Direct FluorometricDeterminationof Urea in Urine

In this quantitative fluorometric method, diacetyl-monoxime is used for the determination of the urea. The products of the reaction of urea with diacetylmonoxime in acid solution exhibit two fluorescence maxima, at 410 and 525 nm. The in-tensity of the 525 nm maximum is linear over a wide range of urea concentrationand the reactionis shown to be practically specific for urinary urea. Additional Keyph rases urea-diacetylmonoxime re-action #{149}fluorometry vs. colorimetry Urea in biological fluids has been determined by a variety of direct and indirect procedures. Some indirect methods depend on the hydrolysis of urea with the enzyme urease to form ammonia, with subsequent quantitation of the ammonia by nesslerization (1-4). Undesirable features of these indirect methods are the use of relatively unstable reagents and loss of ammonia during the procedure. Direct determination of urea depends on the con-densation of urea with some specific reagent such as a-isonitrosopropiophenone (5) and diacetyl derivatives (6-15) in acid media. The direct reaction between urea and diacetyl or diacetyl derivatives appears to offer distinct advantages for urea determination. The products of the reaction of urea with diacetyl-monoxime are fluorescent (16). The fluorescence maxi-mum at 415 nm on optimal excitation at 380 nm has been used for the quantitative determination of urea. However, the fluorescence intensity of the reaction products at this wavelength is not directly proportional to the concentration of urea. I have further examined the reaction of urea with diacetylmonoxime with respect to fluorescence maxima of the products, sample concentration, specificity, and kinetics. The improved method was then applied to determine urea in human and primate urine