Original Article DEVELOPMENT AND VALIDATION OF STABILITY INDICATING RP-HPLC METHOD OF NEPAFENAC AND ITS DEGRADATION PRODUCTS: APPLICATION TO DEGRADATION KINETIC CHHAYA SHRIMALI 1 , MADHURI BAGHEL 1 , SADHANA RAJPUT 2* 1 Quality Assurance Laboratory, Centre for Relevance and Excellence in Novel drug delivery Systems, Pharmacy Department, G. H. Patel Building, Donor’s Plaza, 2 The Maharaja Sayajirao University of Baroda, Vadodara, Gujarat, 390002, India. Email: sjrajput@gmail.com Received: 24 Jul 2014 Revised and Accepted: 25 Aug 2014 ABSTRACT Objective: The objective of present work was to develop and validate simple, precise, accurate and specific stability indicating method for determination of nepafenac in presence of its degradation products and application of the method to study degradation kinetics. Methods: A novel isocratic RP-HPLC method has been developed using C 8 Olyster column, (250X4.6 mm i. d, 5µ particle size) with the mobile phase composition of ACN: 10 mM Ammonium formate buffer (pH 4.0): Methanol (27.5:45:27.5). The flow rate was at 1.0 ml min -1 Results: The degradants peaks were well resolved from Nepafenac peak. Significant degradation was observed in acid, base and oxidative degradation. The drug is relatively stable towards photolysis and dry heat. and effluent was detected at 238 nm. Nepafeac was subjected to stress degradation under acid, base, neutral hydrolysis, oxidation, dry heat, humidity and photolysis, conditions. Kinetic study was also performed. Conclusion: Stability of Nepafenac was determined by stability indicating assay method. Three degradation related impurities was identified by LC- MS. The hydrolytic degradation pathway of nepafenac was postulated. The developed stability indicating method was applied to determine acid, base and oxidative degradation kinetic. Acid hydrolysis and oxidative degradation followed zero order while base hydrolysis followed first order kinetic. Degradation rate constants and half-life were determined. Keywords: Nepafenac, Reverse Phase High Performance Liquid Chromatography (RP-HPLC), Stability Indicating Assay Methods (SIAM’S), Stress Degradation, ICH Q1A (R2), Q2 R1. INTRODUCTION Nepafenac (NEPA), [2-(2-amino 3-benzoyl phenyl) acetamide] (II, Fig. 1) is antiinflammatory (NSAID’s) which is indicated for the treatment of pain and inflammation associated with cataract surgery. NEPA is a pro drug. After penetrating the cornea, NEPA undergoes rapid bioactivation to amfenac, which is a potent NSAID that uniformly inhibits the COX1 and COX2 activity [1]. Amfenac is thought to inhibit the action of prostaglandin H synthase (cyclooxygenase), an enzyme required for prostaglandin production. Preparation of NEPA is disclosed in US patent 4313947 and the scheme is depicted in Fig. 1[2]. I=NEPA, II= 2-amino-benzophenone,III=2-Methylsulfanyl-acetamide, IV= 2-amino-3-benzoyl-α-(methylthio)-benzeneacetamide Fig. 1: Synthetic Scheme of NEPA There is always need for significant SIM’S in modern analytical labs. Environmental factors, such as the temperature, pH,buffer species, ionic strength, light, oxygen, moisture, additives and excipients, can play an important role in the stability of drug substances. Stress testing can help in identifying degradationproducts and provide important information about the intrinsic stability of drug substances [3]. Stability indicating assay is defined as a validated quantitativeanalytical procedure that can detect the changes with time in the pertinent properties (e. g., active ingredient, preservative level) of the drug substance and drug product. A stability-indicating assayaccuratelymeasures the active ingredients without interferencesfrom degradation products, process impurities, excipients, or other potential impurities [4]. No official USP (United States Pharmacopeia Convention) andEP (European Pharmacopoeia) monographs currently exist for NEPA. The literature survey reveals that. Two simple and sensitive visible spectrophotometric methods have been developed for the estimation of NEPA in pure and pharmaceutical dosage forms [5,6]. Determination of NEPA in plasma by RP-HPLC has also been reported [7]. Method for determining chemical purity of NEPA by High performance liquid chromatography with ultraviolet visible detector(HPLC-UV)was disclosed in patent document [8]. The method utilizes gradient elution using mobile phase Ammonium formate buffer (PH 4.25 with formic acid) and acetonitrile (ACN). The separation was carried out on C-18 column. The chromatographic run time was at least 65 min. HPLC-UV studies of the chemical purity or assay of the NEPA was also reported by ElzbietaLipiec-Abramska et al.[9]As in patent document, the method also utilizes gradient elution with mobile phase ammonium formate (pH 4.10 ± 0.02 adjusted with formic acid) and ACN using C18 column at a column temperature of 30 0 MATERIALS AND METHODS C. But the chromatographic run time is 29 min. Hence, the main objective of our work was to develop a simple, accurate and precise, stability-indicating HPLC-UV method for determination of NEPA and its degradation products within a short run time. In contrast to the above reported HPLC-UV methods, the chromatographic run time of the present method is 12 min with isocratic elution. Stress degradation was also performed to determine the most potential degradation related impurities. The validated method was applied to study the rate of degradation of NEPA under acid, base hydrolysis and oxidative degradation. Chemicals, Reagents and Solutions NEPA (Bulk drug) and impurities were kindly provided by Sun Pharmaceuticals. Baroda and were used as received. Nevanac International Journal of Pharmacy and Pharmaceutical Sciences ISSN- 0975-1491 Vol 6, Issue 9, 2014 Innovare Academic Sciences