Doping control analysis of recombinant human erythropoietin, darbepoetin alfa and methoxy polyethylene glycol-epoetin beta in equine plasma by nano-liquid chromatography-tandem mass spectrometry.

This research article presents a method to detect the use of performance-enhancing synthetic drugs in horses using nano-liquid chromatography-tandem mass spectrometry.

Introduction and Background

• The focus of this research is on three synthetic analogues of the endogenous hormone erythropoietin (EPO): recombinant human erythropoietin (rhEPO), darbepoetin alfa (DPO), and methoxy polyethylene glycol-epoetin beta (PEG-EPO).
• Erythropoiesis-stimulating agents, such as those, are clinically used for treating anemia in humans by stimulating red blood cell production. However, they are also used illicitly for enhancing athletic performance by increasing oxygen delivery to muscle tissues. The reported analogues have not been definitively proven to enhance performance in horses as in humans, but they have been covertly used on horses.
• Due to their potential unfair advantages, these protein-based drugs are banned in all types of sports, according to the authorities like the International Olympic Committee (IOC) and the World Anti Doping Agency (WADA).
• WADA’s method for detecting these agents in human urine involves electrophoresis combined with Western blotting. However, this approach does not provide definitive mass spectral data for the confirmation of a positive finding.

Objective of the Research

• The researchers aim to establish a method to detect the presence of rhEPO, DPO, and the newly released PEG-EPO in horse plasma. Previous methods using liquid chromatography-tandem mass spectrometry (LC/MS/MS) have not provided satisfactory sensitivity. They also aim to provide definitive mass spectral data for detection and confirmation.

Research Methodology

• The method involves an immunoaffinity extraction process using anti-rhEPO antibody-coated Dynabeads, followed by trypsin digestion.
• The extract is further purified and concentrated using an on-line trap column, which is part of the nano-LC system.
• Detection and authentication are achieved by monitoring a unique peptide segment of rhEPO/DPO/PEG-EPO using nano-liquid chromatography-tandem mass spectrometry. This mass spectrometry is equipped with a nanospray ionisation source and operates in the selected reaction monitoring mode.

Results

• The method can confirm the presence of rhEPO, DPO and PEG-EPO at concentrations of 0.1, 0.2 and 1.0 ng/mL, respectively, in equine plasma.