A MEPS-UHPLC-MS/MS analytical platform to detect isoprostanoids and specialized pro-resolving mediators in the urinary extracellular vesicles of mountain ultramarathon runners

Oxylipins are powerful signalling compounds derived from polyunsaturated fatty acids (PUFAs) and involved in regulating the immune system response. A mass spectrometry-based method was developed and validated for the targeted profiling of 52 oxylipins (e.g., isoprostanoids, prostaglandins, epoxy- and hydroxy-fatty acids, specialized pro-resolving mediators) and 4 PUFAs in small urinary extracellular vesicles (uEVs). Ultrasound-assisted extraction using a 50:50 v/v MeOH:H₂O mixture ensured optimal analytical performances. Limits of detection ranged between 10 and 400 pg/mL for oxylipins and 0.10–3 ng/mL for PUFAs. Satisfactory recoveries (85–116 %) and good intra- and inter-day precisions (RSD ≤15 %) were obtained for all the analytes. The reliability of the procedure was tested in a real case scenario by monitoring ultramarathon runners during the world Tor des Géants® (TDG) race. Both F₂- and E₂-isoprostanes were detected in small uEVs of the ultramarathon runners, suggesting the onset of an oxidant insult. 5-F_2t-IsoP exhibited significant pre- to post-race variations, thus potentially representing a non-invasive marker of in-vivo lipid peroxidation. The presence of specialized pro-resolving mediators suggests the activation of pro-resolution signalling cascade resolving inflammation. These outcomes may help manage post-exercise recovery and improve training.