浊度法与膜材测定法在评价脂质体柱回收率上的对比研究

A comparative study of evaluating on the recovery of liposomes using the turbidity method and the membrane assay

目的研究浊度法与膜材测定法在评价脂质体柱回收率上的不同。方法采用改良乙醇注入法制备不同粒径的空白脂质体(liposome 1,L1;liposome 2,L2);建立高效液相色谱-蒸发光散射(HPLC-ELSD)法测定脂质体中胆固醇(cholesterol,CH)的含量;采用浊度法和膜材测定法对空白脂质体L1、L2于阳离子交换纤维柱,阳离子交换树脂柱及葡聚糖凝胶柱上的柱回收率进行测定,进而比较上述2种方法在评价相同脂质体于不同微柱回收率上的优劣;对洗脱过程中脂质体的粒径进行了检测。结果制备的空白脂质体L1、L2粒径分别为650.2nm和329.8nm;采用HPLC-ELSD法测定脂质体中CH的含量操作简便、结果准确、重现性好;采用浊度法测定柱回收率时,L1、L2在阳离子交换纤维柱,阳离子交换树脂柱及葡聚糖凝胶柱上回收率达到100%时对应的洗脱次数分别为3、4、4次和2、2、3次;采用膜材测定法测定脂质体柱回收率时,相应的洗脱次数分别为4、4、4次和3、3、3次。结论首次提出脂质体的"表观柱回收率"及"实际柱回收率"的概念,并用其进一步量化浊度法与膜材测定法在测定脂质体柱回收率的不同。浊度法与膜材测定法在评价脂质体柱回收率上存在很大差异,膜材测定法更为科学。

Objective To evaluate the recovery of liposomes using the turbidity method and the membrane assay.Methods The blank liposomes with different sizes（L1,L2） were prepared by modified ethanol injection method.The analytical method was established to determine the cholesterol of liposomes by the high performance liquid chromatography with evaporative light-scatter detector（HPLC-ELSD）.The turbidity method and the membrane assay were employed to determine the recovery of the cation exchange fiber column,the cation exchange resin column and the Sephadex G-50 column,and the differences between two methods were further discussed.The notions of the apparent recovery rate of column and the actual recovery rate of column were proposed for the first time.Moreover,the particle size of the eluent in the elution process was measured.Results The particle sizes of L1 and L2 were 650.2 nm and 329.8 nm,respectively.For the turbidity method,the elution times of the cation exchange fiber column,the cation exchange resin column and the Sephadex G-50 column were 3,4,4 and 2,2,3 to achieve the 100% recovery,w hile the elution times were4,4,4 and 3,3,3 using the membrane assay.In the elution process,the small size of liposomes were first eluted,and the last was the large size of liposomes.Conclusions There is a great difference in the evaluation of the recovery betw een the turbidity method and the membrane assay.It is unscientific to investigate the elution times by the turbidity method,resulting in the erroneous results.The membrane assay is a more scientific and better accurate method.