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.     

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 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.     

Extraction studies of the group IB,IIB, and IIIA–VA elements using 1-(2-pyridylazo)-2-naphthol as chelating reagent

The extraction of PAN chelates of the group IB, IIB and IIIA–VA elements from aqueous solutions of pH 1–10 into chloroform has been studied radiochemically. Re-extraction studies have been made to strip the metal ions from the organic phase into aqueous solutions of KCN, HClO₄ and buffer solutions. The effects of certain masking agents on the extraction of these elements have also been studied. The extraction curves indicate the possibilities of devising group chemical separation procedures for use in activation analysis.     

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.     

Analysis of quinolones by voltage-assisted liquid-phase microextraction combined with LC-MS

The method of liquid-phase microextraction assisted with voltage was developed and applied on determination of quinolones in water sample in this study. Both of the reproducibility and extraction time were improved with the aid of applying voltage. Four analytes in neutral state such as cinoxacin, oxolinic acid, nalidixic acid, and flumequine were extracted from a sample solution at pH 2.0, through a polypropylene hollow fiber which was immobilized with 2-octanone, and then into a 25 μL of the acceptor phase of 40 mM borate buffer at pH 10.0 by applying voltage of 100 V. Subsequently, the acceptor solution was directly subjected to analysis by LC-MS. The performance of the method for four quinolones was also evaluated. Linearity was obtained in the range of 1.0-25.0 ng/mL with R(2) > 0.996. Limits of detection were below 0.6 ng/mL, and recoveries of water sample were ranged from 90.8 to 109.6%.     

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.     

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.     

Liquid–liquid extraction of scandium with nalidixic acid in dichloromethane

The extraction behavior of nalidixic acid (HNA) in CH₂Cl₂ has been studied for various di- and trivalent metal ions such as Cu(II), Fe(II), Ni(II), Mn(II), Sb(II), Co(II), Sc(III), Y(III), Nd(III) and Eu(III) from aqueous buffer solutions of pH 1–7 with 0.1 mol dm⁻³ nalidixic acid in dichloromethane. Separation factors of Sc(III) from these metals has shown that its clean separation is possible at pH 3.4–4. The parameters affecting the extraction of Sc(III) were optimized. The composition of the extracted adduct was determined by slope analysis method that came out to be Sc(NA)₃. Extraction of Sc(III) was studied in the presence of various cations and anions. Among the anions studied only fluoride, citrate and oxalate have significant interference whereas, Fe(III) has reduced the extraction to 53% that can be removed by using ascorbic acid as reducing agent. The proposed extraction system proved good stability up to six extraction-stripping stages for the extraction of Sc(III).     

PHOTOSENSITIZATION BY DRUGS: NALIDIXIC AND OXOLINIC ACIDS

Abstract— The antibacterial drugs, nalidixic acid and oxolinic acid, have been tested as photosensitizers in aqueous solution using 365 nm UV light. Absorption and fluorescence spectra indicate that intramolecular hydrogen bonding stabilizes the unionized form of these compounds in the pH region2–4. The ability of the unionized species to sensitize photooxidation by the type II (singlet oxygen) mechanism was found to be lower than when these drugs were ionized. Comparison withquinoline–3-carboxylic acid and the methyl esters of nalidixic and oxolinic acids emphasised the significance of the hydrogen bonding in relation to the excited state properties. Unionized nalidixic acid undergoes photolysis more readily than the ionized form, apparently by a free radical mechanism, while oxolinic acid is more stable.     

Molecular imprinting-based separation methods for selective analysis of fluoroquinolones in soils

Molecularly imprinted polymers (MIPs) for fluoroquinolone antibiotics (FQs) have been synthesised in one single preparative step by precipitation polymerisation using ciprofloxacin (CIP) as template. Combinations of methacrylic acid (MAA) or 4-vinylpyridine (VP) as functional monomers, ethylene glycol dimethacrylate as crosslinker and dichloromethane, methanol, acetonitrile or toluene as porogens were tested. The experiments carried out by molecularly imprinted solid-phase extraction (MISPE) in cartridges did not allow to detect any imprint effect in the VP-based polymers whereas it was clearly observed in the MAA-based polymers. Among them, the MIP prepared in methanol using MAA as monomer showed the best performance and was chosen for further experiments. The ability of the selected MIP for the selective recognition of other widely used FQs (enoxacin, norfloxacin, danofloxacin and enrofloxacin) and quinolones (Qs) (cinoxacin, flumequine, nalidixic acid and oxolinic acid) was evaluated. The obtained results revealed the high selectivity of the obtained polymer, which was able to distinguish between FQs, that were recognised and retained onto the MIP cartridge, and Qs, which were washed out during loading and washing steps. The MIP was then packed into a stainless steel column (50mmx4.6mm i.d.) and evaluated as chromatography column for screening of FQs in soil samples. The mobile phase composition, flow rate, and the elution profile were then optimised in order to improve peak shape without sacrifying imprinting factor. Finally, under optimised conditions, soil samples spiked with CIP or with a mixture of fluoroquinolones in concentration of 0.5microgg(-1) were successfully analysed by the developed MIP-based procedures.     

Gradient HPLC of antibiotics in urine, ground water, chicken muscle, hospital wastewater, and pharmaceutical samples using C-18 and RP-amide columns

A simple and highly sensitive high pressure liquid chromatographic (HPLC-UV) method has been developed for the determination of ofloxacin, lomefloxacin, cinoxacin, and nalidixic acid, in mobile phase citrate buffer (0.001 M) of pH 4.5 prepared in water (X), methanol (Y), and ACN (Z) using gradient at a flow rate of 1.0 mL/min by direct UV absorbance detection at lambda = 280 nm. Separation of analytes was studied on the C-18 and RP-amide columns and best results were observed on the RP-amide column with LODs (3.3 x S/m) 0.89, 0.55, 0.67, and 1.41 ng/mL for ofloxacin, lomefloxacin, cinoxacin, and nalidixic acid, respectively, and better RSD than the C-18 column. The recovery of Fluoroquinolones (FQs) in urine, ground water, hospital wastewater, and chicken muscle using this method is more than 90%. The method was successfully applied to the analysis of ofloxacin, lomefloxacin, cinoxacin, and nalidixic acid in urine, ground water, pharmaceutical dosage forms, hospital wastewater, and chicken muscle.     

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.     

Determination of nickel,gold and silver in gallium arsenide by radioactivation analysis based on the quantitative isotope dilution principle

A method of radioactivation analysis has been developed for the determination of Ni, Au and Ag impurities in gallium arsenide. The separation and substoichiometric extraction of these elements were studied and analytical procedures are suggested for their determination. All components are separated by suitable procedures and determined by substoichiometric methods. Ni is extracted as diethyldithiocarbamate into toluene, Au as a complex of rhodamine-B in chloroform, and Ag as dithizonate in carbon tetrachloride. The contents of Ni, Au and Ag in a gallium arsenide crystal with a carrier concentration of 1.8·10¹⁶/cm³ were 0.05–0.08, 0.006–0.008 and 0.002–0.005 ppm, respectively.     

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.     

Application of α-benzoinoxime for substoichiometric extractive separation of copper in activation analysis

A method has been developed for the substoichiometric extraction of copper with a solution of α-benzoinoxime in chloroform. The separation can be carried out in the absence or in the presence of tartaric acid at pH 6.8–7.0 and 9.5, respectively. In the presence of tartaric acid no interference has been observed. The method has been applied to the determination of copper in biological material, silicon, aluminium and kaolin by the neutron activation method.     

Complexation of antibacterial quinolonic acid and cinolonic derivatives with Zn(II) and Al(III): application to their determination in human urine

Nalidixic acid, 7-hydroxymethylnalidixic acid, oxolinic acid, pipemidic acid and cinoxacine form complexes with zinc(II) in the presence of acetate buffer of pH 5.5 and oxolinic acid, pipemidic acid and cinoxacine form complexes with aluminium(III) in the presence of chloroacetate buffer of pH 3.0. In all cases, an enhancement of the fluorescence emission was observed. Fluorimetric studies on the spectral characteristics of the complexes were performed. A 1:1 stoichiometry for all the complexes was established. The association constants were calculated, by using the changes in the fluorescence of all antibacterials, that occurred when the complexes were formed. The fluorescence reactions were used to develop methods for the determination of all of the above compounds, showing a higher sensitivity than in the absence of the cationic ions. The methods were satisfactorily applied to the determination of these compounds in urine.     

Development of a rapid extraction procedure for speciation of arsenic in chicken meat

A rapid extraction procedure has been developed for speciation of arsenic in chicken tissue. Water, methanol–water (1:1), and methanol–chloroform (1:1) were tested as extraction media. Individual use of an ultrasonic bath, a microwave oven, or an ultrasonic probe was not sufficient for quantitative recovery of As(III), dimethylarsinate, monomethylarsonate, As(V), and arsenobetaine in spiked samples of chicken tissue. A new extraction procedure using a methanol–water mixture and a microwave oven then an ultrasonic probe enabled extraction of the arsenic species in 7 min with efficiencies ranging from 80 to 100%. HPLC–UV–HG–AFS was used for the determinations. The extraction procedure was 100% efficient when applied to real samples of chicken tissue. AsB (48±5 μg As kg ⁻¹) and one containing-arsenic feed additive, Nitarsone (227±5 μg As kg ⁻¹) were detected.     

On the effect of covalently appended quinolones on termini of DNA duplexes

Quinolones are gyrase inhibitors that are widely used as antibiotics in the clinic. When covalently attached to oligonucleotides as 5'-acylamido substituents, quinolones were found to stabilize duplexes of oligonucleotides against thermal denaturation. For short duplexes, such as qu-T*GCGCA, where qu is a quinolone residue and T is a 5'-amino-5'-deoxythymidine residue, an increase in the UV melting point of up to 27.8 degrees C was measured. The stabilizing effect was demonstrated for all quinolones tested, namely nalidixic acid, oxolinic acid, pipemidic acid, cinoxacin, norfloxacin, and ofloxacin. The three-dimensional structure of (oa-T*GCGCA)2, where oa is an oxolinic acid residue, was solved by two-dimensional NMR spectroscopy and restrained molecular dynamics. In this complex, the oxolinic acid residues disrupt the terminal T1:A6 base pairs and stack on the G2:C5 base pairs. The displaced adenosine residues bind in the minor groove of the core duplex, while the thymidine residues pack against the oxolinic acid residues. The "molecular cap" thus formed fits tightly on the G:C base pairs, resulting in increased base-pairing fidelity, as demonstrated in UV melting experiments with the sequence oa-T*GGTTGAC and target strands containing a mismatched nucleobase. The structure of the "molecular cap" with its disrupted terminal base pair may also be helpful for modeling how quinolones block re-ligation of DNA strands in the active site of gyrases.     

Liquid—liquid extraction of lactate dehydrogenase from muscle using polymer-bound triazine dyes

An extract from porcine muscle containing soluble enzymes has been partitioned between the two liquid phases of an aqueous, biphasic system consisting of dextran, polyethylene glycol, and water. The influence of polymer-bound triazine dyes (Cibacron blue F3G-A and Procion yellow HE-3G) on the partition of lactate dehydrogenase and total protein was studied. The effects of pH and concentrations of polymers and buffer on this so-called affinity partitioning were also determined. The two-phase systems were used in extraction procedures for purification of lactate dehydrogenase to a specific activity of 456–494 U (7.6–8.4 μkat) per mg protein. The use of these systems for extraction of enzymes in technical scale is discussed.