Validation of a LC-MS/MS method for the quantitative analysis of four antibiotics in pig tissues and plasma to assess the risk of transfer of residues to edible matrices after exposure to cross-contaminated feed.

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.

A liquid chromatography coupled to tandem mass spectrometry method for the quantification of spiramycin and its active metabolite neospiramycin in milk of major and minor species: Validation using the accuracy profile.

A liquid chromatography with tandem mass spectrometry (LC-MS/MS) method was developed for the simultaneous quantification of residues of spiramycin, a macrolide antibiotic, and its active metabolite neospiramycin in cow's milk as well as in minor species 'milk, goat and ewe. Spiramycin-d3 was used as internal standard for quantification of both analytes. This analytical method was validated using a global accuracy profile as a graphical decision tool built according to the trueness and the precision of the method. A unique and optimal linear model with logarithm transformation (with a determination coefficient of 0.9991) allowed the measurement of both analytes in the milks of the three animal species, in a wide range from 0.2 to 10 times the Maximal Residue Limit (MRL) (40-2000 µg.kg-1). The limits of detection and quantification were 13 µg.kg-1 and 40 µg.kg-1, respectively. The accuracy profile was established to get 80% of future measurements in routine assays that will fall within the acceptance limits. Trueness of the method, expressed as relative bias, was comprised between -1.6% and 5.7% over the whole range of concentrations. The mean relative standard deviation for repeatability and intermediate precision were comprised between 1.1% and 2.7%; 2.5 and 4.2%, respectively, in all levels of concentration for the three milks. Moreover, a two-order polynomial function was used to model the relative expanded uncertainty with a determination coefficient of 0.834. This function aimed to determine the uncertainty of the future quantifications within the validated dosing range. Overall, the global accuracy profile highlighted the reliability of the method for the routine assays of spiramycin and neospiramycin even in milk from minor species (goat, ewe) by using the most accessible milk (often from cow), while guaranteeing a very high proportion of samples within the fixed acceptance limits. The applicability of this method was tested during a depletion study of spiramycin and neospiramycin in the milk of cow, goat and ewe. The developed analytical method will be useful to assess the distribution profile of the antibiotic and its metabolite in milk of minor species where few studies are available.