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目的:采用高效阴离子交换色谱-脉冲安培(high performance anion exchange chromatography with pulsed amperometric detector,HPAEC-PAD)检测法测定冻干AC群脑膜炎球菌(结合)b型流感嗜血杆菌(结合)联合疫苗成品(以下简称冻干AC-Hib联合疫苗)中游离多糖的含量,并对方法进行验证.方法:合并15支成品用3 kDa超滤离心管浓缩,通过冷酚法分离游离多糖和多糖蛋白结合物,用2 mol·L-1盐酸水解游离多糖,得到对应的单糖;采用HPAEC-PAD法对水解的游离多糖进行分析,根据检测信号峰面积计算对应水解单糖含量,通过化学结构式确定多糖与水解后对应单糖摩尔比,计算出游离多糖的含量.结果:标准曲线R2>0. 99;不同多糖加标回收率均在80％ ～120％;A群多糖、C群多糖和Hib多糖的定量限分别为0. 025,0. 03和0. 05 μg·mL-1;对HPAEC-PAD法检测游离多糖的重复性和中间精密度进行验证,相对标准偏差(RSD)分别小于10％和15％;方法专属性、系统适用性及耐用性良好.结论:冷酚法与HPAEC-PAD法相结合可用于冻干AC-Hib联合疫苗中游离多糖的含量检测,为产品的质量控制和稳定性研究提供参考.“,”Objective: To develop a method for the determination of free polysaccharides in the AC-Hib conjugate vaccine using high performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD). Methods: Fifteen vials of the vaccine product were combined and concentrated with 3 kDa ultrafiltration centrifuge tubes. The sample was treated with cold phenol to remove the polysaccharides conjugated to the carrier protein. The free polysaccharides in the supernatant were hydrolyzed with 2 mol·L-1HCl, and the monosaccharide was analyzed by HPAEC-PAD. The hydrolyzed free polysaccharide was analyzed by HPAEC-PAD method. The content of free polysaccharide was calculated according to the peak area. The mole ratio of monosaccharide after hydrolysis which was used to calculate the content of free polysaccharide was determined through the chemical structure formula. Results: The standard curves generated had a good linearity, R2>0. 99; all the recovery rates of the three polysaccharides were in the range of 80％～120％. The method showed a high repeatability and intermediate precision, with relative standard deviation (RSD)less than 10％ and 15％, respectively. The quantitative limits of A, C and Hib polysaccharides were 0. 025, 0. 03 and 0. 05 μg·mL-1, respectively. Conclusion: The combination of the cold phenol treatment and HPAEC-PAD can be used to determine the free polysaccharides in the AC-Hib conjugate vaccine.