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目的建立A群C群脑膜炎球菌结合疫苗中游离蛋白含量的HPLC检测方法,并对其进行验证。方法采用HPLC法,色谱条件:色谱柱:TSKgel G3000SW(300 mm×7.5 mm,10μm);流动相:0.01 mol/L磷酸盐缓冲液[pH(7.2±0.2)];流速:0.5 ml/min,自动进样100μl;检测波长:280 nm;柱温:室温。对该方法进行专属性、分离度、线性、精密性和准确性验证,确定该方法的定量限和检测限;应用建立的方法检测3批A、C群脑膜炎球菌结合疫苗原液及A群C群脑膜炎球菌结合疫苗成品中游离蛋白含量。结果该方法检测游离蛋白的专属性、分离度良好;蛋白对照品在3~18μg/ml范围内,标准曲线的线性关系良好,R~2=0.998;3批A、C群脑膜炎球菌结合疫苗原液、A群C群脑膜炎球菌结合疫苗成品的加样回收率在82.9%~119.33%之间,RSD小于5%;确定方法检测限为1.0μg/ml,定量限为2.6μg/ml。3批A、C群脑膜炎球菌结合疫苗原液和A群C群脑膜炎球菌结合疫苗成品未检测出游离蛋白。结论建立的HPLC法简便、灵敏、准确度高,可检测A群C群脑膜炎球菌结合疫苗中游离蛋白含量。
Objective To establish an HPLC method for the determination of free protein in group A meningococcal conjugate vaccine and to verify the method. Methods The chromatographic conditions were as follows: column: TSKgel G3000SW (300 mm × 7.5 mm, 10 μm); mobile phase: 0.01 mol / L phosphate buffer [pH 7.2 ± 0.2] Auto-injection 100μl; detection wavelength: 280 nm; column temperature: room temperature. The specificity, resolution, linearity, precision and accuracy of this method were validated to determine the limit of quantification and detection limit of this method. The established method was used to detect three batches of meningococcal conjugate vaccine of Group A and Group C and group A Group of meningococcal conjugate vaccine free protein content. Results The specificity of this method was good and the resolution was good. The calibration curve of the protein was good in the range of 3 ~ 18μg / ml with R ~ 2 = 0.998. Three batches of meningococcal conjugate vaccine The recoveries of the stock solution of the meningococcal group A and group C meningococcal conjugate vaccines ranged from 82.9% to 119.33% with a RSD of less than 5%. The detection limit was 1.0μg / ml and the limit of quantification was 2.6μg / ml. Three batches of meningococcal conjugate vaccine of group A and group C, and the group A meningococcal conjugate vaccine vaccine did not detect free protein. Conclusion The established HPLC method is simple, sensitive and accurate. It can detect free protein in meningococcal group A group C vaccine.