Chapter 4 Instrumental analysis

To get more information in the samples, full scan is preferred on GC/LC-MS. Each scan would collect a mass spectrum to cover the setting mass range. If you narrow down your mass range and keep the same scan time, each mass would gain the collection time and you would get a higher sensitivity. However, if you expand your scan range, the sensitivity for each mass would decrease. You could also extend the collection time for each scan. However, it would affect the separation process.

Full scan is performed synchronously with the separation process. For a better separation on chromotograph, each peak should have at least 10 point to get a nice peak shape. If you want to separate two peaks with a retention time differences of 10s. Assuming the half peak width is 5s, you need to collect 10 mass spectrum within 10s. So the drwell time for each scan is 1s. If you use a high resolution column and the half peak width is 1s, you need to finish a scan within 0.2s. As we discussed above, shorter dwell time would decrease the sensitivity. Thus there is a trade-off between separation and sensitivity. If you use UPLC, the separation could be finished within 20 min while you need to calculate if you mass spectrometry could still show a good sensitivity.

4.1 Column and gradient selection

For GC, higher temperature could release compounds with higher boiling point. For LC, gradient and functional groups of stationary phase would be more important than temperature. Polarity of samples and column should match. More polar solvent could release polar compounds. Normal-phase column will not retain non-polar compounds while reversed-phase will elute polar column in the very beginning. To cover a wide polarity range or logP value compounds, normal phase column should match with non-polar to polar gradient to get a better separation of polar compounds while reverse phase column should match with polar to non-polar gradient to elute compounds. If you use an inappropriate order of gradient, you compounds would not be separated well. If you have no idea about column and gradient selection, check literature’s condition. Meanwhile, the pretreatment methods should fit the column and gradient selection. You will get limited information by injection of non-polar extracts on a normal phase column and nothing will be retained on column.

4.2 Mass resolution

For metabolomics, high resolution mass spectrum should be used to make identification of compounds easier. The Mass Resolving Power is very important for annotation and high resolution mass spectrum should be calibrated in real time. The region between 400–800 m/z was influenced the most by resolution(Najdekr et al. 2016). Orbitrap Fusion’s performance was evaluated here(Barbier Saint Hilaire et al. 2018), as well as the comparison with Fourier transform ion cyclotron resonance (FT-ICR)(Ghaste, Mistrik, and Shulaev 2016). Mass Difference Maps could recalibrate HRMS data (Smirnov et al. 2019).

4.3 Matrix effects

Matrix effects could decrease the sensitivity of untargeted analysis. Such matrix effects could be checked by low resolution mass spectrometry(M. Yu et al. 2017b) and found for high resolution mass spectrometry(Calbiani et al. 2006). Ion suppression should also be considered as a critical issue comparing heterogeneous metabolic profiles(Ghosson et al. 2021).