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

doi:10.5246/jcps.2014.05.044

Journal of Chinese Pharmaceutical Sciences ›› 2014, Vol. 23 ›› Issue (5): 317-323.DOI: 10.5246/jcps.2014.05.044

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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).

Abstract

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

CLC Number:

R962.1

Supporting:

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

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