Abstract
Prenylated (iso)flavonoids are potent bioactive compounds found in the Fabaceae family. Analysis and quantification of this type of phytochemicals is challenging due to their large structural diversity. In this study, the fragmentation of prenylated (iso)flavonoids was investigated using electrospray ionization ion trap mass spectrometry (ESI-IT-MSn) with fragmentation by collision induced dissociation (CID) in combination and Orbitrap-MS (ESI-FT-MS2) with fragmentation by higher energy C-trap dissociation (HCD). With this combination of IT-MSn and high resolution MS (FT-MSn), it was possible to determine the fragmentation pathways and characteristic spectral features of different subclasses of prenylated (iso)flavonoid standards, as well as characteristic fragmentations and neutral losses of different prenyl configurations. Based on our findings, a decision guideline was developed to (i) identify (iso)flavonoid backbones, (ii) annotate prenyl number, (iii) configuration, and (iv) position of unknown prenylated (iso)flavonoids, in complex plant extracts. In this guideline, structural characteristics were identified based on: (i) UV absorbance of the compound, (ii) mass-to-charge (m/z) ratio of the parent compound; (iii) ratio of relative abundances between neutral losses 42 and 56 u in MSn; (iv) retro-Diels-Alder (RDA) fragments, neutral losses 54 and 68 u, and the ratio [M+H–C4H8]+/[M+H]+. Using this guideline, 196 prenylated (iso)flavonoids were annotated in a Glycyrrhiza glabra root extract. In total, 75 skeletons were single prenylated, 104 were double prenylated, and for merely 17 skeletons prenyl number could not unambiguously be annotated. Our prenylation guideline allows rapid screening for identification of prenylated (iso)flavonoids, including prenyl number, configuration, and position, in complex plant extracts. This guideline supports research on these bioactive compounds in the areas of plant metabolomics and natural products.
Highlights
Separation and identification of prenylatedflavonoids in complex plant extracts is performed with a combination of liquid chromatography (LC) with UVeVis spectroscopy and electrospray ionization (ESI) mass spectrometry (MS)
It was proposed that the position of 2,2-DMP prenylation in isoflavones can be putatively assigned based on characteristic neutral loss (NL); a loss of 54 u with >20% relative abundance was characteristic for prenylation on C6, whereas a loss of 54 u with
We hypothesized that prenylatedflavonoids can be identified in complex crude plant extracts based on common spectral features; (i) prenyl number can be defined by the m/z of the parent compound, (ii) prenyl configuration can be defined by the ratio of relative abundances of NLs 42 and 56 u in MSn, (iii) performing FT-MS2 analyses to confirm elemental formulas of prenylrelated fragments can be used to ascertain prenyl configuration, and (iv) prenyl position (A-ring or B-ring prenylation) can be defined by analysis of RDA fragments upon fragmentation
Summary
Separation and identification of prenylated (iso)flavonoids in complex plant extracts is performed with a combination of LC with UVeVis spectroscopy and electrospray ionization (ESI) MS. We hypothesized that prenylated (iso)flavonoids can be identified in complex crude plant extracts based on common spectral features; (i) prenyl number can be defined by the m/z of the parent compound, (ii) prenyl configuration can be defined by the ratio of relative abundances of NLs 42 and 56 u in MSn, (iii) performing FT-MS2 analyses to confirm elemental formulas of prenylrelated fragments can be used to ascertain prenyl configuration, and (iv) prenyl position (A-ring or B-ring prenylation) can be defined by analysis of RDA fragments upon fragmentation
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