Detection of peginesatide in equine serum using liquid chromatography-tandem mass spectrometry for doping control purposes.

The research article discusses the development and validation of a specific method to detect a new drug, peginesatide, which can be used for doping in horse sports, using a proteolytic digestion with subtilisin after protein precipitation and detection via liquid chromatography-tandem mass spectrometry.

Why this research is important

• Peginesatide is a drug approved for the treatment of anaemia in humans with chronic kidney disease. It belongs to a new class of EPO-mimetic peptides (erythropoiesis-stimulating agents) which have been suspected of being illicitly used in horse sports for performance enhancement purposes.
• Because it’s a pegylated dimeric peptide of around 45 kDa and does not have sequence homology to EPO, peginesatide cannot be detected by conventional EPO detection assays. Therefore, it’s crucial to establish a new method of detection for this drug for doping control purposes in horse sports.

A newly developed detection method

• The researchers developed an approach by spiking equine serum with peginesatide, and then applying a proteolytic digestion with subtilisin after protein precipitation.
• The method targets a proteotypic and xenobiotic pentapeptide which can be easily analysed by liquid chromatography-tandem mass spectrometry.

Validation of the method

• The developed method is validated for qualitative purposes and it has been proven to be specific, precise with relative standard deviations below 14%, sensitive with a limit of detection 10 ng mL(-1), and linear.
• The assay is simple, cost-effective, and can be easily transferred to other doping control laboratories.
• Given its feasibility, this mass spectrometric assay is applicable to regular analysis shortly after the drug’s approval, serving as a preemptive measure for anti-doping research.