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 DTacP-sIPV-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。5 μg/ml 范围内标准曲线 r2>0。99，检测下限可达 10 ng/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 DTacP-sIPV-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。
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 amperomet
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 0.1-1.5 μg/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.
HPAEC-PADcombined vaccinepolysaccharide contentHaemophilus influenzae type b
万方数据
维普
