Spectrophotometric Estimation of Sulfadoxine in Pharmaceutical Preparations

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.


Introduction
Sulfadoxine 1 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, 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 ability 2 .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 indicated 3 .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 Spectrophotometry [4][5][6][7][8][9][10][11] , Liquid chromatography [12][13][14][15][16][17][18] , Electrophoresis 19 , Potentiometry 20 and spectrofluorimetry 21 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 diazotized 22 with nitrous acid (NaNO 2 / 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 λ max 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 1.0-5.0µg/mL in method A, 5.0-25.0µg/mL in method B, 5.0-25.0µg/mL in method C and 4.0-8.0µg/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 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.

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.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.*y = mc + a where c is the concentration of sulfadoxine in µg/mL and y is the absorbance at the respective λ max., ¶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).

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.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.

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.

Figure 1 .
Figure 1.Proposed reaction scheme for method A, B, C and D.

Figure 6 .
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 Table2.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.Table2.Evaluation of sulfadoxine in pharmaceutical preparation.

Table 1 .
Optical characteristics and precision.

Table 2 .
Evaluation of sulfadoxine in pharmaceutical preparation.Mean of eight determination, ¶ Mean of nine determinations (three from each level 50, 100 and 150%).† Tablets from different Manufacturers. *