2 Physical Methods and Techniques Part (ii) Organic Mass Spectrometry By J. R. TRAINOR and P. J. DERRICK Institute of Mass Spectrometry and Department of Chemistry University of Warwick Coventry CV4 7AL 1 Introduction To review progress in organic mass spectrometry during the last two years in the space available we have found it necessary to be highly selective and we have been obliged to omit many first-rate papers. We report in three sections the first of which covers technical developments in organic mass spectrometry. The second section covers applications of mass spectrometry and the third reviews fundamental aspects of organic mass spectrometry. As with the previous report,' a strong biological flavour inevitably emerges because so many significant developments over the past five years have concerned large organic molecules of biological importance.2 Technical Developments Ionization Methods.-The mechanisms of the ionization techniques such as fast atom bombardment (FAB) and electrospray' (see below) involving gas-liquid interfaces continued to attract attention. In FAB the effects of acidifying basic mixtures of glycerol-analyte were investigated by Shiea and Sunner3 with the aim of determining the role of preformed ions in the desorption process. Molecule-ion intensities should be enhanced as the concentration of preformed ions in the solution is increased. The study produced mixed results. The authors concluded that acidification of the sample may induce several phase and boundary-layer effects such as changes in the solubility or surface activity of the analyte.The study demonstrates that the desorption process in FAB is controlled by both the chemistry and physics of the solution. The use of benzene as a chemical ionization (CI) reagent for quantitative analysis (quantitation) of hydrocarbon mixtures was investigated by Allgood et aL4 The benzene radical-cation (C,H+Q) produced clean selective ionization of olefinic and benzylic hydrocarbons but did not ionize saturated alkanes. For olefins and alkylben- zenes consistent relative molar sensitivities were achieved for a range of molecular masses. The authors suggested that the reagent has application in quantitation of ' M. A. Baldwin Annu. Rep. Prog. Chem. Sect. B. 1990 86 18-32. M.Sakairi A. L. Yergy K. W. M. Siu J. C. Y. Le Blanc R. Guevremont and S. S. Berman Anal. Chem. 1991 63(14) 1488-90. J. Shiea and J. Sunner Org. Mass Spectrom. 1991 26(1) 38-44. C. Allgood Y. C. Ma and B. Munson Anal. Chem. 1991 63(7) 721-5. 25 26 J. R. Trainor and I? J. Derrick petroleum mixtures where the concentrations of unsaturated aromatics could be determined from a single measurement. Plasma desorption mass spectrometry (PDMS) has been reviewed from the physic- ists' perspective.' For the quantitation of thick layers of polymers PDMS has been shown to offer significant advantages over secondary ion mass spectrometry (SIMS).6 There was a 1-2 order of magnitude increase in the sensitivity of PDMS compared with SIMS and PDMS gave better quantitation of minor components although the major components were detected using both methods.Craig and Bennich' high- lighted the importance of sample preparation in PDMS observing that either bovine serum albumin or lysozyme suppressed the intensities of both melittin and insulin. This was attributed to competition for charge. The sensitivity of plasma desorption ionization was considerably enhanced by using resonance-enhanced multiphoton ionization (REMPI) in a study of adsorbed pyrene. A 100 fold increase in the ions detected following REMPI was observed compared with ions formed by PD alone. Using this method of post-ionization to enhance ion formation in PD the improved sensitivity to pyrene adsorbate deter- mined was sub-femtomole.8 Zubarev et al.have reported a new 'soft' ionization technique exploiting the avalanche of secondary electrons formed in an electron multiplier.' The authors claimed success in desorption/ionization of thermally labile biological molecules of modest molecular masses (up to 1250 Da). Abdel-Baky and Giese" reported high zeptomole ( mole) detection thresholds for electron capture negative ion mass spectrometry which is surely a record for the highest claimed sensitivity. Speir et a/." have used laser desorption to produce neutral peptide molecules in the gas phase and have ionized these molecules by chemical ionization. They were able to correlate the extent of subsequent fragmentation with proton affinity of the reagent species. The photochemistry of small molecules ions has received increasing attention as part of a general increase in the application of laser-based mass spectrometry.In favourable circumstances the injection of energy in quanta of light can be used to distinguish between isomers or influence a reaction path taken when more than one is available.12 Using a C02 laser Wight and Bea~champ'~ distinguished isomers of C4H8 by electron detatchment from C4HI ions trapped for several seconds in an ICR spectrometer. The uses of ICR and FT mass spectrometry are likely to grow as the reactions of trapped ions are studied more dee~1y.l~ Electrospray Ionization (ESI).-Electrospray ionization (ESI) is a recently intro- duced ionization technique which has come of age in a remarkably short time. The potential and limitations of electrospray for the ionization and analysis of biological B.U. R. Sundqvist Anal. Chim. Acta 1991 274(2) 265-75. 'H. Feld A. Leute R. Zurmuehlen and A. Benninghoven Anal. Chem. 1991 63(9) 903-10. ' A. G. Craig and H. Bennich Anal. Chem. 1991 63(4) 352-6. ' D. M. Hrubowchak M. H. Ervin and N. Winograd Anal. Chem. 1991 63(3) 225-32. R. A. Zubarev P. V. Bondarenko A. N. Knush and B. V. Rozynov Rapid Commun. Mass Spectrom. 1991 5( l) 32-4. 10 S. Abdel-Baky and R. W. Giese Anal. Chem. 1991 63(24) 2986-9. J. P. Speir G. S. Gorman D. S. Cornett and I. J. Amster Anal. Chem 1991 63(1) 65-9. l2 M. Bensimon T. Gaeumann and G. Zhao Znt. J. Mass Spectrom. Ion Processes 1990 100 595-609. l3 C. A. Wight and J. L. Beauchamp Int. J. Mass Spectrom.Zon Processes 1990 100 445-55. l4 J. A. Zimmerman C. H. Watson and J. R. Eyler Anal. Chem. 1991,63(4) 361-5. Physical Methods and Techniques -Part (ii) Organic Mass Spectrometry 27 molecules have been reviewed ~ritically.'~ The range of mass analysers to which EN sources have been connected has diversified enormously.'6 ESI interfaced with a quadrupole mass analyser has threatened to eclipse the magnetic-sector mass spectrometer as the instrument for modestly high-mass studies. The new-found power of the otherwise 'low mass' quadrupole mass analyser is available at relatively low cost. Coupling ESI to magnetic sector instruments provides greater resolution but there are questions about sensitivity and reliability which are still being addressed.ESI has been successfully interfaced with Fourier transform/ion cyclotron resonance (IT-ICR) mass spectrometers and quadrupole ion trap (IT) instrument^.'^ FT-ICR has the capability to provide the highest resolution of all mass analysers. ESI produces a very large set of related data which is extremely advantageous for advanced statistical analyses,'* offering exciting potential in differentiating components of complex mixtures. Chowdhury and Chait's analysis of three forms (a-, p-and +-) of bovine trypsin in a native mixture" has illustrated this point. These different trypsin isoforms could be distinguished despite their similar masses (23 293,23 311 and 23 329 Da) owing to differentiation in the principal charge-states adopted by each component.The different charge-states are assumed to reflect the higher-order structure of the isoforms and in particular the number of basic groups available as sites for protonation. Unlike other charged droplet techniques it is significant that electrospray can be successfully performed with solutions having an appreciable ionic strength. A study of the dependence of sensitivity upon the conductivity of the electrosprayed solution was made.20 In this work the increase in ion current obtained was small but more importantly perhaps did not decrease with ionic strength. Acidified aqueous sol- utions have proved difficult to study owing to the high surface tension associated with water; requiring high electric fields to cause desorption of ions. These conditions cause corona discharge terminating ion formation.Organic co-solvents are com- monly used which reduce the surface tension; however because the solution chemistry may be altered by the co-solvent this precludes in vivo and native aqueous studies of biomolecules and their reactions. Chowdhury et aL2' found that they could electrospray pure aqueous solutions by using a sharpened needle-tip. This increases the electric field gradient at the tip sufficiently to maintain the electrospray while reducing the risk of corona discharge. Henry et alZ2described experiments in which an electrospray ion source is interfaced to an FT-ICR. They reported mass accuracy of 0.003% and resolving power better than 60 000 from femtogram quantities of material by utilizing pulsed techniques and extremely long trapping times (in the order of 1000s).They were able to assign unambiguously the charge state of the analyte and by internal calibration obtained mass assignments for overlapping isotopic peaks to an accuracy M. Mann Org. Mass Spectrom. 1990 25( 1 l) 575-87. 16 R. T. Gallagher J. R. Chapman and M. Mann Rapid Commun. Mass Spectrom. 1990 4(10) 369-72. 17 K. D. Henry and F. W. McLafferty Org. Mass Spectrom. 1990 25(9) 490-2; G. J. Vanberkel G. L. Glish and S. A. McLuckey Anal. Chem. 1990,62(13) 1284-95; G. J. Vanberkel S. A. McLuckey and G. L. Glish AnaL Chem. 1991 63(11) 1098-1109. A. G. Femge M. J. Seddon and S. Jarvis Rapid Commun. Mass Spectrom. 1991 5(8) 374-7. 19 S. K. Chowdhury and B. T. Chait Biochem Biophys.Res. Commun. 1990 173(3) 927-31. 20 L. Tang and P. Kebarle Anal. Chem. 1991,63(23) 2709-15. 21 S. K. Chowdhury and B. T. Chait Anal. Chem. 1991 63(15) 1660-4. 22 K. D. Henry J. P. Quinn and F. W. McLafferty J. Am. Chem. Soc. 1991 113(14) 5447-9. 28 J. R. Trainor and P. J. Derrick better than 0.001 YO.Difficulty was experienced in determining the average molecular mass to a consistent agreement with the calculated distribution. It may prove necessary in FT-ICR to determine isotopically averaged molecular masses using much shorter trapping times and lower resolutions. Space charge and any kinetic isotopes effects which may accentuate poor reproducibility are likely to be amplified at long trapping times. Laser Techniques.-Matrix-assisted laser desorption/ionization (MALDI) has become the primary area of interest in laser-based organic mass spe~trometry.~~ MALDI is interfaced mostly with time-of-flight (TOF) mass analysers although success with other combinations have been reported.24 The other important areas of application of lasers concerns the injection of energy into a vaporized ion or molecule in order to induce dissociation or ionization.The mechanism of ion formation in MALDI is actively discussed. Measured velocity distributions have been used to support a jet expansion model for the desorption process in high mass polymers .25 Much effort has been devoted to the development of new matrices26 for MALDI. It is generally accepted that one role of the matrix is as a chromophore.A feature of MALDI spectra is often a dominant matrix peak. By controlling the molar ratio of particular matrices and analytes (e.g nicotinic acid and insulin) the matrix peak has be& demonstrated to be reversibly s~ppressed.~~ Radiation of different wavelengths can be used for MALDI however each different wavelength is likely to have its own set of useful matrices. DNA has been ionized from a frozen aqueous matrix containing metal atoms (copper or sodium). The laser photon energy was tuned to resonant electronic transitions in the metal atoms and efficient production of positive ions was observed?8 The introduction of a liquid matrix (3-nitrobenzyl alcohol) is potentially a very significant development in MALDI.29,30 Using a fibrous substrate for the liquid matrix proteins were detected up to 100 000 Da with 0.1-0.2% mass accuracy.Chan et aZ.,31further reported the detection of a molecular mass in excess of 0.5 MDa. The species was a positive singly charged octamer of bovine albumin. The analyte had been dissolved in O.OIo/~trifhoroacetic acid deposited on a drop of 3-nitrobenzyl alcohol and dehydrated in a warm air flow. Lustig and L~bman~~ described a method of analysis based on post ionization for which they claim three orders of magnitude of linear quantitation in the nanogram-sample range. The sample was introduced via a continuous-flow liquid probe vaporized by a laser and seeded into a supersonic jet. Resonance enhanced multi-photon ionization (REMPI) was performed using a second laser and the ions produced were analysed by time-of-flight (TOF).23 F. Hillenkamp M. Karas R. C. Beavis and B. T. Chait Anal. Chem. 1991,63(24) 1193A-1203A. 24 J. A. Hill R. S. Annan and K. Biemann Rapid Commun. Mass Spectrom 1991 5(9) 395-9. 25 R. C. Beavis and B. T. Chait Chem. Phys. Lett. 1991 181(5) 479-84. 26 S. Zhao K. V. Somayajula A. G. Sharkey D. M. Hercules F. Hillenkamp M. Karas and A. Ingendoh Anal. Chem. 1991 63(5) 450-3. 27 T. W. D. Chan A. W. Colburn and P. J. Derrick Org. Mass Spectrom. 1991 26(4) 342-4. 28 R. W. Nelson R. M. Thomas and P. Williams Rapid Commun Mass Spectrom. 1990 4(9) 348-51. 29 S. Zhao K. V. Somayajula A. G. Sharkey D. M. Hercules and J. Fresenius Anal. Chem. 1990,338(5) 588-92. 30 S. Zhao and Zhongshan Daxue Xuebao Ziran Kexueban 1990 29(3) 96-9.31 T-W. D. Chan A. W. C. Colburn and P. J. Derrick 0%.Mass Spectrom. 1992 27(1) 53-56. 32 D. A. Lustig and D. M. Lubman Rev. Sci. Instrum. 1991 62(4) 957-62. Physical Methods and Techniques -Part (ii) Organic Mass Spectrometry 29 Boernsen et aZ.33*34compared MALDI with reverse-phase high-performance liquid chromatography (HPLC) for the analysis of a range of macromolecules. Using MALDI the TOF spectra of positive and negative ions of sulfonic acids oligonucleotides polysaccharides and peptides were obtained over a mass range up to 200 kDA from as little as 100 femtograms of material with a mass accuracy of 0.2%. Vacuum ultra-violet (VUV) laser photoionization was applied to a series of C6 to C8organic molecules to compare 10.5 eV photoionization with low-energy (12 eV) electron impact i~nization.~~ The results showed VUV photoionization at these energies to be far ‘softer’ giving more abundant molecular ions less fragmentation and enhanced fragment ions formed via low-energy rearrangements.MALDI has been shown able to differentiate between covalent and non-covalent adducts to biomolecules providing a clear advantage over traditional methods of analysis (i. e. liquid chromatography and UV/visible ~pectrometry).~~ Whilst the analytical use of MALDI appears to be predominantly for the determination of relative molecular masses,37 other applications are being found such as quality control e.g. detecting impurities in protein ~ynthesis.~~ It is predicted that the use and versatility of MALDI will increase and develop sharply.Tandem Mass Spectrometry.-Nowadays tandem mass spectrometry can be perfor- med with varying degrees of success on a range of instruments four-sector tandem mass spectrometers double-focusing mass spectrometers with quadrupoles (hybrids),38 trapping devices (FT-ICR and IT) even tandem TOF.39,40 FT-ICR and IT hybrids and triple quadrupoles typically facilitate exploration of low-energy collision regimes. The higher centre-of-mass (CM) collision energies available for collision-induced dissociation studies have made four-sector tandem mass spec- trometers the method of choice for investigating large molecules (molecular masses >1000 Da). This is made all the more efficient when a point-detector (e.g.an electron multiplier) is replaced by an array-detector (e.g.a multi-channel array).41 Low-energy CID of multiply charged biopolymers formed by ESI have been studied42 using a triple quadrupole mass spectrometer. Dissociation could be accom- plished in the nozzle/skimmer interface by adjusting the magnitude of the applied nozzle bias potential. Specific dissociations could be induced in the RF-only collision cell to obtain partial sequence information. The suggestion was made that biopoly- mers can be fingerprinted from the characteristic dissociations of a series of charge states under known conditions as was exemplified by analyses of dissociation of nine charge states of cytochrome C. 33 K. 0. Boernsen M.Schaer and H. M. Widner Chimiu 1990,44(12) 412-16. 34 K.0.Boernsen M. Schaer E. Gassmann and V. Steiner BioL Mass Spectrom 1991,20(8) 471-8. 35 S. E. Van Bramer and M. V. Johnston J. Am. SOC.Muss Spectrorn 1990 1(6) 419-26. 36 M. M. Siegel I. J. Hollander P. R. Hamann J. P. James L. Hinman B. J. Smith A. P. H. Farnsworth A. Phipps D. J. King M. Karas A. Ingendoh and F. Hillenkamp Anal. Chem. 1991,63(21) 2470-81. 37 B. Stahl M. Steup M. Karas and F. Hillenkamp Anal. Chem. 1991 63(14) 1463-6. 38 R.A.Yost and R. K. Boyd Methods Enzymol. 1990 193 154-200. 39 C. Weickhardt R. Weinkauf K. Walter and U. Boesl Znst. Phys. Con$ Ser. 1991 114 169-72. 40 P.J. Demck D. R. Jardine and D. S. Alderdice Org. Muss Spectrom. 1991,26 915-916. 41 F.C. Walls M. A. Baldwin A. M. Falick B. W. Gibson S. Kaur D. A. Maltby B. L. Gillece-Castro K. F. Medzihradszky S. Evans and A. L. Burlingame Biol. Muss Spectrom. Roc. Znt. Symp. Muss Spectrom. Health Life Sci.,2nd 1989 (Pub. 1990) 197-216. 42 R. D.Smith J. A. Loo C. J. Barinaga C. G. Edmonds and H. R. Udseth J. Am. SOC.Muss Spectrom. 1990 1(1) 53-65. 30 J. R. Trainor and P. J. Derrick Tandem mass spectrometry for electrosprayed ions can be favoured by an in- creased efficiency of collision-induced dissociation. Loo et al.43reported an enhance- ment factor of 20 in sequence information obtained by studying a variety of human and animal serum albumin proteins because of the high collision energies accessible. They suggested on the basis of their results that earlier sequences may be in error.FAB MS-MS is increasing its application as a tool to study geometric isomers (e.g. the linkage position in isomeric disaccharide sugarsa). Falick et aZ!5 exposed artefacts in the four-sector tandem mass spectrometry of protonated peptides using FAB as the ionization technique. Artefacts arise from the large background chemical noise and from metastable decompositions in the third field-free region. Likewise has discussed factors concerning precision and accuracy in relation to collision induced dissociation (CID) measurements using hybrid and triple quad- rupole tandem mass spectrometers. A separate investigation to compare hybrid and 4-sector MS-MS using peptides showed full sequence information only when using the latter te~hnology.~~ The combination of tandem mass spectrometry (MS-MS) and liquid chromatogra- phy (LC) has permitted the detection of nucleoside isomers in RNA,48by virtue of the rapid screening capability of LC and the nanogram sensitivity and selectivity of MS-MS.Tandem mass spectrometry has also been used as a method for chiral recognition4’ on carefully prepared samples. Mass Ana1ysers.-Time-ofRight (TOF). There has been a renaissance in the use of time-of-flight (TOF) as a method of mass analysis. Much of the new interest is driven by the developments in laser-based mass spectrometry however TOF has also been used to determine simultaneously the mass and energy of combustion products in rocket fuel using position-sensitive TOF dete~tion.~’ Development of techniques and apparatus to give time-focusing and ion bunching have been given high priority.Brunelle et aims1 described the design and operation of a high-resolution ion mirror constructed from two grids for which they claim second-order time-focusing properties. Improvements in space and time focusing have been described in other ion mirror designs.52i53 Cerezo and Miller54 described the spatial and temporal focusing properties of two Einzel lens designs used in tandem with a toroidal electrostatic sector. A similar study of the optical properties of a reflectron and Einzel lens has been made by 43 J. A. Loo C. G. Edmonds and R. D. Smith Anal. Chem. 1991 63(21) 2488-99. 44 G. E. Hofmeister Z.Zhou and J. A. Leary J. Am. Chem. SOC., 1991 113(16) 5964-70. 4s A. M. Falick K. F. Medzihradszky and F. C. Walls Rapid Commun. Mass Spectrom. 1990,4(9) 318-22. 46 R. I. Martinez J. Am. SOC.Mass Spectrom. 1990 1(3) 272-3. 47 M. F. Bean S. A. Carr G. C. Thorne M. H. Reilly and S. J. Gaskell Anal. Chem. 1991,63(14) 1473-81. 48 T. Hashizume C. C. Nelson S. C. Pomerantz and J. A. McCloskey Nucleosides Nucleotides 1990 9(3) 355-9. 49 G. Hofmeister and J. A. Leary Org. Mass Spectrom. 1991 26(9) 811-12. 50 J. E. Pollard D. A. Lichtin S. W. Janson and R. B. Cohen Rev. Sci. Instrum. 1990,61(10 Pt.2) 3134-6. ” A. Brunelle S. Della-Negra J. Depauw H. Joret and Y. Le Beyec Rapid Commun. Mass Spectrom. 1991,5(1) 40-3. 52 A. N. Kudryavtsev N. V. Nikonenkov B.M.Dubenskii and D. V. Shmikk Rib. Tekh. Eksp. 1990 (2) 140-3. 53 R. Kutscher R. Grix G. Li and H. Wollnik Znt. J. Mass Spectrom. Zon Processes 1991,103(2-31,117-28. 54 A. Cerezo and M. K. Miller Sud Sci. 1991 246(1-3) 450-6. Physical Methods and Techniques -Part (ii) Organic Mass Spectrometry 31 Camus and Melmed.” Several designs for TOF mass analysers have been discussed and compared with the performance of dispersive mass ana1yse1-s.~~~~~ Ion Trap (IT). The quadrupole ion trap (IT) has been developed significantly over the past two particularly in its capabilities for high-mass measurements and for MS-MS. Resolution of 40000-100 000 full-width at half-maximum (FWHM) has been demonstrated using protonated molecule-ions of substance P by modifying the experimental configuration to produce a much slower scan rate.60 Methods for extending mass range have likewise been discussed:61 a calibration using (CsI) Cs+ clusters has been obtained up to (CSI)~~~CS+ (45 000 Da).Despite these exciting developments some problems need attention if there is to be widespread adoption of quadrupole ion traps in mainstream mass spectrometry. Guidugli et al.62described phenomenological ‘black holes’ in their CID spectra produced in an ion trap. At certain values on the Matthieu plot ions were absent or severely reduced in CID spectra. Applications of the IT in chemical studies are growing. Evans et have demonstrated the use of ion-trap mass spectrometry to distinguish isomers of hydroxyindole whose EI spectra are virtually superimposable.Nourse et al.64used the ion trap as ‘reaction vessel’ and analyser to study the methylation of isomeric dihydroxy benzenes. Similarly the identification of the functional double bond location in selected linear alkanes has been made using IT.65 Interfacing Chromatography with Mass Spectrometry.-Much effort has been directed towards coupling of chromatographs with electrospray sources. Covey et al.66have interfaced microbore HPLC directly and full bore HPLC via 20 :1 splitter with an electrospray source fitted to a quadrupole mass spectrometer. They obtained full-scan and MS-MS mass spectra from tryptic digests of peptides of biomedical interest. The suitability of capillary zone electrophoresis as a separator for ESI has been the subject of ~peculation.’~ Verheij et demonstrated the use of FAB with a pseudo-electrochromatography system featuring a larger sample introduction capa- bility than CZE.55 P. P. Camus and A. J. Melmed Surf Sci. 1991 246(1-3) 415-19. 56 T. Bergmann H. Goehlich T. P. Martin H. Schaber and G. Malegiannakis Rev. Sci. Instrum. 1990 61(10 Pt. l) 2585-91; T. Bergmann T. P. Martin and H. Schaber Rev. Sci. Instrum. 1990 61(10 Pt. 11 2592-600; D. C. Hamilton G. Gloeckler F. M. Ipavich R. A. Lundgren R. B. Sheldon and D. Hovestadr Reu. Sci. Insfrum. 1990 61(10 Pt.2) 3104-6. 57 H. Wollnik Nucl. Instrum. Methods Phys. Res. Sect. A 1990 A298(1-3) 156-60. 58 J. F. J. Todd Muss Spectrom. Rev. 1991 10(1) 3-52. 59 R. E. March Org.Mass Spectrom. 1991 26(7) 627-32. 6o J. D. Williams C. A. Cox R. G. Cooks and R. E. Kaiser Rapid Commun.Mass Spectrom. 1991 5(7) 327-9. 61 R. E. Kaiser Jr R. G. Cooks G. C. Stafford Jr J. E. P. Syka and P. H. Hemberger Int. J. Mass Spectrom. Ion Processes 1991 106 79-115. 62 F. Guidugli and P. Traldi Rapid Commun. Mass Spectrom. 1991 5(8) 343-8. 63 C. Evans S. Catinella P. Traldi U. Vettori and G. Allegri Rapid Commun.Muss Spectrom. 1990,4(9) 33 5-40. 64 B. D. Nourse J. S. Brodbelt and R. G. Cooks Org. Muss Spectrom. 1991 26(6) 575-82. 65 J. Einhorn H. I. Kenttamaa and R. G. Cooks J. Am. SOC. Mass Spectrom. 1991 2(4) 305-13. 66 T. E. Covey E. C. Huang and J. D. Henion Anal. Chem. 1991 63(13) 1193-200. 67 E. R. Verheij U. R. Tjaden W.M. A. Niessen and J. Van der Greef J. Chrornutogr. 1991 554(1-2) 339-49. J. R. Trainor and €? J. Derrick 3 Applications Ful1erenes.-The novel species C,' often referred to as 'Buckyballs' (collectively) and higher stable clusters of carbon have been subjected to intensive studies. Ajie et al.68 obtained an improved yield (14%) of carbon clusters from evaporation of graphite by resistive heating which they attributed to the purity of the precursor sample. They estimated the nascent proportion of C,o:C70 to be 85 15 * 3% and discussed formation of mono- and di-cations using electron impact (EI) FAB and LD techniques. A study of the direct laser vaporization of graphite revealed the production of negative-ion clusters comprising up to 13 carbon at0ms.6~ In the low-energy collision-induced dissociation (CID) and ion-molecule reactions of these clusters studied by FT-ICR it was concluded that linear C clusters are formed.Furthermore it was proposed that these species do not exhibit isomeric variation and that the structures are polyethyne for even n and linear cumulene for the odd n clusters. There have been other reports of success in forming and characterizing synthetic carbon clusters derived from coals7' and in plasma di~charge.~' The clusters CL0 and Cf (n = 60,70 and 84) seem to be highly resilient structures resisting dissociation at collision energies as high as 200 eV,72 despite the highly inelastic recoil observed from the target surface. At keV energies in a 4-sector tandem mass spectrometer multiply charged all-carbon clusters show evidence for C2n loss in collisions with helium targets.Accompanying peaks observed in the spectrum have been interpreted as evidence for the incorporation of a helium atom.73 Malhotra and Ross74investigated the natural formation of Buckyballs in various carbonaceous deposits. They reported a general absence of fullerenes (c60and C70) from the mass spectra except in a sample of soot from the low-pressure vaporization of graphite in helium. The results imply that the planar layers of sp2 carbon contained in graphite are important precursors to the formation of fullerenes. Clustered singly and doubly charged carbanions were prepared by laser desorption in an FT-ICR.75 Clusters (c60 C70 and C3') were produced in selected charge states.Ci; ions were distinguished from both CT0 and Cz0 (double harmonic) using isotopic abundance and selective ejection techniques. Organic and Biomolecu1es.-The use of mass spectrometry as a tool in pharmacology for metabolic profiling continues to expand.76 It has been suggested that CID in 68 H. Ajie M. M. Alvarez S. J. Anz R. D. Beck F. Diederich K. Fostiropoulos D. R. Huffman W. Kraetschmer Y. Rubin K. E. Schriver D. Sensharma and R. L. Whetten J. Phys. Chem. 1990,94(24) 8630-3. 69 S. W. McElvany Int. J. Mass Spectrom. Ion Processes 1990 102 81-98. 70 P. F. Greenwood I. G. Dance K. J. Fisher G. D. Willet and L. S. K. Pang Org. Mass Spectrom. 1991 26( lo) 920-2. 71 D. H. Parker P. Wurz K. Chatterjee K.R. Lykke J. E. Hunt M. J. Pellin J. C. Hemminger D. M. Gruen and L. M. Stock J. Am. Chem. SOC. 1991 113(20) 7499-503. 72 R. D. Beck P. St. John M. M. Alvarez F. Diedrich and R. L. Whetten J. Phys. Chem. 1991,95(21) 840-9. 73 T. Weiske D. K. Boehme and H. Schwarz J. Phys. Chem. 1991,95(22) 8451-2. 74 R. Malhotra and D. S. Ross J. Phys. Chem. 1991 95(12) 4599-601. 75 P. A. Limbach L. Schweikhard K. A. Cowen M. T. McDermott A. G. Marshall and J. V. Coe J. Am. Chem. SOC.,1991 113(18) 6795-8. 76 F. P. Abramson Methods Biochem. Anal. 1990 34 (Biomed. Appl. Mass Spectrom.) 289-347; L. M. Harrison and P. V. Fennessey J. Steroid Biochem. 1990 36(5) 407-14; A. Bateman S. Solomon and H. P. J. Bennett J. Biol. Chem. 1990 265(36) 22130-6; J. Claereboudt E.L. Esmans E. G. Van den Physical Methods and Techniques -Part (ii) Organic Mass Spectrometry 33 the high pressure region of ESI sources provides the basis for a low-cost biopolymer sequence in~trument.~~ ESI has changed rapidly into a tool for problem solving with a promising role in elucidating mechanistic steps in enzyme Rules for sequencing peptides from mass spectra are now quite well established. Van Setten et aLgOdiscussed the validity of these rules for a series of isomeric tripeptides. The intensity of immonium ions provided information concerning the positions of certain amino acid residues in the peptide skeleton. Sometimes the analysis of biomolecules can be improved significantly if the analyte is derivatized even trivially." For example the analysis of amino polyaro- matic hydrocarbon-deoxynucleoside adducts by FAB was enhanced by trimethylsila- tion of ether derivatives82 (ie.derivatizing a labile proton with a chemically equivalent massive (CH,),Si-group sometimes called a 'slow proton' and also reducing the ability to form hydrogen bonds). The derivatization was claimed to increase sensitivity and the ability to differentiate isomers of amino-phenan- threneguanine. Thermospray mass spectrometry following HPLC has been shown to be useful for the analysis and characterization of spin adducts complimenting nuclear mag- netic resonance and electron spin resonance techniq~es.'~ Radical additions of 'C(CH3)CN from the thermolysis of azobis(isobutyronitri1e)to molecules containing .~~ a nitrone functional group were detected.Zha et ~ 1 examined the FAB mass spectra of nitrogen heterocycles formed from a glycerol matrix in order to ascertain which molecule ions were formed. A predominance of quasimolecular ions (M-H)+ had been reported in earlier work. They found the protonated molecule (M + H)+ to be predominant however they noted the presence of significant (M + nH)+ (n = 2,3). Horeau and No~aille~~ have used mass spectrometry for the determination of optical activity in alcohols. Treatment of an alcohol with enantiomers with the R R enantiomer being isotopically modified produced labelled diastereomers with different nominal masses. The ratios of the resolved peaks provided the information from which the configuration of the optically active carbon in the alcohol could be deduced.Pruesse and SchwarzS6 reported that Fe+ complexes exhibit sequential metastable loss of olefin followed by molecular hydrogen. From a study of the R,N=CHR2 Eeckhout and M. Claeys Nucleosides Nucleotides 1990,9(3) 333-44; V. Katta and B. T. Chait J. Am. Chem. Soc. 1991,113(22) 8534-5; J. E. Alexander D. F. Hunt M. K. Lee J. Shabanowitz H. Michel S. C. Berlin T. L. MacDonald R. J. Sundberg L. I. Rebhun I. Lionel and A. Frankfurter Roc. Natl. Acud. Sci. U.S.A. 1991 88(11) 4685-9; A. J. Alexander P. Thibault R. K. Boyd J. M. Curtis and K. L. Rinehart Znt. J. Muss Spectrom. Ion Processes 1990,98(2) 107-34; A. M. Bridges P. F. Leadlay W. P. Revill and J. Staunton J. Chem. Soc. Chem. Commun. 1991 (ll) 776-9 (two papers).77 V. Katta S. K. Chowdhury and B. T. Chait Anal. Chem. 1991 63(2) 174-8. 78 B. Ganem Y. T. Li and J. D. Henion J. Am. Chem. Soc. 1991 113(20) 7818-19. 79 A. Shneier C. Kleanthous R. Deka J. R. Coggins and C. Abell J. Am. Chem. SOC. 1991 113(24). 8o D. Van Setten W. Kulik and W. Heerma Biomed. Enuiron. Mass Spectrom. 1990 19(8) 475-80. 81 M. A. Baldwin N. Stahl L. G. Reinders B. W. Gibson S. B. Prusiner and A. L. Burlingame Anal. Biochem. 1990 191( l) 174-82. 82 R. S. Annan R. W. Giese and P. Vouros Anal. Biochem. 1990 191(1) 86-95. 83 E. G. Janzen P. H. Krygsman D. A. Lindsay and D. L. Haire J. Am. Chem. SOC.,1990,112(23) 8279-84. 84 Q. Zha and M. J. Bertrand Org. Muss Spectrom. 1990 25(8) 435-7. 85 A. Horeau and A.Nouaille Tetrahedron Lett. 1990 31(19) 2207-10. 86 T. Pruesse and H. Schwarz Helu. Chim. Acta 1990 73(5) 1163-6. 34 J. R. Trainor and Z? J. Derrick complexes there was evidence for metallacyclic intermediates with some energetic preference for particular ring-size intermediates. Clusters.-The study of novel species exhibiting transitional behaviour between the bulk and molecular properties is part of the attraction of the chemistry of clusters. The study of cluster ions is a very active area of mass spectrometry. Kinetic and thermodynamic properties of metal dication clusters with water have been studied by ele~trospray.'~ Resonance-enhanced multiphoton ionization (REMPI) has been widely used in cluster studies. Gord et ~1.'~used one-colour two-photon REMPI to probe the bonding in heteromolecular clusters of benzene with small polar H-containing molecules capable of forming hydrogen bonds of varying strengths.They correlated this ability to form strong .rr-hydrogen bonds with the fragmentation efficiency in post-ionization of the clusters. Examination of benzene clusters formed in the seeded helium-expansion of C6H6 H20 and CH30H has shown that water has a higher binding energy than methanol. The binding of the CH30H is significantly enhanced when a water molecule has been adsorbed.89 Work on metal carbonyls in the gas phase sheds light on traditional areas of wet chemistry and catalysis. The (C0)5M~+ cation has been shown to bind CH4 with a bond energy of less than 30 kJ m~l-',~'the species formed being chemically distinct from (CO)5Mo+(CH3)H.The latter expelled CO following collisional activation whilst (CO)5Mo+/CH4 expelled CH4. In another study Cr(C0)6 changed its photo- physics when solvated with n methanol molecule^.^^ The photochemistry was highly sensitive to wavelength and sufficiently energetic ionization of the solvated molecule (CH,OH),Cr(CO), revealed a number of competitive channels for reaction dissoci- ation and energy transfer. Multiphoton dissociation of clustered alkyl acrylates led to ionization and dissoci- ation consistent with the charge being localized on a central molecular unit of the alkyl a~rylate.~~ The dissociation observed was loss of alkoxy radical which occurred from an uncharged clustered alkyl acrylate molecule.The number of alkoxy radicals expelled depended on the number of photons absorbed. 4 Fundamental Studies Charge-Remote Fragmentations (CRF).-Charge-remote fragmentations (CRF) refers to fragmentation of an ion particularly following collisional activation in which the charge appears to play no direct role. CRF may yield important structural information as was demonstrated by Crockett et aL93in determining the position of double bonds in a series of fatty acids containing 1,4-diene functional groups. A. T. Blades P. Jayaweera M. G. Ikonomou and P. Kebarle Znt. J. Mass Spectrom. Zon Processes 1990,102,251-67. J. R.Gord A. W. Garrett R. E. Bandy and T. S. Zwier Chem. Phys. Lett. 1990,171(5-6) 443-50. 89 K. 0. Boernsen H. L.Selzle and E. W. Schlag 2. Naturforsch. A 1990,45(9-lo) 1217-18. 90 C. E. C. A. Hop and T. B. McMahon J. Am. Chem. SOC.,1991 113(1) 355-7. 91 W. R. Peifer and J. F. Gamey J. Phys. Chem. 1991,95(3) 1177-83. 92 H. Morita J. E. Freitas and M. A. El-Sayad J. Phys. Chern 1991 %(4) 1664-7. J. S. Crockett M. L. Gross W. W. Christie and R. T. Holman J. Am. SOC. Mass Spectrom. 1990 1(2) 93 183-91. Physical Methods and Techniques -Part (ii) Organic Mass Spectrometry 35 .~~ Contado et ~ 1 discussed mechanisms of allylic cleavage reactions in the light of the CRF concept. Proton Transfer Reactions.-A study of the protonation of ben~onitrile'~ revealed the initial site of proton attachment to be at the nitrile functional group.Interchange of the adduct proton with ring-bound hydrogen was observed with the rates of transfer decreasing in the order ortho > meta > para. The proton affinities of DNA residues have been determined from the dissociations of proton-bound heterocomplexes with amines of known proton affinitie~.~~ Proton affinity scales have been examined in a study of the gas-phase thermochemistry of small organic molecules.97 There was agreement in the lower part of the proton affinity scale for hydrocarbons such as C3H6 and i-C& however in the upper end of the scale proton affinities found for alkyl amines differed significantly from their accepted values. Finding independent confirmation of their upper limit measure- ments it was suggested that the accepted values for proton affinities were in error.Distonic Ions and Ion-Molecule Complexes.-Distonic ions have been recognized in recent years as being important stable ion structures and important reaction inter- mediates. A distonic ion is distinctive as it represents the radical form of a zwitterion or put another way a radical ion with the centres of charge and spin separated. Loss of methyl radical in the low energy reactions of CH,CH=CHCH(CH,)OCH? and CH,=C(CH,)CH(CH,)OCH'; has been shown to involve rearrangement via distonic ion intermediate^^^ where the reaction was driven by the formation of a more stable oxonium ion. The involvement of 1,2-hydrogen shifts in the elimination reactions of distonic ions has been discussed.99 It is clear that more study of distonic ions is required if the products of their reactions are to be predicted confidently.The oxonium CH3CH20+=CHCH2CH3 has been shown by deuterium labelling to eliminate ethene specifically from the ethoxy group via a mechanism involving an ion-molecule complex.'" An investigation was made into the effect of the size of the ion and neutral partners in ion-molecule complex mediated alkane elimination reactions."' Larger ionic partners discriminated against alkane loss in comparison with alkyl loss. Whilst the larger ion decreases the attraction between neutral and the centre of charge a larger neutral increases the attraction lowering the threshold for alkane elimination. The interaction of remote functional groups has been observed in steroid molecules formed by electron impact ionization.'" It has been proposed that functional groups attached to opposite ends of the ion interact in an ion-molecule complex following the detachment of one of the functional groups.Proton transfer occurs within the ion-molecule complex followed by fragmentation. 94 M. J. Contado J. Adam N. J. Jensen and M. L. Gross J. Am. SOC.Mass Spectrom 1991,2(2) 180-3. 95 H. Wincel R. H. Fokkens and N. M. M. Nibbering J. Am. SOC.Mass Spectrom 1990 1(3) 225-32. 96 F. Greco A. Liguori G. Sindona and N. Uccella J. Am. Chem SOC.,1990 112(25) 9092-6. 97 M. Meot-Ner and L. W. Sieck J. Am.Chem. SOC.,1991 113(12) 4448-60. 98 R. D. Bowen and A. W. Wright J. Chem Soc. Chem. Commun.,1991 (15) 1055-7. 99 R. D. Bowen and A.W. Wright J. Chem SOC. Chem. Commun.,1992 (2) 96-8. 100 R. D. Bowen and P. J. Derrick J. Chem. SOC.,Chem Commun.,1990 (21) 1539-41. 101 D. J. McAdoo C. E. Hudson J. C. Traeger A. Grose and L. L. Griffin J. Am. SOC.Mass Spectrom. 1991 2(4) 261-9. 102 P. Longevialle G. Bouchoux and Y. Hoppilliard Org. Mass Spectrom. 1990 25(10) 527-36. 36 J. R. Trainor and P. J. Derrick Chemical Kinetics and Reaction Dynamics.-Brauman Zare and Levine suggested a test for molecular flu~ionality''~ in the reaction coordinate of molecular dissoci- ation. They described an algorithm for the calculation of the statistical distribution of isotopes amongst fragments formed from a precursor molecule prepared with non-statistical atomic labels. Collision-Induced Dissociation (CID).- The theory and use of collision-induced dissociation (CID) has been revie~ed,'~~*'~~ although curiously the pioneering detailed work of Futrell and co-workerslo6 was not covered in either review.A model based on impulsive energy transfer has reproduced the broad features of those collisions between macromolecular ions and inert gases,lo7 which lead to fragmentation and detection of fragment ions. This treatment predicts that the average energy transferred to an ion in a collision should be proportional to the average energy loss suffered by the ion. The implication that large energies are taken up by macromolecular ions in single collisions with inert gases has been supported by measurements of the dependence of energy losses upon pressure of inert gas.'" Charles and Marbury"' discussed optimizing conditions for the collisional dissoci- ation of halogenated dioxins for the purpose of MS-MS.It was claimed that the optimum collision energy is a function of pressure for argon nitrogen and xenon targets but no such dependence was detected when helium was the target gas. At similar pressures the optimum collision energy was higher for argon and nitrogen than for helium and xenon. An upper-limit for the satisfactory dissociation of singly-charged large molecules appears at about 2500 Da.lo7 Above this mass it has been difficult to deposit sufficient energy to dissociate ions and methods have been sought to extend the range. The use of coherent radiation and the formation of multiply charged ions to increase the internal energy deposited was briefly discussed earlier in this article.The use of a solid in replacement for gaseous targets has produced some success11o with spectra having similar salient features to 193 nm photodissociation spectra. Field dissociation is another technique which merits study for the controlled dissociation of large molecules. Charge Reversal.-The charge reversal phenomenon goes one step further than charge neutralization by producing ions of the opposite charge to the precursor on interaction with a buffer gas. Analysis of isomers has been an area of interest where perhaps the most challenging part of the work lies in obtaining consistent results. Charge reversal mass spectrometry"' was applied to the analysis of ten isomeric heptanones and heptanal with mixed results.Collisional activation of enolate ions formed by the OH- abstraction of the acidic P-proton in these substances produced 103 J. I. Brauman R. N. Zare and R. D. Levine Chem. Phys. Lett. 1990 172(3-4) 231-4. 104 J. Bordas-Nagy and K. R. Jennings Int. J. Muss Spectrom. Ion Processes 1990 100 105-31. 105 R. N. Hayes and M. L. Gross Methods Enzymol. 1990 193 237-63. 106 K. Qian A. K. Shukla and J. H. Futrell J. Am. Chem. SOC.1991 113(19) 7121-9. 107 E. Uggerud and P. J. Derrick J. Phys. Chem. 1991 95(3) 1430-6. 108 C. D. Bradley and P. J. Derrick Org. Mass Spectrom. 1991 26 395-401. 109 M. J. Charles and G. D. Marbury Anal. Chem. 1991 63(7) 713-21. 110 E.R. Williams K. D. Henry F. W. Mclafferty J. Shabanowitz and D. F. Hunt J. Am. SOC.Muss Spectrom. 1990 1(5) 413-16. Ill T. Suerig and H. F. Gruetzmacher Org. Muss Spectrom. 1990 25(9) 446-52. Physical Methods and Techniques -Part (ii) Organic Mass Spectrometry 37 characteristic mass spectra for the open-chain isomers. No success was achieved in differentiating between the isomeric cyclic and bicyclic heptanones. Neutralization/Reionization.-Neutralization/ reionization is an important technique which has been used to probe the qualitative structures of gaseous ions and transient neutrals such as hypervalent radicals.l12 Lorquet et al.li3 have suggested that for neutralization/reionization Franck-Condon factors determined for the separated 'free' species are not applicable in accurately modelling experimental observables.They argued that the potential surfaces where reaction occurs in the region of the vertical transition are distorted. They have treated the distortion as coupling of the separated species in terms of induced dipole interactions. Neutralization of the homologous n-alkane radical cations (ethane to hexane) with atomic alkaline earths was studied by Los et al.l14 Dissociations into radicals occurred favouring rupture of the central bond where possible. This behaviour was interpreted as mode selectivity in the cases of n-butane and n-pentane. It was inferred from the kinetic energy releases that the cleavage proceeds on a repulsive state. Interestingly bond selectivity was not observed in n-hexane where each C-C bond could be cleaved.Turecek et a1.1153116 have used neutralization/reionization to investigate the mechanisms of rearrangements in C,H,O+'. Cyclopropanone radical cation was found to be unstable as a transition state for the degenerate isomerization of 'CH2CH2CO+ whilst the CH2=CH:' --OC ion-molecule complex and the dis- tonic ion CH,=C+OCH; were observed to be stable ions. Quite different chemistry was induced by neutralizing 'CH2CH2CO+ the unstable biradical produced dissoci- ated violently leading to the conclusion that neutralization/reionization is much better able to differentiate the isomers of C3H40'' than CID. Ion-Molecule Reactions.-The chemistry of ions trapped for long periods of time (ams) can be significantly different to that of shorter time-scales (aps).Ions which have long lifetimes may dissipate internal energy supplied upon ionization through a variety of physical processes before reaction can occur.The existence of competitive mechanisms for energy dissipation is indicated where excess energy must be supplied to drive a reaction to completion. This may be manifest as collisional cooling where collisions occur or by other competitive relaxation phenomena which are probable on the same time-scale. The threshold for dissociation of C7H71+' to expel I' is some 0.2-0.3 eV in excess of the threshold calculated."' This excess energy was ascribed partly to a straightforward kinetic shift and partly to an intrinsic competitive shift arising from radiative relaxation.This reaction proceeds on the millisecond time- scale where radiative relaxation processes compete for the activation energy. Some of the energy acquired in activation is dissipated by emitting infra-red photons"' before dissociation becomes probable. Thus for an ion to dissociate under these 112 M. George and J. L. Holmes Org. Muss Spectrom. 1990 25(11) 605-8. 113 J. C. Lorquet B. Leyh-Nibant and F. W. McLafferty Inr. J. Mass Spectrom. Zon Processes,1990 100 465-75. 114 J. Los S. Kornig P. G. Kistemaker and J. H. M. Beigersbergen J. Phys. Chem. 1991 95(6) 2143-5. 115 F. Turecek D. E. Drinkwater and F. W. McLafferty J. Am. Chem. Soc. 1991 113(16) 5950-8. 116 F. Turecek D. E. Drinkwater and F. W. McLafferty J. Am. Chem. Soc.1991 113(16) 5958-64. 11' C. Lifshitz 1. Levin S. Kababia and R. C. Dunbar J. Phys. Chem. 1991 95(4) 1667-71. J. R. Trainor and P. J. Derrick competitive conditions it must acquire in activation a minimum excess of energy above that required to cause dissociation due to leakage. Deprotonation of alkyl vinyl ethers with NH has been shown to yield two product carbanions of the general forms CH,=C-OR (i) and -CH=CHOR (ii). Eichinger and Bowie118 used a trisilyl methylated precursor to prepare (i) exclusively. Under CID generic molecules of type (i) exhibited competitive reaction channels leading to elimination of an alkene or a Wittig rearrangement to yield CH2=CRO- from CH2=C-OR. The same group have reported on the reactions of ally1 vinyl ether in which the first step in a complex reaction series is a Claisen rearrangement.In contrast to the alkyl vinyl ethers the Wittig rearrangement is only a minor ~hanne1.l'~ 5 Conclusion Mass spectrometry overall has grown during the years 1990 and 1991 with excep- tional growth in certain areas. The tremendous growth of ESI has induced expansion in the use of single and triple quadrupoles. The development of MALDI is one step behind that of ESI perhaps only because the necessary TOF technology was not commercially available initially. Interest in TOF is now accelerating. Both ESI and MALDI are ionization techniques whose development is stimulated by interest in biomolecules. FT-ICR still holds much potential similarly IT appears to promise great things and has the virtues of cost and simplicity.Quietly tandem mass spectrometry has become an established technique as more attention has focused on ionization techniques. Improvements in MS-MS must be made particularly regarding increased sensitivity and less obviously interpretation of fragmentations. Lasers have steadily become more common place in mass spec- trometry laboratories although a convincing demonstration that laser-induced photodissociation holds the key to better and more controlled fragmentation of ions is still awaited. 118 P. C. H. Eichinger and J. H. Bowie J. Chern. Soc. Perkin Trans. 2,1990 (ll) 1763-8. 119 P.C.H.Eichinger and J. H. Bowie Aust. J. Chem. 1990,43(9) 1479-85.