Further Insights Into the Metabolism of LGD‐4033 in Human Urine. Part 1. Structure Elucidation of Additional Important Metabolites

ABSTRACT This study presents LC‐HRMS/MS analyses of LGD‐4033 post‐administration urine samples, following hydrolysis with β ‐glucuronidase and liquid–liquid extraction, against chemically synthesized molecules that matched previously proposed metabolites, characterized by 1 H and 13 C NMR. Using this targeted metabolic investigation approach and the direct comparison of retention times and mass spectral data (high‐resolution full scan mass accuracy and collision‐induced fragmentation patterns), in accordance with WADA's TD2023IDCR provisions, resulted in unambiguous structural elucidation of additional LGD‐4033 metabolites, including (a) the epi ‐long‐term dihydroxylated metabolite ( M5a ); (b) the epi ‐pyrrolidinone metabolite ( M2d ); (c) the ( R , R )‐diastereoisomer of the ring‐opened hydroxylated metabolite ( M4b ); and (d) one of the two detected tris‐hydroxylated metabolites ( M6a ). Additionally, a new, previously undescribed metabolite, which is a hydroxylated derivative of the pyrrolidinone metabolite M2c , was also detected up to 4 days post‐administration and coded as M7 . Metabolites M5a and M2d are detectable up to 21 days post‐administration and can be considered additional long‐term markers. These findings expand current knowledge of LGD‐4033 metabolism. From a doping control perspective, the proposed synthetic pathways may facilitate the production of reference materials for the detection and identification of a more comprehensive metabolite profile that will increase metabolic certainty in future LGD‐4033 adverse analytical findings.