A Modified Fluorimetric Method for Determination of Hydrogen Peroxide Using Homovanillic Acid Oxidation Principle

Abstract

Hydrogen peroxide (H2O2) level in biological samples is used as an important index in various studies. Quantification of H2O2level in tissue fractions in presence of H2O2metabolizing enzymes may always provide an incorrect result. A modification is proposed for the spectrofluorimetric determination of H2O2in homovanillic acid (HVA) oxidation method. The modification was included to precipitate biological samples with cold trichloroacetic acid (TCA, 5% w/v) followed by its neutralization with K2HPO4before the fluorimetric estimation of H2O2is performed. TCA was used to precipitate the protein portions contained in the tissue fractions. After employing the above modification, it was observed that H2O2content in tissue samples was ≥2 fold higher than the content observed in unmodified method. Minimum 2 h incubation of samples in reaction mixture was required for completion of the reaction. The stability of the HVA dimer as reaction product was found to be >12 h. The method was validated by using known concentrations of H2O2and catalase enzyme that quenches H2O2as substrate. This method can be used efficiently to determine more accurate tissue H2O2level without using internal standard and multiple samples can be processed at a time with additional low cost reagents such as TCA and K2HPO4.