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Mass Spectrometry of DMOX Derivatives


Trienoic Fatty Acids



Methylene-Interrupted Trienoic Fatty Acids

The mass spectra of DMOX derivatives of trienoic fatty acids permit location of the double bonds but less easily than with those of monoenes and dienes. The principles described in the earlier documents in this series apply, so we are in general looking for the diagnostic gaps of 12 amu between significant ions. When the first double bond is close to the carboxyl group, the general fragmentation rule for double bond location is no longer appropriate and the 'fingerprint' spectrum for the relevant monoene derivative is an invaluable guide to its identification.

While identification from first principles can sometimes be problematic, different isomers always seem to have very different spectra so that characterization is possible when spectra of DMOX derivatives of authentic fatty acids can be compared. Unlike the dienes, few model compounds are available for study, so most of the following spectra have been gleaned from analyses of natural products from my own laboratory, and fatty acids with a variety of chain-lengths are described. References are listed when we are aware of prior formal publication of spectra in the scientific literature (please point out any we may have missed).

For example, the mass spectrum of the DMOX derivative of 6,9,12-octadecatrienoate (γ-linolenate or 18:3(n-6)) is illustrated below (Sayanova et al., 1997) -

Mass spectrum of the DMOX derivative of 6,9,12-octadecatrienoate

The double bonds in positions 9 and 12 are easily recognized from the gaps of 12 amu between m/z = 194 and 206, and between 234 and 246, respectively. That in position 6 must be identified by the fingerprint characteristic for an isomer with the first double bond in position 6, i.e. the odd-numbered ion at m/z = 167 (or the triplet at m/z = 167, 180 and 194).

DMOX derivative of 8,11,14-octadecatrienoate (18:3(n-4)) - a minor component of fish oils -

Mass spectrum of the DMOX derivative of 8,11,14-octadecatrienoate

DMOX derivative of the ubiquitous 9,12,15-octadecatrienoate (α-linolenate or 18:3(n-3)) (Zhang et al., 1988) -

Mass spectrum of the DMOX derivative of 9,12,15-octadecatrienoate

In this and the previous example, all three double bonds are easily recognized by the gaps of 12 amu as indicated in the spectrum. Thus in the last, gaps of 12 amu between m/z = 196 and 208, 236 and 248, and 276 and 288 locate the double bonds in position 9, 12 and 15, respectively. Note that with methylene-interrupted double bonds, the gaps of 40 amu between m/z = 196, 236 and 276, between m/z = 208, 248 and 288, and especially between m/z = 182, 222 and 302 are also useful diagnostically as indicated on the spectrum. Comparable ions, 14 amu less, are present in the spectrum of the 8,11,14-isomer illustrated above.

Trienes of the n-1 family of fatty acids have yet to be found in nature, but this spectrum (from a genetically modified plant) is illustrated next for the sake of completeness (Sayanova. et al., 2006). The first two double bonds are easily located but terminal double bonds give problems with all derivative types because of the ease with which a methyl group is lost from the heterocyclic ring. Here again the gaps of 40/41 amu are useful indicators (m/z = 210 to 250 to 290 to 331). DMOX derivative of 11,14,17-octadecatrienoate (18:3(n-1)) -

Mass spectrum of the DMOX derivative of 11,14,17-octadecatrienoate

There are three important eicosatrienoic acids in animal system, the first of which illustrated here is found during essential fatty acid deficiency and is often termed Mead's acid; the DMOX derivative of 5,8,11-eicosatrienoate (18:3(n-9)) is -

Mass spectrum of the DMOX derivative of 5,8,11-eicosatrienoate

The double bond in position 5 must be recognized from the fingerprint ions at m/z = 153 and 166, but the remaining ions are located as marked. All three double bonds are easily recognized by the gaps of 12 amu, as indicated, in the spectra of the two 20:3 isomers that follow. Thus, the DMOX derivative of 8,11,14-eicosatrienoate (20:3(n-6)) -

Mass spectrum of the DMOX derivative of 8,11,14-eicosatrienoate

DMOX of 11,14,17-eicosatrienoate (11,14,17-20:3 or 20:3(n-3)) (Wallis and Browse, 1999) -

Mass spectrum of the DMOX derivative of 11,14,17-eicosatrienoate

We have the spectra of the DMOX derivatives of further methylene-interrupted trienes in our Archive, including four 16:3 isomers, only one of which is illustrated below as an example, i.e. the DMOX derivative of 4,7,10-hexadecatrienoate (16:3(n-6)) -

Mass spectrum of the DMOX derivative of 4,7,10-hexadecatrienoate

The double bond in position 4 must be recognized from the fingerprint ion at m/z = 152, but the remaining ions are located as marked.


Trienoic Fatty Acids with Conjugated Double Bonds

Trienoic fatty acids with only two of the double bonds in conjugation are not often encountered in nature, but 9-cis,11-trans,15-cis-octadecatrienoic acid is formed by biohydrogenation of α-linolenic acid in the rumen of cows and sheep and occurs as a minor component of milk fat, adipose tissue and bile of these species. The mass spectrum of its DMOX derivative is -

Mass spectrum of the DMOX derivative of 9,11,15-18:3

The gap of 12 amu between m/z = 196 and 208 confirms the double bond in position 9, while that for the similar gap between 222 and 234 confirms the double bond in position 11. The outstanding feature of the spectrum is the base ion at m/z = 262, which represents cleavage at the centre of the bis-methylene-interrupted double bond system (see our web page on dienes and below). The gap to m/z = 288 also confirms the double bond in position 15. The mass spectrum does not of course give information on the configurations of the double bonds (Destaillats, F. et al., 2005). In the mass spectrum of the DMOX derivative of cis-6,trans-8,cis-12-octadecatrienoate (illustrated here...), the key diagnostic ion is shifted down to m/z = 220 as might be expected (author, not published previously)

Fully conjugated trienoic acids are well known constituents of certain seed oils and the mass spectrum of the DMOX derivative of punicic or 9-cis,11-trans,13‑cis-octadecatrienoic acid follows.

Mass spectrum of the DMOX derivative of 9,11,13-18:3

In this instance, all the positions of all the double bonds can be recognized from the gaps of 12 amu as illustrated. The mass spectrum of the DMOX derivative of the geometrical isomer α-eleostearate (9c,11t,13t-18:3) is identical to this and has been published by Spitzer (1997). It is noteworthy that the ion formed by cleavage beta to the double bond system at m/z = 288 in this example is highly abundant. Similarly, an analogous ion at m/z = 246 is especially distinctive in the spectrum of the DMOX derivative of 6,8,10-octadecatrienoate (geometry uncertain), as illustrated in the Archive pages here... (author, not published previously). The tropylium ion at m/z = 91 is a small but significant component of the spectrum only with the last example, the conjugated triene, whereas with methyl ester derivatives of fatty acids with three or more double bonds this can be a dominant ion (see the web page on mass spectra of methyl esters of tetraenoic acids, etc).

There seems little doubt that DMOX derivatives are better than 3-pyridylcarbinol esters and pyrrolidides for locating double bonds in conjugated fatty acids. On the other hand, there is a danger of isomerization occurring if the derivatization conditions are too vigorous.


Trienoic Fatty Acids with Bis- and Polymethylene-Interrupted Double Bonds

As described elsewhere for dienes, it has become apparent that bis- and polymethylene-interrupted trienoic fatty acids are more common in nature than may have been supposed. In particular, fatty acids with a 5,9-double bond system or their chain elongation products are present in seed oils from Gymnosperms or in certain marine invertebrates such as sponges.

The spectrum of the DMOX derivative of 5,9,12-octadecatrienoate (pinolenic acid) from a pine species is typical (Fay and Richli, 1991).

Mass spectrum of the DMOX derivative of 5,9,12-octadecatrienoate

As described for (bis-methylene-interrupted dienes, the prominent ion at m/z = 180 represents cleavage at the centre of the bis-methylene-interrupted (5,9) double bond system. Analogous ions are present in the corresponding spectra of pyrrolidides and 3-pyridylcarbinol ('picolinyl') esters. The double bond in position 12 is located by a gap of 12 amu between m/z = 234 and 246. Note that the odd-numbered ion at m/z = 153 is diagnostic for a double bond in position 5, while the fact that the ion at m/z = 113 is so much larger than that at m/z = 126 is also characteristic (see the corresponding web page on monoenes).

The spectrum of the DMOX derivative of 5,9,13-eicosatrienoate (5,9,13-20:3) from a sponge appears to be the only one of its kind to have been discovered so far in that it has two adjacent bis-methylene-interrupted double bond systems -

Mass spectrum of the DMOX derivative of 5,9,13-eicosatrienoate

The ion at m/z = 180 represents cleavage at the centre of the 5,9-double bond system, while that at m/z = 234 is diagnostic for the 9,13-system. However, ions for the individual double bonds are not easily recognized, although this hardly matters with such a unique spectrum.

DMOX derivative of 5,9,19-hexacosatrienoate (5,9,19-26:3) -

Mass spectrum of the DMOX derivative of 5,9,19-hexacosatrienoate

This very-long chain triene is typical of the type of fatty acid component found in sponges, in this instance from Hymeniacidon cinerea. It has the ion at m/z = 180 for the 5,9-double bonds, while the gap of 12 amu later as indicated locates that in position 19.

The spectrum of the DMOX derivative of 7,11,14-eicosatrienoate (7,11,14-20:3) from seed oils of pine species.

Mass spectrum of the DMOX derivative of 7,11,14-eicosatrienoate

In this instance, the spectrum resembles that of 5,9,12-18:3 except that the diagnostic ions are shifted upwards by 28 amu (Wolff et al., 1997)). In particular, the ion representing cleavage at the centre of the bis-methylene-interrupted double bond system is now at m/z = 208.

A few trienoic fatty acids are known in which double bonds are separated by more than two methylene groups, for example from certain conifer species. The spectrum of the DMOX derivative 5,11,14-eicosatrienoate (5,11,14-20:3 or sciadonic) from Pinus contorta seed oil follows (see also Zhang et al., 1988) -

Mass spectrum of the DMOX derivative of 5,11,14-eicosatrienoate

All the double bonds must be identified individually - that in position 5 by the diagnostic ion at m/z = 153, and the others by the gaps of 12 amu.

A few fatty acids with isolated double bonds in position 3 are found in plants, although these are often of the trans rather than the cis configuration as in the example here, the DMOX derivative of 3,9,12-octadecatrienoate from Tanacetum zawadskii seed oil (Tsevegsuren et al., 2003).

Mass spectrum of the DMOX derivative of 3,9,12-octadecatrienoate

The base peak at m/z = 152 is the important diagnostic feature, though this would also be the case if the double bond were in position 2, and it is possible that isomerization has occurred during derivatization (see our web page on DMOX derivatives of monoenes).

We have mass spectra of the DMOX derivatives of further trienoic fatty acids on file and these are illustrated in the Archive Section of these web pages but without detailed interpretation. Most of these have not been formally published elsewhere.


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) - at Science Direct.


Credits/disclaimer Updated: July 13th, 2017 Author: William W. Christie LipidWeb icon