6]Analysis of ascorbic acid and dehydroascorbic acid in biological samples

Abstract

This chapter describes the analysis of ascorbic acid and dehydroascorbic acid in biological samples. The chapter focuses on ascorbate assays because these techniques are substantially more advanced than those for dehydroascorbic acid and most techniques for the latter are based on those of the former. High performance liquid chromatography (HPLC) with electrochemical (EC) detection is the ascorbate assay technique that provides the highest sensitivity, specificity, and accounts for substance interference. The general separation principles of HPLC are utilized to separate ascorbate from other substances. A mobile phase—or the solution for chromatography—is selected that optimizes both separation and detection. Once separation is accomplished, ascorbate is detected by one of two distinct types of EC detectors, amperometric EC detectors, or coulometric detectors. The principle of both detectors is that they pass a voltage across an area. Current generated by the voltage and the components of the mobile phase are measured. Amperometric EC detectors are “flow by” detectors, in which the solution containing ascorbate and the components of the mobile phase for HPLC separation flow around the detector. Coulometric detectors are “flow through” detectors. The mobile phase and ascorbate flow through the detector, which is porous. Coulometric detectors are preferred over amperometric detectors. The chapter concludes with a discussion of the detection of radiolabeled ascorbate and dehydroascorbic acid.