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%.