Application of sulphanilamides disazo dyes with Tropaeolin O for simple, rapid and sensitive spectrophotometric assay of medicines

Article abstract of DOI:10.1016/j.saa.2011.02.036

A rapid, simple and sensitive spectrophotometric method for the determination of some sulphanilamides is described. The method is based on the formation of blue coloured disazo dyes by the diazotization of sulphonamides viz. sulphanilamide (SA), sulphamerazine (SMR), sulphamethazine (SMZ), sulphadimethoxine (SDM), sulphamethoxazole (SMX), sulphadiazine (SDA), sulfathiazole (STZ), sulphaguanidine (SGN), sulphamonomethoxine (SMM), sulphamethoxypyridazine (SMP) in 0.5 M hydrochloric acid media at ice bath followed by the azocoupling reaction with acid monoazo dye Tropaeolin O (TrO) at pH = 10.5. Formed products are stable for 10 h at room temperature. Effective molar absorptivities at absorbance maxima 595 nm for disazo dyes were ～10⁴ M^-1 cm^-1. Stoichiometric ratios of the components of disazo dyes were determined by means of mole ratio and continuous variations methods. Linear ranges for sulphanilamides determination were 0.4-14.0 μg ml^-1. The methods were successfully approved at suphanilamides determination in model solutions and commercial pharmaceutical preparations.

Full text of DOI:10.1016/j.saa.2011.02.036

Spectrochimica Acta Part A 79 (2011) 325–331
Contents lists available at ScienceDirect
Spectrochimica Acta Part A: Molecular and
Biomolecular Spectroscopy
journal homepage: www.elsevier.com/locate/saa
Application of sulphanilamides disazo dyes with Tropaeolin O for simple, rapid
and sensitive spectrophotometric assay of medicines
Maria Boikoa,b,∗, Teodoziya Vrublevskaa, Olha Korkunaa, Grigory Teslyarb
a Ivan Franko Lviv National University, Chemistry Faculty, Analytical Chemistry Department, Kyrylо & Mefodiy Str., 6, 79005 Lviv, Ukraine
b State Scientific Research Control Institute of Veterinary Preparations and Fodder Additives, Donetska Str.,11, 79019 Lviv, Ukraine
a r t i c l e i n f o
a b s t r a c t
Article history:
A rapid, simple and sensitive spectrophotometric method for the determination of some sulphanilamides
is described. The method is based on the formation of blue coloured disazo dyes by the diazoti-
zation of sulphonamides viz. sulphanilamide (SA), sulphamerazine (SMR), sulphamethazine (SMZ),
sulphadimethoxine (SDM), sulphamethoxazole (SMX), sulphadiazine (SDA), sulfathiazole (STZ), sulpha-
guanidine (SGN), sulphamonomethoxine (SMM), sulphamethoxypyridazine (SMP) in 0.5 M hydrochloric
acid media at ice bath followed by the azocoupling reaction with acid monoazo dye Tropaeolin O (TrO)
at pH = 10.5. Formed products are stable for 10 h at room temperature. Effective molar absorptivities
at absorbance maxima 595 nm for disazo dyes were ∼104 M−1 cm−1. Stoichiometric ratios of the com-
ponents of disazo dyes were determined by means of mole ratio and continuous variations methods.
Linear ranges for sulphanilamides determination were 0.4–14.0 ␮g ml−1. The methods were success-
fully approved at suphanilamides determination in model solutions and commercial pharmaceutical
preparations.
Received 8 November 2010
Received in revised form 10 February 2011
Accepted 16 February 2011
Keywords:
Sulphanilamides
Azo dyes
Tropaeolin O
Azocoupling
Disazo dyes
Spectrophotometry
© 2011 Elsevier B.V. All rights reserved.
1. Introduction
of sulphanilamides with trimethoprime and antibiotics of different
groups are more effective [1].
Sulphanilamides (SA) are synthetic chemotherapeutical
medicaments, derivatives of sulphanilic acid, which are able to
depress the development of Gram-positive and Gram-negative
bacteria, some protozoons and pathogenic fungi. In spite of the
fact, that sulphanilamides appeared in medical practice as far
back as the beginning of XX century, this group of medications is
widely used up today for the treatment of infectious pathology
and realized at the pharmaceutical market on a large scale. Over
15 thousand derivatives of sulphanilic acid, about 40 of which are
introduced in medical practice as anti-infectives medicinal, were
synthesized for today.
Sulphanilamides are the analogues of p-aminobenzoic acid
which is the constituent of structure of tetrahydrofolic acid – a
coferment of a few enzymes, which take part in the biosynthesis of
purine and pyrimidine bases, entering into the structure of nucleic
acids. Sulphanilamides’ action on microbal cells is related with the
disturbance of folic and tetrahydrofolic acid synthesis, which are
necessary, in turn, for the biosynthesis of nucleic acids, the end-
point of what is the microorganism’s death. Combined preparations
The harmless and effective use of medications needs a multilevel
control of their quality, in particular, the control of homogeneity
of the dosing units of medicines [2]. Utilizing few different bioac-
tive substances in sulphanilamides composition usually requires
the application of sensitive and selective methods for their deter-
mination both in the prepared drugs dosage forms and their rests
in physiological liquids.
For quantitative sulphanilamides assay a high-performance liq-
uid chromatography [3–6], immunoenzyme analysis [7–9] and
voltammetry [10,11] are often used. However, these methods are
expensive in the use, require the reagents of very high purity,
though their use is reasonable at the analysis of sulphanilamides
rests in animal food, background objects, biological liquids. For the
routine laboratory analysis of sulphanilamides substances and their
preparations nitritometry [12] is more frequently utilized, which is
not very sensitive and requires plenty of the probed samples, and
also it is not selective, because substances which contain primary or
secondary amino groups as well as hydrazides form similar reaction
products.
All spectrophotometric methods, suggested for sulphanilamides
determination in preparations, can be divided into two groups. The
first group of methods is based on SA determination due to own
absorbance in UV spectrum range at ꢀ = 245–270 nm, however,
they are nonselective, because most organic compounds absorb
in the same spectrum range, so these methods are suitable only
∗
Corresponding author at: Ivan Franko Lviv National University, Chemistry Fa-
culty, Analytical Chemistry Department, Kyrylо & Mefodiy Str., 6, 79005 Lviv,
Ukraine. Tel.: +380 322394048.
E-mail address: boiko maria@ukr.net (M. Boiko).
1386-1425/$ – see front matter © 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.saa.2011.02.036

326
M. Boiko et al. / Spectrochimica Acta Part A 79 (2011) 325–331
for the analysis of clean substances [13]. The second group of
spectrophotometric methods is based on absorbance measuring of
products of coloured reaction of SA with different organic reagents,
in particular, aromatic aldehydes, phenols, imines, dinitrobenzene-
furoxanes [14–24]. However, described methods require using
of organic media and they are multistage as well as nonselective
and insensitive. Among the dyes for the spectrophotometric
determination of sulphanilamides only alizarin derivatives are
offered, which form complex compounds with charge transfer
[25], however, methods which are based on such interaction are
low-sensitive and low-contrast.
As primary aromatic amino group is entered to the sul-
phanilamides structure, it can be diazotized and azocoupled
with aromatic compounds. Monoazo dyes are the perspective
reagents for the azocoupling with diazotized sulphanilamides for
the obtaining colour products. Therefore we studied diazotized
sulphanilamides interaction with acid monoazo dye Tropaeolin
O (sodium salt of 2ꢀ,4ꢀ-azophenolsulfonic acid, Acid Yellow, Acid
Orange 6, CAS № 547-57-9, C.I. 14270) with the purpose to elaborate
the spectrophotometric method of SA determination. Spectropho-
tometric utilization of Tropaeolin O is very limited, it is known only
about its use for the determination of palladium(II) [26,27], osmium
(IV) [28] and proteins of albumin and casein [29].
Scheme 1. Reaction sequence for the formation of blue disazo dye under sulphanil-
amide interaction with Tropaeolin O.
2. Experimental
2.1. Reagents and apparatus
All aqueous solutions were prepared using distilled water.
Sulphanilamide,
sulphadimethoxine,
sulphamerazine,
sulphamethoxazole,
sulphamethazine,
sulphadiazine,
sulphathiazole, sulphaguanidine, sulphamonomethoxine, sul-
phamethoxypyridazine were purchased from Sigma (USA).
Solutions of sulphanilamides were prepared by dissolving appro-
priate amounts of the reagents of pharmacopoeia grade in 0.1 M
sodium hydroxide solution because of their slight solubility in
water [30].
Fig. 1. Absorption spectra of Tropaeolin
O
and disazo dyes aqueous
solutions;
C(HCl) = 0.5 M;
C(TrO) = 2.5 × 10−5 M;
C(NaNO2) = 2.5 × 10−4 M;
C(SA) = 1.5 × 10−5 M; pH = 10.5; l = 1 cm.
at 595 nm in 1.0 cm cuvettes. Sulphanilamide concentration was
calculated using the methods of calibration curve and single-point
standardization.
Solutions of Tropaeolin O (Merck, Germany) were prepared by
dissolving appropriate amounts of the reagent of analytical grade
≥97% purity in distilled water.
3. Results and discussion
The solutions of hydrochloric acid, sodium hydroxide and
sodium nitrite were prepared from the chemicals of the analytical
grade.
The interaction of Tropaeolin O (TrO) with ten sulphanilamides
(Table 1), which are more frequently used in medical and veterinary
practice, has been investigated.
Quantitative determination of SA is based on the obtaining of
water soluble coloured products of diazotized sulphanilamides
with Tropaeolin O (Scheme 1). Nitrite-ions diazotize primary aro-
matic amino group of SA (1) in acid media with the formation
of diazotized SA (2), which interact with Tropaeolin O in alkaline
media forming blue coloured disazo dyes (4).
UV–vis measurements were performed with UV-Vis scanning
spectrophotometer CARY.WIN–UV-Vis-50 (Varian, USA) using l cm
cuvettes. All absorbance measurements were performed at ∼20 ◦C.
pH measurements were carried out with a pH-meter, model pH
150M (Gomelsky Plant of Measuring Devices, Belarus), equipped
with a glass electrode. pH of each solution was adjusted using
diluted HCl and NaOH solutions.
2.2. Procedure of sulphanilamides determination
3.1. Absorption spectra
5.0 ml of 0.5 M hydrochloric acid solution was placed into
a 25 ml volumetric flask. Then a sample of solution containing
0.4–14.0 ␮g ml−1 of sulphanilamide in final volume was added.
Next 5.0 ml of 1.25 × 10−2 M sodium nitrite solution was added into
the flask. Obtained solution was stirred and cooled on an ice bath
(∼0 ◦C) for 10 min. Then 1.0 ml of 1.25 × 10−3 M Tropaeolin O solu-
tion was added into the flask. Obtained mixture was neutralized
by adding of 2.5 ml of 1 M sodium hydroxide solution and the pH
value was adjusted to pH = 10.5 and distilled water was added to
the full volume of 25 ml. Then the solution was mixed thoroughly
and the absorbance measurements (at room temperature ∼20 ◦C)
were carried out against all corresponding reagents blank solution
Absorption spectra of disazo dyes (products of diazotized sul-
phanilamides coupling with Tropaeolin O) are shown in Fig. 1.
Colour transition region for Tropaeolin O is pH = 11.1–12.7 from
yellow to reddish-brown; pKa = 11.8. At pH = 11.1 absorption max-
ima of the dye is in the range of waves lengths 420–440 nm
(ε = 3.3 × 104 M−1 cm−1), and at pH = 12.7
– at 475–495 nm
(ε = 2.2 × 104 M−1 cm−1) [31].
As can be seen from absorption spectra (Fig. 1), maximum
absorbance of Tropaeolin is at 420 nm, while the absorbance maxi-
mum of disazo dyes for all tested sulphanilamides are in the range
of waves lengths 589–604 nm, where TrO absorbance is negligi-
ble. More detailed absorbance maxima of all obtained disazo dyes

M. Boiko et al. / Spectrochimica Acta Part A 79 (2011) 325–331
327
Table 1
Molecular structure of investigated sulphanilamides.
O
S
H2N
H3C
NH2
O
Sulphanilamide (SA), streptocidum p-aminobenzenesulphonamide, CAS 1 63-74-1
O
S
N
N
N
H
NH2
NH2
O
Sulphamerazine (SMR) 2-(p-aminobenzenesulphonamido)-4-methylpyrimidine, CAS 1 127-79-7
H3C
O
S
N
N
N
H
O
H3C
H3C
Sulphamethazine (SMZ), sulphadimezine 2-(p-aminobenzenesulphonamido)-4,6-dimethylpyrimidine, CAS 1 57-68-1
Sulphadimethoxine (SDM) 6-(p-aminobenzenesulphonamido)-2,4-dimethoxypyrimidine, CAS 1 122-11-2
O
O
O
S
N
N
N
H
NH2
O
H3C
H3C
O
N
S
NH2
O
N
H
O
S
O
Sulphamethoxazole (SMX) 3-(p-aminobenzenesulphonamido)-5-methyloxazole, CAS 1 723-46-6
Sulphadiazine (SDA), 2-(p-aminobenzenesulphonamido)-pyrimidine, CAS 1 68-35-9
Sulfathiazole (STZ), 2-(p-aminobenzenesulphonamido)-1,3-thiazol, CAS 1 72-14-0
N
N
N
NH2
H
O
O
N
N
S
O
O
NH2
NH2
S
H
HN
N
H
S
H2N
H3C
O
Sulphaguanidine (SGN), N-(p-aminobenzenesulphonamido)-aminoiminomethyl, CAS 1 57-67-0
O
O
S
N
N
NH2
N
H
O
Sulphamonomethoxine (SMM), 4-(p-aminobenzenesulphonamido)-6-methoxypyrimidine, CAS 1 1220-83-3
Sulphamethoxypyridazine (SMP), 6-(p-aminobenzenesulphonamido)-3-methoxypyridazine, CAS 1 000080-35-3
O
H3C
O
N
H
S
NH2
N
N
O
Table 2
Spectroscopic characteristics of the formed disazo dyes and validation results of sulphanilamides spectrophotometric determination with Tropaeolin O; C(HCl) = 0.5 M;
C(NaNO2) = 2.5 × 10−4 M; C(TrO) = 2.5 × 10−5 M; pH = 10.5; l = 1 cm.
Parameters/characteristics
SA
SMR
SMZ
SDM
SMX
SDA
Blue
STZ
SGN
SMM
SMP
Colour
ꢁꢀmax, nm
Stability, h
595–597
0.4–7.0
596–599
0.4–12.0
589–595
0.4–10.0
591–596
0.4–10.0
595–599
0.4–9.0
594–601
10
0.4–9.0
595–601
0.4–8.0
593–604
0.4–13.0
594–600
0.4–12.0
593–598
0.6–14.0
Optimum photometric linear rang,
␮g ml−1
Limit of quantification, ␮g ml−1
Limit of quantification × 107, M
0.19
11
2.7
0.14
5.3
2.0
0.14
5.0
3.1
0.16
5.2
3.1
0.14
5.5
3.0
0.25
10
2.9
0.09
3.5
3.4
0.22
10
2.1
0.24
8.6
2.3
0.22
7.8
2.1
Molar absorptivity, ε595 × 10−4
,
M−1 cm−1
Regression equation (A)a slope (b)
Intercept (a)
0.146
0.001
0.9995
0.66
0.043
0.9994
0.098
0.009
0.9998
0.087
0.010
0.9994
0.109
0.015
0.9997
0.106
0.019
0.9993
0.124
0.011
0.9998
0.077
0.015
0.9995
0.086
0.015
0.9994
0.075
0.015
0.9995
Correlation coefficient (R)
a
A = bc + a, where c is the concentration of sulphanilamide in ␮g ml−1
.

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M. Boiko et al. / Spectrochimica Acta Part A 79 (2011) 325–331
Fig. 2. Effect of hydrochloric acid concentration during SA diazotization on disazo
Fig. 4. Effect of temperature during SA diazotization on disazo dyes formation;
dyes formation; C(TrO) = 2.5 × 10−5 M; C(NaNO2) = 2.5 × 10−4 M; C(SA) = 4 ␮g ml−1
pH = 10.5; l = 1 cm; ꢀ = 595 nm.
;
C(HCl) = 0.5 M; C(TrO) = 2.5 × 10−5 M; C(NaNO2) = 2.5 × 10−4 M; C(SDM) = 4 ␮g ml−1
pH = 10.5; l = 1 cm; ꢀ = 595 nm.
;
Fig. 3. Effect of sodium nitrite concentration during SA diazotization on disazo
dyes formation; C(HCl) = 0.5 M; C(TrO) = 2.5 × 10−5 M; C(SA) = 4 ␮g ml−1; pH = 10.5;
l = 1 cm; ꢀ = 595 nm.
Fig. 5. Effect of time of sulphanilamide diazotization on the disazo dye yield;
C(HCl) = 0.5 M; C(NaNO2) = 2.5 × 10−4 M; C(TrO) = 2.5 × 10−5 M; C(SA) = 4 ␮g ml−1
pH = 10.5; l = 1 cm; ꢀ = 595 nm.
;
are shown in Table 2. Therefore, further investigations have been
carried out at 595 nm.
temperature effect of SA diazotization on the yield of disazo dyes
was investigated (Fig. 4). As follows from experimental data, the
temperature effect is not considerable, however, the yield of di-
azotized sulphanilamides at ∼0 ◦C (solutions were cooled using an
ice bath) increased insignificantly. Moreover, the results, obtained
on cooling during diazotization, were more reproducible, because
relative error of sulphanilamides determination (n = 5, P = 0.95) at
20 ◦C was 4.5%, while at 0 ◦C – 1.5%.
3.2. Conditions of sulphanilamides diazotization
3.2.1. Effect of hydrochloric acid concentration
In order to establish optimal reaction conditions it was impor-
tant to study the effect of hydrochloric acid concentration on the
yield of diazotized sulphanilamides with the following formation
of disazo dyes. The results are presented in Fig. 2.
As follows from Fig. 2, the maximal yield of azocoupling prod-
ucts of all examined SA with TrO are observed at diazotization
in 0.4–1.0 M hydrochloric acid. When the hydrochloric acid with
concentration ≥1.0 M was used for this purpose the yield of azo-
coupling products has been decreased. Therefore we used 0.5 M
HCl for SA diazotization in all following experiments.
3.2.4. Time effect of SA diazotization on disazo dyes formation
According to the experimental results (Fig. 5), SDM diazonium
salt is formed immediately after reagents mixing, however, its
maximal yield as well as azocoupling products respectively has
been observed in 15 min at room temperature and in 10 min at
diazotization on an ice bath, that allows to shorten the analysis
duration. The further increase of diazotization time did not reflect
in disazo dyes yield. Time effect of SA diazotization on disazo dyes
formation for all tested sulphanilamides is the same.
3.2.2. Effect of sodium nitrite concentration
We also investigated the influence of sodium nitrite concen-
tration as diazotizing reagent on the yield of sulphanilamides
diazonium salts as well as disazo dyes accordingly. As it is shown
in Fig. 3, 1 × 10−4 M sodium nitrite solution is optimal for SA
diazotization. High nitrite concentrations, viz. >0.1 M, caused the
decomposition of azo reagent.
3.3. Azocoupling conditions of Tropaeolin O with diazotized
sulphanilamides
3.3.1. Effect of pH
3.2.3. Effect of temperature
In order to establish optimal reaction conditions, the investiga-
tions of pH effect on the analytical signal value of diazotized SA with
TrO were carried out. As follows from Fig. 6, the maximal yield of
disazo dyes is observed in the range of pH = 10–11, that is inherent
A temperature has significant influence on the diazotization of
organic compounds, whereas it is known that reaction of diazonium
salt formation is more effective at low temperatures. Therefore, the

M. Boiko et al. / Spectrochimica Acta Part A 79 (2011) 325–331
329
were consistent and equaled to 1:1 for all tested sulphanil-
amides.
Effective molar absorptivities for all obtained disazo dyes were
calculated using the results obtained in the method of continuous
variations, which are presented in Table 2.
3.3.3. Stability of azocoupling products of diazotized
sulphanilamides with Tropaeolin O
To study the stability of obtained disazo dyes in time we have
measured the absorbance value during one week. As follows from
obtained data, absorbance value of disazo dyes of all examined SA
did not change for 10 h, but in 5 days diminished on 20%.
3.4. Sensitivity and linear range of sulphanilamides
spectrophotometric determination with TrO
Fig. 6. Effect of pH value on Tropaeolin
O azocoupling with diazotized
sulphanilamides; C(NaNO2) = 2.5 × 10−4 M; C(HCl) = 0.5 M; C(TrO) = 2.5 × 10−5 M;
C(SA) = 4 ␮g ml−1; l = 1 cm; ꢀ = 595 nm.
We have investigated that the absorbance of coloured products
linearly depends on SA concentration in the solution. Validation
results of spectrophotometric determination of five sulphanil-
amides by means of TrO are presented in Table 2.
Thus the elaborated methods for the spectrophotometric deter-
mination of all tested sulphanilamides with TrO possess wide linear
ranges (almost one and a half orders of concentration); they are
simple, rapid and sensitive. Sensitiveness of sulphatiazole deter-
mination was the highest, for all remainder SA it was practically
the same. All SA according to the decrease for their determination
sensitivity can be placed in the row, which is shown in Scheme 2.
As follows from Table 2 and Scheme 2 the absorbance value of
products of SA interaction with Tropaeolin O depends on the pres-
ence of the diverse substituents in the sulphanilamide molecule.
Heterocycles in sulphanilamide molecules are electron donor
groups which influence on the absorbance value of azocoupling
products and have a different effect depending on their heteroatom.
In the row of heteroatoms S > O > N electron donor influence of hete-
rocyclic substituents diminishes [33]. The obtained results confirm
this fact, as most sensitive absorbance band at 595 nm belongs to
azocoupling product of diazotized sulphanilamide which contains
sulphur atom in a heterocycle (STZ). The disazo dye of non sub-
stituted sulphanilamide streptocide has the least intensive band.
Besides that the substituents presence in heterocycles influences on
electronegativity of whole cycle, that is why a sensitivity for SMR,
SDM, SMZ is higher than for SMX because they have substituents
in heterocycles.
Fig. 7. The mole ratio curve for disazo dyes of diazotized sulphanilamides
and Tropaeolin O; C(NaNO2) = 2.5 × 10−4 M; C(HCl) = 0.5 M; C(SA) = 1.5 × 10−5 M;
pH = 10.5; l = 1 cm; ꢀ = 595 nm.
The nature of heterocycle substituent as well as substituents
number influences on the sensitivity of SA determination, because
they posses the different electronegativity and the different value
and sign of inductive effect accordingly. So a methyl group is less
electronegative than the methoxyl one. From the obtained results
one can see, that the more substituents are in a heterocyclic ring the
higher is the sensitivity of SA determination, hereby the presence of
group with less electron acceptor ability (methyl) increases a sen-
sitivity. For heterocycles with two heteroatoms both the nature of
heteroatoms and their location as well as the position in which
a sulphanilamide group is present in heterocycle have an influ-
ence. So SMP has the methoxypyridazine substituent (contains
two nitrogen atoms in ortho-position one for one), and SMM has
the methoxypyrimidine substituent (contains two nitrogen atoms
in meta-position one for one), however we cannot compare the
efficiency of substituents influence on the sensitivity value of SA
determination because sulphanilamide group is bonded to hetero-
cycle in different positions towards methoxyl group.
Fig. 8. The continuous variations curve for disazo dyes of diazotized sul-
phanilamides and Tropaeolin O; C(HCl) = 0.5 M; C(NaNO2) = 2.5 × 10−4 M;
C(SA + TrO) = 3 × 10−5 M; pH = 10.5; l = 1 cm; ꢀ = 595 nm.
for azocoupling of diazonium salts with the cresol group of sec-
ond component [32]. All further investigations were carried out at
pH = 10.5.
3.3.2. Effect of Tropaeolin O concentration
According to the experimental results (Fig. 7), maximal value of
disazo dyes absorbance is observed at 1.5-fold reagent excess.
To establish the molar ratio of the components in the com-
pounds of sulphanilamides with TrO, we used mole ratio method
(Fig. 7) and method of continuous variations (Fig. 8). The sto-
ichiometric SA:TrO ratios, estimated by both these methods,
In another hand different maxima value on disazo dyes
absorbance spectra are related to the mesomer effect caused by the
presence of different heteroatomic constituents. The appearance
of the additional peak at 589–604 is related to the absorbance of
new azo group formed at azocoupling, what is characteristically for

330
M. Boiko et al. / Spectrochimica Acta Part A 79 (2011) 325–331
Scheme 2. Series of sensitivity decrease for sulphanilamides determination at azocoupling reaction with Tropaeolin O.
Table 3
Accuracy of spectrophotometric determination of sulphanilamides in model solutions using Tropaeolin O; P = 0.95; n = 5; C(HCl) = 0.5 M; C(NaNO2) = 2.5 × 10−4 M;
C(TrO) = 2.5 × 10−5 M; pH = 10.5; l = 1 cm; ꢀ = 595 nm.
√
Compound
Added, ␮g
Found x S · t˛/ n, ␮g
R.S.D., %
Sulphanilamide
Sulphamerazine
100.0 1.4
100.0 1.2
100.2 0.9
99.9 1.7
100.1 1.0
100.4 1.6
99.7 1.5
100.2 1.2
100.4 1.5
99.8 1.3
1.1
1.0
0.7
1.4
0.8
1.3
1.3
1.0
1.2
1.1
Sulphamethazine
Sulphadimethoxine
Sulphamethoxazole
Sulphadiazine
Sulphamonomethoxine
Sulphathiazole
100
Sulphamethoxypyridazine
Sulphaguanidine
Table 4
Effect of excipients on the sulphanilamides assays by Tropaeolin O; P = 0.95; n = 5.
√
n
Excipient (Exp)
m(SA):m(Exp)a
m(SA):m(Exp)b
% Recovery of SA, x S · t˛/
Sulphanilamide
Sulphamethazine
Sulphadimethoxine
Starch
Gelatine
Aerosil
Calcium stearate
1:0.1
1:0.1
1:0.1
1:0.01
1:25
1:10
1:5
101.8 1.5
98.8 1.5
95.3 2.1
95.4 1.9
98.6 1.7
97.2 1.8
100.3 1.3
96.0 2.2
99.5 1.8
99.5 1.1
97.4 1.5
97.6 1.7
1:1
a
Mass ratios of SA and excipience, which are present in tested drugs [2].
Maxima mass ratios of SA and excipience, which were examined.
b
molecules with the planar location of the conjugate chromophore
system [32]. Presence of two other peaks at 345 and 420 nm is speci-
fied for Tropaeolin O, however they have diverse absorbance value.
This phenomenon can be explained by the conjugation effect of
␲-electron clouds in the double bond of chromophore groups.
The common excipients such as starch, calcium stearate, gela-
tine, aerosil are present in examined pharmaceutical preparations.
Therefore, the effect of common tablet excipient and additives
on the procedure of SA determination by means of Tropae-
olin O was investigated. An error of 5.0% in the absorbance
readings was considered tolerable. The actual SA to exci-
pient ratio is nearly 10- to 100-fold with respect to the excipient
in pharmaceutical preparations. But as follows from Table 4 the
conditions under which the experiments were carried out, viz., the
ratio of SA to excipient is nearly equal to 500–10,000 with respect
to excipient. That means, the excipients were taken in very large
excess for the experiment. Thus the tested common excipients,
which often accompany the pharmaceutical preparations do not
interfere in the present method.
3.5. Application to samples
The accuracy of spectrophotometric determination of sulphanil-
amides with Tropaeolin O was tested on model solutions using the
method “added–found”. SA amounts were calculated using method
of single-point standardization (Table 3).
The obtained results correlate well with the amount of SA added
into the model solutions, and the values of R.S.D. are within the
range of ones for spectrophotometry. Thus the elaborated methods
of sulphanilamides determination by means of TrO can be effec-
tively applied for the analyses of SA substances.
3.5.2. Procedure of tablet preparation
Ten tablets were weighed and finely powdered in porcelain mor-
tar. The accurate amount of powder, equal to ∼500 mg of SA, was
placed into a 100 ml volumetric flask and dissolved in 50 ml of
0.1 M NaOH for obtaining of SA extraction. Then solution was mixed
for 10 min and 0.1 M NaOH was added to complete the volume to
100 ml. Obtained solution was mixed again and filtered through
the fold filter of medium porosity. The filtrate was 50-fold diluted.
Thereto 1.0 ml of filtrate was placed into a 50 ml volumetric flask
and diluted by 0.1 M sodium hydroxide solution to the full volume
of 50 ml. Nominal SA content in solution obtained in such way was
3.5.1. Approbation of the methods during the analysis of drugs
Elaborated method of sulphanilamides determination by
mean of TrO was approved at testing of sulphadimezine,
sulphadimethoxine, sulphanilamide quantity in tablets “Strep-
tocidum”, “Sulfadimesinum”, “Sulfadimethoxine” accordingly.
Amounts of bioactive substances in these drugs vary in the range
0.475–0.525 g according to formal quality control method [12].

M. Boiko et al. / Spectrochimica Acta Part A 79 (2011) 325–331
331
Table 5
Determination of sulphanilamides in pharmaceuticals; P = 0.95; n = 5.
Tablets
Label claim, g
Amount of drug found, mg
Proposed method
Pharmaceutical method
(nitritometry) [12]
√
√
x
S · t˛/
n
R.S.D., %
x
S · t˛/
n
R.S.D., %
Streptocidum (SA)a
0.5
0.5
0.5
501
501
499
6
5
8
1.0
0.8
1.3
500
501
501
8
7
8
1.3
1.1
1.3
Sulfadimesinum (SMZ)b
Sulfadimethoxinum (SDM)a
a
Marketed by VAT “Monfarm”, Ukraine.
SIC “Borshchahivskiy Chemical-Pharmaceutical Plant” CJSC (BCPP), Kyiv, Ukraine.
b
100 ␮g ml−1. For assay solution an aliquot of 1 ml was pick out and
then treated as described above in the recommended procedure for
the sulphanilamides determination.
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The assay results are presented in Table 5.
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According to Table 5, obtained data were comparable to those
obtained using the official methods of nitritometry for each of the
studied drugs.
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4. Conclusions
At first the formation of disazo dyes by the interaction of di-
azotized sulphanilamides with acid monoazo dye Tropaeolin O has
been established. Azocoupling occurs due to the cresol group of
second component (TrO), which has compatibly oriented substi-
tutes. Optimum conditions for sulphanilamide, sulphamerazine,
sulphamethazine, sulphadimethoxine, sulphamethoxazole sulpha-
diazine, sulphathiazole, sulphaguanidine, sulphamonomethoxine,
sulphamethoxypyridazine interaction with Tropaeolin O on the
stages of diazotization as well as azocoupling have been inves-
tigated. Spectroscopic and validation characteristics of sulphanil-
amides determination with Tropaeolin O have been established.
The components ratio in the disazo dyes are SA:TrO = 1:1. The
effective molar absorptivity ε595 is ∼104 M−1 cm−1. The proposed
method is found to be simple, rapid, and economical, allows to
determine wide range of SA concentrations (0.4–14.0 ␮g ml−1) and
competes with most of the spectrophotometric methods available
in literature. The method is advantageous over many spectrophoto-
metric methods with special reference to stability and sensitivity.
The statistical parameters and the recovery study data clearly indi-
cate the reproducibility and accuracy of the method. Elaborated
method has been approved during analysis of model solutions and
commercial pharmaceutical preparations. Obtained data were well
correlated to those obtained using the official methods of nitrito-
metry for each of the studied pills. So the recommended procedure
is well-suited for the assay and evaluation of drugs in pharmaceu-
tical preparations to assure high standard of quality control.
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References
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[2] State Pharmacopoeia of Ukraine Add.2, Kharkiv, 2008.