Bio-analytical Method Development and Validation for Simultaneous Determination of Bictegravir, Emtricitabine, and Tenofovir Alafenamide Fumarate in Human Plasma by LC-MS/MS

Abstract

Abstract: Aim: The current method was developed as a novel and reliable quantitative liquid chromatography-mass (tandem) spectrometry (LC-MS/MS) method for the estimation of analytes like Bictegravir (BIC) Tenofovir Alafenamide Fumarate (TNF), and Emtricitabine (EMT) in plasma of human simultaneously. Materials: Naproxen (NPX) is used as the internal standard for the current study. The ‘Precipitation Extraction technique’ is used for the present study. The Zorbax XDB C18 analytical column (2.1 X 50 and particle size of 5μm) is used for the chromatographic separation with isocratic natured mobile phase, which consists of Acetonitrile: Formic acid (0.1%) in water (70:30, v/v), at a flow rate of 0.15 mL/minute. Methods: The parent→production conversions were observed at m/z 450.1 → 289.1 (BIC), m/z 248.3 → 130.03 (EMT), m/z 477.3 → 270.04 (TNF), and m/z 231.12 → 184.82 (NPX) on a triple quadrupole mass spectrometer, operating in the multiple reaction monitoring (MRM) positive ion mode. The three compounds were found to possess primary groups hence positive manner was selected for the LC-MS/MS study. Results: The subject method validation was performed for the concentration of range 2-500 ng for Bictegravir (BIC), Emtricitabine (EMT), and Tenofovir Alafenamide Fumarate (TNF). The obtained mean recoveries for the three-drug moieties from samples of spiked plasma were found reproducible. Conclusion: Hence, based on the above, the method was proved to be rugged and rapid, with a least total run time of 3.0 min. The current method was successfully validated according to the FDA, EMA, and ICH guidelines, and the stability studies were evaluated accordingly. Keywords: Bictegravir, Emtricitabine, Tenofovir Alafenamide Fumarate, Liquid Chromatography-Mass Spectrometry, Extraction, Stability.