Determination of Ascorbic Acid and Dehydroascorbic Acid in Plasma by High-Performance Liquid Chromatography with Coulometric Detection - Are They Reliable Biomarkers of Oxidative Stress?

The concentrations of the hydrophilic antioxidants ascorbic acid and dehydroascorbic acid in plasma for some time have been considered possible biomarkers of oxidative stress. However, several problems are associated with the accurate measurement of these two compounds. We have developed and validated a selective and reproducible high-performance liquid chromatographic method for the quantification of ascorbic acid and dehydroascorbic acid in plasma. The method meets the requirements of a reliable routine analysis. The plasma samples are stabilized with 5 mM metaphosphoric acid, centrifugated at 4 degrees C before HPLC analysis. For ascorbic acid analysis, the sample pH is adjusted to 2.6, whereas for total ascorbic acid measurement dehydroascorbic acid is reduced to ascorbic acid using dithiothreitol for 5 min at pH 6.2 after which the sample pH is adjusted to 2.6. The samples are analyzed on a reversed-phase system using coulometric detection. Dehydroascorbic acid concentrations ae calculated by subtraction. Within- and between-day coefficients of variation for the complete assay were in the range of 4-8 and 3-6% for ascorbic acid and total ascorbic acid, respectively. The stability of ascorbic acid was monitored under various conditions including storage and the implications as well as the reliability of ascorbic acid as a biomarker are discussed.