A novel tool to screen for treatments with clenbuterol in bovine: Identification of two hepatic markers by metabolomics investigation

•Metabolomics analysis underscored markers of clenbuterol administration.•Clenbuterol administration affect metabolic pathways in liver.•Quantification of liver metabolites indirectly reveal clenbuterol administration.•A diagnostic tool to detect abuse of clenbuterol was developed and validated. Surveillance of illegal use of growth promoters such as β2-agonists in food producing animals rely on the detection of drug residues by LC-MS/MS. Screening strategies focusing on indirect physiological responses following administration of active compounds are promising approaches to strengthen existing targeted methods and ensure food safety. A metabolomics analysis based on LC-HRMS was carried out on liver extracts from bulls experimentally treated with clenbuterol combined with dexamethasone (n = 8) to mimic a potential anabolic practice, and control animals (n = 8). Nicotinic acid and 5′-deoxy-5′-methylthioadenosine were identified as biomarkers of treatment. Ratio values of such markers to others of the same metabolic pathways (nicotinamide or methionine) were used to develop a classification model to assign animals as treated with clenbuterol or non-treated. The classification model was tested on an external validation set comprising 74 animals either treated with different anabolic compounds (β2-agonists, sexual steroids, corticosteroid), or non-treated, showing 100% sensitivity and specificity.