Optimization and Validation of RP-LC/UV-VIS Detection Method for Simultaneous Determination of Fat-Soluble Anti-Oxidant Vitamins, all-trans-Retinol and α-Tocopherol in Human Serum: Effect of Experimental Parameters

A versatile isocratic reversed-phase liquid chromatographic/ultraviolet-visible detection method for simultaneous determination of all-trans-retinol and alpha-tocopherol in human serum was developed and validated after optimization of various chromatographic conditions and other experimental parameters. Analytes were separated on a Kromasil 100 RP(18) (150 x 4.6 mm, 5 mu m) analytical column protected by a Perkin Elmer RP(18) (30 x 4.6 mm, 10 mu m) guard cartridge. The mobile phase, methanol-water (96:04 v/v) was pumped at a flow rate of 2.2 mL min(-1) and the column eluents were monitored at the wavelength of 292 nm using retinyl acetate (1.0 mu g mL(-1)) as the internal standard for both analytes. Sample preparation was based on protein precipitation and stabilization with 2,6-bis(1,1-dimethylethyl)-4-methylphenol/ethanol and a two step extraction process using n-hexane followed by dichloromethane as extraction solvents. Sample size was kept 20 mu L and separation of analytes was achieved in less than 7 min. The present method demonstrated acceptable values for specificity/selectivity, linearity within the expected concentration range, recovery, precision, sensitivity, stability of solutions, robustness, and system suitability specifications and tests. The method was used for monitoring all-trans-retinol and alpha-tocopherol concentrations in human serum samples and could also be applied to other sample matrices such as brain slices and cosmetic products if attention is paid to the extraction procedure.