No horsing around: New peptide drug measured in equine serum

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  • Published: Sep 15, 2012
  • Author: Steve Down
  • Channels: Base Peak
thumbnail image: No horsing around: New peptide drug measured in equine serum

Getting ahead at the races

A peptide-based drug which stimulates the formation of red blood cells and is used illegally in sports doping has been detected in equine serum by a novel LC/MS method.

In the quest for improved performance, many athletes turn to the type of drugs that increase the number of red blood cells in circulation. This is a variation of blood doping, in which red blood cells are transfused directly into the bloodstream but they have the same effect. Extra blood cells increase the oxygen-carrying capacity of the blood, which, in turn, increase the amount of oxygen available to the muscles, so that they can perform more efficiently and improve endurance.

The most common drugs of this type are erythropoietin (EPO) and its analogues. These erythropoiesis-stimulating agents (ESAs) are on the banned list for humans and have also found their way onto the equivalent list for horses. As new ESAs enter the clinical market, they are also added to the prohibited lists and one such example is peginesatide. This was introduced in 2010 as a potential treatment for anaemia and it was approved by the US FDA in 2012 for use with patients who have chronic kidney disease.

Peginesatide has a very different structure to erythropoietin but it mimics its behaviour, increasing the number of red blood cells and haemoglobin levels. But the structural differences mean that the conventional methods for measuring erythropoietin cannot simply be applied to peginesatide. A renowned team of scientists in Germany have devised a mass spectrometric procedure for measuring peginesatide in human serum and now they have expanded that to equine serum, with some modifications.

Peptide marker is key

Ines Möller, Andreas Thomas, Anke Wingender, Marc Machnik, Wilhelm Schänzer and Mario Thevis from the German Sport University at Cologne used the peptide portion of the peginesatide molecule as the basis for their method. The structure consists of two identical 21-amino acid chains linked to a polyethylene glycol chain via several other residues.

In order to produce a small peptide from the long chains, one of the conventional methods would be to break the chain up with the enzyme trypsin. However, that did not work in this case due to the nature of the amino acids present. So, the researchers resorted to subtilisin, a different enzyme which broke down the peptide chain to a pentapeptide which was analysed by mass spectrometry.

A portion of equine serum was mixed with a stable isotope-labelled form of the pentapeptide and processed by a simplified method which eliminated the SPE step employed in the original method for human serum. Instead, the proteins were removed by precipitation as before and the serum was centrifuged, treated with subtilisin, and centrifuged again before analysis.

The final solution was injected into the HPLC system fitted with a C18 column and the eluting peptides were subjected to electrospray ionisation and selected reaction monitoring of the target peptide. Three ion transitions were monitored for validation along with one transition for the internal standard. Although recoveries were low at just 30%, the method showed a wide linear range of 25-1000 ng/mL with a detection limit of 10 ng/mL.

The team expect that concentrations of the drug in horses will be about the same as those in humans. The half-life of peginesatide is about 23 hours, so they estimated that the detection limit will permit doping detection for several days after administration.

They concluded that "Being simple, fast, cost-effective and easily transferable to other laboratories in accordance with the criteria for 'identification by chromatography and mass spectrometry' outlined by the Association of Official racing Chemists [AORC], the method is considered suitable for routine use in the horse sports drug testing arena."

Related Links

European Journal of Mass Spectrometry 2012, 18, 407-412: "Detection of peginesatide in equine serum using liquid chromatography-tandem mass spectrometry for doping control purposes"

Article by Steve Down

The views represented in this article are solely those of the author and do not necessarily represent those of John Wiley and Sons, Ltd.

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