Development and ICH M10-compliant validation of an LC-MS/MS method for quantification of atirmociclib in mouse plasma and its application to preclinical pharmacokinetic studies.

Abstract

Atirmociclib is a selective cyclin-dependent kinase 4 (CDK4) inhibitor currently under clinical investigation for the treatment of advanced malignancies, particularly hormone receptor positive (HR+) or human epidermal growth factor receptor 2 negative (HER2-) breast cancer. Here, we report the first validated liquid chromatography tandem mass spectrometry (LC-MS/MS) bioanalytical method for the quantification of atirmociclib in mouse plasma, validated in compliance with international council for harmonisation-M10 (ICH-M10) guidelines. Sample preparation involved a rapid and straightforward protein precipitation approach using acetonitrile, yielding consistent recovery (>80%) across low to high concentration levels. Chromatographic separation was achieved on a Kinetex C18 column (50 mm length × 2.1 mm internal diameter, 5 μm particle size) using gradient elution with 5 mM ammonium acetate in water (mobile phase A) and 0.1% v/v formic acid in acetonitrile (mobile phase B). The analyte (atirmociclib) and internal standard (warfarin) were detected using Q1/Q3 (m/z) mass transitions 464.2/303.9 and 309.0/163.0 respectively. The method demonstrated excellent selectivity, accuracy, precision, and linearity over a concentration range of 1-1000 ng/mL. Additionally, reliable quantification was maintained for samples exceeding the upper limit of quantification following up to 10-fold dilution. All validation parameters, including stability in biological matrices and solution, met the predefined acceptance criteria. This sensitive, and reproducible method was successfully applied to support preclinical pharmacokinetic study of atirmociclib in mouse, providing a valuable analytical tool.