Development of an LC-MS/MS Approach to Detect and Quantify Three Impurities of Darunavir

Abstract

The In the Darunavir (DAR) synthesis tert-Butyl (2S,3S)-4-chloro-3-hydroxy-1-phenylbutan-2-ylcarbamate (IMP-1), (2S,3S)-1,2-Epoxy-3-(Boc-amino)-4-phenylbutane (IMP-2) and (S)-tert-Butyl 4-chloro-3-oxo-1-phenylbutan-2-ylcarbamate (IMP-3) are generated as intermediates. The presence of Imp-1, Imp-2 and Imp-3 in DAR could potentially affect its effectiveness. The purpose of this investigation was to establish a LC-MS/MS methodology to identify and evaluate Imp-1, Imp-2 and Imp-3 in DAR samples. The method for Imp-1, Imp-2 and Imp-3 analysis was developed on X-Bridge C18 (150 x 4.6 mm, 3.5µm) column with gradient elution using mobile phase consisted of 0.1% formic acid (mobile phase A) and acetonitrile (mobile phase B). Mass spectrometer with electrospray ionization operated in the MRM mode was used in the analysis of Imp-1 (m/z300.2 > 244.1), Imp-2 (m/z264.300 > 120.100) and Imp-3 (m/z298.100 > 241.900). The LC-MS/MS methodology proposed showed a good linearity (0.38 to 1.9ppm), good system precision (RSD = 2.7%, 1.6% and 3.2%), good method precision (RSD = 5.2%, 2.8% and 2.3%), acceptable accuracy (91.8-105.2%, 87.7%-95.3% and 92.8-98.4%), low detection limit (0.127 ppm, 0.125 ppm and 0.12 ppm) and low quantitation limit (0.38 ppm, 0.375 and 0.38 ppm) for Imp-1, Imp-2 and Imp-3 respectively. The LC-MS/MS methodology proposed can be utilized to assess the quality of DAR sample for the presence of Imp-1, Imp-2 and Imp-3.