亚单位流感疫苗中裂解剂壬苯醇醚-9含量HPLC检测方法的建立

Development of HPLC Method for Detection of Cleavage Reagent Nonoxynol-9 Content in Influenza Virus Subunit Vaccine

目的建立亚单位流感疫苗中裂解剂壬苯醇醚-9含量的HPLC检测方法。方法采用4倍体积甲醇沉淀处理亚单位流感疫苗样品,HPLC色谱条件为：色谱柱：Thermo BioBasic-4 C4柱（4.6 mm×150 mm,5μm）,流动相：甲醇∶水=80∶20（v/v）,流速：1 ml/min,紫外检测器检测波长：275 nm,柱温：33℃,上样量：100μl。并对建立的HPLC法进行验证。结果建立的HPLC法检测壬苯醇醚-9标准品在5~2 500μg/ml浓度范围内线性关系良好;检测壬苯醇醚-9的专属性良好;检测壬苯醇醚-9在灭活流感全病毒提取液和亚单位流感疫苗中的回收率约为100%;检测各浓度壬苯醇醚-9标准品溶液和亚单位流感疫苗的日内和日间精密性均小于3%;检测限和定量限均为5μg/ml。结论建立的HPLC法简便、快速、准确,适用于实验室对亚单位流感疫苗中裂解剂含量的常规检测。

Objective To develop a HPLC method for detection of cleavage reagent nonoxynol-9 content in influenza virus subunit vaccine.Methods The test samples of influenza virus subunit vaccine were precipitated with four volumes of methanol.The condition for HPLC was as follows： column： Thermo BioBasic-4 C4 column（4.6 mm × 150 mm,5 μm）;mobile phase： methanol ： water = 80 ∶ 20（v / v）;flow rate： 1 ml / min;detection wavelength of ultraviolet detector： 275 nm;column temperature： 33℃;sample load：100 μl.Results The developed HPLC method showed good linearity for detection of nonoxynol-9 at concentrations of 5 ~ 2 500 μg / ml.The method showed also high specificity for nonoxynol-9.The recovery rates of nonoxynol-9 in inactivated influenza whole virus vaccine extract and in influenza virus subunit vaccine were both about 100%.Both intra-and inter-precision of determination results of nonoxynol-9 standard and influenza virus subunit vaccine were less than 3%.Both the limits of detection and quantification were 5 μg / ml.Conclusion The developed HPLC method was simple,rapid,accurate and suitable for the routine laboratory determination of cleavage reagent content in influenza virus subunit vaccine.