毛细管电泳-激光诱导荧光检测分析重组人促红细胞生成素时不同荧光衍生化方法的比较研究

doi:10.5246/jcps.2014.05.044

中国药学(英文版) ›› 2014, Vol. 23 ›› Issue (5): 317-323.DOI: 10.5246/jcps.2014.05.044

毛细管电泳-激光诱导荧光检测分析重组人促红细胞生成素时不同荧光衍生化方法的比较研究

杨霞¹, 庞楠楠^1,2, 付晓芳¹, 尹红锋³, 廖一平¹, 刘虎威^1*

1. 生物有机与分子工程教育部重点实验室, 北京大学化学与分子工程学院分析化学所, 北京 100871
2. 北京科技大学化学与生物工程学院, 北京 100083
3. 安捷伦实验室, 美国加州 CA 95051

收稿日期:2014-03-07 修回日期:2014-04-20 出版日期:2014-05-23 发布日期:2014-05-05
通讯作者: Tel.: 86-10-62754976; Fax: 86-10-62751708; E-mail: hwliu@pku.edu.cn
基金资助:
National Natural Science Foundation of China (Grant No. 20635001 and 21175005).

Comparative study on different derivatization procedures for analysis of recombinant human erythropoietin by capillary electrophoresis with laser-induced fluorescence detection

Xia Yang¹, Nannan Pang^1,2, Xiaofang Fu¹, Hongfeng Yin³, Yiping Liao¹, Huwei Liu^1*

1. The Key Lab of Bioorganic Chemistry and Molecular Engineering of Ministry of Education, Institute of Analytical Chemistry, College of Chemistry and Molecular Engineering, Peking University, Beijing 100871, China
2. School of Chemical and Biological Engineering, University of Science and Technology Beijing, Beijing 100083, China
3. Agilent Labs, Santa Clara, CA 95051 USA

Received:2014-03-07 Revised:2014-04-20 Online:2014-05-23 Published:2014-05-05
Contact: Tel.: 86-10-62754976; Fax: 86-10-62751708; E-mail: hwliu@pku.edu.cn
Supported by:
National Natural Science Foundation of China (Grant No. 20635001 and 21175005).

摘要/Abstract

摘要：

内源性糖蛋白人促红细胞生成素(hEPO)在控制红细胞生成方面具有重要的作用。而重组EPO(rhEPO)则是一种基因重组药物, 属于高度糖基化的糖蛋白, 在临床上多用于促进术后康复和体能恢复。同时, 作为一种内源性激素, 它对红细胞生成有特异性的刺激作用, 对机体的有氧工作能力有明显的促进作用, 故在一些耐力运动比赛项目中常被滥用。由于其明显的副作用, 国际奥委会已将rhEPO列为违禁药物。又由于rhEPO与人体自然生成的hEPO在序列结构上几乎没有区别, 而且rhEPO的半衰期很短, 在血液和尿液中的浓度非常低, 所以药检难度很大。目前, 国际反兴奋剂委员会的rhEPO检测方法是基于尿检和血检的综合结果。此方法虽然有效, 但仍然存在一些缺点, 比如rhEPO抗体的特异选择性还不是很高, 费用昂贵, 操作步骤繁琐、费时等等。本文采用毛细管电泳-激光诱导荧光检测(CE-LIF)的方法, 分别采用两种荧光衍生试剂FITC和FQ实现完整的rhEPO分子的柱前和柱上衍生化, 并将两种方法的检测灵敏度和糖型分离效率与CE-UV的结果进行了比较。结果证明, FITC和FQ均可用于低浓度rhEPO的荧光标记, 但过量的FITC会干扰标记rhEPO的荧光信号。采用FQ柱前标记能够提高检测灵敏度, 但由于多重标记增加了标记产物的复杂性, 会降低不同糖型rhEPO的分离效果。通过优化FQ柱上衍生反应条件, 能够避免复杂的多重衍生, 可获得高灵敏检测结果及一定程度的糖型分离结果。尽管该方法的检测灵敏度尚达不到检测血液和尿液中的rhEPO的目标, 但仍可能作为rhEPO生产过程中的质量控制方法。

关键词: 重组人促红细胞生成素, 毛细管电泳, 激光诱导荧光检测, 在线荧光标记

Abstract:

Human erythropoietin (hEPO), an endogenous glycoprotein, plays a fundamental role in erythropoiesis controlling the formation of red blood cells. Production of recombinant human erythropoietin (rhEPO) has made it possible for its abuse in competitive sports. In this work, pre-capillary and on-capillary derivatization by 5-furoylquinoline-3-carboxaldehyde (FQ) and fluorescein isothiocyanate (FITC) for the detection of rhEPO by capillary electrophoresis with laser-induced fluorescence detection (CE-LIF) were compared. FQ pre-capillary labeling improves sensitivity but degrades the glycoforms separation due to the inhomogeneity of the reaction products from multiple labeling. Compared with FITC pre-capillary derivatization with the excess fluorescent background, the on-capillary FQ derivatization method can provide shorter analysis time, lower background, and better selectivity. It is demonstrated that, through optimizing reaction conditions of FQ on-capillary derivatization, both high sensitivity and satisfactory resolution for the analysis of the glycoforms of rhEPO could be obtained. This method can be used for the glycoforms profiling and quality control of rhEPO. It may be used as a candidate method for fast screening in doping control analysis.

Key words: Recombinant human erythropoietin, Capillary electrophoresis, Laser-induced fluorescence detection, On-capillary derivatization

中图分类号:

R962.1

Supporting: National Natural Science Foundation of China (Grant No. 20635001 and 21175005).

杨霞, 庞楠楠, 付晓芳, 尹红锋, 廖一平, 刘虎威. 毛细管电泳-激光诱导荧光检测分析重组人促红细胞生成素时不同荧光衍生化方法的比较研究[J]. 中国药学(英文版), 2014, 23(5): 317-323.

Xia Yang, Nannan Pang, Xiaofang Fu, Hongfeng Yin, Yiping Liao, Huwei Liu. Comparative study on different derivatization procedures for analysis of recombinant human erythropoietin by capillary electrophoresis with laser-induced fluorescence detection[J]. Journal of Chinese Pharmaceutical Sciences, 2014, 23(5): 317-323.

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毛细管电泳-激光诱导荧光检测分析重组人促红细胞生成素时不同荧光衍生化方法的比较研究

Comparative study on different derivatization procedures for analysis of recombinant human erythropoietin by capillary electrophoresis with laser-induced fluorescence detection

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