Fit-for-purpose in veterinary drug residue analysis: development and validation of a LC-MS/MS method for screening thirty illicit drugs included in the italian residue detection plan in bovine urine

A selective and sensitive method for screening 31 analytes (nine corticosteroids, eight beta-agonists, seven anabolic steroids, six promazines and zeranol) in bovine urine was validated according to 2002/657/EC guidelines. Upon optimization of sample treatment conditions, the extraction was performed by diethylether at pH 9, after deconjugation. Extraction yields (R%) proved higher than 70% for 19 analytes, 50<R%<70 for 5 analytes, lower than 50% but reproducible for the remaining 6 analytes. The analyses were carried out using HPLC-ESI-MS/MS. The method sensitivity proved high enough to largely exceed the CCbeta requirements of the Italian residue detection plan, ranging from 1-3 ng/mL (20 ng/mL for promazines). The present method allowed the simultaneous analysis of most drugs for which the European legislation prescribes official controls. Its practical applicability was verified on 494 real samples as an alternative to the traditional screening protocols based on multiple immunometric analysis, demonstrating high efficiency and comprehensive investigation capacity, allowing epidemiological assessment of the current trends in cattle breeding drug abuse. Among non-compliant results, nine borderline cases of growth-promoters illegal treatments, making use of long term low dosage administrations and typically yielding urine residues below the cut-off value for immunochemical methods, were detected by using the present LC-MS/MS method.