Investigation of stereoselective metabolism of amphetamine in rat liver microsomes by microdialysis and liquid chromatography with precolumn chiral derivatization

The utility of microdialysis as a quantitative sampling technique for in vitro drug metabolism studies was demonstrated by investigating the stereoselective metabolism of d-, l- and dl-amphetamine by the cytochrome P-450 enzymes. Microdialysates containing the isomers of amphetamine and its metabolite were derivatized with the fluorescent chiral derivatizing agent, (−)-fluorenylethyl chloroformate. The diastereoisomers were isocratically separated by liquid chromatography (LC) on a reversed-phase C 18, 3-μm (100×3.2 mm) column. The intra- and inter-assay relative standard deviation (R.S.D.) was below 10%. Michaelis–Menten parameters, K m and V max were obtained for the formation of both d- and l-hydroxyamphetamine from d-, l- and dl-amphetamine in the concentration range of 10–350 μ M.