title: Reduced fluoresceinamine for peroxynitrite quantification in the presence of nitric oxide.
abstract: 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.
authors.affiliation: Centro de Investigação em Química, Faculdade de Farmácia da Universidade de Coimbra, Pólo das Ciências da Saúde, 3000-548, Coimbra, Portugal.
authors.forename: Eliana F C
authors.initials: EF
authors.lastname: Simões
authors.forename: João M M
authors.initials: JM
authors.lastname: Leitão
authors.forename: Joaquim C G Esteves
authors.initials: JC
authors.lastname: da Silva
mesh_terms: D000818:Animals; D002623:Chemistry Techniques, Analytical / Q000295:instrumentation*; D005452:Fluoresceins / Q000737:chemistry*; D008264:Macrophages / Q000737:chemistry; D051379:Mice; D009569:Nitric Oxide / Q000737:chemistry*; D030421:Peroxynitrous Acid / Q000032:analysis* / Q000097:blood; D013050:Spectrometry, Fluorescence; D013499:Surface Properties
chemical_list: D005452:Fluoresceins; D030421:Peroxynitrous Acid; D009569:Nitric Oxide; C030864:5-aminofluorescein