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 natural products

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-microl 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.