Reactivity of Creatinine With Alkaline 3,5-Dinitrobenzoate: A New Fluorescent Kidney Function Test

Objectives: To develop a new quantitative fluorometric assay for creatinine with enhanced specificity and inherent fluorescence sensitivity. Design and Methods: Creatinine is reacted with 3,5-dinitrobenzoate under alkaline reaction conditions to produce a fluorophore. Alternatively, 3,5-dinitrobenzoyl chloride or methyl-3,5-dinitrobenzoate may be employed as reagents. Fluorophore development was evaluated for Group IA bases under aqueous and mixed solvent conditions. Results: The chemical reaction between creatinine and 3,5-dinitrobenzoate performed under highly alkaline conditions produced a fluorescent product with excitation and emission maxima near 410 and 475 nm, respectively. The reaction proceeds rapidly in the presence of group IA bases under both aqueous and mixed solvent conditions, in particular with water/dimethylsulfoxide and water/ 1,4-butanediol solvent systems containing 1.0 M LiOH. A linear calibration curve was obtained when the fluorescence intensity measured in microamperes was plotted versus creatinine concentration in the range of 5-50 μmol/L. The detection limit for creatinine was well below 1 μmol/L. Conclusions: A simple, sensitive and highly specific fluorometric method for the determination creatinine has been developed