J. Lipid Res.
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A more recent version of this article appeared on October 1, 2005

Papers In Press, published online ahead of print August 1, 2005
J. Lipid Res., doi:10.1194/jlr.D500022-JLR200
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Submitted on May 17, 2005
Revised on July 22, 2005
Accepted on July 23, 2005

A simplified and efficient method for the analysis of fatty acid methyl esters suitable for large clinical studies

Md. Athar Masood, Ken D. Stark, and Norman Salem . Jr

LMBB, NIAAA, NIH, Bethesda, MD 20892-9410

Corresponding Author: nsalem{at}niaaa.nih.gov

Fatty acid profiles, particularly n-3 polyunsaturated status may be an important clinical marker for various chronic diseases. Conventional sample preparation for fatty acid analysis is a complicated and multiple step process and gas chromatography (GC) analysis alone can require more than one hr per sample to resolve fatty acid methyl esters (FAMEs). Fast GC analysis was adapted to human plasma FAME analysis using a modified polyethylene glycol column with smaller internal diameters, thinner stationary phase films, increased carrier gas linear velocity and faster temperature ramping. Our results indicated that fast GC analyses were comparable to conventional GC in peak resolution. A conventional transesterification method based on Lepage and Roy was simplified to a one-step method. All reagents including the internal standard were combined into a stock solution. Samples and the stock solution were combined, vortexed, heated and FAMEs were extracted with hexane without neutralization. A robotics-amenable method was also developed with lower methylation temperatures, and in which 300 mu L of methanol from the stock solution reaction mixture was substituted with toluene. The use of toluene allows the exploitation of open tubes rather than capped tubes, and the extraction by aspiration rather than by vortexing. The simplified methods produced results that were quantitatively similar and with similar coefficients of variation as compared to the original Lepage and Roy method. The present, streamlined methodology is suitable for the direct fatty acid analysis of human plasma and will facilitate such analyses for large clinical trials and is appropriate for research studies.


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