Simultaneous determination of eight β-blockers by gradient high-performance liquid chromatography with combined ultraviolet and fluorescence detection in corneal permeability studies in vitro

A gradient HPLC method with combined ultraviolet and fluorescence detection was developed for the simultaneous determination of eight β-blockers (alprenolol, atenolol, metoprolol, nadolol, pindolol, propranolol, sotalol and timolol) in corneal permeability studies in vitro. Fluorescence detection with excitation wavelength at 230 nm and emission at 302 nm was selective for six of the compounds, whereas UV detection at 205 nm was able to detect all the compounds. Calibration was performed with fluorescence detection for six compounds from 50 or 200 nM to 3 μM, and with UV detection for all the eight compounds from 100 or 200 nM to 30 μM. With optimized fluorescence detection, detection limits between 0.7 and 1.3 nM (0.035–0.065 pmol per 50 μl injection) were obtained for atenolol, metoprolol, nadolol and sotalol. A mixture of eight β-blockers was used in cassette dosing permeability studies with a cultured corneal epithelium. The HPLC method revealed marked differences in the permeation between hydrophilic and lipophilic β-blockers through the corneal epithelial cell culture model.