药学学报, 2013, 48(12): 1823-1828
引用本文:
毕云枫, 刘舒, 张瑞兴, 宋凤瑞, 刘志强. UPLC-MS/MS方法研究中乌头碱的大鼠CYP450代谢产物及途径[J]. 药学学报, 2013, 48(12): 1823-1828.
BI Yun-feng, LIU Shu, ZHANG Rui-xing, SONG Feng-rui, LIU Zhi-qiang. Metabolites and metabolic pathways of mesaconitine in rat liver microsomal investigated by using UPLC-MS/MS method in vitro[J]. Acta Pharmaceutica Sinica, 2013, 48(12): 1823-1828.

UPLC-MS/MS方法研究中乌头碱的大鼠CYP450代谢产物及途径
毕云枫1,2, 刘舒2, 张瑞兴2, 宋凤瑞2, 刘志强2
1. 吉林农业大学食品科学与工程学院, 吉林 长春 130118;
2. 中国科学院长春应用化学研究所, 长春质谱中心, 吉林 长春 130022
摘要:
采用大鼠微粒体与中乌头碱体外孵育,确定中乌头碱经大鼠肝微粒体P450代谢的主要产物。具有高分辨功能的超高效液相色谱-串联质谱联用(UPLC-MS/MS)方法用于分析鉴定中乌头碱的代谢产物。在孵育体系中发现中乌头碱原形和6种代谢产物(M1~M6),利用串联质谱进行结构表征,确定分别为中乌头碱的去甲基、去乙酰基、脱氢及羟基化产物。利用CYP450亚型酶(CYP2C、CYP1A2、CYP2D、CYP3A、CYP2E1)的特异性抑制剂,结合UPLC-MS/MS的多反应监测(MRM)方法,确定了中乌头碱在大鼠肝微粒体中的代谢途径。CYP3A为中乌头碱的主要代谢酶;各代谢产物还可由CYP2C、CYP2D所代谢。CYP2E1和CYP1A2对中乌头碱无代谢作用。
关键词:    中乌头碱      细胞色素P450      代谢      特异性抑制剂探针     
Metabolites and metabolic pathways of mesaconitine in rat liver microsomal investigated by using UPLC-MS/MS method in vitro
BI Yun-feng1,2, LIU Shu2, ZHANG Rui-xing2, SONG Feng-rui2, LIU Zhi-qiang2
1. College of Food Science and Engineering, Jilin Agricultural University, Changchun 130118, China;
2. Changchun Center of Mass Spectrometry, Changchun Institute of Applied Chemistry, Chinese Academy of Science, Changchun 130022, China
Abstract:
Mesaconitine was incubated with rat liver microsomes in vitro. The metabolites of mesaconitine in rat liver microsomes were identified by ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method with high resolution power. A typical reaction mixture of 100 mol·L-1 Tris-HCl buffer (pH 7.4) containing 0.5 g·L-1 microsomal protein and 50 μmol·L-1 mesaconitine was prepared. The above reaction mixture was divided into six groups, and the volume of each group was 200 μL. The incubation mixture was pre-incubated at 37 ℃ for 2 min and the reactions were initiated by adding NADPH generating system. After 90 min incubation at 37 ℃, 200 μL of acetonitrile was added to each group to stop the reaction. The metabolites of mesaconitine were investigated by UPLC-MS/MS method. Mesaconitine and 6 metabolites M1-M6 were found in the incubation system. The structures were characterized according to the data from MS/MS spectra and literatures. The metabolic reactions of mesaconitine in rat liver microsomes included the demethylation, deacetylation, dehydrogenation and hydroxylation. The major metabolic pathways of mesaconitine in rat liver microsomes were determined by UPLC-MS/MS on multiple reaction monitoring (MRM) mode combined with specific inhibitors of cytochrome P450 (CYP) isoforms, including α-naphthoflavone (CYP1A2), quinine (CYP2D), diethyldithiocarbamate (CYP2E1), ketoconazole (CYP3A) and sulfaphenazole (CYP2C), separately. Mesaconitine was mainly metabolized by CYP3A. CYP2C and CYP2D were also more important CYP isoforms for the metabolism reactions of mesaconitine, but CYP1A2 and CYP2E1 haven't any contribution to MA metabolism in rat liver microsomes.
Key words:    mesaconitine    cytochrome P450    metabolism    specific inhibitor probe   
收稿日期: 2013-07-01
基金项目: 国家重点基础研究发展计划(973计划)资助项目(2011CB505300,2011CB505305);国家自然科学基金资助项目(81073040,81274046).
通讯作者: 宋凤瑞
Email: songfr@ciac.ac.cn
相关功能
PDF(691KB) Free
打印本文
0
作者相关文章
毕云枫  在本刊中的所有文章
刘舒  在本刊中的所有文章
张瑞兴  在本刊中的所有文章
宋凤瑞  在本刊中的所有文章
刘志强  在本刊中的所有文章

参考文献:
[1] Chen XG, Lai YQ, Cai ZW. Simultaneous analysis of aconitine and its metabolites by liquid chromatography-electrospray ion trap mass spectrometry[J]. J Chin Mass Spectrom Soc (质谱学报), 2012, 33: 65-73.
[2] Chen PP, Zhao N, Xu XL, et al. Analysis on the metabolites of mesaconitine in the rat urine by liquid chromatography and electrospray ionization mass spectrometry[J]. Acta Pharm Sin (药学学报), 2010, 45: 1043-1047.
[3] Jin J, Chen XY, Zhang YF, et al. Simultaneous determination of erdosteine and its active metabolite in human plasma by liquid chromatography-tandem mass spectrometry with pre-column derivatization[J]. Acta Pharm Sin (药学学报), 2013, 48: 395-400.
[4] Jiang JG, Zhang XY, Zhang TH. Rapid identification 15 effective components of anti common cold medicine with MRM by LC-MS/MS[J]. Acta Pharm Sin (药学学报), 2013, 48: 94-97.
[5] Wang XY, Pi ZF, Liu WL, et al. Biotransformation of aconitum alkaloids before and after the combination of Radix Aconiti Preparata by rat intestinal Flora using semiquantitative analysis method of electrospray ionization mass spectrometry[J]. Chem J Chin Univ (高等学校化学学报), 2011, 32: 1526-1531.
[6] Chen JH, Lee CY, Liau BC, et al. Determination of aconitine-type alkaloids as markers in Fuzi (Aconitum carmichaeli) by LC/(+)ESI/MS3[J]. J Pharm Biomed Anal, 2008, 48: 1105-1111.
[7] Ioannides C, Lewis DF. Cytochromes P450 in the bioactivation of chemicals[J]. Curr Top Med Chem, 2004, 4: 1767-1788.
[8] Ghanayem BI, Hoffler U. Investigation of xenobiotics metabolism, genotoxicity, and carcinogenicity using CYP2E1 (-/-) mice[J]. Curr Drug Metab, 2007, 8: 728-749.
[9] Huttunen KM, Mahonen N, Raunio H, et al. Cytochrome P450-activated prodrugs: targeted drug delivery[J]. Curr Med Chem, 2008, 15: 2346-2365.
[10] Cedric G, Rachel B, Sarah W, et al. Inhibition of human P450 enzymes by multiple constituents of the Ginkgo biloba extract[J]. Biochem Biophys Res Commun, 2004, 318: 1072-1078.
[11] Bi YF, Zhu HB, Pi ZF, et al. Effects of flavonoides from the leaves of acanthopanax on the activity of CYP450 isozymes in rat liver microsomes by using a UPLC-MS/MS and cocktail probe substrates method[J]. Chem J Chin Univ (高等学校化学学报), 2013, 34: 1067-1071.
[12] Otten JN, Hingorani GP, Hartley DP, et al. An in vitro, high throughput, seven CYP cocktail inhibition assay for the evaluation of new chemical entities using LC-MS/MS[J]. Drug Metab Lett, 2011, 5: 17-24.
[13] Duhamel MC, Troncy E, Beaudry F. Metabolic stability and determination of cytochrome P450 isoenzymes' contribution to the metabolism of medetomidine in dog liver microsomes[J]. Biomed Chromatogr, 2010, 24: 868-877.
[14] Wang Y, Liu ZQ, Song FR, et al. Electrospray ionization tandem mass spectrometric study of the aconitines in the roots of aconite[J]. Rapid Commun Mass Spectrom, 2002, 16: 2075-2082.
[15] Ye L, Tang L, Gong Y, et al. Characterization of metabolites and human P450 isoforms involved in the microsomal metabolism of mesaconitine[J]. Xenobiotica, 2011, 41: 46-58.
[16] Wang YG, Wang SQ, Liu YX, et al. Characterization of metabolites and cytochrome P450 isoforms involved in the microsomal metabolism of aconitine[J]. J Chromatogr B Analyt Technol Biomed Life Sci 2006, 844: 292-300.
[17] Tang L, Ye L, Lü C, et al. Involvement of CYP3A4/5 and CYP2D6 in the metabolism of aconitine using human liver microsomes and recombinant CYP450 enzymes[J]. Toxicol Lett, 2011, 202: 47-54.
[18] Ameri A. The effects of aconitum alkaloids on the central nervous system[J]. Prog Neurobiol, 1998, 56: 211-235.