The Analysis of Lipids other than Fatty Acids
Sections C to G GC of Complex Glycerolipids and Other Simple Lipids
C. Diacyl Glycerophospholipids and Diacylglycerols
1. Preparation of diacylglycerols from phospholipids
Molecular species of complex lipids such as glycerophosphatides are most easily separated after the phosphorus moiety has been removed, and this is certainly true for gas chromatography. Normally, dephosphorylation is accomplished by enzymatic hydrolysis with phospholipase C, although other methods are available. While it is technically possible to subject diacylglycerols per se to GC analysis, this is rarely attempted, because of problems of tailing and of acyl migration. The 1,2-diacyl-sn-glycerols obtained by enzymatic hydrolysis are therefore purified and acetylated (or otherwise derivatized) immediately to prevent acyl migration during subsequent separation procedures. In addition, alkyl- and alkenyl-forms may have to be isolated before more detailed studies are commenced. One advantage of this approach is that diacylglycerol acetates and related derivatives from all phospholipid classes may be separated into molecular species by exactly the same procedures. GC methods for the analysis of molecular species of phospholipids in the form of the diacylglycerol derivatives have been reviewed [276,516,584,591,642,744,956]. The technique is also used in profiling lipid classes in tissues (see Section I). In addition, diacylglycerol derivatives are of value in structural analyses of triacylglycerols, although diacylglycerols per se tend to be minor constituents of tissue lipids.
No satisfactory chemical method for removing the phosphorus moiety of glycerophosphatides has been developed. Acetylation with equal mixtures of acetic anhydride and acetic acid at 140°C in a sealed tube has been used for the purpose , but plasmalogens are degraded and acyl migration with formation of a small proportion of 1,3-diacylglycerol acetates takes place. On the other hand, it is argued that this occurs intra- rather than intermolecularly [369,370]. Therefore, the same fatty acids remain in molecular combination and the diacylglycerol acetates so produced are still suited to molecular species analysis (but not for analysis of fatty acid positional distributions), on GC columns packed with nonpolar phases at least. TMS ether derivatives of diacylglycerols have been prepared from glycerophosphatides by subjecting the latter to very high temperatures (approximately 250°C) for brief periods and then silylating [404,405]. Here also, 1,3-derivatives accompany the 1,2-compounds, and the latter do not accurately represent the original composition of the native phospholipid so that their value for structural analysis is limited. Enzymic hydrolysis is perhaps more tedious and time consuming, but it undoubtedly gives the best results.
A variety of enzymes capable of releasing 1,2-diacylglycerols from phosphoglycerides or of ceramide from sphingomyelin, and termed "phospholipase C" (EC 126.96.36.199), has been isolated from microorganisms, but especially from Clostridium welchii and Bacillus cereus. Enzymes from both sources are commercially available, or alternatively highly active preparations of that from B. cereus can be obtained by ammonium sulfate precipitation from the supernatant fluid used as a growth medium for the microorganisms . If the enzyme is to be used in structural studies, little further purification is necessary. The properties and substrate specificities of corresponding enzyme preparations from different sources can vary greatly (reviewed by Brockerhoff and Jensen ) and in structural studies, it is necessary to choose the enzyme from the most appropriate source for a particular phospholipid class. The enzymes do not possess an absolute specificity for a phosphate bond in position 3 of L-glycerol, and that of B. cereus, for example, will react with synthetic phospholipids with the phosphate bonds in positions sn-1, sn-2 and sn-3 ; that of C. welchii hydrolyses D-phosphatidylcholine, but much more slowly than the normal L-isomer . Although there is some evidence that molecular species containing shorter-chain fatty acids are hydrolysed more rapidly than those with longer-chain components, the effect need not be troublesome as the reaction can usually be taken to completion with care.
Phospholipase C preparations from C. welchii are used most often for the preparation of 1,2-diacyl-sn-glycerols from phosphatidylcholine or of ceramides from sphingomyelin. In addition, they can be used to prepare monoacylglycerols from lysophosphatidylcholine  and ceramides from ceramide aminoethylphosphonate . The enzyme is utilised in the following manner .
The phospholipase C of C. welchii (1 mg) in 0.5 M tris(hydroxymethyl)methylamine (Tris) buffer (pH 7.5; 2 mL), 2 × 10−3 M in calcium chloride, is added to phosphatidylcholine (5 mg) in diethyl ether (2 mL). After the mixture is shaken at room temperature for 3 hours, it is extracted three times with diethyl ether (4 mL portions). The ether layer is dried over anhydrous sodium sulfate before it is evaporated in a stream of nitrogen at ambient temperature. Pure 1,2-diacyl-sn-glycerols are obtained by preparative TLC on layers of silica gel G impregnated with boric acid (10%, w/w), with hexane-diethyl ether (50:50, v/v) as solvent system. The appropriate band is located under UV light after spraying with 2',7'-dichlorofluorescein (see Chapter 2), and is eluted from the adsorbent with diethyl ether. (Boric acid, which is also eluted, does not interfere with subsequent stages).
No unnecessary delays are allowable at any stage and the compounds should not be heated or permitted to come in contact with polar solvents, otherwise acyl migration may occur. The diacylglycerols should be acetylated at once with acetic anhydride and pyridine (see Chapter 4 for practical details), as diacylglycerol acetates can be stored indefinitely in an inert atmosphere at low temperatures without coming to harm. In addition, it is advisable that the diacylglycerol acetates be purified by HPLC , by TLC on silica gel G (hexane-diethyl ether, 7:3, v/v, is a suitable solvent system), or more conveniently on a short column of Florisil™ or silica gel (0.3 to 0.5 g) eluted with the same solvent mixture, as this can prolong the life of the GC column in sustained use. A small portion of the diacylglycerols or diacylglycerol acetates should be transesterified so that its fatty acid composition can be compared with that of the original lipid, to ensure that random hydrolysis of molecular species has occurred. TMS or t-butyldimethylsilyl (BDMS) ether derivatives of diacylglycerols are preferred for some purposes, but only the latter are stable during prolonged storage.
Ceramides prepared by this method from sphingomyelin are comparatively stable, but they should be purified on thin layers of silica gel G or by a mini-column procedure with chloroform-methanol (9:1, v/v) as solvent system, before being derivatized and analysed further.
Although C. welchii preparations can be used to hydrolyse phosphatidylethanolamine, provided that some lysophosphatidylcholine  or sphingomyelin  is present to activate the enzyme, better results are obtained with the phospholipase C of B. cereus, which can also be used to prepare diacylglycerols from phosphatidylserine, phosphatidylinositol, phosphatidylglycerol and diphosphatidylglycerol. The author has found the following method to be satisfactory for the purpose [991,1002]. (Note that Tris buffer is not suitable).
Phosphatidylethanolamine (5 mg) and sphingomyelin (3 mg) are mixed with 0.2 M phosphate buffer (pH 7.0; 0.5 mL) containing 0.001 M 2-mercaptoethanol and 0.0004 M zinc chloride, and phospholipase C from B. cereus (1 mg) in the same buffer (0.5 mL) is added. The mixture is shaken vigorously for 2 hours at 37°C, when the diacylglycerols produced are extracted and purified as in the procedure immediately above.
The phospholipase C of Clostridium perfringens is specific for the diacyl forms of phospholipids, yet that of B. cereus hydrolyses the 1-0-alkyl forms of phospholipids three times as quickly as the diacyl or alkenyl forms . The latter enzyme will only hydrolyse sphingomyelin under exceptional circumstances. Phospholipase C preparations that are specific for phosphatidylinositol have been obtained from Staphylococcus aureus  and B. thuringiensis . A related enzyme, found in brain, has been used to prepare diacylglycerols from mono-, di- and triphosphoinositides [396,915]. While hydrolysis does not go to completion, representative diacylglycerols appear to be obtained, although some acyl migration occurs with formation of 1,3-diacylglycerols, probably because of the low pH optimum of the enzyme. Phosphatidic acid has been dephosphorylated by the acidic phosphatase (EC 188.8.131.52) from wheat germ .
With many phospholipid classes, and phosphatidylethanolamine and phosphatidylserine especially, it is advisable to separate alkenylacyl, alkylacyl and diacyl forms (the "diradyl" forms) as the acetate derivatives before proceeding to GC analysis. Until recently, this was a task for TLC. The compounds migrate in the order stated and can be adequately resolved on silica gel layers, with a first development to halfway up the plate with hexane-diethyl ether (1:1, v/v) followed by a full development in the same direction in toluene . Good separations were achieved by Cursted [201,202] with column chromatography on lipophilic Sephadex™, but more recently, Nakagawa and Horrocks  obtained excellent results with HPLC, and this is likely to set the standard. For example, diradylacetylglycerols prepared from the ethanolamine-containing glycerophospholipids of bovine brain were separated into the three forms by adsorption HPLC on silica gel. They were eluted in the same order as on TLC from a column (3.9 × 300 mm) containing μPorasil™, maintained at 36°C, with cyclopentane-hexane-methyl-t-butyl ether-acetic acid (73:24:3:0.03 by volume) as the mobile phase at a flow-rate of 2 mL/min; UV detection at 205 nm was employed. This procedure could also be used with BDMS ether (but not TMS ether) derivatives.
2. Preparation of diacylglycerols from triacylglycerols
The determination of the stereospecific distribution of fatty acids in triacyl-sn-glycerols can be carried out by a number of procedures, all of which are complex and involve sequential hydrolytic and synthetic steps, followed by chromatographic analysis of an array of products [125,167]. One procedure described by Kuksis and co-workers  involves partial hydrolysis of triacylglycerols to rac-1,2-diacylglycerols, which are converted chemically to phosphatidylcholines; these are in turn hydrolysed with the enzyme phospholipase C, which reacts very rapidly with the natural L-form to yield sn-1,2-diacylglycerols and then very slowly with the D-form to produce the sn-2,3-diacylglycerols. Both types of diacylglycerol can be selectively recovered, then derivatized and subjected to GC analysis so that the pairing of the fatty acids in each can be determined. The same methods that are employed to separate diacylglycerols derived from phosphoglycerides are applicable (see below).
3. Separations on packed columns
For much of the early work with packed-column GC, nonpolar stationary phases were used. The nature of the columns and the precise operating conditions that have been recommended vary somewhat from laboratory to laboratory but, as a rough guide, 1 m × 3 mm o.d. columns packed with 2% SE-30™ on a silanized support will give good results. Carrier gas flow-rates of 100-200 mL/min and temperature-programming from 220 to 300°C are commonly quoted. Some deviation from these optimum conditions is permissible, however, and the separations illustrated in Figure 8.7 were obtained on 50 × 0.4 cm. (i.d.) glass columns containing 1% SE-30™, temperature-programmed from 250 to 300°C at 2°C/min, and with the flow-rate of the carrier gas (nitrogen) at 50 mL/min.
Because the elution temperatures are in general lower than with triacylglycerols, the resolutions obtained are sufficient to separate components with combinations of fatty acids differing in chain length by one carbon atom. The separations depend largely on the molecular weight, as with the triacylglycerols in similar circumstances, and the carbon number concept is again used for identification purposes. Distearin has a carbon number of 36 and the diacylglycerol acetate prepared from it has a carbon number of 38 (although the two carbon atoms of the acetate moiety are not counted by some authors in calculating carbon numbers). Components are identified by their retention times (or elution temperatures) relative to authentic materials or to standard triacylglycerols having the same total number of carbon atoms. Quantification is generally relatively straightforward, unlike the situation with triacylglycerols.
Figure 8.7. GC separation of diacylglycerol acetates prepared from the phosphatidylcholine of pig liver. A 50 × 0.4 cm (i.d.) glass column packed with 1% SE-30™ on Chromosorb W™ (acid-washed and silanized; 100 to 120 mesh) was used and was temperature-programmed from 250 to 300°C at 2°C/min; nitrogen was the carrier gas at a flow-rate of 50 mL/min. Trace A before and Trace B after hydrogenation. The numbers above each peak refer to the carbon number of the component.
Some partial separation on the basis of degree of unsaturation may occur but may not be especially desirable. For example, in Figure 8.7(A), the fraction of carbon number 38 consists of two partially resolved peaks; the first contains 16:0 together with 20:4 and the second consists of two C18 acids of varying degrees of unsaturation. On hydrogenation (a suitable procedure is described in Chapter 4), these components merge and the remaining peaks are distinctly sharper with improved resolution (Fig. 8.7(B)); in particular, trace amounts of components containing odd-chain fatty acids become apparent. With these particular columns and operating conditions, recoveries of a wide range of standards were essentially complete whether the compounds were hydrogenated or not, and similar results have been reported from many other laboratories for many different phospholipid classes and with acetate, TMS ether and BDMS ether derivatives (c.f. [220,408,477,478,673,764,966,989,991]). The same technique has been employed for the analysis of diacylglycerols generated from triacylglycerols during stereospecific analyses of the latter [585,645,650,651]. As with the triacylglycerols (Section B), silver ion chromatography used to complement GC permits much more extensive separations.
One novel procedure that may repay attention consisted in selectively deuterating the double bonds in the fatty acids of diacylglycerols derived from phospholipids, prior to conversion to the BDMS ether derivatives for GC separation as above . In this instance, mass spectrometry served to identify and quantify unresolved components in specific peaks.
Acetate, TMS ether and BDMS ether derivatives of diacylglycerols are separable to some extent, both by the chain length and degree of unsaturation of the combined fatty acid constituents, on short columns containing some more polar liquid phases having increased thermal stability. EGSS-X™ was used initially , but improved resolution and column durability were obtained with Silar 5CP™  and Silar 10C™  as stationary phases. GC traces obtained by this means are complex, peaks are poorly shaped and the base-line is noisy so that quantification is difficult. In addition, components are not easily identified, although internal standards help and it is possible to use a series of separation factors similar to those used for fatty acid identification as a guide . Most of the important species from rat liver phospholipids were in fact resolved in this work, and only the species 18:0-18:2 and 16:0-20:4 were poorly separated. However, the peak for 18:0-22:6 was not observed, because of its low abundance and long retention time. The high bleed from polar phases in packed columns rules them out for GC-MS. WCOT columns with polar phases are greatly to be preferred (see below).
Mass spectrometry can be of enormous assistance in identifying unresolved components within a fraction, and one reason for the use of BDMS  and TMS ether  derivatives of diacylglycerols is that they have good mass spectral characteristics. Acetates can also be analysed in this way if need be [369,370]. This is discussed in detail later.
Alkyl- and alkenyl-analogues of diacylglycerols have been prepared and analysed by similar techniques to these [201,203].
The main limitation of all these procedures is that components with two given fatty acids in different positions of the glycerol moiety (e.g. 1-palmito-2-olein and 1-oleo-2-palmitin) are not separated. If this information is required, molecular fractions must be physically isolated by some procedure , in order that they may be subjected to enzymatic hydrolysis with pancreatic lipase or a related enzyme .
4. Separations on WCOT columns
The greater resolving power of WCOT columns has been put to good use for the resolution of diacylglycerol species derived from phospholipids, first with apolar and more recently with polar stationary phases. For example, Gaskell and Brooks  separated the TMS ether derivatives of diacylglycerols from arterial wall phospholipids on a glass WCOT column, coated with OV-1/Silanox™ and maintained isothermally at 300°C, for identification by MS. Others used similar columns to quantify disaturated phospholipid species in animal tissues [295,560] and to resolve synthetic 1,2- and 1,3-diacylglycerols .
Much more impressive separations have come from Kuksis' laboratory. For example, a glass WCOT column (10 m × 0.25 mm) coated with SP-2330™ (a 68% cyanopropyl-32% phenylsiloxane), with hydrogen as the carrier gas and a maximum temperature of 250°C, was used to separate the TMS ether derivatives of diacylglycerols prepared from many different natural lipids . The phosphatidylcholines from rat liver gave about 30 distinct peaks, ranging from the species 16:0-16:0 to 18:1-22:6, and this is illustrated in Figure 8.8. The existence of di-unsaturated species, such as the latter, had not previously been demonstrated in animal tissues. Positional isomers and disaturated species within a given carbon number were not separable, but these cannot be resolved by any chromatographic technique. Subsequently, the technique was applied to the determination of the alk-1-enyl-2-acylglycerol moieties of phospholipids . Applications to the analysis of diacylglycerols derived from triacylglycerols have also been described [523,654]. In order to assist others in identifying molecular species separated in this way, comprehensive lists of relative retention times have been published [646,648].
Figure 8.8. Separation by high-temperature GC of the trimethylsilyl ether derivatives of diacylglycerols, prepared by phospholipase C hydrolysis from the phosphatidylcholines of rat liver . The column was a 10 m × 0.25 mm glass capillary coated with SP-2330™, and was temperature-programmed from 190°C to 250°C at 20°C/min, then was held isothermally at 250°C. Splitless injection was used with hydrogen as the carrier gas. A few only of the major peaks are identified here for illustrative purposes. (Reproduced by kind permission of the authors and of the Canadian Journal of Biochemistry and Cell Biology, and redrawn from the original paper).
Although the column life was found to be short (about 100 analyses), the newer WCOT columns of fused silica with cross-linked and chemically bonded stationary phases would be expected to last much longer. Indeed, some outstanding separations with such a column (SE-54™ as the stationary phase) have recently been published (Myher, J.J., Kuksis, A. and Pind, S. Lipids, 24, 396-407 (1989)). Molecular species with positional isomers in the fatty acid residues were found to be separable.
As the molecular weights of diacylglycerol derivatives are much lower than those of triacylglycerols, there appear to be no difficulties with quantification, and uncorrected detector responses should give comparable results to those obtained by other means.
In a novel application of capillary GC, diastereomeric diacylglycerols of short-chain fatty acids were resolved on a WCOT column coated with a nonpolar phase in the form of the α-methoxy-α-trifluoromethylphenylacetic acid derivatives, and the structures were confirmed by mass spectrometry .
GC analysis of 1-O-alkyl-2-acetyl-phosphorylcholine (platelet-activating factor) is a rather specialised application, and for convenience it is discussed in Chapter 10.
5. Gas chromatography-mass spectrometry
GC-MS has for some time been a favoured technique for the identification of diacylglycerol species, separated in the form of the acetate and TMS or BDMS ether derivatives. Myher  and Saito et al.  have reviewed the topic in some depth and have tabulated much valuable data. Normally it is advisable to use the response of the flame ionisation detector for quantification of the main molecular species, and to use GC-MS for identification and quantification of isomers within a single peak.
The mass spectra of acetate derivatives of diacylglycerols are of course those of triacylglycerols (see Section B.3), except that one of the acyl moieties is an acetyl residue [369,370,478,617]. Published mass spectral data are sparse, but it is apparent that the molecular ion tends to be rather small or nonexistent, although the ion representing loss of water ([M−18]+) can usually be seen. Loss of the acetyl group gives ions at [M−59]+ or [M−60]+, depending on the degree of unsaturation of the residual ion, and this is probably the best marker for determining the molecular weight. In addition, ions are seen for the loss of one or both of the other acyloxy moieties as expected. The higher the degree of unsaturation, the smaller are the ions in the high mass range, but those ions containing a single fatty acid residue are usually sufficiently abundant for identification purposes. Although it has been suggested that ions equivalent to [M−RCOOCH2]+ can serve as an indication of the identity of the fatty acid in position 1 of the glycerol moiety [369,370], Myher  has doubted the practicality of this and suggests that further data are necessary. The procedure has been applied to diacylglycerol acetates derived from egg phosphatidylcholine [369,370] and from the phosphatidylglycerol of Escherichia coli . No mass spectra obtained by chemical ionisation methods appear to have been published for these compounds.
TMS ether derivatives tend to give much better spectra, which permit differentiation of 1,2- and 1,3-diacylglycerols even [82,200]. When the derivatives are prepared by high-temperature hydrolysis followed by silylation, the main products are the 1,3-isomers [404,405]. The principal difference in the spectrum of the latter is the presence of an abundant ion formed as a result of the loss of an acyloxymethylene radical (RCOOCH2); although this might be expected even with a 1,2-isomer, it is not in fact seen to any significant extent. Most analysts use 1,2-diacylglycerol derivatives prepared by milder methods as described above. With mass spectra from electron-impact ionisation, the molecular ion is rarely seen, but ions equivalent to [M−15]+ (loss of a methyl group) and [M-90]+ (loss of the TMS ether moiety) can be used to determine the molecular weight and thence the total carbon number and degree of unsaturation of the acyl moieties. An important diagnostic ion results from the loss of an acyloxy residue, i.e. [M−RCOO]+ or [M−RCOOH]+, if the residual acyl group is unsaturated. Other useful ions are equivalent to [RCO+74], [RCO+90], [RCO] and [RCO−1]. Characteristic ions at m/z = 145 and 129 contain the TMS group and parts of the glycerol backbone. Molecular species from several glycerophospholipids have been examined by GC-MS in this form [201,203,404,405].
It is now apparent that BDMS ether derivatives of diacylglycerols are especially useful, because their greater chemical stability means that they can be subjected to such techniques as silver ion TLC and HPLC in addition to GC-MS. On GC, corresponding fractions elute about two methylene groups later than the TMS ethers. BDMS ethers also give distinctive fragmentations, similar in many ways to the TMS ethers, both with electron-impact and chemical ionisation procedures. The molecular ion tends to be small, but is often measurable if one of the acyl groups is highly unsaturated, and there is always an abundant ion equivalent to [M−57]+ . This is seen at m/z = 671 in the mass spectrum of the BDMS ether derivative of 1-palmitoyl-2-eicosapentaenoyl-sn-glycerol, illustrated in Figure 8.9. Indeed, the ion at [M−57]+ is often sufficiently clear for identification of components present at less than 0.5% of the total. Ions formed by loss of RCOO and RCOOH radicals are of immediate diagnostic value. If the acyl moiety lost is unsaturated, the ion formed by loss of the RCOO radical is much more abundant than that for loss of RCOOH (at m/z = 427 in the figure); if the radical lost is saturated, the two ions are of about the same intensity (at m/z = 472 and 473). The total abundance of ions formed by loss of fragments from position 2 is greater than that from position 1. In addition, there are characteristic ions equivalent to [RCO]+, [RCO+74]+ and [RCO+148]+ for each acyl group. It is possible to use selective ion monitoring of many of these ions for identification and quantification purposes . With chemical ionisation, the intensities of the characteristic ions in the high mass range are enhanced [522-524]. BDMS ether derivatives have been used for the identification of molecular species of phospholipids of animal [477,522-524,653] and microbial origin [220,752]. They have also been employed with diacylglycerols, prepared from phosphoglycerides, and selectively deuterated as an aid to identification .
Figure 8.9. The mass spectrum of the t-butyldimethylsilyl ether derivative of 1-palmitoyl-2-eicosapentaenoyl-sn-glycerol . (Reproduced by kind permission of the authors, and of Analytical Chemistry, and redrawn from the original paper).
Ether analogues of diacylglycerols are readily identified by GC-MS as the TMS or BDMS ether derivatives in a similar manner (reviewed by Egge ). With the former, the molecular weight of a 1-alkyl-2-acylglycerol is given by ions at [M−15]+ and [M−90]+ . For saturated species, the relative abundance of these ions is low, but the [M−90]+ ion especially becomes much more prominent when there is an unsaturated residue. There is an ion of relatively low intensity representing the loss of the alkoxy group, [M−RO]+, one representing loss of the acyloxy moiety, [M−R'COO]+, and others for [R'CO]+ and [R'CO+74]+. The base ion at m/z = 130 contains the TMS ether group and the carbons of the glycerol backbone (c.f. diacylglycerol derivatives where this is at m/z = 129). Mass spectra of some TMS ether derivatives of synthetic dialkylglycerols have been described . In addition, mass spectrometry has been invaluable in determining the structures of the complex ether lipids of Archaebacteria (reviewed elsewhere ).
The TMS ethers of 1-alk-1-enyl-2-acylglycerols are similarly identifiable by GC-MS . Some features are analogous to the alkyl ethers, and for example there are ions equivalent to [M−15]+, [M−90]+, [R'CO]+, [R'CO+74]+, [R'COO+74]+, and a base peak at m/z = 129. Also, ions at [M−RCH=CHO]+ and [R'COO+130]+ are abundant. In the spectra of the relatively common 1-hexadec-1-enyl- and 1-octadec-1-enylglycerols, there are characteristic ions at m/z = 311 and 319, representing [(R'CH=CHO−1)+73]+. The TMS and BDMS ether derivatives of ether lipids of this type from several natural sources have been analysed by GC-MS [201,477,648].
D. Monoacylglycerophospholipds and Monoacylglycerols
Lyso-glycerophospholipids are converted to monoacylglycerols for analysis by GC by the same procedures used to generate diacylglycerols from phospholipids (see Section C.1 above). While 2-monoacylglycerols are usually only trace constituents of tissues, they are generated by the action of pancreatic lipase on triacylglycerols during analyses of positional distributions (reviewed elsewhere [163,167]), and they may also be formed in other circumstances. They can be separated with relative ease by GC after conversion to nonpolar volatile derivatives. For convenience, the alkyl and alkenyl ether analogues are not discussed here, but together with other products of the hydrolysis of ether lipids in Chapter 10.
Wood et al. , for example, were able to separate isomeric 1(3)- and 2-monoacylglycerols from each other as the TMS ether derivatives by GC on packed columns with DEGS as the stationary phase. The 2-isomer eluted first. As monoacylglycerols in the underivatized state isomerize rapidly, the practical value of such separations is doubtful since isomeric compounds differing in degree of unsaturation tend to overlap. By employing more efficient columns and more polar stationary phases, such as Silar 5CP™ or Silar 10C™, much better resolution was obtained, although there were still critical pairs which caused difficulties [411,643,656]. For example, monoacylglycerols such as 1-18:0 and 2-18:1 or 1-18:1 and 2-18:2 overlapped. As a guide to the GC conditions, TMS ether derivatives of monoacylglycerols eluted in reasonable times from a glass column (2 m × 3 mm i.d.) packed with 5% Silar 5C™ on GasChrom Q™ (100-120 mesh) and maintained at 190°C . Carbonate derivatives of 1-monoacylglycerols have been separated under similar conditions . Analysis is greatly simplified if the two isomeric forms of the monoacylglycerols are first resolved by TLC on silica gel impregnated with boric acid, immediately prior to derivatization .
GC of TMS ethers on WCOT columns greatly improves the resolution attainable, although there are still likely to be problems with complex samples [628,772].
The mass spectrometric fragmentations of monoacylglycerol acetate and TMS ether derivatives have been studied systematically both by direct probe insertion and by GC-MS [200,450,656]. With the acetates, there is no simple means of distinguishing the 1(3)- and 2-isomers, although there are differences in the intensities of ions representing loss of the long-chain acyloxy group with position. More characteristic spectra are obtained from the TMS ethers, which are the best derivatives for GC purposes. The molecular ion is usually detectable, especially when the acyl residue is unsaturated. As with the diacylglycerols discussed above, [M−15]+ and [M−90]+ are abundant ions, in addition to those equivalent to [RCO]+ and [RCO+74]+. An ion formed by cleavage between carbons 2 and 3 in the spectra of the 1(3)-monoacylglycerol derivatives at [M−103]+ appears to be absolutely characteristic for this isomer, while a smaller ion at m/z = 205 is of further diagnostic value. Similarly, an ion at m/z = 218 is highly favoured in the spectra of 2-isomers. When isomers are incompletely resolved by GC, they can be still be quantified by selective ion monitoring, by making use of the distinctive ions in the mass spectra. BDMS ethers would no doubt be equally suitable for the purpose. Carbonate derivatives of 1- and 3-monoacyl-sn-glycerols exhibit particularly good molecular ions in MS .
E. Sterol Esters
Fatty acid esters of cholesterol are abundant components of plasma and other animal tissues, while other sterol esters are common if minor constituents of plant lipids and are occasionally found in animals. Cholesterol esters were first successfully separated on nonpolar silicone phases in packed columns, similar to those used initially for triacylglycerol separations (see Section B.1), and they elute at temperatures intermediate between those of diacylglycerol derivatives and triacylglycerols . Components differing by one carbon atom are separable in this way, but useful resolutions of saturated and unsaturated compounds of the same chain length cannot be achieved. Cholesteryl alkyl ethers from bovine heart were subjected to GC under similar conditions . Later, cholesterol esters were successfully separated according to degree of unsaturation on short packed columns containing a thermally stable polar phase, such as Silar 10C™ .
With complex samples, the resolution is still far from ideal, and much better results can now be obtained on modern WCOT columns. For example, cholesterol esters from plasma were well separated according to their chain lengths and partly by degree of unsaturation on WCOT columns of fused silica and coated with a nonpolar phase, OV-1™, temperature-programmed to 330°C; with the polar phase, SP-2330™, better separation by degree of unsaturation was achieved, though peaks were less sharp [861,863]. A report  that hydrogenation can occur on a polar column when hydrogen is the carrier gas has been discounted by others (see Section B.2).
The additional resolution attainable with WCOT columns is essential if the nature of the sterol moiety as well as that of the fatty acid varies. Studies of sterol esters from geochemical samples , plants [249,566], beeswax , dinoflagellates , and plasma in patients with phytosterolemia and xanthomatosis [527,528] illustrate the difficulties that can be encountered. Figure 8.10 shows a separation of sterol esters from the plasma of a patient with phytosterolemia ; fatty acid esters of cholesterol, campesterol and β-sitosterol were identified. In most of the work cited, nonpolar phases were used, although a more polar phenylmethyl silicone (SP-2330™) gave the excellent separation of the figure. Double bonds and other substituents in the sterol moieties contribute to the retention times of the esters, and as these may vary from sample to sample, it is not easy to put forward general rules to predict their behaviour in GC. Although mass spectrometry helps greatly to identify components and to quantify mixtures hidden under a single GC peak, it still appears advisable to pre-fractionate complex samples by reversed-phase HPLC and silver ion chromatography before GC analysis [249,527].
Figure 8.10. GC elution profile of sterol esters from low- and high-density lipoproteins of plasma from a patient with phytosterolemia . A glass WCOT column (10 m × 0.25 mm i.d.), coated with SP-2330™, was maintained isothermally at 250°C with hydrogen as the carrier gas. Abbreviations: c, cholesterol; cam, campesterol; s, β-sitosterol. (Reproduced by kind permission of the authors and of Lipids, and redrawn from the original paper).
With electron-impact ionization in MS, sterol esters rarely give detectable molecular ions . Ions representing loss of the fatty acyl moiety are seen with model compounds, but these are of limited value with unknowns. Nonetheless, valuable structural information can be obtained with some samples . Chemical ionization, however, tends to give base ions representative of the nature of the sterol component . With a suitable choice of reagent gas, and ammonia seems to be the best for the purpose, a good quasi-molecular ion ([M+NH4]+) and ions diagnostic for both the sterol and fatty acid moieties are obtained [566,961]. These findings have been confirmed and extended by others in GC-MS studies of sterol esters from various sources; in particular, it was demonstrated that negative chemical ionisation with ammonia as the reagent gas is to be preferred [248-250]. The EI mass spectrum of cholesteryl hexadecyl ether has also been published .
F. Wax Esters
Natural waxes can consist of a wide range of different lipid classes, including esters of various kinds, hydrocarbons, ketones, hydroxy-ketones, β-diketones, aldehydes, acids and terpenes. With crude mixtures of this kind, it is usually necessary to react them with diazomethane to methylate free carboxyl groups, to acetylate (or to prepare TMS ethers) in order to deactivate free hydroxyl groups and to convert any aldehydes to oximes, prior to GC analysis. The preen glands of birds, for example, contain a wide range of fascinating lipid molecules. However, detailed analyses of these and many other waxy materials is rather a specialised topic, and the reader is referred to reviews that have appeared elsewhere [423,425,928,929]. Esters of long-chain alcohols and fatty acids, the compounds commonly termed "wax esters", are widespread in nature and have some commercial importance, while their analysis presents some general problems that merit attention here. The fatty acids and alcohol constituents from different sources reflect their origins; frequently, both are saturated or monoenoic compounds from 16 to 30 carbon atoms in length, but those of marine origin may be highly unsaturated, for example.
Wax esters tend to have similar molecular weights to diacylglycerol acetates (see Section C above) and are eluted from GC columns under comparable conditions. Initially, short packed columns containing nonpolar silicone phases were employed, and these were easily capable of separating species differing in carbon number by one or two units, where the carbon atoms of the alcohol and fatty acid moieties have equal value . Thus, a 16:0 acid with an 18:1 alcohol and an 18:1 acid with a 16:0 alcohol will co-chromatograph, and both will emerge with species such as 14:0-20:1 and so forth. With marine oils, the high degree of unsaturation in some components can lead to peak broadening, so hydrogenation and/or pre-fractionation by silver ion chromatography is often recommended to improve resolution. Further complications can arise when branched-chain fatty acid or alcohol constituents are present. Several applications of this methodology to wax esters from marine mammals [17,18,152,556] and to the commercial vegetable oil from jojoba [872,874] have appeared. Subsequently, separation of wax esters according to degree of unsaturation was achieved on short packed columns containing polar phases, such as Silar 10C™ [899,901]. Rather complex chromatographic traces were obtained if the sample was not first fractionated by silver ion chromatography, and the high bleed from such columns precluded the use of mass spectrometry for identification purposes.
As with the other lipid classes discussed above, greatly improved resolution of wax esters can now be achieved on WCOT columns. Indeed, short-chain (<C24) esters from the bottlenose dolphin were resolved on a stainless steel WCOT column coated with DEGS and other stationary phases by Ackman and colleagues in 1973 [29,554]. Jojoba wax has been fractionated on a glass WCOT column coated with OV-1™ . More recently, a WCOT column (25 m × 0.2 mm) of fused silica, coated with a methylsilicone phase, was utilised with temperature-programming from 250°C to 350°C for the analysis of the wax esters from the alga, Chlorella kessleri . The separation obtained is illustrated in Figure 8.11; within each carbon number group, species with zero, one and two double bonds are clearly resolved. With on-column injection, good quantification of individual peaks is possible, while mass spectrometry permits estimation of multiple components within a single peak. Excellent resolution by degree of unsaturation was achieved on a glass WCOT column coated with the polar phase SP-2340™ . Within carbon number groups, some separations according to the chain lengths of the individual constituents of the esters were seen. Thus, 14:0-14:0 and 10:0-18:0 were separable, but 14:0-14:0 and 12:0-16:0 were not. It was also observed that the elution temperature had an appreciable effect on the resolution of specific critical pairs.
Figure 8.11. GC separation of the wax esters from the alga, Chlorella kessleri . A fused silica WCOT column (25 m × 0.2 mm i.d.), coated with a methylsilicone phase, was temperature-programmed from 250 to 350°C at 2°C/min, with helium as the carrier gas. (Reproduced by kind permission of the authors and of the Journal of Chromatography, and redrawn from the original paper).
MS is a valuable means of identifying individual wax esters separated by GC. In a systematic study of electron-impact ionisation spectra of a number of model compounds of the form RCOOR' , it was shown that a good molecular ion is always obtained while the base peak is generally the protonated acid ion ([RCOOH2]+), except when the alcohol chain is less than 10 carbon atoms long. The alcohol moiety is indicated by a prominent ion equivalent to [R'−1]+. Relative abundances of all ions are dependent on the chain lengths of the individual aliphatic groups. In order to use MS for quantification of species within each GC peak, a method was developed in which double bonds were reduced with deuterohydrazine, and this was followed by ozonolysis to remove any residual unsaturated species. Relative peak intensities for ions corresponding to [RCOOH]+, [RCOOH2]+ and [R'−1]+ were then measured, since it was observed that the sum of these for each species was a more reliable indicator of the total amount present than were the individual intensities. This approach has been utilised successfully by others [769,872]. In addition, GC-MS has been used to identify phytyl esters in a dinoflagellate , and other wax esters in sediments  and psoriatic nail .
Chemical ionization with butane as the reagent gas gives spectra with much less fragmentation and with the quasimolecular ion ([M+1]+) as the base ion . Others  obtained better results with methane as the reagent gas, although caution was necessary in using the data quantitatively, since the ions for saturated and unsaturated species varied appreciably in intensity. Intact wax esters have been quantified without the need for a chromatographic separation by field desorption [640,820-822] and tandem MS .
Mono- and digalactosyldiacylglycerols are polar molecules of high molecular weight, so they would not be expected to be good candidates for analysis by high-temperature GC. Nonetheless, the TMS ether derivatives of monogalactosyldiacylglycerols from plants were successfully subjected to GC on short packed columns containing methylsilicone phases, under conditions similar to those required for intact triacylglycerols . Sulfoquinovosyldiacylglycerol from plants was separated into molecular species in the same way after methylating the sulfonic acid group with diazomethane and converting the carbohydrate moiety to the TMS ether derivative . In essence, three species were obtained equivalent to the combinations C16-C16, C16-C18 and C18-C18 (irrespective of degree of unsaturation). Later, both mono- and digalactosyldiacylglycerols and their monoacyl equivalents were chromatographed in the same manner, while the products of deacylation were separated as the O-methyl, O-acetyl, O-TMS ether and O-trifluoroacetate derivatives .
A procedure for the release of the diacylglycerol moieties from galactosyldiacylglycerols (including sulfoquinovosyldiacylglycerols) has been described, involving periodic acid oxidation in methanol followed by incubation with 1,1-dimethylhydrazine . The diacylglycerols are then converted to UV-absorbing derivatives for separation by means of HPLC , but equally it should be possible to prepare TMS or BDMS ethers and use the GC conditions developed for the analysis of the equivalent compounds released from phospholipids (see Section C above).
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.
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Updated July 8, 2011