The Mass Spectrometry Primer

In mass spectrometry, the ability to exercise control over experiments is supremely important. Once an ion is created under carefully controlled conditions it must be detected as a discrete event with appropriate sensitivity. The minimal vapor load made GC an ideal early choice as a hyphenated technology but for only some 20% of compounds. Today, we most frequently aerosolize LC eluent as the means of introducing analytes for ionizing within a mass spectrometer, a technique that requires a vacuum environment to ensure control.

An important design element of any mass spectrometer is pumping capacity. Vacuum must be well-distributed in the rarified atmospheric regions of an instrument, and it must be sufficient enough to offset such design necessities as the size of the ion inlet and the amount of vapor needing removal.

LC-MS pumping requirements depend on the interface used. Ultimately this was one of the reasons spurring development of the API source where the vapor is removed before entry to the MS.

The analyzer is an instrument's means of separating or differentiating introduced ions. Both positive and negative ions (as well as uncharged, neutral species) form in the ion source. However, only one polarity is recorded at a given moment. Modern instruments can switch polarities in milliseconds, yielding high fidelity records even of fast, transient events like those typical of ultra performance liquid chromatography (UPLC) or GC separations in which peaks are only about one second wide.

Quadrupoles

In 1953, the West German physicists Wolfgang Paul and Helmut Steinwedel described the development of a quadrupole mass spectrometer. Superimposed radio frequency (RF) and constant direct current (DC) potentials between four parallel rods were shown to act as a mass separator, or filter, where only ions within a particular mass range, exhibiting oscillations of constant amplitude, could collect at the analyzer.

Manufacturers' of today's instruments target them for specific applications. Single quadrupole mass spectrometers require a clean matrix to avoid the interference of unwanted ions, and they exhibit very good sensitivity.

Triple quadrupoles, or tandem, mass spectrometers (MS/MS) add to a single quadrupole instrument an additional quadrupole, which can act in various ways. One way is simply to separate and detect the ions of interest in a complex mixture by the ions' unique mass-to-charge (m/z) ratio. Another way that an additional quadrupole proves useful is when used in conjunction with controlled fragmentation experiments. Such experiments involve colliding ions of interest with another molecule (typically a gas like argon). In such an application, a precursor ion fragments into product ions, and the MS/MS instrument identifies the compound of interest by its unique constituent parts.

The quadrupole analyzer consists of four rods, which are usually arranged in parallel and made of metal such as molybdenum alloys. A tremendous amount of art and science has been invested in developing quadrupole design. Masses are sorted by the motion of their ions, which direct current (DC) and radio frequency (RF) fields induce into an instrument's analyzer. Systematically changing the field strength via the operating software in effect alters which m/z value is filtered or transmitted through to the detector at any given time. Quadrupoles yield a lower resolution than some mass spectrometer designs like time-of-flight (TOF) instruments. Yet quadrupoles are relatively simple, easy-to-use, highly utilitarian, instruments that offer a variety of interfaces at a relatively low cost.

Some terminology, more fully defined later in this primer, is necessary for comparing and describing MS capabilities:

Resolving power

Often abbreviated as "res," resolving power is the ability of a mass spectrometer to separate two masses:

• Low res = unit mass = 1000
• Higher or moderate res = 1000 to 10,000
• High res 10,000+
• Very high res = as much as 3–5 million
• A more detailed examination of resolution and how we measure it appears in the section "Mass Accuracy and Resolution."

Exact Mass is the theoretical exact value for the mass of a compound whereas Accurate Mass is the measured mass value for a compound with an associated error bar like 5ppm. Accurate Mass is also commonly used to refer to the technique rather than the measured mass. Commonly accepted criteria for exact mass work (for instance, to publish in a journal or file a patent) is the ability to derive a measurement on an instrument within 5 ppm of its theoretical mass: 5 ppm at 250 Da is 1.25 mDa (not to be confused with 5 mDa, which would be 20 ppm at 250 Da)

MS/MS

Describes a variety of experiments-multiple-reaction monitoring (MRM) and single-reaction monitoring (SRM). That is monitoring the transition of precursor ions, or fragmentations, to product ion(s), which in general tend to improve the selectivity, specificity, and/or sensitivity of detection over a single-stage-instrument experiment. Two mass analysers in series, or two stages of mass analysis, in a single instrument are used.

In a triple quadrupole mass spectrometer, there are three sets of quadrupole filters, although only the first and third function as mass analyzers. More recent designs have sufficiently differentiated the middle device (replacing the quadrupole of earlier designs) adding increased function so the term triple or tandem quadrupole is often used instead. The first quadrupole (Q1), acting as a mass filter, transmits and accelerates a selected ion towards Q2, which is called a collision cell. Although in some designs Q2 is similar to the other two quadrupoles, RF is imposed on it only for transmission, not mass selection. The pressure in Q2 is higher, and the ions collide with neutral gas in the collision cell. The result is fragmentation by collision-induced dissociation (CID). The fragments are then accelerated into Q3, another scanning mass filter, which sorts them before they enter a detector.

Collision-induced dissociation (CID) also referred to as collisionally activated dissociation (CAD), is a mechanism by which molecular ions are fragmented in the gas phase usually by acceleration by electrical potential to a high kinetic energy in the vacuum region followed by collision with neutral gas molecules such as helium, nitrogen or argon. A portion of the kinetic energy is converted or internalized by the collision which results in chemical bonds breaking and the molecular ion is reduced to smaller fragments. Some similar ‘special purpose' fragmentation methods include electron transfer dissociation (ETD), electron capture dissociation (ECD). See the section on ‘Bio-molecular ionization methods'.

The 237-Da precursor ion entering on the left was fragmented in the MS/MS collision cell. The data system can display only fragments of interest (not all fragments produced) yielding a relatively simple spectrum with respect to the full scan MS spectrum. You can control the extent of fragmentation as you can the choice of precursor ion.

Reversed-Phase HPLC

The term reversed-phase describes the chromatography mode that is just the opposite of normal phase, namely the use of a polar mobile phase and a non-polar [hydrophobic] stationary phase. Figure S-2 illustrates the black three-dye mixture being separated using such a protocol.

In some regulated industries, to meet the specification for positive compound identification, MRM transitions count for 1.5 "identification points," whereas SIR traces count for 1.0. So, assuming sufficient selectivity, to achieve 3 "IPS," you need 2 MRM transitions but three SIR traces.

Magnetic sector, or a sector field mass analyzer, is an early instrument design that persists today, albeit minimally (having been displaced by modern ESI instruments that can operate in the ESI ionization mode). The Waters AutoSpec, for instance, is used universally for extremely high sensitivity dioxin analysis.

Sectors bend the arc-shaped ion trajectories. The ions' "momentum-to-charge" ratios determine the radius of the trajectories, which themselves are determined by an electric and/or magnetic field. Ions with larger m/z ratios proceed through longer paths than those with smaller ones. The paths are controlled by varying the strength of the magnetic field. Double-focusing mass spectrometers combine magnetic and electric fields in various combinations, although the electric sector followed by the magnetic is more common. This earliest of hybridizations uses the electric sector to focus ions by their kinetic energy as they exit the source. Angular focusing preceded by energetic focusing yields separations of ions with the same nominal mass but different chemical formulas.

An ion trap instrument operates on principles similar to those of a quadrupole instrument. Unlike the quadrupole instrument, however, which filters streaming ions, both the ion trap and more capable ion cyclotron (ICR) instrument store ions in a three-dimensional space. Before saturation occurs, the trap or cyclotron allows selected ions to be ejected, according to their masses, for detection. A series of experiments can be performed within the confines of the trap, fragmenting an ion of interest to better define the precursor by its fragments. Fields generated by RF voltages applied to a stacked or "sandwich" geometry (end-cap electrodes at opposing ends) trap ions in space between the two electrodes. Ramping or scanning the RF voltage ejects ions from their secular frequency, or trapped condition. Dynamic range is sometimes limited. The finite volume and capacity for ions limits the instrument's range, especially for samples in complex matrices.

Ion trap instruments were introduced in the 1980s. But limitations imposed by the internal ionization scheme used in those early instruments prevented their use for many applications. Only with the advent of external ionization did the instruments become more universally practical.

The ability to perform sequential fragmentation and thus derive more structural information from a single analyte (i.e., fragmenting an ion, selecting a particular fragment, and repeating the process) is called MSn. GC chromatographic peaks are not wide enough to allow more than a single fragmentation (MS/MS or MS2). Ion trap instruments perform MS/MS or fragmentation experiments in time rather than in space, like quadrupole and sector instruments. So they cannot be used in certain MS/MS experiments like neutral loss and precursor ion comparisons. Also, in MS/MS operation with an ion trap instrument, the bottom third of the MS/MS spectrum is lost, a consequence of trap design. To counter the loss, some manufacturers make available via their software wider scan requirements that necessitate the switching of operating parameters during data acquisition,

The trap design places an upper limit on the ratio between a precursor's mass-to-charge ratio (m/z) and the lowest trapped fragment ion, commonly known as the "one third rule". For example, fragment ions from an ion at m/z 1500 will not be detected below m/z 500 - a significant limitation for the de novo sequencing of peptides. The ion trap has limited dynamic range, the result of space-charge effects when too many ions enter the trapping space. Manufacturers have developed automated scanning, which counts ions before they enter the trap, limiting, or gating, the number allowed in. Difficulty can still be encountered when a relatively small amount of an ion of interest is present in a large population of background ions.

Because of similarities in functional design, quadrupole instruments are hybridized to incorporate the advantages of streaming quadrupole and ion trapping behavior to improve sensitivity and allow on-the-fly experiments not possible with either alone. Such instruments are sometimes called linear traps or Q-traps). The increased volume of a linear trap instrument (over a three-dimensional ion trap) improves dynamic range.

Ion trap instruments do not scan as a quadrupole instrument does so using the single ion monitoring (SIM), or single ion recording (SIR), technique does not improve sensitivity on ion traps as it does on quadrupole and sector instruments.

Fast-fourier transform ion cyclotrons (FTICR) represent the extreme capability of measuring mass with the ability to resolve closely related masses. Although impractical for most applications, a 14.5-tesla magnet can achieve a resolution of more than 3.5 million and thus display the difference between molecular entities whose masses vary by less than the mass of a single electron.

Cyclotron instruments trap ions electrostatically in a cell using a constant magnetic field. Pulses of RF voltage create orbital ionic motion, and the orbiting ions generate a small signal at the detection plates of the cell (the ion's orbital frequency). The frequency is inversely related to the ions' m/z, and the signal intensity is proportional to the number of ions of the same m/z in the cell. At very low cell pressures, a cyclotron instrument can maintain an ion's orbit can for extended periods providing very high resolution measurements.

Sustained off-resonance, irradiation, collision-induced dissociation (SORI-CID) is a CID technique used in Fourier-transform ion cyclotron resonance mass spectrometry. The ions are accelerated in cyclotron motion where increasing pressure results in collisions that produce fragments. After the fragmentation, the pressure is reduced and the high vacuum restored to analyze the fragment ions.

Time-of-flight (TOF) instruments, although developed many years ago, have become the basis for much modern work because of their fast, precise electronics and modern ionization techniques like ESI. A TOF instrument provides accurate mass measurement to within a few parts-per-million (ppm) of a molecule's true mass. A temporally dispersive mass analyzer, the TOF instrument is used in a linear fashion or, aided by electrostatic grids and lenses, as a reflectron. When operated as a reflectron, resolution is increased without dramatically losing sensitivity or needing to increase the size of the flight (or drift) tube.

TOF analyses involve accelerating a group of ions, in a brief burst, to a detector. The ions exit the source, each having received from a "pusher" electrode an identical electrical charge, or potential. Each ion's potential accelerates, or fires, it into a very low pressure tube. Because all similarly charged ions share the same kinetic energy (kinetic energy = ½ mv² where m is the ion mass and v the velocity), those with lower masses evidence greater velocity and a lesser interval before striking the detector. Since mass, charge, and kinetic energy determine the arrival time of an ion at the detector, the ion's velocity can be represented as v = d/t = (2KE/m)^1/2. The ions travel a given distance (d), in time (t), where t depends on the mass-to-charge ratio (m/z). Since all masses are measured for each "push," the TOF instrument can achieve a very high sensitivity relative to scanning instruments.

Today, quadrupole MS systems scan routinely at 10,000 Da, or amu, per second. So a comprehensive scan, even one of short duration-an LC or GC peak of 1 second, for instance-would nevertheless capture each ion 10 times, or more, in each second. The TOF instrument's detector registers ions bombarding the plate within nanoseconds of each other. Such resolution offers the added capabilities of a wide dynamic range and greater sensitivity when compared directly to a scanning instrument such as a quadrupole. Yet the quadrupole instrument is, generally speaking, more sensitive when detecting target analytes in complex mixtures and is, therefore, typically a better quantitation tool. Some instruments, like ion traps, offer a combination of these capabilities. But until the advent of hybrid instruments, no single one could deliver high-order performance in all aspects.

Early MALDI-TOF designs (using matrix assisted laser desorption ionization) accelerated the ions out of the ionization source immediately. Their resolution was relatively poor and their accuracy limited. Delayed extraction (DE), developed for MALDI-TOF instruments, "cools" and focuses the ions for approximately150 nanoseconds after they form. Then it accelerates the ions into the flight tube. The cooled ions have a lower kinetic-energy distribution than uncooled ones, and they ultimately reduce the temporal spread of the ions as they enter the TOF analyzer, resulting in increased resolution and accuracy. DE is significantly less advantageous with macromolecules (for instance proteins >30,000 Da).

Hybrids

The term "hybrid" applies to various mass spectrometer designs that are composites of existing technologies such as double-focusing, magnetic sectors and, more recently, ion traps that "front" cyclotrons. One of the most interesting designs, the quadrupole time-of-flight (QTOF) mass spectrometer, couples a TOF instrument with a quadrupole instrument. This pairing results in the best combination of several performance characteristics: accurate mass measurement, the ability to carry out fragmentation experiments, and high quality quantitation.

Further evolution has produced the coupling of ion mobility measurements and separations with tandem mass spectrometry. Ion mobility mass spectrometry (note: I use ‘IMMS' as an acronym here since imaging mass spectrometry is often abbreviated ‘IMS') is a technique that differentiates ions based on a combination of factors: their size, shape and charge, as well as their mass. IMMS devices are commonly used in airports and hand-held field units for rapidly (20 msec) detecting small molecules whose mobility is known: for example certain narcotics and explosives. When adapted to the higher-order instruments, IMMS provides an orthogonal dimension of separation (to both LC and MS) and some unique, enabling capabilities including these:

• Separation of isomers, isobars, and conformers (from proteins to small molecules) and determination of their average rotational collision-cross section
• Enhanced separation of complex mixtures (by MS or LC-MS) leading to increased peak capacity and sample clean-up (physical separation of ions, especially chemical noise, and ions that interfere with analytes of interest)
• Performance of CID/IMMS, IMMS/CID or CID/IMMS/CID and enhancement of the amount of meaningful information that can be gained from fragmentation experiments in structural elucidation studies.

In all three analytical scenarios, the combination of high-efficiency ion mobility and tandem mass spectrometry can help you overcome analytical challenges that you could not address by other analytical means, including conventional mass spectrometry or liquid chromatography instrumentation.

The review article by H.H. Hill Jr., et al., cited at the end of this section, compares and contrasts various types of ion mobility-mass spectrometers available as of the article's 2007 publication-and describes the advantages of applying them to a wide range of analytes. It targets four methods of ion mobility separation currently used with mass spectrometry:

• Drift-time ion mobility spectrometry (DTIMS)
• Aspiration ion mobility spectrometry (AIMS)
• Differential-mobility spectrometry (DMS), also called field-asymmetric waveform ion mobility spectrometry (FAIMS)
• Traveling-wave ion mobility spectrometry (TWIMS) )

According to the authors "DTIMS provides the highest IMS resolving power, and it is the only (IMMS) method that can directly measure collision cross-sections. AIMS is a low resolution mobility separation method, but it can monitor ions continuously. DMS and FAIMS offer continuous-ion monitoring capability as well as orthogonal ion mobility separations in which high-separation selectivity can be achieved. TWIMS is a novel (IMMS) method whose resolving power is relatively low. Nevertheless, it demonstrates good sensitivity and is well integrated into operation of a commercial mass spectrometer."

Coupled with MS, ion mobility is also being applied to investigate the gas-phase structures of biomolecules. Pringle et al. (cited here) examine the mobility separation of some peptide and protein ions using a hybrid quadrupole/traveling wave ion mobility separator/orthogonal acceleration time-of-flight instrument. Comparing mobility data obtained from the traveling wave (TWIMS) separation device with that obtained using various other mobility separators indicates that "while the mobility characteristics are similar, the new hybrid instrument geometry provides mobility separation without compromising the base sensitivity of the mass spectrometer. This capability facilitates mobility studies of samples at analytically significant levels."

See also:

• Special Feature Perspective: Ion mobility-Mass Spectrometry, A. B. Kanu, P. Dwivedi, M. Tam, L. Matz and H. H. Hill Jr., J. Mass Spectrom. 2008; 43: 1-22 Published online in Wiley InterScience, (https://onlinelibrary.wiley.com/) DOI: 10.1002/jms.1383
  • Why this is important: A concise overview of ion mobility coupled with MS. One hundred and sixty references on ion mobility-mass spectrometry (IMMS) are provided.
    
• An investigation of the mobility separation of some peptide and protein ions using a new hybrid quadrupole/traveling wave IMS/oa-ToF instrument, S. D. Pringle, K. Giles, J. L. Wildgoose, J. P. Williams, S. E. Slade, K. Thalassinos, R. H. Bateman, M. T. Bowers, J. H. Scrivens, Published online (www.sciencedirect.com), International Journal of Mass Spectrometry (2006), doi:10.1016/j.ijms.2006.07.021
  • Why this is important: Describes how IMMS works with biomolecules.