Lenka Kohútová

Capillary electrophoresis‐frontal analysis is one of the most frequently used approaches for the study of plasma protein‐drug interactions as a substantial part of new drug development. However, the capillary electrophoresis‐frontal analysis typically combined with ultraviolet‐visible detection suffers from insufficient concentration sensitivity, particularly for substances with limited solubility and low molar absorption coefficient. The sensitivity problem has been solved in this work by its combination with an on‐line sample preconcentration. According to the knowledge of the authors this combination has never been used to characterize plasma protein‐drug binding. It resulted in a fully automated and versatile methodology for the characterization of binding interactions. Further, the validated method minimalizes the experimental errors due to a reduction in the manipulation of samples. Moreover, employing an on‐line preconcentration strategy with capillary electrophoresis‐frontal analysis using human serum albumin‐salicylic acid as a model system improves the drug concentration sensitivity 17‐fold compared to the conventional method. The value of binding constant (1.51 ± 0.63) · 10 4 L/mol obtained by this new capillary electrophoresis‐frontal analysis modification is in agreement with the value (1.13 ± 0.28) ·10 4 L/mol estimated by a conventional variant of capillary electrophoresis‐frontal analysis without the preconcentration step, as well as with literature data obtained using different techniques.

CE/frontal analysis (CE/FA) is probably one of the most frequently used modes of CE for studying affinity interactions. It is typically performed with classic UV‐Vis detection that suffers from low concentration sensitivity. To overcome this limitation, the applicability of CE/FA in combination with ESI‐MS detection for the investigation of drug–HSA interactions was demonstrated. The developed new method combines the advantages of CE/FA, such as low sample consumption and no labeling or immobilization of interacting partners, with the benefits of MS detection, such as higher selectivity and sensitivity; moreover, it can be used for molecules lacking a fluorophore or chromophore. The binding parameters of tolbutamide (TL) and glimepiride (GLP), first‐ and second‐generation antidiabetics that differ strongly in their solubility in aqueous solutions, were investigated by this CE/FA‐MS method. This method, in contrast to the CE/FA method with the most commonly used UV‐Vis detection, is more sensitive; an almost three times lower LOD was reached. The binding parameters of TL and GLP were investigated by this CE/FA‐MS method and compared with the literature data. The binding constant value of TL obtained by UV‐Vis detection was lower than the value obtained by the method hyphenated with MS detection, which is probably given by the influence of the ESI parameters on the stability of drug–HSA complex. In addition, the ratio of TL and HSA concentrations was divergent in both of the experimental approaches. Finally, it can be concluded that both detection methods have their strengths and weaknesses.