Anal. Chem. 1986, 58, 165R-211R (C15) Davidson, David L. Part. Charact. Technoi. 1984, I , 59-65. (Cl6) Miyoshl, Kazuhlsa; Buckley, Donald H. Ceram. Eng. Sci. Proc. 1983, 4, 874-693. (C17) Glaeser, William A. Wear 1984. 100, 477-467. (C18) Baer, D. R. Appl. Surf. Scl. 1984, 79, 362-396. (Cl9) Menzel, Dletrich lnt. Conf. X-ray Inn .-Shell Processes At., Mol. So/&, Conf. Proc. 1984‘ 487-478; Meisel, A., Finster, J., Eds. (C20) Braue, W.; Dudek, H. J.; Ziegler, G. 81. Ceram. Proc. 1984, 34(Ceram. Surf. Surf. Treat.), 31-44. (C21) Shaw, B. W. Glastech. 8er. 1983, 56(Int. Glaskong., 13th, Band I), 867-682. (C22) Pantano, Carlo G.; Houser, Cheryl A. Proc. SPE-lnt. SOC. Opt. Eng. 1983, 387(Symp. Opt. Surf. Technol. 1983), 130-137. (C23) Kirschner, J. Springer Ser. Opt. Sci. 1984, 43(X-ray Mlcrosc.) 308-3 13. ((224) Cazaux, J. Ultramicrosc. 1984, 72. 321-332. (C25) Powell, C. J. “Electron Beam Interac. Solids Microsc., Microanal. Microllthogr., Proc. Pfferkorn Conf., 1st 1982”; Kyser, Davld F., Niedrig, Heinz, Newbury, Dale E., Eds.; Scanning Electron Microsc.. Inc.: AMF O’Hare, IL, 1984; pp 19-31. (C26) Dwyer, V. M.; Matthew, J. A. D. Vacuum 1983, 33, 787-769. (C27) Tokutaka, H.; Nishimorl, K.; Hayashi, H. Surf. Sci. 1985, 749, 349-365. (C28) Cailler, M.; Barzine, K.; Ganachaud, J. P. Surf. Sci. 1985. 154, 548-574. (C29) Berresheim, K. Fresenlus’ Z. Anal. Chem. 1984, 379, 731. (C30) Pate, B. B.; Oshima, M.; Sllberman, J. A,; Rossi, 0.; Lindau, 1.; Splcer, W. E. J. Vac. Scl. Technol. A 1984, 2 , 957-960. (C31) Lukas, Jaromir; Jezek, Bretislav Coll. Czech. Chem. Commun. 1983, 48, 2909-2913. (C32) Linder, Robert, E.; Mee, Peter B. I€€€ Trans. Magn. 1982, MAG- 78, 1073-1076. (C33) Chambers, S. A.; Greenlee, T. R.; Howell, 0. A.; Weaver, J. H. J. Vac. Sci. Technoi. A 1985, 3 , 1291-1294. (C34) Tokutaka, H.; Nlshimori, K.; Takashlma, K.; Ichlnokawa, T. Surf. Scl. 1983, 133, 547-579. (C35) Garbassi, Fabio Surf. Interface Anal. 1983, 5 , 139-148.
(C36) Datta, M.; Mathieu, H. J.; Landolt, D. Appi. Surf. Sci. 1984, 18, 299-314. (C37) Mathieu, H. J.; Landolt, D. Surf. Interface Anal. 1984, 6 , 82-89. (C38) Mathieu, H. J.; Datta, M.; Landolt, D. J. Vac. Sci. Technol. A 1985, 3. 331-335. ((239) Kovacich, J. A.; Lichtman, D. J. Nectron Spectrosc. Relat. Phenom. 1985, 35, 7-18. (C40) Hammer, G. E.; Shemenskl, R. M. J. Vac. Sci. Techno/. A 1984, 2 , 1132-1 134. (C41) Keenlyside, M.; Stott, F. H.; Wood, G. C. Anal. Proc. 1983, 2 0 , 482-486. (C42) Baudolng, R.; Gaubert, C.; Blanc, E.; Aberdam, D.; Gauthier, Y. Scannlng Nectron Microsc. 1984 (I), 87-102. ((343) Egelhoff, W. F., Jr. J. Vac. Sci. Technol. A 1984, 2 , 350-352. ((244) Cazaux, Jacques Appl. Surf. Sci. 1985, 20, 457-471. (C45) Grant, J. T. J. Vac. Scl. Technol. A 1984, 2 , 1135-1140. (C46) Dwyer, V. M.: Matthew, J. A. D. Surf. Scl. 1984, 143, 57-83. (C47) Nelson, G. C. J. Vac. Sci, Technoi. A 1984, 2 , 1141-1145. (C48) Gaarenstroom, Stephen W.; Waldo, Richard A. Appl. Surf. Sci. 1984, 78, 223-231. (C49) De Bernardez, L. S.; Ferron, J.; Goldberg, E. C.; Buitrago, R. H. Surf. Sci. 1984, 139, 541-548. (C50) Haak, H. W.; Sawatzky, G. A.; Ungier, L.; Gimzewski, J. K.; Thomas, T. D. Rev. Sci. Instrum. 1984, 55, 696-711. (C51) Cazaux. J.; Gramari, D.; Mouze, D.; Nassiopoulos, A. G.; Perrin, J. J. Phys., Colloq., ( C 2 ) 1984, 271-274. (C52) Seah, M. P.; Anthony, M. T. J. Electron Spectrosc. Relat. Phenom. t985935, 145-153. (C53) Seah, M. P.; Mathieu, H. J. Rev. Sci. Instrum. 1985, 56, 703-711. (‘254) Sabbatini, Luigla; Malitesta, Cosimlno; Desimonl, Elio; Zambonin, Pier Glorglo Ann. Chlm. (Rome) 1984, 74, 341-348. (‘255) Peacock, D. C.; Prutton, M.; Roberts, R. Vacuum 1984, 34, 497-507. (C56) Kodama, Y.; Sumitomo, S.;Kato, I.; Jinno, M.;Yamauchi, H. Springer Ser. Chem. Phys. 1984, 36(Second. Ion Mass Spectrom., SIMS 4), 252-254. (C57) Ganschow, 0.; Jede, R.; An L. D.; Manske, E.; Neelsen, J.; Wledmann, L.; Benninghoven, A. J. Vac. Sci. Technoi. A 1885, 7 , 1491-1506.
Mass Spectrometry A. L. Burlingame* Mass Spectrometry Facility, Department of Pharmaceutical Chemistry and the Liver Center, Uniuersity of California, S a n Francisco, California 94143
Thomas A. Baillie Department of Medicinal Chemistry, School of Pharmacy, BC320, Uniuersity of Washington, Seattle, Washington 98195
Peter J. Derrick Department of Physical Chemistry, School of Chemistry, University of New South Wales, Sydney, New South Wales, Australis 2033
OVERVIEW From ita inception, mass spectrometryhas dealt with studies of the chemical reactivity of ionic species in the gas phase and with the exploitation of the knowledge thus gained to carry out structural identification and quantitative analysis. In a rapid revolution, the forefront of the field today has gained a firm foothold in the more complex, challenging, and interesting areas of chemistry and analysis of biological substances in polar condensed phases-media closely resembling the natural aqueous environment of these molecules. This remarkable transition in focus, otential, and utility has brought the mass spectrometrist Ece to face with the nature, complexity, and daily manipulation of ionic chemical reactions and transport and mixing of components of liquid mixtures, their steady-state and interfacial properties, and their solution-to-gas-phaseion transition energetics, including 0003-2700/86/0358-165R$06.50/0
ionic cluster solvation and desolvation. While these fundamental issues involving the details of the phase transition between the solvated neutral/ionic molecule and its charged gas-phase counterpart-ready for mass spectral analysis-are presently not at all well understood, this situation has fortunately not hampered the explosive growth in analytical utility of condensed-phase ionizationf ejection (ejection/ionization) methods. These methods include field desorption, 262Cfplasma desorption, laser desorption, sputtering of substances dissolved in a liquid-phase (matrix) as secondary ions by bombardment of the liquid surface layer with energetic primary ion (LSIMS) or atom (FAB) beams, and thermospray LC/MS coupling. Unlike electron impact (EI) and chemical ionization (CI) ion sources, in which thermal evaporation of the sample is required, these new so-called ”soft-ionization” methods permit desorption/ejection of the sample molecules in ionic form 0 1986 American Chemical Society
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directly from the condensed-phase in which they are present either as the free molecules themselves or in a suitably derivatized form. The initial enthusiasm for these methods stemmed from the fact that highly polar and/or thermally labile biological substances, which previously were completely intractable by mass spectrometric methods of analysis, could now be studied directly in their native, underivatized form. However, it is now clear that appropriate derivatization of such biological substances will develop and be used hand in hand with studies of the free materials in order to derive the maximum amount of structural or sequence information from the particular substance in question. In addition, direct determination of purity, homogeneity, or composition of mixtures will be possible for substances which are inherently difficult to purify or separate by any available chromatographic methods, e.g., glycolipids, complex carbohydrates,components of proteins, and so on. While the sensitivity of these soft ionization techniques cannot compete presently with EI, CI, and GC/MS methodology (in cases where both approaches can, in fact, be employed for a given analyte), the eventual emergence of new generations of ion optical systems holds great promise to bring the sensitivity into the low picomole sample size range rather than the current low nanomole range. The most compelling advance associated with these new methods, in addition to their inherent ability to deal with highly polar, labile substances per se, has been the remarkable progression in the molecular sizes that have become tractable. It seems that every laboratory worth its salt can now run anything up to insulin (5729 daltons) in size, thereby extending mass spectral utility a solid factor of 5 in mass over the last decade-mostly of course, in the last 5 years. Research at the forefront is able to detect resolved cesium iodide clusters at 40 000 daltons and an unresolved signal at 100 000 daltons, while in the protein field the proleolytic enzyme, porcine trypsin, seems currently a good example at 23 406 daltons. Clearly the major present and long-range beneficiaries of this emerging high mass bonus are the biological and biomedical scientists, those concerned with macromolecular constructions and protein expression employing methods of genetic engineering and biotechnology, and those interested in industrial synthetic polymers. As a rule of thumb, in order to derive the optimal performance from each of these soft ionization methods, it is desirable to match their individual operating characteristics with the appropriate type of ion optical mass analyzer and detector. Similarly, it is of crucial importance to determine required mass range, mass resolution, and overall instrument sensitivity as a function of mass range and resolution. At this stage of development of sofbionization methods, the suite of instruments in heavy analytical use are, in general, adaptations and obvious upgrades of instruments conceived and built to meet earlier less-demanding requirements with EIMS. Soft ionization sources present inherently different ion optical trajectory problems and requirements than do E1 and CI sources, such as distribution of ion spatial origins, greater angular spread and distributions of initial ion kinetic energies, and so on. These source characteristics have not as yet been dealt with satisfactorily/optimally in overall instrumental design. In addition, other characteristics are not well-understood, such as ion lifetimes as a function of molecular size or internal energy deposition functions. Further, the optimal physical parameters involving the type of primary beam (angular dependence of ejection of high mass biopolymers, flux, and kinetic energy) have not been determined experimentally. It would seem premature to assume these parameters are satisfactorily described or fully determined by established solid (“static”) SIMS models and theories. This explosive growth in the scope of mass spectrometry is due to the developmentof the “alphabet soup” of ionization techniques for biological and other involatile, thermally labile compounds and, in particular, to those techniques in which ions are ejected from the condensed phase following chargedor neutral-particle bombardment. Although it is apparent that the potential of these techniques for the formation of gaseous ions from biological molecules has not been fully tapped (see section “Innovative Techniques and Instrumentation“), it is less clear ‘ust how much farther in terms of accessible masses they can be taken. There is a crying need for a good theory of formation of the gaseous ions to help address this question of feasible mass range, to guide and direct further development 166R
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of the techniques, and to assist in the choice of experimental conditions within ion sources. The mechanism of ion formation is clearly a multidisciplinary problem, involving solid-state and surface physics, a great deal of physical chemistry, and not a little organic and biochemistry. The sort of synergism, generated by full and enthusiastic cooperation among scientists of differing backgrounds and skills, which could lead to the solutions, is not evident from the recent literature (AI). The obfuscation and confusion over names and acronyms must have contributed to this situation. Analogy with the tower of Babel and the confusion of tongues is not inappropriate (A2). To clarify terminology must a t this stage be beneficial for the field of mass spectrometry, indeed even if only to draw attention away from small differences between techniques to the much more significant characteristics they hold in common. The term “fast atom bombardment (FAB)”,despite its wide popularity, seems to us to have served its purpose. It has proved extremely effective in conveying the message that something of a revolution was occurring in mass spectrometry and that the changes occurring were much more than technical modificationsto a well-establishedsurface science technique. These points have been made, and usage of the term has now become a source of misinformation. Examples present in the most recent literature refer to bombardment of a solid inorganic insulator with kiloelectronvolt Cs+ as SIMS (secondary ion mass spectrometry) (A3),bombardment of a solid inorganic insulator with kiloelectronvolt Ar as FAB ( A 4 ) ,bombardment of a biological molecule in a liquid matrix with kiloelectronvolt Ar as FAB, and bombardment of a biological molecule in a liquid matrix with kiloelectronvolt Cs+ as SIMS. The logical conclusion would be that the charge state of the bombarding particle is the all-important factor, but such a conclusion would be untenable in the case of biological molecules. The evidence is that the charge of the incident particle is not of prime importance with biological molecules in liquid matrices (A5-A7), whereas the incident beam energy, beam flux, and the effect of focusing are significant. Any implication that neutral beams are superior to charged beams as regards the latter characteristics is probably wrong and certainly unproven. Charged beams are probably superior to neutral beams in these respects (A7). The all-important factor for biological samples is now accepted to be the nature of the, usually liquid, matrix in which the sample is dispersed. We recommend that the acronym SIMS be used with appropriate qualification for all the techniques involving bombardment with kiloelectronvolt particles, regardless of whether those particles are neutral or charged. To be quite clear, we are recommending that “SIMS” be used instead of “FAB”, and we have adopted this recommendation in this review. The sorts of qualification we envisage as being occasionally necessary are “SIMS using a liquid matrix”, or the already widely used “liquid SIMS”, and perhaps “atom SIMS” and “ion SIMS”. We see no reason why the megaelectronvolt-bombardment techniques, a t present referred to as “plasma desorption”, “heavy ion induced desorption”, or “fast ion induced desorption”, should not be called ”fast-ion SIMS”. A prefix before “SIMS” is necessary because there may be significant differences between the kiloelectronvolt and megaelectronvolt bombardment as regards the process of ion formation. This point has still to be settled. Nevertheless, it is already clear that there are strong similarities between the techniques with respect to the ion chemistry, as exemplified by identical fragmentation pathways for peptides (see section ”Ionization of Nonvolatiles and Related Surface Phenomena”). The incident particles in megaelectronvolt bombardment are ”fast” according to accepted usage in physics, whereas kiloelectronvolt particles are “slow”,and these megaelectronvolt particles are generally charged. In this review, we refer to the megaelectronvolt-bombardment techniques as “plasma desorption”. Replacing “FAB”with “SIMS was a much clearer case, since as far as results are concerned there is no difference between “ion SIMS” and “atom SIMS” when using a liquid matrix such as glycerol. Moreover, any misunderstanding caused is likely to be less harmful than further reinforcing the false impression that biological samples must be bombarded with neutral atoms if good mass spectra are to be obtained. A case could be made for referring to laser desorption as ”laser SIMS” or ”photon SIMS”; nevertheless we find “laser desorption” an unobjectionable term for the purposes of this review. “Field desorption” is a well-established term applicable to the technique, but probably not to
A. L. Bw!hg*m Is RoleMor 01 Chamisby nnd Pharmaceutical Chemistry in the Department 01 Pharmaceutical Wwmlstry. Unkersny 01 Califanla. Sa" FrancisEo. He Is also Director 01 the NIH-supported National Bio-organic. Biomedical Mars Spectrwnstry FaciiHy and 01 me cwe mass
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,-I 1
spectrometry ISCiMy 01 me Liver center at UCSF. He received his B.S. lrom the Univardiy 01 Rho& Island and his ph.0. hom lhe Massachmens InsMute of rechmlosy in 1962 wlh K. Biemann in determlnatlon 01 the structure 01 indole aikablds. M1 immedateh wried stans Of the Department 01
the mechanism of ion formation (see later section). Electrohydrodynamic ionization, electrospray, and thermospray are reasonably clear terms. The first of these is often also referred to as 'field evaporation", although this seems to he a case of naming the mechanism rather than the technique, and mechanism can he subject to radical change. "Liquid ionization" as used for a specific technique (see later section) seems too general.
SCOPE
me
Thomas Bilill. is currently Assoc$te Relessor of Medicinal Chemistry at the Lh%k&ly 01 Washhglon h %Ilk. WA. He was educated at W a r ~ wUnivermHy. where he earned his B.Sc. (Honr.) in chemlstry in 1970 and Ph.D. in organic chemishy in 1973. HIS graduate studles. canled out under the SUpeNISloII 01 PdeSSar C. J. W. Brooks. dean w a me applicatimn 01 GUMS techniqws to the anahsir 01 steroids and their metabolhes in bmlogicai liuids. Duing me years 1973-1975, he hem a ~ ~ ysock a i ely Fellowship. which was spent in Roles6w J. Sj6vailr labwatory at the Karolinska Instlute in StoCkhoim. Studies carrlsd out during this pastdoctoral perid centered on me metabolic late 01 progesterone in human pregnancy and made extensive use stableisotoplakied t r a m s and OCIMS methods. I n 1975. he was appointed to a lecture ship at me Royal Postgraduate Medical Schwl in London and. in 1978. moved to San Francisco where he was Arsistant Director 01 Or. A. L. Burlingame's Mass Spectrometry Rewurce and A ~ ~ k t a nProfessw t 01 FlwmaCBUticBl Chemistry a1 the UniverrKy 01 Calllwnla. San Francisco. He l w k up in Seattle in 1981. His current research interests le in his p r e ~ ~ poSIimn nt me application 01 mass rpectromelry to studies in me lieid 01 Iwebn corn pound metabOliSm and in the use 01 stable isotopes to investigate molecular mechanisms 01 druginduced toxicnies.
a
Pdw J. Drmdr is Role01 Fhysicai chemrjby and Head 01 me schmi 01 Chem kby st me Universny 01 NOWS& Wales In Sydney. AYShBIla. He received his BSC. (Nons. chemistry. 1966) and ph.0.(physical chemisby. 1969) hom King's C o w .London. HIS d o c t O d Sludb5 Under me SUpBNC *ion 01 R O l e s ~ wA. J. 0. R o b e n ~ ~estab n rshed mat rams of chemical remlons could be measured at limes as shMl as p k o s e mndr: me lednhue erwkyed has corne to be k m 8s "Reid kdzallm klnetks (FIK)". He studied. as a Royal Soclely Ewopaan Fellow 11969-1970). in me Department 01 physks a1 the Royal InstnUte 01 T e d d a g y . Stakhoh. Sweden. He has been a memba 01 stan ot the Space Sciences ~ ~ b a a t universny w. 01 Cainwnla. m e l e y (1971-1972). the ~ e p a n m n t01 Chemistv. Universny Wlege. London (1973-1975). and the Department 01 physical Chemistly. La Trobe Universny. Melbourne. Australia (1975-1981). He t w k up the Chat 01 physical Chemistry at me Universny 01 New Souih Wales in 1961. His research interest^ inci~de~nimlecukarreactions. tern emlsslon. and reactkm 01 macromiec~larand cluster ions. Since the early 1970% he has been cwcerned wnh the development 01 inshumentation and bnilatian techniqws. in panicular field deSarptlon. lor mass spectrometry 01 high molecular weight species (above 10000). His group bUin a forerunner of the new generation 01 double-focusing mass SpectrOmeterS lor high-mass blalogicalstudles and were among the first lo measure such biokgical Samples as paiytoxin and bovine InsJin. He has been awarded the Ramsay Memrlai Fellowship. Rennle Medal 01 the Royal Australian Chemical Institute. and Meld& Medal of the Royal Insthuts of Chemistry.
It has been our intention to bring together in tbii one source the direction of major developments in mass spectrometry and to illustrate these by citing key contributions from both fundamental and applied research. The Review is intended to provide the reader with a sense of the main currents, their breadth and depth, and probable future directions. It is also intended to provide the reader with a glimpse of the diverse discoveries and results that underpin the eventual development of new methods and instruments-the keys to obtaining new insights in all the physical, chemical, and hiological sciences which depend on mass spectrometry at various levels of sophistication. Focal points for future interdisciplinary synergism might he selective quantitative derivatization of large peptides, which would convey properties that direct fragmentation providing specific sequence information, or optimization of LCMS for hiooligomer sequencing and mixture analysis, or the perfect way to control or enhance the internal energy of ions of any size, or many others. The exact topics covered reflect to some extent our own personal interests, although considerable effort has been devoted toward the inclusion of all the important points we felt needed to be made in a general review such as this so that the reader might find ready access to all of the key literature. We have had to be more selective in this review than in the past, first, to limit its size and secondly in recognition of the extensive and detailed reviews on specific topics that are appearing constantly in Mass Spectrometry Reviews (BO. For example, we felt that organic geochemistry was adequately covered by the Vol. 4, No. 1, issue devoted to applications of mass spectrometry to petroleum chemistry. Reviews include Philp on biological markers (B2), Gallegos and Sundararaman on geoporphyrins (831,Schmitter and Arpino on azaarenes (B4),and Teeter on high-resolution masa spectrometric type analysis of complex hydrocarbon mixtures (B.5). In other issues, reviews included computer applications in mass spectral interpretation (B5a), analysis of additives in polymers by Lattimer and Harris (B6),as well as a previous one on polymers themselves by Schulten and Lattimer ( B n ,and even an upcoming one on theory and use of liquid matrices in atom and ion SIMS by De Pauw (B8). which was not available to us a t this writing. It still would appear that an active research knowledge of the field, including an awareness of laboratories with a 'track record" of contributions, and a lot of library footwork is far superior in searching the diverse literature to a subscription to Chemical Abstracts Selects: Mass Spectrometry (B9)or purchase of the expensive volumes 7 or 8 of the Specialist Periodical Reports in Mass Spectrometry (B10, BII). Volume 7 has only appeared in 1985 and covers the literature from 1980 to 1982! Volume 8 covers the literature between July 1982 and June 1984, but WBS unavailable at this Writing. These volumes may have archival value. VG Analytical has sponsored publication of a reference guide to the published collections and literature of mass spectrometric data from 1947 to 1985 (Blla). In addition to these obvious sources, we solicited reprints and preprints of recent papers from wellknown American and international laboratories. Those who responded were certainly of considerable help in providing a glimpse of the most recent findings. The loth International Conference in Mass Spectrometry was held in Swansea in September 1985. The 11th is scheduled for Bordeaux, France, in 1988. Proceedings of the 4th International Conference on Secondary Ion Mass Spectrometry in Osaka, Japan, have been published (R12) and SIMS Vis in press (B13). Proceedings for the 9th and 10th annual meetings of The Japanese Society for Medical Mass Spectrometry have been published (R14, 815). Proceedings of the first China-Japan Joint Symposium in Mass Spectrometry held in Beijing August 21-23,1984, were coedited by Matsuda and Chang (R16). Reports of the 32nd and 33rd United States Annual Conferences on Mass SpecANALYTICAL CHEMISTRY, VOL. 58. NO. 5. APRIL 1986
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trometry and Allied Topics held in San Antonio and San Diego, respectively (B17,B18), have been published and the 34th will be held in Cincinnati in June 1986. An International Symposium on Mass Spectrometry in the Health and Life Sciences was held in San Francisco in September 1984 (B19). A Symposium on Desorption Mass Spectrometry was held in St. Paul, MN October 1984 (B20). The 5th International Symposium on Mass Spectrometry in Life Sciences was held in Gent, Belgium, in May 1984,and the Proceedings have been published (B20a). The 6th meeting in this series is scheduled for August 1986. Watson’s “Introduction to Mass Spectrometry”, 2nd ed., represents a complete revision of his earlier work (B21). Chapman has prepared a new monograph entitled “Practical Organic Mass Spectrometry” (B22)and Message has authored a book on “Practical Aspects of Gas Chromatography/Mass Spectrometry” (B22a). Both of these new volumes should be considered for introductory instructional use by the uninitiated who wish to gain familiarity with the nomenclature and types of methods available, illustrated with examples of the kinds of studies that have been undertaken. While there is, however, no current textbook for advanced organic analytical, biological, and clinical mass spectrometry, the volume published from the San Francisco symposium contains a fairly complete picture in this regard through the eyes of the 30 or so individual invited authors’ chapters (B19). Odham, Larsson, and Mardh have edited a volume on “Gas Chromatography/Mass Spectrometry: Applications in Microbiology”(B23). Facchetti has edited a volume on “Mass Spectrometry of Large Molecules” (B24). Desiderio has prepared a monograph on “Analysis of Neuropeptides by Liquid Chromatography and Mass Spectrometry” (B25). Two volumes will appear in 1986 concerning the preparation and use of stable isotopically labeled compounds and contain several chapters dealing with the use of mass spectrometry (B26, B27). Isotope dilution mass spectrometry has been reviewed in connection with applications to problems in clinical chemistry (B27u). Karaseck has edited a book on “Mass Spectrometry and Environmental Sciences”(B28). And finally, there is a forthcoming book on “Mass Spectrometry and Biomedical Research, edited by Gaskell, which will contain three parts in 26 chapters on analysis of labile and polar compounds, analysis a t high mass, and trace analysis (B29). In addition, there is a complete monograph on “Molecular Orbitals and their Energies Studied by Semiempirical HAM Method” by Lindholm and Asbrink (B30). The contents of this book, which originate from mass spectrometry, include a general theory of semiempirical quantum chemical theory based upon density function theory, a new method to handle self-repulsion, and an embodiment of approximate Hartree-Fock theory including correlation. Parameters for this theory are included also, and most of the book contains applications on photoelectron spectroscopy, UV excitation spectroscopy, electron affinities, and some other phenomena. The IUPAC commission on atomic weights and isotopic abundances has prepared a list of the abundances of the naturally occurring isotopes of the elements 1983 (B31)as well as an element by element review of their accurate atomic weights (B32,B32a). Miller has prepared a chapter on atom LSIMS mass spectrometry and related techniques and its application to organometallic compounds, coordination compounds, and other inorganic systems (B33). Many critical and detailed reviews of specific areas of mass spectrometry have appeared in the past 2 years in journals other than Mass spectrometry Reviews. A number of them are included in the appropriate sections of this review.
INNOVATIVE TECHNIQUES AND INSTRUMENTATION Instrumentation design requirements for SIMS of organic and biological samples in liquid matrices have been laid down by Magee (C1). He states that this field is “still in its infancy [and that] most of the desirable instrumental features outlined in this [his] paper for organic SIMS instruments have yet to be incorporated into present day machines, [that] most instruments currently being used have capabilities for SIMS far below those which are potentially available if currently available ion optical techniques are used.” He calls specific 168R
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attention to the advantages of a focused primary beam and immersion lens for more effective secondary ion extraction and the use of appropriate double focusing ion optics. Independently, Kambara has discussed optimization of the salient instrumental design and operating features necessary to obtain high sensitivity and high-resolution performance in analysis of biological substances using xenon ion LSIMS (C2). He reported results obtained from a new double focusing mass spectrometer (Hitachi M-200) based upon new Matsuda ion optics with low image magnification (1/8),a focused primary beam, wide angular acceptance, and so on, designed to increase the absolute sensitivity for biological substances in the mass range up to 50004000 at unit mass resolution with source slit wide open. The absolute sensitivities were reported for a series of peptides such as bradykinin and a-MSH (1664 daltons) at the 1-pmol level. Others have also reported experimental data to substantiate the advantages of focused primary ion beams (C3-C5) and immersion lens (C3,C5) sources for improvement in the overall performance of existing commercial double focusing mass spectrometers. In general, focusing permits lower primary beam fluxes to be utilized to sputter secondary ions which illuminate primarily the source exit slit (C2). With such a focused cesium primary ion immersion lens type LSIMS source on an MS50 (with 23 kG magnet), sensitivity corresponding to 1.7 nmol for human insulin was obtained for mass resolved single scans of the stable isotope molecular ion cluster (5804-5814 daltons). In addition, one 1.7-nmolsample loading lasted over 10 min in the 1-pA primary cesium beam (C5). Hence, the integrated sensitivity was a factor of 10-20 higher than the commercial Kratos saddle field xenon gun and their source arrangement, while the secondary ion yield was a factor of 5 higher per se. In addition, the sensitivity achievable for a new Wien type high mass instrument built a t UCSF has been found to be in the 1-15 pmol sample range for higher molecular weight carbohydrates and peptides such as MGP (3516 daltons) (C6), the insulins (C6) and epidermal growth factors (C7). In connection with evaluation of the performance of this Wien instrument using (CsI),Cs+ clusters up to n = 70, Aberth has reported that any increases in instrument operating pressure over the vacuum system ambient of 2.6 X torr has a profoundly deleterious effect on the ability to observe the higher mass clusters compared with those of lower mass (C8). This report would indicate that beliefs such as those stated by Magee ( C I ) that “vacuum requirements are not stringent because the presence and residual gas composition are dictated by the organic liquid being used for the sample matrix” are particularly ill-founded for higher mass instrumentation. The sensitivity achievable using SIMS (without any matrix) has been demonstrated by Benninghoven et al. (C9), who, using a recently designed and constructed instrument, have reported detection of femtomoles of material. A variety of new ion guns, but significantly no new atom guns, have been developed with a view to enhancing secondary ion yields in SIMS. The primary beams employed include Au( C I O ) , In+ (C11, C12), Hg+ (C13), Ga+ (C14, C15), and Cs+ (C16). An additional ion source has been designed for SIMS, incorporating the feature of focusing the primary ion beam so as to match the characteristics of the secondary ion beam with the spatial and angular acceptances of the analyzer (C17). The nature of the matrix is crucially important for SIMS of biological samples, and so is the concentration of the sample in the matrix if quantitative analysis is required (C18, C19). In addition, for accurate quantification, it is necessary to utilize a stable isotopically labeled analog of the compound in question as internal standard as illustrated in the case of plasma steroid sulfates (C20). The choice of matrix determines whether sample ions are observed at all and, given that sample ions are obtained, greatly affects the level of background noise, especiallythe “chemical noise”. (See also section on Ionization of Nonvolatiles and Related Surface Phenomena.) The level of background noise is also very much dependent upon the primary beam characteristics and is one reason why it is sensible to focus the primary beam and to operate with the lowest usable primary flux (C2, C5). Gower has compiled a summary of many different liquid matrices that have been used and assessed (C21) (See also sections on specific compound classes). The use of a saturated solution of solids such as glucose, which require heating, has been advocated (C22). Matrices are also important in plasma desorption (fast ion SIMS), as evidenced by the enhanced formation of large
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peptide ions per se as well as their multiply protonated counter parts when using a glutathione (C23) or nitrocellulose matrix (H6). Negative ion LSIMS spectra of ATP and glutathione have been recorded in glycerol crown ether mixtures (C24). The presence of a reactive buffer gas above the surface in SIMS has been shown to raise the relative intensities of molecular ions (compared to fragment ion intensities) (C25, C26); it is not clear whether absolute molecular ion intensities are enhanced or diminished. Potentially the most significant long range development as regards analyzers is probably the increased acceptance of Fourier transform ion cyclotron resonance (FT-ICR) for high-resolutionand high-mass mass spectrometry despite the many unsolved technical problems (see below). Of immediate significance, several large four-sector instruments (C27) are being introduced, having configurations such as VG’s first BE-EB geornetry, i.e., source followed by magnet (B), electric sector (E), collision cell, electric sector (E), and magnet (B), and allowing collisionally activated dissociation (CAD) (or mass spectrometry/mass spectrometry (MS/MS)) to be performed with high mass resolution in both selection of incident “parent” ion and analysis of its fragment ions (see below). This instrument has demonstrated the quality of chemical-noise-free secondary ion mass spectra that can be obtained with high daughter ion mass resolution for a variety of substances including acyl carnitines in patient urine ( C B ) , a 2-aminopyridine derivative of a hexasaccharide, a pcarboethoxyaniline derivative of maltoheptaose (C29), and a variety of peptides (C30)such as renin substrate (1718 daltons) (C31),fibrinopeptide A (1536 daltons) (C31), and C-peptide of human proinsulin (3021 daltons) (C32). As is the situation with normal LSIMS spectra of peptides, in these MS-CADMS spectra particular sequence ion types are observed emphasized, while others may be suppressed depending upon the structure or particular sequence at hand (see also section on peptides and proteins), but at the very least these CAD spectra have separated the trees from the forest and are obviously worth scrutinizing for reproducible sequence-fragmentation pattern correlations. While there is still a paucity of 4-sector MS/MS data available, the quality of these spectra is impressive compared with those reported in the growing literature which are being obtained for lower molecular weight substances using the triple quadrupole instruments (see below) and triple sector instruments such as those of EBE, or BEB type geometries. Indeed, extended mass range (u to 10000-15000 dalton) 4-sector instruments have now Been designed and constructed by both VG and JEOL based upon their ZAB-SE and the HFX-110 double focusing counterparts. The ion trap promises to be significant as regards making mass spectrometry available at low cost, particularly as a detector for gas chromatography (C33). A small high-performance double-focusing mass spectrometer suitable for field work and planetary science has been designed by Nier and Schlutter (C34, C34a). Determination of times-of-flight combined with magnetic dispersion has been proposed as a new MS/MS technique (C35,C36, C36a). Matsuda and coworkers have calculated arrangements of combinations of electronic sectors, which would produce high resolution for time-of-flight (TOF) mass spectrometry (C37). Mass resolution of nearly 5000 has been achieved in linear TOF through using a molecular beam sample source (C38). TOF has enjoyed a surge of renewed interest, being the present method of choice for plasma desorption (fast ion SIMS, see later sections). TOF provides good sensitivity by virtue of high transmission and the fact that it is not a “scanning device”, but the relatively low resolution of TOF will not provide much more than isotopically averaged molecular weight at high masses (C39, see also C40) and a call has been voiced for instruments for higher performance and sensitivity at high mass (C39). Laser desorption (C41) and liquid S M S (1242) have also been combined with TOF. Laser desorption combined with FT-ICR (C43),which promises to become an important technique for high-mass analysis, is covered in later sections. Boerboom et al. (C44) have given a theory of the dodecapole ion lens. Dawson et al. (C45-C48) have reported theoretical and experimental studies of quadrupole performance. Instrumental factors affecting mass spectra at high mass have been discussed by Aberth, and as mentioned above, the pressure in the system is shown to be of particular importance (C8), which is consistent with measurements of the depen-
dence of CAD cross sections upon ion mass (C49). It has been shown that ions as well as electrons play important roles in “postacceleration”detectors (C8, C50), and this finding could be significant for the design of the next generation of detectors for high-mass studies. The use of signal averaging for improving detection limits at high mass has been advocated (C51, C52, see also C53). Ion counting systems making use of microchannel plates we likely to be exploited increasingly in mass spectrometry (C8, C54, C55). A data acquisition/analysis system for processing photoplates has been described (C56). Further work has been reported on the statistical evaluation of accurate mass measurement quality at high resolution (C56a).
The various types of hybrid instruments employed in MS/MS have been reviewed (C57, C58), and attention has been drawn to the problems of artifact peaks (C59). New scan modes have been described using the BEQQ hybrid instrument (C60). The question of calibration of linked scan spectra has been addressed by Boyd et al. (C61) in connection with 4-sector double focusing systems (C27-C32). The advantages of graphical aids for displaying linked scan s ectra have been expounded (C62). It has been shown that BP /E scans are well suited to quantitative analysis when using a reversed geometry instrument (C63). MS/MS has been exploited as a technique for studying polyatomic ion/surface interactions (C64). Biomedical applications of MS/MS are appearing more frequently now than was the case during the previous reporting period. This trend may be attributed, in part, to the increased number of tandem mass spectrometers in the field, but probably also reflects a broader awareness of the capabilities of MS/MS for analytical work. Johnson and Yost (C65)have surveyed recent applications of tandem MS for trace analysis and point out the particular benefits of MS/MS for the study of polar and/or thermally labile compounds ionized by “soft” techniques. Thus, protonated or cationized molecular ion species with low internal energy may be selected by the first mass analyzer for collisional activation and the resulting spectrum of daughter ions analyzed by the second mass analyzer. By this approach, structural information on the analyte may be obtained in cases where the primary mass spectrum is essentially devoid of fragment ions. An added merit of the technique is that when used to analyze ions sputtered from liquid matrices (e.g., SIMS with a glycerol matrix), high background signals derived from the matrix material can, in principle, be effectively removed in the first mass analyzer and a “clean” spectrum of the collisionally activated analyte obtained using the second. This situation may hold true even when the analyte is a minor component of a complex biological extract, as is illustrated by the recent use of SIMS and MS/MS to identify the nigrostriatial toxin, MPP+,in crude extracts of mammalian brain tissue ((266). In view of the rapid developments taking place with SIMS techniques, MS/MS would thus appear to have an especially important role to play in the analysis of involatile and thermally unstable biological substances. A similar case can be made for the use of tandem MS in analyses conducted by thermospray LC/MS. Early reports on the application of MS/MS to biomedical problems utilized direct sample introduction methods and stressed two interrelated features of the technique, viz., (i) its extremely high selectivity which, it was argued, would obviate the need for sample cleanup and chromatographic separation prior to analysis and would provide an alternative to time-consuming GC/MS procedures, and (ii) the rapidity with which direct insertion MS/MS analyses could be performed and the consequent cost effectiveness of the technique. Indeed, several rapid and very sensitive trace analyses of foreign compounds have been carried out by this approach, most of which have been performed on triple quadrupole instruments. An impressive example from the recent literature is to be found in the work of Sanders and co-workers (C67) who developed an MS MS assay for ergotamine in biological fluids, based upon Cd&I with negative ion detection. Ergotamine is thermally unstable and is not, therefore, amenable to GC/MS analysis. By the direct insertion MS/MS technique, however, the compound could be quantified in relatively crude extracts of human plasma down to a concentration of 2 pg mL-l from a 1-mL plasma sample; the CV at this lower limit of detection was 17.5%. When the target analyte is of endogenous origin, however, the situation can be quite different. In most cases, naturally occurring substances present in biological fluids at trace levels will be accompanied by ANALYTICAL CHEMISTRY, VOL. 58, NO. 5, APRIL 1986
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numerous structurally similar or isomeric compounds, some of which may be present in large excess and which, therefore, present potentially major sources of interference in MS/MS analyses performed by direct sample introduction. Furthermore, derivatization of the biological extract in order to enhance the mass spectral properties of the compound-ofinterest may exacerbate the problem in that similar derivatives will likely be produced from a multitude of extraneous components, thereby adding to the possibility of interference and detracting from the selectivity theoretically achievable by MS/MS. Recent MS/MS assays for estradiol-17P in human plasma (C68)and tryptoline in rat brain tissue (C69)illustrate these points; in neither case could satisfactory results have been obtained without sample cleanup and without the aid of efficient capillary GC columns for sample introduction. The examples discussed above represent two extremes of the approach to trace analysis by MS/MS, and there will undoubtedly be many intermediate situations. Much effort at present is directed toward the use of short capillary GC columns for MS/MS work, in which a relatively high degree of chromatographic resolution can be achieved without sacrificing the gain of brief analysis times (C70, C71). Moreover, GC retention data on the compound of interest (an important, although regrettably often overlooked criterion of identity in GC/MS work) is preserved and sensitivity of detection is maximized since the sample is presented to the mass spectrometer in a narrow time interval. In their review, Johnson and Yost (C65)state that recent examples “...have shown the wisdom of not giving up everything we have learned about sample extractions and chromatography, but rather making suitable trade-offs between the selectivity of MS/MS and the selectivity of sample preparation and separation“. We concur heartily with these sentiments, and suggest that the gains in selectivity and sensitivity achievable with GC/MS/MS systems, rather than savings in time, will provide the primary impetus for future biomedical applications of tandem mass spectrometers. Recently, attention has been focused on discussion of the parameters that would lead to faster and more sensitive capillary GC/MS. This has led to optimizing these parameters and demonstration that a 50-pm capillary column coupled directly to the ion source vacuum is beneficial in terms of speed of analysis and sensitivity. E1 spectra were obtained from 50 pg, which could usually be correctly identified (C72). In another study, short open tubular columns with an inner diameter of 0.25 mm were used with emphasis on GC/MS/MS to increase the selectivity of the analysis (C73). The development of a GC/GC/MS apparatus has been described consisting of the combination of a high capacity, high polarity packed first gas chromatographic column followed by a low capacity, low polarity, high-resolution second chromatographic column. The component of interest is switched from the first column into a cold trap and then flash evaporated into the second column, and the technique has been illustrated by analysis of complex mixtures from environmental sources (C74, C75). GC/MS has been employed to determine atmos heric carbon disulfide a t the parts-per-trillion level using ! C34Sz as an internal standard (C76). It has been suggested that (diethy1amino)ethyl esters of carboxylic acids can be used to enhance a specific fragment ion for quantitation using GC/MS (C77). Using a GC double focusing mass spectrometer/ quadrupole instrument, it has been shown that analysis of oestradiol bis(tert-buty1)dimethylsilyl ether gave a detection limit below 10 pg with selected reaction monitoring involving the molecular ion and (M - 57) fragment. With this instrument, parent ion mass resolution (5000) was used to eliminate detection of all components but that which was being analyzed (C78). Work has been described using catalytic transformation of elutin alkenes employing an on-line gas-phase microreactor located ietween a GC column and the mass spectrometer (C79). “Currently, there is no universal LCMS system, and maybe this is too much to hope for. [The] choice of interface depends on the problems to be addressed and laboratories with a wide range of applications may require more than one system. The growth of interest in this area in recent years indicates a bright future for LCMS and exciting further developments in the technique” (C80). One gets the impression that LCMS in particular forms is quite important for certain classes of substances, but it has certainly not yet become the universally important and versatile technique that GC/MS has become. 170R
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Games has outlined all of these points in his recent review with 115 references (C80). There are numerous discussions of the pros and cons of the various strategies that are under active investigation for particular reasons in specific laboratories. A review that concentrates on interfacing microbore HPLC with mass spectrometry discusses the subject with 74 references (C81). Other points of view have been presented on the subject (C82, C83). I t has been pointed out that the acid-base reactions of solvents with solutes in the case of direct liquid introduction LCMS techniques resemble acid-base reactions in the gas phase, not the condensed phase (C84). This paper discusses prediction of ion populations on the basis of gas-phase proton affinities and acidities. Further papers have discussed the direct coupling of micro-HPLC in mass spectrometry (C85) and with atom liquid SIMS (C86). Supercritical fluid chromatography was coupled to a mass spectrometer using a modified direct liquid introduction interface (C87), and supercritical fluid chromatography mass spectrometry was used to characterize the polycyclic aromatic hydrocarbon fraction of a variety of marine diesel fuel samples (C88). By use of a range of compound types it has been shown that the new Finnigan MAT moving belt system is superior to previous interfaces of that type in its ability to provide mass spectral data from low volatility thermally labile samples (C89). A variety of other contributions involving the moving belt interface in LCMS have appeared (C90494). Vestal has reviewed recent applications of thermospray LCMS (C95),and in addition has described a new vaporizor powered by a feedback control triac power supply, which provides both improved performance and better stability over previous interfaces (C96). This paper discusses both the theory of this improved interface and the results of experimental studies of various operating parameters and their effects on performance ((296). Other optimization studies have been carried out in connection with the analysis of herbicides and pesticides using thermospray (C97). The selections of solvents and electrolytes have been discussed in considerable detail (C98-Cl02). The technique has been applied for separation, identification, and quantitation of acyl carnitines in the urine of children with a variety of disorders of metabolism (C103)and has provided the first specific chromatographic method for analyses of these substances intact. In related work, Millington, Roe, and co-workers have reported on the use of both low-resolution(C103u)and high-resolution (C103b) LSIMS techniques for the study of acylcarnitines excreted in metabolic diseases and have demonstrated the great potential of these new methods for diagnostic purposes in clinical research. Other examples for the determination of drugs and their metabolites and biological fluids have been presented (C104). An example of a specific class of substances that are amenable to thermospray LCMS is provided by McCloskey’s studies of the enzymatic hydrolysates of nucleic acids (C105, C106) (see discussion in Nucleic Acid section of this review). Some work has continued using electrospray for LCMS coupling (C107, C108). Hayes and his group have continued to develop and articulate appropriate methodology for determination of stable isotopes such as a double comparison method for mass spectrometric determination of hydrogen and isotopic abundances (Clog),isotopic analyses based on the mass spectrum of carbon dioxide (C110),and practice and principles of isotopic measurements and organic geochemistry (C111). Further discussion of methods of sample preparation for isotopic analysis has also been presented (C112-C115u). Evaluation of a commercial prototype twin mass spectrometer system from VG has been carried out for hydrogen deuterium and 180/160 ratios (C116). A method has been described for computing the relative abundances of unlabeled and monodeuterio-labeled materials in isotopic mixtures for compounds whose mass spectra contains significant (M - H)3peaks (C117). Atom SIMS has been used to measure the 5sFe/56Feisotope ratio in connection with iron bioavailability studies (C118). Fourier transform ion cyclotron resonance mass spectrometry is a subject about which a large number of extensive reviews have appeared within this 2-year period (C118-C126). Many of these are devoted to established aspects of FTICR-MS such as unimolecular processes and photon and multiphoton interactions, electron attachment processes, ion/molecule reactions, thermochemistry, equilibrium studies, reactions involving metal ions, and so on (C120-Cl22, C125). Since these represent more than ample coverage, the reader
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is referred to them for the details, and we will mention here only briefly those aspects that relate to the analysis of polar and labile biological materials. As indicated above, there is a sustained instrumentation development effort under way in attempts to realize the often touted considerable future potentialities and versatility of FT-ICR-MS, which are expected to help address some of the serious analytical problems facing the new biological mass spectrometry. These include the expected benefits that would accrue from features such as high mass analysis at high sensitivity and high resolution with the inherent ability to study collisional activated dissociation processes, in short, high mass, high sensitivity MS/MS. There is a certain suite of incompatibilities between the presently exciting widespread use of liquid matrix ionization techniques and the ultra-high-vacuum systems required for high performance of the FT-ICR systems, especially at high mass. While FT-ICR seems to be emerging from the analytical curiosity stage in this area, it has not quite made it into the forefront of SIMS of either the solid or liquid variety. However, laser techniques may develop into an important role in this regard. The most promising general developments revolve around formation of biological ions in one place and then transferring them into an ultra-high-vacuum ion trap in order to carry out the analyses. Certain successes are occurring using three approaches to solving this problem, one being the differentially pumped dual cell, the second being electrostatic lens to push ions from the outside into the ion trap, and third, the tandem quadrupole ion trap system. Another area that required improvement is in the design and excitation/deexcitation of the ion trap itself (C127-C130). This includes such work as “tailored” and ”stochastic” excitationJdeexcitation (C131,C132).
ENERGETICS Ionization Energies (IEs). A thorough review of electron ionization (EI) of gaseous molecules has been given by Mark (01, see also 02,03).A paradox concerning photoionization (PI) branching ratios of 2C02has been resolved in terms of interaction between specific rotational levels of the [‘2C02]+* B (O,O,O) level and vibronic levels of the A state (04).The adiabatic IE of [NH,]. has been determined to be 11.14 f 0.01 eV, which is 0.3 eV lower than the previously accepted value (05). PI has been achieved of selected single rotational states of Hz ( 0 6 ) and NO (07,see also 0 8 ) . The ionization energy (IE) of CClz has been measured as 9.1 f 0.1 eV using synchrotron radiation (09). Doubly charged ions of di- and triatomic molecules have been studied theoretically (010, D l l ) , and experimentally using synchrotron radiation (012,013). Small doubly charged ions have been subjected to translational spectroscopy (014, 015). The existence of triply charged ions has been the subject of a little controversy (016, Dl 7). IE’s have been measured for 11alkali metal dimers usihg P I (018). PI efficiency curves have been obtained for (Ne)z using synchrotron radiation (019).Appearance energies have been measured for [N3]+and [N4]+-from (N2)2(020).Correlation algorithms have been presented for IE’s of organic molecules (021,see also 022). With PI, the values of IE’s of (Na), cluster ( x = 1-8) have been refined (023)and I E s have been determined for oxidized metal clusters Na,O and K,O ( x = 1-4) (024)(see section “Inorganic Compounds and Metal Clusters”). Heats of Formation (AHf‘s).Lowing and Holmes (025) have shown that stabilization in even-electron carbocations depends primarily on the size of the ion. Heats of formation ( A H i s ) of immonium ions in the literature may contain a systematic error, stemming from reverse energy barriers to a-cleavage in amine molecular ions (026,see also 027). AHf‘s of organic radicals and other unstable neutrals have been see determined from appearance energies of ions (028,029, also 030),and AHis of halomethyleneshave been determined from ion/molecule thermochemistry (031).Similarly, A&’s have been obtained for (E)-and (Z)-prop-l-en-o1(032). AH‘S of a number of organic ions have been determined and in some cases revised (033-035). From a time-resolved photoionization study, AH’, ([CGHS]:) has been placed at 1141 f 10 kJ mol-’, resolving certain discrepancies (036).A H i s have been obtained for the complex [C6H6-HCl]+and the neutral van der Waals precursor (037). RO-H bond energies from gas-phase acidity/electron af-
finity data have been revised (upward by about 8 kJ mol-’) bringing them into line with thermokinetic values (038). Thermodynamics of Ion/Molecule Equilibria. Van’t Hoff plots of the logarithm of the equilibrium constant against the reciprocal of the temperature have been reported by Kebarle et al. for [K]+reacting with various organic molecules (039),alkyl ions reacting with halogen-containing molecules (040),chloride transfer reactions (041),and protonation of glymes and ethers (042).Thermodynamic data (AGO, AS’, AHo) have been obtained from the plots. The general significance of gas-phase measurements on ion/molecule equilibria has been reviewed (043,see also 044,045). Relative gas-phase acidities have been determined for alkanes (046).The effects of methyl substitution upon gasphase acidities of halo-substituted organic acids have been investigated (047).Hydrogen bond energies in bihalide ions have been determined (048).Gas-phase acidities (Le., anion proton affinities (PAS))of molecules containing phosphorusand nitrogen-carbon bonds have been examined theoretically (049,se also 0 5 0 ) . A continuous scale of gas-phase basicities from CHI to HzO has been constructed, correlations between proton affinities and core binding energies are discussed (051). Gas-phase basicities of diols (052),phosphine oxides and phosphoramides (053),and furans (054)have been studied. Proton affinities have been determined for NO2 ( 0 5 5 ) and for CH3NC0, CH3NCS, and CH3SCN (056). The P A of NH3 has been estimated as 859 f 2 kJ mol-’, in an effort to resolve a dispute concerning the exact magnitude of this quantity (057).Both I E s and PA’s have been determined for N,N-dialkylanilines (058). Electron Affinities (EA’s). There have been a number of determinations of electron affinities (EA’s) on the basis of anion-molecule equilibria (059-061). The EA of SFGhas in this way been placed a t 1.05 f 0.1 eV (062).EA’S have also been obtained by studying the unimolecular dissociation of negative ion dimers and considering the competition between moieties for the electron (063).A flowing afterglow study has confirmed the EA of SOz as 1.1f 0.1 eV (064,see also 060), in contradiction to a value of 2.2 eV from ICR. EA’s have been determined for fluorocarbon radicals (065) and for KOH (066). The EA of WFGhas been measured as 3.36 eV (067). Photoelectron spectroscopy has provided EA’Sfor CCO and HCCO (068). The infrared (IR) spectrum of [“I- has been obtained by autodetachment spectroscopy (069).[CH2]-and [CD2]-formed by the flowing afterglow technique have been studied by photoelectron spectroscopy (070,see also 071).
UNIMOLECULAR REACTIONS AND ION STRUCTURES Unimolecular Reaction Rates. Interpreting a mass spectrum remains, at one level or another, an exercise in unimolecular reaction rate theory, and the dynamics of unimolecular reactions is, in the lingua franca of modern chemistry, not a solved problem. The comments of Zare et al. (El) to the effect that even the best potential surface and trajectory calculations still represent only the first step in properly understanding the chemistry in terms of electronic structure could legitimately be extended to unimolecular reactions of polyatomic systems. Nonadiabatic reactions frustrate rigorous quantum mechanical approaches, although, as is clear from the work of Lorquet et al. (E2),significant progress is being made with incorporating semiclassical approximations of nonadiabatic coupling into transition state theory. A “quantitative theory of mass spectral fragmentation patterns”, inspired by multiphoton ionization studies, has been described (E3).The theory does not provide physical insight and appears insufficiently accurate for analytical purposes. The dynamics of unimolecular ionic reactions in the gas phase have been reviewed (E4, E5). Ionization of CF3CHBat very high energies (lo2eV) allows the nature of the fragmentation to be controlled through selection of the ionization site (E6). If this proves to be a general phenomenon, it will be a highly significant finding. Ionized perfluoropropylene fragments from an excited electronic state (E7). The evidence that enol ions isomerize to an acetone structure and decompose to lose the newly formed methyl group preferentially is convincing; the behavior is referred to as nonergodicity (E8, see also E9,E10). It does not, however, necessarily follow that the overall rate of decay ANALYTICAL CHEMISTRY, VOL. 58, NO. 5, APRIL 1986
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of the reactant enol ion cannot be adequately treated by
quasi-equilibrium theory (QET), since the enol and the transition state for isomerization may be in equilibrium. Events subsequent to the transition state in the rate-determining step are neglected in QET calculation of rates; however in this case these events determine the nature of the product. To probe energy randomization through translational energy release ( E l 1 ) necessitates close consideration of possible isotope effects not only in the chemical dynamics but also in instrumental factors determining metastable peak shapes. Lifshitz et al. (E12, E13) have successfully applied timeresolved photoionization mass spectrometry in the millisecond range, at which times emission of infrared radiation becomes a factor to be considered. Iodobenzene (E14),bromobenzene (E15), and phenol (E16) have been studied. Metastable [CF,]+- and [CC14]+.ions have been observed following E1 (El 7). Unimolecular decay of metastable formaldehyde ions appears 2 eV above the first dissociation limit (E18). Deuterium isotope effects in metastable ion decompositions of benzene (El9) and pyridine (E20)have been analyzed on the basis of QET (see also E 2 l ) . The dependence upon internal energy of secondary deuterium isotope effects has been demonstrated (E22). Break-down graphs have been obtained by charge exchange for haloanisoles (E23) and phenylethanol (E241 ions. Rate constants k(E) have been calculated by Brand et al. (E25)from field ionization kinetics (FIK) data (see E26). Charge exchange, angle-resolved CAD and FIK have been compared in the case of pent-3-en-2-01(E27). Photoion-photoelectron coincidence (PIPECO) (E28,E29) is now a widely used technique, finding application with, for example, diatomics (E30,E31),carbonylchromium (E32),and silanes 0333, E34). Rate constants k(E) are determined in many studies, although the restriction to slower decompositions (ME) 5 ca. lo7 s-l) remains. PIPECO measurements have been reported for [C2H$- and [C&]+. (E35),[C5HI0]+(E36),[C&O]+* (E37), [ c H@]+. (E38), [C~HSNO,]’. (E39E41), [C6H50CH3]+-(E42?, and [C4H4N2]+(E43). Earlier measurementa with methvl nitrite have been reassessed (E44). and iodobenzene has been the chosen object of “benchmark measurements” (E45). Arrhenius plots for thermal decomposition of protonated ethers have yielded, for the first time, activation energies and A factors for gas-phase unimolecular reaction of ions (E46, E47). Translational Energy Release. That the numbers of papers on the subject of translational energy release has fallen over the past few years is probably a reflection of a general difficulty in securely interpreting the information in terms of useful chemistry. A factor compounding the problem is that accurate analysis of metastable peak shapes to obtain translational energv release distributions (TERD’s) is not a trivial exercise (E28). TERD’s have been obtained for the major fragment ions from benzene, and, in those cases in which there is no significant energy hump, reproduced by phase space theory (E49, see also E50). Time-resolved TERD’s for loss of CO from phenol ions confirm that energy release increases with increasing time, which is interpreted in terms of the fraction of reverse critical energy partitioned into translation rising at longer times (E51). Limitations of the empirical relationship of Haney and Franklin (T = E*/0.44n) have been demonstrated (E52). Translational energy releases have been studied to elucidate spectroscopicstates of [COz]+-(E531 and structural features of large organic ions (E54-E57). TERD’s for [CrCO]+contain structure reflecting vibrational levels of CO (E58). Deuterium isotope effects on translational energy release in decompositions of protonated formaldehyde and protonated methylimine have been interpreted at a detailed mechanistic level (E59). Deuterium isotope effects with formic acid ions have also been interpreted mechanistically (E60). Ion Structures and Molecular Orbital Calculation. Resorting once more to the lingua franca, “structure“ is said to be a solved problem. In the context of ion structure, this means that energies and geometries of stable structures of small ions calculated using ab initio molecular orbital methods with appropriately sophisticated basis sets are expected to be “accuraten. The identification of transition states by locating cols (stationary points) on the potential surface is a less straightforward, but nevertheless close to routine, procedure (E61, E62). The difficulties and the pitfalls arise when 172R
ANALYTICAL CHEMISTRY, VOL. 58, NO. 5, APRIL 1986
mechanistic conclusions are required from the calculated wells and cols on the many-dimensional surfaces (see section “Reaction Mechanisms”). Structures and relative ener ies of stable ions (i.e,, wells on the potential surfaces) have teen calculated for [C3H70]+ (E63),[CH,O,]+ (E64),chromium cations (E65,E66),and a wide variety of dications (E67-E71). Structures and stabilities of [C6H6]3+.have been calculated by use of ab initio methods (E72). Detailed investigations of electronic structure (i.e., excited states are considered)have been reported for [CH2N](E73),[CH,OH]+. (E74),and [CO]+. (875). Ab initio calculations have been performed on the lowest T state of [CF,]+. (E76). Fundamental vibrations of [CO]+., [C021+.,and [CSz]+. have been calculated using GAUSSIAN 76 with a 4-31G basis set, and good agreement has been obtained with experiment (E77). Calculations of electronic transitions in [C3H3]+have been discussed in the context of laser-induced fluorescence (E78),and there has been a detailed study of ionic states of nitrosyl cyanide (E79). The assignment of qualitative structures to small organic ions observed experimentally has been reviewed by Holmes (E80, see also E81). Structurally complex organic ions involving noncovalent interactions have been reviewed by Meot-Ner (Mautner) (E82,see also E83). By use of various experimental techniques, structures have been characterized or elucidated for [C2H2]+.(E84),[C3H3]+(E85,E86), [CH,N]+ and [CH,N]+ (E87),[CH302]+(E88),[c&o]+. (E89),[c5&]+ (Ego), [C3HsOl+. (E91), [C2H3Sl+ (E9-8, [C$&I+* (E931, [C4H70]+(E94), and [C5H110]+ (E95). Formation of [CH F2C1]+ (E961 and [C12CC1C1]+., [Cl2CC1Br]+., and [Br,tBrCl]+. (E97, see also E98)have been reported. Claims (E99,E100) to have formed [CH2CH4]+.have been questioned (E101). It is implied (El01) that a spectrum originally attributed (E99,E1001 to IC,Ha12+is in fact due to 113C12CHK12+-: a spectrum originally &soc&ed with [CD2CD4j+.coulduiot be reproduced. Lithiated pentacoordinate carbocationshave been proposed to account for observations made using flash vaporization mass spectroscopy (E102). The o-complex (Wheland intermediate) structure has been assigned to gaseous heptaalkylbenzenium ions 03103). Multiply charged [C&6jn+ cations up to n = 6 have been produced by E1 (23104). Doubly charged [C6H6I2+ ions of different structures have been distinguished on the basis of fragmentation initiated by charge exchange (El 05). The preferred sites of protonation have been elucidated for morpholine and related molecules (El%). It was not possible using FT-ICR to detect [SiH,]’., casting doubt on earlier reported observation of this species (E107, see also E108). Formation of chloroethylene anions by e attachment has been studied (El09). Long-lived -[H,01-. - - has been formed in the gas phase (EllO). Reaction Mechanisms. The area of mechanisms of decomposition of positive ions has enjoyed a considerable fillip from the recognition of the possibly widespread role of “distonic ions”. ”Distonic ion” ( E l l l ) ,referring to species with separated charge and radical sites, is jargon within the field of mass spectrometry, since such species are known and discussed apparently without need of this name elsewhere in chemistry (E112). The speed with which the term has been taken up in mass spectrometry, however, could be an indication that it is destined to become accepted chemical nomenclature. As more mechanisms involving distonic ions are postulated and structures of distonic ions established (E113-E117), a degree of doubt seems to grow concerning the importance of ion-molecule complexes as reaction intermediates (E118, E119). If, however, there is such a swing away from ionmolecule complexes toward distonic ions, Longevialle’s work with steroids warns of the need for caution since his studies demonstrate convincingly the role of ion-dipole complexes as intermediates in these very large ions (E120,E121). The growth of ideas such as distonic ions and ion-dipole complexes can be seen as a consequence of the realization that concerted mechanisms are not common (E113,E122, E123) and recognition that many ionic rearrangements are seemingly inexplicable by stepwise mechanisms involving more conventional intermediates. y-Hydrogen rearrangements such as the McLafferty rearrangement are examples where the weight of evidence is in favor of stepwise mechanisms in all cases except possibly one, and that one case is the sub‘ect of some controversy (23113). Roles of distonic ions in t i e fragmentation
MASS SPECTROMETRY
of ionized aliphatic amines (E124) seem particularly clear (El 14, E125-E129), although counterproposals in terms of ion-molecule complexes exist (E130). The role of ion-dipole complexes in ion-molecule reactions is well established, and isomerization of carbonium ions in such complexes formed by collision has been studied (E131). Proton-bound bimolecular complexes, a variation on the ion-dipole theme, have been proposed as a general mechanistic concept for fragmentation followin CI (E132). Mechanistic stucfies by field ionization kinetics (E133), metastable ions (E134),and negative ions (E135)have been reviewed. The question of whether McLafferty rearrangements occur in even-electron ions has been examined (E136). Keto-enol tautomerism in radical cations has received considerable attention, constituting as it does a good stereotype of stepwise rearrangements of positive organic ions (E137E139, see also E140, E141).Other positive organic ions to be studied using metastable ion techniques, isotofic labeling, and CAD include [C2H60]+. (E142),[C3H60]+ (E143,E144), aliphatic esters (E145-E147), [C4HaO]+(E148), [C5HloO]+ (8149, E148, E150-E152), methylbutanols (E153),methylalkanes (E154), dimethylpentene (E155), nitrotoluene (E156), [CgH90]+(E157),hexenone (E158),and trinitrotoluene (E159). As a general phenomenon, ring-opening maintains considerable attraction (E160-E162); in particuIar, significant progress has been made with the cyclopropane [C3H6]+.system (E163). Transition states or intermediates of pyramidal structure have been invoked to explain “scrambling” of C atoms in aseous carbocations (E164). Neutrals formed following E f o f alkanes (E165) and alkenes (E166) have been characterized; the structures conform to reasonable expectations. Stereochemical effects (E167) have been studied with respect to the retro-Diels-Alder fragmentation (E168),cyclohexanols (E169, see also E1 70),cyclohexanes (E171),mesaconates (E172),steroids (E173),and decalyl acetates (E174). It has been concluded that the conformational distribution of decomposing cyclohexylmalonate ions is as would be predicted on the basis of solution chemistry (El75). The retroDiels-Alder reaction in mass spectrometry has been reviewed by TureEek and HanuEi (E176). Substituent effects are studied (E177, E1 78, see also E1 79). The advantages for mechanistic studies of running E1 mass spectra a t low electron energies and low temperatures have been demonstrated (13180). Double 13C labeling has been employed to elucidate loss of ethene from ionized tetrahydrothiophene (E181).Claims of equilibration of oxygen atoms in field-ionized methyl esters have not been corroborated by an E1 study (E182). Through-space interactions appear to be involved in the fragmentation of ionized 1,Bnonadiyne (E183). The proposal that protonated d y l phenyl ether undergoes a fragmentation akin to the Claisen rearrangement has been supported (E184, E185). Triplet [CD O]+ has been formed from [CD30]- by collision-induced ckarge reversal (E186). It has been suggested that unimolecular decomposition of this triplet by loss of Dz gives singlet products (E186). Barriers calculated for 1,Z-H shifts in [HNC]+./[HCN]+. are high (E187, see also E188). Frontier-orbital arguments have been used to explain why there is a significant energy barrier for the addition of H. to [RCO]’ to give [RCOH]+. (E189). Mechanisticstudies resting on quantum chemical calculations (E190) have been undertaken for [CzH401+.(E1911, [CzH2Fz]+.(E192),glycine radical cation (E193),and [C2H20]+.(E194).
SPECTROSCOPY Multiphoton Ionization (MPI). The extensive fragmentation, which can occur in UV/visible multiphoton ionization (MPI), can involve photon absorption by the neutral molecule, by the molecular ion, by neutral fragments formed either directly from the neutral molecule or from the molecular ion, and by fragment ions (F1-F5). This potential complexity creates possibilities for selectivity and control over ionization and fragmentation, and hence there are possibilities of developing specific methods of detecting particular molecules (F6-F9), e.g., azabenzene (FIO) and halogenated aromatics (Fll). Changing the laser pulse width from nanoseconds to picoseconds in MPI of benzaldehyde has demonstrated that “ladder switching” (photon absorption by fragment ions)
decreases as the laser pulse width decreases ( F I 2 ) , since fragmentationof the parent ions (or neutral) must occur within the laser pulse time window if the fragments formed are to absorb photons from the same pulse (F13). “Ladder switching” is thought to be involved in metastable ion decomposition following MPI (8’14) of aniline ( F I 5 ) . The dependence of the decomposition of metastable [C&]+* ions to [C6D5]+and [c6D4]+-upon laser power has been analyzed on the basis that the decompositions occur via a ladder mechanism (i.e., photon absorption occurs in the neutral and in the parent ion, and all fragment ions originate from the parent ion); the ion absor tion cross section has been esticm!(F16, see also F17, F18). Rate mated to be 5 X constants k ( E ) as a function of internal energy have been obtained for [C6H5Cl]+- [C6H6]++ C1. through analysis of distorted fragment ion peak shapes and knowledge of the numbers of photons absorbed (F19). Rate constants accessible are of the order of 105-107s-l, and in the case of chlorobenzene (F19) agree with PIPECO results (see also FZO). The method must suffer from uncertainty in the internal energy due to the departing electron having an undefined kinetic energy (see
-
F21). MPI of ArNO, NeNO, and KrNO van der Waals species has yielded structured excitation spectra in some cases (FZZ, see also F23). The apparent stability of toluene dimer ions with high internal energies, as evidenced by MPI (FZ4),has been explained on the basis that energy transfer between relative motion of the two monomers and their internal motion is severely restricted on account of the mismatch in vibrations (F25, see also F26). [NH3]+.can be formed by MPI with a high degree of vibrational selectivity (FZ7).Vibrational frequencies have been determined for [C6H50H]+.by means of MPI photoelectron spectroscopy (F28). MPI has been applied to detection of neutrals desorbed from surfaces (FZ9). The energy spectra of emitted electrons from MPI of rare gases contain peaks corresponding to various numbers of photons absorbed (F30). Transition-metal carbonyls have been examined using MPI techniques with a view to producing metal or metal-cluster ions (F31-F33). MPI of Cr(CO)6, for example, yields more ground-state [Cr]’ than does EI, the difference is significant in ion-molecule studies (F33). Two-Color Laser Techniques. Two-color techniques, in which two laser beams of different wavelengths are used, are essentially “multiphoton techniques”, but are considered separately because for mass spectrometry they can afford significant advantages over single-color MPI. One laser can be used to produce molecular ions in their ground vibronic state, and a second laser can be used to excite the ions to selected energies (F21, F34). In this way, the uncertainty arising from the energy of the departing electron is removed, since its energy is zero. The results of such experiments with benzene indicate that the decompositions to [C6H5]+,[C6H4]+*, [C4H4]+-,and [C3H3]+are all in competition with each other (F21). El-Sayed et al. (F35-F37) have shown how fast reaction can be studied by two-color techniques through using short laser pulses (picosecond) and investigating the dependence of fragmentation on the time-interval between the first-color and second-color pulses. It has been concluded that the rate of energy distribution in the 2,4-hexadiyne molecular ion is comparableto the delay times (nanoseconds)employed in the experiments (F36) and that excited electronic states of this ion can fragment directly without relaxing to the ground electronic state (R38-F40). In these particular experiments, the internal energies of the molecular ions are uncertain, because ionization with the first laser produces an excited state rather than the ground vibronic state. Two-color techniques have been used to obtain precision and control in photoionization efficiency studies, providing adiabatic IEs and vibrational frequencies of ionic states for aniline (F41),diazabicyclooctane (F42, F43), and NO (F44). IE’s have been determined for van der Waal’s complexes (F45-F48), such as p-xylene clustered with Ar, (F49). The van der Waal’s phenolbenzene molecule has been photodissociated and the products detected using a two-color picosecond technique (F50). There are other spectroscopic techniques related to some of the two-color laser techniques, in the sense that stable gaseous ions are formed and then subjected to laser spectroscopy (F51). For example, laser-induced fluorescence of ANALYTICAL CHEMISTRY, VOL. 58, NO. 5, APRIL 1986
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dihaloacetylene cations has been studied following Penning ionization (F52). Photodissociation (PD). In most laser-ion beam studies of photodissociation (PD), the ions are formed by E1 and excited by photoabsorption, so that, while the internal energies are not fixed absolutely, it is possible to vary the relative internal energies by changing the photon wavelength. This has allowed Russell et al. (F53) to distinguish geometric isomers on the basis of dependence of translational energy release upon wavelength. The conclusions of a laser-ion beam study (F54)of 2,4-hexadiyne seem to be at odds with those of the two-color laser study (F38)as regards involvement of excited electronic states. Other ions formed by EI, whose photodissociation has been studied by laser-ion beam techniques, include molecular ions of halogens (F56),halopropyne ions (F57), and [C4H4]+.(F55). A laser-ion beam study (F58) of n-butylbenzene has found that the increase with photon energy in the ratio of the intensities of m / z 91 and m / z 92 fragment ions is much less dramatic than has been reported previously (see also (F59));the fragmentation of n-butylbenzene is important because of its role as a “calibrant” of energy deposition in CID. Photodissociation of nitrobenzene ions selected by PIPECO has been achieved, thereby ensuring that the internal energy is fixed prior to photoabsorption (F60). It is envisaged (F60) that this development will make possible absorption spectroscopy of excited ions and determination of fast unimolecular rate constants [lo9 s-l]. P D of porphyrin ions produced by FAB selectively characterizes these complex molecules (F61). PD of metal P-diketonate molecular ions has been reported (F62). PD of the dimer ions [(CO,),]+. (F63) and [KrC02]+. (F64)has been studied. Photodissociation of ions trapped in ICR cells has been reported for a number of small organic ions, including (F65),halobenzene ions (F66),[C,H,]+. (F67),and [C5H6N]+* aromatic hydrocarbon ions (F68). Multiphoton absorption (COzlaser) by [C5H10]+.,[C6H12]+.,and [C7HI4]+* trapped in an FT-ICR cell leads to slow decomposition ( k ( E ) 1-50 s-l (F69,see also F70)). Beynon, Harris, et al. (F71)have reported experiments in which high PD yields are obtained by trapping the ions in the space charge within an E1 source. McIver et al. (F72) have shown that PD of oligopeptide ions in an FTICR provides worthwhile structural information. Dunbar et al. (F73, F74) have developed two-pulse techniques in which ions are formed by E1 in an ICR cell, irradiated with one laser pulse, and then after a time-delay irradiated with a second pulse from the same laser. The technique allows collisional and radiative relaxation processes to be characterized and has been applied to halobenzenes (F73, F74). Cross sections for infrared laser photodetachment from vibrationally excited allyl ions are high (F75).
COLLISION PHENOMENA Ion-Molecule Reactions (IMR’s). Cycloaddition reactions have been studied by forming the adducts in a highpressure chemical ionization (CI) source and probing their structures by CAD (G1-G5, see also G6-G8). For the reaction of the 1,3-butadiene cation radical with 1,3-butadiene ( G I ) and for most other reactions studied (G2, G3, G5),the results indicate a stepwise mechanism. In the case of a ketene radical cation and ethylene, it is concluded that the mechanism is a facile [2 + 11cycloaddition (G4). [CC13]+forms a stable adduct with styrene but not with a number of aliphatic olefins (G9). Many condensed species were formed following PI of allene ( G l 0 ) and of butene (G11). Adduct ions have been formed in the collision cell of a triple quadrupole (G12,see also GI3), and fragmentation of anionic adducts has been studied by use of the flowing afterglow technique (G14). The temperatufe dependences of dimensation reactions of [CH2CF2]+-,[C&,] and [C&]+* have been reproduced using statistical phase space theory ((315,see also G16). The formation of the protonated dimer from a trinitrotiazine has been shown to proceed via a t least three different reaction channels (G17). Gas-phase reactions of nucleophiles (G18) have been investigated by ICR (G19),flowing afterglow (GZO),and highpressure MS (G21, G22). Transition state energies have been estimated for SN2reactions [Cll- + RBr + RCl + [Brl- (G22). Reactions of [F]- and [“,Iwith acetates have been studied under CI conditions (G23). A chelation effect has been proe,
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posed to be involved in the binding of [F]- and [ClJ-to certain oxygen-containing fluorocarbons (G24). [OZ]-.oxidizes hydrazines (G25)and catechols (G26)in the gas phase to produce anion radicals and HzOz. CI reactions of double and triple bonds have been sought as means of determining the bonds’ locations (G27). Munson et al. have evaluated selective reagents for CI (G28-G30, see also G31). Relative efficiencies of various monatomic and polyatomic gases for moderating electron energies in negative CI and for collisional stabilization of ions have been determined (G32). Results using the selected ion flow tube (SIFT) technique (G33),in which the ion of interest is separated from other ions and parent gas before entering the flow tube (the “reactor”), are in contradiction to earlier results using the closely related flowin afterglow technique, as regards reactions of [e]+. [CH,] ! , and [C2H2]+.with CzNz(G34,see also G35-G37) and the proton affinity of HC3N (G38). The SIFT technique has been applied to study reactions of [CN]+ (G39),[C,H]+ (G40), and [NH,]+. and [ND,]+. (G41). The mobility of [CN]+ in He has been evaluated over a range of fields and particle densities (G42). The flowing afterglow technique has been used to study reactions of methyl formate and N,N-dimethylformamide (G43)and of trimethyl phosphite (G44)with anions, of the acetyl anion (G45),of organosilicon anions with NzO (G46),of N- and S-containing anions (G47, G48) and of isomeric arylallyl and arylcyclopropyl anions (G49). [CH2XCH3]+. (X = C1, Br) has been shown by ICR to transfer [CH,]’ to nucleophiles and [CHz]+.to electrophiles (G50). Dipole-stabilized carbanions derived from methyl formate and related molecules have been studied by Nibbering et al. (G51,G52, see also G53, G54). Proton exchange between arenium ions and arenes is characterized by a high activation energy between the minima in a double-minimum energy pathway (G55, see also G56). A deuterium isotope effect of 5.5 has been found in the elimination reaction of amide ion with diethyl ether, setting, it is suggested, stringent limits upon the transition state’s energy and vibrational frequencies (G57). IMR’s of [C3H6]+.formed from cyclopropane by charge exchange have been studied by ICR (G58). From an FT-ICR study (G59, see also G60) of alkene radical cations with ammonia, it has been concluded that hydrogen abstraction withy a collision complex is involved in the formation of [c2H6N]. Reactions of alkyl ions with alkylamines have been studied by ICR (G61). The phenylium ion [C6H5]+has been characterized in terms of its lack of reactivity with C2H2,other [C6Hs]+isomers do react (G62). There has been a thorough study of [C6H,]+ using ICR (G63). IMR’s in unsaturated hydrocarbons at near thermal energies have been studied by ICR (G64). .The influence of interfunctional distance upon protonation and subsequent reaction of conformationally stable 0-amino alcohols has been studied using ICR (G65). The formation has been reported of [CHzN]+ from HCN, which is identified as a common impurity for CHI N20, CH4/N2,and C3H8/N20mixtures in ICR cells (G66). MR’s of SiF4 have been investigated by FT-ICR (G67). With MPI, [NH ]+ has been formed in selected levels of the v2 bending mode. The exchange reaction with Dz is enhanced by this vibrational excitation, whereas addition ([NH3D]+ + D) is little affected (G68). By use of beam techniques, reaction dynamics of [H30]+with acetone (G69) and of [CzH3]+with CHI (G70) have been studied. Beam techniques have been reviewed (G71). IMR’s pertinent to combustion have been elucidated using tandem mass spectrometers (G72). Crossed beam studies of [N2]+*/Nzchape transfers at low energy have identified both a dlrect reactlon and a reaction via a collision complex (G73, see also G74). Charge transfer (G75, G76) between [He]+. and N2 can form a predissociative state with a lifetime greater than microseconds (G77). Charge exchange between [NZl2+and Ar has been studied experimentally (G78). With a “guided ion beam” tandem mass spectrometer, an inverse intramolecular isotope effect in HD + [Ar]+. [ArH]+or [ArD]+has been observed a t low energies (G79). Probabilities of scattering beyond a certaill angle (without reference to the possibility of reaction) have been measured for numerous rare-gas ion/atom (or small molecule) collisions (G80). Formation of [H3]+ and its vibrational deactivation have been modeled using a statistical phase space method (G81). Three-body associations of [02]+. and [NO]’ with Kr and Ar at 80 K have been investigated using the SIFT technique (G82). Metastable peaks due to
i
-
MASS SPECTROMETRY
dissociation of [Ar,]+. have been observed using a doublefocusing MS (G83). Chemiluminescence and laser-induced fluorescence of reaction products in flowing afterglow have been shown to be powerful probes of d amics of ion-molecule reactions (G84-G86). Reactions stu ied include [N]+ + CO [CO]' (U = 0-2) + N (G87), [SF,]-* + Ha, D* [SF,]- + HF,DF (G88),and [F]- + HBr,DBr -+ HF,DF + [&I- (G89). Metal and Metal-Containing lons. Using FT-ICR, Freiser et al. (G90-G96) have investigated the reactions of many transition-metal and transition-metal-containing ions with organic and other small molecules. Thus, reactions of [FeCH3]+and [CoCH3]+with aliphatic alkenes and alkynes (G97) and with aliphatic alkanes (G98) and of [FeCH2]+and [CoCH2]+with cyclic hydrocarbons (G99),with olefins (GIOO), with alkanes (G101),and with cyclic alkanes (GlO2) have all been elucidated. Reactions have been examined of [FeCo21+ (G103), [FeCoJ+ (G104), and [FeO]' (G105) with hydrocarbons. [AL] (A = Fe, Co, Ni; L = isobutene, butadiene) react with ferrocene and nickelocene in the gas phase yielding mixed-metal metallocenes by ligand displacement (G106). Bond dissociation energies have been determined for bonds involving transition metals, for example, [Fe-OH]' and [Co-OH]+ (G107), [Fe-Co]' (GlOB),and V-H, Cr-H, Fe-H, Co-H, and Mo-H (G109). [Co]' reacts differently from [Rh]' with toluene, cycloheptatriene, and norbonadiene (GI 10, see also G l I l ) . The paper (G112) presented as concerning reactions of bare tetranuclear transition-metal cluster ions [Sc4]+ has been retracted (G113). The reactant ion has been identified as [Tal+, rather than [Scq]+(Sc 45 daltons, Ta 181 daltons) (G113). The mechanism of reaction of [Fe]+with olefins has been elucidated through studying the structures of the product ions by CAD (G114, see also G115, G116). In some cases, rearrangement occurs to form bis(o1efin) complexes (G114). CAD has also been used to probe the structures of the ionic products of reactions of [Co]+and [Co(ligand) ]+ with butanes (G1171. Reactions of [Au]+,[Ag]+,and [Cu]+*withalcohols have been characterized (G118). A spark source has been used to produce [Co]+(G119)and the other singly charged cations of the fourth period elements (G120,see also G121);the reactions of these ions with organic molecules have been studied (G119, GlZO). The roles of alkali and alkaline-earth metal ions in hydrocarbon flames have been investigated by direct sampling of the flame and MS analysis (G122, G123). Metd-organic negative ions have been reviewed (G124,see also G125). Reactions of N,N'-ethylenebis(acetylacet0niminato)cobalt(II) with radicals is proposed to occur at rates comparable to electron capture and to be responsible for peaks in the negative CIMS above the molecular ion peak (G126). Reactions of [C1Cr02]+with alkenes have been identified using SIFT (G127).
r
---+
+
Collisionally Activated Dissociation (CAD) and Related Beam Techniques. It has been widely supposed that in CAD the importance of electronic excitation relative to direct vibrational excitation becomes greater as the incident ion energy becomes greater, and hence that electronic excitation occurs at kiloelectronvolt energies and vibrational excitation in triple quadrupoles (G128, G129, see also G130). The maximum energy deposition with incident propane and n-butylbenzene ions has been found to occur at 140 eV and 70 eV, respectively, whereas energy deposited in methane and tert-butyl ions increased monotonically with laboratory collision energy (G131, see also G132, (2133). CAD of a proton-bound dimer over a range of laboratory collision energies shows the maximum average energy deposition at 70 eV, but the probability for large energy transfers is highest at kiloelectronvolt energies (G133,see also G134). In CAD of peptide ions up to about mlz 1200, energy deposition increases with increasing incident ion mass at a fixed ion energy (G135). These results (G131, G135) suggest that incident ion velocity (and hence interaction time), rather than incident ion energy, may be the more reliable guide to the mechanism of ion deposition. It has been proposed that in CAD of massive ions (>400 daltons) at kiloelectronvolt incident energies, direct vibrational excitation is the major mode of energy deposition (G136, see also G137, G138);the roposal is supported by molecular mechanics calculations (8139). On the other hand, evidence has been found of the role of excited electronic states in CAD of [O,]'. in a triple quadrupole ((2140). Cross sections in CAD at kiloelectronvolt energies have been found to be comparable to the physical dimensions of the incident ions
(G136-Gl38). The dependence of CAD cross sections upon ion structure has been demonstrated in the case of [cGHs]+. (G141). That angle-resolvedCAD spectra (referred to as "ARMS") are dependent upon scattering angle has been confirmed in a number of studies (GI42, G143, see also G144). The products of higher energy decomposition pathways are broadly favored at larger scattering angles. Translational energy releases in decomposition of nitrobenzene and n-butylbenzene ions have been shown to increase with increasing scattering angle (G145,see G146, G147). A careful examination of the effect of translational energy release upon ARMS has not been able to establish that internal energy deposition is related to scattering angle; ARMS of [C,&H,,]+. could be explicable solely on the basis of translational energy release (G148). Kistemaker et al. (G137) have used a coincidence technique (see G149, G150),in which angles of deflection of both ionic and neutral fragment are measured, to show that scattering during collision and scattering during dissociation (due to energy release) are of comparable importance (see also G151, G152). Neutralization-reionization mass spectrometry (G153) provides information on unstable and reactive neutrals (G154), ion structures and molecular structures. The mass-selected kiloelectronvolt ions are neutralized by collision with a target gas, for which purpose metal vapors have been found to be superior to rare gases (G155, see also G156), contrary to an earlier report (G157). Oxygen has been found to be a particularly efficient target gas for the second collision in which the neutral is reionized (G158). Ionization of neutrals by collision has been used to characterize neutral products of metastable ion decompositions (G159-Gl61); ions are prevented electrostatically from entering the collision region. It has, for example, been shown that [CH20H]- rather than [CH30]-is lost from metastable methyl acetate molecular ions (G162). Translational energies of target gases ionized in the collision with the kiloelectronvolt neutrals have been measured (G163). [HezI2+has been detected using charge stripping ([He2]+. + Nz [Hezl2++ N2 + e-) (G164). Charge stripping has been used to confirm the IE of [NO2]+ (G165) and to examine atomic ions formed by FAB (G166). Triply charged [CS2]3+* (G167)and triply charged aromatic ions (Gl68,see also G169, G170) have been studied using charge stripping techniques. Studies of doublv charged ions combining quantum chemical calculations and charie stripping measGrements have been (G1711, [C2H20]2+(G172),[CH4I2+and reported for [C2F4I2+ [CH5I2+.(G173),and polyacenes (G174). Charge stripping has been used to characterize [C&s]2f and [C,H,]+. (G175, see also G176), and [C5H6]+(G177). Doubly charged ion mass spectra have been reported for small hydrocarbons (G170), alkenes (G178). and ternenes (G179). Vibrational structure'in the [HI+'([D]+) peak from collision-induced charge reversal and fragmentation of [OH]([OD]-) has been successfully analyzed (G180). CAD of negative ions (G181, G182) has been carried out with a triple quadrupole (G183) and with an FT-ICR (G184). Types of reactions encountered in CAD using a triple quadrupole have been surveyed (G185, G186). Correlations have been found between fragment ion intensities in CAD spectra of ketone ions and product enthalpies (G187, see G188).
IONIZATION OF NONVOLATILES AND RELATED SURFACE PHENOMENA Sputtering following Particle Bombardment. The production of gaseous ions from complex biological and other molecules by bombardment with kiloelectronvolt (Hl-H3) or megaelectronvolt (H4-H7) particles is an area that would benefit from a theory with genuine predictive capabilities. At this stage, the basic question of how the primary kiloelectronvolt and megaelectronvolt energies are eventually transformed into the small translational energies encountered in desorption of molecular ions has not been satisfactorily answered (H8-H12). Much reliable information is available about the primary excitation (H14,H10, H l l , H13) and about the desorption of small organic ions (H15, H16). Measurements of translational energies of organic ions desorbed in SIMS are said to be consistent with linear-cascades uttering (HI 7, see also H18). Neutrals desorbed in SIM8 can be ionized and characterized (H19-HZ2). It is the events between ANALYTICAL CHEMISTRY, VOL. 58, NO. 5, APRIL 1986 175R
MASS SPECTROMETRY
the initial energy deposition and the final appearance of ion or neutral that remain clouded. That surface active properties are important in SIMS, when the sample is dissolved in glycerol or certain other liquids, has been emphasized by Ligon et al. (H23-H27, see also H B , H29). The evidence is strong that formation of gaseous molecular ions is assisted if the sample is present at or near the surface rather than in the bulk of the liquid (H30),which is in accord with the broad line of thinking that the molecular ions are “bumped off” the surface by some sort of presumably preferential flow of energy into critical intermolecular interactions. The surface being the desired location of sample is also consistent with the idea that colloidal droplets constitute an intermediate phase in ion production (H31). According to this mechanism, molecular ions are field evaporated from the colloidal droplets. Evidence has been given by Rollgen et al. that in SIMS of glycerol solutions the surface remains “clean” throughout the experiment, essentially because the volume of material sputtered off the liquid target following each particle impact is large (H32, see also H33). It has been shown that, in [Cs]+ kiloelectronvolt bombardment of solid sample, high beam densities favor formation of [M + Na]+ and fragment ions, whereas low beam densities favor formation of [M + H]+ (H34). It has been proposed that these [M + Na]+ and other Na-containing ions are formed by gas-phase ion-molecule reactions in the “selvedge region” (H34). Gaseous ion formation in SIMS of fatty acid monomolecular layers (on Ag) has been proposed to occur by cationization in the “selvedge”vapor phase (H35). Evidence has been put forward that in SIMS, species such as [Li2F]+and [Na2Cl]+are formed in the gas phase (H36). Vickerman et al. (H37-H39) have shown that neutral Ar bombardment of a poorly conducting material such as polystyrene causes less surface damage than [Ar]+ ion bombardment. Michl et al. (H40-H42) have given detailed mechanisms pertaining to kiloelectronvolt bombardment of frozen gases (H43,H44). It has been proposed (H45)that in SIMS using a glycerol matrix the incoming particle initially ionizes or dissociates molecules directly and that these initial radical ions undergo ion-molecule reactions. This mechanism involving radicals runs contrary to most other lines of thinking (H46,H l l , consider however H47). Radical cations are formed from 0-carotene but that this molecule has a low IE and lacks in SIMS (H48), polar sites is probably significant. That chemical reactions occur in liquid matrices in SIMS is well-recognized (H49-H53). With monosaccharides in glycerol, SIMS has been found to give adduct ions but not protonated saccharides (H54). SIMS of trehalose in glycerol has, however, yielded the protonated saccharide as well as adduct ions (H55). It has been demonstrated (H56) that in SIMS of a mixture of peptides in thioglycerol, the basic peptide is favored as regards formation of [M + H]+ ions, whereas the acidic is favored as regards formation of [M - HIions. Similar dehalogenation reactions of halogenated nuand cleosides occur in both SIMS using glycerol matrix (H57) plasma desorption (H58). It has been pointed out that chemical reactions of solid samples can be deliberately induced and products detected by plasma desorption (H59). SIMS of vitamin B12in glycerol has established that intermolecular see also H61, H62). Competition transfer of Co occurs (H60, in SIMS among alkali-metal cations in cationizing organic molecules in liquid matrices does not appear to have an obvious dependence upon any single factor (H63-H65, see also H66-H68).Reduction and oxidation processes have been shown to occur in liquid matrices in SIMS of both inorganics and organics (H69, H47). The presence of [M + 2H] and [M + 3H]+ peaks in SIMS of guanosine has been established by high-resolution measurements; these ions are attributed to reduction of [M 2HI2+and [M + 3HI3+ (H70). [M + 2H]+, [M + 3H]+ and other quasi-molecular ions have also been shown to be intense in SIMS of peptides using a liquid matrix (H71). Sundqvist et al. (H72) have assessed the limitations and possibilities of plasma desorption for the production of gaseous ions from biomolecules (see also H73-H75). Peptide molecular ions in excess of 23OOO daltons have been formed and detected using plasma desorption (H76). A comparison of SIMS using a liquid matrix and plasma desorption has shown that, with small peptides, the fragmentation is almost identical in the two cases (H77). For these peptides, cationized molecules [M Na]+ were intense with plasma desorption but not with
+
+
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SIMS (H77). Using a nitrocellulose backing for the sample support in plasma desorption of peptides has been found to eliminate salt contaminants and also enhance multiply charged ions in the spectra (H78). Aspects of the mechanism of relaxation of electronic energy to translational motion in plasma desorption have been discussed (H79). It has been shown that in plasma desorption the yield of valine ions increases as the square of the primary ion charge (H80). Cluster ions have been said to give increased yields in plasma desorption for the formation of [Cs]’ (H81).Damage cross sections have been measured for plasma desorption of valine and bovine An ion-track model has recently been put insulin “2). forward (2983,2384)for plasma desorption, in which the requirement for desorption of a molecule is that it be “hit” by a number of secondary electrons generated by the fast incident. This number would be about 4 for valine and about 15 for insulin. The model does not indicate the mechanism of desorption, although it has been tentatively suggested that the relevant bonds are broken by the shower of secondary electrons (H83, H84). Liquid Ionization Techniques. The formation of gaseous ions in thermosprayis thought to involve first field evaporation of ions from charged microdroplets, followed by gas-phase ion-molecule reactions (H85-H87, see also H88). The proposed role of the ion-molecule reactions in “transferring the charge” to yield the thermodynamically most favored set of ions has been supported by observations that the relative intensities of positive ions in mass spectra are as expected on the basis of gas-phase basicities (H87-H89). Thermospray spectra have been shown to be similar to SIMS spectra using liquid matrices, except that doubly charged ions are more intense in the former (H90, see also H91). Electrospray (H92),which involves spraying solutions from a capillary at high potential (2-10 kV) into a bath gas, probably also proceeds via field evaporation of ions from charged microdroplets (H93, H94). “Liquid ionization”, in which “hot” argon flows around sample mounted on a heated needle at high potential (1 kV), is said to involve “liquid surface ionization and field assisted thermal desorption”(H95, H96, see also H97). Electrohydrodynamic ionization (H98H102) can be carried out using aqueous solutions (H98);the energy spread of peaks is narrower than when using glycerol (H98). Field Desorption (FD). Measurements of energy deficits of ions in FDMS of saccharides have been taken to show that formation of [M + H]+ is most probable at those ionization sites at which the field strength is highest (H103). A view of ion formation in FD has been proposed in which the creation of a charged sample-vacuum interface and subsequent field evaporation of ions are the key features (H104). It has been suggested (H104)that electrodynamic phenomena observed using an optical microscope in FD from a bare wire of saccharides doped with salts, tartaric acid, and polyvinyl alcohol (H105) may be a “sample loss” process and that one role of the microneedles on activated emitters may be to curtail such phenomena. Claims (H106) that [MI’. of sucrose is intrinsically unstable have been rebutted (H107),on the grounds that under certain circumstances there is a small m/z 342 peak in the FDMS of sucrose. The influence of heating rate on FDMS has been demonstrated with pyridinium oxide salts (HI 08). Oxidation of RuO, ( x = 1-3) has been proposed to occur in pulsed FD of a Ru tip exposed to O2 at 600 O C (H109). Surface reactions involved in producing an amorphous thin Si film from silane by FD have been shown to involve the Gaseous ion formation (e.g., [Na2Cl]+,[K]’) surface (H110). from KCl and NaCl by FD has been shown usmg optical It has microscopy to be related to sample volatility (H111). been found that with bare emitters neutral coordination compounds do not yield ions, whereas cationic complexes do (H112). Isomerization of bradykinin [M H]+ to a novel structure with a propensity to cleave at the Pro-Pro bond has been proposed on the basis of CAD of the pseudomolecular ions in the FDMS (H113). The isomerization presumably occurs in the condensed phase and the new structure presumably involves some sort of ion-dipole bonding. 16N labeling has been used to elucidate mechanisms of decomposition of protonated arginine in FD and SIMS (H114,see also H115).FD has been used to characterize poly(ethy1ene adipate) (HI161 and other polymers (H117, H118).
+
MASS SPECTROMETRY
Laser Desorption (LD). In LD at moderately high power densities (107-108W ern% as used in FT-ICR (H119-H122). the gaseous ions are said ( h 2 3 )to be the result of “a basically thermal process” (H124). LD has been coupled with timeof-flight analysis (H123,H125). Translational energy distributions of [Na]+ and pseudomolecular ions formed by LD have been measured and found to be broader than thermal (H124).The distributions are interpreted in terms of ionmolecule reactions in a dense plasma cloud over the surface (H124).Low power LD (lo2-lo3 W cm-2) of saccharides in a glycerol/salt matrix has shown markedly different binding strengths for different alkali-metal cations (H126). Mechanisms of ion formation at very high power densities (H127)(1011-1013W cm-2)as employed in laser microprobe (LAMMA) are thought to depend upon, among many other factors, sample thickness (HI%).Irradiation of thick samples [20 ~ m ]not , leading to perforation of the support, is said to be a “soft” ionization process, involving a nonthermal shock The prevailing mechanism for thin films appears wave (H128). to remain that involving explosion-likeevaporation and ionmolecule reactions (H129,see also H130,H131). Microprobe LD is said to lead to MS of amino acids similar to CIMS using reagent gases of high PA’s (H132).Contrariwise, evidence has been produced to support a proposal that formation and fragmentation of odd-electron molecular ions play a major role in LAMMA of polycyclic aromatic hydrocarbons, aza heterocyclic and oxygenated analogues, and phenolic compounds (H133).Cluster ions are formed from substituted pyridines (H134).Microprobe LD of zwitterions exhibit similarities with FDMS (H135).Microprobe LD of polyglycols yields mass spectra similar to those obtained with FD (H136,see also H137). It has been found that the threshold ower density is lowered in LD if the sample molecule absorgs at the laser wavelength employed (H138). MACROMOLECULAR AND CLUSTER IONS Physicochemical properties of gaseous massive ions are covered in this section. An extensive review of cluster ions has been given by Mark and Castleman (11,see also 12,13). A review has been given of aspects of the physical chemistry of cluster and macromolecular ions (14). Biological Compounds and Organic Polymers. The number of internal degrees of freedom of a peptide [M + HI+ ion of mass 1620 daltons is sufficiently large that, following deposition of as much as 20 eV internal energy in CAD, fragmentation occurs predominantly at microseconds and longer (15),i.e., there is an enormous “kinetic shift”. Three major reaction channels, which have been identified in CAD of linear peptide [M H]+ ions, lead to N-terminal-containing acylium ions, the acylium ions minus CO, and C-terminal sequence ions (16-18). The last mentioned involves a hydrogen rearrangement. Cyclic peptide [M + HI+ ions under CAD are proposed to be protonated on an amide N, cleave at that N-acyl bond, and eliminate amino acid residues from resulting ring-opened acylium ion (19,see also 110). Significant proportions of molecular ions formed by plasma desorption can be metastable (111-114). In the case of chlorophyll a ions, fragmentation appears to be consistent with QET and rate constants k ( E ) range from > lo9 s-l to lo4 s-l ( I l l ) . The tendency to undergo extensive metastable ion decomposition increases as the mass of the ion increases, a t least up to 6000 daltons (112). This implies that the internal energy of ions formed by plasma desorption increases sharply as the mass of the ion increases. Polyether ions above 2000 daltons from plasma desorption fragment extensive1 , even when the pseudomolecular ions formed are [M + Na]Y (115). Chlorophyll a ions formed by laser desorption (116)and insulin ions formed by SIMS (117)also exhibit metastable ion decomposition (see also 118). Peptide [M + Na]+ ions are proposed to undergo metastable ion decomposition via C-N cleavage with retention of [Na]’ on the N-terminal fragment (113,see also 119,120). In CAD, (M - H)- of carboxylic acid and other functional-group terminated chains are proposed to decompose by loss of CnH2n+2 molecules a t sites remote from the presumed site of charge localization (121,see also 122,123).Polystyrene [MI+. radical-cation chains up to mass 4500 daltons yield only low-mass fragment ions in CAD, which is attributed to random cleavage of the chain followed by sequential depolymerization
+
(124).Polystyrene [MI+-chains decompose unimolecularly to give only high-mass fragment ions, which has been explained in terms of efficient charge delocalization over the chain (125). SIMS has been used to study nylons (126).The question of the extent to which charge is delocalized is likely to be very important in understanding fragmentation of chains and other very large ions. The very strong preferences for formation of certain fragment ions to the exclusion of others in what should be uniformly covalently bonded ions (121,124, 125) shows that at this stage care is called for in interpreting CAD or MIKE spectra of massive ions of unknown structure (127). From photodissociation experiments using an argon ion laser, it was concluded that the only large ions that could be photodissociated at that wavelength (visible) were those containing chromophores (128).Small peptide ions have been photodissociated using an excimer laser (UV) (129). Inorganic Compounds and Metal Clusters. Cluster ions [A,]+ can be formed by sputtering (130-134)from Ag (up to x = 250) Cu, Au, and Ni. Relative metastable peak intensities indicate that clusters of certain sizes (“magic numbers”) are peculiarly stable (130). [Ni(Cu),]+ and [Ni(Au),]+ clusters have been formed by field evaporation from the liquid alloys (135). Ionization efficiency curves have been measured for [K], clusters (3 I x 5 8) in a laser-molecular beam experiment (136,see also 137-142). I E s have been reported for oxidized alkali metal clusters (143).The observation of [Pb,I2+ clusters below the supposed minimum size for such doubly charged clusters has been taken to indicate that these “clusters” have chain structures (144).[Cu,]+ clusters react with isobutane to form adduct ions, whose structures have been probed by CAD (131). Cluster ions of Ni, Fe, and other refractory metals and of alloys have been obtained by PI of laser-vaporized neutral clusters (145-147). Iron oxide clusters have been studied using essentially the same technique (148).Pb, In, and alloy cluster ions have been formed by E1 of clusters formed by adiabatic expansion through a nozzle (149). [Si,]’, [Ge,]+, and [Sn,]’ have been formed by E1 of neutral clusters obtained by quenching the vapors in He (150, see also 151,152).[Si,]’ have been formed by laser-stimulated field desorption (153).Cross sections for photodissociation of [Si,]+ exhibit marked dependence on cluster size (154).One- and two-color ionization of Si, and Ge, has established that both types of clusters have excited electronic states with lifetimes of about 100 ns (155). Such long-lived excited states indicate the existence of energy gaps between round and excited states. A gas-aggregation technique has [een shown to produce [A I, and hence [Ag],+ clusters of controllable selected size (1567. Binary oxide ions can be formed by laser microprobe mass analysis [LAMMA] (157). Rh carbonyl clusters containing up to 10 Rh atoms have been formed by IMRs in an ICR (158). Cationic Fe carbonyl and Cr carbonyl clusters (159)and anionic Fe carbonyl clusters (160)have been created similarly. Cu acetate clusters have been produced by both CI and E1 (161). Large gaseous ions have been produced from many inorganic complexes using SIMS (162,see also 163) and FD. Examples include cationic Fe and Tc complexes (164),Cu chelates (1651,and Mo and W carbonyls (166).Polyoxanions (167),organotin compounds (168), phthalocyanines (169),and chromium oxide clusters (170)have been amenable to study by SIMS (see also 171). FD has given molecular ions for Ru and Os complexes, when SIMS did not give molecular ions but did give structurally diagnostic fragment ions (172). Salt Clusters and van der Waal’sClusters. Alkali halide and other salt cluster ions (173)can be formed either by sputtering (174,175) or by E1 of the neutral clusters from quenching the vapor in He (176-179). [A1Br3],+ (177)and [CuBrl,+ and [(CuBr),Cul+ clusters (178) have been formed using the latter method. Sputtering yields both positive and [(CsI),Csl+ clusters up to 90000 negative clusters (174,175); daltons have been measured (180). That clusters of certain sizes (“magicnumbers”) appear to be especially stable has been treated using various theoretical models (174,180).A proposed (182)quasi-octahedral structure for [M(MX),]+ ions has not which confirms an earlier been supported by calculations (183), proposal that square-planar is the preferred geometry. Relative intensities of metastable peaks elucidate relative see also 186). stabilities of different-sized cluster ions (184,185, Half-lives have been estimated (187) for smaller [Cs(CsI),]+ ANALYTICAL CHEMISTRY, VOL. 58, NO. 5, APRIL 1986
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ions created by sputtering. Large clusters have been formed by sputtering from organic salts (188). Stabilities of [ (NH4Cl),NH4]+ions have been investigated using the thermospray technique (189). van der Waal‘s cluster ions, both positive and negative, now constitute a very active and extensive area of research (190), embracing the inert gases (191-1931,organics (194-196),inorganics (197,198, 1100-1104) and many kinds of mixed clusters (1105-1108).Doubly charged clusters have been reviewed (1109). Doubly charged clusters are generally considered to have minimum sizes, e.g., x = 35 for [(HzO),Hz]2+ (1110).It has been conjectured that the evolution of “magic is a connumbers” for [(HzO),H]+ clusters (1104,1111,199) sequence of cooling by evaporation and solidification into ordered structures (1112,see also 1113). The geometry and binding ener y of mixed propylbenzene/alkane clusters have been studie using two-color laser techniques (1105).MPI techniques have been applied to toluene clusters (1115)and see also 199). Photodissoto ammonia clusters (1116,1117, ciation cross sections have been reported for [(C02),]+.ions (1118,see also 1119). Vibrational spectra have been recorded for [H,]+ cluster (1117). The temperature dependence of nitrile cluster ions has been examined in a study employing an electron storage ring and used to determine enthalpies of formation of neutral clusters (1120).Electron attachment to oxygen clusters (1121)has led to the observation of [(O2),I2and higher homologues (1122).IMR’s of cyclic oligomers of methylenenitramine yield cluster ions (to beyond m / z 4000) (1123,1124); it has been argued on the basis of CAD that these massive species are clusters as distinct from polymers (1125). Thermodynamic properties have been determined for the [Na(C02),+1]+and for equilibrium [Na(C02),]+ COz related CO2/H20clusters (1126). That van der Waal’s clusters may fragment upon ionization has perhaps suprisingly been a matter of controversy, and rather sophisticated experiments have been needed to settle the matter (1127,191).The degree of fragmentation of neutral clusters has been found to decrease in the order of methanol > ammonia > water (1128).Cross-sections have been measured for reactions of [OH(H20),]- with C02and SO2 (1129, see also 1130). It has been proposed that, on account of slow intracluster energy flow, the ether ion in [Ar,.(CH3)zaO]+undergoes unimolecular decomposition without disrupting the argon moiety (1108,see also 1131). [N,.+-, where x is even, exhibit metastability with respect to losses of specific numbers of Nz, while [N,]+, where x is odd, do not (1132).The explanation appears to lie with slower vibrational energy transfer between the central ion in the even clusters [be this [Nz]+-or [N4]+’] and N2 molecules, as compared to [N3]+and N2 molecules in odd clusters (1133).
d
+
-+
CONSTITUENTS OF NUCLEIC ACIDS AND THEIR XENOBIOTICALLY MODIFIED ANALOGUES Mass spectrometry continues to play an increasingly significant role in both the structural characterization of constituents of nucleic acids and the quantitative analysis of specific components at very low levels. Mass spectrometry has even played a central role in the identification of some 50 modified bases naturally occurring in tRNAs, and other covalently modified components formed as a consequence of photochemistry,metabolic activation of xenobiotic substances, and ionizing radiation. In this class of polar labile biological substances, enzymic and chemical hydrolytic methods can be used to obtain the nucleosides themselves where all of the techniques in mass spectrometry still play a balanced role from use of chemical derivatization with electron impact high-resolution mass spectrometry,chemical ionization, GC/MS, and stable isotope dilution at either the monomer level or the base level itself. In addition, field desorption, Californium-252 plasma desorption, and recently liquid matrix sputtering ionization techniques and thermospray LCMS have begun to play an important role as additional techniques in the arsenal for qualitative and quantitative analysis of the free nucleosides/nucleotides and oligonucleotides including protected oligonucleotides. During this period, several authoritative discussions have been presented dealing with both the techniques and examples where they were used effectively. These 178R
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reviews should be considered required reading for the uninitiated and expert alike. McCloskey has summarized the state of our knowledge of the field within the framework of three areas: desorption methods of ionization, identification of components of known structure present in mixtures, and structural characterization (JI).In addition, a review of the use of liquid matrix atom sputtering covers the literature and an accompanying paper describes through mid 1984 (J2), the qualitative comparison of the secondary ion spectra obtained with those previously determined by electron impact and chemical ionization methods on the nucleosides (J3). The putative initiating events in tumorogenesis by a chemical carcinogen involves the metabolic activation by cytochrome P450 of the carcinogen to a reactive electrophile which subsequently forms covalent adducts with cellular DNA. It is of considerableimportance to trace the molecular events that lead to the formation of these adducts in vivo as well as to characterize the detailed structure of the adducts and identify the location of such molecular lesions within the sequence and tertiary structure of native DNA. Since the relative abundance levels are extremely low for these physiologically important covalent lesions, such as one carcinogen per ten to hundred thousand base pairs, the techniques of mass spectrometry have played a crucial role in elucidation of the structures of these xenobiotically modified components of DNA. A recent report discusses the detailed structural analyses carried out on DNA adducts for carcinogens in the aromatic amine class (J4).While mass spectrometry was shown to be a powerful tool for identification of these adducts in vitro, it was noted that analogous studies to be carried out in vivo comprise a relatively untouched but fertile area for future studies. Even from the point of view of identification and monitoring of such adducts occurring as excision products excreted in human urine, judicious application of mass spectrometric quantitative measurement at low levels would provide information necessary to estimate health risk potential for individuals exposed to carcinogenic chemicals (J4a). The structural characterization of both nucleoside and nucleotide covalent DNA adducts has been carried out for the case of dehydroretronecineusing cesium ion liquid SIMS (J5, J6). This substance represents the nucleus of an extensive series of pyrrolizidine alkaloids occurring naturally as monoand diesters in tansy ragwort and other plants. The alkaloids are hepatotoxic to cattle and goats, but also accumulate in honey produced by pollinating bees destined for human consumption. This work establishes that direct analysis of the alkylated nucleotide can be carried out using negative ion LSIMS and thus holds promise for characterization of analogous alkylated oligonucleotidesto obtain possible DNA sequence alkylation specificity. Such studies would permit probing possible inherent directing capability by the tertiary structure of DNA during the intercalation and alkylation events. A further report has discussed the use of atom LSIMS in the negative ion mode for determination of the sequence for small unmodified oligomers of DNA. This paper also presents calculated stabile isotopic cluster profiles for the observed sequence ions in the LSIMS spectra (J7).Taken together the literature cited above contains a rather complete picture of the present and projected scope of usefulness of mass spectrometry in studies of nucleic acid chemistry and, when considered with the previous reviews in this series, provides ready access to all of the significant literature in the field. An extension of previous work using GC ECD analysis of the methylated perfluoroacyl derivatives of cytosine and 5methylcytosine has been carried out by negative ion chemical ionization fused-silica capillary GC/MS in order to demonstrate the usefulness of this method for trace analysis. Using heptachlor as a reference compound, the authors reported a quantitative linear calibration curve in the range 1fg to 1 ng for the cytosine derivatives (J8).Work on quantitative determination of nucleoside-drug analogues using glass capillary GC/MS methods has been reviewed (J8a). GC/MS techniques have been used to identify compounds in urine from normal subjects and cancer patients, such as 5X-methylthioadenosine (J9)and 2XiO’methylguanosine (J10). GCMS studies have been used in the identification and quantitation of 5-(hydroxymethyl)uracil in a dihydropyrimidine dehydrogenase deficiency (J11)and the DNA of y-irradiated cells (J12).Similarly, 8-hydroxyguanine has been found in
MASS SPECTROMETRY
aqueous solutions of DNA (J13)subjected to y irradiation. y-irradiation experiments have been carried out using various DNA bases with and without the presence of tyrosine in order to identify possible base modifications and amino acid cross-linked products by GCMS (514). Recently, thermospray LC/MS enzymatic digests of RNA and DNA have been shown to be potentially useful for the identification of the presence of known modified nucleosides as well as an initial step in the detection and characterization of new unknown constituents (J15). It was found that substantial selectivity is available using the detected masses of the nucleoside and its correspondingbase taken together with chromatographic retention values and UV multiwavelength absorbance ratios despite the fact that components are not necessarily completely resolved from each other or from other components in such mixtures. It was noted that stability of operation and sensitivity of detection depended upon maintenance of constant temperature in the thermospray and that the optimum temperatures were different for nucleosides of different polarities (J16). Thermospray LC/MS has also been used to demonstrate that the quantitative analysis of heteroaromatic mutagens in cooked foods can be carried out in the parts-per-billion range (J17). This technique was then employed in the analysis of 2-amino-3-methylimidazo[4,5-flquinoline and 2-amino-3,4-dimethylimidazo[4,5-flquinoline in broiled salmon (J18). Levels of these two substances were determined in salmon flesh and skin to be in the low partsper-billion range. In addition to these LC/MS results, the techniques of MS/MS have also been used in the analysis of nucleic acid constituents in food products (J19). Further work using liquid chromatography and MS/MS has been applied to the quantification of products of in vitro DNA methylation (JZO). While it is now well established that mass spectra free nucleosides/nucleotides and dinucleotides, etc., can be obtained in both positive and negative mode by sputtering and ionization from viscous liquid matrices, the spectra observed also contained ion currents characteristic of the nature of the matrix and other components or additives, even different types of molecular adduct or cluster ions, depending upon the alkali metal ion content of the sample. An obvious general approach to obtaining the secondary ion mass spectrum of the component of analytical interest only is through the use of some sort of MS/MS technique. In order to evaluate one such MS/MS technique for the case of analysis of nucleosides, some 30 nucleosides, modified nucleosides, and dinucleotides were determined by atom LSIMS using a triple sector instrument which provides an ion kinetic energy spectrum of the component mass selected using the MS MS mode. While these authors found that MS MS and co lisional activation of the parent ion selected di not intensify fragments over those levels observed in the %ormal” secondary ion mass spectra themselves, it was clear that the CAD fragmentation pattern was more significant with respect to that of the background (the chemical noise had been significantly reduced). However, an additional feature of importance was the observation of some new fragments in the negative ion CAD mass spectra which had not been observed in the normal spectrum. While these ion kinetic energy spectra are of clear qualitative analytical usefulness, the low mass resolution (J21)of this EBE geometry in this MS/MS mode limits the molecular size measurable to around 1500-1800 daltons. A further report on the MS MS of dinucleotides has appeared using atom LSIMS in t e negative ion mode (J22)providing information analogous to that obtained earlier by field desorption (J23). Using isotopically labeled queuine analogues, direct evidence has been obtained that queuine is incorporated intact into mammalian tRNA in vivo in the case of mouse fibroblast cells (J24). Electron impact and atom LSIMS have been used to characterize the photo products formed during photolysis of d(T-A) in aqueous solution and ice (J25). Conversion of formycin, a C-nucleoside analogue of adenosine into the isoguanosine analogue 7-amino-3-(~-D-ribofuranosyl)-lHpyrazolo[4,3-d]pyrimidin-5(4H)-oneon UV irradiation in aqueous solution has been established by E1 high-resolution mass spectrometry (J26). A report on an atom LSIMS spectrum of ribosyl-dipthamide has appeared (J27). Atom LSIMS was used to identify the components obtained by HPLC separation of the isomeric 5-fluorocytidine 2/,3/- and 3’,5’-cyclic monophosphates (J28),as well as the cytidine 3/,5’-cyclic monophosphate from rat tissue (J29). Metabolites
l
i,
i
of erythro-9-(2-hydroxy-3-nonyl)hypoxanthinehave been isolated and identified from the urine of rats, dogs, and monkeys (J30). A novel nucleoside, 7-0-D-ribofuranosylhypoxanthine has been identified in the urine of a chronic myelogenous leukemia patient (J31). Mass spectrometry has been used to identify the tetraols formed from hydrolysis of the 7,12-dimethylbenz[a]anthracene-modifiedDNA (J32). The 7-deazapurinenucleoside, tricyclic nucleoside (TCN, NSC 154020), forms a ring-opened bicyclic metabolite in rat liver microsomes (533). Confirmation of a synthesis of some nucleoside cyclic ethylene phosphothionates has been reported (J34). Several new nucleoside antibiotics have been identified from a newly isolated strain of Streptomyces sp. including ascamycin and the corresponding dealanyl derivative (535, J36), guanine 7-N-oxide (J37), and a novel class of uracil nucleoside antibiotics, called liposidomycins, which selectively inhibits bacterial peptidoglycan synthesis (J38). It was noted that the liposidomycins A, B, and C inhibit peptidoglycan synthetase prepared from E coli by an increase of 3 orders of magnitude over tunicamycin but were comparable to moenomycin. In studies of photoaffinity labeling of diphtheria toxin fragment A with NAD atom, LSIMS was used to identify the photoproduct at position 148 as an a-amino(6-nicotinamidy1)butyric acid residue (J39). It is worth reiterating a cautionary point made in McCloskey’s earlier review (J1)that halogenated nucleosides undergo extensive dehalogenation during atom LSIMS in lycerol matrices (J40). Since the dehalogenated substance egins to accumulate as a function of xenon atom primary beam dosage, one might mistakenly conclude that the nonhalogenated analogue of such a nucleoside was indeed present in the original sample. This laboratory has also pointed out that the number of active hydrogens in an organic substance can be determined through use of [hydr~xy-~H,]glycerol in DzO as a sample matrix. The atom LSIMS spectra of a variety of mononucleotides and dinucleotides have been recorded in both positive and negative ion mode (J42),as well as the positive ion spectrum of UpGp (J43). Atom LSIMS was employed to characterize a synthetic octanucleotide containing the EcoRI recognition sequence with a phosphorothioate group at the cleavage site (J44). Routine identification of intermediates in the phosphotriester synthesis of DNA fragments has been carried out by atom LSIMS (J45,546). A method for the analysis of mass spectrally assayed stable isotope-labeling experiments has been reported using 15NH4Cl and ~-[5-’~N]glutamine as precursors in the de novo pyrimidine biosyntheticpathway in isolated rat hepatocytes (J47). Isotope dilution GC MS was used to detect 5-methylcytosine in the genomic DN of the fungus Neurospora crassa (J48). GC/MS method was used to determine the isotopic enrichment of 6-15NHzin adenine nucleotides (549). Atom LSIMS in the negative ion mode has been used to distinguish between (SJ-adenosine 5’-0-(1-thiotriphosphate)containing either an a-nonbridging or an a-0-bridging lSOlabel (J50). In the de novo biosynthesis of tetrahydrobiopterin, GC/MS was used to determine that the C-6 proton is derived from water and not from NADPH (J51). Acetyl-coA synthetase has been shown by atom LSIMS to catalyze the positional exchange of an oxygen-18 of ATP from the 0-nonbridge position to the cup-bridge position in the presence of acetate. However, analogous studies with argininosuccinate synthetase would not catalyze positional isotopic exchange (J52). From the measurement of I3C isotopic content for cerebellar DNA, it has been concluded that a large proportion of DNA in small neurons of the cerebellum does not undergo metabolic turnover during the human life span (J53).
%
d
AMINO ACIDS, PEPTIDES, AND PROTEINS Modified Amino Acids. Carr and Biemann have recently suggested typical strategies and discussed the arsenal of mass spectrometricmethods for the identification of modified amino acids, both as the free substances and as components of peptides (K1). This treatment and the references therein provide an up-to-date overview of the ways in which chemical derivatization methods and the various ionization techniques of mass spectrometry can be brought to bear to identify posttranslationally modified amino acids, including the phenylthiohydantoinsreleased in the stepwise Edman degradation of proteins possessing a free N-terminus. MacKenzie has described developments in the analysis of amino acids in small ANALYTICAL CHEMISTRY, VOL. 58, NO. 5, APRIL 1986
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peptides by GC MS using examples drawn from microbial metabolism an biochemistry (K2). Human whole body leucine metabolism has been carried out in healthy young and elderly adults using [l-'3C]leucine (K3). In this study the effects of intravenous glucose levels were determined on whole body leucine dynamics. GC/MS was used to measure 13C enrichment in plasma leucine (K3). It was used to determine incorporation of deuterium from deuterium oxide into free amino acids of the cotyledons of Sinapis alba L (K4). The mechanism of cleavage of the proenz me from Lactobacillus buchneri was studied using hydroxyl-&Olabeled serine. Mass spectral analysis of the trimethylsilyl derivative of serine derived from this histidine decarboxylase showed that this enzyme arises by nonhydrolytic serinolysis of its proenzyme. By use of ISO enriched water, it has been shown that the trypsin-mediated transformation of pig insulin into an ester of insulin of human sequence involves hydrolysis followed by coupling and is not by direct transpeptidation as reviously assumed by analogy with another system (K6). kbsulfate or ~-[sulfane-~~S]thiocystine was used to establish the metabolic origin of the sulfur atom in thiamin in E. coli (K7). Various LCMS methods have been evaluated for analysis of aromatic amino acids (K8). Chemical ionization mass spectrometry has been used to identify phenylthiohydantoins obtained in studying the amino acids sequence of human plasma apolipoprotein C-I1 (K9). A hereditary defect in the degradation of poly(ADP-ribosylated) proteins has been shown to accumulate glutamyl ribose 5-phosphate (KIO). NMethylated basic amino acids have been characterized in human urine by GC/MS methods (K11). In connection with quantitation of nonenzymatic glycosylation, mass spectrometry has been involved in determination of glucitollysine isolated from plasma protein samples (K12). A novel cyclic amino acid from halophilic phototrophic bacteria of the genus Ectothiorhodospira has been shown by mass spectrometry to be 1,4,5,6-tetrahydro-2-methyl-4-pyrimidinecarboxylic acid (K13). Atom liquid SIMS was used to identify a urinary metabolite of malondialdehyde as Neacetyl-t-(2-propenal)lysine (K14). Atom LSIMS and subsequent MS/MS techniques have established W-[Y-L-(+)glutamyl]-4-carboxyphenylhydrazine in the cultivated mushroom Agaricus bisporus (K15). A borohydride-reducible compound occurring in human aortic elastin has been found to be an unknown cyclic amino acid, 2-amino-6-(6-carboxy3-piperidyl)-6-hydroxyhexanoic acid (K16). This substance was not found to be present in foetal elastin, and its relative amount increased steadily during aging. 5,5-Dichloroleucine was identified by atom LSIMS among other degradation products of the specific plant host toxin, victorin C (K17). Selenium is an essential component of glutathione peroxidase in animal tissues and a number of microbial enzymes. A convenient procedure based on derivatization of selenocysteine by Sanger's reagent has been developed for identification of selenocysteine by mass spectrometry (K18). Peptide Hormones and Enzymic Digests of Proteins. For several years now, mass spectrometry has been playing an increasingly important role in determination of the primary structure (sequence) of relatively small peptides (up to around 10-mers) by chemical derivatization as either the reduced, silylated polyamino alcohols with GC E1 MS, or as the Nacetyl N,O-permethylated analogues using direct probe E1 and CI MS (K1). Both of these established methods are used to guarantee unambiguous sequence information that can be obtained, particularly on N-terminally blocked peptides using sample sizes in the order of 10-50 nmol. While these methods are of established advantage in obtaining clear sequence information of virtually all residues in a given polypeptide, irrespective of the amino acid composition and sequence of the particular peptide, neither of these methods yields reliable determination of molecular size of the peptide in the E1 mode, and the methods may not provide complete knowledge of the C-terminal residue sequence, except with the use of chemical ionization. CI does usually provide molecular weight and C-terminal sequence information on the acetylated permethylated derivatives. With the advent of liquid matrix sputtering ionization 5 years ago, the extreme belief held that peptide sequencing problems were over. Indeed, in the ensuing period, many of the advantages of liquid matrix sputtering ionization on the free and partially derivatized peptides have been established, such as the ease of determining molecular size, molecular weight, and sample homogeneity,
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particularly in the mass range up to 3000-5000 daltons and even to over 10000 daltons, but with considerably more experimental difficulty and sample consumption. While it is clear that the liquid matrix sputtering ionization phenomena produce in general an overabundance of molecular ion species MH' compared to easily discernible and reproducible fragmentation patterns (from which sequence information must be derived), other than the molecular weight per se, chemical noise has become the hallmark and bane of its existence frustrating the unambiguous interpretation of these spectra-especially those of unknowns. Hence, despite the advantages, which could accrue from working with underivatized peptides, relatively large amounts of sample are required to bring the fragmentation pattern up through the chemical noise continuum for spectral interpretation purposes. Thus the sensitivity for molecular weight determination can be in the picomole range whereas sensitivity for obtaining whatever sequence information there is that can be gleaned is in the nanomole range. At this stage, all of the major laboratories involved in developing and applying this methodology to problems in protein chemistry recognize the lack of reliability and the empirical understanding necessary to improve their reliability for obtaining sequence information from secondary ion mass spectra sputtering from liquid matrices ( K I ,K19-K27). Hence there is an overwhelming incentive to somehow ameliorate the situation described above. Since incentive is only one side of this coin, finding solutions expediently is usually dictated by tractability of particular aspects of such a problem. Major aspects of the situation involve (1) understanding and manipulating the matrix, solution, and surface properties to match unknown factors required for the ejection upon sputtering of a particular peptide irrespective of the collective side chain properties; (2) elimination of the chemical noise wihout loss of parent molecular ion selectivity, or mass resolution in the ensuing secondary ion spectrum; (3) the ability to enhance and/or control the internal energy of ejected species, and finally, (4)overall improvement in sensitivity of the method itself, to bring it more in line with the sensitivities obtainable with electron impact and chemical ionization mass spectra. Since mass spectrometry is ion chemistry, unlike other forms of spectroscopy, advancement of our understanding of the role and optimization of matrix factors, which would eventually yield high quality secondary ion mass spectra of only the substance in question (without the chemical noise), is a longer term empirical endeavor not readily tractable, no matter how much attention is paid, in the short term. Hence the whole community is moving toward addressing those tractable factors involving technique and more relevant instrumentation such as MS/MS and collisional activation, refined ion source designs, new ion source and ion optical strategies, and improved ways of addressing control or enhancement of internal energy. As the reader may glean independently by reading the fine print in the many reviews and chapters referenced above, it is the best of times and the worst of times for mass spectrometry and protein chemistry. It is the best of times because molecular weights can be measured directly, up to around mass 10000, and in special cases to 25 000; because peptides covalently modified by posttranslational events, such as glycosylation, phosphorylation, acetylation, acylation, etc. can be measured directly; because there is considerable enthusiasm in the expectation that the sequencing situation will be improved by elimination of the chemical noise using MS/MS and collisional activation techniques; and because selective chemical derivatization may play an important role in directing specific fragmentation processes. It is the worst of times because the sensitivity of liquid matrix sputtering ionization of present mass spectrometry is so disparate compared with the most advanced micro Edman methodology (K28). Hence the forefront of knowledge of new primary protein structure remains experimentally in the hands of those using micro Edman methodology, and by inference in the hands of those determining the cDNA sequences (K28). However, mass spectrometry could become the method of choice for those types of problems where the Edman is either ruled out or experiences difficulties and there are nanomole quantities of peptide or protein with which to work. And in summary, it is clear that the most rapid progress will be made by laboratories that routinely involve both the micro Edman
MASS SPECTROMETRY
and the evolving techniques of mass spectrometry in their overall protein structure elucidation strategies. From a different scientific point of view, the advent of sputterin preformed ionic components directly from the condensetf phase may provide a new method for studying the “actual” ionic distributions present in liquids. Several examples have already been explored in this regard, such as determination of enzyme reaction rates, measurement of chemical equilibria, determination of K,’s and Vmax)sfor tryptic peptide hydrolyses, and formulation of the factors involved in carrying out quantitative analysis of substances such as 4-aminobutyric acid and dansylglutamine (K29-K31). A chapter describing such direct analyses of biochemical reactions in aqueous solutions summarizes these points (K32). Several authors have discussed the details of atom liquid SIMS from the point of view of the present difficulties of sequencing an unknown peptide alluded to above (K33,K34). Another approach has been to use atom LSIMS for the analysis of the molecular products formed by the cleavage of the polypeptide substrate with exopeptidases such as carboxypeptidase Y and leucine aminopeptidase (K35). Several laboratories have reported results of sequence determinations using MSjMS techniques, for example, cyclic peptides (K36), polyamino alcohols (K37),mixtures of tryptic peptides obtained from a recombinant protease inhibitor Eglin C (K38), and human apolipoprotein-B (K39). It has been shown that MS/MS (linked scanning) can be used to distinguish cy- and @-linkedaspartyl peptides (K40). A preliminary report has appeared on the use of collisional activation to obtain sequence information from laser desorption of gramicidin S and a component of gramicidin D using a Fourier-transform ICR mass spectrometry (K41). These spectra were obtained from a single shot from a Tachisto Model 215G pulsed infrared laser, and sample consumption was estimated from the whole size left after a laser shot to be a few picomoles. By use of the moving belt interface HPLC-MS system, it has been shown that acetylated permethylated derivatives yield peptide sequence information, at least up to the octapeptide level (K42). Using the thermospray interface and columns containing the immobilized enzymes, carboxypeptidase Y and trypsin, LCMS has been used for peptide sequencing for a series of peptides ranging in size to about mass 1000, some of which were observed as multiply protonated species (K43, K44). While the presence of alkali metal cations in a liquid matrix secondary ion mass spectrum may be of use in confirming the molecular wei ht of a substance, the disadvantage may be to detract from t i e sensitivity as well as possibly to complicate the fragmentation pattern. Another possibility has been recognized involving depression of the fragmentation pattern for a small peptide (K45). Also, it has been reported that the collisional activated dissociation spectrum of an [M Na]+ ion yields an MS/MS spectrum different from that observed for the corresponding [M + HI+ ion (K46). This latter alteration of the fragmentation pattern has also been observed in the case of sodiated molecular ion MS/MS spectra in maltotetraose compared with [M + HI+ ions (K47). It has been suggested this is due to selective binding of the alkali metal ion with the substance in question (K46, K48). With this in mind, it should be noted that specific derivatization of small peptides can greatly improve relative abundance of sequence ions observed in atom LSIMS spectra (K49). For peptides up to 10-mers the 2-bromo-5-(dimethylamino)benzenesulfonyl derivative directs fragmentation, whereas for larger peptides dansyl derivatives are preferred (K49)although the interpretation of the spectra obtained becomes more difficult for peptides containing amino acid side chain functional groups that are also derivatized. Another suggestion involves sequencing by derivatization to form a quaternary N-terminus followed by subsequent cleavage and detection of the preformed charged N-terminal moiety preferentially (K50). Liquid matrix sputtering techniques have been used to characterize both synthetic and naturally occurring protease inhibitors, in some cases including protecting groups (K51). An alternative approach to N-terminal sequence determination involving omission of the prior acetylation step has been shown of value in the case of blocked N-termini of vesicular-stomatitis-viri N protein, Sendai-virus NP protein, and an intractable immunoglobulin A-light chain (K52). In general, mass spectrometry continues to be the method
+
of choice for determination of N-terminal sequences occurring in N-terminally blocked proteins. Current examples include identification of a formyl blocking group at the N-terminus of the ATPase inhibitor (K53),an N-dimethylproline moiety at the N-terminus of a starfish histone H2B (K54),and acetyl-blocked N-termini of sorbitol and aldehyde dehydrogenases (K55). In studies of nonenzymatic posttranslational glycosylation of certain proteins, it has been shown by atom LSIMS that the product results from addition of the open-chain aldehyde of glucose to amino groups, and that there is no participation of dicarbonyl autoxidation products (K57). The sites of phosphorylation of an homogeneously octaphosphorylated 23-residue tryptic peptide isolated from chicken yolk riboflavin-binding protein was carried out by liquid matrix sputtering ionization in the negative ion mode. This study pointed out that more sequence information was obtained on the dephosphorylated analogue in the positive ion mode in this case than on the phos horylated analogue in the negative ion mode and that an ind)ependent knowledge of the sequence of the dephosphorylatedpeptide together with the exclusive relationship of phosphate and serine were required to interpret the observed negative ion LSIMS spectra unambiguously (K58). An unambiguous molecular ion for the decapeptide ceruletide, formerly caerulein, which contains a tyrosyl-0-sulfate residue, could only be obtained by atom LSIMS in the negative ion mode. The positive ion mode shows peaks resulting from elimination of SO3from both the protonated and the sodiated putative molecular species (K59). Another sulfo-tyrosine peptide that has been determined in the negative ion mode using similar methodology was identified as cholecystokinin-8 (K60). Using a sample of commercial disulfide cross-linked calcitonin, atom LSIMS was used to measure the molecular weight. From this measurement it was concluded that the sample contained a mixture of the oxidized and reduced disulfide linkage. The authors also conclude that their data reflect the composition of the original mixture and that the observed molecular ion profile is not produced as an artifact by atom bombardment of the substance in the liquid matrix (K61). Several reports have recently suggested ways of examining disulfide linkages by direct measurement of the masses of fragments of the protein obtained by chemical or enzymatic degradation under conditions known to minimize disulfide reduction and reshuffling (K62-K64). Gradual reduction of disulfide linkages in oxytocin was observed during continued primary atom bombardment of the sample over a few-minute period (K64). Mass spectrometry was used to identify a volatile mercaptide in its oxidized form as methanesulfonic acid. This is involved in the mixed disulfide of the zymogen of Streptococcal proteinase (K65). Mass spectrometry has made considerablecontributions to qualitative and quantitative studies of endogenous neuropeptides in biological extracts. Two articles review this important field to date (K66,K67). Confirmation of the synthesis of conformationally restricted C-terminal peptides of substance P has been reported (K68). Substance P, substance P 5-11, and substance K have been isolated and characterized in samples of two metastatic ileal carcinoids using atom LSIMS (K69). Atom LSIMS has been used in the determination of primary structure of the califins, three biologically active egg-laying hormonelike peptides from the atrial gland of Aplysia californica (K70). The primary structure of tryptophan-containing peptides from skin extracts of Phyllomedusa rhodei have been established with the use of field desorption mass spectrometry (K71). Some 30 peptides have been characterized from the skin secretions of the South American frog, Xenopus laeuis, with the aid of atom liquid SIMS ranging in size from 300-2700 daltons (K72). Atom liquid SIMS, accurate mass measurement, and linked scan studies have established structures of two cockroach neuropeptides, both containing blocked N-termini (K73). Another peptide hormone with a blocked N-terminus, the adipokinetic hormone of Manduca sexta has been elucidated by atom liquid SIMS and MS/MS techniques (K74). Analogous techniques were employed to determine the sequence of Adipokinetic Hormone I (K75). Opioid-like immunoreactive material isolated from the pituitary of the spiny dogfish shark Squalus acanthias was shown to be endorphin-like peptides containing 30 amino acids (and variants thereof), which show 80% homology with salmon endorphin-I1 (K76). LSIMS and ANALYTICAL CHEMISTRY, VOL. 58, NO. 5, APRIL 1986
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the Edman degradation were used to establish these sequences. A study has been carried out to evaluate the completeness of reactions, the extent of modification of the group of interest, and the extent of other reactions for several reagents commonly used for derivatization of specific sidechain groups of peptides (K77),such as tetranitromethane, BNPS-skatole, diethyl pyrocarbonate, N-acetylimidazole, and iodine monochloride. The use of atom LSIMS for monitoring and optimization of deprotection reactions in peptide synthesis has been discussed (K78). The synthesis and use of 4-(Ntert-butyloxycarbonylaminomethy1)phenylisothiocyanate and its use in microsequencing have been described (K79). Chemical synthesis of a heat-stable enterotoxin produced by enterotoxigenic E. coli strain 18D has been carried out (K80), together with chemical synthesis of fully active and heat-stable fragments of this enterotoxin (K81). Atom LSIMS was used to determine the molecular weights of intermediate products in this synthesis (K81). The atom LSIMS spectrum of ArgArg-Arg-Pro has been reported (K82). Cesium LSIMS has been employed to determine the tryptic peptide map of the N-terminal cyanogen bromide 183 amino acid domain isolated from purified bovine serum albumin monomer (K83). Similar direct measurement of protein digests by atom LSIMS has been carried out for hen and duck egg-white lysozymes (K84). The amino acid sequence of delta haemolysin purified from a canine isolate of S. aureus has been reported (K85). Atom LSIMS mapping was used to characterize human calcitonin gene-related peptide (K86).Atom liquid SIMS was involved in measuring the molecular weights of chymotryptic peptides in the establishment of a sequence for a DNA- and heparin-binding domain isolated by limited thermolysin digestion of human plasma fibronectin (K87, K88). The overall strategy for mass spectrometric verification and correction of the primary structures of proteins deduced from the DNA sequences has been described in detail (K89). Amino acid sequence of the antitumor protein neocarzinostatin has been revised on the basis of mass spectrometric evidence (K90). Spectrometric studies have shown that one monomer of cytoplasmic methionyl-tRNA synthetase from bakers’ yeast has the first methionine removed and the new amino terminus serine acetylated. Atom liquid SIMS was employed to correct the sequence of human myelin basic protein peptide 45-89 (K92). Further work and verification of the DNA predicted amino acid sequence was carried out by mass spectrometry on bacteriophage P22 tail protein (K93). A recent report suggested the use of glycinamidation to eliminate acidic residues to facilitate tryptic digestion of the protein and this also resulted in an improvement in the quality of the liquid SIMS peptide mapping (K94). Assignment of amides in the amino acid sequence of mammalian calmodulin has been reported (K95). Atom LSIMS mapping of proteolytic digests has been carried out to verify protein sequence of protein S, a development-specific protein of Myxococcus xanthus (K96). Reports from three different laboratories have discussed the characterization of human interleukin 2 (K97-K99). Two of these substances were isolated from E. coli cells expressing the human interleukin gene isolated from the T-cell leukemia-derived cell line JURKAT (K97, K98). The third was isolated from the cell supernate of a high producer subclone of the human T-cell line (K99). The primary difference in the data obtained involved in one case the absence of a carbohydrate moiety and in another its presence on the threonine residue position 3, either as an acetylgalactosamine or as a trisaccharide attached to an octapeptide (K99). The latter is a particularly good example of the use of atom liquid SIMS in characterization of modified peptides from different recombinant constructs or cell lines. Tryptic mapping using atom liquid SIMS has been employed fruitfully in the identification of B subunit of Vibrio cholerae classical biotype Inaba 569B toxin (K100). Further application of tryptic peptide molecular ion mapping concerns characterization of normal and variant human hemoglobins (K101-KI03). Identification of formyl-methionyl-leucyl-phenylalanine, a major peptide neutrophil chemotactic factor produced by E. coli, has been reported (K104). Cesium LSIMS has been used in elucidation of the sites of attachment of bilin in the a , p, and y subunits of R-phycoerythrin (K105). Mass spectrometry has played a major role in structural elucidation of methanofuran, a carbon dioxide reduction factor of Methanobacterium thermoautotrophicum (K106). A covalent adduct was isolated as a tetrapeptide containing 3-tryptophanyl-4182R
ANALYTICAL CHEMISTRY, VOL. 58, NO. 5, APRIL 1988
acetylaminobiphenyl from serum albumin isolated from rats after 27-h administration of the carcinogen 4-aminobiphenyl (K107). Atom liquid SIMS, field desorption, and DCI have been carried out and compared on complexes of cis-dichloroplatinum with amino acids and dipeptides (K108). Further examples have been reported on the measurement of the molecular weights of high mass polypeptides using atom liquid SIMS in conjunction with extended ion optical geometries, such as the Kratos inhomogeneous magnet version of the MS-50 using various insulins and bovine proinsulin I (K109, KIIO),and bovine proinsulin I1 (K110). These data show unit mass resolved stable isotope molecular ion clusters with an operating instrument resolution of 7000. The molecular weights of a-EGF and o-EGF were determined by atom LSIMS using 20-40 bg of peptides on a VG ZAB-HF instrument operating at 4 kV and mass resolution of M I A M 800 to maximize the signal (K111). Similar results have been reported subsequently using a 23-kG MS-50 (K112). Recently the molecular weights of bovine and other insulins have been measured on as little as 1-pmol sample size using the newly built Wien mass spectrometer (K113). Mesurements have also been made on mouse a-EGF and cloned EGF on 10 pmol of These total material using this new Wien instrument (K114). Wien results are comparable to the best sensitivities reported for plasma desorption mass spectrometry (K115). Further work has been carried out for the purpose of measurement of molecular weight of peptides of similar size such as atrial gland peptides using multiscan accumulation technique (50 scan) to obtain the nominal mass unresolved isotope cluster for peptide 2 at mass 6072 (K116). The general features of mass spectra obtained from fast heavy ion plasma desorption of peptides and small proteins have been described (K117) and recently reviewed in detail (K118). At least two interesting techniques associated with sample preparations for plasma desorption have been described recently, one having to do with sequestering of alkali-metal and cations in the sample by a nitrocellulose blotting (K119), the second by adding a mixture of reduced and oxidized glutathione to the solution prior to electrospraying on the target (K120). Both these procedures appear to enhance the molecular ion signal and reduce tailing of the unresolved mass profiles while decreasing profile peak widths. In addition, enhancement of multiply charged species also occurs. Careful measurements of molecular ion stable isotope profiles have been carried out between mass 700 and 1700 a t a mass resolution of 15000 (K121)such that the inherent stable isotopic contribution could be subtracted from the observed profile. It was found that the relative amounts of ions (viz. M + 1, 2,3...) other than the protonated molecular ion increased with increasing molecular weight of the peptide, and in the case of peptides containing disulfide linkages, the intensities of [M 21 and [M + 31 were remarkably high. This lends further evidence to support the suggested reduction of the disulfide linkage during primary beam bombardment in the liquid matrrx (K64).- -
+
CARBOHYDRATES AND GLYCOCONJUGATES There is a growing diversity of scientific and medical interests concerned with defining the primary structural nature, and in due course the topology, of the glycosylation associated with all classes of glycoconjugates, including glycoproteins, proteoglycans, and glycolipids. The list of carbohydrate oligomers with well-defined and important biological functions is growing daily and, hence, is attracting the attention of investigators with interests as disparate as cell adhesion, intercellular communication, antigenic determination, modulation of protein receptor function ahd hormone responses, secretory processes, embryonic development, regulation of cell growth, and viral replication. The experts in carbohydrate biochemistry have repeatedly noted that the potentialities for inherent structural complexity and diversity of oligosaccharides (glycosylation) far exceed that which is possible for polypeptides and oligonucleotides and, hence, represent a wealth of possible structural and topological specificity necessary for the putative molecular vocabulary involved in intercellular recognition, communication, control, and regulation. The ultimate goals of molecular structure elucidation of carbohydrate chains are to develop an understanding of the topological features expressed that determine the molecular recognition and specificity in terms of three-dimen-
MASS SPECTROMETRY
sional solution structures. This information could lead to understanding the detailed basis of the interaction with another macromolecule-for example, the topological and energetic basis for antigen antibody recognition and specificity. One will want to know the determinants that control the energetics of the binding in terms of parameters such as hydrophilic/hydrophobic, coulombic, etc. Such detailed characterization of a complex carbohydrate requires isolation of the substance in question, ita purification to structural homogeneity using chromatographic methodology, and structural elucidation using the most advanced methods in both mass spectrometry and nuclear magnetic resonance as well as susceptibility to specific purified glycosyl hydrolases. Recently, complimentary applications of these advanced methods have revolutionized the elucidation of the primary structures of complex carbohydrates and today have begun to shed light on their topology as well. These points are well articulated in a recent overview for nonexperts by Sweeley and Nunez, coverin primarily the period from 1979 to the middle of 1984 (LIT. This review discusses utilization of methylation analysis for determination of composition and linkages of simple oligosaccharides and the recently evolving degradative and analytical strategies for dealing with complex oligosaccharides. A discussion of the details of experimental protocol and spectral interpretation important in the qualitative and quantitative characterization of monosaccharides and determination of their linkage patterns can be found in a recent chapter by Patouraux-Prom6 and Prom6 (L2). Sweeley and Nunez also provide an introduction to the more recent developments of mass spectrometric methodology, which permit characterization of intact oligosaccharides using various derivatization strategies and newer ionization modes, including chemical ionization, field desorption, and ion or atom sputtering from liquid matrices (LSIMS and FAB, see discussion of nomenclature recommended in overview). While these reviews (151,L2),previous ones in this series (L3,L4), and others (L5-L8) will provide the interested reader as well as the expert with the important literature of the last few years of rapid progress, it is certainly clear that the developing usefulness of the ion or atom liquid matrix sputtering ionization techniques in mass spectrometry is making a major revolutionary impact in defining the nature of higher molecular weight, intact complex carbohydrates. This is particularly true for oligomers in the molecular size range where existing column chromatography and HPLC separation, including affinity and lectin separation techniques, do not readily permit purification to homogeneity of the liberated intact carbohydrate moieties. The very recent proliferation of high-performance, high mass ion optical systems coupled with a variety of soft ionization techniques has permitted focusing a new level of scientific attention on optimization of the strategies and experimental factors required for study of the primary structures of unknown complex carbohydrates. Many laboratories not previously involved with the use of double focusin and tandem instrumentation have become involved in stufies aimed at deciphering the types of chemical derivatives and matrices which might be most suitable as chromophores for chromatographic detection and for eventually obtaining high-quality molecular weight, composition, and sequence information on intact oligomers. They are discovering the unusual usefulness of this new mass spectrometric information in scrutinizing the successes and failures of the myriad of “established” chromatographic methodologies currently used to produce “homogeneous” carbohydrate oligomers; in addition, they are learning the limitations of the techniques of nuclear magnetic resonance in establishing molecular homogeneity, especially for high molecular weight carbohydrates. In the last 4-year period, by far the largest expansion of effort engendering the most excitement has centered around liquid matrix sputtering methods-temporarily overshadowing the earlier considerable successes of field desorption and direct chemical ionization methods (L4). Considerable effort has been expended to understand the mechanistic factors controlling the formation of relative abundances of molecular ions (true ion radicals), protonated molecular species, and cationized molecular adducts observed in the processes of field desorption of oligosaccharides (L9). This work in many ways attempts to deal with the same unresolved fundamental issues which are experimentally ob-
served in liquid matrix sputtering and thermospray LCMS. Further work has been carried out on relative effects of the nature of the matrix and the alkali metal cation in influencing the fragmentation which may be used for the identification of (1) stereoisomers of some hexoses (LIO),(2) anomeric (L12), methyl glycosides (L11),(3) 2-acetamido-2-deoxyhexoses and (4) cup-trehalose (L13,L14) using xenon atom sputtering conditions. Using collisional activation (CA), dissociation of the sugar alkali-metal-cation matrix cluster ion [S - M+ - Mx] occurs forming either the cationized sugar [S - M+] species or the cationized matrix [Mx - M+] species. The observed ratio of these two species in different matrices depends upon the relative basicity of the matrix (Mx) used. From such data, it is possible to differentiate these monosaccharide stereoisomers. Further studies involving an analogous monosaccharideproton-matrix cluster ion [S - H+ - Mx] illustrates the extremes of product species behavior observable using collisional activation for glycerol vs. diethanolamine as matrices. The collisional activation (CA) spectrum of [galactose glycerol H+] cluster ion forms gal + H+ and its respective unimolecular fragmentation pattern yielding gal-related structural detail while CA of the [gal + DEA + H+] cluster yields DEA Hiand ita respective DEA-CAD spectrum only. By way of contrast, in the negative ion mode a useful CAD spectrum is obtained on M - H-in a DEA matrix (L15). Further attempts at stereochemical differentiation of monosaccharideshave been carried out using acetone as a chemical ionization reagent gas (L16).The almost ubiquitous occurrence of sodium ion cationization of oligosaccharides in liquid matrix sputtering mass spectrometry has prompted reporting on a successful procedure to sequester the alkali metal cation using a cryptand polymer Kryptofix 222B (E. Merck, Darmstadt (FRG)) such that the sole ion current is the S-H+ species in the case of the oligosaccharide antibiotic of viridopentaose B (L17, L18). The mass spectral behavior of methyl 0-acetyl-0-D-xylopyranosides has been studied systematically (L19).Further work has been reported on the nature of mass spectral characteristics for a variety of oligosaccharides and oligosaccharide derivatives, including oligomers in the cello- (L20) and malto- (L20,L21)series and their alditols (L20)in both positive and negative mode as well as with the addition of sodium iodide. UV and visible absorbing derivatives have been obtained by cyanoborohydridereduction of the imine formed by coupling an aromatic amine and the reducing termini (L22-L24),or analogous covalent coupling with phosphorylethanolamine (L25). Further studies of three pentasaccharide antibiotics, viridopentaoses A, B, and C, have shown that the type of molecular ion species observed is dependent upon the relative proton affinities between the sample and the organic matrix material and the formation of protonated molecular ions or protonated matrix-sampleadducts [Mx - S - H+]. The addition of sodium tended to dominate with the formation of M + Na+ (L26,L27). These studies included use of B/Elinked scanning to obtain the unimolecular dissociation spectra of the various molecular ion species observed under different matrix and salt loading conditions in an effort to establish sequence and structural information for these aminoglycosides. It has been noted that poly(ethy1ene glycol) is a suitable alternative solvent to glycerol (L28). For some carbohydrates, it has the advantage that the protonated matrix cluster ions observed for glycerol are absent. In addition, the PEG ions may be used as an internal standard for accurate mass measurement of unknown carbohydrate fragments (L28). 2-Deoxy-2,3-didehydro-N-glycoloylneuraminic acid has been identified in porcine submandibular gland (L29),while N acetyl-2,3-didehydro-2-deoxyneuraminicacid occurs as a metabolite in mammalian brain (L30). Normal human fibroblasts cultured in a medium enriched with N-acetylmannosamine accumulate large amounts of N-acetylneuraminic acid; hence, these cells may be used as a model for a study of the normal and abnormal metabolism of sialic acid (L31). Evidence for the oxidation of internally linked galactosyl residues has been obtained for Helix pomatia galactogen (L32). A beef liver enzyme preparation has been shown to catalyze isomerization and epimerization of D-erythrose 4phosphate (L33). The product of ADP-ribosyl protein lyase showing was identified as 3-deoxy-D-g~ycero-pentos-2-ulose that the enzyme is not a hydrolase (L34). Ammonium CI GC/MS has shown that l-O-a-2-acetamido-2-deoxy-~-
+
+
+
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galactopyranosyl-myo-inositol and several inositol ditechniques were used to propose structures for three oligosaccharides occur in human urine (L35,,536). l80labeling and saccharides excreted in urine of a patient with early myoclonic atom LSIMS have been used to characterize inositol cyclic epileptic encephalopathy (L61). A series of oligosaccharides phosphate products of polyphosphoinositide cleavage by and their alditols containing an alternating and consecutive phospholipase C (L37, L38). sequence of neutral and acetamido sugars as such, and as the Atom LSIMS spectra of cyanogenic glycosides have been permethylated derivatives, has been carried out by both reported (1539). A series of bitter glycosides from Gentiana positive and negative ion sputtering and electron impact mass have been studied by DCIMS (L40). Structure elucidation spectrometry (156.2).While atom LSIMS can be applied to of a novel branched-chain hexasaccharide glycoside, muscathe nonderivatized oligosaccharides and can be used to esroside B, has been identified (L41). Atom sputtering CAD tablish the molecular weight unambiguously,electron impact spectra have been used to confirm structures of retinyl mass spectrometry of the permethylated or peracetylated phosphate mannose synthesized by liver membranes (L4.2). oligosaccharidesprovides fragment ions formed by cleavage Negative ion LSIMS has been useful in characterizing novel of the glycosidic bonds reproducibly on 5 nmol of sample in glucosylated flavanoids and other phenolic compounds from the tetrameric mass range investigated. This methodology sorghum (L43). Mass spectral data have supported the conwas then applied together with NMR techniques to characclusion that hydroxyl radical is not involved in fungal Cterize a series of 0-glycosidically linked core-region oligosaccharides isolated from human meconium glycoproteins (rr)-C(P) cleavage in the @-1and @-0-4models used to study which express oncofoetalantigens (L63). A tumor-associated the same reaction in lignin (L44). Oxidative degradation of carbohydrate antigen Ca 19-9 has been identified on human monosaccharides by iron(II1) chlorides using near-UV to seminal-plasma glycoproteins (L64). Atom LSIMS and mevisible light has shown the degradation product to be selective thylation analysis were employed to establish four major siproceeding via formation of an iron(II1) monosaccharide alylated oligosaccharide alditols derived from mucus glycocomplex (L45). proteins of human seminal plasma (L65). Major components Methylation Analysis. Further methodological reports of endo-H sensitive glycoproteins of Friend murine leukemia have appeared concerning analysis of partially methylated virus were shown to be oligomannosidic of size classes Man5 alditol acetates (L.2). Retention indices and mass spectral to Mans Identification of novel complex oligosaccharides characteristics have been reported for commonly encountered isolated from caprine P-mannosidosis kidney contain unusual monosaccharides in the analysis of plant polysaccharides using a high polarity (WCOT) column, BP-75, produced by bonding @-linkedmannose residue at the nonreducing termini (C67). the highly polar phase OV-275 on vitreous-silica (L46). EIA unique carbohydrate sequence with a sialidase-resistant and CI-MS of possible constituents of bacterial lipopolysialyl group has been characterized from rainbow trout eggs saccharides have been determined, including the peracetylated by direct probe EIMS (1568).Structural studies of large acidic methyl glycosides of an peracetylated alditols from 2oligosaccharides have been carried out from bovine (L69)and amino-2,4-dideoxy- and 3-amino-3,4-dideoxy-~~-pento- dog erythrocyte (1570) glycophorins. Fourteen carbohydrate pyranoses (L47). Further work on the GC/MS of partially structures have been determined, ten of which not previously methylated aldononitrile peracetates includes determination reported, which were isolated from water soluble human blood of retention indices and fragmentation characteristics for a group active glycoproteinspurified from human ovarian cyst number of monosaccharides (L48). It was noted that these fluid by employing one stage of Smith degradation and derivatives have the advantage of being more volatile than GC/MS analysis of both the permethylated and N-trithe correspondingalditols such that separation can be carried fluoroacetylated and the oligosaccharide alditols (L71). Negative ion LSIMS on a free sialylhexosiminitolgives clear out at lower column temperature. However, the molar response factors are slightly lower than the corresponding alsequence information from both the reducing and nonreducing termini for a substance isolated from urine of a patient with ditols. The use of reductive depolymerization has been dea hexosaminidase deficiency, while the positive ion spectrum veloped to determine polysaccharide linka e using GC MS was of complex origin and difficult to interpret clearly (L72). of the derivatized anhydro alditols (L49). 8C/MS has een Components of Gram-Negative Bacterial Lipopolyused to identify pyruvated monosaccharides among Klebsiella saccharides. Most laboratories still depend heavily on the and Rhizobium species (L50). It has been shown that 3,4di-0-acetyl-1,6-anhydro-2,7-di-O-methyl-~-glycero-~-rnannowell established EL and CI-GC/MS techniques to determine monosaccharide composition and linkage information on heptopyranose occurs during acid hydrolysis of oligomixtures obtained from total hydrolysates of complex carsaccharides having doubly branched L-glycero-D-mannoheptose units substituted by three residues linked through bohydrates obtained from the LPS by mild acidic hydrolysis. A variety of chemical transformation and degradative pro0-3,0-4, and 0-6 (L51). Ammonia CIMS is used to establish tocols is also employed, depending on the nature and the linkages from methylation analysis of three novel phosphocomplexity of the oligosaccharide at hand to obtain simpler inositol-containing sphingolipids from the human pathogenic oligomers or mixtures of smaller oligomers for methylation yeast Histoplasma capsulatum (L5.2). Transient 3-0analysis. However, in the case of characterization of intact methylglucose methylation of dolichyl oligosaccharides has oligomers, it is clear that liquid matrix sputtering techniques been identified in the biosynthesis of halobacterial sulfated coupled with new extended mass range, high sensitivity mass glycoprotein (L53). Structural characterization has been spectrometric instrumentation is beginning to provide totally carried out for a carbohydrate component of Vicia graminea new information about the purity and assembly of the indilectin which is specific for the N blood group (L54). Mevidual sugars and substituents from studies of the fragmenthylation analysis has been used for comparative studies on tation processes observed in the secondary ion mass spectral the polysaccharides of three Cladonia species (Reindeer moss) fragmentation patterns. (L55). Hybrid type glycolipids (lacto-ganglioseries) with novel Atom sputtering has permitted the identification of UDPbranched structures are present in undifferentiated murine 2,3-diacylglucosamine(L73) and, in addition, has permitted leukemia cells (L56). Use of a monoclonal antibody that identification of lipid A tetraacyldisaccharide 1-phosphate defines monofucosyl type 1chains has led to the isolation and products formed from monosaccharide precursors by an enidentification of a glycolipid antigen from human erythrocytes zyme in the cytosol of E . coli (L74). Further studies of temwhich is distinguishable from those from other sources (L57). perature-sensitivemutants of Salmonella typhirnurium, which Nonoxidative and oxidative degradation products of Dare defective in the biosynthesis of 3-deoxy-~-manno-octugalacturonic acid with alkali have been identified by the losonate, have permitted the isolation and characterization pertrimethylsilyl ether derivatives of the sugar acids (L58). of eight lipid A precursors (1575). Laser desorption mass Intact Oligosaccharide Alditols and Their Derivatives. spectrometry using a LAMMA 500 instrument has been used Sweeley and co-workers have used direct probe E1 techniques to elucidate the nature and location of amide-bound (R)-3on permethylated oligosaccharide alditols in order to characyloxyacyl groups in lipid A (L76)and dephosphorylatedfree acterize a variety of structures isolated from human urine lipid A preparations (L77) from lipopolysaccharides of Re (L59). By use of the same derivatives on urinary carbohymutants of various Gram-negative bacteria. Positive atom drates obtained from patients with a-mannosidosis, identiLSIMS was used to provide structural information for comfication of isomeric mixtures from tri- to decaoligosaccharides plete characterization for purified dimethylheptaacylmonohas been reported. Differentiation of components in these phosphoryl lipid A obtained from lipopolysaccharides of mixtures could be carried out on the basis of their distinct Salmonella minnesota R595 (L78). The complete structural fragmentation patterns (L60). Analogous mass spectral
6
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characterization of lipid A components of lipopolysaccharides has been outlined (L79). cu-2+4-inter3-Deoxy-D-manno-o~tulosonic acid disaccharidehas been characterized by GC/MS of the reduced permethylated derivative (L80). By use of a variety of chemical transformations and degradationstogether with methylation analysis and NMR, the structure of the capsular antigen of Neisseria meningitidis serogroup K has been established (L81). Analogous methodology has been employed to establish the structure of the capsular polysaccharide of N . meningitidis serogroup H (L82, L83). The structure of the 0-chain of the phenol-phase soluble cellular lipopolysaccharideof Yersinia enterocolitica serotype 0:9 has been determined (L84). Methylation analysis, Smith degradation, and two-dimensional proton NMR were used to establish repeating trisaccharide of the 0-specific polysaccharide of Citrobacter PCM 1487 (L85). Similar methodology including atom sputtering LSIMS was employed in the identification of the capsular polysaccharide from Streptococcus pneumoniae type 9 (L86). Work on pathogenic clones of E. coli, which cause urinary tract infections involving 0-antigen 6 (L87) and 04K52:H- (L88),have been characterized. Atom LSIMS studies played a key role in establishing that the enterobacterial common-antigen (ECA) is composed of cyclic 4-, 5-, and 6-trisaccharide repeating units (L89). E1 mass spectra of the reduced and permethylated oligosaccharides have been used in the structural characterization of the carboxyl-reduced lipopolysaccharide moiety of Rhodopseudomonas sphaeroides ATCC 17023 (L90). Analyses of alditol acetates have revealed 3-C-branched aldoses in lipopolysaccharide of phase I (Coxiella burnetii (L91). A D-giucosamine-1-7-m-heptosedisaccharide was isolated and identified from the E1 mass spectrom of its methylated alditol (L92). While the heptose residue is not observed under normal hydrolysis conditions, deamination of the complete core permitted its identification in this case. The possible role of periplasmic oligosaccharidesof the cyclic 1--2P-~-glucan type has been invoked as modulators of osmotic adaption by gram-negative bacteria (L93). Glycolipids. Electron impact and chemical ionization mass spectra continue to provide important structural information on permethylated and reduced glycolipids. In addition, field desorption and ion and atom liquid matrix sputtering provide important information including molecular weight, ceramide structure, and sugar sequence on both free and derivatized glycolipids, depending upon whether they are neutral or acidic. In general, field desorption and secondary ion mass spectrometry complement each other and have made it possible to extend the accessible mass range to at least 7000 daltons, a factor of 2 in molecular size over established EI/CI methods. Several overviews, which discuss the general advantages and disadvantages of these techniques and various derivatization strategies, have been published during this period (L6-L8, L94). Trehalose 6-monomycolate has been identified using FD and secondary ion mass spectrometry from a cell-free synthetic system of Bacterionema matruchotii (L95). Positive- and negative-ion atom sputtering and chemical degradative methods have been used to establish the structures of oligosaccharide segments from trehalose-containing lipooligosaccharides of Mycobacterium kansasii and the recognition of a unique N-acylkanosamine-containingepitope (L96,L97). Further work using californium-252 PDMS has established the molecular weights of the intact lipooligosaccharides of M. hansasii (L98). These spectra indicate that the lipooligosaccharide samples are heterogeneous,although no indication of such considerable heterogeneity was observed from examination of the hydrolytically released fatty acids by GC/MS. An intense [M + 281 ion was observed in all spectra and could be accounted for by inclusion of a formyl residue; however, this remains to be established (L99).GC/MS of the alditol acetates was used to establish the nature of a glycolipid antigen specific to Mycobacterium paratuberculosis, which belongs to the polar mycoside C glycopeptidolipid family (L100). The structure of a pyruvylated glycolipid from Mycobacterium smegmatis has been established by atom LSIMS mass spectra, periodate oxidation, and methylation analysis (L101). Reports on the positive and negative secondary ion spectra of a variety of neutral glycosphingolipids have permitted correlations of basic structural moieties including saccharide size, sequence, and ceramide composition (L102-Ll04). Negative ion mode
secondary ion mass spectra obtained using triethylenetetramine of a glycolipid that binds anti-myelin IgM M-proteins was used to postulate a structural sequence of this acidic glycolipid (L105). Permethylated derivatives of N-acetylneuraminic acid containing Gm and N-glycolylneuraminic acid containing Gm,GDI,, and Gib have been determined by direct inlet ammonia CIMS (L106). A number of further studies in blood group glycolipids have been carried out using EI, CI, and secondary ion mass spectrometry, including: a blood group A heptaglycosylceramide with globo-series structure (L107);fucose-containing ceramide pentasaccharidesfrom the plasma of blood group 0 Le(a-b-) nonsecretors (LI08); branched blood group B active glycosphingolipids from human erythrocyte membranes (L109);blood group B active glycosphingolipid in rat bone marrow cells (L110);glycolipids reactive with the monoclonal anti-Lea antibody ( L l I l ) , linear, di-, and triantennary neolactoglycosphingolipidsfrom rabbit erythrocytemembranes (L112). Structures of a series of linear poly-N-acetyllactosaminylceramides isolated from human granulocyteswere structurally characterized by secondary ion mass spectra, methylation analysis, and exo- and endoglycosidase treatment (LI 13). With similar methodology,an M2590 antibody-reactiveantigen isolated from B16 melanoma cells was structurally characterized (L114). By use of a monoclonal antibody ME 311, a glycolipid antigen was obtained by immunizing mice with human metastatic melanoma cell line WM46. Atom sputtering mass spectra revealed an acetyl group on the terminal sialic acid of this glycolipid. Further analysis by proton NMR established the ganglioside to be 9-0-acetyl-GD3(L115). Methylation analysis and direct probe E1 MS established a structure of a new fucoganglioside 6B isolated from a monosialoganglioside fraction of human colonic adenocarcinomas (L116). A com arative study of acidic glycosphingolipidswas carried outy! field desorption in the secondary ion mass spectrometry (L117). An unusual glucuronic acid containing sulfated glycosphingolipid without sialic acid has been identified from reaction with monoclonal IgM in neuropathy and with HNK-1 (L118). Chemical structures of a series of glycosphingolipids isolated from the pupae of Calliphora vicina have been established by direct EIMS and atom LSIMS of the permethylated derivatives (L119, L120). 3-Hydroxybutanoic acid has been identified as a substituent of the acidic extracellular polysaccharide of Rhizobium trifolii 0403 (L121). Oligosaccharides. By use of immobilized @-galactosidase, the disaccharide 6-O-/3-~-galactopyranosyl-2-acetamido-2deoxy-D-galactose was synthesized (L122). Structure of the has been trisaccharide Gal~l+3-(GlcNAc~l-+6)GalNAc-H2 determined by methylation analysis (L123). A series of acidic oligosaccharide alditols have been isolated from salmon egg polysialoglycoproteins and characterized by methylation analysis and secondary ion mass spectra (L124). Two new oligosaccharides have been identified from human milk to be sialylated lacto-N-fucopentaoses (L125). Fukuda, Dell, and co-workers have published a suite of results on the structure of lactosaminoglycan isolated from band 3 glycoprotein of umbilical cord blood erythrocytes (L126),human granulocytes (L127), band 3 glycoprotein of adult human erythrocytes (L128),human neutrophilic granulocytes (L129),human PA1 embryonal carcinoma cells (L130),and human chronic myelogenous leukemia cells (L131). In this series of studies a combination of chemical and enzymatic methods were employed to release the glycosylation from the protein and to carry out degradations. Atom LSIMS was used to characterize the size and the homogeneity/heterogeneity of the oligomers obtained, often as their permethylated derivatives (L132). Similar methodology was used to determine the structures of the main glycans of human amniotic fluid fibronectin revealing for the first time the presence of 0-glycosidically linked oiigosaccharides (L133). Phosphorylated high-mannose oligosaccharides have been isolated from Dictyostelium discoideum, and it has been shown that mannose 6-phosphate residues of the intact oligosaccharides are diesterfied to methyl groups. Both direct chemical ionization and atom sputtering mass spectrometry were employed as well as 31PMR. Interestingly, this methylphosphomannosyl moiety is an acid stable phosphodiester linkage (1234). Recently it has been suggested that it is possible to study a complex formed between an alkyltrimethylammonium ion and a 3-0-methylmannose oligomer by atom LSIMS (L135). ANALYTICAL CHEMISTRY, VOL. 58, NO. 5, APRIL 1986
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Glycopeptides. N-(1-Deoxyhexitol-1-y1)aminoacids have been identified by mass spectrometry as reference compounds for the nonenzymic glycosylation of proteins (L136). The study of the behavior of the 2-acetamido-2-deoxy-a-~-glucopyranosyl residue has been carried out during sequential hydrazinolysis, re-N-acetylation, reduction, and methylation of glycoasparagines and has resulted in the identification of the presence of ei ht byproducts in addition to derivatives (L137). It is postulated that of 2-acetamido-2-~eoxyglucitol these byproducts are formed from the key intermediate, 2acetamido-2-deoxy-~-glucose hydrazone. The storage products resulting from the enzyme defect, canine fucosidosis, have been characterized as fucose-containing glycoasparagines (L138). Glycopeptides from human fibrinogen were analyzed and found to consist of a mixture of equal amounts of monosialylated and disialylated species, but no asialoglycopeptides (L139). Covalently bound inositol has been determined in the hydrophobic membrane-anchoring domain of Torpedo acetylcholinesterase (L140). The glycopeptide antibiotic aridicin A demonstrates the power of liquid matrix sputtering techniques in determining carbohydrate content sequence and overall elemental composition. Accurate mass measurement was used to postulate the presence of a deconoyl substituent (L141). A disulfated oligosaccharide has been identified in bovine lutropin by methylation analysis (L142). Positive and negative ion sputtering spectra can be used to distinguish polymers of the 4-0-sulfate and 6-0-sulfate isomeric type in enzymatic digestive chondroitin sulfate (L143). In an attempt to extend this success, study of another very acidic glycosaminoglycan, heparin, was undertaken, but it was observed that atom LSIMS induces rapid and intense sulfite losses from N-sulfite groups in this biopolymer (LI44). This result has led to chemical modification of heparin fragments by formation of the N-desulfated, N-acetylated derivatives (L144) in an attem t to circumvent this problem. Comparison of positive an negative ion secondary ion mass spectra was carried out for a variety of sulfoglycolipids. Negative ion spectra were found to yield relatively more intense and structurally meaningful fragmentation patterns with which to establish sequence information (L145) and were able to establish the presence and location of the sulfate group (L146). Human interleukin 2(IL2), or T-cell growth factor, has been shown to contain variable glycosylation by secondary ion mass spectrometry (L142). While the carbohydrate appears to have no effect on promotion of T-cell proliferation, its presence may affect the pharmacokinetics of this potential pharmaceutical. The structure of a sleep promoting factor isolated from human urine was investigated by atom LSIMS and shown to be the peptidoglycan, N-acetylglucosaminyl-N-acetylanhydromuramylalanylglutamyldiaminodimelylalanine(L148, L149). The structures of glycopeptides obtained by exhaustive pronase digestion of hi h molecular weight salmon egg polysialoglycoprotein have een shown to be glycotri- and glycotetrapeptides, Thr*-Ser*-Glu and Thr*-Gly-Pro-Ser, respectively. (The asterisk indicates the residue glycosylation site.) The xenon ion LSIMS was obtained on these free glycopeptides (L150).
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DRUG METABOLISM Mass spectrometry has been regarded for many years as an indispensible technique in studies of the metabolic fate and biodisposition of drugs and is employed rotinely both for structure elucidation purposes and for highly sensitive and specific quantitative analyses of drugs and their metabolites in biological fluids. Recent developments in soft ionization techniques, notably LSIMS, have had a major impact in this field in that polar, thermally labile metabolites and drug conjugates may now be analyzed directly by mass spectrometric means. This has led to the identification of previously elusive metabolites and to the detection of products derived from hitherto unrecognized metabolic pathways. The articles reviewed below were chosen from a truly expansive literature to reflect the current role of mass spectrometry in studies of drug metabolism and to highlight developing trends in this very active field of investigation. The reader is referred to a very thorough, although regretably out-of-date, review by Harvey ( M I ) and to a commentary by the same author on applications of mass spectrometry to studies of drugs of abuse 186R
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(M2). Volume 8 of the RSC Specialist Periodical Reports on Mass Spectrometry contains a chapter on drug metabolism, pharmacokinetics, and toxicity, in which the literature from July 1982 to June 1984 is reviewed. At the time of going to press, however, further details of this recent publication were not available. Qualitative Applications. During the period covered by this review, mass spectrometry has contributed directly to the identification of several novel types of drug metabolite. Tinidazole, a 5-nitroimidazolyl antiprotozoal compound, was shown to undergo oxidative metabolism with concomitant migration of the nitro group to the adjacent ((2-4) position (M3). The product of this interesting reaction was not amenable to E1 or CI analysis but yielded an MH+ ion under LSIMS conditions. Oxidation a t both of the heteroatoms in the thiazole ring of chlormethiazole has been found to occur in man and affords an N-oxide, S-oxide product (M4),while metabolic cleavage of the furan moiety in diclofurime, a new calcium antagonist, takes place in the dog and pig (M5). Similar cleavage of a furan ring has been reported for the toxic agents, 2- and 3-methylfuran (M6). Aromatic fluorine substituents are normally considered to be metabolically inert, although replacement of F by both OH and SCH, groups has been reported for the antipsychotic agent flumezapine (M7). A new pathway of nicotine metabolism in vivo, involving methylation of the pyridine nitrogen atom to form a quaternary salt, has been detected in the guinea pig and appears to be specific for the R(+) enantiomer (M8, M9). Identification of the polar N-methyl derivatives of nicotine and nornicotine was accomplished by borohydride reduction and GC/EIMS analysis of the resulting mixture of products. Oxidation of carbon atoms a to nitrogen is a common metabolic pathway for amines, although the resulting carbinolamine derivatives are usually too unstable to be isolated. A recent exception, however, is to be found in a detailed study of the metabolic fate of carpipramine in which three carbinolamines were isolated from urine and characterized by EIMS (MlO). Abbott et al. (MlI) have described the identification of a novel formamide metabolite of methadone in the rat, while Millington and co-workers (M12) reported on the excretion of valproylcarnitine in the urine of pediatric patients receiving valproic acid therapy. The latter paper is of interest in that LSIMS (employed with B/E linked scanning techniques) was used to detect this novel hydrophilic conjugate in urine extracts and thermospray LC/MS was used to distinguish it from ita endogenous isomer, octanoylcarnitine. More recently, administration of the pivaloyloxyether ester of methyldopa to monkeys and humans has been shown to give rise to pivaloylcarnitine in urine, the conjugate again being detected by LSIMS (M13). It would appear, therefore, that conjugation of carboxylic acids with carnitine may be a more common pathway of biotransformation for acidic drugs than suspected previously and that further examples of carnitine conjugation will emerge as SIMS and LC/MS techniques are applied more widely to metabolic studies. The utility of LSIMS in such work is illustrated further by the identification of four quite distinct peptide con‘ugates of phenothiazine in neonatal calf urine, the amino aci composition and sequence of which were deduced by a combination of mass spectrometric and amino acid analysis techniques ( M 1 4 ) . Stable isotope trace methods, employed in conjunction with mass spectrometry, are playing an increasingly important role in studies of drug metabolism (M15--M17).The isotope cluster approach to metabolite identification, in which an equimolar mixture of labeled and unlabeled drug is administered, has been used effectively to investigate the biological fate of agents such as lorcainide (MI@, isopropylantipyrine (M19),suprofen (M20),budesonide (M21),ranitidine (M22),and benfluron (M23). Although deuterium remains by far the most popular isotope for such work, ’ W is employed occasionally for isotope cluster studies (M24). Administration of the labeled compound alone represents a useful technique for the positive identification of drug metabolites that happen to be identical with products of intermediary metabolism; the conversion of benzofenac to p-cresol, for example, was established recently by this method (M25). Differential metabolism of the two enantiomers of a racemic drug may be examined conveniently by the use of so-called “pseudoracemic” mixtures, in which one antipode of the racemate is labeled with a stable isotope. The widely prescribed (?-blockingagent propranolol serves as
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an excellent example of the use of this method and several papers have appeared in the past 2 years describing stereochemical aspects of N-dealkylation (M26,M27) and aromatic ring hydroxylation reactions (M28, M29) with this drug. Metabolism of the chiral barbiturate, hexobarbital, has also been studied recently with a pseudoracemic preparation (M30). The use of stable isotopes to probe the mechanism of various metabolic processes, both in vivo and in vitro, is and deuteria rapidly developing area of application (M31) um-labeled drugs have been used to examine acetylation/ deacetylation reactions (M32,M33) and oxidations catalyzed by cytochromes P-450 (M34-M38) and peroxidase enzymes (M38,M39). Oxygen-18, which is an especially valuable probe of metabolic oxidation processes (M40),has been employed by Thompson and Wand (M41) to investigate the intramolecular transfer of oxygen in a hydroperoxide derived from butylated hydroxytoluene. The same heavy isotope has been used to show that the catechol metabolite of phenytoin derives from two successive aromatic hydroxylations of the parent drug, rather than by dehydrogenation of an intermediate dihydrodiol (M42), and that the 3-hydroxy metabolite of valproic acid can be formed by the microsomal cytochrome P-450 system and is not an exclusive product of mitochondrial enzyme activity, as had been assumed (M43). A major disadvantage inherent in procedures that use stable isotopes for metabolic studies, however, is the lack of a convenient universal detector, comparable to the liquid scintillation counter for radiolabeled compounds, which can be employed in mass balance studies. Hege and co-workers (M44) addressed this problem in their work on the metabolic fate of propafenone in man by administering a deuterium-labeled analogue of the drug orally and subjecting urinary extracts to analysis by GC with a microwave plasma detector. The deuterium-labeled metabolites thus detected were identified subsequently by GC/MS. An alternative approach to assessing mass balance was ado ted by Nakamura et al. (M45)who studied the fate of [13C,lPNz]antipyrine in rats by combusting samples of urine, feces, and blood and analyzing the aseous products, together with samples of breath, for excess 6Nz and 13C02. This work was carried out by isotope ratio MS, as were studies on the conversion of 13C-labeledphenacetin and aminopyrine to l3CO2 by humans as a function of liver disease (M46)and age (M47). Conjugates of drugs and their metabolites with glucuronic and sulfuric acid are widely encountered in metabolism studies and a variety of procedures (mostly based on HPLC) are now available for their isolation from complex biological media in relatively pure form. Many low molecular weight glucuronides have been analyzed successfully, following trimethylsilylation or permethylation, by GC/MS using either E1 or CIMS. Recent examples include the glucuronides of propranolol and alprenolol (M48), denopamine (M49, M50), trans-soberol (M51),and valproic acid (M52). Soft ionization methods, however, are usually required where the conjugate is of higher molecular weight or if the native, underivatized species is to be analyzed (M53). FDMS has proved to be useful in this regard and has been employed recently to characterize glucuronides of the new antitumor anthracycline derivative, epirubicin, and its 13,14-dihydro metabolite (M54)and of two isomeric hydroxylated antipyrine metabolites (M55). As would be expected, LSIMS techniques are well-suited to the study of intact glucuronides and a number of reports on their application have now appeared (M55-M59). Both positive and negative ion spectra generally provide abundant molecular ion species and exhibit fragment ions resulting from cleavage of the labile glycosidic linkage (M56). van Breemen et al. (M60)studied the behavior of several 0-glucuronides under laser desorption conditions and reported that the resultant spectra shared many of the features of the corresponding LSIMS spectra; however, [M + Na]+ and [M + K]’ cationized species, rather than [M + HI+ ions, dominated the molecular ion region. Fenselau and co-workers (M57) compared thermospray LC/MS with atom LSIMS for the analysis of phenolic glucuronides and reported that the former technique produced more fragmentation and doubly charged ions than did the latter. 0-Acylglucuronides have attracted particular attention recently in view of their propensity to undergo pH-dependent rearrangement to P-glucuronidase-resistant forms (M52, M61). In the case of furosemide glucuronide, several such rearrangement products were generated by exposure of the 1-0-acylconjugate to mildly alkaline conditions
and the mixture obtained was analyzed by negative ion thermospray LC/MS (M61);this gave ions for each component corresponding to [M - H]-, the aglycone, and the sugar moiety ( m / z 175) and, in the case of the 1-0-acyl-linked conjugate only, an ion at m / 2 221, which may have diagnostic importance for this isomer. Other drug-sugar conjugates that have been analyzed successfully by MS techniques include phenobarbital N-glucoside (M62, M63) and 1-(2-fluor0-2-deoxy-/3-~arabinofuranosyl)-5-hydroxymethyluracil(M64). The latter example is notable in that the exact mass of the metabolite was determined by 252CfPDMS. LSIMS has had a major impact on the analysis of sulfate conjugates, which traditionally have been very difficult species to characterize directly by MS. Negative ion LSIMS of these compounds yields abundant [M - HI- ions and has been used to identify the sulfate esters of xamoterol, a new cardiac stimulant (M65), 5-hydroxy-6-methyl-2-di-n-propylaminotetralin, a dopamine agonist (M66),and acetaminophen (M59). LSIMS in the positive ion mode has also proved successful with some sulfates (M58, M59, M66) and a report has appeared describing the analysis of the isomeric 3- and 6-sulfates of morphine by laser desorption FTMS (M67). Although the topic of glutathione and related thioether conjugates is treated elsewhere in the review (see under Toxicology), it may be mentioned that both positive and negative ion LSIMS have been found to be well-suited to the analysis of sulfur-containing drug metabolites; the covalent adducts produced by reaction of mesna (2-mercaptoethanesulfonate)with activated cyclophosphamide and ifosfamide may be cited as relevant examples (M68). Although LSIMS techniques have been adopted widely for studies on drug metabolism, applications of MS/MS in this field have been slow to appear. The great potential of tandem MS for screening complex biological samples for specific drug metabolites or classes of metabolite has been discussed by Yost and co-workers (M69, M70) and is illustrated further in a recent report from Straub and Garvie (M59),who employed the technique, in conjunction with LSIMS, to search for polar drug conjugates in biological isolates. Since glucuronides undergo a neutral loss of 176 amu (in both positive and negative LSIMS), a neutral loss scan for 176 amu was employed by these authors to detect glucuronide conjugates in complex sample matrices. Similarly, aryl sulfate esters were detected by performing a neutral loss scan for 80 amu (SO,) in both positive and negative LSIMS; alicyclic sulfate esters, on the other hand, were detected by carrying out a precursor ion scan for m z 97 (HSOJ in the negative ion mode. This approach wou d appear to hold much promise for the rapid “profiling” of polar dry conjugates in complex biological fluids and should be equally applicable to studies with LC MS/MS. Indeed, the metabolic fate in man of the antiarrhyt mic drug ethmozine has been investigated recently by thermospray LC MS/MS (M71). The analysis of pharmaceuticals by atom LS MS and MS MS has been the subject of a commentary (M72) and an M /MS method for the characterization of the trypanocidal agent Suramin and related diarylene ureas by negative ion LSIMS has been published (M73). As a consequence of the developments that have taken place in new ionization and mass analysis techniques, the horizons for MS in studies of drug metabolism have expanded enormously in recent years. Thus, on the one hand, MS can now be applied effectively to characterize polar metabolites which, although of low molecular weight, are refractory to study by conventional E1 or CI methods and, on the other hand, analytical protocols based on MS can now be adopted for studies of the biotransformation of structurally complex and relatively high molecular weight compounds. As an illustration of the latter point, the reader is referred to impressive examples from the recent literature which describe metabolic studies on the and -Blb macrocyclic lactones, 22,23-dihydroavermectin-Bla (M74)and the cyclic undecapeptide, cyclosporine (M75-M79). Only a few years ago, such molecules and their rnetabcjlites would have been considered quite inappropriate for analysis by MS, but today fall well within the capabilities of modern instrumentation. Quantitative Applications. With the increasing potency of many new drugs and the concomitant reduction in their dose and therapeutic blood concentrations, efforts continue to develop assay procedures with extremely high sensitivity and specificity of detection. Numerous SIM GC/MS assays
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have been reported for drugs and their metabolites in biodefined by the use of pseudoracemic mixtures. A new techlogical fluids with detection limits in the low ng mL-l range. nique for the measurement of drug distribution has been In certain cases, where advantage can be taken of the indeveloped by Browne and co-workers (M103)and entails the herently high sensitivity of electron capture NICIMS, even serial administration of multiple stable-isotope-labeled forms greater sensitivities have been achieved. For example, the of the drug and collection of only a single body fluid sample potent antihypertensive agent clonidine has been determined has reviewed the use of for analysis by MS. Wolen (M104) as its 3,5-bis(trifluoromethyl)benzoylderivative at concenstable isotopes to assess drug bioavailability and bioequivatrations as low as 10 pg mL-l plasma by this approach, which lence and several recent bioavailability studies have adopted allowed pharmacokinetic studies to be undertaken in subjects this approach (M81, M82, M105-MllO). It is of interest to given a single oral dose of 50 pg of the drug (M80). Similar note that deuterium, which is the most convenient but least GC/NICIMS assays have been described for other antihydesirable stable isotope for in vivo administration purposes due to the potential for untoward deuterium isotope effects, pertensive drugs, e.g., metoprolol (M81),oxprenolol (M82), was the heavy isotope selected for use in all of these studies. 2-dicyclopropylmethylamino-2-oxazoline(M83)and debrisoWhereas the great majority of pharmacokinetic studies quine (M84);in the latter case, the assay included the 4performed by MS employ GC/MS with E1 or CI ionization, hydroxy metabolite whose rate of formation from debrisoquine alternative approaches are used occasionally. Thus, plasma is known to be genetically determined. levels of the e ipodophyllotoxin derivative, etoposide, were The detection of selected daughter ions of metastable measured by ECf PDMS (MI111 and residues of elemental transitions occurring in the first field-free region of a douplatinum in the renal tissues of dogs given cisplatin were ble-focusing instrument (selected metastable ion monitoring) determined by laser microprobe MS (M112). has been employed in the past to provide enhanced specificity of detection in quantitative MS analyses. Recent examples of the use of this technique in conjunction with GC/MS may TOXICOLOGY be found in studies of the pharmacokinetics of the cannabinoids Al-THC (M85) and AO-THC(M86),where detection A growing awareness of the involvement of chemically relimits from plasma in the low pg mL-' range were reported. active intermediates in the toxic response to a wide spectrum N-Hydroxyphenacetin, a toxic metabolite of the analgesic drug of drugs and environmental chemicals has stimulated interest phenacetin, has been measured in human urine by selected in defining the nature of these short-lived species and in metastable ion monitoring following introduction on the solids understanding their interactions with cellular constituents at probe; TLC was used for preliminary purification of urine the molecular level ( N l ) . As an example of the importance extracts and the methyl ether derivative was employed for of modern MS techniques in this rapidly developing field, the MS (M87). In all of the above examples, deuterium-labeled reader is referred to a series of papers on the metabolic fate analogues of the analytes were employed as internal standards and mechanism of action of the selective nigrostriatial toxin, for quantitative purposes. The use of tandem mass specl-methyl-4-phenyl-l,2,3,4-tetrahydropyridine (MPTP) (N2trometry for trace analysis work has great potential for drug N9). This compound, which is a thermal degradation product metabolism and pharmacokinetic studies (M69, M70, M88, of a mepiridine-type narcotic analgesic, has been found to M89), especially in situations where a polar and or thermally cause parkinsonism in monkeys and humans and is currently labile metabolite is to be quantified in relatively arge numbers the focus of intensive study as a result of its highly selective of biological samples. Straub and Levandoski (M90) have toxic effects on the dopaminergic nigrostriatial system. A described such an approach to the quantitative measurement number of in vitro and in vivo studies have demonstrated that of the N-oxide metabolite of 6-chloro-3-methyl-2,3,4,5-tetra- MPTP undergoes monoamine oxidase catalyzed metabolism hydro-lH-3-benzazepine, a new az-adrenoreceptor antagonist. to yield the dihydropyridine derivative, MPDP, which is oxBy the use of atom LSIMS and MS/MS on a triple quadruidized further to the corresponding pyridinium salt, MPP+. pole instrument, and selected reaction monitorin of the [M This latter species is believed to be the ultimate toxin. While + H]+ [M H - CH,OH]+ transition induced t y collision E1 and/or CIMS have been used to follow the conversion of with Ar, these authors were able to develop a sensitive stable MPTP to MPDP (N6, N7) and to the nontoxic compound, isotope dilution assay for this polar metabolite with a lower N-desmethyl-MPTP (N9),only LSIMS has proved suitable limit of detection corresponding to approximately 100 pmol for the direct analysis of MPP' (N4). As a consequence of analyte on the probe tip. Moreover, sample cleanup was procedures have been developed for the reduction of MPP' limited to a simple extraction step and each assay could be in brain tissue extracts with borohydride (N3)or borodeuteride accomplished in less than 5 min. A second, most impressive, (N5) prior to assay by GC/MS methods. An alternative example of the utility of MS/MS for quantitative drug analysis quantitative approach for tissue-bound MPP' is suggested is to be found in a paper by Tway et al. ( M 9 l )who developed by the elegant LSIMS work of Markey and co-workers (N4, a confirmatory assay for ivermectin in cattle tissue. The N10) who performed MS/MS analysis of MPP' in crude extracts of brain homogenates using a triple quadrupole mass procedure, which involved NH DCI ionization and collisional spectrometer. In any event, LSIMS is likely to play a key role activation of the [M NH;+ adduct ion ( m / z 892) for in future studies on the neurotoxic effects of MPTP and its 22,23-dihydroavermectin-B,,, was carried out with a triple oxidation products. quadrupole instrument and provided reliable detection limits Glutathione (GSH) conjugates of drugs and other foreign of 8-10 ppb at a signal to noise ratio of greater than 1O:l. It was noted by the authors that neither FDMS, atom LSIMS, compounds have long been of toxicological interest since they represent the products of reaction of electrophilic metabolites LC/MS, nor direct insertion EIMS (at a resolution of 10000) with this cellular nucleophile. Although simple adducts, such provided adequate sensitivity and specificity to form the basis as 5'-propyl-GSH (formed during metabolism of the antideof an assay for this compound which would meet regulatory pressant drug iproniazid), may be analyzed by EIMS ( N l l ) , requirements. soft ionization methods are normally required for this class As noted above, stable-isotope-labeled internal standards of conjugate. DCI, using N20 CHI as reagent gas, was used are used widely in quantitative MS assay procedures. The by Paoletti et al. (N12) to i entify the GSH conjugate of influence of such internal standards on factors such as the celiptium, a new antitumor agent, by virtue of its negative ion accuracy, precision, specificity, and limit of detection of MS spectrum. Positive ion LSIMS of GSH conjugates has been assays has been discussed by Garland and Barbalas (M92)and reported to yield abundant MH+ and cationized molecular biomedical applications of stable isotope dilution SIM GC/MS ion species; recent examples of such spectra may be found in procedures have been reviewed by Trager (M93). In addition papers on the metabolism of phenacetin (N13),p-phenetidine to their use in the preparation of internal standards, stable ( N 1 4 ) , and 6-ch1oro-4-oxo-10-propy1-4Hyrano[3,2-g]isotopes are being employed increasingly for a variety of apquinoline-2,8-dicarboxylate (N15). A GSIf conjugate of plications in pharmacokinetic studies. Determination of the morphine has been isolated from rat liver microsomal incufraction of a drug metabolized in a triangular metaboIic bations and analyzed by Cs' ion LSIMS (N16). The likely problem was solved by the use of a new stable isotope method point of attachment of the GSH moiety to the morphine (M94),while "pulse" doses of stable-isotope-labeled drugs have nucleus was deduced by NMR to be loa, which thereby imbeen used to study phenomena such FIB dose-dependent (M95, plicated a quinone methide as being the reactive metabolite M96), time-dependent (M97),and steady-state pharmacoof morphine. However, Yamano et al. (NI 7 ) used EIMS to kinetics (M98, M99). Enantioselective aspects of drug meidentify a conjugate formed in vitro between morphine and tabolism (M100) and disposition (M101, M102) have been
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2-mercaptoethanol as being 8-(2-hydroxyethylthio)dihydromorphinone and concluded that morphinone is a reactive metabolite of this drug. Conjugation of arylnitroso compounds with GSH leads to the generation of chemically unstable adducts. By performing the reaction of 2-nitroso-6-methyldippido[ 1,2-~:3’,2’-d]imidazolewith GSH in a lyceral matrix on the stainless steel target of a LSIMS probe, gaito and Kato (N18)were able to observe the MH’ ions of two short-lived GSH conjugates. The formation of a transient intermediate in the reaction of nitrosobenzene with 1-thioglycerolhas also been detected by LSIMS (N19). Uetrecht (N20) obtained evidence from LSIMS for a GSH sulfinamide conjugate of rocainamide, an antiarrhythmic agent, which appears to have Eeen formed by reaction of the nitroso metabolite of the drug with GSH. Although conjugation of foreign compounds with GSH was thought to represent a pathway for their detoxication, it is now recognized that renal processing of certain GSH adducts may lead to nephrotoxic products (N21). Hexachloro-1,3-butadiene,for example, produces kidney damage, and a GSH conjugate has been identified by FDMS (N22); further metabolism of this conjugate in vivo was proposed to be the source of a novel sulfenic acid urinary metabolite of hexachlorobutadiene, which was characterized as its TMS derivative by EIMS (N22). Further examples of GSH conjugates of nephrotoxic haloalkenes have appeared in studies on tetrafluoroethylene (N23)and chlorotrifluoroethene(N24); negative ions from atom LSIMS were used in the former case, and positive ions from Cs+ LSIMS in the latter. New approaches to the mass spectrometricanalysis of GSH conjugates have included thermospray LC/MS, which succeeded in providing spectral data for the adduct of 3-methylindole where LSIMS had failed (N25). Also, the development of a procedure for the derivatization of GSH conjugates in aqueous media promises to facilitate greatly the extraction and subsequent purification of these conjugates for study by MS (N26). Although apparently not yet applied to the study of xenobiotic-GSH conjugates,252Cf PDMS has been used to determine the molecular weight of a novel endogenous bis(glutathiony1)spermidine cofactor for GSH reductase in trypanosomatids (N27). Cysteine conjugates and their N-acetyl derivatives (mercapturic acids), which represent secondary metabolites of GSH adducts, present less severe problems for MS, and a number of these adducts have been analyzed successfully by E1 and CIMS (N28-N31). Both positive and negative ion LSIMS have been applied to the study of mercapturic acids, and were used to characterize two novel conjugates of 4-cyano-N,Ndimethylaniline (N32-N34). Hydrolysis of a sample of bovine serum albumin to which the reactive metabolite of acetaminophen had become bound covalently yielded a polar, alkali-sensitive amino acid conjugate of the drug, which was (N35). In this identified as 3-cystein-S-yl-4-hydroxyaniline work, stabilization of the labile conjugate was achieved by reaction, in buffered aqueous medium, with ethyl chloroformate and the resulting ethoxycarbonyl derivative was purified by HPLC and characterized by direct insertion CIMS. A modified version of this protocol was later applied to study the nature of hepatic protein-acetaminophen covalent adducts formed in the mouse in vivo (N36);the same cysteine adduct was released during protein hydrolysis and was identified, following conversion to a novel tert-butyldimethylsilyl derivative, by GC/EIMS. These studies on acetaminophen represent one of the few investigations on the molecular nature of drug-protein covalent adducts and demonstrate that reactive drug metabolites may alkylate specific target sites on protein and do not simply bind to nucleophilic centers on macromolecules in an indiscriminate fashion. Metabolic oxidation of aromatic amines is of toxicological interest since many hydroxylamines and nitroso compounds are mutagenic and carcinogenic. Similar products can be formed by metabolic reduction of aromatic nitro groups. EIMS has been applied to a study of compounds related to N-hydroxy-2-aminofluorene (N37, N38) and to products derived from the reductive metabolism of the 5-nitrofuran derivative, furazolidone (N39). Ammonia DCI proved useful in the identification of the hydroxylamine metabolite of procainamide, while the corresponding nitro derivative was detected by GC NICIMS (N40). LSIMS has also been used by Kat0 et al. ( 41) to analyze a series of thermally labile Nhydroxyarylamines which failed to provide satisfactory spectra
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under E1 conditions. N-Hydroxyphenacetin has been identified as a metabolite of phenacetin in human urine and its excretion quantified by a selected metastable ion monitoring Nitrosamines, as a class, continue to attract procedure (M87). much attention in view of their carcinogenic properties and widespread occurrence. Metabolic studies with the cis and (N42)and trans isomers of N-nitroso-2,6-dimethylmorpholine with N-nitrosodiethanolamine(N43)have relied on GC/EIMS methods for metabolite identification. An indirect measure of bioactivation of nitrosamines to their respective genotoxic products involves the detection of lsN2 released from 15N-labeled substrates, In one recent publication, which used this approach, 15N2produced in incubations with isolated hepatocytes was quantified by MS using peak matching at high mass resolution and with 20Neas internal standard (N44). A more comman method for such studies, however, is to quantify 15N2release by the measurement of 14N2:15N2 ratios with an isotope ratio mass spectrometer (N45). Stable isotopes have also been employed to study the products formed in vivo by reaction of nitrosamines with DNA. Thus, Farmer et al. (N46) demonstrated that coadministration of 4-(di[2H3]methylamino)antipyrine and nitrite to rats led to the excretion in urine of deuterated N-7-methylguanine;omission of the nitrite suppressed the formation of this alkylated base, presumably by decreasing the extent of nitrosamine generated in vivo from aminopyrine. Mass spectrometry has been employed with notable success by Kadlubar, Beland, and their co-workers for the identification of covalent adducts between DNA and metabolically activated aromatic amines, and a review of some of their recent work has been published (N47). LSIMS has proved to be a major asset to such studies, since many aromatic amine derivatives are polar, thermally labile, and chemically reactive; the successful identification of Nacetyl-N-sulfonyloxydimethylaminobiphenylby atom LSIMS serves to illustrate this point (N48). Nucleoside and nucleotide covalent adducts of the pyrrolizidine alkaloid dehydroretronecine have been examined by Deinzer and Burlingame (N49), who used Cs+ ion LSIMS for sample ionization. MS MS techniques are also likely to play an important role in t is field. Recently Chang et al. (N50)have characterized the products obtained by exposing calf thymus DNA to Nmethyl-N-nitrosourea by a sequence involving enzymic hydrolysis, HPLC separation, and MS/MS analysis under isobutane CI conditions. These studies, which were performed with a triple quadrupole instrument, were rendered quantitative in nature by the addition of 2H3-methylnucleosides as internal standards. Epoxides formed during metabolism may be chemically reactive and undergo a variety of conjugation processes. The synthesis and elimination reactions of methylsulfonium ions formed from styrene oxide have been studied with the aid of LSIMS (N51) and a novel metabolite produced from ring opening of n-butyl glycidyl ether was shown by LSIMS and NH3 CI/MS to be an N-acetyl-a-amino acid derivative (N52, N53). Although not formed metabolically, teroxirone, an experimental antitumor agent, contains three reactive epoxide functionahties and products of their reaction with diethyldithiocarbamate have been characterized by high-resolution EIMS and by LSIMS (N54). Reactive intermediates may be generated by the metabolic cleavage of heterocyclic ring systems, recent examples of which include the toxic 2- and 3-methylfurans, which are converted to acetylacrolein and methylbutenedial, respectively (M6), and the macrocyclic pyrrolizidine alkaloid senecionine, which gives rise to transIn each case, primary evidence 4-hydroxy-2-hexenal (N55). for the identity of the reactive intermediate was obtained by conventional E1 or CI procedures. Quinones and derivatives thereof have been found to represent electrophilic metabolites of a variety of foreign compounds. 3’-Hydroxyacetanilide,for example, is metabolized in vitro to 2-acetamido-p-benzoquinone which binds extensively to hepatic proteins (N56,N57); this covalent binding, however, which is also evident in vivo, apparently does not lead to liver injury. The antitumor agents m-AMSA (N58) and elliptinium (N59) are metabolized in vitro to reactive quinone imines, as are derivatives of 9-methoxyellipticine (N60). While EIMS was used to identify these quinones, LSIMS was necessary to characterize amino acid adducts of the quinone imine produced from elliptinium (N59). The quinone imine derived from peroxidase-mediated oxidation
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of W-methyl-9-hydroxyellipticiniumhas been shown to arylate purine nucleosides and nucleotides in vitro, leading to regioselective adducts substituted only at the 2’-O-position of the ribose; these adducts were identified by NMR and NH3 DCI techniques (N61). p-Phenetidine is converted to reactive species by peroxidase enzymes and dimeric and trimeric products have been identified by EIMS (N62,N63). Oxidation of the synthetic estrogen, diethylstilbestrol, by peroxidases also generates reactive semiquinone and quinone intermediates and a variety of novel secondary products of the reaction in vitro have been identified by GC/EIMS analysis of their TMS derivatives (N64). In an independent study, the synthetic quinone derivative of diethylstilbestrol has been shown to react with mercaptoethanol via Michael-type addition to afford a stable adduct, which was identified by GC/EIMS as 4-(2hydroxyethylthio)-3,4-di(p-hydroxyphenyl)-2-hexene (N65). The metabolic fate of valproic acid (VPA), an anticonvulsant drug, has received much attention since it has been proposed that the hepatotoxic side-effects of this agent may be mediated by reactive metabolites. GC EIMS techniques have been used extensively in these stu ies and almost 50 metabolites of VPA have now been identified (N66). Of particular interest are the mono- and diunsaturated VPA metabolites which have been found in greatly elevated concentrations in the plasma and urine of patients experiencing adverse side-effects of the drug, and in body fluids of a fatal case of VPA-induced hepatic injury (N67,N68). The terminal olefin metabolite of VPA, 2-n-propyl-4-pentenoicacid, has been shown to undergo further biotransformation in the isolated, perfused rat liver to products that are likely to be hepatotoxic (N69). Incubation of this same olefin with rat liver microsomes led to destruction of cytochrome P-450, and l80labeling methods, in conjunction with GC/EIMS, were employed to investigate the underlying mechanism (N70). Destruction of hepatic microsomal cytochrome P-450 also occurs with allylisopropylacetamide and novonal and the structures of the covalent adducts to prosthetic heme, which are generated during the destructive event, have been identified by NMR and FDMS (N71). A compound designed to destroy cytochrome P-450 through metabolic activation to a cvclobutadiene sDecies has been reDorted and the structure d its covalent adduct to heme agaih characterized by NMR and FDMS (N72). The focus of many of the papers cited above has been to elucidate the molecular mechanisms responsible for the toxic effects of a seemingly diverse array of organic molecules. As en experimental tool for use in such work, stable isotope labeling procedures and mass spectrometry have been adopted with increasing frequency and three brief reviews on their joint applications have been compiled (M31, N73, N74). The mechanism by which aromatic substrates are metabolized to phenols continues to attract attention since reactive arene oxide intermediates are believed to play a key role in the toxic effects of many benzenoid systems. Deuterium isotope effects on these aromatic oxidation processes have been employed as sensitive probes of the reaction mechanism and evidence has been obtained to support the concept of an addition-rearrangement step prior to, or in the absence of, arene oxide formation (N75-N77). In the case of bromobenzene, Monks et al. (N78) used GC/EIMS to show that the [2,4,6-2H3] substrate was converted by rat liver microsomes to obromophenol with 70% retention of all three deuterium atoms; this finding was consistent with the view that o-bromophenol is formed from the 2,3-arene oxide. For benzene itself, deuterium labeling experiments indicated that cyclohexadienone is a major intermediate in the oxidation to phenol (N79). The metabolism of CHC13in kidney homogenates fortified with GSH led to the formation of 2-oxothiazolidine-4-carboxylic acid, which was identified by isobutene CIMS (N80). This finding, together with the observation that CDC13is significantly less nephrotoxic than CHC13 in vivo, was taken as evidence for phosgene being the toxic metabolite of chloroform in the kidney. Deuterium isotope effects have also been used to study the toxicity of tris(2,3-dibromopropyl) phosphate (tris-BP), a flame retardant formerly added to children’s sleepware until it was shown to be mutagenic (N81, N82). Metabolism of tris-BP yields 2-bromoacrolein, itself a potent mutagen, which was identified in hepatic microsomal preparations by GC/EIMS. Interestingly, deuterium substitution in tris-BP at the terminal carbon atoms reduced both the
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mutagenic response and the formation of 2-bromoacrolein. Oxidation of arylacetylenes by cytochrome P-450 leads to the formation of the corresponding arylacetic acids and to inactivation of the enzyme. However, whereas the former event has been shown to be accompanied by a large kinetic deuterium isotope effect when the acetylenic hydrogen is replaced by deuterium (measured by GC/EIMS), enzyme destruction was insensitive to the presence of the heavy isotope (N83). These findings provide a unique insight into the molecular events leading from arylacetylenes to arylacetic acids on the one hand and to P-450 inhibition on the other. Fully deuterated N-nitrosodimethylamine (2H6-NDMA)is less carcinogenic in rats than its unlabeled counterpart and MS techniques have been used to study the origin of this isotope effect. With the aid of a GC/HRMS assay for NDMA, which employed SIM at a mass resolution of 10000 and which used 5N -NDMA as internal standard, Mico et al. (N84)were able to cfemonstrate marked differences in the oral bioavailabilities of the protium and deuterium forms of this carcinogen. Oxygen-18 has also proven to be a valuable tracer in investigations of metabolic activation processes (M40) and examples of its use may be found in studies of the biotransformation of allylisopropylacetamide (N70,N71, N85), acetaminophen (M40,N86), 4-nitroaniline (N87),N-nitrosohexamethyleneimine (N88), and various aromatic compounds which are converted to catechols (N89). A more traditional type of application of mass spectrometry in the field of toxicology has been to the detection of known or suspected toxins in samples of environmental or agricultural origin. A number of very sensitive and highly specific MS screening procedures have been reported for such compounds, including a method for the analysis of nine trichlorothecenes by GC/NICIMS using O2 as reagent gas (N90),and an assay for deoxynivalenol, a mycotoxin, which also utilized negative ion detection but with CH, as CI reagent gas (N91). Isomer-specific determination of tetra- and hexachlorodibenzop-dioxins by capillary GC/NICIMS has been the subject of reports by Oehme and Kirschmer (N92) and Miles et al. (N93), respectively. The former paper reported that amounts of TCDDs as low as 25 pg could be detected against a background of polychlorinated biphenyls and pesticides by SIM of the [M + HO]- adduct ions produced under CH4/N20CI conditions. EI, CI, and NICI have been compared as ionization techniques for use in the GC/MS analysis of 26 polychlorinated dibenzofurans and preliminary results were presented on the NICI spectra obtained using CHI plus small amounts of O2 a s reagent gas (N94). Positive ion NH3 CIMS has been used primarily by Cairns et al. (N95, N96) for the analysis of a variety of different pesticide residues. The thermally labile carbamate pesticide aldicarb and two of its degradation products, aldicarb oxime and aldicarb nitrile, were analyzed successfully by CIMS using a short capillary GC column for sample introduction (N97). This work has been extended recently to include the toxic species aldicarb sulfoxide and aldicarb sulfone (N98). Methylthio-substituted polychlorinated aromatics have been analyzed by GC MS using EI, CI, and NICI methods; the formation of [M - C sulfide anions was found to be a characteristic feature in the NICI spectra of these compounds (N99). Methylthio and methylsulfone derivatives of polychlorinated biphenyls (PCBs) have been detected in patients with “Yushon (PCB poisoning); GC/EIMS analysis revealed the presence of two congeners of methylthio PCBs with 4 chlorine atoms and 16 congeners of methylsulfone PCBs with 3, 4, 5, or 6 chlorines (N100). Methods have been reported for the quantitative determination of diethylstilbestrol in biological samples by GC/MS (N101, N102) and DLI LC/MS (N101),and an interesting paper describing the analysis of enantiomers of indenestrols by chiral HPLC-thermospray MS has appeared (N103). A stable isotope dilution GC/MS assay for the pesticide ethylene dibromide has been published (N104)and is of interest in that the isotope effect on the GC retention time of the internal standard (2H,-ethylene dibromide) was sufficiently large as to effect base line separation of the labeled and unlabeled forms of this compound on capillary columns and thereby permit the analyses to be carried out by GC alone, rather than by GC/MS! Finally, a detailed comparison has been made of the more selective MS techniques, viz., HRMS and tandem MS (both triple quadrupole MS/MS and MIKES), with more conven-
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tional capillary GC/MS methods for the quantitative analysis of hexachlorobenzene and trichlorophenol in human serum and urine (N105)The HRMS and MS/MS procedures, which used relatively short (30 or 50 cm) GC columns for sample introduction, were found to be well-suited to very rapid screening of the target compounds at sub-part-per-billion levels.
EICOSANOIDS The association of MS with research on the arachidonic acid cascade dates back more than 20 years and the use of mass spectrometric techniques for eicosanoid analysis remains a highly active and fruitful area of application (01).Broadly speaking, one can distinguish two principal types of usage of MS, viz., qualitative analyses (mainly by GC/EIMS) of new eicosanoids formed in vitro and in vivo and quantitative stable isotope dilution assays of known compounds by highly sensitive GC/NICIMS methods. The use of direct insertion MS with soft ionization techniques is emerging as a potentially important analytical method for polar, thermally labile species such as members of the leukotriene C, D, and E classes. A popular approach to the quantitative measurement of trace levels of eicosanoids involves preparation of the 0-methyl oxime, trimethylsilyl ether, pentafluorobenzyl ester (MOTMS-PFB) mixed derivatives and carrying out SIM GC/MS of the abundant [M - PFBI- anions (at m z [M - 1811) generated under NH, or CH4 CI conditions ( 2-011). Capillary columns are employed in the majority of cases and detection limits typically are reported to be in the low pg mL-l range. However, packed column GC/MS still has a place in prostaglandin assays, judging from a recent report by Rubio and Garland (011) who improved an earlier capillary GC/MS procedure for the synthetic analogue, acetyltrimoprostil, by reverting to the use of a 1.5-m packed column. The speed of the GC/MS analysis step in eicosanoid assays, which employ capillary columns, can be increased by the use of a high-boiling solvent, e.g., n-dodecane, for sample injection (02, 0 3 ) ; the same technique has been employed effectively for the analysis of steroids by capillary GC/MS (012). The overall selectivity of many of these assays can be enhanced further by employing a selective extraction method for isolation of the target compound($. Fitzgerald and co-workers (09),for example, have reported an elegant method for the selective recovery of thromboxane Bz (TXBz) and its 2,3-dinor metabolite from urine samples, based upon formation of the methoxime derivatives in aqueous media and isolation of the resulting 1,3-diol structures by adsorption onto phenylboronic acid columns. The development of antibody affinity gels for the selective isolation of urinary 6-oxoprostaglandin F1, (6-oxoPGF1,), TXBz, and their dinor metabolites represents an alternative approach to this goal (013), as does double antibody precipitation which has been used in conjunction with HPLC assays (014), but apparently not yet with GC/MS procedures. Several quantitative GC/MS methods for eicosanoids based on E1 ionization have been developed recently and have employed a variety of different types of derivative (015-021). The MO-TMS-PFB derivative cited above has also proven to be useful for E1 work (021), while tert-butyldimethylsilyl(O19,020,022), allyldimethylsilyl(O22),and enol trimethylsilyl ethers (015) have been employed for prostaglandins, thromboxanes, and lipoxygenase products. The known (023) selective reactivity of ester groups at the C-1 position of PGF-M, a dicarboxylic acid metabolite of PGF,, and PGFz,, has been rediscovered and utilized in a GC/EIMS assay for this marker compound in human urine (017). Catalytic hydrogenation of leukotrienes and other products of lipoxygenase pathways has been employed to render these compounds more suitable for GC/MS work. Thus, Woollard and Mallet (020) incorporated a reduction step into their protocol for metabolic profiling of monohydroxy fatty acids in lesional skin of patients with psoriasis. Advantages associated with analysis of the corresponding fully saturated acids are that the sites of hydroxylation in these compounds may be determined readily from the m / z values of intense a-cleavage ions in the E1 spectra of their TMS derivatives (020, 024), and that the gas chromatographic properties of these reduced molecules are superior to those of their unsaturated precursors (019). Internal standards labeled (in the carboxyl group) with oxygen-18, but not with
d
deuterium, are suitable for use in assays where a reduction step is included (019,024). The sulfidopeptide leukotrienes, LTC,,. LTD4, and LTE,, present unique problems for MS assay in that they are not amenable to gas-phase analysis by conventional approaches. Balazy and Murphy (025) have reported a procedure for the sequential reduction of these thioether leukotrienes using 5% Rh/Al2O3and Li/CH3NH2; the resulting fully saturated product from all three leukotrienes, 5-hydroxyeicosanoicacid, is then taken for GC/NICIMS analysis as its TMS-PFB derivative. Specificity of the assay is based upon HPLC separation of LTC4, LTD4, and LTE4 prior to reduction. Alternative approaches to the analysis of sulfidopeptide leukotrienes have been investigated by Blair et al. (026) who have explored the use of thermospray LC/MS and LSIMS for qualitative and quantitative determination of the intact thioethers. LSIMS has, in fact, been used successfullyto identify underivatized PGE2 and PGFz, as products of peroxidase-catalyzed transformations of arachidonic acid; negative ions were monitored in this study and preliminary MS/MS experiments were reported (027). Arachidonic acid monoepoxides, which have been found to be cytochrome P-450 catalyzed oxidation products of arachidonic acid, have attracted considerable attention recently in view of their biological activities and their conversion to other active eicosanoids. Isomers of arachidonic acid epoxide have been analyzed by low-energy charge-exchangeMS with CS2as reagent gas, when ions characteristic of the position of the epoxide function were observed (028). However, no information on the sensitivity of this technique for distinguishing the isomeric epoxides was presented. The metabolic fate of the 5,6-epoxide was studied in ram seminal vesicles and capillary GC/EIMS was used to identify products derived from cyclooxygenase-mediated reactions; two of the major metabolites were found to be stereoisomers of 5-hydroxy-PGI1 (029). The 5,6-epoxides of the prostaglandin endoperoxides, PGGl and PGH1, were also formed in this system (030). The dihydrodiol metabolite of 5,6-epoxyarachidonicacid undergoes secondary biotransformation in seminal vesicles and yields prostaglandins of the E and F series (031). A new technique for the gas-phase analysis of arachidonic acid epoxides, which has been adapted from an early report in the drug metabolism literature (032), involves their conversion to chlorohydrin derivatives by treatment with either HC1 in pyridine or trimethylchlorosilane (033). The chlorine isotope pattern in the chlorohydrin TMS product aids recognition of these derivatives by GC/MS, although a potential drawback of this procedure is that a mixture of positional isomers and diastereoisomeric forms may be generated. Perfusion of the isolated rabbit kidney with prostacyclin (PGIz)was found to lead to the formation of 5-hydroxy-6-oxo-PGF1,, which was identified as its MO-TMS methyl ester derivative by GC EIMS (034). The formation of this metabolite was suggeste to reflect the oxidation of PGIz to an epoxide intermediate via the renal epoxygenase pathway. Recently, Prough and co-workers (035) have demonstrated that the four isomeric arachidonic acid epoxides are substrates for cytosolic glutathione transferases; the GSH adduct produced from 14,15-epoxyarachidonicacid was subjected to analysis by LSIMS and a positive ion spectrum was obtained which exhibited a prominent MH+ ion at m/z 628 and a weak signal at m / z 307, corresponding to the glutathionyl moiety. In an interesting mechanistic study of the reactions leading to arachidonic acid epoxides and their secondary metabolites, Pace-Asciak (036) employed '*O-labeling techniques and GC/NICIMS to demonstrate an intramolecular transfer of the terminal -OH group of the hydroperoxide function in 12-hydroperoxyeicosa-5,8,10,14-tetraenoic acid (12-HPETE). GC/EIMS methods continue to be used widely in studies of the metabolic fate of prostaglandins. A comprehensive investigation of the fate of PGD2in human subjects led to the identification of no less than 25 urinary metabolites, 23 of which had PGF-type structures (037). Most of these PGF derivatives, however, failed to form cyclic boronate esters which indicated that the orientation of the hydroxyl group a t C-11 in these compounds was @, rather than the more commonly encountered a stereochemistry. A related study by the Vanderbilt group confirmed that endogenously synthesized PGDz in humans is converted in substantial part to PGF2 metabolites (0381, while Pugliese et al. (039) have shown that PGDz undergoes reduction in the isolated perfused
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rabbit liver to give 9a,
[email protected] E, and Ez were found to give rise to identical metabolites in rat hepatocyte cultures, viz., dinor-PGE, and tetra-nor-PGE,, these products of @-oxidationbeing identified by GC/EIMS as their MO-tBDMS-methyl esters (040). GC/MS using both E1 and CI ionization was employed to define the metabolic pathways of the stable prostacyclin analogue, iloprost, in the rat (041) and capillary GC/EIMS was used by Newton et al. (042) to identify products of LTB4 metabolism in rat hepatic microsomes as being the (w - 1)-and w-hydroxy acids. A novel series of compounds formed from arachidonic acid in human leukocytes has been reported by Samuelsson and co-workers (043). These compounds, which are trihydroxy-tetraenes, were characterized by classical GC/EIMS methods, used in conjunction with selective derivatization reactions and microscale oxidations and reductions. Similar techniques were employed by the same group to demonstrate that 12-HETE undergoes w-hydroxylation in human polymorphonuclear leukocytes (044). It is noteworthy that these powerful GC MS and microchemical methods for the elucidation of mo ecular structure continue to be employed as effectively in the 1980s as they were in the early days of prostaglandin research in the mid-1960s. Further applications of GC/EIMS to work in the eicosanoid field include the identification of a new group of trihydroxypentaenes formed during the metabolism of eicosapentaenoic acid in porcine leukocytes (045),to the characterization of metabolites of arachidonic acid formed in the rabbit pericardium (046) and by partially purified 5-lipoxygenase (047), and to the identification of products of arachidonyl-CoA elongation reactions in swine cerebral microsomes (048). 14,15-LTA4was detected, as its adduct with methanol, in an incubation of 15-HPETE with purified reticulocyte lipoxygenase (049) and the biomimetic conversion of 15-HPETE to 14,15-LTA4has been investigated with respect to the stereochemistry of hydrogen loss from C-10 (050); both studies again used GC EIMS. The metabolic fate of unsaturated fatty acids with c ain lengths less than (051-054) or greater than (055-057) Czohas also been reported. In all but one case, GC/MS techniques were used, the exception being docosahexaenoic acid (C22:6) whose metabolites were examined by Yergey et al. (056) using both positive and negative ion thermospray LC/MS.
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BIOGENIC AMINES As in the eicosanoid field, mass spectrometry is employed both for the quantitative analysis of trace levels of biogenic amines and their metabolites in biological fluids and tissues and also for the study of the metabolic fate of selected amines in vivo, usually with the aid of stable-isotope-labeled tracers. In the area of quantitative analysis, HPLC coupled to sensitive and specific detection systems (notably the amperometric detectors) has been adopted widely in recent years for routine assays of catecholamines, 5-hydroxyindoles, etc. However, SIM GC/MS procedures remain the most sensitive and specific methods for the determination of biogenic amines in general, and in particular for those compounds that lack readily oxidizable functional groups and that are not, therefore, amenable to analysis by electrochemical detection. A case in point is tryptamine, a pharmacologically active indoleamine which may act as a neuromodulator or neurotransmitter. Two stable isotope dilution GC MS assays for tryptamine in brain tissue have been reported, both of which employed SIM of fluoroacyl derivatives with E1 ionization (PI,P2).Application of the latter assay to measurements of tryptamine in rat brain tissue gave a value of 0.54 pg g-l, which was a factor of 5 less than that reported in the former paper. One approach to studying the functional activity of tryptamine in the central nervous system has been to monitor the excretion of its major metabolite in urine, indole-3-acetic acid, and Gelpi et al. (P3) have compared HPLC and GC/MS methods for this comound and also for tryptophan and its indole metabolites in iological samples (P4). These authors concluded that SIM GC/MS is a superior technique for tryptamine and indole3-acetic acid, while HPLC was preferable for tryptophan, serotonin, and 5-hydroxy indole-3-acetic acid. Both GC/MS and HPLC methods were used effectively to perform comparative ontogenesis studies of tryptamine, serotonin, and tryptophan in rat brain (P5). The extremely low detection
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limits often attainable with electron capture NiCIMS have been exploited in the development of very sensitive assays for selected biogenic amines, and a review of some recent applica' ions, together with a discussion of the underlying practical considerations, has been compiled by Faull and Beck (P6). A method for the quantitative analysis of octopamines and synephrines in human urine has been reported by Williams and co-workers (P7), who carried out capillary GC/ NICIMS assays based on monitoring the M-. or [M - HF1-a anions of the pentafluoropropionyl (PFP) derivatives. This work resulted in the detection of 0- and m-octopamine and m-synephrine, whose presence in human tissues or biological fluids had not been described previously. Higa and Markey (PS)identified 5-methoxyindole-3-aceticacid, a putative pineal metabolite, in human urine and measured its excretion using a packed column GC/NICIMS assay procedure. By monitoring the [M - HF1-s or [M - PFPHI-. ions of the N-PFP, trifluoroethyl ester derivative, the authors were able to detect 0.5 ng of the compound from a 2-mL urine specimen. A useful method for the extractive acylation and MS assay of 3methoxytyramine, normetanephrine, and metanephrine in cerebrospinal fluid has been described by Beck and Faull (P9), who also employed negative ion detection of PFP derivatives and cited limits of detection in the low picogram range. In a collaborative effort by the Stanford and Gainesville groups, Faull, Yost, and co-workers have explored the use of tandem mass spectrometry for the trace determination of tryptolines (tetrahydro-p-carbolines) in relatively crude extracts of rat brain tissue (PIO-PIZ).Interest in these compounds stems from their established pharmacological effects in the central nervous system and from speculation that they may be formed in vivo by condensation reactions between indoleamines and aldehydes. The possibility of artifactual generation of carbolines during sample workup, however, has hampered attempts to assess their true biological significance and prompted the present MS/MS experiments, which were performed with a minimum of sample preparation. Analyses were carried out in the NICI mode, and the reaction m z 348 ([M - HF1-e) m/z 179 (HCOC,F,]--) for the hepta uorobutyryl (HFB) derivative of tryptoline was monitored using a triple quadrupole instrument. Of particular interest is the authors' discussion (P10)of sample introduction techniques. GC/MS/MS with a short packed column (N72)failed to provide the necessary sensitivity of detection, apparently due to competition for thermal electrons in the ion source from extraneous, derivatized material in the injected biological sample. However, with the aid of an efficient capillary GC column and a 15-min temperature program, excellent results were obtained and a detection limit of 0.2 pg was achieved. These results highlight both the advantages of tandem MS techniques for trace analysis and also some of the limitations of the method. Thus, where derivatization of a highly complex mixture is performed, as in the present example, in order to optimize the NICI detection of a trace component thereof, quenching effects in the ion source may compromise seriously the sensitivity and selectivity of the MS/MS analysis if due regard is not paid to adequate sample cleanup prior to ionization. For this reason, the use of GC (and probably LC) inlet systems in conjunction with MS/MS seems likely to develop significantly in the near future, especially when applied to the measurement of trace endogenous components of complex biological matrices. Studies on the metabolic interrelationships of a variety of biogenic amines in vivo have been performed with the aid of stable-isotope-labeled tracers. Nfl-Dimethyltryptamine, labeled with four atoms of deuterium, was found to undergo metabolism in rodent brain to the corresponding deuterated analogues of N-methyltryptamine, tryptamine, 1,2,3,4-tetrahydro-@-carboline,and 2-methyl-l,2,3,4-tetrahydro-/3-carboline (PI3). Stereochemical aspects of the formation of 5hydroxymethyltryptoline in vivo have been examined by administering deuterium-labeled tryptophan to animals and analyzing the urinary tryptoline by capillary GC/MS using these experiments an optically active stationary phase (P14); showed that the two enantiomers of this chiral metabolite were present in unequal amounts, suggesting that at least a portion was produced by enzymatic means. P-Phenylethylamine has been found to be a metabolite in the rat of the antidepressant drug phenelzine (P15), and N-acetylserotonin and 6hydroxymelatonin have been identified as urinary metabolites
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of melatonin in the same species (P16). Both of these studies were performed bf administering deuterated precursors, although a mixed l C, 2H-labeled analogue of ~-threo-3,4-dihydroxyphenylserine (the immediate precursor of norepinephrine) was used to calculate norepinephrine synthesis rates in human subjects with degenerative diseases of the central nervous system (PI 7). Clearly, methodology for quantitatiue metabolic studies of this type should be validated carefully when the administered tracer is to be labeled with deuterium, since unexpected isotope effects can perturb significantly the biological fate of a deuterated tracer relative to that of its unlabeled counterpart (P13). New methods for the derivatization of primary amines in aqueous media have been published (P9,P18-P20) and should prove useful in the development of GC/MS assays for biogenic amines in physiological fluids.
STEROIDS, STEROLS, AND BILE ACIDS Mass spectrometry continues to be used extensively for the identification of steroids, sterols, and bile acids, the great majority of applications being to complex mixture analysis by GC/MS techniques. Shackleton ( & I ) has published an excellent survey of current methodology used in steroid profile analysis, with an emphasis on practical aspects of capillary column GC/MS as applied to the study of urinary steroid metabolites, and has also reviewed (435 references) the present status of mass spectrometry as a research tool in steroid and peptide endocrinology (Q2). Newer procedures for the extraction and purification of steroids and bile acids for subsequent MS analysis have been reviewed by Sjovall (Q3)who emphasizes the value of lipophilic cation and anion exchangers to subfractionate crude extracts according to conjugate class. The topic of steroid profiling by GC/MS has also been addressed in a brief review by Spiteller (Q4). Most authors appear to favor the use of the well-established TMS and MO-TMS derivatives for the analysis of steroids by GC/MS, although a number of alternatives have been proposed recently. Enol-tBDMS and mixed tBDMS-TMS ethers have been employed by Andersson and' Sjovall (Q5, Q6) for the analysis of neutral steroids and bile acids, while the mixed tBDMS-TMS derivatives have also been found to be satisfactory in work with estrogens (Q7). The high chemical stability of the tBDMS ether (and enol-tBDMS ether) group and the presence of abundant [M - C4H9]+ions a t high m / z values in the E1 spectra contribute to the popularity of these silyl derivatives, this being especially true for quantitative SIM GClMS applications. Other examples of the use of enolic derivatives for quantitative analyses of ketosteroids may be found in studies of anabolic steroid metabolites (enol-TMS) (Q8, Q9), progesterone (enol-HFB) ( Q I O ) , androstenedione (enol-PFP) (Q11),dexamethasone (enol-TMS) (Q12),and testosterone (enol-PFP) (811). Octafluorotoluene has been found to react with steroidal ap-unsaturated ketones in dimethylformamide, using CsF as basic catalyst; the resulting pentafluorotolylenol-ethers possess good GC properties, yield structurally informative E1 spectra, and have been employed by Jarman et al. (Q13) to quantify testosterone in human plasma. Cyclic diethylsiliconides have been evaluated for use in the gas-phase analysis of steroidal vicinal- or 1,3-diols and have been found to afford useful E1 spectra and to be considerably more resistant to hydrolysis than their dimethylsiliconide counterparts (Q14). The di-tert-butylsilylene derivatives investigated recently by Brooks and Cole (Q15) should also prove valuable in this regard, although their application to steroid analyses has yet to be described. GC/MS analyses of underivatized urinary steroids are seldom performed, although a procedure has been outlined for the rapid detection of anabolic steroid metabolites in human urine avoiding the formation of derivatives (Q16). An interesting approach to the study of neutral ketosteroids by LSIMS techniques involves reaction of the steroid with Girard's reagents P or T in order to introduce a charged (quaternary amine) functionality into the molecule (Q17, Q18). The LSIMS spectra of the products have been reported (Q17) to contain an abundant ion corresponding to the intact cation at (M + 134) or (M + 1141, where M is the molecular weight of the neutral ketone. Present limitations on the sensitivity attainable with LSIMS, however, would seem to restrict the scope of such a procedure for biomedical applications in the
steroid field, although the technique may prove more useful for drug metabolite analyses (Q18). A variety of both neutral and acidic steroids have been employed as probes of the catalytic function of purified ioszymes of cytochrome P-450, and GC EIMS techniques have been used to identify the products o incubations with substrates such as progesterone (Q19-&21) testosterone (Q19, Q20), androstenedione (Q22), estradiol (Q21), and 18hydroxydeoxycorticosterone (623). Recent work on the testis microsomal enzyme, termed cytochrome P-450,,11, employed oxygen-18 labeling techniques to examine the mechanism by which progesterone is degraded to androstenedione (Q20). Ketoconazole, an antifungal agent, blocks this activity and has been shown to cause an accumulation of 17a,20a-dihydroxypregn-4-en-3-one, which was identified by GC/MS analysis of its n-butylboronate derivative (Q24). Work continues on defining the detailed mechanism of the process by which cholesterol is degraded to pregnenolone (Q25) and Ikekawa and co-workers (Q26) have reviewed the role of SIM GC/MS in such investigations. Further studies on the metabolism of steroids in vitro have dealt with 15- and 16hydroxylation of androgens and estrogens in human fetal liver (Q27), with the fate of testosterone in human sperm and seminal plasma (Q28),with the conversion of 2a-hydroxyprogesterone to 2-hydroxylated corticosteroids by newborn rat adrenal cells (Q29), and with oxidation and reduction processes undergone by progesterone in rat liver epithelial cell cultures (Q30) and in human breast cancer cells (Q31);6ahydroxylated pregnanolone isomers were found to be major products in the latter studies. Houghton et al. (Q32)have partially characterized three C-18 neutral steroids in horse urine, viz., 4-estren-17-01-3-one,estrane-3,17-diol, and a dihydroxylated compound tentatively identified as estr-5(10)ene-3,17-diol. Evidence was resented to suggest that excretion of these steroids may! I e age-dependent. Studies on the biosynthesis of 16-androstenes in enonatal porcine testicular microsomes have demonstrated that 16-dehydropregnenolone serves as an intermediate in the process (Q33), while 6-oxygenated derivatives of 3-oxo-4-enesteroids have been identified as artifacts that appear to result from the hydrolytic cleavage of the corresponding 3-imines formed by reaction of the parent steroids with endogenous primary amines (Q34). Quantitative analyses of steroids and their metabolites by GC/MS techniques are now commonplace and a variety of applications employing SIM for this purpose are to be found in the recent literature (&2,QS-Q9, Q35-Q37). Fused silica capillary columns of 25-30 m length have been adopted by most investigators in this field. Houghton et al. (012) have extended their use of on-column injection techniques with high-boiling solvents to the analysis of anabolic and corticosteroid TMS and MO-TMS derivatives; a solvent effect is maintained by using this approach and the analysis time can be kept to a minimum. Steroids labeled with stable isotopes have been used as tracers for disposition and metabolic studies, recent examples of which include work on the synthetic steroids dexamethasone (Q12) and 17-methyltestosterone (Q38),and on the endogenous compounds progesterone ( Q I O ) , androstenedione ( @ I ) , testosterone (Q11),and cortisol (Q39). Both deuterium and carbon-13 have been used for labeling purposes in these studies. Analogues of testosterone and androstenedione labeled with oxygen-18 were employed as substrates by Beusen and Covey (Q40) to investigate the mechanism by which these steroids are aromatized in human placental microsomes. In neither case was estrogen formation accompanied by loss of l80from the C-3 position, indicating that Schiff base formation does not play a role in the aromatase reaction. Adlercreutz and co-workers have reported on the effect of diet on the urinary estrogen profiles in healthy women (Q41) and have published several papers on the use of GC/EIMS to study the excretion of isoflavonic Dhvtoestrogens and of lignans in human urine (Q42-Q45)aAd *cow milk (943, 944). Gaskell 'and co-workers have reported a series of studies aimed at assessing the value of selective GC/MS procedures for precise quantitative measurements of steroid hormones present at trace levels in human plasma. SIM at high mass resolution (800CklOOOO) provided enhanced selectivity for the detection of cortisol by GC/MS, although the decreased sensitivity associated with this mode of operation led to a fall
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in the precision with which the ratio of analyte to internal standard could be determined (Q46). Selected metastable ion monitoring, on the other hand, was found to provide very high selectivity and sensitivity of detection; when the M+. [M - CdHg]+ fragmentation of estradiol-17P-bis-tBDMS was monitored, a limit of detection of 5 pg was achieved (Q47). Selected reaction monitoring of the same transition using a tandem double focusing/quadrupole instrument gave a detection limit for estradiol of less than 10 pg and, when carried out with a parent ion resolution of 5000, provided extremely high selectivity for the analysis of this estrogen in extracts of human plasma (Q48). The importance, in this example, of gas chromatographic separation of the sample prior to mass spectral analysis was emphasized by the authors, who pointed out that, in general, steroid isomer-specific MS/MS assays will require GC for sample introduction. Mass spectrometry continues to be used widely in research on the biosynthesis and metabolism of vitamin D3 and its congeners. Direct insertion EIMS has been employed to as a new provitamin D identify cholesta-5,7,24-trien-3P-o1 (Q49) and to characterize 10-oxa-19-nor-25-hydroxyvitamin D3 as a novel metabolite of 25-hydroxyvitamin D3 in chick kidney mitochondria (Q50). The significance of the latter finding is that the new compound apparently coelutes exactly with 1,25-dihydroxyvitamin D3 (the major renal metabolite of 25-hydroxyvitamin D3) during normal-phase HPLC with traditional mobile phases and has probably escaped detection earlier for this reason. TMS ether derivatives of vitamin D and its metabolites are well-suited to chromatographic separation by normal-phase HPLC, and off-line LC/MS procedures have been employed to identify 24,25,26,57-tetranor23-hydroxyvitamin D3 as a metabolite of 24,25-dihydroxyvitamin D3 in the perfused rat kidney (Q51) and to study stereochemical aspects of the biosynthesis of 25,26-dihydroxyvitamin D3 (Q52). It has been noted that chromatographic separation of unlabeled and tritiated forms of the TMS ethers of such compounds may take place under normal-phase HPLC conditions and appropriate caution should be exercised,therefore, when collecting column fractions (Q52). The 23S,25R stereoisomer of 25-hydroxyvitamin D3 26,23lactol was identified by Takayama et al. (Q53)as a metabolite of the corresponding 23S,25R,26-trihydroxy compound and was shown to undergo further oxidation to the 26,234actone in chick kidney homogenates. EIMS was used in these studies and again in investigations of the metabolic fate of dihydrotachysterol, in rats (Q54). Chloride ion attachment NICIMS, on the other hand, was employed by Horst and co-workers (Q55) to identify primary and secondary metabolites of 1,25-dihydroxyvitamin D3 in bovine kidney; dichlorodifluoromethane served as reagent gas in this work. In MS studies of a more fundamental nature, Zaretskii et al. (Q56) have examined isomerization reactions of vitamin D and provitamin D in the mass spectrometer and have also reported on the translation energy release for the loss of 18- and 19methyl groups in unsaturated steroids (Q57). Recent applications of mass spectrometry to the identification and quantitative analysis of bile acids in biological fluids have been reviewed in an authoritative article by Sjovall, Lawson, and Setchell (Q58). The growing value of GC/MS techniques for the study of bile acids in a clinical setting is apparent from a recent report from Hofmann and co-workers (Q59)who characterized individual bile acids as their methyl ester, acetate derivatives in 255 duodenal bile samples from patients with gallstones before and during treatment with chenodeoxycholic acid. An approach to the measurement of the endogenous pool size of chenodeoxychloic acid in children has been reported by Norman et al. (Q60)and involves the administration of an oral dose of [11,12-2H2]chenodeoxycholate, followed by analysis of the corresponding compound in bile for deuterium incorporation. Stellaard and Paumgartner (Q6l) have described methodology for the measurement of isotopically labeled bile acids in serum a t concentrations of 0.05-8 KM. Capillary GC/EIMS was used in this work and SIM techniques permitted precise (CV C 1.5%) isotope ratio measurements to be made for chenodeoxycholic, cholic, and deoxycholic acid in a single run at quantities as low as 5 pmol injected on the column. In a study of the dehydroxylation and epimerization reactions which take place when cholic acid is incubated with human intestinal bacteria, Edenharder (Q62) employed hexafluoroisopropyl ester, tri-
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fluoroacetate derivatives for analysis by GC EIMS, while Fromm and co-workers (863)used the more traiitional methyl ester, acetates to demonstrate the formation of Sa-hydroxy$3-chol-6-en-24-oicacid as an intestinal bacterial metabolite of chenodeoxycholic acid in man. The role of peroxisomal enzymes in the metabolism of 3a,7a,12a-trihydroxy-jp-cholestane by rat liver has been studied by Thompson and Krisans (Q64),who employed GC/EIMS to detect a cholestanetetrol derivative produced in vitro; this observation may have significance with respect to the biosynthesis of bile acids from cholesterol and other sterols. Taurine and glycine conjugates of bile acids may be analyzed conveniently by LSIMS techniques and examples have been presented of the utility of LSIMS coupled with collisional activation and MIKES analysis, for the characterization of isomeric bile acid conjugates (Q65, Q66). Tandem MS has also been employed recently for the same purpose (Q67),while the utility of silver adduct ion doublets (due to lo7Agand lo9Ag)for identifying the molecular weight of bile acids studied by LSIMS has been demonstrated by Musselman et al. (868). As discussed in earlier sections of this review, LSIMS techniques have contributed greatly to the analysis of a wide variety of intact glucuronide and sulfate conjugates, and applications in the steroid field have been at the forefront of these developments (869). Both positive and negative ion spectra have been recorded from conjugates of steroids (866, Q69-Q72),vitamin D3and its metabolites (Q70,Q73),steroidal glycosides (Q74,Q75),and a sterol phosphate (Q76). Isomeric conjugates may, in some cases, be distinguished from one another by LSIMS using collisional activation and tandem MS techniques; one recent example of this approach involved and the 3-glucuronides of 5P-pregnane-3a,l7a,21-triol-20-one 5B-pregnane-3a,11@,21-triol-2O-one,whose negative ion spectra afforded intense [M - HI- ions at m / t 527 suitable for collision experiments (Q71). Novel conjugates of steroids with longchain fatty acids have been reported recently and soft ionization techniques have been applied to study the intact esters. Pearlman et al. (Q77) used LSIMS with both conventional and tandem mass analyzers to identify corticosterone 21-oleate as an in vitro metabolite of corticosterone in rat mammary gland tissue, while Raju and colleagues (878) characterized the oleate, linoleate, palmitoleate, palmitate, arachidonate, and sterate esters of androsterone in human breast cyst fluid by HO- ion NICIMS. Steroid and bile acid sulfates have been analyzed successfully by thermospray LC/MS with negative ion detection, and preliminary quantitative results from such studies have been reported (Q79,Q80). Quantitative methods for specific steroid conjugates using LSIMS techniques, on the other hand, are more fully developed and an assay has been published for pregnanediol Sa-glucuronide in human urine based on atom LSIMS with negative ion detection (Q72). Testosterone l7@-glucuronide,a structurally dissimilar steroidal glucuronide, served as internal standard in this assay. In contrast, Shackleton and Chai (869) have reported that accurate quantitative measurement of steroid sulfates in plasma by LSIMS was possible only through the use of stable-isotope-labeled internal standards.
ACKNOWLEDGMENT We are grateful to our many colleagues who so generously responded to our request for their recent reprints and re prints. We thank the UCSF pharmacy students William 8hh, David Myers, and Karen Hobdy for help with our bibliography. We thank Chris Yatsko for production of the final copy of this manuscript. T.A.B. thanks Shannon West for secretarial assistance. We thank Marilyn F. Schwartz for coordination of the schedules and efforts of all those involved in this endeavor. This work was supported by the National Institutes of Health Division of Research Resources Grant RR 01614 (to A.L.B.), Grant AM26743 (to A.L.B.), and Grant GM32165 (to T.A.B.). LITERATURE CITED OVERVIEW
( A I ) Rollgen, F. W.; Giessman, U. Int. J. Mass Spectrom. Ion Processes 1984, 56, 229 (A2) Holy Bible, Genesis, Chapter 11. (A3) Vandervorst, W.; Shepherd, F. R.; Lau, W. M. Nuclear Insfrum. Mefhods Phys. Res. 1985, B9 248. I
MASS SPECTROMETRY (A4) Simko, S.J.; Miller, M. L.; Linton, R. W. Anal. Chem. 1985, 5 7 , 2448. (A5) “Desorption Mass Specrometry. Are SIMS and FAB the Same?”; Lyon, P. A., Ed.; American Chemical Soclety: Washington, DC, 1985. (A6) Magee, C. W. Int. J. Mass Spectrom. Ion Phys. 1983, 49, 211. (A7) Falick, A. M.; Wang, G. H.; Walls, F. C. Anal. Chem., submitted. (A8) Todd, P. J.; Gllsh, G. L.; Christie, W. H. Int. J. Mass Specffom. Ion Processes 1984, 61, 215. SCOPE
(Bl) Mass Spectrom, Rev., Gross, M. L., Ed.; Wiley: New York. (82) Philip, R.-P. Mass Spectrom. Rev. 1985, 4 , 1-54. (83) Gallegos, E. J.; Sundararaman, P. Mass Spectrom. Rev. 1985, 4 , 55-86. (84) Schmitter, J. M.; Arpino, P. J. Mass Spectrom. Rev. 1885, 4 , 87-122. (85) Teeter, R. M. Mass Spectrom. Rev. 1985. 4 , 123-144. (B5a) Martlnson, D. P., Song, E-H. Mass Spectrom. Rev. 1885, 4 , 461-490. (B6) Lattimer, R. P.; Harrls, R. E. Mass Spectrom. Rev. 1985, 4 , 369-390. (B7) Schuben, H A . ; LaHimer, R. P. Mass Spectrom. Rev. 1984, 3 , 23 1-3 16. (88) DePauw, E. I n Mass Spectrom. Rev. 1988, 5 , No. 2, Summer 1986. (89) Chemical Abstracts Selects : Mass Spectrometry; Chemical Abstracts Service: Columbus, OH. (BlO) Specialist Periodical Reports Mass Spectrometry, Voi. 7; R. A. W. Johnstone, Sr. Reptr., The Royal Society of Chemistry: Burlington House, London, 1984. (B11) Specialist Periodical Reports Mass Spectrometry, Vol. 8; M. E. Rose, Sr. Reptr., The Royal Society of Chemistry, Burlington House: London, in press. ( B l l a ) Oliver, R. W. A.; Davis, K. R. “A Guide to, and Commentary on, the Published Collections and Literature of Mass Spectral Data”; VG Analytical: Manchester (U.K.), 1985; 14 pp. (812) Benninghoven, A. I n “Secondary Ion Mass Spectrometry, SIMS IV”; Springer Ser . Chem. Phys 1984. (813) “Secondary Ion Mass Spectrometry, SIMS V“, Springer Ser. Chem. Phys., in press. (814) Proc. Jpn. Med. Mass Spectrom. 1984, 9 . (815) Proc. Jpn. Med. Mass Spectrom. 1985, 10. (Bl6) Matsuda, H., Chang, T.-L., Eds. Proc. of the First ChinaJapan Joint Symposium on Mass Spectrometry, Beijing, 1984. (817) 32nd Annual Conference on Mass Spectrometry and Allied Topics, San Antonio, TX, 1984, 962 pp. (B18) 33rd Annual Conference on Mass Spectrometry and Allied Topics, San Diego, CA, 1985, 1103 pp. (B19) “Mass Spectromety in the Health and Life Sciences”; Burlingame, A. L., Castagnoii, N., Jr., Eds.; Elsevier: Amsterdam, 1985; 638 pp. (620) Lyon, P. A,, Ed., “Desorptlon Mass Spectrometry. Are SIMS and FAB the Same?”; Am. Chem. SOC.: Washlngton, DC, 1985; 298 pp. (B20a) Proc. 5th Internatlonal Symposium on Mass Spectrometry in Liie Sciences, Gent, Belgium, May 1984. Biomed. Mass Spectrom. 1985, 12, 437-576. (821) Watson, J. T. “Introduction to Mass Spectrometry”, 2nd ed.; Raven Press: New York, 1985; 351 pp. (622) Chapman, J. R. I n “Practical Organic Mass Spectrometry”; Wiley: Chichester, 1985; 197 pp. (B22a) Message, G. M. “Practical Aspects of Gas Chromatography/Mass Spectrometry”; Wiley: New York, 1984; 351 pp. (823) Odham, G., Larsson, G., Mardh, P.-A., Eds.; “Gas Chromatography Mass Spectrometry: Applications in Microbiology”; Plenum Press: New York, 1984. (824) “Mass Spectromety of Large Molecules”; Facchetti, S., Ed.; Elsevier: Amsterdam, 1985; p 23. (825) Desiderio, D. M. “Analysis of Neuropeptides by Liquid Chromatography and Mass Spectrometry”; Elsevier: Amsterdam, 1984. (826) Browne, T. R., Baiiiie, T. A,, Eds. J. Clln. Pharmacol. (Suppl.), in press. (827) Muccino, R. R., Ed. “Synthesis and Applications of Isotopically Labelled Compounds”; Eisevler: Amsterdam, in press. (B27a) De Leenheer, A. P.; Lefevere, M. F.; Lambert, W. E.; Colinet, E. S. I n “Advances in Clinlcal Chemistry”; Spiegei, H. E., Ed.; Academic: New York 1985; pp 111-161. (828) Karasek, F., Ed. “Mass Spectrometry in Environmental Sciences”; Plenum Press: New York, 1985; 578 pp. (829) Gaskell, S.,Ed. “MassSpectrometry in Biomedical Research”; Wiley: Chichester, June 1986, in press. (830) Lindhoim, E.; Asbrink, L. “Molecular Orbitals and Their Energies. Studied by Semiempirical HAM Method”; Springer-Veriag: Berlin, 1985; 288 pp. (831) Holden, M. E.; Martin, R. L.; Barnes, I . L. Pure Appl. Chem. 1984, 5 6 , 675-694. (832) Peiger, H. S.;et ai. Pure Appl. Chem. 1984, 5 6 , 695-768. (B32a) DeBikre, P.; Gallet, M. J. Phys. Chem. Ref. Data 1984, 13, 809-891. (833) Miller, J. M. Adv. Inorg. Chem. Radiochem. 1984, 28, 1-27.
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INNOVATIVE TECHNIQUES AND INSTRUMENTATION
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