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

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-retinal and a-tocopherol in human serum using retinyl acetate as internal standard in the concentration of 0.5 mu g/ml. A liquid-phase extraction was applied to the 250 mu l 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 (150 mm x 4.6 mm, 5 mu m), Brownlee analytical (Perkin Elmer) C-18 column (150 mm x 4.6 mm, 5 mu m), and Supelco (Supelcosil) LC-18 column (150 mm x 3 mm, 3 mu m), protected by a Perkin Elmer C-18 (30 mm x 4.6 mm, 10 mu m; Norwalk, USA) pre-column guard cartridge, at 292 nm wavelength, using methanol-water (99:1, v/v), in isocratic mode as mobile phase applied at flow rate of 1.5 ml/min and 1 ml/min for both 5 mu m and 3 mu m columns, respectively. Complete separation of all the analytes was achieved in 3 and 6 min on 3 mu m and 5 mu m columns, respectively by injecting 20 mu l of sample into the HPLC system by autosampler, keeping column oven temperature at 25 degrees 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 3 mu m 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 5 mu m columns. The method was validated and applied for the analysis of all-trans-retinol and a-tocopherol in the serum of human volunteers. (C) 2010 Elsevier B.V. All rights reserved.