Since January 1, 2006, the Food and Drug Administration has required food companies to list the trans fatty acid ("trans fat") content on the Nutrition Facts panel of their products because of an established scientific link between consumption of trans fat and increased risk of coronary heart disease. Trans fatty acids occur naturally to some extent in meat and dairy products but are commonly formed during hydrogenation to manufacture margarine and other hardened-fat products.
There are two types of trans fatty acids—nonconjugated and conjugated. In nonconjugated fatty acids, isolated double bonds along a fatty acid chain are separated by one or more methylene (–CH2–) groups (–C=C–CH2–C=C–), whereas in conjugated fatty acids they are not (–C=C–C=C–). As a result, there are profound structural differences: a fatty acid chain with a cis double bond is bent, while that with a trans double bond is fairly straight, like saturated fatty acid chains.
The health effects are also vastly different, said Magdi M. Mossoba ([email protected]), Research Chemist in the Spectroscopy and Mass Spectrometry Branch at the Food and Drug Administration’s Center for Food Safety and Applied Nutrition in College Park, Md. Whereas nonconjugated trans fats have been shown to increase the risk of coronary heart disease, he said, naturally occurring fatty acids with conjugated double bonds, namely, conjugated linoleic acid (CLA) isomers, are considered beneficial. There are up to 20 CLA isomers, mostly found in dairy products, and some exhibit anticarcinogenic and other beneficial effects in laboratory animals. Accordingly, for labeling purposes, the trans fat content is not to include the content of conjugated fatty acids. As one can tell, he added, there are complex trans fat issues—analytical chemistry, biology, physiology, labeling, and legal—that are far from resolved.
Methods of Analysis
According to Mossoba, although there are various methods approved by AOAC International and the American Oil Chemists’ Society, the most recently validated official methods to identify and quantify trans fatty acids for food labeling purposes are AOAC Official Method 996.06 and AOCS Official Method Ce 1h-05 for capillary gas chromatography (GC) and AOAC Official Method 2000.10 and AOCS Official Method Cd 14d-99 for Fourier transform infrared spectroscopy (FTIR).
• GC. In the GC method, a test sample is first hydrolyzed by addition of acid or base to convert the fatty acids into volatile fatty acid methyl esters, then separated by being carried by a gas through a capillary column coated with highly polar stationary phases. The separated components elute from the column at different times. The trans fatty acid content is the sum of all fatty acids with isolated (nonconjugated) trans double bonds in the chromatogram, with the exception of CLA isomers with conjugated trans double bond geometry.
Problems with GC, Mossoba said, include peak overlap, lack of commercial reference standards, complexity of technique, and difficulty of interpretation, especially identification of minor GC peaks.
Mossoba also mentioned likely improvements being researched. GC improvements, he said, include use of more-optimized GC separation conditions that can reduce (but cannot eliminate) the overlap between GC peaks; other, more powerful separation conditions would take too long (at lower temperatures but longer GC separation times) or require much more work (pre-separation of the trans fat fractions by using thin-layer chromatography or high-pressure liquid chromatography prior to the GC separation step).
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One of the challenges ahead, Mossoba said, is to develop better chromatographic GC columns capable of resolving all or most of the overlapping GC peaks.
• FTIR. In the FTIR method, a test sample is placed between a beam splitter and a detector. When exposed to a high-temperature heating element, it selectively absorbs infrared energy. Changes in the energy intensity output reaching the detector as a function of time yield an infrared spectrum known as an interferogram. When the interferogram is converted from time to frequency by Fourier transform mathematics, a single-beam spectrum is obtained, and trans fat content can be determined by comparison to a trans-fat-free reference.
Isolated double bonds with trans configuration uniquely absorb infrared radiation at a wavelength of 966 cm–1. Since this absorption band occurs on a sloping baseline, Mossoba said, measurement of its height or area becomes increasingly less accurate as the trans levels decrease. Other problems with FTIR, he said, include presence of interfering bands (at 985 and 945 cm–1) due to cis/trans and trans/trans conjugated fatty acids; and presence of very weak interfering bands (at slightly lower energy, near 960 cm–1) due to saturated fatty acids in test samples (e.g., coconut oil) that are high in saturated fats and very low (0.1%) in trans fats.
FTIR Improvements, he said, include generation and measurement of the second derivative of the trans IR band at 966 cm–1 for an isolated trans double bond. The second-derivative band exhibits a narrower band width and hence eliminates potential interferences from any adjacent conjugated or saturated IR bands. Thus, this procedure is more sensitive and more accurate than any other FTIR procedure or official method. Mossoba has organized an international study to validate this procedure and to determine its lower level of quantitation.
FDA Intramural Research
Researchers at FDA’s Center for Food Safety and Applied Nutrition are conducting, with industry and academic consortia partners, two intramural research projects related to trans fat analysis (www.cfsan.fda.gov/~dms/cfsres08.html):
• Project 357, "Evaluating the Measurement of Trans-Fat by Gas Chromatograph." The goal of project 357 is to utilize GC to develop reference materials regarding monounsaturated fatty acids, the most common source of trans fatty acids in partially hydrogenated oils and fats. This work will focus on the development of reference materials and procedures for synthesizing mixtures containing all of the cis/trans isomers of monounsaturated fatty acids and polyunsaturated fatty acids. GC analysis of the mixtures will provide elution patterns that can help improve determination methods of total trans fat content.
Pierluigi Delmonte ([email protected]), Chemist at CFSAN, is principal investigator for the project, which is expected to be completed in 2010.
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• Project 265, "Rapid Determination of Trans-Fats by Developing a Novel Attenuated Total Reflection-Fourier Transform Infrared (ATR-FTIR) Spectroscopic Method." The goal is to develop a rapid (5-min) method for determination of trans fats. Researchers in the United States, Canada, Switzerland, Germany, China, and Australia are participating in an international collaborative study for the validation of the so-called Negative Second Derivative ATR-FTIR procedure. The study participants are mostly researchers from university food science departments and food corporations (ADM and Nestlé). Once validated, this procedure will become an official method for regulatory purposes to ensure accurate trans-fat label information.
Mossoba is the principal investigator for the project, which is expected to be completed by the end of 2009. Once validated, he said, this negative second-derivative ATR-FTIR procedure will make infrared spectroscopy more suitable than ever and a rapid alternative and/or complementary method to GC for the rapid determination of total trans fats for regulatory compliance.
Instrument Suppliers
There are numerous instrument suppliers. Agilent Technology, Santa Clara, Calif. (www.agilent.com), is the major supplier of GC instruments for analysis of trans fatty acids. Among the suppliers of IR instruments are Varian Inc., Palo Alto, Calif. (www.varianinc.com); Thermo Fisher Scientific Inc., Waltham, Mass. (www.thermofisher.com), Bruker Optics Inc., Billerica Mass. (www.brukeroptics.com), and PerkinElmer, Waltham, Mass. (www.perkinelmer.com). A2 Technologies, Danbury, Conn. (www.a2technologies.com), offers portable ATR-FTIR spectrometers.
Speaking at the 2008 IFT Annual Meeting & Food Expo, the researchers said that deep-frying fats undergo chemical reactions, initiated by heat, oxygen, the fats themselves, and compounds contained in the foods being fried. The resulting substances formed are undesirable for sensory and health reasons.
An official parameter for fat degradation is the mass percentage of total polar material (TPM), compounds formed during the deep-frying process. The official method of analysis for TPM uses preparative column chromatography. The fat is separated into polar and non-polar fractions. After solvent evaporation, TPM is measured gravimetrically. The procedure takes several hours, uses toxic and inflammable solvents, and must be carried out by laboratory personnel.
The researchers developed an alternative technique in which the dielectric constant (DC) of the fat is measured with a probe. DC depends on the nature and quantity of polar compounds in the fat. By adequate calibration, the values can be given in mass percentage of TPM. The researchers said that their research results have shown that the values obtained by both methods are comparable. They concluded that the DC method has enormous advantages over the chromatographic method in that it can be carried out by minimally skilled personnel, immediately near the frying apparatus, and without the need for chemicals.
Neil H. Mermelstein, a Fellow of IFT, is Editor Emeritus of Food Technology
[email protected]
