Spectrophotometric Determination of Mebendazole using Diazotization Reaction and Coupling with m-Aminophenol Reagent

A simple and sensitive spectrophotometric method has been developed for the determination of mebendazole (MBZ) in it's pure and tablet form. The method is based on alkaline hydrolysis of MBZ with sodium hydroxide to give primary amine product which reacts with sodium nitrite in an acidic medium (hydrochloric acid) to yield diazotized mebendazole (D-MBZ) which is coupling with m-aminophenol reagent (mAP) to form an azo dye. The absorbance of the azo dye for this suggested method (yellow dye) has been measured at 360 nm. Beer's law for this proposed method is in the range of (0.5-20 μg.ml -1 ), the molar absorptivity value is 1.63×10 4 L.mol -1 .cm -1 and the Sandell's value index is calculated and equal to 0.018 μg.cm 2 . Also, the limit of detection and the limit of quantification are calculated and equal to 0.1718 μg.ml -1 and 0.5726 μg.ml -1 respectively. The ratio of the formed azo dye [D-MBZ: mAP] is [2: 1].


MATERIALS AND SOLUTIONS
All materials used in this proposed research are of high purity. 1-Stock solution of mebendazole (200 μg .ml -1 ) 0.05 g of (MBZ) mebendazole was dissolved with 10 ml of NaOH 1M (with heating at boiling point) and the acidity was adjusted to pH=7 with HCl and completed by distilled water in a volumetric flask to 250 ml.

2-Sodium nitrite (3.39×10 -3 M)
This solution was prepared by dissolving 0.0234 g of sodium nitrite in distilled water then transferred into 100 ml volumetric flasks and completed to the mark with distilled water.

3-Hydrochloric acid (1M)
It was prepared with transfer 8.4ml of concentrated HCl into 100 ml volumetric flask and then completed to 100 ml with distilled water. 4-Diazonium salt reagent (3.39 10 -4 M) D-MBZ solution was prepared by mixing 50 ml of MBZ (stock solution) with 10 ml of sodium nitrite and 1.5 ml of HCl (1M), after shaking well it has completed to the mark with distilled water. 5-m-Aminophenol reagent (0.1%) This m-aminophenol solution was prepared by transfer 0.1 g of m-aminophenol (mAP) into 10 ml of ethanol and completed to the mark with ethanol in 100 ml volumetric flask.

6-Potassium hydroxide (1M)
This solution was prepared by dissolving 5.61 g of KOH in distilled water in a volumetric flask and completed to 100 ml with distilled water. 7-Drug solution (Vermox 100 μg MBZ /ml) Three tablets of MBZ (Vermox 100 mg/tablet) (total weight 1.6992 g) were crushed together and mixed well then weighing 0.0566 g of powder and dissolved with 10 ml of NaOH 1M (with heating at boiling point for 5 minutes) and the acidity was adjusted to pH=7 with HCl then added 10 ml of sodium nitrite (3.39×10 -3 M) and 1.5 ml of HCl (1M) and completed to the mark of the volumetric flask (100 ml) by distilled water.

The Procedure and Calibration Curve:
An aliquot of a sample solution containing (0.05-2.0ml) of 100 µg.ml -1 diazotized MBZ is transferred into a series of 10 ml volumetric flask then 1.5 ml of (0.1% mAP) followed by 1ml of 1M KOH then diluted to the marks with distilled water and measured at 360 nm against blank solution and the results were as in the Fig. (2).

The Optimum Amount of Hydrochloric Acid:
Different amount of 1M HCL solution (0.5-2.0ml) has been studied. 1.5 ml of HCl was enough to obtain a maximum absorbance.

The Effect of Temperature on Diazonium Salt:
The effect of temperature was studied at a various temperature on the diazotization of MBZ (see the results in Table 1). From the previous results in (Table 1) the difference in absorbance is small between the different degrees of temperature so and for ease we had chosen room temperature in suffix experiments.

The Suitable Amount of Reagent (mAP):
The suitable amount of reagent was checked and gave the following results. According to the value of determination coefficient in (Table 2). 1.5 ml of the reagent was chosen as an optimum amount for the next experiments.

Selection of the Optimum Type and Amount of Base:
Many types of base has been checked to make certain of the maximum absorbance (Table 3). According to the results in (Table 3) potassium hydroxide was fixed in the next experiments. The optimum amount of KOH was tested also and it showed that 1 ml is a suitable amount according to the highest absorbance of azo dye and it in subsequent experiments (Table 4).

Order of Addition:
The sequence of additives were studied to choose the suitable sequences (Table 5).  From the previous results the order (I) has been chosen for the next experiments because of its high absorbance.

The stability of Azo Dye with Time:
Effect of time on the azo dye has been studied and the result showed that the dye is stable for at least 60 minutes and the result illustrated in (Table 6). Absorption spectra of a yellow-colored solution (Azo dye) was formed by the coupling of diazotized MBZ with mAP reagent in alkaline medium. This Azo dye has given a maximum absorbance at 360nm against blank solution as shown in the Fig. (3).

Accuracy and Precision:
In this study the accuracy and precision has been calculated through the measurements of recovery, percent relative error (RE%) and relative standard deviation (RSD%) values by performing five replicates to three concentrations within the calibration curve. The results listed in (Table 7).

Application of the Method:
The application of this suggested method has been performed on tablets of mebendazole pharmaceutical (Vermox 100 mg/tablet) through standard addition method by preparing two series of 10 ml volumetric flasks then added increasing amount (0-1.2ml) of standard D-MBZ (100µg/ml) for each one of these flasks and 0.25ml of drug solution (100µg/ml) for each flask of the first series flasks and 0.5ml of the drug for each of the second series flasks then the flasks proceed as mentioned in the standard and curved working method and measured at 360 nm then it gave the following plot and results.  The previous results are very compatible with manufactured drugs results with accepted analytical error.

Comparison of the Methods
The proposed method was compared to another method from literatures (Table 9). Through the results shown in (Table 9) we conclude that the proposed method includes high sensitivity value of MBZ in its drug sample (vermox tablet).

CONCLUSION
After studying the optimal conditions for the proposed method and after applying it to the vermox tablets, we conclude that the proposed method has a high degree of sensitivity in addition to good reproducibility, which confirms its success as a reliable method for the determination of mebendazole in its pure form as well as in the form of a medicinal preparation.