Department of Biochemistry, Faculty of Science, Masaryk University, Kamenice 5, 625 00 Brno, Czech Republic.
Department of Biochemistry, Faculty of Science, Masaryk University, Kamenice 5, 625 00 Brno, Czech Republic.
J Chromatogr A. 2021 Jan 4;1635:461734. doi: 10.1016/j.chroma.2020.461734. Epub 2020 Nov 19.
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）与迁移率变化亲和 CE 一起，是用于研究血浆蛋白-药物相互作用的最常用亲和 CE 模式，这是药物发现早期阶段的重要组成部分。虽然在经典的 CE-FA 设置中，样品是通过在 CE 仪器外部的样品小瓶中离线混合相互作用的伙伴来制备的，并在短孵育期后加载到毛细管中进行分析，但在这项工作中，开发了一种新的方法学方法，将 CE-FA 与直接在毛细管内混合相互作用伙伴结合起来。这种组合除了大大减少所用样品化合物的量之外，还为这些结合相互作用的表征提供了一种全自动且多功能的方法。由于复杂 CE 仪器完全参与进样程序、混合和分离，而不是手动操作，可以最大限度地减少由于实验错误而导致的误差，这是另一个基本优势。在-capillary 混合基于 Krylov 等人引入的层流轮廓扩散方法，使用 Řemínek 等人提出的多区进样修改版。实际上，在方法优化后，发现交替引入 6 个药物和 6 个牛血清蛋白的插头在 BGE 中，每个插头以-10 mbar（-1 kPa）的压力在填充 BGE 的毛细管中注入 3 s，是最佳进样程序。作为模型样品化合物的牛血清白蛋白和普萘洛尔的峰高的重复性计算为 RSDs，优于 3.44%。最后，它的适用性通过测定基本药物普萘洛尔和利多卡因以及酸性药物苯丁唑酮与牛血清白蛋白的表观结合参数来证明。通过新的在线 CE-FA 方法获得的值与经典离线 CE-FA 估计的值以及使用不同技术获得的文献数据一致。
