Observation of exchangeable protons by high-performance liquid chromatography–nuclear magnetic resonance spectroscopy and high-performance liquid chromatography–electrospray ionization mass spectrometry: a useful tool for the hyphenated analysis of natura

The first high-performance liquid chromatography–nuclear magnetic resonance (HPLC–NMR) investigation of exchangeable protons of low-molecular-mass natural products is reported. Model alkaloids or crude plant extracts were dissolved in 2H2O–1H2O–MeCN (deuterium oxide–water–acetonitrile) or 2H2O–MeCN and, after direct injection or chromatographic separation, examined in a 60-μl NMR flow probe. Exchangeable amino protons initially detected by HPLC–electrospray ionization mass spectrometry were subsequently identified and investigated by stop-flow 1H-NMR, two-dimensional (2D) total correlation spectroscopy (TOCSY), and 2D nuclear Overhauser effect spectroscopy (NOESY). These experiments extend the applicability of HPLC–NMR for the investigation and structure elucidation of natural products.