Characterisation of phenylethanoid glycosides by multiple-stage mass spectrometry

Although phenylethanoid glycosides (PhGs) occur widely in plants, their characterisation by liquid chromatography/mass spectrometry (LC/MS) is less well studied than other phenolic glycosides such as flavonoid glycosides. The multiple-stage mass spectrometry (MSⁿ ) experiments required to improve the annotation of common verbascoside-type PhGs are described here.

Deprotonated, ammoniated and sodiated molecules of nine PhGs were subjected to low-energy collision-induced dissociation (CID) in a hybrid ion trap/orbitrap mass spectrometer. Most experiments were recorded at nominal mass using the linear ion trap analyser for wider applicability in the plant metabolomics community. Data interpretation was supported by high-resolution orbitrap scanning of product ions. Comparative data was acquired on the same instrument by performing higher-energy collisional dissociation (HCD) in the C-trap.

Low-energy CID-MS² of the deprotonated and ammoniated molecules generated diagnostic product ions from which the molecular masses of the phenolic acid and phenylethanoid moieties, respectively, could be determined. The sugar at C-3' of the core glucose was preferentially lost from the sodiated molecule following CID-MS² , while CID-MSⁿ produced a sodiated product ion from ring cleavage of the core glucose bearing the sugar at C-6'. Evidence of a disaccharide substitution came from a sodiated disaccharide residue in CID-MSⁿ spectra.

The consistency of PhG dissociation following low-energy CID-MSⁿ of various ions is sufficient to enable annotation of verbascoside-type PhGs in LC/MS analyses of crude plant extracts. This can be achieved on a low-resolution instrument capable of MSⁿ .