LC-MS/MS-BASED MOLECULAR NETWORKING OF THE RESIDUAL METABOLOME OF Rhizopus arrhizus 8.2 FROM Ziziphora pedicellata PAZIJ ET VVED
Abstract
R. arrhizus 8.2 was cultivated in potato dextrose broth for 14 days at 28 °C and 150 rpm. The culture broth was extracted with ethyl acetate, and kojic acid was isolated from the crude extract. The residual fraction was analyzed using an Orbitrap Exploris 120 LC-MS/MS system with an ACE Excel Super C18 column (250 x 4.6 mm, 5 µm). Mobile phases were 0.1% formic acid in water and methanol; data were acquired in positive H-ESI mode over m/z 100-1600. Raw files were converted to mzML, processed in MZmine 3, submitted to GNPS for feature-based molecular networking, and visualized in Cytoscape. The LC-MS/MS profile demonstrated that the post-kojic acid fraction is a chemically complex metabolite pool rather than an inactive by-product. Putatively annotated metabolite families included furocoumarins, phenolic and phenylpropanoid derivatives, tyrosine-derived alkaloid-like compounds, oxidized fatty-acid metabolites, oxygenated heterocycles, and indole-containing pyrrolinone derivatives. Selected metabolites of interest were oxypeucedanin methanolate, hordenine, 5-allyloxypsoralen, heraclenin, salicin, 3,4-dihydroxy-beta-phenylpropionic acid, 5-oxo-6E,8Z,11Z,14Z-eicosatetraenoic acid, and pyrrolinone derivatives. Molecular networking grouped related MS/MS features into metabolite families and improved interpretation of minor compounds that could be overlooked by conventional targeted analysis.
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