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Farms utilizing sewage sludge and manure in their agronomic plant production are recognized as potential hotspots for environmental release of antibiotics and the resulting promotion of antibiotic resistance. As part of the circular economy, the use of biogas digestates for soil fertilizing is steadily increasing, but their potential contribution to the spreading of pharmaceutical residues is largely unknown. Digestates can be produced from a variety of biowaste resources, including sewage sludge, manure, food waste, and fish ensilage. We developed a method for the detection of 17 antibiotics and 2 steroid hormones and applied the method to detect pharmaceutical residues in digestates from most municipal biogas plants in Norway, covering a variety of feedstocks. The detection frequency and measured levels were overall low for most compounds, except a few incidents which cause concern. Specifically, relatively high levels of amoxicillin, penicillin G, ciprofloxacin, and prednisolone were detected in different digestates. Further, ipronidazole was detected in four digestates, although no commercial pharmaceutical products containing ipronidazole are currently registered in Norway. A simplified risk assessment showed a high risk for soil microorganisms and indicates the tendency for antibiotic-resistant bacteria for penicillin G and amoxicillin. For prednisolone and ipronidazole; however, no toxicity data is available for reliable risk assessments. Graphical abstract

The method of voltammetric determination of nitroimidazoles in honey is developed. This method is based on the reduction of nitroimidazoles nitro group on the mercury drop indicator electrode. The obtained values of LODs for nitroimidazoles are as follows: 1.5 × 10−7 M, 8.6 × 10−8 M and 2.0 × 10−7 M for metronidazole, dimetridazole, and ipronidazole, and the linear correlation coefficients are 0.99974, 0.99987 and 0.99849, respectively, which tend towards to 1. The minimum amount of metronidazole that can be detected by means of our method, using simple dissolving of honey sample in water is 300 µg of metronidazole per 1 kg of honey. The developed voltammetric technique’s accuracy was confirmed by the “added-found” method and by HPLC-MS/MS analysis. The calculated amount of metronidazole in the tested model solution is in good agreement with the amount, introduced into honey. The calculated values of texp are 0.52, 0.48 and 0.43 for three different concentrations. Significant advantages of the method are effortless sample preparation, fast procedure of analysis, the possibility of miniaturization, low cost, not necessary use of organic solvents.

Development of visible-light photocatalysts for the removal of nitroimidazoles pharmaceutical residues in the environment has attracted widespread attention. In this respect, a series of nitrogen-containing carbon quantum dots N-CQDs/InNbO4 composites were hydrothermally synthesized for the disposal of ipronidazole. N-CQDs coupled with irregular square shaped InNbO4 was confirmed by XRD, FTIR, TEM and XPS. The as-prepared N-CQDs/InNbO4 composites displayed stronger visible-light absorption from 400 ​nm to 800 ​nm. Owing to photosensitization and electron transfer ability of N-CQDs, the photocatalytic performance of N-CQDs/InNbO4 composites was better than that of InNbO4. The highest removal efficiency of ipronidazole solution was obtained in the presence of 3 ​wt % N-CQDs/InNbO4 composite. Reactive species ●O2−, hole, ●OH and electron all took part in the reaction process, while the removal of ipronidazole was most affected by ●O2−. Moreover, six new transformation products of ipronidazole were found during photocatalytic process.

In this study, a simple, cost-effective, and sensitive HPLC diode-array detection method was developed for the simultaneous determination of six different 5-nitroimidazoles [metronidazole, 2-hydroxymethyl-1-methyl-5-nitro-1H-imidazole, dimetridazole (DMZ), ronidazole, ornidazole, and ipronidazole] in bovine milk samples. A QuEChERS-based sample preparation procedure was optimized by evaluating different cleanup sorbents, including zirconium-based sorbents (Z-Sep and Z-Sep+), C18, and primary–secondary amine (PSA), as well as EMR-Lipid cleanup solution. Acceptable analytical performance for all analytes was observed with recoveries in the range of 45–93% and RSDs of less than 15%. Negligible matrix interference was observed for most of the analytes due to application of PSA sorbent in a dispersive solid-phase extraction cleanup step. Method LOQs (mLOQs) for five of the six investigated analytes were set at a satisfactory low food product value of 2.5 ng/mL. For DMZ only, the mLOQ was set at 10 ng/mL. The procedure was evaluated through the analysis of 10 different natural samples.

In this study, a new method for the determination of 12 nitroimidazoles and their hydroxymetabolites (metronidazole, hydroxymetronidazole, dimetridazole, ronidazole, hydroxydimetridazole, ipronidazole, hydroxyipronidazole, carnidazole, ornidazole, secnidazole, ternidazole, tinidazole) in beeswax has been developed and validated. The optimized sample preparation procedure included melting and dilution of beeswax in a mixture of n-hexane and isopropanol followed by extraction with 2% acetic acid. The extracts were purified on strong cation exchange based solid-phase extraction cartridges and evaporated in a vacuum system with vortex motion. The separation and detection of the nitroimidazoles in the beeswax extracts were achieved within 12 min by liquid chromatography tandem mass spectrometry using a pentafluorophenyl analytical column and applying a gradient elution with acetonitrile and 0.01% acetic acid as mobile phases. The method performance characteristics were evaluated at three concentration levels (1, 2, and 5 μg/kg) and the method was found to be suitable for determination of all tested nitroimidazoles. The limits of detection and quantification were 0.2-0.5 and 0.5-1 μg/kg, respectively. The recoveries varied from 71.2 to 104.9% while the relative standard deviations were less than 13.8% under the intermediate precision conditions.

A sensitive and reliable method for the simultaneous determination of four nitroimidazoles (ipronidazole (IPZ), dimetridazole (DMZ), metronidazole (MNZ) and ronidazole (RNZ)) and three metabolites (IPZ-OH, MNZ-OH and 2-hydroxymethyl-1-methyl-5-nitroimidazole (HMMNI)) in livestock and fishery products was developed. The analytes were extracted from samples with acetone containing acetic acid. The crude extracts were defatted by liquid-liquid partition using acetonitrile and n-hexane followed by solid-phase extraction using a cartridge column packed with divinylbenzene-N-vinylpyrolidone copolymer bearing sulfo groups. The analytes in the eluate from the cartridge column were extracted with ethyl acetate after addition of ammonium sulfate. The solvent was removed from the extract, and the residue was dissolved in 0.1 vol% formic acid. The HPLC separation was performed on a C18 column with a gradient formed from water containing 0.1 vol% formic acid and acetonitrile containing 0.1 vol% formic acid. For detection of the analytes, tandem mass spectrometry with positive ion electrospray ionization was used. The recovery tests were performed on 10 livestock and fishery products. The truenesse ranged from 74.6 to 111.1%, with repeatability of 0.5-8.3 RSD% for the entire procedure. The limit of quantification was 0.0001 mg/kg for IPZ, IPZ-OH, MNZ and MNZ-OH, and 0.0002 mg/mg for DMZ, RNZ and HMMNI.

Nitroimidazoles are not authorised for the treatment of honey bees in the European Union. However, they can be found in honey largely because they are illegally used in apiculture for the treatment of Nosema. The aim of the study was to examine the possible transfer of nitroimidazoles (metronidazole, ronidazole, dimetridazole and ipronidazole) from contaminated beeswax to honey. The wax foundations fortified with a mixture of four nitroimidazoles at three concentration levels (1000, 10,000 and 100,000 μg kg

The misuse of nitroimidazoles (NDZs) can lead to NDZs residues in edible animal tissues, which would be harmful to consumer health. To quickly monitor NDZs residues in edible animal tissues and feed, a monoclonal antibody-based indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) with a simple sample preparation method and clean-up was developed in the present study. At first, a broad-specificity monoclonal antibody, 1D5, against NDZs has been produced, which the IC50 values of the NDZs, dimetridazole, ipronidazole, ronidazole hydroxydimetridazole, and hydroxyipronidazole, were 4.79μgL(-1), 0.47μgL(-1), 5.97μgL(-1), 23.48μgL(-1), and 15.03μgL(-1), respectively. The limit of detection of the method for the NDZ matrix calibration ranged from 4.2μgkg(-1) to 50.3μgkg(-1) in the feed matrices and from 0.11μgkg(-1) to 4.11μgkg(-1) in the edible animal tissues matrices. The recoveries of the NDZs were in the range of 75.5-111.8%. The CVs were less than 14.4%. A good correlation (r=0.9905) between the ELISA and HPLC-MS results of the tissues demonstrated the reliability of the developed ic-ELISA, which makes it a useful tool for screening of NDZs in animal edible tissue and feed.

A multiresidue method for the determination of seven nitroimidazoles and their hydroxy metabolites in milk was developed. Milk samples were extracted with acetonitrile and cleaned up on strong cation-exchange solid phase extraction cartridges. Evaporated to dryness first, the extracts obtained were then reconstituted in 0.1% formic acid and injected onto a liquid chromatography-tandem mass spectrometer (LC-MS/MS). The separation of analytes was achieved on gradient mode using a C18 column and a mobile phase consisting of 0.1% formic acid in acetonitrile and 0.1% formic acid in water. Multiple reaction monitoring mode was set for the MS/MS with positive electrospray ionisation. For quantification, the isotope dilution method was used with isotopically labeled analogues of the target analytes. The method was evaluated entirely in accordance with EU Commission Decision 2002/657/EC and all validation criteria were in the required ranges. The method can easily detect and confirm metronidazole, dimetridazole, ronidazole, ipronidazole, and their hydroxy metabolites below the recommended concentration level of 3 μg/kg. The decision limits and detection capabilities ranged from 0.11 μg/kg to 0.22 μg/kg and from 0.19 μg/kg to 0.37 μg/kg respectively. The overall recoveries were between 96.6% and 105.2% with a good coefficient of variation, less than 8.7% under within-laboratory reproducibility conditions.

A selective method for the determination of fourteen nitroimidazoles and their hydroxy-metabolites in honey was developed based on improved molecularly imprinted solid-phase extraction followed by liquid chromatography–tandem mass spectrometry. The separation of analytes was performed on a C18 column using a mobile phase of 0.1% formic acid in acetonitrile and 0.1% formic acid in water with gradient elution. The method was suitable for metronidazole, hydroxymetronidazole, dimetridazole, ronidazole, hydroxydimetridazole, ipronidazole, hydroxyipronidazole, carnidazole, menidazole, nimorazole, ornidazole, secnidazole, ternidazole, and tinidazole. The procedure was evaluated according to EU Commission Decision 2002/657/EC requirements by determining linearity, specificity, recovery, repeatability, within-laboratory reproducibility, decision limit, detection capability, matrix effects, and stability. The method determined nitroimidazoles and their hydroxy-metabolites below the recommended concentration level o...

An UHPLC-MS/MS method was developed for the quantitative confirmatory analysis of residues of nitroimidazole drugs (dimetridazole, ipronidazole, metronidazole, ornidazole and ronidazole) and their corresponding hydroxy metabolites (HMMNI, ipronidazile-OH and metronidazole-OH) in aquaculture tissue. Samples were extracted by shaking in acetonitrile, water, MgSO4 and NaCl before being defatted with n-hexane pre-saturated with acetonitrile and concentrated under nitrogen. Nitroimidazole residues were determined by UHPLC-MS/MS operating in positive electrospray ionisation mode using a reversed phase BEH C18 column. The method was validated according to the EU Commission Decision 2002/657/EC guidelines. The following performance studies were carried out: specificity, selectivity, linearity, within laboratory repeatability (WLr)/reproducibility (WLR), accuracy, precision, decision limit (CCα), detection capability (CCβ), absolute recovery and stability. The analytical range of the method is 0.1-20 μg kg(-1). Accuracy and precision of the method, under within-laboratory reproducibility conditions, ranged from 83 to 105% and 2.3 to 14.0%, respectively. CCα were 0.07-1.0 μg kg(-1) depending on analyte and matrix. A total of 50 samples can be analysed in a single day using the assay. The method has been extensively evaluated through application to real test samples.

A new and sensitive multi-residue method (MRM) with detection by LC-MS/MS was developed and validated for the screening, determination, and confirmation of residues of 7 nitroimidazoles and 3 of their metabolites in turkey muscle tissues at concentrations ≥ 0.05 ng/g. The compounds were extracted into a solvent with an alkali salt. Sample clean-up and concentration was then done by solid-phase extraction (SPE) and the compounds were quantified by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The characteristic parameters including repeatability, selectivity, ruggedness, stability, level of quantification, and level of confirmation for the new method were determined. Method validation was achieved by independent verification of the parameters measured during method characterization. The seven nitroimidazoles included are metronidazole (MTZ), ronidazole (RNZ), dimetridazole (DMZ), tinidazole (TNZ), ornidazole (ONZ), ipronidazole (IPR), and carnidazole (CNZ). It was discovered during the single laboratory validation of the method that five of the seven nitroimidazoles (i.e. metronidazole, dimetridazole, tinidazole, ornidazole and ipronidazole) and the 3 metabolites (1-(2-hydroxyethyl)-2-hydroxymethyl-5-nitroimidazole (MTZ-OH), 2-hydroxymethyl-1-methyl-5-nitroimidazole (HMMNI, the common metabolite of ronidazole and dimetridazole), and 1-methyl-2-(2'-hydroxyisopropyl)-5-nitroimidazole (IPR-OH) included in this study could be detected, confirmed, and quantified accurately whereas RNZ and CNZ could only be detected and confirmed but not accurately quantified. © Her Majesty the Queen in Right of Canada as Represented by the Minister of Agriculture and Agri-food Canada 2012.

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 (150 × 2.0 mm, 3 μm particle size) at 40°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 μg/kg. The overall recovery of the seven nitroimidazoles ranged from 76.1 to 98.5%; intra- and interassay CV values were

A confirmatory method has been developed to allow for the analysis of fourteen prohibited medicinal additives in pig and poultry compound feed. These compounds are prohibited for use as feed additives although some are still authorised for use in medicated feed. Feed samples are extracted by acetonitrile with addition of sodium sulfate. The extracts undergo a hexane wash to aid with sample purification. The extracts are then evaporated to dryness and reconstituted in initial mobile phase. The samples undergo an ultracentrifugation step prior to injection onto the LC-MS/MS system and are analysed in a run time of 26 min. The LC-MS/MS system is run in MRM mode with both positive and negative electrospray ionisation. The method was validated over three days and is capable of quantitatively analysing for metronidazole, dimetridazole, ronidazole, ipronidazole, chloramphenicol, sulfamethazine, dinitolimide, ethopabate, carbadox and clopidol. The method is also capable of qualitatively analysing for sulfadiazine, tylosin, virginiamycin and avilamycin. A level of 100 microg kg(-1) was used for validation purposes and the method is capable of analysing to this level for all the compounds. Validation criteria of trueness, precision, repeatability and reproducibility along with measurement uncertainty are calculated for all analytes.

Nitroimidazoles (Ronidazole, Dimetridazole, Metronidazole, Ipronidazole) and their hydroxy metabolites are banned substances with antibiotic and anticoccidial activity. They are suspected to be carcinogenic and mutagenic. Since nitroimidazoles showed an inhomogeneous distribution and a rapid degradation in incurred muscle samples, plasma is the preferred target matrix for residue analysis. The analytical method of Polzer et al. [J. Polzer, C. Stachel, P. Gowik, Anal. Chim. Acta 521 (2004) 189] was adapted for liquid chromatography-tandem mass spectrometry detection and was validated in house according to the Commission Decision 2002/657/EC. The method is specific for all nitroimidazole except for Ipronidazole and its metabolite, due to interferences at their retention times in chromatograms of blank plasma and reagents samples. The absence of a matrix effect enables the use of a (linear) calibration curve in solution for quantitation. The apparent recovery (obtained after correction with a deuterated internal standard) is between 93% and 123%, except for the metabolite of Metronidazole (58-63%). The repeatability (CVr=2.49-13.39%) and intralaboratory reproducibility (CVRW=2.49-16.38%) satisfy the Horwitz equation. The obtained values for the detection capacity (CCbeta) range from 0.25 to 1 microg L(-1), while values obtained for the decision limit (CCalpha) are below CCbeta.

A liquid chromatography-mass spectrometry method for the determination of the coccidiostats metronidazole, ronidazole, dimetridazole, secnidazole, tinidazole and ipronidazole in feedstuff was developed. The 5-nitroimidazoles were extracted from animal feed with a pH 2 phosphate buffer solution followed by a solid phase extraction procedure based on HLB cartridges. The liquid chromatographic separation of the extracts was performed on a C18 bonded silica column applying a gradient of elution with pH 4.3 ammonium acetate buffer solution and acetonitrile. Quantification for each 5-nitroimidazole was carried out monitoring its molecular ion [M + H]+ obtained by positive electrospray ionisation mode. A pool of feedstuffs was used to optimise and validate the method. Linearity, quantification limit, intra-day and inter-day reproducibility and accuracy were estimated using this pool of feedstuffs spiked with the drugs. For the confirmatory assay, several fragment ions from each 5-nitroimidazole were obtained and monitored. The method was applied successfully to determine 5-nitroimidazoles in feedstuff at level of 0.05 mg kg−1.

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

Nitroimidazoles in incurred muscle samples showed a considerable inhomogeneity in their distribution. A possible explanation for this phenomenon were the sampling conditions. In an animal study, the influence of sampling after slaughtering was investigated by applying four different ways of treating muscle samples prior to freezing at −24 °C. The muscle samples were analysed using gas chromatography-negative chemical ionisation/mass spectrometry after silylation. The results reaffirmed an inhomogeneous distribution of ipronidazole and ronidazole in muscle samples, irrespective of the sampling procedure. Thermal stress caused a significant loss of analytes, even at +4 °C.

In several animal studies turkeys were treated with different nitroimidazoles (Dimetridazole, Metronidazole, Ronidazole, Ipronidazole). After slaughtering, different matrices (breast muscle, leg muscle, liver, plasma, retina) were analysed for their analyte content, for the percentage of hydroxy-metabolites, for homogeneity, stability and bound, and conjugated residues. The tests showed that for animals treated with Dimetridazole and Ipronidazole, the hydroxy-metabolites (2-hydroxymethyl-1-methyl-5-nitroimidazole (HMMNI) and 1-methyl-2-(2′-hydroxyisopropyl)-5-nitroimidazole (IPZOH)) are the relevant target analytes, whereas for animals treated with Ronidazole and Metronidazole, the parent drug itself is the most relevant analyte. In muscle samples an inhomogeneous analyte distribution was found. Degradation studies showed a rapid decline of the analyte concentration in muscle and liver samples stored at room temperature and a decelerated degradation at 4 °C. In plasma and retina samples, however, the analytes were stable during storage under the same conditions. In these matrices the analytes were found to be present in considerably higher concentrations than in muscle or liver and could be detected for a longer period of time after withdrawal of the medication. Therefore, plasma or retina can be recommended as target matrices for the residue control of nitroimidazoles in turkeys.

Treatment of protozoal pathogens in the reproductive system with chemical agents exposes flagellated sperm cells to potential toxicants. A widely used antiprotozoal agent is metronidazole. Its effect on rabbit and human sperm was compared with a more soluble 5-nitroazole compound, ipronidazole, and with a systemic environmental toxicant, dibromochloropropane (DBCP). The percentages of motile rabbit and human sperm incubated with the compounds, the velocity of sperm, migration of sperm in polyacrylamide gel, young born in rabbits, and penetration of hamster oocytes by treated human sperm were measured in seven experiments. Up to 10 mg/ml metronidazole and 1 mg/ml DBCP had little effect on most sperm characteristics. However, 10 mg/ml metronidazole and 5 mg/ml of ipronidazole increased attachment of human sperm to hamster oocytes, but oocyte penetration was unaffected. Rabbit sperm exposed to 5 mg/ml ipronidazole were infertile. No oocytes were penetrated by DBCP-treated human sperm.

A rapid liquid chromatography–mass spectrometric method for the determination of five nitroimidazoles in water from different sources is described. The extraction procedure was based on HLB solid-phase extraction with acetonitrile followed by an evaporation step. Ternidazol, another nitroimidazole, was used as the internal standard. The liquid chromatographic separation was made on a C18 bonded silica column applying a gradient with an ammonium acetate buffer solution and acetonitrile. Following electrospray ionisation, the protonated molecular ions [M+H]+ were obtained. Quantification for each nitroimidazole was carried out by monitoring its molecular ion. Calibration functions, quantification and detection limits, intra- and inter-day reproducibility and accuracy were estimated. For the confirmatory assay, several fragment ions from each nitroimidazole were obtained and monitored. The method was applied successfully to determine and identify nitroimidazoles in water from different sources at a level of 0.2 μg l−1.

We present a high-throughput confirmatory method for the analysis of 11 nitroimidazoles in muscle samples (metronidazole, MNZ; dimetridazole, DMZ; ronidazole, RNZ; tinidazole, TNZ; carnidazole, CRZ; secnidazole semihydrate, SCZ; ornidazole, ORZ; metronidazole-hydroxy, MNZ-OH; ipronidazole, IPZ; ipronidazole-hydroxy, IPZ-OH; and dimetridazole-hydroxy, HMMNI). Extraction with acetonitrile is efficient and simple, with the majority of recoveries ranging between 65 and 101 %, and without the need for clean-up of the extracts. The chromatographic analysis is performed using UPLC-QqQ-MS in the MRM mode, with an electrospray source operated in positive mode. Total chromatographic analysis time is 12 min. This method has been fully validated in muscle according to Decision 657/2002/CE guidelines, and the CC alpha values obtained were in the range of 0.04-0.4 mu g center dot kg(-1). The method is currently used for the analysis of muscle samples and has been tested in other matrices of animal origin, such as kidney, retina and egg, with excellent results.

A liquid chromatography-tandem mass spectrometry method, recently developed, validated and accredited, was used to screen for metronidazole, ronidazole dimetridazole ipronidazole, ternidazole, tinidazole, ornidazole carnidazole and three hydroxy metabolites (hydroxy-metronidazole, HMMNI and hydroxy-ipronidazole) in Irish retail egg samples. The method used had decision limits (CCα) in the range 0.33-1.26 µg kg(-1) and detection capabilities (CCβ) ranging 0.56-2.15 µg kg(-1) for all analytes. Internal standard-corrected recovery, calculated for the various analytes, ranged 87.2-106.2%, while the coefficient of variance, expressed as % CV, ranged 3.7-11.3%. The method was applied to 160 samples of caged, free range and organic hen and duck eggs available on the Irish retail market as well as two incurred proficiency test egg samples. No nitroimidazole residues were detected in the survey samples above the CCα and the results achieved for the two proficiency test samples were acceptable when compared with the assigned values.

A multi-residue liquid chromatography-mass spectrometry (LC-MS) assay method is described for the determination of four nitroimidazoles in poultry muscle. The extraction procedure is based on liquid-liquid extraction with ethyl acetate followed by an evaporation step. A deuterated internal standard is used. The LC separation was made on a C18 bonded silica column with an aqueous formic acid (0.2%) solution-methanol-acetonitrile (81:13:6) mobile phase. Following electrospray ionization, the protonated molecular ion [M+H]+ is obtained for each compound. Monitoring several ions for each nitroimidazole provides the specificity required for confirmatory assay. Validation of the method was performed to estimate linearity, intra-day and inter-day repeatability, accuracy and detection limit. The present method is capable of identifying nitroimidazole residues in muscle at levels below 5 microg/kg.

A series of monoclonal antibodies were generated that can bind dimetridazole, a nitroimidazole drug used in veterinary medicine. A competition enzyme-linked immunosorption assay (cELISA) is described and is used to characterize the binding of these antibodies to a number of nitroimidazole drugs. The 50% inhibition of control occurred for the most sensitive of these antibodies at 12 ng/mL for dimetridazole, 75 ng/mL for hydroxydimetridazole, 25 ng/mL for ipronidazole, 2000 ng/mL for hydroxyipronidazole, and 20 000 ng/mL for metronidazole. An extraction method for these nitroimidazoles that is compatible with the immunoassay is described. Using this method, as little as 1 ng of dimetridazole could be detected in turkey muscle

A confirmatory method was developed to allow for the analysis of eleven nitroimidazoles and also chloramphenicol in milk and honey samples. These compounds are classified as A6 compounds in Annex IV of Council Regulation 2377/90 (European Commission 1990) and therefore prohibited for use in animal husbandry. Milk samples were extracted by acetonitrile with the addition of NaCl; honey samples were first dissolved in water before a similar extraction. Honey extracts underwent a hexane wash to remove impurities. Both milk and honey extracts were evaporated to dryness and reconstituted in initial mobile phase. These were then injected onto a liquid chromatography-tandem mass spectrometry (LC-MS/MS) system and analysed in less than 9 min. The MS/MS was operated in multiple reaction monitoring (MRM) mode with positive and negative electrospray ionization. The method was validated in accordance with Commission Decision 2002/657/EC and is capable of analysing metronidazole, dimetridazole, ronidazole, ipronidazole and there hydroxy metabolites hydroxymetronidazole, 2-hydroxymethyl-1-methyl-5-nitroimidazole, and hydroxyipronidazole. The method can also analyse for carnidazole, ornidazole, ternidazole, tinidazole, and chloramphenicol. A recommended level of 3 microg l(-1)/microg kg(-1) for methods for metronidazole, dimetridazole, and ronidazole has been recommended by the Community Reference Laboratory (CRL) responsible for this substance group, and this method can easily detect all nitroimidazoles at this level. A minimum required performance level of 0.3 microg l(-1)/microg kg(-1) is in place for chloramphenicol which the method can also easily detect. For nitroimidazoles, the decision limits (CCalpha) and detection capabilities (CCbeta) ranged from 0.41 to 1.55 microg l(-1) and from 0.70 to 2.64 microg l(-1), respectively, in milk; and from 0.38 to 1.16 microg kg(-1) and from 0.66 to 1.98 microg kg(-1), respectively, in honey. For chloramphenicol, the values are 0.07 and 0.11 microg l(-1) in milk and 0.08 and 0.13 microg kg(-1) in honey. Validation criteria of accuracy, precision, repeatability, and reproducibility along with measurement uncertainty were calculated for all analytes in both matrices.

A rapid method has been developed for the determination of dimetridazole (DMZ), ipronidazole (IPZ), and ronidazole (RNZ) in turkey feeds, swine feeds, and premix. The compounds are extracted from samples with warm methanol, the extract is purified over a short alumina column, and an aliquot of the eluate is analyzed by reversed-phase liquid chromatography and UV detection at 309 nm. Alumina cleanup of premixes is not essential, although the resulting chromatograms are cleaner. Recoveries of DMZ from feed formulations ranged from 97 to 103% at the 10.0,50.0, and 100.0 ppm levels, with a standard deviation (SD) of 0.62-3.2%. Recoveries of IPZ ranged from 94.4 to 101.2% at the 5.0,50.0, and 100.0 ppm levels (SD, 0.42-4.4%). RNZ recoveries ranged from 95 to 100.7% at the 6.0,60.0, and 120.0 ppm levels (SD, 1.2-5.33%).

A multiresidue analytical procedure for the determination of four nitroimidazoles (metronidazole, dimetridazole, ronidazole, ipronidazole) and their hydroxy-metabolites in poultry muscle, plasma and egg is presented. The procedure is based on ion-exchange solid phase extraction with acetonitrile as an extractant followed by liquid chromatography-mass spectrometry. The separation of analytes was performed on a C18 column using a mobile phase of 0.1% formic acid in acetonitrile and 0.1% formic acid in water with gradient elution. The electrospray ionization was used to obtain the protonated molecules [M+H](+) and two product ions were monitored for each compound. For the quantification stable isotope-labelled analogues of the analytes were used as internal standards. The whole procedure was evaluated according to EU Commission Decision 2002/657/EC requirements. Specificity, decision limit (CCalpha), detection capacity (CCbeta), recovery and precision were determined during validation process. The overall recoveries ranged between 93 and 103% with a good coefficient of variation, less than 14.0% under within-laboratory reproducibility conditions. CCalpha and CCbeta were 0.05-0.44 and 0.08-0.90microgkg(-1) depending on analyte and matrix.

An ultra performance liquid chromatography (UPLC) coupled to tandem mass spectrometry (MS/MS) procedure was developed for the determination of five 5-nitroimidazoles (dimetridazole, ipronidazole, metronidazole, ronidazole and ternidazole) and three of their metabolites (1-methyl-2-hydroxymethyl-5-nitroimidazole, 1-(2-hydroxyethyl)-2-hydroxymethyl-5-nitroimidazole and 1-methyl-2-(2′-hydroxyisopropyl)-5-nitroimidazole) in egg matrices. Conditions for UPLC separation and electrospray ionization MS/MS in the positive ion mode were optimized. Samples were prepared by liquid-liquid extraction with buffered aqueous 2.5% trichloroacetic acid followed by solid-phase extraction on a Strata-X-C cartridge with reversed-phase and cation-exchange functionalities. The method’s performance was evaluated in accordance with Commission Decision 2002/657/EC, applying the alternative matrix-comprehensive in-house validation approach using specially designed InterVal software. The method was robust against different sample matrix and SPE cartridges, operator change, and changes in sample extract storage. Acceptable apparent recoveries (76 to 109%) were obtained for all analytes. The decision limits (CCα) and detection capabilities (CCα) were within the ranges of 0.19–2.62 and 0.26–4.29 μg kg−1. For all compounds the calibration curve linearity was good with correlation coefficients greater than 0.99. Fifteen eggs and ten whole egg powder samples obtained commercially in Lithuania were analyzed by UPLC-MS/MS; none were found contaminated by 5-nitroimidazoles or their metabolites.

A simple, rapid liquid chromatographic (LC) method that uses UV/VIS detection has been developed for the determination In eggs of residues of the hlstomonostats dlmetridazole (DMZ), ronldazole (RON), Ipronidazole (IPR), and side-chain hydroxylated metabolites of DMZ and RON. Sample pretreatment Includes an aqueous extraction, purification with an Extrelut® cartridge, and acid partitioning with Isooctane. An aliquot of the final aqueous extract Is Injected Into a reversephase LC system; detection Is performed at 313 nm. The limits of determination are in the 5-10 µgkg range. A UV/ VIS spectrum can be obtained at the 10 µg/kg level by using diode-array UV/VIS detection. Recoveries are between 80 and 98% with a coefficient of variation of about 5%. Some 20 samples can be analyzed per day. A side-chain hydroxylated metabolite of IPR can also be detected with this method, as demonstrated with samples from animal experiments. After a single oral dose of the drugs to laying hens, residues of the parent compound and/or the hydroxylated metabolites could be detected In eggs 5-8 days after dosing. Plasma distribution and excretion in feces were established both with and without deconjugatlon. DMZ and IPR were extensively metabolized to hydroxylated nltrolmldazole metabolites; RON was excreted mainly as the parent compound.

A rapid confirmatory method has been developed and validated for the simultaneous identification, confirmation and quantitation of 11 nitroimidazoles in eggs by liquid chromatography tandem mass spectrometry (LC-MS/MS). The method is validated in accordance with Commission Decision 2002/657/EC and is capable of analysing metronidazole (MNZ), dimetridazole (DMZ), ronidazole (RNZ), ipronidazole (IPZ) and their hydroxy metabolites MNZ-OH, HMMNI (hydroxymethyl, methyl nitroimidazole), IPZ-OH. The method is also capable of analysing carnidazole (CRZ), ornidazole (ORZ), tinidazole (TNZ) and ternidazole (TRZ). MNZ, DMZ and RNZ have been assigned a recommended level (RL) of 3 microg kg(-1) by the Community Reference Laboratory (CRL) in Berlin. The developed method described in this study is easily able to detect all the nitroimidazole compounds investigated at this level and below. Egg samples are extracted with acetonitrile, and NaCl is added to help remove matrix contaminants. The acetonitrile extract undergoes a liquid-liquid wash step with hexane; it is then evaporated and reconstituted in mobile phase. The reconstituted samples are analysed by liquid chromatography tandem mass spectrometry (LC-MS/MS). The decision limits (CCalpha) range from 0.33 to 1.26 microg kg(-1) and the detection capabilities (CCbeta), range from 0.56 to 2.15 microg kg(-1). The results of the in ter-assay study, which was performed by fortifying hen egg samples (n=18) on three separate days, show the accuracy calculated for the various analytes to range between 87.2 and 106.2%. The precision of the method, expressed as %CV values for the inter-assay variation of each analyte at the three levels of fortification (3, 4.5 and 6.0 microg kg(-1)), ranged between 3.7 and 11.3%. A Day 4 analysis was carried out to examine species variances in eggs from different birds such as duck and quail and investigating differences in various battery and free range hen eggs.

An immunobiosensor assay was developed for the multi-residue screening of a range of nitroimidazole compounds in various species and sample types including porcine, bovine and ovine kidney, avian liver, serum and eggs and bovine milk. A polyclonal antibody which binds at least seven of the major nitroimidazoles and their metabolites was raised in a sheep after inoculation with a metronidazole protein conjugate. Sample homogenates were extracted into acetonitrile and subjected to micro-centrifugation prior to biosensor analysis. Validation data obtained from the analysis of 20 fortified samples has shown that the method has a detection capability (CCbeta) of less than 1 microgkg(-1) (or microgL(-1)) for dimetridazole (DMZ) in all species and matrices investigated. In addition, cross-reactivity data and the analysis of a small number of fortified samples have shown that the method will also detect a range of other major parent nitroimidazoles and their metabolites including ronidazole (RNZ), ipronidazole (IPZ), metronidazole (MNZ), hydroxymetronidazole (MNZOH), hydroxydimetridazole (DMZOH) and hydroxyipronidazole (IPZOH). The cross-reactivity profile and validation data for the detection of these nitroimidazoles are presented together with the results obtained following the analysis of a small number of incurred samples using the developed method.

A nitroimidazole, molecularly imprinted polymer (MIP) was tested to extract four 5-nitroimidazoles (i.e., dimetridazole (DMZ), ipronidazole (IPZ), metronidazole (MNZ), and ronidazole (RNZ)) and three of their metabolites (i.e., DMZOH, IPZOH, and MNZOH) from egg powder samples. Various MIP templates were produced, and their selectivity was assessed on nitroimidazole standard solutions using liquid chromatography coupled with ultraviolet detection. The optimal cleanup was then used for the extraction of nitroimidazole in egg powder samples, and their quantification was achieved by isotope dilution LC-ESIMS/MS. The sample preparation entails a solubilization of the samples with water and acetonitrile followed by a MISPE cleanup step before LC-ESIMS/MS analysis. Data acquisition was achieved using selected reaction monitoring, and quantification was done with five deuterated analogues (i.e., DMZ- d(3), RNZ- d(3), IPZ- d(3), DMZOH- d(3), and IPZOH- d(3)). DMZOH- d(3) was used to quantify MNZ and MNZOH since they do not have their corresponding internal standards. The method was validated according to the European Union criteria by spiking experiments at concentration levels of 1, 2, and 3 microg/kg. At these three levels and for compounds having their own internal standards, acceptable performance data were obtained, with internal standard corrected recoveries ranging from 91 to 111%, and decision limits (CCalpha) and detection capabilities (CCbeta) were below 0.34 and 0.39 microg/kg, respectively.

An isotope dilution liquid chromatography-electrospray ionization-tandem mass spectrometry method is presented for the simultaneous analysis of several 5-nitroimidazole-based veterinary drugs, which are dimetridazole (DMZ), ronidazole (RNZ), metronidazole (MNZ), ipronidazole (IPZ), and their hydroxylated metabolites (DMZOH, MNZOH, and IPZOH), in egg (fresh egg, whole egg powder, and egg yolk powder) and chicken meat. Data acquisition was achieved by applying multiple reaction monitoring, and quantitation was performed by means of five deuterated internal standards (ISs), namely, DMZ-d3, RNZ-d3, IPZ-d3, DMZOH-d3, and IPZOH-d3, whereas MNZ and MNZOH were quantitated using DMZOH-d3. At the lowest fortification levels (i.e., 0.5 microg/kg for fresh egg and chicken meat and 1.0 microg/kg for other egg-based matrices) and for compounds having their own corresponding deuterated analogue used as an IS, acceptable performance data were obtained (corrected recoveries, 88-111%; decision limits, 0.07-0.36 microg/kg; detection capabilities, 0.11-0.60 microg/kg; and within-lab precision,or = 15%). The method failed to give acceptable quantitative results for MNZ and MNZOH due to the unavailability of the corresponding deuterated ISs. Nevertheless, a reliable identification of these two analytes at levelsor = 1 microg/kg was still feasible.

The results of a validation study of a GC-NCI-MS method for the quantitative determination of 5-nitroimidazoles [1,2-Dimethyl-5-nitroimidazole (dimetridazole, DMZ), 1-methyl-2-[(carbamoyloxy)methyl]-5-nitroimidazole (ronidazole, RNZ), 1-(2-hydroxyethyl)-2-methyl-5-nitroimidazole (metronidazole, MNZ) and 2-isopropyl-1-methyl-5-nitroimidazole (ipronidazole, IPZ)] including the hydroxy metabolites of these agents [2-hydroxymethyl-1-methyl-5-nitroimidazole (HMMNI), 1-(2-hydroxyethyl)-2-hydroxymethyl-5-nitroimidazole (MNZOH), and 1-methyl-2-(2'-hydroxyisopropyl)-5-nitroimidazole (IPZOH)] in turkey and swine muscle are presented. The validation was carried out according to the requirements of the draft for the revision of Commission Decision 93/256/EC, which is expected to be adopted by the European Commission in due course. The determination of the method's performance parameters revealed decision limits (CCalpha) between 0.65 and 2.8 microg/kg for DMZ, RNZ/HMMNI, MNZ and MNZOH. Confirmatory analyses according to the requirements of the forthcoming EC decision are possible for all analytes except for IPZ and IPZOH where already the decision limits (CCalpha) were higher (5.2 microg/kg) than for the above-mentioned nitroimidazoles. The within-laboratory reproducibility and the mean recovery were in an acceptable range for all analytes.

A method was developed for the determination of the nitroimidazole compounds dimetridazole (DMZ) and ronidazole (RNZ) and their common metabolite, 2-hydroxymethyl-1-methyl-5-nitroimidazole (2-OH-M). Extracts obtained from a clean-up process using strong cation exchange (SCX) solid phase extraction (SPE) can be analysed either by high performance liquid chromatography with UV detection (HPLC-UV) or by high performance liquid chromatography with atmospheric pressure chemical ionisation mass spectrometry (HPLC-APCI-MS) as a confirmatory method. Up to 20 samples can be extracted in approximately 4 h. The HPLC-UV analysis had a limit of detection of 0.5 microgram kg-1. Validation in chicken muscle fortified at a concentration of 5 micrograms kg-1 gave recoveries of 75% DMZ, 77% RNZ and 81% 2-OH-M with RSDs of 16.4, 11.3 and 14.0%, respectively (n = 17). Validation in egg fortified at the same concentration gave recoveries of 77% DMZ, 80% RNZ and 80% 2-OH-M, with RSDs of 14.9, 22.0 and 18.2%, respectively (n = 18). The limit of detection of the HPLC-APCI-MS method was 0.1 microgram kg-1 for DMZ and RNZ and 0.5 microgram kg-1 for 2-OH-M. This method gave mean recoveries in fortified egg samples of 65% DMZ, 87% RNZ and 75% 2-OH-M with RSDs of 22, 11 and 14%, respectively (n = 10). The ratios of the peak areas of the molecular ion and a fragment ion were monitored as added confirmation of the presence of the analyte. Both the HPLC-UV screening procedure and the HPLC-APCI-MS confirmatory method have subsequently been used for the analysis of several hundred samples as part of UK surveillance programmes.

A simple, rapid liquid chromatographic (LC) method that uses UV/VIS detection has been developed for the determination in eggs of residues of the histomonostats dimetridazole (DMZ), ronidazole (RON), ipronidazole (IPR), and side-chain hydroxylated metabolites of DMZ and RON. Sample pretreatment includes an aqueous extraction, purification with an Extrelut cartridge, and acid partitioning with isooctane. An aliquot of the final aqueous extract is injected into a reverse-phase LC system; detection is performed at 313 nm. The limits of determination are in the 5-10 microgram/kg range. A UV/VIS spectrum can be obtained at the 10 microgram/kg level by using diode-array UV/VIS detection. Recoveries are between 80 and 98% with a coefficient of variation of about 5%. Some 20 samples can be analyzed per day. A side-chain hydroxylated metabolite of IPR can also be detected with this method, as demonstrated with samples from animal experiments. After a single oral dose of the drugs to laying hens, residues of the parent compound and/or the hydroxylated metabolites could be detected in eggs 5-8 days after dosing. Plasma distribution and excretion in feces were established both with and without deconjugation. DMZ and IPR were extensively metabolized to hydroxylated nitroimidazole metabolites; RON was excreted mainly as the parent compound.

A rapid method for the determination of dimetridazole and ipronidazole in feeds is described. The compounds are extracted from a borate buffer (pH 8.65) with benzene, partitioned into IN HC1, and then partitioned back into benzene from a basic aqueous phase. The benzene extract is concentrated and injected onto a nonpolar (Apiezon L) gas chromatographic column for determination by 63Ni electroncapture detection. Recoveries from feeds of various composition, spiked at 0.2 ppm with both dimetridazole and ipronidazole, ranged from 70 to 115%; for the same feeds spiked at 1 ppm or more, the recoveries were greater than 80%. Carbadox, furazolidone, levamisole, oxytetracycline, chlortetracycline, sulfamethazine, sulfaquinoxaline, arsanilic acid, piperazine, penicillin, and commonly added vitamins and minerals do not interfere. A 2-dimensional thin layer chromatographic system is presented as a means of additional identification.

Determination of drugs in foods . In foods, drugs can come from different sources: animal therapeutics, zootechnical feed supplementation or human food additives. The possible consequences arising from the presence of drugs in food are considered. The quantitative determination of drugs in feeds or in human foods must fulfil various requirements, including specificity, sensitivity, accuracy and rapidity. Spectroscopic, electrochemical and chromatographic techniques are discussed. Some examples reported are ipronidazole, pyrimethamine, diethylstilboestrol, decoquinate and chloramphenicol. The need for quality assurance in analysis is also discussed.

The 5-nitroimidazoles tinidazole (Fasigyn®), ipronidazole (Ro-7-1554), panidazole and ornidazole (Tiberal®, Ro-7-0207) in concentrations of 0.02–1 mM per liter increased the mutation frequency of Klebsiella pneumoniae, Escherichia coli K12 and Citrobacter freundii to streptomycin resistance, including streptomycin dependence, in Luria and Delbruck's fluctuation test. At low concentration (0.1 mM), the increase of the mutation frequency caused by each compound was nearly the same, i.e. 3–4 times the spontaneous mutation frequency. At higher concentrations, considerable differences between the mutagenic activities of the compounds occurred.

Approximately 4000 Large White, day-old turkey poults were used in a series of eight experiments to study the effects of dietary copper supplementation. The hens were grown to 15 or 16 weeks and toms to 24 weeks of age. Either copper as the oxide or sulfate (hydrate) at 120 ppm stimulated growth up to 10% with a concurrent reduction in feed requirement. Levels of 60 ppm were not adequate, whereas 240 ppm were not toxic. There was a greater response to copper in the presence of 4-nitrophenylarsonic acid (4-nitro) or with low protein diets. In two of the eight studies copper enhanced aortic elastin content. Incidence of aortic rupture was greater in the presence of 4-nitro and was reduced by one-half through copper supplementation. Liver copper was not altered by 60 or 120 ppm Cu; 240 ppm caused a significant increase.

1. A major water-soluble metabolite of ipronidazole (1-methyl-2-isopropyl-5-nitroimidazole) has been isolated from the faeces of bile-cannulated rats.2. This metabolite has been identified by spectroscopic and chemical methods as 2,3-dihydro-2-(2-hydroxypropyl)-3-methyl-4-nitro-1H-imidazol-5-ol.

A simple and rapid method is described for the determination of dimetridazole (DMZ) and ipronidazole (IPR) in swine feeds at various levels (0.11-110 ppm). The drugs are released from feed by prewetting with a buffer, followed by extraction with either methanol or methylene chloride, depending on the drug level; if necessary, an acid-base cleanup is used before the liquid chromatographic analysis. The analytes are separated on a C18 column and monitored at 320 nm for detection and quantitation. Recoveries of DMZ from several feed formulations averaged 108% at the 92.8 ppm level with a standard deviation (SD) of 4.00% and a coefficient of variation (CV) of 3.70%, 101% at the 11.2 ppm level with an SD of 11.9% and a CV of 11.8%, and 100% at the 0.112 ppm level with an SD of 9.27% and a CV of 9.25%. Recoveries of IPR averaged 77.1% at the 12.9 ppm level with an SD of 1.75% and a CV of 2.27%; IPR recoveries averaged 35.2% at the 0.129 ppm level with an SD of 3.39% and a CV of 9.63%.