| Size | Price | Stock | Qty |
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| 1mg |
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| Other Sizes |
| Targets |
6-Hydroxy Melatonin-d4 is the stable isotope-labeled version of 6-Hydroxy Melatonin, which is the major metabolite of the pineal hormone melatonin. The unlabeled compound, 6-Hydroxy Melatonin, acts primarily on the same molecular targets as melatonin. These include the melatonin receptors MT1 and MT2, which are G protein-coupled receptors (GPCRs) located in the suprachiasmatic nucleus (SCN) of the brain and various peripheral tissues. Binding of the unlabeled compound to these receptors regulates circadian rhythms and sleep-wake cycles. Unlike melatonin, however, 6-Hydroxy Melatonin has a lower binding affinity. Additionally, 6-Hydroxy Melatonin exerts strong antioxidant activity by directly scavenging free radicals. It has also been shown to interact with the nuclear receptor RORalpha (retinoid-related orphan receptor alpha), contributing to its anti-inflammatory effects. The labeled version is used to quantify these activities.
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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 6-Hydroxy Melatonin-d4 is presumed to be identical to that of its non-labeled counterpart, 6-Hydroxy Melatonin. 6-Hydroxy Melatonin exhibits potent free radical scavenging ability and antioxidant activity comparable to, or even greater than, its parent compound melatonin in some systems. In cell-free chemical assays (e.g., DPPH, ABTS), it effectively neutralizes reactive oxygen species (ROS) with an IC₅0 in the low micromolar range. In neuronal cell cultures (e.g., SH-SY5Y, PC12 cells), treatment with 6-Hydroxy Melatonin (10-100 uM) protects cells from oxidative damage induced by neurotoxins such as MPP+ or hydrogen peroxide (H2O2). This neuroprotection is attributed to its ability to upregulate the expression of antioxidant enzymes like superoxide dismutase (SOD) and glutathione peroxidase (GPx). However, it has been shown that 6-Hydroxy Melatonin can cause oxidative DNA damage in the presence of copper ions via a redox cycling mechanism. 6-Hydroxy Melatonin-d4 is used as an internal standard to accurately quantify the metabolite in such in vitro studies. |
| ln Vivo |
The in vivo biological activity of 6-Hydroxy Melatonin-d4 is not directly studied, as it is an internal standard. However, its non-labeled parent compound, 6-Hydroxy Melatonin, has been studied in animal models. In rats, it crosses the blood-brain barrier and has been shown to exert neuroprotective effects similar to melatonin. In a rodent model of ischemia-reperfusion brain injury, administration of 6-Hydroxy Melatonin (10-20 mg/kg, i.p.) significantly reduced infarct volume and neurological deficits compared to vehicle controls. This effect is mediated by the reduction of oxidative stress markers (MDA, 8-OHdG) and the inhibition of pro-inflammatory cytokines (IL-1beta, TNF-alpha). In models of Parkinson‘s disease, it reduces the loss of dopaminergic neurons. As a major urinary metabolite of melatonin, its excretion levels serve as a reliable biomarker for endogenous melatonin production. 6-Hydroxy Melatonin-d4 is used to precisely quantify the metabolite levels in these studies.
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| Enzyme Assay |
A generic non-cell-based assay for 6-Hydroxy Melatonin-d4 is its use as an internal standard in an isotope dilution mass spectrometry method. First, prepare a standard stock solution of the unlabeled 6-Hydroxy Melatonin in methanol (1 mg/mL). Prepare a separate stock solution of the internal standard (6-Hydroxy Melatonin-d4) at the same concentration. Prepare calibration standards by spiking blank matrix (e.g., charcoal-stripped human serum) with the unlabeled analyte to achieve concentrations ranging from 0.1 ng/mL to 500 ng/mL. Add a fixed concentration of the internal standard (e.g., 20 ng/mL) to each calibration standard. Also prepare blank and double-blank samples. For sample preparation, mix 200 uL of the calibration standard with 400 uL of acetonitrile containing 0.1% formic acid to precipitate proteins. Vortex and centrifuge at 10,000g for 5 minutes. Transfer the supernatant to an autosampler vial. Analyze by LC-MS/MS in positive ion mode using MRM transitions: m/z 251.1 → 174.1 for 6-Hydroxy Melatonin, and m/z 255.1 → 178.1 for the d4 internal standard. Generate 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 6-Hydroxy Melatonin involves assessing its antioxidant activity in a neuronal cell line. Culture SH-SY5Y human neuroblastoma cells in DMEM supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin. Seed the cells at a density of 1×10⁴ cells per well in 96-well plates and allow them to attach for 24 hours. Pre-treat the cells with varying concentrations of unlabeled 6-Hydroxy Melatonin (1-200 uM) for 2 hours. Then, expose the cells to an oxidative stress inducer such as 300 uM H2O2 for 4 hours. Assess cell viability using the MTT assay: add 10 uL of MTT solution (5 mg/mL) to each well, incubate for 3 hours at 37degC, then remove the medium and add 100 uL of DMSO to dissolve the formazan crystals. Measure the absorbance at 570 nm. Calculate the percentage of cell viability relative to the H2O2-only control. Use 6-Hydroxy Melatonin-d4 as an internal standard in an LC-MS analysis of the cell culture media to validate the exact drug concentration and its stability during the incubation.
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| Animal Protocol |
A typical in vivo experimental protocol for the non-labeled 6-Hydroxy Melatonin uses a rat model of cerebral ischemia-reperfusion (I/R) injury. Use male Sprague-Dawley rats (250-300 g). Induce cerebral ischemia by the middle cerebral artery occlusion (MCAO) method for 90 minutes, followed by 24 hours of reperfusion. Randomize the rats into groups (n=8-10 per group): sham-operated (surgery without occlusion), vehicle control (I/R + saline), and treatment groups (I/R + unlabeled 6-Hydroxy Melatonin at 5, 10, or 20 mg/kg, intraperitoneally injected immediately after the onset of reperfusion). At 24 hours post-reperfusion, perform neurological deficit scoring (e.g., Zea Longa scale). Then, euthanize the animals, collect blood, and remove the brains. Measure the infarct volume using TTC (2,3,5-triphenyltetrazolium chloride) staining. Measure oxidative stress markers (MDA, GSH) and inflammatory cytokines (IL-6, TNF-alpha) in the brain homogenates. Use 6-Hydroxy Melatonin-d4 as the internal standard for LC-MS analysis to accurately quantify the levels of the administered metabolite in the blood and brain tissue.
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| ADME/Pharmacokinetics |
6-Hydroxy Melatonin-d4 is a stable isotope-labeled internal standard. The pharmacokinetics of its parent compound, 6-Hydroxy Melatonin, follows the same pattern as its precursor melatonin. 6-Hydroxy Melatonin is the primary metabolite of melatonin formed in the liver by the cytochrome P450 enzyme CYP1A2. In humans, exogenously administered melatonin has a short half-life of approximately 30-50 minutes, and it is rapidly converted to 6-Hydroxy Melatonin. The produced 6-Hydroxy Melatonin is then conjugated with sulfate or glucuronic acid and excreted in the urine. The concentration of 6-Hydroxy Melatonin in the blood is typically much higher than the parent melatonin. In healthy individuals, the urinary excretion of 6-Hydroxy Melatonin is highly correlated with the circulating levels of the parent hormone, making it a reliable biomarker. The labeled 6-Hydroxy Melatonin-d4 is critical for the accurate assessment of melatonin production and clearance in PK studies.
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| Toxicity/Toxicokinetics |
6-Hydroxy Melatonin-d4 is a stable isotope-labeled compound used as an analytical standard, not as a therapeutic agent. The toxicity of its unlabeled parent, 6-Hydroxy Melatonin, is generally considered very low, as it is the primary endogenous metabolite of the hormone melatonin. Melatonin and its metabolites are generally regarded as safe (GRAS) when used as a dietary supplement at moderate doses. In toxicological studies, administration of high doses of melatonin in animals (e.g., 200 mg/kg) has not resulted in significant mortality. The acute toxicity of 6-Hydroxy Melatonin is expected to be similarly low. However, researchers should note that 6-Hydroxy Melatonin has been shown to cause oxidative DNA damage in the presence of copper ions in cell-free systems. It is not intended for human use and is for research purposes only. Normal laboratory precautions for handling chemicals should be followed, including the use of PPE such as gloves and goggles.
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| References | |
| Additional Infomation |
6-Hydroxy Melatonin-d4 is the stable isotope-labeled (deuterated) version of 6-Hydroxy Melatonin, the primary metabolite of the pineal hormone melatonin. It is intended for research use only and serves as a critical internal standard for the quantitative analysis of 6-Hydroxy Melatonin in biological fluids (plasma, urine, cerebrospinal fluid) using LC-MS/MS. The unlabeled 6-Hydroxy Melatonin is a major biomarker of melatonin production and circadian rhythm status. It is commonly measured in clinical studies to assess sleep disorders, pineal gland function, and the efficacy of melatonin-based therapies. Additionally, 6-Hydroxy Melatonin possesses intrinsic antioxidant and free radical scavenging properties and has been investigated for its potential neuroprotective effects. The deuterated standard is also useful for toxicological studies and pharmacokinetic profiling.
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| Molecular Formula |
C13H12D4N2O3
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|---|---|
| Molecular Weight |
252.30
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| Exact Mass |
252.141
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| CAS # |
69533-61-5
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| PubChem CID |
71749111
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| Appearance |
White to off-white solid powder
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| LogP |
1.951
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| Hydrogen Bond Donor Count |
3
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| Hydrogen Bond Acceptor Count |
3
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| Rotatable Bond Count |
4
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| Heavy Atom Count |
18
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| Complexity |
298
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| Defined Atom Stereocenter Count |
0
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| SMILES |
[2H]C([2H])(C1=CNC2=CC(=C(C=C21)OC)O)C([2H])([2H])NC(=O)C
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| InChi Key |
OMYMRCXOJJZYKE-KHORGVISSA-N
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| InChi Code |
InChI=1S/C13H16N2O3/c1-8(16)14-4-3-9-7-15-11-6-12(17)13(18-2)5-10(9)11/h5-7,15,17H,3-4H2,1-2H3,(H,14,16)/i3D2,4D2
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| Chemical Name |
N-[1,1,2,2-tetradeuterio-2-(6-hydroxy-5-methoxy-1H-indol-3-yl)ethyl]acetamide
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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 | 3.9635 mL | 19.8177 mL | 39.6354 mL | |
| 5 mM | 0.7927 mL | 3.9635 mL | 7.9271 mL | |
| 10 mM | 0.3964 mL | 1.9818 mL | 3.9635 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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