Quantification of Endocannabinoids in Serum

AEA, 2-AG, AA, OEA, and PEA were extracted, purified, and quantified in serum by stable isotope dilution liquid chromatography/tandem mass spectrometry (LC-MS/MS) as previously described [22 (link),23 (link),24 (link)]. LC-MS/MS analyses were conducted on a Sciex (Framingham, MA, USA) QTRAP^® 6500+ mass spectrometer coupled with a Shimadzu (Kyoto, Japan) UHPLC system. Liquid chromatographic separation was obtained using 5 μL injections of samples onto a Kinetex 2.6 μm C18 (100 × 2.1 mm) column from Phenomenex (Torrance, CA, USA). The autosampler was set at 4 °C, and the column was maintained at 40 °C during the entire analysis. Endocannabinoids were detected in positive ion mode using electron spray ionization (ESI) and a multiple reaction monitoring (MRM) mode of acquisition, using d₄-AEA as internal standard (IS). The IonDrive^TM Turbo V source temperature was set at 450 °C with an ion spray voltage of 4000 V. The curtain gas was set at 30.0 psi. The nebulizer gas (Gas 1) was set to 40 psi, and the turbo heater gas (Gas 2) was set to 40 psi. The dwell time was set to 30 ms. The collision energy (CE), declustering potential (DP), and collision cell exit potential (CXP) for the monitored transitions are listed in Table 1. The LC-MS/MS chromatogram is presented in Figure 1. The serum levels of AEA, 2-AG, OEA, PEA, and AA were measured in duplicate against standard curves.