Metabolomic Signatures of Commercial Ready-to-Drink Beverages by Dual-Mode Untargeted LC-MS/MS

Background: The rapid expansion of functional ready-to-drink (RTD) beverages-formulated with prebiotic fibers, botanical extracts, and reduced sugar-has outpaced systematic characterization of their small-molecule composition. Methods: We applied dual-mode untargeted high-resolution liquid chromatography-tandem mass spectrometry (LC-MS/MS), integrating hydrophilic interaction (HILIC) and reversed-phase C18 separations, to profile five commercial RTD beverages spanning distinct formulation categories: Coca-Cola^®, Poppi^® Orange, OLIPOP^® Cream Soda, Pure Leaf^® Unsweetened Black Tea, and BeePop™ Peach + Orange Blossom Honey. Results: Across all products, 478 compounds were structurally annotated at Metabolomics Standards Initiative (MSI) Levels 1 and 2, of which 42 matched compounds with reported bioactivity in a curated literature-based reference database. Seventeen compounds-including the NAD+ precursor trigonelline and multiple B vitamins-were detected across all five products. The number and diversity of compounds with reported bioactivity varied substantially by product and correlated with botanical ingredient complexity. Conclusions: This work presents a qualitative molecular survey of the RTD beverage category using standardized, dual-mode untargeted metabolomics, providing a reference dataset for future targeted quantitation studies.