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目的采用高效阴离子交换色谱-脉冲安培法(High-performance anion-exchange chromatography with pulsedamperometric detection,HPAEC-PAD)测定W135群和Y群脑膜炎球菌荚膜多糖中半乳糖和葡萄糖的含量,并对该方法进行验证及初步应用。方法多糖蛋白结合物原液的混合液经HCl水解处理后,进样CarboPac PA10柱,以氢氧化钠-醋酸钠梯度流洗,脉冲积分安培检测器检测。筛选样品的水解条件;对HPAEC-PAD法进行专属性、精密性及准确性验证,并进行初步应用。结果确定的样品水解条件为以2 mol/L HCl于85℃处理1 h。载体蛋白破伤风类毒素对检测结果无干扰;重复性、日内精密性和不同操作者之间精密性试验结果的RSD均小于5%;准确性试验中葡萄糖和半乳糖的回收率在90%～110%之间。HPAEC-PAD法测定多糖蛋白结合物原液的多糖含量与传统间苯二酚比色法测定结果相当;成品疫苗中的W135群和Y群多糖含量测定结果符合《A、C、W135、Y群脑膜炎球菌结合疫苗制造及检定规程草案》的规定(均≥4μg/剂)。结论 HPAEC-PAD法专属性、精密性和准确性良好,可用于A、C、W135、Y群脑膜炎球菌结合疫苗中W135群和Y群多糖含量的测定。
OBJECTIVE To determine the content of galactose and glucose in the capsular polysaccharides of W135 group and Y group by high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) For verification and preliminary application. Methods After the mixture of polysaccharide protein conjugate stock solution was hydrolyzed by HCl, it was injected into CarboPac PA10 column, washed with gradient flow of sodium hydroxide - sodium acetate and detected by pulse integral Ampere detector. Screening samples hydrolysis conditions; HPAEC-PAD method for specificity, precision and accuracy verification, and the initial application. The results of the determination of the sample hydrolysis conditions with 2 mol / L HCl at 85 ℃ for 1 h. Carrier protein tetanus toxoid on the test results without interference; repeatability, intra-day precision and precision between the different operators RSD results were less than 5%; accuracy test glucose and galactose recovery of 90% Between 110%. HPAEC-PAD method polysaccharide protein conjugate solution polysaccharide content and traditional resorcinol colorimetric determination of the results; finished vaccine W135 and Y polysaccharide content determination results in line with "A, C, W135, Y group of meninges The provisions of the draft for the manufacture and verification of the conjugate vaccine for both sexes (all ≥ 4 μg / dose). Conclusion The specificity, precision and accuracy of the HPAEC-PAD method are good and can be used to determine the content of polysaccharide in W135 group and Y group of meningococcal conjugate vaccine of group A, C, W135 and Y.