微柱凝胶离心分离-HPLC法测定克霉唑脂质体的包封率

Determination of entrapment efficiency of clotrimazole liposome with Sephadex G-50 gel minicolumn centrifugation-HPLC

目的:建立克霉唑脂质体包封率的测定方法。方法:采用 HPLC 法测定药物含量,色谱柱为 Hypersil ODS2柱(250mm×4.6 mm,5 μm),检测波长为260 nm,流动相为甲醇-0.025 mol·L^(-1)磷酸氢二钠溶液(90:10),流速为1.0 mL·min^(-1)。采用 Sephadex G-50微柱离心分离脂质体与游离药物,以500 r·min^(-1)离心6s 制备微柱,加样量0.2 mL,洗脱液为pH7.0的磷酸盐缓冲液,2000 r·min^(-1)离心5 min,重复3次。结果:在本色谱条件下克霉唑与辅料分离良好,克霉唑的线性范围为15.1～105.7μg·mL^(-1)(r=0.9991),日内和日间精密度 RSD 均小于0.63%(n=6),平均回收率为97.2%～100.1%(n=3)。Sephadex G-50微柱对克霉唑的平均吸附率大于96.3%,而对空白脂质体的洗脱率大于96.6%。结论:采用 Sephadex G-50微柱离心分离脂质体与游离药物,再用 HPLC 法测定克霉唑脂质体中药物包封率,简便快速,结果重复性好。

Objective：To develop a method for determining the entrapment efficiency of clotrimazole liposome. Method：The content of clotrimazole was determined by HPLC with Hypersil ODS2 column（250mm×4.6mm,5μm） and detected at 260 nm. The mobile phase was composed by methanol -0. 025mol·L^-1 sodium dihydrogen phosphate solution（90： 10）with the flow rate of 1.0 mL·min^-1. The Sephadex G- 50 minicolumn centrifugation was adopted to separate the free clotrimazole from liposome dispersions. The Sephadex G -50 minicolumn was centrifuged at 500 r ·min^-1 for 6 s for removing proper water. The 0. 2 mL sample was centrifuged at 2000 r·min^-1 for 5 rain, and then washed for 3 times with Phosphate buffer solution. Results： It was found that the excipients in the liposome did not interfere with the assay. Clotrimazole had a good linear relation in the range of 15.1 - 105.7 μg· mL^-1 （r =0. 9991 ）. Both the inter - and intra - day RSD were all less than 0. 63% （n =6） ,the recoveries of clotrimazole with blank liposomes were between 97. 2% - 100. 1% （n = 3 ）. By flowing through the Sephadex G -50 minicolumn,the free clotrimazole was adsorbed more than 96.3% ,while the average recovery of blank liposome was more than 96. 6%. Conclusion：This method is technically simple, rapid and efficient to separate and determine entrapment efficiency of clotrimazole liposome.