Abstract: Rapid identification and efficient structural elucidation of metabolites are vitally important in metabonomics, phytochemistry and drug discoveries from the natural sources. In this work, the potentials of the hyphenated HPLC-DAD-SPE-CryoNMR-MS techniques were demonstrated in characterization and structural elucidation using the complex extracts of rosemary (Rosmarinus of ficinalis L.) as an example. 6 metabolites, including 1 phenolic diterpene (carnosol), 2 phenolic acids (rosmarinic acid and caffeic acid), 2 flavonoids (6-methoxyluteolin-7-glucoside and homoplantaginin) and 1 coumaric acid (cis-4-glucosyloxycinnamic acid), were successfully identified from two rosemary extracts with chloroform-methanol (v/v, 3/1) and 50% aqueous methanol on the basis of HPLC, UV, NMR and MS data. Such structural determination benefited from the capability of acquiring the high-quality homo- and heteronuclear 2D NMR spectra with the analytical HPLC separation. The results have not only provided whole set spectroscopic data for these metabolites, but also proved the hyphenated HPLC-DAD-SPE-CryoNMR-MS method to be an important structural elucidation platform for the components in natural products and probably other complex mixtures, even when multiple components in the same chromatographic peak are present with concentration differences in an order of magnitude.

Key words: NMR, structural elucidation, HPLC-DAD-SPE-CryoNMR-MS, rosemary (Rosmarinus of ficinalis L.) 