Electrophoretically mediated microanalysis for characterization of the enantioselective CYP3A4 catalyzed N‐demethylation of ketamine

Abstract

Execution of an enzymatic reaction performed in a capillary with subsequent electrophoretic analysis of the formed products is referred to as electrophoretically mediated microanalysis (EMMA). An EMMA method was developed to investigate the stereoselectivity of the CYP3A4-mediated N-demethylation of ketamine. Ketamine was incubated in a 50 μm id bare fused-silica capillary together with human CYP3A4 Supersomes using a 100 mM phosphate buffer (pH 7.4) at 37°C. A plug containing racemic ketamine and the NADPH regenerating system including all required cofactors for the enzymatic reaction was injected, followed by a plug of the metabolizing enzyme CYP3A4 (500 nM). These two plugs were bracketed by plugs of incubation buffer to ensure proper conditions for the enzymatic reaction. The rest of the capillary was filled with a pH 2.5 running buffer comprising 50 mM Tris, phosphoric acid, and 2% w/v of highly sulfated γ-cyclodextrin. Mixing of reaction plugs was enhanced via application of -10 kV for 10 s. After an incubation of 8 min at 37°C without power application (zero-potential amplification), the capillary was cooled to 25°C within 3 min followed by application of -10 kV for the separation and detection of the formed enantiomers of norketamine. Norketamine formation rates were fitted to the Michaelis-Menten model and the elucidated values for V(max) and K(m) were found to be comparable to those obtained from the off-line assay of a previous study.

In‐capillary reactant mixing for monitoring glycerol kinase kinetics by CE

“…Then, the electrical field is turned‐off so that the reaction continues at zero‐potential for a definite time known as wait time ( t wait ) during which ADP will have enough time to accumulate. The reactants will also continue to mix due to diffusion . The merge time and the wait time were experimentally optimized.…”

“…Several studies have reported the difficulty of mixing more than two reactants by using an electric field as well as the unfeasibility of mixing reactants with identical or very close mobilities. Thus, for mixing three reactant plugs, the mixing process is triggered by the electric field (EMMA) and reactants are then allowed to diffuse at zero‐potential , during a wait time where the product will accumulate . This is the zero‐potential amplification approach .…”

Advances in enzyme substrate analysis with capillary electrophoresis

“…In this approach, on-line mixing was achieved by injecting reactants of different pH values into the capillary using a series of small hydrodynamic injections. With this approach, an enzyme buffered at pH of 7.4 was combined with a separation medium buffered at pH of 2.5 [30,31]. The parabolic shape of consecutive injections, which was a result of the laminar flow, caused each injection band to blend with the surrounding injection bands.…”

“…The mixing of reactants occurs by transverse diffusion, where reactants diffuse towards the wall of the capillary. As a result, this injection mode can be utilized to mix multiple injected reactants for enzymatic reactions where the pH of the enzyme reaction was different than that of the background electrolyte [30,31,41]. …”

Recent advances in CE‐mediated microanalysis for enzyme study

“…The on‐line CE methods for investigation of in vitro drug metabolism implemented from 2002 to 2012 have been summarized by Nowak . Recently, an EMMA method was developed to investigate the stereoselectivity of the CYP 3A4‐mediated N ‐demethylation of ketamine . Partial filling of two incubation buffer plugs was used to protect enzyme and substrates from the pH of the separation buffer as well as the chiral selector in it.…”