Validated Spectrophotometric Method for Determination of Some Benzimidazole Derivatives in Pharmaceutical Formulations Using 1,2-naphthoquinone-4-sulphonate

Aim: To develop and validate a sensitive method for the determination of rapeprazole (RPZ) and omeprazole (OPZ) in bulk and formulations based on nucleophilic substitution reaction of RPZ and OPZ with sodium 1,2-naphthoquinone-4-sulphonate (NQS) in an alkaline medium. Study Design: All variables were studied to optimize the reaction conditions and the reaction mechanism was postulated. Place and Duration of Study: Department of Chemistry, Faculty of Science, Aleppo University, Aleppo, Syria between December 2011 and December 2012. Methodology: The colored products were measured spectrophotometrically at 453 nm using double beam UVD-2960 (Labomed, INC., U.S.A) ultraviolet-visible spectrophotometer with matched 1-cm quartz cells. The reaction time and temperature were 20 min and 25oC for RPZ and OPZ. As per ICH guidelines, the proposed method was validated. The developed method was successfully applied for the estimation of RPZ and OPZ in tablets and capsules and results were compared statistically with the official methods. Research Article International Research Journal of Pure & Applied Chemistry, 3(2): 118-132, 2013 119 Results: The developed method showed a linear Beer's law range from 0.26 to 12.0 and from 0.49 to 12.0 μg mL with limit of detection values of 0.181 and 0.187 μg mL for RPZ and OPZ, respectively. The calculated molar absorptivity values are 7.7×10 and 3.8×10 L mol cm for RPZ-NQS and OPZ-NQS, respectively. The proposed methods were successfully applied to the determination of RPZ and OPZ in formulations and the results tallied well with the label claim. Conclusion: The developed method was linear, sensitive, selective, precise, accurate and robust, being suitable for routine quality control analyses of RPZ and OPZ.

1,2-naphthoquinone-4-sulphonate (NQS) has been used as a chromogenic reagent for the spectrophotometric determination of many pharmaceutical amines. It is a popular spectrophotometric reagent due to its efficient reactivity with both primary and secondary amines, and high reaction rate [38,39]. NQS proved to be a useful and sensitive analytical derivatizing agent for spectrophotometric analysis of pharmaceuticals bearing a primary or secondary amino group, however the use of NQS for spectrophotometric determination of rabeprazole and omeprazole was not reported. Therefore, the present work describes the evaluation of NQS as a chromogenic reagent in the development of simple and rapid spectrophotometric method for determining the content of rabeprazole and omeprazole in pharmaceutical formulations based on the reaction of NQS with amino group of rabeprazole and omeprazole molecules to form orange compounds.

Apparatus
Double beam UVD-2960 (Labomed, INC., U.S.A) ultraviolet-visible spectrophotometer with matched 1-cm quartz cells was used for all the spectrophotometric measurements under the following operating conditions: scan speed medium (400nm/min), scan range 400-600 nm and slit width 0.1 nm. Spectra were automatically obtained by UV-WIN software Ver.5.0.10. Electronic balance (Kern, Germany) was used for weighing the samples and semiautomatic micropipettes were used for measuring the volume of samples.

Stock standard solutions
An accurately weighed 0.05 g standard sample of RPZ and OPZ was dissolved in methanol, transferred into a 100 mL standard flask and diluted to the mark with methanol to obtain 0.5 mg mL -1 . This stock solution was further diluted to obtain working solutions in the ranges of 0.26-12.00 and 0.49-12.00 μg mL −1 for RPZ and OPZ, respectively.

Sodium 1,2-naphthoquinone-4-sulfonate solution
An accurately weighed 0.3 g of NQS was dissolved in double distilled water, transferred into a 100 ml standard flask and diluted to the mark with double distilled water and mixed well to prepare 0.3% w/v. The solution was freshly prepared and protected from light during use.

Alkaline solutions
Sodium hydroxide, disodium hydrogen phosphate, borax and sodium bicarbonate solutions of a concentration range of 0.1-0.6 M were prepared in double distilled water.

General Procedures
Aliquots of standard RPZ (5-240 L, 0.5 mg mL -1 ) and OPZ (9-240 L, 0.5 mg mL -1 ) solutions were transferred into a series of 10mL calibrated volumetric flasks. Then 1.75 mL and 2.0 mL of 0.2 M sodium hydroxide solution was added, followed by 1.0 mL and 1.75 mL of NQS 0.3% (w/v) for RPZ and OPZ, respectively, and then the solutions were allowed to proceed at 25ºC for 20 min for RPZ and OPZ. After that, the volume was made up to the mark with bidistilled water and the absorbance was measured at 453 nm for RPZ-NQS and OPZ-NQS against reagent blank treated similarly under identical conditions.

Tablet (or capsule) sample solutions
Twenty tablets were weighted accurately and crushed to a fine powder. In the case of capsules, the contents of twenty capsules were completely evacuated from shells. An accurately weighed quantity of the powder equivalent to 50 mg of rabeprazole or omeprazole was transferred into a 100 mL calibrated flasks, and dissolved in about 50mL of methanol. The contents of the flask were swirled for 10 min, and then completed to volume with methanol to achieve a concentration of 0.5 mg mL -1 . The contents were mixed well and filtered rejecting the first portion of the filtrate. The general procedure was then followed in the concentration ranges mentioned above.

RESULTS AND DISCUSSION
RPZ and OPZ were found to react with NQS in an alkaline medium at 25ºC producing an orange-colored product of maximum absorption peak (λ max ) at 453 nm ( Fig. 1). Thus, this wavelength was chosen for all further measurements in order to obtain highest sensitivity for the method. It is important to point out that the colorless reagent blank (NQS), in alkaline medium, exhibits negligible absorption at 453 nm. Under the experimental conditions pure drug showed a negligible absorbance at the corresponding maximum.

Optimization of Reaction Variables
Optimum conditions necessary for rapid and quantitative formation of colored product with maximum stability and sensitivity were established by varying the parameters one at a time, keeping the others fixed and observing the effect produced on the absorbance of the colored species. In order to establish the experimental conditions, the effect of various parameters such as volume of NQS, addition of alkaline medium, waiting time and the stability of colored product were studied.

Effect of the solvent nature
The solvent plays an important role in some charge transfer reactions, since it must be able to facilitate the total charge transfer and then allow the complex dissociation and stabilization of the radical anion formed, which is the absorbing specie. The reaction was tested in water, methanol, ethanol, isopropanol, acetone and acetonitrile media. According to the literature, solvents with high dielectric constant are more effective to execute this task. Taking this fact into account and the high solubility of the NQS in water allow its use in the present case. Although the highest dielectric constant of acetonitrile, best sensitivity was achieved with water, probably because of the capacity of this solvent to form stable hydrogen bonds with the radical anion. Then, water was chosen for further experiments.
Maximum absorbance of the orange solutions produced by the reaction between RPZ or OPZ with NQS against reagent blank was observed at 453 nm in water medium (Fig. 1). Thus, this wavelength was chosen for all further measurements in order to obtain highest sensitivity for the method. It is important to point out that the colorless reagent blank (NQS), in water medium, exhibits negligible absorption at 453 nm.

Effect of the NQS volume
The maximum conversion of the analyte into absorbing specie depends on the amount of the reagent available in the solution for reaction and the equilibrium involved. So, the reagent concentration in solution was studied by varying the NQS volume in the range of 0.25 -3.0 mL of 0.3% (w/v) NQS, while the RPZ and OPZ concentration was maintained constant at 12 and 8 μg mL -1 , respectively. The results are shown in the Fig. 2. The study revealed that the reaction was dependent on NQS reagent. The highest absorption was attained when the volume of NQS was 1.75 and 2 mL of 0.3% (w/v) NQS for RPZ and OPZ (Fig. 2). Higher volume of NQS had no effect on the absorption values.

Effect of the alkalinity
To generate the nucleophile from RPZ or OPZ and activate the nucleophilic substitution reaction, alkaline medium was necessary; since the results revealed that RPZ or OPZ have difficulty to react with NQS in acidic media. Different inorganic bases were tested: sodium hydroxide, disodium hydrogen phosphate, and sodium bicarbonate, all prepared as aqueous solution. Best results were obtained in case of sodium hydroxide where with other bases either precipitation of white colloid occurred upon diluting the reaction solution with organic solvent, high blank readings, non reproducible results, and/or weak sensitivity were observed. Studies for optimization of sodium hydroxide concentration revealed that the optimum volume of NaOH was 1 and 0.75 mL of 0.2 M NaOH for RPZ and OPZ, respectively ( Fig. 3). At this value, the amino group of RPZ and OPZ facilitates the nucleophilic substitution reaction. At more concentrations of NaOH, the absorbance of solution obviously decreased. This was attributed probably to the increase in the amount of hydroxide ion that holds back the condensation reaction between RPZ or OPZ and NQS.

International Research Journal of Pure & Applied Chemistry, 3(2): 118-132, 2013
123 sensitivity for the method. It is important to point out that the colorless reagent blank (NQS), in water medium, exhibits negligible absorption at 453 nm.

Effect of the NQS volume
The maximum conversion of the analyte into absorbing specie depends on the amount of the reagent available in the solution for reaction and the equilibrium involved. So, the reagent concentration in solution was studied by varying the NQS volume in the range of 0.25 -3.0 mL of 0.3% (w/v) NQS, while the RPZ and OPZ concentration was maintained constant at 12 and 8 μg mL -1 , respectively. The results are shown in the Fig. 2. The study revealed that the reaction was dependent on NQS reagent. The highest absorption was attained when the volume of NQS was 1.75 and 2 mL of 0.3% (w/v) NQS for RPZ and OPZ (Fig. 2). Higher volume of NQS had no effect on the absorption values.

Effect of the alkalinity
To generate the nucleophile from RPZ or OPZ and activate the nucleophilic substitution reaction, alkaline medium was necessary; since the results revealed that RPZ or OPZ have difficulty to react with NQS in acidic media. Different inorganic bases were tested: sodium hydroxide, disodium hydrogen phosphate, and sodium bicarbonate, all prepared as aqueous solution. Best results were obtained in case of sodium hydroxide where with other bases either precipitation of white colloid occurred upon diluting the reaction solution with organic solvent, high blank readings, non reproducible results, and/or weak sensitivity were observed. Studies for optimization of sodium hydroxide concentration revealed that the optimum volume of NaOH was 1 and 0.75 mL of 0.2 M NaOH for RPZ and OPZ, respectively (Fig. 3). At this value, the amino group of RPZ and OPZ facilitates the nucleophilic substitution reaction. At more concentrations of NaOH, the absorbance of solution obviously decreased. This was attributed probably to the increase in the amount of hydroxide ion that holds back the condensation reaction between RPZ or OPZ and NQS. 123 sensitivity for the method. It is important to point out that the colorless reagent blank (NQS), in water medium, exhibits negligible absorption at 453 nm.

Effect of the NQS volume
The maximum conversion of the analyte into absorbing specie depends on the amount of the reagent available in the solution for reaction and the equilibrium involved. So, the reagent concentration in solution was studied by varying the NQS volume in the range of 0.25 -3.0 mL of 0.3% (w/v) NQS, while the RPZ and OPZ concentration was maintained constant at 12 and 8 μg mL -1 , respectively. The results are shown in the Fig. 2. The study revealed that the reaction was dependent on NQS reagent. The highest absorption was attained when the volume of NQS was 1.75 and 2 mL of 0.3% (w/v) NQS for RPZ and OPZ (Fig. 2). Higher volume of NQS had no effect on the absorption values.

Effect of the alkalinity
To generate the nucleophile from RPZ or OPZ and activate the nucleophilic substitution reaction, alkaline medium was necessary; since the results revealed that RPZ or OPZ have difficulty to react with NQS in acidic media. Different inorganic bases were tested: sodium hydroxide, disodium hydrogen phosphate, and sodium bicarbonate, all prepared as aqueous solution. Best results were obtained in case of sodium hydroxide where with other bases either precipitation of white colloid occurred upon diluting the reaction solution with organic solvent, high blank readings, non reproducible results, and/or weak sensitivity were observed. Studies for optimization of sodium hydroxide concentration revealed that the optimum volume of NaOH was 1 and 0.75 mL of 0.2 M NaOH for RPZ and OPZ, respectively (Fig. 3). At this value, the amino group of RPZ and OPZ facilitates the nucleophilic substitution reaction. At more concentrations of NaOH, the absorbance of solution obviously decreased. This was attributed probably to the increase in the amount of hydroxide ion that holds back the condensation reaction between RPZ or OPZ and NQS.

Effect of the reaction temperature and time
The effect of temperature and time on the reaction of RPZ and OPZ with NQS in alkaline medium was studied at different values (20-75 o C, 0-60 min) by continuous monitoring of the absorbance at 453 nm. It was found that the reaction with NQS was not affected by increasing the temperature, and the reaction at laboratory ambient temperature (25±5 o C) went to completion within 20 min. The results revealed that increasing the temperature had negative effect on the absorption values of the reaction solution. This was probably attributed to the instability of the RPZ-NQS and OPZ-NQS derivative. The optimum reaction time was determined at laboratory ambient temperature. Increase absorbance values were observed from the beginning of the experiment up to 20min (Fig. 4). After this time and up to 60 min, absorbance suffered a slight increase, reaching values up to 2% higher than those observed after 20 min of the reaction. In view of these results, all measurements were carried out after 20 min of mixing of the reagents in order to make the method faster.

Effect of the reaction temperature and time
The effect of temperature and time on the reaction of RPZ and OPZ with NQS in alkaline medium was studied at different values (20-75 o C, 0-60 min) by continuous monitoring of the absorbance at 453 nm. It was found that the reaction with NQS was not affected by increasing the temperature, and the reaction at laboratory ambient temperature (25±5 o C) went to completion within 20 min. The results revealed that increasing the temperature had negative effect on the absorption values of the reaction solution. This was probably attributed to the instability of the RPZ-NQS and OPZ-NQS derivative. The optimum reaction time was determined at laboratory ambient temperature. Increase absorbance values were observed from the beginning of the experiment up to 20min (Fig. 4). After this time and up to 60 min, absorbance suffered a slight increase, reaching values up to 2% higher than those observed after 20 min of the reaction. In view of these results, all measurements were carried out after 20 min of mixing of the reagents in order to make the method faster.

Effect of the reaction temperature and time
The effect of temperature and time on the reaction of RPZ and OPZ with NQS in alkaline medium was studied at different values (20-75 o C, 0-60 min) by continuous monitoring of the absorbance at 453 nm. It was found that the reaction with NQS was not affected by increasing the temperature, and the reaction at laboratory ambient temperature (25±5 o C) went to completion within 20 min. The results revealed that increasing the temperature had negative effect on the absorption values of the reaction solution. This was probably attributed to the instability of the RPZ-NQS and OPZ-NQS derivative. The optimum reaction time was determined at laboratory ambient temperature. Increase absorbance values were observed from the beginning of the experiment up to 20min (Fig. 4). After this time and up to 60 min, absorbance suffered a slight increase, reaching values up to 2% higher than those observed after 20 min of the reaction. In view of these results, all measurements were carried out after 20 min of mixing of the reagents in order to make the method faster.

Stoichiometric Relationship
Under the optimum conditions, the stoichiometry of the reaction between benzimdazole derivatives RPZ or OPZ and NQS was investigated by Job's method of continuous variation [40]. The stoichiometric ratio between NQS and drug was found to be 1:1 (Fig. 5).

[RPZ]+[NQS] = 2×10 -3 M and [OPZ]+[NQS] = 2×10 -3 M.
Based on this ratio, and the presence of only one center (N-H group) in RPZ or OPZ molecule that is available for the nucleophilic substitution reaction, the reaction pathway was postulated to be proceeded as shown in Fig. 6. The formation constant  n of the formed complex is calculated using the data of the continuous variation method by applying Harvey and Manning method [41]: Where A is the absorbance value of the formed complex in the presence of dye concentration C L ; A max is the maximum absorbance value in the presence of excess dye concentration and n is stoichiometric ratio (drug:dye). Logarithmic formation constant is summarized in Table 1.

Stoichiometric Relationship
Under the optimum conditions, the stoichiometry of the reaction between benzimdazole derivatives RPZ or OPZ and NQS was investigated by Job's method of continuous variation [40]. The stoichiometric ratio between NQS and drug was found to be 1:1 (Fig. 5).

[RPZ]+[NQS] = 2×10 -3 M and [OPZ]+[NQS] = 2×10 -3 M.
Based on this ratio, and the presence of only one center (N-H group) in RPZ or OPZ molecule that is available for the nucleophilic substitution reaction, the reaction pathway was postulated to be proceeded as shown in Fig. 6. The formation constant  n of the formed complex is calculated using the data of the continuous variation method by applying Harvey and Manning method [41]: Where A is the absorbance value of the formed complex in the presence of dye concentration C L ; A max is the maximum absorbance value in the presence of excess dye concentration and n is stoichiometric ratio (drug:dye). Logarithmic formation constant is summarized in Table 1.

Stoichiometric Relationship
Under the optimum conditions, the stoichiometry of the reaction between benzimdazole derivatives RPZ or OPZ and NQS was investigated by Job's method of continuous variation [40]. The stoichiometric ratio between NQS and drug was found to be 1:1 (Fig. 5).

[RPZ]+[NQS] = 2×10 -3 M and [OPZ]+[NQS] = 2×10 -3 M.
Based on this ratio, and the presence of only one center (N-H group) in RPZ or OPZ molecule that is available for the nucleophilic substitution reaction, the reaction pathway was postulated to be proceeded as shown in Fig. 6


Where A is the absorbance value of the formed complex in the presence of dye concentration C L ; A max is the maximum absorbance value in the presence of excess dye concentration and n is stoichiometric ratio (drug:dye). Logarithmic formation constant is summarized in Table 1.

Linearity, limits of detection and quantification
In order to test whether the colored species formed in the above methods, adhere to Beer's law the absorbance at appropriate wave lengths of a set of solutions containing varying amounts of RPZ or OPZ and a specified amount of reagent (as given in the recommended procedures for each drug) in aqueous alkaline medium were recorded against the corresponding reagent blank. The Beer's law plots of these systems are recorded graphically. Beer's law range, molar absorptivity, Sandell's sensitivity and Ringbom optimum concentration range for RPZ and OPZ were calculated. Least square regression analysis was carried out for getting the slope, intercept and correlation coefficient values. The results are summarized in Table 1. In the proposed methods, linear plots with good correlation coefficients (more than 0.999) were obtained in the concentration ranges of 0.26-12.0 and 0.49-12.0 μg mL −1 for the RPZ and OPZ, respectively ( Table 1). The minimum level at which the investigated compound can be reliably detected (limit of detection LOD) and quantified (limit of quantification, LOQ) were determined experimentally. The detection limit for the proposed method was calculated by using the following relationship [42]: Where n is the number of the samples; b the slope of line of regression; t is the student's tvalue at 95% confidence level; [43] (A is the experimental value of absorbance; A calc is the absorbance value calculated from the regression equation).
Limits of detection for RPZ and OPZ were found to be 0.181 and 0.187 µg mL -1 , respectively. The limit of quantification (LOQ) was determined as the lowest concentration of investigated compound used in the construction of the corresponding standard curve and defined as 0.26 and 0.49 µg mL -1 for RPZ and OPZ, respectively.

Accuracy and precision
The accuracy and precision of the proposed methods were carried out by six determinations at different concentrations and compared with the official methods for RPZ [36] and OPZ [37]. Percentage relative standard deviation (RSD%) as precision and percentage relative error (Er%) as accuracy of the suggested and official methods were calculated. Table 2 shows the values of RSD% Er% for different concentrations of the drugs determined from the calibration curves. These results are of accuracy and precision show that the proposed method has good repeatability and reproducibility. The values of t-and F-tests obtained at 95% confidence level and five degrees of freedom [44] did not exceed the theoretical tabulated value indicating no significant difference between the methods compared. The proposed method was found to be selective for the estimation of drug in the presence of various tablet excipients. For this purpose, a powder blend using typical tablet excipients was prepared along with the drug and then analyzed. The recoveries were not affected by the excipients and the excipients blend did not show any absorption in the range of analysis.

Application to the Pharmaceutical Dosage Forms
The performance of the proposed methods was assessed by comparison with the reference methods for RPZ [36] and OPZ [37]. Mean values were obtained with a Student's t-and Ftests at 95% confidence limits for four degrees of freedom [44]. The results showed comparable accuracy (t-test) and precision (F-test), since the calculated values of t-and Ftests were less than the theoretical data. The proposed procedures were applied to determine the studied drugs in their pharmaceutical formulations. The results in Table 3 indicate the high accuracy and precision. As can be seen from Table 3, the proposed method has the advantages of being virtually free from interferences by excipients such as glucose, lactose, and starch or from common degradation products. The results obtained were compared statistically by the student's t-test (for accuracy) and the variance ratio F-test (for precision) with those obtained by the reference methods on samples of the same batch ( Table 3). The values of t-and F-tests obtained at 95% confidence level and five degrees of freedom did not exceed the theoretical tabulated value indicating no significant difference between the methods compared. analytical performance and devoid from any potential interference. This gives the advantage of flexibility in performing the analysis on any available instrument. Therefore, this method can be recommended for the routine analysis of RPZ and OPZ in quality control laboratories.