A Procedure for the Separation and Quantitative Analysis of Ascorbic Acid, Dehydroascorbic Acid, Isoascorbic Acid, and Dehydroisoascorbic Acid in Food and Animal Tissue

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Abstract
A procedure is presented for the direct and simultaneous determination of ascorbic acid (AA) and isoascorbic acid (IAA) in food products and animal tissues by reverse phase high-performance liquid chromatography. Two PLRP-S columns in series were used with a pH 2.2 mobile phase containing 20 mM phosphate buffer and 0.17% metaphosphoric acid. An amperometric detector set at 0.7 volt and 20 mA was used. As little as 0.5 ng of each compound could be detected. When the same samples were incubated with homocysteine to reduce dehydroascorbic acid (DHAA) and dehydroisoascorbic acid (DHIAA) to AA and IAA respectively and reinjected into the system, the values for total AA and IAA were obtained. The concentration of the oxidized forms, DHAA and DHIAA, could then be calculated by substraction.