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.     

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.     

Comparison of two extraction methods for the determination of selective serotonin reuptake inhibitors in sewage sludge by hollow fiber liquid-phase microextraction

This paper presents two procedures for the determination of four selective serotonin reuptake inhibitors (citalopram, paroxetine, fluoxetine, and sertraline) and one metabolite (norfluoxetine) in sewage sludge utilizing three-phase hollow fiber liquid-phase microextraction (HF-LPME). First, direct HF-LPME was used for extraction, clean-up, and preconcentration. The pharmaceuticals were extracted from slurry samples into an organic phase and then back-extracted into an aqueous phase in the lumen of the hollow fiber. Second, a procedure combining pressurized hot water extraction and HF-LPME for clean-up and preconcentration was developed for the same analytes and matrix. The extracts were subsequently analyzed by liquid chromatography-mass spectrometry. For direct HF-LPME, limits of detection were between 1 and 12 ng g(-1) (dry weight) and the relative standard deviation (RSD) values were 3-12%. For the second method, limits of detection were approximately 6 ng g(-1) for all the compounds and RSD values were 8-12%. The methods were validated by comparison of results for the same samples. Sewage sludge from a Swedish wastewater treatment plant was analyzed by both methods; average concentrations were similar for citalopram, paroxetine, and fluoxetine with values of approximately 530, 40, and 200 ng g(-1) , respectively.     

Occurrence of selective serotonin reuptake inhibitors in sewage and receiving waters at Spitsbergen and in Norway

A method for the determination of five selective serotonin reuptake inhibitors (citalopram, sertraline, fluoxetine, fluvoxamine and paroxetine) and four of their metabolites (desmethylcitalopram, didesmethylcitalopram, norfluoxetine and desmethylsertraline) in seawater and sewage influents and effluents, has been developed and validated. The method is based on a three-phase hollow-fibre supported liquid phase microextraction of 1.1L samples, followed by high performance liquid chromatography with electrospray ionization and mass spectrometric detection. The detection limits varied between 17 pg/L (citalopram) and 618 ng/L (desmethylsertraline), and the quantification limits between 57 pg/L (citalopram) and 4.1 ng/L (desmethylsertraline). Sampling was done from February to August in 2007 on three different locations with dissimilarities concerning waste water treatment procedures. No significant difference in SSRI cleansing efficiency between merely sieving (Langnes STP, Troms?) and a more advanced sewage treatment (VEAS STP, Oslo) was seen. All the investigated compounds are present in all waste water samples from these STPs, and a total concentration of SSRIs and metabolites up to 840 ng/L has been found. Untreated sewage samples have been collected in the small town Longyearbyen at Spitsbergen. Despite few inhabitants (2000), it was still possible to find traces of SSRIs in the waste water. In Troms? and Longyearbyen the waste water is discharged into the sea, therefore seawater samples have been collected close to the outlets. The results show higher concentrations of SSRIs outside Longyearbyen than Troms?, possibly due to the stronger tidal currents around Troms?. However, the concentrations are quite low, not exceeding total concentrations of 3 ng/L.     

Analysis of anticancer drugs in sewage water by selective SPE and UPLC-ESI-MS-MS

A trace analytical procedure was developed to assay the anticancer drugs methotrexate, azathioprine, doxorubicin, doxorubicinol, vincristine, ifosfamide, cyclophosphamide, etoposide, and procarbazine in water samples from sewage treatment plants. After concentration and purification using Oasis HLB solid-phase extraction cartridges and Oasis WAX cartridges, the analytes were separated using ultra-high performance liquid chromatography coupled with the electrospray ionization tandem mass spectrometry operating in the positive ion mode. The method showed good precision and accuracy. Recoveries of all analytes were in the range of 45.3-108.9% with relative standard deviations between 2.4-24.5%. The limits of detection for influent and effluent sewage water were in the range of 0.6-7.0 ng/L and 0.5-3.5 ng/L, respectively. It is expected that this method will be applied to investigate the environmental occurrence of anticancer drugs in sewage water.     

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.     

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.     

Simultaneous analysis of 16 sulfonamide and trimethoprim antibiotics in environmental waters by liquid chromatography-electrospray tandem mass spectrometry

A sensitive liquid chromatography-electrospray tandem mass spectrometry method combined with solid-phase extraction and silica cartridge cleanup was established for 16 sulfonamides and trimethoprim in various water matrices. Signal suppression of all target analytes in sewage treatment plant influent, effluent and river water was improved by this method developed in this study. The method detection limits for 17 analytes were 20-200 pg/L for influent, 16-120 pg/L for effluent and 8.0-60 pg/L for river water with overall mean recoveries of 62-102% in all studied matrices. This method was used to analyze residual sulfonamides and trimethoprim in wastewater and river samples from Japan, and 8 analytes (0.08 (sulfadimethoxine)-161 ng/L (sulfapyridine) in wastewater and 10 (0.03 (sulfamethizol)-8.9 ng/L (sulfaquinoxaline) in river samples were detected.     

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.     

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.     

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.     

A novel hollow-fibre microporous membrane liquid-liquid extraction for determination of free 4-isobutylacetophenone concentration at ultra trace level in environmental aqueous samples

In this study, a method was developed for determination of the free concentration of 4-isobutylacetophenone, a toxic degradation product of ibuprofen, in river and sewage water samples from Sweden. Sample preparation and analysis were performed by a hollow-fibre microporous membrane liquid-liquid extraction (HF-MMLLE) set-up and gas chromatography-mass spectrometry (GC-MS), respectively. In this novel approach, only the liquid in the membrane pores is utilised for non-depleting extraction. Several parameters were studied, including: type of organic solvent, sample pH, and salt and humic acid content. The optimised method allowed the determination of the analyte at the ng L(-1) level in river and sewage water. A linear plot gave a correlation coefficient better than 0.992 and resulted in a limit of detection of 7 and 14 ng L(-1) for river and sewage water, respectively. The enrichment factor was over 2000 in the fibre and over 300 after dilution. The repeatability and reproducibility were better than 5% and 10%, respectively. For the first time, 4-isobutylacetophenone was found at free concentrations of 40 ng L(-1) or below in sewage waters, while it could not be quantified in a river downstream from a municipal sewage treatment plant.     

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.     

Development of a rapid and sensitive SPE-LC-MS/MS method for the simultaneous estimation of fluoxetine and olanzapine in human plasma

A high‐performance liquid chromatographic assay with tandem mass spectrometric detection was developed to simultaneously quantify fluoxetine and olanzapine in human plasma. The analytes and the internal standard (IS) duloxetine were extracted from 500 μL aliquots of human plasma through solid‐phase extraction. Chromatographic separation was achieved in a run time of 4.0 min on a Hypersil Gold C₁₈ column (50 × 4.6 mm, 5 µm) using isocratic mobile phase consisting of acetonitrile–water containing 2% formic acid (70:30, v/v), at a flow‐rate of 0.5 mL/min. Detection of analytes and internal standard was performed by electrospray ionization tandem mass spectrometry, operating in positive‐ion and multiple reaction monitoring acquisition mode. The protonated precursor to product ion transitions monitored for fluoxetine, olanzapine and IS were m/z 310.01 → 147.69, 313.15 → 256.14 and 298.1 → 153.97, respectively. The method was validated over the concentration range of 1.00–150.20 ng/mL for fluoxetine and 0.12–25.03 ng/mL for olanzapine in human plasma. The intra‐batch and inter‐batch precision (%CV) across four quality control levels was ≤6.28% for both the analytes. In conclusion, a simple and sensitive analytical method was developed and validated in human plasma. This method is suitable for measuring accurate plasma concentration in bioequivalence study and therapeutic drug monitoring as well, following combined administration. Copyright © 2011 John Wiley & Sons, Ltd.     

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.     

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.     

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.     

Development of an analytical system for the simultaneous determination of anabolic macrocyclic lactones in aquatic environmental samples

An analytical procedure that enables routine analysis for trace determination of six anabolic macrocyclic lactones (zearalenone, alpha- and beta-zearalenol, zearalanone, zeranol, and taleranol) in sewage treatment plant (STP) samples has been developed. The method uses solid-phase extraction, followed by high-performance liquid chromatography with on-line tandem mass spectrometry using atmospheric pressure chemical ionization (LC/APCI-MS/MS). The extraction of these compounds from filtered water samples was performed off-line with C(18) solid-phase cartridges. The detection was achieved by isocratic reversed-phase high-performance liquid chromatography coupled with an heated nebulizer (HN) APCI interface operating in negative ion mode. Mean recovery of the analytes in STP effluent samples generally exceeded 81%. This method was used to determine the occurrence of target analytes in the aquatic environment. In the selected STP effluent samples, zearalenone and alpha-zearalenol were detected in the ng/L range.     

Simultaneous quantification of vortioxetine,fluoxetine and their metabolites in rat plasma by UPLC–MS/MS

In this study, a specific and quick ultra-performance liquid chromatography tandem mass spectrometry (UPLC–MS/MS) method was fully developed and validated for simultaneous measurement of the rat plasma levels of vortioxetine (VOR), Lu AA34443 (the major metabolite of VOR), fluoxetine and its metabolite norfluoxetine with diazepam as the internal standard (IS). After a simple protein precipitation with acetonitrile for sample preparation, the separation of the analytes were performed on an Acquity UPLC BEH C18 (2.1 × 50 mm, 1.7 μm) column, with acetonitrile and 0.1% formic acid in water as mobile phase by gradient elution. The detection was achieved on a triple quadrupole tandem mass spectrometer by multiple reaction monitoring mode via an electrospray ionization source. Good linearity was observed in the calibration curve for each analyte. The data of precision, accuracy, matrix effect, recovery and stability all conformed to the bioanalytical method validation of acceptance criteria of US Food and Drug Administration recommendations. The newly developed UPLC–MS/MS method allowed simultaneous quantification of VOR, fluoxetine and their metabolites for the first time and was successfully applied to a pharmacokinetic study in rats.     

Determination of fluoxetine and norfluoxetine enantiomers in human plasma by polypyrrole-coated capillary in-tube solid-phase microextraction coupled with liquid chromatography-fluorescence detection

A sensitive, selective, and reproducible in-tube polypyrrole-coated capillary (PPY) solid-phase microextraction and liquid chromatographic method for fluoxetine and norfluoxetine enantiomers analysis in plasma samples has been developed, validated, and further applied to the analysis of plasma samples from elderly patients undergoing therapy with antidepressants. Important factors in the optimization of in-tube SPME efficiency are discussed, including the sample draw/eject volume, draw/eject cycle number, draw/eject flow-rate, sample pH, and influence of plasma proteins. Separation of the analytes was achieved with a Chiralcel OD-R column and a mobile phase consisting of potassium hexafluorophosphate 7.5 mM and sodium phosphate 0.25 M solution, pH 3.0, and acetonitrile (75:25, v/v) in the isocratic mode, at a flow rate of 1.0 mL/min. Detection was carried out by fluorescence absorbance at Ex/Em 230/290 nm. The multifunctional porous surface structure of the PPY-coated film provided high precision and accuracy for enantiomers. Compared with other commercial capillaries, PPY-coated capillary showed better extraction efficiency for all the analytes. The quantification limits of the proposed method were 10 ng/mL for R- and S-fluoxetine, and 15 ng/mL for R- and S-norfluoxetine, with a coefficient of variation lower than 13%. The response of the method for enantiomers is linear over a dynamic range, from the limit of quantification to 700 ng/mL, with correlation coefficients higher than 0.9940. The in-tube SPME/LC method can therefore be successfully used to analyze plasma samples from ageing patients undergoing therapy with fluoxetine.