Carmoterol is a potent, long-acting β₂-adrenergic agonist originally developed for the treatment of respiratory disease in humans. Although not approved for human or veterinary use, its pharmacological properties suggest potential performance-enhancing effects in horses, making it a substance of concern in equine anti-doping. During routine surveillance, carmoterol was unexpectedly detected in a confiscated material submitted to the laboratory using a non-targeted LC-HRMS approach, prompting a wider investigation.
Screening and confirmation procedures were initially developed for serum, urine and hair samples. For urine and blood, samples were extracted by solid-phase extraction on WCX cartridge following enzymatic hydrolysis and analysis via Thermo Q Exactive mass spectrometer coupled to an Agilent 1260 HPLC system. The initial method achieved low pg/mL sensitivity in blood and urine samples but was later refined using capillary flow-HRMS, using Thermo Vanquish NEO coupled with Thermo Exploris 480, which improved sensitivity approximately ten-fold. Removal of the enzymatic hydrolysis step further enhanced extraction recovery, in serum samples, and reduced detection limits to sub pg/mL levels. Similar improvements were observed with the hair analysis allowing detection limits below 1 pg/mg. Application of the optimized workflows revealed the presence of carmoterol in more than 100 samples across multiple racing jurisdictions. Suspect samples were confirmed in both serum and urine, establishing the method’s suitability for regulatory use.
This represents the first report of the detection of carmoterol in equine sport. The findings highlight the role of non-targeted HRMS in early identification of emerging doping agents and the importance of retrospective sample analysis to capture cases missed by earlier methods. The new methods offer racing laboratories a reliable way to detect carmoterol in different sample matrices, strengthening efforts to safeguard equine welfare and racing integrity.