反相高效液相色谱法分析聚乙二醇修饰的水蛭素

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Hypersil C18，5μm，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。

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

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.