LC-MS/MS based method development for the analysis of florfenicol and its application to estimate relative distribution in various tissues of broiler chicken

Florfenicol, a broad spectrum bacteriostatic antibiotic belonging to amphenicol class, is widely used in poultry and livestock for the treatment of various infections. The major metabolite of florfenicol in different animal species is florfenicol amine which is exploited as the marker residue for the determination of florfenicol. Analysis of florfenicol merely by solvent extraction cannot determine the accurate amount of the drug present in incurred tissues (muscle, liver and kidney) of treated birds, as indicated by this study. Thus the methods solely based on solvent extraction may lead to false negative results. A reliable LC-MS/MS based confirmatory method for the analysis of florfenicol and its metabolites in chicken muscle was developed and validated according to the European Union Commission Decision 2002/657/EC. The method was based on acid hydrolysis to liberate non extractable residues having presumably been covalently bound to tissues, and to convert all the florfenicol residues as well as its metabolites into florfenicol amine. The amine was subsequently recovered with ethyl acetate at pH 10.5, defatted and further cleaned up with dispersive solid phase extraction (dSPE). The LC separation was achieved on reverse phase C-18 column with isocratic elution using acetonitrile/water mobile phase and the analysis was performed on linear ion trap mass spectrometer. Calibration curve was obtained over a concentration range of 25-600 mu g/kg for chicken muscles. The accuracy values ranged from 84 to 101.4% and the precision values for within day and between days ranged from 1.2-11.7%, respectively. Limit of detection (LOD), limit of quantification (LOD), CC alpha and CC beta values were 0.98, 3.2, 113 and 126 mu g/kg, respectively. The developed method was highly robust and was further applied to estimate the relative distribution of solvent extractable against solvent-non-extractable florfenicol drug residues in muscle, liver and kidney samples of broiler chicken after 5 days of oral dosing.