Sample pre-treatments were performed by solid phase extraction (Oasis® MCX μElution technology). Chromatographic separation was performed on a Waters Acquity UPLC® system with diode array detection (DAD) using a gradient of ammonium formate-acetonitrile on BEH C18 2.1x50 mm column.
The analytical methods were validated by using the accuracy profiles approach (β-expectation set at 95%). The methods were successfully validated for sunitinib (10 – 250 ng/mL), N-desethyl sunitinib (15 – 250 ng/mL), axitinib (15 – 250 ng/mL) and pazopanib (20 – 200 μg/mL). The first concentration levels validated were considered as limit of quantification (LOQ).
The validated method will be used in a clinical research study to determine TKI plasma levels and in this way help physicians to optimize the posology in order to achieve the best therapeutic response for their patients.