Hyphenation of solid-phase extraction with liquid chromatography and nuclear magnetic resonance:: Application of HPLC-DAD-SPE-NMR to identification of constituents of Kanahia laniflora

The introduction of on-line solid-phase extraction (SPE) in HPLC-NMR has dramatically enhanced the sensitivity of this technique by concentration of the analytes in a small-volume NMR flow cell and by increasing the amount of the analyte by multiple peak trapping. In this study, the potential of HPLC-DAD-SPE-NMR hyphenation was demonstrated by structure determination of complex constituents of flower, leaf, root, and stem extracts of an African medicinal plant Kanahia laniflora. The technique was shown to allow acquisition of high-quality homo- and heteronuclear 21) NMR data following analytical-scale HPLC separation of extract constituents. Four flavonol glycosides [kaempferol 3-O-(6-O-alpha-L-rhamnopyranoSyl)-beta-D-glucopyranoside; kaempferol 3-O-(2,6-di-O-alpha-L-rhamnopyranosyl)-beta-D-glucopyranoside; quercetin 3-O(2,6-di-O-a-L-rhamnopyranosyl)-beta-D-glucopyranoside(rutin); and isorhamnetin, 3-O-(6-O-alpha-L-rhamnopyranoSyl)-beta-D-gjucopyranoside] and three 5 alpha-cardenolides [coroglaucigenin 3-O-6-deoxy-beta-D-allopyranoside; coroglaucigenin 3-O-(4-O-beta-D-glucopyranosyl)-6-deoxy-beta-D-glucopyranoside; 3'-O-acetyl-3'-epiafroside] were identified, with complete assignments of H-1 and C-13 resonances based on HSQC and HMBC spectra whenever required. Confirmation of the structures was provided by HPLC-MS data. The HPLC-DAD-SPE-NMR technique therefore speeds up the dereplication of complex mixtures of natural origin significantly, by characterization of individual extract components prior to preparative isolation work.