ISSN: 0973-4945; CODEN ECJHAO

E-Journal of Chemistry
http://www.e-journals.net 2010, 7(4), 1246-1253

Spectrophotometric Estimation of
Sulfadoxine in Pharmaceutical Preparations

SANGITA SHARMA*, MADHURJYA NEOG,

VIPUL PRAJAPATI, HIREN PATEL and DIPTI DABHI

Department of Chemistry,
Hemchandracharya North Gujarat University, Patan-384265, Gujarat, India.
researchsharma@yahoo.com

Received 11 December 2009; Accepted 5 February 2010

Abstract: Four simple, sensitive, accurate and rapid visible

spectrophotometric methods (A, B, C and D) have been developed for the
estimation of sulfadoxine in pharmaceutical preparations. They are based on
the diazotization of sulfadoxine with sodium nitrite and hydrochloric acid
followed by coupling with N-(1-naphthyl) ethylenediamine dihydrochloride
(Method A) to form pink coloured chromogen, diphenylamine (Method B) to
form light pink coloured chromogen, chromotropic acid (in alkaline medium)
(Method C) to form orange coloured chromogen, Resorcinol (in alkaline
medium) (Method D) to form light orange coloured chromogen and exhibiting
absorption maxima (λmax) at 536 nm, 524 nm, 520 nm and 496 nm
respectively. The coloured chromogens formed are stable for more than 2 h.
Beer's law was obeyed in the concentration range of 1.0 - 5.0 µg/mL in method
A , 5.0 - 25.0 µg/mL in method B, 5.0 - 25.0 µg/mL in Method C and 4.0 - 8.0
µg/mL in Method D respectively. The results of the three analyses have been
validated statistically and by recovery studies. The results obtained in the
proposed methods are in good agreements with labeled amounts, when
marketed pharmaceutical preparations are analyzed.
Keywords: Diazotization, Visible spectrophotometric, Chromogen, Validation.

Introduction
Sulfadoxine1 is chemically 4-amino-N-(5,6-dimethoxypyrimidin-4-yl) benzenesulphonamide (Molecular
mass 310.33 g/mol). Sulfadoxine is an ultra-long-lasting sulfonamide often used in
combination with pyrimethamine to treat or prevent malaria. It is also used, usually in
combination with other drugs, to treat or prevent various infections in livestock. Both drugs
are antifolates; they inhibit the production of enzymes involved in the synthesis of folic acid
within the parasites. Either drug by itself is only moderately effective in treating malaria,
1247 S. SHARMA et al.

because the parasite Plasmodium falciparum may be able to use exogenous folic acid, i.e.
folic acid which is present in the parasite's environment, while in combination, the two
substances have a synergistic effect which outbalances that ability2. The combination is
considered to be more effective in treating malaria caused by Plasmodium falciparum than
that caused by Plasmodium vivax, for which chloroquine is considered more effective,
though in the absence of a species-specific diagnosis the sulfadoxine-pyrimethamine
combination may be indicated3. Due to side effects, however, it is no longer recommended
as a routine preventative, but only to treat serious malaria infections or to prevent them in
areas where other drugs may not work. It is official in U.S.P, B.P. and European
Pharmacopoeia. Literature survey reveals the estimation of sulfadoxine in pharmaceutical
formulations by various Spectrophotometry4-11, Liquid chromatography12-18, Electrophoresis19,
Potentiometry20 and spectrofluorimetry21 methods. The present work deals with the
development of four simple, low cost and sensitive spectrophotometric methods for the
quantitative estimation of sulfadoxine in bulk and pharmaceutical Preparations.
The aromatic amino group present in sulfadoxine is diazotized22 with nitrous acid
(NaNO2 / HCl) at room temperature and diazonium salt thus formed is coupled with the N-
(1-napthyl) ethylenediamine dihydrochloride (Bratton Marshall reagent) in method A,
diphenylamine in method B, chromotropic acid (in alkaline medium) method C and
resorcinol in method D to form colured chromogens and exhibiting absorptions maxima λmax
at 536 nm, 524 nm, 520 nm and 496 nm respectively. The coloured chromogens formed in
method A, B, C and D are stable for more than 2 h. Beer’s law limits are 1.0-5.0 µg/mL in
method A, 5.0-25.0 µg/mL in method B, 5.0-25.0 µg/mL in method C and 4.0-8.0 µg/mL in
method D respectively. Spectrophotometric parameters are established for standardization of
the method including statistical analysis of data. These methods have been successfully
extended to the pharmaceuticals preparations containing sulfadoxine.
Experimental
A Shimadzu UV / Vis double beam spectrophotometer (model 1700 PC) with 1 cm matched
quartz cells used for all spectral measurements. All chemicals used are of analytical grade.
Sodium nitrite, hydrochloric acid, sodium hydroxide, resorcinol and diphenylamine were
obtained from E. Merck. Sulphamic acid and N-(1-napthyl) ethylenediamine dihydrochloride
were obtained from Qualigens and Acros organics respectively. Conductivity water (pH: 6.32,
Conductivity: 0.92 µ S cm-1) was used for dilution and preparation of all reagents. Sulfadoxin
bulk drug was obtained form Molecule Analytical Laboratory, Ahmedabad, India. Malasulf
(Bombay Tablet Mfg. Co. Pvt. Ltd, Gandhinagar, India) and Reziz (Shreya life Sciences Pvt.
Ltd., Roorkee India) tablets were purchased from the market.
Working standard solution
About 100 mg of sulfadoxine weighed accurately and dissolved in 30 mL of 2 mol
Hydrochloric acid in a 100 mL volumetric flask and diluted up to the mark with water (1000
µg/mL). The final concentration of sulfadoxine was brought to 100 µg/mL with water.
Sample preparation
Two brands of commercial tablets were analyzed by the proposed methods. 20 tablets each
containing 500 mg and 750 mg sulfadoxine were taken and average weight was calculated.
Tablets were crushed thoroughly in a mortar. Tablets powder equivalent to 100 mg of the
drug weighed accurately and dissolved in 30 mL of 2 mol hydrochloric acid in a 100 mL
volumetric flask and allow to sonicate with intermittent shaking for 10 min, cooled and
 Spectrophotometric Estimation of Sulfadoxine 1248

diluted up to the mark with water (1000 µg/mL). The solutions were filtered through
Whatman filter paper No. 41 and the final concentration of sulfadoxine was brought to
100 µg/mL with water.
Assay
Method A
Aliquots of sulfadoxine ranging from 0.1 - 0.5 mL (100 µg/mL) were transferred into a
series of 10 mL volumetric flasks. To each flask 1 mL ice cold sodium nitrite (0.1% w/v),
and 1 mL of 2 mol hydrochloric acid were added at room temperature. After 5 min 1 mL of
sulphamic acid (0.2% w/v) and 1 mL of Bratton Marshall reagent ware added. The volumes
were made up to the mark with distilled water. The absorbance of the pink coloured
chromogen was measured at 536 nm against reagent blank. The amount of sulfadoxine
present in the sample was computed from calibration curve.
Method B
Aliquots of sulfadoxine ranging from 0.5 - 2.5 mL (100 µg/mL) were transferred into a
series of 10 mL volumetric flasks. To each flask 1 mL ice cold sodium nitrite (0.1% w/v),
and 1 mL of 2 mol hydrochloric acid were added at room temperature. After 5 min 1 mL of
sulphamic acid (0.2% w/v) and 0.25 mL of alcoholic diphenylamine (0.3% w/v) were added.
The volumes were made up to the mark with distilled water. The absorbance of the light
pink coloured chromogen was measured at 524 nm against reagent blank. The amount of
sulfadoxine present in the sample was computed from calibration curve.
Method C
Aliquots of sulfadoxine ranging from 0.5 - 2.5 mL (100 µg/mL) were transferred into a
series of 10 mL volumetric flasks. To each flask 1 mL ice cold sodium nitrite (0.1% w/v),
and 1 mL of 2 mol hydrochloric acid were added at room temperature. After 5 min 1 mL of
sulphamic acid (0.2% w/v), 1 mL of aqueous solution of chromotropic (0.2% w/v) acid were
added and 1 mL sodium hydroxide (20% w/v) were added. The volumes were made up to
the mark with distilled water. The absorbance of the orange coloured chromogen was
measured at 520 nm against reagent blank. The amount of sulfadoxine present in the sample
was computed from calibration curve.
Method D
Aliquots of sulfadoxin ranging from 0.4 - 0.8 mL (100 µg/mL) were transferred into a series of
10 mL volumetric flasks. To each flask 0.5 mL ice cold sodium nitrite (0.1% w/v) and 1 mL of 2
mol hydrochloric acid were added at room temperature. After 5 min 1 mL of sulphamic acid
(0.2% w/v), 0.5 mL of aqueous resorcinol (0.5% w/v) and 1 mL sodium hydroxide (20% w/v)
were added. The volumes were made up to the mark with distilled water. The absorbance of
the light orange coloured chromogen was measured at 496 nm against reagent blank. The
amount of sulfadoxin present in the sample was computed from calibration curve.
Results and Discussion
The presence of the aromatic amino group in sulfadoxine, enable the use of diazotization of
the drug with nitrous acid and coupling the resulting diazonium salt with N-(1-naphthy)
ethylenediamine dihydrochloride (Method A), diphenylamine (Method B), chromotropic
acid (in alkaline medium) (Method C) and resorcinol (Method D) to form coloured
chromogens. The proposed chemical reaction are shown in Figure 1.
 1249
S. SHARMA et al.
Figure 1. Proposed reaction scheme for method A, B, C and D.
 Spectrophotometric Estimation of Sulfadoxine 1250

The optical characteristics such as absorption maxima, Beer’s law limit, molar absorptivity
and Sandell’s sensitivity are presented in Table 1. The regression analysis using method of least
squares was made for the slope (m), intercept (a) and correlation (r) obtained from different
concentrations and the results are summarized in Table 1. The percent relative standard deviation
and percent range of error (0.05 and 0.01 level of confidence limits) calculated from the eight
measurements. The result shows that these methods have reasonable precision.
Table 1. Optical characteristics and precision.
Parameters Method A Method B Method C Method D
λ max (nm) 536 524 520 496
Beer’s Law Limits, µg/mL. 1-5 5-25 5-25 4-8
Molar Absorptivity, liter
moles-1 cm-1. 5.45288 × 10 8.81344 × 10 9.66486 × 10 3.48656 × 104
4 3 3

Sandell’s Sensitivity,
0.006 0.035 0.032 0.009
µg/cm2 / 0.001 A.U.
Regression equation (y*)
Slope (m) 0.1609 0.0252 0.0300 0.1102
Intercept (a) 0.0583 0.0634 0.0185 0.0151
Correlation Coefficient (r) 0.9998 0.9974 0.9984 0.9998
% RSD 0.7542 0.4578 0.3824 0.2157
Standard error of mean ± 0.262 ± 0.160 ± 0.135 ±0.076
Range of errors¶
95 % confidence level ± 0.6195959 ± 0.377945 ± 0.319665 ± 0.180277
99 % confidence level ± 0.9169495 ± 0.559326 ± 0.473077 ± 0.266794
*y = mc + a where c is the concentration of sulfadoxine in µg/mL and y is the absorbance at the
respective λmax., ¶For eight measurements.
The developed methods were optimized using different parameters such as sodium nitrite
concentration, hydrochloric acid concentration and concentration of Bratton Marshall reagent
for method A, alcoholic diphenylamine for method B, aqueous chromotropic acid for method
C and aqueous resorcinol for method D for development of maximum colour intensity (Figures
2-5). These experimental variables were studied with 10 µg/mL of sulfadoxine. Optimization
is done by varying one parameter, keeping other constant.
Effect of reagent concentration
The results obtain showed that at least 1.0 mL of N-(1-napthyl) ethylenediamine
dihydrochloride (NEDD) and chromotropic acid (CHROM) are required for maximum
colour development in method A, C. (Figure 2). In method B and D at least 0.25 mL of
diphenylamine (DPA) and 0.5 mL resorcinol (RES) are required respectively (Figure 3). The
amount of sodium nitrite and hydrochloric acid required for optimum color development is
1.0 mL for all the methods (Figure 4 and 5).
Method A
0.8000 (NEDD)
0.7000 Method C
Absorbance

0.6000 (CHROM)
0.5000
0.4000
0.3000
0.2000
0.1000
0.0000
0.00 0.10 0.20 0.40 0.60 0.80 1.00 1.50 2.00

Volume Volume of reagent

of reagent in mL in ml
Figure 2. Effect of N-(1-napthyl) ethylenediamine dihydrochloride (NEDD) and
chromotropic acid (CHROM).
1251 S. SHARMA et al.

Method B
(DPA)
0.9000
Method D
0.8000 (RES)
Absorbance
r ba nc e 0.7000
0.6000
0.5000
0.4000
A bso

0.3000
0.2000
0.1000
0.0000
0.05
0.06
0.07
0.08
0.09
0.10
0.15
0.20
0.25
0.30
0.35
0.40
0.45
0.50
0.60
0.70
0.80
0.90
1.00
Volume of reagent in mL
Figure 3. Effect of diphenylamine (DPA) and resorcinol (RES).
Method A
0.9000
0.8000 Method B
0.7000
Absorbance

0.6000 Method C
0.5000
0.4000 Method D
0.3000
0.2000
0.1000
0.0000
0.00 0.10 0.20 0.40 0.60 0.80 1.00 1.50 2.00
Volume of reagent in mL
Figure 4. Effect of sodium nitrite.
Method A
1.0000

0.8000 Method B
Absorbance

0.6000 Method C
Absorbance

0.4000
Method D
0.2000

0.0000
0.00 0.10 0.20 0.40 0.60 0.80 1.00 1.50 2.00
Volume of reagent in mL
Figure 5. Effect of hydrochloric acid.
Effect of excess nitrous acid
Interference of excess nitrous acid and its effect on the colors of the chromogen are shown in
Figure 6. This interference is minimized by adding sulphamic acid before the coupling reaction.
 Spectrophotometric Estimation of Sulfadoxine 1252

B=Blank preparation, W= Sample preparation without Sulphamic acid, S= Sample preparation with
Sulphamic acid.
Figure 6. Final colour of sample and interference of excess sodium nitrite.
Stability study of the chromogen was carried out by measuring the absorbance values at
time intervals of 10 min and was found to be stable for more than 2.0 h in all the methods.
Moreover, to check the validity of the proposed optimized method, we applied the standard
addition method by adding sulfadoxine to the previously analyzed tablets. The recovery of
each drug was calculated by comparing the concentration obtained from the spiked mixtures
with those of pure drugs. The results are summarized in Table 2. The proposed method was
successfully applied for the determination of sulfadoxine in pharmaceutical dosage forms.
Interference studies reveal that the additives like common excipients and colours that are
usually present in tablets did not interfere at their regularly added levels.
Table 2. Evaluation of sulfadoxine in pharmaceutical preparation.
Labeled Amount, Amount Obtained, % Recovery ±
Method mg/tab Sample† % ± S.D* S.D¶
A 500 1 98.25 ± 0.74 98.65 ± 0.46
750 2 97.96 ± 0.81 98.60 ± 0.23
B 500 1 98.72 ± 0.45 99.95 ± 0.47
750 2 98.65 ± 0.48 99.89 ± 0.50
C 500 1 99.98 ± 0.38 99.41 ± 0.41
750 2 99.82 ± 0.40 99.21 ± 0.61
D 500 1 99.94 ± 0.22 99.61 ± 0.14
750 2 100.11 ± 0.51 99.38 ± 0.15
*Mean of eight determination, ¶ Mean of nine determinations (three from each level 50, 100 and
150%). † Tablets from different Manufacturers.
Conclusion
The developed visible spectrophotometric methods are simple, sensitive, accurate, precise,
reproducible and economical and can be successfully applied for the routine estimation of
Sulfadoxine in bulk and pharmaceutical dosage forms. The value of standard deviation was
satisfactorily low and recovery was close to 100% which indicates the reproducibility and
accuracy of the four methods Table 2.
References
1. British Pharmacopoeia, Her Majesty's Stationary Office British Pharmacopoeia
Commission: London, 2008, 2, 2054-2055.
2. Jeffrey Chulay D, William Watkins M and David Sixsmith G, Am J Trop Med Hyg.,
1984, 33, 325-330.
1253 S. SHARMA et al.

3. Toby Leslie, Ismail Mayan M, Anwar Hasan M, Hanif Safi M, Eveline Klinkenberg,
Christopher J M W and Mark Rowland, J Am Med Assoc., 2007, 297, 2201-2209.
4. Onah J O and Odeiani J E , J Pharm Biomed Anal., 2002, 30, 851-857.
5. Green M D, Mount D L and Todd G D, Analyst, 1995, 120, 2623-2626.
6. Parimoo P, Indian J Pharm Sci., 1987, 49, 28-29.
7. Ansari M T, Ansari T M, Raza A, Ashraf M and Yar M, Chem Analityczna, 2008, 53,
305-313.
8. Raghuveer S, Raju IRK, Vatsa DK and Srivastava C M R, Indian J Pharm Sci.,
1993, 55(2), 69-71.
9. Nagaraja P, Yathirajan HS, Sunitha K R and Vasantha R A, J AOAC Int., 2002, 85(4),
869-874.
10. Rao G R, Murty S S N and Raju I R K, Indian Drugs, 1989, 26, 417-420.
11. Rao G R, Murty S S N and Raju I R K, Indian Drugs, 1989, 26, 237-240
12. Bhoir S I, Bhoir I C, Bhagwat A M and Sundaresan M, J Chromatogr B Biomed Sci
Appl., 2001, 757, 39-47.
13. Lindkvist J, Malm M and Bergqvist Y, Trans R Soc Trop Med Hyg., 2009, 103, 371-376.
14. Guo-Zhen F, Jin-Xing H and Shuo W, J Chromatogr A., 2006, 1127, 12-17.
15. Green M D, Mount D L and Nettey H, J Chromatogr B Biomed Sci Appl., 2002,
767, 159-162.
16. Tao Y, Zhang H, Jin Q, Guo Y and Zhang L, Chin Pharm J., 2006, 8, 41.
17. Bergqvist Y, Hjelm E and Rombo L, Ther Drug Monit., 1987, 09, 203-207.
18. Malm M, Lindkvist J and Bergqvist Y, J Chromatogr Sci., 2008, 46, 837-843.
19. Soto-Chinchilla J J, García-Campaña A M and Gámiz-Gracia L, Electrophoresis,
2007, 28, 4164-4172.
20. Kharitonov S V and Gorelov I P, Pharm Chem J., 2000, 34, 673-676.
21. Cruces Blanco C, Segura Carretero A, Fernández Peinado S, Román Ceba M and
Fernández Gutiérrez A, Fresenius J Anal Chem., 1999, 365, 444-447.
22. Schank K, The Chemistry of Diazonium and Diazo Groups; Patai S, Ed.; Wiley: New
York 1978, Part 2, 645-657.

International Journal of
Medicinal Chemistry
Hindawi Publishing Corporation
http://www.hindawi.com
International Journal of
Carbohydrate
Chemistry
Hindawi Publishing Corporation
http://www.hindawi.com
The Scientific
World Journal
Hindawi Publishing Corporation
http://www.hindawi.com
Journal of
Spectroscopy
Hindawi Publishing Corporation
http://www.hindawi.com
Journal of
Theoretical Chemistry
Hindawi Publishing Corporation
http://www.hindawi.com
Organic Chemistry
International
Hindawi Publishing Corporation
Volume 2014 http://www.hindawi.com
Volume 2014
Volume 2014
International Journal of
Inorganic Chemistry
Hindawi Publishing Corporation
Volume 2014 http://www.hindawi.com
Journal of
Catalysts
Hindawi Publishing Corporation
Volume 2014 http://www.hindawi.com
Photoenergy
International Journal of
International Journal of Advances in
Hindawi Publishing Corporation
Analytical Chemistry
Hindawi Publishing Corporation
Physical Chemistry
Hindawi Publishing Corporation
Volume 2014 http://www.hindawi.com Volume 2014 http://www.hindawi.com Volume 2014 http://www.hindawi.com Volume 2014
Journal of
Quantum Chemistry
Hindawi Publishing Corporation
http://www.hindawi.com Volume 2014
Submit your manuscripts at
http://www.hindawi.com
Journal of
Analytical Methods
in Chemistry
Hindawi Publishing Corporation
http://www.hindawi.com Volume 2014
International Journal of Journal of Bioinorganic Chemistry
Electrochemistry
Hindawi Publishing Corporation
Applied Chemistry
Hindawi Publishing Corporation
and Applications
Hindawi Publishing Corporation
Volume 2014 http://www.hindawi.com Volume 2014 http://www.hindawi.com Volume 2014 http://www.hindawi.com Volume 2014
 Chromatography   Journal of International Journal of
Research International
Hindawi Publishing Corporation
Chemistry
Hindawi Publishing Corporation
Spectroscopy
Hindawi Publishing Corporation
Volume 2014 http://www.hindawi.com Volume 2014 http://www.hindawi.com Volume 2014 http://www.hindawi.com Volume 2014