To Develop And Validate Stability Indicating Assay Of Famciclovir By Using UV And HPLC Method For Pharmaceutical Dosage Form

Famciclovir is a potent antiviral medication primarily used to treat infections caused by herpes viruses, including herpes simplex virus (HSV) and varicella-zoster virus (VZV). It belongs to the nucleoside analogue class of drugs and is structurally similar to acyclovir. Famciclovir is available in oral dosage forms and marketed under various brand names worldwide.

       
           
       
    Figure 01: Structure and IUPAC name: 2-Amino-9H-purine in which the hydrogen at position 9 is substituted by a 4-acetoxy-3-(acetoxymethyl) but-1-yl group.

Overview of Herpes Viruses:

Herpes viruses are a group of DNA viruses that infect humans and other animals, causing a wide range of diseases, including oral and genital herpes, shingles (herpes zoster), and chickenpox (varicella). These viruses establish lifelong infections in the host, with periodic reactivation leading to recurrent outbreaks of symptoms.

Herpes Simplex Virus (HSV):

HSV exists in two primary types: HSV-1 and HSV-2. HSV-1 commonly causes oral herpes, characterized by cold sores or fever blisters around the mouth, while HSV-2 is primarily associated with genital herpes, leading to painful sores in the genital area.

Varicella-Zoster Virus (VZV):

VZV causes chickenpox during primary infection, and later in life, it can reactivate to cause shingles, characterized by a painful rash and blisters along a specific dermatome.

Clinical Manifestations:

The clinical manifestations of herpes virus infections vary depending on the site and type of infection. While some individuals may experience mild symptoms or asymptomatic infection, others may suffer from severe discomfort, pain, and complications. Effective antiviral therapy is essential for managing these infections and reducing their associated morbidity and transmission.

Mechanism of Action:

Famciclovir acts by inhibiting the replication of herpes viruses through its conversion to penciclovir, an active metabolite, by the viral enzyme thymidine kinase. Penciclovir then competitively inhibits viral DNA polymerase, preventing the elongation of viral DNA and thus halting viral replication. This mechanism of action is similar to that of acyclovir.

Development and Advantages:

The development of Famciclovir as a therapeutic agent stemmed from the need for more potent and bioavailable antiviral drugs with improved pharmacokinetic properties compared to acyclovir. Famciclovir offers several advantages over acyclovir, including higher oral bioavailability, longer intracellular half-life, and greater activity against certain herpes virus strains. These attributes contribute to its efficacy in treating herpes infections and preventing recurrent outbreaks.

Pharmacokinetics:

Famciclovir involves rapid and extensive absorption following oral administration, with peak plasma concentrations achieved within 1 to 2 hours. The drug undergoes extensive first-pass metabolism in the liver, primarily mediated by hepatic esterases, leading to the formation of penciclovir. Penciclovir exhibits high protein binding and is primarily eliminated unchanged in the urine via renal excretion.

Clinical Efficacy:

Clinical studies have demonstrated the efficacy of Famciclovir in treating various herpes virus infections, including genital herpes, herpes labialis (cold sores), and herpes zoster. In randomized controlled trials, Famciclovir has been shown to reduce the duration of viral shedding, accelerate lesion healing, and alleviate symptoms associated with herpes outbreaks. Additionally, suppressive therapy with Famciclovir has been effective in reducing the frequency and severity of recurrent genital herpes episodes.

Prophylactic Use:

Famciclovir has been investigated for its potential prophylactic use in certain populations, such as Immuno compromised individuals at risk of herpes virus reactivation. Prophylactic administration of Famciclovir has been shown to reduce the incidence of herpes zoster and herpes simplex virus infections in these high-risk populations, thereby improving their quality of life and reducing healthcare burden.

Safety Profile:

The safety profile of Famciclovir is generally favorable, with most adverse effects being mild to moderate in severity and transient in nature. Common side effects include headache, nausea, diarrhea, and abdominal pain. Rare but serious adverse reactions, such as allergic reactions, thrombotic thrombocytopenic purpura (TTP), and central nervous system effects, have been reported with the use of Famciclovir, although causality is often difficult to establish.

Contraindications: The use of Famciclovir may be contraindicated in certain patient populations, including those with known hypersensitivity to the drug or its components. Caution is advised in patients with renal impairment, as dosage adjustment may be necessary to prevent drug accumulation and potential toxicity. Additionally, Famciclovir should be used with caution during pregnancy and lactation, as limited data are available regarding its safety in these populations.

Experimental:

Chemicals and reagents: HPLC grade Acetonitrile, Methanol, and glacial acetic acid were obtained from Rankem (Mumbai, India). Pure sample of Famciclovir drug was obtained from FDC Limited, Goa, India. Ultra pure water obtained from Milli-Q academic system (Millipore Pvt. Ltd., Bangalore, India) was used to prepare all solutions for the method.  The procured samples were tested to confirm their identity and this included UV-visible wavelength scan, and recording of FT-IR spectra. FT-IR spectra were recorded at ARACOP, Dhule. The sample was prepared as a KBr pellet for recording the spectra. The UV-Visible spectra of Telmisartan were recorded using methanol as solvent was recorded using water as solvent on SICAN 1900 Instrument shown in figure 02.

1. Design and optimize UV and HPLC methods for the quantitative analysis of Famciclovir in the pharmaceutical dosage form.
2. Experiment with different chromatographic conditions, including column type, mobile phase composition, flow rate, and detection wavelength for HPLC method.

• The process was carried out on C18 column (5?m, 250 x 4.6mm, i.d) using the mobile phase consisting of Acetonitrile, methanol, and glacial acetic acid in the ratio 50:20:30 v/v/v respectively at a flow rate of 1 ml min-1. Wavelength was fixed at 310 nm. The mobile phase was filtered through 0.45?m membrane filter and degassed.

c. Determine the optimal conditions for UV analysis, including wavelength selection and sample preparation technique.

• Stock standard solution of the pure drug was prepared by dissolving 100 mg of FCV in 100 ml volumetric flask using water. Then the volume was made up to the mark with the same solvent to give a final concentration of 1000 ?g ml-1. Standard solutions of FCV (1.0, 5.0, 10.0, 20.0, 30.0, and 40.0 ?g ml-1) were prepared by subsequent dilution with mobile phase using Acetonitrile, methanol, and glacial acetic acid in the ratio 50:20:30 v/v/v.

Preparation of Standard Solutions:

1. Prepare stock solutions of Famciclovir of known concentrations to be used for method calibration and validation.
2. Ensure the accuracy and precision of the stock solutions through appropriate dilutions and analytical techniques.

Validation of the method:

The proposed analytical method was validated as per the International Conference on Harmonization (ICH) guidelines Q2 (R1)25: Linearity Precision Robustness Specificity Accuracy Linearity Precision Robustness. Validation of the proposed method Validation of the proposed methods was performed in accordance with the International Conference on Harmonization (ICH) guidelines (2005).

System Suitability Testing:

Perform system suitability tests for the HPLC method to ensure adequate chromatographic performance and resolution. Evaluate parameters such as retention time, peak symmetry, resolution, and column efficiency to ensure the reliability of the method.

Resolution:

The ability of the chromatographic system to separate adjacent peaks. Resolution is determined by the peak width and baseline separation between peaks.

Retention Time:

The time taken for a compound to travel through the chromatographic column and reach the detector. Retention time should be consistent and reproducible for each analyte.

Peak Symmetry:

The shape of chromatographic peaks should be symmetrical, indicating efficient and uniform elution of analytes. Peak asymmetry can indicate issues such as overloading, column degradation, or improper mobile phase composition.

Peak Area:

The area under each peak corresponds to the amount of analyte present in the sample. Peak area should be consistent and proportional to the concentration of analyte injected.

Selectivity:

The ability of the chromatographic method to separate analytes of interest from potential interferences or impurities. Selectivity ensures accurate quantification and identification of target compounds.

Method Validation:

Conduct validation studies according to the developed protocol.

Linearity:

Calibration curves were constructed using three series of standard FCV solutions in the range of 1.0 - 40.0 ?g ml-1. The equation of linear regression and statistical data are shown in the table. The linearity of the calibration curve was validated by the high value of the correlation coefficient.

Limit of detection (LOD) and limit of quantification (LOQ):

The limit of detection and the limit of quantification are defined as LOD and LOQ respectively, where ? denotes standard deviation of y-intercepts of regression lines and s denotes slope of the corresponding calibration curve.17

Precision:

The assay was investigated with respect to system suitability test, method precision and intermediate precision. The system suitability test and method precision were carried out to monitor repeatability and reproducibility. In order to measure repeatability of the system (system suitability test), five consecutive injections were made and the results were evaluated by considering peak area values of FCV. The precision values with their R.S.D. are shown in Table 7.2. The results in Table 2 indicate that the R.S.D. (%) is less than 2%. Three different concentrations of FCV were analyzed in three independent series in the same day (intra-day precision) and three consecutive days (inter-day precision), within each series every sample was injected six times. The R.S.D. values of intra- and inter-day studies varied from 0.17 to 0.57 % showing that the intermediate precision of the method was satisfactory.

Accuracy and recovery studies:

 The accuracy of a method is expressed as the closeness of agreement between the value found and the value that is accepted as a reference value. It is determined by calculating the percent difference (bias %) between the measured mean contents and the corresponding nominal contents.18 Shown in the table results obtained for intra- and inter-day accuracy. The accuracy of the proposed method was also tested by recovery experiments. Recovery experiments were performed by taking different sample concentrations and spiking with FCV at two different concentration levels (50% and 100?V). Six samples were prepared for each recovery level. Samples were treated as described in the procedure for sample preparations. The results obtained are shown in Table from which it is clear that both the recoveries and repeatabilities are excellent.

Robustness:

Robustness relates to the capacity of the method to remain unaffected by small but deliberate variations introduced into the method parameters. Influences of small changes in the mobile phase composition (±10%) and flow rate (±10%) were studied to determine robustness of the method. Peak areas and retention time changes were observed. Peak area values and retention time values varied by less than 2 %. Despite the changes in retention time there was no problem for quantification.

Assay method:

1. 1. API
   2. Pharmaceutical formulation.

Twenty tablets were weighed, crushed and an amount of powder equivalent to100 mg of FCV was accurately weighed, transferred to a 100 ml volumetric flask, made up to volume with water and placed in an ultrasonic bath for 20 min. After filtration through a 0.45?m membrane filter, the solution was suitably diluted with mobile phase to obtain the required concentration. 20?L of solution was injected into the HPLC system to obtain the chromatograms for the standard drug solution and the sample solution. A steady baseline was recorded with the optimized chromatographic conditions. The standard solution of FCV was injected and the chromatogram recorded. The retention time of FCV was found to be 3.56 min. The sample solution prepared from the tablets was then injected and the amount of drug present was calculated from the calibration curve.

Result of Procurement and Confirmation of Identity of Famciclovir:

Figure no. 04: Std. Calibration curve of Famciclovir r2 =0.999

UV-Visible spectra of Famciclovir exhibit absorption at 312 nm. The standard calibration curve for Famciclovir demonstrates strong linearity with an R?2; value of 0.999, indicating reliable quantitative analysis capability.

Method Development:

The process was carried out on C18 column (5?m, 250 x 4.6mm, i.d) using the mobile phase consisting of Acetonitrile, methanol, and glacial acetic acid in the ratio 50:20:30 v/v/v respectively at a flow rate of 1 ml min-1. Wavelength was fixed at 312 nm. The mobile phase was filtered through 0.45?m membrane filter and degassed

Linearity:

Calibration curves were constructed using three series of standard FCV solutions in the range of 0.0 - 40.0 ?g ml-1. The equation of linear regression and statistical data are presented in Table 06 and figure 06.

The limit of detection and the limit of quantification are defined as LOD and LOQ respectively, where ? denotes standard deviation of y-intercepts of regression lines and s denotes slope of the corresponding calibration curve.17

LOD =3.3 ?/s = 0.310, LOQ=10 ?/s = 1.022

The R.S.D. values of intra- and inter-day studies (Table 08) varied from 0.16 to 0.50 % showing that the intermediate precision of the method was satisfactory.18

Accuracy and recovery studies: The results obtained are shown in Table 9 and, 10 from which it is clear that both the recoveries and repeatabilities are excellent.

System Suitability Testing:

Perform system suitability tests for the HPLC method to ensure adequate chromatographic performance and resolution. Evaluate parameters such as retention time, peak symmetry, resolution, and column efficiency to ensure the reliability of the method.

Table no.12: System Suitability Test Parameters for Famciclovir:

Assay method

API and Pharmaceutical formulation. 20?L of solution was injected into the HPLC system to obtain the chromatograms for the standard drug solution and the sample solution. A steady baseline was recorded with the optimized chromatographic conditions. The standard solution of FCV was injected and the chromatogram recorded. The retention time of FCV was found to be 3.56 min. The sample solution prepared from the tablets was then injected and the amount of drug present was calculated from the calibration curve.

 Forced Degradation Studies:

Conduct forced degradation studies to assess the stability-indicating capability of the developed methods. Subject Famciclovir samples to various stress conditions such as heat, light, acid/base hydrolysis, oxidation, and photolysis. Monitor the degradation products using UV and HPLC methods to ensure that the method can accurately quantify Famciclovir in the presence of degradation products.

       
           
       
    Figure 8: Result of normal condition of Famciclovir.

To 10 mL of Famciclovir solution (2mg/mL in distilled water) added 10 mL of 1N HCL and was kept at room temperature for 3 hour. The solution was neutralized. Further dilutions were made with mobile phase to obtain concentration 20g/mL for HPLC analysis.

To 10 mL of Famciclovir solution (2mg/mL in distilled water) added 10 mL of 0.1M NaOH and was kept at room temperature for 3 hour. The solution was neutralized and diluted with mobile phase for further study.

       
           
       
    Figure 10: Result of ne hydrolysis (0.1 M NaOH, 3hr., RT) of Famciclovir.

To 10 mL of Famciclovir solution (2mg/mL in distilled water) added 10 mL distilled water refluxed at 80 0C for 12 hour. The solution was diluted with mobile phase for further study.

To 10 mL of Famciclovir solution (2mg/mL in distilled water) added 10 mL of 30% H2O2 at room temperature for 48 hour.

The pure solid drug substance was spread to about 1mm thickness in petri dish exposed to dry heat at 50 0C for 48 hour in hot air oven. Then, powder equivalent to 25 mg dissolved in 25.0 mL of distilled water. Further dilutions were made in mobile phase to obtain appropriate concentration of Famciclovir.

       
           
       
    Figure 13: Result of Dry heat (500 C, 24hr.) of Famciclovir.

The pure solid drug substance was spread to about 1mm thickness in petri dish exposed to sunlight for 12 hours and then powder equivalent to 25 mg dissolved in 25.0 mL of distilled water. Further dilutions were made in mobile phase to obtain appropriate concentration of Famciclovir.