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.

毛细管电泳-前沿分析（CE-FA）与迁移率迁移亲和CE一起是研究血浆蛋白-药物相互作用的最常用亲和CE模式，这是药物发现早期的重要部分。在经典的CE-FA装置中，样品是通过在CE仪器外部将样品瓶中的相互作用伙伴离线混合制备的，并在短暂的孵育时间后加载到毛细管中并进行分析，在这项工作中，一种新的方法学方法已开发出将CE-FA与直接在毛细管内部混合相互作用伙伴的混合物相结合的技术。这种结合不仅可以大大减少所用样品化合物的量，还可以提供一种用于表征这些结合相互作用的全自动方法。

毛细管内混合基于Krylov等人介绍的层流剖面横向扩散方法。使用，emínek等人提出的多区域注射改进方法。实际上，在方法优化后，将六种药物和六种牛血清蛋白交替注入BGE中，每次在-10压力下注射3 s将BGE填充到毛细管中的压力为mbar（-1 kPa），这是最好的注射程序。以牛血清白蛋白和普萘洛尔为模型样品化合物的平台高的RSD计算的方法重复性优于3.44％。最后通过测定牛血清白蛋白与碱性药物普萘洛尔和利多卡因以及酸性药物苯基丁a的表观结合参数，证明了其适用性。