Hydrophobic deep eutectic solvent-based green sample pretreatment strategy for chromatographic-mass spectrometric...

Anthraquinones from Rheum palmatum L. possess diverse pharmacological activities; however, their intrinsic hydrophobicity limits extraction efficiency using conventional solvents and hampers the simultaneous enrichment of compounds with different polarities. In this study, a green sample pretreatment strategy based on hydrophobic deep eutectic solvents (HDESs) was developed for the enrichment of anthraquinones from Rheum palmatum L. prior to chromatographic-mass spectrometric analysis. Systematic screening of 37 HDESs identified a citral-cinnamyl alcohol (1:1) system with superior solubilization capability toward target anthraquinones, including emodin, rhein, and aloe-emodin. Response surface methodology combined with machine learning analyses identified the solid-to-liquid ratio as the most critical parameter governing extraction efficiency. Under optimized conditions, the total yield of free anthraquinones reached 135.67 mg/g, far exceeding that obtained by conventional methanol extraction (84.79 mg/g). UPLC-Q-TOF-MS profiling showed that the HDES system enabled not only improved enrichment of free anthraquinones but also simultaneous extraction of glycosylated derivatives, thereby expanding the chemical coverage of the extract. Mechanistic analyses suggested that electrostatic complementarity, hydrogen bonding, π-π-related interactions, and van der Waals interactions may collectively contribute to the solubilization and selective enrichment behavior of HDESs. In vitro antioxidant assays demonstrated that HDES extracts exhibited markedly higher radical scavenging activity than methanol extracts in DPPH, ABTS, and hydroxyl radical systems, with a DPPH IC₅₀ value of 16.85 μg/mL compared with 55.81 μg/mL for methanol extracts. Overall, this work establishes a green and efficient HDES-based pretreatment strategy for chromatographic-mass spectrometric analysis of hydrophobic natural products, with support from synergistic noncovalent interactions.