亲和毛细管整体柱在牛血清白蛋白与奈福泮对映体相互作用研究中的应用

Study of interaction between bovine serum albumin and nefopam enantiomers with affinity capillary monolith

利用胃蛋白酶键合的有机聚合物整体柱在电色谱中对手性药物奈福泮的拆分能力,采用前沿分析法同时对奈福泮的两个对映体与牛血清白蛋白(BSA)的相互作用情况进行了考察。经过优化后建立的电色谱条件为:胃蛋白酶修饰的聚(甲基丙烯酸环氧丙酯-乙二醇二甲基丙烯酸酯)毛细管整体柱作为分离通道(32 cm×75μm,有效长度22 cm),运行缓冲液为pH 5.5的15 mmol/L醋酸铵,样品溶剂为pH 7.4的50 mmol/L醋酸铵,运行电压为5.0 kV,电压进样10 kV×6 s,检测波长为215 nm。此时奈福泮两个对映体平台峰彼此完全分离,结合体系中BSA对对映体在电色谱中的分离和检测均无影响,测得两个对映体与BSA的结合常数分别为443和527 L/mol,结合位点数均为1.0,结合位点为Sudlow siteⅡ。

A pepsin modified poly （glycidyl methacrylate-ethyleneglycol dimethacrylate） （poly （GMA-EDMA）） capillary monolith （32 cm× 75μm, effective lenth 22 cm） was applied in exploring the interaction between nero- pare enantiomers and bovine serum albumin （BSA）. Frontal analysis was used to measure the binding constant, number of binding sites and the location of binding sites of BSA to both nefopam enantiomers. Optimal CEC conditions were as follows： 15 mmol/L ammonium acetate was adjusted to pH 5.5 as running buffer, separation voltage was 5.0 kV, detection wavelength was 215 nm, and samples were dissolved witb 50 mmol/L ammonium acetate buffer at pH 7.4 and were injected at 10 kV x6 s. The results indicated that two enantiomers could suc- cessfully separated by CEC on the monolith column. BSA in the binding system showed no effect on the separation and determination of nefopam enantiomers. Frontal analysis demonstrated that BSA has only one binding site with both enantiomers, with binding constants （K） of 443 L/mol and 527 L/mol, respectively. The displacement expe- riments indicated that binding site of both isomers to BSA molecule was Sudlow site Ⅱ.