Original Article HYDROLYTIC DEGRADATION STUDY OF ROXADUSTAT BY RP-HPLC AND HPTLC MRINALINI C. DAMLE 1* , JAY A. SONULE 2 1 Pharmaceutical Quality Assurance, Aissms College of Pharmacy, Pune-411001 Maharashtra India, 2 Master of Pharmacy, Department of Pharmaceutical Quality Assurance, AISSMS College of Pharmacy, Pune-411001 Maharashtra India * Corresponding author: Mrinalini C. Damle; * Email: damle_mc@aissmscop.com Received: 18 May 2023, Revised and Accepted: 13 Jun 2023 ABSTRACT Objective: Simple, rapid RP-HPLC and HPTLC methods have been developed in order to study the degradation of Roxadustat under various stress conditions. The Kinetics of hydrolytic degradation is studied. Methods: Optimum separation of Roxadustat and its degradation products was achieved using the following conditions in HPLC, Agilent eclipse XDB-C8 (150×4.6 mm) column, the mobile phase was composed of methanol: phosphate buffer (pH 5, 0.05 M) (70:30 v/v) with UV detection at 262 nm. The flow rate was at 1.0 ml/min. The RT was 4.6±0.02 min. HPTLC work for Roxadustat was performed on Aluminium plates precoated with silica gel 60 F254, (10 cm × 10 cm with 250 μm layer thickness). The mobile phase was composed of Toulene: Ethyl Acetate: Glacial acetic acid (5:5:0.5 v/v/v) and then scanned. The system was found to give a compact spot for Roxadustat (Rf value of 0.58±0.02). Results: In HPLC the calibration curves plotted were found to be linear over the concentration range of 2.5-25μg/ml, with a correlation coefficient of R 2 =0.9994. In HPTLC the calibration curves plotted were found to be linear over the concentration range of 500-2500 ng/band, with a regression coefficient of R 2 =0.9957. The analytical performance of the proposed methods was validated as per ICH Q2 (R1) guidelines. The degradant peaks were well resolved from the Roxadustat peak. Significant degradation was observed in acid hydrolysis, alkali hydrolysis, and oxidative degradation. The drug is relatively stable towards photolysis, neutral hydrolysis, and thermal conditions. Conclusion: In the current work, simple RP-HPLC and HPTLC analytical methods for the determination of Roxadustat in the presence of its degradation products have been developed. The information presented herein could be very useful while developing formulation procedures to prevent hydrolytic degradation. It can be used as a routine quality control test. Keywords: RP-HPLC, HPTLC, Roxadustat, Hydrolytic degradation, Stress conditions © 2023 The Authors. Published by Innovare Academic Sciences Pvt Ltd. This is an open access article under the CC BY license (https://creativecommons.org/licenses/by/4.0/) DOI: https://dx.doi.org/10.22159/ijpps.2023v15i8.48355. Journal homepage: https://innovareacademics.in/journals/index.php/ijpps. INTRODUCTION Roxadustat is used in the treatment of anemia. It is an oral inhibitor of hypoxia-inducible factor (HIF) prolyl hydroxylase, which promotes erythropoiesis and controls iron metabolism [1, 3]. The chemical name of Roxadustat is 2-[(4-hydroxy-1-methyl-7- phenoxyisoquinoline-3-carbonyl) amino] acetic acid. Its molecular formula is C19H16N2O5 and its molecular weight is 352.3 g/mol, respectively. The chemical structure of Roxadustat is shown in fig. 1. Fig. 1: Structure of roxadustat An important function of the kidneys is the production of erythropoietin, a hormone that stimulates the production of red blood cells [4]. In chronic kidney disease (CKD), the kidneys are not functioning at capacity and there can be a disruption in the production of erythropoietin that can lead to anemia [5, 6]. Anemia is a chronic kidney disease complication associated with increased risks of death and complications [7]. It decreases endogenous erythropoietin production, functional iron deficiency, and inflammation with increased hepcidin levels, among others [8]. Roxadustat stabilizes HIF-α (hypoxia-inducible factor) subunits, which increases HIF transcriptional activity. The increased transcriptional activity produces functional activation of early- response target genes that encode proteins like erythropoietin, erythropoietin receptor, heme biosynthesis enzymes, and proteins that facilitate iron uptake and transport. For the treatment of anemia in patients with chronic kidney diseases who are dependent on dialysis, non-dependent on dialysis, and myelodysplastic syndromes, Roxadustat is a highly effective first-in-class HIF-PHD (hypoxia- inducible factor-prolyl hydroxylase inhibitor) [9, 10]. As per the literature survey for Roxadustat Liquid chromatography- tandem mass spectrometry, UPLC–MS method and UV method were reported, but no stability indicating HPLC and HPTLC method is reported [11, 13]. The present work describes the simple, accurate chromatographic analytical methods for determining Roxadustat. MATERIALS AND METHODS Chemical and reagents Roxadustat was kindly gifted by industry. Other chemicals and reagents like Methanol (HPLC grade), Potassium dihydrogen phosphate (AR grade), Dimethyl sulfoxide (DMSO), Glacial Acetic Acid (AR grade), Toulene (AR grade), Ethyl Acetate (AR grade), Hydrochloric acid (AR grade), Sodium Hydroxide (AR grade), and Hydrogen Peroxide (AR grade) were procured from LOBA CHEMIE PVT. LTD., Mumbai. Instrumentation and chromatographic conditions Instruments used in this method were HPTLC system (CAMAG) comprising of TLC Scanner III, Linomat 5 applicator, Software [win CATS (version 1.4.3)], Microliter syringes [Hamilton (100 µl)], TLC plates (Merck’s aluminum TLC plate precoated with silica gel 60 F254), Twin trough glass chamber. The HPLC was done on HPLC-PDA International Journal of Pharmacy and Pharmaceutical Sciences Print ISSN: 2656-0097 | Online ISSN: 0975-1491 Vol 15, Issue 8, 2023