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目的优化肺炎球菌荚膜多糖中氨基己糖基团的定量检测方法,并进行验证。方法对肺炎球菌荚膜多糖中氨基己糖基团定量检测方法的水解条件(盐酸溶液分别为4、8、10 mol/L,时间分别为0.5、1、2、4、4.5、5、6 h)和乙酰化条件(温度分别为80、90、100℃,时间分别为30、45、60、90、120 min)进行优化,并验证该方法的专属性、线性、准确性、精密性及耐用性。结果最佳水解条件为10 mol/L盐酸溶液水解2 h;最佳乙酰化条件为90℃水浴45 min。该方法可特异性检测肺炎球菌荚膜多糖中氨基己糖的含量;D-盐酸氨基葡萄糖对照品溶液浓度在100~500μg/ml时,与A_(530)值呈良好的线性关系,R~2均>0.99;3次检测11个血清型肺炎球菌荚膜多糖样品的加标回收率均在90%~110%间;该方法的重复性及中间精密度的变异系数(CV)均<2%;6份D-盐酸氨基葡萄糖对照品溶液乙酰化不同时间的CV值为1.3%~5.2%。结论该方法具有良好的专属性、线性、准确性、精密性及耐用性,可用于肺炎球菌荚膜多糖中氨基已糖含量的检测。
Objective To optimize the quantitative determination of hexosaminoglycans in pneumococcal capsular polysaccharide and verify the method. Methods The conditions for the quantitative determination of hexosaminoglycans in pneumococcal capsular polysaccharide were as follows: hydrochloric acid solutions were 4, 8 and 10 mol / L respectively, with time of 0.5,1,2,4,4.5,5,6 h ) And acetylation conditions (temperature of 80, 90, 100 ℃, respectively, the time was 30,45,60,90,120 min) to optimize and verify the specificity of the method, linearity, accuracy, precision and durability Sex. Results The optimal hydrolysis conditions were 10 mol / L hydrochloric acid solution for 2 h. The best acetylation conditions were 90 ℃ water bath for 45 min. This method can detect the content of hexose hexose in pneumococcal capsular polysaccharide. The concentration of D-glucosamine hydrochloride reference substance in the range of 100 ~ 500μg / ml has a good linear relationship with the value of A_ (530) All of them were> 0.99. The spiked recoveries of 11 serotypes of pneumococcal capsular polysaccharide were all between 90% and 110% in 3 times. The coefficient of variation (CV) of repeatability and intermediate precision of the method were both less than 2% ; The CV value of 6 parts of D-glucosamine hydrochloride reference substance solution at different times was 1.3% -5.2%. Conclusion The method has good specificity, linearity, accuracy, precision and durability and can be used for the determination of aminohexose content in pneumococcal capsular polysaccharide.