Open Access Research Article Musirike et al., Pharm Anal Acta 2015, 6:10 10.4172/2153-2435.1000431 Volume 6 • Issue 10 • 1000431 Pharm Anal Acta ISSN: 2153-2435 PAA, an open access journal Abstract A novel high speed, high resolution Reverse phase-UPLC method was developed for the quantification of related substances in Febuxostat drug substance. The separation of drug from the possible impurities was achieved on a Halo C18 column. The innovative approach of using stationary phase with sub 2 µ particles provides a comprehensive combination of selectivity and speed. 10 mM mono basic potassium phosphate buffer at pH 2.7 and acetonitrile mixture was selected as mobile phase. Flow rate and detection were kept at 0.8 mL/min and 320 nm respectively. The developed UPLC method was subjected to validation parameters. System precision, accuracy, specificity, limit of detection, limit of quantification and linearity were established as per the guidelines recommended by ICH. Stability indicating nature of the method was also performed by exposing the sample under various conditions like acid, base, peroxide and photo stability exposures. Total analysis run time 7.0 minutes indicates the speed and cost saving initiation of the developed method. Using the method one can carry out the quantitative estimation of related substances in Febuxostat drug substance, further the same method can be adopted for determination of drug substance assay also. Development and Validation of Reverse Phase-Ultra Performance Liquid Chromatographic Method for Estimation of Related Substances in Febuxostat Drug Substance Maheswara Reddy Musirike*, Hussain Reddy K and Useni Reddy Mallu Department of Chemistry, Sri Krishnadevaraya University, Ananthapur, Andhra Pradesh- 515003, India *Corresponding author: Maheswara Reddy Musirike, Department of Chemistry, Sri Krishnadevaraya University, Kadiri- Ananthapur Hwy, kandukuru, Anantapur, Andhra Pradesh 515591, India, Tel: 085542 55700; E-mail: musirikemahesh@gmail.com Received September 16, 2015; Accepted October 16, 2015; Published October 19, 2015 Citation: Musirike MR, Hussain Reddy K, Mallu UR (2015) Development and Vali- dation of Reverse Phase-Ultra Performance Liquid Chromatographic Method for Estimation of Related Substances in Febuxostat Drug Substance. Pharm Anal Acta 6: 431. doi:10.4172/21532435.1000431 Copyright: © 2015 Musirike MR, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits un- restricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Keywords: Febuxostat; Ultra performance liquid chromatography; Hyperuricemia with gout; Related compounds; Xanthine oxidase inhibitor Introduction Febuxostat is a xanthine oxidase inhibitor indicated for the chronic management of hyperuricemia in patients with gout [1]. e recommended starting dose of drug is 40 mg once daily. For patients who do not achieve a serum uric acid less than 6 mg per dL aſter 2 weeks with 40 mg, Uloric 80 mg is recommended. e primary mode of action of a xanthine oxidase inhibitor, achieves its therapeutic effect by decreasing serum uric acid.e objective of this work was to develop a cost effective ultra fast reverse phase UPLC method, the developed method was validated as per regulatory guidelines and transfer the method to quality control lab for analysis of related substances in Febuxostat. As per the literature Febuxostat Determination was done by spectrophotometric method [2] and RP-HPLC [3,4] methods. LC-MS/MS [5-7] assay method was proposed for bioequivalence and pharmacokinetics evaluation. Since this drug is being marketed in domestic and international market the present investigation by the author was to develop a rapid, accurate and precise RP-UPLC method [8-10] for the determination of related substances. e aim of this paper is to develop a cost effective and fast method. e run time for the newly developed method was 7.0 minutes. e innovative approach of using stationary phase with sub 2 µ particles [11-14] provides a comprehensive combination of selectivity and speed. e validation parameters [15,16] provide valuable information on precision accuracy, limit of detection, limit of quantification and linearity of related substances. e method was subjected to validation according to ICH requirements [17,18] (Figure 1). Materials and Methods Instrumentation and reagents Test samples and reference standards of Febuxostat were donated by Apotex India pvt ltd. Acetonitrile was purchased from Ultra Scan Ltd. Ultra performance liquid chromatography from Waters with gradient elution. MilliQ purification system was used to get HPLC grade water. Halo C18, 100 x 2.1 mm 2.7 µm column purchased from advanced materials technology. Chromatographic conditions e chromatographic separation was achieved on a Halo C18, 100 x 2.1 mm 2.7 µm Column. Mobile phase consists of 10 mM monobasic phosphate buffer with 0.2% Triethyl amine and pH of the solution was adjusted to 2.7. HPLC grade acetonitrile was used as organic modifier. Mobile phase flow rate was kept at 0.8 mL/min. Gradient program was set as Time/% of solution B: 0/40, 3/40, 3.5/70, 4/75, 5/80, 5.5/40, 7/40. Column temperature was maintained at 50 ° C and detection was carried at 320 nm. Sample compartment was maintained at 10 ° C with an injection volume of 1 µL. Preparation of standard and sample A mixture of standard solution was prepared by weighing febuxostat and its related compounds to yield a final concentration of 0.10% of febuxostat and 0.15% of each Impurity A, Impurity B, Impurity C and Impurity D with respect to the sample concentration P h a r m a c e u t i c a A n a l y t i c a A c t a ISSN: 2153-2435 Pharmaceutica Analytica Acta