Validation of LC-MS/MS method for analysis of ruxolitinib and its metabolites in human plasma

The aim of the study was to develop and validate a UPLC-MS/MS method for determining the concentrations of ruxolitinib and its two main metabolites, M18 and M27, in blood plasma.Optimization of chromatographic separation included the selection of the stationary phase, separation temperature, and solvent for sample preparation. For further studies, a Luna Phenyl-Hexyl column and a mobile phase consisting of a mixture of 0.1% formic acid in water and acetonitrile in a gradient flow were used. The separation was performed at 40°C, and sample preparation involved protein precipitation with methanol and filtration. A triple quadrupole mass spectrometer was used for analyte detection after prior electrospray ionization. The full method validation was carried out in accordance with the ICH M10 guidelines. The following validation parameters were calculated: linearity, lower limit of quantification, precision, accuracy, matrix effect, stability.