Development, Optimisation, and Validation of a Stability-Indicating Reverse Phase HPLC Method for Simultaneous Estimation of Tenofovir and Emtricitabine in Drug Substance and Combination Formulations

Abstract

The quantification of impurities and their degradants in the active pharmaceutical ingredients (APIs) of tenofovir and emtricitabine, as well as in their combinations, is essential for quality control analysis and for indicating product stability for product approval. The method was developed and validated in accordance with the International Conference on Harmonisation (ICH) Q2 R2 guidelines. This sensitive method was employed to detect and quantify impurities in both APIs and finished dosage forms for 50 mg/500 mg, 50 mg/850 mg, and 50 mg/1000 mg extended-releasetablet formulations. The same samples of the reference formulations were compared with the generic compositions. The analytical method developed for tenofovir and emtricitabine quantification utilised 50 mM ammonium acetate adjusted pH to 4.7 ± 0.05 with glacial acetic acid as mobile Phase A, consisting of as the mobile phase A, and Mobile Phase B, a mixture of methanol: buffer: acetonitrile in the ratio of 50:40:10%v/v/v/v. Mobile phase C is 100% acetonitrile in a gradient composition. The diluent in the mixing of 25mM ammonium acetate buffer was adjusted to 5.0 ± 0.05, and methanol in the ratio of 80:20 %v/v. This was conducted using a Ghost-Hunter column, 50*4.6mm YMC Pack ODS-AQ, 250*4.6mm, 5µm (Agela & GHC0505-0 A for Ghost Column)  columns with a column oven temperature of 35°C, an injection volume of 10 µL, and a flow rate of 0.8 mL/min at 260 nm, employing The limit of detection was established at 0.05% relative to the test concentrations, ensuring the method's capability to detect all known and unknown impurities in both analytes within the APIs and the finished dosage forms