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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 inhibitory effect of polyphosphorothricin-1,6-diphosphorubicitrate (Hib) conjugate vaccine against Haemophilus influenzae type b (Hib) by high performance anion exchange chromatography with pulsed amperometric detector (HPAEC-PAD) (polyribosylribitol phosphate, PRP) polysaccharide content. Methods The PRP polysaccharide or free polysaccharide in the test vaccine was shaken hydrolyzed at room temperature (16 ± 8) h with a NaOH solution of 0.3 N final concentration. CarboPac anion exchange column PA-10 was used in NaOH / sodium acetate Solution gradient elution conditions hydrolyzate, pulse amp detector detection signal, according to the sample peak area calculated PRP polysaccharide content of the vaccine and free polysaccharide content, the sample was repeated two times to calculate the test results 2 Relative standard deviation (RSD). At the same time, the contents of PRP polysaccharide and free polysaccharides in the same batch of samples were determined by the method of lichenol in Appendix ⅧJ of “Chinese Pharmacopoeia” (2010 edition), and the results obtained by the two methods were compared. Results The same sample was tested twice, the difference between the results was very small, RSD does not exceed 4.6%. The total PRP measured by HPAEC-PAD method was close to that obtained by the phenol method of lichen. The former measured the PRP concentration of the samples S01-S03 at 105%, 104% and 106% of the latter respectively. The HPAEC-PAD method The measured free PRP results differed greatly from the results obtained with the phenol method of lichen. The values of free PRP in the former S01-S03 samples were 126%, 109% and 115% respectively. Conclusion The HPAEC-PAD method has high sensitivity, repeatability and isolation effect for PRP polysaccharide of Hib conjugate vaccine. The sample does not need derivatization and can be used for simultaneous determination of multi-component, which can be used as an alternative to the traditional lichen-phenol method for Hib conjugate vaccine Determination of polysaccharide content.