Analysis of Conjugated Linoleic Acid (CLA)


Abstract: The recognition of conjugated linoleic acid (CLA) as a component or ingredient of foods that has health-giving properties has prompted increased interest in analytical methods for conjugated fatty acids. The application of gas chromatography (GC), ultraviolet spectroscopy and GC-mass spectrometry are briefly summarized. No single method can cover all requirements and a battery of techniques may be required.


Conjugated linoleic acid (CLA) is attracting great interest among nutritionists because it is a natural fat component that appears to have a number of health-giving properties. 9-Cis,11-trans-octadecadienoate is the common natural isomer found in milk and dairy products, where it has arisen as a by-product or intermediate in the microbial biohydrogenation of linoleic acid in the rumen. Synthetic CLA isomers are being produced as health-food supplements, and many natural conjugated fatty acids are available from plant sources, whose therapeutic properties have never been investigated. Synthetic materials are prepared by alkaline isomerization of linoleate and contain mainly 9c,11t- and 10t,12c-18:2, but over-vigour reaction conditions can lead to the production of many more isomers. When humans or other animals ingest these, further conjugated metabolites may be produced. Most aspects of the topic, nutritional, biochemical and analytical, have been reviewed in three monographs published by the American Oil Chemists’ Society, while analysis specifically has been recently reviewed from a practical standpoint [1] or earlier [2] online here...


Gas Chromatography

I first became aware of CLA in the early 1970s, because they interfered with the analysis of essential fatty acids in ruminant animals. Thus, the methyl ester of 9c,11t-18:2 tended to overlap with 18:3(n-3) and 20:1 on the packed gas chromatography (GC) columns of EGSS-X or EGSS-Y, which were widely used in those days [3]. It is perhaps a sign of how science or fashions change, that we might be more concerned now with interference by other fatty acids in the analysis of CLA.

It is essential to note that the method of preparing methyl esters for GC analysis is critical and base-catalysed transesterification appears to be essential [4]; acid-catalysed transesterification procedures (both boron trifluoride/methanol and hydrogen chloride/methanol) caused appreciable isomerization of cis,trans conjugated double bonds to the trans,trans configuration. However, freshly prepared methanolic sulphuric acid can be an excellent methylation reagent for unesterified fatty acids [2].

CartoonWith the high resolution possible with modern capillary columns, the subsequent GC analysis of natural CLA should not present too much of a problem, provided that a suitable standard is available (c.f. Matreya Inc, Pleasant Gap, PA, USA - see our Links page). With conventional 25 metre Carbowax columns, the methyl ester of 9c,11t-18:2 tends to elute long after non-conjugated dienes in an area of the chromatogram free of most potential contaminants. However, the wide range of geometrical and positional isomers in health food supplements containing synthetic CLA are more of a challenge. For example, phases of the Carbowax type will tend to show only the two main peaks for the cis,trans-isomers in commercial CLA isomers, mainly 9c,11t- and 10t,12c-18:2, but these can hide a multitude of positional isomers. Surprisingly, cis,trans- elute ahead of cis,cis- and then of trans,trans-dienes. On the other hand, this type of stationary phase will at least give an approximate figure for the total content of CLA relative to other components.

Many of the isomers may be separable on GC columns of high polarity, but when these are employed in feeding studies other fatty acids may interfere. If we have to consider metabolites of conjugated fatty acids produced in nutritional studies, the technical difficulties multiply. Long columns (100 m) of highly polar phases such as CP-Sil 88TM or BPX-70TM will give much better separations, and the two peaks will split into four (or more), i.e. in the order, 9c,11t-, 8t,10c-, 11c,13t- and 10t,12c-18:2. These are followed by a group of peaks with the cis,cis-configuration and then by the trans,trans group, the order of elution of specific positional isomers within each group varying. Although resolution of the 9c,11t- and 8t,10c-isomers can be achieved in ideal circumstances, it is not easily done on a routine basis for reasons that are not yet clear. The other complicating factor is that small amounts of other fatty acids, such as 21:0 or 20:2 isomers, may contaminate the cis,cis and trans,trans groups from tissue samples.


High-Performance Liquid Chromatography

An important property of conjugated fatty acids is that they have distinctive UV spectra. For example, conjugated dienes exhibit a characteristic absorbance at 234 nm. When the conjugated diene is a major component of a lipid extract, this absorbance can usually be measured with ease. Conjugated fatty acids may be present in tissue lipids at low levels only on the other hand; then the absorbance may only be displayed as a shoulder above a broad band at 200 nm from the end absorption of lipids, and this can hamper analysis. A mathematical technique has been developed to resolve the difficulty in the laboratory of Banni and coworkers, however [5]. By taking the differential of the first derivative spectrum, a second derivative is obtained, which extracts a distinct peak from the shoulder. Improved resolution results, giving narrower bands with minima rather than maxima. Two bands are seen with natural samples, at 233 nm for trans,trans and 242 nm for cis,trans conjugated dienes. Also, quantification is improved as the Beer-Lambert law is unaffected by differentiation. The technique is most often used in tandem with reversed-phase high-performance liquid chromatography to effect separation and identification of conjugated from more conventional fatty acids.

Scottish thistleConjugated fatty acids have some further interesting properties that help to distinguish them. For example, in my early study [2], I observed that a cis,trans conjugated diene behaved in a similar way to a cis-monoene on silver ion thin-layer chromatography, and by a judicious choice of solvents for the mobile phase it could be clearly separated from other fatty acids. In fact the first paper on this topic to my knowledge was published in 1967 (silver resin chromatography), although it has been overlooked by most reviewers [6].

Silver ion high-performance liquid chromatography (HPLC) has proved to be of special value for the separation of positional isomers in the laboratories of Adlof and then of Yurawecz and colleagues. The latter demonstrated superb separations of geometrical and positional isomers of CLA in the form of methyl esters by linking a number of columns in series (up to six), though fortunately two were sufficient for most purposes [7,8]. They used 0.1% acetonitrile in hexane as the mobile phase with UV detection at the specific wavelength of the conjugated double bond system at 233 nm. Trans,trans- eluted before cis,trans- and then cis,cis-isomers, and within each group, positional isomers eluted in the order 11,13-, 10,12-, 9,11- and 8,10-18:2. Recently, my Bulgarian collaborators, Boryana Nikolova-Damyanova and colleagues, have shown that excellent resolution is possible with a single silver ion column if the CLA is in the form of phenacyl esters [9].


Gas Chromatography - Mass Spectrometry

Improved methods have also been developed for gas chromatography-mass spectrometry of CLA isomers. Dimethyloxazoline (DMOX) derivatives, which are more widely used for mass spectrometry of fatty acids in general, are especially valuable for conjugated isomers. Location of the double bonds is a relatively simple matter. DMOX derivatives have similar chromatographic properties to methyl esters so excellent resolution of positional isomers is possible on polar capillary columns. Also, selected ion monitoring can be used to quantify components that are poorly resolved [10,11].

In addition, a derivative developed by Gary Dobson is highly specific for conjugated double bonds, i.e. formation of the Diels-Alder adduct with 2-methyl-1,2,4-triazoline-3,5-dione [12]. The nature of the reaction is shown in Figure 1.

Fig. 1. Reaction of 2-methyl-1,2,4-triazoline-3,5-dione with the methyl ester of a fatty acid with a conjugated double bond system. formulae

A cyclic derivative is formed between the conjugated double bond and the reagent. Although there is a substantial increase in molecular weight, such derivatives can be subjected to analysis by GC-MS and give distinctive and characteristic fragmentations on either side of the ring, permitting unequivocal identification. Indeed the reaction is almost too rapid. It must be controlled by adding only a small molar excess of the reagent, and then stopped almost immediately by addition of 1,3-hexadiene, otherwise the reagent will react with non-conjugated double bond systems. Fortunately, such by-products are highly polar and do not interfere with the GC-MS analysis. A minor disadvantage of the speed of the reaction, is that it does not enable distinction to be made between trans,trans- and cis,trans-dienes; maleic anhydride only reacts with the former, for example, and then under relatively vigorous conditions.


NMR Spectroscopy

Perhaps the single most comprehensive method for commercial CLA preparations has proved to be 13C-NMR spectroscopy [13]; this permitted the identification and quantification of all the positional (7,9- to 11,13-18:2) and geometrical isomers (cis,trans-, trans,cis-, cis,cis- and trans,trans-) present in such samples. This is by far the most complete single analysis of CLA, but unfortunately the methodology is not likely to be applicable to tissue samples at natural levels.


Combined Procedures

While this last procedure has been used to identify and quantify CLA isomers at natural levels, it appears that the methodology is being pushed to its limits. When the ultimate in accuracy is required, it may be advisable to isolate a C18 diene fraction first by reversed-phase HPLC (CLA elutes with linoleate) and then use silver ion chromatography to isolate the CLA (which elutes with the cis-monoene fraction in this instance). All the available GC-MS methods can then be utilized to characterize the isomers as fully as possible. Such procedures were used to identify tetraenoic metabolites (related to arachidonic acid) having a conjugated diene moiety in lipids from rats fed high levels of CLA [14], and to identify the low levels of natural CLA isomers present in cheese [15], for example. This methodology may therefore be important if the mechanism of action of CLA in animal tissues is to be revealed.

As a former professional analyst, I am pleased that there are still tasks where real skill is necessary. In this instance, it seems that a battery of techniques must be employed if we are to make real progress. On the other hand, there are times when I feel that the expectations of lipid analysts are too great. We require our fatty acid analyses to be accurate to about 0.1%, for example. Is such precision essential? Can we accept a lower standard for difficult analyses such as CLA? Would this really influence the interpretation of biological experiments if we did? There are many biochemical fields where researchers look on our standards of accuracy as an impossible pipe dream.


References

  1. Christie, W.W., Dobson, G. and Adlof, R.O. A practical guide to the isolation, analysis and identification of conjugated linoleic acid. Lipids, 42, 1073-1084 (2007).
  2. Christie, W.W., Sébédio, J.L. and Juanéda, P. A practical guide to the analysis of conjugated linoleic acid (CLA). INFORM, 12, 147-152 (2001).
  3. Christie, W.W. The structure of bile phosphatidylcholines. Biochim. Biophys. Acta, 316, 204-211 (1973).
  4. Shantha, N.C., Decker, E.A. and Hennig, B. Comparison of methylation methods for the quantitation of conjugated linoleic acid isomers. J. AOAC Int., 76, 644-649 (1993).
  5. Corongiu, F.P. and Banni, S. Detection of conjugated dienes by second derivative UV spectroscopy. Methods Enzymol., 233, 303-310 (1994).
  6. Emken, E.A., Scholfield, C.R., Davison, V.L. and Frankel, E.N. Separation of conjugated methyl octadecadienoate and trienoate geometric isomers by silver-resin column and preparative gas-liquid chromatography. J. Am. Oil Chem. Soc., 44, 373-375 (1967).
  7. Rickert, R., Steinhart, H., Fritsche, J., Sehat, N., Yurawecz, M.P., Mossoba, M.M., Roach, J.A.G., Eulitz, K., Ku, Y. and Kramer, J.K.G. Enhanced resolution of conjugated linoleic acid isomers by tandem-column silver-ion high performance liquid chromatography. J. High Resol. Chromatogr., 22, 144-148 (1999).
  8. Sehat, N., Rickert, R., Mossoba, M.M., Kramer, J.K.G., Yurawecz, M.P., Roach, J.A.G., Adlof, R.O., Morehouse, K.M., Fritsche, J., Eulitz, K.D., Steinhart, H. and Ku, Y. Improved separation of conjugated fatty acid methyl esters by silver ion-high-performance liquid chromatography. Lipids, 34, 407-413 (1999).
  9. Nikolova-Damyanova, B., Momchilova, S. and Christie, W.W. Silver ion high-performance liquid chromatographic separation of conjugated linoleic acid isomers, and other fatty acids, after conversion to p-methoxyphenacyl derivatives. J. High Resol. Chromatogr., 23, 348-352 (2000).
  10. Mossoba, M.M., Kramer, J.K.G., Yurawecz, M.P., Sehat, N., Roach, J.A.G., Eulitz, K., Fritsche, J., Dugan, M.E.R. and Ku, Y. Impact of novel methodologies on the analysis of conjugated linoleic acid (CLA). Implications of CLA feeding studies. Fett-Lipid, 101, 235-243 (1999).
  11. Fritsche, J., Rickert, R., Steinhart, H., Yurawecz, M.P., Mossoba, M.M., Sehat, N., Roach, J.A.G., Kramer, J.K.G. and Ku, Y. Conjugated linoleic acid (CLA) isomers: formation, analysis, amounts in foods, and dietary intake. Fett-Lipid, 101, 272-276 (1999).
  12. Dobson, G. Identification of conjugated fatty acids by gas chromatography mass spectrometry of 4-methyl-1,2,4-triazoline-3,5-dione adducts. J. Am. Oil Chem. Soc., 75, 137-142 (1998).
  13. Davis, A.L., McNeill, G.P. and Caswell, D.C. Identification and quantification of conjugated linoleic acid isomers in fatty acid mixtures by 13C NMR spectroscopy. Chem. Phys. Lipids, 97, 155-165 (1999).
  14. Sébédio, J.L., Juanéda, P., Dobson, G., Ramilison, I., Martin, J.C., Chardigny, J.M. and Christie, W.W. Metabolites of conjugated isomers of linoleic acid (CLA) in the rat. Biochim. Biophys. Acta, 1345, 5-10 (1997).
  15. Lavillonniere, F., Martin, J.C., Bougnoux, P. and Sébédio, J.L. Analysis of conjugated linoleic acid isomers and content in French cheeses. J. Am. Oil Chem. Soc., 75, 343-352 (1998).

This article has been updated appreciably from two earlier papers (now amalgamated) by the author that first appeared in Lipid Technology (Christie, W.W. Lipid Technology, 9, 73-75 (1997) and Lipid Technology, 12, 64-66 (2000)).


W.W. Christie

Updated: 27/3/2008

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Scottish Crop Research Institute (and MRS Lipid Analysis Unit), Invergowrie, Dundee (DD2 5DA), Scotland

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