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

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Title: Quantitative TLC-based assay for the detection of urease inhibitors in complex mixtures.
Authors: Cotacachi-Latacumba PE; Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Argentina. Electronic address: pcotacachi@fbioyf.unr.edu.ar., Salazar MO; Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Argentina; Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Suipacha 531, Rosario, 2000, Argentina. Electronic address: msalazar@fbioyf.unr.edu.ar., Ramallo IA; Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Argentina; Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Suipacha 531, Rosario, 2000, Argentina. Electronic address: aramallo@fbioyf.unr.edu.ar., Furlan RLE; Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Argentina; Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Suipacha 531, Rosario, 2000, Argentina. Electronic address: rfurlan@fbioyf.unr.edu.ar.
Source: Analytica chimica acta [Anal Chim Acta] 2026 Nov 15; Vol. 1423, pp. 346178. Date of Electronic Publication: 2026 Aug 27.
Publication Type: Journal Article
Language: English
Journal Info: Publisher: Elsevier Country of Publication: Netherlands NLM ID: 0370534 Publication Model: Print-Electronic Cited Medium: Internet ISSN: 1873-4324 (Electronic) Linking ISSN: 00032670 NLM ISO Abbreviation: Anal Chim Acta Subsets: MEDLINE
Imprint Name(s): Publication: Amsterdam : Elsevier
Original Publication: Amsterdam.
MeSH Terms: Urease*/antagonists & inhibitors , Urease*/metabolism , Enzyme Inhibitors*/analysis , Enzyme Inhibitors*/pharmacology, Chromatography, Thin Layer/methods
Abstract: Background: 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.
Results: 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.
Significance: 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.
(Copyright © 2026 Elsevier B.V. All rights reserved.)
Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Contributed Indexing: Keywords: Bioactivity-guided identification; Bioautography; Effect-directed analysis; Mass spectrometry; Natural products
Substance Nomenclature: EC 3.5.1.5 (Urease)
0 (Enzyme Inhibitors)
Entry Date(s): Date Created: 20261006 Date Completed: 20261006 Latest Revision: 20261006
Update Code: 20261007
DOI: 10.1016/j.aca.2026.346178
PMID: 42838678
Database: MEDLINE
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  Data: Quantitative TLC-based assay for the detection of urease inhibitors in complex mixtures.
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  Data: <searchLink fieldCode="AU" term="%22Cotacachi-Latacumba+PE%22">Cotacachi-Latacumba PE</searchLink>; Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Argentina. Electronic address: pcotacachi@fbioyf.unr.edu.ar.<br /><searchLink fieldCode="AU" term="%22Salazar+MO%22">Salazar MO</searchLink>; Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Argentina; Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Suipacha 531, Rosario, 2000, Argentina. Electronic address: msalazar@fbioyf.unr.edu.ar.<br /><searchLink fieldCode="AU" term="%22Ramallo+IA%22">Ramallo IA</searchLink>; Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Argentina; Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Suipacha 531, Rosario, 2000, Argentina. Electronic address: aramallo@fbioyf.unr.edu.ar.<br /><searchLink fieldCode="AU" term="%22Furlan+RLE%22">Furlan RLE</searchLink>; Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Argentina; Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Suipacha 531, Rosario, 2000, Argentina. Electronic address: rfurlan@fbioyf.unr.edu.ar.
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  Data: <searchLink fieldCode="MM" term="%22Urease%22">Urease*</searchLink>/<searchLink fieldCode="MM" term="%22Urease+antagonists+%26+inhibitors%22">antagonists & inhibitors</searchLink> <br /><searchLink fieldCode="MM" term="%22Urease%22">Urease*</searchLink>/<searchLink fieldCode="MM" term="%22Urease+metabolism%22">metabolism</searchLink> <br /><searchLink fieldCode="MM" term="%22Enzyme+Inhibitors%22">Enzyme Inhibitors*</searchLink>/<searchLink fieldCode="MM" term="%22Enzyme+Inhibitors+analysis%22">analysis</searchLink> <br /><searchLink fieldCode="MM" term="%22Enzyme+Inhibitors%22">Enzyme Inhibitors*</searchLink>/<searchLink fieldCode="MM" term="%22Enzyme+Inhibitors+pharmacology%22">pharmacology</searchLink><br /><searchLink fieldCode="MH" term="%22Chromatography%2C+Thin+Layer%22">Chromatography, Thin Layer</searchLink>/<searchLink fieldCode="MH" term="%22Chromatography%2C+Thin+Layer+methods%22">methods</searchLink>
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  Data: Background: 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.<br />Results: 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 (R<superscript>2</superscript> > 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.<br />Significance: 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.<br /> (Copyright © 2026 Elsevier B.V. All rights reserved.)
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  Data: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
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