Thai J. Pharm. Sci.

37 (2013) 171-185 Original article

171

Selective and sensitive assay of mebendazole in pharmaceuticals using bromocresol green by spectrophotometry
N. Swamy and K. Basavaiah*
Department of Chemistry, Manasagangothri, University of Mysore, Mysore 570 006, Karnataka, India *Corresponding author: Tel: +91 8212419659, Fax: +91 8212516133 E-mail address: kanakapurabasavaiah@gmail.com

Abstract:
Three new, simple, rapid and sensitive spectrophotometric methods have been developed for the assay of mebendazole (MBD) in bulk drug, tablets and suspension. First method (method A) is based on the formation of a colored ion-pair complex (1:1 drug/dye) of MBD with bromocresol green (BCG) at pH 1.99 0.01 and extraction of complex into chloroform followed by measurement of yellow ion-pair complex at 430 nm. In second and third methods, drug-dye ion-pair was dissolved either in ethanolic sulphuric acid and resulting acid form of dye was measured at 440 nm (method B) or in ethanolic potassium hydroxide and resulting base form of dye was measured at 600 nm (method C). Under optimized conditions, Beers law was obeyed over 1.0-20.0, 0.5-10 and 0.2-8.0 g mL-1 for method A, method B and method C, respectively, and corresponding molar absorptivity values are 1.55 x 104, 2.95 x 104 and 3.64 x 104 L mol-1 cm-1. Sandell sensitivity, limits of detection and quantification values are also reported for all three methods. Molar ratio of formed ion-pair complex was found to be 1:1 as deduced by Jobs method for method A, and calculated stability constant was also reported. Over linear ranges applicable, accuracy and precision of methods were evaluated on intra-day and inter-day basis. Application of proposed methods to bulk powder, commercial pharmaceutical tablet, suspension and spiked human urine was presented. Keywords: Mebendazole; Bromocresol green; Pharmaceuticals; Spectrophotometry; Assay

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N. Swamy and K. Basavaiah lines, a yellow coloured product formed by the interaction of MBD with KNO3 in concentrated H2SO4 medium was used by Sanghvi and Tandel [36] for the determination of the drug. MBD is reported to reduce Folin-Ciocalteau (FC) reagent in alkaline medium to a blue coloured chromogen (max 700 nm) paving the way for the assay of MBD in dosage forms [37]. Based on the charge-transfer (CT) complex formation reaction with chloranilic acid in dioxane medium, Ajali et al. [38] determined the drug by measuring the absorbance of the complex at 500 nm. Though several reaction schemes [33-38] have been applied for the spectrophotometric assay of MBD, methods based on ion-pair complexation reactions are seldom found in the literature. In continuation to our work on the use of ion-pair reactions for the sensitive and selective determination of several pharmaceuticals [39-46], an attempt was to apply this reaction for the assay of the anthelmintic drug, mebendazole (MBD). In the work presented, the ion-pair formed between MBD and anionic dye, bromocresol green (BCG) in acidic buffer medium was extracted into chloroform and measured at 430 nm. In order to provide highly sensitive methods which could be applied to urine sample, the drug-dye ion-pair was broken in either ethanolic H2SO4 and the acid form of the dye was measured at 440 nm or ethanolic KOH and the base form of the dye was measured at 600 nm. All the three procedures were applied to tablets and oral suspension without interference from the co-formulated substances.

Introduction
Mebendazole (MBD), chemically known as methyl5-benzoyl-2-benzimidazole carbamate, is an anthelmintic and antiinfestive used against hookworm, pinworm, roundworm, tapeworm, threadworm and mixed infestations. It is available in tablet and suspension form. Depending on the type of worm to be treated, the dosage varies in adults and children [1]. MBD in bulk form and tablet dosage form are both included in the USP 23 monographs [2] while the oral suspension is listed in the second supplement [3] of USP 23. The drug is also official in Indian Pharmacopoeia [4] and European Pharmacopoeia [5] and the latter describes a potentiometric titration of 250 mg of MBD in formic acid-acetic acid and methyl ethyl ketone (2-butanone) mixture with acetous perchloric acid. Different analytical techniques available in the literature for the assay of MBD in pharmaceuticals include titrimetry [6-9], uv-spectrophotometry [9-12], phosphorescence method [13], proton nuclear magnetic resonance spectrometry [14], fluorimetry [15-16], thermogravimetry [17, 18], membrane-sensor based potentiometry [19], DC polarography [20], differential pulse polarography [21, 22], high performance liquid chromatography [23-30] and high performance thin layer chromatography [31, 32]. Most of these methods [13-32] are complicated and need sophisticated instruments. Apart from the above, several visible spectrophotometric methods are also found in the literature. When MBD was hydrolysed with KOH, a yellow coloured substance (2-amino-5-benzolyl benzimidazole) was formed, and was measured at 420 nm [33]. MBD on treatment with hydroxylamine, dicyclohexyl carbo-di-imide and FeCl3 produced a red coloured product measurable at 520 nm and served as a basis for the assay [34]. Kar [35] has described a method based on a 2:1 complex formed by MBD with potassium bismuth(III) iodide. The orange complex formed by MBD in acetone was measured at 430 nm. The orange coloured product produced by the reaction of MBD with N-bromosuccinimide was measured at 465 nm and used for the assay of drug by Mohammed et al. [6]. On similar

Materials and Methods

Instruments A Systronics model 166 digital spectrophotometer (Systronics, Ahmedabad, Gujarat, India) with matched 1-cm quartz cells was used for absorbance measurements. A digital pH meter Model Elico L1 120 was used for pH measurements. Standards and reagents Chemicals used were of analytical reagent grade. Spectroscopic grade organic solvents and distilled water were used through out the investigation. Pharmaceutical

Thai J. Pharm. Sci. 37 (2013) 171-185 grade mebendazole (99.8% pure) was gifted from Cipla India Ltd, Mumbai, India, and was used as received. Mebex-100 (Cipla Ltd., Solan, India.) tablets and Mebex suspension (Cipla Ltd, Mumbai central, India.) were purchased from local commercial sources. 1) Bromocresol green, BCG (0.05%) Prepared by shaking 50 mg of BCG dye (Qualigens fine chemicals, Mumbai, India) in 10 mL ethanol to dissolve and made upto mark with water in a 100 mL calibrated flask. 2) Sodium acetate (1 M) Prepared by dissolving 6.8 g of the pure sodium acetate (Merck Pvt. Ltd., Mumbai, India) in 50 mL water. 3) Sodium acetate-hydrochloric acid buffer (pH 1.99) Prepared by mixing 1 M solutions of sodium acetate and hydrochloric acid (Merck Pvt. Ltd., Mumbai, India, sp. gr. 1.18) and the pH was adjusted to 1.99 by drop wise addition of sodium acetate/HCl solution. 4) Ethanolic sulphuric acid, H2SO4 (1%) One mL of conc. H2SO4 (Merck Pvt. Ltd., Mumbai, India, sp. gr. 1.84) was dissolved in 100 mL of ethanol. 5) Ethanolic potassium hydroxide, KOH (1%) One g of the pure KOH (S.D. Fine Chem. Ltd., Mumbai, India) was dissolved in and diluted to 100 mL with ethanol. 6) Standard MBD solution MBD standard solution (200 g mL-1) was prepared by dissolving 10 mg pure MBD in 2 mL 70% perchloric acid (Merck Pvt. Ltd., Mumbai, India), shaken to complete dissolution and made upto mark in a 50 mL standard flask with water. From this, 20 g mL-1 solution was prepared by dilution with water. General procedures Preparation of calibration graph Method A Aliquots of MBD standard solution (20 g mL-1) containing 0.0, 0.5, 1.0, 2.0, 4.0, 6.0, 8.0 and 10.0 mL MBD were measured accurately and transferred into a series of 125 mL separating funnels and the total volume was brought to 10 mL by adding water. To each funnel were added 5 mL each of H2O, and NaOAc-HCl buffer of pH 1.99. Content was mixed and 5 mL of 0.05%

173 BCG dye solution was added to each separating funnel, mixed well and kept aside for 5 min. The drug-dye ion-pair was then extracted with 10 mL of chloroform by shaking for 1 min and the layers were allowed to separate for 2 min. The organic layer was then passed over anhydrous sodium sulphate and absorbance measured at 430 nm against the reagent blank. Method B Into a series of 10 mL volumetric flasks, 0.0, 0.25, 0.5, 1.0, 2.0, 3.0, 4.0 and 5.0 volumes of MBD-BCG complex (20 g mL-1, prepared in method A) equivalent to 0.5 - 10.0 g mL-1 with respect to MBD were transferred. The total volume in each flask was brought to 5 mL with chloroform. After the addition of 1 mL 1% ethanolic H2SO4, the flasks were kept aside for 5 min, then diluted upto the mark with ethanol and absorbance measured at 440 nm against the reagent blank. Method C Different aliquots of 0.0, 0.1, 0.25, 0.5, 1.0, 2.0, 3.0 and 4.0 mL MBD-BCG complex (20 g mL-1, prepared by following the procedure described in method A) equivalent to 0.2-8.0 g mL-1 with respect to MBD were transferred into a series of 10 mL standard flasks and the total volume was brought to 4 mL by adding chloroform. To each flask, 1 mL of 1% ethanolic KOH was added, the content was mixed, and immediately the volume was made upto the mark with ethanol and the absorbance measured at 600 nm against the reagent blank. In all the three methods, standard graph was prepared by plotting the absorbance vs drug concentration, and the concentration of the unknown was read from the calibration graph or computed from the respective regression equation derived using the absorbance-concentration data. Procedure for tablets Twenty tablets each containing 100 mg of MBD were weighed, ground into a fine powder and mixed well. An accurately weighed quantity of finely ground tablet powder equivalent to 5 mg of MBD was accurately weighed into a 50 mL calibrated flask, 30 mL of 4% perchloric acid was added, and the flask was shaken for 20 min; and finally made upto the mark with the

174 same solvent The flask was kept aside for 5 min, and filtered using Whatman No. 42 filter paper. First 10 mL portion of the filtrate was discarded and a suitable aliquot of the filtrate (containing 100 g mL-1 MBD) was diluted with water to get a working concentration of 20 g mL-1 MBD and used for the assay by method A. The ion-pair complex MBD-BCG (20 g mL-1 MBD) from tablet powder was used for the assay by applying the procedure described under method B and method C. Procedure for suspension Five mL of Mebex suspension containing 100 mg MBD was quantitatively transferred into a 50 mL standard flask, added 10 mL of 70% perchloric acid and shaken the content for complete dissolution. Again added 20 mL water, shaken for another 20-30 min, made upto the mark with water and shaken for uniform concentration. The content was filtered using No. 1 filter paper and first 10 mL portion of the filtrate was discarded. The filtrate (2000 g mL-1 in MBD) was diluted to get a working concentration of 20 g mL-1 and assayed using method A. The ion-pair complex (20 g mL-1 in MBD) was employed for assay by method B and method C. Procedure for spiked human urine Five mL of 100 g mL-1 MBD solution was taken in a 25 mL calibrated flask using a micro burette, the flask was made up to the mark with spiked human urine. The content was mixed well for two minutes and 5.0 mL of this spiked urine sample was analyzed using the procedures described earlier in methods A, B and C.

N. Swamy and K. Basavaiah Reaction pathway MBD forms ion-pair complex with BCG, since the drug contains secondary amino group which is protonated in acid medium. Each drug-dye complex molecule, with two oppositely charged ions, behaves as a single unit held together by an electrostatic force of attraction (Scheme 1). In alcoholic acid and alkaline media, this ion-pair complex gets disturbed and it breaks to form yellow and blue colored acid and base forms of the dye and the drug. The mechanism of this breaking is shown in Scheme 2. Optimization of reaction conditions The optimization of the methods was carefully performed to achieve complete ion-pair complex formation, quantitative extraction of the ion-pair complex and maximum sensitivity. For the ion-pair complex formation found by preliminary experiments, reaction conditions such as pH, type of buffer and organic solvent, volume of the dye, and shaking and equilibration time for the extraction of ion-pair complex were optimized. In methods B and C, ethanolic H2SO4 and KOH concentration required for complete breaking of the complex was optimized. Selection of the extracting solvent A number of organic solvents such as chloroform, dichloromethane,1,2-dichloroethane, carbon tetrachloride, hexane, toluene and benzene were examined for extraction of the ion-pair complex in order to provide an applicable extraction procedure. Chloroform was preferred for its efficient and quantitative extraction of ion-pair complex and the stability of the extracted ion-pair, its high sensitivity, and very low absorbance of the reagent blank and shortest time to reach the equilibrium between both phases. Effect of pH on the ion-pair formation The effect of pH of the aqueous phase was studied by extracting the colored complex at pH 0.65-5.20. It was noticed that the maximum absorbance of complex and minimum absorbance of the reagent blank were observed at pH of 1.99 0.01. The results are shown in

Results and Discussion

Spectral characteristics The absorption spectrum of the yellow colored MBD-BCG ion-pair complex which is shown in Fig. 1 has a maximum absorbance (max) at 430 nm. This drug-dye ion-pair complex was broken in ethanolic acid and ethanolic base to yield yellow coloured acid and blue colored base forms of dye with max at 440 and 600 nm, respectively (Fig. 1). In all the cases, the blanks had negligible absorbance.

Thai J. Pharm. Sci. 37 (2013) 171-185 Fig. 2. At pH values greater than 1.99 0.01, a decrease in absorbance of the ion-pair complex was observed and at pH values below 1.99 0.01 an increase in absorbance of the reagent blank was observed. Hence

175 the pH 1.99 0.01 was fixed in all subsequent work. Effect of volume of buffer also studied and it was found that 5.0 mL buffer of pH 1.99 0.01 was optimum (Fig. 3).

Scheme 1

Br
HO

Br OH Br

Br HO Br O HO S O

Br OH Br

Br
HO Br O -O S

Br OH Br
+ H+

Br O O S O BCG (Lactoid ring)

BCG (quinoid ring)

O BCG-anionic form
+ H H2 N N

H N N MBD

H N

O + H+ O
Br Br OH Br N O MBD-protonated form

+ H H2 N N

HO O + Br O -O S O BCG-anionic form

O MBD-protonated form

Br + HO H H 2 N N O Br O N O -O S
MBD-BCG ion-pair O complex measured at 430 nm

Br OH Br

Scheme 2 Br Br + HO OH H 2 H N O N + H+ Br O Br O Method B -O S N O O MBD-BCG ion-pair complex Br O Br O -O O S N O O MBD-BCG ion-pair complex + H H2 N N HO Br Br Br OH HO O Br O Br O + HO S N O O Yellow colour of acid form of MBD-protonated form BCG, measured at 440 nm H H2+ N N Br HO OH Br + OH Method C MBD O H H N N N O O+ Br O-

Br O Br -O S O Blue colour of base form of BCG, measured at 600 nm

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0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 380 410 440 470 500 530 560 590 620 650 680

N. Swamy and K. Basavaiah

Absorbance

Wavelength, nm
Figure 1 Absorption spectra of MBD-BCG ion-pair complex (--) (10 g mL-1 MBD), acid form dye (--) (4 g mL-1 MBD-BCG complex), and base form of the dye (--) (4 g mL-1 MBD-BCG complex), and blanks (- -)

0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 0.65 1.09 1.42 1.99 2.64 3.29 3.79 4.19 4.76 5.20 pH of buffer

Figure 2 Effect of buffer pH (method A, 10 g mL-1 MBD)

Absorbance
Absorbance

0.56 0.52 0.48 0.44 0.4 0 2 4 5 mL, buffer 6 8 10

Figure 3 Effect of volume of buffer (method A, 10 g mL-1 MBD)

Thai J. Pharm. Sci. 37 (2013) 171-185

0.6 0.5 0.4
Absorbance

177

0.3 0.2 0.1 0 2 4 5 6 mL, BCG 8 10

Figure 4 Effect of volume of BCG dye (method A, 10 g mL-1 MBD)

0.55 0.5

Absorbance

0.45 0.4 0 5 10 15 20 25
mL, water

Figure 5 Effect of volume of water (aqueous phase) (method A, 10 g mL-1 MBD)

0.48

Absorbance

0.47

0.46 0.5 1 1.5 mL, acid 2 2.5

Figure 6 Effect of volume of acid (method B, 10 g mL-1 MBD)

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0.51

N. Swamy and K. Basavaiah

Absorbance

0.49

0.47 0.5 1 1.5 mL, KOH 2 2.5

Figure 7 Effect of volume of base (method C, 10 g mL-1 MBD)

0.4 0.3

Absorbance

0.2 0.1 0 0 0.2 0. 4 0.6 0. 8 1 Mole ratio of MBD(VMBD/VMBD+VBCG)

Figure 8 Jobs plot for MBD-BCG ion-pair complex (6.8 x 10-5 M solutions of MBD and BCG)

Different buffers systems of pH 1.99 such as Walpole (1M sodium acetate-1 M hydrochloric acid), Clark and Lubs2 (0.2 M potassium chloride-0.1 M hydrochloric acid), Sorenson (0.1 M sodium chloride-0.1 M hydrochloric acid), Clark and Lubs2 (0.1 M potassium hydrogen phthalate-0.1 M hydrochloric acid), and Mc Ilvaine (0.2 M disodium hydrogenphosphate-0.1 M citric acid) were tried, it was found that Walpole buffer of pH 1.99 was the best fit for complex formation as well as extraction. Effect of dye concentration The effect of the dye concentration was studied in method A by measuring the absorbance of solutions containing a fixed concentration of MBD (10.0 g mL-1) and varied amounts of BCG. It is clear from Fig. 4 that

the maximum absorbance was found with 5.0 mL of 0.05% BCG and beyond that absorbance was nearly constant. Thus, 5.0 mL of 0.05% BCG in a total volume of 25 mL aqueous phase was used for ion-pair formation throughout the investigation (Fig. 4). Effect of the volume of aqueous phase The effect of volume of aqueous phase was studied by using different volumes of aqueous phase (including drug, BCG and buffer) such as 15, 20, 25, 30 and 35 mL and extracting with 10 mL of dichloromethane (Fig. 5). The use of 25 mL of aqueous phase was found to be sufficient to achieve maximum absorbance of measured species and minimum absorbance of reagent blank and hence an aqueous phase of 25 mL was fixed throughout.

Thai J. Pharm. Sci. 37 (2013) 171-185 Effects of contact and shaking time and sequence of addition The effect of contact time between MBD and BCG in the presence of buffer was studied in the time range 0-30 min before extraction and it was found that 5 min was sufficient to achieve maximum absorbance at 430 nm. Shaking times of 0.5-3 min produced a constant absorbance in method A, and hence a shaking time of 1 min was used throughout. In method B, the effect of the time required to break the complex was studied after the addition of ethanolic H2SO4 to the complex and it was found that 5 min was sufficient for that breaking. Immediate breaking was observed in method C, when ethanolic KOH was added to the MBD-BCG ion-pair complex. There was no appreciable change in the absorbance or color of the measured species if the order of addition of the reactants was varied. Effect of study of equilibriation time and number of extractions The time required for the two layers to separate was studied by keeping all other parameters constant and two minutes was adequate for the complete separation of aqueous and organic phases. The number of extraction required for complete removal of the complex from the aqueous phase was examined. One extraction with 10 mL of CHCl3 was sufficient and second extraction gave the absorbance same as the blank absorbance. Effect of volumes of acid and base The volumes of alcoholic H2SO4 and alcoholic KOH required to break the complex were studied in method B and method C by taking a fixed concentration of MBD-BCG complex and it was found that 1.0 mL of acid in method B and 1.0 mL of alkali in method C gave maximum absorbance. (Fig. 6 and 7) Composition of the ion-pair complex The composition of the ion-pair complex formed between MBD and BCG in method A was established by applying Jobs method of continuous variations [47]. In this method, 6.8 x 10-5 M solutions of MBD and BCG

179 were used and mixed in varying volume ratios in such a way that the total volume of each mixture was the same. The absorbance of each solution was measured and plotted against the mole fraction of the drug (Fig. 8). The plot reached a maximum value at a mole fraction of 0.5 indicating that a 1:1 (MBD:BCG) ion-pair complex is formed through the electrostatic attraction between protonated MBD and BCG anion. The conditional stability constant (Kf) of the ion-pair complex was calculated [48] from the data of continuous variations method and the log Kf was found to be 7.63. Stability of the measured species The formation of the ion-pair was rapid and the yellow color extract was stable for at least 4 h without any change in color intensity at room temperature. Also, the absorbance of the yellow color of acid form of the dye and blue color of base form of the dye in method B and method C were stable for more than 12 h and 0.5 h, respectively. Method validation Linearity and sensitivity At described experimental conditions for MBD determination, the absorbance-concentration plots were found to be linear over the concentration ranges stated in Table 1. The regression parameters given in the regression equation calculated from the calibration graphs along with the standard deviations of the slope (Sb) and the intercept (Sa) are also given in Table 1. The linearity of calibration graphs was proved by the high values of the correlation coefficient (r) and the small values of the y-intercepts of the regression equations. The apparent molar absorptivity, Sandell sensitivity, limits of detection and quantification of all the methods were also calculated and recorded in Table 1. Precision and accuracy In order to evaluate the precision of the proposed methods, solutions containing three different concentrations of the MBD were prepared and analyzed in seven replicates. The results obtained from this investigation are summarized in Table 2. The low values

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Table 1 Sensitivity and regression parameters Parameter
max,

N. Swamy and K. Basavaiah

Method A 430 >4 1.0-20.0 1.55 x 104 0.019 0.06 0.17 0.0187 0.0487 9.98 x 10-2 5.60 x 10-3 0.9985

Method B 440 > 12 0.5-10.0 2.95 x 104 0.01 0.01 0.03 0.0109 0.0941 9.98 x 10-2 1.12 x 10-2 0.9994

Method C 600 0.5 0.2-8.0 3.64 x 104 0.008 0.02 0.05 0.0162 0.1055 9.98 x 10-2 1.36 x 10-2 0.9991

nm Colour stability, hour. Linear range, g mL-1 Molar absorptivity (), L mol-1 cm-1 Sandell sensitivity*, g cm-2 Limit of detection (LOD), g mL-1 Limit of quantification (LOQ), g mL-1 Regression equation, Y** Intercept (a) Slope (b) Standard deviation of a (Sa) Standard deviation of b (Sb) Regression coefficient (r)

*Limit of determination as the weight in g mL-1 of solution, which corresponds to an absorbance of A = 0.001 measured in a cuvette of cross-sectional area 1 cm2 and l = 1 cm. **Y = a + bX, where Y is the absorbance, X is concentration in g mL-1, a is intercept and b is slope.

of the relative standard deviation (% RSD) and percentage relative error (% RE) also indicated the high precision and the good accuracy of the proposed methods. The assay procedure was repeated seven times, and percentage relative standard deviation (% RSD) values were obtained within the same day to evaluate repeatability (intra-day precision) and over five different days to evaluate intermediate precision (inter-day precision). Selectivity study Selectivity was evaluated by placebo blank and synthetic mixture analyses. A placebo blank consisting of starch (20 mg), acacia (25 mg), hydroxyl cellulose (20 mg), sodium citrate (30 mg), talc (20 mg), magnesium stearate (25 mg) and sodium alginate (20 mg) was prepared by thorough mixing and its solution was prepared as described under procedure for tablets, by taking about 20 mg, and then subjected to analysis. A synthetic mixture was prepared by adding 10 mg of pure MBD to 10 mg of the above mentioned placebo blank, and the mixture was homogenized. Following the procedure employed for tablets, the synthetic mixture solution was prepared, and a suitable aliquot was subjected to analysis by the methods, after appropriate dilution and no significant interference was observed from these excipients.

Robustness and ruggedness The robustness of the methods was evaluated by making small incremental changes in two selected variables (volumes of buffer and reaction time in method A; volumes of ethanolic H2SO4 and KOH and the breaking times in method B and method C) and the effect of changes was studied on the basis of absorbance of colored systems. The changes had negligible influence on the results as revealed by small RSD % as intermediate precision. The results are tabulated in Table 3. Method ruggedness was studied by having the analysis done by four different analysts, and also by a single analyst performing analysis on four different instruments in the same laboratory. Intermediate precision values in all the methods were in the range 0.63-2.65 indicating acceptable ruggedness. These results are given in Table 3. Application The proposed methods were applied for the determination of MBD in commercial tablets, suspension and spiked human urine. The results were compared with these obtained using an official method [5]. Statistical analysis of the results did not give any significant difference between the performance of the proposed

Thai J. Pharm. Sci. 37 (2013) 171-185

Table 2 Evaluation of Intra-day and inter-day accuracy and precision Intra-day accuracy and precision (n=7) MBD founda RSDb REc -1 (g mL ) % % 6.06 11.92 17.82 3.02 5.99 9.04 1.98 3.99 5.96 0.72 1.36 1.45 0.59 1.46 0.47 1.04 0.56 1.66 0.98 0.73 1.05 0.68 0.15 0.44 1.06 0.12 0.74 Intra-day accuracy and precision (n=7) MBD founda RSDb REc -1 (g mL ) % % 6.09 11.94 17.94 3.08 6.04 9.23 2.04 4.04 6.07 0.34 1.21 1.27 0.83 1.37 0.41 1.12 1.04 1.26 1.54 0.52 0.34 2.56 0.64 2.50 2.13 1.02 1.11

181

Method

MBD taken (g mL-1) 6.0 12.0 18.0 3.0 6.0 9.0 2.0 4.0 6.0

MBD-mebendazole a Mean value of seven determinations; bRelative standard deviation (%); cRelative error (%).

Table 3 Method robustness and ruggedness expressed as intermediate precision (% RSD) Robusiness Parameters altered Volume of buffer / Reaction / ethanolic H2SO4 / breaking ** time ethanolic KOH 1.27 0.85 0.81 1.06 0.72 0.92 Ruggedness interanalysis (%RSD), (n=4) 0.79 0.63 1.54 interinstruments (%RSD), (n=4) 2.65 1.97 2.36

Method A B C

MBD taken (g mL-1) 10 4 4

**In method A, the volumes of buffer were 4, 5 and 6 mL, in method B the volumes of ethanolic H2SO4 added were 0.8, 1.0 and 1.2 mL and in method C the volumes of ethanolic KOH added were 0.8, 1.0 and 1.2 mL. In method A, the reaction times were 4, 5 and 6 min and in method B and method C the breaking times were 4, 5 and 6 min, and 0, 1 and 2 min, respectively. MBD-mebendazole

Table 4 Results of analysis of formulations by the proposed methods and statistical comparison of the results with official method Found* (Percent of label claim SD) Method A Method B 99.7 1.09 t = 1.71 F = 1.43 98.8 0.92 t = 0.47 F = 1.35 100.3 0.43 t = 1.03 F = 2.95 99.5 0.72 t = 0.7 F = 2.21

Tablet brand name aMebex

Nominal amount 100 mg per tablet 100 mg per 5 mL

Official method 100.8 0.91

Method C 101.2 0.72 t = 0.81 F = 1.60 98.6 0.63 t = 0.9 F = 2.88

bMebex

99.1 1.07

suspension

*Mean value of five determinations. (Tabulated t-value at the 95% confidence level and for four degrees of freedom is 2.77). (Tabulated F-value at the 95% confidence level and for four degrees of freedom is 6.39). Marketed by : aCipla Ltd, Solan, India. bCipla Ltd, Mumbai central, India.

182 methods and reference method with respect to accuracy and precision as revealed by Students t-value and variance ratio F-value. The results of assay are given in Table 4. The proposed methods were also applied to the determination of MBD in spiked human urine sample and the results are presented in Table 5. The % recovery was found to be high in spiked human urine sample, this indicates that spiked human urine slightly affect MBD concentration i.e. interfere slightly hence as compared to MBD taken found to be high.

N. Swamy and K. Basavaiah Recovery study To ascertain the accuracy of the proposed methods, recovery experiment was performed via standard addition technique. To a fixed and known amount of MBD in tablet powder (pre-analyzed) or suspension, pure MBD was added at three levels 50, 100 and 150% of the level present in the tablet / suspension and the total was found by the proposed methods. Results of this study are presented in Table 6. In all the cases, the percent found ranged from 99.04 to 100.50 with SD values in the range 0.35 to1.68 and indicate that the co-formulated substances did not interfere in the assay.

Table 5 Mebendazole determination in spiked urine sample (n = 5) Spiked concentration (g mL-1) 10 4 4 Concentration found* (g mL-1) 10.35 4.21 4.26 % Recovery SD* 103.5 0.72 105.3 0.93 106.4 0.63

Method A B C

*Mean value of five determinations of mebendazole.

Table 6 Results of recovery study via standard-addition method Method A Method B Pure Total Pure MBD MBD in Pure Total MBD found, recovered formu- MBD found, added, g mL-1 (% SD*) lation, added, g mL-1 g mL-1 g mL-1 g mL-1 3.0 6.0 9.0 3.0 6.0 9.0 8.96 12.03 14.91 8.90 11.92 14.86 99.37 1.29 100.80 0.35 99.20 0.55 99.07 1.68 99.83 0.91 99.21 1.44 3.01 3.01 3.01 2.98 2.98 2.98 1.5 3.0 4.5 1.5 3.0 4.5 4.51 5.99 7.50 4.47 5.96 7.44 Method C Pure Total Pure MBD MBD found, recovered added, g mL-1 (% SD*) g mL-1 1.0 2.0 3.0 1.0 2.0 3.0 3.01 99.04 1.36 4.01 99.52 0.65 5.02 100.12 0.74 2.97 99.92 1.83 3.95 99.21 0.85 4.98 100.50 0.96

Tablet studied

MBD in formulation, g mL-1 5.98 5.98 5.98 5.93 5.93 5.93

Pure MBD recovered (% SD*) 100.31 1.29 99.34 0.88 99.79 0.53 99.25 1.27 99.38 1.15 99.22 0.81

MBD in formulation, g mL-1 2.02 2.02 2.02 1.97 1.97 1.97

Mebex-100

Mebex Suspension

*Mean value of three determinations.

Thai J. Pharm. Sci. 37 (2013) 171-185

Table 7 Comparison of performance of the present methods with the existing methods Sl. No. 1 2
max

183

Reagent/s used *NBS Potassium bismuth iodide

Methodology Orange product was measured Ion-pair ppt dissolved in acetone and absorbance measured Alkaline hydrolysis product (2-amino-5-benzoylbenzimidazole) measured Measurement of the absorbance of the coloured species in isopropyl alcohol-HCOOH medium Ion-pair complex dissolved in acetone and measured

(nm) 465 325

Linear range (g/mL-1) (L mol-1 cm-1) 0.6-1.6 mg

Remarks Tedious & time consuming, Pptn washing and dissolution steps involved, Less sensitive -

Ref. No. 6 9

KOH

420

33

H2NOH-HCl. *DCC and FeCl3 Potassium bismuth(III) iodide

520

0.4-2.0

Multi step reaction

34

430

0.3-2.1 mg%

6 7 8 9

KNO3 in conc. H2SO4 *F-C reagent *CAA *BCG

Nitrosation product measured Absorbance of blue colored chromogen measured Red coloured C-T complex in dioxan measured a) Measurement of absorbance of extracted MBD-BCG ion pair in CHCl3 b) Acid form of the BCG measured c) Base form of the BCG measured

700 500 430

2-32 1.0-20 1.55 x 104 0.5-10 2.95 x 104 0.2-8.0 3.64 x 104

Tedious & time consuming Pptn washing and dissolution steps involved, Less sensitive Strong H2SO4 medium required -

35

36 37 38

440 600

Sensitive and selective with wide linear dynamic range

Present work

*NBS-N-bromosuccinimide; CAA-chloranilic acid; DCC-dicyclohexyl carbo-di-imide; F-C-Folin-Ciocalteau; BCG-bromocresol green

Conclusion
A significant advantage of the extractive spectrophotometric methods is that it can be applied for the determination of individual compounds in a multi component mixture. The proposed methods make use of simple reagent which an ordinary analytical laboratory can afford, and the procedures do not involve any critical reaction conditions or tedious sample preparation. The methods are highly reliable owing to the stability of the ion-pair complex and acid / base forms of the dye,

which are ultimately measured. Moreover, the methods are accurate, reproducible, adequately sensitive and free from interference caused by the excipients expected to be present in tablets and suspension. The methods were successfully applied to the spiked human urine, and all the methods were demonstrated to be both robust and rugged. The methods offer several advantages over the existing methods in terms of sensitivity, selectivity, linear dynamic range and mild optimum conditions as indicated in Table 7.

184

N. Swamy and K. Basavaiah

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Acknowledgement
Authors express their gratitude to the quality control manager, Cipla India, Ltd., Mumbai for gift sample of pure mebendazole and the authorities of the University of Mysore, Mysore, for permission and facilities.
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