International Journal of Science and Research (IJSR) ISSN (Online): 2319-7064 Index Copernicus Value (2013): 6.14 | Impact Factor (2014): 5.611 Volume 5 Issue 1, January 2016 www.ijsr.net Licensed Under Creative Commons Attribution CC BY RP-HPLC Method Development and Validation for Simultaneous Estimation of Aspirin, Caffeine and Orphenadrine citrate in Tablet Formulation Sanjay Pai P. N. 1 , Sameeksha Gaude 2 , Anusha Palekar 3 Department of Pharmaceutical Analysis (Quality Assurance Group), Goa College of Pharmacy, 18th June Road, Panaji-Goa 403001, India Abstract: A simple, precise and accurate RP-HPLC method has been developed and validated for the simultaneous estimation of a three component mixture - aspirin, caffeine and orphenadrine citrate. The chromatographic separation was achieved with methanol: phosphate buffer (pH 3) in the ratio of 65:35 (v/v) as mobile phase, on ACCLAIM TM 120 C 18 column (5 μm, 4.6 X 250 mm), at a flow rate of 1ml/m using isocratic elution. Detection was carried out at 215 nm. The retention time for aspirin, caffeine and orphenadrine citrate was found to be 4.2, 3.3 and 6.5 m respectively. The linearity range was found to be 10-100 μg/ml for aspirin and 2-20 μg/ml for both caffeine and orphenadrine citrate with r 2 value 0.997, 0.994 and 0.997 respectively. The mean percent recovery was found to be 97.3-97.80% for aspirin, 95-104.3% for caffeine and 96.36-103.4% for orphenadrine citrate. Keywords: Aspirin; caffeine; orphenadrine citrate; RP-HPLC 1. Introduction Simultaneous analysis of drugs present in multi-component dosage forms is challenging when they are present in ratio that varies widely. Aspirin, Caffeine and Orphenadrine citrate is one such combination that is co-administered in dosage form with ratio of their presence of 77: 6: 5 respectively. Aspirin, 2-(acetyloxy) benzoic acid is a prototypical analgesic used in the treatment of mild to moderate pain and has platelet aggregation inhibitory effect [1] . The analgesic, antipyretic and anti-inflammatory effects of aspirin are due to actions by both the acetyl and the salicylate portions of the intact molecule as well as by the active salicylate metabolite. The platelet aggregation-inhibiting activity of aspirin specifically involves the compound’s ability to act as an acetyl donor to cyclooxygenase. Caffeine,(1,3,7-trimethyl-2,3,6,7tetrahydro-1H-purine-2,6- dione) , a methylxanthineis a central nervous system stimulant used with NSAIDs for simple headaches [2] . Caffeine acts as an antagonist at adenosine receptors and thus promotes the release of neurotransmitters. Orphenadrine citrate, [dimethyl({2-[(2-methylphenyl) (phenyl) methoxy]ethyl}) amine is anticholinergic drug of ethanolamine antihistamine class used to treat painful muscle spasms [3] . It binds and inhibits both H1 and NMDA receptors. Literature review reveals a validated stability indicating RP- HPLC [4] method for the simultaneous determination of orphenadrine citrate, caffeine and aspirin in presence of aspirin degradation products, process related impurities and excipients and two derivative spectroscopic methods [5] for the simultaneous estimation of aspirin, caffeine, and orphenadrine citrate. The objective of the present study was to develop and validate a simple, rapid, accurate and precise RP-HPLC method for the simultaneous estimation of aspirin, caffeine and orphenadrine citrate. 2. Materials and Methods Reagents and Chemicals Gift samples of aspirin and caffeine were received from West Coast Laboratories, Mumbai. Orphenadrine citrate gift sample was obtained from RL Fine Chemicals, Bangalore. The tablets were purchased from the market. HPLC grade methanol and milli Q water were used for analysis. Instrumentation HPLC, Thermo scientific - Model Ultimate 3000 equipped with variable wavelength detector was used for the study. Sample injection was manual, through rheodyne injection valve equipped with 20 µl loop.The output signal was monitored and integrated using Chromeleon software. Chromatographic Conditions ACCLAIM TM C18 column (250 X 4.6 mm, 5 µ) was used for separation. The mobile phase containing methanol: phosphate buffer pH 3(65:35)v/v was delivered at the flow rate of 1 ml/m with detection wavelength set at 215 nm. Standard Stock Solutions Standard stock solutions of aspirin, caffeine, and orphenadrine citrate were prepared by dissolving 10 mg each in 10 ml mobile phase, methanol : phosphate buffer pH 3 (65:35)v/v . From the stock solution, working standard solution was prepared by appropriate dilution to 10 ml with the mobile phase. Similarly, the standard mixture of the three drugs was prepared in the mobile phase. Application of the method to the analysis of aspirin, caffeine and orphenadrine citrate in Tablets Tablets, 20 in number manufactured by Eon laboratories as Orphenadrine citrate –Aspirin - Caffeine tablet containing orphenadrine citrate 25 mg, aspirin 385 mg and caffeine 30 mg were used for the study. The average weight of each tablet was determined. Tablets were powdered and an amount equivalent to 10 mg of aspirin was transferred in to 100 ml volumetric flask and extracted with mobile phase by shaking for 20 min. The volume was made up with the same Paper ID: NOV152986 1170