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Determination of ascorbic acid in human urine by high-performance liquid chromatography coupled with fluorimetry
Abstract:
High-performance liquid chromatography on two Asahipak GS-320 hydrophilic gel columns (50 X 0.76 cm I.D.), connected in series, with 0.015 M tartrate buffer (pH 3.0), containing 2 mM ethylenediaminetetraacetate and 0.05% beta-thiodiglycol as eluent allowed the separation of glucose, diketogulonic acid + diketogluconic acid, dehydroisoascorbic acid, dehydroascorbic acid, ascorbic acid, and isoascorbic acid within 55 min. Ascorbic acid in a urine sample was stabilized by the addition of an equal volume of 5% metaphosphoric acid solution, containing 0.5% of beta-thiodiglycol. Filtration of the mixture through a column of Dowex 50W-X8 (H+) facilitated the determination of ascorbic acid and isoascorbic acid in human urine. Samples could be analyzed every 20 min.