胃蛋白酶亲和有机聚合物毛细管整体柱的制备及性能考察

胃蛋白酶亲和有机聚合物毛细管整体柱的制备及性能考察池翠杰;王伟;季一兵

【摘要】The protein modified monolithic column in affinity capillary electrochromatography (CEC)has attracted considerable attention over the past decades because of its great enanti-oseparation ability. A porous polymethacrylate ester-based capillary monolithic column poly (glycidyl methacrylate-co-ethyleneglycol dimethacrylate )( poly( GMA-co-EDMA )) was pre-pared by in situ co-polymerization. The process was initiated thermally by azobisisobutyronitrile (AIBN). The polymerization mixture contained GMA as the function monomer and EDMA as the crosslinking agent with 1,4-butanediol and 1-propanol as the binary porogen solvent. Under the optimized reaction conditions,including the proportion of monomer and porogens,reaction temperature etc,the column exhibited a uniform structure,sufficient permeability and excel-lent pressure resistance. The separation of alkyl benzenes on the column was mainly based on typical reversed-phase chromatographic retention mechanism. The reproducibility and stability were good with RSDs less than 9. 0% . A pepsin functionalized organic polymer monolith was prepared by covalently bonded pepsin to poly(GMA-co-EDMA)monolith with glutaraldehyde as a spacer based on the activity of epoxide group. The enantioseparation performance of the pep-sin affinity monolith for basic enantiomers has been investigated by CEC. Nefopam,amlodip-

ine,citalopram and chlorpheniramine were resolved,and baseline

separations of nefopam, amlodipine,citalopram were achieved. The influences of pH,operating voltage,temperature and sample quantity used on the chiral separation were studied. The chiral recognition mecha-nism

of enantiomers on the monolithic column in CEC is discussed. This work developed a new method for the preparation and application of protein affinity monolith in CEC.%以热引发原位聚合方法制备了聚（甲基丙烯酸缩水甘

油酯（glycidyl methacrylate，GMA）-乙二醇二甲基丙烯酸酯（ethyleneglycol dimethacrylate，EDMA））毛细管整体柱，对整体柱的性能进行了表征。结果

表明，柱内部结构均匀、渗透性好；整体柱能够实现苯等中性小分子化合物的分离，具有反相色谱特征，重现性和稳定性良好。利用整体柱环氧基团的活性，采用间接法，以戊二醛为连接臂制备胃蛋白酶亲和手性整体柱。在毛细管电色谱模式下进行了柱分离性能研究，并对缓冲液 pH 值和运行电压等分离条件进行了考察。结果

表明，亲和整体柱对4种碱性手性药物（奈福泮、氨氯地平、西酞普兰、扑尔敏）有拆分效果，奈福泮、氨氯地平、西酞普兰能达到基线分离。本文为蛋白质亲和毛细管电色谱整体柱的制备和应用提供了新的思路和方法。

【期刊名称】《色谱》

【年(卷),期】2014(000)008

【总页数】7页(P791-797)

【关键词】毛细管电色谱;聚(甲基丙烯酸缩水甘油酯-乙二醇二甲基丙烯酸酯)整体柱;胃蛋白酶亲和手性整体柱;碱性药物;手性分离

【作者】池翠杰;王伟;季一兵

【作者单位】中国药科大学理学院，江苏南京 210009;中国药科大学理学院，江

苏南京 210009;中国药科大学理学院，江苏南京 210009

【正文语种】中文

【中图分类】O658

毛细管电色谱(CEC)具有毛细管电泳(CE)和高效液相色谱(HPLC)的双重分离性能［1］，被广泛用于手性药物、小分子物质、生物样品以及复杂混合物的分离分析［2-7］。毛细管整体柱具有制备方法简单、化学性质稳定、传质效率高、柱容量高等优点，被誉为第4代分离介质［8］。聚丙烯酸酯有机聚合物整体柱，因聚合单体选材范围广，可灵活在固定相中引入需要的活性基团，且使用的pH范围宽，在pH 2～12范围内能稳定存在，而成为目前研究最普遍的CEC 固定相基质之一［9］。

蛋白质类手性选择剂因独特的三维结构而对手性对映体有强大的拆分能力［10］。限于蛋白质作添加剂时背景吸收会干扰分析测定［11］，因此其作为固定相的应

用得到迅速发展［12-14］。但是应用于整体柱CEC手性固定相中的报道目前仅

限于牛血清白蛋白(BSA)和卵粘蛋白(OVM)［15，16］，且拆分的药物非常有限，只实现了色氨酸等氨基酸、华法林、安息香的分离，蛋白质亲和整体柱CEC手性

拆分的优势没有得到充分发挥。因此探寻拆分能力更强的蛋白质手性选择剂，以CEC整体柱为基质制备手性固定相(CSP)是目前较有前景的研究方向。

胃蛋白酶是一种酸性蛋白质，相对分子质量为34 600，等电点约为1，由327个氨基酸组成。其作为手性选择剂适合拆分碱性和中性的手性对映体，具有广泛的应用范围，已成功应用于HPLC和CE对各类光学异构体的分离［17-19］。Fanali

等［17］将胃蛋白酶作为手性添加剂应用于毛细管区带电泳(CZE)实现了普萘洛尔、