Document 82LRXawrwzr60j7jO8az6Nz6K

ADVANCED BIOANALYTICAL SERVICES, INC. ARP&-OI63 3 1 ANALYTICAL REPORT TITLE: Additional Characterization of Metabolites of T-6292, T-6293 and T-6294 from Rat and Human Hepatocytes by TurboIonSpray LC/MS and LC/MS/MS. Semi-Quantitative Analysis of T-6295 in Rat and Human Hepatocytes Incubated with T-6292, T-6293 and T-6294 by LC/MS/MS. DATE: REPORT: 27 January, 1998 98AGKP01.3M AUTHOR: Grace K. Poon, PhD Steve Lowes, PhD PREPARED FOR: 3M Medical Department, Toxicology Services, St. Paul, MN 55133 FEB - 2 ' " J NO. OF PAGES: 69 004058 REPORT: 98AGKP01.3M 15 Catherw ood Road Ithaca, New York 14850 (607) 266-0665 Fax (607) 2 6 6-0749 ADVANCED B IO A N A L Y T IC A L SER VIC ES, INC. ADVANCED BIOANALYTICAL SERVICES SIGNATURE PAGE * z. Title: Additional Characterization of Metabolites of T-6292, T-6293 and T6294 from Rat and Human Hepatocytes by TurboIonSpray LC/MS and LC/MS/MS. Semi-Quantitative Analysis of T-6295 in Rat and Human Hepatocytes Incubated with T-6292, T-6293 and T-6294, by LC/MS/MS. Reported by: i / a u / xWr , V - ace K. Poon, PhD. enior Research Scientist Reviewed and j 0 iJL- Approved by: )f ( : J> ;'V _ {Xfrw-f 1-1______ Mark Lentham, B.A. Auditor Authorized for Release by: '<fc> Steve Lowes, PhD. Scientific Director Advanced BioAnalytical Services Date a _ ,^ x fn \\ . Z- 1 , / 7 / Date g~7 Date 004059 REPORT: 98AGKP01.3M ADVANCED BIO ANALYTIC AL SER VIC ES, INC. TABLE OF CONTENTS 3 SIGNATURE PAGE..............................................................................................................2 TABLE OF CONTENTS...................................................................................................... 3 LIST OF TABLES................................................................................................................ 5 LIST OF FIGURES................................................................................................................6 PART A: Characterization of Metabolites of T-6292, T-6293 and T-6294 from Rat and Human Hepatocytes by TurboIonSpray LC/MS and LC/MS/MS......................10 1. INTRODUCTION.......................................................................... 10 1.1. Backgriound..........................................................................................................10 1.2. Study Objective...................................................................................................... 10 2. EXPERIMENTAL...............................................................................................14 2.1. Chemicals and Materials.....................................................................................14 2.2. Standard Solutions..............................................................................................15 2.3. HPLC Eluent Preparation.................................................................................. 15 2.4. Sample Preparation..............................................................................................16 2.5. LC/MS AND LC/MS/MS INSTRUMENTATION........................................................ 16 2.6. Data Handling.......................................................................................................18 3. RESULTS AND DISCUSSION............................................................ 18 3.1. Control Experiments...........................................................................................19 3.2. Metabolism of T-6292......................................................................................... 19 3.2.1. Metabolism o f T-6292 by Rat Hepatocytes at 0 hour.....................................19 3.2.2. Metabolism of T-6292 by Rat Hepatocytes at 6 hour.....................................20 3.2.3. Metabolism o f T-6292 in Human Hepatocytes at 0 hour...............................21 3.2.4. Metabolism o f T-6292 by Human Hepatocytes at 6 hour..............................21 3.3. Metabolism of T-6293.......................................................................................... 22 3.3.1. Metabolism o f T-6293 by Rat Hepatocytes at 0 hour..................................... 22 3.3.2. Metabolism o f T-6293 by Rat Hepatocytes at 6 hour..................................... 22 3.3.3. Metabolism o f T-6293 by Human Hepatocytes at 0 hour..............................23 3.3.4. Metabolism o f T-6293 by Human Hepatocytes at 6 hour..............................23 3.4. Metabolism of T-6294.......................................................................................... 24 3.4.1. Metabolism o f T-6294 by Rat Hepatocytes at 0 hour......................................24 3.4.2. Metabolism o f T-6294 by Rat Hepatocytes at 6 hour..................................... 24 REPORT: 98AGKP01.3M 004060 ADVANCED B O A N A L Y T IC A L S E R VIC ES, INC. 4 3.4.3. Metabolism o f T-6294 by Human Hpatocytes at 0 hour............................. 25 3.4.4. Metabolism o f T-6294 by Human Hepatocytes at 6 hour............................. 25 3.5. Product-ion Mass Spectra.................................................................................. 25 3.6. Positive Ionization LC/MS Analysis of Human Hepatocytes Incubated with T-6292 and T-6293........................................................................................ 27 PART B: Semi-Quantitative Analysis of T-6295 in Rat and Human Hepatocytes Incubated with T-6292, T-6293 and T-6294 by LC/MS/MS............................. 28 4. INTRODUCTION...............................................................................................28 5. EXPERIMENTAL...............................................................................................28 5.1. Standard Solutions..............................................................................................28 5.2. Preparation of Quenching Solution:................................................................ 29 5.3. HPLC Eluent Preparation:................................................................................. 29 5.4. Sample Preparation..............................................................................................29 5.5. Preparation of Rat Hepatocytes Standard Curve Samples ........................29 5.6. LC/MS/MS Instrumentation............................................................................... 30 5.6.1. HPLC conditions..............................................................................................30 5.6.2. MS conditions...................................................................................................30 5.6.3. Data Handling..................................................................................................31 6. Results and Discussion:.......................................................................................31 7. References:.......................................................................................................... 35 REPORT: 98AGKP01.3M G040S1 ADVANCED BIOANALYTICAL SER VIC ES. INC. LIST OF TABLES < Table I: Summary of Proposed Structures of Metabolites of T-6292 Incubated with Rat and Human Hepatocytes..................................................................................... 12 Table II: Summary of Proposed Structures of Metabolites of T-6293 Incubated with Rat and Human Hepatocytes..................................................................................... 13 Table III: Summary of Proposed Structures of Metabolites of T-6294 Incubated in Rat and Human Hepatocytes......................................................................................14 Table IV: Proposed product-ion fragmentation for Metabolite VI................................... 26 Table V: Proposed product-ion fragmentation for Metabolite VI................................... 27 Table VI: Quantitative Analysis of T-6295 in Rat and Human Hepatocytes after Incubation with T-6292, T-6293 and T-6294.................................................... 32 Table VITQuantitative Analysis of T-6295 in T-6292, T-6293 and T-6294.....................35 004062 REPORT: 98AGKP01.3M --'-. A ADVANCED BIC. A L Y T IC A L SEF 'CES, INC. 6 LIST OF FIGURES Figure 1. Reconstructed ion chromatogram (i), negative ionization mass spectrum (ii) and LC/MS/MS product-ion mass spectrum (iii) of authentic T-6293........... 36 Figure 2. Reconstructed ion chromatogram (i) and LC/MS/MS product-ion mass spectrum (ii) of authentic T-6294...................................................................... 37 Figure 3. Reconstructed ion chromatogram (i), negative ionization mass spectrum (ii) and LC/MS/MS product-ion mass spectrum (iii) of authentic T-6295............ 38 Figure 4. Full scan reconstructed ion chromatograms of control rat hepatocytes incubated for 6 hours with no test articles........................................................ 39 Figure 5. Full scan reconstructed ion chromatograms of control human hepatocytes incubated for 6 hours with no test articles........................................................ 40 Figure 6. Full scan reconstructed ion chromatograms of T-6292 incubated for 6 hours with no hepatocytes............................................................................................41 Figure 7. Full scan reconstructed ion chromatograms of T-6293 incubated for 6 hours with no hepatocytes............................................................................................ 42 Figure 8. Full scan reconstructed ion chromatograms of T-6294 incubated for 6 hours with no hepatocytes............................................................................................ 43 Figure 9. Full scan reconstructed ion chromatograms showing the presence of metabolites after incubating rat hepatocytes with T-6292 at 0 hour................ 44 Figure 10. Full scan reconstructed ion chromatograms showing the prsence of metabolites after incubating rat hepatocytes with T-6292 for 6 hour...............45 Figure 11. Full scan reconstructed ion chromatograms showing the presence of metabolites after incubating human hepatocytes with T-6292 at 0 hr..............46 Figure 12. Full scan reconstructed ion chromatograms showing the presence of metabolites after incubating human hepatocytes with T-6292 for 6 hour......... 47 Figure 13.(i) SRM chromatograms of rat hepatocytes incubated with no test articles; (ii) SRM chromatograms of human hepatocytes incubated with no test articles; (iii) SRM chromatograms of human hepatocytes incubated with no test a rtic le s .............................................................................................................. 48 REPORT; 98AGKP01.3M 004063 ADVANCED BIOANALYTICAL SER VIC ES, INC. / Figure 14.SRM chromatograms showing the absence of metabolite IV in (i) rat hepatocytes incubated with T-6292 at 0 hour; (ii) rat hepatocytes incubated with T-6292 for 6 hour; (iii) human hepatocytes incubated with T-6292 at 0 hour and (iv) human hepatocytes incubated with T-6292 for 6 hour............... 49 Figure 15.SRM total ion chromatograms showing the presence of metabolite VII in (i) T6292 standard solution; (ii) incubation of T-6292 with rat hepatocytes at 0 hour; (iii) incubation of T-6292 with rat hepatocytes at 6 hour; (iv) incubation of T-6292 with human hepatocytes at 0 hour and (v) incubation of T-6292 with human hepatocytes at 6 hour............................................................................. 50 Figure 16.SRM total ion chromatograms showing the presence of metabolite IX in (i) T6292 standard solution; (ii) incubation of T-6292 with rat hepatocytes at 0 hour; (iii) incubation of T-6292 with rat hepatocytes at 6 hour; (iv) incubation of T-6292 with human hepatocytes at 0 hour and (v) incubation of T-6292 with human hepatocytes at 6 hour..............................................................................51 Figure 17.SRM total ion chromatograms showing the presence of metabolite X in (i) T6292 standard solution; (ii) incubation of T-629 with rat hepatocytes at 0 hour; (iii) incubation of T-6292 with rat hepatocytes at 6 hour; (iv) incubation of T6292 with human hepatocytes at 0 hour and (v) incubation of T-6292 with human hepatocytes at 6 hour..............................................................................52 Figure 18. Full scan reconstructed ion chromatograms showing the presence of metabolites after incubating rat hepatocytes with T-6293 for 0 hour.............. 53 Figure 19. Full scan reconstructed ion chromatograms showing the presence of metabolites after incubating rat hepatocytes with T-6293 for 6 hour.............. 54 Figure 20. Full scan reconstructed ion chromatograms showing the presence of metabolites after incubating human hepatocytes with T-6293 for 0 hour........55 Figure 21. Full scan reconstructed ion chromatograms showing the presence of metabolites after incubating human hepatocytes with T-6293 for 6 hour........56 Figure 22. SRM total ion chromatograms showing the presence of metabolite IV in (i) incubation of T-6293 with rat hepatocytes at 0 hour; (ii) incubation of T-6293 with rat hepatocytes at 6 hour; (iii) incubation of T-6293 with human hepatocytes at 0 hour and (iv) incubation of T-6293 with human hepatocytes at 6 hour..................................................................................................................57 REPORT: 98AGKP01.3M 004064 ADVANCED BIOAN ALYTIC AL SERVICES, INC. 8 Figure 23.SRM total ion chromatograms showing the presence of metabolite VII in (i) T6293 standard solution; (ii) incubation of T-6293 with rat hepatocytes at 0 hour; (iii) incubation 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..............................................................................58 Figure 24. SRM total ion chromatograms showing the presence of metabolite IX in (i) T6293 standard solution; (ii) incubation of T-6293 with rat hepatocytes at 0 hour; (iii) incubation 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..............................................................................59 Figure 25. SRM total ion chromatograms showing the presence of metabolite X in (i) T6293 standard solution; (ii) incubation of T-6293 with rat hepatocytes at 0 hour; (iii) incubation 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..............................................................................60 Figure 26. Reconstructed ion chromatograms showing the presence of metabolites after incubating rat hepatocytes with T-6294 for 0 hour........................................... 61 Figure 27.Reconstructed ion chromatograms showing the presence of metabolites after incubating rat hepatocytes with T-6294 for 6 hour........................................... 62 Figure 28. Reconstructed ion chromatograms showing the presence of metabolites after incubating human hepatocytes with T-6294 for 0 hour.................................... 63 Figure 29. Reconstructed ion chromatograms showing the presence of metabolites after incubating human hepatocytes with T-6294 for 6 hour.................................... 64 Figure 30. SRM total ion chromatograms showing the presence of metabolite VII in (i) T6294 standard solution; (ii) incubation of T-6294 with rat hepatocytes at 0 hour; (iii) incubation of T-6294 with rat hepatocytes at 6 hour; (iv) incubation of T-6294 with human hepatocytes at 0 hour and (v) incubation of T-6294 with human hepatocytes at 6 hour............................................................................. 65 Figure 31. SRM total ion chromatograms showing the presence of metabolite X in (i) T6294 standard solution; (ii) incubation of T-6294 with rat hepatocytes at 0 hour; (iii) incubation of T-6294 with rat hepatocytes at 6 hour; (iv) incubation of T-6294 with human hepatocytes at 0 hour and (v) incubation of T-6294 with human hepatocytes at 6 hour..............................................................................66 Figure 32. Negative ionization product-ion mass spectra of (i) metabolite III; (ii) metabolite VI and (iii) metabolite VIII..............................................................67 REPORT: 98AGKP01.3M 004065 ADVANCED BIOANALYTICAL S E R V IC E S , INC. 9 Figure 33.Product-ion mass spectra of (i) Metabolite XIII and (ii) metabolite XI.......... 68 Figure 34. Selected Reaction Monitoring Chromatograms from LC/MS/MS of a Calibration Standard Hepatocyte Extract (2 ng/mL T-6295)...........................69 REPORT: 98AGKP01.3M 004066 ADVANCED BIOAN ALYTIC AL SER VIC ES, INC. 10 PART A: CHARACTERIZATION OF METABOLITES OF T-6292, T-6293 AND T-6294 FROM RAT AND HUMAN HEPATOCYTES BY TURBOIONSPRAY LC/MS AND LC/MS/MS. 1. INTRODUCTION 1.1. Background The metabolism of T-6292, T-6293 and T-6294 in rat hepatocytes was investigated at Advanced BioAnalytical Services, Inc. using liquid chromatography ionspray tandem mass spectrometry (Reference 1). Several metabolites had been identified, namely the glucuronide acid conjugate, the carboxylic acid, the sulfonamido alcohol, the iV-dealkyl sulfonamido alcohol and the sulphonamide of T-6293. 1.2. Study Objective The principle aim of this work was (1) To complete the characterization of the metabolic products of T-6292, T-6293 and T-6294 which were formed by incubating each compound with rat and human hepatocytes; (2) structural characterization of the carboxylic acid and alcohol analogues using tandem mass spectrometry; (3) identification of additional metabolites by LC/MS, using positive ionization ionspray; (4) identification of the aldehyde and iV-dealkyl aldehyde analogues of T-6293; (5) semi-quantitative analysis to determine the concentration of T-6295 in the rat and human hepatocytes after incubating with T-6292, T-6293 and T-6294. REPORT: 98AGKP01.3M 004067 ADVANCED B O A N A L Y T IC A L SER VIC ES. INC. ,, The quenched incubates were directly injected and separated on an HPLC column. Chromatographic separation of the various constituents was accomplished using gradient elution. Metabolites were characterized by LC/MS and LC/MS/MS, and the results are summarized in Tables I - IE. The mass spectrometer was operated in the full scan single MS mode (m/z 150-800). Tandem mass spectrometry was used for further structural elucidation and confirmation. REPORT: 98AGKP01.3M 004068 ADVANCED B O A N A L Y T IC A L SER VIC ES, INC. 12 Table I: Summary of Proposed Structures of Metabolites of T-6292 Incubated with Rat and Human Hepatocytes Proposed Structure CH,--CH, C *F 1 7 ~ 5 0 2 -- N\ CH,--CH,OH c / ~it s o 2~ ~ n \ CH,--CH,0-Glu Abbreviation II (T-6292) m [M-H]' 570 746 Rat 0 hour 6 hour No MS No MS response response X Human 0 hour No MS response 6 hour No MS response X / C H ,-- C H , IV 568 X X X X --:C 8F i7 S O , -- N --C H , C H O CH,--CH, cifIT--SOj--NC V 584 X V X Y CH,--CH,COOH H C,F--SO-- CH,--CH,OH VI 542 X s XS H QiFn ^S O ,-- N VH 540 X trace X trace CH, -- CHO H VIII 556 X X C ,F -- S O -- C H jCO O H H IX 526 Y V S cF,t S O , N `f (T-6294) C H ,-- C H , C H ,-- C H , X 606 X trace X trace C*Fit S O , N 5 0 ,H c8f -- s o -- n h 2 XI 498 V / CgFif SOjH -^h,--CHW C,F,,--SO,--N CH,--CH.O-SO.H xn (T-6295) xm 499 650 S X X XV S Observed X Not observed REPORT: 98AGKP01.3M C040S9 ADVANCED B ID A N A L Y T IC A L SER VIC ES, INC. 13 Table II: Summary of Proposed Structures of Metabolites of T-6293 Incubated with Rat and Human Hepatocytes Proposed Structure Abbreviation [M-H]* --C H , CH , cfn--S:- nC " 9, CH2-- CH .O PO H 0- I (T-6293) 650 Rat 0 hour 6 hour Human 0 hour 6 hour S C BF -- S 0 2-- C,,Fi r - S O -- C gF -- S O -- CKF lT- S O -- C SF -- S O ,-- C,,F--S O -- C H ,-- CH, C H j-- C H ,0H C H 2-- C H , CH; -- CH ,0-G lu --C H , C H , C H ,-- CHO --C H , CH , C H ,-- CH.COOH H C H ,-- CH,OH H C H ,-- CHO C sF l7 - S 0 -- H CHXOOH CF] j S O , H N C H ,-- C H , ^CH 2 CH, "S O ,-- N SO ,H CgF -- SO-- NH, CsF i7 SOjH II (T-6292) m IV V VI vn vm IX (T-6294) X XI xn (T-6295) 570 No MS No MS No MS No MS response response response response 746 X S X 568 X X X X 584 X V X / 542 X X 540 X trace X trace 556 X Y X V 526 X V V S 606 X X X X 498 X V X 499 V / S Observed X Not observed REPORT: 98AGKP01.3M 004070 ADVANCED B O A N A L Y T IC A L SER VIC ES, INC. 14 Table III: Summary of Proposed Structures of Metabolites of T-6294 Incubated in Rat and Human Hepatocytes Proposed Structure H 17 S2 NCuH,--CH,,OH H CF|7 SO, N ' CH, --CHO H --SOj--Nc s F it ^ CHjCOOH H c*F|7 s o 2 N "C CH,--CH, CH,--CH, CKFn~~lSO,--1N^ SO,H CgFp--SO,--NH2 CsF.t- S O 3H Abbreviation VI VII VIII IX (T-6294) X XI XII (T-6295) [M-H]' Rat Human 0 hour 6 hour 0 hour 6 hour 542 X y X y 540 X X X X 556 XyX y 526 y yy y 606 X X X X 498 y y y y 499 y y y y S Observed X Not observed 2. EXPERIMENTAL 2.1. Chemicals and Materials T-6292, T-6293, T-6294 and T-6295 were provided by 3M Medical Department, Toxicology Services, St. Paul, MN 55133. Polypropylene autosampler vial: Polypropylene screw-cap tubes: Ethanol Cat # 66008-014, VWR Scientific Inc., Bridgeport, NJ 08014 16 x 100 mm, Cat # 500 765, Sun Brokers Inc., Wilmington, NC 28402 Cat # 112000200CSPP, Pharmaco Products, Brookefield, CT 06804 REPORT: 98AGKP01.3M 004071 ADVANCED B IO A N A L Y T IC A L SER VIC ES, INC. HPLC Grade Methanol Dimethyl Sulfoxide Ammonium Acetate 2.2. Standard Solutions 15 Cat # 230-4, Baxter/Scientific Products, McGaw Park, IL 60085 Cat # 27043-1, Sigma-Aldrich Inc., Milwaukee, W I53233 Cat # 24019-2, Sigma-Aldrich Inc., Milwaukee, WI 53233 Stock solutions of T-6292, T-6293, T-6294 and T-6295 were prepared at a concentration of 100 pg/mL in dimethyl sulfoxide (DMSO). Working solution (10 pg/mL) of each standard were prepared by diluting lmL of the stock solution into 9 mL ethanol. All stock solutions and working solutions were stored at 4 C and were allowed to equilibrate to room temperature before use. Preparation of Analytical Standard Stock Solutions Stock Solutions of T-6292, T-6293, T-6294 and T-6295 (100 pg/mL): Each test article was accurately weighed on a micro balance and transferred to a 16 x 100 mm polypropylene tube. The solid samples were dissolved in appropriate amount of DMSO to yield a 100 pg/mL solution. Working Solutions of T-6292, T-6293, T-6294 and T-6295 (10 pg/mL): Each standard working solution was prepared by diluting the stock solution (lmL) with ethanol (9 mL) in a 16 x 100 mm polypropylene tube . 2.3. HPLC Eluent Preparation 1 M Ammonium Acetate Solution: Ammonium acetate (77g) was added to NANOpure water (1L) in a volumetric flask. The solution was filtered through a 0.45 pm filter and stored at 4 C. REPORT: 98AGKP01.3M 004072 ADVANCED B IO A N A L Y T IC A L SER VIC ES. INC. 16 2 mM Ammonium Acetate Solution: 1 M ammonium acetate solution (2 mL) was diluted to 1L with NANOpure water. The solution was filtered through a 0.45 mm filter and stored at ambient temperature. A fresh solution was prepared every month. M ethanol: 2 mM Ammonium Acetate (90:10 v/v), Eluent: Methanol (450 mL) was mixed with 2 mM ammonium acetate solution (50 mL). The solution was stored at ambient temperature. A fresh solution was prepared every month. M ethanol: 2 mM Ammonium Acetate (10:90 v/v), Eluent: Methanol (50 mL) was mixed with 2 mM ammonium acetate solution (450 mL). The solution was stored at ambient temperature. A fresh solution was prepared every month. 2.4. Sample Preparation The metabolism study was carried out at SRI International, Menlo Park, CA. The incubates were quenched with an equal volume of ice-cold methanol, centrifuged and shipped in dry ice to Advanced BioAnalytical Services (ABS). The samples were stored at ABS (-20 C). During sample analysis, the supernatant was transferred to an autosampler vial. The autosampler vials were maintained in an ice-water bath at all times except during injection. 2.5. LC/MS and LC/MS/MS Instrumentation Liquid chromatography was performed on Shimadzu LC-10AD pumps and Shimadzu SCL10A gradient controller (Shimadzu Scientific Instruments, Inc., Columbia MD 21046). Samples were introduced with a Perkin-Elmer 200 series autosampler. The HPLC column (Betasil C l8, 2 x 100 mm) was obtained from Keystone Scientific, Inc., Beliefonte, PA 16823. A Sciex API ECTtriple quadrupole mass spectrometer was used for sample analysis. REPORT: 98AGKP01.3M 004073 ADVANCED B IO A N A L Y T IC A L S E R V IC E S , INC. 17 LC Conditions: LC Gradient: Time (min) 0 -5 5-10 10-10.5 10.5-15 %A (10:90 CH3OH:2mM NFLOAc) 80 to 0 % 0% 0 to 80 % 80% %B (90:10 CH3OH:2mM NKLOAc) 20 to 100 % 100% 100 to 20 % 20% Flow rate: 200 pL/min Typical Mass Spectrometer Conditions: MS Mode Source: TurboIonSpray Ion Spray Voltage: -3800 V Orifice: -78 V Declustering Potential: -48 V Ionization Mode: Negative Curtain Gas (U.H.P. N2) 1.2 LPM Nebulizer Gas (N2) 60 PSI Collision Gas Thickness: N/A Collision Energy: N/A Dwell Time: 5 msec Step Size: 1 amu Scan Range: 150-800 amu MS/MS Mode TurboIonSpray -3900 V -60 V -30 V Negative 1.2 LPM 60 PSI 280 x 10*3atoms/square cm 35 eV 200 msec variable Metabolite IV Transitions: m/z 568 -> 269; 526 -> 169; 526 ->119; 526 -> 83; 526 -> 65 Metabolite VII Transitions: m/z 540 -> 269; 540 -> 169; 540 -> 119; 540 -> 83; 540 -> 65 Metabolite IX Transitions: m/z 526 ->419; 526 -> 269; 526 -> 169; REPORT: 98AGKP01.3M 004074 ADVANCED B IO A N A L Y T IC A L S E R V IC E S . INC. Metabolite X Transitions: 526 -> 123; 526 -> 97 m/z 606 -> 269; 606 -> 169; 606 -> 119; 606 -> 83; 606 -> 65 Data System: RAD Version 2.6; LC Tune Version 2.4; Multiview Version 1.2; MacSpec Version 3.3; MacQuan Version 1.4 PPG Calibration Instrument mass axis calibration was performed by infusion of PPG-425 calibration solution (polypropylene glycols, average molecular weight 425, dissolved in 1:1 methanol: 2 mM ammonium acetate containing 0.1 % formic acid and 0.1 % acetonitrile) at a flow rate of 10 pL/min in the positive ion mode of detection. Peak widths were approximately 0.6 amu at half-height in the single MS mode. A calibration check was performed for the analytes on Q1 by infusion of a 1 pg/mL solution in ethanol at a flow rate of 10 pL/min. The mass spectrometric parameters and sensitivity were optimized using 60% B of the HPLC mobile phase at 200 pL/min. 2.6. Data Handling All raw chromatographic and mass spectrometric data were stored on the ABS file server ABS1_FS.DATA in the file hierarchy "RAWDATA:Validation:3MSRI:3MSRI Qualitative". All experimental information was stored in ABS notebook No: 2070. 3. RESULTS AND DISCUSSION T-6293, T-6294 and T-6295 displayed [M-H] ions at m/z 650, 526 and 499 respectively. The reconstructed ion chromatograms and negative ionization product ion mass spectra of compounds T-6293, T-6294 and T-6295 are shown in Figures 1 -3. Their fragmentation patterns were described in details in previous study [reference 1]. As observed before, T-6292 did not show any signal in positive or negative ionization modes REPORT: 98AGKP01.3M 004075 A D VAIN C E D B IO A N A L Y T IC A L S E R V IC E S , INC. 1Q [reference 1]. Therefore, no attempt was made to characterize T-6292 as a parent drug or a metabolite in this study. Rat hepatocytes (reference # 4) and human hepatocytes (Female 1, reference #HH357) were used for the study. 3.1. Control Experiments The full scan reconstructed ion chromatograms of the incubation media which contained rat and human hepatocytes but no test articles are displayed in Figures 4 and 5 respectively. These samples were analysed as control samples. The blank samples did not show any signal at the retention times expected for the T-6292, T-6293 and T-6294 metabolites. The full scan reconstructed ion chromatograms of the incubation media which contained T-6292, T-6293 and T-6294 but no hepatocytes are displayed in Figures 6, 7 and 8 respectively. These samples were analysed as control samples. However, compounds IX, and XI were present in the T-6292 sample (Figure 6). In the T-6293 sample, apart from the parent drug, compound XII was observed (Figure 7). In the T-6294 sample, apart from the parent drug, compound XI was detected (Figure 8). 3.2. Metabolism of T-6292 3.2.1. Metabolism of T-6292 by Rat Hepatocytes at 0 hour The full scan LC/MS reconstructed ion chromatograms of the rat hepatocytes sample incubated with T-6292 for 0 hour are shown in Figures 9. Figure 9 (i - x) displays the ion chromatograms for the following: the parent compound (m/z 570), metabolite III (m/z 746), metabolite V (m/z 584), metabolite VI (m/z 542), metabolite VIE (m/z 556), metabolite IX (m/z 526), metabolite X (m/z 606), metabolite XI (m/z 498), metabolite XI3 (m/z 499) and metabolite XIII (m/z 650). Even though compound IX, XI and XII were detected in this sample, compound IX and XI were observed in the control sample containing T-6292 and no hepatocytes (Figure 6). REPORT: 98AGKP01.3M 004076 3.2.2. ADVANCED B IO A N A L Y T IC A L S E R V IC E S , INC. Metabolism of T-6292 by Rat Hepatocytes at 6 hour 20 The full scan LC/MS reconstructed ion chromatograms of the rat hepatocytes sample incubated with T-6292 for 6 hour are shown in Figures 10, Figure 10 (i - x) displays the ion chromatograms for the following: the parent compound {m/z 570), metabolite HI {m/z 746), metabolite V (m/z 584), metabolite VI (m/z 542), metabolite VIH {m/z 556), metabolite IX (m/z 526), metabolite X {m/z 606), metabolite XI (m/z 498), metabolite XII {m/z 499) and metabolite XIII {m/z 650). This sample contained metabolites HI, V, VI, Vm , IX, XI, XII and XIII. Full scan mass spectra were not obtained for metabolites IV {m/z 568), VII {m/z 540), metabolite IX {m/z 526) and X {m/z 606). As an alternative, a selected reaction monitoring (SRM) analysis was performed. For metabolite IV, the formation of m/z 269, 169, 119, 83 and 65 ions from the precursor ion at m/z 568 were monitored. For metabolite VII, the formation of m/z 269, 169, 119, 83 and 65 ions from the precursor ion at m/z 540 were monitored. For metabolite EX, the formation of m/z 269, 169, 119, 123 and 97 ions from the precursor ion at m/z 526 were monitored. For metabolite X, the formation of m/z 269, 169, 119, 97, 83 and 65 ions from the precursor ion at m/z 606 were monitored. Since these product ions were characteristic of this class of compounds. Tentative identification of metabolites IV and VII was achieved in the study samples when signals were observed at their appropriate transitions. No metabolite IV was detected in the 0 or 6 hour rat study samples (Figure 14 i and ii). Metabolite VII was present in the rat hepatocytes treated with T-6292 for 6 hours (Figure 15 iii), but absent in the 0 hour sample (Figure 15 ii), and the no test article or no hepatocytes control samples (Figures 13 i and 15 i). Figure 16 (ii) and (iii) represent the SRM total ion chromatograms (TIC) of rat hepatocytes incubated with T-6292 for 0 and 6 hour respectively, showing the presence of metabolite IX in the samples. However, this component could also be observed in the control sample containing T-6292 and no hepatocytes (Figure 16 i). REPORT: 98AGKP01.3M 004077 ADVANCED BIOANALYTICAL SER VIC ES. INC. 21 Metabolite X was present in the rat hepatocytes treated with T-6292 for 6 hours (Figure 17 iii) but absent in the 0 hour sample (Figure 17 ii) and the no hepatocytes control sample (Figure 17 i). A potential metabolite (XIII) was observed at 6.1 min and displayed an [M-H]' ion at m/z 650 (Figure 10 x). An addition of 80 mass units relative to T6292 could correspond to the addition of a sulphate moiety. The product-ion mass spectrum of metabolite XIII in Figure 33(i) showed fragment ions at m/z 526, 419 (CgFn ), 319 (CF^*), 269 (C5F 11 ), 219 (C4F9'), 169 (C3F7*), 97 (HSO4' or FNS02 ) and 80 (SO3'). The fragment ions at m/z 80 is consistent with the presence of the sulphate moiety. 3.2.3. Metabolism of T-6292 in Human Hepatocytes at 0 hour The full scan LC/MS reconstructed ion chromatograms of the human hepatocytes sample incubated with T-6292 for 0 hour are shown in Figures 11. Figure 11 (i - x) displays the ion chromatograms for the following: the parent compound (m/z 570), metabolite III (m/z 746), metabolite V (m/z 584), metabolite VI (m/z 542), metabolite VEH (m/z 556), metabolite IX (m/z 526), metabolite X (m/z 606), metabolite XI (m/z 498), metabolite XII (m/z 499) and metabolite XIII (m/z 650). Compounds IX and XI were detected in this sample, as they were observed in the control sample containing T-6292 and no hepatocytes (Figure 6 vii and ix). 3.2.4. Metabolism of T-6292 by Human Hepatocytes at 6 hour The full scan LC/MS reconstructed ion chromatograms of the human hepatocytes sample incubated with T-6292 for 6 hour are shown in Figures 12. Figure 12 (i - x) displays the ion chromatograms for the following: the parent compound (m/z 570), metabolite m (m/z 746), metabolite V (m/z 584), metabolite VI (m/z 542), metabolite VIII (m/z 556), metabolite XI (m/z 498), metabolite IX (m/z 526), metabolite X (m/z 606), metabolite XII (m/z 499) and metabolite XIH (m/z 650). This sample contained the metabolites III, V, VI, Vili, IX, XI, XII and XIII. REPORT: 98AGKP01.3M 004078 ADVANCED B IO A N A L Y T IC A L S E R V IC E S , INC. 22 The SRM total ion chromatograms for metabolites IV (m/z 568), VII (m/z 540) and X were displayed in Figures 14, 15, 16 and 17. No metabolite IV was detected in the study samples (Figure 14 iii and iv). Metabolite VII was present in the human hepatocytes sample treated with T-6292 for 6 hours (Figure 15 v), but absent in the 0 hour sample (Figure 15 iv), and the no test article or no hepatocytes control samples (Figures 13 iii and 15 i). The SRM total ion chromatograms of human hepatocytes incubated with T-6292 for 0 and 6 hour are displayed in Figure 16 (iv and v), respectively, showing the presence of metabolite IX in both samples. However, this component could also be observed in the control sample containing T-6292 and no hepatocytes (Figure 16 i). Metabolite X was observed in the human hepatocytes sample incubated for 6 hours with T-6292 (Figure 17 v), but absent in the 0 hour sample (Figure 17 iv), and the no hepatocytes control sample (Figure 17 i). 3.3. Metabolism of T-6293 3.3.1. Metabolism of T-6293 by Rat Hepatocytes at 0 hour The full scan LC/MS reconstructed ion chromatograms of the rat hepatocytes sample incubated with T-6293 for 0 hour are shown in Figure 18. Figure 18 (i - x) displays the ion chromatograms for the following: the parent compound (m/z 650), metabolite II (m/z 570), metabolite III (m/z 746), metabolite V (m/z 584), metabolite VI (m/z 5.42), metabolite VIII (m/z 556), metabolite IX (m/z 526), metabolite X (m/z 606), metabolite XI (m/z 498) and metabolite XII (m/z 499). Apart from the parent drug, compound XII was detected in this sample. However compound XII was also observed in the control sample containing T-6293 and no hepatocytes (Figure 7x). 3.3.2. Metabolism of T-6293 by Rat Hepatocytes at 6 hour The full scan LC/MS reconstructed ion chromatograms of the rat hepatocytes sample incubated with T-6293 for 6 hour are shown in Figure 19. Figure 19 (i - x) displays the ion chromatograms for the following: the parent compound (m/z 650), metabolite II REPORT: 98AGKP01.3M 004079 ADVANCED B ID A N A L Y T IC A L S E R V IC E S , INC. 23 (m/z 570), metabolite III (m/z 746), metabolite V (m/z 584), metabolite VI (m/z 542), metabolite VIII (m/z 556), metabolite IX (m/z 526), metabolite X (m/z 606), metabolite XI (m/z 498) and metabolite XII (m/z 499). This sample contained metabolites III, V, VI, VIII, EX, XI and XII. SRM analyses were performed for metabolites IV (m/z 568), VII (m/z 540) and IX (m/z 526) and X (m/z 606). No metabolite IV was detected in the study samples (Figure 22 i and ii). Metabolite VII was present in the rat hepatocytes treated with T-6293 for 6 hours (Figure 23 iii), but absent in the 0 hour sample (Figure 23 ii) and the no test article or no hepatocytes control samples (Figures 13 ii and 23 i). Compound EX was found in the T-6293 standard solution and the 6 hour rat hepatocytes sample incubated with T6293 (Figure 24 i, iv and v). No metabolite X was observed in the rat hepatocytes study samples (Figure 25 ii and iii). 3.3.3. Metabolism of T-6293 by Human Hepatocytes at 0 hour The full scan LC/MS reconstructed ion chromatograms of the human hepatocytes sample incubated with T-6293 for 0 hour are shown in Figures 20. Figure 20 (i - x) displays the ion chromatograms for the following: the parent compound (m/z 650), metabolite 13 (m/z 570), metabolite III (m/z 746), metabolite V (m/z 584), metabolite VI (m/z 542), metabolite VIII (m/z 556), metabolite EX (m/z 526), metabolite X (m/z 606), metabolite XI (m/z 498) and metabolite XII (m/z 499). Apart from the parent drug, compound XII was detected in this sample, as was observed in the control sample containing T-6293 and no hepatocytes (Figure 7 x). 3.3.4. Metabolism of T-6293 by Human Hepatocytes at 6 hour The full scan LC/MS reconstructed ion chromatograms of the human hepatocytes sample incubated with T-6293 for 6 hour are shown in Figures 21. Figure 21 (i - x) displays the ion chromatograms for the following: the parent compound (m/z 650), metabolite II (m/z 570), metabolite III (tn/z 746), metabolite V (m/z 584), metabolite VI (m/z 542), metabolite VIII (m/z 556), metabolite EX (m/z 526), metabolite X (m/z 606), metabolite REPORT: 98AGKP01.3M 004030 ADVANCED BIDANALYTICAL S E R VIC ES, INC. 24 XI (m/z 498) and metabolite XII (m/z 499). This sample contained metabolites III, V, VI, VIII, IX, XI and XII. SRM analyses were performed for metabolites IV (m/z 568), VII (m/z 540), IX (m/z 526) and X (m/z 606). No metabolite IV was detected in the study samples (Figure 22 iii and iv). Metabolite VII was present in the human hepatocytes treated with T-6293 for 6 hours (Figure 23 v) but absent in the 0 hour sample (Figure 23 iv) and the no hepatocytes control sample (Figure 23 i). Metabolite IX was found in the 0 and 6 hour study samples (Figure 24 iv and v). No metabolite X was observed in the human hepatocytes study samples (Figure 25 iv and v). 3.4. Metabolism of T-6294 3.4.1. Metabolism of T-6294 by Rat Hepatocytes at 0 hour The full scan LC/MS reconstructed ion chromatograms of the rat hepatocytes sample incubated with T-6294 for 0 hour are shown in Figure 26. Figure 26 (i - vii) displays the ion chromatograms for the following: metabolite VI (m/z 542), metabolite VII (m/z 540), metabolite VIII (m/z 556), the parent compound (m/z 526), metabolite X (m/z 606), metabolite XI {m/z 498) and metabolite XII {m/z 499). Apart from the parent drug, compounds XI and XII were detected in this sample. However compound XI was also observed in the control sample containing T-6294 and no hepatocytes (Figure 8v). 3.4.2. Metabolism of T-6294 by Rat Hepatocytes at 6 hour The full scan LC/MS reconstructed ion chromatograms of the rat hepatocytes sample incubated with T-6294 for 6 hour are shown in Figure 27. Figure 27 (i - vii) displays the ion chromatograms for the following: metabolite VI {m/z 542), metabolite VII {m/z 540), metabolite VIII {m/z 556), the parent compound {m/z 526), metabolite X {m/z 606), metabolite XI {m/z 498) and metabolite XII {m/z 499). This sample contained metabolites VI, VIII, IX, XI and XH. REPORT: 98AGKP01.3M 004031 ADVANCED BIOANALYTIC AL S E R V IC E S , INC. SRM analyses were performed for metabolites VII (m/z 540) and X (m/zl506)7~No 25 metabolite VII was present in the 0 or 6 hours rat hepatocytes samples (Figure 30 ii and iii). No metabolite X was observed in the 0 or 6 hours rat hepatocytes study samples (Figure 31 ii and iii). 3.4.3. Metabolism of T-6294 by Human Hepatocytes at 0 hour The full scan LC/MS reconstructed ion chromatograms of the human hepatocytes sample incubated with T-6294 for 0 hour are shown in Figure 28. Figure 28 (i - vii) displays the ion chromatograms for the following: metabolite VI (m/z 542), metabolite VII (m/z 540), metabolite VIII (m/z 556), the parent compound (m/z 526), metabolite X (m/z 606), metabolite XI (m/z 498) and metabolite XII (m/z 499). Apart from the parent drug, compounds XI and XII were detected in this sample. However compound XI was also observed in the control sample containing T-6294 and no hepatocytes (Figure 8v). 3.4.4. Metabolism of T-6294 by Human Hepatocytes at 6 hour The full scan LC/MS reconstructed ion chromatograms of the human hepatocytes sample incubated with T-6294 for 6 hour are shown in Figure 29. Figure 29 (i - vii) displays the ion chromatograms for the following: metabolite VI (m/z 542), metabolite VII (m/z 540), metabolite VIII {m/z 556), the parent compound (m/z 526), metabolite X {m/z 606), metabolite XI (m/z 498) and metabolite XII {m/z 499). This sample contained metabolites VI, VUI, IX, XI and XII. SRM analyses were performed for metabolites VII (m/z 540) and X (m/z 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]' at m/z 746) is displayed in Figure 32 i. The base peak is at m/z 526 corresponds to the loss of -CH2CH2OGIU fragment. This negative ion mass spectrum contained a fragment ion at m/z 113, due to REPORT: 98AGKP01.3M 004032 ADVANCED B I AN ALYT ICAL S E R V IC E S , INC. internal fragmentation of the gjucuronide acid and it indicates the presence of a glucuronide conjugate. 26 Table IV: Proposed product-ion fragmentation for Metabolite VI Proposed Structure ch2--ch2oh h s o 2" 0II -s- n= ch2 0 II *s-- n =CH-CH2OH o c ,f 7C4F9' c5f ,,C6F,3 C sF ]7 m /z 542 65 92 122 169 219 269 319 419 The product-ion mass spectrum of metabolite VI is displayed in Figure 32 ii and the corresponding fragment ions are illustrated in Table IV. REPORT: 98AGKP01.3M 004033 ADVANCED BIOAN ALYTIC AL SER VIC ES, INC. Table V: Proposed product-ion fragmentation for Metabolite VIII Proposed Structure CgF-- S O -- H C K ,-C O O ' H S 0 2` fso 2- 0 II S-- N=CHCOOH O C4F9 c 5f ,,- gF -- SO-- H m /z 556 65 83 136 219 269 498 27 The product-ion mass spectrum of metabolite VIII is displayed in Figure 32 iii. The fragmentation pattern at m/z 498, 219, 169, 83 and 65 is comparable to that observed for Metabolite VI (Figure 32 ii), and the corresponding fragment ions are shown in Table V. The product-ion mass spectrum of metabolite XI is shown in Figure 33 ii. The fragment ions at m/z 478 and 78 represent a loss of HF and CgFnH moieties from the deprotonated molecule, respectively. 3.6. Positive Ionization LC/MS Analysis of Human Hepatocytes Incubated with T-6292 ANDT-6293 The human hepatocytes incubated with T-6292 and T-6293 for 6 hours were also examined by full scan LC/MS using positive ionization detection. However, the compounds of interest could not be detected in the positive ionization mode, and there were endogenous materials present in the samples at high concentration; therefore neither the parent drug nor the metabolite was observed in this analysis. REPORT: 98AGKP01.3M C040S4 PART B: ADVANCED BIOANALYTIC AL S E R V IC E S . INC. Semi-Quanutative Analysis of T-6295 in Rat and Human Hepatocytes 28 Incubated with T-6292, T-6293 and T-6294 by LC/MS/MS 4. INTRODUCTION T-6292, T-6293 and T-6294 undergo metabolic biotransformation in rat and human hepatocytes to form T-6295. The purpose of this study was to determine the concentration of T-6295 in human and rat hepatocytes after incubating with T-6292, T-6293 and T-6295, using LC/MS/MS analysis. 5. EXPERIMENTAL 5.1. Standard Solutions Preparation of Analytical Standard Solutions for T-6292, T-6293 and T-6294: See Experimental Section 2.2. Preparation of Internal Standard Stock Solution (1 mg/mL): 1H,1H,2H,2H perfluoro octane sulfonic acid Accurately weigh 2 mg of the 1H,1H,2H,2H perfluoro octane sulfonic acid on a micro balance and transfer the chemical to a polypropylene tube. Dilute with an appropriate volume of methanol to yield a 1 mg/mL solution. Store the solution at 4 C and bring to ambient temperature before use. Preparation of Internal Standard Working Solution (8 pg/mL): 1H,1H,2H,2H perfluoro octane sulfonic acid Dilute 0,8 mL of analytical standard stock solution with water in a 100 mL volumetric flask to yield a 8 pg/mL solution. Store the solution at 4 C and bring to ambient temperature before use. Preparation of Internal Standard Spiking Solution (800 ng/mL): 1H,1H,2H,2H perfluoro octane sulfonic acid (10 mL at 8 pg/mL concentration) was diluted with NANOpure water (90 mL) in a 100 mL volumetric flask. 25 pL of this solution was added to each study sample. REPORT: 98AGKP01.3M 004035 5.2'. ADVANCED B IO A N A L Y T IC A L S E R V IC E S , INC. Preparation of Quenching Solution: 29 Methanol was mixed with hepatocytes control media solution for B011-95 (supplied by 3M) in the ratio of 2:1 (v/v). 5.3. HPLC Eluent Preparation: See Experimental Section 2.3. 5.4. Sample Preparation The metabolism study was carried out at SRI International, Menlo Park, CA. The incubates were quenched with an equal volume of ice-cold methanol, centrifuged and shipped in dry ice to ABS. The samples were stored at -20 C. Hepatocytes isolated from Male Rat #2, Female Rat #5, Male Human #341 and Female Human #2, each incubated with 0.1 mM of T-6292, T-6293 and T-6294, respectively, were analysed for T-6295. Rat and Human Hepatocytes incubated with no test articles for 0 and 6 hours were used as control samples. Five stock solutions prepared in DMSO with no hepatocytes (supplied by SRI International): T-6292 from rat 2 and rat 5 studies; T-6293 from rat 2 study; T-6294 from rat 2 and rat 5 studies (10 pL each) were also used as control samples. Each sample (10 pL) containing the analytical standard was diluted with the quenching solution (90 pL), then spiked with the internal standard solution (25 pL). The internal standard solution (25 pL of 1H,1H,2H,2H perfluoro octane sulfonic acid, at 800 ng/mL concentration) was added per study sample (100 pL). 5.5. Preparation of Rat Hepatocytes Standard Curve Samples Appropriate volumes of T-6295 working solution was spiked to control rat hepatocytes according to the scheme outlined below: REPORT: 98AGKP01.3M 0040S6 ADVANCED BIOANALYTICAL S E R V IC E S . INC. 30 Plasma Standard Prep. 7 6 5 4 3 2 1 0 T-6295 Conc. 500 250 100 50 10 2 1 0 Soin # Working Soin Std#7 Std#7 Std#7 Std#4 Std#3 Std#3 0 mL added 0.025 0.150 0.048 0.048 0.096 0.048 0.024 0 Control rat Hpatocytes (mL) 0.475 0.150 0.192 0.432 0.384 0.192 0.216 0.250 Volume (mL) 0.5 0.3 0.24 0.48 0.48 0.24 0.24 0.25 One standard curve was injected at the beginning of the analytical run and a second standard curve was injected at the end of the analytical run. 5.6. LC/MS/MS Instrumentation 5.6.1. HPLC conditions HPLC Column Mobile Phase LC Gradient Flow rate Betasil C18 (2 x 100 mm) 10:90 MeOH:2mM NH4OAc (A) 90:10 MeOH:2mM NKjOAc (B) Time 0 to 2 min 2 to 4 min 4 to 7 min 7 to 8 min 200 (lL/min %B 30% 30 to 100% 100% 100 to 30% 5.6.2. MS conditions Sciex API in +TurboIonSprayTM Interface Ion Spray Voltage: -3900 V Orifice: -60 V Declustering Potential -30 V REPORT: 98AGKP01.3M 0040S7 ADVANCED BIOANALYTIC A L S E R V IC E S , INC. Ionization mode Negative Collision Gas Thickness 280 x 1013 atoms/square cm Collision Energy 35 eV Dwell Time 200 msec Transitions Monitored: m/z 499 -> 99 for T-6295 m/z 499 -> 80 for T-6295 m/z 427 -> 81 for internal standard 31 The product-ion mass spectrum of T-6295 ([M-H]* at m/z 499) produced two fragment ions at m/z 80 and 99 (see Figure 3 iii), which are likely due to ( SO3") and (FSO3') respectively. Both transitions were monitored. Data System RAD Version 2.6; LC Tune Version 2.4; Multiview Version 1.2; MacQuan Version 1.4 5.6.3. Data Handling All raw chromatographic and mass spectrometric data were stored on the ABS file server ABS1_FS.DATA in the file hierarchy "RAWDATA:Validation:3MSRI:3MSRI SemiQuan". All experimental information was stored in ABS notebook No: 2070. 6. RESULTS AND DISCUSSION: One set of male rat hepatocytes, one set of female rat hepatocytes, one set of male human hepatocytes and one set of female human hepatocyte samples were analysed by LC/MS/MS-. The. results are shown in Table VI. The same set of standard calibration curve samples was used to analyse the five stock solutions. The results are shown in Table VII. REPORT: 98AGKP01.3M 0040S8 rCi1 ADVANCED B IO A N A L Y T IC A L S E R V IC E S , INC. 32 Table VI Quantitative Analysis of T-6295 in Rat and Human Hpatocytes after Incubation with T-6292, T-6293 and T-6294 W eighted ( l/x) Intercept = Slope = C orrelation Coeff. Filenam e DB_1 H 341920 H341926 H 341930 H341936 H 341940 H341946 H 341C 920 H341C926 H341C930 H 34IC936 H341C940 H341C946 H 34IN TA 0 H 34IN TA 6 HF2920 HF2926 HF2930 HF2936 HF2940 HF2946 HF2C920 HF2C926 HF2C930 HF2C936 HF2C940 HF2C946 HF2NTA0 HF2NTA6 R2920 R2926 R2930 R2936 R2940 R 2946 R2C920 R2C926 R2C930 * 0.00193 0.00331 0.9961 Sam ple N am e Cone. (ng/m L) D ouble Blank n /a D ouble Blank n/a H 341/T 6292 t=0 n/a H 341/T6292 t=6 n/a H 341/T6293 t=0 n /a H 341/T6293 t=6 n/a H 341/T 6294 t=0 n /a H 34I/T62941=6 n /a H341 N o H ep T6292 t=0 n /a H 3 4 I N o H ep T 6 2 9 2 1=6 n /a H341 N o H ep T 6 2 9 3 t=0 n /a H341 N o H ep T6293 t=6 n /a H341 N o H ep T6294 t=0 n /a H341 No H ep T6294 t=6 n /a H341 N o T est A rticle t=0 n /a H341 No Test A rticle t=6 n /a H um an F em ale 2/T 6292 t=0 n /a H um an Fem ale 2/T62921=6 n /a H um an F em ale 2/T6293 t=0 n /a H um an Fem ale 2/T6293 t=6 n /a H um an Fem ale 2/T6294 t=0 n /a H u m a n F e m a le 2 /T 6 2 9 4 1=6 n /a H um an Fem ale 2 No H ep T6292 t=0 n /a Hum an Fem ale 2 N o H ep T62921=6 n /a H um an Fem ale 2 N o H ep T6293 t=0 n /a H um an Fem ale 2 N o H ep T6293 t=6 n /a H um an Fem ale 2 N o H ep T62941=0 n /a H um an Fem ale 2 N o Hep T62941=6 n /a H um an Fem ale 2 N o Test Article t=0 n /a H um an Fem ale 2 N o T est A rticle t=6 n /a R at M ale 2/T6292 t=0 n /a R at M ale 2/T 6 2 9 2 t=6 n /a R at M ale 2/T6293 t=0 n /a R at M ale 2/T6293 t=6 n /a R at M ale 2/T6294 t=0 R at M ale 2/T6294 t=6 n /a n /a Rat M ale 2 No H ep T6292 t=0 Rat M ale 2 N o H ep T6292 t=6 n /a n /a R at M ale 2 No H ep T6293 t=0 n /a C alc. C one. (ng/m L) n /a n /a 0.36 n/a 7.319 n /a 1.051 n /a n /a n /a 6.574 6.042 0.491 0.603 n /a n /a n /a 15.049 6.946 9 .2 9 4 6.418 5.373 n /a n /a 7.706 8.478 6.739 5.888 n /a n /a 0.993 28.785 10.621 16.526 6.168 5.439 1.041 n /a 6.714 A ccuracy 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 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 REPORT: 98AGKP01.3M 004089 ADVANCED BIOANALYTIC AL SER VIC ES, INC, Table VI Quantitative Analysis of T-6295 in Rat and Human Hepatocytes after ____________ Incubation with T-6292, T-6293 and T-6294 (continued) Filenam e Sam ple N am e C one. C alc. C one. A ccuracy (ng/m L ) (ng/m L) R2C936 R at M ale 2 N o H ep T 6293 t=6 n/a 8.454 n /a R2C940 R at M ale 2 N o H ep T6294 t=0 n/a 4.446 n /a R2C946 R at M a le 2 N o H e p T 6 2 9 4 1=6 n/a 3.615 n/a R2NTA0 Rat M ale 2 No T est Article t=0 n/a n /a n /a R2NTA6 R at M ale 2 N o T est A rticle t=6 n/a n /a n /a R5920 Rat fem ale 5/T62921=0 n/a n/a n /a R5926 Rat fem ale 5/T6292 t=6 n / a 33.934 n /a R5930 Rat fem ale 5/T6293 t=0 n / a 7.862 n /a R5936 Rat fem ale 5/T6293 t=6 n / a 14.147 n / a R5940 Rat fem ale 5/T62941=0 n / a 0.856 n /a R5946 Rat fem ale 5/T 6294t= 6 n / a 8.957 n /a R5C920 Rat fem ale 5 N o Hep T62921=0 n /a n /a n /a R5C926 R at fe m a le 5 N o H ep T 6 2 9 2 1=6 n / a 7.871 n /a R5C930 Rat fem ale 5 N o Hep T62931=0 n / a 7.777 n /a R5C936 R a t fem ale 5 N o H ep T 6293 t=6 n / a 6.96 n /a R5C940 Rat fem ale 5 N o H ep T6294 t=0 n /a n /a n /a R5C946 Rat fem ale 5 No Hep T 6294t=6 n / a 4.095 n /a R5NTA0 Rat fem ale 5 N o Test Article t=0 n /a n /a n /a R5NTA6 Rat fem ale 5 No Test A rticle t=6 n /a n /a n /a Std_l 1 n / a 11.643 n / a S td_l 2 1 1.054 105.367 Std_2 1 2 1.907 95.35 Std_2 2 2 1.732 86.576 Std_3 1 10 9 .9 8 99.799 Std_3 2 10 10.588 105.883 Std_4 1 50 57.979 115.958 Std_4 2 50 54.431 108.862 Std_5 1 100 98.732 98.732 Std_5 2 100 80.054 80.054 Std_6 1 250 259.142 103.657 Std_6 2 250 249.402 99.761 Z ero_l n /a n /a n /a Z ero _ 2 n /a n /a n /a 33 T-6295 was not detected in the control samples which contained only the hepatocytes and no test articles (Table VI). For the rat and human hepatocytes, T-6295 was detected in the control samples which were incubated with T-6293 and T-6294 but with no hepatocytes (Table VI). REPORT: 98AGKP01.3M 004090 ADVANCED BIOANALYTICAL SER VIC ES, INC. For the maie human hpatocytes study samples, T-6295 was present in the 0 hour 34 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 all the samples (Table VII). Since carryover for T-6295 was observed during sample analysis, this affects the values of the second set of standard curve, particularly the calibration standards at low concentrations. REPORT: 98AGKP01.3M 004.091 ADVANCED B IO A N A L Y T IC A L SER VIC ES, INC. Table VII Quantitative Analysis of T-6295 in T-6292, T-6293 and T-6294 Stock Solutions Weighted (1/x) Intercept = Slope = Correlation Coeff. = 0.00150 0.00359 0.9910 Filename DB_1 DB_2 R2T6292Stock R2T6293Stock R2T6294Stock R5T6292Stock R5T6294Stock Std_l 1 Std_l 2 Std_2 1 Std_2 2 Std_3 1 Std_3 2 Std_4 1 Std_4 2 Std_5 1 Std_5 2 Std_6 1 Std_6 2 Zero_l Zero_2 Cone. 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 Calc. Cone. (ng/mL) 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 97.415 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 35 7. REFERENCES : 1 D.E. Mulvana and J. Henion Qualitative investigation of the in vitro metabolism of T-6292, T-6293, T-6294 and T-6295 by rat and human hepatocytes using ion spray LC/MS and LC/MS/MS. Report No. 96ADEM01.3M November 12, 1996. 2 ABS Laboratory Notebook No. 2070 REPORT: 98AGKP01.3M 004092 ADVANCED BIDANALYTICAL S E R V IC E S , INC, Figure 1. Reconstructed ion chromatogram (i), negative ionization mass spectrum (ii) and LC/MS/MS product-ion mass spectrum (iii) of authentic T-6293. 36 XIC (period 1) from 650.0-650.5 amu from T6293 No Hep 5.3.96 (1) Z.O 1 0 70 2.16e5 cps % Intensity 650 [M-H] Spectrum from 6.84 min (27 scans) from 3M T6293 std Prod 650 (3) % Intensity 9.99e3 cps REPORT: 98AGKP01.3M 004093 ADVANCED BIOANALYTICAL SER VIC ES, INC. 37 Figure 2. Reconstructed ion chromatogram (i) and LC/MS/MS product-ion mass spectrum (ii) of authentic T-6294. TIC from 3M T6293 & T6294 Stds Prod (1) 9.60e5 cps Spectrum from 9.49 min (9 scans) from 3M T6293 & T6294 Stds Prod (1), centroided % Intensity 3.54e5 cps [M-H]'526 80- 60- () 40- 20- 65 -~r~ 60 169 219 126 i 120 180 269 240 300 362 419 360 420 480 m/z REPORT: 98AGKP01.3M 004094 ADVANCED BIOAN ALYTIC AL SER VIC ES, INC. 38 Figure 3. Reconstructed ion chromatogram (i), negative ionization mass spectrum (ii) and LC/MS/MS product-ion mass spectrum (iii) of authentic T-6295. XIC (period 1) from 499.0-499.5 amu from T-6295 Std (1) 3.55e6 cps Spectrum from 6.07 min (2 scans) from T-6295 Std (1), subtracted, subtracted, centroided 1.49e6 cps Spectrum from 6.03 min (5 scans) from T6295 Dau 499 (1), centroided % Intensity 4.92e4 cps REPORT: 98AGKP01.3M 004095 Figure 4. ADVANCED BIOAN ALYTIC AL SER VIC ES, INC. Full scan reconstructed ion chromatograms of control rat hepatocytes incubated for 6 hours with no test articles 39 XIC (period I) from 570.0-570.5 amu from B011 R4 N oT A 6h r(l) % Intensity 4.00e4 cps m/z 556.0 - 556.5 For compound VIII 3.60e4 cps m/z 499.0 -> 499.5 For Compound XU (x) 60' 30a 1 3.90 5,22 6,38 7,58 4.60e4 cps 9,34 9 Time, min REPORT: 98AGKP01.3M Figure 5 I ADVANCED I BIOANALYTICAL I S E R V IC E S , INC. 4( Full scan reconstructed ion chromatograms of control human hepalocytes incubated for 6 hours with no test articles XIC (period 1) from 570.0-570.5 amu from B011 FI No TA 6hr (2) % Intensity 1.10 7,20 2.20e4 cps 830 V 6 m/z 650.0 - 650.5 For Compound 1and XIII (ii) 60 301 Av A ./ \ m/z 746.0 -746.5 For Compound III 5,55 6.48 1.40e4 cps 8,79 2.00e4 cps 9.40 AA. m/z 584.0 - 584.5 For Compound V (iv) 60 30 m/z 542.0 - 542.5 For Compound VI 1.26 I1 1 1"i^ 1.10e5 cps 9.29 2.40e4 cps m/z 606.0 - 606.5 For Compound X (viii) 60 30 m/z 498.0 - 498.5 For Compoun XI 1,15 <*> IS 7.80e4 cps 9.18 5.66 3.60e4 cps 8.52 REPORT: 98AGKP01.3M 004097 ADVANCED BIOANALYTIC AL SER VIC ES. INC. 41 Figure 6. Full scan reconstructed ion chromatograms of T-6292 incubated for 6 hours with no hepatocytes XIC (period 1) from 570.0-570.5 amu from T6292 No Hep 5.3.96 (1) % Intensity (i) 60 30 A _ 4.30 7.21 .60e4 cps m/z 650.0 - 650.5 For Compound I and XIII 1.15 (ii) 60 30 6 22 1.40e4 cps 8 80 m/z 746.0 - 746.5 For Compound III m" 1.15 AAS~\ A- _t _ AA /|AAAAA 6.49 2.00e4 cps m/z 584.0 - 584.5 For Compound V (iv) 60 30 7.76 7.20e4 cps 1.15 (vi) 60 30 /-- m/z 526.0 - 526.5 For Compound IX / - ^a/VvAA^~ (vii) 60-1 30 m/z 606.0 - 606.5 For Compound X 9.08 4.44e5 cps 9.41 (IX) 8.91 J 8.20e4 cps REPORT: 98AGKP01.3M 004098 ADVANCED BIOANALYTICAL SER VIC ES, INC. 42 Figure 7. Full scan reconstructed ion chromatograms of T-6293 incubated for 6 hours with no hepatocytes XIC (period 1) from 570.0-570.5 amu from T6293 No Hep 5.3.96 (1) % Intensity j 15 7.20 2.00e4 cps m/z 542.0 - 542.5 For Compound VI 1.37 *i A ,v) " 1 m/z 556.0-556.5 For Compound VIII (vi) 60 30 A/--Aft/VAa m/z 526.0-526.5 For Compound IX 2.20e4 cps 6,70 7.80 2.00e4 cps 9.62 3.80e4 cps m/z 499.0 - 499.5 For Compound XII (x) 60 30---- ^ 1.65 5.72 (XII) 5.40e4 cps 9 Time, min REPORT: 98AGKP01.3M 004099 ADVANCED BIDANALYTICAL SER VIC ES, INC. 43 Figure 8. Full scan reconstructed ion chromatograms of T-6294 incubated for 6 hours with no hepatocytes XIC (period 1) from 542.0-542.5 amu from T6294 No Hep 5.3.96 (1) 2.60e4 cps 1.10 m/z 526.0 - 526.5 For Compound IX 5,44 6.70 w 9.29 ^ 3.5 le6 cps m/z 498.0 - 498.5 For Compound XI (v) 0.27 60 (vi) 1.26 3.19 8.20e4 cps T--------- i 9 Time, min REPORT: 98AGKP01.3M 004100 ADVANCED B IO A N A L Y T IC A L SE R V IC E S . INC. 44 Figure 9. Full scan reconstructed ion chromatograms showing the presence of metabolites after incubating rat hepatocytes with T-6292 at 0 hour. XIC (period 1) from 570.0-570.5 amu from 6.00e4 cps (i) () g 1.15 T ^= ---- r m/z 584.0-584.5 For Compound V 1.24e5 cps 7.75 'vi) 60 30 m/z 606.0-606.5 For Compound X (vii) m/z 498.0-498.5 For Compound XI (viii) 60 30 m/z 499.0-499.5 For Compound XII (ix) 60 30 m/z 650.0-650.5 For compound XIII 1.10 Arj h - ^ 1 ^ 5.17 (EX) 8.87 9.40e4 cps 9.31 5.66 7.31 (XI) 5.71 5.71 (XII) 7.64 2.18e5 cps ? I. rA,r 2.82e5 cps "=r- 2.20e4 cps 004101  H ADVANCED I M M I B HI l C T M I H b i o a n a l y t i c a l s e r v ic e s , in c . Figure 10. Full scan reconstructed ion chromatograms showing the presence of metabolites after incubating rat hepatocytes with T-6292 for 6 hour. XIC (period 1) from 570.0-570.5 amu from B011 R4 T6292 6 hr (2) % Intensity 2.60e4 cps j I 9 miz 650.0-650.5 (x) 60 30 6.11 (XIID 1.15e6 cps 9 Time, min REPORT: 98AGKP01.3M 004102 ADVANCED B IO A N A L Y T IC A L S E R V IC E S . INC. 46 Figure 11. Full scan reconstructed ion chromatograms showing the presence of metabolites after incubating human hepatocytes with T-6292 at 0 hr. XIC (period 1) from 570.0-570.5 amu from B011 FI T6292 Ohr (2) % Intensity ,e 3.00e4 cps 9.34 004103 ADVANCED B IO A N A L Y T IC A L SER VIC ES, INC. 47 Figure 12. Full scan reconstructed ion chromatograms showing the presence of metabolites after incubating human hepatocytes with T-6292 for 6 hour. XIC (period 1) from 570.0-570.5 amu from B011 FI T6292 6hr (2) `M n en sitv l t , 0 2.64 7.20 2.20e4 cps (i) 60 30 m/z 746.0-746.5 00 3600 6.04 (III) 5.94e5 cps m/z 584.0-584.5 ,..., 60 (m) 30. 6.26 (V) 6.58e5 cps m/z 542.0-542.5 (iv), 3600 7.75 (VI) 4.98e5 cps m/z 556.0-556.5 . , 60 (v) 3o > - A * m/z 526.0-526.5 <vi> 60 30 m/z 606.0-606.5 5.88 (VIET) -- y-W w. 7.40e4 cps 1,56e5 cps 9,40 (IX) 8.83 1.20e5 cps m/z 499.0-499.5 (ix) j o r' i 5.71 (XII) 7 1.90e5 cps 8.63 JL m/z 650.0-650.5 (x) 60 30 1 6.10 (XHD 1.76e5 cps 23456789 Time, min REPORT: 98AGKP01.3M 004104 ADVANCED BIOANALYTICAL SE R V IC E S , INC. 48 Figure 13. (i) SRM chromatograms of rat hepatocytes incubated with no test articles; (ii) SRM chromatograms of human hepatocytes incubated with no test articles; (iii) SRM chromatograms of human hepatocytes incubated with no test articles. TIC from R4 No TA 6h MRM 540 b % Intensity 3.37 1,00e2 cps TIC from B011 F) No TA 6h sample 30 MRM2 5.00el cps REPORT: 98AGKP01.3M 004105 ADVANCED B IO A N A L Y T IC A L S E R V IC E S , INC. 49 Figure 14. SRM chromatograms showing the absence of metabolite IV in (i) rat hepatocytes incubated with T-6292 at 0 hour; (ii) rat hepatocytes incubated with T-6292 for 6 hour; (iii) human hepatocytes incubated with T-6292 at 0 hour and (iv) human hepatocytes incubated with T-6292 for 6 hour. TIC from T6292 R4 Ohr m/z 568 % Intensity 2.50el cps TIC from BO11 R4 T6292 6h m/z 568 (1) 1.00e2 cps TIC from B011 Fl T6292 6h m/z 568 (1) 1.00e2 cps REPORT: 98AGKP01.3M 004106 ADVANCED BIO ANALYTIC AL S E R V IC E S , INC. 50 Figure 15. SRM total ion chromatograms showing the presence of metabolite VII in (i) T-6292 standard solution; (ii) incubation of T-6292 with rat hepatocytes at 0 hour; (iii) incubation of T-6292 with rat hepatocytes at 6 hour; (iv) incubation of T-6292 with human hepatocytes at 0 hour and (v) incubation of T-6292 with human hepatocytes at 6 hour. TIC from T6292 STD m/z 540 4.00e 1 cps TIC from T6292 R4 Ohr m/z 540 233 /i 60 (ii) 30 TIC from T6292 R4 6hr m/z 540 i TIC from T6292 FI Ohr m/z 540 2.60e2 cps 4.45e2 cps 7.87 (VII) 3.00e2 cps TIC from T6292 FI 6hr m/z 540 3.55e2 cps REPORT: 98AGKP01.3M 004107 ADVANCED BI CDANALYTIC AL SER VIC ES, INC. 51 Figure 16. SRM total ion chromatograms showing the presence of metabolite IX in (i) T-6292 standard solution; (ii) incubation of T-6292 with rat hepatocytes at 0 hour; (iii) incubation of T-6292 with rat hepatocytes at 6 hour; (iv) incubation of T-6292 with human hepatocytes at 0 hour and (v) incubation of T-6292 with human hepatocytes at 6 hour. TIC from T6292 STD m/z 526 % Intensity 7.20e2 cps TIC from T6292 R4 Ohr m/z 526 1.19e4 cps TIC from T6292 R4 6hr m/z 526 8.96e3 cps TIC from T6292 FI Ohr m/z 526 1-48e4 cps TIC from T6292 F1 6hr m/z 526 9.48e3 cps REPORT: 98AGKP01.3M 004108 ADVANCED BO ANALYTICAL SE R V IC E S , INC. 52 Figure 17. SRM total ion chromatograms showing the presence of metabolite X in (i) T-6292 standard solution; (ii) incubation of T-629 with rat hepatocytes at 0 hour; (iii) incubation of T-6292 with rat hepatocytes at 6 hour; (iv) incubation of T-6292 with human hepatocytes at 0 hour and (v) incubation of T-6292 with human hepatocytes at 6 hour. TIC from T6292 STD m/z 606 3.00el cps TIC from T6292 R4 Ohr m/z 606 0.96 3.50e2 cps TIC from T6292 F I Ohr m/z 606 60- (iv) 30- 1.05 A mh.tl TIC from T6292 FI 6hr m/z 606 3.85e2 cps g.86 (X) 6.00e2 cps REPORT: 98AGKP01.3M 004109 ADVANCED B IO A N A L Y T IC A L SER V IC E S . INC. 53 Figure 18. Full scan reconstructed ion chromatograms showing the presence of metabolites after incubating rat hepatocytes with T-6293 for 0 hour. XIC (period 1) from 650.0-650.5 amu from B011 R4 T6293 Ohr (2) % Intensity 6.15 (T-6293) 1.80e5 cps (i) 60 30 m/z 570.0-570.5 For Compound II 2.40e4 cps m/z 584.0-584.5 For Compound V (iv) 6030' m/z 542.0-542.5 For Compound VI 1.26 9.20e4 cps 9.18 T 2.00e4 cps REPORT: 98AGKP01.3M 004110 ADVANCED BIOANALVTICAL S E R V IC E S , INC. 54 Figure 19. Full scan reconstructed ion chromatograms showing the presence of metabolites after incubating rat hepatocytes with T-6293 for 6 hour. m/z 650.0-650.5 1.44e5 cps m/z 556.0-556.5 4 3O' . '^ r" I " I m/z 526.0-526.5 5 83 (VIET) 1.86e5 cps _ T-* --- V - ikV ---? i7 i iinii1 , I I--, r>w* .. 1 - ^ mmrnm m 3.20e4 cps REPORT: 98AGKP01.3M 004111 ADVANCED B IO A N A L Y T IC A L SERVICES, INC. 55 Figure 20. Full scan reconstructed ion chromatograms showing the presence of metabolites after incubating human hepatocytes with T-6293 for 0 hour. XIC (period 1) from 650.0-650.5 amu from B011 Fl T6293 0 hr (2) % Intensity 6.21 (T6293) 3.30e5 cps 60' (i) 30 m/z 570.0-570.5 For Compound II 1.10 () 60 2^ 2.53 m/z 746.0-746.5 For Compound III 1.15 A A.Aft ftAft m/z 584.0-584.5 For Compound V 6.49 A A, AA A 2.00e4 cps 9.73 2.00e4 cps 9.40 1.64e5 cps 9.23 (iv) 60 m/z 542.0-542.5 For Compound VI 1.32 \ n,m ,mr --s 2.20e4 cps 1.21 :jA, 60 (vu) ^ m/z 606.0-606.5 For Compound X (viii) 60 30 m/z 498.0-498.5 For Compound XI (ix) 60 30 5 m/z 499.0-499.5 For Compound XII 60-1 1-21 2.25 T""~' "" t 5.66 5.72 5.72 (XII) 9.34 9.80e4 cps 7.20e4 cps 8.63 8.00e4 cds 8 9 Time, min REPORT: 98AGKP01.3M 004112 n H H ADVANCED MI MmtfBlfIlHHl b S iq a n a l y t ER V IC ES. ic a l INC. 5 Figure 21. Full scan reconstructed ion chromatograms showing thepresenceof metabolites after incubating human hepatocytes with T-6293 for 6 hour. XIC (period 1) from 650.0-650.5 amu from BOI 1 FI T6293 6 hr (2) % intensity 5.28e5 cps m/z 746.0-746.5 6 05 (III) 8.00e4 cps m/z 542.0-542.5 7 5g (yi) 3.32e5 cps -- ------,--------,----- ,------,----- ------------ ,------, m/z 556.0-556.5 5.88 (VUI) 8.80e4cps m/z 498.0-498.5 For Compound XI (ix) 60 30 7.09 (XI) 5.48e5 cps m/z 499.0-499.5 For Compound XII 5.72 (XII) 7;09 (x) 60 U5 30./vVL '^ av w y v .- 345 =T^ 8 1.10e5 cps T - r" -ri 9 Time, min REPORT: 98AGKP01.3M 004113  ADVANCED PflTI-- I H B I O A N A L Y T IC A L f l 2B s e r v i c e s , i n c . 57 Figure 22. SRM total ion chromatograms showing the presence of metabolite IV in (i)' incubation of T-6293 with rat hepatocytes at 0 hour; (ii) incubation of T- 6293 with rat hepatocytes at 6 hour; (iii) incubation of T-6293 with human hepatocytes at 0 hour and (iv) incubation of T-6293 with human hepatocytes at 6 hour. TIC from T6293 R4 Ohr m/z 568 % Intensity 1.05e2 cps 8.19 TIC from B011 R4 T6293 6h m/z 568 (1) 1.50e2 cps TIC from T6293 FI Ohr m/z 568 g i9 9.00ei cps TIC from BOU Fl T6293 6h m/z 568 (1) 5.00el cps REPORT: 98AGKP01.3M 004114 ADVANCED B D A N A L Y T IC A L S E R V IC E S . INC. 58 Figure 23. SRM total ion chromatograms showing the presence of metabolite VII in (i) T-6293 standard solution; (ii) incubation of T-6293 with rat hepatocytes at 0 hour; (iii) incubation 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 m/z 540 % Intensity 60' (i) 30- DMA jAn11^MJ TIC from T6293 R4 Ohr m/z 540 2.33 3.50el cps 2.55e2 cps TIC from T6293 R4 6hr m/z 540 3.30e2 cps TIC from T6293 FI Ohr m/z 540 1.08 ii 60(iv) 30- TIC from T6293 FI 6hr m/z 540 5.78 2.45e2 cps 3.00e2 cps REPORT: 98AGKP01.3M 004115 ADVANCED BIOA N ALYTIC AL S E R V IC E S . INC. 59 Figure 24. SRM total ion chromatograms showing the presence of metabolite IX in (i) T-6293 standard solution; (ii) incubation of T-6293 with rat hepatocytes at 0 hour; (iii) incubation 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 m/z 526 7.42e4 cps 0.98 (ii) 30 L 'S. TIC from T6293 R4 6hr m/z 526 0.98 60' (iii) 30 T TIC from T6293 FI Ohr m/z 526 0.98 5.76 Aa TIC from T6293 FI 6hr m/z 526 9,27. (IX) 2.02e3 cps 8.75 9 31 (IX) 5.85e2 cps 1.29e3 cps REPORT: 98AGKP01.3M 004116 ADVANCED BIQANALYTICAL SER VIC ES, INC. 60 Figure 25. SRM total ion chromatograms showing the presence of metabolite X in (i) T-6293 standard solution; (ii) incubation of T-6293 with rat hepatocytes at 0 hour; (iii) incubation 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 m/z 606 % Intensity 4.50el cps TIC from T6293 R4 Ohr m/z 606 1.01 3.75e2 cps TIC from T6293 R4 6hr m/z 606 1.01 3.10e2 cps TIC from T6293 FI Ohr m/z 606 0.94 3.50e2 cps TIC from T6293 FI 6hr m/z 606 0.98 60 (v) 30 3.80e2 cps Y ftft 45 iiAr^n/lprwunfW'YwijLl 678 9Time, min REPORT: 98AGKP01.3M 004117 ADVANCED B ID A N A L Y T IC A L S E R V IC E S . INC. 61 Figure 26. Reconstructed ion chromatograms showing the presence of metabolites after incubating rat hepatocytes with T-6294 for 0 hour. XIC (period 1) from 542.0-542.5 amu from B011 R4 T6294 Ohr (1) For Compound VI % Intensity 2.60e4 cps 9.24 m/z 526.0 - 526.5 For Compound IX (T-6294) 3.23e6 cps 8.91 m/z 498.0 - 498.5 For Compound XI (vi) 60 ! --------- I1 i--- ----- m/z 499.0 - 499.5 For Compound XII (vii) 60; --- 7.15(XI) "T 5.72 (XIR 7.15 4.86e5 cps 1,04e5 cps 9 Time, min REPORT: 98AGKP01.3M 004118 ADVANCED BIOANALYTIC AL SER VIC ES, INC. 62 Figure 27. Reconstructed ion chromatograms showing the presence of metabolites after incubating rat hepatocytes with T-6294 for 6 hour. XIC (period 1) from 542.0-542.5 amu from B011 R4 T6294 6 hr (1) % Intensity (i) 60; 1^37 6.81 ' T "" - n - y 7.64 (VI) 6.20e4 cps t -------- r m/z 540.0-540.5 For Compound VII i>i) 60- .1.26 2.40e4 cps 9.29 m/z 556.0-556.5 For Compound VIII :iii) 6Q- 1^ t I m/z 526.0-526.5 For Compound IX (iv) 60- --------- r m/z 606.0-606.5 For Compound X 60- 1.15 T ------- --------T m/z 498.0 - 498.5 For Compound XI (vi) 60; t ----------- 1------------1------------1------------r m/z 499.0 - 499.5 For Compound XII (vii) 60 r 5.99 (VIII) 1.86e5 cps 'i..... I 3.62e6 cps (T-6294) 9.40 i --------r 1 i I I 1.12e5 cps 9.23 8.30 r 5.97e6 cps A7^31 (XI) ~i-------- fr i --------- 1--------- 1 2.28e6 cps 5.72 (XII) J L ~i--------- 1 9 Time, min REPORT: 98AGKP01.3M 004119 ADVANCED B IO A N A L Y T IC A L SER VIC ES. INC. 63 Figure 28. Reconstructed ion chromatograms showing the presence of metabolites' after incubating human hepatocytes with T-6294 for 0 hour. XIC (period 1) from 542.0-542.5 amu from B011 FI T6294 Ohr (1) % Intensity 1.37 (i) 60; 3.00e4 cps 9.13 m/z 540.0 - 540.5 For Compound VII (ii) 60- icry r i m/z 556.0 - 556.5 For Compound VIII 1.10 (iii) 60- 7.76 8.31 2.40e4 cps i i ir 5.45 6.71 3.40e4 cps 8.31 m/z 526.0 - 526.5 For Compound IX 4.52e6 cps 8.86 T-6294 (v) 60- Iiir 9.19 m/z 498.0 -498.5_For Compound XI (vi) 60 "~T 7.32 (XI) 1.82e5 cps m/z 499.0 - 499.5 For Compound XII (vii) 60- 5.73 (xn> 7.32 ----------- 1 1 i----------- 1----------- i----------- 1----------- 1----------- 1 2 34 56 7 1.06e5 cps 8.86 I----------- i----------- 8 9Time, min REPORT: 98AGKP01.3M 004120 ADVANCED BIOANALYTIC AL SER VIC ES, INC. 64 Figure 29. Reconstructed ion chromatograms showing the presence of metabolites after incubating human hepatocytes with T-6294 for 6 hour. XIC (period 1) from 542.0-542.5 amu from B011 FI T6294 6hr (1) % Intensity 3.80e4 cps 7.61 (VI) m/z 556.0 - 556.5 For Compound VIII (m) ^ 1--------- r tAtw m/z 526.0 - 526.5 For Compound IX 5.93 (VHI) 6.72 2.40e4 cps i --"----- T i--------- 1--------- 1--------- 3.3 le6 cps 8.92 (T-6294) (v) 60 m/z 498.0 - 498.5 For Compound XI (vi) 60; m/z 499.0 - 499.5 For Compound XII (v) 60- T- 4 9.14 5.68 7.33 (XI) A 3.88e6 cps 7.33 A5.73 (XH) i---V r 9.68e5 cps 9Time, min REPORT: 98AGKP01.3M 004121  H i ADVANCED W iW W IH bioanalvtical h B h SERVICES. INC. 55 Figure 3U. SKM total ion chromatograms showing the presence of metabolite VII in (i) T-6294 standard solution; (ii) incubation of T-6294 with rat hepatocytes at 0 hour; (iii) incubation of T-6294 with rat hepatocytes at 6 hour; (iv) incubation of T-6294 with human hepatocytes at 0 hour and (v) incubation of T-6294 with human hepatocytes at 6 hour. TIC from T6294 STD m/z 540 % Intensity 3.50el cps 2.33 TIC from T6294 R4 6hr m/z 540 60 (iii) 30 TIC from T6294 FI Ohr m/z 540 1.08 60 (iv) 30 *** - "^\m 2.33 r 5.20 4- TIC from T6294 FI 6hr m/z 540 60(v) 30- 12 2.33 -------- 1-- ------r 34 1,40e3 cps 2.85e2 cps 1.10e3 cps REPORT: 98AGKP01.3M 004122 ADVANCED B IO A N A L Y T IC A L S E R V IC E S , INC. 66 Figure 31- SRM total ion chromatograms showing the presence of metabolite X in (i) T-6294 standard solution; (ii) incubation of T-6294 with rat hepatocytes at 0 hour; (iii) incubation of T-6294 with rat hepatocytes at 6 hour; (iv) incubation of T-6294 with human hepatocytes at 0 hour and (v) incubation of T-6294 with human hepatocytes at 6 hour. TIC from T6294 STD m/z 606 % Intensity 4.00el cps TIC from T6294 R4 6hr m/z 606 1.01 2.90e2 cps TIC from T6294 FI Ohr m/z 606 1.03 60 (iv) 30 ---- TIC from T6294 FI 6hr m/z 606 60 (v) 0.91 ft 3.60e2 cps 3.00e2 cps 1 23456789 Time, min REPORT: 98AGKP01.3M 004123 ADVANCED BIO AN ALYTIC AL SER VIC ES, INC. 67 Figure 32. Negative ionization product-ion mass spectra of (i) metabolite III; (ii) metabolite VI and (iii) metabolite VIII. Spectrumfrom 6.24 rrin (6 scans) from 3M16292 R4 6h PROD % Intensity M etabolite m 80 60 (i) 40 20 113 169 419 526 90 180 270 360 450~ Spectrumfrom 8.48 min (39 scans) fromBOl 1FI 16293 6hr (4) % Intensity MetaboliteVI 80 601 65 (ii) 40 201 92 169 219 269 319 _L 60 120 180 240 300 Spectrumfrom 6.20 min (12 scans) fromBOl 1FI 16293 6hr % Intensity 360 80- 169 M etabolite VDI 60' (iii) 40 65 83 \ 93 201 136 219 540 419 4- 420 60 120 180 *24o" 300 360 420 5.50e4cps [M-H]~ 746 630 720 nVz 7.95e3cps [M-H] 542 480 540 nVz 3.33e3 cps [M-H] 556 498 480 540 nVz REPORT: 98AGKP01.3M 004124 ADVANCED BIDANALYTICAL SER VIC ES, INC. 68 Figure 33. Product-ion mass spectra of (i) Metabolite XIII and (ii) metabolite XI Spectrumfrom6.21 min (25 scans) from 3MT6292 R4 6h PROD65(y498 % Intensity Metabolite XQI 80 60 (i) 40 219 269 319 4{9 240 320 400 Spectrumfrom778.31 min (36 scans) from 3MT6292 R4 6h PROD65CV498 (1) 1.29e5 cps 526 [M-H] 650 480 560 640 rn/z 7.67e4 cps Metabolite XI [M-H]' 498 1i69 478 180 I " 300 360 420 480 rrYz REPORT: 98AGKP01.3M 004125 ADVANCED B IO A N A L Y T IC A L S E R V IC E S , INC. 69 Figure 34. Selected Reaction Monitoring Chromatograms from LC/MS/MS of a Calibration Standard Hepatocyte Extract (2 ng/mL T-6295) XIC (period 1) for 427.0 ->81.1 amu from Std_2 1 % Intensity 80 60 40 20- 1I 0.9 1.8 2.7 3.6 6.96e4 cps 4.88 Internal Standard -i---------- 5.4 6.3 Time,min REPORT: 98AGKP01.3M 004126