Stereoselective metabolism of metoprolol:: Enantioselectivity of α-hydroxymetoprolol in plasma and urine

Direct stereoselective separation on chiral stationary phase was developed for HPLC analysis of the four stereoisomers of alpha-hydroxymetoprolol in human plasma and urine. Plasma samples were prepared using solid-phase extraction columns and urine samples were prepared by liquid-liquid extraction. The stereoisomers were separated on a Chiralpak(R) AD column at 24degreesC with fluorescence detection and a mobile phase consisting of a mixture of hexane:ethanol:isopropanol:diethylamine (88:10.2:1.8: 0.2) for plasma samples and hexane:ethanol:diethylamine (88:12:0.2) for urine samples. Calibration curves for the individual stereoisomers were linear within the concentration range of 2.0-200 ng/ml plasma or 0.125-25 mug/ml urine. The methods were validated with intra- and interday variations less than 15%. The absolute configuration of the pure stereoisomers were assigned by circular dichroism spectra. The methods were employed to determine the concentrations of alpha-hydroxymetoprolol stereoisomers in a metabolism study of multiple-dose administration of racemic metoprolol to hypertensive patients phenotyped as extensive metabolizers of debrisoquine. We observed stereoselectivity in the alpha-hydroxymetoprolol formation favoring the new 1'R chiral center from both metoprolol enantiomers (AUC(1'R/1'S)(0-24) = 3.02). The similar renal clearances (Cl-R) of the four stereoisomers demonstrated absence of stereoselectivity in their renal excretion. (-)-(S)-metoprolol was slightly more alpha-hydroxylated than its antipode (AUC(2S/2R)(0-24) = 1.19), suggesting that this pathway is not responsible for plasma accumulation of this enantiomer in humans.