Original Article DETERMINATION OF 2-CYANO-4’-BROMOMETHYL BIPHENYL GENOTOXIC IMPURITY IN IRBESARTAN DRUG SUBSTANCES USING HPLC TECHNIQUE S. SENTHIL KUMAR a* , RITESH KUMAR SRIVASTAVA b ,V. SRINIVAS RAO a a Faculty of Pharmacy, Pacific Academy of Higher Education and Research University, Udaipur 313004, Rajasthan, India, b Analytical Research and Development, Macleods Pharmaceuticals Limited, R and D Center, Mumbai 400059, India Email: chensenk@gmail.com Received: 12 Aug 2016 Revised and Accepted: 18 0ct 2016 ABSTRACT Objective: The objective of present study was to develop and validate a specific and sensitive HPLC method for the quantitative determination of genotoxic impurity 2-cyano-4’-bromomethyl biphenyl present in irbesartan drug substance. Methods: The development activity was conducted by HPLC with UV as a detector. The impurity was separated on Kromasil C18 250 x 4.6 mm, 5 µm analytical column with a mobile phase consisting of buffer pH 3.2 and acetonitrile in the ratio of 60:40 v/v at a flow rate 1.5 ml/min. The effluent was monitored by UV detection at 258 nm with column temperature maintained at 40 °C and the injection volume 30 μl. Acetonitrile was selected as diluent. Results: Validation activity was planned and completed based on the ICH guideline. The LOD and LOQ value were found to be 0.167 µg/g and 0.506 µg/g and accuracy results were well in the range 98.34 to 103.46 %. The linearity curve showed the correlation coefficient of 0.9999 and method very sensitive. Conclusion: From validation data, it was confirmed that the developed method is specific, sensitive, linear, precise and accurate for the determination of 2-cyano-4’-bromomethyl biphenyl genotoxic impurity in irbesartan drug substances. Keywords: Impurity, HPLC, TTC, Validation, ICH guidelines © 2016 The Authors. Published by Innovare Academic Sciences Pvt Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4. 0/) DOI: http://dx.doi.org/10.22159/ijpps.2016v8i12.14682 INTRODUCTION Irbesartan is a non-peptide tetrazole derivative drug, chemically described as 2-butyl-3-({4-[2-(2H-1,2,3,4-tetrazol-5-yl)phenyl] phenyl} methyl)-1,3diazaspiro [4.4] non-1-en-4-one [1]. Its molecular formula is C25H28N6O and molecular weight 428.53 [2]. The drug is used mainly for the treatment hypertension and commercially available in 75 mg, 150 mg and 300 mg of tablet strength either individually or combination with other antihypertensive drugs [3-5]. 2-cyano-4’-bromomethyl biphenyl is the most important intermediate used in the synthesis of irbesartan [6, 7]. This is identified as a genotoxic impurity in irbesartan according to the guidelines [8, 9]. Based on the threshold of toxicological concern (TTC) and the maximum daily dosage of irbesartan the evaluation limit of its genotoxic impurity is calculated 5.00 µg/g [8]. The chemical structures of irbesartan and 2-cyano-4’-bromomethyl biphenyl are presented in fig. 1 [6, 7]. Fig. 1: Chemical structure of (a) Irbesartan and (b) 2-cyano-4’- bromomethyl biphenyl Detailed literature survey reveals that the many HPLC methods are available for the determination of the drugs individually or in combination with other drugs [10-13]. However, very few analytical methods are available for the determination of genotoxic impurity in irbesartan drug by LC-MS/MS [14] and UPLC [15] technique. There was no HPLC method available for quantitative determination of 2- cyano-4’-bromomethyl biphenyl in irbesartan. Because of literature gap for the determination of 2-cyano-4’- bromomethyl biphenyl genotoxic impurity, we have reported here in a specific and sensitive HPLC analytical method, which separates the impurity from the drug substances and also could quantify the impurity at less than 50 % to the evaluation limit. MATERIALS AND METHODS Chemicals and reagents HPLC grade of water, orthophosphoric acid, methanol, triethylamine, and acetonitrile were purchased from Merck, Mumbai India. All pure drug substances and impurities are used for research purpose were procured in-house Macleods pharmaceutical LTD. Instrumentation The HPLC system consisted of Shimadzu model LC 2010 CHT, UV and PDA detector. The output signals were monitored and integrated using LC solution software. Sartorius analytical balance and Pico + pH meter were used. Chromatographic conditions The chromatographic separation was achieved on Kromasil C18 (250 x 4.6 mm, 5 µm) column using mobile phase consisting of buffer pH 3.2 and acetonitrile in the ratio of 60:40 v/v. The flow rate of the mobile phase was 1.5 ml/min and isocratic program run time was 60 min. The column temperature was maintained at 40 °C and the detection was monitored at 258 nm. The injection volume was 30 µl and acetonitrile used as diluent. Preparation of Buffer pH 3.2: Transferred 5.5 ml of orthophosphoric acid in 1000 ml of water, pH is adjusted to 3.2 with triethylamine. Preparation of standard and sample solutions A stock solution of 2-cyano-4’-bromomethyl biphenyl impurity was prepared by dissolving the appropriate amount of 2-cyano-4’- International Journal of Pharmacy and Pharmaceutical Sciences ISSN- 0975-1491 Vol 8, Issue 12, 2016