高效阴离子交换色谱-脉冲安培法测定W135群和Y群脑膜炎球菌多糖中半乳糖和葡萄糖含量

Determination of galactose and glucose contents in groups W135 and Y meningococcal polysaccharide by high-performance anion-exchange chromatography with pulsed amperometric detection

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 galactose and glucose contents in groups W135 and Y meningococcal polysaccharide by high-performance anion-exchange chromatography with pulsed amperometric detection（HPAEC-PAD）,and verify and preliminarily apply the method.Methods The samples of pooled bulks of polysaccharide-protein conjugate was hydrolyzed with hydrochloric acid and loaded onto CarboPac PA10 column,then eluted by sodium hydroxide-sodium acetate gradient elution and determined by pulse amperometric detection mold.The condition for hydrolysis was optimized,and HPAEC-PAD method was verified for specificity,precision and accuracy,and preliminarily applied.Results The condition for hydrolysis was optimized as treatment with 2 mol / L hydrochloric acid at 85℃ for 1 h.Tetanus toxoid（TT） as a carrier showed no interference to detection result.The RSDs of results of reproducibility test as well as intra-and inter-assays were less than 5%.In accuracy test,the recovery rates of glucose and galactose were 90% ~ 110%.The polysaccharide content in bulk of conjugate determined by HPAEC-PAD was approximately equal to that by routine resorcinol assay.The contents of groups W135 and Y polysaccharides in final product met the Requirements for Groups A,C,W135 and Y Meningococcal Conjugate Vaccine（Draft）（not less than 4 μg / dose）.Conclusion HPAEC-PAD method showed high specificity,precision and accuracy,which might be used for determination of groups W135 and Y meningococcal polysaccharide contents in groups A,C,W135 and Y meningococcal conjugate vaccine.