ANALYTICAL SCIENCES JUNE 2008, VOL.

24 769
2008 © The Japan Society for Analytical Chemistry

Spectrophotometric Determination of Urea in Dermatologic

Formulations and Cosmetics
Jorgovanka BOJIC,*† Blaga RADOVANOVIC,** and Jasmina DIMITRIJEVIC***

*High Polytechnic School, Kosanciceva 36, 37000 Krusevac, Serbia

**Department of Chemistry, Faculty of Natural Science, University of Nis, Visegradska 33, 18000 Nis, Serbia
***Pharmacy, Luke Ivanovica 34, 37000 Krusevac, Serbia

A rapid, relatively sensitive, and low-cost method for the determination of water-soluble urea content in dermatological
therapy products and cosmetics is proposed using a new spectrophotometric assay with water as the only extraction
solvent. Spectrophotometric methods involve addition of a known excess of bromate to urea in an acid medium, followed
by the determination of residual bromine and chlorine reacting with methyl orange and measurement of absorbance at 505
nm. The absorbance increases linearly with urea concentration (r = 0.9998). The systems obey Beer’s law for 6 – 90 mg
ml–1. The calculated apparent molar absorbance values are found to be 4.537 ¥ 103 dm3 mol–1 cm–1 and the Sandell’s
sensitivity is 0.013 mg cm–2. The variables affecting the rate of the reaction were investigated. The relative standard
deviation for five-replication determination of 60 mg ml–1 urea was 2.1% and the detection limit of the method is 0.34 ng ml–1.

(Received August 6, 2007; Accepted December 19, 2007; Published June 10, 2008)

Introduction
Urea is a nitrogen-containing chemical product which is
produced on a scale of some 100000000 ton per year worldwide.
Urea is used in many industrial sectors for numerous functional
uses such as adhesives, binders, sealants, resins, glues, fillers,
catalysts, humectants and dehydrating agents. Urea is also used
as an animal-feed supplement, a fertilizer and an analytical
reagent. In consumer products, urea is found in many
deodorants, shampoos, hair conditioners, hair dyes and dye
removers, liquid soaps, detergents and other cleaning products
and has been extensively used in the treatment of dry skin.
Maximum urea concentration is commonly 1 – 10%.1–3
Urea is an important endogenous product of mammalian
metabolism. This may partly explain why it has not been
rigorously studied with toxicological tests. Nevertheless, urea
appears to cause little or no toxicity to most mammalian species
(ruminants are an exception) and humans at reasonable dose
levels. Although urea generally has a low acute ecotoxicity to
organisms, its well-documented indirect and long-term effects to
the ecosystems, e.g. eutrophication, groundwater pollution, soil
acidification, and ammonia emissions to air, should be
considered.
As one of the major soluble substances of the stratum
corneum, urea has a growing importance in dermatological
therapy and cosmetics. Diseases such as atopic dermatitis or
clinical dry skin are marked by a deficiency of urea. Urea is of
significance for hydration of the stratum corneum. Normal skin
contains approximately one percent urea. Furthermore, urea has
keratolytic and pruritus-easing properties and may be
incorporated as an active ingredient in moisturizers due to its
†
To whom correspondence should be addressed.
E-mail: bjogi@ptt.yu
humectant properties.2,3
Different methods such as potentiometric,3,4 fluorometric,5
enzymatic,6 amperometric,7 spectrophotometric8 and colorimetric
methods9 have also been reported for the determination of urea
and no single technique is dominant in all areas, because of the
diversity of applications. Present methods are often categorized
as direct and indirect methods. The term indirect usually refers
to the enzyme degradation of urea prior to detection. Direct
procedures have been defined as those resulting in a colored
product that does not include prior degradation, but this category
is often expended to include miscellaneous methods that do not
involve enzymatic degradation or colorimetric, such as
manometric, infrared or ultraviolet/visible absorbance
measurements.10
Some of these methods are time-consuming and suffer from
lack of selectivity and short linear dynamic range, involve long
pretreatment steps to remove interfering species, have a higher
limit of detection, require complicated and expensive
instruments, or use reagents that are not commercially available.
For example, the Kjeldahl method is the official method for
determination of urea in dermatologic formulations and
cosmetics11,12 in the Pharmacopoeia of Serbia. This method
require about 3 h for the determination of urea at each sample.
Kinetic methods of analysis are attractive alternatives for the
determination of urea in dermatologic formulations and
cosmetics. The kinetic spectrophotometric method is one of the
most attractive approaches for ultra trace determination of some
species. Its advantage is that only a spectrophotometer is
required as the main instrument.
Methyl orange, such as many acid dyes, is prone to oxidation
to form colorless products in an acid medium, thus providing a
suitable analytical approach for the indirect assay of inorganic
ions,13,14 organic compounds,15–17 and pharmaceuticals.18,19 The
produced bromine and chlorine react with methyl orange and
this reaction causes decolorization of the solution.20,21 However,
770
no bromate–hydrochloric acid reaction has been developed for
the determination of urea.
The present paper describes a sensitive, simple, low-cost, fast
(requiring only 10 min), and relatively selective method for the
determination of urea based on its inhibiting effect on the
reaction of bromate with hydrochloric acid.
The method employed is based on a reaction between bromate
and chloride ions in highly acidic media. Bromate can be
reduced by hydrochloric acid, producing bromine and chlorine:
10Cl– + 2BrO3– + 12H+ æÆ 5Cl2 + Br2 + 6H2O (1)
Decolorization of methyl orange by the reaction products was
used to monitor the reaction spectrophotometrically at 505 nm.
Experimental
Reagents
Distilled water and analytical-reagent grade chemicals were
used. A urea stock solution of 0.010 mol l–1 (e.g. 6 ¥ 10–4 g ml–1)
was prepared by dissolving urea (Alkaloid) in water in a
volumetric flask. A 0.001 mol l–1 (e.g. 1.7 ¥ 10–4 g ml–1)
potassium bromate solution was prepared by dissolving KBrO3
(Merck) in water in a volumetric flask. A solution of methyl
orange 6 ¥ 10–4 mol l–1 (e.g. 1.9 ¥ 10–4 g ml–1) was prepared by
dissolving methyl orange (Merck) in water. Hydrochloric acid
2.33 mol l–1 (e.g. 8.5 ¥ 10–2 g ml–1) was prepared by dilution of
concentrated hydrochloric acid (Merck) with distilled water.
Apparatus
UV/Vis spectra were recorded between 300 and 600 nm,
employing a Perkin-Elmer Lambda 15 UV/Vis spectrophotometer
equipped with two matched 10 mm quartz cuvettes.
A Radiometer PHM 29 b pH-meter and a combined glass-
calomel electrode, GK 2311C, were used to determine the pH-
values of the solutions.
Procedure
The inhibited reaction was monitored spectrophotometrically
by observing the change in the absorbance of the reagents’
solution at 505 nm. An aliquot of the sample solution of urea
was transferred into a 10-ml volumetric flask, and then 1 ml of
2.33 M hydrochloric acid was added, followed by a 1.0 ml
methyl orange solution. The solution was diluted to ca. 5 ml
with water. Then 1.0 ml bromate was added to the solution and
the resulting solution was diluted to the mark with water. The
solution was mixed and a portion of the solution was transferred
to the spectrophotometric cell. The change in the absorbance
with time was measured for 1 – 20 min from the initiation of
addition of the last drop of the bromate solution. All the
solutions were kept at 20˚C. All experiments were carried out in
triplicate for reproducibility of results.
A synthetic cream sample was prepared in the laboratory by
adding known concentrations of urea to the cosmetic
formulations (Belobaza, Belupo, Croatia).
Working solutions of the two urea-containing creams were
freshly prepared: (i) the cosmetic product «Urea cream»,
Pharmacy, Krusevac, Serbia, and (ii) the dermatological product
«Sat–N5», BG-pharm, Belgrade, Serbia. Samples contents were
accurately weighed into a 50-ml volumetric flask and dissolved
in chloroform. They were shaken thoroughly for about 15 – 20
min and then transferred into separating funnels. Distilled water
(10 ml) was added to each of the separating funnels, the contents
were shaken well and then left at room temperature for a few
ANALYTICAL SCIENCES JUNE 2008, VOL. 24
Fig. 1 Absorption spectra of methyl orange aqueous solution: on
natural pH (pH 5.9) (curve 1); in acid solution (pH 0.6) (curve 2); in
acid solution, 5 min after addition of potassium bromate (curve 4); in
acid solution in presence of urea, 5 min after addition of potassium
bromate (curve 3). Conditions: 6 ¥ 10–4 mol l–1 methyl orange, 1 ¥
10–4 mol l–1 bromate, 0.23 mol l–1 HCl, and 60 mg ml–1 urea.
Reference, water.
minutes. The two phases were left to separate, the chloroform
layer was passed through, and extraction was repeated twice.
The aqueous phase was filtered out through a piece of Whatman
filter paper No. 40 to remove the insoluble matter and then
diluted to 100 ml with distilled water. The suitable aliquot of
this solution was analyzed using the procedure described above.
Results and Discussion
The electronic absorption spectrum of methyl orange aqueous
solution before (curve 1) and after addition of hydrochloric acid
(curve 2), as well as after addition of bromate (curve 4) and after
addition of bromate and urea (curve 3) are shown in Fig. 1. It is
observed that the absorption spectrum of methyl orange in water
at pH 5.9 (natural) is characterized by one band in the visible
region, with maxima located at 464 nm, and by two bands in the
ultraviolet region, located at 271 and 199 nm. The chromophore
contain azo linkage has absorption in the visible region, while
the benzene ring and the naphthalene ring have absorptions in
the UV region. The naphthalene ring absorption wavelength is
higher than that of benzene ring. The spectrum recorded after
addition HCl in aqueous solution of methyl orange at pH 0.6,
was characterized by four bands at 505, 312, 272 and 205 nm.
The same bands are present in the spectrum after addition of
bromate and after addition bromate and urea in acid solution of
methyl orange.
The absorbance changes of methyl orange at 505 nm as
functions of reaction time in aqueous solutions, in the presence
of bromate ions at concentrations of 1 ¥ 10–4 and 0.23 mol l–1
hydrochloric acid, are shown in Fig. 2(a). The decrease of
absorption band at 505 nm during the reaction indicates a rapid
degradation of methyl orange. The decrease is also meaningful
with respect to the nitrogen to nitrogen double bond of the azo
dye, as the most active site for oxidative attack. Complete
discoloration of solution was observed after 10 min. At the
same time, a mild increase in the absorbance at 312 nm is
observed. As the change in the absorbance at 312 nm is
considerably less significant than the one at 505 nm, the band in
the visible area is chosen for the spectrophotometric monitoring
of the reaction.
This reaction is monitored spectrophotometrically by
measuring a decrease in the absorbance of the reaction mixture
at 505 nm at 20˚C.
The presence of urea in the medium causes a slower reaction
ANALYTICAL SCIENCES JUNE 2008, VOL. 24 771

Table 1 Analytical parameters for different concentrateions of reagents

Regression equationa
Variable Sab Sbc Sy/xd
Slope (a) Intercept (b) Correlation coefficient

Curea/mol l–1 ¥ 10–4 0 –0.1948 2.0843 –0.9998 0.0082 0.0013 0.0120

0.25 –0.1396 2.2676 –0.9993 0.0116 0.0019 0.0169
0.50 –0.1356 2.3205 –0.9994 0.0106 0.0017 0.0156
0.75 –0.1300 2.3669 –0.9998 0.0046 0.0007 0.0069
1.00 –0.1255 2.4117 –0.9998 0.0046 0.0007 0.0068
1.25 –0.1185 2.4662 –0.9998 0.0040 0.0006 0.0058
1.50 –0.1125 2.5259 –0.9999 0.0035 0.0006 0.0052
CHCl/mol l–1 0.046 –0.0004 2.7639 –0.2901 0.0037 0.0005 0.0049
0.092 –0.0060 2.7587 –0.9862 0.0022 0.0004 0.0032
0.138 –0.00248 2.6283 –0.9996 0.0015 0.0002 0.0022
0.184 –0.0769 2.5759 –0.9995 0.00513 0.0008 0.0075
0.230 –0.1948 2.4843 –0.9998 0.0137 0.0022 0.0201
0.276 –0.4057 2.6919 –0.9998 0.0145 0.0037 0.0156
0.322 –0.7430 2.5537 –0.9986 0.0462 0.0214 0.0302
Cbromate/mol l–1 ¥ 10–4 0.20 –0.0466 2.5483 –0.9997 0.0027 0.0004 0.0039
0.40 –0.0800 2.7327 –0.9991 0.0074 0.0012 0.0108
0.60 –0.1349 2.4747 –0.9994 0.0107 0.0017 0.0156
0.80 –0.1866 2.5981 –0.9999 0.0134 0.0022 0.0084
1.00 –0.1948 2.0843 –0.9998 0.0057 0.0009 0.0120
1.20 –0.2592 2.1302 –0.9995 0.0178 0.0035 0.0228
a. y = ax + b, where x is the time in minutes. b. Standard error of the slope. c. Standard error of the intercept. d. Standard deviation of the fit.

in the solution, the more slowly the decolorization reaction

proceeds.

The influence of variables

Fig. 2 Absorbance change of methyl orange–bromate–HCl–urea
system: (a) 0.00, (b) 15, (c) 90 mg ml–1 urea. Conditions: 6.0 ¥ 10–4
mol l–1 methyl orange, 1.0 ¥ 10–4 mol l–1 bromate, and 0.23 mol l–1
HCl.
which, in the absence of urea, is fairly fast. The inhibition effect
of urea is due to its reaction with produced bromine and
chlorine. This inhibitory effect on the reaction system depends
on the concentration of urea. Figure 2 shows that decolorization
of the reactive mixture containing 15 mg ml–1 of urea occurs
after a 16 min period, whereas the mixture containing six times
as much urea (90 mg ml–1) undergoes complete decolorization
after a 22 min period.
The systems obey Beer’s law for all investigated
concentrations in the range 6 – 90 mg ml–1. The calculated
apparent molar absorptivity was found to be 4.537 ¥ 103 dm3
mol–1 cm–1 and the Sandell sensitivity (S) is 0.013 mg cm–2.
On the basis of the results shown in Table 1, one can observe
that an increase in urea concentration is followed by a decrease
in the value of the slope. The higher the concentration of urea is
The effects of reagent concentrations (concentrations of
methyl orange, bromate, and hydrochloric acid) and temperature
on the reaction system were studied to obtain the best sensitivity
and find optimum conditions.
Hydrochloric acid not only provides a necessary pH of the
reaction system but it also directly participates in the reaction.
It is for this reason that we monitored the influence of changes
in the concentration of the acid upon the reaction speed.
The influence of hydrochloric acid concentration on the
sensitivity was studied over the range of 0.046 – 0.460 mol l–1
with and without urea at 20˚C. Increasing HCl concentration
significantly speeds up the reaction. Within the range 0.046 to
0.138 mol l–1 HCl concentration, the reaction proceeds rather
slowly, whereas the concentrations higher than 0.230 mol l–1
lead to so fast a reaction that cannot be monitored
spectrophotometrically (the time necessary for decolorization of
the system at 0.460 mol l–1 HCl concentration is only 36 s).
The results in Table 1 show when HCl concentration is
increased in the range 0.046 – 0.322 mol l–1, the slope of the
absorbance change for uninhibited reaction increases about 1850
times, and a concomitant increase in the value of the correlation
coefficient R is observed.
In a similar fashion, increased HCl concentrations also affect
the speed of urea-inhibited reaction. Our results shows that, if
the HCl concentration is increased from 0.046 to 0.230 mol l–1,
the difference in absorbance change for uninhibited and
inhibited reaction (DA) in the first 10 min increases, whereas
greater amounts of the acid concentration decrease this
difference. Therefore, the final concentration of 0.230 mol l–1
acid was selected as optimum.
Effect of bromate concentration on the reaction system in the
first 10 min of reaction was studied in the range of 2 ¥ 10–5 – 1.4
¥ 10–4 mol l–1. The results show that an increase in bromate
772
Fig. 3 Effect of methyl orange concentration on the reaction time
uninhibited reaction: (a) 6.0 ¥ 10–4, (b) 1.20 ¥ 10–3 mol l–1.
Conditions: 1 ¥ 10–4 mol l–1 bromate and 0.230 mol l–1 HCl.
concentration in the range of 2 ¥ 10–5 – 1.4 ¥ 10–4 mol l–1 leads
to decreased reaction time.
The analytical parameters shown in Table 1 suggest that
bromate concentration has a significant effect upon the speed of
methyl orange decolorization. When the concentration of
bromate ions in the system is increased in the range of 2 ¥ 10–5 –
1.0 ¥ 10–4 mol l–1, the slope of the absorbance change increases
400 times. A further increase in bromate concentration leads to
so short a reaction time that it is impossible for the reaction to
be monitored spectrophotometrically.
The increase in bromate concentration also contributes a faster
reaction in case urea is present in the system. Differences in the
absorbance for the inhibited and uninhibited reaction
significantly decrease by increasing the bromate concentration
up to 1.0 ¥ 10–4 mol l–1. Therefore, this concentration was
selected for the study.
The influence of methyl orange concentration on the
sensitivity was studied in the range of 3 ¥ 10–5 – 1.20 ¥ 10–3 mol l–1
methyl orange in the presence of 0.230 mol l–1 HCl and 1.0 ¥
10–4 mol l–1 bromate concentration at 20˚C with and without
added urea. The results showed that, if the methyl orange
concentration is increased from 3 ¥ 10–5 to 6.0 ¥ 10–4 mol l–1, the
sensitivity increases, whereas a greater amount of the dye
decreased the sensitivity (Fig. 3). Therefore, the 6.0 ¥ 10–4 mol l–1
methyl orange was selected for the study.
The influence of temperature on the sensitivity was studied
over the temperature range of 20 – 45˚C in the presence of
optimum reagent concentration.
Figure 4 shows the effect of temperature on the reaction time
of uninhibited reaction. The results demonstrate that the
temperature increase within the 20 – 25˚C range has an
insignificant effect on the speed of both the uninhibited and
inhibited reaction, whereas the temperatures higher than 35˚C
significantly increase the speed of the reaction, thereby
shortening the reaction time and affecting the possibility of the
application of the reaction for analytical purposes. When the
temperature was increased up to 20˚C, the reaction time and
sensitivity decreased. Therefore, 20˚C was selected for the
study.
It is worth noting here that the recent trend in urea
determination is based on enzymatic methods, which are
sensitive even to 0.5˚C temperature changes. Difference in the
speed of the investigated reaction at 20 and 25˚C is only 3%. As
room temperature in laboratories is usually between 20 – 25˚C,
ANALYTICAL SCIENCES JUNE 2008, VOL. 24
Fig. 4 Effect of temperature on the reaction time of uninhibited
reaction. Conditions: 9.0 ¥ 10–4 mol l–1 methyl orange, 0.230 mol l–1
HCl, and 1 ¥ 10–4 mol l–1 KBrO3.
it is not necessary to undergo thermostated solution when urea
concentration is determined, a fact that significantly simplifies
the working procedure. Of course, in case temperatures in
laboratories reach 30˚C or higher, the reaction can be speeded
up by 10% or higher and samples must be thermostated.
Validation of the proposed method
After optimum conditions for the investigated reaction were
determined, changes in the absorbance were observed over time
at 6.0 ¥ 10–4 mol l–1 methyl orange, 0.230 mol l–1 HCl, and 1 ¥
10–4 mol l–1 KBrO3 in the presence of urea.
Under the optimum conditions, a linear correlation was found
between the absorbance at 505 nm and the urea concentration.
A good linear relationship was observed over 6 to 90 mg ml–1 of
urea. Solutions of known urea concentrations are utilized for
the standard curve. Under optimum conditions, the best linear
correlation (R = 0.99975) is between DA (DA = A – AB, where AB
is absorbance of blank and A is absorbance of sample) and urea
concentration 5 min after the beginning of the reaction.
The linear plot gave the regression equation DA = 0.17833 +
0.00399x, where A is the absorbance and x the urea
concentration in mg ml–1.
The precision of the proposed method was examined by
determining the relative standard deviation (RSD) of five
replicate analyses on the same solution containing four different
concentration of urea. The intra-assay RSD of the aqueous
solution at urea concentration 25, 50, 60 and 80 mg ml–1 were
2.8, 2.5, 2.1 and 2.2%, respectively. The low values RSD (less
than 3.0%) in different solutions reflect the high precision of the
proposed method.
The accuracy of the proposed method was examined by
performing recovery experiments using same aqueous solutions
containing known amounts of urea. Concentrations of urea in
standard solution were 25, 50, 60 and 80 mg ml–1. Recovery
calculated as 100 – [(added value – found value) ¥ 100]/added
value. High values of recovery imply high accuracy of proposed
method.
The limit of detection defined as DL = 3.3s/S, where s is the
standard deviation of the regression line and S is the slope of the
calibration curve of the analyte.22 In this way, the calculated
determination limit is DA = 0.036, which corresponds to 0.34
mg l–1, that is, 5 ¥ 10–9 mol l–1 urea concentration.
The quantitation limit may be expressed as: QL = 10s/S,
where s is the standard deviation of the regression line and S is
ANALYTICAL SCIENCES JUNE 2008, VOL. 24 773

the slope of the calibration line of the urea at concentration near
the quantitation limit.22 In this way, the calculated quantitation
limit is 0.98 mg l–1, that is, 1.6 ¥ 10–8 mol l–1 urea concentration.
The effects of common excipients and other substances were
tested for possible interferences in the assay. One species was
not considered to interfere if it caused a relative error of less
than 5% for the determination of urea. It was observed that
linoleic acid, benzophenon, glycerin, lanolin, vaseline, isopropyl
mirystate, vitamin A and cholesterol did not interfere with the
determination of urea at the levels found in dermatological and
cosmetic samples. The reason is probably non-solubility or very
low solubility of this substances in water. However, a fairly
large error appeared in the presence of ascorbic acid and B
vitamins. Most inorganic ions did not noticeably affect the
accuracy of the determination of urea at 50 – 100 mg ml–1
concentration under optimum condition. Nitrite, sulfate,
chromate, and dichromate ions interfered severely in small
amounts, when Cl–, I–, ClO3–, and IO3– ions interfere seriously.
The interfering effect of various inorganic ions was completely
removed by using an exchange resin. The results are given in
Table 2.
The method ruggedness was evaluated by five replicate
determinations at different concentrations of urea (25, 50, and
80 mg ml–1) over a period of 5 days. The low values of between-
day RSD for different concentration of urea (less than 5%)
indicate that solutions of KBrO3, HCl and methyl orange has a
very good stability.
The robustness of our proposed method is a measure of its
capacity to remain unaffected by small, but deliberate, variations
in method parameters and provides an indication of its reliability
during normal usage. The most significant variables of the
system (temperature and concentration of bromate, methyl
orange and HCl) were modified ±10% from their optimum
values. Errors lower than 5% were observed in all cases. Thus
the proposed methods were found to be robust for routine
determination of urea in dermatological and cosmetic samples.
Applications
To evaluate the proposed method, we applied it to the
determination of urea in synthetic samples, such as in two
commercial urea-containing creams. The cosmetic formulation
was found to be free from urea; thus, synthetic samples were
prepared by adding known amounts of urea to the formulation.
The recoveries are close to 100%, indicating that there is no
serious interference in synthetic samples. The good agreement
between these results and known values indicate the successful
applicability of the proposed method for the determination of
urea in dermatological and cosmetic samples. For this reason,
we applied the proposed method for determination of urea in
two urea-containing creams. The results are given in Table 3. A
statistical analysis of the results by F and t-test showed no
significant difference in accuracy and precision between the
proposed method and the official11,12 method.
Though further investigations are necessary to confirm the
application of the proposed method to a variety of samples, the
developed procedure is suitable for the routine analysis of urea
in real samples.
References

1. D. Häntschel, G. Sauermann, H. Steinhart, U. Hoppe, and J.

Ennen, J. Cosmet. Sci., 1998, 49, 155.
Table 2 Effect of foreign substances on the determination of 2. K. Bohnsack, Skin Care Forum, http://www.scf.online.com.
urea 3. D. P. A. Correia, J. M. C. S. Magalhães, and A. A. S. C.
Tolerance limit/ Machado, Talanta, 2005, 67, 773.
Ion 4. A. S. Sullari and A. H. Uslan, Talanta, 2002, 57, 1039.
mg ml–1
5. Y. Iida, M. Ikeda, M. Aoto, and I. Sato, Talanta, 2004, 64,
Na+, K+, Mg2+, Ca2+, Al3+, CO32–, CH3COO– 100 1278.
NO3–, SO42–, CrO42–, Cr2O72–, NH4+ 50
6. T. Krawczynski, M. Trojanowicz, and A. Lewenstam,
Cl–, Br–, ClO4–, ClO3–, IO3–, ascorbic acid, <10
Talanta, 1994, 41, 1229.
vitamins B
7. G. G. Guilbault and M. L. Seo, Talanta, 1994, 41, 1029.

Table 3 Determination of urea in synthetic and cosmetics samples

Synthetic sample 1 Synthetic sample 2 Synthetic sample 3 “Urea cream” Pharmacy “Sat-N5” BG-pharm

Claimed/mg g –1
20.0 40.0 60.0 50.0 50.0
Proposed method
Founda/mg g–1 18.6 ± 0.8 38.7 ± 0.7 58.5 ± 0.8 48.8 ± 0.9 48.6 ± 0.8
RSDb, % 4.3 1.7 1.4 1.8 1.7
Errorc, % 7.0 3.2 2.5 2.4 2.8
Recoveryd, % 93.0 96.8 97.5 97.6 97.2
Official method11,12
Founda/mg g–1 18.9 ± 0.9 39.1 ± 0.9 58.9 ± 0.8 49.3 ± 0.8 49.0 ± 0.9
RSDb, % 4.8 2.2 1.4 1.6 1.9
Errorc, % 5.5 2.2 1.8 1.4 2.0
Recoveryd, % 94.5 97.8 98.2 98.6 98.0
F-teste 1.29 1.67 1.05 1.24 1.23
t-testf 0.55 0.81 0.75 0.92 0.72

a. Mean ± standard deviation (n = 5). b. Relative standard deviation. c. Error was calculated for synthetic samples as: [(mean – added value)
¥ 100]/added value; and for cream samples as: [(mean – claimed value) ¥ 100]/claimed value. d. Recovery was calculated for synthetic
samples as: 100 – [(added value – found value) ¥ 100]/added value; and for cream samples as: 100 – [(claimed value – found value) ¥
100]/claimed value. e. Tabulated F-value for (4, 4) degrees of freedom at p(0.95) is 6.39. f. Tabulated t-value for 8 degrees of freedom at
p(0.95) is 2.306.
774
8. R. K. Goo, H. Kanai, V. Inouye, and H. Wakatsuki, J.
Assoc. Off. Anal. Chem., 1980, 63, 985.
9. R. N. Beale and D. Croft, J. Clin. Path., 1961, 14, 418.
10. P. S. Francis, S. W. Lewis, and K. F. Lim, Trends Anal.
Chem., 2002, 21, 389.
11. “Pharmacopoeia of the SRJ IV”, 1984, Federal Office of
Health, Belgrade, 190.
12. “Pharmacopoeia of the SRJ V”, 2001, Federal Office of
Health, Belgrade, 1053.
13. G. Absalan and Y. Alipour, Anal. Sci., 2003, 19, 635.
14. A. Afkhami and F. Mosaed, Anal. Sci., 2002, 18, 667.
15. A. A. Esnafi and H. Movahedinia, Spectrochim. Acta, Part
A, 2003, 59, 3159.
16. A. A. Ensafi, B. Rezaei, and H. Movahedinia, Spectrochim.
Acta, Part A, 2002, 58, 2589.
ANALYTICAL SCIENCES JUNE 2008, VOL. 24
17. A. Afkhami and A. Afshar-E-Asl, Anal. Chim. Acta, 2000,
419, 101.
18. K. Basavaiah, U. Chandrashekar, and P. Nage. Govda, J.
Serb. Chem. Soc., 2005, 70, 969.
19. K. Basavaiah, A. Chandrashekar, and P. N. Govda, J. Serb.
Chem. Soc., 2006, 71, 553.
20. D. F. Boltz and J. A. Howell (ed.), “Colorimetric
Determination of Nonmetals”, 1978, Wiley, New York.
21. D. A. Skoog, D. M. West, and F. J. Holler, “Foundamentals
of Analytical Chemistry”, 1999, Skolska knjiga, Zagreb.
22. International Conference on Harmonization (ICH) of
Technical Requirements for Registration of Pharmaceuticals
for Human Use, Topic Q2 (R1): Validation of Analytical
Procedures: Text and Methodology, Geneva, 2005. http://
www.ich.org/cache/compo/276-254-l.html.