Development and Validation of a Simple and Robust Bioanalytical Method for Simultaneous Quantification of Bictegravir, Emtricitabine, and Tenofovir Alafenamide in Human Plasma Using LC–MS/MS and Its Application in a Pharmacokinetic Study

ABSTRACT A simple, sensitive, and selective LC–MS/MS method has been developed and validated, in accordance with ICH M10 guidelines, for the simultaneous quantification of Bictegravir (BTV), Emtricitabine (ECB), and Tenofovir alafenamide (TAM) in human plasma. Following a straightforward solid‐phase extraction, the analytes were separated using a reverse‐phase C18 column with an isocratic mobile phase consisting of 10 mM ammonium acetate (pH 3.0), acetonitrile, and methanol in a 30:10:60 (v/v/v) ratio. The method demonstrated linear dynamic ranges of 25–1600 ng/mL for BTV, 10.0–6120.1 ng/mL for ECB, and 2.0–803.78 ng/mL for TAM, utilizing a low plasma volume of 200 µL and an injection volume of 5 µL. The validated method has been successfully applied to the analysis of plasma samples from healthy male subjects in pharmacokinetic (PK), bioavailability, and bioequivalence studies of BTV/ECB/TAM tablets (50 mg/200 mg/25 mg). Additionally, the method is suitable for therapeutic drug monitoring, PK evaluations, bioequivalence and bioavailability assessments, toxicity studies, human immunodeficiency virus (HIV) reservoir investigations, and stringent quality control in pharmaceutical manufacturing.