ASCORBATE AND DEHYDROASCORBATE MEASUREMENTS IN AQUEOUS-SOLUTIONS AND PLASMA DETERMINED BY GAS-CHROMATOGRAPHY MASS-SPECTROMETRY

The tert-butyldimethylsilyl derivatives of ascorbic acid and dehydroascorbic acid were characterized by gas chromatography-mass spectrometry, and an isotope dilution assay for ascorbate and dehydroascorbate was developed using [C-13(6)]ascorbic acid and [C-13(6)]- and [6,6-H-2(2)]dehydroascorbate. This assay was used to monitor ascorbic acid loss and the resulting rise of dehydroascorbic acid in aqueous solutions and plasma. Ascorbic acid was shown to rapidly decompose in aqueous solutions containing transition metal ions or when exposed to oxygen. Ethylenediaminetetraacetic acid chelation did not prevent ascorbic acid degradation in aqueous solution, and ascorbate in ethylenediaminetetraacetic acid chelated plasma was converted to dehydroascorbate on freezing. Gas chromatography-mass spectrometry appears to be a satisfactory method for determining the ascorbate and dehydroascrobate content of solutions including human blood plasma, whether or not there is ongoing oxidation of ascorbate in those solutions.