An LC‐MS/MS Method for Quantitative Analysis of Regorafenib and Exploration of Its Intracellular Exposure in Colorectal Cancer Cells
- 1Department of Pharmacy Second Affiliated Hospital of Naval Medical University (Shanghai Changzheng Hospital) Shanghai 200003 China
- 2Department of Hematology Myeloma & Lymphoma Center Second Affiliated Hospital of Naval Medical University (Shanghai Changzheng Hospital) Shanghai 201203 China
- 3College of Traditional Chinese Medicine Shanghai University of Traditional Chinese Medicine Shanghai 201203 China shutcm.edu.cn
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: 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.
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BibTeX
@article{wang2026lcmsms,
title = {{An LC‐MS/MS Method for Quantitative Analysis of Regorafenib and Exploration of Its Intracellular Exposure in Colorectal Cancer Cells}},
author = {Xinran Wang and Wenwen Cai and Zhijun Liu and Jianguo Sun and Zhenhui Jiang and Liya Ye and Zhengyan Liang and Zhipeng Wang and Shouhong Gao and Wansheng Chen},
journal = {International Journal of Analytical Chemistry},
year = {2026},
month = oct,
doi = {10.1155/ianc/5522314},
url = {https://doi.org/10.1155/ianc/5522314},
note = {CC BY 4.0},
}
RIS
TY - JOUR
TI - An LC‐MS/MS Method for Quantitative Analysis of Regorafenib and Exploration of Its Intracellular Exposure in Colorectal Cancer Cells
AU - Xinran Wang
AU - Wenwen Cai
AU - Zhijun Liu
AU - Jianguo Sun
AU - Zhenhui Jiang
AU - Liya Ye
AU - Zhengyan Liang
AU - Zhipeng Wang
AU - Shouhong Gao
AU - Wansheng Chen
PY - 2026
DA - 2026/10/29
JO - International Journal of Analytical Chemistry
DO - 10.1155/ianc/5522314
UR - https://doi.org/10.1155/ianc/5522314
AB - 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: 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.
SN - 1687-8760
SN - 1687-8779
ER -