Simultaneous Bioanalysis of Prodrug Oseltamivir and its Metabolite Oseltamivir Carboxylic Acid in Human Plasma by LC/MS/MS Method and its Application to Disposition Kinetics

Introduction: A selective, sensitive, precise and rapid analytical method using liquid chromatography- tandem mass spectrometry (LC/MS/MS) for simultaneous determination of oseltamivir and oseltamivir carboxylic acid in plasma has been developed and validated, using oseltamivir-D5 and oseltamivir acid-D3 as internal standards. Methods: The analytes were extracted from 300μL of human plasma using solid phase extraction technique. A mixture of methanol and 0.1% formic acid (60:40, v/v) was used as mobile phase at a flow rate of 0.7mL/min, to separate the analytes on Zorbax SB-C18 (50x4.6mm, 3.5μm) analytical column. Results: The calibration curves obtained were linear over the concentration ranges of 0.5-200ng/mL and 2.0-800ng/mL for oseltamivir and oseltamivir carboxylic acid respectively. A run time of 2.5min makes it possible to analyze more than 350 plasma samples in a day, thereby increasing the productivity. Conclusion: The present method was applied successfully to a clinical pharmacokinetic study in South Indian male subjects with 75mg oseltamivir phosphate capsule under fasting conditions and the results were authenticated by incurred sample reanalysis.