Ascorbic acid determination in biological fluids using ascorbate oxidase immobilized on alkylamine glass beads in a flow injection potentiometric system

A flow injection method was developed aimed at the determination of ascorbic acid in biological fluids, particularly fruit juices. The enzyme ascorbic oxidoreductase (EC 1.10.3.3), extracted fromCucurbita maxima, was immobilized onto alkylamine glass beads using glutaral-dehyde as a bifunctional agent. The ascorbic acid concentration was related to oxygen saturation. Fall in oxygen concentration, as a result of ascorbic acid oxidation, was detected by a low cost, homemade oxygen electrode. The calibration graph was linear over the range 0.05 to 3.00 mM (RSD 1%), the maximum number of samples that could be analysed was 90/h. The immobilized enzyme retained its initial activity for 2 mo with more than 600 assays.