Endocannabinoid sample preparation, data acquisition, and processing

Endocannabinoids were isolated by solid phase extraction on 10mg Waters Oasis-HLB cartridges (Milford, MA) as previously described (Luria et al. 2007). Prior to extraction, cartridges were washed with 1 column volume ethyl acetate followed by 2 column volumes methanol, and conditioned with 2mL of 95:5 (v/v) water/methanol (MeOH) with 0.1% acetic acid. The column reservoir was spiked with 5μL anti-oxidant solution, (0.2mg/mL BHT/EDTA in 1:1 MeOH/water), and 10μL 1000nM analytical surrogates. Sample aliquots (250μL media) were then introduced to the column reservoir and diluted with 1 column volume wash solution (5% MeOH, 0.1% acetic acid). The sample was gravity extracted and the sorbent bed was washed with 1 column volume of 20% methanol and 0.1% acetic acid. The solid-phase extraction cartridges were dried by vacuum (@ -7.5 in. Hg for 20 min). Analytes were then eluted by gravity with 0.2mL MeOH, followed by 0.5mL acetonitrile, followed by 0.5mL ethyl acetate into 2mL autosampler vials containing 10μL of a 20% glycerol/MeOH solution. Eluent was dried by vacuum evaporation for 35 min, and residues were re-constituted with 100μL of 100nM internal standard solution containing 1-cyclohexylureido, 3-dodecanoic acid (CUDA), in 50:50 MeOH/acetonitrile. Vials were vortexed for 1 min to dissolve residues, chilled 15 min on wet ice, and extracts transferred to a centrifugal filter (0.1μm Durapore, Millipore, Billerica, MA). After centrifugation (3 min at <4500g and 6°C), the extracts were transferred to 150μL glass inserts in 2mL amber vials, capped, and stored at −20°C until analysis by UPLC-MS/MS. The internal standard was used to quantify the recovery of the deuterated extraction surrogates by ratio response.