pH-dependent separation and identification of saponins from Beta vulgaris L. using high-speed countercurrent chromatography and high-resolution mass spectrometry

This study investigated the saponins from Beta vulgaris L. cv. Cylindra roots using a combination of semi-preparative high-speed countercurrent chromatography (HSCCC) and ultra high-performance liquid chromatography coupled with high-resolution mass spectrometry (UHPLC-HRMS/MS). A new solvent system was developed for the HSCCC purification of saponins under varying pH conditions (3, 5, and 7), which significantly affected their separation. The solvent system consisted of 1-butanol, acetone, acetonitrile, and citrate-phosphate buffer (1.0:0.1:0.05:0.8; v/v/v/v). The pH adjustment in HSCCC noticeably affected separation efficiency, with higher pH values generally leading to faster elution. The separation behavior of saponins depended primarily on the type, number, and arrangement of sugar substituents. In the HSCCC system, compounds bearing more sugar residues tend to be eluted earlier because their greater hydrophilicity enhances interactions with the mobile phase. Despite this overall trend, saponins containing terminal hexose or pentose units were eluted later, indicating that such sugars can significantly extend elution time. Additionally, saponins featuring dioxolane moieties showed a particularly strong pH dependence, with pH adjustments causing reversible shifts in the elution order of isomeric pairs. Ten predominant saponins, including the highly concentrated betavulgaroside IV and betavulgaroside III, were quantified with high accuracy using UHPLC-HRMS/MS. The total saponin content in Cylindra was found to be 7.2 g/kg dry extract (DE), significantly higher than previously reported for other B. vulgaris cultivars. Additionally, the study identified 47 saponins, including the novel aglycone norhederagenin (m/z 455.31), thus expanding the phytochemical profile of beetroot.