Validation of an analytical procedure for the determination of the fluoroquinolone ofloxacin in chicken tissues

A novel analytical procedure was developed for the determination of the fluoroquinolone ofloxacin in chicken kidney, liver, muscle and fat plus skin tissues. The procedure involved a preliminary extraction with 0.15 M HCl followed by solid-phase extraction clean-up. The purification step was performed using a polymeric sorbent coated cartridge. Ofloxacin was analyzed by reversed-phase HPLC using UV detection at 295 nm. The mobile phase used was water–acetonitrile–triethylamine (83:14:0.45, v/v, pH 2.30). The use of triethylamine and the acidic pH modulated the retention of ofloxacin and avoided chemical tailing. The amine modifier and acetonitrile content of the mobile phase were optimized to provide the best selectivity. A flow-rate of 1 ml/min was used and ofloxacin eluted at 5.1 min. HPLC analysis of the tissue samples was performed in 12 min. The procedure was validated according to the International Conference on Harmonisation guidelines across the concentration ranges (100 μg/kg–1.7 mg/kg for kidney and liver tissues and 50 μg/kg–1.0 mg/kg for muscle and fat plus skin tissues). The linearity, the intra- and inter-day accuracies and precisions were determined. The limits of quantification were 50 μg/kg for muscle and fat plus skin tissues and 100 μg/kg for liver and kidney tissues. The procedure was specific and the accuracy values were lower than 20% at the limit of quantitation of spiked samples. The recovery values ranged from 80 to 100%. The limits of detection were established at 60 μg/kg for liver and kidney tissues and at 25 μg/kg for muscle and fat plus skin tissues. Finally, ofloxacin was found to be stable in acidic conditions. The developed procedure is simple, sensitive, accurate and adapted to routine sample analyses such as those carried out for residue depletion studies.