MASS SPECTRA OF METHYL ESTERS OF FATTY ACIDS
Part 6. Tetra-, Penta- and Hexaenoic Fatty Acids
As cautioned in the Introduction to these documents, the mass spectra of methyl esters afford limited information only concerning the structures of unsaturated fatty acids. There are a few key ions that help to identify the common polyenoic fatty acids with methylene-interrupted unsaturation, especially those of the (n-6) and (n-3) families, though they must be interpreted with caution. Thus, it appears that a peak at m/z = 150 is characteristic for fatty acids with an n-6 terminal moiety, while one at m/z = 108 defines an n-3 terminal group (omega ion) (Brauner et al., 1982; Fellenberg et al., 1987). Similarly, the presence of an alpha ion that contains the carboxyl group and the first two double bonds is less often recognised, and tends to be small so easily missed, but is equally important for characterization as discussed at greater length in the web page dealing with methyl esters of trienoic fatty acids. These are illustrated for 20:4(n-3) and 20:4(n-6) -
In comparison to the methylene-interrupted trienes, tetraenes, pentaenes and hexaenes as the methyl esters tend to give molecular ions with very low abundance (if detectable at all), and the ion at [M−31/32]+ for loss of a methoxyl group is only rarely distinguishable. It can therefore be a difficult task to ascertain the molecular weight, and gas chromatographic retention data are especially important as additional aids to identification. The McLafferty ion (m/z = 74) is always small. In the lower molecular weight region, hydrocarbon ions of general formula [CnH2n-5]+ tend to dominate the spectrum with the ion at m/z = 79 as the base peak.
Some other polyunsaturated fatty acids give distinctive fingerprint spectra, although the mechanisms of fragmentation may not be properly understood. Many of the spectra illustrated below will have been published somewhere in the scientific literature, but some are illustrated here for the first time.
Tetraenoic Fatty Acids
Methylene-Interrupted Tetraenes: Of the C18 tetraenoic acids, (stearidonate) is probably the most common in plant and fish tissues at least, and the mass spectrum of its methyl ester is illustrated.
The molecular ion is barely distinguishable but the characteristic ion for the
n-3 structure, at m/z = 108, is apparent as is the alpha ion for Δ6,9 double bonds at
m/z = 194. With fatty acids with four or more double bonds, even when non-conjugated, a tropylium ion at
m/z = 91 becomes a more important component of the spectrum (see the spectrum of methyl parinarate below).
The ion at m/z = 108 is also present in the spectrum of methyl 4,7,10,13-hexadecatetraenoate (16:4(n-3)) (not illustrated), but not in that of methyl 6,9,12,15-hexadecatetraenoate (16:4(n-1)), which has a slightly different and largely uninterpretable fingerprint spectrum. However, an ion at m/z = 221 ([M−41]+) may represent loss of the terminal three carbons. It is also present in the spectrum of the 18:4(n-1), but not in the spectra of analogous trienes (author, unpublished). Similarly, the alpha ion for a Δ6,9 double bond is present in the spectrum of the 16:4(n-1) fatty acid, and for the Δ8,11 double bond as with 18:4(n-1), but not in the spectra of the 16:3 and 18:3 fatty acids of the n-1 family.
More important from a biological standpoint is the essential fatty acid, arachidonic acid (5,8,11,14-20:4 or 20:4(n-6)), and the mass spectrum of methyl arachidonate follows -
Again the molecular ion at m/z = 318 is not very abundant, but the diagnostic ion for the n-6 moiety at m/z = 150 does stand out. The alpha ion for the Δ5,8 double bonds at m/z = 180 is small but distinctive.
There are significant differences from the mass spectrum of methyl 8,11,14,17-eicosatetraenoate (20:4(n-3)), which has the fingerprint illustrated -
The 'diagnostic' ion for the n-3 family of fatty acids at m/z = 108 is indeed present, if somewhat hidden, while there is no ion at m/z = 150. Again, the alpha ion for the Δ8,11 double bonds at m/z = 222 is small but is clearly present.
Methyl 7,10,13,16-docosatetraenoate or 22:4(n-6) has the spectrum -
The characteristic ion at m/z = 150 for the n-6 family of fatty acids does again stand out, and this is also present in the spectrum of 24:4(n-6). The alpha ion for the Δ7,10 double bonds at m/z = 208 is small but can be recognized.
Unusual Tetraenes: The mass spectrum of methyl 5,11,14,17-eicosatraenoate, with four methylene groups between the first two double bonds (from a conifer species) -
While the molecular ion is not very abundant once more, the diagnostic ion for the n-3 moiety at m/z = 108 is prominent, and there are other significant differences (though not necessarily interpretable in structural terms at present) from the spectrum of 20:4(n-3) illustrated earlier. The ion at m/z = 261 may be the alpha ion for the three methylene-interrupted double bonds.
The mass spectrum (probably not published elsewhere) of the conjugated tetraene, methyl parinarate (9c,11t,13t,15c-18:4) is rather different -
The molecular ion is very abundant, and the base peak at m/z = 91 is again presumably a tropylium ion, which does occur frequently in highly unsaturated molecules. The homologous series of ions at m/z = 105, 119, 133, etc, are probably tropylium ions with alkyl substituents. There are no ions that serve to locate double bonds.
Pentaenoic Fatty Acids
The mass spectrum of methyl 5,8,11,14,17-eicosapentaenoate (20:5(n-3) or 'EPA') -
The molecular ion is just discernable with some amplification of the spectrum, but it takes the eye of faith to discern the 'diagnostic' omega ion at m/z = 108 for an n-3 double bond. The alpha ion for the Δ5,8 double bonds at m/z = 180 is small but is in a clear part of the spectrum.
We also have a spectrum for the methyl ester of 21:5(n-3) from a fish oil on file.
Similarly with the mass spectrum of methyl 7,10,13,16,19-docosapentaenoate (22:5(n-3)) -
The molecular ion (m/z = 344) is only seen if this part of the spectrum is magnified, while the remainder of the spectrum is unexciting. Nonetheless both the diagnostic alpha and omega ions can be discerned.
In the mass spectrum of methyl 4,7,10,13,16-docosapentaenoate or 22:5(n-6), the molecular ion is just discernable and the expected omega ion at m/z = 150 and alpha ion at m/z = 166 are seen –
Other than an alpha ion at m/z =180 for Δ5,8 double bonds, there is little useful information in the mass spectrum of the unusual fish oil fatty acid methyl 5,8,11,14,17-octadecapentaenoate (18:5(n-1)) -
Hexaenoic Fatty Acids
The spectrum of methyl 4,7,10,13,16,19-docosahexaenoate (22:6(n-3) or 'DHA') is undistinguished, and the molecular ion is vanishingly small, but it does have the expected omega ion at m/z = 108 and alpha ion at m/z =166.
The same is true of the spectrum of methyl 6,9,12,15,18,21-tetracosahexaenoate or 24:6(n-3) (from a jelly fish).
We have spectra of a few more methyl esters of polyunsaturated fatty acids on file, and they can be accessed (but with no interpretation) from our Archive page.
References
- Brauner, A., Budzikiewicz, H. and Boland, W. Studies in chemical ionization mass spectrometry. 5. Localization of homoconjugated triene and tetraene units in aliphatic compounds. Org. Mass Spectrom., 17, 161-164 (1982).
- Fellenberg, A.J., Johnson, D.W., Poulos, A. and Sharp, P. Simple mass spectrometric differentiation of the n-3, n-6 and n-9 series of methylene interrupted polyenoic acids. Biomed. Environ. Mass Spectrom., 14, 127-130 (1987).
|
||
Updated: 21/4/2008 |
Scottish Crop Research Institute (and MRS Lipid Analysis Unit), Invergowrie, Dundee (DD2 5DA), Scotland
|
 |
