CHAPTER 6

NEW SPECTROPHOTOMETRIC METHODS FOR THE

DETERMINATION OF SULFADOXINE BY FORMATION
OF Co(II) COMPLEXES

! ABSTRACT
II
%
| The enhanced clinical use of sulfadoxine necessitated the study of new methods
I
I for its determination in quality control laboratories. New sensitive visible
I
| spectrophotometric methods are developed for the determination of sulfadoxine.
I
| Proposed methods are based on the reaction of drug with aldehyde followed by cobalt(II)
i
| chloride in acidic medium. The reaction of aldehyde with drug results in the formation of
I$ v
I Schiff base which act as a ligand for the formation of complex with Co(II). Developed |
I I
| methods are successfully applied to the pharmaceutical formulation. |
I
I
I

Sulfadoxine

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6.1. DRUG PROFILE
Sulfadoxine is chemically 4-amino-iV-(5,6-dimethoxypyrimidin-4-yl)benzene-l-
sulfonamide belonging to the class of drug known as sulfanilamides. It is mainly used for
the treatment or prevention of malaria and also used as anti-infective agent [1].
Sulfadoxine has microbial activity similar to that of sulfadiazide. However, it is
principally used for the suppression of malaria caused by chloroquine-resistant
plasmodium falciparum [2]. Sulfadoxine helps to inhibit the enzyme dihydropteroate
synthetase which is an enzyme necessary in the conversion of /?-aminobenzoic acid to
folic acid [3], Folic acid is essential for synthesis and methylation of DNA which is vital
to cell growth in plasmodium falciparum [4]. In such a case because of the nutrient
lacking, parasite find difficulty in reproducing. Sulfadoxine is an ultra-long-lasting
sulfonamide often used in combination with other drugs, to treat or prevent various
infections in livestock, respiratory, urinary tract, and malarial infections [5].

6.2. LITERATURE SURVEY- ANALYTICAL FRAME WORK

Literature survey revealed the estimation of sulfadoxine in pharmaceutical

formulations by various techniques such as electrophoresis [6], potentiometry [7], reverse
phase high performance liquid chromatography (RP-HPLC) [8], liquid chromatography
(LC) [9-10] and spectrophotometry [11-22]. Brief reviews of reported spectrophotometric
methods are described below.
Sharma and Sharma [11] developed a simple extractive spectrophotometric
method for the determination of sulfadoxine in pharmaceutical formulations. These
methods were based on the formation of ion association complexes of the sulfadoxine
with basic methylene blue. The methods were successfully applied to the assay of
sulfadoxine in its tablet formulation and the results were compared with those of a
reference method.
Ansari et al., [12] proposed a new rapid and sensitive spectrophotometric method
for the analysis of amodiaquine and sulfadoxine in bulk and in dosage forms. The
proposed method was based upon the reaction of amino group with sodium nitrite
followed by reaction with /7-naphthol to give a colored product. These species exhibited

144
the maximum absorption at 505 nm for amodiaquine and at 470 nm for sulfadoxine. The
proposed method was applicable for the determination of these drugs in pharmaceutical
formulations.
Syed et al, [13] contributed new spectrophotometric method for the
determination of sulfa drugs namely, sulfanilamide, sulfadoxine and sulfamethoxazole.
The methods were based on the interaction of diazotized sulfa drugs with cardanol to
produce a yellow colored product with a maximum absorption at 415 nm. The color
developed was stable up to 24 hrs,
Balyejusa et al, [14] reported first derivative spectrophotometric method for the
simultaneous separation of sulfadoxine and pyrimethamine. Method was achieved by
applying zero-crossing. Linear calibration graphs of 1 st-derivative values at 273 and 240
nm for sulfadoxine and pyrimethamine respectively with negligible intercepts were
obtained. The authors recommend that it becomes a favorable alternative to the expensive
HPLC method.
Sastry et al., [15] proposed two new spectrophotometric methods for the
determination of sulfaethidole and sulfadoxine in pure samples and in dosage forms. The
1st method was based on the reaction of the primary amino group of the drugs with the
quinonimine produced in situ from metol-Cr(VI) to give a colored charge-transfer
complex with an absorption maximum at 520 nm; the 2 nd method involves the formation
of a colored azo dye (X,max 415 nm) through the coupling of phloroglucinol with each of
the diazotized drugs.
Raghuveer et al, [16] reported a sensitive spectrophotometric method for the
determination of sulfadoxine was based on the reaction with
4- dimethylaminocinnamaldehyde in orthophosphoric acid medium to form a stable and
sensitive chromogen.
Mohamed et al, [17] developed a simple spectrophotometric method for the
determination of 15 sulfonamides in bulk and in dosage forms. The method was based on
the interaction of p-benzoquinone with sulfonamides in 0.1 M HC1. The resulting
chromophore was measured at 500 nm. The effects of different variables on color
development were established.

145
 Rao et al, [18] described spectrophotometric method for the determination of
sulfadoxine and sulfalene in pharmaceutical dosage forms by using sodium
l,2-naphthoquinone-4-sulfonate. Developed colored chromogens were measured at
425-430 nm.
Rao et al, [19] reported spectrophotometric method for the determination of
pindolol and sulfadoxine in pharmaceuticals. Method was based on the treatment of the
drugs with Na2CC>3 soln. followed by reaction with Folin-Ciocalteu reagent and
measurement of absorbance at 765 nm.
Parimoo [20] reported differential spectrophotometric method for the
simultaneously analysis of pyrimethamine and sulfadoxine in syrups by a based on the
measurement of the absorbance in various media at 220-320 nm.
Rao et al, [21] proposed simple spectrophotometric method for the determination
of sulfadoxine and sulfalene based on reaction with metol at pH 2.5 using NaI04 as
oxidant. The chromogens formed had absorption maximum at 520 nm. It was found that
pyrimethamine and trimethoprim present in combination with these drugs did not
interfere.
Rao et al, [22] reported new spectrophotometric method for the determination of
sulfadoxine and sulfalene in combined dosage forms by reaction with o-chloranil at pH
9.0 and measurement of the absorbance of the chromogens at 525 nm.

6.3. AIM AND SCOPE OF THE PRESENT WORK

Quite a few visible spectrophotometric methods have been developed for the
quantification of sulfadoxine in pharmaceuticals. Therefore, developing a selective and
sensitive methods using visible spectrophotometry is of paramount importance. Thus, aim
of the present work is to develop new and simple spectrophotometric methods for the
determination of sulfadoxine.

146
6.4. EXPERIMENTAL

6.4.1. Apparatus

A UV-visible spectrophotometer (SHIMADZU, Model No: UV 2550) with 1 cm

quartz cells was used for the absorbance measurements.

6.4.2. Reagents and Solutions

All solutions were prepared with double distilled water. Chemicals used were of
analytical reagent grade. Solutions of p-dimethylaminobenzaldehyde (PDAB) and
vanillin (VA) (5 * 10'4 M) were prepared in ethanol and cobalt(II) chloride
(1.5403 x 10"4 M) was prepared with water. About 0.1 g of pure sulfadoxine (SFD)
weighed accurately and dissolved in 5 mL of (2 M) hydrochloric acid and diluted to 100
mL with ethanol (1000 pg mL"1).

6.4.3. Assay Procedures

6.4.3.1. Determination of SFD by using PDAB (Method A)

Aliquots containing 40.00-100 pg mL'1 of SFD were transferred into a series of

10 mL volumetric flasks. To each flask, 1 mL of PDAB was added and shaken well at
room temperature. The formation of Schiff base was confirmed by measuring its Xmax at
451 nm (Xmaxof drug was obtained at 272 nm). After 5 min, 0.5 mL of Co(II) solution
was added and the solutions were diluted to 10 mL by using ethanol. The absorbance of
the green colored complex was measured at 672 nm against reagent blank (Figure 6.1).
The amount of SFD present in the sample was computed from calibration curve.

6.43.2. Determination ofSFD by using VA (Method B)

Aliquots containing 20.00-100 pg mL'1 of SFD were transferred into a series of

10 mL volumetric flasks. To each flask, 1 mL of VA was added and shaken well at room
temperature. The formation of Schiff base was confirmed by measuring its X max at 398
nm (X max of drug was obtained at 272 nm). After 5 min, 0.5 mL of Co(II) solution was
added and solutions were diluted to 10 mL by using ethanol. The absorbance of the green
colored complex was measured at 665 nm against reagent blank (Figure 6.1). The
amount of SFD present in the sample was calculated from calibration curve.

147
6.4.33. Assay offormulation

The proposed method was applied for the determination of SFD in tablet
formulation. Tablet weight equivalent to 750 mg of SFD (Reziz forte) were crushed
thoroughly in a mortar and dissolved in 20 mL of 2 M hydrochloric acid and diluted to
100 mL by using ethanol. The solution was filtered through Whatmann No.l filter paper
and diluted quantitatively with ethanol to obtain a suitable concentration for the analysis.

6.5. RESULTS AND DISCUSSION

6.5.1. Chemistry of Colored Species

Compounds containing an azomethine group (-CH=N-), known as Schiff bases

are formed by the condensation of a primary amine with a carbonyl compound [23-24].
In the present work, the drug SFD which contains primary amino group first reacted with
aryl aldehydes to form the corresponding Schiff bases (Scheme 6.1). But, the formed
Schiff bases exhibited their absorbance maxima at lower wavelengths (Table 6.1).
However, it is well known that Schiff bases are good ligands for the preparation of
complexes [25]. So, the Schiff bases of SFD are further treated with cobalt(II) chloride to
form green colored cobalt(II) complexes.

6.5.2. Selection of arayl aldehydes

Different aryl aldehydes are tested, out of which PDAB and VA gave stable green
colored complexes (Table 6.1). The color stability of these complexes may be due to the
presence of electron donating groups in PDAB (-NMe2) and VA (-OH and -OMe) which
helps in the formation of Co(II) complexes. Other aryl aldehydes do not form any colored
complexes.

6.5.3. Optimization of Reaction Conditions

The reaction is carried out at room temperature. It has taken around 5 min for the
complete color development. After the color development absorbance of the complex is
found to be constant up to 6 hrs. The series of solutions containing 60 pg mL'1 of drug
solution is taken and various amount of reagent ranging from 0.5-2.00 mL is added. It is
found that 1 mL of reagent is sufficient to form Schiff base ligand with drug and 0.5 mL

148
of metal solution is found to be optimum to form stable complex with the ligand in both
the cases.

6.5.4. Analytical Data and Method Validation

6.5.4.1. Linearity and sensitivity

Under optimum experimental conditions, linear relations are found between

absorbance and concentration of SFD in the range of 40.00-100.00 pg mL'1 (method A,
Figure 6.2) and 20.00-100.00 pg mL'1 (method B, Figure 6.3). The calibration graph in
each example is described by the equation: Y = a + b X (Where Y = absorbance, a =
intercept, b = slope and X = concentration in pg mL'1) is obtained by the method of least
squares. Correlation coefficient, limit of detection (LOD), limit of quantification (LOQ),
intercepts and slope for the calibration curve are summarized in Table 6.2. The small
values of Sandell’s sensitivity parameters indicate high sensitivity of the proposed
methods.

6.5.4.2. Accuracy and precision

In order to study the accuracy and precision of the proposed methods, three
concentrations of pure SFD within the linearity range are analyzed, each determination
being repeated five times and the percentage relative standard deviation (% RSD) is
found to be less than 2 %. Accuracy of the proposed methods is measured by calculating
the percentage relative error (% RE) and is found to be less than 3 %. The results of this
study indicate the high accuracy and precision of the methods. Detailed results are given
in Tables 6.3A & 6.3B.

6.5.4.3. Selectivity

The effects of wide range of excipients which usually present in the formulations
are studied. It is found that proposed method can be successfully applied for the
determination of SFD in pharmaceutical formulation without any analytical problem due
to the tablet excipients such as glucose, starch and lactose.
The selectivity of the proposed methods is tested by preparing the placebo blank
(solution containing all the tablet excepients except SFD). A solution of analytical
placebo is prepared according to the sample preparation procedure and subjected to the

149
analysis using the procedure described earlier. The absorbance measured is nearly same
as that of the reagent blank which indicates that the change in absorbance with respect to
the reagent blank caused only by the analyte. Non interference by common additives
including SFD is analyzed by preparing the test solution containing following
components: SFD (25 mg), glucose (45 mg), starch (50 mg) and lactose (30 mg). The
entire mixture is transferred to 100 mL calibrated flask, the contents shaken for 20 min;
volume diluted to the mark with distilled water, mixed and filtered. The filtrate after
suitable dilution is analyzed by proposed methods. The result confirms the selectivity of
the proposed methods in the presence of excipients.

6.5.4.4. Robustness

Robustness is examined by evaluating the influence of small variation in some

experimental parameters like concentration and volume of analytical reagents. It is found
that none of these variables had a significant effect on the determination of investigated
drug. This provides an indication of the reliability of the proposed method during normal
usage, so the developed spectrophotometric method is considered robust.

6.6. APPLICATIONS

The proposed methods are successfully applied for the determination of SFD in
dosage form. The results (Table 6.4) shows that the Student’s t tests at 95 % confidence
level are less than the theoretical values, which confirmed the good accuracy of the
methods. It is also clear that the result obtained from the proposed methods is in good
agreement with those obtained from the reported methods.

150
6.7. CONCLUSIONS

❖ The proposed spectrophotometric methods are rapid and easily applicable for the
determination of SFD in tablets.
❖ The proposed methods are free from critical experimental conditions and
complicated procedures such as heating or extraction steps.
❖ The reagents used in the proposed methods are cheap, readily available and the
procedures do not involve any tedious sample preparation.
❖ These advantages encourage the application of the proposed methods in routine
quality control analysis of SFD in pharmaceutical formulation.

151
Table 6.1: Comparison of A^of Schiff bases and Co(II) complexes synthesized from
different aryl aldehydes

^nm for ^-max for

Schiff base Co(ll) complex
Aldehydes R
(nm) (nm)

PDAB 4-N(Me2) 451 672

VA 3-OMe-4-OH 398 665

Syringaldehyde 3,5-(OMe)2-4-OH 325 553*

4-Chlorobenzaldehyde 4-C1 318 552*

4-Flourobenzaldehyde 4-F 298 549*

4-Nitrobenzaldehyde 4- N02 315 554*

4-Bromobenzaldehyde 4-Br 311 552*

■"Wavelength comparable to the X,„,xof Co(II).

Table 6.2: Spectral and statistical data for the determination of SFD

Parameters Method A Method B

X max (nm) 672 665

Beer’s Law Limits (pg ml"1) 40.00 - 100.00 20.00- 100.00

Molar Absorptivity (L mol"1 cm'1) 0.1264 x 104 0.6982 x 104

Sandell’s Sensitivity (pg cm'2) 0.2453 xlO"2 0.4444x1O'2

Limit of Detection* (pg mL'1) 0.2686 0.3465

Limit of Quantification * (pg mL"1) 0.8139 0.3465

Regression Equation** Y=a+bX Y=a+bX

Slope (b) 0.0043 0.0020

Intercept (a) -0.0139 0.0038

Correlation Coefficient (r) 0.9972 0.9990

* Calculated according to ICH guidelines;
** Y is the absorbance and X is the concentration in pg mL'1

152
Table 6.3A: Evaluation of accuracy and precision (Method A)

Amount taken Amount found* RE SD RSD

(pg mL'1) ( pg mL'1) (%) (pg mL1) (%)

40.00 39.71 0.72 0.31 0.78

50.00 49.79 2.10 0.73 1.46

60.00 59.94 0.10 0.54 0.90

* Mean value of five c eterminations

RE - Relative Error; SD - Standard Deviation; RSD - Relative Standard Deviation

Table 6.3B: Evaluation of accuracy and precision (Method B)

Amount taken Amount found* RE SD RSD

(pg mL"1) (l*g mL'1) (%) (pg mL"1) (%)

20.00 20.07 -0.35 0.56 2.82

40.00 39.21 1.97 0.18 0.45

60.00 59.99 0.01 0.51 0.85

* Mean value of five c eterminations

RE - Relative Error; SD - Standard Deviation; RSD - Relative Standard Deviation

Table 6.4: Result of assay of formulation by the proposed method

Brand name Labeled amount Found* ± SD using Found* ± SD using

(mg) Method A Method B

Reziz forte 750 758 ± 0.63 752 ± 0.73

t = 0.30 t = 0.14
♦Mean value of five c eterminations

Tabulated t value at 95 % confidence level is 2.77

153
 o
u>
l/l
CO
d
CM
m
Absorbance
o o
CM
r-i
in
o o

350 400 450 500 550 600 650 700 750

Wavelength (nm)

Figure 6.1: Absorption maximum for (i) Co(II), (ii) method A and (iii) method B
CM
in
o
fN
Absorbance
Absorbance

Hm
O
oin

0 --------- 1---------- 1---------- 1

oo
in
o

in
o

0 50 100 150
tH

Concentration (fig ml/1) Concentration (fig ml/1)

Figure 6.2: Calibration curve for method A Figure 6.3: Calibration curve for method B

154
Scheme 6.1: Reaction of PDAB and VA with SFD followed by the complex formation

155
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