Liquid Chromatography-Tandem Mass Spectrometry Method for Simultaneous Quantification of Four Endocannabinoids and...

The endocannabinoid system regulates immune function, inflammation, and metabolism and has gained increasing attention in clinical research. However, the instability, low abundance, and physicochemical complexity of endocannabinoids (ECs) and EC-like substances (EC-like) make their quantification in plasma analytically challenging.

The authors developed and validated a liquid chromatography-tandem mass spectrometry method for the simultaneous quantification of N-arachidonoylethanolamide (AEA), 2-arachidonoylglycerol, and its isomer 1-arachidonoylglycerol (2-AG/1-AG), oleoylethanolamide (OEA), and palmitoylethanolamide (PEA) in human plasma.

Extraction was performed using liquid-liquid extraction combined with liquid chromatography-tandem mass spectrometry. The method achieved lower limits of quantification of 0.1 mcg/L for AEA, 0.2 mcg/L for 2-AG/1-AG, and 0.5 mcg/L for OEA and PEA, with calibration curves showing high linearity (R 2 ≥ 0.995). Intra-assay and interassay accuracy and precision were both within 15%. Additional validation parameters, including selectivity, recovery, carryover, matrix suitability, and dilution integrity, fulfilled regulatory criteria. Preanalytical handling significantly influenced concentrations: Delayed centrifugation and postthaw storage increased AEA, OEA, and PEA levels. The validated method was applied to plasma samples from patients coinfected with HIV and hepatitis C virus, enabling reproducible quantification of ECs and EC-like substances in a clinically relevant cohort.

The study demonstrates that biological and technical factors markedly affect plasma EC and EC-like concentrations. Standardized preanalytical processing is therefore essential for accurate measurement, and the proposed method provides a robust tool for clinical and pharmacological research.