HPLC-SPE-NMR hyphenation in natural products research: optimization of analysis of Croton membranaceus extract

摘要

The HPLC-SPE-NMR technique was used for the analysis of a root-bark extract of Croton membranaceus. The components of the extract were separated on an analytical-size reversed-phase HPLC column, the chromatographic peaks were trapped on SPE (solid-phase extraction) cartridges after post-column dilution of the eluate with water and the compounds were eluted from the cartridges with acetonitrile-d(3) into a 30 mu l 600 MHz NMR probe in a fully automated procedure. The trapping efficiency of scopoletin (1), the major extract constituent, was much higher on a GP (general phase, a polystyrene-type polymer) SPE phase than on a C18 phase. Thus, under the conditions used, up to 100 mu g of scopoletin per cartridge could be accumulated linearly after repeated trappings. The maximum achievable NMR signal-to-noise ratio using the GP cartridges was at least four times higher than that achievable with the C18 cartridges. It was shown that excessively long T-1 relaxation times may compromise experiments in which acetonitrile-d3 is used as the cartridge eluent. Nevertheless, the sensitivity gain provided by the HPLC-SPE-NMR technique through repeated peak trappings allowed the acquisition of good-quality proton-detected 2D NMR spectra without the need for solvent suppression. Copyright (c) 2005 John Wiley & Sons, Ltd.