INTRODUCTION:

Famciclovir chemically known as 9-[4-acetoxy-3-(acetoxymethyl) but-1-yl]-2-aminopurine is prodrug of penciclovir. Famciclovir is of antiviral category used in treatment of herpes zoster and genital and mucocutaneous herpes^1-4. Literature survey reveals UV-Visible spectrophotometric method^5-7,colorimetric method^8-9, HPLC^5,10 determination of famciclovir from pharmaceutical dosage forms, but forced degradation details were not presented in these articles. The reported stability assay method¹¹ using acetonitrile and phosphate buffer of 0.05 M as mobile phase component with mild stress conditions whereas another methodology¹² explains gradient mode for separation. One view to develop stability indicating method is to use previous reported assay methods to check their ability to serve as stability indicating method, and then the organic modifier can be chosen accordingly^13,14.The same approach explored here to use methanol: KH₂PO₄ buffer (pH 3.0) as mobile phase for estimation of famciclovir in bulk and pharmaceuticals. The aim of this work was to develop simple, isocratic RP-HPLC analytical method for determination of famciclovir in the presence of its degradation products in bulk and pharmaceuticals.

The novelty of proposed method over the earlier method^11,12 was that (1) the use of methanol as organic modifier preferred due to its low cost as compared to acetonitrile and use of 0.01 M buffer concentration (2) the method is isocratic one (3) separation of degradation products from famciclovir main peak to prove stability indicating ability of the method. The samples had been degraded using different stress conditions like acid, alkali, neutral hydrolysis, peroxide, thermal and photolytic stress. The method had been validated as per the ICH guidelines¹⁵.

MATERIAL AND METHOD:

Chemicals and Reagents:

Famciclovir as gift sample obtained from Cipla. Methanol (HPLC grade), potassium hydrogen phosphate, ortho phosphoric acid are of reagent grade. Double distilled water was used in preparation of mobile phase. Local commercial tablet formulation of Famciclovir named Famtrex-250 of CIPLA LTD. containing 250 mg of famciclovir was used.

Instrumentation:

The HPLC chromatographic system Agilent Technologies 1200 series, a G1315D quaternary pump, a G1315D diode array detector, and a rheodyne injector fitted with a 20-μL loop. Data were recorded and evaluated by use of EZ Chrome Elite software.

Chromatographic Conditions:

An appropriate separation was achieved on Eclipse Plus column (250 X 4.6 mm i. d. X 5 micron) as a stationary phase from Agilent. Detection was performed at 242 nm using photo-diode array detector. The mobile phase was composed of methanol: KH₂PO₄ buffer (0.01 M) (pH 3.0, with ortho phosphoric acid) (35:65, v/v). The flow rate was 1 ml/min. Before use, the mobile phase was filtered through a 0.22 μm Nylon filter.

Preparation of Stock solutions:

The standard stock solution of famciclovir was prepared by dissolving 25 mg of famciclovir in 25.0 mL of distilled water.

Calibration curve:

Calibration curve were prepared by taking 0.1 to 0.5 mL of standard famciclovir by micropipette to different 10.0 mL volumetric flask and diluted up to the mark with mobile phase to obtain final concentrations of 10-50μg/mL of famciclovir. Standard solutions (n=3) were injected through 20 μL loop system and chromatogram obtained with 1.0 mL flow rate. Calibration curve was constructed by plotting average peak area against concentration and regression equation was calculated.

Validation of the method:

The developed method was validated in terms of linearity, accuracy, limit of detection, limit of quantification, precision and repeatability of measurement.

Sample Preparation:

A total of 20 tablets were weighed, the average weight of tablet calculated. An amount of tablet powder equivalent to 25 mg of famciclovir was transferred to 25.0 mL volumetric flask; 15 mL of distilled water was added and flask was kept in an ultrasonic bath for 10 min. The volume was made up to the mark with water. Further dilutions of solutions were made with mobile phase to obtain final concentration of 20 μg/mL. The chromatogram obtained for standard and tablet is depicted in fig. no.1and 2.

Fig. 1. HPLC chromatogram of std. Famciclovir (Rt – 7.17 min)

Forced degradation conditions:

Forced degradation study was performed to check specificity, stability indicating ability of the proposed method.

Fig. 2. HPLC Chromatogram obtained from Famciclovir in tablet (Rt- 7.18 min.)

Acid degradation studies:

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

Alkali degradation studies:

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 2 hour. The solution was neutralized and diluted with mobile phase for further study.

Neutral degradation studies:

To 10 mL of Famciclovir solution (2mg/mL in distilled water) added 10 mL distilled water refluxed at 80⁰C for 12 hour. The solution was diluted with mobile phase for further study.

Oxidation studies:

To 10 mL of Famciclovir solution (2mg/mL in distilled water) added 10 mL of 30% H₂O₂ at room temperature for 24 hour.

Thermal studies:

The pure solid drug substance was spread to about 1mm thickness in petri dish exposed to dry heat at 50⁰C for 24 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.

Photo degradation studies:

The pure solid drug substance was spread to about 1mm thickness in petri dish exposed to sunlight for 6 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.

After the stress studies, the samples were analyzed as per the chromatographic conditions reported. Fig. no. 3 shows the degradation behavior of famciclovir under different stress conditions.

Fig. 3. Typical chromatograms obtained from Famciclovir degraded by

(A) Acid hydrolysis (1M HCL, 2 hr., RT)

(B) Alkaline hydrolysis (0.1 M NaOH, 2 hr., RT)

(D) Oxidation studies ( 30 % H₂O_2,24 hr., RT)

(E) Dry heat (50⁰C, 24hr.)

(F) Photo degradation (Sunlight exposure, 6 hr)

RESULTS AND DISCUSSION:

Optimization of chromatographic conditions:

To develop the accurate, precise, specific, isocratic and stability indicating RP-HPLC method for the estimation of famciclovir, various compositions of solvents were tried. The use of reported mobile phases containing acetonitrile, methanol, phosphate buffer, use of different pH values of mobile phase was also tried. The proposed mobile phase satisfies the system suitability parameters with separation of degradant from famciclovir with good resolution and capacity factor value as reported in Table no.1.

Table no.1 System Suitability Test Parameters for Famciclovir

Analyte

RSD of Replicate Injections

Tailing Factor

No. of Theoretical Plates

Capacity factor

(< 2)

(> 2000)

(>0.5)

Famciclovir

(20 mcg/mL)

0.8549

1.0272

8363.83

1.87

Forced degradation of famciclovir:

The results of the stress studies indicate the specificity and stability indicating ability of the method developed. Famciclovir was degradated in 1M HCl and 0.1M NaOH when kept at room temperature for 2 hr. The neutralization of degraded sample is important step in analysis. The degraded products have the good resolution from the famciclovir main peak. The results of forced degradation studies are given in Table no.2.

Method validation:

The proposed method was validated as per ICH guidelines.

Accuracy:

The accuracy of the method was determined by recovery studies. The standard bulk drug was added at 80%, 100% and 120% to pre-analyzed tablet powder. These samples were estimated by developed RP-HPLC method. The results are given in Table no. 3.

Table no. 3 Accuracy (at 80, 100 & 120 % level)

Accuracy (Recovery studies)	Pure drug added	Recovery (%)(n=3, ± SD)
	80%	99.75± 0.2680
	100%	99.73 ± 0.1404
	120%	99.86 ± 0.1571

Precision:

Precision was studied by measuring intra-day variation (repeatability) and intermediate (by injection of samples over three consecutive days) precision of the method. Percentage relative standard deviation (% RSD) was found to be less than 2% shows that the method is precise. The results are given in Table no.4.

Table no.4 Results from determination of the precision of the method (n = 6)

Concentration	Intra-day precision		Inter-day precision
Concentration	Mean area ± SD	RSD (%)	Mean area ± SD	RSD (%)
20μg mL−1	789994.2± 7154.59	0.9056	789534.3± 8180.65	1.1122

Linearity and Range:

Linearity study was carried out by preparing standard solutions at different concentration levels from 10 to 50 μg mL^−1.The regression equation was y = 39363x + 8288.9 with correlation coefficient value 0.9997.

Specificity:

The proposed method is able to estimate famciclovir in presence of its degradation products with good resolution.

Robustness of the method:

The small deliberate variations in the optimized mobile phase were made such as change is flow rate of mobile phase, change in mobile phase composition ratio. The effect of change of the parameter was studied. It was found to be unaffected by small changes in parameter with small SD value.

Use of the Method for Analysis of Marketed Famciclovir Formulations:

The validated method was used to estimate the famciclovir content of commercially available brands of tablet Famtrex -250, containing 250 mg famciclovir. The results of the assay indicated the method is selective for analysis of famciclovir without interference from the excipients present. The results are given in Table no.5.

Table no.5 Result of famciclovir in marketed formulation

Marketed Formulation	Drug	% amount found ± SD	% RSD
Famtrex -250 (tablet)	Famciclovir	100.19% ± 1.0452	1.0432

ACKNOWLEDGEMENTS:

The authors are highly grateful Dr. S. J. Surana, Principal, R.C. Patel Institute of Pharmaceutical Education and Research for providing necessary facilities to carry out work.

REFERENCES:

1) Budavari, S., Editor., The Meck Index 13 th Edn., Merck and Co., Inc., Whitehouse Station, NJ, 1997, 3960, pg. no. 695.

2) Clarke’s Analysis of Drugs and Poisons, third edition, edited by L.Y. Galichet, Pharmaceutical Press 2004, London Volume 2, 1014-1015.

3) Sweetman SC, Martindale, 33 rd Edn., The Complete Drug Reference, Pharmaceutical Press, London, 2002, 620.3.

4) Vajpayee M, Malhotra N. Antiviral drugs against Herpes infections. Indian Journal of Pharmcology 2000; 32: 330-338.

5) Srinubabu G., et al. Development and validation of Liquid Chromatographic and UV derivative spectrophotometric methods for the determination of Famciclovir in Pharmaceutical dosage forms. Chem. Pharm. Bull. 2006; 54(6), 819-822.

6) Nizamuddin N., et al. Spectrophotometric estimation of Famciclovir in bulk and tablet dosage form. Indian Journal of Pharmaceutical Sciences 2007; May-June, 451-453.

7) Prabahakar G., Kapse G. K. and Appala Raju S. UV Sepectrophotometric determination of Famciclovir. Asian Journal of Chemistry, 2005; 17 (2), 1319-1321.

8) Reddy P. V. and Sudha Rani B. Estimation of Some Antiviral Drugs Using Tpooo as Analytical Reagent. E-Journal of Chemistry, 2006 July; 3(12), 154-158.

9) Krishna M.V., Latha P.V.M. and Sankar D.G. Spectrophotometric Determination of Famciclovir and Racecodotril Using 2, 6-Dichloroquinone–Chlorimide. E-Journal of Chemistry. 2007 January; 4(1), 50-52.

10) Vishnumulaka S., et. al, Development and Validation of LC Method for the Determination of Famciclovir in Pharmaceutical Formulation Using an Experimental Design. E-Journal of Chemistry. 2008 January; 5 (1), 58-67.

11) Narasimha Raju Ch. B. V., Panda G., Rao G.N. Stability Indicating LC Assay Method for the Determination of Famciclovir in Bulk Drug and Pharmaceutical Dosage Forms. Chromatographia. 2008; 68, 837–841.

12) N.V.V.S.S. Raman, et al., Development and validation of a stability-indicating RP-LC method for famciclovir, Journal of Pharmaceutical and Biomedical Analysis. 2009; 50,797–802.

13) Bakshi M., Saranjit Singh. Development of validated stability-indicating assay methods-critical review. Journal of Pharmaceutical and Biomedical Analysis, 2000; 28, 1011-1040.

14) Singh S. and Bakshi M., Guidance on conduct of stress tests to determine inherent stability of drugs. Pharmaceutical Technology On-Line. 2000April.

15) International Conference on Harmonization (ICH), Validation of Analytical Procedures: Text and Methodology Q2 (R1), November 2005.

Received on 09.12.2009 Modified on 20.01.2010

Research J. Pharm. and Tech.3 (3): July-Sept. 2010; Page 762-765

Analyte (Famciclovir)	Retention time of Famciclovir (min.)	Retention time of Degradation Products (min)
Normal condition	7.17 (100%)	-
Acidic hydrolysis 1M HCL, 2 hr, RT	7.11 (7.77%)	2.56 (56.27%), 3.55 (35.97%)
Basic hydrolysis 0.1M NaOH, 2 hr, RT	-	2.587 (100%)
Neutral hydrolysis, 12 hr reflux at 80⁰C	7.52 (86.63%)	2.63 (0.50%), 3.65 (4.86 %), 4.10 (8.01 %),
Oxidative condition 30% H₂O₂, RT, 24 hr	6.91 ( 89.46 % )	2.41 (7.65%), 4.08 (1.27), 4.98 (1.62%)
Dry heat studies 50⁰C, 24 hr	6.90 (96.22%)	4.14 (3.78% )
Photo degradation study Sunlight, 6 hr.	7.56 (100%)	-