Label-free high-throughput screening assay for the identification of norepinephrine transporter (NET/SLC6A2) inhibitors.

The human norepinephrine transporter (NET) is an established drug target for a wide range of psychiatric disorders. Conventional methods that are used to functionally characterize NET inhibitors are based on the use of radiolabeled or fluorescent substrates. These methods are highly informative, but pose limitations to either high-throughput screening (HTS) adaptation or physiologically accurate representation of the endogenous uptake events. Recently, we developed a label-free functional assay based on the activation of G protein-coupled receptors by a transported substrate, termed the TRACT assay. In this study, the TRACT assay technology was applied to NET expressed in a doxycycline-inducible HEK 293 JumpIn cell line. Three endogenous substrates of NET-norepinephrine (NE), dopamine (DA) and epinephrine (EP)-were compared in the characterization of the reference NET inhibitor nisoxetine. The resulting assay, using NE as a substrate, was validated in a manual HTS set-up with a Z' = 0.55. The inhibitory potencies of several reported NET inhibitors from the TRACT assay showed positive correlation with those from an established fluorescent substrate uptake assay. These findings demonstrate the suitability of the TRACT assay for HTS characterization and screening of NET inhibitors and provide a basis for investigation of other solute carrier transporters with label-free biosensors.

Magnetic solid-phase extraction coupled with UHPLC-MS/MS for four antidepressants and one metabolite in clinical plasma and urine samples.

Aim: Routine therapeutic drug monitoring is highly recommended since common antidepressant combinations increase the risk of drug-drug interactions or overlapping toxicity. Materials & methods: A magnetic solid-phase extraction by using C18-functionalized magnetic silica nanoparticles (C18-Fe3O4@SiO2 NPs) as sorbent was proposed for rapid extraction of venlafaxine, paroxetine, fluoxetine, norfluoxetine and sertraline from clinical plasma and urine samples followed by ultra-HPLC-MS/MS assay. Results: The synthesized C18-Fe3O4@SiO2 NPs showed high magnetization and efficient extraction for the analytes. After cleanup by magnetic solid-phase extraction, no matrix effects were found in plasma and urine matrices. The analytes showed LODs among 0.15-0.75 ng ml-1, appropriate linearity (R ≥ 0.9990) from 2.5 to 1000 ng ml-1, acceptable accuracies 89.1-110.9% with precisions ≤11.0%. The protocol was successfully applied for the analysis of patients' plasma and urine samples. Conclusion: It shows high potential in routine therapeutic drug monitoring of clinical biological samples.

Space-Resolved Tissue Analysis by Solid-Phase Microextraction Coupled to High-Resolution Mass Spectrometry via Desorption Electrospray Ionization.

It is hard to overstate the tremendous utility of desorption electrospray ionization (DESI) and its various configurations for rapid and high-throughput analyses or spatially resolved imaging of heterogeneous systems. However, there have been few attempts to employ this technique in spatially resolved mode with solid substrates featuring extractive and analyte-enrichment properties. This study documents the development of a platform that combines solid-phase microextraction (SPME) with desorption electrospray ionization mass spectrometry (DESI-MS) for unidimensional investigation of the heterogeneous distribution of compounds in semisolid systems (i.e., depth profiling across the fiber axis), with the ultimate end of employing it for brain tissue analysis. To this end, a DESI interface and a custom holder accommodating SPME probes were built in house, with the latter contributing to reduction of mechanical sources of signal instability. The system was evaluated through the quantitative reconstruction of the laminar and radial concentration gradients of xenobiotics introduced in multilayer gel arrangements and surrogate brain tissue models. Good quantitative capability was achieved by employing a strategy that combined signal correction via preloading internal standard onto SPME fibers and signal integration in scan-by-scan mode. The proposed technique's suitability for characterizing more complex systems, such as rat brains ex vivo, was also evaluated. The proposed approach allows for fast and noninvasive probing of three-dimensional objects without the need for their slicing, and the space-resolved mode reduces the number of required probe insertions, allowing in vivo applications. We foresee suitability of this setup for examining the spatial patterns of local drug release in the brain and the extent of the resultant physiological responses.

Microextraction by packed sorbent followed by ultra high performance liquid chromatography for the fast extraction and determination of six antidepressants in urine.

The growing use of antidepressants in recent years has led to their increasing presence in forensic analyses. In this work, microextraction by packed sorbent followed by ultra-performance liquid chromatography with photodiode array detection provided a fast method for determining the antidepressants mirtazapine, venlafaxine, escitalopram, fluoxetine, fluvoxamine, and sertraline in human urine. The microextraction conditions (viz., type of sorbent, number of draw-eject extraction cycles or strokes, sample volume and pH, and type and volume of washing solution and eluent) were optimized by using an experimental design. The ensuing analytical method was validated in terms of linearity (25-1000 ng/mL urine), limit of detection (lower than 7.1 ng/mL), limit of quantification (25 ng/mL), precision (4.7-15.1% as relative standard deviation), and accuracy (80.4-126.1% as mean recovery for four replicate determinations). The proposed method allowed the six target antidepressants to be determined at concentrations from therapeutic to toxic levels. The application to small volumes (300 µL) of urine afforded fast extraction of the analytes and provided results on a par with those of existing clinical and forensic alternatives.

Indirect enantioseparation of fluoxetine in mouse serum by derivatization with 1R-(-)-menthyl chloroformate followed by ultra high performance liquid chromatography and quadrupole time-of-flight mass spectrometry.

Here we describe a simple and sensitive analytical method for the enantioselective quantification of fluoxetine in mouse serum using ultra high performance liquid chromatography with quadrupole time-of-flight mass spectrometry. The sample preparation method included a simple deproteinization with acetonitrile in 50 µL of serum, followed by derivatization of the extracts in 50 µL of 2 mM 1R-(-)-menthyl chloroformate at 45ºC for 55 min. These conditions were statistically optimized through response surface methodology using a central composite design. Under the optimized conditions, neither racemization nor kinetic resolution occurred. The derivatized diastereomers were readily resolved on a conventional sub-2 µm C18 column under a simple gradient elution of aqueous methanol containing 0.1% formic acid. The established method was validated and found to be linear, precise, and accurate over the concentration range of 5.0-1000.0 ng/mL for both R and S enantiomers (r(2) > 0.993). Stability tests of the prepared samples at three different concentration levels showed that the R- and S-fluoxetine derivatives were relatively stable for 48 h. No significant matrix effects were observed. Last, the developed method was successfully used for enantiomeric analysis of real serum samples collected at a number of time points from mice administered with racemic fluoxetine.

Development of a novel mixed hemimicelles dispersive micro solid phase extraction using 1-hexadecyl-3-methylimidazolium bromide coated magnetic graphene for the separation and preconcentration of fluoxetine in different matrices before its determination by fiber optic linear array spectrophotometry and mode-mismatched thermal lens spectroscopy.

This study aims at developing a novel, sensitive, fast, simple and convenient method for separation and preconcentration of trace amounts of fluoxetine before its spectrophotometric determination. The method is based on combination of magnetic mixed hemimicelles solid phase extraction and dispersive micro solid phase extraction using 1-hexadecyl-3-methylimidazolium bromide coated magnetic graphene as a sorbent. The magnetic graphene was synthesized by a simple coprecipitation method and characterized by X-ray diffraction (XRD), Fourier transform infrared (FT-IR) spectroscopy and scanning electron microscopy (SEM). The retained analyte was eluted using a 100 µL mixture of methanol/acetic acid (9:1) and converted into fluoxetine-ß-cyclodextrin inclusion complex. The analyte was then quantified by fiber optic linear array spectrophotometry as well as mode-mismatched thermal lens spectroscopy (TLS). The factors affecting the separation, preconcentration and determination of fluoxetine were investigated and optimized. With a 50 mL sample and under optimized conditions using the spectrophotometry technique, the method exhibited a linear dynamic range of 0.4-60.0 µg L(-1), a detection limit of 0.21 µg L(-1), an enrichment factor of 167, and a relative standard deviation of 2.1% and 3.8% (n = 6) at 60 µg L(-1) level of fluoxetine for intra- and inter-day analyses, respectively. However, with thermal lens spectrometry and a sample volume of 10 mL, the method exhibited a linear dynamic range of 0.05-300 µg L(-1), a detection limit of 0.016 µg L(-1) and a relative standard deviation of 3.8% and 5.6% (n = 6) at 60 µg L(-1) level of fluoxetine for intra- and inter-day analyses, respectively. The method was successfully applied to determine fluoxetine in pharmaceutical formulation, human urine and environmental water samples.

Low environmental levels of neuro-active pharmaceuticals alter phototactic behaviour and reproduction in Daphnia magna.

Assessing the risks of emerging contaminants, such as pharmaceuticals in the environment requires an understanding of their exposure regime and their effects at environmentally relevant concentrations across species. Daphnia magna represents an excellent invertebrate model species to study the mode of action of emerging pollutants, allowing the assessment of effects at different biological levels. The present study aims to test the hypothesis that different families of neuro-active pharmaceuticals at low environmentally relevant concentrations may lead to similar phenotypic responses in D. magna. Phenotypic traits included reproduction and behavioural responses. Selected pharmaceuticals were carbamazepine, diazepam and propranolol, three widely prescribed compounds, already detected at considerable levels in the environment (ng to few µg/L). Fluoxetine was also included in behavioural assays. The three tested neuro-active pharmaceuticals were able to enhance reproduction at 1ng/L of propranolol, 0.1µg/L of diazepam and 1µg/L of carbamazepine. Fluoxetine, carbamazepine and diazepam increased positive phototactic behaviour at concentrations ranging from 1, 10 and 100ng/L, respectively. Reported responses were nonmonotonic, which means that eco-toxicity testing of pharmaceuticals need to assess effects at the ng/L range.

MicroQuEChERS-nanoliquid chromatography-nanospray-tandem mass spectrometry for the detection and quantification of trace pharmaceuticals in benthic invertebrates.

Due to industrialization and the use of chemical products in everyday life, various types of drugs and pesticides are present in our environment, which threaten and cause negative impacts on aquatic ecosystems. The consequences of these pollutants are gradually becoming visible. Recent evidence confirms that long term exposure to environmental pharmaceutical concentrations can induce adverse effects in aquatic vertebrates and invertebrates such as reproductive impairments and collapse wild populations. Consequently, one of the challenges of environmental science is to evaluate the associated risks. In this context, a new methodology has been developed using nano-LC-nano-ESI MS/MS to quantify traces of two pharmaceuticals (a neuropharmaceutical drug, fluoxetine, and an anticonvulsant drug, carbamazepine) in two molluscs, Potamopyrgus antipodarum and Valvata piscinalis, which are both prosobranch gastropods. A simple and quick extraction method was developed based on a modified and miniaturized version of the QuEChERS method. The procedure involves the extraction of approximately 10 mg of wet mollusc tissue by 500 µL of a mixture of acetonitrile/water/hexane (50/20/30) and 100 mg of buffer salt. Thus, the extraction step was carried out on an individual scale. The sensitivity of this method allowed for the detection of levels as low as 18 ng/g and 128 ng/g for carbamazepine and fluoxetine, respectively, with recoveries of greater than 85% for the two targeted compounds. This method was then applied to both gastropod species exposed to fluoxetine under laboratory conditions. The results provide evidence of bioaccumulation in both P. antipodarum and V. piscinalis and reveal the inter-species differences.

Liquid chromatographic enantioseparation of (RS)-mexiletine and (RS)-fluoxetine using chiral derivatizing reagents synthesized with (S)-naproxen moiety.

Enantiomeric separation of racemic mexiletine and fluoxetine was achieved using three chiral derivatizing reagents (CDRs) based on (S)-naproxen. Diastereomers were synthesized by reaction of mexiletine or fluoxetine with the CDRs and were separated on a C18 column under reversed-phase conditions using a binary mixture of acetonitrile and triethylammonium phosphate/water, with UV detection at 230 and 226 nm. The results obtained for enantioseparation of the two drugs using the three CDRs were compiled and compared. The conditions for derivatization and chromatographic separation were optimized. The method was validated for linearity, repeatability, limit of detection and limit of quantification.

Parallel electromembrane extraction in the 96-well format.

The repeatability and extraction recoveries of parallel electromembrane extraction (Pa-EME) was thoroughly investigated in the present project. Amitriptyline, fluoxetine, and haloperidol were isolated from eight samples of pure water, undiluted human plasma, and undiluted human urine, respectively; in total 24 samples were processed in parallel. The repeatability was found to be independent of the different sample matrices (pure water samples, human plasma, and water) processed in parallel, although the respective samples contained different matrix components. In another experiment seven of the 24 wells were perforated. Even though the perforation caused the total current level in the Pa-EME setup to increase, the intact circuits were unaffected by the collapse in seven of the circuits. In another approach, exhaustive extraction of amitriptyline, fluoxetine, and haloperidol was demonstrated from pure water samples. Amitriptyline and haloperidol were also isolated exhaustively from undiluted human plasma samples; the extraction recovery of fluoxetine from undiluted human plasma was 81%. Finally, the sample throughput was increased with the Pa-EME configuration. The extraction recoveries were investigated by processing 1, 8, 68, or 96 samples in parallel in 10min; neither the extraction recoveries nor the repeatability was affected by the total numbers of samples. Eventually, the Pa-EME was combined with ultra performance liquid chromatography (UPLC) to combine high-throughput sample preparation with high-throughput analytical instrumentation. The aim of the present investigation was to demonstrate the potential of electromembrane extraction as a high throughput sample preparation platform; and hopefully to increase the interest for EME in the bioanalytical field to solve exisiting and novel analytical challenges.

Fast evaluation of enantioselective drug metabolism by electrophoretically mediated microanalysis: application to fluoxetine metabolism by CYP2D6.

In this work, a capillary electrophoretic methodology for the enantioselective in vitro evaluation of drugs metabolism is applied to the evaluation of fluoxetine (FLX) metabolism by cytochrome 2D6 (CYP2D6). This methodology comprises the in-capillary enzymatic reaction and the chiral separation of FLX and its major metabolite, norfluoxetine enantiomers employing highly sulfated ß-CD and the partial filling technique. The methodology employed in this work is a fast way to obtain a first approach of the enantioselective in vitro metabolism of racemic drugs, with the additional advantage of an extremely low consumption of enzymes, CDs and all the reagents involved in the process. Michaelis-Menten kinetic parameters (Km and Vmax ) for the metabolism of FLX enantiomers by CYP2D6 have been estimated by nonlinear fitting of experimental data to the Michaelis-Menten equation. Km values have been found to be 30 ± 3 µM for S-FLX and 39 ± 5 µM for R-FLX. Vmax estimations were 28.6 ± 1.2 and 34 ± 2 pmol·min(-1) ·(pmol CYP)(-1) for S- and R-FLX, respectively. Similar results were obtained using a single enantiomer (R-FLX), indicating that the use of the racemate is a good option for obtaining enantioselective estimations. The results obtained show a slight enantioselectivity in favor of R-FLX.

Removal of trace organic chemical contaminants by a membrane bioreactor.

Emerging wastewater treatment processes such as membrane bioreactors (MBRs) have attracted a significant amount of interest internationally due to their ability to produce high quality effluent suitable for water recycling. It is therefore important that their efficiency in removing hazardous trace organic contaminants be assessed. Accordingly, this study investigated the removal of trace organic chemical contaminants through a full-scale, package MBR in New South Wales, Australia. This study was unique in the context of MBR research because it characterised the removal of 48 trace organic chemical contaminants, which included steroidal hormones, xenoestrogens, pesticides, caffeine, pharmaceuticals and personal care products (PPCPs). Results showed that the removal of most trace organic chemical contaminants through the MBR was high (above 90%). However, amitriptyline, carbamazepine, diazepam, diclofenac, fluoxetine, gemfibrozil, omeprazole, sulphamethoxazole and trimethoprim were only partially removed through the MBR with the removal efficiencies of 24-68%. These are potential indicators for assessing MBR performance as these chemicals are usually sensitive to changes in the treatment systems. The trace organic chemical contaminants detected in the MBR permeate were 1 to 6 orders of magnitude lower than guideline values reported in the Australian Guidelines for Water Recycling. The outcomes of this study enhanced our understanding of the levels and removal of trace organic contaminants by MBRs.

Characterization of the Subcritical Water Extraction of Fluoxetine-Hydrochloride.

The characteristics of using Subcritical Water Extraction (SWE) to recover Fluoxetine-Hydrochloride from both standard solutions and the contents of commercial capsule formulations were investigated. Analysis of solutions and extracts was done by HPLC with UV detection at 254 nm. Standard solutions of Fluoxetine-Hydrochloride were exposed to a variety of SWE operating conditions, including temperatures from 125 to 275°C and periods ranging from 5 to 30 min. Fluoxetine-Hydrochloride could be quantitatively recovered from standard solutions (1.0mg/mL) that were heated up to 175°C for 30 min, up to 200°C for 15 min, or up to 225°C for 10 min. At higher temperatures and/or times, Fluoxetine-Hydrochloride recoveries were generally incomplete and often produced decomposition by-products during the process. By comparison, the concentration of Fluoxetine-Hydrochloride in the standard solution had relatively little effect on recovery. Considering these parameters, an SWE method was developed to extract Fluoxetine-Hydrochloride from the contents of Prozac(®) capsules. It was found that Fluoxetine-Hydrochloride could be quantitatively extracted from the capsule contents in 8 min at a temperature of 200°C using 3.5 mL of water as the extraction solvent. Gelatinization of the starch excipient in the capsule contents was also observed to occur temporarily during the capsule extractions, before ultimately disappearing again. The period of this phenomenon was dependent on both temperature and sample size. The results indicate that SWE can be a very useful method for Fluoxetine-Hydrochloride extraction and suggest that it may be interesting to explore other pharmaceuticals using this method as well.

Trace analysis of fluoxetine and its metabolite norfluoxetine. Part II: Enantioselective quantification and studies of matrix effects in raw and treated wastewater by solid phase extraction and liquid chromatography-tandem mass spectrometry.

The isotope-labeled compounds fluoxetine-d5 and norfluoxetine-d5 were used to study matrix effects caused by co-eluting compounds originating from raw and treated wastewater samples, collected in Uppsala, Sweden. The matrix effects were investigated by the determination of matrix factors (MF) and by a post-column infusion method. The matrix factors were determined to be 38-47% and 71-86% for the enantiomers of norfluoxetine-d5 and fluoxetine-d5, respectively. The influence of matrix effects when quantifying the enantiomers of the active pharmaceutical ingredient and the metabolite in wastewater samples with LC-MS/MS is discussed and methods to overcome the problem are presented. The enantiomeric concentrations of fluoxetine and its human metabolite norfluoxetine, quantified by a one-point calibration method, were 12-52 pM (3.5-16 ng L⁻¹) in raw wastewater and 4-48 pM (1.2-15 ng L⁻¹) in treated wastewater. Furthermore, the calculated enantiomeric fractions (EF) of the substances were found to be between 0.68 and 0.71 in both matrices. Neither the EF values for fluoxetine nor those for norfluoxetine were significantly different in the raw wastewater compared to the treated wastewater. Interestingly, the concentration of (S)-fluoxetine was found to be higher than the concentration of (R)-fluoxetine in both raw and treated wastewater. These results are different from other results presented in the literature, which shows that the relative concentrations of the enantiomers of a chiral active pharmaceutical ingredient might be significantly different in wastewater samples from different treatment systems. We report, for the first time, the concentrations of the enantiomers of norfluoxetine in wastewater samples. The concentrations of (S)-norfluoxetine were found to be higher than the concentration of (R)-norfluoxetine in the raw as well as in the treated wastewater samples.

Spectrofluorimetric determination of DL-N-methyl-3-phenyl-3-[(α,α,α-trifluoro-p-tolyl)oxy]propylamine in pharmaceuticals and chitosan solution.

A spectrofluorimetric method for the determination of D,L-N-methyl-3-phenyl-3-[(α,α,α-trifluoro-p-tolyl)oxy]propylamine, fluoxetine (F), in pharmaceuticals was evaluated in the 50.0-500.0 µg ml⁻¹ range. Linearity, sensibility, quantification and detection limit, and precision values are satisfactory. The method does not need pre-treatment and was successfully applied to the determination in pharmaceuticals and chitosan (Ch) solution. Ch has an ability to carry and absorb fat and may eventually be used together with F in slimming diets, and then interactions of Ch-F may occur. This work seeks to study these interactions by monitoring the photophysics of a drug in the presence of Ch. The results warn about the care that must be taken when both compounds are prescribed together.

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.

Validation and use of in vivo solid phase micro-extraction (SPME) for the detection of emerging contaminants in fish.

A variety of emerging chemicals of concern are released continuously to surface water through the municipal wastewater effluent discharges. The ability to rapidly determine bioaccumulation of these contaminants in exposed fish without sacrificing the animal (i.e. in vivo) would be of significant advantage to facilitate research, assessment and monitoring of their risk to the environment. In this study, an in vivo solid phase micro-extraction (SPME) approach was developed and applied to the measurement of a variety of emerging contaminants (carbamazepine, naproxen, diclofenac, gemfibrozil, bisphenol A, fluoxetine, ibuprofen and atrazine) in fish. Our results indicated in vivo SPME was a potential alternative extraction technique for quantitative determination of contaminants in lab exposures and as well after exposure to two municipal wastewater effluents (MWWE), with a major advantage over conventional techniques due to its ability to non-lethally sample tissues of living organisms.

Extraction of fluoxetine from aquatic and urine samples using sodium dodecyl sulfate-coated iron oxide magnetic nanoparticles followed by spectrofluorimetric determination.

A new method based on the combination of magnetic solid phase extraction (MSPE) and spectrofluorimetric determination was developed for isolation and preconcentration of fluoxetine form aquatic and biological samples using sodium dodecyl sulfate (SDS) coated Fe(3)O(4) nanoparticles (NPs) as a sorbent. The unique properties of Fe(3)O(4) NPs including high surface area and strong magnetism were utilized effectively in the MSPE process. Effect of different parameters influencing the extraction efficiency of fluoxetine including the amount of Fe(3)O(4) and SDS, pH value, sample volume, extraction time, desorption solvent and time were optimized. Under optimized condition, the method was successfully applied to the extraction of fluoxetine from water and urine samples and absolute recovery amount of 85%, detection limit of 20 µg L(-1) and a relative standard deviation (RSD) of 1.4% were obtained. The method linear response was over a range of 50-1000 µg L(-1) with R(2)=0.9968. The relative recovery in different aquatic and urine matrices were investigated and values of 80% to 104% were obtained. The whole procedure showed to be conveniently fast, efficient and economical for extraction of fluoxetine from environmental and biological samples.

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.

High-performance liquid chromatographic enantioseparation of (R,S)-fluoxetine using Marfey's reagent and (S)-N-(4-nitrophenoxycarbonyl) phenylalanine methoxyethyl ester as chiral derivatizing reagents along with direct thin-layer chromatographic resolution and isolation of enantiomers using L-tartaric acid as mobile phase additive.

Chiral assay of enantiomers of fluoxetine was achieved in pharmaceutical formulations using direct and indirect methods. L-tartaric acid was used as a mobile phase additive in thin-layer chromatography; the enantiomers were separated and isolated and were used to determine the elution order in HPLC. (R,S)-flouxetine was derivatized with (S)-N-(4-nitrophenoxycarbonyl)phenylalanine methoxyethyl ester [(S)-NIFE], Marfey's reagent and 1-fluoro-2,4-dinitrophenyl-L-methionine amide (FDNP-L-Met-NH2. The diastereomers were separated using RP-HPLC. The effect of flow rate and TFA concentration on resolution was studied. The diastereomers obtained by derivatization with FDNP-L-Met-NH2 were also separated by RP-TLC.