The Lipid Library

MASS SPECTRA OF OXYGENATED FATTY ACIDS

Part 1A. 2- to 8-Hydroxy Acids

In this webpage, mass spectra are arranged and discussed according to the positions of hydroxyl groups in the fatty acyl chains. Spectra of methyl esters, picolinyl esters, DMOX derivatives and pyrrolidides are all described here (when available). None of these have unique advantages, and there are too many gaps in this account to discuss which is best in any systematic way. However, it does appear that methyl ester derivatives are perfectly satisfactory for saturated fatty acids at least (the definitive paper is by Ryhage and Stenhagen (1960)). Preparation of the trimethylsilyl (TMS) ether derivatives of the hydroxyl groups can be helpful (see Nicolaides et al. (1983)). Pyrrolidides are a good choice for 2- and 3-hydroxy isomers, especially.

The choice of examples in this section may appear rather eccentric. As with my other documents on mass spectrometry, this is a subjective account that details only those oxygenated fatty acids encountered during our research activities here and for which we have spectra available for illustration purposes. However, I trust that we have a sufficiently wide range of spectra to give the flavour of the topic. My colleagues and I have never worked with eicosanoids, so no spectra of such compounds can be discussed here. Many of the spectra have not been published elsewhere, but I cite references to previous publications when these are known.

A common feature of mass spectra of hydroxy acids is fragmentation leading to the loss of the elements of water, especially corresponding to [M-17]⁺ or [M-18]⁺.

2-Hydroxy Fatty Acids

2-Hydroxy long-chain saturated fatty acids occur frequently in sphingolipids of plant and animal origin. The mass spectrum of methyl 2-hydroxy-palmitate is -

The ion at m/z = 90 may be the McLafferty rearrangement ion (cleavage between C2 and C3), and that at m/z = 227 ([M-59]⁺) may represent expulsion of the three-carbon fragment comprising C2 to C4 (Ryhage and Stenhagen, 1960). Analogous features are present in the spectra of methyl esters of saturated or monoenoic 2-hydroxy-fatty acids with 10 to 27 carbon atoms (not illustrated here, but see the appropriate Archive section of this website).

Trimethylsilyl ethers of hydroxy esters are often prepared to reduce the polarity of the compounds and facilitate GC analysis. The mass spectrum of methyl 2-hydroxydocosanoate, after further derivatization to the trimethylsilyl ether, follows -

The molecular ion is of low abundance, and this is followed by an ion at m/z = 427, representing loss of a methyl group from the trimethylsilyl ether moiety. The base peak at m/z = 383 ([M-59]⁺) may be the result of cleavage between carbons 1 and 2 (see also Capella et al., 1968). Again, we have spectra of a number of related homologues on file that show similar features.

We have been able to obtain poor yields only of the picolinyl ester, and the spectrum of picolinyl 2-hydroxy-octadecanoate is -

The relative proportions of ions in the high mass region are low. Following the molecular ion, there is a gap of 17 amu for loss of a hydroxyl group, but successive ions thereafter are 14 amu apart for cleavage at each methylene group. The McLafferty ion has moved from m/z = 151 to 167, and must contain the oxygen on carbon 2, while the usual ion at m/z = 164 has moved to m/z = 180.

After several attempts, we have not succeeded in preparing DMOX derivatives from saturated 2-hydroxy acids.

On the other hand, the pyrrolide derivatives were easy to prepare from the methyl esters of 2-hydroxy-fatty acids, and they can be recommended for structural analysis purposes. Tulloch (1985) has described the mass spectra of the pyrrolidides of the complete series of hydroxy-octadecanoates and their TMS ethers. The pyrrolidide of 2-hydroxy hexadecanoate has the distinctive mass spectrum illustrated next -

The key ion is the base peak at m/z = 129, which reflects cleavage between carbons 2 and 3 adjacent to the carbon with the hydroxyl group. Other useful diagnostic ions are those at m/z = 142, 152, 171 and 184. In the high molecular weight region, the last significant ion before the molecular ion is at m/z = 308, representing a loss of 17 amu or the hydroxyl moiety. Analogous features are present in the spectra of pyrrolidides of saturated 2-hydroxy-fatty acids with 12 to 26 carbon atoms (not illustrated here, but see the appropriate Archive pages on this website).

The mass spectrum of the pyrrolidide of 16-methyl-2-hydroxy-heptadecanoate -

This fatty acid is present in wool wax and its mass spectrum has not been published elsewhere. It differs significantly from the previous (apart from the molecular weight) mainly in that the last significant ion before the molecular ion is at m/z = 338, representing a loss of 15 (rather than 17) amu for the terminal methyl group, presumably. A gap for the loss of the iso-methyl branch is apparent.

Monoenoic 2-hydroxy-long-chain fatty acids are also found in ceramides, and the mass spectrum of methyl 2-hydroxy-tetracos-9-enoate is -

The spectrum is not very different from that of the corresponding saturated fatty acid. The 2-hydroxyl group is recognized by the ion at m/z = 90, but there is nothing to indicate the position of the double bond. Preparation of the TMS ether derivative adds little in mass spectrometric, as opposed to chromatographic, terms. We have no spectra of picolinyl ester, DMOX or pyrrolidide derivatives for comparison purpose, but I would expect the last of these to be the most useful.

2-Hydroxy-octadeca-9,12,15-trienoic acid is a minor component of the seed oil of Thymus vulgaris, but the spectrum of its methyl ester is not very informative in terms of the position of the hydroxyl group, although the ion at m/z = 108 is a reminder that it is an (n-3) fatty acid.

Although, we failed with saturated 2-hydroxy acids, we were able to prepare the DMOX derivatives of 2-hydroxy-octadeca-9,12,15-trienoate, which gave a particularly distinctive mass spectrum -

The ion at m/z = 142 locates the hydroxyl group definitively, while the double bonds are best located using the approach favoured for picolinyl esters, i.e. to look for the gaps of 26 amu for each of the double bonds as illustrated. The pyrrolidide has a similar spectrum, except that the ions in the high mass range are much less abundant.

3-Hydroxy Fatty Acids

3-Hydroxy acids are found in certain microbial lipids (rhamnolipids), avian waxes and occasionally in animal tissues, sometimes as by-products of β-oxidation The mass spectrum of methyl 3-hydroxy-tetradecanoate (from a commercial standard) is -

The molecular ion (m/z = 258) can only be seen by magnifying the appropriate part of the spectrum, and identification of homologues can be difficult without access to authentic spectra. However, the base peak at m/z = 103 is produced by a characteristic cleavage alpha to the carbon with the hydroxyl group and defines its position (Ryhage and Stenhagen, 1960).

The TMS ether derivative prepared from the methyl ester has a poor molecular ion also, but the base peak at m/z = 315 is equivalent to [M-15]⁺, enabling determination of chain-length. The hydroxyl group is located by the ion at m/z = 175.

Once more, after several attempts, we did not succeeded in preparing either picolinyl esters or DMOX derivatives from 3-hydroxy acids. 

The pyrrolide derivative is easy to prepare from the methyl ester, and 3-hydroxy-tetradecanoate has the distinctive spectrum illustrated -

The characteristic ion is that at m/z = 142, representing cleavage between carbon atoms 3 and 4, as shown (Tulloch, 1985).

5-Hydroxy Fatty Acids

4- and 5-Hydroxy acids tend to form lactones spontaneously, i.e. γ- and δ-lactones, respectively, and they are most often encountered in this form. They are important flavour components of dairy products.

The δ-lactone from 5-hydroxy-tetradecanoate, present in cow's milk, has the mass spectrum -

The molecular ion is not discernable, but there is a distinctive fragmentation at the lactone ring (the base ion).

6-Hydroxy Fatty Acids

When the hydroxy group is in a more central part of the molecule, the main fragmentation is still beta to the oxygen atom or alpha to the carbon to which it is linked, although an ion that reflects further fragmentation by the loss of the elements of methanol (32 amu less) may be even more abundant. The molecular ion is not easily detected, and ions equivalent to [M-31]⁺ or [M-17]⁺ must be used to determine the molecular weight. Thus, in the spectrum of methyl 6-hydroxy-tetradecanoate -

- the key fragmentation ion is at m/z = 145, while that at m/z = 113 is formed from this by further fragmentation as illustrated.

8-Hydroxy Fatty Acids

8-Hydroxy-hexadecanoic acid is also a minor component of milk fat and its methyl ester exhibits similar features to the previous, except that the key diagnostic ions are shifted upwards by 28 amu as expected.

Thus, the key fragmentation ion is at m/z = 173, while that at m/z = 141 is formed from this by further fragmentation with the loss of methanol. In addition, the ion at m/z = 144 is formed by a similar cleavage alpha to the carbon carrying the hydroxyl group, following protonation (as is the ion at m/z = 116 in the previous spectrum).

Spectra of further hydroxy fatty acids are available, but without interpretation, in the Archive Sections of these web pages, i.e. for methyl esters -- picolinyl esters -- DMOX derivatives -- pyrrolidides.

References

• Capella, P., Galli, C. and Fumagalli, R. Hydroxy fatty acids from cerebrosides of the central nervous system: gas-liquid chromatography determination and mass spectrometry identification. Lipids, 3, 431-438 (1968).
• Nicolaides, N., Soukup, V.G. and Ruth, E.C. Mass spectrometry fragmentation patterns of the acetoxy and trimethylsilyl derivatives of all the positional isomers of the methyl hydroxypalmitates. Biomed. Mass Spectrom., 10, 441-449 (1983).
• Ryhage, R. and Stenhagen, E. Mass spectrometric studies. VI. Methyl esters of normal chain oxo-, hydroxy-, methoxy- and epoxy-acids. Arkiv Kemi, 15, 545-574 (1960).
• Tulloch, A.P. Mass spectrometry of pyrrolidides of oxo, hydroxy and trimethylsilyloxy octadecanoic acids. Lipids, 20, 652-663 (1985).

Updated: 16/5/2008 Credits/disclaimer	Scottish Crop Research Institute (and MRS Lipid Analysis Unit), Invergowrie, Dundee (DD2 5DA), Scotland