Doping control analysis of seven bioactive peptides in horse plasma by liquid chromatography-mass spectrometry.

The researchers developed an inexpensive and efficient method of detecting specific bioactive peptides—substances that could potentially be misused for doping—in horse plasma. Using a specialized extraction process and high-resolution mass spectrometry, they were able to detect extremely low amounts of seven target peptides and their metabolites.

Research Method

• The research centred around the development of a detection method for seven specific peptides (GHRP-1, GHRP-2, GHRP-6, ipamorelin, hexarelin, CJC-1295, and N-acetylated LKKTETQ, an active ingredient of TB-500) that could potentially be misused for doping purposes in horses.
• Instead of using the traditional immunoaffinity purification method—which can be cost-prohibitive because it requires often expensive monoclonal or polyclonal antibodies—the researchers used a simpler extraction method involving a mixed-mode anion exchange solid-phase cartridge.
• The extracted peptides and their in vitro metabolites were later analyzed through ultra-high-performance liquid chromatographic separation coupled with a hybrid high-resolution mass spectrometer.

Results and Findings

• The detection method they developed was able to identify all seven target peptides at incredibly low concentrations, with the limits of detection estimated to be under 50 pg/mL.
• The researchers validated their method with regard to its ability to differentiate the target peptides from others (specificity), its consistency of results (precision), and its efficiency and accuracy in extract recovery.
• The method also proved applicable in a real-world scenario. The researchers successfully detected the peptide N-acetylated LKKTETQ and its metabolite N-acetylated LK in plasma samples from two thoroughbred geldings to whom they had administered TB-500.
• The researchers suggested that this method could be modified to detect more bioactive peptides, such as dermorphin, β-casomorphin, and desmopressin.
• By utilizing high-resolution mass spectrometry, they can also retrospectively process full-scan data to look for peptides and their metabolites that weren’t targeted at the time of the original analysis.
• The research marks the first identification of in vitro metabolites of all the studied peptides, except for TB-500, in horses.

Significance of the Study

• The study represents significant progress in advancing methods that allow for more efficient, inexpensive, and extensive detection of potentially misused substances in the equine sporting world.
• The new method offers an alternative to laboratories for detecting doping substances in equine and potentially human samples without the need for costly antibodies.
• These results expand the knowledge on doping control, providing new insights on techniques for the detection and analysis of specific bioactive peptides, which can support implementation and enforcement of anti-doping regulations.