Simultaneous determination of ammonium and urea in a single matrix using colorimetric methods: application to urease inhibitors

Urea hydrolysis is a key process governing nitrogen availability and losses in agricultural systems, yet its accurate assessment remains constrained by the lack of simple methodologies for simultaneous determination of urea and ammonium in a single matrix. This limitation complicates the evaluation of urease activity and inhibitor efficiency. The aim of this study was to develop and validate a fast and reliable colorimetric method for the simultaneous quantification of urea and ammonium in 2 M KCl extracts, enabling integrated monitoring of nitrogen transformations. The method combines the diacetyl monoxime reaction for urea with the salicylate-Berthelot reaction for ammonium, allowing both analytes to be determined using standard UV–Vis spectrophotometry. The approach demonstrates excellent linearity (R 2 > 0.999) within the working range, with limits of detection of 0.7 mmol L −1 for urea and 0.3 mmol L −1 for ammonium. Validation experiments confirmed high accuracy and precision, with recoveries close to 100% and relative standard deviations below 3%. No significant mutual interference between the analytes were observed. The method also maintained a consistent nitrogen mass balance (100 ± 7%), supporting its robustness for monitoring urea hydrolysis. Application of the method to urease inhibitors in vitro revealed that N-(n-butyl) thiophosphoric triamide (NBPT) and acetohydroxamic acid (AHA) produced strong inhibition of urea hydrolysis, reducing ammonium formation by up to 73–77% after 72 h. In contrast, potassium thiosulfate and pyoverdine exhibited limited inhibition, while Metschnikowia pulcherrima extracts showed variable performance, with inhibition values up to 63%. Further studies using interaction with agricultural soils are needed. This method provides a simple, cost-effective, and robust approach for the simultaneous determination of urea and ammonium in a single extract, enabling direct monitoring of substrate transformation and product formation. Its application facilitates the evaluation of urease inhibitors in vitro and supports improved nitrogen management strategies, contributing to enhanced efficiency and more sustainable agricultural practices.