微流控芯片毛细管电泳法在酶法工艺青霉素类产品中残留蛋白检测的应用探讨  

Determination of residual proteins in the penicillin antibiotics produced by enzymatic process with microfluidic chip-based capillary electrophoresis

作　　者：王琰[1] 姚尚辰[1] 尹利辉[1] 胡昌勤[1] 孟红梅[2] 张彦民 邹文博[1] 许明哲[1] WANG Yan;YAO Shang-chen;YIN Li-hui;HU Chang-qin;MENG Hong-mei;ZHANG Yan-min;ZOU Wen-bo;XU Ming-zhe(National Institutes for Food and Drug Control,Beijing 102629,China;Department of Neurology and Neuroscience Center,First Hospital of Jilin University,Changchun 130021,China;School of Pharmacy,Health Science Center,Xi'an Jiaotong University,Xi'an 710061,China)

机构地区：[1]中国食品药品检定研究院,北京102629 [2]吉林大学白求恩第一医院神经内科和神经科学中心,长春130021 [3]西安交通大学医学部药学院,西安710061

出　　处：《中国新药杂志》2020年第15期1770-1777,共8页Chinese Journal of New Drugs

基　　金：国家“重大新药创制”科技重大专项资助项目(2017ZX09101001-007)。

摘　　要：目的:蛋白残留量是影响酶法工艺生产半合成类抗生素的关键质量属性之一,它不仅影响药品质量,还会威胁人用药安全。本文尝试采用微流控芯片毛细管电泳(microfluidic chip-based capillary elec-trophoresis,MCCE)技术对青霉素类产品中的蛋白残留量进行分析和测定,并对MCCE法的影响因素和测定准确性进行了探讨。方法:以酶法工艺典型青霉素类药品-阿莫西林为例,采用Agilent高灵敏蛋白250型试剂盒分别对催化酶蛋白和阿莫西林样品进行前处理,再将处理后的溶液上样至蛋白芯片(5 cm×5 cm×0.8 cm),最后采用Agilent生物分析仪2100进行分析与检测。该法蛋白分子量检测范围为10~250 kDa;分析时间为30 min;样品量为9μL。结果:牛血清白蛋白(BSA)在5~500 ng·mL^-1(r=0.99,n=4)的浓度范围内线性关系良好;蛋白分子量测定准确度约为100.6%(n=4×7),不同浓度BSA在阿莫西林溶液中得到的加标回收标准曲线与BSA标准曲线之间没有显著性差异(P=0.28>0.05,n=4),显示准确度良好;检测限和定量限分别约为3 ng·mL^-1(1.5 ppm)和10 ng·mL^-1(5.0 ppm);定量日内和日间精密度均<15%(n=4);BSA在阿莫西林溶液中6 h内基本稳定(RSD<3.4%)。结论:本研究建立MCCE法可用于酶法工艺产品阿莫西林中残留蛋白的测定,方法简便快速、灵敏度高且准确性好,为酶法工艺半合成抗生素中残留蛋白的测定提供了一种不同原理的检测手段。Objective:The residual protein was one of critical quality attributes influencing semi-synthetic antibiotics produced by enzymatic process,which not only affects the quality of drugs but also threatens the safety of drug application.In this study,a microfluidic chip-based capillary electrophoresis(MCCE)method was established for the determination of residual proteins in penicillin products.Several effect factors and detecting accuracy for MCCE method were also investigated.Methods:Taking amoxicillin-one of typical penicillins-as an example,Agilent high sensitivity protein 250 kit was adopted for the preparation of penicillin G acylase(PGA)and amxocillin.Solutions were loaded into the protein chips(5 cm×5 cm×0.8 cm).Agilent Bioanalyzer 2100 was used for the analysis and detection.The detection range of protein molecular weight were 10~250 kDa.The analysis time was 30 min and the sample volume was 9μL.Results:The linear range of bovine serum albumin(BSA)was 5~500 ng·mL^-1(r=0.99).The accuracy of protein sizing was 100.6%(n=4×7).There was no significant difference(P=0.28>0.05,n=4)between the standard curves of BSA in amoxicillin test solution and BSA in water.The limits of detection and quantitation were 3 ng·mL^-1(1.5 ppm)and 10 ng·mL^-1(5.0 ppm),respectively.The quantitation RSD of inter-and intra-day was less than 15%(n=4).BSA in amoxicillin test solution was basically stable within 6 hours(RSD<3.4%).Conclusion:The MCCE method developed in this study,which is simple,fast,highly-sensitive and accurate,could be utilized for the determination of residual proteins in amoxicillin produced by enzymatic process.It provided a detection method with different principles for the detection of residual proteins in the semi-synthetic antibiotics produced by enzymatic process.

关 键 词：微流控芯片毛细管电泳 酶法工艺 阿莫西林 残留蛋白 含量测定 

分 类 号：R917[医药卫生—药物分析学]