Development of an ELISA screening test for nitroimidazoles in egg and chicken muscle

An antibody was generated that can bind metronidazole (MNZ), a nitroimidazole drug used in veterinary medicine to treat poultry for coccidiosis and histomoniasis. A direct competitive enzyme-linked immunosorbent assay (cELISA) is described. It was used to characterise binding of this antibody to a number of nitroimidazole drugs. It displayed cross-reactivity with dimetridazole (DMZ), ronidazole (RNZ), hydroxydimetridazole (DMZOH), and ipronidazole (IPZ).Egg and chicken muscle samples were extracted with acetonitrile and de-fatted by washing with hexane. Detection capabilities (CCβ) were determined: dimetridazole, <1 ppb (egg) and <2 ppb (muscle); metronidazole, <10 ppb; ronidazole and hydroxydimetridazole, <20 ppb; ipronidazole, <40 ppb.     

Simultaneous determination of six quinolones in pig muscle by liquid chromatography-atmospheric pressure chemical ionisation mass spectrometry.

An LC-MS method is described for the confirmation of six quinolones (enrofloxacine, ciprofloxacine, marbofloxacine, danofloxacine, sarafloxacine and difloxacine) in pig muscle. The quinolones were extracted from muscle (2 g) with phosphate buffer (pH 7.4). After centrifugation, the extract was purified on a C18 solid-phase extraction cartridge. Samples were analysed by LC with gradient elution on a C18 column and detected by MS via an atmospheric pressure chemical ionisation interface. For each compound, an intense pseudo-molecular ion [M+H]+ is obtained. The assay is specific and reproducible and allows the confirmation of the six quinolones at the 7.5 micrograms kg-1 level in pig muscle.     

Determination of florfenicol amine in channel catfish muscle by liquid chromatography

A method for quantifying florfenicol amine (FFA) in channel catfish muscle was validated according to U.S. Food and Drug Administration guidelines. FFA is the proposed marker residue for the veterinary antibiotic florfenicol in catfish muscle for regulatory surveillance purposes. The method includes acid hydrolysis followed by sample cleanup with ethyl acetate extraction, basification, solid-phase extraction, and quantitation by liquid chromatography with UV detection. The assay was validated at 5 concentrations in the range of 0.075-35 microg/g muscle. The overall mean recovery of FFA from fish tissues fortified at these concentrations ranged from 85.7 to 92.3%, 4.8-17.2% relative standard deviation (RSD). The assay limit of detection was 0.044 microg/g muscle based on analysis of control muscle. Catfish muscle samples containing incurred florfenicol residues were analyzed in quintuplicate with RSD < 5%. Acid hydrolysis has previously been demonstrated to convert florfenicol and its known metabolites to FFA and to release a significant amount of FFA from nonextractable florfenicol residues in tissues containing incurred residues in other species. By using acid hydrolysis, this method should yield a more accurate estimate of the total florfenicol-related residue level in muscle tissue from florfenicol-treated catfish than could be achieved by solvent extraction alone.     

Green methodology based on dispersive liquid–liquid microextraction and micellar electrokinetic chromatography for 5‐nitroimidazole analysis in water samples

Dispersive liquid–liquid microextraction has been proposed as an extraction technique combined with micellar electrokinetic chromatography (MEKC) for the analysis of eight 5‐nitroimidazole compounds, including some metabolites, in water samples. Determination has been carried out using a diode array detector, employing 20 mM sodium phosphate and 150 mM SDS as separation buffer. Separation has taken place under a voltage of 25 kV and a temperature of 20°C. Samples were prepared in a buffer without micelles and they were hydrodynamically injected at 50 mbar for 25 s, producing a sweeping effect on the analytes for increasing sensitivity. Different factors involved in the dispersive liquid–liquid microextraction procedure were optimized, such as sample pH, nature, and volume of extraction and dispersive solvents in the mixture, percentage of NaCl added to sample and shaking time after the injection of the extraction and dispersive solvents. The method was characterized for water samples, achieving detection limits lower than 2.4 μg/L. Trueness was checked in river, tap, and bottled water. Dispersive liquid–liquid microextraction combined with MEKC constitutes an easy, cheap, and green alternative for 5‐nitroimidazole analysis in environmental water samples.     

Determination of quinolones in animal tissues and eggs by high-performance liquid chromatography with photodiode-array detection

A rapid, specific reversed-phase HPLC method is described, with solid-phase extraction, for assaying five quinolones (ciprofloxacin, difloxacin, enrofloxacin, norfloxacin and marbofloxacin) with confirmative diode-array detection in samples of bovine kidney, muscle and eggs. The least efficient extraction was marbofloxacin from kidney tissue (64%). The lower detection limit for each quinolone was: enrofloxacin and ciprofloxacin, 1 ng; norfloxacin and difloxacin, 2 ng; marbofloxacin, 4 ng injected. The intra-day relative standard deviations were lower than 7.9% and lower than 8.6% for inter-day assays. These results indicate that the developed method had an acceptable precision.     

Determination of three nitroimidazole residues in poultry meat by gas chromatography with nitrogen-phosphorus detection.

A method was developed for the determination of the nitroimidazole compounds dimetridazole (DMZ), ronidazole (RNZ) and metronidazole (MNZ) by gas chromatography with nitrogen phosphorus detection. Nitroimidazole compounds were extracted with acetonitrile, followed by acidification using acetic acid and cleanup using strong cation-exchange (SCX) SPE column. Validation in chicken muscle fortified at a concentration of 5 microg/kg gave mean recoveries of 85% DMZ, 90% RNZ, 80% MNZ with RSDs of 13.0, 14.3, 11.2%, respectively (n=6). The method is suitable for statutory residue testing and is used as a quick screening method in the National Residue Surveillance Plan in China.     

The development of a multi-nitroimidazole residue analysis assay by optical biosensor via a proof of concept project to develop and assess a prototype test kit

An assay based on optical biosensor technology has been developed to detect a broad range of nitroimidazole drug residues and their metabolites (dimetridazole (DMZ), metronidazole (MNZ), ronidazole (RNZ), hydroxymetronidazole (HO-MNZ) and hydroxydimetridazole (HO-DMZ)) in chicken muscle. The detection limit for the procedure was determined as 0.5 ppb for DMZ and detection capabilities (CCβs) ranged from <1 ppb for DMZ, MNZ and RNZ to <2 ppb for HO-MNZ and HO-DMZ. Intra-assay variation (n = 6) was calculated as 11.6% at a concentration of 1 ppb DMZ and 4.7% at a concentration of 2 ppb DMZ. Inter-assay variation (n = 3) was determined to be 14.2% at a concentration of 1 ppb DMZ and 3.5% at a concentration of 2 ppb DMZ.A prototype kit based on this assay was produced and a multinational study was undertaken to independently evaluate its performance. The resulting data showed that the kit can be implemented with little difficulty in laboratories of varying expertise and is sensitive enough to meet the standards required by international law. Feedback from this study led to the incorporation of some minor improvements to the kit. The commercial partner in the project, XenoSense Ltd., was consulted with regards to producing a commercial test kit based on the prototype assay. As feedback from the collaborative study had been positive with respect to speed, ease of use and performance of the kit, the decision to commercialise the kit was taken. In conclusion, the prototype nitroimidazole kit was shown to offer numerous advantages over existing analytical techniques.     

A high performance liquid chromatographic method for the determination of albuterol enantiomers in human serum using solid phase extraction and chemical derivatization.

A high performance liquid chromatographic method was developed for the simultaneous assay of R(-)- and S(+)-albuterol in human serum. The assay involves solid phase extraction as a sample clean-up step and derivatization of racemic albuterol to its diastereomeric thioureas with 2,3,4,6-tetra-O-acetyl-alpha-D-glucopyranosyl isothiocyanate. Chromatographic separation was accomplished under isocratic conditions using an octadecylsilane column and a mobile phase consisting of 29:71 acetonitrile:distilled water containing 0.1% triethylamine, pH 4.0 (adjusted with concentrated phosphoric acid) at a flow rate of 0.8 mL/min. The diastereomers were detected using a fluorescence detector set at 223 nm excitation and no emission filter. Racemic bamethane was used as internal standard. Drug to internal standard peak-height ratios were linear over a 2-20 ng/mL range for each enantiomer. The limit of detection of each analyte was 1.0 ng/mL (S/N = 3).     

High-performance liquid chromatographic analysis of oxytetracycline in chinook salmon following administration of medicated feed.

A high-performance liquid chromatographic assay was developed to detect oxytetracycline (OTC) in chinook salmon muscle tissue. A solid-phase extraction protocol was used to recover OTC and the internal standard, epitetracycline hydrochloride, from the salmon tissue samples. OTC was analyzed using a mobile phase of methanol-0.02 M phosphate buffer, pH 2.25 (60:190), an ultraviolet detection wavelength of 365 nm and 250 mm x 4.6 mm I.D. Ultrasphere ODS column. A linear calibration curve (r2 = 0.999) of OTC in salmon muscle tissue from 0.05 to 3.0 ppm was obtained. Using a signal-to-noise ratio of 5:1, the OTC detection limit was 0.5 ppm in salmon muscle tissue. OTC recovery (74.4%) and intra-assay variability (2.3%) were optimized for salmon muscle tissue. An in vivo feeding study was performed by administrating OTC-medicated feed for a period of 10 days, followed by a 42-day sampling period. The half-life for the elimination of OTC in chinook salmon muscle tissue was found to be 5.4 days.     

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.     

Determination of nitroimidazole antibiotics based on dispersive solid-phase extraction combined with capillary electrophoresis

For a long time, the detection of nitroimidazole antibiotics (NIABs) has been a research focus in environmental analytical chemistry. In this work, a novel technique for the analysis of nitroimidazoles was established based on capillary electrophoresis (CE). UiO-66, synthesized using a solvothermal method, was utilized as an adsorbent in the dispersive solid-phase extraction (DSPE) of five different NIABs. The separation and detection of NIABs in environmental water samples were accomplished using the CE diode array detection method. The optimal extraction conditions were obtained after systematically studying the effects of adsorption time, the amount of extractant, and elution solvent on extraction efficiency. According to the results of the study, the limit of detections of the five NIABs were between 16 and 97 ng/mL, the relative standard deviations were between 0.32% and 0.55%, and the spike recoveries were between 87.43% and 104.8%. This study presents a novel technique for measuring NIABs in complex water samples.     

Cryogenic grinding pre-treatment improves extraction efficiency of fluoroquinolones for HPLC-MS/MS determination in animal tissue

An efficiency extraction of fluoroquinolones in chicken muscle was achieved by pulverizing it in a freezer mill before treatment with NaOH (10mM)/MeCN (1:1). The improvement of cryogenic grinding in the extraction was demonstrated for the same piece (whole leg) of four chickens treated with enrofloxacin in equal doses. A confirmatory method based on high-performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) was used to analyze the extracts. The chromatographic separation was achieved in 5min with a Synergi Fusion-RP 80A (50 x 2mm, 4μm) column filled with a hybrid polymer. The HPLC was coupled with a detector based in a quadrupole–linear ion trap Q-TRAP that allows a confirmatory detection according to the European legislation. The specificity of the method was assessed by testing a number of representative blank muscle samples (n = 10) to verify the absence of potential interfering compounds. The limits of detection and quantitation were 2 and 5ng g^-1 of quinolones in muscle samples, respectively. The chromatographic method was demonstrated to be linear for the range studied (5–500ng g^-1) with the P value for lack-of-fit in the ANOVA table greater or equal to 0.10 (calibration coefficient 0.9998 and 0.9996 for ciprofloxacin and enrofloxacin, respectively). The mean intra-day relative standard deviation (RSD) (n = 6, c = 50ng g^-1) was 6%; inter-day assay gave a RSD of 12%. The extraction and clean-up were carried out in one step with very satisfactory recovery data (between 65 and 101%).     

Supercritical fluid extraction of the fluoroquinolones norfloxacin and ofloxacin from orally treated-chicken breast muscles

Supercritical fluid extraction (SFE) of the fluoroquinolones norfloxacin and ofloxacin from chicken breast muscles was examined. A liquid chromatography with fluorescence detection was used for the determination of the fluoroquinolones. Extraction conditions of the SFE were optimized by determining the extraction parameters to achieve a sufficiently high recovery of each fluoroquinolone in fortified-muscle samples. Recovery values for the extraction of the fluoroquinolones using the SFE ranged from 70 to 87%. Chickens were treated orally with each fluoroquinolone and their muscles were extracted at set time intervals for time-course determination of the fluoroquinolones in chickens. The SFE combined with liquid chromatographic analysis showed that the concentrations of the fluoroquinolones decreased gradually with time in the chicken muscles after oral treatment, giving a concentration less than 5 ng/ml in 120 h. No further sample cleanup procedures were required after the SFE. These results suggest that SFE method is an extraction method for the determination of norfloxacin and ofloxacin in chicken muscle.     

Determination of amoxicillin in trout by liquid chromatography with UV detection after derivatization

A liquid chromatographic (LC) method based on solid-phase extraction was developed for determination of amoxicillin in muscle tissue of rainbow trout. The compound was extracted in an aqueous solution by precipitation of organic material with a mixture of sulfuric acid and sodium tungstate. The extract was processed by solid-phase extraction on an end-capped phenyl sorbent, and concentrated on a divinylbenzene-co-N-vinylpyrrolidone polymeric sorbent. The extract was derivatized and analyzed by reversed-phase gradient LC on a C18 column with UV detection at 323 nm. The method detection limit was 2.9 micrograms/kg. Mean recovery in muscle was 80.5% (range 10-200 micrograms/kg). The method was applied to fillets from trout offered feed containing amoxicillin in an aquaculture pilot plant. Amoxicillin was detected in muscle tissue shortly after administration but not 3 weeks later. The relative repeatability standard deviation for incurred residues in muscle tissue was 6.4% (range 11-143 micrograms/kg).     

Synthesis and characterization of a hypoxia-sensitive MRI probe

Tissue hypoxia occurs in pathologic conditions, such as cancer, ischemic heart disease and stroke when oxygen demand is greater than oxygen supply. An imaging method that can differentiate hypoxic versus normoxic tissue could have an immediate impact on therapy choices. In this work, the gadolinium(III) complex of 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) with a 2-nitroimidazole attached to one carboxyl group via an amide linkage was prepared, characterized and tested as a hypoxia-sensitive MRI agent. A control complex, Gd(DO3A-monobutylamide), was also prepared in order to test whether the nitroimidazole side-chain alters either the water proton T(1) relaxivity or the thermodynamic stability of the complex. The stabilities of these complexes were lower than that of Gd(DOTA)(-) as expected for mono-amide derivatives. The water proton T(1) relaxivity (r(1)), bound water residence lifetime (τ(M)) and rotational correlation time (τ(R)) of both complexes was determined by relaxivity measurements, variable temperature (17) O?NMR spectroscopy and proton nuclear magnetic relaxation dispersion (NMRD) studies. The resulting parameters (r(1) =6.38?mM(-1) s(-1) at 20?MHz, τ(M) =0.71?μs, τ(R) =141?ps) determined for the nitroimidazole derivative closely parallel to those of other Gd(DO3A-monoamide) complexes of similar molecular size. In vitro MR imaging experiments with 9L rat glioma cells maintained under nitrogen (hypoxic) versus oxygen (normoxic) gas showed that both agents enter cells but only the nitroimidazole derivative was trapped in cells maintained under N(2) as evidenced by an approximately twofold decrease in T(1) measured for hypoxic cells versus normoxic cells exposed to this agent. These results suggest that the nitroimidazole derivative might serve as a molecular reporter for discriminating hypoxic versus normoxic tissues by MRI.     

鱼肉中微囊藻毒素的高效液相色谱法分析

应用反相高效液相色谱法分析了鱼肉中藻毒素的含量。用BDS C18色谱柱,以水（含1％三氟乙酸）：甲醇＝30：70（V／V）溶液为流动相,238nm紫外检测。鱼肉样品用甲醇－水和丙酮混合溶剂提取,经正已烷萃取后,将有机相弃去,水相用固相萃取柱净化后进行高效液相色谱分析。该法检测限为10ng/g,峰面积标准曲线在50－250ng范围内有良好线性关系,平均回收率为85．1％－88．2％。     

High-performance liquid chromatographic analysis of Romet-30 in salmon following administration of medicated feed

A sensitive and selective high-performance liquid chromatographic assay was developed for the simultaneous quantitation of sulphadimethoxine (SDM) and ormetoprim (OMP) in chinook salmon muscle tissue. SDM and OMP were extracted from tissue samples using a solid-phase extraction technique. Resolution of both drugs was accomplished using an Ultrasphere ion-pair column (250 x 4.6 mm I.D.) and a mobile phase of acetonitrile-methanol-0.1 M phosphate buffer, pH 4 (17:10:73) with ultraviolet detection at 280 nm. The calibration curve in salmon muscle tissue was linear over the concentration range 0.2-20 ppm for both SDM (r2 = 0.9974) and OMP (r2 = 0.9956). The minimum detectable quantity of SDM and OMP in salmon muscle tissue was 0.2 ppm at a signal-to-noise ratio of 5:1. An in vivo feeding experiment was undertaken where chinook salmon were administered Romet-30-medicated feed for a 10-day period, followed by a 42-day wash-out period. The rate of tissue uptake and decline of SDM and OMP was shown to differ.     

A reversed phase high-performance liquid chromatography method for determination of cephalexin in human plasma

A reversed-phase HPLC method for the analysis of cephalexin (7-[(aminophenylacetyl)amino]-3-methyl-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid) by isocratic separation is described. A comparison is made between the ultrafiltration land the extraction procedure developed in our laboratory. The extraction procedure, based on the deproteinization of plasma with perchloric acid followed by the extraction with dichloroethane and separation of cephalexin on a reversed phase column, gave better recovery. Quantitative validation of our method has been performed by an external standard technique. The relative standard deviations were between 1.1 and 1.9% in the within-day assay and between 1.2 and 2.2% in the inter-day assay. The limit of detection was 0.14ug/ml and the limit of quantification was 0.28ug/ml. This method is rapid, sensitive, simple, and reproducible. The LC MS/MS technique was successfully used for identification and quantification of cephalexin in human plasma samples.     

Detection of streptomycin and dihydrostreptomycin residues in milk,honey and meat samples using an optical biosensor

Immuno-biosensor inhibition assays for the detection of streptomycin and dihydrostreptomycin residues in whole cows' milk, honey, pig kidney and pig muscle are reported. The antibody showed high cross-reactivity with dihydrostreptomycin in various foodstuffs (buffer 103%, milk 96%, honey 84%, kidney extract 129% and muscle extract 98%). There was no significant cross-reaction with other aminoglycosides or commonly used antibiotics. A streptomycin derivative was used to prepare a stable, reusable sensor chip surface. The assay allowed the direct analysis of bovine whole milk (fat content approximately 3.5%). Honey samples required dilution with buffer, while kidney and muscle samples from pigs were homogenized in an aqueous extraction buffer and clarified by centrifugation. The limit of detection for each assay was determined from known streptomycin-free samples (n = 20; mean - (3 x standard deviation)) and the results were as follows: milk 30 microg kg(-1), honey 15 microg kg(-1), kidney 50 microg kg(-1) and muscle 70 microg kg(-1). Repeatability (or relative standard deviation) between runs were calculated (n = 3) at the respective Community maximum residue limits (MRL) and 0.5 x MRL with the exception of honey since no European MRL exists at present. Results were determined as 4.3% (200 microg kg(-1)) and 2.8% (100 microg kg(-1)) in milk, 13.3% (40 microg kg(-1)) and 9.5% (20 microg kg(-1)) in honey, 7.1% (1000 microg kg(-1)) and 7.6% (500 microg kg(-1)) in kidney and 7.1% (500 microg kg(-1)) and 11% (250 microg kg(-1)) in muscle.     

Rapid determination of nitroimidazole residues in honey by liquid chromatography/tandem mass spectrometry

We developed a rapid and efficient means of determining residues of four nitroimidazoles-i.e., dimetridazole, ipronidazole, metronidazole, and ronidazole-and three hydrophilic metabolites- i.e., 2-hydroxymethyl-1-methyl-5-nitroimidazole, 1 -methyl-2-(2'-hydroxyisopropyl)-5-nitroimidazole, and 1-(2-hydroxyethyl)-2-hydroxymethyl-nitroimidazole--in honey. We applied a QuEChERS (Quick, Easy, Cheap, Effective, Rugged, and Safe) procedure improved to suit a nitroimidazole analysis, which is fast (approximately 30 min) and uses less organic solvent. The procedure involves initial single-phase extraction of 5 g of honey with acetonitrile containing 1% acetic acid, followed by liquid-liquid partitioning involving the addition of 5 g sodium chloride, 1.5 g trisodium citrate dihydrate, and 4 g magnesium sulfate. Moreover, matrix from honey was reduced by an SPE method with an alumina-N cartridge. The samples were analyzed using LC/MS/MS. Chromatographic separation of these nitroimidazoles and metabolites was performed in the gradient mode on a pentafluorophenylpropyl-bonded silica column (150x2.0 mm, 3 pm particle size) at 40 degrees C. The mobile phase consisted of a 0.01% acetic acid solution and acetonitrile, and the flow rate was 0.2 mL/min. The method was validated using honey spiked with these nitroimidazoles from 0.1 to 0.5 microg/kg. The overall recovery of the seven nitroimidazoles ranged from 76.1 to 98.5%; intra- and interassay CV values were <9.5 and <14.2%, respectively. The LOQ ranged from 0.1 to 0.5 microg/kg. LC/MS/MS coupled with the QuEChERS method showed good potential as a method for determining nitroimidazole residues in honey.