Document B5XYQ6ORbO87gyJa6eeDdnqpj

ADVANCED BIOANALYTICAL SERVICES, INC. ANALYTICAL RIEPORT TITLE: AdditionalCharacterizationof Metabolitesof T-6292, T-6293 and T-6294 from Rat and Human Hepatocytes by TurbolonSpray LC/MS and LC/MS/MS. Semi-Quantitative Analysisof T-6295 in Rat and Human Hepatocytes Incubated with T-6292,T-6293 and T-6294 by LC/MS/MS. DATE: REPORT: AUTHOR: PREPARED FOR: 27 January, 1998 98AGKPOI.3M Grace K. Poon, PhD Steve Lowes, PhD 3M Medical Department, Toxicology Services, St.Paul, MN 55133 FB .2 NO. OF PAGES: 69 REPORT: 98AGKPOI.3M 15 Catherwood Road o Ithaca.New York 14850 n (607) 266-0665 n Fax (607) 266-0/-49 AOVANCEO BIOANALYTICAL SERVICES, INC. ADVANCED BIOANALYTICAL SIGNATURE PAGE SERVICES Title: AdditionalCharacterizatioonfMetabolitesofT-6292,T-6293 and T6294 from Rat and Human Hepatocytesby TurbolonSpray LC/MS and LC/MS/MS. Sen-d-QuantitatiAvnealysisofT-6295 inRat and Human HepatocytesIncubated withT-6292,T-6293 and T-6294,by LC/MS/MS. Reported by: J6;@eau /9@v acieorK. Poon, PhD. @e n' ResearchScientist zqzn Date Reviewed and Approved by: /,It1 Mark Lentham bate Auditor Authorizedfor Release by: Steve Low*@, PhD. Date ScientifiDcirector Advanced BioAnalyticalServices REPORT: 98AGKPOI.3M ADVANCEO 3 BIOANALYTICAL SERVICES, INC. TABLE OF CONTENTS SIGNATURE PAGE ..........................................................2............... TABLE OF CONTENTS .......................................................3.............. LIST OF TABLES ............................................................5.............. LIST OF FIGURES ...........................................................6............... PART A: CharacterizatioonfMetabolitesofT-6292, T-6293 and T-6294 from Rat and Human Hepatocytesby TurbolonSpray LC/MS and LC/MS/MS ............1.0....... I INTRODUCTION ...................................................1.0.............. 1.1. 1.2. 2. BACKGRIOUND .................................................................1.0................. STUDY OBJECTTVE ..............................................................1.0................ EXPERIMENTAL ...................................................1.4.............. 2.1. 2.2. 2.3. 2.4. 2.5. 2.6. 3. CHEMICALS AND MATERIALS .............................................1.4.............. STANDARD SOLUTIONS ..................................................1.5.............. HPLC ELUENT PREPARATION ............................................1.5.............. SAMPLE PREPARATION ..................................................1.6.............. LC/MS AND LC/MS/MS INSTRUMENTATION ...............................1.6.............. DATA HANDLING .......................................................1.8.............. RESULTS AND DISCUSSION ........................................1.8.............. 3.1. CONTROL EXPERIMENTS ................................................1.9.............. 3.2. METABOLISM OF T-6292 ................................................1.9.............. 3.2.1. Metabolism of T-6292 by Rat Hepatocytes at 0 hour ......................1.9............ 3.2.2. 3.2.3. Metabolism of T-6292 by Rat Hepatocytes at 6 hour .......................2.0............ Metabolism of T-6292 in Human Hepatocytes at 0 hour ...................2.1.......... 3.2.4. Metabolism of T-6292 by Human Hepatocytes at 6 hour ...................2.1......... 3.3. METABOLISM OF T-6293 ................................................2.2.............. 3.3.1. Metabolism of T-6293 by Rat Hepatocytes at 0 hour .....................2.2............. 3.3.2. Metabolism of T-6293 by Rat Hepatocytes at 6 hour .....................2.2............. 3 3.3. Metabolism of T-6293 by Human Hepatocytes at 0 hour ..................2.3.......... 3.3.4. Metabolism of T-6293 by Human Hepatocytes at 6 hour .................2.3........... 3.4. METABOLISM OF T-6294 ................................................2.4.............. 3.4.1. Metabolism of T-6294 by Rat Hepatocytes at 0 hour .....................2.4............. 3.4.2. Metabolism of T-6294 by Rat Hepatocytes at 6 hour .....................2.4............. REPORT: 98AGKPOI.3M AOVANCEO SIOANALYTICAL 4 SERVICES, INC. 3.4.3. MetabolisomfT-6294byHuman Hepatocytaets0hour.............2.5........... 3.4.4. MetabolisomfT-6294byHuman Hepatocytaets6 hour.............2.5........... 3.5. PRODUCT-ION MASS SPECTRA ...........................................25.............. 3.6. POSITIVE IONIZAT7[ON LC/N4S ANALYSIS OF HUMAN HEPATOCYTES INCUBATED wrrh T-6292 AND T-6293 .............................................2.7.............. PART B: Semi -QuantitativAenalysisof T-6295 inRat and Human Hepatocytes IncubatedwithT-6292,T-6293 and T-6294 by LC/MS/MS ................2.8........... 4. INTRODUCTION ................................................2.8.............. 5. EXPERIN4ENTAL ................................................2.8.............. 5.1. 5.2. STANDARD SOLUTIONS ................................................2.8.............. PREPARATION OF QUENCHING SOLUITON: ........................................2.9................. 5.3. HPLC ELUENT PREPARATION: ................................................2.9................ 5.4. 5.5. SAMPLE PREPARAT71ON ................................................2.9.............. PREPARATION OF RAT HEPATOCYTES STANDARD CURVE SAMPLES ..............2.9....... 5.6. LC/MS/MS INSTRU-MENTANON .........................................3.0............. 5.6.1. HPLC conditions..................................................3.0.............. 5.6.2. MS conditions.....................................................3.0.............. 5.6.3. Data Handling ....................................................3.1.............. 6. Resultsand Discussion:............................................3.1.............. 7. References ......................................................3.5.............. REPORT: 98AGKPOI.3M ADVANCED BIOANALYTICAL SERVICES, INC. LISTOF TABLES Table1: SummaryofProposeSdtructuroefsMetabolitoefsT-6292IncubatewdithRat and Human Hepatocytes..............................................12............... Table II: Summary of Proposed Structuresof Metabolitesof T-6293 Incubatedwith Rat and Human Hepatocytes..............................................1.3.............. Table III:Summary of Proposed Structuresof Metabolitesof T-6294 Incubatedin Rat and Human Hepatocytes..............................................14............... Table IV: Proposed product-ionfragmentationforMetaboliteVI ....................2.6.............. Table V: Proposed product-ionfragmentationforMetaboliteVI ....................2.7.............. TableVI:QuantitativeAnalysis of T-6295 in Rat and Human Hepatocytes after Incubationwith T-6292, T-6293 and T-6294 ............................3.2.............. Table VII:QuantitativeAnalysisof T-6295 inT-6292,T-6293 and T-6294 ............3.5....... REPORT: 98AGKPOI.3M A E E) 6 BICA:,'@,,@',LYTICA:@L MMM SE=',:CES, INC. LISTOF FIGURES Figur1e.Reconstruicotncehdromatog(ria)nm,egatiivoenizatmiaosnsspectr(uimi) and LC/MS/MS product-iomnass spectrum(iiio)fauthentiTc-6293 .......3.6... Figure2. Reconstructedion chromatogram (i) and LC/MS/MS product-ionmass spectrum(iio)f authentiTc-6294.....................................3.7............. Figure3. Reconstructeidon chromatogram (i),negativeionizatiomnass spectrum(ii) and LC/MS/MS product-iomnass spectrum(iiio)fauthentiTc-6295........3.8... Figure4. Full scan reconstructeidon chromatograms of controlrat hepatocytes incubatedfor6 hours withno testarticle.s.............................3.9............. Figure5. Fullscan reconstructeidon chromatograms of controlhuman hepatocytes incubatedfor6 hours withno testarticle.s.............................4.0............. Figure6. Fullscan reconstructeidonchromatograms of T-6292 incubatedfor6 hours with no hepatoc),t.e.s.............................................4.1.............. Figure7. Fullscanreconstructeidon chromatograms of T-6293 incubatedfor6 hours with no hepatocytes...............................................4.2.............. Figure8. Fullscanreconstructeidon chromatograms of T-6294 incubatedfor6 hours with no hepatocytes...............................................4.3.............. Fi0-ure9. Full scan reconstructeidon chromato9rams showing the presence of metaboliteasfterincubatingrathepatocyteswithT-6292 at0 hour..........4.4..... FigureIO.Fullscan reconstructeidon chromatograms showing the pr'esenceof metaboliteasfterincubatingrathepatocyteswithT-6292 for6 hour.........4.5.... Figure11.Full scan reconstructeidon chromatograms showing the presence of metabolitesafterincubatinghuman hepatocyteswithT-6292 at0 hr........4.6..... Figure 12.Full scan reconstructedion chromatograms showing the presence of metabolitesafterincubatinghuman hepatocyteswithT-6292 for6 hour.....4.7.. Figure13.(iS)RM chromatogramsof rathepatocyteisncubatedwith no testarticle(si;i) SRM chromatograms of human hepatocytesincubatedwith no testarticles; (iiiS)RM chromatogramsof human hepatocytesincubatedwith no test article.s........................................................4.8............. REPORT: 98AGKPOI.3M AOVANCEO BIOANALYTICAL 7 SERVICES, INC. Figure14.SRM chromatograms showing the absence of metaboliteIV in (i)rat hepatocytesincubatedwith T-6292 at 0 hour;(ii)rathepatocytesincubated with T-6292 for 6 hour;(iiih)uman hepatocytesincubatedwith T-6292 at 0 hour and (iv)human hepatocytesincubatedwith T-6292 for6 hour..........4.9.... Figure 15.SRM totalionchromatograms showina thepresenceof metaboliteVII in(i)T6292 standardsolution;(ii)incubationof T-6292 with rathepatocytesat 0 hour;(iiii)ncubationof T-6292 with rathepatocytesat6 hour;(iv)incubation ofT-6292 with human hepatocytesat0 hour and (v)incubationof T-6292 with human hepatocytesat6 hour..........................................5.0................ Figure 16.SRM totalion chromatograms showing the presenceof metaboliteIX in (i)T6292 standardsolution(;ii)incubationof T-6292 with rathepatocytesat 0 hour;(iiii)ncubationof T-6292 with rathepatocytesat 6 hour;(iv)incubation of T-6292 with human hepatocytesat0 hour and (v)incubationof T-6292 with human hepatocytesat6 hour...........................................5.1................ Figure 17.SRM totalion chromatograms showing the presence of metaboliteX in (i)T6292 standardsolution;(iii)ncubationofT-629 with rathepatocytesat0 hour; (iiii)ncubationof T-6292 with rathepatocytesat6 hour;(lv)incubationof T6292 with human hepatocytesat 0 hour and (v) incubationof T-6292 with human hepatocytesat6 hour...........................................5.2............... Figure18.Full scan reconstructedion chromatograms showing the presence of metabolitesafterincubatingrathepatocyteswithT-6293 for0 hour.........5.3.... Fic,ur1e9.Fullscan reconstructedion chromatograms showing the presence of metabolitesafterincubatingrathepatocyteswith T-6293 for6 hour.........5.4.... Figure20.Full scan reconstructedion chromatograms showing the presence of metaboliteasfteirncubatinhguman hepatocytewsithT-6293 for0 hour.....5.5.. Figure21.Full scan reconstructedion chromatograms showing the presence of metabolitesafterincubatinghuman hepatocyteswith T-6293 for6 hour.....5.6.. Figure22.SRM totalion chromatograms showing the presenceof metaboliteIV in (i) incubationof T-6293 with rathepatocytesat0 hour;(ii)incubationof T-6293 with rat hepatocytesat 6 hour; (iii)incubationof T-6293 with human hepatocytesat0 hour and (iv)incubationof T-6293 with human hepatocytesat 6 hour.............................................................5.7............... REPORT: 98AGKPOI.3M ADVANCED SIOANALYTICAL 8 SERVICES, INC. Figure23.SRM totalionchromatograms showing thepresenceof metaboliteVH in (i)T6293 standardsolution;(ii)incubationof T-6293 with rat hepatocytesat 0 hour;(iiii)ncubationof T-6293 with rathepatocytesat6 hour;(iv)incubation of T-6293 with human hepatocytesat0 hour and (v)incubationof T-6293 with human hepatocytesat6 hour...........................................5.8............... Figure24.SRM totalionchromatograms showing thepresenceof metaboliteIX in (i)T6293 standardsolution;(ii)incubationof T-6293 with rathepatocytesat 0 hour;(iiii)ncubationof T-6293 withrathepatocytesat6 hour;(iv)incubation of T-6293 with human hepatocytesat0 hour and (v)incubationofT-6293 with human hepatocytesat6 hour...........................................5.9............... Figure25.SRM totalion chromatograms showing thepresenceof metaboliteX in (i)T6293 standardsolution(;ii)incubationof T-6293 with rathepatocytesat 0 hour;(iiii)ncubationof T-6293 withrathepatocytesat6 hour;(iv)incubation of T-6293 with human hepatocytesat0 hour and (v)incubationof T-6293 with human hepatocytesat6 hour...........................................6.0............... Figure26.Reconstructedion chromatograms showing the presence of metabolitesafter incubatingr,athepatocyteswith T-6294 for0 hour........................6.1............... Figure27.Reconstructedion chromatograms showing the presence of metabolitesafter incubatinsr!athepatocyteswith T-6294 for6 hour........................6.2............... Figure28.Reconstructedion chromatograms showing the presence of metabolitesafter incubati1n. ahuman hepatocyteswith T-6294 for0 hour....................6.3............... Figure29.Reconstructedion chromatograms showing the presence of metabolitesafter incubatin-human hepatocyteswith T-6294 for6 hour....................6.4............... Figure30.SRM totalionchromatograms showing thepresenceof metaboliteVH in (i)T6294 standardsolution(;iii)ncubationofT-6294 with rathepatocytesat0 hour;(iiii)ncubationof T-6294 withrathepatocytesat6 hour;(iv)incubation of T-6294 with human hepatocytesat0 hour and (v)incubationof T-6294 with human hepatocytesat6 hour ..........................................6.5.............. Figure3 1.SRM totalionchromatograms showing thepresenceof metaboliteX in (i)T6294 standardsolution;(ii)incubationof T-6294 with rathepatocytesat 0 hour; (iiii)ncubationof T-6294 with rathepatocytesat6 hour;(iv)incubation of T-6294 with human hepatocytesat0 hour and (v)incubationof T-6294 with human hepatocytesat6 hour...........................................6.6.............. Figure32.Negat]N,eionizationproduct-ionmass spectraof (i)metaboliteIII;(ii) metaboliteVI and (iiim)etabolitVeIH ..................................6.7.............. REPORT: 98AGKPOI.3M AD\IANCEO 9 BIOANALYTICAL SERVICES, INC. Figure33.Product-ionmass spectraof (i)MetaboliteXIIIIand (iim)etaboliteXI .......6.8... Figure34.Selected Reaction Monitoring Chromatograms from LC/MS/MS of a CalibrationStandardHepatocyteExtract(2 ng/mL T-6295) ................6.9.......... REPORT: 98AGKPOI.3M AEDV-^ %CEr--) 10 BIOA',@ALY L@ICAL ELM SEPV'=ES, INC. PARTA: CHARACTERIZATIOONF METABOLITESOF T-629T2-,6293 AND T-629F4ROM RAT AND HUMAN HEPATOCYTESBY TL-RBOIONSPRALYC/MSAND LC/MS/MS. 1. INTRODUCTION 1.1.BACKGROL-ND The metabolism of T-6292,T-6293 and T-6294 inrathepatocyteswas investigateadt Advanced BioAnalyticalServicesI,nc.usingliquidchromatographyionspraytandem mass spectrometry(Reference1).Severalmetabolitehsad been identifienda,mely the glucuronideacidconju-atet,hecarboxylicacid,thesulfonamidoalcohol,theN-dealkyl sulfonamidoalcoholand thesulphonamideof T-6293. 1.2. STUDY OBJECTIVE The principlaeim ofthiswork was (1) To completethecharacterizatioofnthemetabolicproductsofT-6292,T-6293 and T-6294 which were formed by incubatingeachcompound withratand human hepatocytes; (2) structuraclharacterizatioofnthecarboxylicacidand alcoholanaloguesusing tandem mass spectrometry; (3) identificatioofnadditionamletabolitebsy LC/MS, usingpositivieonization ionspray; (4) identificatioofnthealdehydeand N-dealkylaldehydeanaloguesofT-6293; (5) semi-quantitatiavnealysistodeterminetheconcentratioonf T-6295 intheratand human hepatocytesafterincubatingwith T-6292,T-6293 and T-6294. REPORT: 98AGKPOI.3M AOVANCEO BIOANALYTICAL SERVICES, INC. The quenched incubateswere directlyinjectedand separatedon an HPLC column. Chromatographic separationof the variousconstituentwsas accomplished using gradient elution.Metaboliteswere characterizedby LC/N4S and LC/MS/MS, and theresultsare summarized inTables I - IH. The mass spectrometerwas operatedinthe fullscan single MS mode (nVz 150-800).Tandem mass spectrometrywas used forfurtherstructural elucidationand confirmation. REPORT: 98AGKPOI.3M ADVANCED 12 BIOANALYTICAL SERVICES, INC. Table1:Summary ofProposeSdtructuroefsMetabolitoefsT-6292Incubatewdith Rat and Human Hepatocytes Proposed Structure CH, -CH /- 3 C,F,,-SO,-N "'CH,--CHOH CH,-CH, C,F,,--SO,--N/ - "CH,-CH.P-Glu Abbreviation [M-Hl- Rat 0 hour 6 hour 11 570 No MS No MS (T-6292) response response 111 746 x V/ Human 0 hour No MS response 6 hour No MS response x /@CH,-CH, IV 568 x x x C,F,f-SO,-N 'CH, --CHO CH, --cm, / v 584 x V/ x 'CH, --CHCOOH H C,F,T-So,-N / CH,-CH,OH vi 542 x H VH C,F,,-SO,-N 'CH, --CHO 540 x H / C,F,-F--SO.;-N ". CHCOOH viu 556 x H / C,FIT-SO,--N 'I@ CH,-CH, ix 526 (T-6294) CH, --CH, x CxFI7--SOI--N 606 x V/ x trace x x trace x C8FI7-SO27-NH2 xi 498 V/ V, C 8F 17---S0 _@H xn 499 x (T-6295) xm 650 x x C.F,,---SO.,-N 'cli@-CH.0-SO,H x V, trace trace Observed x Not observed REPORT: 98AGKPOI.3M 13 BIOA.-%,ALYTICAL SEPVICP--S. INC. Table II:Summary ofProposed StructuresofMetabolitesofT-6293 Incubatedwith Rat and Human Hepatocytes Proposed Structure /CH2--CH@, 0 0I- 'CH,---CH,O@-OH Abbreviation [M-H]- 1 (T-6293) 650 Rat 0 hour 6 hour V/ Human 0 hour 6 hour V/ C H, -CH, C,F,7-SO,-N CH,-CH,OH C,F,5-SOi--N /CH2-CH, CH,-CH,O-Glu C,F,T-SO@-N CH,-CH, / CH,-CHO CH,-CH, C,F,-7r--SO,--N C14.-CH,COOH H C,F,7-SO,- 11 CH,-CH.OH C,F,T-SO@--N H / CH,-CHO C,F,5- H / SO.@-- N CH.COOH H / C,F,F-SO2-- N "I CH,-CH3 CRF17--SO2--N CH, --CH, / "SO,H c 8 FIT-so,----NH2 r8F 17-S 0 @H IV/ Observed x Not observed 11 (T-6292) in IV 570 No MS No MS No MS No MS response response response response 746 x x I 568 x x x x v 584 x V/ x V/ vi 542 x x vii 540 x trace x trace Villi 556 x x DC 526 x (T-6294) x 606 x x -x x xi 498 x x xii 499 v (T-6295) REPORT: 98AGKPOI.3M Ar--'V,a.NCEE) 14 BIOANALYTICAL SERVICES, INC. TableIII:Summary ofProposedStructureosfMetabolitoefsT-6294Incubateidn RatandHuman Hepatocytes ProposeSdtructure Abbreviation H I/ 8F 17 "CH,--CHOH CF,@--SO,-N H / 'CH, ---CHO H / C,F,,--S02--N @-CH2COOH C,F,7-SO2-N H / 'CH, -CH, CH,-CH3 / "-solh CgF 1-7 -So,---NH, C F 8 17--So @H vi vn vni ix (T-6294) x xi XIII (T-6295) (M-H]- Rat Human 0 hour 6 hour 0 hour 6 hour 542 x x 540 x x x x 556 x 526 x V/ 606 x x x x 498 V/ V/ I/ V/ 499 V/ V/ V" V/ V/ Observed x Not observed 2. EXPERIMENTAL 2.1. CliEMICALS AND MATERULS T-6292, T-6293, T-6294 and T-6295 were providedby 3M Medical Department, Toxicology Services,St.Paul,MN 55133. Polypropyleneautosamplervial: Polypropylenescrew-cap tubes: Ethanol Cat # 66008-014,VWR ScientifiIcnc.,Bridgeport, NJ 08014 16 x 100 mm, Cat # 500 765, Sun Brokers Inc., Wilmington, NC 28402 Cat # I12000200CSPP, PharinacoProducts, Brookefield,CT 06804 REPORT: 98AGKPOI.3M C)VANCED E31CANALYTICAL SERVICES, INC. HPLC Grade Methanol Dimethyl Sulfoxide Ammonium Acetate 15 Cat # 230-4,Baxter/ScientifPircoducts,McGaw Park,IIL60085 Cat # 27043-1,Sigma-AldrichInc.,Milwaukee, Wl 53233 Cat # 24019-2,Sigma-AldrichInc.,Milwaukee, WI 53233 2.2. STANDARD SOLUTIONS StocksolutionsofT-6292,T-6293,T-6294 and T-6295 were preparedata concentration of 100 gg/mL indimethylsulfoxid(eDMSO). Working solutio(n10jig/mL)ofeach standardwere preparedby dilutinIgmL ofthestocksolutioinnto9 mL ethanol.All stocksolutionsand workingsolutionwsere storedat4' C and were allowedtoequilibrate toroom temperaturebeforeuse. Preparation of AnalyticalStandard Stock Solutions Stock Solutionsof T-6292, T-6293,T-6294 and T-6295 (100 gg/mL): Each testarticlweas accuratelyweighed on a micro balanceand transferretdo a 16 x 100 mm polypropylenetube.The solidsampleswere dissolvedinappropriataemount of DMSO toyielda 100 [tg/mLsolution. Working SolutionsofT-6292,T-6293,T-6294 andT-6295 (10jig/mL): Each standardworkingsolutionwas preparedby dilutintghestocksolution(ImL) with ethanol(9mL) ina 16 x 100 mm polypropylenteube. 2.3. HPLC ELUENT PREPARATION 1 M Ammonium AcetateSolutionA:mmonium acetat(e77g)was addedtoNANOpure water(IL)ina volumetricflask.The solutiownas filteretdhrouC)gha 0.45gm filtearnd storedat4' C. REPORT: 98AGKPOI.3M ADVANCED BIOANALYTICAL 16 SERVICES, INC. 2 mM Ammonium AcetatSeolutionI:M ammoniumacetastoeluti(o2nmL) was dilutedto IL withNANOpure water.The solutionwas filteretdhrougha 0.45mm filter and storedatambienttemperature.A freshsolutionwas preparedevery month. Methanol : 2 mM Ammonium Acetate (90:10v/v),Eluent:Methanol (450mL) was mixed with2 mM ammonium acetatesolution(50mL). The solutionwas storedat ambient temperature.A freshsolutionwas preparedevery month. Methanol: 2 mM Ammonium Acetate (10:90v/v),Eluent:Methanol (50mL) was mixed with2 mM ammonium acetatesolution(450 mL). The solutionwas storedat ambienttemperature.A freshsolutionwas preparedeverymonth. 2.4. SAMPLE PREPARATION The metabolismstudywas carriedout atSRI InternationaMle,nlo Park,CA. The incubateswere quenched withan equalvolume of ice-coldmethanol,centrifugedand shippedindry icetoAdvanced BioAnalyticaSlervices(ABS). The sampleswere stored atABS (-20'C). Duringsample analysist,hesupernatanwtas transferretdoan autosamplervial.The autosamplervialswere maintainedinan ice-watebrathatalltimes exceptdurinc@ginjection. 2.5. LC/MS AND LC/MS/MS INSTRUMEENTATION Liquidchromatographywas performedon Shimadzu LC-IOAD pumps and Shimadzu SCLIOA gradientcontrolle(rShimadzuScientifiIcnstrumentsI,nc.,Columbia MD 21046).Samples were introducedwith a Perkin-Elmer200 seriesautosamplerT.he HPLC column (BetasilC18, 2 x 100 mm) was obtained from Keystone Scientific,Inc., BellefonteP,A 16823.A SciexAPI IR' triplqeuadrupolemass spectrometewras used for sample analysis. REPORT: 98AGKPOI.3M AOVANCEO 17 SIOANALYTICAL MMM SERVICES, INC. LC Conditions: LC Gradient: Time %A (min) (10:90CH30H:2mMNH4OAc) 0-5 80 to0 % 5-10 0% 10-10.5 0 to80 % 10.5- 15 80% %B (90:IOCH30H:2mMNH4OAc) 20 to 100 % 100% 100 to 20 % 20% Flow rate: 200 gumin TypicalMass SpectrometerConditions: MS Mode Source: TurbolonSpray Ion Spray Voltage: -3800 V Orifice: -78 V DeclusterincP,otential: -48 V IonizationMode: Negative CurtainGas (U.H.P.N,) 1.2LPM NebulizerGas (N,) 60 PSI CollisionGas Thickness: N/A CollisionEnergy: N/A Dwell Time: 5 msec Step Size: lamu Scan Range: 150-800 amu MS/MS Mode* TurboIonSpray -3900 V -60 V -30 V Negative 1.2LPM 60 PSI 280 x 1013 atoms/squarecm 35 eV 200 msec variable MetabolitIeV Transitions:nVz 568 ->269;526 526 -> 83; 526 -> 65 MetaboliteVII Transitions: nVz 540 -> 269; 540 540 -> 83; 540 -> 65 MetaboliteIX Transitions: ni/z526 -> 419; 526 169;526 -> 119; 169;540 -> 119; 269; 526 -> 169; REPORT: 98AGKPOI.3M AOVANCEO BIOANALYTICAL 18 SERVICES, INC. 526 123;526 97 MetaboliteX Transitions: m/z 606 -> 269;606 -> 169;606 -> 119; 606 -> 83;606 -> 65 Data System: RAD Version2.6;LC Tune Version2.4; MultiviewVersion1.2;MacSpec Version3.3; MacQuan Version 1.4 PPG Calibration Instrumentmass axiscalibratiownas performedby infusionof PPG-425 calibration solution(polypropyleneglycols,averagemolecularweight425,dissolvedin 1:1 methanol :2 mM ammonium acetatecontaining0.1 % formicacidand 0.1 % acetonitrile) ata flow rateof 10 gumin inthepositivieon mode ofdetectionP.eak widthswere approximately0.6amu athalf-heighitnthesingleMS mode. A calibratiocnheck was performedfortheanalyteson Ql by infusionof a I @ig/mLsolutioninethanolata flow rateof 10 gumin. The mass spectrometripcarametersand sensitivitwyere optimized using60% B oftheHPLC mobilephase at200 pumin. 2.6. DATA HA-NDLING All raw chromatographicand mass spectrometridcatawere storedon theABS fileserver ABSI-FS.DATA inthefilheierarch"yRAWDATA:Validation:3MSRI:3MSRI QualitativeA"l.lexperimentalinformationwas storedinABS notebookNo: 2070. 3. RESULTS AND DISCUSSION T-6293,T-6294 and T-6295 displayed[M-Hl- ionsatni/z650,526 and 499 respectively. The reconstructeidonchromatograms and negativeionizatiopnroductionmass spectra of compounds T-6293,T-6294 and T-6295 areshown inFigures1-3.Their fragmentationpatternswere describedindetailisnpreviousstudy[reference1].As observedbefore,T-6292 did notshow any signalinpositiveor negativeionizatiomnodes REPORT: 98AGKPOI.3M AOVANCEO BIOANALYTICAL 19 SERVICES, INC. [referenIc]e.Thereforneo,attempwtasmadetocharacterTi-z6e292asaparendtrugor a metaboliteinthisstudy. Rat hepatocytes(referenc#e 4) and human hepatocytes(Female 1,reference#HH357) were used forthestudy. 3.1. CONTROL EXPERU,4ENTS The fullscanreconstructeidonchromatogramsof theincubationmedia which contained ratand human hepatocytesbut no testarticleasredisplayedinFigures4 and 5 respectivelyT.hese samples were analysedas controlsamples.The blank samples did not show any signalattheretentiotnimesexpectedfortheT-6292,T-6293 and T-6294 metabolites. The fullscanreconstructeidonchromatogramsof theincubationmedia which contained T-6292, T-6293 and T-6294 but no hepatocytesaredisplayedinFigures6,7 and 8 respectivelyT.hese sampleswere analysedas controlsamples. However, compounds IX, and XI were presentintheT-6292 sample(Figure6). IntheT-6293 sample,apartfrom theparentdrug,compound XII was observed(Figure7). IntheT-6294 sample,apart from theparentdrug,compound XI was detected(Figure8). 3.2. METABOLISM OF T-6292 3.2.1. Metabolism of T-6292 by Rat Hepatocytes at0 hour The fu.lslc-anLC/MS reconstructeidonchromatograms of therathepatocytessample incubatedwithT-6292 for0 hourareshown inFigures9. Figure9 (i- x)displaysthe ion chromatograms for the following: the parent compound (nvz 570), metabolite M (in/z746),metabolitVe ("Vz584),metabolitVeI (nVz542),metaboliteVEII(nVz556), metaboliteIX (nVz526),metaboliteX (rn/6z06),metabolitXeI (nVz498),metabolitXeII (rnlz499)and metaboliteXIII(ndz650).Even thoughcompound DC,XI and XII were detectedinthissample,compound IX and XI were observedinthecontrolsample containingT-6292 and no hepatocytes(Fioure6). REPORT: 98AGKPOI.3M AOVANCEO BIOANALYTICAL 20 SERVICES, INC. 3.2.2. MetabolismofT-6292by Rat Hepatocyteast6 hour The fullscanLC/.MSreconstructieodnchromatogramosftherathepatocytseasmple incubatedwith T-6292 for6 hour areshown inFigures10. Figure 10 (i- x) displaysthe ion chromatograms forthefollowing:theparentcompound (tnl,5-70),metaboliteIII (rn/z746),metaboliteV (rnlz584),metaboliteVI (nilz542),metaboliteVIII(nVz556), metaboliteIX ("z/z526),metaboliteX (nilz606),metaboliteXI (nvz498),metaboliteXII (nVz499) and metaboliteXIII (nVz 650).This sample containedmetabolitesIIIV,, VI, VIII,IX,XI,XII and XIB. Fullscanmass spectrawere notobtainedformetaboliteIsV (nil5z68),VII (nVz540), metabolitIeX (mlz526)and X (nVz606).As an alternativaes,electerdeaction monitoring-(,SRM) analysiswas performed. For metaboliteIV,theformationof "t/z269, 169, 119,83 and 65 ionsfrom theprecursorion atin/z568 were monitored.For metaboliteVII,the formationof "z/z269, 169, 119,83 and 65 ionsfrom theprecursorion atnVz 540 were monitored.For metaboliteDC, theformationof"z/z269, 169,119, 123 and 97 ionsfrom the precursorion atnilz526 were monitored.For metaboliteX, the formationof nVz 269, 169, 119,97, 83 and 65 ionsfrom theprecursorion at"I/z606 were monitored. Since theseproduct ionswere characteristoifcthisclassofcompounds. Tentativeidentificatiofn metabolitesIV and VH was achievedinthe studysamples when signalswere observed attheirappropriatetransitions. No metaboliteIV was detectedinthe0 or 6 hour ratstudysamples (Figure14 i and ii). MetaboliteVII was presentinthe rathepatocytestreatedwithT-6292 for6 hours(Figure 15 iii)b,ut absentinthe 0 hour sample (Figure15 ii)a,nd theno testarticloer no hepatocytescontrolsamples (Figures13 iand 15 i). Figure16 (ii)and (iiir)epresentheSRM totalionchromatograms(TIC)ofrat hepatocytesincubatedwith T-6292 for0 and 6 hour respectivelys,howing thepresence of metaboliteIX inthesamples. However, thiscomponent couldalsobe observedinthe controlsample containingT-6292 and no hepatocytes(Figure16 i). REPORT: 98AGKPOI.3M AOVANCEO BIOANALYTICAL 21 MMM SERVICES. INC. MetaboliteX was presentin therathepatocytestreatedwithT-6292 for6 hours (Figure 17 iiib)ut absentinthe0 hour sample(Figure17ii)and theno hepatocytecsontrol sample (Fl-ure17 i). A potentiamletabolit(eXIII)was observedat6.1min and displayedan [M-H]- ionat nVz 650 (Figure10 x).An additionof80 mass unitsrelativteoT6292 couldcorrespond totheadditionof a sulphatemoiety. The product-ionmass spectrumof metaboliteXEE[ inFigure33(i)showed fragment ionsatnLIz526,419 (C8F]7-)3,19 (C6FI3-), 269 (C5F, -), 219 (C4F9-), 169 (C3F7-), 97 (HS04- or FNS02-) and 80 (S03-). The fragmentionsatnVz 80 isconsistenwtiththepresenceof thesulphatemoiety. 3.2.3. Metabolism of T-6292 in Human Hepatocytes at 0 hour The fullscan LC/MS reconstructeidonchromatograms of thehuman hepatocytessample incubatedwithT-6292 for0 hourareshown inFigures11.FigureII (i- x)displaysthe ionchromatograms forthefollowingt:heparentcompound (rwz570),metabolitIeII (rn/7z46),metabolitVe (inlz584),metabolitVeI (nil5z42),metabolitVeIII(inl5z56), metabolitIeX (nt/-7526m)e.tabolitXe (ml-6,06),metabolitXeI (tWz498),metabolitXeII (nil4z99) and metabolitXeHI (rn/6z50).Compounds IX and XI weredetectedinthis sample,as theywere observedin thecontrolsample containingT-6292 and no hepatocyte(sFl-Ure6 viland ix). 3.2.4. Metabolism of T-6292 by Human Hepatocytesat6 hour The fullscanLC/MS reconstructeidonchromatogramsofthehuman hepatocytesample incubatedwithT-6292 for6 hour areshown inFigures12. Figure12 (i- x) displaysthe ionchromatograms forthe followingt:heparentcompound (nLIz570),metaboliteIII (n-L7l4z6), metabolite V (7?z/5z84), metabolite VI (nilz542), metabolite VIII (nilz556), metabolitXeI (in/4z98),metabolitIeX (rnl5z26),metabolitXe (rnl6z06),metabolitXeII (nVz499) and metabolitXeIH (nVz650).ThissamplecontainedthemetaboliteIsIIV,, VI,VIII,IX,XI,XII and XEII. REPORT: 98AGKPOI.3M AOVANCED SIOANALYTICAL 22 SERVICES, INC. The SRM totailonchromatogramfsormetabolitIeVs(nVz568),VH (rnl5z40)andX were displayedinFigures 14, 15, 16 and 17. No metaboliteFV was detectedin thestudy samples (Figure14 illand iv).MetaboliteVE was presentin thehuman hepatocytes sample treatedwith T-6292 for6 hours (Figure15 v),but absentinthe0 hour sample (Figure15 iv),and theno testarticloer no hepatocytescontrolsamples (Figures13 iii and 15 i). The SRM totalion chromatograms ofhuman hepatocytesincubatedwith T-6292 for0 and 6 hour aredisplayedin Figure 16 (ivand v),respectivelys,howing thepresenceof metaboliteIX inboth samples. However, thiscomponent couldalsobe observed inthe controlsample containingT-6292 and no hepatocytes(Figure16 i). MetaboliteX was observed in thehuman hepatocytessample incubatedfor6 hours with T-6292 (Figure17 v), but absentinthe0 hour sample (Figure17 iv),and theno hepatocytescontrolsample (Figure17 i). 3.3. METABOLISM OF T-6293 3.3.1. Metabolism of T-6293 by Rat Hepatocytes at 0 hour The fullscan LC/MS reconstructedion chromatograms of therathepatocytessample incubatedA-ithT-6293 for0 hour areshown inFigure18. Figure 18 (i- x) displaysthe ion chromatograms forthe following:theparentcompound (nz/z650),metabolite11 (nLIz570),metaboliteIII(mlz746),metaboliteV (nilz584),metaboliteVI (nilz5@42), metaboliteVEII(nVz556),metaboliteDC (nilz526),metaboliteX (nvz606),metabolite XI (nilz498) and metaboliteXH (nVz499). Apart from theparentdrug,compound XII was detectedinthissample. However compound XII was alsoobserved inthe control sample containingT-6293 and no hepatocytes(Figure7x). 3.3.2. Metabolism of T-6293 by Rat Hepatocytes at 6 hour The fullscan LC/MS reconstructeidonchromatograms of therathepatocytessample incubatedwithT-6293 for6 hour areshown inFigure 19. Figure 19 (i- x) displaysthe ionchromatograms forthefollowing:theparentcompound (nvz 650),metaboliteIl REPORT: 98AGKPOI.3M AOVANCEO BIOANALYTICAL 3 SERVICES, INC. (nz/5z70),metabolitIeII(nzl7z46),metaboliVte(nVz584),metaboliVtIe (rnl5z42), metabolitVeIE (nVz556),metabolitIeX (nVz526),metaboliXte(nV-6-06),metabolite XI (inl4z98)andmetabolitXeII(nVz499).ThissamplecontainemdetabolitHeIs,V, VI, VIH, IX, XI and XII. SRM analyseswere performed formetaboliteIsV (nVz568),VU (?Wz540) and IX (nVz526) and X (nVz606). No metaboliteIV was detectedinthestudysamples(Figure 22 iand ii).MetaboliteVII was presentintherathepatocytestreatedwithT-6293 for6 hours (Figure23 iii)b,ut absentinthe0 hour sample (Figure23 ii)and theno testarticle or no hepatocytescontrolsamples (Figures13 iiand 23 i).Compound IX was found in theT-6293 standardsolutionand the6 hour rathepatocytessample incubatedwith T6293 (Figure24 i,iv and v).No metaboliteX was observed inthe rathepatocytesstudy samples (Figure25 iiand iii). 3.3.3. Metabolism of T-6293 by Human Hepatocytes at 0 hour The fullscanLC/MS reconstructeidonchromatograms ofthehuman hepatocytessample incubatedwithT-6293 for0 hour areshown inFigures20. Figure20 (i- x) displaysthe ionchromatograms forthefollowing:theparentcompound (nvz 650),metaboliteII (nVz570),metaboliteHI (nz/z746),metaboliteV (nz/z584),metaboliteVI (inlz542), metaboliteVIII(?n/z556),metaboliteIX (mlz 526),metaboliteX (nVz606),metabolite XI (rnlz498) and metaboliteXH (rnlz499). Apartfrom theparentdrug,compound X]EI was detectedinthissample, aswas observed inthe controlsample containingT-6293 and no hepatocytes(Figure7 x). 3.3.4. Metabolism of T-6293 by Human Hepatocytesat 6 hour ThefulslcanLCIMS reconstructieodnchromatogramosfthehuman hepatocytseasmple incubatedwithT-6293 for6 hour areshown inFigures21. Figure21 (i- x) displaysthe ionchromatograms forthefollowing:theparentcompound (nVz650),metaboliteII (nVz570),metaboliteIII(inlz746),metaboliteV (nz/z584),metaboliteVI (inlz542), metaboliteVIII(inl.5@5-6),metaboliteIX (nVz526),metaboliteX (in/z606),metabolite REPORT: 98AGKPOI.3M AOVANCED MIMM BIOANALYTICAL SERVICES, INC. 24 XI (in/z498) and metaboliteXII (nVz499). This sample containedmetabolitesIII,V, VI, VIH, IX,XI andXII. SRM analyseswere performedformetaboliteIsV (nVz568),VII (iWz540),IX (nVz526) and X (nil6z06).No metabolitIeV was detectedinthestudysamples(Figure22 iiiand iv).MetaboliteVH was presentinthehuman hepatocytetsreatedwithT-6293 for6 hours(Figure23 v)butabsentinthe0 hour sample(Figure23 iv)and theno hepatocytes controlsample (Ficure23 i).MetaboliteIX was found inthe0 and 6 hour studysamples (Figure24 ivand v). No metaboliteX was observedinthehuman hepatocytesstudy samples(Figure25 ivand v). 3.4. METABOLISM OF T-6294 3.4.1. Metabolism of T-6294 by Rat Hepatocytes at 0 hour The fullscan LC/MS reconstructeidonchromatograms oftherathepatocytessample incubatedwithT-6294 for0 hourareshown inFigure26. Ficure26 (i- vii)displaysthe ionchromatograms forthefollowingm:etabolitVeI (n7/5z42),metabolitVeIE[("L/5z40), metaboliteVIII(tnlz556),theparentcompound (nVz526),metaboliteX (nVz606), metaboliteXI (n4-498) and metabolitXeII (nVz499). Apartfrom theparentdrug, compounds XI and XII were detectedinthissample.However compound XI was also observedin thecontrolsample containingT-6294 and no hepatocytes(Figure8v). 3.4.2. Metabolism of T-6294 by Rat Hepatocytesat6 hour The fullscanLCVMS reconstructeidonchromatogramsof therathepatocytesample incubatedwithT-6294 for6 hourareshown inFigure27. Figure27 (i- vii)displaysthe ionchromatogramsforthefollowingm:etabolitVeI (nVz542),metabolitVeII (nVz540), metaboliteVIII(inl-5756),theparentcompound (nil5z26),metabolitXe (nVz606), metaboliteXI (nil4z98)and metabolitXeII (nVz499).Thissamplecontained metabolitesVI,VIH, IX,XI and XIE[. REPORT: 98AGKPOI.3M ADVANCEO 25 BICANALYTICAL SERVICES, INC. SRM anaiysewserepeFfonnefZorImetabolitesVii (tw@ 54u) and X (rwz out)).INIO metabolite VH was present in the 0 or 6 hours rathepatocytes samples (Figure 30 iiand iii).No metabolite X was observed in the 0 or 6 hours rathepatocytes study samples (Figure31 iiand iii). 3.4.3. Metabolism of T-6294 by Human Hepatocytes at 0 hour ThefulslcanLCF.MSreconstrucitoencdhromatograomfsthehumanhepatocytseasmple incubated with T-6294 for 0 hour are shown in Figure 28. Ficure 28 (i- vii)displays the ion chromatograms for the following:metaboliteVI (nLIz542), metaboliteVII (nvz 540), metabolite VIII (rnlz556), the parent compound (rn/z526), metaboliteX (nVz 606), metabolite XI (nVz 498) and meta@t)olitXeII (?Wz 499). Apart from the parent drug, compounds XI and XH were detected in thissample. However compound XI was also observed in the control sample containingT-6294 and no hepatocytes (Figure 8v). 3.4.4. Metabolism of T-6294 by Human Hepatocytes at 6 hour The fullscan LC/.MS reconstructedion chromatograms of the human hepatocytes sample incubated with T-6294 for 6 hour are shown inFigure 29. Figure 29 (i- vii)displaysthe ion chromatograms for the followin-:metaboliteVI (nilz542), metaboliteVH (nVz 540), metabolite VIII (??z/5z56), the parent compound (inlz526), metaboliteX (nilz606), metabolite XI (nVz 498) and metaboliteXII (rn/z499). This sample contained metabolites VI, VIII,IX, XI and XII. SRM analyses were performed for metabolitesVII (nVz 540) and X (nVz 606). No metabolite VII was present in the 0 or 6 hours human hepatocytes samples (Figure 30 iv and v). No metabolite X was observed in the 0 or 6 hours human hepatocytes study samples (Figure 31 iv and v). 3.5. PRODUCT-ION MASS SPECTRA The product-ion mass spectrum of metabolite III([M-H]- atnvz 746) isdisplayed in Figure 32 i.The base peak isatnz/z526 corresponds to the lossof -CH,CH20GIu fragment. This negative ion mass spectrum contained a fragment ion atnvz 113, due to REPORT: 98AGKPOI.3M ADVANCED 26 BIOANALYTICAL SERVICES, INC. intemaifragmentationof tneglucuroni(laecictand itindicatesthepresenceof a glucuronideconjuaate. Table IV: Proposed product-ionfragmentationforMetaboliteVI Proposed Structure nvz 542 8FI7--S02--N 'CH2 -CH20H 65 HS02- 0 11 -1S1 -N=CH, - 92 0 0 -1S1 -N=CH-CH,OH- 122 0 C3F7- 169 C4F9- 219 CeFll- 269 C6F,3' 319 CsF17' 419 The product-ionmass spectrum of metaboliteVI isdisplayedinFigure 32 iiand the correspondingfragment ionsare illustratiendTable IV. REPORT: 98AGKPOI.3M AOVANCEO BIOANALYTICAL 7 SERVICES, INC. Table V: Proposed product-ionfragmentationforMetaboliteVIII ProposedStructure M/Z CSF 17--S02 -N H / "I CH2-Coo- 556 65 HS02- FS02- 83 0 -11 S11 -N=CH,COOH 136 0 C4F9- 219 C5F,I- 269 498 SF 17-S02- NH The product-ionmass spectrum of metaboliteVIIIisdisplayedin Figure32 iii.The fragmentationpatternatm/z 498, 219, 169,83 and 65 iscomparable to thatobserved for MetaboliteVI (Figure32 ii)a,nd thecorrespondingfragmentionsareshown inTable V. The product-ionmass spectrum ofmetaboliteXI isshown inFigure33 ii.The 6agment ions at?Wz 478 and 78 representa lossof HF and C8F17H moietiesfrom thedeprotonated molecule,respectively. 3.6. POSITIVE IONIZATION LC/MS WITH T-6292 AND T-6293 ANALYSIS OF HUMAN HEPATOCYTES INCUBATED The human hepatocyteisncubatedwithT-6292andT-6293for6 hourswere also examined by fullscan LC/MS usingpositiveionizatiodnetection.However, the compounds of interesctould not be detectedinthepositiveionizationmode, and there were endogenous materialspresentinthe samples athigh concentrationt;hereforeneither the parentdrug nor the metabolitewas observed inthisanalysis. REPORT: 98AGKPO1.3M AOVANCE[D 28 BIOANALY-- ICAL SERVIC;---S. INC. FAK 1 13: Nemi-Quantitative alysisof 1-62.9:i)n K-aTand Human Hepatocytes Incubatedwith T-6292, T-6293 and T-6294 by LC/MS/MS 4. INTRODUCTION T-6292, T-6293 and T-6294 undergo metabolicbiotransformatioinnratand human hepatocytestoform T-6295. The purposeof thisstudywas todeterrriitnhee concentrationof T-6295 inhuman and rathepatocytesafterincubatingwith T-6292, T-6293 and T-6295, usingLCIMS/MS analysis. 5. EXPERIMENTAL 5.1. STANDARD SOLUTIONS Preparation of AnalyticalStandard Solutionsfor T-6292, T-6293 and T-6294: See Experimental Section2.2. Preparation of InternalStandard Stock Solution(1mg/mL): 1H,lH,2H,2H perfluoro octane sulfonicacid Accuratelyweigh 2 mg of the IH, IH,2H,2H perfluorooctane sulfonicacidon a micro balance and transfetrhechemical to a polypropylenetube. Dilutewith an appropriate volume of methanol toyielda I mg/mL solution.Storethesolutionat4' C and bringto ambient temperaturebefore use. Preparation of Internal Standard Working Solution (8 geml): IH,IH,2H,2H perfluoro octane sulfonicacid Dilute0.8 mL of analyticasltandardstocksolutionwith water ina 100 mL volumetric flasktoyielda 8 gg/mL solution.Storethesolutionat4' C and bringto ambient temperaturebefore use. Preparation of Internal Standard Spiking Solution (800 ng/mL): IH, 1H,2H,2H perfluorooctane sulfonicacid(10 mL at8 )ig/mL concentration)was dilutedwithNANOpure water(90mL) ina 100 mL volumetricflask.25 gL of this solutionwas added toeach studysample. REPORT: 98AGKPOI.3M ACDVANCEO 29 BIOANALYTICAL SERVICES, INC. 5.2. FREPARATION OF QUENCHING SOLUTION: Methanol was mixed withhepatocytescontrolmedia solutionforB01 1-95(suppliedby 3M) intheratiof2:1(v/v). 5.3. HPLC ELUENT PREPARATION: See ExperimentalSection2.3. 5.4. SAMPLE PREPARATION The metabolismstudywas carriedoutatSRI InternationaMle,nlo Park,CA. The incubateswere quenched with an equalvolume of ice-coldmethanol,centrifugedand shippedindry iceto ABS. The sampleswere storedat-20'C. Hepatocytesisolatefdrom Male Rat #2,Female Rat #5, Male Human #341 and Female Human #2, each incubatedwith0.1 mM ofT-6292,T-6293 and T-6294,respectively, were analysedforT-6295. Rat and Human Hepatocytesincubatedwithno testarticlefsor0 and 6 hours were used ascontrolsamples. FivestocksolutionspreparedinDMSO with no hepatocytes(suppliedby SRI InternationalT)-:6292 from rat2 and rat5 studiesT;-6293 from rat2 study;T-6294 from rat2 and rat5 studies(10 pL each)were alsoused as controlsamples.Each sample (10 @tL)containingtheanalyticasltandardwas dilutedwiththequenchingsolution (90 @tL),thenspikedwith theinternasltandardsolution(25pL). The internasltandardsolution(25 gL of 1H,lH,2H,2H perfluoroctanesulfonicacid,at 800 ng/mL concentrationw)as added per studysample(100 pL). 5.5. PREPARATION OF PAT HEPATOCYTES STANDARD CURVE SAMPLES Appropriatevolumes ofT-6295 wo rkingsolutionwas spikedtocontrolrathepatocytes accordingto the scheme outlined below: REPORT: 98AGKPOI.3M ADVANCEO 30 BIOANALYTICAL SERVICES, INC. Plasma StandardPrep. 7 6 5 4 3 2 1 0 T-6295 Soln Conc. 500 Working Soln 250 Std#7 100 1 Std#7 50 Std#7 10 Std#4 2 Std#3 1 Std#3 0 0 mL added 0.025 Controlrat Hepatocytes(mL) 0.475 Volume (ML) 0.5 0.150 0.048 0.048 0.096 0.048 0.024 1 0 0.150 0.192 0.432 0.384 0.192 0.216 0.250 0.3 0.24 0.48 0.48 0.24 1 0.24 0.25 One standardcurvewas injectedatthebeginningof theanalyticarlun and a second standardcurve was injectedattheend oftheanalyticarlun. 5.6. LC/NIS/NIS INSTRUMENTATION 5.6.1. HPLC conditions HPLC Column Mobile Phase LC Gradient Flow rate BetasilC18 (2x 100mm) 10:90MeOH:2mM NH40Ac (A) 90:10MeOH:2mM NH40Ac (B) Time 0 to2 min 2 to4 min 4 to7 min 7 to8 min 200 RLJmin %B 30% 30 to 100% 100% 100 to 30% 5.6.2. MS conditions Sciex API III'TurboIonSprayTMInterface Ion Spray Voltage: -3900 V Orifice: -60 V DeclusterincP.o,tential -30 V REPORT: 98AGKPOI.3M AOVANCEO 31 BIOANALYTICAL SERVICES, INC. ionizationmo?le Negative CollisionGas Thickness 280 x 1013 atoms/squarecm CollisionEnergy 35 eV Dwell Time 200 msec TransitionsMonitored: rnlz499 99 forT-6295 nVz 499 80 forT-6295 in/z427 81 forinternalstandard The product-ionmass spectrum of T-6295 ([M-H]- atnVz 499) produced two fragment ionsatnVz 80 and 99 (seeFigure 3 iii)w,hich arelikelydue to (*S03-) and (FS03-) respectivelyB.oth transitionwsere monitored. Data System RAD Version2.6;LC Tune Version2.4; Multiview Version 1.2;MacQuan Version 1.4 5.6.3. Data Handling All raw chromatogc@raphicand mass spectrometridcatawere storedon theABS fileserver ABS I-FS.DATA inthefilehierarchy"RAV*rDATA: Validation3:MSRI: 3MSRI SemiQuan". All exper-imentailnformationwas storedinABS notebook No: 2070. 6. RESULTS AND DISCUSSION: One setof male rathepatocytes,one setof female rathepatocytes,one setof m@le human hepatocytesand one setof female human hepatocytesamples were analysedby LC/MS/MS-. The@resultsare shown inTable VI. The same setof standardcalibratiocnurve samples was used toanalysethefivestock solutionsT.he resultsareshown inTable VH. REPORT: 98AGKPOI.3M MMM AOVANCEO 32 BIOANALYTICAL SERVICES. INC. Table VI Weighted(I/x) Intercept= Slope= CorrelatioCnoeff. Filename DB-l DB-2 H341920 H341926 H341930 H341936 H341940 H341946 H341C920 H341C926 H341C930 H341C936 H341C940 H341C946 H341NTAO H341 NTA6 HF'-1920 HF--1926 HF--1930 HF--1936 HF--)940 HF-1946 HT:'-)C920 HF'-)C926 HF--7C930 HF2C936 Hfn-C940 HF2C946 HF2NTAO Hfz'-NTA6 R2920 R2926 R2930 R2936 R2940 R2946 R2C920 R2C926 R2C930 QuantitativeAnalysisofT-6295 inRat and Human Hepatocytesafter IncubationwithT-6292,T-6293 and T-6294 0.00193 0.00331 0.9961 S2mple Name Cone. (ng/mL) Double Blank- n/a DoubleBlank n/a H341IT6292 t--O n/a H341/76292 t--6 n/a H34 I/T6293 t---o n/a H341rr6293 t=6 n/a H341/T6294 t--o n/a H341/T6'-194t=6 n/a H341 No Hep T6292 t--O n/a H341 No Hep T6292 t--6 n/a H341 No Hep T6293 t--O n/a H341 No Hep T6293 t--6 n/a H341 No Hep T6294 t--O n/a H341 No Hep T6294 t=6 n/a H341 No TestArticlte=O n/a H341 No TestArticlte=6 n/a Human Female 2/T6292 t--O n/a Human Female 2/T6292 t--6 n/a Human Female 2/T6293 t--O n/a Human Female2rr6293t--6 n/a Human Female 2fT6294 t--O n/a Human Female 2fT6294 t--6 n/a Human Female 2 No Hep T6292 t--O n/a Human Female 2 No Hep T6292 t--6 n/a Human Female 2 No Hep T6293 t---O n/a Human Female 2 No Hep T6293 t=6 n/a Human Female 2 No Hep T6294 t--o n/a Human Female 2 No Hep T6294 t--6 n/a Human Female 2 No TestArticlte--O n/a Human Female 2 No TestArticlte=6 n/a Rat Male 2jT6292 t--O n/a RatMale YF6292 t--6 n/a RatMale 2fr6293t--O n/a RatMale 2/T6293 t--6 n/a RatMale 2rr6294t--O n/a RatMale 2/T6294t=6 n/a Rat Male 2 No Hep T6292 t--O n/a RatMale 2 No Hep T6292 t=6 n/a Rat Male 2 No Hep T6293 t--O n/a Calc. Conc. (ng/mL) n/a n/a 0.36 n/a 7.319 n/a 1.051 n/a n/a n/a 6.574 6.042 O@491 0.603 n/a n/a n/a 15.049 6.946 9.294 6.418 5.373 n/a n/a 7.706 8.478 6.739 5.888 n/a nta 0.993 28.785 10.621 16.526 6.168 5.439 1.041 n/a 6.714 Accuracy n/a n/a n/a n/a n/a n/a nla n/a n/a n/a n/a n/a n/a n/a n/a n/a n/a n/a n/a n/a n/a n/a n/a n/a n/a n/a n/a n/a n/a n/a n/a n/a n/a nla n/a n/a n/a nla n/a REPORT: 98AGKPOI.3M 4OW AOVANCEO 33 BIOANALYTICAL MMM SERVICES, INC. iadie i Filen2ffie QuantitativAenMysis of i-6y95 inKat and Human liepatocyteasfter IncubationwithT-6292,T-6293 and T-6294 (continued) Sample Name Conc. Caic.Conc. Accuracy (ng/mL) (ng/niL) R2C936 R2C940 Rat Male 2 No Hep T6293 t--6 Rat Male 2 No Hep T6294 t--O n/a 8.454 n/a n/a 4,446 n/a R2C946 Rat Male 2 No Hep T6294 t=6 n/a 3.615 n/a R2NTAO R2NTA6 Rat Male 2 No Test Articl1e=0 Rat Maic 2 No TestArticlte=6 n/a n/a n/a nla n/a n/a R5920 R5926 Rat femaleS/T6292 t=O Rat female5/T6292 t=6 n/a n/a n/a n/a 33.934 n/a R5930 Rat female5/T6293 (=O n/a 7.862 n/a R5936 Rat female5fr6293 t=6 n/a 14.147 n/a R5940 Rat female5/T6294 t=O n/a 0.856 nla R5946 Rat female5rr6294 t=6 nla 8.957 n/a R5C920 Rat female5 No Hep T6292 t=O n/a n/a n/a R5C926 Rat female5 No Hep T6292 t=6 n/a 7.871 n/a R5C930 Rat female5 No Hep T6293 t--O n/a 7.777 n/a R5C936 Rat female5 No Hep T6293 t=6 n/a 6.96 n/a R5C940 Rat female5 No Hep T6294 t--O n/a n/a n/a R5C946 Rat fenial5e No Hep T6294 t--6 n/a 4.095 n/a R5NTAO Rat female5 No TestArticlet=O n/a nla n/a R5NTA6 Rat female5 No TestArticlet--6 n/a n/a n/a Std-I I n/a 11.(>43 n/a Std-I2 Std-2 1 1 I.O.S4 105.367 2 1.907 95.35 Std-2 2 2 1.732 86.576 Std-3 1 10 9.98 99.799 Std-32 10 10.588 105.883 Std-4 1 Sid-42 50 57.979 115.958 50 54.431 108.862 Std-5 I Std-52 too 98.732 98.732 too 80.054 80.054 Std-6 1 250 259.142 103.657 Std-62 250 249.402 99.761 Zero-[ n/a n/a nla Zero-2 n/a n/a n/a T-6295 was not detectedin thecontrolsampleswhich containedonlythehepatocytesand no testarticles(TableVI). For theratand human hepatocytesT,-6295 was detectedinthecontrolsampleswhich were incubatedwithT-6293 and T-6294 butwithno hepatocyte(sTableVI). REPORT: 98AGKPOI.3M MMM ADVANCEO 34 BIOANALYTICAL SERVICES, INC. I-orThe-male liuman hepatocytes study samples, 1-6295-was present in the 0 hour samples incubated with T-6292, T-6293 and T-6294. T-6295 was not observed in the 6 hour samples. For the female human hepatocytes study samples, T-6295 was present in the 0 hour sample incubated with T-6292, 0 and 6 hour samples incubated with T-6293 and T-6294, and was not observed in the 6 hour samples incubated with T-6292 (Table VI). For the male rat hepatocytes study samples, T-6295 was detected in the 0 and 6 hour samples incubated with T-6292, T-6293 and T-6294 (Table VI). For the female rat hepatocytes study samples, T-6295 was present in the 6 hour sample incubated with T-6292, 0 and 6 hour samples incubated with T-6293 and T-6294, and was not observed in the 0 hour sample incubated with T-6292 (Table VI). LC/MS/MS analyses of the stock solutions showed T-6295 was present at high concentration in allthe samples (Table VII). Since carryover for T-6295 was observed during sample analysis,thisaffects the values of the second set of standard curve, particularly the calibrationstandards at low concentrations. REPORT: 98AGKPOI.3M ADVANCED 35 E310ANALYTICAL SERVICES, INC. Table VII QuantitativAenalysisofT-6295inT-6292,T-6293and T-6294 Stock Solutions Weighted(I/x) Intercept Slope = CorrelatioCnoeff. 0.00150 0.00359 0.9910 Filename DB 1 DB 2 R2T6292Stock R2T6293Stock R2T6294Stock R5T6292Stock R5T6294Stock Std-I 1 Std-I2 Std-2 1 Std-2 2 Std-3 1 Std-3 2 Std_4 1 Std_.@42 Std-5 1 Std-5 2 Std-6 1 Std-6 2 Zero 1 Zero 2 Conc. 0 0 n/a n/a n /a n /a n /a 1 n /a 2 n/a 10 n/a 50 50 100 100 250 250 0 0 Catc.Conc. (ng/niL) n/a n/a 125.713 961.102 671.542 160.661 210.365 1.256 5.563 1.569 4.772 9.307 12.66 53.853 59.95 97.,115 70.035 245.415 274.2 n/a 3.096 Accuracy (ng/mL) n/a n/a n/a n/a n/a n/a n /a 125.56 n /a 78.46 n/a 93.07 n/a 107.71 119.9 97.41 70.04 98.17 109.68 n/a n/a 7. REFERENCES: I D.E. Mulvana and J.Henion Qualitativienvestigationf theinvitrometabolismof T-6292,T-6293,T-6294 and T-6295 by ratand human hepatocytesusingionspray LC/MS and LC/MS/MS. ReportNo. 96ADEMOI.3M November 12,1996. 2 ABS LaboratoryNotebook No. 2070 REPORT: 98AGKPOI.3M ADVANCEO 36 BIOANALYTICAL MMM SERVICES. INC. kigureI. Reconstructedionchromatogram (i)n,egativeionizatinomass spectrum(iia)nd LC/MS/MS product-ionmass spectrum(iiio)f authenticT-6293. XIC (period1)from 650.0-650.5amu from T6293 No Hep 5.3.96(1)2.6 T6293 % Ia ensity 80. 60 40 20 2.16e5cps 1 2 3 4 5 6 7 8 Spectrum from 6.46n-dn(8 scans)from T6293 No Hep 5.3.96(1),subtracteds,ubtracted % Intensity 650 [M-H] 9 Time, min 8.97e4 cps 80 60 (ii) 311 40 20 503 240 320 400 480 560 Spectrum from 6.84 min (27scans)from 3M T6293 stdProd 650 (3) % Intensity 80 60 40 169 20 97 123 79 80 160 269 319 240 320 419 400 695 745 640 526 720 m/z 9.99e3 cps [M-H] 480 560 640 nvz REPORT: 98AGKPO1.3M MMM ADVANCED 37 BIOANALYTICAL SERVICES. INC. Figure2. Reconstructedionchromatogram (i)and LC/MS/MS product-ionmass spectrum (iio)f authenticT-6294. TIC from 3M T6293 & T6294 StdsProd (1) % In@.,nsity 9.6Oe5 cps T-6294 9 47 8060' 409.11 20, A- .-.I "E" 4.8 4.6 6.4 7.2 8.0 8'8 Spectrum from 9.49 min (9 scans)from 3M T6293 & T6294 StdsProd (1),centroided % In.ensity L 9.6 Time, min 3.54e5 cps [M-Hl- 526 806040* 20- 60 19 219 126 120 180 269 240 300 362 419 360 420 480 m/z REPORT: 98AGKPOI.3M ADVANCED 38 BIOANALYTICAL SERVICES, INC. Figure 3. Reconstructedion chromatogram (i)n,egativeionizationmass spectrum (ii)and LC/MS/MS product-ionmass spectrum (iiio)f authenticT-6295. XIC (period1)from499.0-499.a5mu fromT-6295Std (1) % Intensity 6.05 T-6295 3.55e6cps 80- 604020- 1 2 3 4 5 6 7 8 Spectrum from 6.07 min(2 scans) from T-6295 Std (1),subtractesudb,tracted,centroided % Intensity 4 9 Time,min 1.49e6 cps 80- 60 40- 20, 240 320 400 480 560 6@O Spectrum from 6.03 min (5 scans) from T6295 Dau 499 (1),centroided % Intensity 8) 99 80. 60 40 169 130 20- 720 800 nVz 4.92e4 cps (M-H]- 499 60 120 180 240 300 360 420 480 nVz REPORT: 98AGKPOI.3M AOVANCEO 39 BIOANAI-YTI@AL MMM SERVICES, l@NC. Figur4e. Fujsicanreconstru!cotnecdliromatogorracmosntr th--pe-Faoc-yfi incubatfeodr6hourwsithnotesatrticles XIC (perio1d)from570.0-570a.m5ufromBOIIR4 No TA 6hr(1) % Intensity 1.15 6 (i) 30 ol, nVz650.-06 .5 For Compound 1.15 I and XIII (ii) 60 2.69 30 rlvz746.0 - 746.5 For Compound III 1.21 (iii6)0 - 30. rtvz584.0 - 584.5 For Compound V 7.75 6.93 (iv)60 30 1.3- nVz 542.0 - 542.5 For Compound VI 5 60. (v) 30. rp@2.14@@@@52..3194@ 6.43 8.13 A 5.39 A@ 4.OOe4 cps 3.OOe4 cps 3.8Oe4 cps 9.8Oe4 cps 9.18 3.2Oe4 cps 9 12 nVz556.-0556.F5orcompounVdIII 1.15 (vi)60 30 3 24@4 nVz 526.0 - 526.5 For Compound IX (vii)60 30 6- m/z 606.0 -606.5 For Compound X 1 15 . (viii63)00. 8@1 @ 44 l1@88 5.66 703 3.6Oe4 cps 55@.66 @6 4.8Oe4 cps 901 69898@ 774477@8 83300@@9 0' 3.OOe4 cps 9.* 88@ 594 74422@@ 9 nVz 498.0 - 498.5 For Compound XI 3.90 (ix)60 30 165 3 30 m/z 499.0 499.5 For Compound XII 390 (x) 60 30 2.36 1 2 3 4 4.OOe4 cps 9. 4 522 7 58 522 638 758 4.6Oe4 cps 934 5 6 7 Time, n'@n REPORT9:8AGKPOI.3M M ADVANCED In BIOANALY-ICAL SERVICES. INC. k igureb.-F-uTF-scraenconstructeiiolncliromatogr incubatedfor6 hourswithno testarticles XIC (perio1d)from570.0-570a.m5ufromBOIIFl No TA 6hr(2) % Intensity . 1.10 (i)60 30'-A,,- 275 3.85 IV, 650.0-650.5ForCompound Iand XIII . (ii)60 30' A. . - fl@@ -j\ a 55 m, rrjz746.0-746.5For Compound III 6.48 1 15 (iii)60* 30' A@ ni/z584.0 -584.5 ForCompound V 40 Ihuman hepatiTc--Yfg- 2.2Oe4 cps 70 83300@ 9@95566 1.4Oe4 cps 879 2.OOe4 cps 9.40 1.1Oe5 cps 9.29 (iV) 60 30 m/z 542.0 -542.5For Compound VI 1.26 (V) 60 30'__ _A, 253 A A 2.4Oe4 cps m/z 556.0 -556.5For Compound VIII . (v i) 6 0' 1.10 1 .36 30'_ nVz 526.0- 526.5For Compound IX 1.21 (vii)60 30'-L, nVz 606.0 -606.5 For Compound X (viii6)0 - 30' trvz498.0 -498.5 ForCompoun XI jA@@ 1 15 (ix) 60. 30"\.WA 2 A -a m/z 499.0 -499.5 For Compound XII . 0.99 1.87 (x) 60 30--@,A 1 2 3 5.44 5.11 824 .70 0 77..442 2@ 6-.92 8.24 2.8Oe4 cps 2.4Oe4 cps 9.40 5.66 4.23 4.23j.V5O.606 4 5 6 7.8Oe4 cps 9.18 3.6Oe4 cps 8.52 3.8Oe4 cps 8.57 7 8 9 Time, n-@n REPORT: 98AGKPOI.3M ADVANCEO 41 In BIOANAL@Y-TICAL SERVICES, INC. Figure 6. Fullscan reconstructedionchromatograms ofT-6292 incubatedfor6 hours with no hepatocytes XIC (period1)from 570.0-570.5amu from T6292 No Hep 5.3.96(1) % int nsity 7.21 4.30 60 30 L:@o@ 1.60e4 cps nVz 650.0 -650.5 For Compound Iand XIII 1.15 60 30 Ar-\ 5.61 5.12 6122 1.4Oe4 cps ago nmvlz.46.0 746.5 For Compound 111 60' 1.15 30' A A A^ QAAA A A Ao 6.49 2.OOe4 cps nilz v) 60 30 1-orCompound V 7.76 7.2Oe4 cps nvz -42.0 - 542.5 For Compound VI 12 (v)60' 30' 1 2 3 ni/z556.0 -556.5 For Compound 1.15 2.20 (vi) 60 30 A VIII nVz 526.0 - 526.5 For Compound IX (vii) 60 30' nVz 606.0 -606.5 For Compound X (viii) 60 30' nVz 498.0 - 498.5 For Compound XI 60' 30 ni/z499.0 -499.5 For Compound XII (x) 60 30- 121 2 7.65 3.2Oe4 cps 9.30 4 5 6 7 8 9 Time, n-dn 1.60e4 cps 5.45 6.66 5.45 5.67 A 4.44e5 cps 9.41 (M 8.9 8.20e4 cps 9.35 a 7 32 (Xl) 2.04e5 cps 4.41 A 4 5 6 765 3.08e5 cps 7 8 REPORT: 9 Time, min 98AGKPO I.3M ADVANCED 42 BIOANALYTICAL SERVICES, INC. Figure 7. Fullscan reconstructeidon chromatograms ofT-6293 incubatedfor6 hours with no hepatocvtes XIC (period1)from 570.0-570.5amu from T6293 No Hep 5.3.96(1) % Intensity 1.15 7.20 2.OOe4 cps 60 304 -k nvz 50.0-650.5For Compound Iand XIII 60 30 rril7z46.0 -746.5 For Compound 111 60 30 . 1.10 4 A AA A A nV5z84-.5084F.o5CrompouVnd 1.26 (iv)60 A& l@@ rn/z542.0 -542.5 For Compound Vi 1.37 (v) 60 3() A 6.27(T-6293) 649 AIA@ 2.16e5cps 1.6Oe4 cps 9.23 1.8Oe4 cps 9.23 2.2Oe4 cps ni/z 56.0-556.5or ompound VIII 670 (vi)60 30 nVz 526.0-526.5For Compound IX 1 21 60 0.22 2.80 3() 5.17 4.07 nVz 606.0-606.5 For Compound X (viii6)(1 566 0 A@ ,-664@ t ni/z498.0 -498.5For Compound XI I 572 1.65 (ix)6c@ 3(l@,@A 2.OOe4 cps 9.62 3.8Oe4 cps 923 1.60e4 cps 5.40e4 cps m/z 499.0 -499.5 For Compound XII 5.72(XIII) 5.40e4 cps 1.65 (x) 6rl 3.90 3(1 r 1 2 3 4 5 6 7 8 9 Time,min REPORT: 98AGKPOI.3M M A[)VANCED BIOANALYTICAL 43 SERVICES, INC. Figure 8. Fullscan reconstructedion chromatograms ofT-6294 incubatedfor6 hours with no hepatocytes XIC (period1)from 542.0-542.5amu from T6294 No Hep 5.3.96(1) % Intensity 1.32 60 2.6Oe4 cps 9.23 nVz 556.0 -556.5 For Compound VIII 1.10 60 Nk@-@ NAM, m/z 526.0 - 526.5 For Compound IX 60 (iii) 5.44 6. 0 1.80e4 cps 9.29 3.5 1e6 cps 8.85 T-6294) nVz606.-0606.F5orCompounXd 1.15 60 (iv) V-\AW 5.66 AAAA /\rA@n nVz 498.0 -498.5 For Compound )U 60 (v) nVz 499.0 - 499.5 For Compound 1.26 0 27 60 (vi) MI 3.19 1 2 3 4 5.06 5 6 1.40e4 cpS 9.07 7.31(XI) 8.2Oe4 cps 2.8Oe4 cps 7 8 9 Time, min REPORT: 98AGKPOI.3M ADVANCED 44 BIOANALYTICAL SERVICES, INC. Figure9. Fullscanreconstructieodnchromatogramshowingthepresencoefmetabolites afterincubatinrgathepatocytewsithT-6292at0 hour. XIC (perio1d)from570.0-570a.m5u from 6.OOe4 cps % int nsity 7.20 1.15 60' 30' nVz 746.0-746.5For Compound IH . 60 1.15 30' 8.4Oe4 cps 6.48 nVz 584.0-584.5 For Compound V 60 1.32 30" nVz 542.0-542.5 For Compound VI (iv)60' 1.15 30' nVz 556.0-556.5 For Compound VIII 1.10 (v) 60 30',@_ nVz 526.0-526.5 For Compound IX :vi) 60 30' nVz 606.0-606.5For Compound X (vii) 60 30' r-__ nVz 498.0-498.5For Compound XI (viii6)0 30 1.24e5 cps 7.75 9.20 6.70 7.64 7.6Oe4 cps 4.OOe4 cps 5.44 8.30 5.17 5.66 5.71 (IEK)8. 2.98e5 cps 9.@6 9.4Oe4 cps 9.31 7.31(XI) 2.]8e5 cps A mlz 499.0-499.5 For Compound XII (ix)60' 30' ffi6/5z0.0-650F.o5rcompoundXIII 1.10 (x) 60 30" 75 1 2 3 4 5.71 (Xlll) AA- - 6.76 7.(>4 2.82e5 cps A2.2Oe4 cps 8.41 5 6 7 8 9 Time, ff@n REPORT: 98AGKPOI.3M ADVANCEO 45 BIOANALYTICAL SERVICES, INC. 14 igure 10.k uiiscan reconst-ru--cTIeo-nTc -na-Fogra-msshowing the presence of metabolites afterincubating rat hepatocytes with T-6292 for 6 hour. XIC (perio1d)from570.0-570a.m5u fromBOIIR4 T62926hr(2) % Intensity 1.15 275 (i) 60 30 7.21 2.6Oe4 cps 11%.@@ nVz 746.0-746.5 (ii)60 30- 6.16 (Ell) 9.1OeS cps nVz 584.0-584.5 (iii6)0. 30' 6.27(V) 2.45e6 cps m/z 542.0-542.5 (iv)60 30 7.70 (VI) 1.70e6 cps rrvz556.0-556.5 5.89(VUI) 1.42e5cps (V) 60' 30' nVz 526.0-526.5 60 (v')3(l nVz 606.0-606.5 6c@ (vii3)(), 1.15 nVz 498.0-498.5 60 (viii3)0 -- nVz 499.0-499.5 60 (ix)30 1.78e5cps 9.41 (IX)8.91 5.18 1.28e5cps 9 13 7.15(Xr) 5.72 A@ A- 5.72(XM 7.15 1.15e6 cvs 5.02e5 cps rrvz650.0-650.5 1 (x) 63((11 2 6.11(XIH) 1.15e6 cps 4 5 6 7 8 9 Time. rain REPORT: 98AGKPOI.3M ADVANCEED 46 BIOANALYTICAL SERVICES, INC. Figur1e1.Fullscanreconstrucitoencdhromatograsmhsowintghepresencoefmetabolites afteirncubatihnugmanhepatocywtietshT-629a2t0 hr. XIC (perio1d)from570.0-570a.m5ufromBOIIFlT6292Ohr(2) % Intensity 1.15 (i) 60. 30 AA- 7.20 6.70 3.OOe4 cps nVz 746.0-746.5For Compound 111 3.OOe4 cps 6.48 1.15 (ii)60 30', nVz 584.0-584.5For Compound V (iii6)0- 30' 1.44e5 cps 7.75 9 ni/z542.0-542.5For Compound Vi 1.15 (iv) 60 30 m/z 556.0-556.5For Compound VIII (v) 60 30' _A 1.15 nVz 526.0-526.5For Compound IX 3.4Oe4 cps 7.64 5.44 6.70 4.2Oe4 cps 8.24 3.06e5 cps 9.40 (vi)60 30r nVz606.0-60F6o.r5CompoundX 1.50e5 cps 9.34 (vii6)0 30r m/z498.0-498F.o5rcompoundXI (vii6i0)' 39, 4=::- mlz 499.0-499.5For Compound XII (ix) 630r m/z650.0-65F0o.r5CompoundXIII 1.10 (x) 60' 30'P 1 O&JE@=@ A^ 2 3 4 5.60 4= A 5 6 (Xl) 2.06e5 cp; 3.52e5 cps 7 69 2.OOe4 cps 9.56 7 8 9 Time, rmn REPORT: 98AGKPOI.3M ADVANCEO 47 SIOANALYTICAL SERVICES, INC. Figure 12. Fullscanreconstructedion chromatograms showing thepresenceof metabolitesafterincubatinghuman hepatocyteswithT-6292 for6 hour. XIC (period1)from 570.0-570.5amu fromBO II Fl T6292 6hr(2) % Intensity 1.10 2.64 7.20 2.2Oe4 cps 601 (i) 30 nVz 746.0-746.5 60 (ii)30' nVz 584.0-584.5 (iii60) 30' 6.04(IH) 6.26(V) 5.94e5 cps 6.58e5 cps rniz542.0-542.5 60. (iv)30" (VI) 4.98e5 cps nVz 556.0-556.5 60' (v) 30', nVz 526.0-526.5 (vi)60. 30' nVz 606.0-606.5 (vii)6030* nVz 498.0-498.5 (viii6)0. 30' nVz 499.0-499.5 (ix)60' 30' 5.88(VIM 7.4Oe4 cps 1.56e5 cps 940 (EK) 8.83 A 1.20e5 cps 9.12 - 7.31(XI) 9.86e5cps 731 5.71(XII) 1.90e5 cps 8.63 nVz 650.0-650.5 6.10 (XHI) 1.76e5cps (x) 60. 30' 1 2 3 4 5 6 7 8 Time, min REPORT: 98AGKPO1.3M AE)VANCE:D 48 BIOANALYTICAL SERVICES, iNC. Figure 13. (i)SRM chromatograms of rathepatocytesincubatedwith no testarticles; (iiS)RM chromatograms of human hepatocytesincubatedwith no test articles(;iiiS)RM chromatograms of human hepatocytesincubated with no testarticles. TIC from R4 No TA 6h MR@M 540 b % Intensity 3.37 l.OOe2 cps 80. 60- 4.90 40- 20- TIC from BO II F I No TA 6h sample 30 MRM2 80604020- TIC from Fl No TA 6 h sample 30 MRM540 b 1.18 8060- 40 20 1 2 3 4 5 6 Time, min 5.00el cps 1.50e2 cps 7 8 9 REPORT: 98AGKPOI.3M MMM AOVANCEO 49 BIOANALYTICAL SERVICES. INC, Figure 14. SRM chromatograms showing theabsence of metaboliteIV in (i)rat hepatocytesincubated with T-6292 at 0 hour; (iir)athepatocytes incubated with T-6292 for6 hour; (iiih)uman hepatocytesincubated with T-6292 at 0 hour and (iv)human hepatocytesincubated with T-6292 for 6 hour. TIC from T6292 R4 Ohr rrvz568 % Intensity 90 (i) 60 30 Iiiii III TIC from BO I I R4 T6292 6h nVz 568 (1) 90 (ii)60 30 2.50el cps l.OOe2 cps TIC from T6292 Fl Ohr nVz 568 90 (iii)60 30 ^,A A 2.2Oe2 cps 7.38 T'ICfrom BOI I Fl T6292 6h nVz 568 (1) 90 (iv) 60 30 l.OOe2 cps 1 2 3 4 5 6 7 8 9 Time, min REPORT: 98AGKPOI.3M 50 SERVICEE@, INi:@ - --- ---- - ----------- ...... Figure 15. SRM totalion chromatograms showing the presenceof metabolite@'llin (i)T-6292 standard solution;(ii)incubationofT-6292 with rathepatocytes at 0 hour-,(iiii)ncubationof T-6292 with rathepatocytesat 6 hour, (iv) incubationof T-6292 with human hepatocytesat 0 hour and (v)incubation of T-6292 with human hepatocytesat 6 hour. TIC fi-omT629'-STD nVz 540 % Int nsity @'ps 6( Tl('I'rorn'F6292R4 Ohr in@z540 2 @33 6('3(@-@ '171(f@r.om 1'6292 R4 ()hr 5,4(,) 6(@ (Ili3)0 -!A -A@A@- Tl( from 1'6292 1-'1()Iirrii@ri? 1 08 2 .@')5 60(iv)30 - 5.7(.) ic2 cps )e2 (@ps TI(.from 1'6292 f,'Ol@irtii/i54@) 60 (N,) 30 4.15 4 e.2cps 5.78 7,8 X,'1]) miri RI i'OR,I(')8i@CiKi)()l,3M ADVANCEO 51 BIOANALYTICAL SERVICES, INC. Figure16. SRM totalionchromatograms showing thepresenceofmetaboliteIX in (i) T-6292 standardsolution(;iii)ncubationofT-6292 with rathepatocytesat 0 hour;(iiii)ncubationofT-6292 withrathepatocytesat6 hour;(iv) incubationof T-6292 withhuman hepatocytesat0 hour and (v)incubation ofT-6292 with human hepatocytesat6 hour. TIC from T6292 STD nVz 526 % Intensity 6030- 7.2Oe2 cps 9.31 (IEK) TIC from T6292 R4 Ohr m/z 526 60- 30- 0.98 Z@- TIC from T6292 R4 6hr nVz 526 (iii6)030- 0.98 6.14 1.19e4 cps 9.31(IX) 8.96e3 cps 9.33 (IX) TIC from T6292 Fl Ohr m/z 526 60(iv)30- TIC from T6292 Fl 6hr m/z 526 60- (v) 30- 1 2 1.48e4 cps 9 31 (IX) 9.48e3 cps 9 31 (IX) 6.14 88..777 71 4 5 6 7 8 9 Time, min REPORT: 98AGKPOI.3M AOVANCEO 52 BIOANALY-RICAL SERVICES, INC. Figur1e7. SRM totailonchromatograsmhsowintghepresencoefmetaboliXte in(iT)-6292standarsdolutio(ni;ii)ncubatioofnT-629withrat hepatocytaet0shour;(iiiin)cubatioofTn-6292withrathepatocytes at6 hour;(ivi)ncubatioofnT-629w2ithhumanhepatocytaet0shour and(v)incubatioofnT-629w2ithhumanhepatocytaet6shour. TIC from T6292 STD m/z 606 % In ensity 6030- 3.00e I cps TIC from T6292 R4 Ohr nVz 606 0.96 6030- 6.60 3.5Oe2 cps TIC from T6292 R4 6hr ngz 606 6030- 6.86 (X) 5.7Oe2 cps TIC from T6292 Fl Ohr nVz 606 1.05 60(iv) 30- AA 3.85e2 cps TIC from T6292 Fl 6hr nVz 606 60(v)30- 0.96 6.86 (X) 6.OOe2 cps 9.38 1 2 3 4 5 6 7 8 9 Time,min REPORT: 98AGKPOI.3M ADVANCED 53 BIOANALYTICAL SERVICES, INC. Figure 18.Fullscan reconstructedionchromatograms showing thepresenceof metabolitesafterincubatingrathepatocyteswithT-6293 for0 hour. XIC (period1)from 650.0-650.5amu from BOI I R4 T6293 Ohr (2) % Intensity 6.15(T-6293) 1.80c5 cps (io (i) 301 m/z 570.0-570.5ForCompound II 1.15 2.4Oe4 cps 7.20 (ii)6o m/z 746.0-746.5For Compound III 6.48 1.15 (iii61)0 30 @A A m/z 584.0-584.5ForCompound V (iv)60 30'- m/z 542.0-542.5For Compound VI 1.26 60 (v) 30"--AA-f@@@ nVz 556.0-556.5 For Compound VIII 1 10 (vi)6300.', nVz 526.0-526.5For Compound IX 1.15 5.11 (vii) (io 30@.A6.@ nVz 606.0-606.5For Compound X (viii6)0. 30' l@15 2.OOe4 cps 9.45 9.2Oe4 cps 9.18 2.OOe4 cps 2.OOe4 cps 8.24 3.OOe4 cps 945 6.60e4 cps 912 nVz 498.0-498.5ForCompound Xi 1.15 (ix) 60. 30.A- rrvz499.0-499.5ForCompound XII (x) 1.15 5.72 5.72 (XM 5.2Oe4 cps 6.OOe4 cps 2 3 4 5 6 7 8 9 Time, min REPORT: 98AGKPOI.3M AOVANCEO 54 SIOANALY'TICAL SERVICES, INC. Figure19. Fullscan reconstructeidonchromatograms showingthepresenceofmetaboutes afterincubatingrathepatocytewsithT-6293for6 hour. rri/6z50.0-650.5 % Intensity 6 II (T-6293) 1.44e5 cps (i) 60 30 1 rrL/z570.0-570,.5 1.15 A@@ 2.4Oe4 cps (ii)60 30' -AA rri/4z6.0-746.5 60' 30' 1.64e5 cps nVz 584.0-584.5 (iv)60 30' nVz 542.0-542.5 (V) 60 30' 6 2 (V) 1.84e5 cps 9.24 4.7Oe5 cps ni/z556.0-556.5 (vi) 60F 30'_ ni/z 526.0-526.5 60' 30' 5.83 (VE[F) 12 5.78 1.86e5 cps 3.2Oe4 cps (IX) ni/z606.0-606.5 (viii6)0' 30' 7.OOe4 cps 9.08 m/z498.0-498.5 (ix)60' 3or 7.10 (XI) 5.38e5 cDs 5.61 nVz 499.0-499.5 5.61 (XH) 1.64e5 cps (x) 60' 3or -A 7 11 1 2 3 4 5 6 7 8 9 Time, min REPORT: 98AGKPOI.3M ADVANCEO 55 BIOANALYTICAL SERVICES, INC. Figure20. Fullscan reconstructedionchromatograms showing thepresenceof metabolitesafterincubatinghuman hepatocyteswithT-6293 for0 hour. XIC (period1)from 650.0-650.5amu from B01 IFl T6293 0 hr (2) % Intensity 6.21(T6293) 3.3Oe5 cps (i) 60 30' M/z 570.0-570.5For Compound 11 1.10 2.53 (ii6)0 30 nVz 746.0-746.5For Compound III 1.15 (iii6)0 30 -L .. ;@. I\- nVz 584.0-584.5ForCompound V A. A@ 6.49 2.OOe4 cps 9.73 2.OOe4 cps 9.40 1.64e5 cps 9.23 (iV) 60 30' nVz 542.0-542.5For Compound Vi 1.32 1.98 (v) 60 30 2.2Oe4 cps nVz 556.0-556.5For Compound VIII (Vi) 59 30 8.19 3.6Oe4 cDs nvz 526.0--1@-,6.5For Compound IX 60 (vii) 30 2.6Oe4 cps 93.344@ ni/z606.0-606.5ForCompound X (vii6io0)l 3 nVz 498.0-498.5For Compound XI (ix)60 25 30'Z- 6 m/z 499.0-499.5ForCompoundXII 60' 1.211 2.25 30 I?@-@ -A CA 2 3 /1, . ^ v 4 5.66 5.72 9.8Oe4 CDs 7.2Oe4 cps 8.63 5.72(XII) - A 5 - - -ql- 6 7 8.OOe4 cr)s 8.63 ==:@ 8 9 Time,min REPORT: 98AGKPOI.3M ADVANCEO 56 BIOANALYTICAL MMM SERVICES, INC. Figur2e1. Fulsicanreconstru!cotnecildiromatogrsatnilo@winegpresenci-@-@--6f metaboliatfetseirncubatihnugmanhepatocywtietshT-629f3or6hour. XIC(peri1o)dfrom650.0-65a0m.u5fromBOIIFlT62936 hr(2) % Intensity 6.15(r) 60 (i)30' ni/z570.0-570.5 1.15 60 30 At\ A 5.2ge5 cps 2.2Oe4 cps nVz 746.0-746.5 60' 30' 6.05(U 8.OOe4 cps m/z 584.0-584.5 (iv)60. 30' nVz 542.0-542.5 60 (v) 30' ffi/5z56.0-556.5 % I tensity (vi) 60' 30' ni/z526.0-526.5For Compound IX 1.32 6300.'L ni/z606.0-606.5For Compound X (viii) 60 1 15 30t- A- - mlz 498.0-498.5For Compound XI (ix)60 30' 6.10 (V) 2.02e5 cps 7.58(VI) 3.32e5cps 5.88(VIrl) 8.8Oe4 cps 2.6Oe4 cps (IX)8.85 4.8Oe4 cps 879 5.66 7.09 (X][) 5.48c5 cps rrvz499.0-499.5For Compound XII (x) 60' 30-_,,\6 _ 1 15 1 @t% @- 2 - %^- A 4 3 4 5.72(Xll) 7.09 1.1Oe5 cps 5 7 8 9 Time, min REPORT: 98AGKPOI.3M AOVANCEO 57 BIOANALYTICAL SERVICEES, INC. r lgur2e2.SJKMtotaiioncliromatograsmtsiowintghepresencoefmetaboliItVeTn-Ciyincubationof T-6293 with rathepatocytesat0 hour; (iii)ncubationof T- 6293 with rathepatocytesat6 hour; (iiii)ncubationofT-6293 withhuman hepatocytesat0 hour and (iv)incubationofT-6293 withhuman hepatocytesat6 hour. TIC from T6293 R4 Ohr ffL/z568 % I ensity 90. 1 32 60 - 30 AA 8.19 1.05e2 cps TIC from BO I I R4 T6293 6h nVz 568 (1) 90. 6030. 1.50e2 cps T TIC from T6293 Fl Ohr rn/z568 90. 6030 8.19 766 9.00e I cps TIC from BOIJ F] T6293 6h ffi/5z68 (1) 90. 5.00e I cps (iv)60- 30- t 1 2 3 4 5 6 7 8 9 Time, min REPORT: 98AGKPOl.3M ADVANCEO SIOANALYTICAL 58 SERVICES, INC. Figure 23. SRM totalion chromatograms showing the presence of metabolite Vil in (i)T-6293 standard solution;(ii)incubation of T-6293 with rat hepatocytes at 0 hour; (iiii)ncubation of T-6293 with rat hepatocytes at 6 hour; (iv) incubation of T-6293 with human hepatocytes at 0 hour and (v)incubation of T-6293 with human hepatocytes at 6 hour. TIC from T6293 STD nVz 540 % Irtensity 60- 30.L I : L4" TIC from T6293 6030- R4 Ohr nVz 540 2.33 3.50eI cps 2.5 e2 cps 6.36 TIC from T6293 R4 6hr m/z 540 6030- -A@ TIC from T6293 F I Ohr nVz 540 1.08 60, ('v)30- ",AA TIC fromT6293 Fl 6hrnVz 540 60- _10 1.088 (v) 30- 1 2 3 4 3.3Oe2 cps 7.85(VII) 5.18 2.45e2 cps 3.OOe2cps 5.78 7.,8"5(VH) 5 6 7 8 9 Time, min REPORT: 98AGKPOI.3M AOVANCEO 59 BIOANALYTICAL SERVICES, INC. Figure24.SRM totailonchromatogramshowingthepresencoefmetabolitIeX in(i) T-6293standardsolutio(ni;ii)ncubationfT-6293withrathepatocyteast 0 hour;(iiin)cubatioonfT-6293withrathepatocyteast6 hour;(iv) incubatioonfT-6293withhuman hepatocytaets0 hourand(v)incubation ofT-6293withhuman hepatocytaets6 hour. TIC fromT6293 STD nVz 526 % Intensity 60(i)30- 7.42e4 cps 9.31(EX) TIC from T6293 R4 Ohr ffi/z526 0.98 60- 30- TIC from T6293 R4 6hr nVz 526 0.98 60, 30- JL TIC from T6293 Fl Ohr mJz 526 0.98 60- ('v)30- 5.76 A\ 6.20 2.08e3 cps _L.2_(@IX) T 2.02e3 cps 8.75 9.31(]X) 5.85e2 cps 9.29(IX) TIC from T6293 Fl 6hr m/z 526 60(v) 30. 1.06 1 2 3 4 6.10 5 6 7 1.29e3 cps 8.75 9.33(IX) 9 Time, min REPORT: 98AGKPOI.3M ADVANCEO -BIOANALYTICAL 60 SERVICES, INC. Figure25.SRM totailonchromatogramsshowingthepresencoefmetaboliXtein(i) T-6293standarsdolutio(ni;ii)ncubatioofnT-6293withrathepatocytaets 0 hour;(iiiin)cubationfT-6293withrathepatocytaets6 hour;(iv) incubatiofnT-6293withhuman hepatocytaets0 hourand(v)incubation ofT-6293withhuman hepatocytaets6 hour. TIC fromT6293 STD m/z606 % Intensity 4.50eI cps 60' 30, 1 TIC from T6293 R4 Ohr nVz 606 1.01 60 30 TIC from T6293 R4 6hr nVz 606 1.01 6030' 3.75e2 cps 9.38 3.1Oe2 cps 9.45 TIC from T6293 F I Ohr m/z 606 0.94 60(iv) 30- 3.5Oe2 cps 9.52 TIC from T6293 F I 6hr nVz 606 0.98 3.8Oec2ps 60, (v) 30, A@ A -1, ...r I, m. beraitlmei- 1 2 3 4 5 6 7 8 9Time, min REPORT: 98AGKPOI.3M MMM ADVANCEO 61 BIOANALYTICAL SERVICES, INC. Figure26. Reconstructedionchromatograms showing thepresenceofmetabolites afterincubatingrathepatocyteswithT-6294 for0 hour. XIC (period1)from 542.0-542.5amu from BO IIR4 T6294 Ohr(1)For Compound VI % Intensity 60 nVz 540.0 -540.5 For Compound VII 1.15 60 1.87 M/z 556.0 -556.5 For Cc-ipound VIII 1.10 60. 5.44 6.71 2.6Oe4 cps 9.24 2.8Oe4 cps 9.57 3.4Oe4cps 8.31 nVz 526.0-526.5For Compound IX (T-6294) 60 (iv) nVz 606.0 -606.5 ForCompound X 60 1.15 (v) nVz 498.0 - 498.5For Compound XI (vi)60 3.23e6cps 8.91 5.66 7.15(XI) 8.OOe4 cps 9.19 4.86e5 cps nVz 499.0 -499.5 For Compound XII (vii60) 4@q@@ l@ A @ 1 2 3 4 5.72(XII) 7.15 6 7 8 1.04e5cps 9 Time,min REPORT: 98AGKPOI.3M AOVANCEO 62 BIOANALYTICAL SERVICES, INC. Figure27. Reconstructedionchromatograms showing thepresenceofmetabotites afterincubatingrathepatocyteswithT-6294 for6 hour. XIC (period1)from 542.0-542.5amu from BOI 1 R4 T6294 6 hr (1) % Intensity (i) 60 1.37 6.81 7.64(VI) 6.2Oe4 cps m/z 540.0-540.5For Compound VII 1.26 60L-A-A 2.4Oe4 cps 9.29 m/z 556.0-556.5For Compound VIII 60 M/z 526.0-526.5For Compound IX iv) 60 5.99(VM) 1.86e5 cps 3.62e6 cps (T-6294)9.40 M/z 606.0-606.5For Compound X v) 60 1.15 1.12e5 cps .2 8.30 ni/z498.0 - 498.5 For Compound )U ',Vi6)0 7.31 1) 5.97e6 cps nl/z499.0-499.5For Compound XII t 2.28e6cps 7.31 (vii6)0 5.72(Xll) - I I I i I 41 1 1 2 3 4 5 6 7 8 9Time,min REPORT: 98AGKPOI.3M ADVANCEO 63 BIOANALYTICAL SERVICES, INC. Figure28. iKeconstrucitoMncliro togramssliowintghepresencoelmetabojife-9-- afteirncubatihnugman hepatocytweisthT-6294for0 hour. XIC (perio1d)from542.0-542.a5mu fromB01 IFl T6294 Ohr(1) % Intensity (i) 60 1.37 1.92 5.18 1 1 1 1 1 1 3.OOe4 cps 9.13 1 1 m/z 540.0 - 540.5 For Compound VII (ii6)0 7.76 8.31 2.4Oe4 cps m/z 556.0 - 556.5 For Compound VIII 1.10 (iii6)0- 5.45 6.71 3.4Oe4 cps 8.31 m/z 526.0 - 526.5 For Compound IX (iv) 60 1 m/z 606.0 - 606.5 For Compound X (v) 60. 4.52e6 cps 8.86 T-6294 z@i9.OOe4 cps 9.19 M/z 498.0 -498.5-ForCompound )U (vi)60 1.82e5 cps 7.32 (XI) 5.73 m/z 499.0 - 499.5 For Compound XII (vii6)0. 1 2 3 4 5.73(XII) 7.32 1.06e5 cps 8.86 5 6 7 8 9Time, min REPORT: 98AGKPOI.3M AOVANCEO 64 MMM BIOANALYTICAL SERVICES, INC. Figure 29. Reconstructedion chromatograms showing thepresenceofmetabolites afterincubatinghuman hepatocyteswithT-6294 for6 hour. XIC (period1)from 542.0-542.5amu from BOI I Fl T6294 6hr (1) % Intensity (i)60 3.8Oe4 cps 7.61(VI) m/z 540.0 - 540.5 For Compound VII 5.84 (ii)60 2.4Oe4 cps m/z 556.0 - 556.5 For Compound VIII (iii6)0 m/z 526.0 - 526.5 For Compound IX (iv)60 m/z 606.0 - 606.5 For Compound X (v) 60 I .. . .. I I nVz 498.0 - 498.5 For Compound XI (vi)60 1 m/z 499.0 - 499.5 For Compound Y@H (v) 60 2 3 4 5.93(VM) 6.72 2.4Oe4 cps 3.31e6 cps 8.92 (T-6294) 5.68 I ^. 1^ 7.33(XI) a 7.33 5.73(XH) 5 6 7 8 7.6Oe4 cps 9.14 3.88e6 cps 9.68e5 cps 9Time, min REPORT: 98AGKPOI.3M AOVANCED 65 BIOANALYTICAL SERVICES, INC. k igure -iU. SKM total!on chromatograms showing thepresence of metaboliteVirin (i)T-6294 standard solution;(iii)ncubationof T-6294 with rat hepatocytesat 0 hour; (iiii)ncubationof T-6294 with rat hepatocytes at 6 hour; (iv)incubationof T-6294 with human hepatocytesat 0 hour and (v)incubation ofT-6294 with human hepatocytesat 6 hour. TIC fromT6294STD nVz540 % Intensity 3.50eI cps 6030- TIC from T6294 R4 Ohr m/z 540 2.33 6030- 1.75e2cps TIC from T6294 R4 6hr nVz 540 5.20 6030- 1.40e3cps TIC from T6294 F I Ohr rn/z540 1.08 60(iv) 30- L133 2.33 A 2.85e2cps TIC from T6294 Fl 6hr nVz 540 60- (v)30- 2.33 5.78 5.22 1.1Oc3 cps 1 2 3 4 5 6 7 8 9 Time, min REPORT:98AGKPOI.3M ADVANCED 66 BIOANALYTICAL SERVICES, INC. Figure31.SRM totailonchromatogramsshowingthepresencoefmetaboliXtein(i) T-6294standarsdolutio(ni;ii)ncubatioofnT-6294withrathepatocytaets 0 hour;(iiin)cubatiofnT-6294withrathepatocytaets6 hour;(iv) incubatiofnT-6294withhuman hepatocytaets0 hourand(v)incubation ofT-6294withhuman hepatocytaets6 hour. TIC fromT6294 STD m/z606 % Intensity 4.00eI cps 60(i) 30- TIC from T6294 R4 Ohr nVz 606 0.96 6030- -AKAA-M 3.2Oe2 cps TIC from T6294 R4 6hr ni/z606 1.01 6030- 2.9Oe2 cps TIC from T6294 Fl Ohr m/z 606 1.03 6030- A A- A 3.6Oe2 cps TIC from T6294 Fl 6hr m/z 606 0.91 3.OOe2 cps 60(v) 30- 1 2 3 4 5 6 7 8 9 Time, min REPORT: 98AGKPOI.3M ADVANCED 67 BIOANALYTICAL SERVICES, INC. Figure32. Negativeionizatiopnroduct-ionmass spectraof(i)metabolite111;(ii) metaboliteVI and (iiim)etabolitVeIII. Specamfrorn624 % Intffisity 80' rrii(n6scam) frorn3MT6292R46h FROD NidabolitMe 60' 40 219 419 20' 113 169 90 180 270 Spectrumfrom 8.48nin (39scam) fromBOI IF] T6M % ity I 8060- 65 92 450 6hr(4) NktabffitMe 40169 20- 219 269 319 60 12D 180 240 300 360 Spectrum from 6.2Drrin(12scans)from BOI IFl 16293 6hr % Intm4 80' 169 NUabolite VIH (iu)60' 65 136 219 40' 83 93 20* -60 - 120 180 240 3bO 3@O 5 5@o 419 420 4@O 5-5Oa4 cps D4-K- 746 6@O 720 nYz 7.95e3 cps D,4-H -542 48D --@o nyz 3.33e3 cps [NI." 556 498 4@O 5@o nYz REPORT: 98AGKPOI.3M AOVANCEO 68 BIOANALY-TICAL MMM SERVICES, INC. Figure33. Product-ionms, sspectraof (i)MetaboliteXHI and (iim)etaboliteXI Specnm % 80- from 6.21rrin(25 scans)from 3M T6292 R4 6h PRCD 65(Y498 NietaboliMtBe 60. 40- 20 110 80 169 219 2ffl 419 80 160 240 320 4M Spwtrumfiom78.31ryin(36scam)froi3nM T6292R4 6hPROD 65(Y498(1) 80. NktWmflteM 60- 40 20 11 119 169 60 120 180 240 3@O 526 4M 5@O 3@O 420 1.29e5cps 64oniz 7.67e4cps 178 480 rdz REPORT: 98AGKPOI.3M AOVANCEO 69 BIOANALYTICAL SERVICES, INC. Figure34. SelectedReaction Monitoring Chromatograms from LC/MS/MS of a CalibrationStandard HepatocyteExtract(2 ng/mL T-6295) XIC (period1)for499.0 99.1amu from Std-2 I % Intensity .98 T-6295 4.55e2cps 80- 60- 40- 20, A. L,1--U -ILA.L.&A 0.9 1.8 2.7 3.6 4.5 5.4 6.3 Time,min XIC (period1)for427.0 81.1amu from Std-2 I % I ensity 6.96e4cps 4.88 InternaSltandard 8060 4020- 0.9 1.8 @.7 3.6 @.5 5.4 6.3Time,min REPORT: 98AGKPOI.3M