Abstract
An in‐line screening and an off‐line chiral CE method were developed to determine the stereoselectivity of flavin‐containing monooxygenase (FMO) isoforms using cimetidine (CIM) as a substrate. The S‐oxygenation of CIM was investigated using achiral chemical oxidants and (human supersomes) enzymatic metabolism procedures. In the off‐line setup, the chiral selector sulfobutylether‐β‐CD was chosen to separate the CIM S‐oxide (CSO) metabolites. The electrophoretic migration order of CSO was confirmed to be (+) before (−) through the use of single enantiomers obtained by preparative chromatography. For the electrophoretically mediated microanalysis method, the in‐line enzymatic reaction was performed in 100 mM phosphate reaction buffer (pH 8.3), whereas 50 mM phosphate buffer with 30 mM chiral selector (pH 2.5) was used as a BGE. During the screening of FMO isoenzymes by the electrophoretically mediated microanalysis method, formation of the new chiral center on the CIM sulfur was found to be stereoselective. FMO1 produces more (−)‐CSO‐enantiomer, while FMO3 generates mainly (+)‐CSO‐enantiomer. On the other hand, FMO5 shows no activity. The kinetic constants of FMO1 and FMO3 were measured by the off‐line method. A K~m~=4.31 mM for the formation of the (+)‐CSO‐enantiomer and a K~m~=4.56 mM for the (−)‐CSO‐enantiomer are reported for the first time for FMO1.