Liquid chromatography–tandem mass spectrometric assay for ponatinib and N-desmethyl ponatinib in mouse plasma

• Published in: Journal of chromatography. B, Analytical technologies in the biomedical and life sciences Vol. 1023-1024; pp. 24 - 29
• Main Authors: Sparidans, Rolf W., Kort, Anita, Schinkel, Alfred H., Schellens, Jan H.M., Beijnen, Jos H.
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 15.06.2016
• Subjects: ambient temperature; Animals; Assaying; Chromatography; Chromatography, Liquid - methods; Drugs; evaporation; Female; females; Imidazoles - blood; Imidazoles - chemistry; ionization; isotope labeling; LC−MS/MS; leukemia; Limit of Detection; Linear Models; Liquid chromatography; Liquid-liquid extraction; Liquids; mass spectrometry; Metabolites; Mice; monitoring; Mouse plasma; N-desmethyl ponatinib; oral administration; pharmacokinetics; Ponatinib; Pyridazines - blood; Pyridazines - chemistry; Reproducibility of Results; reversed-phase liquid chromatography; Spectroscopy; Tandem Mass Spectrometry - methods; ALL; LLOQ; CML; DMP; BCR-ABL; N-desmethyl ponatinib; IS; CYP3A4; Ponatinib; LC−MS/MS; SRM; Liquid-liquid extraction; QC; FVB; tBME; TKI; Ph; ESI; Mouse plasma
• ISSN: 1570-0232; 1873-376X; 1873-376X
• DOI: 10.1016/j.jchromb.2016.04.049

•The first validated bioanalytical LC–MS/MS assay for ponatinib and its active metabolite.•Liquid-liquid extraction showed a selective treatment for the mouse plasma samples.•The assay has successfully been validated in the 5–5000 and 1–1000ng/ml ranges.•The assay was successfully used for a pharmacokinetic pilot study in mice. Ponatinib is a multi-targeted third generation BCR−ABL1 tyrosine-kinase inhibitor approved for specific types of leukemia. A bioanalytical assay for this drug and its N-desmethyl metabolite in mouse plasma was developed and validated using liquid chromatography-tandem mass spectrometric (LC−MS/MS) with liquid-liquid extraction as sample pre-treatment procedure. After extraction with tert-butyl methyl ether of both analytes with their isotopically labeled internal standards and evaporation and reconstitution of the extract, compounds were separated by reversed-phase liquid chromatography under alkaline conditions. After electrospray ionization, both compounds were quantified in the selected reaction monitoring mode of a triple quadrupole mass spectrometer. The linear assay was validated in the ranges 5–5000ng/ml for ponatinib and 1–1000ng/ml for N-desmethyl ponatinib. Within-run (n=18) and between-run (3 runs; n=18) precisions were 10% and 12% at the lower limit of quantification for the metabolite, all other precisions were ≤8% for the metabolite and ≤6% for ponatinib. Accuracies were between 92 and 108% for both compounds in the whole calibration range. The drug was sufficiently stable under most relevant analytical conditions, only ponatinib showed more than 15% hydrolytic degradation after storage for 6h and longer at ambient temperature in mouse plasma. Finally, the assay was successfully applied to determine plasma drug levels and study pharmacokinetics after oral administration of ponatinib to female FVB mice.