Flow-injection stopped-flow spectrofluorimetric kinetic determination of total ascorbic acid based on an enzyme-linked coupled reaction

A flow-injection enzymatic kinetic spectrofluorimetric procedure for determining total ascorbic acid in solution by combining the merging zones principle with the stopped-flow technique is described. It is based on the oxidation of ascorbic acid catalysed by laccase to dehydroascorbic acid, which then reacts with o-phenylenediamine to form a fluorescent quinoxaline. Total ascorbic acid is determined in the range of 0.025–1.0 μg ml−1 at 30 determinations per h. The procedure is applied to the determination of total ascorbic acid in wine, beer, urine and pharmaceutical preparations.