Part 1. Saturated and Branched-Chain Fatty Acids

Mass Spectrometry of 3-Pyridylcarbinol Esters

Formula of 3-pyridylcarbinol tetradecanoateAlthough I do not wish to be dogmatic on the subject, in my opinion, 3-pyridylcarbinol ('picolinyl') esters are the best derivatives available for identification of fatty acids by gas chromatography-mass spectrometry (GC-MS), at least in mass spectrometry terms. Their GC properties are far from ideal, but many of the modern polar stationary phases are sufficiently thermally stable to afford good resolution. That said, dimethyloxazoline (DMOX) and pyrrolidide derivatives are also extremely useful, and can be better than 3-pyridylcarbinol esters in some circumstances. For example, DMOX derivatives have much better GC properties and are particularly suited to location of conjugated double bond systems. Rather than considering them as competitors for the title 'ideal method', I prefer to treat them as providing useful complementary data. Faced with a novel sample, it is always useful to prepared more than one type of derivative for analysis by GC-MS.

It should be noted that for most of the 30 years since they were first described, 3-pyridylcarbinol esters have been incorrectly termed ‘picolinyl’ esters. The correct trivial name is ‘nicotinyl’ esters, but all dubiety is removed if they are given the more systematic name ‘3-pyridylcarbinol’ (picolinyl alcohol is in fact 2-pyridylcarbinol).

This and the next few web pages describe the use of 3-pyridylcarbinol esters specifically for structure determination of fatty acids. In addition to analysis by GC, they can be separated by reversed-phase HPLC provided that a base-deactivated stationary phase is employed if a small amount of pyridine is added to the mobile phase - a useful technique to enrich minor components (Christie, 1998) (see our web page on concentration of minor components). Methods for preparing the derivatives are described elsewhere in our website in the section Preparation of derivatives.

D.J. Harvey was responsible for much of the early work on 3-pyridylcarbinol ('picolinyl') esters, and his review article is still very valuable (Harvey, 1992).

 

Straight-Chain Saturated Fatty Acids

The mass spectrum of 3-pyridylcarbinyl palmitate (hexadecanoate or 16:0) (Harvey, 1982) is illustrated -

Mass spectrum of 3-pyridylcarbinyl palmitate

It is typical in that it has prominent ions at m/z = 92, 108,151 and 164, which are all fragments about the pyridine ring (if any of these ions is missing from a spectrum it may be indicative of a functional group adjacent to the carboxyl moiety). The molecular ion (m/z = 347) is easily distinguished and it is always odd-numbered, because of the presence of the nitrogen atom, but most other ions are even numbered. Ions below m/z = 92 can usually be ignored.

In interpreting such spectra, the simplest approach is to start with the molecular ion and progress downward, as if one were unzipping the molecule one methylene group at a time. Thus there is loss of a methyl group to m/z = 332, followed by a series of ions 14 amu apart for loss of successive methylene groups, i.e. m/z = 318, 304, 290, 276 and so forth. There is little sign of the complex rearrangement ions that can be found with such fatty acid derivatives as methyl esters.

Mass spectral fragmentations for 3-pyridylcarbinyl palmitate

The main fragmentations are often portrayed simplistically as cleavages at the points shown, but in reality those close to the carboxyl group can involve some rearrangement with specific hydrogen abstractions (Harvey, 1992; Hamilton and Christie, 2000; Yang et al., 2006). In particular, Yang and colleagues (2006) have shown that the ion at m/z = 151 is not formed simply by the well-known McLafferty rearrangement as is usually proposed (see our web pages on Methyl esters of Saturated Fatty Acids for a discussion of the McLafferty mechanism). However, those ions further down the saturated chain probably represent simple radical-induced cleavage. As these web pages are not intended as detailed mechanistic accounts, fragmentations are generally represented in a simple manner here. Those with an interest in fragmentation mechanisms will find that a study of the spectra of the saturated deuterated derivatives in our Archive page will be a useful exercise.

There follow spectra for 3-pyridylcarbinyl decanoate (10:0) -

Mass spectrum of 3-pyridylcarbinyl decanoate

- and 3-pyridylcarbinyl tetracosanoate (24:0), which show essentially the same features -

Mass spectrum of 3-pyridylcarbinyl tetracosanoate

We have spectra on file for 3-pyridylcarbinol esters of straight-chain fatty acids from 2:0 to 30:0, including nearly all the odd-chain ones, and others labelled with stable isotopes. These can be accessed (but without interpretation) from our Archive page. Only a few of them have been published formally elsewhere.

 

Monomethyl-Branched-Chain Saturated Fatty Acids

The majority of naturally occurring monomethyl-branched fatty acids have saturated alkyl chains, and the most common of these are the iso- and anteiso-methyl isomers. They are produced by bacteria mainly, but they can enter animal tissues via the food chain, for example. The mass spectrum of 3-pyridylcarbinyl iso-methyl-octadecanoate (17-methyl) follows (Harvey, 1982) -

Mass spectrum of 3-pyridylcarbinyl iso-methyl-octadecanoate

The spectrum resembles that of a straight-chain saturated fatty acid except for the very obvious gap of 28 amu between m/z = 346 and 374, which represents loss of carbon-17 and its attached methyl group, locating it definitively. This is one example where 3-pyridylcarbinol esters have a distinct advantage over other types of derivative.

In the mass spectrum of 3-pyridylcarbinyl anteiso-methyl-octadecanoate (16-methyl), this gap is shifted to between m/z = 332 and 360 as -

Mass spectrum of 3-pyridylcarbinyl anteiso-methyl-octadecanoate

With that of 3-pyridylcarbinyl 15-methyloctadecanoate (not published elsewhere), the gap is shifted to m/z = 318 to 346 -

Mass spectrum of 3-pyridylcarbinyl 15-methyloctadecanoate

With that of 3-pyridylcarbinyl 11-methyloctadecanoate (not published elsewhere), the gap is between m/z = 262 and 290 -

Mass spectrum of 3-pyridylcarbinyl 11-methyloctadecanoate

With that of 3-pyridylcarbinyl 10-methyloctadecanoate (tuberculostearate) (not published elsewhere), the gap is between m/z = 248 and 276 -

Mass spectrum of 3-pyridylcarbinyl 10-methyloctadecanoate

The mass spectrum of 3-pyridylcarbinyl 3-methylpentadecanoate is interesting in that the usual ion at m/z = 164 is shifted to 178, and there is a gap of 27 amu between m/z = 151 and 178 to locate the branch point.

Mass spectrum of 3-pyridylcarbinyl 3-methylpentadecanoate

Of course, it is easy to predict what the diagnostic features in the spectra of other isomers or homologues not illustrated or archived here will be.

We have spectra of many more 3-pyridylcarbinol esters of branched-chain saturated fatty acids on file, and they can be accessed (but without interpretation) from our Archive page. Only a few of these have been published elsewhere.

 

Di- and Polymethyl-Branched-Chain Saturated Fatty Acids

Similar principles to the above apply in the interpretation of mass spectra of 3-pyridylcarbinol esters of di- and polymethyl-branched-chain fatty acids. For example, the 3-pyridylcarbinol ester of an unusual dimethyl-branched fatty acid from a sponge, i.e. 8,10-dimethyl-hexadecanoate follows (Nechev et al., 2002).

Mass spectrum of 3-pyridylcarbinyl 8,10-dimethyl-hexadecanoate

Gaps of 28 amu between m/z = 220 and 248, and between m/z = 262 and 290, locate the branch points.

Polymethyl-branched fatty acids derived from isoprene units via phytol are more common in nature, especially as minor components of fish oils. For example, the spectrum of the 3-pyridylcarbinol ester of 4,8,12-trimethyltridecanoate is -

Mass spectrum of 3-pyridylcarbinyl 4,8,12-trimethyltridecanoate

There are clear gaps of 28 amu between ions at m/z = 164 and 192, 234 and 262, and 304 and 332, which locate the methyl groups on carbons 4, 8 and 12, respectively (Christie, 1997).

In the spectrum of the 3-pyridylcarbinol ester of the homologous fatty acid 5,9,13-trimethyl-tetradecanoate (not published elsewhere), the diagnostic gaps are all 14 amu higher as anticipated -

Mass spectrum of 3-pyridylcarbinyl 5,9,13-trimethyl-tetradecanoate

The mass spectrum of 3-pyridylcarbinyl 2,6,10,14-pentadecanoate (pristanate) has some similar features (not published elsewhere) -

Mass spectrum of 3-pyridylcarbinyl 2,6,10,14-pentadecanoate

- for example, the methyl branches in positions 6, 10 and 14 are located by gaps of 28 amu between m/z = 206 and 234, 276 and 304, and 346 and 374 respectively. However, the presence of the 2-methyl group is recognized as the the ion usually found at m/z = 151 is shifted to 165, while the ion normally at m/z = 164 is shifted to 178.

In comparison, in the spectrum of the homologous 3,7,11,15-tetramethylhexadecanoate (phytanate), the gaps for the methyl branches on carbons 7, 11 and 15 are shifted up by 14 amu as expected (Christie, 1989).

Mass spectrum of 3-pyridylcarbinyl 3,7,11,15-tetramethylhexadecanoate

The methyl group on carbon 3 is located by the fact that the ion at m/z = 151 is again substantial, while that normally at m/z = 164 has shifted to 178.

 

References

  • Christie, W.W. Gas Chromatography and Lipids (Oily Press, Dundee) (1989).
  • Christie, W.W. Structural analysis of fatty acids. In: Advances in Lipid Methodology - Four, pp. 119-169 (edited by W.W. Christie, Oily Press, Dundee) (1997).
  • 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).
  • Harvey, D.J. Picolinyl esters as derivatives for the structural determination of long chain branched and unsaturated fatty acids. Biomed. Mass Spectrom., 9, 33-38 (1982) (DOI: 10.1002/bms.1200090107).
  • Harvey, D.J. Mass spectrometry of picolinyl and other nitrogen-containing derivatives of lipids. In: Advances in Lipid Methodology - One, pp. 19-80 (edited by W.W. Christie, Oily Press, Ayr) (1992).
  • Nechev, J., Christie, W.W., Robaina, R., de Diego, F.M., Ivanova, A., Popov, S. and Stefanov, K. Chemical composition of the sponge Chondrosia reniformis from the Canary Islands. Hydrobiologia, 489, 91-98 (2002) (DOI: 10.1023/A:1023206620304).
  • Yang, S., Minkler, P., Hoppel, C. and Tserng, K.-Y. Picolinyl ester fragmentation mechanism studies with application to the identification of acylcarnitine acyl groups following transesterification. J. Am. Soc. Mass Spectrom., 17, 1620-1628 (2006) (DOI: 10.1016/j.jasms.2006.07.004).

Updated: August 5th, 2013 

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