Optimization of a sensitive and reliable UPLC-MS/MS method to simultaneously quantify almonertinib and HAS-719 and its application to study the interaction with nicardipine

Abstract Almonertinib, a novel third-generation epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor, was selected by the Chinese Society of Clinical Oncology as a first-line therapy for EGFR T790M mutated non-small cell lung cancer in 2021. Almonertinib is primarily metabolized by CYP3A4, so it could interact with a variety of drugs metabolized by CYP3A4, leading to the changes of systemic exposure. For the purpose of this experiment, an ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) assay with accuracy and simplicity was optimized and fully validated for the simultaneous quantitative determination of almonertinib and its metabolite HAS-719, and drug-drug interactions (DDIs) between almonertinib and nicardipine in vivo and in vitro was researched. After one-step precipitation of protein with acetonitrile, chromatographic separations of almonaitinib, HAS-719 and gefitinib (internal standard, IS) were achieved by gradient elution with 0.1% formic acid aqueous solution and acetonitrile. Detection of analytes was achieved by MS/MS coupled with multiple reaction monitoring (MRM) in the positive ion mode with ion transitions of m/z 526.01 → 72.04 for almonertinib, m/z 512.18 → 455.08 for HAS-719, and m/z 447.16 → 128.11 for IS. There was favorable linearity in the 0.5–200 ng/mL calibration range for almonertinib and 0.5–100 ng/mL for HAS-719. The lower limit of quantification (LLOQ) for both analytes were 0.5 ng/mL. The precision, accuracy, stability, matrix effect, and extraction recovery required for methodological validation were consistent with the requirements of FDA guideline. Then, the UPLC-MS/MS assay was employed successfully on the interactions of almonertinib and nicardipine in vivo and in vitro. The half-maximal inhibitory concentration (IC50) was 1.19 µM in rat liver microsomes (RLM), where nicardipine inhibited the metabolism of almonertinib with a mixed inhibitory mechanism. In pharmacokinetic experiments of rats, it was observed that nicardipine could significantly alter the pharmacokinetic profiles of almonertinib, including AUC(0−∞), AUC(0−t) and Cmax, but had no effect on the metabolism of HAS-719. According to the findings, it was indicated that nicardipine could inhibit the metabolism of almonertinib in vitro and in vivo.