Gas Chromatography-Mass Spectrometry and Fatty Acids

A.  Introduction

In recent years, gas chromatography in combination with mass spectrometry (MS) has become one of the most powerful tools in the hands of lipid analysts. Simple GC-MS systems, such as the Finnigan MAT "ion trap" detector and the Hewlett Packard "mass selective detector", have become less costly, more reliable and simpler to use; the author has made use of the latter instrument in his own research and in preparing many of the figures for this chapter. It is therefore likely that even more laboratories will acquire this facility. The basic principles of an electron-impact (EI) mass spectrometer are described in Chapter 3. For fatty acid identification especially, it could be argued that this basic system will have the capacity to provide answers to most problems. The main difficulties arise with compounds with labile functional groups, which may not always be subjected safely to GC and where it may not be easy to recognise the molecular ion, because excessive fragmentation has occurred. Reducing the ionization potential from the standard 70 eV can sometimes help.

Other "soft" ionisation procedures are then often the preferred alternative. For example, in chemical ionisation (CI) procedures, the sample is mixed with a large excess of a reagent gas such as methane or butane, which is ionised. The reagent ions interact with the sample, transferring the charge and some energy, and bringing about fragmentation; the resulting ions are separated and analysed. Field ionisation and field desorption are similar methods in which the molecules are subjected to a powerful electrical field, which brings about ionisation with a relatively small transfer of energy, so that the amount of fragmentation is reduced. In the field desorption method, the sample is ionised without prior volatilisation and this is the mildest method of all. Some of these ionisation procedures are more compatible with GC separation than are others. Fast atom bombardment (FAB) MS is a solid-phase ionisation technique, utilising production of charged particles from the surface of a liquid matrix, and it has proved of great value for structure determination of complex ionic glycolipids and phospholipids; it is not appropriate to discuss it further in the context of this chapter. This is also true of tandem mass spectrometry of lipids, which has been reviewed elsewhere [30].

Methyl ester derivatives of fatty acids are not always the most useful for identification purposes using MS, and pyrrolidides and picolinyl esters especially have advantages in many circumstances (see Chapter 4 for methods of preparation). For example, stable ions may be produced with the latter that assist in locating double bonds in unsaturated fatty acids. It is also advantageous in some instances to prepare specific adducts of double bonds and derivatives of other functional groups to assist both with the GC separation and with identification by MS. Often this results in a considerable increase in mass with a corresponding change in GC retention characteristics. On fused silica WCOT columns coated with polar and non-polar phases, picolinyl esters tend to elute at a temperature 5°C higher than the corresponding pyrrolidides, which in turn elute 40 to 50°C higher than the methyl esters [173]. Non-polar stationary phases, such as cross-linked methyl- or methylphenylsilicones, stand up to the required conditions better, but some loss of resolution is inevitable. Analogous effects are seen with many other types of derivative, especially those used in the location of double bonds.

In this chapter, most emphasis is on practical GC-MS procedures, and mechanistic aspects of the fragmentation process are not treated in depth. Perhaps even more than with other spectroscopic techniques, interpretation of mass spectra is dependent on experience gained with model compounds, and it is not possible to draw up simple rules to guide the reader. This chapter should not be read in isolation; it should be remembered that other chromatographic procedures can give simplified fractions that are more easily analysed subsequently by GC-MS than are the intact samples (see Chapter 6). GC-MS, as opposed to MS with sample introduction by other means, gives the analyst information of two kinds on a given compound - its mass spectrum and GC retention time. The latter can often be of crucial importance as an aid to identification with closely related isomers. Aspects of mass spectrometry of fatty acid derivatives have been reviewed by several authors [45,446,569,612,704,809]. Much more information is available on the specialist section of this website here..

Note added subsequent to publication. Most emphasis here is given to mass spectra of picolinyl esters, but dimethyloxazoline derivatives may now be favoured in many circumstances.

 

B.  Saturated Stright-Chain Fatty Acids

The methyl ester derivatives of long-chain saturated fatty acids are easily identified by electron-impact MS, and their spectra are characterised by a prominent molecular ion (M+), and other significant ions equivalent to m/z = M−31 (loss of methanol) and M−43 (loss of C2, C3 and C4 as a result of a complex rearrangement), together with a series of ions of general formula - [CH3COO(CH2)n]+, often with intensity maxima at m/z = 87, 143 and 199 [781]. The base ion at m/z = 74 is often termed the "McLafferty rearrangement ion", and is formed after cleavage of the parent molecule beta to the carboxyl group [574]. (The mechanism of its formation is one of the most widely studied processes in mass spectroscopy). CI mass spectra, in contrast, have a prominent quasi-molecular ion (MH+), and minor ions only at [MH−32]+ and [MH−32−18]+ [631].

Mass spectra of pyrrolidine derivatives of saturated fatty acids have prominent molecular ions, and a base peak at m/z = 113, equivalent to the McLafferty ion [48]. Similarly, picolinyl ester derivatives have spectra with distinctive molecular ions, in addition to abundant ions containing the pyridine ring at m/z = 93, 108, 151 (the McLafferty ion) and 164 [359]. The spectrum of picolinyl palmitate is illustrated in Figure 7.1. In the high mass range, there is a regular series of ions 14 atomic mass units (amu) apart, i.e. at m/z = 332, 318, 304, 290, 276 and so forth, representing cleavage between successive methylene groups.

mass spectrum of picolinyl hexadecanoate

Figure 7.1.  The mass spectrum of picolinyl hexadecanoate.

 

Mass spectrometry has long been the favoured method for the analysis of deuterated fatty acids (reviewed elsewhere [570,934]). Methyl ester derivatives and electron impact MS are not the best combination for locating deuterium atoms on specific carbon atoms in saturated fatty acids, because of the complex nature of the rearrangements that happen. Pyrrolidine derivatives of deuterated fatty acids are better in some circumstances [48,50], but isotope effects can occur within the mass spectrometer, and elimination reactions take place that confuse the results [484]. Conversely, specifically deuterated fatty acids are excellent substrates for the study of ion fragmentations, and the trimethylsilyl (TMS) esters of deuterated decanoic acids have been used for this purpose, for example [935].

In measurements of isotopic purity, field desorption MS (of the free acids) has considerable potential, since the molecular ion is the base peak [539]. It is also possible to use t-butyldimethylsilyl esters of fatty acids (see Chapter 4 for details of preparation) with EI-MS, since these give a particularly prominent [M−C4H9]+ ion from which the precise molecular weight can be determined [692,709,1004].

 

C.  Unsaturated Fatty Acids

1.  Methyl ester derivatives

Procedures for locating double bonds in fatty acids were reviewed comprehensively by Minnikin [612], and the more recent literature has been assessed by Schmitz and Klein [809]. EI mass spectra of unsaturated fatty acids are very different from those of their saturated analogues, and they also vary a little according to degree of unsaturation. The mass spectrum of methyl oleate is illustrated in Figure 7.2. Again, there is a distinct molecular ion with prominent ions for loss of methanol ([M−32]+, the base peak with some instruments [338]) and of a methoxyl radical [M−31]+. [M−74]+ and [M−116]+ ions stand out in the high mass range. In the spectra of dienes and trienes, the molecular ion is more pronounced, while those representing losses of 32, 74 and 116 amu are less so.

mass spectrum of methyl octadec-9-enoate

Figure 7.2.  The mass spectrum of methyl octadec-9-enoate.

 

Unfortunately, there are no ions that serve to indicate the location or stereochemistry of the double bonds in positional isomers. This is believed to be because double bond migration occurs when the molecular ion is formed, resulting in a range of common intermediate products, which in turn give common fragment ions. The only exception is the 2-isomer, which has a characteristic ion at m/z = 113, the base ion for the cis-isomer [780]. Similarly, in the mass spectra of the series of isomeric methylene-interrupted octadecadienoates, only that of the 2,5-isomer differs from the remainder in having the ion at m/z = 113 together with one at m/z = 139 [180].

With the spectra of polyenoic fatty acids, the intensities of some ions vary, but they are not readily interpretable in terms of the positions of the double bonds. Nonetheless, there are specific ions in the spectra of the main families of polyenes that may be of diagnostic value [253]. Methyl esters of a number of different fatty acids of the (n-3) family are reported to give a characteristic fragment at m/z = 108 (27 to 66% of the base peak), while those of the (n-6) series give a prominent ion at m/z = 150 (9 to 30% of the base peak). These ions are believed to represent fragments from the terminal region of the molecule. Fatty acids of the (n-9) family would therefore be expected to have an abundant ion at m/z = 192, and this was indeed present in the spectrum of 20:3(n-9) (11% of the base peak). Features of this kind were used for the provisional identification of long-chain (C26 to C38) fatty acids of the (n-6) family with four or five double bonds in tissues of patients with the Zellweger syndrome [733,838], and of unusual fatty acids of the (n-3) series in ram spermatozoa [732]. In these examples, it is necessary to assume that all the double bonds are methylene-interrupted.

While it is obviously disappointing not to be able to obtain complete information on a fatty acid from its EI mass spectrum, the capacity to determine accurate molecular weights together with GC retention time data can be of considerable value to the analyst. Two GC-MS studies selected from many to have been published testify to this [39,655].

Acetylenic fatty acids, as the methyl ester derivatives, have complex and distinctive EI mass spectra, which vary with the position of the triple bond and can be used to distinguish isomers [480].

With CI procedures, the molecular ion is more readily seen with polyunsaturated components [631], and in some circumstances more information can be obtained on the location of double bonds. Usually, however, it is necessary to have the free acid or a salt form. For example, collision-activated decomposition spectra of negative ions obtained by chemical ionisation gave distinct fragments corresponding to cleavage allylic to the double bond of elaidic acid [918]. Analogous methods have been described that make use of iron, lithium and other alkali metal salts of monoenoic acids as the substrates [32,447,700]. Similarly, by making use of a suitable reagent gas, it may be possible to locate isolated double or triple bonds [139]. Such techniques are not of course always compatible with gas chromatography.

 

2.  Pyrrolidine derivatives

Various types of amide derivatives of fatty acids give distinctive EI mass spectra from which many functional groups, including double bonds, can be located. It appears that the reason for this is that the charge on the molecular ion is sited on the nitrogen-containing functional group predominantly, rather than being localised at the double bond. Acyl pyrrolidides were the first to be employed extensively for the purpose, and their use has been reviewed by Andersson [45]. The method of preparation is described in Chapter 4 (or better in the mass spectrometry pages of this site). On electron impact, pyrrolidides fragment to produce a series of ions as a result of successive cleavage of carbon-carbon bonds induced by radicals. In comparison to methyl esters, there is less scrambling of hydrogen atoms.

The EI mass spectrum of N-octadec-9-enoylpyrrolidine is illustrated in Figure 7.3 [45,51]. In addition to the base peak at m/z = 113 (observed with saturated derivatives (Section B)) and the molecular ion, there is a series of ions 14 amu apart in general, except in the vicinity of the double bond, where there is an interval of 12 amu., i.e. occurring between m/z = 196 and m/z = 208, and corresponding to fragmentation between carbons 8 and 9 in the aliphatic chain.

mass spectrum of N-octadec-9-enoylpyrrolidine

Figure 7.3. The mass spectrum of N-octadec-9-enoylpyrrolidine.

 

For the spectra of the pyrrolidides of the isomeric 5- to 15-octadecenoic acids, the following rule was formulated:

"If an interval of 12 amu, instead of the regular 14, is observed between the most intense peaks of clusters of fragments containing n and n+1 carbon atoms in the acid moiety, a double bond occurred between carbons n and n+1 in the molecule".

The pyrrolidides of the remaining octadecenoic acid isomers also have distinctive spectra, but they do not fit the rule. The technique has been utilised with vicinal di-deuteriated mono-ethylenic compounds [49]. All the spectra are available on this website here..

Methylene-interrupted polyunsaturated fatty acids, as the pyrrolidides, have characteristic spectra that can usually be interpreted in terms of the positions of the double bonds with some modification to the above rule [45,47]. Similarly, a conjugated diene, 9,11-octadecadienoate, has been identified by this means in the lipids of human tissues [417]. It must be admitted, however, that it is not easy to identify unknowns from first principles using such spectra. Nonetheless, a good molecular ion is almost always obtained; with access to the spectra of model compounds and to natural mixtures the fatty acids of which have been identified by other means, it is possible to make good use of pyrrolidides in the analysis of unknown samples. For example, they have been used to identify fatty acids in the bovine lens [941], marine organisms [70,207,452,534,843], bacteria [909] and algae [770].

Isomeric mono-acetylenic fatty acids in the form of the pyrrolidides have distinctive mass spectra, which can be used for identification purposes [942].

Note added subsequent to publication. By coincidence 4,4-dimethyloxazoline (DMOX) have the same molecular weight as pyrrolidides and display very similar mass spectral fragmentations. As they have superior GC properties, they are now favoured by many analysts.

 

3.  Picolinyl ester derivatives

Harvey [359] first proposed the use of picolinyl (3-hydroxymethylpyridine) ester derivatives for the identification of fatty acids containing double bonds and other functional groups. Like the pyrrolidides, they fragment under electron impact by radical-induced cleavage at each carbon-carbon bond. The method of preparation is described in Chapter 4 (or better in the mass spectrometry pages of this site). The author recently took part in a comparative study of the utility of pyrrolidides and picolinyl esters for the recognition of unsaturated fatty acids in natural mixtures of animal and marine origin [173]. Without doubt, picolinyl esters are to be preferred and give spectra from which both the number and the positions of double bonds can be deduced; pyrrolidides have marginally better chromatographic properties. Harvey [359] obtained better results by operating his mass spectrometer at an ionisation potential of 25 eV, but the author obtained satisfactory spectra at 70 eV; instrumental differences may explain the discrepancy.

The EI mass spectrum of the picolinyl ester derivative of oleic acid is illustrated in Figure 7.4, and details of the spectra of the complete series of isomeric octadecenoate derivatives (2- to 17-) have been published [172]. That shown differs from those published by Harvey [359,361] in that the base ion is at m/z = 92 rather than at 108, possibly a consequence of the different ionisation potentials utilised in the two studies. There is a particularly prominent molecular ion (m/z = 373), and the ions containing the pyridine ring at m/z = 92, 108, 151 and 164 are all very evident as with saturated compounds (Section B).

Points of cleavage where characteristic fragmentations occur that permit the location of the double bond are illustrated in Figure 7.5a, and data for each of the isomers is listed in Table 7.1. In nearly all the spectra, a distinctive feature is a doublet of prominent ions 14 amu apart, representing cleavage at points A and B on the distal side of the double bond. In the spectrum of the 9-isomer, they are seen at m/z = 274 and 288; such ions in exactly the same place are found in the spectra of the picolinyl derivatives of 9-14:1, 9-16:1, 9-17:1, 9-20:1 and 9-22:1 fatty acids [173,175,184]. Their formation was rationalised in terms of an initial abstraction of allylic hydrogens on either side of the double bond with production of conjugated diene systems, which are relatively stable [361]. When natural samples containing fatty acids in which the double bonds are two carbon atoms apart are examined by GC-MS in the form of the picolinyl ester derivatives (as in the sample illustrated in Figure 6.4 for example), this doublet can usually be picked out for diagnostic purposes even when the isomers are imperfectly resolved. When the double bonds are near either end of the molecule, these ions can be less obvious (Table 7.1) but there are then other distinguishing features.

mass spectrum of picolinyl octadec-9-enoate

Figure 7.4.  The mass spectrum of picolinyl octadec-9-enoate.

Mass spectral fragmentations of a picolinyl ester

Figure 7.5.  The bonds where characteristic mass spectrometric fragmentation occurs for (a) picolinyl octadec-9-enoate and (b) picolinyl 9,12-octadecadienoate.

 

Distinctive fragmentations at the double bond are also apparent for each isomer. In the spectrum of picolinyl oleate again, there is a gap of 26 amu between m/z = 260 and 234 for fragmentations adjacent to the double bond. This gap is seen in the spectra of most of the isomers, although a gap of 40 amu between points C and E (i.e. between m/z = 260 and 220 in this instance) is often easier to locate. Difficulties arise mainly with the 3- and 4-isomers, but there are other diagnostic features with these. On either side of the double bond, regular series of ions are found 14 amu apart for cleavage between successive methylene groups. Thus for oleate once more, these are seen at m/z = 358, 344, 330, 316, 302, 288 and 274 on the distal side of the double bond, and at m/z = 220, 206, 192, 178 and 164 on the carboxyl side. Analogous features are seen in the spectra of the other isomers. All the spectra are available on this website here..

Table 7.1.

Relative abundancesa of the molecular ion and of ions characteristic of the picolinyl moiety, and m/z valuesb and relative abundances of ions characteristic of double bond positions in mass spectra of picolinyl octadecenoates [172]. To open this in a separate window - right click here...

 

Harvey [361] also demonstrated distinctive features in the spectrum of the picolinyl ester of linoleic acid, but that illustrated in Figure 7.6 is from the author's laboratory (again it differs significantly only in that the base peak is at m/z = 92 rather than at 108). Data for the complete series of isomeric methylene-interrupted octadecadienoates (2,5- to 14,17-) have been published [172], and some of this is listed in Table 7.2 as it has proved invaluable in the author's laboratory for the interpretation of the spectra of unknown polyenoic fatty acid derivatives. Returning to Figure 7.6, ions corresponding to cleavage on either side of the terminal double bond are seen at m/z = 300 and 274 (26 amu apart), there is then a gap of 14 amu to 260, then one of 26 amu for the internal double bond to 234. When examining the spectra of unknowns, however, it is often easier to locate gaps of 40 amu between points A and C and C and E (Figure 7.5b), i.e. in this instance between m/z = 300 and 260, and between 260 and 220. Regular series of ions 14 amu apart on either side of the double bond confirm that there are no further functional groups in these regions of the molecule. The spectra of the remaining isomers have corresponding ions or other distinctive features. All the spectra are available on this website here..

mass spectrum of picolinyl 9,12-octadecadienoate 

Figure 7.6.  The mass spectrum of picolinyl 9,12-octadecadienoate.

 

Table 7.2.

Relative abundancesa of the molecular ion and of ions characteristic of the picolinyl moiety, and m/z valuesb and relative abundances of ions characteristic of double bond positions in mass spectra of picolinyl octadecadienoates [172]. To open this in a separate window - right click here...

 

With dienoic acids with more than one methylene group between the double bonds, the spectra of the picolinyl ester derivatives are less easy to interpret in terms of the positions of the double bonds [171]. The terminal bond can be identified with relative ease, and there are ions diagnostic of the internal bond provided that model spectra are available for comparison purposes. With such assistance, the author was able to identify the dienes shown in Figure 6.4 in Chapter 6 [175].

Interpretation of the spectra of the picolinyl ester derivatives of tri- and tetraenoic fatty acids containing methylene-interrupted double bonds presents little problem. Gaps of 26 amu for each of the double bonds are usually seen as with the dienes, although again it is often easier to detects gaps of 40 amu between the terminal end of each double bond and the methylene group on the carboxyl side. Full spectra of 18:3(n-3) [361], 20:3(n-9), 20:3(n-6) and 20:3(n-3) [173], 16:4(n-1) [175], 20:4(n-6) and 22:6(n-3) [361] have been published, together with brief details of many more. With the last and with isomers with 5 double bonds, the spectra are much more complex and have to be examined with care, but the spectrum of the picolinyl ester derivative of 22:5(n-6) is readily distinguished from that of 22:5(n-3) and both are as expected [173].

As an example, the mass spectrum of picolinyl 5,8,11,14,17-eicosapentaenoate (20:5(n-3)) is illustrated in Figure 7.7. There is an adequate molecular ion, a gap of 15 amu for the terminal methyl group to m/z = 378 one of 14 to m/z = 364; the gaps of 26 amu for the double bonds between m/z= 364 and 338, 324 and 298, 284 and 258, 244 and 218, and 204 and 178 are not always easy to pick out, but the associated gaps of 40 amu for each double bond and the attached methylene group on the carboxyl side, i.e. for m/z = 364 to 324 to 284 to 244 to 204 to 164, are also diagnostic. The spectra of many more polyunsaturated fatty acids are available in the specialist mass spectrometry areas of this website.

mass spectrum of picolinyl 5,8,11,14,17-eicosapentaenoate

Figure 7.7. The mass spectrum of picolinyl 5,8,11,14,17-eicosapentaenoate.

 

The picolinyl ester of a mono-acetylenic fatty acid gave a spectrum which resembled that from its monoethylenic analogue except that there was a gap of 24 amu for fragmentations on either side of the triple bond [172]. A series of di-acetylenic fatty acids gave spectra which were rather complex, but the terminal triple bond generally exhibited a fragmentation with a gap of 24 amu [174].

It is the author's opinion then that picolinyl esters are the best general purpose derivative for locating double bonds by GC-MS. They have been used in analyses of fatty acids of animal tissues [173,184,363,365,979], a fish oil [173,184], marine invertebrates [175] and bacteria [960].

 

4.  Addition compounds

While the EI mass spectra of pyrrolidine and picolinyl ester derivatives of unsaturated fatty acids can often yield sufficient information for structure determination, there are circumstances when the result is not clear or where confirmation by a different method is desirable. The preferred method then is to form some addition compound with the double bonds that gives a distinctive fragmentation pattern. Very many different methods have been described for the purpose, and as they have been reviewed in detail elsewhere [612,809] only the more important are considered here. Methods of preparation are described in Chapter 4.

Potentially the simplest method consisted in deuteration of polyunsaturated fatty acids with deuterodiimide, followed by conversion to the pyrrolidine derivative for examination by GC-MS [472]. The positions of the original double bonds are deduced from the ions containing deuterium atoms. In a critical examination of the method, Klein and Schmitz [484] concluded that good results could be obtained with fatty acids containing up to six double bonds, although great care was necessary in interpreting the spectra because of the occurrence of unexpected elimination reactions. There were also problems with some instruments. Nonetheless, it is one of the few methods of this type that is easily applied to polyunsaturated fatty acids. It might be of interest to ascertain how picolinyl esters would fare in this approach. One disadvantage of the procedure is that it can only be applied to pure fatty acids or to rather simple mixtures.

If simplicity is a virtue, then a further excellent method for locating double bonds, especially in monoenoic acids, consists in preparing the dimethyl disulfide adducts, since a single reagent and a one step reaction is required for the preparation (see Chapter 4) [261]. The EI mass spectrum of the dimethyl disulfide adduct of methyl oleate is illustrated in Figure 7.8. 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 (either of these can be the base peak, probably dependent on instrumental factors). There is also a prominent ion at m/z = 185, corresponding to the latter fragment with the loss of methanol. In addition, these derivatives give good molecular ions. As the adduct adds substantially to the molecular weight of the original ester, it tends to elute at a temperature about 40°C higher than the former from a GC column containing a non-polar silicone phase. Adduct formation is entirely stereospecific, presumably by trans addition, so that erythro- and threo-derivatives are formed from cis-and trans-isomers, respectively [143]. Although the different geometrical isomers have virtually identical spectra, they are eluted separately from GC columns containing either polar or non-polar phases, that derived from the cis-isomer eluting first [143,540]. The procedure has been used with a variety of monoenoic fatty acids [143,233,540,667,701,841,952] and with some polyenes from natural sources [841,952].

The mass spectrum of the dimethyl disulfide adduct of methyl octadec-9-enoate

Figure 7.8. The mass spectrum of the dimethyl disulfide adduct of methyl octadec-9-enoate.

 

A number of workers have approached the problem of double bond location by oxidising it to a vicinal diol with alkaline permanganate or osmium tetroxide and then derivatizing further to a non-polar form (see Chapter 4). The stereochemistry is also retained in this way, so that geometrical isomers can be resolved. Isopropylidene derivatives of the diols were used first for the purpose [572], but more use has been made of the TMS ethers [57,146,239,240]. The former is more useful for establishing the configuration of the original double bond, while the latter gives more intense fragments for locating it. Figure 7.9 illustrates the EI mass spectrum of the TMS ether of the vicinal diol prepared from methyl oleate. Ions in the high mass range are of very low intensity and the molecular ion is not seen, but two ions at m/z = 215 and 259, which represent cleavage between the carbon atoms of the original double bond, are particularly prominent. Indeed these ions are sufficiently intense to be of use in estimating mixtures of positional isomers of unsaturated fatty acids. When softer chemical ionisation procedures are used, a small molecular ion is then seen and the ions for fragmentation between the functional moieties are still sufficiently intense for structure determination both with monoenes and dienes [58,632,717]. Indeed the geometry of the original double bond can be determined from differences in the intensities of the fragment ions. Although there are difficulties in handling polyhydroxy compounds, prepared from polyenoic fatty acids, the TMS ether derivatives give good spectra from which the original structures can be deduced [225,442,443,637,808,810].

mass spectrum of the trimethylsilyl ether derivative of the vicinal diol prepared
from methyl octadec-9-enoate

Figure 7.9. The mass spectrum of the trimethylsilyl ether derivative of the vicinal diol prepared from methyl octadec-9-enoate.

 

Because so many TMS ether groups add substantially to the molecular weight of a fatty acid and can in prolonged use lead to deterioration of the ion source, other workers have preferred to prepare polymethoxy derivatives [534,893]. These give excellent mass spectra when chemical ionisation is employed.

One further procedure worth mentioning briefly here consists in preparing methoxy- or methoxyhalogeno-derivatives, via the mercuric acetate adducts, for identification by MS [611-613,834]. Oxymercuration has also been employed to convert triple bonds to keto groups, which can be located in this form or after reduction to hydroxyl derivatives with sodium borohydride [140,480].

 

D.  Branched-Chain Fatty Acids

Mass spectrometry has been used more than any other technique for the location of alkyl branches in fatty acids. The methyl ester derivatives have been used extensively, although they are certainly not the best in all circumstances, as a branch-point does not provide a centre for charge localisation. Mass spectral data for a wide range of simple methyl-branched isomers, as the methyl esters, have been published [6,56]. Unfortunately, the hardest to identify are the most commonly occurring iso- and anteiso-isomers. An iso-methyl fatty ester can be distinguished from the corresponding straight-chain compound by the presence of a small ion at [M−65]+ and a doublet at [M−55]+ and [M−56]+ (all less than 1% of the base peak), and by its rather different GC retention time. In the spectrum of an anteiso-isomer, the ion for [M−29]+ is greater than that for [M−31]+. Small ions at [M−61]+, [M−60]+ and [M−79]+ are also of diagnostic value. Related esters with a single double bond in the aliphatic chain have characteristic spectra [118]. Chemical ionisation [953] and other soft ionisation techniques [445] may simplify identification.

When branching occurs near the centre of a chain, an ion of the type [CH3OOC(CH2)n]+, designated the 'a' ion and representing cleavage adjacent to the carbon carrying the methyl group, together with a+1 and a+2, and ions representing a similar fragment but with cleavage on the remote side of the methyl carbon, designated the 'b' ion, are of diagnostic value. As the principal ions are also present in the spectra of the corresponding straight-chain compounds, interpretation is based mainly on changes in relative intensity and comparison with spectra of model esters. However, the triplet of a, a+1 and a+2 is often distinctive, and in the spectrum of methyl 10-methylstearate for example, they occur at m/z = 171, 172 and 173. In addition, ions derived from a and b by loss of the elements of water or methanol can assist with interpretations. When the methyl branch is on carbons 2, 3 and 4, shifts in the ions at m/z = 74 (to 88 in the 2-isomer) and 87 (to 101 in the 2- and 3-isomers), and equivalent to [M−43]+ (to [M−57]+ in all three isomers) and [M−29]+ (to [M−43]+ in the 2- and 3-isomers) serve for identification. Similar mass spectral features are used to identify dimethyl- and ethyl-branched fatty acids. Applications of GC-MS to isoprenoid fatty acids have been reviewed [562]. Those studies cited here are representative of many applications to natural samples [232,238,423,425,668,852,853].

Pyrrolidine [52] and picolinyl ester [359] derivatives of branched-chain fatty acids give especially distinctive spectra, and are certainly to be preferred with samples that are relatively simple in composition, i.e. such that the bulky nitrogen-containing moiety does not impair GC resolution excessively. Again, it is the author's experience that the latter derivative has some advantages. With both types of derivative, the spectra superficially resemble those of the corresponding straight-chain compounds, but there are characteristic fragmentations on either side of the carbon atom linked to the methyl group. The result is that a diagnostic gap of 28 amu appears in the spectrum. Thus in the spectrum of the picolinyl ester of iso-methylheptadecanoate (M+ = 375), there is a gap from m/z = 332 to 360; in the spectrum of the corresponding anteiso-isomer, the gap is from m/z = 318 to 346. This feature is also seen in spectra from isoprenoid fatty acids, and that of the picolinyl ester derivative of phytanic acid (3,7,11,15-tetramethylhexadecanoate) is illustrated in Figure 7.10 (W.W. Christie and E.Y. Brechany, unpublished). There is a good molecular ion at m/z = 403, then gaps of 28 amu are seen between m/z = 151 and 178, 220 and 248, 290 and 318, and 360 and 388 for each of the methyl groups. When the methyl group is in position 2, the typical ions at m/z = 151 and 164 are shifted to 165 and 178 (the base peak) respectively [361]. Both pyrrolidides [95,147,206,296,770] and picolinyl esters [96,173,175,184,363,365,979] have been used for the identification of branched-chain esters in natural mixtures.

mass spectrum of picolinyl 3,7,11,15-tetramethyl-hexadecanoate

Figure 7.10. The mass spectrum of picolinyl 3,7,11,15-tetramethyl-hexadecanoate.

 

E.  Carbocyclic Fatty Acids

1.  Cyclopropanoid fatty acids

Mass spectrometric procedures for the location of cyclopropane rings were reviewed by Minnikin [612]. The methyl ester derivatives of cyclopropanoid fatty acids are not readily distinguished from mono-unsaturated fatty acids with a similar total number of carbon atoms by MS, apparently because on ionisation the cyclopropane ring opens up to form such a compound [179,997]. If necessary, methyl esters can be used if the ring is first opened by vigorous catalytic hydrogenation (with formation of two methyl-branched fatty acids) [571] or by reaction with boron trifluoride-methanol (with formation of two methoxymethyl fatty acids) [610].

Pyrrolidine derivatives of cyclopropanoid fatty acids give more useful spectra, although the diagnostic ions are not as clear as with the analogous monoenes [45,292,293]. The technique was used to identify 17-methyl-cis-9,10-methyleneoctadecanoic acid in a protozoan, for example [397].

Much better results are obtained with picolinyl ester derivatives. Harvey [360] showed that, with the picolinyl ester derivative of cis-9,10-methyleneoctadecanoic acid, characteristic fragments from either side of the ring are found, i.e. at m/z = 234 and 274. These can be seen in Figure 7.11 (spectrum obtained at 70 eV), together with an even more distinctive ion at m/z = 247, which represents a fragment containing carbon 9 in the ring, together with the remainder of the molecule on the same side as the picolinyl ester group (it differs from that published elsewhere, and obtained at an ionisation potential of 25 eV [360], only in that m/z = 92 rather than 108 is the base peak). Spectra which are interpretable in terms of the positions of the rings are also obtained with bis-cyclopropanoid fatty acid derivatives. The technique was used to identify 11,12-methyleneoctadecanoic acid in the lipids of a bacterium [960]. Further details are available in our mass spectrometry pages.

mass spectrum of picolinyl 9,10-methylene-octadecanoate

Figure 7.11. The mass spectrum of picolinyl 9,10-methylene-octadecanoate.

 

2.  Cyclopropenoid fatty acids

Cyclopropenoid fatty acids (methyl ester derivatives) are rather labile and not easily subjected to GC (see Chapter 5). Nonetheless, mass spectra of the methyl ester derivatives of some naturally occurring fatty acids of this kind have been published [698]. These are rather different from the spectra of most other unsaturated esters, but it is doubtful whether they would assist greatly in the identification of an unknown. One of the most popular methods for the analysis of cyclopropenoid fatty acids involves GC of the methoxy- and keto-derivatives formed by reaction with silver nitrate. Such derivatives produce rather complex spectra which can with care be interpreted in terms of the position of the original ring [37,241]. One of the first procedures in which MS was utilised involved ozonolysis or permanganate-periodate oxidation of the cyclopropene ring to form a diketo fatty acid, which has a distinctive mass spectrum [398]. On the other hand, a simpler method is the addition of methanethiol to the cyclopropene ring to yield non-polar derivatives with good GC [746] and MS [398] properties.

 

3.  Cyclopentenyl and related fatty acids

The methyl ester derivatives of cyclopentenyl fatty acids give electron-impact mass spectra with important structural information [183]. The base peak in methyl hydnocarpate (11-cyclopent-2-enylundecanoate) is at m/z = 67, presumably for the cyclopentene ring itself, while there are significant fragments at m/z = 82 and 185 for cleavage beta to the cyclopentene ring. The McLafferty rearrangement ion is not prominent, although this is the base ion in the spectrum of the corresponding cyclopentyl (i.e. saturated) fatty ester. With the latter, there is a prominent ion at m/z = 199 for cleavage alpha to the cyclopentyl ring.

When in addition to a cyclopentenyl double bond there is a further double bond in the aliphatic chain, as with methyl gorlate (13-cyclopent-2-enyltridec-6-enoate), the base ion is still at m/z = 67, and there is another abundant ion at m/z = 80, formally equivalent to a dihydrofulvene ion; there are no ions diagnostic for the position of the double bond in the chain.

Pyrrolidine derivatives give good spectra with conspicuous molecular ions, and distinctive ions in the high mass range for the loss of fragments corresponding to the cyclopentenyl ring and successive methylene groups [842]. In the spectrum of the gorlic acid derivative, there are small but significant ions diagnostic for the aliphatic double bond. Even better spectra are obtained with the picolinyl ester derivatives (Christie, W.W., Brechany, E.Y. and Shukla, V.K.S. Lipids, 24, 116-120 (1989)). The base ion in this instance is again at m/z = 67 for the cyclopentenyl ring, and ions in the high mass range characteristic for any aliphatic double bonds are usually clearly seen. It is not known what effect variation of the position of the double bond in the ring would have on mass spectrometric fragmentation with any of these derivatives.

Mass spectra of fatty acids with terminal cyclohexyl [215,812], cyclobutyl and cycloheptyl rings [215] have been described. Cyclic fatty acids are produced in oxidised oils, and these also have been subjected to structural analysis by GC-MS [368,705].

 

F.  Oxygenated Fatty Acids

1.  Hydroxy fatty acids

The methyl ester derivatives of hydroxy fatty acids give good EI mass spectra in which there are ions diagnostic of the position of the hydroxyl group [783]. The most characteristic ions are associated with cleavages beta to the oxygen atom (or alpha to the carbon carrying this atom); they are accompanied by ions 32 amu lower formed by the elimination of methanol. Unfortunately. there is rarely a significant molecular ion, although ions equivalent to [M−OH]+ and [M−OCH3]+ are usually prominent. Chemical ionisation, with ammonia as the reagent gas, gives a good [M+1]+ ion, however [717].

Because of the high polarity of such esters, it is more usual to convert the hydroxyl group to a trimethylsilyl ether or other derivative for separation by GC, and these also affect the fragmentation patterns observed. This has been discussed in part in relation to addition compounds for the location of double bonds in Section C.4 above. Perhaps the most useful study of this type is one of the acetoxy and trimethylsilyl ether derivatives of all the positional isomers of the methyl hydroxy-palmitates [670]. As with the hydroxy esters, fragmentation occurs alpha to the carbon containing the functional group. With the acetoxy derivatives, the main diagnostic ion generally represents a cleavage on the side remote from the carboxyl group, while with the trimethylsilyl ethers, the main cleavage is on the side adjacent to the carboxyl group. Neither derivative gives a good molecular ion, but there is usually an acceptable ion equivalent to [M−CH3]+ for the TMS ether.

The spectrum of the trimethylsilyl ether derivative of methyl 12-hydroxystearate is illustrated in Figure 7.12 as an example. No molecular ion (at m/z = 386) is evident, although the molecular weight can be ascertained from a characteristic ion at m/z = 371 (M−15, loss of a methyl group) and = 355 (M−31, loss of methanol). The base peak at m/z = 301 is the fragment formed by cleavage of the molecule between carbons 12 (carrying the TMS ether group) and 13 and containing the carboxyl moiety. The abundant ion at m/z = 187 is formed by cleavage between carbons 11 and 12, and contains the terminal region of the molecule including the TMS ether group. The McLafferty rearrangement ion at m/z = 73 is almost as large as the base peak. (The CI mass spectrum of this compound has been published elsewhere [717]). CI-MS with methane or isobutane as the reagent gas is reported to give better spectra of the TMS ethers of polyhydroxy fatty acid methyl esters than does EI-MS [878].

mass spectrum of the TMS ether derivative of methyl 12-hydroxystearate

Figure 7.12. The mass spectrum of the TMS ether derivative of methyl 12-hydroxystearate.

 

Among a large number of reports, TMS ethers have been used with EI GC-MS to identify the 2-hydroxy fatty acids from sphingolipids [145,531], for the hydroxy fatty acids in royal jelly [543], for mono-, di- and trihydroxy fatty acids from plant cutins [239,932] and for mycolic acids (3-hydroxy) [87,272,457,919]. Cyclic di-tert-butylsilene derivatives have been used for GC-MS identification of 2- and 3-hydroxy fatty acids [132]. In addition, natural 2-acetoxy [68] and 2-methoxy [69] fatty acids have been identified in marine organisms using GC-MS.

The positions of hydroperoxy groups formed during the oxidative deterioration or degradation of lipids are most commonly determined by reduction of the hydroperoxide to a hydroxyl group and hydrogenation of any double bonds, followed by methylation and conversion to the TMS ether derivative for GC-MS. This approach is also of value with the eicosanoids derived from arachidonic acid. Those papers cited here are representative of many [223,263,604,833,879,911,984,1005]. Similar results are obtained with phenyl esters [371].

Pyrrolidine derivatives of the complete series of methyl hydroxy-stearates and of their TMS ether derivatives have been prepared and their mass spectra described [936]. The principal mode of fragmentation is enhanced and is again alpha to the carbon carrying the hydroxyl group, although the spectra of the 2-, 3- and 4-isomers are very different from the rest. Additional complications result when keto groups are present [932]. Picolinyl esters of hydroxy acids as the TMS derivatives also have very distinctive mass spectra [361].

When a double bond is present in a molecule in addition to a hydroxyl group, it is essential to prepare the TMS ether of the methyl ester derivative if interpretable mass spectra are to be obtained [481,833]. The position of the double bond relative to that of the TMS ether greatly affects the fragmentation pattern. With esters in which the two groups are allylic, no fragmentation occurs between them but cleavage does occur on either side of the system of functional groups; a similar effect is seen with conjugated diene or enyne systems allylic to a hydroxyl group. When there is one methylene group between the double bond and the carbon linked to the TMS ether, the ions caused by fragmentation alpha to the TMS group on the side of the double bond are most abundant. Where the functional moieties are separated by two methylene groups, the two alpha-cleavage ions are of approximately equal intensity. Procedures of this kind have been used for naturally occurring fatty acids from seed oils [481] and for hydroxy fatty acids derived from peroxidation products [406]. In these circumstances, the preparation of pyrrolidine derivatives is not helpful, because oxygen-containing ions are so enhanced that there is little fragmentation adjacent to the double bonds [235].

Hydroperoxides from unsaturated fatty acid derivatives have been examined by direct-probe chemical ionisation MS [716,717].

 

2.  Keto fatty acids

The methyl ester derivatives of keto fatty acids give characteristic EI mass spectra with fragmentations alpha and to some extent beta to the keto group [473]. Mass spectra of some keto-hydroxy esters [932] and of derivatives of unsaturated keto fatty acid from natural sources [481,717,833,968] have been published. In addition, mass spectra for the pyrrolidides of the complete series of oxo-stearates [936] and of some hydroxy-oxo fatty acids [932] have been described. With the former, the most abundant ion tends to be that representing cleavage beta to the keto group and containing the pyrrolidine moiety.

 

3.  Cyclic oxygen-containing fatty acids

Epoxy fatty acids (as the methyl esters) give EI mass spectra which are not easily interpreted in terms of the position of the oxygen atom (especially when double bonds are also present), although it is possible with some effort if suitable model compounds are available [116,473,481]. It is generally recommended that the epoxide be isomerised to a mixture of keto compounds [473], or that the ring should be opened with boron trifluoride-methanol reagent to a mixture of methoxy-hydroxy derivatives [481,833] or with lithium aluminium hydride to hydroxy derivatives followed by trimethylsilylation [940], for identification by GC-MS. MS was used to study the incorporation of 18O into the oxirane ring of 9,10-epoxyoctadecanoic acid in wheat tissues [488]. If derivatization is undesirable, more useful spectra are obtained with chemical ionisation [474,717].

Methyl esters of furanoid fatty acids give good EI mass spectra with characteristic ions being produced by fragmentations alpha to the furan ring [790].

Cyclic hydroperoxides have been characterised by chemical ionisation MS with a direct exposure probe [262].

 

G.  Some Miscellaneous Fatty Acids

Methyl esters of dibasic acids give EI mass spectra which are more complex than those of the monobasic equivalents [782]. The molecular ion is not easily found, but the molecular weight can be obtained from an ion representing [M−31]+. There is usually a prominent and diagnostic ion at [M−73]+, and a series of ions at m/z = 84 + 14n appears to be typical. Straight-chain, branched-chain and unsaturated dibasic acids have been identified by this means in royal jelly [543]. Other workers have preferred to use TMS ester derivatives for identification by GC-MS, although rearrangement ions complicate the picture [229,663]. Once more, picolinyl ester derivatives would appear to be preferable for the purpose, since they give good molecular ions and diagnostic fragments in the hydrocarbon chain [361].

Two novel brominated fatty acids, (5Z,9Z)-6-bromo-25-methyl-5,9-hexacosadienoic acid and the isomeric 24-methyl compound, were identified in the form of the pyrrolidine derivatives by GC-MS, although it was necessary to replace the bromine atom in each with deuterium to establish its position [975].

 

Abbreviations

The following abbreviations are employed at various points in the text of these chapters:

Amu, atomic mass units; BDMS, tert-butyldimethylsilyl; BHT, 2,6-di-tert-butyl-p-cresol; CI, chemical ionisation; DNP, dinitrophenyl; ECL, equivalent chain-length; ECN, equivalent carbon number; EI, electron-impact ionisation; FCL, fractional chain-length; GC, gas chromatography; GLC, gas-liquid chromatography; HPLC, high-performance liquid chromatography; IR, infrared; MS, mass spectrometry; NMR, nuclear magnetic resonance; PAF, platelet-activating factor; ODS, octadecylsilyl; TLC, thin-layer chromatography; TMS, trimethylsilyl; UV, ultraviolet.

 

This document is part of the book "Gas Chromatography and Lipids" by William W. Christie and published in 1989 by P.J. Barnes & Associates (The Oily Press Ltd), who retain the copyright.

Updated: July 6th, 2011