Measurement of ascorbic acid and dehydroascorbic acid in mammalian tissue utilizing HPLC and electrochemical detection

Abstract

Reliable measurement of the reduced and oxidized forms of ascorbic acid (AA) is challenging because they are highly reactive and unstable compounds. Detection of small amounts of AA and dehydroascorbic acid (DHAA) is essential for determining the biochemical function of the vitamin. While a variety of techniques exist for measurement of AA with detection limits in the millimolar range, a need exists for highly reliable assessment of picomolar levels of AA and DHAA in tissues. The present study presents a method for measuring AA and DHAA that combines high performance liquid chromatography with the advantages and increased detection limits and selectivity available with coulometric electrochemical detection. The difference between AA and "total AA" in tissue samples gives an assessment of DHAA concentration. Verification of the reliability of the assay is by the successful linear recovery of exogenously added AA and DHAA in tissue homogenate. Optimal conditions for reducing DHAA in tissue samples include a pH of 7.2, reaction time of 10 min, reaction temperature equal to room temperature, and a 10 mM concentration of the reducing agent, beta-mercaptoethanol. AA and DHAA are measured in several mammalian tissues using the method presented.