Assessment of the N-oxidation of deprenyl, methamphetamine, and amphetamine enantiomers by chiral capillary electrophoresis: an in vitro metabolism study.
A chiral capillary electrophoresis method using hydroxypropyl-beta-cyclodextrin as chiral selector was developed and validated for the quantification of the N-oxygenated metabolites of deprenyl, methamphetamine, and amphetamine enantiomers, formed in vitro. The influence of various parameters (selector concentration, buffer pH, temperature, polymer additive, etc.) on the simultaneous separation of the optical isomers of the parent drugs and their metabolites has been evaluated. The buffer pH had the greatest impact on the separation selectivity of the N-oxygenated compounds. Linear calibration curves were obtained over the concentration range of 2.5-50 microM for the enantiomers of amphetamine-hydroxylamine, methamphetamine-hydroxylamine, and deprenyl-N-oxide. The inter- and intra-assay precision and accuracy varied by less than 15% for all analytes at concentrations of 5, 10, and 30 microM, and less than 20% at the lower limit of quantitation (2.5 microM). The sample extraction recovery ranged between 109 and 129% at the three concentration levels. The drug enantiomers were incubated with recombinant human flavin-containing monooxygenase enzymes (FMO3 and FMO1), and human liver microsomes, respectively. The enantioselectivity of the substrate preference, as well as the stereoselective formation of the new chiral center upon the oxidation of the prochiral tertiary nitrogen of deprenyl were assessed. FMO1, the extrahepatic form of the enzyme in man, was shown to be more active in the N-oxygenation of both deprenyl and methamphetamine isomers than FMO3. Deprenyl enantiomers and S-methamphetamine were substrates of human recombinant FMO3. Conversion of amphetamine to its hydroxylamine derivative could not be observed on incubation with either FMO1 or FMO3. Formation of the new chiral center on the nitrogen, during N-oxidation of the tertiary amine deprenyl, was found stereoselective. The two FMO isoforms have shown opposite preference in the formation of this chiral center. Methamphetamine-hydroxylamine formed from methamphetamine was further transformed by FMO, amphetamine-hydroxylamine was identified as the product of a demethylation reaction.
Szöko
Eva
E
Department of Pharmacodynamics, Semmelweis University, Budapest, Hungary. szokeva@net.sote.hu
Tábi
Tamás
T
Borbás
Tímea
T
Dalmadi
Balázs
B
Tihanyi
Károly
K
Magyar
Kálmán
K
D000073738:2-Hydroxypropyl-beta-cyclodextrin; D000661:Amphetamine / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D002021:Buffers; D019075:Electrophoresis, Capillary / Q000379:methods* / Q000706:statistics & numerical data; D006801:Humans; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D008694:Methamphetamine / Q000031:analogs & derivatives / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D008862:Microsomes, Liver / Q000378:metabolism; D015394:Molecular Structure; D010084:Oxidation-Reduction; D010105:Oxygenases / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D012642:Selegiline / Q000031:analogs & derivatives / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D013237:Stereoisomerism; D047392:beta-Cyclodextrins
D002021:Buffers; D011994:Recombinant Proteins; D047392:beta-Cyclodextrins; D000073738:2-Hydroxypropyl-beta-cyclodextrin; D012642:Selegiline; D008694:Methamphetamine; D000661:Amphetamine; D010105:Oxygenases; C020066:dimethylaniline monooxygenase (N-oxide forming)