Following ionization, analytes are accelerated into the vacuum chamber of the mass spectrometer. Here, the mass analyzer filters and (optionally) fragments the charged ions. The filtered ions hit detectors, their signals are amplified by detection multipliers, and the final outputs are analyzed by computer software systems. Alternately, they may be fragmented and filtered a second or even third time prior to analysis. Ions may pass through several mass analyses following multiple rounds of fragmentation, which is often abbreviated as MS/MS or MSn.
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A quadrupole system uses four cylindrical magnets that are set parallel to each other and function to filter ions based on their mass-to-charge ratio (m/z). The analyzer consists of two pairs of like charged magnets that oppose each other and keep the ions within the ion path of the quadrupole under vacuum. Ions are filtered based on their masses as they traverse the linear ion path.
When a linear series of three quadrupoles is used, the resulting triple stage quadrupole analyzer is able to both filter and fragment the ion stream. In most cases, the first (Q1) and third (Q3) quadrupoles act as mass filters, while the second (Q2) quadrupole dissociates ions by having them collide with argon, helium or nitrogen gas.
Quadrupole-based mass analyzers excel at tracking single ions or reactions for extended periods of time. This is why they are preferentially used in the targeted analysis of compounds, especially known compounds such as drugs and pollutants. This is also why quadrupole mass analyzers are often used in the fields of food safety, environmental analysis, clinical and forensic toxicology studies.
The triple quadrupole (QQQ) mass spectrometer (MS) consists of a series of three quadrupoles and selects ions of specific mass-to-charge ratios (m/z) when a specific DC/RF voltage combination is applied. The first and third quadrupoles (Q1) act as mass filters, while the Q2 acts as a collision cell.
Triple quadrupole MS systems can be operated in a tandem MS/MS assay called Selected Reaction Monitoring (SRM) (sometimes also called Multiple Reaction Monitoring (MRM)) mode. SRM is a highly selective mode whereby a fixed set of DC and RF voltages is applied to the quadrupole, permitting only one precursor ion, which is measured by its m/z, to pass. After the Q1 filters that specific precursor ion, the Q2 produces product ions via collision of the precursor ion with a neutral gas (e.g., nitrogen) in a process called collision-induced dissociation (CID). Product ions progress to the Q3, where only a specific m/z is permitted to pass. By breaking the ion apart into its component fragments, a given molecular species can be identified not only by its mass but by product identity. In this way, SRM reduces noise and increases selectivity.
Most ion traps used in mass spectrometry are based on the model invented by Wolgang Paul (who shared in the 1989 Nobel Prize). The Paul-based ion trap uses a combination of direct current (DC) and radio frequency (rf) electrical fields to trap charged ions for analysis. Through the application of direct and alternating electrical currents, the ions inside of the static and oscillating electrical fields become “trapped.”
Ion traps create a three-dimensional analogue of the quadrupole mass analyzer by virtue of their construction. Here, three electrodes with hyperbolic surfaces function to trap charged ions: two endcap electrodes and a central ring electrode. Because ions are subjected to electrical fields from three directions, not two, they do not move.
Such stability offers the advantages of high sensitivity and high resolution; it also enables the selection and study of individual ion/molecule reactions through MSn. As a result, ion traps are preferably used for the discovery and targeted analysis of molecules and molecular reactions in fields such as metabolomics and lipidomics, as well as post-translational modification analysis.
The Orbitrap is a modified Kingdon ion trap that consists of two endcap electrodes and a central “spindle” electrode. A DC current applied to the Orbitrap spindle electrode results in a high static voltage between the endcap electrodes. Ions entering the Orbitrap are trapped by their attraction to the spindle electrode, which is contrasted by their inertia, and begin to orbit around the spindle electrode.
As the ions orbit, they oscillate between the two outer electrodes at different frequencies. Eventually, the ions separate into discrete bands that are determined by their different masses. These different oscillation frequencies enable mass-to-charge measurement and mass spectra reporting using fast Fourier transforms (FFTs).
The Orbitrap’s ability to trap and separate small amounts of ions enables its high-resolution and accurate-mass detection characteristics. Orbitrap-based mass spectrometers are preferentially used for non-targeted analysis of unknown compounds in areas of biomarker discovery, proteomics and metabolism.
Ions of different masses can be separated based on their flight times because lighter ions travel faster than heavier ones. A time-of-flight (TOF) analyzer accelerates samples through the mass analyzer and measures their flight time once they reach the detector. Based on flight time differences, which depend on ion velocities and kinetic energies as well as when the ions started their acceleration and ended their journey at the detector, the ion masses are then calculated.
Several mass analyzers are often combined in one mass spectrometer to achieve different separation, resolution and detection goals.
For example, the Thermo Scientific™ Q Exactive™ Focus mass spectrometer contains a hyperbolic quadrupole mass filter in order to better select precursor ions prior to their injection into and detection by an Orbitrap mass analyzer.
The Orbitrap, meanwhile, enables the resolution of spectral peaks having almost the same m/z values. Furthermore, this instrument contains an HCD collision cell to effectively fragment larger ions and molecules for effective detection and confirmation of molecular identities.
There are many other hybrid mass spectrometers that combine different mass analyzers, including the following:
These hybrid mass spectrometers combine the best features of each mass analyzer, providing additional precision, resolution, robustness, etc. For example, the Q Exactive™ Hybrid Quadrupole-Orbitrap™ Mass Spectrometer couples high resolution and accurate mass measurement with precursor selection and compound fragmentation.