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

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 PAK C₈ DD(4.6 mm×250 mm,5 μm);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1.0 mL·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0.41 μg·mL^-1;最低定量限为1.39 μg·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.