Anandamide-d4 targets the same receptors as anandamide: the cannabinoid CB1 receptor (CB1R) and CB2 receptor (CB2R). CB1R is predominantly expressed in the central nervous system and regulates neurotransmitter release, pain perception, appetite, and memory. CB2R is primarily expressed in immune cells and modulates inflammation. Anandamide is an endogenous agonist (endocannabinoid) with higher affinity for CB1 (Kᵢ ~10-50 nM) than for CB2 (Kᵢ ~500-1000 nM). It is also a partial agonist at the transient receptor potential vanilloid type 1 (TRPV1) channel and a weak agonist at the GPR55 receptor. Anandamide is synthesized from N-arachidonoyl-phosphatidylethanolamine (NAPE) by NAPE-PLD and degraded by FAAH to arachidonic acid and ethanolamine. The deuterated form has the same receptor binding and functional activity (as a tracer), but it is not used for pharmacological studies (due to cost). Instead, it is used as an internal standard for quantifying endogenous anandamide levels in biological samples (plasma, brain, urine). The target for analysis is the compound itself, measured by mass spectrometry. The compound does not have a therapeutic target; it is a stable isotope-labeled standard.

Stable heavy isotopes of hydrogen, carbon, and other elements have been incorporated into drug molecules, largely as tracers for quantitation during the drug development process. Studies involving the human use of drugs labeled with deuterium suggest that these compounds may offer some advantages when compared with their nondeuterated counterparts. Deuteration has gained attention because of its potential to affect the pharmacokinetic and metabolic profiles of drugs. Deutetrabenazine is the first deuterated drug to receive Food and Drug Administration approval. This deuterated form of the drug tetrabenazine is indicated for the treatment of chorea associated with Huntington's disease as well as tardive dyskinesia. Ongoing clinical trials suggest that a number of other deuterated compounds are being evaluated for the treatment of human diseases and not merely as research tools.

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In vitro, anandamide-d4 is not used for biological activity assays; it is used as an internal standard for LC-MS/MS. For lipid extraction, add 10-100 ng of anandamide-d4 to 100 uL plasma or tissue homogenate (brain) as an internal standard. Extract with ethyl acetate or hexane:isopropanol (3:2). Separate on a C18 column (2.1 × 100 mm) with mobile phase: water:acetonitrile:formic acid (0.1%). Detection in positive ion mode: parent [M+H]+ m/z 352.3 (for d4) vs. 348.3 (for unlabeled anandamide). The retention time for anandamide-d4 is the same as that for anandamide (∼3-4 min). The calibration curve for anandamide (0.1-100 ng/mL) is prepared using anandamide-d4 as internal standard. The method has an LLOQ of 0.1 ng/mL. In enzyme assays, anandamide-d4 can be used as a substrate for FAAH. Incubate 1 uM anandamide-d4 with recombinant FAAH (10 ng) in 50 mM Tris-HCl (pH 7.5), 1 mM EDTA at 37degC for 15 min. The product, arachidonic acid-d4 and deuterated ethanolamine, is measured by LC-MS. The deuterated ethanolamine (mass shift +4) distinguishes it from endogenous ethanolamine. For NAPE-PLD activity, incubate 1 uM NAPE substrate with anandamide-d4 as internal standard. The compound is stable in plasma at -80degC for years. In cell-based assays, anandamide-d4 (0.1-10 uM) could, in principle, activate CB1 receptors, but due to its cost, it is not used for such purposes. For receptor binding (non-cellular), anandamide-d4 can be used as a radioligand substitute for [3H]-CP-55,940 or [3H]-WIN-55,212-2, but this is not standard practice. For research, it is exclusively an analytical standard.

Deuterated compounds may, in some cases, offer advantages over nondeuterated forms, often through alterations in clearance. Deuteration may also redirect metabolic pathways in directions that reduce toxicities. The approval of additional deuterated compounds may soon follow. Clinicians will need to be familiar with the dosing, efficacy, potential side effects, and unique metabolic profiles of these new entities.

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In vivo, anandamide-d4 is not administered to animals; it is used as an internal standard for measuring endogenous anandamide levels in tissues. For example, in a mouse model of chronic pain, brain and spinal cord tissues are collected, homogenized, and extracted with organic solvent. Anandamide-d4 (10 ng) is added to each sample before extraction to correct for recovery and matrix effects. The ratio of endogenous anandamide to anandamide-d4 is used to calculate the concentration. In pharmacokinetic studies of anandamide itself, the d4 internal standard is added after sample collection (not dosed). The deuterated compound is stable and does not undergo deuterium exchange. For quantification, the method is validated in accordance with FDA guidelines for bioanalytical methods. The compound is not used for imaging or therapy. Its LD₅0 is not determined, as it is not administered. For research, the compound is considered safe if handled properly. It is not a controlled substance.

General protocol for in vitro use (as internal standard): Prepare stock solution of anandamide-d4 in methanol (1 mg/mL). Dilute to a working concentration of 100 ng/mL in methanol. For plasma samples (100 uL), add 10 uL of working solution (1 ng). Add 1 mL of ethyl acetate:hexane (1:1), vortex for 2 min, centrifuge at 10,000×g for 5 min, transfer the organic layer to a new tube, evaporate under nitrogen, and reconstitute in 100 uL methanol:water (1:1). Inject 10 uL onto LC-MS/MS. Column: C18 (2.1 × 50 mm, 1.7 um). Mobile phase A: 0.1% formic acid in water; B: 0.1% formic acid in acetonitrile. Gradient: 30% B to 95% B over 5 min. Detection: MRM: anandamide (m/z 348.3 → 62.1), anandamide-d4 (m/z 352.3 → 66.1). The retention time is ~3.5 min. The calibration curve for anandamide (0.1, 0.5, 1, 5, 10, 25, 50, 100 ng/mL) is prepared in surrogate matrix (charcoal-stripped plasma) or in buffer. Linear regression with 1/x2 weighting. The LLOQ is 0.1 ng/mL. For brain tissue, homogenize 50 mg tissue in 500 uL of water, add 10 uL of internal standard, and extract with ethyl acetate as above. For FAAH activity assay, incubate anandamide-d4 (1 uM) with FAAH (10 ng) in 100 uL assay buffer (50 mM Tris-HCl pH 7.5, 1 mM EDTA) for 15 min at 37degC. Stop with 200 uL of acetonitrile containing 0.1% formic acid. Centrifuge and inject onto LC-MS. The product arachidonic acid-d4 (m/z 307.3 → 289.3) and ethanolamine-d4 (m/z 66.1) are monitored. For NAPE-PLD, incubate NAPE-d4 (or unlabeled NAPE with d4 internal standard). For stability, store anandamide-d4 in ethanol at -80degC; it is stable for at least 2 years. Avoid freeze-thaw cycles (degrade after 5 cycles). For handling, wear gloves and work in a fume hood. The compound is not classified as hazardous.

General protocol for in vivo animal experiments: Anandamide-d4 is not administered in vivo; it is used as an ex vivo internal standard. For a typical study, treat mice with a drug that modulates the endocannabinoid system (e.g., the FAAH inhibitor URB597, 10 mg/kg, IP). After 2 h, euthanize mice, perfuse with PBS, and collect brain regions (e.g., hippocampus, prefrontal cortex). Homogenize 100 mg tissue in 1 mL of 0.1 M HCl (to inhibit FAAH). Add 10 uL of anandamide-d4 (100 ng/mL) as internal standard. Extract with 3 mL of chloroform:methanol (2:1). Centrifuge, collect the organic phase, evaporate, and reconstitute. Quantify by LC-MS/MS. The concentration of anandamide is expressed as ng/g tissue. In a chronic pain model (e.g., CFA-induced inflammatory pain), measure anandamide levels in the periaqueductal gray (PAG) or spinal cord. For pharmacokinetic studies of anandamide itself, administer unlabeled anandamide (5 mg/kg, IV) to rats, collect plasma at various times, and analyze using anandamide-d4 as internal standard. The half-life of anandamide is very short (t1/2 <5 min) due to rapid hydrolysis by FAAH. For FAAH activity in vivo, administer the FAAH inhibitor, then quantify the anandamide-d4 added ex vivo. All animal work requires IACUC approval.

General pharmacokinetic properties: Anandamide-d4 has the same physicochemical properties as anandamide: MW 351.56, LogP ∼5.5. When used as an internal standard, it is not dosed. For information, anandamide (unlabeled) has a plasma half-life of 5-10 min in rodents due to rapid hydrolysis by FAAH. It is distributed to the brain, liver, and lungs. Volume of distribution (Vd) is high (5-10 L/kg). Oral bioavailability is low (<10%). The compound is not a drug; it is a standard. For storage, anandamide-d4 is supplied in ethanol (100 ug/mL) or as a solid. Store at -80degC, protect from light. Avoid repeated freeze-thaw cycles. The compound is stable for at least 1 year at -80degC. For LC-MS, the internal standard is stable in solution at -20degC for 1 month. For research use only; not for clinical or therapeutic applications.

General toxicity profile: Anandamide-d4 is a stable isotope-labeled compound with low toxicity. At the concentrations used as an internal standard (ng/mL), it is non-toxic. In vitro, anandamide-d4 at 1 uM does not affect cell viability (MTT >95%). In acute toxicity studies in mice, administration of anandamide (unlabeled) at 50 mg/kg (IV) causes hypothermia, sedation, and catalepsy, but no mortality. The LD₅0 is >100 mg/kg. For the deuterated version, similar toxicity is expected, but it is not used in vivo. The compound is not a controlled substance (anandamide is not scheduled in most countries). Standard safety precautions (gloves, lab coat) are sufficient. For research use only.

Anandamide-d4 (AEA-d4) is also known as N-(2-hydroxyethyl-1,1,2,2-d4)-5Z,8Z,11Z,14Z-eicosatetraenamide. The molecular formula is C22H33D4NO2, MW 351.56. The isotopic purity is >98% atom % D. The compound is supplied as a solution in ethanol (100 ug/mL) or as a solid. It is used as an internal standard for the quantification of anandamide in biological samples. Anandamide is an endocannabinoid involved in pain regulation, appetite, and mood. The deuterated standard is essential for accurate LC-MS/MS quantification. For research use only. Not for clinical or therapeutic use.

C22H33D4NO2

351.56

351.308

946524-40-9

Anandamide; 94421-68-8

24762159

Liquid

2

16

25

408

0

[2H]C([2H])(C([2H])([2H])O)NC(=O)CCC/C=C\\C/C=C\\C/C=C\\C/C=C\\CCCCC

LGEQQWMQCRIYKG-KALLKTRHSA-N

InChI=1S/C22H37NO2/c1-2-3-4-5-6-7-8-9-10-11-12-13-14-15-16-17-18-19-22(25)23-20-21-24/h6-7,9-10,12-13,15-16,24H,2-5,8,11,14,17-21H2,1H3,(H,23,25)/b7-6-,10-9-,13-12-,16-15-/i20D2,21D2

(5Z,8Z,11Z,14Z)-N-(1,1,2,2-tetradeuterio-2-hydroxyethyl)icosa-5,8,11,14-tetraenamide

AEA-d4; Arachidonoyl ethanolamide-d4

2934.99.9001

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.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~100 mg/mL (~284.45 mM; with sonication)

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.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*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).

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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)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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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).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)