A Transformation-Guided Mass Spectrometry Workflow for Expanding In Vivo Metabolite Coverage and Revealing Noncanonical Metabolism of Epigallocatechin Gallate

In vivo metabolism of dietary bioactive compounds generates extensive chemical diversity, but much of this metabolite space remains inaccessible to current metabolomics workflows because of limited acquisition depth, database dependence, and insufficient structural validation. Here, we present a closed-loop analytical workflow for the systematic in vivo discovery, annotation, and validation of bioactive metabolites. The workflow integrates iterative DeepScan acquisition to improve MS2 coverage, diagnostic fragment-guided screening to prioritize scaffold-related metabolites, transformation-driven annotation with fragment ion search (FISh) prediction, multistage MSn evidence chaining, and structured tracing of unresolved metabolites with verified precursor origin. Using (−)-epigallocatechin gallate (EGCG), a major green tea bioactive component, as a model compound, this approach substantially expanded scaffold-linked precursor coverage, with later DeepScan injections (IDs 02−05) contributing 75% of the recovered precursor candidates, while 63% of annotated metabolites required transformation-rule expansion. A total of 702 EGCG-related candidate metabolites were detected, including 343 annotated metabolites assigned at Levels 1, 2b, and 3. Among the 209 metabolites assigned at Levels 1 and 2b, 111 had not been previously reported in vivo. In addition to resolving multiple noncanonical metabolic pathways, the workflow established a curated database of 359 traceable but structurally unresolved candidate metabolites for progressive refinement of the accessible chemical space. For structural validation, EGCG-derived reference products were generated through 21 different in vitro transformations. This analytical strategy addresses major limitations of database-centric metabolomics and provides a transferable framework for in vivo metabolite discovery and structural characterization of dietary bioactive compounds and related small-molecule exposures.