Fluorophotometric Determination of Hydrogen Peroxide and Other Reactive Oxygen Species with Fluorescein Hydrazide (FH) and Its Crystal Structure

Methods for the fluorophotometric determination of hydrogen peroxide (H2O2) and other reactive oxygen species (ROS) were proposed by using the fluorescence reaction between H2O2 or other ROS and fluorescein hydrazide (FH). In the determination of H2O2, the calibration curve exhibited linearity over the H2O2 concentration range of 2.1—460 ng ml−1 at an emission wavelength of 527 nm with an excitation of 460 nm and with the relative standard deviations (n=6) of 4.06%, 1.78%, and 2.21% for 3.1 ng ml−1, 30.8 ng ml−1, and for 308 ng ml−1 of H2O2, respectively. The detection limit for H2O2 was 0.7 ng ml−1 due to three blank determinations (ρ=3). The calibration curves for ROS-related compounds were also constructed under the optimum conditions. This method was successfully applied in the assay of H2O2 in human urine. In addition, we performed the characterization of FH, and interesting information was obtained with regard to the relationship between the chemical structure and fluorescence.