Detection and Excretion Profiling of Semaglutide Use in Human Urine by UHPLC-HRMS for Anti-Doping Purpose

The gastric-emptying and satiety effects of semaglutide may lead to its misuse by athletes in weight-class sports, especially as the approved oral formulation is more convenient than subcutaneous injections. This study aimed to establish a simple ultrahigh-performance liquid chromatography-high resolution mass spectrometry (UHPLC-HRMS) method for monitoring semaglutide abuse based on urinary metabolites U6 and U7, and elucidate the urinary excretion profile of the oral formulation. Multidimensional optimization determined three key factors for reliable trace detection (recovery > 90%), with enhanced hydrophobic interactions dominating the enrichment process: (i) the adsorbent, (ii) the loading pH, and (iii) the washing solvent. Orbitrap-HRMS operating in dual acquisition modes enabled both specific identification and retrospective analysis. Systematic qualitative and quantitative validation was performed, and a single oral microdose of semaglutide (3 mg) was administered to a healthy volunteer, followed by continuous urine collection for 1 week. The optimized method provided satisfactory sensitivity (limit of detection of 0.03 ng/mL) and quantitative performance (linearity range: 0.1-20 ng/mL, R² > 0.995; accuracy > 85%) for both U6 and U7. Excretion profiling of 25 postdose samples showed that U6 and U7 were first detectable at 21 h, peaked at 0.15 and 0.10 ng/mL, respectively, and remained detectable for up to 28 h. Additionally, five abundant metabolites of the absorption enhancer salcaprozate sodium were identified (detection window up to 7 days), serving as sensitive screening indicators for oral semaglutide. These findings demonstrate the effectiveness of the developed method and provide the first perspective on the urinary excretion profile of oral semaglutide.