Measurement of lenvatinib using UPLC-MS/MS

The plasma concentration of lenvatinib was measured using UPLC-MS/MS [18]. Calibration, quality control (QC), and internal standard (IS) solutions were prepared by diluting the stock solution in 100% methanol. Deuterated lenvatinib (lenvatinib-d₄) was used as an IS. For sample pretreatment, solid-phase extraction with an OASIS^® MCX µElution plate was used. For the calibration or QC sample, 330 µL of 4% aqueous phosphoric acid solution, 20 µL of IS (100 ng/mL), 100 µL of calibration or QC solution, and 100 µL of blank plasma were added to a 2-mL polypropylene tube and vortexed for 20 s. The patient plasma sample (100 µL) was mixed with 330 µL of 4% aqueous phosphoric acid solution, 20 µL of IS (100 ng/mL), and 100 µL of 100% methanol for volume adjustment. The MCX µElution plate was conditioned and equilibrated by adding 200 µL of 100% methanol and 200 µL of ultrapure water to each well. Then, 500 µL of the above mixture containing calibration, QC, or patient sample was added to each well. Each well was washed with 200 µL of 2% aqueous formic acid solution followed by 200 µL of 100% methanol. After the washing step, the analyte was eluted with 100 µL of 1.25% ammonium solution (25% aqueous ammonia/100% methanol = 5/95) into a round-well collection plate (Waters). Subsequently, 100 µL of ultrapure water was added to each well, and the contents were mixed by pipetting. The plate was sealed with an adhesive seal (Waters). The injection volume was 10 µL. Lenvatinib was separated using an ACQUITY UPLC^® BEH C18 column. The MS/MS transitions monitored in the positive ionization mode for lenvatinib and lenvatinib-d₄ were m/z 427.02 → m/z 370.05 and m/z 431.09 → m/z 370.05, respectively. The mobile phase was a gradient of 0.1% aqueous formic acid solution:0.1% formic acid/acetonitrile solution. Validation was performed according to the US Food and Drug Administration guidelines.