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

摘要:

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,3.5 μm),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20 μL。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。

Abstract:

Objective:To establish a simple,feasible and precise quality control strategy for determination 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 ultrafiltration,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 ultrafiltration 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,ultrafiltration 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.