HPLC法测定人用皮卡狂犬病疫苗和皮卡佐剂中硫酸卡那霉素的含量

HPLC determination of kanamycin sulfate in PIKA rabies vaccine for human use and PIKA adjuvant

目的:建立测定人用皮卡狂犬病疫苗和皮卡佐剂中硫酸卡那霉素含量的HPLC方法。方法:色谱柱为CAPCELL PAKC8 DD(4.6 mm×250 mm,5μm);流动相为磷酸二氢钾缓冲液(0.02 mol.L-1,pH 7.5)-甲醇-乙腈(23∶7∶40);流速为1.0mL.min-1;柱温为45℃;采用柱前衍生化法,衍生化试剂为2,4,6-三硝基苯磺酸(TNBS);检测波长为350 nm。结果:在0.01～1.0 mg.mL-1范围内,硫酸卡那霉素的浓度与峰面积呈现良好的线性关系(A=3×106C-3313.9,r=1.000,n=7);在80%,100%,120%3个浓度下的平均回收率(n=3)分别为82.5%(RSD=1.6%),90.3%(RSD=2.0%),81.6%(RSD=1.8%);方法的最低检测限为0.41μg.mL-1;最低定量限为1.39μg.mL-1;供试品溶液在8 h内稳定(RSD=1.1%)。结论:本方法准确、快速,通用性良好。

Objective：To establish an HPLC method for determining the content of kanamycin sulfate in PIKA rabies vaccine for human use and PIKA adjuvant.Methods：A C8 column（CAPCELL PAK C8 DD,4.6 mm×250 mm,5 μm） was used,and the mobile phase was 0.02 mol·L-1 of potassium dihydrogen phosphate（adjusted with 1 mol·L-1 of potassium hydroxide to pH 7.5）-methanol-acetonitrile（23∶7∶40）.The flow rate was 1.0 mL·min-1,and the column temperature was kept at 45 ℃.The method was based on a pre-column derivatization of kanamycin sulfate with a 2,4,6-trinitrobenzenesulphonic acid reagent,and the detection wavelength was set at 350 nm.Results：The linear range was 0.01-1.0 mg·mL^-1,A=3×106C-3313.9,r =1.000（n =7）.The average recoveries at the concentration of about 80%,100% and 120% were 82.5%（n=3,RSD=1.6%）,90.3%（n=3,RSD=2.0%） and 81.6%（n=3,RSD=1.8%）,respectively.The LOD was 0.41 μg·mL^-1 and the LOQ was 1.39 μg·mL^-1.The sample solution was stable for at least eight hours（RSD=1.1%）.Conclusion：The method is accurate,rapid and can be used widely.