Effective cleanup for the determination of six quinolone residues in shrimp before HPLC with diode array detection in compliance with the European Union Decision 2002/657/EC

A high‐performance liquid chromatographic method was developed for the determination of six quinolone residues (ciprofloxacin, enrofloxacin, sarafloxacin, oxolinic acid, nalidixic acid, and flumequine) in shrimp tissue samples. Separation was carried out by a LiChrospher® 100 RP‐8e column, running at a 22 min gradient elution program, and the mobile phase consisted of citric acid (0.4 mol/L), acetonitrile and methanol. Detection was achieved by a diode array detector, monitoring at 255 and 275 nm. Sample preparation included initial extraction with citric acid solution and further clean‐up by solid‐phase extraction, employing Lichrolut RP‐18 cartridges. Validation was performed according to the European Union Decision 2002/657/EC. The detection capability was 127.2 μg/kg for ciprofloxacin, 115.2 μg/kg for enrofloxacin, 126.2 μg/kg for sarafloxacin, 113.1 μg/kg for oxolinic acid, 125.2 μg/kg for nalidixic acid, and 239.0 μg/kg for flumequine. Recoveries ranged between 83.0 and 121.6%. The Youden test was applied to study the method ruggedness.     

Novel enrofloxacin imprinted polymer applied to the solid-phase extraction of fluorinated quinolones from urine and tissue samples

A new molecularly imprinted polymer, prepared following a non-covalent approach, was synthesised using enrofloxacin as a template molecule. The imprinting effect of the polymer was verified by chromatographic evaluation and, interestingly, this evaluation also revealed that the imprinted polymer showed a high degree of cross-reactivity for ciprofloxacin, the major metabolite of enrofloxacin. The molecularly imprinted polymer was then applied as a selective sorbent in a two-step solid-phase extraction method focussing upon complex biological matrices, specifically human urine and pig liver. This two-step solid-phase extraction protocol, in which a commercial Oasis HLB cartridge and a molecularly imprinted solid-phase extraction cartridge were combined, allowed enrofloxacin and ciprofloxacin to be determined by liquid chromatography coupled to a UV detector at levels below the maximum residue limits established by the European Union. The quantification and detection limits in tissue samples of enrofloxacin and ciprofloxacin were established at 50 μg kg⁻¹ and 30 μg kg⁻¹, respectively.     

Simultaneous determination of trimethoprim,enrofloxacin, and ciprofloxacin in blood serum of poultry

A method is proposed for the simultaneous determination of trimethoprim, enrofloxacin, and ciprofloxacin in blood serum of poultry using HPLC. Samples were prepared using protein precipitation. The chromatographic separation of substances was attained on a C18 column using a mixture of a 50 mM acetate buffer solution (pH 3.0) with acetonitrile (86: 14) as a mobile phase. Detection was performed at 278 nm. Linearity was observed in the concentration range 50–5000 ng/mL for trimethoprim and 10–5000 ng/mL for ciprofloxacin and enrofloxacin. Precision was ≤2.1% for enrofloxacin, ≤1.6% for ciprofloxacin, and ≤14% for trimethoprim. Accuracy was ≤10.2% for enrofloxacin, ≤9.9% for ciprofloxacin, and ≤11.9% for trimethoprim. The method was applied to pharmacokinetic studies of complex antibacterial drugs containing trimethoprim and enrofloxacin as active substances.     

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.     

反相高效液相色谱法同时测定4种氟喹诺酮类药物在鸡可食性组织中的残留

建立了一种可同时测定鸡可食性组织中环丙沙星、达氟沙星、恩诺沙星及沙拉沙星等多种残留的反相高效液相色谱 -荧光分析法。鸡的肌肉、皮和脂、肝、肾等4种组织经不同pH值的磷酸二氢钾缓冲溶液匀浆提取,上清液通过C18固相萃 取柱净化,以流动相洗脱。洗脱液经液相色谱分离后,用荧光检测器进行检测(激发波长280 nm, 发射波长450 nm),外标 法定量。对鸡的4种组织进行添加回收率测定,结果显示方法在添加水平为20~300 μg/kg时药物的回收率约为53.9%～93.4%,批间回收率测定值的相对标准偏差低于23%;环丙沙星、恩诺沙星、沙拉沙星 的定量检出限为20 μg/kg,达氟沙星为4μg/kg。方法简单、快速,能满足常规兽药残留检测的需要。     

Quantitation of nine quinolones in chicken tissues by high-performance liquid chromatography with fluorescence detection

A simple reversed-phase high-performance liquid chromatographic method was developed and validated for simultaneous analysis of nine quinolones (ciprofloxacin, danofloxacin, difloxacin, enrofloxacin, flumequine, marbofloxacin, nalidixic acid, oxolinic acid, sarafloxacin) in chicken tissue. The analytes were extracted from homogenized muscle using an acetonitrile basic solution. After centrifugation and partial evaporation, direct injection was possible. Three different HPLC conditions were applied to quantify the residual quinolones. Separation was achieved on a PLRP-S column and detection was performed with a monochromator fluorescence detector. The recovery, the limit of detection, the limit of quantification, the accuracy and the precision of the method were evaluated from spiked tissue samples at concentration levels ranging from 15 microg kg(-1) to 300 microg kg(-1) according to the maximum residue limit of each quinolone. This method is also suitable for porcine, bovine, ovine and fish muscle tissue.     

Analysis of cephalexin from canine skin biopsy by liquid chromatography with ultraviolet-visible photodiode-array detection

A sensitive and selective ion-paired liquid chromatographic method with UV-VIS photodiode-array detection was developed to measure cephalexin in skin biopsy samples. The method involved a sonication of minced canine skin with ethanol-acetonitrile-water (30:20:50, v/v/v) and ultrafiltration of received extract through 10,000 daltons. Separation of cephalexin from other components was by liquid chromatography using a reversed-phase column which was eluted with an ion-paired acetonitrile-water solution. Detection was achieved with a UV-VIS photodiode-array detector scanning from 230 to 320 nm. Cephalexin in the eluate was quantitated at its wavelength maximum of 260 nm. The evaluation of chromatographic peak homogeneity was performed by absorbance ratios, contour maps, first-derivative spectra and a three-dimensional spectrochromatogram. Additionally, the cephalexin peak identity was confirmed by liquid chromatography-mass spectrometry.     

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.     

Determination of residues of enrofloxacin and its metabolite ciprofloxacin in chicken muscle by capillary electrophoresis using laser-induced fluorescence detection

A method for the residue analysis of the veterinary antimicrobial agent enrofloxacin and its active desethyl metabolite ciprofloxacin in chicken muscle tissue has been developed and validated. The detection of the analytes was performed by laser-induced fluorescence (LIF) detection using a HeCd laser (lambda(ex) = 325 nm) providing an enhancement in sensitivity and selectivity compared to conventional UV detection. The assay has been validated with satisfying results. The limits of quantification for enrofloxacin and ciprofloxacin were 5 microg/kg and 20 microg/kg, respectively, with a fivefold preconcentration yielded by a sample clean-up with a simple liquid-liquid extraction procedure. Calibration graphs were linear from 5 to 1000 microg/kg for enrofloxacin and from 20 to 1000 microg/kg for ciprofloxacin. The assay allows the detection of contaminated muscle samples at the required maximum residue limit of the European Union, which is 100 microg/kg for the sum of enrofloxacin and ciprofloxacin.     

铽增敏高效液相色谱柱后衍生荧光检测法同时检测鸡肉中的三种氟喹诺酮类药物残留

基于氟喹诺酮类药物与铽离子形成配合物后的荧光增强作用,建立了同时检测鸡肉中氟喹诺酮类(FQs)药物环丙沙星、诺氟沙星和恩诺沙星残留的Tb3+增敏高效液相色谱（HPLC）柱后衍生荧光检测方法。优化的实验条件如下：流动相为0.05 mol/L 醋酸/醋酸钠缓冲液(pH 6.0)-乙腈(体积比为89∶11),色谱柱为Hypersil BDS-C18,柱温40 ℃,流速0.8 mL/min;Tb3+浓度为8×10-5 mol/L;衍生反应温度40 ℃,衍生泵流速0.5 mL/min;荧光检测激发波长271 nm,发射波长545 nm。实验结果表明,将上述3种药物以1.0,10.0,50.0,100.0 ng/g水平添加到鸡肉后的回收率范围为66.3%~88.0%,相对标准偏差（RSD）均小于15.0%。定量分析的线性范围为0.1~500 ng/mL,方法的日内和日间RSD均小于13.0%;最低检出限分别为0.05(环丙沙星)、0.05(诺氟沙星)和0.08(恩诺沙星)ng/g,比前人报道的非衍生高效液相色谱荧光检测法检测FQs药物的灵敏度有极大的提高。该项研究为FQs药物多残留检测提供了灵敏度更高的新方法。     

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.     

Screening of quinolone residues in pig muscle by planar chromatography

Summary A high-performance thin-layer chromatographic method based on solid-phase extraction has been developed for the qualitative determination of seven quinolones (enrofloxacin, ciprofloxacin, danofloxacin, norfloxacin, flumequine, oxolinic acid and nalidixic acid) in pork muscle. After preparation of the samples by extraction and clean-up by solid-phase extraction on reversed-phase cartridges, extracts were spotted and eluted on silica gel plates. The plate is first inspected under UV illumination at 312 nm, then sprayed with terbium chloride solution and again monitored under 312 nm UV. The method has been validated to a level of 15 μg kg⁻¹ for enrofloxacin, ciprofloxacin, danofloxacin, norfloxacin and 5 μg kg⁻¹ for flumequin, oxolinic acid and nalidixic acid.     

Determination of ceftazidime in dolphin serum by liquid chromatography with ultraviolet-visible detection and confirmation by thermospray liquid chromatography-mass spectrometry.

A simple and sensitive liquid chromatographic method has been developed for the determination of therapeutic levels of ceftazidime in dolphin serum. The method involved an ultrafiltration of diluted serum with an equal amount of acetonitrile-ethanol-water (40:40:20, v/v/v) through a 10,000 daltons molecular mass cut-off filter. Separation of ceftazidime from the other serum components was performed by ion-paired (dodecanesulfonate) liquid chromatography using a reversed-phase column eluted with acetonitrile-water solution. The ultraviolet absorbance of the column effluent was monitored in the 200-340 nm range of a photodiode-array detector or at 258.8 nm on a variable-wavelength ultraviolet-visible detector. Recoveries of ceftazidime from dolphin serum spiked with 20 and 2 micrograms/ml were 92.9 and 91.1% with coefficients of variation of 5.5 and 5.7%, respectively. A correlation coefficient of 0.9994 occurred with ceftazidime in aqueous solutions (n = 6, in duplicates). The limit of detection for this antibiotic was estimated to be approximately 50 ppb (ng/ml). The unbound ceftazidime concentrations in dosed dolphin serum were determined to calculate the protein bindings of this antibiotic which yielded 32 +/- 2%. The ceftazidime peak identity in dosed dolphin serum was confirmed by thermospray liquid chromatography-mass spectrometry. The thermospray mass spectrum of ceftazidime exhibited only the fragment ions, involving the opening of the beta-lactam ring, at m/z 237, 255 and 315 when positive-ion detection mode was employed and the fragment ions at m/z 235, 253 and 313 when negative-ion detection mode was used.     

Determination of fluoroquinolones in edible animal tissue samples by high performance liquid chromatography after solid phase extraction

In the present work, a rapid, accurate, and sensitive method has been developed for the quantitative determination of five fluoroquinolones (enoxacin, ofloxacin, norfloxacin, ciprofloxacin, and enrofloxacin) in edible animal tissues (muscle tissue, liver, kidney, and eggs). The separation was accomplished on an Inertsil (250 x 4 mm) C8, 5 microm, analytical column, at ambient temperature within 15 min. The mobile phase consisted of a mixture of citric acid (0.4 mol L(-1))-CH3OH-CH3CN (87:9:4% v/v). UV detection at 275 nm yielded the following limits of detection: 100 pg per 20 microL injected volume for enoxacin, norfloxacin, and ciprofloxacin, 20 pg for ofloxacin, and 200 pg for enrofloxacin. Peaks in real samples were identified by means of a photodiode array detector. The method was validated in terms of intra-day (n = 8) and inter-day (n = 8) precision and accuracy. Tissue samples were purified from endogenous interference by solid-phase extraction using Oasis HLB cartridges. The solid-phase extraction protocol was optimized in terms of retention and elution. Recovery rates at fortification levels of 40, 60, and 80 ng/g ranged from 82.5% to 111.1%. The applicability of the method was examined using real samples from a chicken treated orally with the five studied fluoroquinolones.     

Use of the diphasic dialysis as a new extraction procedure in the determination of enrofloxacin and ciprofloxacin in egg

We have developed a new method for the analysis of the fluoroquinolones enrofloxacin and ciprofloxacin in eggs using a diphasic dialysis procedure as extraction and purification method. High pressure liquid chromatography-mass spectrometry (HPLC-MS) was used for the confirmatory determination of these compounds. The method was found to be linear between 10 and 800 ng g⁻¹ for enrofloxacin, and between 20 and 1600 ng g⁻¹ for ciprofloxacin. The recovery percentages were in the 70-104% range for enrofloxacin, and 55-97% for ciprofloxacin. The assay described was repeatable and reproducible with a limit of quantitation of 2 and 4 ng g⁻¹ for enrofloxacin and ciprofloxacin from egg, respectively.     

胶束毛细管电泳在线推扫技术分离检测猪肉组织中痕量喹诺酮类药物

利用胶束毛细管电泳法结合在线推扫富集技术对组织中残留的痕量环丙沙星、氧氟沙星和恩诺沙星进行了检测, 弥补了毛细管电泳检测灵敏度低的缺点, 大大减化了操作过程, 为动物食品组织中残留的痕量药物检测提供了一种新的简便可靠的方法.     

Determination of quinolones and fluoroquinolones in fish tissue and seafood by high-performance liquid chromatography with electrospray ionisation tandem mass spectrometric detection

A reversed-phase high-performance liquid chromatographic method with tandem mass-spectrometric detection was developed and validated for the simultaneous analysis of eight quinolones and fluoroquinolones (oxolinic acid, flumequine, piromidic acid, enrofloxacin, ciprofloxacin, danofloxacin, sarafloxacin and orbifloxacin) in trout tissue, prawns and abalone. The analytes were extracted from homogenised tissue using acetonitrile and the extracts subjected to an automated two-stage solid-phase extraction process involving polymeric reversed-phase and anion-exchange cartridges. Good recoveries were obtained for all analytes and the limit of quantification was 5 microg/kg (10 microg/kg for ciprofloxacin). The limit of detection was 1-3 microg/kg, depending on the analyte and matrix. Confirmation of the identity of a residue was achieved by further tandem mass-spectrometric analysis. A procedure for estimating the uncertainty associated with the measurement is presented.     

Development and validation of an HPLC confirmatory method for residue analysis of ten quinolones in tissues of various food-producing animals, according to the European Union Decision 2002/657/EC

The aim of this work was to develop an HPLC method for the simultaneous determination of ten quinolones: enoxacin, ofloxacin, norfloxacin, ciprofloxacin, danofloxacin, enrofloxacin, sarafloxacin, oxolinic acid, nalidixic acid, and flumequine, in various tissues of food-producing animals. Separation was achieved on a PerfectSil Target column (250 mm x 4 mm, ODS-3, 5 microm), by MZ-Analysentechnik (Germany), at room temperature. The mobile phase consisted of 0.1% TFA-CH(3)OH-CH(3)CN and was delivered by a gradient program of 35 min. The detection and quantitation was performed on a photodiode array detector at 275 and 255 nm. Caffeine (7.5 ng/microL) was used as the internal standard (IS). Analytes were isolated from tissue samples by 0.1% methanolic TFA solution. SPE, using LiChrolut RP-18 cartridges, was applied for further purification. The extraction protocol was optimized and the final recoveries varied between 92.0 and 107.4%. The method was fully validated according to Commission Decision 2002/657/EC. Limits of quantitation for the examined quinolones extracted from each tissue were much lower than the respective Maximum Residue Levels, ranging between 30 and 50 microg/kg for bovine tissue, between 30 and 55 microg/kg for ovine tissue, and between 40 and 50 microg/kg for porcine tissue.     

Multiresidue determination of fluoroquinolones in shrimp by liquid chromatography-fluorescence-mass spectrometry

An efficient multiresidue method for analysis of fluoroquinolones in shrimp has been developed in which quantitation by fluorescence and confirmation by Multiple Stage Mass Spectrometry (MS) is achieved simultaneously. In this method, shrimp tissue is extracted with ammoniacal acetonitrile and the extract is defatted and then evaporated. After dissolution in basic phosphate buffer, fluoroquinolones in the extract are separated by liquid chromatography and quantitated, taking advantage of their intense fluorescence. Eluate from the fluorescence detector enters the MS, which allows for confirmation by monitoring ratios of 2 prominent product ions in the MS3 or MS2 spectrum. Using this method, 8 fluoroquinolones have been analyzed in shrimp samples fortified at 10, 25, 50, or 100 ppb levels. Recoveries for desethyleneciprofloxacin, norfloxacin, ciprofloxacin, danofloxacin, enrofloxacin, orbifloxacin, sarafloxacin, and difloxacin ranged from 75 to 92%, with relative standard deviation values of <6%. The limits of quantitation ranged from 0.1 to 1 ng/g. Enrofloxacin and ciprofloxacin were also successfully determined in enrofloxacin-incurred shrimp using this method.     

Determination of the antibacterial enrofloxacin by differential-pulse adsorptive stripping voltammetry

Two new methods for the determination of enrofloxacin in commercial formulations and canine urine samples, based on adsorptive stripping voltammetry (AdSV), are proposed. One of the proposed method uses univariate calibration to analyse enrofloxacin in commercial formulations and the other applies principal component regression (PCR) to the voltammetric measurements to determine enrofloxacin in the presence of its metabolite ciprofloxacin. The linear concentration ranges of application were 4-25 and 18-55 ng ml⁻¹ by using an accumulation potential of −0.3 V and a 180 or 60 s accumulation time, respectively for the univariate method. The first concentration range was used for the multivariate method. Both methods were successfully applied to the analysis of commercial formulations and spiked canine urine samples, respectively.