Detection of gonadotropin‐releasing hormone and its analogues in equine and canine urine by high‐resolution data‐independent acquisition

The abuse of prohibited agents including peptides and basic small‐molecule drugs is an area of great concern in horseracing due to their high potential to act as doping agents. These compound classes include agents such as growth hormone‐releasing peptides, peptide analgesics, beta‐2‐adrenergic receptor agonists, and quaternary ammonium drugs that can be challenging to detect and regulate because of 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 the development and validation of methodology capable of the detection of over 50 different peptide‐based doping agents, related secretagogues, quaternary ammonium drugs, and other challenging small molecules in equine urine following solid‐phase extraction using a mixed mode weak cation exchange sorbent. Following sample extraction, the compounds were analyzed using LC–MS with chromatographic separation via a reverse phase gradient and detection via selective reaction monitoring 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 sub‐ng/mL ranges with adequate precision and accuracy sufficient for an initial testing procedure. Stability studies indicated that most compounds were sufficiently stable to allow for effective screening using conditions commonly utilized in drug testing laboratories.

Section: Limitations and Suggested Improvementsmentioning

confidence: 99%

Detection of doping peptides and basic drugs in equine urine using liquid chromatography–mass spectrometry

Flores,

Hargrave,

Clifford

et al. 2023

Analytical advances in horseracing medication and doping control from 2018 to 2023

Gray,

Lubbock,

Love

et al. 2024

The analytical approaches taken by laboratories to implement robust and efficient regulation of horseracing medication and doping control are complex and constantly evolving. Each laboratory's approach will be dictated by differences in regulatory, economic and scientific drivers specific to their local environment. However, in general, laboratories will all be undertaking developments and improvements to their screening strategies in order to meet new and emerging threats as well as provide improved service to their customers. In this paper, the published analytical advances in horseracing medication and doping control since the 22nd International Conference of Racing Analysts and Veterinarians will be reviewed. Due to the unprecedented impact of COVID‐19 on the worldwide economy, the normal 2‐year period of this review was extended to over 5 years. As such, there was considerable ground to cover, resulting in an increase in the number of relevant publications included from 107 to 307. Major trends in publications will be summarised and possible future directions highlighted. This will cover developments in the detection of ‘small’ and ‘large’ molecule drugs, sample preparation procedures and the use of alternative matrices, instrumental advances/applications, drug metabolism and pharmacokinetics, the detection and prevalence of ‘endogenous' compounds and biomarker and OMICs approaches. Particular emphasis will be given to research into the potential threat of gene doping, which is a significant area of new and continued research for many laboratories. Furthermore, developments in analytical instrumentation relevant to equine medication and doping control will be discussed.

High‐Throughput Equine Doping Controls on a Trapped Ion Mobility Quadrupole‐Time‐of‐Flight Mass Spectrometer: Technical Considerations of dia/slice/prmPASEF Applied to the Long‐Term Detection of Monoclonal Antibodies

Delcourt,

Pinetre,

Chabot

et al. 2024

Data‐independent acquisition (DIA) methods employing a scanning quadrupole were recently described across multiple platforms. These strategies display remarkable performances in untargeted proteomics studies thanks to rapid duty cycles, leading to ultrashort liquid chromatography gradients while maintaining enough data points per peaks when coupled to fast‐scanning mass analyzer. In this article, we perform the evaluation of three data acquisition strategies named diaPASEF,slicePASEF, and prmPASEF on a trapped ion mobility spectrometry quadrupole‐time‐of‐flight (TIMS‐Q‐TOF) mass spectrometer for high‐throughput doping control screening analyses. We report that slicePASEF outperforms diaPASEF and is almost as sensitive as prmPASEF in detecting humanized monoclonal antibodies for several weeks in equine plasma after administration. We observed that diaPASEF is still providing the best performances in untargeted proteomics studies employing high amounts of input materials, which is linked with the high complexity of slicePASEF data and current processing algorithms.