Liquid chromatography with ultraviolet absorbance-mass spectrometric detection and with nuclear magnetic resonance spectroscopy: a powerful combination for the on-line structural investigation of plant metabolites.

In order to discover new bioactive compounds from plant sources which could become new leads or new drugs, extracts should be submitted at the same time to chemical screening and to various biological or pharmacological targets. Metabolite profiling using hyphenated techniques such as LC/UV, LC/MS and more recently LC/NMR, quickly provides plenty of structural information, leading to a partial or a complete on-line de novo structure determination of the natural products of interest. As a complement to this approach, bioassays performed after LC/microfractionation of the extracts allow efficient localisation of the bioactive LC-peaks in the chromatograms. The combination of metabolite profiling and LC/bioassays provides the possibility of distinguishing between already known bioactive compounds (dereplication) and new molecules directly in crude plant extracts. Thus, the tedious isolation of compounds of low interest can be avoided and targeted isolation of new bioactive products or constituents presenting novel or unusual spectroscopic features can be undertaken. Several examples of rapid localisation of bioactive compounds, based on post-chromatographic bioautographic testing of LC/NMR microfractions and subsequent on-line identification will be illustrated. Application of hyphenated techniques for the efficient characterisation of labile constituents or constituents difficult to separate at the preparative scale will also be mentioned. The possibilities and limitations of LC/UV/NMR/MS and LC/bioassay as well as future development expected in this field will be discussed.