Liquid chromatographyâ¿¿tandem mass spectrometric assay for therapeutic drug monitoring of the B-Raf inhibitor encorafenib, the EGFR inhibitors afatinib, erlotinib and gefitinib and the Oâ¿¿desmethyl metabolites of erlotinib and gefitinib in human plasma

⿢The first validated bioanalytical assay for encorafenib.⿢The first combined assay for a B-Raf inhibitor and EGFR inhibitors.⿢The assay has successfully been validated for all target compounds.⿢The assay was successfully used for therapeutic drug monitoring.⿢Recent new insights in erlotinib desmethylation could be confirmed.A quantitative bioanalytical liquid chromatography⿿tandem mass spectrometric (LC⿿MS/MS) assay for tyrosine kinase inhibitors afatinib, erlotinib, gefitinib and encorafenib and the O⿿desmethyl metabolites of erlotinib and gefitinib was developed and validated. Plasma samples were pre-treated using protein precipitation with acetonitrile containing stable isotopically labeled internal standard of afatinib, erlotinib, gefitinib and OSI-420. After dilution, the extract was directly analyzed by a reversed-phase liquid chromatography⿿tandem mass spectrometric system. Both O⿿desmethyl-erlotinib isomers, OSI⿿420 and OSI⿿413 could be separated, OSI-413 was only determined semi-quantitatively.The assay was completely validated for human plasma in a 10⿿10,000 ng/ml range for encorafenib, 5⿿5000 ng/ml for erlotinib, 2⿿2000 ng/ml for gefitinib and 1⿿1000 ng/ml for the other compounds. Calibration models were only linear when the corresponding labeled internal standard was used for quantification. Within-run precisions (n = 18) and between-run precisions (3 runs; n = 18) all were ⿤12.3% and accuracies were between 93 and 108% for each analyte over the tested concentration ranges. All compounds were sufficiently stable under all relevant analytical conditions. Finally, the assay was successfully applied to determine drug levels in plasma from patients treated with a kinase inhibitor.