title: Enantioselective in-line and off-line CE methods for the kinetic study on cimetidine and its chiral metabolites with reference to flavin-containing monooxygenase genetic isoforms.
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-beta-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.
authors.affiliation: Laboratory for Pharmaceutical Analysis, Department of Pharmaceutical Sciences, Katholieke Universiteit Leuven, Leuven, Belgium.
authors.forename: Xin
authors.initials: X
authors.lastname: Hai
authors.forename: Erwin
authors.initials: E
authors.lastname: Adams
authors.forename: Jos
authors.initials: J
authors.lastname: Hoogmartens
authors.forename: Ann
authors.initials: A
authors.lastname: Van Schepdael
mesh_terms: D002138:Calibration; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D002927:Cimetidine / Q000031:analogs & derivatives* / Q000032:analysis* / Q000138:chemical synthesis / Q000378:metabolism; D003840:Deoxycholic Acid / Q000737:chemistry; D019075:Electrophoresis, Capillary / Q000295:instrumentation / Q000379:methods*; D007700:Kinetics; D010105:Oxygenases / Q000378:metabolism*; D020033:Protein Isoforms / Q000378:metabolism; D013237:Stereoisomerism; D013379:Substrate Specificity
chemical_list: D020033:Protein Isoforms; D003840:Deoxycholic Acid; D002927:Cimetidine; D010105:Oxygenases; C020066:dimethylaniline monooxygenase (N-oxide forming); C034042:cimetidine sulfoxide