Resolution refers to the ability of a mass spectrometer to distinguish ions with similar masses (with a mass difference of Δm). There are two main methods for calculating resolution: 10% peak-to-valley ratio and full-width at half-maximum.
The 10% peak-to-valley calculation method is as shown in the figure below (the source of the figure is from the Internet):
The calculation method for the full-width at half-maximum is shown in the following figure (image source: internet):
Now let’s learn some terminology as we look at the diagram.
Mass charge ratio：The ratio of the mass of an ion to its charge is called the mass-to-charge ratio, abbreviated as m/z. For example, for the lipid CL 72:7 | CL 18:1_18:2_18:2_18:2, with the molecular formula C81H144O17P2, its mass-to-charge ratio is 1449.9818 for [M-H]- and 724.4877 for [M-2H]2-, as shown in the following figure.
Abundance of ions：The intensity of the ion signal detected by the detector.
Relative abundance of ions：The intensity of other ion peaks normalized to the strongest peak within a specified mass-to-charge ratio range in the mass spectrum, where the strongest peak is set as 100%.
Base peak: In a mass spectrum, the ion peak with the highest intensity within a specified mass-to-charge ratio range is called the base peak. The relative abundance of the base peak is 100%.
Total ion current (TIC): A plot of the sum of all ion intensities within a selected mass range over time or scan number.
Extracted ion chromatogram (EIC): A chromatogram of a specific mass-to-charge ratio extracted from a full scan mass spectrum.
Mass chromatogram: A plot of the ion intensity at a specified mass-to-charge ratio over time or scan number, also commonly referred to as a mass spectrum.
Next, let’s take a look at the powerful application of high-resolution mass spectrometry through an example. Taking the example of measuring the metabolite glutamic acid in glutamine-13C5-15N2 cultured cells. The molecular formula of glutamic acid is C5H9NO4, with an average molecular weight of 147.1293 and a monoisotopic molecular weight of 147.05316. The theoretical calculation of the [M+H]+ mass-to-charge ratio (m/z) is 148.0604343.
The upper figure in the image is the extracted ion chromatogram (EIC) of glutamic acid (retention time 8.79 min), and the lower figure is the mass spectrum of glutamic acid. Only one peak can be seen in the chromatogram, while the mass spectrum at 8.79 min shows multiple isotopic labeled forms of glutamic acid (see the labels in the figure). From the figure, we can see that we cannot determine the multiple isotopic labeling of glutamic acid using chromatography alone.
The figure below shows the mass spectra of glutamic acid-13C3 ([M+H]+ theoretical value is 151.0705, experimental value is 151.0704, with an error of 0.66 ppm) and glutamic acid-13C2-15N1 ([M+H]+ theoretical value is 151.0642, experimental value is 151.0643, with an error of 0.66 ppm) measured by high-resolution mass spectrometry (resolution 70,000). If we were to use low-resolution mass spectrometry, we would only observe a single peak at m/z 151.07, without being able to distinguish the two peaks corresponding to different isotopic labeling.
High-resolution mass spectrometry can also provide specific MS/MS spectra for targeted ions, which allows for rapid compound identification. Taking 2-hydroxybutyric acid and 3-hydroxybutyric acid tested under HILIC conditions as an example, it can be determined through the MS/MS spectrum of m/z=103.0405 that 2-hydroxybutyric acid has a characteristic ion at 57.0347, while 3-hydroxybutyric acid has a characteristic ion at 59.0139. Therefore, it is possible to quickly determine that 2-hydroxybutyric acid is at 2.41 min and 3-hydroxybutyric acid is at 3.42 min.
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