【摘要】 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1~10μg·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≥10μg)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≥4μg)。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。更多还原

【Abstract】 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 by1H and13C 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- 1TFA 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 as-say. 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.更多还原