A unified approach for high-throughput quantitative analysis of the residues of multi-class veterinary drugs and pesticides in bovine milk using LC-MS/MS and GC–MS/MS

A simple, selective and fast multi-residue method was developed to determine 18 veterinary drugs in milk by ultra-high-pressure liquid chromatography coupled to tandem quadrupole mass spectrometry. The selected antibiotics include quinolones, sulphonamides, macrolides, anthelmintics and one tetracycline. An extraction procedure based on QuEChERS methodology (quick, easy, cheap, effective, rugged and safe), consisting of a liquid extraction of the milk samples with acetonitrile without sample clean up was performed. The extract was centrifuged and the supernatant was filtered prior to chromatographic analysis. Several chromatographic conditions were optimized in order to obtain a fast separation (less than 10 min). The antibiotics were detected by electrospray ionization in positive ion mode with multiple reaction monitoring (MRM) and mass spectrometric conditions were optimized in order to increase selectivity and sensitivity. The developed method was validated in terms of linearity, trueness, precision, limits of detection (LODs) and quantification. Mean recoveries ranged from 70% to 110% and interday precision was lower than 21%. LODs ranged from 1 to 4 microg/kg. Finally, the method was applied to real samples and only traces of tylosin and fenbendezol were detected in two samples.

Ultra-performance liquid chromatography combined with time-of-flight mass spectrometry (UPLC–ToF-MS) has been used for screening and quantification of more than 100 veterinary drugs in milk. The veterinary drugs represent different classes including benzimidazoles, macrolides, penicillins, quinolones, sulphonamides, pyrimidines, tetracylines, nitroimidazoles, tranquillizers, ionophores, amphenicols and non-steroidal anti-inflammatory agents (NSAIDs). After protein precipitation, centrifugation and solid-phase extraction (SPE), the extracts were analysed by UPLC–ToF-MS. From the acquired full scan data the drug-specific ions were extracted for construction of the chromatograms and evaluation of the results. The analytical method was validated according to the EU guidelines (2002/657/EC) for a quantitative screening method. At the concentration level of interest (MRL level) the results for repeatability (%RSD < 20% for 86% of the compounds), reproducibility (%RSD < 40% for 96% of the compounds) and the accuracy (80–120% for 88% of the compounds) were satisfactory. Evaluation of the CCβ values and the linearity results demonstrates that the developed method shows adequate sensitivity and linearity to provide quantitative results. Furthermore, the method is accurate enough to differentiate between suspected and negative samples or drug concentrations below or above the MRL. A set of 100 samples of raw milk were screened for residues. No suspected (positive) results were obtained except for the included blind reference sample containing sulphamethazine (88 μg/l) that tested positive for this compound. UPLC–ToF-MS combines high resolution for both LC and MS with high mass accuracy which is very powerful for the multi-compound analysis of veterinary drugs. The technique seems to be powerful enough for the analysis of not only veterinary drugs but also organic contaminants like pesticides, mycotoxins and plant toxins in one single method.

A fast and straightforward generic procedure for the simultaneous extraction of various classes of pesticides, mycotoxins, plant toxins, and veterinary drugs in various matrixes has been developed, for subsequent analysis by liquid chromatography with mass spectrometric detection. As a first step, four existing multianalyte procedures and three newly proposed methods were compared for a test set of 172 pesticides, mycotoxins, and plant toxins spiked to a feed matrix. The new procedures, which basically involved extraction/dilution of the sample with water and an acidified organic solvent (methanol, acetonitrile, or acetone), were most promising. The three new generic extraction methods were further tested for applicability to other matrixes (maize, honey, milk, egg, meat). Overall, the best recoveries were obtained for acetone, followed by acetonitrile. With respect to matrix effects, acetonitrile was the most favorable solvent and methanol was the worst. The occurrence of matrix effects decreased for the matrixes in the order of feed > maize > meat > milk > egg > honey. The extraction method selected as the default procedure (water/acetonitrile/1% formic acid) was also evaluated for applicability to multiple classes of veterinary drugs in all six matrixes, with satisfactory results. Finally, the generic extraction procedure was validated for 136 pesticides, 36 natural toxins, and 86 veterinary drugs in compound feed and honey at three levels (0.01, 0.02, and 0.05 mg/kg) using ultraperformance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) for analysis of the extracts. For over 80% of the analytes, recoveries were between 70 and 120% and precision (expressed as relative standard deviation) was mostly in the range of 5-10% (except for feed at 0.01 mg/kg; adequate recoveries for 62% of the analytes). The limits of detection were from <0.01 to 0.05 mg/kg for most analytes, which is usually sufficient to verify compliance of products with legal tolerances. The results clearly demonstrate the feasibility of the generic approach proposed. Application of the method in routine monitoring programs would imply a drastic reduction of both effort and time.