DEVELOPMENT AND VALIDATION OF RP-HPLC METHOD FOR SIMULTANEOUS DETERMINATION OF RELATED SUBSTANCES PRESENT IN THE PHARMACEUTICAL DOSAGE FORM OF EMTRICITABINE, TENOFOVIR ALAFENAMIDE AND DOLUTEGRAVIR

Abstract

A novel, rapid and an accurate reversed phase high-performance liquid chromatography (RP-HPLC) method was developed and validated for the simultaneous quantification of emtricitabine and tenofovir related impurities namely emtricitabine s-oxide (emtricitabine impurity-A) and tenofovir (S)-propanol (tenofovir impurity-A), respectively in the combined pharmaceutical dosage form of emtricitabine, dolutegravir and tenofovir alafenamide. The separation was accomplished on Inertsil ODS C18 (250  4.6 mm, 5μ) column using 0.5 M potassium dihydrogen phosphate buffer: acetonitrile (80: 20, v/v), pH 4.5 ± 0.1 maintained by 0.1 % o-phosphoric acid as mobile phase delivered at a flow rate of 1.0 mL/min. Detection was carried out at 260 nm. Emtricitabine impurity-A and tenofovir impurity-A analytes were eluted at retention times of 10.5 and 16.6 min, respectively. The developed method was successfully validated for various parameters like system suitability, linearity, accuracy, precision, robustness, limit of detection and quantification in accordance with ICH guidelines. The calibration curves were linear over the concentration range of 0.125-7.500 μg/ml for emtricitabine impurity-A and tenofovir impurity-A with correlation coefficients  0.998. The validated method was successfully applied for the simultaneous estimation of emtricitabine s-oxide (emtricitabine impurity-A) and tenofovir (S)-propanol (tenofovir impurity-A) in fixed-dose combination comprising of emtricitabine, dolutegravir and tenofovir alafenamide.