HPLC–UV Analysis of Fruquintinib in Plasma of a Patient with Colorectal Cancer

Fruquintinib is a small-molecule tyrosine kinase inhibitor of vascular endothelial growth factor receptors (VEGFR) used in patients with metastatic colorectal cancer. Fruquintinib reportedly inhibits the phosphorylation of VEGFR-2 by >80% at plasma trough concentrations >176 ng/mL. Therefore, maintaining a stable trough concentration of fruquintinib is clinically necessary. To date, the only reported method for quantifying fruquintinib concentrations in human plasma is LC-MS/MS, which is challenging to use routinely in clinical settings given its high cost and technical demands. Therefore, in this study, we aimed to develop a method for determining fruquintinib levels in human plasma and validate it for therapeutic drug monitoring (TDM). For the analysis, a 50-μL human plasma sample was obtained, and protein precipitation was performed using acetonitrile extraction. Fruquintinib and acetanilide (internal standard) were separated on a reversed-phase column using a mobile phase consisting of 0.5% KH₂PO₄ (pH 2.6) and acetonitrile (69/31, v/v), pumped at a flow rate of 1.0 mL/min. The detection wavelength was set at 237 nm. The calibration curves for fruquintinib were linear (r² = 0.999) in the range of 50-2000 ng/mL. The accuracy and precision in all validation experiments adhered to the guidelines of the U.S. Food and Drug Administration. Using the validated method, we successfully measured fruquintinib concentrations in the plasma of a patient. This article presents a simple, novel, and sensitive HPLC-UV-based method for determining the plasma concentration of fruquintinib and confirms its applicability in the TDM of fruquintinib in the clinical setting.