Advantages of a Multi-Platform Workflow Integrating Biphasic Extraction, Chromatography, and NMR for Characterization of Metabolites in Extracts of Malakabi Ajwa Date Seeds.

Background/Objectives: Date seeds contain a wide range of biologically active constituents and have been associated with multiple health benefits. Our study aimed to evaluate the utility of biphasic aqueous methanol/chloroform extraction for separating diverse metabolites from powdered Malakabi Ajwa date seeds and to identify the most effective chromatographic and spectroscopic techniques for their comprehensive profiling and structural characterization. Methods: Biphasic extraction generated organic, aqueous, and insoluble phases. Proper chromatography-mass spectrometry and spectroscopy analytical tools were then utilized to profile the metabolites in the aqueous and organic phases. Results and Conclusions: In the organic phase, fatty acids and their methyl esters were identified using GC-MS and confirmed by 1H NMR, while the absence of an O-H band in the FT-IR spectrum, together with 1H NMR data, supported the natural esterification of these fatty acids. In the aqueous phase, UV-Vis spectroscopy and LC-MS revealed anthocyanin pigments, and LC-MS additionally detected four amino acids. HPLC was used to quantify and validate the sugar composition, and 1H 1D and 2D NMR spectra enabled the assignment of α- and β-glucopyranose, β-fructopyranose, and sucrose signals. 31P NMR indicated that orthophosphate monoesters were the predominant phosphorus-containing compounds in both organic and aqueous phases. Overall, our findings show how an integrated chromatographic and spectroscopic workflow provides a robust strategy for comprehensive metabolite profiling of natural products such as Malakabi Ajwa date seeds.