Combination of on-line capillary electrophoretic assay withmass spectrometry detection for the study of drug metabolism bycytochromes P450
Electrophoresis(2015)
摘要
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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