IDENTIFICATION OF METABOLITES OF MEISOINDIGO FROM INTERSPECIES MICROSOMES AND IN VIVO BIOLOGICAL FLUIDS IN RATS BY LIQUID CHROMATOGRAPHY TANDEM MASS SPECTROMETRY HUANG MENG NATIONAL UNIVERSITY OF SINGAPORE 2009 IDENTIFICATION OF METABOLITES OF MEISOINDIGO FROM INTERSPECIES MICROSOMES AND IN VIVO BIOLOGICAL FLUIDS IN RATS BY LIQUID CHROMATOGRAPHY TANDEM MASS SPECTROMETRY HUANG MENG (M.Sc., PEKING UNION MEDICAL COLLEGE) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF PHARMACY NATIONAL UNIVERSITY OF SINGAPORE 2009 ACKNOWLEDGEMENTS I am deeply indebted to my supervisor Prof Ho Chi Lui, Paul for his continuous encouragement, wise advice and kind support throughout the period of my PhD study Without him, this thesis would not have been possible Furthermore, I wish to thank Dr Goh Lin Tang from Waters Asia Limited, Singapore, for his kind assistance with UPLC-QTOF experiments, as well as Dr Mohammed Bahou from Singapore Synchrotron Light Source, National University of Singapore, for his assistance with synchrotron IR experiments I am also grateful to the technical assistance rendered by laboratory officers, Ms Ng Sek Eng, Ms Ng Swee Eng, Ms Quek Mui Hong in our department Especially, I would like to express my heartfelt gratitude to my dear friends, who never failed to give me great suggestions in numerous different ways They are Dr Lin Haishu, Dr Su Jie, Dr Liu Xin, Dr Wu Jinzhu, Dr Ong Pei Shi, Dr Yau Wai Ping, Dr Lim Fung Chye Perry, Mr Zou Peng, Ms Zheng Lin, Ms Wang Chunxia, Ms Yin Min MaungMaung, Ms Nway Nway Aye, Ms Yong Hong, Ms Anahita Fathi-Azarbayjani, Mr Zhang Yaochun, Mr Ling Hui, Mr Wang Zhe, Ms Cheong Han Hui, Mr Tarang Nema, Ms Choo Qiuyi, Ms Yang Shili, Mr Li Fang Last but not least, I would like to extend my appreciation to my family for their support all along i TABLE OF CONTENTS ACKNOWLEDGEMENTS…………………………………………………………i TABLE OF CONTENTS…………………………………………………………ii SUMMARY…………………………………………………………………………vi LIST OF PUBLICATIONS………………………………………………………ix LIST OF TABLES……………………………………………………………………x LIST OF FIGURES…………………………………………………………………xi LIST OF SYMBOLS………………………………………………………………xiv CHAPTER Introduction 1.1 Fundamentals of drug metabolism……………………………………………2 1.2 LC-MS/MS in drug metabolism studies………………………………………5 1.3 Background of the anti-leukemic agent - meisoindigo………………………12 1.3.1 Discovery of meisoindigo…………………………………………13 1.3.2 Mechanism of action………………………………………………15 1.3.3 Clinical indications and side effects………………………………16 1.4 Previous work on meisoindigo metabolism and its limitations………………17 1.5 Research objectives …………………………………………………………21 CHAPTER Identification of stereoisomeric metabolites of meisoindigo in rat liver microsomes by achiral and chiral LC-MS/MS ii 2.1 Introduction…………………………………………………………………23 2.2 Materials and methods………………………………………………………24 2.2.1 Chemicals…………………………………………………………24 2.2.2 Liver microsomal preparation……………………………………25 2.2.3 Liver microsomal incubation……………………………………25 2.2.4 Achiral LC-MS/MS analysis………………………………………26 2.2.5 Isolation of metabolites by preparative HPLC and MS/MS/MS…29 2.2.6 Chiral LC-MS/MS analysis………………………………………29 2.2.7 Metabolite synthesis and purification……………………………30 2.2.8 NMR spectroscopy and Synchrotron IR spectroscopy……………31 2.2.9 Comparative study on incubations under normoxic and hypoxic conditions…………………………………………………………31 2.3 Results…………………………………………………………………33 2.3.1 Protonated metabolites at m/z 279………………………………36 2.3.2 Protonated metabolites at m/z 265………………………………43 2.3.3 Protonated metabolites at m/z 295………………………………45 2.3.4 Incubations under normoxic and hypoxic conditions…………51 2.4 Discussion…………………………………………………………………52 CHAPTER Metabolism of meisoindigo in rat, pig and human liver microsomes by UFLC-MS/MS 3.1 Introduction…………………………………………………………………58 3.2 Materials and methods……………………………………………………60 iii 3.2.1 Chemicals…………………………………………………………60 3.2.2 Liver microsomal preparation and origin…………………………61 3.2.3 Liver microsomal incubations…………………………………62 3.2.4 UFLC-MS/MS analysis……………………………………………63 3.2.5 Metabolite identification…………………………………………64 3.2.6 Metabolic stability calculations and statistical analysis…………65 3.2.7 Metabolite formation………………………………………………66 3.3 Results………………………………………………………………………66 3.3.1 Metabolite identification…………………………………………66 3.3.2 Metabolic stability………………………………………………77 3.3.3 Metabolite formation………………………………………………80 3.4 Discussion…………………………………………………………………82 CHAPTER Characterization of metabolites of meisoindigo in rat kidney microsomes by HPLC-MS/MS 4.1 Introduction…………………………………………………………………88 4.2 Materials and methods………………………………………………………89 4.2.1 Chemicals…………………………………………………………89 4.2.2 Preparation of rat kidney microsomes……………………………90 4.2.3 Metabolite identification…………………………………………90 4.2.4 Metabolic stability and metabolite formation……………………93 4.3 Results and discussion………………………………………………………95 4.3.1 Metabolite identification…………………………………………95 iv 4.3.2 Metabolic stability and metabolite formation……………………104 CHAPTER Identification of circulatory and excretory metabolites of meisoindigo in rat plasma, urine and feces by HPLC-MS/MS 5.1 Introduction…………………………………………………………………109 5.2 Materials and Methods……………………………………………………110 5.2.1 Chemicals……………………………………………………110 5.2.2 Rat plasma, urine, feces collection and sample preparation……111 5.2.3 Chromatography and mass spectrometry conditions for rat plasma and feces………………………………………………………113 5.2.4 Chromatography and mass spectrometry conditions for rat urine……………………………………………………………116 5.3 Results and Discussion……………………………………………………120 5.3.1 Circulatory metabolites in rat plasma……………………………120 5.3.2 Excretory metabolites in rat urine………………………………125 5.3.3 Excretory metabolites in rat feces………………………………134 CHAPTER Conclusions…………………………………………………………139 BIBLIOGRAPHY…………………………………………………………………146 APPENDICES……………………………………………………………………158 v SUMMARY Meisoindigo has been a routine therapeutic agent in the clinical treatment of chronic myelogenous leukemia in China since the 1980s However, information relevant to the metabolism of meisoindigo is limited In this thesis, in vitro metabolism studies were firstly carried out in rat, pig and human liver microsomes of different genders by LC-MS/MS The qualitative metabolite identification was accomplished by integration of MRM with conventional full MS scan followed by MS/MS methodology, together with chiral chromatography, proton NMR spectroscopy and synchrotron infrared spectroscopy The semi-quantitative metabolic stability and metabolite formation were simultaneously measured by MRM The in vitro metabolic pathways of meisoindigo in three species were proposed as stereoselective 3,3’ double bond reduction, stereoselective reduction followed by N-demethylation, both stereoselective and regioselective reduction followed by phenyl mono-oxidation, and regioselective phenyl mono-oxidation Besides, N-demethylation was another in vitro metabolic pathway in only pig and human In particular, two metabolites undergone reduction followed by phenyl mono-oxidation at positions 4, 5, or 7, as well as one metabolite undergone phenyl mono-oxidation were found to be uniquely present in human The in vitro t1/2 and in vitro CLint values of meisoindigo were calculated Statistical analysis showed there were no significant vi differences in the metabolic stability profiles of meisoindigo among three species, and gender effect on the metabolic stability of meisoindigo was negligible Formation profiles of the most significant reductive metabolites were obtained in the three species Secondly, in vitro extrahepatic metabolism of meisoindigo in rat kidney microsomes was qualitatively and quantitatively investigated by LC-MS/MS The major metabolic pathways in rat kidney microsomes were proposed as 3,3’ double bond reduction, whereas the minor metabolic pathway was phenyl mono-oxidation The in vitro half-life (t1/2) values of meisoindigo in male and female rat kidney microsomes were calculated, respectively There were no statistically significant gender differences in the metabolic stability profiles of meisoindigo The reductive metabolite formation profiles of meisoindigo in male and female rat kidney microsomes were plotted semi-quantitatively Thirdly, in vivo circulatory metabolites of meisoindigo in male rat plasma, as well as excretory metabolites in male rat urine and feces were identified by LC-MS/MS The major metabolic pathway in rat plasma was proposed as 3,3’ double bond reduction, whereas the minor metabolic pathways were reduction followed by N-demethylation, and reduction followed by phenyl mono-oxidation The major metabolic pathways in the rat urine were proposed as reduction followed by phenyl mono-oxidation, and its glucuronide conjugation and sulfate conjugation, whereas the minor metabolic pathways were 3,3’ double bond reduction, N-demethylation, reduction followed by N-demethylation, phenyl di-oxidation, phenyl mono-oxidation and its glucuronide vii conjugation and sulfate conjugation The major metabolic pathway in the rat feces was proposed as reduction followed by phenyl mono-oxidation, whereas the minor metabolic pathways were reduction followed by N-demethylation, and reduction followed by phenyl di-oxidation The phase I metabolic pathways showed a significant in vitro-in vivo correlation in rat This study shed new light in the metabolic profiles of meisoindigo in rat, pig and human The findings may allow clinicians and researchers better understanding on the biopharmaceutical properties of meisoindigo viii and nonspecific binding to microsomes Drug Metab Dispos 27: 1350-1359 Obach RS, Baxter JG, Liston TE, Silber BM, Jones BC, MacIntyre F, Rance DJ and Wastall P (1997) The prediction of human pharmacokinetic parameters from preclinical and in vitro metabolism data J Pharmacol Exp Ther 283: 46-58 Ohashi N, Furuuchi S and Yoshikawa M (1998) Usefulness of the hydrogen-deuterium exchange method in the study of drug metabolism using liquid chromatography-tandem mass spectrometry J Pharm Biomed Anal 18: 325-334 Peng Y and Wang MZ (1990) Screening for in vitro metabolites of meisoindigo using RP-HPLC-DAD with gradient elution Acta Pharmaceutica Sinica 25: 208-214 Prakash C, Shaffer CL and Nedderman A (2007) Analytical strategies for identifying drug metabolites Mass Spectrom Rev 26: 340-369 Pritchard JF, Jurima-Romet M, Reimer MLJ, Mortimer E, Rolfe B and Cayen MN (2003) Making better drugs: decision gates in non-clinical drug development Nature Rev Drug Discov 2: 542-553 Robb DB, Covey TR and Bruins AP (2000) Atmospheric pressure photoionization: an ionization method for liquid chromatography-mass spectrometry Anal Chem 72: 3653 Seglen PO (1976) Preparation of isolated rat liver cells Methods Cell Biol 13: 29-83 Sharifi N and Steinman RA (2002) Targeted chemotherapy: chronic myelogenous leukemia as a model J Mol Med 80: 219-232 Shevchenko A, Chernushevich I, EnsW, Standing KG, Thomson B,Wilm M and Mann 154 M (1997) Rapid ‘de novo’ peptide sequencing by a combination of nanoelectrospray, isotopic labeling and a quadrupole/time-of-flight mass spectrometer Rapid Commun Mass Spectrom 11: 1015-1024 Shou WZ, Magis L, Li AC, Naidong W and Bryant MS (2005) A novel approach to perform metabolite screening during the quantitative LC-MS/MS analyses of in vitro metabolic stability samples using a hybrid triple-quadrupole linear ion trap mass spectrometer J Mass Spectrom 40: 1347-1356 Sinz MW and Podoll T (2002) The mass spectrometer in drug metabolism, In Mass Spectrometry in Drug Discovery, (RossiDT, SinzMW, eds) Marcel Dekker: New York, pp 271–336 Steinman RA (2002) Cell cycle regulators and hematopoiesis Oncogene 21: 3403-3413 Van de Kerkhof EG, de Graaf IAM, de Jager MH and Groothuis GMM (2007) Induction of phase I and II drug metabolism in rat small intestine and colon in vitro Drug Metab Dispos 35: 898-907 Wang LG, Liu XM and Chen RH (2003) Derivatives of isoindigo, indigo and indirubin and use in treating cancer PCT Int Appl WO03/051900 A1 Weidolf L and Covey TR (1992) Studies on the metabolism of omeprazole in the rat using liquid chromatography/ionspray mass spectrometry and the isotope cluster technique with [34S]omeprazole Rapid Commun Mass Spectrom 6: 192-196 Williams RL, Brater DC and Mordenti J (1990) Rational therapeutics: a clinical pharmacologic guide for the health professional pp 104 Informa Healthcare 155 Wu KM, Zhang MY, Fang Z and Huang L (1984) Synthesis of N1-substituted derivatives of indirubin, an antileukemic compound Acta Pharm Sin 19: 513-518 Wu KM, Zhang MY, Fang Z and Huang L (1985) Potential antileukemic agents, synthesis of derivatives of indirubin, indigo, and isoindigotin Acta Pharm Sin 20: 821-826 Xiao ZJ, Hao YS, Liu BC and Qian LS (2002) Indirubin and meisoindigo in the treatment of chronic myelogenous leukemia in China Leuk Lymphoma 43: 1763-1768 Xiao ZJ, Qian LS, Liu BC and Hao YS (2000) Meisoindigo for the treatment of chronic myelogenous leukemia Br J Haematol 111: 711-712 Xiao ZJ, Wang Y, Lu L, Li ZJ, Peng Z, Han ZC and Hao YS (2006) Anti-angiogenesis effects of meisoindigo on chronic myelogenous leukemia in vitro Leuk Res 30: 54-59 Xu X, Ziegler RG, Waterhouse DJ, Saavedra JE and Keefer LK (2002) Stable isotope dilution high-performance liquid chromatography-electrospray ionization mass spectrometry method for endogenous 2- and 4-hydroxyestrones in human urine J Chromatogr B Anal Technol Biomed Life Sci 780: 315-330 Zhang SX (1982) Studies on the chemical constituents of Isatis indigotina root Chin Trad Herb Drugs 11: 247-248 Zheng QT, Lu DJ and Yang SL (1979) Pharmacological studies of indirubin I Antitumor effect Comm Chin Herb Med 10: 35-39 Zheng QT, Qi SB and Cheng ZY (1979) Pharmacologic studies of indirubin II 156 Absorption distribution and excretion of 3H-indirubin Comm Chin Herb Med 10: 19-21 Zuber R, Anzenbacherova E and Anzenbacher P (2002) Cytochromes P450 and experimental models of drug metabolism J Cell Mol Med 6: 189-198 157 APPENDICES APPENDIX UPLC-QTOF RESULTS FOR MEISOINDIGO AND ITS IN VITRO METABOLITES IN MALE RAT LIVER MICROSOMES Control Control Sample Sample Appendix 1-1 Base Peak Intensity (BPI) and extracted ion chromatogram (XIC) of control and sample for protonated meisoindigo at accurate m/z 277.0977 158 Control Control Sample Sample Appendix 1-2 Base Peak Intensity (BPI) and extracted ion chromatogram (XIC) of control and sample for protonated metabolites at accurate m/z 279.1134 Control Control Sample Sample Appendix 1-3 Base Peak Intensity (BPI) and extracted ion chromatogram (XIC) of control and sample for protonated metabolites at accurate m/z 265.0977 159 Control Control Sample Sample Appendix 1-4 Base Peak Intensity (BPI) and extracted ion chromatogram (XIC) of control and sample for protonated metabolites at accurate m/z 295.1083 160 APPENDIX MS/MS/MS (MS3) RESULTS FOR MEISOINDIGO AND ITS IN VITRO METABOLITES +MS3 (277.10),(218.10): 32 MCA scans from Sample (MS3-218-CE-47-AF2-40) of MS3-218-CE-47-AF2-40.wiff (Turbo Spray) Max 1.0e6 cps 218.3 1.03e6 1.00e6 9.50e5 9.00e5 8.50e5 8.00e5 7.50e5 7.00e5 6.50e5 6.00e5 5.50e5 5.00e5 4.50e5 4.00e5 3.50e5 3.00e5 2.50e5 190.2 2.00e5 219.8 1.50e5 1.00e5 217.2 221.2 5.00e4 50 60 70 80 90 100 110 120 130 140 150 160 170 m/z, amu 180 190 200 210 220 230 240 250 260 270 280 Appendix 2-1 MS3 spectrum of the product ion at m/z 218 generated from protonated meisoindigo at m/z 277 +MS3 (277.10),(234.20): 32 MCA scans from Sample (MS3-234-CE-45-AF2-45) of MS3-234-CE-45-AF2-45.wiff (Turbo Spray) Max 1.5e7 cps 206.2 1.5e7 1.5e7 1.4e7 1.4e7 1.3e7 1.3e7 1.2e7 1.2e7 1.1e7 1.1e7 1.0e7 9.5e6 9.0e6 8.5e6 8.0e6 7.5e6 7.0e6 6.5e6 6.0e6 5.5e6 5.0e6 4.5e6 4.0e6 234.2 3.5e6 205.2 3.0e6 2.5e6 216.1 2.0e6 207.6 1.5e6 179.3 1.0e6 165.1 5.0e5 50 60 70 80 90 100 110 120 130 146.0 140 178.3 191.2 204.0 152.2 150 160 170 m/z, amu 180 219.1 208.2 190 200 210.1 210 233.2 217.2 220 235.7 238.2 230 240 250 260 270 280 Appendix 2-2 MS3 spectrum of the product ion at m/z 234 generated from protonated meisoindigo at m/z 277 161 +MS3 (279.10),(147.10): 32 MCA scans from Sample (MS3-147-CE-41-AF2-25) of MS3-147-CE-41-AF2-25.wiff (Turbo Spray) Max 3.7e5 cps 118.0 3.6e5 3.4e5 3.2e5 147.2 3.0e5 2.8e5 2.6e5 2.4e5 2.2e5 66.4 2.0e5 1.8e5 68.6 1.6e5 1.4e5 1.2e5 1.0e5 68.273.6 76.2 78.0 146.2 8.0e4 79.2 6.0e4 4.0e4 50 60 70 80 90 100 110 120 130 140 150 160 170 m/z, amu 180 190 200 210 220 230 240 250 260 270 280 Appendix 2-3 MS3 spectrum of the product ion at m/z 147 generated from protonated metabolites at m/z 279 +MS3 (279.10),(251.10): 32 MCA scans from Sample (MS3-251-CE-23-AF2-25) of MS3-251-CE-23-AF2-25.wiff (Turbo Spray) Max 6.3e5 cps 233.3 6.3e5 6.0e5 223.2 5.5e5 5.0e5 4.5e5 220.2 4.0e5 3.5e5 3.0e5 2.5e5 236.3 2.0e5 249.4 1.5e5 134.3 251.3 158.2 1.0e5 222.2234.6 238.7 5.0e4 50 60 70 80 90 100 110 120 130 140 150 160 170 m/z, amu 180 190 200 210 220 230 240 250 260 270 280 Appendix 2-4 MS3 spectrum of the product ion at m/z 251 generated from protonated metabolites at m/z 279 162 APPENDIX HPLC-MS/MS PROFILE OF HYDROGENATION PRODUCTS 11.60 4.3e9 4.2e9 12.54 Synthetic products 4.0e9 3.8e9 3.6e9 3.4e9 3.2e9 M1+M2 (M279-1) 3.0e9 2.8e9 M3+M4 (M279-2) 2.6e9 2.4e9 2.2e9 15.40 2.0e9 1.8e9 1.6e9 1.4e9 1.2e9 13.97 1.0e9 14.50 8.0e8 6.0e8 11.27 4.0e8 10.40 2.0e8 0.0 10 11 12 13 14 Time, 15 14.80 2.0e8 Rat microsomal sample 1.9e8 16 17 18 19 20 21 22 23 24 25 23 24 25 15.10 12.51 1.8e8 1.7e8 1.6e8 M3+M4 (M279-2) 1.5e8 1.4e8 1.3e8 1.2e8 17.92 1.1e8 16.52 1.0e8 11.53 17.54 M1+M2 (M279-1) 9.0e7 8.0e7 14.30 13.00 7.0e7 20.41 21.06 6.0e7 5.0e7 4.0e7 9.93 3.0e7 10.73 2.0e7 1.0e7 0.0 10 11 12 13 14 Time, 15 16 17 18 19 20 21 22 Appendix 3-1 Total ion chromatograms (TIC) of EMS for synthetic product and rat liver microsomal sample Diluted hydrogenation products after two hours reaction showed the same retention times and fragmentation patterns on LC-MS/MS as the two metabolites at m/z 279 from rat microsomal sample 163 APPENDIX HPLC-MS/MS RESULTS OF FULL SCAN SPECTRA (EMS) AND UV CHROMATOGRAMS AT 254 NM FOR THREE MALE SPECIES TIC of +EMS: from Sample (40-3-S) of 40-3-S.wiff (Turbo Spray) Max 2.0e8 cps 2.0e8 Parent 12.51 1.8e8 Rat EMS 1.6e8 1.2e8 1.0e8 Interference M279-2 Minor metabolites 1.4e8 14.80 15.10 M279-1 11.53 15.67 14.30 8.0e7 13.00 6.0e7 4.0e7 10.73 9.93 2.0e7 0.0 8.0 8.5 9.0 9.5 10.0 10.5 11.0 11.5 12.0 Time, 12.5 13.0 13.5 Channel from Sample (40-3-S) of 40-3-S.wiff -84.0 -84.5 Rat UV 14.5 15.0 15.5 16.0 Max -24.2 % Minor metabolites -85.0 14.0 Parent M279-1 M279-2 -85.5 -86.0 -86.5 8.0 8.5 9.0 9.5 10.0 10.5 11.0 11.5 12.0 Time, 12.5 13.0 13.5 14.0 14.5 15.0 15.5 Appendix 4-1 EMS and UV for male rat liver microsomal samples after incubation for 60 with substrate concentration of 50 uM 164 16.0 TIC of +EMS: from Sample (40-P-S) of 40-P-S.wiff (Turbo Spray) Max 2.2e8 cps 15.05 12.47 2.2e8 2.0e8 Pig EMS 1.8e8 14.76 Parent M279-2 Interference 1.6e8 1.4e8 M279-1 1.2e8 1.0e8 11.51 8.0e7 6.0e7 4.0e7 2.0e7 0.0 8.0 8.5 9.0 9.5 10.0 10.5 11.0 11.5 12.0 Time, 12.5 13.0 13.5 14.0 14.5 15.0 15.5 Channel from Sample (40-P-S) of 40-P-S.wiff M279-1 Pig UV -96.2 Parent M279-2 -95.8 -96.0 16.0 Max -14.8 % -96.4 -96.6 -96.8 -97.0 -97.2 8.0 8.5 9.0 9.5 10.0 10.5 11.0 11.5 12.0 Time, 12.5 13.0 13.5 14.0 14.5 15.0 15.5 16.0 Appendix 4-2 EMS and UV for male pig liver microsomal samples after incubation for 60 with substrate concentration of 50 uM TIC of +EMS: from Sample (40-H-S) of 40-H-S.wiff (Turbo Spray) Max 3.1e8 cps 14.75 3.0e8 M279-2 2.5e8 2.0e8 Human EMS Parent Interference 12.51 1.5e8 M279-1 1.0e8 12.89 13.43 13.97 11.49 5.0e7 0.0 8.0 8.5 9.0 9.5 10.0 10.5 11.0 11.5 12.0 Time, 12.5 13.0 13.5 14.0 14.5 15.0 15.5 Channel from Sample (40-H-S) of 40-H-S.wiff -88.0 Parent -88.5 -89.0 16.0 Max -8.9 % Human UV -89.5 -90.0 M279-1 -90.5 M279-2 -91.0 -91.5 -92.0 -92.5 8.5 9.0 9.5 10.0 10.5 11.0 11.5 12.0 Time, 12.5 13.0 13.5 14.0 14.5 15.0 15.5 16.0 Appendix 4-3 EMS and UV for male human liver microsomal samples after incubation for 60 with substrate concentration of 50 uM 165 APPENDIX HPLC-MS/MS RESULTS OF CIRCULATORY AND EXCRETORY METABOLITES OF MEISOINDIGO IN RAT PLASMA, URINE AND FECES 20.47 1.9e4 1.8e4 1.7e4 1.6e4 1.5e4 1.4e4 1.3e4 19.72 1.2e4 1.1e4 1.0e4 9000.0 8000.0 7000.0 6000.0 5000.0 4000.0 3000.0 2000.0 1000.0 21.43 0.0 10 11 12 13 Ti 14 15 16 17 18 19 20 21 24.09 22 23 24 25 26 i Appendix 5-1 Extracted ion chromatogram (XIC) of MRM transition 279.1→147.1 for circulatory metabolites of meisoindigo in rat plasma at h postdose 147.2 295.1 550 500 450 400 350 375.1 300 267.1 250 200 240.2 150 100 148.3 50 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 / Appendix 5-2 EPI mass spectra of protonated excretory metabolites of meisoindigo at m/z 375 in rat urine collected from to 24 h postdose 166 420 22.60 5200 5000 4800 4600 4400 4200 4000 3800 3600 21.27 3400 3200 3000 2800 2600 2400 2200 2000 1800 1600 1400 1200 1000 17.93 800 600 400 14.56 200 10 12 14 15.57 16 Time, 25.47 18 20 22 24 26 28 30 Appendix 5-3 Extracted ion chromatogram (XIC) of MRM transition 279.1→147.1 for excretory metabolites of meisoindigo in rat urine collected from to 24 h postdose 21.39 9840 9500 9000 8500 8000 7500 7000 6500 6000 5500 5000 4500 4000 19.25 3500 3000 2500 2000 1500 1000 500 20.94 10 12 14 16 Time 18 20 22 24 26 28 30 Appendix 5-4 Extracted ion chromatogram (XIC) of MRM transition 265.1→133.1 for excretory metabolites of meisoindigo in rat urine collected from to 24 h postdose 167 15.22 7293 7000 6500 6000 5500 5000 4500 4000 3500 3000 2500 13.71 2000 1500 12.38 1000 12.04 500 14.84 10 Ti 11 12 13 14 16.65 15 16 17 18 19 20 i Appendix 5-5 Extracted ion chromatogram (XIC) of MRM transition 265.1→133.1 for excretory metabolites of meisoindigo in rat feces collected from to 48 h postdose 168 .. .IDENTIFICATION OF METABOLITES OF MEISOINDIGO FROM INTERSPECIES MICROSOMES AND IN VIVO BIOLOGICAL FLUIDS IN RATS BY LIQUID CHROMATOGRAPHY TANDEM MASS SPECTROMETRY HUANG MENG (M.Sc., PEKING... profile was almost absent in the literatures and therefore, the in vitro and in vivo metabolism of meisoindigo were evaluated in this thesis Before introducing the background of meisoindigo in. .. excretory 119 metabolites in rat urine H-NMR data for meisoindigo, M1+M2, and M3+M4 meisoindigo 42 and its 115 x LIST OF FIGURES FIG 1-1 Chemical structures of indirubin and meisoindigo 14 FIG