title: Pitfalls in the sample preparation and analysis of N-acylethanolamines.
abstract: N-acylethanolamines (NAEs) are a group of lipid mediators synthesized in response to a number of physiological and pathological stimuli. Because of the low tissue concentrations of NAEs, analyses often include liquid extraction followed by solid-phase extraction and subsequent quantitation by LC/MS or GC/MS. Reported levels of NAEs vary considerably, however, and often no explanation is given for these discrepancies. Brought on by difficulties encountered during method development, the effects of using four different brands of silica-containing solid phase extraction (SPE) columns and five different brands of chloroform for sample preparation were investigated. Considerable variation in the retention and recoveries of seven NAEs and 2-arachidonoylglycerol existed between the SPE columns. Furthermore, it was found that some chloroforms contained quantifiable amounts of N-palmitoylethanolamine and N-stearoylethanolamine. Finally, it was found that use of one of the chloroforms resulted in a loss of N-oleoylethanolamine from solution due to addition of chlorine to the ω-9 bond. The identity of this reaction product was confirmed by LC-MS/MS and NMR. It is recommended that these aspects of sample preparation and analysis should be thoroughly validated during method development and the relevant information on specific brands used be reported in future communications in order to better estimate the validity of reported quantitative data.
authors.affiliation: Department of Pharmaceutics and Analytical Chemistry, Faculty of Pharmaceutical Sciences, University of Copenhagen, Copenhagen, Denmark. cs@farma.ku.dk
authors.forename: Christian
authors.initials: C
authors.lastname: Skonberg
authors.forename: Andreas
authors.initials: A
authors.lastname: Artmann
authors.forename: Claus
authors.initials: C
authors.lastname: Cornett
authors.forename: Steen Honoré
authors.initials: SH
authors.lastname: Hansen
authors.forename: Harald S
authors.initials: HS
authors.lastname: Hansen
mesh_terms: D001095:Arachidonic Acids; D002853:Chromatography, Liquid / Q000379:methods; D063388:Endocannabinoids; D004983:Ethanolamines / Q000032:analysis*; D008401:Gas Chromatography-Mass Spectrometry; D005989:Glycerides; D013058:Mass Spectrometry; D052616:Solid Phase Extraction / Q000379:methods*
chemical_list: D001095:Arachidonic Acids; D063388:Endocannabinoids; D004983:Ethanolamines; D005989:Glycerides; C022203:N-acylethanolamines; C094503:glyceryl 2-arachidonate
references.citation: Vet Immunol Immunopathol. 2010 Jan 15;133(1):9-15
references.pmid: 19625089
references.citation: Chem Phys Lipids. 2000 Nov;108(1-2):135-50
references.pmid: 11106787
references.citation: J Pharmacol Exp Ther. 2006 Dec;319(3):1051-61
references.pmid: 16997973
references.citation: Biochem Pharmacol. 2009 Sep 15;78(6):553-60
references.pmid: 19413995
references.citation: Synapse. 1999 Sep 1;33(3):181-91
references.pmid: 10420166
references.citation: Biochem Biophys Res Commun. 1999 Mar 16;256(2):377-80
references.pmid: 10079192
references.citation: Science. 1992 Dec 18;258(5090):1946-9
references.pmid: 1470919
references.citation: J Chromatogr B Biomed Sci Appl. 1997 Mar 7;690(1-2):7-13
references.pmid: 9106024
references.citation: Anal Biochem. 2003 Mar 1;314(1):8-15
references.pmid: 12633597
references.citation: J Neuroendocrinol. 2008 May;20 Suppl 1:94-9
references.pmid: 18426507
references.citation: Anal Biochem. 2000 Apr 10;280(1):87-93
references.pmid: 10805525
references.citation: J Chromatogr B Analyt Technol Biomed Life Sci. 2009 Sep 15;877(26):2909-23
references.pmid: 19414283
references.citation: Anal Sci. 2003 Dec;19(12):1593-8
references.pmid: 14696920
references.citation: J Neurosci. 2000 May 1;20(9):3401-7
references.pmid: 10777802
references.citation: J Biol Chem. 1957 May;226(1):497-509
references.pmid: 13428781
references.citation: Anal Biochem. 2008 Feb 15;373(2):220-8
references.pmid: 17981259
references.citation: Chem Phys Lipids. 2000 Feb;104(2):185-91
references.pmid: 10669310
references.citation: J Neuroendocrinol. 2008 May;20 Suppl 1:39-46
references.pmid: 18426498
references.citation: J Neurochem. 2007 Dec;103(5):1907-16
references.pmid: 17868306
references.citation: Curr Opin Chem Biol. 2009 Jun;13(3):321-31
references.pmid: 19497779
references.citation: J Chromatogr B Analyt Technol Biomed Life Sci. 2009 Jul 15;877(22):2052-60
references.pmid: 19535304
references.citation: Biochem J. 2002 Aug 15;366(Pt 1):137-44
references.pmid: 12010121
references.citation: Prostaglandins Leukot Essent Fatty Acids. 1995 Oct;53(4):301-8
references.pmid: 8577784
references.citation: Am J Physiol Regul Integr Comp Physiol. 2008 Jul;295(1):R45-50
references.pmid: 18434444
references.citation: Mass Spectrom Rev. 2003 Mar-Apr;22(2):81-152
references.pmid: 12820273
references.citation: Chem Phys Lipids. 2002 Dec 31;121(1-2):111-34
references.pmid: 12505695
references.citation: J Neurochem. 2000 Dec;75(6):2434-44
references.pmid: 11080195
references.citation: Anal Biochem. 2007 Jan 15;360(2):216-26
references.pmid: 17141174
references.citation: Biochim Biophys Acta. 2008 Apr;1781(4):200-12
references.pmid: 18316044
references.citation: Prostaglandins Other Lipid Mediat. 2006 Dec;81(3-4):106-12
references.pmid: 17085319
references.citation: Neurochem Int. 2009 Mar-Apr;54(3-4):199-204
references.pmid: 19100800
references.citation: Anal Biochem. 2009 Dec 1;395(1):68-76
references.pmid: 19665986