Part 2. Branched-Chain Fatty Acids

As cautioned in the 'Introduction' to these documents, the mass spectra of methyl esters sometimes afford limited information only concerning the structures of fatty acids. The molecular weight is usually obtainable and is an important piece of information. GC retention data are also highly relevant. However, the more common saturated methyl-branched fatty acids can often be identified from the mass spectra of their methyl ester derivatives, especially when spectra of model compounds are available for comparison purposes. Here, only spectra encountered in my own research can be described in detail.


Monomethyl-Branched Fatty Acids

Very many natural branched-chain fatty acids have been identified by means of mass spectrometry of the methyl ester derivatives, but generally this means that subtle differences only from the spectra of the straight-chain analogues must be utilized for identification purposes. Pyridinylcarbinol (‘picolinyl’) esters and pyrrolidides are the best derivatives in this instance (see the appropriate sections of these web pages), but dimethyloxazoline derivatives are less suitable, especially for the iso- and anteiso-methyl derivatives, which are most often encountered in nature. The definitive paper on the mass spectra of methyl esters of mono-methyl-branched fatty acids has data for the complete series of (2- to 17-methyl)-octadecanoates (Apon and Nicolaides, 1975), although much of the data for the more important acids from a biological standpoint were published earlier (Ryhage and Stenhagen, 1959). Representative spectra are illustrated below with brief details of interpretation only.

The two most common type of branched-chain fatty acids are those with methyl branches in the iso or anteiso positions and representative spectra are shown below. Unfortunately, the former has the least distinctive spectra of all the isomers, and it is helpful to know that these two elute after most other isomers from all GC phases; the iso-isomer elutes before the anteiso (but earlier than the linear fatty acid with the same number of carbons). If in doubt, hydrogenation will remove unsaturated components that might confuse identification.

The mass spectrum of methyl iso-methylhexadecanoate(or 15-methylhexadecanoate) -

Mass spectrum of methyl iso-methylhexadecanoate

In this instance, the features that may distinguish the spectrum from that of the straight-chain analogue and other branched isomers are not always easy to see. The [M-15]+ ion (m/z =269) may be larger in the spectrum of an iso-isomer than in the straight-chain analogue, and there is usually a doublet of ions for [M-31/2]+ (see the papers cited above). Any other significant ions are rather small (<1% of the base peak), and equivalent to [M-65]+, [M-55]+ and [M-56]+, or here at m/z = 219, 229 and 228, respectively, but these may not apply uniformly across the chain-length range. As with normal saturated fatty acids, the McLafferty rearrangement ion at m/z = 74 is the base peak.

As a further example, the mass spectrum of methyl anteiso-methyl-hexadecanoate(or 14-methyl-hexadecanoate) is shown next -

Mass spectrum of methyl anteiso-methyl-hexadecanoate

The molecular ion is clearly seen and the important distinguishing feature from that of the straight-chain analogue is that an ion at [M-29]+ (m/z = 255) is more abundant than that equivalent to [M-31]+. Also, an ion at [M-61]+ (m/z = 223) is small but distinctive (sometimes with ions at [M-60]+ and [M-79]+) (again see the papers cited above). We have found these ions in the C14, C16 and C18 analogues, but not C20 and C24 (number of carbons in the straight chain).

Fatty acids with methyl branches in other positions are found in bacterial lipids and can also be produced by animal tissues, e.g. in ruminants or in human sebaceous secretions and vernix caseosa (the greasy secretion on the newborn baby), when methylmalonate is utilized instead of malonate in fatty acid biosynthesis. The methyl esters may then give distinctive fragments ('a' and 'b', together with 'a+1' and 'a+2') on either side of the branch as shown -

Fragmentation of a branched ester

In addition, there may be diagnostic ions for 'b' after the loss of methanol, or strictly speaking a methoxyl group plus a hydrogen atom ([b-32]+), and then for a further loss of a water molecule ([b-50]+).

As mentioned earlier, the examples illustrated below below were selected from the limited range available to us from our past research efforts. Most were found in sponges, where they were probably derived from bacteria ingested as part of their diet.

Thus, in the mass spectrum of methyl 15-methyl-docosanoate, ion 'a' is at m/z = 241, and ion 'b' at m/z = 269.

Mass spectrum of methyl 15-methyl-docosanoate

In the mass spectrum of methyl 14-methyl-eicosanoate, ion 'a' is at m/z = 227, and ion 'b' at m/z = 255.

Mass spectrum of methyl 14-methyl-eicosanoate

In the spectrum of methyl 13-methyl-eicosanoate, ion 'a' is at m/z = 213 and ion 'b' at m/z = 241.

Mass spectrum of methyl 13-methyl-eicosanoate

In the spectrum of methyl 11-methyl-octadecanoate, ion 'a' is at m/z = 185 and ion 'b' at m/z = 213.

Mass spectrum of methyl 11-methyl-octadecanoate

In the spectrum of methyl 10-methyl-hexadecanoate(a homologue of 10-methyl-octadecanoate or 'tuberculostearate' – a marker for the pathogen Mycobacterium tuberculosis), ion 'a' is at m/z = 171 and ion 'b' at m/z = 199.

Mass spectrum of methyl 10-methyl-hexadecanoate

In the spectrum of methyl 9-methyl-tetradecanoate, ion 'a' is at m/z = 157 and ion 'b' at m/z = 185.

mass spectrum of methyl 9-methyl-tetradecanoate

However, it is evident that many of the same ions occur in these various mass spectra, differing only in relative abundance. It is important to note that it might be difficult to identify an unknown unequivocally from such data without access to spectra of model compounds.

The spectrum of methyl 3-methylpentadecanoateis rather different. Ion 'b' at m/z = 101 (replacing that normally found at m/z = 87) is especially distinctive.

Mass spectrum of methyl 3-methylpentadecanoate

In all of these spectra, ions equivalent to [M-43]+ and [M-29]+, which are presumed to be due to the loss of fragments consisting of carbons 2 to 4 and 2 to 3, respectively, are prominent. The spectrum of the 3-isomer has characteristic ions at [M-57]+ and [M-53]+, presumably because the fragments incorporate the methyl branch (see the web pages on normal saturated fatty acids for a discussion of the mechanism).


Multi-Methyl Branched Fatty Acids

Isoprenoid fatty acids, derived from metabolism of the phytol of chlorophyll, are significant components of marine oils and of ruminant fats. Phytanic acid can accumulate in plasma of patients suffering from Refsum's syndrome. A number of different isomers have been found in nature, and the mass spectra of the three most common of these are illustrated below.

The mass spectrum of methyl 4,8,12-tridecanoate(from a fish oil) -

Mass spectrum of methyl 4,8,12-tridecanoate

The McLafferty ion at m/z = 74 is no longer the base peak in the spectrum, because of the presence of the methyl branch on carbon 4; there is thus only one hydrogen atom available for abstraction (see the web pages on mass spectra of normal saturated fatty acids for a discussion of the mechanism).

The mass spectrum of methyl pristanateor 2,6,10,14-tetramethylpentadecanoate is -

Mass spectrum of methyl pristanate or 2,6,10,14-tetramethylpentadecanoate

The mass spectrum of methyl phytanateor 3,7,11,15-tetramethylhexadecanoate is -

Mass spectrum of methyl phytanate or 3,7,11,15-tetramethylhexadecanoate

Note that the McLafferty ion at m/z = 88 is the base peak in the spectrum of methyl pristanate as it now contains the methyl group on carbon 2, while the ion at m/z = 101 for a fragment containing the methyl branch in position 3 dominates the spectrum of methyl phytanate. I will leave the reader to sort out the remaining 'a' and 'b' ions for the these spectra - I am content to consider them as fingerprints. A review by Lough (1975)discusses these spectra in some detail.

Spectra of many more methyl esters of branched-chain fatty acids can be accessed from our Archive pages (without interpretation).

Note.Those with access to instruments with a facility for collisional dissociation of molecular ions generated by electron ionization (tandem mass spectrometry) will be able to obtain additional data that should remove any dubiety regarding interpretation (Ran-Ressler et al., 2012).



  • Apon, J.M.B. and Nicolaides, N. The determination of the position isomers of the methyl branched FA esters by capillary gas chromatography/mass spectrometry.J. Chromatogr. Sci., 13, 467-473 (1975).
  • Lough, A.K. The chemistry and biochemistry of phytanic, pristanic and related acids. Prog. Chem. Fats other Lipids., 14, 5-48 (1975) (DOI: 10.1016/0079-6832(75)90001-4).
  • Ran-Ressler, R.R., Lawrence, P. and Brenna, J.T. Structural characterization of saturated branched chain fatty acid methyl esters by collisional dissociation of molecular ions generated by electron ionization. J. Lipid Res., 53, 195-203 (2012) (DOI: 10.1194/jlr.D020651).
  • Ryhage, R. and Stenhagen, E. Mass spectrometric studies. IV. Esters of monomethyl-substituted long chain carboxylic acids. Arkiv Kemi, 15, 291-315 (1959).

Updated January 14, 2013