Establishment and application of a rapid method for the determination of both total and unbound anlotinib concentrations by high-performance liquid chromatography-tandem mass spectrometry.

The unbound concentration of anlotinib is closely associated with its therapeutic efficacy and adverse reactions. In this study, we established an accurate and rapid liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the detection of total and unbound concentrations of anlotinib, which was subsequently applied to clinical samples. The separation of unbound and protein-bound anlotinib was achieved through filtration-ultrafiltration (CF-UF). Anlotinib-d5 served as the internal standard, and protein precipitation was utilized for sample preparation. The final method was thoroughly validated over a concentration range of 0.5-200 ng/mL according to related regulatory guidelines. Additionally, we demonstrated the clinical value of this method by analyzing blood samples from 39 lung cancer patients to quantify both total and unbound anlotinib concentrations. This method provides a foundation for further research into the relationship between anlotinib concentrations, therapeutic efficacy, and adverse reactions, ultimately facilitating the optimization of treatment strategies for patients.