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.
Highlights
N-Acylphosphatidylethanolamines (NAPEs) are triacylated phospholipids derived from phosphatidylethanolamine (PE) with a third, N-linked fatty acyl chain [1]
Acetonitrile, methyl-tert-butyl ether (MTBE), dichloromethane, palmitoyl chloride, heptadecanoyl chloride, stearoyl chloride, oleoyl chloride, 4-dimethylaminopyridine, ammonium hydrogen carbonate, ammonium formate, 2propanol, formic acid, and acetic acid were purchased from Sigma–Aldrich (St Louis, MO, USA), and pyridine was purchased from VWR (Radnor, PA, USA)
NAPE standards were synthesized as described by Guo et al [21], modified after Hofle et al [35]. 1 mL dichloromethane was mixed with 0.25 mol L-␣-phosphatidylethanolamine and 25 L pyridine diluted 1:25 with dichloromethane, and the mixture was vortexed for 10 s. 6.25 mol of 4dimethylaminopyridine and 6.5 mol of the respective acyl chloride were added and the reaction was allowed to take place for 18 h at 21ЊC
Summary
N-Acylphosphatidylethanolamines (NAPEs) are triacylated phospholipids derived from phosphatidylethanolamine (PE) with a third, N-linked fatty acyl chain [1]. Postprandial increase of NAPE levels and inhibition of food intake by their administration, may suggest a potential anorectic role [10, 11], this is subject of controversy and has not yet been clearly shown [1]
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