Metabolite Norfluoxetine In Plasma Research Articles

Rapid and direct measurement of free concentrations of highly protein‐bound fluoxetine and its metabolite norfluoxetine in plasma

Fluoxetine (F) and its active N-demethylated metabolite, norfluoxetine (NF), are selective serotonin re-uptake inhibitors that bind extensively to plasma proteins. Development and validation of a novel method for measuring free concentrations of F and NF in plasma are reported here. The plasma filtrate was prepared by a high-speed short-duration ultrafiltration (UF) and then submitted directly to a short-column liquid chromatography/tandem mass spectrometric (LC/MS/MS) assay. There was no significant matrix effect on the analysis, and non-specific binding of the analytes to the UF devices was negligible. For validation of the method, the recovery of the free analytes was compared to that from an optimized equilibrium dialysis method, and analyte stability was examined under conditions mimicking the sample storage, handling, and analysis procedures. The linearity range was 0.37-12 ng/mL for F and NF; the within-run and between-run relative standard deviations were less than 11.9%, and accuracies across the assay range were 100 +/- 10.3%. This new method was then further validated in a pharmacokinetic (PK) study in beagle dogs receiving a single oral dose of fluoxetine hydrochloride. The integrity of the resulting PK data of free F and NF was absolute. The PK data indicate that the novel method is accurate and reliable. To our knowledge this is the first report describing a rapid and reliable method for direct measurement of free concentrations of F and NF in plasma, which will be useful for clinical pharmacokinetic/pharmacodynamic studies of F. Furthermore, the strategies described herein may be applied to the development and validation of methods for measuring the free concentrations of other drugs in plasma.

Determination of fluoxetine and norfluoxetine in human plasma by high-performance liquid chromatography with ultraviolet detection in psychiatric patients

A rapid high-performance liquid chromatographic method is described for the simultaneous determination of the widely used antidepressant drug, fluoxetine and its principal metabolite norfluoxetine in plasma. After liquid–liquid extraction the compounds were separated in a reversed-phase column and assayed by ultraviolet absorption at 226 nm. The analytical interference from psychoactive drugs and their metabolites was also studied. The extraction recoveries were 93 and 87% for norfluoxetine and fluoxetine, respectively. The limit of quantitation under the described conditions was 14 nmol/l for both compounds. The method was found to be reproducible with coefficients of variation less than 10%. A great variability in plasma concentrations of fluoxetine and norfluoxetine as well as in fluoxetine/norfluoxetine ratios was found among the 29 patients studied. This result suggests the implication of genetically polymorphic enzymes, presumably CYP2D6, CYP2C9 and CYP2C19 in the metabolism of fluoxetine to norfluoxetine. Therapeutic drug monitoring should thus be useful in patients treated with regular doses.

Sensitive liquid chromatographic–tandem mass spectrometric method for the determination of fluoxetine and its primary active metabolite norfluoxetine in human plasma

A sensitive method for the simultaneous determination of fluoxetine and its major active metabolite norfluoxetine in plasma was developed, using high-performance liquid chromatographic separation with tandem mass spectrometric detection. The samples were extracted from alkalised plasma with hexane–isoamyl alcohol (98:2, v/v) followed by back-extraction into formic acid (2%). Chromatography was performed on a Phenomenex ® Luna C 18 (2) 5 μm, 150×2 mm column with a mobile phase consisting of acetonitrile–0.02% formic acid (340:660, v/v) at a flow-rate of 0.35 ml/min. Detection was achieved by a Perkin-Elmer Sciex API 2000 mass spectrometer (LC–MS–MS) set at unit resolution in the multiple reaction monitoring mode. TurboIonSpray ionisation was used for ion production. The mean recoveries for fluoxetine and norfluoxetine were 98 and 97%, respectively, with a lower limit of quantification set at 0.15 ng/ml for the analyte and its metabolite. This assay method makes use of the increased sensitivity and selectivity of mass spectrometric (MS–MS) detection to allow for a more rapid (extraction and chromatography) and sensitive method for the simultaneous determination of fluoxetine and norfluoxetine in human plasma than has previously been described.

Sensitive and Selective Liquid-Chromatographic Assay of Fluoxetine and Norfluoxetine in Plasma with Fluorescence Detection After Precolumn Derivatization

We determined fluoxetine (Prozac) and its major metabolite norfluoxetine in plasma by liquid chromatography with fluorescence detection. After liquid-liquid extraction from 1 mL of plasma, the extract was derivatized at room temperature with dansyl chloride, and the highly fluorescent derivatives were chromatographed with a reversed-phase C18 column and a mobile phase of phosphate buffer and acetonitrile. Dansylated fluoxetine, norfluoxetine, and the internal standard were eluted in less than 14 min with no interference from endogenous material. The calibration curve was linear over the concentration range 25-800 micrograms/L with inter- and intra-assay imprecision (CV) of less than 10%. Validity of the assay was checked by comparing results for 110 patients' samples with those by a liquid-chromatographic method with ultraviolet detection (r = 0.993 for fluoxetine, 0.957 for norfluoxetine). The identity of the dansylated derivatives was verified by positive chemical ionization mass spectroscopy. The lower limit of detection was approximately 3 micrograms/L. Because no major antidepressant, neuroleptic, or respective drug metabolites interfere with the quantification of fluoxetine and norfluoxetine, this is a useful procedure for pharmacokinetic studies and in clinical settings.