Patent ID: 6153399
Filing Date: 2000-11-28
Classification: C12Q,G01N,Y10T

Abstract:
A method for quantitative determination of ascorbic acid in a sample, comprising the steps of:(a) selecting ascorbate oxidase which catalyzes the reaction of L-ascorbic acid with oxygen to form dehydroascorbic acid and hydrogen peroxide;(b) selecting a chromogen represented by formulae (I) or (II): ##STR7## wherein Y represents a hydrogen atom or a group represented by formula (III): ##STR8## wherein Z represents oxygen or sulfur; and X represents hydrogen, alkyl, alkenyl, aryl, mono-substituted amino or unsubstituted amino;R.sup.1 represents hydroxy, mono- or di-substituted amino or unsubstituted amino;R.sup.2 represents hydrogen, hydroxy, alkyl, alkoxy, aryl, alkenyl, mono- or di-substituted amino or unsubstituted amino;R.sup.3, R.sup.4, R.sup.5 and R.sup.6 independently represent hydrogen, alkyl, alkenyl, alkanoyl, aroyl, aryl, halogen, nitro, sulfo, carboxy, hydroxy, alkoxy or a group represented by formulae (IV), (V), (VI), (VII) or (VIII): ##STR9## wherein A.sup.1 represents alkylene; A.sup.2 represents hydrogen, hydroxy, alkyl, alkoxy, aryl, alkenyl, alicyclic alkyl, mono- or di-substituted amino or unsubstituted amino; and B.sup.1, B.sup.2, B.sup.3, B.sup.4, B.sup.5 and B.sup.6 independently represent hydrogen, alkyl, alkenyl, alkanoyl, aroyl, aryl, halogen, nitro, sulfo, carboxy, hydroxy, alkoxy or hydroxy-substituted alkyl; orR.sup.3 and R.sup.4, or R.sup.5 and R.sup.6 are combined together to form alkenylene; andJ represents oxygen, sulfur or a group represented by formula (IX) or (XI): ##STR10## wherein R.sup.7 and R.sup.8 independently represent hydrogen, alkyl or alkenyl;(c) reacting in an aqueous medium ascorbic acid in the sample with oxygen in the presence of the ascorbate oxidase, chromogen and peroxidase wherein pigment formation catalyzed by peroxidase from the chromogen and H.sub.2 O.sub.2 which is produced from L-ascorbic acid in the sample by ascorbate oxidase is not substantially affected by presence of L-ascorbic acid;(d) measuring an absorbance of the aqueous medium, wherein the absorbance is indicative of the quantity of ascorbic acid in the sample; and(e) comparing the absorbance with a calibration curve obtained using known amounts of ascorbic acid.