The Essential Guide to Fatty Acid Analysis
Understanding the Nomenclature
Fatty acids are carboxylic acids which are typically found in lipids (fats and oils) in plant and animal tissue. These acids are generally named according to the number of carbon atoms in the chain, and the number of double bonds in the chain, for example,C18:1, octadecaenoic acid contains 18 carbon atoms with a single double bond in the chain. Saturated fatty acids contain no double bonds. Mono unsaturated fatty acids contain one double bond, poly unsaturated fatty acids contain two or more. Trans fatty acids are unsaturated fatty acids in which at least one double bond is in the trans position as opposed to the more typical cis position. Trans fatty acids occur infrequently in nature; most trans fatty acids in food are the result of partial hydrogenation of the fat. Most fatty acids in food are bound to glycerol molecule as triglycerides, but several other moieties are common: monoglycerides, diglycerides, phospholipids, free acids etc. The US Nutrition Labeling Education Act (NLEA) requires total fat to be expressed as triglyceride equivalents; saturated fatty acids and trans fatty acids must be expressed as free acids.
How Do You Analyze Fatty Acids?
Fatty acids are commonly analyzed by gas chromatography (GC) after conversion to fatty acid methyl esters (FAMEs) which are more easily separated and quantified than either triglycerides or free fatty acids. In most methods the fat is saponified, which liberates the fatty acids from triglycerides, phospholipids, etc.—producing free acids. The free acids are trans-esterified to form fatty acid methyl esters. Matrices that are not pure fats and oils require an extraction step to liberate the fat for analysis. Most solid samples are hydrolyzed by strong acid and/or akali, then extracted with organic solvents. In order to accurately quantify the fatty acid content of the sample as a weight percentage of sample, a synthetic fatty acid (typically C13:0, C19:0, C21:0 or C23:0) is added to the sample prior to extraction as an internal standard. The use of the internal standard compensates for variability in both the preparation and analysis of the sample.
The fatty acid methyl esters are then separated on the GC and quantified using a flame ionization detector (FID). Separations are performed with wax type capillary columns when only basic chain length and saturation are needed. In order to quantify cis versus trans isomerization specialized, highly-polar capillary columns are used. The FID burns the FAMEs producing ions generating an electrical current which is measured and plotted as the response in the chromatogram.
Fatty Acid Methodology
At the Eurofins Nutrition Analysis Center (ENAC) we perform three general types of fatty acids analysis. Our basic fatty acid profile quantifies fatty acids from C8:0 to C24:1, and separately quantifies the Omega 6 and Omega 3 isomers that are commonly found in most vegetable and marine oils.
The second profile is our Full Fatty Acids –NLEA profile which quantifies fatty acids from C4:0 to C24:1. It includes separation of the cis and trans fatty acids as required for NLEA regulations. It also includes quantification of several different positional isomers (Omega 3, Omega 6, Omega 7, Omega 9 etc.)
The third profile is the GOED (Global Organization for EPA and DHA Omega 3) method. This method is also known as the CRN method and is essentially the same US and European Pharmacopeia methods for EPA and DHA in fish oil supplements. This method is considered the gold standard for analysis of EPA (C20:5 Omega 3) and DHA (C22:6 Omega3), but it is specialized for the quantification of these fatty acids and only quantifies Omega 3 fatty acids.
Fatty Acid Results and Reports
Most fatty acids are bound to glycerol as triglycerides and may exist in various other molecules (for example monoglycerides or phospholipids), which may contain one or more different fatty acids. However, fatty acids are typically quantified as individual fatty acid methyl esters. This leads to various methods for reporting the final quantified results. Traditionally fatty acid results have been reported as % of the total area for all peaks. This method works well enough for refined vegetable oils where the fatty acid content is >95% of the total mass. The method works less well for oils that contain significant amounts of non-triglyceride constituents. It is also prone to greater variability between laboratories depending on the exact conditions of the analysis. Eurofins Food Testing Laboratories will report results on this basis only when specifically requested.
By default results are reported as individual fatty acids as a % weight/weight of the sample. Reporting as fatty acid methyl esters may be requested; this is the form that is actually analyzed on the GC. Reporting as triglyceride equivalents may also be requested; this is the form that most fat is in especially for refined oils and some supplements. Results may also be reported as fatty acid ethyl esters; this is primarily used for fish oil based supplements in which the fatty acids have been converted to fatty acid ethyl esters.
Saturated fat and trans fat for nutrition labels are required to be listed as fatty acids per NLEA regulations. Total fat is required to be reported as triglyceride equivalents.
Omega fatty acids have been linked to some of the most exciting new nutritional research in the industry. Proper quantification of fatty acids provides an unbeatable edge in marketing your products by identifying and emphasizing specific beneficial fatty acids. Understanding the scientific approach for fatty acid analysis is integral to the development of new products and market application. As a world leader in fatty acid analysis, our laboratory personnel includes American Oil Chemist Society (AOCS) Approved Chemists who are accomplished in facilitating our clients’ analytical needs.
