Validation of a liquid chromatography/tandem mass spectrometry method for the simultaneous quantification of Sotrastaurin and its metabolite N-desmethyl-sotrastaurin in human blood

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• 作者：Annamaria Jakab^a ; ^{annamaria.jakab@novartis.com} ; Serge Winter^a ; Bertrand Guy^a ; Marc Raccuglia^a ; Frank Picard^a ; John M. Kovarik^a ; Jayraj Chudasama^b ; Swati Guttikar^b ; Puran Singhal^b ; Olivier Kretz^a
• 关键词：Sotrastaurin ; N-desmethyl-sotrastaurin ; AEB071 ; Metabolite ; LC&ndash ; MS/MS ; Human blood
• 刊名：Journal of Chromatography B ; Analytical Technologies in the Biomedical and Life Sciences
• 出版年：2012
• 期刊代码：124_15700232
• 类别：ch
• 出版时间：15 May, 2012
• 卷：897
• 期：Complete
• 页码：10-16
• 文件大小：546 K

摘要

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) 鈥淕uidance for Industry, Bioanalytical Method Validation鈥? Chromatographic separation was performed using an RP C₁₈ (50 mm 脳 4.6 mm, 5 渭m) column at 40 卤 3.0 掳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 渭L, low inter-run bias and variability (for Sotrastaurin, 鈭?.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 鈮?0%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.