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

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 1Tris-HCl buffer(pH 7.4) containing 0.5 g·L 1microsomal protein and 50 μmol·L 1mesaconitine 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.