Original Article DEVELOPMENT AND VALIDATION OF DEFERASIROX-RELATED SUBSTANCE METHOD IN SOLID DOSAGE FORMS USING HPLC RUJUTA TRIVEDI 1* , BHANUBHAI SUHAGIA 2 1 Department of Pharmacy, Dharmsinh Desai University, Nadiad-387001, Gujarat, India. 2 Faculty of Pharmacy, Dharmsinh Desai University, Nadiad-387001, Gujarat, India * Corresponding author: Rujuta Trivedi; * Email: rujuta.trivedi@yahoo.co.in Received: 19 Dec 2024, Revised and Accepted: 07 Feb 2025 ABSTRACT Objective: A simple, sensitive, and reproducible Reverse-Phase High-Performance Liquid Chromatography (RP-HPLC) method was developed to quantitatively determine related substances in bulk and pharmaceutical dosage forms of Deferasirox (DFS). The method's performance was validated according to the present International Council on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) guidelines for specificity, limit of detection (LOD), limit of quantification (LOQ), linearity, accuracy, precision, and Robustness. The developed method can be used for routine analysis of the determination of DFS in the presence of degradation products and impurities. Methods: Chromatographic separation was achieved on Shimadzu shim pack GIS (150*4.6) mm5μm using isocratic elution mode, which included 0.1 % Formic acid in Water: Acetonitrile in gradient mode as mobile phase at a flow rate of 1.0 ml/min. And column temperature of 40 °C. Detection was performed at a wavelength of 254 nm coupled with an Ultra Violet (UV) detector. The Injection volume was 10 μl. The drug was exposed to various stress conditions in degradation studies, such as acidic, basic, oxidative, hydrolytic, photolytic, and thermal degradation. Results: The retention time of DFS was 8.1 min. The resolution of DFS and four impurities was greater than 2.0 for all pairs of components. The high correlation coefficient (r2>0.9990) values indicated clear correlations between the investigated compound concentrations and their peak areas within the test ranges. The Repeatability and intermediate precision expressed by the Relative Standard Deviation (RSD) were less than 10%. The accuracy evaluated by performing recovery studies via a spike method was 50-150%. Stress Degradation studies were carried out; however, no significant degradation was observed in any of the stress degradation conditions, implying a chemically stable drug formulation and the method used to measure stability is robust to minor variations; hence, the product is likely to maintain its quality and efficacy throughout its shelf life. Conclusion: Hence, a suitable, reliable, accurate, precise, and economical HPLC method was developed, which is ideal for analyzing DFS in both bulk and commercial formulations. Keywords: Deferasirox, Related substances, Impurity, Validation, Forced degradation, High-performance liquid chromatography © 2025 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/ijap.2025v17i2.53465 Journal homepage: https://innovareacademics.in/journals/index.php/ijap INTRODUCTION Iron is essential in all living organisms and involves many physiological processes, such as oxygen transport and energy transduction. It is estimated that more than a quarter of the human population is affected at some stage of their life by abnormalities of iron metabolism, particularly iron deficiency anemia. Similarly, millions of people suffer from iron overload, including hereditary hemochromatosis and Thalassemia Intermedia (TI), which are caused by increased iron absorption. Excess iron is toxic in these and other iron-loading conditions, causing increased morbidity and mortality due to tissue damage. DFS is an oral iron chelator. Its primary use is to reduce chronic iron overload in patients receiving long-term blood transfusions for conditions such as beta- thalassemia and other chronic anemia [1-6]. Iron chelators, such as deferoxamine or deferasirox, are commonly used to treat iron overload disorders. If the iron chelator or its impurities degrade, the degradation products may become toxic or cause harmful side effects. For example, impurities might lead to adverse patient reactions, such as liver damage, kidney issues, gastrointestinal discomfort, and increased toxicological burden due to unintended or harmful by-products from degradation or contamination. A reliable method can be developed and validated to identify and quantify these impurities; manufacturers can ensure that their product is safe for patient use. Regulatory agencies such as the FDA, EMA, and ICH have stringent guidelines for the development, validation, and testing of drugs, particularly in the case of impurities. The ICH Q3A and Q3B guidelines (on impurities in new drug substances and products) provide detailed standards for identifying, quantifying, and controlling impurities. Proper method development and validation of the analytical technique (such as HPLC, GC, or UV spectrophotometry) help ensure compliance with regulatory standards. This is essential for obtaining approval for the drug and maintaining it in the market [9]. By creating specific impurities, researchers can validate analytical methods for their ability to detect, quantify, and separate these impurities from the API. This ensures that the method is robust and reliable. For regulatory approval, methods must be validated to demonstrate their accuracy, precision, specificity, sensitivity, and Robustness [10]. Various factors such as experimental design and analysis conditions such as matrix type, column type, mobile phase, elution modes, detection wavelengths, and therapeutic category influence the separation and identification of the impurities and degradation products [11]. Hence, an attempt was made to develop an RP-HPLC method that is simple, rapid, accurate, precise, specific, economical, sensitive, and stable, indicating the estimation of DFS and related substances in bulk and pharmaceutical tablet formulation. Deferasirox is a white to slightly yellow powder. It is the first oral medication approved in the USA for this purpose. It was approved by the United States Food and Drug Administration (FDA) in November 2005 [7]. It is approved in the European Union by the European Medicines Agency (EMA) for children 6 years and older for chronic iron overload from repeated blood transfusions. Its low molecular weight and high lipophilicity allow the drug to be taken orally, unlike deferoxamine, which must be administered by intravenous infusion. Along with deferiprone, DFS seems capable of removing iron from cells (cardiac myocytes and hepatocytes) and iron from the blood. Chemically it is 4- [3,5-bis(2-hydroxyphenyl),4-[3,5-bis(2-hydroxyphenyl)-1,2,4-triazol-1- yl] benzoic acid 4-[3,5-bis(2-hydroxyphenyl)-1,2,4-triazol-1-yl] benzoic acid [8]. The structure of DFS is shown in fig. 1. International Journal of Applied Pharmaceutics ISSN- 0975-7058 Vol 17, Issue 2, 2025