<![CDATA[Abstract. Luliana S, Saputri FC, Abdullah S, Alwi RS, Mahayasih PGMW, Yan CC, Mun’im A. 2026. Comparative phytochemical profiling of leaves from three Indonesian Vitex species using spectrophotometry, HP-TLC, and LC-HRMS. Biodiversitas 27 (9): d270906. https://doi.org/10.13057/biodiv/d270906. The genus Vitex is widely used in traditional medicine, yet comparative phytochemical data integrating quantitative assays and metabolite profiling for Indonesian collections remain limited. This study compared leaf extracts from three Indonesian Vitex collections representing Vitex pinnata, Vitex negundo, and Vitex trifolia. Leaves were extracted with 70% ethanol using microwave-assisted extraction. Extraction yield, Total Phenolic Content (TPC), and Total Flavonoid Content (TFC) were determined from three independent extraction batches (n = 3), while HP-TLC and LC-HRMS were used for qualitative phytochemical profiling. V. pinnata showed the highest extraction yield (34.77±1.54%), TPC (424.31±24.48 mg GAE/g extract), and TFC (32.03±2.01 mg QE/g extract). HP-TLC revealed distinct chromatographic fingerprint patterns among the three collections. LC-HRMS yielded 79 putatively annotated features (MSI Level 2), comprising 43 annotations in V. pinnata, 28 in V. negundo, and 39 in V. trifolia. All three collections shared seven features, while 10 were shared only by V. pinnata and V. trifolia, four by V. pinnata and V. negundo, and three by V. negundo and V. trifolia; 22, 14, and 19 features were unique to the respective collections. Flavonoids were most prominent in V. pinnata, iridoid-related annotations were comparatively abundant in V. negundo, and V. trifolia showed relatively high phenolic and terpenoid annotation counts. These findings provide a preliminary comparative phytochemical reference for Indonesian Vitex materials. Because each species was represented by a single geographically distinct collection, the observed differences should be interpreted at the collection level. Multisite sampling and confirmation using authentic standards are required to establish robust species-associated markers.]]>
