Development and Validation of an LC–MS/MS Method for Simultaneous Quantification of Pazopanib, Cabozantinib, Sorafenib, Axitinib, Tivozanib, Sunitinib, and the Metabolites Sorafenib N-Oxide and Desethyl-Sunitinib in Human Plasma

Background: Pazopanib, cabozantinib, sorafenib, axitinib, tivozanib, and sunitinib are widely used tyrosine kinase inhibitors (TKIs) targeting the vascular endothelial growth factor receptor signaling pathway. Considerable interindividual pharmacokinetic variability, together with established exposure–toxicity relationships, underlines the need for reliable analytical methods for therapeutic drug monitoring (TDM). The aim of this study was to develop and validate an LC–MS/MS method for the simultaneous determination of these six TKIs and the two metabolites, sorafenib N-oxide and desethyl-sunitinib, in human plasma and to investigate their stability under relevant preanalytical conditions. Methods: Plasma samples were prepared by protein precipitation and analyzed using LC–MS/MS with positive electrospray ionization and multiple reaction monitoring. Validation included assessments of linearity, accuracy, precision, lower limit of quantification, dilution integrity, carry-over, and stability under different storage conditions. As proof of concept, the method was applied to plasma samples from patients receiving TKI treatment. Results: All analytes demonstrated linear responses across the validated calibration ranges. Validation parameters met predefined acceptance criteria for all analytes, except for sorafenib N-oxide, which exhibited CV% values slightly above the acceptance limit (15%). All compounds remained stable during storage at −80 °C for at least five years. Furthermore, all analytes were stable at room temperature for 24 h, except sorafenib N-oxide, which showed instability under these conditions. Analysis of patient samples confirmed the suitability of the method for clinical application. Conclusions: We established and validated a fast, sensitive, and accurate LC–MS/MS method enabling simultaneous quantification of six TKIs and two metabolites in human plasma. The analytical method was successfully applied to plasma samples from patients in treatment with the six TKIs, demonstrating proof of concept for its application in TDM.