In-vitro metabolism of isotetrandrine, a bisbenzylisoquinoline alkaloid, in rat hepatic S9 fraction by high-performance liquid chromatography-atmospheric pressure ionization mass spectrometry

Abstract

Abstract The objective of this study was to investigate the in-vitro metabolism of isotetrandrine, a bisbenzylisoquinoline alkaloid, using rat hepatic S9 fraction and to profile and identify its metabolites using high-performance liquid chromatography-atmospheric pressure ionization mass spectrometry (HPLC-MS) and tandem mass spectrometry (MS/MS). Isotetrandrine was incubated at a concentration of 100 μg mL−1 with male rat hepatic S9 fraction in the presence of an NADPH generating system (Tris buffer, pH 7.4, 37°C). Samples were removed at 60 min after reaction initiation. Unchanged isotetrandrine (∼63% of the sample) and four metabolites were profiled, characterized and tentatively identified using solvent extraction, methyl derivatization, and HPLC-MS and MS/MS techniques. Isotetrandrine metabolites were mainly formed via two main pathways, N-demethylation and isoquinoline ring oxidation. The first pathway produced a major metabolite, N-desmethyl isotetrandrine (∼16% of the sample). The second pathway produced three minor oxidized metabolites, hydroxy-isotetrandrine (∼6% of the sample), oxo-isotetrandrine (∼7% of the sample), and oxo-hydroxy-isotetrandrine (∼7% of the sample). Diazomethane treatment of these metabolites did not produce any methyl derivatives and therefore the hydroxylated sites of the metabolites were tentatively assigned at the heterocyclic moieties of the isoquinoline rings. In conclusion, isotetrandrine is substantially metabolized in this in-vitro rat hepatic system.