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AOVANCEO BIOANALYTICAL SERVICES, INC. ,)95MYHWOI.3M A Ion Spray LC/MS Determinationof PerfluoroAnalyticalStandards Provided by 3M Medical Department Prepared for 3M MedicalDepartment,ToxicologyServices, St. Paul,MN 55133-3220 Henry Y. Wu, Ph.D. Jack D. Henion,Ph.D. Advanced BioAnalyticaSlervicesI,nc. Date:August 30, 1995 AOVANCEO BIOANALYTICAL SERVICES, INC. TABLE OF CONTENT 95MYHWOI.3M Page SIGNATURE PAGE .........................................i ....... SUMMARY .............................................i.i...... 1.INTRODUCTION ........................................I........ 1.1 Study Objectives .....................................I ....... 1.2Chemical StructuresN,ames or 3M Identificatioannsd, MolecularWeights ..... I 2.MATERLKLS AND METHODS .................................2...... 2.1 Chemicals and Materials ................................2....... 2.2LC/MS Instrumentation.................................3...... 2.3Preparationof Stock and Test Solutions ........................5.... 3.RESLJLTASND DISCUSSION.................................5.. 3.1T-6295..........................................6....... 3.2T-6294 ..........................................6....... 3.3T-6293 ..........................................7 ....... 3.4Fose Amide .......................................7........ 3.5Perfluorooctanamide...................................8 ...... 4.CONCLUSION ..........................................8....... 5.FIGURES .............................................9........ Figure1.TotalIon Chromatogram (TIC)fortheInjectioonf 1 liLofDMSO ...... 9 Figure2.TIC fortheInjectionosf 1 gL of Top, Middle and Bottom Day Zero SolutionofT-6295 .................................10....... Figure3.TIC fortheInjectionosf I pL of Top,Middle and Bottom Day Seven SolutionofT-6295 .................................I.I...... Figure4.ExtractedIon Chromatograms fortheInjectioonf 1 liLof Day Zero Top Solutionof T-6295 .................................1.2 ...... Figure5. Full-ScanMass Spectrum ofPeak I inFigure4 ................1.3 . ELM ADVANCEO BIOANALYTICAL SERVICES, INC. 95MYHWOI.3M Figure 6. Full-ScanMass Spectrum ofPeak 2 inFigure4 .................1.4 Figure 7. Full-ScanMass Spectrum ofPeak 3 inFigure4 .................1.5 Figur8e.Full-SMcasnsSpectroufmPeak4 inFigur4e .................16. Figure 9. Full-ScanMass SpectraofPeaks 5a and 5b inFigure4 .............17 Figure 10. Full-ScanMass Spectrum of Peak 6 inFigure4 ................1.8. Figure 11. Full-ScanMass Spectrum of Peak 7 inFigure4 ................1.9. Figure 12.TIC fortheInjectionosf I gL of Top, Middle and Bottom Day Zero Solutionof T-6294 ...................................20..... Figure 13.TIC fortheInjectionosf 1 gL of Top, Middle and Bottom Day Seven Solutionof T-6294 ...................................21..... Figure 14.ExtractedIon Chromatograms fortheInjectionof I liL of Day Seven Top SolutionofT-6294 ................................22.... Figure 15. Full-ScanMass Spectrum of Peak I inFigure 14 ................2.3 Figure 16. Full-ScanMass Spectrum of Peak 2 inFigure 14 ................2.4 Figure 17. Full-ScanMass Spectrum ofPeak 3 inFigure 14 ................25. Figure 18. Full-ScanMass Spectrum ofPeak 4 inFigure 14 ................2.6 Figure 19. Full-ScanMass Spectrum ofPeak 5 inFigure 14 ................2.7 Figure20.TotalIon Chromatogram (TIC)fortheInjectioonf I pL of Water ......28 Figure 21. TIC forthe Injectionosf I gL of Top, Middle and Bottom Day Zero Solutionof T-6293 ...................................29..... Figure22. TIC fortheInjectionosf I pL of Top, Middle and Bottom Day Seven Solutionof T-6293 ...................................3.0 .... Figure 23. ExtractedIon Chromatograms fortheInjectionof I liLof Day Seven Bottom Solutionof T-6293 ..............................3.1 ... Figure24. Full-ScanMass Spectrum ofPeak I inFigure23 ................32. Figure25. Full-ScanMass Spectrum ofPeak 2 inFigure23 ................3.3 Figure26. Full-ScanMass Spectrum of Peak 3 inFigure23 ................3.4 Figure 27. TIC fortheInjectionosf I gL of Top, Middle and Bottom Day Zero SolutionofFose Amide ................................35.... Figure 28.TIC fortheInjectionosf I gL of Top, Middle and Bottom Day Seven SolutionofFose Amide ................................36.... Figure29. ExtractedIon Chromatograms fortheInjectioonf I pL of Day Zero Top SolutionofFose Amide ................................37.... Figure 30. Full-ScanMass Spectraof Peaks Ia and 1b inFigure 29 ............38 AOVA NCEO BIOANALYTICAL SERVICES, INC, 95MYHWOI.3M Figure 3 1. Full-ScanMass Spectrum of'Peak2 inFigure 29 ................3.9 Figure32. Full-ScanMass Spectrum ofPeak 3 inFigure 29 ................4.0 Figure33. Full-ScanMass Spectrum of Peak 4 inFigure 29 ................4.1 Figure34. Full-ScanMass Spectrum of Peak 5 inFigure 29 ................4.2 Figure 35. Full-ScanMass Spectrum of Peak 6 inFigure 29 ................4.3 Figure 36. Full-ScanMass Spectrum of Peak 7 inFigure 29 ................4.4 Figure 37.TIC fortheInjectionosf I pL of Top, Middle and Bottom Day Zero Solutionof Perfluorooctanamide...........................4.5 ... Figure 38.TIC fortheInjectionosf 1 pL of Top, Middle and Bottom Day Seven Solutionof Perfluorooctanamide...........................4.6... Figure39. ExtractedIon Chromatograms fortheInjectioonf I p.Lof Day Zero Top Solutionof Perfluorooctanamide ...........................4.7... Figure40. Full-ScanMass Spectrum of Peak I inFigure39 ................48. Figure 4 1. Full-ScanMass Spectrum of Peak 2 inFigure 39 ................4.9 Figure42. Full-ScanMass Spectrum of Peak 3 inFigure 39 ................5.0 Figur4e3.Full-ScanMass Spectrum of Peak 4 inFigure 39 ................5.1 Figur4e4.Full-ScanMass Spectrum of Peak 5 inFigure 39 ................5.2 Figure45. Full-ScanMass Spectrum of Peak 6 inFigure 39 ................5.3 Figure46. Full-ScanMass Spectrum of Peak 6 inFigure39 ................5.4 A[DVANCEO BIOANALYTICAL SERVICES, INC. ADVANCED BIOANALYTICAL SERVICES 95MYHWOI.3M SIGNATURE PAGE Title:Ion Spray LC/MS Determinationof PerfluoroAnalyticalStandardsProvided by 3M Medical Department Reported by: Henry Y. Wu, Ph.D. Date SeniorResearch Scientist Reviewed by: Mary *nnsgsgw od S. QM@UtyityAassuran Manager /Date Authorizedfor Releaseby: <:fJa-cTHenion,Ph.D. ABS LaboratoryDirector (@ate ADVANCEO BIOANALYTICAL SERVICES, INC. SUMMARY 95MYHWOI.3M Five perfluoro compounds including T-6293, T-6294, T-6295, fose amide and perfluorooctanamidweere analyzedby full-scainonsprayLC/MS forcompositionidentification and ambienttemperaturestabiliitnyDMSO and water(onlyforT-6293). The on-lineLC/MS determinationof the listedtestarticlewsas carriedoutunderunitmass resolutiocnonditions followingmass axis calibratiounsing PPG referencecompounds. Chromatography was performedon a 2 x 100 mm DeltabondTmC8 column usinglineargradientelutionwith acetonitrilewaterfrom 5:95 to 95:5in 15 minutes.The mass spectrometewras operatedin thenegativeion mode. The LC/MS experimentsshowed thatallfiveperfluorocompounds containsome impuritiesI.n additiona,llfiveperfluorocompounds arestableinDMSO and waterforatleasta week atambienttemperatureata concentratioonf 10 mM. No apparentdegradationwas observed by LC/MS analysesof thesesamples. Attemptsto obtainmolecularion informationfor compounds T-6292 and butylfoseby ion sprayand atmosphericpressurechemicalionization (A,PCI)were unsuccessful.However, poor responsefrom bothionizatiomnodes didnot provide sufficienstensitiviftoyrtheLC/MS analysisofthesetwo compounds. ifi ADVANCED BIOANALYTICAL SERVICES, INC. 95MYHWOI.3M 1. INTRODUCTION Seven perfluorocompounds includinTg-6292,T-6293,T-6294,T-6295,foseamide,butylfose and perfluorooctanamidweere the targetmoleculesforqualitativceompositionand stability determinationA.mong them,fiveperfluorocompounds includingT-6293,T-6294,T-6295,fose amide and perfluorooctanamidweere analyzedby full-scainon sprayLC/MS forcomposition identificatiaonnd ambienttemperatursetabiliitnyDMSO and water(onlyforT-6293).The on-line LC/MS determinationof the listedtestarticlewsas carriedout under unitmass resolution conditionsfollowingmass axiscalibratiounsingPPG referencecompounds. Chromatography was performed on a 2 x 100 mm DeltabondTmC8 column usinglineargradientelutionwith acetonitrile-watferrom 5:95to 95:5 in 15 minutes.The mass spectrometerwas operatedin the negativeion mode. The LC/MS experimentsshowed thatallfiveperfluorocompounds contain some impuritiesI.n additiona,llfiveperfluorocompounds arestableinDMSO and waterforat leasta week atambient temperatureata concentratioonf 10 mM. No apparentdegradationwas observedby LC/MS analysesof thesesamples.Attemptstoobtainmolecularioninformationfor compounds T-6292 and butylfoseby ion sprayand atmosphericpressurechemicalionization (APCI) were unsuccessfulP.oor responsefrom bothpositivaend negativeionizatiomnodes did notprovidesufficienstensitiviftoyrtheLC/MS analysiosfthesetwo compounds. 1.1 Study Objectives The objectiveosf thisstudywere: toqualitativedleyterminethecompositionofeachperfluorcoompound. toqualitativedleyterminethestabiliotfyeachcompound inDMSO orwater(forT-6293 only) asa stocksolutionat10 mM concentration. 1.2 Chemical Structures, Names or 3M Identificationsa,nd Molecular Weights 0 C8F17 - 1 S- 0-K+ II 0 T-6295 MW = 538.22 anion m/z = 499 ADVANCED BIOANALYTICAL SERVICES, INC. C8F17 0 11 S-N 11 0 CH2CH3 / '-H C8FI7 0 11 S---N 11 ,-,,CH2CH3 0 ICH2CH20_p_O-NIHI ,+ 0 1 0 NH4+ 0 11 C8F]7 -S-N 11 0 0 1-10, CH 2CH 3 N@-I CH2CH20H C8 F17 11 S- NH2 11 0 0 II C7F,:5 -C- Nf'2 0 11 C8F17 - S- 11 0 -,,CH2CH2CH2CH3 N N*-, CH2CH20H 95MYHWOI.3M T-6294 MW = 527.20 anion m/z = 526 T-6293 MW = 685.29 monoanion m/z 650 T-6292 MW = 571.25 foseamide MW = 499.14 anion m/z = 498 Perfluorooctanamide MW = 413.09 anion m/z = 412 butylfose MW = 599.31 2. MATERIALS AND METHODS 2.1 Chemicals and Materials Allsevenperfluorcoompoundswereprovidedby 3M MedicalDepartmentT,oxicologSyervices, St.Paul,MN 55133-3220. Methyl sulfoxide(DMSO, Cat #27,043-1)was obtainedfrom SigmaAldrichChemicals,Milwaukee, WI 53233. Acetonitril(eCat #015-4) was obtainedfrom 2 ADVANCEO BIOANALYTICAL SERVICES, INC. 95MYHWOI.3M Baxter/ScientifPircoducts,McGaw Park,IIL60085. Ammonium Acetate(Cat #JT0596-01) was purchasedfrom J.T.Baker,Danvers,MA 01923. High purity(NANOpure) water was made by Bamstead Model D7331 UltrapureWater System,Dubuque, IA 52001. All materialsand equipment may be substitutebdy equivalentmaterialsand equipment. FisherVortex Genie @@, Cat # 12-812,FisherScientificS,pringfieldN,J 07081 Microbalance,Model MT5, Mettler-ToledoI,nc.,Hightstown,NJ 08520 SiliconizedGraduated Tubes, 2 niL,Cat #M-720, Phenix Research Products,Hayward, CA 94545 PipettesC,at #P-1000, P-200, P-100, P-20,Rainin InstrumentCo.,Woburn, MA 01888 SiliconizedGreen SlickTips,I mL, Cat #TGS-415BRS, Phenix Research Products,Hayward, CA 94545 SiliconizedClearTips,250 gL, Cat #T-051 BRCS, Phenix Research Products,Hayward, CA 94545 Polypropyleneautosamplervials(siliconizeidn house),Cat #500 102, Sun Brokers,Inc., Wilmington, NC 28402 Snap Cap, II nim, Cat #69158-P, ScientifiRcesources Inc.,Eatontown, NJ 07724 SolventFiltratioAnpparatus,Cat #32-006 10,KrackelerScientificA,lbany, NY 12201 Vacuum Pump, Cat #54-907,VWR ScientificR,ochester,NY 14603 0.45 gm Nylon Titan Membrane FiltersC,at #74547-NN, ScientificResources Inc., Eatontown, NJ 07724 Liquid Nitrogen,Cat #SS 180N-NIT4052, Empire Airgas,Elmira,NY 14901 U.H.P. Nitrogen,Empire Airgas,Elmira,NY 14901 U.H.P. Argon, Empire Airgas,Elmira,NY 14901 Fused-silica1,00 g i.d.x 170 g o.d.,Polyn-iicrToechnologies,Phoenix, AZ 85023 PEEK tubing,0.007" i.d.x 1/16"o.d.,Cat.#1536, Upchurch ScientificI,nc.,Oak Harbor, WA 98277 2.2 LC/MS Instrumentation The liquidchromatography partof the system includeda Hewlett-PackardModel 1090L pump, Hewlett-Packard,Avondale, PA 19311. DeltabondTM C8 columns,5 )im,2 x 100 mm, and DeltabondTm C8 guard cartridge(s5gm, 2 x 10 mm) were obtainedfrom Keystone ScientificI,nc., 3 ELM AOVANCEO BIOANALYTICAL SERVICES, INC. 95MYHWOI.3M BellefonteP,A 16823. The mass spectrometewras a PE-SCIEX API III'from PE-SCIEX, ThornhillO,ntario. HPLC Conditions: Flow Rate Column Temperature InjectioVnolume Column Guard Cwtddge Pressure Eluents t(min) 0 15 200 pumin Ambient IPL 2 x 100mm Deltabone C8 2 x 10 mm DeltabondTm C8 70 bar %acetonitrile 5 %water 95 95 5 This program was used fortheanalysesof T-6294, T-6295, foseamide and perfluorooctanamide. For theanalysisof T-6293 samples,waterwas replacedby 10 mM annnonium acetatewith the same gradientprogram. Mass SpectrometerConditions: Ion Spray Voltage OrificeVoltage DeclusteringPotential MultiplieVroltage CurtainGas (U.H.P.N 2 NebulizerGas (N 2) InterfaceHeater Turbospray Temperature Turbospray Gas Flow Dwell Time Mass Scan Range Scan Rate -4200V -90V (-70V forT-6293) -60V (-40V forT-6293) 4000 V 1.4Umin 50-70 psi 60 *C 500 *C 4.0L/min I msec 100-900 Da 1.69sec/scan 4 OVANCEO SIOANALYTICAL SERVICES, INC. 95MYHWOI.3M Count Controlle(rCC) 10 Mass calibratioonf Q Iin therangeof 100-900was performeddailyby theinfusionof a 10-4 M PPG (polypropylengelycolsa,verageMW 425, 1000 and 2000)calibratiosnolution(PE-SCEEX API IH" operator'msanual)ata flow of 10 gL/min. Calibratiownas performed on nVz 59.0, 325.3,500.38 and 906.7. The mass spectrometripcarameters,MS/MS peak widths and sensitivitayreoptimizeddaily.Peak widthsareapproximately0.7amu athalf-heighitnthesingle MS mode. 2.3 Preparation of Stock and Test Solutions Stock Solutions:Accuratelyweigh approximately5 mg of one of the perfluorocompounds (assumingpurity100%) on a microbalanceina siliconizegdraduatedtube(2mL). Dissolvethe solidsample inan appropriataemount of DMSO orwater(forT-6293 only)toyielda 10 MM solution.Storethesolutionatambienttemperature. TestSolutionsC:arefullytake10 p.Lofthe10 mM stocksolutiofnrom thetop,middle and bottom partsof thesolutiona,nd diluttehem withDMSO or water(forT-6293 only)by 10-to I00-foldto givetestsolutionwsithconcentrationosf 100-1000gM. Vortex-mixthedilutedsolutionsa,nd analyzethem immediatelyafterpreparation. The testsolutionswithsame concentrationfsorthe analyteswere preparedon day zero and day sevenin thesame way from thesame stocksolution.The stocksolutionwsere storedatambient temperatureon thelabbench withoutdisturbance. 3. RESULTS AND DISCUSSION As requestedby 3M, thepolypropylenetubesand vialsused in thiswork were siliconized.The pipettetipsused inthiswork were alsosiliconizedT.he LCIMS conditionsfortheanalysesof differenptartsof a solutionon bothday zero and day sevenwere thesame. To avoid cross contaminations,olventblanks(DMSO orwater)were injectebdeforetheinjectioonf a different type of testsolution.No LC/MS datawere obtainedforcompounds T-6292 and butylfose, becausethemolecularionsignalwas tooweak tobe used toperformLCIMS experiments. The on-lineLC/MS determinatioonfthefivecompounds testedwas carriedoutunder unitmass 5 MMM ADVANCED BIOANALYTICAL SERVICES, INC. 95MYHWOI.3M resolutionconditionsfollowingmass axiscalibratiounsingPPG referencecompounds. To achievesufficientlfyastfull-scaancquisitiosncan ratesfortheserelativelhyighermolecularweight compounds, a compromise of 0.5 Da stepsizeacquisitiownas used intheseexperiments. These conditionsproduce plotsofcorrespondingmass spectrafrom theproprietaryPE SCIEX software thatdo not includethe isotopicpeaks. Mass spectralplotsof individualtestarticlmeass spectra from these acquisitionshow reliablemolecular weight measurements. In particular, differentiatioofn molecularweights differinbgy only one dalton,e.g.the sulfonamidevs.the sulfonicacidatm/z 498 and 499,may be reliabldyeterminedunder theseconditions. 3.1 T-6295 A typicaltotalionchromatogram (TIC) from the injectioonf I gL ofDMSO isshown inFigure 1. The TIC from theinjectionosf top,middle and bottom partsof theT-6295 solutionon day zero and day seven were sunnnarizedinFigures2-3. Extractedion chromatograms from the injection of I gL of day zero testsolution(100 gM) dilutedfrom thetop partof the stocksolutionare shown in Figure4. Full-scansingleMS spectraof thecomponents detectedfrom Figure 4 are shown in Figures5-11. The identicalmass spectrainFigure 9 for peaks 5a and 5b suggestthat thesetwo peaks arelikelyfrom two structurailsomers.By carefulleyxamining theTIC from the injectionosf solventblank and differenptartsof the T-6295 solutionson both day zero and day seven,thereisno apparentdifferenceamong thethreepartsof the solutionand thereisno apparent change inthecompositionof thestocksolutionatambient temperaturefora week. Although trace impuritiesat m/z 299, 349, 399,449, 549 and 607 aredetectedin T-6295, the major species presentisthenVz 499 anion. 3.2 T-6294 The TIC from the injectionsof top,middle and bottom partsof the T-6294 solutionon day zero and day seven were summarized in Figures 12-1.3.Extractedion chromatograms from the injectioonf I gL ofday seventestsolution(100 liM) dilutedfrom thetop partof thestocksolution are presentedin Figure 14. Full-scansingleMS spectraof the components detectedfrom Figure 14 areshown inFigures 15-19. By carefullyexamining theTIC from theinjectionosf solvent blank and differenptartsof theT-6294 solutionsi,tappearsthatthebottom partof thesolution containsfewer impurities.In addition,ionsatm/z 297.5,311.5,325.5 and 339.5 showed higher relativreesponses(compared to the ion at m/z 526 for [M-H]-of T-6294) for the day seven 6 ADVANCEE) BIOANALYTICAL SERVICES, INC. 95MYHWOI.3M solutiontshanthoseobtainedfortheday zerosolution(sSeeFigures12 and 13 in theregionsof 5.6-8.5niin.).Again,them/z 526 anionisthemajor speciesinthesample. 3.3 T-6293 T-6293 was not solublein DMSO. Therefore,water was used to preparethe stock and test solutionfsorT-6293. A typicaTlIC from theinjectioonf I liLof waterisshown inFigure20. Totalionchromatograms from theinjectionosf top,middle and bottom partsof the T-6293 solutionon day zero and day seven are summarized in Figures21-22. Extractedion chromatogramsfrom theinjectioonf I pL of day seventestsolution(I mM) dilutedfrom the bottom partof thestocksolutionarepresentedinFigure23. Full-scansingleMS spectraof the components detectedfrom Figure23 areshown in Figures24-26. Components atnL/z650,630 and 369 were detected.Based on infusionexperimentst,heion atM/z 526 in Figure25 isa fragmentof nVz 650. By carefulleyxaminingtheTIC from theinjectionosf solventblankand differenptartsof theT-6293 solutionson both day zeroand day seven,thereisno apparent differencaemong thethreepartsof thesolutionand thereisno apparentchange inthecomposition of thestocksolutionatambienttemperaturefora week, althoughthechemicalbackground forthe day seven solutionseems tobe noisieprossiblydue tolowersensitiviotfythemass spectrometer. For unknown reasons,thechemicalbackground fortheday zerotop and middle solutionsare somewhat higherthanthatfortheday zerobottom solution(seeFigure2 1). 3.4 Fose Amide The TIC from theinjectionosf top,middle and bottom partsof thesolutionof foseamide on day zero and day seven were summaried inFigures27-28. Extractedion chromatograms from the injectioonf 1 gL ofday zerotestsolutio(n100liM)dilutefdrom thetoppartofthestocksolution arepresentedin Figure29. Full-scansingleMS spectraof thecomponents detectedinFigure29 are shown inFigures30-36. Components atm/z 606, 499,498, 398, 339.5,325.5,and 311.5 were detected.Peaks marked withan X arefrom thechemicalbackground of theLC eluent(see Figures34 and 36). Interestingltyh,etop portionoftheday zerosolutionshowed much higher relativreesponsefortheionsatm/z 499, 339.5,325.5 and 311.5 thanthoseobtainedfrom the middle and bottom portionsof thesame day solution.On thecontraryt,he day seven solution showed theoppositetrendfortheresponsesof thoseions.The ionat ni/z499 appearedattwo differenrtetentiotnimes(seepeak Ia and Ib inFigure29).Peak Ia ispossiblyfrom an impurity, whilepeak lb istheisotopifcorm ofthemajorcomponent atnl/z498 (seeFigures30 and 35).By 7 ELM AOVANCEO BIOANALYTICAL SERVICES, INC. 95MYHWOI.3M carefulleyxamining theTIC from theinjectionOsf solventblankand differenptartsof the solutions of foseamide on both day zero and day seven,thereisno significandtifferencebetween the three partsof the solutionand thereisno apparentchange in the compositionof the stock solutionat ambient temperaturefora week. 3.5 Perfluorooctanamide The TIC from theinjectionosf top,middle and bottom partsof thesolutionof perfluorooctanamide on day zeroand day seven are summarized inFigures37-38. Extractedion chromatogratns from theinjectioonf I pL ofday zerotestsolution(ImM) dilutedfrom thetop partof thestocksolution are presentedin Figure39. Full-scansingleMS spectraof thecomponents detectedin Figure39 are shown in Figures40-46. Major components atm/z 412, 392, 369, 339.5,325.5,311.5 and 297 were detected.Peaks marked with an X are from thechemical background of the LC eluent (seeFigure 46). Again, thetop portionof the day zero solutionshowed much higherrelative responsefor the ions at ni/z339.5,325.5 and 311.5 than thoseobtainedfrom the middle and bottom portionsof thesame day solution.The relativreesponsesforthe ionsatm/z 339.5,325.5 and 311.5 forthe day seven solutionwere significantlyower thanthoseobtainedfortheday zero solution.Itisclearfrom Figure39 thatthissample has severalcomponents presentinadditionto thetestarticloef interest. 4. CONCLUSION Five perfluorocompounds were analyzedby ion sprayLC/MS. In general,allfivecompounds are stableinDMSO or water(forT-6293 only).No significandtegradationproductswere detectedfor any ofthe compounds analyzed.However, itappearsthatthelow molecularweight impuritiesare not uniformlydistributeidnsome solutions. ADVANCEID BIOANALYTICAL SERVICES, INC. S. FIGURES 95MYHWOI.3M Figure 1.Total Ion Chromatogram (TIC) for the Injectioonf 1 JILof DMSO TIC of allmasses, 100 to 900 DMSO LC/MS-3- 7/5t95- 10:45AM 1 uL ofDMSO,deftabond C8,ACN/H20 gradient(5%to95% in15 min) 100 55,000,000 7& 50. 2& 1 101 201 301 401 501 0.0 2.8 5.7 8.5 11.3 14.1 Scan/Time (min) ADVANCECD BIOANALYTICAL SERVICES, INC. 95MYHWOI.3M Figure 2. TIC for the Injectionosf 1 liLof Top, Middle and Bottom Day Zero Solutionof T-6295 100' Top 57,800,000 75' fA 50, cr_ 2& 0- 10a Middle 76 55,000,000 50, 25- c@ 100- Bottom 7& 53,500,000 (D 'E 50r (D CD cc 25- 1 101 201 301 401 501 0.0 2.8 5.6 8.5 11.3 14.1 ScanfTime (min) 10 MMM ADVANCEO BIOANALYTICAL SERVICES, INC. 95MYHWOI.3M Figure 3. TIC for the Injectionosf 1 gL of Top, Middle and Bottom Day Seven Solutionof T-6295 100 Top 30,700,000 75* CD E 50@ (D cr 25- o@ 100' Middle 29,200,000 75" 50' (D (D cc 25- 0. 10(@ Bottom 75- CD 31,700,000 (D 25- L2 1 101 0.0 2.8 201 301 401 501 5.6 8.5 11.3 14.1 Scan[Time (min) AOVANCEO BIOANALYTICAL SERVICES, INC. 95MYHWOI.3M Figure 4. Extracted Ion Chromatograms for the Injectionof I AL of Day Zero Top Solution of T-6295 T-6295 LC/MS-1 -7/5/95- 11:11AM 1 uL(100 uM/lop)T-6295,deltabonCd8,ACN/H20(5% to95% in15 min) 100/900 5,400,000 T-1C 299/299 20,000 349/349 2 399/399 449/449 a) (D 499/499 549/549 3 4 5b 5a 6 607/607 7 0.0 2.5 5.0 7.5 10.0 1 90 178 267 356 Time (min)/Scan 60,000 130,000 100,000 2,100,000 30,000 30,000 12.5 15.0 444 533 12 ADVANCEO BIOANALYTICAL SERVICES, INC. Figure S. Full-Scan MASS Spectrum of Peak 1 in Figure 4 -Profil0e1 SCAN T-6295 LC/MS-1/Sca4n0s-42 Scans 40-42 minus 1-35& 45-700Time=1.13min T-6295 LC/MS-1 -7/5195- 11:11AM 1 uL(100uM/top)T-6295,deltaboCn8d,ACNIH20(5% to95% in15min) 2 peaks 185.0 100. 75. 50- 'Zi (D cc 25- 299.0 95MYHWOI.3M 9970 200 460 600 8'00 m/Z 13 MMM AOVANCEID BIOANALYTICAL SERVICES. INC. Figure 6. Full-ScanMass Spectrum of Peak 2 in Figure 4 T-6295 LCIMS-1/Scans 57-63 -ProfilQel SCAN Scans 57-63 minus 1-54& 72-700 Time=1.66 min T-6295 LC/MS-1 -7/5/95- 11:11AM 1 uL(l00 uM/top)T-6295,deftabonCd8,ACN/H20(5% to95% in15 min) 1 peak 100- 349.0 95MYHWOI.3M 22,857 75- 16 50- (D (D a: 25- 200 400 600 800 m/z 14 MMM AOVANCEO BIOANALYTICAL SERVICES, INC. Figure 7. Full-Scan Mass Spectrum of Peak 3 in Figure 4 T-6295 LC/MS-1/Scans 143-159 -Profil0e1 SCAN Scans 143-159 minus 1-135& 178-700Time=4.23 min T-6295 LC/MS-1 -7/SM5 - 11:11AM 1 uL(100uM/top)T-6295,deltaboCn8d,ACN/H20(5% to95% in15 min) 1 peak 399.0 100. 95MYHWOI.3M 74,118 75. 50- 4) a: 25- 0 200 400 600 860 M/Z 15 15 CatherwooRdoad a IthacaN.ew York14850o (607)266-0665n Fax(607)266-0749 ADVANCED BIOANALYTICAL SERVICES, INC. Figure 8. Full-ScanMass Spectrum of Peak 4 in Figure 4 T-6295 LCIMS-I/Scans 197-205 -Profil0e1 SCAN Scans 197-205minus 1-178& 214-700 Time=5.64 min T-6295 LCIMS-1 -7/5/95- 11:11AM 1 uL(l00 uM/top)T-6295,deltaboCn8d,ACN/H20(5% to95% in15 min) 2 peaks 449.0 100. 95MYHWOI.3M 63,333 75- (D 'E 50- CD (D cr 25461.0 0 200 400 600 800 m/z 16 15 Catherwood Road n Ithaca.New York 14850 a (607)266-0665 n Fax (60-l)266-0749 ADVANCED BIOANALYTICAL SERVICES, INC. 95MYHWOI.3M Figure 9. Full-Scan Mass Spectra of Peaks 5a and 5b in Figure 4 T-6295 LC/MS-1/Scans 220-225 -ProfilQeLSCAN Scans 220-225 minus 1-196& 285-712 Time=6.25 min T-6295 LC/MS-1 -7/5/95- 11:11AM 1 uL(l00 uM/top)T-6295,deftaboCn8d,ACN/H20(5% to95% in15 min) 1 peak loo lpeak 5a 1 499.0 1,204,860 Z-*@, 75 .2:, 4) 16 50 (D cc 25 0. 200 400 600 rn/z T-6295 LC/MS-1/Scans 228-236 -ProfilQeLSCAN Scans 228-236 minus 1-180& 400-700Time=6.51 min T-6295 LC/MS-1 -7/5/95- 11:11AM 1 uL(l00 uMttop)T-6295,deftaboCn8d,ACN/H20(5% to95% in15 min) 1 peak FP-e-a-k5b7 499.0 100 8@o 1,758,889 75 ..2a:r_ CD 50 (D 25 0' 200 400 600 8@o rrvz 17 15 CatherwoodRoad m lihaca.New York 14850 v (607) 266-0665 n Fax (607,)266-0749 MMM ADVANCED BIDANALYTICAL SERVICES, INC. Figure 10. Full-Scan Mass Spectrum of Peak 6 in Figure 4 T-6295 LC/MS-1/Scans243-245 -PmfileQl SCAN Scans 243-245minus 1-240& 248-712Time--6.85min T-6295 LCtMS-1 -7/5/95- 11:11AM 1 uL(l00 uM/top)T-6295,deltabCo8n,dACNIH20(5% to95% in15 min) 4 peaks 183.0 100. 95MYHWOI.3M 156,683 75- a) E soci 0 cc 25. 311.5 549.0 431.5 0 200 400 600 800 M/Z 18 15 Catherwood Road m lllhaca,New York 14850 m (607) 266-0665 m Fax (60@@)266-01-49 ADVANCED BIOANALYTICAL SERVICES, INC. Figure 11. Full-Scan Mass Spectrum of Peak 7 in Figure 4 -Profile01 SCAN T-6295 LC/MS-1/Scans 260-262 Scans 260-262 minus 1-256 & 268-700 Time=7.33 min T-6295 LC/MS-1 -7/5/95- 11:11 AM 1 uL(l00 uMAop)T-6295,deftabond C8,ACN/H20(5% to 95% in 15 min) 3 peaks 100. 183.0 95MYHWOI.3M 101,898 75- 50- 4D 25- 325.5 607.0 0 200 400 600 800 m/z 19 15 Catherwood Road o I'Lhaca.New York 14850 n (607) 266-0665 o Fax (607) 266-0-1740- AOVANCED BIOANALYTICAL SERVICES, INC. 95MYHWOI.3M Figure 12. TIC for the Injectionsof 1 gL of Top, Middle and Bottom Day Zero Solution of T-6294 100, Top 50,900,000 75' 50@ a: 25- D 100, Middle 75' 62,200,000 59 (D cr_ 25- 0. 100 Bottom 75 54,600,000 50r (D cc 25- 1 101 201 301 401 501 0.0 2.8 5.6 8.5 11.3 14.1 Scanrrime (min) 20 15 Catherwood Road n I'thaca,New York 14850 a (607) 266-0665 Fax (607) 266-0749 MMM AOVANCED BIOANALYTICAL SERVICES, INC. 95MYHWOI.3M Figure 13. TIC for the Injectionsof I gL of Top, Middle and Bottom Day Seven Solution of T-6294 1oo, Top 43,500,000 76 (D 50 a: 25- 100@ Middle 76 39,300,000 (D cr 25- a 100- Bottom 75- 50@ 25- 33,900,000 1 101 201 301 401 501 0.0 2.8 5.6 8.5 11.3 14.1 ScanrTime (min) 21 15 Catherwood Road n Ithaca.New York 14850 a (6071)266-0665 a Fax (607) 266-0-/49 AOVANCED BIOANALYTICAL SERVICES, INC. 95MYHWOI.3M Figure 14. ExtractedIon Chromatograms for the Injectionof 1 RL of Day Seven Top Solutionof T-6294 T-6294 LC/MS-1 -7/12/95-1:08PM 1 ulofT-6294(l00 umttop),delCt8a,ACN/H20(5%ACN to95%ACN in15 min) 100/900 - 10 TIC 50 (D ir 297.5/297.5 100 50. m 0.1 311.5/311.5 2 10(@ 50. ol 325.5/325.5 10(@ 3,710,000 40,000 250,000 190,000 a: 0-1 339.5/339.5 10(> 5(> a: 526/526 10& 50 cr 0. 0.0 2.5 1 90 70,000 1,460,000 5.0 7.5 10.0 12.5 15.0 178 267 356 444 533 Time (min)/Scan 22 15 Catherwood Road o Ithaca.New York 14850 o (601@)266-0665 n Fax (607) 266-0749 ADVANCED BIOANALYTICAL SERVICES, INC. Figure 15. Full-ScanMass Spectrum of Peak 1 in Figure 14 T-6294 LC/MS-1/Scans212-221 -Profil0e1 SCAN Scans 212-221minus 1-210& 225-712Time--6.08min T-6294 LC/MS-1 -7/12/95- 1:08PM 1 ulofT-6294(l00 uM/top),delCt8a,ACN/H20(50/oACNto950/*ACNin15 min) 2 peaks loo. 183.0 95MYHWOI.3M 36,447 75- 50a: 25- 297.5 0- r 200 400 600 800 m/z 23 15 Catherwood Road a Ithaca,New York 14850 n (607)266-0665 n Fax (607) 266-0-49 MMM ADVANCED BIOANALYTICAL SERVICES, INC. Figure 16. Full-Scan Mass Spectrum of Peak 2 in Figure 14 -Profil0e1 SCAN T-6294 LCIMS-1/Scans 230-234 Scans 230-234 minus 1-220 & 245-712'rime--6.5m1in T-6294 LC/MS-1 -7/12/95- 1:08PM 1 ulofT-6294(l00 umttop),deftCa8,ACNIH20(50/oACN to950/oACNin15 min) 3 peaks 311.5 100. 95MYHWOI.3M 163,658 75. 183.0 50a: 25. 269.0 0260 p 4'00 60'0 80'0 m/z 24 15 Catherwood Road o Ithaca.New York 14850 s (607) 266-0665 o Fax (607.)266-0749 AOVANCED BIOANALYTICAL SERVICES, INC. Figure 17. Full-Scan Mass Spectrum of Peak 3 in Figure 14 -Profile0 1SCAN T-6294 LC/MS-1/Scans 243-247 Scans 243-247 minus 1-232 & 258-712 7ime--6.88min T-6294 LC/MS-1 -7/12195- 1:08 PM 1 ulofT-6294(100 uM/top),deltCa8,ACNIH20(5%ACN to950/cACN in15 min) 4 peaks too. 325.5 95MYHWOI.3M 158,440 75- 183.0 50- 25- 269.0 343.0 200 400 600 Soo mlz 25 15 Catherwood Road a Ithaca.New York 14850 a (607) 266-0665 a Fax (607) 266-0749 AOVANCEO BIOANALYTICAL SERVICES, INC. Figure 18. Full-Scan Mass Spectrum of Peak 4 in Figure 14 T-6294 LC/MS-1/Scans257-260 -QlSCAN Scans 257-260 minus 1-250& 270-712Time=7.26min T-6294 LC/MS-1 -7/12195-1:08PM 1 ulofT-6294(100uM/top),delCt8a,ACN/H20(5%ACN to95*/*ACNin15 min) 3 peaks 339.5 95MYHWOI.3M 59,865 7& 50- cc 183.0 25- 343.0 0. 200 400 600 800 mtz 26 15 Catherwood Road o Ithaca.New York 14850 a (60-172)66-0665 m Fax (607) 266-0-14@G MMM AOVANCEO BIOANALYTICAL SERVICES, INC. Figure 19. Full-Scan Mass Spectrum of Peak 5 in Figure 14 -Profil0e1 SCAN T-6294 LC/MS-I/Scans432-435 Scans 432-435 minus 1-410& 460-712Time=l2.20 min T-6294 LC/MS-1 -7/12/95- 1:08PM 1 ulofT-6294(100uM/top),deftCa8,ACN/H20(5*/oACN to95%ACN 3 peaks 100. 526.0 in15 min) 95MYHWOI.3M 1,232,500 75- 50- 25169.0 219.0 oa_ 200 400 600 800 mjz 27 15 Catherwood Road n Ithaca. New York 14850 a (607) 266-0665 n Fax f,607)266-0749 AOVANCED BIOANALYTICAL SERVICES, INC. 95MYHWOI.3M Figure 20. Total Ion Chromatogram (TIC) for the Injectionof I RL of Water TIC of allmasses, 100 to900 H20 LC/MS-4 -7/13/95- 11:56 AM 1 ulofH20,delta C8,ACN/H20 gradient(S%ACN to950/oACN in15 min) 100 15,000,000 7& 5& CD a: 2& - - - -- - - - - - 1 101 201 301 401 501 0.0 2.8 5.6 8.5 11.3 14.1 ScarvTime (min) 28 15 Catherwood Road o I'Lhaca.New York 14850 o (60-172)66-0665 n Fax (607) 266-0749 MMM AOVANCEO BIOANALYTICAL SERVICES, INC. 95MYHWOI.3M Figure 21. TIC for the Injectionosf 1 gL of Top, Middle and Bottom Day Zero Solutionof T-6293 100 Top 39,800,000 75, A 50, cc 2 0- 100 Middle 7& c 50' (D cc 25- 24,400,000 0- 83' Bottom 6Z 30,700,000 42- cr 21' 1 101 201 301 401 501 0.0 2.8 5.6 8.5 11.3 14.1 Scan/'rime(min) 29 ADVANCEO BIOANALYTICAL SERVICES, INC. 95MYHWOI.3M Figure 22. TIC for the Injectionosf I gL of Top, Middle and Bottom Day Seven Solutionof T-6293 100 TOP 19,500,000 75" 50. cc 25 0. 100 Middle 75' 15,600,000 5(Y a: 25 100 Bottom 75- 50, CD a: 2& 0' 1 101 0.0 2.8 17,900,000 201 301 401 501 5.6 8.5 11.3 14.1 Scanrrime (min) 30 AOVANCEO BIOANALYTICAL SERVICES, INC. 95MYHWOI.3M Figure 23. ExtractedIon Chromatograms for the Injectionof 1 gL of Day Seven Bottom Solutionof T-6293 T-6293 LC/MS-3 -7/13/95- 11:34AM 1 ulofT-6293(l mM inH20/boftom),deltaC8,ACN/H20 gradient(50/*ACtNo95%ACN in15 100/900 10 TIC 7& 'E 5a 2& 1,260,000 369/369 100 75, 5D 2 50,000 650/650 100 75- 50- (D a: 2 630,000 2 630/630 100, 7& 50, c: 2 0.0 2.5 1 90 3 300,000 5.0 7.5 10.0 12.5 15.0 178 267 355 444 532 Time (min Scan 31 ADVANCEO BIOANALYTICAL SERVICES, INC. 95MYHWOI.3M Figure 24. Full-ScanMass Spectrum of Peak I in Figure23 T-6293 LC/MS-3/Scans 284-285 -Profil0e1 SCAN Scans 284-285 minus 1-278 & 289-712 Time=8.00 min T-6293 LC/MS-3 -7/13/95 -11:34 AM 1 ulofT-6293(l mM inH20/bottom),deltCa8,ACN/H20 gradient(5%ACN to95%ACN 1 peak 100 369.0 in15 40,000 75- 50- a: 2& 200 400 600 800 nVz 32 AOVANCEO BIOANALYTICAL SERVICES, INC. 95MYHWOI.3M Figure 25. Full-ScanMass Spectrum of Peak 2 in Figure 23 T-6293 LC/MS-3/Scans 314-323 -Profil0e1 SCAN Scans 314-323 minus 1-284 & 450-712 7ime--8.96min T-6293 LC/MS-3 -7/13195- 11:34AM 1 ulofT-6293(l mM inH20/boftom),deftCa8,ACNIH20 gradient(50/oACtNo95%ACN 2 peaks 100- 650.0 in15 min) 505,068 75- 50- . 19 0 a: 25- 526.0 0. 200 460 600 800 m/z 33 AOVANCED BIOANALYTICAL SERVICES, INC. Figure 26. Full-ScanMass Spectrum of Peak 3 in Figure 23 95MYHWOI.3M T-6293 LC/MS-3/Scans 412-420 -Profile0 1SCAN Scans 412-420 minus 1-391 & 440-712 Time--l1.72min T-6293 LC/MS-3 -7/13/95- 11:34AM 1 ulofT-6293(l mM inH20/bottom),deftCa8,ACN/H20 gradient(50/cACtNo950/cACN in15 min) 2 peaks 100- 630.0 218,889 75- 50cr 25- 419.0 0 200 400 600 800 m/z 34 ADVANCED BIOANALYTICAL SERVICES, INC. 95MYHWOI.3M Figure 27. TIC for the Injectionosf 1 gL of Top, Middle and Bottom Day Zero Solutionof Fose Amide loor Top 50,400,000 75' 50, ci (D cc 2& 0. 100, Middle 75* 48,900,000 50@ cc 25- 010(@ Bottom 7& 44,600,000 50, 25- LM 1 0.0 101 201 301 401 501 2.8 5.6 8.5 11.3 14.1 Scan/Time (min) 35 MMM ADVANCED BIOANALYTICAL SERVICES, INC. 95MYHWOI.3M Figure 28. TIC for the Injectionosf 1 gL of Top, Middle and Bottom Day Seven Solutionof Fose Amide 1Oor Top 48,500,000 75' cc 2& ioa Nfiddle 75, 50r a) a) cc 25- 010D Bottom 75- "E 50 (D CD cc 25- oll 1 101 0.0 2.8 jv6ow@ 46,300,000 43,900,000 201 301 401 501 5.6 8.5 11.3 14.1 ScanfTime (min) 36 MMM ADVANCEED SIOANALYTICAL SERVICES, INC. 95MYHWOI.3M Figure 29. ExtractedIon Chromatograms for the Injectionof 1 ;IL of Day Zero Top Solutionof Fose Amide Fose Amide LC/MS- 1-7/5/95 -4:32 PM 1 uL of Fose Amide(100 uwtop),deltaC8,ACNIH20(5% to95% in15 min) 100/900 TIC 4,360,000 499/499 311.5/311.5 325.5/325.5 339.5/339.5 CD la 2 A . 3 A 4 lb 410,000 120.000 A 170,000 110,000 398/398 50,000 5 498/498 1,850,000 6 raO6/606 0.0 2.5 1 90 7 50 7.5 10.0 12.5 178 267 355 444 Time (min)/Scan 30,000 15.0 533 37 AOVANCED SIOANALYTICAL SERVICES, INC. 95MYHWOI.3M Figure 30. Full-ScanMass Spectraof Peaks la and lb in Figure 29 Fose Amide LC/MS-1/Scans 221-225 -Profil0e1SCAN Scans 221-225 minus 1-196 & 440-712 Time--6.26min Fose Amide LC/MS-1 - 7/5/95-4:32 PM 1 uL ofFose Amide(l00 uM/top),deftCa8,ACN/HgO(5% to95% in15 min) 3 peaks [P-eakla 1 L---------i-- 141,722 100 183.0 75 50 499.0 25 297.5 ol 200 4;M 600 8@o rrl/z -Profil0e1SCAN Fose Amide LC/MS-1/Scans 390-397 Scans 390-397 minus 1-178 & 405-712 Time=l 1.07min Fose Amide LCIMS-1 - 7/5/95- 4:32 PM 1 uL ofFose Amide(l00 uM/top),deltCa8,ACN/H20(5% to95% in15 min) 1 peak FP-e-a-k lb7 100 498.0 1,482,110 75 50 cr 25 L 200 400 600 800 nvz - 38 ADVANCED SIOANALYTICAL SERVICES, INC. Figure 31. Full-ScanMass Spectrum of Peak 2 in Figure 29 Fose Amide LC/MS-1/Scans 236-245 -Profil0e1 SCAN Scans 236-245 minus 1-231 & 250-712 Time--6.76min Fose Amide LCIMS-1 -7/5/95-4:32 PM 1 uL ofFose Amide(100 umttop),deltCa8,ACN/H20(5% to95% in15 min) 2 peaks loo. 183.0 95MYHWOI.3M 393,298 75. (D "E 50- (D cc 25. 311.5 0 200 460 600 800 m/z 39 MMM ADVANCEO SIOANALYTICAL SERVICES, INC. Figure 32. Full-Scan Mass Spectrum of Peak 3 in Figure 29 -Profil0e1 SCAN Fose Amide LC/MS-1 /Scans 250-258 Scans 250-258 minus 1-235 & 270-712 Time=7.14 min Fose Amide LCIMS-1 -7/5/95-4:32PM IuL ofFoseAmide(I00 uM/top),delCtBa,ACN/H20(5% to95% in15 min) 2 peaks loo. 183.0 95MYHWOI.3M 349,901 75- rA 50. c(rD 25. 325.5 0 200 400 600 800 m/z . 40 MMM AOVANCEO BIOANALYTICAL SERVICES, INC. Figure 33. Full-ScanMass Spectrum of Peak 4 in Figure 29 Fose Amide LC/MS-1/Scans 262-271 -Profil0e1 SCAN Scans 262-271 minus 1-250 & 277-712 Time=7.49 min Fose Amide LCIMS-1 -7/5/95-4:32 PM 1 uL ofFose Amide(l00 uM/top),deftCaB,ACNIH20(5% to95% in15 min) 2 peaks loo. 183.0 95MYHWOI.3M 77,834 75- 15 50. (D cc 25. 339.5 0 200 400 600 800 m/z 41 AOVANCEE) BIOANALYTICAL SERVICES, INC. Figure 34. Full-ScanMass Spectrum of Peak 5 in Figure 29 -profil0e1 SCAN Fose Amide LC/MS-1/Scans 343-344 Scans 343-344 minus 1-338 & 355-712 Time=9.66 min Fose Amide LC/MS- 1-7/5/95-4:32 PM 1 uL of Fose Amide(100 uM/top),deltCa8,ACN/H20(5% to95% in15 min) 2 peaks 398.0 100 95MYHWOI.3M 45,000 75- (D 5& (D cc 25- 249.0 n 200 400 600 800 miz 42 AOVANCEO BIOANALYTICAL SERVICES, INC. Figure 35. Full-ScanMass Spectrum of Peak 6 in Figure 29 -Profil0e1 SCAN Fose Amide LC/MS-1/Scans 388-398 Scans 388-398 minus 1-200 & 420-712 Time=l 1.06min Fose Amide LCIMS-1 -7/5/95-4:32 PM 1 uL of Fose Amide(100 uM/top),deltCa8,ACN/H20(5% to95% in15 min) 1 peak 498.0 100. 95MYHWOI.3M 1,269,023 75. 50- cc 25. 200 460 600 860 m/z 43 ADVANCEO BIOANALYTICAL SERVICES, INC. Figure 36. Full-ScanMass Spectrum of Peak 7 in Figure 29 -Profil0e1 SCAN FoseAmide LC/MS-I/Sc4a2n5s-426 Scans 425-426 minus 1-420 & 430-712 Time=l 1.98 min Fose Amide LCIMS-1 -7/5195-4:32 PM 1 uL ofFoss Amide(100 uM/top),deltCa8,ACN/H20(5% to95% in15 min) 3 peaks 606.0 IOD 95MYHWOI.3M 25,000 7,5 CD 50- cc 2& 249.0 526.0 n- 200 400 600 800 rrl/z 44 AOVANCEO BIOANALYTICAL SERVICES, INC. 95MYHWOI.3M Figure 37. TIC for the Injectionosf I liLof Top, Middle and Bottom Day Zero Solutionof Perfluorooctanamide Iocr TOP 17,500,000 75' 50 4) cc 2-rr 0- 1OCY Middle 75' 25,400,000 50, a: 25- 0- 100, Bottom 75- 17,300,000 cr 25- ol 1 101 201 301 401 501 0.0 2.8 5.6 8.5 11.3 14.1 Scanrrime (min) 45 ADVANCEO BIOANALYTICAL SERVICES, INC. 95MYHWOI.3M Figure 38. TIC for the Injectionosf 1 gL of Top, Middle and Bottom Day Seven Solutionof Perfluorooctanamide 1O(Y Top 15,100,000 75' ..26:, 50, (D 2& 0- 100, Nflddle 75' 14,700,000 50(D 25- 0- 100 Bottom 75- 16,000,000 5a . ig (D cr 25- ol 1 101 201 301 401 501 0.0 2.8 5.6 8.5 11.3 14.1 Scan/Time (min) 46 MMM AEDVANCED BIOANALYTICAL SERVICES, INC. 95MYHWO1.3M Figure 39. ExtractedIon Chromatograms for the Injectionof 1 JIL of Day Zero Top Solutionof Perfluorooctanamide C7Fl 5CONH2 LC/MS-2- 7/5/95-6:25 PM 1 uL ofC7Fl 5CONH2(l mM/top),deltCa8,ACN/H20(5% to95% in 15 min' 100/900 990,000 TCA 369/369 220,000 297/297 2 20,000 311.5/311.5 325.51325.5 a: 339.5/339.5 392/392 412/412 0.0 2.5 1 90 3 4 A 5 6 70,000 90,000 40,000 120,000 370,000 7 5.0 7.5 10.0 12.5 15.0 178 267 354 443 531 Time (min)/Scan 47 MMM [DVANCEO BIOANALYTICAL SERVICES, INC. Figure 40. Full-ScanMass Spectrum of Peak 1 in Figure 39 C7Fl 5CONH2 LC/MS-2/Scans 224-226 -Profil0e1 SCAN Scans 224-226 minus 1-224 & 227-712 Time--6.32min C7FI SCONH2 LC/MS-2 -7/5/95- 6:25 PM 1 uL ofC7Fl5CONH2(l mM/top),deltCa8,ACN/H20(5% to95% in15 min) 2 peaks loo. 183.0 95MYHWOI.3M 84,243 75- 50cc 25. 297.0 0- 260 400 660 800 m/z 48 MMM AOVANCED BIOANALYTICAL SERVICES, INC. Figure 41. Full-ScanMass Spectrum of Peak 2 in Figure 39 -Profil0e1 SCAN C7Fl5CONH2 LC/MS-2/Scans 164-167 Scans 164-167 minus 1-140 & 200-712 Time=4.64 min C7Fl5CONH2 LCIMS-2 -7/5(95-6:25PM 1 uL ofC7FI 5CONH2(l mM/top),deftCa8,ACN/H20(5% to95% inIS min) 4 peaks loo. 169.0 95MYHWOI.3M 221,740 75. uv cc I 119.0 25 219.0 369.0 0- 200 400 600 800 m/z . 49 MMM ADVANCEO BIOANALYTICAL SERVICES, INC. Figure 42. Full-ScanMass Spectrum of Peak 3 in Figure 39 -Profil0e1 SCAN C7Fl 5CONH2 LC/MS-2/Scans 239-245 Scans 239-245 minus 1-235 & 250-500 Time--6.80min C7Fl5CONH2 LC/MS-2 -7/5/95- 6:25PM I uL ofC7Fl 5CONH2(l mmttop),deftCa8,ACNIH20(5% to95% in15 min) 2 peaks loo. 183.0 95MYHWOI.3M 180,925 75. 504) cr 25. 311.5 200 400 600 800 m/z 50 MMM ACDVANCEO BIOANALYTICAL SERVICES, INC. Figure 43. Full-ScanMass Spectrum of Peak 4 in Figure 39 C7Fl5CONH2 LC/MS-2/Scans 251-261 -Profil0e1 SCAN Scans 251-261 minus 1-240 & 360-712 Time=7. 19 min C7Fl 5CONH2 LC/MS-2 -7/5/95- 6:25 PM 1 uL ofC7Fl5CONH2(l mM/top),deftCaB,ACN/H20(5% to95% in15 min) 2 peaks 183.0 100 95MYHWOI.3M 169,107 75. So'Zi 0 a: 25. 325.5 0- 200 400 600 800 m/z 51 MMM AOVANCED BIOANALYTICAL SERVICES, INC. Figure 44. Full-ScanMass Spectrum of Peak 5 in Figure 39 -Profil0e1 SCAN C7Fl5CONH2 LC/MS-2/Scans 265-271 Scans 265-271 minus 1-262 & 274-712 Time=7.53 min C7Fl5CONH2 LCIMS-2 -7/5/95-6:25PM 1 uL ofC7FI 5CONH2(l mmttop),deftCa8,ACN/H20(5% to95% in15 min) 2 peaks 183.0 100. 95MYHWOI.3M 45,726 75- (D 50- (D a: 25 339.5 0L 200 400 600 800 m/z 52 MMM AOVANCEO BIOANALYTICAL SERVICES, INC. Figure 45. Full-ScanMass Spectrum of Peak 6 in Figure 39 C7Fl5CONH2 LC/MS-2/Scans 356-357 -ProfilQel SCAN Scans 356-357 minus 1-354 & 370-712 Time=l 0.07 min C7Fl5CONH2 LCAAS-2 -7/5/95-6:25 PM 1 uL ofC7Fl 5CONH2(l mM/top),deftCa8,ACNIH20(5% to95% in15 min) 3 peaks 100. 223.0 95MYHWOI.3M 105,000 75- 50. 4D cc 25. 154.0 392.0 200 400 600 800 m/z 53 MMM ADVANCEED BIOANALYTICAL SERVICES, INC. Figure 46. Full-ScanMass Spectrum of Peak 7 in Figure 39 C7Fl5CONH2 LC/MS-2/Scans366-367 -ProfilQel SCAN Scans 366-367 minus 1-362 & 380-712 Time--l0.35 min C7FI 5CONH2 LCfMS-2- 7/5/95-6:25 PM 1 uL ofC7FI5CONH2(l mM/top),delCt8a,ACN/H20(5%to95% in15min' 2 peaks 412.0 100. 95MYHWOI.3M 329,613 7& (D 18 5(@ (D 25249.0 200 400 600 800 M/Z 54