title: Simultaneous determination of a novel KDR kinase inhibitor and its N-oxide metabolite in human plasma using 96-well solid-phase extraction and liquid chromatography/tandem mass spectrometry.
abstract: To support pharmacokinetic studies, a selective and sensitive liquid chromatography/tandem mass spectrometry (LC-MS/MS) method has been developed and validated for the simultaneous determination of a novel KDR kinase inhibitor (1) and its active metabolite (2) in human plasma. The method is fully automated using a Packard MultiPROBE II system and a TomTec Quadra 96 liquid handling workstation to perform sample preparation and solid-phase extraction (SPE). Following the extraction on a mixed-mode SPE using Oasis MCX 96-well plate, the analytes were separated on a Aquasil C18 column (50 mm x 2.1 mm, i.d., 3 microm) with a mobile phase consisting of acetonitrile/ammonium acetate buffer (5 mM, pH 5.0) (60/40, v/v). The run time for each injection was 4.5 min with the retention times of approximately 2.0 and 2.7 min for 1 and 2 respectively, at a flow rate of 0.25 mL/min. A tandem mass spectrometric detection was conducted using multiple reaction monitoring (MRM) under the positive ion mode with a turbo ion-spray interface. The linear ranges of the calibration curves were 0.05-400 ng/mL for 1 and 0.1-400 ng/mL for 2 on a PE Sciex API 4000 LC-MS/MS system. The lower limits of quantitation (LLOQ) of the assay were 0.05 and 0.1 ng/mL for 1 and 2 respectively, when 0.4 mL of plasma was processed. Intra-day assay precision (using five standard curves prepared by spiking compounds to five lots of plasma) was less than 4.9% for 1 and less than 9.6% for 2 on each concentration. Assay accuracy was found to be 95.1-104.6% of nominal for 1 standards and 93.5-105.6% for 2 standards. QC samples were stable when kept at room temperature for 4 h, at -70 degrees C for 10 days, and after three freeze-thaw cycles. The extraction recoveries were 80%, 83% and 84% for 1 and 2 and I.S. respectively, and no significant matrix effects were observed. The method was successfully applied to plasma samples from clinical studies after oral administration of compound 1.
authors.affiliation: Merck Research Laboratories, Department of Drug Metabolism, WP75A-303, West Point, PA 19486, USA. yang_xu@merck.com
authors.forename: Yang
authors.initials: Y
authors.lastname: Xu
authors.forename: Lihong
authors.initials: L
authors.lastname: Du
authors.forename: Eric D
authors.initials: ED
authors.lastname: Soli
authors.forename: Matthew P
authors.initials: MP
authors.lastname: Braun
authors.forename: Dennis C
authors.initials: DC
authors.lastname: Dean
authors.forename: Donald G
authors.initials: DG
authors.lastname: Musson
mesh_terms: D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000097:blood*; D013058:Mass Spectrometry / Q000379:methods*; D010087:Oxides / Q000378:metabolism*; D012015:Reference Standards; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
chemical_list: D047908:Intracellular Signaling Peptides and Proteins; D010087:Oxides; C015482:protein kinase modulator