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Mass Spectrometry of Methyl Esters


Hydroxy Fatty Acids - Trimethylsilyl Ether Derivatives



In this document, mass spectra 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 independently of degree of unsaturation. Preparation of these derivatives can be helpful both in terms of chromatographic resolution and of interpretation of mass spectra (see Nicolaides et al., 1983); a suitable laboratory protocol 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. Many of the spectra have not been published elsewhere, but I cite references to previous publications when these are known to me. 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 -

Mass spectrum of the TMS derivative of methyl 2-hydroxydocosanoate

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]+) 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 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. Pyrrolidides are a good alternative choice for 2- and 3-hydroxy isomers.

The TMS ether derivative prepared from methyl 3-hydroxy-tetradecanoate, like the underivatized compound, has a poor molecular ion, but the base peak at m/z = 315 is equivalent to [M‑59]+, 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.

Mass spectrum of methyl 3-hydroxy-tetradecanoate as the TMS ether


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.

Mass spectrum of methyl 9-hydroxy-heptadecanoate as the TMS ether

Fragment ion for m/z = 230There 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.

Mass spectrum of methyl 9-hydroxy-octadec-12-enoate as the TMS ether

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 also 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.

Mass spectrum of methyl 10-hydroxy-octadecanoate - TMS derivative

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) -

Mass spectrum of the TMS ether of methyl ricinoleate

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 also have the spectrum of the TMS ether derivative of 14-hydroxy-eicos-11-enoic (lesquerolic acid), the C20 analogue of ricinoleic acid, available in our Archive pages, and these are useful for comparison purposes.

The next three spectra were obtained from minor components (differing in chain-length) of cow's milk fat.

Methyl 13-hydroxy-octadecanoate - TMS ether derivative -

Mass spectrum of 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 are also evident and 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 -

Mass spectrum of methyl 14-hydroxy-hexadecanoate - TMS ether derivative

Methyl 15-hydroxy-octadecanoate - TMS ether derivative -

Mass spectrum of methyl 15-hydroxy-octadecanoate - TMS ether

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).

Mass spectrum of the TMS ether derivative of methyl 15-hydroxy-linoleate

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 unfavorable terminal (omega) or omega-1 carbons. Similar fatty acids are found in waxes and plant cutins, which were the source of the following examples. 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 -

Mass spectrum of TMS ether of methyl 15-hydroxyhexadecanoate

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. However, the distinctive feature is the base peak 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 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 peak 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).

Mass spectrum of the TMS ether of methyl 16-hydroxyhexadecanoate

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. Also, ions in the lower mass range are now much more abundant.

Mass spectrum of TMS ether of methyl 18-hydroxy-octadec-9-enoate


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. As an example, the mass spectrum of the TMS ether derivative of methyl 9,10,18-trihydroxy-octadecanoate is illustrated. There is a simple cleavage between the vicinal hydroxyl groups as illustrated. The ion at m/z = 332 may be produced by a rearrangement involving migration of the TMSO group to the carboxyl moiety.

Mass spectrum of TMS ether derivative of methyl 9,10,18-trihydroxy-octadecanoate

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).

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 also 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


I also 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.


Credits/disclaimer Updated: July 12th, 2017 Author: William W. Christie LipidWeb icon