UHPLC-TIMS-qTOF-MS/MS expands metabolite annotation and identifies distinct cultivar-specific metabolite profiles in American elderberry flowers

American elderberry ( Sambucus nigra L. subsp. canadensis (L.) Bolli) is a deciduous shrub native to North America. It is widely cultivated for its fruits and flowers which have been traditionally used to treat ailments such as colds and flu. However, the detailed study of American elderflower metabolite profiles and natural product composition is still missing. Here we report the use of a Bruker TIMS-TOF mass spectrometer coupled to a Waters UPLC system to perform untargeted metabolomics analyses. First, LC-MS/MS data with either TIMS Off or TIMS On were collected and compared. Significantly more tandem (MS/MS) spectra were acquired using UHPLC-TIMS-QTOF-MS/MS in the PASEF mode (5619 MS/MS spectra) when compared to UHPLC-QTOF-MS/MS (1679 MS/MS spectra), leading to more metabolite identifications. UHPLC-TIMS-QTOF-MS/MS was then used to profile and compare flowers from five American elderberry genotypes, e.g. , Cherokee, Dandy, Bob Gordon, Barber, and Ranch. A total of 6722 mass features were detected, out of which 262 were annotated. The identified metabolites included phenolics (173), triterpenes (20), carbohydrates (21), fatty acids/lipids (33), iridoids (6), and other classes of metabolites (9). Distinct genotype-specific metabolite profiles were observed among the genotypes as revealed by PCA and PLS-DA analyses. Phenolics, fatty acids/lipids, and iridoids were found to be significantly higher in Bob Gordon. Triterpenes were higher in Dandy and carbohydrates were more abundant in Barber. A strong negative correlation was observed between mite damage and the total triterpene level (and select but specific individual triterpenes). In contrast, no such correlations were observed between mite damage and phenolics (or other classes of metabolites). The data demonstrate that elderflowers are a rich source of health promoting metabolites and have potential nutraceutical applications.