title: Validation of a liquid chromatography/tandem mass spectrometry method for the simultaneous quantification of Sotrastaurin and its metabolite N-desmethyl-sotrastaurin in human blood.
abstract: A liquid chromatography/tandem mass spectrometry (HPLC-MS/MS) method was validated for the quantification of Sotrastaurin (AEB071) and N-desmethyl-sotrastaurin in human blood. The validation of the analytical procedure was performed according to the latest Food and Drug Administration (FDA) "Guidance for Industry, Bioanalytical Method Validation". Chromatographic separation was performed using an RP C₁₈ (50 mm × 4.6 mm, 5 μm) column at 40±3.0 °C with a mobile phase consisted of 2 mM ammonium acetate in water (pH 4.5):methanol:acetonitrile (25:15:60, v/v) of a flow rate of 1 mL/min followed by quantification with tandem mass spectrometer, operated in electrospray ionization (ESI) positive ion mode and applying multiple reaction monitoring (MRM). The validated method described in this paper presents high absolute recovery, with a sensitivity of 3.00 ng/mL as lower limit of quantitation using a sample volume of 300 μL, low inter-run bias and variability (for Sotrastaurin, -4.4 to 0.4% and 1.8 to 2.5% and for N-desmethyl-sotrastaurin, ranged from 1.6 to 2.3% and 2.7 to 3.9%, respectively) with a short runtime of 3.5 min. The method was validated using K₃EDTA as specific anticoagulant and cross-validated using Li-Heparin and Na-Heparin. The method was specific for Sotrastaurin and N-desmethyl-sotrastaurin within the given criteria of acceptance (apparent peak area for Sotrastaurin and N-desmethyl-sotrastaurin in zero samples ≤ 20% of mean peak area at LLOQ) in human blood. The method was fully validated for the quantitative determination of Sotrastaurin and its metabolite N-desmethyl-sotrastaurin in human blood between the range of 3.00 ng/mL and 1200 ng/mL.
authors.affiliation: Novartis Institutes for BioMedical Research, DMPK, Fabrikstrasse 14, CH-4056 Basel, Switzerland. annamaria.jakab@novartis.com
authors.forename: Annamaria
authors.initials: A
authors.lastname: Jakab
authors.forename: Serge
authors.initials: S
authors.lastname: Winter
authors.forename: Bertrand
authors.initials: B
authors.lastname: Guy
authors.forename: Marc
authors.initials: M
authors.lastname: Raccuglia
authors.forename: Frank
authors.initials: F
authors.lastname: Picard
authors.forename: John M
authors.initials: JM
authors.lastname: Kovarik
authors.forename: Jayraj
authors.initials: J
authors.lastname: Chudasama
authors.forename: Swati
authors.initials: S
authors.lastname: Guttikar
authors.forename: Puran
authors.initials: P
authors.lastname: Singhal
authors.forename: Olivier
authors.initials: O
authors.lastname: Kretz
mesh_terms: D002851:Chromatography, High Pressure Liquid / Q000379:methods* / Q000592:standards; D004355:Drug Stability; D006801:Humans; D016018:Least-Squares Analysis; D011758:Pyrroles / Q000097:blood* / Q000737:chemistry / Q000378:metabolism; D011799:Quinazolines / Q000097:blood* / Q000737:chemistry / Q000378:metabolism; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D053719:Tandem Mass Spectrometry / Q000379:methods
chemical_list: D011758:Pyrroles; D011799:Quinazolines; C543528:sotrastaurin