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| Targets |
As an isotope-labeled compound, the target of 2-Chloroaniline hydrochloride-13C6 is the analytical instrument itself, such as GC-MS or LC-MS. It is not designed to have biological activity but rather mimics the physicochemical behavior of its non-labeled counterpart (2-chloroaniline). The primary role of this deuterated compound is to act as a precise internal standard. It provides a distinct mass shift of +6 Da that does not interfere with the analyte's quantitation signal. This allows it to correct for variables in sample preparation, matrix effects, ion suppression, and fluctuations in instrumental response. In environmental and toxicological research, the unlabeled 2-chloroaniline is a known metabolite of various herbicides and azo dyes. It is considered a priority pollutant and serves as a biomarker for exposure to certain toxic substances.
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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 2-Chloroaniline hydrochloride-13C6 itself has not been reported, as it is used solely as an analytical internal standard. The in vitro activity of its unlabeled counterpart, 2-chloroaniline, has been studied. In HepG2 human liver cancer cell lines, 2-chloroaniline induces cytotoxicity with an IC₅0 value ranging from 250-500 uM after 24 hours of exposure. Mechanistically, it has been shown to increase levels of reactive oxygen species (ROS), induce lipid peroxidation, and cause DNA damage as measured by the Comet assay. Additionally, it has been shown to alter the expression of various genes involved in xenobiotic metabolism, particularly CYP1A1 and CYP1A2, which are involved in its biotransformation. In environmental microbiology, it can inhibit the growth of certain bacterial strains at millimolar concentrations. |
| ln Vivo |
In vivo, the biological activity of 2-Chloroaniline hydrochloride-13C6 itself has not been characterized because it is not a pharmacologically active drug. It is used exclusively as an analytical standard. However, its parent compound, 2-chloroaniline, has known systemic toxicity in animals. Following oral exposure, it is readily absorbed and distributed throughout the body. In rats, it has been shown to induce methemoglobinemia due to its ability to oxidize hemoglobin to methemoglobin, leading to hypoxia. Subchronic exposure results in lesions in the spleen and liver. It is also a suspected carcinogen; in a 2-year bioassay, oral administration of 2-chloroaniline hydrochloride to rats and mice led to an increased incidence of hemangiosarcomas and hepatocellular adenomas. The labeled version is used in tracer studies to understand the metabolism and distribution of 2-chloroaniline without confounding background.
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| Enzyme Assay |
A generic non-cell-based assay protocol for 2-Chloroaniline hydrochloride-13C6 involves its use as an internal standard in a matrix-matched calibration curve. First, prepare a standard stock solution of the unlabeled 2-chloroaniline hydrochloride in methanol at 1 mg/mL. Prepare separate stock solutions of the internal standard (2-Chloroaniline hydrochloride-13C6) at the same concentration. Prepare calibration standards by serially diluting the unlabeled analyte in control matrix (e.g., blank river water, urine, or soil extract) to achieve concentrations ranging from 0.5 to 500 ng/mL. To each calibration standard, add a fixed concentration of the internal standard (e.g., 50 ng/mL). Also prepare blank and double-blank samples. Mix thoroughly. Proceed with liquid-liquid extraction (using ethyl acetate) or solid-phase extraction (SPE). Analyze all samples by LC-MS/MS in MRM mode monitoring the specific mass transitions for both the analyte and the internal standard. Construct the calibration curve by plotting the peak area ratio (analyte/IS) vs. nominal concentration.
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| Cell Assay |
A standard in vitro cell-based assay for the unlabeled 2-Chloroaniline involves assessing its cytotoxicity in the HepG2 human hepatoma cell line. First, culture HepG2 cells in DMEM supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin in a 5% CO2 incubator at 37degC. Seed the cells at a density of 1×10⁴ cells per well in 96-well plates and allow them to attach overnight. Treat the cells with increasing concentrations of unlabeled 2-chloroaniline hydrochloride (10 uM to 10 mM) or vehicle control (PBS) for 24 hours. After the exposure period, measure cell viability using the MTT assay: add 10 uL of MTT solution (5 mg/mL) to each well, incubate for 3 hours, then solubilize the formazan crystals in 100 uL of DMSO. Measure absorbance at 570 nm using a microplate reader. Calculate the half-maximal inhibitory concentration (IC₅0) from the dose-response curve using GraphPad Prism. Use the deuterated compound as an LC-MS internal standard to confirm exposure concentrations in the cell culture medium.
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| Animal Protocol |
A typical in vivo animal experiment for 2-Chloroaniline hydrochloride-13C6 is used to study the metabolism and distribution of its parent compound. A reference protocol uses male Sprague-Dawley rats (200-250 g). Dose the animals orally with a single dose of unlabeled 2-chloroaniline hydrochloride (e.g., 50 mg/kg) suspended in corn oil. Include a control group receiving vehicle only. Collect blood samples via tail vein at various time points (0, 0.5, 1, 2, 4, 8, 12, and 24 hours) post-dosing into heparinized tubes. Immediately separate plasma by centrifugation. At the end of the time course, euthanize the animals and collect liver, kidney, and brain tissues. Homogenize the tissues in phosphate-buffered saline (PBS). Extract 2-chloroaniline and its metabolites from plasma and tissue homogenates using solid-phase extraction (SPE). Use 2-Chloroaniline hydrochloride-13C6 as the internal standard and analyze all samples by LC-MS/MS to quantify the total and free concentrations of the compound and to identify its major metabolites.
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| ADME/Pharmacokinetics |
The pharmacokinetic profile of 2-Chloroaniline hydrochloride-13C6 is expected to be identical to that of its non-labeled analog, as it is an internal standard and not a therapeutic agent. The PK of unlabeled 2-chloroaniline has been studied in animal models. In rats, following oral administration, it is rapidly absorbed with peak plasma concentrations (Tmax) occurring between 0.5 to 2 hours. It exhibits a moderate distribution volume and is extensively bound to plasma proteins (approx. 70-80%). The compound is extensively metabolized, primarily via CYP450 enzymes, to form 2-amino-5-chlorophenol, 4-amino-3-chlorophenol, and various sulfate or glucuronide conjugates. The elimination half-life ranges from 6 to 12 hours in rodents. Excretion occurs primarily via urine (approx. 70% of the dose within 48 hours). The labeled compound (¹3C₆) is used as an internal standard to accurately determine these parameters in mass spectrometry assays.
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| References | |
| Additional Infomation |
2-Chloroaniline hydrochloride-13C6 is a stable isotope-labeled compound used exclusively for research purposes, primarily as an internal standard for the quantification of 2-chloroaniline in complex matrices via GC-MS or LC-MS. The unlabeled 2-chloroaniline is a high-production-volume chemical intermediate. It is a known environmental contaminant commonly found in wastewater from textile and dye industries. The compound is considered a priority pollutant by the EPA due to its potential toxicity. It is a primary metabolite of several herbicides (e.g., propanil) and azo dyes. The compound has been shown to cause methemoglobinemia and is a suspected carcinogen. Toxicological studies have reported an oral LD₅0 of 256 mg/kg in rats. When handling this material, researchers should follow standard safety procedures due to its toxic nature, and it should be stored at 4degC away from moisture.
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| Molecular Formula |
C6H7CL2N
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| Molecular Weight |
169.988408327103
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| Exact Mass |
169.015
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| CAS # |
1261170-86-8
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| PubChem CID |
162642404
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| Appearance |
White to off-white solid powder
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| Hydrogen Bond Donor Count |
2
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| Hydrogen Bond Acceptor Count |
1
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| Rotatable Bond Count |
0
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| Heavy Atom Count |
9
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| Complexity |
74.9
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| Defined Atom Stereocenter Count |
0
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| SMILES |
[13CH]1=[13CH][13CH]=[13C]([13C](=[13CH]1)N)Cl.Cl
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| InChi Key |
DRGIDRZFKRLQTE-BVNCJLROSA-N
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| InChi Code |
InChI=1S/C6H6ClN.ClH/c7-5-3-1-2-4-6(5)8;/h1-4H,8H2;1H/i1+1,2+1,3+1,4+1,5+1,6+1;
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| Chemical Name |
6-chloro(1,2,3,4,5,6-13C6)cyclohexa-1,3,5-trien-1-amine;hydrochloride
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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: Please store this product in a sealed and protected environment, avoid exposure to moisture. |
| 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) |
DMSO: 250 mg/mL (1470.67 mM)
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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 | 5.8827 mL | 29.4135 mL | 58.8270 mL | |
| 5 mM | 1.1765 mL | 5.8827 mL | 11.7654 mL | |
| 10 mM | 0.5883 mL | 2.9413 mL | 5.8827 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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