title: Validation and implementation of a liquid chromatography/tandem mass spectrometry assay for quantitation of the total and unbound RO4929097, a γ-secretase inhibitor targeting Notch signaling, in human plasma.
abstract: A reversed-phased liquid chromatography with tandem mass spectrometry (LC-MS/MS) method was developed and validated for quantitation of the total and unbound RO4929097, a γ-secretase inhibitor targeting Notch signaling, in human plasma. Sample preparation involved a liquid-liquid extraction with ethyl acetate. Chromatographic separation was achieved on a Waters X-Terra™ MS C(18) column with an isocratic mobile phase consisting of methanol/0.45% formic acid in water (60:40, v/v) running at a flow rate of 0.2 ml/min for 6 min. The lower limits of quantitation (LLOQs) were 5 ng/ml for the total RO4929097 in plasma and 0.5 ng/ml for the unbound drug in phosphate buffer solution (PBS). Calibration curves were linear over RO4929097 concentration range of 5-2000 ng/ml in plasma for the total drug and 0.5-200 ng/ml in PBS for the unbound drug. The intra-day and inter-day accuracy and precision were within the generally accepted criteria for bioanalytical method (<15%). The method has been successfully employed to characterize the total and unbound plasma pharmacokinetics of RO4929097 after its oral administration in cancer patients.
authors.affiliation: Karmanos Cancer Institute, Wayne State University, Detroit, MI 48201, USA.
authors.forename: Jianmei
authors.initials: J
authors.lastname: Wu
authors.forename: Richard
authors.initials: R
authors.lastname: Wiegand
authors.forename: Patricia
authors.initials: P
authors.lastname: LoRusso
authors.forename: Jing
authors.initials: J
authors.lastname: Li
mesh_terms: D053829:Amyloid Precursor Protein Secretases / Q000037:antagonists & inhibitors; D000704:Analysis of Variance; D001552:Benzazepines / Q000097:blood* / Q000493:pharmacokinetics; D002851:Chromatography, High Pressure Liquid; D056148:Chromatography, Reverse-Phase / Q000379:methods*; D004355:Drug Stability; D004791:Enzyme Inhibitors / Q000097:blood / Q000493:pharmacokinetics; D006801:Humans; D016018:Least-Squares Analysis; D011485:Protein Binding; D051880:Receptors, Notch / Q000037:antagonists & inhibitors / Q000378:metabolism; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D015398:Signal Transduction / Q000187:drug effects; D053719:Tandem Mass Spectrometry / Q000379:methods*; D005466:Fluorocarbons
chemical_list: D053829:Amyloid Precursor Protein Secretases; D001552:Benzazepines; D004791:Enzyme Inhibitors; D051880:Receptors, Notch; C545185:2,2-dimethyl-N-(6-oxo-6,7-dihydro-5H-dibenzo(b,d)azepin-7-yl)-N'-(2,2,3,3,3-pentafluoropropyl)malonamide; D005466:Fluorocarbons