UPLC-MS／MS方法研究中乌头碱的大鼠CYP450代谢产物及途径

Metabolites and metabolic pathways of mesaconitine in rat liver microsomal investigated by using UPLC-MS/MS method in vitro

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一串联质谱联用（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、CYPlA2、CYP2D、CYP3A、CYP2E11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CYPlA2对中乌头碱无代谢作用。

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 tool -L l Tris-HC1 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 gL. The incubation mixture was pre-incubated at 37 ℃ for 2 rain and the reactions were initiated by adding NADPH generating system. After 90 min incubation at 37 ℃, 200 gL 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 a-naphthoflavone （CYP1A2）, quinine （CYP2D）, diethyldithiocarbamate （CYP2E1）, ketoconazole （CYP3A） and sulfaphenazole （CYP2C）, separately. Mesaconitine was mainly metabolized by CYP3A. CYP2C and CYP2D were also moreimportant CYP isoforms for the metabolism reactions of mesaconitine, but CYP1A2 and CYP2E1 haven＇t any contribution to MA metabolism in rat liver microsomes.