Combined detection of inhibitors of the activin receptor signaling pathways (IASPs) by means of LC-HRMS/MS for human doping control

Members of the transforming growth factor beta superfamily such as myostatin, activin A, and GDF-11, are dimeric cytokines signaling through activin receptors. They play important regulative roles in different biological processes as the formation of muscle and red blood cells. Therefore, inhibitors of the activin receptor signaling pathways (IASPs) are potential performance-enhancing agents in sports, which are included in sections S2 ("Peptide hormones, growth factors, related substances and mimetics") and S4 ("Hormone and metabolic modulators") of the WADA Prohibited List. Within this research project, a multiplexed detection assay for nine IASPs in doping control serum/plasma samples by means of (immuno-)affinity purification, tryptic digestion and LC-HRMS/MS was developed. The method was validated and proved to be specific and sensitive (LOD: 10-50 ng/mL). Additionally, it was modified to allow for using urine. As proof-of-concept, authentic Luspatercept serum and urine and Sotatercept serum post-administration samples were successfully analyzed. Luspatercept could be detected in both matrices up to 70 days after the initial and 7 weeks after the second dose. Sotatercept was successfully detected in a serum sample collected 43 h following injection. The presented method can be employed in doping control routine analysis as a qualitative initial testing procedure.