Reduced Fluoresceinamine for Peroxynitrite Quantification in the Presence of Nitric Oxide

摘要

A new fluorescent analytical methodology for the quantification of peroxynitrite (ONOO-) in the presence of nitric oxide (NO) was developed. The quantification of ONOO- is based in the oxidation of the non-fluorescent reduced fluoresceinamine to a high fluorescent oxidized fluoresceinamine in reaction conditions where the interference of NO is minimized. Screening factorial experimental designs and optimization Box-Behnken experimental design methodologies were used in order to optimize the detection of ONOO- in the presence of NO. The factors analysed were: reduced fluoresceinamine concentration (C Fl ); cobalt chloride concentration (C CoCl2 ); presence of oxygen (O 2 ); and, the pH (pH). The concentration of sodium hydroxide (C NaOH ) needed to diluted the initially solution of ONOO- was also evaluated. An optimum region for ONOO- quantification where the influence of NO is minimal was identified - C Fl from 0.50 to 1.56 mM, C CoCl2 from 0 to 1.252 × 10−2 M, pH from 6 to 8 and C NaOH 0.10 M. Better results were found in the presence of NO at pH 7.4, C Fl 0.5 mM, without oxygen, without cobalt chloride and with a previous dilution of peroxynitrite solution with C NaOH 0.1 M. This methodology shows a linear range from 0.25 to 40 μM with a limit of detection of 0.08 μM. The bioanalytical methodology was successfully applied in the ONOO- quantification of fortified serum and macrophage samples.