Development and Validation of UPLC-ESI-MS/MS Technique for the Determination of 2-Isopropyl-4-(chloromethyl)thiazole in Ritonavir

Abstract

The objective of this work was to develop and validate a rapid, highly sensitive ultra performance liquid chromatography-tandem mass spectrometry (UPLC-ESI-MS/MS) method for the quantification of 2-isopropyl-4-(chloromethyl)thiazole in ritonavir. Chromatographic conditions of this impurity were achieved on an AQUITY UPLC column HSS (high strength silica) T3 column (100 mm long, 2.1 mm internal diameter, 1.8 μm diameter) using a gradient elution with 0.1% formic acid in water and methanol at a flow rate of 0.3 mL/min. LCMS/MS was operated under the multiple reaction mode (MRM) using electrospray ionization technique in positive ion mode and the transitions of m/z 176.1[M+H]⁺→140.1 for quantifier, 176.1[M+H]⁺→71.0 for qualifier were used to measure the impurity, respectively. The total chromatographic run time was 10 min. Full validation of the analytical method was carried out, including its system precision, selectivity, linearity, accuracy, recovery, ruggedness, stability and robustness. A linear response function was achieved in the concentration range of 0.12-1.86 μg/g with r > 0.99. The detection limit and quantitation limit of this impurity were 0.06 and 0.12 μg/g, respectively. Consistent recoveries were obtained during intra- and inter-day precision experiments in validation ranged from 80-120%. The developed method could be helpful not only for quality control and also for risk management of potential genotoxicity of this impurity in ritonavir drug substance.