Measurement of ascorbic acid and dehydroascorbic acid in biological samples.

Ascorbic acid and dehydroascorbic acid are commonly used biomarkers of oxidative stress in a variety of experimental models. However, the accurate measurement of these labile compounds remains a challenge both in terms of sample collection and analysis. Determination of dehydroascorbic acid most commonly involves indirect measurement. The concentration is calculated by subtraction of the measured ascorbic acid concentration from that of total ascorbic acid analyzed after reduction of the dehydroascorbic acid present; a method referred to as the subtraction method. Consequently, successful determination of dehydroascorbic acid is dependent upon proper sample handling, quantitative reduction of the compound, and accurate quantification of both ascorbic acid and total ascorbic acid. The unit presents a detailed introduction to ascorbate analysis in biological samples and discusses common problems and pitfalls. The analytical method described is based on reversed-phase HPLC with coloumetric detection. This method includes co-analysis of isoascorbic acid and uric acid. Where applicable, uric acid can conveniently be used as an endogenous intrasample standard that significantly improves the accuracy of the subsequent dehydroascorbic acid calculation.