Fatty Alcohols. 1. Nicotinates Derivatives

Fatty alcohols can be characterized by GC-MS most readily as the nicotinate derivatives, first described by Vetter and Meister (1981). These are structurally analogous to 3-pyridylcarbinol esters and fragment in much the same way. Indeed, with alkyl moieties of the same basic structure, nicotinates and 3-pyridylcarbinol esters have identical molecular weights. The two most abundant ions at m/z = 107 and 124 correspond to fragmentation at the carboxyl moiety. Thereafter, interpretation of spectra is very similar to that for 3-pyridylcarbinol esters. Nicotinates are also very useful for structural determination of diacylglycerols and related lipids.

Formula of the nicotinate derivative of hexadecan-1-ol

D.J. Harvey was responsible for much of the early work on nicotinates, and his review article is most valuable (Harvey, 1992). The preparation procedure used in our laboratory is described below. The mass spectra of the nicotinates of fatty alcohols illustrated below were obtained in a study of fish waxes (Joh et al., 1995).

 

Saturated and Branched-Chain Fatty Alcohols

The mass spectrum of the nicotinate derivative of hexadecanol is illustrated first -

Mass spectrum of the nicotinate derivative of hexadecanol

As with 3-pyridylcarbinol esters, interpretation is easiest if we start with the molecular ion and work backwards (see the web pages on 3-Pyridylcarbinol esters esters of saturated and branched-chain fatty acids). There is a gap of 15 amu for loss of the terminal methyl group to m/z = 332, and then a series of ions 14 amu apart for fragmentations at successive methylene groups, until the characteristic ions at m/z = 124 and 107 are reached.

The same features are seen in the mass spectrum of the nicotinate derivative of octadecanol next -

Mass spectrum of the nicotinate derivative of octadecanol

Branched fatty alcohols can also be identified by this means in a similar manner to 3-pyridylcarbinol esters, thus in the spectrum of the nicotinate derivative of 14-methyl-hexadecanol (anteiso-isomer) -

Mass spectrum of the nicotinate derivative of 14-methyl-hexadecanol

- the gap of 28 amu between m/z = 304 and 332 reflects cleavage on either side of the carbon carrying the methyl group. As might be expected, this gap is shifted to between m/z = 318 and 346 in the spectrum of the iso-isomer (15-methyl-hexadecanol) next -

Mass spectrum of the nicotinate derivative of 15-methyl-hexadecanol

 

Monoenoic Fatty Alcohols

The mass spectrum of the nicotinate derivative of octadec-9-en-1-ol follows -

Mass spectrum of the nicotinate derivative of octadec-9-en-1-ol

Again, the spectrum resembles that for a 3-pyridylcarbinol ester (see the web pages on 3-Pyridylcarbinol esters of monoenoic fatty acids). The double bond is located by the gap of 28 amu between m/z = 234 and 260, while the doublet at m/z = 274 and 288 are invaluable guides (identical ions are present in the spectrum of 3-pyridylcarbinyl oleate).

The mass spectrum of the nicotinate derivative of octadec-11-en-1-ol -

Mass spectrum of the nicotinate derivative of octadec-11-en-1-ol

Here, the diagnostic ions are shifted upwards by 28 amu, i.e. for the double bond to between m/z = 262 and 288, with the doublet at m/z = 392 and 316. A further shift upwards of 28 amu for the diagnostic ions is then seen in the spectrum of the nicotinate of eicos-13-en-1-ol -

Mass spectrum of the nicotinate derivative of eicos-13-en-1-ol

 

Di- and Polyenoic Fatty Alcohols

The spectra reported here were from the first definitive finding of such fatty alcohols in a fish oil (Joh et al. (1995)). The mass spectrum of the nicotinate derivative of octadeca-9,12-dien-1-ol (the analogue of linoleic acid) is -

Mass spectrum of the nicotinate derivative of octadeca-9,12-dien-1-ol

The double bonds in positions 9 and 12 can be located by the gaps of 26 amu between m/z = 234 and 260, and 274 and 300, respectively. On the other hand, the limited information available suggests that a gap of 12 amu, analogous to that found with DMOX derivatives may be more useful with nicotinates, i.e. from m/z = 234 to 246, and 274 to 286, respectively.

The mass spectrum of the nicotinate derivative of octadeca-11,14-dien-1-ol -

Mass spectrum of the nicotinate derivative of octadeca-11,14-dien-1-ol

In this instance, the diagnostic ions are again shifted upwards by 28 amu from the previous example.

The mass spectrum of the nicotinate derivative of eicos-5,8,11,14,17-pentaen-1-ol -

Mass spectrum of the nicotinate derivative of eicos-5,8,11,14,17-pentaen-1-ol

Here, only the terminal double bonds can be located with confidence, and these by the gaps of 40 amu for the methylene groups followed by the double bond, i.e. from m/z = 284 to 324, and 324 to 364, for the double bonds in positions 14 and 17 respectively.

The mass spectrum of the nicotinate derivative of docos-7,10,13,16,19-pentaen-1-ol -

Mass spectrum of the nicotinate derivative of docos-7,10,13,16,19-pentaen-1-ol

Again, the double bonds in positions 13, 16 and 19 only can be located from the gaps of 40 amu between m/z = 272 and 312, 312 and 352, and 352 and 392 respectively.

There has been a suggestion in the literature that rather than preparing 3-pyridylcarbinol esters of fatty acids for structural analysis, better results might be obtained by reducing them to the aliphatic alcohols and then preparing nicotinates for mass spectrometry. From my admittedly limited experience of nicotinates relative to 3-pyridylcarbinol esters, I have to disagree.

 

Alkan-2-ols

From the limited number of mass spectra available to us, those of nicotinates of alkan-2-ols are almost indistinguishable in terms of which ions are formed from those of alkan-1-ols. For example, the mass spectrum of the nicotinate of hexadecan-2-ol is illustrated next -

Mass spectrum of the nicotinate derivative of hexadecan-2-ol

Essentially the same ions are present in the spectra of both isomers, but their relative abundances in the spectrum of the 2-alkanol derivative are lower in the high mass range. In contrast, 1- and 2-alkanols are readily distinguishable as the trimethylsilyl ether derivatives (see the web page dealing with TMS derivatives of alkanols).

 

Diacylglycerols Derived from Phospholipids

A common approach to the analysis of phospholipids consists in enzymatic hydrolysis to the diacylglycerols followed by conversion to UV-absorbing derivatives for separation by liquid chromatography. While most such derivatives offer little useful structural information when subjected to mass spectrometry, nicotinates are an important exception. Not only do they permit identification of which fatty acid is esterified to each position, but they allow location of the double bonds in the fatty acyl chains. Separations can also be monitored by UV absorption. The following spectra were obtained by separation of molecular species by reversed-phase HPLC on a base-deactivated ODS column, with particle-beam electron-impact mass spectrometry (Dobson et al., 1998). The spectra below have been adapted from this paper, where much more detailed interpretation is offered than is appropriate here.

For example, the mass spectrum of the nicotinate derivative of 1,2-diolein is illustrated first -

mass spectrum of a diacylglycerol nicotinate (18:1-18:1)

There is an abundant molecular ion, together with the ions at m/z = 106/7 and 124 for fragmentations at the nicotinoyl moiety. Ions at m/z = 444 ([M-RCOO]+) and 462 ([M-RCO+2]+) establish the size of the acyl moieties. The distinctive fragments shown serve to locate the double bonds in the chain. It is noteworthy that there is no dubiety about the molecular ion.

Next follow the mass spectra of the nicotinate derivatives of both 1-palmitoyl-2-oleoyl-glycerol and 1-oleoyl-2-palmitoyl-glycerol -

mass spectrum of a diacylglycerol nicotinate (16:0-18:1)

mass spectrum of a diacylglycerol nicotinate (18:1-16:0)

The spectra are qualitatively similar, but differ in the relative abundance of specific ions that serve to to determine the nature of the acyl-chains, specifically at m/z = 436 (M-RCO+2), and 461 (M-R'CO+1; not at m/z = 462 as for diolein).

Mass spectrum of nicotinate derivative of 1,2-palmitoyl/linoleoyl-glycerol in which the reverse isomers may be present, although only one formula is illustrated -

mass spectrum of a diacylglycerol nicotinate (16:0-18:2)

- and lastly, this may be true also for the nicotinate of 1,2-linoleoyl/linolenoyl-glycerol -

mass spectrum of a diacylglycerol nicotinate (18:2-18:3)

 

Preparation of Nicotinates

The following method was used to prepare the nicotinate derivatives for the above spectra (Dobson et al. (1998)). It was adapted from a procedure by Zollner and Schmid (1996).

Protocol:

At 0°C, N,N'-dicyclohexylcarbodiimide (Caution - carcinogenic!) (20 mg) was added to a solution of fatty alcohols (up to 2 mg), nicotinic acid (10 mg) and 4-dimethylaminopyridine (2 mg) in dichloromethane (3 mL). After 5 minutes, the mixture was allowed to warm to room temperature and left overnight. Hexane (2 mL) was then added and the product filtered through a cotton wool plug pre-washed with hexane. After taking to dryness, the product was purified by solid-phase extraction through a bonded NH2 column. After pre-washing the column with hexane, the product was applied in hexane and washed with hexane (8 mL). The required product was recovered by elution with hexane-acetone (95:5, v/v; 10 mL).

 

We have some spectra of further nicotinate derivatives of fatty alcohols on file also in our Archive pages, but without interpretation.

 

References

  • Dobson, G., Itabashi, Y., Christie, W.W. and Robertson, G.W. Liquid chromatography with particle-beam electron-impact mass spectrometry of diacylglycerol nicotinates. Chem. Phys. Lipids, 97, 27-39 (1998) (DOI: 10.1016/S0009-3084(98)00089-9).
  • 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).
  • Joh, Y.-G., Brechany, E.Y. and Christie, W.W. Characterization of wax esters in the roe oil of the amber fish Seriola aureovittata, by silver ion high-performance liquid chromatography. J. Am. Oil Chem. Soc., 72, 707-713 (1995) (DOI: 10.1007/BF02635660).
  • Vetter, W. and Meister, W. Nicotinates as derivatives for the mass spectrometry investigation of long chain alcohols. Org. Mass Spectrom., 16, 118-122 (1981) (DOI: 10.1002/oms.1210160304).
  • Zollner, P. and Schmid, R. Utility of nicotinoyl derivatives in structural studies of mono- and diacylglycerols by GC/MS. Part 3. Application to acylglycerols with methyl branchings and epoxy and cyclopropyl rings. J. Mass Spectrom., 31, 411-417 (1996) (DOI: 10.1002/(SICI)1096-9888(199604)31:43.0.CO;2-2).

Updated September 2, 2013

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