title: Validated UPLC-MS/MS method for determination of moclobemide in human brain cell supernatant and its application to bidirectional transport study.
abstract: A simple and sensitive analytical method based on ultraperformance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) has been developed for determination of moclobemide in human brain cell monolayer as an in vitro model of blood-brain barrier. Brucine was employed as the internal standard. Moclobemide and internal standard were extracted from cell supernatant by ethyl acetate after alkalinizing with sodium hydroxide. The UPLC separation was performed on an Acquity UPLC(TM) BEH C18 column (50 × 2.1 mm, 1.7 µm, Waters, USA) with a mobile phase consisting of methanol-water (29.5:70.5, v/v); the water in the mobile phase contained 0.05% ammonium acetate and 0.1% formic acid. Detection of the analytes was achieved using positive ion electrospray via multiple reaction monitoring mode. The mass transitions were m/z 269.16 → 182.01 for moclobemide and m/z 395.24 → 324.15 for brucine. The extraction recovery was 83.0-83.4% and the lower limit of quantitation (LLOQ) was 1.0 ng/mL for moclobemide. The method was validated from LLOQ to 1980 ng/mL with a coefficient of determination greater than 0.999. Intra- and inter-day accuracies of the method at three concentrations ranged from 89.1 to 100.9% for moclobemide with precision of 1.1-9.6%. This validated method was successfully applied to bidirectional transport study of moclobemide blood-brain barrier permeability.
authors.affiliation: Clinical Pharmacy and Pharmacology Research Institute, The Second Xiangya Hospital, Central South University, 139 Middle Renmin Road, Changsha, Hunan Province 410011, PR China.
authors.forename: Dai
authors.initials: D
authors.lastname: Li-Bo
authors.forename: Yan
authors.initials: Y
authors.lastname: Miao
authors.forename: Li
authors.initials: L
authors.lastname: Huan-De
authors.forename: Fang
authors.initials: F
authors.lastname: Ping-Fei
authors.forename: Wang
authors.initials: W
authors.lastname: Feng
authors.forename: Deng
authors.initials: D
authors.lastname: Yang
mesh_terms: D001812:Blood-Brain Barrier / Q000378:metabolism*; D001923:Brain Chemistry; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004355:Drug Stability; D006801:Humans; D016014:Linear Models; D020912:Moclobemide / Q000032:analysis* / Q000493:pharmacokinetics*; D008954:Models, Biological; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D020912:Moclobemide
keywords: UPLC-MS/MS; bidirectional transport; blood-brain barrier; moclobemide