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  • An optimized and validated RP-HPLC/UV detection method for simultaneous determination of all-trans-retinol (vitamin A) and alpha-tocopherol (vitamin E) in human serum: comparison of different particulate reversed-phase HPLC columns.

An optimized and validated RP-HPLC/UV detection method for simultaneous determination of all-trans-retinol (vitamin A) and alpha-tocopherol (vitamin E) in human serum: comparison of different particulate reversed-phase HPLC columns.

Journal of chromatography. B, Analytical technologies in the biomedical and life sciences (2010-08-11)
Abad Khan, Muhammad I Khan, Zafar Iqbal, Yasar Shah, Lateef Ahmad, David G Watson
ABSTRACT

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 alpha-tocopherol in human serum using retinyl acetate as internal standard in the concentration of 0.5 microg/ml. A liquid-phase extraction was applied to the 250 microl 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 microm), Brownlee analytical (Perkin Elmer) C(18) column (150 mm x 4.6 mm, 5 microm), and Supelco (Supelcosil) LC-18 column (150 mm x 3 mm, 3 microm), protected by a Perkin Elmer C(18) (30 mm x 4.6 mm, 10 microm; 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 microm and 3 microm columns, respectively. Complete separation of all the analytes was achieved in 3 and 6 min on 3 microm and 5 microm columns, respectively by injecting 20 microl 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 microm 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 microm columns. The method was validated and applied for the analysis of all-trans-retinol and alpha-tocopherol in the serum of human volunteers.

MATERIALS
Product Number
Brand
Product Description

Supelco
Retinyl Acetate (Vitamin A Acetate), Pharmaceutical Secondary Standard; Certified Reference Material
Retinol acetate, European Pharmacopoeia (EP) Reference Standard
Supelco
SUPELCOSIL LC-18-S HPLC Column, 5 μm particle size, L × I.D. 15 cm × 4.6 mm
Supelco
SUPELCOSIL LC-18-S HPLC Column, 5 μm particle size, L × I.D. 25 cm × 2.1 mm
Supelco
Retinyl acetate, analytical standard
Supelco
SUPELCOSIL LC-NH2-NP HPLC Column, 5 μm particle size, L × I.D. 25 cm × 4.6 mm
Supelco
SUPELCOSIL LC-18-S HPLC Column, 5 μm particle size, L × I.D. 25 cm × 4.6 mm
Supelco
SUPELCOSIL LC-NH2-NP Supelguard Cartridge, 5 μm particle size, L × I.D. 2 cm × 4 mm
Supelco
SUPELCOSIL LC-18 Supelguard Cartridge, 5 μm particle size, L × I.D. 2 cm × 4 mm
Sigma-Aldrich
Retinyl acetate, synthetic, crystalline solid or supercooled liquid
Supelco
SUPELCOSIL LC-18 Supelguard Cartridge, 5 μm particle size, L × I.D. 2 cm × 4 mm
Supelco
SUPELCOSIL LC-18 Supelguard Cartridge, 5 μm particle size, L × I.D. 2 cm × 3 mm
Supelco
SUPELCOSIL LC-18 Supelguard Cartridge, 5 μm particle size, L × I.D. 2 cm × 2.1 mm
Sigma-Aldrich
Retinyl acetate, solid or viscous liquid, BioReagent, synthetic, suitable for cell culture
Sigma-Aldrich
Retinyl acetate, synthetic, matrix dispersion, 475,000-650,000 USP units/g