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Accurately weigh a portion of uniform sample containing an estimated amount of free urea
between 0.0150g and 0.0300g into a 100 ml volumetric flask. Use ultrasound for three minutes
to dissolve and then dilute to volume with mobile phase solution and shake well to assure
homogeneity. (Note: For solid samples or fluids containing substantial undissolved solids,
ultrasound for 5 minutes shake periodically over a period of about 15 minutes to ensure that all
urea has an opportunity to dissolve.) Filter a portion through a 0.45 µm porosity (or finer) filter
before injecting onto HPLC column. Samples should be analyzed on the same day as prepared.
Determination
(a)
Inject 10 µl of each urea standard until two consecutive injections of each give the same peak
area within ± 2% for the same standard. Average the peak areas for the accepted standard
determinations.
(b)
Inject 10 µl of prepared sample. Identify the urea peak by retention time relative to a urea
standard and note if the peak area falls within the range of the high and low standards. If not,
prepare a new sample with the weight adjusted to permit peak to fall within the standard range.
(c)
Perform sufficient sample injections (minimum of two) from the same sample flask such that at
least two consecutive determinations yield peak areas which agree with each other to a
precision of at least ± 2%.
2
Determine the average value of agreeing peak areas.
Calculations
Calculate the average instrument urea working standard response from n standards as:
Urea Factor = �� ( ) � /
Where AP is the average response of 2 or more agreeing peak area for the working standard r, W
r
is the
weight of urea in the 100 ml working standard and n is the number of agreeing working standards used
in the calculation.
AOAC Official Method 959.03
1
Reagents
Either fresh commercial 1% urease solution was used or the urease solution was prepared by dissolving
1 gram of urease powder in 100 ml of distilled water, or one gram jack bean meal was transferred into
100 ml of distilled water and shaken for 5 minutes. Ten ml of this solution was transferred into a 250 ml
Erlenmeyer flask, and diluted with 50 ml distilled water. The enzyme activity was determined by
titration with 0.1 N HCl in the presence of methyl purple (Fisher Scientific, Suwanee, GA) to a reddish
purple color, then back titrated with 0.1N NaOH to a green color. The amount of 0.1 N hydrochloric acid
required to neutralize the remainder of solution was calculated and added to this solution. The enzyme