Supplementary Material for: Determination of the Endocannabinoids Anandamide and 2-Arachidonoyl Glycerol with Gas Chromatography-Mass Spectrometry: Analytical and Preanalytical Challenges and Pitfalls Lanz C. Mattsson J. Stickel F. Dufour J.-F. Brenneisen R. 10.6084/m9.figshare.6264632.v1 https://karger.figshare.com/articles/dataset/Supplementary_Material_for_Determination_of_the_Endocannabinoids_Anandamide_and_2-Arachidonoyl_Glycerol_with_Gas_Chromatography-Mass_Spectrometry_Analytical_and_Preanalytical_Challenges_and_Pitfalls/6264632 <b><i>Background:</i></b> The endocannabinoids anandamide (<i>N</i>-arachidonoyl ethanolamide [AEA]) and 2-arachidonoyl glycerol (2-AG) are involved in the regulation of neuronal, immune, metabolic, vascular, and reproductory functions. <b><i>Methods:</i></b> The development and validation of an analytical method for the determination of AEA and 2-AG in human plasma based on liquid-liquid extraction and gas chromatography-mass spectrometry after silylation is described and (pre)-analytical pitfalls are identified. <b><i>Results:</i></b> In contrast to 2-AG, AEA was unstable in whole blood and increased by a factor of 2.3 within 3 h on ice. AEA was stable in plasma on ice for 4 h while 2-AG tended to decrease. Excellent stability at room/ambient temperature was found for both derivatized compounds over 45 h. Furthermore, 3 freeze-thaw cycles revealed a complex pattern: endogenous AEA was stable in plasma but slightly increased in spiked samples (+12.8%), while endogenous 2-AG concentrations increased by 51% and declined by 24% in spiked samples. A long-term study over 4 weeks at –80°C showed that low endogenous AEA and spiked 2-AG concentrations were stable. However, spiked AEA tended to increase (+19%) and endogenous 2-AG significantly increased by 50% after 2 weeks. Food intake 2 h before blood collection showed no effect on AEA concentrations, whereas 2-AG increased significantly by a factor of 3. <b><i>Conclusions:</i></b> Overall, limited in vitro and/or in vivo/ex vivo chemical stability of endocannabinoids has to be taken into account. 2018-06-12 06:38:33 Endocannabinoids Anandamide 2-Arachidonoyl glycerol Human plasma Gas chromatography-mass spectrometry Pitfalls