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

Abstract

Background: The endocannabinoids anandamide (N-arachidonoyl ethanolamide [AEA]) and 2-arachidonoyl glycerol (2-AG) are involved in the regulation of neuronal, immune, metabolic, vascular, and reproductory functions.

Methods: 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.

Results: 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.

Conclusions: Overall, limited in vitro and/or in vivo/ex vivo chemical stability of endocannabinoids has to be taken into account.

Keywords: 2-Arachidonoyl glycerol; Anandamide; Endocannabinoids; Gas chromatography-mass spectrometry; Human plasma; Pitfalls.

Fig. 1

Gas chromatography-mass spectrometry chromatograms obtained in selected ion monitoring mode with the plasma of a healthy volunteer with concentrations of AEA and AG being 0.69 and 1.98 ng/mL, respectively (a), and pooled plasma spiked with AEA and 2-AG at 3 ng/mL (total concentrations of AEA and AG: 3.57 and 3.52 ng/mL, respectively) (b). For AEA m/z 420.2 is depicted as the quantifier ion (to the left of the dotted line), whereas m/z 433.2 is shown for 2-AG and 1-AG (to the right of the dotted line). For experimental conditions, see online supplementary material. AEA, anandamide; AG, arachidonoyl glycerol.

Fig. 2

Stability of AEA (black bars) and AG (grey bars) in whole blood. Depicted are mean values + SD of EC concentrations from plasma samples of 3 individuals. Blood samples were kept on ice for 15, 75, and 195 min, referred to as sampling time points 1, 2, and 3, respectively, before centrifugation to obtain plasma. AEA, anandamide; AG, arachidonoyl glycerol; EC, endocannabinoid. * p < 0.05.

Fig. 3

Benchtop stability over 4 h (a) and autosampler stability over 45 h (b) of AEA (black bars) and AG (grey bars) in plasma spiked with AEA and 2-AG at 3 ng/mL. Depicted are mean values + SD of total EC concentrations of 3 samples prepared in the same plasma for each time point. AEA, anandamide; AG, arachidonoyl glycerol; EC, endocannabinoid.

Fig. 4

Freeze-thaw stability of AEA (black bars) and AG (grey bars) over 3 cycles obtained with pooled blank plasma (a) and the same plasma spiked with AEA and 2-AG at 3 ng/mL (b). Depicted are mean values + SD of 3 samples. Total EC concentrations are given for the spiked samples. AEA, anandamide; AG, arachidonoyl glycerol; EC, endocannabinoid. * p < 0.05 compared with the concentrations at the beginning (cycle 0).

Fig. 5

Long-term stability of AEA (black bars) and AG (grey bars) before and 2 and 4 weeks after storing the samples at −80°C obtained with pooled blank plasma (a) and the same plasma spiked with AEA and 2-AG at 3 ng/mL (b). Mean values + SD of 3 samples are presented for each time point. Total EC concentrations are given for the spiked samples. AEA, anandamide; AG, arachidonoyl glycerol; EC, endocannabinoid. * p < 0.05 compared with the concentrations at the beginning (before freezing).

Fig. 6

Influence of food intake on AEA (black bars) and AG (grey bars) plasma concentrations. Depicted are mean values + SD of fasting EC concentrations obtained from 5 healthy subjects (fasting) and the EC concentrations measured from the same subjects 2 h after a regular lunch (postprandial). AEA, anandamide; AG, arachidonoyl glycerol; EC, endocannabinoid. * p < 0.05.