Analysis of Fatty Acids in Food Lipids

There are basically two mechanisms to convert the fatty acids in a complex lipid to fatty acid methyl esters (FAMEs) methylation following hydrolysis of the fatty acids from the complex lipids, or direct transesterification. The first mechanism involves saponification (alkaline hydrolysis) in which the ester bond is cleaved between the fatty acid and the glycerol moiety (e.g., triacylglycerols and phospholipids) under heat and in the presence of an alkali (usually sodium hydroxide), followed by methylation performed in the presence of an acidic catalyst in methanol. Direct transesterification is usually a one-step reaction involving alkaline or acidic catalysts. 

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  

Analysis of Fatty Acids in Food Lipids Equation D1.2.1 

Extracted lipid sample in organic solvent (see Support Protocol) 

10% to 14% boron trifluoride ( BF3) in methanol (Sigma or Supelco also see recipe) 

---

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

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. 

Published applications of TLC or HPTLC are dedicated to different topics in food analysis. With approximately 20% of all published method in the last 50 years, the analysis of dyes and pigments are the largest part of application followed by analysis of fatty acids, lipids, phospholipids and mycotoxins [162, 163], Although neither paper nor thin-layer chromatography plays a major role for routine analysis of berry anthoeyanins the value of these methods for research oriented work should never be imderestimated. 

Ackman, R.G. (1992b) Application of gas-liquid chromatography to lipid separation and analysis qualitative and quantitative analysis, in Fatty Acids in Foods and Their Health Implications, ed. C.K. Chow, Marcel Dekker, New York, pp. 47-63. 

The methods used for determination of fat or oil in food are often based on extraction with either ethyl ether or petroleum ether and gravimetric determination of the extraction residue. These methods may provide unreliable or incorrect results, particularly with food of animal origin. As shown in Table 14.20, where a corned beef sample was analyzed, the amount and composition of fatty acids in the fat residue were influenced greatly by the analytical methods used. In addition to the accessible free lipids, the emulsifiers present and the changes induced by autoxidation affect the amount of extractable lipids and the lipid-to-nonlipid ratio in the residue. The use of a standard method still does not eliminate the disadvantages shown by analytical methods of fat analysis. Therefore, in questionable cases, quantitative determination of fatty acids and/or glycerol is recommended. 

These methods are based on lipid (substrate) oxidation and specihc to the analysis of oxidation that occurs in food lipids. The tests employed strongly correlate to the conditions that oils and fats are subjected to during processing, food preparation, and storage. The substrate is a model compound that could be a pure triacylgly-cerol, fatty acid methyl ester, or an actual edible oil/lipid. Favorable conditions for substrate oxidation (e.g., high temperature) are provided to facilitate increased rate of oxidation reactions in a controlled environment. The end point is determined... 

Lipase-catalyzed hydrolysis of the vitamin esters in food samples in SCCO2 has been investigated as a pretreatment step in the analytical determination of vitamins in food samples (216, 217). Lipase-catalyzed transesterification of oils with methanol have been used for the determination of total fat content in lipid-containing samples such as oilseeds and meat samples (218, 219) and for the determination of fatty or resin acid content of tall oil products (220). Esterification of fatty acids with methanol in SCCO2 has been reported for total fatty acid analysis of soapstock (221). 

One of the most interesting fields of application of MAE in food analysis is the extraction of lipids. This step, traditionally performed with conventional Soxhlet extraction, has been performed with the focused microwave-assisted Soxhlet extractor prototype of Figure 2B. Extraction of oil from olives, srm-flower seeds, and soyabeans extraction of the lipid fraction of dairy products (milk and cheese) and extraction of fatty acids from precooked and sausage foods have significant advantages over conventional methods, including dramatically reduced extraction times, lower degradation of thermolabile analytes, and acceleration of other analytical steps such as hydrolysis in milk samples, in addition to completeness of analyte extraction, which is not always achieved with conventional methods. 

Pasha, M. K. and Ahmad, F. (1992) Analysis of triacylglycerols containing cyclopropene fatty acids in Sterculia foetida (Linn.) seed lipids. J. Agric. Food Chem., 40, 626-9. 

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. 

Accurate determination of lipids in foods is required for nutritional labeling, certification, or for evaluation of standard of identity and uniformity, as well as examination of their effects on functional and nutritional properties of foods. Following lipid extraction and precise quantitative analysis, lipids so obtained may be used for analysis of other lipid characteristics and properties provided that nondestructive and mild extraction procedures are employed that retain the integrity of lipids. Thus, determination of lipid classes, fatty acid composition (unit du), and oxidative state of lipids (Chapter D2), amongst others, may be pursued following the extraction process. 

---