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

Hydrogen peroxide (H 2 O 2 ) level in biological samples is used as an important index in various studies. Quantification of H 2 O 2 level in tissue fractions in presence of H 2 O 2 metabolizing enzymes may always provide an incorrect result. A modification is proposed for the spectrofluorimetric determination of H 2 O 2 in 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 K 2 HPO 4 before the fluorimetric estimation of H 2 O 2 is performed. TCA was used to precipitate the protein portions contained in the tissue fractions. After employing the above modification, it was observed that H 2 O 2 content 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 H 2 O 2 and catalase enzyme that quenches H 2 O 2 as substrate. This method can be used efficiently to determine more accurate tissue H 2 O 2 level without using internal standard and multiple samples can be processed at a time with additional low cost reagents such as TCA and K 2 HPO 4 .