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

Yoondam Seo, Jisoo Park, Hyeon Jeong Lee, Minyoung Kim, Inseon Kang, Junghyun Son, Min Kyu Oh, Hophil Min

Research output: Contribution to journalArticlepeer-review

3 Citations (Scopus)


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.

Original languageEnglish
Article number3860
JournalScientific reports
Issue number1
Publication statusPublished - 2023 Dec

Bibliographical note

Funding Information:
This work was supported by an intramural Grant (2V09270) of the Korea Institute of Science and Technology (KIST), and a National Research Foundation of Korea (NRF) Grants (NRF-2021R1C1C1006989) funded by the Korean Government (MSIT).

Publisher Copyright:
© 2023, The Author(s).

ASJC Scopus subject areas

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