Fast Atom Bombardment Ionization in Mass Spectrometry

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Fast Atom Bombardment Ionization in Mass Spectrometry Magda Claeys and Jan Claereboudt, University of Antwerp (UIA), Belgium Copyright © 1999 Academic Press

Introduction The mass spectrometry community uses 1981 as the starting point of fast atom bombardment (FAB) when Barber and co-workers published their first paper. The basis of FAB was laid down in the mid1970s through pioneering research on static secondary ion mass spectrometry (SIMS) by Benninghoven. At the time of its discovery FAB meant a significant breakthrough in the analysis of polar biomolecules, such as peptides. Until then other ionization techniques such as field desorption, SIMS using ion beams and 252Cf radiation, and laser desorption of ions from surfaces had been applied to the analysis of polar biomolecules with varying degrees of success and experimental difficulty. Since the development of FAB new powerful ionization techniques have emerged, namely matrix-assisted laser desorption ionization (MALDI) and electrospray ionization (ESI), which are routinely used nowadays and have greatly extended the applicability of mass spectrometry to polar biomolecules of high molecular mass (Mr > 200 000). Despite the enormous successes of these last two ionization techniques, it is fair to state that FAB still enjoys an important place in bioanalytical laboratories involved in the analysis of molecules of medium polarity, including, for example, most natural products. A survey of the literature in a representative journal on natural product research, The Journal of Natural Products, for the years 1996 and 1997 revealed that of the articles in which mass spectrometric analysis was reported, FAB was used in 44% of the studies. The gas-phase ionization methods, electron impact (EI) and chemical ionization (CI), are still employed in 65% and 15% of the studies respectively, while the use of ESI and MALDI was rather limited (i.e. 5% and 0.7% respectively). In FAB the sample ions are formed by bombardment of the sample in a liquid matrix with a high-energy beam of atoms (xenon or argon) or ions (caesium). The defining attribute of FAB is the use of a viscous liquid matrix to obtain long-lasting spectra and to ‘soften’ the sputtering process. The liquid matrix is able to provide continuous surface renewal

MASS SPECTROMETRY Methods & Instrumentation so that intense primary beams may be used. The overall result is that secondary ion beams with a useful intensity for scanning mass spectrometers may be prolonged to periods of 20 min or more. Interestingly, the importance of the liquid matrix in FAB was not realized by Barber and co-workers when they discovered the technique. In their first article emphasis was laid on the use of fast atoms (argon) instead of ions as energetic bombarding particles and the term ‘fast atom bombardment’ was introduced to describe the new technique. However, similar spectra to those obtained using a fast neutral atom beam were achieved with better sensitivity using caesium or mercury ions as bombarding particles. The latter technique is known as ‘liquid secondary-ion mass spectrometry’ (LSIMS), but very often and also in this review the acronym FAB is used to refer to LSIMS.

Instrumentation The experimental set-up for FAB MS analysis is basically very simple and is illustrated in Figure 1. A beam of fast atoms is produced in a saddle field discharge source, called a ‘FAB gun’, by first ionizing an inert gas (Xe or Ar) to generate ions (Xe+ or Ar+) and accelerating these ions into an appropriate medium where the fast ions can capture an electron, thereby being converted from fast ions (having a kinetic energy due to acceleration) to fast atoms with energies as high as 10 keV. The beam of bombarding particles contains neutral atoms but also ions in various charge states. As illustrated in Figure 1 the beam of bombarding particles is usually maintained at a large angle relative to the axis of the beam of secondary ions extracted by the ion optics. The intersection of the primary atom–ion beam and the secondary-ion beam is at the focal point of the ion optics. The sample film is mounted on a clean metal tip of the FAB probe which is introduced into the mass spectrometer through a vacuum-lock assembly so that the sample surface rests at the focal point of the instrument. The experimental set-up for LSIMS analysis is very similar; the only difference is the replacement

506 FAST ATOM BOMBARDMENT IONIZATION IN MASS SPECTROMETRY

the following sequence of events:

Figure 1

Experimental set-up for FAB.

of the fast atom gun by a caesium ion source which produces a focused beam of fast Cs+ ions (up to 35 keV). The caesium ions are thermionically emitted from an alkali aluminosilicate solid maintained at high temperature (approximately 1000°C). It is worth mentioning that caesium ion impact has largely replaced FAB in modern instruments. Advantages of the caesium ion source compared to a fast atom gun are that the energy distribution of the beam is narrow, beam focusing can be accomplished and the very low gas load introduced into the ion source.

Mechanism of ion formation It should be noted that whilst most of the particleinduced desorption techniques are simple to perform, they are only partially understood in theory, i.e. there exists no clear understanding of the mechanisms involved in ion formation from organic molecules in the condensed phase. It is, however, striking that different desorption ionization (DI) techniques [including FAB and LSIMS but also plasma desorption (PD) and laser desorption (LD)] produce reasonably similar mass spectra from nonvolatile organic molecules. Similarities in the spectra produced with incident beams of keV atoms or ions, or MeV particles and photons, can only be explained by similar ion formation and ion dissociation processes occurring after the initially very different physical excitation process. A general mechanism for the DI process can be presented, at least schematically, by

It is evident that the primary processes involved in the energy deposition step are dependent on the type of physical excitation used, and some of the properties of the intermediate phase must depend on the nature of these primary processes. However, the final step, leading to the production of molecular ions and fragment ions, appears to be independent of the primary processes occurring in the condensed phase. It is reasonable to propose that in the processes of ion formation and stabilization, chemical reactions strongly influence the end-products. These reactions are believed to take place in the vibrationally excited, disturbed surface layer, known as the ‘selvedge’ region. Several models describing the mechanism of FAB are available in the literature. It can be stated that there are almost as many models as there are theoreticians studying the problem. A unified model for desorption ionization

A comprehensive qualitative model for the DI process, as exemplified in FAB, LSIMS, PD and LD, has been proposed. The main features of this unified model are: (1) irrespective of its origin, the energy deposited at the sample surface is converted from its original form into vibrational energy; (2) desorption of intact molecules and preformed ions which can be described as a vibrational or thermal process (although no equilibrium is implied); (3) ion–molecule reactions (e.g. protonation, cationization and cluster ion formation) and EI occurring in the selvedge region; and (4) dissociation of energetic (metastable) ions well removed from the surface (i.e. in the vacuum region). A schematic illustration of this unified model is given in Figure 2. In the unified model the processes of desorption and ionization are considered separately. Another characteristic of the model is that desorption is followed by chemical reactions of two types occurring in two distinct regions. First, in the selvedge region, fast atom(ion)–molecule reactions and EI can take place. Secondly, in the free vacuum, unimolecular dissociations occur which are governed by the internal energy

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Figure 2 Unified model for desorption ionization. Reprinted with permission from Cooks RG and Busch KL (1983) Matrix effects, internal energies and MS/MS spectra of molecular ions sputtered from surfaces. International Journal of Mass Spectrometry and Ion Physics 53: 111–124. Copyright Elsevier Science.

of the parent ion, the characteristic timescale of the instrument and the structures of the gas phase ions. Ion formation mechanisms

The four major mechanisms for the formation of ions operating during FAB are: (1) direct desorption of preformed ions; (2) cationization (including protonation) and anionization in which a neutral analyte (M) is observed as adduct with a cation, i.e. [M+Cat]+ or with an anion, i.e. [M+An]−, (3) ion-beam induced processes leading to [M–H]+ and M+• ions; and (4) cluster ion formation. These four mechanisms cover most of the processes observed in FAB and largely describe the majority of ions detected in FAB spectra. Molecular-ion like species which are commonly encountered in FAB spectra are listed in Table 1. Possible mechanisms for the formation of positive ions are given in Table 2; analogous mechanisms can be formulated for negative ions. That ion formation is the difficult, energetically demanding step in FAB is indicated by the observation that production of ions in the gas phase is much easier from samples that exist as preformed ions in the condensed phase than it is for neutral molecules. The mechanism by which preformed ions are converted into gaseous ions is conceptually the most straightforward: the molecular agitation generated by the bombarding particle, a process called ‘sputtering’, releases preformed ions into the gas phase. The types of compound that comprise preformed ions are inorganic salts, organic salts, and strong acids and bases. An

artist’s conception of the violent activity at the surface of a matrix solution of the analyte is presented in Figure 3. The next type of ion for which the formation can be easily rationalized is the [M+Cat]+ ions; [M+Na]+ is used as a typical example. However, attachment of other metal ions, especially alkali ions, also readily occurs if these ions are present in the sample. Na+ may be present in the sample as a salt impurity or it may be added deliberately by the analyst; it is a preformed ion which can attach to a neutral molecule (M) either in the gas phase or in the condensed phase. The [M+H]+ ions can be generated by a mechanism analogous to that proposed for [M+Na]+; in this case the preformed ion is the proton. Free Table 1

Molecular-ion like species in FAB spectra

Positive ions

Negative ions

Cat+ from salt Cat+ An−

An− from salt Cat+ An−

[M + H]+

[M − H]–

[M + Cat]+, e.g. [M + Na]+, [M + K]+

[M + An]–, e.g. [M + Cl]–

[M + 2Na − H]+ and analogues

[M + Na − 2H]– and analogues

M+ •

M– • +

[M − H]

[M + H]–

Clusters:

Clusters:

[Cat n + 1 Ann ]+

[Catn Ann + 1]−

[M(M + H)] , e.g. sample– sample or sample–matrix clusters

[M(M − H)]−

+

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Table 2

Possible ion formation mechanisms in FAB

Ions formed

Mechanism

Preformed ions Sputtering e.g. Cat+ Cat+An–(cond.) → Cat+(gas) + An−(gas) [M + Na]+

Attachment of Na+ in the gas phase Na+ (cond.) → Na+(gas) M(cond.) → M(gas) M(gas) + Na+(gas) → [M + Na]+(gas) Attachment of Na+ in the condensed phase followed by sputtering M(cond.) + Na+ (cond.) → [M + Na]+(cond.) [M + Na]+ (cond.) → [M + Na]+(gas)

[M + H]+

Attachment of H+ in the gas phase analogous to Na+ attachment Attachment of H+ in the condensed phase followed by sputtering, analogous to Na+ attachment Disproportionation

M+•

Disproportionation 2M(cond.) → M+•(cond.) + M–•(cond.) M+•(cond.) → M+•(gas) Fast atom–ion beam-induced reactions M(cond.) – H• → [M–H]• (cond.) [M−H]•(cond.) + H+ (cond.) → M+• (cond.) M+•(cond.) → M+•(gas)

[M – H]+

Fast atom–ion beam-induced reactions M(cond.) – H2 → [M–H2] (cond.) [M–H2] (cond.) + H+ (cond.) → [M–H]+ (cond.) [M–H]+(cond.) → [M–H]+ (gas)

cond. = condensed phase.

protons are probably produced during particle bombardment at the impact site, or can be added deliberately in the sample in the form of a strong acid. Another mechanism for the formation of protonated molecules which has been considered is a dispropor-

tionation reaction, i.e. perturbation in a hydrogenbonded system caused by the bombarding particle can result in the sputtering of a [M+H]+ ion and its complementary [M–H]− anion. True odd-electron molecular ions (M+•) are also formed, especially from compounds with low ionization potentials. M+• ions are usually obtained for polyunsaturated molecules with conjugated systems, for example the fullerenes C60 and C70. The study of the mechanism of ion formation in FAB is still a research topic of interest. There is no firm evidence that M+• ions are produced by direct EI. Current mechanistic studies focus on molecular hydrogen and hydrogen radical loss from protonated M which yield [M–H]+ and M+• ions respectively. [M–H]+ and odd-electron M+• ion formation in FAB was formerly attributed to gasphase ionization-like processes but recent studies have demonstrated that fast atom–ion beam induced processes should be considered. The ion species of the types [M+H]+, [M−H]− and [M+Cat]+ are very useful for molecular mass determination, which is of key importance in the structure elucidation of natural products. A literature survey in The Journal of Natural Products for the years 1996 and 1997 revealed that in 52% of the studies in which FAB was used, additional accurate mass measurement at high resolution was performed on molecular ion species to obtain the precise molecular composition. Energy deposition

FAB is generally regarded as a ‘soft’ ionization technique but the question may be asked: what is meant by ‘soft’? Molecular-ion like species formed during FAB show little fragmentation, suggesting that they are formed with a low internal energy. Energy deposition during FAB has been addressed by several workers and it has been established that ions are formed with energies varying between 1 and 4 eV, revealing a maximum at 1 eV and a high-energy tail. The classification ‘soft’ should, therefore, be used with some caution.

Matrix selection and properties The defining attribute of FAB is the use of a viscous liquid matrix. Consequently, many studies have been devoted to selection of suitable matrices in FAB. General matrix requirements concerning the solvent properties of the matrix are summarized as follows:

Figure 3 Artist’s conception of the violent activity taking place at the surface of a matrix solution during FAB.

(1) the samples must be soluble in the matrix; (2) only low vapour pressure solvents can be considered in the vacuum of the mass spectrometer;

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(3) the matrix must be electrically conductive to avoid charging of the surface layer; (4) ions from the matrix itself must not interfere with analyte ions in the FAB mass spectrum; (5) the matrix must be chemically inert. Lists of useful matrices for FAB are available and their physical and chemical properties have been compiled. In the authors’ laboratory where FAB is still routinely applied to the analysis of plant secondary metabolites such as saponins, flavonoid glycosides, fatty acid derivatives and small synthetic peptides (Mr < 3000), two matrices are mainly used: glycerol, in the analysis of polar hydrophilic compounds and m-nitrobenzylalcohol (m-NBA) for lipophilic compounds. When glycerol is selected the sample is first dissolved in a cosolvent, methanol or a methanol–water mixture, while in the case of m-NBA dichloromethane is employed as cosolvent to facilitate addition of the sample to the matrix. It is a misconception that FAB can only be applied to the analysis of polar analytes; lipophilic compounds such as fatty acids and their derivatives are well amenable to FAB analysis if a lipophilic matrix is selected. Other matrices that have often been employed in peptide analysis include thioglycerol and a eutectic mixture of dithiothreitol and dithioerythritol (3:1, w/w), known as ‘magic bullet’. For negative ion FAB the basic matrices di- and triethanolamine have also been used. Evaporation of the liquid matrix in the vacuum of the mass spectrometer has to be considered because it can result in significant changes of the physical state of the sample solution. The effect of matrix evaporation on secondary ion formation has been investigated for samples dissolved in glycerol with and without a cosolvent. Depending on the residence time of the sample solution in the vacuum, the bulk and surface concentration of the analyte in the matrix can become supersaturated, analyte molecules can precipitate, and as such the conditions for secondary ion formation can be altered. Fortunately, the solid layers formed by precipitation of analyte molecules at the surface of sample solutions can be removed by sputtering, thereby making the underlying layer amenable to FAB analysis. In other models where evaporation of the liquid matrix was not taken into account, replenishment of the surface layer has been rationalized by diffusion of analyte molecules from the bulk to the surface layer. It is not likely that diffusion, which is not a rapid mechanism, is as important as previously thought to obtaining long-lasting analyte signals in FAB.

Sample preparation for FAB analysis Impure samples of biological origin cannot be directly submitted to FAB analysis. Polar hydrophilic samples are usually isolated from a biological matrix containing alkali salts by chromatographic procedures employing buffers. When buffers are required for the isolation, preference is given to systems composed of volatile salts, acids and bases. Alkali salts can be eliminated from samples by resorting to simple desalting procedures based on the use of reversed phase cartridges. In the presence of Na+, for example, acidic compounds containing carboxylic or phenolic groups give rise to multiple sodiated molecular species (i.e. [M + Na]+, [M – H + 2Na]+ and [M – 2H + 3Na]+) leading to decreased detection sensitivity. In order to improve the detection of peptides it is common practice to add a volatile strong acid such as trifluoroacetic acid to the analyte–matrix mixture whereby neutralization of the carboxylate part of the peptide zwitterionic structure results in an enhanced [M + H]+ ion formation. Derivatization strategies to increase the solubility of the analyte in the liquid matrix or to convert the analyte into a salt form with better desorption properties may also be considered. Molecules containing labile groups which are prone to hydrolysis in FAB can be stabilized by using a Li+-containing matrix.

Continuous-flow FAB FAB is the basis for an effective coupling technique for liquid chromatography (LC), namely, continuous-flow FAB. A set-up for continuous-flow FAB is given in Figure 4. In this technique, a liquid flow of typically 5–10 µL min–1 obtained by splitting the LC flow, is introduced into a heated FAB source via a narrow-bore fused silica capillary. The glycerol matrix is added to the LC effluent to a concentration of approximately 0.5% and subsequently the mixture is directed into the FAB source through a metallic frit. The liquid flow ensures that there is a continuous flow on the probe tip in which previously eluted sample is continuously removed from the area where sputtering occurs. In other designs there is no metal frit but then a cotton wick is used to disperse the solvent and to obtain stable operating conditions. In addition to coupling to LC, continuous-flow FAB has been shown to be a useful technique for the introduction of flow-injected samples and effluents of capillary electrophoresis and microdialysis.

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Figure 4

Experimental set-up for continuous-flow FAB.

Combination of FAB with collision-induced dissociation and tandem mass spectrometry In order to obtain structural information on biomolecules it is common practice to use FAB in combination with collision-induced dissociation (CID) and tandem mass spectrometry. As discussed above FAB is a soft ionization technique producing mainly molecular-ion like species with a low internal energy. These species can be fragmented by CID during which the internal energy of the ions is increased. CID can be performed at low and high collision energies generally leading to different and complementary structural information. The FAB spectrum of compounds of biological origin is usually quite complex for several reasons: peaks due to the FAB matrix are always present but, in addition, impurities and salt forms of the molecular ions are often encountered making the interpretation of a FAB mass spectrum particularly difficult. Using CID and tandem mass spectrometry it is possible to obtain a spectrum of one well-defined molecular ion species allowing the corresponding fragment ions to be determined unambiguously. Figure 5 illustrates first-order FAB spectra using glycerol as liquid matrix obtained for a flavonoid glycoside, kaempferol3-O-rutinoside, which was isolated from the leaves of an African medicinal plant Morinda morindoides, with and without subsequent desalting. Without desalting several molecular ion species of kaempferol-3-O-rutinoside are observed with low intensity, namely, [M + H]+, [M + Na]+, [M – H + 2Na]+ and [M – 2H + 3Na]+. The presence of Na+ in the sample is also evident from the matrix peaks including abundant sodiated species. Using desalting a sufficiently intense [M + H]+ signal could be obtained which was amenable to CID and tandem mass

spectrometry. This methodology showed that the ions at m/z 287, 449 and 461 are fragment ions of the protonated molecule.

Selected applications A number of reviews and overviews are available in the literature. For comprehensive surveys of the literature the biennial reviews of mass spectrometry in the journal Analytical Chemistry can be consulted. As it is impossible to review all applications of FAB only selected applications will be mentioned here. FAB has been particularly useful for the analysis of peptides and proteins, providing molecular mass information for large peptides with an upper mass limit of approximately 24 000 Da. It can be stated that in the field of peptides and proteins FAB has now largely been overtaken, but not entirely replaced by, MALDI and ESI. Other biomolecules that have been successfully analysed using FAB are glycoconjugates, including glycopeptides, nucleotides, terpenoid and flavonoid glycosides, and complex lipids such as glycosphingolipids, gangliosides, phospholipids and steroids. The relatively frequent use of FAB compared to other soft ionization techniques (i.e. CI, ESI and MALDI) in natural product research can be attributed to the reliable molecular mass and composition that can be obtained which is of key importance in the structure characterization of unknown compounds. FAB has also been applied in areas that are not of direct biochemical interest, such as the analysis of dyestuffs, organometallics, quaternary ammonium salts, triphenyl phosphonium salts, surfactants and chiral complexes. With regard to this last application FAB has been found to be more suitable than ESI for studying enantioselective intermolecular interactions.

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Figure 5 First-order FAB spectra obtained using glycerol (G) as the liquid matrix for kaempferol-3-O-rutinoside, isolated from a medicinal plant, with and without subsequent desalting. Reprinted with permission from Li QM, Dillen L and Claeys M (1992) Positive ion FAB analysis of flavonoid glycosides. Simple procedures for desalting and control of sodium salt contamination. Biological Mass Spectrometry 23: 408–410. Copyright John Wiley and Sons Ltd.

See also: Biochemical Applications of Mass Spectrometry; Fragmentation in Mass Spectrometry; Ion Energetics in Mass Spectrometry; Ionization Theory; IR Spectroscopy Sample Preparation Methods;

MS-MS and MSn; Organometallics Studied Using Mass Spectrometry; Peptides and Proteins Studied Using Mass Spectrometry; Plasma Desorption Ionization in Mass Spectrometry; Spectroscopy of Ions.

512 FIBRE OPTIC PROBES IN OPTICAL SPECTROSCOPY, CLINICAL APPLICATIONS

Further reading Barber M, Bordoli RS, Sedgwick RD and Tyler AN (1981) Fast atom bombardment of solids (F.A.B.): a new ion source for mass spectrometry. Journal of the Chemical Society, Chemical Communications 325–327. Barber M, Bordoli RS, Elliott GJ, Sedgwick RD and Tyler AN (1982) Fast atom bombardment mass spectrometry. Analytical Chemistry 54: 645A–657A. Caprioli RM and Suter MJF (1992) Continuous-flow fast atom bombardment: recent advances and applications. International Journal of Mass Spectrometry and Ion Processes 118/119: 449–476. Claeys M, Li QM, Van den Heuvel H and Dillen L (1996) Mass spectrometric studies on flavonoid glycosides. In: Newton RP and Walton TJ (eds) Applications of Modern Mass Spectrometry in Plant Science Research, pp 182–194. Oxford: Oxford Science Publications. Cook KD, Todd PJ and Friar DH (1989) Physical properties of matrices used for fast atom bombardment. Biomedical and Environmental Mass Spectrometry 18: 492–497. Cooks RG and Busch KL (1983) Matrix effects, internal energies and MS/MS spectra of molecular ions sputtered from surfaces. International Journal of Mass Spectrometry and Ion Physics 53: 111–124.

De Pauw E (1986) Liquid matrices for secondary ion mass spectrometry. Mass Spectrometry Reviews 5: 191–212. De Pauw E, Agnello A and Derwa F (1991) Liquid matrices for liquid secondary ion mass spectrometry-fast atom bombardment: an update. Mass Spectrometry Reviews 10: 283–301. Gower JL (1985) Matrix compounds for fast atom bombardment mass spectrometry. Biomedical Mass Spectrometry 12: 191–196. Hemling M (1987) Fast atom bombardment mass spectrometry and its application to the analysis of some peptides and proteins. Pharmaceutical Research 4: 5–5. Junker E, Wirth KP and Röllgen FW (1992) Effects of matrix evaporation during continuous sputtering in fast atom bombardment. International Journal of Mass Spectrometry and Ion Processes 122: 3–23. Li QM, Dillen L and Claeys M (1992) Positive ion FAB analysis of flavonoid glycosides. Simple procedures for desalting and control of sodium salt contamination. Biological Mass Spectrometry 23: 408–410. Sawada M (1997) Chiral recognition detected by fast atom bombardment mass spectrometry. Mass Spectrometry Reviews 16: 73–90.

Fibre Optic Probes in Optical Spectroscopy, Clinical Applications Urs Utzinger and Rebecca R Richards-Kortum, The University of Texas at Austin, TX, USA Copyright © 1999 Academic Press

Introduction In the clinical environment, optical techniques have been used for decades (microscope, colposcope, ophthalmoscope, endoscope, laparascope). Integration of spectroscopic devices into existing procedures is an obvious task. Fibre optic cables provide a flexible solution for adequate optical interfacing between the optical and spectroscopic device and the sample to be interrogated in situ. Fibre optic probes can be inserted into cavities and tubular structures, put in contact with epithelial surfaces and also inserted into structures that can be punctuated by rigid devices such as needles. Fibre optic devices for optical spectroscopy can be manufactured as flexible catheters with an outer diameter not exceeding 0.5 mm. We will present in this article fibre optic solutions for

ELECTRONIC SPECTROSCOPY Applications

fluorescence and both elastic and inelastic scattering detection. After describing the fibre optic interface, we will present probes for fluorescence spectroscopy followed by probes for reflectance measurements and side looking. Diffuser tips, refocusing and designs for Raman spectroscopy are discussed towards the end of the article.

The fibre optic interface A spectroscopic system incorporates a light source, an optical analyser with detector, and a light transport conduit which in many cases is made of fibre optic cables. A separate illumination and collection channel minimizes background signals produced in the illumination fibre (Figure 1A). The excitation or illumination light source is typically a laser or a filtered