Capillary electrophoresis-frontal analysis (CE-FA) together with mobility shift affinity CE is the most frequently used mode of affinity CE for a study of plasma protein-drug interactions, which is a substantial part of the early stage of drug discovery. Whereas in the classic CE-FA setup the sample is prepared by off-line mixing of the interaction partners in the sample vial outside the CE instrument and after a short incubation period loaded into the capillary and analysed, in this work a new methodological approach has been developed that combines CE-FA with the mixing of interacting partners directly inside the capillary. This combination gives rise to a fully automated and versatile methodology for the characterization of these binding interactions besides a substantial reduction in the amounts of sample compounds used. The minimization of possible experimental errors due to the full involving of sophisticated CE instrument in the injection procedure, mixing and separation instead of manual manipulation is another fundamental benefit. The in-capillary mixing is based on the transverse diffusion of laminar flow profile methodology introduced by Krylov et al. using its multi-zone injection modification presented by Řemínek at al.. Actually, after the method optimization, the alternate introduction of six plugs of drug and six plugs of bovine serum protein in BGE, each injected for 3 s at a pressure of -10 mbar (-1 kPa) into the capillary filled by BGE, was found to be the best injection procedure. The method repeatability calculated as RSDs of plateau highs of bovine serum albumin and propranolol as model sample compounds were better than 3.44 %. Its applicability was finally demonstrated on the determination of apparent binding parameters of bovine serum albumin for basic drugs propranolol and lidocaine and acid drug phenylbutazone. The values obtained by a new on-line CE-FA methodology are in agreement with values estimated by classic off-line CE-FA, as well as with literature data obtained using different techniques.
- MeSH
- keratitida diagnostické zobrazování MeSH
- konfokální mikroskopie * metody MeSH
- lidé MeSH
- nemoci rohovky * diagnostické zobrazování diagnóza MeSH
- rohovka diagnostické zobrazování MeSH
- úbytek endoteliálních buněk rohovky diagnostické zobrazování MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- přehledy MeSH
Diabetes is one of the most widespread diseases characterized by a deficiency in the production of insulin or its ineffectiveness. As a result, the increased concentrations of glucose in the blood lead not only to damage to many of the body's systems but also cause the nonenzymatic glycation of plasma proteins affecting their drug binding. Since the binding ability influences its pharmacokinetics and pharmacodynamics, this is a very important issue in the development of new drugs and personalized medicine. In this study, capillary electrophoresis-frontal analysis was used to evaluate the affinities between human serum albumin or its glycated form and the first generation of sulfonylurea antidiabetics, since their inadequate concentration may induce hypoglycaemia or on the contrary hyperglycaemia. The binding constants decrease in the sequence acetohexamide > tolbutamide > chlorpropamide > carbutamide both for normal and glycated human serum albumins, with glycated giving lower values. These results provide a more quantitative picture of how these drugs bind with normal and modified human serum albumin and indicate capillary electrophoresis-frontal analysis to be another tool for examining the changes arising from modifications of albumin, or any other protein, with all its benefits like short analysis time, small sample requirement, and 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.
