| Size | Price | Stock | Qty |
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| 1mg |
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| Other Sizes |
| Targets |
Norfluoxetine-d5 is a stable isotope-labeled internal standard. Its unlabeled parent compound, Norfluoxetine, is the primary active metabolite of the SSRI antidepressant Fluoxetine (Prozac). Norfluoxetine targets the serotonin transporter (SERT), which is located in the presynaptic membrane of serotonergic neurons. By binding to SERT, Norfluoxetine inhibits the reuptake of serotonin (5-HT) from the synaptic cleft back into the presynaptic neuron. This leads to an increased concentration of serotonin in the synapse, enhancing serotonergic neurotransmission. Norfluoxetine is equipotent to Fluoxetine as a SERT inhibitor and has a much longer half-life. Additionally, Norfluoxetine has a moderate affinity for the norepinephrine transporter (NET). The labeled version (d5) is used to trace this interaction for analytical purposes, not for therapeutic effect.
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| ln Vitro |
Drug compounds have included stable heavy isotopes of carbon, hydrogen, and other elements, mostly as quantitative tracers while the drugs were being developed. Because deuteration may have an effect on a drug's pharmacokinetics and metabolic properties, it is a cause for concern [1].
The in vitro biological activity of Norfluoxetine-d5 is not directly studied, as it is an internal standard. The unlabeled Norfluoxetine is a potent and selective serotonin reuptake inhibitor (SSRI). In vitro studies using rat brain synaptosomal preparations have shown that Norfluoxetine inhibits serotonin (5-HT) uptake with a Ki (inhibition constant) of 44.7 nM. In isolated human platelets, it inhibits 5-HT uptake with an IC₅0 of approximately 15 nM. In cell lines expressing the human serotonin transporter (hSERT), Norfluoxetine binds to the transporter with an affinity (Ki) similar to that of Fluoxetine. Furthermore, in PC12 cell cultures (a neuronal cell line), Norfluoxetine (1-10 uM) increases the extracellular concentration of serotonin and activates downstream signaling cascades, including the MAPK/ERK pathway. The labeled Norfluoxetine-d5 is used as an internal standard for LC-MS analysis to accurately quantify Norfluoxetine levels in the cell culture medium. |
| ln Vivo |
Norfluoxetine-d5 is a stable isotope-labeled internal standard. The in vivo activity of its unlabeled parent, Norfluoxetine, is the primary driver of the antidepressant effect of Fluoxetine. In animal models of depression, such as the forced swim test (FST) in rats, administration of Norfluoxetine (10-20 mg/kg, i.p.) significantly reduces immobility time, indicating an antidepressant-like effect. This effect is mediated by the inhibition of SERT in the raphe nuclei and forebrain. In vivo microdialysis studies in freely moving rats have shown that systemic injection of Norfluoxetine leads to a rapid and sustained increase (up to 300-400% of baseline) in extracellular serotonin levels in the prefrontal cortex and hippocampus. Norfluoxetine is also known to antagonize the 5-HT depletion induced by p-chloroamphetamine. The labeled Norfluoxetine-d5 is used as an internal standard to accurately quantify the drug‘s concentration in the brain and plasma in these animal studies.
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| Enzyme Assay |
A generic non-cell-based assay for Norfluoxetine-d5 involves its use as an internal standard in an LC-MS/MS method for quantifying Norfluoxetine and Fluoxetine in human plasma. Prepare a standard stock solution of unlabeled Norfluoxetine hydrochloride in methanol (1 mg/mL). Prepare a separate stock solution of the internal standard (Norfluoxetine-d5 hydrochloride) at the same concentration. Prepare calibration standards by spiking the unlabeled analyte into a blank matrix (e.g., charcoal-stripped human plasma) to achieve concentrations ranging from 0.1 to 50 ng/mL. Add a fixed concentration of the internal standard (e.g., 5 ng/mL) to each calibration standard. Also prepare blank and double-blank samples. For sample preparation, perform liquid-liquid extraction with ethyl acetate/hexane (1:1). Transfer the organic layer to a clean tube and evaporate under nitrogen. Reconstitute the residue in mobile phase. Analyze the samples by LC-MS/MS in positive ion mode. Monitor the mass transitions: m/z 296.2 → 134.1 for Norfluoxetine, and m/z 301.2 → 139.1 for Norfluoxetine-d5. Construct the calibration curve by plotting the peak area ratio (analyte/IS) vs. the nominal concentration.
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| Cell Assay |
A standard in vitro cell-based protocol for the unlabeled Norfluoxetine involves the measurement of serotonin uptake inhibition. For this protocol, culture a cell line that stably expresses the human serotonin transporter (hSERT), such as HEK293-hSERT cells. Grow the cells in DMEM supplemented with 10% FBS and antibiotics at 37degC in a 5% CO2 incubator. Seed the cells in 24-well plates at a density of 1×10⁵ cells/well and allow them to attach for 48 hours. On the day of the assay, wash the cells twice with pre-warmed uptake buffer (25 mM HEPES, 120 mM NaCl, 5.4 mM KCl, 1.2 mM CaCl2, 1.2 mM MgSO4, 5 mM glucose, 1 mM ascorbic acid, pH 7.4). Pre-incubate the cells with increasing concentrations (0.1 nM to 10 uM) of unlabeled Norfluoxetine in uptake buffer for 10 minutes. Then, add a fixed concentration of [3H]-serotonin (50 nM) to the wells and incubate for another 10 minutes at 37degC. Terminate the reaction by rapidly washing the cells three times with ice-cold uptake buffer. Lyse the cells with 0.5 mL of 0.1 N NaOH and quantify the accumulated radioactivity by liquid scintillation counting. Use Norfluoxetine-d5 as an internal standard in an LC-MS analysis to confirm the drug concentration in the buffer solution. Calculate the IC₅0 for serotonin uptake inhibition.
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| Animal Protocol |
A standard in vivo animal protocol for Norfluoxetine-d5 is a pharmacokinetic (PK) study in male Sprague-Dawley rats. Use rats weighing 200-250 g (n=5 per time point). Administer a single oral dose of unlabeled Norfluoxetine hydrochloride (10 mg/kg) dissolved in saline. Collect blood samples from the tail vein at various time points (0, 1, 2, 4, 6, 8, 12, 24, 48, 72 h) into heparinized tubes. Centrifuge the samples to obtain plasma and store at -80degC. For bioanalysis, spike 50 uL of plasma with a fixed amount of Norfluoxetine-d5 as the internal standard. Perform protein precipitation by adding 150 uL of ice-cold acetonitrile. Vortex and centrifuge at 14,000g for 10 minutes. Inject 5 uL of the supernatant into the LC-MS/MS system. Quantify the plasma concentrations of Norfluoxetine. Calculate the PK parameters (Cmax, Tmax, AUC(0-t), AUC(0-∞), t½, and clearance (CL)) using non-compartmental analysis with Phoenix WinNonlin software. This protocol is widely used to characterize the long-acting properties of Norfluoxetine.
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| ADME/Pharmacokinetics |
Norfluoxetine-d5 is a stable isotope-labeled internal standard, and its PK is the same as its unlabeled parent. Norfluoxetine is the primary active metabolite of Fluoxetine. It has a very long elimination half-life in humans, ranging from 4 to 16 days, which is significantly longer than the parent drug (Fluoxetine has a half-life of 1-3 days). This long half-life is due to its high lipophilicity (large volume of distribution, Vd ~ 20-40 L/kg) and extensive tissue binding. It is primarily metabolized by the liver via CYP2D6 and CYP2C9 to norfluoxetine glucuronide and other metabolites. The drug and its metabolites are then excreted in the urine. Because of the long half-life, steady-state concentrations are reached only after several weeks of dosing. The labeled Norfluoxetine-d5 is critical for quantifying the low and variable plasma levels of this metabolite in PK studies and for therapeutic drug monitoring in a clinical research context.
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| Toxicity/Toxicokinetics |
Norfluoxetine-d5 is a research-grade internal standard, not a drug. The toxicity of its unlabeled parent, Norfluoxetine, is similar to that of Fluoxetine. These compounds are generally considered safe at therapeutic doses but have a range of dose-dependent adverse effects. In humans, side effects include nausea, headache, insomnia, somnolence, sexual dysfunction, and agitation. High doses can lead to serotonin syndrome, a potentially life-threatening condition characterized by autonomic instability and neuromuscular abnormalities. In animal toxicology studies, the oral LD₅0 of Norfluoxetine is approximately 300-500 mg/kg in rats. Chronic exposure in rats has been associated with reductions in body weight and food consumption. For laboratory personnel, standard safety precautions should be used when handling Norfluoxetine-d5 to avoid direct exposure. It should be stored as a powder at -20degC.
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| References | |
| Additional Infomation |
Norfluoxetine-d5 is the stable isotope-labeled (deuterated) version of Norfluoxetine, the primary active metabolite of the widely prescribed SSRI antidepressant Fluoxetine (Prozac). It is intended for research use only and serves as a sensitive internal standard for the accurate quantification of Norfluoxetine and Fluoxetine in biological samples by LC-MS/MS. Norfluoxetine is equipotent to Fluoxetine as an inhibitor of the serotonin transporter (SERT) and has a significantly longer elimination half-life (4-16 days in humans), making it a major contributor to the drug‘s prolonged therapeutic effect and the potential for drug-drug interactions. This labeled compound is an essential tool for pharmacokinetic studies, bioequivalence studies, and neuropsychiatric research focused on serotonin signaling. It is not for diagnostic or therapeutic use.
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| Molecular Formula |
C16H12D5CLF3NO
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| Molecular Weight |
336.79
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| Exact Mass |
336.126
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| CAS # |
1185132-92-6
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| PubChem CID |
45040123
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| Appearance |
White to off-white solid powder
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| LogP |
5.676
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| Hydrogen Bond Donor Count |
2
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| Hydrogen Bond Acceptor Count |
5
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| Rotatable Bond Count |
5
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| Heavy Atom Count |
22
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| Complexity |
294
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| Defined Atom Stereocenter Count |
0
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| SMILES |
[2H]C([2H])(C([2H])([2H])N)C([2H])(C1=CC=CC=C1)OC2=CC=C(C=C2)C(F)(F)F.Cl
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| InChi Key |
GMTWWEPBGGXBTO-KDPCSTGBSA-N
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| InChi Code |
InChI=1S/C16H16F3NO.ClH/c17-16(18,19)13-6-8-14(9-7-13)21-15(10-11-20)12-4-2-1-3-5-12;/h1-9,15H,10-11,20H2;1H/i10D2,11D2,15D;
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| Chemical Name |
1,1,2,2,3-pentadeuterio-3-phenyl-3-[4-(trifluoromethyl)phenoxy]propan-1-amine;hydrochloride
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| HS Tariff Code |
2934.99.9001
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| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month |
| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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| Solubility (In Vitro) |
May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples
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| Solubility (In Vivo) |
Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.
Injection Formulations
Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)(e.g. IP/IV/IM/SC) *Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution. Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline) Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil) Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals). View More
Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)] Oral Formulations
Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium) Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals). View More
Oral Formulation 3: Dissolved in PEG400 (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.9692 mL | 14.8460 mL | 29.6921 mL | |
| 5 mM | 0.5938 mL | 2.9692 mL | 5.9384 mL | |
| 10 mM | 0.2969 mL | 1.4846 mL | 2.9692 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
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