www.megazyme.

com

UREA/AMMONIA
(Rapid)

ASSAY PROCEDURE
K-URAMR 04/20

(For the rapid assay of urea and ammonia in all

samples, including grape juice and wine)

(*50 Assays of each per Kit)

* The number of tests per kit can be doubled if all volumes are halved

© Megazyme 2020
INTRODUCTION:
Urea and ammonia are widely occurring natural compounds. As
urea is the most abundant organic solute in urine, and ammonia is
produced as a consequence of microbial protein catabolism, these
analytes serve as reliable quality indicators for food products such as
fruit juice, milk, cheese, meat and seafood. Ammonium carbonate
is used as a leaven in baked goods such as quick breads, cookies
and muffins. Unlike some other kits, this kit benefits from the
use of a glutamate dehydrogenase that is not inhibited by tannins
found in, for example, grape juice and wine. In the wine industry,
ammonia determination is important in the calculation of yeast
available nitrogen (YAN). YAN is comprised of three highly variable
components, free ammonium ions, primary amino nitrogen (from free
amino acids) and the contribution from the sidechain of L-arginine.1
For the most accurate determination of YAN, all three components
should be quantified, and this is possible using Megazyme’s
L-Arginine/Urea/Ammonia Kit (K-LARGE) and NOPA Kit
(K-PANOPA). Urea determination can be important in preventing
the formation of the known carcinogen ethyl carbamate (EC) in
finished wine.

PRINCIPLE:
Urea is hydrolysed to ammonia (NH3) and carbon dioxide (CO2) by
the enzyme urease (1).

(urease)
(1) Urea + H2O 2NH3 + CO2

In the presence of glutamate dehydrogenase (GlDH) and reduced

nicotinamide-adenine dinucleotide phosphate (NADPH), ammonia (as
ammonium ions; NH4+) reacts with 2-oxoglutarate to form
L-glutamic acid and NADP+ (2).

(GlDH)
(2) 2-Oxoglutarate + NADPH + NH4+ L-glutamic acid + NADP+ + H2O

The amount of NADP+ formed is stoichiometric with the amount of

ammonia. For each mole of urea reacted, two moles of NADPH are
consumed. NADPH consumption is measured by the decrease in
absorbance at 340 nm.2

1
SPECIFICITY, SENSITIVITY, LINEARITY AND PRECISION:
The assay is specific for urea and ammonia. In the analysis of reagent grade
urea and ammonium sulphate, results of approx. 100% can be expected.
The smallest differentiating absorbance for the assay is 0.005 absorbance
units. This corresponds to 0.018 mg of ammonia (or 0.031 mg urea)/L
of sample solution at the maximum sample volume of 2.00 mL. The
detection limit is 0.071 mg of ammonia (or 0.1258 mg of urea)/L, which
is derived from an absorbance difference of 0.020 with the maximum
sample volume of 2.00 mL.
The assay is linear over the range of 0.2 to 7 μg of ammonia (0.3 to
14 μg of urea) per assay (Figure 1, page 12). In duplicate determinations
using one sample solution, an absorbance difference of 0.005 to 0.010
may occur. With a sample volume of 2.00 mL, this corresponds to an
ammonia concentration of approx. 0.018 to 0.035 mg/L (or 0.031 to
0.063 mg of urea/L) of sample solution. If the sample is diluted during
sample preparation, the result is multiplied by the dilution factor, F. If,
in sample preparation, the sample is weighed, e.g. 10 g/L, a difference of
0.02 to 0.05 g/100 g can be expected.

INTERFERENCE:
If the conversion of urea and ammonia has been completed within
the times specified in the assays, it can be generally concluded that
no interference has occurred. However, this can be further checked
by adding ammonia [approx. 4 μg in 0.1 mL (not supplied)] or urea
(approx. 7 μg in 0.1 mL) to the cuvette on completion of the reaction. A
significant decrease in the absorbance should be observed.
Interfering substances in the sample being analysed can be identified by
including an internal standard. Quantitative recovery of this standard
would be expected. Losses in sample handling and extraction are
identified by performing recovery experiments, i.e. by adding ammonia or
urea to the sample in the initial extraction steps.
In alkaline buffer solution, protein fragments may slowly release ammonia
which can lead to a slow creep reaction. This is not a problem
because the reaction is completed so quickly. Tannins in fruit juice can
lead to significant inhibition of GlDH from beef liver, the enzyme
employed in Ammonia and Urea/Ammonia kits supplied by others.
However, the enzyme used in the K-URAMR kit does not suffer from
this limitation (Figure 2, page 12).

SAFETY:
The general safety measures that apply to all chemical substances should
be adhered to.

2
For more information regarding the safe usage and handling of this
product please refer to the associated SDS that is available from the
Megazyme website.

KITS:
Kits suitable for performing 50 assays (of each) are available from
Megazyme. The kits contain the full assay method plus:
Bottle 1: Buffer (18 mL, pH 8.0) plus 2-oxoglutarate and
sodium azide (0.02% w/v) as a preservative.
Stable for > 2 years at 4°C.
Bottle 2: NADPH. Lyophilised powder.
Stable for > 5 years below -10°C.
Bottle 3: Glutamate dehydrogenase suspension (1.1 mL).
Stable for > 2 years at 4°C.
Bottle 4: Urease solution (2.7 mL).
Stable for > 2 years below -10°C.
Bottle 5: Urea control powder (~ 2 g).
Stable for > 2 years; store sealed at room temperature.

PREPARATION OF REAGENT SOLUTIONS:

1. Use the contents of bottle 1 as supplied.
Stable for > 2 years at 4°C.
2. Dissolve the contents of bottle 2 in 12 mL of distilled water.
Stable for ~ 4 weeks at 4°C or stable for > 2 years below
-10°C (to avoid repetitive freeze/thaw cycles, divide into
appropriately sized aliquots and store in polypropylene tubes).
3. Use the contents of bottle 3 as supplied. Store the bottle in an
upright position. Swirl the bottle to mix contents before
use. Stable for > 2 years at 4°C.
4. Use the contents of bottle 4 as supplied. Store the bottle in an
upright position. Stable for > 2 years below -10°C.
5. Accurately weigh approx. 70 mg of urea into a 1 L volumetric
flask, fill to the mark with distilled water and mix thoroughly.
Prepare fresh before use. This control solution is stable for ~ 3
months below -10°C.

NOTE: The urea standard solution is only assayed where there is

some doubt about the accuracy of the spectrophotometer being used
or where it is suspected that inhibition is being caused by substances in
the sample. The concentrations of ammonia and urea are determined
directly from the extinction coefficient of NADPH (page 6).

3
EQUIPMENT (RECOMMENDED):
1. Glass test tubes (round bottomed; 16 x 100 mm).
2. Disposable plastic cuvettes (1 cm light path, 3.0 mL).
3. Micro-pipettors, e.g. Gilson Pipetman® (100 μL).
4. Positive displacement pipettor, e.g. Eppendorf Multipette®
- with 12.5 mL Combitip® [to dispense 0.5 mL aliquots of
NADPH buffer (solution 2)].
- with 25 mL Combitip® (to dispense 2.0 mL of distilled water).
5. Analytical balance.
6. Spectrophotometer set at 340 nm.
7. Vortex mixer (e.g. IKA® Yellowline Test Tube Shaker TTS2).
8. Stop clock.
9. Whatman No. 1 (9 cm) filter papers and GF/A (9 cm) glass fibre
filter papers.

4
PROCEDURE:
Wavelength: 340 nm
Cuvette: 1 cm light path (glass or plastic)
Temperature: ~ 25°C
Final volume: 2.62 mL (ammonia)
2.67 mL (urea)
Sample solution: 0.2-7.0 μg of ammonia per cuvette
or 0.3-14.0 μg urea per cuvette
(in 0.1-2.0 mL sample volume)
Read against air (without a cuvette in the light path) or against water

Pipette into cuvettes Blank Sample

distilled water (at ~ 25°C) 2.10 mL 2.00 mL

sample - 0.10 mL
solution 1 (buffer) 0.30 mL 0.30 mL
solution 2 (NADPH) 0.20 mL 0.20 mL
Mix*, read the absorbances of the solutions (A1) after approx. 2 min
and start the reactions immediately by addition of:
suspension 3 (GlDH) 0.02 mL 0.02 mL
Mix* and read the absorbances of the solutions (A2) after approx. 5
min. Then add**:
solution 4 (Urease) 0.05 mL 0.05 mL
Mix* and read the absorbances of the solutions (A3) at the end of
the reaction (approx. 5 min). If the reaction has not stopped after
5 min continue to read the absorbances at 1 min intervals until the
absorbances remain the same.

* for example with a plastic spatula or by gentle inversion after sealing

the cuvette with a cuvette cap or Parafilm®.
This reaction sequence is shown in Figure 3 (page 13).

5
CALCULATION:
Determine the absorbance difference (A1-A2) for both blank and
sample. Subtract the absorbance difference of the blank from the
absorbance difference of the sample, thereby obtaining ΔAammonia.

Determine the absorbance difference (A2-A3) for both blank and

sample. Subtract the absorbance difference of the blank from the
absorbance difference of the sample, thereby obtaining ΔAurea.

The values of ΔAammonia and ΔAurea should as a rule be at least 0.100

absorbance units to achieve sufficiently accurate results.

The concentration of ammonia and urea can be calculated as follows:

c = V x MW x ΔA [g/L]
ε x d x v

where:
V = final volume [mL]
MW = molecular weight of the substance assayed [g/mol]
ε = extinction coefficient of NADPH at 340 nm
= 6300 [l x mol-1 x cm-1]
d = light path [cm]
v = sample volume [mL]

It follows for ammonia:

c = 2.62 x 17.03 x ΔAammonia [g/L]

6300 x 1.0 x 0.10

= 0.07082 x ΔAammonia [g/L]

For urea:

c = 2.67 x 60.06 x ΔAurea [g/L]

6300 x 1.0 x 0.10 x 2

= 0.1273 x ΔAurea [g/L]

NOTE: These calculations can be simplified by using the Megazyme

Mega-CalcTM, downloadable from where the product appears on
the Megazyme website (www.megazyme.com).

6
If the sample has been diluted during preparation, the result must be
multiplied by the dilution factor, F.

When analysing solid and semi-solid samples which are weighed out
for sample preparation, the content (g/100 g) is calculated from the
amount weighed as follows:

Content of ammonia
= cammonia [g/L sample solution] x 100 [g/100 g]
weightsample [g/L sample solution]

Content of urea
= curea [g/L sample solution] x 100 [g/100 g]
weightsample [g/L sample solution]

SAMPLE PREPARATION:
1. Sample dilution.
The amount of urea (ammonia) present in the cuvette (i.e. in the
0.1 mL of sample being analysed) should range between 0.3 and
14 μg (0.2 and 7 μg). The sample solution must therefore be diluted
sufficiently to yield a urea (ammonia) concentration between 0.02 and
0.14 g/L (0.01 and 0.08 g/L).

Dilution Table

Estimated concentration of Dilution Dilution

urea (ammonia) (g/L) with water factor (F)
< 0.14 (< 0.07) No dilution required 1
0.14-1.4 (0.07-0.7) 1 + 9 10
1.4-14 (0.7-7.0) 1 + 99 100

If the value of ΔAammonia or ΔAurea is too low (e.g. < 0.100), weigh out
more sample or dilute less strongly. Alternatively, the sample volume
to be pipetted into the cuvette can be increased up to 2.00 mL, making
sure that the sum of the sample and distilled water components in the
reaction is 2.10 mL and using the new sample volume in the equation.

2. Sample clarification:
Carrez reagents cannot be used for deproteinisation as their
use results in significantly reduced recoveries. Perchloric or
trichloroacetic acid are used as alternatives [see point (h)
Samples containing protein, on page 8].

7
3. General considerations.
(a) Liquid samples: clear, slightly coloured and approximately
neutral, liquid samples can be used directly in the assay.
(b) Acidic samples: if > 0.1 mL of an acidic sample is to be used
undiluted (such as wine or fruit juice), the pH of the solution should
be increased to approx. 8.0 using 2 M NaOH, and the solution
incubated at room temperature for 30 min.
(c) Carbon dioxide: samples containing significant quantities of
carbon dioxide, such as beer, should be degassed by increasing the
pH to approx. 8.0 with 2 M NaOH and gentle stirring, or by stirring
with a glass rod.
(d) Coloured samples: an additional sample blank, i.e. sample with
no GlDH, may be necessary in the case of coloured samples.
(e) Strongly coloured samples: if used undiluted, strongly
coloured samples should be treated by the addition of 0.2 g of
polyvinylpolypyrrolidone (PVPP)/10 mL of sample. Shake the tube
vigorously for 5 min and then filter through Whatman No. 1 filter
paper.
(f) Solid samples: homogenise or crush solid samples in distilled
water and filter if necessary.
(g) Samples containing fat: extract such samples with hot water
at a temperature above the melting point of the fat, e.g. in a 100 mL
volumetric flask at 60°C. Adjust to room temperature and fill the
volumetric flask to the mark with distilled water. Store on ice or in a
refrigerator for 15-30 min and then filter. Discard the first few mL of
filtrate and use the clear supernatant (which may be slightly opalescent)
for assay.
(h) Samples containing protein: deproteinise samples containing
protein by adding an equal volume of ice-cold 1 M perchloric acid with
mixing. Centrifuge at 1,500 g for 10 min and neutralise the supernatant
with 1 M KOH. Alternatively, use trichloroacetic acid as described in
sample preparation example (b) below.

SAMPLE PREPARATION EXAMPLES:

(a) Determination of urea and ammonia in grape juice/must
and wine.
Generally, the concentration of urea and ammonia in white and red
grape juice/must and wine can be determined without any sample
treatment (except filtration and dilution according to the dilution
table, if necessary). If volumes greater than 25 μL of red wine are to
be analysed, it may be necessary to remove some of the colour with
activated PVPP as described in “General considerations - (e) strongly
8
coloured samples”. However, typically, no dilution is required and a sample
volume of 25-50 μL is satisfactory.

(b) Determination of urea in milk.

In a glass test-tube, accurately mix 1 mL of milk with 3 mL of
0.3 M trichloroacetic acid. Incubate at room temperature for 5 min to
ensure complete precipitation of protein and then centrifuge at room
temperature for 3 min at 2,000 g. Use the clear supernatant directly
for the assay. Typically, no further dilution is required and a sample volume
of 0.1 mL is satisfactory.

(c) Determination of urea and ammonia in meat and meat

products.
Accurately weigh approx. 5 g of representative material into a 100 mL
Duran® bottle. Add 20 mL of 1 M perchloric acid and homogenise
for 2 min using an Ultra-turrax® or Polytron® homogeniser (or
equivalent). Quantitatively transfer to a 40 mL glass beaker and
adjust the pH to approx. 8.0 using 2 M KOH. Quantitatively transfer
to a 100 mL volumetric flask and adjust to the mark with distilled
water (ensuring the fat containing layer is “above” the mark, and
the aqueous layer is “at” the mark). Incubate at 4°C for 20 min to
precipitate potassium perchlorate and allow separation of the fat.
Filter, discarding the first 3-5 mL, and use the clear filtrate for the
assay. Typically, no further dilution is required and a sample volume of
0.5 mL is satisfactory.

(d) Determination of urea and ammonia in water (e.g.

swimming pool water).
The urea and ammonia concentration of water can generally be
determined without any sample treatment (except dilution according
to the dilution table). Typically, no dilution is required and sample
volumes up to 2.0 mL will be required.

(e) Determination of urea and ammonia in baking products.

Accurately weigh approx. 10 g of representative material into a 100 mL
Duran® bottle. Add 20 mL of 1 M perchloric acid and homogenise for
2 min using an Ultra-turrax® or Polytron® homogeniser (or
equivalent). Quantitatively transfer to a 40 mL glass beaker and
adjust the pH to approx. 8.0 using 2 M KOH. Quantitatively transfer
to a 100 mL volumetric flask and adjust to the mark with distilled
water (ensuring the fat containing layer is “above” the mark, and the
aqueous layer is “at” the mark). Store on ice for 20 min to precipitate
potassium perchlorate and allow separation of the fat. Filter, discard
the first 3-5 mL, and use the clear filtrate for the assay. Typically, no
further dilution is required and a sample volume of 0.5 mL is satisfactory.

9
(f) Determination of urea and ammonia in fruit juices.
Adjust 25 mL of fruit juice to approx. pH 8.0 with 2 M KOH,
quantitatively transfer to a 50 mL volumetric flask and fill to the mark
with distilled water. Transfer the solution to a 100 mL beaker, add 1 g
of PVPP and stir the suspension for 2 min on a magnetic stirrer. Filter
an aliquot of the suspension and use the clear, slightly turbid solution
for the assay. Typically, no further dilution is required and a sample volume
of 0.1 mL is satisfactory.

(g) Determination of urea and ammonia in liquorice

products.
Homogenise approx. 3 g of sample using a pestle and mortar and
accurately weigh approx. 1 g of representative material into a 100 mL
volumetric flask. Add 60 mL of distilled water and incubate at 70°C
for 10 min, or until fully dissolved. Allow to equilibrate to room
temperature and fill to the mark with distilled water. Filter and use
the slightly coloured filtrate for the assay. Typically, no further dilution is
required and a sample volume of 0.5 mL is satisfactory.

(h) Determination of urea and ammonia in whole blood

samples.
a. Solutions:
Concentrated Carrez I solution. Dissolve 30 g of potassium
hexacyanoferrate (II) {K4[Fe(CN)6].3H2O} (Sigma cat. no. P9387) in
200 mL of distilled water. Store at room temperature.
Concentrated Carrez II solution. Dissolve 60 g of zinc sulphate
{ZnSO4.7H2O} (Sigma cat. no. Z4750) in 200 mL of distilled water.
Store at room temperature.
b. Procedure:
Heat 1 mL of whole blood sample at approx. 80°C for 20 min in a
microfuge tube then centrifuge at 13,000 x g for 10 min and recover
the supernatant. Add 20 µL Carrez Reagent II and mix thoroughly,
then add 20 µL Carrez Reagent I and mix thoroughly. Centrifuge
the sample again at 13,000 x g for 10 min and recover the clarified
supernatant for use in the assay. If required, dilute the sample
appropriately in distilled water for the assay.
Note: The final volume of the clarified supernatant will be
approximately one quarter of the starting volume of the original
sample. Therefore adjust the volume of the starting material as
required to obtain sufficient volume of clarified sample for the test.
(i) Determination of urea and ammonia in biological tissue
samples.
Accurately weigh approx. 5 g of representative biological tissue
into a 100 mL Duran® bottle. Add 20 mL of 1 M perchloric acid

10
and homogenise for 2 min using an Ultra-turrax® or Polytron®
homogeniser (or equivalent). Quantitatively transfer to a 40 mL
glass beaker and adjust the pH to approx. 8.0 using 2 M KOH.
Quantitatively transfer to a 100 mL volumetric flask and adjust to the
mark with distilled water (ensuring the fat containing layer is “above”
the mark, and the aqueous layer is “at” the mark). Store on ice for
20 min to precipitate potassium perchlorate and allow separation of
the fat (if present). Centrifuge an appropriate volume of the sample
at 13,000 x g for 10 min and recover the clarified supernatant for use
in the assay, alternatively filter through Whatman No. 1 filter paper,
discarding the first 3-5 mL, and use the clear filtrate for the assay.
If required, dilute the sample appropriately in distilled water for the
assay.
Note: The amount of starting material and volumes used can be
adjusted accordingly depending on the amount of analyte present in
the sample.
(j) Determination of urea and ammonia in biological fluid
samples (e.g. urine and serum).
For some biological fluid samples it may be sufficient to test
them directly without any sample preparation other than
appropriate dilution in distilled water. If this is not adequate then
deproteinisation with either perchloric acid or trichloracetic acid may
be required.
Deproteinise biological samples by adding an equal volume of ice-cold
1 M perchloric acid with mixing. Centrifuge an appropriate volume
of the sample at 1,500 x g for 10 min and recover the supernatant
for use in the assay, alternatively filter through Whatman No. 1 filter
paper, discarding the first 3-5 mL, and use the filtrate for the assay.
If required, dilute the sample appropriately in distilled water for the
assay. Alternatively, use 50% (w/v) trichloroacetic acid instead of
perchloric acid.

REFERENCES:
1. Martin, O., Brandriss, M. C., Schneider, G. & Bakalinsky, A. T.
(2003). Improved anaerobic use of arginine by Saccharomyces
cerevisiae. App. Env. Microbiol., 69, 1623-1628.
2. Kerscher, L. & Ziegenhorn, J. (1990). Urea. “Methods of
Enzymatic Analysis” (Bergmeyer, H. U., ed.), 3rd ed., Vol. VIII,
pp. 444-453, VCH Publishers (UK) Ltd., Cambridge, UK.

11
 Absorbance, 340 nm

Incubation time, min

Figure 1. Decrease in absorbance at 340 nm on incubation of 1-7 μg

of ammonia with glutamate dehydrogenase in the presence of
NADPH.
Absorbance, 340 nm

Time, min

Figure 2. Decrease in absorbance at 340 nm on incubation of

untreated red must preparation with glutamate dehydrogenase in the
presence of NADPH. A. blank; B. 0.025 mL of red must sample;
C. 0.05 mL of red must sample.

12
13
 A
Absorbance, 340 nm

B
+ Glutamate
dehydrogenase + Urease

Incubation time, min

Figure 3. Decrease in absorbance at 340 nm on incubation of a urea/

ammonia standard with glutamate dehydrogenase and urease in the
presence of NADPH. A. blank; B. 4 μg of ammonia plus 7 μg of
urea. Glutamate dehydrogenase and urease were added at the points
shown by the arrows.

NOTES:

14
13
NOTES:

15
14
 Bray Business Park, Bray,
Co. Wicklow,
A98 YV29,
IRELAND.
Telephone: (353.1) 286 1220
Facsimile: (353.1) 286 1264
Internet: www.megazyme.com
E-Mail: [email protected]

WITHOUT GUARANTEE
The information contained in this booklet is, to the best of our knowledge, true and accurate, but
since the conditions of use are beyond our control, no warranty is given or is implied in respect of
any recommendation or suggestions which may be made or that any use will not infringe any patents.

15
16