Determination of endocannabinoids and endocannabinoid-like substances in human K3EDTA plasma – LC-MS/MS method validation and pre-analytical characteristics

• Published in: Talanta (Oxford) 2019-11, Vol.204, p.386-394
• Main Authors: Gurke, R., Thomas, D., Schreiber, Y., Schäfer, S.M.G., Fleck, S.C., Geisslinger, G., Ferreirós, N.
• Format: Article
• Language: English
• Subjects: Anticoagulants - chemistry; Arachidonic Acids - blood; Arachidonic Acids - chemistry; Chromatography, High Pressure Liquid - methods; Edetic Acid - chemistry; Endocannabinoid; Endocannabinoids - blood; Endocannabinoids - chemistry; Ethanolamines - blood; Glycerides - blood; Glycerides - chemistry; Human plasma; Humans; LC-MS/MS; Liquid-Liquid Extraction - methods; Method validation; Oleic Acids - blood; Palmitic Acids - blood; Polyunsaturated Alkamides - blood; Pre-analytical sample handling; Specimen Handling; Tandem Mass Spectrometry - methods
• ISSN: 0039-9140; 1873-3573
• DOI: 10.1016/j.talanta.2019.06.004

The determination of endocannabinoids and endocannabinoid-like substances in biological human samples is a vibrant field of research with great significance due to postulated relevance of these substances in diseases such as Alzheimer's disease, multiple sclerosis, cancer and cardiovascular diseases. For a possible use as biomarker in early prediction or diagnosis of a disease as well as examination of a successful treatment, the valid determination of the analytes in common accessible human samples, such as plasma or serum, is of great importance. A method for the determination of arachidonoyl ethanolamide, oleoyl ethanolamide, palmitoyl ethanolamide, 1-arachidonoyl glycerol and 2-arachidonoyl glycerol in human K3EDTA plasma using liquid-liquid-extraction in combination with liquid chromatography-tandem-mass spectrometry has been developed and validated for the quantification of the aforementioned analytes. Particular emphasis was placed on the chromatographic separation of the isomers 1-arachidonoyl glycerol and 2-arachidonoyl glycerol, arachidonoyl ethanolamide and O-arachidonoyl ethanolamine (virodhamine) as well as oleoyl ethanolamide and vaccenic acid ethanolamide. During the validation process, increasing concentrations of 1-arachidonoyl glycerol and 2-arachidonoyl glycerol while storing plasma samples were observed. In-depth investigation of pre-analytical sample handling revealed rising concentrations for both analytes in plasma and for arachidonoyl ethanolamide, oleoyl ethanolamide and palmitoyl ethanolamide in whole blood, dependent on the period and temperature of storage. Prevention of the increase in concentration was not possible, raising the question whether human K3EDTA plasma is suitable for the determination of endocannabinoids and endocannabinoid-like substances. Especially the common practice to calculate the concentration of 2-arachidonoyl glycerol as sum of 1-arachidonoyl glycerol and 2-arachidonoyl glycerol is highly questionable because the concentrations of both analytes increase unequally while storing the plasma samples in the fridge. [Display omitted] •UHPLC separation of isomers 1-AG and 2-AG, OEA and VEA as well as AEA and O-AEA.•Full method validation in accordance with EMA and FDA.•Pre-analytical sample handling has a dramatic impact on analyte concentration.•2-AG and 1-AG should not be calculated as sum parameter.