Part 1. Saturated and Branched-Chain Fatty Acids

Mass Spectrometry of DMOX Derivatives

 

Formula of a DMOX derivative4,4-Dimethyloxazoline (DMOX) derivatives give excellent mass spectra of fatty acids with electron-impact ionization that frequently permit unequivocal identification. Any dubiety that may remain can be removed if the analyst has access to authentic spectra, and many of those illustrated below and in the other documents in this website should prove useful for this purpose. The gas chromatographic properties of DMOX derivatives are better than those of 3-pyridinylcarbinol ('picolinyl') esters and pyrrolidides, and many analysts consider them the best single derivative available for identification of fatty acids in mass spectrometric terms. This could be debated, especially when there are functional groups near the terminal end of the molecule, but in much of our research, we have used more than one type of derivative as they provide useful complementary data. One drawback of DMOX derivatives is that they are not very stable chemically; ring opening occurs rapidly on storage in the presence of traces of moisture, although this problem can be ameliorated by storing them in the presence of desiccant. However, there is no simple cleanup step, as is available for other derivatives.

DMOX derivatives can also be separated on a micro-preparative scale by reversed-phase HPLC if a base-deactivated stationary phase is employed (Christie, 1998), and this can be a useful means of enriching minor components for further analysis.

DMOX derivatives are very similar in their fragmentation properties in mass spectrometry to pyrrolidides. Indeed, in spite of the differences in structure, the two have identical molecular weights and the McLafferty ions from each are in the same place, for example. Although pyrrolidides appear to have fallen out of fashion, they are more stable chemically and have advantages in some circumstances, especially when functional groups are near the terminal carbon atom. DMOX derivatives are better with unsaturated fatty acids.

Chinese scientists were responsible for much of the early work on DMOX derivatives, and their papers are listed in the Bibliography Section of these pages. A definitive review of the topic, including mechanistic aspects, has been published by Spitzer (1997). References are listed when we are aware of prior formal publication of spectra in the scientific literature, but many of the following spectra will not have been published elsewhere.

Methods for preparing the derivatives are described elsewhere in our website in the document on Preparation of derivatives.

 

Straight-Chain Saturated Fatty Acids

DMOX derivatives are not at their best with saturated fatty acids as the ions of higher mass tend to be of low intensity, and rearrangement ions occur in greater abundance than with unsaturated fatty acid derivatives. However, they do at least give a usable molecular ion. This together with GC retention data is usually sufficient for definitive identification purposes. The mass spectrum of the DMOX derivative of palmitic acid is illustrated first (for example, see Zhang et al., 1988).

Mass spectrum of the DMOX derivative of palmitic acid

The McLafferty ion at m/z = 113 is usually the base peak (see the web pages on methyl esters of saturated fatty acids for a mechanistic discussion), accompanied by a prominent ion at m/z = 126. In general, the best approach to interpretation of the mass spectra of DMOX derivatives is to start with the molecular ion and work backwards. The molecular ion (m/z = 309) is small but recognizable in this instance, and then there is a gap of 15 amu to m/z = 294, followed sequentially by ions 14 amu apart (m/z = 280, 266, 252, and so forth), which can be considered simplistically as cleavage at successive methylene groups (but see the section on branched-chain acids below). Similar features are seen in the spectra of related fatty acids, as illustrated for the DMOX derivatives of dodecanoate (12:0) and docosanoate (22:0) –

Mass spectra of the DMOX derivatives of dodecanoic and docosanoic acids

We also have mass spectra on file for the DMOX derivatives of many more saturated fatty acids (6:0 to 30:0), including a number labelled with stable isotopes (Hamilton and Christie, 2000). These are illustrated without interpretation in the Archive section of these web pages.

 

Mono-Methyl-Branched-Chain Saturated Fatty Acids

DMOX derivatives are not the best for locating methyl-branched in fatty acids, especially in the most common fatty acids of this type with iso and anteiso-branches, although they have been used for the purpose by identifying ‘local intensity minima’ (Yu et al., 1988). This is one example, at least, where 3-pyridinylcarbinol esters (and pyrrolidides) are undoubtedly much better. The explanation lies in the fact that with saturated fatty acids, a methyl group can be lost readily from the heterocyclic ring to give two series of ions, which further fragment and confound any interpretation, especially in the higher molecular weight region of the spectrum (Hamilton and Christie, 2000). However, if taken in conjunction with GC retention data, these spectra may suffice for identification.

DMOX derivative of 15-methyl-hexadecanoate (iso-methyl-16:0) -

Mass spectrum of the DMOX derivative of 15-methyl-hexadecanoate

DMOX derivative of 14-methyl-hexadecanoate (anteiso-methyl-16:0) -

Mass spectrum of the DMOX derivative of 14-methyl-hexadecanoate

The spectra of the above two fatty acids resemble those of the corresponding saturated fatty acids, other than minor changes in ion intensity, with no features that locate the branch point, c.f. the spectra of the corresponding 3-pyridinylcarbinol esters or pyrrolidides, in contrast .

DMOX derivative of 11-methyl-octadecanoate -

Mass spectrum of the DMOX derivative of 11-methyl-octadecanoate

In this and other isomers, where the methyl branch is more central, it can be located by the gap of 28 amu between m/z = 224 and 252, for the loss of carbon 11 and the associated methyl group. However, as the diagnostic ions are of relatively low abundance, interpretation can be difficult when faced with an unknown.

In the mass spectrum of the DMOX derivative of 10-methyl-octadecanoate (tuberculostearate), the gap of 28 amu is now between m/z = 210 and 238 -

Mass spectrum of the DMOX derivative of 10-methyl-octadecanoate

The spectrum of the DMOX derivative of 9-methyl-tetradecanoate has the gap of 28 amu between m/z = 196 and 224.

Mass spectrum of the DMOX derivative of 9-methyl-tetradecanoate

The mass spectrum of the DMOX derivative of 3-methyl-pentadecanoate (unpublished) is unusual if not unexpected in that the customary ion at m/z = 126 is missing and there is a gap of 27 amu between m/z = 113 to 140 to locate the branch point.

Mass spectrum of the DMOX derivative of 3-methyl-pentadecanoate

We have mass spectra on file for the DMOX derivatives of many more branched-chain fatty acids. These are illustrated without interpretation in the Archive section of these web pages.

 

Multi-Methyl-Branched-Chain Saturated Fatty Acids

Isoprenoid fatty acids, derived from phytol, are found at low levels in many animal species, but especially those of marine origin. Mass spectra of the three most common of these are now illustrated. The DMOX derivative of 4,8,12- trimethyl-tridecanoate -

Mass spectrum of the DMOX derivative of 4,8,12- trimethyl-tridecanoate

The branch points are indicated by the gaps of 28 amu between the ions marked, although that for the methyl group in position 12 is not distinct.

DMOX derivative of 2,6,10,14-tetramethyl-pentadecanoate or pristanate -

Mass spectrum of the DMOX derivative of 2,6,10,14-tetramethyl-pentadecanoate

Although the diagnostic ions tend to be small, each of the branch-points can be located from the spectrum. The methyl branch in position 2 is easily seen as the expected ions at m/z = 113 and 126 are shifted upwards to m/z = 127 and 140, respectively. The remaining branches are located by the gaps between m/z = 168 and 196, 238 and 266 and 310 and 336 (the origin of the ion at m/z = 310 is not known).

DMOX derivative of 3,7,11,15-tetramethyl-hexadecanoate or phytanate -

Mass spectrum of the DMOX derivative of 3,7,11,15-tetramethyl-hexadecanoate

In this instance, only the ion at m/z = 126 is shifted upwards by 14 amu (c.f. the spectrum for 3-methyl-15:0 above), and the gaps of 28 amu between m/z = 182 and 210, 252 and 280, and 322 and 350 serve to locate the methyl branches in positions 7, 11 and 15, respectively. It is interesting that the iso-methyl branch in the last fatty acid can be clearly located, although this is not possible for mono-methyl-substituted fatty acids.

We also have the mass spectrum of the DMOX derivative of a non-isoprenoid dimethyl fatty acid (8,10-dimethyl-16:0) in our Archive section here...

 

References

  • Christie, W.W. Gas chromatography-mass spectrometry methods for structural analysis of fatty acids. Lipids, 33, 343-353 (1998) (DOI: 10.1007/s11745-998-0214-x).
  • 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).
  • Spitzer, V. Structure analysis of fatty acids by gas chromatography - low resolution electron impact mass spectrometry of their 4,4-dimethyloxazoline derivatives - a review. Prog. Lipid Res., 35, 387-408 (1997) (DOI: 10.1016/S0163-7827(96)00011-2).
  • Yu, Q.T., Liu, B.N., Zhang, J.Y. and Huang, Z.H. Location of methyl branchings in fatty acids: Fatty acids in uropygial secretion of Shanghai ducks by GC-MS of 4,4-dimethyloxazoline derivatives. Lipids, 23, 804-810 (1988) (DOI: 10.1007/BF02536225).
  • Zhang, J.Y., Yu, Q.T., Liu, B.N. and Huang, Z.H. Chemical modification in mass spectrometry IV. 2-Alkenyl-4,4-dimethyloxazolines as derivatives for double bond location of long-chain olefinic acids. Biomed. Environ. Mass Spectrom., 15, 33-44 (1988) (DOI: 10.1002/bms.1200150106).

 Updated September 11, 2013

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