Work

An LC‐MS/MS Method for Quantitative Analysis of Regorafenib and Exploration of Its Intracellular Exposure in Colorectal Cancer Cells

Year: 2026

Type: article

Abstract: Objective: 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: > 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.

Source: International Journal of Analytical Chemistry

DOI: 10.1155/ianc/5522314