Ascorbic and dehydroascorbic acids measured in plasma preserved with dithiothreitol or metaphosphoric acid

Abstract

We describe a rapid method for accurately and precisely measuring ascorbic acid and dehydroascorbic acid in plasma. Total analysis time is <10 min, replicate analyses of a single pool provide precisions ≤2%, and values measured in supplemented samples agree with known concentrations of 4.68 and 11.83 mg/L. The stability and homogeneity of lyophilized plasma samples supplemented with ascorbic acid and dithiothreitol are documented. We also describe a procedure in which metaphosphoric acid (50 g/L) is used to prepare a reference material for the measurement of ascorbic acid and dehydroascorbic acid. The procedure for both acids consists of first measuring the native ascorbic acid, then reducing the dehydroascorbic acid, at neutral pH, with dithiothreitol, and finally measuring the total ascorbic acid; dehydroascorbic acid is then determined by difference. The metaphosphoric-acid-treated samples were stable at -70 °C, but stability decreased with temperature over the range examined, 4-50 °C.