Part 4. Polyunsaturated Fatty Acids

Mass Spectra of Pyrrolidine Derivatives of Polyunsaturated

As was described for dienes, the interpretation of mass spectra of pyrrolidides is similar to that for monoenes in that it is necessary to look for the gap of 12 amu that locates each double bond. However, it is not always easy to locate the first double bond especially, and then the 'fingerprint' of the appropriate monoene is a useful guide. As with the other pyrrolidides, it is always helpful to have access to an authentic spectrum, as I have never yet found two positional isomers with the same mass spectrum. With four or more double bonds, I usually advise that the spectrum be treated as a fingerprint to be compared those of standards. It is my general impression that pyrrolidides are less useful than DMOX (or 3-pyridylcarbinol) derivatives when trying to find the key diagnostic ions in the spectrum of an unknown polyenoic fatty acid, although they have their merits in other circumstances. We have fewer spectra of pyrrolidides available here than of DMOX derivatives, but in general the two are usually very similar in the key diagnostic areas. I have been unable to find many illustrations of mass spectra of pyrrolidides of the common range of polyunsaturated acids in the scientific literature.

 

Trienoic Fatty Acids

With higher polyunsaturated fatty acids, the picture becomes even less clear than with mono- and dienes, as there is more noise around the diagnostic ions that hampers recognition. Thus, the earlier rule was extended by Andersson et al. (1975) as -

"The presence of a peak relatively more intense than the peak clusters which flank it and which are involved in probable intervals of 12 amu indicates the presence of a methylene-interrupted system. If the prominent peak contains m carbons of the fatty acid residue, the methylene carbon in the molecule is at position m+1".

The mass spectrum of the pyrrolidine derivative of 6,9,12-octadecatrienoate (or γ-linolenate or 18:3(n-6)) (Das Gupta et al., 1992) is -

Mass spectrum of the pyrrolidide of 6,9,12-octadecatrienoate

The ions in the higher mass range have all been magnified five fold as an aid to identification. The double bond in position 6 is best recognized via the spectrum of the 6-monoene (see our web page on pyrrolidides of monoenes) by the characteristic ions at m/z = 140, 154 and 166. Those in positions 9 and 12 are located by the gaps of 12 amu between 194 and 206, and between 234 and 246, respectively.

The mass spectrum of the pyrrolidine derivative of 9,12,15-octadecatrienoate (or α-linolenate or 18:3(n-3)) (Andersson et al., 1975) is -

Mass spectrum of the pyrrolidide of 9,12,15-octadecatrienoate

The gaps of 12 amu between m/z = 196 and 208, 236 and 248, and 276 and 288, define the double bonds in positions 9, 12 and 15, respectively. However, from a practical standpoint, it is just as well we have the standard spectrum to consult. It also helps to remember that for methylene-interrupted fatty acids, the equivalent diagnostic ions for each double bond should be 40 amu apart (196 – 236 – 276, and 208 – 248 – 288).

The mass spectrum of the pyrrolidine derivative of 5,8,11-eicosatrienoate (or 20:3(n-9) or Mead's acid), a fatty acid that assumes importance in essential fatty acid deficiency is -

Mass spectrum of the pyrrolidide of 5,8,11-eicosatrienoate

The double bond in position 5 is located by the characteristic fingerprint of ions at m/z = 140, 153 and 166 (a small but significant difference from a 6-isomer), while those in positions 8 and 11 are defined by the gaps of 12 amu between m/z = 180 and 192, and 220 and 232, respectively (Valicenti et al., 1978).

The mass spectrum of the pyrrolidine derivative of 8,11,14-eicosatrienoate (or 20:3(n-6)) is -

Mass spectrum of the pyrrolidide of 8,11,14-eicosatrienoate

The important diagnostic ions denote the gaps of 12 amu between m/z = 182 and 194, 222 and 234, and 262 and 274, for the double bonds in positions 8, 11 and 14, respectively.

A few trienoic acids with bis-methylene-interrupted double bond systems are known, and 5,9,12-octadecatrienoic (pinolenic) acid is found in the lipids of pine species. The mass spectrum of its pyrrolidide derivative is -

Mass spectrum of the pyrrolidide of 5,9,12-octadecatrienoate

The distinctive feature is an ion at m/z = 180 formed by cleavage at the centre of the bis-methylene-interrupted double bond system (c.f. the spectra of 5,9-18:2 and of the DMOX derivative). The double bond in position 5 would otherwise not be easy to locate from first principles, but those in positions 9 and 12 are confirmed by the gaps of 12 amu between m/z = 194 and 206, and 234 and 246, respectively.

Rather more unusual is a fatty acid with two bis-methylene-interrupted double bond systems, and the mass spectrum of the pyrrolidide of 5,9,13-eicosatrienoate (5,9,13-20:3) from a sponge is -

Mass spectrum of the pyrrolidide of 5,9,13-eicosatrienoate

The ion at m/z = 180 for cleavage between carbons 7 and 8, seen in the previous spectrum, is again evident, while the distinctive ion at m/z = 234 represents cleavage between carbons 11 and 12 (data first published by Carballeira and Medina, 1994).

Trienoic acids with more than two methylene groups between double bonds are less common in nature, but 3,9,12-octadecatrienoate is occasionally found in seeds of Chrysanthemum and related species, although the double bond in position 3 is usually of the trans rather than the cis configuration as here. Its pyrrolidide has the mass spectrum -

Mass spectrum of the pyrrolidide of 3,9,12-octadecatrienoate

The double bond in position 3 is defined by the abundant ion at m/z = 152 (for some reason, we were unable to prepare the derivative from cis-3-18:1 for comparison purposes, although there was no such problem with the trans-3 isomer). The remaining double bonds are located as marked. On the other hand with further experience, it now appears that the double bond in position 3 has migrated to position 2 during the derivatization step, so that the above is probably the spectrum of the 2,9,12-isomer in fact.

5,11,14-Eicosatrienoate (or 5,11,14-20:3) is found in seeds from some species of Gymnosperms and the mass spectrum of its pyrrolidide is -

Mass spectrum of the pyrrolidide of 5,11,14-eicosatrienoate

As is usual with double bonds in position 5, the ions in the higher mass range are of rather low abundance and a ten-fold magnification is needed to see them clearly. The double bonds in the 11 and 14 positions are located by the gaps of 12 amu between m/z = 222 and 234, and 262 and 274, respectively.

Further complex trienoic fatty acids are found in sponge lipids (see our Archive pages for more spectra, but without interpretation).

 

Tetraenoic Fatty Acids

The mass spectrum of the pyrrolidide of 6,9,12,15-octadecatrienoate (stearidonate or 18:4(n-3)) -

Mass spectrum of the pyrrolidide of 6,9,12,15-octadecatrienoate

The double bond in position 6 is identified from the characteristic fingerprint ions at m/z = 140, 154 and 160, while the double bonds in positions 9, 12 and 15 are recognized by the gaps of 12 amu between m/z = 194 and 206, 234 and 246, and 274 and 286, respectively.

The mass spectrum of arachidonoylpyrrolidine (5,8,11,14-20:4 or 20:4(n-6)) follows -

Mass spectrum of the pyrrolidide of arachidonate

Gaps of 12 amu between m/z = 180 and 192, 220 and 232, and 260 and 272, locate the double bonds in positions 8, 11 and 14, respectively. That in position 5 must be inferred, as the expected diagnostic ion at m/z = 153 is submerged among many small ions (Valicenti et al., 1978).

A further useful mass spectrum of a tetraene is that of 7,10,13,16-docosatetraenoate (or 22:4(n-6)) -

Mass spectrum of the pyrrolidide of 7,10,13,16-docosatetraenoate

The spectrum is too confused to serve as an absolute guide to locate double bond position, but at least is a useful fingerprint. While the expected ions are indeed present, other ions could confound any interpretation without further evidence.

 

Penta- and Hexaenoic Fatty Acids

The following spectra are all best considered as fingerprints, without trying to pick out the key diagnostic ions, which are usually confused in all polyenes. No interpretation is offered therefore, and the spectra are left to speak for themselves. Although the molecular ions are small, they are easier to identify than in the corresponding methyl esters.

Mass spectrum of the pyrrolidide of 5,8,11,14,17-eicosapentaenoate (20:5(n-3) or EPA) -

Mass spectrum of the pyrrolidide of 5,8,11,14,17-eicosapentaenoate

Mass spectrum of the pyrrolidide of 4,7,10,13,16-docosapentaenoate (or 22:5(n-6)) -

Mass spectrum of the pyrrolidide of 4,7,10,13,16-docosapentaenoate

Mass spectrum of the pyrrolidide of 4,7,10,13,16,19-docosahexaenoate (22:6(n-3) or DHA) -

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

Mass spectrum of the pyrrolidide of 6,9,12,15,18,21-tetracosahexaenoate (24:6(n-3)) -

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

Spectra of a few more pyrrolidine derivatives of polyunsaturated fatty acids are available on our Archive pages, but without interpretation.

 

References

  • Andersson, B.A., Christie, W.W. and Holman, R.T. Mass spectrometric determination of positions of double bonds in polyunsaturated fatty acid pyrrolidides. Lipids, 10, 215-219 (1975) (DOI: 10.1007/BF02532483).
  • Carballeira, N.M. and Medina, J.R. New Δ5,9 fatty acids in the phospholipids of the sea anemone Stoichactis helianthus. J. Nat. Prod., 57, 1688-1695 (1994) (DOI: 10.1021/np50114a011).
  • Dasgupta, A., Banerjee, P. and Malik, S. Use of microwave irradiation for rapid transesterification of lipids and accelerated synthesis of fatty acyl pyrrolidides for analysis by GC-MS: study of fatty acid profiles of olive oil, evening primrose oil, fish oils and phospholipids from mango pulp. Chem. Phys. Lipids, 62, 281-291 (1992) (DOI: 10.1016/0009-3084(92)90065-W).
  • Valicenti, A.J., Chapman, C.J., Holman, R.T. and Chipault, J.R. Mass spectrometry identification of C20 fatty acids in bovine lens using the pyrrolidide derivative. Lipids, 13, 190-194 (1978) (DOI: 10.1007/BF02533398).

Updated November 14, 2013

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