Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)A method for the simultaneous determination of flumequine, oxolinic acid, sarafloxacin, danofloxacin, enrofloxacin, and ciprofloxacin in tilapia (Orechromis niloticus) fillet, using high-performance liquid chromatography with fluorescence detection (HPLC-FLD) is presented. The quinolones were extracted from the food matrix with a solution of 10% trichloroacetic acid and methanol (80:20 v/v). Clean-up of the extract was performed using polymeric solid-phase extraction cartridges. Identification of the quinolones was confirmed by liquid chromatography-tandem mass spectrometry. The HPLC-FLD method was validated in-house and the following analytical parameters were obtained: linearity higher than 0.99 for all the quinolones; intra- and inter-assay precisions were lower than 3.5% and 10.9%, respectively; and recoveries ranged from 73% to 110%. The limit of quantification was below the maximum residue limit established by the Joint Expert Committee on Food Additives (JECFA), which indicates that the method is appropriate for the determination of quinolones in fish fillet.261013311340Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)