Improvement of a capillary electrophoresis/frontal analysis (CE/FA) method for determining binding constants: discussion on relevant parameters.

Drug-plasma protein interactions have a significant impact on both pharmacokinetics (i.e., absorption, distribution, metabolism, and excretion) and pharmacodynamics (pharmacological effects). Therefore, it is of high interest to evaluate this binding during the drug development process. Capillary electrophoresis (CE) is an interesting analytical tool for drug-protein binding characterization because it consumes a relatively low amount of reagents and enables assays that can be carried out under near-physiological conditions. The most interesting mode of CE for the study of biomolecular interactions is CE/frontal analysis (CE/FA). However, some confusion in how to conduct CE/FA experiments has emerged in the literature. The present study examines, using research into drug-albumin interactions as an example, the most important steps to take into consideration when building up new CE/FA binding assays. These include the following: choosing the buffer and applied voltage; evaluating protein adsorption onto the capillary wall; choosing the injection volume; choosing the drug and protein concentrations; and, finally, verifying the co-migration of the protein and drug-protein complex. The experimental part of the present report can serve as a checklist for developing the key parameters that need to be addressed for successful and reliable interaction studies. In a second time, short-end injection was used to enhance throughput. The strengths of the binding constants (K(a)) for nine selected drugs (basic, neutral, and acidic substances) to albumin, which is the most important plasma protein, were from logK(a) 2.9 to 5.4. These values were compared to those obtained with validated methods and good agreement was achieved.