The Lipid Library

FATTY ACIDS AND MASS SPECTROMETRY

Introduction

What You Will Find Here

This series of documents is a practical guide to structure determination of natural fatty acids by mass spectrometry with electron-impact ionization, as opposed to a mechanistic review, and it is illustrated with many more mass spectra than would be possible in a conventional review - over 580 at the last count. I hope the old adage that "a picture is worth a thousand words" applies here. These do not include our Archive pages, where all our spectra are now displayed (but without interpretation) -  over 1700 so far. To view them, visit our Archive of methyl esters --- picolinyl esters --- DMOX derivatives --- pyrrolidides --- other derivatives/lipids.

We have spectra of picolinyl ester derivatives of more than 470 different fatty acids on file, as well as 250 dimethyloxazoline (DMOX) derivatives and 350 methyl esters, not to mention pyrrolidides (over 200) and many other derivatives (and related lipids). We are unique in having spectra of all the possible cis-18:1 isomers, from 2-18:1 to 17-18:1, as picolinyl esters, DMOX derivatives and pyrrolidides, and also for all the methylene-interrupted 18:2 isomers (2,5-18:2 to 14,17-18:2), most of which are illustrated in these web pages. To this can be added, branched-chain, cyclic, oxygenated, sulfur-containing, halogenated, allenic, non-methylene-interrupted dienes and polyenes, etc. I have tried to illustrate spectra of C18 fatty acids wherever possible to simplify comparisons, but fragmentations can usually be extrapolated easily to other chain-lengths. Note that in interpreting these mass spectra, the positions of double bonds relative to the carboxyl group are more often important than those relative to the terminal methyl group.

All the spectra illustrated have been obtained as part of the lipid chemistry research effort at the Scottish Crop Research Institute, and many have not been published elsewhere. This is therefore a personal account, as spectra obtained by others cannot be reproduced easily here. An extensive bibliography is provided on this website, listing more than a thousand references, but I have kept citations in the text to the absolute minimum.

These pages are revised and improved frequently as new information and spectra become available.

Why Mass Spectrometry is Invaluable

The common fatty acids of animal and plant origin have even-numbered chains of 16 to 22 carbon atoms with zero to six double bonds of the cis configuration; methylene-interrupted double bond systems predominate. Nature provides countless exceptions, however, and odd- and even-numbered fatty acids with up to nearly a hundred carbon atoms exist. In addition, double bonds can be of the trans configuration, acetylenic and allenic bonds occur, and there can be innumerable other structural features, including branch points, rings, oxygenated functions, and many more. More than a thousand different fatty acids of natural origin must exist, as well as others produced as artefacts when fats are used in commerce and in cooking, for example.

It is essential to have simple rapid methods for determination of fatty acid structures and for isolation of pure components of mixtures for further analysis. In particular, new methods involving gas chromatography-mass spectrometry (GC-MS), GC linked to Fourier-transform infrared spectroscopy (FTIR), and silver ion and reversed-phase high-performance liquid chromatography (HPLC) are available, amongst others. I have described the current state of the methodology in published reviews (see below). Of these methods, GC-MS is especially useful. Straightforward derivatization procedures are required that utilize readily available reagents and have simple glassware requirements. A feature of particular importance with GC-MS is that it is rarely necessary to isolate components in a pure form, as may be required by other spectroscopic methods (e.g. NMR spectroscopy) or by chemical degradative procedures.

Fatty acids are usually analysed by GC as methyl ester derivatives, but their mass spectra may not always contain ions indicative of structural features; the positions of double bonds in the aliphatic chain, for example, cannot be determined unequivocally. However, there are times when it is convenient to analyse such esters by mass spectrometry, for example for confirmatory purposes or as a guide to what further work may be required. Molecular weight and retention times are useful analytical parameters, some limited structural information may be available, and indeed definitive spectra can be obtained often with branched-chain fatty acids or those with additional oxygenated functional groups.

In the most useful approach to structure determination, the carboxyl group is derivatized with a reagent containing a nitrogen atom. When the molecule is ionized in the mass spectrometer, the nitrogen atom not the alkyl chain carries the charge, and double bond ionization and migration is minimized. Radical-induced cleavage occurs evenly along the chain and gives a series of relatively abundant ions of high mass from the cleavage of each C-C bond. When a double bond or other functional group is reached, diagnostic ions usually occur. The first useful nitrogen-containing derivatives, i.e. pyrrolidides, were described over thirty years ago. They give useful spectra and should not be discounted (indeed I believe they have been greatly under-valued, especially for labile fatty acids, such as those with epoxide rings, or with terminal functional moieties). However, most analysts now prefer either picolinyl (3-hydroxymethylpyridinyl) ester or 4,4-dimethyloxazoline (DMOX) derivatives.

Other nitrogenous derivatives have been described that may have excellent mass spectrometric properties, but there are relatively few published spectra and none are readily available to us for discussion here.

Both picolinyl ester and DMOX derivatives have their merits in mass spectrometry terms, and neither should be neglected. Each has advantages for particular types of fatty acid, and they are best considered as providing complementary information. With difficult samples, I have prepared both types of derivative, and often pyrrolidides also. As methyl esters are usually available for other purposes, it is often convenient to analyse these by GC-MS simply for confirmatory or record purposes. Picolinyl esters and pyrrolidides tend to give spectra that are easier to interpret than those of DMOX derivatives when functional groups are near the terminal end of the fatty acyl chain (see Hamilton, J.T.G. and Christie, W.W. Chem. Phys. Lipids, 105, 93-104 (2000)). On the other hand, DMOX derivatives may have advantages for functional groups in positions 4 to 6. While an objective comparison of DMOX derivatives, pyrrolidides and other simple amides would seem desirable, a very large number of different fatty acid types would be needed for meaningful results.

In choosing a derivative for mass spectrometry, good chromatographic properties are also important. One advantage of DMOX derivatives is that they are only slightly less volatile than methyl esters; they can be subjected to GC analysis on polar stationary phases under comparable conditions and give equivalent resolution. Pyrrolidides also have reasonable chromatographic properties, and can give some unexpected separations. Picolinyl esters, on the other hand, require column temperatures about 50°C higher than for methyl esters, and that meant initially that they had to be separated on non-polar phases, such as DB-5^TM, which gave relatively poor resolution. From time to time, we still find a DB-5^TM column to be of value, for example for fatty acids of high molecular weight. With the introduction of new polar phases, which are stable to high column temperatures and have low-bleed characteristics for MS analysis, such as BPX-70^TM or even some of those of the Carbowax type, such as Supelcowax 10^TM, the problem of GC resolution of picolinyl esters is greatly lessened and only very-long-chain fatty acids (>C₂₄) tend to cause problems. We only have experience of these specific columns, so cannot comment on other commercial phases that may be available. Some further information is included in our page on preparation of methyl esters.

It is worth noting that methyl esters, picolinyl esters and pyrrolidide derivatives are prepared under relatively mild conditions, and they are stable chemically so can be stored for long periods at –20ºC. DMOX derivatives require harsh conditions for preparation, and precautions are necessary to prevent hydrolysis on storage, but at least it is a simple one-pot method that can be applied to most lipid types. Of course, there are times when it is necessary to prepare derivatives other than of the carboxyl group.

In these webpages, I concentrate on practical applications of the various derivatives to structure determination of fatty acids as opposed to mechanistic concepts, and these are illustrated with many more mass spectra than would be possible in a book or conventional publications. Also, for practical reasons, I have included some spectra of natural lipids and analytical artefacts that may be encountered from time to time in fatty acid samples.

Of course, other chromatographic and spectroscopic methods than GC-MS may have to be used for characterization purposes. In particular, we have found high-performance liquid chromatography in the reversed-phase and silver ion modes to be especially useful for simplifying mixtures prior to GC-MS (see our webpage on this aspect of the topic).

What You Will Not Find Here

Many alternative types of fatty acid derivatives to those described in these pages have been published in the literature. As we have no experience of most of these, we cannot discuss them here. Any new derivatives described should have a better combination of chromatographic and mass spectrometric fragmentation properties than the existing ones if they are to be taken seriously.

In the last few years, some very interesting papers have appeared dealing with acetonitrile-chemical-reaction tandem mass spectrometry in the gas phase for locating double bonds in fatty acid methyl esters (from the laboratory of Professor J.T. Brenna mainly). The technique appears very promising, but in comparison with more established methods, only a handful of model compounds have been analysed by this means may not be suited to functional groups other than double bonds. Mass spectrometry with atmospheric-pressure chemical ionization (APCI) in conjunction with liquid chromatography has also been used in a number of labs to characterize long-chain fatty acids, and papers in which electrospray ionization is used for the purpose are beginning to appear. I have no personal experience of these techniques and cannot comment further at present. A bibliography of the relevant published papers is available on this website.

Similarly, we have no experience of remote-site fragmentation methods involving tandem mass spectrometry or collisional activation of carboxylate anions or alkali metal-cationized fatty acids. These appear to be useful techniques for locating double bonds especially, but they require more sophisticated and expensive instrumentation than the electron-impact methods described here.

What to Read

For general information on fatty acid analysis, I recommend that readers consult my book - Lipid Analysis (3rd Edition) - listed below - or "Gas Chromatography and Lipids", the latter now available on this site. The first two reviews below give more detailed information on our GC-MS methodology -

• Christie, W.W. Structural analysis of fatty acids. In Advances in Lipid Methodology - Four, pp. 119-169 (1997) (edited by W.W. Christie, Oily Press, Dundee).
• Christie, W.W. Gas chromatography-mass spectrometry methods for structural analysis of fatty acids. Lipids, 33, 343-353 (1998).
• Christie, W.W. Lipid Analysis (3rd edition). (Oily Press, Bridgwater) (2003).
• Murphy, R.C. Mass Spectrometry of Lipids (Handbook of Lipid Research, Vol. 7) (Plenum Press, N.Y.) (1993).

The last of these is most useful for those interested in mechanistic aspects of lipid mass spectrometry. Many more review articles are listed in the Bibliography - review articles section of these pages.

Updated: 11/4/2008 Credits/disclaimer	Scottish Crop Research Institute (and MRS Lipid Analysis Unit), Invergowrie, Dundee (DD2 5DA), Scotland