Mass spectra of complex molecules I. Chemical ionization of sphingolipids

CHEMISTRY AND PHYSICSOF LIPIDS 12 (1974) 182-200. NORTH-HOLLANDPUBL. CO.

MASS S P E C T R A O F C O M P L E X M O L E C U L E S I. C H E M I C A L I O N I Z A T I O N O F S P H I N G O L I P I D S

S.P. MARKEY and D.A. WENGER B.F. Stolinsky Research Laboratory, Departrnent of Pediatrics, Univ. of Colorado Medical Center, Denver, Colorado 80220, USA

Received April 4, 1973 Accepted July 29, 1973

Methane chemical ionization spectra of acetylated and perdeutero-acetylated ceramides, cerebrosides, and ceramide dihexosides have been analyzed and compared with electron impact ionization spectra of the same compounds. Abundant fragment ions for the loss of acetic acid from the protonated molecular ion in C.I. spectra readily enable the determination of the molecular weights of the principal species of sphingolipid mixtures. Complete structures can be elucidated by the combination of E.I. and C.I. mass spectra.

I. Introduction The structure determination of complex, polyfunctional organic compounds is of interest in many areas of biomedical research, most notably protein and carbohydrate chemistry. In the past few years, mass spectrometry has been successfully applied to the characterization of many classes of derivatized compounds with molecular weights below 1500 amu. Limitations on extending the mass range for the study of higher molecular weight compounds have been volatility requirements, the fragmentation of complex molecules upon electron impact, and instrumentation. Polar, polyfunctional molecules may have vapor pressures too low for mass spectral analysis. Derivitization, such as permethylation of peptides [1 ] or trimethylsilylation of carbohydrates [2], is frequently used to substantially increase vapor pressure by eliminating intermolecular hydrogen bonding. While useful for many analyses, derivatization invariably increases the molecular weight - 14 amu per methyl, 42 amu per acetyl, 72 amu per trimethylsilyl, and 96 amu per trifluroacetyl group. For a polyfunctional molecule, it is apparent that the molecular weight of the compound may be quickly increased beyond the usable mass range o£a mass spectrometer. Derivitization also requires that the compound be stable to the reaction conditions employed, and that yields are quantitative so that by-products do not confuse the analysis and necessitate separation techniques. Using commonly

S.P. Markey, D.A. Igenger,Massspectraof sphingolipids

183

employed derivatives, 1500 amu is generally the upper limit for mass spectral analo ysis because the heat required (200-300°C) to produce sufficient vapor pressure at 10 -7 mm may well promote pyrolytic degradation. Electron impact induced ionization at 70 eV imparts excess energy to the newly formed ions resulting in fragmentation in order to stabilize the charge. One corollary of the quasi-equilibrium theory of mass spectrometry is that larger molecules will have more possible competing fragmentation reactions than small molecules. If the rates of these decomposition reactions are similar to those of small model compounds containing the same functional groups, then the probability of observing the intact molecule (molecular ion) diminishes. Unfortunately, electron impact with energies approximating the ionization potential of organic compounds is inefficient, resulting in significant sensitivity losses. For those high molecular weight molecules successfully studied and reported in the literature, it is not uncommon to see the electron impact mass spectra reported with scale expansion factors above the low m/e region. Most organic magnetic deflection mass spectrometers have been designed to have a useful mass range of 1-2000. It is possible with 8-10 kV accelerating voltage instruments to lower the accelerating voltage and extend the mass range to 4000. Fales has reported the mass spectrum of a perfluoroalkyl phospholonitrilate with an intense molecular ion at m/e 3629, obtained on a high resolution double focusing mass spectrometer [3]. Although decreased resolution may result from lowered accelerating voltage, it is instrumentally practicable to obtain mass spectral data on compounds with molecular weights up to 4000. Mass identification may be difficult at high m/e values, but some marker compounds have been described which are potentially valuable above m/e 1000 [4]. It is possible to peak match [5 ], extrapolate from photographic plates [6], or use computer recording techniques to assist in high m/e identification [7, 8]. In spite of these techniques, it remains a difficult technical problem to reliably identify high m/e values. Chemical ionization (C.I.) mass spectrometry has previously been shown to have potential in the study of compounds difficult to analyze by electron impact (E.I.) techniques. For example, Kiryushin et al. have compared the E.I. and C.I. mass spectra from some permethylated peptides [9]. Not only were the fragmentations more readily interpretable in the C.I. spectra, but the percentage of total ion current above m/e 200 was significantly higher in the C.I. spectra. Thus it would appear that C.I. eliminates some of the problems involved in the analysis of complex molecules by mass spectrometry. An investigation of the use of C.I. for the structure determination of sphingolipids is summarized in this report. In our studies on the chemistry of inborn errors of metabolism, the ability to characterize and determine the primary structure of small quantities of complex sphingolipids such as ganghosides would be most valuable. Consequently, sphingolipids of varying degrees of complexity were chosen for this study. Initially, permethylation using dimethylsulfinyl carbanion-methyl iodide was chosen as the means of derivitization. The requirements of compound

184

s.P. Markey, D.A. Wenger,Mass spectra of sphingolipids

stability to derivitization procedures and avoidance of by-products were not met for monosianogangliosides. Acetylation with acetic anhydride-pyridine has proven to be a satisfactory alternative, although increasing the molecular weight more than permethylation. Recently, Andersson et al. have reported the E.I. spectra of acetylated cerebrosides which coincide with our results [ 10]. Most of the previous mass spectral studies of sphingolipids which have been recently reviewed by Odham and Stenhagen have utilized trimethylsilyl derivatives [11 ].

II. Materials and methods A. Sources o f lipids

Hydroxy fatty acid ceramide (I) was purchased from Applied Science Laboratories, State College, Pennsylvania. Galactocerebroside (IV) (galactosyl ceramide, GL-lb) was isolated from bovine brain white matter after chloroform-methanol (2:1, v/v) extraction. Purification was obtained on a column of silicic acid followed by a silica gel column. The material that eluted from the silica gel column with chloroformmethanol (9:1, v/v) was galactocerebroside with a variety of fatty acid esters. Two orcinol positive spots were detected in the region of monohexosylceramide on thin layer chromatography in chloroform-methanol-water (60:35: 8, v/v). Psychosine (II) (galactosyl/31~' 1 sphingosine) was prepared from this mixture of natural beef brain white matter galactocerebrosides by refluxing for four hours with 0.05 M KOH in n-butanol-water (10:1. v/v).* After dialysis of the reaction mixture against cold water over-night, the retained solution was lyophilized. The resulting residue was applied to a column of silica gel where the psychosine was clearly separated from unhydrolyzed galactocerebroside. The ratio of hexose to sphingosine was determined to be 1 to 0.96. Hexose was measured according to the method of Hildebrand et al. [12] and sphingosine by the method of Lauter and Trams [13]. Mixed bovine brain gangliosides (type III, Sigma, St. Louis, Missouri) were hydrolized with 0.1 NHC1 in a boiling water bath for 1.5 hr). The acid solution was partitioned with 5 volumes of chloroform-methanol (2:1, v/v), and the lower phase was isolated. Glucosyl ceramide (Ill) and dihexosyl ceramide (V) were purified by silica gel column chromatography followed by preparative thin layer chromatography in chloroform-methanol-water (60: 35: 8, v/v). The chromatographically pure lipids were found to have the expected hexose to sphingosine ratio. Acid hydrolysis of the dihexosylceramide produced equal amounts of glucose and galactose when visualized on thin layer chromatography in n-butanol-acetone-water (20: 25:

5, v/v). The compounds above were acetylated with pyridine-acetic anhydride 1: 1, v/v at room temperature overnight, evaporated to dryness at 60°C with nitrogen, and * N.S. Radin, personal communication.

S.P. Markey, D.A. lCenger,Mass spectra of sphingolipids

185

redissolved in chloroform for evaporation on the mass spectrometer solid probe. Deuteroacetylation was performed with acetic anhydride-d6 (Merck & Co., Inc., Rahway, N.J.) Permethylation of several milligrams of I was accomplished by a modification of Hakomori's procedure [14]. Following extraction of the product into CHC13, traces of dimethylsulfoxide and side products are removed by chromatography on LH-20 in CHC13-MeOH 1 : 1. v/v. Mass spectra were recorded on a AEI MS-12 (Manchester, England) medium resolution mass spectrometer using an on-line computer recording system [7] based upon a DEC PDPSi (Maynard, Mass.) All E T spectra were obtained with a standard ion source operated at 160-250°C depending upon compound volatility. Accelerating voltage was reduced to 5 - 6 kV for compounds with molecular weights above 1000. Trap current was maintained at 100/aA and electron energy was kept at 70 eV for all E T spectra. C.I. spectra were obtained on the same MS-12 modified for high ion source pressure as described previously [ 15]. Attempts to obtain greater sensitivity using an enclosed filament sealed in quartz were abandoned during these studies when it was observed that sensitivity dropped markedly after 3 - 4 h of filament life. Pyrolysis products and polymerization of carrier gas led to a buildup of conducting layers and leakage paths between the filament and ion source block. Deterioration of the filament (pitting) when operated at high pressures was also apparent. The spectra reported in this study were obtained with a conventional rhenium wire filament insulated from the ion source block and maintained at 500 eV. Filament current was emission regulated at 500/aA. The ion source temperature was regulated between 100-200°C, and compounds were observed to volatalize at temperatures 10-30°C below those used for E.I. work. Methane was the C.I. reagent gas used in all spectra descussed.

HI. Results All of the mass spectra reported were determined by low resolution techniques. Except for those compounds in which deuterium labelling could be employed (deuteromethylation or acetylation) the suggested fragmentations are based only on comparison of spectra and the mass shifts observed. A generalized formula for sphingolipids is shown in fig. 1. Fragment ions common to many of the spectra are indicated by the letters a-j. A. Ceramides

Both hydroxy and non-hydroxy fatty acid ceramides were studied in the course of these investigations. The fragmentations of the non-hydroxy fatty acid ceramides were closely analogous to those observed for hydroxy fatty acid ceramides. Conse-

186

S.P. Markey, D.A. Wenger, Mass spectra of sphingolipMs

b

CH3(CH2]I~--- C H ~-'~CH--C H I

o~

f

g

C H ---)- CH 2 ~ -

I

l'r'"

0 ---J- S u g a r s

L"h ~"J ,,2

N--RZI~'Cd

. . . .

C~O

/ e

I

H - - C --OR I I (CH2) n I CH 3

Fig. 1. Generalized formula showing the fragmentations observed and the nomenclature used for the mass spectrometry of sphingolipids.

quently, the hydroxy fatty acid ceramides are used in the following discussion to illustrate the general principles. Several underivatized sphingolipids were run using C.I. and E.I. to determine whether derivitization is necessary in all cases. Figs. 2 and 3 show the E.I. and C.I. spectra of hydroxy fatty acid ceramide (1). As is the case for all of the spectra studied, the abundance of higher molecular weight fragments in the C.I. spectrum is obvious. The E.I. spectrum has been scale expanded above m/e 80 for ease of comparison of the spectra. The E.I. spectrum does contain a significant amount of structural information. A weak ion is observed for the loss of water from the molecular ion. The strong peak at m/e 325 derives from a loss of OH from m/e 342, arising from cleavage of the bond between C - 2 and C - 3 of the long chain base to produce fragment ion b. The ions at m/e 426,409,398 and 381 probably entail the losses of hydrocarbon fragments. The C.I. spectrum of I (fig. 2) is less ambiguous. Ions at m/e 648, 620, 592 and 564 indicate that I is a mixture containing C24 , C22 , C20, and C18 (major component) hydroxy fatty acid ceramides. The loss of water from the protonated ion is a favoured process in the C.I. spectra of alcohols, but a weak MH+ ion is observed for the major component. One specific site of protonation is not particularly favored as is evidenced by the structurally significant fragments a-e. From the relative intensity of fragment a and the absence of homologous ions 28, 56, and 84 amu higher, it can be determined that the variation in molecular species is due to variation in the hydroxy fatty acid portion of the molecule rather than in the sphingosine base. Although many of the ions in figs. 2 and 3 have the same nominal m/e ratio, the modes of fragmentation leading to them may be profoundly different. Electron impact induced ionization always proceeds via the loss of one electron from an atom with a low ionization potential. Chemical ionization with a proton transfer reagent gas involves the protonation of some site (not necessarily the most basic) followed by rearrangement and/or cleavage to yield the most stable ion. For example, the origins of ions b in figs. 2 and 3 are shown in scheme 1. The appearance of ions at

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Fig. 2. E.I. mass spectrum of underivatized hydroxy fatty acid ceramides, scale expanded above

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220

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Fig. 3. C.I. mass spectrum of underivatized hydroxy fatty acid ceramides.

120

I

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HO" "(CH2)nCH3

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OH

,,, i ,LL,~,,,I,,~.I,,,,L,,.i~hl+..,,..,!L,,..~.1LL,.;.,,~.q.L..,.,.~......'~'.'-,-.v '"h

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S.P. Markey,D.A. ICenger,Massspectra of sphingolipids

El

OH i R_CH= CH_CH.¢ CH_CHiO H _<-,~i ~-NH I C=O ! R'

~

189

OH i R-CH= CH-CH

4CH-CHiOH +NH I C=O

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OH

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+

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i

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R-c.= ~:NH ! C=O 6

R'

Scheme 1.

m/e 30_0 and 282 in both hydroxy and non-hydroxy fatty acid ceramide suggests the rearrangement in scheme 2, similar to that proposed by Kiryushkin et al. for peptides [9]. The net result is a protonated ammonium species, OH

OH

I

I

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R-C H - C H - C H20 H I

H~I~-H

+NH s

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C= O

m/e 300

f R'

MH +

Scheme 2. 2 amu higher than expected from simple cleavage. When I is permethylated (R 1=R2---CH3), the C.I. and E.I. spectra become complimentary. Fig. 4 shows the E.I. spectrum of permethylated 1, which has been normalized above m/e 200 in order to emphasize key fragment ions. Loss of methanol to give an ion at m/e 605, as well as M-CH2OMe (ion f, m/e 592) identify the molecular weight of the principal species. Ions a and e determine the sphingosine base and hydroxy fatty acid lengths, confirmed by ion b. In contrast, the C.I. spectrum

S.P. Markey, D.A. Wenger,Mass spectra of sphingolipids

190 El t013-

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441]

Fig. 4. E.I. mass spectrum of permethylated hydroxy fatty acid ceramides above role 200. of permethylated I in fig. 5 is dominated by the protonated molecular ion and its loss of methanol. The lack of any other specific fragment ions, and the high chemical background due to the derivitization procedure suggest that permethylated ceramides are not particularly useful for C.I. structure determinations, except perhaps for molecular weight determinations. Even for conventional E.I. studies, acetylated or trimethylsilyated ceramides are more likely to remain the derivatives of choice. Mass spectra of acetylated I (R 1=CH 3 CO, R=H) are readily interpreted. The E.I. spectrum in fig. 6 exhibits ions for the sequential losses of acetic acid from the molecular ion, as well as the structural ions b, d, and e. The ion at m/e 264 is probably the long-chain base fragment described by Andersson et al. in the mass spectra of acetylated cerebrosides [10]. The C.I. Spectrum in fig. 7 is dominated by the protonated molecular ion and its loss of acetic acid. Fragment ion b (scheme I) is the most significant ion containing structural information. Upon deuteroacetylation, shifts in MH ÷ and MH ÷ - A c O H peaks are clear indications of the molecular weight and the presence of three hydroxy functions. Fragment ions b and b-AcOH shift six and three amu respectively, revealing the presence of a hydroxy fatty acid. The ease of preparing acetates and the sensitivity of C.I. recommends these derivatives for microscale procedures.

S.P. Markey, D.A. Wenger, Mass spectra of sphingolipids

CI

I0 n-

191

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2,o

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Fig. 5. C.I. massspectrum ofperme~ylated hydroxy fatty acid ceramidesabove m ~ 200.

B. Pyschosine Like underivatized ceramides which yield simple mass spectra, simple glycolipids such as psychosine (II) may not require derivitization for C.I. The E.I. spectra of II revealed pyrolysis products, and charred organic material remained on the probe tip following the run. The same problem was found with C.I., but an interpretable spectrum was produced. A protonated molecular ion (m/e 462, 18%) and an ion for the loss of water (m/e 444, 4-5%) are of surprising intensity considering the residue remaining after analysis. The base peak, ion a at m/e 239 identifies the sphingosine residue, and is complemented by an ion corresponding to a-H20), m/e 221, 30%. Ions at m/e 163 (29%), 145 (33%), and 127 (28%) probably arise from the hexose portion/dehydrated to various extents. While it is possible to obtain the C.I. spectrum of II, the obvious temperature dependence of underivatized II makes it unlikely that C.I. will be used analytically'for underivatized glycolipids. Acetylation of psychosine produced the E.I. and C.I. spectra in figs. 8 and 9. The effect of acetylation in directing ionization is apparent from the absence of type a fragments in either the E.I. or C.I. spectra, an effect seen also with ceramides. Type a and/fragment ions would have the same nominal role 331, but the shift of 12 amu observed upon perdeuteroacetylation confirms that m/e 331 arises from

S.P. Markey, D.A. Wenger,Mass spectra of sphingolipids

192

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El

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Fig. 6. E.I. mass spectrum of acetylated hydroxy fatty acid ceramides above role 250 (role 43, base peak). the hexose residue. Weak type b ions combined with MH + serve to identify the sphingosine base by difference. Type g and h ions serve to confirm the structural assignment. Clearly, the determination of a structure from the E.I. spectrum of acetylated II alone would be difficult, unless it was known that the compound had a molecular weight approximating 700. A molecular ion of 0.015% relative intensity was barely detectable, but Andersson reported a molecular ion of 0.5% [ 10]. Ease of preparation of perdeuteroacetates to clarify the mass spectra makes these attractive derivatives for E.I. or C.I. From the shifts of type b and/' ions, the proximity of the acetylated nitrogen to the hexose is derivable. Type g and b ions which are at even role ratios identify the sphingosine base. The intense MH+-AcOH in the C.I. spectrum is obviously of diagnostic advantage over that seen with E.I. Many of the ions below m/e 160, not shown in figs. 8 or 9, arise from the acetylated hexose residue and are of marginal diagnostic value. Ions at m/e 169, 211, and 217 have been documented for peracetylated monosaccharides [ 16]. Loss of ketene from/'1 is of moderate intensity in the C.I. spectrum (m/e 289, 17%), and is also present in the E.I. spectrum (5%). As with ceramides, a sphingosine base generated ion is present at role 264 in the E.I. spectrum.

S.P. Markey, D.A. lCenger,Massspectra of sphingolipids

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Fig. 7. C.I. Mass spectrum of aeetylated hydroxy fatty acid ceramides above role 250.

C Cerebrosides Underivatized cerebrosides could not be analyzed by either E.I. or C.I. methods only pyrolysis products were observed. E.I. mass spectra of homogeneous cerebroside acetates have been described in the literature, and our results parallel those reported [ 10]. Molecular ions are below 0.1%, and M-59 peaks range from 1-5%. The C.I. spectrum of a homogeneous C18 fatty acid glucosyl ceramide (III) revealed a weak MH ÷ (m/e 938, 3.0%) and a very intense MH+-AcOH ion (m/e 878, 93%) in relation to the base peak], (m/e 331). No type b ions, valuable in the distinction between fatty acid residues and sphingosine variations could be detected. Weak ions of the appropriate m/e for type b were observed, and were found to shift 15 amu on perdeuteroacetylation, identifying them as arising from trace quantities of psychosine impurities. Because the C.I. spectra of peracetylated gluco- and galactocerebrosides are dominated by MH+-60 ions, it is simple to determine the molecular species present by comparison of the spectra produced from the non-labelled and perdeuteroacetylated derivatives. In contrast, E.I. produces weak ions of high m/e (M +, 0.5%; M-60, 2% for C18 galactocerebroside), as well as low intensity smaller fragments which might identify the fatty acid variations. In a naturally obtained mixture, it

194

S.P. Markey, D.A. Wenger,Mass spectra oJ sphingolipids

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Fig. 8. E.I. mass spectrum of acetylated psychosine, normalized above m/e 150. would be most difficult to determine which species are present, even using high resolution E.I. methods. The C.I. spectrum of a mixture of acetylated galactocerebrosides (IV) is shown in fig. 10. The m/e shifts observed upon perdeuteroacetylation are indicated next to the principle MH +-AcOH ions. Analysis of psychosine from this mixture revealed only a single sphingosine base. Thus, these ions correspond to C18 , C18 hydroxy, C24 monounsaturated, C24 hydroxy, and C24 monounsaturated hydroxy fatty acids, which agree well with the principal species known to be present [ 17,18]. Relative quantitation of the fatty acids would be possible using C.I. if several scans were measured and averaged either by manual or computer recording techniques [ 19].

195

S.P. Markey, D.A. lCenger,Mass spectra of sphingolipids

Cl io n

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Acetylated lactosyl ceramides with C18 and C20 fatty acids (V) have molecular weights of 1225 and 1253, respectively• Upon E.I. analysis, these compounds yield spectra similar to those described for acetylated cerebrosides. Fig. 11 is an E.I. spectrum obtained from perdeuteroacetylated V. Weak peaks are present for the loss of deuteroacetate from the molecular species• Peaks at role 640 and 577 represent j 2 and the loss of deuterated acetic acid from this fragment. Shifts due to the deuteroacetylated hexoses are readily apparent

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S.P. Markey, D.A. Wenger, Mass spectra of sphingolipMs C I

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Fig. 12. C.I. mass spectrum of acctylated ceramide dihexosides. when compared to fig. 8, particularly those at role 343 (331 + 12), and 173, 172 (169+4 or 3). The C.I. spectrum of acetylated V in fig. 12 does not contain as many structurally informative ions as the E.I. spectrum. Intense ions are present for the loss of acetic acid from MH +. As with cerebrosides, it is evident that C.I. can be used to determine molecular species present in mixtures. Aside from the type ]1 and ]2 ions, there are no characteristic peaks at low role. Using C.I. and E.I. spectra together, it is possible to elucidate the structures of either pure compounds or mixtures of ceramide dihexosides.

IV. Discussion

Examining the mass spectra produced with this series of increasingly complex sphingolipids, the potential of C.I. combined with E.I. data becomes clear. Chemical

s.P. Markey, D.A. Wenger,Mass spectra of sphingolipids

199

ionization markedly increases the sensitivity of mass spectrometry to detect molecular species or simple losses of neutral molecules (water, methanol, acetic acid) from the protonated molecular ion. The high degree of fragmentation encountered with electron impact induced ionization can be avoided, and high molecular weight, polyfunctional compounds can be studied in the gas phase at temperatures below those used for E.I. analyses. However, C.I. data alone is insufficient in most cases to determine other than the molecular species. Structural subtleties must be derived from a variety of techniques, including E.I. analyses. CT like E.I., will not permit more than primary structure determination of complex molecules. For example, it is not possible to distinguish glucosyl from galactosyl ceramide, or whether ct or/3 configurations of the glycosidic bonds are present. For the analysis of micrograms of mixtures derived from natural sources, chemical ionization affords a significant amount of structural information with minimum effort. The limitations of chemical ionization analyses of complex sphingolipids are not known. Mass spectral resolution was the limiting factor in not pursuing these studies with more complex molecules. Higher molecular weight compounds were run using CT, but the m/e ratios could not be reliably determined. Whereas it is not difficult to obtain a resolution of 5000 with a single focusing mass spectrometer in the E.I. mode, resolution drops remarkedly in the C.I. mode, apparently due to a wider kinetic energy spread of ions leaving the ion source. In these studies, the resolution dropped to 1500 and was further decreased when operating at reduced accelerating voltages. Future efforts will employ C.I. with a double focusing mass spectrometer. Choice of appropriate derivatives for C.I. and E.I. work with high molecular weight compounds is not easily resolved. The simplicity of acetate formation, lack of side products from the reaction, stability of the products, moderate gain in molecular weight when compared to other derivatives, and the diagnostic assistance of perdeuteroacetylation to determine the origins of fragment ions are distinct advantages in the analysis of glycosphingolipids. Trimethylsilyl derivatives, however, offer the additional feature of directing fragmentation upon electron impact to produce fragments characteristic of base and fatty acid portions of cerebrosides [20]. One additional variable in C.I. work is the choice of reagent gas. Methane was chosen for these studies, but Hunt et al. have recommended a variety of reagent gas mixtures for different functional group analyses [21]. It may be possible to produce the desired fragmentations by changing the reagent gas rather than changing from acetates to derivatives which effect greater weight gain.

Acknowledgments This work was supported by NIH grants HD-04870, HD-04024 and MCH Special Project 252.

200

S.P. Markey, D.A. Wenger,Mass spectra of sphingolipids

References [ 1] K. Biemann, in: Biochemical applications of mass spectrometry, ed. by G. Waller. Wileyinterscience, New York (1972) ch. 15 [2] T. Radford and D.C. DeJongh, in: Biochemical applications of mass spectrometry, ed. by G. Waller. Wiley-lnterscience, New York (1972) ch. 12 [3] H.M. Fales, Anal. Chem. 38 (1966) 1058 [4] T. Aczel, Anal. Chem. 40 (1968) 1917 [5] J.T. Watson, in: Biochemical applications of mass spectrometry, ed. by G. Waller. WileyInterscience, New York (1972) ch. 2 [6] C. Hignite and K. Biemann, Org. Mass Spectrom. 2 (1969) 1215 [7] J.R. Plattner and S.P. Markey, Org. Mass Spectrom. (1971) 463 [8] G. Waller et al., in- Biochemical applications of mass spectrometry, ed. by G. Waller. Wiley-Interscience, New York (1972) ch, 3 [9] A.A. Kiryushkin, H.M. Fales, T. Axenrod, E.J. Gilbert and G.W.A. Milne, Org. Mass Spectrom. 5 (1971) 19 [10] B.A. Andersson, K.-A. Karlsson, I. Pascher, B.E. Samuelsson and G.O. Steen, Chem. Phys. Lipids 9 (1972) 89 [11] G. Odham and E. S tenhagen, in: Biochemical applications of mass spectrome try, ed. by G. Waller. Wiley-Interscience, New York (1972) ch. 9 [12] J. Hildebrand, P. Stryckmans and P. Stoffyn, J. Lipid Res. 12 (1971) 361 [13] C.J. Lauter and E.G. Trams, J. Lipid Res. 3 (.1962) 136 [14] S. Hakomori, J. Biochem. 55 (1964) 205 [15] S.P. Markey, R.C. Murphy and D.A. Wenger, Proc. 20th Ann. Conf. on Mass Spectrometry and Allied Topics, Dallas, Texas (1972) [ 16] H. Budzikiewicz, C. Djerassi and D.H. Williams, Structure elucidation of natural products by mass spectrometry, vol. 2. Holden-Day, San Francisco (1964) ch. 27 [17] A.J. Aeher and J.N. Kanfer, J. Lipid Res. 13 (1972) 139 [18] E. Moscatelli, Lipids 7 (1972) 268 [19] B.H. Albrecht, J.R. Plattner, D. Hagerman, S.P. Markey and R.C. Murphy, Proc. 20th Ann. Conf. on Mass Spectrometry and Allied Topics, Dallas, Texas (1972) [20] K.-A. Karlsson, I. Pascher, B.E. Samuelsson and G.O. Steen, Chem. Phys. Lipids 9 (1972) 230 [21] D.F. Hunt, C.N. McEwen and R.A. Upham, Tetrahedron Letters No. 47 (1971) 4539.