Oral Presentation 24th International Conference of Racing Analysts and Veterinarians 2026

Beyond the parent drug: Expanding tramadol metabolite detection using high-resolution mass spectrometry and molecular networking  (130359)

Cheka Kehelpannala 1 , Adam Cawley 1 , Harry Foster 1 , Stuart Paine 2 , Steven Karamatic 3 , Thusitha Rupasinghe 4
  1. Racing Analytical Services Ltd, Flemington, VIC, Australia
  2. School of Veterinary Medicine and Science, University of Nottingham, Leicestershire, United Kingdom
  3. Greyhound Racing Victoria, West Melbourne , VIC, Australia
  4. SCIEX Oceania, Melbourne, VIC, Australia

Integrating drug metabolites into routine doping control enhances detection capabilities and supports the interpretation of drug use regarding timing, dosage, and administration route. Despite advances in untargeted metabolomic techniques using mass spectrometry, comprehensive metabolite identification remains challenging. Molecular networking (MN) is a recent development to organise and visualise compounds based on tandem mass spectrometry data for discovery of novel metabolites.

In this study, we present a combined targeted and untargeted approach for analysing tramadol and its metabolites in canine plasma and urine. Initially, elimination and excretion profiles of the opioid analgesic tramadol and its primary metabolites – N-desmethyltramadol (N-DMT) and the active major metabolite O-desmethyltramadol (O-DMT) – were investigated following administration of Tramal®SR (5 mg/kg tramadol hydrochloride) twice daily for five days to six greyhounds.

Liquid chromatography-high resolution mass spectrometry (LC-HRMS) using an Orbitrap Exploris™ 120 in targeted MS2 mode was employed to analyse plasma and urine samples with a validated quantitative method. Appropriate pharmacokinetic analysis was performed. Tramadol and N-DMT were detectable in plasma up to 48 hours and in urine for up to 192 hours post-final dose. O-DMT was detected for 24 hours in unhydrolysed plasma, 48 hours in hydrolysed plasma, and 72 hours in hydrolysed urine. These results highlight the value of monitoring N-DMT, alongside tramadol and O-DMT, for improved detection in both matrices. Subsequently, we performed an untargeted LC-HRMS analysis using an Orbitrap Exploris™ 240 in data-dependent MS2 mode. The data were processed via the Global Natural Products Social platform and Compound Discoverer software independently to generate MN. This approach enabled the identification of more than 10 tramadol-related metabolites, including previously unreported compounds. Electron activated dissociation (EAD) of tramadol and its primary metabolites on the Sciex ZenoToF 7600 was also explored to obtain additional diagnostic information for structural characterisation.