The Analysis of Lipids other than Fatty Acids

Miscellaneous Separations of Lipids by Gas Chromatography

A.  Fatty Alcohols

1.  Isolation and GC analysis
Fatty primary alcohols with similar aliphatic chains to fatty acids tend to occur in the free state in tissues at low concentrations only, but they may be of some metabolic importance as precursors of alkyl lipids, as plant growth regulators and as insect pheromones, for example. In addition, they are found in esterified form in wax esters, which are substantial components of many natural materials. Secondary alcohols may be present in plant surface waxes, together with aliphatic diols which are common constituents of skin lipids. In mammalian tissues, the primary alcohols are saturated or monoenoic, but never di- or polyunsaturated; in wax esters of marine origin, the alcohol constituents are often closely related in structure to the fatty acids from which they may derive biosynthetically. The occurrence, chemistry and metabolism of fatty alcohols [577] and their chromatographic properties [577,579] have been reviewed.

Alcohols may be released from the esterified form by any of the hydrolytic or transesterification procedures described in Chapter 4. If a pure wax ester fraction is hydrolysed, the alcohols are obtained simply by solvent extraction of the alkaline solution. On the other hand, when other lipids are present, it is advisable to isolate them as a class by adsorption chromatography. TLC on layers of silica gel G with the elution system described for simple lipid separations in Chapter 2, i.e. with hexane-diethyl ether-formic acid (80:20:2, by volume) as the mobile phase, is usually used. With such a system, any secondary alcohols migrate ahead of primary alcohols, which in turn are slightly less polar than cholesterol; diols migrate just in front of monoacylglycerols. If cholesterol is present in an extract, it may be necessary to re-run the plate in the same direction to obtain additional resolution and ensure that primary alcohols and cholesterol are fully separated. Procedures of this kind were utilised to isolate trace levels of fatty alcohols from animal tissues, for example [108,662,904]. When wax esters are transesterified, the methyl esters and free alcohols can be separated on a mini-column of Florisil™ or silica gel; hexane-diethyl ether (9:1, v/v) elutes methyl esters, while diethyl ether is utilised to recover the free alcohols. Methods have been described for the simultaneous analysis of alcohol and fatty acid derivatives from specific samples by GC, without such a preliminary separation (see below), but it remains to be determined whether they have wider applicability.

As fatty alcohols are comparable in structure and molecular weight to fatty acid methyl esters, they are usually subjected to GC on the same stationary phases and under near-identical conditions. It is certainly possible to separate alcohols in the free form by GC, especially on modern WCOT columns of fused silica, but sharper peaks are obtained if less polar derivatives such as the acetates, trifluoroacetates or TMS ethers are prepared. Suitable preparation procedures are described in detail in Chapter 4. Jamieson and Reid [438] studied the relative retention times of many different saturated and unsaturated fatty alcohols in the free form and as the acetates on packed GC columns containing polar polyester phases, and concluded that very similar separation factors applied as with the equivalent fatty acid methyl esters. The order of elution was - methyl ester < alcohol acetate < free alcohol. A TMS ether derivative would be expected to have a lower retention time than an acetate, but the separation factors for double bonds in the alkyl chain in this instance were found to be lower than with the acetates and resolution in general was poorer; some changes in retention sequence for specific isomers was noted, depending on the type of derivative [439]. In contrast, the free alcohol eluted before derivatized forms on nonpolar phases [944]. It is therefore possible to use equivalent chain-length data for the provisional identification of fatty alcohols in the same way as with methyl ester derivatives of fatty acids (see Chapter 5).

In practice, acetate derivatives appear to have been preferred by most analysts. They have been employed in GC analyses of alcohols from animal tissues [108,662,904], from wax esters of marine origin [17,29,422,757], from microorganisms [657], from avian uropygial glands [424,426] and from vegetable oils such as jojoba [344]. (These are representative analyses from many that could have been cited in which packed columns or the older stainless steel WCOT columns were utilised.) Acetates have also been used in more recent work with WCOT columns of fused silica or glass. For example, the resolution of positional and configurational isomers of some insect pheromones (C14 and C16 olefinic primary alcohols) was studied with four different stationary phases in this way [377].

Jojoba fatty acids and alcohols were analysed simultaneously as the ethyl ester and acetate derivatives on a glass WCOT column coated with OV-1™, from which the ester eluted before the structurally related acetate [302]. As an alternative, a Grignard reagent (ethyl magnesium bromide) was utilised to hydrolyse the fatty acids and alcohols of jojoba wax to tertiary alcohols and free primary alcohols, respectively, and these were analysed simultaneously without further derivatization on a WCOT column of fused silica coated with a phase of the Carbowax™ type [714]. The nature of the separation is illustrated in Figure 10.1, from which it can be seen that the primary alcohols give sharp peaks which emerge well before the tertiary alcohols of related alkyl structure. This methodology may be suitable for waxes of mammalian origin also, but wax esters from fish might be too complex. Nonetheless, free alcohols and methyl ester derivatives of fatty acids from wax esters of marine copepods have been analysed simultaneously on a glass WCOT column coated with the polar phase Silar 10™ [470]. Alcohols eluted before esters of analogous structure, and certainly the main components appeared to be well resolved. None of the alcohols were di- or polyunsaturated, however.

Figure10-01.png

Figure 10.1. GC separation of primary (P) and tertiary (T) alcohols derived from Grignard reduction of the alcohols and fatty acids, respectively, from jojoba wax [714]. A fused silica WCOT column (20 m × 0.317 mm. i.d.), coated with Carbowax 20M™, was maintained at 220°C. (Reproduced by kind permission of the authors and of Lipids, and redrawn from the original paper.)

 

Alkane-2,3-diols from the uropygial glands of birds have been separated on packed columns (SE-30™) in the form of the isopropylidene derivatives [492], and on glass WCOT columns coated with the same phase in the form of the TMS ethers (both threo and erythro isomers) [3]. In addition, a number of synthetic 1,2- and 1,3-diols have been subjected to GC analysis in the form of cyclic di-tert-butylsilene derivatives [132]. Racemic 2-alkanols have been resolved in the form of D-phenylpropionate [349], N-(1-phenylethyl) urethane [340] and R-(+)-2-phenylselenopropionate [608] derivatives by GC.

 

2.  Other separatory procedures and structure determination
Fatty alcohols may be separated and analysed by many of the procedures described in detail in Chapter 6 for fatty acid derivatives. For example, silver ion chromatography has been much used as an aid to the isolation and identification of specific components. Saturated and monoenoic fractions were isolated as the acetates from mullet roe by a column procedure [422], but TLC has been used more often; hexane-diethyl ether (9:1, v/v) as the mobile phase in silver ion TLC will separate these fractions [344]. The effect of double bond position on the migration of all the isomeric cis-octadecenols and the corresponding acetates on TLC with layers impregnated with silver nitrate was examined [327]. Like the methyl ester derivatives of the analogous fatty acids, the Rf values of isomeric acetates fall on a sinusoidal curve with a minimum around the 5- and 6-isomers, but the free alcohols hardly show this effect. Similarly, HPLC in the reversed-phase mode has been employed for the isolation of fatty alcohols, such as the insect sex hormones, under conditions similar to methyl esters. Thus, various unsaturated aliphatic acetates were resolved on an ODS phase with acetonitrile-water (7:3 or 93:7, v/v) as the mobile phase and with UV detection at 205 nm (or at 235 nm for isomers with conjugated double bond systems) [53].

Determination of double bond position in fatty alcohols or acetates, isolated by silver ion chromatography and preparative GC in combination, has been carried out by means of periodate-permanganate oxidation [344] and by ozonolysis [422,757]. Chain lengths can be established by hydrogenating and comparing GC retention times with those of authentic standards.

13C NMR spectroscopy has been utilised to assign double bond configurations to unsaturated alkanols [776].

 

3.  Gas chromatography-mass spectrometry
Free alcohols are not ideal substrates for GC-MS with electron-impact ionization as they do not in general give a molecular ion, the first significant ion in the high mass range to be observed usually being equivalent to [M−18]+. This is followed by a characteristic ion at [M−46]+, equivalent to (M−H2O−CH2=CH2). Little other structural information is gleanable from the spectra, and in general they resemble those given by mono-alkenes. Nevertheless, triacontanol, a naturally occurring plant growth regulator, was identified by MS in the free form [771]. The spectrum of hexadecanol is illustrated in Figure 10.2. The molecular ion is only detectable on amplification, but the ions for [M−18]+ and [M−46]+ are reasonably abundant. With negative-ion MS, a good molecular ion is obtained, but there is little other structural information [754]. Secondary alcohols in which the hydroxyl group is located approximately centrally in the alkyl chain are found in some insect waxes. On electron impact, these give characteristic cleavages between the carbon containing the hydroxyl group and the adjacent carbon atom [113]. Collisional activation decomposition spectra may yield further information, but this technique is not compatible with GC [31].

Figure10-02.png

Figure 10.2. The mass spectrum of hexadecan-1-ol.

 

In contrast, acetate derivatives can give adequate molecular ions, although they are not always detected. There is usually a distinctive ion at m/z = 61, equivalent to [CH3COOH2]+, together with the corresponding fragment at [M−60]+ [837,868]. Thus, in the mass spectrum of hexadecyl acetate, illustrated in Figure 10.3, the molecular ion is recognisable (m/z = 284), and there are ions at m/z = 224 ([M−60]+), equivalent to loss of the acetyl group, and at m/z = 196, representing the further loss of an ethylene group. Saturated 1- and 2-alkoxyacetates have spectra that are similar in the high mass range, but small ions at m/z = 73 and 116 are only seen with the former, while ions at m/z = 87 and 102 are found only with the latter [657]. iso- and anteiso-Methyl branch points in the aliphatic chain are differentiated by the presence of an ion equivalent to [M−60−29]+ in the spectrum of the latter, while the base ions are at m/z = 56 and 70, respectively [364]. It is claimed that differences in double bond position in alkenyl acetates give rise to small but significant variations in the intensities of specific ions [402,541]; whether these data can be readily translated to other laboratories and instruments is problematical, however. Tandem mass spectrometry in the negative-ion mode certainly does give information on double bond position in alkenyl acetates, but again the technique may not be used in conjunction with GC [906].

Figure10-03.png

Figure 10.3. The mass spectrum of the acetate derivative of hexadecan-1-ol.

 

TMS ethers, as might be expected, give good EI mass spectra; the molecular ion may be small, but the ion representing loss of a methyl group is often the base peak [364,836]. Spectra of deuterated decanol TMS ethers gave information on the mechanisms of the fragmentation processes [935]. TMS ethers were utilised with GC-MS to identify fatty alcohols (including diols) in cutin monomers [964]. In addition, related derivatives suited to electron capture detection have been described [134].

N-Alkyl-2-pyrrolidone derivatives, analogous to fatty acid pyrrolidides (see Chapter 6), have given useful spectra with substituted alkanols [95]. However, it soon became evident that alkyl nicotinates, analogous to fatty acid picolinyl esters, give much more informative spectra in which methyl-branch points and double bond positions are readily determined [96,364,950]. These derivatives were used in conjunction with GC-MS for the identification of the aliphatic alcohols from meibomian glands [363,365]. The mass spectrum of the nicotinate derivative of anteiso-nonadecanol, obtained at an ionisation potential of 25 eV, is illustrated in Figure 10.4 [364]. The molecular ion (m/z = 389) is small but measurable, and the position of the methyl branch is indicated by a gap of 28 amu between m/z = 360 and 332 in the high mass range. The base ion (m/z = 124) represents protonated nicotinic acid. Equally informative spectra are given by nicotinate derivatives of diols.

Figure10-04.png

Figure 10.4. Mass spectrum (25 eV) of the nicotinate derivative of anteiso-methyl-octadecan-1-ol.

 

Regrettably, these are not the easiest of compounds to prepare and this led Harvey [362] to investigate picolinyldimethylsilyl ether derivatives, which also proved to give excellent spectra. The required reagent is not yet available commercially, but can be synthesised without too much difficulty.

Other approaches to locating functional groups in the aliphatic moieties of alcohols have involved more extensive chemical reaction or derivatization prior to GC-MS. For example, branched-chain primary alcohols have been oxidised to the corresponding acids and methylated for analysis, since the mass spectra of methyl esters are well documented [424,426]. Others prepared pyrrolidides, after oxidation to the acids, as these give spectra which are more readily interpreted [46]. Similarly, secondary alcohols have been oxidised to ketones as an aid to identification [113]. Double bonds in alkyl chains of alcohols have been located by MS after the preparation of suitable chemical adducts, similar to those described for fatty acids in Chapter 7. Oxidation to diols and conversion to the TMS ethers is one method [638], but synthesis of dimethyl disulfide adducts from alcohol acetates is a one-step reaction (see Chapter 4) and is now preferred [143,540]. On the other hand, it may be too much to expect that a single method will provide all the information desired on a given sample; it required partial hydrogenation, coupled with GC-MS and GC/Fourier-transform IR (to identify trans-double bonds), to determine the structure of a trienoic insect trail pheromone [1006].

 

B.  The Hydrolysis Products of Ether Lipids

1.  Isolation and identification of ether lipids
Ether lipids are widespread in nature, and only in the higher plants are they rarely encountered. In any complete analysis of lipid samples, the ether forms must be quantified and the alkyl and alkenyl moieties must be identified. Suitable procedures have been reviewed briefly by the author [163,168] and more comprehensively by others [202,588,801,865,875,957]. One important distinguishing feature of alkyl as opposed to acyl lipids is that the ether bonds are stable to alkali.

Alkyl- and alk-1-enyldiacylglycerols sometimes accompany triacylglycerols in tissues, and they can be isolated by a preparative TLC procedure, although some care is required. They tend to migrate close to each other in the order stated and a double development with mobile phases such as toluene-methanol (199:1, v/v) [763] or hexane-diethyl ether (95:5, v/v) [803] can give satisfactory results. The various ether forms of phospholipids are less easily resolved, especially in the native form, but this is discussed in Chapter 8 (C.1) and Chapter 9 (D.3). When they have been separated by TLC, triacylglycerols and the ether analogues can be quantified by procedures such as charring followed by densitometry, or by GC of the methyl esters of the fatty acid components of the compounds with a suitable internal standard. Basic transesterification procedures should be used to avoid disruption of vinyl ether bonds, which would result in contamination of the methyl esters by free aldehydes or dimethyl acetals; any other hydrolysis products can be removed by a mini-column procedure (see Chapter 4). In calculating the results, the differing molar proportions of fatty acids in each lipid class must be taken into account.

Plasmalogens may be detected by spraying TLC plates with 2,4-dinitrophenylhydrazine (0.4%) in 2 M hydrochloric acid; aldehydes are released which show up as yellow-orange spots on warming the plates [804]. Alternatively, aldehydes released by exposure to fumes of hydrochloric acid appear as purple spots on spraying with a 2% aqueous solution of 4-amino-5-hydrazino-1,2,4-triazole-3-thiol in 1 M sodium hydroxide [745]. The total amount of plasmalogen in a lipid sample can be determined by preparing the p-nitrophenylhydrazone derivatives of the aldehydes released under acidic conditions and estimating these spectrophotometrically at 395 nm relative to a suitable blank [255,755]. They are prepared from the aldehydes or directly from the natural plasmalogens by the following procedure.

To a solution of the lipids (1-5 mg) in 95% ethanol (1.6 mL) is added freshly prepared 0.02 M p-nitrophenylhydrazine in the same solvent (0.2 mL), followed by 0.5 M sulfuric acid (0.2 mL). After heating at 70°C for 20 min, the solution is cooled, water (1 mL) and hexane (2 mL) are added, and the mixture is thoroughly shaken. The hexane layer is washed with water (2 × 2 mL) and dried over anhydrous sodium sulfate, before the solvent is evaporated to yield the required product.

The plasmalogen content can also be measured by a method involving specific binding of mercury salts to the vinyl ether bond [148].

Unfortunately, there is no simple spot test for the identification of alkyl lipids and the ether linkage is not easily disrupted. They must be identified by their chromatographic behaviour on TLC adsorbents relative to authentic standards or by the chromatographic behaviour of their hydrolysis products, i.e. free fatty acids and 1-alkylglycerols. The latter tend to migrate with or just ahead of monoacylglycerols on TLC adsorbents, but they cannot be hydrolysed further, and they react with periodate-Schiff reagent [163].

With plasmalogens, spectroscopic aids to identification are useful; the ether-linked double bond exhibits a characteristic band in the IR spectrum at 6.1 μm, while the olefinic protons adjacent to the ether bond produce a doublet centred at 5.89τin the NMR spectrum [802]. The ether bond in alkyldiacylglycerols gives a characteristic sharp band at 9 μm in their IR spectrum [90]. In the NMR spectra of the compounds, the protons on the carbon atom adjacent to the ether group give rise to a distinctive signal in the form of a triplet centred on 3.4τ[832]. Mass spectrometric methods of identification are discussed below.

Lipid samples containing plasmalogens should not be stored for long periods in solvents containing acetone, methanol or glacial acetic acid, as some rearrangement or other degradation may occur.

 

2. GC analysis of alkyl- and alk-1-enylglycerols and fatty aldehyde derivatives

As an alternative to or to complement an estimation by fatty acid analysis, ether lipids can be quantified by determination of the alkyl or alk-1-enyl moieties and indeed in a complete analysis, the individual components should be characterised. Methods are available that are suited to pure lipids with one type of ether moiety, isolated as described in the previous section, or that can be applied to more complex samples containing all of the radyl forms. 1-Alkylglycerols are released from alkyldiacylglycerols both by saponification and transesterification, but better recoveries are obtained if hydrogenolysis with lithium aluminium hydride is used, and this also brings about the release of alk-1-enylglycerols from plasmalogens [914,1001]. Although the technique has been used to remove the phosphorus group from phosphoglycerides, better results are reportedly obtained by hydrogenolysis with Vitride reagent (70% sodium bis-(2-methoxyethoxy) aluminium hydride in benzene) [867,886]. The following method is recommended -

Vitride reagent (0.5 mL) is added to the lipid (1 to 10 mg) in diethyl ether-benzene (2.5 mL; 4:1, v/v) in a test tube, and the solution is heated with occasional shaking for 30 min at 37°C. On cooling, water-ethanol (10 mL; 5:1,v/v) is added cautiously, then the products are extracted with diethyl ether (3 × 6 mL portions); hexane (10 mL) is added and the solution is dried over anhydrous sodium sulfate. After filtering, the solvent is removed from the combined extracts in a rotary evaporator. The samples are dissolved in a little chloroform and applied to a silica gel G TLC plate, which is developed in diethyl ether-hexane (4:1, v/v). The products are identified by their Rf values relative to standards, alkyl ethers migrating just ahead of the alk-1-enyl analogues, and they are eluted from the adsorbents with several volumes of diethyl ether.

Once the alkyl- and alk-1-enylglycerols are recovered from the adsorbent, they may be analysed separately by appropriate procedures. The GC methods used to identify individual components can also yield information on the total amount of sample if suitable internal standards are added. Various alternative procedures for determining the total amount of each ether form have been described, and the simplest appears to be acetylation with 14C-acetic anhydride, followed by separation of the acetate derivatives for liquid scintillation counting [921]. Alk-1-enylglycerols are not normally analysed as such, but are converted to aldehyde derivatives as described below. The fatty acid components of the sample are reduced to fatty alcohols during the reaction, but they can be analysed independently from a further sample of the lipid.

Alkylglycerols must be converted to less-polar volatile derivatives such as isopropylidene compounds, TMS ethers or acetates for GC analysis (see Chapter 4 for details of preparation) and of these, isopropylidene derivatives appear to be generally favoured. In this form, they may be separated both according to chain length and to the number of double bonds in the alkyl chain on GC columns packed with similar polyester liquid phases as are used to separated methyl esters, e.g. EGS, EGSS-X™, EGSS-Y™ and related polyesters (c.f. [643,656,999]). Because of the higher molecular weights, slightly higher column temperatures are necessary. It is a common practice to add an internal standard, such as the heptadecylglycerol derivative so that the amount of alkyl ether lipid in the sample can be estimated, when the nature of the aliphatic moieties is determined [688,886]. Nonpolar phases can also be used as, for example, when the ethers have branched and multi-branched alkyl moieties. Figure 10.5 illustrates a separation of the isopropylidene derivatives of 1-O-alkylglycerols from the glycerol ether diesters of the skin surface lipid of the guinea pig on a column packed with 3% SE-30™ and temperature-programmed from 150 to 300°C [228]; the straight-chain compounds are separated from those with a single methyl branch, and these are in turn separable from isomers with several methyl branches.

Figure10-05.png

Figure 10.5. GC separation of the isopropylidene derivatives of 1-O-alkylglycerols from the glycerol ether diesters of skin surface lipids from the guinea pig [228]. A glass column (2 m × 4 mm), packed with 3% SE-30™, was temperature-programmed from 150 to 300°C at 3°C/min. In groups of two peaks, the first component is multi-branched and the second is straight-chain; a third central component has a single methyl branch. (Reproduced by kind permission of the authors and of Biochimica Biophysica Acta, and redrawn from the original paper.)

 

Although it does not appear to have been taken up to any significant degree, one other of the many methods of analysing alkylglycerols that have been described deserves further consideration, i.e. the preparation under basic conditions of thionocarbonate derivatives [749]. These may be estimated by their absorbance at 235 nm, and they are also suited to GC analysis (this may be also true for alk-1-enylglycerol derivatives).

From preparations of alkylglycerols or their derivatives from natural sources, individual isomers or homologues can be isolated by procedures related to those described for fatty acids in Chapter 6, for example, for determination of the positions of double bonds by oxidation with permanganate-periodate [352] or by ozonolysis [750] with GC identification of the fragments. The IR and NMR spectra of alkylglycerols and their derivatives are similar to those of the lipids from which they are derived, except that the free hydroxyl residues or specific functional groups in the derivatives introduce additional features. Mass spectrometry of these compounds is discussed below. Racemic short-chain alkylglycerols, as the isopropylidene derivatives, have been resolved on a WCOT column of fused silica coated with a chiral phase [807].

In order to determine the composition of the alk-1-enyl moieties, it is necessary to liberate the aldehydes quantitatively from the plasmalogens and this is usually achieved simply by treatment with acid. The perfect method for the release of aldehydes has yet to be devised, and Anderson et al. [44] have critically examined some of the acidic hydrolysis procedures that have been described and recommend the following:

"The plasmalogens (0.2 to 2 mg) in diethyl ether (1.5 mL) are shaken vigorously for 2 min with conc. hydrochloric acid (1 mL). The ether layer is removed and the aqueous phase is extracted once more with ether (2 mL) and once with hexane (2 mL). The combined extracts are washed with distilled water before the solvent is evaporated in a stream of nitrogen. The free aldehydes are obtained by preparative TLC on silica gel G layers, with hexane-diethyl ether (90:10, v/v) as the mobile phase. Aldehydes migrate to just below the solvent front, and they can be recovered from the adsorbent for further analysis by elution with diethyl ether. The other products of the reaction are found much further down the plate."

It is now known that, although complete hydrolysis of the vinyl ether bond occurs with this method, only 80% recovery of aldehydes is likely to be attained, although these are probably representative in composition of the alk-1-enyl moieties originally present in the natural compound [992]. Addition of an odd-chain aldehyde to the reaction medium for use as an internal standard will correct for a proportion of these losses. Generation of aldehydes can also be effected as part of a more comprehensive scheme for the analysis of ether lipids (see below).

The properties of fatty aldehydes have been reviewed [303,576]. In the native form, they can be analysed by GC on similar columns to those used for fatty acid analysis, and they can be identified and estimated by analogous procedures. Standard aldehyde mixtures are available from commercial sources, or they can be prepared from the corresponding fatty acids by a number of simple methods. Aldehydes have been reported to be stable for long periods, if stored at −20°C in solution in carbon disulfide or other inert solvents such as pentane or diethyl ether [990], but they should not be kept in contact with other lipids, especially those containing ethanolamine, which catalyses a condensation reaction in which 2,3-dialkylacroleins are formed [805].

On the other hand, because free aldehydes have some tendency to polymerise on standing, especially in the presence of traces of alkali, it is more usual to convert them to more stable derivatives. Of these, acetals are the most popular, especially dimethyl acetals which are easy to prepare, although cyclic acetals (of 1,3-propanediol in particular) are also used because of their greater stability. Dimethyl acetals are prepared by heating the aldehydes or alk-1-enylglycerols under reflux with 5% methanolic hydrogen chloride in the same manner as was described earlier for the preparation of methyl esters (Chapter 4). They can also be prepared directly from plasmalogens, but prior to GC analysis it may be desirable to separate them from the methyl ester derivatives of the fatty acids, which are formed at the same time, by means of adsorption chromatography (see Chapter 4 also) or by saponification of the esters. On the other hand, the resolving power of modern WCOT columns with polar stationary phases is such that the common range of aldehydes found in animal tissues is well resolved from the methyl esters. The C16 and C18 dimethyl acetals emerge clearly ahead of the corresponding esters in a region of the chromatographic trace that tends to be comparatively empty (c.f. [91]).

Cyclic acetals or 1,2-dioxolanes are prepared by condensing 1,3-propanediol with aldehydes in the presence of an acidic catalyst. They have greater thermal stability and are sometimes favoured for GC analysis [889]. They are prepared as follows:

The aldehydes or plasmalogens (up to 10 mg) are heated with p-toluenesulfonic acid (0.5 mg) and 1,3-propanediol (50 mg) in chloroform (5 mL) in a sealed tube at 80°C for 2 hr. On cooling, chloroform (3 mL), methanol (4 mL) and water (3 mL) are added, and the mixture is shaken. The lower solvent layer, which contains the required derivatives, is evaporated.

Hydrazone derivatives of aldehydes can be converted directly to acetals by heating them with the required alcohol and an acid catalyst in the presence of acetone, which serves as an exchanger [580]. All acetal derivatives are stable to alkaline conditions, but they are hydrolysed by aqueous acid. Although decomposition of dimethyl acetals to methyl enol ethers tended to occur on packed columns in the early days of the technique [583,881], this should not be a problem with modern packing materials. On the other hand, there is a report of some decomposition of dimethyl acetals on a modern WCOT column of fused silica, when a "hot needle" injection technique was employed [135]; this should not happen with on-column injection. Like the free aldehydes, dimethyl acetals can be separated according to chain length and the number of double bonds, under comparable GC conditions as are used with the analogous methyl esters, and similarly they can be identified by their retention times relative to authentic standards or by using equivalent chain-length (ECL) values, as illustrated earlier (Chapter 5) for methyl esters. In addition, individual components can be isolated and characterised by the same procedures as those used to determine the structures of fatty acids [689].

The IR spectra of free aldehydes are similar to those of the related fatty acid esters, except that the distinctive frequency for the carbonyl functions is at 5.9 μm with an additional band at 3.7 μm [581]. In the NMR spectra of aldehydes, a triplet at 9.7τ is characteristic of the proton on the carbonyl group [576]. Mass spectra of aldehydes have been recorded and these are described in the next section. The elution properties of isomeric monounsaturated aldehydes on silver ion chromatography have been investigated [327].

An integrated method for the simultaneous determination of both the alkyl and alk-1-enyl moieties of lipids is obviously desirable. In one such [886], the plasmalogens are converted to cyclic acetals in the presence of heptadecanal as an internal standard, before the products are submitted to hydrogenolysis with Vitride reagent to release the alkylglycerols in the presence of heptadecylglycerol as a further internal standard. The alkylglycerols are converted to the isopropylidene derivatives, and each type of product is isolated by TLC prior to identification and quantification by GC. As an alternative, the products of the Vitride reaction are acetylated, and acidified to release the aldehydes; the two types of compound are then analysed by GC simultaneously, the aldehydes emerging well ahead of the alkylglycerol acetates [920]. The Vitride reaction is described above in detail, a procedure for acetylation with acetic anhydride and pyridine is given in Chapter 4, and the method for the release of aldehydes with acid is also given above. The nature of the GC separation is illustrated in Figure 10.6. A packed column, with Silar 5CP™ as the stationary phase, was used and was maintained isothermally at 220°C. If temperature-programming and a modern WCOT column were employed, a much tidier chromatogram would be anticipated.

Figure10-06.png

Figure 10.6. "Typical" GC separation of alkyldiacetylglycerols and aldehydes derived from plasmalogens [920]. A glass column (200 × 0.5 cm), packed with 10% Silar 5CP™ on GasChrom Q™, was maintained at 220°C. The chart speed was reduced from 20 to 5 mm/min after elution of the aldehydes. (Reproduced by kind permission of the author and of Fette Seifen Anstrichmittel, and redrawn from the original paper.)

 

3.  Gas chromatography-mass spectrometry
Mass spectrometry of ether lipids has been reviewed by Egge [237] and Myher [642]. There have been a number of electron-impact spectra published for isopropylidene derivatives of alkylglycerols, and that for the C16 compound is illustrated in Figure 10.7 [106,337,339,758,868]. The molecular ion is rarely detectable, but there is usually a sufficiently abundant ion equivalent to [M−15]+ (i.e. at m/z = 341, loss of methyl from the isopropylidene group) for determination of molecular weight. The base peak is at m/z = 101 and represents cleavage between carbons 1 and 2 of the glycerol moiety, the charge being retained by the fragment containing the isopropylidene group. Among other fragments obviously derived from the molecular ion are [M−31]+, [M-59]+ (m/z = 297), [M-76]+, [M-103]+ (m/z = 253) and [M−132]+ (m/z = 224). The diagnostic ions are more easily seen in spectra obtained at an ionisation potential of 25 eV [237]. Mass spectra of 2-methoxy- [337,339] and 2-hydroxyalkylglycerols [629] in the form of isopropylidene derivatives have also been published. In many respects, the mass spectra of isopropylidene derivatives of thioglycerol ethers are similar to those of the related O-alkyl compounds, but they differ in that the former tend to have a somewhat greater molecular ion while the ion at [M−15]+ is smaller [106,254].

Figure10-07.png

Figure 10.7. The mass spectrum of the isopropylidene derivative of hexadecyl-1-glycerol.

 

With TMS ether derivatives of 1-O-alkylglycerols, the molecular ion is again of low intensity but ions equivalent to [M−15]+, [M−90]+, [M−103]+, [M−147]+ and [M−180]+ are significant [643,656]. The base ion is usually at m/z = 205 for cleavage between carbons 1 and 2 of the glycerol moiety. Acetate derivatives tend to give a small but significant molecular ion, especially with higher homologues, and there are characteristic ions at [M−43]+, [M−60]+ (loss of an acetic acid moiety), [M−73]+, [M−103]+ (often the base peak), [M−120]+ and [M−145]+ [237]. A 2-hydroxyalkylglycerol in the Harderian gland of the rabbit was identified as the acetate derivative; its mass spectrum resembled that of an unsaturated compound because the 2-acetyl group was lost so readily [774].

With none of these derivatives can the positions of double bonds and methyl branches in the alkyl chain be located. Nicotinates have been employed successfully with other diols to fix the positions of such substituents [364], and there would appear to be no reason why they should not be used here. This would certainly appear to be simpler than an alternative approach in which aldehydes were cleaved to form iodides, which were in turn converted to nitriles, thence to acids and finally to pyrrolidide derivatives for identification by GC-MS [859].

Alk-1-enylglycerols have been subjected to mass spectrometry in the form of the TMS ether derivatives, and they give distinctive spectra with a good molecular ion [656]. However, most analysts have preferred to study aldehydes and their derivatives prepared by acid hydrolysis of plasmalogens. An important paper on the subject was published by Christiansen et al. [156]. Saturated aldehydes give a small but significant molecular ion, and there is a series of peaks of the form 68 + 14n, where n = 0, 1, 2 and so on. In the high mass range, there are characteristic peaks at [M−18]+, [M−28]+, [M−44]+ and [M−46]+, which are not seen in the spectra of unsaturated isomers, although these have somewhat greater molecular ions. With dimethyl acetals, molecular ions are not observed in the electron-impact mass spectra, but an ion equivalent to [M−32]+ (loss of methanol) permits the determination of molecular weight. An ion at [M−64]+ represents loss of a vinyl methyl ether moiety. In homologues at the higher end of the molecular weight range, the base peak is at m/z = 71. O-Methyl- and O-t-butyldimethylsilyloximes tend to give much better molecular ions, especially when chemical ionisation is employed [710], and reduction with lithium aluminium hydride to alcohol derivatives has also been recommended [624]. Similarly, negative-ion MS tends to give an excellent molecular ion with aldehydes [754]. On the other hand, none of these methods give any information on the positions of double bonds or methyl branches in the alkyl chain, so a more informative approach might be to oxidise the aldehyde to an acid for conversion to a picolinyl ester derivative for mass spectrometric analysis. Indeed, methodology of this kind has been employed in the analysis of long-chain bases (see Section E.1 below).

 

4.  Platelet-activating factor
1-Alkyl-2-acetyl-sn-glycerophosphorylcholine or "platelet-activating factor" (commonly abbreviated to PAF) is present in minute concentrations in platelets and certain other cells, yet exerts profound physiological effects. Its chemistry and biochemistry have been reviewed [353,866] and here... It is such a polar molecule that HPLC in the adsorption mode is required for its isolation. As the author and others [107,168] have reviewed these methods in some detail, it need only be mentioned here as an example that it elutes between sphingomyelin and lysophosphatidylcholine from a column of silica gel with a gradient of hexane-isopropanol-water in which the water content is increased [110]. No existing method is sufficiently sensitive to demonstrate the separation at natural concentrations, however, other than by incorporating a radioactive label by biosynthetic means (note added later - electrospray mass spectrometry may now be the best method).

The most satisfactory method of quantification and of identifying isomers of PAF in which the nature of the alkyl-chain varies is probably GC or GC-MS, after collection of the appropriate fraction from an HPLC column. Interestingly, intact ether-linked equivalents of lysophosphatidic acid can be subjected to GC separation after conversion to the TMS ether derivative [917], but this does not appear to be a practical approach to the analysis of PAF. The preferred method is to hydrolyse PAF to 1-O-alkyl-2-acetylglycerol with the phospholipase C of B. cereus or C. welchii (see Chapter 8 for detailed procedures), then to convert to a suitable derivative for GC analysis. With synthetic samples, GC conditions similar to those described above for alkyl ether derivatives can be employed. With PAF at the concentrations existing in natural tissues, the problem of detection is technically demanding. GC-MS with selective ion monitoring of BDMS [793,794] and TMS ether [672] derivatives of 1-alkyl-2-acetylglycerols has given good results with some samples.

What is probably the most sensitive method involves preparation of the pentafluorobenzoyl derivative of 1-alkyl-2-acetylglycerols, which gave 92% of the total ion current as the molecular ion when subjected to GC-MS with negative-ion chemical ionisation [753]. It was necessary to add deuterium-labelled PAF as an internal standard for quantification purposes, and as little as 100 fg could be measured. Others prepared a heptafluorobutyrate derivative from the 1-alkyl-2-acetylglycerols derived from PAF, then employed GC on a glass WCOT column with highly sensitive electron-capture detection; amounts as low as 20 pg were determined [120]. PAF [916] and lyso-PAF [358] have been analysed by GC-MS with somewhat lower sensitivity, after chemical hydrolysis, as propionyl and isopropylidene derivatives respectively.

 

C.  Cholesterol

Free and esterified cholesterol can be measured during the determination of a total lipid profile by the methods described in Chapter 8. On the other hand, so important is the absolute concentration of cholesterol in plasma as a diagnostic marker in disease states believed to be that a number of methods have been developed for the rapid determination of cholesterol alone by various means. For routine clinical applications, all such methods, including GC, should be capable of a high degree of automation. The procedures available have been reviewed and compared elsewhere [336,659,1011]. Enzymatic and colorimetric methods appear to be favoured in most routine clinical applications (commercial kits are available for the purpose), but GC procedures have certain advantages in terms of precision and specificity, and may also permit detection of sterols other than cholesterol.

All such methods require that the lipids be extracted from the plasma or other tissue, and an appropriate internal standard is added at this stage. If the total cholesterol concentration is required, as well as that of the free cholesterol, a hydrolysis step is inserted. Finally, the products are derivatized for GC analysis. To simplify the first steps, it is possible to hydrolyse a plasma sample directly. In most of the published papers, the internal standard has been cholestane, octadodecane, desmosterol or epicoprostanol (3α-hydroxy-5β-cholestane), while the GC separation has been effected on a column (50 to 100 cm long) packed with 3% SE-30™ and operated isothermally in the range 200 to 240°C; preparation of TMS ethers gives sharper peaks and improved quantification. Innumerable procedures, varying in minor details, have been described for the purpose. The following candidate reference method is recommended [214].

The serum sample (about 0.2 mL) is weighed into a screw-capped vial, and a solution of epicoprostanol, in an amount equivalent to the concentration of cholesterol expected, in ethanol (0.6 mL) is added, followed by ethanol (1 mL) and 0.4 mL of ethanolic potassium hydroxide solution (4.6 mole of KOH in 0.3 L of water and 1 L of ethanol). The mixture is left at 37°C for 3 hours, it is cooled and water (2 mL) and hexane (4 mL) are added. The whole is shaken thoroughly for 15 min, centrifuged and the organic layer recovered. An aliquot of this is taken to dryness and the residue is converted to the TMS ether derivatives (see Chapter 4 for a method).

While many different GC columns could be used, a glass WCOT column (25 m × 0.3 mm), coated with OV-1™ and operated isothermally at 240°C, gave satisfactory results in the work cited; the TMS ether derivatives of epicoprostanol and cholesterol eluted in 17.7 and 20 min, respectively [214]. Of course, the procedure must be calibrated carefully with suitable standards, and the original publication should be consulted for the fine detail of the protocol. Other workers have obtained results adequate for many purposes with packed columns (1.5 to 2 m × 4 mm i.d.) and similar nonpolar silicone phases (e.g. 3% SE-30™ relative to the support). In routine use, a coefficient of variation of 0.35 to 0.5% was obtained. Even higher accuracy is claimed for "definitive" procedures in which [3,4-13C]-cholesterol [273,703] and deuterated cholesterol [187,983] are employed as internal standards and GC-MS is used for quantification.

There is an enormous body of work on the analysis of steroids other than cholesterol by chromatographic means (reviewed elsewhere [381]), and detailed discussion is outside the scope of this book. It may be worth noting, however, that GC on WCOT columns has been utilised to study the products of oxidation of cholesterol [691] and to identify phytosterols accumulating in the plasma of patients suffering from phytosterolaemia [527].

 

D.  Glycerol

Free glycerol or that released by hydrolysis of triacylglycerols is readily estimated by enzymatic or chemical means using one of the many kits that are available commercially for the purpose (reviewed elsewhere [659]). GC methods may also be used, and the following is suited to the determination of the glycerol content of simple lipids [390].

The glycerolipid (1 to 10 mg) and a known amount of a suitable methyl ester as an internal standard, say methyl pentadecanoate, are dissolved in dry diethyl ether (2 mL), and an ethereal solution of lithium aluminium hydride (20 mg in 3 mL) is added in portions of 0.1 mL until the boiling stops. After addition of a one volume excess of the lithium aluminium hydride solution, the mixture is refluxed for 1 hour. Acetic anhydride is added dropwise with cooling to destroy the excess reagent, followed by additional acetic anhydride (2.5 mL) and xylene (3 mL). The ether is removed by evaporation and the residue is refluxed for 6 hours; the temperature should reach 110°C if no ether remains. Finally, the reagents are removed on a rotary evaporator and the products are taken up in dry diethyl ether for analysis by GC.

The procedure may be scaled down appreciably if this is required. A somewhat different method is recommended for determining the glycerol in phosphoglycerides [389], because lithium aluminium hydride is insufficiently vigorous, but replacement of this reagent with Vitride (see Section B.1 above) should suffice. On a packed column of EGS, triacetin elutes just before octadecanyl acetate (fatty alcohol acetates are also produced in the reaction), but it elutes nearer octanyl acetate on a nonpolar stationary phase, such as Apiezon L™ or SE-30™. An alternative method for simple glycerides, involving alkaline hydrolysis followed by determination of the glycerol as the TMS ether derivative on a WCOT column, has recently been described [138].

 

E.  Long-Chain Bases

1.  Isolation, derivatization and GC separation
The long-chain or sphingoid bases are the characteristic structural components of sphingolipids and very many different compounds, including homologues and isomers, can exist in a single natural source (see Chapter 2 (or our specific webpage) for a brief description) [460,974,981]. Before these constituents of sphingolipids can be analysed, it is first necessary to hydrolyse any glycosidic linkage or phosphate bond as well as the amide bond to the fatty acyl group. Ideally, this should be accomplished by a procedure in which no degradation or rearrangement of the bases occurs, but the perfect method has not yet been devised. Base-catalysed hydrolysis has been advocated by many analysts, and the following method appears to give much less degradation than others to have been described [625].

The sphingolipids (up to 5 mg) are dissolved in warm dioxane (2.5 mL), 10% aqueous barium hydroxide solution (2.5 mL) is added and the mixture is heated in a sealed tube at 110°C for 24 hours. On cooling, water (10 mL) is added, and the solution is extracted with chloroform (2 × 15 mL). After drying over anhydrous sodium sulfate, the solvent is evaporated to yield the required long-chain bases.

Some degradation of trihydroxy bases especially may be caused by even this procedure, but it is troublesome only if these are present in small amounts.

In other laboratories, acid-catalysed hydrolysis has been used, although rearrangement and substitution at C-3 and C-5 inevitably occurs to a certain extent, thereby altering the configuration of the bases from the erythro to the threo form. In addition, O-methoxy artefacts are formed in the presence of methanol, and compounds containing a tetrahydrofuran ring may be formed from trihydroxy bases. A recently described procedure [454], in which aqueous hydrochloric acid in acetonitrile (an aprotic solvent) is employed for hydrolysis, reportedly produces fewer artefacts than earlier methods, in which methanol is the solvent. It gives particularly good yields of long-chain bases from gangliosides. The detailed method is -

The hydrolysis reagent consists of 0.5 M HCl and 4 M water in acetonitrile (0.3 mL), and is added to the glycolipids (up to 200 micrograms) in a Teflon-lined screw-capped tube, which is flushed with nitrogen, sealed and heated at 75°C for 2 hours. The solvents are evaporated in a stream of nitrogen, chloroform (5 mL) is added followed by 0.05 M sodium hydroxide in methanol-0.9% saline solution-chloroform (48:47:3 by volume) (1 mL), and the mixture is shaken thoroughly before being centrifuged at 3000g. The lower phase is washed with three further portions of the sodium hydroxide solution, and then with two portions of the same solvents but without sodium hydroxide. Finally, the lower phase is evaporated in a stream of nitrogen to recover the required bases."

With sphingolipids other than gangliosides, somewhat milder hydrolysis conditions were preferred, i.e. sphingomyelins were reacted for 1 hour in 1 M HCl in water-methanol (2:1 by volume) at 70°C for 16 hours, while cerebrosides required 3 M HCl in water-methanol (1:1 by volume) at 60°C for 1.5 hours [573]. (A related procedure, but for the isolation of the fatty acid constituents of sphingolipids for further analysis, has also been described [65] (see Chapter 4).) An objective experimental comparison of the above base- and acid-catalysed methods with a range of different substrates would now appear to be desirable.

HPLC procedures for the analysis of long-chain bases have been described and are reviewed elsewhere [168]. These appear to be sensitive, and some of the separations are impressive, but the methods have as yet been applied to a limited range of relatively simple samples only. Much more experimental work with GC methods has been published. As with other lipids with polar functional moieties, it is necessary to prepare volatile nonpolar derivatives, and most analysts have made use of O-TMS or N-acetyl-O-TMS ether derivatives. Acetylation is carried out as follows [280]:

The long-chain bases (0.1 to 0.2 mg) are reacted with freshly prepared acetic anhydride in methanol (1:4, v/v; 50 μL) at room temperature overnight. n-Butanol (2 mL) is added to facilitate the removal of the excess acetic anhydride during evaporation in a stream of nitrogen.

A silylation reagent consisting of hexamethyldisilazane (2.6 mL), dry pyridine (2 mL) and trimethylchlorosilane (1.6 mL) has been recommended for long-chain bases [149], but any of the more powerful silylating reagents described in recent years (see Chapter 4) should give good results.

The sphingoid bases from plasma sphingomyelin, for example, were separated as N-acetyl-O-TMS ether derivatives on a packed column (2 m × 3 mm) packed with 3% SE-30™, maintained isothermally at 230°C [724]. Saturated and unsaturated isomers are only partly resolved by this means. More recently, other workers used a WCOT column (25 m), coated with OV-101 and operated isothermally at 260°C, for similar separations [356]. In this instance, base-line resolution of sphingosine and dihydrosphingosine derivatives was possible. Simple O-TMS ethers (i.e. not N-acetylated) tend to elute at slightly lower temperatures, but peaks tail somewhat, and these derivatives may not be quite so useful for mass spectrometric identification purposes (see below) [461]. As an example, a separation of the straight- and branched-chain dihydroxy bases from the cerebrosides of the Harderian gland of the guinea pig is illustrated in Figure 10.8 [1008]. Again a packed column containing a nonpolar phase, OV-101™ was employed to effect the separation.

Figure 10.8. GC separation of the TMS ether derivatives of long-chain bases from the cerebrosides of the Harderian gland of the guinea pig [1008]. A glass column (2 m × 3 mm i.d.), packed with 1% OV-1™, was maintained at 220°C with nitrogen at 30 mL/min as the carrier gas. (Reproduced by kind permission of the authors and of the Journal of Biochemistry (Tokyo), and redrawn from the original paper.) Figure10-08.png

Such procedures may be suitable for the analysis of samples containing a relatively simple range of long-chain bases, but some natural lipid extracts are very complex. Alternative complementary techniques must then be employed to obtain the resolution needed. The most widely used approach is to stabilise the amino group by conversion to the dinitrophenyl (DNP) derivative, for separation into different classes by TLC procedures; these compounds are yellow in colour so are easily seen on a TLC plate. DNP derivatives can later be degraded to aldehydes by periodate oxidation for GC analysis in this form [464,465]. The procedure for the preparation of N-DNP derivatives of sphingoid bases is:

The sphingoid bases (up to 5 mg) are reacted with 1-fluoro-2,4-dinitrobenzene (5 mg) in methanol (1 mL), with addition of potassium borate buffer (pH 10.5; 4 mL) dropwise followed by heating at 60°C for 30 min. After cooling, the mixture is partitioned between chloroform, methanol and water in the ratio of 8:4:3 by volume, and the lower phase is collected and evaporated. The products are purified by chromatography on a short column of silicic acid (1 g), from which nonpolar impurities are eluted first with hexane-diethyl ether (7:3; 20 mL), while the required DNP derivatives are recovered with the same solvents in the ratio 1:1.

They can be separated into three groups on layers of silica gel G impregnated with 2% boric acid, i.e. saturated dihydroxy-, unsaturated dihydroxy (with a trans double bond in position 4) and trihydroxy bases, with chloroform-hexane-methanol (5:5:2, by volume) as the mobile phase [465]. When acidic hydrolysis procedures are utilised in the preparation of the bases, unnatural threo-isomers of the unsaturated dihydroxy bases are found just below the natural erythro compounds on the TLC plate. Each of the fractions separated by TLC can be recovered from the adsorbent by elution with chloroform-methanol (2:1, v/v), but the eluate should be washed with one quarter the volume of water to remove boric acid which is also eluted. If need be, the bases can be further resolved by silver ion TLC [462] or by HPLC in the reversed-phase mode (reviewed elsewhere [168]). It is possible that HPLC in the adsorption or silver ion modes could also contribute to the problem of analysis, but these do not appear to have been tried.

To simplify the resolution of saturated, unsaturated and branched-chain isomers, Karlsson [464,465] and others have oxidised the DNP derivatives of each group of bases, separated by TLC, to aldehydes. As similar compounds are produced from both di- and trihydroxy bases of the threo or erythro configurations, it is essential to carry out the TLC separation prior to oxidation. Aldehydes are readily separated by chain length and degree of unsaturation on GC columns similar to those used for the separation of methyl esters of fatty acids (see also Section B above). Either periodate or lead tetraacetate may be used for the oxidation step, and the following method can be recommended [466].

The long-chain bases (2 mg) are oxidized by reaction with lead tetraacetate (30 mg) in benzene (0.5 mL) (caution!) at 50°C for 1 hour. Water (5 mL) and hexane (5 mL) are added, the mixture is shaken thoroughly and the solvent layers are dried over anhydrous sodium sulfate before the solvent is evaporated. The aldehydes are analysed immediately by GC.

Aldehydes are more easily identified than are the parent compounds, since a wide range of standards is available from commercial sources or can be prepared synthetically from other lipids. As an example of the full application of this methodology, more than 30 different bases were detected in the sphingolipids of bovine kidney [469]. Mass spectrometry can be utilised as an aid to identification of aldehydes (see also Section B above), although some workers have preferred to reduce them to fatty alcohols and then to prepare acetate or TMS ether derivatives for this purpose [624]. In addition, all the methods for the location of double bonds in fatty acids, such as ozonolysis or hydroxylation with osmium tetroxide and preparation of TMS ethers for MS, have been utilised with aldehydes prepared from sphingoid bases [464,465].

One further approach to identification has been to oxidize the aldehydes to fatty acids by the following procedure [887,888,971].

The aldehydes (up to 1 mg) are oxidized in tetrahydrofuran-water (9:1, v/v; 2 mL) to which silver oxide (30 mg) is added. The mixture is shaken gently for 24 hours at room temperature, then the solvent is removed in a stream of nitrogen, 6 M nitric acid (1 mL) is added, and the products are extracted with diethyl ether (8 mL). The extraction is repeated and the combined ether layers are washed with water, dried over anhydrous sodium sulfate and evaporated under nitrogen.

If methyl ester derivatives are prepared for GC analysis, it may be necessary to remove some residual aldehydes (in the form of dimethyl acetals) by preparative TLC (see Chapter 4). These will not be troublesome if picolinyl ester derivatives are prepared for identification by GC-MS (see Chapter 7).

Note that aldehyde derivatives prepared from trihydroxy bases will be one carbon shorter than those from equivalent dihydroxy bases, and the number of hydroxyl groups must be determined from the TLC behaviour of the base or its DNP derivative. The proportions of the various isomers within each class of base can be determined with reasonable accuracy by GC, but artefact formation during the hydrolysis stage may distort the apparent relative proportions of the various classes of base to each other.

Recently, it has been demonstrated that 13C NMR spectroscopy may be used to determine the configuration of long-chain bases while they still form part of intact natural lipids [792]. For the first time, it was shown unequivocally that they were exclusively of the erythro configuration.

 

2.  Gas chromatography-mass spectrometry
It is not necessary to consider the degraded forms of sphingoid bases, such as aldehydes, here as equivalent compounds are dealt with above (Section B.3). Mass spectrometry is certainly the most powerful tool available to the analyst for identifying long-chain bases, although appropriate derivatives must be prepared for the purpose. Karlsson [464,465] favours the preparation of TMS ether or methyl ethers of the DNP derivatives, with hydroxylation of double bonds and similar derivatization of the resulting hydroxyl groups. Certainly, this approach gives definitive spectra, but the molecular weights are then frequently too high for GC-MS. Pure individual compounds are then required, and direct insertion probes into the instrument must be utilised. Similar types of derivative have been employed with HPLC coupled to mass spectrometry, and this has been discussed elsewhere [168].

O-TMS ether and N-acetyl-O-TMS derivatives are better suited to analysis by GC-MS. Of these, the latter have been most used and the details of the fragmentation processes involved with electron-impact ionisation have been well worked out, principally in Sweeley's laboratory [280,495,724]. The mass spectrum of bis-O-trimethylsilyl-N-acetylsphinganine is illustrated in Figure 10.9 [280,495]. There is no detectable molecular ion (expected at m/z = 487), but the molecular weight is clearly indicated by an ion equivalent to [M−15]+ at m/z = 472. There are small peaks at [M−59]+ (m/z = 428), representing loss of the acetamido group, and at [M−90]+ (m/z = 397), for the loss of a trimethylsilanol moiety. The ion at m/z = 384 is the part of the molecule remaining after the loss of the terminal methylene and its TMS ether group. That at m/z = 313 represents cleavage between carbons 2 and 3 of the molecule, but the corresponding fragment from the remainder of the molecule at m/z = 174 is small (c.f. the spectra of unsaturated isomers). In contrast, an ion at m/z = 157 is particularly abundant, while the base peak is at m/z = 73.

Figure10-09.png

Figure 10.9. The mass spectrum of bis-O-trimethylsilyl-N-acetyl-sphinganine [495]. (Reproduced by kind permission of the authors and of Chemistry and Physics of Lipids, and redrawn from the original paper.)

 

Many features in the mass spectrum of the N-acetyl-O-TMS ether derivative of sphinga-4,14-dienine, shown in Figure 10.10 [724], are similar to this, but the ion for [M−59]+ at m/z = 424 is more abundant, and there are major peaks for both fragments formed by cleavage between carbons 2 and 3, i.e. at m/z = 309 and 174. Derivatives of this kind have been employed with many natural samples [374,375,600,888,971]. The positions of double bonds or of methyl branches cannot be deduced from such spectra, and this is one reason for approaching the problem of structure determination via the degradative route discussed in the previous section. On the other hand, these compounds can be subjected to hydroxylation of the double bonds and formation of TMS ethers for identification, although the molecular weights are thereby increased very substantially [375,724].

Figure10-10.png

Figure 10.10. The mass spectrum of the N-acetyl-O-trimethylsilyl ether derivative of sphinga-4,14-dienine, isolated from plasma sphingomyelin [724]. (Reproduced by kind permission of the authors and of Biochemistry, and redrawn from the original paper.)

 

The TMS ethers of the base with a free amine group have also been employed for GC-MS [461]. Again there is no molecular ion, although the molecular weight is indicated by an ion equivalent to [M−15]+, and there are prominent fragments for cleavage between carbons 1 and 2 and between carbons 2 and 3. The structures of several natural sphingoid bases have been determined with such derivatives [601,602,811,887].

One further type of derivative of sphingoid bases for GC-MS purposes is worthy of note, i.e. cyclic boronates [277]. Usually, it is necessary to protect the amine group, but a bis-boronate derivative is formed from trihydroxy bases. Model compounds, prepared from standards, had excellent GC properties and also exhibited distinctive fragmentation properties on mass spectrometry. For example, they gave molecular ions in reasonable abundance. Unfortunately, they do not appear to have been applied to the analysis of natural samples.

 

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 12, 2011