ANALYTICAL BIOCHEMISTRY 70, 377-380 (1976)

A Sensitive Assay for Allantoin

JOHN ABRAHAM, FIORINDO A . SIMEONE,

AND ROBERT W . HOPKINS

Division of Biological and Medical Sciences of Brown University, and

the Department of Surgery, The Miriam Hospital,
Providence, Rhode Island
Received January l, 1975; accepted September 19, 1975

Allantoin is separated by thin-layer chromatography (TLC) and sprayed with an

acidic solution ofp-dimethylaminobenzaldehyde. The yellow color produced is
read in a densitometer and compared with that of a standard. The lower limit of
quantitation is 0.1 ~g per spot (0.5 mg/100 ml). The method can be utilized for the
estimation of allantoin in serum, lymph, and urine.

Several reports (1-9) have been published on the determination of

allantoin. Most of these methods have been based on the application of the
Rimini- Schryver color reaction for formaldehyde to the aldehyde group of
glyoxylic acid which is formed by the hydrolysis of allantoin. Several
compounds, including uric acid, ergothioneine, and glucose may interfere
with the assay. Therefore, Christman et al. (5) have pointed out the
desirability of the quantitative separation of allantoin from other
constituents of blood prior to the analysis. Cline et al. (10) have
investigated the utilization of the color reaction between p-dimeth-
ylaminobenzaldehyde and allantoin on paper chromatograms for
the quantitation of allantoin. Based on this, Zelck (11) has reported a
procedure to determine concentrations of allantoin in urine. Though the
method was specific, the sensitivity of the assay was such that the lowest
amount of allantoin that could be determined was 20/xg (200 mg/100 ml).
The reported values (5) for the concentration of allantoin in the blood of
various animals range between 1 and 3 rag/100 ml. This work was
undertaken to develop a method which would be specific and sensitive
enough to measure the concentration of allantoin in serum and lymph of
mongrel dogs in studies of hemorrhagic shock.

METHODS
Reagents and materials. Thin-layer chromatography (TLC) plates.
Flexible cellulose-coated plates, 20 x 20 cm (J. T. Baker Chemical Co.)
are used.
Allantoin standard. A stock solution containing 1.0 mg/ml is prepared by
dissolving allantoin (Nutritional Biochemical Corporation) in distilled

377
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All rights of reproductionin any formreserved.
378 ABRAHAM, S I M E O N E AND HOPKINS

water heated to 50°C. Working standards are prepared by the proper

dilution of the stock solution. It is desirable to use a fresh standard because
of the possibility of decomposition of allantoin in neutral or acidic media
(3).
Spraying solution. This is p r e p a r e d by dissolving 1.0 g
p-dimethylaminobenzaldehyde (certified, Fisher Scientific Company) in 3
ml of concentrated hydrochloric acid and then diluting to 15 ml with
1-Butanol (certified, Fisher Scientific Company). This solution is sufficient
to spray two TLC plates and is prepared fresh every day.
Apparatus. The Photovolt TLC densitometer Model 530 equipped with
an integrator is used to scan the plates.
Procedure. Large amounts of protein in serum and lymph causes tailing
of the spots when the TLC plate is developed. Most of the proteins are
removed by adding 0.4 ml of serum or lymph to 0.2 ml of 1.0 N perchloric
acid and centrifuging. Urine samples are diluted at least 100 times and used
without deproteinization.
TLC analysis ofallantoin. Ten-microliter aliquots of the solutions to be
analyzed are spotted with 10/xl disposable pipets on a thin-layer plate. In
addition, 10-/zl aliquots of standard solutions, which are to be compared
with the samples, are spotted on the same plate. The plate is developed
using a mobile phase of 1.0 N acetic acid:ethanol:l-Butanol, 2:2:7 by
volume. The length of the development is approximately 17 cm, which
requires about 2.5 hr.
After the development, the TLC plate is air-dried until the solvent has
evaporated. It is then dried in an oven at 75°C for 15 min. The plate is then
sprayed with a solution ofp-dimethylaminobenzaldehyde,dried in air for 5
min, and again dried for another 5 min in an oven at 75°C. The plate is then
allowed to equilibrate for 25 min in a closed chamber. The color densities of
the spots are read in a densitometer using the 445 nm filter. By comparing
the densities of spots of the sample solutions with those of the standards,
the concentration of allantoin in the solution can be calculated. This may be
converted to the concentration of allantoin in serum, lymph, or urine by
using the appropriate dilution factor.
Results. The Rs value of allantoin in the solvent system described above
was 0.23.
The reproducibility of the method has been ascertained by determining
allantoin in different standard solutions. The results of these determina-
tions are given in Table 1.
In order to determine the recovery of the added allantoin, known
amounts of allantoin were added to dog's serum and the total allantoin
content was determined. The results are given in Table 2. The average
recovery of added allantoin was 93.2%.

DISCUSSION
As shown in Table 1, amounts as small as 0.1/xg (0.5 mg/100 ml) can be
accurately determined by this procedure. In the method described by
 ASSAY F O R A L L A N T O I N 379

TABLE 1

ANALYSIS OF STANDARD ALLANTO1N SOLUTIONS

Volume of solution Concentration of Concentration of

in each spot allantoin given allantoin found a
(p,I) (mg/100 ml) (mg/100 ml)

20 0.5 (0.10) r' 0.45 _+ 0.004 (0.09)

10 1.0 (0.10) 0.9 _+ 0.009 (0.09)
10 3.0 (0.30) 3.2 -+ 0.12 (0.32)
10 4.0 (0.40) 4.1 _+ 0.32 (0.41)

Each result is average of seven determinations, and the error is expressed as standard
erFor.
Values in parentheses are the amounts of allantoin (/~g) in each spot.

Zelck (11), amounts smaller than 20 /xg (200 mg/100 ml) could not be
assayed. Thus, the technique described has improved the sensitivity of the
assay about 400-fold. The color produced is found to obey Beer's law in the
range 0.1 to 1.0/zg of allantoin. Deviation from Beer's law is observed when
the amount of allantoin increased above 1.0 tzg per spot. Under those
circumstances the sample should be appropriately diluted.
The conditions and precautions necessary for the increased sensitivity
are discussed below. As has been demonstrated by Cline (10), we observed
that the color produced in the reaction between allantoin and
p-dimethylaminobenzaldehyde is dependent on the amount of acid and
p-dimethylaminobenzaldehyde in the spray. Of the several concentrations
~Aed, the amount of hydrochloric acid andp-dimethylaminobenzaldehyde
recommended in our procedure was found to produce the best results. The
solvent in the spray also affects the color development. 1-Butanol was
found to be superior to other solvents, including methanol, chloroform,
ether, and acetone.
The intensity of the color reaction on the plate is also influenced by the

TABLE 2

RECOVERY OF ALLANTOIN A D D E D TO SERUM

Volume of serum Amount of allantoin Total allantoin

taken added to serum found a Recovery
(ml) (/xg) (/xg) (%)

0.40 -- 16.2b + 0.85 --

0.40 10.0 24.9 _+ 0.57 95.0
0.40 20.0 32.3 _+ 0.76 89.2
0.40 30.0 44.1 _+ 1.2 95.5

~' Each result is an average of four determinations.

b This is the amount of allantoin found in 0.4 ml serum of a mongrel dog, and corresponds
to a concentration of 4.05 mg/100 ml.
380 ABRAHAM, SIMEONE AND HOPKINS

relative humidity of the atmosphere. Elaborate arrangements would be

required to keep the humidity low and constant. The effect of this variable
is minimized by running standards simultaneously with the unknown in
each TLC plate.
Other factors that influenced the color density are the drying conditions
after the spraying. Drying the plate at high temperature for long periods
produced high background readings. The best results are obtained when
the plate is heated at 75°C for 5 min. When the plate is left exposed to air
after the spraying and drying, the background readings increased, probably
due to the slow air-oxidation of the reagent p-dimethylaminobenzaldehyde.
This effect is minimized by storing the plate in a closed chamber until it is
read. Under these circumstances, the color was stable for at least 1 hr.
The sensitivity of the present assay should permit the determination of
allantoin in any biological fluid in which the concentration is above 1.5
mg%. However, when allantoin concentration is lower than 1.5 rag%, the
volume of the sample spotted on the plate must be increased to 20/xl. No
attempt was made to increase further the volume of solution in each spot.
Since allantoin is separated by TLC before the color reaction, interference
from uric acid, urea, and other amido compounds is eliminated. The
method is specific and selective. This assay is applicable to any fluids
containing allantoin, provided the concentration of all other substances
would not cause tailing of the spots when TLC is developed. Several
samples can be analyzed in a relatively short time. This method is suited for
the routine determination of allantoin.
We have applied this method to the measurement of allantoin in blood,
thoracic duct lymph, and urine of dogs subjected to a period of oligemia (12).

ACKNOWLEDGMENTS
This work was supported by The Miriam Hospital and by PHS Research Grant No. GM
19930 from the National Institutes of Health.

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