Stopped flow kinetic determination of nalidixic acid and norfloxacin based on lanthanide-sensitized fluorescence

Nalidixic acid and norfloxacin were determined in this work for the first time by applying kinetic methodology to complex formation reactions involving energy-transfer processes. By using initial-rate measurements, a fluorescence-based method based on the formation of the complexes of these compounds with terbium(III) in the presence of Tris buffer was developed. The formation rate of these complexes is very high, so the stopped-flow mixing technique is required in order to make kinetic measurements, which, in addition, allows the method to be automated. A second ligand, EDTA, is also used to decrease the reaction rate of both systems via ligand-exchange, which is necessary in order to obtain appropriate kinetic data. The reaction rate is obtained within only 0.1–0.2 s after the reactants are mixed, which allows ready application of the proposed method to routine analyses. The calibration graph is linear over the range 0.02–7.0 μg ml−1 nalidixic acid and 0.4–9.0 μg ml−1 norfloxacin. The detection limit for nalidixic acid and norfloxacin is 0.006 and 0.13 μg ml−1, respectively. The selectivity and precision of the method were also studied. The proposed kinetic method was satisfactorily applied to the direct determination of these compounds in serum samples.