Trace Level Quantification of the (−)2-(2-amino-5-chlorophenyl)-4-cyclopropyl-1,1,1-trifluoro-3-butyn-2-ol Genotoxic Impurity in Efavirenz Drug Substance and Drug Product Using LC–MS/MS

Efavirenz is a non-nucleoside reverse transcriptase inhibitor used in the treatment of human immunodeficiency virus type-1 (HIV). (2S)-(2-Amino-5-chlorophenyl)-4-cyclopropyl-1,1,1-trifluoro-3-butyn-2-ol (AMCOL), used as an intermediate in the synthesis of efavirenz and a degradation impurity, has an aminoaryl derivative which is a well-known alerting function for genotoxic activity. Upon request from a regulatory agency, a selective and sensitive liquid chromatography–tandem mass spectrometry (LC–MS/MS) method was developed for trace level quantitative determination of AMCOL related compound of efavirenz, for a risk assessment and comparison of impurity levels with the commercially available innovator product (brand name: Sustiva). The method provided excellent sensitivity at a typical target analyte level of <2.5 ppm, an established threshold of toxicological concern (TTC), when the drug substance and drug product samples were prepared at 15.0 mg/mL. The AMCOL sample was analyzed on a Luna C18 (2) (100 mm × 4.6 mm, 3 µm) column interfaced with a triple quadrupole tandem mass spectrometer operated in a multiple reaction monitoring (MRM) mode. Positive electrospray ionization (ESI) was employed as the ionization source and the mobile phase used was 5.0 mM ammonium acetate-methanol (35:65, v/v). The calibration curve showed good linearity over the concentration range of 0.2–5.0 ppm with a correlation coefficient of >0.999. The limit of detection (LOD) and limit of quantification (LOQ) were found to be 0.07 and 0.2 ppm, respectively. The developed method was validated as per international council on harmonization (ICH) guidelines in terms of LOD, LOQ, linearity, precision, accuracy, specificity, and robustness.