OBJECTIVE To establish a ultra-high-performance liquid chromatography-mass spectrum/mass spectrum(UPLC-MS/MS) method for the determination of anlotinib in human plasma and assessment of clinical application.

METHODS Zanubrutinib was used as internal standard and the extraction process was performed through protein precipitation method using acetonitrile, followed by separation on an Ultimate XB-C₁₈(100 mm×2.1 mm, 3.0 μm) column using acetonitrile and 10 mmol·L⁻¹ ammonium acetate-0.1% formic acid step-elution gradient. The flow rate was 0.6 mL·min⁻¹ and injection volume was 5 μL. The mass analysis was performed by positive ion electrospray ionization in multiple-reaction monitoring mode, and the mass spectrometer was set at m/z 408.1→339.1 for anlotinib and m/z 472.2→290.1 for internal standard, respectively. The specificity, standard curve and lower limit of quantification, precision and recovery, matrix effect and stability of the method and clinical application were investigated.

RESULTS The method was validated over the concentration range of 1.0−100.0 ng·mL⁻¹, with R²=0.998 4. The precision RSD was<9%, the recovery and matrix effect were 104.81%−107.32% and 102.54%−105.26%, respectively, and this method had good stability and was not affected by matrix effect. The method had been used for determined 52 advanced non-small cell lung cancer patients treated with anlotinib. The trough plasma concentration (C_trough) was measured on day 43 after initiation of anlotinib treatment. Anlotinib C_trough were higher than lower limit of quantitation (1.0 ng·mL⁻¹) from 52 patients. The plasma concentration of anlotinib C_trough was (11.38±4.29)ng·mL⁻¹ with 37.66% coefficients of variation, which were shown large inter-patient variability.

CONCLUSION This method is high sensitivity, specificity and accurate, and suitable for determination of anlotinib in human plasma.