~ 60 ~ The Pharma Innovation Journal 2018; 7(8): 60-72 ISSN (E): 2277- 7695 ISSN (P): 2349-8242 NAAS Rating: 5.03 TPI 2018; 7(8): 60-72 © 2018 TPI www.thepharmajournal.com Received: 04-06-2018 Accepted: 06-07-2018 Alka N Choudhary Quality Assurance Research Laboratory, Department of Pharmaceutical Sciences, Shri Guru Ram Rai University, Patel Nagar, Dehradun, India Ajay Chaudhary Quality Assurance Research Laboratory, Department of Pharmaceutical Sciences, Shri Guru Ram Rai University, Patel Nagar, Dehradun, India Kamlesh K Dutta Quest Pharmaceuticals Private Limited, Birganj, Nepal Correspondence Alka N Choudhary Quality Assurance Research Laboratory, Department of Pharmaceutical Sciences, Shri Guru Ram Rai University, Patel Nagar, Dehradun, India Analytical method development for simultaneous estimation for drug content and release of levodopa, Carbidopa and Entacapone in combined dosage form by rp-HPLC Alka N Choudhary, Ajay Chaudhary and Kamlesh K Dutta Abstract Objective: The present study was designed to develop a RP-HPLC method which is capable of estimating the content and release of Levodopa, Carbidopa and Entacapone simultaneously in combined dosage form. Methods: Selection of diluents for a clean sample was based on the solubility to provide sink condition for the target analytes in common. C18 column was selected as the stationary phase. Gradient Scouting Technique was opted to screen whether isocratic or gradient method is the possibility with different mobile phases. Detection wavelength was selected with help of PDA detector screening over the wavelength 200 – 400 nm. Strength & pH of buffer, column temperature, flow rate and injection volume were optimized by performing trials at different buffer strengths & pHs, column temperatures, flow rates and injection volumes respectively. Results: Simultaneous separation of Levodopa, Carbidopa and Entacapone was achieved on a Sunshell C18 column (150 mm x 4.6 mm x 2.6 μm) as stationary phase with combination of mobile phases; Phosphate Buffer, pH 3.0 and Methanol in gradient mode at flow rate of 1.0 ml/min, column temperature kept at 35ºC, PDA/UV detector at 280 nm and injection volume 5 μL. The diluents used for sample preparation was Phosphate Buffer, pH 5.5. The retention time of Levodopa, Carbidopa and Entacapone were found to be 2.27, 3.43 and 11.77 minutes respectively with tailing factor ≤ 1.5, resolution > 2 and NTP > 2000 for each analyte peak. Conclusion: Simple, economical and rapid method was developed and further validated successfully as per ICH: Q2 (R1). Keywords: Parkinson’s Disease, levodopa, Carbidopa, Entacapone, rp-HPLC, method development, validation, ICH: q2 (r1) 1. Introduction Parkinson’s disease (PD) is a type of movement disorder that can affect the ability to perform common, daily activities. The disease developes as cell loss occurs in a very specific region of the brain called substantia nigra. The nerve cells, or neurons, in this region produce a specific type of neurotransmitter (a chemical messenger that allows neurons to communicate) called dopamine. The neurotransmitter dopamine helps to regulate movement [1, 2] . Symptoms of Parkinson`s disease are related to depeletion of dopamine. But administration of dopamine is ineffective in the treatment of Parkinson’s disease. This is because it does not cross blood-brain barrier. However, Levodopa, the metabolic precursor of dopamine, does cross the blood-brain barrier, and presumably is converted to dopamine in the brain [3] . Hence, Levodopa is used in the treatment of Parkinson’s disease. Levodopa is the most effective medicine for relieving symptoms of Parkinson’s disease. However, Levodopa, is extensively metabolized to various metabolites and only small portion of a given dose is transported unchanged to the central nervous system. Two major pathways of metabolism are decarboxylation by dopa decarboxylase (DDC) and O-methylation by catechol-O- methyltransferase (COMT) [4] . Levodopa when administered concomitantly with Carbidopa and Entacaopne, plasma levels of Levodopa are greater and more sustained than after administered of Levodopa alone [5] . Carbidopa is an inhibitor of dopa decarboxylase (DDC), and Entacapone is an inhibitor of catechol-O-methyltransferase (COMT) preventing decarboxylation and O-methylation of Levodopa outside of the central nervous system respectively providing greater and sustained plasma level of Levodopa available to cross