Optimized extraction of 2-arachidonyl glycerol and anandamide from aortic tissue and plasma for quantification by LC-MS/MS.
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.
Department of Pharmaceutical Sciences, Bill Gatton College of Pharmacy, East Tennessee State University, Johnson City, TN, USA.
Christopher
C
Garst
Department of Biomedical Sciences, Center for Inflammation, Infectious Disease and Immunity, Quillen College of Medicine, East Tennessee State University, Johnson City, TN, USA.
Makenzie
M
Fulmer
Department of Biomedical Sciences, Center for Inflammation, Infectious Disease and Immunity, Quillen College of Medicine, East Tennessee State University, Johnson City, TN, USA.
Doug
D
Thewke
Department of Pharmaceutical Sciences, Bill Gatton College of Pharmacy, East Tennessee State University, Johnson City, TN, USA.
Stacy
S
Brown
2-AG; 2-Arachidonoylglycerol; AEA; Anandamide; Endocannabinoids; LC-MS; Liquid Chromatography; Mass Spectrometry; Toluene Extraction