Differentiation and identification of recombinant human erythropoietin and darbepoetin Alfa in equine plasma by LC-MS/MS for doping control.

This research article presents a new method for identifying and differentiating two protein-based drugs, recombinant human erythropoietin (rhEPO) and darbepoetin alfa (DPO), which are sometimes misused in equine sports for performance enhancement. The method uses liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) on equine plasma samples, providing a potential tool in the fight against blood doping in the horse racing industry.

Understanding Protein-Based Drugs

• Recombinant human erythropoietin (rhEPO) and darbepoetin alfa (DPO) are protein-based drugs used to treat anemia in humans.
• These drugs function by stimulating the production of erythrocytes, a type of blood cell that transports oxygen in the body.
• However, because of their ability to enhance performance, these drugs are often misused in both human and equine sporting arenas.

Method Development

• The researchers developed a novel method to differentiate and identify rhEPO and DPO in the blood plasma of horses through the use of liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS).
• The process involved analyte extraction and enrichment, dual digestion by trypsin and peptide-N-glycosidase F (PNGase F), and subsequent analysis using LC-MS/MS.
• Two distinctive deglycosylated tryptic peptides were employed to differentiate and identify the two drugs.

Application and Benefits

• This is the first method developed to help identify and differentiate the presence of these two drugs in equine plasma.
• The limit of identification for the drugs was lower than previous techniques, making it incredibly sensitive and accurate.
• The researchers were successful in identifying DPO in plasma samples collected from a research horse post-DPO administration, and from racehorses out of competition in North America.
• This method brings a promising addition to current strategies against blood doping in the horse racing industry.

Resolving Challenges

• In the process, the researchers encountered and solved the analyte carryover problem by adding 20% acetonitrile to the solvent of the sample digest to increase solubility of the peptides.