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

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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.