Shibukawa A, Nagao M, Kuroda Y, Nakagawa T
Faculty of Pharmaceutical Sciences, Kyoto University, Japan.
Anal Chem. 1990 Apr 1;62(7):712-6. doi: 10.1021/ac00206a013.
A new on-line high-performance liquid chromatography system was developed for the stereoselective determination of free drug concentration in drug-protein binding equilibrium. When a 40-300-microL portion of a sample solution containing 50-200 microM racemic warfarin (Wf) and 100-550 microM human serum albumin was directly injected into the internal-surface reversed-phase silica column, Wf gave a trapezoidal peak exhibiting a plateau region. The concentration in the plateau region was equal to the free Wf concentration in the initial sample solution. By the delivery of a portion (90 microL) of the eluent in the plateau region into the chiral separation column (Chiral AGP column) by column switching, the free concentrations of respective enantiomers of Wf were determined. The results agreed well with those obtained by the conventional ultrafiltration method. The precision was also confirmed by the within-run and day-to-day reproducibilities (coefficient of variation less than or equal to 3.05%, n = 5). The present method is simple and rapid and four sample solutions can be analyzed within 1 h without pretreatment.
开发了一种新型在线高效液相色谱系统，用于立体选择性测定药物 - 蛋白质结合平衡中游离药物的浓度。当将40 - 300微升含有50 - 200微摩尔消旋华法林（Wf）和100 - 550微摩尔人血清白蛋白的样品溶液直接注入内表面反相硅胶柱时，Wf给出一个呈现平台区的梯形峰。平台区的浓度等于初始样品溶液中游离Wf的浓度。通过柱切换将平台区的一部分洗脱液（90微升）输送到手性分离柱（Chiral AGP柱）中，测定了Wf各对映体的游离浓度。结果与通过传统超滤法获得的结果非常吻合。通过批内和日间重现性（变异系数小于或等于3.05%，n = 5）也证实了精密度。本方法简单快速，无需预处理即可在1小时内分析四个样品溶液。
