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| Targets |
Formononetin-d3-1 is a deuterated isoflavone used as an analytical internal standard. The unlabeled parent compound, Formononetin, is an FGFR2 inhibitor and a phytoestrogen that binds to estrogen receptors (ERalpha and ERbeta) with moderate affinity, exhibiting estrogenic or anti-estrogenic effects depending on the context. Formononetin also targets NF-kappaB, PI3K/Akt, and other pathways. The deuterated form has no independent biological activity.
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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 impact on a drug's pharmacokinetics and metabolic profile, it has drawn attention [1].
The deuterium-labeled form (d3-1) is not studied for independent in vitro bioactivity as it is an analytical internal standard. Unlabeled Formononetin, however, has multiple biological activities: it induces osteogenic differentiation of mesenchymal stem cells via FGFR2 signaling, inhibits cancer cell proliferation, exhibits antioxidant and anti-inflammatory effects, and suppresses TNF-alpha and IL-6 production in macrophages. When metabolized in the rumen, formononetin transforms into a potent estrogen (equol). |
| ln Vivo |
In vivo, unlabeled Formononetin has been studied for bone health, cardiovascular protection, and anti-cancer activities in animal models. It reduces ovariectomy-induced bone loss in rats, lowers blood glucose in diabetic models, and inhibits tumor growth in xenograft models. The deuterated form (d3-1) is not administered in vivo. Instead, it is used as an internal standard in pharmacokinetic studies of formononetin or formononetin-containing herbal extracts.
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| Enzyme Assay |
Formononetin-d3-1 is designed for LC-MS/MS applications rather than cell-free biochemical assays. For typical use: Prepare stock solutions of formononetin-d3-1 in methanol or DMSO at 1 mg/mL. Add a fixed concentration (e.g., 10-100 ng/mL) of the deuterated internal standard to each calibrator, quality control, and study sample. Extract samples by protein precipitation with acetonitrile or by solid-phase extraction. Separate on a C18 reverse-phase column. Detect using multiple reaction monitoring in negative ion mode: m/z 271.28 → 268.26 (unlabeled) and m/z 274.28 → 271.26 (labeled, +3 Da). Calculate concentrations based on peak area ratios.
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| Cell Assay |
Cell-based assays are not performed with the deuterated internal standard. For studies of formononetin's activity, culture bone marrow-derived mesenchymal stem cells (BMSCs), osteoblasts, or cancer cell lines (e.g., MCF-7, PC-3, A549) in appropriate medium. Treat cells with unlabeled formononetin at concentrations of 1-100 uM for 24-72 hours. Assess cell viability by MTT or CCK-8. Measure osteogenic differentiation by ALP activity and Alizarin Red S staining. Assess estrogenic activity by luciferase reporter assays in ER-expressing cells. Use formononetin-d3-1 as internal standard for LC-MS/MS quantification of intracellular formononetin levels.
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| Animal Protocol |
Animal studies are not performed with Formononetin-d3-1 itself. For PK studies of formononetin, administer unlabeled formononetin to rats or mice via oral gavage (10-100 mg/kg) or intravenous injection (1-10 mg/kg). Collect blood at multiple time points (0-24 hours). Harvest plasma, add formononetin-d3-1 as internal standard, extract, and analyze by LC-MS/MS to determine formononetin concentrations. Calculate PK parameters such as Cmax, Tmax, t1/2, AUC, and oral bioavailability. Tissue distribution studies can also be performed.
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| ADME/Pharmacokinetics |
Formononetin-d3-1 (Molecular Weight: 271.28, Formula: C16H9D3O4) is a stable isotope-labeled compound. As an internal standard, it is not studied for pharmacokinetic properties in vivo. Unlabeled formononetin has moderate oral bioavailability (approximately 20-30% in rodents), with Cmax reached within 1-2 hours post-administration. It is extensively metabolized by phase II conjugation (glucuronidation, sulfation) and has a terminal half-life of 2-6 hours. The deuterium label may slightly alter PK due to kinetic isotope effects. Storage at -20degC, protected from light.
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| Toxicity/Toxicokinetics |
Formononetin-d3-1 is considered safe for laboratory use under standard chemical safety guidelines. The compound is not intended for human consumption. Formononetin (unlabeled) is generally recognized as safe (GRAS) at dietary intake levels from soy and other legumes. At high doses in animal studies, no significant toxicity was observed. No specific toxicity data for the deuterated form are available. Standard laboratory precautions including gloves, lab coat, and safety glasses should be used. Avoid inhalation and skin contact. Dispose of waste according to institutional guidelines.
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| References | |
| Additional Infomation |
Formononetin-d3-1 is a research-grade compound exclusively for in vitro analytical applications. It is used as an internal standard for the accurate quantification of Formononetin in biological matrices (plasma, urine, tissues) by LC-MS/MS. Formononetin (7-hydroxy-4′-methoxyisoflavone) is a bioactive isoflavone found in Trifolium pratense (red clover), Astragalus membranaceus, and Glycyrrhiza species. The d3 isotope labeling creates a 3.03 Da mass shift for precise quantification. The compound is for research use only and not for diagnostic or therapeutic applications.
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| Molecular Formula |
C16H9D3O4
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| Molecular Weight |
271.28
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| Related CAS # |
Formononetin;485-72-3
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| Appearance |
Typically exists as solid at room temperature
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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 | 3.6862 mL | 18.4311 mL | 36.8623 mL | |
| 5 mM | 0.7372 mL | 3.6862 mL | 7.3725 mL | |
| 10 mM | 0.3686 mL | 1.8431 mL | 3.6862 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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