João Manuel Martins Leitão

Abstract The effect on the fluorescence of the europium:tetracycline (Eu:Tc), europium:oxytetracycline (Eu:OxyTc) and europium:chlortetracycline (Eu:ClTc) complexes in approximately 2:1 ratio of nitric oxide (NO), peroxynitrite (ONOO-), hydrogen peroxide (H2O2) and superoxide (O-2 (center dot-)) was assessed at three ROS/RNS concentrations levels, 30 A degrees C and pH 6.00, 7.00 and 8.00. Except for the NO, an enhancement of fluorescence intensity was observed at pH 7.00 for all the europium tetracyclines complexes-the high enhancement was observed for H2O2. The quenching of the fluorescence of the Tc complexes, without and with the presence of other ROS/RNS species, provoked by NO constituted the bases for an analytical strategy for NO detection. The quantification capability was evaluated in a NO donor and in a standard solution. Good quantification results were obtained with the Eu:Tc (3:1) and Eu:OxyTc (4:1) complexes in the presence of H2O2 200 mu M with a detection limit of about 3 mu M (Eu:OxyTc).

Abstract A fluorescence flow injection analysis (FIA) methodology for nitric oxide (NO) quantification was optimized by factorial analysis for the lowest limit of detection of nitric oxide. This methodology is based on the reaction of the NO with the non-fluorescent reduced fluoresceinamine given a high fluorescent oxidized fluoresceinamine. Box-Behnken and central composite optimization experimental design methodologies were used. The factors initially analysed by a screening experimental design methodology were the flow rate of the pump (Q), loop volume (L), reactor length (R), reduced fluoresceinamine concentration (C-Fl) and cobalt chloride concentration (C-CoCl2). The response variables under analysis were the maximum fluorescence intensity, response repeatability and peak width. The optimum conditions were: one flow stream FIA configuration, Q = 0.60 mL min(-1), L = 100 mu L, R = 2 m, C-Fl = 1.50 mM and without CoCl2. A linear working range between 5 to 40 mu M was evaluated with a limit of detection of 1.20 mu M. Hydrogen peroxide, superoxide, nitrite and nitrate did not interfere with the NO detection. Good results were found in the quantification of NO liberated by a NO donor at pH 7.4 and in fortified serum samples.

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 x 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 mu M with a limit of detection of 0.08 mu M. The bioanalytical methodology was successfully applied in the ONOO- quantification of fortified serum and macrophage samples.

Abstract A fluorescent derivatization reaction for Diltiazem drug quantification based on the condensation reaction of citric or malonic acid with acetic anhydride, catalyzed by the tertiary amine group of Diltiazem, was developed. Excitation emission matrices (EEMs) of fluorescence of the pure solvent (ethanol), standard and sample solutions following a standard addition methodology were analysed by PARAFAC based methods (PARAFAC, PARAFAC2 and PARALIND) to obtain robust calibration methodologies. The quantification results of the sample were compared with the official US Pharmacopeia high performance liquid chromatography-ultraviolet method (USP HPLC-UV). Although the experimental sets of EEM show linearity deviations all the PARAFAC based methods allow correct robust estimation of Diltiazem concentration in pharmaceutical formulations. The closest results were: derivatization with citric acid and PARAFAC2 six components non-negativity constraint model with a detection limit of 0.088 ppm; and, derivatization with malonic acid and PARAFAC six components non-negativity constraint model with a detection limit of 0.066 ppm for the malonic acid was observed. The simultaneous utilization of the three PARAFAC methods gives further information about the intrinsic structure of the data sets under analysis, i.e., it works as an efficient diagnostic tool for the existence of non-linearity and colinearity.

Abstract Silica based nanostructured composite materials doped with luminol and cobalt(II) ion were synthesized and characterized, resulting in a highly chemiluminescent material in the presence of hydrogen peroxide. A detection system with the CL light guided from the reaction tube to the photomultiplier tube using a one millimeter glass optical fiber was developed and assessed. A linear response was observed using a semi-logarithm calibration between 50-2000M hydrogen peroxide with 1M as the limit of detection.