Characterisation of phenylethanoid glycosides by multiple‐stage mass spectrometry

Rationale: 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. Methods: 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. Results: 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. Conclusions: 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