Selective sample preparation for the analysis of (fluoro)quinolones in baby food: molecularly imprinted polymers versus anion-exchange resins

In this work, an analytical method for simultaneous analysis of several quinolones (cinoxacin, oxolinic acid, nalidixic acid, and flumequine) and fluoroquinolones (norfloxacin, enrofloxacin, enoxacin, ciprofloxacin, and danofloxacin) in baby-food samples is described for the first time. The method is based on isolation of these analytes by ultrasound-assisted extraction procedure followed by a solid-phase extraction sample clean-up step and final determination of the analytes by HPLC using UV detection. For the extraction step, 2 g baby food was mixed with methanol in a centrifuge tube and one single extraction cycle of 15 min at room temperature was carried out. After centrifugation, supernatant was collected and two different solid-phase extraction procedures were developed and evaluated for sample clean-up. The first was based on use of strong anion-exchange cartridges whereas the second was based on use of a ciprofloxacin-imprinted polymer. Both sample clean-up procedures had their own advantages and drawbacks, and the analytical performance and applicability of each procedure was established and properly discussed. The anion-exchange resin-based method enabled simultaneous determination of quinolones and fluoroquinolones, reaching limits of detection ranging from 0.03 to 0.11 μg g⁻¹. In contrast, the use of a ciprofloxacin-imprinted polymer did provide selectivity towards fluoroquinolones, leading to chromatograms free from co-extractives reaching limits of detection one order of magnitude lower than those obtained by the first approach.     

Multiresidue determination of quinolone and fluoroquinolone antibiotics in fish and shrimp by liquid chromatography/tandem mass spectrometry

A multiresidue method was developed to measure low levels of 8 fluoroquinolones (norfloxacin, ofloxacin, danofloxacin, ciprofloxacin, desethylene ciprofloxacin, enrofloxacin, sarafloxacin, and difloxacin) and 4 quinolones (oxolinic acid, flumequine, nalidixic acid, and piromidic acid). Method detection limits range from 0.1 ng/g for quinolones to 0.4 ng/g for fluoroquinolones. Average recoveries range from 57 to 96%, depending on analyte and commodity; relative standard deviations are all less than 18%. The drugs are extracted from tissues using a mixture of ethanol and 1% acetic acid, diluted in aqueous HCI, and defatted by extraction with hexane. The compounds are further isolated using cation-exchange solid-phase extraction and measured using liquid chromatography with electrospray tandem mass spectrometry detection. The method has been evaluated and applied to the analysis of salmon, trout, and shrimp. Detectable residues were observed in 10 out of 73 samples, at concentrations ranging from 0.28 to 16 ng/g.     

Determination of eight fluoroquinolones in groundwater samples with ultrasound-assisted ionic liquid dispersive liquid–liquid microextraction prior to high-performance liquid chromatography and fluorescence detection

An ultrasound-assisted ionic liquid dispersive liquid–liquid microextraction (US-IL-DLLME) procedure was developed for the extraction of eight fluoroquinolones (marbofloxacin, norfloxacin, ciprofloxacin, lomefloxacin, danofloxacin, enrofloxacin, oxolinic acid and nalidixic acid) in groundwater, using high-performance liquid chromatography with fluorescence detection (HPLC-FD). The ultrasound-assisted process was applied to accelerate the formation of the fine cloudy solution using a small volume of disperser solvent (0.4 mL of methanol), which increased the extraction efficiency and reduced the equilibrium time.     

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.     

Simultaneous determination of eleven quinolones in animal feed by liquid chromatography with fluorescence and ultraviolet absorbance detection

A rapid and simple multi-residue procedure is described for assaying eleven quinolones (cinoxacin, ciprofloxacin, danofloxacin, difloxacin, enrofloxacin, flumequine, marbofloxacin, nalidixic acid, norfloxacin, oxolinic acid and sarafloxacin) in feeds at sub-additive levels (1–5 mg kg⁻¹). Five grams of sample were extracted by a metaphosphoric acid/acetonitrile mixture (70/30, v/v) and purified onto OASIS HLB cartridges. The determination was achieved by liquid chromatography (LC) using a GEMINI C18 analytical column both with fluorescence detection (FD) and photodiode-array (DAD). Limits of detection for each drug were in the range 0.04–0.8 mg kg⁻¹. Above the limit of quantification (LOQ), in poultry feed the recoveries were from 69 to 98% with relative standard deviations less than or equal 10%. Finally the measurement uncertainty was estimated using the bottom-up approach.     

Micropreparative fractionation of DNA fragments on metathesis-based monoliths: influence of stoichiometry on separation

New antibiotics were recently developed, among which are the (fluoro)quinolones. This paper presents an analytical method which allows the determination of 11 (fluoro)quinolones in swine kidneys: norfloxacin, ofloxacin, cinoxacin, oxolinic acid, nalidixic acid, flumequine, enrofloxacin, enoxacin, ciprofloxacin, danofloxacin and marbofloxacin. The procedure involves a rapid and efficient pre-treatment by solid-phase extraction (recoveries 83-98%), followed by the sensitive and selective determination of all compounds in a single run using LC-ESI-MS-MS. Multiple reaction monitoring (MRM) was used for selective detection of each (fluoro)quinolone. Quinine was selected as internal standard. The accuracy of the method, expressed as recovery, was between 89 and 109%; the repeatability had a maximum RSD lower than 15%. The limits of detection (LOD) were much lower than the respective Maximum Residue Limits (MRL)/4.     

Multiresidue determination of eleven quinolones in milk by liquid chromatography with fluorescence detection

A liquid chromatographic method with fluorescence detection was developed for simultaneous determination of norfloxacin, ofloxacin, ciprofloxacin, pefloxacin, lomefloxacin, danofloxacin, enrofloxacin, sarafloxacin, difloxacin, oxolinic acid, and flumequine in milk The samples were extracted with 10% trichloroacetic acid/acetonitrile (9 + 1, v/v) and cleaned by Strata-X reversed-phase solid-phase extraction cartridges. The 11 quinolones were separated on a reversed-phase C18 column (Hypersil BDS-C18) with mobile phase gradient elution and detected with fluorescence by means of a wavelength program. The recoveries for milk fortified with the 11 quinolones at 3 levels were 69-88% with acceptable relative standard deviations of <9% (intraday) and <14% (interday). The limits of detection were 23 microg/L for enrofloxacin, and 1-9 microg/L for the other 10 quinolones.     

高效液相色谱-串联质谱法测定蜂蜜中残留的19种喹诺酮类药物

建立了高效液相色谱-电喷雾串联质谱联用测定蜂蜜中恩诺沙星、环丙沙星、诺氟沙星、氧氟沙星、双氟沙星、恶喹酸、氟甲喹、沙拉沙星、司帕沙星、丹诺沙星、氟罗沙星、马波沙星、伊诺沙星、奥比沙星、吡哌酸、培氟沙星、洛美沙星、西诺沙星和萘啶酸等19种喹诺酮类药物残留的方法。比较酸性溶液阳离子固相萃取(PCX柱)、近中性缓冲溶液反相固相萃取（HLB柱）和碱性溶液阴离子固相萃取(PAX柱)3种不同提取净化方法的提取效果,最终选择使用碱性溶液溶解蜂蜜样品,强阴离子固相萃取柱一步富集净化。以甲醇和0.1%甲酸溶液作为流动相,C18作为分析色谱柱,采用梯度洗脱方式进行液相色谱分离,选择离子反应监测模式检测19种喹诺酮类药物,内标方法定量。在1～100 μg/L范围内,19种喹诺酮类药物的线性相关系数均大于0.991。通过实际样品的添加回收试验,方法的定量限（S/N=10）为1.0 μg/kg,3个添加水平的回收率为71%～118%,相对标准偏差为4.2%～6.7%。     

Room temperature ionic liquid-mediated molecularly imprinted polymer monolith for the selective recognition of quinolones in pork samples

A novel molecularly imprinted polymer monolith was prepared by the room temperature ionic liquid-mediated in situ molecular imprinting technique, using norfloxacin (NOR) as the template, methacrylic acid as the functional monomer, ethylene glycol dimethacrylate as the cross-linker. The optimal synthesis conditions and recognition properties of NOR-imprinted monolithic column were investigated. The results indicated that the imprinted monoliths exhibited good ability of selective recognition against the template and its structural analog. Using the fabricated material as solid-phase extraction sorbent, a sample pre-treatment procedure of molecularly imprinted solid-phase extraction coupling with HPLC was developed for determination of trace quinolone residues in animal tissues samples. The recoveries ranging from 78.16 to 93.50% for eight quinolones antibiotics such as marbofloxacin, NOR, ciprofloxacin, danofloxacin, difloxacin, oxolinic acid, flumequine and enrofloxacin were obtained.     

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.     

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.     

液相色谱-串联质谱法检测水产品中15种喹诺酮类药物残留量

建立了液相色谱-串联质谱技术同时检测水产品中15种喹诺酮类药物(氟罗沙星、氧氟沙星、依诺沙星、诺氟沙星、环丙沙星、恩诺沙星、洛关沙星、单诺沙星、奥比沙星、双氟沙星、沙拉沙星、司帕沙星、口恶喹酸、萘啶酸、氟甲喹)残留量的方法.试样中残留的喹诺酮类药物采用乙腈提取,提取液经正已烷液液分配脱脂后,以强阳离子固相萃取小柱净化,液相色谱.串联质谱法测定.对液/质分离条件与样品前处理条件进行了优化,并对喹诺酮类药物在分析过程的稳定性进行了研究.15种喹诺酮类药物在1.0～100 μg/L范围内线性关系良好,相关系数为0.9924～0.9992.在0.002～0.04 mg/kg浓度范围内,平均加标回收率在79.9%～93.8%;相对标准偏差为4.8%～14.6%.方法可满足水产品中喹诺酮类药物多残留检测与确证的需要.     

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.     

Determination of fluoroquinolone residues in poultry muscle in Portugal

A total of 98 poultry samples, including chicken and turkey muscle, were analysed, using a sensitive and reliable analytical method based on liquid chromatography (LC) with spectrofluorimetric detection, for simultaneous determination of four fluoroquinolone (FQ) antibiotics, namely enrofloxacin (ENRO), ciprofloxacin (CIPRO), norfloxacin (NOR), and sarafloxacin (SARA). The method involved extraction with 0.15 mol L⁻¹ HCl and clean-up by solid-phase extraction using Oasis HLB cartridges. Chromatographic separation was carried out on a C₁₈ TSK gel column, in isocratic mode, with 0.025 mol L⁻¹ H₃PO₄ solution, adjusted to pH 3.0 with tetrabutylammonium hydroxide-methanol (78:22) as mobile phase. Good linearity over the investigated concentration range was observed, with mean values of correlation coefficients higher than 0.9989 for all the analytes studied. The limits of quantification (LOQ), expressed as the lowest fortification level with acceptable precision were 15 μg kg⁻¹ for ENRO, CIPRO, and NOR, and 30 μg kg⁻¹ for SARA; these values are in compliance with requirements for monitoring of maximum residues levels (MRLs). Overall recoveries from spiked samples ranged from 80% to 92% with relative standard deviations (RSD) lower than 6.1%. Of the chicken and turkey samples analysed, 44.2% and 37.8%, respectively, were contaminated. The levels found in the analysed poultry samples, collected from markets of Oporto and Coimbra, located in the north and central zones of Portugal, respectively, were lower than 114.2 and 87.6 μg kg⁻¹ in chicken and turkey muscle samples, respectively. One positive chicken sample was contaminated with ENRO at levels higher than the MRL.     

High-throughput screening for multi-class veterinary drug residues in animal muscle using liquid chromatography/tandem mass spectrometry with on-line solid-phase extraction

A rapid qualitative method using on-line column-switching liquid chromatography/tandem mass spectrometry (LC/MS/MS) was developed and validated for screening 13 target veterinary drugs: four macrolides - erythromycin A, josamycin (leucomycin A3), kitasamycin (leucomycin A5), and tylosin A; six (fluoro)quinolones - ciprofloxacin, danofloxacin, enrofloxacin, flumequine, oxolinic acid, and sarafloxacin; and lincomycin, virginiamycin M1, and trimethoprim in different animal muscles. Clindamycin, norfloxacin, nalidixic acid, oleandomycin, ormetoprim, and roxithromycin were used as the internal standards. After simple deproteination and analyte extraction of muscle samples using acetonitrile, the supernatant was subjected to on-line cleanup and direct analysis by LC/MS/MS. On-line cleanup with an extraction cartridge packed with hydrophilic-hydrophobic polymer sorbent followed by fast LC using a short C18 column resulted in a total analysis cycle of 6 min for 19 drugs. This screening method considerably reduced the time and the cost for the quantitative and confirmatory analyses. The application of a control point approach was also introduced and explained.     

Comparison of adsorbents for on-line solid-phase extraction of polycyclic aromatic hydrocarbons before liquid chromatography with UV detection

Summary On-line solid-phase extraction (SPE) coupled with reversed-phase liquid chromatography and UV detection at 254 nm has been used for the determination of trace-level polycyclic aromatic hydrocarbons (PAH) in soil extracts. Five commercially available adsorbents (C₈, C₁₈, PLRP-S, PRP-1, and Bond-Elut Env) were evaluated. Results showed that recovery of the PAH decreased with increasing molecular weight, because of their poorer solubility. Recovery of high-molecular-weight PAH was significantly improved by addition of 10% (v/v) acetonitrile to the sample before loading of the SPE adsorbent. PAH recovery ranged from 64.0 to 108% when a 50 mL sample spiked with 1 μg L⁻¹ was applied to these adsorbents. Determination of PAH was possible with detection limits below 0.05 μg L⁻¹, which corresponds to 0.2 μg kg⁻¹ soil. The method was successfully used to determine PAH in soil extracts.     

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.     

Development of an HPLC multi-residue method for the determination of ten quinolones in bovine liver and porcine kidney according to the European Union Decision 2002/657/EC

A sensitive multi-residue analytical method was developed for the determination of ten quinolones: enoxacin, ofloxacin, norfloxacin, ciprofloxacin, danofloxacin, enrofloxacin, sarafloxacin, oxolinic acid, nalidixic acid, and flumequine in bovine liver and porcine kidney. A simple liquid extraction step followed by a solid phase extraction clean up procedure was applied for the extraction of quinolones from liver and kidney tissues. Recoveries of the extraction varied between 82 and 88% for bovine liver and 92 and 95% for porcine kidney. Separation was performed on an ODS-3 PerfectSil Target (250 x 4 mm) 5 microm analytical column at 25 degrees C. The mobile phase consisted of a mixture of TFA 0.1%-CH(3)CN-CH(3)OH, delivered at a flow rate of 1.2 mL/min according to a gradient program. Elution of quinolones and the internal standard (caffeine, 7.5 ng/microL) was complete within 27 min. Photodiode array detection was used for monitoring the eluants at 275 and 255 nm. The method was fully validated according to the European Union Decision 2002/657/EC, determining linearity, selectivity, decision limit, detection capability, accuracy, and precision. The LODs of the specific method of quinolone determination in bovine liver varied between 3 and 7 microg/kg and in porcine kidney between 3 and 4 microg/kg.     

Separation and simultaneous determination of quinolone antibiotics by capillary zone electrophoresis

Summary The potential of capillary zone electrophoresis has been investigated for the separation and quantitative determination of some quinolone antibiotics. The influence of different conditions, such as the nature and concentration of the electrophoretic electrolyte, on migration time, peak symmetry, efficiency and resolution was studied. A buffer consisting of 100mm HEPES adjusted to pH 8.5 containing 10% (v/v) acetonitrile was found to furnish a very efficient and stable electrophoretic system for the separation of exoxacin, ciprofloxacin, ofloxacin, oxolinic acid, nalidixic acid and pipemedic acid. A linear relationship between concentration and peak area for each compound was obtained in the concentration range 0.25–40 μg mL⁻¹; detection limits were approximately 0.25 ng mL⁻¹. It was demonstrated that the method can be used for the simultaneous determination of these six antibiotics in serum and urine samples.     

Extension of multi-residue methodology to include the determination of quinolones in food.

The multi-residue procedure for basic drugs described elsewhere by this laboratory has been evaluated for quinolone and fluoroquinolone antibiotics. The fluoroquinolones are a relatively new class of drug and an increasing number of licensed products containing these compounds are becoming available for use in animal husbandry. This, along with the possibility of the development of antibiotic resistant human pathogens, make it an important class of drug for which methodology is required for the monitoring of residues in food. Validation data are presented for a range of compounds including the quinolones; oxolinic acid and nalidixic acid, and the fluoroquinolones; flumequine, ciprofloxacin, danofloxacin, enoxacin, enrofloxacin, lomefloxacin, marbofloxacin, norfloxacin, ofloxacin and sarafloxacin. Foods for which the method was validated included poultry muscle (chicken and turkey), egg, chicken liver, honey, cattle muscle and pig muscle. This application of the multi-residue procedure further demonstrates the importance and wide-ranging usefulness of the technique.