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Salinomycin (SAL) and lasalocid (LAS) are widely used as ionophore antibiotics for coccidiosis control. However, their common use as feed additives has led to the occurrence of feed cross-contamination, which has toxic effects on non-target animals. There have been few reports on multiple-residue detection for SAL and LAS in recent years. In this study, two single-chain antibody fragments (scFvs) capable of specifically recognizing SAL and LAS were constructed. Using LAS-scFv and SAL-scFv as parent antibodies, a complete bispecific single-chain diabody (scDb) against both LAS and SAL was built using splicing by overlap extension polymerase chain reaction (SOE-PCR). In addition, the key amino acid sites and interaction energy of antibody variable regions for small-molecule recognition were preliminarily studied by homology modeling and molecular docking. Finally, IC 50 values of 12.9 and 8.6 ng/mL, with a linear range of 6.9-24.0 and 4.7-16.0 ng/mL, were obtained for LAS-scFv and SAL-scFv, respectively. An indirect competitive enzyme-linked immunosorbent assay (icELISA) method was established using scDb to obtain an IC 50 of 3.5 ng/mL for LAS and 4.1 ng/mL for SAL, which showed better sensitivity and specificity than those of the parent scFv antibodies. The recoveries of LAS and SAL in chicken liver were 89.2-92.7%(CV<4.7%) and 88.6-90.2% (CV<6.8%)), respectively., (© 2021. Springer-Verlag GmbH Germany, part of Springer Nature.)

The surface-enhanced activities of size- and shape-controlled gold nanoparticles (AuNPs) with superior chemical stability were investigated to explore a possible development of a simple and non-destructive spectroscopic method to help the regulatory agency's analytical services for rapid detection and characterization of selected antimicrobials in animal feeds. Feed samples spiked at different concentration ranges of antimicrobials were evaluated using AuNPs as a surface-enhanced Raman spectroscopy (SERS) agent. The collected SERS spectra were mathematically preprocessed for further analysis. The classification models obtained 100% predictive accuracy with zero or little misclassification. The first two canonical variables (p = 0.001) could explain >95% of the variability in preprocessed spectral data. Most chemometric models for predicting MON, DEC, and LAS concentrations showed a high predictive accuracy (r 2  > 0.90), lower predictive error (<20 mg/kg), and satisfactory regression quality (slope close to 1.0). The statistical results showed no statistically significant difference between the reference and SERS predicted values (p > 0.05). The findings and implications from the study indicate that SERS would be a powerful and efficient technique possessing a great potential serving as an excellent monitoring and screening tool for antimicrobial contaminated samples in the on-site analysis., (Published by Elsevier Ltd.)

An analytical method was developed and validated for the determination of three polyether ionophores (monensin, lasalocid, and salinomycin) in 60 samples of Brazilian Minas Frescal cheese by UHPLC-MS/MS. Linearity ranged from 1 to 8 μg kg -1 for monensin and salinomycin, and from 0.50 to 4 μg kg -1 for lasalocid. Limits of detection and quantitation were 0.50 μg kg -1 and 1 μg kg -1 , respectively, for both monensin and salinomycin, and 0.25 μg kg -1 and 0.50 μg kg -1 , respectively, for lasalocid. Recoveries were between 69% and 84% with coefficients of variation up to 16.28% for repeatability and 13.79% for intermediate precision. A total of 60 samples of Minas Frescal cheese were analysed and only monensin residues were found. Monensin was detected in 55% of the samples and quantified in 5 of them at mean levels varying from 1.00 to 1.73 μg kg -1 . The proposed method demonstrated the suitability for monitoring these substances in cheese.

A method for the simultaneous determination of three commonly used coccidiostats in chicken liver was developed, comprising a multi-residue QuEChERS (quick, easy, cheap, effective, rugged and safe) extraction step, and a liquid chromatography-ultra violet-fluorescence (HPLC-UV/FL) analysis. The QuEChERS extraction was optimized using an experimental design approach that includes a screening step to obtain the critical variables, an optimization step using multiple response surface analysis and the calculation of a desirability parameter. The optimized method was validated with fortified samples, reaching an average recovery of 91% and an overall precision of 5.5% (mean of three analytes at three levels). Limits of detection calculated on fortified samples were 20 µg kg -1 for lasalocid, 15 µg kg -1 for nicarbazin and 120 µg kg -1 for diclazuril. These values resulted at least one order of magnitude lower than the maximum allowed residue limit (MRL) of the studied coccidiostats for chicken liver., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Widespread use of coccidiostats, in spite of beneficial control of protozoan infections in poultry, implies a risk of residues in edible tissues, and there is increasing interest in the development of strategies for prevention of veterinary drugs residue in food-producing animals. The aim of this study is assigned to clarify the impact of silymarin addendum in the diet on lasalocid concentration in the liver and breast muscles from the broiler. Four groups of chickens received a feed with lasalocid at levels between 75 and 200 mg kg -1 . Other four groups received a feed with lasalocid (75-200 mg kg -1 ) plus silymarin. Significant differences of lasalocid concentrations between the liver and breast muscles were observed. Moreover, the chickens from the groups supplemented with silymarin shown significant decreases of lasalocid concentrations in the analysed tissues. The herbal substance did not counteract the ionophore in the treatment of coccidiosis and did not change biochemical parameters of blood. These findings suggest that silymarin might be used in chicken feeding in order to reduce the risk from lasalocid contamination of the broiler edible tissues.

A confirmatory multi-residue method was developed for the determination in milk of 19 coccidiostats (amprolium, arprinocid, clazuril, clopidol, decoquinate, diclazuril, ethopabate, halofuginone, lasalocid, maduramicin, monensin, narasin, nicarbazin, nequinate, robenidine, salinomycin, semduramicin, toltrazuril sulfone and toltrazuril sulfoxide). Sample preparation utilising extraction with organic solvent and clean up by SPE and freezing was found reliable and time-efficient. Optimised chromatography and MS conditions with positive and negative ESI achieved sufficient sensitivity and selectivity. Validation experiments has proven method usefulness for routine analysis of coccidiostats in milk samples. An on-farm study conducted on dairy cows fed with experimentally contaminated feed with salinomycin and lasalocid showed negligible transfer to milk. No residues of lasalocid were found in collected samples. Salinomycin was found only in 5 of 168 samples analysed, while the concentrations of salinomycin in those samples (0.119-0.179 µg kg - 1 ) was significantly below the limit of salinomycin in milk set by European Union legislation. Such low concentrations of both coccidiostats cannot be explained by conjugation during dairy cows' metabolism, as shown by experiments with enzymatic hydrolysis.

Ionophore antimicrobials are heavily used in the livestock industries, both for preventing animal infection by coccidia protozoa and for increasing feed efficiency. Ionophores are excreted mostly unmetabolized and are released into the environment when manure is land-applied to fertilize croplands. Here, an analytical method was optimized to study the occurrences of five ionophore residues (monensin, lasalocid, maduramycin, salinomycin, and narasin) in dairy manure after solid-liquid separation and further treatment of the liquid manure by a membrane-based treatment system. Ionophore residues from the separated solid manure (dewatered manure) and suspended solids of manure slurry samples were extracted using ultrasonication with methanol, followed by sample clean-up using solid phase extraction (SPE) and subsequent analysis via liquid chromatography-tandem mass spectrometry (LC-MS/MS). The use of an ethyl acetate and methanol (1:1 v:v) mixture as an SPE eluent resulted in higher recoveries and lower method quantitation limits (MQL), when compared to using methanol. Overall recoveries from separated solid manure ranged from 73 to 134%. Liquid manure fractions were diluted with Nanopure™ water and cleaned up using SPE, where recoveries ranged from 51 to 100%. The developed extraction and LC-MS/MS methods were applied to analyze dairy manure samples subjected to an advanced manure treatment process involving a membrane-based filtration step (reverse osmosis). Monensin and lasalocid were detected at higher concentrations in the suspended solid fractions (4.40-420 ng/g for lasalocid and 85-1950 ng/g for monensin) compared to the liquid fractions (

Lasalocid is a veterinary ionophore antibiotic used for prevention and treatment of coccidiosis in poultry. It is excreted from the treated animals mostly in its active form and enters the environment with the use of contaminated manure on agricultural land. To properly assess the risk that lasalocid poses to the environment, it is necessary to know its environmental concentrations as well as the rates of its degradation in manure and dissipation in soil. These values are still largely unknown. A research was undertaken to ascertain the rate of lasalocid degradation in manure under different storage conditions (aging in a pile or composting) and on agricultural soil after using lasalocid-contaminated manure. The results have shown that there is considerable difference in lasalocid degradation between aging manure with no treatment (t1/2=61.8±1.7 d) and composting (t1/2=17.5±0.8 d). Half-lives in soil are much shorter (on average 3.1±0.4 d). On the basis of the measured concentrations of lasalocid in soil after manure application, we can conclude that it can potentially be harmful to soil organisms (PEC/PNEC ratio of 1.18), but only in a worst-case scenario of using the maximum permissible amount of manure and immediately after application. To make certain that no harmful effects occur, composting is recommended., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

A method for the determination of lasalocid, an antibiotic and coccidiostat, in grease and fat is described. The manufacture of lasalocid produces a grease-like residue as a waste byproduct. Recently this byproduct has been shown to have been illegally introduced into the animal feed chain. Therefore, a quantitative and confirmatory procedure to analyse for lasalocid in this matrix is needed. A portion of grease/oil sample was extracted into hexane-washed acetonitrile, and a portion of the extract was then applied to a carboxylic acid solid-phase extraction (SPE) column for concentration and clean-up. The SPE column was washed with additional hexane-washed acetonitrile and ethyl acetate/methanol, after which lasalocid was eluted with 10% ammoniated methanol. The eluate was evaporated to dryness, redissolved in (1:1) acetonitrile-water and filtered through a PTFE syringe filter. Confirmation and quantitation of lasalocid in the final extract employed a triple quadrupole LC-MS/MS. The method was applied to grease and oil samples containing from 0.02 to 34,000 mg kg(-1) of lasalocid.

Campylobacter bacteria are foodborne pathogens that can colonize the gut of food animals. Limited in their ability to ferment sugars, Campylobacter can derive energy for growth via amino acid catabolism. The objectives of the present studies were to test whether supplemental distillers grains containing high amounts of rumen-undegradable intake protein or supplemental lasalocid may, by promoting amino acid flow to the lower bovine gut, increase intestinal carriage of Campylobacter. In study one, 10 steers (5 per treatment) were adapted to diets formulated to achieve 0 or 30% dried distillers grains. After an initial 14-day adaptation to the basal diet, control and treated steers were fed their respective diets for 23 days, after which time they were fed supplemental lasalocid for an additional 8 days, followed by a 5-day withdrawal. In study two, 24 steers preacclimated to a basal diet were adapted via 3-day periodic increases to dietary treatments formulated to achieve 0, 30, or 60% wet corn distillers grains with solubles. Analysis of Campylobacter bacteria cultured from duodenal and fecal samples in study one and from fecal samples in study two revealed no effect of dried distillers grains or wet corn distillers grains with solubles on the prevalence or concentrations of duodenal or fecal Campylobacter. The results from study one indicated that colonized steers, regardless of treatment, harbored higher Campylobacter concentrations when transitioned to the basal diet than when coming off pasture. Campylobacter carriage was unaffected by lasalocid. These results provide no evidence that feeding distillers grains high in rumen-undegradable intake protein or supplemental lasalocid contributes to increased intestinal carriage of Campylobacter in fed cattle.

A confirmatory method for the determination of 11 regulated coccidiostats including the ionophore antibiotics lasalocid, maduramicin, monensin, narasin, salinomycin, and semduramicin and the chemical coccidiostats decoquinate, diclazuril, halofuginone, nicarbazin, and robenidine in animal feed was developed and validated. The procedure was intended for the identification and quantification of the coccidiostats at concentrations relating both to the unintentional carryover as stated in Regulation 574/2011 and to the authorized levels in target feed. The analytes were determined by LC/MS/MS in the positive or negative electrospray ionization mode. The method performance characteristics were estimated in the relevant application field from 0.003 to 200 mg/kg. Validation criteria of linearity, specificity, trueness, precision, LOD, and LOQ along with measurement uncertainty were estimated for all analytes. Absolute and relative matrix effects were also studied. The results proved that the method performance was satisfactory, and it was successfully applied to carryover control by analyzing 165 feed samples collected within regulatory monitoring plans. Finally, since the carryover phenomenon in feed may result in the presence of residues in food products of animal origin, a survey has been carried out on the occurrence of coccidiostats in 167 eggs and animal muscles.

The usefulness of Tb(4)O(7) nanoparticles (NPs) as analytical reagents using sensitized luminescence as a detection system is described for the first time, and the results obtained are compared with those obtained using Tb(III) ions. Two drugs used in veterinary practice, namely, lasalocid (LAS) and salicylate (SAL), have been chosen as model analytes to carry out this study. The experimental conditions for these systems have been optimized, and their analytical features were obtained. The detection limits obtained for LAS and SAL using Tb(4)O(7) NPs were 1.0 and 4.0 ng mL(-1), respectively, which were comparable to those obtained using Tb(III) ions: 1.8 and 1.0 ng mL(-1), respectively. However, precision data, with relative standard deviation values in the range 2.3-3.8% using the NPs and 3.5-6.5% using Tb(III) ions, were slightly better for LAS with Tb(4)O(7) NPs. The practical analytical usefulness of Tb(4)O(7) NPs as luminescent reagents has been shown by performing the determination of LAS in tap water, feed premix, and egg samples, obtaining recoveries in the range of 80.0-105.0%.

The single-laboratory validation (SLV) of an LC-MS/MS method for determination and confirmation of two ionophores, narasin and monensin, in animal tissues is described. The data demonstrated linearity of matrix-matched calibration curves using a weighted (1/x) regression and selectivity of the method for narasin and monensin in the presence of lasalocid, salinomycin, maduramycin, nicarbazin, and sulfadiazine. Recoveries varied from 86.2 to 103.5% for narasin and 89.1 to 105.1% for monensin. Intertrial repeatability precision [relative standard deviation of repeatability (RSDr)] varied from 3.9 to 13.8% for narasin and 3.3 to 16.3% for monensin in fortified tissue. Precision of the method was verified in incurred tissues. The LOQ of the method was validated and ranged from 0.45 ng/g in milk, to 4.0 ng/g in chicken fat, but was 0.75 ng/g for most tissues. Two confirmatory ions for each analyte were examined across all matrixes, resulting in estimated false-negative rates of 0.00% (95% confidence interval of 0.00-0.68%) for monensin ions (540 samples) compared to the U.S. and European Union (EU) acceptance criteria. The confirmatory ions for narasin demonstrated 0.00% false-negative rates (95% confidence interval of 0.00-0.58%) when compared to either the U.S. or EU criteria in 630 samples. The method was robust when small changes in method parameters were made and stability of fortified tissues, extracts, and calibration solutions were estimated. The data satisfy the requirements of the AOAC Stakeholder Panel on Veterinary Drug Residue for SLV studies, and the method was adopted Official Methods of Analysis First Action 2011.24 by the AOAC Expert Review Panel on Veterinary Drug Residues.

A simple and highly sensitive LC/MS method was developed for the simultaneous determination of six ionophores--lasalocid, monensin, laidlomycin, maduramycin, salinomycin, and narasin--in feed. The procedure involved extraction of 1 g of feed with 4 mL of methanol-water (9 + 1, v/v) by shaking on a platform shaker for 45 min. After centrifugation, the extracts were diluted with methanol-water (75 + 25, v/v) and analyzed without any cleanup. The analysis was performed on a Betasil C18 column (150 x 4.6 mm id, 5 pm particle size) connected to an LC/MS system operated in the atmospheric pressure chemical ionization (APCI) mode. We believe this to be the first method that uses the APCI mode for the analysis of ionophores. The mobile phase consisted of 50 mM ammonium acetate as solvent A and acetonitrile-methanol (7 + 3, v/v) as solvent B in a gradient run. Excellent recoveries of 81-120% were found for all compounds at fortification levels of 1-200 microg/g, with RSD < or =15% (except 17% for maduramycin at 2 and 5 microg/g, and 16% for salinomycin at 1 microg/g). At 0.5 microg/g, recoveries of 87-119% were obtained, with RSD < or =20%. However, recovery of lasalocid was 133% and salinomycin 79% in sow and horse feed, respectively. Average RSD values of lasalocid and salinomycin were 22 and 21%, respectively. Finally, proficiency test samples analyzed with the method demonstrated favorable agreement with the certified values.

Recent legislation has addressed the unavoidable carry-over of coccidiostats and histomonostats in feed, which may lead to the presence of residues of these compounds in eggs. In this study, laying hens received cross-contaminated feed at a ratio of 2.5%, 5% and 10% of the therapeutic dose of monensin and lasalocid for broilers. The eggs were collected during the treatment and depletion period and were analysed using liquid chromatography-tandem mass spectrometry. The different egg matrices were separated and analysed during the plateau phase. High lasalocid concentrations, which exceeded the maximum residue level, and low monensin concentrations were found in whole egg. Plateau levels were reached at days 7-9 for lasalocid and at days 3-5 for monensin. For lasalocid, the highest concentrations were measured in egg yolk; residue concentrations in egg white were very low.

Lasalocid is a polyether ionophoric coccidiostat used for the prevention of coccidiosis in poultry at a prescribed concentration and during a certain time interval. Due to a public health concern about the presence of coccidiostat residues in poultry, the aim of the present study was to determine the levels of lasalocid residues in the edible tissues of broiler chickens (breast muscle, thigh muscle, heart, liver, gizzard, kidneys and skin/fat) fed commercially produced feed containing 100 mg kg⁻¹) of lasalocid in complete feed throughout the 5-day withdrawal period (WP). The residues were investigated by liquid chromatography coupled with electrospray ionisation (ESI) tandem mass spectrometry (MS/MS) with triple quadrupole. The limit of detection (LOD) and the limit of quantification (LOQ) of the method were 0.47 and 1.44 µg kg⁻¹, respectively. The average recovery based on the matrix-fortified calibrations for chicken tissues ranged between 79% and 98%. Lasalocid was found to accumulate in the liver, followed by the heart, skin/fat, kidneys, thigh muscle and gizzard. The lowest concentrations of lasalocid residues were found in the breast muscle. On day 5 of the WP, residue concentrations of lasalocid did not decline below the LOQ of the method, but were far below the maximum residue level (MRL) established for lasalocid in poultry from 20 to 100 µg kg⁻¹ by European Commission Regulation (EU) No. 37/2010. The results confirmed that the WP established for lasalocid is sufficient to ensure the decline of its residues in the tissues of broiler chickens to the safe residue level.

Lasalocid is an ionophore antibiotic extensively used as a coccidiostat in poultry production. Lasalocid should not be fed to egg-laying hens as it accumulates in the eggs, and residues have often been found in eggs. Other ionophores are toxic to humans, but the exact level of lasalocid toxicity to humans has not been established. Approximately 250 egg samples were analysed for lasalocid each year from the 10 billion eggs consumed annually in the UK. A census of the 32 Scottish Local Authority Environmental Health Departments assessed awareness of lasalocid residues in eggs, and the results indicated that awareness of lasalocid was very low and no local authorities tested for lasalocid. The example of lasalocid revealed weaknesses in the current sampling regime surrounding foods of animal origin. Conclusions are drawn that central government should raise awareness within local authorities and provide financial support on local authority sampling to achieve proper representation.

Lasalocid is a highly atypical polyether ionophoric antibiotic, firstly because it contains a type of aromatic ring normally associated with fungal polyketides, and secondly because the formation of its tetrahydropyran ring appears to contravene Baldwin's rules, which predict the kinetically preferred routes for cyclisation reactions in organic chemistry. The lasalocid biosynthetic gene cluster has been cloned from Streptomyces lasaliensis, and the las locus (73,533 bp) was found to contain seven modular polyketide synthase (PKS) genes, including all the activities necessary for the synthesis of the aromatic moiety. Specific deletion from the gene cluster of the flanking lasC gene, which is predicted to encode a flavin-linked epoxidase, abolished production both of lasalocid and of the minor cometabolite iso-lasalocid without leading to accumulation of an identifiable intermediate; this suggests that oxidative cyclisation to form the polyether rings takes place on the PKS before release of the full-length polyketide product. Meanwhile, a mutant in which the adjacent epoxide hydrolase lasB had been deleted produced iso-lasalocid only. Iso-lasalocid differs from lasalocid in the replacement of the tetrahydropyran ring by a tetrohydrofuran ring and represents the kinetically favoured product of cyclisation. The LasB epoxide hydrolase is therefore directly implicated in control of the stereochemical course of polyether ring formation during lasalocid biosynthesis.

A liquid chromatographic (LC) method for the analysis of lasalocid sodium in premixes, complete animal feeds, and trace-level feeds was collaboratively studied. The method employs a 0.5% HCI acidified methanol extraction followed by 20 min sonication in a water bath heated to 40 degrees C. Samples are then shaken on a mechanical shaker for 1 h and stored overnight, followed by an additional 10 min shaking the following morning. Sample extracts are diluted if necessary with extractant, filtered, and injected onto an LC system. Determination of all lasalocid homologs is by reversed-phase LC with fluorescence detection at 314 nm excitation and 418 nm emission. Eight samples of drug premixes, medicated feeds, and mineral supplements, along with 2 samples for trace-level analysis were sent to 20 collaborators in the United States, Canada, and The Netherlands. Study data were returned by 17 laboratories. Two additional supplemental trace-level samples and a blank feed were provided to 15 of the collaborating laboratories, and test data were received from all 15 participants. For the drug premixes, medicated feeds, and mineral supplements, RSDr values (within-laboratory repeatability) ranged from 1.2 to 19.9%, RSDR values (among-laboratory reproducibility) ranged from 3.4 to 32.3%, and HorRat values ranged from 0.35 to 3.73. For the trace-level samples, only lasalocid A, the predominant homolog comprising > 90% of the sum of all homolog peak area, was quantified. All laboratories correctly identified the analyte. Although some instrument response was reported by a number of laboratories for the blank feed, all but one laboratory's results were well below the 1 mg/kg limit of quantification. RSDr values for the initial 2 trace-level samples were excessive, ranging from 51.6 to 64.4%. RSDR values ranged from 51.6 to 75.7%, and HorRat values ranged from 3.6 to 4.0. Data for the initial trace-level samples indicated that the test samples were improperly prepared to ensure homogeneity, and a new set of supplemental samples was provided to collaborators, with significantly improved results. RSDr values for the 2 supplemental trace-level samples ranged from 1.6 to 2.5%, RSDR values ranged from 5.6 to 9.2%, and HorRat values ranged from 0.43 to 0.62.

Monensin and lasalocid are polyether ionophores commonly used in the beef and poultry industries for the prevention of coccidial infections and promotion of growth. These ionophores can exhibit higher toxicity than many other antibiotics; thus, evaluating their fate in the environments associated with concentrated feed operations is important. Sorption of monensin and lasalocid was measured in eight soils of varying physiochemical composition. Organic carbon-normalized sorption coefficients (log Koc) ranged from 2.1 to 3.8 for monensin and from 2.9 to 4.2 for lasalocid and were inversely correlated to equilibrium soil-solution pH. Degradation of lasalocid and monensin in two contrasting soils with and without manure amendment was measured in moist soils at 23 degrees C and 0.03 MPa moisture potential. The half-life of both compounds in the fresh nonsterile soils was less than 4 d, for which monensin degraded slightly faster than lasalocid. Fresh liquid manure amendments did not significantly alter degradation of either compound. Based on parallel 60Co-sterilized soil experiments, some abiotic degradation of monensin was apparent, whereas lasalocid only degraded in the presence of microbes. Analysis of beef-derived lagoon effluent used for irrigation confirmed that monensin can be present at low-ppb to low-ppm concentrations in the aqueous and suspended solids fractions, respectively; however, subsequent analysis of drainage water in a nearby ditch suggested that attenuation by soil after land application will greatly reduce the amount entering surface waters.

A published confirmatory method for the quantitative determination of four ionophoric coccidiostats (lasalocid, monensin, salinomycin and narasin) in eggs and broiler meat has been further developed. It is proposed for replacement of liquid chromatography methods previously used in analysis of ionophoric coccidiostats. The samples were extracted with acetonitrile and purified on a silica solid phase extraction column. Purified samples were analysed by liquid chromatography-mass spectrometry and the method, was validated according to the Commission Decision 2002/657/EC. The validation parameters selectivity, linearity, specificity, precision, recovery, decision limit (CCalpha) and detection capability (CCbeta) were determined. The recoveries of coccidiostats analysed ranged from 64-99% in eggs and 62-100% in broiler meat. CCalpha varied from 0.8-1.4 microg/kg in eggs and from 1.5-2.5 microg/kg in broiler meat. CCbeta varied from 0.9 microg/kg to 2.0 microg/kg in eggs and from 1.7-3.2 microg/kg in broiler meat.

A survey of the presence of residues of anticoccidials was performed. Three hundred and twenty egg samples, purchased in eight different European countries, were analysed for the presence of nine different compounds: dimetridazole, diclazuril, halofuginone, robenidine, nicarbazin, narasin, salinomycin, lasalocid and monensin. Analyses were performed by LC-MS/MS. Of the samples analysed, 114 (35.6%) contained one or more of the nine anticoccidials in concentrations ranging from 0.1 to 63 microg kg-1. Salinomycin and lasalocid account for more than 60% of all positive samples. Almost 90% of all positive samples contained less than 2 microg kg-1. Results were put into perspective of the farming method and country of origin.

The gas phase stabilities of Group I metal complexes of the polyether ionophore antibiotics lasalocid and monensin were investigated by collision induced dissociation mass spectrometry. Electrospray ionization was used with a triple quadrupole mass spectrometer for the determination of threshold dissociation energies upon application of increasing collision energies. Various data analysis techniques for the determination of dissociation energies are discussed to assess the most suitable method for determining the stabilities of the ionophore-metal complexes studied here. In all cases only the relative stabilities of different complexes may be obtained by the method presented in this study, which does not assess absolute gas phase dissociation energies. Correction factors have been applied, however, to account for the energy conversion during collisions of different metal complexes and the varying degrees of freedom of different sized ligands, allowing for the comparison of the stabilities of different ionophores with like-metals. The measured threshold dissociation energies were compared with respect to the ionic radius of the metal cation, revealing a maximum stability for the K+ complexes of both lasalocid and monensin. A striking decrease in the stabilities of the Rb+ and Cs+ complexes was observed and is believed to be related to a decreasing degree of coordination that the ionophores can accomplish with the larger metals.

A sensitive and selective liquid chromatographic tandem mass spectrometric method (LC/MS/MS) for the simultaneous detection of the ionophoric coccidiostats narasin, monensin, lasalocid and salinomycin in whole eggs has been developed. A very simple sample preparation consisting of an extraction with an organic solvent was carried out. Sample extracts were injected into the LC/MS/MS system on a C18 column and an isocratic elution was performed. Nigericin was used as internal standard. The precursor ions produced by electrospray positive ionisation were selected for collisional dissociation with argon into product ions. Validation of the methods was performed based on Commission Decision 2002/657/EC.1 CC(alpha) was found to be 1 microg/kg for all four compounds. Monitoring of Belgian egg samples in 2004 revealed that residues of salinomycin, lasalocid and monensin could be found., (Copyright 2005 John Wiley & Sons, Ltd.)

Monoclonal antibodies (MAbs) against lasalocid and semduramicin were prepared using keyhole limpet hemocyanin conjugates for the immunization of mice. With these MAbs, we developed quantitative enzyme-linked immunosorbent assay (ELISA) methods for lasalocid and semduramicin. The ELISAs were quantitative in the ranges of 0.1-50 ng/mL for lasalocid and 0.05-12.5 ng/mL for semduramicin, and showed 50% inhibition concentrations of 1.2 ng/mL for lasalocid and 0.5 ng/mL for semduramicin. The coefficient of variations (CV%) of lasalocid were 0.3-4.4% for intra-assay and 0.5-5.1% for inter-assay and those of semduramicin were 0.1-4.6% for intra-assay and 0.3-5.2% for inter-assay. The detection limits for lasalocid and semduramicin were 10 ng/g and 5 ng/g in chicken liver and muscle, respectively. Based on the immunochromatographic method, rapid test kits for lasalocid and semduramicin were also developed. With these kits, the detection limits of lasalocid were 50 ng/mL for standard solution and 125 ng/g for chicken muscle, and those of semduramicin were 10 ng/mL for standard solution and 100 ng/g for chicken muscle.

A rapid, accurate, and selective method was developed for the forensic determination of ionophore antibiotics in animal feeds. A simple extraction procedure and liquid chromatography/tandem mass spectrometry (LC/MS/MS) in the selected reaction monitoring (SRM) mode were used for rapid identification and confirmation of monensin and lasalocid in feed samples and for quantitation of monensin. Extracts from a homogenous portion of ground feeds were prepared using liquid-solid extraction and liquid-liquid extraction techniques. Feed extracts were further purified by a simple defatting and solvent wash step and then concentrated to dryness. Feed extract residues were reconstituted in 1 mL LC mobile phase and a 2 microL aliquot injected into the SRM LC/MS system. The latter system used a C18, 100 x 2.0 mm, LC column coupled to a PE-Sciex API 2000 tandem triple quadrupole mass spectrometer equipped with a TurbolonSpray LC/MS interface. Feed samples were extracted and analyzed for the determination of monensin and lasalocid within a couple of hours. Control feed samples fortified with monensin at concentrations from 50 ppb to 5 ppm provided a linear response and calibration curve across this range with a correlation coefficient of 0.996.

Methodology has been developed for the determination of lasalocid in analytically 'difficult' matrices such as processed and spiced foods. The procedure was based on an existing silica-based solid-phase extraction (SPE) clean-up to which was added a novel NH2 SPE step before HPLC with fluorescence detection. Use of the additional step enabled the determination of lasalocid in matrices such as baby food, meat pies ('pasties'), etc. Analysis of these matrices was not possible using the standard clean-up on its own. Chromatography showed a massive reduction in the amount of co-extractives and interferences. Validation data were obtained down to the 10-40 mg kg(-1) level for a range of products. Recoveries ranged from 74% at 10 microg kg(-1) for pork sausages to 96% at 40 microg kg(-1) for meat pies.

A liquid chromatographic/mass spectrometric (LC/MS) electrospray confirmation method has been developed to confirm 4 ionophores (monensin, lasalocid, salinomycin, and narasin) in a variety of animal feeds using a single quadrupole mass spectrometer. The sodium ions of these compounds are dominant in the electrospray mass spectrum. Using optimized "in-source" collision induced dissociation, characteristic fragment ions seen previously using MS/MS can be observed. The drugs were extracted from the feed matrix using hexane-ethyl acetate and isolated using a silica solid-phase extraction cartridge. These ionophores were confirmed in both medicated feeds and nonmedicated feeds fortified with these drugs at the 1-50 ppm level. In addition, this method was used to confirm residues of monensin in a nonmedicated feed that was collected from a feed mill immediately after the production of a similar feed that was medicated with high levels of monensin.

A simple and rapid method has been developed for the extraction of lasalocid from chicken muscle, eggs and liver and kidney from chicken, pig, sheep and calf. This method allows the screening of a large number of samples, i.e. 30-40 within a working day, and has an overall analysis time of 90 min. Lasalocid standard solution can be detected at 1 ng ml-1 by both HPLC-fluorescence (HPLC-F) and LC-MS-MS; the limit of quantification in fortified samples by the described method is 1 ng g-1. Results show good repeatability and mean 'spiked' recoveries by HPLC-F in the range of 10 to 200 ng g-1 (ppb) of 103, 87, 107, 97, 97, 103, 93, 109 and 100% in chicken muscle, chicken liver, egg, pig liver, pig kidney, sheep liver, sheep kidney, calf liver and calf kidney, respectively. For concentrations between 1 and 6 ng g-1 of spiked lasalocid in eggs and chicken liver by LC-MS-MS, the average recoveries were 76 and 59%, respectively.

A rapid and very effective analytical procedure for the simultaneous determination of four polyether antibiotics (PEs) lasalocid, monensin, salinomycin and narasin in poultry feeds was tested. PEs were extracted from samples using methanol and without and clean-up derivatized with 2,4-dinitrophenylhydrazine (DNP) in an acidic medium at 55 degrees C. The derivatization mixture was analyzed directly on an ODS column (150 x 4.6 mm, 5 microns) with methanol--1.5% aqueous acetic acid (90:10, v/v) as eluent and UV detection was carried out at 305/392 nm. The recoveries of the PEs from spiked samples were 85-100% with RSDs of 4-10% in a concentration range of 50-150 mg/kg.

We undertake the determination of a wide range of veterinary drug residues in a range of animal products. Various screening analyses are employed, followed by HPLC-API (atmospheric pressure ionisation)-MS for the unequivocal confirmation of significant positives. EU legislation for the use of GC-MS as a confirmatory technique requires the successful monitoring of at least four diagnostic ions and although no such requirement exists for HPLC-MS confirmation, a similar requirement would seem appropriate. Until recently, reports describing the electrospray MS confirmation of residues of the polyether ionophores have been based on monitoring one or two ions. We have found that the addition of ammonium acetate to the HPLC mobile phase, in conjunction with 'cone voltage' or 'skimmer' assisted fragmentation, is a convenient way of producing additional diagnostic ions from polyether ionophore compounds, without compromising the overall sensitivity. Results for lasalocid, the most widely used compound, are presented. Electrospray MS data and acquisition parameters for lasalocid, monensin, narasin and salinomycin are described. The advantage of this analytical approach is that it may be used to generate confirmatory data using a single quadrupole MS system, without the need for advanced MS instrumentation, e.g., MS-MS.

Monensin, salinomycin and narasin were detectable in six, two and one, respectively, out of 161 eggs surveyed in Northern Ireland in 1994. In all cases, the concentrations detected were less than 2.5 ng/g. Lasalocid was detectable in 107 eggs at concentrations ranging from 0.3 to 129 ng/g. Cross-contamination of unmedicated feeds with monensin during feed manufacture (up to eight batches of unmedicated feed contaminated with monensin) was similar to that previously observed for lasalocid (up to nine batches contaminated). Therefore differences in the incidence in eggs could not be explained by differential carry-over during feed manufacture. In a feeding trial it was shown that the relative ability of monensin, salinomycin and lasalocid to accumulate in eggs was in the ratio 0.12:3.3:63 ng/g egg per mg/kg feed, respectively. This indicated that the potential for monensin and salinomycin to cause residues in eggs was very low, by comparison with lasalocid. In 1995, a granular formulation of the lasalocid premix was introduced into the United Kingdom that decreased the carry-over of this drug from medicated to unmedicated feed. Six months after the introduction of this formulation, the incidence of lasalocid residues in eggs (21%) was lower than that found (66.5%) in an earlier survey (1994) carried out, and published, by this laboratory.

Lasalocid is a coccidiostat licensed for use in poultry, but not for use in egg-laying birds. Lasalocid residues were determined in an egg sample from each of 161 egg producers in Northern Ireland using liquid chromatography-electrospray ionization mass spectrometry, following reports from the Veterinary Medicines Directorate concerning the incidence of lasalocid residues in the United Kingdom. Approximately 66% of the eggs contained lasalocid residues at concentrations in excess of 0.3 ng/g. There was no apparent difference in the incidence of lasalocid residues between free range and battery eggs. Carry-over of lasalocid from medicated to unmedicated batches of both premix and feed, during milling processes, was identified as a possible cause of contamination. Subsequently, egg-laying birds were fed meal containing a range of lasalocid concentrations, similar to those found as a result of unintentional contamination at a feed mill (0.1-5.0 mg/kg). The concentrations of lasalocid, measured in their eggs, were similar to that found in the survey. Lasalocid persisted in eggs for 10 days after withdrawal of medicated feed and replacement with lasalocid-free feed.

A liquid chromatographic method for determining residues of ionophores in bovine, porcine, and avian tissues is described. Tissues were extracted with iso-octane-ethyl acetate and the extracts purified on silica solid-phase extraction columns. Monensin, narasin and salinomycin were detected by UV absorbance following post-column derivatization with vanillin, and lasalocid was detected underivatized using fluorescence. In muscle, kidney and fat, all drugs were determined from a single sample extract. In liver, a separate extraction was done to determine lasalocid. The detection limit for lasalocid, narasin and salinomycin was 5 ppb; for monensin it was 2 ppb. Average recoveries were: lasalocid-71.0%; monensin-94.3%; salinomycin-97.2%; narasin-94.1%.

A method is presented for the determination of the polyether ionophore, lasalocid, in eggs. Samples are extracted under acidic conditions using acetonitrile and the extracts are cleaned up using a simple liquid-liquid extraction step. Lasalocid is detected and quantified using liquid chromatography-electrospray mass spectrometry on a quadrupole bench-top instrument. The technique is highly sensitive, with a detection limit of about 0.5 ng g-1 of lasalocid in homogenized egg samples.

Polyclonal antibodies against the coccidiostat, lasalocid, were raised in sheep. The antisera were applied in an enzyme-linked immunosorbent assay (ELISA) for lasalocid, which was validated for chicken serum, liver and muscle. Bridge homology in the ELISA was overcome by absorbing unspecific antisera onto a conjugate between salinomycin and chicken serum albumin, which was immobilized onto Biosilon beads. The described assay is highly specific for lasalocid, and is capable of detecting lasalocid at concentrations of less than 0.15 ng/g, depending on the tissue. Residual concentrations of lasalocid in broilers fed medicated feed containing 90 mg/kg lasalocid were measured during a 7 day withdrawal period. The half-life of lasalocid was 11, 36 and 41 h in serum, liver and muscle, respectively. Lasalocid concentrations in liver were approximately 10 ng/g 7 days withdrawal of the medicated feed.

A high-performance liquid chromatographic (HPLC) method for the determination of the ionophore coccidiostat lasalocid in poultry muscle and eggs was developed. The drug was extracted from tissue with acetonitrile. The extract was partitioned between saturated salt and carbon tetrachloride and the organic layer evaporated to dryness. Clean-up was by solid-phase extraction on a silica column. HPLC analysis was carried out on either a polymeric PLRP-S or a porous graphitic carbon Hypercarb column with a basic mobile phase and fluorescence detection with excitation at 310 nm and emission at 420-430 nm. Average recoveries from poultry muscle at the 0.002, 0.010 and 0.050 mg kg-1 levels were 65.7, 72.0 and 77.9%, respectively. Average recoveries from egg at the 0.010 and 0.100 mg kg-1 levels were 76.2 and 76.4%, respectively.

A collaborative study has been completed on a spectrofluorometric method for determining lasalocid sodium in finished poultry feeds. After a brief treatment of feed samples with pH 4.7 buffer, the drug is extracted with ethyl acetate. The ethyl acetate extract is further cleaned up by treatment with HCI and NaOH and quantitatively measured by spectrofluorometry. Nonspecific fluorescence is corrected for via the lasalocid-boric acid complex. The method is rapid, sensitive, and free of interference. A number of other feed additives, including monensin and ethoxyquin, do not interfere. Nine collaborators participated in the study and agreement between laboratories was satisfactory. The average recoveries of lasalocid sodium from the mash feeds added at levels of 0.0064, 0.0080, and 0.0096% were 100, 98, and 99%, respectively, and the corresponding coefficients of variation were 12.8, 8.2, and 8.0% (all results included). The method has been adopted as official first action.

The development of a tissue residue method for an antibiotic is dependent on the history of the overall problem; the current requirements that must be met for regulatory approval; the chemistry of the compound; its physical/chemical parameters; and its microbiological spectrum for the best possible fit of all of these to ensure a procedure that will work reliably in the originator's laboratory and to the same degree of effectiveness in anyone's laboratory. A note is made of those factors that must be included as controls on the procedure and those experiments that must be included to differentiate matrix from method influences.

Methods for the analysis of polyether antibiotics in animal tissues and fluids are described. For monensin and nigericin, only methods based on bioautography are available. For lasalocid, in addition to TLC bioautography for quantitation in chicken skin and fat, LC methods based on fluorescence detection have been developed for quantitation in animal blood, milk, liver, skin, and fat. In addition, a confirmatory method for lasalocid is described; this is based on purification by LC followed by silylation and pyrolysis gas chromatography-positive chemical ionization mass spectrometry.

The microbiological cylinder plate assay method for lasalocid sodium reported earlier was studied in a slightly modified form by 7 laboratories. Assay values paralleled recovery values and all data were corrected by the corresponding daily recovery factor for the feed type. Agreement between laboratories for mashes and pellets containing 0.0064, 0.0080, and 0.0096% lasalocid sodium was satisfactory. Coefficients of variation for mashes were 14.6, 9.8, and 10.7% and for pellets, 15,2, 10.9, and 15.6%. The average of the values found for the 3 mashes was 99.4% of the calculated value and for the 3 pellets, 86.5%. The method has been adopted as official first action.

An analytical method has been developed that can reliably measure the metabolic marker residue of lasalocid. The method monitors this marker residue in food samples to ensure that the total residue of toxicological concern is not being exceeded. Interlaboratory studies of the liquid chromatographic determinative procedure and the gas chromatographic/mass spectrometric confirmatory procedure for lasalocid sodium at the 0.7 ppm level and higher were successful.

A liquid chromatograph was interfaced to an atomic absorption spectrometer for the detection and quantitation of maduramicin in feed matrixes at the 1-8 ppm level. Ionophores in general form strong 1:1 products with various metal cations, yielding complexes that are insoluble in water but very soluble in organic solvents. Maduramicin, a carboxylic, polyalcohol, polyether antibiotic, is labeled with the sodium cation and analyzed by atomic absorption spectroscopy (AAS). The lower limit of detection is approximately 100-200 ng maduramicin sodium salt. Feeds containing 1-8 ppm maduramicin are extracted with acetone, the extract is passed through an alumina column, the column is eluted with acetonitrile-water (90 + 10), and the eluate is analyzed for maduramicin by liquid chromatography-AAS after concentration and conversion of maduramicin to the sodium salt. Recoveries of maduramicin averaged 89.5%. Liquid chromatography with AAS detection has been shown to be a sensitive and highly specific technique for the determination of ionophores in general and maduramicin in particular.

A GLC method is presented for the determination of lasalocid and its bromo analog. The method is based on the quantitative trimethylsilylation of the compounds without any molecular cleavage, followed by chromatography on a nonpolar silicone column. Silylation was carried out directly without any extraction or prior cleanup, despite the complexity of the dosage forms. This procedure was used for the assay of pure substances, pellets, premixes, experimental ampul solutions, and mycelial filter cakes. The results were in good agreement with data obtained by microbiological procedures.

The U.S. Food and Drug Administration (FDA) sponsored an interlaboratory study of a liquid chromatographic determinative procedure for lasalocid sodium in chicken skin with adhering fat. Four laboratories analyzed 35 dosed tissue samples and 82 fortified tissue samples containing lasalocid at levels ranging from 0.1 to 0.6 ppm. Samples were homogenized with acetonitrile, washed with hexane, and partitioned into the mobile phase prior to analysis liquid chromatography with fluorescence detection. The results of the interlaboratory study showed good reproducibility for fortified samples. Fortification levels, average recoveries, and interlaboratory percent coefficients of variation were as follows: 0.6 ppm, 0.57 ppm, and 9.7; 0.3 ppm, 0.25 ppm, and 9.1; and 0.15 ppm, 0.14 ppm, and 7.0, respectively. Data for analysis of the dosed tissue also showed good agreement among the laboratories.