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The present work was to develop a validated a new spectrophotometric method for the determination of Promethazine based on a charge transfer complexation reaction of heterocyclic ring containing drug. In this method, the oxidation of Promethazine was done by a known excess amount of cerium IV sulphate in acidic medium, the unreacted cerium IV sulphate was treated with Iron II Sulphate. After 5 min the resultant Iron III Sulphate solution was treated with Ammonium thiocyanate, immediately it forms blood red colour of Iron III Sulphate-thiocyanate drug complex. This blood red coloured complex formed under standardized conditions was measured at 480 nm. Beer's law was obeyed for 10-40 µg / ml-2 of Promethazine. Results of analysis were validated statistically and by recovery studies. The procedures described were successfully applied for the determination of Promethazine in bulk drugs and in tablet formulations.
Key Words: Promethazine, cerium IV sulphate, Iron III Sulphate, Ammonium thiocyanate.
Promethazine is chemically N, N - dimethyl - 1 -phenotheazin - 1 ayl - propam - 2 - amine. It is freely soluble in water whose structure given in Fig. 1. It is used in the treatment of Psychiatric diseases.
Survey of Literature revealed that various methods were reported for the estimation of Promethazine in pharmaceutical formulations. This drug can be analyzed based on solid-phase extraction (SPE) and UV Spectrophotometry and simultaneous determination of promethazine, chloromazine and perphenazine by multivasiate calibration methods. But no attempts have been reported in determining promethazine via oxidative complaining reaction.
The present investigation was under taken with the aim of developing new, simple, rapid and accurate Method. This spectrophotometric method based on a charge transfer complexation reaction.
MATERIALS AND METHODS
A Specronics 1001 Spectrophotometer with 10 mm Matched quartz cuvettes used for absorbance values of the drug solution.
All chemicals were of analytical reagent grade. Double distilled water was used through out the investigation.
Standard solution of Promethazine solution: An accurately weighted 50 mg of Promethazine was dissolved in double distilled water. Then this drug solution was made up to the mask in the 50 ml standard flask with double distilled water, to give a standard working solution.
0.05M Cerric Ammonium Sulphate: 2.9826 g of AR Cerric Ammonium Sulphate was dissolved in double distilled water and further diluting to 100 ml with double distilled water.
0.02M Ammonium Ferrous Sulphate Solution:
0.7842 g of A.R.Ammonium Ferrous Sulphate was dissolved in distilled water and diluting to 100 ml with water.
1M Ammonium thiocyanatic Solution: A 1M Ammonium thiocyanate solution was prepared by dissolving '7' g of the reagent in water and diluting to 100 ml with water.
5N Hydrochloric acid solution: Hydrochloric acid solution (5N) was prepared by diluting the requisite volume of concentrated AR hydrochloric acid (Ranbaxy make) with double distilled water i.e., 45.45 ml of concentrated Ar hydrochloric acid AR, and diluting 100 ml of double distilled water.
Spectrophotometry: The estimation of chloro-promazine by cerimetric method. In this method is based on the oxidation of the drug by a known excess amount of cerric IV Sulphate in acid and oxidation product. When unreacted ce IV sulphate, it oxidized from iron II sulphate to Iron III sulphate After 5 minutes the iron III sulphate solution was treated with (1M) ammonium thiocyanate, immediately it forms blood-red coloured complex. i.e. Iron III sulphate - thiocyanate complex solution. This sample solution was measured at 480 nm against a blank solution. The blank were prepared for this study, the reagent blank containing optimum concentrations of the reagents expect drug.
The absorbance was found to decrease linearly with increasing concentrations of Promethazine and this forms the basis for the determination of drug. Finally the estimation of the drug was made through the calibration curve.
Reaction Scheme showing formation of measured colour:
Proposed assay procedure
A Series of 25 ml of volumetric flasks 1 ml, 2ml, 3ml and 4ml of the working standard solution of the drug was pipetted into each flask. 1ml of 0.05N carric ammonium sulphates IV and 1 ml of 5N Hydrochloric acid solution and requisite volume of double distilled water were added. The flasks were let stand for 5 min with occasional shaking, subsequently 1 ml of 0.02N ammonium ferrous sulphate was added to each flask and the contents were mixed well, unreacted CeIV Sulphate converts Iron II to Iron III. Then after 5 min this solution was treated with 3 ml of 1M ammonium thiocyanate solution. It forms blood-red coloured solution of Iron III sulphate thiocyanate complex. The absorbance of the blood-red coloured solution in each flask was measured at 480 nm by using spectrophotometer against a blank solution. The Absorption Spectrum of Promethazine was represented in fig. 2. The Promethazine curve was obtained by plotting absorbance values against the amount of standard drug. The amount of Promethazine present in the sample was computed from the calibration curve were represented in fig. 3 and the result was given in Table 1.
Validation of the Method: This method was validated in terms of linearity, accuracy, precision, specificity and reproducibility of the sample applications. The linearity of this method was investigated by serially diluting the stock solutions of Promethazine and measured the absorbance value at 480 nm by Spectronics 1001 spectrophotometer. Calibration curves were constructed by plotting the absorbance difference values against the amount of drug in µg / ml.
Statistical analysis: A Statistical analysis was performed on the statistically significant variables using the statistical software and coefficient of variation and student t-test was determined.
The Standard deviation and ttest and coefficient of variation of the Promethazine were calculated from five measurements of replicate samples. The values of standard deviation and coefficient variation, tcal were shown Table 3.
The values of standard deviation and coefficient of variation were low, indicated high accuracy and reproducibility of this method. The data of assay values of commercial formulations was subjected to statistical evaluation for student 't' test to study the proposed method. The calculated 't' values were less than 't' theoretical values with 4 (n-1=5-1) degrees of freedom at 5% level of significance indicate that there is no significant difference between proposed method and standard method.
RESULTS AND DISCUSSION
The present study was carried out to develop a simple, rapid, sensitive, precise, reproducible and accurate spectrophotometric method for the estimation of Promethazine in pharmaceutical dosage forms.
In this method is based on the oxidation of drug by a known excess amount of cerric IV sulphate in acidic medium, it produced unreacted CeIV sulphate and oxidation product. When unreacted CeIV Sulphate was treated with Ammonium Ferrous Sulphate, it oxidized form Iron II Sulphate to Iron III Sulphate. After 5 min the Iron III Sulphate solution was treated with 1 M ammonium thiocyanate immediately it form blood-red coloured Iron III Sulphate thiocyanate complex and measured at 480 nm against a blank solution. The standard deviation, coefficient variation and tcal of the Promethazine were calculated from five measurements of replicate samples. The values of standard deviation and coefficient of variations are low, indicates high accuracy and reproducibility of this method. The recovery values of tablets were shown in Table 2.
The data of assay values of commercial formulations was subjected to statistical evolution for student 't' test to study the proposed method. The calculated 't' values are less than 't' theoretical values with 4(n-1=5-1) degrees of freedom at 5% level of significance indicate that there is no significant difference between proposed method and standard method.
Table 1: spectral data for calibration curve
Amount of Drug (µg)
Table 2: The recovery values of tablets
Table 3: Statistical analysis of the Promethazine in tablets
* Average of five determinations based on label claim
ttab= Tabulated Value; tcal= Calculated Value