定量核磁共振氢谱法测定肺炎球菌荚膜多糖中C多糖杂质的含量  

Determination of C-Polysaccharide Content in Streptococcus Pneumoniae Capsular Polysaccharides by Quantitative Proton Magnetic Resonance Spectroscopy

作　　者：王丽娟 马庆华 张秀[1] 扶晖 WANG Li-Juan;MA Qing-Hua;ZHANG Xiu;FU Hui(Analytical Instrumentation Center,College of Chemistry and Molecular Engineering,Peking University,Beijing 100871,China;Beijing Zhifei Lvzhu Biopharmaceutical Co.,Ltd.,Beijing 100176,China)

机构地区：[1]北京大学化学与分子工程学院,北京大学分析测试中心,北京100871 [2]北京智飞绿竹生物制药有限公司,北京100176

出　　处：《分析化学》2024年第5期735-742,共8页Chinese Journal of Analytical Chemistry

摘　　要：建立了核磁共振氢谱(^(1)H NMR)法测定肺炎球菌荚膜多糖中C多糖杂质(C-polysaccharide,C-Ps)含量的方法。通过二维^(1)H-^(15)N异核多键相关(HMBC)谱确定δ_(H) 3.24为C-Ps的特征峰。以3种血清型肺炎球菌荚膜多糖6A、6B和10A为样品、二甲基亚砜为内标物,建立了C-Ps绝对定量分析方法,并进行了方法学验证。结果表明,C-Ps浓度在2.5~198μg/mL范围内线性关系良好(R2>0.999),检测方法的定量限为2.5μg/mL;加标回收率在102%~109%之间;重复性相对标准偏差(RSD)小于3%,5 d内稳定性RSD小于1%。本分析方法操作简单,重复性和普适性良好,可作为肺炎球菌荚膜多糖研发和生产中C-Ps杂质的检测方法,为质检控制提供了新方法和新思路。A quantitative nuclear magnetic resonance(qNMR)method to analyze the content of residual C-polysaccharide(C-Ps)in the Streptococcus pneumoniae capsular polysaccharide was developed.The characteristic peak for C-Ps was confirmed atδ_(H) 3.24 by 2D ^(1)H-^(15)N heteronuclear multiple bond correlation(HMBC).By using pneumococcal serotypes 6A,6B and 10A capsular polysaccharids as model samples and dimethyl sulfone as internal standard,the proton qNMR(^(1)H qNMR)absolute quantitation method was established and validated.The linear detection range for C-Ps was 2.5-198μg/mL(R2>0.999),the limit of quantification was 2.5μg/mL,and the spiked recoveries were 102%‒109%.The relative standard deviation(RSD)of repeatability and the RSD of 5 days stability of this method were lower than 3%and 1%,respectively.The established ^(1)H qNMR method could be successfully used to determine the absolute C-Ps contents of Streptococcus pneumoniae capsular polysaccharides.This method showed many advantages such as simple operation,good repeatability and robustness,and could be easily adopted for the quality control of Streptococcus pneumoniae capsular polysaccharides during the research and development stages.

关 键 词：定量核磁共振氢谱 肺炎球菌荚膜多糖 C多糖杂质 磷酸胆碱 质量控制 

分 类 号：R392-33[医药卫生—免疫学] O657.2[医药卫生—基础医学]