高效阴离子色谱-脉冲安培检测法测定脑膜炎球菌结合疫苗中A群多糖含量

目的：采用高效阴离子交换色谱-脉冲安培检测法（HPAEC-PAD）测定A群脑膜炎球菌多糖及脑膜炎球菌多糖蛋白结合疫苗中6-磷酸甘露糖胺的含量，并对该方法进行验证，实现微量A群脑膜炎球菌多糖的定量检测。方法：A群脑膜炎球菌多糖酸水解处理后，经¹H、¹³C NMR及COSY谱图分析，产物结构为6-磷酸甘露糖胺单体，然后进样CarboPac® PA10柱，氢氧化钠-醋酸钠梯度洗脱，脉冲安培检测。筛选样品的水解条件;对HPAEC-PAD法进行专属性、精密度及准确性验证，并进行初步应用。结果：确定样品的水解条件为2 mol·L^-1三氟乙酸煮沸5 h。 载体蛋白破伤风类毒素对检测无干扰；在1～10 μg·mL^-1范围内，标准曲线线性关系良好（r > 0.999），加样回收率在95.73%～102.4%之间;试验内和试验间精密度试验结果RSD小于5%。 HPAEC-PAD法测定疫苗原液的多糖含量与Chen法比色测定结果相当，成品疫苗的A群多糖含量测定符合《A、C群脑膜炎球菌结合疫苗制造及检定规程草案》（每剂均≥10 μg）和《A、C、W135、Y群脑膜炎球菌结合疫苗制造及检定规程草案》（每剂均≥4 μg）。结论：HPAEC-PAD法专属性、精密度和准确性良好，可用于A群脑膜炎球菌多糖含量测定，尤其是脑膜炎球菌结合疫苗中A群多糖含量测定。

Objective: To determine the mannosamine-6-phosphate content in group A meningococcal polysaccharides and conjugate vaccine by high-performance anion-exchange chromatography with pulsed amperometric detection(HPAEC-PAD)and verify the method. Methods: The hydrolysate of group A meningococcal polysaccharides hydrolyzed with trifluoracetic acid(TFA)was proved to be mannosamine-6-phosphate by ¹H and ¹³C NMR and COSY spectra analysis,which eluted by sodium hydroxide and sodium acetate,followed by chromatographic separation on an CarboPac® PA10 column coupled to the sensitive pulsed amperometric detector.The condition for hydrolysis was optimized,and the HPAEC-PAD method was verified for specificity,precision and accuracy,and was preliminarily applied. Results: The condition for hydrolysis was optimized as treatment with 2 mol·L^-1 TFA at 100℃ for 5 h.Tetanus toxoid(TT) as a carrier showed no interference to the detection result.The standard curve demonstrated a good linear relationship(r=0.9994)in the range of 1-10 μg·mL^-1.In accuracy test,recovery rate was between 95.73%-102.4%;the RSD of repeatability and intermediate precision was less than 5%.The polysaccharide content in bulk of conjugate determined by HPAEC-PAD was approximately equal to that by routine Chen assay.The content of group A polysaccharides in the final product met the Requirements for Groups A,C Meningococcal Conjugate Vaccine(Draft)(no less than 10 μg per dose)and the Requirements for Groups A,C,W135 and Y Meningococcal Conjugate Vaccine(Draft)(no less than 4 μg per dose). Conclusion: The HPAEC-PAD method showed high specificity,precision and accuracy,which might be used for determination of group A meningococcal polysaccharides in group A meningococcal conjugate vaccine.