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A novel, simple, sensitive, specific and rapid reverse-phase liquid chromatography-electrospray ionization-mass 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 analyzed 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 chromatography-mass 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.

A Novel LC-MS/MS Method Development and Validation for Ultra-trace Level Determination of Three Genotoxic Efavirenz Impurities