Marta Zeisbergerová

Development of electrophoretically mediated microanalysis method for the kinetics study of flavin-containing monooxygenase in a partially filled capillary†

An electrophoretically mediated microanalysis method with a partial filling technique was developed for flavin‐containing monooxygenase, form 3 (FMO3). The in‐line enzymatic reaction was performed in 100 mM phosphate reaction buffer (pH 7.4) whereas 150 mM phosphate buffer (pH 3.3) was used as a background electrolyte. A long plug of cofactor NADPH dissolved in reaction buffer was hydrodynamically injected into a fused‐silica capillary, followed by enzyme and substrate solution. The reaction was initiated at 37°C in the thermostated part of the cartridge by the application of 9 kV for 0.9 min. The voltage was turned off to increase the product amount (zero‐potential amplification) and again turned on at a constant voltage of 10 kV to elute all the components. Direct detection was performed at 191 nm. The developed electrophoretically mediated microanalysis method was applied for the kinetics study of FMO3 using clozapine as a substrate probe. A Michaelis–Menten constant (Km) of 410.3 µM was estimated from the corrected peak area of the product, clozapine N‐oxide. The calculated value of the maximum reaction velocity (Vmax) was found to be 1.86 nmol/nmol enzyme/min. The acquired FMO3 kinetic parameters are in accordance with the published literature data.

On‐line drug metabolites generation and their subsequent target analysis by capillary zone electrophoresis with UV‐absorption detection

This study presents the in‐capillary enzymatic biotransformation of dextromethorphan, an antitusive drug and opioid receptor antagonist, and subsequent electrophoretic separation of its products. The study includes the optimization of separation parameters to fulfill the requirements of an online microreaction. The analyses were performed in a bare fused‐silica capillary using 100 mM sodium tetraborate (pH 10.0) mixed with linear polyacrylamide (20%, v/v) and 2‐propanol (10%, v/v). This BGE was suitable for monitoring both off‐line and in‐capillary incubations. The partial filling technique enabled the enzymatic reaction to be carried out in its optimal environment (20 mM sodium phosphate, pH 7.4). Finally, in‐capillary microreaction in the presence of cytochrome P450 3A4 gave satisfactory outcomes.

New Capillary Electrophoretic Method for On-line Screenings of Drug Metabolism Mediated by Cytochrome P450 Enzymes

A new method for the determination of kinetic and inhibition parameters of cytochromes P450 reactions by means of on‐line CE was developed. It is based on transverse diffusion of laminar flow profiles methodology introduced by Krylov et al. that injection procedure was modified. The solutions of an enzyme and its substrates are injected by hydrodynamic pressure as a series of repeated consecutive plugs. Proposed injection of three plugs of enzyme surrounded with plugs of substrates represents a certain trade‐off to obtain the reaction mixture with the satisfying homogeneity by the short‐injection procedure as possible. Mathematical modeling confirmed the assumption of a consistent distribution of reactants in the final reaction mixture. Kinetic and inhibition studies of cytochrome P450 isoform 2C9s reaction with diclofenac as a probe substrate and sulfaphenazole as a probe inhibitor were conducted in order to prove the practical applicability of the proposed method for on‐line screenings of drug metabolism mediated by cytochrome P450 enzymes. As a result, an apparent Michaelis constant of 2.66 ± 0.18 μM, apparent maximum reaction velocity of 7.91 ± 0.22 nmol min−1 nmol−1, Hill coefficient of 1.59 ± 0.16, half maximal inhibitory concentration of 0.94 ± 0.04 μM and apparent inhibition constant of 0.39 ± 0.07 μM were determined. All these values are in agreement with literature data obtained using different techniques. In addition, less than 30 nL of cytochrome P450 2C9 solution was consumed per analysis in the kinetic and inhibition studies using this method.

Various instrumental approaches for determination of organic acids in wines.

Biosensors based on lactate oxidase, sarcosine oxidase and mixture of fumarase and sarcosine oxidase were used for monitoring of organic acids in wine samples. Additionally, tartaric acid was determined by modified colorimetric method based on formation of the vanadate-tartrate complex. The above mentioned methods were used for the analysis of 31 wine samples and obtained data were compared with the results from capillary electrophoresis as a basic standard method. This comparison showed a certain degree of correlation between biosensors and capillary electrophoresis. The provided information pointed to the potential uses of biosensors in the field of winemaking.

Integration of on‐line protein digestion by trypsin in CZE by means of electrophoretically mediated microanalysis

In this work, electrophoretically mediated microanalysis (EMMA) was applied to the in‐capillary tryptic digestion of proteins for proteomic purposes. Compared with classical in‐solution tryptic digestion or the trypsin reactor commonly used for this purpose, the EMMA‐based method is rapid, can be automated and requires only a small amount of trypsin preparation. Moreover, the protein digestion and the analysis of the resulting peptides are integrated into one procedure. A combination of the EMMA methodology with a partial filling technique was used in this study, since the pH optimum of the trypsin reaction differs strongly from the best pH for the CZE separation of peptides. In this set‐up, a part of the capillary is filled with the best buffer for the tryptic digestion (50 mM Tris‐HCl buffer, pH 8.5) whereas the rest of the capillary is filled with the BGE optimal for peptide separation (0.1 M phosphate buffer, pH 2.5). As the proteins differ in their isoelectric points, a sandwich type of injection was used. The analysed protein is thus injected between two trypsin zones, which ensures their mixing and digestion. The analysis of one protein comprising both the digestion and the peptide separation is then completed in 1 h using a commercial instrument for CE with no modifications.