Mass spectrometry

Abstract

Basic concepts and definitions

MS is a branch of analytical chemistry that deals with all aspects of instrumentation and the applications of this technique. ^, Molecular mass (sometimes referred to as molecular weight ) is measured in unified atomic mass units (u), also known as the dalton (Da), equal to ¹ ⁄ ₁₂ of the atomic mass of the most abundant isotope of a carbon atom in its lowest energy state.

In MS, the mass-to-charge ratio ( m/z ) is mass of the ion divided by its charge, where m is the molecular weight of the ion and z is the number of charges present on the molecule. Small molecules (<1000 Da) are typically single charged, and therefore the m/z value is the same as the mass of the molecular ion. However, when larger molecules (e.g., proteins, peptides) are ionized, they typically carry multiple charges and therefore the z value is an integer greater than 1. In such cases, the m/z value will be a fraction of the mass of the ion.

All MS techniques require an initial ionization step in which an ion is produced from a neutral atom or molecule. Ions are formed in the ion source of the mass spectrometer. Some ion sources require the targeted analyte to be present in solution in the form of ions, in these cases the ion source function is to transfer the ions from a condensed phase to the gas phase. The development of versatile ionization techniques has allowed MS to become the robust broad-spectrum analytical methodology it is nowadays; this was highlighted in 2002, when John Fenn and Koichi Tanaka shared the Nobel Prize for their development of electrospray and laser desorption ionization techniques, respectively.

In the most frequently used ion sources of commercially produced MS instruments, ionization in positive ion mode typically results from the addition of one (or more) proton(s) to the basic site(s) on the molecule. This process is referred to as protonation and leads to formation of a positively charged ion. The mass of a single charged protonated ion is greater than the mass of the uncharged neutral molecule by the added mass of one proton, approximately 1 Da. Negatively charged ions (negative ion mode of MS operation) can be generated by the loss of a proton or addition to the molecule of a negatively charged moiety.

Ions may also be produced by removal of one or more electrons from a molecule using EI. Historically, this ionization method was the dominant technique used in MS [most commonly in gas chromatography–mass spectrometry (GC-MS) instruments], and is still used in most of the methods using GC-MS instruments, but other ionization techniques are now more frequently used in clinical MS-based methods.

Ions formed in the ion source are separated according to m/z values in a mass analyzer, and may undergo fragmentation, whereby energy is imparted into the ionized analyte, causing internal bonds to break and resulting in the production of multiple fragments of the molecule. Fragmentation may take place within different regions of the mass spectrometer; it may occur due to the deliberate action of the operator, or excessive energy imparted into the parent molecule , as it is being ionized or passes through the vacuum region of the mass analyzer. An unfragmented ion of the intact molecule is referred to as the molecular ion , whereas the species that occur on fragmentation of the molecular ion are called the fragment ions.

If the ionization of the analyte in the source produces little or no fragmentation, it is referred to as being soft , and the most abundant peak in the mass spectrum (the base peak) is often the molecular ion. If the ion source produces extensive fragmentation, it is referred to as hard ionization, and the base peak in the resulting spectra may be one of the produced 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 dissociation cell (also known as the collision cell) inside a tandem mass spectrometer are known as product ions , and the technique is called tandem mass spectrometry (MS/MS) . Ions that give rise to the product ions are known as precursor ions . A tandem mass spectrometer consists of two mass spectrometers operated in sequence (MS/MS in space) or a single mass spectrometer capable of sequential fragmentation and detection of ions within a single region of space but separated by time (MS/MS in time). Most commonly in the MS-based clinical diagnostic methods, precursor ions are dissociated into product ions between the two stages of m/z analysis (MS/MS in space).

A mass spectrum is represented by the relative abundance of the detected ions, plotted as a function of m/z ( Fig. 20.1 ). As mentioned earlier, for small molecules, usually the ions are singly charged ( z = 1); thus the m/z ratio is equal to the mass of the ion, and if ionization occurs by protonation, then the mass of the ion is approximately 1 Da (accurate mass 1.00728 Da) greater than the neutral molecule from which the ion is formed. However, in some cases, the charge may be represented by an integer number greater than 1, in which case the m/z ratio is not equal to the mass of the ion but rather is a fraction of the mass of the ion.

An ion may be positively charged, in which case the number of electrons in the ion is less than the sum of the number of protons in all nuclei of the ion, or negatively charged, in which case the number of electrons is greater than the number of protons. By convention, in MS, z is taken as an absolute value (e.g., z = 1 for Na ⁺ and Cl ⁻ ).

Chemical interferences, as well as higher background noise, are more common for analytes with m/z 200 to 500 than for m/z less than 200 and m/z greater than 500. Monitoring ions with higher m/z often results in greater signal-to-noise ratio, because of the lower background noise and lower occurrence of isomers and isobars of the targeted molecules.

A peak in a mass spectrum can be characterized by its resolution [( m/z )/(Δ m/z )], where Δ m/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. Typically, the width of the peak is measured at 50% of the height of the peak and is referred to as the full width at half height (FWHH) or full width half maximum (FWHM) resolution; another frequently encountered definition for resolution is based on the 10% valley (Δ m/z as the distance between two peaks of equal intensity with the valley between the peaks of 10% of the peak height) ( Fig. 20.2 ). The 10% valley is a more conservative definition than FWHM because, for a given quoted resolution (e.g., 2000), the peaks are narrower under the 10% valley definition, hence better separated. High resolution is a desirable property in MS because it can help reduce interferences from nearby peaks in the mass spectrum, thereby allowing to achieve a higher specificity.

By setting the relative abundance of the base peak to 100%, relative abundance of the fragment ions can be compared among multiple instruments. Because fragmentation at specific bonds depends on their chemical nature and strength of the bonds, information from the mass spectrum can be used for interpreting molecular structure of the analyte. In some cases, the partial or even complete molecular structure can be deduced (or at least reconciled) with features found in the mass spectrum.

Computer-based libraries of mass spectra are also available to assist with identification of the analyte(s) based on fragmentation pattern. 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 . In general, an unknown is considered to be identified if the relative abundances of three or four ion fragment ions agree within ±20% of those from a reference compound; and the relative abundances of the fragments, monoisotopic and isotopic ions of the molecular ion, are in agreement with the relative abundances of the ions in a reference mass spectrum.

When interfaced to a liquid or gas chromatograph, the mass spectrometer functions as a powerful detector, able to provide structural information on peaks of the analytes. Depending on the operating characteristics of the mass spectrometer and the chromatographic peak width, multiple mass spectral scans can be acquired across the peak. The data also can be displayed as a function of time to yield a total ion chromatogram (TIC), where each time point corresponds to the total abundance of all acquired m/z .

The mass spectrometer can be considered close to a universal detector, because molecules of many classes (e.g. small molecules, peptides, proteins, lipids) may be ionized and then detected in a mass spectrometer. Furthermore, there are different MS operation modes and different types of fragmentation can be applied to provide different types of data, allowing to obtain complementary information about the measured compound(s). Finally, the instrument data system can analyze and display the collected data in various modes, allowing the operator to selectively process information from the acquired data.

For example, it is possible to display only chromatograms of ions with a preselected m/z ; the data that come from this preselection is called an extracted ion chromatogram (EIC) and is displayed as the intensity of signal on the y -axis, plotted against time on the x -axis. The height or area of the peaks can be obtained from the data and used for quantitative analysis. Furthermore, the EIC allows selecting data corresponding to the analyte of interest, as identified by its m/z, while disregarding data corresponding to the other m/z acquired during the analysis. With high-resolution instruments, specificity of analysis can be enhanced by use of narrow m/z windows for plotting EIC. Such data processing results in a reduced number of overlapping chromatographic peaks from ions of nearby m/z and cleaner baseline , thus improving the quantitative accuracy and the specificity ( Fig. 20.3 ).

Sample preparation is critical for obtaining high-quality MS data, particularly when dealing with highly complex sample matrices, commonly encountered in clinical chemistry. This typically involves one or more of the following steps: (1) protein precipitation followed by centrifugation or filtration, (2) solid-phase extraction, (3) liquid-liquid extraction, (4) affinity enrichment, and (5) derivatization, or combination of these techniques (see Chapters 21 and 23 ).

Derivatization is the process of chemically modifying (addition of a functional group) to the target compound(s), to have more favorable properties for chromatographic separation and/or MS analysis. The goals of derivatization vary, depending on the application, but typically include (1) increased volatility (in the case of GC-MS), (2) greater thermal stability (in the case of GC-MS), (3) modified chromatographic properties (in the case of either GC-MS or LC-MS), (4) greater ionization efficiency (in the case of LC-MS), (5) favorable fragmentation properties (in the case of either GC-MS or LC-MS), or a combination of these.

Analysis by MS can be used to target specific known compounds ( targeted analysis ) or seek to identify one or more unknown compounds in a sample ( screening ). When only one or a few targeted analytes are of interest for quantitative analysis and their mass spectra are known, the mass spectrometer is set to monitor only those ions of interest. This detection technique is known as selected ion monitoring (SIM). Because SIM collects data on a limited number of m/z , more data points are collected for the selected m/z, which results in a greater specificity, improved signal-to-noise ratio for the analyte of interest, and greater sensitivity and enables more accurate quantitation with greater precision. One drawback of SIM is related to specificity of detection. Most biological samples are highly complex, and thus it is not uncommon to have multiple compounds with very close or identical masses to be present in the sample matrix. In those cases, chromatography can aid in separation of these isobars; however, they still can affect a SIM result if a peak of interfering substance is not fully separated from the analyte of interest.

By using a triple-quadrupole mass analyzer, a method known as selected reaction monitoring (SRM; or multiple reaction monitoring [MRM], if used for the simultaneous detection of number of ions), can be used to help alleviate such potential issues. In a triple-quadrupole mass analyzer, the first quadrupole mass analyzer is set to transmit the m/z of the molecular ion, the analyte gets fragmented in the collision cell (the second quadrupole), and the third quadrupole mass analyzer is set to transmit the m/z of one or more known fragment ions of the targeted molecule. In this manner, data similar to those gathered by SIM can be acquired but with added specificity from the use of the targeted molecular ion and fragment ions. A more detailed description of MRM mode of data acquisition is given in the section of this chapter describing tandem MS.

Analytical screening methods are used in clinical chemistry laboratories less commonly than the analysis of target compounds. The main task for screening methods is qualitative identification of unknowns in a sample. In most cases, this is performed by matching chromatographic retention time and fragment ion patterns ( m/z and relative abundance) of either fragment ions generated in the ion source of a single-stage mass spectrometer, or product ions formed in a collision cell in a tandem mass spectrometer.

A chemical element may be composed of a single isotope or multiple isotopes. Isotopes of an element have the same number of protons but different numbers of neutrons. For example, naturally occurring carbon is composed primarily of two isotopes: ¹² C, whose nuclei contain six protons and six neutrons, and ¹³ C, whose nuclei contain six protons and seven neutrons (abundance of ¹⁴ C isotope for the purposes of routine MS is negligible, as compared with the other two isotopes). The natural abundance of ¹² C is approximately 98.9%, and the natural abundance of ¹³ C is approximately 1.1%. Some elements, such as phosphorous and arsenic, have only a single isotope in the naturally occurring state, whereas other elements, such as tin, may have as many as 10 naturally occurring isotopes.

For molecules consisting of multiple atoms, the isotope pattern is a combination of the isotope patterns of the individual atoms. ^, As an example, carbon monoxide (CO) has the following combinations of isotopes ¹² C ¹⁶ O (molecular weight 28), ¹³ C ¹⁶ O (molecular weight 29), ¹² C ¹⁷ O (molecular weight 29), ¹² C ¹⁸ O (molecular weight 30), and ¹³ C ¹⁸ O (molecular weight 31).

Nitrogen (N ₂ ) is isobaric with CO; that is, it has nearly the same mass. However, the accurate masses of the isotope peaks of isobars may differ. For example, the monoisotopic mass of ¹² C ¹⁶ O ⁺ (the isotopic peak composed of the most abundant atomic isotopes) has an accurate mass of 27.9944 Da, whereas N ₂ ⁺ has an accurate molecular mass of 28.0056 Da. The accurate mass can be used to infer the chemical formula of a compound or to confirm the identity of a target compound. This technique requires a mass analyzer capable of high mass accuracy (few parts per million), is limited to compounds with molecular weight of a few hundred daltons or less, and is unable to discriminate isomers (compounds that have the same chemical formulas but different molecular structure).

Isotopic information also can be used to infer the chemical formula of an unknown or to confirm chemical identity of a target compound. Using CO ⁺ and N ₂ ⁺ as examples, the monoisotopic and the next two isotopic peaks of CO ⁺ have a relative abundance of 0.986, 0.011, and 0.002, whereas the monoisotopic and the next two isotopic peaks of N ₂ ⁺ have relative abundances of 0.993, 0.007, and 0.000. This technique requires accurate measurement of relative isotopic peak abundances and, if used in conjunction with accurate mass measurements, can be powerful.

A distinct advantage of the mass spectrometer is that it can distinguish between ions of the same chemical formula that have different masses, because of the different isotopic composition. To illustrate with a simple example, ¹² C ¹⁶ O ⁺ has a different mass than ¹² C ¹⁸ O ⁺ , and these two forms can be separated and detected in a mass spectrometer. One can take advantage of this fact by using synthetically produced forms of a target analyte. The labeling consists of substitution of one or more monoisotopic atoms with isotopic atoms (e.g., substituting ² H for ¹ H, ¹³ C for ¹² C, or ¹⁵ N for ¹⁴ N). The stable isotope-labeled analog of the targeted molecule can be chemically synthesized and added to the samples as an internal standard, which behaves nearly identically to the native compounds during sample preparation and chromatographic separation. ^, In this respect, ¹³ C or ¹⁵ N is generally preferred over ² H labeling, because ² H-labeled compounds sometimes exhibit chromatographic shifts compared with unlabeled compounds, whereas ¹³ C- or ¹⁵ N-labeled compounds generally do not. A quantitative analysis can then be carried out by comparison of the signal from the native compound, relative to the stable isotope labeled version of the compound, added into the samples during the sample preparation.

An internal standard should be selected to have a sufficient number of isotopic atoms so that no naturally occurring isotopes (e.g., ² H or ¹³ C) of the analyte of interest would significantly contribute to the signal of the internal standard. As an example, for the methamphetamine derivatives shown in Fig. 20.4 A, an internal standard with at least three ² H or ¹³ C atoms is preferred, because contribution of the natural abundance of these isotopes to the molecular ion [(M + 3) ⁺ ] would be negligible (<0.1%). The position of the stable isotope atoms within the molecule and the number of isotopic ions within the structure are also important for adequate performance of the methods. ^, For example, the m/z 204 ion for methamphetamine represents the aliphatic portion of the molecule (loss of the aromatic ring). If three deuterium atoms were located on the aromatic ring of the pentafluoropropionyl derivative of methamphetamine, the native and the isotope-labeled molecules would both yield the m/z 204 ion. This m/z 204 ion would therefore fail to distinguish the native compound from the isotope-labeled compound and would therefore not be useful as an internal standard. On the other hand, if ² H labeling were to occur in the aliphatic portion of the molecule, the fragment ion analogous to the m/z 204 would contain three ² H ( m/z 207), and the ion could be useful as an internal standard. The same comments apply to the compound illustrated in Fig. 20.4 B.

The concepts of internal standard selection are different when applied to tandem MS. For example, it is possible for a native compound and the internal standard to have product ions of the same m/z, because the precursor ion m/z is different for the targeted analyte and the internal standard.

When using deuterium ( ² H) labeling, the isotopic ions must be located in the positions within the molecule where it will not be exchangeable with hydrogen atoms (in solution or in gas phase). For example, deuterium labeling of an acidic hydrogen position would be useless because the ² H would easily exchange with protons in the matrix, making the original labeling moot. Certain other labeling positions within a molecule, where ² H could exchange with hydrogen (alcohols, amines, amides, and thiols), also must be avoided.

A technique of quantitative analysis of compounds relative to their isotopic analogs added to the samples at known or fixed concentration is called isotope dilution analysis or isotope dilution mass spectrometry (IDMS). The IDMS technique is widely used in methods for analysis of clinically relevant biomarkers.

MS is often considered as a highly sensitive technique. Sensitivity is a somewhat problematic term because it is used in two different ways. In an official definition it means the slope of a calibration curve (or more generally, a change in signal vs. the change in concentration), but more commonly it is used to signify the ability to detect or quantify an analyte at very low concentration; that is, a more sensitive technique would be able to detect or quantify a lower concentration of the target analyte.

Instrumentation

A mass spectrometer consists of the following components: (1) ion source, (2) vacuum system, (3) mass analyzer, and (4) detector ( Fig. 20.5 ). Most modern mass spectrometers also include a computer for instrument control, data acquisition, and data processing.

Electron ionization

In EI, gas-phase molecules are bombarded by electrons emitted from a heated filament, causing extensive fragmentation of the molecules ( Fig. 20.6 ). To make the process robust, prevent filament oxidation, and minimize scattering of the electron beam, the ionization must occur in a vacuum. EI is typically performed using electrons with a kinetic energy of 70 eV; collision of electrons having such energy with most molecules results in formation of radical cations (i.e., a molecular fragment that is both a positively charged ion and a radical). A radical is a molecule or ion containing an unpaired electron. The radical ion then often undergoes intramolecular rearrangement and dissociation to produce a cation and an uncharged radical:

AB ^+• → A ⁺ + B ^•

Positive ions are drawn out of the ionization chamber by an electrical field and electrostatically focused and introduced into the mass analyzer. EI is primarily used as an ion source in GC-MS. Because the same ion energy (70 eV) is used in all commercial EI-GC-MS instruments and because the fragmentation pattern is only weakly dependent on small deviations from 70 eV, fragmentation patterns observed using an EI source are reproducible and relatively unique for each chemical compound. The fragmentation pattern is therefore often used as a fingerprint to identify compounds by matching mass spectra of unknown compounds to the entries in the mass spectral libraries.

Chemical ionization

CI is a soft ionization technique in which a proton is transferred (or abstracted from) through a gas phase by reaction with a molecule such as methane, ammonia (NH ₃ ), isobutane, or water vapor. The reagent gas is supplied into a CI ion source at a pressure of approximately 0.1 torr. ( N ote : For practical purposes, torr is equivalent to millimeter of mercury and is a unit commonly used in the field of MS). An electron beam produces reactive species through a series of ion-molecule reactions, resulting in formation of reactive intermediates (e.g., methonium [CH+5] if methane is the CI reagent gas), leading to ionization of the gas phase molecules, typically via attachment of a proton. In most cases, relatively little fragmentation occurs during this process, and for the majority of the molecules, only molecular ions (in the form of a protonated molecule) are observed in the mass spectra. The lack of fragmentation enhances sensitivity of detection, because the signal is not spread out over a large number of molecular fragments. Although this process enhances sensitivity, it does not allow one to obtain adequate mass spectral information to confirm identity of the analyte.

Negative ion electron capture CI has become popular for quantification of drugs, such as benzodiazepines. Negative ions are formed when thermalized electrons are captured by electronegative functional groups or atoms within the molecule (e.g., chlorine, fluorine). Negative ion CI often enables high sensitivity detection for the molecules, which may efficiently capture an electron.

Electrospray ionization

ESI, the most frequently used ionization technique in clinical MS, is a soft ionization technique in which a sample is ionized at atmospheric pressure before introduction into the mass analyzer. ^, An effluent from a separation device, typically an HPLC, is passed through a narrow metal or fused silica capillary to which a 1- to 5-kV voltage has been applied ( Fig. 20.7 A ). The applied high voltage causes mechanical instability in the liquid, leads to formation of an electrospray cone (also known as Taylor cone), creates aerosol and causes expulsion of charged droplets ( Fig. 20.8 ). In some variations of commercial ESI sources, a nebulizing gas aids in spray formation, and directs and speeds up the evaporative process. As droplets evaporate, while migrating through the atmospheric pressure region, they expel smaller droplets as the charge-to-volume ratio of the droplets exceeds the Raleigh instability limit, leading to expulsions of ions from the droplets. In most cases, the ionization process produces protonated species as the result of in-solution acid/base chemistry. However, other ionization products are sometimes observed, such as metal ion (Na ⁺ or K ⁺ ) or NH ₄ ⁺ adducts, or ions formed by redox processes. Ions then pass through an orifice, sampling cone and one or more extraction cones (skimmers) before entering the high-vacuum region of the mass analyzer.

One 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 to 15 amino acid residues in a protein. For example, for a molecule of mass 20,000, 20 charges supplied by the addition of 20 protons, resulting in an ion with m/z of approximately 1000 [or more correctly, m/z 1001 = (20,000 + 20)/20]. The phenomenon of multiple charging results in the formation of a series of peaks in the mass spectrum, with each peak corresponding to a different number of added protons and as a result, extends the accessible mass range of instruments. In addition to proteins and peptides, multiply charged ions are also observed for oligonucleotides in negative ion mode.

It should be noted that Figs. 20.7 and 20.8 represent 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 analyzer, many modern hardware configurations offset the probe relative to the orifice of the sampling cone; in most of the commercial instruments, the spray is orthogonal to the sampling cone.

ESI tends to be an efficient ion source for polar compounds or for molecules that are present as ions in solution, which includes a majority of biomolecules. ESI and APCI are the most commonly used ion sources in clinical applications of MS.

As already mentioned, ESI is considered a soft ionization source ; however, it is possible to generate fragment ions before mass analysis, by applying a higher than typical voltage gradient in the low-pressure region of the electrospray interface, causing collisional heating and fragmentation of the ions.

Atmospheric pressure chemical ionization

In APCI ion sources, as in ESI, the ionization takes place at atmospheric pressure, involves nebulization and desolvation, and uses the same design of the ion extraction cone as ESI. However, in APCI, no high voltage is applied to the inlet capillary. Instead, the mobile phase from the separation device gets evaporated and the vapor passes by a corona discharge needle. ^, Somewhat analogously to the processes occurring in a CI source, ions generated by the corona discharge undergo variety of ion-molecule reactions such as the following:

CH ₃ OH + H ⁺ → CH ₃ OH ₂ ⁺

A(analyte) + CH ₃ OH ₂ ⁺ → AH ⁺ + CH ₃ OH

H ₂ O + H ⁺ → H ₃ O ⁺

A(analyte) + H ₃ O ⁺ → AH ⁺ + H ₂ O

Because solvent molecules from the evaporated mobile phase (e.g., water, methanol, acetonitrile) are present in the vapor in excess, relative to the sample constituents, they are predominantly ionized early in the ion molecule cascade of reactions and then act as a reagent gas that reacts secondarily to ionize analyte molecules (see Fig. 20.7 B ). The products of these secondary reactions may contain clusters of solvent and analyte molecules. A countercurrent flow of heated inert gas, such as nitrogen, is applied in the direction opposite to the direction of the ions entering vacuum region of the mass analyzer, to assist with evaporation of solvent from the sprayed droplets and to minimize number of noncharged molecules entering the vacuum region of the mass analyzer. A decluttering potential is applied at the region where the pressure transitions from atmospheric to the vacuum. This potential causes acceleration of ions entering the vacuum region of the mass analyzer; when the ions acquire enough energy, the adducts break away from the ion, leaving a bare analyte ions.

As with ESI, APCI is a soft ionization technique, resulting in relatively little fragmentation; however, unlike ESI, APCI typically requires use of higher temperature, which may cause pyrolysis of the thermally labile compounds and may cause issues with quantitative performance of the assays (e.g., deuterium/ hydrogen exchange in deuterium labeled internal standard molecules).

When compared with EI, the mass spectra produced by APCI, ESI, and other soft ionization techniques typically have fewer fragments and are less useful for analyte identification. However, because of the little fragmentation, APCI and other soft ionization sources are well matched to the requirements of tandem MS (discussed later) and are well suited for quantitative analysis. APCI and ESI are the most commonly used ion sources for quantitative analysis in clinical MS. In the case of nonpolar compounds, such as many steroids and some drug molecules, APCI could provide a higher ionization efficiency than ESI.

Inductively coupled plasma

ICP, similarly to ESI and APCI, is an atmospheric pressure ionization method. However, unlike most atmospheric pressure ionization methods, which are soft (i.e., produce little fragmentation), ICP is the ultimate in hard ionization, typically leading to complete atomization of the molecules present in a sample during ionization. Consequently, its primary use is for elemental analysis. In the clinical laboratory, ICP-MS is particularly useful for trace element analysis in biological samples. ICP-MS is extremely sensitive (parts per trillion limits of detection) and is capable of a wide dynamic range of measurements.

After sample preparation, which typically includes the addition of an internal standard and in some cases an acid digestion step, the sample is introduced into the ion source, usually via a nebulizer fed by a peristaltic pump. The nebulized sample is transmitted into argon plasma, generated by inductively coupling power into the plasma using a high-powered, radiofrequency (RF) generator ( Fig. 20.9 ). The temperature of the plasma is typically 6000 to 10,000 K (comparable with the temperature on the surface of the Sun). The sample is introduced into the plasma, and the generated ions are transmitted into the mass analyzer. 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.

Compared with MS equipped with other atmospheric pressure ionization sources, ICP-MS is subject to less frequent occurrence of interference. Most interfering species in ICP-MS methods are polyatomic ions formed in the torch via ion-molecule reactions. For example, argon oxide (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 region in front of the mass analyzer, with a reactant gas, such as NH ₃ , supplied into the reaction cell. The reactant gas reacts with polyatomic interferences and fragments them before introduction into the mass analyzer. A related technique uses a nonreactive collision gas, which removes interferences using collisions, relying on differences in collision cross-sections between polyatomic ions and monoatomic ions. 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. For example, the masses of ArO ⁺ and ⁵⁶ Fe ⁺ differ by 0.022 Da—a difference that may be resolved using a high-resolution mass spectrometer.

Matrix-assisted laser desorption ionization

MALDI is another type of soft ionization technique in which ions are produced through energy transfer from a pulsed laser beam to the sample. Samples for MALDI ionization are prepared as dry spots, consisting of the sample mixed with a matrix (small molecular weight ultraviolet [UV]-absorbing compound), applied on a target and dried. A pulsed laser irradiates the dried spots, triggering ablation and desorption of the sample and matrix material; ions produced in the process are accelerated and enter into the mass analyzer ( Fig. 20.10 ). In other applications, a layer of the solid matrix is deposited on the target and allowed to crystalize, and then the sample is applied on top of the matrix. Application of the liquid sample causes partial solubilization of the matrix followed by recrystallization. With this approach, the sample is maintained in the outer layer of the matrix, in some cases allowing enhancement of the sensitivity and reduction of the background noise.

Ambient ionization

Ambient ionization (AI) is a type of ionization in which ions are formed directly from a sample, without, or after minimal sample preparation. Desorption electrospray ionization (DESI) was the first described AI technique. DESI uses an electrospray source to generate charged droplets directed to the tested sample; these charged droplets cause formation of secondary ions, which are introduced into a mass spectrometer. Since the time this technique was introduced, more than 50 other AI-MS approaches have been described. In the described AI methods, ions are formed by one of the following techniques: (a) thermal desorption from a sample, followed by CI; (b) laser ablation, accompanying by ionization; (c) deposition of a drop of solvent on the surface of a sample, followed by transfer of the droplet into an ion source and subjecting the droplet to ESI; or (d) introduction of a sample in plasma, which results in formation of metastable atoms and reactive ions.

One of the tasks performed by pathologists is microscopic evaluation of tissue cell morphology. AI-MS techniques enable imaging capabilities, which could allow determination of the molecular composition of tissue samples. Such information could be useful for evaluation of surgical margins during cancer surgeries, especially if performed in real time, while the tissue is resected. Several designs of such devices have been developed and evaluated.

Rapid evaporative ionization MS (REIMS or iKnife) was the first described AI-MS technique. Using this device, ions are produced during thermal ablation of the resected tissues and transmitted into a mass spectrometer in real-time during the surgery; the device allowed special resolution of 0.5 to 2 mm. In another design of AI-MS, a drop of water is applied on the tissue during a surgical procedure; after a brief exposure, the droplet is transferred into an ion source of a mass spectrometer, where the sample constituents subjected to ESI. In the evaluated prototype of the device, cycle time from sampling to obtaining the real time mass spectral information was less than 5 seconds.

In the studies performed to date, AI techniques allowed for discrimination between normal and abnormal tissues, with the following classes of molecules serving as biomarkers: (a) fatty acid, (b) lipids, (c) cholesterol metabolites, (d) carbohydrates, and (e) peptides. The ability of the AI-MS to determine marginal regions between pathologic and normal tissue was demonstrated for breast, brain, kidney, prostate, bladder, stomach, and colon cancer tissues. If the technique and the devices will prove to be sufficiently robust and will be commercialized, they could enable surgeons to use real-time information on tumor margins, while performing cancer surgeries, potentially leading to better treatment outcomes.