Oxylipin Extraction and Analysis

Free oxylipins were analysed as previously described (Kutzner et al. 2019 (link); Rund et al. 2018 (link),2019 (link)). In brief, 10 µL of internal standards, butylated hydroxy toluene and enzyme inhibitors were added to ~ 20–50 mg tissue. The samples were homogenized in 300 µL methanol using a ball mill. Samples were centrifuged, the supernatants were collected and loaded onto the pre-conditioned solid phase extraction (SPE) cartridges. Oxylipins were extracted by SPE using a C8/anion exchange cartridge material (Bond Elut Certify II, Agilent, Waldbronn, Germany). The eluate was evaporated and reconstituted in methanol containing second internal standards. Samples were analysed by means of LC–MS/MS (QTRAP, Sciex, Darmstadt, Germany) with negative electrospray ionization. Detection was carried out in scheduled selected reaction monitoring mode.