Effective Separation and Simultaneous Determination of Four Fluoroquinolones in Milk by CE with SPE

A simple and sensitive capillary electrophoresis method with solid phase extraction was developed for the determination of sarafloxacin, ciprofloxacin, enrofloxacin and flumequine in milk. Solid-phase extraction with Oasis HLB cartridge column was used for the isolation of four fluoroquinolones in raw milk from a farm and fresh milk sample. Separation conditions of CE, including running buffer, voltage and temperature, were investigated and optimized. Baseline separation was achieved for the four fluoroquinolones under the developed conditions. Correlation coefficients greater than 0.9998 were obtained for all fluoroquinolones with a dynamic range from 1 up to 100 mg L^?1. The intra-day precision was less than 5%, and the inter-day precision was less than 6%. The method recoveries of four fluoroquinolones were in the range of 70.9–90.6%. The detection limits for sarafloxacin, ciprofloxacin, enrofloxacin and flumequine was 19.8, 15.2, 13.3 and 15.9 μg kg^?1, respectively, which allows positive detection of the fluoroquinolones at the targeted maximum residue levels in milk samples.     

Determination of levofloxacin,ciprofloxacin, moxifloxacin and gemifloxacin in urine and plasma by HPLC–FLD–DAD using pentafluorophenyl core–shell column: Application to drug monitoring

Concentrations of fluoroquinolones, which are used in the treatment of many bacterial infections, should be monitored in biological fluids as they exhibit concentration-dependent bactericidal activity. In this study, a liquid chromatography method for the determination of levofloxacin, ciprofloxacin, moxifloxacin and gemifloxacin in human urine and plasma was developed for the first time. The efficiency of five different columns for the separation of these fluoroquinolones was compared. Experimental parameters that affect the separation, such as percentage of organic solvent, pH, temperature, gradient shape and detector wavelength, were optimized by a step-by-step approach. Using a pentafluorophenyl core–shell column (100 × 4.6 mm, 2.7 μm), the separation of four analytes was accomplished in <7.5 min. The developed method was validated for the determination of analytes in both urine and plasma with respect to sensitivity, specificity, linearity (r ≥ 0.9989), recovery (79.46–102.69%), accuracy, precision and stability (85.79–111.07%). The intra- and inter-day accuracies were within 89.55–111.94% with relative standard deviations of 0.35–8.05%. The feasibility of method was demonstrated by analyzing urine and plasma samples of patients orally receiving levofloxacin, ciprofloxacin or moxifloxacin. The developed method is suitable for therapeutic drug monitoring of these fluoroquinolones and can be applied to pharmacokinetic and toxicological studies.     

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.     

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.     

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.     

Micellar nanotubes dispersed electrokinetic chromatography for the simultaneous determination of antibiotics in bovine milk

A method to determine four antibiotics for veterinary use (ciprofloxacin, enrofloxacin, florfenicol, and chloramphenicol) of different families (fluoroquinolones and amphenicols) in bovine milk was developed. The determination of the analytes was carried out using micellar electrokinetic capillary chromatography (MEKC) with a common sodium borate-SDS buffer solution containing single-walled carbon nanotubes (SWCNTs). In this way, a great improvement in the electrophoretic resolution and the separation efficiency was achieved compared to MEKC. An online reverse electrode polarity-stacking mode (REPSM) was carried out to enhance sensitivity. This step was performed in only 2 min and it allowed a stacked percentage of 103. That means that all the amount of injected analytes is effectively stacked. When this stacking procedure was combined with an off-line preconcentration step, based on SPE, analytes could be detected in lower concentration than the established maximum residue limits (MRLs). The LODs for the four compounds were between 6.8 and 13.8 μg L(-1) and the RSD values were between 1.1% and 6.6%. The whole method was applied to spiked real samples with acceptable precision and satisfactory recoveries.     

Determination of Fluoroquinolones in Animal Feed by Ion Pair High-performance Liquid Chromatography with Fluorescence Detection

A sensitive and simple method is described for the determination of fluoroquinolones by high-performance liquid chromatography (HPLC) using a C18 column and fluorescence detection. The mobile phase was acetonitrile and 0.025M phosphate acid with 0.0025M sodium 1-heptanesulfonate monohydrate in water with a gradient program. The chromatographic conditions were optimized for the determination of ciprofloxacin, enrofloxacin, sarafloxacin, and flumequine in animal feed. The samples were extracted by a hydrochloric acid and acetonitrile followed by dilution in 0.01M oxalic acid at pH 4.0 and purified by solid-phase extraction. The procedure was validated by fortifying feed at 200, 1000, and 2000?µg/kg. The limits of detection and quantification were from 28.5 to 74.7 and 31.7 to 94.6?µg/kg, respectively. The average recoveries for fluoroquinolones were from 89.7 to 100.3%. The method was validated and shown to be efficient and precise for quantification of fluoroquinolones in animal feed. The results demonstrate the feasibility of the method for routine use to monitor these substances in feed.     

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.     

Use of ionic liquid based chitosan as sorbent for preconcentration of fluoroquinolones in milk,egg, fish,bovine, and chicken meat samples by solid phase extraction prior to HPLC determination

The possibility of using ionic liquid based chitosan sorbent for the separation and preconcentration of fluoroquinolone antibiotics (marbofloxacin, enoxacin, ofloxacin, ciprofloxacin, and enrofloxacin) has been studied. For this reason, different ionic liquids were prepared and coated on the chitosan sorbent. The conditions of the preconcentration of fluoroquinolones on a microcolumn have been optimized and the extraction efficiencies of the prepared sorbents have been compared. The compounds were eluted with 5 mL of 20% NH₃ (v/v, MeOH) solution and determined by HPLC with diode array and fluorescence detector. The limits of detection were found as 4.23 µ g L^?1 for marbofloxacin, and 1.09 µg L^?1 for enoxacin; 3.23 × 10^?3 µg L^?1 for ofloxacin; 8.39 × 10^?3 µg L^?1 for ciprofloxacin; and 19.50 × 10^?3 µg L^?1 for enrofloxacin. The developed method was applied for the analysis of fluoroquinolone in milk, egg, fish, bovine, and chicken samples and the recoveries were obtained in the range 70–100%.     

Laser induced fluorescence coupled to capillary electrophoresis for the determination of fluoroquinolones in foods of animal origin using molecularly imprinted polymers

A method for the simultaneous determination of four fluoroquinolones of veterinary use (ciprofloxacin, danofloxacin, enrofloxacin and sarafloxacin) in two complex matrixes, such as bovine raw milk and pig kidney, has been established and validated. The method is based on the use of capillary electrophoresis (CE) coupled with a very sensitive detection mode, such as laser induced fluorescence (LIF) detection, due to the fact the all the compounds selected show native fluorescence. In order to achieve high selectivity in the sample treatment procedure, a commercially available molecularly imprinted polymer has been used for the solid phase extraction of the analytes. Once the retention and elution processes were optimized, the final extract was analyzed by CE-LIF using a 325 nm He–Cd laser. Optimum separation was obtained in a 70 cm × 75 μm capillary using a 125 mM phosphoric acid solution at pH 2.8 with 36% methanol as background electrolyte. The method provided very low detection limits, ranging from 0.17 to 0.98 μg/kg for milk and 1.10 to 10.5 μg/kg for kidney, with acceptable precision and satisfactory recoveries.     

Molecularly Imprinted Monolithic Column for Selective On-Line Extraction of Enrofloxacin and Ciprofloxacin from Urine

A simple on-line molecularly imprinted solid-phase extraction coupled with liquid chromatographic separation was developed for the simultaneous determination of enrofloxacin and its active metabolite ciprofloxacin from urine samples. The molecularly imprinted monolithic columns were synthesized in water-containing systems using norflorxacin as dummy template and methanol–water (5:1, v/v) as porogenic solvent, which reveal high affinity to enrofloxacin and ciprofloxacin in the aqueous environment. Due to the unique porous structure and flow-through channels existing in the network skeleton of the imprinted monolith, urine samples could be injected directly into the imprinted column, proteins and other biological matrix were quickly washed out and the analytes were selectively retained and enriched. Good linearity was obtained from 0.05 to 200 mg L^?1 with relative standard deviations less than 3.1%. The recoveries were higher than 87% at three different concentrations and the limit of detection of the method was 0.01 mg L^?1. Moreover, by increasing the injection volume, the sensitivity of the method could be further improved.     

Molecularly imprinted polymers for the solid‐phase extraction of four fluoroquilones from milk and lake water samples

A method based on molecular crowding and ionic liquids as reaction solvents has been used for the synthesis of molecularly imprinted polymers. Levofloxacin was selected as the template, polymethyl methacrylate was the molecular crowding agent, and 1‐butyl‐3‐methylimidazolium tetrafluoroborate (ionic liquid) was selected as the reaction solvent and porogen. The optimized proportion for the mixed porogen was dimethyl sulfoxide/ionic liquid/polymethyl methacrylate 1:1.6:5 in chloroform (150 mg mL^?1). The morphology and chemical composition of levofloxacin imprinted polymers were assessed by scanning electron microscopy and Fourier transform infrared spectroscopy. The absorption experiments demonstrated that the levofloxacin imprinted polymers possess high selective recognition property to levofloxacin and analogs, including enrofloxacin, ciprofloxacin and gatifloxacin, which all belong to fluoroquinolones. An extraction method using levofloxacin imprinted polymers as sorbent followed by high‐performance liquid chromatography analysis was optimized for the determination of four fluoroquinolones in milk and lake water samples. Under the optimized conditions, good linearity was observed in a range of 5–1000 ng g^?1 with the limit of detection between 0.3 and 0.5 ng g^?1 for the four fluoroquinolones. The recoveries at three spiked levels ranged 82.4–98.3% with the relative standard deviation ≤4.9.     

Fluoroquinolone antibiotic determination in bovine,ovine and caprine milk using solid-phase extraction and high-performance liquid chromatography-fluorescence detection with ionic liquids as mobile phase additives

This paper describes the use of 1-ethyl-3-methylimidazolium tetrafluoroborate (EMIm-BF₄) as mobile phase additive for the analysis by high-performance liquid chromatography with fluorescence detection of a group of seven basic fluoroquinolone antibiotics (i.e. fleroxacin, ciprofloxacin, lomefloxacin, danofloxacin, enrofloxacin, sarafloxacin and difloxacin) in different milk samples. EMIm-BF₄ was found superior to 1-butyl-3-methylimidazolium tetrafluoroborate for the separation of the analytes from chromatographic interferences of the sample matrix. The optimized method was applied to the analysis of ovine, caprine and bovine milk, in the last case in either skimmed, semi-skimmed and full-cream milk after suitable acidic deproteination followed by a solid-phase extraction procedure. Recovery values between 73% and 113% were obtained for the three types of bovine milk samples, as well as for ovine and caprine milk (RSDs below 16% in all cases), which clearly demonstrates the applicability of the method to the three types of milk irrespective of the fat content of the samples. Limits of detection were in the range of 0.5–8.1 μg/L (approximately 0.5–25.9 μg/kg), well below the maximum residue limits established for these compounds by the current European legislation. A screening study of 24 different milk samples was also developed. In none of the samples, residues of the selected antibiotics were found.     

Combination of microwave-assisted micellar extraction with liquid chromatography tandem mass spectrometry for the determination of fluoroquinolone antibiotics in coastal marine sediments and sewage sludges samples

In this work, a method for the analysis of fluoroquinolone antibiotics using microwave-assisted micellar extraction combined with liquid chromatography-mass spectrometry detection has been developed. Different surfactants were tested for use as extractants in the isolation of the analytes from solid samples, and several experimental designs were evaluated for the determination and optimization of the variables that affect recovery from the matrix. Under optimal conditions, we obtained recoveries greater than 73% with relative standard deviations below 8%. Compounds were detected by liquid chromatography-electrospray ionization tandem mass spectrometry with detection limits between 0.15 and 0.55 ng g(-1) and quantification limits between 0.49 and 1.85 ng g(-1) . Finally, the optimized method was applied in the determination of antibiotics in real solid samples. Four fluoroquinolones (levofloxacin, norfloxacin, ciprofloxacin and enrofloxacin) were found in coastal marine sediments taken close to a marine outfall and in sewage sludge samples from a wastewater treatment plant. Concentrations ranged between 0.81 and 34.3 ng g(-1) in the sediments and 3.43 and 206.1 ng g(-1) in the sludge.     

Solid-phase microextraction with micellar desorption and HPLC-fluorescence detection for the analysis of fluoroquinolones residues in water samples

A sensitive and useful method based on solid-phase microextraction with micellar desorption (SPME-MD) coupled to HPLC with fluorescence detection was developed for the determination of five fluoroquinolones (levofloxacin, norfloxacin, ciprofloxacin, enrofloxacin, and sarafloxacin) in environmental water matrices. The SPME extraction efficiency was optimized with regard to time, temperature, pH, and ionic strength using a CW-TPR fiber. A detailed study about the optimum conditions for micellar desorption (surfactant type, concentration, and desorption time) were made. Among different surfactants studied, Polyoxyethylene 10 lauryl ether showed the best responses to extract fluoroquinolones using SPME-MD. Relative standard deviations of the developed method were below 9%. Limits of detection and quantification were between 0.01–0.2 and 0.03–0.6 ng mL⁻¹, respectively. The recoveries achieved for all five compounds were in the 81–116% range. The proposed method was compared using conventional desorbing agent as methanol. Finally, the SPME-MD methodology was applied to the determination of these target analytes in several environmental liquid samples, including seawater, groundwater, and wastewater samples with excellent results.     

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.     

Multiresidue analysis of quinolones and fluoroquinolones in soil by ultrasonic-assisted extraction in small columns and HPLC-UV

In this work, a new and simple analytical methodology for the simultaneous analysis of several quinolones (cinoxacin, oxolinic acid, nalidixic acid and flumequine) and fluoroquinolones (norfloxacin, enrofloxacin, enoxacin, ciprofloxacin and danofloxacin) in soil samples is presented. The method is based on the extraction of these analytes by an ultrasonic-assisted extraction in small columns and their subsequent quantification by HPLC using UV detection. The observed strong sorption of quinolones and fluoroquinlones to soil together their different acid-base properties made necessary an exhaustive optimisation of the extraction step. The optimum extraction procedure, based on the formation of antibiotic-Mg(II) complexes, allowed to desorb and quantitatively extract both groups of antibiotics in a single step, which was not possible by using conventional organic solvents. The proposed method was validated and the limits of detection achieved were in the low μg g⁻¹ concentration range proving its suitability for the determination of quinolones and fluoroquinolones in soil samples at realistic environmental concentration level.     

Multiresidue determination of quinolones regulated by the European Union in bovine and porcine plasma. Application of chromatographic and capillary electrophoretic methodologies

This paper presents the multiresidue determination of the series of quinolones regulated by the European Union (marbofloxacin, ciprofloxacin, danofloxacin, enrofloxacin, sarafloxacin, difloxacin, oxolinic acid and flumequine) in bovine and porcine plasma using capillary electrophoresis and liquid chromatography with ultraviolet detection (CE‐UV, LC‐UV), liquid chromatography–mass spectrometry and –tandem mass spectrometry (LC‐MS, LC‐MS/MS) methods. These procedures involve a sample preparation by solid‐phase extraction for clean‐up and preconcentration of the analytes before their injection into the separation system. All methods give satisfactory results in terms of linearity, precision, accuracy and limits of quantification. The suitability of the methods to determine quinolones was evaluated by determining the concentration of enrofloxacin and ciprofloxacin in real samples from pig plasma and cow plasma. Copyright © 2010 John Wiley & Sons, Ltd.     

Freeze–thaw approach: A practical sample preparation strategy for residue analysis of multi‐class veterinary drugs in chicken muscle

Seven drugs from different classes, namely, fluoroquinolones (enrofloxacin, ciprofloxacin, sarafloxacin), sulfonamides (sulfadimidine, sulfamonomethoxine), and macrolides (tilmicosin, tylosin), were used as test compounds in chickens by oral administration, a simple extraction step after cryogenic freezing might allow the effective extraction of multi‐class veterinary drug residues from minced chicken muscles by mix vortexing. On basis of the optimized freeze–thaw approach, a convenient, selective, and reproducible liquid chromatography with tandem mass spectrometry method was developed. At three spiking levels in blank chicken and medicated chicken muscles, average recoveries of the analytes were in the range of 71–106 and 63–119%, respectively. All the relative standard deviations were <20%. The limits of quantification of analytes were 0.2–5.0 ng/g. Regardless of the chicken levels, there were no significant differences (P > 0.05) in the average contents of almost any of the analytes in medicated chickens between this method and specific methods in the literature for the determination of specific analytes. Finally, the developed method was successfully extended to the monitoring of residues of 55 common veterinary drugs in food animal muscles.     

Determination of enrofloxacin and ciprofloxacin in milk using molecularly imprinted solid-phase extraction

A molecularly imprinted solid-phase extraction procedure was developed for the simultaneous identification of enrofloxacin and its active metabolite, ciprofloxacin, in milk samples. Water-compatible molecularly imprinted polymers synthesized in a water-methanol system show a high degree of cross-reactivity for enrofloxacin and ciprofloxacin in aqueous environments. The imprinted particles were applied as selective sorbents in a solid-phase extraction process focusing upon complex milk matrices, which allowed the matrix compounds present in milk samples to be removed effectively. The extracts were sufficiently clean for further chromatographic analysis, and no interference originating from the biological matrix was observed. The mean recoveries of enrofloxacin and ciprofloxacin from milk sample were 82.6-93.5% and 81.2-94.8%, respectively, with the RSD less than 7.5%. This method is simple and sensitive, and is therefore an alternative tool to the existing HPLC methods for analyzing residual enrofloxacin and ciprofloxacin in biological samples.