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Development of UPLC- MS/MS Method to Quantify Baseline Levels of Endocannabinoids in Mouse Brain

Alexandria Senetra
Florida State University

Co-Authors: Samantha L Penman1, Panayotis K Thanos1, Christopher R McCurdy2, Abhisheak Sharma2
1University of New York at Buffalo, 2University of Florida

Background: The endocannabinoid (eCB) system is comprised of endogenous cannabinoids, anandamide (AEA) and 2-arachidonoyl glycerol (2-AG), their respective metabolic enzymes, fatty-acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL), and the cannabinoid type-1 (CB1) and type-2 (CB2) receptors (Lu et al, 2016). The eCB has been identified as an important neuromodulatory system responsible for many physiological functions, such as pain, neuroprotection, cognitive function, and motor activity (Zou et al, 2018).

Objective: To develop a sensitive bioanalytical method to obtain baseline levels of anandamide and 2-AG in the brain of C57BL/6 mice and stabilize further metabolism of 2-AG during sample processing and storage.

Methods: A bioanalytical method to quantify baseline levels of AEA and 2-AG in mouse brain homogenates, was developed using a Waters Acquity I-Class ultra performance-liquid chromatography coupled with a Xevo TQ-XS triple quadrupole mass spectrometer (UPLC-MS/MS). Whole mouse brains were harvested, flash-frozen, and stored at -80˚C until analysis. Samples were thawed on ice and homogenized at a ratio of 1:3 (mg/μL) with 50 mM Tris Buffer + 50 nM NF1819 (pH-7.4±0.1).

Results: Charcoal-stripped plasma was used as a surrogate matrix to ensure complete removal of endogenous AEA and 2-AG. A range of 1-250 ng/mL was found to be linear for AEA and 2-AG, with a correlation value of >0.99 using a 1/x2 weighing factor. Patel et al. stated that degradation of 2-AG after collection of brain sample decreases, while AEA appeared unchanged during sample preparation. Thus, we used 50 mM Tris buffer with 50 nM NF1819 (MAGL inhibitor), to help prevent the metabolism of 2-AG while processing brain samples. Concentration ranges of AEA in whole mouse brains were found to be 30.8-45.8 ng/g in males and 35.9-53.5 ng/g in females. The 2-AG concentration ranges in male and female mouse brains were 695.3-2344.2 and 709.5-1957.7 ng/g, respectively. There were no significant sex differences observed between baseline levels of AEA or 2-AG in mice.

Conclusions: This sensitive and selective bioanalytical method was successfully applied for the quantification of AEA and 2-AG brain levels in male and female C57BL/6 mice and could be used to understand the effect of different treatments on baseline endocannabinoid levels. By utilizing the MAGL inhibitor during sample processing, we were able to prevent the degradation of 2-AG and achieve a linearity range required for the quantification of endogenous endocannabinoids in whole mouse brains.