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Optimized extraction of 2‐arachidonyl glycerol and anandamide from aortic tissue and plasma for quantification by LC‐MS/MS
Author(s) -
Garst Christopher,
Fulmer Makenzie,
Thewke Doug,
Brown Stacy
Publication year - 2016
Publication title -
european journal of lipid science and technology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.614
H-Index - 94
eISSN - 1438-9312
pISSN - 1438-7697
DOI - 10.1002/ejlt.201500115
Subject(s) - anandamide , chemistry , endocannabinoid system , chromatography , extraction (chemistry) , analyte , mass spectrometry , cannabinoid receptor , solid phase extraction , receptor , biochemistry , agonist
Atherosclerosis is a disease characterized by plaque formation due to an accumulation of fat, cholesterol, and immune cells in the walls of arteries. If a plaque ruptures, an occlusive thrombosis may form that causes either a heart attack or stroke. Macrophages express CB‐2 receptors, and are one type of immune cell that plays a role in plaque destabilization and rupture. Endocannabinoids anandamide (AEA) and 2‐arachidonyl glycerol (2‐AG) have been found to have activity on CB‐1 and CB‐2 receptors throughout the body and immune system. In this study, we investigated several sample preparation options for the LC‐MS quantification of AEA and 2‐AG from plasma and aortic tissue. The extractions considered included liquid–liquid (LLE), solid‐phase (SPE), and supported liquid (SLE). Some extraction protocols yielded high analyte recovery and prevention of 1‐AG/2‐AG isomerization. Our results indicate that a liquid‐liquid extraction using toluene yields the highest recovery for both analytes, coupled with low ionization suppression in the mass spectrometer. This extraction and corresponding LC‐MS/MS assay provides a simple, high throughput mechanism for the quantification of 2‐AG and AEA in matrices relevant to the study of endocannabinoids' role in atherosclerosis. Practical applications: We developed an extraction method for AEA and 2‐AG from plasma and aortic tissue samples and an LC‐MS/MS assay for quantification of these compounds to help understand the role of endocannabinoids and CB‐2 receptors in atherosclerosis. This assay can be applied toward the measurement of endocannabinoids, AEA and 2AG, in aortic tissue and plasma. Mass chromatogram of AEA and 2‐AG extracted from spiked aortic tissue (10 mcg/mL) using toluene LLE protocol and analyzed by LC‐MS/MS running in +ESI mode.