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.

Lipidomics: Prospects from a technological perspective

“…This allows for either separation of phospholipids from other lipids [17], or separation of phospholipids or sphingolipids into individual classes [18,19]. Another benefit of SPE in lipidomics is the selective enrichment of low-abundance analytes of interest, such as long-chain base phosphates [20], steroids [21], or N-acylphosphatidylethanolamines [22].…”

Lipidomic Study of Precursors of Endocannabinoids in Freshwater Bryozoan Pectinatella magnifica

“…For example, the bacterium Bdellovibrio contains NAPtdEtn in which at least one of the three FA at the sn ‐1, sn ‐2, or N ‐acyl positions is C17 or C19 cyclopropane FA (Muller, Beck, Strauch, & Linscheid, ). Triebl, Weissengruber, Trotzmuller, Lankmayr, and Kofeler () described dozens of molecular species in the rat brain, where N ‐acyl is formed by either saturated FA (16:0 or 18:0) or PUFA (20:4 or 22:6). In another work on rat brain, some N ‐arachidonoyl‐PtdEtn were quantitated.…”

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“…Targeted approaches are performed by applying a predefined set of compounds with known fragmentation behaviors and ideally also retention times. They can be carried out using a triple quadrupole MS in SRM mode, with just a few or sometimes even only one lipid class considered per HPLC run [46,47]. Such a focused analysis also results in a high degree of flexibility for developing custom lipid-classspecific chromatography techniques.…”