An UPLC–ESI–MS/MS Bioanalytical Methodology for the Quantification of Gilteritinib in Human Liver Microsomes: Application to In Vitro and In Silico Metabolic Stability Estimation

Abstract

Gilteritinib (Xospata®) is a tyrosine kinase inhibitor (TKI) that works by inhibiting numerous receptor tyrosine kinases, involving AXL and FMS-like tyrosine kinase 3 (FLT3). Gilteritinib (GTB) was approved (28 November 2018) by the USFDA for the treatment of refractory or relapsed (R/R) acute myeloid leukemia (AML) with a FLT3 mutation. In the current study, a fast, highly sensitive, and specific ultra-performance liquid chromatography tandem mass spectrometry (UPLC–MS/MS) analytical methodology was created for GTB determination in human liver microsomes (HLMs) utilizing an electrospray ionization (ESI) source. The developed methodology (UPLC–ESI–MS/MS) was utilized in the assessment of GTB metabolic stability. The UPLC–ESI–MS/MS methodology was validated following the rules of the FDA that include selectivity, linearity, accuracy, precision, matrix effect, stability, and extraction recovery. The generated data of the optimized validation parameters of the current UPLC–ESI–MS/MS methodology were acceptable as reported in the FDA guidelines. GTB parent ions were generated in the ESI source (positive mode) and GTB daughter ions (two) were quantified in the mass analyzer utilizing multiple reaction monitoring (MRM) modes. The plotted GTB calibration curve showed a wide range of linearity from 1 ng/mL to 3000 ng/mL in HLMs matrix (y = 1.7298x + 3.62941 and r2 = 0.9949). The intraday and interday precision and accuracy outcomes of the current UPLC–ESI–MS/MS methodology were 0.35–11.39% and 0.27–4.32%, respectively. GTB and encorafenib (EFB) (internal standard; IS) were resoluted utilizing a reversed stationary phase (ZORBAX Eclipse plus C18 column; 1.8 μm PS, 2.1 mm ID, and 50 mm L) at 22 ± 2 °C. The calculated lower limit of quantification (LLOQ) was 0.94 ng/mL, revealing the UPLC–ESI–MS/MS methodology sensitivity. The two metabolic stability factors including in vitro half-life (t1/2) and intrinsic clearance (Clint) of GTB were 14.32 min and 56.64 mL/min/kg, respectively, predicting the moderate extraction ratio and good bioavailability of GTB. The current UPLC–ESI–MS/MS methodology is fast, sensitive and exhibits a wider range of linearity (1 to 3000 ng/mL) compared to other reported methods and is considered the first validated methodology for the determination of GTB metabolic stability.