Method Development of Pegmolesatide for Doping Analysis: A Novel Synthetic Erythropoietin‐Mimetic Agent

ABSTRACT Pegmolesatide, a novel synthetic erythropoietin‐mimetic agent, was developed by the Hansoh Pharmaceutical Manufacturing Company Ltd. (Jiangsu, China). In late 2023, it was approved in China for the treatment of anemia in both dialysis and non‐dialysis patients with chronic kidney disease, with the advantages of reduced immunogenicity and extended duration of action. The aim of this study was to develop a strategy for detecting pegmolesatide in doping analysis. Here, we present a bottom‐up nano‐liquid chromatography–high‐resolution tandem mass spectrometry approach for qualitative analysis of pegmolesatide using erythropoietin receptor coupled magnetic beads, followed by trypsinization and subsequent detection of characteristic peptides. Using full scan and data‐dependent MS/MS (ddMS2) modes, two characteristic peptide segments of pegmolesatide were identified. An analytical method using product ion scan mode was developed to detect the identified characteristic peptides. Both peptide segments were analyzed for the initial testing procedure, whereas one characteristic peptide segment obtained from complete trypsinization was analyzed for the confirmation procedure. After full validation, the selectivity, reliability, limit of detection, limit of identification, carryover, and stability were evaluated. The results demonstrate that our developed method can be a fit‐for‐purpose analytical method for the antidoping of pegmolesatide.