Determination of Ciprofloxacin,Enrofloxacin, and Marbofloxacin in Bovine Urine,Serum, and Milk by Microextraction by a Packed Sorbent Coupled to Ultra-High Performance Liquid Chromatography

This article reports new, easy, and rapid microextraction by packed sorbent (MEPS)–ultra high performance liquid chromatography with photodiode array detection for the simultaneous determination in bovine urine, serum, and milk of three antibiotics belonging to the class of the fluoroquinolones, namely ciprofloxacin, enrofloxacin, and marbofloxacin, approved for veterinary and human use (ciprofloxacin). The chromatographic separation of the analytes and all aspects influencing the MEPS performance were optimized for the extraction from the considered biological samples. The optimized procedure required simple sample pretreatment, a short (<8?min) isocratic elution, and provided sufficient sensitivity for the determination of the analytes at trace levels in compliance with current legislation. Limits of quantitation were in the range from 0.002 (ciprofloxacin, urine) to 0.048?μg/mL (enrofloxacin, milk). Recoveries from 79% (enrofloxacin, milk) to 88% (ciprofloxacin, urine/serum) were obtained on spiked samples. The within-day (n?=?6) and between-day (n?=?6 over 3?days) relative standard deviation percentages in bovine urine, serum, and milk samples ranged from 2.2 (ciprofloxacin, urine) to 2.5 (enrofloxacin, serum) and from 3.1 (ciprofloxacin, urine) to 3.7 (enrofloxacin, milk), respectively, and were not concentration dependent. To the best of our knowledge, this is the first study describing a fast and simple method for the determination of fluoroquinolones in bovine biological samples.     

Multiresidue HPLC analysis of ten quinolones in milk after solid phase extraction: validation according to the European Union Decision 2002/657/EC

A rapid and sensitive analytical method was developed for the residue analysis of ten quinolones (enoxacin (ENO), ofloxacin (OFL), norfloxacin (NOR), ciprofloxacin (CIP), danofloxacin (DAN), enrofloxacin (ENR), sarafloxacin (SAR), oxolinic acid (OXO), nalidixic acid (NAL), and flumequine (FLU)) in cow's milk. The analytes were extracted from milk by a deproteinization step followed by a simple SPE cleanup procedure using LiChrolut RP-18 Merck cartridges. Recoveries varied between 75 and 92%. HPLC separation was performed at 25 degrees C using an ODS-3 PerfectSil Target (250 x 4 mm(2)) 5 microm analytical column (MZ-Analysentechnik, Germany). The mobile phase consisted of a mixture of TFA 0.1%-CH(3)CN-CH(3)OH, delivered by a gradient program at the flow rate of 1.2 mL/min. Elution of the ten analytes and the internal standard (caffeine, 7.5 ng/microL) was completed within 27 min. Column effluent was monitored using a photodiode array detector, set at 275 and 255 nm. The developed method was validated according to the criteria of Commission Decision 2002/657/EC. The LODs of the specific method of quinolones' determination in milk varied between 1.5 and 6.8 ng/microL.     

Determination of enrofloxacin and its metabolite ciprofloxacin in goat milk by high-performance liquid chromatography with diode-array detection. Optimization and validation

A high-performance liquid chromatography-diode array detection method (HPLC-DAD) combined with liquid chromatography-mass spectrometry was developed for the determination of enrofloxacin and its metabolite ciprofloxacin in goat milk. The HPLC-DAD method validation was compliant with the "DG SANCO 1805/2000" European regulation. The residues were extracted from milk with phosphate buffer, purified on a C18 Speedisk cartridge SPE (Baker) and then analysed using HPLC-DAD set at 277 nm. The decision limit (CCa) calculated by spiking samples at 100 microg/kg with both analytes, taking into account the maximum residue limit (MRL) of 100 microg/kg established by the European Union for the sum of enrofloxacin and its metabolite ciprofloxacin in milk, was 105.3 microg/kg for enrofloxacin and 105.5 microg/kg for ciprofloxacin. The detection capability (CCbeta) was 110.7 and 110.9 microg/kg for enrofloxacin and ciprofloxacin, respectively. The mean recoveries of the method, calculated by spiking samples at 50, 100 and 150 microg/kg were 84% for enrofloxacin and 88% for ciprofloxacin. The limit of quantification was 20 microg/kg for both analytes. The HPLC-DAD validated method was successfully applied for the first time in goats milk, and proved to be suitable for the sensitive and accurate quantification and confirmation analysis of enrofloxacin and ciprofloxacin for regulatory purposes.     

Extraction of quinolones from milk samples using bentonite/magnetite nanoparticles before determination by high‐performance liquid chromatography with fluorimetric detection

In this work, bentonite magnetic nanoparticles synthesized by a typical coprecipitation method were used as the adsorbent for the magnetic solid‐phase extraction of six quinolones (ciprofloxacin, difloxacin, enrofloxacin, norfloxacin, sarafloxacin, and lomefloxacin) from milk samples followed by high‐performance liquid chromatography with fluorimetric detection. Under the optimized conditions, the linear quantitation range for the six quinolones was 0.3–200 ng/mL, and the correlation coefficients of the calibration curves ranged from 0.9994 to 0.9999. The detection limit of the method was 0.1 ng/mL. Recoveries of quinolones from pure and low‐fat spiked milk samples varied from 80.4 to 92.7% and from 81.3 to 93.5%, respectively. These results demonstrated that the proposed method for the determination of six quinolones in milk samples was rapid, reliable, and efficient.     

Determination of quinolone residues in infant and young children powdered milk combining solid-phase extraction and ultra-performance liquid chromatography-tandem mass spectrometry

The present work describes a method based on solid-phase extraction (SPE) and ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) for the simultaneous determination of three quinolones (pipemidic acid, oxolinic acid and flumequine) and twelve fluoroquinolones (marbofloxacin, fleroxacin, pefloxacin, levofloxacin, norfloxacin, ciprofloxacin, enrofloxacin, danofloxacin, lomefloxacin, difloxacin, sarafloxacin, and moxifloxacin) in different infant and young children powdered milks. After suitable deproteination of the reconstituted powdered samples, a SPE procedure was developed providing recovery values higher than 84% (RSDs lower than 13%) for all the analytes, with limits of detection between 0.04 and 0.52 μg/kg. UPLC-MS/MS analyses were carried out in less than 10 min. Sixteen infant and young children powdered milk samples of different origin, type and composition bought at Spanish markets were analyzed. Residues of the selected antibiotics were not detected in any of the analyzed samples.     

Determination of quinolone antibiotics in urine by capillary electrophoresis with chemiluminescence detection

A novel and simple method is presented for the determination of norfloxacin, ciprofloxacin, and ofloxacin by capillary electrophoresis with chemiluminescence detection. This method is based on the enhancing effect of quinolones on the chemiluminescence reaction of the Ce(SO₄)₂–Ru(bpy)₃²⁺–HNO₃ system. Three quinolones were successfully separated and detected under optimum conditions. The obtained detection limits were 2.3×10^–7 mol/L, 5.2×10^–8 mol/L, and 7.8×10^–8 mol/L for ciprofloxacin, norfloxacin, and ofloxacin, respectively. The RSD of migration time and peak area were less than 1.8 and 3.8% (n = 5), respectively. The applicability of the proposed method was illustrated in the determination of ofloxacin in eye drops and of norfloxacin in human urine samples, and the monitoring of pharmacokinetics for norfloxacin.     

Water-compatible molecularly imprinted polymer for the selective recognition of fluoroquinolone antibiotics in biological samples

A novel water-compatible molecularly imprinted polymer (MIP), prepared with enrofloxacin (ENR) as the template, has been optimised for the selective extraction of fluoroquinolone antibiotics in aqueous media. The results of a morphological characterisation and selectivity tests of the polymer material for ENR and related derivatives are reported. High affinity for the piperazine-based fluoroquinolones marbofloxacin, ciprofloxacin, norfloxacin and ofloxacin was observed, whereas no retention was found for nonrelated antibiotics. Various parameters affecting the extraction efficiency of the polymer have been optimised to achieve selective extraction of the antibiotics from real samples and to reduce nonspecific interactions. These findings resulted in a MISPE/HPLC-FLD method allowing direct extraction of the analytes from aqueous samples with a selective wash using just 50% (v/v) organic solvent. The method showed excellent recoveries and precision when buffered urine samples fortified at five concentration levels (25–250 ng mL⁻¹ each) of marbofloxacin, ciprofloxacin, norfloxacin, enrofloxacin and sarafloxacin were tested (53–88%, RSD 1–10%, n = 3). Moreover, the biological matrix of the aqueous samples did not influence the preconcentration efficiency of the fluoroquinolones on the MIP cartridges; no significant differences were observed between the recovery rates of the antibiotics in buffer and urine samples. The detection limits of the whole process range between 1.9 and 34 ng mL^–1 when 5-mL urine samples are processed. The developed method has been successfully applied to preconcentration of norfloxacin in urine samples of a medicated patient, demonstrating the ability of the novel MIP for selective extraction of fluoroquinolones in urine samples.     

Ionic liquid‐based microwave‐assisted surfactant‐improved dispersive liquid–liquid microextraction and derivatization of aminoglycosides in milk samples

A green and simple method, ionic liquid‐based microwave‐assisted surfactant‐improved dispersive liquid–liquid microextraction and derivatization was developed for the determination of aminoglycosides in milk samples. Nonionic surfactant Triton X‐100 and ionic liquid 1‐hexyl‐3‐methylimidazolium hexafluorophosphate were used as the disperser and extraction solvent, respectively. Extraction, preconcentration, and derivatization of aminoglycosides were carried out in a single step. Several experimental parameters, including type and volume of extraction solvent, type and concentration of surfactant, microwave power and irradiation time, concentration of derivatization reagent, and pH value and volume of buffer were investigated and optimized. Under the optimum experimental conditions, the linearities for determining the analytes were in the range 0.4–10.0 ng/mL for tobramycin, 1.0–25.0 ng/mL for neomycin, and 2.0–50.0 ng/mL for gentamicin, with the correlation coefficients ranging from 0.9991 to 0.9998. The LODs for the analytes were between 0.11 and 0.50 ng/mL. The present method was applied to the analysis of different milk samples, and the recoveries of aminoglycosides obtained were in the range 96.4–105.4% with the RSDs lower than 5.5%. The results showed that the present method was a rapid, convenient, and environmentally friendly method for the determination of aminoglycosides in milk samples.     

Determination of fluoroquinolone antibiotics in surface waters from Mondego River by high performance liquid chromatography using a monolithic column

A novel LC-fluorescence detection method based on the use of a monolithic column for the determination of norfloxacin, ciprofloxacin, and enrofloxacin antibiotic residues in environmental waters was developed. Fluoroquinolones (FQs) were isocratically eluted using a mobile phase consisting of 0.025 M phosphoric acid solution at pH 3.0 with tetrabutylammonium and methanol (960:40, v/v) through a Chromolith Performance RP-18e column (100x4.6 mm) at a flow rate of 2.5 mL/min and detected at excitation and emission wavelengths of 278 and 450 nm, respectively. After acidification and addition of EDTA, water samples were extracted using an Oasis HLB cartridge. Linearity was evaluated in the range of 0.05 to 1 microg/mL and correlation coefficients of 0.9945 for norfloxacin, 0.9974 for ciprofloxacin, and 0.9982 for enrofloxacin were found. The limit of quantification was 25 ng/L for the three FQs. The recovery of FQs spiked into river water samples at 25, 50, and 100 ng/L fortification levels ranged from 76.5 to 91.0% for norfloxacin, 78.5 to 97.2% for ciprofloxacin, and 79.4 to 93.6% for enrofloxacin. This method was successfully applied to the analysis of water samples from the Mondego River, and ciprofloxacin and enrofloxacin residues were detected in eight water samples.     

Development of multiple monolithic fiber solid‐phase microextraction and liquid chromatography–tandem mass spectrometry method for the sensitive monitoring of aminoglycosides in honey and milk samples

A simple and sensitive method for the simultaneous extraction and determination of six aminoglycosides in honey and milk samples was developed using multiple monolithic fiber solid‐phase microextraction and liquid chromatography with tandem mass spectrometry. The multiple monolithic fibers based on poly(methacrylic acid‐co‐ethylenedimethacrylate) monolith as the extraction medium was used to concentrate target analytes. Because there were abundant carboxyl groups in the monolith, the monolithic fibers could extract aminoglycosides effectively through cation‐exchange and hydrophobic interactions. To obtain optimum extraction performance, several extraction parameters including desorption solvent, adsorption and desorption time, pH value and ionic strength in sample matrix, were investigated in detail. Under the optimized extraction conditions, the limits of detection of the proposed method were 0.10–0.30 and 0.23–0.59 μg/kg for honey and milk samples, respectively. Satisfactory linearity was achieved for analytes with the coefficients of determination above 0.99. At the same time, the developed method showed acceptable method repeatability and reproducibility. Finally, the proposed method was successfully applied to the determination of aminoglycosides in real honey and milk samples. Recoveries obtained for the determination of six target analytes in spiking samples ranged from 67.9 to 110%, and the relative standard deviations were in the range of 1.2–11%.     

Development and Validation of a Dispersive Solid Phase Extraction Liquid Chromatography Mass Spectrometry Method with Electrospray Ionization for the Determination of Multiclass Pesticides and Metabolites in Meat and Milk

A dispersive solid phase extraction–liquid chromatography tandem mass spectrometry method with electrospray ionization was validated in food of animal origin for the determination of multiclass pesticide residues and their metabolites. A simple and low-cost sample preparation procedure using freezing as the clean-up step was used to identify and quantify analytes belonging to 39 different chemical classes in meat and milk matrices. Mean recoveries in the range of 70–120% with relative standard deviations <10% were obtained for the majority of the analytes. The limit of quantification of the method was 10 µg/kg. The matrix effects were statistically evaluated and the quantification of the analytes was conducted using calibration curves constructed with matrix matched calibration standards covering concentrations from 5 to 200 µg/kg. The proposed method was applied in 86 samples of animal origin taken from the Greek market, two of which were found positive for pesticides.     

Core-shell magnetic porous organic polymer for magnetic solid-phase extraction of fluoroquinolone antibiotics in honey samples followed by high-performance liquid chromatography with fluorescence detection

By monomer-mediated in-situ growth synthesis strategy, with hydroquinone and 1,3,5-tris(4-aminophenyl)benzene as monomers, a core-shell magnetic porous organic polymer was synthesized through a simple azo reaction. Based on this, a magnetic solid-phase extraction–high-performance liquid chromatography–fluorescence detection method was proposed for the analysis of fluoroquinolones in a honey sample. With ofloxacin, ciprofloxacin, enrofloxacin, lomefloxacin, and difloxacin as target analytes, factors affecting the extraction efficiency had been optimized. The LODs were 1.5–5.4 ng/L (corresponding to 0.23–0.81 μg/kg in honey). The linear range was 0.005–20 μg/L for difloxacin, 0.01–20 μg/L for ofloxacin, ciprofloxacin and lomefloxacin, and 0.02–20 μg/L for enrofloxacin. The enrichment factor was 84.4–91.7-fold with a high extraction efficiency of 84.4–91.7%. The method was assessed by the analysis of target fluoroquinolones in honey samples, and the recoveries for the spiked samples were 79.3–95.8%. The results indicated that the established magnetic solid-phase extraction–high-performance liquid chromatography–fluorescence detection method is efficient for the analysis of trace fluoroquinolones in honey.     

Direct determination of five fluoroquinolones in chicken whole blood and in veterinary drugs by HPLC

A direct, accurate, and sensitive chromatographic analytical method for the quantitative determination of five fluoroquinolones (enoxacin, ofloxacin, norfloxacin, ciprofloxacin, and enrofloxacin) in chicken whole blood is proposed in the present study. For quantitative determination lamotrigine was used as internal standard at a concentration of 20 ng/microL. The developed method was successfully applied to the determination of enrofloxacin, as the main component of commercially available veterinary drugs. Fluoroquinolone antibiotics were separated on an Inertsil (250 x 4 mm) C8, 5 microm, analytical column, at ambient temperature. The mobile phase consisted of a mixture of citric acid (0.4 mol L(-1))-CH3OH-CH3CN (87:9:4% v/v) leading to retention times less than 14 min, at a flow rate 1.4 mL min(-1). UV detection at 275 nm provided limits of detection of 2 ng/mL per 20 microL injected volume for enoxacin, norfloxacin, and ciprofloxacin, 0.4 ng/mL for ofloxacin, and 4 ng/mL for enrofloxacin. Preparation of chicken blood samples is based on the deproteinization with acetonitrile while the pharmaceutical drug was simply diluted with water. Peaks of examined analytes in real samples were identified by means of a photodiode array detector. The method was validated in terms of within-day (n=6) precision and accuracy after chicken whole blood sample deproteinization by CH3CN. Using 50 microL of chicken blood sample, recovery rates at fortification levels of 40, 60, and 80 ng ranged from 86.7% to 103.7%. The applicability of the method was evaluated using real samples from chicken under fluoroquinolone treatment.     

Determination of antibiotics in feedstuff samples by microemulsion electrokinetic chromatography using fullerene as additive

A microemulsion electrokinetic chromatography method was developed for the simultaneous detection and quantification of ciprofloxacin, norfloxacin, sulfamethoxazole, tetracycline, and oxytetracycline in feedstuff samples. The BGE composition was optimized by applying a Taguchi parameter design and consisted of phosphate 30 mmol/L, Tween‐80 0.01 mmol/L, and fullerene 3 μmol/L, and adjusted to pH 8.0; the addition of surfactant and fullerene modifies the mobility of the analytes improving their resolution. Theoretical studies showed π‐π and van der Waals interactions between antibiotic molecules and fullerene used as a pseudostationary phase. Under optimal conditions, limits of detection ranged from 0.7 to 1.5 μg/g; the analytes were separated in less than 6 min. The methodology proposed is useful for controlling and monitoring antibiotic residues in feedstuff samples.     

QuEChERS-超高效液相色谱-串联质谱法测定牛奶中6种头孢菌素类抗生素残留

建立了QuEChERS-超高效液相色谱-串联质谱（UPLC-MS/MS）同时测定牛奶中6种头孢菌素类抗生素的分析方法。牛奶样品加入含1%（v/v）醋酸的乙腈溶液提取,于45℃氮吹浓缩,经100 mg C18吸附剂净化后,采用HSS T3柱（100 mm×2.1 mm,1.8 μm）分离,以0.1%（v/v）甲酸水溶液和乙腈为流动相进行梯度洗脱,在电喷雾离子源、正离子模式（ESI⁺）下电离,多反应监测（MRM）模式下检测,外标法定量。结果表明,6种头孢菌素类抗生素在2~200 μg/L质量浓度范围内均呈良好的线性关系,相关系数（r）均大于0.999,方法的检出限为0.2~0.6 μg/kg、定量限为0.8~2.0 μg/kg。在8、16和80 μg/kg的加标水平下,6种头孢菌素类抗生素的回收率为75.1%~94.4%,相对标准偏差为0.63%~8.3%。该方法简单快捷,准确可靠,适用于牛奶中头孢菌素类抗生素残留的同时测定。     

Dispersive liquid-liquid microextraction and gas chromatography-mass spectrometry determination of polychlorinated biphenyls and polybrominated diphenyl ethers in milk

An effective multi‐residue pretreatment technique, solid‐phase extraction (SPE) combined with dispersive liquid–liquid microextraction (DLLME), was proposed for the trace analysis of 14 polychlorinated biphenyls (PCBs) and polybrominated diphenyl ethers (PBDEs) in milk samples using gas chromatography–mass spectrometry (GC‐MS). Interesting analytes in milk samples were extracted with hexane after protein precipitation. The hexane extracts were loaded on an LC‐Florisil column to isolate analytes from the milk matrix. The elutes were dried and dissolved in acetone, which was used as the disperser solvent in subsequent DLLME procedures. The effects of several important parameters on the extraction efficiency were evaluated. Under the optimized conditions, a linear relationship was obtained in the range of 0.02–10.00 μg/L (PCBs) and 0.5–100.00 μg/L (PBDEs). The LOD (S/N=3) and relative standard deviations (RSDs, n=5) for all analytes were 0.01–0.4 μg/L and 0.6–8.5%, respectively. The recoveries of the standards added to raw bovine milk samples were 74.0–131.8%, and the repeatabilities of the analysis results were 1.12–17.41%. This method has been successfully applied to estimating PCBs and PBDEs in milk samples.     

Determination of metoserpate,buquinolate, and diclofenac in pork,eggs, and milk using liquid chromatography–tandem mass spectrometry

In this work, a method was developed for the simultaneous determination of residual metoserpate, buquinolate and diclofenac in pork, milk, and eggs. Samples were extracted with 0.1% formic acid in acetonitrile, defatted with n‐hexane, and filtered prior to analysis using liquid chromatography–tandem mass spectrometry. The analytes were separated on a C₁₈ column using 0.1% acetic acid and methanol as the mobile phase. The matrix‐matched calibration curves showed good linearity over a concentration range of 5–50 ng/g with coefficients of determination (R²) ≥0.991. The intra‐ and inter‐day accuracies (expressed as recovery percentage values) calculated using three spiking levels (5, 10, and 20 μg/kg) were 80–108.65 and 74.06–107.15%, respectively, and the precisions (expressed as relative standard deviation) were 2.86–13.67 and 0.05–11.74%, respectively, for the tested drugs determined in various matrices. The limits of quantification (1 and 2 μg/kg) were below the uniform residual level (0.01 mg/kg) set for compounds that have no specific maximum residue limit (MRL). The developed method was tested using market samples and none of the target analytes was detected in any of the samples. The validated method proved to be practicable for detection of the tested analytes in pork, milk, and eggs.     

Direct determination of four fluoroquinolones,enoxacin, norfloxacin,ofloxacin, and ciprofloxacin,in pharmaceuticals and blood serum by HPLC

A rapid, accurate and sensitive method has been developed for the quantitative determination of four fluoroquinolone antimicrobial agents, enoxacin, norfloxacin, ofloxacin and ciprofloxacin, with high in-vitro activity against a wide range of Gram-negative and Gram-positive organisms.A Kromasil 100 C(8) 250 mm x 4 mm, 5 microm analytical column was used with an eluting system consisting of a mixture of CH(3)CN-CH(3)OH-citric acid 0.4 mol L(-1) (7:15:78 %, v/v). Detection was performed with a variable wavelength UV-visible detector at 275 nm resulting in limits of detection: 0.02 ng per 20 microL injection for enoxacin and 0.01 ng for ofloxacin, norfloxacin and ciprofloxacin. Hydrochlorothiazide (HCT) was used as internal standard at a concentration of 2 ng microL(-1). A rectilinear relationship was observed up to 2 ng microL(-1) for enoxacin, 12 ng microL(-1) for ofloxacin, 3 ng microL(-1) for norfloxacin, and 5 ng microL(-1) for ciprofloxacin. Separation was achieved within 10 min. The statistical evaluation of the method was examined by performing intra-day (n=8) and inter-day precision assays (n=8) and was found to be satisfactory with high accuracy and precision. The method was applied to the direct determination of the four fluoroquinolones in human blood serum. Sample pretreatment involved only protein precipitation with acetonitrile. Recovery of analytes in spiked samples was 97+/-6% over the range 0.1-0.5 ng microL(-1).     

Determination of (fluoro)quinolones in environmental water using online preconcentration with column switching linked to large sample volumes and fluorescence detection

An analytical method based on online enrichment using coupled-column liquid chromatography with fluorescence detection has been developed to determine marbofloxacin, ciprofloxacin, danofloxacin, enrofloxacin, norfloxacin, lomefloxacin, oxolinic acid, and nalidixic acid at trace levels in surface water. The sample containing the pharmaceuticals was pumped through a short C18 column in such a way that the analytes were retained on the column, whereas polar interferences, eluting at the first of the chromatogram, were discarded to waste. Then, the analytes were transferred by the chromatographic mobile phase to a second C18 analytical column, where they were separated following a conventional chromatography. The optimized approach allowed to preconcentrate 15 mL of sample volume adjusted at acid pH with phosphoric acid and modified with 5% of methanol, at a flow rate of 1.5 mL/min in 10 min. R(2) values were between 0.994 and 0.998, detection and quantitation limits ranged between 0.001 and 0.080 and between 0.002 and 0.100 μg/L, respectively, and the interday precision was below 9.8%. Recoveries in three different surface water samples, spiked at concentration levels between 0.002 and 0.500 μg/L (n = 3 for each spiking level), ranged from 82.1 to 125.8% with the relative standard deviation lower than 12%.     

Simple Multiresidue Determination of Fluoroquinolones in Bovine Milk by Liquid Chromatography with Fluorescence Detection

Abstract

A simple and sensitive method for the simultaneous determination of five fluoroquinolones (ciprofloxacin, enrofloxacin, danofloxacin, difloxacin, and sarafloxacin) in bovine milk was developed. Protein precipitation from milk samples was achieved by the addition of acetonitrile and o‐phosphoric acid. Acetonitrile was removed with dichloromethane, leaving the fluoroquinolones in the acid aqueous extract. The aqueous extract was analyzed by liquid chromatography with fluorescence detection (LC–FD). The mobile phase was composed of acetonitrile and 10 mM citrate buffer solution of pH 4.5, with an initial composition of acetonitrile‐water (12∶88, v/v) and using linear gradient elution. Norfloxacin was used as an internal standard. The limits of detection found ranged from 1 to 6 ng · mL^?1 and were below the maximum residue limits (MRLs) established by the European Union. The proposed method was applied to the determination of these compounds in different bovine milk samples. Method validation was carried out by a recovery assay.