Oxidative Coupling Reaction for Micro Trace Analysis of Mebendazol Residual with p-bromoaniline in Presence of n- bromosuccinimide

Rapid, reproducible and accurate method has been developed for the assay for of mebendazol (MBZ) residual assay. The method is based on alkaline hydrolysis of MBZ with sodium hydroxide then oxidation with N-bromosuccinimide (NBS) followed by coupling with 4-Bromoaniline (4-BA) to yield a highly colored product absorbed at maximum 434 nm. Regression analysis of linearity range was found (0.6-2.8) μg.ml -1 . The optimum conditions that affect the oxidation were studied. The developed method was found to be precise with mean value of relative standard deviation (1.1531.303) and accurate with relative error (0.5940-1.7821) .The calculated molar absorptivity and sandal sensitivity values of (29825 L.mol -1 .cm -1 ), 0.0099 μg.cm -2 respectively. The limit of detection and limit of quantitation were of 0.04696, 0.156548 μg.ml -1 respectively .The suggested method showed good recovery with a mean value of 100.77% for analysis of dosage forms.


Introduction:
Mebendazol (MBZ) methyl 5-benzoylbenzimLdazole-2-carbamate is one of the anthelmintic drugs (Fig.1). The principle mode of action for Mebendazole is by its inhibitory effect on tubulin polymerization which results in the loss of cytoplasmic microtules.It is used to treat hookworm, pinworm, round worm and mixed of infections. MBZ is highly used to treat gastrointestinal helming in both humans and animals (1). Mebendazole causes degenerative changes in intestinal blood cells and worm intestinal cells by binding to the collagen-sensitive topline site, thus inhibiting polymerization or aggregation in microtubules. The loss of microtubules, cytoplasm, impairs the absorption of glucose in the larval and adult stages of sensitive parasites and depletes the glycogen stock. It is available in tablet and suspension form. Depending on the type of worm to be treated, the dosage varies in adults and children (2).
Various analytical method is found for the assay of MBZ in different formulation dosage including UV-visible spectrometry (3-7), highperformance liquid chromatography (8-11), selective membrane sensor (12)(13)(14) and potentiometric analysis (14). The present work stand on oxidation of hydrolyzed MBZ in the presence of NBS then coupling with 4-BA to get a paled orang complex absorbed at 434 nm at pH 9.

Reagents
All chemicals used are of highest purity available. MBZ pharmaceutical -grade was gifted from" State Company for Drug Industries and Medical Appliances Samara-Iraq (SDI)" Stock Solution of Mebendazol (100 µg.mL -1 ) 0.05 g of MBZ was dissolved with 10 mL 1 M NaOH and the volume was adjusted to 500 mL to the mark with distilled water in 500 mL volumetric flask.

4-Bromo Aniline (5.8× 10 -3 M)
Reagent solution was prepared by dissolving 0.1 g from 4-BA in 10 ml of methanol then completed the volume to the mark with distilled water in 100 ml volumetric flask. N-Bromosuccinimide Solution (1.1× 10 -2 M) 0.1 g from NBS was dissolved with distilled water then complete the volume to the 50 mL in a volumetric flask.. Sodium Hydroxide Solution (1M) 4 g was weighted from NaOH to prepare 1 M in 100 ml volumetric flask. MBZ Syrup Formulation (100 µg.mL -1 ) This solution was prepared by taking0.5 ml from each: Veromx (20 mg.ml -1 Belgium) and, mebandazol S-awa (20 mg.ml -1 Iraq) dissolved with 10 ml of 1 M NaOH and complete the volume in 100 ml volumetric flask.

Recommended Procedure:
In 5 ml volumetric flask different aliquots (0.6-2.8) µg.ml -1 from MBZ were added then 0.2 ml from 5.8× 10 -3 M 4-NBA followed by 0.3 ml from1.1× 0 -2 M NBS was added and the solutions were left for about 5 min in 15 °c then dilute it to the mark with methanol and recorded the absorbance of the colored complex at 434 nm against blank solution.

Procedures for Pharmaceutical Preparations (Suspensions):
To apply the suggested spectral method to pharmaceutical preparations, equivalent volumes of (1, 1.6, 2) µg.ml -1 of MBZ commercial were transferred to 5 ml volumetric flasks and supplemented by applying optimal conditions.

Spectral Characteristics
The procedure contain basic hydrolysis of MBZ followed by the addition of 4-BA and the oxidation reagent to form orange color derivatives having λmax at 434 nm. The other reagent blank have practically zero absorbance at this wavelength as shown in Fig. 2. The formation of a color complex with the reagents was shown in Scheme 1. Alkaline hydrolysis of BNZ yields primary amino group with a bright yellow color solution.
The oxidation and coupling reaction was studied at different pH media, pH 9 was suitable for best absorption values at 434nm (Fig.3).For complete development procedure it was found that best volume of 4-BA is 0.2 mL from (5.8× 10 -3 M) (Fig.4) , the optimum time to complete oxidation is 5 min (Fig.5), with 0.3 ml of (1.1× 10 -2 M )NBS (Fig. 6 ) at 15°C. To study the effect of the order of addition for reagents to obtain the best results, it was clear from the study that the order of mentioned method gave the best results (drug, reagent and oxidative reagent) (Fig.7), otherwise the solution was colorless. The study investigated different solvents to dilute the solutions, methanol appear a suitable for color development

Calibration and Analytical Data
Applying the best condition on the work to investigate calibration and optical characteristics for different concentration (0.6-2.8) µg.ml -1 (A) versus absorbance was done and the residual plot showed random distribution of error and no presence of systematic error (B), (C) represent the comparison between real and predicated absorbance value as showed in Fig. 8, the statistical data can be seen in Table 1.

The Nature of the Colored Product
The stoichiometry of drug with the reagent was studied by Job's method. The method required to prepare same concentration (1.700×10 -3 M) for each standard MBZ and 4-BA reagent solution. In Job's method, a series of volumetric flasks (5 ml), different volumes of the drug solution ranging from (1-9) mL and (9-1) mL of reagent solution were mixed. A 0.3ml of (NBS) (1×10 -2 M) were added and volumes are completed to the mark with methanol. The absorbance was measured at 434 nm against the blank. The results in Fig. 9 shows that the ratio is 1:2.

Precision and Accuracy of the Suggested Method
The accuracy and precision value of the MBZ were evaluated by performing five replicate to three concentrations within the calibration curve to measure both relative error and standard deviation for both accuracy and precision respectively the result listed in Table 2 .

Interference.
Tables 3shows the effect of some interferences substances when added to the drug solution and its effect is measured by the absorption value of the drug solutions have 2 μg / mL of MBZ and different quantity of different species in a final volume of 5 ml.  It is noted from this study that there is a large overlap shown by the citrate group (magnesium citrate, sodium citrate) and stearic acid in determination of MBZ because of the incompatibility between the active substance of the drug and this group, it causes increasing the dissolution of the drug, so it is preferable not to add it to the drug as a lubricating agents (added to improve manufacturability of the drug products) therefore it was neglected. Also it shows in table3 an overlap of the substance of the starch and all sugars in its high concentrations, this has been confirmed by previous studies. (15)

Analytical Application of Dosage Forms of MBZ
The suggested method was examined to quantitative analysis of MBZ, using the selected experimental conditions. In this method, different concentration (1, 1.6, 2) µg.ml -1 of a dosage formulation solution (20mg.ml -1 ) were spiked to 5ml volumetric flasks and were treated similar to working range. Five times replicate was done to measure absorbance at 434nm. RSD and Recovery were estimated and the results are shown in Table (4). Estimation of 1ppm of MBZ in dosage forms was applied with standard addition as it appear in Fig.10 and 11 with recovery of 90%.

Conclusions:
This study was done to estimate MBZ in pure and dosage forms. This method is considered simple, sensitive, selective and rapid. The process deals with alkaline hydrolysis of MBZ drug then oxidative coupling reaction takes place with a 4-NBA in presence of NBS. The proposed method showed that it is more appropriate for estimating the low concentration of MBZ, while in high concentrations, less sensitivity it obtained because the interaction between the reagent and oxidized agent. The method is applied successfully in pharmaceuticals dosage and good recovery with high compatibility and accuracy values.