and Litchfield, C , Lipids 8,661 (1973). 13. Kuksis, A. and Breckenridge, W.C., J. Amer. Oil Chem. Soc. 42,978 (1965). 14. Bugaut, M. and Bezard, J., J. Chromatog. Sci. 8, 380 (1970). 15. Kuksis, A. and Ludwig, J., Lipids /, 202 (1966). 16. Breckenridge, W.C. and Kuksis, A., Lipids 5,342 (1970). 17. Matsui, M., Watanabe, T. and Ikekawa, N., Bull. Jap. Soc. Sci. Fisheries 39,367 (1973). 18. Ackman, R.G., Eaton, C.A., Kinneman, J. and Litchfield, C , Lipids 10,44 (1975). 19. Litchfield, C , Harlow, R.D. and Reiser, R., Lipids 2, 363 (1967). 20. Ikekawa, N., Matsui, M., Yoshida, T. and Watanabe, T., Bull. Jap. Soc. Sci. Fisheries 38,1267 (1972). 21. Marai, L., Breckenridge, W.C. and Kuksis, A., Lipids 4, 562(1969).

22. Ackman, R.G., Eaton, C.A. and Litchfield, C , Lipids 6, 69(1971). 23. Litchfield, C , Ackman, R.G., Sipos, J.C. and Eaton, C.A., Lipids 6,674 (1971). 24. Harlow, R.D., Litchfield, C , and Reiser, R., Lipids /, 216(1966). 25. Bojesen, I., Lipids 9, 835 (1974). 26. Wood, R., Harlow, R.D., and Lambremont, E.N., Lipids 4,159(1969). 27. Bezard, J.A., Lipids 6,630 (1971). 28. Bezard, J.A., Bugaut, M., and Clement, G., J. Amer. Oil Chem. Soc. 48,134(1970). 29. Imai, C , Watanabe, H., Haga, N. and Ii, T., J. Amer. Oil Chem. Soc. 57,326 (1973). 30. Litchfield, C , Miller, E., Harlow, R.D., and Reiser, R., Lipids 2,345 (1967). 31. Eckert, W.R., Fette Seifen Anstrichm. 75,150(1973).

Analysis and Quantification of Ether Lipids by Chromatographic Methods Harald H.0. Schmid, Patricia C. Bandi and Kwel Lee Su, University ot Minnesota, The Hormel Institute, Austin, Minnesota 55912

Abstract Chromatognphlc methods, especially thin-layer chromatography [TLC] and gas-liquid chromatography [GLC] are widely used In Investigations of the occurrence, molecular structure and metabolism of ether lipids. The application of such techniques to structural analysis and quantification, In combination with methods for the degradation and dertvatlzatJon of ether Npids, is discussed.

Introduction Lipids containing ether bonds occur widely in nature. The most common structures are the 1-0-alkyl and l-O-alk-l'-enyl analogs of triglycerides and glycerophosphatides. H2C-O-CH2-CH2-R

91

HjC-O-CH-CH-R R'CO-CH |

R'CO-CH 1

H2C-OCR" l-O-Alkyl-2,3-diacyl-

1 0. H^-OCR" 1-O-Alk-l'-enyl-2,3-diacylan-glycerol

•n-glycerol (Glycerol ether dieater) H2C-O-CHj-CH2-R

(Neutral plaamalogen) H2C-O-CH-CH-R R'CO-CH |

R'CO-CH

19

HjC-OPOCHjCHjNHj OH l-O-Alkyl-2-acyl-«nglycero-3-phoaphoethanolamine

OH 1-O-Alk-l'-enyl-2-acyl-snglycero-3-phoaphoethanolaplne (Ethanolamine plasmalogen)

478 •OCTOBER 1975

The corresponding choline glycerophosphatides are also present in relatively high amounts in many mammalian and avian tissues, whereas N-methylethanolamine and serine glycerophosphatides are prevalent in certain bacteria. Other polar ether lipids such as the 2-aminoethylphosphonate analogs or N-acetyl and N-acyl derivatives of ethanolamine glycerophosphatides are less common. Ether lipids containing substituted 0-alkyl moieties or having a polyol backbone other than glycerol usually occur only in small amounts. In the past decade, interest in ether lipids, often referred to as alkoxylipids, has centered on structural investigations, on their biosynthesis and catabolism and on their use as model compounds in the study of various biochemical, biophysical and physiological phenomena. An excellent summary of progress in the ether lipid field is available in a monograph consisting of a series of comprehensive reviews (1). Much of this progress was made possible by advances in chromatographic techniques (2,3). It is our aim to present here a brief summary of degradative and chromatographic methods used in ether lipid analysis and quantification. We do not intend to present a comprehensive review, but to limit the discussion to applications of thin-layer and gas-liquid chromatography, the most useful and versatile chromatographic techniques. We hope that this will stimulate interdisciplinary interest in ether lipids and further research in their physiological role. We also believe that the presence of alkoxylipids or their degradation products in lipid extracts and lipid preparations should be taken into account by anyone involved in lipid analysis. Emphasis is placed on naturally occurring ether lipids. However, the chromatographic behavior of various synthetic isomers and analogs of the natural alkoxylipids will be described, since such materials are frequently used as model compounds. Methods for the chemical synthesis of alkoxy

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lipids and their derivatives have been reviewed by Baumann (1), Gigg(l)andPaltauf(4).

Separation of Lipid Classes Neutral Ether Lipids Prior to the development of TLC, the presence of ether lipids in neutral lipid fractions was demonstrated by indirect methods, for example, by the detection of 0-alkyl glycerols among the unsaponifiable material (5-8) and by the formation of free aldehydes upon acidic hydrolysis (9-12). The first separation (13) of alkyl diacyl glycerols from triacyl glycerols was accomplished by TLC on Silica Gel G (Merck) using hexane-diethyl ether-acetic acid (90:10:1) as developing solvent. It was later shown (14) that double development of the chromatogram with hexane-diethyl ether (95:5) gives complete separation of alk-1-enyl diacyl, alkyl diacyl and triacyl glycerols. Quantitative estimations can be carried out on the chromatoplate after charring by comparing known amounts of ether lipid standards side by side with the total lipid mixture (14,15). Because neutral ether lipids usually occur in much, smaller amounts than triglycerides, their isolation on a preparative scale tends to be time-consuming and difficult (14). Therefore, it was found to be of advantage to prepare a "concentrate" of neutral ether lipids (16) prior to their isolation. An initial separation of the total neutral lipids on a layer of Silica Gel H (Merck) 2mm thick is carried out. A fraction above and including the leading edge of the triglyceride fraction is scraped off and eluted with water-saturated diethyl ether. Purification of the minor lipid constituents is then achieved by repeated preparative TLC. Such methods were also used for the preparation of large amounts of pure alk-1-enyl diacyl glycerols from shark liver oil (17,18). Figure 1 illustrates the concentration and subsequent isolation of neutral alkoxylipids and other minor constituents present in a lipid extract of bovine heart muscle that had been stored for about one year (16).

As is evident from Figure 1, a number of neutral lipid classes and artifacts produced during storage may exhibit migration rates in TLC similar to those of neutral ether lipids. Therefore, it is advantageous to use various solvent systems of slightly different polarity (19), e.g., hexane-diethyl ether (95:5, or 90:10, or 80:20) with authentic standards. Dialkyl monoacyl glycerols and trialkyl glycerols which are not normally found as natural lipids are less polar than alk-1-enyl diacyl glycerols and are separable from them by TLC (20). Interest in neutral ether lipids has increased considerably since relatively high amounts of alkyl diacyl glycerols have been discovered in neoplastic tissues (21-23). Special care must be taken in estimating the amounts of alkyl diacyl glycerols on a chromatoplate because some common solvent systems, such as hexane-diethyl ether (90:10), do not separate this lipid class from fatty acid methyl esters. We have found (24) that from tissues which are rich in free fatty acids, such as many neoplasms, small amounts of fatty acid methyl esters are obtained in the course of lipid extraction (25). Polar Ether Lipids Subfractionation of intact phospholipids into alk-1-enyl acyl, alkyl acyl and diacyl species by chromatographic methods has not been accomplished so far. However, such fractionations by TLC are possible after "masking" the polar groups by diazomethanolysis of the intact phospholipid (26,27) or by methylation (28) of phosphatidic acid, produced by phospholipase D hydrolysis. Ethanolamine glycerophosphatides can be fractionated as their 0-methylated N-dinitrophenyl derivatives (29). Subfractionation of alk-1-enyl acyl, alkyl acyl and diacyl derivatives is achieved (27,29) by TLC using multiple development of the chromatogram. Unfortunately, diazomethane used for the methylation appears to react to some extent with the alk-1-enyl ether leading to an artifact (29). Although glycerophosphatides can be fractionated according to their total number of double bonds by argentation TLC (30,31), the application of this technique to the subfractionation of choline (32) or ethanolamine (33) glycerophosphatides containing alk-1-enyl acyl and alkyl acyl analogs produced fractions more or less enriched in alkoxylipids, but not very useful for analytical or preparative purposes. In general, cleavage of the phosphoester bond followed by fractionation of the acetylated substituted glycerols, as described below, appears to be the most convenient and reliable and, therefore, the most widely used technique.

Degradative Methods Purification ofPlasmalogens through Selective Degradation

i i Figure 1. Thin-layer chromatogram of neutral lipids from a stored extract of bovine heart muscle. Adsorbent: Silica Gel G (Merck); Solvent: hexanediethyl ether (95:5, v/v), developed twice. (I) stored extract; (II) concentrate; (1) unidentified; (2) steryl esters; (3) 2,3-dialkyl acroleins; (4) aldehydes; (5) alk-1-enyl diacyl glycerols; (6) alkyl diacyl glycerols; (7) triacyl glycerols. [H.H.O. Schmid et al., J. Lipid Res. 8, 692 (1967). Reproduced by permission of Lipid Research, Inc.]

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The fact that alk-1-cnyl acyl and alkyl acyl glycerophosphatides behave differently than their diacyl analogs in certain chemical and enzymic reactions has been utilized for preparative purposes. For example, mild alkaline hydrolysis proceeds more readily with diacyl choline and ethanolamine glycerophosphatides (34-36). Similarly, phospholipase A2 (phosphatide acylhydrolase, EC 3.1.1.4) from Crotalus atrox venom attacks the 2-acyl moieties of diacyl choline glycerophosphatides more readily than the corresponding acyl groups of the alk-1-enyl acyl analogs (37). Commercial preparations of phospholipase D (EC 3.1.4.4) from cabbage were found to attack diacyl (38) choline glycerophosphatides quite readily, but the alk-1-enyl acyl analogs are more resistant and can be obtained in relatively pure form (38). Phospholipase C (EC 3.1.4.3) from Clostridium welchii may be used in the same way (39).

OCTOBER 1975*479

Selective hydrolysis of the 1-acyl moieties of choline and ethanolamine glycerophosphatides by a purified pancreatic lipase (40,41) was used for the preparation of the corresponding plasmalogens in high yield (42). A commercially available lipase (Boehringer Mannheim GmbH, Germany) from Rhizopus arrhizus delemar (42a) was found to be equally applicable (F. Paltauf, personal communication). In general, however, the chemical and enzymic degradations referred to above are of limited use for lipid analysis, since they are neither completely specific nor do they remove any alkyl acyl analogs. The following procedures are, therefore, employed for analytical purposes. Hydrolysis ofAlk-1-enyl Glycerols It has long been known that long-chain aldehydes are produced from alk-1-enyl glycerolipids upon treatment with HgCl2 (43) or acid (44). When both long-chain aldehydes and 2-acyl lysophosphatides are to be recovered, mild conditions of hydrolysis such as 90% acetic acid at 36-38°C are used (4548). Under these conditions, some of the aldehydes undergo condensation to 2,3-dialkylacroleins, especially in the presence of ethanolamine glycerophosphatides (49). Acidic hydrolysis of lysoplasmalogens also produces cyclic acetals of glycerol (50,51). The formation of cyclic acetals is minimized by the use of trichloroacetic acid-HgC^ reagent (52); a mixture of acetic acid, hydrochloric acid and HgCl2 has also been used successfully (53). Hydrolytic cleavage to obtain aldehydes can be carried out by suspending the lipids in diethyl ether saturated with cone. HC1 (54,55). Hydrolysis of the alk-1-enyl ether bond can also be accomplished on a chromatoplate by exposing it to HC1 fumes (19). This fact allows the use of a "separation-reaction-separation" technique by which neutral lipids (19) or phospholipids (56,57) are first fractionated by TLC, alk-1-enyl glycerols are then cleaved by exposure to HC1 fumes and the reaction products are subsequently resolved by TLC in a direction perpendicular to the first development. Alternatively, a spray reagent containing HgCl2 (58) or 12% HC1 in methanol (59) can be used for hydrolysis. Hydrogenolysis

Hydrolysis of Phosphoester Bonds Removal of the phosphoryl-base group and acetylation of the resulting diglyceride analogs produces derivatives which can be readily separated by TLC into alk-1-enyl acyl, alkyl acyl and diacyl compounds. Acetolysis (67) of intact phospholipids has been used (68) to prepare the disubstituted glycerol acetates. However, some acyl migration (69,70) and some destruction of plasmalogens (71) occurs. This makes the use of phospholipase C, followed by the acetylation of the hydrolysis products, the method of choice. Phospholipase C (EC 3.1.4.3) from Clostridium welchii hydrolyses choline glycerophosphatides, but does not attack acidic phospholipids. Ethanolamine glycerophosphatides are hydrolyzed only in the presence of choline glycerophosphatides or sphingomyelin (71,72) or, preferably, choline lysophosphatides (73). The enzyme obtained from Bacillus cereus (74) hydrolyses both choline and ethanolamine, as well as serine and inositol, glycerophosphatides (75). Acetylation of the disubstituted glycerols with acetic anhydride in pyridine yields a mixture of alk-1-enyl acyl, alkyl acyl and diacyl glycerol acetates which are separable by TLC using toluene (76,77) or multiple development (73) with hexane-diethyl ether (85:15). Acyl migration was found to be minimal and the method has found wide application in both structural and metabolic investigations (1). In combination with other degradative procedures, such as phospholipase A2 (EC 3.1.1.4) hydrolysis and acidic hydrolysis of alk-1-enyl glycerol, it can be used for the analysis of the long-chain moieties at carbons 1 and 2 of glycerol (47,78) and to determine the incorporation of radioactivity into these structures (48). The alk-1-enyl acyl, alkyl acyl and diacyl glycerol acetates can be further fractionated according to their number of double bonds (68,78) by argentation chromatography (79) or according to their molecular weight (76-78, 80, 81) by "high temperature" GLC (82,83). Disubstituted glycerol acetates can be prepared from neutral alkoxylipids after hydrolysis with pancreatic lipase and were used for the analysis of molecular species (77). The fractionation of intact alkyl diacyl glycerols by GLC according to their molecular weight is possible (78), whereas alk-1-enyl diacyl glycerols apparently are not stable enough under these conditions.

Analysis of Aliphatic Moieties Reduction of ionic (60) or neutral (18) ether lipids with lithium aluminum hydride in diethyl ether, followed by destruction of the excess LiAlH4 with water, yields alkyl and alk-1-enyl glycerols as well as long-chain alcohols derived from acyl moieties. The reaction products can be separated by TLC (61,62) using hexane-diethyl ether (20:80). Treatment of the lithium aluminum complex with an excess of acetic anhydride (63) yields diacetates of alkyl and alk-1-enyl glycerols and long-chain alkyl acetates (18). When the lithium aluminum complex is destroyed with mineral acid, long-chain aldehydes are obtained instead of alk-1-enyl glycerols. Usually, small amounts of 2,3-dialkylacroleins (49) and longchain cyclic acetals of glycerol, i.e., substituted dioxanes and dioxolanes (64), are also produced. Nevertheless, LiAlH4 reduction has been widely used in structural and metabolic studies of neutral and ionic alkoxylipids (1). More recently, Vitride reagent (sodium bis [ 2-methoxyethoxy] aluminum hydride, 70% in benzene) has become available; it was reported to give more quantitative recovery of alk-1-enyl glycerols (65) and was found to be more convenient in certain experiments (66).

480 •OCTOBER 1975

Alk-1-enyl Glycerols Intact alk-1-enyl glycerols as constituents of neutral lipids, phospholipids, or of products derived from them, can be detected in TLC fractions by their bright yellow color after spraying the chromatoplate with a saturated ethanolic solution of 2,4-dinitrophenylhydrazine containing 10% sulfuric acid. 2,3-Dialkylacroleins migrate ahead of aldehydes in TLC and give a deep orange color with 2,4-dinitrophenylhydrazine (49). To distinguish alk-1-enyl diacylglycerols from free aldehydes, the use of solvent systems of different polarity or the "separation-reaction-separation" technique (19) is recommended. Recently, 4-amino-5-hydrazino-l,2,4-triazole-3-thiol (2% in aqueous IN NaOH), a color reagent specific for aldehydes, has been described (84). As little as l^g of palmitaldehyde can be detected as a purple spot on a chromatoplate. Hydrolytic cleavage of alk-1-enyl glycerols yields aldehydes which can be isolated by TLC and fractionated into individual components by GLC (85,86) on a polar stationary phase such as ethylene glycol succinate, commonly employed for the

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analysis of fatty acid methyl esters. Cyclic acetals, prepared by reacting alk-1-enyl glycerols or aldehydes with a short-chain diol in the presence of /Moluenesulfonic acid (87) are more stable than free aldehydes and well suited as derivatives for GLC analysis. Dimethyl acetals of long-chain aldehydes are obtained from alk-1-enyl glycerolipids by acidic methanolysis (86). They are separable from fatty acid methyl esters by TLC using xylene (88), toluene (89) or dichloroethane (90) as developing solvent. Although dimethyl acetals are stable during storage, they can form methyl vinyl ethers in GLC on an acidic stationary phase (91,92) or under certain methylation conditions (93). Reduction of long-chain aldehydes with LiAlJfy yields alcohols which are separable by GLC as alkyl acetates (14,86). Alkyl acetates are very stable and can be subjected to catalytic hydrogenation to eliminate double bonds prior to GLC analysis. However, care has to be taken in the purification of the aldehydes prior to reduction since any free fatty acids or acyl groups are also reduced to alcohols and may, thus, lead to erroneous results. Catalytic hydrogenation of alk-1-enyl glycerolipids yields alkyl glycerolipids. Alkyl Glycerols The 0-alkyl glycerol bond is resistant to all but the most drastic degradative procedures. Removal of acyl moieties by acidic or alkaline hydrolysis of neutral alkoxylipids, or hydrogenolysis of either neutral or polar ether lipids with LiAlH4 or Vitride, yields alkyl glycerol. Volatile derivatives have to be prepared to make possible analysis of alkyl glycerols by GLC. Ketalation with acetone (94) to yield isopropylidene derivatives is the most widely used method. The reaction is carried out at room temperature in anhydrous acetone in the presence of catalytic amounts of 70% perchloric acid. Other less commonly used derivatives are the diacetates (95), dimethyl ethers (96), bistrifluoroacetates (TFA) (97) and the bistrimethylsilyl (TMS) ethers (97). Both TFA-derivatives (97) and TMS-derivatives (97a,b) of 1-alkyl glycerols can be separated by GLC from those of the 2-alkyl isomers. Cleavage of the vicinal hydroxy groups with sodium metaperiodate in pyridine (98) affords 0-alkyl glycolaldehydes in quantitative yield, which are separable by GLC (99).

Special Techniques

Argentation TLC (79) can be applied to the fractionation of ether lipids or their derivatives according to their number of double bonds. A procedure for the separation of various classes of neutral alkoxylipids according to their functional groups and number of double bonds on a plate partially impregnated with AgNO3 has been described (100). The fact that arsenite and borate ions can complex with vicinal hydroxy groups has been applied to the successful separation of 1-alkyl from 2-alkyl glycerols on impregnated TLC plates (101,102). Since most naturally occurring alkyl and alk-1-enyl glycerols are saturated and monounsaturated (1), reductive ozonolysis gives simple fragments which can be analyzed by GLC. This method has been used to identify various monounsaturated isomers present in naturally occurring alkyl and alk-1-enyl glycerols (103-105). The position of double bonds and of cyclopropane groups in alk-1-enyl moieties has also been determined by GLC using an open capillary column (106).

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Quantification Alk-1-enyl Glycerolipids Acidic hydrolysis of alk-1-enyl glycerols to yield aldehydes provided the first methods of quantification (107) using fuchsin-sulfurous acid (107,108). The long-chain aldehydes may also be quantified as /?-nitrophenylhydrazones (109) or 2,4-dinitrophenylhydrazones (110). These measurements are made spectrophotometrically. An alternative method for the quantification of intact alk-1-enyl glycerolipids is the specific iodination of the alk-1enyl ether bond to form the iodoacetal (111-113). Excess iodine is titrated with sodium thiosulfate. This method was modified (114) for potentiometric determination of the endpoint during the titration of excess iodine. Accuracy and sensitivity of the method were thereby increased to quantify 0.5 j^mole of plasmalogens within ±2%. The iodination method, coupled with spectrophotometric determination of excess iodine (115), can be used to quantify as little as 0.01 jimole of plasmalogen. Two-dimensional "separation-reaction-separation" TLC has been used to quantify alk-1-enyl acyl glycerophosphatides by determining the phosphorus content of the lysophospholipids obtained by hydrolytic cleavage (56,58). Quantifications were also accomplished by determining the phosphorus content of the water soluble fragments obtained by consecutive alkaline and acidic hydrolysis (116-118). Alkyl Glycerolipids Alkyl glycerols, derived from ether lipids by hydrogenolysis or other methods, can be quantified by periodate cleavage of the vicinal hydroxy groups followed by colorimetric determination of the formaldehyde (8), by UV estimation of their cyclic thionocarbonate derivatives (119), or by hydroiodic acid cleavage of the ether bond to form alkyliodides from saturated alkyl moieties and alkyl diiodides from monounsaturated alkyl moieties (103,119,120). Alkyl iodides and alkyl diiodides are separable by TLC and can be quantified by their absorbance at 257 nm with a sensitivity of 1/imole of total alkyl glycerol (121). A sensitive and reliable method for the quantification of alkyl glycerols as constituents of complex lipids is the degradation by hydrogenolysis with LiAlFfy or Vitride, and subsequent analysis of l-alkyl-2,3-isopropylidene glycerols by GLC using an internal standard (122,123). Alk-1-enyl and Alkyl Glycerolipids Because of their metabolic relationship (1), it is desirable to quantify alk-1-enyl and alkyl glycerolipids simultaneously. Hydrogenolysis with LiAlH4 followed by TLC separation of alkyl and alk-1-enyl glycerols has been used for this purpose (62). In this method, alk-1-enyl and alkyl glycerols are quantified by photodensitometry (124) after charring, using a synthetic alkyl glycerol as standard. Although the method is convenient and sensitive, and it has been widely used (1), the results obtained with it have not been in agreement with those obtained by other methods. It appears that the TLC-photodensitometry method gives high values for alkyl glycerols and very low values for alk-1-enyl glycerols (62,66). A better procedure (66) involves chemical degradation, the preparation of derivatives, and quantification by GLC using internal standards. The method is applicable to the quantification of alkyl and alk-1-enyl glycerols in total lipids or in indivi-

OCTOBER 1975* 481

dual lipid fractions. The lipid sample, together with known amounts of 1,1-dimethoxyheptadecane and 1-heptadecylglycerol, is suspended in dry benzene and reacted with 1,3-propanediol in the presence of p-toluenesulfonic acid at reflux temperature while water is removed azeotropically. Alk-1-enyl moieties and 1,1-dimethoxyheptadecane are thus quantitatively converted to 1,3-dioxanes. The reaction mixture is then cooled to room temperature and transferred to a separating funnel. The lower phase, consisting of excess 1,3propanediol, is discarded and the upper phase is added dropwise at room temperature to Vitride solution. After 1 hour, aqueous sulfuric acid and diethyl ether are added and the clear ether solution is washed and dried. The reaction products, long-chain 1,3-dioxanes and alkyl glycerols, are isolated by TLC using hexane-diethyl ether (40:60), isopropylidene derivatives of the alkyl glycerols are prepared, and analysis by GLC is carried out as demonstrated in Figure 2.

Unusual Ether Lipids Most naturally occurring ether lipids contain straight-chain saturated and monounsaturated alkyl and alk-1-enyl moieties of 16 and 18 carbon atoms (1). Relatively large amounts of branched-chain alkyl and alk-1-enyl glycerols of the "iso and anteiso" types are found in ruminant tissues (47,126-128); they are separable from their straight-chain isomers by GLC as are branched-chain derivatives having methyl branches near the ether bond (129). Alkyl and alk-1-enyl moieties containing 20 or more carbon atoms occur in marine species (1,18) and have occasionally been found in large amounts in mammalian tissues (53,90,130,131). Cyclopropane analogs are major components of the alk-1-enyl glycerols of some anaerobic bacteria (132,133). Lactate fermenting anaerobic bacteria contain unusually large amounts of serine plasmalogens (134,135). 2-Aminoethyl phosphonate derivatives of alkyl acyl glycerols occur in protozoa (136), N-acetyl ethanolamine plasmalogens were found in bovine and human brain and placenta (137), and the corresponding N-acyl derivatives in bovine erythrocytes (137a).

Substituted Alkyl Glycerols

Figure 2. Gas chromatograms of alkyl dioxanes (A) and isopropylidene derivatives of alkyl glycerols (6) derived from total lipids of Novikoff hepatoma, including internal standards (shaded areas). Victoreen 4000 instrument with hydrogen flame detector; aluminum column 180 x 0.4 cm inside diameter, 10% EGSS-X on Gas Chrom P, 100/120 mesh (Applied Science Laboratories); 200°C; helium at 40 psi. (K.L. Su and H.H.O. Schmid, Lipids 9, 208 (1974). Reproduced by permission of American Oil Chemists'Society.)

Quantification is based on comparison of the total peak areas with those of the internal standards. It is facilitated by the fact that natural mixtures of alkyl and alk-1-enyl glycerols usually are not very complex, consisting mainly of 16:0, 18:0 and 18:1 compounds (1). The method is reliable since the degradation reactions were found to be quantitative. The comparisons to internal standards are independent of recoveries from the reaction mixtures and chromatoplates, and high values due to the presence of interfering substances are avoided. In addition, the composition of the constituent alkyl and alk-1-enyl moieties is obtained. As little as 0.1 fimole of alkoxylipid has been determined with an accuracy of £2.5%. Very recently, another spectrophotometric method was published (125) which involves degradation of phospholipids by Vitride reduction or by phospholipase C (B. cereus) hydrolysis coupled with saponification, to obtain alkyl and alk-1enyl glycerols. Periodate cleavage of the vicinal hydroxy groups of alkyl and alk-1-enyl glycerols produces alkyl glycolaldehydes and long-chain aldehydes which are quantified by a spectrophotometric assay of their fuchsin chromophore compounds. Aldehydes produced from alk-1-enyl glycerols by acid hydrolysis are measured in a separate sample and the amount of alkyl glycerols is calculated by the difference. Care has to be taken because periodate cleavage also occurs with other vicinal diols which may be present in the sample or may be produced from a-hydroxy fatty acids by Vitride reduction.

482 •OCTOBER 1975

2'-Methoxy substituted 1-0-alkyl glycerols were isolated from the unsaponifiable material of shark liver oils, and were characterized by chromatographic as well as by chemical and spectroscopic methods (138). These structures were found to occur as neutral lipids and phospholipids in a variety of marine species (139) and, in smaller amounts, in mammalian tissues (140). The corresponding 2'-hydroxy substituted 1-0alkyl glycerols were also recently detected in the unsaponifiable material of shark liver oil (141). Both 2'-hydroxy and 2'-oxo substituted 1-0-alkyl glycerols were found in the choline and ethanolamine phosphatides of rat brain after administration of a long-chain 1,2-alkanediol (142-144) or 1,2-alkaneketol (145). Neither 2'-hydroxy nor 2'oxo substituted phospholipids are separable by TLC from the unsubstituted analogs. After phospholipase C hydrolysis, l-0-2'-hydroxyalkyl-2-acyl glycerols and l-0-2'-oxoalkyl-2acyl glycerols are separable by TLC from one another and from 1,2-diacyl glycerols (142-145). Various derivatives of hydroxy and oxo substituted alkyl glycerols were prepared and characterized by chromatographic and spectroscopic methods (146). Relatively large amounts of hydroxy substituted alkyl glycerolipids were found in the pink portion of the rabbit harderian gland (147). 0-Hexadecyl moieties with hydroxy groups at C-10 and C-ll and 0-octadecyl moieties with hydroxy groups at C-ll and C-12 were identified. These structures occur as constituents of a neutral lipid, in which all hydroxy groups are esterified. This lipid was found to be slightly more polar than triacylglycerols (147) and was isolated by TLC using double development of the chromatogram with benzene. Rabbit harderian gland also contains alkyl diacyl glycerols having an isovaleroyl group at carbon-3 of glycerol (148), a fact that makes this lipid more polar than the normal alkyl diacyl glycerol and, thus, separable by TLC.

Diphytanyl Glycerols A unique alkoxylipid has been detected as constituent of phospholipids and glycolipids of extremely hal'ophilic bacteria (149). Its structure was identified as 2,3-di-0-phytanyl-s/iglycerol (150). Analysis of the lipids of Halobacterium cutirubrum showed the most abundant polar lipid to be the

JOURNAL OF CHROMATOGRAPHIC SCIENCE • VOL. 13

diphytanyl ether analog of phosphatidylglycerophosphate (151) and the second most abundant to be the sulfate ester of a triglycosyl diphytanyl glycerol (152). A smaller amount of the diphytanyl analog of phosphatidylglycerol was also detected. In general, it was observed that all diphytanyl lipids had a higher mobility on silicic acid impregnated paper than their diester analogs. Present knowledge of the structure and metabolism of these interesting lipids has been reviewed by Kates (1). 1 -0-Phytanyl glycerol has been found in the unsaponifiable fraction of cod liver oil (139) and was formed in mammalian brain from intracerebrally administered dihydrophytol (153). Alkoxylipids Not Containing Glycerol Neutral lipids and phospholipids having a short-chain alkanediol backbone occur widely in nature (154). 0-Alkyl diols (155,156) and 0-alk-l-enyl diols (156-159) were found in neutral lipids (155-158) and phospholipids (159) of mammalian tissues (155,157,159) and of marine invertebrates (156, 158). Although such lipids can occasionally (155) be isolated by TLC, they are usually not sufficiently different in their physical properties from glycerolipids to permit their separation on the basis of polarity. Hence, separation methods on the basis of molecular size by gel permeation chromatography were developed (160,161). Migration rates in adsorption and reversed phase partition TLC of synthetic ethers, esters and ether esters of various diols and of glycerol have been compared (162), as illustrated in Figure 3.

10

Figure 3. Thin-layer chromatogram of long-chain derivatives of glycerol, 1,3-propanediol and 1,2-ethanediol. Adsorbent: Silica Gel G (Merck); Solvent: hexane-diethyl ether (90:10, v/v). (1) 1,2,3-trialkyl glycerol; (2) 1-acyl-2,3-dialkyl glycerol; (3) 1-alkyl-2,3-diacyl glycerol; (4) 1,2,3-triacyl glycerol; (5) 1,3-dialkyl propanediol; (6) 1-alkyl-3-acyl propanediol; (7) 1,3diacyl propanediol; (8) 1,2-dialkyl ethanediol; (9) 1-alkyl-2-acyl ethanediol; (10) 1,2-diacyl ethanediol. [W.J. Baumann et al., Biochim. Biophys. Acta 144,355 (1967). Reproduced by permission of Elsevier Scientific Publishing Company.]

Both adsorption (61,163) and argentation (163,164) TLC have been used in the synthetic (163,164) and semi-synthetic

JOURNAL OF CHROMATOGRAPHIC SCIENCE • VOL. 13

(61) preparation of alk-1-enyl ethanediols and their derivatives. Separations by GLC of monoethers and monocsters of 1,2-ethanediol as their trifluoroacetates, acetates and trimethylsilyl derivatives on polar and nonpolar stationary phases have been described (165). Mass spectrometric analysis of alkyl and alk-1-enyl diols and their esters (166,167) aids greatly in their identification. GLC-mass spectrometry was found to be of particular importance for the identification and quantification of short-chain diols as they occur in neutral and polar lipids (168) containing an acyl diol or alk-1-enyl diol backbone. Alkyl ethanediol phosphocholines and phosphoethanolamines are separable from choline and ethanolamine glycerophosphatides by TLC (169) using multiple development of the chromatogram with chloroform-methanol-acctic acid-water (50:25:4:2). Long-chain alkyl ethers of cholesterol have been synthesized and characterized (170). The presence of small amounts of alkyl (171) and alk-1-enyl (172) cholesterols in heart muscle has been reported.

Conclusion Since ether lipids are common constituents of neutral lipids and of phospholipids, their presence must be considered in lipid analysis. Neutral ether lipids usually are found only in trace amounts except in shark liver and other marine organisms. Alkyl diacyl glycerols are found in increased amounts in various mammalian neoplasms. Alk-1-enyl diacyl and alkyl diacyl glycerols can be separated from each other and from triacyl glycerols by TLC both on an analytical and preparative scale. Polar ether lipids, especially alk-1-enyl acyl glycerophosphatides, often occur in large amounts. Therefore, derivatives such as dimethyl acetals produced from the alk-1-enyl moieties by acidic methanolysis, may be encountered when fatty acid methyl esters are analyzed by GLC. Although dimethyl acetals of long-chain aldehydes have shorter retention times than the corresponding fatty acid methyl esters on the polar stationary phases most commonly used, their presence may lead to misinterpretation of analytical results. Therefore, separation of fatty acid methyl esters and dimethyl acetals by TLC should always precede GLC analysis of methyl esters. The relative instability of alk-1-enyl glycerols during extraction, storage, fractionation or degradation of lipids is also a factor to be considered. Cleavage of the alk-1-enyl glycerol produces not only aldehydes and their condensation products, but also 2-acyl lysophosphatides or diacyl glycerols whose presence in a tissue extract or subcellular preparation may be misinterpreted as that of a genuine metabolite. Subfractionation of natural alk-1-enyl acyl, alkyl acyl and diacyl glycerophosphatides is not possible. Specific degradation and derivatization is used for structural analysis and quantification. It appears that quantification of suitable derivatives of alkyl and alk-1-enyl glycerols by GLC based on internal standards is the most useful and reliable method of determining the amounts of ether lipids present in a lipid extract or in an individual lipid fraction.

Acknowledgments This work was supported in part by PHS Research Grants Nos. CA 13113 and NS 10013, PHS Research Grant No. HL 08214 from the Program Projects Branch, Extramural Programs, National Heart and Lung Institute, and by The Hormel Foundation.

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Application of Open-Tubular Column/Gas-Liquid Chromatography to the Analysis of Complex Mixtures of Branched-Chain Fatty Acids A. Smith and A.K. Lough, Rowett Research Institute, Bucksbum, Aberdeen, AB2 9SB, United Kingdom

Abstract Methyl and butyl esters of branched-chain fatty acids of subcutaneous triacylglycerols from barley-fed lambs were subjected to urea-adduction and the resulting fractions were analyzed as methyl esters by gas-liquid chromatography on wall-coated open-tubular columns. The chromatogram of each fraction was less complex than that of the original and there was evidence to suggest that, in the original mixture of branched-chain acids, components resistant to urea-adduction were present, but their occurrence was obscured in chromatograms by the presence of monomethyl-substituted acids.

Introduction The advent of gas-liquid chromatography (GLC) has played a major role in the rapid advances which have been made in the chemistry and biochemistry of lipids in the last 15 to 20 years. The scope of GLC was extended as a result of the development and commercial availability of open-tubular columns. The literature relating to the use of these columns has been reviewed by Ettre (1) and by Ackman (2). The greatly improved resolution of open-tubular or capillary columns has been of particular advantage in the separation and identification of many hitherto unknown fatty acids. This report is concerned with the application of open-tubular column chromatography to the analysis and identification of mixtures of branched-chain fatty acids of ruminant origin. 486 •OCTOBER 1975

Lambs fed on a diet containing a high proportion of rolled barley have abnormally soft subcutaneous adipose tissue (3). Analyses of the fatty acids of triacylglycerols of subcutaneous and perinephric tissue of these lambs revealed the presence of branched-chain fatty acids in greater proportions (up to 13%) than exist in the depot lipids of conventionally-fed lambs. Analysis of the methyl esters of these fatty acids by conventional GLC showed that the mixture of branched-chain components was too complex for meaningful identifications to be made by this technique (4,5). When concentrates of the methyl esters of the branchedchain fatty acids were analyzed using a highly efficient opentubular column coupled to a mass spectrometer (GC/MS) it was found that the component fatty acids varied in chain length from 10 to 17 carbon atoms and included mono-, diand trimethyl substituted components (6). The monomethyl acids which constituted the major proportion of the total branched-chain fatty acids were, for the most part, methyltetradecanoic and methyl hexadecanoic acids in which substittution occurred on a carbon atom designated by an even number in relation to the carboxyl group. The dibranched acids had a chain length of 11 to 15 carbon atoms with methyl substitution at the 4 and 8 positions. A tribranched acid (2,6,10 trimethyltetradecanoic acid) was also identified. In relation to the foregoing observations, it was reported that difficulty had been experienced in obtaining mass spectra which represented single components (6). Though it was suggested that this might be indicative of some loss of resolution in the gas chromatographic aspect of the analysis, it is likely that the problem of incomplete resolution was, to some extent, JOURNAL OF CHROMATOGRAPHIC SCIENCE • VOL. 13