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

Abstract

Conventional sample preparation for fatty acid analysis is a complicated, multiple-step process, and gas chromatography (GC) analysis alone can require >1 h 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 with the elimination of the neutralization and centrifugation steps. A robotics-amenable method was also developed, with lower methylation temperatures and in an open-tube format using multiple reagent additions. The simplified methods produced results that were quantitatively similar and with similar coefficients of variation as compared with the original Lepage and Roy method.The present streamlined methodology is suitable for the direct fatty acid analysis of human plasma, is appropriate for research studies, and will facilitate large clinical trials and make possible population studies. Conventional sample preparation for fatty acid analysis is a complicated, multiple-step process, and gas chromatography (GC) analysis alone can require >1 h 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 with the elimination of the neutralization and centrifugation steps. A robotics-amenable method was also developed, with lower methylation temperatures and in an open-tube format using multiple reagent additions. The simplified methods produced results that were quantitatively similar and with similar coefficients of variation as compared with the original Lepage and Roy method. The present streamlined methodology is suitable for the direct fatty acid analysis of human plasma, is appropriate for research studies, and will facilitate large clinical trials and make possible population studies. The consumption of n-3 polyunsaturated fatty acids (PUFAs), particularly eicosapentaenoic acid (20:5n-3, EPA) and docosahexaenoic acid (22:6n-3, DHA), is implicated in various aspects of human health (1Salem Jr, N. Omega-3 fatty acids: molecular and biochemical aspects.in: Spiller G. Scala J. New Protective Roles of Selective Nutrients in Human Nutrition. Alan R. Liss, New York1989: 263-317Google Scholar, 2Simopoulos A.P. Essential fatty acids in health and chronic disease.Am. J. Clin. Nutr. 1999; 70: 560-569Google Scholar). Presently, analysis of the fatty acid composition of human blood and plasma samples involves tedious and time-consuming extraction and transesterification procedures (3Dacremont G. Vincent G. Assay of plasmalogens and polyunsaturated fatty acids (PUFA) in erythrocytes and fibroblasts.J. Inherit. Metab. Dis. 1995; 18: 84-89Google Scholar, 4Garces R. Mancha M. One-step lipid extraction and fatty acid methyl esters preparation from fresh plant tissues.Anal. Biochem. 1993; 211: 139-143Google Scholar, 5Lepage G. Roy C.C. Improved recovery of fatty acid through direct transesterification without prior extraction or purification.J. Lipid Res. 1984; 25: 1391-1396Google Scholar, 6Lepage G. Roy C.C. Direct transesterification of all classes of lipids in a one-step reaction.J. Lipid Res. 1986; 27: 114-120Google Scholar, 7Morrison W.R. Smith L.M. Preparation of fatty acid methyl esters and dimethylacetals from lipids with boron fluoride-methanol.J. Lipid Res. 1964