A Spectrophotometric Method For Determination of Urea

CLINICA CHIMICA ACTA 295
A SPECTROPHOTOMETRIC METHOD FOR DETERMINATION

OF UREA
G. CERIOTTI AND L. SPANDRIO
Ce)ztre for the Study of Tunmrrs, and Cli+zical Laborutovies,

St. Joseph’s Hospital, Busto Arsizio (Italy)
(Received June ISth, 1962)
SUMMARY
If phenazone is substituted for perchloric acid or arsenic pentoxide in the diace-

tylmonoxime reaction for urea determination, a colored product is formed, apparently
from a double condensation of phenazone with diacetylmonoxime and urea. The
color has a maximum absorption at 460 m,u. Beer’s law is followed for concentrations
of urea ranging from 0.1 to 2 ,&ml in the final reaction mixture. The sensitivity is
very high (Ei& s.goo), the color is stable, and the reaction seems to be specific.
It is very simple and is routinely performed on an amount of filtrate corresponding
to IO ,ul of blood.
The determination of urea by FEARON’S condensation reaction’ with diacetyl-

monoxime, is very simple and sensitive. However, it may be criticized from several
points of view, also in its more recent modifications2. The hydroxylamine formed
during the reaction tends to destroy the color; its action is usually counteracted by
oxidants, e.g. arsenic pentoxide or perchloric acid, but these reagents also affect the
color which is partly destroyed and rendered unstable especially in presence of light.
Furthermore, the action of the oxidizing agents is not constant, but increases with
decreasing concentrations of urea. The color produced is therefore dependent on the
equilibrium of several different opposing factors, so that the calibration curve is not
linear but sigmoidal. In order to avoid these inconveniences, an attempt has been
made to remove the hydroxylamine without the use of oxidants. One method of
doing so would be to cause the hydroxylamine to react with an unsaturated car-
boxylic acid giving rise to the corresponding hydroxyamino acid3 acccording to the
reaction :
NH,OH+COOH-CH=CH-COOH+COOH-CHNH,-CHOH-COOH.
Another method would be to add a ketone that competes for hydroxylamine with the
ketone produced by condensation between urea and diacetylmonoxime.
Both possibilities were examined using maleic acid and several ketones. Phena-
zone (antipyrine) appeared to be the most successful reagent, although the mechanism
is probably partially different from that postulated above.
C/in. C&u. Acta, 8 (1963) zgs-ggg

296 I>.WANDRIO, G. CEKIOTTI
ESI'EIUMENTAI
Reageuts. Urea was obtained from Merck, phenazone from Bayer, and diacetylmon-
oxime and 4-aminophenazone (4-aminoantipyrine) from BDH. Other reagents were
obtained from Carlo Erba.
Solution I, diacetylmonoxime (0.5 g) dissob-ed in IOO ml of 5”,,) (v/v) acetic acid.
Solution II antipyrine (0.4 g) dissolved in IOO ml of 40?~, (v/v) sulfuric acid
(analytical grade). The solution is stable.
Recommended pvoceduve. The blood is deproteinized by trichloroacetic acid
or according to the procedure described previously for blood glucose determina-
tionl. The latter procedure allows the same filtrate to be used for both urea and glu-
cose determinations. To LJ ml of acid cadmium sulfate solution (13 g 3CdS0, - SH,O
dissolved in 63.5 ml I ,V sulfuric acid and diluted to I 1 with water) in a centrifuge
tube, is added 0.1 ml of osalated blood. After a few minutes, when the solution has
become brown, 5.9 ml of sodium borate buffer (6.2 g boric aciddissolvedin rsoml IN
sodium hydroxide and diluted to I 1 with water) are added. The mixture is shaken
and after a few minutes centrifuged for a short time at about 2000 rev./min. To I ml
of the clear supernate, distilled water is added to a volume of 5 ml; then I ml of solu-
tion I and 4 ml of solution II are added; the solutions are mixed and heated in a
boiling water bath for jo min. When the contents of the test tubes are mixed, it is
necessary to use rubber gloves or to employ test tubes with ground stoppers to avoid
contact with hands, because traces of urea from the skin may affect the results.
4fter the tubes hale been cooled under running water, the color developed is
measured on a Beckman DL or on an Unicam spectrophotometer at 460 rnp against
a blank treated in the same manner. The color given by the blank is a \ery faint
yellow. A common clinical photometer may be used, provided that a standard curve
is made on the same apparatus; we used an Elvi photometer with filter 490. The
absorbance curve has a sharp peak at 460 rnp (Fig, I). The color is stable for at least
20 h even when exposed to direct light. The E:‘:;,, is 5.900. Beer’s law is followed
between 0.1 and 2 /&g/mlin the final solution, thus, with I ml of filtrate, a range
of blood urea between 0.1 and 2 g"/,.,is covered.
Choice of the couditions for the veactiou

In order to establish the best conditions for the reaction, se\Teral variables were
investigated.
(r) Time of boiling. ‘IXe color production increased up to 50 nun; it remained
stable up to ho min and then tended to decrease, hence a time of 50 min was chosen.
(2) Concclztvatiovt of diacetylmonosime. A sharp increase of color occurred up to
a concentration of 0.5'7~of diacetylmonosime; there was almost no variation between
0.5 and I",; and then a slow decrease.
(3) Cowewtvatiorh of pheuazone. The color increased sharply with increasing con-
centrations of phenazone up to 0.2~~. The absorption curve showed a maximum with
no appreciable variations between 0.2 and 0.47X; a slow decrease of color formation
occurred with further increase of phenazone concentration
(4) Influesce of acidity. The sulfuric acid concentration had an important in-
SPECTROPHOTOMETRIC DETERMINATION OF UREA 297
fluence on the color produced. The best results were obtained at a 40’31 concentration
(v/v).
Sf~ecijcity of the reaction

Several substances were tested for possible interference : arginine, sodium,
barbiturate, adenine, hypoxanthine, guanosine, uracil, santhine, adenosine, cytidylic
acid, ribo- and desosyribonucleic acid did not interfere even at concentrations of
rooj&ml.
0.9
r A
.b
400 450 500 1 2 3 4 5 6 7 8 9 10

Wavelength kry.~) concn.Of urea (/ug/ml~
Fig. T. Absorption spectra of the Fig. 2. Standard curves of the diacet~lmono~ime

colors produced by the reaction of reaction for urea performed under various conditions:
urea with diacetylmonosime and 13 reaction with perchloric acid; 8 reaction with mal-
phenazone ( ); of dia- eic acid: cl reaction with dimedone; L reaction with
cetylmonoxime and phenazone at phenazone; A reaction with 4-aminophenazone.
ten times the normal concentra-
tion, without urea (- - - -) ; of urea
and diacetylmonoxime in presence
of perchloric acid, maleic acid, di-
medone or 4-aminophenazone
(.-.-. ).
Recovery exfieriments
To I ml of blood filtrate, corresponding to 0.01 ml of blood, various concentra-
tions of urea ranging from 1-12 ,ug in I ml of water were added; the mixture was
diluted to 5 ml with water and the reaction performed. A mean recovery of 98.7
f 4.4% was obtained.
298 L. SPANDRIO, G. CERIOTTI
Sttvdy of other procedures fey the urea-diacetytylmonoxime reactiolz

A description of the other procedures which were studied for the urea-diacctyl-
monoxime reaction is of little practical importance but is of some interest for thct
sake of comparison and for a better understanding of the antipyrine reaction.
I. Reaction with perclzloric acid. The reaction was performed exactly as described
by E(ITAMUR.4’. The curve obtained (Fig. 2) shows the characteristic S-shape des-
cribed by KITAMURA. The sensitivity, compared to the antipyrine reaction, varied
from 0.1-0.2 depending on the urea concentrations. The absorption spectrum had ;I
sharp maximum at 480 rnp.
II. Reaction ~~it~~~~~ei~ ncid. This reaction was performed under the same
conditions as those used with antipyrine, except that a solution of 0.3T:h maleic acid
in 40% sulfuric acid was used. The results obtained are shown in Fig. 2. The shape
of the curve is analogous to that obtained with perchloric acid, although a little better;
the absorption spectrum (Fig. I) is the same. Possibly not only hydroxyaspartic but
also aspartic acid is formed, thus giving rise to oxygen that affects the color developed.
III. Reactiola with dimedo?Le (dimethylcyclohexane-r,3-dione). The conditions
used were the same as for the reaction with antipyrine; but the concentration of
dimedone had to be kept much lower because at equimolar concentrations with
diacetylmonoxime an intense turbidity appeared on boiling. At 0.029:& concentrations
the solutions remained perfectly clear; as seen from Fig. 2, a sigmoidal curve was
again obtained, though it was a little less curved than those given by maleic acid and
perchloric acid. The concentration of dimedone was probably too low to remove all
the hydroxylamine formed. The absorption curve had its maximum at 480 mp.
IV. Reaction with 4-arn~no~nt~pyyi~~e. This reaction is particularly interesting
because the compound under study differs from antipyrine only in having the position
4 occupied by an amino-group. The reaction performed under the same conditions
as with antipyrine gave an absorption spectrum with a maximum at 480 rnp, identical
to that obtained by perchloric acid, maleic acid and dimedone. However, there was
a linear relationship between concentration and absorption (Fig. 2). Probably the
amount of ketone added (0.4%) was sufficient to block all the hydroxylamine formed.
CONCLUSIONS
Substitution of unsaturated carboxylic acids or of ketones for oxidizing agents

in the reaction between urea and diacetylmonoxime was shown to be convenient.
In the less favourable cases a standard curve analogous to that given by perchloric
acid was obtained. In the case of 4_aminophenazone, Beer’s law was obeyed. The
color formed was the same, irrespective of the reagent used to remove hydroxylamine.
Phenazone must be considered separately; in this case, not only was a linear standard
curve obtained, but the absorption increased strongly (Fig. 2) and the absorption
spectrum was different (Fig. I).This proves that the colored product is different from
that formed in the common urea-diacetylmonoxime reaction. When diacetylmonox-
ime and phenazone reacted under the same conditions as in the test reaction but at
concentrations ten times higher and in absence of urea, a yellow color was formed,
but its absorption spectrum had a sharp maximum at 450 rnp, so that the product
was different from that obtained when urea was present (Fig. I).This was probably
responsible for the very faint yellowish color that appeared in the blank. Therefore
SPECTROPHOTOMETRIC DETERMINATION OF UREA 299
it may be supposed that, under the conditions used, urea does not react directly with
diacetylmonoxime but with the product formed by interaction between phenazone
and diacetylmonoxime. This is proved by the completely different behaviour of 3-
aminophenazone, whose 4-position is occupied.
From a practical point of view, the reaction with antipyrine offers several
advantages. It has a very high sensitivity (Et”&, 5.900), the color is stable, and
the standard curve follows Beer’s law over a rather large range (between 0.1 and
2 pg/ml), so that small samples of blood can be used and a single test can cover varia-
tions of blood urea ranging between 0.1 and 2 g o/oo. In the routine clinical laboratory
the filtrate from 0.1 ml of blood may serve for repeated tests both for urea and for
blood glucose determinations. Simply by reducing the amounts of the reagents, the
test could be easily adapted for samples of IO ~1 of blood.
REFERENCES
1 W. R. FEARON, B&hem. J., 33 (1939) gor.

2 M. KITAMURA AND I.IUCHI, Clin.Chim. Acta, q(1g5g) 701.
3 J. HOUBEN, Die Methoden der organ&hen Chemie, Vol. I, G. Thieme, Leipzig, 1941, p. 403
4 G. CERIOTTI, Clin.Chim. Acta, 8 (1963) 157.

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CLINICA CHIMICA ACTA 295

A SPECTROPHOTOMETRIC METHOD FOR DETERMINATION

G. CERIOTTI AND L. SPANDRIO

Ce)ztre for the Study of Tunmrrs, and Cli+zical Laborutovies,

If phenazone is substituted for perchloric acid or arsenic pentoxide in the diace-

The determination of urea by FEARON’S condensation reaction’ with diacetyl-

C/in. C&u. Acta, 8 (1963) zgs-ggg

Choice of the couditions for the veactiou

Sf~ecijcity of the reaction

400 450 500 1 2 3 4 5 6 7 8 9 10

Fig. T. Absorption spectra of the Fig. 2. Standard curves of the diacet~lmono~ime

Sttvdy of other procedures fey the urea-diacetytylmonoxime reactiolz

Substitution of unsaturated carboxylic acids or of ketones for oxidizing agents

1 W. R. FEARON, B&hem. J., 33 (1939) gor.

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