口服六价重配轮状病毒减毒活疫苗(Vero细胞)中蔗糖含量高效液相色谱检测方法的建立及验证  

Development and verification of a high-performance liquid chromatography method for determination of sucrose content in oral hexavalent reassortant live attenuated rotavirus vaccine(Vero cells)

作　　者：何亮 徐海燕 申瑷琳 石艳红 罗敏 施金荣 HE Liang;XU Haiyan;SHEN Ailin;SHI Yanhong;LUO Min;Shi Jinrong(Quality Control Department,Wuhan Institute of Biological Products Co.,Ltd.,Wuhan 430207,Hubei Province,China)

机构地区：[1]武汉生物制品研究所有限责任公司质量控制室,湖北武汉430207

出　　处：《中国生物制品学杂志》2024年第11期1367-1372,共6页Chinese Journal of Biologicals

基　　金：国家重点研发计划(2020YFC0842100)。

摘　　要：目的建立口服六价重配轮状病毒减毒活疫苗(Vero细胞)中蔗糖含量的高效液相色谱(high-performance liquid chromatography,HPLC)检测方法,并进行验证,以期为以蔗糖为辅料疫苗中蔗糖含量的测定提供可靠的方法。方法采用HPLC法测定口服六价重配轮状病毒减毒活疫苗(Vero细胞)中蔗糖含量,色谱柱为Luna^(R)5μm NH2100?(LC Colum 250 mm×4.6 mm),示差检测器温度为30℃,流动相为乙腈,流速为0.5 mL/min,进样体积为10μL,柱温箱温度为30℃。验证方法的系统适用性、专属性、线性范围、精密性、准确性及耐用性。同时对HPLC法与旋光度法测得供试品中蔗糖含量结果的一致性进行比较。结果系统适用性溶液中乳糖和蔗糖的分离度良好,乳糖不干扰蔗糖的含量测定;枸橼酸、枸橼酸钠、氯化锌氯化钙混合物对蔗糖含量测定无干扰;蔗糖浓度在6~14 mg/mL范围内与峰面积具有良好的线性关系,线性方程为:y=142189 x+2182.4,相关系数(r)=0.9999;重复性及中间精密性验证相对标准偏差(RSD)分别为0.55%和0.49%;80%、100%、120%浓度样品平均回收率均在99%~101%之间;流速、柱温、流动相比例、检测器温度发生微小变动或更换不同批号色谱柱对检测结果无影响,与原条件测得供试品蔗糖含量的相对偏差(RD)均<1%。采用HPLC法与旋光法测得多批供试品中蔗糖含量的RD均<1%。结论建立的HPLC法具有良好的专属性,精密性、准确性及耐用性,可用于口服六价重配轮状病毒减毒活疫苗(Vero细胞)中蔗糖含量的检测。Objective To develop and verify a high-performance liquid chromatography(HPLC)method for the determination of sucrose content in oral hexavalent reassortant live attenuated rotavirus vaccines(Vero cells),in order to provide a reliable method for the determination of sucrose content in vaccines with sucrose as an excipient.Methods A HPLC method for the determination of sucrose content in oral hexavalent reassortant live attenuated rotavirus vaccines(Vero cells)was developed.The chromatographic column was Luna^(R)5μm NH2100?(LC Colum 250 mm×4.6 mm),the differential detector temperature was 30℃,the mobile phase was acetonitrile,the flow rate was 0.5 mL/min,the injection volume was 10μL,and the column temperature was 30℃.The system suitability,specificity,linear range,precision,accuracy and robustness of the method were verified.At the same time,the consistency of the results of HPLC and polarimetry method for determining the sucrose content in the test sample was analyzed.Results The separation of lactose and sucrose in system suitability test solution was good,and the lactose did not interfere with the determination of sucrose content.The citric acid,sodium citrate,zinc chloride and calcium chloride showed no interference to the determination of sucrose content.The sucrose concentration at the range of 6-14 mg/mL showed a good linear relationship with the peak area,and the linear equation was:y=142189 x+2182.4,with the correlation coefficient(r)of 0.9999.The RSDs of reproducibility and intermediate precision verification were 0.55%and 0.49%respectively.The average recovery rates of samples with the concentration of 80%,100%,and 120%were all between 99%and 101%.Minor changes in flow rate,column temperature,mobile phase ratio and detector temperature,or replacement of chromatographic columns of different batches had no effect on the test results,and the relative deviations(RDs)with the sucrose content measured under the original conditions were all less than 1%.The RDs of sucrose content in multiple batches of

关 键 词：口服六价重配轮状病毒减毒活疫苗(Vero细胞) 蔗糖含量 高效液相色谱法 

分 类 号：R917[医药卫生—药物分析学]