A high-performance thin-layer chromatography-bioautography-mass spectrometry approach for screening β-lactamase...

A high-performance thin-layer chromatography-bioautography (HPTLC-bioautography) method was developed for the rapid screening of β-lactamase inhibitors based on nitrocefin chromogenic hydrolysis. By integrating chromatographic separation with on-plate enzymatic detection, the proposed approach enables direct localization of β-lactamase inhibitory constituents from complex matrices while effectively minimizing matrix and sample color interference. Key experimental parameters were systematically optimized, and the method exhibited good flexibility by allowing screening against four different classes of β-lactamases through enzyme selection. Quantitative analysis was achieved by densitometric scanning at 525 nm, corresponding to the maximum absorption of the hydrolysis product. The method showed a limit of detection of 2 ng and a limit of quantification of 8 ng for the reference inhibitor tazobactam, along with satisfactory precision, repeatability, accuracy, and stability. The applicability of the method was demonstrated by screening extracts from twelve herbal medicines, leading to the identification of β-lactamase inhibitory zones in Perilla folium, Lonicera japonica Flos, and Lonicera Flos. Two active constituents were further characterized as rosmarinic acid and isochlorogenic acid A using online HPTLC-mass spectrometry, and their activities were verified by molecular docking and microplate-based enzymatic assay. Overall, the proposed HPTLC-bioautography strategy provides an efficient and selective analytical platform for the screening and characterization of β-lactamase inhibitors in complex natural products.