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

Quantification of polysaccharide in serogroup W135 and Y in ACYW135 polysaccharide-protein conjugates by HPAEC-PAD

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.