12_chapter 6.pdf

CHAPTER 6

NEW SPECTROPHOTOMETRIC METHODS FOR THE DETERMINATION OF SULFADOXINE BY FORMATION OF Co(II) COMPLEXES

! II % | I I I | I | i | I$ I I | I I I

ABSTRACT The enhanced clinical use of sulfadoxine necessitated the study of new methods for

its

determination

in

quality

control

laboratories.

New

sensitive

visible

spectrophotometric methods are developed for the determination of sulfadoxine. Proposed methods are based on the reaction of drug with aldehyde followed by cobalt(II) chloride in acidic medium. The reaction of aldehyde with drug results in the formation of v

Schiff base which act as a ligand for the formation of complex with Co(II). Developed | I methods are successfully applied to the pharmaceutical formulation. |

Sulfadoxine

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6.1. DRUG PROFILE Sulfadoxine is chemically 4-amino-iV-(5,6-dimethoxypyrimidin-4-yl)benzene-lsulfonamide 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 Schiff base (nm)

^-max for Co(ll) complex (nm)

Aldehydes

R

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

672

665

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

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

X max (nm) Beer’s Law Limits (pg ml"1)

Regression Equation**

* 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

Reziz forte

Labeled amount

Found* ± SD using

Found* ± SD using

(mg)

Method A

Method B

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

o

m

CM

CM

o o

o

r-i

in

Absorbance

d

CO

350

400

450

500 550 600 Wavelength (nm)

650

700

750

Figure 6.1: Absorption maximum for (i) Co(II), (ii) method A and (iii) method B

in

CM

O

Absorbance

Hm

Absorbance

fN

o

oin --------- 1---------- 1---------- 1

0 tH

o

in

oo

o

in

0

50

100

150

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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157