Design and validation of a robust RP-UPLC approach for simultaneous quantification of emtricitabine, tenofovir, and nelfinavir in active pharmaceutical ingredients and finished dosage products

Abstract

Background: Fixed-dose combinations of antiretroviral agents such as Emtricitabine (EMT), Tenofovir (TEN), and Nelfinavir (NEL) are central to Human Immunodeficiency Virus (HIV) therapy. Reliable quantification of these drugs is essential for pharmaceutical quality assurance and therapeutic drug monitoring (TDM) in clinical research. Methodology: A reverse-phase ultra-performance liquid chromatography (RP-UPLC) method was developed to simultaneously estimate EMT, TEN, and NEL in bulk and combined dosage forms. Separation was performed on a Waters CHS C18 column using potassium dihydrogen phosphate buffer and acetonitrile (65:35 v/v, pH 3.0) at a flow rate of 0.3 mL/min, with UV detection at 260 nm. Method validation was conducted in accordance with ICH guidelines for linearity, accuracy, precision, sensitivity, robustness, and stability. Forced degradation studies were conducted under acidic, alkaline, oxidative, thermal, and photolytic conditions. Results and Discussion: Retention times were 1.004 min (EMT), 1.310 min (TEN), and 1.870 min (NEL), with excellent peak resolution. Recovery studies demonstrated high accuracy, 99.74% (EMT), 99.47% (TEN), and 100.33% (NEL). The method showed strong linearity (r² > 0.999), low LOD/LOQ values, and robustness. Stability-indicating potential was confirmed via forced degradation studies. The technique proved to be rapid, precise, and stability-indicating, making it suitable for routine pharmaceutical quality control. Its sensitivity and robustness suggest potential utility in biomedical applications, including TDM and pharmacokinetic studies in HIV patients. Conclusion: The validated RP-UPLC method enables accurate, precise, and rapid estimation of EMT, TEN, and NEL, supporting its application in both pharmaceutical and clinical research settings.