Lipid Extraction and Analysis by HPLC-MS

Tissues were homogenized in CHCl₃ (10 ml), then MeOH (5 ml), H₂O (2.5 ml) and HCl 2 N were added and the lipids extracted by vigorous mixing. The organic layer was recovered and dried under N₂. The resulting lipid fraction was pre-purified by solid-phase extraction over silica, and NAPEs were eluted with CHCl₃-MeOH (6:4, v/v). The resulting lipid fraction was analyzed by HPLC-MS using a LTQ Orbitrap mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA) coupled to an Accela HPLC system (Thermo Fisher Scientific). Analyte separation was achieved by using a C-18 Kinetex C-18 column (5 μm, 4.6 × 150 mm; Phenomenex, Utrecht, Netherlands) and a C-18 pre-column. Mobile phases A and B were composed of MeOH-H₂O-NH₄OH (75:25:0.1, v/v/v) and MeOH-NH₄OH (100:0.1, v/v), respectively. The gradient (0.5 ml min⁻¹) was as follows: from 100% A to 100% B in 15 min, followed by 10 min at 100% B and subsequent re-equilibration at 100% A. Mass spectrometry analysis in the negative mode was performed with an ESI source. The measurement of eCB were generated as previously described32 (link), and the data were normalized to tissue sample weight.