Fluorophotometric Determination of Hydrogen Peroxide with Fluorescin in the Presence of Cobalt (II) and Reaction Against Other Reactive Oxygen Species

A fluorophotometric method for the determination of hydrogen peroxide (H2O2) using fluorescin was developed. This method was based on the oxidative reaction of fluorescin, a colorless, non-fluorescent lactoid fluorescein, by H2O2 to give highly fluorescein fluorescence emission. In the determination of H2O2, the calibration curve exhibited linearity over the H2O2 concentration range of 1.5–310 ng mL−1 at an emission wavelength of 525 nm with an excitation of 500 nm and with relative standard deviations (n = 6) of 2.51%, 2.48%, and 1.31% 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 1.9 ng mL−1 six blank determinations was performed (ρ = 6). This proposed method was applied to detection of other reactive oxygen species and nitrogen species (ROS/RNS) such as singlet oxygen (1O2), hydroxyl radical (•OH), peroxynitrite (ONOO−) etc., and it was possible to detect them with a high sensitivity. In addition, this proposed method was applied to the recovery tests of H2O2 in calf serum, human saliva, rain water, and wheat noodles; the results were satisfactory.