title: Quantitative analysis of N-acylphosphatidylethanolamine molecular species in rat brain using solid-phase extraction combined with reversed-phase chromatography and tandem mass spectrometry.
abstract: A novel method for the sensitive and selective identification and quantification of N-acylphosphatidylethanolamine molecular species was developed. Samples were prepared using a combination of liquid-liquid and solid-phase extraction, and intact N-acylphosphatidylethanolamine species were determined by reversed-phase high-performance liquid chromatography coupled to positive electrospray tandem mass spectrometry. As a result of their biological functions as precursors for N-acylethanolamines and as signaling molecules, tissue concentrations of N-acylphosphatidylethanolamines are very low, and their analysis is additionally hindered by the vast excess of other sample components. Our sample preparation methods are able to selectively separate the analytes of interest from any expected biological interferences. Finally, the highest selectivity is achieved by coupling chromatographic separation and two N-acyl chain specific selected reaction monitoring scans per analyte, enabling identification of both the N-acyl chain and the phosphatidylethanolamine moiety. The validated method is suitable for the reliable quantification of N-acylphosphatidylethanolamine species from rat brain with a lower limit of quantification of 10 pmol/g and a linear range up to 2300 pmol/g. In total, 41 N-acylphosphatidylethanolamine molecular species with six different N-acyl chains, amounting to a total concentration of 3 nmol/g, were quantified.
authors.affiliation: Core Facility for Mass Spectrometry, Center for Medical Research, Medical University of Graz, Graz, Austria.
authors.forename: Alexander
authors.initials: A
authors.lastname: Triebl
authors.affiliation: Institute of Analytical Chemistry and Food Chemistry, Graz University of Technology, Graz, Austria.
authors.forename: Sabrina
authors.initials: S
authors.lastname: Weissengruber
authors.affiliation: Core Facility for Mass Spectrometry, Center for Medical Research, Medical University of Graz, Graz, Austria.
authors.forename: Martin
authors.initials: M
authors.lastname: Trötzmüller
authors.affiliation: Institute of Analytical Chemistry and Food Chemistry, Graz University of Technology, Graz, Austria.
authors.forename: Ernst
authors.initials: E
authors.lastname: Lankmayr
authors.affiliation: Core Facility for Mass Spectrometry, Center for Medical Research, Medical University of Graz, Graz, Austria.
authors.forename: Harald
authors.initials: H
authors.lastname: Köfeler
mesh_terms: D000818:Animals; D001921:Brain; D056148:Chromatography, Reverse-Phase; D010714:Phosphatidylethanolamines / Q000032:analysis*; D051381:Rats; D052616:Solid Phase Extraction; D053719:Tandem Mass Spectrometry
chemical_list: C000609813:N-acylphosphatidylethanolamine; D010714:Phosphatidylethanolamines
keywords: Mass spectrometry; N-acylphosphatidylethanolamine; Solid-phase extraction