In vitro enantioselective human liver microsomal metabolism and prediction of in vivo pharmacokinetic parameters of tetrabenazine by DLLME-CE

A new capillary electrophoresis method for the enantioselective analysis of cis- and trans- dihydrotetrabenazine (diHTBZ) after in vitro metabolism by human liver microsomes (HLMs) was developed. The chiral electrophoretic separations were performed by using tris-phosphate buffer (pH 2.5) containing 1% (w/v) carboxymethyl-β-CD as background electrolyte with an applied voltage of +15 kV and capillary temperature kept at 15 °C. Dispersive liquid-liquid microextraction was employed to extract the analytes from HLMs. Dichloromethane was used as extraction solvent (75 μL) and acetone as disperser solvent (150 μL). The method was validated according to official guidelines and showed to be linear over the concentration range of 0.29-19.57 μmol L−1 (r = 0.9955) for each metabolite enantiomer. Within- and between-day precision and accuracy evaluated by relative standard deviation and relative error were lower than 15% for all enantiomers. The stability assay showed that the analytes kept stable under handling, storage and in metabolism conditions. After method validation, an enantioselective in vitro metabolism and in vivo pharmacokinetic prediction was carried out. This study showed a stereoselective metabolism and the observed kinetic profile indicated a substrate inhibition behavior. DiHTBZ enantiomers were catalyzed mainly by CYP2C19 and the predicted clearance suggests that liver metabolism is the main route for TBZ elimination which supports the literature data.