Quantification of Upadacitinib, Dasatinib, Ruxolitinib, Imatinib, and Cabozantinib in Plasma With Liquid Chromatography-Tandem Mass Spectrometry.

Background

Tyrosine kinase inhibitors (TKIs) are increasingly used to treat hematological malignancies and solid tumors. As pharmacokinetic variability is substantial among patients, therapeutic drug monitoring may help optimize treatment efficacy and minimize toxicity. However, simultaneous quantification of multiple TKIs with markedly different therapeutic concentration ranges is challenging. A sensitive and selective liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated for simultaneous quantification of upadacitinib, dasatinib, ruxolitinib, imatinib, and cabozantinib in human plasma.

Methods

Proteins were precipitated in methanol containing stable isotope-labeled internal standards. Rapid chromatographic separation (5 minutes) was achieved on a BEH C18 column using a gradient of 0.025% ammonium hydroxide in water and methanol. TKIs were detected using a triple-quadrupole mass spectrometry in positive electrospray ionization mode. EMA ICH M10 guidelines were used for method validation.

Results

All analytes showed excellent linearity (R2 ≥ 0.9992) over calibration ranges of 0.1-10,000 mcg/L depending on the analyte. Lower limits of quantification were 0.1 mcg/L for upadacitinib and dasatinib, 0.5 mcg/L for ruxolitinib, 2.25 mcg/L for cabozantinib, and 10 mcg/L for imatinib. Within-run and between-run precision were ≤4.7% and ≤3.1%, respectively, and accuracy ranged from 0.1% to 13.2%. No significant matrix effects, carryover, or interference were observed. All analytes were stable during short-term storage (3 days at 5°C and 20°C), freeze-thaw cycles, and long-term storage (12 months at -20°C and -80°C).

Conclusions

This simple, rapid, sensitive, and robust LC-MS/MS method enables simultaneous quantification of 5 TKIs at different concentrations with minimal sample volume and excellent analytical performance, supporting its use in therapeutic drug monitoring and pharmacokinetic studies of kinase inhibitors in clinical practice.