As the extracts used in the initial screening were not available in sufficient quantity for further fractionation, dried plant material from the six active plant parts was extracted at a larger scale using maceration with EtOAc, the solvent used to generate the initial 1,600-extract library . To confirm the presence of rotenone and deguelin analogs in the newly obtained extracts, we used UHPLC-DDA-HRMS 2 -based profiling for metabolite annotation. The analysis of the new extracts was performed using a better chromatographic resolution than was used when obtaining the initial screening metabolomics data (100 mm vs. 50 mm column, longer elution gradient), and charged aerosol detector (CAD) detection was added to obtain semi-quantitative information about the composition of the extracts (Figure B) . The data obtained from this improved metabolite profiling were subjected to Ion-Identity (IIN) Feature-Based Molecular Networking (FBMN) combined with CANOPUS chemical class annotation to highlight compounds of interest (Figure ). Four extracts covering a maximum of the chemical diversity from the six active extracts, one per species, were selected to further isolate the targeted compounds. These were isolated using one-step high-resolution semi-preparative HPLC and characterized using HRMS, NMR, and chiroptical methods (Figure D and E) . Finally, the cytotoxicity of the compounds and their activity against T. cruzi were evaluated in vitro (Figure ). LC-MS peaks corresponding to rotenoids were retrieved using the CANOPUS chemical class annotations. Using the PDA and CAD data, we could confirm they were UV-reactive and present in a relative amount sufficient for isolation. D) The HPLC gradient was optimized for better separating the targeted compounds for the four selected extracts, one by species. E) Finally, targeted compounds were obtained using semi-preparative HPLC in a single chromatography step. Pure compounds' structural elucidation was performed using NMR, HRMS, and chiroptical methods, and finally (F) their bioactivity against T. cruzi was assessed in vitro.