2h4 aea

Isofluorane was used to anesthetize animals at time of tissue harvest (0900 to 1100 h), following 24 h food deprivation or ad-libitum feeding. Blood was collected by cardiac puncture and stored in EDTA-lined tubes on ice, then plasma was obtained by centrifugation (1500 g for 10 minutes, maintained at 4°C). Jejunum was rapidly collected, washed with phosphate-buffered saline (PBS) on ice, sliced longitudinally on a stainless steel plate on ice, scraped with a glass slide to obtain mucosa, then snap-frozen in liquid N_2. All samples were stored at −80°C until processing. Frozen tissues were weighed and subsequently homogenized in 1.0 mL of methanol solution containing the internal standard, [²H₅] 2-AG and [²H₄]-AEA (Cayman Chemical, Ann Arbor, MI, USA). Lipids were extracted with chloroform (2 mL) and washed with water (1 mL). Lipids were similarly extracted from plasma samples, with the exception of a 0.9 % saline wash replacing water (0.1 mL plasma at the expense of saline). Organic phases were collected and separated by open-bed silica gel column chromatography as previously described [28 (link)]. Eluate was gently dried under N₂ stream (99.998% pure) and resuspended in 0.1 mL of methanol:chloroform (9:1), with 1μL injection for analysis by ultra-performance liquid chromatography coupled to tandem mass spectrometry (UPLC/MS/MS).