Ultra Trace Level Detection and Quantification of Identified Genotoxic Impurity Ethyl (1R,5R,6R)-7-(tert-butyl)-5-(pentan-3-yloxy)-7-azabicyclo[4.1.0]hept-3-ene-3-carboxylate in Oseltamivir Phosphate Drug Substance by Liquid Chromatography-Mass Spectroscopy

Abstract

A new rapid run time LC-MS method was developed and validated for detection (0.03 ppm) and quantification (0.1 ppm) in ultra-trace level of genotoxic impurity (GTI) ethyl-(1R,5R,6R)-7-(tertbutyl)- 5-(pentan-3-yloxy)-7-azabicyclo[4.1.0]hept-3-ene-3-carboxylate in oseltamivir phosphate active pharmaceutical ingredient (API). The method is price effective, time redeemable and capable to confirm the parent and daughter ion masses through mass spectrometry and tandem mass spectrometry for further fragmentation. An isocratic program and YMC Pack Pro C4 reverse phase column (150 mm × 4.6 mm × 3.0 μm) was used to achieve separation between oseltamivir phosphate and ethyl-(1R,5R,6R)- 7-(tert-butyl)-5-(pentan-3-yloxy)-7-azabicyclo[4.1.0]hept-3-ene-3-carboxylate impurity. Mobile phase-A used was 0.01 M ammonium acetate in water and mobile phase-B used was acetonitrile in the ration of 40:60 v/v. Diluent was used methanol. The chromatographic conditions were used, injection volume: 20 μL, flow rate: 1.0 mL/min, oven temperature: 50 ºC, auto sampler: 5 °C and run time 8.0 min. The detection and quantification levels found at 0.03 and 0.1 ppm, respectively. Ethyl (1R,5R,6R)- 7-(tert-butyl)-5-(pentan-3-yloxy)-7-azabicyclo[4.1.0]hept-3-ene-3-carboxylate impurity is linear from 0.1 to 15 ppm levels with regression coefficient 0.9994. The recoveries were found in the range of 93.8% to 105.0%.