Part 1. Introduction, Saturated and Branched-Chain Acids

Introduction to Pyrrolidide Derivatives

Formula of a pyrrolidideFatty acid pyrrolidine derivatives or pyrrolidides or acyl-pyrrolidines were the first of the nitrogen-containing derivatives to be recognized as offering new possibilities for the structural analysis of fatty acids by mass spectrometry. Indeed, they may have been described too soon to be of immediate practical value, as the packed GC columns then available were not stable enough to high temperatures and afforded insufficient resolution to handle fatty acid pyrrolidides satisfactorily. Following their discovery (Vetter et al., 1971), there was a series of papers from Anderson and Holman describing results with model compounds, followed by many applications from others to natural samples (see our web page with a literature survey of the topic). When 3-pyridylcarbinol ('picolinyl') ester and dimethyloxazoline (DMOX) derivatives were described a decade later, capillary columns of fused silica with thermally stable phases and high resolution together with a new generation of simple bench-top mass spectrometers had just become available. The new derivatives caught the eye of analysts, and pyrrolidides were relatively neglected, perhaps unjustly.

Nonetheless, they have been used extensively by a dedicated band of analysts, especially those interested in marine lipids. However, although a great deal of tabulated mass spectral data has been published in the literature, relatively few spectra have been represented pictorially. I have used the old adage that 'a picture is worth a thousand words' elsewhere in these web pages and this philosophy continues here. Though it is now somewhat out of date, a useful review of mass spectrometry of pyrrolidides has been published by Andersson (1978).

Comparison with DMOX derivatives

By an interesting and useful coincidence, the pyrrolidide of a given fatty acid has exactly the same molecular weight as the corresponding DMOX derivative, in spite of the very different structures. As with DMOX derivatives, the base peak is usually the McLafferty ion at m/z = 113, with the ion at m/z = 126 also being very abundant. At least for the common range of fatty acids, the fragmentation mechanisms for acyl-pyrrolidines are essentially the same as for DMOX derivatives and indeed were worked our first for the former. In analysing mass spectra of pyrrolidines, we are often looking for exactly the same diagnostic ions as for the corresponding DMOX derivatives.

One important potential advantage is the fact that pyrrolidides lack the methyl groups attached to the ring that can hinder the interpretation of spectra from DMOX derivatives (Hamilton and Christie, 2000). My present subjective impression is that DMOX derivatives may be better than pyrrolidides for locating double bonds. Pyrrolidides are certainly better when functional groups, e.g. methyl branches or ring structures, are near the terminal end of the molecule. They are also better for sensitive fatty acids such as epoxides.

Pyrrolidide derivatives are prepared under much milder conditions than DMOX derivatives, directly from methyl esters, and are more stable chemically (see our web-pages - Preparation of derivatives for mass spectrometry).

Ions in the high mass range that are required for diagnostic purposes tend to be of low abundance in the mass spectra of pyrrolidides (and with DMOX derivatives sometimes), but it should be remembered that this is only a relative feature. The convention in mass spectrometry is to set the most abundant ion (base peak) at 100%, with others adjusted in relation to this. Is the base peak at an especially high abundance in the mass spectra of pyrrolidides, or are the remaining peaks at low abundance? It is not difficult with modern mass spectrometry software to magnify minor ions, and provided that this is clearly indicated, there should be no objection.

Gas Chromatography

Pyrrolidides elute from GC columns at somewhat higher temperatures than DMOX derivatives, but this is no longer a disadvantage, because of the wider availability of polar stationary phases that have greater thermal stability. Indeed, the author has observed that some astonishingly good separations of isomeric pyrrolidides. For example, pyrrolidides of 5-, 7-, 9-, 11- and 13-18:1 fatty acids were all resolved to the baseline on a 25 m column of Supelcowax 10™, with the 5-isomer actually eluting before the 18:0 derivative (Christie, 2002), as illustrated in the figure below.

GC separation of pyrrolidides

The pyrrolidine moiety can change the selectivity of the separation appreciably, and I have observed reversals in the order of elution of certain polyunsaturated components in the form of the pyrrolidides as compared to the methyl esters. A comprehensive study of the gas chromatographic properties of pyrrolidides might prove illuminating.

Summary

Pyrrolidides are very similar in their mass spectrometric properties to DMOX derivatives of fatty acids with advantages in some circumstances and disadvantages in others. They are a useful alternative to DMOX derivatives and complement 3-pyridylcarbinol esters in studies of fatty acid structure.

As with other sections on this website, the following account is a subjective one that only uses those mass spectra that were available from my personal researches. In this section, spectra of the common range of fatty acids are described. Spectra of more unusual fatty acids are described (together with other derivatives in the 'Miscellaneous' section of these documents. Mechanistic studies of mass spectra of pyrrolidides of normal saturated fatty acids (labelled with stable isotopes) have been published by Andersson et al. (1975, 1982).

 

Straight-Chain Saturated Fatty Acids

The mass spectrum of palmitoyl pyrrolidine (16:0) is illustrated first -

Mass spectrum of palmitoyl pyrrolidine

As mentioned earlier, the base peak is the McLafferty rearrangement ion at m/z = 113, while that at m/z = 126 is also relatively abundant (as with DMOX derivatives). It was necessary to magnify the ions in the high mass region at least four fold to visualize the significant ions sufficiently. Following the molecular ion at m/z = 309 (odd numbered as with all nitrogen-containing derivatives), the ions in the high mass range are uniformly 14 amu apart (and even numbered) for loss of successive methylene groups, i.e. at m/z = 294, 280, 266, 252, 238, etc.

Pyrrolidides - MS fragmentations

As a further example, the mass spectrum of the pyrrolidide of tetracosanoate (24:0) is -

Mass spectrum of the pyrrolidide of tetracosanoate

- in which all the significant ions in the high mass range are 14 amu apart as expected, although it was now necessary to magnify the ions in the higher mass region ten-fold to see them clearly.

The mass spectrum of the pyrrolidide of hexanoate (6:0) -

Mass spectrum of the pyrrolidide of hexanoate

Pyrrolidides seem to be especially useful for shorter-chain fatty acids, because of the convenience of the method of preparation.

We have mass spectra on file for pyrrolidides of many more saturated fatty acids, including some labelled with stable isotopes, and these can be found (but without interpretation) in our Archive web page.

 

Monomethyl-Branched Fatty Acids

The mass spectra of pyrrolidide derivatives of fatty acids with anteiso- and iso-methyl branches, in contrast to those of DMOX derivatives, are distinctive and can be used for characterization purposes (see our web pages on 'Mass spectrometry of DMOX derivatives. Part 1. Saturated and branched-chain fatty acids'). Few if any of the following spectra have been published formally elsewhere. The first paper to be published on the topic was by Andersson and Holman (1975), who demonstrated the ease of distinguishing iso and anteiso-isomers in particular. To illustrate the effect of the position of the branch points on fragmentation, it has been necessary to chose examples of fatty acids of differing chain-length, because of the limited range of material available to us from natural sources.

The mass spectrum of the pyrrolidide of 13-methyl-tetradecanoate (iso isomer) -

Mass spectrum of the pyrrolidide of 13-methyl-tetradecanoate

The methyl branch is easily located by the gap of 28 amu between m/z = 252 and 280 for loss of carbon 13 with its methyl group. In this and most of the following spectra, all the ions in the higher mass range have been magnified five fold to simplify the interpretation.

Similarly, in the mass spectrum of the pyrrolidide of 12-methyl-tetradecanoate (anteiso isomer) -

Mass spectrum of the pyrrolidide of 12-methyl-tetradecanoate

- the gap of 28 amu is now shifted downwards as expected to between m/z = 238 and 266 for loss of carbon 12 and its methyl group.

The mass spectrum of the pyrrolidide of 17-methyl-tetracosanoate -

Mass spectrum of the pyrrolidide of 17-methyl-tetracosanoate

- the diagnostic gap of 28 amu is between m/z = 308 and 336 for loss of carbon 17 and its methyl group.

The mass spectrum of the pyrrolidide of 14-methyl-heptadecanoate -

Mass spectrum of the pyrrolidide of 14-methyl-heptadecanoate

- now the diagnostic gap of 28 amu is shifted to between the ions at m/z = 266 and 294.

The mass spectrum of the pyrrolidide of 11-methyl-octadecanoate -

Mass spectrum of the pyrrolidide of 11-methyl-octadecanoate

- the diagnostic gap of 28 amu is shifted to between the ions at m/z = 224 and 252.

The mass spectrum of the pyrrolidide of 9-methyl-tetradecanoate -

Mass spectrum of the pyrrolidide of 9-methyl-tetradecanoate

- as expected the diagnostic gap of 28 amu is between the ions at m/z = 196 and 224.

The mass spectrum of the pyrrolidide of 3-methyl-tetradecanoate -

Mass spectrum of the pyrrolidide of 3-methyl-tetradecanoate

The gap of 27/28 amu for carbon-3 and its associated methyl group is between m/z = 113 and 140, and the usual ion at m/z = 126 can only be seen because of the considerable magnification.

We have mass spectra on file for pyrrolidides of many more branched-chain fatty acids, and these can be found (but without interpretation) in our Archive web page.

 

Multi-Methyl-Branched Fatty Acids

An unusual dimethyl-branched fatty acid, 8,10-dimethyl-hexadecanoate, has been found in a sponge and the mass spectrum of its pyrrolidine derivative is -

Mass spectrum of the pyrrolidide of 8,10-dimethyl-hexadecanoate

The methyl groups are located by the two gaps of 28 amu as indicated.

The most common multi-methyl-branched fatty acids encountered in nature are the isoprenoid compounds derived from phytol, such as 4,8,12-trimethyltridecanoic acid, which is often encountered as a minor component of marine oils, and the mass spectrum of its pyrrolidide is illustrated next. Again, each of the methyl groups is located by the gaps of 28 amu as indicated (the last one is not clear in this instance).

Mass spectrum of the pyrrolidide of 4,8,12-trimethyltridecanoic acid

Finally, the best known of such acids is probably phytanic (3,7,11,15-tetramethyl-hexadecanoic) acid, and the mass spectrum of its pyrrolidide is -

Mass spectrum of the pyrrolidide of 3,7,11,15-tetramethyl-hexadecanoic acid

The gaps of 27/28 amu between m/z = 113 and 140, 182 and 210, 252 and 280, and 322 and 350 locate the methyl branches in positions 3, 7, 11 and 15, respectively.

 

References

  • Andersson, B.A. Mass spectrometry of fatty acid pyrrolidides. Prog. Chem. Fats other Lipids, 16, 279-308 (1978) (DOI: 10.1016/0079-6832(78)90048-4).
  • Andersson, B.A. and Holman, R.T. Mass spectrometric localization of methyl branching in fatty acids using acylpyrrolidines. Lipids, 10, 716-718 (1975) (DOI: 10.1007/BF02532767).
  • Andersson, B.A., Dinger, F. and Dinh-Nguyen, N. Mass spectrometry studies of stable isotope labelled carboxylic acid derivatives. I. Normal chain saturated deuterium and carbon-13 compounds. Chem. Scripta, 8, 200-203 (1975).
  • Andersson, B.A., Dinger, F. and Dinh-Nguyen, D. Mass spectrometry studies of stable isotope-labelled carboxylic acid derivatives. IV. Pyrrolidides of vicinal and geminal dideuterated n-octadecanoic acids and the omega trideuterated analogue. Chem. Scripta, 19, 118-121 (1982).
  • Christie, W.W. Unusual gas chromatographic properties of fatty acid pyrrolidides. Eur. J. Lipid Sci. Technol., 104, 69-70 (2002) (DOI: 10.1002/1438-9312(200201)104:13.0.CO;2-G).
  • Hamilton, J.T.G. and Christie, W.W. Mechanisms for ion formation during the electron impact-mass spectrometry of picolinyl ester and 4,4-dimethyloxazoline derivatives of fatty acids. Chem. Phys. Lipids, 105, 93-104 (2000) (DOI: 10.1016/S0009-3084(99)00133-4).
  • Vetter, W., Walther, W. and Vecchi, M. Pyrrolidides as derivatives for structural analysis of aliphatic and alicyclic fatty acids by mass spectrometry. Helv. Chim. Acta, 54, 1599-1605 (1971) (DOI: 10.1002/hlca.19710540611).

Updated November 6, 2013 

pdf