Chemical profiling of botanical extracts obtained in NADES systems using centrifugal partition chromatography combined with 13C NMR dereplication—Hypericum perforatum as a case study.

Introduction: Natural deep eutectic solvents (NADES) have emerged as interesting extractants to develop botanical ingredients. They are nontoxic and biodegradable, nonflammable, easy to prepare, and able to solubilize a wide range of molecules. However, NADES extracts remain difficult to analyze because the metabolites of interest stay highly diluted in the nonvolatile viscous NADES matrix. Objective: This study presents a robust analytical workflow for the chemical profiling of NADES extracts. It is applied to Hypericum perforatum aerial parts extracted with the neutral mixture fructose/glycerol/water (3/1/1, w/w/w), and compared to the chemical profiling of a classical dry methanol extract. Methods: Exploiting polarity differences between metabolites, the H. perforatum NADES extract was partitioned in a liquid–liquid solvent system to trap the hydrophilic NADES constituents in the lower phase. The upper phase, containing a diversity of secondary metabolites from H. perforatum, was fractionated by centrifugal partition chromatography. All fractions were chemically investigated using a 13C NMR dereplication method which involves hierarchical clustering analysis of the whole NMR dataset, a natural metabolite database for metabolite identification, and 2D NMR analyses for validation. Liquid chromatography–mass spectrometry (LC‐MS) analyses were also performed to complete the identification process. Results: A range of 21 metabolites were unambiguously identified, including glycosylated flavonols, lactones, catechins, phenolic acids, lipids, and simple sugars, and 15 additional minor extract constituents were annotated by LC‐MS based on exact mass measurements. Conclusion: The proposed identification process is rapid and nondestructive and provides good prospects to deeply characterize botanical extracts obtained in nonvolatile and viscous NADES systems. Botanical ingredients developed in NADES remain difficult to analyze because the metabolites of interest are diluted in the nonvolatile viscous NADES matrix. A robust analytical workflow was developed on Hypericum perforatum aerial parts extracted with the neutral mixture fructose/glycerol/water (3/1/1, w/w/w). The extract was partitioned in a biphasic system to trap hydrophilic NADES constituents in the lower phase. The upper phase was chemically profiled by NMR dereplication. 21 metabolites were unambiguously identified and 15 minor compounds were annotated by LC‐MS. [ABSTRACT FROM AUTHOR]