title: Simultaneous determination of a p38 MAP kinase inhibitor and its amide hydrolyzed metabolite in Cynomolgus monkey plasma by LC-MS/MS, and its application to a toxicokinetic study.
abstract: A LC-MS/MS method was developed for the determination of a p38 MAP kinase inhibitor (Compound I) and its amide hydrolyzed metabolite (M7) in Cynomolgus monkey plasma over the concentration range of 1.00-1000ng/mL. Stable isotope labeled compounds (d(3)-Compound I and d(3)-M7) were used as internal standards (IS). Samples were prepared using protein precipitation in the 96-well format with a 30μL plasma sample volume. Chromatographic separation was performed with reversed-phase liquid chromatography on a Varian Monochrom C(18) (100mm×2.00mm, 5μm) analytical column. The mobile phases were 5mM ammonium formate in acetonitrile/water (95/5, v/v) pH 7.0 and 5mM ammonium formate in acetonitrile/water (5/95, v/v) pH 7.0. Gradient elution, at a flow rate of 550μL/min, was used to separate Compound I and M7. Positive atmospheric pressure chemical ionization was utilized with detection by multiple reaction monitoring (MRM). Total run time was about 3.2min. This method was validated following the current Food and Drug Administration (FDA) guidance for bioanalytical method validation. The intra- and inter-day precision (% CV) and accuracy (% bias) at all concentrations tested were below 15% for both analytes. The mean recoveries for Compound I, M7, d(3)-Compound I, and d(3)-M7 were 106%, 107%, 108% and 105%, respectively. The method was successfully applied to support a GLP toxicokinetic study in Cynomolgus monkeys after oral administration of Compound I. A total of 48 samples (∼12.5% of the total number of samples) were selected for incurred sample reanalysis (ISR). The % difference between the reassay concentrations and the original concentrations were all less than 20% of their mean values and met the acceptance criteria for ISR.
authors.affiliation: Department of Pharmacokinetics and Drug Metabolism, Amgen, Thousand Oaks, CA 91320-1799, United States.
authors.forename: Anna
authors.initials: A
authors.lastname: Akrami
authors.forename: Faye
authors.initials: F
authors.lastname: Hsieh
authors.forename: Valerie
authors.initials: V
authors.lastname: Almon
authors.forename: Bernd A
authors.initials: BA
authors.lastname: Bruenner
authors.forename: Christopher
authors.initials: C
authors.lastname: James
authors.forename: Philip
authors.initials: P
authors.lastname: Wong
mesh_terms: D000097:Acetonitriles / Q000737:chemistry; D000284:Administration, Oral; D000577:Amides / Q000737:chemistry*; D000818:Animals; D019540:Area Under Curve; D002138:Calibration; D002623:Chemistry Techniques, Analytical; D002853:Chromatography, Liquid / Q000379:methods; D004791:Enzyme Inhibitors / Q000032:analysis* / Q000097:blood; D005260:Female; D005561:Formates / Q000737:chemistry; D007477:Ions; D008252:Macaca fascicularis; D008297:Male; D011786:Quality Control; D015203:Reproducibility of Results; D053719:Tandem Mass Spectrometry / Q000379:methods; D014867:Water / Q000737:chemistry; D048051:p38 Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors*
chemical_list: D000097:Acetonitriles; D000577:Amides; D004791:Enzyme Inhibitors; D005561:Formates; D007477:Ions; D014867:Water; C030544:formic acid; D048051:p38 Mitogen-Activated Protein Kinases; C032159:acetonitrile