毛细管电泳法快速测定大鼠血浆中丙吡胺游离药物和总药物浓度及血浆蛋白结合率

目的：建立一个全新、简单、快速的高效毛细管电泳方法，不需要对血浆样品进行任何前处理，血浆样品直接进样测定丙吡胺在大鼠血浆中的游离药物浓度和总药物浓度及血浆蛋白结合率。方法：采用40.2 cm未涂层石英毛细管（75 μm×375 μm，有效长度30 cm），分别以pH 7.4的20 mmol·L^-1磷酸盐缓冲溶液和含有50 mmol·L^-1三羟甲基氨基甲烷（Tris）的20 mmol·L^-1磷酸盐缓冲溶液作为缓冲体系，测定游离药物浓度和总药物浓度。采用3.447 kPa压力直接进血浆样品5 s，分离电压15 kV，温度20℃，检测波长190 nm。结果：方法的专属性强，在线测定血浆中总药物浓度时，丙吡胺与蛋白的结合得到充分破坏并与血浆蛋白成分在5 min内达到基线分离，丙吡胺在血浆中总质量浓度分别在1.0~20.0 μg·mL^-1和20.0~200.0 μg·mL^-1的低、高浓度区间内呈现良好的线性关系，相关系数分别为0.999 2和0.997 7，回收率为93.8%~110.7%。在体外血浆生物样品中，当预温孵的丙吡胺质量浓度为100、50、25、12.5、10 μg·mL^-1时，血浆蛋白结合率平均值为86.7%、86.3%、86.4%、78.3%、74.7%。在体内血浆生物样品中，丙吡胺血浆蛋白结合率平均值范围在39.3%~91.5%。测定结果表明丙吡胺与大鼠血浆蛋白的结合具有一强、一弱2个结合位点，结合常数分别为5.3×104 mol^-1·L和1.2×102 mol^-1·L。绘制了大鼠在给药后24 h体内丙吡胺游离和总药物浓度的药时曲线。结论：利用所建立的新方法，将含丙吡胺的血浆生物样品直接进样，高效、快速地测定了大鼠体内外血浆生物样品中丙吡胺的游离药物和总药物浓度。新建立的方法灵敏，高效快速，操作简单，耗样量少，价廉，有潜在的广泛的应用价值。

Objective:To establish a novel and fast high-performance capillary electrophoresis method for the determination of free and total drug concentrations of disopyramide in rat plasma in vivo and in vitro without any sample preparation process,and also to detect the plasma protein-binding rate.Methods:The separation column was 40.2 cm separation capillary(75 μm)with 15 kV separation voltage.The sample injection time was 5 s with the pressure of 3.447 kPa.20 mmol·L^-1 potassium phosphate buffer and 20 mmol·L^-1 potassium phosphate buffer containing 50 mmol·L^-1 Tris were adopted for the determination of free drug concentrations and total drug concentrations,respectively.Both buffers' pH value were equal to 7.4.Results:Under online total drug concentration measurement condition,good linear relationships(R₁²=0.999 2,R₂²=0.997 7)were achieved within the concentration of 1.0-20.0 μg·mL^-1 and 20.0-200.0 μg·mL^-1,respectively.The recoveries were 93.8%-110.7%.For the disopyramide precubation concentrations of 100,50,25,12.5,10 μg·mL^-1 in vitro,the corresponding plasma protein binding rates(PPB)were 86.7%,86.3%,86.4%,78.3%,74.7%,respectively.The average plasma protein binding rate range was 39.3%-91.5% in vivo.Two binding sites could be found between disopyramide and rat plasma protein.The strong and weak bound sites' binding constants were 5.3×104 mol^-1·L and 1.2×102 mol^-1·L,respectively.Lastly,the free and total drug concentration-time curves in 24 h were drawn.Conclusion:The novel method was applied to detect the free and total drug concentrations of disopyramide in rat plasma in vivo and in vitro.The novel method is simple,fast,cheap,effective and has a prospect of being widely used.