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Anal. Chem. 1985, 57, 1208-1210
In four-layer structures, this technique can be utilized to excite dopants in one layer perferentially, while in three-layer structures molecular concentration profiles are amenable to measurement. Registry No. SOz,7631-86-9PVA (homopolymer),9002-89-5; polystyrene, 9003-53-6. LITERATURE CITED (1) Hatta, A.; ashima, T.; Suetaka, W. Appl. Phys. A 1982, A29, 71. (2) Tompklns, H. G. Appl. Spectrosc. 1974, 28, 335. (3) Blackwell, C. S.;Degen, P. J.; Osterholtz, F. D. Appl. Spectrosc. 1978, 32, 480. (4) Palik, E. D.; Gllson, J. W.; Holm, R. T.; Hass, M.; Braunsteln, M.; Garcia, 13.Appl. Opt. 1978, 77, 1776. (5) Iwamoto, R.; Miya, M.; Ohta, K.; Mima, S.J . Chem. Phys. 1981, 74, 4780. (6) Tompkins, H. G.; Allara, D. L. Rev. Scl. Instrum. 1974, 4 5 , 1221. (7) Boerlo, F. J.; Chen, S. L. Appl. Spectrosc. 1979, 33, 121. (8) Aliara, D. L.; Baca, A,; Pryde, C. A. Macromolecules 1978, 7 1 , 1215. (9) Ishitanl, A.; Ishlda, H.; Soieda, F.; Nagasawa, Y. Anal. Chem. 1982, 5 4 , 682. (IO) Golden, W. 0.; Saperstein, D. D. J . Nectron Spectrosc. Relat. Phenom, 1983, 3 0 , 43. (11) Zhizhin, G. N.; Moskalova, M. A.; Sigarev, A. A.; Yakovlev, V. Opt. Commun. 1982, 4 3 , 31. (12) Aravind, P. K.; Rendell, R. W.; Metiu, H. Chem. Phys. Lett. 1982, 85,
396. (13) Yamada, H. Appl. Spectrosc. Rev. 1981, 77, 227. (14) Bhasln, K.; Bryan, D.; Alexander, R. W.; Bell. R. J. J . Chem. Phys. 1978, 6 4 , 5019. (15) Pockrand, I.;Swalen, J. D.; Gordon, J. G., 11; Phllpott, M. R. Surf. Sei. 1977, 7 4 , 237. (16) Gordon, J. G., 11; Swalen, J. D. Opt. Commun. 1977, 2 2 , 374. (17) Weber, W. H.; Eagen, C. F. Opt. Lett. 1979, 4 , 236. (18) Chabal, Y. J.; Severs, A. J. J . Vac. Scl. Techno/. 1978, 15, 638. (19) Benner, R. E.; Dornhaus, R.; Chang, R. K. Opt. Commun. 1979, 3 0 , 145
(20) Girlando, A.; Phllpott, M. R.; Heitmann, D.; Swalen, J. D.; Santo, R. J . Chem. Phys. 1980, 72, 5187. (21) Weltz, D. A.; Garoff, S.; Hanson, C. D.; Gramila, T. J.; Gersten, J. I. ODt. Left. 1982. 7.89. (22) Kholl, W.; Phllpott, M. R.; Swalen, J. D.; Glrlando, A. J. Chem. Phys. 1982, 77, 2254.
(23) Swalen, J. D.; Tacke, M.; Santo, R.; Rleckhoff, K. E.; Fischer, J. Helv. Chim. Acta 1978, 67, 960. (24) Rabolt, J. F.; Santo, R.; Swalen, J. D. Appl. Spectrosc. 1979, 33, 549. (25) Rabolt, J. F.; Santo, R.; Swalen, J. D. Appl. Spectrosc. 1980, 3 4 , 517. (26) Stegeman, G. I.; Fortenberry, R.; Karaguleff, C.; Moshrefzadeh, R.; Hetherington, W. M., 111; VanWyck, N. E.; Slpe, J. E. Opt. Lett. 1984, 8 , 295. (27) Hetherington, W. M., 111; Van Wyck, N. E.; Koenlg, E. W.; Stegemann, G. I.; Fortenberry, R. M. Opf.Lett. 1984, 9,88. (28) Schlotter, N. E.; Rabolt, J. F. Appl. Spectrosc. 1984, 38, 208. (29) Tlen, P. K. Appl. Opt. 1971, 70, 2395. (30) Kogelnlk, H.;Weber, H. P. J . Opt. SOC.Am. 1974, 6 4 , 174. (31) Swalen, J. D.; Santo, R.; Tacke, M.; Fischer, J. IBM J . Res. Dev. 1977, 2 7 , 168. (32) Reisinger, A. Appl. Opt. 1973, 12, 1015. (33) Ulrlch, R.; Torge, R. Appl. Opt. 1973, 72, 2901. (34) Rabolt, J. F.; Santo, R.; Schlotter, N. E.; Swalen, J. D. IBM J . Res. Dev. 1982, 2 6 , 209. (35) Polky, J. N.; Mitchell, G. L. J . Opt. SOC.Am. 1974, 6 4 , 274. (36) Tlen, P. K. Rev. M o d . Phys. 1977, 4 9 , 381. (37) Rabolt, J. F.; Schlotter, N. E.; Swalen, J. D. J . Phys. Chem. 1981, 85, 4141. (38) Ulrich, R. Appl. Opt. 1970, 6 0 , 1337. (39) Swalen, J. D.; Rleckhoff, K. E.; Tacke, M. Opt. Commun. 1978, 2 4 , 146. (40) Tiefenthaler, K.; Lukosz, W. Opt. Lett. 1984, 10, 137. (41) Marcuse, D. "Light Transmlsslon Optics"; Van Nostrand Relnhold: New York, 1972;Chapter 8. (42) Bracewell, R. N. "The Fourier Transform and Its Applications"; McGraw Hill: New York, 1978;p 46. (43) Han, 0.H.; Bohn, P. W., unpublished results. (44) Winograd, N. Prog . Solid State Chem. 1982, 13, 285. (45) Swingle, R. S.,11; Riggs, W. M. CRC Crit. Rev. Anal. Chem. 7975, 5,267. (46) Holm, R Scannlng Nectron Mlcrosc. 1982, 7, 1043
RECEIVED for review November 1,1984. Accepted February 4,1985. Support of this work by the donors of the Petroleum Research Fund, administered by the American Chemical Society, and the National Science Foundation Grant DMR83-16981 is gratefully acknowledged.
Tandem Mass Spectrometry with Fast Atom Bombardment Ionization of Cobalamins I. Jonathan Amster and Fred W. McLafferty* Department of Chemistry, Cornel1 University, Ithaca, New York 14850
Artifact peaks in mass spectra from fast atom bombardment (FAB) lonlzatlon can be characterized by tandem mass spectrometry. FAB of vitamin B,, (molecular weight 1354) produces a m / z 1388 peak whose abundance grows at the H)' with continuing bombardment. The expense of (M secondary mass spectrum of m / z 1388 produced by colllsionally actlvated dissociation (CAD) indicates that It Is formed by Intermolecular transfer of a cobalt atom from one molecule to the phosphate group of another. The CAD mass spectra of m / z 1329 formed by loss of the axlal substituent on Co from four different cobalamins are virtually identical, evidence that matching CAD reference spectra to characterize ion structures is also applicable to much larger fragment ions.
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Fast atom bombardment (FAB) has shown itself to be a powerful ionization method for the mass spectral analysis of high molecular weight compounds ( I , 2). Labile organic molecules as large as human proinsulin, molecular weight 9390,
have been found to produce detectable molecular ion species (3). For many compounds, FAB produces both molecular and fragment ion species that give important molecular weight and substructural information. When FAB ionization does not form appreciable fragment ions, as in the case of the heptadecapeptide gastrin ( 4 ) ,tandem mass spectrometry (5) can supply substructural information; the molecular ion species separated by the first mass analyzer (MS-I) is made to undergo collisionally activated dissociation (CAD) producing fragment ions which are mass analyzed in MS-I1 (6-10). Another application of tandem mass spectrometry to an important problem of FAB, spurious peaks, is described here (9). In FAB, the sample is desorbed from a solution, usually glycerol, by irradiation with an intense atom beam for several minutes, so that sample degradation or chemical reactions between sample and solute can occur (11,12). Solvent-molecular ion complexes have been observed in the FAB mass spectrum of folic acid (13),(M 12n)+ions in the FAB spectra of amines (14), amine replacement o f a halogen atom by a solvent proton in the FAB spectra of halogen substituted nucleotides (151, and hydrogenation by glycerol in the FAB
0003-2700/85/0357-1208$01.50/00 1985 American Chemical Society
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($Hz)zCO"z
I 1329
GO"?
1355
F;"
11
ses 1388
Figure 1. FAB mass spectrum (molecular ion region) of vitamin B,,, recorded within 1 min of (upper), and 5 min after (lower), turning on
M
the fast atom beam.
spectra of several compounds (16). Such peaks, which can be misleading when interpreting the FAB spectrum of an unknown compound, can be individually characterized by further measuring their CAD spectra. This is illustrated here using an artifact (M H - CN 59)' peak and a fragment (M H - CN - 59)+ peak present in the FAB mass spectrum of vitamin Blz and of m / z 1329 peaks from vitamin BIZ and three related cobalamins.
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EXPERIMENTAL SECTION The experiments were performed on a tandem mass spectrometer (5) consisting of a Hitachi RMH-2 double-focusing instrument as MS-I (source acceleration voltage 10 kV), a molecular beam collision region, and an electrostatic malyzer as MS-11. The instrument was fitted with an Ion Tech fast atom (6 kV xenon) gun, with samples introduced on an axially mounted direct insertion probe with a stainless steel tip. The sample (10 pg) was deposited on the probe as a methanol solution, the methanol allowed to evaporate, and the residual dissolved in 1p L of glycerol. For CAD spectra the helium collision gas pressure was adjusted to attenuate the precursor beam to 30% of its normal intensity. For increased sensitivity an MS-I resolution of -600 was used, so that the precursor ions included a variable amount of neighboring (usually isotope) peaks. The CAD spectra exhibited a resolution of 100 (20% valley) and a mass accuracy of h2 after approximate correction for energy loss on collision (IO);mass assignments in the CAD spectra of m / z 1355 and 1329 from vitamin Blz agree with those of its FAB fragment ions (17). The samples of cyanocobalamin, hydroxocobalamin, methylcobalamin, and coenzyme vitamin Blz were obtained from Sigma Chemical Co.
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P
1270
1329
1388 971
Figure 2. CAD spectra of m l z 1388 (lower), m l z 1329 (middle), and m l z 1270 (upper) ions from the FAB mass spectrum of vitamin B,2.
RESULTS AND DISCUSSION The FAB mass spectrum of vitamin B12reported by Barber et al. (17)exhibits clusters of ions having higher mass values, although lower abundance, than the molecular ion species. Our FAB spectrum of Blz also shows these ions (Figure l), but their abundance5 increase rapidly after even short exposure to the FAB beam. After 5 min of exposure the most abundant cluster of peaks centered a t m / z 1388 has grown in absolute intensity to be larger than the (M + H)+ peak a t mlz 1355, while this and m / z 1328 have decreased. The m / z 1388 (fl) ions were selected by MS-I and made to undergo collisionally activated dissociation (CAD), producing the CAD spectrum of Figure 2. In comparison to the previously reported (4)CAD spectrum of the m / z 1329 ion (M + H - CN)+ from vitamin B12,the spectra show many of
the same neutral losses; these are similar to the lowes found in the FAB spectrum (17). These losses, as illustrated in Figure 2, are due to cleavages in the long axial side chain, with the exception of the 59 dalton loss discussed below. The similarity of the CAD spectrum of mlz 1388 suggests that this ion has the camposition (M + H - CN + 59)+,with a cobalt atom or an acetamide moiety (CzH5NO)as the most likely identities for the mass 59 fragment. The CAD spectrum of m / z 1388 also gives information about the site of attachment of this adduct. Although the peaks corresponding to m / z 1270, 1183,1126, 1069, and 1051 of the m / z 1329 CAD spectrum now appear shifted 59 mass units higher in the mlz 1388 CAD spectrum, peaks appear at mlz 971 and 989 in both spectra; the m / z 1128 (1069 59) of the m / z 1388 CAD spectrum is
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ANALYTICAL CHEMISTRY, VOL. 57, NO. 7, JUNE 1985
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H5N0 losses. The original proposal ( 4 ) that CzH5N0 loss produces the m / z 1270 has been recently confirmed (19) by an exact mass measurement of the m / z 1270 ion. The utility of CAD spectra for structure assignment of smaller fragment ions by matching CAD reference spectra is well established (20). To see if this technique can be applied to large substructures, the CAD spectra of m / z 1329 ions produced by FAB from the related compounds cyanocobalamin, methylcobalamin, hydroxocobalamin, and coenzyme Blz were collected (Figure 3). As can be seen, the spectra are identical within experimental error.
ACKNOWLEDGMENT We are indebted to M. A. Baldwin, M. T. Cheng, C. J. Proctor, C. Wesdemiotis, and E. R. Williams for helpful advice and assistance. Registry No. Cyanocobalamin, 68-19-9;hydroxocobalamin, 13422-51-0;methylcobalamin, 13422-55-4;coenzyme vitamin B,,, 13870-90-1.
LITERATURE CITED
Figure 3. CAD spectra of m l r 1329 ions from the FAB mass spectra of (top to bottom, respectively) coenzyme B,,, hydroxocobalamin,
methyl cobalamin, and cyanocobalamin. also much more abundant than its counterpart m/z 1069 peak in the m/z 1329 CAD spectrum. This indicates that the mass 59 fragment is attached to the oxygens of the phosphate group, the best remaining ligand site, which would be expected to bind cobalt (18). This assignment is supported by recent independent results of Grotjahn et al. (19),who show that the FAB mass BpeCtrUm of cupribalamin, the Cu analog of vitamin Blzwithout the CN ligand, exhibits a corresponding (M + H + Cu)' ion. The loss of 59 mass units from the precursor ions m/z 1329 and 1388 is anomalous in that it is the only major ion present in either CAD spectrum that is not a result of fragmentation of the long axial chain of the molecule. This loss corresponds to the m/z 1270 ion in the FAB mass spectrum of vitamin BIZ, for which were proposed (17)two possible compositions, (M + H - CN - CH3CONHz)+and (M + H - CN - Co)'. The CAD spectrum of the m / z 1270 ion (Figure 2) also shows the same neutral losses as the m / z 1329 ion, including loss of 59 daltons. However, this ion could lose 59 daltons as cobalt only one time, while there are three acetamide and three propionamide groups in vitamin B12 as possible origins for Cp-
Barber, M.; Bordoli, R. S.;Sedgwlck, R . D.; Tyler, A. N. J . Cbem. Soc., Chem. Commun. 1981, 325-327. Devienne, F. W.; Roustan, J. C. Org. Mass Spectrom. 1982, 17, 173-181. Barber, M.; Bordoli, R. S.; Elliot, G. J.; Horock, N. J. Biocbem. Biophys. Res. Cornmuti. 1983, 110 (3), 753-757. Amster, I.J.; Baldwin, M. A.; Cheng, M. T.; Proctor, C. J.; McLafferty, F. W. J . Am. Chem. SOC. 1983, 105, 1654-1655. Todd, P. J.; McGilvery, D. C.; Baldwin, M. A,; McLafferty, F. W. I n "Tandem Mass Spectrometry"; McLafferty, Ff. W., Ed.; Wiiey: New York, 1983; Chapter 13. McLafferty, F. W.; Amster, I.J.; Baldwin, M. A,; Barbalas, M. P.; Cheng, M. T.; Cohen, S.L.; Danis, P. 0.; Kruppa, G. H.; Proctor, C. J.; Turecek, F. Int. J . Mass Spectrom. Ion Phys. 1982, 4 5 , 323-329. Hunt, D. F.; Bone, W. M.; Shabanowitz, J.; Rhodes, J.; Baliard, J. M. Anal. Chem. 1981, 53, 1704-1706. Tomer, K. B.; Crow, F. W.; Gross, M. L.; Koppel, K. D. Anal. Chem. 1984, 56, 880-886. Amster, I. J.; Baldwin, M. A.; Cohen, S. L.; Danis, P.0.; McGiivery, D. c.; Proctor, C. J.; McLafferty, F. W. 31st Annual Conference on Mass Spectrometry and Allied Topics, Boston, 1983; p 201. Neumann, G. M.; Derrick, P. J. Org. Mass Spectrom. 1984, 19, 165-1 70. Smith, L. A.; Caprioli, R. M. 8iOmed. Mass Spectrom. 1983, 10, 98-102. Field, F. H. J . Pbys. Cbem. 1982, 8 6 , 5115. Melli, J . ; Selbl, J. Inf. J . Mass Spectrom. Ion Pbys. 1983, 4 6 , 367-370. Parry, H.; Costello, C. E.; Biemann, K. 32nd Annual Conference on Mass Spectrometry and Allied Topics, San Antonio, 1984; p 751. Sethi, S. K.; Nelson, C. C.; McCloskey, J. A. 32nd Annual Conference on Mass Spectrometry and Allied Topics, San Antonio, 1984; p 620. Meili, J.; Selbl, J. Org. Mass Spectrom. 1984, 19, 581-582. Barber, M.; Bordoll, R. S.;Sedwlck, R . D.; Tyler, A. N. Biomed. Mass Spectrom. 1981, 8 , 492-495. Cornelius, R. D.; Hart, P. A.; Cleland, W. W. Inorg. Cbem. 1977, 16, 2799. Grotjahn, L.; Koppenhagen, V. 8.; Ernst, L. Z Naturforscb. 1984, 396, 248-25 1. Zwinselman, J. J.; Nibbering, N. M. M.; Ciommer, B.; Schwarz, H. I n "Tandem Mass Spectrometry"; McLafferty, F. W., Ed.; W h y : New York, 1983; Chapter 4.
RECEIVED for review December 3,1984. Accepted February 19,1985. This work was supported by the National Institutes of Health, Grant GM-16609.
