Sensitive LC–MS/MS Method for Simultaneous Quantification of Alectinib and Its Major Metabolite M4 in Limited‐Volume Human Plasma

Alectinib is an oral anaplastic lymphoma kinase inhibitor, and M4 is its major pharmacologically active metabolite. Simultaneous quantification of alectinib and M4 in limited-volume plasma is important for pediatric clinical studies, particularly when concentrations are low or exceed the calibration range. An LC-MS/MS method was developed and validated for simultaneous quantification of alectinib and its major metabolite, M4, in human plasma. Alectinib and M4 were extracted from 20 μL of plasma by protein precipitation and quantified using stable isotope-labeled internal standards. Calibration ranges were 0.100-20.0 ng/mL for alectinib and 1.00-200 ng/mL for M4, with lower limits of quantification of 0.100 and 1.00 ng/mL, respectively. Full validation demonstrated acceptable selectivity, linearity, matrix effects, dilution reproducibility, stability, and carryover. Inter-day accuracy (RE) ranged from -9.9% to 5.1%, and inter-day precision (CV) was ≤ 8.3% for both analytes. Partial validation after analytical column replacement also met predefined acceptance criteria. The method was successfully applied to plasma samples obtained from one patient enrolled in an investigator-initiated clinical trial of alectinib, with both alectinib and M4 remaining quantifiable up to 72 h after a single oral dose. This sensitive LC-MS/MS method supports the simultaneous quantification of alectinib and M4 in limited-volume pediatric clinical study samples.