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Fast evaluation of enantioselective drug metabolism by electrophoretically mediated microanalysis: Application to fluoxetine metabolism by CYP 2 D 6
Author(s) -
AsensiBernardi Lucía,
MartínBiosca Yolanda,
EscuderGilabert Laura,
Sagrado Salvador,
MedinaHernández María José
Publication year - 2013
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.201300267
Subject(s) - enantioselective synthesis , enantiomer , metabolite , metabolism , chemistry , sulfation , cytochrome p450 , drug metabolism , capillary electrophoresis , unspecific monooxygenase , racemic mixture , chromatography , stereochemistry , biochemistry , cyp1a2 , catalysis
In this work, a capillary electrophoretic methodology for the enantioselective in vitro evaluation of drugs metabolism is applied to the evaluation of fluoxetine ( FLX ) metabolism by cytochrome 2 D 6 ( CYP 2 D 6). This methodology comprises the in‐capillary enzymatic reaction and the chiral separation of FLX and its major metabolite, norfluoxetine enantiomers employing highly sulfated β‐ CD and the partial filling technique. The methodology employed in this work is a fast way to obtain a first approach of the enantioselective in vitro metabolism of racemic drugs, with the additional advantage of an extremely low consumption of enzymes, CD s and all the reagents involved in the process. M ichaelis– M enten kinetic parameters ( K m and V max ) for the metabolism of FLX enantiomers by CYP 2 D 6 have been estimated by nonlinear fitting of experimental data to the M ichaelis– M enten equation. K m values have been found to be 30 ± 3 μM for S ‐ FLX and 39 ± 5 μM for R ‐ FLX . V max estimations were 28.6 ± 1.2 and 34 ± 2 pmol·min −1 ·(pmol CYP ) −1 for S ‐ and R ‐ FLX , respectively. Similar results were obtained using a single enantiomer ( R ‐ FLX ), indicating that the use of the racemate is a good option for obtaining enantioselective estimations. The results obtained show a slight enantioselectivity in favor of R ‐ FLX .