Biosynthesis and Identification of Clenbuterol Metabolites in Urine and In Vitro Microsome Incubation Samples Using UHPLC‐Q‐Exactive Orbitrap Mass Spectrometry: A Comparison Between Human and Bovine Metabolism

ABSTRACT Clenbuterol (Clb) is a β2‐agonist drug included in the list of substances prohibited during and out of competition by the World Anti‐Doping Agency (WADA‐AMA). Several adverse analytical findings have been detected by accredited WADA laboratories, but athletes often claim that results are due to dietary contamination. In this contribution, bovine microsomal incubation and the excretion of bovine and human urinary metabolites of Clb were analyzed and compared using liquid chromatography electrospray Q‐Exactive‐Orbitrap mass spectrometry to determine differences in Clb metabolism. Urine samples were processed by solid‐phase extraction prior to electrospray analysis in both the positive and negative ion modes. MS/MS experiments were obtained by parallel monitoring reaction (PRM) triggered by an inclusion ions list. The strategy for metabolite identification involved the search for typical biotransformation based on accurate mass shifts using diagnostic fragment ions from the parent drug. This approach successfully identified eight metabolites, including a novel N ‐methylated form of Clb, reported here for the first time. Additionally, four metabolites found exclusively in bovine urine offer significant potential for further research aimed at distinguishing unintentional doping.