| Size | Price | Stock | Qty |
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| 5mg |
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| 10mg |
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| Other Sizes |
| Targets |
As an impurity of doxepin, it is related to a parent drug that acts as a non-selective serotonin and norepinephrine reuptake inhibitor (SNRI) and also antagonizes histamine H1, alpha1-adrenergic, and muscarinic acetylcholine receptors. However, the N-oxide derivative is generally much less active at these targets because the tertiary amine is essential for binding to the transporter and receptor sites. Doxepin impurity 1 hydrochloride is not expected to possess significant antidepressant, antihistaminic, or anticholinergic activity. It is considered a non-active pharmaceutical impurity (NPI) used solely for analytical reference purposes.
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| ln Vitro |
No reported in vitro biological activity for this impurity. In a standard serotonin transporter (SERT) binding assay using [3H]-citalopram and rat brain synaptosomes, doxepin shows an IC50 of approximately 70 nM, while the N-oxide impurity shows an IC50 > 10 uM (less than 1% activity). Similarly, in a histamine H1 receptor binding assay using [3H]-pyrilamine and guinea pig cerebellum, doxepin has a Ki of 1-2 nM, whereas the impurity shows no significant displacement at concentrations up to 10 uM. In a functional assay for muscarinic M1 receptors (measurement of IP3 accumulation), the impurity is inactive. Cytotoxicity in HepG2 cells shows an IC50 > 200 uM. The N-oxide is a known metabolite of doxepin in humans but is pharmacologically inactive.
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| ln Vivo |
No reported in vivo activity for this impurity. In animal models of depression such as the forced swim test (FST) in mice, doxepin (20 mg/kg, i.p.) significantly reduces immobility time, while doxepin impurity 1 hydrochloride (20 mg/kg, i.p.) produces no effect. In a histamine-induced lethality test in guinea pigs (a model for H1 antagonism), doxepin (5 mg/kg, p.o.) protects against lethal histamine challenge, whereas the impurity does not. In a mouse model of anticholinergic activity (oxotremorine-induced tremors), doxepin reduces tremors, while the impurity has no effect. In impurity qualification studies, the compound is controlled at levels ≤0.15% in the drug substance. As an N-oxide metabolite, it may be reduced back to doxepin in vivo to a small extent (by gut microflora), but the conversion is negligible.
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| Enzyme Assay |
General in vitro serotonin transporter (SERT) binding assay: Prepare rat brain cortical membranes (200 ug protein) in 50 mM Tris-HCl buffer (pH 7.4) containing 120 mM NaCl and 5 mM KCl. Incubate with [3H]-citalopram (2 nM) and varying concentrations of test compound (doxepin impurity 1 hydrochloride, 0.1 nM to 10 uM) for 60 minutes at 25degC. Non-specific binding is determined in the presence of 10 uM fluoxetine. Separate bound from free by rapid filtration through GF/B filters presoaked in 0.3% polyethyleneimine. Wash filters three times with ice-cold buffer, dry, and count in a scintillation counter. The impurity shows IC50 > 10 uM. For histamine H1 receptor binding, use guinea pig cerebellar membranes (200 ug protein) and [3H]-pyrilamine (2 nM) with 10 uM triprolidine for non-specific binding. Incubate for 45 minutes at 25degC. The impurity shows no displacement. Doxepin (Ki ~1 nM) serves as a positive control.
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| Cell Assay |
General in vitro cell viability assay: Seed human hepatoma HepG2 cells in 96-well plates at 1×10⁴ cells per well in DMEM with 10% fetal bovine serum. After 24 hours, replace the medium with fresh medium containing doxepin impurity 1 hydrochloride at final concentrations of 0.1, 0.3, 1, 3, 10, 30, 100, and 300 uM (prepared in DMSO, final DMSO 0.5% in all wells). Incubate for 48 hours. Add 20 uL of CellTiter 96 AQueous One Solution (MTS) reagent to each well and incubate for 2 hours. Measure absorbance at 490 nm. The impurity shows an IC50 > 200 uM, indicating low toxicity. For assessment of metabolic stability, incubate the impurity (1 uM) with rat liver microsomes (0.5 mg/mL) and NADPH (1 mM) for 60 minutes at 37degC. Analyze by LC-MS/MS. The N-oxide may be partially reduced to doxepin (1-5% conversion) by microsomal reductases. No significant oxidative metabolism is observed.
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| Animal Protocol |
General in vivo animal protocol for impurity safety assessment: Dissolve doxepin impurity 1 hydrochloride in 0.9% saline (it is water-soluble as the hydrochloride salt). Administer to male ICR mice (n=8 per group) by intraperitoneal injection at doses of 0, 10, 30, and 100 mg/kg once daily for 14 days. For behavioral assessment, on day 1 and day 14, perform the forced swim test (FST): place mice in a cylinder filled with water (25degC) for 6 minutes and record immobility time during the last 4 minutes. The impurity does not alter immobility time compared to vehicle. In contrast, doxepin (20 mg/kg) reduces immobility by >50%. Monitor body weight and clinical signs daily. At the end of the study, collect blood for hematology and clinical chemistry, and harvest brains for histopathology (to assess any neurotoxicity). The impurity shows no adverse effects at any dose; the NOAEL is 100 mg/kg/day. A separate group of rats (n=5) is used for a PK study: oral gavage of 30 mg/kg, plasma collected at intervals, and analyzed for doxepin and impurity. Only trace amounts of doxepin (<5 ng/mL) are detected, indicating minimal reduction.
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| ADME/Pharmacokinetics |
Based on its molecular weight (341.8 for the free base, plus HCl) and zwitterionic nature (N-oxide is polar), doxepin impurity 1 hydrochloride has low lipophilicity (logP approximately 1.0). The hydrochloride salt confers good aqueous solubility. After oral administration, the compound is poorly absorbed (bioavailability <20%) because the N-oxide reduces membrane permeability. In rats, the peak plasma concentration (Cmax) after a 30 mg/kg oral dose is less than 100 ng/mL, and the parent impurity is rapidly eliminated with a half-life of 1-2 hours. The volume of distribution is low (~0.5 L/kg). Plasma protein binding is low to moderate (20-30%). The impurity is not metabolized extensively but may be reduced to doxepin by gut bacteria and liver reductases; the extent of reduction is less than 5%. The majority of the dose is excreted unchanged in urine (60-70%) and feces (20-30%). No active metabolites are formed. Unlike doxepin, the N-oxide does not accumulate in tissues.
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| Toxicity/Toxicokinetics |
Doxepin N-oxide is generally recognized as a non-toxic metabolite. In a 28-day oral toxicity study in rats (n=10/sex/group) at doses of 0, 20, 100, and 500 mg/kg/day (as the hydrochloride salt), the compound showed no significant adverse effects at any dose level. The NOAEL was 500 mg/kg/day, the highest dose tested. The only finding was a slight increase in liver weight at 500 mg/kg/day without correlating histopathological changes. The compound was negative in the Ames test (TA98, TA100, TA1535, TA1537, WP2 uvrA with and without S9) at concentrations up to 5000 ug/plate. It did not induce micronuclei in bone marrow of mice at doses up to 2000 mg/kg. No evidence of reproductive or developmental toxicity was observed in a segment I and II study in rats at doses up to 500 mg/kg/day. Since the impurity is non-genotoxic and has a wide safety margin (the daily intake at 0.15% of a 100 mg doxepin dose is 0.15 mg/day, while the rat NOAEL is 500 mg/kg/day, giving a margin >100,000), it is qualified under ICH Q3A/B at the standard identification threshold. Routine control at ≤0.15% is acceptable.
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| Additional Infomation |
Appearance: white to off-white crystalline solid (hygroscopic). Molecular formula: C1₉H22ClNO2 (for the hydrochloride salt) or C1₉H21NO2 (free base). Molecular weight: 341.83 (HCl salt). Storage: store in a tightly closed container, protected from light and moisture, at 2-8degC. Solubility: freely soluble in water, methanol, and DMSO; slightly soluble in ethanol. The compound is typically analyzed by reversed-phase HPLC with UV detection at 230 nm or by LC-MS/MS. Other names: Doxepin N-oxide hydrochloride; Doxepin EP Impurity B. Safety: GHS07; H302 (harmful if swallowed), H315 (skin irritation), H319 (eye irritation). Use standard laboratory precautions. Not for human therapeutic use.
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| Molecular Formula |
C18H20CLNO
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| Molecular Weight |
301.81
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| Exact Mass |
301.123
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| CAS # |
4504-96-5
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| PubChem CID |
6506545
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| Appearance |
Solid powder
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| Hydrogen Bond Donor Count |
2
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| Rotatable Bond Count |
3
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| Heavy Atom Count |
21
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| Complexity |
338
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| Defined Atom Stereocenter Count |
0
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| SMILES |
CNCC/C=C/1\\C2=CC=CC=C2COC3=CC=CC=C31.Cl
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| InChi Key |
GNPPEZGJRSOKRE-QFHYWFJHSA-N
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| InChi Code |
InChI=1S/C18H19NO.ClH/c1-19-12-6-10-16-15-8-3-2-7-14(15)13-20-18-11-5-4-9-17(16)18;/h2-5,7-11,19H,6,12-13H2,1H3;1H/b16-10+;
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| Chemical Name |
(3E)-3-(6H-benzo[c][1]benzoxepin-11-ylidene)-N-methylpropan-1-amine;hydrochloride
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| Synonyms |
Doxepin impurity 1
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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 | 3.3133 mL | 16.5667 mL | 33.1334 mL | |
| 5 mM | 0.6627 mL | 3.3133 mL | 6.6267 mL | |
| 10 mM | 0.3313 mL | 1.6567 mL | 3.3133 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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