MASS SPECTROMETRY & SPECTROSCOPY
Separation without hyphenation using two-dimensional mass spectrometry (2DMS)
Steven Wright, Verdel Instruments Introduction
In most laboratory applications, mass spectrometers are coupled to chromatography systems that separate the compounds in a sample so that they may be analysed one at a time. Liquid chromatography (LC), gas chromatography (GC) and ion chromatography (IC) are the most common separation techniques to be coupled with mass spectrometry (MS), giving rise to the so-called hyphenated techniques of LC-MS, GC-MS, and IC-MS. However, method development in chromatography is a skilled art, and an unwelcome bottleneck in the analytical workflow. Even in the hands of an experienced operator, separation of all the components in a complex sample is highly unlikely, resulting in co-elution of analytes.
Method development in chromatography is a skilled art, and an unwelcome bottleneck in the analytical workflow.”
Tandem mass spectrometers (or MS/MS instruments) are capable of fragmenting precursor ions and mass-analysing the resulting product ions. This is a powerful route to determining both the structure and identity of an analyte. However, it is difficult to analyse mixtures of analytes; the task of determining structure and identity by assigning fragments to a precursor quickly becomes intractable if more than one analyte is present. The problem of co-eluting analytes is overcome in a tandem mass spectrometer by using a quadrupole filter to selectively transmit only one precursor. In a targeted analysis, the analytes of interest are known, and precursor selection may be programmed in advance. For untargeted analysis, more complex methods must be employed. Data dependent analysis (DDA) refers to techniques that dynamically select precursors based on their peak intensities, while data independent analysis (DIA) is an umbrella term for methods that attempt unbiased analysis of all the components in a mixture.
Two-dimensional mass spectrometry (2DMS) is a DIA technique that is unfamiliar to most (even in the field of MS) but has the potential to become a routine and important analytical tool. Its current obscurity is a consequence of its origins in the field of Fourier Transform Ion Cyclotron (FTICR) MS, a highly specialist technique, largely confined to academia and high-end industrial applications. In this note, we show that 2DMS can now be performed using Quadrupole Time-of-Flight (QToF) instruments. These may be found in many analytical laboratories and are widely used for routine high-resolution analyses. Crucially, 2DMS does not require chromatography – the sample mixture is injected by direct infusion. The task of separating out the signals due to all the precursors and their corresponding fragments is performed by the mass spectrometer using a novel Fourier Transform technique.
Interpretation of a
2D mass spectrum For those new to the technique, a 2D mass spectrum can be confusing, largely as a result of previous exposure to 2D contour plots from LC-MS and other hyphenated techniques. The output from 2DMS can also be displayed as a contour plot (or 3D equivalent) but the orthogonal axes are both m/z axes – one corresponding to the precursor and the other to the fragments. When LC-MS data is displayed in this way, only one axis is m/z, the other is retention time,
Figure 1: Idealised representation of a typical 2D mass spectrum. Precursors and their associated fragments are colour coded. Intact precursors appear on the diagonal autocorrelation line while correlated product ions are arranged in horizontal lines with the same y-axis value as their parent precursor ion.
and the problem of co-eluting analytes remains.
Correlation is a fundamental concept in 2DMS. There are strong parallels with 2D NMR, a technique that assists structural determination by exposing correlations between spin-spin coupled or nearby nuclei. A 2D mass spectrum reveals correlations between fragments and precursors that allow the structure and identities of all the precursors present to be determined. Figure 1 shows an idealised 2D mass spectrum of five compounds. There is a diagonal line corresponding to precursor ion m/z = product ion m/z. This is called the autocorrelation line. Peaks found on this line represent ions that are unfragmented and are therefore precursors, by definition. Off-diagonal peaks correspond to product ions. All the product ion peaks appearing on the same horizontal line are correlated with the precursor ion peak at the intercept with the autocorrelation line.
Basic principles
and instrumentation 2DMS is implemented on a QToF instrument by repurposing the quadrupole as an ion trap. This is simply accomplished by alternating the voltages applied to lenses either end of the quadrupole between low and high values to trap and release ions. Ions in a quadrupole oscillate or orbit as if trapped in a potential well. The frequency of this oscillation (known as the secular frequency) is dependent on the m/z value of the ion. Light ions oscillate at high frequencies; heavy ions oscillate at low frequencies. Ions of a particular m/z may be resonantly excited. For example, if an m/z 100 ion has a secular frequency of 200 kHz, applying a 200 kHz waveform across two opposing quadrupole rods will excite all m/z 100 ions to higher orbits but leave ions of, say, m/z 150 unaffected. Consequently, a broadband pulse comprising many frequencies with varying amplitudes will excite different ions to different orbital radii. These pulses can be specifically constructed to sinusoidally modulate the ion orbital radii with pre-determined encoding frequencies.
A necessary component of 2DMS is the coupling of precursor ion radius modulation with a radius-dependent fragmentation technique. If the fragmentation efficiency depends on the precursor ion orbital radius, then the fragment yield will modulate at the same frequency as the precursor signal; precursors and their fragments become inextricably linked by their common modulation frequency. Collision induced dissociation (CID) and ultraviolet photodissociation (UVPD) are examples of radius-dependent fragmentation methods that can be implemented in a quadrupole. During data acquisition, ion signals are seen to modulate. The modulation frequencies can be recovered in post-processing by applying the Fourier transform to every ion signal. Construction of a 2D spectrum starts by plotting modulation frequency on the y-axis and the m/z values recorded by the ToF analyser on the x-axis. A precursor ion and its correlated product ions have the same modulation frequency and therefore the same y-axis value, resulting in peaks arranged in horizontal lines. The y-axis is then converted to m/z (of the parent precursor) using the known relationship between encoding frequency and m/z.
2DMS is currently available as a retrofitted upgrade kit for Bruker QToF instruments. Kits for instruments supplied by
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INTERNATIONAL LABMATE - FEBRUARY 2026
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