spectrophotometric estimation of sulfadoxine in...

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.

[email protected]

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




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




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.

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

12

49

S. S

HA

RM

A et a

l.

Volume of reagent in mL

Ab

sorb

ance


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 × 104 8.81344 × 10

3 9.66486 × 10

3 3.48656 × 10

4

Sandell’s Sensitivity, µg/cm

2 / 0.001 A.U.

0.006 0.035 0.032 0.009

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).

Figure 2. Effect of N-(1-napthyl) ethylenediamine dihydrochloride (NEDD) and

chromotropic acid (CHROM).

0.0000

0.1000

0.2000

0.3000

0.4000

0.5000

0.6000

0.7000

0.8000

0.00 0.10 0.20 0.40 0.60 0.80 1.00 1.50 2.00

Volume of reagent in ml

Method A

(NEDD)

Method C

(CHROM)

Ab

sorb

ance



Ab

sorb

ance


Ab

sorb

ance


Figure 3. Effect of diphenylamine (DPA) and resorcinol (RES).

Figure 4. Effect of sodium nitrite.

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.

0.0000

0.1000

0.2000

0.3000

0.4000

0.5000

0.6000

0.7000

0.8000

0.9000

0.0

5

0.0

6

0.0

7

0.0

8

0.0

9

0.1

0

0.1

5

0.2

0

0.2

5

0.3

0

0.3

5

0.4

0

0.4

5

0.5

0

0.6

0

0.7

0

0.8

0

0.9

0

1.0

0

Ab

sor

ba

nc

e

Method B

(DPA)

Method D

(RES)

0.0000

0.1000

0.2000

0.3000

0.4000

0.5000

0.6000

0.7000

0.8000

0.9000

0.00 0.10 0.20 0.40 0.60 0.80 1.00 1.50 2.00

Method A

Method B

Method C

Method D

0.0000

0.2000

0.4000

0.6000

0.8000

1.0000

0.00 0.10 0.20 0.40 0.60 0.80 1.00 1.50 2.00

Ab

so

rba

nc

e

Method A

Method B

Method C

Method D


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.

Method Labeled Amount,

mg/tab Sample

†

Amount Obtained, % ± S.D

*

% Recovery ± 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.

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