A validated assay for the simultaneous quantification of six tyrosine kinase inhibitors and two active metabolites in human serum using liquid chromatography coupled with tandem mass spectrometry

• Development and validation of an assay for quantifying 6 TKIs and 2 metabolites.
• The combination of TKIs is unique by incorporating regorafenib and pazopanib.
• Due to large differences in detection ranges, the assay is challenging.
• The method is used to explore the value of TDM and to support IIS proposals.

A sensitive, sophisticated and practical bioanalytical assay for the simultaneous determination of six tyrosine kinase inhibitors (imatinib, sunitinib, nilotinib, dasatinib, pazopanib, regorafenib) and two active metabolites (N-desmethyl imatinib and N-desethyl sunitinib) was developed and validated. For the quantitative assay, a mixture of three stable isotopes as internal standards was added to human serum, standards and controls. Thereafter, samples were pre-treated using protein precipitation with methanol. The supernatant was diluted with water and injected into an ultra pressure liquid chromatographic system with an Acquity TQ tandem mass spectrometry detector. The compounds were separated on an Acquity BEH C18 analytical column (100 mm × 2.1 mm ID, 1.7 μm particle size) and eluted with a linear gradient system. The ions were detected in the multiple reaction monitoring mode. The lower limit of quantification and the linearity of all compounds generously met with the concentrations that are to be expected in clinical practice. The developed bioanalytical assay can be used for guiding TKI therapy in daily clinical practice as well as for investigator-initiated research.