Digitized liquid chromatography-mass spectrometry metabolomics to explore chemical space of Acanthospermum glabratum (DC.) wild

Ethnopharmacological relevance: Acanthospermum glabratum (DC.) Wild. is used by rural communities in KwaZulu-Natal, South Africa, to manage diarrhoea and other gastrointestinal disorders. However, its phytochemical composition remains largely undocumented, limiting scientific validation of its traditional therapeutic applications. This study aimed to enhance metabolome coverage of A. glabratum using multiple extraction solvents and to comprehensively characterise its phytochemical landscape through untargeted LC-MS/MS-based metabolomics Leaves were extracted using methanol, water, ethyl acetate, and petroleum ether. Extracts were analysed on an LC-qTOF-MS platform to obtain both MS and MS/MS data. Spectral processing was performed using MS-DIAL, and metabolite annotation was achieved through GNPS molecular networking. Principal Component Analysis (PCA) was used to assess metabolic variation across solvents, identify clustering patterns, and determine discriminatory metabolites. Thirty-nine metabolites were annotated, including phenolic acids such as caffeic acid, rosmarinic acid, flavonoids (peonidin 3-galactoside, apigenin-7-O-glucuronide), triterpenoids (enoxolone), and alkaloids (quinine). Methanol extracts yielded the broadest chemical diversity, while water and non-polar solvents enriched hydrophilic and lipophilic compounds, respectively. GNPS molecular networking revealed distinct solvent-specific molecular families, several with known anti-diarrhoeal, antimicrobial, and anti-inflammatory relevance. This first comprehensive metabolomic investigation of A. glabratum uncovers substantial chemical diversity that supports its traditional use for diarrhoea. The solvent-dependent metabolic signatures and annotated bioactive candidates provide a foundational metabolomic atlas for future pharmacological validation. Targeted isolation, mechanistic studies, and integration of network pharmacology and multi-omics are recommended to further elucidate therapeutic pathways and safety profiles.