Combination of on-line capillary electrophoretic assay with mass spectrometry detection for the study of drug metabolism by cytochromes P450

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Authors

LANGMAJEROVÁ Monika ŘEMÍNEK Roman PELCOVÁ Marta FORET František GLATZ Zdeněk

Year of publication 2015
Type Article in Periodical
Magazine / Source Electrophoresis
MU Faculty or unit

Faculty of Science

Citation
Doi http://dx.doi.org/10.1002/elps.201400394
Field Biochemistry
Keywords capillary electrophoresis; mass spectrometry; enzymatic reaction
Description A new CE-MS method with enzymatic reaction inside the capillary was developed for the study of drug metabolism by cytochromes P450. This automated method, based on the transverse diffusion of laminar flow profiles methodology, is comprised of the injection of substrates and enzyme, their mixing, incubation and separation of the reaction products, all performed by CE, and their detection, identification and quantification by MS. The developed and validated method was finally used to conduct a kinetic study of cytochrome P450 isoform 2C9 or human liver microsomes with diclofenac in order to demonstrate its practical functionality. All the estimated kinetic values – apparent Michaelis constants and apparent maximum reaction velocities were in agreement with literature data obtained using other techniques. In addition, the consumption of reactants was in the tens of nL per analysis. The method’s usability was further demonstrated on tolbutamide, the other probe substrate of cytochrome P450 isoform 2C9. As a result, the method is conceptually applicable for the screening of any other cytochromes P450 isoform and its substrates and inhibitors after adapting the incubation and separation conditions.
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