Determination of coumarin via a two-step chromogenic reaction coupled with capillary electrophoresis in legume seeds.

Coumarin is a naturally occurring secondary metabolite found in many plants and exhibits various biological activities. However, robust and reliable techniques for the simultaneous qualitative and quantitative analysis of coumarin are still lacking. This study focused on optimizing the chromogenic reaction of coumarin with the Gibbs reagent by utilizing borate as a chelating agent to enable sensitive detection through capillary electrophoresis (CE). It was observed that alkaline hydrolysis of coumarin at pH 12.1 produced the strongest color response in the presence of borate, which was further enhanced by incubation at 60 °C. In the chromogenic phase, a pH of 8.8 was associated with both high reaction intensity and greater stability of the colored product relative to higher or lower pH values. After optimizing the color development process, CE analysis of coumarin exhibited a strong linear correlation within the concentration range of 10-200 mg L-1. Using this method, coumarin concentrations in Medicago sativa and Trifolium pratense seeds were determined to be 608.4 and 26.8 mg kg-1, respectively. Spike-and-recovery rates of 93-98% confirmed the accuracy of the method. This study provides an alternative and efficient tool for qualitative and quantitative determination of coumarin.