The Lipid Library

MASS SPECTRA OF METHYL ESTERS OF FATTY ACIDS

Part 7. Derivatization of the Double Bonds

Information on preparation of methyl esters per se with suitable protocols is available here, and the topic is discussed from a theoretical standpoint in some detail here. Sometimes it is necessary or helpful to derivatize methyl esters of fatty acids further by reaction at the double bonds in order to locate these by GC-MS. Preparation and mass spectral properties of dimethyl disulfide (DMDS) and of 4-methyl-triazoline-3,5-dione (MTAD) adducts are described below, together with the useful techniques of hydrogenation and deuteration.

Dimethyl Disulfide Adducts

To get round the problem of locating double bonds, it is possible to prepare specific derivatives of unsaturated fatty acids that 'fix' the double bond. The most useful of these for monoenes are the dimethyl disulfide adducts, as they have excellent mass spectrometric properties and are prepared in a simple one-pot reaction (Francis, 1981).

Protocol: The monoenes (1 mg) are dissolved in dimethyl disulfide (0.2 mL) and a solution (0.05 mL) of iodine in diethyl ether (60 mg/mL) is added. The mixture is stirred for 24 hours, then hexane (5 mL) is added, and the mixture is washed with dilute sodium thiosulfate solution, dried over anhydrous sodium sulfate and evaporated to dryness. The product is taken up in fresh hexane for injection directly onto the GC column.

The mass spectrum of the dimethyl disulfide adduct of methyl oleate is illustrated first -

Cleavage occurs between the carbons that originally constituted the double bond to yield two substantial fragment ions, i.e. that containing the terminal methyl part of the molecule at m/z = 173 and that with the carboxyl group at m/z = 217. A further prominent ion at m/z = 185 corresponds to the latter fragment with the loss of the elements of methanol.

The mass spectrum of the dimethyl disulfide adduct of methyl 11-octadecenoate (cis-vaccenate) follows -

The spectrum differs from the previous one in that the diagnostic ions are shifted by 28 amu as expected.

Dimethyl disulfide adducts have been utilized to identify monoenoic fatty acid isomers in a wide range of natural samples, and they have also been used for dienes in some circumstances. The review articles cited in the Introduction pages on mass spectrometry provide further details, and there is a comprehensive list of references in the Bibliography section of the website. There is also a separate article on the topic ( here...).

Diels-Alder (MTAD) Adducts for Conjugated Double Bonds

A useful derivative specific for determination of double bond positions in conjugated dienes is to form the Diels-Alder adduct of the fatty acid methyl ester by reaction with the reagent, 4-methyl-1,2,4-triazoline-3,5-dione (MTAD). Such derivatives have excellent mass spectrometric properties, enabling determination of structures in such samples as commercial conjugated linoleic acid (CLA) and the metabolites formed from this in animal tissues.

Reaction occurs almost instantaneously at room temperature and must be stopped immediately by adding 1,3-hexadiene (Dobson, 1998).

Protocol: The CLA methyl ester (220 μg; 1.15mM) and MTAD (425 μg; 5.8 mM) in dichloromethane (650 μL) are mixed in a test-tube at 0°C by agitating for less than 10 seconds. The reaction is immediately stopped by addition of 1,3-hexadiene, followed by agitation for a few seconds. Excess reagents are removed a stream of nitrogen at 30°C, and the sample is re-dissolved in dichloromethane for analysis by GC-MS.

The mass spectrum of the MTAD adduct of methyl 9-cis,11-trans-octadecadienoate is illustrated below -

Cleavage occurs on either side of the six-membered ring, enabling simple location of the carbons that originally constituted the conjugated double bond system, i.e. at m/z = 250 and 322. Confirmatory evidence comes from the ion representing loss of methanol from the ion containing the carboxyl moiety, i.e. at m/z = 290 in this instance. Indeed, these ions can be used with selective ion monitoring to quantify positional isomers in the presence of mixtures.

As an example, the spectrum of the MTAD adduct of the 10,12-18:2 isomer follows -

In this instance, the carbons that were part of the original conjugated double bond system can be located by the ions at m/z = 336 and 236, with that at 304 representing loss of methanol from the carboxyl-containing ion.

We have spectra of more MTAD adducts of conjugated dienes on file, and they can be accessed (but with no interpretation) from our Archive page.

When the reagent is reacted with a conjugated triene, such as punicic acid (9,11,13-octadecatrienoate), two possible products are formed.

Both of these products are separated by GC and give diagnostic spectra, first with the 9,11-double bonds reacting -

The key diagnostic ions are expected to be at m/z = 248 and 322, but with the latter further fragmentation has occurred with loss of a methoxyl group, so that the ion at m/z = 291 helps to define the structure.

Then with the 11,13-double bonds reacting -

In this instance interpretation is less straight forward, and the original article by Dobson cited above should be consulted. However, the important diagnostic ions at m/z = 222 and 348 are present, as is the latter less the elements of methanol (m/z =316).

The method can also be used with care to locate conjugated dienes in the presence of non-conjugated double bonds, e.g. for 5,8,11,13-eicosatetraenoate (see Sebedio et al., 1997), and the spectrum is shown below.

However, the reagent is best used with fractions enriched in conjugated fatty acids if possible, as a small amount of reaction with methylene-interrupted double bonds, moving them into conjugation, can sometimes occur.

Hydrogenation

Catalytic hydrogenation is a simple procedure that provides invaluable structural information regarding fatty acid identity, when combined with GC or GC-MS analysis. It is best carried out with methyl esters, but then there may be considerable advantages in conversion to picolinyl ester or DMOX derivatives for mass spectrometry.

Some needlessly complex procedures involving high pressures and temperatures are sometimes described in the literature, but details of a convenient practical procedure are described below (Christie, 2003).

Protocol: The unsaturated ester (1-2 mg) in a test-tube is dissolved in methanol (1 mL) and Adams' catalyst (platinum oxide; 1 mg) is added. The tube is connected via a two-way tap both to a reservoir of hydrogen (e.g. in a balloon or football bladder) at or just above atmospheric pressure, and to a vacuum pump. The tube is alternatively evacuated and flushed with hydrogen several times to remove any air, then it is shaken vigorously while an atmosphere of hydrogen at a slight positive pressure is maintained for 2 hr. At the end of this time, the hydrogen supply is disconnected, the tube is flushed with nitrogen and the solution is filtered to remove the catalyst. The solvent is evaporated under reduced pressure, and the required saturated ester is taken up in hexane or diethyl ether for GLC analysis.

At its simplest, hydrogenation is used merely to determine the chain length of components. By eliminating all unsaturated centres in fatty acid methyl esters from most samples of natural origin, a simple set of peaks is obtained for the saturated even-numbered homologous series and these can be compared with authentic standards. However, the presence of anomalous peaks may be an indication of novel structures. In samples of animal origin, small amounts of odd-chain fatty acids may be detected in the GC trace, together with methyl-branched fatty acids, usually iso- closely followed by anteiso-isomers, which are best identified as the picolinyl ester or pyrrolidide derivatives.

Deuteration

While hydrogenation eliminates unsaturation and aids identification and location of other functional groups, selective deuteration will assist both in locating unsaturation and characterizing other moieties. Indeed, deuteration has been used since the early days of mass spectrometry of lipids as a means of locating double bonds, and for unravelling fragmentation mechanisms, but the value of the procedure for structure determinations was limited with methyl ester derivatives as the wide range of rearrangement ions formed led to some scrambling of the deuterium atoms in the alkyl chain. However, by using nitrogen-containing derivatives, which give clean radical-induced fragmentations with minimal rearrangement, most problems have been eliminated. Again, the reaction is best carried out with methyl esters prior to conversion to picolinyl ester or DMOX derivatives for mass spectrometry.

Deuteration with deuterium gas and Wilkinson's catalyst (tris(triphenylphosphine)-rhodium(I) chloride) is usually employed for the purpose, and can be recommended. Gaseous deuterium is available commercially in small cylinders, or it can be generated in situ by reaction of deuterium chloride with sodium borodeuteride. It is essential to have a good excess of deuterium so that the reaction goes rapidly to completion, otherwise some isomerization of the double bonds and scrambling of the hydrogen atoms is possible (author, unpublished). The following method is based on that of Dickens et al. (1982).

Protocol: Methyl esters of unsaturated fatty acids are subjected to deuteration with deuterium gas and Wilkinson's catalyst. The fatty ester (up to 2 mg) and Wilkinson's catalyst (5 mg) in dioxane (1 ml) are degassed with helium in a tube fitted with a septum. The vessel is purged with five volumes of deuterium with constant stirring, and then is left with an atmosphere of deuterium at 60ºC for 2h. The solvent is removed in a stream of nitrogen and the required ester is obtained by adsorption chromatography on a small column of Florisil™ (0.5 g), eluted with hexane-acetone (96:4, v/v).

The technique was first used in conjunction with pyrrolidide derivatives, and has also been used with DMOX derivatives, but picolinyl esters appear best for the purpose, as they give cleaner radical-induced fragmentations with fewer rearrangement ions. Also, they give more abundant ions of high molecular weight with saturated fatty acids. On mass spectral analysis, clear diagnostic ion fragments are obtained that permit the determination of the positions of the original double bonds in the alkyl chain.

As an example, an usual fatty acid with two double bonds and a triple bond in conjugation in the seed oil of Tanacetum corymbosum was identified as octadeca-8,10-dien-12-ynoic acid by deuteration of the methyl ester derivative prior to conversion to the picolinyl ester for analysis by GC-MS (Tsevegsuren et al., 1998). The spectrum is -

Mass spectra of picolinyl esters are described elsewhere in these web pages, suffice for the moment to point out that one deuterium atom is added to each carbon of the double bonds and two to each carbon of the triple bonds, and these are easily identified from the mass spectrum.

A further example was the proof of structure of 12-oxo-octadec-9-enoic acid from milk fat (Brechany and Christie, 1994). Methodology of the kind described above has enabled us to identify a large number of different fatty acids of marine, plant and animal origins.

References

• Brechany, E.Y. and Christie, W.W. Identification of the unsaturated oxo fatty acids in cheese. J. Dairy Res., 61, 111-115 (1994).
• Christie, W.W. Lipid Analysis (3rd edition) (Oily Press, Bridgwater) (2003).
• Dickens, B.F., Ramesha, C.S. and Thompson, G.A. Quantification of phospholipid molecular species by coupled gas chromatography-mass spectrometry of deuterated samples. Anal. Biochem., 127, 37-48 (1982).
• Dobson, G. Identification of conjugated fatty acids by gas chromatography mass spectrometry of 4-methyl-1,2,4-triazoline-3,5-dione adducts. J. Am. Oil Chem. Soc., 75, 137-142 (1998).
• Francis, G.W. Alkylthiolation for the determination of double-bond position in unsaturated fatty acid esters. Chem. Phys. Lipids, 29, 369-374 (1981).
• Sebedio, J.L., Juaneda, P., Dobson, G., Ramilison, I., Martin, J.C., Chardigny, J.M. and Christie, W.W. Metabolites of conjugated isomers of linoleic acid (CLA) in the rat. Biochim. Biophys. Acta, 1345, 5-10 (1997).
• Tsevegsuren, N., Christie, W.W. and Lösel, D. Tanacetum (Chrysanthemum) corymbosum seed oil: a rich source of a novel conjugated acetylenic acid. Lipids, 33, 723-727 (1998).

Updated: 21/4/2008 Credits/disclaimer	Scottish Crop Research Institute (and MRS Lipid Analysis Unit), Invergowrie, Dundee (DD2 5DA), Scotland