A 1‐Min UPLC–MS/MS Assay for the Wee1 Inhibitor Adavosertib in Human Liver Microsomes: Validation and Metabolic Stability Assessment

ABSTRACT Adavosertib (ADB) is an investigational oral Wee1 kinase inhibitor evaluated in acute myeloid leukemia and solid tumors. Reported ADB assays all use hydrophilic interaction or aqueous normal‐phase chromatography because the analyte is poorly retained under reversed‐phase conditions, and all target plasma, blood, or tissue rather than microsomal incubations. This work establishes reversed‐phase retention of ADB and applies the assay to substrate‐depletion metabolic stability in human liver microsomes (HLMs). ADB was retained on an Eclipse Plus C8 phase (2.1 × 50 mm, 1.8 µm) using 10 mM ammonium formate at pH 3.2 in water/acetonitrile (75:25, v/v) at 0.35 mL/min, eluting at 0.75 min against fedratinib (internal standard [IS]) at 0.52 min. Validation gave linearity from 1 to 4000 ng/mL under 1/ x weighting ( y = 3.1389 x − 6.7996; r 2 = 0.9973). Intraday precision and accuracy were 0.59%–3.12% RSD and −1.11% to −9.00% error; inter‐day, 0.70%–3.22% RSD and −2.20% to −12.00% error. The method cuts cycle time 5‐fold and mobile‐phase use roughly 11‐fold. Because the IS is not stable‐isotope‐labeled, compensation for HLM matrix effects may be less complete than with a labeled analog. In vitro t 1/2 was 26.56 min, and intrinsic clearance was 30.53 mL/min/kg, indicating high clearance. Greenness scored 0.63 (analytical GREEnness metric [AGREE]).