Introduction

What You Will Find Here

This series of documents is primarily 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 1900 so far. To view them, visit our Archive of methyl esters --- 3-pyridylcarbinol ('picolinyl') esters --- DMOX derivatives --- pyrrolidides --- other derivatives/lipids.

We have spectra of 3-pyridylcarbinol ('picolinyl') ester derivatives of more than 480 different fatty acids on file, as well as 320 dimethyloxazoline (DMOX) derivatives and 460 methyl esters, not to mention pyrrolidides (over 250) and many other fatty acid 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 3-pyridylcarbinol 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 the mass spectra of nitrogen-containing derivatives especially, the positions of double bonds relative to the carboxyl group are more often important than those relative to the terminal methyl group. The reverse is sometimes true for methyl ester derivatives of polyunsaturated fatty acids.

In addition, a few spectra are described of aliphatic and alicyclic compounds other than fatty acid derivatives (e.g. acetals, alcohols and sterols), as these are often encountered in fatty acid preparations. Some common contaminants are also discussed.

All the spectra illustrated have been obtained as part of the lipid chemistry research effort at the James Hutton Institute, and many have not been published elsewhere. This is therefore a personal account. That said, I am grateful to a number of scientists who have provided samples for analysis or mass spectrometry files obtained on Agilent instruments, and they are acknowledged on the relevant web pages. An extensive bibliography is provided on this website, listing more than a thousand relevant references, but I have kept citations in the text to an essential minimum. These pages are revised and improved as new information and spectra become available.

I have NOT described the use of mass spectrometry for quantitative analysis of fatty acids in these web pages. Then quite different problems arise and methyl ester derivatives may be as good as any other for the purpose. I will leave that topic for someone else to discuss, but readers should be aware that gas chromatography (GC) with flame-ionization detection is by far the simplest and arguably the most accurate approach to quantitative analysis when the composition of a sample is known..

 

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. Many 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 with electron-impact ionization 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 key structural features; the positions of double bonds in the aliphatic chain, for example, can only rarely 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 forty 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 3-pyridylcarbinol ('picolinyl') ester or 4,4-dimethyloxazoline (DMOX) derivatives.

 

Derivatives for mass spectrometry

Other nitrogenous derivatives have been described that may have excellent mass spectrometric properties, but there are relatively few published spectra and and we have none from our own lab for discussion here.

Both 3-pyridylcarbinol 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 rather than simply as alternatives. 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. 3-Pyridylcarbinol 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 similar conditions and can give comparable resolution. Pyrrolidides also have reasonable chromatographic properties, and can give some unexpected separations. 3-Pyridylcarbinol 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-5TM, which gave relatively poor resolution. From time to time, we still find a DB-5TM 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-70TM or even some of those of the Carbowax type, such as Supelcowax 10TM, the problem of GC resolution of 3-pyridylcarbinol 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 web page on preparation of methyl esters.

It is worth noting that methyl esters, 3-pyridylcarbinol 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. However, milder methods are now available. Derivative preparation is discussed in detail on a separate web page. Of course, there are times when it is necessary to prepare derivatives other than of the carboxyl group. For example, trimethylsilyl ethers are invaluable for hydroxy fatty acids.

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).

 

Alternative Techniques

Many alternative types of fatty acid derivatives to those described in these pages have been published in the literature, but as we have no experience of them, 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. While simple oxazoline derivatives, as opposed to DMOX, look excellent on paper, there is no body of published spectra for comparison purposes (Kuklev, D.V. and Smith, W.L. A procedure for preparing oxazolines of highly unsaturated fatty acids to determine double bond positions by mass spectrometry. J. Lipid Res., 44, 1060-1066 (2003)).

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 mainly from the laboratory of Professor J. Thomas Brenna, who has provided an article of the topic HERE... 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 appearing in significant numbers. I have no personal experience of these techniques and cannot comment further at present. Some interesting results have been obtained by direct infusion or ‘shotgun’ methods, but they seem only suitable for rapid screening in my estimation at least. A bibliography of relevant published papers is available on this website.

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

 

What to Read

For general information on fatty acid analysis or lipid analysis in general, I recommend that readers consult my book - Lipid Analysis (4th Edition) - listed below - or "Gas Chromatography and Lipids", the latter now available on this site. The first two reviews listed below, though now somewhat dated, 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) (DOI: 10.1007/s11745-998-0214-x).
  • Christie, W.W. and Han, X. Lipid Analysis - Isolation, Separation, Identification and Lipidomic Analysis (4th edition), 446 pages (Oily Press, Bridgwater, U.K.) (2010) - http://store.elsevier.com/product.jsp?locale=en_US&isbn=9780955251245.
  • 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 our Bibliography - review articles section. On the other hand, readers will find that these web pages contain much more practical information and vastly more illustrations of mass spectra than are available elsewhere.

Updated May 5, 2014

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