Detection of salinomycin and lasalocid in chicken liver by icELISA based on functional bispecific single-chain antibody (scDb) and interpretation of molecular recognition mechanism.

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, IC50 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 IC50 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.

A rapid and convenient screening method for detection of restricted monensin, decoquinate, and lasalocid in animal feed by applying SERS and chemometrics.

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 (r2 > 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.

Polyether ionophores residues in Minas Frescal cheese by UHPLC-MS/MS.

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.

Multiple response optimization of a QuEChERS extraction and HPLC analysis of diclazuril, nicarbazin and lasalocid in chicken liver.

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.

The decrease of lasalocid residue in the edible tissues by silymarin supplementation of chicken diet.

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.

Coccidiostats in milk: development of a multi-residue method and transfer of salinomycin and lasalocid from contaminated feed.

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.

Method development for the analysis of ionophore antimicrobials in dairy manure to assess removal within a membrane-based treatment system.

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 (

Avaliação da eficácia da lasalocida e de alguns fatores epidemiológicos de Eimeria spp. parasitando bezerros Nelore mantidos em regime de pastejo / Effectiveness of lasalocid assessment and some epidemiological factors of Eimeria spp. parasitizing Nellore calves kept on pasture

A principal importância da eimeriose em bovinos, se deve ao baixo desempenho produtivo que os animais demonstram quando esta enfermidade apresenta-se sob a forma sub-clínica. Como objetivos, o presente trabalho avaliou a eficácia do uso da lasalocida sódica contra espécies de Eimeria spp. parasitando bezerros; avaliou também o desempenho ponderal dos animais submetidos aos diferentes tratamentos e analisou alguns fatores epidemiológicos que possam interferir na infecção por Eimeria nos bezerros. Foram utilizados 288 bezerros no dia 0 do estudo. Os animais pertencentes ao tratamento 01 receberam sal mineral proteinado de baixo consumo sem adição de lasalocida, enquanto que os bezerros do Tratamento 02 sal mineral proteinado de baixo consumo, com adição de lasalocida sódica, administrado via oral para bezerros dos quatro/cinco/seis meses até dez meses de idade. Colheita de fezes e pesagem dos animais foram realizadas nos dias 0 (antes do início do experimento), na desmama, 30 e 60 dias após desmama (DPD). A avaliação de alguns fatores epidemiológicos que pudessem ser relacionados com a infecção por Eimeria spp nos bezerros, como o desmame, sexo e época do ano, foram analisados neste estudo, levando-se em consideração os resultados encontrados durante todo estudo, para os 144 animais pertencentes ao grupo controle. Foram identificadas nove espécies de Eimeria nos bezerros em ordem decrescente: E. brasiliensis, E. wyomingensis, E. bovis, E. canadenses, E. zuernii, E. auburnensis, E. ellipsoidalis, E. pellita e E. cylindrica. Inesperadamente, diminuição na carga parasitária dos animais pode ser observada após o desmame. Mesmo a fazenda não adotando medidas de manejo que visam maior produtividade como a Inseminação Artificial em Tempo Fixo, que por sua vez acaba aumentando o número de nascimentos e unidade animal/hectare em uma determinada época do ano, elevado parasitismo pelo coccídio em questão foi diagnosticado nos bezerros pertencentes ao grupo controle. Talvez a época do ano em que o estudo foi realizado pode ter influenciado neste aspecto. As contagens de oocistos por grama (OoPG) de fezes para Eimeria dos animais tratados com lasalocida foram estatisticamente inferiores (P≤ 0,05) as do grupo controle após o início do estudo. O composto alcançou eficácia ≥ 95% contra o parasito em questão. No final do estudo, os animais que receberam lasalocida ganharam em média, 7,2kg a mais (P≤ 0,05) que os bezerros pertencentes ao grupo controle. Em propriedades que tem como objetivo a venda de bezerros logo após a desmama, recomenda-se o início do tratamento com a lasalocida, junto ao creep-feeding, a partir de três messes de idade, uma vez que diferencial no ganho em peso médio dos bezerros tratados foi significativamente (P≤ 0,05) mais elevado, em comparação ao grupo controle, após cinco meses de tratamento com o referido composto. Apesar de a lasalocida ser utilizada como um aditivo alimentar para animais, a diferença no ganho em peso vivo médio entre animais tratados com a lasalocida, em comparação a animais pertencentes ao grupo controle, também pode ser relacionada, em partes, a infecção dos animais por Eimeria spp., conforme discutido neste artigo, entretanto, futuros estudos devem ser conduzidos para comprovar esta hipótese.(AU)

The main importance of eimeriosis in cattle is due to lower performance shown with the disease in its sub-clinical form. This study evaluated the efficacy of lasalocid used against Eimeria spp. parasitizing calves. We also evaluated the weight gain of calves submitted to different treatments and analyzed some epidemiological factors that might interfere with Eimeria infection; 288 calves were used in the study. The calves of treatment 1 received protein mineral salt in low consumption without lasalocid, while the calves of treatment 2 received protein mineral salt on low consumption with lasalocid, administered orally to 4 to 10-month-old calves. Harvest of feces and weight control was made on days 0 (before the start of the experiment), at weaning, and 30 and 60 days after weaning (DAW). Evaluation of some epidemiological factors which could be related to infection by Eimeria spp. of the calves, such as weaning, sex and time of year, were analyzed, taking into account the results regarding the 144 calves of the control group. Nine species of Eimeria were identified in descending order: E. brasiliensis, E. wyomingensis, E. bovis, E. canadian, E. zuernii, E. auburnensis, E. ellipsoidalis, E. pellita and E. cylindrica. Unexpectedly, decrease in parasite load could be observed after weaning. Even the farm did not adopt management measures aimed for greater productivity, as Artificial Insemination in Fixed Time, which in turn ends up with increase of the number of births and animal unit per hectare at a certain period of year, high parasitism of coccidia was diagnosed in calves of the control group. Oocyst counts per gram (OPG) of calves treated with lasalocid were significantly lower (P≤ 0.05) in the control group. The compound achieved ≥ 95% efficacy against the parasite in question. At the end of the study, calves fed lasalocid gained on average 7.2kg (p≤ 0.05) more than calves in the control group. For a farm that aims to sell calves soon after weaning, is recommended to start treatment with lasalocid, with the creep-feeding, from an age of three months on, since the weight gain calves treated with lasalocid was significantly (p=0.05) higher compared with the weight gain of the control group after five months of treatment. The difference in weight gain of calves treated with lasalocid compared with caves in the control group may also be partially related to the infection by Eimeria spp., as discussed in this paper.(AU)

Analysis of lasalocid residues in grease and fat using liquid chromatography-mass spectrometry.

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.

Degradation and dissipation of the veterinary ionophore lasalocid in manure and soil.

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.

Effect of distillers feedstuffs and lasalocid on Campylobacter carriage in feedlot cattle.

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.

Development and validation of a multiresidue liquid chromatography/tandem mass spectrometry method for 11 coccidiostats in feed.

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.

Application of Tb(4)O(7) nanoparticles for lasalocid and salicylate determination in food analysis.

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%.

Transfer of the coccidiostats monensin and lasalocid from feed at cross-contamination levels to whole egg, egg white and egg yolk.

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.

Determination of narasin and monensin in bovine, swine, and chicken tissues by liquid chromatography with tandem mass spectrometry: first action 2011.24.

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.

Rapid method for the simultaneous determination of six ionophores in feed by liquid chromatography/mass spectrometry.

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.

Determination of lasalocid residues in the tissues of broiler chickens by liquid chromatography-tandem mass spectrometry.

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 awareness and sampling in Scotland.

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.

Analysis of specific mutants in the lasalocid gene cluster: evidence for enzymatic catalysis of a disfavoured polyether ring closure.

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.

Determination of lasalocid sodium in animal feeds and premixes by reversed-phase liquid chromatography: collaborative study.

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.