Simultaneous UPLC–MS/MS quantification of the endocannabinoids 2-arachidonoyl glycerol (2AG), 1-arachidonoyl glycerol (1AG), and anandamide in human plasma: Minimization of matrix-effects, 2AG/1AG isomerization and degradation by toluene solvent extraction

• Published in: Journal of chromatography. B, Analytical technologies in the biomedical and life sciences Analytical technologies in the biomedical and life sciences, 2012-02, Vol.883-884 (3), p.161-171
• Main Authors: Zoerner, Alexander A., Batkai, Sandor, Suchy, Maria-Theresia, Gutzki, Frank-Mathias, Engeli, Stefan, Jordan, Jens, Tsikas, Dimitrios
• Format: Article
• Language: English
• Subjects: 2AG; Adolescents; AEA; Arachidonic Acids - blood; Arachidonic Acids - chemistry; Arachidonic Acids - isolation & purification; Arachidonic Acids - urine; Chemical Fractionation - methods; Children; chromatography; Chromatography, High Pressure Liquid - methods; Clinical studies; creatinine; Endocannabinoids; ethyl acetate; evaporation; Glycerides - blood; Glycerides - chemistry; Glycerides - isolation & purification; Glycerides - urine; Glycerols; Human; Humans; ionization; Isomerisation; Isomerism; isomerization; Limit of Detection; Linear Models; Matrix-effects; Patients; phospholipids; Plasma; Polyunsaturated Alkamides - blood; Polyunsaturated Alkamides - chemistry; Polyunsaturated Alkamides - isolation & purification; Polyunsaturated Alkamides - urine; quality control; Quantification; Reproducibility of Results; Solvents; Tandem mass spectrometry; Tandem Mass Spectrometry - methods; Toluene; Toluene - chemistry; Toxicity; urine
• ISSN: 1570-0232; 1873-376X
• DOI: 10.1016/j.jchromb.2011.06.025

Analysis of the endocannabinoid (EC) system's key molecules 2-arachidonoyl glycerol (2AG) and arachidonoyl ethanolamide (anandamide, AEA) is challenging due to several peculiarities. 2AG isomerizes spontaneously to its biologically inactive analogue 1-arachidonoyl glycerol (1AG) by acyl migration and it is only chromatographically distinguishable from 1AG. Matrix-effects caused primarily by co-extracted phospholipids may further compromise analysis. In addition, 2AG and 1AG are unstable under certain conditions like solvent evaporation or reconstitution of dried extracts. We examined effects of different organic solvents and their mixtures, such as toluene, ethyl acetate, and chloroform-methanol, on 2AG/1AG isomerisation, 2AG/1AG stability, and matrix-effects in the UPLC–MS/MS analysis of 2AG and AEA in human plasma. Toluene prevented, both, 2AG isomerisation to 1AG and degradation of 2AG/1AG during evaporation. Toluene extracts contain only 2% of matrix-effect-causing plasma phospholipids compared to extracts from the traditionally used solvent mixture chloroform–methanol. Toluene and all other tested organic solvents provide comparable 2AG and AEA extraction yields (60–80%). Based on these favourable toluene properties, we developed and validated a UPLC–MS/MS method with positive electrospray ionization (ESI+) that allows for simultaneous accurate and precise measurement of 2AG and AEA in human plasma. The UPLC–MS/MS method was cross-validated with a previously described fully-validated GC–MS/MS method for AEA in human plasma. A close correlation (r2=0.821) was observed between the results obtained from UPLC–MS/MS (y) and GC–MS/MS (x) methods (y=0.01+0.85x). The UPLC–MS/MS method is suitable for routine measurement of 2AG and AEA in human plasma samples (1mL) in clinical settings as shown by quality control plasma samples processed over a period of 100 days. The UPLC–MS/MS method was further extended to human urine. In urine, AEA was not detectable and 2AG was detected in only 3 out of 19 samples from healthy subjects at 160, 180 and 212pM corresponding to 12.3, 14.5 and 9.9pmol/mmol creatinine, respectively.