Lipid Extraction and Quantification

Plasma was sonicated and extracted with chloroform/methanol/Tris-HCl 50 mmol/L pH 7.5 (2 : 1 : 1, vol/vol) containing internal standards ([H₂]8 AEA 5 pmol; [H₂]5 2-AG, [H₂]5 PEA, and [H₂]4 OEA 50 pmol each) for EC quantification as well as 1,2-heptadecanoin (Larodan AB, Malmo, Sweden) for DAG measurement. The lipid-containing organic phase was dried down, weighed, and prepurified by open-bed chromatography on silica gel with 99 : 1, 90 : 10, and 50 : 50 (v/v) chloroform/methanol. The 90 : 10 fraction was used for EC and N-acylethanolamine quantification by LC-APCI-MS (LCMS-2020, Shimadzu) as previously reported [54 (link)]. DAG levels were measured by LC-MS-MS using an LC20AB coupled to a hybrid detector IT-TOF (Shimadzu Corporation, Kyoto, Japan) equipped with an ESI interface [55 (link)].