Abstract: One of the two 5 mL reservoir syringes of a stopped-flow module of a fluorescence spectrometer was filled with a solution containing 0.15 mM borate buffer of pH 9.2, 2 mM o-phthaldialdehyde (OPA) and 3 mM N-acetylcysteine and the other with 0.15 mM borate buffer and an aminoglycoside antibiotic, amikacin (I), kanamicin (II) or tobramycin (III) at a final concentration of 0.05-30 µg/ml. A 0.15 mL portion from each syringe was transferred to a mixing chamber (1 cm path length) at 25°C and the fluorescence measured at 455 nm (excitation at 335 nm) throughout the reaction. The initial-rate procedure, based on the measurement of the slope of the kinetic curve fluorescence intensity vs. time, which is proportional to the analyte concentration was applied. Calibration graphs were linear for 0.05-30 µg/ml of I. Three equations were required to cover the concentration ranges 0.05-1 µg/ml, 1-10 µg/ml and 10^-30 µg/ml. The detection limit was 0.02 µg/ml. RSD (n = 11) were 1.8 and 0.9% for 0.5 and 12 µg/ml of I. I/interferent ratios of 1:50 and 1:4 were tolerated for penicillins and tetracyclines respectively. The mean recovery was 98.7%.

Abstract: The sample was deproteinized with methanol, which contained an internal standard (sissomicin or netilmicin). After centrifugation, a counter-ion reagent, containing disodium ethane-1,2-disulfonate and Na octanesulfonate, was added to the supernatant solution and an aliquot was subjected to HPLC on a column (10 cm x 8 mm) of Radial-Pak C18 with a mobile phase of water - methanol (16:9) containing disodium ethane-1,2-disulfonate and Na octanesulfonate (pH 3.5) and detection at 440 nm. Tobramycin, netilmicin and sissomicin were determined by using continuous-flow post-column derivatization with phthalaldehyde. The limit of detection was 0.3 µg mL-1. For tobramycin, a coefficient of variation (n = 10) of <2.5% and good correlation of results with those of a microbiological assay (r = 0.956) and of a homogeneous enzyme immunoassay (r = 0.988) were achieved.