An 1100-LC system coupled to a 1946A-MS detector (Agilent Technologies, Inc., Palo Alto, CA, USA) equipped with an electrospray ionization interface was used to measure anandamide and 2-AG levels in each tissue punch. Lipids were separated using a XDB Eclipse C18 column (50 × 4.6 mm i.d., 1.8 μm, Zorbax), eluted with a gradient of methanol in water (from 75% to 85% in 2.5 min, to 90% in 7.5 min, to 100% in 14 min, and to 75% in 20 min) at a flow rate of 1.0 mL/min. Column temperature was kept at 40 C. MS detection was in the positive ionization mode, capillary voltage was at 3 kV, and fragmentor voltage varied from 120V. N2 was used as drying gas at a flow rate of 13 L/min and temperature of 350 °C. Nebulizer pressure was set at 60 PSI. Quantifications were conducted using an isotope-dilution method (Moise et al., 2008, Astarita and Piomelli, 2009) by monitoring Na+ adducts of the molecular ions ([M+Na]+) in the selected ion-monitoring mode. Quantification limits were 0.08 pmol for anandamide and 0.4 pmol for 2-AG.
