Identification of stereoisomeric metabolites of meisoindigo in rat liver microsomes by achiral and chiral liquid chromatography/tandem mass spectrometry.

N-methylisoindigotin, abbreviated as meisoindigo, has been a routine therapeutic agent in the clinical treatment of chronic myelogenous leukemia in China since the 1980s. However, information relevant to in vitro metabolism of meisoindigo is limited. In this study, in vitro stereoisomeric metabolites of meisoindigo in rat liver microsomes were identified for the first time by achiral and chiral liquid chromatography/tandem mass spectrometry, together with proton NMR spectroscopy and synchrotron infrared spectroscopy. The major in vitro phase I metabolites of meisoindigo were tentatively identified as stereoselective-reduced meisoindigo, which comprised a pair of (3-R, 3'-R) and (3-S, 3'-S) enantiomers with lower abundance, as well as another pair of (3-R, 3'-S) and (3-S, 3'-R) enantiomers with higher abundance. One type of minor in vitro metabolites was tentatively identified as stereoselective N-demethyl-reduced meisoindigo including a pair of (3-R, 3'-R) and (3-S, 3'-S) enantiomers, as well as one meso compound. Another type of minor in vitro metabolites was tentatively identified as both stereoselective and regioselective monohydroxyl-reduced meisoindigo. Based on the metabolite profiling, three parallel metabolic pathways of meisoindigo in rat liver microsomes were proposed.