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

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 microl 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 microg 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-d(3) 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.