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.     

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.     

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.     

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.     

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.     

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.     

Determination of fluoxetine and norfluoxetine in rat plasma by HPLC with pre-column derivatization and fluorescence detection

Both fluoxetine (FLX) and its N-demethylated metabolite, norfluoxetine (NFLX), have been reported to be potent serotonin-reuptake inhibitors. A sensitive and reliable method that allows simultaneous quantification of their plasma levels would be valuable and was developed in this work. The procedure included extraction of FLX and NFLX from plasma, fluorescence derivatization with 4-(N-chloroformylmethyl-N-methyl) amino-7-nitro-2,1,3-benzoxadiazole (NBD-COCl), separation of the derivatives on an octadecylsilica column with acetonitrile-water (55:45,v/v) as mobile phase and fluorescence detection with emission at 537 nm and excitation at 478 nm. The calibration curves were linear for FLX and NFLX concentration over the range of 10-1000 nM (r = 0.9992 and r = 0.9997) and the limits of quantitation were 10 nM in 100 micro L of plasma. Precision of intra- and inter-day RSD of less than 12% and accuracy of intra- and inter-day RE within -6.0-13% were achieved. The method described was applied to analysis of the plasma samples from rats treated with FLX hydrochloride and to the pharmacokinetic study.     

Determination of paclitaxel in human and rat blood samples after administration of low dose paclitaxel by HPLC-UV detection

A simple and sensitive HPLC-UV method was developed for the determination of paclitaxel (TXL) in human and rat blood samples. 4-Hydroxybenzoic acid n-hexyl ester was used as an internal standard. TXL was extracted by a liquid-liquid extraction with tert-butylmethyl ether. The disturbing peaks in the case of serum sample were removed by pre-extraction with hexane. The separation of TXL was achieved within 25 min using an ODS column with 50% acetonitrile aqueous solution as a mobile phase at a flow rate of 1.0 mL/min. The eluent was monitored at 230 nm, and the resulted retention times of TXL and IS were 11.2 and 20.4 min. The detection limits of TXL for human plasma, serum and rat plasma samples at a signal-to-noise ratio of 3 were 10, 9.5 and 7.5 ng/mL, respectively. The proposed methods were applicable to the determination of TXL in human patients' plasma ranging from 15 to 27 ng/mL. Furthermore, monitoring of the time course of TXL after its single administration to rat could be demonstrated.     

Enantiomeric separation of fluoxetine and norfluoxetine in plasma and serum samples with high detection sensitivity capillary electrophoresis

A capillary electrophoresis method was optimized for the stereoselective analysis of the antidepressant drug fluoxetine and its main demethylated metabolite norfluoxetine using a cyclodextrin-modified sodium phosphate buffer at pH 2.5. The combination of a neutral and a negatively charged cyclodextrin, dimethylated-beta- and phosphated-gamma-respectively, provided the baseline enantiomeric separation of the two compounds. The very low concentrations of chiral selectors employed together with the use of a high sensitivity detection cell of special design (zeta-shaped) in a diode array UV detector allowed us to reach a limit of detection of 0.005 and 0.01 microg/mL for fluoxetine and norfluoxetine, respectively. Analysis of fluoxetine and norfluoxetine standard mixtures showed a reproducibility of migration times and peak area and linearity in the concentration range of 0.1-2.0 microg/mL. The optimized method was applied to the analysis of clinical serum and plasma samples of patients under depression therapy. In all the analyzed samples the enantiomeric forms of fluoxetine and norfluoxetine were easily identified. The fluoxetine and metabolite enantiomeric ratio confirmed the stereoselectivity of the metabolic process of the fluoxetine drug in accordance with the literature data.     

Simultaneous determination of fluoxetine and norfluoxetine enantiomers in biological samples by gas chromatography with electron-capture detection.

An electron-capture gas chromatographic procedure was developed for the simultaneous analysis of the enantiomers of fluoxetine and norfluoxetine. The assay involves basic extraction of these enantiomers from the biological samples, followed by their conversion to diastereoisomers using the chiral derivatizing reagent (S)-(-)-N-trifluoroacetylprolyl chloride. The method was utilized to detect and measure the quantity of these enantiomers in plasma and urine of patients and in liver and brain tissue of rats treated with (R,S)-fluoxetine.     

Determination of drug-protein interactions by combined microdialysis and high-performance liquid chromatography

Summary A simple and rapid method for determination of the parameters of the interaction between drugs and protein, including the association constant and the number of binding sites, has been developed by use of a microdialysis sampling technique combined with high-performance liquid chromatography. The drug and protein (carbamazepine (5H-dibenz[b,f]flazepine-5-carboxamide, CBZ) and human serum albumin (HSA) were used as examples) were mixed in different molar ratios in 0.067 M potassium phosphate buffer, pH 7.4, and incubated at 37°C in a water-bath. The microdialysis probe was the used to sample the mixed CBZ-HSA solution at a perfusion rate of 1 μL min⁻¹. The concentration of CBZ in the microdialysate was determined by reversed-phase high-performance liquid chromatography. Relative recovery (R), determined in vitro under similar conditions, was approximately 42.7%; theRSD ofR was approximately 1.85%. The estimated association constant (K) and the number of the binding sites,n, on one molecule of HSA were 1.06×10⁴ M⁻¹ and 0.880, respectively, which is in good agreement with the literature values determined by high-performance frontal analysis. The potential use of microdialysis is also discussed.     

Determination of tryptophan and kynurenine in brain microdialysis samples by capillary electrophoresis with electrochemical detection

Capillary electrophoresis with electrochemical detection (CEEC) is evaluated for the determination of tryptophan and kynurenine in microdialysis samples obtained from rat brain. These compounds were separated from all other electroactive metabolites of tryptophan. Limits of detection for both compounds were in the low attomole range. The response was linear for kynurenine between 4.9 and 980 fmol injected with a correlation coefficient of 0.9992 (n = 12). The system was evaluated for monitoring tryptophan and kynurenine in the extracellular fluid of the rat brain following systemic administration of tryptophan.     

In vivo microdialysis determination of collagen-induced serotonin release in rat blood

We developed a sensitive microbore HPLC method coupled with an on-line microdialysis system to simultaneously measure endogenous 5-hydroxytryptamine (serotonin; 5-HT) and its major metabolite 5-hydroxyindoleacetic acid (5-HIAA) in the rat blood in vivo. A dialysis tube was placed in the right jugular vein. The validity of the procedure is demonstrated because analysis of the aggregating agents, collagen (I mg/kg) plus epinephrine (0.3 mg/kg) after intravenous injection, showed that they induced an increase in 5-HT and 5-HIAA levels in the jugular vein of the rat.     

Characterization of interaction property of multicomponents in Chinese Herb with protein by microdialysis combined with HPLC

Interaction of traditional Chinese Herb Rhizoma Chuanxiong and protein was studied by microdialysis coupled with high performance liquid chromatography. Compounds in Rhizoma Chuanxiong, such as ferulic acid, senkyunolide A and 3-butylphthalide, were identified by HPLC, HPLC-MS and UV-vis. Microdialysis recoveries and binding degrees of compounds in Rhizoma Chuanxiong with human serum albumin (HSA) and other human plasma protein were determined: recoveries of microdialysis sampling ranged from 36.7 to 98.4% with R.S.D. below 3.1%; while binding to HSA ranged from 0 to 91.5% (0.3 mM HSA) and from 0 to 93.5% (0.6 mM HSA), respectively. Compared with HSA, most of compounds bound to human blood serum more extensively and the results showed that binding of these compounds in Rhizoma Chuanxiong was influenced by pH. Two compounds were found to bind to HSA and human blood serum, their binding degrees were consistent with ferulic acid and 3-butylphthalide, the active compounds in Rhizoma Chuangxiong.     

Derivatization chemistries for determination of serotonin, norepinephrine and dopamine in brain microdialysis samples by liquid chromatography with fluorescence detection

The present paper provides an overview on currently developed derivatization chemistries and techniques for determination of monoamine neurotransmitters serotonin (5-HT), norepinephrine (NE) and dopamine (DA) in microdialysis samples by microbore liquid chromatography with fluorescence detection. In mild alkaline conditions, 5-hydroxyindoles and catecholamines react with benzylamine (BA), forming highly fluorescent 2-phenyl-4,5-pyrrolobenzoxazoles and 2-phenyl(4,5-dihydropyrrolo) [2,3-f]benzoxazoles, respectively. However, for derivatization of DA a higher fluorescence intensity was achieved for reaction with 1,2-diphenylethylenediamine (DPE) rather than with BA, therefore for simultaneous determination of 5-HT, NE and DA in brain microdialysates, a two-step derivatization with BA followed by DPE was developed. The detection limits for 5-HT, NE and DA were 0.2, 0.08 and 0.13 fmol, respectively, in an injection volume of 20 microL, which corresponds to concentrations of 30, 12 and 19.5 pm, respectively in standard solution prior to derivatization. The experimental data presented demonstrate the ability of the technique to simultaneously monitor neuronally releasable pools of monoamine neurotransmitters in the rat and mouse brains at basal conditions and following pharmacological treatments or physiological stimuli. These techniques play an important role in drug discovery and clinical investigation of psychiatric and neurological diseases such as depression, schizophrenia and Parkinson's disease.     

Determination of dopamine in rat striatum by microdialysis and high-performance liquid chromatography with electrochemical detection on a functionalized multi-wall carbon nanotube electrode

High performance liquid chromatography coupled with microdialysis sampling and electrochemical detection (HPLC–ECD) has been used to determine dopamine (DA). In the HPLC–ECD a multi-wall carbon nanotube electrode chemically modified with carboxyl groups (MWNT-COOH CME) was used as the working electrode for determination of DA. The results indicated that the MWNT-COOH CME enabled efficient electrocatalytic oxidation of DA with relatively high sensitivity and stability and long life. Peak currents for dopamine were linearly dependent on concentration in the range 5.0×10⁻⁹ to 5.0×10⁻⁵ mol L⁻¹ and the calculated detection limit (S/N=3) was 2.5×10⁻⁹ mol L⁻¹. The method had been successfully used to measure dopamine in rat striatal microdialysate. To study the physiological effect of nitric oxide (NO) on striatal release of DA, 0.5 mmol L⁻¹ sodium nitroprusside (SNP) was a continuously perfused into rat striatum. This resulted in a 46% increase in DA basal level.     

Determination of fluoxetine in plasma by gas chromatography-mass spectrometry using stir bar sorptive extraction

This article presents a method employing stir bar sorptive extraction (SBSE) with in situ derivatization, in combination with either thermal or liquid desorption on-line coupled to gas chromatography-mass spectrometry for the analysis of fluoxetine in plasma samples. Ethyl chloroformate was employed as derivatizing agent producing symmetrical peaks. Parameters such as solvent polarity, time for analyte desorption, and extraction time, were evaluated. During the validation process, the developed method presented specificity, linearity (R² > 0.99), precision (R.S.D. < 15%), and limits of quantification (LOQ) of 30 and 1.37 pg mL⁻¹, when liquid and thermal desorption were employed, respectively. This simple and highly sensitive method showed to be adequate for the measurement of fluoxetine in typical and trace concentration levels.     

Determination of Unbound Ampicillin in Rat Blood by Microdialysis and Liquid Chromatography

ABSTRACT

An in vivo microdialysis sampling method coupled to high-performance liquid chromatography has been applied for continuous monitoring of unbound ampicillin in rat blood. A microdialysis probe was inserted into the jugular vein/right atrium of Sprague-Dawley rats, and doses of 100 and 200 mg/kg ampicillin were then administered via the femoral vein. Dialysates were collected and directly injected into a liquid chromatographic system. Isocratic elution of ampicillin was achieved within 10 min using the liquid chromatographic system. The chromatographic mobile phase consisted of methanol-100 mM monosodium phosphoric acid (25:75, v/v, pH 5.5). The wavelength of the UV detector was set at 230 nm. The calibration curves from 0.25 to 50 μg/ml were linear with correlation coefficients of 0.995. The method provides a simple technique for rapid analysis of unbound ampicillin in rat blood for used in pharmacokinetic study.     

Determination of the adsorption isotherm of fluoxetine by adjustable factor h-root method (AF-HRM)

Summary Conventional methods to determine competitive adsorption isotherm are tedious, and require large quantities of pure isomers. In some cases pure enantiomers are not available, thereby making the full experiments to determine the competitive adsorption isotherm of enantiomers impossible. A new method, adjustable factor h-root method (AF-HRM), had been presented for the measurement of langmuir equilibrium coefficients. In the new approach, the retention factors and the frontal retention factors are required to determine the parameters of isotherms. As the ultimate goal is to separate enantiomers of fluoxetine on a preparative scale, a convenient method to determine the adsorption isotherm is essential. In this paper, we have demonstrated that AF-HRM can be applied to determine the parameters in the extended Langmuir model. Experimental results agree well with the prediction.