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目的利用离子色谱法即高效阴离子交换柱层析—脉冲安培检测法(HPAEC-PAD),测定ACYW135脑膜炎球菌多糖蛋白结合物中Y群和W135群多糖含量的方法,并加以验证。方法将ACYW135脑膜炎球菌多糖蛋白结合物中Y群和W135群多糖用三氟乙酸(TFA)水解为特异性单糖(葡萄糖、半乳糖),并去除水解液中残留的TFA,用HPAEC-PAD分析检测,用PA10糖分析柱分离单糖,电化学检测器测定葡萄糖及半乳糖含量,用Chromeleon色谱工作站记录并分析数据。对上述方法进行专属性、准确性、重复性验证,确定该方法的检出限和定量限。结果 Y群和W135群多糖在TFA2.5 mol/L(终浓度)、90℃、4 h可完全水解为葡萄糖、半乳糖。对照品葡萄糖及半乳糖在0.10~60.00μg/m L范围内,质量浓度和色谱峰面积呈较好的线性关系,r均大于0.99,回收率为87.94%~109.80%,相对标准偏差(RSD)为1.00%~2.00%,检出限为0.05μg/m L(信噪比3∶1),定量限为0.10μg/m L(信噪比10∶1)。结论离子色谱法可同时检测脑膜炎球菌多糖蛋白结合物中Y群和W135群糖含量,该方法操作简便、灵敏、快速,干扰小,重现性好,适用于对相关疫苗生产过程中的质量控制。
Objective To determine the content of polysaccharide in Y group and W135 group of ACYW135 meningococcal polysaccharide protein conjugate by ion chromatography (HPAEC-PAD), and to verify the method. Methods The polysaccharide of Y group and W135 group in ACYW135 meningococcal polysaccharide protein conjugate was hydrolyzed to specific monosaccharide (glucose, galactose) by trifluoroacetic acid (TFA), and the residual TFA was removed from the hydrolyzate. HPAEC-PAD Analysis and detection, separation of monosaccharides with PA10 sugar analytical column, electrochemical detection of glucose and galactose content, with Chromeleon chromatography workstation to record and analyze the data. The above method for specificity, accuracy, reproducibility verification, to determine the method of detection limits and quantitative limits. Results The polysaccharides of group Y and group W135 were completely hydrolyzed to glucose and galactose at TMA 2.5 mol / L (final concentration) and 90 ℃ for 4 h. The calibration curves of glucose and galactose in the range of 0.10 ~ 60.00μg / m L showed a good linear relationship with the peak area of the chromatograms, r> 0.99 and the recoveries of 87.94% -109.80%. The relative standard deviations (RSDs) The detection limit was 0.05μg / m L (signal to noise ratio 3:1). The limit of quantification was 0.10μg / m L (signal to noise ratio 10:1). Conclusion Ion chromatography can simultaneously detect the content of Y and W135 glycoconjugates in the meningococcal polysaccharide conjugate. The method is simple, sensitive, rapid, sensitive and reproducible. It is suitable for the quality control of the related vaccine production process control.