反相高效液相色谱法分析聚乙二醇修饰的水蛭素  

Analysis of PEGylated Hirudin with Reverse-Phase High Performance Liquid Chromatography

作　　者：肖凤君[1] 王晓娜[1] 毕建进[1] 于爱平[1] 

机构地区：[1]军事医学科学院放射与辐射医学研究所,北京100850

出　　处：《生物技术通讯》2009年第1期78-80,共3页Letters in Biotechnology

摘　　要：目的:采用反相高效液相色谱法(RP-HPLC)对聚乙二醇(PEG)修饰的水蛭素进行分析分离,用以分析修饰产物中不同修饰度水蛭素的组成和比例。方法:色谱柱为HypersilC18,5μm,4.6mm×150mm;流动相A为H2O+0.01%的三氟乙酸,流动相B为乙腈+0.01%的三氟乙酸。40min内由10%～50%流动相B进行梯度洗脱,洗脱流速1mL/min,上样量50μL,检测波长为214nm。结果:在单甲基化PEG-丙酸琥珀酰亚胺和水蛭素摩尔比不同的的反应产物中,PEG1-水蛭素、PEG2-水蛭素均可以达到基线分离,且不同批次的反应产物进行RP-HPLC的重复性良好。结论:RP-HPLC可以有效地对PEG修饰的水蛭素产物进行分析分离,为PEG化水蛭素的长效、缓释剂型的开发提供技术支持。Objective： To analyze the composition and proportion of the modification products of hirudin with polyethylene glycol（PEG） by reverse-phase high performance liquid chromatograpby（RP-HPLC）. Methods： Chromatography column Hypersil C18, 5μm, 4.6mm×150mm was used in the assay. H2O＋0.01% trifluoroacetic acid was used as mobile phase A and aeetonitrile＋0.01% trifluoroaeetic acid was used as mobile phase B. Gradient elution was performed with 10%-50% mobile phase B in 40 min. The flow rate was 1 mL/min and the detection wavelength was 214 nm. Results： PEG1-hirudin and PEG2-hirudin in different reaction systems could be successfully isolated at baseline by RP-HPLC. High reproducibility was reached for two batches of modification products. Conclusion： PEGylated hirudin can be well analyzed with RP-HPLC, which helps for the development of the PEGylated and sustained-releasing dosage form of hirudin.

关 键 词：反相高效液相色谱 水蛭素 聚乙二醇 

分 类 号：Q503[生物学—生物化学]