DEVELOPMENT OF SPECTROFLUORIMETRIC METHODS FOR DETERMINATION OF ERYTHROMYCIN AND KANAMYCIN AND THEIR APPLICATION IN THE YELLOW FEVER VACCINE
The antibiotics erythromycin and kanamycin, members of the macrolide andaminoglycoside classes respectively, have their importance as preservative agentsfor the preparation process and during the using of the yellow fever vaccine, fivedoses presentation, produced in Technology on Immunobiologicals Institute, Bio-Manguinhos, FIOCRUZ, RJ. In this work two new approaches for thespectrofluorimetric determination of these antibiotics are presented, taking intoconsideration their selectivity performance for this specific matrix (vaccine). Apreliminary study was performed to evaluate the luminescent behavior(phosphorescence and fluorescence) of these substances in different experimentalconditions aiming to find the best ones that would lead to the development of theanalytical methods. For erythromycin, which does not present nativeluminescence, a photochemical derivatization was employed using analitesolutions prepared in sulfuric acid. As a result, a photo-product was obtained,which presented fluorescence at 412/465 nm. The experimental conditions wereoptimized aiming the maximization of the fluorescent signal. In this case, thestudied parameters were the UV irradiation time, the type and concentration of theacid utilized, and the time and temperature used for the heating step. The methodwas partially validated, indicating limits of detection and quantification of0.25 µg mL-1 and 0.85 µg mL-1, respectively. The linear dynamic range of themethod extended up to 200 µg mL-1 and the parameters related to precision androbustness were very satisfactory. The method was applied in the analysis of asimulated yellow fever vaccine and in commercial pharmaceutical formulations.Recovery percent between 98.2 and 105.2 % were achieved. The methodologydeveloped for kanamycin, which also do not presented natural fluorescence, wasbased on the oxirreduction reaction of this compound with Ce (IV), producing Ce(III), a strongly fluorescent species (255/360 nm). The fluorescence intensities, measured in the excitation/emission pair of Ce (III), was directly proportional tothe kanamycin concentration. The reaction conditions were studied by theevaluation of the amount of Ce (IV), the type and concentration of the acidutilized in reactional medium and heating time and temperature. Large linearrange (up to 1000 µg mL-1) was obtained, with detection and quantification limitsof 1.22 µg mL-1 and 4.08 µg mL-1, respectively. The recovery percent obtained inthe analysis of a diluted vaccine was 109.8 % while the recovery achieved fordiluted spiked urine was 103.4 %.
Advisor:MARCIA ARISSAWA; MARCIA ARISSAWA; CLAUDIA HAMACHER; RICARDO QUEIROZ AUCELIO; RICARDO QUEIROZ AUCELIO; ADERVAL SEVERINO LUNA
Source Type:Master's Thesis
Date of Publication:04/29/2005