title: Liquid chromatography-tandem mass spectrometric assay for the tyrosine kinase inhibitor afatinib in mouse plasma using salting-out liquid-liquid extraction.
abstract: A quantitative bioanalytical liquid chromatography-tandem mass spectrometric (LC-MS/MS) assay for afatinib, an irreversible inhibitor of the ErbB (erythroblastic leukemia viral oncogene homolog) tyrosine kinase family, was developed and validated. Plasma samples were pre-treated using salting-out assisted liquid-liquid extraction (SALLE) with acetonitrile, magnesium chloride and a stable isotopically labeled internal standard. After dilution, the extract was directly injected into the reversed-phase liquid chromatographic system. The eluate was transferred into the electrospray interface with positive ionization and compounds were detected in the selected reaction monitoring mode of a triple quadrupole mass spectrometer. The assay was completely validated for plasma in a 0.5-500ng/ml calibration range with r(2)=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.
authors.affiliation: Utrecht University, Faculty of Science, Department of Pharmaceutical Sciences, Division of Pharmacoepidemiology & Clinical Pharmacology, Universiteitsweg 99, 3584CG Utrecht, The Netherlands. Electronic address: R.W.Sparidans@uu.nl.
authors.forename: Rolf W
authors.initials: RW
authors.lastname: Sparidans
authors.affiliation: The Netherlands Cancer Institute, Department of Molecular Oncology, Plesmanlaan 121, 1066CX Amsterdam, The Netherlands. Electronic address: s.v.hoppe@nki.nl.
authors.forename: Stephanie
authors.initials: S
authors.lastname: van Hoppe
authors.affiliation: Utrecht University, Faculty of Science, Department of Pharmaceutical Sciences, Division of Pharmacoepidemiology & Clinical Pharmacology, Universiteitsweg 99, 3584CG Utrecht, The Netherlands. Electronic address: J.J.M.Rood@uu.nl.
authors.forename: Johannes J M
authors.initials: JJ
authors.lastname: Rood
authors.affiliation: The Netherlands Cancer Institute, Department of Molecular Oncology, Plesmanlaan 121, 1066CX Amsterdam, The Netherlands. Electronic address: a.schinkel@nki.nl.
authors.forename: Alfred H
authors.initials: AH
authors.lastname: Schinkel
authors.affiliation: Utrecht University, Faculty of Science, Department of Pharmaceutical Sciences, Division of Pharmacoepidemiology & Clinical Pharmacology, Universiteitsweg 99, 3584CG Utrecht, The Netherlands; The Netherlands Cancer Institute, Department of Clinical Pharmacology, Plesmanlaan 121, 1066CX Amsterdam, The Netherlands. Electronic address: j.schellens@nki.nl.
authors.forename: Jan H M
authors.initials: JH
authors.lastname: Schellens
authors.affiliation: Utrecht University, Faculty of Science, Department of Pharmaceutical Sciences, Division of Pharmacoepidemiology & Clinical Pharmacology, Universiteitsweg 99, 3584CG Utrecht, The Netherlands; The Netherlands Cancer Institute, Department of Clinical Pharmacology, Plesmanlaan 121, 1066CX Amsterdam, The Netherlands; Slotervaart Hospital, Department of Pharmacy & Pharmacology, Louwesweg 6, 1066 EC Amsterdam, The Netherlands. Electronic address: jos.beijnen@slz.nl.
authors.forename: Jos H
authors.initials: JH
authors.lastname: Beijnen
mesh_terms: D000077716:Afatinib; D000818:Animals; D002853:Chromatography, Liquid / Q000379:methods*; D005260:Female; D016014:Linear Models; D059625:Liquid-Liquid Extraction / Q000379:methods*; D051379:Mice; D047428:Protein Kinase Inhibitors / Q000097:blood*; D011799:Quinazolines / Q000097:blood*; D015203:Reproducibility of Results; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D047428:Protein Kinase Inhibitors; D011799:Quinazolines; D000077716:Afatinib
keywords: Afatinib; LC–MS/MS; Mouse; Plasma; SALLE