Dereplication of Natural Extracts Diluted in Glycerin: Physical Suppression of Glycerin by Centrifugal Partition Chromatography Combined with Presaturation of Solvent Signals in 13C-Nuclear Magnetic Resonance Spectroscopy

Abstract

For scientific, regulatory, and safety reasons, the chemical profile knowledge of natural extracts incorporated in commercial cosmetic formulations is of primary importance. Many extracts are produced or stabilized in glycerin, a practice which hampers their characterization. This article proposes a new methodology for the quick identification of metabolites present in natural extracts when diluted in glycerin. As an extension of a 13C nuclear magnetic resonance (NMR) based dereplication process, two complementary approaches are presented for the chemical profiling of natural extracts diluted in glycerin: A physical suppression by centrifugal partition chromatography (CPC) with the appropriate biphasic solvent system EtOAc/CH3CN/water 3:3:4 (v/v/v) for the crude extract fractionation, and a spectroscopic suppression by presaturation of 13C-NMR signals of glycerin applied to glycerin containing fractions. This innovative workflow was applied to a model mixture containing 23 natural metabolites. Dereplication by 13C-NMR was applied either on the dry model mixture or after dilution at 5% in glycerin, for comparison, resulting in the detection of 20 out of 23 compounds in the two model mixtures. Subsequently, a natural extract of Cedrus atlantica diluted in glycerin was characterized and resulted in the identification of 12 metabolites. The first annotations by 13C-NMR were confirmed by two-dimensional NMR and completed by LC-MS analyses for the annotation of five additional minor compounds. These results demonstrate that the application of physical suppression by CPC and presaturation of 13C-NMR solvent signals highly facilitates the quick chemical profiling of natural extracts diluted in glycerin.