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 C18, 3-micron (100 x 3.2 mm) column. The intra- and inter-assay relative standard deviation (R.S.D.) was below 10%. Michaelis-Menten parameters, K(m) and Vmax were obtained for the formation of both D- and L-hydroxyamphetamine from D-, L- and DL-amphetamine in the concentration range of 10-350 microM.