LC–MS/MS assay for the quantitation of the ATR kinase inhibitor VX-970 in human plasma

• Published in: Journal of pharmaceutical and biomedical analysis 2017-11, Vol.146, p.244-250
• Main Authors: Kiesel, Brian F., Scemama, Jonas, Parise, Robert A., Villaruz, Liza, Iffland, Andre, Doyle, Austin, Ivy, Percy, Chu, Edward, Bakkenist, Christopher J., Beumer, Jan H.
• Format: Article
• Language: English
• Subjects: Ataxia Telangiectasia Mutated Proteins - antagonists & inhibitors; Biological Assay - methods; Chromatography, Liquid - methods; Clinical; Formates - chemistry; Humans; Isoxazoles - chemistry; Plasma - chemistry; Pyrazines - chemistry; Reproducibility of Results; Spectrometry, Mass, Electrospray Ionization - methods; Tandem mass spectrometry; Tandem Mass Spectrometry - methods; VX-970
• ISSN: 0731-7085; 1873-264X
• DOI: 10.1016/j.jpba.2017.08.037

[Display omitted] •VX-970 is a first in human inhibitor of ATR, a DNA damage response protein.•VX-970 is being studied in oncology Phase I and Phase II trials.•An LC–MS/MS assay from 3 to 5000ng/mL in 0.05mL plasma was validated.•This assay is currently utilized to quantitate triapine in clinical samples. DNA damaging chemotherapy and radiation are widely used standard-of-care modalities for the treatment of cancer. Nevertheless, the outcome for many patients remains poor and this may be attributed, at least in part, to highly effective DNA repair mechanisms. Ataxia-telangiectasia mutated and Rad3-related (ATR) is a key regulator of the DNA-damage response (DDR) that orchestrates the repair of damaged replication forks. ATR is a serine/threonine protein kinase and ATR kinase inhibitors potentiate chemotherapy and radiation. The ATR kinase inhibitor VX-970 (NSC 780162) is in clinical development in combination with primary cytotoxic agents and as a monotherapy for tumors harboring specific mutations. We have developed and validated an LC–MS/MS assay for the sensitive, accurate and precise quantitation of VX-970 in human plasma. A dilute-and-shoot method was used to precipitate proteins followed by chromatographic separation with a Phenomenex Polar-RP 80Å (4μm, 50×2mm) column and a gradient acetonitrile-water mobile phase containing 0.1% formic acid from a 50μL sample volume. Detection was achieved using an API 4000 mass spectrometer using electrospray positive ionization mode. The assay was linear from 3 to 5,000ng/mL, proved to be accurate (94.6–104.2%) and precise (