Endogenous ethanolamide analysis in human plasma using HPLC tandem MS with electrospray ionization

• Published in: Journal of chromatography. B, Analytical technologies in the biomedical and life sciences Vol. 877; no. 22; pp. 2052 - 2060
• Main Authors: Palandra, Joe, Prusakiewicz, Jeff, Ozer, Josef S., Zhang, Yanhua, Heath, Timothy G.
• Format: Journal Article
• Language: English
• Published: Amsterdam Elsevier B.V 15.07.2009; Elsevier
• Subjects: Analysis; Analytical, structural and metabolic biochemistry; Arachidonic Acids - blood; Biological and medical sciences; Biomarker validation; Cannabinoid Receptor Modulators - blood; Chromatography, High Pressure Liquid - methods; Electrospray mass spectrometry; Endocannabinoids; Endogenous analysis; Fundamental and applied biological sciences. Psychology; General pharmacology; Humans; Linoleic Acids - blood; Medical sciences; Multiplexing; Pharmacology. Drug treatments; Polyunsaturated Alkamides - blood; Quantitative analysis; Spectrometry, Mass, Electrospray Ionization - methods; Tandem Mass Spectrometry - methods; Multiplexing; Endogenous analysis; Electrospray mass spectrometry; Biomarker validation; Quantitative analysis; Human; Validation; Biological fluid; Tandem mass spectrometry; HPLC chromatography; Biological marker; Endogenous substance; Electrospray; Blood plasma; Endogenous; Mass spectrometry
• ISSN: 1570-0232; 1873-376X
• DOI: 10.1016/j.jchromb.2009.05.043

A sensitive and selective liquid chromatography tandem mass spectrometry (LC\MS\MS) method has been developed for the simultaneous quantification in human plasma of the endocannabinoid anandamide (AEA) and three other related ethanolamides, linoleoyl ethanolamide (LEA), oleoyl ethanolamide (OEA), and palmitoyl ethanolamide (PEA). The analytical methodology requires 50μL of human plasma which is processed via protein precipitation using a 96-well protein precipitation plate. Chromatographic separation of plasma extract was achieved with a Phenomenex Gemini C6-Phenyl HPLC column (2.1mm×50mm, 5μm) at a flow rate of 0.30mL/min using gradient elution and a mobile phase consisting of acetonitrile and 5mM ammonium formate. All four fatty acid ethanolamides were quantified by positive ion electrospray ionization tandem mass spectrometry, with the detection of ion current signal generated from the selected reaction monitoring (SRM) transition of [M+H]+→m/z 62. Deuterated anandamide (AEA-d8) was used as an internal standard for all four ethanolamides. The lower limit of quantitation was 0.05ng/mL for AEA and LEA, 0.5ng/mL for OEA and 1.0ng/mL for PEA. Inter-assay precision and accuracy were typically within 12% for the four endogenous analytes and overall extraction recoveries ranged between 40% and 100%.