Fast-heating mass spectrometry of phosphatidylcholine, lysophosphatidylcholine, phosphatidylethanolamine, and sphingomyelin

ANALYTICALBIOCHEMIBTRY

142,43-52(1984)

Fast-Heating Mass Spectrometry of Phosphatidylcholine, Lysophosphatidylcholine, Phosphatidylethanolamine, and Sphingomyelin DANIEL DESSORT, MARCEL MERSEL, PIERRE LEPAGE,* AND ALAIN VAN D~RSSELAER* Centre de Neurochimie du CNRS and +Laboratoire de Chimie Organique des Substances Natwelles, 5 Rue Blaise Pascal, 67084 Strasbourg Cedex, France Received September 27, 1983 A fast-heating probe and chemical ionization have been used to obtain mass spectra of the synthetic glycerophospholipids 1,2diacyl-sn-glycero-3-phosphocholine, 1,2diacyl-sn-glycero-3phosphoethanolamine, and I-monoacyl-2-lyso-sn-glycero-3-phosphocholine. The phospholipids investigated Save quasimolecular peaks and fragment ions, with preferential cleavage of the CO bond @ position to the phosphorus atom) and loss of phosphoethanolamine or phosphocholine. This technique makes possible the analysis of mixtures of intact phosphatidylethanolamine, phosphatidylcholine, 2+sophosphatidylcholine, and sphingomyelin isolated from natural sources such as egg yolk or brain, Only minor and inexpensive modifications of a standard mass spectrometer are required. 8 1984 .k-kmic b Inc. KEY WORDS: mass spectrometry; fast heating; chemical ionization; selected ion monitoring; natural phospholipids.

The analysis of glycerophospholipids of biological origin involves some difficulties, especially due to the great complexity of the mixtures of molecular species differing only in the nature and position of the fatty acyl chains. This is one of the reasons why it is difficult to achieve separation and characterization of the molecular species by classical methods without chemical or biochemical modifications. A significant advance was achieved using high-performance liquid chromatography, which permitted separation of classes of phospholipids and of some molecular species (1). Low-resolution MS’ (with accuracy from 0.1 to 1 amu) should permit the analysis of a mixture of molecular species based on mass separation as long as volatilization processes

occurring in the ion source of the mass spectrometer do not damage the sample, and lead to spectra simple enough to be interpreted. Since electron-impact MS (2,3) and chemical-ionization MS (4) have shown very early generation of molecular or quasimolecular ions on intact 1,2dioleoyl GPC, one can predict that MS could become a powerful method for investigating phospholipids. Field-desorption MS produces intense quasi-molecular ions from phosphatidylcholine (6,7), and produces reliable mass spectra on egg yolk lecithin (8). Recent reports on fielddesorption MS of natural mixtures of phospholipids (9-l 3) have shown that this technique is now mature for these investigations, especially by producing quasimolecular ions and informative fragment ions at higher emitter temperatures for fatty acid profile studies (1 l), and by being very sensitive (0. l1 kg material per spectrum was required) (11). Secondary-ion MS ( 14) and fast-atom bombardment MS (12,15,16) were also used to investigate natural mixtures. The major disadvantage of these soft-ionization tech-

’ Abbreviations used: MS, mass spectrometty; GPC, glycerophosphocholine; PC, phosphatidylcholine; PSC, phosphatidylsulfocholin~ D/CL, desorption/chemical ionization; PE, phosphatidylethanolamine; LPC, 2-lysophosphatidylethanolamine; SM, sphingomyelin; amu, atomic mass unit; GPE, glycerophosphoethanolamine; EI, electron impact; GC, gas chromatography. 43

0003-2697184

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Copyri@tt Q 1984 by Academic Press, Inc. All ri&s of reproduction in any form rcscwed.

44

DESSORT ET AL.

niques is that the related devices for field- from various biological sources are now roudesorption MS and fast-atom bombardment tinely analyzed at the microgram level in our MS cannot be readily fitted on some mass laboratory using the described method. We spectrometers, and are expensive; in addi- complete here our MS investigation of phostion, these techniques require some experi- pholipids by the analysis of some natural ence (17). mixtures of phosphatidylethanolamine, 2-lyA recent report (18) on ammonia chemicalsophosphatidylcholine, and sphingomyelin. ionization MS of intact PC has shown the For diacylglycerophospholipids (PE and PC), ability of a classical direct-inlet solid probe selected-ion monitoring experiments were to produce quasimolecular ions of this class carried out which permitted semiquantitative of compounds, but the related sensitivity did determination in natural mixtures. not appear very satisfactory since 20 to 50 pg material per run was required (even though MATERIALS AND METHODS this was nearly the sensitivity recently announced for fast-atom bombardment MS Instrumental. All experiments were carried experiments on such compounds). out on a Finn&an 4021 quadrupole mass Flash vaporization (5) and closed-probe spectrometer equipped with the Incas data techniques (19) were proposed to provide system and fitted with the standard unmodsignificant improvements of mass spectra of ified EI/CI ion source. Chemical ionization lipids. We now propose a convenient alter- was carried out using 0.30 Torr ammonia or native to field-desorption MS and fast-atom methane in the ion source as reagent gases. bombardment MS of phospholipids. We have The temperature of the source block was already shown that the fast-heating method maintained at 12O’C. The scanning rate of was able to produce reliable electron-impact the quadrupole during the acquisition process was set to 1 amu/msec. The sample (1 pg in mass spectra on PC (20), and chemicalionization mass spectra on PSC and PC (2 1). solvent) was loaded on the filament of the The fast-heating probe used consisted of a fast-heating probe, using a lo-p1 Hamilton modified desorption/chemical ionization (22) syringe. After evaporating the solvent, the or direct-exposure probe (23,23) in order to probe was inserted into the mass spectromeattain the optimal desorption temperature of ter, so that the filament bearing the sample the sample in less than 1 s; thus, the thermal entered the ion source (“in-beam” configudegradation of fragile molecules is limited. ration). The sample was volatilized by a 500An “in-beam” configuration of the sample mA current in the filament. Desorption of holder was necessary to obtain reliable mass the sample occurs almost immediately. The spectra (20). The spectra produced showed fast-scanning capacity of the quadrupole inquasimolecular peaks and informative frag- strument allowed the recording of several ment ions. The advantages of the method mass spectra. are (i) its relative simplicity, (ii) the possibility Chemicals. Egg yolk LPC (type I), bovine brain SM, egg yolk PE and PC, 1,2dipalof obtaining electron-impact and chemicalGPC, and ionization mass spectra of samples of low mitoyl GPE, 1-oleoyl-Zpalmitoyl volatility, (iii) a sensitivity (1 pg or less of 1-palmitoyl-Zlyso GPC were purchased from material per run) comparable to the best Sigma, St. Louis, Missouri. Trifluoroacetic anhydride was purchased from Janssen reported for field desorption with phospholipids (1 l), and (iv) the low requirement for Chimica, Beerse, Belgium, and silicic acid from Mallinckrodt, St. Louis, Missouri. experience. High sensitivity is very important for routine analyses, because samples from Extraction and pur@ation of rat brain biological origin may often be available only PC. The lipids of adult rat brains were extracted according to Folch et al. (25). The in very small amounts. PSC and PC mixtures

MASS SPECTROMETRY

extract was washed with 20% 0.125 M NaCl, and the organic phase was evaporated to dryness using a rotary evaporator. The lipids were dissolved in chloroform and chromatographed on a silicic acid column as reported by Freysz et al. (26). The fraction containing the choline phospholipids was further submitted to thin-layer chromatography on silica gel G plates (Merck) using chloroform:methanol:27% ammonia (65:35:5), v/v/v). The PC spot was scraped into a column and eluted with 20 ml of the same solvent. The solvent was evaporated, and the PC was dissolved in chlorofornumethanol (9: 1) before mass spectrometric analysis. RESULTS AND DISCUSSION

Using the experimental conditions described above, we have obtained reproducible mass spectra of the related phospholipids and of the corresponding natural mixtures. Although electron impact, with the fast-heating, “in-beam” sample holder, allowed the generation of protonated molecular ions from PC (20), PSC, and PE, we have chosen in most cases to work with the chemical ionization mode because this was found to be more sensitive. Mass Spectrum of Unprotected Synthetic 1,2-Dipalmitoyl GPE Fielddesorption MS has already been used to investigate this substance (8,13), whose

300

400

45

OF PHOSPHOLIPIDS

spectra were mainly characterized by the generation of an intensive protonated molecular ion, followed by a peak resulting from the loss of the phosphoethanolamine portion (m/z 551). For the present study, ammonia and methane were used as reagent gases. The mass spectrum produced with ammonia showed two quasimolecular peaks; a protonated molecular [M + H]+ peak and a [M + NH$ peak. This may complicate mass spectra of natural mixtures; for this reason, we preferred methane as the reagent gas for PE analysis. The methane CI spectrum of 1,2-dipalmitoyl GPE (Fig. 1, M, 69 l), was characterized by the presence of an intense protonated molecular ion (m/z 692), followed by peaks at m/z 586, 569, and 551 corresponding to the loss of the phosphoethanolamine portion [M + H - 141]+. The peak at m/z 569 should correspond to the diacylglycerol moiety. In the electron-impact mass spectrum, characteristic fragment ions containing the carbon backbone of one of the fatty acid chains (symbolized by “R”) can be recognized, assuming similar fragmentation processes as for PC under electron impact conditions (2,3); m/z 239 (RCO), m/z 313 (RCO + 74), m/z 367 (RCO + 128), and m/z 423 (RCO + 184). The peaks at m/z 239 (RCO) and 313 (RCO + 74) were also present in the methane CI spectrum. PE can easily be derivatized before mass spectrometric analysis using tri5uoroacetic anhydride as the N-acyl-

500

600

FIG. 1. Chemical ionization mass spectrum of synthetic 1,2dipalmitoyl used as reagent gas.

760

I D

WC (M, 691). Methane was

46

DESSORT ET AL. 1

FIG. 2. Methane CI mass spectrum (molecular region) of unprotected egg yolk PE.

ating reagent ( 10 min at room temperature). The ammonia chemical ionization spectrum of 1,2dipalmitoyl GPE (N-trifluoroacetylated) was characterized by a strong [M + NH$ peak at m/z 805. This produces a mass shift of 113 amu, assigned to the protonated molecular peak produced with methane on the unprotected sample.

their natural abundance. This has been previously explained by some differences in the stability of the fragment ions in the case of electron-impact experiments on PC (2). A classical GC/MS assay of the fatty acid methyl esters is probably the best method for determining accurate qualitative and quantitative fatty acid profiles of phospholipids. A disadvantage of the method is that no information

Analysis of Egg Yolk PE The analysis of PE from soybean oil was recently performed using fielddesorption MS (11) and fast-atom bombardment MS (16). Figure 2 shows the molecular ion region of the methane chemical-ionization mass spectrum which we have obtained with underivatized egg yolk PE. The generation of intense protonated molecular ions allows the differentiation between the 13 major molecular species (listed in Table 1) readily determined in this mixture. These data are in agreement with those reported by Holub et al. (27). In the ammonia CI mass spectrum of the Ntrifluoroacetylated sample, the molecular species profile was not significantly modified. Consequently, it seems unnecessary to derivatize the sample, but this procedure may help to enhance spectra of contaminated samples (i.e., containing triglycerides). As for synthetic compounds, electron impact was used to determine the fatty acid profile of the mixture, but we found that the intensity of the peaks for the fatty acids did not reflect

TABLE I ANALYSISOFEGG YOLK PHO~PHATIDYLETHANOLAMINE

[M + H]+

Molecular species

Relative amounts @)

714 716 718

c34:3 C34:2 c34: I

2.29 12.33 18.22

740 742 744 746

C36:4 C36:3 C36:2 C36: 1

4.09 6.56 18.01 14.89

764 766 768

C38.6 C38:5 C38:4

1.54 5.03 13.52

792 794 796

Cti6 C405 c4Oz4

0.85 1.44 1.23

Note. Methane was wed as reagent gas. Molecular species were determined using the spectrum of Fig. 2, and relative amounts were calculated after integrating the selected ion monitoring traces from Fig. 3.

MASS SPECTROMETRY

OF PHOSPHOLIPIDS

47

process of the signal, more accurate area measurements may be computed, thus giving more accuracy and better signal-to-noise ratios than single mass peak-height measurements. Mass Spectrum of I-Oleoyl2-Palmitoyl GPC PC is probably the class of phospholipids most recently investigated by MS. Electron impact MS (2,3,20,28), chemical-ionization MS (4,18,2 1,28), fielddesorption MS (6MS (11, FIG. 3. Plot of the SIM traces of intact egg yolk PE. 8,l I- 13), fast atom-bombardment Detection of [M + H]+ species. Methane was used as 12,14- 16) and secondary-ion MS (14) were reagent gas. Desorption of the sample occurs almost used to investigate synthetic compounds. immediately after switching the heating current in the The CI mass spectrum of I-oleoyl-2-palfilament at zero time (x axis); duration of the signal, 2 mitoyl GPC (Fig. 4, M, 759) shows a protons; y axis, ion current; 2 axis, m/z values. ated molecular peak at m/z 760, followed by a strong peak at m/z 7 18 [M + H - 42]+, on the fatty acid position may be deduced which may correspond to the generation of the corresponding protonated PE by ammofrom the EI or CI spectra. We have attempted a semiquantitative nia substitution (21). Results from the invesanalysis of the molecular species of egg yolk tigation of deuterated PC and the use of [“Nlammonia CI have recently shown that PE by means of selected ions monitoring. The use of this well-known technique, cur- this peak results from the loss of the trimethgroup and the addition of rently used in GC/MS for quantitative mea- ylammonium surements, allows the enhancement of detec- NH: from the reagent gas, and the peak at m/z 6 12 was interpreted as the ammonium tion sensitivity and a greater reproductibility adduct of diacylglycerol (18). Loss of the of measurement. Figure 3 shows the selectedion monitoring experiment carried out using phosphocholine portion is indicated by the 1 pg unprotected sample and monitoring the peak at m/z 577 [M + H - 183]+. A peak protonated molecular ions. Each peak being should be noted at m/z 8 17 [M + 58]+ and is presumably due to ion-molecule reactions defined by several data during the acquisition 1OOd %

710

6l2

0 mfz

400

000

000

700

FIG. 4. Ammonia CI mass spectrum of synthetic I-oleoyl-2-palmitoyl

000

GPC (MW 759).

DESSORT ET AL. 766.

650

m/L

FIG.

700

760

l

600

prbtonated

molecular

ion

660

900

5. Ammonia CI mass spectrum (molecular region) of rat brain PC.

(proposed structure: [M + CH2 =N(CH&]‘) (21). This peak was encountered in all PC we have studied up to now under fast-heating conditions (electron-impact or chemical-ionization modes). Determining the origin and structure of various ions in the mass spectra of PC requires the standard tools of MS (labeling, tandem MS, MIKE, and high res-

olution), but some of them cannot achieved by a quadrupole instrument. Analysis of Natural Mixtures

be

of PC

Natural mixtures of PC have been previously investigated by field desorption of PC from egg yolk (8,11,12), myelin (13), and soybean oil (11,12), and by fast atom-bom-

TABLE 2 ANALYSIS•

FRATBRAINANDEGGYOLK

PHOSPHATIDYLCHOLINE

Relative amounts (96) ml2

values

Egg yolk rat brain (4

(a)

(b)

(4

690 692

3.30 28.10

N.D. N.D.

N.D. N.D.

N.D. N.D.

813 815 817 819

714 716 718 720

N.D. b 1.00 41.90 11.50

N.D. 22.20 45.80 N.D.

N.D. 25.90 42.40 N.D.

N.D. 16.60 44.40 N.D.

782 784 786 788

839 841 843 845

740 742 744 746

N.D. N.D. 1.50 11.40

2.70 3.00 10.20 12.30

N.D. 2.70 15.30 9.50

3.10 1.80 8.60 19.70

806 810

863 867

764 768

N.D. 1.30

N.D. 3.90

N.D. 4.20

N.D. 4.90

Molecular species

MH+

M + 58

C32: 1 C32:O

732 134

789” 791

c343 C34:2 C34:l c34:o

756 758 760 762

C36:4 C36:3 C36:2 C36:l C38:4 C38:2

MH-42

Note. Ammonia was used as reagent gas. Molecular and relative amounts were determined using the SIM was made using different techniques: (a) described in (2%. 0 This peak is not distinguishable from the natural C36: 1). b N.D., not determined.

species were determined using the mass spectra of mixtures, traces of Fii. 6. Comparison of data obtained for egg yolk PC this article, (b) FDMS measurement (1 I), (c) GLC/MS assay isotopic contribution peak from m/r 788 (M + H peak from

MASS SPECTROMETRY

bardment MS on egg yolk PC (11,12,15,16). We have encountered relatively few difficulties in the analysis of the mixtures of PC from biological origins. The ammonia chemical-ionization mass spectra of rat brain (Fig. 5) and egg yolk PC can easily be interpreted because of the simultaneous presence of [M + HI+, [M + 58]+ (odd mass number), and [M + H - 42]+ peaks. Table 2 lists the molecular species found. The fatty acid profile was determined using methane as reagent gas, and was based upon the presence of (RCO + 74) peaks. The following major fatty acids were readily detected in rat brain and egg yolk PC: palmitoleoyl (m/z 3 1 I), palmitoy1 (m/z 313), linoleoyl (m/z 337), oleoyl (m/z 339), and stearoyl (m/z 341). The analysis by selected-ion monitoring of both mixtures was carried out using 1 pg sample per run (Fig. 6). The relative abundances of molecular species are reported in Table 2,

EGG

YOLK

RAT

OF PHOSPHOLIPIDS

49

and are in agreement with the data previously found under field desorption conditions (11) or CC/MS (29). Although the duration of the signal we report here did not exceed 4 s, it was possible to increase the observation time up to lo- 12 s by lowering the preset heating current. With increased observation times, lower relative intensities of quasimolecular peaks were observed, and the sensitivity was obviously decreased. It may be noted that some relatively minor molecular species (1% of total amount, about 15 pmol) were detected in mixtures with a good signal/noise ratio.

Ammonia Chemical Ionization Mass Spectrum of I-Palmitoyl-2-lyso GPC In the FD mass spectra of synthetic LPC, protonated molecular peaks and ions above [M + H]+ such as [M + Na]+, [M + K]+, [M + choline]+, and [M + H2P04-CH2CH2-

BRAIN

FIG. 6. Plot of SIM traces of egg yolk PC and rat brain PC (detection of protonated molecular ions). Ammonia was used as reagent gas. Duration of the signal, 3.6 s after switching the heating current in the filament at zero time (x axis); y axis, ion current; 2 axis, m/z values.

50

DESSORT ET AL.

[M+ZH-C”,]”

m,z

482

HO c

O&SCHpC.:(”

InlL

484

100 x

!%I

0

FK. 7. Ammonia CI mass spectrum and propsxed structure of some fragment ions of synthetic I-palmitoyl-2-lyso GPC (MW 495).

N(CH&]+ were reported (8). The ammonia for PC), m/z 692 (M + 197), and m/z 734 CI mass spectrum we have obtained (Fig. 7) (M + 239). This latter peak may suggest conshows a protonated molecular peak at m/z tamination of the sample by dipalmitoyl PC 496 and some fragment ions, the structures (Mr 733) or generation of this compound by of which may be compared to those described ion-molecule reactions occurring on the sup in the ammonia CI mass spectra of l-Oport during the fast-heating process or in hexadecyl GPC reported by Polonsky et al. the gas phase after volatilization of the sample. (30). The peak at m/z 348 may correspond to the ammonium adduct of the acylglycerol Ammonia Chemical Ionization Mass moiety. Spectrum of Egg Yolk LPC Other unexpected peaks above [M + H]+ were found when larger amounts of sample The molecular region of the ammonia CI were used (i.e. 5 pg): m/z 553 (M + 58, as mass spectrum is shown in Fig. 8. The major ‘P

50

0 mrz FIG.

450

500

550

8. Ammonia CI mass spectrum (m/z 400 to m/z 600) of egg yolk LFC.

MASS SPECTROMETRY

51

OF PHOSPHOLIPIDS

TABLE 3 ANALYSIS

Molecular species

MH+

OF EGG YOLK

MH-14

LYSOPHOSPHATIDYLCHOLINE

MH-32

MH-42

MH-72

MH-86

Cl6:l

494

480

C16:O

496

482

464

452 454

422 424

410

Cl&l C18:O

522 524

508 510

490 492

480 482

450 452

436 438

Note. Ammonia was used as reagent gas. Molecular species were determined using the spectrum of Fig. 8.

molecular species we found for these mixtures are: palmitoyl LPC, palmitoleoyl LPC, stearoyl LPC, and oleoyl LPC. The simultaneous presence of the protonated molecular peaks and fragment ions allows the identification of these species (Table 3). An unexpected peak was found at m/z 553, which was considered to result from ion-molecule reactions [M(C16:0) + 581. This peak at m/z 553 was previously found in fast atom-bombardment experiments on mixtures of LPC (15), and it was interpreted by interaction with the glycerol sample matrix. Ammonia CZ Mass Spectrum of Bovine Brain Sphingomyelin SM from bovine erythrocytes has already been successllly analyzed by fielddesorption MS and fast atom-bombardment MS (11). The ammonia CI mass spectrum we have

FIG.

obtained from bovine brain SM (Fig. 9) shows two main molecular species, which were recognized by the presence of their protonated molecular peak. They are stearoyl SM (C18:O SM, M, 730) and nervonyl SM (C24: 1 SM, M, 812). The loss of the phosphorylcholine portion was indicated by peaks at mass [M + H - 183]+: m/z 548 (731183) and m/z 630 (813-183). As for LPC, the investigation of fatty acid profiles is not required for SM, since the molecular species of these classes of phospholipids differ only by the nature of the single branched fatty acid chain per molecular species; the values of the molecular weight distribution of these phospholipids allow the unambiguous characterization of the fatty acids. Consequently, their accurate quantitative analysis can be achieved using CC/MS analysis of the corresponding fatty acid derivatives, but the method allows purity control of these compounds.

9. Ammonia CI mass spectrum of bovine brain SM.

52

DESSORT ET AL. ‘.

Some major disadvantages reported by authors for field&sorption MS and fast atombombardment MS investigations of phospholipids were not encountered for the present fast-heating experiments. For example, the analysis of PC by field desorption needed very careful cleanup of the sample to avoid cationization phenomena (8). Fast atombombardment MS does not appear to be a method of choice for studying natural mixtures of phospholipids, as [M + H - 21 ions are produced, thus leading to possible confusions with unsaturated species, as has been reported by Fenwick et af. (16). ACKNOWLEDGMENTS We are grateful to Professor P. Mandel and Professor G. Ourisson for helpful discussions and reading the manuscript.

11. Lehmann, W. D., and Kessler, M. (1983) Chem. Phys. Lipids 32, 123-135. 12. Catlow, D. A. (1983) In?. J. Mass Spectrom. fan. Phys. 46, 387-390. 13. Sugatani, J., Kino, M., Saito, K., Matsuo, T., Matsuda, M., and Katakuse, I. (1982) Biomed. Mass Spectrom. 9, 293-301. 14. Aberth, W., Straub, K. M., and Burlingame, A. L. (1982) Anal. Chem. 54, 2029-2034. 15. May, H. E., and Desiderio, D. M. (1983) J. Chem. Sot. Chem. Commun. 2,72-73. 16. Fenwick, G. R., Eagles, J., and Self, R. (1983) Biomed. Mass Spectrom. 10, 382-386. 17. Martin, S. A., Costello, C. E., and Biemann, K. (1982) Anal. Chem. 54,2362-2368. 18. Crawford, C. G., and Plattner, R. D. ( 1983) J. Lipid Res. 24, 456-460. 19. Klein, R. A. (1978) Chem. Phys. Lipids 21, 29l312. 20. Dessert, D., Van Dorsselaer, A., Tian, S. J., and Vincendon, G. ( 1982) Tetrahedron Lett. 23, I3951398. 21. Dessert, D., B&ret, P., Nakatani, Y., Ourisson, G., and Kates, M. (1983) Chem. Phys. Lipids 33, 323-330.

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