Spectrophotometric Determination of Sulfamethoxazole and Sulfadiazine in Pure and Pharmaceuticals Preparation

Abstract

A rapid and sensitive spectrophotometric method is proposed for the determination of sulfamethoxazole and sulfadiazine. This method is based on the diazotization of sulfonamide with sodium nitrite and a coupling reaction of the diazo-compound with thymol. The optimum reaction conditions and other analytical parameters were evaluated. The linear ranges for the determination of sulfamethoxazole and sulfadiazine are 1-10 μg mL -1 and 1-7 μg mL -1 and their detection limits are 0.008 μg mL -1 and 0.007 μg mL -1 respectively. There was no interference from commonly utilized tablet excipients. The method has been used to determine sulfamethoxazole and sulfadiazine in pharmaceuticals preparation without separation.

Full text

INTRODUCTION
Sulfonamides are a class of drugs commonly used for
their bacteriostatic activity especially in the treatment of urinary-
tract infections1. Sulfadiazine and sulfamethoxazole (Scheme-I)
are now available as widely used pharmaceutical products and
veterinary practices. They are kinds of sulfonamides used in
the treatment of urinary-tract infectious, pneumocystis pneu-
monia, chronic bronchitis, meningococcal meningitis, acute
otitis media and toxoplasmosis2.
S
H2N
O
O
N
N
HN
H2NS NH
O
ONO
CH3
S
u
l
f
a
d
i
a
z
i
n
e
Sulfamethoxazole
Scheme-I: Structural formulas of sulfadiazine and sulfamethoxazole
The aromatic amines were determined spectophotometry
by a diazotization reaction. It is based on the conversion of
free primary aryl amine into a diazonium salt by a reaction
with nitrous acid, on coupling the salt then rapidly forms an
azo-dye with a chromogenic reagent, such as N-(1-napthyl)-
ethylenediamine (NED)3, α-naphthylamine4 and 8-hydroxy-
quinoline5. The procedure requires the removal of excess
nitrous acid by sulfamic acid, the stabilization of intermediary
diazonium salt at low temperature. Various methods have been
Spectrophotometric Determination of Sulfamethoxazole and
Sulfadiazine in Pure and Pharmaceuticals Preparation
SAADIYAH A. DHAHIR* and AMAL H. MHEMEED
Chemistry Department, College Science for Women, Baghdad University, Baghdad, Iraq
*Corresponding author: E-mail: sadiataher@yahoo.com
Asian Journal of Chemistry; Vol. 24, No. 7 (2012), 3053-3058
(Received: 5 July 2011; Accepted: 13 February 2012) AJC-11064
A rapid and sensitive spectrophotometric method is proposed for the determination of sulfamethoxazole and sulfadiazine. This method is
based on the diazotization of sulfonamide with sodium nitrite and a coupling reaction of the diazo-compound with thymol. The optimum
reaction conditions and other analytical parameters were evaluated. The linear ranges for the determination of sulfamethoxazole and
sulfadiazine are 1-10 µg mL-1 and 1-7 µg mL-1 and their detection limits are 0.008 µg mL-1 and 0.007 µg mL-1 respectively. There was no
interference from commonly utilized tablet excipients. The method has been used to determine sulfamethoxazole and sulfadiazine in
pharmaceuticals preparation without separation.
Key Words: Spectrophotometry, Sulfamethoxazole, Sulfadiazine, Diazotization, Thymol.
developed and used for the separation and quantitative esti-
mation of sulfonamides sulfadiazine and sulfamethoxazole
in food matrices, biological samples and veterinary products
and different final dosage forms. These methods include titri-
metric method6,7, high-performance liquid chromatographic
(HPLC) technique7-10. Thin layer chromatography11,12,capillary
zone electrophoresis13, liquid chromatography with post column
fluorescence derivatization14 and micellar electrokinetic capillary
chromatographic method15. In the present study, we succeeded
in developing a novel coupling agent for sensitive and selective
spectrophotometric determination of the sulfonamide class of
drugs based on the coupling of their diazotized form with
thymol, which results in the formation of coloured products
in alkaline medium.
EXPERIMENTAL
A UV/VIS spectrophotometer (T60U with 1 cm matched
quartz cells) was used. Sulfamethoxazole and sulfadiazine
chemical reference substances were used from state company
for drug Industries and Medical Appliance-(SDI) Samara-Iraq
(BDH), standard solutions of pure reference sulfamethoxazole
and sulfadiazine100 µg mL-1 were freshly prepared bydissolving
0.01 g both of them in 10 mL absolute ethanol and then diluted
with distilled water to 100 mL.
Standard solution of 100 µg mL-1 of thymol was freshly
prepared by dissolving 0.01 g of thymol with distilled water
to 100 mL. All other reagents and solvents used were of analy-
tical grade without further purification. Sodium nitrite (99.8 %

purity) and standard solution of 1 % was prepared. 1 M
sodium carbonate of (98 % purity) was prepared, 100 µg mL-1
of varies interferences and 1 M both of HCl, sulfuric acid,
acetic acid nitric acid phosphoric acid, sodium hydroxide,
potassium hydroxide, potassium carbonate and ammonium
hydroxide were used.
Aliquots of standard sulfonamide solutions (sulfametho-
xazole and sulfadiazine) were transferred into 10 mL calibrated
flasks followed by 0.5 mL hydrochloric acid to each. After
cooling in an ice bath, 0.5 mL of sodium nitrite (1.0 % m/V)
was added under swirling. The solutions were allowed to stand
for 5 min, then 0.5 mL of sulphamic acid was added for excess
of nitronuim ion the solutions were allowed to stand for 5 min,
then 0.5 mL of thymol was added, with 0.5. mL of sodium
hydroxide (1 M) for sulfamethoxazole or 0.5. mL of potassium
carbonate. (1 M) for sulfadiazine The solution was made up
to the mark with distilled water mixed thoroughly and after
5min the absorbance was measured at 417 nm for sulfame-
thoxazole and 469 nm sulfadiazine against a reagent blank
and the calibration graph was constructed.
Analysis of dosage forms
Metheprim tablets: These formulations were purchased
from local commercial sources, the state company for drugs
industrial and medical application Ninawa (N.D.I) and used
for the analysis: Each tablet contain 80 mg trimethoprim and
400 mg sulfamethoxazole. Twenty tablets were powdered and
mixed thoroughly. An amount equivalent to 100 mg of powdered
was then dissolved in 5 mL ethanol and complete to 100 mL
with distill water. and appropriate aliquots of the solution were
treated as mentioned above in the general procedure.
Oral solution: Co-trimoxazole suspension from Pharaonia
Pharmaceuticals Egypt, Alexandria, this drug contain 40 mg
trimethoprim and 200 mg sulfamethoxazole each 5 mL of
drugs contain 200 mg of sulfamethoxazole, 0.25 mL was trans-
ferred into 100 mL volumetric flasks dissolved in 5 mL ethanol
and filtered and diluted up to the mark with distilled water.
Working standard was prepared by suitable dilution and the
recommended procedure was used for sulfamethoxazole for
its determination.
Gel cream: Canting silver sulfadiazine from India
Pharmaceuticals, 1 g from drug was transferred in to separation
funnel and shaking with 5 mL ethanol for 15 min and then
added 3 mL ethanol and shaking for 15 min and added 2 mL
and shaking for 15 min, then the organic layer was separated
from the water layer and dissolved in 100 mL volumetric
flasks 50 mL ethanol and diluted up to the mark with distilled
water.
RESULTS AND DISCUSSION
A new spectrometric method was developed for the
determination of sulfadiazine and sulfamethoxazole. The
method depends upon diazotization of the sulfa drugs followed
by coupling with thymol in basic solution.
Absorption spectra: Sulfa drugs sulfamethoxazole and
sulfadiazine could be readily diazotized in acidic medium and
the diazonium cation would then coupling with a molecule of
thymol in basic medium as a coupling agent to produce a yellow
coloured azo product.
The coloured reaction products were developed as
mentioned in the general procedure and the absorption maxima
were found to be 417 nm for sulfamethoxazole and 469 nm
for sulfadiazine drug. Figs. 1 and 2 are shows the absorption
spectrum of azo dye of sulfamethoxazole and sulfadiazine.
The results coloured product was found to be stable for about
more one day. The value of absorbance decreased above 70 ºC.
Hence, room temperature was preferred for the experiments.
Fig. 1. Absorption spectra of (A) thymol versus distilled water, (B)
sulfamethaxazole versus distilled water and (C) Azo dye against
reagent blank
Fig. 2. Absorption spectra of (A) thymol versus distilled water, (B)
sulfadiazine versus distilled water and (C) Azo dye against reagent
blank
Optimization of the reaction conditions: The effect of
various parameters on the absorption intensity of the dye
formed was studied and the reaction conditions are optimized.
The factors affecting colour development, reproducibility,
sensitivity and conformity with Beer's law were investigated
with sulfamethoxazole and sulfadiazine.
Effect of acid concentration: The effect of acidity on
the diazotization reaction was studied in for sulfamethoxazole,
and sulfadiazine in the range of 0.1-1 mL from 1 M HCl, HNO3,
H2SO4, CH3COOH and H3PO4. The maximum diazotization
was obtained in 0.5 mL of HCl and the formation of the azo-
dye was reached its maximum absorbance after about 5 min;
so 0.5 mL of 1 M HCl, was used in this study for sulfame-
thoxazole, and sulfadiazine. As had been noticed previously16.
Effect of base in this study was investigated the effect
0.1-1 mL from 1 M of NaOH, KOH, Na2CO3, K2CO3, NH4OH
on the intensity of the produced product. For sulfamethoxazole
and sulfadiazine. It was found that the presence of 0.8 mL of
200 400 600 800 900
2.200
1.650
1.100
0.550
0.000
Abs
Wavelength (nm)
BC
A
BC
A
200 400 600 800 900
Wavelength (nm)
1.000
0.750
0.500
0.250
0.000
Abs
3054 Dhahir et al. Asian J. Chem.

1 M of NaOH led to increase the intensity of the produced
product for sulfamethoxazole and0.6 mL of 1 M K2CO3 for
sulfadiazine. Therefore these bases which give high sensitivity
were selected in subsequent experiments.
Effect of sodium nitrite: The optimum concentration of
sodium nitrite solution was found to be 0.3 mL of 1 % solution
of sodium nitrite for sulfamethoxazole and sulfadiazine.
Effect of temperature: The effect of different temperature
on diazotization and coupling was studied for two selected of
sulfa drugs. It was found that diazotization at 0 ºC and at room
temperature produced the same colour intensity. Therefore,
working at room temperature (25 ºC) was preferred. It was
found at higher temperatures the absorbance value decrease,
indicating the dissociation of the product on prolonged heating.
Effect of diazotization and coupling time: Since diazoti-
zation for 3 min or more gave the same results, 3 min was
selected. The azo dye was formed almost instantaneouslyafter
the addition of thymol and reached its maximum absorbance
after 5 min; then 5 min developing time was used in this study.
The colour obtained was stable for at least 24 h.
Effect of coupling agent: The effect of varying the concen-
tration of coupling reagent was studied using the proposed
procedure and adding 0.1-1 mL of 100 µg mL-1 of thymol to a
series of drug solutions of both sulfamethoxazole and sulfa-
diazine. It was found that maximum and stable colours were
formed with 0.5 mL of thymol solution for both sulfamethoxazole
and sulfadiazine in final volume of 10 mL.
Effect of organic solvents: The effect of organic solvents
such as methanol, ethanol, acetone and distilled water were
studied by using in the dilution and measuring the absorbance
the absorbance were found 0.186, 0.155, 0.143 and 0.671
respectivelydistilled water found to be the best.
Effect of order of addition: To obtain optimum results
the order of addition of reagents should be followed as given
under the procedure for two sulfa drugs, otherwise a loss in
colour intensity was observed.
Effect of interference: The effects of some foreign ions
which often accompany this drug in pharmaceutical products
were studied by adding different amounts of foreign ions to
10 µg/mL of for sulfamethoxazole and sulfadiazine. The colour
was developed following the recommended procedure
described earlier. It was observed that the Arabic gum, glucose,
starch, fructose, acetate, urea, NaCl, benzoic acid, salicylic
acid, naphthylamine, m-cresol, 2,4-dichloro aniline and
o-cresol were not interfering with the determination at levels
found in dosage form.
Calibration graph: Employing the conditions described
in the procedure, a linear calibration graph for sulfamethoxazole
and sulfadiazine is obtained, Figs. 3 and 4 shows that Beer's
law is obeyed over the concentration range of 1-10 µg/mL for
sulfamethoxazole and 1-7 µg/mL sulfadiazine with correlation
coefficient of 0.9996 and 0.9997 and an intercept of 0.0471
and 0.0208 respectively. The conditional molar absorptivity
of the yalow product formed was found to be (2.6 × 104, 2.1 ×
104) mol-1 cm-1. l for sulfadiazine and sulfamethoxazole,
respectively. The per cent relative standard deviations based
on five replicates were 0.492, 0.657 for sulfamethoxazole and
sulfadiazine other optimal characteristics and statistical data for
the sulfadiazine and sulfamethoxazole were listed in Table-1.
y = 0.0803x + 0.0208
R2 = 0.9997
0
0.2
0.4
0.6
0.8
1
0 2 4 6 8 10 12
Concentration µg/ml
Absorbance
Fig. 3. Calibration graph of sulfamethoxazole
y = 0.1037x + 0.0471
R2 = 0.9996
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
0 2 4 6
8
Concentration µg/ml
Absorbance
Fig. 4. Calibration graph of sulfadiazine
TABLE-1
OPTIMAL CHARACTERISTICS AND STATISTICAL DATA
FOR THE SULFADIAZINE AND SULFAMETHOXAZOLE
Parameter
Sulfadiazine
developed
method
Sulfamethoxazole
developed
method
Colour Yellow Yellow
λmax (nm) 469 473
Stability constant 3.4 x1011 5. 6 × 1010
Beer’s law limit a (µg mL-1) 1-7 1-10
Molar absorptivity (mol-1cm-1 L) 2.6 × 104 2.1 × 104
Sandell’s sensitivity (µg cm-2) 0.0096 0.012
Slope 0.1037 0.0803
Intercept 0.0471 0.0208
Correlation coefficient 0.9996 0.9997
Limit of quantization (mg mL–1) 0.025 0.029
LOD (µg mL-1) 0.077 00.087
aRSD (%) 0.657 0.492
Average recovery (%) 100.57 100.12
aAverage of five determination
Reaction mechanism of the dye: The stoicheiometry of
the reaction between both sulfamethoxazole and sulfadiazine
with thymol were investigated using job's method17 and mole
ratio method. The results obtained in Figs. 5 and 6 show that
1:1 drug to reagent was formed at 473 nm for sulfamethoxazole
and 469 nm sulfadiazine.
The product formed was water soluble, the stability
constant was calculated by comparing the absorbance of a
solution containing stoicheiometric amount of 6 × 10-4 M both
y = 0.0803x + 0.0208
R2 = 0.9997
Concentration (µµ
µµ
µg/mL)
0 2 4 6 8 10 12
Absorbance
1.0
0.8
0.6
0.4
0.2
0.0
y = 0.1037x + 0.0471
R2 = 0.9996
Concentration (µµ
µµ
µg/mL)
0 2 4 6 8
Absorbance
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
Vol. 24, No. 7 (2012) Spectrophotometric Determination of Sulfamethoxazole and Sulfadiazine 3055

Fig. 5. (a) Job's plot method and (b) molar ratio method of sulfamethoxazole-
thymol
Fig. 6. (a) Job's plot method and (b) molar ratio method of sulfadiazine-
thymol
sulfamethoxazole, sulfadiazine and thymol with that of solution
containing the optimum amount of thymol (6 ×10-4 M). The
average conditional stability constant of the dye in water under
the described experimental conditions was 3 × 106 L2 mol-2.
For sulfamethoxazole-thymol dye and 6.4 × 106 L2 mol-2 for
sulfadiazine-thymol dye. The proposed mechanism of reac-
tion between thymol and the sulfonamide drug is illustrated in
Scheme-II.
NH2
N
O
H3C
NHSO2
N
O
H3C
NHSO2N+N
S
H2N
O
O
N
N
HN
HCl
NaNO2
S
N
OO
N
N
HN
N
+
Diazonuim of Sulfadiazine
CH3
CH3
HO
H3C
S
N
OON
N
HN
N
H3CCH3
HO
CH3
N
O
H3C
NHSO2N
CH3
CH3
OH
H3C
Thymol
Diazonuim of Sulfamethoxazole
Sulfadiazine
Sulfamethoxazole
NaoH
K
2
CO
3
Azo dye
Scheme-II: Scheme of the proposed reaction mechanism
Precision and accuracy: sulfamethoxazole and sulfadi-
azine were determined at three different concentrations the
results shown in Table-2. A satisfactory precision and accuracy
could be obtained with the proposed method.
TABLE-2
ACCURACY AND PRECISION OF THE PROPOSED METHOD
Pure
drugs
Taken
(µg/mL)
Found
(µg/mL)
Recovery
(%)
*Average
recovery (%)
*RSD
(%)
8 8.2 102.50 0.434
9 8.9 98.88 0.487
SFMx
10 9.9 99.00
100.12
0.555
5 5.1 102.0 0.771
6 5.9 98.33 0.645
SFD
7 7.1 101.40
100.57
0.555
*Average of five determinations. SFMx = Sulfamethaxazole;
SFD = sulfadiazine;
Analysis of pharmaceutical preparations: Two types
of drug containing sulfamethaxazole (tablet and oral solution)
and sulfadiazine (cream) have been analyzed and they gave
good accuracy and precision these applications (Table-3).
The excellent sensitivity than other spectroscopic methods
in literature for the oxidative coupling reaction of sulfametha-
xazole and sulfadiazine as showed in Table-4.
Evaluate the results of the proposed method: For the
evaluating the results of the proposed method comparing with
the standard method to determine the efficiency and success
in the estimate due to unavailable of the standard method in
the British pharmacopoeia, there for standard addition method
was used for determination of both sulfamethoxazole and sulfa-
diazine in pure and pharmaceutical preparation. The results
0.0 0.2 0.4 0.6 0.8 1.0
[R] / [R] + [s]
1.0
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.0
Absorbance
(A)
[R] / [s]
(B)
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.0
Absorbance
0 1 2 3 4 5 6
0.0 0.2 0.4 0.6 0.8 1.0
[R] / [R] + [s]
1.0
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.0
Absorbance
(A)
[R] / [s]
(B)
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.0
Absorbance
0 2 4 6
N
O
H
3
C
NHSO
2
N
C
H
3
C
H
3
O
H
H
3
C
C
S
N
O
O
N
N
H
N
N
H
3
C CH
3
HO
CH
3
3056 Dhahir et al. Asian J. Chem.

shown in Figs. 7-9 shows that the results of standard addition
method agree well with the proposed method, indicating that
the method is selective and free from interference.
TABLE-3
APPLICATION OF THE PHARMACEUTICAL PREPARATIONS
OF DETERMINATION OF SULFAMETHAXAZOLE
AND SULFADIAZINE DRUGS
Sample
preparation
Taken
(µg/mL)
Found
(µg/mL)
Recovery
(%)
Average
recovery
(%)
*RSD
(%)
8 8.6 107.5 0.956
9 8.7 96.66 0.858
Tablets a
metheprim 10 9.8 98.00
100.72
1.260
8 7.9 98.75 0.645
9 9.1 101.11 0.771
Oral
solution
metheprim 10 9.7 97.00
98.95
1.260
5 4.9 98.00 1.260
6 5.9 98.33 0.956
Sulfadiazine
cream 7 7.1 101.42
99.25
0.771
*Average of five determinations
0
0.5
1
1.5
2
-9 -6 -3 0 3 6 9
12
A
b
s
o
r
b
a
n
c
e
µg
/
ml
Fig. 7. Standard addition method for determination of sulfamethoxazole
tablets
0
0.5
1
1.5
2
-9 -6 -3 0 3 6 9
12
A
b
s
o
r
b
a
n
c
e
.
µg
/
ml
Fig. 8. Standard addition method for determination of sulfamethoxazole
oral suspension
Fig. 9. Standard addition method for determination of sulfadiazine cream
Conclusion
The proposed methods were found to be simple, econo-
mical, selective and sensitive. The statistical parameters and
recovery study data clearly indicate the reproducibility and
accuracy of the methods. Analysis of the samples containing
sulfamethoxazole and sulfadiazine showed no Interference
from the common excipients. Hence, these methods could be
considered for the determination of sulfamethoxazole and
sulfadiazine in the quality control laboratories.
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TABLE-4
COMPARISON OF SULFAMETHOXAZOLE AND SULFADIAZINE DETE RMINATION
IN THE PROPOSED METHOD AND OTHER LITERATURE METHODS
Drugs Reagent λmax (nm) ε
(L mol-1 cm-1 )
Beer’s law
range (ppm)
Colour of
dye RSD (%) LOD Ref.
8-Hydroxyquinoline 500 - o.1-7.0 Red 0.1-0.5 0.03-0.05 5
Salicy laldehyde 445 - 5-40 Yellow - 0.06 18
2-Nap hthol 482 1.34 × 104 0.21-0.66 - - - 19
Orcinol 390 - 2-10 - - - 20
o-Phtha laldehyde 340 - 0.01-0.24 - 1.95-2.08 - 21
Sulfamethoxazole
Present met hod 473 2.1 × 104 1-10 Yellow 0.492 0.0087
α-Naph thalamine - - 0.2-20 - - 0.06 4
8-Hydroxyquinoline 500 - 0.1-7.0 Red 0.1-0.5 0.03-0.05 5
Glutaraldehyde - - - - 3.2-4.6 3.1 22
Acetylacetone formald ehyde 400 - 4-72 Yellow 1.07 - 23
Sulfadiazine
Present met hod 469 2.6 × 104 1-7 Yellow 0.657 0.007
-9 -6 -3 0 3 6 9 12
µµ
µµ
µg/mL
2.0
1.5
1.0
0.5
0
Absrobance
-9 -6 -3 0 3 6 9 12
µµ
µµ
µg/mL
Absrobance
2.0
1.5
1.0
0.5
0
Absrobance
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0
-7 -6 -5 -4 -3 -2 -1 0 1 2 3 4 5 6 7 8
µµ
µµ
µg/mL
Vol. 24, No. 7 (2012) Spectrophotometric Determination of Sulfamethoxazole and Sulfadiazine 3057

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