Confirmative Structural Annotation for Metabolites of ()-7,3'-Dihydroxy-4'-methoxy-8-methylflavane, A Natural Sweet Taste Modulator, by Liquid Chromatography-Three-Dimensional Mass Spectrometry.

Flavonoids occupy the largest family of natural products and possess a broad spectrum of health benefits. Their metabolites are sometimes the truly effective molecules . It is still challenging, however, to unambiguously identify flavonoid metabolites using conventional LC-MS/MS. Herein, we aimed to pursue auxiliary structural clues to / values in both MS and MS spectra through LC coupled to three-dimensional MS (LC-3D MS). MS, as the first dimension, was in charge of suggesting theoretical molecular formulas, MS, the as second dimension, was responsible for offering substructures, and exactly, online energy-resolved MS (ER-MS), as the third dimension, provided optimal collision energies (OCEs) that reflected the linkage manners among the substructures. Metabolic characterization of a natural sweet taste modulator, namely, ()-7,3'-dihydroxy-4'-methoxy-8-methylflavane (DHMMF), was conducted as a proof-of-concept. Extensive efforts, such as full MS and MS scans on IT-TOF-MS and predictive selected-reaction monitoring mode on Qtrap-MS, were made for in-depth metabolite mining. Seventeen metabolites (-) were captured from DHMMF-treated biological samples, including 17 (-), 10 (, , , , and ), and 2 ( and ) metabolites from urine, plasma, and feces, respectively. Their structures were configured by integrating MS, MS, and OCE information. Except , all metabolites were new compounds. LC-MS/MS-guided chromatographic purification yielded three glucuronyl-conjugated metabolites (, , and ), and NMR spectroscopic assays consolidated the structures transmitted from LC-3D MS. Demethylation, glucuronidation, and sulfation occurred as the primary metabolic pathways of DHMMF. Above all, LC-3D MS bridged LC-MS/MS from putatively structural annotation toward confidence-enhanced identification, beyond the metabolite characterization of flavonoids.