A Beginner's Guide to Mass Spectrometry of Fatty Acids
Part 1. Methyl Esters
At its simplest, mass spectrometry (MS) is a technique in which organic molecules are bombarded by electrons or other ionic species causing them to ionize and fragment for separation by a magnetic field. We use the fragmentation patterns as a means of identification. 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 "electrospray ionization", "chemical ionization", "fast-atom bombardment" and others, but in the more basic mass spectrometers linked to gas chromatography (GC-MS), electron impact ionization is encountered most often. It is the one most relevant to this specific topic and is the only one considered here. The various ionic species produced from a given organic compound by electron impact ionization are separated according to mass (strictly speaking mass/charge (m/z) ratio in which z = 1) in a magnetic field in the mass spectrometer, 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 ion), which is usually normalized to 100% for record, comparison and publication purposes.
In this and the next article, I discuss the basic principles only of identification of fatty acid structures by mass spectrometry. More detailed information with many more spectra illustrated is available in the other articles in the mass spectrometry pages of this website, and two review articles [1,2] or a book [3] can be recommended.
Jigsaw Puzzles and Bricks
Interpretation
of a mass spectrum is often compared to doing a jigsaw puzzle.
We have many different pieces or fragments, and we must try to put them together in a sensible way
to find the picture or to 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 more 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 in the aliphatic chain.
Methyl esters are the derivative of choice in most applications to the analysis of fatty acids, but when we subject them to electron impact ionization in a mass spectrometer, the charged aliphatic chain breaks up into so many fragments that a branch-point or other structural feature cannot be identified easily. A double bond can carry the charge and become mobile; it moves up and down the chain, so that its original position cannot be determined. This is especially troublesome with mono- and dienoic fatty acids. Using the above analogy, we have taken a sledgehammer to break up the molecule. This is something of an exaggeration, as we can usually settle on a restricted number of possible structures from mass spectra of methyl esters, although we may not obtain a definitive answer. An important detail is the molecular weight of the fatty acid ester, which is obtained from the molecular ion as this tells us the number of carbon, hydrogen and oxygen atoms. We therefore know if the fatty acid is saturated or unsaturated, and often an experienced eye can tell from close examination of the spectrum if there is a branch point or other substituent and where that substituent is located in the aliphatic chain. It helps that there are many authentic mass spectra of methyl esters available in the scientific literature for comparison purposes, not least in this website here...
Interpretation of the mass spectrum of a simple saturated isomer such as methyl palmitate is straight-forward (Figure 1). There is a clear molecular ion at the end of the high mass range at m/z = 270. The ion at m/z = 239 represents a loss of 31 mass units (CH3O) as shown, and this confirms that we are dealing with a methyl ester, while the ion at m/z = 74 is also diagnostic for a methyl ester and is most abundant with saturated fatty acids. The latter is the so-called 'McLafferty ion' and is displayed as a simple cleavage here, although in reality it is formed by a rearrangement involving the ester moiety (more detail here..). The long homologous series of related ions (14 amu apart) at m/z = 87, 101, 115, 129, 143, 157, 199, etc. of general formula [CH3OCO(CH2)n]+ is evidence that there are unlikely to be other functional groups in the chain. Unfortunately, the spectrum of a comparable branched chain derivative can look very similar to this, although there can be minor but significant differences.
The mass spectrum of methyl oleate is illustrated next (Figure 2). The molecular ion is at m/z = 296, i.e., two less than for methyl stearate (or -2H). We usually lose one or two 'bricks' neatly, and an ion representing loss of 32 mass units (methanol from the ester moiety) and another of 74 units (both m/z = 74 per se and [M‑74]+ at m/z = 222) confirm that we really do have a methyl ester, although there are no ions that help us to determine the position or configuration (cis/trans) of the double bond.
Strictly speaking, as the molecular weight is two less than for a fully saturated fatty acid derivative, we could have either a double bond or more rarely a cyclic structure present, but the correct assignment is usually possible from close examination relative to standards or from simple experiments, for example by attempting hydrogenation. The spectrum is 'busier' than for the methyl ester of a saturated fatty acid, i.e., there are ion clusters rather than relatively solitary ions.
Further invaluable information that we should have as an aid to identification is the retention time relative to standards of the fatty acid methyl ester (or equivalent chain-length value) on the GC column of the GC-MS system. This together with the molecular weight and mass spectrometry data will usually suggest that some structures are more plausible than others.
Double Bond Location in Polyunsaturated Fatty Acid Methyl Esters
We usually 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. Although we cannot do this directly with monoenes and dienes, it is fortunate that with the conventional series of polyunsaturated fatty acids, i.e., with three or more methylene-interrupted double bonds, we have some useful characteristic ions of diagnostic value. These can be seen in the mass spectrum of methyl 6,9,12‑octadecatrienoate (γ-linolenate) illustrated in Figure 3.
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, while the ion at m/z = 74 again confirms that we do indeed have a methyl ester. These ions tend to be much smaller than in spectra from more saturated esters. Ions from the hydrocarbon part of the molecule of general formula [CnH2n‑5]+ tend to dominate the spectrum with that at m/z = 79 as the base ion, 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 methyl esters of all polyunsaturated fatty acids from the n‑6 biosynthetic family [4]. To completes the identification, there is a fragment (‘alpha’ ion) from the carboxyl end of the molecule at m/z = 194, although the position of this ion will vary according to the position of the first double bond in fatty acids of the n-6 family. The latter ion was best defined in a paper by Brauner and coworkers [5] (more details here...).
In mass spectra of methyl esters of fatty acids of the n-3 family, the omega ion stands out at m/z = 108 (while that at m/z = 150 is negligible), and for the minor (n-9) and (n-4) biosynthetic families of fatty acids, the relevant ions are at m/z = 192 and 122, respectively. It should be stressed that these ions are valuable but not infallible guides, and they are not reliable when the double bonds are not methylene interrupted.
That said, it is surprising how often mass spectra from methyl esters of different polyunsaturated fatty acids are sufficiently distinctive to be considered as ‘fingerprints’, which can be identified by comparison with the spectra of standards. Unfortunately, when there are five or more double bonds, the molecular ion can be of low abundance and difficult to identify.
Chemical Derivatization for Double Bond Location
If we want to have definitive information on double bond positions, we must 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 is the topic of the Part 2 of my "Beginner’s Guide". Alternatively, with monoenoic fatty acids, 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 were described, usually involving oxygenation followed by further derivatization, but most of these fell by the wayside when dimethyl disulfide adducts were described [6]. This is a simple single-step (if odoriferous) reaction involving reaction of dimethyl disulfide with an unsaturated ester in the presence of iodine as catalyst. The nature of the reaction is shown in Figure 4.
With methyl oleate as illustrated, 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 substantial 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 (the full spectrum is available here..). This technique has now been applied to identify many monoenoic fatty acids (and other aliphatic compounds) in natural samples in numerous laboratories, and new applications continue to appear in the scientific literature. (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, although the reaction can be used usefully even with methylene-interrupted dienes, such as linoleic acid, if the reaction is carried out under mild conditions. 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 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, as discussed in Part 2.
Be Wary of Computerized Identification Techniques!
Most GC-MS systems are sold with software that enables comparison of spectra of unknowns with a library of spectra of standards, and this can indeed be very useful. That said, beginners should be very careful of how this facility is used for many reasons. While the library may contain 100,000 spectra or more, this is still a small number in comparison to the number of organic compounds in general and fatty acid derivatives in particular that might be encountered. Then, the identification is made from the intensities of a relatively small number of ions, and there can be some variability depending on such factors as the make and model of the instrument and its condition/age. A comparison with the spectrum of methyl oleate, for example, may suggest many possible positional or configurational isomers, all with a confidence of 99%. The computer does not recognize that isomeric methyl octadecenoates of this kind have spectra that are identical for all practical purposes. Analysts should have a sufficient understanding of the principles of mass spectrometry to know when to ignore the computer and make informed decisions.
Quantification
I do not discuss quantification by GC-MS in these pages, mainly because I believe that GC with flame-ionization detection and quantification is robust methodology with an appreciable linear range and with a relatively simple requirement for standards [3]. While GC-MS can be used for the purpose, it requires careful calibration with a wide range of standards [8]. I view quantification and structure determination as separate tasks.
Liquid Chromatography-Mass Spectrometry (LC-MS)
LC-MS with electrospray ionization and other soft ionization methods is being used increasingly for determination of fatty acid compositions with derivatives of various kinds, including methyl esters. In my opinion, which is given with the caveat that I have no direct practical experience, it offers no advantages over GC-MS (when this is available) for analysis of the common types of fatty acid that are likely to be encountered in animal and plant tissues. However, it is undoubtedly the best approach for many oxylipins, which are relatively unstable chemically.
References
- 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).
- Christie, W.W. Gas chromatography-mass spectrometry methods for structural analysis of fatty acids. Lipids, 33, 343-353 (1998); DOI.
- Christie, W.W. and Han, X. Lipid Analysis - Isolation, Separation, Identification and Lipidomic Analysis (4th edition), 446 pages (Oily Press, Woodhead Publishing and now Elsevier) (2010) - see Science Direct.
- 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); DOI.
- 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); DOI.
- Francis, G.W. Alkylthiolation for the determination of double bond position in unsaturated fatty acid esters. Chem. Phys. Lipids, 29, 369-374 (1981); DOI.
- 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 disulfide adducts. Chem. Phys. Lipids, 60, 39-50 (1991); DOI.
- Quehenberger, O., Armando, A.M. and Dennis, E.A. High sensitivity quantitative lipidomics analysis of fatty acids in biological samples by gas chromatography-mass spectrometry. Biochim. Biophys. Acta, 1811, 648-656 (2011); DOI.
A pdf 
file of the two parts of this beginner's
guide is available here..
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