Detection and Excretion Profile of Retatrutide in Human Plasma and Urine by LC‐HRMS: Implications for Antidoping Analysis

ABSTRACT Rationale Retatrutide (LY3437943) is a novel long‐acting triple incretin receptor agonist that targets the glucagon‐like peptide‐1 receptor (GLP‐1R), glucose‐dependent insulinotropic polypeptide receptor (GIPR), and glucagon receptor (GCGR). Its pronounced effects on body composition and metabolic health, combined with increasing off‐label use among physically active populations, raise concerns about potential misuse in sport. Despite growing clinical interest and anecdotal reports about use among amateur athletes, information on the detection and elimination profile of retatrutide in humans remains unexplored. Methods A method based on liquid chromatography–high‐resolution mass spectrometry (LC‐HRMS) dedicated to monitor retatrutide was developed and validated in human plasma and urine in accordance with World Anti‐Doping Agency (WADA) criteria. Detection was based on the [M + 3H] 3+ precursor ion by targeted selected‐ion monitoring (t‐SIM), with confirmatory analysis via parallel reaction monitoring (PRM) of structurally diagnostic fragment ions. From four retatrutide users who self‐administered 2 mg subcutaneously on Days 1 and 7, blood and urine samples were collected at predefined time points following self‐administration. Results The method was proved fit‐for‐purpose regarding selectivity, reliability, robustness, and stability. Limits of detection (LOD) were evaluated using both empirical ≥ 95% detection rate criteria and sigmoidal logistic regression modeling, yielding 6.14 ng/mL in plasma and 2.44 ng/mL in urine. Neither intact retatrutide nor metabolite‐derived signals were detected in urine samples throughout the entire collection period, despite systematic application of multiple extraction strategies. Plasma concentration–time profiles exhibited nonmonotonic behavior, consistent with prolonged subcutaneous absorption and depot‐like release kinetics following repeated dosing. Conclusion These findings suggest that plasma represents the appropriate matrix for intact retatrutide detection compared with urine. The extended plasma detection window, approximately 56 days after the first dose, supports plasma‐based testing as a suitable analytical strategy for this and related long‐acting therapeutic peptides in antidoping testing context.