An LC-MS/MS-Based Quantification of Intracellular Anlotinib Reveals Exposure-Toxicity Relationships in Endothelial...

Local drug exposure is closely associated with both therapeutic efficacy and adverse reactions. Anlotinib is approved for the treatment of chemotherapy-refractory or metastatic non-small cell lung cancer; its clinical application is limited by severe adverse reactions, particularly hypertension and diarrhea, and the relationship between intracellular exposure and toxicity remains unclear. In this study, a rapid and sensitive UHPLC-MS/MS method was developed using an Agilent 1290-6460A ultra-high performance liquid chromatography coupled to a triple quadrupole mass spectrometer to quantify anlotinib in human intestinal epithelial cells (HIECs) and human umbilical vein endothelial cells (HUVECs). Pazopanib-d3 was employed as an internal standard, and anlotinib was accurately quantified in 10-4000 ng/mL in positive ionization mode, with acceptable specificity, linearity, accuracy, precision, stability, and dilution integrity. The method was successfully applied to determine intracellular and extracellular anlotinib concentrations in HIECs and HUVECs. Notably, significant differences were observed between intracellular and extracellular concentrations, and intracellular anlotinib concentrations, rather than extracellular concentrations, were positively correlated with cytotoxicity in both cell types. Collectively, this study established a robust analytical approach for intracellular anlotinib quantification and provided experimental evidence linking intracellular exposure to cytotoxicity, offering a foundation for further investigation of anlotinib-induced adverse reactions.