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 (rho = 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.
We have reported on a simple, highly sensitive, and highprecision fluorophotometric determination of H2O2 and other reactive oxidant species (ROS) by using a redox reaction with fluorescein hydrazide (FH) and cobalt(II) in a dodecyltrimethylammonium chloride (DTAC) micellar medium. 1 In this study, we acetylated FH in order to apply this determination method to intracellular imaging of H2O2 and other ROS. At this time, an excellent crystal of FHTA had been obtained. For the purpose of clarifying the structure of FHTA, the mechanism of the determination method and the relation between the fluorescence and the chemical structure, we carried out an X-ray analysis.A modification of a procedure of Akita 2 for the synthesis of FH and FHTA was employed. FH was synthesized by the hydrazide derivatization of fluorescein with hydrazine monohydrate. Also, equivalent molar amounts of FH and sodium acetate were dissolved in 10 ml of acetic anhydride, and heated for 30 min. The mixture was poured into ice water, and a yellowish-white substance was subsequently filtered. The product, FHTA is shown in Fig. 1.Crystals of FHTA were grown from an aqueous ethanol solution. A crystal having a size of 0.4 ¥ 0.2 ¥ 0.1 nm was used for diffraction experiments (Table 1). The structure was solved by a direct method with SHELXS97. 3 Non-hydrogen atoms were refined anisotropically. Hydrogen atoms were calculated at the ideal positions and isotropically included in calculations of the structure factors. In this analysis, the R factor was large compared with the usual analysis because there was a small disorder in the region of a large temperature factor of end. As shown in Fig. 2, FHTA was formed with the benzene ring through hydrazine for a xanthene scaffold to have a plane structure, and a spirolactam ring was formed with a connection that was almost right angled at 87.45(4)˚. We found that hydrazation fluorescein would force this platform to adopt a
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