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

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 ¹H and ¹³C 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.