Comprehensive identification of polyphenolic metabolites in aspen knotwood by combination of 2D NMR and HPLC‐HRMS

Abstract Introduction European aspen ( Populus tremula L.) knotwood contains large amounts of polyphenolic metabolites, mainly flavonoids, and can be considered as a promising industrial‐scale source of valuable bioactive compounds. Valorization of knotwood extractives requires detailed information on their chemical composition and a relevant analytical methodology. Objective This study proposes combined analytical strategy for non‐targeted screening and identification of polyphenolic plant metabolites and is aimed at comprehensive characterization of knotwood extractives. Materials and methods Aspen knotwood acetone extract with determined antioxidant activity was an object of the study. Two‐dimensional NMR spectroscopy with Structure Elucidator expert system was used for preliminary search of major components and specific structures. Liquid chromatography–high‐resolution mass spectrometry (HPLC‐HRMS) with data‐dependent MS/MS spectra acquisition was used as a complementary technique providing molecular‐level characterization and identification of the detected metabolites. Results Twenty‐eight phenolic metabolites were found and identified. Among them, flavonoids, aromadendrin and naringenin, as well as their glycosylated derivatives (mainly O ‐glucosides) and methyl ethers, dominated. Taxifolin and its 7‐ O ‐glucoside were detected as minor components. Other detected compounds are represented by p ‐coumaric acid and its rutinoside and small amounts of glycosylated ferulic acid. Nineteen of the detected compounds were discovered in aspen knotwood for the first time. The results were confirmed by preparative isolation of individual compounds and NMR studies. Conclusion The proposed analytical strategy based on 2D NMR and HPLC‐HRMS can be considered a powerful tool in the analysis of plant extractives and allowed for the identification and semi‐quantification of a large number of polyphenols in aspen knotwood.