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Abstract

A SELECTIVE AND SENSITIVE LIQUID CHROMATOGRAPHIC/ TANDEM MASS SPECTROMETRIC METHOD FOR SIMULTANEOUS ESTIMATION OF OSELTAMIVIR AND ITS METABOLITE OSELTAMIVIR CARBOXYLIC ACID IN HUMAN PLASMA FOR BIOAVAILABILITY OR BIOEQUIVALENCE STUDIES

Shankrappa Janiwarad*, Kashif Ul Haq, Dinesh Choudhary

ABSTRACT

Selective and sensitive liquid chromatographic/tandem mass spectrometric method was developed and validated for the simultaneous estimation of Oseltamivir and its metabolite Oseltamivir carboxylic acid in Human plasma containing CPDA as an anticoagulant. A plasma sample of 0.5ml was extracted by single step solid phase extraction method using Oasis® HLB (30 mg/cc) cartridges and peak of interest on Hydrosphere column C18 (150 x 4.6 mm, particle size 5μm, pore size 12nm) using a isocratic flow of Acetonitrile and 0.1% Formic acid (90:10, v/v) as mobile phase. The quantitation was carried out using a triple quadruple API-3200 LC/MS/MS with positive electro spray ionization in multiple reaction-monitoring (MRM) mode. The parent to product ion transitions for Oseltamivir, Oseltamivir carboxylic acid & Oseltamivir-d3 (internal standard) were m/z 313.20 > 166.00; 285.30 > 138.20; 316.40 > 228.01, respectively. The method was validated as per US FDA guidelines to establish selectivity, sensitivity, matrix factor, linearity of response, accuracy, precision, recovery, stabilities, dilution integrity, ruggedness reinjection reproducibility and extended batch verification. The weighted (1/x2) calibration curves were linear over a range of 2.08 ng/mL to 241.12 ng/mL for Oseltamivir and 10.8 ng/mL to 1251.8 ng/mL for Oseltamivir carboxylic acid with coefficient of correlation (r) >0.9994 for Oseltamivir and >0.9982 for Oseltamivir carboxylic acid in a course of precision & accuracy batches. The mean % recovery of spiked quality control samples was >83% and >72% for Oseltamivir and Oseltamivir carboxylic acid, respectively, while the % recovery of Oseltamivir-d3 in spiked samples was >72%.

Keywords: Oseltamivir, Oseltamivir carboxylic acid, LC-MS/MS, Method validation.


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