Quantification of Endocannabinoids and Oxylipins

Endocannabinoids and oxylipins were isolated from 250 µL of plasma using solid phase extraction and quantified by liquid chromatography tandem mass spectrometry as previously described [1 (link)]. Briefly, plasma was thawed on ice, and mixed with deuterated endocannabinoid and oxylipin internal standards in the head space of 60 mg Oasis-HLB solid phase extraction column (Waters Corp., Milford, MA, USA), where they were up-diluted to 20% methanol/0.1% acetic acid, and gravity loaded onto the columns, followed by vacuum to remove solvent. The columns were then wetted with 0.2 mL methanol and gravity eluted with 1.5 mL ethyl acetate. Solvent was removed by vacuum and residues were reconstituted in 50 µL methanol containing the internal standard 1-cyclohexyl-3-dodecyl-urea (Sigma, Aldrich, St. Louis, MO, USA). The resulting samples were filtered and analyzed by UPLC-(ESI)MS/MS by back-to-back (+)-mode/(−)-mode injections for endocannabinoid and oxylipin levels, respectively.