Development and Validation of Two LCMS/MS Methods for Simultaneous Estimation of Oseltamivir and its Metabolite in Human Plasma and Application in Bioequivalence Study

Oseltamivir phosphate is licensed for the treatment of patients with influenza virus infection. Two LCMS/MS methods for simultaneous quantification of Oseltamivir and its active metabolite, oseltamivir carboxylate in human plasma were described here. After solid phase extraction sample was separated either on a reversed phase C18 column with a stepwise gradient using 0.05% formic acid and methanol or a cation-exchange column using an isocratic mobile phase (7 mM Ammonium formate, pH 3.5 ± 0.2: Methanol: 50:50, v/v). Flow rate of 1 mL/min was maintained in both cases. A triple quadrupole mass spectrometer operating in the positive ionization mode was used for detection and drug quantification. Both methods were validated over a range of 0.52ng/ml to 207.00 ng/ml for Oseltamivir and 4.08 ng/ml to 1200.00 ng/ml for Oseltamivir Carboxylate. Deuterated Oseltamivir and Oseltamivir carboxylate were used as internal standards. The accuracies and precisions for Oseltamivir were between 91-102% and 0.9 - 13.7% for all concentration levels. The accuracies and precisions for Oseltamivir carboxylate were between 88-109% and 0.5 - 8.2% at all levels. Furthermore, Oseltamivir and its metabolite were stable in plasma ex vivo for at least 191 days when stored at -20°C or below.