1,0-Hr sample. 34 Figure 19. Totalion and extractepdarent(nVz 650) and proposed metaboliteion chromatogramsforT-6293 obtainedfrom Rat 1,6-Hr sample. 35 Figure20. PossiblestructurefsortheobservedmetaboliteosfT-6293 with [M-H]- of 542 and 556 amu from Rat 1. 36 Figure 2 1. Extractedionchromatogramsof sulfonamide(nVz498) and sulfonate(nVz 499) ionsforT-6293 fromRat 1,0-Hr (top)and6-Hr (bottom)samples. 37 Figure22. Extractedionchromatogramsof proposed metabolitewsithnVz 327 and 419 forT-6293 from Rat 1,0-Hr(top)and 6-Hr(bottom)samples. 38 AOVANCED BIOANALYTICAL SERVICES, INC. REPORT: 96ADEMOI.3M 6 Figure 23. Total ion and extractedparent(m/z 526) and proposed metaboliteion chromatograms forT-6294 obtainedfrom Rat ControlSample 13. 39 Figure 24. Total ion and extractedparent(m/z 526) and proposed metaboliteion chromatograms forT-6294 obtainedfrom Rat 1,0-Hr sample. 40 Figure25. Total ion and extractepdarent(m/z 526) and proposed metaboliteion chromatograms forT-6294 obtainedfrom Rat 1,6-Hr sample. 41 Figure 26. Product ion scan for perfluorooctanesulfonamide metabolite (CF3(CF2)7So2NH2, m/z 498) of T-6294 inRat 1,6-Hr sample and proposed fragment structures. 42 Figure 27. Totalion and extractepdarent(m/z 499) and homolog ion chromatograrnsfor T-6295 obtainedfrom Rat ControlSample 13. 43 Figure 28. Totalion and extractedparent(nVz499) and homolog ion chromatograms for T-6295 obtainedfrom.Rat 1,0-Hr sample. 44 Figure29. Totalion and extractedparent(m/z499) and homolog ion chromatograms for T-6295 obtainedfrom Rat 1,6-Hr. 45 AOVANCED BIOANALYTICAL SERVICES, INC. REPORT: 96ADFMOI.3M 7 INTRODUCTION 1.1 Introduction The invitrometabolismofT-6292,T-6293,T-6294 and T-6295 in ratand human hepatocytiencubateswas investigatuesdingion spray LCFMS and LC/MS/MS. The quenchedincubatewsere directliynjecteadnd separatedon an HPLC column. Chromatographicresolutioonf thevariousconstituentwsas accomplishedusing gradientelutionwithmethanoland eitherwateror 2 mN4 ammonium acetatein water.The mass spectrometewras operatedinthefullscan(M/z 100to800) single MS mode and peaks of interest'wetrheenstudiedby MS/MS techniquesfor confirmatioonfselectemdetabolitpeeaks. 1.2 Study Objective The objectivoef thisstudywas todetectand characteriztehemetabolismofT-6292, T-6293,T-6294 and T-6295 by ratand human hepatocytes. 2. EXPERIMENTAL 2.1 Chemicals and Materials T-6292,T-6293,T-6294 and T-6295 (Figure1)were providedby 3M Medical Department,ToxicologyServicesS,t.Paul,MN 55133. Siliconizepdolypropylenaeutosamplervialc:at# 500 102,Sun Brokers,Inc., WilmingtonNC 28402.Siliconizbeyd EaglePicherIndustrieIsn,c.M,iami, OK 74354 Siliconizepdolypropylensecrew-captubes:16 X 101 mm, Cat# 60.541,Sarstedt Inc.,Newton, NC 28658,siliconiziend-house. 2.2 LC/MS Instrumentation Liquid chromatographywas performedusing eithertwo Shimadzu LC-IOAD pumps (ShimadzuScientifIincstrumentsI,nc.,Columbia,MD 21046) with a WatersWISP 717 plusautosample(rWaters,MilfordMA 01757)or a HewlettPackard1090L liquicdhromatograph(Hewlett-PackarAdv,ondale,PA 19311).A BetasilC18 column,2 mm x 100 mm, was obtainedfrom KeystoneScientific, AOVANCEO BIOANALYTICAL SERVICES. INC. REPORT: 96ADF-MOI.3M 8 Inc.,BellefonteP,A 16823. The mass spectrometerussed were an API III'and API 300 from PE-SCIEX, Concord,Ontario. 2.3 Standard Solutions Sincecertificatoefsanalysiswere not availablfeor the testcompounds, 100% puritywas assumed,althoughinthecaseofT-6293,thepuritywas known todiffer significantflryom thisvalue. Stocksolutionosf T-6292,T-6293,T-6294 and T6295 were preparedata concentrationf 10 mM indimethylsulfoxide.Working solution(s10 gg/mL) of each standardwere preparedin 1:1 acetonitrilmeM:2 ammonium acetate.AU stocksolutionasnd working solutionswere storedat4'C and were allowedtoequilibratteoroom temperaturebeforeuse. Preparation of AnalyticalStandard Stock Solutions Stock Solutionsof T-6292, T-6293, T-6294 and T-6295 (10 mM): Accuratelyweigh eachtestarticloen a micro balanceand transfeirtto a 16 X 101 nun siuconizepdolypropylenetube. Dissolvethe solidsample in an appropriate amount ofDMSO toyielda 10mM solution. Working Solutionsof T-6292, T-6293, T-6294 and T-6295 (10 gg/mL): Dilutean appropriataeliquootf 10mM stocksolutiotno 10 mL with 1:1 2 mM ammonium acetataec:etonitrilnea 16 X 101 mm siliconizepdolypropylene tube. 2.4 HPLC EluentPreparation 2 mM Ammonium acetateA:nunonium acetate(154.2 mg) was weighed and transferreidntoa I litegrraduatedcylinderA.fteraddingwatertothe I-L mark, the stiffesdolutionwas filtereudnder vacuum througha 0.45gm filter. Methanol:Methanol was filtereudnder vacuum througha 0.45Jimfilter. 2.5 Sample Preparation The metabolismstudywas carriedout atSRI InternationaMle,nlo Park,CA. The incubateswere quenched with an equalvolume of ice-coldmethanol,centrifuged and shippedpacked indry iceto Advanced BioAnalyticaSlervices(ABS). The sampleswere storedatABS at-20*C and were removed only for transfeorf [)VANCEO BIOANALYTICAL SERVICES, INC. REPORT: 96ADEMOI.3M 9 supematant to an autosamplervial. Aftersampling,theautosamplervialswere maintainedinan ice-waterbathatalltimesexceptduringinjection. 2.6 Sample Analysis The samples analyzed includedRat I (alltestarticles0, and 6-Hr and control sample 13 (no testarticlea)n)d Human #H-1 16 (alltestarticles0,and 6-Hr). These were extensivelystudiedunder a varietyof chromatographicand mass spectrometricconditions. HPLC Conditions: InitiaHlPLC analyseswere successfullpyerformed using gradientelutionwith waterand methanol only. Laterinthestudy,itwas found that ammonium acetatewas requiredfor elutionof T-6293. Thus, water was replaced by 2 mM ammonium acetateforfurtheranalyticawlork. Because two different gradientpumps were used forthiswork, itwas difficultto reproducethe gradient obtainedusing theBP-1090L with theShimadzu LC-IOAD, thus for consistency withingradientsystems,the same gradientprogram was used for both systems. Although thisresultedin some variabilitiyn retentiontimes between days, depending on the system used,allcomparable samples were analyzedwith the same system. GradientProgram: Time (Min) 0 5 9 10 15 %Methanol 20 95 95 20 20 %Water (2mM Amm.Acetate) 80 5 5 80 80 Mass SMtrometer Conditions: The mass spectrometerwas operatedin the ion spray mode with negativeion detection.The curtaingas was ultra-higphuritynitrogen. Although MS and MS/MS conditionswere variedfor differenatnalytest,hemain differencewsere in ion sprayvoltage,orificveoltageand collisioennergy.No positiveionizationwork was performed,as theanalytesof interesdtid not ionizeunder theseconditions.It AOVANCED BIOANALYTICAL SERVICES, INC. REPORT: 96ADEMOI.3M 10 is conceivablethatadditionamletabolitecsould be detectedusing positive ionizationa,nd thisshouldbe a partoffurtherexperimentation. The approximateMS and MS/MS acquisitiopnarametersused fortheAPI HI' are listebdelow. Similarparameterswere used forthework performedon theAPI 300. MS Mode MS/MS Mode IonSpray Voltage -3600 V -3600 V OrificVeoltage -70V DeclusterinPgotential 42V -70V 42V CurtainGas (U.H.P.N2) 1.2LPM 1.2LPM NebulizerGas (N2) 60 PSI 60 PSI CollisioEnnergy 18 eV CGT Scan Range 100-800 amu 250 variable Step Size seeNote below variable Dwell Time 3 msec 4 msec Note: InitiaLlC/MS stepsizesof I amu were usedtolocatethechromatographic peaks of potentiamletabolitesO.nce located,stepsizesof0.1 amu were used for mass confirmation. Instrumentmass axiscalibratiownas performedby infusionofPPG-425 calibration solutio(npolypropylengelycols,averagemolecularweight425, dissolvedin 1:1 methanol:2niM ammonium acetatecontaining0.1% fomiic acid and 0.1% acetonitrialtea)flowrateof 10 pumin inthepositivieo'mnode of detection.Peak widthswere approximatel0y.6 amu at half-heighitn thesingleMS mode. A calibratiocnheckwas performedfortheanalyteson Ql and Q3 by infusionof a 1 ggftnlsolutionin 1:1 acetonitrilmeM:2 arrunoniumacetataeta flow rateof 10 gumin. The mass spectrometripcarametersand sensitiviwteyre optimizedusing thissolutioant50 pljhiin. 2.7 Data Handling Allraw chromatographiacnd mass spectrometridcatawere storedon theABS file serverABSI-FS.DATA inthefilehierarch"yRA)VDATA: Development:3M: 3M AOVANCED BIOANALYTICAL SERVICES, INC. REPORT: 96ADEMOI.3M Metabolism".All pertinenetxperimentalinformationwas storedin ABS notebook No. 1067. 3. RESULTS AND DISCUSSION Initiailnvestigatioonfstheinvitrometabolismofthetestarticlewsere performed usingdirectinjectioonf thequenched hepatocyteincubatesratherthan sample extraction.The reasonforthisisthat,due tothepolarnatureofthesecompounds and suspectedmetabolitesi,twas feltthatextractiornecoverieswould be low and importantmetabolitescould be missed. Most ofthemass spectrometricinformation obtainedinthisstudywere from Q I scanningexperiments,followedby production scans.Precursorion scansof logicalCID productionswere used but resultedonly inconfirmationofpreviouslyfound metabolitespresentathighlevels.Neutralloss experimentswere even lesssensitivuender theconditionsused and provided little information. 3.1 Mass Spectrometric Analysis Results for Test Articles Structuresof T-6292, T-6293, T-6294 and T-6295 are shown in Figure 1. The negativeionizatiomnass spectraobtainedfrom infusionof the individualanalytes areshown inFigure2. Itshouldbe noted thatT-6292 could notbe ionizedineither thenegativeionizatiomnode, sinceithas only a very weakly acidicalkylhydroxyl group or thepositiveionizatiomnode, sincethemost basicsiteisa tertiarnyitrogen of a sulfonamide. The factthatT-6293 existsmainly in the diphosphoestefrorm' (K =1204), comprising82% of T-6293 solids,accountsfor the reducedresponse seen for the monoester (M, = 65 1),when compared toT-6294 and T-6295. The diesterwas not detectableunder theconditionsused inthisstudy.The negativeionizatiocnollision induceddissociatio(nCID) full-scasnpectraof the compounds are presentedin Figure3. T-6295 was found tobe extremelystableand requiredboth a high orifice voltageand high collisioennergyforfragmentation.A summary descriptioonf the proposed plausibleCIODfragmentationpathways areshown in Figures4-6. Several of thesefragmentswere diagnosticformetaboliteidentification. 3.2 Metabolism by Rat Hepatocytes Metabolism ofthetestarticlebsy rathepatocyteswas examined using a Sciex API III'mass spectrometer. in M AOVANCEO BIOANALYTICAL SERVICES, INC. REPORT: 96ADEMOI.3M 12 3.2.1Metabolism of T-6292 by Rat Hepatocytes AlthoughT-6292 did not ionizeunderthe conditionussed,and was thus not detectablien theseexperiments,severalpotentiamletaboliteswere nonetheless found. When comparingthe totalion chromatogram(TIC) obtainedfrom the controlratsample (Figure7, top;no testarticlaedded)with thatobtainedfrom the Rat 1,0-Hr sample (Figure8, top,reactioqnuenchedatapproximatelytime0), severallargepeaks areobservedinthelatteorver therangeof 7.2to 8.5 min which areabsentin the controlsample. Itshouldbe noted thatthereis a 0.5 min differencbeetweentheTICS, due to operatorerror;thecontrolTIC peaks are0.5 min earlierT.he 0-Hr peaks were'foundtocontainmasses in a homologous series which are compound relatedbut which may alsobe impuritieosr degradation products.Each of theobservedpeakscontainsmasses which differby 80 and 107 amu. An example isgiveninFigure9. Itissuspectedthatthedifferenitonswithin each chromatographicpeak were generatedby CID inthesamplingorificreegionof themass spectrometersinceno collisiognas was used. In theexample given,the lowestmass ion,rri/3z19, isa characterismtaiscs forperfluoroalkyclompounds, namely, C6F,3, The second ion,ni/z426, is 107 amu higher,which has been previouslydescribedforthesecompounds as a lossofEtNS02 (Figure5). The ion observedatnVz 506 is 80 amu higher,which correspondto S03, a common loss under theseconditions.Thus,theproposedstructureosf thisand theotheranalogs areshown inFigure10a. Itmay'be somewhat surprisintghat0-Hr sample would containmetaboliteastsignificalnetvelsh;owever, forrapidreactionst,hesampling and quenchingatthistimepointmay be crucial. Summary extractedionchromatogramsof thefragmentions analogousto nitz319, differinognly by CF2 groups,are shown in Figure 8. As predictedn,o correspondingpeak appearsfornilz469 (C9F,9),sincethiswould exceed the perfluoroalkyclhainlengthofthepar-enmtolecule.Chromatographicpeak splitting isevidentin thelateelutingpeaks,probablydue to chromatographicresolutioonf thebranchedisomersofthesecompounds. CorrespondingspectrafortheRat 1, 6Hr sample (Figure11)indicatae decreasedresponseforthesecompounds, except for the parentCF,7 fragment(nVz 419). The controlsample containedno significanltevelsof theseions(Figure7). An additionahlomologous seriesof proposedmetaboliteosf theparentcompound and impuritiehsas been observedwhich appearto be glucuronicacidconjugates. MM M AOVANCED BIOANALYTICAL SERVICES, INC. REPORT: 96ADEMOI.3M 13 The masses ofthesecompounds match thoseof the0-glucuronides(Figure10b)of thesecompounds. Figure12 shows theextracteidonchromatogramsforthe [MH]- of the suspectedintacgtlucuronideisn the seriesfor Rat 1, 6-Hr. The glucuronideosf theC3 analogthroughtheparentcompound, the C8 analog,are presented.By comparison,the peaks are greatlydiminishedin the 0-Hr sample (Figure13)and absentinthecontrolsample(datanotshown). No evidencewas found forany of thepossibleN-glucuronides. An additionamlajor metabolitweas theN-di-dealkylatpeedrfluoroalkyslulfonamide (Figure14)withan [M-H]-ofm/i 498. For thiscompound, a homologous series was alsoobserved,butonlyfortheC7 and C6 homologs (datanot shown). A carboxyliaccidmetabolitweas alsofound (Figure14)with an [M-H]- of nVz 584. A homologous serieswas found for thismetabolitwehich includedthe C4-C8 homologs (datanotshown). Extractedionchromatogramsof theC8 homologs of thesemetabolitefsrom Rat 1,0 and 6-Hr arepresentedinFigure15. To support theproposed structuroef thecarboxylicacidmetabolitea,n MS/MS production scanwas performedforprecursorion nVz 584 (Figure16). The fragmentions formedwere consistenwtiththestructuren.e parention isnot observedbut a conceivablleossof thethree-memberedlactoneCH2C02 from thecarboxylicacid anionwould producetheionatnVz 526. 3.2.2Metabolism of T-6293 by Rat Hepatocytes T-6293 alsounderwent significanmtetabolismby rathepatocytes.'Me TICs obtainedfrom thecontrolsample,Rat 1,0-Hr and Rat 1,6-Hr arepresentedatthe topof Figures17,18 and 19,respectivelyA.s withT-6292,a 0.5 n-@ndifference existsbetween theTICS,due to operatorerror;thecontrolTIC peaks are0.5 n-iin earlierS.everalpossiblemetabolitewsere found intheratsamples. The extracted ionchromatogrwnsfortheanionsof thesemetaboliteasrealsoshown in Figures 17-19.None of themetabolitefsound in the6-Hr samplewere found ineitherthe 0-Hr or controlsamples.The parentanionatm/z 650 was found inboth the0 and 6-Hr samples,althoughreducedby halfinthelatter. PossiblestructurefsornVz 542 and 556 areshown inFigure20. Very littl"eup front"CID occurredforthesepeaks,thusMS/MS experimentswould be required to supportthe proposed structures.The observedretentiotnimesare consistent with theproposedstructuresw,ith the acidelutingearlietrhanthe alcohol.The ADVANCEO BICANALYTICAL SERVICES, INC. REPORT: 96ADEMOI.3M 14 metaboliteobservedwithan [M-H] ofm/z 584 was shown tobe thecarboxylicacid previouslyseenforT-6292 (seeFigure14)by aMS/MS production scan (datanot shown). The glucuronidperoposedforT-6292 was alsoseen in T-6293 (Figure 19,m/z 746). The ions498 and 499 were presentatsignificanltevelsinboth 0 and 6-Hr samples, althoughtheybothhad higherresponsesinthe6-Hrsample(Figure2 1).The Rely structureosf thesecompounds areperfluorooctanesulfon(amt/ez 499, T-6295) and perfluorooctanesulfonam(imd/ez 498). From Figure21, itcan be seen thatfor both0 and 6-Hr samples,thelatemr/z 499 peak coincideswiththe498 ion.This is believedtobe an artifadcute to theisotopicontributionf the ni/z498 ion and incompletemass resolutiobnetween m/z 498 and m/z 499. Additionallyt,he observedretentiotnimeforthefirspteak inthenVz 499 ion chromatogram iscloser to thatroutineloybservedfor perfluorooctanesulfontahtaen for the second (see Figure28). Thus, itisassumed thatthesecondnVz 499 ion is actuallMy/z 498 being detectedas nL/z499.The nVz 499 ion was found atsignificanthliygh levels in allcontrolsamplesexamined (datanot shown) atthesame retentiotnime as in thesesamples. Thus, whileitmay alsobe a productof metabolism,thereisan endogenous backgroundlevelas well. Two additionailonswere found intheTIC, m/lz327 and nVz 419 (Figure22).The ionatnVz 419 isa common fragmention (seeFigure4) and an easilyconceivable productof degradatioannd/ormetabolismwhile m/z 327 is more difficultto rationalizaend may be a productof"up front"CID witha parentionhigherthanthe scan range used. Furtherexperimentatiownould be required.In the control sample,neitherionwas detected(notshown). 3.2.3 Metabolism of T-6294 by Ra't Hepatocytes The TICs and extracteidonchromatogramsfortheproposed metaboliteosf T-6294 from thecontrol0,-Hr and 6-Hr samplesforRat I arepresentedin Figures23-25, respectivelyT.he parentcompound ([M-H]-= 526) was observedinboth 0 and 6Hr samplesatapproximatel8y.7 min. Severalmetabolitewsere found in therat hepatocytesamples for T-6294. The main metabolitewas perflurorooctane sulfonamide([M-H]-= 498,8.0min),theN-dealkylatiopnroduct.This metabolite was observedin boththe0 and 6-Hr samples,althoughitwas much largerin the latterA. MS/MS productionscanwas performedon the6-Hr sample forprecursor MMM ADVANCE[) BIOANALYTICAL SERVICES, INC. REPORT: 96ADEMOI.3M 15 ion m/z 498 (Figure26),and allobservedfragmentsupportedthe proposed structure.The stnicturefsor the radicalspeciesshown in Figure 26 are not proposed tobe themost stableconfigurationsT.he metaboliteobserved atnvz 483 (6.6min) ispostulatedto be thesame as thefragmentionproposed inFigure 16. The remainingmetaboliteastm/z 542 and 556 arepostulatedto be those in Figure 20,with oxidationoccurringon theterminalmethylgroup. 3.2.4 Metabolism of T-6295 by Rat Hepatocytes The controlblank,0-Hr and 6-Hr TICs for T-6295 in Rat I are presented in Figures27-29,respectivelyA.s previouslystated(Section3.2.2)t,he499 ion was observed observedin allcontrolsamples. The remainingions in the extractedion chromatograms aremembers of thehomologous seriesseenpreviouslyfor T-6292.. Itisdifficultto believethattheseareproductsof metabolismsincetheirformation would requiredeletionof CF2 unitsfrom the chain. Apparently,T-6295 is metabolizedminimallyby rathepatocyteincubates,asjudged by the high levelsof parentinboththe 0 and 6-Hr samples. 3.3 Metabolism by Human Hepatocytes Metabolism of the testarticlebsy human hepatocyteswas similarto thatof the rat but fewer typeswere seen.The human samples were examined using a Sciex API 300 mass spectrometerh,owever, the investigatiownas lessextensivethanthatof theratsamples. Furtherwork iscertainlwyarranted. Since no new metabolism was discoveredforthehuman samples,theresultswillonly be discussedusing text;no figuresarepresented. 3.3.1 Metabolism of T-6292 by Human Hepatocytes The homologous seriespresentedin Figure 10a was not observed in the human. Interestinglhyo,wever, theglucuronideseriespresentedin Figure 10b was seen in the 6-Hr samples. The sulfonamideinFigure 14 was observed atvery low levels and the acidinFigure 14 was not detectedatall. 3.3.2 Metabolism of T-6293 by Human Hepatocytes The parent ion was the only compound-related moiety seen in these samples; however, with optimizatioonf themass spectrometerfocused on T-6293, further informationshouldbe attainableB.ased solelyon abundance the parention isone- ADVANCED BIOANALYTICAL SERVICES, INC. REPORT: 96ADFMOI.3M 16 thirdlower inthe6-Hr sample thaninthe0-Hr sample, thus some metabolism may have occurred. 3.3.3 Metabolism of T-6294 by Human Hepatocytes The parent and sulfonamide metabolite(nVz 498, see Figure 14) were the only compound-relatedionsobserved in the samples. The abundance of the parention in the 0-Hr sample was reducedapproximatelyby halfin the 6-Hr sample and, conversely,the abundance of the sulfonamidein the 0-Hr sample was approximatelydoubled inthe6-Hr sample. 3.3.4 Metabolism of T-6295 by Human Hepatocytes There were no discemable differencebsetween the0 and 6-Hr sample forT-6295. 4. CONCLUSIONS Severalpotentiamletabolitesand impuritieswere determinedfrom theLC/MS data in the study. FurtherMS/MS experimentationp,ossiblyin conjunctionwith a sample cleanupand concentratiosntep,would be requiredto furthersupport the postulatedstructureisn theratsamples. Additionallyf,or the human samples, furtherhigh sensitivitdyetectionexperimentatioins required for complete preliminaryinvestigatiofno,llowedby MS/MS experimentatiofnor confirmationof the metabolitestructures. 5. REFERENCES 1. Gordon, StevenC.,Ph.D.,D.A.B.T.,HealthHazard Summary of A=onium SaltsofMono-, Di- and Tri[N-ethyl(perfluorooctane)sulfonarnidoethyplh)osphate(FC-807). September 14,1994. Suppliedby 3M Company. AOVANCEED BIOANALYTICAL SERVICES, INC. REPORT: 96ADEMOI.31%1 17 Figure 1. Structuresof the study testarticles T-6292 Mr= 571 0 F3C-CF2-CF2-CF2-CF2-CF2 CF2-CF2-S-< 11 0 CH2-CH3 CH2-CH2-OH T-6293 Mr = 651 0 ll_ F3C-CF2-CF2-CF2-CF2-CF2-CF2-CF2-19 < 11 0 CH2-CH3 0 11 CH2-CH2-0-P-OH 1 Uri T-6294 Mr= 527 0 1< F3C-CF2-CF2-CF2-CF2-CF2-CF2-CF2-S- 11 0 CH2-CH3 H T-6295 Mr= 500 0 11 F3C-CF2-CF2-CF2-CF2-CF2-CF2-CF2-S-OH ADVANCE[) BIOANALYTICAL SERVICES. INC. REPORT: 96ADEMOI.3M 18 Figure 2. Negative ionizationmass spectra obtained from the testarticles T-6293 ([M.Hl- = 650.1), T-6294 ([M-HI" = 526.1) and T6295 ([M-Hl- = 498.9) Spectrum from T-6293 ql.Ol UCL) l.8e6. 220.0 1.2e6.E 6.Oe5- 255.2 650.1 1.95e7 cps 200 360 400 Spectrum from T-6294 ql.02 co 9.Oe6CLL) 6.Oe6- 3.Oe6- Soo 600 m/z, amu 526.1 700 1.96e7 cps 2@O 380 400 5;0 600 m/z, amu Spectrum from T-6295 ql.03 9.Oe6- 498.9 CL 6.Oe6- -E 3.Oe6220.0 399.1 200 300 460 560 600 m/z, amu 700 1.93e7 cps 700 ADVANCED BIOANALYTICAL SERVICES, INC. REPORT: 96ADEMOI.3M 19 Figure 3. Product ion spectraobtainedfrom the deprotonatedtestarticles T-6293, T-6294, and T-6295. T-6293 Product Ion Scan 10() Z@ 7550- 251 123 cc 0 1 4 li 100 200 495,000 526 650 419 I- 300 400 500 600 700 rrl/z T-6294 Product Ion Scan 10D 75- 50CD 25 126 100 169 219 200 269 300 ni/z 419 400 405,000 526 500 600 T-6295 Product Ion Scan ioa 2t, 75. 50. (D 25 a: 0 1 80 99 1, 1 100 130 11 169 11 230 I- 1 - 200 280 -, i 300 rrvz 330 41. 40* 0 1,620,000 499 I 500 ADVANCEO BIOANALYTICAL MMM SERVICES, INC. REPORT: 96ADEMOI.3M 20 Figure 4. Proposed CID fragmentationmechanism for T-6293. T-6293 m/z 650 F3C-CF2-CF2-CF2-CF2-CF 0 1 CH2-CH3 0 2-CF2 -CF2-S-< ii 11 -CH2-0-P-OH 0 PC@H (Ox (124amu) m/z 526 F3C-CF2-CF2-CF2-CF2-CF2-CF2-CF2-S Et-N=S02 (107 am u) 0 CH -CH3 -N 0 m/z 419 F3C-CF2-CF2-CF2-CF2-CF2-CF2-CF2 AOVANCEO BIOANALYTICAL SERVICES. INC. REPORT: 96ADEMOI.3M 21 Figure 5. Proposed CID fragmentationmechanisms for T-6294. T-6294 m/z = 526 F3C-CF2-CF2-CF2-CF2-CF2-CF2-CF2-S 11 14-@0 H2-CH3 0 F-S-N CH2-CH3 Et-N=S02 (107amu) M/z 126 (pathway notshown) m/z = 419 d@ c b@ a@ F3C-C;F.2;-CfF,;I-rC?F, -CF -CF2-CF2-CF2 d c F3C-CF2 M/z = 119 F3C-CF2-CF2- m/z = 169 \a a b F3C-CF2-CF2-CF2-CF2 F3C-CF2-CF2-CF@- m/z = 269 m/z 219 ADVANCED BIOANALYTICAL SERVICES. INC. REPORT: 96ADF-MOI.3M 22 Figure 6. Proposed CID fragmentationmechanisms for T-6295. T-6295 m/z 499 F3C-CF2-CF2 -CF2-CF 2-C,IF2-CF2- 0 11 *CF',2 -)s 0 - Pathway A Pathway B: Homolyticcleavages along perfluoroalkcyhlain S03 (80 amu) m/z = 419 d b@ a F3C-CF CF CF CF -CF2-CF2-CF2 /dd c F3C-CF2 m/z= 119 F3C-CF2-CF2- m/z = 169 \aa b F3C-CF2-CF2-CF2-CF2 F3C-CF2-CF2-CF@- M/z 269 m/z = 219 CF2(CF2)4SCF3 m/z = 330 CF2(CF2)3SCF3' M/z = 280 -CF2(CF2@S03- m/z = 230 CF2CF2SO@ CF2SO3 -S03 m/z = 1'80 m/z = 130 M/z = 80 0 11 F-S-0 0 m/z = 99 (pathwaynotshown) ADVANCED BIOANALYTICAL SERVICES, INC. REPORT: 96ADEMOI.3M 23 Figure 7. Total ion and extractedfragment ion chromatograms for T6292 obtainedfrom Rat Control Sample 13. TIC ofallmasses, 100 to 800 controlsample 13,no TA too- 75- 50- > 25- n 1 101 0.0 2.5 controlsample 13,no TA 169/169 201 301 4.9 7.3 Scanrrime(min) 2,805,000 401 9.7 590,000 219/219 110,000 269/269 A@ 319/319 . - . 45,000 10,000 369/369 590,000 419/419 - - 469/469 0.0 2.0 1 84 ACDVANCED BIOANALYTICAL SERVICES. INC. 55,000 i -A boAcibmiL 590,000 4.0 6.0 165 248 Time (min)/Scan 8.0 10.0 331 414 REPORT: 96ADEMOI.3M 24 Figure 8. Total ion and extractedfragment ion chromatograms for T6292 obtained from Rat 1, 0-Hr sample. TIC of allmasses@ 100 to800 T-6292 Rat 1,0-Hr,lo ulini 100. 75- 50- 25 1.4 cc n- c--1 0.0 1691169 3. 101 2.4 5.1 39 201 4.8 ScanMme (min) 8.4. 75 5,090,000 301 401 7.2 9.6 7.25 820,000 219/219 7.54 765,000 269/269 319/319 7.75 8.09 485,000 645,000 369/369 4191419 469/469- 833 FL 185,000 605,000 820,000 0.0 2.0 4.0 6'0 8.0 10.0 1 84 167 250 333 416 Time (min)/Scan AOVANCED BIOANALYTICAL SERVICES, INC. REPORT: 96ADEMOI.3M 25 Figure 9. Extractedion chromatograms of fragment peaks for T-6292 homologous seriesin Rat 1, 0-Hr sample. T-6292 Rat 1,0-Hr,10 ulini 319/319 10(@ 7EP cc 25 426/426 1 00@ 75 5G cc 29 506/506 100 7.5 50 cc 2 0.0 2.0 1 84 4.0 6.0 8.0 167 250 333 7ime (min)/Scan 645,000 125,000 280,000 10.0 416 ADVANCECD BIOANALYTICAL SERVICES, INC. REPORT: 96ADEMOI.3M 26 Figure 10a. Proposed structure of parent compound homologous seriesfrom Figures 8 and 11. for T-6292 0 1 F3C-(CF2)n-CIF'2-S< 11 0 CH2-CH3 03- Mass ofParentAnion n 356 1 406 2 456 3 506 4 556 5 606 6 CorrespondingFragment Ion in FiL-ures8 and II 169 219 269 319 369 419 Figure 10b. Proposed structureof glucuronide for homologous series from Figures 12 and 13. 0 11 F3C-(CF2)n-CF2-S-< 11 0 CH2-CH3 CH2-CH2-0--GIU- Mass of Parent Anion n 496 1 546 2 596 3 646 4 696 5 746 6 ADVANCED BIOANALYTICAL SERVICES, INC. REPORT: 96ADEMOI.3M 27 Figure 11. Totalion and extractedfragmention chromatograms for T. 6292 obtainedfrom Rat 1, 6-Hr sample. TIC ofallmasses, 100 to800 T-6292 Rat 1,6-Hr,10 ulini 100 75 50, 25, cc n- 1 101 0.0 2.4 169/169 201 301 4.8 7.2 ScarvTime(min) 7.27 4,295,000 401 9.6 575,000 219/219 269/269 319/319 A k,4@- 7.49 IL IL 8.05 195,000 160,000 170,000 369/369 4191419 469/469 8.33 A=A 200,000 840,000 840,000 0.0 2.0 4.0 6.0 80 10.0 1 84 167 250 333 416 Time (min)/Scan ADVANCE[D BIOANALYTICAL SERVICES, INC. REPORT: 96ADEMOI.3M 28 Figure 12. Extractedion chromatograms of putativeglucuronidepeaks for T-6292 homologous seriesin Rat 1, 6-Hr sample. T-6292 Rat 1, 6-Hr,10 ulinj 496/496 lo 50 a: 0 546/546 lool 50cc 0. 596/596 0 50 cc 0 646/646 lo 50 0 696/696 10 50 cr_ 0 746/746 lool 50, CD 0- 0.0 2.0 1 84 5.56 5.97 6.21 6.43 6.57 6.69 4.0 6.0 8.0 167 250 333 7ime (min)/Scan 155,000 180,000 50,000 100,000 10,000 90,000 10.0 416 MMM AOVANCED BIOANALYTICAL SERVICES, INC. REPORT: 96ADEMOI.3M 29 Figure 13. Extractedion chromatograms of putativeglucuronidepeaks for T-6292 homologous seriesin Rat 1, 0-Hr sample. T-6292 Rat 1,0-Hr,1o ulini 496/496 10 50 0 546/546 105,000 45,000 ioo50, 0. 596/596 100. 50- 10,000 0. 646/646 25,000 100, 50. ir 0. 696/696 10 50 01 746/746 105,000 10,000 10 50 cc 0.0 2.0 4.0 6.0 8.0 10.0 1 84 167 250 333 416 Time (min)/Scan ADVANCEO BIOANALYTICAL SERVICES, INC. REPORT: 96ADEMOI.3M 30 Figure 14. Proposed structuresof the anions of the sulfonamide and carboxylicacid metabolitesfor T-6292 from Rat 1. Sulfonamide Metabolite(m/z498) 0 11 F3C-(CF2)6-CF2-S-NH CarboxylicAcid Metabolite(m/z584) 0 I F3C-(CF2)6 -CF 2-S-< 11 0 CH2-CH3 CH2- 0 0 ADVANCED BIOANALYTICAL SERVICES. INC. REPORT: 96ADEMOI.3M 31 Figure 15. Extractedion chromatograms of sulfonamide (m/z 498) and carboxylicacid (ni/z584) metabolitesfor T-6292 from Rat 1, 0-Hr (top)and 6-Hr (bottom)samples. T-6292 Rat 1,0-Hr,10 ulini 498/498 100. 50 (D cc 584/584 100. 5D cr_ 0.0 2.0 1 84 T-6292 Rat 1,6-Hr,10 ulinj 498/498 10& 50 a: 5841584 10(@ Ile 5D 0.0 2.0 1 84 4.0 6.0 167 250 7ime (min)/Scan 4.0 6.0 167 250 Time (min)/Scan 55,000 30,000 8.0 10.0 333 416 425,000 490,000 8.0 10.0 333 416 AOVANCEE) BIOANALYTICAL SERVICES, INC. REPORT: 96ADEMOI.3M 32 Figure 16. Product ion scan for carboxylicacid metaboliteof T-6292 in Rat 1, 6-Hr sample and proposed fragment structures. Product IonScan , Parent=584 T-6292 Rat 1,6-Hr.10 ul ini 100 19,615 419 526 219 75 c 5D 169 ID a: 25 269 83 483 o- .- - I I I I I . .. 1DO 200 300 400 500 600 m/z CarboxylicAcid MetaboliteProductIonAssignments rn/z(amu) 169,219,269,419,526 483 Descril2tion See Figure5 0 11 F3C-(CF2)6-CF2-S - 0 83 AOVANCEED BIOANALYTICAL SERVICES, INC. 0 11 F-S 0 REPORT: 96ADEMOI.3M 33 Figure 17. Total ion and extractedparent (m/z 650) and proposed metaboliteion chromatograms for T-6293 obtained from Rat Control Sample 13. TICofallmasses,100 to800 controslample13, no TA 100 75- 50-'@O-i0.31636 25- 4.43 2.50 3.49 L 55..1100 55.-887@ 7-.2 cc 1 0.0 101 201 301 2.5 4.9 7.3 Scanrrim(emin) controslample13,no TA 542/542 2,805,000 8.64 9*.87 401 9.7 0 556/556 0 584/584 0 650/650 0 746/746 0 0.0 2.0 4.0 6.0 8.0 10.0 1 84 165 248 331 414 Time(min)/Scan ADVANCED BIOANALYTICAL SERVICES, INC. REPORT: 96ADF-MOI.3M 34 Figure 18. Total ion and extractedparent (m/z 650) and proposed metaboliteion chromatograms for T-6293 obtained from Rat 1, 0-Hr sample. TIC ofallmasses 100 to 800 Rat 1,0-Hr,10 ulinj 100- 75- 50- CD 25- (D a: Of 1 0.0 1.40 Rat 1,0-Hr, 10 ulinj 642/542 2.94 101 2.4 4.89 4.02 L 5.55 6.55 6.70 7.74 201 301 4.8 7.2 Scan/1-ime(min) 2,680,000 8.89 8"89 401 9.6 315,000 556/556 315,000 584/584 315,000 650/650 746/746 0.0 - 2.0 1 84 6.70 4.0 6.0 8.0 167 250 333 Time (min)/Scan 315,000 315,000 10.0 416 AOVANCED BIOANALYTICAL SERVICES, INC. REPORT: 96ADF-MOI.3M 35 Figure 19. Total ion and extractedparent (ni/z650) and proposed metaboliteion chromatograms for T-6293 obtainedfrom Rat 1, 6-Hr sample. TIC of allmasses 100 to800 T-6293 Rat 1,6-Hr,10 ulinj ..7 100, 75- 50. 25- cc 0 1 0.0 f1l2.@288 101 2.4 T-6293 Rat 1,6-Hr,10 ulinj 542/542 4.89 850 3..3377@ 6@46.14.1 5u 201 301 4.8 7.2 Scan/Time (min) 7.95 5561556 6 6.48 584/584 6.67 2,910,000 401 9.6 310,000 -lp 65,000 M=96 165,000 650/650 6.70 140,000 746/746 0.0 2.0 1 84 6.63 490 6.0 8.0 167 250 333 rime (min)/Scan 125,000 10.0 416 ACDVANCE[D SIOANALYTICAL SERVICES, INC. REPORT: 96ADEMOI.3M 36 Figure 20. Possiblestructurefsor the observed metabolitesof T-6293 with [M-H]- of 542 and 556 amu from Rat 1. Mr = 543 [M-H]- 542 0 CH2-CH20H 1-< F3C-CF2-CF2-CF2-CF2-CF2-CF2-CF2-S 11 0 mr 557 [M-H]- 556 0 CH2-COOH ll-< F3C-CF2-CF2-CF2-CF2-CF2-CF2-CF2-8 11 H 0 ADVANCED BIOANALYTICAL SERVICES, INC. REPORT: 96ADEMOI.3M 37 Figure 21. Extractedion chromatograms of sulfonamide (M/z 498) and sulfonate(m/z 499) ions for T-6293 from Rat 1, 0-Hr (top) and 6-Hr (bottom)samples. Rat 1,0-Hr,10 ulinj 498/498 1 260,000 50. 0i 499/499 100 100,000 50. CD cc 0 Li 0.0 2.0 4.0 6.0 8.0 10.0 1 84 167 250 333 416 7ime (min)/Scan T-6293 Rat 1,6-Hr,10 ulinj 498/498 100 645,000 so. a: 0 499/499 ioa 50. 265,000 ol 16 0.0 2.0 4.0 6.0 8.0 10.0 1 84 167 250 333 416 Time (min)/Scan ACDVANCEO BIOANALYTICAL MM SERVICES, INC. REPORT: %ADEMOI.3M 38 Figure 22. Extractedion chromatograms of proposed metaboliteswith m/z 327 and 419 for T-6293 from Rat 1, 0-Hr (top) and 6-Hr (bottom) samples. Rat 1,0-Hr,10 ulinj 327/327 100. 85,000 50. ol 419/419 10& d@OA 110,000 50. Al &A A A ol 0.0 2.0 4.0 6.0 8.0 10.0 1 84 167 250 333 416 Time (min)/Scan T-6293 Rat 1,6-Hr,10 ulinj 327/327 10& 50- cc A 0. 419/419 100. 50- cc 0 0.0 2.0 1 84 A 4.0 6.0 8.0 167 250 333 Time (min)/Scan 215,000 370,000 10.0 416 MMM ADVANCE[) BIOANALYTICAL SERVICES, INC. REPORT: 96ADEMOI.3M 39 Figure23. Total ion and extractedparent (m/z 526) and proposed metaboliteion chromatograms for T-6294 obtained from Rat Control Sample 13. TIC of allmasses, 100 to 800 T-6294, Control,no testarticle1,0 ulinj1,090 100 75 50 2 a: 0 1 0.0 7.3 5.0 1.5 3.2 4.2 5.0 101 201 301 2.4 4.9 7.3 ScanMme (min) T-6294,Control,no testarticle1,0 ulinj1,090 483/483 2,905,000 401 9.7 10,000 498/498 10,000 526/526 10,000 542/542 10,000 556/556 10,000 0.0 2.0 4.0 6.0 8.0 1 83 166 245 328 412 Time (min)/Scan ADVANCED BIOANALYTICAL SERVICES, INC. REPORT: 96ADEMOI.3M 40 Figure 24. Total ion and extractedparent (m/z 526) and proposed metaboliteion chromatograms for T-6294 obtainedfrom Rat 1, 0-Hr sample. TIC ofallmasses, 100 to 800 T-6294, Ratl, 0-hr,10 ulinj,1090 10D 75 50 2& cc n 1 0.0 3 .1 101 2.4 T-6294, Rati, 0-hr,10 ulinj,1090 483/483 5.0 4.1 201 4.8 Scan/Time (min) 5,295,000 8.6 7.4 301 401 7.2 9.6 1,895,000 498/498 526/526 542/542 7.90 180,000 8.64 1,895,000 10,000 556/556 0.0 2.0 1 84 4.0 @.0 167 250 Time (min)/Scan 1,895,000 8.0 10.0 333 416 MMM ADVANCE[D BIOANALYTICAL SERVICES, INC. REPORT: 96ADEMOI.3M 41 Figure 25. Total ion and extractedparent (m/z 526) and proposed metaboliteion chromatograms for T-6294 obtainedfrom Rat 1, 6-Hr sample. TIC ofallmasses, 100 to800 T-6294, Ratl,6-hr,lo ulini,109o 1 oc@ 75- 50. 3.0 *Pc!i 25 a.- - --4l"1".5 - - a4:) 0. 1 101 0.0 2.4 T-6294, Ratl,6-hr,10 ulinj,1090 483/483 e.r 4.9 -A - 201 4.9 Scan/Time (min) 8.1 7.3 8.7 301 7.3 7,595,000 401 9.7 6.58 80,000 49a/498 526/526 3,535,000 B. 3 8.73 1,350,000 542/542 556/556 0.0 2.0 1 83 8K 6.75 A 4.0 6.0 80 165 248 331 Time (min)/Scan 30,000 50,000 10.0 414 AOVANCEED BIOANALYTICAL SERVICES, INC. REPORT: 96ADEMOI.3M 42 Figure 26. Product ion scan for perfluorooctanesulfonamidmeetabolite (CF3(CF2)7SO,NH., m/z 498) of T-6294 in Rat 1, 6-Hr sample and proposed fragment structures. -PmfileDAUGHTER Parent 498 T-6294 Rat 1,6-Hr,10 ulinj 1 DO- 398,750 498 7578 r 50- cc 25- 169 219 259 100 200 qw 460 nVz N-DealkylatedMetaboliteProductIonAssignments m/z (amu) 169,219 478 259 Description See Figure5 0 F3C-(CF2)g-6F-11-N 11 0 0 11 F2C-(CF2)2-CF-S-N 11 0 478 500 0 78 11 S=N - 11 ADVANCED BIOANALYTICAL SERVICES, INC. REPORT: 96ADEMOI.3M 43 Figure 27. Totalion and extractedparent(m/z 499) and homolog ion chromatograms for T-6295 obtainedfrom Rat Control Sample 13. TIC of allmasses, 100 to800 controlmedia (nota),10 ul,gradient7 100 - 1.09 75- E 5025- 22..6688@ a: 1 101 0.0 2.4 6.80 4.73 201 301 4.8 7.2 Scanfrime(min) 720,000 8.94 401 9.6 controlmedia (no ta),10 ul,gradien7t 4991499 449/449 399/399 349/349 6.80 6.58 6.34 299/299 0.0 2eO 4.0 6.0 8.0 1 84 167 250 333 Time (min)/Scan 440,000 15,000 50,000 440,000 440,000 10.0 416 MMM ADVANCECD BIOANALYTICAL SERVICES, INC. REPORT: 96ADEMOI.3M 44 Figure 28. Total ion and extractedparent (m/z 499) and homolog ion chromatograms for T-6295 obtainedfrom Rat 1, 0-Hr sample. TIC ofallmasses, 100 to 800 T-6295,ratl,0-hr,10 ul,gradient7 :::z.- IOD 75 50 1.06 2Ll.o6 (D cc 1 101 0.0 2.4 T-6295,ratl,0-hr,10 ul,gradien7t 499/499 6.86 4.91 201 301 4.8 7.2 Scanfrime(min) 6.86 2,060,000 8.96 401 9.6 1,420,000 449/449 180,000 6.62 399/399 375,000 6.36 349/349 135,000 6.00 299/299 5.25 25,000 0.0 2.0 4.0 6.0 8.0 10.0 1 84 167 250 333 416 Time (min)/Scan MMM ADVANCED BIOANALYTICAL SERVICES, INC. REPORT: 96ADEMOI.3M 45 Figure 29. Total ion and extractedparent (nVz 499) and homolog ion chromatograms for T-6295 obtainedfrom Rat 1, 6-Hr. TIC ofallmassea 100 to800 T-6295,ratl,6-hr,10 ul,gradien7t .e ico 75- .594 (D 25 108 cc 0 1 101 0.0 2.4 T-6295,ratl,6-hr,10 ul,gradien7t 4991499 6.94 6.0366@.9 39 4.69,.j 6.03 A 201 301 4.8 7.3 ScanMme (min) 6.94 2,830,000 401 9.7 1,960,000 449/449 275,000 6.66 399/399 11 349/349 6.39 k 6.03 535,000 220,000 29gi299 5.24 65,000 0.0 2.0 4.0 6.0 8.0 10.0 1 84 167 249 332 415 Time (min)/Scan MMM ACDVANCED BIOANALYTICAL SERVICES. INC. REPORT: 96ADEMOI.3M