Fluorimetric determination of total ascorbic acid by a stopped-flow mixing technique

A simple, rapid and automatic fluorimetric method for the determination of total ascorbic acid is described. The method makes use of the stopped-flow mixing technique in order to achieve the rapid oxidation of ascorbic acid by dissolved oxygen to dehydroascorbic acid, which then reacts with o-phenylenediamine to form a fluorescent quinoxaline. The initial rate and fluorescence signal of this system are directly proportional to the ascorbic acid concentration. The calibration graph was linear over the range 0.1-30 microg ml(-1) (kinetic method) and 0.25-34 microg ml(-1) (equilibrium method). The precision (% RSD) was close to 0.5%. The method has been used for the determination of ascorbic acid in pharmaceutical formulations, fruit juices, soft drinks and blood serum.