肺炎球菌多糖结合物原液中二甲基亚砜残留量检测方法的建立与验证  

Establishment and validation of a determination method of dimethyl sulfoxide residue content in pneumococcal polysaccharide conjugate bulk

作　　者：邓海清[1] 杜闪 王斌 贾松华 郭龙静 朱留强 王艺霞 刘建凯[1] 尹珊珊[1] Deng Haiqing;Du Shan;Wang Bin;Jia Songhua;Guo Longjing;Zhu Liuqiang;Wang Yixia;Liu Jiankai;Yin Shanshan(Beijing Engineering Technology Research Center for New Conjugate Vaccine,Beijing Minhai Biological Technology Co.Ltd.,Beijing 102600,China)

机构地区：[1]北京民海生物科技有限公司结合疫苗新技术研究北京市重点实验室,北京102600

出　　处：《国际生物制品学杂志》2024年第5期306-311,共6页International Journal of Biologicals

基　　金：国家重点研发计划(No.2023YFC2308000)。

摘　　要：目的建立并验证肺炎球菌多糖结合物原液中二甲基亚砜(dimethyl sulfoxide, DMSO)的检测方法。方法通过优化反相高效液相色谱法(reversed phase high performance liquid chromatography, RP-HPLC)中的流动相、柱温以及流速, 建立最优检测方法, 并对该方法的专属性、线性、准确度、精密度、稳定性等进行验证。结果以10%乙腈为流动相、流速为0.6 ml/min, 柱温35 ℃时, RP-HPLC检测方法最优。该方法检测干扰成分试验组和对照组中DMSO含量的相对误差在-2.6%~2.1%, 分离度均≥1.5;浓度与峰面积呈良好的线性关系, 回归决定系数>0.990 0;各浓度水平检测所得的偏倚值均在-5.31%~-0.89%, 95%的偏倚置信上限也低于8.0%;不同浓度精密度在0.70%~4.45%, 95%的精密度置信上限不超过8.0%;方法综合能力评价显示在4~50 μg/ml范围内方法总变异不超过7.0%, 95%/90%容忍区间和95%预测区间均在80%~120%范围内;检测供试品溶液在20 h内峰面积均值的相对标准偏差均≤2.0%。结论成功建立了肺炎球菌多糖结合物原液中DMSO的检测方法, 该方法的专属性、线性、准确度、精密度、稳定性良好, 综合评价能力高。Objective To establish and validate a method for the detection of dimethyl sulfoxide(DMSO)in pneumococcal polysaccharide conjugate bulk.Methods The mobile phase,column temperature and flow rate in the reversed phase high performance liquid chromatography(RP-HPLC)were optimized to establish the optimal detection method.The method was validated in terms of specificity,linearity,accuracy,precision and stability.Results The RP-HPLC method was optimized with 10%acetonitrile as the mobile phase at a flow rate of 0.6 ml/min and a column temperature of 35℃.The relative errors of this method for the detection of DMSO content in the test and control groups of the interfering components ranged from-2.6%to 2.1%,and the resolutions were all≥1.5;the concentrations showed good linearity with the peak areas,and the regression coefficient of determination was≥0.9900;the biases of different concentration levels ranged from-5.31%to-0.89%,and the upper limit of bias in 95%was≤8.0%;the precision of different concentrations ranged from 0.70%to 4.45%,and the upper limit of confidence of the precision in 95%was<8.0%.The evaluation of the comprehensive ability of the method showed that the total method variation was≤7.0%in the range of 4-50μg/ml.Both the 95%prediction interval and the 95%/90%tolerance interval were in the range of 80%-120%.The relative standard deviation of the mean peak area of the test solution within 20 h were≤2.0%.Conclusion The method for the determination of DMSO in pneumococcal polysaccharide conjugate bulk is successfully established,which shows good specificity,linearity,accuracy,precision,stability and high comprehensive evaluation ability.

关 键 词：肺炎球菌多糖 结合物 色谱法 高效液相 二甲基亚砜 方法验证 

分 类 号：R927.2[医药卫生—药学]