Fatty acid methyl ester analysis

L. M. Mallory and G. S. Sayler, Application of FAME (fatty acid methyl ester) analysis in the numerical taxonomic determination of bacterial guild structure, Mi-croh. Ecol. t0 l%2 (1984). 

Gas Chromatography for Fatty Acid Methyl Ester Analysis... 

In Chapter 8 a leading expert in the detection of adulteration of oils and fats, Rossell, explains the economic advantages which tempt unscrupulous dealers to add cheap oils to high-value oils such as olive oil. He then describes a technique which he and his coworkers at Bristol have pioneered where stable carbon isotope ratio (SCIR) measurement has simplified the task of detecting adulteration. He compares sterol and fatty acid methyl ester analysis with SCIR and shows how clearly the latter detects maize oil. 

FATTY ACID METHYL ESTER ANALYSIS (FAME)... 

Many standard compendium methods (ASTM, EPA, FAAM) are based on GC analysis. Examples are the GC-FID determination of fatty acid methyl esters (FAME ... 

The use of both ozonation and ozonolysis is reviewed32. Ozonation leads to ozonide and ozonolysis leads to oxidized fragments, showing the use of both oxidative (AgN03) or reductive [(CH3)2S or PI13P] methods to produce the FAME (fatty acids methyl esters) that by subsequent GC analysis enabled determination of the position of the double bonds in the original molecule (equations 2-4). 

Lipids can be identified and quantified using thin-layer chromatography (TEC) and gas chromatography (GC) (Galliard, 1968). Extraction of lipids is achieved by homogenizing potato tubers with isopropanol in a blender, followed by a series of filtrations and extractions with chloroform-methanol (2 1). Chloroform is removed by rotary evaporation and the residue is redissolved in benzene-ethanol (4 1). This extract is passed through a DEAE-cellulose column, and the fractions collected are subjected to TEC on 250 p,m layers of silica gel G, using three solvent systems. Fatty acid methyl esters for GC analysis are prepared by transmethylation of the parent lipids, or by diazomethane treatment of the free fatty acids released by acid... 

The analysis of essential fatty acids involves hydrolysis of the ester bonds and subsequent formation of the fatty acid methyl esters, which can be separated by gas chromatography (GC) [10]. By accident, the plasmalogens are hydrolysed in the same reaction and the methylation reaction transforms them into dimethylacetals, which appear in the GC run of the fatty acid methyl esters [4]. 

The fatty acid glycerol esters and the plasmalogens are transmethylated by adding methanolic HC1 to the sample and heating the mixture in a closed vial at 90°C for 4 h. After cooling the sample, the fatty acid methyl esters and the dimethylacetals are extracted with hexane. The concentrated hexane solution is ready for analysis by GC. 

D Holme and H Peck, Analytical Biochemtstiy, 3rd ed (1998), Addison Wi li Longman (New York), pp 406-442 Structure, function, and analysis of lipids C Matthews, K van Holde, and K Ahern, Biochemistry, 3rd ed (2000), Benjamin/ Cummings (San Francisco), pp 315-357 Lipid structure and function G Patton, S Cann, H Brunengraber, and J. Lowenstein, in Methods in Enzymology, Vol 72, J Lowenstein, Editor (1981), Academic Press (New York), pp 8-20 Separation of fatty acid methyl esters by gas chromatography on capillary columns N Radin, in Methods in Enzymology, Vol 72, J M Lowenstein, Editor (1981), Academic Press (New York), pp 5-7 Extraction of lipids with hexane-isopropanol L Stryer, Biochemistry, 4th ed (1995), W H Freeman (New York), pp 263-270, 603-606 Lipid structure and function... 

D1.2 Analysis of Fatty Acids in Food Lipids Basic Protocol 1 Preparation of Fatty Acid Methyl Esters from Lipid Dl.2.1... 

BASIC PROTOCOL I PREPARATION OF FATTY ACID METHYL ESTERS FROM LIPID SAMPLES CATALYZED WITH BORON TRIFLUORIDE IN METHANOL In this method, lipid samples are first saponified with an excess of NaOH in methanol. Liberated fatty acids are then methylated in the presence of BF3 in methanol. The resulting fatty acid methyl esters (FAMEs) are extracted with an organic solvent (isooctane or hexane), and then sealed in GC sample vials for analysis. Because of the acidic condition and high temperature (100°C) used in the process, isomerization will occur to those fatty acids containing conjugated dienes, such as in dairy and ruminant meat products, that contain conjugated linoleic acids (CLA). If CLA isomers are of interest in the analysis, Basic Protocol 2 or the Alternate Protocol should be used instead. Based on experience, this method underestimates the amount of the naturally occurring cis-9, trans-11 CLA isomer by -10%. The formulas for the chemical reactions involved in this protocol are outlined in Equation D1.2.1 Saponification RCOO-R + NaOH, RCOO-Na + R -OH v 100°C DC Esterification RCOO-Na + CH,OH r 3 v RCOO-CH, + NaOH ioo°c ... 

Sample preparation is probably the most important step in any analytical procedure. Poor preparation of lipid samples will only yield inferior or questionable results. Some commonly performed sample-preparation procedures for gas-liquid chromatographic (GC) analysis of fatty acids in food samples are introduced in this unit. Since the introduction of gas chromatography in the 1950s, significant progress has been made in fatty acid analysis of lipids however, fatty acid methyl esters (FAMEs) are still the most commonly used fatty acid derivative for routine analysis of food fatty acid composition. 

Bannon, C.D., Craske, J.D., Hai, N.T., Harper, N.L., and O Rourke, K.L. 1982. Analysis of fatty acid methyl esters with high accuracy and reliability. II. Methylation of fats and oils with boron tri-... 

Fatty acids, see also Fats conjugated bonds, 524 (fig.) fatty acid methyl esters (FAMEs) analysis by GC, 445-450 preparation using boron trifluoride,... 

If the analysis of fatty acid methyl esters gives a large amount of information on the composition of fats, there remain some unsolved problems, related to the two major flaws of the approach. The transesterification of TGs derived from polyunsatured acids is not always quantitative. A more important and general problem is that the method does not provide any information regarding the actual composition of the TGs. 

Figure 5. GC/FTD analysis of the phospholipid-derived fatty acid methyl esters from derivatization/SFE extraction of Bacillus suhtilis. The front chromatogram shows the second extraction of the same sample (I.S.=internal standard).

Fatty acid methyl ester (FAME) analysis... 

Figure 4.1 GC/MS analysis of methyl esters prepared from a whole cell lipid extract of the YEpOLEX-PDesat-TnD11Z-transformed ole1 strain of Saccharomyces cerevisiae (A) total ion spectrum of fatty acid methyl esters resolved by capillary GLC (B) mass spectrum of the degradation products of the DMDS adduct of Z11 -16 Me in A. The diagnostic m/z values of the DMDS adduct of Z11-16 Me are labeled. (Reproduced with permission from Knipple et al., 1998. 1998 by The National Academy of Sciences.)...

An expression construct consisting of the open reading frame of the TnFB A9Ds cDNA inserted into the yeast desaturase expression vector YEpOLEX was used to transform the olel strain of S. cerevisiae as described above. Many transformant colonies were obtained on medium lacking unsaturated fatty acids, indicating complementation of the olel mutation by the encoded T. ni desaturase. GC/MS analysis of the fatty acid methyl esters obtained from the transformants showed that the TnFBA9Ds cDNA encoded a A9 desaturase that produced oleic acid (Z9-18 Acid) and palmitoleic acid (Z9-16 Acid) (Liu et al., 1999). Quantitation of these unsaturated fatty acids under standard conditions as described above revealed about three times more of the former than the latter (Rosenfield et al., 2001). 

Vine, J. (1980). Analysis of fatty acid methyl esters by high resolution gas chromatography— chemical ionisation mass spectrometry. J. Chromatogr. 196, 415-424. 

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