Lipid Analysis Protocol for Endocannabinoids

Extraction, purification and quantification of AEA, 2-AG, PEA and OEA from tissues require several biochemical steps as described previously [27 (link)]. N = 5 mice were used for these measurements. First, tissues were dounce-homogenized and extracted with chloroform/methanol/Tris-HCl 50 mM pH 7.5 (2:1:1, v/v) containing internal deuterated standards for AEA, 2-AG, PEA and OEA quantification by isotope dilution ([²H]₈AEA, [²H]₅2AG, [²H]₄ PEA, [²H]₄ OEA (Cayman Chemicals, MI, USA), as well as 1,2-heptadecanoin (Larodan AB, Malmo, Sweden), and 1-palmitoyl-2-oleoyl-N-heptadecanoyl phosphatidylethanolamine) for DAG and N-acyl-phosphatidylethanolamine measurement, respectively (see below). The lipid-containing organic phase was dried down, weighed and pre-purified by open bed chromatography on silica gel. Fractions were obtained by eluting the column with 99:1, 90:10 and 50:50 (v/v) chloroform/methanol. The 90:10 fraction was used for AEA, 2-AG, PEA and OEA quantification by liquid chromatography-atmospheric pressure chemical ionization-mass spectrometry (LC-APCI-MS), as previously described and using selected ion monitoring at M + 1 values for the four compounds and their deuterated homologues, as described in [1 (link),31 (link)].