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La lactancia materna puede afectarse en bebés con fisura de paladar, debido a diversos factores asociados a la alimentación. El objetivo del estudio fue relacionar el tiempo de lactancia materna y el tiempo de extracción de leche materna con factores asociados a la alimentación en bebés de entre 0 y 6 meses portadores de fisura de paladar. Se revisaron registros electrónicos de atenciones de 36 bebés portadores de fisura de paladar de un hospital público de Chile. Fueron extraídas características sociodemográficas de la madre y padre, antecedentes perinatales y postnatales de los bebés y características generales de la alimentación hasta los 6 meses. Se compararon los datos con el Test de la U de Mann-Whitney y se establecieron relaciones con las correlaciones de Pearson y Spearman. En el tiempo de lactancia materna directa (días) se observaron diferencias significativas en: los bebés que utilizaron sonda nasogástrica (p = 0.009), la indicación de extracción de leche materna (p = 0.017), la presencia de extracción manual de leche materna (p = 0.005) y la presencia de extracción eléctrica de leche materna (p = 0.002). Además, se registró una correlación positiva (p =

Vicerrectoria de Investigacion y Desarrollo (VID) of the Universidad de Chile PROA004/18

The presence of antimicrobial and pesticide residues in products sourced from peasant family farms selected from eight regions of Chile was determined. A total of 204 samples were analyzed from family farm products, including honey, lettuce, tomato, strawberry, raspberry, lamb meat, and cow's milk. Pesticide residues were found in 43 of 107 samples tested for them, but only 4 samples had concentrations that exceeded the maximum residue limits (MRLs) set by Chilean regulations. As for animal products, 2 of 15 cow's milk samples tested positive for permethrin residues; however, these concentrations did not exceed Chilean MRLs. No pesticide residues were detected in honey samples. As for antimicrobial drugs, 4 of 14 lamb meat samples and 11 of 79 cow's milk samples tested positive for different classes of antimicrobial residues, such as tetracyclines, macrolides, aminoglycosides, and β-lactams. Among these, only three samples exceeded the MRLs for these matrices. Traces of tetracyclines and sulfonamides were detected in 7 of 29 honey samples. These findings show that these chemical contaminants are present in trace concentrations in foodstuffs produced by peasant family farms in Chile; however, most residues did not exceed the regulatory limits. This study is the first assessment of the presence of residues from antimicrobial drugs and pesticides in food products sourced from peasant family farms in Chile. Data about the current state of drug residues in this segment of food products provide a baseline for efforts to close possible gaps in current surveillance schemes.

Recent studies have detected different antimicrobial residues in broiler chicken feathers, where they persisted for longer periods of time and at greater concentrations than in edible tissues. However, until today, lincomycin behaviour in this nonedible tissue has not been assessed yet. Considering this, an analytical methodology to detect and quantify this antibiotic concentration in feathers, muscle, and liver tissues from broiler chickens was implemented and in-house validated. The methodology will allow the determination of the bioaccumulation of this highly persistent antibiotic in feathers of treated birds. For this purpose, 98% lincomycin and 95% lincomycin D3 standards were used. Methanol was selected as the extraction solvent, and Chromabond® Florisil® cartridges were used for the clean-up stage. The separation of analytes was performed through the analytical column SunFire C18 with a running time of 4 minutes, and the instrumental analysis was performed through an LC-MS/MS, with a liquid chromatograph Agilent® 1290 Infinity, coupled to an AB SCIEX® API 5500 mass spectrometer. An internal protocol for an in-house validation was designed based on recommendations from Commission Decision 2002/657/EC and the Guidance document on the estimation of limit of detection and limit of quantification for measurements in the field of contaminants in feed and food. The average retention time for lincomycin was 2.255 min (for quantifier ion, 126.0). The calibration curves showed a coefficient of determination (r2) greater than 0.99 for all matrices, while recovery levels ranged between 98% and 101%. The limit of detection (LOD) calculated was of 19, 22, and 10 μg·kg−1, and the limit of quantification (LOQ) was of 62, 73, and 34 μg·kg−1 in feathers, muscle, and liver, respectively. This method detects lincomycin in the studied matrices, confidently and accurately, as it is required for designing analytical studies of drug residues in edible and nonedible tissues, such as feathers.

Shiga toxin-producing Escherichia coli (STEC) are zoonotic pathogens responsible for causing food-borne diseases in humans. While South America has the highest incidence of human STEC infections, information about the genomic characteristics of the circulating strains is scarce. The aim of this study was to analyze genomic data of STEC strains isolated in South America from cattle, beef, and humans, predicting the antibiotic resistome, serotypes, sequence types (STs), clonal complexes (CCs) and phylogenomic backgrounds. A total of 130 whole genome sequences of STEC strains were analyzed, where 39.2% were isolated from cattle, 36.9% from beef, and 23.8% from humans. The ST11 was the most predicted (20.8%) and included O-:H7 (10.8%) and O157:H7 (10%) serotypes. The successful expansion of non-O157 clones such as ST16/CC29-O111:H8 and ST21/CC29-O26:H11 is highlighted, suggesting multilateral trade and travel. Virulome analyses showed that the predominant stx subtype was stx2a (54.6%), most strains carried ehaA (96.2%), iha (91.5%) and lpfA (77.7%) genes. We present genomic data that can be used to support the surveillance of STEC strains circulating at the livestock-food-human interface in South America, in order to control the spread of critical clones “from farm to table”.

Although the relationship between the use of antimicrobials and the development of resistant bacteria is well established, information about the use of antimicrobials in companion animals has been poorly described, which represents a risk to public health. The aim of this study was to describe the antimicrobial prescription patterns of Chilean companion animal veterinarians. A nationally distributed survey targeted at companion animal veterinarians was designed. The survey included questions about the veterinarian&rsquo, s demographics, bacterial diseases treated, prescribed antimicrobials, and the use of laboratory diagnostic tools. Three hundred twenty-three responses were collected, most of the respondents were female (59.4%). The most frequently reported bacterial diseases were pyoderma (17.2%), followed by otitis and abscesses (7.4%). The antimicrobials most used corresponded with critically or highly important drugs for veterinary and human use, including &beta, lactams (65.3%), quinolones (36.2%) and tetracyclines (23.2%). Only 15% of the veterinarians reported the use of laboratory diagnostic tests, although 67% declared they were aware of the official antimicrobial classification schemes. Our results describe for the first time the usage of antimicrobials by veterinarian practitioners in Chile to treat different diseases in companion animals. The data presented here provide a baseline that could help to promote the implementation of clinical guidelines and regulations in order to improve current treatments.

Salmonella Infantis is a zoonotic pathogen that causes gastroenteritis in humans and animals, with poultry being its main reservoir. In Chile, there are no data to characterize S. Infantis strains in poultry production. In this study, 87 S. Infantis strains were isolated from chicken meat for sale in supermarkets in Santiago, Chile, and characterized according to their virulence genes, biofilm formation abilities, antibiotic susceptibility, and resistance genes. Through polymerase chain reaction or PCR, the strains were analyzed to detect the presence of 11 virulence genes, 12 antibiotic resistance genes, and integrase genes. Moreover, disc diffusion susceptibility to 18 antimicrobials and the ability to form biofilm in vitro were evaluated. Results demonstrated six different virulence gene profiles. Ninety-four percent of the strains were multi-resistant to antibiotics with weak biofilm formation abilities, 63.2% of the strains were broad spectrum &beta, lactam resistant, and the bla CTX-M-65 gene was amplified in 13 strains. Only 3.4% of the strains were fluoroquinolone resistant, and the qnrB gene was amplified in two strains. Colistin resistance was exhibited in 28.7% of the strains, but mrc genes were not amplified in any strain under study. The isolated S. Infantis strains are pathogenic and antibiotic multi-resistant, and thus, this Salmonella serotype should be under surveillance in the poultry food production chain with the aim of protecting public health.

Simple Summary Eggs are a readily available and important food source for low income families that raise chicken in their households. It is therefore important to monitor if these products are safe for human consumption and contain no antibiotic residues that could lead to allergic reactions, intoxication, or antimicrobial resistance. However, little is known about the antimicrobial content of eggs consumed in rural households in Chile. Consequently, the aim of this study was to collect chicken eggs from rural households in the central region of Chile and screen them for antimicrobial activity. The results indicate that most collected eggs (73% of the 83 surveyed households), exhibited antimicrobial activity for at least one of the four tested antimicrobials. These results indicate that household members who consume these eggs are in danger of developing antibiotic-related illnesses and could contribute to antimicrobial resistance. Therefore, further studies are required to identify the exact compounds used to treat the chickens and to establish preventive measures to eradicate antimicrobial presence in their food supply. Abstract Eggs are the main product generated from backyard poultry production systems (BPS) because they can quickly be consumed and sold to meet essential family needs. Nevertheless, antimicrobial residues can accumulate in this product. The objective of this study was to evaluate the presence of antimicrobial residues in eggs produced by poultry kept in BPS in central Chile. To assess this, eggs were obtained from 83 BPS and analysed to evaluate the presence of antibiotic residues (families: tetracyclines, beta-lactams, aminoglycosides and macrolides), using a Four-Plate Test screening method for the detection, based on a bacterial growth inhibition method. Results show a lack of biosecurity procedures at BPS level, making these systems susceptible to the dissemination of antimicrobial residues. These include intensive animal production units in the proximity, and the presence of shared watercourses with other farms. Furthermore, 66% of the surveyed owners are indicated as giving pharmacological treatments to their chickens. Eggs from 61 BPS were positive for at least one antimicrobial. Fifty-three BPS were positive for more than one antimicrobial, and one BPS was positive for all four antimicrobials tested. Consequently, there is a risk that poultry eggs produced in BPS in central Chile carry residues of different families of antimicrobials.

Successful treatment of diseases that sicken food-producing animals requires the use of antimicrobials. However, these drugs could result in serious consequences for human, animal, and environmental health. The emergence of antimicrobial resistance requires better communication with consumers to inform them about the risks associated with the use of these drugs, as well as the benefits gained from their rational use. This work studied consumer perceptions about the use of antimicrobials in the production of animal origin foods by questioning 72 consumers in nine focus groups. The participants were all inhabitants of the Metropolitan Region, Chile, and were assigned to focus groups of 5 to 12 individuals each, according to their age: age groups I, II, and III spanned age ranges of 25 to 35, 36 to 50, and older than 50 years, respectively. Consumers regarded the use of antimicrobials in farm animals as a relevant and necessary practice, as long as it was done adequately by veterinarians to ensure productivity and to guarantee food quality and safety. Even though most consumers identified antimicrobial use as a potential risk to their health, only a few recognized that antimicrobial resistance could be transferred from animals to humans via food consumption. Also, consumers complained that they did not have access to enough information about foods of animal origin. Consequently, they stated that food industry and regulatory entities should ensure transparency of production processes and also educate the population on this topic. Consumer perceptions about responsible and judicious use of antimicrobials may become an ally in the effort to reduce and improve the use of these drugs in animal production systems.

The objective of this research study was to estimate the withdrawal time (WT) for oxytetracycline (OTC), administered intraperitoneally to two species of salmonids, rainbow trout (Oncorhynchus mykiss) and Atlantic salmon (Salmo salar). This was done in salmonid farms during the fattening phase in seawater, under field conditions. Seven cages from different marine farms were included, each as an experimental unit, treated with an intraperitoneal dose of OTC (in a range from 20 to 35 mg kg−1). From each sea cage, 10 fishes were randomly selected for laboratory analysis at each sampling point after treatment, which were variable according to the harvest of each cage. All selected fish were treated with a single dose of two different commercial OTC formulations; Formulation A (OTC 30%) and Formulation B (OTC 20%), both extra label. Study was performed according to VICH GL 57 guideline. Samples were kept cold until laboratory analysis. Homogenized samples were analyzed using a mass triple quadrupole spectrometer (Waters®, Xevo TQ-S micro). The analytical method was optimized for use in the laboratory and an internal validation procedure was performed following European Commission Decision 2002/657/EC for linearity, recovery and precision. The detection range, including the limit of detection (LOD) and limit of quantification (LOQ), were calculated according to VICH GL49 guidance. According to the degree days obtained from the residue depletion study and those observed in the last sampling, most of the sampled cages had concentrations that allow the veterinary drug to be suitable for a market with a maximum residue limit (MRL) value of 200 μg kg−1, but not for a market with an MRL of 10 μg kg−1, as might be expected, regardless of the dose and commercial veterinary drug used. Depending on the considered MRL, withdrawal times ranged from 5390 to 1676- and 4061–1641-degree days, for rainbow trout and Atlantic salmon, respectively. These estimates, obtained from field samples, can provide references regarding the WTs that must be considered in order to avoid the presence of OTC + 4-epi-OTC residues in Atlantic salmon and rainbow trout fillets, and thus protect the consumer health. Further studies are needed to assess the impact of other factors on WT under field conditions in different salmonid species.

Antimicrobials are currently used in poultry for disease treatment. However, their excretion in bird feces may contaminate the environment. Considering this, the objective of this work was to quantify antimicrobials residues concentrations in therapeutically treated broiler chicken droppings throughout the post-treatment period. For this aim a multiresidue method using high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) was validated. Forty-eight male broiler chickens were distributed and treated with commercial formulations of 5 different antimicrobials. Results showed that oxytetracycline and 4-epi-oxytetracycline, presented the highest concentrations during all sampling period, detecting concentrations of 1471.41 µg kg−1 at the last sampling point (day 22 post-treatment). Florfenicol, tylosin, enrofloxacin, and ciprofloxacin were eliminated and detected in treated chicken droppings until d 18 post-treatment. Sulfachloropyridazine decrease gradually during post-treatment period until day 30. Results demonstrate that studied antimicrobials in treated chicken droppings were eliminated for prolonged periods, therefore becoming a significant route of residues dissemination into the environment.

Simple Summary After administration of antimicrobials, poultry excrete significant concentrations of antimicrobials through their droppings, which accumulate in the litter where poultry are housed. Poultry litter, which consists mainly of wood shavings, feathers, and droppings, is widely used as an agricultural fertilizer worldwide. The period that antimicrobials persist in agricultural soils could present various environmental and public health concerns. Thus, in this research, a method to identify different families of antimicrobials in poultry litter was developed. Results show that HPLC–MS/MS can reliably detect nine different compounds from three families of antimicrobials. This method was used to identify antimicrobials from commercial poultry flocks, providing a valuable and specific tool to monitor these residues in poultry litter prior to its use as an agricultural fertilizer. Abstract Tetracyclines, sulphonamides, and quinolones are families of antimicrobials (AMs) widely used in the poultry industry and can excrete up to 90% of AMs administrated, which accumulate in poultry litter. Worryingly, poultry litter is widely used as an agriculture fertilizer, contributing to the spread AMs residues in the environment. The aim of this research was to develop a method that could simultaneously identify and quantify three AMs families in poultry litter by high-performance liquid chromatography–tandem mass spectrometry (HPLC–MS/MS). Samples of AMs free poultry litter were used to validate the method according to 657/2002/EC and VICH GL49. Results indicate that limit of detection (LOD) ranged from 8.95 to 20.86 μg kg−1, while limits of quantitation (LOQ) values were between 26.85 and 62.58 µg kg−1 of tetracycline, 4-epi-tetracycline, oxytetracycline, 4-epi-oxytetracycline, enrofloxacin, ciprofloxacin, flumequine, sulfachloropyridazine, and sulfadiazine. Recoveries obtained ranged from 93 to 108%. The analysis of field samples obtained from seven commercial poultry flocks confirmed the adequacy of the method since it detected means concentrations ranging from 20 to 10,364 μg kg−1. This provides us an accurate and reliable tool to monitor AMs residues in poultry litter and control its use as agricultural fertilizer.

The objective was to gather information on the status of antimicrobial surveillance in the Asia Pacific region and suggest control strategies. Twenty-one economies of the Asia Pacific region participated in this initiative. A survey was conducted on antimicrobial use and surveillance throughout the region. A workshop was carried out to create awareness about the issue and discuss the implementation of control strategies. Based on the survey results and workshop conclusions, it can be established that there is better understanding of the implications of antimicrobial resistance in the human medicine area. Only few economies take actions to control antimicrobial resistance on a veterinary/agricultural level. To confront antimicrobial resistance, it is critical to raise awareness; cooperation between all countries is needed to apply international standards, to be able to have harmonized public policies. Countries must align and improve their systems for surveillance and monitoring of antimicrobial resistance in human, animals, and the environment.

A suitable analytical method is required to study the behaviour of florfenicol (FF) and its metabolite florfenicol amine (FFA) in broiler’s feathers. An LC-MS/MS method was developed, assessed and intra-laboratory-validated for FF and FFA analyses. We chose cloramphenicol-d5 as an internal standard, acetone as a solvent for the extraction of the analytes and dichloromethane for the clean-up. Through LC-MS/MS analysis, we established a detection limit of 20 μg kg–1, as well as calculated quantification limits of 24.4 and 24.5 μg kg–1 for FF and FFA, respectively. Validation parameters such as linearity, recovery and precision were calculated following Commission Decision 2002/657/EC. For linearity, all standard curves showed a standard coefficient greater than 0.99. Recoveries ranged from 99% to 102% for all studied concentrations. The results show that this analytical method is precise and reliable. For the depletion study, 64 Ross 308 broilers were treated with a therapeutic dosage of 10% FF duri...

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.

Salmonid Rickettsial Septicemia (SRS) is the disease of greatest economic importance in the Chilean salmon farming industry, causing high mortality in fish during the final stage of their productive cycle at sea. Since current, commercially available vaccines have not demonstrated the expected efficacy levels, antimicrobials, most commonly florfenicol, are still the main resource for the treatment and control of this pathogen. The aim of this study was to determine the most appropriate single dose of florfenicol, administered through medicated feed, for the treatment of Piscirickettsia salmonis (P. salmonis), using pharmacokinetic/pharmacodynamic (PK/PD) models. Previously, Minimum Inhibitory Concentrations (MICs) of florfenicol were determined for 87 P. salmonis isolates in order to define the epidemiological cut-off point (COWT). The most commonly observed MIC was 0.125 μg mL-1 (83.7%). The COWT value was 0.25 μg mL-1 with a standard deviation of 0.47 log2 μg mL-1 and 0.36 log2 μg mL-1, for Normalized resistance interpretation (NRI) method and ECOFFinder method, respectively. A MIC of 1 μg mL-1 was considered the pharmacodynamic value (PD) to define PK/PD indices. Three doses of florfenicol were evaluated in fish farmed under controlled conditions. For each dose, 150 fish were used and blood plasma samples were collected at different time points (0–48 hours). PK parameters were obtained from curves representing plasma concentrations as a function of time. The results of Monte Carlo simulation indicate that at a dose of 20 mg/Kg l.w. of florfenicol, administered orally as medicated feed, there is 100% probability (PTA) of achieving the desired efficacy (AUC0-24h/MIC>125). According to these results, we suggest that at the indicated dose, the PK/PD cut-off point for florfenicol versus P. salmonis could be 2 μg mL-1 (PTA = 99%). In order to assess the indicated dose in Atlantic salmon, fish were inoculated with P. salmonis LF-89 strain and then treated with the optimized dose of florfenicol, 20 mg/Kg bw for 15 days.

Tetracyclines, sulfonamides and amphenicols are broad spectrum antimicrobial drugs that are widely used in poultry farming. However, a high proportion of these drugs can be excreted at high concentrations in droppings, even after the end of a therapy course. This work intended to assess and compare concentrations of florfenicol (FF), florfenicol amine (FFa), chlortetracycline (CTC), 4-epi-chlortetracycline (4-epi-CTC), and sulfachloropyridazine (SCP) in broiler chicken droppings. To this end, 70 chickens were housed under controlled environmental conditions, and assigned to experimental groups that were treated with therapeutic doses of either 10% FF, 20% CTC, or 10% SCP. Consequently, we implemented and designed an in-house validation for three analytical methodologies, which allowed us to quantify the concentrations of these three antimicrobial drugs using liquid chromatography coupled to mass spectrometry (LC-MS/MS). Our results showed that FF and FFa concentrations were detected in chicken droppings up to day 10 after ceasing treatment, while CTC and 4-epi-CTC were detected up to day 25. As for SCP residues, these were detected up to day 21. Noticeably, CTC showed the longest excretion period, as well as the highest concentrations detected after the end of its administration using therapeutic doses.

Antimicrobial residues might persist in products and by-products destined for human or animal consumption. Studies exploring the depletion behavior of florfenicol residues in broiler chicken claws are scarce, even though claws can enter the food chain directly or indirectly. Hence, this study intended to assess the concentrations of florfenicol (FF) and florfenicol amine (FFA)&mdash, its active metabolite&mdash, in chicken claws from birds that were treated with a therapeutic dose of florfenicol. Furthermore, concentrations of these analytes in this matrix were compared with their concentrations in edible tissues at each sampling point. A group of 70 broiler chickens were raised under controlled conditions and used to assess residue depletion. Sampling points were on days 5, 10, 20, 25, 30, 35, and 40 after ceasing treatment, thus extending beyond the withdrawal period established for muscle tissue (30 days). Analytes were extracted using HPLC-grade water and acetone, and dichloromethane was used for the clean-up stage. Liquid chromatography coupled to mass spectroscopy detection (LC&ndash, MS/MS) was used to detect and quantify the analytes. The analytical methodology developed in this study was validated in-house and based on the recommendations described in the Commission Decision 2002/657/EC from the European Union. Analyte concentrations were calculated by linear regression analysis of calibration curves that were fortified using an internal standard of chloramphenicol-d5 (CAF-d5). The depletion time of FF and FFA was set at 74 days in claws, based on a 95% confidence level and using the limit of detection (LOD) as the cut-off point. Our findings show that FF and FFA can be found in chicken claws at higher concentrations than in muscle and liver samples at each sampling point.

Analytical methodology for the detection of sulfachloropyridazine (SCP) in samples of feathersvia LC-MS/MS was implemented based on techniques previously published by other authors: 1- Hindle R. A validated atmospheric pressure chemical ionization method for analysing sulphonamides in pork muscle. 2013. Available at http://www.youngin.com/application/0411-0045EN-E.pdf. Accessed October 12, 2017. 2- Renew JE, Huang CH. Simultaneous determination of fluoroquinolone, sulfonamide, and trimethoprim antibiotics in wastewater using tandem solid phase extraction and liquid chromatography–electrospray mass spectrometry. J Chromatogr A 2004; 1042: 113-21. 3- Shao B, Dong D, Wu y, Hu J, Meng J, Tu X, Xu S. Simultaneous determination of 17 Sulfonamide residues in porcine meat, kidney and liver by solid phase extraction and liquid chromatography-tandem mass spectrometry. Anal Chim Acta 2005; 546:174-81. 4- Pang G, Cao YZ, Zhang JJ, Jia GQ, Fan CL, Li XM, Liu YM, Li ZY, Shi YQ. Determination of sulfonamides in honey by liquid chromatography- tandem mass spectrometry. J AOAC Int 2005; 88:1304-11. 5- Stubbings G, Bigwood T. The development and validation of a multi-class liquid chromatography tandem mass spectrometry (LC– MS/MS) procedure for the determination of veterinary drug residues in animal tissue using a QuEChERS (QUick, Easy, CHeap, Effective, Rugged and Safe) approach. Anal Chim Acta 2009; 637:68-78. 6- Bedendo GC, Jardim IC, Carasek E. A simple hollow fiber renewal liquid membrane extraction method for analysis of sulphonamides in honey samples with determination by liquid chromatography-tandem mass spectrometry. J Chromatogr A 2010; 1217:6449-54. 7- Yu H, Tao Y, Chen D, Wang Y, Huang L, Peng D, et al. Development of a high-performance liquid chromatography method and a liquid chromatography-tandem mass spectrometry method with the pressurized liquid extraction for the quantification and confirmation of sulphonamides in the foods of animal origin. J Chromatogr B 2011; 879:2653-62. The method is based on a solid-liquid extraction with organic solvents. The analyte was concentrated using a water bath at 40-50°C under a mild nitrogen flow.For the instrumental analysis, a Symmetry C8 analytical column of 3.5μm and 2.1 x 100mm (Waters®) was fitted in an Agilent 1290 infinity series liquid-chromatograph equipment, coupled to an API 3200 (AB Sciex, Darmstadt, Germany) triple-quadrupole mass-spectrometer. The analytical data was then integrated using the Analyst® version 1.5 softwarepackage (SCIEX, Framingham, Massachusetts).

Analytical methodology for the detection of sulfachloropyridazine (SCP) in samples of feathersvia LC-MS/MS was implemented based on techniques previously published by other authors: 1- Hindle R. A validated atmospheric pressure chemical ionization method for analysing sulphonamides in pork muscle. 2013. Available at http://www.youngin.com/application/0411-0045EN-E.pdf. Accessed October 12, 2017. 2- Renew JE, Huang CH. Simultaneous determination of fluoroquinolone, sulfonamide, and trimethoprim antibiotics in wastewater using tandem solid phase extraction and liquid chromatography–electrospray mass spectrometry. J Chromatogr A 2004; 1042: 113-21. 3- Shao B, Dong D, Wu y, Hu J, Meng J, Tu X, Xu S. Simultaneous determination of 17 Sulfonamide residues in porcine meat, kidney and liver by solid phase extraction and liquid chromatography-tandem mass spectrometry. Anal Chim Acta 2005; 546:174-81. 4- Pang G, Cao YZ, Zhang JJ, Jia GQ, Fan CL, Li XM, Liu YM, Li ZY, Shi YQ. Determination of sulfonamides in honey by liquid chromatography- tandem mass spectrometry. J AOAC Int 2005; 88:1304-11. 5- Stubbings G, Bigwood T. The development and validation of a multi-class liquid chromatography tandem mass spectrometry (LC– MS/MS) procedure for the determination of veterinary drug residues in animal tissue using a QuEChERS (QUick, Easy, CHeap, Effective, Rugged and Safe) approach. Anal Chim Acta 2009; 637:68-78. 6- Bedendo GC, Jardim IC, Carasek E. A simple hollow fiber renewal liquid membrane extraction method for analysis of sulphonamides in honey samples with determination by liquid chromatography-tandem mass spectrometry. J Chromatogr A 2010; 1217:6449-54. 7- Yu H, Tao Y, Chen D, Wang Y, Huang L, Peng D, et al. Development of a high-performance liquid chromatography method and a liquid chromatography-tandem mass spectrometry method with the pressurized liquid extraction for the quantification and confirmation of sulphonamides in the foods of animal origin. J Chromatogr B 2011; 879:2653-62. The method is based mainly on a solid-liquid extraction with organic solvents. The analyte was concentrated using a water bath at 40-50°C under a mild nitrogen flow.For the instrumental analysis, a Symmetry C8 analytical column of 3.5μm and 2.1 x 100mm (Waters®) was fitted in an Agilent 1290 infinity series liquid-chromatograph equipment, coupled to an API 3200 (AB Sciex, Darmstadt, Germany) triple-quadrupole mass-spectrometer. The analytical data was then integrated using the Analyst® version 1.5 softwarepackage (SCIEX, Framingham, Massachusetts).

Analytical methodology for the detection of sulfachloropyridazine (SCP) in samples of feathersvia LC-MS/MS was implemented based on techniques previously published by other authors: 1- Hindle R. A validated atmospheric pressure chemical ionization method for analysing sulphonamides in pork muscle. 2013. Available at http://www.youngin.com/application/0411-0045EN-E.pdf. Accessed October 12, 2017. 2- Renew JE, Huang CH. Simultaneous determination of fluoroquinolone, sulfonamide, and trimethoprim antibiotics in wastewater using tandem solid phase extraction and liquid chromatography–electrospray mass spectrometry. J Chromatogr A 2004; 1042: 113-21. 3- Shao B, Dong D, Wu y, Hu J, Meng J, Tu X, Xu S. Simultaneous determination of 17 Sulfonamide residues in porcine meat, kidney and liver by solid phase extraction and liquid chromatography-tandem mass spectrometry. Anal Chim Acta 2005; 546:174-81. 4- Pang G, Cao YZ, Zhang JJ, Jia GQ, Fan CL, Li XM, Liu YM, Li ZY, Shi YQ. Determination of sulfonamides in honey by liquid chromatography- tandem mass spectrometry. J AOAC Int 2005; 88:1304-11. 5- Stubbings G, Bigwood T. The development and validation of a multi-class liquid chromatography tandem mass spectrometry (LC– MS/MS) procedure for the determination of veterinary drug residues in animal tissue using a QuEChERS (QUick, Easy, CHeap, Effective, Rugged and Safe) approach. Anal Chim Acta 2009; 637:68-78. 6- Bedendo GC, Jardim IC, Carasek E. A simple hollow fiber renewal liquid membrane extraction method for analysis of sulphonamides in honey samples with determination by liquid chromatography-tandem mass spectrometry. J Chromatogr A 2010; 1217:6449-54. 7- Yu H, Tao Y, Chen D, Wang Y, Huang L, Peng D, et al. Development of a high-performance liquid chromatography method and a liquid chromatography-tandem mass spectrometry method with the pressurized liquid extraction for the quantification and confirmation of sulphonamides in the foods of animal origin. J Chromatogr B 2011; 879:2653-62. The method is based on a solid-liquid extraction with organic solvents. The clean up is carried outthroughtaromatic sulfonic acid (Bakerbond spe™) disposable extraction columns of 6 mL. The analyte is concentrated using a water bath at 40-50°C under a mild nitrogen flow.For the instrumental analysis, a Symmetry C8 analytical column of 3.5μm and 2.1 x 100mm (Waters®) was fitted in an Agilent 1290 infinity series liquid-chromatograph equipment, coupled to an API 3200 (AB Sciex, Darmstadt, Germany) triple-quadrupole mass-spectrometer. The analytical data was then integrated using the Analyst® version 1.5 softwarepackage (SCIEX, Framingham, Massachusetts).

Several antimicrobials are routinely used by the poultry farming industry on their daily operations, however, researchers have found for some antimicrobials that their residues persist for longer periods in feathers than they do in edible tissues, and at higher concentrations, as well. But this information is not known for other classes of antimicrobials, such as the sulfonamides. Therefore, this work presents an accurate and reliable analytical method for the detection of sulfachloropyridazine (SCP) in feathers and edible tissues from broiler chickens. This method was also validated in-house and then used to study the depletion of sulfachloropyridazine in those matrices. The experimental group comprised 54 broiler chickens, who were raised under controlled conditions and then treated with a commercial formulation of 10% sulfachloropyridazine for 5 days. Samples were analyzed via LC-MS/MS, using 13C6-sulfamethazine (SMZ-13C6) as an internal standard. Aromatic sulfonic acid solid phase extraction (SPE) cartridges were used to clean up the samples. The Limit of Detection (LOD) for this method was set at 10 μg kg-1 on feathers and liver; and at 5 μg kg-1 on muscle. Within the range of 10-100 μg kg-1, the calibration curves for all matrices presented a determination coefficient greater than 0.96. Our results show, with a 95% confidence level, that sulfachloropyridazine persisted in feathers for up to 55 days after ceasing treatment, and its concentrations were higher than in edible tissues. In consequence, to avoid re-entry of antimicrobial residues into the food-chain, we recommend monitoring and inspecting animal diets that contain feather derivatives, such as feathers meals, because they could be sourced from birds that might have been medicated with sulfachloropyridazine.

Tylosin is one of the most commonly used antimicrobial drugs from the macrolide family and in broiler chickens it is used specially for the treatment of infectious pathologies. The poultry industry produces several by-products, among which feathers account for up to 7% of a chicken's live weight, thus they amount to a substantial mass across the whole industry. Feathers have been repurposed as an animal feed ingredient by making them feather meal. Therefore, the presence of high concentrations of residues from antimicrobial drugs in feathers might pose a risk to global public health, due to re-entry of these residues into the food chain. This work aimed to characterise the depletion behaviour of tylosin in feather samples, while considering its depletion in muscle and liver tissue samples as a reference point. To achieve this goal, we have implemented and validated an analytical methodology suitable for detecting and quantifying tylosin in these matrices. Sixty broiler chickens, raised under controlled conditions, received an oral dose of 32 mg kg-1 of tylosin for 5 days. Tylosin was quantified in muscle, liver and feathers by liquid chromatography coupled with a photodiode array detector (HPLC-DAD). High concentrations of tylosin were detected in feather samples over the whole experimental period after completing both the therapy and the recommended withdrawal time (WDT). On the other hand, tylosin concentrations in muscle and liver tissue samples fell below the limit of detection of this method on the first sampling day. Our results indicate that the WDT for feather samples is 27 days, hence using feather meal for the formulation of animal diets or for other agricultural purposes could contaminate with antimicrobial residues either other livestock species or the environment. In consequence, we recommend monitoring this matrix when birds have been treated with tylosin, within the context of poultry farming.

For the detection of tetraciclines in feathers, muscle and liver, an internal protocol was designed for in-house validation of two methods, through high performance liquid chromatography with tandem mass spectrometric (HPLC-MS/MS) techniques. This protocol was based on the recommendation of the decision 2002/657/EC from the European Community as well as the food and drug administration (FDA) VICHGL49. Limit of detection (LOD) was set at 20 μg kg-1 and the limit of quantification (LOQ) ranged from 21.5 to 24.2, 21.2 to 21.6, and 25.0 to 27.7 μg kg-1 in feahers, muscle and liver samples, respectively, for all analytes. The calibrations curves show a coefficient of determination (R2) above 0.98, 0.99 and 0.96 for feathers, muscle and liver samples, respectively. Analyte recovery ranged from 92 to 108%. In conclusion, these methods can be deemed accurate and reliable, and their validation is a fundamental step to be performed in depletion studies on these matrices.

Antibiotics are widely used in poultry production for the treatment of bacterial diseases. However, residues may remain in products and by-products destined for human consumption or animal feeding. The claws of chickens, which are a by-product of the poultry industry, can directly or indirectly enter the food chain as meals destined to feed other productive animals. Thus, it becomes necessary to determine and quantify antimicrobial residues present in this matrix. The objective of the study was to assess the depletion of oxytetracycline (OTC) and its metabolite 4-epi-OTC in broiler chicken's claws. Claws of 32 broilers treated with a therapeutic dosage of 10% OTC during 7 days were analysed. Samples were taken at days 3, 9, 15 and 19 post-treatment. As for the control group, eight broiler chickens were raised under the same conditions. Extraction was carried out through EDTA-McIlvaine buffer, and clean-up employed a SPE C-18 Sep-Pak®. Instrumental analysis was performed through LC-MS/MS. The concentrations of both analytes were determined in claw samples until day 19 post-treatment. Average concentrations were within the LOD (20 μg kg-1) and LOQ (22 µg kg-1) for OTC and 84 μg kg-1 for 4-epi-OTC. Withdrawal times (WDTs) of 39 days for OTC and 54 days for 4-epi-OTC were established in claws based on 95% confidence. These findings demonstrate that claws can be a source of antimicrobial residue entry into the food chain, since the results showed that OTC and its metabolite can be found in chicken's claws for long periods, even exceeding the average lifespan of a broiler chicken.