Liquid chromatography-tandem mass spectrometric assay for the ALK inhibitor crizotinib in mouse plasma

A quantitative bioanalytical liquid chromatography-tandem mass spectrometric (LC-MS/MS) assay for the ALK inhibitor crizotinib was developed and validated. Plasma samples were pre-treated using protein precipitation with acetonitrile containing crizotinib-¹³C₂-²H₅ as internal standard. The extract was directly injected into the chromatographic system after dilution with water. This system consisted of a sub-2μm particle, trifunctional bonded octadecyl silica column with a gradient using 0.1% (v/v) of ammonium hydroxide in water and methanol. The eluate was transferred into the electrospray interface with positive ionization and the analyte was detected in the selected reaction monitoring mode of a triple quadrupole mass spectrometer. The assay was validated in a 10-10,000ng/ml calibration range with r²=0.99980±0.00014 for double logarithmic linear regression (n=5). Within day precisions (n=6) were 3.4-4.8%, between day (3 days; n=18) precisions 3.6-4.9%. Accuracies were between 107% and 112% for the whole calibration range. The drug was sufficiently stable under all relevant analytical conditions. Oxidative metabolites of crizotinib were monitored semi-quantitatively. Finally, the assay was successfully used to assess drug pharmacokinetics in mice.