Investigation of doxycycline residues in bones after oral administration to broiler chickens.

Introduction: Chicken bones, a by-product of the poultry industry, can directly or indirectly enter the food chain. Bone meal and bone products could be sources of many contaminants. Considering the wide range of uses made of bones in the culinary and food industries, this material needs to be safe and antibiotic residue-free. To determine if such is the case, the concentration of doxycycline in chicken bones was investigated, this antimicrobial being one of the most commonly used in poultry production. Material and Methods: Ross 308 broilers were grouped into three experimental and one control group. Doxycycline was administered in drinking water at therapeutic and sub-therapeutic doses, as well as via spray treatment. The concentration of doxycycline in bones was determined post slaughter by ultra-high performance liquid chromatography-tandem mass spectrometry. Results: Doxycycline was quantified at 135 µg/kg 22 days after the last day of antibiotic administration at therapeutic doses; 2,285 µg/kg after sub-therapeutic treatment for 27 days and 9.62 µg/kg 22 days after the end of spray application. Conclusion: High concentrations and long persistence of doxycycline in bones were found in this study. Doxycycline can contaminate all bone-derived products in the food and fertiliser industries.

Simultaneous determination of 68 antimicrobial compounds in pigs oral fluid by ultra-high performance liquid chromatography-tandem mass spectrometry.

Improper use of antimicrobials in veterinary medicine can lead to residues in food of animal origin. Post-mortem monitoring of antibiotics in animal products is carried out as part of official EU programmes on food safety and consumer health. Oral fluid testing is a promising surveillance method to monitor appropriate treatment in pigs and to avoid residues in edible tissues. Oral fluid analysis can be implemented in an antibiotic residue control programme, thus preventing economic losses due to meat disposal as a result of drug detection in tissues after the withdrawal period. An analytical method was developed for the analysis of 68 compounds from 12 groups (penicillins, cephalosporins, sulfonamides, macrolides, fluoroquinolones, tetracyclines, aminoglycosides, pleuromutilins, diaminopyrimidines, lincosamides, polypeptides and sulfones) in pig oral fluid. Extraction of antibacterials was performed with 0.5 % formic acid. Analyses were carried out by ultra-high performance liquid chromatography with triple quadrupole mass spectrometry (UHPLC-MS/MS) detection. The chromatographic separation was achieved on a Zorbax analytical column (2.1 × 50 mm) with a mobile phase consisting of acetonitrile and heptafluorobutyric acid (HFBA). The total run time was 7 min. The method was validated as a confirmatory method according to the Commission Implementing Regulation (EU) 2021/808. The reliability of the method was verified by testing real samples from pig farms.

Analysis of Aminoglycoside Antibiotics: A Challenge in Food Control.

Aminoglycosides are a widely used group of antibiotics in veterinary medicine. However, misuse and abuse of these drugs can lead to residues in the edible tissues of animals. Due to the toxicity of aminoglycosides and the exposure of consumers to the emergence of drug resistance, new methods are being sought to determine aminoglycosides in food. The method presented in this manuscript describes the determination of twelve aminoglycosides (streptomycin, dihydrostreptomycin, spectinomycin, neomycin, gentamicin, hygromycin, paromomycin, kanamycin, tobramycin, amikacin, apramycin, and sisomycin) in thirteen matrices (muscle, kidney, liver, fat, sausages, shrimps, fish honey, milk, eggs, whey powder, sour cream, and curd). Aminoglycosides were isolated from samples with extraction buffer (10 mM NH4OOCH3, 0.4 mM Na2EDTA, 1% NaCl, 2% TCA). For the clean-up purpose, HLB cartridges were used. Analysis was performed using ultra-high-performance liquid chromatography coupled with tandem mass spectrometry (UHPLC-MS/MS) with a Poroshell analytical column and a mobile phase of acetonitrile and heptafluorobutyric acid. The method was validated according to Commission Regulation (EU) 2021/808 requirements. Good performance characteristics were obtained for recovery, linearity, precision, specificity, and decision limits (CCα). This simple and high-sensitivity method can determine multi-aminoglycosides in various food samples for confirmatory analysis.

Analysis of Antimicrobials in Muscle and Drinking Water in Terms of Reducing the Need of Antimicrobial Use by Increasing the Health and Welfare of Pig and Broiler.

Antimicrobial residues may pose harmful effects on the health of consumers. At the same time, an adequate quality of drinking water for animals is one of the important element to ensure animal welfare and food without antibacterials. The presented study is aimed at estimating the residue levels of antibacterial compounds, such as penicillins, cephalosporin, macrolides, tetracyclines, quinolones, sulphonamides, aminoglycosides, diaminopirymidines, pleuromutilines and lincosamides in meat and on-farm drinking water samples using liquid chromatography-tandem mass spectrometry (LC-MS/MS), as a part of a surveillance system on pig and broiler farms within the project Healthy Livestock. A total of 870 samples of muscle from pig and broiler, as well as 229 water samples were analysed for antibiotic residues. Samples were collected from farms in EU countries in two steps, before and after implementation of a tailor-made health plan. In muscle samples, the detected concentrations of doxycycline in the post-intervention step (15.9-70.8 µg/kg) were lower than concentrations in the pre-intervention step (20.6-100 µg/kg). In water samples, doxycycline in an average concentration of 119 µg/L in the pre- and 23.1 µg/L in the post-intervention step, as well as enrofloxacin at concentrations of 170 µg/L in the pre- and 1.72 µg/L in the post-intervention step were quantified. Amoxicillin was only present before intervention. The obtained results confirm the effectiveness of the intervention actions. The concentrations of antibiotics in muscles and water were lower after implementation of a health plan on the farms.

Does Deoxynivalenol Affect Amoxicillin and Doxycycline Absorption in the Gastrointestinal Tract? Ex Vivo Study on Swine Jejunum Mucosa Explants.

The presence of deoxynivalenol (DON) in feed may increase intestinal barrier permeability. Disturbance of the intestinal barrier integrity may affect the absorption of antibiotics used in animals. Since the bioavailability of orally administered antibiotics significantly affects their efficacy and safety, it was decided to evaluate how DON influences the absorption of the most commonly used antibiotics in pigs, i.e., amoxicillin (AMX) and doxycycline (DOX). The studies were conducted using jejunal explants from adult pigs. Explants were incubated in Ussing chambers, in which a buffer containing DON (30 µg/mL), AMX (50 µg/mL), DOX (30 µg/mL), a combination of AMX + DON, or a combination of DOX + DON was used. Changes in transepithelial electrical resistance (TEER), the flux of transcellular and intracellular transport markers, and the flux of antibiotics across explants were measured. DON increased the permeability of small intestine explants, expressed by a reduction in TEER and an intensification of transcellular marker transport. DON did not affect AMX transport, but it accelerated DOX transport by approximately five times. The results suggest that DON inhibits the efflux transport of DOX to the intestinal lumen, and thus significantly changes its absorption from the gastrointestinal tract.

UHPLC-MS/MS Analysis of Antibiotics Transfer and Concentrations in Porcine Oral Fluid after Intramuscular Application.

The monitoring of antibiotic use in animals is a crucial element to ensure food safety. The main goal of this study was to analyse the distribution of selected antibiotics to porcine oral fluid, as well as to demonstrate that an oral fluid is an alternative to other biological matrices used in the control of antibacterials. Therefore, an animal study with pigs treated using seven different antibiotics was performed. Sulfadoxine (SDX) with trimethoprim (TRMP), lincomycin (LIN), tiamulin (TIAM), tylosin (TYL), amoxicillin (AMX) and penicillin G (PEN G) were injected intramuscularly to pigs, and concentrations of these analytes in the oral fluid were assessed. Ultra-high-performance liquid chromatography coupled with mass spectrometry (UHPLC-MS/MS) was used to quantify the analytes. On the first day of medication, the highest concentrations for SDX and TRMP at the level of 22,300 µg/kg and 14,100 µg/kg were found, respectively. The concentrations of LIN (10,500 µg/kg) and TIAM (7600 µg/kg) were also relatively high. The peak of TYL was recorded on the second day of drug administration. Most of the analytes were present in oral fluid for 30 days, apart from TYL, which was detected for 27 days. It was found that AMX and PEN G were quantified only for 5 and 8 days, respectively, at very low concentrations. It was found that oral fluid can be used for the verification of antibiotics on pig farms.

Risk to Public Health Regarding Doxycycline Residues in Chicken Claws.

Doxycycline residues in claws may pose a potential risk to humans. A study with broiler chickens was performed to find the concentrations of doxycycline in claws after treatment. For the quantification of the antibiotic of interest in claws, an ultrahigh-performance liquid chromatography-tandem mass spectrometry method was developed. Doxycycline was detectable at 3043 µg/kg at sub-therapeutic treatment, 223 µg/kg after therapeutic doses, and 14 µg/kg after spray application on the last day of the experiment (day 22). Almost 70 days is required for the concentration of doxycycline to reach zero. The obtained results show that monitoring of doxycycline in claws should be implemented.

Transfer of enrofloxacin, ciprofloxacin, and lincomycin into eggshells and residue depletion in egg components after multiple oral administration to laying hens.

Regardless of whether antimicrobial drugs are administered to laying hens legally or illegally, residues of these drugs may be present in the eggs. Even if the eggs are not intended for human consumption, byproducts/biowaste, such as eggshells, may contain residues of the drugs used, which may pose a risk to human health and the environment. In the presented research, 2 different groups of laying hens received enrofloxacin (10 mg/kg body weight) and lincomycin (20 mg/kg body weight) once daily for 5 d. Eggs were collected daily and the concentration of enrofloxacin, its metabolite ciprofloxacin, and lincomycin residue in the eggshells, whole eggs, egg yolks, and egg whites were determined by ultra-high-performance liquid chromatography-tandem mass spectrometry. This study demonstrates the transfer of enrofloxacin, ciprofloxacin, and lincomycin into the eggshells and provides evidence for the distribution into the eggshells after administration of these drugs to laying hens. The enrofloxacin residues were detected in the eggshell for 10 d after cessation of treatment, ciprofloxacin and lincomycin were rapidly eliminated and 2 d after finish drugs administration they were no longer detected in the eggshell.

Multi-Class Procedure for Analysis of 50 Antibacterial Compounds in Eggshells Using Ultra-High-Performance Liquid Chromatography-Tandem Mass Spectrometry.

In this work, for the first time, Ultra-High-Performance Liquid Chromatography-Tandem Mass Spectrometry (UHPLC-MS/MS) method was developed for qualitative and quantitative analysis of veterinary antibiotics (cephalosporins, diaminopyrimidines, fluoro(quinolones), lincosamides, macrolides, penicillins, pleuromutilins, sulfonamides, tetracyclines, and sulfones) in hen eggshells. The sample preparation method is based on a liquid-liquid extraction with a mixture of metaphosphoric acid, ascorbic acid, EDTA disodium salt dihydrate, and acetonitrile. The chromatographic separation was performed on Luna® Omega Polar C18 10 column in gradient elution mode and quantitated in an 8 min run. Validation such as linearity, selectivity, precision, recovery, matrix effect, limit of quantification (LOQ), and limit of detection (LOD) was found to be within the acceptance criteria of the validation guidelines of the Commission Decision 2002/657/EC and EUR 28099 EN. Average recoveries ranged from 81-120%. The calculated LOQ values ranged from 1 to 10 µg/kg, the LOD values ranged from 0.3 to 4.0 µg/kg, depending on analyte. The developed method has been successfully applied to the determination of antibacterial compounds in hen eggshell samples obtained from different sources. The results revealed that enrofloxacin, lincomycin, doxycycline, and oxytetracycline were detected in hen eggshell samples.

Doxycycline transfer from substrate to white button mushroom (Agaricus bisporus) and assessment of the potential consumer exposure.

The presence of antibiotic residues in the food chain may pose a serious risk to human health. Locating and evaluating new sources of consumer exposure to antibiotic residues in food is a very important element of health protection. The possibility of doxycycline uptake from the substrate for mushroom cultivation by the white button mushroom (Agaricus bisporus) fruit body was investigated. Mushrooms were experimentally cultivated on substrate contaminated with 8 different doxycycline concentrations in substrate and analyte concentrations in mushroom samples were measured using ultra-high performance liquid chromatography - triple quadrupole tandem mass spectrometry (UHPLC-MS/MS) The obtained results clearly indicated that doxycycline transfers from contaminated substrate to mushrooms at concentrations ranging from 0.87 to 72.3 µg/kg, depending on substrate contamination concentration level and order of harvesting.

Multi-residues UHPLC-MS/MS analysis of 53 antibacterial compounds in poultry feathers as an analytical tool in food safety assurance.

Under the EU official monitoring programs, the antibiotics are controlled in tissues of food producing animals. However, regarding the overuse of medicines in veterinary practice, there is a strong need to find an alternative to post-mortem analysis of antibiotics. The use of feathers, as an unconventional matrix, enable to control of birds treatment during breeding. Thus, a novel ultra-high performance liquid chromatography-tandem mass spectrometry method for the analysis of 53 compounds from 11 groups of antibacterials, including penicillins, cephalosporins, macrolides, tetracyclines, quinolones, sulfonamides, aminoglycosides, pleuromutilins, diaminopirymidynes, diaminopirymidynes derivatives and lincosamides in chicken feathers has been developed. The isolation of analytes by solvent extraction method with oxalic acid, Na2EDTA and acetonitrile was performed. The extracts were cleaned by filtration with OASIS HLB cartridges. Chromatographic separation was achieved on a Zorbax SB-C18 analytical column using mobile phase consisting of 0.025% heptafluorobutyric acid and acetonitrile. All compounds were successfully validated, with good sensitivity, accuracy and precision. The developed method gives an opportunity to effective monitoring, whether chickens have been given antibiotics, as well as let to identify the medicines that were used.

Simultaneous determination of 45 antibacterial compounds in mushrooms - Agaricus bisporus by ultra-high performance liquid chromatography-tandem mass spectrometry.

A multi-residue method was developed for the determination of 45 antibacterial compounds in mushrooms - Agaricus bisporus. The method is based on liquid-liquid extraction with acetonitrile with addition of trichloroacetic acid and filtration through sodium sulphate, followed by the identification and quantification of the analyte residues by ultra-high performance liquid chromatography triple quadrupole tandem mass spectrometry (UHPLC-MS/MS). Satisfactory linearity was obtained for all compounds with regression coefficients greater than 0.99. The mean recoveries of antibacterial agents were in the range of 73% - 118%. Repeatability and intra-lab reproducibility were lower than 10% and 15%, respectively. The limit of detection ranged from 0.3-3.0 µg kg-1 and the limit of quantification was in the range of 1.0 - 10. 0 µg kg-1 depending on analyte. The proposed method can be successfully applied for the determination of antibiotics in commercially available mushrooms.

Contamination of wild boars' (Sus scrofa) muscles with tetracycline antibiotic from oral-delivered rabies vaccine baits.

Tetracycline (TC) is used as a biomarker for rabies vaccine bait intended for foxes. However, there is a high probability of intake of the vaccine by other species living in the forest ecosystem, including wild boars (Sus scrofa), and TC residues can occur in the animals' tissues. In this study, muscle samples from 144 animals were tested for the presence of TC, collected after rabies vaccine distribution. For the quantitative analysis of TC and its 4-epi form, a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed. The results of this study show that TC was found in 53 samples, which represents 37% of all tested animals. The concentrations were in the range 5-286 µg/kg. The preliminary results suggest that the risk exists of contamination of muscle tissue of wild boars with TC from oral-delivery rabies vaccine baits containing the antibiotic. Control should be considered of TC residues in wild boar meat for human consumption.

Tetracycline antibiotics transfer from contaminated milk to dairy products and the effect of the skimming step and pasteurisation process on residue concentrations.

The presence of antibiotics in raw milk and milk derivatives poses a threat to human health and can negatively affect the dairy industry. Therefore, the main object of this study was to investigate the transfer of oxytetracycline (OTC), tetracycline (TC), chlortetracycline (CTC) and doxycycline (DC) from raw, experimental milk contaminated with tetracyclines (TCs) to different dairy products: cream, butter, buttermilk, sour milk, whey, curd and cheese. Additionally the effect of the skimming process on TCs concentrations was tested, as well as the influence of low-temperature long-time pasteurisation. The analyses of TCs in milk and dairy products were performed by an LC-MS/MS method. In order to determine TCs residues in dairy products, an analytical method was developed with the same extraction step for all matrices. TCs molecules were inhomogenously distributed between the milk derivative fractions. The highest concentrations were determined in curd and cheese in the ranges 320-482 µg/kg and 280-561 µg/kg, respectively. Low levels of TCs in butter and whey were observed (11.8-41.2 µg/kg). TCs were found in sour milk (66.0-111 µg/kg), cream (85.0-115 µg/kg) and buttermilk (196-221 µg/kg) at much higher levels than in butter and whey, but lower than in curd and cheese. During the skimming process, the highest yield of cream was obtained after the raw milk was held at 2-8°C for 24 h. The differences in concentrations of TCs between whole milk and skimmed milk, expressed as percentages of recovery, were below 19% (recoveries in excess of 81%). The highest content was observed in milk and cream skimmed at 2-8°C. The degradation percentages for TCs during the pasteurisation process (63°C for 30 min) were below 19%.