A single-plate strategy for simultaneous chemical and biological profiling of natural products via TLC-bioautography-ambient mass spectrometry.

In conventional natural product research, the separation of bioactivity screening from chemical characterization often creates a disconnect, hindering the direct identification of active constituents. To bridge this gap, we present an integrated single-plate strategy that combines thin layer chromatography-bioautography with ambient ionization mass spectrometry (AIMS) for direct in situ identification. This approach utilizes two complementary techniques, graphite-assisted laser ablation direct analysis in real-time mass spectrometry (LA-DART-MS) and thin-layer chromatography-spray ionization mass spectrometry (TLC-SI-MS), to enable the rapid characterization of bioactive zones. The versatility of this platform was demonstrated by successfully profiling diverse bioactivities in herbal matrices, including acetylcholinesterase, lipase and α-glucosidase inhibitors, as well as antioxidant and antibacterial agents. The complementary use of AIMS techniques yielded robust datasets for these complex samples. Notably, the system enabled the discovery of differential antioxidant markers between northern and southern hawthorn, with structural validation confirmed by nuclear magnetic resonance (NMR) spectroscopy. These findings provide a scientific basis for the quality control of hawthorn. This single-plate strategy serves as an efficient tool for the integrated screening and structural characterization of natural products, effectively linking biological function with chemical composition.