The Lipid Library

Beginners' Guide to Mass Spectrometry of fatty acids.

2. General Purpose Derivatives

Abstract: Simple nitrogen-containing derivatives of fatty acids, such as picolinyl esters or 4,4-dimethyloxazolines, make identification of most fatty acids by mass spectrometry a relatively simple task.

In the first contribution dealing with this topic, I tried to persuade you that there was no need to run for cover when the words "mass spectrometry" (MS) were spoken. Basic gas chromatography (GC) equipment linked to a mass spectrometer, which can handle most fatty acid derivatives, is available for about the cost of a luxury car and it will take up considerably less space. Running costs are less than those of an expensive car, and provided it is maintained properly, it will run without trouble for 10 years or so. You do not need a specialized technician to operate it, and interpretation of the results can be relatively straight forward. Therefore, I felt justified in introducing the topic of mass spectrometry to a non-specialist audience.

Fatty Acid Derivatives

I have used an analogy in which mass spectrometry is compared to demolishing and re-assembling a brick wall [1]. If we take it apart a few bricks at a time, it is possible to reassemble it easily. We will know the correct dimensions and where any door or window should be placed. With a fatty acid derivative, in comparison, we need to confirm that it is indeed a fatty acid, determine the molecular mass and then locate any double bonds or other functional groups. Methyl ester derivatives are not suitable, but there are invaluable alternatives. All of these contain nitrogen atoms in close proximity to the carboxyl group. With such compounds the nitrogen, rather than the aliphatic chain (as with methyl esters), carries the charge when the molecule is ionized in the mass spectrometer. Rather uniform fragmentation occurs along the aliphatic chain, and with a little experience it is easy to pick out functional groups such as double bonds, methyl branch points or cyclic structures.

Three derivatives have been widely used for the purpose: pyrrolidides, picolinyl esters and 4,4-dimethyloxazoline (DMOX) derivatives (Figure 1). Pyrrolidides were the first derivatives of this type to be described about 30 years ago, but they have been overtaken by others, presumably because their gas chromatographic properties are less than ideal. Picolinyl esters have better mass spectrometric properties than pyrrolidides, and DMOX derivatives are excellent derivatives for chromatography, so these two are now favoured in most laboratories.

Figure 1. Chemical formulae for pyrrolidide, picolinyl ester and dimethyloxazoline derivatives.

In addition to the review articles cited in the previous document, definitive reviews on the use of pyrrolidides [2], picolinyl esters [3] and DMOX derivatives [4] have been published.

Picolinyl Esters

Picolinyl esters can be prepared best from free fatty acids so it is often necessary to first hydrolyse an intact lipid sample or methyl ester. The original method involved dissolving the fatty acid in an excess of thionyl chloride to form the acid chloride, which was then reacted with a 1% solution of 3-hydroxymethylpyridine in acetonitrile to form the picolinyl ester for direct analysis by GC-MS. In my laboratory, we prefer a mild quantitative method involving the formation of an imidazolide by reacting the fatty acid with 1,1'-carbonyldiimidazole in dichloromethane prior to reaction with the picolinyl reagent in triethylamine in the presence of 4-pyrrolidinopyridine as a catalyst [4].

As an example, the mass spectrum of the picolinyl ester derivative of (gamma-linolenic acid (6,9,12-18:3) is illustrated in Figure 2. There are large ions at m/z = 92, 108, 151 and 164, which contain the pyridine ring and various elements of the carboxyl group and serve mainly to indicate that the compound is indeed a picolinyl ester. It is more instructive to consider the high molecular weight part of the spectrum. There is a good molecular ion (m/z = 369), useful confirmation that we have a C₁₈ fatty acid with three double bonds. Then there is a uniform series of ions 14 atomic mass units (amu) apart, representing loss of each successive methyl and methylene group from the terminal end of the molecule, until we reach the ion at m/z = 298. Then there is a gap of 26 amu for the carbons constituting the terminal double bond to m/z = 272, a further gap of 14 amu for the methylene group at carbon-11, then another gap of 26 amu between m/z = 234 and 258, a gap of 14 amu for the methylene group at carbon-8, and so forth. The double bond nearest to the carboxyl group nearest to the carboxyl group is not always easily spotted from first principles, but with a little experience it is not too difficult to define. Of course, it always helps to have access to spectra of standards.

Figure 2.Mass spectrum of the picolinyl ester derivative of 6,9,12-octadecatrienoate.

All spectra are therefore interpreted in the same way, by starting with the molecular ion and working backwards one methylene group at a time until a functional group is reached. Thus if there is a methyl branch, for example, there will be a gap of 28 amu for loss of the methylene and its attached methyl group.

4,4-Dimethyloxazoline (DMOX) Derivatives

DMOX derivatives can be prepared simply by reacting the free fatty acid (or the methyl ester or even an intact lipid) with 2-amino-2-methyl-1-propanol (AMP) in a micro-reaction vial at 180°C for 16 hours in a nitrogen atmosphere [5]. However, we have observed that the product must be stored under strictly anhydrous conditions otherwise partial hydrolysis can occur.

The mass spectrum of the DMOX derivative of oleic acid is illustrated in Figure 3. In this instance the ions at m/z = 113 and 126 confirm that we have indeed formed the DMOX derivative. Again, there is a clear molecular ion at m/z = 335, followed by gaps of 14 amu for the loss of each successive methylene group (m/z = 320, 306, 292, 278, etc), until we find a gap of 12 amu which is indicative of the presence of the double bond, between m/z = 196 and 208. To locate this precisely, we must use the "12 mass rule", i.e.

"if a there is an interval of 12 amu between the most intense peaks of clusters of ions containing n and n-1 carbon atoms, there is a double bond between carbon n and n+1 in the molecule".

This may seem rather convoluted, but works remarkably well in practice. Other functional groups, such as branch points or ring structures, are located as with picolinyl esters.

Figure 3. Mass spectrum of the DMOX derivative of oleate.

Which is Best?

How do we decide when to use picolinyl esters and when DMOX derivatives? DMOX derivatives have excellent chromatographic properties so can be easily resolved on all the common polar stationary phases used in GC analysis. In my opinion, the mass spectral characteristics in the high mass range are not as good as with picolinyl esters in general, although DMOX derivatives do appear to be especially useful with cyclic and conjugated fatty acids. Picolinyl esters require much higher temperatures than the equivalent DMOX or methyl ester derivatives to elute them from GC columns, and at first they could only be analysed on non-polar stationary phases. However, we have used the thermally stable polar BPX-70 and Supelcowax 10 columns with some success with picolinyl esters of fatty acids with up to 20 carbon atoms.

Pyrrolidides should not be forgotten. In spite of the marked differences in structure, pyrrolidides have exactly the same molecular weight as the corresponding DMOX derivatives and they give very similar fragmentation patterns in mass spectrometry. They can be preferable to DMOX derivatives for fatty acids with terminal functional groups.

New MS methods involving acetonitrile-chemical ionization look interesting, but are more limited in their potential range of application than the derivatives described above.

Finally in answering the question of which is best, I prefer to not to take a rigid stance. I prefer picolinyl esters when I know that I am facing samples containing novel fatty acids structures, although I almost always prepare DMOX derivatives for confirmation or for rapid screening of straight-forward samples. The various types of derivative should be considered as complementary to each other - not as simple alternatives.

References

1. Christie, W.W. Beginners' guide to mass spectrometry of fatty acids. 1. The nature of the problem. Lipid Technology, 8, 18-20 (1996).
2. Andersson, B.A. Mass spectrometry of fatty acid pyrrolidides. Prog. Chem. Fats other Lipids, 16, 279-308 (1978).
3. Harvey, D.J. Mass spectrometry of picolinyl and other nitrogen-containing derivatives of fatty acids. In: Advances in Lipid Methodology - One, pp. 19-80 (W.W. Christie, ed., Oily Press, Dundee) (1992).
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).
5. Balazy, M. and Nies, A.S. Characterization of epoxides of polyunsaturated fatty acids by mass spectrometry via 3-pyridinylmethyl esters. Biomed. Environ. Mass Spectrom., 18, 328-336 (1989).
6. Yu, Q.T., Liu, B.N., Zhang, J.Y. and Huang, Z.H. Location of methyl branches in fatty acids: Fatty acids in uropygial secretion of Shanghai ducks by gas chromatography-mass spectrometry of 4,4-dimethyloxazoline derivatives. Lipids, 23, 804-810 (1988).

This article was first published in Lipid Technology, 8, 64-66 (1996) and has now been substantially re-written.

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Updated: 1/5/2007 Credits/disclaimer	Scottish Crop Research Institute (and MRS Lipid Analysis Unit), Invergowrie, Dundee (DD2 5DA), Scotland	Right click on the icon to open in a new window