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目的优化现行胸腺肽质量标准中控制高分子量(Mw)物质的检测方法,解决该方法存在的对照品Mw大小与洗脱时间矛盾的问题。方法采用高效液相凝胶过滤色谱法(HPGFC),改变流动相的组成,建立高Mw物质的分析方法,并进行方法学验证。结果确定三氟乙酸-乙腈-水(0.05∶45∶55)作为流动相的HPGFC分析方法,在该洗脱条件下,对照品的洗脱顺序符合Mw变化的规律,经过方法学验证,改进后的方法更适于检测高Mw物质的含量。结论优化后的高Mw物质检测方法可用于胸腺肽制剂的质量控制,并为其他生化制剂高Mw蛋白检测提供了方法学参考。
OBJECTIVE To optimize the detection method for the control of high molecular weight (Mw) substances in the current thymosin quality standards and to solve the problem that the method has the contradiction between the size of reference substance Mw and the elution time. Methods High-performance liquid gel filtration chromatography (HPGFC) was used to change the composition of the mobile phase. The analytical method of high-MW material was established and validated by methodology. Results The HPLC analysis method of trifluoroacetic acid-acetonitrile-water (0.05:45:55) as the mobile phase was established. Under the conditions of elution, the elution order of the reference substance accorded with the rule of Mw variation. After verification by methodology, Method is more suitable for detecting the content of high-Mw substance. Conclusion The optimized method for detecting high Mw substances can be used for the quality control of thymosin preparations and provides a methodological reference for the detection of other high-Mw proteins in biochemicals.