Spectrophotometric method for the determination of Valacyclovir in Pharmaceutical formulations

Valacyclovir—chemically identified as L-valine-2-[(2-amino-1,6-dihydro-6-oxo-9$H$-purin-9-yl) methoxy] ethyl ester—is the $L$-valyl ester prodrug of the antiviral agent acyclovir. It exhibits potent activity against Herpes Simplex Virus types 1 (HSV-1) and 2 (HSV-2), as well as the Varicella-Zoster Virus (VZV). As an antiviral prodrug, Valacyclovir inhibits viral replication; however, it is not curative, as the underlying viruses remain latent within the host between outbreaks. Therapeutically, Valacyclovir reduces the severity and duration of episodes, accelerates the healing of lesions, prevents the formation of new vesicles, and alleviates associated pain and pruritus. Furthermore, it has been shown to reduce the incidence and duration of post-herpetic neuralgia. A review of the existing literature reveals several analytical techniques for the estimation of Valacyclovir in both pharmaceutical dosage forms and biological fluids. These reported methods include various spectrophotometric methods^1–10, as well as high-performance liquid chromatography (HPLC)^11–17 and reversed-phase HPLC (RP-HPLC)^18-21. This investigation was undertaken to develop simple, rapid, and accurate methods for the analysis of valacyclovir in both bulk drug form and pharmaceutical formulations, addressing the limitations of existing techniques.

Absorption Spectrum of the Valacyclovir  for the maximum wavelength

The maximum absorbance wavelength for Valacyclovir treated with Cerium(IV) sulfate was determined as follows: 1.0 mL of Valacyclovir solution (100 µg/mL) was transferred into a volumetric flask, followed by the addition of 1.0 mL of 5 N hydrochloric acid and 1.0 mL of Cerium(IV) sulfate. The mixture was diluted to 5.0 mL with distilled water and allowed to stand for 15 minutes with occasional shaking. Subsequently, 1.0 mL of ammonium ferrous sulfate was added and mixed thoroughly. After 1 minute, 3.0 mL of 1 M ammonium thiocyanate was added, and the solution was diluted to the mark. The resulting blood-red solution was scanned in the range of 360–650 nm against a reagent blank. As shown in Fig.1 the solution exhibited maximum absorbance at 480 nm; therefore, all subsequent measurements were performed at this wavelength.

Fig.1.  Absorption spectrum of the Valacyclovir–Cerium(IV) reaction product

MATERIALS AND METHODS

Instrumentation

Spectronic 1000 plus UV Visible Spectrophotometer with 1 cm matched quartz cells was used for all spectral and absorbance measurements. AR grade chemicals are used for preparation of reagents and solutions in the present investigations

Cerric ammoniune sulphate (0.05M): Exactly 2.9826 g of AR cerium(IV) ammonium sulfate was weighed and dissolved in a small volume of double-distilled water. The resulting solution was then adjusted to a final volume of 100 mL in a standard flask.

Ammonium ferrous sulphate Solution (0.02M):  A 0.02 M solution was prepared by dissolving 0.7842 g of AR grade ammonium ferrous sulfate in distilled water. The mixture was transferred to a 100 mL volumetric flask and diluted to the mark with distilled water.

Ammonium Thiocyanate Solution (1.0 M): A 1.0 M solution was prepared by dissolving 7.0 g of AR grade ammonium thiocyanate in double-distilled water. The solution was then transferred to a 100 mL volumetric flask and diluted to the mark with the same solvent..

Standard Valacyclovir solution: 50 mg of Acyclovir is dissolved in 50 ml methanol. 1.0 ml of the above stock solution is further diluted to 10 ml with methanol to get working concentration of 100 mg/mL

Assay Procedure: Aliquots of Valacyclovir (0.5–2.5 mL) were transferred into a series of 10 mL volumetric flasks. To each flask, 1.0 mL of 5 M hydrochloric acid and 1.0 mL of Cerium(IV) sulfate were added. The total volume was adjusted to 5.0 mL with distilled water, and the solutions were allowed to stand for 15 minutes with occasional shaking. Subsequently, 1.0 mL of ammonium ferrous sulfate was added to each flask and mixed thoroughly. After 1 minute, 3.0 mL of 1 M ammonium thiocyanate was added, and each flask was diluted to the mark with water. The absorbance of the resulting solutions was measured at 480 nm against a reagent blank. The drug concentration in the unknown samples was determined by referencing the standard calibration curve (Fig. 2).

Fig.2: Calibration curve of valacyclovir

Analysis of Valacyclovir in Pharmaceutical Formulations

For the analysis of Valacyclovir, an accurately weighed portion of the sample, equivalent to 50 mg of the drug, was transferred into a 50 mL volumetric flask containing 25 mL of chloroform. The mixture was sonicated for 20 minutes to ensure complete dissolution, and the final volume was adjusted to the mark with chloroform. Appropriate aliquots were then further diluted with chloroform and subjected to spectrophotometric analysis using the procedure described previously. The drug content in the sample was determined using the respective calibration curve, and the results are summarized in Table 2

RESULTS AND DISCUSSION

The proposed method is based on the oxidation of Valacyclovir by a measured excess of Cerium(IV) sulfate in an acidic medium. The residual oxidant is reduced by a fixed amount of Iron(II), followed by the formation of a red Iron(III)-thiocyanate complex, which is measured at 480 nm. As the concentration of Valacyclovir increases, the concentration of Cerium(IV) decreases proportionally. Consequently, the unreacted oxidant produces less Iron(III), resulting in a linear decrease in the absorbance of the Iron(III)-thiocyanate complex. This inverse relationship forms the basis of the assay.

Statistical analysis of five replicate measurements showed low standard deviation values, indicating high accuracy and reproducibility (Table.2.). The assay data for commercial formulations were subjected to Student’s t-test for validation. At a 5% significance level with four degrees of freedom (n-1=4), the calculated t-values  were found to be less than the theoretical t-values. This indicates no significant difference between the proposed method and the standard reference method. Thus, the described method is rapid, reliable, and suitable for routine quality control analysis.

Table.1: Optical characteristics of the proposed methods

*Y = a+bX, where Y is the absorbance and X concentration in μg/ml

Table.2:Pharmaceutical analysis of valacyclovir

^*Average of five determination