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Animal waste is a potential pollution hazard as it can harbour contaminants, such as antimicrobial residues, mycotoxins, and pesticides, becoming a risk to the public, animal, and environmental health. To assess this risk, 15 experimental broiler chickens orally received contaminants to evaluate excretion levels. An analytical method was previously developed to detect 18 substances in poultry droppings using high-performance liquid chromatography coupled to a tandem mass spectrometer (HPLC-MS/MS). Contaminants including tetracycline, 4-epi-tetracycline, oxytetracycline, 4-epi-oxytetracycline, chlortetracycline, 4-epi-chlortetracycline, tylosin, erythromycin, enrofloxacin, ciprofloxacin, flumequine, florfenicol, sulfachloropyridazine, sulfadiazine, 2,4-dichlorophenoxyacetic acid, zearalenone, alpha- and beta-zearalenol, were extracted with EDTA-McIlvain and acetonitrile. This method showed a p -value < 0.05, RSD < 25%, and R 2  > 0.95 in the calibration curves linearity for all analytes. The limit of quantification, selectivity, decision limit for confirmation, matrix effect, precision, and recovery parameters were validated according to European Union document 2021/808/EC, technical report CEN/TR 16059, SANTE/11813/2017 and according to the Veterinary International Conference on Harmonization: VICH GL2 and GL49. This method confirmed the detection of most analytes 12-36 h post-administration and simultaneously detected and quantified mixed contaminants. Thereby, poultry droppings are a potential matrix for spreading contaminants in animal production before slaughter and their control will minimize environmental impacts and mitigate antimicrobial resistance.

To explore potential factors contributing to high fluoroquinolone resistance levels, it is essential to develop analytical methods capable of detecting residues and trace amounts of antibiotic use in broilers. The aim of the present study was to develop and in-house validate a sensitive UHPLC-MS/MS method capable of determining enrofloxacin (ENR) and flumequine (FLU) residues at slaughter age (day 45) when the animals were treated with these antimicrobials one day after hatching. Residue depletion of ENR and FLU in feathers was also assessed. Two experimental trials were performed, both consisting of 5 different treatment groups. In the first trial animals were treated with ENR and in the second one with FLU. The developed method was successfully validated and was found to be sensitive enough to detect residues of fluoroquinolones in the feathers up until slaughter age in all treatment groups. Average ENR concentration on day 45 was 10 ng g -1 feather after drinking water treatment, with all concentrations above the limit of quantification (LOQ) of 5 ng g -1 feather. For FLU average concentration on day 45 after drinking water administration was 4 ng g -1 feather, with an LOQ of 1 ng g -1 feather. Therefore, the method is suited for application to monitor fluoroquinolone use in broilers.

This study developed a highly sensitive microbiological method utilizing a novel microtiter plate to screen 10 sulfonamides in chicken muscles, eggs, and prawns. This plate was fabricated from agar incorporating trimethoprim and spread with Bacillus megaterium . After residue detection by bioassay, the same test solutions were analyzed by LC-MS/MS for accurate identification and quantification. It also proved eco-friendly compared to using other quantitative methods. The residual drugs were extracted with McIlvaine buffer and purified using an Oasis ® MCX cartridge. A triethylamine/methanol/water (0.5:75:24.5, v/v/v) mixture was used as the eluate. The obtained LOD values of the bioassay ranged from 5 to 25 µg kg- 1 allowing the detection of the target drugs at the MRLs established in Japan. Adhering to ISO/IEC 17025 standards, the performance of the bioassay was evaluated. Based on the inhibition zone size in bioassay results, quality control yielded a Z score within ±2, indicating reasonable control over the screening process. Proficiency testing of a chicken muscle sample spiked with sulfadimidine demonstrated the inhibition zone detection of the bioassay and quantified value alignment of LC-MS/MS with reference values. In a surveillance study of 91 samples, sulfamethoxazole was detected in one prawn sample.

This study aimed to present a sensitive, accurate, and precise analytical method for the determination of 32 antibiotics from 5 groups (sulfonamides, macrolides, aminoglycosides, tetracyclines, and quinolones) and some individual antibiotics (lincomycin, griseofulvin, and 5-hydroxy-flunixin) in 63 honey samples collected from Tehran market. In the presented method, the samples were hydrolyzed by 1% HFBA (hepta fluoro butyric acid) in water, purified on Strata XL polymeric reversed-phase cartridges, and finally analyzed by reversed-phase ion-pair liquid chromatography-electrospray ionization tandem mass spectrometry (RP-IP-LC-ESI-MS/MS). Good performance characteristics were gained for recovery, precision, range, and linearity, the limit of detections (LODs), and the limit of quantifications (LOQs). According to the presented results and considering the absence of permissible limits for antibiotics in honey, 74.6% of the tested samples had antibiotic residues more than the LOQ of the method. The results show that the validated method is suitable for simultaneously detecting antibiotic residues in honey.

Monitoring of antimicrobials residues in food of animal origin is performed by control laboratories to ensure public health, and knowledge of the stability of antimicrobials during storage is essential for the reliability of results. For stability studies, analysis of incurred samples is preferential to fortified samples due to the possible conversion of antimicrobial metabolites back to parent compounds during sample preparation, storage, and analysis of the incurred samples, resulting in an increased concentration of the analyte. We have analyzed the concentrations of 13 antimicrobials from 8 groups (tetracyclines, fluoroquinolones, phenicols, sulfonamides, aminoglycosides, penicillins, macrolides, and nitroimidazoles) at different time points of freeze-storage (1 week; 1, 2, and 3 months) using HPLC-MS/MS. Incurred samples were prepared from muscle tissue, liver, kidneys, eggs, and milk taken from different animals (cows, pigs, poultry, goats, and fish). Incurred and fortified samples of honey were investigated as well. The results have shown that all analytes in all samples were stable during the investigated periods regardless of animal species, matrix, and concentration levels.

Brazil plays an important role in ensuring its position on the international market by assuring high food safety standards for its products, and all products should meet the requirements for residues from veterinary drugs and contaminants in animal products. Statutory monitoring provides insights into the compliance of the Brazilian industry regarding these legal requirements. The objective of this study was to provide insight into the safety of Brazilian animal products by reporting the occurrence of residues from veterinary drugs and contaminants according to an analysis of an 11-year report published by the Brazilian Ministry of Agriculture, Livestock and Food Supply (MAPA). Between 2010 and 2021, 166,647 samples from animal-derived products were analyzed in Brazil, and 624 of those samples were non-compliant (0.37%) exceeding maximum residue limits (>MRLs) or showed the presence of prohibited substances. The most common types of substances found in the non-compliant samples were heavy metals, parasiticides, and antimicrobials, accounting for 82% of all documents from the MAPA. Among Brazilian products, the challenge related to occurrence of substances varied across the food supply chain, with highest incidence rates observed in the fish chain, followed by eggs, milk, equids, sheep/goat, honey, bovine, swine, and broilers chains in decreasing order. Considering the type of substance, heavy metals were found to be more prevalent in fish products, mainly arsenic in wild fish. The prevalence of contaminants and heavy metals decreased, while that of veterinary drugs increased in Brazilian products from 2010 to 2021. From these results, it can be concluded that the number of accidental incidents including those associated with environmental contaminants decreased over the last decade, opposed to those involving human adversaries and deliberate illegal actions, such as the abuse of veterinary drugs, increased. Future monitoring plans need to take this paradigm shift into account.

Nowadays, incorrect apply of antibiotics to treat infections in honey has led to health risks for humans and antibiotic resistance. Current systematic review and meta-analysis conducted to study antibiotic residues in honey. Data were obtained through searching the databases, including Scopus, Web of Science, PubMed, and other internal databases. The pooled concentration of antibiotic residues was 5.032 (µg/kg) that ranged from 4.72 to 5.33 (µg/kg). The ranking of antibiotics concentration was found in order of fluoroquinolone (8.59 µg/kg) > tetracycline (5.68 µg/kg) > sulfonamides (5.54 µg/kg) > macrolides (4.19µg/kg), respectively. Liquid chromatography-mass spectrometry (LC-MS) method (37.9.7%), high-performance liquid chromatography (HPLC) method (34.4%), and enzyme-linked immunosorbent assay (ELISA) method (27.5.8%) were the most used methods in various studies. In order to avoid contamination, proper use of antibiotics, placement of hives at a suitable distance from agricultural environment, and regular control of antibiotic residues in honey seems to be necessary.

Analyte stability is more commonly a confounding factor in analytical chemistry than many analysts recognize. In this study, we assessed the stability of 31 common veterinary drugs in water and final extracts of bovine (milk and kidney/liver) and chicken (muscle and egg) matrices. Two different sample preparation methods were evaluated for one-month storage of the final extracts at typical room, refrigerator, and freezer temperatures. Liquid chromatography - mass spectrometry (LC-MS) by triple quadrupole and high-resolution techniques was used for analysis of the extracts spiked at different relevant concentrations for general regulatory purposes (10-1000 ng/g sample equivalent). Comparison of results between two labs demonstrated that stable drugs (≤20% loss) at all tested conditions consisted of danofloxacin, enrofloxacin, florfenicol, flubendazole, hydroxy-flubendazole, flumequine, flunixin, 5-hydroxy-flunixin, lincomycin, and meloxicam. The tested drugs found to be the most unstable (>20% loss at room temperature within a matter of days) consisted of the β-lactams (ampicillin, cefalexin, cloxacillin, and penicillin G). Curiously, the following antibiotics (mostly macrolides) were apparently more stable in sample extracts than water: emamectin, erythromycin, ivermectin, lasalocid, monensin, tilmicosin, tulathromycin, and tylosin. Those and the other drug analytes (ciprofloxacin, doxycycline, florfenicol amine, 2-amino-flubendazole, oxytetracycline, sulfadiazine, sulfadimethoxine, sulfamethazine, and trimethoprim) were mostly stable for a month in refrigerated extracts, especially at higher concentrations, but not in all cases. In practice, freezer storage of extract solutions was found to be acceptable for at least a month, with a few exceptions.

The aim of this study was to investigate the transfer of residues of five β-lactam antibiotics (ampicillin, penicillin G, cloxacillin, dicloxacillin and cephalexin) and two tetracyclines (tetracycline and oxytetracycline) in the processing of cheese and whey powder, evaluating the effect of the processes and the final concentration in each product generated. Raw milk was fortified at two concentration levels with the seven antibiotics. The first concentration level (C1) was chosen according to the maximum residue limit (MRL) of each antibiotic (ampicillin and penicillin G: 4 µg kg -1 ; cloxacillin and dicloxacillin: 30 µg kg -1 ; cephalexin, tetracycline and oxytetracycline: 100 µg kg -1 ). The second concentration level (C2) was spiked as follows according to each antibiotic: 0.5 MRL (cloxacillin, dicloxacillin, cephalexin), 0.1 MRL (tetracycline and oxytetracycline) and 3 MRL (ampicillin and penicillin G). The antibiotics were analyzed by LC-MS/MS. No ampicillin or penicillin G residues were found in cheese or whey powder, although they were detected in whey at concentrations similar to those added to raw milk. Cephalexin was mostly distributed in whey between 82% and 96%, being the antibiotic that presented the highest concentration in whey powder (784 ± 98 µg kg -1 ) when milk was spiked at the MRL. The whey distribution of cloxacillin and dicloxacillin ranged from 57% to 59% for cloxacillin and from 46% to 48% for dicloxacillin, and both concentrated in whey powder. Tetracyclines were the antibiotics that concentrated in cheese, with retentions between 75% and 80% for oxytetracycline and between 83% and 87% for tetracycline. The distribution of antibiotics in the dissimilar stages of the cheese and whey powder production processes, as well as their concentration in the final products, depend on each type of antibiotic. Knowledge of the transfer of antibiotic residues during the process and final disposal is an input for the risk assessment of their consumption.

In this study, an immunochromatographic test (using the Charm QUAD2® Test) was used to screen for residual macrolides and lincosamides in raw cow's milk. The validation parameters (selectivity/specificity, detection capability (CCβ), and ruggedness) were in agreement with the requirements of[EC] 2021. The selectivity of the immunochromatographic test was verified by the negative results of microbiological tests. The false-positive rate was 0%. The CCβ values of the immunochromatographic test for various antibiotics in milk were as follows: erythromycin 0.02 mg/kg, spiramycin 0.1 mg/kg, tilmicosin 0.025 mg/kg, tylosin 0.05 mg/kg, lincomycin 0.15 mg/kg, and pirlimycin 0.15 mg/kg. The determined CCβ values were lower than the respective maximum residue limits (MRLs; regulatory limits in Japan) for milk, except for lincomycin (equal to the MRL). The presence of antibiotic groups other than macrolides and lincosamides did not interfere with the specificity of the test. It showed no significant difference in lot-to-lot repeatability. The results obtained by the two researchers showed no significant differences. Finally, the test was applied to milk samples obtained from a tylosin-treated cow. The outcome was positive and in agreement with the results of the chemical analytical and microbiological methods. Therefore, this validated immunochromatographic test is expected to be suitable for routine analysis to ensure milk safety.

Cross-contamination between medicated and non-medicated feed can occur during production, processing, transport or storage of animal feed. This may lead to the presence of low concentrations of antibiotics in supposedly drug-free feed for food production animals, which potentially could also harm consumers due to residues. In addition, consumption of sub-therapeutic concentrations of antibiotics may increase the risk of emergence of resistant bacteria. In this study, LC-MS/MS methods were developed to quantify four antibiotics (sulfadimethoxine, oxytetracycline, trimethoprim and amoxicillin) in several pig matrices, i.e. plasma, muscle, liver, kidneys and faeces. All methods were validated using the accuracy profile, except for amoxicillin in faeces, for which extraction could not be optimised for low concentrations. These methods were then applied as part of an animal study during which several pigs received contaminated feed at a concentration corresponding to 2% of therapeutic dose, in order to evaluate the risk of the presence of residues in animal faeces and tissues. The results showed that sulfadimethoxine is well absorbed and accumulates in the muscle, kidneys and liver, where concentrations were higher than the maximum residue limits (MRLs) authorised in EU legislation. Conversely, oxytetracycline was mostly found in faeces as its oral absorption is very low. Trimethoprim concentrations were slightly higher than the tolerated MRL in the kidneys, but they were below this level in the other tissues. Finally, amoxicillin concentrations remained below the lower limit of quantification of the methods in all matrices.

Treatment of food-producing animals with veterinary medicinal products (VMPs) can result in residues in foodstuffs (e.g. eggs, meat, milk, or honey) representing a potential consumer health risk. To ensure consumer safety, worldwide regulatory concepts for setting safe limits for residues of VMPs e.g. as tolerances (US) or maximum residue limits (MRLs, EU) are used. Based on these limits so-called withdrawal periods (WP) are determined. A WP represents the minimum period of time required between the last administration of the VMP and the marketing of foodstuff. Usually, WPs are estimated using regression analysis based on residue studies. With high statistical confidence (usually 95% in the EU and 99% in the US) the residues in almost all treated animals (usually 95%) have to be below MRL when edible produce is harvested. Here, uncertainties from both sampling and biological variability are taken into account but uncertainties of measurement associated with the analytical test methods are not systematically considered. This paper describes a simulation experiment to investigate the extent to which relevant sources of measurement uncertainty (accuracy and precision) can impact the length of WPs. A set of real residue depletion data was artificially 'contaminated' with measurement uncertainty related to permitted ranges for accuracy and precision. The results show that both accuracy and precision had a noticeable effect on the overall WP. Due consideration of sources of measurement uncertainty may improve the robustness, quality and reliability of calculations upon which regulatory decisions on consumer safety of residues are based.

The intensification of aquaculture in Cameroon requires efficient screening methods to control veterinary drug residues in fish. This is why the charm II radio receptor technique for the detection of antimicrobial residues in aquaculture fish initiated in Belgium was transferred to Cameroon according to Commission Decision 2002/657/EC. The validation parameters included the following tests: repeatability, reproducibility and robustness in addition to the mandatory characteristics, detection capability and specificity. Selected veterinary drug-free fish samples of tilapia ( Oreochromis niloticus ), catfish ( Clarias gariepinus ), carp ( Cyprinus Carpio ) and kanga ( Heterotis niloticus ) were spiked at different target concentrations with different antimicrobials (β-lactams, tetracycline, sulfonamides, macrolides and chloramphenicol). The detection capabilities (CCβ) were at MRPL (0.3 µg/kg for chloramphenicol), or below the regulatory limits in a range of 0.25-0.5 MRL with 0% false-negative results. β-lactams (penicillin G), tetracyclines (tetracycline, chloretetracycline and oxytetracycline) and macrolides (erythromycine A) were detected at half MRL (25, 50 and 100 µg/kg respectively), while sulfonamides (sulfamethazine) was detected at 25 µg/kg (0.25 MRL). The detection capabilities (CCβ) obtained were satisfactory as the cut-off factors (Fm) were less than the mean values of blank fish readings (B) (Fm < B). Furthermore, the method was applicable since the cut-off factor was less than the positivity threshold (Fm < T). The variability of the data under repeatability and reproducibility conditions was acceptable, with a relative standard deviation less than 15%. Results were unaffected by delaying the reading time from 0 h to 24 h after the addition of scintillation fluid, with a precision below 16%. Likewise, non-target drugs were not detected even at high concentrations (100 MRL) in a cross-reactivity study. From the overall results, the performance characteristics (detection capabilities, precision, robustness and specificity) were suitable and comparable to the initial validation results, indicating that the transfer to Cameroon laboratory was valid, the method was reliable and could be used in aquaculture fish quality monitoring programs in Cameroon.

Some florfenicol (FF) metabolites have a strong binding affinity towards biomolecules in the edible tissues of some food animals. These bound FF residues cannot be extracted directly from edible tissues with organic solvents and are present in higher concentrations even than solvent extractable residues. In this study, an ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was established to detect the total residues of FF in eggs, by quantifying the metabolite florfenicol amine (FFA). The sample was hydrolyzed at 95-100 °C for 4 hours to release sample-matrix bound residues and convert them all into FFA. The hydrolyzed sample was washed with ethyl acetate to remove interfering substances, extracted with ethyl acetate under alkaline conditions, purified by solid phase extraction and quantified by UPLC-MS/MS. The recoveries of FFA in eggs ranged from 91.2 to 102.4%, with an RSD ≤ 10.9%. The LOD and LOQ were 0.5 and 1.0 μg/kg, respectively. This method can be applied to the quantification of total FF residues in eggs.

Sulphonamides (SAs) are widely used in animal husbandry. In our work, based on multi-walled carbon nanotubes, a novel residue method was developed for highly sensitive and determination trace levels of sulfamethoxazole, acetyl sulfamethoxazole and aditoprim in edible swine tissues by LC-MS/MS with magnetic solid-phase extraction. The samples were extracted using 2% ammoniated acetonitrile and purified by magnetic solid phase extraction (MSPE). Under the optimal conditions, good linearity was obtained ranging from 5 to 160 μg kg -1 . The limits of detection (LOD) and quantification (LOQ) were 2 μg kg -1 and 5 μg kg -1 respectively. The average recoveries were 73.9-94.8% at different spiking levels. The inter-day RSDs were 6.2-10.7% and the intra-day RSDs were 2.4-5.4%. MSPE based on multi-walled carbon nanotubes was a simple and efficient method to enrich and separate the analyses and could be successfully applied for extraction of sulfamethoxazole, acetyl sulfamethoxazole and aditoprim residues in swine tissues.

A qualitative and quantitative liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for the sensitive and exhaustive analysis of residues from triarylmethane dyes, triarylmethane-derivative dyes, phenothiazines, phenoxazines and xanthenes in aquaculture samples. For a wider and more robust detection of dye misuse on farms, other residue markers were also included the leuco forms of brilliant green, crystal violet and malachite green; one direct metabolite of Victoria pure blue BO and methylene blue and three bile acids, which are endogenous markers of the effects of dye contamination in fish. We optimised the extraction method by comparing several extraction solvents and sample solvents reported in the literature to have the best extraction efficiency. The residues were determined using a positive electrospray ionisation source. We assessed the parameters of this LC-MS/MS method by evaluating the matrix effects, identification and quantitative parameters according to the criteria stipulated in the European Commission Decision No. 2002/657/EC. A study on the applicability of the method was conducted on various aquaculture species and on a positive catfish.

β-agonists have been illegally used for growth promoting purposes in animal husbandry, leading to residue concentrations capable of inducing acute toxic reactions among consumers of animal-based food. There is not only a need for detecting β-agonist residues at low concentrations, but also to increase the number of compounds to be monitored. It was therefore the aim of this paper to develop a unified method capable of detecting a wide range of different β-agonists (20 analytes including some metabolites) in a variety of matrices (muscle, liver, plasma, milk and urine). The developed procedure permits the quick processing of samples with limited labour input and consumption of consumables. The method has been validated according to the Commission Decision 98/536/EC. Detection is based on ultrahigh-performance chromatography coupled to high-resolution mass spectrometry. Validation was performed on two different instruments (Orbitrap and time of flight). The obtained limit of quantification (0.05 to 0.5 μg/kg and the average recovery of 78% in the most complex matrix (liver) satisfies current regulatory requirements.

Moxidectin (MXD), an antiparasitic drug, is effective for a variety of external and internal parasites in companion and farm animals. This study aimed to calculate the withdrawal period by investigating the residue depletion of MXD in swine edible tissues after pouring at the dosage of 2.5 mg/kg B.W. The concentrations of MXD in swine edible tissues were determined by a modified preparation procedure based on HPLC-FLD. The method was validated giving LOD and LOQ of 0.5 μg/kg and 1 μg/kg respectively with measured recoveries ranging from 62.9%-89.2% at three different concentrations and a precision (RSD) of less or equal to 15.7%. The muscle, liver, kidney and fat tissues were collected at 0.5, 5, 10, 20, 25 d after administration. The results showed that fat was the target tissue with the highest concentration for MXD. The withdrawal period was 26 days for the MRL of 500 μg/kg in fat. The results provide fundamental information to ensure food safety and establishment of a rational medication regimen.

Antibiotic residues have become a major concern worldwide as food contaminants due to the risk that they may pose to human health. The presence of these residues in food is due to improper veterinary practices. Consequently, rapid and cost-effective clean-up methods prior to analysis for these residues in food matrices are increasingly becoming necessary in order to ensure food safety. Miniaturised extraction and pre-concentration techniques have been developed as alternatives to conventional extraction procedures in recent years. Furthermore, the current trends in analytical sample preparation favour extraction techniques that comply with the principles of green analytical chemistry. Solid phase micro-extraction, stir bar sorptive extraction, stir cake sorptive extraction and fabric phase sorptive extraction methods are very promising sorbent-based sorptive micro-extraction techniques, and they are compliant to the principles of green chemistry. This review critically discusses the application of these techniques in the extraction and pre-concentration of antibiotic residues from food samples in the years 2015 to 2020.

An immunobiosensor assay was developed for multi-residue screening in bovine milk of the parent amphenicols, thiamphenicol and florfenicol, along with the metabolite florfenicol amine. A polyclonal antibody raised in a rabbit after immunisation with a florfenicol amine-protein conjugate was employed in the assay. Milk samples were subjected to acetonitrile extraction, reconstituted in buffer and diluted prior to biosensor analysis. Validation data obtained from the analysis of fortified samples has shown that the method has a detection capability of less than 0.25 µg kg -1 for florfenicol and less than 0.5 µg kg -1 for florfenicol amine and thiamphenicol. The cross-reactivity profile and validation data for the detection of these amphenicols is presented together with results obtained following the analysis of florfenicol incurred samples using the developed screening method along with a comparison of results obtained from the analysis of the same incurred samples using an MRM 3 UPLC-MS/MS confirmatory method. Results are also presented obtained from the analysis of samples from both treated and non-treated animals which were co-housed and which show the potential for cross-contamination.

Diclazuril, a broad-spectrum anticoccidial drug, may be accumulated in edible tissues of animals through illegal use, which poses potential threats to human health through the food chain. In this study, an innovative hapten was designed and an immunogen of diclazuril was successfully synthesised with keyhole limpet haemocyanin as carrier protein; then a monoclonal antibody with high specificity was obtained. Furthermore, based on the novel antibody, a one-step indirect competitive enzyme-linked immunosorbent assay (icELISA) was established for rapid and specific detection of diclazuril residues. Compared with the traditional icELISA method, this method saves at least 0.5 hours and one washing step. Under the optimal conditions, the one-step icELISA for diclazuril exhibited good performance with a 50% inhibition concentration (IC 50 ) value of 0.952 μg/kg. The average recoveries of the icELISA ranged from 73.1% to 115.5% with the coefficient of variation lower than 12.7%, which was evaluated by detecting spiked animal-origin food samples. Finally, the one-step icELISA shows a good correlation with an ultra-high liquid chromatography-tandem mass spectrometry method. Those results demonstrate that the one-step icELISA developed for diclazuril detection is time-saving, low-cost, specific, sensitive, and reliable. It shows good potential for social, environmental, and economic benefits in future use.

Betamethasone is a glucocorticoid authorised in cattle for the treatment of metabolic and inflammatory diseases, but, in Europe, it is illegally employed to improve productive performances. LC-MS/MS is the official control method of veterinary drugs residues in food of animal origin. An experimental study was developed to evaluate the feasibility of proton magnetic resonance spectroscopy (1H-MRS) as a potential alternative approach to detect the presence of betamethasone residues. Eight rat liver samples were collected 24 h post-betamethasone-treatment from experimental and control animals and were analysed by 1H-MRS using a 7-Tesla MRI scanner. 1H-MR reference spectra both of the Bentelan formulation used for treatment, and of three solutions of betamethasone in dimethyl sulphoxide (DMSO) at 5, 10 and 100 mM, respectively, were acquired to fit analyte-peaks in the liver samples spectra. Betamethasone-peaks were found only in the 100 mM betamethasone in DMSO solution spectrum. Betamethasone residues were not detected in any of the tissue samples analysed, probably related to the low concentration of injected drug. These findings allow us to establish, for the first time in the literature, the detection limit (in the range 10-100 mM) of betamethasone for the 7-Tesla MRI scanner used here. Given this very-low sensitivity, we conclude that the evaluated 1H-MR spectroscopy approach is not suitable for the detection of betamethasone residues in edible tissues, since the maximum residue limit imposed by Commission Regulation (EC) 37/2010 for betamethasone in the liver, and metabolic concentrations required to be detected in animal samples from livestock, are far below the detection limit we found.

Colistin is a polypeptide antibiotic mainly used in porcine and poultry to treat gastrointestinal infections. It has been included by the World Health Organisation (WHO) in the list of critically important human antibiotics of high priority for antimicrobial resistance since 2017. Therefore, it is necessary to develop specific and sensitive screening methods for this molecule. Screening for colistin with immunoassays is an interesting alternative to LC-MS/MS screening methods. The performance of three commercially available ELISA kits was evaluated in poultry and porcine muscles for the detection of colistin in regards to its European maximum residue limit (MRL) (150 µg/kg). The applicability of the three ELISA kits to the detection of colistin at or below the MRL in porcine and poultry muscles was demonstrated. The detection capabilities (CCβ) of two kits were or lower than or equal to the MRL (150 µg/kg). The lowest detection capability (30 µg/kg) was achieved with the third ELISA kit. The specificity of the three kits was very satisfactory (false positive rates 0%). The three kits are very specific for the detection of colistin (colistin A and B) and polymyxin B.

Herein, we have successfully established a novel, rapid, and simple lateral-flow immunoassay based on time-resolved fluorescence and biotin-streptavidin to detect the residues of various antibiotics in milk. The fluorescence signal and sensitivity of immunochromatography were enhanced through biotinylated antibody coupled with streptavidin europium microspheres. Moreover, due to the use of a QR Code and fluorescent reader, quantitative detection and real-time data uploading can be achieved. Under the optimal conditions, the various antibiotic residues were detected in the milk samples. The results showed that the limits of detection of tylosin, lincomycin and doxycycline were 0.10, 0.06, and 0.27 ng/mL, respectively. The recoveries of the spiked milk samples were 88.9%~127%, with coefficients of variation less than 11%, and the test strip can be stored at room temperature for 12 months. This study shows that the proposed time-resolved fluorescence immunoassay is sensitive, rapid and reliable, and has the potential to be used for detection of veterinary antibiotic residues in food safety fields.

Selective androgen receptor modulators (SARMs) are a class of new emerging "designer" steroid compounds gaining popularity over more well established anabolic-androgenic steroids (AAS) amongst both non-professional and elite athletes. Moreover, due to their anabolic activity, SARM compounds may also potentially be abused in livestock animals to increase meat production. Consequently, SARM residues should be monitored as a part of routine testing employed within both anti-doping and drug residue laboratories. Since only a limited amount of information on SARM compound stability is currently available within the peer-reviewed literature, this study reports a practical approach to assess optimal storage conditions for 15 SARM compounds in solvent solutions (standard stock and intermediate mixed standard solutions) stored at -20°C for up to 1 year, as well as in a range of urine test matrices (bovine, equine, canine and human) under frozen (-20°C, -80°C) storage for up to 20 weeks and post freeze-thaw cycles. Moreover, SARM storage stability within solvent extracts was assessed at -20°C (0-4 weeks) and 4°C (0-2 weeks). Findings demonstrate that SARM analytes are stable in reference solutions when stored at -20°C, apart from PF-06260414 (stock solution) which should be stored at lower temperatures (e.g. -80°C). A limited degree of compound instability was observed for a number of SARM analytes in urine both when stored at -20°C, and after repeated freeze-thaw cycles. Moreover, SARM compounds within reconstituted urine solvent extracts were found to be effectively stable when stored for up to 4 weeks at -20°C and for 2 weeks at 4°C. The long-term stability testing data reported here will inform the more timely and effective development and validation of analytical methods for SARM residue detection and analysis, ensuring confidence in findings from monitoring of livestock animals and anti-doping processes.

A liquid chromatography-high resolution mass spectrometry (LC-HRMS) method was developed and validated for the determination of residual peptide antibiotics (bacitracin A, colistin A and B, enramycin A and B, virginiamycin M1 and S1) in bovine milk. LC-HRMS accurate mass data provided the necessary selectivity and sensitivity to quantitate and identify these important antibiotics in milk at residue levels without extensive sample preparation. Milk samples were extracted using 0.3% formic acid in acetonitrile with 0.06% trifluoroacetic acid added to improve peptide recoveries. Sample clean-up was minimal with an aliquot of the extract evaporated and reconstituted in a formic acid/water-acetonitrile mixture and then filtered. LC separation was performed with 0.3% formic acid in the gradient to improve the peak shape and reproducibility of the peptide analytes. A Quadruple-Orbitrap HRMS instrument with full-scan MS1 data collection followed by all-ion-fragmentation was used to obtain the exact mass of the precursor and confirmatory product ions. One advantage of LC-HRMS is that a combination of multiple precursor ions, including different charge states or adducts, can be used for quantification. The method was validated at four concentration levels ranging from 12.5 to 200 ng/g in three types of bovine milk. For bacitracin A, colistins and enramycins, the average recoveries compared to solvent standards ranged between 70% and 120%. Average recoveries for virginiamycin residues in milk extracts were unacceptably high (up to 138%) using solvent standards, but recoveries using matrix-matched calibration were determined to be 90-115%. Matrix effects were found to be less than 25% for the other analytes when internal standard correction was used for the colistins. Intra-day relative standard deviations were generally below 15%. The method detection limits for the peptide antibiotic residues in milk (0.5 to 5.5 ng/g) were well below regulatory levels of concern.

The study aimed to evaluate the effects of heat on macrocyclic lactone residues in cows' milk. Ivermectin, abamectin, doramectin, eprinomectin and moxidectin were added to raw milk in three concentrations. The milk was then pasteurised (40 seconds at 74°C or 1 minute at 80°C) and boiled (10 minutes at 100°C). The analyses were performed with a validated method: LC-MS/MS. Thermal treatment resulted in a statistically significant decrease in the abamectin, eprinomectin, and moxidectin concentrations in the milk; however, the residues did not completely degrade. Boiling resulted in a greater decrease in the moxidectin concentrations than was observed with pasteurisation. The high pasteurisation and boiling processes had a greater effect on the eprinomectin residues than did the low pasteurisation process. The pasteurisation and boiling processes did not have an effect on the doramectin and ivermectin. The study concluded that the macrocyclic lactones are generally resistant to such processes.

Salicylates are among the most known anti-inflammatory drugs, used both in human and veterinary medicine. They also occur naturally in plants. Residues of salicylic acid in tissues and eggs may occur after drug administration or exposure of animals to feed material with high salicylate content. An animal study was performed on laying hens. The birds received sodium salicylate or acetylsalicylic acid (10 mg/kg b.w.) for 7 days or were given corn containing 1.18 mg/kg of salicylic acid. Samples of liver, muscle and plasma were collected at 0, 4, 8, 24 and 72 h after treatment; eggs were collected daily for 14 days. Salicylic acid and its metabolites: gentisic acid, salicyluric acid and gentisuric acid were determined using liquid chromatography coupled with tandem mass spectrometry. In both liver and muscle, the residues after administration of sodium salicylate were initially higher than for acetylsalicylic acid but they depleted at the same time. The deposition and depletion profile of salicylic acid in eggs was similar for groups receiving both drugs; the plateau level reached 248 ± 61.5 μg/kg and 275 ± 82.1 μg/kg. The concentration of salicylic acid in tissues and eggs of animals receiving salicylic acid was low. Gentisic acid was found in individual samples of liver, muscle and eggs from all treated groups. The exposure of hens to the salicylates at feed additive levels and to naturally occurring salicylates results in low residue concentrations and fast depletion of salicylic acid. The eggs do not pose any risk to consumers sensitive to salicylates.

Imprudent and superfluous use of antibiotics contributes to the selection of resistant bacteria, which is a large threat to human health. Therefore analytical procedures have been implemented in the poultry production sector to check if antibiotic treatments are registered, aiming to achieve more prudent use of antibiotics. These methods rely on the analysis of feathers, a matrix in which antibiotic residues persist. However, other routes besides direct administration, through which poultry feathers could contain antibiotic residues, should also be taken into account. In this research the vertical transmission from parent hen to broiler was investigated through a controlled animal study for the antibiotics enrofloxacin, doxycycline and sulfachlorpyridazine. Vertical transmission was observed for all antibiotics to both egg and egg shell. Also it is demonstrated that the transferred antibiotics from parent hen to chick are subsequently excreted via the chick's droppings. Through this route, the broilers' environment is contaminated. If eggs are hatched that were taken during treatment of the parent hen, this indirect route and/or the direct vertical transmission can eventually result in the detection of low concentrations of antibiotic residues in the broilers' feathers at greater age: <50 µg kg -1 for freely extractable residues and <10 µg kg -1 for non-freely extractable residues. No antibiotics were detected in the broilers' muscle or kidney from 4 weeks of age. This research provides relevant information regarding the possible amount of residues originating from vertical transmission when monitoring matrices such as feathers and broiler droppings in order to stimulate correct use and registration of antibiotics in the poultry sector.

A recombinant gene CCS consisting of three single genes (CAP-ScFv, CPFX-ScFv and SM 2 -ScFv) were constructed by PCR with three pairs of primers. The length of CCS gene fragment was 2,260 bp. A recombinant plasmid (pGEX-CCS) was obtained in pGEX-6p-1. pGEX-CCS was induced in E.coli BL21(DE3), and the molecular weight of recombinant fusion protein (GST-CCS) was 108.87 kDa. GST-CCS was successfully applied to analysis of CAP-OVA (CAP-ovalbumin conjugate), CPFX-OVA and SM 2 -OVA simultaneously. The sensitivity of GST-CCS against three veterinary drugs was tested by indirect ELISA at dilution ratio 1:8000. These findings provide a foundation for the construction of fusion genes with linkers and for the potential development of a rapid screening method for the simultaneous detection of veterinary drug residues.

Due to the lack of drugs regulated for aquaculture, we have evaluated the use of albendazole (ABZ) - a potential drug to be regulated for fish - under food safety perspectives assessing the depletion profile of ABZ and its main metabolites (albendazole sulphoxide - ABZSO, albendazole sulphone - ABZSO 2 and albendazole amino sulphone - ABZ-2-NH 2 SO 2 ) in fish fillets (muscle and skin) after single dose oral administration of 10 mg ABZ kg -1 body weight. For the drug administration, a suitable procedure for ABZ incorporation into fish feed was employed, obtaining good homogeneity of ABZ concentration among feed pellets (CV<4.1%) and low drug leaching when medicated feed remained in the water for up to 60 min (<2.7%). After medication, fish were euthanised at 8, 12, 24, 48, 72, 96 and 120 h and fillets collected. Depletion studies in various fish species (patinga and tilapia) were conducted simultaneously, under water temperature at 30.4 ± 0.3 °C and pH 6.8 ± 0.1. The highest concentrations for the sum of residues (ABZ, ABZSO, ABZSO 2 and ABZ-2-NH 2 SO 2 ) in fish fillet were 1210 ng g -1 in patinga and 637 ng g -1 in tilapia. Under the employed rearing conditions, the obtained results did not indicate a requirement for a minimum withdrawal period to be proposed for tilapia considering the maximum residue limit of 100 µg g -1 , since the determined residual concentration was -1 ) from 24 h post medication treatment. For patinga, the estimated minimum withdrawal period was 3 days. With the well-recognised demands for new alternative veterinary drugs for aquaculture use by fish farmers, this study offers important evidence for consideration of ABZ use in tilapia and patinga taking into account food safety issues.

An ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) based multiclass multi-residue method for the simultaneous analysis of 5 polyether ionophores, 4 tetracycline and 10 sulfonamides in animal and aquaculture fish tissues was developed and validated. Sample extraction and clean-up were based on a modified QuEChERS method. The method was validated using an in-house validation based on performance characteristics modified from Commission Decision 2002/657/EC. Both matrix effect and uncertainties associated with sample preparation and instrumental analysis were minimised by the use of matrix-matched calibrations. Recoveries of analytes were generally satisfactory and typically fell between 80% and 113%. The repeatability and intermediate reproducibility measured as relative standard deviations were in most cases less than 15% (n = 63). The decision limit (CCα) and detection capability (CCβ) ranged from 110.7 to 125.8 and 121.5 to 151.7 µg kg -1 for tetracyclines, 113.4 to 118.3 and 116.8 to 126.5 µg kg -1 for sulfonamides and 50.8 to 52.4 and 51.5 to 55.6 µg kg -1 for polyether ionophores, respectively. The method displayed its fitness for purpose through satisfactory results obtained in international proficiency testing schemes. The method was applied to animal and aquaculture fish tissues obtained from different sources in South Africa. Polyether ionophores were predominantly detected in samples in the range 4.26-290.10 µg/kg. Oxytetracycline was found in one porcine liver sample; however, none of the targeted analytes were present above the detection limit in the aquaculture samples.

Chloramphenicol (CAP) is a broad-spectrum antibiotic used widely both in human and in veterinary medication but due to adverse health effects is not authorised anymore for use in food-producing animals in many countries. CAP molecule contains two asymmetric centers resulting in four para-CAP stereoisomers, but only the RR-CAP enantiomer is bioactive with significant antimicrobial activity. In this study the detection of the four CAP stereoisomers was tested by five commercial ELISA kits and the Charm® II Chloramphenicol Test. These immunoassay tests are commonly used and widely accepted for screening of CAP residues in foods of animal origin, including honey. The test results definitely show that SS-CAP residues are not detected; even high SS-CAP concentrations are missed due to the lack of any cross reactivity and the high specificity of the CAP antibodies to RR-CAP. In former studies chiral LC-MS/MS analysis indicated clearly that honey samples with raised CAP concentrations often contain the SS-CAP enantiomer in addition to the bioactive RR-CAP. According to this study, the investigated screening tests carry the risk of systematically false-compliant (false negative) results for CAP and a discrepancy between LC-MS/MS and ELISA/Charm® test results. As a consequence of this study, it is recommended that immunoassay manufacturers develop and use CAP antibodies which also bind SS-CAP. The origin of SS-CAP residues in honey samples is discussed and general toxicological and regulatory aspects of CAP stereoisomers are raised.

Lincomycin is the first antimicrobial agent described for the lincosamide class and it is commonly used for the treatment of infectious enteric and respiratory diseases in poultry. Maximum residue limits (MRLs) in edible tissues have been established for this antimicrobial, however, no regulation has been proposed yet for by-products that are not intended for direct human consumption. Feathers are a by-product from poultry farming that might be used as an ingredient for diets fed to other farm animal species. The presence of antimicrobial residues in them is not monitored in spite of the fact that several studies have proved that they can persist in feathers. Currently though, no evidence has been presented regarding the behaviour of lincomycin in this matrix. Hence, this work intended to assess the depletion of lincomycin residues in feathers of birds treated with therapeutic doses and compare them with those detected in muscle and liver samples. Samples were collected for several days after ceasing treatment from a group of broiler chickens treated with a 25% lincomycin formulation. Methanol and Florisil® columns were used to extract and retain the analyte, and samples were analysed using a triple quadrupole mass spectrometer (API 5500, AB SCIEX™). On day 1 after ceasing treatment, average concentrations of lincomycin detected in feather samples reached up to 8582 μg kg -1 and by day 16, these had only declined by 63%, to an average of 3138 μg kg -1 . Lincomycin residues were detected in feathers at every sampling point, even after they were not detectable in edible tissues. Depletion time was 98 days for feathers, considering the LOQ established for the methodology as cut-off value for the calculations. Data showed that lincomycin is highly persistent in feathers, which may result in this matrix becoming a re-entry route for its residues into the food chain.

A liquid chromatography high resolution mass spectrometry (LC-HRMS) screening method was developed previously to analyze for veterinary drug residues commonly found in different types of aquaculture products. This method has been further evaluated for its feasibility to detect several other classes of compounds that might also be a concern as possible contaminants in farmed tilapia, salmon, eel and shrimp. Some chemicals could contaminate water sources used in aquaculture production through agricultural run-off. These compounds include several widely used triazine herbicides, organophosphate and carbamate pesticides, as well as various discarded human pharmaceuticals. Other possible contaminants investigated were selected disinfectants, some newer antibiotics, growth promoters, and various parasiticides. The sample preparation consisted of an acidic acetonitrile extraction followed by solid-phase extraction clean-up. Data were collected with a quadrupole-Orbitrap MS using both non-targeted and targeted acquisition. This rapid clean-up procedure and HRMS detection method described previously for veterinary drug residues also worked well for many other types of compounds. Most analytes had screening limit levels between 0.5-10 ng/g in the matrices examined using exact mass identification criteria. The strategy described in this paper for testing the performance of additional analytes will help expand the applicability of the HRMS procedure as aquaculture samples can now be analyzed for a wider range of contaminants.

Four analytical methods were developed and validated for the determination of veterinary drug residues and environmental pesticide residues in honey: (a) GC-MS method for the analysis of amitraz and all metabolites containing the 2,4-dimethylaniline moiety; (b) GC-MS method for the analysis of thymol, chlorfenvinphos and coumaphos; (c) GC-MS method for the analysis of 75 active substances; (d) LC-MS/MS method for the analysis of 60 active substances. Between the GC-MS (method c) and the LC-MS/MS method (method d) there was no overlap among active substances, meaning that using both methods 135 active substances originating from the environment in total were included and validated. The first method involved hydrolysis of amitraz and its metabolites containing the 2,4-dimethylaniline moiety to 2,4-dimethylaniline and extraction of 2,4-dimethylaniline to n-hexane. The other three methods had the same extraction procedure with a mixture of solvents: acetone, dichloromethane and petroleum ether. All 4 methods were tested in practice. Sixty samples of honey were analysed: 22 from organic and 38 from conventional production. Overall, residues were mainly higher than reported in literature but did not exceed MRLs. Risk assessment confirmed that the analysed samples are of no cause for concern for consumers.

A simple and reliable method using liquid chromatography with diode array detector was developed for the simultaneous determination of florfenicol and thiamphenicol in medicated feed. The analytes were extracted from the minced feed with methanol and ethyl acetate (1:1, v/v). Next, the extract was further cleaned up by dispersive solid phase extraction using anhydrous magnesium sulfate, PSA and C18 sorbents. Finally, 1 mL of extract was evaporated, the residue resuspended in Milli-Q water, and filtered. The method was validated in-house at medicated levels, in the concentration range 10-300 µg/mL (50-1500 mg/kg). Values of <6.5% and <6.0% were found, respectively, for repeatability and within-laboratory reproducibility. The LODs for the two fenicols were 2.4-5.3 mg/kg, while the LOQs were 3.8-5.6 mg/kg. The expanded uncertainty was estimated to be in the range of 10.0-14.5%, depending on the analyte. Recoveries varied from 81.7% to 97.5%. The methodology was applied to the analysis of animal feedingstuffs collected from poultry and pig farms.

This paper presents the results from a residue study conducted on a statistically representative number of chicken broilers that were individually orally treated with the selected nitroimidazoles (metronidazole, ornidazole and ipronidazole) in an appropriate amount close to the theoretical therapeutic dose. A mutual persistence comparison of the monitored analytes in feathers, serum, muscle and shanks was performed and attention was also paid to selected metabolites (hydroxymetronidazole and hydroxyipronidazole). An analytical LC/MS/MS method using SupelMIP SPE nitroimidazoles cartridges was developed for the determination of nitroimidazoles residues in poultry feathers, serum, muscle and shanks and the method was validated according to Commission Decision 2002/657/EC. High concentrations of nitroimidazoles residues in feathers were observed 19 days after the broilers' treatment unlike the muscle and serum samples, where nitroimidazoles depletion was significantly faster (residue concentrations were below detection limits in 5 days in muscle and in 12 days in serum). Shanks (chicken claws) also proved to be a very useful matrix for the detection of nitroimidazoles drugs misuse due to the longer persistence of these drugs residues and their metabolites in this matrix (determinable concentrations were observed 19 days after the broilers' last treatment). Feathers and shanks appear to be suitable matrixes for the screening of various nitroimidazoles in poultry because long-term persistence of residues enables reliable detection of the illegal use of nitroimidazoles compounds in official checks.

Violative residues of florfenicol (FF) in porcine edible tissues pose a potential risk for human health. In this study, urine was selected as target matrix for routine residue monitoring of FF in pig, and a thin layer chromatography (TLC)-high-performance liquid chromatography (HPLC) method was developed for simultaneously determining FF and florfenicol amine (FFA) in porcine urine. The urine samples were extracted with ethyl acetate under alkaline environment. The extracts were enriched through evaporation, purified by TLC and analysed by HPLC at 225 nm. A Waters Symmetry C 18 column was used for the separation of the two analytes. The mobile phase was acetonitrile-phosphate buffer mixtures (33.3: 66.7, v/v), and was pumped at 0.6 mL/min. The TLC-HPLC method was well validated and successfully applied to residue depletion study. Good analytical specificity was confirmed by the lack of interfering peaks at the retention times of FF and FFA. The standard curves showed good linearity (FF: y = 143064x - 1045.3, r = 0.9999; FFA: y = 275826x + 1888.8, r = 0.9999) over the range of 0.0625-8 μg/mL. The precision ranged from 0.83% to 11.66% and 2.19% to 8.75% for intraday and interday determination, respectively. The corresponding accuracy ranged from -13.38% to 10.78% and -12.15% to 7.14%, respectively. The limits of quantification (LOQs) for FF and FFA were 0.125 μg/mL. The residue depletion study showed that the concentrations of FF and FFA in urine were higher than those in edible tissues at three time points. This method was reliable, simple and cost efficient, and could be used to monitor FF residues in porcine edible tissue without slaughtering animals. TLC showed excellent purification efficiency and is expected to solve matrix interferences in veterinary drug residue analysis.

The available information on drug residue stability in chicken egg is scarce. The objective of this study was to evaluate the stability of drug residues in egg under different traditional cooking procedures. Fresh eggs were spiked with different drug concentrations of albendazole (ABZ) and its albendazole sulphoxide (ABZSO) and albendazole sulphone (ABZSO 2 ) metabolites; flubendazole (FLBZ) and its reduced flubendazole (R-FLBZ) and hydrolyzed flubendazole (H-FLBZ) metabolites; amoxicillin (AMX); and enrofloxacin (EFX) and its ciprofloxacin (CFX) metabolite. The egg samples were cooked in different ways, namely, boiling, microwaving, and omelette making. Drug residue concentrations in egg were quantified by HPLC with UV or fluorescence detectors. ABZ and ABZSO concentrations in egg were not affected by boiling and microwaving, while the omelette processing significantly reduced these molecules. Residues of ABZSO 2 in egg were stable or increased after all cooking procedures. In contrast, FLBZ and its metabolites FLBZ-H and FLBZ-R residues in egg decreased after all treatments. The residue concentration quantified for EFX and CFX did not show significant changes after any cooking method. AMX residues were unstable, with extremely significant drug reduction after all cooking processes. Conventional methods of egg cooking cannot be considered a tool to eliminate all veterinary drug residues.

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.

In the present study, a multi-class multi-residue method for the simultaneous detection and determination of antibiotics in European sea bass (Dicentrarchus labrax) was developed and validated. The method based on UHPLC-MS/MS proved to be a rapid, highly selective and sensitive tool, requiring minimum sample preparation, for screening and detection of 47 compounds from eight different classes. The validation was performed according to EU regulation 2002/657/EC, proving the method's suitability for application in routine analysis. The method was applied to the analysis of 30 samples of farmed European sea bass purchased in different supermarkets in Portugal. Antibacterial residues were detected in 6 of the 30 analysed samples, namely enrofloxacin and oxytetracycline, in concentrations ranging from 0.1 to 12 µg kg -1 .

In many countries, ractopamine hydrochloride (RAC) is allowed to be used in animal production as a β-agonist, which is an energy repartitioning agent able to offer economic benefits such as increased muscle and decreased fat deposition, feed conversion improvement and an increase in average daily weight gain. However, some countries have banned its use and established strict traceability programmes because of pharmacological implications of β-agonist residues in meat products. In Brazil, commercial RAC is controlled (5-20 mg kg -1 ) and only added to pig diet during the last 28 days before slaughter. However, the control is more difficult when co-products, like meat and bone meal (MBM), which can be produced from RAC treated animals, are part of the feed composition. Therefore, a study was undertaken to evaluate the presence of RAC residue concentrations in urine and tissues of gilts (n = 40) in four dietary groups: 0%, 7%, 14% and 21% (w/w) of MBM-containing RAC (53.5 µg kg -1 ). The concentration of RAC residues in MBM, pig tissues and urine was determined by LC-MS. Low RAC concentrations were detected in muscle, kidney, liver and lungs (limit of detection = 0.15, 0.5, 0.5 and 1.0 µg kg -1 , respectively); however, no RAC residues were quantified above the limit of quantification (0.5, 2.5, 2.5 and 2.5 µg kg -1 , respectively). In urine, the RAC concentration remained below 1.35 µg L -1 . These data suggest that MBM (containing 53.5 µg kg -1 RAC) added to diet up to 21% (w/w) could hamper the trade where RAC is restricted or has zero-tolerance policy.

Salmon consumption is increasing year by year. Salmon aquaculture is the fastest growing food production system in the world, and often uses feed mixed with antibiotics or other drugs. Feed can be also contaminated by environmental contaminants like persistent organic pollutants and organophosphorus pesticides that usually accumulate in fatty tissue, or emerging contaminants such as perfluoroalkyl substances (PFASs), that instead bioaccumulate in protein tissues. Therefore, there is the need to investigate the presence of antibiotics and environmental contaminants, with multi-class and multi-residue liquid chromatography-high resolution mass spectrometry and gas chromatography tandem mass spectrometry methods to monitor a broad spectrum of residues comparing between wild and farmed salmons. The presence of residues was encountered at a concentration range of 0.35-51.52 ng g -1 for antibiotics only in farmed salmon, 0.19-34.51 ng g -1 for PFASs and 0.26-9.01 ng g -1 for (polybrominated diphenyl ethers) PBDEs, and 0.19-5.91 ng g -1 for organochlorine pesticides with higher frequencies and concentrations in farmed fish. Finally, the risk deriving from salmon intake is low, being of minor concern only for PBDE 99 and perfluorooctanoic acid.

The Infiniplex for milk® (IPM) kit is a quick method for the simultaneous and qualitative detection of more than 100 molecules including antibiotic residues, mycotoxins, anti-inflammatories and antiparasitic drugs into a single test that does not require milk treatment. The IPM® kit was validated according to the European decision EC/2002/657 and according to the European guideline for the validation of screening methods (2010). Our validation was focused only on antibiotic residues. The washing step was identified as the most critical step of the assay. Insufficient washes could cause a significant background noise that prevents imaging. Positive controls have to be freshly prepared each day (insufficient stability). The method was specific with a low false-positive rate of 1.7% on 5 discrete test regions (DTR) ((beta-lactams, lincomycin, virginiamycin, quinolones and sulphonamides)) and a false-positive rate of 0% on the 26 other DTR. During our validation, the 42 determined detection capabilities CCβ for 12 antibiotic families (aminoglycosides, cephalosporins, lincosamides, macrolides, miscellaneous antibiotics, penicillins, phenolated polymixins, polypeptide antibiotics, quinolones, sulphonamides, tetracyclines) were at between once and twice the decision levels stated by the manufacturer. Forty CCβ determined were lower than the respective regulatory limits (i.e. MRL, RC, MRPL) in milk, except for tilmicosin (1.5 times the MRL) and neospiramycin (>1.25 times the MRL). The estimated CCβ of thiamphenicol, cloxacillin, danofloxacin, sulphathiazol, ceftiofur and sulphamonomethoxine were lower than or at the MRL. However, it was difficult to approach an accurate CCβ with only qualitative results. It is impossible to know whether or not we were close to the cut-off value. The software could be improved by differentiating between low-positive and high-positive results. The results of our participation in three qualitative proficiency tests in 2016 and 2017 for the detection of quinolones, tetracyclines and sulphonamides in cows' milk were very satisfactory.

A general solid-phase extraction (SPE) method using ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) for the determination of moroxydine residues in pig and chicken samples has been developed. After extraction and purification of real samples, moroxydine residues were detected using a hydrophobic interaction liquid chromatography column with an optimised mobile phase composition. The extraction reagents, the kind of SPE columns and the type of eluents were optimised to achieve the maximum extraction efficiency. The matrix effects from the animal tissue influenced the quality of the quantitative data obtained. Under the optimised conditions, the moroxydine residues in pig and chicken samples spiked at three levels (1.0 μg/kg, 5.0 μg/kg and 10.0 μg/kg) were determined with good recoveries (61.5%-105.4%) and adequate relative standard deviations (3.2%-13.0%). In pig and chicken samples, the limit of detection (LOD) was 0.3 μg/kg, and the limit of quantification (LOQ) was 1.0 μg/kg. A sufficiently linear relationship in the range of 1.0 μg/kg-20.0 μg/kg was achieved with a good correlation coefficient (R 2  ≥ 0.99).

The residue depletion of niclosamide (NIC) in freshwater fish including blunt snout bream (Megalobrama amblycephala), yellow catfish (Pelteobagrus fulvidraco) and channel catfish (Ietalurus Punetaus) were investigated under laboratory conditions. NIC concentrations in fish were determined by Liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) with heated electrospray ionisation. The mean recoveries were in the range of 75.5-98.5%, with relative standard deviations ranging from 1.0 to 9.4%. The depletion results showed that the long half-life of NIC was 8.8-22.1 days. Risk assessment of NIC residue in edible tissues (muscle and skin) of the three species of freshwater fish was performed using the quotient (RQ), because of no maximum residue limit (MRL) has been set for NIC in fish. The results demonstrated that the RQ values were all significantly lower than 1. Therefore, the effect of NIC residues in the three species of freshwater fish at the immersion concentration was negligible to Chinese people.

A simple, reliable and sensitive liquid chromatography-tandem mass spectrometry-based confirmatory method was redeveloped and validated for the simultaneous determination of chloramphenicol, thiamphenicol, florfenicol and florfenicol amine in chicken muscles. The analytes were extracted from minced chicken muscle with acetonitrile and ammoniated water mixture. A second extraction with ethyl acetate was followed by evaporation and dissolution of the residue in ammoniated methanol before defatting with n-hexane. Finally, the extract was further cleaned up by dispersive solid phase extraction using C-18 end-capped dispersive material. The validation protocol was adapted from the European Commission Decision 2002/657/EC and all the performance characteristics were successfully satisfied. The recoveries of all the analytes were found to be in the range of 86.4-108.1% and the precision values, within day and between days, ranged from 2.7% to 11% and 4.4% to 16.3%, respectively. The method was tested in various incurred samples and applied to analyse a wide range of random poultry market samples (n = 120) collected from three cities of the Punjab, Pakistan. Chloramphenicol and florfenicol residues were detected at low levels in less than 11% of the samples. Chloramphenicol was detected only in 4 samples with the concentration range of 0.17-0.477 µg kg -1 , whereas the levels of florfenicol/florfenicol amine residues detected in 9 samples ranged from 8.7 to 32.8 µg kg -1 . Moreover, most of the florfenicol residues were identified as tissue bound, extractable only after strong acid hydrolysis.

Demand for honey is increasing, especially if it is organic and if its nutritional properties are linked to untreated environments in order to guarantee quality for health. Sources of contamination of honey can be divided into environmental and apicultural. Therefore, the distribution of persistent organic pollutants, pesticides and antibiotic residues from geographical areas with different contamination sources (high anthropic impact, intensive farming, husbandry and low anthropic impact) was investigated in order to confirm the potential transfer of xenobiotics into the supply chain and to give beekeepers tools for the selection of areas dedicated to organic production. The presence of polychlorinated biphenyls, polybrominated diphenyl ether and polycyclic aromatic hydrocarbons was confirmed, not only in proximity to highly urbanised centres, where the concentrations were higher, but in all environment contexts, confirming their ubiquity. No antibiotics or neonicotinoids were detected in 95 organic honeys, demonstrating the absence of apicultural treatments and consequently the good quality of honey of different areas. These results are important due to the undefined regulatory European situation on honey antibiotic limits.

In this work, high affinity polyclonal antibodies for ribavirin (RBV) from new haptens were prepared and were used to analyse RBV residues in chicken muscle, eggs and duck muscle. The new haptens were synthesised with different spacers, and the best antibody was obtained with an IC 50 value as low as 0.61 ng/mL in indirect competitive enzyme-linked immunosorbent assay (ELISA). The cross-reactivities with another five antiviral drugs including amantadine, rimantadine, moroxydine, zanamivir and oseltamivir were less than 0.1%, which indicated the good specificity of the antibody. An ELISA was developed based on the antibody and applied to detect RBV in multi-food matrices. The sample preparation prior to detection only needed simple dilution after trichloroacetic acid extraction. The limits of detection were 1.07, 1.18 and 1.03 μg/kg in chicken muscle, eggs and duck muscle, respectively. Recoveries ranged from 89.0% to 112.7% with coefficients of variation below 13.0%. Ten blind samples of chicken muscle were analysed simultaneously by ELISA and liquid chromatography-tandem mass spectrometry, and a good correlation between the methods was observed. The results indicated that the high affinity antibody could be applied for the simple and fast detection of RBV in multi-food matrices.