Tandem mass spectrometry (MS/MS) instrumentation

Tandem mass spectrometry (MS/MS) instrumentation

Richard A . Yost and Dean D . Fetterolf Department of Chemistry. University of Florida. Gainesville. Florida 32611

I . Introduction ....................................................... 1 A . Evolution of MSMS instruments ................................. 2 B . Operationalmodes .............................................. 3 C . Applicability .................................................... 5

I1 . Instrumental components ........................................... 6 A . Sample introduction and ionization ............................... 6 B . Massanalysis ................................................... 9 C . Ionactivation ................................................... 11 D . Iondetection ................................................... 16 E . Instrument control and data handling ............................. 16

19 A . Tandem quadrupole instruments ................................. 20 B . Tandem sector instruments ...................................... 23 C . Other tandem instruments ....................................... 28

IV . Additional resolution elements ...................................... 32 32

B . ChromatographyMSMS and M S M S M S .......................... 33 V . Range of applications ............................................... 35

A . Mixtureanalysis ................................................ 35 B . Structure elucidation ............................................ 39 C . Fundamental studies ............................................ 39

VI . Future prospects ................................................... 40 VII . Nomenclature ..................................................... 41

JX . References ........................................................ 42

III . MSMS systems ....................................................

A . Energy resolution ...............................................

VIII . Acknowledgments ................................................. 41

I . INTRODUCTION

Tandem mass spectrometry is a rapidly developing field of mass spectrometry based upon a new generation of instruments with two (or more) mass analyzers employed in sequence for both separation and identification operations . Its rapid growth is due largely to the use of this added dimension of mass spectrometric information for the analysis of complex mixtures. wherein a sample is ionized and one component is separated by the first stage of mass analysis. fragmented by collision with a gas. and identified by the second stage of mass analysis . Mass spectrometry/mass spectrometry

Mass Spectrometry Reviews 1903. 2. 1 4 5 0 1983 John Wiley & Sons. Inc . CCC 0277-7037/83/010001-45$05.50

2 YOST AND FETTEROLF

(MUMS) is analogous to combined gas chromatographylmass spectrometry (GUMS) in which the separation is based on the chromatographic retention behavior rather than the molecular weight of each component. MS/MS is particularly useful for the rapid detection of specific components in a complex mixture with minimal sample preparation. In addition, MSMS can be used in conjunction with GC to provide higher selectivity than that possible with GUMS or MS/MS alone.

MSNS is the subject of a timely new book edited by McLafferty (1). Included are several chapters on MSNS instrumentation authored by the researchers who have been most involved in the instrumental developments. Tandem mass spectrometry has been reviewed for broad audiences recently in Science (2) and Chemical and Engineering N m s (3). Its development has also been critically assessed in the most recent biannual review of mass spectrometry in Analytical Chemistry (4).

A. Evolution of MS/MS instruments

Despite the fact that the early commercialization of mass spectrometry in the 1940s was spurred by interest in quantitative mixture analysis by the petroleum industry, the single mass spectrometer is not well suited to mixture analysis. The coupling of a gas chromatograph for separation with the mass spectrometer for identification in the late 1950s made possible the sensitive, selective analysis of mixtures.

Although MSNS is most commonly thought of today as a mixture analysis technique, it has its roots in the use of so-called “metastable” ions to aid in structural elucidation (5). Decoupling of the sectors in a normal-geometry (electric sector before magnetic sector) instrument made it possible to observe these metastable ions without interference from the normal “stable” ions (6). A more versatile instrument for studies of metastable ions was realized by reversing the double-focusing geometry (magnetic sector first), providing for ”Mass-Analyzed Ion Kinetic Energy Spectrometry” or MIKES (7). A scan of all metastable ions arising from a given parent ion (selected by the magnetic sector) was obtained simply by scanning the electric sector voltage. A significant increase in the variety and intensity of fragment ions was obtained by collisionally activated dissociation (CAD) (8). The extension of these techniques into mixture analysis opened the way for the widespread use of MS/ MS (9). During this same period a great variety of tandem mass spectrometers were developed for fundamental studies of iodmolecule reactions (10).

As the analytical applications of MSMS for mixture analysis have been demonstrated, new tandem instruments specifically designed for rapid, sensitive, and selective analysis have been developed. These have included multiple-quadrupole (11) and multiple-sector (12,13) MSNS instruments. These and other high-performance tandem mass spectrometers, most with sophisticated data systems, are now available from a number of sources (14-19).

MSMS INSTRUMENTATION 3

B. Operational modes

In contrast to the two-dimensional nature of conventional MS data (ion mass and abundance), the information derived from a MSMS experiment is three dimensional (parent ion mass, daughter ion mass, and abundance), as shown in Fig. 1 (20). The various operational modes in MSMS can be thought of as giving access to specific two-dimensional subsets of this three- dimensional data field. The simplest operational modes are the four single- scan modes: normal MS scan, daughter scan, parent scan, and neutral loss scan.

The information in a normal mass spectrum is the row of peaks along the front edge of the plot in Fig. 1. This scan can be implemented by either coupling the two mass analyzers together (parent mass = daughter mass), using an intermediate detector after the first mass analyzer, or (in the case of tandem quadrupoles) putting one of the quadrupoles in total-ion (RF- only) mode. Note that for a two-sector double-focusing instrument, this results in high resolution.

A daughter scan produces the spectrum of all the daughters of a selected parent ion. A daughter spectrum in the three-dimensional representation of Fig. 1 extends rearward (parallel to the right-hand edge) from any of the parent ions in the normal mass spectrum. This mode is easiest to implement if the first mass analyzer is a quadrupole, magnet, or double-focusing stage. In these cases only the second mass analyzer need be scanned to identify all the daughter ions formed from the selected parent ion. In other MSMS instruments, both analyzers must be scanned together (linked scans). A daughter spectrum is particularly useful in mixture analysis since it gives the mass spectrum of a selected component.

A parent scan yields the spectrum of all the parent ions which produce the selected daughter ion. In Fig. 1, these are lines of constant daughter mass, proceeding left to right from any of the daughter ions in the normal spectrum, parallel to the rear edge. Only if the second mass analyzer is a quadrupole can these scans be easily implemented without linked scanning of both analyzers. In mixture analysis, a parent spectrum shows all the components which fragment to give a characteristic fragment ion.

A neutral loss scan shows all the parent ions which lose a selected mass upon fragmentation. The neutral loss spectrum is shown as a line parallel to the front rank, but shifted by a mass equal to the neutral loss, in Fig. 1. The use of the term ”neutral loss scan” for this mode is rather unfortunate, since the neutral loss is constant rather than scanned. This mode is easiest to imelement on tandem quadrupoles due to the quadrupole’s linear scan function and its independence with regard to ion energy. On multiple-sector instruments it can involve quite complex linked scanning of the analyzers.

A fifth operational mode is selected reaction monitoring (SRM), in which the two mass analyzers are set to monitor specific parentldaughter pairs, analogous to selected ion monitoring in GCMS. This corresponds to acquiring the ion abundance at one (or several) points in the parent mass/

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daughter mass plane of Fig. 1. This is useful when screening a mixture for a set of targeted compounds at maximum sensitivity.

Whereas conventional mass spectrometry usually involves repeating the same scan over and over, in MSMS it is desirable to vary the sequence of scan modes. To obtain the three-dimensional data of Fig. 1, for instance, it is helpful to obtain a normal mass spectrum and then automatically obtain a daughter scan for each parent ion detected. When screening a mixture by SRM, it is valuable to be able to automatically obtain a complete daughter spectrum for confirmation of any ”positives” (selected reactions with signal intensity above a threshold). New analytical methods can be realized by computer control of this variety of operational modes.

C. Applicability

The tandem mass spectrometer has a wide range of applicability to analytical as well as fundamental studies. It has been pointed out that the modern MSMS instrument is a complete chemical laboratory with capabilities for synthesis, separation, reaction, and analysis all in sequence (21).

For mixture analysis, MSMS offers a wide range of applicability, comple- menting that of GCMS. For instance, thermally labile and low-volatility samples may be analyzed directly by MSMS. In general, GUMS is preferable to MS/MS when a large number of unknown components in a mixture are to be identified. Analysis for targeted compounds in a mixture, however, can be performed extremely rapidly by MUMS because of the essentially simultaneous access to mixture components. The enhanced selectivity often makes it possible to analyze for trace components with little or no sample preparation, and with improved limits of detection due to the reduction in ”chemical noise.” For example, chlorinated compounds in human serum and urine at the 100 parts per trillion level have recently been detected, with 100 samples analyzed per hour (22).

For the elucidation of the structures of gas-phase ions and ultimately entire compounds, MSMS offers an entirely new dimension of information. A major limitation of a conventional mass spectrum is that it does not indicate the genetic relationships among the ions in the spectrum. The consequences of this missing information are far-reaching. The interpretation of a mass spectrum is often a difficult and time-consuming process based on cataloged fragmentation reactions and intuition gained through experience. The only information on each piece of the puzzle (a fragment ion) is its mass and relative abundance. MSiMS provides a great deal more information about each piece and its neighbors: where it came from (its precursors) and where it goes (its fragments), as shown in Fig. 1. The potential of the MSMS technique for structural elucidation has been explored (20,23,24). Comput- erized interpretation of MS/MS data for the elucidation of structure shows promise (2,25).

Fundamental studies of ion/molecule reactions, the earliest application of tandem mass spectrometry, are increasingly important because of the wide-


spread analytical use of MStMS. They should not only increase our understanding of the processes involved, but also illuminate new approaches for analysis, such as the use of reactive collisions (26). These fundamental studies will benefit from the commercial availability of sophisticated new computer- controlled MSMS instruments.

11. INSTRUMENTAL COMPONENTS

Before considering complete MSMS systems, it is useful to review the individual components of such systems. The variety of techniques available for the production, activation, analysis, and detection of ions is reflected in a wide range of MS/MS systems. Of particular importance in MS/MS is the instrument control and data-handling system.

A. Sample introduction and ionization

In MSMS, as in conventional mass spectrometry, the type of sample and the information desired dictate the methods for sample introduction and ionization. For mixture analysis by MS/MS, for instance, the direct insertion probe is widely used because it permits the rapid introduction of samples with little or no sample preparation when chromatographic separation is not desired. Samples as complex as plant material (27), blood serum (22), and industrial sludge (28) have been introduced with the probe without sample cleanup. Rapid derivatization, on the probe, of polycyclic aromatic hydro- carbons (PAHs) to nitro-PAHs with N204, for example, has been performed prior to analyzing the sample (29). Limited separation by microdistillation off the probe is also possible as the probe is heated (ballistically or under computer control). A modified direct insertion probe is used for introduction of labile or involatile samples in combination with ionization techniques such as desorption chemical ionization, secondary ion mass spectrometry, and fast atom bombardment. Since these techniques often provide only molecular weight information, MS/MS offers the advantage of obtaining structural information by fragmentation of the molecular ion.

The use of chromatography for sample introduction, either gas chromatography (GC) or liquid chromatography (LC), provides an additional resolution element in MSMS. Chromatographic separation, for instance, can separate isomers, which cannot be separated in the first stage of mass analysis. For the MSMS analysis of a complex mixture for several components, a short (<20 in.) packed GC column directly coupled to the ion source provides an extremely rapid (up to 100 sampledh) method for introduction of volatile samples (22). The use of chromatography/MS/MS will be discussed in more detail in Section IV B.

The best method of ionization in MS/MS depends upon the application and type of sample. For elucidation of structure of pure compounds, ionization by electron impact is the method of choice. Each ion produced in the ion source can then be mass selected and fragmented by CAD. This generates a complete three-dimensional structural map (Fig. 1) or a fragmentation tree


6 5

51

39

30

27

Figure 2. Computer-generated fragmentation tree for nitrobenzene ( M W = 123) showing only the most intense fragmentation pathways. Plotted on the Finnigan MAT triple quadrupole MSMS instrument (15).

(Fig. 2) of the compound. For mixture analysis, however, it is desirable to form only one ion for each component, characteristic of its molecular weight. Chemical ionization (CI) is typically the method of choice. An added advantage of CI is the ability to enhance the ionization of the compounds of interest with respect to the sample matrix by selection of positive or negative ions and the choice of an appropriate reagent gas. For maximum sensitivity in mixture analysis, fragmentation of the molecular ion ideally should produce only one abundant, characteristic daughter ion. For qualitative analysis, as in structure elucidation, however, multiple fragment ions are desirable.

Because of its widespread use for MS/MS mixture analysis, CI merits further attention here. Since its introduction by Munson and Field in 1966 (30), a wide variety of CI methods have been developed. Most commonly these involve the use of gas-phase Bronsted acids such as (CH,)+, (C,H,)+, and (NH,)+ to convert the sample molecules to positively charged even- electron ions (M + H) + or (M - H) +. These reagent ions are produced by bombarding methane, isobutane, or ammonia, respectively, at pressures of approximately 1 torr with high-energy electrons (100-400 eV). If nonselective ionization is desired, (CH,)+ (with its low proton affinity) will produce pro- tonated molecular ions for most compounds. Ionization by (NH,)+ (with a higher proton affinity) is more selective. It will protonate nitrogen-containing compounds and form (M + NH,)+ adduct ions with oxygen-containing compounds (31). This added selectivity could be used in MSMS, for example, for the analysis of nitrogen compounds in coal liquids. The nucleophilic addition of NO+ to organosulfur compounds has been investigated for the


analysis of crude oils by triple quadrupole M S N S (32). The (M + NO)+ adduct ions formed can be detected by a neutral loss scan for NO (30 u).

Negative-ion chemical ionization (CI) is very selective and sensitive for compounds with high electron affinity. This added selectivity helps reduce background interference from the sample matrix, making possible the analysis for pollutants in untreated sample matrices at the trace level by M S N S (28). Although the ionization efficiency of negative-ion CI can be significantly greater than that of positive-ion CI, the greater stability of negative ions often results in a lower fragmentation efficiency and fewer daughter ions. The selection of the optimum ionization technique for trace analysis by M S N S must therefore include consideration of ionization and fragmentation efficiencies, as well as the level of background "chemical noise" from the sample matrix. The ability to alternate rapidly the acquisition of positive and negative ion data with quadrupole instruments is a particularly powerful tool for mixture analysis by MSNS.

A very important ionization technique for mixture analysis by M S N S is atmospheric pressure chemical ionization (33). It can provide extremely selective and sensitive analysis for targeted compounds in a variety of samples (34)-

The ionization techniques discussed above are generally applicable to volatile and thermally stable samples. The analysis of compounds with thermally labile, polar functional groups (OH, COOH, and NH,) or of high molecular weight (>600 u) requires the use of specialized ionization techniques. These ionization techniques often produce only a molecular or pseu- domolecular ion such as (M + H)+ or (M + Na)+. Combining these techniques with MSMS permits structural information to be obtained by fragmentation of these ions.

The simplest of these methods, desorption chemical ionization (DCI) (35), has not yet been reported for use with MSMS. Field desorption has been combined with MSMS for several biomedical applications, including peptide sequencing (36) and the study of the covalent binding of benzo[a]pyrene with DNA (37). The combination of secondary ion mass spectrometry (SIMS) with MSMS has been shown to be well suited for the analysis of complex solid samples (38). Fast atom bombardment (FAB) has emerged recently as an alternative method for the ionization of involatile samples, in which a beam of atoms replaces the ion beam in SIMS (39). The unique feature of FAB is the use of a viscous liquid matrix (usually glycerol) for the sample. The matrix produces an intense background of signals at almost every mass; the use of MS/MS can help eliminate this chemical noise. Recently, Hunt and co-workers have employed FAB with MSMS for the sequencing of underivatized peptides (40). Other sample ionization techniques which have been coupled with MSMS include pyrolysis (41) and laser desorption (42).

It should be noted that, with the exception of laser desorption, hardware to permit use of all the ionization techniques discussed above is commercially available as either standard or optional equipment on various M S N S instruments.


A final consideration in sample introduction and ionization for MSMS is ion source cleanliness. Contaminants that would normally be separated from the sample during extraction or by the chromatography step in GCMS are often introduced into the ion source on a direct insertion probe. Repeated introduction of "dirty" samples such as blood serum or oil directly into the ion source can rapidly lead to increased background and decreased sensitivity. A partial solution to this problem is provided by the new Finnigan MAT 4500 ion source, in which changing ion volumes is as rapid as changing samples on the probe (15).

B. Mass analysis

After sample introduction and ionization, the parent ions can be mass analyzed (directly or indirectly) with any one of a number of analyzers. After activation of the selected parent ion, the resulting daughter ions can be separated by a second stage of mass analysis.

In order to understand the advantages and limitations of MSMS instruments, it is important to consider the characteristics of each type of analyzer as it is used in a tandem mass spectrometer. Table I lists the various types of analyzers together with their upper mass limit and their effective mass resolution when used for either the first or second stage of mass analysis. The question of resolution for several of these analyzers in MSMS has been addressed (43). The most commonly used analyzers in MSMS are the quadrupole mass filter, the magnetic sector, and the electric sector. We have also considered the double-focusing analyzer (electric sector/magnetic sector or magnetic sector/electric sector), which may be used to provide a single stage of mass analysis in multiple-sector MSMS instruments. A single Fourier transform ion cyclotron resonance (FTICR) analyzer can provide two or more stages of sequential mass analysis for MSMS. The time-of-flight analyzer also has potential for use in tandem MS instrumentation. Each of these analyzers is considered in more detail below. In addition to mass resolution

Table I. Characteristics of mass analvzers for MS/MS.

Mass analyzer Quadrupole Magnetic sector Electric sector Two sectors

FTICR double-focusing

Maximum effective mass resolution As first analyzer

2 x unit (a mass)

As second analyzer

2 x unit (a mass) (in hundreds) (in hundreds)

30 2" 1 1"

200 < unitb

200 100'

Practical (and ideal) upper mass limit

(in thousands)

Time-of-flight 5d Id 5 i20i 'Can be improved with pre- or postacceleration. bEjection of all ions except selected parent generally < unit mass resolution. cAt rn = 100 (a Urn). dEstimates based on early experiments (115).


and range, other important characteristics, including ion energy effects, transmission, and scan speed, are discussed. The combination of two (or more) analyzers in tandem is covered in Section 111.

Not only the type of mass analyzers, but also their order, is critical in MS/ MS. When an ion dissociates, kinetic energy is released and the daughter ions acquire a range of velocities. If separation in the second analyzer is velocity dependent, this will lead to peak broadening and decreased resolution. In quadrupoles, ions are separated directly according to their mlz ratios, so ion velocity has little effect on the mass resolution. In magnetic sectors, however, analysis is a function of the ion momentum mvlz; in electric sectors, it is based on kinetic energy mu%, so even more broadening is observed, and unit mass resolution cannot be achieved. The time-of-flight analyzer should behave similarly.

The quadrupole mass filter separates low-energy ions ( 4 0 0 eV) according to their mlz ratios, The characteristics of the quadrupole mass filter have been described by Dawson (44). Several features of the quadrupole that have popularized it for GC/MS, such as unit mass resolution, rapid scanning (>lo3 d s ) , and ease of computer control, are equally important in MSMS. Quad- rupoles are usually tuned to provide unit mass resolution (proportional to mass) over the entire mass range. It is possible to vary the resolution elec- tronically, however, so that a given ion and its isotopically labeled analog may be passed simultaneously (45). If daughter ions are formed at relatively high (>lo0 eV) kinetic energies, resolution may be degraded in the quadrupole that follows. The ion activation process produces daughter ions of the same velocity (as the parent ion), in contrast to the ion source, which produces ions of the same energy. Hence higher-mass daughter ions will have higher kinetic energies than those of low mass, requiring that the ion energy be scanned together with the mass.

The upper mass limit for most quadrupoles is 103 u, although lower [Sciex (18), 5 x 102 u] and higher [Nermag (46), 1.5 x lo3 u, and Finnigan MAT 4600 (15), 1.8 X lo3 u] limits are also available. Although quadrupoles have been modified for use at masses up to 60 x lo3 u (47,48) unit mass resolution at such high masses is not currently possible.

Magnetic and electric sectors are the major alternative to quadrupoles for MSMS instruments. Ion energy in the sectors is typically three orders of magnitude greater (3-10 keV) than in quadrupoles. The magnetic sector can generally provide higher mass resolution (3 x lo3) than the quadrupole when used as the first analyzer. As the second analyzer, however, the resolution is much worse (200), owing to kinetic energy release. With the advent of high-field magnets, a practical upper mass limit of 3 x lo3 u can be obtained. Traditionally, the scan rates of magnetic sectors have been rather slow. Newer, laminated magnets can be scanned as rapidly as 0.5 s/decade.

The electric sector, because it is a kinetic energy analyzer, cannot be used to separate ions before fragmentation. Because of the kinetic energy release upon fragmentation, even an electric sector with high energy resolution (5 x lo3) can yield only poor mass resolution (100).


An electric sector and a magnetic sector in tandem can provide a single stage of high mass resolution (20 x lo3) for MSMS, with the other stage being a third sector, a quadrupole, or even two more sectors in tandem. The sectors in the double-focusing analyzer can be arranged in normal geometry (electric sector first) or reversed geometry (magnet first). The kinetic energy release should not affect significantly the resolution of a double-focusing second analyzer. Note that scanning the daughter ion mass on such an analyzer will be more complex than in a normal mass spectrometer, however, since the daughter ions will all have the same velocity, not the same energy.

Tandem mass spectrometry can also be implemented at low energies with an ion cyclotron resonance (ICR) mass spectrometer. Incorporation of Fourier transform techniques (49,50) offers several advantages over conventional ICR. These advantages include high mass range (5 x lo3) and resolution (10 x lo3 at mass 100) and rapid data acquisition. The ICR can be combined with other analyzers in tandem for MSMS studies. A more elegant approach, however, is to employ sequential (tandem in time) mass analysis. The parent ion is selected by ejecting all other ions from the cell. The bandwidth of the ejection pulse usually precludes unit mass resolution. The parent ion may then be accelerated (with better resolution) by irradiating it at its cyclotron frequency. Daughter ion mass resolution can be high if the cell pressure is kept low.

The time-of-flight mass analyzer is of particular interest in MSMS because of its potential to provide a complete mass spectrum lo4 times a second. However, no electronics system capable of collecting and processing the data at this rate has yet been produced. For parent ions, all of the same energy from the pulsed ionization source, the ions will be separated in time, with mlz proportional to t2. For inorganic ions, mass resolution of up to 2000 has been achieved. For organic ions, unimolecular decomposition along the flight tube, and the concomitant kinetic energy spread, limit the mass resolution to 500. Daughter ions, however, cannot be directly separated by time of flight, since they travel with the same velocity as their parent. Only by retarding or reaccelerating the daughter ions can they be separated by time of flight. Mass resolution would be limited in either case by kinetic energy release, A unique feature of the time-of-flight technique is that it can be combined with essentially any other analyzer in order to provide another stage of mass analysis. All that is required is a pulsed ion source and time- resolved ion detection (see Section I11 C).

C. Ion activation

Following ionization and mass selection of the parent ions by the first analyzer, daughter ions (of different mlz) can be obtained by a variety of ionactivation processes. Table I1 lists several ion-activation methods which produce positive ions. Collisionally activated dissociation (CAD) is by far the most commonly used in MSMS.

Metastable ions are ions which are stable enough to leave the ion source


Table 11. Ion activation processes in MS/MS. Unimolecular (metastable)

Collisionally activated dissociation (CAD)”

ml+ --j m2+ + m3

ml+ Z m2+ + m3

Photodissocia tion h v

ml+ --* mz+ f m3 0 -

Electron excitation ml+ -+ m2+ + ma

Charge stripping m,+ -+ m12+ + e

Charge inversion m,-+ m,+ + 2e

Charge exchange ml+ m, + N +

N

N

N Associative iodmolecule reactions m, + m l N +

’Often called collision-induced dissociation (CID).

but have adequate internal energy to decompose unimolecularly before reaching the detector. These transitions are observed in normal-geometry sector mass spectrometers as broad peaks at apparent mass m* = mYml. Pioneering studies of metastable ions were performed by ion kinetic energy spectroscopy. This technique has been discussed in detail by Cooks and CO- authors (5). IKES permits measurement of peak shapes for these transitions due to the kinetic energy release upon fragmentation. This information is valuable for elucidation of reaction pathways and for structural elucidation of isomers. The energetics and mechanisms of metastable-ion decomposition have also been discussed by Beynon and Gilbert (51). Unfortunately, these decompositions do not occur in appreciable abundance and are often associated with structural rearrangements (8). In order to increase the number of peaks and their magnitude, sufficient energy must be added to the parent ion to cause it to fragment. This can be carried out by any of the methods listed in Table 11.

In photodissociation the amount of energy added to the ion is selected by varying the excitation wavelength. This can provide more precise energy control than can varying ion energy in CAD. Griffith and co-workers (52) have compared the photodissociation spectra of n-butyl- and n-pentylben- zenes with the high-energy CAD spectra. The photodissociation of small ions has been studied with triple quadrupole instruments by Vestal and Futrell(53) and by McGilvery and Morrison (54).

Ions trapped in an ICR spectrometer can be excited by electrons. By varying the potential difference between the filament and the cell, the excitation energy can be controlled. Comparisons between electron and collisional excitation have been published (55).

The most common method of adding internal energy to an ion in MS/MS, however, is collisional excitation with a neutral gas N:

AB+ % (AB+)* + A+ + B

MS/MS INSTRUMENTATION 13

This collisional excitation is generally camed out in one of two different energy regimes because of the kinetic energy requirements of different mass analyzers. Collisions at high energy (>1 keV) in sector instruments involve a two-step process, excitation followed by unimolecular decay. These glanc- ing high-energy collisions lead to direct electronic excitation (56) and are characterized by minimal momentum transfer and small scattering angles. The scattering angle of a given product ion is a function of the energy transferred, and can be investigated by angle-resolved mass spectrometry (57,58). Several other processes are observed with high-energy collisions. Charge exchange and charge inversion are normally limited to sector instruments because of the high kinetic energy requirements of these reactions. Charge inversion has proven to be useful in the MSMS analysis of PAHs in solvent-refined coal (59).

In contrast to these high-energy collisions, collisional excitation in quadrupole and ICR instruments is generally performed at low collision energies (0-100 eV). These low-energy ”billiard ball” collisions involve vibrational excitation by momentum transfer (56,60) and are characterized by significant momentum transfer and large scattering angles. The function of the center quadrupole in triple-quadrupole MSMS instruments is to focus the daughter ions scattered through large angles. It has been proposed that the low-energy CAD mechanism involves a two-step process, vibrational excitation followed by unimolecular decay (61). However, at least in the case of associative ion/ molecule reactions at very low energies, a one-step mechanism involving the formation of a collision complex predominates (62).

Daughter ions arising from charge exchange and associative iodmolecule reactions can be observed only with a quadrupole or ICR as the second analyzer, because these ions are formed with low (and indeterminate) kinetic energy. They cannot conveniently, therefore, be mass analyzed with sector or time-of-flight analyzers. The associative iodmolecule reactions are particularly valuable because of their selectivity (see Section V B). Hybrid instruments which combine both high- and low-energy analyzers [such as a magnetic sector and a quadrupole (63)] permit ions with a wider range of kinetic energy to be studied.

In order to control the energy transferred in the collision process, two experimental variables can be adjusted. The effective ion kinetic energy (in center-of-mass coordinates) can be varied either by changing the collision cell potential relative to the source, or by changing the collision gas. This provides an added resolution element in MSMS as discussed in Section V A. The second method involves controlling the average number of collisions in the collision cell by varying the pressure (64).

A variety of collision cells are used for CAD in MSMS instruments, de- pending on the kinetic energy and the mass analyzers. The majority of sector instruments use a collision chamber 1 3 cm in length with entrance and exit slits for the ion beam, pressurized with helium. The main disadvantage of this type of cell is the effusion of target gas through the slits, resulting in a


significant number of collisions occurring outside the optimum focal region. This leads to an overall CAD efficiency of 4% (8,65,66).

McLafferty and co-workers (13) recently have described a nozzle/skimmer collision system located at the focal point between two double-focusing analyzers. With the skimmer at the entrance to a 7 x lo3-L/s diffusion pump, a CAD efficiency for methane of 11% at 25 keV was obtained. Recently, Glish and Todd (67) have designed a simpler ”molecular beam” collision region which uses a hypodermic needle as a means of introducing collision gas. These ”beam” designs have led to a tenfold improvement in the efficiency of the high-energy CAD process.

In the case of tandem quadrupole MSMS systems, three different approaches to cell design have been taken. The most common collision region consists of an RF-only quadrupole (60), enclosed within a gas-tight cylinder that can be pressurized with collision gas. An alternative design (68,69) uses a ”leaky dielectric” tube, extending into the two mass filters, as a collision cell which simulates an RF-only quadrupole. This cell can also be pressurized with a collision gas. A third design (70) consists of a ”90% gas-transparent” quadrupole with a collision gas jet. The area surrounding this collision region is pumped cryogenically. The overall CAD efficiency for quadrupole systems with an RF-only collision cell ranges from 10 to 65% (71). A fourth design, using a short field-free collision chamber (63,72), is less efficient for CAD.

An important concept in MSMS, the efficiency of the CAD process, de- serves further attention. Table I11 shows a series of equations developed by Yost and Enke (60,71) to describe the efficiency in terms of the fragment ion abundance (F) and the parent ion abundance at the entrance (Po) and the exit (P) of the collision cell. Figure 3 shows the typical relationship between the fragmentation, collection, and overall efficiencies as a function of collision gas pressure (11).

Because of the differences in collision cell design, location and type of pressure gauges, and calibration problems associated with changing collision gases, it has been suggested (13,67) that parent ion transmission (PIP,) or attenuation (1 - P/Po) be reported rather than pressure. In this way, efficiency curves plotted versus PIP, could be compared more easily for different instruments. Figure 4 shows the data from Fig. 3 replotted in this manner.

Table 111. Fragmentation efficiency: E, = CF/(BF + P )

Expressions for efficiency of CAD process.=

Collection efficiency: Ec = (BF + P)/Po = P/[Po(l - EF)] Overall efficiency: EO = E, x E , = 8F/Po = PEF/[Po(l - E F ) ]

Parent ion transmission: PIP, = Ec - E, = Ec(1 - E,) Parent ion attenuation: = 1 - Ec-1 - E,) 1 - (PIPo)

’BF = summed abundance of all fragment ions; Po = parent ion abundance at entrance of collision cell; P = parent ion abundance at exit of collision cell.


I O - ~ I O - ~ PRESSURE ( T O R R )

Figure 3. Typical CAD efficiency curves, plotted as a function of collision gas pressure: (a) collection efficiency; (b) fragmentation efficiency; (c) overall efficiency (11).

100 80 60 40 20 0 P I po (70)

Figure 4. Typical CAD efficiency curves, plotted as a function of parent ion transmission, P/Po: (a) collection efficiency; (b) fragmentation efficiency; (c) overall efficiency.


Unfortunately, this plot hides the true nature of the collection efficiency E, and therefore the overall efficiency Eo. From Table 111, we see that E, = (P/ PJ(1 - EF) and E , = E , x EF = (P/P,)EF/(l - EF); plotting either of these versus PIP, gives curves that are dependent only on the fragmentation efficiency E,. The presentation of the data versus pressure, as in Fig. 3, is preferable, even if the pressure axis needs scaling for comparison.

D. Ion detection

The ion detectors used in most tandem mass spectrometers are discrete dynode (Cu-Be) or continuous dynode channeltron multipliers. An intermediate detector is often used in a multiple-sector MSMS instrument to facilitate its use as a single-stage mass spectrometer. This is unnecessary in tandem quadrupole MSMS instruments, since either the first or second mass filter can be put into RF-only mode to transmit all ions.

These detectors can be used with a high-gain electrometer amplifier for current measurement, or with a pulse amplifier/discriminator for ion counting. Because ion abundances may vary by four or more orders of magnitude from parent ion to daughter ions (see Fig. l), a large linear dynamic range andor automated control of the detector sensitivity is desirable. For trace analysis applications, the reduction in chemical noise afforded by MSMS makes ion counting particularly useful.

For the simultaneous detection of positive and negative ions, the multiplier may be fitted with conversion dynodes operated at high, opposite polarities (73). When the dynode is struck by an ion, ions or electrons are ejected and detected by the multiplier. This eliminates the need for two multipliers, or the inconvenience of floating the multiplier for negative ion detection.

The use of a detection system which can record simultaneously an entire mass spectrum increases the scan speed or sensitivity of MSMS techniques. These advantages were recognized in early metastable-ion studies which used photoplate detection systems (74). More recently, Louter and co-workers (75) have employed a channeltron electron multiplier array for simultaneous detection in MSMS. The time array detector proposed by Stults and co-workers (76) provides the detection of a complete time-of-flight spectrum for MSMS.

Finally, note that the FTICR system does not have an ion detector per se. Rather, image currents arising from the motion of the ions in the cell are detected on the cell plates (50).

E. Instrument control and data handling

In order to realize the tremendous potential of MSMS, computerized control of the instrument and computerized handling of the data are necessary. The limitations of a manually controlled MSMS system were summarized by Hwang and Kiser (77):


The many weeks needed to locate and study systematically all the ob- servable metastable transitions in a mass spectrum is clearly a serious drawback to the ready use of this technique in defining the ionic fragmentation pathways of a molecule. [77]

With modern computerized MSMS instruments, this type of study can be completed in a few minutes instead of many weeks. The techniques for computerized control and data processing obviously deserve our attention.

The control of an MSMS instrument is significantly more complex than that of a single mass spectrometer. Several challenges are presented by this increased complexity. The first is the development of a simple and easy-to- use method of controlling the various operating modes. This is probably best accomplished through the use of microprocessor control and hardware scanning, since this frees the main processor to perform data acquisition and processing at a much higher rate. The second challenge comes about from the fact that each different operational mode produces a spectrum requiring a different interpretation. Furthermore, by rapidly switching between operating modes, the analyst can obtain an enormous wealth of information about the sample in a short period of time. The data system must be flexible enough to allow the automated control of any sequence of scans during data acquisition, and then be able to separate out the different spectra and present them to the operator. In addition to controlling the operating modes, the data system should be able to control accurately (or at least monitor) other experimental variables such as amplifier gain, ion energy in the collision region, collision gas pressure, and sample inlet temperature. If the potentials on the ion source and various lenses are controlled as well, computerized optimization of the entire system is possible.

Often, the operator will want to define a set of predetermined operating modes and instrument conditions to be cycled through during data acquisition. Table IV shows an example of such an experiment performed on a Finnigan MAT triple-quadrupole system. The sequence involves acquiring both positive- and negative-ion normal mass spectrum with quadrupole 1 with a preamplifier gain of lo6 V/A. Collision gas is then turned on, and a sequence of daughter, parent, and neutral loss scans is specified with various collision energies ( - 30 to + 30 V), mass ranges, ion polarities, and preamplifier gains.

For many applications, rather than use a predetermined sequence of scans, it would be preferable to use continuous feedback from the data to permit the data system to intelligently select the best instrument configuration for acquiring the next set of data. An example of this would involve the screening of complex mixtures for several targeted compounds using single-reaction monitoring (SRM). If one or more of the compounds is found in the SRh4 screening step, the data system could immediately acquire a full set of daughter spectra of the suspect parent ions. It could then compare these with a library to confirm the presence of these compounds. This capability is also available on the Finnigan MAT system.

Tab

le IV.

Exa

mpl

e of

an o

pera

tor-

defi

ned

MSM

S se

quen

ce.'

Cal

ibra

tion

tabl

es:

QU

AD

KA

LQ

I Q

UA

D 3

/CA

LQ3

Syst

em d

escr

ipto

rs:

Q1

43

En

try

Mas

s Ti

me

Mas

s In

stru

men

tb

No.

se

t Sc

an

(4

win

dow

s co

nfig

urat

ion

1 -

Q1

1.00

80

-700

1 +

MS: Q

1 M

ASS

SPE

C/ +

/GA

IN: 6

CG

IN: T

UR

N C

OLL

ISIO

N G

AS

ON

2

-

3 4 28

3 Q

3 0.

50

40-3

00

D28

3: D

AU

GH

TER

S/ - /G

AIN

: 7/C

E: 2

5.0

5 21

9 Q

3 0.

50

40-2

50

D12

9: D

AU

GH

TER

S/ + /

GA

IN: 7

/CE:

- 25

.0

6 28

3 Q

1 0.

50

100-

700

l'283

: PA

REN

TS/ -

/GA

IN:

7/C

E: 3

0.0

7 69

Q

1 0.

50

50-7

00

P 69

: PA

REN

TS/ +

/GA

IN: 7

/CE:

- 30

.0

8 69

Q

1 +

43

0.

50

5-65

0 N

69 : N

EUTR

AL

LOSS

/ - /G

AIN

: 7/C

E: 3

0.0

1.00

80

-700

1 -

MS:

Q1

MA

SS S

PEC

/-/G

AIN

: 6

-

Q1 -

2.00

-

0.50

54

50

N 5

0: N

EUTR

AL

LOSS

/ +/G

AIN

: 7/

CE:

- 30

.0

-

CG

OT:

TU

RN

CO

LLIS

ION

GA

S O

FF

9 50

Q

1 +

43

'Def

ined

on

the

Finn

igan

MA

T tr

iple

-qua

drup

ole

MS/

MS

inst

rum

ent.

-

2.00

10

-

bGA

IN: 6

= 1

0-6 A

N; C

E: 2

5.0-

V c

ollis

ion

ener

gy.

c(

4

Lb 9 3 2 ?li

E G 0


This level of computer control necessitates use of a system with fore- groundbackground capabilities, so that data acquisition can continue un- interrupted while real-time data processing and display take place. In addition to the automated control of the instrument, the system should allow manual control for optimization, tuning, and trouble-shooting.

A portion of the data handling for MS/MS data can be performed with familiar GUMS data-processing programs, including background subtrac- tion, quantitation, and plotting. For many such programs (library searching, for example) unit mass resolution of the MS/MS data is required. In addition to the standard data-handling programs, special programs are necessary to process the multidimensional data that are produced by MSMS instrumentation. These include graphic display of various two- and three-dimensional slices of data out of the multidimensional space. Particularly useful are programs which display the interconnections or “genetic relationships” between ions for elucidation of fragmentation pathways and molecular structure. An example from such a program is presented in Section V B.

The comparison and sharing of MSMS data among different laboratories is hampered not only by the variability arising from different instrument conditions, but also by variations in the format of stored data. A standard method for formatting computer files of MSMS data has been proposed (78). Widespread acceptance of such a standard would facilitate the sharing of data from different laboratones and instruments.

111. MS/MS SYSTEMS

There is a wide range of MSMS implementations, using all types of mass analyzers (see Section I1 B) in tandem. A limited number of systems are currently in widespread use, but many new ones are being investigated.

In comparing MSMS instruments, it is helpful to consider the performance characteristics required for various applications. The use of MSMS for the analysis of complex mixtures requires sensitivity, selectivity, and speed. Sensitivity requires high ion transmission and an efficient CAD process; selectivity requires good mass resolution in both stages of mass analysis; and speed requires rapid scanning as well as rapid switching of mass and operational modes for efficient monitoring of multiple reactions. For structure elucidation studies, good mass resolution and precise control of collision conditions are important. For nearly all applications, the availability of sophisticated computer control is critical to the success of MSMS. The type and order of mass analyzers are important for many reasons besides resolution, mass range, and scan speed. The ease of implementing the various operational modes is one such consideration.

If the two stages of mass analysis are independent (as in tandem quadrupole instruments), then parent and daughter scans require scanning only one analyzer; otherwise, the analyzers must both be scanned in concert, linked to obey a given scan function. Good mass resolution is often difficult to achieve in the second stage of mass analysis because of the kinetic energy


release in the ion activation process. While this is not a problem with mass analyzers which are essentially independent of ion energy (quadrupoles and double-focusing analyzers), it severely limits resolution in instruments using magnetic sectors (momentum analysis) and electric sectors (energy analysis), as shown in Table I. A further problem is the presence of "artifact peaks" due to ion dissociation in other areas of the instrument. These peaks are eliminated if a quadrupole or double-focusing analyzer is used for mass analysis.

In the sections which follow, we will concentrate on those implementations already put into practice. Other possible MSMS instruments, proposed or under investigation, will then be discussed briefly.

A. Tandem quadrupole instruments

The use of quadrupoles for both stages of mass analysis in MSMS leads to several important features. Mass analysis in quadrupoles requires that the ions have relatively low kinetic energies, in the range of 2-20 eV if good resolution is to be maintained. The CAD process is extremely efficient at these low energies, particularly if an RF-only quadrupole is employed as a collision chamber to focus the ions scattered by the collision process. Many of the features of quadrupoles which have led to their widespread use in GUMS are equally important in MSNS. These include small sue, simplicity and low cost, rapid scanning, ease of computer control, and tolerance of high pressures. These characteristics of quadrupoles are the keys to the advantages (and limitations) of quadrupole MSMS instruments.

The earliest tandem quadrupole instruments were designed for studies of iodmolecule reactions. A tandem quadrupole instrument was reported in 1972 by Lampe of Pennsylvania State University (79). It employed a field- free collision cell between two homemade quadrupole mass filters with unit mass resolution up to mass 200. The second quadrupole could also be mounted after the collision cell at right angles to detect ions scattered at 90". A similar system was reported by Iden, Lairdon, and Koski of Johns Hopkins Uni- versity (80) for investigation of the angular distribution of iodmolecule reaction products. The selected ion flow tube system described recently (81) employs tandem quadrupoles with a 1-m drift tube in between for the study of these reactions.

The concept of employing an RF-only quadrupole as a fragmentation chamber between two quadrupole mass filters was originated in the early 1970s by McGilvery and Morrison of La Trobe University (54) together with Vestal and Futrell(53) of the University of Utah. Both goups designed triple quadrupole instruments for studies of photodissociation of ions. Even at pressure down to 1W torr in the center quadrupole, ions from CAD greatly exceeded those from photodissociation. These early instruments proved to be extremely useful for studies of iodphoton interactions, but the CAD "interference" was not recognized as analytically useful.

The concept of a computer-controlled tandem quadrupole instrument us-


ing CAD for mixture analysis was developed in 1975 by Yost and Enke of Michigan State University. Their early studies were performed on the La Trobe instrument, modified for CAD (60,71). The use of the Michigan State triple-quadrupole instrument for mixture analysis and structure elucidation was then demonstrated (11,20). Shortly thereafter, Hunt, Shabanowitz, and Giordani at the University of Virginia (28) assembled a triple-quadrupole instrument and applied it to mixture analysis. Zakett, Cooks, and Fies of Purdue University (72) subsequently assembled a double-quadrupole instrument, with the assistance of Finnigan Corp.

In the past few years, three companies (Finnigan MAT, Sciex, and Ex- tranuclear Laboratories) have developed triple-quadrupole MS/MS systems. These systems range in price from $150,000 for a manual system to $350,000-$500,000 for a fully computer-controlled instrument. Over 40 of these systems have been sold.

The company which has sold the most triple-quadrupole instruments is Finnigan MAT (15). The triple-stage quadrupole is shown in Fig. 5 (82). It includes a dual CUE1 ion source and mass filters with mass range to lo3. One of the outstanding features of the Finnigan instrument is the sophisticated computer system for automated control and data handling. It includes interactive real-time control of all MSMS operating modes, and special programs to process and display MSMS data. Continuous feedback of the data permits automatic control of the scan sequence. The system includes a gas chromatograph, solids probe, and, as options, a desorption probe and FAB.

Sciex offers a triple-quadrupole system (TAGA 6000) with several unique features (18). It employs atmospheric pressure chemical ionization (APCI), which has many advantages for ambient air analysis. Various inlets are available, including solids probe and GC interface. The instrument uses close coupling of the three quadrupoles (without lenses) with a gas jet introducing

ELECTRON ION SOURCE OUADRUPOLE 1 OUADRUPOLE 2 OUADRUPOLE 1 MULTIPLIER

I I 2nd STAGE

MASS I I

1.1 STAGE MASS COLLISION,

I IONIZATION SEPARATION FOCUSSING SEPARATION DETECTION

L 50 cm I

Figure 5. quadrupole MSMS instrument (15).

Scale drawing (side view) of the Finnigan MAT TSQ triple-stage

22 YOST AND FE’ITEROLF

the collision gas through the gas-transparent rods of the center quadrupole (70). Detection is by ion counting. The computer system controls essentially all instrumental parameters, and provides real-time control of operating modes. MSNS spectra obtained on this instrument often show less than unit mass resolution, due perhaps to the large energy spread of the ions from the APCI source (70).

Two tandem quadrupole systems are available from Extranuclear Labo- ratories (14). One employs the common center quadrupole collision cell; the other uses two quadrupoles with a cylindrical tube of ”leaky dielectric” material extending from inside the first quadrupole to inside the second (69). This resistive material attenuates the dc fields of the quadrupoles much more than the RF fields, approximating the effects of an RF-only center quadrupole. These systems are available with mass ranges up to lo3, EI, dual CU EI, and APCI sources, solids probe, pyrolysis probe, and GC. Also available is a data system designed by Teknivent (83).

The major advantages of tandem quadrupole instruments include high CAD efficiency, unit mass resolution in both stages, high scan speed, efficient computer control, and potentially low cost. The high CAD efficiency (60), combined with high transmission, provides the sensitivity necessary for trace analysis, as demonstrated with the achievement of subpicogram detection limits (11,22,34). The capability for unit mass resolution in both mass analyzers not only provides enhanced selectivity for mixture analysis, but also permits conventional mass spectral processing approaches such as library searching to be employed. The high scan speeds make it practical to obtain limited MSMS data on each GC peak (see Section IV B); rapid switching between masses makes multiple-reaction monitoring of 50 or more targeted compounds possible for a sample rapidly desorbed from a probe (84). Tan- dem quadrupole instruments are particularly amenable to the high level of computer control required in many applications. Each quadrupole has a linear scan function and may be scanned independently of the others, making all the operating modes (including neutral loss scans) easy to implement. Even in fundamental studies, this high degree of computer control is ad- vantageous. The energy-resolved breakdown curve in Fig. 6, with data taken at 125 ion energies between 0 and 30 eV, took less than 2 min to acquire under computer control. Research is under way at Michigan State University (85) and Lawrence Livermore National Laboratory (86) to develop the next generation of advanced computer-controlled tandem quadrupole systems. Finally, tandem quadrupole systems have the potential of being mass produced at a cost as low, if not lower, than that of instruments for GUMS. This should open new areas for routine analysis of complex mixtures in process control and clinical and environmental applications. Tandem quadrupole MSMS is rapidly becoming an important complement to GUMS because of its great versatility, wide range of applicability, and its potential to analyze a thousand or more samples a day.

The most serious limitation of the tandem quadrupole implementation of MSMS is the limited mass range. At high masses (near 1000) the transmission of the quadrupole is also lower than that of a sector instrument at equivalent


I00

75

3? al c v 2 50 m 2 m 0 U -

2 5

5 10 15 20 25 30 ION ENERGY (ev )

Figure 6. Energy-resolved breakdown curve for the (C7H7)+ (mlz 91) ion from toluene showing the parent ion and its four major fragment ions produced by CAD (the mlz 63 ion data coincides with that for the mlz 51 ion).

resolution. Furthermore, isobaric ions from a mixture cannot be resolved by the first quadrupole. Finally, some extra dimensions of information available with high-energy CAD (charge inversion and stripping, and kinetic energy release) are generally unavailable on quadrupole instruments. In contrast, quadrupoles provide added dimensions of information in collision energy resolution, associative reactions, and rapid switching between positive and negative ions.

B. Tandem sector instruments

The earliest MS/MS studies were performed with tandem sector instruments because of the availability of two-sector high-resolution mass spectrometers. By "uncoupling" the two sectors, fragmentations occumng before or between the sectors could be observed, albeit with very limited mass resolution.

The initial uncoupling experiments were performed in 1964 by Barber and Elliott (6) on a normal-geometry (electric sector/magnetic sector) double- focusing instrument. This "accelerating voltage scan," as well as other scanning modes of two-sector MSMS instruments, is summarized in Table V. In this parent scan, the electric and magnetic sectors are set to pass a selected

Tab

le V.

two

sect

ors.

” Impl

emen

tati

on o

f op

erat

ing

mod

es o

n tw

o-se

ctor

MSI

MS

inst

rum

ents

wit

h ac

tivat

ion

befo

re b

oth

sect

ors

or b

etw

een

the

Inst

rum

ent

Dau

ehte

r sc

an

Pare

nt s

can

Neu

tral

loss

sca

n ~

~

Act

ivat

ion

befo

re s

ecto

rsb

(Acc

eler

atin

g vol

tage

sca

n)

(ele

ctric

lmag

netic

or

V c

onst

ant

E an

d B

cons

tant

V

con

stan

t m

agne

ticle

lect

ric)

B an

d E

scan

ned

at c

onst

ant

V s

cann

ed (

16)

B an

d E

scan

ned

at c

onst

ant

(90)

BI

E (8

7)

B(l

- E

)”’

E

or

or

B co

nsta

nt

V c

onst

ant

E an

d V

sca

nned

at c

onst

ant

B an

d E

scan

ned

at c

onst

ant

B21E

E’

IV

(91)

(9

2)

Act

ivat

ion

betw

een

sect

ors‘

(M

IKES

) V

con

stan

t V

con

stan

t (m

amet

idel

ectr

ic on

lv)

V a

nd B

con

stan

t B

and

E sc

anne

d at

con

stan

t BZE

B

and

E sc

anne

d at

con

stan

t v

,I

(95)

B2

(1 - g) (96)d

E

scan

ned

(94)

\ L

V/

.(

c3 0, 3 3

“E =

ele

ctric

sect

or v

olta

ge; B

= m

agne

tic s

ecto

r fie

ld s

tren

gth;

V =

acc

eler

atin

g vo

ltage

; rE

= e

lect

ric se

ctor

radi

us.

bGen

eral

ly po

or p

aren

t io

n m

ass

reso

lutio

n.

Gen

eral

ly p

oor

daug

hter

ion

mas

s re

solu

tion.

>

*B sc

anne

d an

d E

com

pute

r co

ntro

lled,

pre

sum

ably

to

obey

this

fun

ctio

n. S

ee a

lso

foot

note

on

p. 2

5.

crl 8 E;;

0


daughter ion m2 formed in the field-free region between the ion source and the sectors. The accelerating voltage V is than scanned upward from its normal value V, in order to detect all possible parent ions m,, where V = (ml/m2)Vfl. Although this mode is simple to implement, it has a few disad- vantages: Changing the accelerating voltage gives rise to source defocusing and varying sensitivity; it may also be scanned over only limited range (typically <4:1).

If the fragmentation process occurs in the field-free region before both sectors, it does not matter in what order the sectors are arranged. Both daughter and neutral loss scans are also possible in this case, although they require linked scanning of two parameters (the magnetic field strength B , the electric sector voltage E , or V). Scanning V will have the defocusing problem discussed above, and a limited range. Linked scanning of B, on the other hand, requires a thermostatted Hall probe for precise control of the magnetic field. The daughter scan is most commonly implemented by scanning B and E so that the ratio BIE is constant, with V fixed (87). Neutral loss spectra were first obtained by iterative manual adjustment of E as B was slowly scanned (88). The scan function for neutral loss spectra was not calculated until 1979 (89), however, and first implemented in 1980 (90). The accelerating voltage is held constant and the magnetic and electric sectors are scanned (under computer control) in such a way that ( B I E ) ( l - E)’” is constant. Other scan functions for various operating modes involving fragmentation before the two sectors have been reported, including daughter scans at constant E2/V (91) and parent scans at constant B2/E (92). A number of other potential scan functions, as yet not implemented, have been calculated (92). Generally, in all scans in which fragmentation occurs before the sectors, daughter ion resolution will be good, but parent ion resolution will be poor. This often leads to ambiguity in identifying the parent ion yielding a particular daughter ion.

It was recognized early in the evolution of MSNS that a reversed-geometry two-sector instrument (magnetic sector/electric sector) was preferable, since a daughter spectrum of fragments formed between the sectors could be obtained simply by scanning the electric sector field strength E such that E = (m2/m,)E, (93,94). This has been called MIKES (Mass-analyzed Ion Kinetic Energy Spectrometry) (94) by most and DAD1 (Direct Analysis of Daughter Ions) (93) by others. More recently, parent scans have been implemented by scanning at constant B2E using an analog circuit (95). Computer-controlled parent and neutral loss scans were also recently reported (96), in which the electric sector voltage was controlled as the magnetic field was scanned. Although not specified, the parent scan presumably obeyed the B2E function (95) and the neutral loss scan probably was at constant BZ(l - (rEE/2V)] with V constant, where rE is the radius of the electric sector.*

*For fragmentation between the sectors, B = (2Vml/er$)1’2 and E = (2m,/r,m,)V, with V constant. For a neutral loss scan, rn, - m, is constant. Solving for it, we get rn, - m2 = B2 [l - (rEE/2V)] = const, giving us the scan function for B and E. The other scan functions can be calculated in the same manner. (Y, and yB are the radii of the electric and magnetic sectors, respectively.)


Generally, in scans in which fragmentation occurs between the sectors, parent ion resolution is high, but daughter ion resolution is quite limited. This leads to the now familiar MIKES spectra which show broad daughter ion peaks with adjacent masses unresolved. These broad peaks do carry information on kinetic energy release, however, which can provide an added dimension of information. Normal-geometry double-focusing mass spectrometers, many with collision cells before the sectors, are available from VG, Kratos, and Finnigan MAT (15,16,19). Reversed-geometry instruments are available from Finnigan MAT (MAT 212 and 312) and from VG (ZAB- 2F), the latter designed specifically for MSMS work. These instruments are all available with various inlets, gas chromatographs, liquid chromatographs, and data systems. They range in price from $200,000 to $600,000.

The major advantages of two-sector MSMS instruments are their widespread availability and their ability, with the sectors normally coupled, to function also as high-resolution mass spectrometers. Note, however, that in MSMS, two complementary dimensions of information are provided about the samples; in high-resolution mass spectrometry, only a single dimension of information is available, albeit at higher resolution. The chief limitations of the two-sector instruments are their inability to provide unit mass resolution in both stages of mass analysis, the complexity of many of the linked scan functions, and the presence of artifact peaks in the spectra. These arise from fragmentations in the other field-free regions or in the electric sector. They have been treated fully by Lacey and Macdonald (97) using three- dimensional plots.

One two-sector instrument which reduces some of these limitations is the tandem magnetic sector instrument developed by Louter and co-workers (75) at the FOM Institute in Amsterdam. The instrument can simultaneously record all daughter ions over a 4:l mass range using a channeltron electron multiplier array after the second magnet. It also employs postacceleration of the ion beam (after fragmentation between the sectors) to 30 keV in order to decrease the relative kinetic release and improve the second-stage mass resolution. Indeed, mass resolution of up to 600 has been obtained in both stages. Because of the simultaneous detection system, the instrument is particularly well suited to studying short-lived phenomena such as pulsed laser desorption.

Many of the capabilities of two-sector instruments can be enhanced, and the limitations reduced, by adding one or more sectors. First we will consider three-sector instruments. The earliest example of such a study made use of the small electric sector empioyed on the Kratos MS-30 double-beam mass spectrometer to deflect one of the beams to its detector (98). Aside from these instruments, only a handful of three-sector instruments are currently in use. Maquestiau and co-workers (99) at the State University of Mons were the first to construct a triple-sector instrument (by adding an electric sector to the rear of a normal-geometry two-sector AEI MS902) for MSMS studies. An advantage of the three-sector assembly is the elimination of artifact peaks in most scans (100). They were soon followed by Russell, McBay, and Mueller


at Oak Ridge National Laboratory (101), who designed an electric/magnetid electric sector instrument to provide high resolution in the first stage of mass analysis. The first commercial triple-sector instrument, the Kratos MS-50TA, has been installed at the NSF Midwest Center for Mass Spectrometry at the University of Nebraska. This instrument demonstrates that not all two-sector instruments are easy to modify; in order to add a second electric sector after the magnet, the instrument is supported several feet above the floor. VG has also announced a three-sector instrument, the ZAB 3F, with a magnetid electridmagnetic sector configuration. The commercial three-sector instruments cost from $500,000 to $800,000.

The major advantage of these instruments is the availability of high resolution in the first stage of mass analysis in order to separate isobaric ions from mixtures. In that configuration, however (fragmentation between the second and third sectors), they provide less than unit mass resolution in the second stage. If fragmentations between the first two sectors are observed, then high resolution is available for the daughter ions instead of the parents.

The ultimate MSMS instrument would provide high resolution in both stages; that is, it would be a four-sector instrument, with two double-focusing instruments in tandem. Such an instrument was constructed in the 1960s for studying iodmolecule reactions by Futrell and Tiernan of the Wright- Patterson Aerospace Research Laboratory (102). Similar instruments have also been designed by White and Forman at Rensselaer Polytechnic Institute (103) and others (104) to provide extreme mass resolution for selectivity in determining nuclidic abundances. McLafferty and co-workers at Cornell Uni- versity (105) have built a four-sector (electridmagnetidelectridmagnetic) instrument specifically for MSMS studies, as shown in Fig. 7. This instrument

1st STAGE

MASS SEPARATION

ION

I I

t L t C I HUN

MULTIPLIER

- -__ MAGNET,"\\'

SECTOR I SECTOR I

U SOURCE COLLISION

GAS INLET

, 50 cm ,

I I

2nd STAGE MASS SEPARATION

Figure 7. Scale drawing (top view) of the four-sector MSMS instrument constructed at Cornell (13).


was proposed in 1977 (105), and first tested as a three-sector instrument, minus the final magnet (13). The four-sector instrument has recently been completed (2), providing high resolution in both the first (50 x lo3) and second (20 x lo3) stages of mass analysis. The use of inhomogeneous high- field magnets makes the instrument particularly well suited for high-mass studies. “Preacceleration” of the ions (prior to collision) to 30 keV and a collision cell employing a He molecular beam have been used to increase CAD efficiency and improve resolution. This design provides CAD efficiencies of up to 10%. A simpler molecular beam design has recently been in- corporated (67) in a three-sector instrument (12).

There is no four-sector MSMS system available commercially; in view of the high cost ($l,OOO,OOO?) such a commercial instrument is unlikely to be offered. The capability of enhanced selectivity for both parent and daughter ions is valuable for special problems in trace mixture analysis.

The major advantages of all the tandem sector implementations of MS/ MS include their utility as high-resolution mass spectrometers and their higher mass range and greater sensitivity at high mass compared with quadrupoles. In addition, it is possible to study two or more sequential fragmentation steps, even with only two sectors (see Section IV B on MSMS/ MS). In many operating modes, sector MSMS instruments provide useful kinetic energy release information on the fragmentation process and permit observation of charge stripping and charge inversion reactions. This com- plements the added dimension of information provided in quadrupole MS/ MS through energy resolution and associative collisions. Note that sector instruments may also be modified to provide angular resolution; see Section IV A for a detailed discussion of these added resolution elements.

The most serious limitation of sector MSMS instruments (except for the tandem double-focusing system) is the lack of simultaneous unit mass resolution of parent and daughter ions. Similarly, except for the four-sector implementation, artifact peaks may lead to ambiguities in many operating modes. The efficiency of the CAD process in tandem sector instruments is generally lower by an order of magnitude than that in tandem quadrupoles (see Section I1 C). The general lack of sophisticated computer control, the complexity of many of the linked-scan modes, and the lower scan speeds are also limitations. Perhaps the biggest disadvantage, however, is the cost of these instruments.

C. Other tandem instruments

A number of other implementations of MSMS can be envisioned; a few of these are currently available or under development. Not surprisingly, a variety of other tandem instruments have been employed over the years for fundamental studies in chemistry and physics.

The most interesting new approach to MSMS implementations is the combination of sectors and quadrupoles together to produce “hybrid” MS/


MS instruments. As early as 1964, von Zahn and Tatarczyk used such a hybrid for metastable ion studies (106). It consisted of a small double-focusing sector instrument, followed by a 3-m RF-only quadrupole for unimolecular decomposition and a final quadrupole mass filter. More recent instruments for the study of iodmolecule reactions have employed tandem magnetic sector/quadrupole designs (107-109).

The first hybrid instrument designed specifically for analytical MSMS studies was reported in 1981 by Cooks and co-workers at Purdue University (63,110). It incorporates a magnetic sector followed by an RF-only quadrupole collision cell and a quadrupole mass filter. It may also be assembled without the quadrupole collision cell, and with the analyzers reversed (quadrupole/ magnetic sector). Early results with these other configurations appeared recently (63). A rather unusual approach to ion acceleration is used for the instrument. The ion source is held near ground potential, and the magnetic sector is floated at high voltage. As a result, the quadrupoles can be operated at ground. Furthermore, since source focusing is not dependent on the accelerating voltage, parent spectra can be obtained by scanning the accelerating voltage with the magnetic field strength fixed.

A significant advantage of the tandem magnetic sector/quadrupole implementation over tandem sector designs is the unit mass resolution obtained in the second stage of mass analysis. Artifact peaks are also eliminated by the acceleratioddeceleration scheme and the use of a quadrupole. Compared with tandem quadrupole instruments, the hybrid provides better mass resolution in the first stage and allows collisions to be observed at collision energies up to a few hundred volts. CAD efficiencies of a few percent were reported using the quadrupole collision cell; with a field-free collision cell, efficiencies were about two orders of magnitude less.

Because of the radically different ion kinetic energy requirements for mass analysis by sectors and quadrupoles, an acceleration and/or deceleration scheme must be employed between the analyzers. This means that one of the analyzers must float at high voltage. These considerations for various hybrid instrument configurations have been discussed in detail by Glish et al. (63) and by Beynon et al. (43). If a quadrupole is used as the second mass analyzer, then the ion beam must be retarded to a fraction m2/m, of its collision energy before entering the quadrupole. That means that not only must the quadrupole potential be floated, it must also be scanned as the daughter ion mass m2 is changed. If the ions are decelerated to a reasonably low energy before collision, this problem may be ignored. If, however, the collisions occur at high energy (before deceleration), mass analysis with the quadrupole is impossible. If the ion energy is set so that daughter ions m2 will enter the quadrupole with low energy, higher-mass daughter ions (and the parent ion) will have such large energies that they will pass right through the quadrupole, causing gross background interference. If a quadrupole is used as the first analyzer, there is some danger that the spread of ion energy and position at the exit of the quadrupole will make it impossible for the accelerating lens assembly to provide a good sensitivity and resolution in sub-


sequent sectors. In any case, ion fragmentation at some energy intermediate between that of the quadrupole and the sector produces daughter ions with such a wide spread of kinetic energies that the second stage of mass analysis is complicated or even impossible. Despite these problems, hybrid instruments offer a great deal of potential for MSMS studies.

A wide range of possible hybrid sector/quadrupole implementations have been proposed and their relative merits evaluated (43,63). Commercial instruments are on the way. Cooks and co-workers, in collaboration with Finnigan MAT, are constructing a computerized hybrid with a MAT 312 reversed-geometry double-focusing mass spectrometer followed by a quadrupole collision chamber and a quadrupole mass analyzer. VG has announced the 70-70 EQ (Fig. 8) which combines a normal geometry double- focusing system with a quadrupole collision cell and a quadrupole analyzer (111). The quadrupoles float at a voltage near the accelerating voltage on the source. CAD efficiency of up to 4% has been reported, with a maximum ion collision energy of 500 eV. Resolution of up to 25 x lo3 in the first stage and unit mass resolution up to mass 1.5 x lo3 in the second stage are possible.

Another type of ”hybrid” instrument is the “time-of-flighvmagnetic sector” system (not actually a tandem instrument) recently reported by Stults and co-workers (76). This implementation requires, at its simplest, only the modification of a single-focusing magnetic sector instrument (in this case, an LKB 9000) to allow the ion beam from the source to be pulsed and the flight time of ions to be determined. With the magnetic field strength B and the accelerating voltage V fixed, a single pulse of the source will provide all the parent ions dissociating to form daughter ions of constant momentum. Note that this is not one of the conventional linear scans (at constant parent

1st STAGE MASS SEPARATION

I 1 QUAORUPOLE I

1 4 COLLISION I

I QUAORUPOLE I

MULTIPLIERS

ION SOURCE

50 cm

Figure 8. twoquadrupole MSMS instrument (19).

Scale drawing (top view) of the VG 70-70 EQ hybrid two-sector/


ion m,, daughter ion m2, or neutral loss) in the three-dimensional map of Fig. 1, but rather a parabolic scan, at constant mgm,. The proposed addition of a computerized time array detector would permit all the data along that scan to be acquired for each pulse of the source. Complete three-dimensional MSMS data could be obtained rapidly, then, by successive scans at constant mYm, as the magnetic field is scanned. Because such a parabolic scan will “miss” the center of many of the MSMS peaks, a greater number of scans than normal would be needed. The ability to obtain all MSMS data in a single scan of the magnetic field will be particularly useful for GCMSMS. Assignment of parent ion mass could be ambiguous because of kinetic energy release. Also, as in sector MSMS, artifact peaks will be produced from fragmentations occurring in the accelerating region and the magnetic field.

The use of a modified time-of-flight mass spectrometer for the study of metastable ions was proposed by several groups in the 1960s (112-115). The approach used by Haddon and McLafferty (then at Purdue University) bears consideration (115). The parent ion [and its unimolecular or collisional (116) fragment ions, which will travel with the same velocity] are selected by pulsing a grid midway along the flight tube. A retarding field then separates the daughter ions in time, according to their mass. Successive daughter scans could be observed on the oscilloscope display by manually adjusting the parent ion gate time. Although mass resolution is limited with this technique, it has the potential to provide MS/MS data very rapidly. McLafferty (2) has recently proposed the construction of a tandem time-of-flight MS/MS system employing a two-dimensional array detector based on this earlier work.

The Fourier transform FTICR technique has been applied recently for analytical MSMS studies by McIver of the University of California, Irvine (117), and Cody and Freiser of Purdue University (118). Although ICR has been traditionally employed for iodmolecule reaction studies, it was the recent introduction of FTICR that made it practical for MSMS. In this “double- resonance” technique all ions except the parent ion of interest are ejected from the cell. The parent ion is then excited by irradiation at its cyclotron frequency, and the resulting daughter spectrum recorded in the usual FT way. MSA4S with the FTICR instrument does not require two analyzers in tandem; rather, because the ions are trapped in the cell, the two stages of mass analysis are ”tandem in time,” one after the other. Therefore, two or more sequential ion activation steps (MS/MS/MS) may be studied by a sequence of double-resonance pulses. Because of the temporal nature of the MSMS process, the only single-scan function possible is the daughter ion scan. Because the ionization and CAD processes occur in the same region, sample, reagent gas, and collision gas are all present simultaneously, com- plicating the CAD process. The mass resolution available in FTICR mass spectrometry can be extremely high, given a low enough cell pressure and long enough analysis time (119). It is difficult to eject all ions except the selected parent ion because of the bandwidth of the ejection pulse. It is possible, however, to excite the parent with relatively high resolution. A ETICR system with MSMS capabilities is available from Nicolet (17).


IV. ADDITIONAL RESOLUTION ELEMENTS

MSMS can be considered to provide an added dimension of information compared with conventional mass spectrometry. There are a number of ways to further increase the analytical resolution of MSMS. These additional “resolution elements” are made possible by variations of the sample introduction/ ionization or the ion activation processes.

The use of a separation technique in or before sample introduction can greatly increase the selectivity of the MSMS technique. This can be accomplished by microdistillation off the probe or by combined chromatography/ MSMS, or even with another mass separation stage as discussed below. The mode of ionization can be another resolution element. The choice of reagent gas in both conventional CI and APCI can provide ionization selectivity, as can the choice of ion polarity.

The ion activation step can give added resolution if reactive collision gases are used, as shown in Section V B. A particularly useful resolution element is the energy involved in the ion activation process. This can include variation of the collision energy or the scattering angle at which daughter ions are observed, as well as measurement of energy release.

A. Energy resolution

Control of ion internal energy (energy resolution) provides added resolution in MSMS. From a fundamental standpoint the effect of ion kinetic energy can give us a better understanding of the CAD process. Analytically it provides additional structural information and enhanced selectivity.

In MSMS systems such as tandem quadrupoles, in which collisions occur at low energies, the effects of ion kinetic energy on the CAD process are often more pronounced than for collisions at high energies. In both cases, however, it is observed that, at increased collision energy, ions of higher appearance energies increase in abundance at the expense of ions of lower appearance energies (8,62). The changes in relative abundance of the daughter ions as a function of internal energy can be presented as breakdown curves. Theoretical breakdown curves may be calculated using the quasi- equilibrium theory (QET) (120).

The effects of ion kinetic energy on the relative abundance of ions can be observed readily in tandem quadrupole instruments by scanning the offset potential on the collision cell relative to that on the ion source. Figure 6 shows the energy-resolved CAD breakdown curve for (C,H,) + produced from toluene by 70-eV electron impact. This complete breakdown curve was obtained in less than 2 min under computer control. Breakdown curves obtained in a similar manner have been compared to QET and angle-resolved data (58). The breakdown curve data are analytically useful both for isomer differentiation (62,121) and for optimization of CAD conditions for trace analysis.

At the high kinetic energies commonly employed in sector MSMS instru-


ments, the scattering angle (usually 4") of a given daughter ion is a function of the energy deposited in the CAD process. This is the basis of angle- resolved mass spectrometry (57). Breakdown curves obtained by this technique agree well with the QET breakdown curves (58). In sector MSMS instruments, the observed peak broadening is due to kinetic energy release. The shapes of these peaks give information on the energetics of fragmentation, which can be useful for the elucidation of reaction pathways and ion structure (5).

Studies of the energetics of the ion activation process continue to be of fundamental importance to the physical chemist. It is apparent, however, that they can also provide enhanced capabilities to the analytical chemist.

B. Combined chromatography/MS/MS and MSNSIMS

Combining chromatography, particularly capillary GC, with MSMS would require extremely rapid scanning if complete MSMS information were to be acquired for each chromatographic peak. Monitoring one or two parent/ daughter pairs in GCMSMS (selected reaction monitoring, or SRM) provides much greater sensitivity, albeit at the expense of selectivity. SRM is still more selective than the analogous single ion monitoring (SIM) in normal GCMS, since the selected parent ion must fragment to form the selected daughter ion in order to be detected. GCMSMS (with SRM) may offer better limits of detection than GCMS (with SIM) because of enhanced selectivity. This reduction in chemical noise is particularly important in GCMSMS, since column bleed is often the major limitation on GCMS detection limits (122). For example, a detection limit of 100 pg/mL was obtained for deacetylme- tipranolol in serum by capillary GCMSMS, by using SRM and an isotopically labeled internal standard (82).

MSMS can provide a molecular weight profile for all compounds with a specific functional group through the use of neutral loss and parent scans. This makes the MSMS a versatile "selective" GC detector. By selection of the operating mode, the MSMS can idenhfy the molecular ion for each GC peak. The MSMS now becomes a selective detector for phthalates (parent scan for 149+ or 148-), halogenated compounds (parent spectrum for X- or neutral loss of X or HX), or carboxylic acids (neutral loss of COOH), to name but a few.

Figure 9 compares the GCMS and GCMSMS analysis of a l-wL mixture containing 50 ng of each of the 47 (base + neutral)-extractable priority pollutants (123). Separation was performed on a 20-m SE-54 capillary column, followed by positive CI. The upper display shows a conventional GCMS reconstructed ion chromatogram (FUC) trace. The lower RIC trace is the GCI MSMS analysis for a neutral loss of T I . This "Cl-specific" scan shows 12 of the 15 C1-containing compounds. The baseline in the GCMSMS trace is reduced by a factor of lo3 compared with the GCMS trace. This increased selectivity and decrease in noise often results in an overall improvement in the detection limits.


1004 a

20 0 400 600 800 I000 1200 1400

SCAN

Figure 9. Chromatograms from the analysis of a mixture of the 47 base- neutral priority pollutants (50 ng each): (a) GCMS analysis, 20-m capillary column, methane positive ion CI; (b) GC/MSMS analysis conditions as in (a), but neutral loss scan for 35Cl. Twelve of fifteen chlorinated compounds identified: (1) 1,3-dichlorobenzene; (2) 1,4-dichlorobenzene; (3) 1,2-dichlorobenzene; (4) 2-chloroethyl vinyl ether; (5) hexachloroethane; (6) 1,2,4-tri- chlorobenzene; (7) hexachlorobutadiene; (8) hexachlorocyclobutadiene; (9) 2- chloronaphthalene; (10) 4-chlorophenyl phenyl ether; (11) hexachloroben- zene; (12) 3,3'-dichlorobenzidine. Not found were bis(2-chloroisopropyl) ether, bis(2-chloroethyl) ether, and bis(2-ch1oroethoxy)methane.

MSNS offers the ability to overcome two major drawbacks of the common direct liquid introduction (DLI) method of LCMS. In the DLI approach, the solvent acts as the reagent gas for chemical ionization. The abundant reagent ions create chemical noise that precludes scanning below approximately 100 u. The CI spectra also lack the structural information needed for peak identification. In LC/MS/MS, however, fragmentation of the molecular ion by CAD permits identification. The LCMSNS analyses of aromatic acids (124) and sulfa drugs (125) have been carried out using a DLI split interface with triple-quadrupole systems. A triple quadrupole system employing a moving belt LC interface has also been described (126).

Several implementations of tandem mass spectrometry allow more than two sequential stages of mass analysis. This ability to observe two or more consecutive reactions:


brings us MSNSNS (and presumably MS/MS/MS/MS . . . ). Although two sequential fragmentations may be observed in double-sector instruments (127,128), the presence of artifact peaks can lead to ambiguous interpreta- tions. Two consecutive fragmentations have also been studied with three- sector instruments (100,101,129).

MSNSNS is also possible with FTICR instruments, with fragmentations sequential in time (117). Sequential fragmentations have also been studied on triple-quadrupole MSMS instruments, with fragmentation occumng in the declustering lenses before the first quadrupole as well as in the center quadrupole (70).

V. RANGE OF APPLICATIONS

Analytical applications of MSiMS were pioneered in the laboratories of Cooks, McLafferty, Beynon, and Jennings. Within the last few years, however, dramatic innovations and improvements in MSNS instrumentation have greatly increased the number of laboratories utilizing these techniques, and generated on impressive range of applications (see, for instance, Table 1 in ref. 2, and refs. 1 and 3). Some of these are discussed briefly in the subsections below.

A. Mixture analysis

Much of the current interest in MSNS is due to its ability to rapidly provide sensitive and selective analysis of complex mixtures. The technique is rapid not only owing to the elimination of the chromatographic separation step, but also because samples can often be analyzed with little or no preparation. A second advantage of MSNS for mixture analysis is the reduction of chemical noise provided by two sequential stages of mass analysis.

We often think of sensitivity, defined in Table VI, as the best figure of merit for evaluating analytical methods. The response of the detector in mass spectrometry, however, is not the limiting factor; detecting a single ion is routine. In order to take advantage of this sensitivity, high selectivity must be achieved, so that signal from the analyte can be differentiated from that due to other compounds in the mass spectrometer background or in the sample itself. The practical figure of merit for MS/MS mixture analysis, therefore, is the limit of detection, defined in Table VI, a function both of sensitivity and selectivity. The increase in selectivity provided by two stages of mass

Table VI. Figures of merit for analytical methods. Slope of d Signal

[Amount for which Noise Limit of detection = signal/noise =

36 YOST AND FETI'EROLF

Table VII. triple-quadrupole MSiMS (11).

Calculation of ultimate detection limits for single-reaction monitoring with

Semi tivity Ion source efficiency Ql/Q2/Q3 Transmission CID Efficiency

i t 5

1 t 2 lo-'

Overall efficiency" 1 P

Noise Electrical noise Chemical noise

A (<1 ionis) <it13 A

Limit of detectionb For signalhoise = 3 9 ions required

= lo9 molecules = lW5 rnol

"Detection system sensitivity = 1@'* A for 1 ionis. bAt the extreme sensitivity limit, the peak height is quantized by the number of ions reaching

the detector. The dectection of n ions gives a signahoise ratio of nl".

analysis often yields improved (lower) limits of detection, even though some sensitivity is lost in the second stage.

The ultimate detection limits for MSMS (Table VII) were calculated in the early analytical studies of triple-quadrupole MS/MS (11). The calculations indicate that, as long as the selectivity of SRM is adequate to keep the chemical noise low, the limit of detection should require detecting only enough ions to give an adequate signalhoise ratio (9 ions for signalhoise = 3). Based on the sensitivity of that instrument, femtomole (W5 mol) detection limits were predicted. In many cases, detection limits close to these levels have been achieved (1 1,22,34,130,131). Furthermore, increases in ion source efficiency and ion transmission lead directly to improvements in detection limits when chemical noise is not limiting.

For many mixture analysis applications, the capabilities of MSMS and GC/MS are complementary. Some MSMS applications, however, take advantage of the special features of tandem mass spectrometry to provide unique capabilities. An example of this is the rapid identification of drug metabolites in physiological samples (132). Metabolites are addition or deg- radation products of the original drug, and should therefore retain many of the same characteristic substructures, as shown by the same daughter ions and neutral losses. The MSMS technique used to identify all metabolites in a physiological sample involves a series of steps, as shown in Figs. 10 and 11 for the antiepileptic drug primidone (132): (i) A normal (positive or negative ion) CI mass spectrum of the pure drug is obtained, as shown in Fig. 10(a). (ii) Characteristic substructures of the drug, both daughter ions and neutral losses, are identified by recording daughter spectra of the molecular ions and major fragment ions in the CI mass spectrum. Figure 10(b) shows the sum of the daughter spectra of six of these ions. (iii) Next, for a sample


PURE DRUG (PRIMIDONE)

NORMAL SPECTRUM (POSITIVE C I )

(M + H)+ 2 19

247

, , , /,,'59,

100 150 200 2 5 0 mlz

62

SUMMED DAUGHTER SPECTRA OF

162, 174, 190, 219, 247. 2 5 9

119 91 I

100 150 200 250 mlz

Figure 10. Spectra obtained in a study of the metabolites of the drug primidone: (a) the positive (CH,) CI mass spectrum of the pure drug showing the (M + H)+ ion at m/z 219 as well as (M + 29)+, (M + 41)+, and fragment ions; (b) the summed daughter spectra of six ions in (a).

of serum, urine, or tissue, all ions which contain the drug's characteristic substructures are identified by acquiring a parent spectrum for each selected daughter ion and a neutral loss spectrum for each selected neutral loss. Each of the parent ions seen in these scans corresponds to a potential metabolite. Figure ll(a) shows one such scan, a parent spectrum for the characteristic 162 daughter ion, from an extract of serum from a patient being treated with

SERUM EXTRACT

1 162

190 I

n 1 I 2 3 3

l 124726~ 235

, ,

P A R E N T SPECTRUM OF 162

219

~

207 i

rnlz 8 , 162

DAUGHTER SPEC TRUM O F 2 0 7

119 I

rnlz

PURE METABOLITE (PEMA)

C

DAUGHTER SPECTRUM OF 2 0 7

119

1 151

, , ?,,,I;; ,I, ;,, , ,

100 150

rnlz

5 2

.lil 2 0 0

Figure 11. (a) Parent spectrum of an extract of a patient’s serum for the mlz 162 daughter ion characteristic of primidone, showing (M + H)+ ions for the drug (mlz 219) and two metabolites (m/z 207 and m/z 233); (b) the daughter spectrum of the 207 ion from the serum extract; (c) the daughter spectrum of the 207 ion from pure phenylethylmalonamide (PEMA) for comparison with (b).

38


the drug. The (M + H)+ ions are seen for the parent drug (at m/z 219), plus two possible metabolites (at m/z 207 and 233). In addition, the (M + 29)+ and (M + 41)+ ions are observed for each compound. (iv) A daughter spectrum of each potential metabolite’s molecular ion is obtained in order to determine the metabolite structure. The daughter spectrum of the mlz 207 ion is shown in Fig. ll(b). It may be interpreted directly, or compared with the daughter spectrum of the authentic metabolite [Fig. ll(c)] in order to confirm the structure. In this case two metabolites (phenylethylmalonamide and phenobarbital) are identified. This example demonstrates the potential of employing the variety of MS/MS operating modes to develop new analytical methods.

B. Structure elucidation

The application of MSiMS to elucidate the structure of gas-phase ions (8) and therefore that of the original molecule (23) is widely appreciated. It permits the analyst to separate a portion of a molecule and fragment it, enabling him to build up the structure piece by piece. If this process is carried out systematically, all pathways for the formation or fragmentation of every ion in the mass spectrum are determined. This enormous amount of information is a promising data base for computerized structure elucidation, as shown by the computer-generated fragmentiition tree in Fig. 2.

It is not uncommon for two ions of different structure to give CAD spectra which are too similar to permit their differentiation. The use of energy resolution (Section IV A) is one way to help discriminate between two such isomeric ion structures. Another is the use of selective collision gases to form adduct ions at low energy (26). The daughter spectra of two isomeric (C,H,)+ ions, with isobutane as the collision gas, are shown in Fig. 12. The 1-penten- 3-yne ion readily forms a number of adduct ions with the collision gas, making it easy to differentiate from the cyclopentadiene ion, which does not. The normal daughter spectra of these ions, with nitrogen as the collision gas, are nearly indistinguishable.

There is some controversy surrounding the use of CAD for determination of ion structures. Bass and Bowers have shown recently (133) that calculation of the relative contribution of two isomeric structures based on linear inter- polation of peak height ratios in CAD spectra is not necessarily valid. Fur- thermore, they have shown that the practice of ignoring those CAD peaks which also arise by unimolecular decomposition (8) can pose problems. Con- tinuing studies in several laboratories should address these problems, help- ing to more firmly establish the use of MSMS for ion structure determination.

C. Fundamental studies

Although the current interest in MSiMS is primarily the result of its analytical potential, MSMS continues to serve as a powerful tool for the study of fundamental kinetics and energetics. Kinetics studies can yield thermo-

40

66 M+ "'1 a

40 50 60

[Oj

YOST AND FETTEROLF

(M f C3H 7 j

(M * C H,+f

10 00 90 100 110 120 rnlz

Figure 12. Comparison of the daughter spectra for two isomeric (C,H,)' ions reacted with 6 mtorr of isobutane at a collision energy of 2.6 eV: (a) the cyclopentadiene ion (from dicyclopentadiene) spectrum; (b) the l-penten-3- yne ion spectrum showing addition products.

chemical values, such as the relative proton affinities of two species combined in a proton-bound dimer (134). Similarly, MSMS can be used to study, in the gas phase, reactions analogous to those observed in traditional con- densed-phase organic chemistry (135). These studies are of importance both for the fundamental information they generate, as well as for their role in the development of new MSMS techniques for analysis.

VI. FUTURE PROSPECTS

Tandem mass spectrometry is a rapidly evolving field, with particularly dramatic advancements being made in instrumentation. Continued studies of the underlying fundamentals promise further improvements in MSMS for analytical applications.

Although the triple-quadrupole and the multiple-sector instruments rep- resent the bulk of MSMS instrumentation in use today, new hybrid instruments have significant potential. The use of time-of-flight measurements in


conjunction with another mass analyzer may open the door for extremely rapid but inexpensive MSMS. For particularly demanding problems, the continued development of GCMSMS, LCMSMS, and MSMSMS holds promise. Instruments with improved sensitivity are required in order to take advantage of the reduced chemical background made possible by MSMS. This will be effected not only by optimization of the CAD process, but also by increasing the efficiency of ionization sources. Source improvements will also need to address both the advantages and the problems (matrix effects) of ion source selectivity.

Perhaps the most important advance in MS/MS will involve those improvements in computer control and instrumental design necessary to make low-cost ($50,000-$150,000) computerized tandem mass spectrometers available commercially. This will make MS/MS viable for rapid mixture analysis in applications ranging from the clinical laboratory to process control. Struc- ture elucidation applications will benefit from new computerized techniques as well as desorption ionization and higher-mass-range analyzers for handling large biomolecules. MSMS promises to continue to be an exciting field of research.

APCI CAD CI DCI EI FAB FTICR GCMS ICR IKES LC MIKES MSIMS RF SIMS SRM

VII. NOMENCLATURE

Atmospheric pressure chemical ionization Collisionally activated dissociation Chemical ionization Desorption chemical ionization Electron impact Fast atom bombardment Fourier transform ion cyclotron resonance Gas chromatography/mass spectrometry Ion cyclotron resonance Ion kinetic energy spectroscopy Liquid chromatography Mass-analyzed ion kinetic energy spectrometry Mass spectrometrylmass spectrometry Radiofrequency Secondary ion mass spectrometry Selected reaction monitoring

VIII. ACKNOWLEDGMENTS

The MS/MS work at the University of Florida has been supported by the National Science Foundation and the Petroleum Research Fund of the Amer- ican Chemical Society.


IX. REFERENCES

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Elsevier: Amsterdam, 1973. 6. Barber, M.; Elliott, R. M. Presented at ASTM Conference on Mass Spectrometry,

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Conference on Mass Spectrometry, Vienna, Austria, August 1982. 23. Bozorgzodeh, M. H.; Morgan, R. P.; Beynon, J. H. Analyst 1978, 203, 613-622. 24. McLafferty, F. W. Philos. Trans. R. SOC. London Ser. A 1979, 293, 93. 25. Johnson, J. V.; Yost, R. A. Presented at International Conference on Computers

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Tandem mass spectrometry (MS/MS) instrumentation