A rapid and sensitive UPLC-MS/MS method for simultaneous determination of four potential mutagenic impurities at trace levels in ripretinib drug substance.

In the synthesis of ripretinib, a new oral tyrosine kinase inhibitor, impurities could arise directly from starting materials, reagents and intermediates. Among these process impurities, four specific intermediate impurities were found to contain the structural alerts of primary aromatic amine and aldehyde groups, triggering the concern of potential mutagenic impurities (PMIs). Two complementary (quantitative) structure-activity relationship [(Q)SAR] evaluation systems (expert rule-based and statistics-based) were subsequently employed to assess and classify the mutagenic risk of the four known impurities. The Sarah prediction results of these four impurities were all positive and they were categorized as class 3, where the threshold of toxicological concern (TTC) of 1.5 μg d would apply. Hereby, a rapid and sensitive UPLC-MS/MS method was developed for the simultaneous and trace level quantification of the four PMIs in ripretinib drug substance. The separation was achieved on a C18 column under the optimized gradient elution program consuming only nine minutes and the four PMIs were all well separated from ripretinib so that they could be easily diverted to waste a switch valve. The time-segmented multiple reaction monitoring (MRM) mode further improved the sensitivity and allowed for the quantification of the four PMIs as low as 10% of the acceptable limit. The method was fully validated, and proved sufficient in terms of selectivity, sensitivity, linearity, precision and accuracy. The factors involved in the method development and pathways for fragment ions of the four PMIs were also discussed and the study will contribute to risk management of PMIs present in ripretinib.