Development of a monoclonal antibody-based time-resolved fluorescence immunochromatographic assay strip for sensitively detecting florfenicol residues in milk and eggs: Theoretical chemical insights into unexpected high specificity.

Florfenicol (FF), with its broad-spectrum antibacterial activity, is frequently abused in the livestock and poultry industries and has aroused the growing public concern. Owing to structural similarities and varying maximum residue limits between florfenicol and other chloramphenicol (CAP)-type antibiotics, including thiamphenicol (TAP) and chloramphenicol (CAP), there is an urgent need for a rapid and effective immunoassay method to distinguish them, in order to minimize the risk of false positives. Fortunately, a highly specific monoclonal antibody (mAb), named as SF11, has been developed using hybridoma technology. Molecular simulations have revealed that the mAb SF11's specificity in recognizing florfenicol stems from the π-π stacking interaction between florfenicol and the mAb SF11 binding pocket. Using this highly specific mAb, a sensitive time-resolved fluorescence immunochromatographic assay (TRFICA) strip for rapid florfenicol detection has been developed. Under optimal conditions, this TRFICA demonstrated good analytical performance for the detection of florfenicol in milk and eggs samples, with the half-maximal inhibition concentration (IC50) values of 1.89 and 2.86 ng mL-1, the limit of detection (LOD) of 0.23 and 0.48 ng mL-1, the cut-off values of 62.50 and 31.25 ng mL-1, and the testing time of approximately thirteen minutes. Spiked recoveries in the milk and eggs samples ranged from 104.7 % to 112.3 % and 95.3 % to 116.4 %, respectively, with no obvious cross-reactions with the other analogues observed. The TRFICA results correlated well with those of high-performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) for real samples, indicating that the developed TRFICA method was sensitive, accurate and adapted for the rapid determination of florfenicol in milk and egg samples.

Fluorescent probe of quantum dots and zinc oxide in a highly selective polymer simultaneously determined florfenicol and sparfloxacin.

A nanocomposite fluorescent probe was fabricated for the simultaneous determination of florfenicol and sparfloxacin based on fluorescence quenching. The probe was synthesized by integrating nitrogen-doped graphene quantum dots (N-GQDs), cadmium telluride quantum dots (CdTe QDs) and zinc oxide nanoparticles (ZnO) into a molecularly imprinted polymer (MIP). The determination was based on the quenching of fluorescence emissions from N-GQDs by florfenicol, detected at 410 nm, and the quenching of fluorescence emissions from CdTe QDs by sparfloxacin, detected at 550 nm. The fluorescent probe was highly sensitive and specific with good linear relationships for florfenicol and sparfloxacin in the range  0.10 to 100.0 µg L-1. The limits of detection for florfenicol and sparfloxacin were 0.06 and 0.10 µg L-1, respectively. The fluorescent probe was used to determine florfenicol and sparfloxacin in food samples and the results agreed well with the results of chromatographic determination. Recoveries of spiked milk, egg and chicken samples reached 93.3-103.4% with good precision (RSD < 6%). The advantages of the nano-optosensor include high sensitivity and selectivity, simplicity, rapidity, convenience, good accuracy and precision.

Carboxylated fluorescent microsphere based immunochromatographic test strip enabled sensitive and quantitative on-site detection for florfenicol in eggs.

Florfenicol (FF), used popularly in prevention and treatment of virus infections in livestock and poultry, has widely been found in eggs and harmful to human health. In this work, a sensitive and quantitative on-site detecting solution, monoclonal antibody-based carboxylated fluorescent microsphere immunochromatographic test strip assay (FM-ICTS), is design and applied for FF detection. The proposed method can sensitively detect FF in low detection limit of 0.030 ng/g and quantitatively measure its concentration from 0.1 ng/mL to 8.1 ng/mL (R2 = 0.9991) with high repeatability (CV<8.0 %). In addition, the established FM-ICTS method exhibited high measurement accuracy in FF samples as compared with HPLC-MS analysis and demonstrated satisfied recoveries (99.1-101.3 %). More importantly, the quantitative FF test strip demonstrate ultra-high stability, which presents approximately equivalent detection ability to the fresh one after stored at 4 °C for more than one year or stored at 37 °C for 60 days. Therefore, the proposed method is a promising solution for rapidly and sensitively quantitative determination of FF in eggs.

A magnetic relaxation switching and colorimetric dual-mode aptasensor for the rapid detection of florfenicol and florfenicol amine in eggs.

It is important to establish a sensitive and rapid screening detection method for Florfenicol (FF) residue in eggs. A magnetic relaxation switch (MRS) and colorimetric aptasensor were developed for the detection of FF based on aptamer-modified Au@Fe3O4 nanoparticles (NPs). Apt-Au@Fe3O4 NPs were played as a "switch" between dispersion and aggregation, with a concomitant change in the R2 (T2 relaxivity, 1/T2W) and the UV-vis absorption spectra. To improve the sensitivity and stability of the method, the aptamers modification, salt inducing aggregation, and reaction conditions were optimized. The molar ratio of aptamers to Au, the incubation time of aptamers modification, the molar ratio of NaCl to Au, the dilute ratio of Apt-Au@Fe3O4, and reaction time were optimized to be 2:1, 3 h, 15:1, 1:300 and 15 min, respectively. The working range and LOD of MRS analysis are 0.1-10 nM and 1.10 nM for Florfenicol amine (FFA), 4-40 nM and 5.65 nM for FF. Noticeably, the colorimetric analysis can also qualitatively analyze the FF and FFA. The working ranges and LOD were 5-40 µM (5 µM) and 10-40 µM (10 µM), respectively. Hence, the results indicated that this aptasensor could be a potential tool for the rapid detection of FF residue in food.

MRM3-based UHPLC-MS/MS method for quantitation of total florfenicol residue content in milk and withdrawal study profile of milk from treated cows.

Florfenicol is a broad spectrum antibacterial, licensed globally for treatment of animal and aquaculture diseases. In the EU, Canada and US it is not permitted for use in animals producing milk or eggs. There are no published methods for analysis of total florfenicol content in milk/milk products as these lack a hydrolysis step, failing to meet the marker residue definition. A method for determining total florfenicol content in milk that meets this definition is reported for the first time. Use of a UHPLC-MS/MS multiple reaction monitoring-cubed method improved the selective detection and quantitation of lower levels of florfenicol amine in milk compared to MRM only. Single laboratory validation data and withdrawal profile in bovine milk are presented. A withdrawal period of over 50 days is indicated in case of off-label use. Requirement for hydrolysis is demonstrated.

Screening method for the detection of residues of amphenicol antibiotics in bovine milk by optical biosensor.

An immunobiosensor assay was developed for multi-residue screening in bovine milk of the parent amphenicols, thiamphenicol and florfenicol, along with the metabolite florfenicol amine. A polyclonal antibody raised in a rabbit after immunisation with a florfenicol amine-protein conjugate was employed in the assay. Milk samples were subjected to acetonitrile extraction, reconstituted in buffer and diluted prior to biosensor analysis. Validation data obtained from the analysis of fortified samples has shown that the method has a detection capability of less than 0.25 µg kg-1 for florfenicol and less than 0.5 µg kg-1 for florfenicol amine and thiamphenicol. The cross-reactivity profile and validation data for the detection of these amphenicols is presented together with results obtained following the analysis of florfenicol incurred samples using the developed screening method along with a comparison of results obtained from the analysis of the same incurred samples using an MRM3 UPLC-MS/MS confirmatory method. Results are also presented obtained from the analysis of samples from both treated and non-treated animals which were co-housed and which show the potential for cross-contamination.

Depletion of florfenicol and florfenicol amine in eggs of laying hens and growing pullets after oral administration.

In this study, we carried out two experiments to evaluate depletion of florfenicol (FF) and its metabolite florfenicol amine (FFA) in eggs from growing pullets and laying hens. Eggs were collected, and the egg white and yolk were separated. FF and FFA were analysed by liquid chromatography-tandem mass spectrometry. In the first experiment, 30 laying hens were given FF capsules at 50 mg/kg·bw-1 daily for 5 d. FF + FFA was detectable in egg white (1,190 µg/kg) on day 1 of treatment and increased slowly thereafter. After treatment, the residues decreased rapidly and were not detected by day 11. In yolk, residues were detected at a lower concentration on day 1 and increased dramatically to 3308 µg/kg at the end of treatment. The residues remained steady over the next 4 days post-treatment, followed by a rapid drop. Residues were not detectable on day 15 post-treatment. In the second experiment, four groups (B1 through B4) of growing pullets were treated in the same manner for 25, 20, 15, and 10 days before egg primiparity. FF and FFA were not detectable in the eggs of group B1; however, they were detectable in egg whites and yolks of groups B2, B3, and B4. The highest total concentrations of FF and FFA detected in egg white and yolk of group B4 were 3,190 µg/kg and 3,214 µg/kg, respectively. Thereafter, concentrations decreased until no more residues were detected in egg whites or yolks on days 17 and 21 post-treatment, respectively. Therefore, drug treatment should be stopped at least 21 d before primiparity of growing pullets to guarantee food safety.

Fraudulent antibiotic products on the market for aquaculture use.

Antibiotics in aquaculture are used to treat bacterial infections. In order for these products to work effectively fish need to be properly dosed. One of the emerging issues in aquaculture is under-dosing large populations of fish with antibiotics. This happens inadvertently for a number of reasons including the use of fraudulent medications. In this study we evaluated 17 antibiotic products (8 florfenicol and 9 oxytetracycline brands purchased in Asia) by HPLC to determine if the product labels accurately reflected the active pharmaceutical ingredient (API) in the package. We determined authenticity scores for different batches of products at two separate laboratories by comparing the observed API to the label API concentration. We found that 48 % of the antibiotic batches had authenticity scores below 80 % (i.e. observed API in package was at least 20 % less than the label API concentration). Further, there were 9 or the 31 batches of drugs tested had no measureable API. Some products had variation in their authenticity scores between batches making it difficult to rely on a brand. The price of florfenicol products may help identify products with low authenticity scores, but in the case of oxytetracycline, the price of all the products tested was relatively similar. The findings in this study suggest that not all florfenicol and oxytetracycline antibiotic products on the market in Asia have API concentrations indicated on their labels. This could be problematic for medicating fish on aquaculture farms.

The bacterial microbiota in florfenicol contaminated soils: The antibiotic resistome and the nitrogen cycle.

Soil antibiotic resistome and the nitrogen cycle are affected by florfenicol addition to manured soils but their interactions have not been fully described. In the present study, antibiotic resistance genes (ARGs) and nitrogen cycle genes possessed by soil bacteria were characterized using real-time fluorescence quantification PCR (qPCR) and metagenomic sequencing in a short-term (30 d) soil model experiment. Florfenicol significantly changed in the abundance of genes conferring resistance to aminoglycosides, ß-lactams, tetracyclines and macrolides. And the abundance of Sphingomonadaceae, the protein metabolic and nitrogen metabolic functions, as well as NO reductase, nitrate reductase, nitrite reductase and N2O reductase can also be affected by florfenicol. In this way, ARG types of genes conferring resistance to aminoglycosides, ß-lactamases, tetracyclines, colistin, fosfomycin, phenicols and trimethoprim were closely associated with multiple nitrogen cycle genes. Actinobacteria, Chlorobi, Firmicutes, Gemmatimonadetes, Nitrospirae, Proteobacteria and Verrucomicrobia played an important role in spreading of ARGs. Moreover, soil physicochemical properties were important factors affecting the distribution of soil flora. This study provides a theoretical basis for further exploration of the transmission regularity and interference mechanism of ARGs in soil bacteria responsible for nitrogen cycle.

Determination of Chloramphenicol, Thiamphenicol and Florfenicol in Chinese Gelatin Medicines using Dispersive Solid-Phase Extraction Coupled with Ultra High-Performance Liquid Chromatography-Mass Spectrometry.

This study established a rapid and reliable method to determine chloramphenicol (CAP), thiamphenicol (TAP) and florfenicol (FF) residues in Chinese gelatin medicines. CAP, TAP and FF were extracted from medicine samples using 2% (v/v) ammonium hydroxide in acetonitrile. Trypsin was used to eliminate the matrix effect caused by protein components in gelatin medicines, whereas anhydrous sodium sulfate, C18-N and NH2-PSA adsorbents were applied to reduce matrix effect induced by other components. The analytical method of these drugs was optimized on ultra high-performance liquid chromatography-mass spectrometer (UHPLC-MS/MS) through the analysis of their standard linearity and regression. The optimized extraction and analytical method were validated in one Chinese gelatin medicine sample (Colla corii asini, E Jiao) with three fortification levels (2, 5 and 10 µg/kg), and the recoveries of these drug residues ranged of 87.6-102.7%. The limit of detection and quantification of CAP, TAP and FF in the sample were 0.2 and 0.5 µg/kg, 0.4 and 1.5 µg/kg, and 0.5 and 1.5 µg/kg, respectively. A total of 30 Chinese gelatin medicine samples were analyzed using the established method. No drug residues were found in these samples except for one Testudinis Carapacis et Plastri (1.67 µg/kg FF) and one turtle shell glue (2.55 µg/kg FF).

A reliable and cost-efficient TLC-HPLC method for determining total florfenicol residues in porcine edible tissues.

It is still a challenge to solve the matrix interferences in veterinary drug residue analysis. In this study, we reported a thin layer chromatography (TLC)-high-performance liquid chromatography (HPLC) method for determining total florfenicol (FF) residues, expressed as florfenicol amine (FFA), in porcine edible tissues. The tissue homogenate were acid-hydrolyzed to liberate the bound residues and convert them into FFA. The hydrolysates were washed with ethyl acetate and subsequently extracted with ethyl acetate under alkaline conditions. The supernatants were concentrated through evaporation, defatted with hexane, purified by TLC and analyzed by HPLC at 225 nm. The optimal developing solvent for TLC purification was ethyl acetate-acetone-ammonium hydroxide mixtures (2:8:0.5, v/v/v). The method was fully validated according to decision 2002/657/EC, and could be used for the routine monitoring of FF residues in pig. TLC showed excellent purification efficiency, and was expected to solve the matrix interferences in veterinary drug residue analysis.

Capped cadmium sulfide quantum dots with a new ionic liquid as a fluorescent probe for sensitive detection of florfenicol in meat samples.

The new ionic liquid capped CdS quantum dots (IL-CdS QDs) as a fluorescent probe was successfully synthesized by a hydrothermal method in a one step process and used for the facile and sensitive determination of florfenicol (FLF) in aqueous media. The new ionic liquid 3-(2-[(5-amino-1,3,4-thiadiazol-2-yl)thio]ethyl)-1-methyl-1H-imidazol-3-ium chloride (IL) was synthesized by introducing 5-amino-1,3,4-thiadiazole-2-thiol as a ligand onto the alkyl chain of the 1-chloroethyl-3-methylimidazolium chloride ILs. This task specific ionic liquid reagent was used for the capping of CdS QDs which played the role of recognition element of FLF. The IL-CdS QDs were characterized by Ultra Violet-Visible absorption -spectroscopy (UV-Vis absorption spectroscopy), Fourier transform infrared (FT-IR) spectroscopy, X-ray diffraction (XRD), and transmission electron microscopy (TEM). Quenching of fluorescence intensity of the IL-CdS QDs was in proportion to the addition of FLF concentration. Under the optimum conditions, the fluorescence intensity ratio of IL-CdS QDs in the presence and absence of FLF versus FLF concentrations gave a linear response according to the Stern-Volmer equation from 0.1 to 20 µg mL-1 (0.3 to 56 µmol L-1) with a limit of detection 0.035 µg mL-1 (0.098 µmol L-1). The developed method was applied to the determination of FLF in fish and chicken meats with satisfactory results. This method revealed some advantages such as high sensitivity, precision and wide linear range to FLF. The proposed method can be utilized for rapid screening the quality of meat products.

Mechanistic insight into the oxazoline decomposition of DFC-M, a synthetic intermediate of florfenicol.

2-(dichloromethyl)-5[4-(methylsulfonyl)-phenyl]-4-(fluoromethyl)-oxazoline (DFC-M, 1) is a key oxazoline-containing intermediate in commercial process for the synthesis of Florfenicol (3), a marketed broad spectrum veterinary antibiotic. DFC-M was not stable in solution due to the presence of oxazoline moiety, which provided further hindrance for analytical sample preparation and HPLC analysis. Hence, the mechanistic study on the in-solution degradation of DFC-M was carried out via online and offline UPLC-HR-ESI-MS as well as in-situ NMR, and the degradation pathways were proposed. This mechanistic information, together with the follow-up solution stability study, provided crucial information regarding the solution handling and mobile phase selection for DFC-M analysis during commercial processing.

Development of an Accelerated Solvent Extraction-Ultra-Performance Liquid Chromatography-Fluorescence Detection Method for Quantitative Analysis of Thiamphenicol, Florfenicol and Florfenicol Amine in Poultry Eggs.

A simple, rapid and novel method for the detection of residues of thiamphenicol (TAP), florfenicol (FF) and its metabolite, florfenicol amine (FFA), in poultry eggs by ultra-performance liquid chromatography-fluorescence detection (UPLC-FLD) was developed. The samples were extracted with acetonitrile-ammonia (98:2, v/v) using accelerated solvent extraction (ASE) and purified by manual degreasing with acetonitrile-saturated n-hexane. The target compounds were separated on an ACQUITY UPLC® BEH C18 (2.1 mm × 100 mm, 1.7 µm) chromatographic column using a mobile phase composed of 0.005 mol/L NaH2PO4, 0.003 mol/L sodium lauryl sulfate and 0.05% trimethylamine, adjusted to pH 5.3 ± 0.1 by phosphoric acid and acetonitrile (64:36, v/v). The limits of detection (LODs) and limits of quantification (LOQs) of the three target compounds in poultry eggs were 1.8-4.9 µg/kg and 4.3-11.7 µg/kg, respectively. The recoveries of the three target compounds in poultry eggs were above 80.1% when the spiked concentrations of three phenicols were the LOQ, 0.5 maximum residue limit (MRL), 1.0 MRL and 2.0 MRL. The intraday relative standard deviations (RSDs) were less than 5.5%, and the interday RSDs were less than 6.6%. Finally, this new detection method was successfully applied to the quantitative analysis of TAP, FF and FFA in 150 commercial poultry eggs.

HPLC-DAD analysis of florfenicol and thiamphenicol in medicated feedingstuffs.

A simple and reliable method using liquid chromatography with diode array detector was developed for the simultaneous determination of florfenicol and thiamphenicol in medicated feed. The analytes were extracted from the minced feed with methanol and ethyl acetate (1:1, v/v). Next, the extract was further cleaned up by dispersive solid phase extraction using anhydrous magnesium sulfate, PSA and C18 sorbents. Finally, 1 mL of extract was evaporated, the residue resuspended in Milli-Q water, and filtered. The method was validated in-house at medicated levels, in the concentration range 10-300 µg/mL (50-1500 mg/kg). Values of <6.5% and <6.0% were found, respectively, for repeatability and within-laboratory reproducibility. The LODs for the two fenicols were 2.4-5.3 mg/kg, while the LOQs were 3.8-5.6 mg/kg. The expanded uncertainty was estimated to be in the range of 10.0-14.5%, depending on the analyte. Recoveries varied from 81.7% to 97.5%. The methodology was applied to the analysis of animal feedingstuffs collected from poultry and pig farms.

Multimedia fate modeling of antibiotic sulfamethoxazole, lincomycin, and florfenicol in a seasonally ice-covered river receiving WWTP effluents.

As a result of the widespread use of antibiotics, a large amount of excretions from human and animals, containing antibiotic residues, is discharged into aquatic environments, leading to potential adverse effects on the ecosystems' health. These residues' impact on seasonally ice-covered rivers remains under investigated. To understand the environmental fate of antibiotics with high-detection frequencies and concentration levels, sulfamethoxazole, lincomycin, and florfenicol were used as models in the present study. A Level IV fugacity model was established and applied to a seasonally ice-covered river receiving municipal wastewater treatment plant (WWTP) effluents, the Songhua River in Northeast China. Model validation and sensitivity analysis suggested that the fugacity model could successfully simulate the monitoring concentration within an average difference of one logarithmic unit. The advection process played a major role in the transport and attenuation of the antibiotics in the ice-covered river receiving WWTP effluents. The scenario simulation indicated that increasing the targeted antibiotic concentrations in WWTP effluents to µg L-1 could keep the targeted antibiotic concentrations higher than 10 ng L-1 in the receiving river from the WWTP discharge source to 25 km downstream. This finding also demonstrates that the depth of water and ice, as well as flow velocity, play key roles in the fate of antibiotics in the ice-covered river receiving WWTP effluents. To our best knowledge, this is the first major study to combine experimental investigation with modeling to explore the environmental behaviors and fate of antibiotics in such a river.

Development and application of a population physiologically based pharmacokinetic model for florfenicol and its metabolite florfenicol amine in cattle.

Florfenicol (FF) is used in cattle to treat respiratory diseases but could result in tissue residues. This study aimed to develop a population physiologically based pharmacokinetic (PBPK) model to predict the concentrations of FF and its metabolite, florfenicol amine (FFA), in cattle after four different routes of administration, and to calculate and compare the withdrawal intervals (WDIs) with approved withdrawal times based on different marker residues and their MRLs or tolerances. A flow-limited PBPK model including both FF and FFA sub-models were developed with published data using acslXtreme. This model predicted FF and FFA concentrations in tissues and plasma/serum after intramuscular or subcutaneous administration. Based on the model, the WDIs of 46 and 58 days were calculated to ensure that total residue concentrations (FF + FFA) in 95th percentile of the population after intramuscular and subcutaneous administration were below the MRL, respectively. WDIs were calculated as 44 and 47 days to ensure that FFA concentrations after intramuscular and subcutaneous administration fell below tolerances in 99th percentile of the population, respectively. WDIs were longer than the corresponding label in China, US, and EU. This model provides a useful tool to predict tissue residues of FF and FFA in cattle to improve food safety.

Development and comparison of liquid-liquid extraction and accelerated solvent extraction methods for quantitative analysis of chloramphenicol, thiamphenicol, florfenicol, and florfenicol amine in poultry eggs.

Accelerated solvent extraction was investigated as a novel alternative technology for the separation and quantitative analysis of chloramphenicol, thiamphenicol, florfenicol, and florfenicol amine from poultry eggs, and the results were compared with the results of liquid-liquid extraction. Rapid quantification of the target compounds was carried out by ultra-performance liquid chromatography-electrospray ionization tandem triple quadrupole mass spectrometry. This optimized method was validated according to the requirements defined by the European Union and the United States Food and Drug Administration. Finally, the new approach was successfully applied to the quantitative determination of these analytes in 90 commercial poultry eggs from local supermarkets.

Determination of total florfenicol residues as florfenicol amine in bovine tissues and eel by liquid chromatography-tandem mass spectrometry using external calibration.

A reliable liquid chromatography-tandem mass spectrometry method was developed to determine total florfenicol residues in bovine tissues and eel. Florfenicol and its metabolites (florfenicol amine, monochloroflorfenicol, florfenicol oxamic acid, and florfenicol alcohol) were analyzed as the marker residue, florfenicol amine, as defined by several regulatory agencies. After hydrolysis with hydrochloric acid, samples were defatted and subjected to solid-supported liquid extraction and Oasis MCX-cartridge cleanup before analysis. The method was validated for florfenicol and its metabolites at two levels in eel and bovine muscle, fat, and liver. Excellent recoveries were obtained (93-104%), with relative standard deviations of <6% for all compounds. Negligible matrix effects and minimal analyte loss during sample preparation enabled accurate quantification by external calibration using solvent standards. No interfering peaks were observed around the retention time of florfenicol amine, indicating the high selectivity of the method. Retention times in the spiked samples corresponding to that of the calibration standard in solvent did not exceed ±0.1 min. Ion ratios from the spiked sample were within ±10% (relative) of the calibration standards. Calibration curves were linear in the range of 0.5 to 100 ng/mL, with coefficients of determination higher than 0.998. The limits of quantification and limits of detection of the proposed method were estimated to be 0.01 mg/kg and 0.0005 mg/kg, respectively, in all food samples. Thus, the developed method is considered reliable and suitable for regulatory use.

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.