Zeitschrift für Chromatographie und Trenntechniken
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Fluorometric Determination of Vitamin Constituents in Human Plasma Using Ultra Performance Liquid Chromatography

Edward C Bell, Mathew John, Rodney J Hughes and Thu Pham

Purpose: A rapid, selective and sensitive ultra performance liquid chromatography method has been developed for the detection and quantification of several vitamins in human plasma. Methods: Alpha tocopherol, gamma tocopherol, and retinol are assayed using fluorescence detection. Excitation/emission wavelengths are 295 nm/330 nm and 325 nm/470 nm for the analysis of both tocopherols and retinol, respectively. Retinol acetate is employed as the internal standard. The reversed phase method incorporates gradient elution with a mobile phase consisting of methanol and acetonitrile. Separation of vitamin compounds is achieved using a bridged ethyl-hybrid C18 column. Results: The retention times for retinol, retinol acetate, gamma tocopherol and alpha tocopherol are 1.6 minutes, 1.8 minutes, 3.9 minutes and 4.3 minutes, respectively. The limits of quantification for retinol, gamma tocopherol and alpha tocopherol, were 0.02 μg/ml, 0.02 μg/ml, and 0.1 μg/ml, respectively. Conclusion: The assay method is suitable for the analysis of these vitamins in human plasma following the ingestion of foods fortified with these fat soluble vitamins.