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目的建立脑膜炎球菌结合疫苗中A群游离多糖含量的定量检测方法,并进行验证。方法采用高效阴离子色谱-电导法(high-performance anion-exchange chromatography with conductivity detection,HPAEC-CD),分析柱:IonPacTMAS11 Analytical(4 mm×250 mm),22 mmol/L Na OH流洗,流速为1 ml/min,25μl自动进样,电导检测。筛选样品的氧化反应最佳温度、双氧水浓度及干烤时间,确定HPAEC-CD的检测限和定量限,并进行专属性、准确性及精密度验证。应用建立的方法检测3批多糖蛋白结合物原液A-破伤风类毒素(A-tetanus toxoid,A-TT)多糖及游离多糖含量,并与《中国药典》三部(2010版)附录ⅦA磷测定法中的传统比色法进行比较;同时检测3批A、C群及A、C、W135、Y群脑膜炎球菌多糖蛋白结合疫苗的多糖及游离多糖含量。结果确定样品的氧化条件为:100μl/ml H2O2,220℃干烤90 min。磷标准品在0.1~1μg/ml范围内,峰面积和浓度线性关系良好(r=0.999 877);TT、C-TT、W-TT及Y-TT对测定无干扰;该方法的检测限为0.007μg/ml,定量限为0.024μg/ml;A群脑膜炎球菌多糖原液的加样回收率在97.56%~102.95%之间;重复性及不同操作者间的精密度试验结果 RSD均小于5%。A-TT的多糖含量及游离多糖含量与传统比色法测定结果差异较小;3批A、C群及A、C、W135、Y群脑膜炎球菌多糖蛋白结合疫苗的多糖含量均符合《A、C群脑膜炎球菌结合疫苗制造及检定规程草案》及《A、C、W135、Y群脑膜炎球菌结合疫苗制造及检定规程草案》要求,游离多糖含量分别为24.31%、23.04%、24.44%和19.07%、17.80%、19.46%。结论采用H2O2氧化处理,HPAEC-CD检测,除了能对脑膜炎球菌多糖蛋白结合疫苗中微量A群多糖含量进行定量检测外,还能够进行游离多糖含量的检测。该方法灵敏度和精密度良好,操作简单、安全,对操作者及环境提供了保护。
Objective To establish a quantitative detection method of free polysaccharide in group A meningococcal conjugate vaccine and verify the results. Methods High-performance anion-exchange chromatography with conductivity detection (HPAEC-CD) was used. The analytical column was IonPacTMAS11 (4 mm × 250 mm) and 22 mmol / L NaOH ml / min, 25μl automatic injection, conductivity detection. The optimum oxidation reaction temperature, hydrogen peroxide concentration and dry roasting time of the samples were screened to determine the detection limit and the limit of quantification of HPAEC-CD, and the specificity, accuracy and precision verification. The contents of polysaccharides and free polysaccharides of A-tetanus toxoid (A-TT) were detected by the established method and were compared with those of the Chinese Pharmacopoeia (2010) Method of traditional colorimetric method for comparison; simultaneous detection of three batches of A, C and A, C, W135, Y meningococcal polysaccharide protein conjugate vaccine polysaccharide and free polysaccharide content. As a result, it was confirmed that the oxidation conditions of the sample were: 100 μl / ml H2O2, dry roasted at 120 ° C for 90 min. The calibration curve was linear over the range of 0.1 ~ 1μg / ml (r = 0.999 877). TT, C-TT, W-TT and Y- 0.007μg / ml, the limit of quantification was 0.024μg / ml; the recoveries of the meningococcal group A polysaccharide solution were between 97.56% and 102.95%; the RSDs of repeatability and precision between different operators were less than 5 %. A-TT polysaccharide content and free polysaccharide content and the traditional colorimetric determination of the difference is small; 3 batches A, C group and A, C, W135, Y group meningococcal polysaccharide protein conjugate vaccine polysaccharide content are in line with “A , C draft meningococcal conjugate vaccine manufacturing and testing protocols ”and“ A, C, W135, Y group meningococcal conjugate vaccine manufacturing and testing protocols draft ”requirements, free polysaccharide content were 24.31%, respectively, 23.04%, 24.44% And 19.07%, 17.80% and 19.46% respectively. Conclusion H2O2 oxidation and HPAEC-CD detection can not only detect the content of trace A polysaccharide in meningococcal polysaccharide conjugate vaccine, but also detect the content of free polysaccharide. The method is sensitive and precise, easy to operate, safe and provides protection to the operator and the environment.