Accelerated Stability Studies on Valacyclovir ...

1 INTERNATIONAL JOURNAL OF PHARMACEUTICAL AND CHEMICAL SCIENCES ISSN: 2277 5005. Research Article Accelerated Stability Studies on Valacyclovir Hydrochloride by RP-HPLC. VMK. Gowtham Potnuru, C. Jothimanivannan*, CH. Arjun, M. Jambulingam, S. Ananda Thangadurai and D. Kamalakannan Swamy Vivekanandha College of Pharmacy, Department of Pharmaceutical Analysis. Elayampalaym, Tiruchengode, Namakkal, Tamilnadu, India. ABSTRACT. A specific, accurate, precise and sensitive RP-HPLC method has been developed for the determination of Valacyclovir hydrochloride in the presence of its degradation products by Accelerated Stability Studies . As per ICH guideline Q1A (R2), drug was subjected to all stress conditions such as hydrolysis (acidic and alkaline), oxidation (30% H2O2 v/v), photolysis, (As per ICH guideline Q1B), thermal degradation and humidity study. All stressed samples were successfully analyzed on C18 column using mobile phase water: methanol: perchloric acid in the ratio of 95:4:1v/v with a flow rate of ml/min and detection was made at 255 nm.

2 Moderate change in Stability of drug was shown in thermal and oxidative Studies whereas drug was found to be stable in acid, alkali, humidity and photolysis Studies . The developed method was validated over the linearity, precision, accuracy and specificity as per ICH. guideline Q2B. The major degradants was identified as acyclovir and D- Valacyclovir hydrochloride through comparison with the standard. The developed method with good separation of all degradation products from drug could be successfully applied for the determination of Valacyclovir hydrochloride in the presence of its degradation products in formulation. It can be used for analysis of samples during Stability testing. Keywords: Valacyclovir hydrochloride, RP-HPLC, Forced Degradation. INTRODUCTION integral part of the systematic approach to Valacyclovir is, L-Valine 2-[(2 amino 1, Stability evaluation. Stress testing of the 6 dihydro 6 oxo 9H-purin 9yl) methoxy] drug substance can help identify the likely ethyl ester (Fig 1).

3 After oral degradation products, which can in turn administration Valacyclovir is rapidly help establish the degradation pathways converted into acyclovir which has and the intrinsic Stability of the molecule demonstrated antiviral activity, against and validate the Stability indicating power herpes simplex virus type I (HSV-1) and 2 of the analytical procedures used. The (HSV-II), Varicella zoster virus (VZV). nature of the stress testing will depend on Valacyclovir is available as tablet dosage the individual drug substance and the type form in the market. Few HPLC methods of drug product involved. Stress testing is were reported for the determination of likely to be carried out on a single Valacyclovir in its pharmaceutical substance10. The main objective was to formulation and in serum. Previously develop a suitable method of analysis reported HPLC methods using C18, C8 with which is Stability indicating and to get an flow rate 3, 1 ml/min respectively our idea of how drug substance or product reported liquid chromatographic method degrades, degenerates and behaves determined Valacyclovir in presence of under changing conditions by keeping all acyclovir using C18 with flow rate this in view, it was thorough worthwhile to ml/min1-8.

4 The ICH guideline Q1A (R2)9 develop Stability indicating HPLC method emphasizes that the testing of those for Valacyclovir pure drug and its tablet features which are susceptible to change dosage form. Literature survey revealed during storage and are likely to influence that few methods are reported for the quality, safety and efficacy, must be done determination of Valacyclovir in the by validated Stability indicating testing presence of its degradation product in method. As per Q1A (R2) information on pharmaceutical dosage form. the Stability of the drug substance is an Vol. 2 (1) Jan-Mar 2013 110. INTERNATIONAL JOURNAL OF PHARMACEUTICAL AND CHEMICAL SCIENCES ISSN: 2277 5005. EXPERIMENTAL solution in to a 10 ml dry, clean volumetric Chemicals and Reagents: flask and the volume was made up to Valacyclovir hydrochloride (standard) is mark with mobile phase (120 g/ml). The obtained from Aurobindo Pharma Ltd., solution was filtered and sonicated to Hyderabad, , India.

5 Methanol, remove water bubbles. Twenty micro liters Perchloric acid, Water, Hydrogen of solution was injected into system, the peroxide, Hydrochloric acid, Sodium chromatogram obtained was shown in hydroxide etc., which are of analytical Figure 2. grade reagent were purchased from E. Merck Ltd., Mumbai, India. Double distilled Sample preparation water was used throughout the study. Ten tablets were accurately weighed and Pharmaceutical dosage form (tablet) was powdered. The equivalent weight of 60 mg obtained from local market, manufactured of tablet powder was transferred into a by Cipla Pharma Ltd., as a brand name of 100 ml dry, clean volumetric flask and the Valcivir 500 mg which was used as volume made up to the mark with diluent sample. (600 g/ml). From this 2 ml was pipetted out in to a 10 ml dry, clean volumetric Instrumentation flask and the volume was made up to Analysis was performed on a mark with mobile phase (120 g/ml). The chromatographic system of Waters 2629 solution was filtered and sonicated to equipped with an auto injector with remove water bubbles.

6 UV/Visible detector (UV-2489). A. chromatographic separation was achieved Forced degradation Studies (stress on Crownpak C18 column (5 m, 250mm x testing). , ) analytical column. Data Acid degradation acquisition was made with Empower-2 The reaction was initiated by adding 10 ml software. Analytical Balance (BSA224S- of Valacyclovir hydrochloride to 100 ml CW, Sartorius), pH Meter (Eutech) from volumetric flask containing 5 M. Shimadzu were used for the study. hydrochloric acid. The volume made to Thermal oven and humidity desiccator 100 ml with 5 M hydrochloric acid. The were used to find Stability of drug. degradation was carried out in thermostatically controlled water bath, Chromatographic conditions protected from light. The degradation was Valacyclovir hydrochloride chromatogram carried at 850c temperature for 60. was developed on Crownpak C18, (250mm minutes. The chromatogram obtained was x 5 m, mm, ) column, using a shown in Figure 4. mobile phase containing water: methanol: perchloric acid in the ratio of 95:4:1v/v at Base degradation ambient temperature.

7 The flow rate was The reaction was initiated by adding 10 ml maintained at ml/ min throughout of Valacyclovir hydrochloride to 100 ml analysis. Initially the method was volumetric flask containing 1 M sodium developed for standard drug then it was hydroxide. The volume made to 100 ml extended to stress samples. The standard with 1 M sodium hydroxide. The and all stress samples were prepared in degradation was carried out in mobile phase. thermostatically controlled water bath, protected from light. The degradation was Preparation of Standard Stock carried at ambient temperature for 60. Solutions minutes. The chromatogram obtained was The quantity containing 60 mg of shown in Figure 5. Valacyclovir hydrochloride was accurately weighed and transferred into a 100 ml dry, Peroxide degradation clean volumetric flask and the volume was The reaction was initiated by adding 10 ml made up to mark with diluent (600 g/ml). of Valacyclovir hydrochloride to 100 ml 2 ml was pipetted out from the above volumetric flask containing 30 % hydrogen Vol.

8 2 (1) Jan-Mar 2013 111. INTERNATIONAL JOURNAL OF PHARMACEUTICAL AND CHEMICAL SCIENCES ISSN: 2277 5005. peroxide. The volume made to 100 ml with Validation of the method 30 % hydrogen peroxide. The degradation Method and system precision was carried out in thermostatically Precision of the method was verified by controlled water bath, protected from light. repeatability (system precision) and The degradation was carried at intermediate precision (method precision). temperature 850C for 30 minutes. The Studies . Repeatability Studies were chromatogram obtained was shown in performed by six replicate injections of Figure 6. 120 g/ml of Valacyclovir hydrochloride on the same day. The Studies were replicated Thermolytic degradation on different days to determine method The thermal Stability of Valacyclovir precision. The data is quoted in Table 2. hydrochloride was studied by heating it, both as powder and in mobile phase Accuracy solution at temperature 105 C for 24 Accuracy of the method was carried out hours.

9 The chromatogram obtained was by applying the method to drug sample to shown in Figure 7. which known amount of Valacyclovir hydrochloride standard powder Photolytic degradation corresponding to 50 %, 100 % and 150 %. Valacyclovir hydrochloride (60 mg, of label claim had been added (standard accurately weighed) was dissolved in 100 addition method), the solutions are ml mobile phase. The solution was analyzed by optimized method. In our exposed to light (UV lamp) for 120 hrs. study, the percentage recovery of The chromatogram obtained was shown in Valacyclovir hydrochloride was found to Figure 8. be , , and % from 50. %, 100 % and 150 % sample solutions Humidity degradation respectively. The obtained percentage Valacyclovir hydrochloride sample was recovery of both drugs was found to be kept under 250C temperature at 90 % RH within the range. This indicates the for 120 hrs time in humidity chamber proposed method was more accurate than under observation.

10 The chromatogram the existing methods. The results were obtained was shown in Figure 9 displayed in Table 3. Method validation Linearity Validation of developed analytical method From the standard stock solution , 1, was performed as per ICH guideline , 2, and ml was transferred to five Q2B11, over the linearity, accuracy, 10 ml flasks and made up the volume with precision and specificity. mobile phase. The concentrations of VAL. were found to be 30-150 g/ml. Twenty RESULTS AND DISCUSSION micro liter of the each standard solution Analysis of stressed samples was injected and chromatograms were Analysis of all stressed samples was recorded. The correlation coefficient (r2). performed using a mobile phase value of drug was found to be The containing water: methanol: perchloric equation of regression line for Valacyclovir acid in the ratio of 95:4:1v/v. The retention hydrochloride was found to be y = time of degraded sample was found to be same as that of undegraded sample for acid, base, photolysis and humidity Specificity conditions.