Combination of on‐line CE assay with MS detection for the study of drug metabolism by cytochromes P450

Abstract

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 cytochrome P450 isoform and its substrates and inhibitors after adapting the incubation and separation conditions.

Recent advances in CE‐MS coupling: Instrumentation, methodology, and applications

“…The method of multisegment injection is based on sample loading between BGE spacers followed by CE‐MS and represents very promising and elegant way of throughput increase. An innovative approach for faster analysis of substrate‐enzyme reactions represents repetitive loading of substrate and enzyme in layers in to the separation capillary . After the loading, the transverse diffusion and laminar flow profiles induce merging of the layers, followed by enzyme‐substrate reaction.…”

Recent advances in CE mediated microanalysis for enzymatic and derivatization reactions

“…However, it is also important to know the diffusion profiles of the reactants to approximately estimate the pressure and the time used for the injection of the plugs. Recently Langmajerová et al used TDLFP mixing to perform on‐line CE‐MS to study the metabolism of diclofenac by CYP2C9 and tolbutamide by human liver microsomes. Combination of on‐line CE‐HPLC‐MS to study cytochrome P450 has been done before but incorporating TDLFP to mix the reactants has been described in this study .…”

Sphingomyelin ability to act as chiral selector using nanodisc electrokinetic chromatography

“…Capillary electrophoresis (CE) is a separation and analysis technique, which allows for quick analysis, high theoretical plate counts and resolution using nanoliter sample volumes. CE has become a valuable technique for studying biomolecule affinity 8–10 , and membrane characterization 11–14 as well as pharmaceutical absorption 15–17 and metabolism 18–20 . We recently introduced nanodisc electrokinetic chromatography (NEKC), an adaptation of CE using lipid bilayer nanodiscs as additives, as a technique for the study of small molecule interactions with lipid bilayers 13,14 .…”