药物代谢研究
摘要
药物代谢转化研究在新药研究中占有重要地位，是了解药物作用过程的重要环节，开展药物的代谢转化规律研究可确定药物代谢途径及结构-代谢-活性／毒性间相关性，为药物结构改造和设计新的剂型提供依据，并有助于了解药物代谢酶代谢机制，药物代谢酶催化活性增高的内在原因等。
本论文选择了目前我所正在深入进行研究的抗抑郁新药沉香呋喃类化合物AF-5及抗肿瘤药海南新碱衍生物HH07A进行药物代谢转化研究，为在新药研究过程中，深入进行药效，药代动力学和毒理等方面的研究提供可靠依据。
本论文分为二部分：
第一部分沉香呋喃类化合物AF-5的药物代谢转化研究。
第一章结合多离子选择性检测和无分流进样技术应用GC-MS联用仪建立了生物样品中微量和痕量AF-5的分析方法，确定AF-5在靶位器官脑中动态分布及其组织分布和排泄的特征。
第二章应用肝微粒体体外代谢法探讨AF-5的代谢，先应用高效薄层色谱法确定AF-5及其代谢物分离提取条件，然后建立GC-MS色谱法分离鉴定肝微粒体体系中AF-5及其代谢物。并探讨奎尼丁、香豆素、罂粟碱、酮康唑、红霉素选择性抑制剂，苯巴比妥钠诱导剂及底物浓度等对AF-5代谢的影响。确定AF-5代谢酶促动力学特征，结果表明：AF-5在肝微粒体体系能够代谢成初级及次级十几种代谢物，PB能诱导代谢AF-5的P450同功酶，AF-5在肝微粒体体系中代谢涉及P4502A6，P4502D6及P4503A4同功酶，并呈现多相动力学特征。
第三章根据AF-5在肝微粒体体系中代谢酶促动力学特征，优化代谢体系，并应用柱色谱、薄层色谱及半制备高效液相色谱法分离制备纯化八种代谢物，用NMR、MS、IR和UV鉴定其结构，经合成确认其中五种代谢物及其构型，并探讨AF-5在肝微粒体体系中代谢位点及生成代谢产物立体选择性特征，结果表明：双键能激活烯丙位代谢活性，其代谢物呈现部分立体选择性，2位优先代谢为α构型羟基是β构型近3倍，1’位主要代谢为R构型羟基，在9位主要是R构型代谢物。
第四章在体外代谢研究基础上建立体内生物样品中微量及痕量代谢物分离分析的GC-MS法，并结合代谢物标准品和衍生化技术，分离鉴定脑、血、胆汁和尿中代谢物，确定其结构，存在形式及其动态分布。并系统对比血、胆汁、脑及尿中代谢物特征，明确AF-5在体内代谢特征及其排泄途径，结果表明：胆汁、血、脑中代谢物相似，并与肝微粒体体系相似，尿中主要为次生代谢物。体内过程是AF-5经肝代谢后，代谢物以游离型经血传递分布，在1.5h左右脑、血等中代谢物量达到最大值，然后经胆汁、尿等排泄，存在分布和消除二相特征。AF-5主要以代谢物形式排泄，其中胆汁中代谢物主要是以结合型形式排泄，尿中代谢物一部分以结合型排泄，一部分以游离型排泄。给药方式和剂型探讨表明：相对油剂和腹腔注射口服卵磷脂时AF-5代谢程度高。此外AF-5在体内2位代谢为β构型羟基是α构型的1.5倍，呈现部分立体选择性。
第二部分海南粗榧新碱衍生物HH07A代谢转化研究
第一章先系统探讨HH07A在C_2、C_8、C_(18)键合固定相上的色谱行为，并建立分析尿、胆汁及粪等生物样品中HH07A的高效液相色谱法，确定其排泄特征。
第二章应用肝微粒体体外代谢法研究HH07A的代谢转化，建立在线同时分离检测HH07A及其代谢物的反相HPLC-DAD色谱法，分离和鉴定了肝微粒体系中HH07A的两个代谢物，并优化代谢体系，用减压色谱、薄层色谱和半制备高效液相色谱分离制备了一个代谢物，应用NMR、MS、IR和UV鉴定其结构。
第三章结合β-环糊精手相添加剂建立HH07A两异构体分离的高效毛细管电泳法，并应用该法初探HH07A在肝微粒体体系中代谢立体选择性和分离检测代谢物。
第四章在体外代谢研究基础上，结合硅醚化衍生方法建立生物体液中微量代谢物分离鉴定的GC-MS法，分离鉴定尿中四种代谢物，推测其代谢途径。
以上研究表明：合理应用HPCE、GC-MS、HPLC-DAD及TLC等现代色谱法，先探讨药物一般药代特征，然后在体外代谢体系中研究药物代谢酶促动力学特征，优化代谢体系，分离制备鉴定代谢物，在此基础上再开展体内药物代谢转化研究，能够有效地确定代谢物结构，代谢途径，排泄途径，代谢立体选择性及其存在形态等特征，为药物结构改造和剂型选择启迪思路，为确定药物药理活性及其毒副作用提供科学依据。
The metabolism of a drug has important consequences on its therapeutic efficacy or on its toxicity.The investigation of drug biotransformation therefore plays an important role in the process of new drug research .By investigating biotransformation of a drug , the structures of metabolites are identified,and metabolic pathways of the drug can also be clarified.moreover the relationship can be established between the structure.metabolism and toxicity. These are very useful to improve the drug structure and select the proper pharmaceutical preparation form . This dissertation is mainly concerned with the studies on in vitro and in vivo biotransformation of AF-5 and HH07A, two compounds with new chemical structures.The dissertation is divided into two parts . The first part is entitled'The study on in vitro and in vivo biostransformation of AF-5".Chapter1:A GC-MS method is developed for'the analysis of small amounts of AF-5 in tissue samples.and the distribution and excretion of AF-5 are investigated, moreover the concentration variation with time in rat brain is discussed in detail.Chapter 2: The in vitro metabolism of AF-5 was studied by rat hepatic microsomes system.Both GC-MS and TLC methods were developed for the on-line analysis of AF-5 and fourteen metabolites.then several factors affecting the metabolism of AF-5 in vitro such as inducer ,inhibitor. AF-5 concentration and so on were investigated in detail ,and the enzyme kinetic characteristics of metabolism of AF-5 in vitro were confirmed.Chapter 3: According to the enzyme kinetic study of metabolism of AF-5 in vitro, the constitution of the incubation system was optimized. Column chromatography .thin layer chromatography and preparative high performace liquid chromatography were then used for the isolation and purification of the eight major metabolites , and their structures were elucidated by UV.IR, MS and NMR data.The metabolic sites and stereoselectivity of AF-5 in vitro were also discussed.Chapter4:A solid-phase extraction method with Amberlite XAD-2 was developed to extract and separate the metabolites from the biological fluids of rat such as plasma .urine, bile and brain . The in vivo metabolites of AF-5 in biological fluids were investigated by GC-MS. The mass spectra of the metabolites are presented and major fragmention pathways are discussed. Furthermore .the in vivo characteristics of the metabolites such as elimination , excretion, existing form , dymamic behavior and metabolic stereoselectivity were investigated.The second part of this dissertation is entitled "The study on in vivo and in vitro biotransformation of HH07A".Chapter 1 :The chromatographic behavior of HH07A was investigated in detail on different reversed-phase media such as C2, C8, C18 bonded silica. A HPLC method for determining HH07A in biological samples was developed, and the excretion characteristics of HH07A were discussed.
Chapter 2: The in vitro metabolism of HH07A was studied by rat hepatic microsomes system, The constitution of the incubation system was optimized and a RP-HPLC- DAD method was developed for the on-line analysis of HH07A and its two metabolites. The thin layer chromatography, column chromatography and preparative HPLC were used for the isolation and purification of a major metabolite.and its structure was identified by UV,IR,MS and NMR data.
Chapter 3:A method for the separation of HH07A enantiomers by high performance capillary electrophoresis was developed. The working solution is composed of 0.04mol/LTris-H3PO4(pH=3)-0.0065mol/Lβ-cyclodextrin. The method has been used to examine the HH07A metabolism in rat hepatic microsomes system.
Chapter 4:The metabolism of HH07A In vlvo has been Investigated by GC-MS.After oral administration of HH07A, the rat urine was passed through a macroporous resin XAD-2 column, which was then eluted with methanol After hydrolyzed with glucuronidase, the eluate was extracted with dichloromethane,concentrated,TMS derivatized and analyzed with GC-MS. The mass spectra of the metabolites and their derivatives were presented.The structures of the metabolites were proposed and the in vivo metabolic pathway was discussed.
The above experimental results suggested that according to the chemical and physical characteristics of the drug, specific and sensitive chromatographic methods were first developed to detect the small amount of the drug in biological samples, and the ADME of the drug in vitro was investigated.Chromatographic technologies were used to isolate and purify the metabolites in vitro,and their structures were identified by UV,IR,MS and NMR data. On these bases, the metabolism of the drug in vivo was investigated .The metabolites in biological fluids were identified and detected by GC-MS or HPLC-DAD method.Thus the in vivo metabolic characteristics of the drug were elucidated.This is a convenient and effective way in the investigation of the biotransformation of drugs with new chemical structures.
本论文选择了目前我所正在深入进行研究的抗抑郁新药沉香呋喃类化合物AF-5及抗肿瘤药海南新碱衍生物HH07A进行药物代谢转化研究，为在新药研究过程中，深入进行药效，药代动力学和毒理等方面的研究提供可靠依据。
本论文分为二部分：
第一部分沉香呋喃类化合物AF-5的药物代谢转化研究。
第一章结合多离子选择性检测和无分流进样技术应用GC-MS联用仪建立了生物样品中微量和痕量AF-5的分析方法，确定AF-5在靶位器官脑中动态分布及其组织分布和排泄的特征。
第二章应用肝微粒体体外代谢法探讨AF-5的代谢，先应用高效薄层色谱法确定AF-5及其代谢物分离提取条件，然后建立GC-MS色谱法分离鉴定肝微粒体体系中AF-5及其代谢物。并探讨奎尼丁、香豆素、罂粟碱、酮康唑、红霉素选择性抑制剂，苯巴比妥钠诱导剂及底物浓度等对AF-5代谢的影响。确定AF-5代谢酶促动力学特征，结果表明：AF-5在肝微粒体体系能够代谢成初级及次级十几种代谢物，PB能诱导代谢AF-5的P450同功酶，AF-5在肝微粒体体系中代谢涉及P4502A6，P4502D6及P4503A4同功酶，并呈现多相动力学特征。
第三章根据AF-5在肝微粒体体系中代谢酶促动力学特征，优化代谢体系，并应用柱色谱、薄层色谱及半制备高效液相色谱法分离制备纯化八种代谢物，用NMR、MS、IR和UV鉴定其结构，经合成确认其中五种代谢物及其构型，并探讨AF-5在肝微粒体体系中代谢位点及生成代谢产物立体选择性特征，结果表明：双键能激活烯丙位代谢活性，其代谢物呈现部分立体选择性，2位优先代谢为α构型羟基是β构型近3倍，1’位主要代谢为R构型羟基，在9位主要是R构型代谢物。
第四章在体外代谢研究基础上建立体内生物样品中微量及痕量代谢物分离分析的GC-MS法，并结合代谢物标准品和衍生化技术，分离鉴定脑、血、胆汁和尿中代谢物，确定其结构，存在形式及其动态分布。并系统对比血、胆汁、脑及尿中代谢物特征，明确AF-5在体内代谢特征及其排泄途径，结果表明：胆汁、血、脑中代谢物相似，并与肝微粒体体系相似，尿中主要为次生代谢物。体内过程是AF-5经肝代谢后，代谢物以游离型经血传递分布，在1.5h左右脑、血等中代谢物量达到最大值，然后经胆汁、尿等排泄，存在分布和消除二相特征。AF-5主要以代谢物形式排泄，其中胆汁中代谢物主要是以结合型形式排泄，尿中代谢物一部分以结合型排泄，一部分以游离型排泄。给药方式和剂型探讨表明：相对油剂和腹腔注射口服卵磷脂时AF-5代谢程度高。此外AF-5在体内2位代谢为β构型羟基是α构型的1.5倍，呈现部分立体选择性。
第二部分海南粗榧新碱衍生物HH07A代谢转化研究
第一章先系统探讨HH07A在C_2、C_8、C_(18)键合固定相上的色谱行为，并建立分析尿、胆汁及粪等生物样品中HH07A的高效液相色谱法，确定其排泄特征。
第二章应用肝微粒体体外代谢法研究HH07A的代谢转化，建立在线同时分离检测HH07A及其代谢物的反相HPLC-DAD色谱法，分离和鉴定了肝微粒体系中HH07A的两个代谢物，并优化代谢体系，用减压色谱、薄层色谱和半制备高效液相色谱分离制备了一个代谢物，应用NMR、MS、IR和UV鉴定其结构。
第三章结合β-环糊精手相添加剂建立HH07A两异构体分离的高效毛细管电泳法，并应用该法初探HH07A在肝微粒体体系中代谢立体选择性和分离检测代谢物。
第四章在体外代谢研究基础上，结合硅醚化衍生方法建立生物体液中微量代谢物分离鉴定的GC-MS法，分离鉴定尿中四种代谢物，推测其代谢途径。
以上研究表明：合理应用HPCE、GC-MS、HPLC-DAD及TLC等现代色谱法，先探讨药物一般药代特征，然后在体外代谢体系中研究药物代谢酶促动力学特征，优化代谢体系，分离制备鉴定代谢物，在此基础上再开展体内药物代谢转化研究，能够有效地确定代谢物结构，代谢途径，排泄途径，代谢立体选择性及其存在形态等特征，为药物结构改造和剂型选择启迪思路，为确定药物药理活性及其毒副作用提供科学依据。
The metabolism of a drug has important consequences on its therapeutic efficacy or on its toxicity.The investigation of drug biotransformation therefore plays an important role in the process of new drug research .By investigating biotransformation of a drug , the structures of metabolites are identified,and metabolic pathways of the drug can also be clarified.moreover the relationship can be established between the structure.metabolism and toxicity. These are very useful to improve the drug structure and select the proper pharmaceutical preparation form . This dissertation is mainly concerned with the studies on in vitro and in vivo biotransformation of AF-5 and HH07A, two compounds with new chemical structures.The dissertation is divided into two parts . The first part is entitled'The study on in vitro and in vivo biostransformation of AF-5".Chapter1:A GC-MS method is developed for'the analysis of small amounts of AF-5 in tissue samples.and the distribution and excretion of AF-5 are investigated, moreover the concentration variation with time in rat brain is discussed in detail.Chapter 2: The in vitro metabolism of AF-5 was studied by rat hepatic microsomes system.Both GC-MS and TLC methods were developed for the on-line analysis of AF-5 and fourteen metabolites.then several factors affecting the metabolism of AF-5 in vitro such as inducer ,inhibitor. AF-5 concentration and so on were investigated in detail ,and the enzyme kinetic characteristics of metabolism of AF-5 in vitro were confirmed.Chapter 3: According to the enzyme kinetic study of metabolism of AF-5 in vitro, the constitution of the incubation system was optimized. Column chromatography .thin layer chromatography and preparative high performace liquid chromatography were then used for the isolation and purification of the eight major metabolites , and their structures were elucidated by UV.IR, MS and NMR data.The metabolic sites and stereoselectivity of AF-5 in vitro were also discussed.Chapter4:A solid-phase extraction method with Amberlite XAD-2 was developed to extract and separate the metabolites from the biological fluids of rat such as plasma .urine, bile and brain . The in vivo metabolites of AF-5 in biological fluids were investigated by GC-MS. The mass spectra of the metabolites are presented and major fragmention pathways are discussed. Furthermore .the in vivo characteristics of the metabolites such as elimination , excretion, existing form , dymamic behavior and metabolic stereoselectivity were investigated.The second part of this dissertation is entitled "The study on in vivo and in vitro biotransformation of HH07A".Chapter 1 :The chromatographic behavior of HH07A was investigated in detail on different reversed-phase media such as C2, C8, C18 bonded silica. A HPLC method for determining HH07A in biological samples was developed, and the excretion characteristics of HH07A were discussed.
Chapter 2: The in vitro metabolism of HH07A was studied by rat hepatic microsomes system, The constitution of the incubation system was optimized and a RP-HPLC- DAD method was developed for the on-line analysis of HH07A and its two metabolites. The thin layer chromatography, column chromatography and preparative HPLC were used for the isolation and purification of a major metabolite.and its structure was identified by UV,IR,MS and NMR data.
Chapter 3:A method for the separation of HH07A enantiomers by high performance capillary electrophoresis was developed. The working solution is composed of 0.04mol/LTris-H3PO4(pH=3)-0.0065mol/Lβ-cyclodextrin. The method has been used to examine the HH07A metabolism in rat hepatic microsomes system.
Chapter 4:The metabolism of HH07A In vlvo has been Investigated by GC-MS.After oral administration of HH07A, the rat urine was passed through a macroporous resin XAD-2 column, which was then eluted with methanol After hydrolyzed with glucuronidase, the eluate was extracted with dichloromethane,concentrated,TMS derivatized and analyzed with GC-MS. The mass spectra of the metabolites and their derivatives were presented.The structures of the metabolites were proposed and the in vivo metabolic pathway was discussed.
The above experimental results suggested that according to the chemical and physical characteristics of the drug, specific and sensitive chromatographic methods were first developed to detect the small amount of the drug in biological samples, and the ADME of the drug in vitro was investigated.Chromatographic technologies were used to isolate and purify the metabolites in vitro,and their structures were identified by UV,IR,MS and NMR data. On these bases, the metabolism of the drug in vivo was investigated .The metabolites in biological fluids were identified and detected by GC-MS or HPLC-DAD method.Thus the in vivo metabolic characteristics of the drug were elucidated.This is a convenient and effective way in the investigation of the biotransformation of drugs with new chemical structures.
引文
1.Singin A S.Masko A S ,Moldovancva L K.Chem.-Pharm. J.,1985,9:1046
2. Masko A S,Singin A S.Serebriakov N G.Chem.-Pharm. J. 1987.3:262
3.Phillips D H .Milter J A.Miller E C,et al .Cancer Research ,1981,41:176
4.Wong S H Y,Ther.Drug Monit.,1993,15(6)576
5. Hezarl M, Davis P J, Drug Metab. and Disp.,1993,21(2):259
6. Swart P J,Oelen WEM,Bruins A P ,et al ,J. Anal.Toxicol.,1994,18(2):71
7. Van't Klooster G A E ,Woutersen-Van Nijianten FAM,Kolker H J ,et al, J. Chromatogr. Biomed. Appl. ,1992,23(8):158
8. Dogteron P, Drug Metab. and Disp.,1993,21(4),699
9. Lhost G, Maton N.Laurent H ,et al,J. Pharm. Biomed.Anal.,1994,12(2):285
10 .Murthy S S,Shetty H U ,Nelson W L,Nelson W L ,et al,Biomed.Anal.,1994, 40:1637
11. Malltz D S , Nelson W L .and Shen D D.Nelson W L.et al,Drug Metab.and Disp.,1996,24(4):513
12 . Marathe P H ,Shen D D .Nelson W L,et al .Drug Metab. and Disp.,1994,22(2)237
13. Shou M G, Yang S K, Drug Metab. and Disp.,1996,24(5):595
14. Wang R W.Liu L.Cheng H Y.Drug Metab. and Disp.,1996,24(7):786
16.Rush W R ,et al ,Drug Metab. and Disp.,1987,15:571
16. Zbalda S, Brewer C F.Levine W G ,Drug Metab. and Disp.,1994,22(3):412
17.Lavrijsen K.,et al,Drug Metab. and Disp.,1990,18:704
18. Fitzpatrick F A,Murphy R C,Pharmacolog.Rev. 1988,40(4),229
19. Schmalix W A,Lang D,Schneider A,Drug Metab. and Disp.,1998,24(12):1314
20.Sewer M B ,Koop D R ,Morgan E T,Drug Metab. and Disp.,1996,24(4),401
21. Horning M G,Drur Metabolism and Drug Toxicity,Raven Press,New York,1984,P3S3
22.Sheurer J, Moore C M,J.Anal. Toxicol.,1992,16(4):264
23 . Nayor S,Kajbaf M, Lamb J H, Biol. Mass Spectrom.,1993,22(7),388
24.Steven H. Hoke,R. Graham Cooks ,J. Natural Products ,1994,57(2):277
25. Paml G.Eur. J. Drug Metab. Pharm.,1984,9:311
26.Schoun J,Dulery B,Haegele K D,Biol.Mass Spectrom.,1992,21(2),625
27.Benoit E.Jaussaud P,Besse S,et al,J Chromatogr. Biomed.Appl.,1992,583:167
28. Vanluchene E,et al, J. Chromatogr,1983,279:573
29.Machida M.Tanaka S.Nakamori K.Biomed. Chromatogr..1993,7(2):82
30.Mengeters M J B, J. Liq. Chromatogr.,1993,161:257
31.Nobilis M.Anzenbacher P, Pastera J.et al,J.Chromatogr.Biomed.Appl.,1996,68(1):143
32.Bernert J T,Powell M K!J.Anal.Toxicol..1996,20(3):179
33.Poo G K,Chul Y C,Mccague R,et al,Drug Metab.Disp.,1993,21(6):1119
34.Tomlinson A J, Benson L M,Naylers,LC-GC,1994,12(2):122
35.Trenerry V C,J. Chromatogr.,1996,718(1):217
36. Pommier F,J Chromatogr.,Biomed. Appl.,1995,674(2):155
37. Li F,J.Chromatogr.,Biiomed.Appl.,1995,675(1):277
38. Weinz C,J Chromatogr.,Biomed.Appl.,1995,674(2):287
39. Kobayashi Y,et al, Biol. Pharm.Bull.,1995,18(11):1614
40. Karas M,et al,Anal. Chem.,1988,60:2299
41. Sanwald P, David M,et al, J.Chromatogr.Biomed.Appl.,1996,678(1):53
42. Taylor W G, Haii T W, Vedres D D, Drug Metab.and Disp.,1996 23(11):1188
43. Lowry O H,et al, J. Biol. Chem.,1956,193:265
44．杨峻山，陈玉武，药学学报，1983，18：191
45．杨峻山等，药学学报，1986，21(7)：516
46. Garg D C,et al,Clin. Pharmacol. Ther.,1983,33(4):455
47．孙南君，梁晓天，药学学报，1981，16：24
48．叶玉梅，徐承熊等，药学学报，1995，30：12
49．叶玉梅，徐承熊等，药学学报，1995，30：487
50. Ariens E S,et al,Biochem. Pharmacol.,1988,37:9
51. Magang Shou,Shen K.Yang, Drug Metab.and Disp.,1996,24(5):595
52. Kyle J. Garton, Peter Yuen, Jerrold Meinwald,Kenneth E. Thummel and Evan D.Kharasch ,Drug Metab. and Disp.,1995,23(12):1426
2. Masko A S,Singin A S.Serebriakov N G.Chem.-Pharm. J. 1987.3:262
3.Phillips D H .Milter J A.Miller E C,et al .Cancer Research ,1981,41:176
4.Wong S H Y,Ther.Drug Monit.,1993,15(6)576
5. Hezarl M, Davis P J, Drug Metab. and Disp.,1993,21(2):259
6. Swart P J,Oelen WEM,Bruins A P ,et al ,J. Anal.Toxicol.,1994,18(2):71
7. Van't Klooster G A E ,Woutersen-Van Nijianten FAM,Kolker H J ,et al, J. Chromatogr. Biomed. Appl. ,1992,23(8):158
8. Dogteron P, Drug Metab. and Disp.,1993,21(4),699
9. Lhost G, Maton N.Laurent H ,et al,J. Pharm. Biomed.Anal.,1994,12(2):285
10 .Murthy S S,Shetty H U ,Nelson W L,Nelson W L ,et al,Biomed.Anal.,1994, 40:1637
11. Malltz D S , Nelson W L .and Shen D D.Nelson W L.et al,Drug Metab.and Disp.,1996,24(4):513
12 . Marathe P H ,Shen D D .Nelson W L,et al .Drug Metab. and Disp.,1994,22(2)237
13. Shou M G, Yang S K, Drug Metab. and Disp.,1996,24(5):595
14. Wang R W.Liu L.Cheng H Y.Drug Metab. and Disp.,1996,24(7):786
16.Rush W R ,et al ,Drug Metab. and Disp.,1987,15:571
16. Zbalda S, Brewer C F.Levine W G ,Drug Metab. and Disp.,1994,22(3):412
17.Lavrijsen K.,et al,Drug Metab. and Disp.,1990,18:704
18. Fitzpatrick F A,Murphy R C,Pharmacolog.Rev. 1988,40(4),229
19. Schmalix W A,Lang D,Schneider A,Drug Metab. and Disp.,1998,24(12):1314
20.Sewer M B ,Koop D R ,Morgan E T,Drug Metab. and Disp.,1996,24(4),401
21. Horning M G,Drur Metabolism and Drug Toxicity,Raven Press,New York,1984,P3S3
22.Sheurer J, Moore C M,J.Anal. Toxicol.,1992,16(4):264
23 . Nayor S,Kajbaf M, Lamb J H, Biol. Mass Spectrom.,1993,22(7),388
24.Steven H. Hoke,R. Graham Cooks ,J. Natural Products ,1994,57(2):277
25. Paml G.Eur. J. Drug Metab. Pharm.,1984,9:311
26.Schoun J,Dulery B,Haegele K D,Biol.Mass Spectrom.,1992,21(2),625
27.Benoit E.Jaussaud P,Besse S,et al,J Chromatogr. Biomed.Appl.,1992,583:167
28. Vanluchene E,et al, J. Chromatogr,1983,279:573
29.Machida M.Tanaka S.Nakamori K.Biomed. Chromatogr..1993,7(2):82
30.Mengeters M J B, J. Liq. Chromatogr.,1993,161:257
31.Nobilis M.Anzenbacher P, Pastera J.et al,J.Chromatogr.Biomed.Appl.,1996,68(1):143
32.Bernert J T,Powell M K!J.Anal.Toxicol..1996,20(3):179
33.Poo G K,Chul Y C,Mccague R,et al,Drug Metab.Disp.,1993,21(6):1119
34.Tomlinson A J, Benson L M,Naylers,LC-GC,1994,12(2):122
35.Trenerry V C,J. Chromatogr.,1996,718(1):217
36. Pommier F,J Chromatogr.,Biomed. Appl.,1995,674(2):155
37. Li F,J.Chromatogr.,Biiomed.Appl.,1995,675(1):277
38. Weinz C,J Chromatogr.,Biomed.Appl.,1995,674(2):287
39. Kobayashi Y,et al, Biol. Pharm.Bull.,1995,18(11):1614
40. Karas M,et al,Anal. Chem.,1988,60:2299
41. Sanwald P, David M,et al, J.Chromatogr.Biomed.Appl.,1996,678(1):53
42. Taylor W G, Haii T W, Vedres D D, Drug Metab.and Disp.,1996 23(11):1188
43. Lowry O H,et al, J. Biol. Chem.,1956,193:265
44．杨峻山，陈玉武，药学学报，1983，18：191
45．杨峻山等，药学学报，1986，21(7)：516
46. Garg D C,et al,Clin. Pharmacol. Ther.,1983,33(4):455
47．孙南君，梁晓天，药学学报，1981，16：24
48．叶玉梅，徐承熊等，药学学报，1995，30：12
49．叶玉梅，徐承熊等，药学学报，1995，30：487
50. Ariens E S,et al,Biochem. Pharmacol.,1988,37:9
51. Magang Shou,Shen K.Yang, Drug Metab.and Disp.,1996,24(5):595
52. Kyle J. Garton, Peter Yuen, Jerrold Meinwald,Kenneth E. Thummel and Evan D.Kharasch ,Drug Metab. and Disp.,1995,23(12):1426