Abstract：
In the early stages of drug discovery, beyond the biological activity screening, determining the physicochemical properties that affect the distribution of molecules in the human body is an essential step. Plasma protein binding (PPB) is one of the most important investigated endpoints. Nevertheless, the methodology for measuring %PPB is significantly less popular and standardized than other physicochemical properties, like lipophilicity. Here, we proposed how to modify protocols presented by Valko into column safety conditions and evaluated their robustness using fractional factorial design. For robustness testing, four factors were selected: column temperature, mobile phase flow rate, maximum isopropanol concentration in the mobile phase, and buffer pH. Elaborate methods have been applied for the analysis of HSA affinity for three groups of antibiotic-oriented substances that vary in chemical structure: fluoroquinolones, sulfonamides, and tetrazole derivatives. Furthermore, based on the reversed-phase chromatography the workflow of pilot studies was proposed to select molecules that have high affinity to HSA and can not be eluted from the HSA column using the concentration of organic modifier recommended by the column manufacturer.
摘要：
在药物发现的早期阶段，超越生物活性筛选，确定影响分子在人体内分布的物理化学性质是必不可少的步骤。血浆蛋白结合（PPB）是最重要的研究终点之一。然而，测量%PPB的方法比其他物理化学性质明显不受欢迎和标准化，比如亲脂性.这里,我们提出了如何将Valko提出的方案修改为色谱柱安全条件,并使用分数阶乘设计评估了其稳健性.对于稳健性测试，选择了四个因素：柱温，流动相流速，流动相中的最大异丙醇浓度，和缓冲液pH值。详细的方法已用于分析HSA对三组化学结构不同的抗生素导向物质的亲和力：氟喹诺酮，磺胺类药物,和四唑衍生物。此外，基于反相色谱，提出了中试研究的工作流程，以选择对HSA具有高亲和力并且无法使用色谱柱制造商推荐的有机改性剂浓度从HSA色谱柱中洗脱的分子。
