高效液相色谱法同时测定离体角膜灌流液中8个普萘洛尔衍生物

Simultaneous determination of eight derivatives of propranolol in cornea perfusate in vitro by high performance liquid chromatography

为了解促渗透剂对不同物质的促渗透能力,建立了采用高效液相色谱分离测定8个普萘洛尔衍生物的分析方法。采用盒式给药技术在离体角膜上皮侧给予普萘洛尔衍生物,内皮侧接收灌流液,用甲醇沉淀蛋白、高速离心后进样,采用Agilent Zorbax Extend色谱柱,以乙腈(A)-0.03%(v/v)磷酸水溶液(B)为流动相,在3%A～20%A间梯度洗脱,柱温30℃,检测波长205 nm。结果表明,该方法可在31 min内实现离体角膜灌流液中8个衍生物的完全分离和定量,线性范围为0.2(0.1)～40.0μmol/L,线性关系良好,相关系数均不小于0.997 0。其加样回收率为91.12%～105.73%,日内精密度(相对标准偏差(RSD))为1.00%～11.63%,日间精密度为1.18%～18.58%,样本冻存、冻融稳定性良好。该法适用于离体角膜灌流、细胞小室培养等跨膜吸收研究中普萘洛尔衍生物的检测。

A high performance liquid chromatographic method was developed for the simultaneous determination of eight derivatives of propranolol.Cassette dosing method was used in the epithelium side of cornea in vitro to get the effect of penetrant,and the perfusate was collected in the side of endothelium.The protein in the sample was precipitated and discarded by high speed centrifugation before injection.An Agilent Zorbax Extend column（150 mm×3 mm,5μm） was used at 30 ℃.The mobile phase system contained acetonitrile and 0.03%（v/v） phosphoric acid aqueous solution and the percentage of acetonitrile changed between 3% and 20%（v/v） in a linear gradient elution.The samples were detected by an ultraviolet（UV） detector at 205 nm.The results showed that the eight derivatives of propranolol were completely separated and determined in 31 min.The correlation coefficients were above 0.997 0 and good linear relationships were obtained in the range of 0.2（0.1）-40.0 μmol/L.Under the optimized conditions,the recoveries of the derivatives were in the range of 91.12%-105.73%.The intra-day relative standard deviations（RSDs） were in the range of 1.00%-11.63%,and the inter-day RSDs were in the range of 1.18%-18.58%.The sample showed stability under room temperature,freeze and three cycles of freeze-thaw conditions.This method is fast and accurate for the quantitative analysis of the derivatives of propranolol in transmembrane absorption such as cornea perfusion in vitro or transwell cell system.