Spectrophotometric Assay of Metoclopramide Hydrochloride in Pharmaceutical Preparations via Arsenazo III-Cerium (III) Reaction

A simple, rapid, accurate and precise spectrophotometric method is proposed for determining metoclopramide – hydrochloride (MCPH) in pure form and in pharmaceutical preparations .The method is based on oxidation reduction reaction between metoclopramide hydrochloride and cerium (IV) ion, then the subsequent reaction of cerium (III) and arsenazo III reagent in acidic medium to produce a blue-violet complex which is stable, water-soluble and has a maximum absorption at 654nm with a molar absorptivity of 9.36x10 l.mol.cm and Sandell's sensitivity of 0.0038 μg.cm. Beer's law is obeyed in concentration range from 1-30 μg metoclopramide hydrochloride in final volume of 25 ml, (0.04-1.2) ppm with a relative standard deviation (RSD) ( ±1.80% ±3.98%) and the limit of detection (LOD) is 0.0095 μg/ml and the limit of quantitation (LOQ) is 0.0317 μg/ml . The method is suitable for the determination of metoclopramide hydrochloride in the presence of other ingredients that are usually present in dosage forms and the recoveries are obtained in the range of 97.6-101.0 % . The proposed method has been applied successfully to the determination of the intended compound in its pharmaceutical preparations ( tablet and injection).

Metoclopramide was used as a treatment of nausea and vomiting in association with migraine and severe headache. In this setting, the combination of metoclopramide with an analgesic proved to be very efficacious with a short delay of action. The drug was used for the control of sickness due to radiation therapy and chemotherapy, and for the prevention and treatment of postoperative nausea and vomiting (PONV). Metoclopramide also has a gastrointestinal prokinetic effect through cholinergic stimulation.This effect consists of an increased tension in the lower esophageal sphincter and the gastric fundus, an increase in gastric and small intestinal motility, and a relaxation of the pylorus and duodenum during contraction of the stomach. The gastro-kinetic effect is mediated by an antagonism at the D 2 and 5-HT 3 receptors and by activation of the 5-HT 4 receptor ( Donnerer, 2003).
The chromatographic methods are a lot cost, they are consuming of time and limiting of application and also the flow-injection chemiluminescent are often typically less sensitive and have their own intrinsic disadvantages such as technical complexity or require an expensive instrumentation (Al-Arfaj, 2004).
The aim of the present work is to develop a simple, sensitive, specific spectrophotometric method for the determination of MCPH in bulk as well as pharmaceutical formulation.

Apparatus
All spectral and absorbance measurements were performed on double-beam Shimadzu UV-Visible-160 recording spectrophotometer with 1.0 cm matched glass cells. pH measurements were preformed using HANNA 301 pH meter.

Reagents
All chemicals used were of analytical grade reagents.
Standard solution of MCPH (100 µg / ml). It was prepared by dissolving 0.0100 g of MCPH (N.D.I) in distilled water and the volume was completed to 100 ml with distilled water in a volumetric flask. The solution was then transferred to a dark bottle and is stable for at least one month.
Working solution of MCPH (25µg/ ml). It was prepared by the appropriate dilution of the stock solution with distilled water.

Ammonium ceric sulphate dihydrate [cerium(IV)ion solution],(4x10 -4 M).
This solution has been prepared by dissolving 0.0253g of ammonium ceric sulphate dihydrate in sulphuric acid (0.05N) and the volume was completed to 100 ml with sulphuric acid (0.05N) in a volumetric flask.
Sulphuric acid solution,(0.05N).This solution is prepared by dilution of 1.4 ml of the concentrated sulphuric acid (35.5 N) solution to 1000 ml with distilled water in a volumetric flask.
Sodium hydroxide solution (1M).This solution is prepared by appropriate dilution of the concentrated volumetric (Fluka) solution with distilled water to 1000 ml in a volumetric flask and then transferred to a plastic bottle.
Sodium hydroxide solution (0.01M). This solution is prepared by dilution of 1.0 ml of the concentrated NaOH (1M) solution to 100 ml with distilled water in a volumetric flask.

Recommended procedure and calibration curve
An aliquot of standard solution (1-50) µg of MCPH was transferred into a series of 25ml volumetric flasks. To each flask, 2.5 ml of sodium hydroxide (0.01M) solution and 0.5 ml ammonium ceric sulphate dihydrate solution were added. The reaction mixture was allowed to stand for 20 min after mixing thoroughly. A 0.7 ml of arsenazo III was added and the contents were diluted to the mark with distilled water and mixed well. The absorbance of the formed coloure (blue-violet) was measured at 654 nm against the corresponding reagent blank. A linear calibration graph was obtained over the concentration range of (1-30) µg of MCPH /25ml,(0.04-1.2) ppm. Higher concentrations show a negative deviation from Beer's law (Fig.1). The apparent molar absorptivity has been found to be 9.36×10 4 l.mol -1 .cm -1 and Sandell's sensitivity is 0.0038 µg.cm -2 and the limit of detection (LOD) is 0.0095 µg/ml and the limit of quantitation (LOQ) is 0.0317 µg/ml.

Procedure for dosage forms
For tablets: at least ten tablets (5 mg MCPH /tablet ) of the drug were weighed, powdered and mixed well. A portion equivalent to 0.01 g of MCPH was weighed and dissolved in 100 ml of distilled water, shaken well, filtered and diluted with water to 100 ml in a volumetric flask. An aliquot of the diluted drug solution was then treated as done in a recommended procedure.
For injection: the content of the container of MCPH injection (10 mg MCPH /2 ml) was mixed well and transferred into 100 ml volumetric flask and completed to the mark with distilled water. An aliquot of the diluted drug solution was then treated as done in recommended procedure.

The Principle of colour reaction
Under the reaction conditions, metoclopramide hydrochloride (MCPH) was treated with ammonium ceric sulphate dihydrate in acidic medium, which undergoes oxidationreduction reaction to give Ce (III) ions. The cerium III formed was quantitatively and rapidly converted into the stable arsenazo III -Ce (III) coulered complex which exhibited absorption maxima at 654 nm against reagent blank solution. The intensity of the formed complex has been found to be proportional to the amount of MCPH originally present in solution.

Optimization of variables
The effect of various parameters on the absorption intensity of the coloured complex is studied and the reaction conditions have been optimized. The preliminary investigation showed that the complex formed have a maximum absorption at 654 nm against reagent blank.

Effect of pH
The effect of pH on the intensity of the coloured complex is examined. Different volumes (0.5-3.5 ml) of 0.01M sodium hydroxide are added to an aliquot of solution containing 25 µg MCPH/25 ml. The absorbances are read against the reagent blank. The results are shown in The results shown in Table 1 indicate that the pH of 3.36 (pH≈3.4) is considered optimum. A pH 3.4 is selected for subsequent investigation as it gives maximum absorbance for the coloured product. Four buffer solutions of pH 3.4 with different compositions have: tartaric acid-NaOH (B 1 ), citric acid-NaOH (B 2 ), KH Phthalate-HCl (B 3 ), and glycine-HCl (B 4 ) ( Perrin and Dempsey, 1974).The results indicate that all types of buffer solutions decrease the intensity and stability of absorption of the coloured complex, so the use of buffer solutions is not recommended. A 2.5 ml of 0.01M NaOH solution has been recommended for subsequent experiments.

Effect of oxidizing agent [cerium(IV) ion] amount
Different amounts of cerium(IV) ion solution are added to 2.5-30 µg of MCPH/25ml and the optimum amount which gives higher intensity of coloured complex and higher value of correlation coefficient (Table 2) has been selected The results shown in Table 2 indicate that the volume of 0.5 ml of 4x10 -4 M cerium (IV) ion solution gives good correlation coefficient and the lower blank value(0.106) compared with 1.5 ml of the oxidant gives (0.289) absorbance value.

Effect of reaction time
The effect of time needed for the complete reduction of cerium (IV) to cerium (III) ion is studied by standing of the solutions after adding cerium (IV) ion solutions for different times, then the reagent is added and the absorbance are measured against the reagent blank and the results indicate that a complete reduction of cerium (IV) ion occurred after 20 minutes. Therefore, the time (20 minutes) is recommended for the subsequent experiments.

Effect of arsenazo III reagent amount
The effect of the amount of arsenazo III reagent on the maximum formation of the coloured complex is investigated for 2.5-30 µg of MCPH/25ml. The results are shown in Table 3. The results shown in Table 3 indicate that 0.7 ml of arsenazo III reagent solution gives the good sensitivity and good value of correlation coefficient, therefore it has been selected for subsequent experiments.

Effect of surfactant
The effect of surfactant was studied by the addition of 3 ml of various types of surfactant (positive, negative and neutral charged) to the medium of reaction with different orders of addition. The results are shown in Table 4. The results in Table 4 indicate that all types of surfactants solutions decrease the intensity of absorption of the coloured complex, so that the use of surfactants solutions is not recommended.

Effect of time
The effect of time on the development and stability of coloured complex for different amounts of MCPH is investigated under the optimum experimental conditions established. Complete colour formation occurs immediately after all reaction mixtures are added and the absorbance of the complex remains constant for at least 60 minutes (Table 5)  The results shown in Table 5 indicate that the stability period is sufficient to allow several measurements to be performed sequentially.

Absorption spectra
Absorption spectra of the coloured complex formed from the reaction between cerium III ion with arsenazo III reagent in acidic medium against its corresponding reagent blank show maximum absorption at 654 nm in contrast to the arsenazo III reagent blank which shows a weak absorption at the same wavelength (Fig.2).

Accuracy and precision
To check the accuracy and precision, MCPH was determined at three different concentrations. The results are shown in Table 6 indicate that the method is satisfactory.

Nature of the reaction between MCPH and cerium IV ion
Job's method has been used in the determination of the reaction ratio of MCPH with cerium(IV) ion. The obtained results in (Fig.3) showed that a1:1 MCPH to cerium IV ion ratio is obtained.

Fig. 3 : Job's plot for MCPH-Cerium(IV) ion
The probable mechanism of the reaction may be according to the following :

Nature of arsenazo III-cerium (III) ion complex
The stoichiometry of the reaction is investigated using the Job's and mole ratio methods (Delevie,1997) under the optimized conditions. The obtained results showed that a 1:1 arsenazo III to cerium (III) ion ratio is obtained, this result is identical with that in the literature (Sandell and Onishi, 1978) The probable mechanism of the reaction might be as the following: or

Effect of interferences
In order to test the efficiency and selectivity of the proposed method , the effect of some foreign substances (e.g., acacia, glucose, lactose, sorbitol, and starch) that usually present in dosage forms were studied by adding different amounts of foreign substances to 25 µg MCPH / 25 ml. It was observed that the studied foreign species did not interfere in the present method (Table 7).

Application of the method
The proposed method was successfully applied to the determination of MCPH in its pharmaceutical preparations (tablet and injection). The results which are shown in Table 8 indicate that good recoveries were obtained. The performance of the proposed method was a ssessed by calculating the student's ttest and F-test (Christian, 2004) compared with the literature method (Khalil, 2010). The results in Table 9 show that the calculated values of t-test and F-test did not exceed the theoretical values at the 95% confidence indicating that there is no significant difference between the proposed method and the literature method.  Table 10, shows the comparison between some of the analytical variables for the present method with that of other literature spectrophotometric methods. The results indicate that the proposed method has a good sensitivity and has a wide application part in determination of the drug under investigation in its pharmaceutical preparations.

CONCLUSION
The proposed method for the spectrophotometric determination of metoclopramidehydrochloride in pharmaceutical preparations is simple, sensitive, rapid, accurate and precise. The method is based on oxidation -reduction reaction between metoclopramide hydrochloride and cerium (IV) ion (ceric ion), then the subsequent reaction of cerium (III) with arsenazo III reagent in acidic medium to produce blue-violet complex which is stable, water soluble and has a maximum absorption at 654nm. the proposed method has been applied successfully to the determination of the intended compound in its pharmaceutical preparations (tablet and injection).