title: Simultaneous determination of fluoxetine and norfluoxetine enantiomers using isotope discrimination mass spectroscopy solution method and its application in the CYP2C9-mediated stereoselective interactions.
abstract: In this study, we developed an LC-MS/MS method based on an isotope discrimination mass spectroscopy solution (IDMSS) technology to simultaneously quantify enantiomers of fluoxetine (FLX) and norfluoxetine (NFLX) in a CYP2C9 incubation mixture. S-FLX and S-NFLX were labeled to form S-FLX-d5 and S-NFLX-d5. The method has several advantages over conventional chiral separation methods, in terms of the analysis period, resolution, and lower limit of quantification. The primary advantage of the method is that the two enantiomers can always be simultaneously determined by mass spectroscopy regardless if they are separated on column or not, owing to which it has high throughput and high sensitivity. The lower limit of quantification (amount on column) is 12.5 and 1.25 pg for FLX and NFLX, respectively. The retention time of FLX, NFLX, and the internal standard is only 1.9 min. The calibration curves were linear over the concentration range of 0.1-100 ng/ml for NFLX and 1-1000 ng/ml for FLX with an accepted reproducible (RSD<10%) and accurate (CV<10%). No significant kinetic isotope effect was found in the metabolism of S-FLX-d5 catalyzed by CYP2C9*1 and CYP2C9*2. The half-maximal inhibitory concentration values between R-FLX and S-FLX catalyzed by CYP2C9*1 and CYP2C9*2 were determined in this study. The inhibitory effects of R- to S-FLX were stronger than those of S- to R-FLX in both CYP2C9*1 and CYP2C9*2. The IDMSS technology is useful for stereoselective study of chiral compound in vitro.
authors.affiliation: College of Pharmaceutical Sciences, Zhejiang University, 886 Yuhangtang Road, Hangzhou, Zhejiang 310058, China.
authors.forename: Lushan
authors.initials: L
authors.lastname: Yu
authors.forename: Shengjia
authors.initials: S
authors.lastname: Wang
authors.forename: Huidi
authors.initials: H
authors.lastname: Jiang
authors.forename: Hui
authors.initials: H
authors.lastname: Zhou
authors.forename: Su
authors.initials: S
authors.lastname: Zeng
mesh_terms: D001189:Aryl Hydrocarbon Hydroxylases / Q000378:metabolism*; D002853:Chromatography, Liquid / Q000379:methods*; D065729:Cytochrome P-450 CYP2C9; D004355:Drug Stability; D005473:Fluoxetine / Q000031:analogs & derivatives* / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D007553:Isotope Labeling; D016014:Linear Models; D015203:Reproducibility of Results; D013237:Stereoisomerism; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D005473:Fluoxetine; C450260:CYP2C9 protein, human; D065729:Cytochrome P-450 CYP2C9; D001189:Aryl Hydrocarbon Hydroxylases; C036139:norfluoxetine