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RAD140 is a selective androgen receptor modulator which has been abused in sporting competitions. Its use is prohibited by the World Anti-Doping Agency (WADA) for athletes at all times. In addition to its illicit use, adverse analytical findings of RAD140 in doping control samples might result from other scenarios, e.g., the ingestion of contaminated dietary supplements. The differentiation between samples resulting from such contamination scenarios and intentional doping presents a considerable challenge, as little is known about the metabolism and elimination behavior of RAD140 in humans. In this study, six micro-dose excretion studies with five adult male volunteers each were conducted, and urine samples were analyzed by means of LC-HRMS/MS. Multiple metabolites, firstly detected in human urine, are described in this study. The sample preparation included an enzymatic hydrolysis step, which facilitated the estimation of RAD140 concentrations in urine. The elimination profiles and detection times for six metabolites as well as the intact drug are presented. The method was extensively characterized and deemed fit-for-purpose. The metabolite ratios were investigated for their predictive power in estimating the dose of RAD140 intake. The presented data will aid in better case result management in future doping cases involving RAD140.

Rationale: The synthetic β-adrenoreceptor agonist zilpaterol is legitimately used as an animal feed supplement in selected countries due to its known effects on lipolysis and protein biosynthesis. These pharmacological characteristics of zilpaterol have contributed to its classification as doping agent in sport by the World Anti-Doping Agency. However, the use as a feed supplement can lead to residues of the drug in edible tissues and, possibly, also in the urine of consumers.Methods: To provide urinary elimination profiles of microdosed zilpaterol and to determine whether the ingestion of zilpaterol below or at the acceptable daily intake level of 0.04 μg/kg bodyweight can result in an adverse analytical finding (AAF) in doping controls, healthy volunteers were administered single or multiple oral doses of 0.5 μg or 3 μg zilpaterol to mimic ingestion of contaminated cattle meat. Urine samples were collected and analyzed using a validated high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry (HPLC-ESI-MS/ MS) method and a newly developed chiral high-performance liquid chromatographyatmospheric pressure chemical ionization-tandem mass spectrometry (HPLC-APCI-MS/MS) method.Results: Urinary peak concentrations of zilpaterol were observed for all volunteers 1.5-12.5 h after ingestion, and maximum levels >5 ng/mL, which would constitute an AAF in doping controls, were found after the intake of 3 μg of zilpaterol on five consecutive days in one out of five study participants. Noteworthy, the enantiomeric ratio of excreted zilpaterol remained constant over time. Conclusion:This study provides first insights into the urinary excretion of microdosed zilpaterol. Furthermore, a method was successfully developed and applied for the separation of the zilpaterol enantiomers with mass spectrometric detection.

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