Quantitative TLC-based assay for the detection of urease inhibitors in complex mixtures

Urease inhibitors are increasingly relevant in agricultural and food chemistry because urease activity influences nitrogen-use efficiency, ammonia emissions, microbial survival, and food quality. Although numerous solution-based assays are available, no quantitative thin-layer chromatography (TLC)-based bioautographic method has been reported for detecting and localizing urease inhibitors directly within complex mixtures. This study aimed to develop and validate a quantitative TLC bioautographic assay suitable for effect-directed analysis of food-related matrices.

The assay combines chromatographic separation, urease immobilization in an agar layer, and ammonia detection through the Berthelot reaction to quantitatively visualize urease inhibition zones while avoiding false-positive responses associated with pH indicator-based detection. Validation showed linearity for thiourea over 0.47-15 μg/spot (R² > 0.999), with LOD and LOQ values of 0.15 ± 0.08 and 0.46 ± 0.25 μg/spot, respectively, and intra-plate and intraday RSDs of 7.6-11.8% and 9.9%, respectively. Parallel screening of essential oils enabled rapid comparative evaluation of urease-inhibitory activity, while direct gas chromatography-mass spectrometry analysis of active chromatographic zones allowed microscale identification of eugenol as the principal bioactive constituent without preparative isolation.

This work presents the first quantitative TLC-based bioautographic assay for urease inhibitors and establishes an integrated effect-directed analytical workflow that combines quantitative activity localization with direct molecular identification. The methodology provides a simple and cost-effective platform for screening and characterizing urease inhibitors in complex food- and agriculture-related matrices, facilitating the discovery of bioactive compounds with potential applications in food preservation, quality control, and sustainable agricultural practices.