Simultaneous determination of endocannabinoids in murine plasma and brain substructures by surrogate-based LC-MS/MS:...

The endocannabinoids (eCBs), N-arachidonoylethanolamine (anandamide, AEA) and 2-ararchidonylglycerol (2-AG) have been identified as main endogenous ligands for cannabinoid receptors. Developing a sensitive and robust method to determine AEA and 2-AG has been shown to be essential to understand their effects in stress regulation and the pathogenesis of affective disorders. Since eCBs are endogenous molecules, there is no true blank matrix available to construct calibration curves, thus, it has been a challenge to determine eCBs in plasma and brain matrix. A liquid chromatography tandem mass spectrometry (LC-MS/MS) method is developed to determine the concentrations of AEA and 2-AG in murine plasma and different brain substructures (prefrontal cortex, hippocampus and hypothalamus). To overcome the endogenous interference, a "surrogate analyte" approach was adopted using stable isotope-labeled standards as surrogates of authentic analytes to generate calibration curves in biological matrix. The mobile phase, composed of formic acid 0.1% in water-acetonitrile (40:60, v/v), was optimized to separate 2-AG and its non-bioactive isomer 1-AG. The analytes were extracted with ethyl acetate/n-hexane (9:1, v/v) and separated on an Xbridge C18 (2.1 × 100 mm, 3.5 μm) column using N-Oleoylethanolamine-d2 (OEA-d2) as the internal standard. Detection was performed in multiple reaction monitoring (MRM) mode with an electrospray ionization source operated in positive ion mode. The method was applied to assess plasma and brain eCBs in tumor-bearing mice.