Mass Spectrometry

Alan L. Rockwood, Ph.D., D.A.B.C.C., Thomas M. Annesley, Ph.D. and Nicholas E. Sherman, Ph.D.*

Mass spectrometry (MS) is a powerful qualitative and quantitative analytical technique that is used to measure a wide range of clinically relevant analytes. When MS is coupled with gas or liquid chromatographs, the resultant analyzers have expanded analytical capabilities with widespread clinical applications. In addition, because of its ability to identify and quantify proteins, MS is a key analytical tool that is used in the emerging field of proteomics.

We begin this chapter with a discussion of the basic concepts and definitions of MS followed by discussions of MS instrumentation and clinical applications, and we end the chapter with a discussion of logistic/operational/quality issues. In this chapter, it is impossible to cover all concepts in a field as vast as mass spectrometry, even if one limits one’s focus to clinical mass spectrometry. The Clinical and Laboratory Standards Institute (CLSI) has published recommendations on clinical mass spectrometry that can serve as a good next step for study of this topic and another gateway into the extensive literature on this topic.²⁷

Basic Concepts and Definitions

MS is the branch of science that deals with all aspects of mass spectrometers and the results obtained with them. Molecular mass (sometimes referred to as molecular weight) is measured in units of unified atomic mass unit (u), also known as the dalton (Da). The mass of a carbon atom in its lowest energy state is defined as 12 Da. Although the term atomic mass unit (amu) has been regarded as equivalent to the Da, it is only approximately equal to the Da and now is considered an obsolete unit; its use to refer to the Da is strongly discouraged. A mass spectrometer is an analytical instrument that first ionizes a target molecule and then separates and measures the mass of a molecule or its fragments. Mass analysis is the process by which one or more ionic species are identified according to the mass-to-charge ratio (m/z).¹¹⁷ The analysis is qualitative, quantitative, and extremely useful for determining the elemental composition and structure of both inorganic and organic compounds.

All MS techniques require an ionization step in which an ion is produced from a neutral atom or molecule. In fact, the development of versatile ionization techniques caused MS to become the excellent analytical tool it is today. In 2002, John Fenn and Koichi Tanaka shared the Nobel Prize for their development of electrospray 43,44 and laser desorption^72,92,115 ionization, respectively.

Ions may undergo fragmentation in a mass spectrometer. An unfragmented ion of the original molecule is called the molecular ion. If the ionization source is soft, meaning that it produces little fragmentation, the most abundant peak in the mass spectrum (the base peak) may be the molecular ion. Ions that are formed by fragmentation of a molecular ion in the ion source are known as fragment ions. If the ion source is hard, meaning that it produces extensive fragmentation, the base peak may be one of the fragment ions. By convention, the base peak in a mass spectrum is assigned a relative abundance value of 100%.

Fragment ions that are formed in a separate dissociation cell in a tandem mass spectrometer are known as product ions. A tandem mass spectrometer consists of two mass spectrometers operated in sequence, or a single mass spectrometer capable of sequential measurement of ions. Generally, ions are dissociated into product ions between the two stages of m/z analysis.

A mass spectrum is represented by the relative abundance of each ion plotted as a function of its m/z ratio (Figure 14-1). Usually, each ion has a single charge (z = 1); thus the m/z ratio is equal to the mass. However, in some cases, the charge may be represented by some other integer number, in which case the m/z ratio is not equal to the mass, but rather is some fraction of the mass.

Figure 14-1 Mass spectrum of the pentafluoropropionyl (A) and carbethoxyhexafluorobutyryl (B) derivatives of D-methamphetamine.

Monitoring ions with higher mass usually results in lower limits of detection because fewer background ions are present. A peak in a mass spectrum can be characterized by its resolution, [(m/z)/(Δm/z)], where Δm/z is the width of the mass spectral peak. This parameter characterizes the ability of a mass spectrometer to separate nearby masses from each other.

By setting the relative abundance of the base peak to 100% and therefore using the relative, rather than absolute, abundance of each ion fragment, instrument-dependent variability is minimized, and the mass spectrum can be compared with spectra obtained on other instruments. Because fragmentation at specific bonds depends on their chemical nature, the mass spectrum can be interpreted in terms of the molecular structure of the analyte. In some cases, the chemical structure of the analyte can be deduced, or at least reconciled with features found in the mass spectrum. Computer-based libraries of spectra are also available to assist in identification of the analyte(s). In some applications, the mass spectrum of an analyte may be matched against mass spectra in a database, thereby identifying the analyte by its mass spectral fingerprint.

When interfaced to a liquid or gas chromatograph, the mass spectrometer functions as a powerful detector, providing structural information in real time on individual analytes as they elute from a chromatographic column. Depending on the operating characteristics of the mass spectrometer and the chromatographic peak width, several mass spectral scans can be acquired across the peak. The sum of all ions produced is displayed as a function of time to yield a total ion chromatogram.

The mass spectrometer is considered to be a “universal detector” because all compounds have mass. The data system can also be programmed to display chromatograms of only preselected ions acquired during data acquisition. The resultant display is called an extracted ion chromatogram, sometimes referred to as an extracted ion profile. Both of these displays have the appearance of a chromatogram with signal intensity plotted as a function of time. Retention times can be measured and peak heights or peak areas can be integrated for use in quantitative analysis.

Sample preparation is critical to successful mass spectrometry, particularly when one is dealing with complex matrices, such as are commonly encountered in clinical chemistry. This typically involves one or more of the following steps: (1) protein precipitation, (2) solid-phase extraction, (3) liquid-liquid extraction, or (4) derivatization. Derivatization is the process of chemically modifying the target compound(s) to be more favorably analyzed by mass spectrometry. Derivatization usually involves the addition of some well-defined functional group. The goals of derivatization vary, depending on the application, but typically include one or more of the following: (1) increased volatility, (2) greater thermal stability, (3) modified chromatographic properties, (4) greater ionization efficiency, or (5) favorable fragmentation properties.

When only a few analytes are of interest for quantitative analysis and their mass spectrum is known, the mass spectrometer is programmed to monitor only those ions of interest. This selective detection technique is known as selected ion monitoring (SIM). Because SIM focuses on a limited number of ions, more ion signal is collected for each selected m/z. This increases the signal-to-noise ratio of the analyte and improves the lower limit of detection. In general, an unknown is considered identified if the relative abundances of three or four ions agree within ±20% of those from a reference compound.

A chemical element may be composed of a single isotope or multiple isotopes. Each isotope of an element has the same number of protons in its nucleus but different numbers of neutrons. For example, naturally occurring carbon is composed primarily of two isotopes: ¹²C, whose nuclei contain six protons and six neutrons, and ¹³C, whose nuclei contain six protons and seven neutrons. Some elements, such as arsenic, have only a single isotope in the naturally occurring state.

A distinct advantage of the mass spectrometer is that it can simultaneously differentiate and quantify a compound with a normal abundance of isotope from an analog enriched with a stable isotope [e.g., (1) ²H relative to ¹H, (2) ¹³C relative to ¹²C, (3) ¹⁵N relative to ¹⁴N, (4) ¹⁸O relative to ¹⁶O]. A compound labeled with a stable isotope is used as an internal standard because it behaves nearly identically to the native compound during sample preparation and chromatographic analysis. The internal standard must have a sufficient amount of heavy isotope so that no naturally occurring isotope (such as ²H or ¹³C) contributes significantly to the ion current. For the methamphetamine derivatives shown in Figure 14-2, an internal standard with at least three ²H or ¹³C atoms is preferred, because at this point the natural abundance of these isotopes is ≈0.1% at 3 mass units above the molecular ion [(M+3)⁺]. Placement of the stable isotope atoms in the structure is also important. For example, the m/z 204 ion for methamphetamine represents the aliphatic portion of the molecule (loss of the aromatic ring). If five deuterium atoms were present on the aromatic ring of methamphetamine, the pentafluoropropionyl derivatives of both native and isotope-labeled drug would yield the m/z 204 ion. Similarly, the stable isotope must be located so that it will not exchange with solvent molecules. The ability to quantify a compound relative to an isotopic species of known or fixed concentration is known as isotope dilution analysis, and the specific mass spectrometric technique is known as isotope dilution mass spectrometry (IDMS). The IDMS technique has been used to develop definitive methods for a number of clinically relevant analytes including drugs of abuse.

Figure 14-2 Fragmentation patterns for the pentafluoropropionyl (A) and carbethoxyhexafluorobutyryl (B) derivatives of methamphetamine (R = CH₃) and amphetamine (R = H; masses in parentheses). Compare the predicted masses with the spectrum shown in Figure 14-1. Note that for the pentafluoropropionyl derivative, only one ion (204, 190 m/z) is characteristic of the aliphatic portion of the molecule.

Instrumentation

A mass spectrometer consists of (1) an ion source, (2) a vacuum system, (3) a mass analyzer, (4) a detector, and (5) a computer (Figure 14-3).

Figure 14-3 Block diagram of the components of a chromatograph–mass spectrometer system. The mass analyzer and the detector are always under vacuum. The ion source may be under vacuum or under near-atmospheric pressure conditions, depending on the ionization mode. The computer system is an integral part of data acquisition and output.

Ion Source

Many approaches have been used to form ions in both high-vacuum and near-atmospheric pressure conditions. Electron ionization (EI) and chemical ionization (CI) are ionization techniques used when gas-phase molecules are introduced directly into the analyzer from a gas chromatograph. In other analyses, such as high-performance liquid chromatography-mass spectrometry (HPLC-MS), electrospray ionization (ESI), atmospheric pressure chemical ionization (APCI), and atmospheric pressure photoionization (APPI) interfaces are used as ionization sources.19,41,50,60 Other ionization techniques include (1) inductively coupled plasma (ICP), (2) matrix-assisted laser desorption ionization (MALDI), (3) atmospheric pressure matrix-assisted laser desorption (AP-MALDI), and (4) a number of other less well-known sources. This chapter will limit its discussion to ion sources of interest to clinical chemical analysis. The CLSI document C-50A contains recommendations for matching the capabilities of different ion source technologies to various application classes.²⁷

Electron Ionization

In EI, gas-phase molecules are bombarded by electrons emitted from a heated filament and attracted to a collector electrode (Figure 14-4). This process must occur in a vacuum to prevent filament oxidation. A potential difference of 70 eV generates electrons with sufficient energy that a near collision with most organic molecules produces a radical cation that is both an ion and a radical.¹¹⁷ The radical ion then often undergoes unimolecular rearrangement and dissociation to produce a cation and a radical:

Figure 14-4 Electron impact ion source. The small magnets are used to collimate a dense electron beam, which is drawn from a heated filament placed at a negative potential. The electron beam is positioned in front of a repeller, which is at a slightly positive potential compared with the ion source. The repeller sends any positively charged fragment ions toward the opening at the front of the ion source. The accelerating plates strongly attract the positively charged fragment ions.

Positive ions are repelled or drawn out of the ionization chamber by an electrical field. The cations are then electrostatically focused and introduced into the mass analyzer. EI is primarily used as an ion source in gas chromatography–mass spectrometry (GC-MS). As determined by their chemical stability, the relative proportions of molecular ions and fragment ions produced in an EI source are reasonably reproducible. The fragmentation pattern is often used as a fingerprint to identify compounds by comparison with mass spectral libraries.

Chemical Ionization

CI is a “soft” ionization technique in which a proton is transferred to, or abstracted from, a gas-phase analyte by a reagent gas molecule such as methane, ammonia, isobutane, or water. The reagent gas is bled into a special CI source so the source pressure is increased to about 0.1 torr. (Note: For virtually all practical purposes “torr” is equivalent to “mm Hg” and is the more customary term used in the field of mass spectrometry.) An electron beam produces reactive species (such as for methane). Through a series of ion-molecule reactions, analyte ions become charged, usually via attachment of a proton. In most cases, relatively little fragmentation occurs. Negative ion electron capture CI is also possible and has become popular for quantification of drugs such as benzodiazepines. Negative ion formation occurs when thermalized electrons are captured by an electronegative substituent, such as chlorine or fluorine on the analyte. When applicable, negative ion CI has very low limits of detection. CI also is used as an ion source in GC-MS.

Electrospray Ionization

ESI is a soft ionization technique in which a sample is ionized at atmospheric pressure before introduction into the mass analyzer.121,126 The sample, typically an HPLC effluent, is passed through a narrow metal or fused silica capillary to which a 3- to 5-kV voltage has been applied (Figure 14-5, A). The partial charge separation between the liquid and the capillary results in instability in the liquid that in turn results in expulsion of a series of charged droplets from a Taylor cone, which forms at the tip of the capillary. A coaxial nebulizing gas, which is used in several types of ESI sources, helps direct the charged droplets toward a counter-electrode. The droplets evaporate as they migrate through the atmospheric pressure region, expelling smaller droplets as the charge-to-volume ratio exceeds the Raleigh instability limit. The proton- or ammonium-adduct of the molecule, which may be associated with solvent molecules, is “desolvated” to form “bare” ions and is typically formed in the ionization process. However, other ionization products are sometimes observed, such as metal ion adducts with analyte molecules, or ions formed by redox processes. Ions then pass through a sampling cone and one or more extraction cones (skimmers) before entering the high-vacuum region of the mass analyzer.

Figure 14-5 Schematics of (A) electrospray and (B) atmospheric pressure chemical ionization sources. Note the different points where ionization occurs, as described in the text.

One unusual feature of ESI is the production of multiple charged ions, particularly from peptides and proteins. It is common to observe approximately one charge for every 10 amino acid residues in a protein. For example, because a molecule of mass 20,000 can yield 20 charges, it can be detected at m/z 1000 (20,000/20) on a lower resolution and with a less expensive analyzer. This greatly extends the accessible mass range of such an instrument. In these cases, one usually observes a series of peaks, with each peak corresponding to a different number of charges. Nucleic acid polymers can also produce a series of multiply charged ions, particularly when ESI mass spectra are acquired in negative ion mode.

It should be noted that Figure 14-5 is a simplified illustration of the probe being directed toward the sampling cone of the mass detector. To enhance performance and minimize contamination of the mass detector, many modern hardware configurations have offset the probe and/or the mass detector relative to the sampling cone.

ESI tends to be an efficient ion source for polar compounds or for compounds that are ionized in solution, which include a high percentage of medically interesting compounds. ESI, along with APCI, allows an effective interface between a liquid chromatograph and a mass spectrometer. These have become the most widely used ion sources in clinical mass spectrometry.

Atmospheric Pressure Chemical Ionization

APCI is similar to ESI in the sense that ionization takes place at atmospheric pressure, involves nebulization and desolvation, and uses the same sample and extraction cones as ESI. The major difference lies in the mode of ionization. In APCI, no voltage is applied to the inlet capillary. Instead, a separate corona discharge needle ¹⁷ is used to generate a corona discharge. Somewhat analogously to the processes occurring in a CI source, ions generated by the corona discharge undergo ion-molecule reactions such as the following:

Because eluent solvent molecules (e.g., water, methanol) are present in excess relative to analytes in the sample, they are predominantly ionized early in the ion molecule cascade and then act as a reagent gas that reacts secondarily to ionize analyte molecules (Figure 14-5, B). Because the products of these secondary reactions may contain clusters of solvent and analyte molecules, a heated transfer tube or a countercurrent flow of a curtain gas, such as nitrogen, is used to decluster the ions. As with ESI, APCI is a soft ionization technique that produces relatively little fragmentation. Thus, compared with EI, the mass spectrum produced by APCI and other soft ionization techniques is less useful for analyte identification by mass spectral fingerprinting. However, because the ion current is concentrated into a single mass spectral peak (or relatively few mass spectral peaks if isotope peaks are considered), APCI and other soft ion sources are well matched to the requirements of tandem mass spectrometry (discussed later) and the combination of the two used for quantitative analysis. APCI and ESI are generally useful for a similar range of compounds. However, APCI is often a more efficient ion source than ESI for relatively nonpolar compounds.

Atmospheric Pressure Photoionization

APPI is a relatively new and less frequently used ion source in clinical chemistry that provides a complementary approach to ESI or APCI. The physical configuration of an APPI source is similar to that for APCI, but an ultraviolet photon flux is used instead of a corona discharge needle to generate ions in the gas phase.100,111,112 In APPI, an ionizable dopant, such as toluene or acetone, is often infused coaxially to the nebulizer to provide a source of ions that provide charge or proton transfer to analytes, thus increasing the efficiency of analyte ionization. APPI has a similar range of application to APCI but is often more useful than APCI for compounds of very low polarity, such as many steroids.

Inductively Coupled Plasma

As with ESI, APCI, and APPI, ICP is an atmospheric pressure ionization method. However, unlike most atmospheric pressure ionization methods, which are “soft” (i.e., producing little fragmentation), ICP is the ultimate in “hard” ionization, typically leading to complete atomization of the sample during ionization. Consequently, its primary use is for elemental analysis. In the clinical laboratory, it is particularly useful for trace metal and heavy metal analysis in tissue or body fluids. ICP is extremely sensitive (e.g., parts per trillion) and is capable of extremely high dynamic ranges.

Following sample preparation, which generally includes the addition of an internal standard such as yttrium and sometimes includes an acid digestion step, the sample is introduced into the ion source via a nebulizer fed by a peristaltic pump. The nebulized sample is transmitted into hot plasma generated at atmospheric pressure by inductively coupling power into the plasma using a high-powered, radiofrequency (RF) generator. A small orifice samples the plasma, and ions are transmitted to the mass analyzer through a series of differential pumping stages. The atmospheric sampling apparatus is conceptually similar to that of other atmospheric pressure ion sources, such as electrospray, except that the device must withstand the extremely high temperatures generated by the plasma.

ICP-MS is comparatively free from most interference. However, some interfering species can be extremely troublesome. Most interfering species are small polyatomic ions formed in the torch via ion-molecule reactions. For example, ArO⁺ interferes with iron at m/z 56. One solution to this problem is to use a reaction cell, which consists of a moderate-pressure gas placed before the m/z analyzer.¹² A reactant gas, such as NH₃, is bled into the reaction cell. The reactant gas reacts with polyatomic interferences and removes them before introduction into the m/z analyzer. Another approach to removing interferences of the same nominal mass is to use a high-resolution mass spectrometer, which is capable of resolving species with similar nominal mass.¹² For example, the masses of ArO⁺ and ⁵⁶Fe⁺ differ by 0.022 Da—a difference that is easily resolved using a high-resolution mass spectrometer.

Matrix-Assisted Laser Desorption/Ionization

MALDI and related techniques rely on energy transfer processes from a pulsed laser beam to the sample for ion generation. In most cases, the analyte is dissolved in a solution of matrix, a small molecular weight UV-absorbing compound, and this solution is placed on a target that is introduced into the mass spectrometer. A UV laser vaporizes small amounts of matrix and analyte into a plume of ions that is directed into a mass analyzer (Figure 14-6). MALDI is another of the “soft” ionization methods. Related techniques include atmospheric pressure matrix-assisted laser desorption/ionization, in which the MALDI process occurs at atmospheric pressure rather than reduced pressure, and surface-enhanced laser desorption ionization (SELDI), which uses a MALDI target surface modified with some type of affinity capture property [hydrophobic, ionic, immobilized metal affinity chromatography (IMAC), DNA, antibody, etc].

Figure 14-6 A generic view of the process of matrix-assisted laser desorption ionization. Cocrystallized matrix and analyte molecules are irradiated with an ultraviolet (UV) laser. The laser vaporizes the matrix, producing a plume of matrix ions, analyte ions, and neutrals. Gas-phase ions are directed into a mass analyzer.

Ionization Methods of Potential Interest

Sonic spray ionization is one of the lesser used ionization methods with potential utility for clinical mass spectrometry.53,54 It is as yet uncertain whether this technology has sufficient advantages over ESI and APCI to find frequent application, although it may find application for the analysis of thermally labile compounds. Desorption electrospray ionization (DESI)¹¹⁴ and direct analysis in real time (DART)²⁸ are two relatively new ionization methods that exemplify the generation of ions from surfaces at atmospheric pressure. Most applications of DESI and DART to date have been directed toward minimal sample preparation.

Ionization Methods of Historical Interest

The older literature includes several ionization methods or sample introduction methods that, although promising, or even widely used at one time, hold little interest for current practice in clinical chemistry. These include (1) fast atom bombardment (FAB), (2) thermospray (TSI), (3) direct liquid introduction (DLI), (4) plasma desorption, (5) field ionization (FI), (6) field desorption (FD), (7) surface ionization mass spectrometry (SIMS), (8) laser desorption (LD), and others. ESI and APCI ion sources have largely rendered these techniques obsolete.

Vacuum System

With the exception of certain ion trap mass spectrometers, ion separation in any of the mass analyzers requires that the ions do not collide with any other molecules during their interaction with magnetic or electric fields. This requires the use of a vacuum from 10⁻³ to 10⁻⁹ torr, depending on mass analyzer type. The length of the ion path in the analyzer must be less than the mean free path length, unless collisions play a role in mass analysis. Fourier transform ion cyclotron resonance (FT-ICR) requires the lowest pressure (10⁻⁹ torr). The quadrupole ion trap (QIT) tolerates the highest pressure (10⁻³ torr), a pressure range in which some collisions occur between ions and background gas. Routine quality assurance checks for vacuum leaks should include evaluation of air and water in the mass analyzer.

Efficient high-vacuum pumps generally do not operate well near atmospheric pressure. Thus the vacuum system must have a mechanical vacuum pump to evacuate the system to a pressure at which the high-vacuum pumps are effective. Mechanical pumps require routine maintenance, such as ballasting and replacing the pump oil. The diffusion pump is the least expensive and most reliable high-vacuum pump. Turbomolecular pumps and cryopumps are also used on mass analyzers, having largely replaced diffusion pumps. These high-vacuum pumps also require routine maintenance for optimal operation. A key consideration in construction of the vacuum system is pumping speed. The ability of the pump to maintain the vacuum by removing any gas (or solvent vapor) that enters the system determines the flow rate of gas or liquid that is introduced into the mass spectrometer. In general, higher pump capacities are associated with lower detection limits because noise arising from the gas background is reduced.

Mass Analyzers, Tandem Mass Spectrometers, and Ion Detectors

The term mass spectrometry is somewhat a misnomer because mass spectrometers do not measure molecular mass, but rather they measure mass-to-charge ratio. This fact is fundamental to the physical operating principles of mass spectrometers and consequently affects all aspects of instrumentation design and operation and interpretation of results. The symbol m/z is used to denote mass-to-charge ratio and conventionally has been defined as a dimensionless quantity.⁹⁷ However, a “dimensionless” mass-to-charge ratio is not consistent with equations of ion motion in the presence of electric and magnetic fields, which require units of mass divided by charge. Furthermore, the m/z scale sometimes is loosely discussed in terms of daltons (Da, also known as unified atomic mass units, u), although strictly speaking, Da is a unit of mass, not mass-to-charge ratio. Despite these nomenclature issues, the present chapter often follows conventional practices by discussing m/z in terms of mass and Da.

To help avoid some of the confusion surrounding the use of m/z, it has been proposed that it should be defined explicitly as quantity having units of mass-to-charge ratio, with mass specified in daltons and charge specified in elementary charges, and that this unit should be called the Thomson (Th) in honor of one of the pioneers of MS.29,36 This terminology is sometimes seen in the literature but has not yet been widely adopted.

General Classes of Mass Spectrometers

Mass spectrometers are broadly classified into two groups: beam-type instruments and trapping-type instruments. In a beam-type instrument, the ions make one pass through the instrument and then strike the detector, where they are destructively detected. The entire process, from the time an ion enters the analyzer until the time it is detected, generally takes microseconds to milliseconds.

In a trapping-type analyzer, ions are held in a spatially confined region of space through a combination of magnetic and/or electrostatic and/or RF electrical fields. The trapping fields are manipulated in ways that allow m/z measurements to be performed. Trapping times may range from a fraction of a second to minutes, although most clinical applications are at the low end of this range. Detection of the ions in a trapping-type instrument may be destructive or nondestructive, depending on the specific type of mass spectrometer used. In this context, “destructive” means that ions are destroyed in the detection process. Additional discussions of mass analyzers, tandem mass spectrometers, and ion detectors are available,35,107 and the CLSI document C-50A contains recommendations for matching the capabilities of different m/z analyzers to various application classes.²⁷

Beam-Type Designs

The main beam-type mass spectrometer designs are (1) quadrupole, (2) magnetic sector, and (3) time-of-flight (TOF). It is convenient to categorize beam-type instruments into two broad categories, those that produce a mass spectrum by scanning the m/z range over a period of time (quadrupole and magnetic sectors) and those that acquire successive instantaneous snapshots of the mass spectrum (TOF). This categorization is not hard and fast. Certain instrument designs have been adapted to scanning or nonscanning operations. Nevertheless, the categorization is a useful one because it covers the majority of instruments currently available, and because scanning and nonscanning instruments are adapted for different optimal usages.

Quadrupole: Quadrupole mass spectrometers, sometimes known as quadrupole mass filters (QMFs), are currently the most widely used mass spectrometers, having displaced magnetic sector mass spectrometers as the standard instrument. Although these instruments lag behind magnetic sector instruments in terms of (1) sensitivity, (2) upper mass range, (3) resolution, and (4) mass accuracy, they offer an attractive and practical mix of features that accounts for their popularity, including (1) ease of use, (2) flexibility, (3) adequate performance for most applications, (4) relatively low cost, (5) noncritical site requirements, and (6) highly developed software systems.

A quadrupole mass spectrometer consists of four parallel electrically conductive rods arranged in a square array (Figure 14-7). The four rods form a long channel through which the ion beam passes. The beam enters near the axis at one end of the array, passes through the array in a direction generally parallel to the axis, and exits at the far end of the array. The ion beam entering the quadrupole array may contain a mixture of ions of various m/z values, but only ions of a very narrow m/z range (typically Δm/z<1) are successfully transported through the device to reach the detector. Ions outside this narrow range are ejected radially. The Δm/z range represents a pass band, analogous to the pass band of an interference filter in optics (see Chapter 10), which is why quadrupole mass spectrometers are often referred to as mass filters rather than mass spectrometers.

Figure 14-7 Diagram of quadrupole mass filter, including the radiofrequency (RF) part of voltages applied to the quadrupole rods.

Quadrupole mass spectrometers rely on a superposition of RF and constant direct current, or DC potentials applied to the quadrupole rods. DC voltages are applied to the electrodes in a quadrupolar pattern. For example, a positive DC potential is applied to electrodes 1 and 3, as indicated in Figure 14-8, and an equivalent negative DC potential is applied to electrodes 2 and 4. The DC potentials are relatively small, on the order of a few volts. Superimposed on the DC potentials are RF potentials, also applied in a quadrupolar fashion. RF potentials range up to the kilovolt range, and frequency is on the order of 1 MHz. The frequency is typically fixed and highly stable, although variable frequency operation is possible in principle.

Figure 14-8 Direct current (DC) voltages applied to quadrupole rod assembly.

The physical principles underlying the operation of a quadrupole mass spectrometer are rigorously described by solutions of a complicated differential equation, the Mathieu equation.³³ When an ion is subjected to a quadrupolar RF field, its trajectory is described qualitatively as a combination of fast and slow oscillatory motions. For descriptive purposes, the fast component will be ignored here. The slow component oscillates about the quadrupolar axis; this resembles the motion of a particle in a fictitious harmonic pseudopotential. The frequency of this oscillation is sometimes called the secular frequency.

The effective force associated with the pseudopotential is directed inward toward the quadrupolar axis and is proportional to the distance from the axis. It therefore acts as a confining force, preventing ions from being ejected radially from the quadrupolar assembly. Figure 14-9, A, shows an example of an ion confined by an RF-only quadrupole. Below a certain m/z cutoff frequency (which depends on the frequency and amplitude of the RF field), ions are ejected rather than confined. Figure 14-9, B, shows an example of an ion ejected by an RF-only quadrupolar field. This establishes the low mass cutoff for the m/z pass band. The effective confining force is strongest just above the low m/z cutoff, and then decreases asymptotically toward zero at high m/z.