MICELLE ENHANCED SPECTROFLUOROMETRIC DETERMINATION OF L-ASCORBIC-ACIDBASED ON LACCASE-LINKED COUPLING REACTION USING FLOW-INJECTION STOPPED-FLOW TECHNIQUE

A novel spectrofluorimetric method for determination of L-ascorbic aci d is described. It is based on the inhibition of L-ascorbic acid on th e formation df 2,3-diaminophenazine, which is an oxidation product of o-phenylenediamine catalyzed by laccase, in the presence of Brij-35, w hich shows strong enhancement on the fluorescence(at lambda em/lambda ex=530nm/430nm) of the product, while no effect on the laccase-catalyz ed reaction. The mechanism of o-phenylenediamine oxidation reaction ca talyzed by laccase in the presence of L-ascorbic acid is discussed. L- ascorbic acid is determined in the range of 0.02 similar to 2.0 mu g/m l with a detection limit of 0.014 mu g/ml. The method is applied to th e determination of L-ascorbic acid in pharmaceuticals, the results agr ee well with the nominal values and those obtained by the reference me thod.