Abstract: Objective To establish an HPLC-TRIPLE QUAD-MS/MS method for determination of related substances in efavirenz tablets and to deduce their mass spectrometry fragmentation pathways. Methods The separation was carried out on a C₁₈ column (4.6 mm×100 mm, 3 μm). The mobile phase was 0.1% formic acid (containing 2 mmol·L^-1 ammonium formate)-methanol (gradient elution). An ion source ESI was used and quantification was in a positive ion mode in a multi-reaction monitoring mode (MRM). Results The mass spectra of efavirenz and four of its impurities were analyzed before the fragmentation pathways were deduced. The determination method was validified methodologically. Impurities A, B, and C had good linear relationships within the range of 1-500 ng·mL^-1, while impurity D had a good linear relationship within the range of 5-500 ng·mL^-1. The limits of quantification ranged from 1.0 to 5.0 ng·mL^-1, while the average recovery ranged from 89.5% to 95.3%. Conclusion The established HPLC-TRIPLE QUAD-MS/MS method has a strong specificity, high sensitivity, and good accuracy. It is suitable for simultaneous quantitative determination of related substances in efavirenz tablets.

Key words: Efavirenz Tablets, Related Substances, HPLC-TRIPLE QUAD-MS/MS, Mass Spectrometry Quantification