Abstract: AIMTo study the stereoselectivity in O-demethylation of trans tramadol. METHODSWith or without quinine and quinidine as inhibitors, rat liver microsomes were incubated in vitro with the enantiomers or the racemate of trans tramadol. The concentrations of the enantiomers of trans tramadol and O-demethyltramadol in the incubates were determined by high performance capillary electrophoresis. The O-demethylation processes were assayed by using the enzyme kinetic analysis method. RESULTSAfter incubation, the concentrations of (-)-O-demethyltramadol were higher than those of (+)-enantiomer in all rat liver microsomal incubates. Enzyme kinetic analysis showed that the K_m of the formation of the enantiomers of O-demethyltramadol were similar; The V_max and Cl_int of the formation of (-)-O-demethyltramadol were significantly higher than those of the formation of (+)-enantiomer. When the racemate of trans tramadol was used as the substrate, there was interaction between the two enantiomers. The K_m of the formation of the enantiomers of O-demethyltramadol increased, the V_max of the formation of (+)-O-demethyltramadol decreased, the V_max of the formation of (-)-O-demethyltramadol increased slightly. The O-demethylation of the enantiomers of trans tramadol was shown to be inhibited competitively by quinine and quinidine. The K_i of quinine and quinidine were 1.6 and 10.8 μmol·L^-1 to the formation of (-)-O-demethyltramadol, 0.8 and 3.4 μmol·L^-1 to the formation of (+)-O-demethyltramadol, respectively. Furthermore, quinine and quinidine were found to have stereoselective inhibition on the formation of O-demethyltramadol, both mainly inhibited the formation of (+)-O-demethyltramadol. CONCLUSIONThe O-demethylation of trans tramadol was found to be stereoselective in rat liver microsomes in vitro, preferentially metabolized (-)-enantiomer. The stereoselectivity could be influenced by the interaction between the two enantiomers and the enzyme selective inhibitors.