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| Targets |
Serotonin-d4, as an isotopically labeled internal standard, is not designed to bind to specific biological receptors. However, the non-labeled parent compound, Serotonin (5-HT), is an endogenous neurotransmitter that exerts its effects by binding to a family of G protein-coupled receptors (5-HT1, 5-HT2, 5-HT3, 5-HT4, 5-HT5, 5-HT6, 5-HT7) and the ligand-gated ion channel 5-HT3 receptor. Serotonin is involved in the regulation of mood, appetite, sleep, memory, and learning. Serotonin also acts as a hormone in the periphery, regulating gastrointestinal motility and platelet aggregation. Additionally, Serotonin is an inhibitor (blocker/antagonist) of catechol O-methyltransferase (COMT) with a Ki of 44 microM. COMT is an enzyme that degrades catecholamines such as dopamine, epinephrine, and norepinephrine. The deuterated version is used solely as an analytical standard and does not engage in biological interactions.
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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].
As a deuterium-labeled internal standard, Serotonin-d4 itself does not possess intrinsic in vitro biological activity. It is used exclusively for analytical quantification purposes. However, the non-labeled Serotonin (5-HT) exhibits a wide range of in vitro biological activities. Serotonin acts as an agonist at 5-HT receptors, which are expressed in various cell types. In cultured neurons, Serotonin can modulate neuronal excitability, neurotransmitter release, and gene expression. In platelets, Serotonin induces aggregation. In smooth muscle cells, Serotonin causes contraction. Serotonin also inhibits the activity of catechol O-methyltransferase (COMT) in cell-free assays, with a Ki of 44 microM. The effects of Serotonin are concentration-dependent and can be blocked by specific 5-HT receptor antagonists. The deuterated version is used as an internal standard for the accurate quantification of Serotonin in biological samples, including cell culture supernatants, cell lysates, and tissue homogenates. |
| ln Vivo |
Serotonin-d4 does not exhibit in vivo biological activity because it is an analytical standard. However, the non-labeled Serotonin (5-HT) is a critical neurotransmitter in the central nervous system and a hormone in the periphery. In vivo, Serotonin is synthesized from tryptophan and stored in vesicles in serotonergic neurons. Upon stimulation, it is released into the synaptic cleft and binds to 5-HT receptors on postsynaptic neurons. Serotonin is then taken back up into the presynaptic neuron by the serotonin transporter (SERT). Dysregulation of the serotonergic system is implicated in numerous psychiatric disorders, including depression, anxiety, obsessive-compulsive disorder, and eating disorders. Selective serotonin reuptake inhibitors (SSRIs), such as fluoxetine, are widely used to treat these disorders by increasing the concentration of Serotonin in the synaptic cleft. As an internal standard, the deuterated form may be administered in tracer amounts in animal studies to enable precise quantification of Serotonin levels in the brain and other tissues.
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| Enzyme Assay |
A standard non-cellular protocol for using Serotonin-d4 as an internal standard involves its incorporation into the sample preparation workflow for LC-MS analysis. A stock solution of the internal standard (1 mg/mL) is prepared in methanol, water, or 0.1% formic acid. For plasma samples, 50 uL of plasma is transferred to a microcentrifuge tube. Then, 10 uL of the internal standard solution (diluted to a working concentration of 100 ng/mL) is added. Proteins are precipitated by adding 150 uL of ice-cold acetonitrile containing 0.1% formic acid. The mixture is vortexed for 1 minute and centrifuged at 14,000 rpm for 10 minutes at 4degC. The supernatant (150 uL) is transferred to an autosampler vial and mixed with 150 uL of water containing 0.1% formic acid. For tissue samples (e.g., brain), the tissue (50 mg) is homogenized in 500 uL of 0.1 M perchloric acid containing 0.1% EDTA and the internal standard. After centrifugation, the supernatant is filtered and analyzed by LC-MS/MS. The sample (5-10 uL) is injected onto an LC-MS/MS system operated in positive ion mode with multiple reaction monitoring (MRM) for the specific transitions of Serotonin and Serotonin-d4.
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| Cell Assay |
A typical in vitro cellular protocol for using Serotonin-d4 as an internal standard involves the quantification of Serotonin in cultured neurons or other cell types. Cells (e.g., 1×10⁶ differentiated SH-SY5Y neuroblastoma cells) are cultured in appropriate medium for 24 hours. After treatment with test compounds (e.g., SSRIs or other serotonergic agents), the culture medium is collected. Cells are washed twice with ice-cold PBS and harvested by scraping. Cell pellets are resuspended in 200 uL of PBS, and 10 uL of the internal standard solution (100 ng/mL) is added. Cells are lysed by sonication (3×10 seconds on ice). Proteins are precipitated by adding 800 uL of acetonitrile containing 0.1% formic acid. After centrifugation, the supernatant is analyzed by LC-MS/MS. The concentration of Serotonin in the culture medium and cell lysate is quantified using a calibration curve prepared with Serotonin standards and normalized to total protein content. The internal standard corrects for extraction efficiency and matrix effects.
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| Animal Protocol |
A typical in vivo animal protocol for using Serotonin-d4 as an internal standard involves the quantification of Serotonin in brain tissue and plasma. Male Sprague-Dawley rats (200-250 g) are administered a test compound (e.g., an SSRI) via oral gavage or intraperitoneal injection. Blood samples (200 uL) are collected via the tail vein into EDTA-coated tubes at various time points (0, 0.5, 1, 2, 4, 6, 8, 12, 24 hours) post-dose. Plasma is separated by centrifugation (2,000 g, 10 minutes, 4degC). At the final time point, rats are euthanized, and the brain is removed, dissected into specific regions (e.g., hippocampus, prefrontal cortex, striatum), and snap-frozen in liquid nitrogen. Tissue samples (50-100 mg) are homogenized in 500 uL of 0.1 M perchloric acid containing 0.1% EDTA and Serotonin-d4 internal standard (10 ng/mL). After centrifugation, the supernatant is filtered and analyzed by LC-MS/MS. The concentration of Serotonin in plasma and brain tissue is quantified using a calibration curve. The internal standard corrects for variations in sample preparation and matrix effects.
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| ADME/Pharmacokinetics |
As an analytical internal standard, Serotonin-d4 is not characterized by typical pharmacokinetic parameters. However, the non-labeled Serotonin (5-HT) has been extensively studied. Serotonin is synthesized in the central nervous system and enterochromaffin cells of the gastrointestinal tract. After release, Serotonin is rapidly taken up into presynaptic neurons by the serotonin transporter (SERT) and metabolized by monoamine oxidase (MAO) to 5-hydroxyindoleacetic acid (5-HIAA). The plasma half-life of Serotonin is very short (minutes) due to rapid uptake and metabolism. The compound does not cross the blood-brain barrier. Most of the body's Serotonin is synthesized in the periphery and is not available to the central nervous system. The deuterated internal standard is used to accurately quantify Serotonin in biological samples, including plasma, urine, and tissue homogenates, to study the dynamics of the serotonergic system under various physiological and pathological conditions.
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| Toxicity/Toxicokinetics |
Toxicity data specific to Serotonin-d4 are not available. Serotonin (non-labeled) is an endogenous neurotransmitter and is not considered toxic at physiological concentrations. However, excessively high levels of Serotonin in the body can lead to serotonin syndrome, a potentially life-threatening condition characterized by agitation, confusion, rapid heart rate, high blood pressure, dilated pupils, loss of muscle coordination, muscle rigidity, and hyperthermia. Serotonin syndrome can occur as a result of overdoses of serotonergic drugs, such as SSRIs, MAOIs, or combinations thereof. Standard laboratory safety precautions should be followed when handling Serotonin-d4, including the use of gloves, lab coats, and safety glasses. Avoid inhalation, ingestion, and direct skin or eye contact. The compound should be stored at -20degC or 4degC, protected from light and moisture. It is intended for research use only and should not be used in humans or animals for therapeutic or diagnostic purposes.
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| References |
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| Additional Infomation |
Serotonin-d4 (CAS# 58264-95-2) is a stable isotope-labeled compound with a molecular weight of 180.24. The molecular formula is C10H8D4N2O, and it is also known as 5-Hydroxytryptamine-d4, 5-HT-d4, and 3-(2-aminoethyl-1,1,2,2-d4)-1H-indol-5-ol. The isotopic purity is typically >98%, and the chemical purity is ≥95-98%. Serotonin is a monoamine neurotransmitter in the central nervous system and an endogenous 5-HT receptor agonist. It is also an inhibitor (antagonist) of catechol O-methyltransferase (COMT) with a Ki of 44 microM. The deuterated version is intended for use as an internal standard for the quantification of Serotonin by GC- or LC-MS. This product is for research use only and is not approved for clinical or diagnostic applications. The compound should be stored at -20degC or 4degC, protected from light and moisture.
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| Molecular Formula |
C10H12N2O
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| Molecular Weight |
180.239728927612
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| Exact Mass |
180.12
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| CAS # |
58264-95-2
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| PubChem CID |
71752180
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| Appearance |
White to light yellow solid powder
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| Hydrogen Bond Donor Count |
3
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| Rotatable Bond Count |
2
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| Heavy Atom Count |
13
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| Complexity |
174
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| Defined Atom Stereocenter Count |
0
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| SMILES |
N([2H])([2H])C([2H])([2H])CC1C2C(=CC=C(C=2)O)NC=1
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| InChi Key |
QZAYGJVTTNCVMB-KHORGVISSA-N
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| InChi Code |
InChI=1S/C10H12N2O/c11-4-3-7-6-12-10-2-1-8(13)5-9(7)10/h1-2,5-6,12-13H,3-4,11H2/i3D2,4D2
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| Chemical Name |
3-(2-amino-1,1,2,2-tetradeuterioethyl)-1H-indol-5-ol
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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 Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture. |
| 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 | 5.5482 mL | 27.7408 mL | 55.4816 mL | |
| 5 mM | 1.1096 mL | 5.5482 mL | 11.0963 mL | |
| 10 mM | 0.5548 mL | 2.7741 mL | 5.5482 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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