Ultrasound-assisted dispersive liquid-liquid microextraction coupled with field-amplified capillary electrophoresis for sensitive and quantitative determination of fluoxetine and norfluoxetine enantiomers in biological fluids.

A rapid, simple, and sensitive technique for the quantitative detection of fluoxetine and norfluoxetine enantiomers in biological fluids was developed based on the combination of field-amplified sample stacking (FASS)-related capillary electrophoresis (CE) with ultrasound-assisted dispersive liquid-liquid microextraction (UA-DLLME). The extraction efficiency of UA-DLLME was strongly related to extraction time, salt concentration, type of extraction and dispersion solvents, and volume of extraction and dispersion solvents. The extracted fluoxetine and norfluoxetine enantiomers in a mixture of 50% methanol and 50% deionized water were efficiently stacked using FASS and then separated using cyclodextrin-modified CE. Under optimal conditions of FASS (chiral selector, 3 mM trimethyl-ß-cyclodextrin; and background electrolyte, 100 mM phosphate buffer) and UA-DLLME (extraction solvent, 200 µL of acetone; and dispersed solvent, 50 µL of C2H2Cl4 in 1 mL of the sample solution), the obtained enrichment factors of fluoxetine and norfluoxetine enantiomers reached approximately 2000. The linear ranges for the quantification of fluoxetine and norfluoxetine enantiomers were 0.3-150 and 0.6-150 nM, respectively. The relative standard deviations in peak areas and migration time for four analytes were less than 3.3% and 6.3%, respectively. The proposed system provided limits of detection (signal-to-noise ratio of 3) for four analytes corresponding to 0.1 nM. The precision and accuracy for urine and serum samples were less than 6.8 and 8.3%, respectively. These findings suggested that the proposed system exhibited a high potential for the reliable determination of fluoxetine and norfluoxetine enantiomers in clinical samples. Graphical abstract.

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.

An in vivo evaluation of the ontogeny of stereoselective fluoxetine metabolism and disposition in lambs from birth to one year of age.

1. The objective was to determine the ontogeny of stereoselective fluoxetine (FX) disposition in postnatal sheep from newborn to adulthood. 2. Catheters were implanted in a carotid artery and jugular vein. FX was administered intravenously, followed by serial arterial blood and cumulative urine collection. The concentrations of R,S-FX and R,S-norfluoxetine (R,S-NFX) in samples were measured using a validated enantioselective LC/MS/MS analytical method. 3. The metabolism of FX at 4.2 ± 0.4 days was limited compared to adults, but had developed compared to the fetus. Total body clearance (ClTB) did not significantly increase up to 33.6 ± 0.9 days, but significantly increased at 98.5 ± 2.0 days, with no further changes up to 397.3 ± 8.5 days. Up to 13.4 ± 0.8 days, the disposition of FX included Phase I metabolism to NFX and trifluoromethylphenol (TFMP), and renal elimination. At 32.9 ± 0.9 days, metabolism included Phase II conjugates of FX and NFX. Renal elimination of these compounds was low. 4. The elimination of FX increased in a non-linear manner during the first year in sheep. The metabolism and disposition of FX and NFX in plasma and urine were stereoselective and this appeared due to both stereoselective protein binding and metabolism.

Optimization and validation of an SBSE-HPLC-FD method using laboratory-made stir bars for fluoxetine determination in human plasma.

Depression is the largest cause of disability worldwide, affecting 350 million people. Notwithstanding that clinical trials demonstrate antidepressants efficacy, the efficient response can vary individually concerning therapeutic dosage. Although important, plasma levels monitoring remains an analytical challenge whereas clean-up and pre-concentration represent critical steps. Therefore, this study aims to develop, optimize and validate a method for fluoxetine determination in human plasma, employing a laboratory-made device consisting of a PDMS stir bar sorptive for extraction, coupled with high-performance liquid chromatography-fluorescence detection (SBSE-HPLC-FD). Optimization involved sorption-desorption steps. For sorption, temperature and time were assessed by factorial and central composite design approaches, taking into account the desirability and the response surface results, with stirring speed also examined. For desorption kinetics and ultrasonic and magnetic stirring mode were evaluated. The proposed method after validation was robust, linear (25.00-1000.00 ng mL-1 , R2 > 0.98) and presented good intra- (RSD 4.18%) and inter-day-assay (RSD 11.60%) precision and accuracy (recovery 109.60%), allowing reliable quantitation without interference. The method was successfully applied to real samples. SBSE-HPLC-FD could represent a feasible alternative with good cost-benefit for low-volume samples and therapeutic drug monitoring, as well as contributing to correlation studies between plasma fluoxetine levels and clinical response, which is still little studied.

Experimental and Clinical Pharmacokinetics of Fluoxetine and Amitriptyline: Comparative Analysis and Possible Methods of Extrapolation.

The pharmacokinetics of two fluoxetine capsulated dosage forms and two amitriptyline tablet forms after a single oral intake was studied in dogs and healthy volunteers. High significant correlations were detected between plasma concentrations of fluoxetine (r=0.96, p<0.00001, n=11) and amitriptyline (r=0.78, p<0.0224, n=8) in dogs and volunteers. A correlation of medium strength (though insignificant) was detected between nortriptyline concentrations in the plasma of dogs and volunteers (r=0.69, p<0.199, n=5). The bioavailability parameters of the test drugs in dogs and volunteers did not differ. Similar trends of fluoxetine and amitriptyline pharmacokinetic parameters were revealed in volunteers and animals. Methods for extrapolation of experimental pharmacokinetics parameters of fluoxetine and amitriptyline obtained on dogs for humans are proposed and validated.

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.

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.

Therapeutic Drug Monitoring of Selective Serotonin Reuptake Inhibitors in Elderly Patients.

BACKGROUND: Elderly patients are at increased risk for elevated serum concentrations from treatment with selective serotonin reuptake inhibitors (SSRIs). The aim of this study was to examine the use of therapeutic drug monitoring (TDM) of SSRIs in elderly compared with younger patients. METHODS: All serum concentration measurements of SSRIs (escitalopram, citalopram, fluoxetine, fluvoxamine, paroxetine, and sertraline) performed at our laboratory in 2011 were included. The use of TDM (relative frequency) in older versus younger patients was examined by comparing the use of TDM in patients aged 60 years or older with that in patients younger than 60 years and by evaluating the use of TDM relative to age (age groups in decennials). The number of patients with an SSRI dispensed by prescription in the same region and period (the Norwegian Prescription Database) was used as reference. Additionally, the number of samples above the upper limit of the recommended reference range in patients aged 60 years or older and patients younger than 60 years was evaluated. RESULTS: TDM of an SSRI had been performed in 6333 patients. For all SSRIs, the use of TDM was significantly lower (8.2% versus 10.6% for citalopram, 10.0% versus 13.8% for escitalopram, 8.6% versus 17.0% for fluoxetine, 5.6% versus 10.3% for paroxetine, and 8.1% versus 15.0% for sertraline) in patients aged 60 years or older compared with those younger than 60 years (P < 0.001). There was a gradual decline in the use of TDM with increasing age, with a 3-fold difference between the youngest (10-19) and oldest (90+) patients (P < 0.0001). The percentage of samples above the upper limit of the recommended reference range was 2-fold higher in patients aged 60 years or older (6.7%) compared with patients younger than 60 (3.4%) years (P < 0.0001). CONCLUSIONS: Clinical follow-up of patients with TDM of SSRIs is less frequent in older patients compared with younger patients. This is in contrast to the general guidelines for TDM where patients of advanced age are considered of particular importance to monitor closely.

Poor Man's Methadone: A Case Report of Loperamide Toxicity.

Loperamide, a common over-the-counter antidiarrheal drug and opioid derivative, is formulated to act upon intestinal opioid receptors. However, at high doses, loperamide crosses the blood-brain barrier and reaches central opioid receptors in the brain, leading to central opiate effects including euphoria and respiratory depression. We report the case of a young man found dead in his residence with a known history of drug abuse. At autopsy, the only significant findings were a distended bladder and bloody oral purge. Drug screening found nontoxic levels of alprazolam, fluoxetine, and marijuana metabolites. Liquid chromatography time-of-flight mass spectrometry found an unusual set of split isotope peaks consistent with chlorine. On the basis of autopsy and toxicological findings, loperamide toxicity was suspected because of its opioid properties and molecular formula containing chlorine. A sample of loperamide was analyzed by liquid chromatography time-of-flight mass spectrometry, resulting in a matching mass and retention time to the decedent's sample. Subsequently, quantitative testing detected 63 ng/mL of loperamide or more than 6 times of therapeutic peak concentration. Cause of death was determined as "toxic effects of loperamide with fluoxetine and alprazolam." Because of its opioid effects and easy accessibility, loperamide is known as "poor man's methadone" and may go undetected at medical and forensic drug screening.

Effect of fluoxetine on induced tooth movement in rats.

INTRODUCTION: Fluoxetine is a widely used antidepressant. Its various effects on bone mineral density are well described. The aim of this study was to evaluate the effect of fluoxetine on induced tooth movement. METHODS: Seventy-two Wistar rats were divided into 3 groups: M (n = 24; 0.9% saline solution and induced tooth movement), FM (n = 24; fluoxetine, 10 mg/kg, and induced tooth movement), and F (n = 24; fluoxetine, 10 mg/kg only). After 30 days of daily saline solution or fluoxetine administration, an orthodontic appliance (30 cN) was used to displace the first molar mesially in groups M and FM. The animals were killed 3, 7, and 14 days after placement of the orthodontic appliances. The animals in group F did not receive induced tooth movement but were killed at the same times. We evaluated tooth movement rates, collagen neoformation rates by polarization microscopy, numbers of osteoclast by tartrate-resistant acid phosphatase, and trabecular bone modeling by microcomputed tomography of the femur. RESULTS: The tooth movement rates were similar in groups M and FM at all studied time points (P >0.05). The rate of newly formed collagen had a reverse pattern in groups M and FM, but the difference was not statistically significant (P >0.05). There were significantly more osteoclasts in group FM than in group F on day 3 (P <0.01). The trabecular spacing was significantly larger in group F compared with group M on day 14 (P <0.05). CONCLUSIONS: Fluoxetine did not interfere with induced tooth movement or trabecular bone in rats.

Effects of selective serotonin reuptake inhibition on neural activity related to risky decisions and monetary rewards in healthy males.

Selective serotonin reuptake inhibitors (SSRIs) such as fluoxetine are commonly prescribed antidepressant drugs targeting the dysfunctional serotonin (5-HT) system, yet little is known about the functional effects of prolonged serotonin reuptake inhibition in healthy individuals. Here we used functional MRI (fMRI) to investigate how a three-week fluoxetine intervention influences neural activity related to risk taking and reward processing. Employing a double-blinded parallel-group design, 29 healthy young males were randomly assigned to receive 3 weeks of a daily dose of 40 mg fluoxetine or placebo. Participants underwent task-related fMRI prior to and after the three-week intervention while performing a card gambling task. The task required participants to choose between two decks of cards. Choices were associated with different risk levels and potential reward magnitudes. Relative to placebo, the SSRI intervention did not alter individual risk-choice preferences, but modified neural activity during decision-making and reward processing: During the choice phase, SSRI reduced the neural response to increasing risk in lateral orbitofrontal cortex, a key structure for value-based decision-making. During the outcome phase, a midbrain region showed an independent decrease in the responsiveness to rewarding outcomes. This midbrain cluster included the raphe nuclei from which serotonergic modulatory projections originate to both cortical and subcortical regions. The findings corroborate the involvement of the normally functioning 5HT-system in decision-making under risk and processing of monetary rewards. The data suggest that prolonged SSRI treatment might reduce emotional engagement by reducing the impact of risk during decision-making or the impact of reward during outcome evaluation.

Effect of CYP2D6, CYP2C9 and ABCB1 genotypes on fluoxetine plasma concentrations and clinical improvement in children and adolescent patients.

There is little known about pharmacogenetic of fluoxetine in children and adolescents. In this study, we evaluate, for the first time, the influence of CYP2D6, CYP2C9 and ABCB1 genotypes on the steady-state plasma concentrations of fluoxetine and its active metabolite (S)-norfluoxetine, and on the clinical improvement in children and adolescent patients receiving fluoxetine treatment. The assessment was performed in 83 patients after 8 and 12 weeks of treatment. Fluoxetine/(S)-norfluoxetine ratio was negatively correlated with the number of active CYP2D6 alleles (r: -0.450; P<0.001). Regarding the G2677T ABCB1 polymorphism, T allele carriers showed significantly higher improvements on the majority of scales including the Clinical Global Impression-Improvement scale (P<0.001). Our results confirm the influence of CYP2D6 genetic variants in fluoxetine pharmacokinetics and provide evidence for the potential effect of the ABCB1 genotype on the clinical improvement in children and adolescent patients treated with fluoxetine.

Fluoxetine dose and administration method differentially affect hippocampal plasticity in adult female rats.

Selective serotonin reuptake inhibitor medications are one of the most common treatments for mood disorders. In humans, these medications are taken orally, usually once per day. Unfortunately, administration of antidepressant medications in rodent models is often through injection, oral gavage, or minipump implant, all relatively stressful procedures. The aim of the present study was to investigate how administration of the commonly used SSRI, fluoxetine, via a wafer cookie, compares to fluoxetine administration using an osmotic minipump, with regards to serum drug levels and hippocampal plasticity. For this experiment, adult female Sprague-Dawley rats were divided over the two administration methods: (1) cookie and (2) osmotic minipump and three fluoxetine treatment doses: 0, 5, or 10 mg/kg/day. Results show that a fluoxetine dose of 5 mg/kg/day, but not 10 mg/kg/day, results in comparable serum levels of fluoxetine and its active metabolite norfluoxetine between the two administration methods. Furthermore, minipump administration of fluoxetine resulted in higher levels of cell proliferation in the granule cell layer (GCL) at a 5 mg dose compared to a 10 mg dose. Synaptophysin expression in the GCL, but not CA3, was significantly lower after fluoxetine treatment, regardless of administration method. These data suggest that the administration method and dose of fluoxetine can differentially affect hippocampal plasticity in the adult female rat.

Influence of gasoline inhalation on the enantioselective pharmacokinetics of fluoxetine in rats.

Fluoxetine is used clinically as a racemic mixture of (+)-(S) and (-)-(R) enantiomers for the treatment of depression. CYP2D6 catalyzes the metabolism of both fluoxetine enantiomers. We aimed to evaluate whether exposure to gasoline results in CYP2D inhibition. Male Wistar rats exposed to filtered air (n = 36; control group) or to 600 ppm of gasoline (n = 36) in a nose-only inhalation exposure chamber for 6 weeks (6 h/day, 5 days/week) received a single oral 10-mg/kg dose of racemic fluoxetine. Fluoxetine enantiomers in plasma samples were analyzed by a validated analytical method using LC-MS/MS. The separation of fluoxetine enantiomers was performed in a Chirobiotic V column using as the mobile phase a mixture of ethanol:ammonium acetate 15 mM. Higher plasma concentrations of the (+)-(S)-fluoxetine enantiomer were found in the control group (enantiomeric ratio AUC((+)-(S)/(-)-(R)) = 1.68). In animals exposed to gasoline, we observed an increase in AUC(0-∞) for both enantiomers, with a sharper increase seen for the (-)-(R)-fluoxetine enantiomer (enantiomeric ratio AUC((+)-(S)/(-)-(R)) = 1.07), resulting in a loss of enantioselectivity. Exposure to gasoline was found to result in the loss of enantioselectivity of fluoxetine, with the predominant reduction occurring in the clearance of the (-)-(R)-fluoxetine enantiomer (55% vs. 30%).

A validated microfluidics-based LC-chip-MS/MS method for the quantitation of fluoxetine and norfluoxetine in rat serum.

An increasing number of quantitative bioanalyses need to be performed on samples available in limited volumes, such as pharmacokinetic studies on small animals. In this context, microfluidic systems as the LC-chip device coupled to a mass spectrometer combine small sample volume requirements and high sensitivity. In this study, we present the development of a microfluidics-based method for fluoxetine (FLX) and norfluoxetine (NFL) quantitation dedicated to pharmacokinetic investigations in the rat serum. Using the methodology of experimental design, LC parameters were optimised in terms of peak resolution, analysis time, and sensitivity. An SPE method was then developed for serum samples on miniaturised 96-well plates containing a mixed-mode strong cation exchanger that provided very clean extracts with good analyte recovery (≥66.0%). The total SPE-LC-MS/MS process required only 20 µL per sample and the method provided a good sensitivity in a total run time of 12 min. Finally, the developed method for FLX and NFL quantitation in rat serum was fully validated. After having selected the most appropriate regression model on the basis of the accuracy profiles, method selectivity, trueness, precision, accuracy and linearity were demonstrated.

Therapeutic drug monitoring of children and adolescents treated with fluoxetine.

INTRODUCTION: Information about therapeutic serum levels of fluoxetine (FLX) and its major metabolite norfluoxetine (NORFLX) in children and adolescents is scarce. METHODS: Therapeutic drug monitoring (TDM) of FLX was routinely performed in 71 subjects treated for a major depressive disorder (MDD) (10-60 mg/d FLX, median: 20 mg/d). Correlations between serum concentration and dosage, age, gender, smoking habits and adverse events were analysed. RESULTS: Serum concentrations of the active moiety (FLX + NORFLX) ranged from 21 to 613 ng/mL (mean concentration of 213 ± 118 ng/mL, median: 185 ng/mL). High inter-individual variability in serum concentrations of the active moiety of FLX at each dosage level was observed and no relationship between serum concentration and clinical outcome was found. Apart from smoking, none of the factors tested had a significant eff ect on the serum concentration. DISCUSSION: It was shown that serum concentrations of the active moiety of FLX in children and adolescents seem to be similar to those in adults, with a high level of inter-individual variation. The proportion of patients who showed benefits from treatment with a dose of 20 mg/d FLX was high.

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.

Antidepressant exposure as a predictor of clinical outcomes in the Treatment of Resistant Depression in Adolescents (TORDIA) study.

This paper examines the relationship between plasma concentration of antidepressant and both clinical response and adverse effects in treatment-resistant depressed adolescents. Adolescents (n = 334) with major depression who had not responded to a selective serotonin reuptake inhibitor (SSRI) were randomized to 1 of 4 treatments: switch to another SSRI (fluoxetine, citalopram, or paroxetine), switch to venlafaxine, switch to SSRI plus cognitive behavior therapy, or switch to venlafaxine plus cognitive behavior therapy. Adolescents who did not improve by 6 weeks had their dose increased. Plasma concentrations of medication and metabolites were measured at 6 weeks in 244 participants and at 12 weeks in 204 participants. Adolescents treated with citalopram whose plasma concentration was equal to or greater than the geometric mean (GM) showed a higher response rate compared to those with less than the GM, with parallel but nonsignificant findings for fluoxetine. A dose increase of citalopram or fluoxetine at week 6 was most likely to result in response when it led to a change in concentration from less than the GM at 6 weeks to the GM or greater at week 12. Plasma levels of paroxetine, venlafaxine, or O-desmethylvenlafaxine were not related to clinical response. Exposure was associated with more cardiovascular and dermatologic side effects in those receiving venlafaxine. Antidepressant concentration may be useful in optimizing treatment for depressed adolescents receiving fluoxetine or citalopram.

Pharmacokinetics of fluoxetine in rhesus macaques following multiple routes of administration.

BACKGROUND/AIMS: Fluoxetine (Prozac) is a selective serotonin reuptake inhibitor currently used to treat depression and mood disorders. It has been widely studied clinically and preclinically, yet there is limited knowledge of its pharmacokinetics in nonhuman primates. METHODS: The present study characterized the pharmacokinetics of fluoxetine and its active metabolite norfluoxetine in rhesus macaques following both acute (1, 3, 5.6 and 10 mg/kg) and chronic doses (5.6 and 10 mg/kg/day) via different routes of administration (intravenous, subcutaneous, intramuscular, and oral). Blood samples were collected at multiple time points following administration and analyzed using mass spectrometry. RESULTS: Fluoxetine had a half-life of 11-16 h and norfluoxetine had a half-life of 21-29 h. Potentially functionally significant serum concentrations of norfluoxetine were present at 24 h even after a single administration of fluoxetine. Similar to observations in humans under steady state conditions, norfluoxetine accounted for the greater percentage of active drug in the blood stream. CONCLUSION: A daily dose of 10 mg/kg administered orally maintained serum concentrations in the human clinical range over the course of 6 weeks. Given the long half-lives of fluoxetine and norfluoxetine observed in this study, precautions should be taken when designing preclinical studies to prevent accumulation of drug serum concentrations.

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.