Determination of Polypeptide Antibiotic Residues in Food of Animal Origin by Ultra-High-Performance Liquid Chromatography-Tandem Mass Spectrometry.

A novel UHPLC-MS/MS method for the determination of polypeptide antibiotic residues in animal muscle, milk, and eggs was developed and validated. Bacitracin A, colistin A, colistin B, polymyxin B1, and polymyxin B2 were extracted from the samples with a mixture of acetonitrile/water/ammonia solution 25%, 80/10/10 (v/v/v), and put through further evaporation, reconstitution, and filtration steps. The chromatographic separation was performed on a C18 column in gradient elution mode. Mass spectral acquisitions were performed in selective multiple reaction monitoring mode by a triple quadrupole mass spectrometer. The method was validated according to the criteria of Commission Decision 2002/657/EC. The method quantifies polypeptides in a linear range from 10 to 1000 µg kg-1, where the lowest concentration on the calibration curve refers to the limit of quantification (LOQ). The recoveries ranged from 70 to 99%, the repeatability was below 13%, and within-laboratory reproducibility was lower than 15%. The decision limit (CCα) and detection capability (CCß) values were calculated, and ruggedness and stability studies were performed, to fulfill the criteria for confirmatory methods. Moreover, the developed method may also be used for screening purposes by its labor efficiency.

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.

Quantification of twenty pharmacologically active dyes in water samples using UPLC-MS/MS.

This study presents a multi-compound method for the determination of 20 pharmacologically active dyes from 5 different chemical classes in environmental water samples. These compounds, including triphenylmethane dyes (malachite green, crystal violet, brilliant green, ethyl violet, methyl violet 2B, pararosaniline, victoria blue B, victoria blue R, victoria pure blue BO), phenothiazine dyes (methylene blue, azure A, azure B, azure C, new methylene blue, thionine), phenoxazine dye (nile blue A), acridine dyes (acriflavine, proflavine) and xanthene dyes (rhodamine B, rhodamine 6G) constitute pharmacologically active substances (PASs). For the optimisation of sample preparation, different solid-phase extraction (SPE) sorbents and a wide range of pH (from 2 to 12) of water samples were tested. Finally, water samples were preconcentrated and cleaned up on diol SPE cartridges. Extracts were analysed by ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) operating in the positive electrospray ionisation (ESI+) mode. The chromatographic separation of the 20 pharmacologically active dyes was achieved within 5 min by using a pentafluorophenyl (F5) analytical column and mobile phases of ammonium acetate buffer (0.05 M, pH = 3.5) and acetonitrile with gradient elution. The developed method was validated proving to be suitable for the determination of all tested compounds. Limits of quantification were 0.01-0.1 µg/l, are sensitive enough to quantify very low concentration levels of the dyes in environmental water samples. The obtained recovery values for all tested analytes were between 71.2 and 104.9% with a good RSD, less than 14 % at all fortification levels. The application of the developed method to water samples allows the detection of dyes such as crystal violet, rhodamine B, and methyl violet in two wastewater samples in concentration range from 0.017 to 0.0043 µg/l).

The influence of cooking procedures on doxycycline concentration in contaminated eggs.

Doxycycline (DC) is forbidden compound in laying hens. Most information about drug residues in eggs concern their concentrations in raw matrix and the data about the influence of cooking on antibiotics residues in eggs are limited. Thus, the residues concentration of DC in eggs after different cooking methods was investigated. Analyses of DC were assayed by liquid chromatography - tandem mass spectrometry method. The stability of DC in eggs were depended upon the type and time of cooking procedure. By microwaving DC was reduced most effective with concentrations decreased by 53% and 50.3% after 4min of microwaving without cover and microwaving with cover, respectively. In fried eggs, DC was reduced by 39.8% in 6min. By the boiling cooking, the smallest reduction was observed with the concentration decreased by 29.8% after 8min. The obtained results show that ordinary cooking does not eliminate the all DC residues present in eggs.

Oral Fluid as a Biological Material for Antemortem Detection of Oxytetracycline in Pigs by Liquid Chromatography-Tandem Mass Spectrometry.

The presence of antibiotic residues in pig tissues requires a search for new methods for their antemortem detection. To find an alternative for postmortem pig carcass analysis, an oral fluid was tested. To prove the suitability of oral fluid for the detection of antibiotics administered by injection, oxytetracycline was chosen. Research was conducted on two groups of animals: group 1, 100% treated; and group 2, 50% treated and 50% untreated. Oxytetracycline was assayed by a high-performance liquid chromatography-tandem mass spectrometry method. The antibiotic was detectable 2 h post administration in group 1 and group 2 at the concentrations of 10653 ± 1421 µg/kg and 7457 ± 1145 µg/kg, respectively. At withdrawal period (21st day), oxytetracycline concentrations in oral fluid (30.8 ± 9.4 µg/kg in group 1 and 11.6 ± 5.6 µg/kg in group 2) were similar to those determined in muscle (34.5 ± 8.2 µg/kg). The concentrations of oxytetracycline in liver and kidney were 76.8 ± 22 µg/kg and 204 ± 49 µg/kg, respectively. The results of this study indicate that oral fluid analysis can be used for antemortem oxytetracycline detection in pigs, even if the half of animals in one pen are treated.

Multiresidue determination of veterinary medicines in lyophilized egg albumen with subsequent consumer exposure evaluation.

The process of lyophilization causes that the veterinary drugs residues present in egg albumen do not decompose, as it takes place during the process of high-temperature drying. Thus, lyophilized albumen may be a potential source of their residues for consumers. As a consequence, reliable methods for the determination of veterinary medicinal products from egg albumen are needed. The method for the determination of 85 analytes in lyophilized egg albumen was developed and successfully validated. The recoveries were between 84 and 110%, within laboratory repeatability and reproducibility - in the range of 3.29-16.8% and -5.93 to 19.3%. The presence of enrofloxacin and doxycycline was confirmed in real egg albumen samples. The concentrations ranged from 5.65-596µg/kg for doxycycline to 0.89-134µg/kg for enrofloxacin. Nevertheless, the evaluated daily intake and % of the ADI (Acceptable Daily Intake) received by the consumers' were at a toxicologically accepted level.

Influence of enrofloxacin traces in drinking water to doxycycline tissue pharmacokinetics in healthy and infected by Mycoplasma gallisepticum broiler chickens.

Most of antibiotics, administrated in the treatment of poultry diseases are dissolved in drinking water, and it can lead to water supply systems contamination, especially when the regular cleaning is not using. This situation can lead to unconscious administration of low doses of antibiotics to untreated animals. The aim of this study was to clarify the impact of the exposure of enrofloxacin traces (500 µg l(-1)) to doxycycline pharmacokinetics in healthy and experimentally Mycoplasma gallisepticum infected broiler chickens., Two experimental groups, received of enrofloxacin in water and all groups, received 20 mg kg(-1) bw of doxycycline. The compounds concentrations in muscles and livers were determined by LC-MS/MS. The maximum drug tissue concentration (Cmax) of doxycycline was highest in liver obtained from infected chickens which, received enrofloxacin traces (ENR + DC/MG). It was about 40% higher than in healthy chickens from group I which received only doxycycline. It was found that the concentration-time curve AUC(0-t) values in group ENR + DC/MG were almost 75% higher than in the group (DC) and 35% higher than in group (ENR + DC) which also received enrofloxacin traces. The constant exposure of broiler chickens on enrofloxacin traces as well as infection, may significantly influenced on doxycycline tissue pharmacokinetic profile.

Pharmacokinetics of tulathromycin in edible tissues of healthy and experimentally infected pigs with Actinobacillus pleuropneumoniae.

The aim of this study was the comparison of the tissue pharmacokinetics of tulathromycin in healthy pigs and pigs experimentally infected with Actinobacillus pleuropneumoniae (App). Tulathromycin was given to 24 healthy and 24 infected pigs by intramuscular injection at a single dosage of 2.5 mg kg(-1) body weight (b.w.). Pigs were euthanised at each group and then samples of liver, kidney, muscle, injection site and skin with fat were taken at scheduled time points. Drug concentrations were determined by LC-MS/MS. In this study, higher values of the area under the concentration-time curves (AUC) were calculated in all tissue samples taken from infected than healthy pigs. In pigs with App the AUCs of liver, kidney, muscle, skin with fat and injection site were 1111, 1973, 235, 181 and 2931 mg kg(-1) h, while in pigs without inflammation they were 509, 1295, 151, 111 and 1587 mg kg(-1) h, respectively. Maximum drug tissue concentrations (Cmax) in infected animals were 2370, 6650, 2016, 666 and 83,870 µg kg(-1), while in healthy pigs they were 1483, 6677, 1733, 509 and 55,006 µg kg(-1), respectively. The eliminations half-times (T1/2) were respectively longer in all tissue samples taken from infected animals (from 157.3 to 187.3 h) than in healthy ones (from 138.6 to 161.2 h). The tulathromycin tissue concentrations were significantly higher (p < 0.05) in all tissue samples of the infected pigs compared with the healthy animals at 360 h (from 0.0014 to 0.0280) and at 792 h (from 0.0007 to 0.0242) after drug administration. The results suggest that the tissue pharmacokinetic properties and residue depletion of tulathromycin can be influenced by the disease state of animals.

Determination of (fluoro)quinolones in eggs by liquid chromatography with fluorescence detection and confirmation by liquid chromatography-tandem mass spectrometry.

A multiresidue analytical procedure for determination of seven fluoroquinolones (marbofloxacin, norfloxacin as internal standard, ciprofloxacin, danofloxacin, enrofloxacin, sarafloxacin and difloxacin), and three quinolones (oxolinic acid, nalidixic acid and flumequine) in eggs is presented. The procedure is based on dispersive solid-phase extraction technique with acetonitrile as extractant. Norfloxacin and ciprofloxacin - d8 were used as internal standards to quantify the (fluoro)quinolones. Analyses were realised by LC-FLD for screening and LC-MS/MS for confirmatory purposes. The whole procedure was evaluated according to the Commission Decision 2002/657/EC. Specificity, decision limit (CCα), detection capacity (CCß), recovery (absolute and relative), precision (repeatability and reproducibility) were determined during validation process. Recoveries (relative) for the LC-FLD screening determination ranged from 85% to 93%, repeatability and reproducibility were in the range of 5-9% to 9-16%, respectively. CCα and CCß were 13-37 and 17-43 µg/kg pending on analite. For the LC-MS/MS confirmatory method, the relative recoveries were satisfactory (92-99%) with repeatability and reproducibility in the range of 4-7% to 6-12%, respectively. CCα and CCß were 3-7 and 7-11 µg/kg depending on the analite. The results of both prepared methods showed these analytical procedures simple, rapid, sensitive and suitable for routine control of eggs.