Part 6. Tetra-, Penta- and Hexaenoic Fatty Acids

As cautioned in the Introduction to these documents, the mass spectra of methyl esters obtained with electron-impact ionization afford limited information only concerning the structures of unsaturated fatty acids. On the other hand, there are a few key ions that help to identify the common polyenoic fatty acids with methylene-interrupted unsaturation, especially those of the biologically important (n-6) and (n-3) families, though these must be interpreted with caution. Thus, it appears that an ion 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) (Holman and Rahm, 1971; 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 fragmentations are illustrated for 20:4(n-3) and 20:4(n-6) -

Mass spectral fragmentations in relation to double bond positions

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, so gas chromatographic retention data are especially important as additional aids to identification. This is rarely a problem with nitrogen-containing derivatives, such as the pyrrolidides, etc. Similarly, the McLafferty ion (m/z = 74) is always small in the mass spectra of methyl esters of polyunsaturated fatty acids. 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. Some of the spectra illustrated below will have been published somewhere in the scientific literature (though we have not checked thoroughly), but others are illustrated here for the first time.


Tetraenoic Fatty Acids

Methylene-Interrupted Tetraenes:

Of the C18 tetraenoic acids, (stearidonate or 18:4(n-3)) is probably the most common in plant and fish tissues at least, and the mass spectrum of its methyl ester is illustrated.

Mass spectrum of methyl 6,9,12,15-octadecatetraenoate

Formula of a tropylium ionThe 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. The ion at m/z = 108 is also present in the spectrum of methyl 4,7,10,13-hexadecatetraenoate (16:4(n-3)) (illustrated separately). With fatty acids with four or more double bonds, even when non-conjugated, a tropylium rearrangement ion at m/z = 91 becomes a more important component of the spectrum (see the spectrum of methyl parinarate below).

In the mass spectrum of methyl 5,8,11,14-octadecatetraenoate (18:4(n-4)) from fish oils, the alpha ion is at m/z = 180 and the omega ion at m/z = 122 (illustrated here..).

The spectrum of methyl 6,9,12,15-hexadecatetraenoate (16:4(n-1)) is different and is also interpretable in terms of the double bond positions. An ion at m/z = 221 ([M−41]+) may represent loss of the terminal three carbons. Analogous ions are present in the spectra of 18:4(n-1), 20:4(n-1) and 18:5(n-1) (see below), but not in those of related trienes (author, unpublished observation). Similarly, the alpha ion for a Δ6,9 double bond is present in the spectrum of the 16:4(n-1) fatty acid, for the Δ8,11 double bond in 18:4(n-1), and for the 10,13-double bonds in 20:4(n-1), but not in the spectra of the 16:3 and 18:3 fatty acids of the n-1 family.

Mass spectrum of methyl 6,9,12,15-hexadecatetraenoate

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 -

Mass spectrum of methyl arachidonate

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)) illustrated next -

Mass spectrum of methyl 8,11,14,17-eicosatetraenoate

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 -

Mass spectrum of methyl 7,10,13,16-docosatetraenoate

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. Similarly, the spectrum of 22:4(n-3) has the expected alpha and beta ions at m/z = 250 and 108, respectively.


Unusual Tetraenes:

5,9,12,15-Octadecatetraenoic acid, with a bis-methylene-interrupted double bond system, is only rarely encountered in nature (in some conifer species mainly) and it its methyl ester has the spectrum -

Mass spectrum of methyl 5,9,12,15-octadecatetraenoate

The omega ion at m/z = 108 for the (n-3) double bond system is present, as are ions representing cleavage at the centre of the bis-methylene-interrupted double bond system at m/z = 141 and 149. There is no obvious alpha ion, but the ion at m/z = 241 ([M-49]+, found in the spectra of methyl esters of other fatty acids with a Δ5,9 double bond system (see our web page on methyl esters of dienes, for example), is present if in low abundance.

The mass spectrum of methyl 5,11,14,17-eicosatraenoate, with four methylene groups between the first two double bonds (from a conifer species) -

Mass spectrum of methyl 5,11,14,17-eicosatraenoate

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 of the conjugated tetraene, methyl parinarate (9c,11t,13t,15c-18:4), is rather different. Like many here, it has probably not been published elsewhere.

Mass spectrum of methyl parinarate

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. While these ions are also significant in the spectra of non-conjugated polyenes, they are much more abundant here. 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') -

Mass spectrum of methyl 5,8,11,14,17-eicosapentaenoate

The molecular ion is just discernible with some amplification of the spectrum, but it takes the eye of faith to see 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 part of the spectrum that is otherwise clear. We also have a spectrum for the methyl ester of 21:5(n-3) from a fish oil on file here...

Similarly with the mass spectrum of methyl 7,10,13,16,19-docosapentaenoate (22:5(n-3)) -

Mass spectrum of methyl 7,10,13,16,19-docosapentaenoate

The molecular ion (m/z = 344) is very small. However, the diagnostic alpha (m/z = 208) and omega (m/z = 108) ions can both 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 –

Mass spectrum of methyl 4,7,10,13,16-docosapentaenoate

3,6,9,12,15-octadecapentaenoic acid (18:5(n-3)) is occasionally found as a minor component of some marine algae, and its methyl ester has the mass spectrum –

Mass spectrum of methyl 5,8,11,14,17-octadecapentaenoate

It has the expected omega ion at m/z = 108 and the alpha ion at m/z = 152, with no significant molecular ion.

One further C18 isomer is known, but other than an alpha ion at m/z = 180 for Δ5,8 double bonds, there is little useful structural information in the mass spectrum of the unusual fish oil fatty acid methyl 5,8,11,14,17-octadecapentaenoate (18:5(n-1)) -

Mass spectrum of methyl 5,8,11,14,17-octadecapentaenoate

There is a small ion at m/z = 247 ([M-41]+), which may represent loss of the terminal three carbons as described above for the (n-1) tetraenes).


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.

Mass spectrum of methyl 4,7,10,13,16,19-docosahexaenoate

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), except that the alpha ion is now at m/z = 194.

Mass spectrum of methyl 6,9,12,15,18,21-tetracosahexaenoate

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.



  • 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) (DOI: 10.1002/oms.1210170403).
  • 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) (DOI: 10.1002/bms.1200140306).
  • Holman, R.T. and Rahm, J.J. Analysis and characterization of polyunsaturated fatty acids. Prog. Chem. Fats Other Lipids, 9, 15-90 (1971) (DOI: 10.1016/0079-6832(71)90024-3).

Updated June 26, 2013