Zdeněk Glatz

Analysis of Thiocyanate in Biological Fluids by Capillary Zone Electrophoresis

A new sensitive and simple method has been developed for the determination of thiocyanate in human serum, urine and saliva. The determinations were performed in a fused-silica capillary [64.5 cm (56 cm effective length) x 75 microm] using 0.1 M beta-alanine-HCl (pH 3.50) as a background electrolyte, separation voltage 18 kV (negative polarity), temperature of capillary 25 degrees C and direct detection at 200 nm. Serum samples were 10-times diluted with deionised water and deproteinised with acetonitrile in the ratio 1:2. Urine and saliva samples need only 20-fold dilution with deionised water. The proposed method was successfully applied to the determination of thiocyanate in various human serum, saliva and urine samples.

Application of short‐end injection procedure in CE

CE is well known for its many advantages, including the speed of the analyses; most of which can be accomplished in less than 10 min. However many potential fields of application such as pharmaceutical research and industry and clinical diagnostics require analyses of large numbers of samples and so need an even shorter analysis time to be practical. There are generally three main options for enhancing the throughput of electromigration methods – using microchip or multi‐capillary systems or modifying the method preformed on single capillary instruments. The latter option will probably be the method of choice for most laboratories, since they are only equipped with classical commercial CE instrumentation. Although several approaches can be used – higher electric field, EOF modification, external pressure application during analysis, this article reviews one of the simplest and at the same time most used, the so‐called short‐end injection procedure.

Determination of lignans in Schisandra chinensis using micellar electrokinetic capillary chromatography.

Micellar electrokinetic capillary chromatography (MEKC) has been developed as a promising method for the determination of lignans in plant samples. The separation conditions have been optimized with respect to the different parameters including sodium dodecyl sulfate (SDS) and acetonitrile concentration, pH of the background electrolyte, separation voltage, and capillary temperature. The background electrolyte consisting of 40 mM SDS and 35% acetonitrile in 10 mM tetraborate buffer (pH 9.3) was found to be the most suitable electrolyte for this analysis. The applied voltage of 28 kV (positive polarity) and the capillary temperature 25°C gave the best separation of lignans. The interday reproducibility of the peak areas and the migration times was below 2.0%. The results of MEKC analyses were compared with those obtained by capillary electrochromatography (CEC) and reversed‐phase high‐performance liquid chromatography (RP‐HPLC). The possibilities of using this method for the determination of lignans in drug and in serum samples were also tested.

Analysis of artichoke (Cynara cardunculus L.) extract by means of micellar electrokinetic capillary chromatography.

The main constituents of artichoke extract were separated by micellar electrokinetic chromatography (MEKC), using a buffer consisting of 100 mM sodium dodecyl sulfate (SDS) in 20 mM sodium dihydrogen phosphate, 20 mM disodium tetraborate (pH 8.6) as background electrolyte. Optimum separation voltage of 28 kV (positive polarity) and a capillary temperature of 25°C gave the best analysis. The UV detection was performed at 200 nm. The method was successfully used to analyze plant and drug samples as well as for the study of artichoke antioxidant activity. The quantitative MEKC results were in good agreement to those obtained previously by reversed‐phase high‐performance liquid chromatography (RP‐HPLC).

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.

Optimized on‐line enantioselective capillary electrophoretic method for kinetic and inhibition studies of drug metabolism mediated by cytochrome P450 enzymes

Pharmacokinetic and pharmacodynamic properties of a chiral drug can significantly differ between application of the racemate and single enantiomers. During drug development, the characteristics of candidate compounds have to be assessed prior to clinical testing. Since biotransformation significantly influences drug actions in an organism, metabolism studies represent a crucial part of such tests. Hence, an optimized and economical capillary electrophoretic method for on‐line studies of the enantioselective drug metabolism mediated by cytochrome P450 enzymes was developed. It comprises a diffusion‐based procedure, which enables mixing of the enzyme with virtually any compound inside the nanoliter‐scale capillary reactor and without the need of additional optimization of mixing conditions. For CYP3A4, ketamine as probe substrate and highly sulfated γ‐cyclodextrin as chiral selector, improved separation conditions for ketamine and norketamine enantiomers compared to a previously published electrophoretically mediated microanalysis method were elucidated. The new approach was thoroughly validated for the CYP3A4‐mediated N‐demethylation pathway of ketamine and applied to the determination of its kinetic parameters and the inhibition characteristics in presence of ketoconazole and dexmedetomidine. The determined parameters were found to be comparable to literature data obtained with different techniques. The presented method constitutes a miniaturized and cost‐effective tool, which should be suitable for the assessment of the stereoselective aspects of kinetic and inhibition studies of cytochrome P450‐mediated metabolic steps within early stages of the development of a new drug.

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.

Comparison of various capillary electrophoretic approaches for the study of drug–protein interaction with emphasis on minimal consumption of protein sample and possibility of automation†

The binding ability of a drug to plasma proteins influences the pharmacokinetics of a drug. As a result, it is a very important issue in new drug development. In this study, affinity capillary electrophoresis, capillary electrophoresis with frontal analysis, and Hummel Dreyer methods with internal and external calibration were used to study the affinity between bovine serum albumin and salicylic acid. The binding constant was measured by all these approaches including the equilibrium dialysis, which is considered to be a reference method. The comparison of results and other considerations showed the best electrophoretic approach to be capillary electrophoresis-frontal analysis, which is characterized by the high sample throughput with the possibility of automation, very small quantities of biomacromolecules, simplicity, and a short analysis time. The mechanism of complex formation was then examined by capillary electrophoresis with frontal analysis. The binding parameters were determined and the corresponding thermodynamic parameters such as Gibbs free energy ΔG(0), enthalpy ΔH(0), and entropy changes ΔS(0) at various temperatures were calculated. The results showed that the binding of bovine serum albumin and salicylic acid was spontaneous, and that hydrogen bonding and van der Waals forces played a major role in the formation of the complex.

Determination of the kinetic parameters of rhodanese by electrophoretically mediated microanalysis in a partially filled capillary.

Electrophoretically mediated microanalysis (EMMA) was applied for the study of kinetic parameters of the bisubstrate enzymatic reaction of rhodanese. The Michaelis constants (Km) for both substrates and the effect of temperature on rhodanese reaction were evaluated by means of the combination of the EMMA methodology with a partial filling technique. In this setup, the part of the capillary is filled with the buffer best for the enzymatic reaction whereas, the rest of the capillary is filled with the background electrolyte optimal for separation of substrates and products. The enzymatic reaction was performed in 25 mM N‐(2‐hydroxymethyl)piperazine‐2’‐(2‐ethanesulfonic acid) (HEPES) buffer (pH 8.5) while the low pH background electrolyte 100 mM β‐alanine‐HCl (pH 3.5) was used for separation of substrates and products that are the inorganic anions. The estimated value of Km for thiosulfate of 1.30×10−2 M was consistent with previously published values; the Km for cyanide of 7.6×10−3 M was determined for the first time. In addition, the type of kinetic mechanism of enzymatic reaction was also elucidated. The finding of the double displacement (ping‐pong) mechanism is in accordance with previous literature data. Also, the experimentally determined temperature optimum of the rhodanese‐catalyzed reaction around 20–25°C agreed with literature values.

On‐capillary derivatisation as an approach to enhancing sensitivity in capillary electrophoresis

Separation technologies play an important role in revealing biological processes at various omic levels, in pharmacological and clinical research. In this context, CE is a strong candidate for analyses of samples with rapidly increasing complexity. Even though CE is well known for its many advantages in this regard, the sensitivity of CE analyses is insufficient for many applications. Accordingly, there are generally three main options for enhancing the sensitivity of CE analyses – using special detection techniques, using sample pre‐concentration and derivatisation. Derivatisation is often the method of choice for many laboratories, since it is simple and provides several advantages such as small sample volume demand and the possibility of automation. Although it can be performed in different ways depending on where the reaction takes place, this article reviews one of the simplest and at the same time most useful approaches on‐capillary derivatisation. Even if in many cases the use of on‐capillary derivatisation alone is enough to improve the detection sensitivity, on other occasions it needs to be employed in combination with the other above‐mentioned strategies. After a simple discussion of derivatisation in general, special attention is focused on the on‐capillary approach and methodologies available for on‐capillary reactant mixing. Its applications in various fields are also described.