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.

Fig. 1.

Structures of the key compounds in this study: PEA, SEA, and OEA.

Fig. 2.

Evaluation of SPE columns from Supelco (open square), Waters (open triangle), Isolute (closed circle), and Phenomenex (x) on the recoveries of PEA, SEA, OEA, and LEA (top row, left to right) and AEA, EPEA, DHEA, and 2-AG (bottom row, left to right; values are mean with SEM, n = 5).

Fig. 3.

Levels of PEA (A) and SEA (B) found in different brands of chloroform (calculated concentration of µM in injected sample; mean with range; n = 2).

Fig. 4.

Results from incubation of OEA in five different chloroforms stabilized with either EtOH or amylene (mean with SEM; n = 6, from two separate experiments). A: Percent OEA remaining, calculated relative to the standard solution of OEA in MeCN. B: Formation of m/z 396 (9,10-dichloro-SEA) in the respective solvents, calculated as percent relative to Merck LiChrosolv with amylene.

Fig. 5.

A: Profile spectrum showing the isotope pattern of OEA in MeCN (top) (full scan m/z 320–335, FWHM 0.25) and simulated Gaussian profile spectrum of C₂₀H₄₀NO₂⁺ (bottom). B: Profile spectrum showing the isotope pattern of the reaction product between OEA and Merck LiChroSolv chloroform stabilized with amylene (top) (full scan m/z 390–405, FWHM 0.25), and simulated Gaussian profile spectrum of C₂₀H₄₀NO₂Cl₂⁺ (bottom).

Fig. 6.

A: MS² of m/z 326.25, 10 µM OEA standard in MeCN. B: MS² of m/z 396.25 from incubation of OEA with Merck LiChroSolv chloroform. Curved lines indicate that chlorines are lost in most fragments.

Fig. 7.

¹H-NMR of 9,10-dichloro-SEA.