A novel LC-MS/MS method development and validation for ultra-trace level determination of three genotoxic efavirenz impurities

Abstract

A novel, simple, sensitive, specific and rapid reverse-phase liquid chromatography-electrospray ionizationmass spectrometry method was established and validated for the ultra-trace analysis of efavirenz-related compound C, 8-hydroxy efavirenz and its impurity 1, which are potential genotoxic impurities associated with efavirenz. These were separated on high performance liquid chromatography and detected with tandem mass spectrometry in the positive ionization mode with MRM transitions, m/z 314.15>244.10, 212.10>57.10, 305.70>69.05 and 364.15>291.25. A gradient method was developed to separate the impurities using 0.01 M ammonium acetate buffer and methanol at 1 ml/min flow-rate. Samples were analysed using a Hypersil C-18 column (5 μm, 250×4.6 mm). The calibration curves have shown good linearity in the concentration range of 2.5-78 ppb and a correlation coefficient of 0.99. Mean intra and inter day precision was 2.6-3.2 ppm and 2.5-2.6 ppm, respectively. For the 3 genotoxic impurities the limit of detection and limit of quantitation were 0.04 ppm and 0.125 ppm, respectively. The liquid chromatographymass spectrometry/mass spectrometry method developed was specific, sensitive, precise and accurate. The developed method could be used for quantification and monitoring of genotoxic impurities of efavirenz.