Liquid chromatography-tandem mass spectrometric assay for the tyrosine kinase inhibitor afatinib in mouse plasma using salting-out liquid-liquid extraction

The assay was completely validated for plasma in a 0.5-500 ng/ml calibration range with r2 = 0.995 ± 0.002 (n = 6) using linear regression with the inverse square of the concentration as the weighting factor for the calibration. Within-run precisions (n = 18) were 2.7-11.7% and between-run (3 runs; n = 18) precisions 3.0-14.5%. Accuracies were between 96-109% for the whole calibration range. The drug was sufficiently stable under all relevant analytical conditions. Finally, the assay was successfully applied to determine plasma drug levels and study pharmacokinetics after oral administration of afatinib to female FVB mice.