HPLC法测定重组溶葡球菌酶及相关物质的研究

HPLC determination of recombinant lysostaphin and its related substances

目的用HPLC法测定重组溶葡球菌酶及其相关物质,进行相关验证和研究。方法高压液相色谱法,色谱柱为SOURCETM5RPC ST4.6/150,流动相系为水-乙腈缓冲液,检测波长为280 nm,重复进样6次,验证方法的重复性;分析重组溶葡球菌酶及其相关物质,验证方法的分离度;不同浓度样品进样,确定该方法的线性范围。结果重复进样变异系数〈5%,重组溶葡球菌酶和其主要杂质的分离度R〉1.5,重组溶菌酶在250～5 000 U/ml范围内呈良好的直线关系。结论该方法重复性好,专属性强,准确性高,可以很好的满足溶葡球菌酶原料药HPLC的纯度检测。

Objective To establish a HPLC method for the determination of active pharmaceutical ingredients,recombinant lysostaphin and its impurities.Methods Chromatography was performed by using a SOURCETM 5RPC ST column,with mobile phase of water-acetonitrile system.The monitoring wavelength was 280 nm.The samples were repeated for 6 times to validate the repeatability of the method.Recombinant lysostaphin and its impurities were analyzed to make sure the chromatographic resolution.Different concentration samples were analyzed to find the linear range.Results The coefficient of variation was less than 5%.The chromatographic resolution of recombinant lysostaphin and its impurities was greater than 1.5.The correlation coefficient of linear calibration curves was over 0.99 in the concentration range of 250～5 000 U/ml.Conclusion The proposed method is reproducible and highly selective.It can be used for the analysis of recombinant lysostaphin and its impurities.