Trans Fatty acids analysis methods

General Analysis of trans fatty acids NMR spectra of lipids Guidelines for interpretation of 13C NMR spectra Applications of high-resolution methods to oils and fats Structure determination of polyunsaturated triacylglycerols 31P NMR for profiling of phospholipids 1998 (210) 1996(211) 1998(10, 18) 1999(212,213) 1999(213) 1998 (10) 1996(214) 1996(215)... 

Olive oil is often illegally adulterated with other less expensive vegetable oils. Oils widely used for this purpose include olive pomace oil, corn oil, peanut oil, cottonseed oil, sunflower oil, soybean oil, and poppy seed oil. °° Among the varions chemical and physical methods employed toward the detection of the adulteration of olive oil by low-grade olive oils and seed oils are (a) Sterol analysis (presence of stigmasterol and 3-sitosterol), (b) alkane analysis (C27, C29, and C31), (c) wax and aliphatic alcohol analysis, (d) fatty acids/(with HPLC) trans fatty acid, and (e) Triacylglycerol. 

To obtain a total fatty acid profile, to include co3 fatty acids, and other potentially beneficial fatty acids such as GLA, the approach is normally straightforward and involves extraction of lipids in the presence of an appropriate, derivatization of fatty acids to methyl esters, and analysis by GC. Appropriate extraction methods, depending on the type of food and lipids presents, must be employed to extract the total lipids efficiently. An alternative is to hydrolyze the sample directly to release all the fatty acids in the free form followed by derivatisation. An appropriate methylation step is required. Alkaline methods are milder, but will only derivatize EFA. Acidic methods are used to derivatize samples containing FFA with or without EFA. Separation of FAME can normally be achieved on Carbowax type columns of medium length, but samples such as milk fats or PHVO, containing complex mixtures of trans fatty acids, require longer polar columns and may require the use of additional techniques. Milk fats also contain SCFA and specific precautions or adaptations of protocols are necessary for their analysis. 

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 ... 

This method is more efficient in the resolution of cis and trans conformational isomers of fatty acids than the usually used GC methods with packed columns, and there are no problems with derivatization of short-chain fatty acids or heat-labile polyunsaturates. Another advantage over GC methods is that the separated fatty acids are not destroyed during their detection, which enables further analysis to be performed. The trans isomers are generally eluted after the corresponding cis isomers. Positioning of the double bonds in the proximity of the carboxy group of an acid usually leads to a shift in the direction of the methyl end of the carbon chain. 

Although most Ag-HPLC analyses have traditionally been done using CLA methyl esters, improved separation and quantitation of CLA isomers has been noted when phenacyl or p-methoxyphenacyl ester rather than methyl ester derivatives were analyzed (58). Nikolova-Damyanova et al. (59) demonstrated improved separation of the p-methoxyphenacyl derivatives of the cis/trans 8,10- through 11,13-18 2 isomers in a commercial sample of CLA when compared with CLAME. Only a single Ag-HPLC column was required. Resolution of the cis/ trans isomers was similar to that obtained for CLAME using Ag-HPLC systems with two or three Ag-HPLC columns connected in series (60). A stepwise solvent gradient of 100% hexane/dichloromethane/ACN (40 60 0.2 vol/vol/vol 30 min) to 100% dichloromethane/ACN (100 1) over 10 min with UV detection at 270 nm (for phenacyl esters) yielded a semiquantitative estimation of fatty acid composition comparable to, but more detailed than that obtainable by GC analysis as FAME. This method was also applied to detamine the CLA isomer composition of a sample of beef/pig fat, but prefractionation by RP-HPLC was required before Ag-HPLC analysis. The prefractionation step was required to concentrate the CLA isomers and to remove octadecenoate and other fatty acids that might interfere with Ag-HPLC analysis. 

Aliquots of the total lipids, total phospholipids, and separated phospholipid classes were subjected to methano-lysis at 80 C for 3 hours in 5% methanolic HCl. Resultant fatty acid methyl esters were extracted with hexane and analyzed by gas chromatography using a bonded fused silica column (cyanopropylmethylsilicone, 0.25 mm i.d. X 50 m, 0.25 jam film, Tokyo Kasei Kogyo). The fatty acid methyl esters were identified by comparing their retention times with those of standards. The double-bond positions and the cis and trans configurations of fatty acid methyl esters were determined by the pyrrolidide method (6) and IR analysis (7), respectively. 

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