Polar and non-polar discrimination: why some molecules are ‘naturally’ difficult to ionise

In mass spectrometry, ‘polarity discrimination’ refers to the systematic preference of ionisation methods for molecules of different polarities: certain ionisation techniques are more effective at ionising highly polar molecules that readily gain or lose charge, whilst the ionisation efficiency for weakly polar and non-polar molecules is low, resulting in weak signals or even complete undetectability. Consequently, both the mass spectra and imaging maps are biased towards polar compounds and do not accurately reflect the distribution of all types of molecules in the sample. This is not a fault with the instrument, but rather a consequence of the selectivity inherent in the ionisation mechanism itself—whether a molecule can be ‘activated’ depends on how easily it can acquire a charge under this mechanism.

The root cause of polarity discrimination lies in the principle of ionisation. Taking electrospray-based techniques (including DESI) as an example, these rely on charge transfer from charged droplets to induce ionisation; this process favours polar molecules with high proton affinity, which are easily protonated or readily undergo addition reactions; for weakly polar or non-polar compounds found in lipids—such as neutral lipids, terpenes, flavonoids and glycosides—it is difficult for a charge to ‘attach’, resulting in a very weak response. For those working in metabolomics and lipidomics, this means that a large class of important molecules is systematically underestimated.

‘Non-polar discrimination’ detection, however, requires an ionisation mechanism that does not depend on molecular polarity. Neo Source INSTRUMENT’s MSI DPI (DESI + PI) and MSI LDPI (laser desorption + PI) incorporate photochemical secondary ionisation following primary desorption: The energy of vacuum ultraviolet photons is sufficient to ionise the vast majority of organic molecules; whether ionisation occurs depends primarily on the molecule’s ionisation energy rather than its polarity. Consequently, this method is effective for polar, weakly polar and non-polar molecules alike, thereby addressing the shortfall of traditional DESI in the detection of non-polar compounds.

This difference is very evident in real samples. According to data from the official website, compared with commercially available imaging sources, MSI DPI can ‘detect up to a hundred additional secondary metabolites, such as lipids, terpenoids, flavonoids, amino acids and glycosides, in both positive and negative ion modes’ in animal tissues and plant leaves, with an overall signal enhancement of 1–3 orders of magnitude; the signal for theanine in tea leaves increased 14.5-fold, that for caffeine 61-fold, and that for cholesterol in mouse brains approximately 205-fold—many of these are components that were previously easily ‘missed’ due to polarity discrimination. Both sources can be fitted to mass spectrometers from Agilent, AB SCIEX and Thermo.