title: LC-MS/MS assay for the simultaneous quantitation of the ATM inhibitor AZ31 and the ATR inhibitor AZD6738 in mouse plasma.
abstract: The ATM kinase inhibitor AZ31 and ATR kinase inhibitor AZD6738 are in various phases of preclinical and clinical evaluation for their ability to potentiate chemoradiation. To support the preclinical evaluation of their pharmacokinetics, we developed and validated an LC-MS/MS assay for the simultaneous quantification of AZ31 and AZD6738 in mouse plasma. A "dilute and shoot" method was used to precipitate proteins from a sample volume of 50μL. Chromatographic separation was achieved using a Phenomenex Polar-RP column and a gradient mobile phase consisting of methanol-water with 0.1% formic acid. Detection was accomplished using a Waters Quattro Micro mass spectrometer in positive ionization mode. The assay utilizing 50μL sample was linear from 10 to 5000ng/mL and determined to be both accurate (-8.2 to 8.6%) and precise (<5.4% CV) and achieved the criteria for U.S. FDA guidance for bioanalytical method validation. Quantification was achieved in mouse tissue homogenate using a separate 200μL sample preparation. This LC-MS/MS assay will be essential for determining the tissue distribution and pharmacokinetics in future mouse studies.
authors.affiliation: Cancer Therapeutics Program, University of Pittsburgh Cancer Institute, Pittsburgh, PA, USA; Department of Pharmaceutical Sciences, School of Pharmacy, University of Pittsburgh, Pittsburgh, PA, USA.
authors.forename: Brian F
authors.initials: BF
authors.lastname: Kiesel
authors.affiliation: Department of Radiation Oncology, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA.
authors.forename: Jeffrey C
authors.initials: JC
authors.lastname: Shogan
authors.affiliation: Cancer Therapeutics Program, University of Pittsburgh Cancer Institute, Pittsburgh, PA, USA.
authors.forename: Madani
authors.initials: M
authors.lastname: Rachid
authors.affiliation: Cancer Therapeutics Program, University of Pittsburgh Cancer Institute, Pittsburgh, PA, USA.
authors.forename: Robert A
authors.initials: RA
authors.lastname: Parise
authors.affiliation: Department of Radiation Oncology, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA.
authors.forename: Frank P
authors.initials: FP
authors.lastname: Vendetti
authors.affiliation: Department of Radiation Oncology, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA; Department of Pharmacology and Chemical Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA.
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Bakkenist
authors.affiliation: Cancer Therapeutics Program, University of Pittsburgh Cancer Institute, Pittsburgh, PA, USA; Department of Pharmaceutical Sciences, School of Pharmacy, University of Pittsburgh, Pittsburgh, PA, USA; Division of Hematology-Oncology, Department of Medicine, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA. Electronic address: beumerj@gmail.com.
authors.forename: Jan H
authors.initials: JH
authors.lastname: Beumer
mesh_terms: D000497:Alloys / Q000737:chemistry*; D000818:Animals; D064007:Ataxia Telangiectasia Mutated Proteins / Q000037:antagonists & inhibitors*; D001681:Biological Assay / Q000379:methods; D002853:Chromatography, Liquid / Q000379:methods*; D004355:Drug Stability; D005561:Formates / Q000737:chemistry; D007211:Indoles; D000432:Methanol / Q000737:chemistry; D051379:Mice; D009025:Morpholines; D010949:Plasma / Q000737:chemistry*; D011743:Pyrimidines / Q000097:blood / Q000737:chemistry*; D015203:Reproducibility of Results; D013449:Sulfonamides; D013454:Sulfoxides / Q000097:blood / Q000737:chemistry*; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D000497:Alloys; D005561:Formates; D007211:Indoles; C586533:Mg-Al-Zn-Mn-Si-Cu alloy; D009025:Morpholines; D011743:Pyrimidines; D013449:Sulfonamides; D013454:Sulfoxides; C030544:formic acid; C000611951:ceralasertib; C576897:Atr protein, mouse; D064007:Ataxia Telangiectasia Mutated Proteins; D000432:Methanol
keywords: ATM; ATR; AZ31; AZD6738; Assay; Validation