www.ijpsonline.com guinea pig liver microsomes was 0.084 nmol/min/ Marcel Dekker Inc: 1999. p. 109-30. nmol P450. In contrast, metabolism of coumarin to 5. Williams DA. . In: Foye WO, Lemke TL, Williams DA, editors. Principles of Medicinal Chemistry. 4th ed. New Delhi; BI 7-hydroxycoumarin was not observed in rat liver Waverly Pvt. Ltd: 1999. p. 83-140. microsomes indicating the absence of this activity. 6. Iyer KR, Sinz, MW. Characterization of Phase I and Phase II hepatic This is not a new finding and it has been reported drug metabolism activities in a panel of human liver preparations. Chem Biol Interact 1999;118:151-69. previously that CYP2A6-like activity shows species 7. Bhopale AK. Synthesis of metabolites of probe substrates of CYP2A6 differences17. Coumarin metabolism in monkey, and CYP2C9. M Pharm Sci Thesis. University of Mumbai: October mouse, hamster and rabbit seems to be most similar 2001. 8. Schenkman J, Cinti DT. Preparation of microsomes with calcium. Meth 17 to humans . In mouse, the isoform CYP2A5 is Enzymol 1978;52:84-7. responsible for coumarin 7-hydroxylation while in rats, 9. Omura T, Sato R. The carbon monoxide binding pigment of liver coumarin is mainly metabolized by opening of lactone microsomes. J Biol Chem 1964;239:2370-7. 10. Tassaneeyakul W, Birkett DJ, Veronese ME, McManus ME, Tukey RH, ring via hydrolysis by esterase. It is suggested that Quttroachi LC, et al. SpeciÞ city of substrates and inhibitor probes for this may be the reason for coumarin hepatotoxicity human cytochromes. J Pharmacol Exp Ther 1993;265:401-7. in rats (which is not observed in case of guinea pigs 11. Chauret N, Gauthier A, Martin J, Nicoll Griffith DA. In vitro 17 comparison of cytochrome P450 mediated metabolic activities in human, or humans) where less than 1% of coumarin is dog, cat, and horse. Drug Metab Dispos 1997;25:1130-43. metabolized to 7-hydroxycoumarin. 12. Rodrigues AD, Lin JH. In vitro models for early studies of drug metabolism. In: Testa B, Van de Waterbeemd H, Folkers G, Guy R, editors. Pharmacokinetic optimization in drug research-biological, The above studies indicate the utility of this assay physicochemical and computational strategies. 1st ed. Germany; Wiley for determination of coumarin 7-hydroxylase activity VCH: 2001. p. 217-44. is animal liver microsomes and potentially, CYP2A6 13. Gillette JR. Techniques for studying drug metabolism in vitro. In: La Du BN, Mandel HG, Way LE, editors. Fundamentals of drug activity in human liver microsomal preparations. metabolism and disposition. 1st ed. Baltimore; Waverly Press Inc: 1997. p. 400-16. ACKNOWLEDGEMENTS 14. Hayes RN, Pool WF, Sinz MW, Woolf TF. Recent developments in drug metabolism methodology. In: Welling PG, Tse FL, editors. . 2nd ed. New York; Marcel Dekker Inc: 1995. p. The authors would like to extend special thanks to 201-34. Dr. M.L. Kubal, Wockhardt Ltd., Aurangabad for 15. Sinz MW, Iyer KR. The role and practice of drug metabolism in drug discovery and development. Asian Chem Lett 2001;5:93-104. providing HPLC columns used in this work. This 16. Clarke SE, Jones BC. Human cytochrome P450s and their role in work was conducted with the support of AICTE grant metabolism based drug-drug interactions. In: Rodrigues AD, editor. - 8019/RDII/R&D/PHA(213)/2001-02. Drug-drug interactions. 1st ed. New York; Marcel Dekker Inc: 2000. (www.medknow.com).p. 55-88. 17. Spatzenegger M, Born SL, Halpert JR. Cytochrome P450 in laboratory REFERENCES animal species. In: Lee JS, Obach RS, Fisher MB, editors. Drug This PDF is available for freemetabolizing download enzymes – Cytochrome P450 and other enzymes in drug discovery and development. Netherlands; Fontis Media SA: 2003. p. 1. Mannering GJ. Microsomal enzyme systems which catalyze drug 179-209. metabolism. In: La Du BN, Mandal HG, Way LE, editors. Fundamentals of drug metabolismfrom and disposition. a site 1st ed. Baltimore;hosted Waverly by Press Medknow Publications Inc: 1972. p. 206-45. 2. Parkinson A. Biotransformation of xenobiotics. In: Klassen CD, Amdur MO, Doull J, editors. Toxicology - The basic science of poisons. 5th ed. Accepted 6 June 2007 New York; McGraw Hill: 1996. p. 139-63. Revised 29 November 2006 3. Wrighton SA, Stevens JC. The human hepatic cytochrome P450s Received 17 February 2005 involved in drug metabolism. Crit Rev Toxicol 1992;22:1-21. Indian J. Pharm. Sci., 2007, 69 (3): 448-451 4. Ortiz de Montellano PR. The cytochrome P450 oxidative system. In: Woolf TW, editor. Handbook of drug metabolism. 1st ed. New York;

Spectrophotometric Estimation of Famciclovir in Bulk and Tablet Dosage Form

S. NIZAMUDDIN, B. M. GURUPADAYYA*, M. C. RAVI, Y. N. MANOHARA AND S. APPALA RAJU1 Department of Pharmaceutical Analysis, National College of Pharmacy, Shimoga - 577 201, India, 1Department of Pharmaceutical Analysis, H. K. E’s College of Pharmacy, Gulbarga - 585 105, India.

May - June 2007 Indian Journal of Pharmaceutical Sciences 451 www.ijpsonline.com

Two, simple, accurate, rapid and sensitive methods have been developed for the estimation of famciclovir in tablet dosage forms. Method A is based on the nucleophillic substitution product with Folin’s reagent to form colored chromogen exhibiting absorption maximum at 454 nm with apparent molar absorptivity of 3.99×104 l/mol.cm and obeyed Beer’s law in the concentration range of 2-10 µg/ml. Method B is based on diazotisation and coupling reaction with resorcinol to form colored chromogen exhibiting absorbance maximum at 386 nm with apparent molar absorptivity of 3.15×104 l/mol.cm and obeyed Beer’s law in the concentration range of 7-21 µg/ml.

Famciclovir chemically 2-[2-(2-Amino-9H-purin-9- into 100 ml volumetric flask containing 50 ml of yl)ethyl]trimethylene diacetate1,2 is an acyclic methanol and ß ask was kept for ultrasonication for 5 nucleoside analog, it is a new generation min, then it was diluted upto the mark with methanol which is active in vitro and in vivo against herpes and the solution was Þ ltered through Whatman Þ lter simplex virus types 1 and 2 and against varicella- paper No. 41. From the above solution 10 ml was zoster virus3-6. It is not ofÞ cial in any Pharmacopoeia. pipetted out into a 100 ml volumetric ß ask and the A few analytical methods have been reported for its volume was made up to the mark with methanol. The quantitative estimation in pharmaceutical formulations Þ nal concentration of famciclovir was brought to 100 that include estimation in plasma and urine by HPLC7 µg/ml with methanol and used for the analysis. and UV8 spectrophotometric estimation in methanol. In method A aliquots of famciclovir ranging from 0.2- The present work describes two simple colorimetric 1.2 ml portion of standard solution were transferred methods for the estimation of famciclovir in into a series of 10 ml volumetric ß asks. To each ß ask pharmaceutical dosage forms. Method A involves 0.2 ml of sodium 1,2-naphthaoquinone-4-sulphonate nucleophillic substitution product with Folin’s reagent (1% w/v) and 2 ml of NaOH (5% w/v) solutions to form colored chromogen exhibiting absorption were added to each flask and kept for 20 min at maximum at 454 nm against reagent blank. Method lab temperature. The solutions were made upto the B is based on diazotisation and coupling reaction mark with distilled water. The absorbances were with resorcinol to form colored chromogen exhibiting measured at 454 nm against a reagent blank prepared absorbance maximum at 386 nm against reagent simultaneously. The amount of the drug in a sample blank. was calculated from the calibration graph.

A Shimadzu UV/Vis double beam spectrophotometer(www.medknow.com). In method B aliquots of famciclovir ranging from (model 1601) withThis 1 cm PDF matched is quartzavailable cells was for 0.7–2.1free download ml were transferred into a series of 10 ml used for all spectral measurements. Solution of Folin’s volumetric flasks. To each 1 ml of HCl (1M) and reagent [sodium (1,2-naphthaoquinone-4-sulphonate] 0.5 ml of NaNO2 (1%w/v) were added at room (1% w/v), NaOH (5% w/v), HCl (1M), NaNO (1% temperature. After 5 min, 1.5 ml of resorcinol (0.2% from a site hosted 2by Medknow Publications w/v), resorcinol (0.2% w/v) and NaOH (1M) were w/v) and 0.5 ml of NaOH (1M) were added. The freshly prepared in distilled water. All chemicals volumes were made up to the mark with distilled used were of AR grade from S. D. Fine Chemicals, water. The absorbance of the yellow colored Mumbai. chromogen was measured at 386 nm against reagent blank. The amount of famciclovir present in the Standard solution of famciclovir was prepared by sample was computed form calibration curve. dissolving 100 mg in 100 ml and diluting 10 ml of this solution to 100 ml with methanol (100 µg/ml). For method A, the use of Folin’s reagent >0.2 ml The method was extended for determination of resulted in decrease the absorbance and <0.7 ml famciclovir in tablets. Famtrex, Cipla Ltd. containing resulted in cloudiness and use 5% NaOH, >4 ml and 250 and 500 mg strength were taken. Twenty tablets <2.5 ml was found to disturbs the Beer’s law. For were weighed and powdered. The tablet powder method B, 1 ml of 1M HCl and 1% NaNO2 was equivalent to 100 mg of famciclovir was transferred necessary for completion of reaction. 0.5 ml of 1M NaOH gives the good linearity and 1.5 ml of 0.2% *For correspondence resorcinol gives good result, addition of more than 2 E-mail: [email protected] ml resulted in non-linear color production.

452 Indian Journal of Pharmaceutical Sciences May - June 2007 www.ijpsonline.com

TABLE 1: ANALYSIS DATA OF FAMCICLOVIR IN TABLET FORMULATION Tablets Labeled Reference Amount obtained* (mg) % Recovery**±SD amount (mg) method Proposed method A B A B Brand 1 250 249.6±0.03 251.32±0.03 248.12±0.03 100.53±0.03 99.25±0.02 Brand 1 500 500.8±0.02 501.2±0.04 499.32±0.02 100.24±0.02 99.86±0.04 *Average of eight determinations. **Mean and standard deviation of eight determinations.

TABLE 2: OPTICAL CHARACTERISTICS AND repeatability and accuracy of these methods were PRECISION OF THE METHODS found to be good, which is evident by low standard Parameters Method Method A B deviation values (0.1142 for method A and 0.21 for method B). The percentage recovery value obtained λmax (nm) 454 386 Beer’s law limits (μg/ml)(C) 2-10 7-21 (100.24-100.53 for method A and 99.25-99.86 for Molar extinction 3.9903×104 1.1568×104 coefÞ cient (l/mol.cm) method B) indicates no interference from excipients Sandell’s sensitivity 0.0082 0.028 used in the formulation. Thus the developed methods (μg/ cm2/0.001 A.U.) are simple, sensitive, accurate, and precise and can Regression equation (Y*) Slope (b) 0.1231 0.0357 be successfully applied for the routine estimation Intercept (a) 0.0019 0.0003 of famciclovir in pharmaceutical dosage forms. Correlation co-efÞ cient (r) 0.9999 0.9994 Percentage RSD 0.1142 0.21 Range of errors** ACKNOWLEDGEMENTS ConÞ dence Limits 0.00071 0.0009 with 0.05 level ConÞ dence limits 0.00105 0.0014 The authors wish to thank the Cipla Ltd, Daman, for with 0.01 level providing gift sample of famciclovir. *Y= a+bc, where C is the concentration of famciclovir in µg/ml and Y is the absorbance at the respective λ . **For eight measurements. max REFERENCES To test the accuracy and reproducibility of the 1. Saltzman, R., Jurewicz, R. and Boon, R., Antimicrob. Agents proposed method, adding known amounts of the drug Chemother., 1994, 38, 2454. to the preanalysed formulation and reanalyzing the 2. Sweetman, S.C., In; Martindale, The Complete Drug References, 33rd mixture by proposed method carried out recovery Edn., Pharmaceutical Press., London (U.K.), 2002, 620. experiments. The results are shown in Table 1. 3. Maryadele J.O’Neil. Eds., In; Merck Index; An Encyclopedia of (www.medknow.com).Chemicals Drugs and Biologicals, 13th Edn., Merck, and Co., INC., Stability study of the chromogen was carried out by Whitehouse Station, NJ., 2001, 3960. measuring the absorbanceThis PDF values isat timeavailable intervals of for 4.freeHarden, download M.R., J. Med. Chem ., 1989, 32, 1738. 5. Vere Hodge, R.A., Antiviral Chem. Chemother., 1993, 4, 67. 10 min-4 h and it was found to be stable for 30 min 6. Circelli, R., Antivir. Res., 1996, 29, 141 for both methods. The optical characteristics such 7. McMeekin, J.R., Anal. Proc., 1992, 29, 178. as absorption maxima,from Beer’s a site law limits,hosted correlation by Medknow 8. Appala Raju, Publications S., Prabakar, V.K. and Kapse, G.K., Asian J. Chem., coefficient (r), slope (m), y-intercept (c), molar 2005, 17, 1319. absorptivity and Sandell’s sensitivity calculated from 8 replicates readings are incorporated in Table 2. The molar absorptivity and Sandell’s sensitivity values Accepted 10 June 2007 Revised 13 December 2006 show the sensitivity of both the methods. The analysis Received 31 March 2006 results of marketed formulations are in good agreement Indian J. Pharm. Sci., 2007, 69 (3): 451-453 with the official methods. The reproducibility,

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