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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

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Abstract

A versatile isocratic reversed-phase liquid chromatographic/ultraviolet–visible detection method for simultaneous determination of all-trans-retinol and α-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 RP18 (150 × 4.6 mm, 5 μm) analytical column protected by a Perkin Elmer RP18 (30 × 4.6 mm, 10 μm) 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 μg 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 μL 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 α-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.

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Authors and Affiliations

  1. Department of Pharmacy, University of Peshawar, Peshawar-25120, Pakistan

    Muhammad I. Khan, Abad Khan, Zafar Iqbal, Lateef Ahmad & Yasar Shah

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  1. Muhammad I. Khan
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  2. Abad Khan
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  3. Zafar Iqbal
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  4. Lateef Ahmad
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  5. Yasar Shah
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Correspondence to Zafar Iqbal.

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Khan, M.I., Khan, A., Iqbal, Z. et al. 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. Chroma 71, 577–586 (2010). https://doi.org/10.1365/s10337-010-1486-0

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