Low-level determination of 4-chlorobutyl-(S)-[4-chloro-2-(4-cyclopropyl-1,1,1-trifluoro-2-hydroxy-but-3yn-2-yl)phenyl] carbamate (4-CTHC) in efavirenz drug substance by LC-MS.

A rapid, simple, sensitive, and selective liquid chromatography-tandem mass spectrometry (LC-MS) test method was developed and validated for the trace level determination of 4-chlorobutyl-(S)-[4-chloro-2-(4-cyclopropyl-1,1,1-trifluoro-2-hydroxy-but-3yn-2-yl)phenyl] carbamate (4-CTHC). 4-CTHC is a potential genotoxic impurity in efavirenz drug substance and the acceptable level is 2.5 μg ml-1 with respect to analyte concentration according to ICH M7(R1) Multidisciplinary Guidelines M7(R1). The LC-MS/MS analysis of 4-CTHC impurity was carried out on a Kinetex C18 (150 × 4.6 mm, 5.0 μm) column. In this test procedure, the mobile phase was prepared with buffer (0.1% formic acid in water) and acetonitrile in the ratio of 1:3 (v/v). The method set flow rate was 0.4 ml min-1 . The method was developed with a short run time of <10 min. Selective ion monitoring acquisition mode and negative polarity electrospray ionization mode were used as an MS method to quantify genotoxic impurities at 422.25 Da. The method showed linearity in a range of 0.64-3.71 μg ml-1 with a correlation coefficient of 0.9992. The RSD for intra-day and inter-day precision was found to be <5%. The method's accuracy was in the range of 106.5-112.4% for the genotoxic impurity of 4-CTHC. The procedure was validated as per the current ICH Q2 (R1) guidelines and proved suitable for stability testing in the quality control laboratory for pharmaceutical preparations.