Sara Esmaeilzadeh1, Hadi Valizadeh2, Parvin Zakeri-Milani3. 1. Faculty of Pharmacy and Research Center for Pharmaceutical Nanotechnology, Tabriz University of Medical Sciences, Tabriz, Iran.; Student Research Committee, Faculty of Pharmacy, Tabriz University of Medical Sciences, Tabriz, Iran. 2. Drug Applied Research Center and Faculty of Pharmacy, Tabriz University of Medical Sciences, Tabriz, Iran. 3. Liver and Gastrointestinal Diseases Research Center and Faculty of Pharmacy, Tabriz University of Medical Sciences, Tabriz, Iran.
Abstract
PURPOSE: The main goal of this study was development of a reverse phase high performance liquid chromatography (RP-HPLC) method for flutamide quantitation which is applicable to protein binding studies. METHODS: Ultrafilteration method was used for protein binding study of flutamide. For sample analysis, flutamide was extracted by a simple and low cost extraction method using diethyl ether and then was determined by HPLC/UV. Acetanilide was used as an internal standard. The chromatographic system consisted of a reversed-phase C8 column with C8 pre-column, and the mobile phase of a mixture of 29% (v/v) methanol, 38% (v/v) acetonitrile and 33% (v/v) potassium dihydrogen phosphate buffer (50 mM) with pH adjusted to 3.2. RESULTS: Acetanilide and flutamide were eluted at 1.8 and 2.9 min, respectively. The linearity of method was confirmed in the range of 62.5-16000 ng/ml (r(2) > 0.99). The limit of quantification was shown to be 62.5 ng/ml. Precision and accuracy ranges found to be (0.2-1.4%, 90-105%) and (0.2-5.3 %, 86.7-98.5 %) respectively. Acetanilide and flutamide capacity factor values of 1.35 and 2.87, tailing factor values of 1.24 and 1.07 and resolution values of 1.8 and 3.22 were obtained in accordance with ICH guidelines. CONCLUSION: Based on the obtained results a rapid, precise, accurate, sensitive and cost-effective analysis procedure was proposed for quantitative determination of flutamide.
PURPOSE: The main goal of this study was development of a reverse phase high performance liquid chromatography (RP-HPLC) method for flutamide quantitation which is applicable to protein binding studies. METHODS: Ultrafilteration method was used for protein binding study of flutamide. For sample analysis, flutamide was extracted by a simple and low cost extraction method using diethyl ether and then was determined by HPLC/UV. Acetanilide was used as an internal standard. The chromatographic system consisted of a reversed-phase C8 column with C8 pre-column, and the mobile phase of a mixture of 29% (v/v) methanol, 38% (v/v) acetonitrile and 33% (v/v) potassium dihydrogen phosphate buffer (50 mM) with pH adjusted to 3.2. RESULTS:Acetanilide and flutamide were eluted at 1.8 and 2.9 min, respectively. The linearity of method was confirmed in the range of 62.5-16000 ng/ml (r(2) > 0.99). The limit of quantification was shown to be 62.5 ng/ml. Precision and accuracy ranges found to be (0.2-1.4%, 90-105%) and (0.2-5.3 %, 86.7-98.5 %) respectively. Acetanilide and flutamide capacity factor values of 1.35 and 2.87, tailing factor values of 1.24 and 1.07 and resolution values of 1.8 and 3.22 were obtained in accordance with ICH guidelines. CONCLUSION: Based on the obtained results a rapid, precise, accurate, sensitive and cost-effective analysis procedure was proposed for quantitative determination of flutamide.
Entities:
Keywords:
Acetanilide; Flutamide; High pressure liquid chromatography; ICH guidelines; Protein binding
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