An LC‐MS/MS Method for Quantitative Analysis of Regorafenib and Exploration of Its Intracellular Exposure in Colorectal Cancer Cells
Xinran Wang, Wenwen Cai, Zhijun Liu, Jianguo Sun, Zhenhui Jiang, Liya Ye, Zhengyan Liang, Zhipeng Wang, Shouhong Gao, Wansheng ChenObjective
Regorafenib (Reg) is a multikinase inhibitor for treating metastatic colorectal cancer (CRC); however, plasma concentrations may not fully predict clinical response. This study aimed to develop and validate an LC‐MS/MS method for quantifying Reg in CRC cells and to preliminarily characterize its temporal and spatial distribution.
Methods
Separation of Reg and the internal standard (selinexor) was achieved using an Agilent Poroshell 120 EC‐C18 column (3.0 × 100 mm, 2.7 μm) with isocratic elution using acetonitrile and 0.1% aqueous formic acid (70:30, v/v). Detection was performed in multiple reaction monitoring mode using an Agilent 1260‐6460 UHPLC‐MS/MS system. HCT116 and SW480 cells were treated with Reg at their respective IC50 concentrations for 12, 24, 36, and 48 h, and Reg exposure in whole‐cell, nuclear, and mitochondrial fractions was measured.
Results
The UHPLC‐MS/MS method showed good linearity ( R 2 > 0.99) and a lower limit of quantification of 1.0 ng/mL. Precision (intraday RSD% < 11.39%; interday RSD% < 9.11%) and accuracy (RE% within ±6.80%) were within the acceptance criteria. The recovery ranged from 86.03% to 106.41%, and consistent matrix effects were observed. Reg exposure in whole‐cell samples increased in a time‐dependent manner, while nuclear and mitochondrial fraction‐associated exposures showed distinct temporal patterns between the two CRC cell lines.
Conclusion
The validated UHPLC‐MS/MS method enables sensitive and reliable quantification of Reg in CRC cells and isolated subcellular fractions. The observed temporal differences in intracellular and fraction‐associated Reg exposure may be associated with cellular response and may support future exposure–response assessments beyond plasma‐based pharmacokinetics.