Abstract
Masitinib (MST) is a selective tyrosine kinase inhibitor. Validated liquid chromatography tandem mass spectrometric method (LC–MS/MS) was developed for the quantification of MST in rat liver microsomes (RLMs) matrix. The developed method was applied to metabolic stability and excretion rate studies. Reversed phase liquid chromatography was used for resolution of MST and bosutinib (IS) using C18 (50 mm × 2.1 mm, 1.8 μm). Binary solvent system consisted of 35% solvent A (0.1% formic acid in H2O, pH: 3.2) and 65% solvent B (acetonitrile) used as mobile phase at flow rate of 0.25 mL with a total run time of 5 min. Injection volume was 5 µL. Generation of ions was done in positive ESI source and quantification of MST and IS were done using MRM mode. The developed method showed a linearity in the range of 5–200 ng/mL (r2 ≥ 0.9992) with LOQ and LOD of 0.25 and 0.76 ng/mL in RLMs. The intra- and inter-day precision and accuracy ranged from 0.95 to 1.49 and − 5.22 to 1.13%, respectively in RLMs. Rate of disappearance of MST during incubation with RLMs was almost linear allover incubation time. In vitro t1/2 was 50.38 min and CLin was 3.11 ± 0.2. The developed method was applied also to measure the rate of masitinib excretion in rat urine. The method can used for further pharmacokinetic studies of MST.
Highlights
IntroductionMST is registered for the treatment of mast cell tumors in dogs under the trade name of Masivet in Europe (since 2009) and Kinavet in USA (since 2011) [1, 2] _ENREF_11_ENREF_2
The developed method was applied to measure the rate of masitinib excretion in rat urine
Validated LC–MS/MS method was developed for assaying MST in rat liver microsomes (RLMs) matrices
Summary
MST is registered for the treatment of mast cell tumors in dogs under the trade name of Masivet in Europe (since 2009) and Kinavet in USA (since 2011) [1, 2] _ENREF_11_ENREF_2 It acts selectively targeting mainly wild type forms and mutated c-Kit R, PDGFRα/β, Lck, LYn, FGFR3 and FAK. It is considered the first approved anticancer veterinary therapy for the treatment of unresectable canine mast cell tumors (CMCTs), which harbors activating c-KitR mutations at dose of 12.5 mg/kg per day [3] _ENREF_1. Based on MST metabolic stability data, secondary pharmacokinetic parameters such as hepatic clearance (CLH), bioavailability and in vivo t1/2 can be calculated which is important for creating in vivo–in vitro correlation for proper metabolic stability study. If the studied drug is rapidly metabolized, in vivo bioavailability will be low [7]
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