离子色谱法测定ACYW135群脑膜炎球菌结合物中Y群及W135群多糖含量

摘要

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Chrome-leon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/mL 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/mL(信噪比3∶1),定量限为0.10μg /mL(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 set up a determination and verification of meningococcal polysaccharides ( PS) in serogroup W135 and Y in ACYW135 tetravalent polysaccharide-protein conjugates by means of HPAEC-PAD ( High performance ani-on exchange chromatography-pulsed amperometric detection). Methods The polysacchrides from the tetravalent conju-gates was hydrolyzed by TFA into monosacharide such as, glucose ( Gal)/galactose ( Glc) , which is characteristic of PSY/PSW respectively, and then analyzed by HPAEC-PAD followed with residual TFA removing from hydrolysis solution. Fur-thermore, Gal/Glc was determined by separation of PA10 anion exchange column, followed by detection with electrochemi-cal detector and Chromeleon workshop analysis. The specificity, accuracy and repeatability was verified for the test , re-spectively, and detection limit and quantitative limit were determined. Results The Gal/Glc was completely released un-der an optimized hydrolysis including, TFA with a final concentration 2. 5 mol/L, 90℃, and 4 hs. A good linear relation-ship was obtained from the concentration and peak’s area, with r>0. 99, within the range of 0. 10-60. 00 μg/mL for con-trols Gal and Glc. The average recovery rate was in 87. 94% ~109. 80%, and RSD (Relative standard deviation) was in 1. 00%-2. 00%. The detection limit was 0. 05μg/mL(Signal to noise ratio, SNR 3 ∶ 1) , and quantitative limit was 0. 10μg/mL (SNR 3 ∶ 1). Conclusion The content of polysaccharide in serogroup W135 and Y could be determined in the same time in the tetravalent conjugates by means of HPAEC-PAD, and the method was sensitive with a good repeatability associated with less interference, simple and fast in performance. It should be suitable to quality control of the related vaccine production.