Simultaneous determination of fluoxetine and its major active metabolite norfluoxetine in human plasma by LC‐MS/MS using supported liquid extraction

A rapid, sensitive and selective bioanalytical method was developed for the simultaneous determination of fluoxetine and its primary metabolite norfluoxetine in human plasma. Sample preparation was based on supported liquid extraction (SLE) using methyl tert‐butyl ether to extract the analytes from human plasma. Chromatography was performed on a Synergi 4 μ polar‐RP column using a fast gradient. The ionization was optimized using ESI (+) and selectivity was achieved by tandem mass spectrometric analysis using MRM functions, m/z 310 → 44 for fluoxetine, m/z 296 → 134 for norfluoxetine and m/z 315 → 44 for fluoxetine‐d₅ (internal standard). The method is linear over the range of 0.05–20 ng/mL (using a human plasma sample volume of 0.1 mL) with a coefficient determination of greater than 0.999. The method is accurate and precise with intra‐batch and inter‐batch accuracy (%bias) of <±15% and precision (%CV) of <15% for both analytes. A run time of 4 min means a high throughput of samples can be achieved. To our knowledge, this method appears to be the most sensitive one reported so far for the quantitation of fluoxetine and norfluoxetine and can be used for routine therapeutic drug monitoring or pharmacokinetic studies. Copyright © 2011 John Wiley & Sons, Ltd.     

Hollow-fibre supported liquid membrane extraction for determination of fluoxetine and norfluoxetine concentration at ultra trace level in sewage samples

In this study, a method was developed for determining the concentration of the pharmaceutical fluoxetine and its metabolite, norfluoxetine, in sewage water samples. Sample preparation was performed by hollow-fibre supported liquid membrane (HF-SLM) extraction with final analysis using liquid chromatography with UV detection. Several parameters were studied including type of organic solvent, sample and acceptor pH, and salt and humic acid content. The optimised method allowed determination of the analyte at the ng/L level in sewage water. A linear plot gave a correlation coefficient better than 0.991 for both analytes and resulted in limits of detection in sewage water of 11 and 12 ng/L, for fluoxetine and norfluoxetine, respectively. The enrichment factor was over 1700 for both analytes in sewage water. The repeatability and reproducibility were better than 8% and 17%, respectively. The developed methodology was used to study daily variations of fluoxetine and norfluoxetine in municipal sewage streams. Norfluoxetine has been detected for the first time in sewage water and a preliminary analysis gave average concentrations of 150 and 225 ng/L for norfluoxetine and fluoxetine, respectively.     

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 C18 (2) 5 microm, 150x2 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. TurbolonSpray 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 specific liquid chromatography-tandem mass spectrometry method for assay of fluoxetine and its metabolite norfluoxetine in human plasma and application of method to pharmacokinetic analysis

A simple, specific and sensitive high-performance liquid chromatography — electrospray tandem mass spectrometry method is developed for the simultaneous determination of fluoxetine and its metabolite norfluoxetine in human plasma. Plasma samples were simply treated with acetonitrile to precipitate and remove proteins and the isolated supernatants were directly injected into the high-performance liquid chromatography — electrospray tandem mass spectrometry system. Chromatographic separation of the analytes was achieved on a Discovery C₁₈ (100 × 2.1 mm I.D., particle size 3.0 μm) column using 0.1% formic acid in water — acetonitrile (40: 60) as mobile phase with a flow rate of 0.2 mL/min. Diazepam was used as the internal standard. The compounds were ionized in the electrospray ionization source of the mass spectrometer and were detected by selected reaction ion monitoring of the transitions of m/z 310 → m/z 44.3 for fluoxetine, m/z 296 → m/z 134 for norfluoxetine and m/z 285 → m/z 193 for the internal standard. The method has low limit of detection (LOD) of 0.02 ng/mL and 0.03 ng/mL for fluoxetine and norfluoxetine, respectively. The inter- and intra-run precision was measured to be below 5.3% (relative standard deviation) for both fluoxetine and norfluoxetine. The developed method was successfully used to investigate plasma concentrations of fluoxetine and norfluoxetine in the pharmacokinetic study of Chinese volunteers who received fluoxetine orally.     

Determination of fluoxetine and its N-desmethyl metabolite in human plasma by high-performance liquid chromatography

A rapid and sensitive high-performance liquid chromatographic method has been developed for the simultaneous determination of the antidepressant fluoxetine and its active metabolite norfluoxetine in human plasma using paroxetine as internal standard. After liquid-liquid extraction, the compounds were separated on a C18 column using as mobile phase acetonitrile and 40 mM potassium dihydrogen phosphate buffer (pH 2.3) in the ratio 31:69 (v/v). The quantification of fluoxetine and norfluoxetine was made by fluorescence detection at Ex/Em 230/312 nm. The assay for each analyte was linear over the ranges 1-39 and 0.9-36 ng/ml, respectively. For both compounds intra- and inter-day accuracy and precision ranged between −7.9-12.4 and 0.7-14.7%, respectively. The method was applied to the analysis of plasma samples obtained from healthy subjects treated with one single oral dose of 40 mg fluoxetine.     

Analysis of paroxetine, fluoxetine and norfluoxetine in fish tissues using pressurized liquid extraction, mixed mode solid phase extraction cleanup and liquid chromatography-tandem mass spectrometry

Recent studies have shown that selective serotonin reuptake inhibitors (SSRIs) such as fluoxetine are accumulated in the tissues of fish as a result of discharges of pharmaceuticals into surface waters from municipal wastewater treatment plants. In this study, an analytical method based on liquid chromatography with atmospheric pressure chemical ionization and tandem mass spectrometry (LC-APCI-MS/MS) was developed and validated for the determination of residues of paroxetine, fluoxetine and its active metabolite, norfluoxetine, in fish tissue. The procedure for sample preparation includes extraction of tissue by pressurized liquid extraction (PLE), followed by cleanup on a mixed-mode solid phase extraction (SPE) cartridge, Oasis MCX. With the optimized method, matrix interferences were reduced and recoveries >85% were obtained. The limits of quantitation (LOQ) determined by analysis of spiked fish tissue were 0.24, 0.07, and 0.14 ng/g wet weight for paroxetine, fluoxetine and norfluoxetine, respectively. This method was successfully applied to the analysis of samples of fish collected from Hamilton Harbour in Ontario, Canada, which is an urbanized and industrialized embayment of Lake Ontario. These analyses showed that the three analytes were present in fish tissues at concentrations up to approximately 1 microg/kg wet weight.     

Improved HPLC determination of fluoxetine and norfluoxetine in human plasma

Summary An HPLC method with fluorescence detection has been developed for the determination of fluoxetine and its main metabolite norfluoxetine in human plasma. Pretreatment of the biological samples by liquid-liquid extraction was used to improve the sensitivity of a previously published SPE procedure. The method uses 200 μL plasma and recovery is good for both analytes. On a C₈ column with a mixture of perchlorate buffer and acetonitrile as mobile phase fluoxetine, norfluoxetine and the internal standard (paroxetine) were eluted in less than 9 min, without interference from the biological matrix. Response for both analytes was linearly dependent on concentration over the range 2.5–500 ng mL⁻¹, and repeatability (RSD%) was <4%. The limit of detection was 1 ng mL⁻¹ for both fluoxetines. Application to plasma samples from depressed patients treated with fluoxetine gave good results. There was no interference from other common CNS drugs. This method seems to be a useful tool for clinical monitoring, because it requires small plasma samples and is highly sensitive and highly selective.     

Solid-phase extraction of fluoxetine and norfluoxetine from serum with gas chromatography-electron-capture detection

A rapid, selective, and sensitive method is described for the purification and analysis of fluoxetine and norfluoxetine using a solid-phase extraction column and gas chromatography-electron-capture detection. Linear quantitative response curves for fluoxetine and norfluoxetine are generated over a concentration range of 20-200 ng/ml. Overall extraction efficiency of the extraction procedure is found to be greater than 90% and greater than 75% with correlation coefficients of 0.997 and 0.993 for fluoxetine and norfluoxetine, respectively.     

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

A rapid and sensitive method was developed for the simultaneous determination of fluoxetine and its primary metabolite, norfluoxetine, in plasma. It was based on a column-switching approach with a precolumn packed with large size particles coupled with a liquid chromatography–electrospray ionisation–mass spectrometry (LC-ESI-MS). After a simple centrifugation, plasma samples were directly injected onto the precolumn. The endogenous material was excluded thanks to a high flow rate while analytes were retained by hydrophobic interactions. Afterwards, the target compounds were eluted in back flush mode to an octadecyl analytical column and detected by ESI-MS. The overall analysis time per sample, from plasma sample preparation to data acquisition, was achieved in less than 4 min. Method performances were evaluated. The method showed good linearity in the range of 25–1000 ng mL^–1 with a determination coefficient higher than 0.99. Limits of quantification were estimated at 25 ng mL^–1 for fluoxetine and norfluoxetine. Moreover, method precision was better than 6% in the studied concentration range. These results demonstrated that the method could be used to quantify target compounds. Finally, the developed assay proved to be suitable for the simultaneous analysis of fluoxetine and its metabolite in real plasma samples.     

Trace analysis of fluoxetine and its metabolite norfluoxetine. Part I: development of a chiral liquid chromatography-tandem mass spectrometry method for wastewater samples

An enantioselective method for the determination of fluoxetine (a selective serotonin reuptake inhibitor) and its pharmacologically active metabolite norfluoxetine has been developed for raw and treated wastewater samples. The stable isotope-labeled fluoxetine and norfluoxetine were used in an extended way for extraction recovery calculations at trace level concentrations in wastewater. Wastewater samples were enriched by solid phase extraction (SPE) with Evolute CX-50 extraction cartridges. The obtained extraction recoveries ranged between 65 and 82% in raw and treated wastewater at a trace level concentration of 50 pM (15-16 ng L⁻¹). The target compounds were identified by the use of chiral liquid chromatography tandem mass spectrometry (LC-MS/MS) in selected reaction monitoring (SRM) mode. The enantiomers were successfully resolved on a chiral α₁-acid glycoprotein column (chiral AGP) with acetonitrile and 10 mM ammonium acetate buffer at pH 4.4 (3/97, v/v) as the mobile phase. The effects of pH, amount of organic modifier and buffer concentration in the mobile phase were investigated on the enantiomeric resolution (R_s) of the target compounds. Enantiomeric R_s-values above 2.0 (1.03 RSD%, n = 3) were achieved for the enantiomers of fluoxetine and norfluoxetine in all mobile phases investigated. The method was validated by assessing parameters such as cross-contamination and carryover during SPE and during LC analysis. Cross-talk effects were examined during the detection of the analytes in SRM mode. In addition, the isotopic purity of fluoxetine-d₅ and norfluoxetine-d₅ were assessed to exclude the possibility of self-contamination. The interassay precision of the chromatographic separation was excellent, with relative standard deviations (RSD) equal to or lower than 0.56 and 0.81% in raw and treated wastewaters, respectively. The method detection and quantification limits (respectively, MDL and MQL) were determined by the use of fluoxetine-d₅ and norfluoxetine-d₅. The MQL for the single enantiomers ranged from 12 to 14 pM (3.6-4.3 ng L⁻¹) in raw wastewater and from 3 to 4 pM (0.9-1 ng L⁻¹) in treated wastewater. The developed method has been employed for the quantification of (R)-fluoxetine, (S)-fluoxetine and the enantiomers of norfluoxetine in raw and treated wastewater samples to be presented in Part II of this study.     

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

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.     

Enantioseparation of cetirizine by sulfated-beta-cyclodextrin-mediated capillary electrophoresis

A sulfated beta-cyclodextrin (sulfated beta-CD)-mediated capillary electrophoresis method is described for the enantioseparation of cetirizine using achiral cefazolin as an internal standard. The enantioseparation of the drug was performed in a borate buffer (5 mM, pH 8.7) with 1% sulfated beta-CD (w/v) as chiral selector at 10 kV. Several parameters affecting the separation were studied, including the pH and the concentration of borate buffer and chiral selector. Under optimized conditions, a baseline separation of two enantiomers was achieved in less than 7 min. Using cefazolin as an internal standard (IS), the linear range of the method for the determination of levocetirizine was over 1.0 to 50.0 microg/mL; the detection limit (signal-to-noise ratio = 3) of levocetirizine was 0.5 microg/mL. The method allowed the enantioseparation of cetirizine in bulk samples and enantiomeric purity evaluation of levocetirizine (R-enantiomer) in pharmaceutical tablets (Xyzal), and it was also found to be suitable for enantioseparation in human plasma.     

Sensitive micromethod for column liquid chromatographic determination of fluoxetine and norfluoxetine in human plasma.

A rapid, selective and sensitive micromethod has been developed for the determination of fluoxetine (FLU) and its demethylated metabolite norfluoxetine (N-FLU) using a 250-microliters plasma sample and column liquid chromatography with ultraviolet detection at 226 nm. The limit of detection is 2.0 ng/ml for both FLU and N-FLU. Peak-height ratios are linear over a concentration range of 10-800 and 10-1000 ng/ml for FLU and N-FLU, respectively. Acceptable coefficients of variation are demonstrated for both within-run and day-to-day assays. Selected drugs were checked for interference. The method, which requires a very small volume of plasma, is sensitive enough for pharmacokinetic studies in animals, clinical pharmacology studies and drug monitoring in children or adult patients.     

Rapid liquid chromatography-tandem mass spectrometry method for the determination of a broad mixture of pharmaceuticals in surface water

Herein, a new method for the detection of 13 different pharmaceuticals and one metabolite in surface water at low ng/L levels is described. The method utilizes ultra performance liquid chromatography-tandem mass spectrometry and a solid-phase extraction sample preparation. Mean method detection limits were low (4.10 ng/L) and overall solid-phase extraction recovery and reproducibility was adequate (mean recovery, 77.9%; mean RSD, 7.3%). The method allows for quick run times and minimal solvent use as compared with other previously reported high performance liquid chromatography-based methods. Application of this method for the detection of pharmaceuticals in Tennessee River surface water determined that caffeine, sulfamethoxazole, and carbamazepine were frequently detected (100% of samples). Trimethoprim was moderately detected (30% of samples); acetaminophen, atorvastatin, and lovastatin were infrequently detected (10% of samples); and ciprofloxacin, diltiazem, fluoxetine, levofloxacin, norfluoxetine, ranitidine, and sertraline were not detected. This study reports the first detection of lovastatin in surface water.     

Development and validation method for determination of fluoxetine and its main metabolite norfluoxetine by nonaqueous capillary electrophoresis in human urine

A simple, rapid and sensitive procedure using nonaqueous capillary electrophoresis (NACE) to measure fluoxetine and its main metabolite norfluoxetine has been developed and validated. Optimum separation of fluoxetine and norfluoxetine, by measuring at 230 nm, was obtained on a 60 cm × 75 μm capillary using a nonaqueous solution system of 7:3 methanol-acetonitrile containing 15 mM ammonium acetate, capillary temperature and voltage 25 °C and 25 kV, respectively and hydrodynamic injection. Paroxetine was used as internal standard. Good results were obtained for different aspects including stability of the solutions, linearity, and precision. Detection limits of 10 μg L⁻¹ were obtained for fluoxetine and its metabolite. This method has been used to determine fluoxetine and it main metabolite at clinically relevant levels in human urine. Before NACE determination, the samples were purified and enriched by means of extraction-preconcentration step with a preconditioned C₁₈ cartridge and eluting the compounds with methanol.     

Fluoxetine and norfluoxetine analysis by direct injection of human plasma in a column switching liquid chromatographic system

A column switching LC method is presented for the analysis of fluoxetine (FLU) and norfluoxetine (NFLU) by direct injection of human plasma using a lab-made restricted access media (RAM) column. A RAM-BSA-octadecyl silica (C-18) column (40 mm x 4.6 mm, 10 microm) is evaluated in both backflush and foreflush elution modes and coupled with a C-18 lab-made (50 mm x 4.6 mm, 3 microm) analytical column in order to perform online sample preparation. Direct injection of 100 microL of plasma samples is possible with the developed approach. In addition, reduction of sample handling is obtained when compared with traditional liquid-liquid extraction (LLE) and SPE. The total analysis time is around 20 min. A LOQ of 15 ng/mL is achieved in a concentration range of 15-500 ng/mL, allowing the therapeutic drug monitoring of clinical samples. The precision values achieved are lower than 15% for all the evaluated points with adequate recovery and accuracy. Furthermore, no matrix interferences are found in the analysis and the proposed method shows to be an adequate alternative for analysis of FLU in plasma.     

On-line sample stacking and short-end injection CE for the determination of fluoxetine and norfluoxetine in plasma: Method development and validation using experimental designs

A short-end injection CE method combining field-amplified sample stacking (FASS) is presented for the analysis of fluoxetine (FL) and norfluoxetine in plasma. In this study, FASS enhanced the sensitivity about 1100-fold, while short-end injection reduced the analysis time to less than 4 min. Parameters involved in the separations were investigated using a central composite design (CCD) and response surface methodology to optimize the separation conditions in a total of only 32 runs. Samples injected into the capillary for 99.9 s at a voltage of -5 kV were stacked in a water plug (0.5 psi, 9 s). Baseline resolution of FL and its major metabolite was achieved using a BGE formulation consisting of phosphate-triethanolamine at low pH, and a separation voltage of -10 kV. Five percent methanol was added as organic modifier to enhance selectivity and resolution. The linear range was between 10 and 500 ng/mL (r >0.9946), covering the expected plasma therapeutic ranges. The LOD in plasma were 4 ng/mL (S/N = 3), a value comparable to that obtained using LC-MS, showing the success of the on-line stacking technique. Our method was also successfully validated in quantification and pharmacokinetic studies with three volunteer plasma samples and could be applied to pharmacogenetic studies.     

Optimization of alcohol‐assisted dispersive liquid–liquid microextraction by experimental design for the rapid determination of fluoxetine in biological samples

A sensitive and rapid method based on alcohol‐assisted dispersive liquid–liquid microextraction followed by high‐performance liquid chromatography for the determination of fluoxetine in human plasma and urine samples was developed. The effects of six parameters on the extraction recovery were investigated and optimized utilizing Plackett–Burman design and Box–Benken design, respectively. According to the Plackett–Burman design results, the volume of disperser solvent, extraction time, and stirring speed had no effect on the recovery of fluoxetine. The optimized conditions included a mixture of 172 μL of 1‐octanol as extraction solvent and 400 μL of methanol as disperser solvent, pH of 11.3 and 0% w/v of salt in the sample solution. Replicating the experiment in optimized condition for five times, gave the average extraction recoveries equal to 90.15%. The detection limit of fluoxetine in human plasma was obtained 3 ng/mL, and the linearity was in the range of 10–1200 ng/mL. The corresponding values for human urine were 4.2 ng/mL with the linearity range from 10 to 2000 ng/mL. Relative standard deviations for intra and inter day extraction of fluoxetine were less than 7% in five measurements. The developed method was successfully applied for the determination of fluoxetine in human plasma and urine samples.     

Enantioselective Analysis of Fluoxetine and Norfluoxetine by LC in Culture Medium for Application in Biotransformation Studies Employing Fungi

A liquid chromatography method is described for the analysis of fluoxetine and norfluoxetine enantiomers in fungi cultures. The analytes were separated simultaneously by LC employing a serial system. The resolution was performed using a mobile phase of ethanol: 15 mM ammonium acetate buffer solution, pH 5.9: acetonitrile (77.5:17.5:5, v/v/v). UV detection was at 227 nm. Hexane: isoamyl alcohol (98:2, v/v) was used as extractor solvent. The calibration curves were linear over the concentration range of 12.5–3,750 ng mL^?1 (r ≥ 0.996). The values for intra- and inter-day precision and accuracy were ≤10% for all analytes. The validated method was used to evaluate fluoxetine biotransformation to its mammalian metabolite, norfluoxetine, by selected endophytic fungi. Although the desired biotransformation was not observed in the conditions used here, the method could be used to evaluate the biotransformation of fluoxetine by other fungi or to be extended to other matrices with adequate procedures for sample preparation.     

Assay of urinary alpha-fluoro-beta-alanine by gas chromatography-mass spectrometry for the biological monitoring of occupational exposure to 5-fluorouracil in oncology nurses and pharmacy technicians

The validation of an analytical method for the measurement of the unnatural amino acid alpha-fluoro-beta-alanine (AFBA), the main metabolite of the antineoplastic drug 5-fluorouracil (5FU), in urine for the biological monitoring of the exposure of hospital workers to the drug when preparing the therapeutical doses and administering to cancer patients is described. The method employed a two-step extractive derivatization of the analyte from urine to the N-trifluoroacety-n-butyl ester derivative and detection by selected-ion monitoring gas chromatography-mass spectrometry of structurally specific fragments. The limit of detection was 20 ng/mL with quantification accuracy better than +/-20% and precision (CV%) better than +/-20% in the range 0.020-10 microg/mL. Norleucine was used as the internal standard and the sample-to-sample analysis time was less than 15 min. The validated method has been applied to the biological monitoring of some hospital workers potentially exposed to 5FU and to matched control subjects. On a total number of 65 analyzed urine samples from control and exposed subjects, only three, obtained from exposed subjects, were found to be positive, with values of 20, 30 and 1150 ng/mL, respectively.