Covering: 2013 to 2019 The exploration of the chemical diversity of extracts from various biological sources has led to major drug discoveries. Over the past two decades, despite the introduction of advanced methodologies for natural product (NP) research (e.g., dereplication and high content screening), successful accounts of the validation of NPs as lead therapeutic candidates have been limited. In this context, one of the main challenges faced is related to working with crude natural extracts because of their complex composition and the inadequacies of classical bioguided isolation studies given the pace of high-throughput screening campaigns. In line with the development of metabolomics, genomics and chemometrics, significant advances in metabolite profiling have been achieved and have generated high-quality massive genome and metabolome data on natural extracts. The unambiguous identification of each individual NP in an extract using generic methods remains challenging. However, the establishment of structural links among NPs via molecular network analysis and the determination of common features of extract composition have provided invaluable information to the scientific community. In this context, new multi-informational-based profiling approaches integrating taxonomic and/or bioactivity data can hold promise for the discovery and development of new bioactive compounds and return NPs back to an exciting era of development. In this article, we examine recent studies that have the potential to improve the efficiency of NP prioritisation and to accelerate the targeted isolation of key NPs. Perspectives on the field's evolution are discussed.

The isolation of pure compounds from extracts represents a key step common to all investigations of natural product (NP) research. Isolation methods have gone through a remarkable evolution. Current approaches combine powerful metabolite profiling methods for compounds annotation with omics mining results and/or bioassay for bioactive NPs/biomarkers priorisation. Targeted isolation of prioritized NPs is performed using high-resolution chromatographic methods that closely match those used for analytical profiling. Considerable progress has been made by the introduction of innovative stationary phases providing remarkable selectivity for efficient NPs isolation. Today, efficient separation conditions determined at the analytical scale using high- or ultra-high-performance liquid chromatography can be optimized via HPLC modelling software and efficiently transferred to the semi-preparative scale by chromatographic calculation. This ensures similar selectivity at both the analytical and preparative scales and provides a precise separation prediction. High-resolution conditions at the preparative scale can notably be granted using optimized sample preparation and dry load sample introduction. Monitoring by ultraviolet, mass spectrometry, and or universal systems such as evaporative light scattering detectors and nuclear magnetic resonance allows to precisely guide the isolation or trigger the collection of specific NPs with different structural scaffolds. Such approaches can be applied at different scales depending on the amounts of NPs to be isolated. This review will showcase recent research to highlight both the potential and constraints of using these cutting-edge technologies for the isolation of plant and microorganism metabolites. Several strategies involving their application will be examined and critically discussed. Graphical abstract