Development And Validation of RP-HPLC Method For Determination of Valacyclovir by Quality by Design Approach

Valacyclovir hydrochloride is L-valine, 2- [(2-amino-1, 6-dihydro-6-oxo-9H-purin-9-yl) methoxy] ethyl ester, monohydrochloride. It is an antiviral agent which is used in the treatment of herpes zoster and herpes simplex virus. (1) Valacyclovir is converted rapidly, and virtually completed, to acyclovir after oral administration in healthy adults. This conversion is thought to result from first-pass intestinal and hepatic metabolism through enzymatic hydrolysis (2) It inhibits viral DNA synthesis. Valacyclovir is converted by esterase to the active drug acyclovir via hepatic first-pass metabolism (3) The ICH guideline Q1A (R2) emphasizes that the testing of those features which are susceptible to change during storage and are likely to influence quality, safety and efficacy, must be done by validated stability indicating testing method. As per Q1(R2) information on the stability of the drug substance is an integral part of the systematic approach to stability evaluation.(4) The mechanism of action of Valacyclovir is inhibition of the viral DNA polymerase and also involved in the viral DNA chain termination.(5) Valacyclovir is available as a tablet dosage form in the market. Few HPLC methods have been reported for determining valacyclovir in pharmaceutical formulations, biological fluids, and spectrophotometric methods.. (6)Valacyclovir is converted by esterase to active drug valacyclovir via hepatic first-pass metabolism Viral thymidine kinase phosphorylates acyclovir triphosphate, inhibiting herpes viral DNA replication by competitive inhibition of viral DNA polymerase and termination of the growing viral DNA chain. The goal of creating and verifying an analytical technique is to make sure it is more specialized, accurate, and exact for a given analyte. The major goal of that is to make improvements to the requirements and parameters that must be adhered during the development and validation procedures. (9).

Figure 1. Structure Of Valacyclovir

A literature survey revealed that a number of methods like UV(10)(11) (12) HPLC(13) (14) (15) stability indicating HPLC (16) (17) (18) that have been reported for the estimation of valacyclovir in combination with other drugs, but still RP-HPLC method by Quality by Design for estimation of valacyclovir is not present. So, aim of this work is to develop simple, fast, and sensitive method with less run time and good peak symmetry to ensure the established method with respect to linearity, specificity, precision, accuracy, robustness, LOD and LOQ.

MATERIALS AND METHODS:   `          

MATERIALS:

The drug sample of Valacyclovir HCL was obtained from Reliables Shree Industrial Training Centre, Jalgaon. Formulation (Valcivir-500 mg tablet, Cipla Pharmaceutical Ltd., Sikkim, India.) was purchased from a local Pharmacy for research purpose. All other chemicals and reagents were used in the study of analytical grade. An Isocratic High-Pressure Liquid Chromatograph (Agilent 1100 & software is Chemstation) with autosampler injector, variable wavelength programmable UV-Vis. detector Analytical Technologies Limited 2080 system & operating software UV-Vis Analyst was used.

Method: The analysis was performed using high-performance liquid chromatography (Agilent tech gradient system) with a PDA detector having chemstation software, UV-visible spectrophotometer (model 2080), pH meter (VSI1-B), Electronic balance (WENSER High Resolution Balance), Ultra Sonicator. The symmetry C18 Column (150 mm X4.6 mm and 5µm) was used as a stationary phase with a flow rate of 1.1ml /min.

Instruments and reference standards:

The HPLC study was carried out on WATERS-2695 with a Photodiode array detector (PDA). C-18 column (250mm×4.6mm×5μm particle size) was used at ambient temperature. other equipment’s Sonicator (ePEI ultrasonic generator), Analytical balance (Mettler Toledo), vortex meter (IKA Vortex), and Hot air oven (Yorco scientific). pH meter (Eutech instruments pH tutor, pH meter, India) was used.

QBD software:

Design Expert® (13.0.0.5) modelling software (Stat-Ease Inc., Minneapolis, MN, USA) was used for generation of contour plots and 3D space.

Preparations of solutions:                                                            

Preparation of Standard stock solution:

Accurately weighed 10 mg of valacyclovir was transferred to 50 ml volumetric flasks, 3/4th of diluents was added and sonicated for 10 minutes. Flasks were made up of diluents and labeled as Standard stock solution (1000 µg/ml Valacyclovir) 1ml from the above solution was pipetted out and taken into a 10 ml volumetric flask and made up with diluent (100 µg/ml of valacyclovir).

Preparation of Sample stock solution:

Accurately weighed 20 tablets and the average weight of each tablet was calculated, then the weight equivalent to the tablet was transferred into a 10 ml volumetric flask, 5ml of diluents was added and sonicated for 25 min, further the volume was made up with diluent and filtered by HPLC. (1000 µg/ml of valacyclovir) 1ml of filtered sample stock solution was transferred to a 10 ml volumetric flask and made up with diluent (100 µg/ml). 

Determination of detection wavelength:

Between 200 to 400 nm, the standard solution was scanned as shown in Fig. 2, and the wavelength of maximum absorption for the drug was determined to be 255 nm.

Figure 2 UV Spectrum of Valacyclovir

Chromatographic conditions:

The Discovery C-18 column (250 mm x 4.6 mm particle size) was equilibrated with a mobile phase methanol: OPA in the ratio of (41:59). The detection wavelength was 255nm with a flow rate of 1.1ml/min at 25ºC, and the sample size is 20µl.

Initial method development by QbD approach

A Quality by Design with Design of Experiments approach to the development of an analytical method mainly involves two phases as follows:

a) Screening Phase

b) Statistical Analysis and Final Optimization

Screening phase:

The experimental design was constructed using design expert software version 13 (13.0.0.5) for the study of different variables (% organic phase, flow rate and temperature) and to verify method performances. The levels of these variables are as given in Table 2. The retention time, resolution, theoretical plates, and asymmetry were used as a response in experimental design as a controlling response, which is expected to affect and control method responses. A 2 factorial design consisting of two factors and four responses was considered for the experimental Plan. Initially and after confirming that the process is a non-linear, the Box-Behnken design was used. The experimental observations along with the Design (DOE) plan are shown in Table 3

Table 1. Factors And Levels of Independent Variables

Table 2 Central Composite Design and Response

Statistical Analysis and Final Optimization:

The responses obtained after carrying out the above trial runs were fed back to Design Expert software and plots like 3D-response surface plots and Graph plots were plotted. These graphs demonstrated how important procedure factors affected the chosen quality criteria. The analysis of these plots was used to estimate which method parameter gave the most acceptable responses. Thus, based on these observations, the final critical method parameters of the method were determined and the optimized chromatographic conditions were finalized. Additionally, the significance of each method parameter chosen for the study was determined using a statistical analysis tool like ANOVA for each individual response using the p-value (probability).

Validation of the optimized Method:

Linearity:

The linearity of peak areas versus different concentrations was evaluated for valacyclovir over the range of 10-50 µg/ml and for all the related substances over the range of 0.3μg/ml to 6μg/ml. The correlation coefficient (r2) for valacyclovir and each related substance was calculated.

Accuracy (% Recovery):

The accuracy of the method was confirmed by a recovery study from marketed formulation at 3 levels of standard addition. The accuracy was determined by the standard addition method. Three different levels (80%, 100%, and 120%) of standards were spiked to commercial tablets in triplicate. The mean of percentage recoveries and the % RSD was calculated. The percentage recovery of valacyclovir is 80%.

Precision:

The Precision is reported in terms of Relative Standard deviation (RSD). There are three levels of precision: repeatability, reproducibility, and intermediate precision. It is carried out on a sample API.

• Repeatability (intraday precision)
• Reproducibility
• Intermediate precision (interday precision)

Limits of detection and quantitation

Limits of detection (LOD) and Limit of quantitation (LOQ) were determined from the signal-to-noise ratio. The detection limit was referred to as the lowest concentration level resulting in a peak area of three times the baseline noise. The quantitation limit was referred to as the lowest concentration level that provided a peak area with a signal-to-noise ratio higher than ten.

LOD = 3:3 δ/S; LOQ = 10 δ/S.

Robustness

For robustness studies, 80 μg/ml of Valacyclovir was used. To demonstrate the robustness of the method, the following optimized conditions were slightly varied.

• (53.8%) 0.1% OPA: Acetonitrile (46.2%) ratio of mobile phase
• 1.1 ml/min of flow rate
• 25⁰C of temperature.

RESULTS AND DISCUSSION:

Preliminary studies on Valacyclovir:

• Melting point:

The procured reference standard of Valacyclovir was found to melt in the range of 159-162⁰C respectively.

• Solubility:

The drug was found to be freely soluble in water, DMSO, Methanol and insoluble in ether.

Statistical Analysis of Experimental Data by Design-expert Software:

Analysis of variance (ANOVA) was applied to study the significance of the model generated for the five responses shown in Tables 4-8. 2D Contour and 3D Surface plots were analyzed to visualize the effect of factors and their interactions on the Design Expert® software's responses. The regions shaded in dark blue represent lower values, and shaded in dark red represent higher values. The regions shaded in light blue, green, and yellow represent intermediate values.

Table 3. ANOVA Table for Retention time using CCD

The model's F-value of 5977.94 indicates that it is significant. An F-value this large might arise owing to noise only 0.02% of the time. Model terms with P-values less than 0.0500 are significant. In this example, A and B are important model terms. Values larger than 0.1000 indicate that the model terms are unimportant. Model reduction may improve your model if there are many inconsequential model terms (except those required to enable hierarchy).

Table 4. ANOVA Table for peak area using CCD

The Model F-value of 331.30 implies the model is significant. There is only a 0.01% chance that an F-value this large could occur due to noise. P-values less than 0.0500 indicate model terms are significant. In this case, B is a significant model term. Values greater than 0.1000 indicate the model terms are not significant. If there are many insignificant model terms (not counting those required to support hierarchy), model reduction may improve your model.

The model's F-value of 17.73 indicates that it is significant. An F-value this large might arise owing to noise just 0.54% of the time. Model terms with P-values less than 0.0500 are significant. B is an important model term in this scenario. Values larger than 0.1000 imply that the model terms are unimportant. Model reduction may improve your model if there are many inconsequential model terms (except those required to enable hierarchy).

The Model F-value of 21.79 implies the model is significant. There is only a 0.34% chance that an F-value this large could occur due to noise. P-values less than 0.0500 indicate model terms are significant. In this case, A, and B are significant model terms. Values greater than 0.1000 indicate the model terms are not significant. If there are many insignificant model terms (not counting those required to support hierarchy), model reduction may improve your model.

Figure. 3 Contour Plot for RT of Valacyclovir against Mobile phase and Flow rate

Figure. 4 Contour Plot for Peak Area of Valacyclovir against Mobile phase and Flow rate

Figure. 5 Contour plot for Theoretical plates of Valacyclovir against Mobile phase and Flow rate

Figure. 6 Contour plot for Telling Factor of Valacyclovir against Mobile phase and Floe rate

From the above 2D Contour and 3D Surface plots of retention time, it shows the two-dimensional contour plot as a function of Column temperature, Flow rate and organic ratio. Based on the Colour code, the working region can be easily identified. Retention time maps represent the value of the retention time, with warm “red” colours indicating larger retention time, cold “blue” colours lower and light green to yellow colour represent intermediate retention time.

Design Validation

From the actual versus predicted plots (Fig. 4) for the five responses, it was observed that the selected models for the respective responses were suitable for the selected design. It was further evidenced by the ANOVA.

Figure. 7 Colour point by the value of retention time Predicted vs Actual

Figure. 8 Colour point by the value of peak area Predicted vs Actual

Figure. 9 Colour point by the value of theoretical plates Predicted vs Actual

Figure. 10 Colour point by the value of tailing factors Predicted vs Actual

Table 7. Final optimized HPLC chromatographic conditions

Figure. 11 Chromatogram of Final Optimized Method

Method validation:

The developed method was linear over the concentration range with 10-50 µg/ml of valacyclovir with a correlation coefficient of 0.999 respectively. The accuracy studies at 50, 100, and 150% levels were conducted to ensure drug recovery within 98-102%. Intermediate precision, reproducibility, and repeatability were carried out, and the % RSD values were found to be less than 2%. The LOD and LOQ values for valacyclovir were determined to be 0.29 µg/ml and 0.89 µg/ml. The method's robustness was verified by minor adjustments to experimental conditions, revealing a 2% RSD value for the peak area, as detailed in Table 7. The summary of the method validation parameters is shown in Table 8.

Table 8. Results of the Validation Parameters