疫苗中铝佐剂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 aluminium adjuvant content in vaccine

作　　者：邵天舒 李潇 李玉立 赵璇 梅婕 何欢 李文东 SHAO Tianshu;LI Xiao;LI Yuli;ZHAO Xuan;MEI Jie;HE Huan;LI Wendong(Laboratory for Safety Research and Evaluation of Innovative Drugs,Beijing 102206,China)

机构地区：[1]北京市药品检验研究院国家药品监督管理局创新药物安全研究与评价重点实验室,北京102206

出　　处：《中国生物制品学杂志》2024年第1期58-64,共7页Chinese Journal of Biologicals

基　　金：北京市科技新星计划(Z21110000212101007)。

摘　　要：目的 建立疫苗中铝佐剂含量的高效液相色谱(high performance liquid chromatography,HPLC)测定方法,并进行验证及初步应用。方法 采用8-羟基喹啉衍生化方法进行测定。色谱柱为苯基-己基色谱柱[Luna 5u Phenyl-Hexyl(250 mm×4.6 mm)],流动相为乙酸铵-8-羟基喹啉乙腈溶液(60∶40),并添加终浓度为20 mg/L的抗坏血酸,荧光检测器激发波长380 nm,发射波长520 nm,流动相流速为1.0 mL/min,柱温为40℃,进样量为50μL,等度洗脱。对方法的专属性、线性范围、准确性、重复性、稳定性和耐用性进行验证。采用建立的方法测定12批疫苗中的铝含量,并与《中国药典》三部(2020版)通则3106的滴定法测定结果进行比较。结果 样品色谱图中无干扰色谱峰,非铝佐剂疫苗成分、磷酸盐缓冲液对测定均无干扰。铝标准品在6.25~100μg/mL浓度范围内线性关系良好,r=0.999 6;准确性验证试验中甲型肝炎灭活疫苗、重组乙型肝炎疫苗、吸附无细胞百白破疫苗及肠道病毒71型灭活疫苗铝含量平均加样回收率分别为98.32%、100.85%、101.09%和99.31%;4种疫苗同批次平行6份供试品溶液中铝含量检测结果的相对标准偏差(relative standard deviation,RSD)分别为1.09%、1.42%、0.97%和1.30%;4种疫苗样品溶液室温放置0、2、4、6、8 h,铝含量检测结果的RSD分别为0.82%、0.73%、0.40%和0.48%;流动相中乙酸铵溶液与8-羟基喹啉乙腈溶液比例在±5%范围改变时,4种疫苗铝含量测定结果波动范围均不超过2%。建立的HPLC法测定12批疫苗样品中铝含量结果与《中国药典》三部(2020版)滴定法结果无明显差异。结论 成功建立了疫苗中铝佐剂含量HPLC测定方法,该方法专属性、线性、准确性、重复性、稳定性、耐用性良好,操作简便且自动化程度高,人为因素干扰少,可实现单支样品测定,为快速大批量测定疫苗成品中铝含量,以及生产过程中监控半成品分装提供了有效手段�Objective To develop a high performance liquid chromatography(HPLC)method for determination of aluminium adjuvant content in vaccine,and verify and preliminarily apply the method.Methods The 8-hydroxyquinoline derivatization method was used for determination.The chromatographic column was phenyl-hexyl column[Luna 5u PhenylHexyl(250 mm × 4.6 mm)],and the mobile phase was composed of ammonium acetate solution-acetonitrile(with 8-hydroxyquinoline)(60 ∶ 40)containing 20 mg/L ascorbic acid,while eluted at a flow rate of 1.0 mL/min with the isocratic eluent.The excitation wavelength and the emission wavelength of the fluorescence detector were 380 nm and 520 nm respectively.The column temperature was 40 ℃,and the sample injection was 50 μL.The developed method was verified for the specificity,linear range,accuracy,repeatability,stability and durability,and used to determine the aluminum content in 12 batches of vaccines.The results were compared with those determined by titration in general principle 3106of Chinese Pharmacopoeia(VolumeⅢ,2020 edition).Results No interference peaks appeared in the sample chromatogram,and the non-aluminum adjuvant vaccine components and phosphate buffer had no interference with the determination.The linearity of aluminum standard was good in the concentration range of 6.25 ~ 100 μg/mL,r = 0.999 6.The average results of spike recoveries of aluminum content in inactivated hepatitis A vaccine,recombinant hepatitis B vaccine,adsorbed acellular DTP vaccine and inactivated enterovirus 71 vaccine were 98.32%,100.85%,101.09% and 99.31%,respectively in the verification for accuracy.The relative standard deviations(RSDs) of the determination results of aluminum content in the solution of six samples of the four vaccines in the same batch were 1.09%,1.42%,0.97% and1.30%,respectively.The RSDs of aluminum content of four vaccine samples stored at room tempe-rature for 0,2,4,6 and8 h were 0.82%,0.73%,0.40% and 0.48%,respectively.When the ratio of ammonium acetate solution to 8-hydroxyquinoline

关 键 词：疫苗 铝佐剂 高效液相色谱 

分 类 号：R917[医药卫生—药物分析学]