高效阴离子交换色谱-脉冲安培检测法测定吸附无细胞百(三组分)白破灭活脊髓灰质炎和b型流感嗜血杆菌(结合)联合疫苗的多糖含量  

Quantitative determination of polysaccharide content in diphtheria tetanus acellular component pertussis and inactivated poliomyelitis and Haemophilus type b conjugate combined vaccine by high performance anion-exchange chromatography with pulsed amperometric detector

作　　者：杨柏峰 吕溪琳 吴丽洁 赵明 李世慧 YANG Bai-feng;LYU Xi-lin;WU Li-jie;ZHAO Ming;LI Shi-hui(Beijing Institute of Biological Products Company Limited,Beijing 100176,China)

机构地区：[1]北京生物制品研究所有限责任公司细菌类疫苗研究室,100176

出　　处：《中国医药生物技术》2024年第1期37-44,共8页Chinese Medicinal Biotechnology

摘　　要：目的 采用高效阴离子交换色谱-脉冲安培检测法测定吸附无细胞百(三组分)白破灭活脊髓灰质炎和b型流感嗜血杆菌(结合)联合疫苗(以下简称DTac P-s IPV-Hib)中多糖含量,并对方法进行验证。方法 在1 ml样品中加入0.02 g柠檬酸钠,振荡混匀后,置于37℃保温24 h,4000×g离心5 min收集上清液,稀释后加入6 mol/L盐酸,100℃下加热2 h,冰浴10 min后加入0.8 ml氢氧化钠(1 mol/L)溶液用于终止酸水解。采用高效阴离子交换色谱-脉冲安培检测法根据检测信号峰面积计算对应水解荚膜多糖(PRP)含量,通过PRP占多糖含量干重的41.3%计算出总的多糖含量。结果 核糖醇参考品浓度在0.1~1.5μg/ml范围内标准曲线r2>0.99,检测下限可达10ng/ml。核糖醇加标回收率均在95%~105%;对高效阴离子交换色谱-脉冲安培检测法检测多糖的重复性和中间精密度进行验证,相对标准偏差(RSD)均小于5%,方法专属性和耐用性良好。结论 使用柠檬酸钠作为解吸附剂,与高效阴离子交换色谱-脉冲安培检测法相结合可用于DTac P-s IPV-Hib中Hib多糖的含量检测,为产品的质量控制和稳定性研究提供参考。Objective High performance anion-exchange chromatography with pulsed amperometric detector method(HPAEC-PAD)was used to determine the polysaccharide content in diphtheria tetanus acellular component pertussis and inactivated poliomyelitis and Haemophilus type b conjugate combined vaccine(DTacP-sIPV-Hib),and the validation of method was performed.Methods 0.02 g of sodium citrate was added to 1 ml of sample.It was shaked and mixed well,and then incubated at 37℃for 24 h.After centrifugation at 4000×g for 5 min,the supernatant was collected,diluted and then added with 6 mol/L hydrochloric acid.After heating at 100℃for 2 h and ice-bathing for 10 min,0.8 ml of sodium hydroxide(1 mol/L)solution was added to stop the acid hydrolysis.The HPAEC-PAD was used to calculate the corresponding hydrolyzed PRP content according to the peak area of the detection signal.The total polysaccharide content was calculated by PRP accounting for 41.3%of the dry weight of the polysaccharide content.Results When the concentration of ribitol reference is in the range of 0.1-1.5μg/ml,the r2 of the standard curve is greater than 0.99,and the detection limit can reach 10 ng/ml.The spiked recovery of ribitol was in the range of 95%-105%.The repeatability and intermediate precision of the HPAEC-PAD for detecting polysaccharides were validated,and the relative standard deviation(RSD)was less than 5%.The specificity and durability of the method are good.Conclusion The HPAEC-PAD method with sodium citrate as desorbent could be used for detection of the polysaccharide content in DTacP-sIPV-Hib combined vaccine,thus providing a reference for product quality control and stability research.

关 键 词：高效阴离子交换色谱-脉冲安培检测法 联合疫苗 多糖含量 B型流感嗜血杆菌 

分 类 号：R392-33[医药卫生—免疫学]