UC Davis

The potential abuse of small molecule drugs and peptides in horseracing is an area of great concern due to their high potential to act as doping agents. These compounds include classes of agents as growth hormone-releasing peptides (GHRPs), beta-2-agonists and quaternary ammonium drugs that are a challenge to detect and regulate due to their chemical properties and potential rapid elimination following administration. The use of highly sensitive and selective analytical techniques such as liquid chromatography – mass spectrometry (LC-MS) is necessary to provide coverage of these substances and their potential metabolites. This study describes development and validation of methodology capable of the detection of over fifty different peptide-based doping agents, related secretagogues, quaternary ammonium drugs, and other challenging small molecules in equine urine following solid phase extraction using a weak cation exchange sorbent. Following sample extraction the compounds were detected using LC-MS with chromatographic separation via a reverse phase gradient at 0.4 ml/min and selective reaction monitoring based detection following introduction to a triple-stage quadrupole mass spectrometer using positive mode electrospray ionization. Validation parameters including limits of detection and quantitation, accuracy, precision, linear range, recovery, stability, and matrix effects were determined. Briefly, the limits of detection for most compounds were in the 0.05 – 1.0 ng/mL range with adequate precision and accuracy sufficient for an initial testing method. Stability studies indicated that most compounds were sufficiently stable to allow for effective screening using conditions commonly utilized in drug testing laboratories.