Control of Antimicrobials in Feed Using Liquid Chromatography-Tandem Mass Spectrometry: Assessment of Cross-Contamination Rates at the Farm Level.

Antimicrobial cross-contamination of animal feed may occur during feed manufacturing, because shared production lines can be used for the production of medicated and nonmedicated feeds, and also during feed transport, storage at the farm level, and usage. This is a major issue in the current context in which antimicrobial usage must be controlled to maintain their effectiveness. The purpose of this study was to assess the antimicrobial cross-contamination rate of feed at the farm level. Here, we optimized a liquid chromatography-tandem mass spectrometry method for the determination of 11 antimicrobials in feed for pigs, poultry, and rabbits, which were strategically chosen. The method was validated according to European regulations in terms of mass spectrometry identification criteria and quantification criteria (linearity, trueness, precision, limit of quantification, and limit of decision). The results were in compliance with these regulations except for doxycycline, which may be quantified with higher uncertainty. This method was applied to the analysis of 192 nonmedicated pig, poultry, and rabbit feed samples that were collected directly from farms to assess antimicrobials animal exposure. Cross-contamination rates were relatively high with 44% of the samples being contaminated at a concentration above the quantification limit of 0.125 mg/kg and 15% of the samples being contaminated above 1 mg/kg. This result suggests that the current regulations and feed processing recommendations need to be improved, taking into account the risks arising from these contaminations.

A non-targeted LC-HRMS approach for detecting exposure to illegal veterinary treatments: The case of cephalosporins in commercial laying Hens.

Cephalosporins are of particular importance in human medicine and should be reserved for second-line curative treatment in the veterinary field to avoid any emerging antimicrobial resistance. Due to misuse of ceftiofur in the poultry sector in France, it is now recommended to completely stop using cephalosporins in this sector. Methods currently used for the control of veterinary practices are mostly based on liquid chromatography coupled to mass spectrometry in a targeted mode, including parent compounds and any major metabolites. The aim of the present study was to evaluate the relevance of untargeted metabolomic approaches to highlight a possible exposure of laying hens to cephalosporins using a predictive model including selected treatment biomarkers. An experimentation carried out on living animals involved the administration of cefquinome and ceftiofur. Three biological matrices-droppings, eggs and liver-were investigated. Metabolites were extracted and analysed by liquid chromatography coupled to high resolution mass spectrometry in a full scan mode. Metabolites impacted by the treatment were selected by using univariate and multivariate statistical analyses. Predictive models built from the potential biomarkers selected in the "droppings" matrix were validated and able to classify "treated" and "control" hens. PLS-DA and logistic regression models were compared and both models gave satisfactory results in terms of prediction. Results were of less interest for other matrices in which only biomarkers of exposure to cefquinome were detected.

Validation approach for a fast and simple targeted screening method for 75 antibiotics in meat and aquaculture products using LC-MS/MS.

An approach is described to validate a fast and simple targeted screening method for antibiotic analysis in meat and aquaculture products by LC-MS/MS. The strategy of validation was applied for a panel of 75 antibiotics belonging to different families, i.e., penicillins, cephalosporins, sulfonamides, macrolides, quinolones and phenicols. The samples were extracted once with acetonitrile, concentrated by evaporation and injected into the LC-MS/MS system. The approach chosen for the validation was based on the Community Reference Laboratory (CRL) guidelines for the validation of screening qualitative methods. The aim of the validation was to prove sufficient sensitivity of the method to detect all the targeted antibiotics at the level of interest, generally the maximum residue limit (MRL). A robustness study was also performed to test the influence of different factors. The validation showed that the method is valid to detect and identify 73 antibiotics of the 75 antibiotics studied in meat and aquaculture products at the validation levels.

Validation of a liquid chromatography-high-resolution mass spectrometry method for the analysis of ceftiofur in poultry muscle, kidneys and plasma: A unique accuracy profile for each and every matrix.

A robust, selective and specific liquid chromatography-high resolution mass spectrometry (LC-HRMS) method was developed for the quantification of total residues of ceftiofur, an antibiotic belonging to the 3rd generation cephalosporins in plasma, muscle and kidney of poultry. Ceftiofur and conjugates in samples were firstly hydrolyzed with dithioerythritol into desfuroylceftiofur, which was then stabilized by derivatization with iodoacetamide into desfuroylceftiofur acetamide. Sample were then submitted to a solid phase extraction followed the accurate mass analysis of desfuroylceftiofur acetamide by LC-HRMS in full scan mode using a linear trap quadrupole (LTQ)-Orbitrap mass spectrometer with a resolving power 60,000 full width at half maximum (FWHM). The method was fully validated over a dosing range between 100 and 2000 µg kg(-1) (or µg L(-1)) using the total error approach. Accuracy profiles a graphical decision-making tool were built by computing results of validation procedure with acceptance limits set at ±60%, and ß-expectation tolerance intervals, i.e. the interval assuming to contain a ß % of future measurements (ß=90% in this study). Total measurement error including trueness, repeatability and intermediate precision were evaluated. Relative bias of trueness was never exceeding the threshold of 6% in all matrices at all level of concentration. The mean relative standard deviation for repeatability was lower than 16% at all levels of concentration for all matrices; the mean relative standard deviation for intermediate precision was lower than 25% at all levels of concentration for all matrices. This validation approach proved that the method is reliable for the quantification in each and every matrix (i.e. plasma, kidneys and muscle of chicken) thanks to only one single regression model (i.e. linear) obtained from external calibration standards (without matrix) with deuterated labelled internal standard. The developed method was applied during a depletion study of ceftiofur in chicken tissues and plasma.

The Monitoring of Triphenylmethane Dyes in Aquaculture Products Through the European Union Network of Official Control Laboratories.

Aquaculture has been the fastest growing animal production industry for the past four decades, and almost half of the fish eaten in the world are now farmed fish. To prevent diseases in this more intensive aquaculture farming, use of therapeutic chemicals has become a basic choice. The monitoring of malachite green, a triphenylmethane dye and one of the oldest and widely used chemicals in fish production, has gained more interest since the mid 1990s when this substance was finally proven to be toxic enough to be prohibited in seafood products destined for human consumption. The enforcement of the European Union (EU) regulation of this banned substance along with some other triphenylmethane dye congeners and their metabolites in its domestic production and in seafood imports was undertaken through the National Residue Monitoring Plans implemented in nearly all of the 28 EU member states. The reliability of the overall European monitoring of this dye contamination in aquaculture products was assessed by using the results of proficiency testing (PT) studies provided by the EU Reference Laboratory (EU-RL) in charge of the network of the EU National Reference Laboratories (NRLs). The proficiency of each NRL providing analytical support services for regulating dye residues was carefully checked during three PT rounds. In the process, the analytical methods developed and validated for this purpose have gradually been improved and extended over the last two decades.

European Proficiency testing of national reference laboratories for the confirmation of sulfonamide residues in muscle and milk.

Two interlaboratory studies were organized in 2002-2003 in order to check the proficiency of laboratories in confirming the presence of sulfonamide residues in muscle and milk. These studies involved 25 EU National Reference Laboratories (NRLs) from 21 different European Countries in charge of statutory monitoring of antimicrobial residues in food of animal origin at a national level. The study was conducted according to international and national guidelines by the Community Reference Laboratory (CRL) in charge of antimicrobial substances. Four different test matrices of sheep muscle and four different test matrices of bovine milk containing different sulfonamide substances were prepared and sent to the participants. Each participant was asked to use his own routine confirmatory method and to analyse each sample in triplicate within a period of about six weeks during which the stability of the materials was checked by the organizer. The sulfonamide content of each material was determined by calculating the robust means of all the results and the deviation of the results from the assigned values was assessed by calculating Z-scores. Overall, results were satisfactory, particularly considering that it was the first proficiency test dealing with sulfonamides organised by the Community Reference Laboratory.