Energy & Fuels 1990,4, 737-741
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Fast Atom Bombardment Mass Spectrometric and Tandem Mass Spectrometric Studies of Some Functionalized Tetrapyrroles Derived from Chlorophylls a and b B. J. Keely and J. R. Maxwell* Organic Geochemistry Unit, School of Chemistry, University of Bristol, Cantock's Close, Bristol BS8 lTS,U.K. Received June 19,1990. Revised Manuscript Received October 1, 1990
Fast atom bombardment (FAB) mass spectra of a number of functionalized tetrapyrroles derived from chlorophylls a and b have been obtained and MS/MS studies used to investigate aspects of the fragmentation behavior of some of them. The application of this approach is demonstrated by the assignment of pheophytin b isolated from a lake sediment where the amount and purity were insufficient for 'H nuclear magnetic resonance spectroscopic studies.
Introduction Chlorophyllsand functionalized chlorins are the putative precursors and intermediates, respectively, on the degradative pathway to sedimentary porphyrins.' Although many sedimentary porphyrin structures have been determined through the concerted use of electron ionization mass spectrometry (EIMS) and 'H nuclear magnetic resonance (NMR) spectroscopy,2 it is only recently that sedimentary chlorins have been characterized in a similar manner.*s Where it is not possible to isolate components in sufficient quantity or purity for characterization by NMR studies, assignment has to rely on high-performance liquid chromatography (HPLC) coelution and MS comparison with standards. E1 spectra can be obtained for pheophorbide~?~ but the more functionalized components, chlorophylls and pheophytins, often do not yield significant molecular ion information as a result of a variety of thermal transformations induced by the high probe and source temperatures required.6*8Fast atom bombardment mass spectrometry (FABMS) has previously been demonstrated to yield molecular ion information from chlorophyllsg10 and has also assisted in the assignment of two sedimentary chlorins derived from chlorophyll a.11J2 Furthermore, FABMS will allow recognition of allomeric (oxidative) degradation products (single or double oxygen incorporation in ring E), which may be present as im(1) Treibs, A. Angew. Chem. 1936,49, 682-686. (2) Chicarelli, M. I.; Kaur, S.; Maxwell, J. R. In Metal Complexes in Fossil Fuels: ACS Svmuosium Series 344: American Chemical Societv: Washington; DC, lg8187;'pp40-67. (3) Keely, B. J.; Maxwell, J. R. Org. Geochem. Submitted for publication. (4) Keely, B. J.; Maxwell, J. R. J. Chem. Soc., Perkin T r a m . 1. Submitted for publication. ( 5 ) Keely, B. J.; Maxwell, J. R. Energy Fuels, in this issue. (6) Budzikiewicz, H. In The Porphyrins; Dolphin, D., Ed.; Academic Press: New York, 1978; pp 395-461. (7) Smith, K. M. In Porphyrins and Metalloporphyrins; Smith, K. M., Ed.; Elsevier: Amsterdam, 1975; pp 381-398. (8) Bazeaz, M. B.; Bradley, C. V.; Brereton, R. G. Tetrahedron Lett. 1982.23. 1211-1214. .~ .~~ (9) Barber, M:; Bordoli, R.; Elliott, G. J.; Sedgewick, R. D.; Tyler, A. Anal. Chem. . . - . 1982.54.645A-657A. ..- -, .., . . .. .. .. (10) Brereton, R. G.; Bazzaz, M. B.; Santikarn, S.;Williams, D. H. Tetrahedron Lett. 1983,24, 5775-5778. (11) Keely, B. J.; Brereton, R. G.; Maxwell, J. R. In Adoances in Organic Geochemistry 1987; Mattavell i, L.,Novelli, L.;Eds.; Pergamon Press: Oxford, 1988; 0,rg. Geochem., 1988, 13,801-805. (12) Keely, B. J. Ph.D. Ther,is, University of Bristol, 1989. ~
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purities in standards prepared from ~hlorophylls,'~ and have been suggested as possible intermediates in the formation of sedimentary etioporphyrin~.'~Accordingly, we have examined the FAB spectra of a number of chlorin standards (1-4 in Figure 1 ), whose counterparts occur, or might be expected to occur, in sediments. Although it has been recognized that extensive fragmentation is often a feature of FAB spectra, including (13) Hynninen, P. H.
J. Chromatogr. 1979,175, 75-88. (14) Baker, E. W.; Louda, J. W. In Aduances in Organic Geochemistry 1981; Bjoray, et al., Ed.; Wiley: Chichester, 1983; pp 401-421. 0 - 1990 American
Chemical Societv
738 Energy & Fuels, Vol. 4, No. 6, 1990
Keely and Maxwell
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those of chlorophyll u9 and pheophytin~,'~ the understanding of these fragmentation processes is limited. We describe here the use of MS/MS techniques to investigate aspects of the fragmentation behavior of a number of the above standards (1, 2a, 2b, 3a, and 4a in Figure 1) and their application to the recognition of three chlorins in a Recent lake sediment.
Experimental Section Chlorophyll transformation products were prepared from chlorophylls a and b isolated from spinach, using previously described meth~ds.'~J' The components were purified by use of reversed-phase HPLC, after which purity and compound identifications were established by use of analytical HPLC and 'H NMR spectroscopy.12 Mass spectra were acquired on a Finnigan TSQ 70 instrument. Concentrated acetone solutions of individual components (2150pmol) were mixed with the matrix, which had previously been applied to the probe tip. The matrix used was either 2,2'-thiodiethanol [bis(2-hydroxyethyl)sulfide] or 3-nitrobenzyl alcohol (NBA). Samples were bombarded, at an incident angle of 45", with a xenon atom beam provided by an Ion Tech FAB gun operating at 8 keV with a current of 1mA. MS/MS experiments were performed using argon (1.3 mTorr) for collision activation and with a potential of 45 eV applied to
the collision cell. E1 mass spectra were recorded at 70 eV (350 mA) using a direct insertion probe heated ballistically from am-
(15) Grese, R. p.; Cerny, R. L.; Gross, M. L.; Senge, M. J. Am. SOC. Mass. Spectrom. 1990,1, 72-84. (16) Furhop, J.-H.; Smith, K. M. In Porphyrins and Metalloporphyrins; Smith, K. M., Ed.; Elsevier Scientific: New York, 1975; pp
757-869.
(17) Pennington, F. C.; Strain, H. H.; Svec, W. A,; Katz, J. J. J. Am. Chem. SOC.1964,86, 1418-1426.
bient temperature to 360 "C. Spectra shown were produced by summation of a number of scans without background subtraction.
Results and Discussion In the cases (la-c, 2a-c, 3b, and 4b) where FAB mass spectra were acquired with both thiodiethanol and NBA used as matrices, the spectra generated by using thiodiethanol showed, with the exception of la, a predominance of the protonated molecule ([M + HI+) over the molecular ion (M+),as is often the case in spectra generated by using FAB ionization. In contrast, spectra obtained with NBA showed M'+ as the dominant species in the molecular ion region. A dominance of Me+ in FAB spectra of chlorophylls and pheophytins obtained from NBA was recently reportedI5 and was attributed to a lower acidity of NBA than for matrices in which [M + H]+ dominates. In the present study, more intense spectra, particularly of the pheophytins (lb-4b, IC, %e),were observed using NBA rather than thiodiethanol as matrix. This feature appears to be largely the result of solubility differences in the matrix, since it was clearly observed when spectra were obtained from each matrix using the same amount of pheophytin u (lb). Interpretation of the FAB spectra in terms of MS/MS fragmentation is complicated, firstly, because of the difference in the nature of the resultant spectra (the MS/MS spectra being collisionally induced) and, secondly, because of differences in the stabilities of the two principal molecular ion species ([M + H]+ being an even-electron species and Me+being an odd-electron species). Finally, consideration must also be given to the contribution of the 13C-containingM'+ at m/z corresponding to the [M + HI+.
Energy & Fuels, Vol. 4, No. 6, 1990 739
FABMS and MSIMS of Tetrapyrroles a
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Figure 3. Pheophytin a (lb) FAB mass spectra (nitrobenzyl alcohol matrix): (a) partial spectrum; (b) neutral loss of 278; (c) daughter spectrum of m / t 592 (odd-electron fragment ion); (d) daughter spectrum of m / z 593 (even-electron fragment ion).
For these reasons we have made a direct comparison of E1 and FAB spectra using pheophorbides (la-4a),which are amenable to both ionization techniques. As an example, FAB ionization of pyropheophorbide a methyl ester (2a; Figure 2a) gave rise to all of the principal fragment ions observed in the E1 spectrum (Figure 2b), but having different relative abundances. In addition, the FAB spectrum reveals, more readily than does the E1 spectrum, the presence of a minor impurity consistent with a single oxygen allomer (Me+= m/z 564). The principal EI-derived fragment ions of pyropheophorbide a methyl ester have been rationalized previously6 as arising from @-cleavageof the C-17 substituent ( m / z 461) with further expulsion of CO giving rise to m / z 433. As a result of stability differences between M'+ and [M + HI+, the absolute intensities of fragment ions in the two daughter ion FAB spectra (Figure 2c,d) do not directly relate to the relative contributions of fragment ions seen in the FAB spectrum (Figure 2a). A better indication of the contribution from each of the molecular ion species probably comes from a comparison of the relative intensities of the various fragment ions within each of the daughter ion spectra. Thus, the ions a t m/z 461 and 433 are both observed to arise mainly from the molecular ion (M*+;Figure 2c) and can be rationalized on the same basis as in the E1 spectrum. The ions a t m / z 447 and 419 arise predominantly from the protonated molecule ([M + HI+; Figure 2d). Although their origins are not clear, and determination of the fragmentations giving rise to these ions would require detailed mass spectral studies, they are consistent with loss of the substituents a t both C-17 and C-18 to give mlz 447 and with m / z 419 arising from additional loss of CO. Among the even-electron fragment ions derived from m J z 549 in Figure 2d the ion at m f z 462, being odd electron, appears
to arise from the 13C-containingM'+ (Le., is analogous to m / z 461 derived from the 12C-containingM + in Figure 2c). The ion at m / z 521 (Figure 2d) is consistent with loss of CO from [M HI+. Like pyropheophorbide a methyl ester (2a)the major fragment ions in the E1 mass spectrum6 of pheophorbide a methyl ester (la)also occur in the FAB spectrum (not shown). In the FAB spectrum loss of the carbomethoxy substituent to give m/z 547 from both M'+ (m/z 606) and [M H]+ are major fragmentation process, as indicated by MS/MS daughter ion spectra. These fragmentations are discussed below in relation to the pheophytins. Other fragment ions (mlz 403,417,431,445,459, and 519) occur at 2 daltons (Da) lower than in the FAB spectrum of pyropheophorbide a methyl ester (2a). These ions can be rationalized in a formal sense as resulting from fragmentations similar to those observed for pyropheophorbide a methyl ester (2a)but with additional loss of the carbomethoxy substituent. The FAB spectra (like the E1 spectra) of the mesopheophorbide a methyl esters (ðyl components, i.e., 3a, 4a) show the same principal fragmentation losses as their pheophorbide counterparts la and 2a,resulting in virtually identical spectra, but shifted to 2 Da higher. It is apparent, therefore, that the major fragmentations observed in components la-4a do not include losses involving the C-3 (vinyl or ethyl) substituent. The pheophytins lb-4b yield FAB spectra that exhibit a number of ions in common with their pheophorbide counterparts (la-4a). For example, a number of ions in the range mlz 400-460 in the spectrum of pheophytin a (lb;Figure 3a) are also observed in the spectrum of pheophorbide a (la;see above). A major fragmentation of'the pheophytins, which have phytol as the esterifying alcohol, is loss of 278 Da. This loss was demonstrated, by
+
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740 Energy & Fuels, Vol. 4, No. 6,1990
Keely and Maxwell
daughter ion MS/MS and by neutral loss scanning (Figure 3b), to arise from both Me'+ to give m/z 592 and [M + H]+ to give m/z 593 and corresponds to loss of the phytyl chain (as phytadiene) with back-transfer of a proton to the charge-retaining fragment. Thus, components that pcwsess a phytyl chain may be identified. Previously,"J2 we were unable to determine whether the phytyl chain of pheophytins and chlorophylls was lost from the protonated molecule with back-transfer of a proton ([M H]+ - 278) or from the molecular ion through a double hydrogen rearrangementla (Me+- 277). From the results shown here, it is apparent that this loss occurs predominantly through a rearrangement process involving loss of 278 Da from both Me+and [M + HI+. Fragment ions corresponding to loss of the phytyl chain as a radical to give m/z 591 (M'+ - 279) and to loss involving double hydrogen rearrangement? to give m / z 593 (M'+ - 277) were also observed in MS/MS daughter spectra, and the latter case was identified as a minor process by neutral loss scanning. In the case of l b and 3b loss of the C-132carbomethoxy occurs from oddelectron ions as a fragmentation process to give, for example, m/z 533 (m/z 592 - 'C02Me) in Figure 3c and from even-electron ions as a rearrangement involving abstraction of an additional proton to give, for example, m/z 533 (m/z 593 - [C02Me+ HI) in Figure 3d. Similarly, loss of the whole C-17 substituent with additional loss of the C-132 carbomethoxy substituent gives rise to m / z 459 (Figure 3c) and m / z 460 (Figure 3d) through fragmentation and rearrangement processes, respectively. The same ions, resulting from losses of both the C-17 and C-132substituents, were also observed for pheophorbide a methyl ester (la), which contains a carbomethoxy substituent a t the latter position. Pyro components (2a,b; 4a,b),which lack the C-132carbomethoxy substituent, cannot undergo this loss, and consequently they contain fragment ions a t 2 mass units higher than their "parent" compounds (la,b; 3a,b). In addition, meso components (3a,b;4a,b) show the same losses as their C-3 vinyl counterparts (la,b; 2a,b) resulting in virtually identical spectra, but shifted to 2 Da higher. A very recent report15 of the FAB spectra of chlorophylls and pheophytins identified charge-remote fragmentation within the phytyl chain. Under the conditions used here, we have not observed this phenomenon. In summary, the FAB mass spectra of pheophytins and pheophorbides examined above contain structural information that can be revealed by the use of MS/MS techniques. An example of the application of this approach is given as follows. Previously, we identified pheophytin a (lb) and pyropheophytins a and b (2b,c) in a Recent lake sediment, on the basis of comparison of their FAB and 'H NMR spectra with those of standards." Subsequently, we fully characterized the more abundant of these components [pheophytin a (lb) and pyropheophytin a (2b)] using NMR spectroscopic techniques (COSYand NOE).3 Because of the low amounts of material, it was not possible to purify pyropheophytin b (2c) and pheophytin b (lc),which were also isolated,12in sufficient amounts for an analogous level of characterization by 'H NMR. In particular, the assignment of pheophytin b (IC) relied only on HPLC coelution with a standard19 and the recognition of a weak molecular ion in a FAB spectrum of poor quality because of the low amount of material available and the presence
+
(18) McLafferty, F. W. Interpretation of Mass Spectra; University Science Books: Mill Valley, CA, 1980. (19) Keely, B. J.; Brereton, R. G.In Advances in Organic Geochemistry 1985; Leythaeuser,D., Rullkbtter,J., Eds.;Pergamon Prese: Oxford. 1986; Org. Geochem. 1986, IO, 975-980.
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Figure 4. Pheophytin b (IC) FAB mass spectra (nitrobenzyl alcohol matrix): (a) impure sedimentary component; (b) MS/MS daughter spectrum of m/z 884 (M'+) of sedimentary component; ( c ) MS/MS daughter spectrum of m / t 884 (M*+)of standard.
of impurities (Figure 4a).12 In the daughter spectrum of the molecular ion (Figure 4b) characteristic fragment ions that are present allow confirmation of the tentative assignment. Hence, m/z 606 and 605 ( M +- 278, M + - 279) result from loss of the phytyl chain. The ions a t m / z 547 and 545 result from additional loss of the C-132 carbomethoxy substituent (by way of fragmentation and fragmentation with rearrangement respectively). Similarly, loss of the whole (2-17 substituent from M'+ occurs to give m/z 533, and m/z 473 corresponds to loss of both the C-17 and C-13*substituents (cf. lb). Furthermore, the daughter spectrum of the molecular ion (Figure 4b) compares favorably with that of the standard (Figure 4c) and allows further confirmation of the assignment even though the sedimentary component is relatively impure.
Conclusions Although the presence of both Me+and [M + HI+ complicate the spectra of the pheophorbides and pheophytins examined, the spectra contain useful structural information especially when used in conjunction with MS/MS techniques. Tandem mass spectrometry shows that both M'+ and [M + H]+ undergo fragmentations that appear to obey the even-electron rule. Hence, the protonated molecule was only observed to undergo fragmentations involving
Energy & Fuels 1990, 4, 741-747
rearrangement processes. FABMS and especially FABMS/MS offer a means of structural assignment of sedimentary chlorins when amounts or purities are too low for characterization using 'H NMR spectroscopic techniques. Furthermore, since a number of ions are common to the spectra of all of the components examined, possibilities exist for selecting the components from relatively crude mixtures by using specific fragment ions (e.g., parent ions of m / z 433). In particular, interpretation of the FABMS fragmentation processes through the use of MS/MS provides a basis for understanding the fragmen-
741
tation behavior of these components under LC/MS conditiom20 Acknowledgment is made to the donors of the Petroleum Research Fund, administered by the American Chemical Society, for partial support of this research. We also thank the Natural Environment Research Council for MS facilities (GR3/2951 and GR3/3758) and Mr. J. F. Carter for technical assistance. (20) Eckardt, C. B.; Carter, J. F.; Maxwell J. R. Energy Fuels, in this issue.
Combined Liquid Chromatography/Mass Spectrometry of Tetrapyrroles of Sedimentary Significance C. B. Eckardt,* J. F. Carter, and J. R. Maxwell Organic Geochemistry Unit, School of Chemistry, University of Bristol, Cantock's Close, Bristol BS8 1TS, U.K. Received April 27, 1990. Revised Manuscript Received J u n e 13, 1990
The on-line coupling of a high-performance liquid chromatograph to a mass spectrometer (LC/MS) has allowed the analysis of a number of tetrapyrrole pigments of sedimentary significance. Mass spectra were obtained under conditions of discharge ionization and are characterized by the presence of protonated ion species (positive-ion mode) or molecular ions (negative-ion mode), respectively. Fragmentation processes were found to be restricted to the periphery of the structures. In the positive-ion mode the extent of fragmentation could be manipulated by alteration of the repeller voltage in the ion source, allowing molecular weight information to be obtained, as weli as specific fragmentation patterns. These findings have suggested a number of applications of LC/MS in the investigation of sedimentary tetrapyrrole distributions. In particular, the assignment of individual components in mixtures without resort to their isolation is possible from comparison of mass spectra with those of authentic standards and from coinjection with the standards.
Introduction The investigation of sedimentary tetrapyrrole distributions can yield information relating to the assessment of biological input, depositional paleoenvironment, and thermal maturity of sedimentary organic matter.' Among other techniques, high-performance liquid chromatography (HPLC) of porphyrin free bases, and of chlorophylls and their derived intermediates on the degradative pathway to the porphyrins, has proved to be a rapid and reliable method for separation of complex mixtures with reproducibly good resolution.24 Typically, the analysis provides a UV/visible-monitored chromatogram, allowing assignments from relative retention time comparisons and coinjections with authentic standards. The method has also been applied extensively to the isolation of individual components for full structure determination, for example, by 'H NMR experiment^.^ (1) Baker, E. W.; Louda, J . W. In Biological Markers in the Sedimentary Record; Johns, R. B., Ed.; Elsevier: Amsterdam: 1986, pp 125-155. (2) Barwise, A. J. G.; Evershed, R. P.; Wolff, G. A.; Eglinton, G.; Maxwell, J. R.J . Chromatogr. 1986, 368, 1-9. (3) Chicarelli, M. I.; Wolff, G. A.; Maxwell, J. R. J. Chromatogr. 1986, 368,ll-19. (4) Keely, B.J. Ph.D. Thesis, University of Bristol, UK, 1989. (5) Chicarelli, M. I.; Maxwell, J. R. Trends Anal. Chem. 1987, 6, 158-164, and references therein.
The mass spectrometric detection of tetrapyrroles, performed via direct insertion of a mixture or a single compound, is frequently applied using electron impact (EI/MS),6v7reactant gas plasma (CI/MS),g'o or fast atom bombardment (FAB/MS)4JJ1ionization. Under EI/MS or FAB/MS conditions, the mass spectra are generally characterized by abundant molecular or pseudomolecular ions, respectively, and fragmentation appears to occur mainly within the substituents on the macrocycle and to depend on the degree of functionalization. More extensive structural information, based on cleavage of the macrocycle at the meso positions, can be obtained using chemical ionization (DCI/MS) conditions with certain reactant gases such as a m m ~ n i a . ~ J ~ J ~ The use of chromatographic separation directly prior to mass spectrometric analysis in the investigation of complex ~~
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( 6 ) Baker, E. W. J . Am. Chem. Soc.1966, i%, 2311-2315. (7)Castro, A. J.; van Berkel, G. J.; Doolittle, F. G.; Filby, R. H. Org. Geochem. 1989, 14, 193-202. (8) Shaw, G. J.; Edinton. G.; Quirke, J. M. E. Anal. Chem. 1981.53, 2014-2020. (9) Tolf, B. R.;Xiang Yu-Yiang;Wegmann-Szente, A.; Kehres, L. A.; Bunnenberg, E.; Djerassi, C. J. Am. Chem. SOC.1986, 108, 1363-1374. (10) van Berkel, G. J.; Glish, G. L.; McLuckey, S . A. Org. Ceochem. 1989, 14, 203-212. (11) Keely, B. J.; Maxwell, J . R. Energy Fuels, accompanying paper in this issue. (12) Eckardt, C. B. Ph.D. Thesis, University of Aachen, FRG, 1989.
0 1990 American Chemical Society
