An optimized and validated RP-HPLC/UV detection method for simultaneous determination of all-trans-Retinol (Vitamin A) and α-Tocopherol (Vitamin E) in human serum: Comparison of different particulate reversed-phase HPLC columns

摘要

A novel, simple and fast reversed-phase HPLC/UV method was developed, optimized for various chromatographic conditions, and validated according to international guidelines for simultaneous determination of all-trans-retinol and α-tocopherol in human serum using retinyl acetate as internal standard in the concentration of 0.5μg/ml. A liquid-phase extraction was applied to the 250μl of serum with n-hexane-dichloromethane mixture (70:30, v/v), in two steps, using ethanol-methanol mixture (95:5, v/v) for protein precipitation and BHT (butylated hydroxy toluene) as stabilizer for sample preparation. Both analytes were analyzed on Kromasil 100 C_(18) column (150mm×4.6mm, 5μm), Brownlee analytical (Perkin Elmer) C_(18) column (150mm×4.6mm, 5μm), and Supelco (Supelcosil) LC-18 column (150mm×3mm, 3μm), protected by a Perkin Elmer C_(18) (30mm×4.6mm, 10μm; Norwalk, USA) pre-column guard cartridge, at 292nm wavelength, using methanol-water (99:1, v/v), in isocratic mode as mobile phase applied at flow rate of 1.5ml/min and 1ml/min for both 5μm and 3μm columns, respectively. Complete separation of all the analytes was achieved in 3 and 6min on 3μm and 5μm columns, respectively by injecting 20μl of sample into the HPLC system by autosampler, keeping column oven temperature at 25°C. Different particulate reversed-phase chromatographic columns were evaluated in order to select the best column in terms of sensitivity, selectivity, resolution and short run time of both the analytes and it was concluded that 3μm columns are better to be used in clinical set up as well as in laboratories for the separation of these analytes in a shorter time as compared with 5μm columns. The method was validated and applied for the analysis of all-trans-retinol and α-tocopherol in the serum of human volunteers.