Spectrophotometric Determination of Chlorpromazine Hydrochloride Using 4-Nitroanilline by Oxidative Coupling Reaction

A simple, rapid spectrophotometric method has been established for the determination of chlorpromazine hydrochloride (CPZ) in its pure form and in a tablet formulations. The  suggested  method  is  based  on  the  oxidative coupling  reaction  with4-nitroainlline using KIO3 in acidic solution to produce a violet colored product with maximum absorption at λ=526 nm.The  analytical data  obtained  throughout  this  study  could  be  summarid  as  follows:  1ml of 1M HCl (pH=2.2), 1 ml  of  4-nitroanilline (1x10-2M), and 1.5ml  of (1x10-2)KIO3 per 25 ml reaction medium. The order of additions, coupling reaction time, and temperature in addition to the type of solvent were studied. 
The Beer′s law is obeyed over the concentration range of(5–40) µg ml-1, but the detection limit and quantification limit are 0.34 besides 1.03 µg ml-1 respectively. The correlation coefficient (r) for the calibration graph was found to be 0.9980, molar absorptivity of 10. 25 × 103 L.mol -1.cm-1, and Sandell′s sensitivity index of 0.03467 µg.cm-2. The precision and accuracy of the method were tested by calculating the percentage of relative standard deviation (RSD%) (<1.831%) and the average recovery percent (99.22%) average error percent Erel%(0.558). 
Direct and standard addition procedures were applied to both standards and specimens of pharmaceutical and the results indicate that the suggested method was successfully applied for the determination of CPZ.

In this work, a study of the spectrophotometric determination of CPZ-HCl relying on the oxidative coupling of the drug with 4-nitroaniline using potassium iodate as oxidation reagent in acidic medium.

Materials and Methods:
T90 UV-Visible Spectrophotometer PG Instrumental Ltd, UK with 10 mm quartz cell was used for all spectrophotometric quantities, Jenway 3310 pH meter was used to check the pH of solutions, and Sartorius Balance 210S kern was used to perform all weight measurements.

Reagents
Fully chemicals used were of analytical grade and chlorpromazine.HCl typical material was provided from State Company for Drug Industries and Medical Appliance (SDI) Samarra-Iraq.Distilled water was used to prepare all solutions.
A 1000 µg/ml (2.814 × 10 -3 M))CPZ of solution was prepared by dissolving 0.100gm in100ml distilled water and stored in dark and used, for at least one month, as stock solution.More dilute working solutions of the drug were prepared by serial dilutions with distilled water.4-nitoanilline solution (1.000 × 10 -2 M ) was freshly ready daily by dissolving0.0345gm in 5ml ethanol and completed by distilled water to 25ml , while the calculated weight of KIO 3 0.2140gm dissolved in 100ml distilled water was required to prepare (1.000 × 10 -2 M) solution.1.000 × 10 -1 M solution of each of sodium hydroxide and hydrochloric acid was prepared and used.

General procedure
In a 25 ml volumetric flask, 1 ml of 1.000 × 10 -2 M 4-nitroanilline (R) solution is mixed with 1.500 ml of 1.000x10 -2 M KIO 3 .To this mixture 3 ml aliquot of CPZ solution is added and the mixture is rendered acidic by the addition of 1. 000ml of 1.000 M HCl (11.8N).The mixture is then diluted and the value of absorbance is measured at 526 nm after letting the mixture to stand for 15 minutes in the dark.

Application to various dosage forms
A portion equivalent to 0.1075 gm of a homogenized powder of 10 tablets Largactil (Aubrey Pharmaceutical Industries -Aleppo/Syria with distinction from the company Avnitis -France 25mg/tablet) was weighed and dissolved in 100 ml water.Dissolution of the drug was assisted by means of magnetic stirring and an ultrasonic bath.The resulted solution was cleaned through a Whatman filter paper No.1.and the volume was made up to the dent with distilled water in 100ml calibrated flask.The drug gratified of an aliquot of this solution was found by applying the general procedure as pronounced above.

Results and Discussion:
When CPZ was treated according to the recommended procedure, the recorded absorption spectrum of the formed reaction product for the range of 450 to 600 nm against reagent blank shows an extreme absorption at 526 nm, while, the blank does not have any significant absorbance in this region, as it is shown in (Fig. 2).

Optimization of reaction conditions:
The effect of the amount of HCl, 4-nitroanilline, and potassium periodate was investigated.The results show that use of 1.000 ml of 1.000 M hydrochloric acid (i.e.pH=2.2) with 1.500ml of 1.000 × 10 -2 M of KIO 3 , and 1ml of 1.000 × 10 -2 M of coupling reagent give best results (Fig. 3 a, b and c respectively).The order of addition of the reactants should be followed, as cited in the recommended procedure.Moreover, optimum time for full color development was found to be 10minutes, and room temperature15C ο was found the most favorable for the color development of the reaction product, Fig. 4 (a and b respectively).Furthermore, water was found to be the best solvent among different solvents (viz: water, ethanol, acetone and diethyl ether) tried to solvate the reaction medium and to obtain the perfect absorbance, Fig. 5.

Calibration graph and the statistical data
Using the obtained optimum conditions, a calibration curve was built (Fig. 6).The graph shows that the violet colored product obeys Beer's law in the range of concentration of 5 -40 μg/ml of CPZ.Table 1.depicts the statistical information of the calibration curve of spectrophotometric determination of CPZ.

precision and Accuracy
The capability of the method was statistically evaluated via measuring accuracy as relative error percentage (E rel %), and precision as relative standard deviation percent of the proposed methods.Table 2 illustrates that the results obtained for seven replicates at three concentration levels of CPZ were satisfactory which indicate that the proposed methods have a good precision and accuracy.

Table2. Evaluation of precision and accuracy for determination of CPZ .
*

Stoichiometry of reaction
The stoichiometry of the response between CPZ and the mixture was investigated using Job's method and mole-ratio method.The consequences obtained via the two methods indicate that the stoichiometry of the water-soluble coupling product between drug and the reagent is 1:1 (Fig. 7 and Fig. 8)(15).Accordingly, a mechanism for formation of the colored product upon the reaction between CPZ and reagent in presence KIO 3 is founded in scheme 1: Scheme 1.The suggested mechanism for formation of the colored product.

Analysis of CPZ in pharmaceutical preparations
Two procedures (direct calibration and standard additions) using the proposed method to determine CPZ in Largactil tablets.The obtained results are shown in Table 3 and in Fig. 9. Good agreement in results was found for both procedures.

Conclusion:
A rapid, simple and precise spectrophotometric method has been suggested for the determination of chlorpromazine hydrochloride in aqueous solution based on oxidation with 4-nitroanilline and KIO 3 in the acidic solution.The suggested method does not require temperature control or the solvent extraction step, the method was applied, successfully for the determined of amounts commercial CPZ drug.

Figure 1 .
Figure 1.The structure of chlorpromazine hydrochloride.

Figure 2 .
Figure 2. The absorption spectra of 30 µg/ml CPZ-R coupling product against reagent blank solution, 30 µg/ml CPZ -R coupling product against distilled water, and reagent blank solution against distilled water.

Figure 3 .
Figure 3.Effect of (a) HCl, (b) KIO 3 , and (c) 4nitroanaline on the on the formation of the colored product.

Figure 4 .
Figure 4. Effect of (a) coupling time, (b) Temperature, on the formation of the colored product.

Figure 6 .
Figure 6.Calibration curve for the determination of chlorpromazine hydrochloride under optimal condition.