Development and validation of a method for analyzing the sialylated glycopeptides of recombinant erythropoietin in urine using LC–HRMS

Abstract

Erythropoietin (EPO) is a glycoprotein hormone that stimulates red blood cell production. It is produced naturally in the body and is used to treat patients with anemia. Recombinant EPO (rEPO) is used illicitly in sports to improve performance by increasing the blood’s capacity to carry oxygen. The World Anti-Doping Agency has therefore prohibited the use of rEPO. In this study, we developed a bottom-up mass spectrometric method for profiling the site-specific N-glycosylation of rEPO. We revealed that intact glycopeptides have a site-specific tetra-sialic glycan structure. Using this structure as an exogenous marker, we developed a method for use in doping studies. The profiling of rEPO N-glycopeptides revealed the presence of tri- and tetra-sialylated N-glycopeptides. By selecting a peptide with a tetra-sialic acid structure as the target, its limit of detection (LOD) was estimated to be < 500 pg/mL. Furthermore, we confirmed the detection of the target rEPO glycopeptide using three other rEPO products. We additionally validated the linearity, carryover, selectivity, matrix effect, LOD, and intraday precision of this method. To the best of our knowledge, this is the first report of a doping analysis using liquid chromatography/mass spectrometry-based detection of the rEPO glycopeptide with a tetra-sialic acid structure in human urine samples.

Whispers in the Blood: Leveraging MicroRNAs for Unveiling Autologous Blood Doping in Athletes

“…In the quest for additional data to enhance the detection of ABT, beyond relying solely on hematological markers and total blood miRNAs, urine presents an overlooked biospecimen. Urine samples are routinely gathered in anti-doping controls and are authorized for detecting rhEPO [ 67 ]. Physical activity and an individual’s overall physical condition can influence blood volume and potentially alter total blood miRNA profiles due to exercise-induced leukocytosis [ 68 ].…”