Mass Spectrometry of Methyl Esters
Hydroxy Fatty Acids - Trimethylsilyl Ether Derivatives
In this document,
mass spectra (electron-impact ionization) of methyl esters of hydroxy fatty acids in the form
of the trimethylsilyl (TMS) ether derivatives are discussed
in relation to the positions of hydroxyl groups in the alkyl chains in fatty acids of varying degrees of unsaturation.
Preparation of these derivatives can be helpful both in terms of improved chromatographic resolution and of interpretation of mass spectra
(see Nicolaides et al., 1983);
a suitable laboratory protocol for their preparation is described here....
Spectra of 3‑pyridylcarbinol esters and
DMOX derivatives together with pyrrolidides are described in separate documents.
A separate web page (Part 1) deals with mass spectra of methyl esters of hydroxy acids
without further derivatization, and as there, only those hydroxy fatty acids encountered during our research activities
and for which we have spectra available for illustrative purposes can be described here (no eicosanoids or related oxylipins unfortunately).
Many of the spectra have not been published elsewhere, but I cite references to previous publications when these are known to me.
Methyl esters of hydroxy acids can be analysed by GC-MS as their tert.-butyldimethylsilyl ether derivatives in complex mixtures
(Chance et al., 1998), but we have no spectra available for illustrative purposes.
This is a practical as opposed to a mechanistic tutorial.
There is a web page dealing with the occurrence, chemistry and biochemistry of hydroxy acids on this site
here...
2- and 3-Hydroxy Fatty Acids
The mass spectrum of methyl 2-hydroxydocosanoate after further derivatization to the trimethylsilyl ether is -
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 (TMS) moiety. The base ion at m/z = 383 ([M‑59]+) is presumably the result of cleavage between carbons 1 and 2 (see Capella et al., 1968) and serves to locate the hydroxyl group.
The spectra of TMS derivatives of methyl esters of monoenoic 2-hydroxy-long-chain fatty acids, which are also found in sphingolipids, in the form of the TMS ethers display similar diagnostic ions to the saturated analogues, except that ions in the low mass range are much more abundant relative to those in the high mass range, although there is a distinct molecular ion at least. There are no ions that serve to locate the double bond, of course. For example, the spectrum of the TMS derivative of methyl 2-hydroxy-tetracos-15-enoate is -
The TMS ether derivative prepared from methyl 3-hydroxy-tetradecanoate, like the underivatized compound, has a poor molecular ion, but the base ion 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, while that at m/z = 73 is the trimethylsilyl moiety.
Pyrrolidides are an alternative choice for 2- and 3-hydroxy isomers, but we have not been able to prepare 3‑pyridylcarbinol ester or DMOX derivatives of the common 2- or 3-hydroxy acids for comparison purposes (for which we have no explanation).
9- to 15-Hydroxy Fatty Acids
9-Hydroxy-heptadecanoic acid is a minor component of bovine milk fat and its methyl ester as the TMS ether has the following spectrum.
There
is an insignificant molecular ion, and we have to use the [M‑15]+ ion to confirm the molecular weight.
However, the ions that locate the derivatized hydroxyl groups for fragmentation on either side of carbon-9
at m/z = 215 and 259 could not be more distinctive.
The ion at m/z = 230 is formed by a complex rearrangement involving transfer
of the TMS group to the carboxyl group (as illustrated).
Similarly, a small ion at m/z = 146 in the spectra of trimethylsilyl ethers of methyl esters of this and other hydroxy acids
is formed by a transfer of the trimethylsilyl group to the McLafferty ion (Kleiman and Spencer, 1973).
9-Hydroxy-octadec-12-enoic acid is present in the seed oil of Nerium oleander and the mass spectrum of the methyl ester derivative after conversion to the trimethylsilyl ether derivative has been published by Kleiman and Spencer (1973). Their paper is still invaluable both as a practical and mechanistic guide to mass spectra of TMS ether derivatives of unsaturated fatty acids, and I am indebted to them for some of the interpretations of mass spectra that follow. The intensities of the various ions are dependent on the relative positions of the hydroxyl group and the double bonds.
A small molecular ion together with ions reflecting the consecutive loss of the methyl (m/z = 369) and methoxyl (m/z = 337) groups are again apparent, while the ions for fragmentation on either side of the carbon linked to the TMSO group at m/z = 227 and 259 are easily distinguished. The ion at m/z = 294 or [M‑90]+ represents the loss of HOSi(CH3)3.
10-Hydroxy-octadecanoic acid is produced by several microorganisms and is a minor component of cow's milk fat, which was the origin of the spectrum of the trimethylsilyl ether derivative of methyl 10-hydroxy-octadecanoate illustrated here. It gives a distinctive and relatively simple fragmentation, with the two main ions formed by cleavage on both sides alpha to the carbon carrying the TMS group standing out clearly. The ion at m/z = 244 is that involving transfer of the TMS group to the carboxyl group as described for the 9-isomer.
12-Hydroxy-octadec-9-enoic (ricinoleic) acid is the main component of castor oil (from Ricinus communis), which is a major agricultural and industrial product. The trimethylsilyl ether derivative of the methyl ester has a highly characteristic spectrum (Kleiman and Spencer, 1973) -
The ions at m/z = 187 and 299 represent cleavage on either side of the hydroxyl group. That at m/z = 270 is formed by the complex rearrangement involving transfer of the TMS group to the carboxyl group as described above. We have the spectrum of the TMS ether derivative of 14-hydroxy-eicos-11-enoic (lesquerolic acid), the C20 analogue of ricinoleic acid, and that of the C22 homologue in our Archive pages, and these are useful for comparison purposes.
The next three spectra were obtained from minor components of cow's milk fat that differ in chain-length as well as the position of the hydroxyl groups.
Methyl 13-hydroxy-octadecanoate - TMS ether derivative -
Fragments adjacent to the TMS ether are as expected, while the ions formed by the complex rearrangement involving transfer of the TMS group to the carboxyl group stand out because they are even numbered. Note the increase in the abundance of the ion from the distal portion of the molecule relative to that from the carboxyl end. The spectra of the next two isomers are illustrated without further comment.
Methyl 14-hydroxy-hexadecanoate - TMS ether derivative -
Methyl 15-hydroxy-octadecanoate - TMS ether derivative -
15-Hydroxy-octadeca-9,12-dienoic acid (15-hydroxy-linoleate or 'avenoleate') is found in the monogalactosyldiacylglycerol fraction of oat seed lipids. In nature, the hydroxyl group is esterified with a further molecule of linoleate, i.e., it is an estolide. The trimethylsilyl ether derivative of methyl 15-hydroxy-linoleate has a rather distinctive spectrum (see Hamberg and Hamberg, 1996).
The molecular ion (m/z = 382) can just be detected, but the outstanding feature is the base ion at m/z = 145 which represents cleavage between carbons 14 and 15, i.e., the terminal part of the molecule, but the ion from the carboxyl end of the molecule (m/z = 339) is just detectable. The ion at m/z = 310 is presumably one of the complex rearrangement ions discussed earlier.
(ω-1)- and ω-Hydroxy Fatty Acids
In animal tissues, a family of enzymes termed cytochromes P450s are involved in fatty acid oxidation, hydroxylating with high specificity at the energetically unfavourable terminal (omega) or omega-1 carbons. Comparable fatty acids are found in waxes and plant cutins, which were the source of the spectra that follow. There are striking differences between the spectra from methyl esters of these two positional isomers in the form of the TMS ether derivatives.
As example of an (ω-1)-hydroxy derivative, the spectrum of the TMS ether of methyl 15-hydroxyhexadecanoate is -
The molecular ion is not easily discerned, but there are ions for successive loss of the methyl and methoxyl groups, i.e., at m/z = 343, 327 and 311. The distinctive feature is the base ion at m/z = 117, which may simply represent an alpha cleavage although other rearrangements are possible. The ion at m/z = 146 may be the McLafferty ion with the TMSO group attached as mentioned earlier. This ion is also present in the spectrum of the C18 analogue, as is the ion representing [M‑44]+ at m/z = 314 in this instance (see here... and Nicolaides et al., 1983).
On the other hand, the trimethylsilyl ether of methyl 16-hydroxyhexadecanoate (an ω-hydroxy acid) has an equally simple but very different mass spectrum. The molecular ion is not seen, and the first significant ion at m/z = 343, represent the loss of a methyl group from the TMS moiety. The base ion at m/z = 311 is for a further loss of the methoxyl group from the carboxyl end of the molecule (see Nicolaides et al., 1983). There is no ion in the low mass region equivalent to that which so dominated the previous spectrum (at m/z = 117).
When there is a double bond in the molecule, as in the spectrum of the TMS ether of methyl 18-hydroxy-octadec-9-enoate, there is now a respectable molecular ion, followed by ions for the loss of successive methyl and methoxyl groups. Ions in the lower mass range are now much more abundant.
Among many relevant publications, mass spectra of oxidized fatty acids from seed oils by both electron impact ionization and positive chemical ionization have been described (Xia and Budge, 2018).
Di- and Polyhydroxy Acids
Di- and trihydroxy acids are common constituents of plant cutins. Because of their high polarity, they are best analysed by gas chromatography in the form of the trimethylsilyl ether derivatives. The mass spectrum of the TMS ether derivative of methyl 9,10,18‑trihydroxy-octadecanoate is illustrated where a simple cleavage between the vicinal hydroxyl groups is clearly seen. The ion at m/z = 332 may be produced by a rearrangement involving migration of the TMSO group to the carboxyl moiety.
Hydroxylation of double bonds followed by preparation of TMS ethers has been used as a means of structural identification of unsaturated fatty acids (Argoudelis and Perkins, 1968; Capella and Zorzut, 1968), although the spectrum of the TMS ether derivative of methyl 9,10-dihydroxy-octadecanoate illustrated in the Archive was of a minor natural component of castor oil.
Only a few of the spectra we have on file can be illustrated here. They are representative of a large number of isomers and homologues of methyl esters of hydroxy acids, available for consultation but without interpretation in our Archive pages. There are a few spectra of acetate derivatives here, but they are of little value for characterization purposes.
Part 1 of this topic deals with mass spectra of methyl esters of hydroxy acids without further derivatization.
References
- Argoudelis, C.J. and Perkins, E.G. Determination of double bond position in mono-unsaturated fatty acids using combination gas chromatography mass spectrometry. Lipids, 3, 379-381 (1968); DOI.
- Capella, P. and Zorzut, C.M. Determination of double bond position in monounsaturated fatty acid esters by mass spectrometry of their trimethylsilyloxy derivatives. Anal. Chem., 40, 1458-1463 (1968); DOI.
- 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); DOI.
- Chance, D.L., Gerhardt, K.O. and Mawhinney, T.P. Gas-liquid chromatography mass spectrometry of hydroxy fatty acids as their methyl esters tert.-butyldimethylsilyl ethers. J. Chromatogr. A, 793, 91-98 (1998); DOI.
- Hamberg, M. and Hamberg, G. 15(R)-Hydroxylinoleic acid, an oxylipin from oat seeds. Phytochemistry, 42, 729-732 (1996); DOI.
- Kleiman, R. and Spencer, G.F. Gas chromatography-mass spectrometry of methyl esters of unsaturated oxygenated fatty acids. J. Am. Oil Chem. Soc., 50, 31-38 (1973); DOI.
- 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); DOI.
- 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); DOI.
- Xia, W. and Budge, S.M. GC-MS characterization of hydroxy fatty acids generated from lipid oxidation in vegetable oils. Eur. J. Lipid Sci. Technol., 120, 1700313 (2018); DOI.
I can recommend - 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) - from Science Direct.
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