Supplementary Material for: Determination of the Endocannabinoids Anandamide and 2-Arachidonoyl Glycerol with Gas Chromatography-Mass Spectrometry: Analytical and Preanalytical Challenges and Pitfalls

<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.

<b><i>背景：</i></b> 内源性大麻素类物质花生四烯酸乙醇胺（anandamide, N-arachidonoyl ethanolamide [AEA]）与2-花生四烯酰甘油酯（2-arachidonoyl glycerol, 2-AG）参与神经元、免疫、代谢、血管及生殖功能的调控。<b><i>方法：</i></b> 本研究建立并验证了一种基于液液萃取、结合甲硅烷基化衍生后气相色谱-质谱联用法的分析方法，用于测定人血浆中的AEA与2-AG，并识别了分析前与分析阶段的潜在陷阱。<b><i>结果：</i></b> 与2-AG不同，AEA在全血中稳定性较差，于冰浴条件下3小时内浓度升至初始值的2.3倍。AEA在冰浴保存的血浆中可稳定4小时，而2-AG则呈现浓度下降趋势。两种经甲硅烷基化衍生的化合物在室温/环境温度下均可稳定存在超过45小时。此外，3次冻融循环实验呈现出复杂的变化模式：内源性AEA在血浆中保持稳定，但在加标样本中浓度小幅升高（+12.8%）；内源性2-AG在血浆中浓度升高51%，而在加标样本中则下降24%。一项为期4周的-80℃长期稳定性实验显示，低浓度内源性AEA与加标2-AG的浓度保持稳定。然而，加标AEA浓度呈上升趋势（+19%），且内源性2-AG在2周后浓度显著升高50%。采血前2小时进食对AEA浓度无显著影响，而2-AG浓度则显著升高3倍。<b><i>结论：</i></b> 总体而言，研究需考虑内源性大麻素类物质有限的体外以及体内/离体化学稳定性。