Polarity-Gradient NADES-Aqueous Biphasic Extraction Platform for Enhancing Honey Volatile Metabolite Profiles

This study presents an integrated analytical strategy combining a gradient natural deep eutectic solvent-based aqueous biphasic system (NADES-ABS) and comprehensive metabolomic profiling to efficiently extract and analyze honey VOCs. A novel polarity-gradient NADES-ABS was developed by combining a sugar-based NADES with five small-molecular alcohols of varying degrees of polarity. The phase behavior and partitioning characteristics were then systematically evaluated. Single-factor experiments and response surface methodology (RSM) were used to determine the optimal extraction conditions: temperature 46.68 °C, pH 7.67, alcohol concentration 32.32 wt %, honey concentration 0.29 g/mL, NADES concentration 20 wt %, and extraction time 20 min. Density functional theory (DFT) and molecular dynamics (MD) simulations provided molecular-level insights into the partitioning mechanisms. GC-MS-based metabolomic analysis enabled detailed VOC profiling of four major types of Chinese honey. The environmental sustainability of the method was quantitatively evaluated using the AGREEprep (0.71) and analytical GREEnness (0.70) metrics. This integrative workflow improves our understanding of the volatile metabolome of honey and provides a reliable tool for authentication, quality control, and the discovery of bioactive markers.