Identification of Candidate Blood Biomarkers of Recombinant Human Erythropoietin Administration Using Targeted Polar Metabolomics by HILIC‐MS/MS

Increasing oxygen transport through elevated hemoglobin concentration and red blood cell mass is a key objective of blood doping, commonly achieved via recombinant human erythropoietin (rHuEPO) administration or blood transfusions. While the Athlete Biological Passport (ABP) offers an effective indirect tool for detecting such manipulations, its sensitivity and specificity may be limited, particularly in cases involving microdoses or confounding physiological factors. To address these limitations, the identification of novel biomarkers that complement current ABP markers is essential. This study presents a targeted metabolomics approach to discover candidate biomarkers of rHuEPO administration by analyzing polar metabolites in plasma and serum from two administration studies: one involving a single CERA injection, and the other using multiple doses of epoetin delta. Hydrophilic interaction chromatography hyphenated with tandem mass spectrometry enabled the selective and sensitive detection of a panel of polar endogenous metabolites. Following data normalization and stringent quality control, generalized least squares models were applied to evidence temporal changes in metabolite signals. Among the most responsive and concordant markers across both studies were hypoxanthine and inosine, which showed significant and marked increases following rHuEPO administration. Notably, the relative increase of these metabolites coincided with the maximum in reticulocyte percentages, reflecting maximal erythropoietic activity. As intermediates in purine metabolism, their increases are likely tied to augmented purine turnover during red blood cell production. These findings suggest that hypoxanthine and inosine are promising candidate biomarkers to complement existing ABP parameters. However, further validation is required to confirm their reliability and applicability within the ABP framework.