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

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.