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).