The quantification is based on the method described in: Kyllingsbæk, A.(1975). Extraction and colorimetric determination of urea in plants. Acta Agriculturæ Scandinavica 25, 109-112.
Colour reagent
TSC-solution: 27.4 mM thiosemicarbacide
DAM-solution: 250 mM diacetylmonoxime (2.3-butandione monoxime) and 200 mM KNO3
Acid solution: 175 ml HCl (37%) and 100 ml H2PO4 (85%), made up to 500 ml with water
Immediately before the assay the three stock solutions are mixed in a relation of 1:2.5:37.5 (TSC:DAM:Acid), giving the color reagent.
Assay
20 microliters of aquous leaf extracts and 180 microliters of water are mixed in a microfuge tubes.
600 microliters of freshly prepared colour reagent is added.
The microfuge tubes are closed, inverted several times, and placed in a water bath at 80°C for 30 min. The bath was covered with tin foil to prevent light exposure of the samples (the colour formed is light sensitive).
Standards are prepared by mixing 200 microliters of 18.8, 37.5, 75, 150 microM urea solutions with 600 microliters colour reagent processing in parallel to the samples.
Samples are briefly cooled in a room-temperature water bath and spectrophotometric measurements are performed at 527 nm. Light exposure should be kept low.
Remarks
The inclusion of potassium nitrate into the DAM solution of the Kyllingsbæk reagents increases the sensitivity of the colorimetric urea determination. The method was found to be about four times more sensitive than a similar method described in Bremner, J.M. In: Methods of Soil Analysis, Part 2. Nitrogen-urea.
The calibration with pure urea solutions is only sufficiently accurate if not more than 20 microliters leaf extract (potato) are used in the assay. Greater volumes interfere with the level of urea detection. However, calibration solutions may be made by mixing urea standards with leaf extracts from which (ideally) urea was removed (by urease treatment). [Since the leaf urea concentration in untreated (not urea sprayed) potato leaves was found to be very low (close to the detection limit) and most of the low signal obtained from these leaves was not due to urea, the urease treatment might not be necessary.]
