Micelle Enhanced Spectrofluorimetric Determination of L-ascorbic Acid Based on Laccase-linked Coupling Reaction Using Flow- injection Stopped-flow Technique

A novel spectrofluorimetric method for determination of L-ascorbic acid is described. It is based on the inhibition of L-ascorbic acid on the formation of 2, 3-diaminophenazine, which is an oxidation product of o-phenylenediamine catalyzed by laccase, in the presence of Brij-35, which shows strong enhancement on the fluorescence(at λem/λex=530nm/430nm) of the product, while no effect on the laccase-catalyzed reaction. The mechanism of o-phenylenediamine oxidation reaction catalyzed by laccase in the presence of L-ascorbic acid is discussed. L-ascorbic acid is determined in the range of O.02-2.Oμg/ml with a detection limit of 0.014μg/ml. The method is applied to the determination of L-ascorbic acid in pharmaceuticals, the results agree well with the nominal values and those obtained by the reference method.