The Lipid Library

Beginners' Guide to Mass Spectrometry of fatty acids.

1. The Nature of the Problem

Abstract: Mass spectrometry is an accessible technique for fatty acid characterization that should hold no fears for lipid analysts.

In most of my articles, I have tried to concentrate on chromatographic analysis, especially gas-liquid and high-performance liquid chromatography, of lipids, as I have assumed that most of my readers have access to this equipment or at least aspire to have such access. I know that mention of the words "mass spectrometry" (MS) causes many of you to switch off. However, MS is no longer a technique for millionaires - it is possible to purchase a gas chromatography (GC)-MS facility for little more than the cost of a good HPLC system. Nor do you need a specialised technician to operate the equipment and interpret the results nowadays, at least for the more basic instrumentation. Therefore, I feel that I can introduce some topics suited to such equipment here. For those who require more detailed information, two review articles can be recommended [1,2].

At its simplest, MS is a technique in which organic molecules are bombarded by electrons or other ionic species causing them to ionize and fragment. If you pick up a journal dealing with mass spectrometry, there will be a host of technical terms dealing with methods of ionization especially, such as "fast-atom bombardment", "tandem techniques", "chemical ionization", but in the more basic mass spectrometers, electron impact ionization is the only one that is relevant to my topic and need be considered here. The various ionic species produced by electron impact are separated according to mass (strictly speaking mass/charge (m/z) ratio) in a magnetic field, and a spectrum is obtained that in effect is a bar diagram showing the masses of the fragment ions and their abundances relative to the most abundant ion (base peak) given a value of 100%.

Jigsaw Puzzles and Bricks

Interpretation of the spectrum is often compared to doing a jigsaw puzzle. You have to try to put the pieces together in a sensible way to find the picture, or describe the molecule. I prefer an analogy, in which mass spectrometry is compared to demolishing and re-assembling a brick wall. If we use a sledgehammer to create a pile of rubble, we will know the total mass present but reassembling the wall is impossible. If we can take the wall apart cleanly a few bricks at a time, it will be possible to reassemble it easily. We will know the correct dimensions and where any door or window should be placed. With a fatty acid derivative, in comparison, we need to confirm that it is indeed a fatty acid, determine the molecular weight and then locate any double bonds or other functional groups.

Methyl esters are the derivative of choice in most analytical applications of lipids. However, when we subject them to electron impact in a mass spectrometer, the charged aliphatic chain breaks up into an indeterminate number of fragments, so a branch-point or ring structure is not easily identified, and a double bond becomes mobile and moves up and down the chain, so its original position cannot reliably be determined. We have taken a sledgehammer to break up the molecule. Yet all is not lost, as the molecular weight of the fatty acid ester is obtained (from the molecular ion), and therefore the number of carbons, hydrogens and oxygens. This tells us if the fatty acid is saturated, and often an experienced eye can tell if there is a branch point.

We usually lose one or two "bricks" neatly. An ion representing loss of 32 mass units (methanol) and another of 74 units from a rearrangement involving the ester moiety confirm that we really do have a methyl ester. If the molecular weight is two less than for a fully saturated fatty acid, we may have a double bond or a cyclic structure present, and this can usually be confirmed by simple experiments, for example by attempting hydrogenation.

The other information we may have as an aid to identification is the relative retention time of the fatty acid derivative (or equivalent chain-length value) on the GC component of the GC-MS system. This together with the molecular weight data may suggest that some structures are more plausible than others.

Double Bond Location in Polyunsaturated Fatty Acids

More often than not we wish to know where double bonds are located in an aliphatic chain, and the mass spectrum of the methyl esters are not always of assistance. We cannot do this directly with monoenes and dienes.

Fortunately, with the conventional series of polyunsaturated fatty acids, that is with three or more methylene-interrupted double bonds, we have some useful characteristic ions. These are illustrated in Figure 1, which shows the mass spectrum of methyl 6,9,12-octadecatrienoate (γ-linolenate).

First, the molecular ion at m/z = 292 is 6 units less than for methyl stearate (18:0), which tells us that there are three double bonds. There is an ion at m/z = 264, representing the loss of methanol, which together with one at m/z = 74 confirms that we do indeed have a methyl ester. These ions tend to be much bigger in spectra from more saturated esters. Ions from the hydrocarbon part of the molecule of general formula [C_nH_2n-5]⁺ tend to dominate the spectrum with the ion at m/z = 79 as the base peak, but these tell us little about the detailed structure.

However, an ion at m/z = 150 (‘omega’ ion) formed by a fragmentation at the terminal end of the molecule is characteristic of all polyunsaturated fatty acids from the n-6 biosynthetic series [3]. Also, there is a fragment (‘alpha’ ion) from the carboxyl end of the molecule at m/z =194, which completes the identification, although the position of this ion will vary according to the position of the first double bond in fatty acids of the n-6 series. The latter ion was defined in a paper by Brauner and coworkers [4], which until recently had been overlooked by most experts in this field.

In mass spectra of methyl esters of fatty acids of the n-3 series, the omega ion stands out at m/z = 108 (and that at m/z = 150 is negligible). For the minor (n-9) and (n-4) biosynthetic families the relevant ions are at m/z = 192 and 122, respectively. It should be stressed that these ions are not infallible guides, and are not reliable when the double bonds are not methylene interrupted.

Chemical Derivatization for Double Bond Location

If we want to have definitive information on double bond positions, we have to get away from the sledgehammer approach. There are two ways this can be done. One is to prepare particular nitrogen-containing derivatives of fatty acids, and this will be the topic of my next article. Alternatively, with monoenoic fatty acids especially, it is possible to "fix" the double bonds by reacting them with appropriate reagents to give chemical derivatives that give distinctive fragmentations in the mass spectrometer.

A host of such derivatives have been described, usually involving oxygenation followed by further derivatization, but most of these fell by the wayside when dimethyl disulphide adducts were described [5]. This is simple single-step (if odoriferous) reaction involving reaction of dimethyl disulphide with an unsaturated ester in the presence of iodine as catalyst. The nature of the reaction is shown in Figure 2.

For example with methyl oleate, the molecular weight increases substantially (from 296 to 390) but this is still in a comfortable range for GC analysis. The mass spectrum gives a good molecular ion but the most abundant ions represent cleavage at carbon atoms that were originally linked by the double bond (m/z = 173 and 217 as shown), and this is located unequivocally. This technique has now been applied to identify many monoenoic fatty acids (and other aliphatic compounds) in natural samples in a number of laboratories. (This is the best test of any method - often a new procedure is described with one or two model compounds and then falls into oblivion).

The technique is less straight-forward if there is more than one double bond in the molecule, as bis-adducts are only formed quantitatively if there are more than three carbons between double bonds, for example [6]. However, the reaction can be used usefully even with methylene-interrupted dienes, such as linoleic acid, since two mono-adducts (one double bond derivatized and one not) are then formed and these are easily separable by GC for analysis by MS [7]. (There is a now more substantial article on this aspect of the topic on the website here..)

Such methods add an extra complication to analysis, and it would obviously be much easier if a single derivative could be used for all purposes - my next contribution.

References

1. Christie,W.W. Structural analysis of fatty acids. In Advances in Lipid Methodology - Four, pp. 119-169 (edited by W.W. Christie, Oily Press, Dundee) (1997).
2. Christie, W.W. Gas chromatography-mass spectrometry methods for structural analysis of fatty acids. Lipids, 33, 343-353 (1998).
3. Fellenberg, A.J., Johnson, D.W., Poulos, A. and Sharp, P. Simple mass spectrometric differentiation of the n-3, n-6 and n-9 series of methylene interrupted polyenoic acids. Biomed. Environ. Mass Spectrom., 14, 127-130 (1987).
4. Brauner, A., Budzikiewicz, H. and Boland, W. Studies in chemical ionization mass spectrometry. 5. Localization of homoconjugated triene and tetraene units in aliphatic compounds. Org. Mass Spectrom., 17, 161-164 (1982))
5. Francis, G.W. Alkylthiolation for the determination of double bond position in unsaturated fatty acid esters. Chem. Phys. Lipids, 29, 369-374 (1981).
6. Vincenti, M., Guglieilmetti, G., Cassani, G. and Tonini, C. Determination of double bond position in diunsaturated compounds by mass spectrometry of dimethyl disulphide derivatives. Anal. Chem., 59, 694-699 (1987).
7. Yamamoto, K., Shibahara, A., Nakayama,T. and Kajimoto,G. Determination of double bond positions in methylene-interrupted dienoic fatty acids by GC-MS as their dimethyl disulphide adducts. Chem. Phys. Lipids, 60, 39-50 (1991).

This article was first published in Lipid Technology, 8, 18-20 (1996) and has now been substantially re-written.

The second part of this beginner's guide to mass spectrometry of fatty acids can be accessed here..

Or, go to our main mass spectrometry pages here..

Updated: 1/5/2007 Credits/disclaimer	Scottish Crop Research Institute (and MRS Lipid Analysis Unit), Invergowrie, Dundee (DD2 5DA), Scotland