Substitution of peroxidase in Trinder's reagent with iron(II) for the determination of hydrogen peroxide in enzymic reactions by applying flow injection

Abstract

The kinetics and mechanism of the reactions between 4-aminoantipyrine and/or 3,5-dichloro-2-hydroxybenzenesulfonic acid with Fe²⁺ in the presence of H₂O₂ have been investigated. A differential spectrophotometric flow injection (Fl) method for the determination of H₂O₂ has been developed, by substituting horseradish peroxidase (PO) in Trinder's reagent with Fe²⁺. This method has been applied to the determination of glucose in human serum, with good results, by using an enzymic reactor with immobilized glucose oxidase and an automated Fl system, after optimizing its parameters. The agreement between the results of this method with those of the routine method used by a hospital clinical laboratory for the determination of glucose in human serum was very good. The correlation line of the two methods shows a slope of 1.03 and a correlation coefficient (r) of 0.9997. The method was also applied to the determination of H₂O₂. The working curve, with Fe²⁺ as promoter, was linear for 10–400 µmol l_–1 H₂O₂ compared with 5–170 µmol l^–1 H₂O₂ when PO was used. The slopes of the curves for Fe²⁺ and PO are 1.69 and 4.35 A l mmol^–1, respectively, showing a reduced sensitivity for the proposed method compared with the PO method.