高效阴离子交换色谱-脉冲安培法测定b型流感嗜血杆菌结合疫苗的多糖含量

Determination of polysaccharide content in Haemophilus influenzae type b conjugate vaccines by high performance anion exchange chromatography-pulsed amperometric detector

目的采用高效阴离子交换色谱-脉冲安培检测法(high performance anion exchange chromatography withpulsed amperometric detector,HPAEC-PAD)测定b型流感嗜血杆菌(haemophilus influenzae type b,Hib)结合疫苗的多聚磷酸核糖基核糖醇(polyribosylribitol phosphate,PRP)多糖含量。方法用终浓度为0.3 N的NaOH溶液将待测疫苗中的PRP多糖或分离得到的游离多糖在室温下振荡水解(16±8)h,采用CarboPac誖PA-10阴离子交换柱在NaOH/醋酸钠溶液梯度洗脱条件下分离水解产物,用脉冲安培检测器检测信号,根据样品的峰面积计算得到待测疫苗中的PRP多糖含量及游离多糖含量,样品均重复检测2次,计算2次检测结果的相对标准偏差(RSD)。同时采用《中国药典》三部(2010版)附录ⅧJ中的地衣酚法测定相同批次样品的PRP多糖含量及游离多糖含量,并对两种方法得到的结果进行比较。结果相同样品重复检测2次,结果间的差异非常小,RSD不超过4.6%。HPAEC-PAD法测得的总PRP结果与地衣酚法测得的结果接近,前者测得的S01～S03样品的PRP浓度值分别为后者的105%、104%和106%;HPAEC-PAD法测得的游离PRP结果与地衣酚法测得的结果差别较大,前者测得的S01～S03样品的游离PRP浓度值分别为后者的126%、109%和115%。结论 HPAEC-PAD法测定Hib结合疫苗的PRP多糖含量灵敏度高,重复性和分离效果好,样品不需要衍生处理,可同时测定多组分,可作为传统地衣酚法的替代方法用于Hib结合疫苗中多糖含量的测定。

Objective To determine the polyribosylribitol phosphate（PRP） content in Haemophilus influenzae type b conjugate vaccine by high performance anion exchange chromatography-pulsed amperometric detector（HPAEC-PAD）.Methods The PRP in test samples of Hib conjugate vaccine or isolated free polysaccharide was hydrolyzed with 0.3 mol / L sodium hydroxide at room temperature in oscillation for（16 ± 8） h,then separated by CarboPac PA-10 anion exchange column,eluted by sodium hydroxide / sodium acetate gradient elution,detected by pulsed amperometric detector,and calculated according to the peak area of test samples.The samples were tested for 2 times,and the relative standard deviation（RSD） of test results was calculated.Meanwhile,the PRP content in test sample or free PRP content of the same batch of vaccine were determined by orcinol method in the appendix of Chinese Pharmacopoeia（Volume Ⅲ,2010 edition）,and the result was compared with that by HPAEC-PAD.Results The RSD between 2 test results of the same sample was not more than 4.6%.The total PRP contents in samples S01~S03 determined by HPAEC-PAD were 105%,104% and 106%,while the free PRP contents were 126%,109% and 115% of those by orcinol method,respectively.Conclusion HPAEC-PAD method showed high sensitivity,reproducibility and separation effectiveness for PRP content in Hib conjugate vaccine,by which the samples needed no derivation,and multiple components were determined at the same time,therefore may be used as an alternative method of traditional orcinol method for deter-mination of PRP content in Hib conjugate vaccine.