肺炎球菌荚膜多糖去氧胆酸钠残留量测定方法的优化及验证

Optimization and verification of determination method for residual deoxysodium cholate content in pneumococcal polysaccharide

目的 优化肺炎球菌荚膜多糖中去氧胆酸钠残留量的检测方法,并进行验证。方法 采用《中国药典》三部（2010版）甲型肝炎灭活疫苗制造与检定规程附录Ⅰ中去氧胆酸钠残留量测定法,对肺炎球菌荚膜多糖原液中去氧胆酸钠残留量测定方法进行适用性确认;比较对照品在70℃水浴不同时间（20、40、60、120 min）及80℃水浴30 min的吸光度值,确定最佳反应温度及时间;验证优化后方法的线性、重复性及准确度。结果2批肺炎球菌荚膜多糖原液70℃水浴20 min后检测去氧胆酸钠残留量,回收率在8.6%～240.8%之间,适用性不合格;优化后的反应条件为80℃水浴30 min。去氧胆酸钠浓度在10～80μg/ml范围内,与A值呈良好的线性关系,R2〈0.99;3批去氧胆酸钠对照品溶液的重复性测定结果的CV值均小于8%;23种肺炎球菌荚膜多糖原液分别添加低、中、高浓度的去氧胆酸钠对照品溶液回收率在80%～120%之间。结论 优化后的肺炎球菌荚膜多糖去氧胆酸钠残留量测定方法操作简单,线性好,结果准确,重复性好,可用于肺炎球菌荚膜多糖去氧胆酸钠残留量的检测。

Objective To optimize the determination method for residual deoxysodium cholate content in pneumococcal polysaccharide. Methods The determination method for residual deoxysodium cholate content in bulk of pneumococcal polysaccharide was verified for suitability by the method in Chinese Pharmacopoeia （Volume Ⅲ, 2010 edition）. The absorbance of reference after incubation in 70 ℃ water bath for 20, 40, 60 and 120 rain and in 80 ℃ water bath for 30 min were determined, based on which the reaction temperature and time were optimized. The optimized method was verified for linearity, reproducibility and accuracy. Results The residual deoxysodium cholate content in two batches of bulks of pneumococcal polysaccharide after incubation in 70 ℃ water bath for 20 rain were determined, and the result showed recoveries of 8. 6% -240. 8%, indicating that the suitability was unqualified. The condition for reaction was optimized as incubation in 80 ℃ water bath for 30 min. The deoxysodium cholate contents at a range of 10 - 80 μg/ml showed good linearity with the absorbances, with a R2 value of more than 0. 99. The CVs of determination results of three batches of references in reproducibility test were less than 8%. The recovery rates of deoxysodium cholate at low, moderate and high concentrations, added to 23 kinds of bulks of pneumococcal polysaccharide, were 80% - 120%. Conclusion The optimized method was simple, which showed good linearity, accuracy and reproducibility, and might be used for determination of residual deoxysodium cholate content in pneumococcal polysaccharide.