异亚丙基莽草酸脂质体的包封率测定方法比较

Comparative Study of Entrapment Efficiency Determination for Isopropylidene Shikimic Acid Liposome

目的:研究异亚丙基莽草酸脂质体含量和包封率的测定方法,建立一套准确度好、简便可行的质控方案。方法:采用Agilent Eclipse Plus C18色谱柱(4.6 mm×100 mm,3.5μm),流动相乙腈-0.05%磷酸溶液(10∶90),检测波长220 nm,流速1.0 mL·min-1,柱温30℃,进样量20μL。采用HPLC测定脂质体中异亚丙基莽草酸的含量;比较超滤法、凝胶色谱法和透析法用于包封率的测定。结果:异亚丙基莽草酸脂质体采用4倍量甲醇破乳并高速离心沉淀后,取上清液进行测定,药物专属性良好,线性范围1.004～150.6 mg·L-1(r=0.999 9),加样回收率(102.01±1.18)%,准确度和精密度良好,异亚丙基莽草酸对照品溶液于6 h内稳定;超滤法、葡聚糖凝胶法及透析法测得的包封率分别为(92.96±1.91)%,(91.23±2.23)%,(73.66±7.10)%。结论:所建立的HPLC稳定可靠、准确度良好,可用于异亚丙基莽草酸脂质体的质量控制和体外分析;超滤法测定包封率简便快捷、测定结果与凝胶色谱法相当,可作为本品包封率测定的常规方法;透析法由于测定条件的限制,其测定结果受脂质体溶液稳定性、药物在脂质体中存在状态的影响较大,可能会造成测定结果偏低。

Objective： To establish a simple, feasible and precise quality control strategy for determiiaation of concentration and entrapment efficiency of isopropylidene shikimic acid liposome. Method： Chromatographic conditions were： Agilent Eclipse Plus C18 column （4.6 mm × 100 mm, 3.5 μm）, column temperature of 30 ℃ , mobile phase of acetonitrile-0. 05% phosphoric acid solution （ 10： 90） , flow rate of 1.0 mL · min^-1, injection volume of 20 μL and detection wavelength at 220 nm. The content of ISA in liposomes was determined by HPLC; Encapsulation efficiency was determined by uitrafiltration, gel chromatography and dialysis method, respectively, and results were compared with each other. Result： After demulsification by adding 4 times the amount of methanol into ISA liposome and high speed centrifugal sedimentation, supernatant was used to determine, determination results showed a good specificity, precision and accuracy, regression equation showed a good linearity （ r = 0. 999 9） within range of 1. 004-150.6 mg.L - 1, recovery was （ 102. 01 ± 1.18 ） % , reference substance solution of ISA was stable in 6 h; Entrapment efficiency of ISA liposome determined by ultrafihration method, gel permeation method and dialysis method were （92.96 ±1.91）%, （91.23 ±2.23）%, （73.66 ±7.10） % , respectively. Conclusion： This established HPLC method was stable and reliable, it could be used for quality control and in vitro analysis; In determination of encapsulation efficiency, uhrafiltration method was simple and efficient, its results were close to gel permeation chromatography; Due to limit of measurement conditions,determination result of dialysis method might be lower for its time-consuming process and higher temperature, and also depending on stability of liposome solution and existence state of drugs in liposome.