Systematic assessment of column selectivity for the isolation of subcordatine and other flavonoid glycosides from Strychnos subcordata spruce ex Benth using off-line single heart-cutting two-dimensional liquid chromatography.

The genus Strychnos has gained worldwide recognition due to its role in the preparation of curare, a traditional arrow poison used by Indigenous peoples in South America, and its potential as a promising source of bioactive natural products. Although the phytochemical content of several Strychnos species has been extensively investigated, others, such as S. subcordata, remain largely unexplored. In this study, we describe for the first time the systematic screening of the selectivity of selected columns (C18, C8, PFP, phenyl-hexyl, CN and biphenyl) for the isolation of target compounds from the methanol fraction of S. subcordata leaves based on the off-line single heart-cutting two-dimensional liquid chromatography (2D-LC) approach. Among these compounds, an unknown isoflavone, herein named subcordatine, and two rare flavonoid glycosides containing a terminal apiosyl unit were isolated and elucidated using 1D and 2D NMR analyses, as well as MS. Among the tested configurations, the combination of a C18 column in the first dimension (1D) and a biphenyl column in the second dimension (2D) proved particularly effective for isolating the target compounds in higher yields. Subcordatine, in particular, exhibited stronger retention on phenyl-type columns, especially the biphenyl column, likely due to conformational similarities between isoflavones and the biphenyl moieties in the stationary phase. These findings underscore the importance of specific molecular interactions in chromatographic selectivity and highlight the potential of off-line heart-cutting 2D-LC methodologies for isolating structurally complex natural products.