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| 1mg |
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| Other Sizes |
| Targets |
Levomefolic acid-13C,d3 serves as an internal standard and does not exert its own biological effects in analytical contexts. However, the unlabeled parent compound, Levomefolic acid (5-MTHF), is the biologically active form of folate and plays crucial roles in one-carbon metabolism. It serves as a methyl donor in the conversion of homocysteine to methionine, a reaction catalyzed by methionine synthase. This reaction requires vitamin B12 as a cofactor. Methionine can then be converted to S-adenosylmethionine (SAMe), the primary methyl donor for numerous methylation reactions, including DNA methylation. Levomefolic acid is also a cofactor for the synthesis of purines and pyrimidines, which are essential for DNA replication and repair. Its biological targets include methionine synthase and various enzymes involved in folate metabolism.
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| ln Vitro |
Drug compounds have included stable heavy isotopes of carbon, hydrogen, and other elements, mostly as tracers that influence measurement during the drug development process. It's possible that the pharmacokinetics and functional range of medications contribute to the concern over mutagenesis [1].
Levomefolic acid-13C,d3 itself does not exhibit in vitro biological activity because it is used as an internal standard. However, the unlabeled Levomefolic acid (5-MTHF) is a biologically active form of folate that has been extensively studied. In vitro, Levomefolic acid is essential for the growth and proliferation of human cells. It is required for nucleotide synthesis, DNA methylation, and the maintenance of genomic stability. In cell culture, folate deficiency leads to cell cycle arrest, increased DNA damage, and apoptosis. Levomefolic acid can also reduce the production of homocysteine in vitro, potentially protecting against homocysteine-induced cellular toxicity. The compound has been shown to support the proliferation of various cell types, including lymphocytes, fibroblasts, and hematopoietic stem cells. The deuterated version is used as an internal standard to accurately quantify Levomefolic acid in cell culture experiments. |
| ln Vivo |
Levomefolic acid-13C,d3 does not exhibit in vivo biological activity because it is an analytical standard. However, the unlabeled Levomefolic acid (5-MTHF) is an orally active, brain-penetrant natural active form of folic acid. In vivo, Levomefolic acid is the primary circulating form of folate and is transported into tissues via specific folate receptors and transporters. It plays a critical role in one-carbon metabolism, including the synthesis of nucleotides and the regulation of homocysteine levels. Levomefolic acid is used as a dietary supplement and has been studied for its potential role in preventing neural tube defects during pregnancy, reducing the risk of cardiovascular disease by lowering homocysteine levels, and improving cognitive function in the elderly. The compound is also being investigated for its potential benefits in depression, as it may enhance the synthesis of neurotransmitters such as serotonin, dopamine, and norepinephrine.
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| Enzyme Assay |
A typical non-cellular protocol for using Levomefolic acid-13C,d3 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 water or methanol containing 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 1 ug/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. 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 Levomefolic acid and the labeled internal standard. Quantification is achieved by calculating the peak area ratio of the analyte to the internal standard against a calibration curve.
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| Cell Assay |
A typical in vitro cellular protocol for using Levomefolic acid-13C,d3 as an internal standard involves the quantification of Levomefolic acid in cultured cells. Cells (e.g., 1×10⁶ primary hepatocytes or neuronal cells) are cultured in folate-free medium for 24 hours to deplete endogenous folate. After treatment with test compounds, 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 (1 ug/mL) is added. Cells are lysed by three freeze-thaw cycles (liquid nitrogen for 3 minutes, then 37degC water bath for 5 minutes). Proteins are precipitated by adding 800 uL of acetonitrile containing 0.1% formic acid. After centrifugation, the supernatant is analyzed by LC-MS/MS. Levomefolic acid levels are 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 Levomefolic acid-13C,d3 as an internal standard involves the quantification of Levomefolic acid in plasma and tissues. Male Sprague-Dawley rats (200-250 g) are administered Levomefolic acid (non-labeled) via oral gavage at a dose of 1-10 mg/kg. 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). For tissue analysis, rats are euthanized at the final time point, and liver, kidney, brain, and other tissues are harvested, rinsed with PBS, snap-frozen in liquid nitrogen, and stored at -80degC. Tissues are homogenized in PBS (100 mg tissue/1 mL PBS) using a tissue homogenizer. For extraction, 50 uL of plasma or tissue homogenate is mixed with 10 uL of internal standard solution (1 ug/mL) and 150 uL of acetonitrile. After centrifugation, the supernatant is analyzed by LC-MS/MS. The internal standard corrects for variations in sample preparation and matrix effects.
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| ADME/Pharmacokinetics |
As an analytical internal standard, Levomefolic acid-13C,d3 is not characterized by typical pharmacokinetic parameters. However, the unlabeled Levomefolic acid has been studied for its pharmacokinetic properties. After oral administration, Levomefolic acid is well-absorbed and has high oral bioavailability, as it does not require enzymatic reduction like folic acid. Peak plasma concentrations (Cmax) are reached within 1-2 hours (Tmax). The plasma elimination half-life (t1/2) is approximately 2-4 hours. Levomefolic acid is distributed to various tissues, including the brain, due to active transport via folate receptors and transporters. It is metabolized to other folate derivatives, including 5,10-methylenetetrahydrofolate and 10-formyltetrahydrofolate, which are involved in one-carbon metabolism. The compound is eliminated primarily in the urine, with significant enterohepatic recirculation. The deuterated internal standard is used to accurately quantify Levomefolic acid in biological samples.
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| Toxicity/Toxicokinetics |
Toxicity data specific to Levomefolic acid-13C,d3 are not available. The unlabeled Levomefolic acid is the biologically active form of folate and is generally recognized as safe (GRAS). It is an essential nutrient that is required for normal cellular function. Toxicity from folate is rare because it is water-soluble and excess amounts are excreted in the urine. However, very high doses (e.g., >15 mg/day) may cause gastrointestinal disturbances, sleep disturbances, and skin reactions. It is important to note that supplementation with folic acid (the synthetic form) can mask vitamin B12 deficiency, which can lead to neurological damage. Levomefolic acid is less likely to mask B12 deficiency because it does not require conversion. The compound should be stored at -20degC or as recommended by the supplier, protected from light and moisture. It is for research use only and should not be used in humans for therapeutic or diagnostic purposes without appropriate approval.
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| References | |
| Additional Infomation |
Levomefolic acid-13C,d3 (CAS# 1356019-94-7) is a stable isotope-labeled compound with a molecular weight of 463.47. The molecular formula is C1913CH22D3N7O6, and it is also known as 5-methyltetrahydrofolate-13C,d3 and 5-MTHF-13C,d3. Levomefolic acid is the biologically active form of folic acid, essential for DNA synthesis, repair, and methylation. It is an orally active, brain-penetrant natural active form of folic acid and is one of the most widely used folic acid food supplements. The isotopically labeled version is used as an internal standard for the quantification of Levomefolic acid by LC-MS. This product is for research use only and is not approved for clinical or nutritional supplement applications. It should be stored under recommended conditions, typically at -20degC or -80degC, protected from light and moisture.
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| Molecular Formula |
C20H25N7O6
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| Molecular Weight |
463.466943502426
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| Exact Mass |
463.208
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| CAS # |
1356019-94-7
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| Related CAS # |
Levomefolic acid;31690-09-2
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| PubChem CID |
136242415
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| Appearance |
White to off-white solid powder
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| LogP |
-0.5
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| Hydrogen Bond Donor Count |
7
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| Hydrogen Bond Acceptor Count |
10
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| Rotatable Bond Count |
9
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| Heavy Atom Count |
33
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| Complexity |
865
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| Defined Atom Stereocenter Count |
2
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| SMILES |
O=C1C2=C(N([H])C(N([H])[H])=N1)N([H])C([H])([H])[C@]([H])(C([H])([H])N([H])C1C([H])=C([H])C(C(N([H])[C@]([H])(C(=O)O[H])C([H])([H])C([H])([H])C(=O)O[H])=O)=C([H])C=1[H])N2[13C]([2H])([2H])[2H]
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| InChi Key |
ZNOVTXRBGFNYRX-OWXPWHJLSA-N
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| InChi Code |
InChI=1S/C20H25N7O6/c1-27-12(9-23-16-15(27)18(31)26-20(21)25-16)8-22-11-4-2-10(3-5-11)17(30)24-13(19(32)33)6-7-14(28)29/h2-5,12-13,22H,6-9H2,1H3,(H,24,30)(H,28,29)(H,32,33)(H4,21,23,25,26,31)/t12-,13-/m0/s1/i1+1D3
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| Chemical Name |
(2S)-2-[[4-[[(6S)-2-amino-4-oxo-5-(trideuterio(113C)methyl)-3,6,7,8-tetrahydropteridin-6-yl]methylamino]benzoyl]amino]pentanedioic acid
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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: This product requires protection from light (avoid light exposure) during transportation and storage. |
| 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 | 2.1576 mL | 10.7882 mL | 21.5764 mL | |
| 5 mM | 0.4315 mL | 2.1576 mL | 4.3153 mL | |
| 10 mM | 0.2158 mL | 1.0788 mL | 2.1576 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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