Urea reagents were prepared as described in Jung et al. (1975) (link). The final Jung working reagent consisted of 100 mg/L o-phthalaldehyde, 215 mg/L N-(1-naphthyl)ethylenediamine, 2.5 mol/L sulfuric acid, 2.5 g/L boric acid, and 0.03% Brij-35. The modified reagent used 513 mg/L primaquine bisphosphate in place of the 215 mg/L N-(1-naphthyl)ethylenediamine reagent. The urea standard was prepared in double-distilled water and contained 5.00 mg/dL urea. To perform the assay, 50 μL of water, 50 μL of the 5.00 mg/dL standard, and 50 μL samples were transferred into separate wells of a clear flat-bottom 96-well plate. Then to each well, 200 μL of freshly prepared working reagent was added and mixed quickly by gently rocking the plate. The reaction was incubated for 1 h at room temperature. Optical densities (OD) at 430 and 505 nm were measured on the plate reader for assays using the modified reagent and the original Jung reagent, respectively.
The calibration curve (Fig. 1A) shows that the assay is linear between 0.00 and 5.00 mg/dL urea. For calculation of the sample urea concentration, the experimenter can choose either to use the slope of the standard curve or to use a single urea concentration (see below). We found that it is sufficient to use one blank (water) and one single urea concentration (5.00 mg/dL) to calculate the sample urea concentrations. In this work, urea concentration in the sample was calculated from the OD values:
[Urea]=ODSAMPLE−ODBLANKODSTANDARD−ODBLANK×n×[Standard](mg/dL) where ODSAMPLE, ODSTANDARD, and ODBLANK are OD430 nm values of the sample, standard, and water blank, respectively. [Standard] is the concentration of the urea standard (5.00 mg/dL or 0.83 mmol/L) and n is the dilution factor. Dilution of samples in distilled water is necessary when sample OD430 nm values are higher than the OD430 nm value for the 5.00 mg/dL urea standard.