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目的建立测定多糖结合疫苗中残余1-氰基-4-二甲氨基-吡啶四氟化硼(1-cyano-4-dimethylaminopyridinium tetrafluoroborate,CDAP)的高效液相色谱法(high performance liquid chromatography,HPLC),为多糖结合疫苗的质量监控提供依据。方法 HPLC色谱条件:色谱柱:TSK gel ODS-100V(4.6 mm×250 mm,5μm);流动相:乙腈-水-三氟乙酸(60∶40∶0.1);检测波长:300 nm;流速:1 ml/min;进样体积:15μl;理论塔板数按CDAP峰面积计算不低于2 000。以峰面积对浓度进行线性回归,确定该方法的线性范围,并对该方法进行专属性、检测限、定量限、精密度、稳定性及准确度验证。结果该方法检测CDAP专属性良好,最低检测限和定量限分别为35 ng/ml和40 ng/ml;CDAP浓度在160~4 000 ng/ml范围内,标准曲线的线性关系良好,R~2=0.999 9;供试品和CDAP标准品连续6次进样,RSD分别为0.26%、0.57%;供试品在8 h内稳定,平均加样回收率为94.5%,RSD为2.5%。结论建立了测定多糖结合疫苗中残余CDAP含量的HPLC测定方法,该方法准确、简便、可靠,能有效控制多糖结合疫苗的质量。
Objective To establish a high performance liquid chromatography (HPLC) method for the determination of residual 1-cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP) in polysaccharide conjugate vaccine. , Provide the basis for the quality control of polysaccharide conjugate vaccine. Method HPLC chromatographic conditions: column: TSK gel ODS-100V (4.6 mm × 250 mm, 5 μm); mobile phase: acetonitrile- water- trifluoroacetic acid (60:40:0.1); detection wavelength: 300 nm; ml / min; injection volume: 15μl; theoretical plate number calculated by CDAP peak area of not less than 2 000. The peak area of the concentration of linear regression to determine the linear range of the method, and the specificity of the method, limit of detection, limit of quantification, precision, stability and accuracy verification. Results The specificity of this method for detection of CDAP was good, with the lowest limit of detection (LOD) and limit of quantification (35 ng / ml and 40 ng / ml, respectively). The calibration curve was linear over a range of 160-4000 ng / = 0.999 9; The samples and CDAP standards were injected 6 times in a row with the RSDs of 0.26% and 0.57%, respectively. The samples were stable within 8 h with an average recovery of 94.5% and a RSD of 2.5%. Conclusion The HPLC method for the determination of residual CDAP in polysaccharide conjugate vaccine was established. The method is accurate, simple and reliable and can effectively control the quality of polysaccharide conjugate vaccine.