反相高效液相色谱法测定兔眼玻璃体腔环孢素A浓度的研究

Study of RP-HPLC on determination of the concentration of cyclosporin A in vitreous of rabbit eyes

目的 探讨反相高效液相色谱法 (RP- HPL C)测定兔眼玻璃体腔环孢素 A(Cs A)浓度的可行性。方法 兔眼玻璃体标本 0 .3ml经乙醚单步提取后 ,甲醇溶解回收 ,以 C1 8硅胶键合相为固定相 ,乙腈 :水 (76 :2 4 )为流动相 ,流速 :0 .2 m l/m in,75℃柱温下进行色谱分析 ,紫外检测波长为 2 0 0 nm。结果 Cs A兔眼玻璃体浓度在 2 5～4 0 0 ng/ml范围内呈良好的线性关系 ,r=0 .9981,该方法的平均回收率为 97.7% ,绝对回收率为 88.3% ,日内和日间精密度 RSD均 <10 % (n=5 ) ,最低检测限为 10 ng/ml。结论 RP- HPL C检测兔眼玻璃体腔 Cs A浓度结果可靠 ,适用性强 。

Objective To explore a practical RP-HPLC method to determine the concentration of cyclosporin A(CsA) in vitreous of rabbit eyes.Methods CsA was extracted from vitreous with diethyl ether and separated using a C 18 column and a mobile phase of acetonitrile-purified water (76∶24). The column temperature was maintained at 75℃ and CsA was detected with ultraviolet ray at 200nm at the flow rate of 0.2 ml/min.Results A good linear relationship was obtained between the amount of CsA from 25 ng/ml to 400 ng/ml, r=0.9981.The average method recovery and absolute recovery of CsA was 97.7% and 88.3% respectively, and the relative standard deviation(RSD) within-day and between-day were less than 10%(n=5).The detection limit of CsA was 10 ng/ml.Conclusions The improved method can enhance the detective limitation significantly without causing damage to the column and decreasing the accuracy.The procedure is simple, sensitive and suitable for the determination of CsA in vitreous with good practicability.