注射用两性霉素B脂质体效价测定法的改进

Improvement of microbiological potency determination of amphotericin B liposome for injection

目的建立准确测定注射用两性霉素B脂质体效价含量的生物测定方法,解决现有质控标准中培养体系下无法形成抑菌圈的难点问题。方法效价测定方法:管碟法,沙氏培养基(含1%氯化钠)pH6.0~6.2,磷酸盐缓冲液(pH7.0,含1%聚山梨醇),检定菌为啤酒酵母(ATCC9763)。桥接液相方法:色谱柱:CAPCELLPAK MGⅡC18(250mm×4.6mm,5μm);流动相:乙腈:磷酸溶液(pH1.00±0.05)(370:630, V/V);流速:0.8mL/min;柱温:25℃;检测波长:383nm;进样量:10μL。结果改进后的方法:抑菌圈边缘清晰,且其抑菌圈直径大小符合生物测定法的方法学要求;在1.0~11.9u/mL浓度范围内线性良好(r=0.9995);可信限率符合中国药典要求(不大于5%);通过HPLC间接桥接方式验证,效价含量测定结果准确,可满足预期用途。结论改进后的效价测定方法通过了方法学验证,可作为注射用两性霉素B脂质体的日常质控方法。

Objective To establish a bioassay method for accurately determining the titer of amphotericin B liposome for injection and solve the difficult problem that the inhibition zone cannot be formed under the culture system in the existing quality control standards. Methods The cylinder pate method with sabouraud medium(containing 1% sodium chloride, pH 6.0~6.2), phosphate buffer(pH 7.0, containing 1% polysorbate) was used. The test organism was Saccharomyces cerevisiae(ATCC9763). The HPLC method: column: CAPCELLPAK MG IIC18(250 mm×4.6 mm, 5μm), mobile phase: Acetonitrile: Phosphoric acid solution(pH 1.00±0.05)(370: 630, V/V), flow rate: 0.8 mL/min, column temperature: 25°C, detection wavelength: 383 nm, and injection amount: 10μL. Results The improved method showed that the inhibition zone was suitable in size and the edge was clear. The linearity was good in the concentration range of 1.0~111.9 u/mL(r=0.9995). The credibility rate is in line with the requirements of the Chinese Pharmacopoeia(not more than 5%). Conclusion The improved method for determining the titer content has passed the methodological verification, which can be used as a daily quality control method for amphotericin B liposome for injection.