Method development of a targeted glycopeptide analysis of recombination erythropoietin for doping control using liquid chromatography-high resolution mass spectrometry

Abstract

Recombinant EPO, designated by the World Anti-Doping Agency as a prohibited drug, has been misused and abused to improve athletic performance in sports. It has a glycan structure of a tetra-sialic acid, unlike endogenous EPO. We aimed to identify this structure in a recombinant EPO using the Biological Reference Preparation for erythropoietin (BRP) as a working standard. Thus, in order to analyze the recombinant EPO, a LC？MS method using a glycopeptide enrichment technique on the intact recombinant EPO glycopeptide was established. In addition, a detection experiment was performed in urine samples through the optimization process of the recombinant EPO detection assay. By optimizing the analysis method, the experimental time was reduced to less than one day, and the process of distinguishing recombinant EPO from endogenous EPO was more efficiently and less time consuming. In this study, we were able to analyze an intact glycopeptide with a tetra-sialic acid glycan structure present only in recombinant EPO through an LC？MS analysis. This rapid assay allows an effective detection of recombinant EPO, a prohibited substance in sport competitions.