高效阴离子交换色谱-脉冲安培检测法测定b型流感嗜血杆菌结合疫苗多糖及游离多糖的含量

目的：建立高效阴离子交换色谱-脉冲安培检测法（HPAEC-PAD）测定b型流感嗜血杆菌（Hib）结合疫苗的多糖及游离多糖含量。方法：将疫苗中的有效抗原成分-Hib多糖，其结构为多聚磷酸核糖基核糖醇（polyribosylribitol phosphate，PRP），用6 mol·L^-1盐酸进行水解，得到其核糖醇（ribitol）单体；使用CarboPac^® MA1阴离子交换柱（4 mm×250 mm），以580 mmol·L^-1氢氧化钠溶液为流动相，流速为0.4 mL·min^-1，柱温35℃，进样体积100 μL，脉冲安培检测器收集信号，核糖醇的含量以峰面积计算得到，再根据核糖醇占PRP干重的质量分数（41.3%），计算出Hib结合疫苗中PRP及游离多糖（Free PRP）的含量，并评估该方法的重复性和再现性。结果：核糖醇质量浓度在0.075~1.05 μg·mL^-1范围内，与峰面积响应值呈良好的线性关系（r=0.999 9），最低检测限为2.5 ng·mL^-1；标准品供试溶液连续进样6次，峰面积及保留时间的RSD均小于5%；核糖醇加标回收率在80%~120%之间；对来自5个企业的4个不同批次的Hib结合疫苗的PRP及Free PRP含量进行测定，结果均符合规定。结论：本方法在测定Hib结合疫苗的PRP及Free PRP含量时，样品前处理简单，分离效果好，且灵敏度高，结果稳定，可靠，对控制该疫苗的质量具有重要意义。

Objective: To establish a detection method for polysaccharide and free polysaccharide contents of Haemophilus influenzae type b(Hib)conjugate vaccine by high performance anion exchange chromatography-pulsed amperometric detector(HPAEC-PAD). Methods: The polyribosylribitol phosphate(PRP) in the test sample of Hib conjugate vaccine was hydrolyzed with 6 mol·L^-1 hydrochloric acid, generating ribitol monomer. The chromatographic separation was achieved on a CarboPac^® MA1 anion-exchange column (4 mm×250 mm). The mobile phase was a water solution containing 580 mmol·L^-1 sodium hydroxide with isocratic elution at a flow rate of 0.4 mL·min^-1. The column temperature was set at 35℃ and the injection volume was 100 μL. Based on the ribitol monomer,the result of PRP and Free PRP contents was achieved by pulsed ampere detector. The method was tested for determination of ribitol using peak area,and the results were expressed as ribitol concentration. To obtain the PRP content,it was necessary to multiply the dilution factor,taking into consideration that ribitol accounted for 41.3% of the PRP dry weight. The repeatability and reproducibility of the analytical method were evaluated. Results: The ribitol concentration in range of 0.075-1.05 μg·mL^-1 showed good linear relationship with the peak area (r=0.999 9),while the quantitative limit was 2.5 ng·mL^-1;the ribitol control was loaded for 6 times,of which the RSDs of peak area and retention time were less than 5%. The results showed that the recovery of standard addition was 80%-120%. The results of PRP content and Free PRP content of 4 batches from 5 enterprises all accorded with the national standard. Conclusion: The method has the advantages of easy pretreatment,good distinguishability and high sensitiveness,which are of important significance in quality control of the vaccine.