*4.10. Lipidomic Analysis*

For oxylipins determination plasma and urine samples were extracted essentially as described by Wolfer et al. 2015 [60]. Briefly plasma samples were prepared by transferring 100 μL to the preparation plate after thawing and brief vortexing. A volume of 20 μL of IS working solution and 30 μL of 2% formic acid solution in water were added, and the plate was capped and gently mixed. The SPE plate was conditioned using 200 μL of MeOH and the sorbent equilibrated with 200 μL of H2O. Samples were transferred from the preparation plate to the SPE, the preparation plate was further washed with 50 μL of MeOH/H2O 1:1, and the rinsing solution will be added to the SPE plate. Following aspiration, the SPE plate will washed with 200 μL of H2O + 2% NH4OH and 200 μL of H2O/ACN 1:1. Oxylipins will be eluted with 4 × 25 μL of MeOH + 2% formic acid. The elution fraction was evaporated under N2 and the residues reconstituted in 120 μL of MeOH/H2O. Urine samples were prepared by mixing 50 μL of urine with 50 μL of MeOH and 20 μL of IS working solution following thawing and brief vortexing.

Oxylipins determination was carried out with ultrahigh performance liquid chromatography (UHPLC) coupled with mass spectrometry (triple quadrupole, q-tof or orbitrap instrument) the methods and conditions of ionization were chosen to achieve the best results in order of quantification and reproducibility.
