Fluorometry of hydrogen peroxide using oxidative decomposition of folic acid

Hydrogen peroxide (H₂O₂) is one of the most important reactive oxygen species. In the present study, a fluorometry method for detecting H₂O₂ utilizing folic acid was evaluated. Folic acid was decomposed by H₂O₂ in the presence of Cu(II) into pterine-6-carboxylic acid, leading to strong fluorescence enhancement. In the absence of the metal ion, superoxide and H₂O₂ could not decompose folic acid. Also, H₂O₂ plus sodium hypochlorite (a source of singlet oxygen) could not induce fluorescence enhancement. These results demonstrate that H₂O₂ can be selectively detected using folic acid plus Cu(II). The limit of detection (LOD; at S/N=3) for H₂O₂ is 0.5 μM. This method based on the fluorescence enhancement of folic acid was applied in order to determine small amounts of H₂O₂ generated through the autooxidation of semicarbazide (generation rate: ∼0.01 μM min⁻¹), a carcinogenic compound.