Liquid chromatography/tandem mass spectrometry for the determination of fluoxetine and its main active metabolite norfluoxetine in human plasma with deuterated fluoxetine as internal standard

Abstract

Fluoxetine (F) and its N-demehylated metabolite norfluoxetine (NF) are selective inhibitors of serotonin reuptake in humans. A new sensitive rapid method for the simultaneous determination of F and NF in plasma was established and validated, and was further applied to assess the bioequivalence of two oral formulations of F in 22 healthy Chinese male volunteers who received a single oral dose of each formulation (containing 20 mg of fluoxetine hydrochloride). The new method involves using liquid chromatography/tandem mass spectrometry (LC/MS/MS) in multiple reaction monitoring mode with deuterated fluoxetine (DF) as internal standard. High levels of analytical sensitivity and specificity of MS/MS detection enabled use of a simple liquid-liquid extraction procedure. The combination of a simple sample clean-up procedure and short chromatographic run-time (5 min) considerably increased the productivity of the analytical method. The method was validated for the plasma concentration range 0.27-22 ng/mL for both of the test compounds, and the calibration curves were linear with coefficients of correlation >0.999. The limit of detection was 0.1 ng/mL for plasma F and NF. Taking the plasma sample size (200 micro L) into account the new method for determination of F and NF is more sensitive than those described previously.

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

“…Duplicate 20 mL aliquots of the filtrate were then spiked with 10 mL IS spiking solution and directly subjected to LC/MS/MS analysis. Total drug concentrations were measured as previously described 30 with some modifications. In brief, each plasma sample (50 mL) was mixed with 10 mL IS spiking solution and 20 mL 1 M KCl/1 M NaOH buffer (25/20, v/v, pH 12), and then extracted with 1 mL EtOAc.…”

“…[26][27][28] Previously, analytical methods for F and NF in plasma were developed for measuring their total concentrations. [29][30][31][32][33] However, no significant correlation has been identified between the total concentrations of F and NF in serum and the therapeutic response in depressed patients. [34][35][36][37][38][39] Holladay et al reported that transgenic mice over-expressing serum alpha-1-acid glycoprotein exhibited increased total serum concentrations of F, but the antidepressant activity of the drug was significantly reduced relative to the normal mice, which was consistent with the lower brain drug levels in the transgenic mice.…”

A validated enantioselective assay for the simultaneous quantitation of (R)-, (S)-fluoxetine and (R)-, (S)-norfluoxetine in ovine plasma using liquid chromatography with tandem mass spectrometry (LC/MS/MS)

“…Several fluoxetine analytical methodologies have been reported in the literature. Methods with non-chiral chromatographic conditions using either liquid or gas chromatography offered only information for racemic fluoxetine and/or norfluoxetine, but not for specific enantiomers [12][13][14][15][16][17][18][19][20]. Methods with non-chiral chromatographic conditions that were enantioselective for the (R)-, (S)-enantiomers of fluoxetine and/or norfluoxetine required a derivatization step during sample preparation to yield their respective diastereomers, which were separated more readily using non-chiral chromatographic conditions.…”

Rapid analysis of fluoxetine and its metabolite in plasma by LC-MS with column-switching approach

“…19, 20]. In order to achieve high sensitivity, several approaches including a preconcentration step [11,12,15,16,17,18], large injection volume [15,16], derivatisation procedures [21] or the use of more specific and sensitive detectors such as mass spectrometry (MS or MS/MS) [22,23] were found necessary. More recently, column miniaturisation has evolved as a very suitable alternative for sensitivity improvement [24].…”