MASS SPECTRA OF SOME MISCELLANEOUS LIPOPHILIC COMPONENTS
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During the analysis of natural fatty acid samples, various less common lipids may be encountered. They may be present naturally in the samples or they can be formed as artefacts, or indeed by mistake if a derivatization reaction is carried out incorrectly. For example, sterols can interfere in GC traces if not removed from samples of fatty acid esters, and it can be helpful to have a spectrum of cholesterol available as a check. Of course, analysis of sterols per se is an important task outwith our general research interests. The following have cropped up during our work. Most of these spectra are offered for comparison or record purposes, and I have added cursory descriptions only. You may find some further relevant information in our pages on artefacts and additives.
Free Fatty Acids
Free (unesterified) fatty acids are rarely analysed as such by GC-MS, but they have distinctive mass spectra and the following are illustrated as examples. I presume that they will have been published elsewhere in the literature by someone somewhere, but I have only been able to find a paper dealing with short-chain fatty acids. Brief comments only are offered on interpretation.
Mass spectra of saturated fatty acids are especially interesting and that of palmitic acid is -
The most abundant peaks are at m/z = 60, the McLafferty rearrangement ion, and 73 in the lower molecular weight range, but the molecular ion is clearly abundant, and there are ions representing fragmentations between methylene groups of the form [HOOC(CH2)n]+ from m/z = 115 to 255. An ion at m/z = 239 ([M-17]+) presumably reflects a loss of OH- from the carboxyl group.
The mass spectrum of stearic acid -
As might be expected the spectra of unsaturated fatty acids are rather different, with hydrocarbon ions (general formula [CnH2n-1]+]) being most abundant, and that of oleic acid is -
Ions in the low mass range predominate, and as expected, there is nothing that might serve to locate the double bond. In contrast to saturated fatty acids, the ion representing the loss of the elements of water from the carboxyl group ([M-18]+, m/z = 264) is more abundant than the molecular ion. The McLafferty ion at m/z = 60 is relatively small. How far this is typical of monounsaturated acids is unclear, as the mass spectrum of petroselinic acid (6-18:1) is different, at least in terms of the relative abundances of particular ions -
Ions in the high mass range are more abundant in this spectrum. The spectrum of linoleic acid is also dominated by ions in the low mass range. In this instance, the molecular ion is more abundant than any representing the loss of elements of water from the carboxyl group.
Spectra of many more free fatty acids are available in our Archive pages, but without interpretation.
Dimethylacetals of Aliphatic Aldehydes
When the plasmalogen forms of phospholipids, common in animal tissues and in some microorganisms (but not plants), are treated with acidic transesterification reagents, the vinyl ether bond is broken and aldehydes are generated which are immediately converted to dimethyl acetals. These are almost exclusively saturated and monounsaturated (C16 and C18 in chain length), and they tend to elute just before 16:0 and 18:0 methyl esters on most GC phases. The mass spectra of the three common isomers follow. |
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First the dimethyl acetal of hexadecan-1-al -
The spectrum is not at all exciting. The base peak is the McLafferty rearrangement ion at m/z = 75, but the molecular ion can only be seen if this region of the spectrum is greatly magnified. The first significant ion in the high mass range, at m/z = 255, represents the loss of a methoxyl ion.
Mass spectrum of the dimethylacetal of octadecan-1-al -
Mass spectrum of the dimethylacetal of octadec-9-en-1-al -
No further comment seems necessary.
Fatty Acid Ethyl Esters
Fatty acid ethyl esters can be found naturally in animal tissues at very low levels in certain circumstances, and they can be a useful marker for excessive alcohol consumption in humans. I have encountered them as artefacts when methylating reagents have been accidentally contaminated with ethanol, and I am aware that this has happened to others. In addition, purified fatty acid preparations intended for pharmaceutical applications are often converted to ethyl esters prior to administration or encapsulation. The mass spectrum of ethyl palmitate is -
The spectrum resembles that of the methyl ester, except that the McLafferty ion is at m/z = 88 instead of 74. In the high mass range, following the molecular ion, there is an ion at m/z = 255 for loss of the ethyl group, then one at m/z = 241 from a rearrangement reaction involving expulsion of a three-carbon fragment (C2 to C4). An ion at m/z = 239 adjacent to this represents loss of the ethoxide ion. Similar features are seen in the spectra that follow as further examples. As with other simple esters, the McLafferty ion becomes less abundant with increasing unsaturation.
Mass spectrum of ethyl oleate -
In this instance, the ion for less of the ethyl group (m/z = 282) is much less abundant than that for the loss of the ethoxide moiety (m/z = 264).
Mass spectrum of ethyl linoleate -
Mass spectrum of ethyl 5,8,11,14,17-eicosapentaenoate (20:5(n-3) or 'EPA') -
Note that the ion at m/z = 108, typical of fatty acids of the (n-3) family in the spectra of methyl esters, is independent of the nature of the alcohol moiety so is also prominent in this spectrum.
Mass spectra of many more ethyl esters are available in our Archive pages, but without interpretation.
n-Propyl, i-Propyl and n-Butyl Esters
Occasionally, it may be necessary to prepare esters other than methyl for specific purposes, and some examples follow. Propan-2-ol esters may have a slight advantage over methyl esters in mass spectrometric and chromatographic terms as regards geometric isomers and highly unsaturated fatty acids (see our webpage on alternatives to methyl esters).
Mass spectrum of n-propyl hexadecanoate (16:0) -
The ion at m/z = 239 ([M-59]+) reflects the loss of a propyloxy ion, while that at m/z = 257 ([M-41]+) is presumably due to the loss of a propenyl moiety. The McLafferty ion is at m/z = 102.
Mass spectrum of n-propyl 9,12-octadecadienoate (linoleate) -
As in the spectrum of methyl linoleate, hydrocarbon ions predominate. The ion representing the loss of the propyloxy (m/z = 263) ion is prominent, but in contrast to the previous spectrum that for loss of a propenyl ion (m/z = 279) is not.
Fatty acid esters of iso-propanol (propan-2-ol) have excellent gas chromatographic properties, and for example they elute appreciable before most higher aliphatic esters in general and the analogous n-propyl esters in particular. They have been recommended for the separation of positional and geometrical isomers of mono- and polyenoic fatty acids (c.f. Wolff, R.L. J. Chromatogr. Sci., 30, 17-22 (1992) and an article in our Topics pages).
Mass spectrum of i-propyl hexadecanoate (16:0) -
The spectrum is somewhat different from that of the corresponding n-propyl derivative, though mainly in the relative abundances of ions. The base peak is at m/z = 256 for loss of a propenyl moiety (m/z = 256 ([M-42]+ rather than ([M-41]+).
Similarly, the mass spectrum of i-propyl 9,12-octadecadienoate (linoleate) differs in minor ways only from that of the n-propyl ester above -
On the other hand, the ion for loss of a propyl unit (now at [M-43]+ or m/z = 327) is apparent even in the spectrum of i-propyl docosahexaenoate, so that the molecular weight can at least be determined.
In comparison, it is often difficult to determine the molecular weight of methyl esters of polyunsaturated fatty acids. As isopropyl esters are reputed to have excellent GC properties, it might be of value to study them further.
Butyl esters are sometimes favoured for the analysis of short-chain fatty acids such as those in milk fat. The mass spectrum of n-butyl hexadecanoate (16:0) is -
By analogy with the previous spectra, the ion at m/z = 239 ([M-73]+) reflects the loss of a butyloxy ion, while that at m/z = 257 ([M-55]+) is presumably due to the loss of a butenyl moiety. The McLafferty ion is at m/z = 116.
Similarly, the mass spectrum of n-butyl 9,12-octadecadienoate (linoleate) has comparable features to that of the n-propyl ester above -
In this instance, the first significant ion in the high mass region at m/z = 279 ([M-57]+)represents loss of a butyl rather than a butenyl moiety. Mass spectra from many more n-propyl, i-propyl and n-butyl esters are available in our Archive pages, but without interpretation.
Cholesterol
Cholesterol is by far the most abundant sterol in animal tissues. It tends to elute from GC columns long after the methyl ester derivatives, but it does elute eventually as a broad hump in chromatograms - disrupting subsequent analyses of the latter. In practice, it is best eliminated by adsorption chromatography before methyl esters or other derivatives are analysed by GC. Sterols per se are best analysed separately on non-polar phases either in the free form or better as the trimethylsilyl ethers.
The mass spectrum of free cholesterol -
- and that of its trimethylsilyl ether derivative -
Detailed interpretation is a matter for sterol specialists, and the spectra are offered here simply for record purposes. There are many more spectra of sterols from animal tissues, plants and yeasts in our Archive pages, again without interpretation.
Squalene
The hydrocarbon squalene tends to be a very minor component of animal tissues, where it is the biosynthetic precursor of sterols. However, it can be a major constituent of fats and oils of marine origin on occasion, for example in shark oils. Its mass spectrum follows, but no attempt is made at detailed interpretation.
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Updated: 30/8/2007 |
Scottish Crop Research Institute (and MRS Lipid Analysis Unit), Invergowrie, Dundee (DD2 5DA), Scotland
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