Quantitative and qualitative profiling of endocannabinoids in human plasma using a triple quadrupole linear ion trap mass spectrometer with liquid chromatography

Abstract

Owing to the large implication of endocannabinoids (ECs) in many physiological and pathophysiological processes, a rapid liquid chromatography/electrospray ionisation triple quadrupole linear ion trap mass spectrometric assay (LC/ESI-QqQ(LIT)) was developed for the detection and characterization of anandamide (AEA), 2-arachidonoyl glycerol (2-AG), virodhamine (VA), noladin ether (2-AGE), and N-arachidonoyl dopamine (NADA) in human plasma. The ECs were extracted from 500 microL of plasma by liquid-liquid extraction (LLE) and separated by using an XTerra C18 MS column (50 x 3.0 mm i.d., 3.5 microm) with gradient elution. The mobile phase was composed of a mixture of acetonitrile, water, and formic acid (0.1%). For confirmatory analysis, an information-dependent acquisition (IDA) experiment was performed with selected reaction monitoring (SRM) as survey scan and enhanced product ion (EPI) as dependent scan. The assay was found to be linear in the concentration range of 0.1-5 ng/mL for AEA, 0.3-5 ng/mL for VA, 2-AGE, and NADA and 1-20 ng/mL for 2-AG using a 0.5 mL aliquot of plasma. Repeatability and intermediate precision were found less than 15% over the tested concentration ranges. The developed method thus provided the rapid, highly sensitive and highly selective requirement for assess quantitation, and identification of ECs in plasma.

A quantitiative LC-MS/MS method for the measurement of arachidonic acid, prostanoids, endocannabinoids, N-acylethanolamines and steroids in human plasma

“…Furthermore, our method was not able to detect endogenous concentrations of A-GABA, A-GLY, A-SER, ANDRO, PGE 2 EA and NADA in human blood plasma. In agreement with our findings, two studies have tried to measure NADA and A-GLY in human plasma without success [25,27]. So far, A-GABA and A-SER have only been identified in the mouse brain [58].…”

“…The LLOQ reported for NE by [25] (0.3 ng/mL) was lower than the LLOQ reported here (3.2 ng/mL). These studies used an API 4000 QTrap mass spectrometer in positive mode (as used here), but a different extraction procedure (single extraction with n-heptane/ ethyl acetate (1/1, v/v)), solvent composition for the LC (ACN and water both with 0.1% formic acid) and MRM transition (365/273).…”

“…Interestingly, two sensitive methods have been validated for the measurement of NE in human plasma, but neither of them reported endogenous levels [25,27]. The LLOQ reported for NE by [25] (0.3 ng/mL) was lower than the LLOQ reported here (3.2 ng/mL).…”

“…It has been widely used for the analysis of ECs and similar lipids [24][25][26][27][28]. However, it has only recently been used for the analysis of steroids [29][30][31].…”

Endogenous cannabinoid receptor CB1 activation promotes vascular smooth-muscle cell proliferation and neointima formation

“…Analyses were performed on an LC-MS/MS system consisting of an Ultimate 3000 RS (Dionex, CA) LC system and a 5500 QTrap triple quadrupole/linear ion trap mass spectrometer equipped with a TurboIon-Spray interface (AB Sciex; ON, Canada). Data acquisition and analysis were performed using Analyst software version 1.5.1 (AB Sciex) ( 21 ).…”