UPLC-MS/MS determination of florfenicol and florfenicol amine antimicrobial residues in tilapia muscle.

Abstract

Despite the benefits to fish farmers, the use of antimicrobials in aquaculture has concerned consumers and competent authorities. The indiscriminate use of such substances promotes the emergence of resistant microorganisms, decreases the effectiveness of treatments, and causes possible toxic effects in humans. In Brazil, florfenicol is the only antimicrobial registered for use in aquaculture and is often used in tilapia in cage creation. Thus, this study aimed to develop a method for determination of florfenicol residues and its metabolite florfenicol amine in tilapia fillet by UPLC-MS/MS. Analytes were extracted with ethyl acetate, followed by liquid-liquid partition clean-up with hexane and SPE. The sorbents C18, phenyl and HLB-Oasis were evaluated by SPE. Phenyl sorbent showed the best results, and the extraction conditions were optimized in the sample matrix with fractional factorial design 24-1. The analytes were separated on a C18 chromatographic column (50×2.1mm×1.7μm) using water (A) and acetonitrile (B) as mobile phase at a flow rate of 0.3mLmin-1 with a linear gradient (in% B): 0-2.0min: 20%; 2.0-2.5min: increase to 90%; 2.5-3.5min: 90%; 3.0-3.5min: decrease to 20%; 4.0-5.0min: 20%. The analytes were monitored in a MS/MS triple quadrupole system by MRM mode with transitions at m/z 356.1>336.1 (florfenicol) and m/z 248.1>130.1 (florfenicol amine). The optimized method was validated obtaining LOQ values of 3 and 25ngg-1 for florfenicol and florfenicol amine, respectively, precision between 20 and 36%, absolute extraction efficiency between 38 and 80%, and adequate linearity. The method was applied to samples intended for human consumption, and within the 15 evaluated samples, only one showed florfenicol residue at 30ngg-1, which is below the maximum residue limit established in Brazil.