Discovery of active components in herbs using chromatographic separation coupled with online bioassay

•The current analytical techniques for the discovery of bioactive compounds were reviewed.•The different subtypes of online bioassay will be discussed.•Examples are presented to illustrate the strengths and limitations of different methods.

Discovery of bioactive compounds from complex mixtures is a challenge. In past decades, several strategies were developed and implemented for rapid and effective screening and characterization of bioactive components in complex matrices. This review mainly focused on the online strategies, which integrated the separation science, mass spectrometry, and bioactivity screening in a single platform, allowing simultaneous screening and characterization of active compounds from complex matrices, especially from the herbs. The online screening methodologies, including pre-column affinity-based screening and post-column bioassay, were discussed and their applied examples were also presented to illustrate the strengths and limitations of these approaches.