Reaction of Lipids Containing Hydroxy Groups with Trimethylsulfonium Hydroxide: Formation of O-Methyl Derivatives

Klaus Vosmann a, Erhard Schulte b, Erika Klein c, and Nikolaus Weber c,* ~lnstitut for Chemie und Physik der Fette, BAGKF, D-48147 MOnster, blnstitut flit Lebensmittelchemie

der Universit~it, D-48147 MOnster, and Clnstitut for Biochemie und Technologie der Fette, H.P. Kaufmann-lnstitut, BAGKF, D-48147 MOnster, Germany

ABSTRACT: Base-catalyzed transesterification of acyl lipids with trimethylsulfonium hydroxide (TMSH) is an easy and con- venient method for the preparation of fatty acid methyl esters (FAME) for gas chromatography (GC) analyses. We have found, however, that lipids containing hydroxy groups are partially converted to the corresponding O-methyl ether derivatives which may interfere with FAME in GC separations. For exam- ple, long-chain alcohols are found to be converted to alkyl methyl ethers, rac-l-O-alkylglycerols to the corresponding 2- O- and 3-O-monomethyl ethers, as well as 2,3-di-O-methyl ethers, hydroxy fatty acids to methoxy FAME, and cholesterol to cholesteryl 313-methyl ether. From our results, it is obvious that TMSH derivatization method is not recommended without limi- tation for lipids containing hydroxy groups; it may be, however, of some diagnostic value for the analysis of such lipids by GC/mass spectrometry. Lipids 31, 349-352 (1996).

Fatty acid methyl esters (FAME) are the most common deriv- atives for the analysis of fatty acids by gas chromatography (GC) (1,2). Recently, a very convenient method has been de- scribed for the derivatization of acyl lipids to FAME by using trimethylsulfonium hydroxide (TMSH) as the reagent (2-5). This method is based on earlier publications which have drawn attention to the advantage o f this mild methylation reagent for the preparation of FAME for GC analysis (6,7). It was also, shown that FAME are formed by base-catalyzed transesterification, of acyJ~ lipids, such as, triacylglycerols, whereas unesterified fatty acids are methylated by pyrolytic decomposition of the corresponding TMSH salts (7). Here we report about the limitations of the use of TMSH in derivatiza- tion of lipids for GC analyses in the case that appreciable pro- portions of lipids containing hydroxy groups are present in the sample.

* To whom correspondence should be addressed at Institut ftir Biochemie und Technologie der Fette, H.P. Kaufmann-lnstitut, BAGKF, Piusallee 68, 48147 MUnster, Germany. Abbreviations: FAME, fatty acid methyl esters; GC, gas chromatography; GC/MS, gas chromatography/mass spectrometry; TMSH, trimethylsulfo- nium hydroxide.

MATERIALS AND METHODS

Chemicals. Long-chain alcohols, cholesterol, and methyl methanesulfonate were purchased from E. Merck (Darmstadt, Germany). Hydroxy and epoxy fatty acids, as well as choles- teryl 3~-methyl ether, were products of Sigma-Aldrich (Deisen- hofen, Germany). TMSH reagent (0.2 M TMSH in methanol) was a product of Macherey-Nagel (Dtiren, Germany), Mono- methylethers of rac-l-O-alkylglycerols were obtained from Calbiochem-Novabiochem (Bad Soden, Germany) and Berch- told (Bern, Switzerland).

Preparation of standards. FAME were prepared by the reac- tion of free fatty acids with diazomethane (8). rac-l-O-Alkyl- glycerols, rac- 1-O-alkyl-2,3-di-O-methylglycerols, and methyl ethers of long-chain alcohols were prepared according to the procedure of Baumann and Mangold (9) by the use of alkyl methanesulfonates or methyl methanesulfonate. Methoxy FAME were prepared from the corresponding hydroxy FAME by the reaction with diazomethane in the presence of silica gel (10).

Derivatizationfor GC. To a solution of 0.8 mg lipid contain- ing hydroxy groups in 40 ~ t-butyl methyl ether, 20 BL TMSH reagent were added. The derivatization mixture was used im- mediately for GCo

GC. GC of was carried out using a Hewlett-Packard (B6blin- gen, Germany) HP-5890 Series Ilgas chromatograph fitted with a flame-ionization detector. O-Methyl ether derivatives of lipids were separated on a 0.15 Bm FFAP-CB fused silica capillary column (Macherey-Nagel), 25 m x 0.25 mm i.d. The alkyl methyl ethers were separated using nitrogen as carrier gas (lin- ear velocity 20 cm/s) initially at 160~ for 6 min, followed by linear programming from 160 to 240~ at 4~ per min. The final temperature was kept constant for l0 min. The split ratio was l:10; the injector as well as flame-ionization detector tem- perature was 270~ GC peaks were assigned by comparison of their retention times with those of known standards. Methoxy FAME as well as O-methyl derivatives of rac-1-O-alkylglyc- erols were separated under similar conditions using a linear tem- perature program from 160 to 240~ at 8~ per min. The final temperature was kept constant for 20 min. Cholesteryl 313- methyl ether was separated isothermically at 240~

Copyright �9 1996 by AOCS Press 349 Lipids, Vol. 31i no. 3 (1996)

350 COMMUNICATION

GC/mass spectrometry (MS). GC/MS of O-methyl deriva- tives was performed at electron impact mode (70 eV) and chemical ionization mode (230 eV; methane as reagent gas, 160 Pa) on a Hewlett-Packard instrument Model 5890 Series 11/5989 A (Palo Alto, CA). The GC instrument was equipped with a 0.23 ~tm Permabond OV-1 fused silica capillary col- umn (Macherey-Nagel) 25 m x 0.32 mm i.d. The carrier gas was He at a flow rate of 1.0 mL/min. The column tempera- ture was initially kept at 150~ for 5 min and then pro- grammed from 150 to 270~ at 4~ the final tempera- ture was held for 5 min. Other operating conditions were split/splitless injector in splitless mode (temperature 300~ interface temperature 280~ and ion source temperature 200~ Structural assignments were made for purchased gen- uine methoxy lipid standards and methoxy derivatives pre- pared from authentic samples by established procedures. Peaks of methoxy derivatives of hydroxy lipids obtained by

the reaction with TMSH were assigned a structure if ions cor- responding to all the important mass fragments of Table 1 were present, and no other high mass ions were detected.

RESULTS A N D DISCUSSION

Single compounds or mixtures of lipids containing hydroxy groups, such as long-chain alcohols, alkylglycerols, hydroxy fatty acids, and cholesterol, were reacted with TMSH under pyrolytic conditions in order to study the possible formation of artefactual derivatives which may interfere with the GC analysis of FAME.

Figure 1 shows the gas chromatogram of the reaction prod- ucts of a mixture of 1-hexadecanol and 1-octadecanol with TMSH. Obviously around 30% of the alcohols were trans- formed to the corresponding 1-O-methyl ethers, i.e., hexade- cyl methyl ether and octadecyl methyl ether, which were

TABLE 1 Electron Impact Mass Spectral Data of O-Methyl Derivatives of Various Hydroxy Group Containing Lipids Formed by the Reaction with Trimethylsulfonium Hydroxide O-Methyl ether derivatives of hydroxy group containing lipids Important mass fragments (re~z) a

Hexadecyl 1 -O-methyl 224 (14.3) 169 (5.0) 168 (2.3) ether b [M - CHgOH] + [M - (CH3OH + C2H4)] + [M - (CH3OH + C4H8)] +

Octadecyl 1-O-methyl 252 (13.0) 224 (4.5) 196 (1.8) ether b [M - CH3OH] + [M - (CH3OH + C2H4)] + [M - (CH3OH + C4H8)] +

cis-9-Octadecenyl 282 (1.3) 250 (8.2) 222 (1.6) 194 (2.2) 1-O-methyl ether b [M] + [M - CHgOH] + [M - (CH3OH + C2H4)] + [M - (CH3OH + C4H8)] +

rac-1-O-Hexadecyl- 345 (0.3) 312 (1.9) 280 (1.9) 253 (7.2) 2,3-di-O-methylglycerol c [M + 1 ]+ [M - CH3OH] + [M - C2H802 ]+ [M - C4H 1102 ]+

rac-1 -O-octadecyl 373 (0.2) 340 (2.6) 308 (2.1) 281 (9.2) 2,3-di-O-methylglycerol c [M + 1 ]+ [M - CH3OH] + [M - C2H802 ]+ [M-C4H 1102 ]+

rac-1 -O-Octadec-9'-enyl- 370 (1.1) 293 (2.1) 267 (1.5) 250 (6.1) 2,3-di-O-methylglycerol c [M] + [M - C3H902 ]+ [M - C5Hl lO2 ]+ [M - C5H1203 ]+

rac-l-O-Hexadecyl- 331 (0.4) 285 (0.8) 241 (1.3) 222 (3.1) 2-O-methylglycerol c [M + 1 ]+ [M - C 2 H 5 0 ] + [M - C4H902 ]+

rac-l-O-Octadecyl- 359 (0.2) 313 (1.5) 269 (1.6) 250 (4.6) 2-O-methylglycerol c [M + 1 ]+ [M - C2H50] + [M - C4H902] +

rac-1-O-Octadec-9'-enyl- 356 (0.4) 293 (1.4) 267 (3.6) 250 (11.3) 2-O-methylglycerol c [M ]+ [M - C2H702 ]+ [M - C4H902 ]+ [M - C4H1003] +

rac-l-O-Hexadecyl- 331 (1.0) 285 (1.7) 253 (6.7) 225 (6.3) 3-O-methylglycerol c [M + 1 ]+ [M - C 2 H 5 0 ] + [M - C3H902 ]+ [M - C4H903 ]+

rac-1 -O-Octadecyl- 359 (0.2) 313 (1.1) 281 (5.8) 253 (5.0) 3-O-methylglycerol c [M + 1 ]+ [M - C2H50] + [M - C3H902 ]+ [M - C4H903] +

rac-1-O-Octadec-9'-enyl- 356 (0.9) 293 (0.7) 267 (1.4) 250 (9.1) 3-O-methylglycerol c [M + 1 ]+ [M - C2H702 ]+ [M - C4H902 ]+ [M -' C4H1003 ]+

12-Methoxystearic 243 (65.3) 211 (15.8) 174 (9.8) 129 (66.3) acid methyl ester d [M - C6H13 ]+ [243 - CH3OH] + [243 - (2 x CHgOH)] + CsH 17 ~

12-Methoxyoctadec-9-enoic 294 (1.6) 129 (84.2) 97 (84.7) acid methyl ester d [M - CHgOH] + CsH 17 ~ [129 - CH3OH] +

Cholesteryl 400 (50.5) 368 (99.4) 353 (46.8) 255 (32.9) 3[3-O-methyl ether e [M] + [M - CH~OH] + [M - C;tHTO]+ [M - (CH~OH + C~H 17)] +

alntensities relative to base peak. bBase peaks of alkyl 1-O-methyl ethers: 16:0, m/e 55; 18:0, re~e55; 18:1, m/e 57. CBase peaks of isomeric mono- and dimethyl 1-O-alkylglyceryl ethers, respectively: 16:0, m/e 57 and 58; 18:0, m/e 57 and 58; 18:1, m/e 55 and 57. dBase peaks of methoxy fatty acid methyl esters: 18:0, m/e 55; 18:1, m/e 55. eBase peak of cho!P~t~ry| 24~-O-methyl ether: m/e 55. - -

Lipids, Vol. 31, no. 3 (1996)

COMMUNICATION 351

a Ia_

300

200

100

i .

4

I I I I I I I i I

0 2 4 6 81012141618

Time (min)

FIG. 1. Gas chromatogram of the reaction products of a mixture of 1 - hexadecanol and 1 -octadecanol with trimethylsulfonium hydroxide (1, hexadecyl 1-O-methyl ether; 2, octadecyl 1-O-methyl ether; 3, 1-hexa- decanol, not reacted; 4, 1-octadecanol, not reacted); FID, flame-ioniza- tion detection.

identified by comparison with standards as well as by GC/MS (Table 1). The most important mass fragments for the struc- tural assignment of the primary long-chain alcohols were the [M-CH3OH] § ions which are typical of the O-methyl ether structure. Other structurally important intense ions have not been found. These observations are consistent with the litera- ture (11). Molecular weight was determined in all samples, i.e., alkylmethyl ethers, alkylmethylglyceryl ethers, methoxy FAME, and cholesteryl 3~-O-methyl ether, by chemical ion- ization MS.

The gas chromatogram of the reaction products formed from a mixture of rac- l-O-hexadecylglycerol and rac- 1-O-

octadecylglycerol with TMSH is shown in Figure 2. The six reaction products observed in addition to the nonreacted alkylglycerols were identified by comparison with authentic standards as well as by GC/MS (Table 1) as the isomeric 2- O-methyl and 3-O-methyl 1-O-aikylglycerols, respectively, and thecorresponding 2,3-di-O-methyl ether derivatives. In most electron impact spectra, small proportions of [M + 1 ]+ ions have been observed. Earlier studies have also shown [M + 1 ]+ ions for saturated long-chain alcohols (11), whereas [M + 1] § peaks have not been detected for alkylglyceryl ethers (12). Fragments with two, three, or four carbon atoms were found to be typical cleavage products of the glycerol backbone of alkyl(methyl)glyceryl ethers (Table I) which is consistent with the literature (13). It is interesting to note that the 3-O-methyl derivatives are formed in higher proportions than the 2-O-methyl derivatives (ratio around 1:0.8; yield of mono- plus dimethyl derivatives around 80%), suggesting that different sterical effects are operative in the O-methyla- tion at positions 2 and 3 of 1-O-alkylglycerols.

Figure 3 shows the gas chromatogram of the reaction prod- ucts of a mixture of 12-hydroxystearic acid and ricinoleic acid with TMSH. Obviously, the free fatty acids were converted

i,

200

100-

2..... _ _ 1

I" 6

I

4

7 8

I I I I I I

0 5 10 15 20 25 30

Time (min)

FIG. 2. Gas chromatogram of the reaction products of a mixture of rac- 1-O-hexadecylglycerol and rac-l-O-octadecylglycerol with trimethyl- sulfonium hydroxide (1, rac-1-O-hexadecyl-2,3-di-O-methylglycerol; 2, rac-1-O-octadecyl-2,3-di-O-methylglycerol; 3, rac-1-O-hexadecyl-3- O-methylglycerol; 4, rac-1-O-hexadecyl-2-O-methylglycerol; 5, rac-1- O-octadecyl-3-O-methylglycerol; 6, rac-l-O-octadecyl-2-O-methyl- glycerol; 7, rac-l-O-hexadecylglycerol, not reacted; 8, rac-1-O-octade- cylglycerol, not reacted); see Figure 1 for abbreviation.

completely to the methyl esters. In addition, small proportions of methyl 12-methoxystearate and methyl 12-methoxyoleate (around 3 to 5%, each) are formed as confirmed by compari- son with standards as well as by GC/MS (Table 1). The loss of CH3OH fragments is typical of lipids containing methyl ether and methyl ester groups (14); the fragments at m/e 243, 211, 197, and 97 clearly show the position of the methoxy group at the acyl backbone. Surprisingly, both methyl ery- thro- and threo-9,10-dihydroxyoctadecanoates were con-

20.

C3 ,T 10-

0

I I I I i I I I I

0 2 4 6 8 1012141618

Time (min) FIG. 3. Gas chromatogram ot the reaction products or a mixture of 12- hydroxy stearic acid and ricinoleic acid with trimethylsulfonium hy- droxide (1, 12-methoxy stearic acid methyl ester; 2, 12-methoxy oc- tadec-9-enoic acid methyl ester; 3, 12-hydroxy stearic acid methyl ester plus ricinoleic acid methyl ester); see Figure 1 for abbreviation.

Lipids, Vol. 31, no. 3 (1996)

352 COMMUNICATION

150

--- loo LI.

50

..Jl ~ A

I I I I I I

0 5 10 15 20 25 30

Time (min)

2

L

FIG. 4. Gas chromatogram of the reaction products of cholesterol with trimethylsulfonium hydroxide (1, cholesteryl 3[3-O-methyl ether; 2, cho- lesterol, not reacted). See Figure 1 for abbreviation.

verted to the monomethoxy plus dimethoxy derivatives to an extent of around 60% (data not shown), demonstrating a pos- itive effect of the vicinal hydroxy groups on O-methyl ether formation. A similar effect was observed for the vicinal hy- droxy groups of 1-O-alkylglycerols.

It is evident from Figure 4 that cholesteryl 3]]-O-methyl ether is formed in high proportions (around 40%) by reacting cholesterol with TMSH. The 3-O-methyl derivative was iden- tified by comparison with an authentic standard as well as by GC/MS (Table 1). In addition, mass spectrum of cholesteryl 3l]-O-methyl ether agreed with that given in the literature (15).

In conclusion, our data clearly show that O-methyl ether derivatives are formed, in part, from lipids containing hy- droxy groups during, pyrolytic reaction conditions in the GC injector, whereas the hydroxy groups do not react with the TMSH reagent at room temperature (data not shown). In GC, peaks of the O-methyl derivatives may interfere partially with peaks of FAME and, therefore, lead to erroneous results. The use of TMSH is not generally recommended, therefore, for the analysis of lipids containing hydroxy groups. It can be of some diagnostic value, however, for the detection of these lipids in samples. In contrast to lipids withhydroxy groups, epoxy fatty acids such as cis- and trans-9,10-epoxystearic acids are converted to the corresponding FAME without fur- ther derivatization (data not shown).

Studies are in progress concerning methods which enable us to circumvent the disturbing effects of O-methyl ether for-

mation on FAME analyses of, e.g., wax esters, hydroxy fatty acids, and animal lipids with high proportions of ether glyc- erolipids.

REFERENCES

1. Christie, W.W. (1993) Preparation of Ester Derivatives of Fatty Acids for Chromatographic Analysis, in Advances in Lipid Methodology--Two (Christie, W.W., ed.) pp. 69-111, The Oily Press, Dundee.

2. Schulte, E. (1993) Gas Chromatography of Acylglycerols and Fatty Acids with Capillary Columns, in Analysis of Lipids (Mukherjee, K.D., and Weber, N., eds.) pp. 139-148, CRC Press, Boca Raton, FL.

3. Schulte, E., and Weber, K. (1989) Schnelle Herstellung der Fetts~turemethylester aus Fetten mit Trimethylsulphonium-hy- droxid, Fat Sci. Technol. 91, 181-183.

4. Miiller, K.D., Husmann, H., Nalik, H.P., and Schomburg, G. (1990) Transesterification of Fatty Acids from Microorganisms and Human Blood Serum by Trimethylsulfonium Hydroxide (TMSH) for GC Analysis, Chromatographia 30, 245-248.

5. E1-Hamdy, A.H., and Christie, W.W. (1993) Preparation of Methyl Esters of Fatty Acids with Trimethylsulphonium Hy- droxide-An Appraisal, J. Chromatogr. 630, 438-441.

6. Butte, W., Eilers, J., and Kirsch, M. (1982) Trialkylsulfonium and Trialkylselenonium Hydroxides for the Pyrolytic Alkyla- don of Acidic Compounds, AnaL Lett. 15A, 841-850.

7. Butte, W.L (1983) Rapid Method for the Determination of Fatty Acid Profiles from Fats and Oils Using Trimethylsulphonium Hydroxide for Transesterification, J. Chromatogr. 261, 142-145.

8. Christie, W.W. (1982)Lipid Analysis. Isolation, Separation, Identification, and Structural Analysis of Lipids, 2nd edn., pp. 54-55, Pergamon Press, Oxford.

9. Baumann, W.J., and Mangold, H.K. (1965) Reactions of Aliphatic Methanesulfonates. I. Syntheses of Long-Chain Glyc- eryl-(1) Ethers, J. Org. Chem. 29, 3055-3057. Lutz, A., KnOrr, W., and Spiteller, G. (1991) Produkte des reduktiven Abbaus von ~-(Acyloxy)plasmalogenen aus Rindergewebe-Lipiden mit LiA1H 4, Liebigs Ann. Chem., 1151-1155. McLafferty, F.W. (1973) Interpretation of Mass Spectra, 2nd edn., pp. 113-116, 146-147, W.A. Benjamin, Reading. Ryhage, R., and Stenhagen, E. (1960) Mass Spectrometry in Lipid Research, J. Lipid Res. 1,361-390. Egge, H. (1983) Mass Spectrometry of Ether Lipids, in Ether Lipids. Biochemical and Biomedical Aspects (Mangold, H.K., and Paltauf, F., eds.) pp. 141-159, Academic Press, New York.

14. Murphy, R.C. (1993) Mass Spectrometry of Lipids, in Handbook of Lipid Research (Snyder, F., ed.) Vol. 7, pp. 71-130, Plenum Press, New York.

15. Wiley PBM Database (1992) Wiley, New York.

10.

11.

12.

13.

[Received February 21, 1995; accepted December 21, 1995]

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