Metabolic fingerprinting reveals a novel candidate biomarker for prednisolone treatment in cattle

The use of glucocorticoids as growth promoters for meat-producing animals is strictly regulated within the European Union. However, in the past few years, a higher frequency of non-compliant bovine urine samples for prednisolone has been noticed, which could not be directly related to fraudulent use of prednisolone. As such, questions have risen about the origin of this compound. Unfortunately, at present, no decisive strategy has been established to discriminate between endogenous and exogenous prednisolone. In this study, an untargeted metabolomics strategy, based on Orbitrap and QqTOF mass spectrometry, was deployed to reveal urinary biomarkers, which are indicative for the exogenous administration of the synthetic glucocorticoid prednisolone. For this purpose, prednisolone was administered intramuscularly and per os to 12 bovines and a total of 2700 urine samples were collected before, during and after treatment. Multivariate statistical data analysis (i.e. OPLS-DA) revealed four differentiating metabolites that allowed discrimination between urine samples collected before and during prednisolone administration. None of these compounds were present in urine containing endogenous prednisolone, of which the formation was induced by the administration of a synthetic analogue of adrenocorticotropic hormone. Only one metabolite was retained as a highly suitable biomarker during growth-promoting and therapeutic prednisolone treatment, with 93.4 % sensitivity and 96.3 % specificity. Besides, this compound could be detected up to 4 days after a single therapeutic per os prednisolone administration. Based on accurate mass, isotope pattern, and MS/MS spectra, this compound was putatively annotated and is suggested as an actionable biomarker for exogenous prednisolone administration.