Bioanalytical Method Development and Validation for Simultaneous Estimation of Atanavir and Ritonavir in Human Plasma using Liquid Chromatography coupled with Tandem Mass Spectrometry

Abstract

Background: Two common antiretroviral medications used to treat HIV infection are atazanavir (ATZ) and ritonavir (RTV). For their simultaneous measurement in human plasma to assist pharmacokinetic, bioequivalency, and therapeutic drug monitoring studies, a trustworthy bioanalytical technique is necessary. Goal: Using Saquinavir as the internal standard (IS), this work sought to develop and test a straightforward, quick, sensitive, and repeatable liquid chromatography–tandem mass spectrometry (LC-MS/MS) approach for the simultaneous measurement of ATZ and RTV in human plasma. Methods: Using Strata X-C 96-well cartridges and solid-phase extraction (SPE), human plasma samples were extracted. Using an isocratic mobile phase of 20 mM ammonium formate buffer and acetonitrile containing formic acid, chromatographic separation was accomplished on a Hypurity Aquastar C18 column (150 × 4.6 mm, 5 μm). Using multiple reaction monitoring (MRM), detection was carried out on an API 3000 triple quadrupole mass spectrometer running in positive electrospray ionization mode. The FDA's bioanalytical technique validation standards were followed in the validation process. Results: Over concentration ranges of 49.7–5967.3 ng/mL for ATZ and 49.7–5966.4 ng/mL for RTV, the technique demonstrated excellent linearity (r² > 0.99). With extraction recoveries of 83.85% for ATZ and 81.32% for RTV, precision and accuracy satisfied FDA acceptance requirements. Under benchtop, freeze-thaw, post-preparative, and long-term storage settings, both analytes maintained their stability. Conclusion: The validated LC-MS/MS approach is suited for pharmacokinetic, bio equivalency, and therapeutic medication monitoring applications because it is selective, accurate, precise, robust, and repeatable for the simultaneous detection of ATZ and RTV in human plasma.