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