A facile and intelligent detection method for diclazuril based on a stable dual emissive Eu3+-dopped metal-organic framework.

Diclazuril (DIC) is a broad-spectrum anti-coccidiosis drug of the triazine class, widely used in poultry farming. The overuse of DIC may lead to its accumulation in animal bodies, which may enter the food chain and threaten human health. In this work, we fabricated a stable Eu3+-doped UiO-66 fluorescence sensor (EuUHIPA-30) for the sensitive detection of DIC. Among 20 veterinary drugs, the fluorescence of EuUHIPA-30 selectively responds to DIC, with a low detection limit (0.19 µM) and fast response (10 s). EuUHIPA-30 is recyclable and can detect DIC in chicken and eggs with good recoveries. Moreover, a smartphone-integrated paper-based sensor enables the instrument-free, rapid, visual, and intelligent detection of DIC in chickens and eggs. This work provides a promising candidate for practical fluorescent DIC sensing in animal-derived food to promote food safety.

A solid-liquid microextraction approach from a spot in an adsorbent layer of a chromatographic plate.

A new approach to extracting substances from a spot on a chromatographic plate for subsequent liquid chromatography-mass spectrometry analysis is described. This method involves extraction in a solid phase (an adsorbent layer of a chromatographic plate) - a liquid system using a simple device. For a single extraction of six selected coccidiostats from the adsorbent layer on the chromatographic plate with silica gel, 50 µL of methanol was used for 5 min. The data from the extraction experiments and liquid chromatography-mass spectrometry measurements demonstrated a good correlation between the ratio of the peak areas of the coccidiostats to the internal standard and the concentration of the substances in the range of two orders of magnitude. The coefficients of determination for the mentioned correlations range from 0.962 to 0.999. Moreover, the repeatability and reproducibility, expressed as the percentage values of relative standard deviation, do not exceed 7.5 % for any of the coccidiostats.

Simultaneous determination of selected ionophoric coccidiostats and amino acids in feed premixes using high-performance liquid chromatography-ultraviolet detection method.

The combination of ionophoric coccidiostats and amino acids (AAs) is important in poultry feeding to enhance immunity and improve the growth and feed efficiency of birds suffering from coccidiosis. A simple, rapid, and economical high-performance liquid chromatography-ultraviolet detection (HPLC-UV) method for the simultaneous determination of three ionophoric coccidiostats, namely salinomycin (SAL), maduramicin (MAD), and monensin (MON) in addition to three AAs; L-tryptophan (L-TRP), alpha-ketoleucin (KLEU), and L-valine (L-VAL) in feed premixes was developed and validated. Chromatographic separation was achieved in less than 12 min using a phenyl hexyl column with a mobile phase consisting of acetonitrile/methanol/water (25:20:55, v/v/v) adjusted to pH 3 using phosphoric acid. Isocratic elution was performed at a flow rate of 1 mL/min with UV detection at 210 nm. The method showed good linearity in the ranges 0.50-5.0 mg/mL for MON, 0.20-2.0 mg/mL for MAD and SAL, 10.0-100.0 µg/mL for L-TRP and KLEU, and 50.0-500.0 µg/mL for VAL. The developed method was successfully applied to determine the studied analytes in feed premixes with good recoveries and precision. The good validation criteria of the proposed method allow its utilization in quality control laboratories.

Analysis of authorized coccidiostats in chicken feces and environmental water by ultra-performance liquid chromatography-tandem mass spectrometry based on dispersive solid-phase extraction and lyophilization.

A simple, sensitive, and efficient method based on ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was developed for the determination of 8 coccidiostats in chicken feces and environmental water (including sewage, pond water, and lake water) surrounding the farm. Target analytes in chicken feces were extracted with 2% acetic acid in acetonitrile solution, followed by a dispersive solid-phase extraction (DSPE) cleanup step using the mixture of PSA and C18 adsorbents. Environmental water samples were pretreated using a lyophilization approach. Analysis was carried out on a UPLC-MS/MS with the combination of methanol and 0.1% formic acid aqueous solution as the mobile phase under multiple reaction monitoring in positive and negative ionization modes. Results showed that 8 coccidiostats were linear with correlation coefficients higher than 0.99. Method validation was performed using fortified samples, reaching satisfactory recoveries of 75.9%-97.8% in chicken feces and 71.9%-108.2% in environmental water. Limits of detection for 8 analytes in chicken feces and environmental water were 0.03∼2 µg/kg and 0.005∼1 µg/L, respectively. Matrix effects were calculated and strong signal suppression (>50%) for some coccidiostats was observed. The developed method was successfully applied to analyze coccidiostats in chicken feces and environmental water collected from local chicken farms.

Multi-class, multi-residue determination of 132 veterinary drugs in milk by magnetic solid-phase extraction based on magnetic hypercrosslinked polystyrene prior to their determination by high-performance liquid chromatography-tandem mass spectrometry.

A quantitative multi-class multi-residue analytical method was developed for the determination of veterinary drugs in milk by high-performance liquid chromatography - tandem mass spectrometry (HPLC-MS/MS). A total of 132 veterinary drugs investigated belonged to almost 15 classes including sulfonamides, ß-lactams, tetracyclines, quinolones, macrolides, nitrofurans, nitroimidazoles, phenicols, lincosamides, pleuromutilins, macrocyclic lactones, quinoxaline antibiotics, benzimidazoles, anthelmintics, coccidiostats and some others. A magnetic solid-phase extraction procedure was developed using magnetic hypercrosslinked polystyrene (HCP/Fe3O4) for the sample preparation prior to HPLC-MS/MS without deproteinization step. The results indicated recoveries of 85-107% for 14 sulfonamides, 85-120% for 13 ß-lactams, 89-115% for 4 tetracyclines, 82-119% for 14 quinolones, 82-115% for 8 macrolides, 97-109% for 4 nitrofurans, 84-115% for 10 nitroimidazoles, 89-114% for 3 phenicols, 86-111% for 3 lincosamides, 97-102% for 2 pleuromutilins, 72-88% for 4 macrocyclic lactones, 87-104% for 4 quinoxaline antibiotics, 76-119% for 21 benzimidazoles, 79-115% for 12 anthelmintics, 81-118% for 12 coccidiostats and 75-119 % for 5 unclassified drugs, with relative standard deviations (RSDs) of less than 20%, and the LOQs ranged from 0.05 to 1 µg kg-1. This methodology was then applied to field-collected real milk samples and trace levels of some veterinary drugs were detected.

DART mass spectrometry: a rapid tool for the identification of feed additives containing coccidiostats as active substances.

Feed additives require pre-market authorisation prior to their use in the EU. For the group of coccidiostats, the EU regulations authorising these products include specifications for these substances and the major components of the feed additive formulations. Feed business operators can use only feed additives that meet these criteria. The traceability of these products is supported by the allocation of specific identification numbers that need to be printed on the feed additive label along with other information. In the present study, Direct Analysis in Real Time mass spectrometry (DART-MS) was applied to investigate the correct characterisation of feed additives that contain coccidiostats as active substances. The results of the study demonstrated that this technique allows an unequivocal identification of the active substances in the feed additive formulations when combining DART with high-resolution mass spectrometry. In addition, two feed additives containing the same coccidiostat, but different excipients could be correctly classified according to their composition. The method protocol is very simple and comprises two steps, namely the extraction of the feed additives with an organic solvent and the subsequent measurement with DART-MS. For the evaluation of the MS spectra, chemometrics was applied offering an effective method for classification. Chemometric models were established with nominal masses obtained from the analysis of the samples, thus showing that these feed additives could be correctly classified even using low-resolution mass spectrometry. Moreover, we demonstrated that molecule-specific stable isotope patterns obtained with low-resolution mass spectrometry could be used as an alternative tool for the confirmation of the active substance.

Determination of pharmaceuticals and heavy metals in groundwater for human and animal consumption and crop irrigation in Galicia.

Pharmaceuticals and heavy metals are contaminants present in groundwaters, which are the main source of drinking water in most parts of the world. In the northwest region of Spain, Galicia, groundwater harvesting is a common practice for drinking water supply, crop irrigation, cattle watering, as well as recreational use such as filling pools. In order to assess the quality of Galician groundwaters, the presence of 21 pharmaceuticals and 10 heavy metals was analysed by UPLC-MS/MS and ICP/MS methods, respectively, in a total of 118 groundwater samples from private wells. Seventeen of the 21 compounds studied were detected in 28% of the samples, with the highest presence of pharmaceuticals belonging to the antimicrobial group (52%), specifically the sulphonamides group in a range of concentration between 21 and 14.9 ng/L. In addition, 30% of the samples contained at least one heavy metal (Mn, As and Fe) above the legally permitted levels. Evaluation of the risk associated with the consumption of the analysed groundwater indicated no human risk for any of the detected pharmaceuticals but high cancer risk for children due to Cd, Cr and As concentrations was observe.

A new sample treatment strategy based on simultaneous supramolecular solvent and dispersive solid-phase extraction for the determination of ionophore coccidiostats in all legislated foodstuffs.

A single-step sample treatment, for the simultaneous extraction and clean-up for the determination of ionophore coccidiostats in EU legislated foodstuffs, is here proposed. The treatment is based on the combination of: (i) a supramolecular solvent with restricted access properties (SUPRAS-RAM), spontaneously formed by the addition of hexanol, water and THF to the sample; and (ii) dispersive solid phase extraction (dSPE). The SUPRAS-RAM extract was directly compatible with LC-MS/MS and no further re-extraction, evaporation or cleanup procedures were necessary. SUPRAS-RAM efficiently extracted the ionophores (recoveries in milk, eggs, fat, liver, kidney, and chicken and beef muscle were in the range 71-112%) and removed proteins and carbohydrates, whereas dSPE removed fats and other lipophilic compounds. The method was validated following the European Commission Decision 2002/657/EC. Detection limits (0.004-0.07 µg kg-1) were far below the maximum residue limits (1-150 µg kg-1). Method analytical and operational characteristics were suitable for routine determination of ionophores.

Microwave method synthesis of magnetic ionic liquid/gold nanoparticles as ultrasensitive SERS substrates for trace clopidol detection.

Clopidol is one of the most widely used anti-coccidiosis drugs. Its residues in poultry products and the environment pose a serious threat to human health. In this work, microwave-assisted synthesis of magnetic ionic liquid/gold nanoparticles (MIL-Au NPs) as the SERS substrates were first designed for sensitive and reliable determination of clopidol residue in egg samples. The experiment shows that MIL(1-methyl-3-hexyl imidazole ferric tetrachloride ([C6mim]FeCl4)) and microwave play a key role in the dispersion and morphology of Au NPs. Under the optimal conditions, the as-prepared MIL-Au NPs were applied to the SERS detection of clopidol in methanol and egg solution and its detection limits can be as low as to 0.5 µg/kg (equal to 0.5 ppb) in both solutions. The standard curves with regression coefficients of 0.9298 and 0.93496 were constructed in the linear range of 100-1000 ppb and 0.5-50 ppb for clopidol in egg solutions. Moreover, satisfactory recoveries (97.5-103.2%) were obtained for egg samples. The developed SERS method provides a way for quantitation of clopidol and can be applied for the convenient, reliable, and highly sensitive detection of antibiotic residues in food and environment, which has great potential in food safety and biological monitoring. Graphical abstract.

A new sensitive method for the simultaneous chromatographic separation and tandem mass spectrometry detection of anticoccidials, including highly polar compounds, in environmental waters.

A sensitive and selective method was developed and validated for the determination of 26 anticoccidial compounds (six ionophores and twenty chemical coccidiostats) in surface and groundwater samples at parts-per-quadrillion (pg L-1) to parts-per-trillion (ng L-1) levels by ultra-high performance liquid chromatography with tandem mass spectrometry detection (UHPLC-MS/MS). A range of different analytical columns and mobile phase compositions were evaluated to enhance selectivity and retention of a number of highly polar and basic anticoccidials along with other non-polar coccidiostats. A combined separation, including these problematic polar compounds, was achieved on a phenyl-hexyl column, by binary gradient elution with water/acetonitrile using ammonium formate and formic acid as additives. The anticoccidial residues were extracted from raw, unfiltered, water samples (250 mL) using polymeric divinylbenzene solid phase extraction (SPE) cartridges, with subsequent elution (methanol:acetonitrile:ethyl acetate, 40:40:20, v/v) and concentration prior to determination. The method recovery (at a concentration representative of realistic expected environmental water concentrations based on literature review) ranged from 81% to 105%. The method was successfully validated for 26 anticoccidials, at four concentration levels, in accordance to Commission Decision 2002/657/EC and SANTE/11813/2017 guidelines. Trueness and precision, under within-laboratory reproducibility conditions, ranged from 88% to 111% and 0.9% to 10.3% respectively.

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.

Multi-residue analysis using liquid chromatography tandem mass spectrometry for detection of 20 coccidiostats in poultry, livestock, and aquatic tissues.

In this study, we developed a novel analysis method based on liquid chromatography/tandem mass spectrometry (LC-MS/MS) to allow the simultaneous identification of 20 coccidiostats in eight matrix categories, including the muscles of chicken, swine, cow, and fish as well as chicken eggs, bovine milk, and porcine viscera. In the pretreatment procedure, acetonitrile/methanol (95:5, v/v) containing 1% formic acid, 5 g of sodium acetate, and 6.0 g of anhydrous magnesium sulfate was used for extraction, followed by a clean-up procedure using n-hexane saturated with ACN to facilitate the elimination of analytes from high lipid samples. Chromatographic separations were achieved using a Poroshell 120SB C18 column and operated with a gradient mobile phase system consisting of methanol (with 0.1% formic acid) and 5 mM ammonium formate, and the MS detection was monitored simultaneously. The method was validated in accordance with the Guidelines for the Validation of Food Chemical Methods by the Taiwan Food and Drug Administration. The limit of quantitation among 8 matrices were 0.5-2 ng g-1. The proposed method proved highly effective in detecting the presence of targeted veterinary drugs, providing a high degree of precision and accuracy over a broad range of matrices.

A screen-printed electrode modified with tungsten disulfide nanosheets for nanomolar detection of the arsenic drug roxarsone.

A sensitive electrochemical (voltammetric; DPV) sensor has been developed for the determination of coccidiostat drug (roxarsone) based on the use of an SPCE (screen-printed carbon electrode) modified with tungsten disulfide nanosheets (WS2 NSs). The electrochemical detection of roxarsone on the WS2-modified SPCE was examined by electrochemical strategies. XPS, XRD, Raman, SEM, TEM, EDS and EIS were used to characterize the nanosheets. The effects of scan rate, pH values (phosphate buffer) and buffer concentration were optimized. A selective roxarsone sensor was developed that works best at -0.64 V (vs. Ag/AgCl) and performs much better than the bare SPCE. Features include (a) a wider linear range (0.05 to 490 µM), (b) a nanomolar detection limit (0.03 µM) and (c) high sensitivity (29 µA·µM-1·cm-2). The modified SPCEs have been successfully applied to the determination of roxarsone in spiked meat samples where they gave high accuracy and good recoveries. Graphical abstract Synthesis of WS2 nanosheets and electrochemical detection of roxarsone.

Multi-residue methodology for the determination of 16 coccidiostats in animal tissues and eggs by hydrophilic interaction liquid chromatography - Tandem mass spectrometry.

A simple, sensitive and efficient confirmatory method was developed and validated for the determination of 16 coccidiostats in animal tissues and eggs using hydrophilic interaction liquid chromatography-tandem mass spectrometry (HILIC-MS/MS). The sample preparation consisted of a solid-liquid extraction with ACN and dispersive SPE cleanup with MgSO4 and C18. Analysis was realized in an Acquity BEH HILIC silica column, in SRM mode. Both positive and negative ionization was performed, using polarity switching. Isocratic elution was used with a mobile phase of ACN: aqueous ammonium formate 1 mM with 0.1% formic acid (80:20, v/v). Method validation was performed in eggs, poultry, bovine, ovine, porcine and rabbit tissue and exceptionally low LODs were achieved, varying from 0.004 µg kg-1 (decoquinate in porcine tissue) to 0.560 µg kg-1 (halofuginone in eggs). The developed methodology was applied in 82 muscle and egg samples through the Greek National Residue Control Plan for coccidiostats.

Pleurotus ostreatus extract inhibits Eimeria species development in naturally infected broiler chickens.

Coccidian organisms of poultry have proved very hard to control due to their abilities to resist most anticoccidial drugs. Pleurotus ostreatus (Fr.) Jacq. ex (Pleurotaceae), a medicinal mushroom, was investigated in vivo against Eimeria spp. Ninety-six broilers (day-old) naturally infected with Eimeria spp. were divided into eight groups (12 birds per group). Group A was infected untreated (negative control) and group B was treated with toltrazuril (positive control) while groups C-H were gavaged with graded doses of P. ostreatus extract at 100, 200, 300, 400, 500, and 600 mg/kg, respectively. The phytochemical analysis of the aqueous extract of P. ostreatus which revealed saponins, flavonoids, anthraquinones, and alkaloids was evaluated for anticoccidial activity by assessing the inhibition of oocyst output, lesion score, faecal score, weight differences, haematological parameters, and leucocyte differential counts. The acute toxicity study showed extract of P. ostreatus to be non-toxic at 600 mg/kg. The weight of the groups treated with the extracts and toltrazuril increased significantly (P < 0.05) compared with the untreated control. Treated groups significantly (P < 0.05) reduced oocyst output except groups C and D. The therapeutic best-fit ED50 value for the extract was 448 mg/kg. The post-treatment mean packed cell volume and red blood cell count were significantly higher (P < 0.05) than the untreated group, while the WBC count was significantly higher (P < 0.05) in the untreated group. Pleurotus ostreatus therefore could be a potential source of new anticoccidial medicine which could find application in the control of avian coccidiosis.

Development of molecularly imprinted electrochemical sensor with reduced graphene oxide and titanium dioxide enhanced performance for the detection of toltrazuril in chicken muscle and egg.

A molecularly imprinted electrochemical sensor for toltrazuril (TZR) detection based on molecularly imprinted polymers (MIPs) immobilized on reduced graphene oxide (rGO) and titanium dioxide (TiO2) modified platinum (Pt) electrode surface was fabricated for the first time. The synergistic fast electron transfer ability, large electroactive surface area and high catalytic activity from rGO and TiO2 contribute to amplify the electrochemical signal and consequently improve the sensitivity of the sensor. The cyclic voltammetry (CV), differential pulse voltammetry (DPV) and electrochemical impedance spectroscopy (EIS) tests were used to evaluate the performance of the electrochemical sensor. The results showed that the electrochemical sensor possessed high sensitivity, good selectivity and anti-interference ability toward TZR. By using the DPV, the electrochemical sensor displayed a wide linear concentration range from 0.43 to 42.54 µg/L, with a limit of detection of 0.21 µg/L (S/N = 3). Moreover, the recoveries and relative standard deviations (RSD) were 85.0%-97.0% and 3.5%-6.4% at three concentration levels, respectively, implying that the established sensor is promising for the accurate detection of TZR at trace levels in chicken muscle and egg samples.

Development and optimization of confirmatory liquid chromatography-Orbitrap mass spectrometry method for the determination of 17 anticoccidials in poultry and eggs.

A new sensitive and selective multi-residue method based on liquid chromatography - Orbitrap high resolution mass spectrometry (LC-Orbitrap-HRMS) was developed and validated for the determination of 17 anticoccidials in poultry and eggs. Instrumental parameters were optimized by the means of statistical experimental designs to improve the sensitivity, precision, and repeatability of the method. Further optimization of auto-tuned MS parameters led to an increase of signal intensity by 10% to 99% for 16 out of 17 analytes. The sample preparation procedure included extraction from muscle tissue and egg samples with acetonitrile, followed by preconcentration, reconstitution, and filtration. Validation was performed according to the Commission Decision 2002/657/EC. The occurrence of anticoccidials in eggs and poultry was assessed by using the developed analytical procedure within the Latvian national monitoring program, revealing quantifiable residues for 6 analytes (marker residue of nicarbazin - 4,4'-dinitrocarbanilide (DNC), salinomycin, narasin, toltrazuril, and its two metabolites).

Exposure of Brazilian soil and groundwater to pollution by coccidiostats and antimicrobial agents used as growth promoters.

The World Health Organization has identified antimicrobial resistance as one of the most important threats to global health. Brazil is one of the world's leading meat producers and the Brazilian use of veterinary antimicrobials as therapeutic agents and prophylactic or growth promoters in animal production remains problematic. Many antimicrobials are not completely metabolized and their excretion represents a significant source of environmental exposure. The aim of this work was to estimate the exposure risk of soil and groundwater to pollution by growth promoters (GPs) and anticoccidial additives (AAs) in Brazil by using a method based on a geographical information system (GIS). The principle adopted is that the greater the amount of animals, the greater the quantity of antimicrobials present, and the greater the soil vulnerability to pollution. Our research showed that GPs and AAs are extensively used in the Brazilian animal production system. An analysis of market data showed that zinc bacitracin, monensin, salinomycin, colistin and tylosin are representative GPs and AAs. This study presents a qualitative approach for risk assessment based on worst-case scenarios. First, the probable environmental concentration was estimated using a correlation between the number of heads of the herds of poultry and cattle, and the amounts of drug released. The leaching risk potential was characterized for each compound, as proposed in ISO 15175. The potential of soil pollution was evaluated for each antimicrobial as a function of its binding and dissipation rates. These rates were calculated using georeferenced data of organic carbon, average temperature, water balance and hydro-geological parameters. The consequences were modeled based on Brazilian soil usage. Finally, the risk was calculated by combining the different maps generated using spatial multi-criteria decision analysis. Higher risk was found for the midwest, southeast and south regions of Brazil. Groundwater was found to be more vulnerable than soil.

Development and comparison of HPLC-MS/MS and UPLC-MS/MS methods for determining eight coccidiostats in beef.

A high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method and an ultraperformance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method for determining eight coccidiostat (halofuginone, lasalocid, maduramicin, monensin, narasin, nigericin, robenidine and salinomycin) residues in beef were developed and compared. Samples were extracted with a mixture of acetic acid, acetonitrile and ethyl acetate and were then purified on a C18 solid-phase extraction (SPE) column. The purified samples were analyzed by HPLC-MS/MS and UPLC-MS/MS, using 0.1% formic acid-water solution (A) and pure methanol (B) as the mobile phase. The samples were fractionated on a C18 column using different gradient elution procedures, followed by qualitative analysis using a mass spectrometer operated in multiple reaction monitoring (MRM) mode with positive electrospray ionization; the external standard method was used for quantitation. At spiked levels that ranged from the limit of quantification (LOQ) to 100 µg/kg, the average recoveries were 71.96%-100.32% and 71.24%-89.24%, with relative standard deviations (RSDs) of 2.65%-12.38% and 2.98%-14.86% for UPLC-MS/MS and HPLC-MS/MS, respectively. The limits of detection (LODs) and LOQs of the eight coccidiostats were 0.14-0.32 µg/kg and 0.43-1.21 µg/kg, respectively, for UPLC-MS/MS analysis and 0.16-0.58 µg/kg and 0.53-1.92 µg/kg, respectively, for HPLC-MS/MS analysis. Both methods had good accuracy and precision, but UPLC-MS/MS had higher sensitivity than HPLC-MS/MS.

Simultaneous analysis of coccidiostats and sulphonamides in non-target feed by HPLC-MS/MS and validation following the Commission Decision 2002/657/EC.

Taking into consideration the maximum level (ML) for coccidiostats included in the European Regulation 574/2011 and the fact that the presence of residues of sulphonamides in non-target feed is forbidden, the aim of this article is to present an analytical method based on HPLC-MS/MS for the identification and quantification of sulphonamides and coccidiostats in non-target feeds. The method was validated following Commission Decision 2002/657/EC, and recovery, repeatability and reproducibility were within the limits established in the Decision. For coccidiostats, the decision limit and detection capability were calculated for the different species taking into account the ML allowed in Regulation 574/2011. The applicability of the method was investigated in 50 feed samples collected from dairy farms, 50 obtained from feed mills and 10 interlaboratory feed samples.