Applications of a simultaneous assay of ascorbic acid, dehydroascorbic acid and ascorbic sulphate in biological materials

A modified spectrophotometric assay for ascorbic acid and its derivatives based on their reaction with 2,4-dinitrophenylhydrazine (DNPH) is described. Using standard ascorbic acid or ascorbic sulphate solutions, together with animal tissue or compound diet extracts, the conditions for ascorbic acid degradation were determined. For the differential measurement of reduced ascorbic acid (AA), dehydroascorbic acid (dAA) and ascorbic sulphate (AS), five series of simultaneous determinations were performed. These included the use of (1) KBrO3 for the hydrolysis of AS, (2) 2,6-dichlorophenolindophenol as an oxidant, (3) DNPH to form a hydrazone derivative with dAA and (4 and 5) two blanks (where ascorbate was degraded) to correct for interfering substances. A variety of vertebrate and invertebrate tissues were examined for their ascorbate content, and the advantages of the modified procedure over currently available assays are discussed. The results suggest that the Artemia cyst is a unique material in which ascorbic sulphate is present in large amounts whereas fish tissues do not contain this form of vitamin C.