Mass Spectrometry of Fatty Acid Pyrrolidides
Introduction and Saturated Straight-Chain Fatty Acids
Introduction to Pyrrolidide Derivatives
Fatty 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 by Vetter et al. (1971),
there was a series of papers from Andersson and Holman describing results with model compounds,
and these were succeeded 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 become available.
The new derivatives caught the eye of analysts, and there was a tendency to neglect pyrrolidides, perhaps unjustly.
Nonetheless, they have been used extensively by a dedicated band of analysts, especially those interested in marine lipids, and 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 Though it is now somewhat out of date, a useful review of mass spectrometry of pyrrolidides has been published by Andersson (1978).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 the same molecular weight as the corresponding DMOX derivative, in spite of the great difference in structure. 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 almost 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, although the relative intensities can differ somewhat.
One important potential advantage is the fact that pyrrolidides do not have methyl groups attached to the ring, the loss of which 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, but pyrrolidides are a better choice when functional groups, e.g., methyl branches or ring structures, are near the terminal end of the molecule.
The standard procedure for preparing pyrrolidide derivatives involves much milder conditions than that for DMOX derivatives, and pyrrolidides are more stable chemically so are better suited for chemically sensitive fatty acids such as epoxides.(see our web-pages - Preparation of derivatives for mass spectrometry).
Although 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), it should be remembered that this is only a relative feature, as the convention in mass spectrometry is to set the most abundant ion (base ion) at 100%, with others adjusted in relation to this. Is the base ion at an especially high abundance in the mass spectra of pyrrolidides, or are the remaining ions at low abundance? It is not difficult with modern mass spectrometry software to magnify minor ions, and if this is clearly indicated, there should be no objection when spectra are illustrated in publications.
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, and 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.
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 more 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 structures.
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 research programme. 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). Because of their polarity, an advantageous analytical strategy can be to prepare concentrates by chromatography or other means prior to GC-MS as described on a separate web page; this is often best accomplished as the methyl ester derivatives before preparing the pyrrolidides.
Straight-Chain Saturated Fatty Acids
The mass spectrum of palmitoyl pyrrolidine (16:0) is illustrated first -
The main points of fragmentation are illustrated simplistically as this is not intended to be a mechanistic treatise.
As mentioned earlier, the base peak is the McLafferty rearrangement ion at
m/z = 113, while that at m/z = 126 is relatively abundant (as with DMOX derivatives).
It has been necessary to magnify the ions in the high mass region at least four-fold relative to the base ion
to visualize the significant ions sufficiently in this instance.
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 and so forth.
There is usually a small ion at m/z = 70 representing a fragmentation at the pyrrolidine ring (not seen with DMOX derivatives).
The ion representing [M‑15]+ from loss of the terminal methyl group is small in comparison to that in the corresponding DMOX derivative in which this ion is derived from loss of a methyl group from the heterocyclic ring. Thus, in the spectrum of the pyrrolidide of 2H35‑octadecanoate -
- there is a small ion at m/z = 254 or [M-18]+ for the loss of the loss of the terminal methyl group (trideuterocarbon), but there is no such ion in the spectrum of the corresponding DMOX derivative. There are no significant ions representing loss of 2 and 3 carbon units (carbons 2 to 4) in the high mass range, which can cause problems of interpretation on occasion with DMOX derivatives, so pyrrolidides are better than DMOX derivatives for fatty acids with terminal functional groups.
As a further example, the mass spectrum of the pyrrolidide of the very-long-chain tetracosanoate (24:0) is -
- 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 relative to the base ion to see them clearly.
Pyrrolidides seem to be especially useful for shorter-chain fatty acids, because of the convenience of the method of preparation and their relatively low volatility. The mass spectrum of the pyrrolidide of hexanoate (6:0) -
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.
References
- Andersson, B.A. Mass spectrometry of fatty acid pyrrolidides. Prog. Chem. Fats other Lipids, 16, 279-308 (1978); DOI.
- 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 ω-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.
- 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.
- 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.
I can 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.
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