Development and validation of qualitative methods for the detection of GLP-1 receptor agonists in serum and dried blood spots by LC-HRMS/MS for doping control purposes.

The increasing use of glucagon-like peptide-1 (GLP-1) receptor agonists for weight management has raised concerns about their potential misuse in sports, where effects such as weight loss, glucose-dependent insulin secretion, and enhanced mitochondrial biogenesis may offer performance advantages. Although currently not prohibited, semaglutide and tirzepatide have been included in the World Anti-Doping Agency (WADA) Monitoring Program to assess potential misuse. In response to these concerns, this study aimed to develop detection methods for five GLP-1 receptor agonists as well as the endogenous metabolite GLP-1(9-36)-NH2 in serum and dried blood spot (DBS) samples by liquid-chromatography and high-resolution tandem mass spectrometry (LC-HRMS/MS). For analytes with expected low blood concentrations (exenatide, lixisenatide, GLP-1(9-36)-NH2), affinity purification using magnetic beads coupled to an anti-GLP-1 antibody and a GLP-1 receptor fusion protein achieved limits of detection (LODs) ranging from 9.4 pg/mL (exenatide) to 48 pg/mL (lixisenatide and GLP-1(9-36)-NH2) in 100 μL of serum. Serum analysis provided superior sensitivity compared to DBS and was therefore prioritized for these analytes. For higher-abundant, lipidated peptides (liraglutide, semaglutide, tirzepatide), mixed-mode anion-exchange solid-phase extraction was employed, allowing for LODs ranging from 0.48 ng/mL (tirzepatide) to 4.7 ng/mL (liraglutide and semaglutide) in serum, and 0.95 ng/mL (liraglutide and semaglutide) to 3.8 ng/mL (tirzepatide) in DBS. Both sample matrices were suitable for reliable detection. In addition, post-administration samples from humans treated with semaglutide and tirzepatide, as well as murine samples following administration of exenatide, lixisenatide, and liraglutide, confirmed the suitability of the developed methods for sports drug testing.