Journal of Al-Nahrain University

Vol.14 (3), September, 2011, pp.9-16

Science

Spectrophotometric Method for Determination of Sulfamethoxazole in

Pharmaceutical Preparations by Diazotization-Coupling Reaction
*

Raghad Sinan and Wasan A. Al-Uzri

Chemistry Department, College of Science, Baghdad University, Al-Jadiria,
BaghdadIraq.
*
To Whom Correspondence Should be Addressed.
Abstract
A simple, rapid and sensitive spectrophotometric method has been developed for the
determination of sulfamethoxazole (SMZ) in pure form and pharmaceutical preparations. The
proposed method is based on the diazotization reaction of SMZ with sodium nitrite in hydrochloric
acid medium to form diazonium salt, which is coupled with 1-naphthol in sodium hydroxide
medium to form azo dye, showing absorption maxima at 525 nm. Linearity was observed from
214 g mL-1 SMZ. Statistical analysis of the results and comparison with results by BrattonMarshalls method are also reported.
Keywords: Sulfamethoxazole; Spectrophotometric; 1-Naphthol; Diazotization-Coupling Reaction.
Dizotization and coupling reactions
were used for determination of SMZ
by diazotization reaction of SMZ and
coupling with different coupling agents
such as primaquine phosphate[13], NED[14] ,
iminodibenzyl[15],
1-naphthylamine[16] , 3aminophenol[17] and pyrogallol[18]. These
reactions required removing of excess of
sodium nitrite by sulfamic acid or ammonium
sulfamate, or required diluting the azo dye by
alcohol, heating step and buffer solution for
coupling reaction.
In this work, a rapid and sensitive method
using spectrophotometric detection at 525 nm
was proposed for determination of SMZ in
pharmaceutical preparations. The method is
based on the diazotization reaction of SMZ
with sodium nitrite in hydrochloric acid
medium; the formed diazonium salt is then
coupled with 1-naphthol in sodium hydroxide
medium to form a purplish violet watersoluble mono azo dye. This method does not
need to get rid of excess sodium nitrite (by
addition sulfamic acid or ammonium
sulfamate) because of the low concentration of
sodium nitrite used by adding equimolar
solution of SMZ and sodium nitrite. The
proposed method has been successfully
applied to the determination of SMZ in
pharmaceutical preparations. The method is
safe, simple, sensitive, selective and accurate.

Introduction
Sulfamethoxazole (SMZ), 4-amino-(5methyl-3-isoazolyl) benzensulfonamide, is a
white crystalline powder[1]. It is short- to
medium-acting agents used almost exclusively
to treat urinary tract infections. The usual adult
dosage is 1 g of SMZ two or three times daily.
The fixed-drug combination of trimethoprimSMZ is the drug of choice for infections such
as Pneumocystis jiroveci (formerly P carinii)
pneumonia, toxoplasmosis, nocardiosis, and
occasionally other bacterial infections[2].
Various methods for SMZ determination
have been described, including differential
scanning calorimetry[3], spectrofluorimetry[4],
potentiometry titration[5], sequential injectionchemiluminescence[6] ,
capillary
zone
[7]
electrophoresis ,
reverse
phase-high
performance
liquid
chromatography[8],
micellar electrokinetic chromatography[9],
spectrophotometry[10] and nuclear magnetic
resonance[11].
The Bratton and Marshalls method was
considered to be the most commonly used
colorimetric method for sulfa drugs. In
this method, SMZ was diazotized with
dilute hydrochloric acid and sodium nitrite,
and the excess of nitrite was destroyed
with ammonium sulfamate. A pink color
was
formed
by
adding
N-(1naphthyl)ethylenediamine
dihydrochloride
(NED). The color intensity was measured at
545 nm [12] .
9

Raghad Sinan

Experimental
Apparatus
All spectral and absorbance measurements
were performed on a Shimadzu UV - VIS 160
(Tokyo, Japan) digital double-beam recording
spectrophotometer using 1 cm quartz cells.

Pharmaceutical preparations of
sulfamethoxazole
Pharmaceutical preparations were obtained
from commercial sources.
1- Metheprim Tablets (SDI, Samarra-Iraq):
400 mg sulfamethoxazole and 80 mg
trimethoprim for each tablet.
2- Supreme
Tablets
(Micronova-India):
400 mg sulfamethoxazole and 80 mg
trimethoprim for each tablet.
3- Co-trimoxazole Suspensions (Pharaonia
Pharmaceuticals,
Alexandria-Egypt):
200 mg sulfamethoxazole and 40 mg
trimethoprim for each 5 mL of suspension
(100 mL).
4- Cotrim Suspensions (Asia Pharmaceutical
Industries-Syria):200 mg sulfamethoxazole
and 40 mg trimethoprim for each 5 mL of
suspension (100 mL).

Reagents
All chemicals were of analytical reagents
grade.
1- Sulfamethazole (SMZ) (253.3 g mol-1)
stock standard solution (500 g mL-1) was
prepared by dissolving 0.0500 g of pure
SMZ (SDI) in 5 mL of ethanol and made
up to 100 mL volumetric flask with
distilled water. Working standard solutions
were prepared by suitable dilution of the
stock standard solution.
2- Sodium nitrite (69 g mol-1) solution
(510 -3 M) was prepared by dissolving
0.0690 g of sodium nitrite (Merck) in
distilled water and diluting to the marked
in 200 mL volumetric flask. Then,
3.95 10 -4 M was prepared by diluting
19.75 mL of sodium nitrite solution
(5 10-3 M) with distilled water in
250 mL volumetric flask.
3- Hydrochloric acid solution (1 M) was
prepared by diluting 43 mL of 11.64 M of
concentrated hydrochloric acid (BHD)
with distilled water in 500 mL volumetric
flask.
4- 1-Naphthol (144.17 g mol1) solution
(0.1% w/v) was prepared by dissolving
0.1000 g of 1-naphthol (BHD) in ethanol
(BHD) and diluting to the marked with
same solvent in 100 mL volumetric flask.
The solution of 3.95 10 -4 M 1-naphthol
was prepared by diluting 14.2 mL of
1-naphthol solution (0.1% = 6.93610 -3 M)
with distilled water in 250 mL volumetric
flask.
5- 1-Naphthol solution (3.95 10 4 M) was
prepared by dissolving 0.01 g of
1-naphthol (BHD) in ethanol (BHD) and
diluting to the marked with same solvent in
100 mL volumetric flask.
6- Sodium hydroxide solution (4 M) was
prepared by dissolving 16.0000 g of
sodium hydroxide (BHD) in distilled water
and diluting to the marked in 100 mL
volumetric flask.

Analytical procedure for calibration

An aliquot of a standard solution
(100 g mL-1 = 3.95 10 -4 M) containing
0.1 3.5 mL of SMZ was transferred into a
series of 25 mL calibrated flasks. To this
solution was added equimolar of sodium nitrite
solution (3.95 10 -4 M) and the acidity was
adjusted with 3 mL of 1 M hydrochloric acid
solution. The solution was shaken thoroughly.
Then, 2 mL of 0.1% 1-naphthol and 3 mL of
4 M sodium hydroxide solutions were added
and the contents were diluted to the mark with
distilled water and mixed well. After 5 min,
the absorbance of the colored azo dye was
measured at 525 nm against the corresponding
reagent blank.
Procedure for the assay of pharmaceutical
preparations
1- Tablets solution (500 g mL-1)
Weigh and finally powdered of 10 tablets,
extract an accurately weighed portion of the
powder equivalent to about 50 mg of SMZ in
5 mL of ethanol and filtered into a 100 mL
volumetric flask, the residue was
washed with distilled water and finally the
volume was diluted to the marked with
distilled water. Further appropriate solution
(100 g mL-1) was made by using distilled
water. Three different concentrations of this
tablets solution were analyzed in five
replicate by analytical spectrophotometric
procedure.
10

Journal of Al-Nahrain University

Vol.14 (3), September, 2011, pp.9-16

2- Suspension solution (500 g mL-1)

An aliquot corresponding to 100 mg of SMZ
was dissolved in 5 mL of ethanol. The
solution was filtered into 200 mL volumetric
flask; the residue was washed with 5 mL of
ethanol and finally the volume was diluted to
the marked with distilled water.
Further
appropriate
solutions
of
pharmaceutical preparations were made by
using distilled water.

Science

Optimization of the experimental conditions

The effects of various parameters on the
absorption intensity of the formed product
were optimized.
In the subsequent experiments, 1 mL of
SMZ solution (100 g mL-1 = 3.95 10-4 M)
with equimolar of sodium nitrite solution
(1 mL of 3.95 10 -4 M) was taken in 25 mL
final volume and mixing with 3 mL of acid
(1 M), 3 mL of 1-naphthol (0.1%) and 2 mL of
base (4 M) and diluting to the marked with
distilled water. The absorbance of a series of
solutions were measured by varying one and
fixing the other parameters at 525 nm versus
reagents blanks.
This method does not need to get rid of
excess sodium nitrite (by addition of sulfamic
acid or ammonium sulfamate) because of the
low concentration of sodium nitrite used in
equimolar solution of SMZ and sodium nitrite.
The diazotization reaction of SMZ was
formed in acidic medium. Therefore, the
effects of different acids solutions (1 M) were
studied such as hydrochloric acid, sulfuric
acid, phosphoric acid and acetic acid. It was
found that hydrochloric acid the most suitable
acidic medium for a maximum absorbance and
was used in all subsequent experiments.
The coupling reaction of diazotized SMZ
with 1-naphthol was formed in alkaline
medium. Therefore, the effects of different
alkaline solutions (4 M) were studied such as
sodium
hydroxide,
sodium
carbonate,
potassium
hydroxide
and
ammonium
hydroxide. It was found that sodium hydroxide
the most suitable alkaline medium for a
maximum absorbance and was used in all
subsequent experiments.
The effects of different volumes
(0.05 5 mL) of hydrochloric acid (1 M),
(0.05 5 mL) of 1-naphthol (0.1% w/v) and
(0.5 5 mL) of sodium hydroxide (4 M) were
examined on the maximum absorbance of the
formed product. Fig. (2) shows that 3 mL of
HCl (1 M), 2 mL of 1-naphthol (0.1%) and
3 mL of NaOH (3 M) were enough to obtain
the maximum absorbance.
The effect of temperature on the color
intensity of the azo dye [it produce from
mixing 1 mL of SMZ (100 ppm = 3.95
10-4 M), 1 mL of sodium nitrite (3.95
10-4 M), 3 mL of hydrochloric acid (1 M),

Results and Discussion

Preliminary studies
Throughout the preliminary study on the
diazotization reaction of SMZ, with sodium
nitrite in hydrochloric acid medium; the
formed diazonium salt is then coupled with
1-naphthol in sodium hydroxide medium, a
purplish violet water-soluble azo dye was
obtained with a maximum absorbance at
525 nm [Fig. (1)]. The absorbance of the azo
dye solution [it produce from mixing 1 mL of
SMZ (100 ppm = 3.95 10 -4 M), 1 mL of
sodium nitrite (3.95 10-4 M), 3 mL of
hydrochloric acid (1 M), 2 mL of 1-naphthol
(0.1%), and 3 mL of sodium hydroxide (4M)
and diluted to 25 mL with distilled water]
measured versus reagent blank [it contain all
components above except of SMZ] which has
negligible absorbance at this wavelength.

Fig. (1) Absorption spectra of the azo dye

against reagent blank (A) and reagent blank
against distilled water (B).

11

Raghad Sinan

2 mL of 1-naphthol (0.1%), and 3 mL of

sodium hydroxide (4 M) and diluted to 25 mL
with distilled water] was studied. In practice,
high absorbance was obtained when the color
was developed at room temperature (25C)
that when in ice bath (5C) or in water bath
(45C).

about 2 min at room temperature. The color

was stable for a period of 2 h.
The stoichiometry of the product was
investigated using the mole ratio and
continuous variation methods.
In mole ratio method, increased volumes
(0.5 4 mL) of 3.95 10 -4 M 1-naphthol
solution (VR) were added to a 2 mL of
3.95 10 -4 M of SMZ (VD) were diazotized
using 2 mL of sodium nitrite (3.95 10 -4 M)
and 3 mL of hydrochloric acid (1 M) and
coupled according to analytical procedure in
3 mL of sodium hydroxide (4 M) and diluted
to 25 mL with distilled water.
In continuous variation method, volumes
(1 6 mL) of 3.95 10 -4 M portions of SMZ
(VD) were diazotized using equimolar of
sodium nitrite (3.95 10 -4 M) and 3 mL of
hydrochloric acid (1 M) and coupled according
to analytical procedure with the corresponding
complementary volume of 3.95 10 -4 M
1-naphthol solution (VR) to give a total volume
of 6 mL for VD + VR in 3 mL of sodium
hydroxide (4 M) and diluted to 25 mL with
distilled water.
The results obtained in Fig. (3) and
Fig. (4) show that a 1:1 azo dye was formed
between
diazotized
SMZ
(D)
and
1-naphthol (R).

Fig. (3) Mole ratio plot.

Fig. (2) Optimum conditions for

determination of sulfamethoxazole.
The stability of the dye was studied for 2 h
following the mixing of the reagents. The
colored azo dye developed rapidly after
mixing and attained maximum absorbance
Fig. (4) Continuous variation plot.
12

Journal of Al-Nahrain University

Vol.14 (3), September, 2011, pp.9-16

excipient, none of these substances interfered

seriously [Table (1)].

For the diazotization process; it would be

expected that NH2 group in SMZ would be
readily diazotized in a hydrochloric acid
medium, and that diazonium group would then
react with a molecule of 1-naphthol by
electrophilic substitution at the 4-position of
the coupling agent to produce an intense
purplish violet azo dye in sodium hydroxide
medium. An investigation of the continuous
molar variation of diazotized SMZ and
1-naphthol showed that diazotized SMZ
interacts with 1-naphthol in the ratio of 1 : 1.
A reaction sequence based on the above results
is shown in Scheme (1).
_ +
Cl N

NH 2

Table (1)
Determination of 4 g mL-1 of
SMZ in the presence of excipients.
Excipient,
40 g mL-1

Mg-stearate
Lactose
Talc
Starch
PVP
TMP

HCl

ONa

1-Naphthol
Purplish violet azo dye

O
H 3C _
_

O O
_ S_

RSD,
%

4.002
4.030
4.003
3.913
3.986
4.007

+ 0.550
+ 0.750
+ 0.075
2.175
0.350
+ 0.175

100.550
100.750
100.075
97.825
99.650
100.175

1.143
1.355
0.740
1.794
0.971
0.575

Analytical characteristics of
spectrophotometric method
For proposed method, the calibration graph
was obtained by the procedure described
previous and a series of standard solutions was
analyzed in triplicate to test the linearity. The
molar absorptivity (), the Sandells sensitivity
(S), the slope (a), the intercept (b), the
correction coefficient (r) and correlation of
determination (r2) were evaluated by a least
squares regression analysis and are included in
Table (2).
Statistical evaluation[20] of the regression
line gave the values of standard deviations for
residuals (Sy/x), slope (Sa) and intercept (Sb) at
95% confidence are shown in the same Table.
These small figures point out to the high
precision of the proposed method. The limit of
detection (LOD) and limit of quantitative
(LOQ) were determined using the formula:
LOD or LOQ = k Sy/x / b, where k = 3 for
LOD and 10 for LOQ. The LOD and LOQ
values are shown in Table (2).

NaOH

Rec.,
%

Diazotized SM Z (I)
OH

Erel, %

*Average of five determination, Erel = relative error,

Rec. = recovery, RSD = relative standard deviation.

SMZ

Concn.
of SMZ,
g mL-1
Found*

NaNO 2

Science

_
H

Scheme (1) Reaction sequence.

The product formed was soluble in water.
The apparent stability constant was calculated
by comparing the absorbance of a solution
containing stoichiometric amount of SMZ and
1-naphthol (3.95 10 -4 M) (AS) that of a
solution containing a five-fold excess of
1-naphthol reagent (Am) and according to
analytical procedure. The stability constant
(K)[19] [K = (1-) / 2 C; = (Am As) / Am;
Am = 0.522, As = 0.367; C = 1.58 10 -5 M]
of the product in water under the
described experimental of conditions was
5.059 10 5 L mol-1
In order, assess the possible analytical
applications of the proposed method. The
effects of some common excipients and
compounds frequently found with SMZ in
pharmaceutical
preparations,
such
as
magnesium stearate, lactose, talc, starch, poly
vinyl pirrolidone (PVP) and trimethoprim
(TMP) were studied by analyzing. Synthetic
sample solutions containing 4 g mL-1 of SMZ
and excess amounts (10-fold excess) of each
13

Raghad Sinan

Table (3)
Accuracy and precision of the proposed
method.

Table (2)
Data for calibration graph for SMZ using the
proposed method.
Parameter

Value

Linearity range, g mL-1

2 - 14

0.9998

r2

Concn. of SMZ,
g mL-1

Erel, %

Rec.,
%

RSD,
%

Present

Found*

0.9996

4.000

3.975

0.625

99.375

1.524

, L mol-1 cm-1

3.3714 104

8.000

7.981

0.238

99.762

0.360

S, g cm-2

7.5132 10 -3

12.000

11.759

2.008

97.992

0.325

-1

a, mL g

0.1331

0.0034

*Average of five determination.

Sy/x

1.0403 10

Sa

7.2721 10 -4

Sb

5.4876 10 -3

LOD, g mL-1

0.2345

-1

0.7816

LOQ, g mL

Pharmaceutical Applications
The proposed method was applied
successfully to analysis of different
pharmaceutical preparations containing SMZ
and the results are summarized in Table (4).
For all preparations examined, the assay
results of proposed method were in good
agreement with the declared content.
The results obtained by the proposed
method were compared with BrattonMarshalls method [12] [Table (5)] by applying
the F-test and t-test at 95% confidence level.
The calculated values for F (5.527) and
t (0.069) for proposed method did not exceed
the critical values of F4,4 = 9.605 and t = 2.306
(n1 + n2 2 = 8). These confirming that there
are no significant differences between the
proposed method with Bratton-Marshalls
method with respect to precision and accuracy
in the determination of SMZ in pharmaceutical
preparations.

-2

Accuracy and precision of the proposed

spectrophotometric method
The accuracy and precision of the
proposed method were tested by analyzing five
replicate of SMZ by proposed spectrophotometric
method
for
three
different
concentrations of SMZ. The low values of
Erel% summarized in Table (3). The RSD%
was found to be less. These values indicated
the high accuracy and precision of the
proposed method.

Table (4)
Application of the proposed method for determination of SMZ in pharmaceutical preparations.
Pharmaceutical preparation
Metheprim
Tablets
Supreme
Tablets
Co-trimoxazole Suspensions
Cotrim
Suspensions

Concn. of SMZ, g mL-1

Present
Found*
4.000
8.000
12.000
4.000
8.000
12.000
4.000
8.000
12.000
4.000
8.000
12.000

3.918
7.921
11.997
3.985
8.005
11.991
4.005
8.014
12.088
4.032
8.026
12.000

* Average of five determinations.

14

Erel,
%

Rec.,
%

RSD, %

2.050
0.988
0.025
0.375
+ 0.063
0.075
+ 0.125
+ 0.175
+ 0.733
+ 0.800
+ 0.325
0.000

97.950
99.012
99.975
99.625
100.063
99.925
100.125
100.175
100.733
100.800
100.325
100.000

2.180
1.407
0.886
1.835
0.457
0.262
1.970
0.432
2.414
1.741
0.627
0.365

Journal of Al-Nahrain University

Vol.14 (3), September, 2011, pp.9-16

Science

Table (5)
Comparison of the proposed method with Bratton-Marshalls method for
determination of pharmaceutical preparations of SMZ.
Recovery*, %
Pharmaceutical preparation

Proposed
method

Bratton-Marshalls method

Pure SMZ

100.000

100.000

Metheprim Tablets

98.979

99.700

Supreme Tablets

99.871

99.925

Co-trimoxazole Suspensions

100.344

100.225

CotrimSuspensions

100.375

99.625

* Average of five determination.

sulfamethoxazole
in
pharmaceutical
preparations containing trimethoprim by
spectrofluorimetry after derivatization with
fluorescamine, Fresenius J. Anal. Chem.,
Vol. 365, No. 5, 1999, pp. 444-447.
[5] N.M.A.K. Nazer, T.K. Shabeer and P.
Riyazuddin,
Indirect
potentiometric
titration of sulphamethoxazole in the
presence of trimethoprim in co-trimazole
tablets using copper based mercury film
electrode, Chem. Pharm. Bull., Vol. 49,
No. 3, 2001, pp. 278-281.
[6] H. Pasekov, M. Polek, J.F. Cigarro and
J. Dolejov, Determination of some
sulphonamides by sequential injection
analysis
with
chemiluminescence
detection, Anal. Chim. Acta, Vol. 438,
No. 1-2, 2001, pp. 165-173.
[7] J.J.B. Nevado, G.C. Penalvo and F.J.G.
Bernardo, Determination of sulfamethoxazole, sulfadiazine and associated
compounds in pharmaceutical preparations
by capillary zone electrophoresis, J.
Chromatogr. A, Vol. 918, No. 1, 2001,
pp. 205-210.
[8] C. Akay and S.A. Ozkan, Simultaneous
LC determination of trimethoprim and
sulphamethoxazole
in
pharmaceutical
formulations, J. Pharm. Biomed. Anal.,
Vol. 30, No. 4, 2002, pp. 1207-1213.
[9] J.M.L. Gallego and J.P. Arroyo,
Determination of prednisolone and the
most important associated compounds in
ocular and cutaneous pharmaceutical
preparations by micellar electrokinetic

Conclusions
The proposed method offers clear
advantages for the fast determination of SMZ
in the presence of the related compounds, such
as TMP in pharmaceutical preparations. The
method was found to be very simple, rapid,
low cost, and fairly selective than some of the
reported colorimetric methods. They had an
advantage of being accurate, did not require
the removal of excipients, any chemical
sample pretreatment, temperature control, pH
control, solvent extraction step, and expensive
reagents and solvents.
The proposed method was applied to the
analysis of SMZ in tablets and suspensions
solutions, suggesting that they used as a
reliable and advantageous alternative to the
other previously exported methods for routine
analysis of SMZ in these samples.
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