DEVELOPMENT AND VALIDATION OF A STABILITY-INDICATING RP-HPLC METHOD FOR THE DETERMINATION OF XIPAMIDE IN PURE AND DOSAGE FORMS Original Article HEBA M. EL-SAYED* a , SOAD S. ABD EL-HAY a,b a Analytical Chemistry Department, Faculty of Pharmacy, Zagazig University, Zagazig 44519,Egypt, b Pharmaceutical Chemistry Department, Faculty of Pharmacy, King Abdul-Aziz University, Saudi Arabia Email: heba328@yahoo.com Received: 25 Feb 2016 Revised and Accepted: 08 Apr 2016 ABSTRACT Objective: A simple, selective, precise and stability-indicating RP-HPLC-method was developed and validated for the determination of xipamide (XIP). Methods: Stability tests were done through exposure of the analyte solution to thermal, photolytic, hydrolytic and oxidative stress conditions. The chromatographic separation was carried out in less than five min on a RP stainless-steel C-18 analytical column (150 mm ×4.6 mm ID, 5 µm) with an isocratic elution system of 0.023 M orthophosphoric acid of pH 2.6 and acetonitrile as the mobile phase in the ratio of 60: 40 at 1.5 ml/min flow rate at room temperature. A diode array UV was used at 220 nm for detection. Results: The degradation products were well separated from the pure drug. The elution time of XIP was found to be 4.561±0.024 min. The method was validated in terms of linearity, accuracy, precision, limit of detection (LOD), limit of quantitation (LOQ) and robustness. Good linearity was found in the concentration range of 1–100 µg/ml with a correlation coefficient of 0.9999. Intraday and interday precision were within 1.4%. LOD and LOQ were 0.088 μg/ml and 0.267 μg/ml, respectively and percentage recovery of XIP was found to be 99.92±1.02 %. Conclusion: The proposed method was successfully applied to the determination of XIP in pure form and in its pharmaceutical preparation without interference from its degradation products. Keywords: Xipamide, Stability indicating RP-HPLC, Stress degradation, Pure form, Dosage forms © 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/) INTRODUCTION XIP is 4-chloro-2, 6-dimethyl-5-sulfamoylsalicylanilide [1]. It has a moderately powerful diuretic action and is used for the treatment of high blood pressure and edema [2]. It decreases active reabsorption of sodium and accompanying chloride by binding to the chloride site of the electroneutral Na + /Cl - co-transport system and inhibiting its action [3]. The structural formula of XIP is shown in fig. 1. Fig. 1: Chemical structure of XIP Despite its wide use, few procedures have been developed for its determination. These include spectrophotometry [4-6], spectro- fluorimetry [6], TLC-densitometry [5] and HPLC [7-12]. However, there is no reported LC method for stability determination of XIP, so the development of a simple, reproducible and selective analytical HPLC method would greatly aid in XIP stability determination either in pure or dosage forms. The present manuscript describes the degradation behavior of XIP under acidic, basic, oxidative and photolytic conditions. Optimization of LC conditions to separate the drug from its degradation products on a RP C18 column in less than 5 min and method validation were also achieved. MATERIALS AND METHODS Chemicals and reagents Pharmaceutical grade XIP (99.09%) was kindly provided by EIPICO Pharmaceutical Industries Company, Cairo, Egypt. Analytical reagent grade chemicals were used in all experiments. HPLC grade methanol, acetonitrile (TEDEA, Fairfield, USA). HCl, NaOH and H2O2 (Merck, USA), orthophosphoric acid 85% (Burdick & Jackson, USA) were used. Fresh double distilled water was used throughout the analysis. The pharmaceutical preparation used was Epitens ® (EIPICO, Egypt, 10 mg and 30 mg triamterene/tablet). Instrumentation and chromatographic conditions HPLC apparatus (Agilent 1100) consisted of quaternary pump G1310A with solvent cabinet quaternary, vacuum degasser G1322A and a four-channel gradient pump; autosampler G1313A, variable wavelength detector (VWD) G1314A with the standard flow cell 10 mm path length, 14 μl volume, 40 bar maximum pressure. LC separations were performed on an Agilent 5 µm Hypersil BDS C18 column (150 mm x 4.6 mm ID). pH meter (Metrohm, USA) was used. For the determination of XIP and its degradation products, an isocratic mobile phase consisting of 0.023 M orthophosphoric acid of pH 2.6 and acetonitrile as the mobile phase in the ratio of 60: 40 was used, 1.5 ml/min flow rate at room temperature. pH is adjusted using H3PO4 and NaOH. 10 µl of sample was injected each run. Detection was achieved at 220 nm. Preparation of stock solutions Standard stock solution of XIP (0.1 mg/ml) was prepared by dissolving 10 mg of the drug in methanol, sonicated and completed to 100 ml with methanol in a 100 ml volumetric flask. The standard working solutions were prepared by diluting aliquots of the stock solution with methanol to obtain concentrations ranging from 1–140 µg/ml. Construction of calibration curves The calibration graph was constructed by plotting the peak areas obtained at wavelength 220 nm against the corresponding injected concentrations of XIP. Assay of tablets A total of 10 tablets of Epitens ® were finely powdered. An accurately weighed quantity of the powder equivalent to 10 mg XIP was International Journal of Pharmacy and Pharmaceutical Sciences ISSN- 0975-1491 Vol 8, Issue 5, 2016