METHOD DEVELOPMENT AND VALIDATION OF THE CHROMATOGRAPHIC ANALYSIS OF FLUTICASONE PROPIONATE AND SALMETEROL XINAFOATE COMBINATION IN SOLUTIONS AND HUMAN PLASMA USING HPLC WITH UV DETECTION Original Article MOHAMMAD JAMAL A. SHAMMOUT 1* , HAMMAM B. YOUSEF 2 , KHALID H. ABU-SHANDI 2 , MOHAMMAD I. ALOMARI 3 , MOHAMMAD R. HASAN 4 , ATEF O. AL-OTHMAN 5 , WALEED A. MANASREH 6 1 Department of Basic and Applied Sciences, Zarqa University College, Al-Balqa Applied University, Al-Salt 19117, Jordan, 2 Department of Chemistry and Chemical Technology, Faculty of Science, Tafila Technical University, Tafila, Jordan, 3 Department of Chemistry, Faculty of Arts and Sciences, University of Petra, Amman, Jordan, 4 Bioanalytical Lab, Jordan Center for Pharmaceutical Research (JPCR), Amman, Jordan, 5 Faculty of Applied Medical Sciences, Al-Ahliyya Amman University, Amman, Jordan, 6 Department of Chemistry, Faculty of Science, Mutah University, Karak, Jordan * Received: 03 Apr 2021, Revised and Accepted: 29 May 2021 Email: dr.mj-shammout@bau.edu.jo ABSTRACT Objective: A simple, Rapid, and sensitive HPLC method utilizing UV detection was developed and validated for the simultaneous estimation of Fluticasone propionate (FP) and Salmeterol xinafoate (SX) in solutions and in vitro human plasma. Methods: Chromatographic analysis was done on SUPELCO ® RP-C18 column (150 x 4.6 mm, 5 μm particle size) with an isocratic mobile phase composed of methanol, acetonitrile, and water (50:20:30, v/v) mixture while flow rate was set to 1 ml/min. Detection with UV at maximum absorbance wavelength ( ʎ max Results: Method was accurate and precise over a linear (R ) values of 236 and 252 for FP and SX, respectively. Spiked plasma samples were liquid-liquid extracted by diethyl ether and reconstituted using methanol. 2 The developed method was successfully applied for the analysis of FP and SX in spiked human plasma samples. The method is considered to be accurate and precise over a linear (R >0.995) range of (0.067-100 µg/ml) and (0.0333-50 µg/ml) for FP and SX, respectively. LOD/lOQ values were 0.13/0.6 and 0.06/0.3 µg/ml for FP and SX, respectively. 2 Conclusion: This validated method revealed simple and cheap extraction procedures and detectors, non-buffered mobile phase, and short retention times with excellent resolution. >0.9969) range of (6.67-66.67 µg/ml) and (3.33-33.3 µg/ml) for FP and SX, respectively. Extraction efficiency was approved by recovery values of (94.98–102.46 %) and (96.54–102.62 %) for FP and SX, respectively. Keywords: Fluticasone propionate, Salmeterol xinafoate, HPLC UV, Method validation, Human plasma © 2021 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.2021v13i4.41678. Journal homepage: https://innovareacademics.in/journals/index.php/ijap INTRODUCTION Glucocorticosteroids and corticosteroids are classes of steroid hormones naturally synthesized in the adrenal cortex from cholesterol; regulate many aspects of metabolism and immune functions [1]. Fluticasone propionate (FP) (fig. 1a), a new generation of glucocorticosteroids, is considered as one of the important medications for asthma diseases. It has a strong therapeutic effect against bronchi inflammation. Salmeterol Xinafoate (SX) (fig. 1b) is a new long-acting β2-agonist used in the treatment of nocturnal airway obstruction and has proved to be highly effective in this aspect as well [2, 3]. Fig. 1: Chemical structure of fluticasone propionate (a) and salmeterol Xinafoate (b) The two drugs are formulated as dry powder inhalers or pressurized metered-dose inhalers individually or in the combined formulation. However, it is not yet known whether using Salmeterol xinafoate (SX) alone or in combination with Fluticasone propionate (FP) constitutes the best treatment [4, 5]. Several chromatographic techniques have been published in the literature for analysis purposes of fluticasone propionate (FP) and/or, salmeterol xinafoate (SX) in their pharmaceutical preparations and different matrices. For example these methods include UV- spectrometric methods [6, 7], HPLC with UV detection methods [8-13], HPLC-MS/MS methods [14, 15], UPLC-MS/MS methods [16, 17], UPLC- PDA method [18], and HPTLC method [19, 20]. In this article, a new validated method has been developed for the simultaneous estimation of fluticasone propionate (FP) and salmeterol xinafoate (SX), combined in spiked human plasma samples using High-Performance Liquid Chromatography (HPLC) with UV detection. The method includes a simple solvent extraction technique for recovering fluticasone propionate (FP) and salmeterol xinafoate (SX) from spiked human plasma samples. According to the literature survey, No HPLC method with UV detection for simultaneous assay of fluticasone propionate (FP) and salmeterol xinafoate (SX) in the human plasma was reported. MATERIALS AND METHODS Reagents The studied drugs, Fluticasone Propionate (FP), Salmeterol Xinafoate (SX), and β-Estradiol (βE) were supplied by SIGMA and used without any further treatment. Highly pure Methanol, Acetonitrile (HPLC grade) were bought from TEDIA. Deionized International Journal of Applied Pharmaceutics ISSN- 0975-7058 Vol 13, Issue 4, 2021