Exposure of tilapia (Oreochromis niloticus) to the antibiotic florfenicol in water: determination of the bioconcentration factor and the withdrawal period.

The demand for healthier foods with high nutritional value has resulted in intensive fish farming. In this production system, high-frequency infections occur, and antibiotics are administrated for control. Only two antibiotics are allowed for use in Brazilian aquaculture, one of which is florfenicol. In this work, a bioconcentration assay was performed to assess the accumulation of florfenicol in the muscle of Nile tilapia (Oreochromis niloticus). Tilapia was evaluated as it is the most produced fish species in Brazil. The fish were exposed to florfenicol at a nominal concentration of 10 mg/L, through the water. Muscle and water were collected at 0, 1.5, 3, 6, 24, and 48 h during the exposure phase and at 1.5, 3, 6, 24, 48, and 120 h during the depuration phase. Quantification was performed using an LC-MS/MS. The results showed rapid absorption and elimination of the antibiotic (half-life, t1/2 = 5 h), with low potential for accumulation of florfenicol in tilapia muscles. The study was performed to determine the bioconcentration factor (BCF) and withdrawal period of florfenicol, being 0.05 mL/µg and 1.8 h, respectively. The results contribute to set protocols for the safe use of florfenicol in tilapia transport, avoiding residues in fish that may pose risks to human health.

Residue Depletion of Florfenicol and Florfenicol Amine in Broiler Chicken Claws and a Comparison of Their Concentrations in Edible Tissues Using LC⁻MS/MS.

Antimicrobial residues might persist in products and by-products destined for human or animal consumption. Studies exploring the depletion behavior of florfenicol residues in broiler chicken claws are scarce, even though claws can enter the food chain directly or indirectly. Hence, this study intended to assess the concentrations of florfenicol (FF) and florfenicol amine (FFA)-its active metabolite-in chicken claws from birds that were treated with a therapeutic dose of florfenicol. Furthermore, concentrations of these analytes in this matrix were compared with their concentrations in edible tissues at each sampling point. A group of 70 broiler chickens were raised under controlled conditions and used to assess residue depletion. Sampling points were on days 5, 10, 20, 25, 30, 35, and 40 after ceasing treatment, thus extending beyond the withdrawal period established for muscle tissue (30 days). Analytes were extracted using HPLC-grade water and acetone, and dichloromethane was used for the clean-up stage. Liquid chromatography coupled to mass spectroscopy detection (LC⁻MS/MS) was used to detect and quantify the analytes. The analytical methodology developed in this study was validated in-house and based on the recommendations described in the Commission Decision 2002/657/EC from the European Union. Analyte concentrations were calculated by linear regression analysis of calibration curves that were fortified using an internal standard of chloramphenicol-d5 (CAF-d5). The depletion time of FF and FFA was set at 74 days in claws, based on a 95% confidence level and using the limit of detection (LOD) as the cut-off point. Our findings show that FF and FFA can be found in chicken claws at higher concentrations than in muscle and liver samples at each sampling point.

Single-laboratory validation of an LC-MS/MS method for determining florfenicol (FF) and florfenicol amine (FFA) residues in chicken feathers and application to a residue-depletion study.

A suitable analytical method is required to study the behaviour of florfenicol (FF) and its metabolite florfenicol amine (FFA) in broiler's feathers. An LC-MS/MS method was developed, assessed and intra-laboratory-validated for FF and FFA analyses. We chose cloramphenicol-d5 as an internal standard, acetone as a solvent for the extraction of the analytes and dichloromethane for the clean-up. Through LC-MS/MS analysis, we established a detection limit of 20 µg kg-1, as well as calculated quantification limits of 24.4 and 24.5 µg kg-1 for FF and FFA, respectively. Validation parameters such as linearity, recovery and precision were calculated following Commission Decision 2002/657/EC. For linearity, all standard curves showed a standard coefficient greater than 0.99. Recoveries ranged from 99% to 102% for all studied concentrations. The results show that this analytical method is precise and reliable. For the depletion study, 64 Ross 308 broilers were treated with a therapeutic dosage of 10% FF during 5 consecutive days and their feathers were then analysed. Samples were drawn on days 5, 10, 15, 20, 25, 30, 35 and 40 post-treatments. As for the control group, 16 broiler chickens were raised under the same regime. Throughout the whole study, the detected concentrations of FF and FFA in feather samples were above 100 µg kg-1. In fact, even on day 30 post-treatment we detected concentrations of 221.8 and 28.8 µg kg-1 for FF and FFA, respectively. Based on these results, we conclude that these analytes will persist for a long time and will deplete slowly in feathers of treated broiler chickens.

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

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.