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 mu M. 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: similar to 0.01 mu M min(-1)), a carcinogenic compound.