Anal. Chem. 1984, 56,417 R-467 R
Mass Spectrometry A. L. Burlingame* and Joanne 0. Whitney Mass Spectrometry Facility, Department of Pharmaceutical Chemistry, and Liver Center, University of California, San Francisco, California 94143
David H. Russell Department of Chemistry, Texas A&M University, College Station, Texas 77843
OVERVIEW
have been quick to realize the potential of these techniques in solving those problems which have been intractable by classical approaches. The search is on now for methods to retain the production of abundant molecular ions, but to increase and control fragmentation in liquid ionization processes in order to produce fragment ions which can be used for pertinent and unambiguous structural determination. These issues may be resolved when a better understanding of the mechanisms of secondary ion production is achieved. The present period has been characterized by anecdotal, scenario construction, but serious exploration and a rational approach to the problem should lead to a comprehensive elucidation of both the solution and the gas phase chemistry involved. An additional concern on the part of many laboratories, particularly those involved in biomedical research, has been the feasibility of quantitation by liquid secondary ion mass spectrometry (LSIMS). Although several studies have shown that quantitation may be achieved using stable isotopes as internal standards, the dynamic range appears to be very limited-plagued by competitive and suppressive processes which limit or invalidate quantitation. In spite of the dire predictions which always occur when innovations appear on the scene, the older soft ionization techniques such as CI, FD, and 252Cf-plasmadesorption have not diminished in importance but have assumed a larger and more critical role in structural elucidation. Both positive and negative CI and, particularly, desorption CI are well suited for the analysis of polar compounds in the lower mass range which now can be adjusted up to (and often above) 1000 daltons. Spectra contain abundant ions related to molecular weight as well as fragment ions eminently suitable for structure determination. Background (viz., chemical noise) is generally lower than that produced by LSIMS or FAB ionization and the technique produces very sensitive quantitative analysis in the femtomole to picomole sample range. Field desorption must still be considered the most appropriate method for large, lipophilic polar molecules. Although much of the literature contains statements concerning the technical difficulties associated with FD, a laboratory with a modicum of talented personnel can smoothly produce useful information from a complex molecule in little more than an hour. The “unlimited” mass range of 252CfPDMS, coupled with time-of-flight instruments, continues to have unparalleled present advantages for high mass unknowns, although other high mass analyzers such as the Wien filter and the Fourier transform ion cyclotron resonance technique with superconductingsolenoids are being considered as practical, even superior, alternatives. Undoubtedly xenon neutral atom (FAB) and cesium ion bombardment using liquid matrices have made the greatest contribution to the analysis of polar, charged molecules at both low and high molecular mass measurement. There is almost no major class of compounds which has not been subjected to these techniques, and diverse means to maximize information have been employed including changes in target size, liquid matrix, bombarding atoms or ions, and derivatization to direct fragmentation. This review period has certainly entailed a learning situation for most laboratories. It is axiomatic that what has been learned is that sputtering methods are not a panacea for all
Mass spectrometryis a complex and encompassing scientific discipline which has become increasingly involved in the most diverse and demanding fields of study at the molecular level. Knowledge of the physical chemistry of reactions in the gas phase, of advanced instrument design and use, of computer-oriented processes, and of fragmentation mechanisms is critical but is no longer sufficient to solve the pressing and difficult problems a t the forefront of modern organic and biological chemistry. The mornings when a mass spectrometrist is handed a group of samples to be applied on a direct probe for low resolution E1 spectra are becoming few. Even the uninitiated are aware of high resolution mass spectrometry (HRMS) coupled with high resolution gas chromatography (HRGC)/ HRMS, chemical ionization (CI), field desorption (FD), fast atom bombardment (FAB),high field magnets, “hyphenated” techniques (liquid chromatography (LC)/MS, MS/MS, Fourier transform (FT) MS, etc.), and the permutations and combinations of the a vanced armamentarium available to determine mass and unravel molecular structure. The typical sample is likely to have been months in the purification, to be in low microgram quantity r t h e world‘s supply” (Al)], and to be not amenable to further purification or structural determination by other analytical techniques. It is likely that it is of relatively high mass (-1000-3000 daltons) and that complete structural information as well as assessment of purity is demanded. For successful practice of his art, the mass spectrometrist must know the history of the sample, its provisional composition and mass, ita solubility characteristics, and as much about its chemistry as is humanly possible. Since acquisition of this knowledge is difficult, if not impossible, after the fact, the mass spectrometrist has become a member of a team suggesting and many times dictating the orderly evaluation of experimental detail to ensure the eventual receipt of a sample which will “work”and which will have the greatest chance for yielding the most detailed and complete information desired. This situation has arisen naturally through the rather amazing dispersion and availability of innovative methodology and instrumentation and from an ever growing and evolving experience with a complex suite of molecules derived from biological, medical, geochemical, and environmental disciplines. Without doubt, the greatest direct impetus has been the development and dissemination of soft ionization techniques and high field magnets. Attempts to obtain molecular weight and structural information at high mass have been universally accepted as a most desirable goal; every laboratory with a high field magnet and the appropriate ion source can now obtain a spectrum of the molecular ion region of insulin around 6000 daltons or for that matter of proinsulin at around 9000 daltons. However, it is abundantly clear that a new generation of advanced ion optical instrumentation purposefully designed to optimize sensitivity throughout its extended or high mass range is required to match high mass separation (resolution) and ionization advances with detection capabilities. The capital costs will be large-probably in the range of $300000 to $900 000 per installation. Scientists working in other disciplines, particularly in protein and carbohydrate chemistry,
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the mass spectrometrist’s problems, but when used in a disciplined, systematic manner, taking cognizance of the accumulated knowledge of sample chemistry which has guaranteed success in the past, they are most useful and effective techniques. One aspect of FAB for structural determination of higher molecular weight compounds which has received increasing attention is the difficulty of determining exact elemental composition because of the complexity of stable natural abundance isotopic composition. Moreover, it has been repeatedly pointed out that the use of stable isotopes for confirmation of fragmentation mechanisms fails because of the difficulty of determining isotopic distribution at high mass. Originally, there was some disagreementabout the importance of high resolution at high mass since it was supposed that all that would be necessary would be the experimental observation of mass unresolved clusters around the predicted molecular weight of a substance, whose composition could then be intellectually derived from previous chemical analysis and known molecular weight increments. It has become increasingly evident that this view was short sighted and that both molecular and fragment ion composition will be necessary for absolute confirmation of both structure and fragmentation processes. The desirability of on-line LC MS to resolve complex mixtures is obvious and considerab e progress has been made, although the limiting factor in its application appears to be low and substance-specific sensitivity, particularly at high mass. There is, moreover, an element of uncertainty about the direction which LC/MS interfaces will take since no consensus has been reached concerning the relative long range merits of the moving belt, DCI, thermospray, or other variations. MS/MS remains a great hope for resolution of complex mixtures without chromatography, and its resolving power and ability to confirm structure have been demonstratedmainly with samples of known composition. Since spectacular innovations in analytical separatory systems seem unlikely in the near future (capillary columns for GC and microbore columns for LC have almost reached their theoretical limits of resolving power), MS/MS or some version of it has the greatest potential for the next great advances in mixture analysis. Its promise is even brighter with the advent of soft ionization techniques since its successful use does not depend upon the polarity or composition of the sample, because initial ionization can be produced by E1 or CI, FAB, LC/MS, or 252Cf PDMS. The major problem remaining before widespread and targeted use of MS MS in actual problem solving is achieved is its failure to pro uce structurally significant fragmentation at high mass. Parallels between solution chemistry and gas-phase chemistry continue to appear in the literature. Morton (A2) has written an extensive review of gas-phase ion-molecule reactions and, where possible, made comparisons with analogous solution-phase reactions. This review includes data from ion-molecule reactions (cations and anions), field ionization kinetics (FIK), metastable ion studies, and neutral products from reactions of gaseous ions. Specific examples of gas-phase solvolysis include phenyl ethers, “onium ions”, and leaving group effects. Finally, this current overview should be considered backto-back with the one from our previous biennial review (A3) since we believe the latter could have been reprinted here as a completely cogent contemporary assessment of mass spectrometry today.
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SCOPE The number of papers on the subject of mass spectrometry has increased again since our last review two years ago. This is due to the widespread application of innovative techniques as well as to the almost universal acceptance by the scientific community of mass spectrometry as the “sine qua nonn for structural identification and analysis. Although we limited coverage to organic (as opposed to organometallic and inorganic) substances,many papers still had to be eliminated-not because they were unsound or uninteresting, but because trends in the field could be better illustrated by other publications. In addition, there has been a sharp increase in reviews of both mass spectral techniques and applications, sometimes of work less than a year old. These contain numerous references and we feel that they can be consulted if 418R
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the reader desires a more exhaustive listing of references than is presented here. The burden of preparation of this review has been made less palatable by the increasing tendency of authors to prepare multiple publications of essentially the same data-first in proceedings of meetings, then in journals devoted to mass spectrometry, again in specialist journals, and finally in reviews. The sources consulted in preparing this review have included our active participation in the research fields themselves, a knowledge of the activities of the most productive laboratories, and a monthly search of the most respected journals, particularly those not solely dedicated to mass spectrometricresearch. The listings in Current Contents (B1) have been helpful. Reliance upon Chemical Abstracts Selects: Mass Spectrometry (B2)would have resulted in the exclusion of many of the most innovative and important papers. In addition to the more traditional sources, this year for the first time we solicited reprints of recent papers from well-known American and international laboratories and received an ovewhelmingly enthusiastic response. The information obtained in this manner aided in producing a more complete and definitive survey than would have been possible without the help of the many contributors. Sources of mass spectral data and applications involving the use of mass spectrometry in biochemical, medical, and bioorganic analyses are abundant. The next scheduled volume of the Specialist Periodical Reports in Mass Spectrometry [published by the Chemical Society (England) (R3)] is not available for consideration as yet, but previous issues contained excellent seminars and insightful opinions on fundamental mass spectrometric theory and applications. However, in common with the Mass Spectrometry Bulletin (England) (B4) and Gas Chromatography-Mass Spectrometry Abstracts (B5), coverage of the literature is slow in appearing. The journal Mass Spectrometry Reviews is now in its third year and, although its quality and scope have been variable, it promises to be one of the more valuable sources of prompt information on selected topics. The Ninth International Conference on Mass Spectrometry was held in Vienna in 1982 and the Tenth will take place in Swansea, Wales, in 1985. A Workshop on SIMS was held in Munster, Germany, in 1982, and the SIMS IV Conference took place in Osaka, Japan, in 1983. The 7th and 8th Annual Meetings of the Japanese Society for Medical Mass Spectrometry were held in Tokyo in 1982 and in Tottori in 1983, respectively. A course on Mass Spectrometry of Large Molecules was given by the Joint Research Centre of the EEC at Ispra, Italy, in September 1983. The 7th International Workshop on Glycoconjugates in Ronneby-Lund, Sweden, 1983, was distinguished by many contributions dealing with mass spectrometry. The 30th and 31st United States Annual Conferences on Mass Spectrometry and Allied Topics were held in Honolulu and Boston, respectively, and the 32nd will be held in San Antonio, TX, in May 1984. The first Texas A & M Symposium on Particle Induced Mass Spectrometry of Volatile Biomoleculeswas a success in 1983 and a second will occur in May 1984. The 3rd Asilomar (CA) Conference in September 1983 covered “Structure Identification of Gaseous Ions” and the 4th, to be held in September 1984, will be related to “Ionization from Surfaces”. An International Symposium on Mass Spectrometry in the Life and Health Sciences will be held in San Francisco in September 1984 (B6). An interesting phenomenon on the American mass spectrometry scene has been the proliferation and success of regional discussion groups. At least 15 groups, representing all geographical areas of the country, meet monthly to hear specialist lectures and tour facilities of major research laboratories. McLafferty’s “Interpretation of Mass Spectra” (3rd edition) (B7) and Howe, Williams and Bowen’s “Mass Spectrometry: Principles and Applications” (2nd edition) (B8)remain valid classroom texts; however, since they were published in 1980 and 1981, respectively, they do not have up-to-date information on the newer soft ionization techniques. Such information must be obtained from the current literature and reveals the obvious need for an up-to-date treatment of mass spectrometry suitable for instructional purposes. “Tandem Mass Spectrometry” (B9) edited by McLafferty summarizes instrumental developments in MS/MS as well as providing
MASS SPECTROMETRY
INNOVATIVE TECHNIQUES AND INSTRUMENTATION
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Chemistry and space sciences labaratmy of me University of California. BBrkeiey. and wa5 Assistant Professor 01 ChemlstrV Unlll 1968. He became ASSOClaIe Research Chemist in 1966 and Research Chemist in 1972. He assumed his current respensiblliiies in 1978. From 1984 to 1973. he was a member of several interdisciplinary scientific IeamS and WmmmeeS snhusted with lb planning and conduct of me lunar S C h U , program and me preliminary examination and distribution of lunar m m p k hem me U.S. Apclio and U.S.S.R. Luna sample return missions. h i m a mls time. as director 01 mass specnornay m n , he pb-red d e v * p m l of real-tlme. hi+sensWity. higkesolutkx' mass spectrometry. field bnbaHon kinetics. and deuterium difference ~pectroswpyin NMR. Dwha 1970-72 he was awarded a -"helm Memorial Felbwship vhich was Spent On biochemical-biomedical applicatbn~of mass ~pectromelrywMh J. S@vall at me KarOlhskka Inrtihlte. Stockholm. His research progam e n c m pess85 me MeS 01 diverse techniques of mass Spectrometry lo probe n w k u l a r nature of biohlcai function and dyslvnnion In the wntext of blwnsdicai. dlnlcai. and environmentsl research. His current interests focus on daveWment of techniqws for ~nuclurslstudies of polar. lablle bloOHgOmers Of intermediate mokdaar size ( ~ 1 0 0 0 0daii0"S) using field dew p t b n and energelk bn and atom spunering b n sowces. ~
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Joanne m y ls Adjunct AssocbIe Rolessa of Chemishy and Pharmaceutical c h e m w in me mpenmem 01 ~harmalkai chrrmstry.Universny 01 CBIHwnla, San Frsnclsw. She is also Deputy Director of the NIKSuppated Natbnal Bio-Drganlc. .y W i c a l Mars Spectrometry Facilny and .. is Director of the High Performance Wrmtography Facility of the Liver Center SI W S F . She recetved her B.S. from Malymamt. Manhattan COlbw and har ph. D. hom Duke Unkernsity In 1967. *e she was a James B. Duke Fellow in Zmloey. Her c ~ r e n tresearch inlerests include bile mlt metabolkm in perinatal deuebpment. L. I perllc"la* me use Of ma58 rpecbomeay in diagnosis 01 ne+ natal heptobiliary and gastrointestinal disw&rs and structural characterlrallm by mass spectromehy 01 antigenic determinants in membrane vesicles Iran hepatlcdetived t u r n .
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d Is an Apslstant Rofsssar 01 8 M Vnlverslty and mrdlnator of organic analysis for me TAMU Center for Trace Charannilallm. He ,-ked NS B.S. unkasity A r k a n ~ ~ s - L t tRock k in 1974 and his RID. b m me University 01 Nsbraska-Unwb h 1978. Hls pduate work. vnder me supervision of Rofessa M. L. & o s . invdved studias Of h - m o k u l a ,eactbms "Sing ion Cyclotron mass spectrometry and unlmdecular reacthms 01 garphase bns. He span1 2 years at Oak R&e Nalkml Laboratmy as a research sclentkt in me Analytical Chnmishy IXvkbn w a * k g on davelopmant of tandsm mass s p e c n m h y lor me plLllmrwuLcu d)ssoclaUm reacHons and g a m s physical organic chemlsay studbs. His p r m t msearch interests hclvJe use 01 lasers In mass specnomeay as both bnilal!a sources and a means to induce diSSDCiallDn 01 gas-phase kx'k systems. and lb uy1 of Fourier h a n s f m mars rpectmmetry (FTMS) lor invsst~lgatlons01 garphase bn-molec~lereactions and as an anabvsis mmod lor hioh molecular (>m/z 5000) mokuies. He is a member of the Amrkan Chemical Society and me Amerlcan Society fa Mass Specnomehy. UVMllgtry at Texas A
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representative selections of applications. There are many thoughtful and critical reviews of specific areas of mass spectrometry which have a peared in the past 2 years and a number of them are covererfin the appropriate sections of this review.
In virtually every area of ma88 spectrometry, the development of new instruments and instrumental methods continues to consume a large fraction of the research efforts. Over the past 2 years most of these efforts have been directed toward increasing the performance of existing instrument designs. This contrasts sharply with the period of the last review when a number of new instrument designs appeared in the literature, viz. instruments for tandem mass spectrometry and for high mass applications. However, this does not indicate that instrument development has become stagnant. New instruments Specifically for high masslhigh resolution are presently in the design stage by two commercial vendors. Although the two instrument designs differ (i.e., one vendor has incowrated inhomogeneous magnetic fields to increase the mass range while the other has chosen to utilize a larger radius magnet system), both instruments are designed to operate in the m a s range 7o(Ht15000. In addition, it is thought that both instruments should achieve high resolution at these extended mass limits. The previously developed major present alternative for high mass analysis is the time-of-flight (TOF) instrument. In terms of mass range and ion transmission properties, this instrument type has an inherent advantage over existing high field double focusing instruments. However, the severe limitation of mass resolution has disadvantages in determining fragmentation patterns for high molecular weight substances. The stragetic seriousness of the limited resolution must he questioned in light of the number of important problems which have been solved using this approach, and the fact that other ion optical systems with competitive m a s ranges are yet to become available in research laboratories. (This situation will change in the immediate future). Certainly, Macfarlane's research TOF methodology continues to challenge the entire field of high mass molecular weight determination, and this group is developing highly novel uses of computer technology for enhancing data acquisition and processing ( C I ) . This technology is being developed and utilized in other laboratories (e.& Fales' laboratory at NIH); Danigel has recently described the system in use at Marhurg, W. Germany (CZ), and Sundqvist and co-workers have developed a versatile system using fast &e., 90 MeV) heavy ions from the Uttsala tandem accelerator (C3). New instrument designs for high mass applications are being developed at Texas A & M University using Fourier transform mass spectrometry (FTMS). Following the pioneering work of Comisarow and Marshall in the field of FTMS ( C 4 ) , Russell has designed a heavy ion source for desorption ionization with mass analysis using FTMS (CS).The major advantage of this approach is the inherently high m a s resolution and extended mass range. In addition, the sensitivty of this method should be comparable to time-of-flight systems since the ion transmission characteristics are superior to present sector type instruments. T o date, ions of greater than rnlr 3000 are routinely detected and ions up to rn/z 5OOO should be feasible using existing instrumentation, while minor instrument modifications should extend this to 1OOOO. The addition of an 8-T magnet system in the fall of 1984 could extend the FTMS mass range to greater than rn z 15000. An alternate ionization method for high mass MS is the use of laser desorption ionization. Gross and co-workers have demonstrated this method and are continuing further development in this area (C6). Both the heavy ion and laser desorption ionization approach are well suited to FTMS as is the use of plasma desorption ionization ( C l ) . The investigations in this area hold considerable promise for major developments in the high resolution/high mass problem areas In the area of particle desorption ionization, Aberth et al. have described the use of keV Cs+ ions as an alternative to neutrals for ionization from liquid matrices (C7). This approach produces spectra of nonvolatile molecules comparable to fast-atom bombardment (FAB). A similar system has been described by McEwen (CS). Todd and co-workers have described a new ion source for thermal emission mass spectrometry (C9)and this ion source has been used for studying thermal ionivltion of organic salts (CIO).The source produces ample signal for studying the thermally desorbed ions by using tandem mass spectrometry methods. These studies should be highly informative with respect to the mechanistic aspects
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of laser desorption ionization (LDI). For example, Cotter has used time-resolved laser desorption ionization to probe the thermal contribution to LDI (C11). In addition, Kistemaker et al. have discussed the thermal aspeds of LDI (C12). Yergey and Cotter have examined the thermal desorption of organic salts (C13) and concluded that an important mechanism for ionization involves thermal processes. The thermal aspects of ionization of large molecules play an important role but it is becoming more apparent that other effects are also very important. For example, Wood and Sun have studied the enhancement of field desorption ionization by adding polyhydroxyl materials (C14); the additives used are pentaerythritol and cis-inositol. It is reported that these additives result in both more stable and markedly enhanced ion yields. Cooks and co-workers have shown that the addition of ammonium chloride increases the absolute signal intensity for the intact cation by using secondary ion desorption ionization (C15). Absolute sensitivities of (0.5-1) x C/pg are reported. The use of diethanolamine for enhancing SIMS of oligosaccharides has been suggested; however, the spectra are complicated by the formation of adducts ions with the matrix materials (CIS). Busch et al. have described methods for sample preparation for SIMS and laser desorption ionization (C17). The methods described are designed to convert the sample to a precharged state prior to exposure to the ionizing radiation. Kloppel et al. have suggested that highly porous targets be employed for SIMS, and the authors claim that these targets avoid rapid depletion of sample by the incident radiation (C18). Martin et al. have critically examined operating parameters for optimizing FAB ionization (C19). These studies include the evaluation of the various sample support surfaces, solvents, cleaning of the support surface, etc. The sensitivity of FAB ionization to the examined parameters is significant and illustrates the complexity of the various matrix effects. Macfarlane and co-workers have recently reported on the use of modified surfaces, e.g., ion-containing polymers such as Nafion, to control the site of molecule absorption (C20). The utility of this method is demonstrated using [Bleomycin-B1' CuICl; the spectrum is similar to that obtained without Nafion. Developments and new applications of SIMS continue to appear in a variety of chemistry and physics journals. Some of the recent achievements have been reviewed (C21, (222). In this work data are reported for peptides and proteins in the mass range 1000-14000, and emphasis is placed on metastable decay processes, neutral components, multiply charged ions, and cluster formation. Campana and co-workers have discussed the experimental data for alkali iodide clusters in terms of the cluster structure (C23). Correlations between bulk crystal structure and structure of the gas phase ionic clusters are discussed. McLafferty et al. have reported on the metastable and collision-induceddissociation spectra of cesium iodide clusters (C24). The results are discussed in terms of relative stabilities of different clusters. Positive and negative ion SIMS spectra for neuropeptides leucine- and methionine-enkephalin were reported by Westmore et al. (CW). The spectra provide molecular weight information as well as structure and sequence information. SIMS has also been applied to studies of small peptides and the octapeptide angiotensin I1 (C26). Ens et al. have used SIMS to characterize protected diribonucleoside monophosphate in both the positive and negative ion mode (C27). Chait and Field have reported on the use of fission-fragment or plasma desorption ionization for the analysis of polypeptide antibiotics (CZ8). This same method was used to examine covalent adducts of DNA with polynuclear aromatics (C29). PDMS has also been used to study the composition of solid coal; although abundant ions are observed at m / z less than 900, virtually no ions were detected at higher m / z (C30). The mass spectrometry of chlorophyll a was investigated using PDMS ((231). The major emphasis of this work was the extent of slow decomposition reactions, which the authors suggest is a highly efficient means for depleting the molecular ion population. Kamensky et al. have compared molecule ion yields for low and high energy particle-induced desorption ionization (C32). The study suggests that as the molecular weight of the sample is increased, the yield for high energy particles relative to low energy particles increases. McNeal et al. have described the use of PDMS for sequencin fully protected oligonucleotides; results for both positive ($3) and negative (C34) ions are
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reported. The mass spectrum of bovine insulin was recorded using 90-MeV "'1, and both molecular ions and fragment ions were detected (C35). Chan and Cook have discussed the use of multiply charged ions for extending the mass range of sector instruments; using electrohydrodynamic ionization, charge states as high as f4 are detected (C36). Fast-atom bombardment ionization has been used to characterize a number of difficult samples and in particular a large number of biomolecules extremely difficult to analyze using other mass spectrometric methods. Grigsby and coworkers have used FAB to characterize the nitrogen containing compounds in fossil fuel extracts (C37). Nibbering and coworkers have studied the fragmentation of methionine and compared the results from FAB with the fragmentation observed using other ionization methods, viz. SIMS, LDI, and FDMS (C38). Fenwick et al. have used FAB to characterize a crude plant extract containing a complex mixture of glucosinoates (C39). Also, these authors discuss the FAB spectra of glucosinolates obtained in both the positive and negative ion mode. The FAB spectra of a series of penicillins have been studied using both the free acids and alkali metal salts (C40). In addition to abundant molecular ion yields, structurally informative fragment ions are observed. The fragmentation reactions of positive and negative ions from 6-0-methylglucose polysaccharide have been investigated using both glycerol and monothioglycerol matrices ((241). Smith and Caprioli have discussed the use of FAB for following enzyme catalysis in real time (C42). The reaction was monitored by observing the decrease in (M + H)+of the substrate and observing a corresponding increase in the product ion signals. Desiderio et al. have used FAB in conjunction with tandem mass spectrometry to measure leucine enkephalin in caudate nucleus tissue (C43). This procedure is applicable to low level monitoring (450 pmol/g tissue) and is highly specific. Kamerling and co-workers have studied a series of underivatized oligosaccharidesand glycopeptides derived from glycoproteins of the N-glycosidic type (C44). The positive and negative ion spectra contain molecular weight and sequence information. Oligopeptides have been sequenced using a combination of enzymatic hydrolysis and FAB; the FAB spectra were recorded by using 1-5 nmol samples (C45). The angiotensin peptides I and I1 were sequenced using FAB and tandem MS methods; both positive and negative ion spectra were utilized for the analysis (C46). FAB was used to study the large oligopeptides melittin, glucagon, and the a-chain of bovine insulin (C47). The intact bovine insulin molecular ion has been detected using FAB (C48),and more recently the FAB spectrum of proinsulin has been reported (C49). Multiscan averaging techniques have been employed to resolve the molecular ion stable isotope cluster for bovine insulin as well as methyl glucose polymer (C50). Wada and co-workers have used FAB to pinpoint single amino acid substitution which creates abnormality associated with human hernoglobulin variants (C51). The analysis requires microgram quantities of tryptic hydrolysates of purified hemoglobins. The mechanism(s) of ion formation from surfaces exposed to high energy excitation continues to perplex and challenge the physicist and chemist. A large volume of data now exists on the influence of various experimental parameters on the yields of secondary ions and on the nature of the sputtered ions. The current status of these problems is outlined and discussed in the recent volume edited by Benninghoven (C52). The contributors to this volume represent the leaders in the field of desorption ionization and the volume covers field desorption, plasma desorption, SIMS including FAB, and laser desorption. Garrison has performed calculations using classical dynamics to model energetic Ar particles bombarding an ordered benzene/nickel surface (C53). The calculations estimate the internal energy of ejected benzene molecules is such that little fragmentation of the ions should occur; this result is consistent with the available experimental data. The difficulty associated with applying theoretical models developed for clean, well-ordered surfaces to problems dealing with disordered systems, Le., an organic sample of questionable purity on a solid surface or liquid matrix, is not straightforward and one should not expect close correlations. Cook and Chan have discussed the deposition of internal energy associated with desorption ionization from liquid matrices, viz. FAB and electrohydrodynamic ionization (C54). The greater degree of the fragmentation associated with FAB is interpreted as this
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process’ increasing the internal energy of the sample ions. Laxhuber and co-workers have used a simple model to explain the sputtering phenomena (C55). Using this model the authors suggest that the sputtering yield is as high for organics as it is for inorganics; however, the damage cross section for organics is much higher. Similar effects may account for the differences in ion yields for low and high energy particle bombardment (C32). Tantsyrev has examined the ion yield from sputtered benzene deposited on a nickel surface and explained the results in terms of ion-molecule reactions occurring in a “hot spot” generated by the inelastic impact (C56). Magee has discussed the sputtered ion yield in terms of the momentum transfer process and the amount of radiation damage incurred at the sample surface (C57). Moon and Winograd have presented a simplified scheme to distinguish between surface and gas phase fragmentation of molecule ions in SIMS and FABMS (C58). The scheme is based on measuring the distribution angle of ejected parent and fragment ions; examples are presented for chlorobenzene, benzene, and pyridine supported on Ag. Kawano et al. have discussed the thermionic and thermophysical properties of various surfaces employed for negative ion surface ionization (C59). The major emphasis of the paper is the instrumentation and operating conditionsand a large volume of literature is utilized to bring the experimental results in perspective. The SIMS spectrum of glycerol has been studied and the suggestion made that formation of (M + H)+ occurs by a radical cation intermediate (C60). The role of the liquid matrix used in FAB has also been discussed in view of the slow rate of mixing found for deuterated glycerols (C61). Field has also discussed the SIMS spectrum of glycerol and the extent of radiation damage by the incident radiation (C62). It is proposed that some of product ion yield can be rationalized by a free radical mechanism. Secondary ion mass spectrometry of solids at cryogenic temperatures provides insight into the fundamental processes associated with sputtering at keV energies. Michl and coworkers have examined several systems as a function of the nature and energy of the primary ions. In the cases of solid COz, COS, and CS2 the SIMS spectra is dominated by ions at masses higher than the parent ion (033). The SIMS spectra of C02is characterized by well-defined cluster ions; whereas, the CS2 and COS are dominated by a complex pattern of positive and negative cluster ions. SIMS of the solid nitrogen oxides produces a complex spectrum of clusters which the authors interpret as evidence for ion-neutral and neutralneutral reactions occurring before the cluster ion reaches the mass analyzer ((264). The authors propose a qualitative model for this process. The same authors observe an intense cluster series for the oxygen system, [03n+2]+, n = 1, 2, etc. (C65). These results are discussed using the same model proposed earlier (C64). In a recent paper, Michl has discussed the available information on SIMS at cryogenic temperatures and given extensive rationalization of the most commonly used model for interpretation of the data (C66). The general mechanistic implications of this model for both keV and MeV particle-induced desorption ionization are discussed. Newer methods for performing surface ionization are being developed with the major emphasis being the development of large particles to enhance the momentum transfer. One involves the formation of metal ions using field emission (C67). Clearly this approach provides a new method for generating the primary bombarding ion, but the advantages gained are rather small. A similar ion source developed by Noda et al. appears to be more versatile and have better operating characteristics (C68, C69). Knabe and Krueger have designed a scheme for producing massive charged particles (109-1015 daltons) using dust particles (C70, C71). This approach is highly novel and may ultimately result in large gains in secondary ion yields (see ref 32). The amount of work reported using field desorption has diminished significantly since the introduction of FAB. This is related to the technical difficulties associated with FD in comparison to FAB, but it is clear that for certain classes of molecules the FD method is still the method of choice. McRae and Derrick have examined the degree of fragmentation induced during field desorption of polyethylene glycol and explained the results in terms of field induced dissociation (C72). That is, the large potential field influences the energy barriers to dissociation. Odom et al. have described a pulsed FD
spectrometer for studying the FD ion formation mechanism (C73). The mass resolution of the time-of-flight spectrometer is increased using a reflection lens. Fraley and co-workers have characterized the temperature variation across various FD emitters and the temperature dependence of the fragmentation process (C74). Desiderio and Yamada have described a method for measurement of picomole amounts of leucine enkephalin in canine spinal cord tissue using FDMS (C75). Ala-leucine enkephalin is employed as an internal standard. Lattimer and Schulten have reported on the use of FDMS for the characterization of hydrocarbon polymers (C76). The characterization of primary beams used for liquid and solid-state SIMS and the nature of the sputtered ions has been the subject of several reports. Bentz and Gale have discussed a simple method for obtaining information about the site and rate at which sputtering occurs (C77). This report is a first attempt at developing a method for specifying experimental conditions used for organic work. Dahling and co-workers have discussed the proton abstraction reaction observed in negative ion field desorption ionization (C78). The authors suggest that the ions are not “preformed” but are instead formed by ionmolecule reactions in the space charge region of the ion source. On the other hand, Caprioli suggests that the relationships between the pK of organic acids and the corresponding FAB spectra are representative of the ionic states of the solution prior to particle-induced ionization (C79). Stoll and Rollgen have suggested, based on laser desorption ionization measurements, that attachment of alkali metal cations occurs by a gas phase ion-molecule reaction process (C80). These processes have been discussed and compared for several ionization methods (C81). The occurrence of doubly charged ions in desorption ionization has been discussed by Heller et al. (C82). Benninghoven has discussed various mechanistic aspects of SIMS from the solid state and the application of the method to a large number of sample types (C83). The use of these methods for pico- and femtomole range samples is the subject of a very recent publication (C84). New methods for sample introduction, particularly for samples of low volatility, have been reported by several workers. Traldi has discussed the use of samples supported on gold wires and inserted directly into the electron impact ionization source (C85). Using this approach, the sample is admitted directly into the electron beam by electron excitation followed by EI. Also, the spectra are not characteristic of desorption ionization in that the molecule ion is an odd electron species. Feigl and co-workers have reported on the use of ultrashort electric pulses to produce ions from organic solids (C86). Using the apparatus described, the energy is delivered to the support surface on the nanosecond timescale, and the spectra are similar to those obtained using other desorption methods. Guiochon et al. have reported on the production of negative ions of nonvolatile materials by liquid solution chemical ionization MS (C87). The technique is demonstrated using samples of vitamin B12 and erythromycin A. Reinhold and Carr have discussed the use of direct CIMS using polyimide-coated wires (C88). This study illustrates the problems associated with sample interactions with the support material and sample volatilization associated with other probe introduction methods. Jungelas et al. have described the combination of liquid chromatography and time-of-flight mass spectrometry for characterization of low volatility samples; the technique utilizes plasma desorption ionization (C89). A versatile and practical method for performing LCMS appears to be the approach used by Vestal, viz. thermal-spray liquid droplet formation (C90). This approach appears to overcome many of the problems associated with other LCMS methods; however, to date this method has not been utilized for complex biomolecule mixtures. Having successfully ionized the sample, the mass spectroscopist is still faced with the problem of sample characterization. In this area there seem to be several approaches that may be taken. One method of course is to interpret the mass spectrum obtained by simply scanning the mass-tocharge ratio in the manner dictated by the instrument type. A highly versatile approach is the method that is now commonly referred to as tandem mass spectrometry. A variety of instrument types may be utilized for this approach to sample characterization, and a recent volume has appeared that describes both the applications of the technique and the various instruments commonly used (C91). For further disANALYTICAL CHEMISTRY, VOL. 56, NO. 5, APRIL 1984
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cussion of this mass spectrometric method, the reader should refer to the review entitled “Tandem Mass Spectrometry”. Variations of the general technique are presently being employed by a number of workers. These include the use of sample introduction using chromatographic techniques (C92) and the dynamics of the dissociation process such as the correlation of energy partitioning and ion structure (C93). Voyksner an co-workers have made comparisons of GC/ HRMS with tandem mass spectrometry for the characterization of polychlorinated biphenyls and tetrachlorodibenzofuran (C94). In particular, these authors address the various advantages and limitations associated with each experimental approach. A similar comparison has been made for direct liquid introduction LC tandem mass spectrometry (C95). Caldecourt et al. have described the use of atmospheric pressure ionization tandem mass spectrometry for characterization of an odor constituent in an organophosphorothioate sample (C96). Cooks and co-workers have used tandem mass spectrometry in conjunction with desorption ionization to characterize the desorbed ions (C97), and this same research group has discussed the use of tandem mass spectrometry for the characterization of diesel particulates (98). Fourier transform mass spectrometry (FTMS) is beginning to emerge as a viable analytical mass spectrometer. It has now been 10 years since Comisarow and Marshall ((24) introduced the concept of FTMS and numerous workers have contributed to its continued development. McIver’s research group has made major contributions to this development, especially in the area of understanding the ion trapping process. In a recent paper this group has discussed the mechanism of nonreactive ion loss, and a model consistent with the experimental observations has been developed ((299). In this same vein, McIver et al. have described an elongated trap cell for use in FTMS, and the authors demonstrate that this cell is less sensitive to trapping voltages than is the standard cubic cell (ClOO). The cell gives improved mass measurement accuracy and increased dynamic range. Marshall et al. have recently presented the theory for stochastic excitation for FTMS (C101). This method for ion excitation promises to give an increased resolution, especially for MS/MS type experiments. Giancaspro and Comisarow have performed a systematic study of the interpolation of Fourier transform spectra (C102). The applications of these results to FT spectroscopy in general and FTMS in particular are discussed. Wilkins et al. have discussed exact mass measurements using FTMS (C103) and have also discussed various analytical applications of FTMS (C104). White and Wilkins have reported on the use of capillary column GC/MS using FTMS methods (C105). Although this approach is not seriously limited by the basic vacuum requirements of FTMS, the recently introduced use of pulsed valves for GC/FTMS appears to be a viable means for improving the mass resolution and sensitivity (C106). Hsu et al. have used FTMS for the characterization of complex organometallic clusters which are difficult to analyze using other mass spectrometers due to sample lability (C107). In particular, the problem associated with ion source arcin so frequently encountered with high voltage instruments oes not affect FTMS. Similar results are reported for the analysis of boroxime-supported triosmiumoxymethylidyne clusters (C108). McIver et al. have discussed the use of FTMS for studies on multiphoton ionization; the two techniques are highly compatible since both techniques are pulsed methods (C109). Freiser and co-workers continue to utilize FTMS for studies on collision-induceddissociation (CID) (C110). This group has used CID-FTMS for the study of the consecutive dissociation process (C111), and the abilit to perform high resolution meawrements on the CID pro uct ions has been reported (C112). White and Wilkins have demonstrated that high resolution on CID product ions can be performed at sufficient resolution to do exact mass measurements (C113). Bricker and co-workershave demonstrated the feasibility of performing high energy (keV) CID-FTMS,and the observation of charge-stripping reactions confirms the occurrence of the high energy reactions (C114). Noest and Kort have discussed several aspects of FTMS computer software (CllS-Cl17). Wronka and Ridge have described a frequency-scanned ion cyclotron resonance system (C118). Although this system is not as elaborate as the standard FTMS system, the apparatus is well suited for ion molecule reaction studies, and the system is demonstrated using cluster ion formation in transition metal
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carbonyl systems. Kemper and Bowers have described an improved tandem ICR spectrometer and demonstrated the performance using previously studied ion molecule chemistry (C119). Examples are also presented for the use of this apparatus for studying the dependence of ion molecule reactions on the kinetic energy of the reactant ion. The flowing afterglow apparatus has proven to be a versatile method for studying ion molecule reactions. A number of systems have been studied using this experimental apparatus, and a recent study has utilized penning ionization which has some advantages over other ionization methods (C120). Other studies have included the chemistry of H2P- (C121), the chemistry of negative ions with alkyl nitrites (C122),synthesis and reactions of H2NS- (C123), and the reactions of trimethylchlorosilane with a variety of nucleophiles (C124). A timely overview of this technique and its applications to problems in gas phase ion chemistry has been prepared by DePuy and co-workers (C125). A number of additional reports on instrumentation have appeared. Ryba and Mains have described an einzel lens system for improving the resolution of ion kinetic energy spectra (C126), and van Koppen et al. have described an improved high pressure temperature variable ion source with coaxial electron beam ion exit slit (C127). Dawson and Fulford have discusse the effective containment of ions in triple quadrupoles used for CID (C128). Ion containment is examined under a variety of experimental conditions and the results aid in developing standard operating parameters for CID. Chen and Boerboom have described a new electostatic analyzer for wide beams and discussed the ion optics of this device (C129). Several publicationson the theory of ion optics have appeared (C130) and the third order terms associated with crossed toroidal electric and inhomogeneous magnetic fields have been presented (C131, C132).
d
TANDEM MASS SPECTROMETRY The field of tandem mass spectrometry or MS/MS continues to attract interest from a wide range of research areas. The MS/MS experiment is performed using a wide range of mass analyzers for the initial mass selection process and for mass analysis of the product ions generated by dissociation of the primary ions. The various instrument configurations presently in use include low mass resolution devices, e.g., quadrupoles and magnetic analyzers, and ultrahigh resolution, double focusing spectrometers. Each instrument configuration possesses certain characteristics which in a particular application may have specific advantages over another configuration. For example, the tandem quadrupole systems have similar characteristics to quadrupole GC/MS systems: rapid sample analysis, ease of computer interfacing/control, low maintenance cost, and relatively high ion transmission characteristics. In addition, nominal mass resolution is achieved for the MS/MS spectrum. The sector instruments on the other hand have specific capabilities not available on the quadrupole systems, viz. high mass resolution, collisioninduced dissociation (CID) at higher energy (keV energies), and larger mass range. McLafferty has edited a book in which a large number of MS/MS practioners have described the instrumentation and representative applications to a variety of analytical problems (El). A substantial fraction of this book is dedicated to instrument considerations and the specific performance capabilities of these various instruments. In addition, the use of tandem mass spectrometry with a wide range of ionization sources is discussed and representative examples presented. Previously in this review the question of the role of tandem mass spectrometryin the analytical laboratory has been raised. In particular, the advantages/disadvantages of the various instrument types were raised in regard to the role of high resolution mass selection followed by low resolution or high resolution product ion analysis. In the analysis of highly complex mixtures (e.g., fossil fuel extracts, plant materials, biological matrices) it is questionable whether low resolution mass separation of the primary ion beam is adequate for the characterization of the specific component. For example, in the characterization of a methylpyrene in the presence of a substituted benzofuran (cyclohexyl-2,3-dihydro-2-methylbenzofuran), both molecules have the same nominal mass (216) but the exact masses differ by 0.057 unit, requiring mass resolution of ca. 4000 for complete separation. Direct ioni-
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zation of this hypothetical mixture and MS/MS analysis would not be an acceptable characterization procedure; Le., the MS/MS spectrum would be a composite of both components. It may be argued that selective ionization can be employed to differentiate the two components, and several workers have used chemical ionization to enhance ionization selectivity. However, in the particular example given, electron impact ionization is advantageous because both molecules have large molecular ion yields and relatively simple mass spectra. In addition, one does not always have the flexibility of choosing a specific ionization method. For example, for polar molecules one may be limited to FAB ionization and, in addition to sample impurity, one is confronted with a method with high chemical noise, e.g., the background ion current from the glycerol oligomers or other sample impurities. This is a particular area where high resolution MS/MS may prove to be the method of choice. In a paper that will appear in print very soon Gross and co-workers describe such a problem (E2). In the FAB spectrum of 5’-(2-~henyletheneyl)tubercidin the molecular ion (M H) overlaps with the tetramer of glycerol (M4 H)+ (both nominal m / z 369) and in the negative ion spectra the (M - €4)- ions overlap at nominal mass 367. However, at 10000 resolving power the two ions are resolved and a “clean” MS/MS spectrum is obtained. Thus, this example illustrates the advantage of this capability; however, at this point there are relatively few examples which actually require this elaborate instrumental approach. Some general applications of high resolution MS/MS have been discussed in a recent review by Gross et al. (E3). Over this review period there have been numerous reports of MS/MS studies which illustrate the power of the method for sample Characterization; however, many of these reports deal with the characterization of small model systems and these studies are discussed in the section on Ion Structure; the reader is referred to this section for further discussion and examples. In the area of more analytical problems, Hunt and co-workers have used MS/MS for the characterization of organosulfur compounds (E4). The work utilizes constant neutral loss scans monitoring the loss of HS from aromatic compounds in the presence of aliphatic and alkylated thiophenes. The analysis requires ca. 20 min/sample and relatively high specificity is obtained. Hunt has also discussed the use of MS/MS for environmental studies (E5).For a variety of specific analyses the time required is 25-30 min. The determination of drug metabolites of primidone, cinromide, and phenytoin in urine and plasma extracts was reported (E6). Although prior sample preparation was employed, the sample workup procedure was relatively simple and fast. Weber and co-workers have used MS/MS in conjunction with field desorption ionization for the characterization of surfactants (E7).This study deals with cationic, anionic, and neutral surfactants without prior sample separation, and the authors report specific determination of chain length and branching of the alkyl chains. It is of interest to note that interpretation of the data for neutral surfactants was more difficult. Tomer has described use of tandem MS for mixture analysis using fast-atom bombardment ionization (E@. The characterization of fentanyl derivatives (“China White”) has been reported and the MS/MS spectra compared with reference spectra; the bulk of the structurally important data was obtained from the even-electron, secondary fragment ions (E9). Hunt and co-workers have described the use of MS/MS for metabolic profiling of urinary carboxylic acids (E10). Steinauer and Schluneggar have compared the amino acid sequence ions for several tripeptides using CID spectra obtained in both the first and second field free regions of a reversed geometry spectrometer ( E l l ) . Other than for analysis of amino acid mixtures there seems to be little advantage over the normal E1 spectra. Field desorption in con’unction with MS/MS was used to characterize six protonated dinucleoside monophosphates (E12). Detailed structural information was reported and it was noted that isomeric sequences could be identified for both ribo- and deoxyribonucleotides. Barbdas et al. have reported on the characterization of penicillins and other p-lactams by using MS/MS (E13).The compounds are characterized using a prominent fragment ion common to the compound class, viz. C7HI2NO2S+; this ion shows characteristic fragmentation of the p-lactam ring and substituent type and position, but not the stereochemistry. Cheng et al. have discussed the results of MS/MS studies on steroids in terms
+
+
of structurelstereochemical information (7314). These workers suggest that absolute stereochemical assignments can be made for D-ring substituted 3-hydroxy steroids for both the hydroxyl group and the A/B ring junction; this is illustrated for 3’hydroxy-5’-pregnan-2O-one.Tomer et al. have reported on a method for locating double bonds in fatty acids by using negative ion MS/MS (E15). The sample was ionized by FAB from triethanolamine and, on collisional activation, specific fragmentation allylic to the double bond was observed. The analysis is complicated when the fatty acid has two sites of unsaturation; however, even in these cases a clear preference for allylic cleavage is observed. Voyksner has utilized MS/MS to study the cluster ions formed on ionization of two reverse phase solvents used for direct liquid introduction LC/MS (E16).The solvent effects on sample protonation and fragmentation are discussed. Liquid ion evaporation ionization has been performed on a triple quadrupole system and the resulting ions studied by using MS/MS (E17). MS/MS spectra for acids, salts, drugs, amino acids, peptides, nucleosides, and nucleotides are reported. The utility of this approach for molecular weight and structure information is discussed. The molecular ion of dimethylmorpholinophosphoramidate was used to evaluate the performance of triple quadrupole instruments for MS MS characterization of relatively large molecules (E18). he effect of the center-of-masscollision energy on the fragmentation patterns and sequential decomposition is also discussed. McLafferty has evaluated the performance of a triple sector magnetic system for the analysis of high molecular weight systems ranging from ca. m / z 1100 (lys-bradykinin) to m/z 2096 (15-met-gastrin); also included in this study are cluster ions of CsI (CS3&+; m / z 8966) (E19). Although the MS/MS sensitivity is considerably less than for small molecules, respectable signalto-noise levels can be achieved by usin signal averaging. Studies such as these are beginning to asdress a highly important aspect of MS/MS, i.e., the use of MS/MS with a system rich in chemical noise-FAB ionization. Also, the efficiency of the collisional activation process to produce structurally significant fragmentation can be questioned. It is clear that the full analytical utility of MS/MS, especially for large molecules, will not be realized until major breakthroughs are made which enhance the ion collection efficiency and mass resolution. The efficiency of translational-to-internal energy conversion via bimolecular collision processes is a significant limitation in large molecule studies. To date, there appear to be no examples where MS/MS structural characterization is as informative as the fragmentation reactions observed for ion source decomposition processes, and certainly the utility of low resolution MS/MS spectra (e.g., less than unit resolution) may be questioned. The use of B / E linked MS/MS scans on forward geometry high resolution instruments, viz. Kratos MS-50, provides increased resolution; however, the poorly defined collision region results in poor sensitivity (E20). For example, the ion collection efficiency for the (M + H)+ion of bradykinin is 100 times less for B/E scans than for MS/MS scans by using third field-free region CID. In the past the instruments used for studies of metastable and collision-induceddissociation processes have been complicated and required the presence of the true instrument specialist. In the last few years a number of commercial systems have appeared which are fully controlled by the instrument data system and require little attention from the instrument operator. In a recent paper, Jennings’ group has described a metastable mapping system using a commercial system operated via computer which requires very little operator interaction (E21). The reactions occur in the first field-free region of a forward geometry high resolution instrument and the reaction products are focused by using B/E scans. The capabilities of this approach are demonstrated using relatively complex chemical systems. Boyd and coworkers have described the methods for linked MS/MS scans to detect decompositionreactions in the first field-free region of a reverse geometry high resolution instrument (E22). These scan laws are applicable to single-step and consecutive dissociation reactions. The occurrence of artifact peaks and the resolving power are discussed. The possible applications of this approach to accurate mass measurement for product ions are also discussed. The use of metastable mapping for sequence information of small peptides has been discussed by
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Farncombe and co-workers (E23). The use of linked-scans for quantitative determinations was discussed by Walther et al. (E24); deuterium analogues are proposed as internal standards, and possible interferences from isotope peaks are discussed. The method is illustrated for the quantitation of caffeine in beverages. Katakuse and Desiderio have used linked-scan methods to investigate the positive and negative ions produced by FAB ionization of leucine enkephalin (E25). It is suggested that this method facilitates amino acid sequencing at the nanomole level and provides high specificity for the determination of endogenous peptides. Longstaff and Rose have discussed the use of linked-scans and GC MS for characterization of boronic acid esters (E26). There as been some discussion as to the proper method for calculation of isomeric composition of gaseous ions based on the CID spectra. Bowers (E27) has questioned the methods used by McLafferty (E%) to evaluate the proportion of benzyl and tropylium ions formed by direct ionization of several different precursors. The question centers upon the assumption of linear superposition of CID spectra. Although McLafferty (E28) finds no evidence that previous studies utilizin this assumption provided gross errors, the points raised by owers have solid foundations and more work should be evaluated and the linear superposition postulate tested further. The mechanistic and dynamic aspects of converting translational energy into internal energy of the primary ion via energetic bimolecular collisions, the most common method used for MS MS, continues to be an active area of investigation. SUC studies not only provide fundamental information on collision dynamics of polyatomics but also provide a basis for improved sensitivity and selectivity of the CID process. Douglas has studied the CID of halogen containing benzene as a function of target gas pressure and primary ion energy (E29). The collision energy dependence of the cross section and fragment energies are discussed in terms of the two-step model for CID; i.e., the excitation process and the fragmentation process are separable. He further suggests that a t the center-of-mass energies employed for these studies ((100 eV) the dominant excitation mode is translation-tovibrational. Kim has developed a probability model to account for the effects of sequential collision processes (E30, ,531). Based on this theory it is suggested that sequential collisions are important for collision processes performed at high energy (ca.8 kV). McLuckey and co-workers utilized energy-resolved tandem mass spectrometry and Fourier transform mass spectrometry to study the collision energy dependence of CID spectra (E32). The CID energy dependence over the energy range 1-50 eV is compared with PIPECO breakdown graphs and reasonably good agreement is obtained in all cases except the molecular ion of n-butylbenzene. Although the authors suggest this deviation arises from incomplete energy equilibrium, i.e., nonergodic behavior, this seems rather unlikely based on previous data on this system. A more probable explanation lies in the dynamics of collisional excitation/ deexcitation processes over this energy range. Yost has studied the energy dependence of CID and compared the results with QET breakdown data and angle-resolved CID (E33). The associative product ions are also studied as a function of collision energy and the use of energy-resolved studies for isomer distinction discussed. Bricker et al. have studied the FIMS-CID reactions of several systems at keV energies and discussed the results in terms of vibrational vs. electronic excitation (E34). In particular, the systems studied are not observed to dissociate when collisional activation is performed at low energy (
