... of Pharmaceutical Chemistry, SAFA College of Pharmacy, Kurnool, Andhra .... Tripathi, Essentials of Medical Pharmacology, 6th ed, Jaypee Brothers Medical.
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Scholars Research Library Archives of Applied Science Research, 2011, 3 (1):328-332
(http://scholarsresearchlibrary.com/archive.html) ISSN 0975-508X CODEN (USA) AASRC9
Spectrophotometric determination and validation of Acyclovir S. Ashok Reddy1, Raja Chakraborty2*, Saikat Sen3, B. Parameshappa3 1
Department of Pharmaceutical Chemistry, SAFA College of Pharmacy, Kurnool, Andhra Pradesh 2 Department of Pharmaceutical Chemistry and 3Department of Pharmacology, C.E.S. College of Pharmacy, Kurnool, Andhra Pradesh ______________________________________________________________________________ ABSTRACT Acyclovir, an antiviral drug commonly used in the treatment of Herps Simplex virus and Herps Zoster virus infection. A simple and sensitive method for determination of Acyclovir has been developed. The method is based on formation of Schiff’s base by the reaction of an amine group and aldehyde group in acidic pH forming a yellow colored chromogen with absorption maxima 470 nm. Beer’s law limit found in range between 2-10 µg/mL. Intraday precision (RSD < 2%) and accuracy (98.8-101.5 ± 0.85%) for the developed method was evaluated. This method was successfully evaluated for the estimation of commercially available acyclovir. Key words: Acyclovir, Schiff’s Base, vanillin, Beer’s Law ______________________________________________________________________________ INTRODUCTION Acyclovir is antiviral drug having extremely selective and low in cytotoxicity. Acyclovir’s selective inhibition of viral DNA synthesis depends on interaction with HSV thymidine kinase and DNA polymerase. Cellular enzymes convert the acyclovir to acyclovir triphosphate, which is present in higher concentrations in HSV-infected than in uninfected cells and competes for endogenous deoxyguanosine triphosphate. Viral DNA polymerase is competitively inhibited by Acyclovir triphosphate. Acyclovir triphosphate also acts as a chain terminator when incorporated into viral DNA, because it lacks a 3a-hydroxyl group. Acyclovir causes suicide inactivation when the terminated DNA template containing acyclovir binds the viral DNA polymerase and leads to its irreversible inactivation [1,2]. Survey of literature shows different UV-Vis Spectrophotometric method has been developed to determine different antiviral drug i.e. valacyclovir, gancyclovir, famcyclovir [3,4,5]. Several methods have been developed for spectrophotometric evaluation of acyclovir. These methods are 328 Scholar Research Library
Raja Chakraborty et al Arch. Appl. Sci. Res., 2011, 3 (1):328-332 _____________________________________________________________________________ based on oxidation with either cerium ammonium sulfate (Method A) or potassium persulfate (Method B). The oxidation products in both methods are coupled with 3-methylbenzothiazolin 2one hydrazone, a deep blue color produce with a maximum absorption wavelength at 630 nm [6]. Another method was developed based on its oxidative coupling reaction with 3methylbenzothiazolin-2-one hydrazone (MBTH) in the presence of FeCl3 as an oxidant to produce deep-green colored species measurable at 616 nm [7]. In another method Acyclovir was reacted with copper (II) and cobalt (II) using borax or sodium pH 9 hydroxide buffers using nonaqueous medium using 1% pyridine in methanol, respectively. The formed complexes had absorbtion maxima at 290 nm [8]. The absorbance–concentration plot is linear over the range 20–200 µg/ml with minimum detectability of 1.06 µg/ml. In the present study, we succeeded in developing a novel reaction for sensitive and selective spectrophotometric determination of the acyclovir in bulk formulation. MATERIALS AND METHOD Apparatus Shimadzu UV-1700 UV/VIS spectrophotometer with 1 cm matched quartz cells was used for spectral and absorbance measurements. The entire chemicals used were of AR grade. Acyclovir was as a gift sample from Star Tech Labs. The commercially available tablets were procured from the local market. Freshly prepared 5N nitric acid, 0.1N hydrochloric acid and ethanolic solution of vanillin were used in the present investigation. Preparation of standard solution Aliquots of acyclovir 0.2-1.0 ml of stock solution (Concentration 100µg/ml) were transferred into a series of 10ml volumetric flask. To each flask 1.5 ml 1% w/v of ethanolic vanillin solution and 1.0 ml of 5 N nitric acid were added, the solution was heated on a boiling water bath for 10 min, cooled to room temperature. The volume of the solution was making up to 10ml by using 0.1 N hydrochloric acid. The concentration of the acyclovir estimated was 2-10µg/mL. The absorbance of the yellowed colored chromogen was measured at 470nm against reagent blank. . Figure 1 represents the absorption maximum of acyclovir at 470nm.
Figure 1: Determination of absorption maxima
329 Scholar Research Library
Raja Chakraborty et al Arch. Appl. Sci. Res., 2011, 3 (1):328-332 _____________________________________________________________________________ The reaction is depends on the concentration of nitric acid, optimum result was found at concentration of 5 N HNO3. Vanillin concentration also effect on the reaction 0.1% w/v was found to be ideal for the reaction. The yellow color only develop after heating the mixture at 70°C for 10 min. Methanol, ethanol, water, chloroform and carbon tetrachloride were tested for dissolving vanillin but highest reading was observed when ethanol was used for solubilizing and dilution. The stability of the chromogen was found for 48 hrs Application of the proposed procedure for the determination of dosage form Commercial tablets from different batches of brand were analyzed by the proposed methods. Twenty Acyclovir tablets taken each containing 200 mg was weighted and powdered by using mortar and pastel. Weigh of the tablet powder equivalent to 100mg of the drug was taken in 10 0ml volumetric flask. The solution was prepared and make up the volume by using 0.1 N hydrochloric acid. Further dilution was done to prepare stock solution concentration of 100 µg/ml by using 0.1 N hydrochloric acid. Aliquots of acyclovir concentration ranging from 2-10 µg/ml solution was prepared by taking 0.2-1.0 ml of stock solution into a series of 10.0 ml volumetric flask. To each flask 1.5 ml of 1% w/v eathanolic solution of vanillin and 1.0 ml 1 N HNO3 was mixed properly and the solution was heated on a boiling water bath for 10min, cooled at temperature and make up the volume with 0.1 N HCl. The absorbance of the yellow colored chromogen was measured at 470 nm against reagent blank. Figure 2 represents the calibration curve of acyclovir with vanillin.
Figure 2: Calibration curve of acyclovir and vanillin.
Validation of the method The reproducibility of the proposed method was determined by replicate analysis of six separate solutions of the working standard at five concentration level (2-10 µgmL-1). The method gave satisfactory result; RSD did not exceed 2% represents the good reproducibility of the method. The accuracy of the proposed method was evaluated by standard addition method. The recovery values of the added concentration were 96.6-99.3 ± 0.84%, indicating the accuracy of the proposed method. Robustness was examined by evaluating the influence of small variation of method variables i.e. concentration of analytical reagent, reaction time. In these experiments, one 330 Scholar Research Library
Raja Chakraborty et al Arch. Appl. Sci. Res., 2011, 3 (1):328-332 _____________________________________________________________________________ parameter was changed and others were kept unchanged and the recovery percentage was calculated each time. It was found that small variation of recovery value was 98.8-101.5 ± 0.85%. The data indicates the reliability of the proposed method. Ruggedness also tested by using two different instruments at two different laboratories and different elapsed time keeping the other parameters unchanged. Results obtained from lab-to-lab and day-to-day variation was reproducible as the relative standard deviation (RSDs) did not exceed 2%. RESULT AND DISCUSSION Spectrophotometry methods development for the determination of drugs has increased considerable in recent years because of their importance in pharmaceutical analysis. A new method has been developed for the spectrophotometric estimation of acyclovir. The maximum absorption of acyclovir when it is treated with vanillin acidic media was found to be 470 nm. The regression coefficient was found to be 0.998 and the percentage recovery range of 98.8% to 101.5% was indicates the accuracy of method. From the proposed method, it was found that acyclovir obeys linearity within the concentration range of 2-10 µg/ml. The data given in Table 2. Reproducibility of the method was confirmed as the % RSD is less than 2. All the parameters have been tabulated in Table 1. The method validation parameters are within the specific limit. The above method was applied on commercially available brand of acyclovir (Acivir) and the found results showed that the drug content of this formulation was 198.2mg in accordance with the labelled claim 200mg. The percentage recovery was found 99.1. Table 1: Validation parameters Parameters Accuracy Precision Ruggedness Linearity and range
Value 0.969 0.969
