Dietrich A. Volmer

This tutorial illustrates the most important definitions used in high-resolution mass spectrometry and clarifies the misconceptions surrounding some of the relevant terms used in this field, such as mass resolution, mass accuracy, and mass defect. Examples are given for recent advanced applications of accurate mass measurements, mass defect labels for protein identification, and mass defect filtering techniques in drug metabolism studies.

Today's use of mass spectrometry (MS) is very different from applications just 10 years ago, because the availability of modern hybrid mass analyzers, such as quadrupole-time-of-flight (Q-TOF), quadrupole-Fourier-transform ion cyclotron resonance (FT-ICR), or linear-ion trap-orbitrap instruments, has led to a multitude of selective scan functions, data processing routines, and structural identification tools. Mass-based structural identification of unknown compounds such as drug metabolites from complex biological matrices often involves high-resolution MS measurements because tandem mass spectrometry (MS-MS) spectra from regular triple-quadrupole or ion-trap instruments usually do not confirm unambiguously the identity of a compound in a sample. A precise, high-resolution measurement of the accurate mass of the molecule, on the other hand, can provide the empirical formula of that compound, aiding the structural identification process tremendously. In these experiments, it is important to measure the mass-to-charge ratio (

) with the smallest mass uncertainty possible. Ideally, the analytical protocol provides sufficient resolution and mass accuracy to yield a single empirical formula for every compound analyzed by comparing the experimental results to the calculated exact masses. Of course, the situation becomes even more complicated when isobaric interferences are present because overlapping signals lead to shifts in the measured 

 values. Except for isomeric ions, isobaric signals in mass spectra will always lead to inhomogeneous peak broadening in the spectra because of the different mass defects of the elements, if the mass spectrometer has an insufficiently high resolving power. On the other hand, knowing the mass defect of expected compounds in a complex sample can be used to simplify the obtained results by using so-called mass defect filters. 

This last paragraph is probably confusing for beginners to MS, but the use of as many of the relevant terms common in high-resolution MS as possible was intentional. The underlying principles and theories are not trivial and often not fully understood, even by more experienced mass spectrometrists. For example, while measurements of high mass accuracy (i.e., how close is the experimentally determined accurate mass to the calculated exact mass) are performed almost always on instruments with high resolving power, it is sometimes possible to obtain very accurate mass measurements from low-resolution instruments. Moreover, high mass accuracy is available only from properly mass-calibrated instruments, while resolving very close species in a spectrum only requires sufficient resolving power. Simply stated, the use of high resolution alone does not guarantee accurate results. To correctly interpret the results from such experiments, it is vital to have a keen understanding of the theory and instrumentation involved in the process.  

It is the goal of the present article to provide a basic introduction to the most important definitions and principles used in high-resolution MS and to clarify some of the misconceptions surrounding these terms. Further, we will explain briefly the most common mass analyzers used for high-resolution mass measurements and describe some recent advanced applications of accurate mass measurements, such as mass defect labels for protein identification and mass defect filtering techniques used in drug metabolism studies. 

The unit of measurement used in MS is the unified atomic mass unit (u); defined as 1/12 the mass of a 

C atom is exactly 12 u. This selection is simply a convention and, interestingly, before 1961, 

O was used for the same purpose. Two important terms are 

. The nominal mass of a molecule is defined as the sum of the integer masses of the most abundant isotopes in a molecule. For example, let us consider molecular nitrogen, N

. These molecules have different empirical formulae; however, their associated nominal masses are the same: N

, 2 × 12 + 4 × 1 = 28 u. Mass spectrometers with insufficient mass resolving power such as quadrupole or ion-trap MS will not be able to distinguish these two molecules after ionization. The two resulting ions also are said to be 

The monoisotopic mass of a molecule differs from the nominal mass in that the sum of the exact masses of the most abundant (not the lightest!) isotopes of the constituent atoms is used instead of the integer masses. The exact mass of an atom is the result of the masses of its nucleons, while taking into account its nuclear binding energy (this will be addressed in detail in the next section). In the literature, the term 

 is used more commonly instead of monoisotopic mass. For the earlier example, the calculated monoisotopic (exact) masses are 2 × 14.00307 = 28.00614 and 2 × 12 + 4 × 1.007825 = 28.03130 for N

, respectively. It is clear that while these molecules have the same nominal mass, they have different monoisotopic masses. It follows that, due to the noninteger monoisotopic masses, different empirical formulas will never result in the same monoisotopic mass.  

 includes the weighted average of all the naturally occurring isotopes of the constituting elements of the molecule. For larger molecules, such as peptides and proteins, the average mass can be significantly larger than the monoisotopic mass. Importantly, the monoisotopic mass is not necessarily the most abundant peak in the mass spectrum. 

The mass defect is simply the difference between the exact mass and the nominal mass. This defect is characteristic for every atom. For example, the 

O atom is composed of eight protons, eight neutrons, and eight electrons with individual masses of 1.007276470, 1.008664904, and 0.000548579903 u, respectively, adding up to a total mass of 16.131919633 u, which is larger than the monoisotopic value of 15.994915 u. To explain this mass defect, we must look at the nuclear binding energy. Binding energy is required to separate the protons and neutrons in the atomic nucleus, and the observed mass defect arises from the relativistic loss of mass occurring when this binding energy is released during the formation of a stable atomic nucleus. Figure 1 illustrates the binding energy and the associated mass defect for the first 90 elements of the periodic table.  

The two forces acting on the protons and neutrons that compose the nucleus are the attractive nuclear force and coulombic repulsion. These forces act in unison and generate the resulting binding energy and observed mass defect. The nuclear force is a relatively strong attractive force that acts to bind together the protons and neutrons to form a stable atomic nucleus. Although it is a strong attractive force, the nuclear force has an effective range of only a few proton radii. Opposing the nuclear force is coulombic repulsion, existing between the positively charged protons. The coulombic force is substantially weaker than the nuclear force; however, it is effective over a much longer range. Coulombic repulsion increases quadratically with the addition of protons compared with the stronger nuclear force that increases linearly with increasing numbers of protons and neutrons. This interplay of forces allows the elements to be placed into two broad categories, as observed in Figure 1. Light elements with atomic numbers of less than 56 (iron) have nuclei in which the nuclear force is dominant, allowing for the formation of stable atoms (large binding energy). In this region, there is a rapid increase in the binding energy with each addition of protons and neutrons to form larger elements. Elements with atomic numbers greater than 56 have sufficient numbers of protons in their associated nuclei so that the repulsive coulombic force begins to destabilize the nucleus, in turn lowering the overall stability (binding energy) as the atomic mass increases.  

From Figure 1, we observe that the first five elements before 

C in the periodic table exhibit positive mass defects compared with the negative defects seen for larger atoms. This is simply the result of the selection of 

C as the reference point for the unified atomic mass unit. As we will see in a later section, this characteristic mass defect can be used as a means of identifying and characterizing specific molecules in complex samples. 

A slight variation of the mass defect definition leads to the 

. The Kendrick mass scale was first developed in 1963 as a way of simplifying the analysis of complex mixtures of organic substances containing compounds with extensive methylene (CH

) repetitions (1). To reduce the complexity of these data sets, a mass scale based upon CH

 is defined as exactly 14 u as opposed to 14.01565 u in the 

C scale. The Kendrick mass of a molecule is then calculated as follows: 

The Kendrick mass defect is the difference between the exact and the nominal Kendrick mass. The prime advantage of the Kendrick mass scale is that members of a homologous series differing only in the degree of alkylation will all exhibit the same mass defect. A plot of Kendrick mass defect versus nominal Kendrick mass greatly simplifies complex MS data by providing information about the class of compounds from the 

 axis. Marshall and coworkers have demonstrated a number of interesting applications of this technique in combination with high resolution FT-ICR data (Figure 2).  

Accurately Measuring Masses and Determining Elemental Formulae

 refers to how close the mass measured by the mass spectrometer comes to the calculated exact mass of an ion. Importantly, if the mass measurement can be made with sufficient accuracy, a unique empirical formula can be assigned. The number of possible empirical formulae calculated from the data decreases rapidly with increasing mass accuracy (Figure 3). In the absence of a unique empirical formula, analysis can be highly ambiguous. Often, the experimentally determined accurate mass measurements do not exhibit the required measurement certainty needed for unambiguous elemental formula assignment within a window of accepted mass uncertainties. In these cases, a series of potential molecular formulae are possible and it is usually left to experienced operators, or software programs, to select the most likely candidate by applying additional means of identification, such as isotope ratios.  

The mass accuracy of a measurement is reported commonly as a relative mass error or mass uncertainty (parts per million), as shown in the following equation:  

Unique elemental formulae often can be found for very small molecules with moderate mass measurement uncertainties because the relative error is mass dependent. This is illustrated by considering our now-familiar example of N

, differing by only 0.02516 u or 25.16 millimass units (mmu) between their calculated exact masses. At the nominal mass of 

 28 and considering C, H, N, and O as possible atoms in the positive ion mode, no species come within 400 ppm of each other (N

 versus CO), and there is almost 900 ppm between N

. The problem is, of course, that the required mass accuracy increases exponentially with increasing mass, as seen in Figure 3. 

Accurate mass measurements are performed most often using high-resolution instruments. High-resolution experiments do not, however, automatically guarantee accurate results. First of all, the use of high-resolution instruments can ensure that the peak associated with a particular 

 value is free of other interfering species, which could otherwise lead to peak shifts and inaccurate results. Importantly, accurate 

 measurement capability is possible only if the mass analyzer can resolve adjacent peaks in very complex samples. Figure 4 illustrates an example in which an FT-ICR instrument was able to resolve four adjacent species in an MS-MS spectrum. Sometimes even low-resolution mass spectrometers can be used for this purpose. Naturally, for an instrument to generate accurate mass data, the instrument must be properly mass-calibrated.  

The ability of an instrument to separate closely spaced peaks is termed 

 is calculated from the acquired data and is used to quantify the separation between peaks in a mass spectrum. In other words, an instrument can be said to have high resolving power, whereas the resulting spectra are said to be of high resolution.  

There are two common methods for determining the resolution in a spectrum. The first method is used for spectra generated on magnetic sector instruments: the 10% valley definition. In this definition, two peaks of equal intensity are said to be resolved when the overlapping region between the peaks is equal to 10% or less of the intensity of the original peaks. The resolution 

 is the mass difference between the two species. The second method, now routinely applied, uses the full width at half maximum (FWHM) of the peak. For FWHM calculations, resolution is reported as 

divided by the width of the peak at half its height. It is important to realize that for a given spectrum, the two definitions do not give the same values. For perfectly Gaussian peaks, FWHM values are 1.8× larger than those obtained from the 10% valley definition. 

The criteria for whether spectra are considered low- or high-resolution are not defined clearly. Generally, low-resolution spectra exhibit 

 < 2000, and high-resolution spectra show 

 > 5000. Of course, there is a trade-off between resolution and sensitivity, and the desired resolution usually is the minimum required to solve the specific problem under investigation. Modern high-resolution instruments can produce spectra with resolutions in the tens of thousands and in some cases, as we will see in the next section, the hundred thousands to million regions. 

Instruments for Accurate Mass Measurements

This section briefly summarizes the most important mass analyzers for high resolution and accurate mass measurements, namely, magnetic sector, time-of-flight (TOF), Fourier transform-ion cyclotron resonance (FT-ICR), and orbitrap instruments. The interested reader is referred to textbooks on mass spectrometry such as Gross's excellent 

 (2) for a more detailed description of the instruments. 

In the past, high-resolution experiments were carried out using double-focusing magnetic sector instruments, consisting of an electrostatic analyzer and a magnetic sector. The two sectors focus ions based upon different principles. The double-focusing instrument consists of an electrostatic analyzer operating as a kinetic energy separator coupled to momentum separation carried out via the magnetic sector. These instrument designs still are quite useful in specialized applications, and resolving powers >70,000 are possible with modern double-focusing machines with mass uncertainties between 1 and 5 ppm (3). 

The TOF mass analyzer is the most straightforward design among the instruments discussed here. This analyzer takes advantage of the fact that ions of different mass (

) will take differing times to traverse a certain distance, given that they both experience an equal initial accelerating force. Light ions will reach the detector first, with heavier ions requiring longer periods to travel the same distance. Modern TOF instruments usually are equipped with a reflectron to enhance resolution. Also, the TOF analyzer is often the final stage in the common hybrid design of Q-TOF instruments. Resolving powers of up to 20,000 have been demonstrated, and the mass accuracies can be within less than 5 ppm with appropriate mass calibration (2).  

The ion cyclotron resonance mass analyzer is based upon the trapping of ions using both magnetic and electrostatic fields. In FT-ICR mass spectrometry, a trapping cell is placed in the center of a spatially uniform magnetic field 

. This magnetic field is provided by a superconducting magnet similar to those used in nuclear magnetic resonance (NMR) spectroscopy. Ions are trapped by combined magnetic and electrostatic fields, which cause the ions to undergo cyclotron motion characteristic of their 

 value. The frequency of this cyclotron motion is measured and converted from the time domain to the frequency domain using the Fourier transform. Very high resolving powers have been reported (up to 3,300,000) with mass errors of often less than 1 ppm (4). 

The orbitrap is a relatively new instrument, and its operation is somewhat similar to that of the quadrupole ion trap. The orbitrap makes use of a static electrostatic field to trap ions, however, as opposed to the dynamic radio frequency field used in quadrupole ion traps. The orbitrap consists of a two-electrode system composed of a thin wire inner electrode and an electrically isolated coaxial outer electrode. A static electromagnetic field is generated, which traps the ions radially in stable trajectories. The motion of trapped ions can be described as a simple harmonic oscillator with a characteristic frequency that is dependent upon the 

 value of the ion. As with FT-ICR, the resulting time domain data are converted to a frequency domain spectrum using a Fourier transformation. Resolutions of up to 150,000 have been reported along with sub-part-per-million relative mass uncertainties (5). 

Although mass analyzers for accurate mass measurements are usually high-resolution instruments, sometimes low-resolution mass spectrometers can be used for measuring 

 values with high accuracies (6–8). Several studies have demonstrated relative mass uncertainties of <10 ppm for molecules with 

 between 200 and 6000 on triple-quadrupole and ion-trap instruments. Of course, because one never knows in advance whether potential interferences are present in unknown samples, only high mass resolving power can give sample-independent, reliable mass measurements. 

Some Interesting Applications Using Mass Defect

Mass Defect Labeling for Peptides and Proteins

Most peptides and proteins exhibit similar mass defect characteristics because amino acids contain principally only five elements: C, H, O, N, and S. Their mass defects are 0, 0.0078, -0.0051, 0.0031, and -0.0279, respectively, generating a net mass defect for a protein molecule. Of course, carbon does not affect the mass defect of a protein, and the combined mass defects of oxygen and nitrogen tend to cancel each other, resulting in virtually no net mass defect shifts. Furthermore, because sulfur is not very abundant in proteins, it does not influence the total mass defect greatly. As a result, hydrogen is the dominating element in the mass defect for the entire protein molecule. Proteins can have large mass defects, increasing by approximately 0.05 u for every 100 u increase in mass. Amster and coworkers have shown that despite these large mass defects, the distribution of masses is quite narrow (Figure 5). This example demonstrates that peptides are clustered tightly within each individual unit mass value. Because of the large amount of overlap within these clusters, the ability to unambiguously identify a peptide from accurate mass data alone is very difficult.  

Several methods have been developed to incorporate mass defect labels into either peptides or proteins, with the goal of altering the mass defect of a peptide through added groups that exhibit significant mass defects. The incorporated label will shift the mass to unpopulated regions of the unit mass scale, thereby reducing the complexity of these regions. This shift will decrease possible interferences and enhance the number of identified proteins. 

In the following section, we discuss two interesting examples for mass defect labels. The first involves bromine for shifting the mass defect of either peptides or intact proteins. In the second example, rare earth metals are utilized. 

Amster and colleagues developed a method for the identification of peptides generated from a tryptic digest of whole cell lysates of 

. In their experiments, the side chain of cysteine was derivatized with 2,4-dibromo-(2'-iodo) acetanilide. Figure 6a shows the distribution of the relative mass defects for all tryptic peptides. Figure 6b demonstrates the effect of cysteine labeling, with singly labeled cysteine-containing peptides centered on the left of the main distribution and the doubly labeled species on the right. This shift to unpopulated regions of the unit mass scale increases the number of identified derivatized peptides compared with underivatized peptides and should increase the number of identified proteins. To demonstrate the effectiveness of their technique, the authors examined the 

proteome using matrix-assisted laser desorption–ionization (MALDI) FT-ICR MS, which enabled them to identify 304 proteins in a cysteine-labeled sample compared with 268 proteins in an unlabeled sample.  

A similar technique was developed by Schneider and colleagues (9,10). Their technique was to label intact proteins with a bromine-tag by coupling the protein N-terminus with 3-bromo-1-(5-carboxy-pentyl)-pyridium bromide-NHS ester (BDOPP). This method differed from the previous method in that tandem MS experiments on the labeled proteins were used for protein identification. The technique was used for differential expression experiments, in which the ability to detect whether a protein in a sample is up- or down-regulated is probed through the use of isotopically pure "light" and "heavy" labels. In this case, the light label includes a 

Br. As with the previous method, proteins were detected after imparting a characteristic mass defect that aids in identification and helps to reduce chemical interferences. 

Finally, Meares and colleagues developed a label for cysteine side chains, utilizing bromoacetamidobenzyl-1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetra-acetic acid ligands bound to one of three possible rare earth metals: terbium (Tb), yttrium (Y), and lutetium (Lu) (Figure 7). After derivatization, the labeled peptides were first bound to an antibody column, allowing the removal of interferences from unlabeled species. After elution, the labeled peptides were submitted to MS analysis. As with the previous methods, the rare earth element labels shifted the mass defect of the peptides by a characteristic amount. Because three different metals were available, the resulting mass defect could be fine-tuned depending upon the complexity of the biological samples.  

Articles

This tutorial illustrates the most important definitions used in high-resolution mass spectrometry and clarifies the misconceptions surrounding some of the relevant terms used in this field.

Dealing With the Masses: A Tutorial on Accurate Masses, Mass 32 Uncertainties, and Mass Defects

Mass spectrometry (MS) instrumentation has undergone several improvements over the years, including increased sensitivity, ease-of-use, and possibilities for more sophisticated and time-efficient experiments. This article illustrates the technical principles and typical applications of the most common mass analyzers used in bioanalytical laboratories today. There are four basic types of mass analyzers found in modern MS: time-of-flight (TOF), quadrupole and a recent derivative, quadrupole linear ion trap (LIT), which were covered in Part I of this series (1), and quadrupole ion trap (QIT), and Fourier transform ion cyclotron resonance (FT-ICR), which are discussed in this installment. Rather than simply explaining the underlying physical principles, we concentrate on each mass analyzer's characteristic features, their limitations, and their applications. In addition, several unique techniques and unusual or exotic mass analyzer designs are highlighted throughout the text. We have chosen examples from our own research in small molecule applications. Therefore, some interesting recent hybrid designs (with mostly proteomics applications) only are described briefly. 

The analyzers described in Part I of this article are ion beam-type instruments (except for the linear ion trap). In this installment, we will concentrate on ion-trapping devices, which allow the storage of ions using electric potentials (ion trap) or magnetic fields (ion cyclotron resonance, ICR) with subsequent mass analysis in the same space. This section explains the principles of quadrupole ion trap (QIT) MS. 

The ion trap can be envisioned as a regular linear quadrupole wrapped around itself. It is composed of two end-cap electrodes and one ring electrode. By applying appropriate potentials on these electrodes, a pseudo-potential well, to which ions are confined, is formed within the trap, and ions can be stored for long intervals in this instrument. The ion trap is kept at high pressures relative to other mass analyzers, because of the presence of a damping gas, usually helium, which causes collisional cooling of the analyte ions with subsequent confinement in the center of the trap. The usual pressure inside the trap is ~10

Mass analysis in the ion trap is achieved by means of the so-called 

. In the mass selective instability scan, ions of specific 

 values are ejected from the trap selectively by linearly increasing the rf amplitude on the ring electrode. Detection of the ions occurs following the ejection process. The resonance ejection technique extends the mass range of the ion trap by exciting the ions in the trap via a supplemental potential applied to the end caps. If the amplitude of this additional potential is large enough, ions are ejected from the ion trap at ring electrode rf voltages lower than those required for the mass selective instability scan. 

Furthermore, specific scans can be used selectively to eject all unwanted ions from the trap. A selected precursor, for instance, can be isolated by applying a broadband waveform consisting of all frequencies except that of the secular frequency of the ion of interest. It is important to realize that an experiment on an ion trap is composed of several steps occurring sequentially, in the same space. This chain of events is called a 

. The separated time periods include ion injection, isolation, excitation, and analysis. In addition, very often an initial pre-scan is performed to determine the ideal injection time, thereby reducing 

 effects. This is important because a fundamental limitation of the ion trap is the dependence of ion stabilities upon the number of ions present in the trap. As the amount of ions is increased, space charging becomes an important factor. The electrostatic field is distorted by having too many ions in the trap, thus causing broadening of peaks, mass scale shifts, and loss in sensitivity. Eventually, ions are no longer trapped when the ion density becomes too large. Therefore, the pre-scan assesses the amount of ions entering the trap, and certain parameters, such as the injection time, are adjusted consequently to correct for possible space charging. Interestingly, quadrupole linear ion traps (1) also exhibit space charge effects, but they have larger ion storage capacity (2). 

After the ions are injected into the trap from the source, a suitable rf voltage on the ring electrode confines them to stable trajectories. An isolation scan can be performed subsequently in order to accumulate a specific ion or a range of ions selectively. The next possible step is ion excitation, in the case of tandem MS. Finally, ions are analyzed by ejection from the ion trap to the detector through openings in the end caps. Figure 1 exhibits representative scan functions in an ion trap. The type of scan dictates the length of time spent on each step. The differences in the operation of ion traps and quadrupoles have a direct effect upon the duty cycle of the instruments, and we see that ion traps are very sensitive in a full-scan experiment, but do not gain very much sensitivity, as one would imagine, using the selected ion monitoring (SIM) mode. On the other hand, a SIM experiment in a quadrupole instrument has much higher sensitivity than it does for a full scan experiment (1). 

 Figure 1. The events during an QIT micro-scan are: 1. ion injection, 2. isolation, 3. excitation, 4. mass analysis. (a) Full-scan MS: a waveform is used to eject unwanted ions from the trap and to isolate a mass range of interest during ion injection; the rf voltage is ramped during mass analysis to eject the ions out of the trap sequentially. (b) SIM: a waveform is used to isolate a narrow m/z range of interest [the waveform has a notch present, corresponding to that of the secular frequency of the ion of interest] for selective accumulation. All unwanted ions are removed by resonant ejection; the rf ramping during mass analysis is very short for the limited m/z range. (c) MSn: a waveform is used to isolate only the precursor ion; the resonance excitation step dissociates the precursor ion using an auxiliary rf voltage at the end caps. Product ions are analyzed subsequently.			

Further important differences between ion trap and quadrupole MS are seen in tandem MS experiments. The QIT can be described as a 

 for a triple quadrupole) and allows multiple stages of tandem mass spectrometry (MS

 ), whereby precursor ions can be activated selectively and dissociated to form product ions. The product ions are detected by subsequent ejection from the trap. The ion activation of the isolated precursor usually is achieved via "on-resonance" excitation by applying a small (tickle) potential, corresponding to the secular frequency of the ion, across the end caps. As ion activation times in the order of tens of milliseconds can be used without significant ion losses, multiple collisions occur during the excitation period. Because of this relatively long time-scale, this excitation technique falls in the category of so-called "slow heating" processes (3). For a slow-heating process, excitation in an ion trap still is quite fast, due to the high pressure of helium present in the trap. There are other slow-heating methods, such as "sustained off-resonance irradiation" (SORI, see next paragraph) with longer excitation periods. MS

  experiments are quite advantageous for the elucidation of fragmentation pathways, which in turn aid in the analysis of CID spectra for identifying molecular structures for precursors as well as product ions. The results are, in principle, similar to those obtained in quadrupole linear ion traps. 

Figure 2. Extremely high resolution can be achieved using an FT-ICR mass spectrometer. This figure shows a small m/z range from a crude phytoplankton extract analyzed in full scan on a 9.4-T FT-ICR. Under lower resolution conditions, these three peaks would coalesce into one unresolved peak.			

Sometimes, mass spectrometrists point out that ion traps yield CID spectra with only a limited number of fragment ions, in comparison to triple quadrupole spectra. This is, in fact, quite true and often one needs to conduct several stages of MS

  to generate a sufficient number of product ions. The reason for this limitation is, of course, based upon the resonance excitation used for ion activation, which does not excite the product ions, because of their different secular frequencies (that is, they are no longer "in resonance" with the excitation frequency and therefore do not pick up further energy). In fact, molecules that tend to cleave off a large number of unspecific small molecules (mostly H

O) are sometimes difficult to analyze by MS

 , because structurally informative backbone cleavages are hard to reach. The selective activation procedure, however, offers a tremendous advantage with its ability to yield exact relationships between precursor and product ions, allowing the "genealogical" mapping (4) of the entire fragmentation process. Triple quadrupole CID experiments, on the other hand, are much less selective, as the product ions tend to undergo further collisions, to give additional second or even higher-generation product ions. The linked information between precursor and different generation product ions is difficult to determine in such cases. 

The analytical figures of merit of QIT mass spectrometers are somewhat comparable to quadrupole mass analyzers (that is, mass ranges of up to 

 4000 can be analyzed, at unit mass resolution). Higher mass resolution can be achieved with special slow scanning routines, for much smaller 

 ranges. The detection sensitivity, as outlined earlier, generally is very good in the full-scan mode because of the ion-trapping abilities. The duty cycle in the SIM or MRM modes of the QIT-MS is much lower than on the quadrupole MS, because of the significant overhead contributions to the microscan sequence (Figure 1). The sensitivity enhancements in the SIM mode in comparison to the full scan mode, therefore, are not very pronounced.  

Finally, the QIT also has been combined recently with a TOF analyzer (5). In QIT-TOF, the QIT is used as a precursor ion selection and/or MS

  stage, prior to high-resolution, accurate mass TOF analysis. Such an instrument allows accurate mass data to be obtained for product ions of each individual stage of MS

 . This instrument originally had been available only with a MALDI source; therefore, most current applications include large molecules such as proteins and polysaccharides. 

The Fourier Transform Ion Cyclotron Mass Analyzer

FT-ICR MS has experienced a remarkable increase in use by analytical chemists recently. This instrument was employed historically for fundamental physics and physical chemistry applications. Recently, however, new developments such as efficient ion-transport devices have allowed the use of external ionization sources (particularly ESI and MALDI), significantly increasing the versatility of FT-ICR mass spectrometers. Furthermore, quadrupole and linear ion-trap analyzers have been implemented for precursor ion selection and collisional activation, before FT-ICR mass analysis. Consequently, FT-ICR has become an invaluable tool for biological MS, primarily for its flexibility in ion-activation techniques available and its extremely high resolution and mass accuracy.  

While the principles of mass analysis by ICR date back to 1932, it was not until 1974 that Marshall and Comisarow (6) added the use of Fourier transform pulse techniques to ICR. Before we illustrate the unique properties of FT-ICR MS, a basic description of this analyzer allows one to understand how it achieves these important advantages over other instruments. For readers interested in a more thorough treatment of ICR fundamentals, we suggest reference 7. 

 entering the magnetic field, will bend the ion's path into a circular motion, as follows: 

 are vectors, perpendicular to each other, 

 is the balancing centripetal force. If the ion is under extremely low pressure conditions, it does not experience any collisions with residual gas molecules in the cell. Consequently, the speed of an ion in the FT-ICR cell is constant. With ω= 

, it follows that the angular velocity is given by 

This is the fundamental equation for FT-ICR, where the ion's 

 value can be directly measured by its ion cyclotron frequency ω

 and no dependence on the ion's initial velocity upon injection into the cell (that is, no energy focusing is required for the precise determination of 

). The ion's cyclotron frequency ranges from kHz to MHz values, depending upon its mass, for small organic molecules to biomolecules in the kDa range. It also is informative to look at the radius of the cyclotron motion, 

 (see Equation 1). As illustrated by Marshall and coworkers (7), cyclotron radii between 0.08 mm for 

 10,000 are assumed by the ions at room temperature and a magnetic field strength of 3 T (tesla). 

In order to measure the cyclotron frequency, and thus the mass, of an ion (via Equation 2), ions must be accelerated coherently to a new radius. This is required because initially, not all ions are at the same point on their circular orbits. Therefore, their motions are incoherent, and their frequencies thus cancel out and cannot be measured. For all ions of a specific 

 value to become coherent, an rf field, rotating with the ICR frequency of the specific ion, is applied. After this excitation, the radius of the ion no longer depends upon its 

 is the peak-to-peak magnitude of the rf electric field, 

 is the distance between the plates, and 

 is the excitation time. Note that by exciting all ions of different 

 values with a broadband rf field, they all end up at the same radius after the excitation process. The resulting ions all have become spatially coherent, but ions of different 

 values still have a characteristic cyclotron frequency. Each 

 value can be differentiated based upon the speed at which they orbit the common radius occupied by all ions. 

A unique and quite interesting feature of FT-ICR mass spectrometers is the way the frequency measurement is performed. FT-ICR analyzers do not have a detector, where ions are discharged physically, as is the case with most other mass spectrometers. Rather, detection is based upon measuring the 

 that individual ion packages induce when they pass the detector plates repeatedly, at their cyclotron frequencies. In a typical FT-ICR experiment, the ions in the cell are excited simultaneously, and a composite image current for all ions is measured. Next, this transient signal is converted from the time domain to the frequency domain using a fast Fourier transform algorithm. The individual frequencies then can be transformed to 

 values by means of Equation 2. Note that this method of detection is non-destructive. Ions can be measured for extended transient times, if required, thereby increasing the mass resolution. The transient signals diminish eventually, because of collisions in the ICR cell slowing down the orbiting ions. 

Hybrid Instruments for Biological Applications			

It is evident that the strength of the magnetic field is an important factor in several parameters. First, the initial radius of an ion is inversely proportional to 

, so that for a higher magnetic field, a higher mass range can be analyzed. Ions of larger 

 occupy larger radii and there is a mass range limit based upon the size of the ICR cell and the magnetic field. Furthermore, as 

 increases, so does the cyclotron frequency of an ion, indicating that higher magnetic fields yield higher mass resolution. However, there is a reduction in the lower limit of 

 values with a higher magnetic field, because their frequencies become correspondingly higher and thus more difficult to measure. The most striking feature of FT-ICR MS is its outstanding mass resolution, significantly larger than any other MS technique available currently. (Figure 2 shows an illustrative example for biotoxins; additional detail is given in the sidebar "Mass Resolution Versus Mass Accuracy.") As seen with quadrupole ion traps, FT-ICR also is affected by space charge effects, if too many ions are present in the cell. The interactions between the ions lead to dephasing of the ion cloud and peak coalescence.  

As mentioned earlier, one of the primary advantages of FT-ICR is the flexibility in ion activation techniques for MS-MS experiments available. As isolation and excitation take place in the same confined space, this instrument belongs to the tandem-in-time category. SORI often is applied for collisional activation in FT-ICR instruments. In SORI, a neutral gas pulse is introduced into the ICR cell and the precursor ion is excited at a frequency slightly higher than the natural cyclotron frequency. Ions undergo multiple acceleration/deceleration cycles as they increase and decrease their orbit radii repeatedly in the FT-ICR cell before dissociation takes place. The activation times often are in the hundreds of milliseconds, sometimes up to seconds, and a large number of collisions take place. This approach is somewhat similar to quadrupole ion trap MS

  experiments. An alternative to SORI is 

 (IRMPD), which is available for all commercial FT-ICR instruments. IRMPD activates ions in the ICR cell by irradiation with a low-power CO

 infrared laser (10.6 µm) for tens to hundreds of milliseconds per experiment, resulting in the stepwise absorption of photons, followed by subsequent dissociation of the ion. This process is rather non-selective, therefore, all trapped ions are excited and secondary product ions also can be observed. This feature of IRMPD can be convenient, because the resulting spectra often are similar to low-energy CID spectra measured on triple quadrupole instruments (8). Finally, a fundamentally different technique of activation also is available on all commercial FT-ICR instruments, 

 (ECD). ECD involves the capture of low-energy electrons, with charge-state reduction and subsequent fragmentation. It is therefore only applicable to multiply charged cationic species, usually peptides or proteins. ECD is assumed to occur much faster than dissociation in other activation techniques, permitting the occurrence of direct bond cleavages only. 

As with other hybrid mass spectrometers, MS-MS experiments are conducted increasingly outside the ICR cell, either with a linear ion trap or a Qq arrangement, coupled to the FT-ICR mass analyzer (see the sidebar "Hybrid Instruments for Biological Applications"). They offer increased compatibility with fast chromatography runs, as ion activation experiments directly in the ICR cell often take too long to be compatible with chromatography experiments. 

In this tutorial, we have presented a short overview of the operating principles and selected applications for the most common mass analyzers used in biological MS today, namely TOF, quadrupole, ion trap, and FT-ICR. As illustrated, the way these analyzers function and their characteristic features are quite different. The TOF analyzer is an ideal match for pulsed-ion sources such as MALDI. Because of its high speed, it also is very useful for extremely fast chromatography applications. It offers high transmission efficiency and a very high mass range. On the other hand, the TOF has limits with respect to duty cycle, dynamic range, and precursor ion selection in MS-MS. Quadrupoles are rugged and versatile. They can be used as mass filters, ion guides, and collision cells. On the other hand, hyphenation with pulsed-ionization sources is less than ideal and their resolving power is limited. Quadrupole ion traps offer high sensitivity in the full-scan mode as well as the ability to perform multiples stages of MS-MS. Similar to quadrupoles, the resolving power is limited. In addition, careful tuning of multiple operating parameters is required to avoid space charge effects, to obtain meaningful MS-MS spectra, and to use it in quantitative applications. Finally, the FT-ICR offers the highest resolving power and mass accuracy of any analyzer available today. Because the ion-detection method is non-destructive, remeasurements for extremely high sensitivity, resolution, and accuracy are possible readily. FT-ICR is very flexible with respect to ion-activation techniques as well. To obtain reliable results, however, it requires careful adjustment of many operational parameters. The hyphenation with chromatography is more complicated because the scan sequences for high-resolution spectra can be time-consuming, sometimes too slow for acquiring a sufficient number of data points across chromatographic peaks. For all the analyzers, we also have shown that there is an increasing trend toward hybrid designs to increase versatility by combining the intrinsic advantages of the individual analyzers. 

Finally, we cannot finish this article without mentioning an exciting new type of mass spectrometer that has recently emerged, promising resolving powers similar to FT-ICR without the need for superconducting magnets. This mass analyzer is called the orbitrap, and its operating principles are summarized in the sidebar "The Orbitrap Mass Analyzer." 

2. D.J. Douglas, A.J. Frank, and D. Mao, 

4. R.J. Strife, L.C. Robosky, G. Garret, M.M. Ketcha, J.D. Shaffer, and N. Zhang, 

The second installment of this two-part series illustrates further technical principles and applications of the most common mass analyzers used in bioanalytical laboratories today, as well as novel techniques and mass analyzer designs. Examples are based upon the authors' research in small molecule applications.

Tutorial — Mass Analyzers: An Overview of Several Designs and Their Applications, Part II

Mass spectrometry (MS) instrumentation has undergone several improvements over the years, including increased sensitivity, ease-of-use, and possibilities for more sophisticated and time-efficient experiments. These developments have led to an increase in the number of biological applications. In the 1990s and until recently, new instrumental innovations in organic MS had focused mainly upon front-end techniques — ionization interfaces specifically. In particular, electrospray ionization (ESI), atmospheric-pressure chemical ionization (APCI), and matrix-assisted laser desorption–ionization (MALDI) have evolved from these developments and have seen significant performance improvements over the years. Along the way, a few other interesting liquid chromatography–mass spectrometry (LC–MS) interfaces (for example, thermospray, particle-beam) have paved the way for modern LC–MS, but have then disappeared quickly from research laboratories. Also, a promising new ionization source, atmospheric-pressure photoionization (APPI) was introduced just a few years ago. As a result of all these innovations, rather good and stable MS sample introduction devices are available for a wide range of compound classes and most researchers seem quite satisfied with what they presently have available at their disposal. Continuing research on these ionization sources currently is aimed at enhancing the performance of such devices, through miniaturization, automation, and parallel-analyses. Lately, however, MS instrumentation research is dominated by improvements in the performance of the actual mass analyzers. Moreover, there is a growing trend toward combining different analyzer designs in order to increase the versatility and allow multiple experiments to be performed simultaneously using a single instrument. One of the most recent examples of such a novel hybrid instrument is the linear ion trap-Fourier transform ion cyclotron resonance mass spectrometer (LIT-FT-ICR), which will be discussed subsequently in more detail. This instrument allows fast, multistage tandem MS (MS-MS) experiments as well as ultra-high resolution and accurate mass determinations. The driving force behind this recent trend of improving analyzer designs mainly has been scientists from biotechnology and pharmaceutical research laboratories. Their time is costly and their analytical protocols now routinely include the use of multiple MS instruments. 

This article illustrates the technical principles and typical applications of the most common mass analyzers used in bioanalytical laboratories today. The classical mass spectrometer — the magnetic sector instrument — is not covered by this tutorial. Although these instruments allow very selective scan modes, their implementation is much less routine and is beyond the scope of this article. Moreover, magnetic sector instruments seem to have reached the apex of their technological development, while improvements in instrument technologies based upon other mass analyzers continue to evolve and develop in new directions. Magnetic sector instruments still are employed for specialized analyses, for example, in the petroleum industry or for the analysis of dioxins by gas chromatography–mass spectrometry (GC–MS). 

There are four basic types of mass analyzers found in modern MS: time-of-flight (TOF), quadrupole and a recent derivative, quadrupole linear ion trap (LIT), quadrupole ion trap (QIT), and FT-ICR. The first part of this article will cover TOF and quadrupole analyzers; Part II will address QIT and FT-ICR. More interestingly, however, there are several very advanced permutations of these analyzers, which through the combination of different analyzers create enhanced MS-MS capabilities, novel scan modes, or superior mass resolution and accuracy. For example, the quadrupole-time-of-flight (QqTOF) instrument has developed into one of the most successful hybrid MS designs, allowing the generation of triple quadrupole–like MS–MS spectra, combined with high resolution and accurate mass capability in the TOF analyzer. This combined potential, together with flexible data-dependent acquisition routines, has been proven to be of significant importance for proteomics MS. Rather than just explaining the underlying physical principles, we will concentrate on their characteristic features, their limitations, and their applications (more details, in particular on the fundamental operating principles, can be found in textbooks on mass spectrometry; for example [1]). In addition, several unique techniques and unusual or exotic mass analyzer designs are highlighted throughout the text. We have chosen all of the examples from our own research; as a result, this overview is biased toward applications to small molecules. Therefore, some interesting recent hybrid designs with mostly proteomics applications, such as ion trap-TOF, only are described briefly. 

A linear TOF mass spectrometer is the simplest mass analyzer that exists today, consisting only of an ion-accelerating region, a flight tube, and a detector. These linear instruments are used primarily in combination with the MALDI technique for analysis of large biomolecules. In theory, all ions experience the same potential difference during acceleration and thus have the same kinetic energy at the start of the flight tube, and thus different velocities depending upon their mass. Therefore, their arrival time at the detector is proportional to their mass and they reach the detector in order of increasing mass. The mass-to-charge ratios (

) of the ions relate to the flight times 

 is the acceleration potential. For example, the electrosprayed antibiotic ciprofloxacin (MW = 331 g/mol; MH+  at 

 332) arrives at the detector 9.3 μs after being accelerated with 20 kV into a 1-m long flight tube, whereas the much heavier singly protonated bovine insulin molecule (

 5734) needs 65.1 Âµs to reach the end of the tube. These flight times translate into the 

 values via Equation 2. The differences of flight times are, of course, the basis for resolving ions of different 

 in the TOF analyzer and the mass resolution depends on the flight time differences, which are proportional to 

The major deficiency of a simple linear TOF instrument is its insufficient mass resolution, resulting from flight time variations of ions of the same 

. First, the laser pulses used for ion generation in MALDI-TOF instruments are fairly long, resulting in a wide distribution of starting times of the same ions. Further, the ionization process adds a certain amount of initial kinetic energy to the molecules before acceleration. Finally, the different spatial positions in the source from where the ions are formed lead to varying values of 

 (Equation 2) and thus to flight time variations. As a result, modern TOF instruments commonly employ two techniques to enhance resolution, 

A reflectron TOF (RETOF) is used to focus ions of the same 

 but of different kinetic energy. There are different designs of reflectrons but often they consist of a series of rings or grids. It is located after the drift tube, creating a retarding field that the ions penetrate. Depending upon their kinetic energy, they enter this field at different depths and then are reflected back into the flight tube, where they drift to the detector, which is placed close to the ion source. Consider the case of ions of the same 

 but significant energy spread, which would be detected as broad peaks in the linear TOF: the ion with a higher kinetic energy penetrates deeper and spends more time in the reflectron. The slow ion, on the other hand, returns to the flight tube faster, but with the same lower kinetic energy. The faster ion will catch up with the slow one at the detector, thereby improving mass resolution greatly. 

The delayed extraction technique can be used on both linear and RETOF instruments, to compensate for the velocity distributions of ions. In this technique, which also is sometimes called 

, ions are allowed to drift free of electric fields after formation for a certain delay time. The ions then are accelerated into the flight tube. By allowing them to drift, faster ions move farther away from the sample target than the slower ions. After the delay, these faster ions then will experience less of the accelerating voltage between the sample target and the extraction plate than the slow ones. This procedure compensates for the initial energy distributions of ions with the same 

There are several benefits that TOF analyzers offer when combined with ionization sources such as electrospray or MALDI. In addition to the high mass range that can be analyzed (with a linear TOF instrument) and the high ion transmission, there is the unique advantage of quasisimultaneous detection of all ions, resulting in high sensitivity "full-scan" analyses (of course, the term "full scan" obviously is wrong in the context of TOF-MS, as a TOF-MS is not a scanning device, but the common terminology is used here for the sake of convenience). Thus, they are ideal in qualitative applications such as the identification of unknown compounds, in which the acquisition of an entire mass spectrum is required. Figure 1 shows an example for the analysis of metabolites of an anticancer drug, analyzed on an ESI-TOF instrument, demonstrating the high resolving power and mass accuracy of a TOF analyzer. In addition, TOF analyzers exhibit very fast acquisition times and thus are ideal for hyphenation with capillary electrophoresis and fast LC. Of course, the pulsed nature of TOF analysis makes the hyphenation with a continuous ion source such as electrospray more difficult than with MALDI, which inherently is compatible with TOF analysis. The necessary "slicing" of the ion beam coming from the ESI source can be performed axially or orthogonally. For organic LC–MS, the design is usually an orthogonal acceleration (oa) TOF type. The ions are focused into the orthogonal accelerator as a narrow ion beam and a slice of it is pushed down into the TOF tube. The amount of ions entering the TOF tube (that is, the length of the "slice") relative to the total amount of ions in the orthogonal accelerator determines the 

 of the instrument (see the sidebar "Duty Cycle in MS" for further explanation). After the heaviest ions have reached the detector, the next pulse pushes another slice of ions into the drift tube. 

 Figure 1. Metabolism of doxorubicin in mouse liver microsomes was analyzed by ESI-TOF for the determination of accurate masses of the metabolites formed in vitro. Idarubicin was used as an internal standard in this experiment and two main metabolites were characterized with the aid of the high resolution afforded by the TOF mass analyzer (with internal calibration).			

The downside of TOF applications is the limited dynamic range; that is, the ratio of the maximum to the minimum observable ion intensities or concentrations over which a linear response is obtained from the detector. This is the direct result of the types of detectors that are employed for handling the large number of high-resolution spectra, making the application of TOF instruments to quantitative analyses less attractive. There is another weakness encountered with TOF analyzers: it is technically difficult to select a precursor ion for MS–MS experiments. In MS–MS, a specific precursor ion is isolated and then made to undergo dissociation to fragment ions, which are analyzed subsequently to obtain structural information. It is important for the precursor ion of interest to be isolated from interfering species; in other words, one needs to be able to select a very narrow 

 range. In a RETOF instrument, a TOF gate can provide only a very coarse precursor selection (over several 

 units wide), to study metastable compositions. This technique is used in combination with postsource decay (PSD) and is quite popular for sequencing of peptides. 

It is much more elegant, however, to utilize a separate mass spectrometer for precursor ion selection, allowing the isolation of a single 

 value. One option is to use another TOF drift tube together with a time selector, and such a design is available commercially for specialized proteomics applications (see the sidebar "TOF–TOF Instruments for High Energy CID"). Much more common, however, is the implementation of a quadrupole mass filter prior to the TOF tube. In fact, this can be taken one step further, by adding a second, radio frequency (rf)-only quadrupole as a collision cell (Q

 TOF). The TOF analyzer then is used to provide accurate mass data for fragment ions formed in q

 . Before we describe the QqTOF instrument, however, we will take a look at the basic operating principles of a quadrupole. 

TOFâTOF Instruments for High-Energy CID			

The quadrupole is the most common analyzer found in analytical laboratories today. It is not only the standard analyzer for GC–MS (which will not be discussed here) but also is part of various designs of LC–MS instruments. Quadrupoles are not used only as mass analyzers; they also are implemented frequently as ion transfer optics, collision cells, and linear ion traps (

). This mass analyzer works on the basis of electric fields generated between a set of four axial rods through which ions pass on their way to the detector. The voltages applied to these rods consist of direct current (dc) and rf components, which together create a quadrupolar field, allowing only a certain 

 range to pass through for a given combination of potentials; ions outside that 

 region hit the rods and are discharged. The quadrupole therefore is referred to also as a 

, into which ions are accelerated from the ion source by a small potential. Positive ions will be attracted to negative rods, and vice versa. The rf component, however, causes the electric field to alternate at a frequency in the megahertz range. The applied potential to the rods is of the form: 

 is the rf potential of frequency ω/2π. Two rf waveforms are applied to two pairs of opposing parallel rods, with a 180° phase shift. As a result, the trajectory of an ion fluctuates constantly as it travels between the rods until its detection.  

In a full-scan experiment, the dc and rf components are ramped at a constant ratio, and ions entering from the ion source are enabled to pass through the rod assembly successively. For a specific combination of dc and rf, only a very small 

 range can pass through, the size of which determines the resolution of the instrument. Quadrupoles usually are operated at unit resolution throughout the mass range (often up to 

 1500 and 1501, for example. Higher mass resolution, however, is available on a recent commercial quadrupole instrument produced by Thermo Electron (Waltham, MA). The sequential detection in the full-scan mode of any quadrupole analyzer results in a low duty cycle, typically of the order of 0.1%, depending upon the monitored 

 range. The duty cycle (and the resulting sensitivity) is much higher in the 

 (SIM) mode of the quadrupole (close to 100%). In this case, the dc and rf potentials are held constant, so only a specific 

 ratio can pass through. One also can jump between different voltages, to allow more than one 

 ratio to be detected sequentially, with a corresponding reduction in duty cycle. 

Single quadrupole instruments usually are limited to measuring intact species generated by the ionization source, resulting in limited selectivity in comparison to many of the advanced designs described in the following. They have, nevertheless, found a wide application range in LC–MS and GC–MS. Such instruments often are considered more of a selective HPLC detector today than a mass spectrometer. Typical applications of single quadrupoles include purity assessments, molecular weight confirmations, and automated preparative HPLC fractionations. Figure 2 shows an example for a mass-triggered fractionation experiment from a semipreparative HPLC run, where a single quadrupole was used to supply trigger signals to the fraction collector, to start collecting the chromatographic peaks of interest. 

Figure 2. Mass-triggered fractionation chromatogram using a single quadrupole instrument set-up with specific m/z values for triggering the fraction collector. A crude phytoplankton extract was purified into several fractions for further analysis of marine toxins by mass spectrometry. Arrows above show the start of individual triggers (with the m/z responsible for the trigger).			

In a single-quadrupole instrument operating with an ESI or APCI source, selectivity can be increased when fragmentation is induced in the ion source region ("in-source CID"). This procedure refers to the activation of ions in the region between the ion source and the analyzer, in which fragmentation can be initiated by collisions with residual gas molecules at intermediate pressures. The generated fragment ions can be used for limited structure elucidation or confirmation purposes. One must be careful, however, as no precursor ion selection takes place and fragment ions cannot be linked specifically to the precursor ion of interest. In other words, fragment ions could originate from several compounds for coeluted peaks in LC–MS, making structure assignments difficult (similarly, quantitative analysis in the SIM mode would be difficult when isobaric interferences contribute to the chromatographic peaks). 

The most common mass analyzer for quantitative bioanalytical analyses is the 

. This MS–MS instrument consists of three sequential quadrupoles (Q

 ; Q refers to a mass-resolving quadrupole, q to an rf-only quadrupole; 

). This configuration allows additional ion activation in q

 , after the ion of interest has been selected in Q

  is operated in the rf-only mode, in which only an rf voltage is applied to the quadrupole rods (no dc component), thus effectively becoming a wide-band pass for the ions. It can be filled with a neutral gas such as N

  or Ar, acting as a collision gas. The ions leaving Q

  with offset voltages between 0 and 100 V, typically. The results of the 

 (CID) can be analyzed with the mass analyzer Q

 . This is the simplest form of an MS–MS experiment on a triple-quadrupole mass spectrometer, and it is called a 

. In addition, several other selective MS–MS modes are available, depending upon which mode Q

This tutorial illustrates the technical principles and typical applications of the most common mass analyzers used in bioanalytical laboratories today, as well as novel techniques and mass analyzer designs. Examples are based upon the authors' research in small molecule applications.

Dietrich A. Volmer

Articles

Dealing With the Masses: A Tutorial on Accurate Masses, Mass 32 Uncertainties, and Mass Defects

Tutorial — Mass Analyzers: An Overview of Several Designs and Their Applications, Part II

Tutorial — Mass Analyzers: An Overview of Several Designs and Their Applications, Part I

Recent Advances in Bioanalytical Mass Spectrometry and Their Application to Marine Toxins (PDF)