HPLC−NMR On-Line Coupling Including the ROESY Technique: Direct Characterization of Naphthylisoquinoline Alkaloids in Crude Plant Extracts

Abstract

Directly coupled 600-MHz HPLC−1H NMR was used for the first time to investigate the potential of this technique in the screening of tropical plants for naphthylisoquinoline alkaloids. A leaf extract of Ancistrocladus guineënsis was analyzed both in the on-flow mode and in the stop-flow mode using reversed-phase isocratic and gradient compositions of acetonitrile and D2O on a C18 column. On-flow experiments proved the occurrence of naphthylisoquinoline alkaloids in the extract. Stop-flow measurements confirmed the presence of ancistrotectorine and two new diastereomeric alkaloids. With 2D TOCSY experiments performed in the stop-flow mode, it was possible to deduce their constitution. For the elucidation of the relative configuration, a 2D HPLC−NMR ROESY experiment was developed by supplying a conventional ROESY sequence with a double presaturation block during the relaxation delay. Application of the optimized ROESY method confirmed the constitution of the two new alkaloids and showed relative cis and trans configurations of the methyl groups at the isoquinoline moieties. These results underline the value of this approach for a rapid and reliable phytochemical screening of plant extracts for naphthylisoquinoline alkaloids. The identification of known alkaloids as well as the characterization of new structures is possible without any purification step. The results were used as the basic information for the selective isolation of the new structures.