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). A potential genotoxic impurity in Efavirenz drug substance and acceptable levels is 2.5 μg mL-1 with respect to analyte concentration as followed ICH M7(R1) Multidisciplinary Guidelines M7(R1). The LC-MS/MS analysis of 4-CTHC impurity was carried out on Kinetex, C-18 (150 mm x 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 fewer than 10 minutes. A selective ion monitoring (SIM) acquisition mode and negative polarity electrospray ionization (ESI- ) mode was used as an MS method to quantify genotoxic impurity at 422.25 Da. The method was shown linearity from 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 less than 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 lab for pharmaceutical preparations.