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.

Dereplication of Natural Extracts Diluted in Propylene Glycol, 1,3-Propanediol and Glycerin. Comparison of Leontopodium alpinum Cass. (Edelweiss) Extracts as a Case Study

“…Details of each fractionation are presented in Table 1. The method employed for the processing of the glycerinated extract has been previously described in [7]. First, solvent systems were prepared in a separatory funnel by mixing in the required amounts of pure solvents.…”

“…The aim of this research work was to develop an original and efficient workflow for the rapid analysis of the major compounds in natural extracts diluted in propylene glycol or propanediol, two carrier solvents largely present in marketed cosmetic ingredients. The adopted strategy is based on the one used in our previous works on the dereplication of glycerinated natural extracts [7]. Nevertheless, the physical removal of carrier solvent by CPC fractionation using the biphasic solvent system EtOAc/CH 3 CN/water 3:3:4 (v/v/v), as used for glycerinated extracts, cannot be applied as such to extracts prepared in propanediols.…”

“…The dereplication workflow was thus started on the concentrated edelweiss extracts using the previously described procedure involving among others a CPC fractionation step with the biphasic solvent system EtOAc/CH 3 CN/water 3:3:4 (v/v/v) in the isocratic ascending elution mode [7]. Although the preliminary partition tests showed a good distribution of the sample compounds between the two phases of the biphasic solvent system (Figure S4), the fractionation by CPC was not satisfactory.…”

“…This article presents an original and efficient workflow for the rapid identification of the main compounds in natural extracts diluted in propylene glycol and propanediol, which complements the recently published work with a glycerinated extract [7]. The original identification procedure, published in 2014 [8], has already been used to characterize hundreds of dry extracts [9][10][11][12][13] both for academic or industrial purposes.…”

“…Secondly, it makes NMR analysis more complex due to the very intense solvent peaks and due to the presence of numerous artefacts they induce in the spectra. These issues were recently overcome for glycerinated extracts by optimizing the original procedure through a combination of a physical suppression of glycerin by CPC and of a spectroscopic suppression of glycerin signals by presaturation in 13 C NMR [7]. Moreover, the presaturation in 13 C NMR solvent signals was already reported for the signals of the two propanediols, leading to an intensity decrease by at least 94% and to the suppression of the related artefacts [14].…”